KR100845450B1 - New combination of serotonin agonist 5ht2 and antagonist 5ht6 as pharmaceutical formulation - Google Patents

Abstract

The present invention is a human being or requiring the 5-HT _2c receptor and the 5-HT ₆ sufficient amount of the 5-HT _2c receptor agonist and 5-HT ₆ receptor antagonists in the prevention or treatment of the receptor-associated diseases, indicating the therapeutic effect It relates to a method comprising administering to an animal.

The present invention also relates to a pharmaceutical composition containing an effective amount of a combination of 5-HT _2c receptor agonist and 5-HT ₆ receptor antagonist and, if necessary, a pharmaceutically acceptable carrier.

Description

New combination of serotonin agonists (5HT2) and antagonists (5HT6) as pharmaceutical formulations {NEW COMBINATION OF SEROTONIN AGONIST (5HT2) AND ANTAGONIST (5HT6) AS PHARMACEUTICAL FORMULATION}

The present invention relates to the prevention or treatment of 5-HT _2C and 5-HT ₆ receptor related diseases. The present invention also provides a therapeutic pharmaceutical composition comprising a 5-HT _2C receptor agonist and a 5-HT ₆ receptor antagonist.

Serotonin (5-hydroxytrytamine or 5-HT) is an important neurotransmitter of the peripheral and central nervous system (PNS and CNS), and has a variety of sensations, exercises and behaviors including diet, sleep, body temperature, blood pressure, emotion and cognitive control. It is related to the function. More than 14 specific serotonin receptor subtypes are expressed in mammalian PNS and CNS and are formally classified (Glennon et al . , Neurosci. Biobehav. Rev. 1990 , 14 , 35-37; and D. Hoyer et al . , Pharmacol. Rev. 1994, reference 46, 157-203). Serotonin agonists and antagonists include anxiety, depression, hypertension, migraine, obesity, substance abuse and addiction, obsessive compulsive disorder, schizophrenia, autism, neurodegenerative diseases (eg Alzheimer's disease, Parkinson's disease, and Huntington's chorea), and It has been proposed for the treatment of a wide range of diseases including chemotherapy-induced vomiting.

The 5-HT ₂ receptor subfamily consists of three subtypes, namely 5-HT _2A , 5-HT _2B and 5-HT _2C receptors. Serotonin 5-HT _2C receptors are expressed in many brain regions and are involved in the regulation of dietary intake (Dourish, CT Obes. Res , 1995 , 3 , Suppl . 4 , 449S-462S; Bickerdike, MJ et al. , Diabetes, Obes. Metab. 1999 , 1 , 207-214). In contrast to non-specific 5-HT _2C 5-HT _2C receptor agonists m- chlorophenyl piperazine (m-CPP) reduces the food intake in mice that express the normal 5-HT _2C receptors, like the receptors, 5-HT _2C It has been found to lack activity in mice expressing mutant inactive forms of the receptor (Tecott, LH et al ., Nature 1995 , 374 , 542-546).

In addition, m-CPP and the recently identified Aptinoindole U-22394A identified as 5-HT _2C receptor agonist (unpublished data) have been reported to reduce body weight after 2 and 9 weeks of treatment in humans (Walsh, AES, Psychopharmacology 1994 , 116 , 120-122; Sargent, PA et al., Psychopharmacology 1997 , 133 , 309-312 and Gallant, DM et al . , Curr. Ther. Res. 1967 , 9 , 579-581).

Recently, a series of pyrrolo [3,2,1- ij ] quinoline derivatives have been identified as 5-HT _2C receptor agonists with selectivity over 5-HT _2A receptors (Isaac M. et al. , Bioorg, Med. Chem. Lett. 2000 , 10 , 919-921). The compound has been proposed as a novel attempt to treat obesity and epilepsy.

