DE19622502A1 - Truncated versions of exendin peptide(s) for treating diabetes - Google Patents

Abstract

Peptides (I) and (II), their salts and esters, and derivatives with residues 1, 2, 28, 29 and/or 30 deleted, are new: R1R2N-HSDGTFTSDLSKQMEEEAVRLFIEWLKNGX-COR3 (I) R1R2N-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGX-COR3 (II) R1 = hydrogen, acetyl, trifluoroacetyl, adamantyl, fluorenyloxymethyl, benzyloxymethyl, butoxycarbonyl, Alloc (not defined), 1-6C alkyl, 2-8C alkenyl or 7-9C aralkyl R2 = R1 except that 1-6C alkyl is replaced by 4-6C alkyl R3 = OR4 or NR4R5 R4 = H or 1-6C alkyl R5 = H or 1-6C alkyl. Also new are derivatives of (I) and (II), with up to 11 specific modifications in the peptide chain.

Description

This invention relates to novel exendin analogs useful in the therapy of diabetes mellitus can be used, process for their preparation and containing them Drug.

Background of the invention

A functional connection between small intestine and exocrine pancreas has been reported in the Sixties proved after the accurate determination of insulin in the plasma became possible. The insulin response after oral glucose administration is much stronger than those after intravenous glucose administration, even if identical glucose plasma levels be achieved. This "incretin effect" is explained in the functional combination of entero-insular axis. This effect is caused by intestinal hormones, which after Meals released from the small intestine, increased measurably circulate in the plasma and the Enhance glucose-induced insulin release. In addition to the classic incretin hormone "Gastric inhibitory polypeptide I" (GIP), is today "glucagon-like peptide-1" (GLP-1) in the foreground of interest. In a relatively short time GLP-1 is from physiologically most interesting incretin hormone candidates as a potential alternative For the treatment of diabetes mellitus type II matured. The present invention describes new substances that bind to the naturally occurring GLP-1 molecule in the Effect are modeled, but have been changed so that the stability in improved effectiveness of the peptide improved.

Antidiabetogenic effect

Infusion and subcutaneous injection of GLP-1 cause in patients with diabetes Type II mellitus a significant increase in insulin secretion and inhibition of insulin secretion Glucagon release (Gutniak, M. (1992); Kreymann, B. (1987); Nathan, D.M. (1992);  Nauck, M.A. (1993a & b)). Both are from a therapeutic point of interest and at the hypoglycemic effect of GLP-1 involved: insulin promotes its Intestinal tissue is the glucose uptake, and by the lower glucagon levels is the Suppressed gluconeogenesis. The inhibition of glucagon secretion must be considered indirect GLP-1 activity, since glucagon-producing A cells are not GLP-1 Express receptors (Komatsu, R. (1989)). Rather, it seems the increased Insulin and somatostatin release to be crucial. Both hormones are as Inhibitors of glucagon release known.

Certainly, two molecular mechanisms contribute to GLP-1-induced Insulin release in diabetes mellitus type II at. In addition to the direct reinforcement of Glucose-induced insulin release sensitizes GLP-1 to a subset of B cells to the key stimulus "glucose" (Fehmann, H.C. (1991)) and possibly also against other stimuli, so that a total of more B cells secrete insulin. This "priming" effect most likely explains the fact that GLP-1, despite its relative short plasma half-life leads to a prolonged insulin release.

This effect is dependent on increased glucose levels (<108 mg / dl) (Göke, R. (1993a)). It distinguishes GLP-1 in principle from the sulfonylureas, which are the Influencing Insulin Secretion Regardless of Glucose Plasma Levels. Is that sinking? Glucose value below 108 mg / dl, the insulin secretion will dry up even with intravenous Infusion of GLP-1. Therefore, the therapeutic use of GLP-1 hardly To expect hypoglycemia. In fact, they have been in previous clinical trials also not described.

From a therapeutic point of view, in every case the synthesis is more stable and more effective GLP-1 analog peptides desirable. It has now been based on the original synthesized from the venom of lizard isolated molecule exendin peptide analogues, with the goal of improved therapeutics for the treatment of diabetes mellitus type II develop. These peptides are therefore similar to GLP-1 and exendin, But they differ significantly from both hormones.  

The novel peptide sequences described as the subject of the invention show activity on insulin synthesis and delivery as well as effect on the insulin effect in the target tissues Muscle and fat tissue and gastric emptying.

Subject of the invention

The present invention is based on the sequence of exendin-3 and exendin-4, Peptides derived from the secretions of Heloderma horridum and Heloderma suspectum, respectively (Eng, J. et al. (1990, 1992)). The amino acid sequence and effect of Both peptides at the pancreas have already been published by several authors (Eng, J. et al. (1990); Raufman, J.P. (1992); Göke, R. (1993b); Thorens, B. (1993)). object of this invention are new truncated exendin fragments containing the Amino acid sequences of exendin-3- (1-30), or exendin-4- (1-30), wherein the C-terminal end of these sequences shortened by up to 3 amino acids or by up to extended to 8 amino acids and the N-terminal end by up to 1 amino acid can be shortened. Thus, peptide fragments with the following are particularly preferred sequences:

wherein the amino acids at position 1 or 28 and following depending on the given Chain length can be part of the sequence. The peptides are from the N-terminus to the C-terminus numbered.

The carboxyl group COR₃ of the amino acid at the C-terminal end can be in free form (R₃ = OH) or in the form of a physiologically acceptable alkali metal or alkaline earth metal salt, such as As the sodium, potassium or calcium salt. The carboxyl group can also be esterified with primary, secondary or tertiary alcohols, such as. B. Methanol, branched or unbranched C₁-C₆-alkyl alcohols, in particular Ethyl alcohol or tert-butanol. The carboxyl group can also be used with primary or be amidated secondary amines, such as. As ammonia, branched or unbranched C₁-C₆-alkylamines or C₁-C₆-di-alkylamines, in particular methylamine or Dimethylamine.

The amino group of the amino acid NR₁R₂ at the N-terminal end can be in free form (R₁, R₂ = H) or in the form of a physiologically acceptable salt, such as. B. of Chlorides or acetate are present. The amino group can also be acetylated with acids be such that R₁ = H and R₂ = acetyl, trifluoroacetyl, adamantyl, or by the common amino protecting groups of peptide chemistry, such as. Fmoc, Z, Boc, Alloc be protected, or N-alkylated with R₁ and / or R₂ = C₁-C₆-alkyl or C₂-C₈- Aralkyl or alkenyl.

