DE19637230A1 - 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. The new substances are characterized by increased Stability while maintaining efficacy.

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 Target tissues the glucose uptake and an inhibition of gluconeogenesis. Furthermore, in GLP-1 analogues an increase in glucose uptake in the Periphery expected. The inhibition of glucagon secretion must be as indirect GLP-1 Effect, 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. However, the pharmacokinetic problems are problematic Properties of GLP-1. Due to its very short half-life, the duration of action is only limited.

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  to the goal of improved degradation-stabilized therapeutics with extended duration of action Develop diabetes mellitus treatment. These peptides have the same pharmacological action such as GLP-1, but surprisingly have a clear longer half-life.

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 particular the Glucose uptake in the target tissues muscle and adipose tissue, as well as the Gastric emptying.

Subject of the invention

The present invention is based on the sequence of exendin-3 and exendin-t, 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 shortened the C-terminal end of these sequences by up to 3 amino acids, preferably at most 1 amino acid, and the N-terminal end by up to 2, preferably at most 1 amino acid, may be shortened. Particularly preferred that is, peptide fragments having the following sequences; in particular, the Peptide Fragments Based on Exendendin-3- (1-30) (SEQ ID NO: 1):

the amino acids being in position 1, 2, 28, 29 or 30 depending on the desired Chain length can be part of the sequence. The peptides are from the N-terminus to Numbered C-terininus. X₁ represents a proteogenic or nonproteogenic Amino acid except glycine.

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- Alkenyl or C₇-C₉-aralkyl.

Alkyl radicals are straight-chain, branched or optionally cyclic Alkyl radicals understood, preferably methyl, ethyl, isopropyl and cyclohexyl.

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 a maximum of eleven the modifications listed under (a) to (p) were carried out. Preferred are those exendin fragments which are maximally 9, more preferred are those which are maximal 5 have the modifications listed under (a) to (p).

• (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 in 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 α-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 acids at positions 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 24, 28, 29 are 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 replaced by a neutral one for stabilization L- or D-amino acid, except L-leucine, preferably by Nle, D-Nle, Ala, D-Ala, Ile, D-Ile, Val or D-Val, 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, preferably 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 non-proteinogenic D- or L-amino acid except glycine, preferably Arg, D-Arg, Tyr or D-Tyr, especially preferred 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 acids is located.

The invention, in addition to new truncated and stabilized exendin-3 and Exendin-4 analogues also methods for preparing 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 glycine at position 30, the exendin-3 or exendin-4 sequence was raised against a other proteogenic or non-proteogenic amino acid exchanged to aid in the synthesis after cleavage of the amino-terminal protecting group, no diketopiperazine formation to have available.

The exendin (1-30) analogs and fragments are opposite to the exendins-1 (1-39) advantageous because the shorter sequences make these analogues easier and higher Yields can be synthesized.

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:

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 nonproteinogenic amino acids are listed below with their 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 Citruilin Cit cyclohexylalanine Cha α, γ-diaminobutyric acid Dab α, β-Diaminoprnpionsäure 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 is absorbed in aqueous solution and at physiological pH Proton and consequently carries a positive charge.

Neutral: The amino acid is in aqueous solution and at physiological pH in one uncharged condition.

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 Table 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 give rise to non- Insulin-dependent diabetes mellitus (NIDDM) significantly improved Metabolic condition. In particular, regardless of the insulin secretory Effect increased the glucose uptake in muscle and fat tissues. Due to the The inhibitory effect on glucagon release is also the administration of the Exendin analogues in insulin-dependent diabetes mellitus makes sense. Across from Glucagon-like peptide-1 (GLP-1) and the known exendin-3 and exendin-4 Sequences possess the novel exendin analogues a surprisingly higher Effectiveness in the various test systems, making them suitable for a therapeutic Application are more suitable than GLP-1, exendin-3 or exendin-4. The advantages of  new exendin analogs are in particular the following: higher stability Degradation and metabolism, longer duration of action, efficacy at lower doses. Particular preference is given to analogs based on exendin-3, which are particularly long Show efficacy or efficacy at a particularly low dose.

