WO2002022122A1

WO2002022122A1 - Use of compounds for the treatment of obesity

Info

Publication number: WO2002022122A1
Application number: PCT/DK2000/000514
Authority: WO
Inventors: Erik Bardrum Nielsen; Søren TULLIN; Carsten Enggaard Stidsen; Nanni Din; Kirsten Raun; Christian Fledelius; Harrie C. M. Boonen; Heike A. Wieland; Edward Leon Barsoumian; Seiichiro Nishimura
Original assignee: Novo Nordisk A/S; Boehringer Ingelheim International, Gmbh
Priority date: 2000-09-15
Filing date: 2000-09-15
Publication date: 2002-03-21
Also published as: AU2000272704A1

Abstract

The present invention relates to the use of compounds that induce Ca2+ release through the type 1 Ryanodine receptor for the treatment of obesity. The present invention also embraces pharmaceutical compositions comprising these compounds and methods of using the compounds and their pharmaceutical compositions.

Description

USE OF COMPOUNDS FOR THE TREATMENT OF OBESITY

FIELD OF INVENTION

The present invention relates to the use of compounds that induce Ca²⁺ release through the type 1 Ryanodine receptor for the treatment of obesity. The present invention also embraces pharmaceutical compositions comprising these compounds and methods of using the compounds and their pharmaceutical compositions.

BACKGROUND OF THE INVENTION

Obesity is a well known risk factor for the development of many very common diseases such as atherosclerosis, hypertension, Type 2 diabetes (non-insulin dependent diabetes mellitus (NIDDM)), dyslipidaemia, coronary heart disease, and osteoarthritis and various malignan- cies. It also causes considerable problems through reduced motility and decreased quality of life. The incidence of obesity and thereby also these diseases is increasing throughout the entire industrialised world. Except for exercise, diet and food restriction no convincing treatment for reducing body weight effectively and acceptably currently exist. However, due to the important effect of obesity as a risk factor in serious and even mortal and common diseases it will be important to find pharmaceutical compounds useful in prevention and treatment of obesity.

The term obesity implies an excess of adipose tissue. In this context obesity is best viewed as any degree of excess adiposity that imparts a health risk. The distinction between normal and obese individuals can only be approximated, but the health risk imparted by obesity is probably a continuum with increasing adiposity. However, in the context of the present invention, individuals with a body mass index (BMI = body weight in kilograms divided by the square of the height in meters) above 25 are to be regarded as obese.

Even mild obesity increases the risk for premature death, diabetes, hypertension, atherosclerosis, gallbladder disease and certain types of cancer. In the industrialised western world the prevalence of obesity has increased significantly in the past few decades. Because of the high prevalence of obesity and its health consequences, its prevention and treatment should be a high public health priority. When energy intake exceeds energy expenditure, the excess calories are stored in adipose tissue, and if this net positive balance is prolonged, obesity results, i.e. there are two components to weight balance, and an abnormality on either side (intake or expenditure) can lead to obesity.

The ryanodine receptor type 1 (RyR1) is also known as the skeletal muscle sarcoplasmic re- ticulum calcium release channel. Two other isoforms exist: RyR2, which predominantly is expressed in the cardiac muscle and in the brain, and RyR3, which has a wide tissue distribution. All three ryanodine receptor subtypes consist of about 5000 amino acids and are ac- cordingly amongst the largest ion channels characterised to date (Zucchi, R. and Ronca- Testoni, S. (1997), Pharmacol. Rev. 49(1), 1-51).

