Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/16—Amides, e.g. hydroxamic acids
  • A61K31/18—Sulfonamides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
  • A61K31/404—Indoles, e.g. pindolol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/4965—Non-condensed pyrazines
  • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
  • A61P27/06—Antiglaucoma agents or miotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention relates to the prophylaxis or treatment of a 5-HT 2 c and a 5-HT 6 receptor-related disease.
• the invention provides a pharmaceutical composition containing a 5-HT 2 c receptor agonist and a 5-HT 6 receptor antagonist for therapeutic use.
• Serotonin (5-hydroxytryptamine or 5-HT) is a key neurotransmitter of the peripheral and central nervous system (PNS and CNS) and has been 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 serotonin receptor subtypes are expressed in the mammalian PNS and CNS and have been formally classified; see Glennon, et al., Neiirosci 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-HT 2 subfamily of receptors is composed of three subtypes, the 5-HT2A > 5- HT2B and 5-HT 2 c receptors. Serotonin 5-HT2C receptors are expressed in many brain regions and have been implicated in the regulation of food intake (Dourish, C.T. Obes. Res. 1995, 3, Suppl 4, 449S-462S; Bickerdike, M.J., et al. Diabetes, Obes. Metab. 1999, 1, 207-214).
• the 5-HT2C receptor 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. IDrugs 1999, 2, 109-120).
• the 5-HT 2 c receptor subtype has further been suggested to be involved in urinary disorders such as urinary incontinence (Leysen, D.C.M. IDrugs 1999, 2, 109-120).
• 5-HTg 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 CNS disorders.
• 5-HTg receptor has been linked to generalized stress and anxiety states (Yoshioka et al. Life Sci. 1998, 17/18, 1473-1477).
• a 5-HT 2 c receptor agonist and a 5-HTg receptor antagonist reduces food intake by more than the administration of either agonist or antagonist alone.
• Such combined administration of a 5-HT 2 c receptor agonist and a 5-HTg 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 comprising an effective amount of a combination of a 5-HT 2 c receptor agonist and a 5-HT6 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-HTg receptor, comprising administering to a human or animal subject in need thereof a 5-HT 2 c receptor agonist and a 5-HT 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-HT 2 c receptor agonist and a 5-HTg receptor antagonist for the manufacture of a medicament for the treatment of a disease related to the 5-HT 2 c receptor and the 5-HT6 receptor.
• Another aspect of the invention provides a process for preparing a pharmaceutical composition, wherein a 5-HT ; receptor agonist and a 5-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 ⁇ HT 2 c receptor agonist and a 5-HTg receptor antagonist as a combined preparation for simultaneous, separate or sequential use in therapy of a disease, in particular obesity, related to the 5-HT 2 c receptor and the 5-HTg receptor.
• Figure 1 shows the effect on food intake in ob/ob mice following combined administration with a 5-HT 2 c 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 effect of each agonist and antagonist alone.
• Figure 2 shows the effect on food intake in ob/ob mice following combined administration of a 5-HT 2C receptor agonist (BVT.2938F; 5 mg/kg sc) and a 5-HT 6 receptor antagonist (BVT.5182C; 3 mg/kg sc), as well as the effect of each agonist and antagonist alone.
• BVT.2938F 5-HT 2C receptor agonist
• BVT.5182C 5-HT 6 receptor antagonist
• the present invention is based on the unexpected finding that combined administration of a 5-HT 2 c receptor agonist and a 5-HTg receptor antagonist reduces food intake more than either agonist or antagonist alone.
• Such combined administration of a 5-HT 2 c receptor agonist and a 5-HTg receptor antagonist may also offer 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-therapy.
• the lower doses required by the combined administration may reduce the risk of adverse events.
• the lower doses required by the combined administration may reduce the risk of tolerance development and abuse liability.
• therapy based on two targets may increase the individual therapeutic efficacy relative to therapy based on one target.
• the risk of non-responsive efficacy 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, post- traumatic stress syndrome, and sleep disorders as well as for treatment of urinary incontinence (or more generally overactive bladder), sexual dysfunctions, gastrointestinal disorders and glaucoma.
• CNS disorders such as, depression, mania, schizophreniform disorders, anxiety, memory disorders (such as Alzheimer's disease) migraine headache, drug addiction, convulsive disorders, personality disorders, post- traumatic stress syndrome, and sleep disorders as well as for treatment of urinary incontinence (or more generally overactive bladder), sexual dysfunctions, gastrointestinal disorders and glaucoma.
• 5-HT 2C receptor agonist refers to a compound that causes activation of the serotonin 5-HT 2 c receptor.
• the 5-HT 2 c receptor agonist preferably has an affinity constant, Kj, of less than 50 nM, preferably less than 20 nM, and an in vitro intrinsic activity, measured as intracellular Ca 2+ levels, greater than 20%, preferably greater than 50%, relative to 5-HT (1 ⁇ M).
• the term "5-HT ⁇ receptor antagonist” as used herein refers to a compound that causes blockade of the serotonin 5-HT6 receptor mediated responses.