In addition, the 5-HT _2C receptor subtype has been suggested to be involved in CNS diseases such as depression and anxiety (Jenck, F. et al . , Expert Opin. Invest.Drugs 1998 , 7 , 1587-1599; Leysen, DCM IDrugs 1999 , 2 , 109-120). Furthermore, the 5-HT _2C receptor subtype has been suggested to be involved in urinary diseases such as urinary incontinence (Leysen, DCM IDrugs 1999 , 2 , 109-120).

5-HT ₆ 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) It is related to dietary intake and CNS disease.

Thus, for example, Bentley JC et al . , Br. J. Pharmacol . 1999 , 126 , 66P] show that dietary uptake in rats is indicated by administration of 5-HT ₆ antagonists. In addition, the number of antidepressants and non-typical antipsychotic drugs wherein exhibit high affinity for 5-HT ₆ receptor, which suggested the involvement of 5-HT ₆ receptors in schizophrenia (Roth, etc., J. Pharmacol. Exp. Ther, 1994 , 268 , 1403-1410; Sleight et al . , Expert Opin.Ther . Patents 1998 , 8, 1217-1224; Bourson et al., Br. J. Pharm. 1998 , 125 , 1562-1566; Boess et al . , Mol.Pharmacol . 1998 , 54 , 577-583; Sleight et al. Br . J. Pharmacol. 1998 , 124 , 556-562). In addition, 5-HT ₆ receptors are associated with general stress and anxiety (Yoshioka et al., Life Sci . 1998 , 17/18 , 1473-1477).

Summary of the Invention

In accordance with the present invention, it was unexpectedly found that the combined administration of 5-HT _2c receptor agonists and 5-HT ₆ receptor antagonists further reduced dietary intake compared to the administration of agonists or antagonists alone. Such combination administration of 5-HT _2c receptor agonists and 5-HT ₆ receptor antagonists may provide a therapeutic advantage over treatment with agonists or antagonists alone.

Accordingly, one aspect of the present invention provides a pharmaceutical composition containing an effective amount of a combination of 5-HT _2c receptor agonist and 5-HT ₆ receptor antagonist, and if necessary a pharmaceutically acceptable carrier.

Another aspect of the invention is a method for the prevention or treatment of 5-HT _2c receptor and 5-HT ₆ receptor related diseases, particularly obesity, in which a sufficient amount of 5-HT _2c receptor agonists and 5-HT ₆ receptors exhibit therapeutic effects. A method comprising administering to an animal or human in need of an antagonist (simultaneously or sequentially).

Another aspect of the present invention provides the use of 5-HT _2c receptor agonists and 5-HT ₆ receptor antagonists for the manufacture of drugs for the treatment of 5-HT _2c receptor and 5-HT ₆ receptor related diseases.

Another aspect of the present invention provides a method of preparing a pharmaceutical composition, wherein the combined therapeutic amount of 5-HT _2c receptor agonist and 5-HT ₆ receptor antagonist is directly mixed with a pharmaceutically acceptable carrier.

Another aspect of the invention provides a product comprising a 5-HT _2c receptor agonist and a 5-HT ₆ receptor antagonist simultaneously, separately or sequentially to treat 5-HT _2c receptor and 5-HT ₆ receptor related diseases, in particular obesity. It is provided as a combined formulation for use as.

1 shows diet in ob / ob mice after the combined administration of 5-HT _2c receptor agonist (PNU-183933F; 50 mg / kg oral) and 5-HT ₆ receptor antagonist (PNU-186053A; 50 mg / kg subcutaneous) Ingestion effect and the sole effect of each agonist and antagonist are shown.

FIG. 2 shows diet in ob / ob mice after combined administration of 5-HT _2c receptor agonists (BVT.2938F; 5 mg / kg subcutaneous) and 5-HT ₆ receptor antagonists (BVT.5182C; 3 mg / kg subcutaneous) Ingestion effect and the sole effect of each agonist and antagonist are shown.

As mentioned above, the present invention is due to the unexpected finding that the combination administration of 5-HT _2c receptor agonist and 5-HT ₆ receptor antagonist further reduces dietary intake compared to the administration of agonist or antagonist alone. This combination administration of 5-HT _2c receptor agonists and 5-HT ₆ receptor antagonists may also provide several advantages over the treatment of agonists or antagonists alone, for example in the treatment of obesity.