Alkyl radicals are straight-chain, branched or optionally cyclic Understood alkyl radicals.

All exendin fragments can be present as fully or partially protected derivatives.

The invention also relates to exendin fragments with the abovementioned Properties and sequence lengths, in which additionally at least one but maximum thirteen of the modifications listed under (a) to (p) were carried out.  

Preferred are those exendin fragments which are at most 9, particularly preferred those having a maximum of 5 of the listed under (a) to (p) modifications.

• (a) The α-amino acid in position 1 is D-His, Ala, D-Ala, Gly, Lys or D-Lys, wherein Ala, Gly or Lys are particularly preferred; or
• (b) The α-amino acid in position 2 is Ser, D-Ser, Thr, D-Thr, Gly, Ala, D-Ala, Ile, D- Ile, Val, D-Val, Leu or D-Leu, preferably Ser, Thr, Gly, Ala, Val, Ile or Leu; or
• (c) The α-amino acid in position 3 is Glu, D-Glu, Asp, D-Asp, Ala or D-Ala, preferably Glu, Asp or Ala; or
• (d) The amino acid in position 4 is Ala, D-Ala or β-Ala, preferably Ala; or
• (e) The α-amino acid in position 5 is Ser, Tyr or Ala; or
• (f) The α-amino acid at position 6 is Ala, Ile, Val, Leu, Cha or Tyr Ala, Ile, Val, Leu or Tyr; or
• (g) The α-amino acid in position 7 is Ala, D-Ala, Tyr, D-Tyr, Ser, D-Ser or D-Thr, preferably Ala, Tyr or Ser; or
• (h) The amino acid in position 8 is Ala, Tyr or Thr; or
• (i) The α-amino acid in position 9 is Ala, D-Ala, Glu, D-Glu or D-Asp, preferably Ala or Glu; or
• (j) The amino acid in position 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 24, 28, 29, 31-38 is independently a proteinogenic or non-proteinogenic D- or L-amino acid, preferably a proteinogenic L-amino acid; or
• (k) The α-amino acid in position 13 is a neutral L-amino acid, preferably one neutral proteinogenic L-amino acid; or
• (l) The α-amino acid in position 14 is stabilized by a neutral L- or D-amino acid replaced, preferably by Nle, D-Nle, Ala, D-Ala, Ile, D-Ile, Val, D-Val, Leu or D-Leu, particularly preferred are Ile, Val or Ala; or
• (m) The α-amino acid in position 22 is D-Phe, Tyr, D-Tyr, Leu, D-Leu, Val, D- Val, L-Cha, D-Cha, β-1-Nal, β-2-Nal or β-1-D-Nal, preferred are Tyr, Leu or Val; or  
• (n) The α-amino acid in position 23 is Leu, D-Leu, D-Ile, Val, D-Val, L-Cha, D- Cha, Tyr, D-Tyr, Phe or D-Phe, preferred are Leu, Val, Tyr or Phe; or
• (o) The α-amino acid in position 25, 26 or 27 is a neutral L- or D- Amino acid, preferably a neutral, proteinogenic L-amino acid; or
• (p) The α-amino acid at position 30 is a proteinogenic or nonprotinogenic D or L-amino acid, preferably Arg, D-Arg, Tyr or D-Tyr, more preferably are Arg or Tyr.

Among the new exendin fragments, those which are particularly preferred are those which are adjacent the already mentioned properties and sequence lengths, at position 10 the amino acid Leucine and / or at position 19 the amino acid valine, at position 14 instead of Methionine contain the amino acid isoleucine or alanine and at position 30 arginine. Also particularly preferred are those modifications of exendin fragments which, in addition to the mentioned particularly preferred amino acids to the Positions 10, 14, 19 and 30, at position 2 one of the 20 known proteinogenic L-amino located acids.

The invention is in addition to new truncated and stabilized exendin-3 and Exendin-4 analogs also methods of making these analogs which include the Analogs in solid-phase synthesis from protected, contained in the analogues Amino acids, which are coupled together in the order which the Amino acid sequences correspond in the analogues and which optionally by corresponding amino acids not found in the natural exendin peptides were supplemented.

The abbreviations and definitions of the amino acids used were described in Pure Appl. Chem. 31, 639-45 (1972) and ibid. 40, 277-90 (1974) and agree with the PCT Rules (WIPO Standard St. 23: Recommendation for the Presentation of Nucleotide and Amino Acid Sequences in Patent Applications and Published Patent Documents). The one- or three-letter codes are as follows:  

amino acid abbreviations

The abbreviations stand for L-amino acids, if no other specifications such as D- or D, L- are indicated. D-Amino acids are in single-letter code with small Letters written. Certain amino acids are natural and unnatural achiral, z. B. glycine. In the representation of all peptides is the N-terminal end on the left and the C-terminal end on the right.  

Examples of non-proteinogenic amino acids are listed below with theirs Abbreviations indicated:

β-alanine β-Ala o-amino benzoic acid oab m-aminobenzoic acid Mab p-aminobenzoic acid Pab m-aminomethylbenzoic acid Amb ω-aminohexanoic acid Ahx ω-aminoheptanoic ahp ω-aminooctanoic acid AOC ω-aminodecanoic Ade ω-Aminotetradecansäure Atd citrulline Cit cyclohexylalanine Cha α, γ-diaminobutyric acid Dab α, β-diaminopropionic acid Dap Methionine sulfoxide Met (O) C ^α -methyl-alanine Aib N-methyl-glycine (sarcosine) Sar naphthylalanine N / A norleucine Never ornithine Orn 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid Tic

All amino acids can be subdivided into the following three main classes according to their physicochemical properties:
Sauer: The amino acid releases a proton in aqueous solution and at physiological pH and consequently carries a negative charge.
Basic: The amino acid absorbs a proton in aqueous solution and at physiological pH and consequently carries a positive charge.
Neutral: The amino acid is in an uncharged state in aqueous solution and at physiological pH.

