Title of Invention	A PHARMACEUTICAL COMPOSITION COMPRISING 5-HT2c RECEPTOR AGONIST AND 5-HT6 RECEPTOR ANTOGONIST
Abstract	A phannaceutical composition comprising an effective amount of a combination of a 5-HT2c receptor agonist and a 5-HT6 receptor antagonist, or a salt, or enantiomer of the said agonist and/or antagonist, and a pharmaceutically acceptable carrier.

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TECHNICAL FELX) The present invention relates to the prophylaxis or treatment of a 5-HT2c and a S-HTg receptor-related disease. In addition, the invention provides a pharmaceutical coniposition containing a S-HTac receptor agonist and a S-HTe receptor antagonist for therapeutic use. BACKGROUND ART Serotonin (5-hydroxytryptamine or 5-HT) is a key neurotransmitter of the peripheral and central nervous system (PNS and CNS) and has heen implicated in a variety of sensory, motor and behavioral functions such as regulation of eating, sleeping, body temperature, blood pressure, emotions and cognition. At least 14 distinct serotouiu receptor subtypes are expressed in the mammalian PNS and CNS and have been formally classified; see Glennon, et al., Neurosci. Biobehav. Rev. 1990,14,35-37; and D. Hoyer, et al., Pharmacol Rev. 1994, 46,157-203. Serotoninergic agonists and antagonists have been suggested for the treatment of a wide range of disorders, including anxiety, depression, hypertension, migraine, obesity, drug abuse and addiction, compulsive disorders, schizophrenia, autism, neurodegenerative disorders (e.g. Alzheimer's disease. Parkinsonism, and Huntington's chorea), and chemotherapy-induced vomiting. The 5-HT2 subfamily of receptors is composed of three subtypes, the 5-HT2A» 5-HT2B and 5-HT2C receptors. Serotoniu 5-HT2c receptors are expressed hi many brain regions and have been unplicated in the regulation of food intake (Douiish, C.T. Obes. Res, 1995,5, Suppl 4,449S-462S; Bickerdike, M.J., et al. Diabetes, Obes, Metab, 1999, i, 207-214). It has been demonstrated tiiat the non-specific 5-HT2C recqjtor agonist m-chlorophenylpiperazine (m-CPP), which has some preference for the 5-HT2C receptor, reduces food mtake in mice that express the normal 5-HT2C receptor while the compound lacks activity in mice expressiag the mutated inactive form of the 5-HT2C receptor (Tecott, L.H., et al. Nature 1995, 374, 542-546). . Moreover, it has been reported that m-CFP aad the azepinoindole U-22394A, the latter recently identified to be a 5-HT2c receptor agonist (unpublished observation), reduce body weight ID humans following two and nine weeks of treatment, respectively (Walsh, A. E. S., Psychopharmacology 1994,116,120-122; Sargent, PA., et al. Psychopharmacology 1997, 735, 309-312 and Gallant, DM., et al. Curr. Ther. Res. 1967,5,579-581). Recently, a series of pyrrolo[3,2,l-f/]quinoline derivatives was identified to be 5-HT2C receptor agonists having selectivity over the 5-HT2A. receptor (Isaac M., et al., Bioorg. Med Chem. Lett. 2000,10, 919-921). Tte compounds are said to offer a novel approach to the treatment of obesity and epilepsy. The 5-HT2C i^&ceptor subtype has also been suggested to be involved in CNS disorders, such as depression and anxiety (Jenck, F., et al. Expert Opin. Invest. Drugs 1998, 7,1587-1599; Leysen, D.C.M. iZJrwgs 1999,2,109-120). The 5-HT2C receptor subtype has further been suggested to be involved in urinary disorders such as urinary incontinence (Leysen, D.C.M. mrugs 1999,2,109-120). Also the 5-HT5 receptor (identified in 1993 - Monsma et al., Mol. PharmacoL^ 1993, 43, 320-327 and Ruat, M. et al. Biochem. Biophys. Res, Commun. 1993,193,269-276) has been implicated in the regulation of food intake and CMS disorders. Thus, for example, Bentley, J. C, et al., Br J. Pharmacol. 