Functionally, RyR1 is involved in skeletal muscle excitation-contraction coupling. Upon depolarisation of the skeletal muscle plasmamembrane (sarcolemma) a conformational change in the voltage sensitive dihydropyridine receptor (DHPR) is transmitted to RyR1 , which then releases calcium from the sarcoplasmic reticulum (SR) into the cytoplasm. Alternatively, calcium entering the cell through DHPR could induce calcium release from SR (via RyR1) through a mechanism known as calcium-induced calcium release (CICR). Regardless of the mechanism, however, depolarisation of the plasmamembrane causes calcium release through RyR1 , thereby causing an increase in the cytoplasmic calcium concentration, which again triggers cross-bridge cycling and muscle contraction. The released calcium is ultimately pumped back into the SR by the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA pump) in an ATP consuming process. During contraction, calcium cycling is responsible for about 20-50% of the ATP consumed (Clausen, T., Hardeveld, C. van and Everts, M. E. (1991), Physiol. Rev. 71(3), 733-773.). A modest depolarisation and thereby a modest increase in the cytoplasmic calcium concentration induced by increasing the extracellular potassium concentration to about 20 mM can, furthermore, increase the oxygen consumption of isolated skeletal muscle 10 times or more without eliciting contraction (Erlij, D., Shen, W. K., Reinach, P. and Schoen, H. (1982), Am. J. Physiol. 243, C87-C95 and Hardeveld, C. van and Clausen, T. (1984), Am. J. Physiol. 247, E421-E430.) The latter effect can be inhibited by the RyR1 antagonist dantrolene. An increased calcium release through RyR1 and consequently an increased pumping activity and ATP consumption of the SERCA pump is accordingly responsible for the increase in energy expenditure (Erlij, D., Shen, W. K., Reinach, P. and Schoen, H. (1982), Am. J. Physiol. 243, C87-C95). It should finally be mentioned that a number of intracellular proteins - e.g. FK506 12kDa binding protein (FKBP12), Calmodulin, the dihydropyridin receptor (DHPR), Sorcin, Calseques- trin, Annexin VI, Triadin, Junctin, S100s etc. interact with and modulate the Ca²⁺ releasing activity of RyR1 (Mackrill, J. J. (1999), Biochem. J. 337, 345-361, and Zucchi, R. and Ronca- Testoni, S. (1997), Pharmacol. Rev. 49(1), 1-51).

One object of the present invention is to provide compounds which can effectively be used for the treatment of obesity by increasing energy expenditure through stimulation of the intracellular calcium cycling described above.

SUMMARY OF THE INVENTION

The present invention provides the use of a compound that induces Ca²⁺ release through the type 1 Ryanodine receptor for the preparation of a pharmaceutical composition for the treat- ment of obesity.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the present invention provides the use of a compound that induces Ca²⁺ release through the type 1 Ryanodine receptor, or pharmaceutically acceptable salts thereof, for the manufacture of a medicament for the treatment of obesity.

In one embodiment the compound is a RyR1 agonist.

In a further embodiment the compound induces Ca²⁺ release through RyR1 by affecting the interaction between an intracellular protein and RyR1. In one embodiment the intracellular protein is FKBP12. In a further embodiment the intracellular protein is Calmodulin. In a still further embodiment the intracellular protein is the dihydropyridin receptor (DHPR). In a further embodiment the intracellular protein is Sorcin. In a still further embodiment the intracellular protein is Calsequestrin. In a further embodiment the intracellular protein is Annexin VI. In a still further embodiment the intracellular protein is Triadin. In a further embodiment the intracellular protein is Junctin. In a still further embodiment the protein is S 00s. In a still further embodiment the compound has a more pronounced effect on Ca²⁺ release through RyR1 as compared to its effects on Ca²⁺ release through RyR2 and RyR3.

In a further embodiment the compound is a Bastadin or a derivative thereof, especially Bastadin 5 or Bastadin 10 or a derivative thereof.

In a still further embodiment the compound is Ryanodine or a derivative thereof.

In a further embodiment the compound is 2-phenyl-4,7-dioxobenzothiazole or a derivative thereof.

In a still further embodiment the compound is 3-(3,4,5-Trimethoxy-phenyl)-but-2-enoic acid (4-methoxy-phenyl)-amide

and pharmaceutically acceptable salts thereof.