The definition "carries a positive / negative charge" or "is uncharged" applies in this case if, statistically, a significant number of Amino acids of a class (at least 25%) is in the stated state.

In addition to the 20 so-called proteinogenic amino acids, their incorporation into proteins by The information of the genetic code is regulated, can not be proteinogenic incorporate into peptide sequences via the synthetic methods described. An enumeration of proteinogenic amino acids and their classification into the above three classes is given in Scheme 1. Non-proteinogenic amino acids are not genetically encoded. Examples of non-proteinogenic amino acids and their classification into acidic, basic or neutral amino acids are given in Table 1.

Table 1

The exendin analogs which are the subject of this invention have advantageous therapeutic properties. So they lead to a stimulation of insulin release from the endocrine pancreas, to an increase in Insulinbiosynthese as well as to a increased peripheral glucose utilization. Because these effects only at the same time  high blood sugar levels are not observed after their administration the occurrence of hypoglycaemia. Furthermore, the exendin analogs inhibit the glucagon release from the endocrine pancreas and thus lead to a Lowering gluconeogenesis. The exendin analogues do not work insulin-dependent diabetes mellitus (MDDM) to a marked improvement in Metabolic condition. Due to the inhibitory effect on the Glucagon release is also the administration of the exendin analogs Insulin-dependent diabetes mellitus makes sense. Against glucagon-like peptide-1 (GLP-1) and the known exendin-3 and exendin-4 sequences have the Exendin analogues of the invention surprisingly higher activity in the different test systems, making them better for a therapeutic application are suitable as GLP-1, exendin-3 or exendin-4. The benefits of the new exendin In particular, analogs are: higher stability to degradation and Metabolism, longer duration of action, efficacy at lower doses.

Solid phase and liquid phase synthesis is a common method of preparation of peptides. To the process for the production of a certain product in the It is to optimize the purity of the crude product and the yield required, the process parameters and the materials used, for example the Carrier material, the reagents which are to bring the groups to react, the Materials for blocking the groups that should not react, or the Reagents which split off blocking materials to the product to be prepared, adapt to the intermediates or starting materials to be prepared. These Adaptation is not easy given the interpendency of the many process parameters.

Medicaments containing the peptides of the invention individually or together as active Active ingredient in addition to conventional auxiliaries and additives are preferably administered parenterally (subcutaneously, intramuscularly or intravenously). In question but come also all usual application methods such as oral, rectal, buccal (including sublingual), pulmonary, transdermal, iontophoretic, vaginal and intranasal. The Medicines have an insulin-regulating effect and thereby promotes beneficial  Way the balance of blood sugar. Advantageous for the application of Medicament is when it contains the active ingredient in amounts of 150 nmol-500 nmol. A good effect is achieved in particular if the dose is between 10.0-50.0 pmol / kg / min when infused.

The exendin analogs according to the invention or pharmacologically acceptable salts Of these are preferably stored as sterile lyophilisates and before injection with a suitable isotonic solution. In this form the analogues can then injected, infused or possibly also absorbed through the mucous membranes become. As solvents may be the usual, for injection or infusion suitable isotonic, aqueous systems, the usual in injection solutions Additives such as stabilizers and solubilizers are used. Physiological saline or optionally isotonic buffered Solutions are preferred in this case.

Additives are z. B. tartrate or citrate buffer, ethanol, complexing agents (such Ethylenedianmintetraessigsäure and their non-toxic salts), high molecular weight Polymers (such as liquid polyethylene oxide) for viscosity control. Liquid carriers for injection solutions must be sterile and are preferably in ampoules bottled. Solid carriers are z. Starch, lactose, mannitol, methylcellulose, Talc, highly dispersed silicic acids, higher molecular weight fatty acids (such as stearic acid), Gelantine, agar-agar, calcium phosphate, magnesium stearate, animal and vegetable Fat, solid high molecular weight polymers (such as polyethylene glycol); for oral administration suitable preparations may, if desired, contain flavorings and sweeteners. For nasal application, surfactants may improve absorption the nasal mucosa may be added, e.g. B. cholic acid, taurocholic acid, Chenodeoxycholic acid, glycolic acid, dehydrocholic acid, deoxycholic acid and Cyclodextrins.  

The daily dose to be administered is in the range of 150-500 nmol. The determination the biological activity is based on measurements against international Reference preparations for glucagon-like peptide-1, exendin-3 or exendin-4 in one common test procedure for glucagon-like peptide-1.

The exendin analogues according to the invention can be synthesized according to the methods described in the Peptide Synthesis customary processes are prepared, as z. In J. M. Steward and J. D. Young "Solid Phase Peptide Synthesis", 2nd Ed., Pierce Chemical Co., Rockford, Illinois (1984) and J. Meienhofer "Hormonal Proteins and Peptides", Vol. 2, Academic Press, New York, (1973) for solid-phase synthesis and in E. Schroder and K. Lubke "The Peptides, Vol. 1, Academic Press, New York, (1965) for liquid phase synthesis have been described.

General Methods for Peptide Synthesis

In general, in the synthesis of peptides protected amino acids to a growing peptide chain added. The first amino acid is either on the amino group or the carboxyl group as well as any reactive group in the side chain protected. This protected amino acid becomes either an inert carrier coupled or can also be used in solution. The next amino acid in the Peptide sequence is suitably protected under conditions involving the formation of a Favor amide bond, given to the first. After all the desired Amino acids are coupled in the proper sequential order, the Cleaved protective groups and optionally the carrier phase. The obtained raw Polypeptide is reprecipitated and preferably chromatographically to the final product cleaned.

A preferred method for displaying analogs of physiologically active polypeptides, having less than about forty amino acids, involves solid phase peptide synthesis. In this method, the α-amino functions (N ^α ) and any reactive side chains are protected with acid- or base-labile groups. The protecting groups used should be stable under the conditions of amide bond formation, but readily split off without adversely affecting the resulting polypeptide chain. Suitable protecting groups for the α-amino function include, but are not limited to, the following groups: t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z), o-chlorobenzyloxycarbonyl, bi-phenylisopropyloxycarbonyl, tert -amyloxycarbonyl (Amoc), α, α-Dimetyl-3,5-di-methoxybenzyloxycarbonyl, o-nitrosulfenyl, 2-cyano-t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc), 1- (4,4-dimethyl-2,6-dioxo-cylohex-1-ylidene) -ethyl (Dde) and similar. Preferably, 9-fluorenylmethoxycarbonyl (Fmoc) is used as the N ^α -protecting group.