1999,126, 65P describes food intake reduction in rats by the administration of a S-HTg antagonist Also, several antidepressants and atypical antipsychotics display high affinity for the 5-HT5 receptor which have suggested the involvement of the 5-HT6 receptor in schizophrenia (Roth et al. 1 Pharmacol Exp. Ther 1994,268, 1403-1410; Sleight et al. Expert Opin. Ther Patents 1998, 8,12174224; Bourson et al. Br J. Pharm, 1998,125, 1562-1566; Boess et al. Mol. Pharmacol. 1998, 54, 577-583; Slei^t et al. Br J. Pharmacol 1998,124, 556-562). In addition, the 5-HT5 receptor has been linked to generalized stress and anxiety states (Yoshioka et al. Life Sci 1998,17/18,1473-1477). SUMMARY OF THE INVENTION According to the present invention it has now unexpectedly been found that the combined administration of a 5-HT2C receptor agonist and a 5-HT5 receptor antagonist reduces food intake by more than the administration of either agonist or antagonist alone. Such combined administration of a 5-HT2c receptor agonist and a 5-HT5 receptor antagonist may offer therapeutic advantages as compared to treatment with either agonist or antagonist alone. One aspect of the present invention therefore provides a pharmaceutical composition coiiq)rising an effective amount of a combination of a 5-HT2c receptor agonist and a 5-HT5 receptor antagonist, and optionally a pharmaceutically acceptable carrier. Another aspect of the invention provides a method of preventing or treating a disease, in particular obesity, related to the 5-HT2C receptor and the 5-HT5 receptor, con^rising administering to a human or animal subject in need thereof a 5-HT2c receptor agonist and a 5-HT5 receptor antagonist (simultaneously or sequentially) in sufficient amounts to provide a therapeutic effect Still another aspect of the invention provides the use of a 5-HT2C receptor agonist and a S-HTg receptor antagonist for the manufacture of a medicament for the treatment of a disease related to the 5-HT2C receptor and the S-HTg receptor. Another aspect of the invention provides a process for preparing a pharmaceutical composition, wherein a 5-HT2C receptor agonist and a S-HTg receptor antagonist in a combined therapeutic amount are intimately mixed with a pharmaceutically acceptable carrier. Yet another aspect of the invention provides a product containing a 5-HT2c receptor agonist and a S-HT^ receptor antagonist as a combined preparation for simultaneous, separate or sequential use in therapy of a disease, in particular obesity, related to the 5-HT2c receptor and the S-HTg receptor. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the effect on food intake in ob/ob mice following combined administration with a 5-HT2C receptor agonist (PNU-183933F; 50 mg/kg po) and a 5-HTg receptor antagonist (PNU-186053 A; 50 mg/kg sc), as well as the eSdCt of each agonist and antagonist alone. Figure 2 shows the effect on food intake in ob/ob mice following combined administration of a 5-HT2c receptor agonist (BVT.2938F; 5 mg/kg sc) and a S-HTg receptor antagonist (BVT.5182C; 3 mg/kg sc), as well as the effect of each agonist and antagonist alone. DETAILED DESCRIPTION OF THE INVENTION As naentioned above, the present invention is based on the unexpected finding that combined administration of a 5-HT2c receptor agonist and a S-HTg receptor antagonist rediK:es food intake more than eithea: agonist or antagonist alone. Such combined administration of a 5-HT2c receptor agonist and a 5-HT^ receptor antagonist may also ofifea: several benefits, for instance in the treatment of obesity, as compared to treatment with either agonist or antagonist alone. Firstly, the combined administration requires lower doses of each compound to yield similar or improved reduction of food intake than