In a still further embodiment the compound is selected from the group consisting of ryanodine, 9-methyl-7-bromoeudistomin D (MBED), sulmazole, anthraquinones, milrinone, suramin, 4-chloro-m-cresol, δ-hexachlorocyclohexane, FK-506, rapamycin, bastadins (e.g. Bastadin 5 and Bastadin 10), quinolidomicin A1, heparin, miotoxin a, thimerosal, dithiodipyridine, mesotetra-(4-N-methylpyridyl)-porphine tetraiodide (TMPyP), disulfonic stilbene derivatives, palmitoyl carnitine, 2-phenyl-4,7-dioxobenzothiazole, and adenine nucleotides, (Zucchi, R. and Ronca-Testoni, S. (1997), Pharmacol. Rev. 49(1), 1-51) and pharmaceutically acceptable salts thereof.

The induction of Ca²⁺ release through the type 1 Ryanodine receptor can e.g. be measured by the use of the assays described in examples 1 and 2.

In one embodiment the Ca²⁺ release, at the therapeutic dose of the compound, is below the threshold for eliciting contraction in the skeletal muscle. In a further embodiment EC₅₀ for the induction of Ca²⁺ release through RyR1 is at least ten times lower than EC₅₀ for the induction of Ca²⁺ release through RyR2 and RyR3.

The above mentioned compounds are available from commercial suppliers and derivatives and analogues may be prepared according to conventional methods.

The present compounds may have one or more asymmetric centres and it is intended that stereoisomers (optical isomers), as separated, pure or partially purified stereoisomers or racemic mixtures thereof, are included in the scope of the invention.

The present invention also encompasses pharmaceutically acceptable salts of the present compounds. Such salts include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts. Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric acids and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methane- sulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids and the like. Further examples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable salts listed in J. Pharm. Sci. 1977, 66, 2, which is incorporated herein by reference. Examples of metal salts include lithium, sodium, potassium, magnesium salts and the like. Examples of ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium, tetramethylammonium salts and the like.

Also intended as pharmaceutically acceptable acid addition salts are the hydrates which the present compounds are able to form.

Within the context of the present invention, a RyR1 agonist is understood to refer to any compound which at a given concentration induces Ca²⁺ release through the type 1 Ryano- dine receptor. Within the context of the present invention, treatment of obesity is to be understood as treatment and/or prevention of obesity.

In a further aspect, the present invention relates to a method for the treatment of obesity which method comprises administering an effective amount of a compound as defined above to a patient in need of such a treatment.

In one embodiment the effective amount of the compound is in the range from about 0.05 to about 2000 mg, preferably from about 0,1 mg to about 1000 mg and especially preferred from about 0.5 to about 500 mg per day.

In a still further aspect, the invention relates to the use of a compound as defined above for the manufacture of a medicament for the reduction of BMI.

In a further aspect, the invention relates to the use of a compound as defined above for the manufacture of a medicament for increasing the energy expenditure.

In a still further aspect, the invention relates to a pharmaceutical composition comprising, as an active ingredient, a compound as defined above or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier. In one embodiment the composition comprises from about 0.05 to about 1000 mg of the compound or a pharmaceutically acceptable salt thereof. In a further embodiment the compound is ryanodine. In a further embodiment the compound is 9-methyl-7-bromoeudistomin D (MBED). In a still further embodiment the compound is sulmazole. In a further embodiment the compound is anthraquinones. In a still further embodiment the compound is milrinone. In a further embodiment the compound is suramin. In a still further embodiment the compound is 4-chloro-m-cresol. In a still further embodiment the compound is δ-hexachlorocyclohexane. In a further embodiment the compound is FK-506. In a still further embodiment the compound is rapamycin. In a further embodiment the compound is a bastadin (e.g. Bastadin 5 or Bastadin 10). In a still further embodiment the compound is quinolidomicin A1. In a further embodiment the compound is heparin. In a still further embodiment the compound is miotoxin a. In a further embodiment the compound is thimerosal. In a still further embodiment the compound is dithiodipyridine. In a further embodiment the compound is mesotetra-(4-N-methylpyridyl)-porphine tetraiodide (TMPyP). In a still further embodiment the compound is a disulfonic stilbene derivative. In a further embodiment the compound is palmitoyl camitine. In a still further emboodiment the compound is 2-phenyl-4,7-dioxobenzothiazole. In a further embodiment the compound is an adenine nucleotide.

