NOVEL PEPTIDE

Abstract: A pentapeptide having the peptide sequence H-Val-Pro-Gly-Pro-Arg-OH and addition salts thereof. The pentapeptide has appetite regulating activity.

Description: Novel peptide Field of the Invention The present invention relates to a new pentapeptide and addition salts thereof having appetite regulating activity, processes for its preparation, pharmaceutical compositions containing it and a method for its pharmacological use. The object of the invention is to provide a pentapeptide with appetite regulating activity. Such a substance will be useful in the reduction of obesity. Background of the Invention From EP 269 595 it is known that the petide sequence X-Pro-Y-Pro-Arg wherein a) X is Ala and Y is Gly, or b) X is Val and Y is Asp, and derivatives thereof have an effect on the regulation of the appetite. Disclosure of the Invention According to the present invention it has been found that pentapeptide of the peptide sequence H-Val-Pro-Gly-Pro-Arg-OH or its addition salts with organic or inorganic acids and bases is a potent appetite regulating agent. Acid addition salts with organic or inorganic acids may be prepared by lyophilisation from aqueous solutions contain- ing acids such as acetic acid, trifluoroacetic acid, ben- zoic acid, hydrochloric acid, sulphuric acid etc. Addition salts with organic bases, such as secondary or tertiary amines, may also be prepared through lyophilisation from aqueous solutions containing such bases. Alternatively, ion exchange chromatography can be employed in order to obtain salts of the peptide. Such methods are well known in the art. Particularly preferred are the salts with acetic acid, trifluoroacetic acid, dicyclohexylamine or ammonia. The salt-forming agent may also be an α-amino acid. The a ino acids making up the pentapeptide of the invention are all of the L-isomer form. Obesity can be associated with serious and life threatening disorders such as diabetes mellitus, arteriosclerosis, hypertension, angina pectoris, thrombosis and pickwickian syndrome. Reduction of obesity by appetite regulation can therefore be useful in the treatment of such disorders. Methods of preparation The pentapeptide of the present invention can be prepared using the standard solid phase sequential coupling tech¬ nique utilizing an automatic peptide synthesiser. The preparation starts from the C-terminal amino acid which can be obtained grafted to a methylbenzhydrylamine, benzhydrylamine or chloromethylated resin. The other amino acids are grafted step by step, after having protected the side chains thereof. Protective groups for the side chains of amino acids are well known in the art. The whole protected peptide is released either from the chloro¬ methylated resin by ammoniolysis to obtain the protected amide, or by hydrofluoric acid treatment to obtain the acid, or from the methylbenzhydrylamine or benzhydrylamine resins by treatment with hydrofluoric acid at 0°C, to obtain, at the same time, the release from the resin and the deprotection. The t-butoxycarbonyl (BOC) group is preferably used to protect the α-amino group. The choice principle used to select the protective groups for the side chains is that said groups must not be removed when BOC is removed from the α-amino group, step by step, during the preparation. The peptide can also be obtained via stepwise solution phase coupling using the appropriately derivatised amino acids. The same protective groups as in the solid phase strategy may be used here. Systematic experiments on rats have demonstrated that they lose their appetite and weight when given injections of the pentapeptide. The rats given the pentapeptide show satiety. The peptide according to the invention can be used as a drug, for instance in the form of pharmaceutical compositions. The pharmaceutical compositions can be administered orally, for instance in the form of tablets, film coated tablets, dragees, hard and soft gelatine capsules, solutions, emulsions or suspensions. The peptide can also be enclosed in microcapsules. The administration can also be rectally, for instance in the form of suppositories or parenterally, for instance in the form of solutions for injection. The peptide can also be administered via inhalation or as a nose spray or powder. Finally the administration as sublinguett should be mentioned. For the preparation of tablets, film coated tablets, dragees and hard gelatine capsules the peptide can be mixed with pharmaceutically inert, inorganic or organic excipients. For tablets, dragees and hard gelatine WO,2 ,388 ° „ PCT/SE92/00027 capsules such excipients can be lactose, corn starch or derivatives thereof, talc, stearin acid or salts thereof, etc. 5 For soft gelatine capsules vegetable oil, wax, fats, semisolid and liquid polyols etc, are suitable as excipients. For the preparation of solutions and sirups water, polyol, 10 saccharose, invert sugar, glucose and the like are suitable as excipients. For solutions for injection water, alcohol, polyol, glycerine, vegetable oil etc are suitable as excipients. 15 For suppositories natural or hardened oil, wax, fat, semiliquid or liquid polyol and the like are suitable as excipients. 20 The pharmaceutical compositions can also contain preservatives, stabilizers, e ulgators, sweeteners, colouring matters, flavourings, salts for changing the osmotic pressure, buffers, sugar coatings or antioxidants. They can also contain further therapeutically valuable 25 agents. The dosage of the pentapeptide according to the invention to homo should lie within the interval 1-100 mg/day, preferably 1-20 mg/day. The pentapeptide should be given 30 at each meal. The invention is illustrated by the following non-limiting examples. _ rCr/SE92/0«,27 EXAMPLES Preparation of H-Val-Pro-Gly-Pro-Arg-OH acetate: The resin used was Applied Biosystems Inc. Boc-Arg(Mts)- 5 PAM resin (806 g, 0.5 mmol) . Sequencial coupling was per¬ formed on an ABI model 43OA instrument using the standard Boc-peptide synthesis protocol. Briefly, each cycle con¬ sisted of the following steps: wash (CH 2 C1 2 ) , cleavage (TFA, 33% in CH 2 C1 2 , then 50% in CH-,C1 2 ) , wash (CH^l-.), 0 wash (DMF), neutralisation (Diisopropylethylamine, 10% in DMF, wash (DMF), coupling using the preformed symmetrical protected amino acid anhydride in DMF. The last coupling cycle (valine) was followed by deprotection of the α-amino 5 group as above and neutralisation, washing with CH-,C1 2 , and drying under a stream of nitrogen and finally in vacuo. Cleavage and removal of the side chain protecting groups was accomplished on an HF-reaction apparatus type 1 from Peptide Institute Inc. using 8 mL of concentrated HF 0 containing 1 mL dimethyl sulphide and 1 g of 4- methylphenol. Cleavage was performed at -5 - 0°C for 2 h. The HF was evaporated and the residue thoroughly washed with diethyl ether and extracted with 20% acetic acid, which was diluted to 10% and lyophilised. The crude 5 pentapeptide was purified by preparative HPLC using a YMC S-5 120A ODS column (20 mm x 250 mm), using gradient elution (10 mL/min) with a gradient from 10-20% of B in A during 15 min (A = 0.1% TFA and 0.015% NH-. in H 2 0; B = 70% CH 3 CN, 0.085% TFA and 0.015% NH-. in H 2 0) (t R = 12 min). 0 Evaporation of most of the eluent and lyophilisation from 10% acetic acid gave the title pentapeptide as colourless solid. FAB-MS (VG Trio-II instrument): 525 (MH^); 271 (C" 3 ); 368 (C" 4 ); 426 (Y" 4 ); 329 (Y" 3 ). Regulation of the appetite Systematic experiments on rats have demonstrated that they lose their appetite and weight when given injections of the pentapeptide. The rats were fasting for 17 hours. 15 minutes before the food was presented the rats were given an intraperitoneal injection containing peptide or saline, respectively, and the cumulated food intake was measured at times 0, 30 minutes, 60 minutes, 2 hours, 4 hours and 6 hours. The rats were given saline one day, peptide the following day, saline etc. The results are shown in the following tables, Table 1 Time Intake of food (g) Intake of food (g) (hours) Control Peptide (20 μg) n = 7 n = 7 0 1/2 5.94 + 0.14 5.04 + 0.27 1 9.39 + 0.30 6.44 + 0.38 2 12.10 + 0.38 9.01 + 0.42 4 14.56 + 0.33 11.36 + 0.49 6 15.59 + 0.49 10.36 + 0.28 Pharmaceutical compositions The peptide according to the invention can be administered in ordinary pharmaceutical compositions. Below is presented some proposals for compositions wherein the peptide is a constituent.