mono-ther£5>y. Secondly, the lower doses required by the combined administration may reduce the risk of adverse events. Thirdly, the lower doses required by the combined administration may reduce the risk of tolerance development and abuse liability. Fourthly, therapy based on two targets may increase the individual therapeutic efficacy relative to tfaer^y based on one target The risk of non-responsive efficacy (non-responders) may be reduced as well. The beneficial effects of the combined administration of this invention is useful not only for the modulation of eating behavior, and for treating over-weight and obesity, but may also be useful for the treatment of CNS disorders such as, depression, mania, schizophreniform disorders, anxiety, memory disorders (such as Alzheimer's disease) migraine headache, drug addiction, convulsive disorders, personality disorders, posttraumatic stress syndrome, and sleep disorders as well as for treatment of urinary incontinence (or more generally overactive bladder), sexual dysfunctions, gastrointestinal disorders and glaucoma. The term "5-HT2c rec^tor agonisf' as used herein refers to a compound that causes activation of the serotonin 5-HT2c receptor. The 5-HT2c receptor agonist preferably has an affinity constant, Kf, of less tban 50 nM, preferably less than 20 nM, and an in vitro intrinsic activity, measured as intracellular Ca^+ levels, greater than 20%, preferably greater than 50%, relative to 5-HT (1 |iM). The term "5-HT5 receptor antagonist" as used herein refers to a compound that causes blockade of the serotonin 5-HT5 receptor mediated responses. The S-HTg receptor antagonist preferably has an afBnity constant, Kj, of less than 50 nM, preferably less than 20 nM, and an in vz^ro intrinsic activity, measured as intracellular cAMP levels, less than 50%, preferably less than 20%, relative to 5-HT (1 pM). In vitro assays that may be used for determining the afSnity and the intrinsic activity, respectively, of 5-HT2c receptor agonists and S-HTg receptOT antagonists are known in the art and are also given in the Experimental Part below, as are assays for determining affinity to 5--HT2A a^d 5-HT2B receptors. Generally, the 5-HT2c receptor agonists and S-HTg receptor antagonists should be sufficiently selective not to cause any substantial adverse side effects. The terms "selective" and "substantial" in this context are, however, to be interpreted broadly, the meanings thereof being readily apparent to the skilled person. The 5-HT2C receptor agonist preferably has a selectivity for the 5-HT2c receptor of at least 5, preferably at least 10 and more preferably at least 20, relative to the 5-HT2A> 5-HT23 and S-HTg receptors, respectively (measured as the afBnity ratios 5- HT2A/5-HT20 5-HT2B/5-HT2C and 5.Hr6/5-Hrr2c)- The 5-HT5 receptor antagonist preferably has a selectivity for the 5-HT5 receptor of at least 5, preferably at least 10 and more preferably at least 20, relative to the 5-HT2J^, 5-HT2B and 5-HT2c receptors, respectively (measured as the affinity ratios 5-HT2A/5-HT6' 5-HT2B/5-HT5 and 5-HT2C/5-HT6). Relevant tests to determine whether a compound is a selective 5-HT2C receptor agonist or a selective 5-HT5 receptor antagonist are known in the art, and are, as mentioned above, also outlmed in the Experimental Part below. Compounds known to be 5-HT2C receptor agonists are, for example, azetidine and pyrrolidine derivatives of the type described in EP-A-0863136; tricyclic pyrrole derivatives of the type described in EP-A-0657426; 1-aminoethylindoles of the type described in EP-A-0655440; pyrazinoindoles of the type described in EP-A-0572863; piperazinylpyrazines of the type described in US 4,081,542; indoline derivatives of the type described in WO 00/12475; pyrroloindoles, pyridoindoles