First, combination administration requires smaller doses of each compound to exhibit a similar or advanced reduction in dietary intake than single administration.

Second, lower doses required for combination administration can reduce the risk of side effects.

Third, lower doses required for combination administration can reduce the risk of developing resistance and the tendency of abuse.

Fourth, treatment described in two goals may increase individual therapeutic efficacy compared to one described in one goal. In addition, the risk of non-reactive efficacy (non-responders) can be reduced.

The advantages of the combination administration of the present invention are not only useful for the adjustment of dietary behavior, treatment of overweight and obesity, but also for depression, mania, schizophrenia, anxiety, memory disorders (such as Alzheimer's disease), migraine, drug addiction, obsessive compulsive disorder, Central nervous system disorders such as personality disorders, post-traumatic stress disorder, sleep disorders, as well as treatment of urinary incontinence (or more common overactive bladder), sexual dysfunction, gastrointestinal disorders and glaucoma.

As used herein, the term "5-HT _2c receptor agonist" refers to a compound that activates serotonin 5-HT _2c receptor. Preferably, the 5-HT _2c receptor agonist has an affinity constant (K _i ) of less than 50 nM, preferably less than 20 nM, and the in vitro native activity measured at intracellular Ca ²⁺ levels is 5-HT ( 1 μM), more than 20%, preferably more than 50%.

As used herein, the term "5-HT ₆ receptor antagonist" refers to a compound that interferes with a serotonin 5-HT ₆ receptor mediated response. Preferably, the 5-HT ₆ receptor antagonist has an affinity constant (K _i ) of less than 50 nM, preferably less than 20 nM, and intrinsic activity in vitro as measured by intracellular cAMP levels is 5-HT (1 μM). It is less than 50%, preferably less than 20% compared to.

In vitro assays that can be used to determine the affinity and intrinsic activity of 5-HT _2c receptor agonists and 5-HT ₆ receptor antagonists, respectively, are known and are also given in the Examples section below, 5-HT _2A and 5 The same is true for assays that determine affinity for the -HT _2B receptor.

In general, 5-HT _2c receptor agonists and 5-HT ₆ receptor antagonists should be sufficiently selective so as not to cause any substantial side effects. However, the terms " optional " and " substantial " herein are repeated extensively, so the meaning is apparent to those skilled in the art.

Preferably, the 5-HT _2c receptor agonist is 5-HT _2A, 5-HT _2B and It has selectivity for at least 5, preferably at least 10 and more preferably at least 20 5-HT _2c receptors relative to each of the 5-HT ₆ receptors (5-HT _2A / 5-HT _2c , 5-HT _2B / 5 -HT _2c and 5-HT ₆ / 5-HT _2c measured in affinity ratios).

5-HT ₆ receptor antagonists include 5-HT _2A, 5-HT _2B and It has selectivity for at least 5, preferably at least 10 and more preferably at least 20 5-HT ₆ receptors relative to each of the 5-HT _2c receptors (5-HT _2A / 5-HT ₆ , 5-HT _2B / 5 -HT ₆ and 5-HT _2c / 5-HT ₆ measured in affinity ratios).

Relative tests to determine whether a compound is a selective 5-HT _2c receptor agonist or a selective 5-HT ₆ receptor antagonist are known and, as discussed above, are shown in the Examples section below.