Claims: Claims 1. A pentapeptide having the peptide sequence H-Val-Pro-Gly-Pro-Arg-OH or its addition salts with organic or inorganic acids and bases. 2. A process for the preparation of the pentapeptide according to claim 1, characterized by a) coupling the amino acids sequentially in solid phase starting from the C-terminal amino acid utilizing an automatic peptide synthesiser, or b) coupling the amino acids stepwise in a solution phase using appropriately derivatised amino acids. 3. A pharmaceutical preparation comprising as active ingredient a pentapeptide according to claim 1. 4. A pharmaceutical preparation according to claim 3 in dosage unit form. 5. A pharmaceutical preparation according to claims 3-4 comprising the active ingredient in association with a pharmaceutically acceptable carrier. 6. A pentapeptide according to claim 1 for use of a therapeutically active substance. 7. Use of a pentapeptide according to claim 1 for the preparation of medicaments with appetite regulating activity. 8. A method for the treatment of obesity, in mammals including man, characterized by administration to a host in need of such treatment of an effective amount of a pentapeptide according to claim 1.

Inventors: ANDERSSON CARL-MAGNUS ALEXANDE, ERLANSON-ALBERTSSON CHARLOTTE, ANDERSSON, CARL-MAGNUS, ALEXANDER, ERLANSON-ALBERTSSON, CHARLOTTE
Applicants: ASTRA AB, AKTIEBOLAGET ASTRA
Application year: 1992
Main classification: A61K37
IPC codes: A61K38, A61P3, C07K1, C07K7