and azepinoindoles of the type described in WO 00/12510; indazole derivatives of the type described in WO 00/12482; pyrroloquinolines of the tj'pe described in WO 00/12502; 23,4,4a-tetrahydro-lH-pyra2ino[l,2"a]quinoxalin-5(6i?)ones of the type described in WO 00/35922; indazolylpropylammes of the type described in WO 00/12481; indazoles of the type described in WO 00/17170; piperazinylpyrazines of the type described in WO 00/76984 and in Swedish patent applications Nos. 0004244-0 and 0004245-7, filed on 20 Noveniber 2000; heterocycle fused y-carbolines of ttie type described in WO 00/77001, WO 00/77002 and WO 00/77010; benzofurylpiperazines of the type described in WO 01/09111 and WO 01/09123; benzofurans of the type described in WO 01/09122; benzothiophenes of tbe type described in 01/09126; pyridinylpiperazines of the type described in EP 370560; pyrroloquinolines of the type described in Bioorg. Med. Chenx Lett 2000,10,919-921; aminoalkylindazoles of the type described in WO 98/30548; indoles of the type described in WO 01/12603; indolines of the type described in WO 01/12602; pyrazino(aza)indoles of the type described in WO 00/44753; tricyclic pyrroles or pyrazoles of the type described in WO 98/56768. Currently preferable 5-HT2c receptor agonists are of the arylpiperazine and piperazinylpyrazine compound classes, in particular compounds disclosed in WO 00/76984 and in Swedish patent applications Nos. 0004244-0 and 0004245-7, filed on 20 November 2000. Compounds known to be 5-HT6 receptor antagonists are, for example, piperazinylbenzenesulfonamides of the type described in WO 99/37623; sulfonylbenzene derivatives of the type described in EP-A-0930302; sulfonamide derivatives of the type described in WO 99/02502; sulfonamide derivatives of the type described in WO 99/42465; sulfonamide derivatives of the type described in WO 98/27081; carboxamide derivatives of the type described in WO 98/27058; sulfonamide derivatives of the type described in EP-A-0815861; pyrrolidonomethylindole derivatives of the type described in WO 99/47516; bicyclic piperidine and piperazine derivatives of the type described in WO 99/65906; pyrazolopyrimidine and pyrazolotriazdne derivatives of the type described in H^-A-0941994; aiylsulfone- substituted hexahydroazepinoindoles of the type described in WO 01/05793; oxazinocarbazoles of the type described in WO 01/09142; aminoalkoxycarbazoles of the type describedin WO 01/17963; diphenylsulfones of the type described in the ^ intemadonal patent application PCT/USOO/30177, filed on June 20, 2000; and arj^lsulfonylindoles of the type described in the Swedish patent application No. ~ 0003810-9. filed on October 20» 2000. Currently preferable 5-HT5 receptor antagonists include the azepinoindole compound class, such as the class of arylsulfone-substituted hexahydroazepinoindoles conrpounds disclosed in WO 01/05793. Other preferred 5-HT5 receptor antagonists include the arylsulf onylindole compound class, such as the compound class described in the Swedish patent application No. 0003810-9. The 5-HT2C receptor agonists and the 5-HT6 receptor antagonists may be the compounds as such or where appropriate the pharmaceutically acceptable salts (acid or base addition salts) thereof or stereochemically isomeric forms thereof (including optical isomers, such as enantiomers and racemates). The pharmaceutically acceptable addition salts as mraitioned above are meant to comprise the flierapeutically active non-toxic acid and base addition salt forms which the compounds are able to form. Compounds which have basic properties can be converted to their pharmaceutically acceptable acid addition salts by treating the base form with an ^propriate acid. Exemplary acids include inorganic acids, such as