Suitable side chain protecting groups include, but are not limited to, the following these are limited to: acetyl, allyl (all), allyloxycarbonyl (alloc), benzyl (bzl), Benzyloxycarbonyl (Z), t-butyloxycarbonyl (Boc), benzyloxymethyl (Bom), o- Bromobenzyloxycarbonyl, t-butyl (tBu), t-butyldimethylsilyl, 2-chlorobenzyl, 2- Chlorobenzyloxycarbonyl (2-ClZ), 2,6-dichlorobenzyl, cyclohexyl, cyclopentyl, 1- (4,4-di methyl-2,6-dioxo-cyclohex-1-ylidene) ethyl (Dde), isopropyl, 4-methoxy-2,3,6-tri methylbenzylsulfonyl (Mtr), 2,2,5,7,8 pentamethylchroman-6-sulfonyl (Pmc), pivalyl, Tetrahydropyran-2-yl, tosyl (Tos), 2,4,6-trimethoxybenzyl, trimethylsilyl and trityl (Trt).

In solid-phase synthesis, the C-terminal amino acid becomes the first to be suitable Carrier material coupled. Suitable carrier materials are those which are inert to the Reagents and the reaction conditions of the stepwise condensation and Cleavage reactions are, and which are not in the reaction media used to solve. Examples of commercially available carrier materials include Styrene / divinylbenzene copolymers which react with reactive groups and / or Polyethylene glycol were modified, as well as chloromethylated styrene / divinylbenzene Copolymer, hydroxy or aminomethylated styrene / divinylbenzene copolymer and similar. With 4-benzyloxybenzyl alcohol (Wang anchor (Wang, S. S. 1973)) or 2- Chlorotrityl chloride (Barlos, K. et al., 1989) derivatized polystyrene (1%) divinylbenzene  or TentaGel® (Rapp Polymere, Tübingen) is preferably used, if the Peptide acid should be displayed. If it is the peptide amide, so that is with 5- (4'-aminomethyl) -3 ', 5'-dimethoxyphenoxy) valeric acid (PAL anchor) (Albericio, F. et. al. 1987) or the p- (2,4-dimethoxyphenylaminomethyl) phenoxy group (Rink Amide anchor (Rink, H. 1987)) derivatized polystyrene (1%) divinylbenzene or TentaGel® preferred.

The attachment to the polymeric supports can be achieved by reaction of the C-terminal Fmoc-Ge protected amino acid, with the addition of an activating reagent, in ethanol, Acetonitrile, N, N-dimethylformamide (DMF), dichloromethane, tetrahydrofuran, N- Methylpyrrolidone or similar solvents, preferably in DMF, with the Carrier material at room temperature or elevated temperatures, eg. B. between 40 ° C. and 60 ° C, preferably at room temperature, and reaction times from 2 to 72 Hours, preferably about 2 × 2 hours.

The coupling of the N ^α -protected amino acid, preferably the Fmoc amino acid, to the PAL, Wang or Rink anchors can be achieved, for example, with the aid of coupling reagents such as N, N'-dicyclohexylcarbodiimide (DCC), N, N'-diisopropylcarbodiimide ( DIC) or other carbodiimides, 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) or other uronium salts, O-acyl ureas, benzotriazol-1-yl oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) or other phosphonium salts, N-hydroxysuccinimides, other N-hydroxyimides, or oximes, in the presence or absence of 1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole, preferably with Assistance of TBTU with the addition of HOBt, with or without the addition of a base such as diisopropylethylamine (DIEA), triethylamine or N-methylmorpholine, preferably diisopropylethylamine, at reaction times of 2 to 72 hours, preferably 3 hours, in a 1.5 to 3 times Excess of aminos and the coupling reagents, preferably in a 2-fold excess and temperatures between about 10 ° C and 50 ° C, preferably at 25 ° C, in a solvent such as dimethylformamide, N-methylpyrrolidone or dichloromethane, preferably dimethylformamide. Instead of the coupling reagents, the active ester (eg pentafluorophenyl, p-nitrophenyl or the like), the symmetrical anhydride of the N ^α -Fmoc-amino acid, its acid chloride or fluoride can also be used under the conditions described above.

The coupling of the N ^α -protected amino acid, preferably the Fmoc amino acid, to the 2-chlorotrityl resin is preferably carried out in dichloromethane with the addition of DIEA at reaction times of 10 to 120 minutes, preferably 20 minutes, but is not limited to Limited use of this solvent and this base.

The successive coupling of the protected amino acids can be carried out by the methods customary in peptide synthesis, typically in a peptide synthesizer. After cleavage of the N ^α -Fmoc protecting group of the coupled amino acid on the solid phase by treatment with piperidine (10% to 50%) in dimethylformamide for 5 to 20 minutes, preferably 2 x 2 minutes with 50% piperidine in DMF and 1 x 15 minutes with 20% piperidine in DMF, the next protected amino acid is added in a 3 to 10-fold excess, preferably in a 10-fold excess, in an inert, non-aqueous, polar solvent such as dichloromethane, DMF or mixtures of both, preferably DMF, and temperatures about 10 ° C and 50 ° C, preferably at 25 ° C, coupled to the previous one. Suitable coupling reagents are the reagents already mentioned in the coupling of the first N ^α -Fmoc amino acid to the PAL, Wang or Rink anchors. Again, alternatively, active esters of the protected amino acid, their chlorides or fluorides or their symmetrical anhydrides can be used.

At the end of the solid phase synthesis, the peptide is cleaved off from the carrier material simultaneous cleavage of side chain protecting groups. The splitting can with Trifluoroacetic acid or other strongly acidic media with the addition of 5% -20% v / v Scavengers such as dimethyl sulfide, ethyl methyl sulfide, thioanisole, thiocresol, m-cresol,  Anisole Ethanedithiol, phenol or water, preferably 15% v / v dimethyl sulfide / Ethanedithiol / m-cresol 1.1: 1, within 0.5 to 3 hours, preferably 2 hours, respectively. Fully protected peptides in the side chains are prepared by cleavage of the 2- Chlortritylankers with glacial acetic acid / trifluoroethanol / dichloromethane 2: 2: 6. The Protected peptide can be purified by chromatography on silica gel. Is this Peptide connected via the Wang anchor with the solid phase, and is said to be a peptide with C-ter Minaler alkylamidation can be obtained, so the cleavage on a Aminolysis be carried out with an alkylamine or Fluoroalkylamin. The Aminolysis is carried out at temperatures between about -10 ° C and 50 ° C, preferably about 25 ° C, and reaction times between about 12 and 24 hours, preferably about 18 Hours, performed. Furthermore, the peptide can also by transesterification, for. B. with Methanol, are split by the carrier.