• the 5-HTg receptor antagonist preferably has an affinity constant, Kj, of less than 50 nM, preferably less than 20 nM, and an in vitro intrinsic activity, measured as intracellular cAMP levels, less than 50%, preferably less than 20%, relative to 5-HT (1 ⁇ M).
• the 5-HT2C receptor agonists and 5-HT6 receptor antagonists should be sufficiently selective not to cause any substantial adverse side effects.
• selective and substantially in this context are, however, to be interpreted broadly, the meanings thereof being readily apparent to the skilled person.
• the 5-HT 2 c receptor agonist preferably has a selectivity for the 5-HT 2 c receptor of at least 5, preferably at least 10 and more preferably at least 20, relative to the 5- HT 2A , 5-HT 2 B and 5-HTg receptors, respectively (measured as the affinity ratios 5- HT 2A /5-HT 2C , 5-HT 2B /5-HT 2C and 5-HT 6 /5-HT 2C ).
• the 5-HTg receptor antagonist preferably has a selectivity for the 5-HTg receptor of at least 5, preferably at least 10 and more preferably at least 20, relative to the 5-HT 2j > 5-HT 2J3 and 5-HT 2 c receptors, respectively (measured as the affinity ratios 5-HT 2A /5-HT 6 , 5-HT 2B /5-HT 6 and 5-HT 2c /5-HT 6 ).
• Relevant tests to determine whether a compound is a selective 5-HT 2 c receptor agonist or a selective 5-HTg receptor antagonist are known in the art, and are, as mentioned above, also outlined in the Experimental Part below.
• Compounds known to be 5-HT 2C 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 type described in WO 00/12502; 2,3,4,4a- tetrahydro-lH-pyrazino[l,2-a]
• 5- ⁇ T 2 c 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-HTg 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 pyrazolotriazine derivatives of the type described in EP-A-0941994; ary
• preferable 5-HTg receptor antagonists include the azepinoindole compound class, such as the class of arylsulfone-substituted hexahydroazepinoindoles compounds disclosed in WO 01/05793.
• Other preferred 5-HTg receptor antagonists include the arylsulfonylindole compound class, such as the compound class described in the Swedish patent application No. 0003810-9.
• the 5-HT 2C receptor agonists and the 5-HTg 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 mentioned above are meant to comprise the therapeutically 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 appropriate 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.
• organic acids such as acetic acid, propanoic acid, hydroxyacetic acid, lactic acid, pyruvic acid, glycolic acid, maleic acid, malonic acid, oxalic acid, benzenesulfonic acid, toluenesulf
• Exemplary base addition salt forms are the sodium, potassium, calcium salts, and salts with pharmaceutically acceptable amines such as, for example, ammonia, alkylamines, benzathine, and amino acids, such as, e.g. arginine and lysine.
• the term 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-HT 2 c receptor agonists and the 5-HTg receptor antagonists may also be prodrugs or forms that may release the active ingredient in question after metabolic tranformation in vivo. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs" ed. H.
• the 5-HT2C receptor agonists and the 5-HTg 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 receptor agonist unit dosage form and the 5-HTg receptor antagonist unit dosage form in the same package, for example in the same blister.
• the 5-HT 2 c receptor agonists and the 5-HTg receptor antagonists in the form of free bases or salt, can be brought 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.
• 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 invention comprise an effective amount of a 5-HT 2 c receptor agonist and a 5-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 material, organic or inorganic, suitable for oral, enteral, rectal, percutaneous, subcutaneous or parenteral administration, such as: water, gelatin, gum arabicum, lactose, macrocrystalline cellulose, starch, sodium starch glycolate, calcium hydrogen phosphate, magnesium stearate, talcum, colloidal silicon 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.
• 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.
• each of the specific 5-HT 2 c receptor agonist and 5-HTg 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 weight, 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 example, range from about 0.001 mg to about 150 mg per kilo of body weight, preferably from 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-HT 2 c receptor agonist and of the 5-HTg receptor antagonist, administered singly or multiply in doses, e.g. dosages of from about 0.01 mg to about 1 g each.
• 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.
• the 5-HT 6 receptor antagonist 6-methyl-9-(phenylsulfonyl)-l, 2,3, 4,5,6- hexahydroazepino[4,5-b]indole, hydrochloride (“PNU- 186053 A”) was prepared as described in WO 01/05793.
• the 5-HT 2C receptor agonist (2R)-l-(3- ⁇ 2-[(2-ethoxy-3-pyridinyl)oxy]ethoxy ⁇ -2- pyrazinyl)-2-methylpiperazine, fumarate (“B VT.2938F”) was prepared as described in WO 00/76984.
• BVT.5182C The 5-HT 6 receptor antagonist l-(phenylsulfonyl)-4-(l-piperazinyl)-lH-indole, hydrochloride
• BVT.5182C was prepared as described in Swedish patent application 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-piperazinoindole (compound 1) that undergoes steps (a) to (c) to afford 1- (phenylsulfonyl)-4-(l-piperazinyl)-lH-indole, hydrochloride (yield 80%).