Compounds known as 5-HT _2c receptor agonists include, 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-aminoethylindole of the type described in EP-A-0655440; Pyrazinoindoles of the type described in EP-A-0572863; Piperazinylpyrazine of the type described in US 4,081,542; Indolin derivatives of the type described in WO 00/12475; Pyrroloindole, pyridoindole and azepineindole 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-1H-pyrazino [1,2-a] quinoxalin-5 ( 6H ) one of the type described in WO 00/35922; Indazolylpropylamine of the type described in WO 00/12481; Indazoles of the type described in WO 00/17170; Piperazinylpyrazine of the types described in WO 00/76984 and in Swedish Patent Applications No. 0004244-0 and 0004245-7, filed November 20, 2000; Heterocyclic fused γ-carbolines of the type described in WO 00/77001, WO 00/77002 and WO 00/77010; Benzofurylpiperazine of the types described in WO 01/09111 and WO 01/09123; Benzofuran of the type described in WO 01/09122; Benzothiophenes of the type described in WO 01/09126; Pyridinylpiperazine of the type described in EP 370560; Bioorg. Med. Chem. Lett. 2000, 10, 919-921 pyrroloquinoline of the type described in; Aminoalkylindazoles of the type described in WO 98/30548; Indole of the type described in WO 01/12603; Indolin 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 preferred 5-HT _2c receptor agonists are arylpiperazine and piperazinylpyrazine compounds, in particular Swedish patent applications No. 0004244-0 and 0004245-7, filed WO 00/76984 and 20 November 2000. Compounds disclosed in

Compounds known as 5-HT ₆ receptor antagonists include, 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 pyreazine derivatives of the type described in WO 99/65906; Pyrazolopyrimidine and pyrazolotriazine derivatives of the type described in EP-A-0941994; Arylsulfone substituted hexahydroazinoindoles of the type described in WO 01/05793; Oxazinocarbazoles of the type described in WO 01/09142; Aminoalkoxycarbazoles of the type described in WO 01/17963; Diphenylsulfones of the type described in international patent application PCT / US00 / 30177 filed Jun. 20, 2000; And arylsulfonylindoles described in Swedish Patent Application (October 20, 2000) No. 0003810-9.

Currently preferred 5-HT ₆ receptor antagonists include azepineindole compounds, for example arylsulfon substituted hexahydroazinoindole compounds disclosed in WO 01/05793. Other preferred 5-HT ₆ receptor antagonists include arylsulfonylindole compounds, such as those described in Swedish Patent Application No. 0003810-9.

5-HT _2c receptor agonists and 5-HT ₆ receptor antagonists include the compound or a pharmaceutically acceptable salt (acid or base addition salt) of the compound or stereoisomers thereof (such as enantiomers and racemates) Can be).

Such pharmaceutically acceptable addition salts include forms of therapeutically active non-toxic acid and base addition salts that the compound may form. Basic compounds can be converted into pharmaceutically acceptable acid addition salts by treating the base form with a suitable acid. Illustrative acids include inorganic acids such as hydrogen chloride, hydrogen bromide, hydrogen iodide, sulfuric acid, phosphoric acid; And 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, Organic acids such as salicylic acid, p-aminosalicylic acid, pamoic acid, benzoic acid, ascorbic acid and the like. Illustrative base addition salt forms include sodium, potassium, calcium salts, and salts having pharmaceutically acceptable amines such as ammonia, alkylamines, benzine, and amino acids such as arginine and lysine. As used herein, the term "addition salt" also includes compounds and sorbates that the salts can form, such as hydrates, alcoholates, and the like.

5-HT _2c receptor agonists and 5-HT ₆ receptor antagonists may also be in forms or prodrugs capable of releasing the active ingredient after metabolic conversion in vivo. Conventional procedures for the preparation and selection of suitable prodrug derivatives are described, for example, in "Design of Prodrugs" [H. Bundgaard, edited by Elsevier, 1985.

The 5-HT _2c receptor agonist and the 5-HT ₆ receptor antagonist can be formulated in various pharmaceutical forms for administration, in the same pharmaceutical dosage form as the same tablet or in separate pharmaceutical dosage forms. In the latter case, however, it may be advantageous to place the 5-HT _2c receptor agonist unit dosage form and the 5-HT ₆ receptor antagonist unit dosage form in the same package, eg, the same blister.