The acid solution obtained is mixed with 3-20 times the amount of cold ether or n- Hexane, preferably a 10-fold excess of diethyl ether to the peptide to precipitate and thus from the scavengers remaining in the ether and cleaved off Separate protective groups. Another cleaning can be done by multiple reprecipitation of the peptide from glacial acetic acid. The resulting Precipitat is in water or tert-butanol or mixtures of the two solvents, preferably a 1: 1 Mixture of tert-butanol / water, taken up and freeze-dried.

The resulting peptide may be purified by any or all of the following chromatographic Methods are: ion exchange over a weakly basic resin in the Acetate form; hydrophobic adsorption chromatography on underivatized polysty rol / divinylbenzene copolymers (eg Amberlite® XAD); adsorption on silica gel; Ion exchange chromatography on carboxymethylcellulose; Partition chromatography, e.g. To Sephadex® G-25; countercurrent distribution; or high pressure liquid chromatography (HPLC), in particular "reversed-phase" HPLC on octyl or octadecylsilylsilica (ODS) phases.  

In summary, a part of the present invention includes methods for Preparation of polypeptides and their pharmaceutically acceptable salts. These Methods which result in physiologically active truncated homologues and analogues of Exendin-3 or Exendin-4, with the preferred chain lengths mentioned above and Modifications result from sequential condensation processes Protected amino acids on a suitable carrier material, to cleave the Carrier and the protective groups, and for purification of the obtained crude peptides together.

The amino acid analysis was carried out with an amino acid analyzer 420A from the company Applied Biosystems (Weiterstadt). 50 to 1000 pmol of the to be analyzed Sample was applied to the sample carrier in 10 to 40 μl solution and then fully automatically hydrolyzed in the gas phase at 160 ° C with 6N hydrochloric acid for 90 minutes, derivatized with phenylisothiocyanate and analyzed on-line via a Microbore-HPLC. Mass spectroscopic studies were performed on an API III triple Quadrupole mass spectrometer (SCIEX, Thornhill, Canada) equipped with ion spray Ion source performed.

The protected amino acid derivatives may, for. B. from Novabiochem GmbH (Bad Soden).  

example 1 HGEGTFTSDLSKQ-Nle-EEEAVRLFIEWLkNGR-NH₂ (SEQ ID NO: 3) [Nle¹⁴, Arg³⁰] -Exendin-4- (1-30) -NH₂

Example 1 was carried out in a 0.02 mmol by the solid phase method on 5- (4'-amino methyl) -3 ', 5'-dimethoxyphenoxy) valerianyl-alanyl-aminomethyl-polystyrene (1%) divinylbenzene (loading: 0 , 5 mmol / g) were synthesized on a Multiple Peptide Synthesizer SyRo II from MultiSynTech (Bochum). The α-amino functions of the amino acids were protected 9-fluorenylmethoxycarbonyl (Fmoc). As side-chain protecting groups, t-butyl (tBu) for Asp, Glu, Ser and Thr, trityl (Trt) for Asn, Gln and His, t-butyloxycarbonyl (Boc) for Lys and Trp, and 2,2,5,7,8- Pentamethylchroman-6-sulfonyl (Pmc) used for Arg. The sequential coupling of the protected amino acids was carried out in 10-fold excess with double couplings of 2 times 40 minutes duration and with N, N-diisopropylcarbodiimide / 1-hydroxybenzotriazole as activating reagents. The cleavage of the peptide from the polymeric support with simultaneous deprotection was carried out in trifluoroacetic acid (85%) in the presence of 15% ethaniticbiol / dimethyl sulfide / m-cresol (1: 1: 1 v / v / v) for 120 minutes at room temperature. Subsequently, the peptide was precipitated with anhydrous diethyl ether and washed to complete removal of the thiols several times with anhydrous diethyl ether. Freeze-drying of the precipitate from water / tert-butanol (1: 1) gave 62 mg of the crude peptide. The crude peptide was purified by reversed-phase HPLC with a gradient of 37% to 42% acetonitrile / 0.9% TFA in 30 minutes. The eluate was concentrated, lyophilized to give a yield of 29 mg of a white solid with a purity of 97%.
Amino Acid Analysis: Ala 1.08 (1); Asx 1.91 (2); Glx 6,10 (6); Phe 1,78 (2); Gly 3.10 (3); His 1.00 (1); Ile 0.88 (1); Lys 2.02 (2); Lei 3.24 (3); Nile 1.10 (1); Arg 1.98 (2); Ser 2.04 (2); Thr 1.99 (2); Val 0.91 (1); Trp 0.87 (1).
ESI MS: 3488.2

Example 2 HGEGTFTSDLSKQ-Nle-EEEAVRLFIEWLKNGY-NH₂ (SEQ ID NO: 4) [Nle¹⁴, Tyr³⁰] -Exendin-4- (1-30) -NH₂

Example 2 was synthesized in a 0.0076 mmol batch by the solid phase method on TentaGel® (Rapp Polymere, Tübingen) which was reacted with the Rink amide anchor (4- (2 ', 4', -dimethoxyphenyl-aminomethyl) - phenoxy group) was derivatized (loading: 0.18 mmol / g) on a multiple peptide synthesizer SyRo II from MultiSynTech (Bochum) synthesized. The protected amino acids used were analogous to Example 1. The sequential coupling of the protected amino acids was carried out in 8-fold excess with single couplings of 40 minutes duration, at 40 ° C and with stirring. Activation reagents used were 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetra-methyluronium tetrafluoroborate (TBTU) / 1-hydroxybenzotriazole with the addition of diisopropylethylamine. The cleavage and purification of the peptide was carried out analogously to Example 1. There were obtained 18.1 mg of a white solid with a purity of 95%.
Amino Acid Analysis: Ala 1.03 (1); Asx 1.90 (2); Glx 6,24 (6); Phe 1.94 (2); Gly 3,12 (3); His 1.02 (1); Ile 1.09 (1); Lys 2.01 (2); Leu 3.06 (3); Nle 1.08 (1); Arg 0.97 (1); Ser 1.98 (2); Thr 1,80 (2); Val 0.93 (1); Trp 1.01 (1); Tyr 0.90 (1).
ESI MS: 3494.8