In a further aspect, the invention relates to a pharmaceutical composition for the treatment of obesity, the composition comprising as an active ingredient, a compound as defined above or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier.

In a further aspect of the invention the present compounds may be administered in combination with further pharmacologically active substances e.g. an antidiabetic or other pharmacologically active material, including other compounds for the treatment and/or prevention of insulin resistance and diseases, wherein insulin resistance is the pathophysiological mechanism. Suitable antidiabetics comprise insulin, GLP-1 derivatives such as those disclosed in WO 98/08871 to Novo Nordisk A/S which is incorporated herein by reference as well as orally active hypoglycaemic agents.

The orally active hypoglycaemic agents preferably comprise sulphonylureas, biguanides, oxadiazolidinediones, thiazolidinediones, glucosidase inhibitors, glucagon antagonists, GLP- 1 agonists, potassium channel openers such as those disclosed in WO 97/26265 and WO 99/03861 to Novo Nordisk A/S which are incorporated herein by reference, insulin sensitizers, hepatic enzyme inhibitors, glucose uptake modulators, compounds modifying the lipid metabolism, compounds lowering food intake, PPAR and RXR agonists and agents acting on the ATP-dependent potassium channel of the β-cells.

Furthermore, the compounds according to the invention may be administered in combination with antiobesity agents or appetite regulating agents.

Such agents may be selected from the group consisting of CART agonists, NPY antagonists, MC4 agonists, orexin antagonists, H3 antagonists, TNF agonists, CRF agonists, CRF BP antagonists, urocortin agonists, β3 agonists, MSH agonists, CCK agonists, serotonin re-uptake inhibitors, mixed serotonin and noradrenergic compounds, 5HT agonists, bombesin agonists, galanin antagonists, growth hormone, growth hormone releasing compounds, TRH agonists, uncoupling protein 2 or 3 modulators, leptin agonists, DA agonists (bromocriptin, doprexin), lipase/amylase inhibitors, PPAR modulators, RXR modulators or TR β agonists. In a still further aspect the invention relates to a method of identifying a RyR1 agonist characterised by the use of skeletal muscle microsomes or membrane preparations from cell lines that express RyR1 , recombinantly or endogenously, for screening out compounds that affect the binding of ³H-ryanodine to RyR

In a further aspect the invention relates to a method of identifying a RyR1 agonist characterised by the use of cell lines that express RyR1 , recombinantly or endogenously, for screening out compounds that induce Ca²⁺ release through RyRl

The RyR agonist halothane can induce Ca²⁺ release through recombinant RyR's when expressed in e.g. HEK293 cells. Furthermore, the dissociation konstant (K_d) for ³H-ryanodine binding to recombinant RyR1 is identical to the K_ϋ obtained with native skeletal muscle RyR1 (Du, G. G., Imredy, J. P. and MacLennan, D. H. (1998), J. Biol. Chem. 273 (50), 33259- 33266). In conclusion, the recombinant RyR's seem to preserve the characteristics of the na- tive RyR's.

PHARMACEUTICAL COMPOSITION

The compounds of the invention may be administered alone or in combination with pharmaceutically acceptable carriers or excipients, in either single or multiple doses. The pharmaceutical compositions according to the invention may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy,19^th Edition.Oennaro, Ed., Mack Publishing Co., Easton, PA, 1995.