• the intermediate 2 (1.0 eq) was dissolved in DMF and Na ⁇ (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 BOC group on intermediate 3 was removed by dissolving the compound in methanol followed by addition of ether saturated with HC1 gas.
• the HC1 salt (4) was filtered and dried.
• the active ingredients 1 and 2 are mixed with ingredients 3, 4, 5 and 6 for about 10 minutes.
• the magnesium stearate (7) is then added, and the resultant mixture is mixed for about 5 minutes and compressed into tablet form with or without film-coating.
• 5-HT 2 c receptor affinity is determined in competition experiments, where the ability of a compound in serial dilution to displace 3 H-labeled 5-HT, bound to membranes prepared from a transfected HEK293 cell line stably expressing the human 5-HT 2 c receptor protein, is monitored by Scintillation Proximity Assay (SPA) technology. Non-specific binding is defined using 5 ⁇ M mianserin.
• 5-HT 2A receptor affinity is determined in competition experiments, where the ability of a compound in serial dilution to displace 3 H-labeled ketanserin or lysergic acid diethylamide (LSD), bound to membranes prepared from a transfected CHO cell line stably expressing the human 5-HT 2 A receptor protein, is monitored by measuring the radioactivity of filtered membrane homogenates on glass fiber filters in a scintillation counter. Non-specific binding is defined using 5 ⁇ M mianserin.
• 5-HT2B receptor 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 membranes prepared 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 ⁇ M mianserin.
• SPA Scintillation Proximity Assay
• the agonist efficacy at the 5-HT 2 c receptor is determined by the ability of a compound to mobilise intracellular calcium in transfected HEK293 cells, stably expressing the human 5-HT 2 c 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 serotonin at 1 ⁇ M.
• the radioligand binding assay uses [ 3 H]-lysergic acid diethylamide (LSD).
• LSD [ 3 H]-lysergic acid diethylamide
• the assay is carried out in 96-well sample plates by the addition of 11 ⁇ l of the test compound at the appropriate dilution (the assay employs 11 serial concentrations of samples run in duplicate), 11 ⁇ l of radioligand, and 178 ⁇ l of a washed mixture of WGA-coated SPA beads and membranes in binding buffer prepared from HEK293- cells containing cloned human 5-HT5 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 IC 50 values are converted to Kj (affinity constant) values using the Cheng- Prusoff equation (Cheng, Y. C. et al., Biochem. Pharmacol. 1973, 22, 3099-3108).
• the antagonist potency at the 5-HTg receptor is determined by the ability of a compound to antagonize the increase in cAMP induced by 5-HT in HEK293 cells, stably expressing the human 5-HTg receptor protein, using a cAMP SPA direct screening assay system (RPA559, Amersham Pharmacia Biotech, Uppsala, Sweden).
• 5-HT2C receptor agonists (2i?)-methyl-l- ⁇ 3-[2-(3-pyridinyloxy)ethoxy]-2- pyrazinyl ⁇ piperazine, fumarate (“PNU-183933F”) and (2 ⁇ )-l-(3- ⁇ 2-[(2-ethoxy-3- pyridinyl)oxy]ethoxy ⁇ -2-pyrazinyl)-2-methylpiperazine, fumarate (“BVT.2938F”) were dissolved in saline (0.9% NaCl) and diluted in the same vehicle to the appropriate concentration.
• mice Male mice 8-9 weeks old (C57BL/6JBom-Lep ob (ob/ob), Bomholtsgaard,
• mice were housed singly in cages at 23+l°C, 40-60% humidity and had free access to water and standard laboratory chow.
• the 12/12 h light/dark 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 pellets weighing 20 mg each).
• the compounds were administered between 4.20 and 5.00 p.m. before dark onset.
• Three groups of animals received (i) 5-HTg antagonist in 25% cyclodextrin; (ii) 5-HT 2 c agonist in saline; and (iii) the combination 5-HT 2 c agonist/5-HT6 antagonist, respectively.
• 5-HT6 antagonist or saline was administered 30 min before administration of the 5-HT 2 c 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.
• 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).
• Fig. 1 The results shown in Fig. 1 indicate that combined treatment with the 5-HTg receptor antagonist "PNU- 186053 A" (50 mg/kg subcutaneously) and the 5-HT2C receptor agonist "PNU-183933F” (50 mg/kg per orally) decreased food consumption significantly more than the compounds given alone.
• the results shown in Fig. 2 indicate that combined treatment with the 5-HT2 receptor agonist "BVT.2938F” (5 mg/kg subcutaneously) and the 5-HTg receptor antagonist "BVT.5182C” (3 mg/kg subcutaneously) decreased food consumption, at 12 and 21 hours following administration, significantly more than the compounds given alone.
• a 5-HT2C receptor agonist and a 5-HTg receptor antagonist reduces food intake more efficiently as compared to treatment with either agonist or antagonist alone.

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