The 5-HT _2c receptor agonist and the 5-HT ₆ receptor antagonist in free base or salt form are in the form of suitable gallens, such as compositions for oral, injectable, nasal spray administration, etc., depending on acceptable pharmaceutical procedures. You can do Such pharmaceutical compositions according to the invention may contain effective amounts of 5-HT _2c receptor agonists and 5-HT ₆ receptor antagonists together with known formulated pharmaceutically acceptable carrier materials, or diluents. The carrier may be any inert, organic or inorganic substance suitable for oral, enteric, rectal, transdermal, subcutaneous or injection administration, for example water, gelatin, gum arabic, lactose, microcrystalline cellulose, starch, sodium starch Glycolate, calcium hydrogen phosphate, magnesium stearate, talcum, colloidal silicon dioxide, and the like. Such compositions may also contain pharmacologically active substances and conventional additives such as stabilizers, wetting agents, emulsifiers, flavoring agents, buffers, and the like.

Compositions according to the invention are in solid or liquid form for oral administration such as tablets, pills, capsules, powders, syrups, elixirs, dispersed granules, cachets, suppositories, and the like, emulsions, suspensions, sterile solutions for parenteral administration. It may be prepared in the form of a, a nasal spray, such as a spray, a transdermal agent such as a patch.

The specific dosage levels of each of the 5-HT _2c receptor agonist and the 5-HT ₆ receptor antagonist, and the frequency of administration of the specific combination, depend on the efficacy of each specific compound used, the metabolic stability and duration of action of the compound, the age and weight of the patient. It will vary depending on various factors, including general health, sex, diet, method and time of administration, rate of excretion, drug combination, and severity of treatment status. The daily dosage is, for example, from about 0.001 mg to about 150 mg per kilo of body weight, preferably 1 kilo of body weight, for each of the 5-HT _2c receptor agonist and the 5-HT ₆ receptor antagonist administered alone or together. From about 0.01 mg to about 100 mg per sugar, especially from about 0.1 to about 50 mg per kilogram of body weight, for example from 0.01 mg to 1 g each. Generally, such combined dosages are given orally, but parenteral or rectal administration may also be selected, for example. Exemplary tablet combination formulations include (A) two separate tablets, one tablet comprising 10 mg, 20 mg or 50 mg of 5-HT _2c receptor agonist, and 10 mg of 5-HT ₆ receptor antagonist, Another tablet comprising 20 mg or 50 mg; Or (B) a combination tablet comprising 10 mg, 20 mg or 50 mg of 5-HT _2c receptor agonist and 10 mg, 20 mg or 50 mg of 5-HT ₆ receptor antagonist,

The invention will be explained in more detail by the following non-limiting examples.

A. Preparation of Test Compound

5-HT _2c receptor agonist (2R) -methyl-1- {3- [2-3-pyridinyloxyethoxy] -2-pyrazinyl} piperazine, fumarate, as described in WO 00/76984 (" PNU-183933F ") was prepared. The free base was converted to its fumarate salt.

mp 126-129 ° C., MS m / z 315 (M) ⁺ . Anal. (C ₁₆ H ₂₁ N ₅ O ₂ .C ₄ H ₄ O ₄ ) C, H, N.

5-HT ₆ receptor antagonist 6-methyl-9- (phenylsulfonyl) -1,2,3,4,5,6-hexahydroazino [4,5-b] indole as described in WO 01/05793 , Hydrochloride ("PNU-186053A") was prepared.

5-HT _2c receptor agonist (2R) -1- (3- {2- [2-ethoxy-3-pyridinyl) oxy] ethoxy} -2-pyrazinyl) -as described in WO 00/76984 2-Methylpiperazine, fumarate ("BVT, 2938F) was prepared.