The pharmaceutical compositions may be specifically formulated for administration by any suitable route such as the oral, rectal, nasal, pulmonary, topical (including buccal and sublin- gual), transdermal, intracisternal, intraperitoneal, vaginal and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) route, the oral route being preferred. It will be appreciated that the preferred route will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated and the active ingredient chosen. Pharmaceutical compositions for oral administration include solid dosage forms such as capsules, tablets, dragees, pills, lozenges, powders and granules. Where appropriate, they can be prepared with coatings such as enteric coatings or they can be formulated so as to provide controlled release of the active ingredient such as sustained or prolonged release ac- cording to methods well-known in the art.

Liquid dosage forms for oral administration include solutions, emulsions, suspensions, syrups and elixirs.

Pharmaceutical compositions for parenteral administration include sterile aqueous and non- aqueous injectable solutions, dispersions, suspensions or emulsions as well as sterile powders to be reconstituted in sterile injectable solutions or dispersions prior to use. Depot injectable formulations are also contemplated as being within the scope of the present invention.

Other suitable administration forms include suppositories, sprays, ointments, cremes, gels, inhalants, dermal patches, implants etc.

A typical oral dosage is in the range of from about 0.001 to about 100 mg/kg body weight per day, preferably from about 0.01 to about 50 mg/kg body weight per day, and more preferred from about 0.05 to about 10 mg/kg body weight per day administered in one or more dosages such as 1 to 3 dosages. The exact dosage will depend upon the frequency and mode of administration, the sex, age, weight and general condition of the subject treated, the nature and severity of the condition treated and any concomitant diseases to be treated and other factors evident to those skilled in the art.

The formulations may conveniently be presented in unit dosage form by methods known to those skilled in the art. A typical unit dosage form for oral administration one or more times per day such as 1 to 3 times per day may contain of from 0.05 to about 1000 mg, preferably from about 0.1 to about 500 mg, and more preferred from about 0.5 mg to about 200 mg.

For parenteral routes, such as intravenous, intrathecal, intramuscular and similar administration, typically doses are in the order of about half the dose employed for oral administration.

The compounds of this invention are generally utilized as the free substance or as a pharmaceutically acceptable salt thereof. One example is an acid addition salt of a compound having the utility of a free base. When a compound of the invention contains a free base such salts are prepared in a conventional manner by treating a solution or suspension of a free base of the compound with a chemical equivalent of a pharmaceutically acceptable acid, for example, inorganic and organic acids. Representative examples are mentioned above. Physiologically acceptable salts of a compound with a hydroxy group include the anion of said compound in combination with a suitable cation such as sodium or ammonium ion.

For parenteral administration, solutions of the present compounds in sterile aqueous solution, aqueous propylene glycol or sesame or peanut oil may be employed. Such aqueous solutions should be suitable buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. The aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. The sterile aqueous media employed are all readily available by standard techniques known to those skilled in the art.

Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solution and various organic solvents. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid or lower alkyl ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phosphol- ipids, fatty acids, fatty acid amines, polyoxyethylene or water. Similarly, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax. The pharmaceutical compositions formed by combining the compounds of the invention and the pharmaceutically acceptable carriers are then readily administered in a variety of dosage forms suitable for the disclosed routes of administration. The formulations may conveniently be presented in unit dosage form by methods known in the art of pharmacy.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules or tablets, each containing a predetermined amount of the active ingredient, and which may include a suitable excipient. These formulations may be in the form of powder or granules, as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion.

If a solid carrier is used for oral administration, the preparation may be tabletted, placed in a hard gelatine capsule in powder or pellet form or it can be in the form of a troche or lozenge. The amount of solid carrier will vary widely but will usually be from about 25 mg to about 1 g. If a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gela- tine capsule or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution.

A typical tablet which may be prepared by conventional tabletting techniques may contain:

Core:

Active compound (as free compound or salt thereof) 5 mg

Colloidal silicon dioxide (Aerosil) 1.5 mg

Cellulose, microcryst. (Avicel) 70 mg Modified cellulose gum (Ac-Di-Sol) 7.5 mg

Magnesium stearate q.s.