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 usual 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 Drug is when blood levels between 20 and 50 pmol / l are achieved. To are infusion rates of 0.4-1.2 pmol / kg / min. required. In subcutaneous or buccal Depending on the galenic form and the desired duration of action, the application amount of substance of 5-500 nmol required.

The exendin analogs according to the invention or pharmacologically acceptable salts thereof are preferably stored as sterile lyophilisates and before the application mixed 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, for example, 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-dimethoxybenzyloxycarbonyl, o-nitrosulfenyl, 2-cyano-t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc), 1- (4,4-dimethyl-2,6-dioxy-cylohex-1-ylidene) -ethyl ( Dde) and similar. Preferably, 9-fluorenylmethoxycarbonyl (Fmoc) is used as N ^α -protecting groups.

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- dimethyl-2,6-dioxo-cyclohex-1-ylidene) ethyl (Dde), isopropyl, 4-methoxy-2,3,6- trimethylbenzylsulfonyl (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 Abspaltungsraktionen 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 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'-dicyclohexylcarbodlimide (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 the aid of 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-fold 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 in a 3 to 10-fold excess, preferably in a 10-fold excess, in an inert, nonaqueous, polar solvent such as dichloromethane, DMF or mixtures of both, preferably DMF, and temperatures between 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 linked to the solid phase via the Wang anchor, and is said to be a peptide with C Terminal amination 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 split off Separate protective groups. Another cleaning can be done by multiple reprecipitation of the peptide from ice-vinegar. The resulting Precipitat is in water or tert-butanol or mixtures of the two solvents, preferably 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 the Novabiochem. GmbH (Bad Soden).

The following examples are only an illustrative selection of the inventive idea and no limitation of the subject invention.  

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 batch by the solid phase method on 5- (4'-aminomethyl) -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% ethanedithiol / dimethylsulfide / 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 of the company 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-tetramethyluronium 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 20 (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 HGEGTFTSDLSKOMEEEAVRLFIEWLKNGR-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 were 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 exendin derivatives were prepared in high purity. (Ex-4 = exendin-4, ex-3 = exendin-3)

Biological data Peptide metabolism in ectopeptidase preparations or on 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.

Ectopeptidase 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 incubated 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 the IIPLC analysis, a system with the following components was used:
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 carried out via Lichrospher C-8, 5 μ, 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 onto the column equilibrated with mobile phase A, the incubation products were eluted with a linear gradient from 0% to 80% B 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 control was GLP1- (7-36) -NH2. The results are shown in Table 1.

Degradation rate [mM / 100 ng / ml NEP24.11 / min] GLP1- (7-36) -NH₂ 0.0586 [Nle¹⁴, Arg³⁰] -Ex-4-1-30) -NH₂ Ex. 1 0.0083

Measurement of the 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 of growth on plastic cell culture plates are the subconfluent cells after a single rinse 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. Algorhythm introduced by Tsien and co-workers (Grynkiewicz, G., 1985):

[Ca²⁺] _cyt = ((FF _min ) / (F _max -F)) × K _D

(F: fluorescence of the respective measuring point;
K _D : 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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Barlos, K., Gatos, D., Kapolos, S., Paphotiu, G., Schafer, W., and Wengqing, Y. (1989) Tetrahedron Lett. 30, 3947-3950.
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Claims (5)

1. peptide, characterized in that it has the sequence 1 or 2 wherein X represents a proteogenic or non-proteogenic amino acid other than glycine, the amino acids in position 1, 2, 28, 29 or 30 may independently or separately be part of the sequence and the N-terminus represented by NR₁R₂, wherein
R₁ is hydrogen, acetyl, trifluoroacetyl, adamantyl, Fmoc, Z, Boc, Alloc, C₁-C₆-alkyl, C₂-C₈ alkenyl or C₇-C₉ aralkyl,
R₂ is hydrogen, acetyl, trifluoroacetyl, adamantyl, Fmoc, Z, Boc, Alloc, C₄-C₆ alkyl, C₂-C₈ alkenyl or C₇-C₉ 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 11 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 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 α-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 acids at position 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 24, 28, 29 are independently a proteinogenic or non-proteinogenic D or L amino acid 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 except L-leucine, preferably by Nle, D-Nle, Ala, D-Ala, Ile, D-Ile, Val or D- Val, especially 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 at 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 other than glycine, 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.