Example 3 HSDGTFTSDLSKQ-Nle-EEEAVRLFIEWLKNGR-NH₂ (SEQ ID NO: 5) [Nle¹⁴, Arg³⁰] -Exendin-3- (1-30) -NH₂

Example 3 was synthesized analogously according to the method described for Example 2. There was obtained 17.6 mg of a white solid with a purity of 99%.
Amino acid analysis: Ala 0.99 (1); Asx 2.98 (3); Glx 5,16 (5); Phe 2.08 (2); Gly 2,16 (2); His 0.95 (1); Ile 1.03 (1); Lys 2.04 (2); Leu 2.91 (3); Nle 1.05 (1); Arg 1.04 (1); Ser 3.00 (3); Thr 2.05 (2); Val 1.01 (1); Trp 1.18 (1); Tyr 0.98 (1).
ESI MS: 3504.4

Example 4 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGR-NH₂ (SEQ ID NO: 6) [Arg³⁰] -Exendin-4 (1-30) -NH₂

Example 4 was synthesized analogously according to the method described for Example 1. There was obtained 17.9 mg of a white solid with a purity of 96%.
Amino Acid Analysis: Ala 0.96 (1); Asx 2.01 (2); Glx 6.00 (6); Phe 1.80 (2); Gly 3.21 (3); His 0.96 (1); Ile 1.07 (1); Lys 1.92 (2); Leu 2,98 (3); Met 1.06 (1); Arg 1.90 (2); Ser 1.91 (2); Thr 2.09 (2); Val 0.97 (1); Trp 0.84 (1).
ESI MS: 3508.4

Example 5 GEGTFTSDLSKQ-Nle-EEEAVRLFIEWLKNGR-NH₂ (SEQ ID NO: 7) [Nle¹⁴, Arg³⁰] -Exendin-4- (2-30) -NH₂

Example 5 was synthesized analogously according to the method described for Example 2. There was obtained 13.2 mg of a white solid with a purity of 97%.
Amino Acid Analysis: Ala 1.04 (1); Asx 1.98 (2); Glx 6.08 (6); Phe 1.86 (2); Gly 2.91 (3); Ile 0.96 (1); Lys 1.84 (2); Leu 2,98 (3); Nle 1.04 (1); Arg 1.90 (2); Ser 1.94 (2); Thr 1.92 (2); Val 0.96 (1); Trp 0.85 (1).
ESI-MS: 3350.8

Example 6 HGEGTFTSDLSKQMEEEAVRAFIEWLKNGR-NH₂ (SEQ ID NO: 8) [Ala²¹, Arg³⁰] -Exendin-4- (1-30) -NH₂

Example 6 was synthesized analogously according to the method described for Example 1. There were obtained 11.1 mg of a white solid having a purity of 95%.
Amino Acid Analysis: Ala 2.08 (2); Asx 1.93 (2); Glx 6.07 (6); Phe 1.74 (2); Gly 2.97 (3); His 0.98 (1); Ile 0.87 (1); Lys 2:15 (2); Leu 2.02 (2); Met 0.96 (1); Arg 2,13 (2); Ser 1.87 (2); Thr 2.07 (2); Val 1.04 (1); Trp 0.87 (1).
ESI MS: 3466.3

Example 7 HGEGTFTSDLSKQMEEEAVRLFIEWLKAGR-NH₂ (SEQ ID NO: 9) [Ala²⁸, Arg³⁰] -Exendin-4- (1-30) -NH₂

Example 7 was synthesized analogously according to the method described for Example 1. There was obtained 15.0 mg of a white solid having a purity of 97%.
Amino acid analysis: Ala 1.98 (2); Asx 0.98 (1); Glx 6,22 (6); Phe 1.92 (2); Gly 3.03 (3); His 0.99 (1); Ile 1.03 (1); Lys 2.05 (2); Leu 3.03 (3); Met 0.96 (1); Arg 1.84 (2); Ser 1.98 (2); Thr 2.09 (2); Val 1.01 (1); Trp 0.72 (1).
ESI MS: 3465.4

Example 8 HGEGTFTSDLSKQMEEEAVRAFIEWLKAGR-NH₂ (SEQ ID NO: 10) [Ala ^21,28, Arg³⁰] -Exendin-4 (1-30) -NH₂

Example 8 was synthesized analogously according to the method described for Example 1. There was obtained 18.4 mg of a white solid with a purity of 95%.
Amino Acid Analysis: Ala 3,12 (3); Asx 0.99 (1); Glx 6,04 (6); Phe 1.80 (2); Gly 3.00 (3); His 0.96 (1); Ile 1.02 (1); Lys 1.84 (2); Lei 1.97 (2); Met 0.98 (1); Arg 2.03 (2); Ser 1.91 (2); Thr 1,88 (2); Val 0.99 (1); Trp 0.99 (1).
ESI MS: 3423.3

Example 9

In an analogous manner, the following high purity exendin derivatives were prepared.

Biological data Peptide metabolism in ectopeptidase preparations or kidney microvilli membrane preparations Preparation of brush border microvilli membranes

By subcellular fractionation using the Differential centrifugation method (Booth and Kenny (1975)) Isolated microvillimembrans of the rat and Schweierenierencortex. To judge the Purity and the yield of the membranes are 4 brush border Ectopeptidases measured by fluorimetry and other marker enzymes by colorimetry.

Ektopeptidase preparations

Purified human neutral endopeptidase 24.11 was used in the recombinant form of Genentech (San Francisco, U.S.A.), dipeptidyl peptidase IV, was isolated as an isolate from human Placenta purchased from Calbiochem (Bad Soden).