5-HT ₆ receptor antagonist 1- (phenylsulfonyl) -4- (1-piperazinyl) -1H-indole, hydro, as described in Swedish Patent Application No. 0003810-9 (filed Oct. 20, 2000 ) Chloride ("BVT.5182C") was prepared. In short, BVT.5182C is prepared according to the general procedure depicted in Scheme 1 below, starting with commercially available 4-piperazinoindole (Compound 1) and then through steps (a) to (c) 1- ( Phenylsulfonyl) -4- (1-piperazinyl) -1 H -indole, hydrochloride was obtained (yield 80%). HPLC purity>95%; ¹ H NMR (DMSO- d6 ) δ9.64 (br s, 2H), 8.00-7.85 (m, 3H), 7.79 (d, J = 3.77 Hz, 1H), 7.70-7.65 (m, 1H), 7.63- 7.60 (m, 3H), 7.27-7.22 (m, 1H), 6.95 (d, J = 3.76 Hz, 1H), 6.81-6.77 (m, 1H), 3.30-3.20 (m, 4H); ¹³ C NMR (DMSO- d6 ) δ 144.79, 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).

Scheme 1

Step (a): BOC Protection of Piperazine N4 Nitrogen

4-piperazinoindole (1 equiv), DMAP (0.1 equiv) and Et ₃ N (4 equiv) were dissolved in DMF. (BOC) ₂ O (1.1 equiv) was added and the reaction mixture was stirred at rt (12 h). DMF was evaporated and the residue was purified by silica gel chromatography using a mixture of chloroform, methanol and ammonia as eluent. HPLC: 100% purity. MS m / z 302.2 (M + H).

Step (b): Preparation of Intermediate 3

After intermediate 2 (1 equiv) was dissolved in DMF and NaH (1.3 equiv) was added, the suspension was stirred for 0.5 h under nitrogen atmosphere. Benzenesulfonyl chloride (1.2 equiv) was added and the reaction stirred at rt overnight. The volatiles were evaporated. The residue was dissolved in DCM, washed with saturated NaHCO ₃ solution, dried (MgSO ₄ ), filtered and concentrated to give an oily residue, and the mixture was purified by silica gel chromatography using hexane and ethyl acetate mixture (7: 3) as an eluent. Purification gave tert-butyl 4- [1- (benzenesulfonyl) -1 H -indol-4-yl)]-1-piperazinecarboxylate (3). HPLC 100%. The structure was confirmed by NMR ( ¹ H and ¹³ C) and MS analysis.

Step (c): removal of the BOC protector

Intermediate 3 was dissolved in methanol and ether saturated with HCl gas was added to remove the BOC group of intermediate 3. HCl salt (4) was filtered off and dried.

B. Preparation of Pharmaceutical Composition

refine

ingredient mg / tablet

1.5-HT _2c Receptor Agonist 10.0

2. 5-HT ₆ Receptor 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

Active ingredients 1 and 2 were mixed with components 3, 4, 5 and 6 for 10 minutes. Then magnesium stearate (7) was added and the resulting mixture was mixed for 5 minutes and then compressed into tablet form with or without film-coating.

C. Receptor Affinity and Efficacy Analysis

5-HT _2c Receptor affinity analysis

5-HT _2c receptor affinity was determined in a competition experiment, in which a stepwise diluted compound bound to a membrane prepared from a transformed HEK293 cell line stably expressing human 5-HT _2c receptor protein was bound to a ³ H-labeled 5- The ability to replace HT was monitored by Scintillation Proximity Assay (SPA) technology. Non-specific binding was defined using 5 μM mianserin.

 5-HT _2A Receptor affinity analysis

5-HT _2A receptor affinity was measured in a competition experiment, in which a stepwise diluted compound bound to a membrane prepared from a transformed CHO cell line stably expressing a human 5-HT _2A receptor protein, ³ H-labeled ketan The ability to replace serine or resergic acid diethylamide (LSD) was monitored by measuring the radioactivity of the filtered membrane homogenate on a glass fiber filter in a scintillation counter. Non-specific binding was defined using 5 μM Mianserine.

5-HT _2B Receptor affinity analysis

5-HT _2B receptor affinity was measured in a competition experiment, in which a stepwise diluted compound bound to a membrane prepared from a transformed CHO cell line stably expressing human 5-HT _2B receptor protein was bound to ³ H-labeled 5- The ability to replace HT was monitored by flash proximity search technique. Non-specific binding was defined using 5 μM Mianserine.

5-HT _2C Receptor Efficacy Analysis

Calcium-kill a decorated using the fluorescent dye FLUO-3 (Sigma, St. Louis , MO, USA), 5-HT 2c transformed to the agonist potency for the receptor stably expressing human 5-HT _2c receptor protein It was measured by the ability of the compound to move intracellular calcium in HEK293 cells. The relative potency (%) relative to the efficacy of 1 μM serotonin was measured.

5-HT ₆ Receptor affinity analysis

Radioligand binding assays were performed using [ ³ H] -riseric acid diethylamide (LSD). Prepared from HEK293 cells containing 11 μl of test compound appropriately diluted in 96-well sample plate (in this assay, 11 concentrations of samples were used in duplicate), 11 μl of radioligand, and cloned human 5-HT ₆ receptor. The analysis was performed by adding 178 μl of the wash mixture of membrane and WGA-encapsulated SPA beads in binding buffer. The plate was shaken for 5 minutes and then incubated for 1 hour at room temperature. The plate was then placed in a counting cassette and counted with a scintillation counter. The specific binding cpm obtained was applied to a single-site binding model using GraphPad Prism ver.2.0.

The estimated IC ₅₀ values were converted to K _i (affinity constant) values using the Cheng-Prusoff equation (Cheng, YC et al. , Biochem. Pharmacol. 1973 , 22 , 3099-3108).

5-HT ₆ Receptor Efficacy Analysis

using the cAMP SPA direct search analysis system (RPA559, Amersham Pharmacia Biotech, Uppsala , Sweden), the 5-HT ₆ HEK293 5-HT of cells stably expressing the human 5-HT ₆ receptor protein of the antagonist potency for the receptor It was measured by the ability of the compound to offset the cAMP increase induced by.

D. Dietary Intake Test

Test compound

5-HT _2c receptor agonist ( 2R ) -methyl-1- {3- [2- (3-pyridinyloxy) ethoxy] -2-pyrazinyl} pyrrazine, fumarate ("PNU-183933F") and ( 2R ) -1- (3- {2- [2-ethoxy-3-pyridinyl) oxy] ethoxy} -2-pyrazinyl) -2-methylpiperazine, fumarate ("BVT, 2938F) It was dissolved in saline (0.9% NaCl) and diluted to the appropriate concentration in the same medium.

5-HT ₆ receptor antagonist 6-methyl-9- (phenylsulfonyl) -1,2,3,4,5,6-hexahydroazino [4,5- b ] indole, hydrochloride ("PNU-186053A ") And 1- (phenylsulfonyl) -4- (1-piperazinyl) -1 H -indole, hydrochloride (" BVT.5182C ") were dissolved and diluted in 25% cyclodextrin.

Fresh solutions were prepared on the day of treatment.

animal

8-9 week old male mice (C57BL / 6JBom-Lep ^ob (ob / ob), Bomholtsgaard, Denmark) with an average body weight of 45 g were used. Animals were housed 1 per cage and bred at 23 ± 1 ° C., 40-60% humidity, freely fed with water and standard laboratory food. The 12/12 hour light / dark cycle was set to dark at 5 pm. Animals were conditioned for at least 1 week before the start of the study. During the experiment, animals were fed a special diet (20 mg of dust free precision pellets each of BioServ, Frenchtown, NJ, USA).

Experimental part

First, the animals were transferred to a specific cage "Habitest Modular Animal Behavior Test System" (Colbourn Instr, Allentown, PA, USA). The cage consists of a feeder with a sensor for measuring dietary intake, an optical lickometer to indicate water intake and an infrared based monitor to record overall general athletic activity. The monitor is connected to the computer and the results are continuously controlled and monitored. Dietary pellets were weighed in the amount required for one full study, filled and weighed with fresh tap water. Animals were allowed to adjust their condition for three days to ensure default values. Animals were weighed at the start and last 3 pm of the study. Compounds were administered at 4: 20-5 pm before dark. Three groups of animals each were (iv) 5-HT ₆ antagonists in 25% cyclodextrin; (Ii) 5-HT _2C agonists in saline; And (iii) a 5-HT _2C agonist / 5-HT ₆ antagonist combination. In combination, 5-HT ₆ antagonist or saline was administered 30 minutes prior to 5-HT _2C agonist or 25% cyclodextrin administration. The media used were each administered to the fourth group in the same manner. The study was completed on Day 5. The computer was weighed using an Mettler-Toledo PR5002 / PR802 balance that was assisted.

Evaluation of the result

Each dose group consisted of 12-16 animals. Data were corrected for the dietary losses described in Weighing Loss for 22 hours and assumed to be proportional to time. Calculations were performed on pre- and post-treatment data. 3 hours (5 pm-8 pm), 6 hours (5 pm-11 pm), 12 hours (5 pm-5 am), 21 hours (5 pm-2 pm) before and after treatment Values are expressed as% of baseline dietary intake (mean ± SEM) relative to differences in dietary intake.

The results shown in FIG. 1 show that the combination treatment of 5-HT ₆ receptor antagonist "PNU-186053A" (50 mg / kg subcutaneous) and 5-HT _2c receptor agonist "PNU-183933F" (50 mg / kg oral) resulted in a compound Significantly reduced dietary consumption compared to administration alone. Correspondingly, the results shown in FIG. 2 show a combination of 5-HT _2c receptor agonist “BVT.2938F” (5 mg / kg subcutaneous) and 5-HT ₆ receptor antagonist “BVT.5182C” (3 mg / kg subcutaneous) Treatment shows a significant reduction in dietary consumption at 12 and 21 hours post-dose than the compound administered alone. Thus, it is evident that the combined treatment of 5-HT _2c receptor agonists and 5-HT ₆ receptor antagonists reduces dietary intake more efficiently than treatment with agonists or antagonists alone.

Claims (16)

(2R) -methyl-1- {3- [2- (3-pyridinyloxy) ethoxy] -2-pyrazinyl} piperazine fumarate and (2R) -1- (3- {2-[(2 6-methyl-9- (phenylsulfonyl) and 5-HT _2c receptor agonists selected from -ethoxy-3-pyridinyl) oxy] ethoxy} -2-pyrazinyl) -2-methylpiperazine fumarate -1,2,3,4,5,6-hexahydroazino- [4,5-b] indole hydrochloride and 1- (phenylsulfonyl) -4- (1-piperazinyl) -1H-indole hydro Of overweight, obesity, depression, anxiety, memory disorders, sleep disorders, gastrointestinal disorders, urinary incontinence or glaucoma, containing an effective amount of a combination of 5-HT ₆ receptor antagonists selected from chloride, and a pharmaceutically acceptable carrier as needed. Pharmaceutical compositions for the treatment. delete delete delete delete A method of preparing a pharmaceutical composition according to claim 1, wherein the combined therapeutic amounts of 5-HT _2c receptor agonist and 5-HT ₆ receptor antagonist are directly mixed with a pharmaceutically acceptable carrier. As a combination preparation for simultaneous, separate or sequential use in the treatment of diseases selected from overweight, obesity, depression, anxiety, memory disorders, sleep disorders, gastrointestinal disorders, incontinence and glaucoma, (2R) -methyl-1- {3 -[2- (3-pyridinyloxy) ethoxy] -2-pyrazinyl} piperazine fumarate and (2R) -1- (3- {2-[(2-ethoxy-3-pyridinyl) oxy ] Ethoxy} -2-pyrazinyl) -2-methylpiperazin fumarate, 5-HT _2c receptor agonist, and 6-methyl-9- (phenylsulfonyl) -1,2,3,4, 5-HT ₆ receptor selected from 5,6-hexahydroazino [4,5-b] indole hydrochloride and 1- (phenylsulfonyl) -4- (1-piperazinyl) -1H-indole hydrochloride Products containing antagonists. delete delete delete delete delete delete delete delete delete

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