hydrogen chloride, hydrogen bromide, hydrogen iodide, sulphuric acid, phosphoric acid; and organic acids such as acetic acid, propanoic acid, hydroxyacetic acid, lactic acid, pyruvic acid, glycolic acid, maleic acid, malonic acid, oxalic acid, benzenesulfonic acid, toluenesulfonic acid, methanesulfonic acid, trifluoroacetic acid, fumaric acid, succinic acid, malic acid, tartaric acid, citric acid, salicylic acid, p-aminosalicylic acid, pamoic acid, benzoic acid, ascorbic acid and the like. Exemplary base addition salt forms are the sodium, potassium, calcium salts, and salts with pharmaceutically acceptable ammes such as, for example, ammonia, alkylamines, benzathine, and amino acids, such as, e.g. arginine and lysine. The terra addition salt as used herein also comprises solvates which the compounds and salts thereof are able to form, such as, for example, hydrates, alcoholates and the like. The 5-HT2C receptor agonists and the S-HT^ receptor antagonists may also be prodrugs or forms that may release the active ingredient in question aft^ metabolic tranformation in vivo. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in *T)esign of Prodrugs" ed. H. Bundgaard, Elsevier, 1985. The 5-HT2C receptor agonists and the 5-HT5 receptor antagonists may be formulated into various pharmaceutical forms for administrative purposes, either in the same pharmaceutical dosage form, such as in the same tablet, or in separate pharmaceutical dosage forms. In the latter case, however, it may be advantageous to put the 5-HT2C rcceptor agonist unit dosage form and the 5-HT6 receptor antagonist unit dosage form in the same package, for example in the same blister. The 5-HT2C receptor agonists and the 5-HT5 receptor antagonists, in the form of ftee bases or salt, can be brou^t into suitable galenic forms, such as compositions for oral use, for injection, for nasal spray administration or the like, in accordance with accepted pharmaceutical procedures. Such pharmaceutical compositions according to the invCTition coioprise an effective amount of a 5-HT2c receptor agonist and a S-HTg receptor antagonist in association with compatible pharmaceutically acceptable carrier materials, or diluents, as are well known in the art. The carriers may be any inert matKial, organic or inorganic, suitable for oral, enteral, rectal, percutaneous, subcutaneous or parenteral administration, such as: water, gelatin, gum arabicum, lactose, microcrystalline cellulose, starch, sodium starch glycolate, calcium hydrogen phosphate, magnesium stearate, talcum, colloidal sUicon dioxide, and the like. Such compositions may also contain other pharmacologically active agents, and conventional additives, such as stabilizers, wetting agents, emulsifiers, flavoring agents, buffers, and the like. The compositions according to the invention can e.g. be made up in solid or liquid form for oral administration, such as tablets, pills, capsules, powders, syrups, elixirs, dispersable granules, cachets, suppositories and the like, in the form of sterile solutions, suspensions or emulsions for parenteral administration, sprays, e.g. a nasal spray, transdermal preparations, e.g. patches, and the like. The dose level of each of the specific 5-HT2C receptor agonist and S-HT^ receptor antagonist, and the frequency of dosage of the specific combination will vary depending on a variety of factors including the potency of each specific compound employed, the metabolic stability and length of action of that compound, the patient's age, body wei^t, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the condition to be treated). The daily dosage may, for exan:5)le, range from about 0.001 mg to about 150 mg per kilo of body wei^t, preferably fix)m about 0.01 mg to about 100 mg per