Coating:

HPMC approx. 9 mg

*Mywacett 9-40 T approx. 0.9 mg

*Acylated monoglyceride used as plasticizer for film coating.

The compounds of the invention may be administered to a mammal, especially a human in need thereof. Such mammals include also animals, both domestic animals, e.g. household pets, and non-domestic animals such as wildlife.

If desired, the pharmaceutical composition of the invention may comprise the compound of the invention in combination with further pharmacologically active substances such as those described in the foregoing.

PHARMACOLOGICAL METHODS

Compounds that induce Ca²⁺ release through the RyR1 may be evaluated in vitro and in vivo for their ability to increase skeletal muscle metabolism and energy expenditure, and such evaluation may be performed as described below.

Identification of compounds that increase the oxygen consumption of muscle preparations in vitro

Rat paired soleus muscles are isolated and mounted in vitro between a displacement device and an isometric force transducer and superfused with oxygenated physiological salt solution at 30°C. Force development and skeletal muscle oxygen consumption (measured using fibre optic oxygen sensors) is monitored continuously. Concentration response curves for RyR1 agonists and/or partial agonists are generated in order to determine the concentration required to induce a half maximal effect in muscle resting tension, active twitch contraction and oxygen consumption. The compounds which increase the oxygen consumption at a concentration where there is no effect on contraction are investigated further.

Identification of compounds RyR1 agonists and/or partial agonists that increases energy expenditure in vivo The effects of the RyR1 agonists and/or partial agonists on energy expenditure in rats, hamsters, mice, or guinea pigs is determined by indirect calorimetry. The animals are placed in airtight acrylic Oxymax chambers and O₂ and CO₂ concentrations in the air led to and from (inlet and outlet air) the chambers are recorded and the consumption of O₂ and production of CO₂ is calculated. Based on the amount of O₂ consumed and CO₂ produced, energy expenditure is calculated. Compounds which at a given dose increase whole body energy expenditure without obvious deleterios effects are investigated further.

The present invention is further illustrated by the following examples which, however, are not to be construed as limiting the scope of protection.

The features disclosed in the foregoing description and in the following examples may, both separately and in any combination thereof, be material for realising the invention in diverse forms thereof. EXAMPLES

Example 1

Identification of active compounds

Active compounds were identified in a cell-based Ca²⁺-release assay, using the FLIPR₃₈₄ (Molecular Devices Corp., Sunnyvale, CA, USA) High Throughput Screening (HTS) system. Briefly, RyR1 expressing CHO cells were cultured in 384-well microtiter plates. Prior to the experiment cells were loaded with the Ca²⁺ sensitive dye Fluo4-AM (Molecular Probes Inc., Eugene, OR, USA). The effects of the test compounds on the intracellular calcium concentration in RyR1 expressing CHO cells were subsequently evaluated using the FLIPR s according to the manufacturers descriptions. As an example the following compound was found to increase the intracellular calcium concentration in the RyR1 expressing CHO cells: 3-(3,4,5-Trimethoxy-phenyl)-but-2-enoic acid (4-methoxy-phenyI)-amide

Compounds which increased the intracellular calcium concentration in the RyR1 cells (such as the compound shown above) were further evaluated as described in example 2.

Example 2 Identification of compounds which preferentially induce Ca²⁺ release through RyR1

Dose-response curves for the effects of the compounds from example 1 on the intracellular calcium concentration in non-transfected CHO cells, RyR1 expressing CHO cells, RyR2 expressing CHO cells as well as RyR3 expressing cells were generated using the FLIPR₃₈₄. Only compounds which at the EC₅₀ concentration for induction of Ca²⁺ release through RyR1 have little if any effect on the intracellular calcium concentration in non-transfected CHO cells, RyR2 expressing CHO cells and RyR3 expressing cells are investigated further. It should, furthermore, be mentioned that partial RyR1 agonists are particularly interesting since such compounds should possess superior safety characteristics in vivo. However, the most promising compounds are ultimately being tested with regard to their in vitro effects on contraction and oxygen consumption (or glucose utilisation) of isolated skeletal muscle preparations and their in vivo effect on energy expenditure. Compounds that at a given dose/concentration increase energy expenditure in vivo or in vitro without affecting contraction will be investigated further.