Incubation protocol

Microvilli membranes (0.5-1 μg protein) or the respective ectopeptidase preparation (60-300 ng) were mixed with 10 μg of peptide (about 3 nmol) in 100 μl of HEPES buffer (50 mM, pH 7.4) containing 50 mM NaCl. At predetermined times  (Duration up to 1 hour), the reactions were stopped by boiling. Subsequently, the samples were centrifuged (10,000 × g) with 150 μl of 0.1% TFA diluted and analyzed by reversed phase (RP) HPLC. Each sample was duplicated certainly.

HPLC analysis

For HPLC analysis, a system was used with the following components: A "2248" low pressure pump (Pharmacia-LKB, Freiburg), a WISP 10B Autoinjector (Millipore-Waters, Eschborn), a UV detector SP-4 (Gynkotec, Berlin), a low-pressure mixing system (Pharmacia-LKB, Freiburg) and a "Program Manager" Software control (Pharmacia-LKB, Freiburg). The separations were over Lichrospher C-8, 5μ, 4 × 124 mm (Merck, Darmstadt) with a binary gradient with the eluents A: 0.1% trifluoroacetic acid (TFA) and B: acetonitrile: water: TFA (70: 29,9: 0, 1). After injection of 244 μl of the sample solution to that with eluent A equilibrated column, the incubation products were incubated with a linear gradient of 0% to 80% B eluted in 80 min and detected at 215 nm UV absorption.

Calculation of proteolysis rates

For each incubation period of each peptide, two measurements were taken and plotted the mean peak height of the substrate peak versus time. On the example of GLP-1 showed that the peak height was linearly proportional to the quantity of the Peptides in the sample solution is. Within the first hour of incubation with the Microvilli membranes or the peptidases also showed a linear decrease in the Peak height can be observed over time. The proteolysis rate is thus determined by the Determination of the height of the substrate peak and in [μmol substrate / mg Protein / minute].

Degradation stability of exendin analogues

[Nle¹⁴, Arg³⁰] -Exendin-4- (1-30) -NH₂ (SEQ ID NO: 3) was neutralized Endopeptidase 24.11 was incubated as described above and the rate of degradation was determined.  

The controls were GLP1- (7-36) -NH₂ and exendin-4- (1-39). The results are in Table 1 listed.

degradation rate [mM / 100 ng / ml NEP 24.11 / min] GLP1- (7-36) -NH₂ 0.0586 Exendin-4 0.00009305 [Nele1, Arg3 +] -Ex-4- (1-30) -NH2 _{Ex. 1} 0.0083

Measurement of increase of cytosolic calcium concentration in B cells of the endocrine pancreas (clonal B-cell line INS-1) Breeding of INS-1 cells (Asfari, M., 1992)

INS-1 cells are incubated in RPMI 1640 medium with 10% FCS, 10 mM HEPES buffer (pH 7.4), 2 mM L-glutamine, 100 i.U. Penicillin / ml, 100 μg streptomycin / ml, 1 mM pyruvate (Sodium salt) and 50 μM 2-mercaptoethanol, at 37 ° C, in an atmosphere of 95% air and 5% CO₂. After 6 to 8 days growth on plastic Cell culture plates become the subconfluent cells after rinsing once with PBS (phosphate-buffered saline) by incubation for four minutes at 37 ° C. with 0.025% Trypsin and 0.27 mM EDTA in isoosmotic saline solution detached from the pad.

Preparation of the cells for calcium measurements

The detached cells are in spinning medium (culture medium as above, but with 5% FCS and 25 mM HEPES) and incubated at 37 ° C for two and a half hours in a spinner bottle with stirring bar incubated. Then remove the medium by Centrifugation and resuspension of cells in spinner medium. Then for 30 minutes Incubation at 37 ° C with 2 μM fura-2 / acetoxymethyl ester, under the same Conditions as before. The fura loading of the cells is done by washing once  the cells in spinning medium (room temperature) ended. After that, the cells are in Spinning medium resuspended at room temperature (2 x 10⁷ cells / ml). From this Suspension, the cells are removed for calcium measurements.

Measurements of cytosolic calcium concentration

The measurements are carried out at 37 ° C in a modified Krebs-Ringer buffer (KRBH) with 136 mM NaCl, 4.8 mM KCl, 2 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 5 mM NaHCO₃, 10 mM glucose, 250 μM sulfinpyrazone (to inhibit fura-2 efflux in the medium) and 25 mM HEPES buffer (with NaOH to pH 7.4). The cell concentration is 1-2 × 10⁶ / ml. The measurements are carried out in a stirred cuvette in a spectrofluorimeter at 37 ° C, with 1.5 ml of cell suspension. The excitation wavelength is 340 nm, emission wavelength 505 nm. At the end of the measurements, 50 μM MnCl₂ and then 100 μM DTPA (dieethylenetriamine pentaacetate) are added to quench the fluorescence from extracellular fura to measure the extracellular fluorescence indicator To be able to determine fluorescence. After the addition of DTPA, the conversion of the entire furas is first to a calcium-saturated and then a calcium-free state, to determine the calibration values F _max (calcium-saturated) and F _min (calcium-free) for the respective measurement. For this purpose, the cells are lysed by addition of 0.1% Triton X-100. Contact with the high extracellular calcium concentration saturates the dye with calcium. Thereafter, 5 mM EGTA (ethylenebis (oxyethylenenitrilo) tetraacetate) and 20 mM Tris solution are added to completely convert the dye into the calcium free form.

The cytosolic calcium ion concentration is calculated according to the method of R. Algorithm introduced by Tsien and co-workers (Grynkiewicz, G., 1985):

[Ca ²⁺ ] _cyt = ((F-F _min ) / (F _max -F)) × K _D

(F: fluorescence of the respective measuring point;
KD: dissociation constant of calcium complex of fura-2, 225 nM (Grynkiewicz, G., 1985)).

(Prior to this calculation, compensation for the presence of extracellular fura is performed by first subtracting the amount of fluorescence (extracellular fura) determined by manganese addition from the fluorescence values of the measurement points, then correcting F _max by subtracting the same amount F _min is determined. for this purpose, the particular by addition of manganese fluorescence amount is divided by the value 2.24. the value of 2.24 was determined as the device's own proportionality between the fluorescence of calciumgesättigtem and calcium free Fura-2 at an excitation wavelength of 340 nm (measured with unesterified , free fura-2) The correction amount thus obtained was subtracted from F _min .)
The peptides investigated were added from thousandfold concentrated solutions (10 ^-5 M) in KRBH without CaCl₂ and glucose.