kilo of body weight, especially from about 0.1 to about 50 mg per kilo of body weight of each of the 5-HT2C receptor agonist and of the 5-HT5 receptor antagonist, administered sin^y or multiply in doses, e.g. dosages of from about 0.01 mg to about 1 g each. Usually, such a combined dosage is given orally but e.g. parenteral or rectal administration may also be chosen. An exemplary tablet combination formulation .may be in the form of either (A) two separate tablets, i.e. one tablet containing 10 mg, 20 mg or 50 mg of a 5-HT2c recq>tor agonist, and one tablet containing 10 mg, 20 mg or50 mg of a 5-HT6 receptor antagonist; or (B) a combined tablet containing 10 mg, 20 mg or 50 mg of a 5-HT2C receptor agonist and 10 mg, 20 mg or 50 mg of a 5-HT6 receptor antagonist The invention will now be illustrated further by the following non-limiting Experimental Section. EXPERIMENTAL SECTION A. Preparation of test compounds The free base of the 5-HT2C receptor agonist (2R)-methyI-l-{3-[2-(3-pyridinyloxy) ethoxy]-2-pyrazinyl}piperazine, fumarate CTNU-183933F') was prepared as described in WO 00/76984. The free base was converted to its fumarate salt, m.p. 126-129°C. MS miz 315 (M)-". Anal. (C16H21N5O2 • C4H4O4) C, H, N. The 5-HT6 receptor antagonist 6-methyU9-(pheiTylsulforiyl)--l,2,3,4,5^6- hexdhydroazepino[4,5-b]indole, hydrochloride C*PNU-186053A") was prepared as described m WO 01/05793. The 5-HT2C receptor agonist (2R)-l'(3'{2'[(2-ethoxy-3-pyridi7tyl)oxy]ethoxy}-2-pyradnyl)-2~methylpiperazine, fionarate ("BVT.2938F') was prepared as described in WO 00/76984. The 5-HT6 receptor antagonist l'(phenylsidfoTQfl)-4'Q-pipeTazmyl)-lH4ndole, hydrochloride ("BVT.5182C") was prepared as described in Swedish patent ^)piication No. 0003810-9. filed on October 20,2000. Briefly, BVT.5182C was prepared according the general procedure depicted in Scheme 1, below, starting from commercially available 4-piperazinoiDdole (compound 1) that undergoes steps (a) to (c) to afford 1-(phenylsulfonyl)-4-(l"'piperazinyl)-l-ff-indole, hydrochloride (yield 80%). HPLC purity >95%; ^HNMR (pUSO-d6) 5 9.64 (br s, 2H), 8.00-7.85 (m, 3 H), 7.79 (d, J= 3.77 Hz, 1 H), 7.70-7.65 (m, 1 H), 7.63-7.60 (m, 3 H), 7.27-7.22 (m, 1 H), 6.95 (d, J= 3.76 Hz, 1 H), 6.81-6.77 (m, 1 H), 3.30-3.20 (m, 4 H); ^^C NMR (DMS0-d6) 5144.79, 137.02,135.22,134.62,129.82.126.85,125.63,125.54,123.49,111.15,107.87, 107.76,47.81,42.86; MS (posES-FIA) m/z 342 (M+H). Step (a): BOCprotection ofthepiperazine N4 nitrogen 4-Piperazinoindole (leq), DMAP (0.1 eq) and EtsN (4 eq) were dissolved in DMF. (B0C)20 (1.1 eq) was added and the reaction mixture was stirred at room temperature (12 h). DMF was evaporated and the residue was purified by chromatography on silica gel using a mixture of chloroform, methanol and ammonia as eluent HPLC: 100 % purity. MS miz 302.2 (M+H). Step (b): Preparation of intermediate 3 The intermediate 2 (1.0 eq) was dissolved in DMF and NaH (1.3 eq) was added and the suspension was stirred for 0.5 h under nitrogen atmosphere. Benzenesulfonyl chloride (1.2 eq) was added and the reaction was stirred overnight at room temperature. The volatiles were evaporated. The residue was dissolved in DCM, washed with a saturated solution of NaHCOs, dried (MgS04), filtered and concentrated to give an oily residue that was purified by chromatogrq)hy on silica gel using a mixture of hexane and ethylacetate (7:3) as eluent to give tert butyl 4-[l-(benzenesulfonyl)-lif-indol-4-yl)]-l- piperazinecarboxylate (3). HPLC 100 %. NMR (^H and ^^C) and MS analyses support -■ the stated structure. Step (c): Removal of the BOC protecting group The BOC group on intermediate 3 was removed by dissolving the compound in methanol followed by addition of ether saturated with HCl gas. The HQ salt (4) was filtered and dried, B. Preparation of a pharmaceutical composition Tablet Ingredients mg/tablet 1. 