FURTHER EXAMPLES DESCRIBING ALTERNATIVE WAYS TO IDENTIFY ACTIVE COMPOUNDS

Example 3

Identification of compounds that affect the binding of ³H-ryanodine to RyR1

Many but not all compounds that induce Ca²⁺ release through RyR1 are known to increase the binding of ³H-ryanodine to RyR1. It is therefore possible to use an assay, based on binding of ³H-ryanodine to RyR1 containing membranes/microsomes, for the identification of compounds that induce Ca²⁺ release through RyRl

Briefly, microsomes are prepared according to standard protocols from either skeletal muscle (Campbell, K. P., and MacLennan, D. H. (1981), J. Biol. Chem. 256, 4626-4632) or cell lines which endogenously or recombinantly express RyR1 (Chen, S. R., Vaughan, D. M., Airey, J. A., Coronado, R., and MacLennan, D. H. (1993), Biochemistry 32, 3743-3753). ³H-ryanodine is obtained from DuPont NEN and is used at a concentration approximately equal to one third the dissociation constant, K_D (approximately 3 nM). Binding of ³H-ryanodine to the RyR1 containing microsomes, in the presence of various test compounds, is subsequently measured as previously described (Du, G. G. and MacLennan, D. H. (1998), J. Biol. Chem. 273 (48), 31867-31872; and Du, G. G., Imredy, J. P. and MacLennan, D. H. (1998), J. Biol. Chem. 273 (50), 33259-33266). Preferably, however, a high throughput screening (HTS) set- up based on e.g. the scintillation proximity assay (SPA) or 96-well filter plate (e.g. from Milli- pore, Bedford, MA, USA) technology is used for the identification of compounds, which affect the binding of ³H-ryanodine to RyR1. Compounds which, at a concentration of ≤ 50 μM increase the binding of ³H-ryanodine to RyR1 more than 50% are investigated further (see example 2 and 3). Example 4

Identification of compounds that preferentially affect the binding of ³H-rvanodine to RyR1

Herein it is investigated whether the compounds from example 1 affect the binding of ³H- ryanodine to RyR2 and RyR3. RyR2 containing microsomes are prepared (Campbell, K. P., and MacLennan, D. H. (1981), J. Biol. Chem. 256, 4626-4632; and Chen, S. R., Vaughan, D. M., Airey, J. A., Coronado, R., and MacLennan, D. H. (1993), Biochemistry 32, 3743-3753) from either the cardiac muscle or from cell lines which endogenously or recombinantly express RyR2. RyR3 containing microsomes are prepared from either the brain (does also con- tain RyR2) or from cell lines which endogenously or recombinantly express RyR3^2,3. Again a ³H-ryanodine concentration equal to about _D/3 is used in the binding assay, which otherwise is performed as described in example 1. Compounds are tested at a concentration that increase the binding of ³H-ryanodine 50% in the RyR1 binding assay. Compounds which change the binding of ³H-ryanodine to RyR2 and RyR3 less than 25% (thereby showing some selectivity towards RyR1) are selected for further investigation.