Activity of the exendin analogues

Several exendin analogs were used in the above-described calcium assay on INS-1 cells tested for their biological activity. The data are exemplary in Figure 1 as also shown in Table 2.  

Table 2

literature

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Asfari, M., Janjic, D., Meda, P., Li, G., Halban, P.A. and Wollheim, C.B. (1992) Endocrinology 130, 167-178.

Barlos, K., Gatos, D., Kapolos, S., Paphotiu, G., Schafer, W., and Wengqing, Y. (1989) Tetrahedron Lett. 30, 3947-3950.

Booth, and Kenny, (1975) Biochem. J 142, 575-581.

Eng, J., Andrews, P.C., Kleinman, W.A., Singh, L., and Raufman, J.-P. (1990) J. Biol. Chem. 265, 20259-20262.

Eng, J., Andrews, P.C., Kleinman, W.A., Singh, L., Singh, G., and Raufman, J.-P. (1992) J. Biol. Chem. 267, 7402-7405.

Fehmann, H. C., Göke, R., Göke, B., Bachle, R., Wagner, B. and Arnold, R. (1991) Biochim. Biophys. Acta 1091, 356-63.

Göke, R., Wagner, B., Fehmann, H.C. and Göke, B. (1993a) Res. Exp. Med. Berl. 193, 597-103

Göke, R., Fehman, H.C., Linn, T., Schmidt, H., Eng, J. and Göke, B. (1993b) J. Biol. Chem. 268, 19650-19655.

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Gutniak, M., Orskov, C., Holst, J.J., Ahren, B. and Efendic, S. (1992) N. Engl. J. Med. 326, 1316-1322.

Komatsu, R., Matsuyama, T., Namba, M., Watanabe, N., Itoh, H., Kono, N. and Tarui, S. (1989) Diabetes 38, 902-905.

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SEQUENCE LISTING

Claims (5)

1. peptide, characterized in that it has the sequence 1 or 2 wherein the amino acids in position 1 or in positions 28 to 38 independently or separately may be part of the sequence and the N-terminus is represented by NR₁R₂, wherein R₁ is hydrogen, acetyl, trifluoroacetyl, adamantyl, Fmoc, Z, Boe, Alloc , C₁-C₆-alkyl, C₂-C₈-alkenyl or C₆-C₈-aralkyl,
R₂ is hydrogen, acetyl, trifluoroacetyl, adamantyl, Fmoc, Z. Boc, Alloc, C₁-C₆ alkyl, C₂-C₈ alkenyl or C₆-C₈ aralkyl, and the C-terminus is represented by COR₃, wherein
R₃ is equal to OR₄ or NR₄R₅
with R₄ is hydrogen or C₁-C₆-alkyl
where R₅ is hydrogen or C₁-C₆-alkyl
means, and their physiologically acceptable salts and esters. 2. Peptides according to claim 1, characterized in that at least one, but at most 13 of the following modifications to the amino acid chain are carried out

• (a) The α-amino acid in position 1 is D-His, Ala, D-Ala, Gly, Lys or D-Lys, with Ala, Gly or Lys being particularly preferred; or
• (b) The α-amino acid in position 2 is Ser, D-Ser Thr D-Thr Gly, Ala, D-Ala, Ile, D-Ile, Val, D-Val, Leu or D-Leu, preferably Ser, Thr , Gly, Ala, Val, Ile or Leu; or
• (c) The α-amino acid in position 3 is Glu, D-Glu, Asp, D-Asp, Ala or D-Ala, preferably Glu, Asp or Ala; or
• (d) The amino acid in position 4 is Ala, D-Ala or β-Ala, preferably Ala; or
• (e) The α-amino acid in position 5 is Ser, Tyr or Ala; or
• (f) The α-amino acid in position 6 is Ala, Ile, Val, Leu, Cha or Tyr, preferably Ala, Ile, Val, Leu or Tyr; or
• (g) The α-amino acid in position 7 is Ala, D-Ala, Tyr, D-Tyr, Ser, D-Ser or D-Thr, preferably Ala, Tyr or Ser; or
• (h) The amino acid in position 8 is Ala, Tyr or Thr; or
• (i) The α-amino acid in position 9 is Ala, D-Ala, Glu, D-Glu or D-Asp, preferably Ala or Glu; or
• (j) The amino acid at position 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 24, 28, 29, 31-38 is independently a proteinogenic or non-proteinogenic gene D or L. Amino acid, preferably a proteinogenic L-amino acid; or
• (k) The α-amino acid in position 13 is a neutral L-amino acid, preferably a neutral proteinogenic L-amino acid; or
• (l) The α-amino acid in position 14 is replaced by a neutral L or D amino acid for stabilization, preferably by Nle, D-Nle, Ala, D-Ala, Ile, D-Ile, Val, D-Val, Leu or D-Leu, particularly preferred are Ile, Val or Ala; or
• (m) The α-amino acid at position 22 is D-Phe, Tyr, D-Tyr, Leu, D-Leu, Val, D-Val, L-Cha, D-Cha, β-1-Nal, β-2 -Nal or β-1-D-Nal, preferred are Tyr, Leu or Val; or
• (n) The α-amino acid in position 23 is Leu, D-Leu, D-Ile, Val, D-Val, L-Cha, D-Cha, Tyr, D-Tyr, Phe or D-Phe, preferably Leu , Val, Tyr or Phe; or
• (o) The α-amino acid in position 25, 26 or 27 is a neutral L or D amino acid, preferably a neutral, proteinogenic L-amino acid; or
• (p) The α-amino acid in position 30 is a proteinogenic or non-proteinogenic D or L amino acid, preferably Arg, D-Arg, Tyr or D-Tyr, more preferably Arg or Tyr.

3. Peptide according to claim 1 or 2, characterized in that it is the Stimulates insulin release. 4. Medicaments containing in addition to conventional carriers and excipients at least one A peptide according to claim 1, 2 or 3. 5. Use of peptides according to claim 1, 2 or 3 for the preparation of pharmaceutical compositions for the treatment of diabetes.