5-HT2C receptor agonist 10.0 2. 5-HT6 recq)tor antagonist 10.0 3. Cellulose, microcrystalline 57.0 4. Calcium hydrogen phosphate 15.0 5. Sodium starch glycolate 5.0 6. Silicon dioxide, colloidal 0.25 7. Magnesium stearate 0.75 The active ingredients 1 and 2 are mixed with ingredients 3, 4,5 and 6 for about 10 minutes. ITie mz^esium stearate (7) is then added, and the resultant mixture is mixed for about 5 minutes and compressed into tablet fonn with or without film-coating. C. Receptor affinity and efficacy assays 5'HT2C receptor affimty assay 5-HT2C receptor affinity is determined in competition experiments, where the ability of a compound in serial dilution to displace ^H-labeled 5-HT, bound to membranes prepared from a transfected HEK293 cell line stably expressing the human 5-HT2C receptor protein, is monitored by Scintillation Proximity Assay (SPA) technology. Non-specific binding is defined using 5 |iM mianserin. 5'tLT2A Tecepior ajjuiuy iuisay S-KH^K 3^^&ptor affinity is determined in competition experiments, where the ability of a compound in serial dilution to displace ^H-labeled ketanserin or lysergic acid diethylamide (LSD), boxmd to membranes prepared from a transfected CHO cell line stably expressing the human 5-HT2A receptor protein, is monitored by measming the radioactivity of filtered membrane homogenates on glass fiber filters in a scintillation counter. Non-specific binding is defined using 5 fiM mianserin. S'HT2B recepUiT affinity assay 5-HT2B receptor affinity is determined in competition experiments, where the ability of a compound in serial dilution to displace ^H-labeled 5-HT, bound to membrane pr^ared from a transfected CHO cell line stably expressing the human 5-HT2B receptor protein, is monitored by Scintillation Proximity Assay (SPA) technology. Non-specific binding is defined using 5 pM mianserin. 5'HT2C receptor efficacy assay The agonist efficacy at the 5-HT2c receptor is determined by the ability of a compound to mobilise intracellular calcium in transfected HEK293 cells, stably expressing the human 5-HT2c receptor protein, using the calcium-chelating fluorescent dye FLUO-3 (Sigma, St. Louis, MO, U.S.A.). Relative efficacy (%) is measured relative to that of SCTotonin at 1 ^iM. S-HTe receptor affinity assay The radioligand binding assay uses [^H]-lysergic acid diethylamide (LSD). The assay is carried out in 96-well sample plates by the addition of 11 pi of the test compound at the ^propriate dilution (the assay employs 11 serial concentrations of samples run in duplicate), 11 pi of radioligand, and 178 pi of a washed mixture of WGA-coated SPA beads and membranes in binding buffer prepared fi-om HEK293-cells containing cloned human 5-HTg receptor. The plates are shaken for about 5 minutes and then incubated at room temperature for 1 hour. The plates are then loaded into counting cassettes and counted in a scintillation counter. The specifically bound cpm obtained are fit to a one-site binding model using GraphPad Prism ver. 2.0. Estimated IC50 values are converted to Kj (affinity constant) values using the Cheng-Pnisoff equation (Cheng, Y. C. et al., Biochem. Phca-macol 1973, 22, 3099-3108). S'HT^ receptor efficacy assay The antagonist potency at the S-HTg receptor is detennined by the ability of a compound to antagonize the increase in cAMP induced by 5-HT in HEK293 cells, stably expressing the human 5-HT6 receptor protein, using a cAMP SPA direct screening assay system (RPA559, Amersham Pharmacia Biotech, Uppsala, Sweden). D« Food intake test Test compounds 5-HT2C receptor agonists (2J?)-methyl-l-{3-[2-(3-pyTidinyloxy)ethoxy]-2-pyrazinyi}piperazme, fumarate C*PNU-183933F') and (2^)-l-(3-{2-[(2-ethoxy-3-p)Tidinyl)oxy]ethoxy}-2-pyrazinyl)-2-methylpiperazine, fumarate C'BVT.2938F') were dissolved in saline (0.9% NaQ) and diluted in the same vehicle to the appropriate concentration. S-HTgreceptor antagonists 6-methyl-9-(phenylsulfonyl)-l,2,3,4,5,6-hexahydroazepmo[4,5-fc]indole, hydrochloride C*PNU"186053A") and 1-(phenylsulfonyI)-4-(l-piperazinyI)-lH-indole, hydrochloride (S-HTg receptor antagonist ("BVT.5182C") were dissolved and diluted in 25% cyclodextrin. Fresh solutions were prepared on the day of treatment Aninuds Male mice 8-9 weeks old (C57BL/6JBom-Lep°^ (ob/ob), Bomholtsgaard, Denmark) with an average body weight of 45 g were used. The animals were housed singly in cages at 23±1°C, 40-60% humidity and had free access to water and standard laboratory chow. The 12/12 h light/daric cycle was set to lights off at 5 p.m. The animals were conditioned for at least one week before start of study. During experimental sessions, the animals obtained special chow (BioServ, Frenchtown, NJ, USA dust-free precision peUets weighing 20 mg each). Experimental section At the start of &e study the animals were transferred to special cages "operant test cages" (Habitest Modular Animal Behavior Test System; Colboum Instr, Allentown, PA, USA). These cages consist of a feeder trough with sensors for measurement of food intake, an optic lickometer for registration of water intake and an infrared-based monitor for recording overall general motor activity. The monitors are coupled to a computer, which controls and monitcff events continuously. Food pellets were weired to the amount needed for one whole study and water bottles were filled with fresh tap water and weighed. The animals were conditioned to tiieir new CTvironment for fliree days to establish baseline values. The animals were weighed at 3 p,m. at the start and at the end of the study. The compounds were administered between A^Ja and 5.00 p.m. before dark onset Three groups of animals received (i) S-HTg antagonist in 25% cyclodextrin; (ii) 5-HT2C agonist in saline; and (iii) the combination 5-HT2C agonist/5-HT6 antagonist, respectively. When combined, 5-HT6 antagonist or saline was administered 30 min before administration of the 5-HT2C agonist or 25% cyclodextrin. A fourth group received respectively vehicle administered in the same way. The study ended on the fifth day. Weighing was performed with a computer-assisted Mettler-Toledo PR5002/PR802 balance. Evaluation of results Each dose group consisted of 12-16 animals. Data were corrected for food spillage based on the weighed spillage during 22 hours and assumed to be proportional over time. Calculations were performed for the data before and after treatment The values were expressed as % of basal food intake (mean ± SEM) for the difference between food intake before treatment and 3 h (5 pm - 8 pm), 6 h (5 pm - 11 pm), 12 h (5 pm - 5 am), 21 h (5 pm - 2 pm). The results shown in Fig. 1 indicate that combined treatment with the S-HTg receptor antagonist 'TPNU-186053 A" (50 mg/kg subcutaneously) and the 5-HT2C receptor agonist *'PNU-183933F* (50 mg/kg per orally) decreased food consumption sigmficantly more than the compounds given alone. Correspondingly, the results shown in Hg. 2 indicate that combined treatment with tiie 5-HT2C receptor agonist "BVT.2938F* (5 mg/kg subcutaneously) and the 5-HT6 receptor antagonist •'BVT.5182C' (3 mg/kg subcutaneously) decreased food consumption, at 12 and 21 17. A pharmaceutical composition substantially as herein described with reference to the accompanying drawings. 18. A process for preparing a pharmaceutical composition substantially as herein described with reference to the accompanying drawings.

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