Example 5

Additional example describing identification of active compounds

As a further example, active compounds can be identified by their effect on cellular energy expenditure in e.g. a cell-based glucose utilisation assay. Briefly, Cells which endogenously or recombinantly express RyR1 are cultured in 24-well (or 96-well) plates over night, followed by an 1 hour incubation in the presence of ¹⁴C-glucose and test compound. The ¹⁴CO₂ gen- erated during the 1 hour incubation is measured in a ¹⁴CO₂ capture assay as previously described (Collins, C. L, Bode, B. P., Souba, W. W., and Abcouwer, S. F. (1998), Biotech- niques 24 (5), 803-808). Compounds that in RyR1 expressing cells increase the cellular CO₂ production more than 10% are subsequently tested on cells which do not express RyR1 (same assay). Only compounds which have no effect on the cellular CO₂ production in the latter cell type are further investigated with respect to in vitro effect on contractility and glucose utilisation of isolated skeletal muscle and in vivo effect on basal metabolic rate etc.

Claims

1. The use of a compound that induces Ca²⁺ release through the type 1 Ryanodine receptor, or pharmaceutically acceptable salts thereof, for the manufacture of a medicament for the treatment of obesity.

2. The use according to claim 1, wherein the compound is a RyR1 agonist.

3. The use, according to claim 1 , wherein the compound induces Ca²⁺ release through RyR1 by affecting the interaction between an intracellular protein and RyR1.

4. The use according to any of the claims 1-3, wherein the compound is a Bastadin or a derivative thereof.

5. The use according to any of the claims 1-4, wherein the compound is Bastadin 5 or Bastadin 10 or a derivative thereof.

6. The use according to any of the claims 1-2, wherein the compound is Ryanodine or a derivative thereof.

7. The use according to any of the claims 1-2, wherein the compound is 2-phenyl-4,7- dioxobenzothiazole or a derivative thereof.

8. The use according to claim 1 , wherein the compound is 3-(3,4,5-Trimethoxy-phenyl)-but-2-enoic acid (4-methoxy-phenyl)-amide

and pharmaceutically acceptable salts thereof.

9. The use according to claim 1 , wherein the compound is selected from the group consisting of Ryanodine, 9-methyI-7-bromoeudistomin D (MBED), sulmazole, anthraquinones, milrinone, suramin, 4-chloro-m-cresol, δ-hexachlorocyclohexane, FK-506, rapamycin, Bastadins (such as Bastadin 5 and Bastadin 10), quinolidomicin A1, heparin, miotoxin a, thimerosal, dithiodipyridine, mesotetra-(4-N-methylpyridyl)-porphine tetraiodide (TMPyP), disulfonic stilbene derivatives, palmitoyl carnitine, 2-phenyl-4,7-dioxobenzothiazole, and adenine nucleotides, and pharmaceutically acceptable salts thereof.

10. A method for the treatment of obesity which method comprises administering an effective amount of a compound as defined in any one of the claims 1-9 to a patient in need of such a treatment.

11 The method according to claim 10 wherein the effective amount of the compound is in the range from about 0.05 to about 2000 mg, preferably from about 0,1 mg to about 1000 mg and especially preferred from about 0.5 to about 500 mg per day.

12. A method of identifying a RyR1 agonist characterised by the use of skeletal muscle mi- crosomes or membrane preparations from cell lines that express RyR1 , recombinantly or endogenously, for screening out compounds that affect the binding of ³H-ryanodine to RyRl

13. A method of identifying a RyR1 agonist characterised by the use of cell lines that express RyR1 , recombinantly or endogenously, for screening out compounds that induce Ca²⁺ re- lease through RyR1.

14. The use of a compound as defined in any one of the claims 1-9 for the manufacture of a medicament for the reduction of BMI.

15. The use of a compound as defined in any one of the claims 1-9 for the manufacture of a medicament for increasing the energy expenditure.

16. A pharmaceutical composition comprising, as an active ingredient, a compound as defined in any of the claims 1-9 or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier.

17. The composition according to claim 16 in unit dosage form, comprising from about 0.05 to about 1000 mg of the compound or a pharmaceutically acceptable salt thereof.

18. A pharmaceutical composition for the treatment of obesity, the composition comprising as an active ingredient, a compound as defined in any of the claims 1-9 or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier.