WO2007113205A1

WO2007113205A1 - Glp-1 peptide agonists

Info

Publication number: WO2007113205A1
Application number: PCT/EP2007/053023
Authority: WO
Inventors: Janos Tibor Kodra; Marie Skovgaard; David A. Liberles
Original assignee: Novo Nordisk A/S
Priority date: 2006-04-03
Filing date: 2007-03-29
Publication date: 2007-10-11
Also published as: EP2004213A1; JP2009532422A

Abstract

The present invention relates to novel glucagon-like-peptide-1 (GLP-1) peptide agonists that are able to activate the GLP-1 receptor and that exhibit enhanced enzymatic stability relative to GLP-1 (7-37). The invention further relates to methods of making and using such peptides.

Description

GLP-1 Peptide Agonists

FIELD OF THE INVENTION

The present invention relates to novel glucagon-like-peptide-1 (GLP-1 ) peptide agonists that are able to activate the GLP-1 receptor and that exhibit enhanced enzymatic stability relative to human GLP-1 (7-37). The invention further relates to methods of making and using such peptides.

BACKGROUND OF THE INVENTION

Diabetes mellitus is a metabolic disorder in which the ability to utilize glucose is partly or completely lost. About 5% of all people suffer from diabetes and the disorder approaches epidemic proportions. Since the introduction of insulin in the 1920^'s, continuous efforts have been made to im- prove the treatment of diabetes mellitus.

One peptide expected to become very important in the treatment of diabetes is glucagon-like peptide-1 (GLP-1 ). Human GLP-1 is a 37 amino acid residue peptide originating from preproglucagon which is synthesized La. in the L-cells in the distal ileum, in the pancreas and in the brain. GLP-1 is an important gut hormone with regulatory function in glucose metabolism and gastrointestinal secretion and metabolism. GLP-1 stimulates insulin secretion in a glucose-dependant manner, stimulates insulin biosynthesis, promotes beta cell rescue, decreases glucagon secretion, gastric emptying and food intake. Human GLP-1 is hydrolysed to GLP-1 (7-37) and GLP-1 (7-36)-amide which are both insulinotropic peptides. A simple system is used to describe fragments and analogues of this peptide. Thus, for example, [Gly⁸]GLP-1 (7-37) designates an analogue of GLP-1 (7-37) formally derived from GLP-1 (7-37) by substituting the naturally occurring amino acid residue in position 8 (Ala) by GIy. Similarly, (N^ε34-tetradecanoyl)[Lys³⁴]GLP-1 (7-37) designates GLP-1 (7-37) wherein the ε-amino group of the Lys residue in position 34 has been tetradecanoylated.

In the last decade a number of peptides have been isolated from the venom of the GiIa monster lizards (Helodetma suspectum and Helodetma horridum). Exendin-4 is a 39 amino acid residue peptide isolated from the venom of Heloderma suspectum, and this peptide shares 52% homology with GLP-1 (7-37) in the overlapping region. Exendin-4 is a potent GLP-1 receptor agonist which has been shown to stimulate insulin release and ensuing lowering of the blood glucose level when injected into dogs. The group of exendin-4(1-39), certain fragments thereof, analogs thereof and derivatives thereof, are potent insulinotropic agents. Most importantly the group of exendin-4(1-39), insulinotropic fragments thereof, insulinotropic analogs thereof and insulinotropic derivatives thereof. SUMMARY OF THE INVENTION

The present invention relates to peptides that are agonists of the GLP-1 receptor and that exhibit exhibit enhanced chemical stability relative to native GLP-1 (7-37). In some embodiments, the peptides of the invention may also carry a net positive charge at physiological pH.

The peptides of the invention may comprise the amino acid sequence of formula I:

Xaa₇-Xaa₈-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂o-Glu-Xaa₂₂-Xaa₂₃-Ala- Xaa₂₅-Xaa₂₆-Xaa₂₇-Phe-I Ie-Xaa₃o-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄o-

Xaa4i-Xaa42-Xaa43-Xaa44-Xaa45-Xaa46

Formula (I) (SEQ ID No: 1 ) wherein

Xaa₇ is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, N^α-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine, 3-pyridylalanine,

2-pyridylalanine or 4-pyridylalanine;

Xaa₈ is Ala, GIy, VaI, Leu, lie, Lys, Aib, (1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl) carboxylic acid, (1- aminocycloheptyl) carboxylic acid, or (1-aminocyclooctyl) carboxylic acid; Xaa₁₆ is VaI or Leu;

Xaa₁₈ is Ser, Lys or Arg;

Xaa₂o is Leu or Met;

Xaa₂₂ is GIy, GIu or Aib; Xaa₂₃ is GIn, GIu, Lys or Arg;

Xaa₂₅ is Ala or VaI;

Xaa₂₆ is Lys, GIu or Arg;

Xaa₂₇ is GIu or Leu;

Xaa₃₀ is Ala, GIu or Arg; Xaa₃₃ is VaI or Lys;

Xaa₃₄ is Lys, GIu, Asn or Arg;

Xaa₃₅ is GIy or Aib; Xaa₃₆ is Arg, Gly or Lys;

Xaa₃₇ is GIy, Ala, GIu, Pro, Lys, amide or is absent;

Xaa₃₈ is Lys, Ser, amide or is absent.

Xaa₃₉ is Ser, Lys, amide or is absent; Xaa₄₀ is GIy, amide or is absent;

Xaa₄₁ is Ala, amide or is absent;

Xaa₄₂ is Pro, amide or is absent;

Xaa₄₃ is Pro, amide or is absent;

Xaa₄₄ is Pro, amide or is absent; Xaa₄₅ is Ser, amide or is absent;

Xaa₄₆ is amide or is absent ; where if Xaa₃₈, Xaa₃₉, Xaa₄₀, Xaa₄₁, Xaa₄₂, Xaa₄₃, Xaa₄₄, Xaa₄₅ or Xaa₄₆ is absent then each amino acid residue downstream is also absent and where the total number of basic residues

(Lys residues÷ Arg residues) in amino acids 7-37 is greater than the total number of acidic resi- dues (Asp residues + GIu residues) in amino acids 7-37.

The peptides of the invention may also comprise the amino acid sequence of the formula (IV)

Xaa₇-Xaa₈- Xaa₉-Gly-Thr- Phe-Thr-Ser-Asp-Xaa₁₆- Xaa₁₇-Xaa₁₈-Gln-Leu- Xaa₂₁-Xaa₂₂-Xaa₂₃-Ala- Xaa₂₅-Xaa₂6-Xaa₂₇-Phe-lle-Xaa₃o-Trp-Leu-Xaa₃₃-Asn-Xaa₃5-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃9-Xaa₄o- Xaa₄-|-Xaa₄₂-Xaa₄₃-Xaa₄₄-Xaa₄₅~Xaa₄Q

Formula (IV) (SEQ ID No: 4)

2-pyridylalanine or 4-pyridylalanine;

Xaa₈ is Ala, GIy, VaI, Leu, lie, Lys, Aib, (1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl) carboxylic acid, (1- aminocycloheptyl) carboxylic acid, or (1-aminocyclooctyl) carboxylic acid;

Xaa₉ is GIu or Asp;

Xaa₁₆ is VaI or Leu;

Xaa₁₇ is Ser or Thr; Xaa₁₈ is Ser, Lys or Arg;

Xaa₂₁ is GIu or Asp; Xaa₂₂ is GIy, GIu or Aib;

Xaa₂₃ is GIn, Lys or Arg;

Xaa₂₅ is Ala or VaI;

Xaa₂₆ is Lys, or Arg; Xaa₂₇ is GIu or Leu;

Xaa₃₀ is Ala, GIu or Arg;

Xaa₃₃ is VaI or Lys;

Xaa^ is Lys, GIu, Asn or Arg;

Xaa₃₅ is GIy or Aib; Xaa₃₆ is Arg, GIy or Lys;

Xaa₃₇ is GIy, Ala, GIu, Pro, Lys, amide or is absent;

Xaa₃₈ is Lys, Ser, amide or is absent.

Xaa₃₉ is Ser, Lys, amide or is absent;

Xaa₄₀ is GIy, amide or is absent; Xaa₄₁ is Ala, amide or is absent;

Xaa₄₂ is Pro, amide or is absent;

Xaa₄₃ is Pro, amide or is absent;

Xaa₄₄ is Pro, amide or is absent;

Xaa₄₅ is Ser, amide or is absent; Xaa₄₆ is amide or is absent ; where if Xaa₃₈, Xaa₃₉, Xaa₄₀, Xaa₄₁, Xaa₄₂, Xaa₄₃, Xaa₄₄, Xaa₄₅ or Xaa₄₆ is absent then each amino acid residue downstream is also absent.

The peptides of the invention may also comprise the amino acid sequence of the formula (V)

Xaa₇-Xaa₈-Glu-Gly-Thr- Leu-Thr-Ser-Asp-Xaa₁₆- Xaa₁₇-Xaa₁₈-Xaa₁₉-Xaa₂o- Xaa₂₁-Xaa₂₂-Xaa₂₃-

Ala-Xaa₂₅-Xaa₂₆-Xaa₂₇-Phe-lle-Xaa₃o-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-

Xaa₄o-Xaa₄-|-Xaa₄₂-Xaa₄₃-Xaa₄₄-Xaa₄₅-Xaa₄g

Formula (V) (SEQ ID No: 5)

Xaa₇ is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, N^α-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine; Xaa₈ is Ala, GIy, VaI, Leu, lie, Lys, Aib, (1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl) carboxylic acid, (1- aminocycloheptyl) carboxylic acid, or (1-aminocyclooctyl) carboxylic acid;

Xaa₁₆ is VaI or Leu;

Xaa₁₈ is Ser, Lys or Arg;

Xaa₁₉ is Tyr or GIn;

Xaa₂o is GIu or Asp;

Xaa₂₂ is GIy, GIu or Aib or Lys;

Xaa₂₃ is GIn, GIu, Lys or Arg;

Xaa₂₅ is Ala or VaI;

Xaa₂₆ is Lys, GIu or Arg;

Xaa₂₇ is GIu or Leu; Xaa₃₀ is Ala, GIu, Asp or Arg;

Xaa₃₃ is VaI, Ne or Lys;

Xaa^ is Lys, Asn or Arg;

Xaa₃₅ is GIy or Aib;

Xaa₃₆ is Arg, GIy or Lys; Xaa₃₇ is GIy, Ala, GIu, Pro, Lys, amide or is absent;

Xaa₃₈ is Lys, Ser, amide or is absent.

Xaa₃₉ is Ser, Lys, amide or is absent;

Xaa₄₀ is GIy, amide or is absent;

Xaa₄₁ is Ala, amide or is absent; Xaa₄₂ is Pro, amide or is absent;

Xaa₄₃ is Pro, amide or is absent;

Xaa₄₄ is Pro, amide or is absent;

Xaa₄₅ is Ser, amide or is absent;

Xaa₄₆ is amide or is absent ; where if Xaa₃₈, Xaa₃₉, Xaa₄₀, Xaa₄₁, Xaa₄₂, Xaa₄₃, Xaa₄₄, Xaa₄₅ or Xaa₄₆ is absent then each amino acid residue downstream is also absent

The present invention further relates to formulations comprising the peptides of the invention and to the use of these formulations in treating various diseases and/or conditions such as type 2 diabetes.

DEFINITIONS In the present specification, the following terms have the indicated meaning: The term "polypeptide" and "peptide" as used herein means a compound composed of constituent amino acids connected by peptide bonds. The constituent amino acids may be from the group of the amino acids encoded by the genetic code and/or they may be natural amino acids which are not encoded by the genetic code, as well as synthetic amino acids. Natural amino acids which are not encoded by the genetic code are e.g. hydroxyproline, v- carboxyglutamate, ornithine, phosphoserine, D-alanine and D-glutamine. Synthetic amino acids comprise amino acids manufactured by chemical synthesis, i.e. D-isomers of the amino acids encoded by the genetic code such as D-alanine and D-leucine, Aib (α-aminoisobutyric acid), Abu (α-aminobutyric acid), Tie (tert-butylglycine), β-alanine, 3-aminomethyl benzoic acid, an- thranilic acid.

The term "agonist" as used herein means a compound which is an agonist of the human GLP-1 receptor, i.e. a compound that stimulates the formation of cAMP in a suitable medium containing the human GLP-1 receptor. The potency of a peptide of the invention as an agonist is determined by calculating the EC₅₀ value from the dose-response curve as described below.

Baby hamster kidney (BHK) cells expressing the cloned human GLP-1 receptor (BHK^67-12A) are grown in DMEM media with the addition of 100 IU/mL penicillin, 100 μl_/mL streptomycin, 10% fetal calf serum and 1 mg/mL Geneticin G-418 (Life Technologies). Plasma membranes are prepared by homogenisation in buffer (10 mM Tris-HCI, 30 mM NaCI and 1 mM dithiothreitol, pH 7.4, containing, in addition, 5 mg/L leupeptin (Sigma, St. Louis, MO, USA), 5 mg/L pepstatin (Sigma), 100 mg/L bacitracin (Sigma), and 16 mg/L aprotinin (Calbiochem-Novabiochem, La JoIIa, CA). The homogenate is centrifuged on top of a layer of 41 w/v% sucrose. The white band between the two layers is diluted in buffer and centrifuged. Plasma membranes are stored at - 80°C until used.

The functional receptor assay is carried out by measuring cAMP as a response to stimulation by the peptide. Incubation are carried out in 96-well microtiter plates in a total volume of 140 μL and with the following final concentrations: 50 mM Tris-HCI, 1 mM EGTA, 1.5 mM MgSO₄, 1.7 mM ATP, 20 mM GTP, 2 mM 3-isobutyl-1-methylxanthine (IBMX), 0.01 % Tween- 20, pH 7.4. Compounds to be tested for agonist activity are dissolved and diluted in buffer. GTP is freshly prepared for each experiment : 2.5 μg of membrane is added to each well and the mixture is incubated for 90 min at room temperature in the dark with shaking. The reaction is stopped by the addition of 25 μL of 0.5 M HCI. Formed cAMP is measured by a scintillation proximity assay (RPA 542, Amersham, UK). Dose-response curves are plotted for the individual compounds and EC₅₀ values are calculated using GraphPad Prism software. The method for determination of plasma elimination half-life of a peptide of the invention in man is: The peptide is dissolved in an isotonic buffer, pH 7.4, PBS or any other suitable buffer. The dose is injected peripherally, preferably in the abdominal or upper thigh. Blood samples for determination of active peptide are taken at frequent intervals, and for a sufficient duration to cover the terminal elimination part (e.g. Pre-dose, 1 , 2, 3, 4, 5, 6, 7, 8, 10, 12, 24 (day 2), 36 (day 2), 48 (day 3), 60 (day 3), 72 (day 4) and 84 (day 4) hours post dose). Determination of the concentration of active peptide is performed as described in Wilken et al., Diabetologia 43(51 ):A143, 2000. Derived pharmacokinetic parameters are calculated from the concentration-time data for each individual subject by use of non-compartmental methods, using the commercially available software WinNonlin Version 2.1 (Pharsight, Cary, NC, USA). The terminal elimination rate constant is estimated by log-linear regression on the terminal log-linear part of the concentration- time curve, and used for calculating the elimination half-life.

The term "enzymatic stability" as used herein in reference to a polypeptide means a polypeptide which exhibits enhanced resistance relative to GLP-1 (7-37) to the plasma peptidase dipeptidyl aminopeptidase-4 (DPP-IV).

Resistance of a peptide to degradation by dipeptidyl aminopeptidase IV (DPP-IV) is determined by the following degradation assay :

Peptide stability towards DPP-IV was tested using porcine DPP-IV. The peptides were dissolved in a 20 mM Tris-HCI (pH8.0) buffer and 1 m-units of porcine DPP-IV were added per 4.5 nmol peptide. The peptides were incubated at 37° C and samples were quenched with half a volume 10% triflouroacetic acid. Products are separated and quantified using HPLC analysis.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to GLP-1 peptides where such peptides are agonists of the GLP-1 receptor as demonstrated in a cAMP production assay and exhibit enhanced enzymatic stability relative to GLP-1 (7-37).

In one embodiment, the peptides of the invention comprise an amino acid sequence of formula I:

Xaa₇-Xaa₈-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂o-Glu-Xaa₂₂-Xaa₂₃-Ala- Xaa₂5-Xaa₂6-Xaa₂₇-Phe-lle-Xaa30-Trp-Leu-Xaa33-Xaa3₄-Xaa35-Xaa3₆-Xaa3₇-Xaa₃₈-Xaa39-Xaa₄0- Xaa4"i-Xaa42-Xaa43-Xaa44-Xaa45-Xaa4g Formula (I) (SEQ ID No: 1 ) wherein Xaa₇ is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, N^α-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine, 3-pyridylalanine,

2-pyridylalanine or 4-pyridylalanine;

Xaa₁₆ is VaI or Leu;

Xaa₁₈ is Ser, Lys or Arg;

Xaa₁₉ is Tyr or GIn; Xaa₂₀ is Leu or Met;

Xaa₂₂ is GIy, GIu or Aib;

Xaa₂₃ is GIn, GIu, Lys or Arg;

Xaa₂₅ is Ala or VaI;

Xaa₂₆ is Lys, GIu or Arg; Xaa₂₇ is GIu or Leu;

Xaa₃₀ is Ala, GIu or Arg;

Xaa₃₃ is VaI or Lys;

Xaa₃₄ is Lys, GIu, Asn or Arg;

Xaa₃₅ is GIy or Aib; Xaa₃₆ is Arg, GIy or Lys;

Xaa₃₇ is GIy, Ala, GIu, Pro, Lys, amide or is absent;

Xaa₃₈ is Lys, Ser, amide or is absent.

Xaa₃₉ is Ser, Lys, amide or is absent;

Xaa₄₀ is GIy, amide or is absent; Xaa₄₁ is Ala, amide or is absent;

Xaa₄₂ is Pro, amide or is absent;

Xaa₄₃ is Pro, amide or is absent;

Xaa₄₄ is Pro, amide or is absent;

In a further embodiment of the invention, the peptide of the invention comprises an amino acid sequence of formula (II): Xaa7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Xaa₁₈-Tyr-Leu-Glu-Xaa22-Xaa23-Ala-Ala-Xaa26-

Glu-Phe-lle-Xaa₃o-Trp-Leu-Val-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈

Formula (II) (SEQ ID No: 2) wherein Xaa₇ is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, N^α-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine, 3-pyridylalanine,

2-pyridylalanine or 4-pyridylalanine;

Xaa₁₈ is Ser, Lys or Arg;

Xaa₂₂ is GIy, GIu or Aib;

Xaa₂₃ is GIn, GIu, Lys or Arg;

Xaa₂₆ is Lys, GIu or Arg; Xaa₃₀ is Ala, GIu or Arg;

Xaa₃₄ is Lys, GIu or Arg;

Xaa₃₅ is GIy or Aib;

Xaa₃₆ is Arg or Lys;

Xaa₃₇ is GIy, Ala, GIu or Lys; Xaa₃₈ is Lys, amide or is absent.

In a further embodiment of the invention, the peptide of the invention comprises an amino acid sequence of formula (III):

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Xaa₁₈-Tyr-Xaa₂o-Glu-Gly-Xaa23-Ala-Val-Xaa₂6-

Leu-Phe-lle-Ala-Trp-Leu-Xaa₃₃-Xaa₃₄-Gly-Xaa₃₆-Gly-Xaa₃₈ Formula (III) (SEQ ID No: 3) where

Xaa₁₈ is Lys or Arg;

Xaa₂o is Leu or Met;

Xaa₂₃ is GIn or Asn;

Xaa₂₆ is Lys or Arg; Xaa₃₃ is Lys or Arg

Xaa₃₄ is Lys or Arg;

Xaa₃₆ is Arg or Lys;

Xaa₃₈ is Lys, amide or is absent. In yet another embodiment of the invention, the peptide of the invention comprises the amino acid sequence of the formula (IV) (SEQ ID NO:4)

Xaa₇-Xaa₈- Xaa₉-Gly-Thr- Phe-Thr-Ser-Asp-Xaa₁₆- Xaa₁₇-Xaa₁₈-Gln-Leu- Xaa₂i-Xaa₂₂-Xaa₂₃-Ala- Xaa25-Xaa26-Xaa2₇-Phe-lle-Xaa30-Trp-Leu-Xaa33-Asn-Xaa35-Xaa36-Xaa₃₇-Xaa38-Xaa39-Xaa40- Xaa4-|-Xaa42-Xaa4₃-Xaa44-Xaa45~Xaa4Q

Formula (IV) (SEQ ID No: 4)

2-pyridylalanine or 4-pyridylalanine;

Xaa₉ is GIu or Asp;

Xaa₁₆ is VaI or Leu;

Xaa₁₈ is Ser, Lys or Arg;

Xaa₂₂ is GIy, GIu or Aib;

Xaa₂₃ is GIn, Lys or Arg;

Xaa₂₅ is Ala or VaI;

Xaa₂₆ is Lys, or Arg;

Xaa₂₇ is GIu or Leu; Xaa₃₀ is Ala, GIu or Arg;

Xaa₃₃ is VaI or Lys;

Xaa₃₄ is Lys, GIu, Asn or Arg;

Xaa₃₅ is GIy or Aib;

Xaa₃₈ is Lys, Ser, amide or is absent.

Xaa₃₉ is Ser, Lys, amide or is absent;

Xaa₄₀ is GIy, amide or is absent;

Xaa₄₁ is Ala, amide or is absent; Xaa₄₂ is Pro, amide or is absent;

Xaa₄₃ is Pro, amide or is absent; Xaa₄₄ is Pro, amide or is absent; Xaa₄₅ is Ser, amide or is absent; Xaa₄₆ is amide or is absent ; where if Xaa₃₈, Xaa₃₉, Xaa₄₀, Xaa₄₁, Xaa₄₂, Xaa₄₃, Xaa₄₄, Xaa₄₅ or Xaa₄₆ is absent then each amino acid residue downstream is also absent.

In another embodiment, peptides of the invention may comprise the amino acid sequence of the formula (V)

Xaa₇-Xaa₈-Glu-Gly-Thr- Leu-Thr-Ser-Asp-Xaa₁₆- Xaa₁₇-Xaa₁₈-Xaa₁₉-Xaa2o- Xaa₂i-Xaa22-Xaa₂3- Ala-Xaa₂₅-Xaa₂₆-Xaa₂₇-Phe-lle-Xaa₃₀-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉- Xaa₄o-Xaa₄-|-Xaa₄₂~xaa₄₃-Xaa₄₄-Xaa₄₅-Xaa₄g

Formula (V) (SEQ ID No: 5)

2-pyridylalanine or 4-pyridylalanine;

Xaa₁₆ is VaI or Leu;

Xaa₁₇ is Ser or Thr;

Xaa₁₈ is Ser, Lys or Arg; Xaa₁₉ is Tyr or GIn;

Xaa₂o is GIu or Asp;

Xaa₂₂ is GIy, GIu or Aib or Lys;

Xaa₂₃ is GIn, GIu, Lys or Arg; Xaa₂₅ is Ala or VaI;

Xaa₂₆ is Lys, GIu or Arg;

Xaa₂₇ is GIu or Leu;

Xaa₃₀ is Ala, GIu, Asp or Arg;

Xaa₃₃ is VaI, Ne or Lys; Xaa₃₄ is Lys, Asn or Arg;

Xaa₃₅ is GIy or Aib; Xaa₃₆ is Arg, Gly or Lys;

Xaa₃₇ is GIy, Ala, GIu, Pro, Lys, amide or is absent; Xaa₃₈ is Lys, Ser, amide or is absent. Xaa₃₉ is Ser, Lys, amide or is absent; Xaa₄₀ is GIy, amide or is absent; Xaa₄₁ is Ala, amide or is absent; Xaa₄₂ is Pro, amide or is absent; Xaa₄₃ is Pro, amide or is absent; Xaa₄₄ is Pro, amide or is absent; Xaa₄₅ is Ser, amide or is absent; Xaa₄₆ is amide or is absent ; where if Xaa₃₈, Xaa₃₉, Xaa₄₀, Xaa₄₁, Xaa₄₂, Xaa₄₃, Xaa₄₄, Xaa₄₅ or Xaa₄₆ is absent then each amino acid residue downstream is also absent.

In one embodiment of the invention, the total number of basic residues (Lys residues÷

Arg residues) in amino acid positions 7-37 in the sequence of a peptide of the invention is greater than the total number of acidic residues (Asp residues + GIu residues) in amino acid positions 7-37 in the sequence of the peptide.

In another embodiment of the invention, the peptides of the invention comprise no more than ten amino acid residues which have been exchanged, added or deleted as compared to GLP-1 (7-37).

In yet another embodiment of the invention, the peptides of the invention comprise no more eight amino acid residues which have been exchanged, added or deleted as com- pared to GLP-1 (7-37).

In yet another embodiment of the invention, the peptides of the invention comprise no more six amino acid residues which have been exchanged, added or deleted as compared to GLP-1 (7-37).

In yet another embodiment of the invention, the peptides of the invention comprise no more four amino acid residues which have been exchanged, added or deleted as compared to GLP-1 (7-37).

In yet another embodiment of the invention, the peptides of the invention comprise no more than 4 amino acid residues which are not encoded by the genetic code.

In yet another embodiment of the invention, the peptides of the invention comprise no more than 3 amino acid residues which are not encoded by the genetic code. In yet another embodiment of the invention, the peptides of the invention comprise no more than 2 amino acid residues which are not encoded by the genetic code.

In yet another embodiment of the invention, the peptides of the invention comprise no more than one amino acid residue which is not encoded by the genetic code. In yet another embodiment of the invention, the peptides of the invention comprise three lysine residues.

In yet another embodiment of the invention, the peptides of the invention comprise two lysine residues.

In yet another embodiment of the invention, the peptides of the invention comprise only one lysine residue.

In yet another embodiment of the invention, the peptides of the invention have an isoelectric point of greater than 7.

In yet another embodiment of the invention, the peptides of the invention have an isoelectric point of greater than 8. In yet another embodiment of the invention, the peptides of the invention have an isoelectric point of greater than 9.

In yet another embodiment of the invention, the peptides of the invention have an isoelectric point of greater than 10.

In a further embodiment of the invention, the peptide of the invention comprises an amino acid sequence selected from the group consisting of: formula (Vl) (SEQ ID No: 6)

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Tyr-Met-Glu-Gly-Gln-Ala-Val-Arg-Leu-Phe- Ile-Ala-Trp-Leu-Lys-Lys-Gly-Arg-Gly-Xaa₃₈ Where Xaa₃₈ is amide or is absent;

formula (VII) (SEQ ID NO:7):

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Gln-Leu-Glu-Glu-Gln-Ala-Ala- Lys-Glu-Phe-lle-Glu-Trp-Leu-Val-Asn-Gly-Gly-Pro-Ser-Xaa₃₉;

Where Xaa₃₉ is amide or is absent

Formula VIII (SEQ ID NO: 8) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Thr-Gln-Tyr-Leu-Glu-Glu-Lys-Ala-Ala- Lys-Glu-Phe-lle-Asp-Trp-Leu-lle-Asn-Gly-Arg-Pro-Lys-Xaa₃₉ Where Xaa₃₉ is amide or is absent and

Formula IX (SEQ ID NO: 9)

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Thr-Ser-Tyr-Leu-Glu-Glu-Lys-Ala-Ala- Lys-Glu-Phe-lle-Asp-Trp-Leu-lle-Lys-Gly-Arg-Pro-Lys- Xaa₃₉

Where Xaa₃₉ is amide or is absent

The present invention also includes peptides that are analogues of the peptides of the invention having the sequences shown in SQ ID NOs: 1-9 where by "analogues" as used herein is meant peptides whose sequences have conservative substitutions to the sequences shown in SQ ID NOs: 1-9 where by "conservative substitutions" is intended swaps within groups such as replacement of the aliphatic or hydrophobic amino acids Ala, VaI, Leu and lie; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and GIu; replacement of the amide residues Asn and GIn, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and GIy The present invention further includes derivatives of the peptides or analogues of the invention where the term "derivative" as used herein in relation to a peptide or analogue means a chemically modified peptide or an analogue thereof, wherein at least one substituent is not present in the unmodified peptide or an analogue thereof, i.e. a peptide which has been covalently modified. Typical modifications are amides, carbohydrates, alkyl groups, acyl groups, polyethylene glycol groups, esters and the like. An example of a derivative of GLP- 1 (7-37) is Arg³⁴, Lys²⁶(N^ε-(γ-Glu(N^α-hexadecanoyl)))-GLP-1 (7-37).

In one embodiment, the derivatives are peptides or analogues that have been modified with lipophilic substituents such as acyl groups where modification of GLP-1 peptide with lipophilic substituents is described in WO 96/29342, WO 98/08871 , WO 99/43708 and WO 00/34331 , the contents of each of which are hereby incorporated in their entirety.

In another embodiment, the derivatives are peptides or analogues that have been modified with polyethylene glycol moieties where such modifications are described in WO 00/66629 and WO 03/40309, the contents of each of which are hereby incorporated in their entirety.

The peptides of the invention can be produced by classical peptide synthesis, e.g. solid phase peptide synthesis using t-Boc or F-Moc chemistry or other well established techniques., see the examples and e.g. Houben-Weyl, Methods of organic Chemistry, Volume E 22a, E 22b and E 22 c; Green and Wuts, "Protecting Groups in Organic Synthesis", Jogn Wiley & Sons, 1999.

The peptides of the inventions may also be produced by a method which comprises cul- turing a host cell containing a DNA sequence encoding the polypeptide and capable of express- ing the polypeptide in a suitable nutrient medium under conditions permitting the expression of the peptide, after which the resulting peptide is recovered from the culture.

The medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to pub- lished recipes (e.g. in catalogues of the American Type Culture Collection). The peptide produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatog- raphy, gel filtration chromatography, affinity chromatography, or the like, dependent on the type of peptide in question.

The DNA sequence encoding the therapeutic polypeptide may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the polypeptide by hybridisation using synthetic oligonu- cleotide probes in accordance with standard techniques (see, for example, Sambrook, J, Fritsch, EF and Maniatis, T, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989). The DNA sequence encoding the polypeptide may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981 ), 1859 - 1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801 - 805. The DNA sequence may also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683,202 or Saiki et al., Science 239 (1988), 487 - 491.

The DNA sequence may be inserted into any vector which may conveniently be sub- jected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated. The vector is preferably an expression vector in which the DNA sequence encoding the peptide is operably linked to additional segments required for transcription of the DNA, such as a promoter. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or het- erologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA encoding the peptide of the invention in a variety of host cells are well known in the art, cf. for instance Sambrook et ai, supra.

The DNA sequence encoding the peptide may also, if necessary, be operably connected to a suitable terminator, polyadenylation signals, transcriptional enhancer sequences, and trans- lational enhancer sequences. The recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.

The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate.

To direct a parent peptide of the present invention into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector. The secretory signal sequence is joined to the DNA sequence encoding the peptide in the correct reading frame. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the peptide. The secretory signal sequence may be that normally associated with the peptide or may be from a gene encoding another secreted protein.

The procedures used to ligate the DNA sequences coding for the present peptide, the promoter and optionally the terminator and/or secretory signal sequence, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al.., supra).

The host cell into which the DNA sequence or the recombinant vector is introduced may be any cell which is capable of producing the present peptide and includes bacteria, yeast, fungi and higher eukaryotic cells. Examples of suitable host cells well known and used in the art are, without limitation, E. coli, Saccharomyces cerevisiae, or mammalian BHK or CHO cell lines. Pharmaceutical compositions

Pharmaceutical compositions containing a compound according to the present invention may be prepared by conventional techniques, e.g. as described in Remington's Pharmaceutical Sciences, 1985 or in Remington: The Science and Practice of Pharmacy, 19^th edition, 1995.

One object of the present invention is to provide a pharmaceutical formulation comprising a peptide according to the present invention. In one embodiment, the peptide is present in the formulation at a concentration of from about 0.1 mg/ml to about 25 mg/ml. In another embodiment, the peptide is present in the formulation at a concentration of from about 1 mg/ml to about 10 mg/ml.

In another embodiment, the formulation has a pH from 4.0 to 10.0.

In another embodiment of the invention, the pH of the formulation is selected from the list consisting of 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1 , 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1 , 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.0.

In another embodiment, the formulation has a pH from 4.0 to 7.0.

In yet another embodiment, the formulation has a pH from 7.0 to 10.0.

In yet another embodiment, the formulation has a pH from 7.0 to 8.0. The formulation may further comprise a buffer system, preservative(s), isotonicity agent(s), chelating agent(s), stabilizers and surfactants. In one embodiment of the invention the pharmaceutical formulation is an aqueous formulation, i.e. formulation comprising water. Such formulation is typically a solution or a suspension. In a further embodiment of the invention the pharmaceutical formulation is an aqueous solution. The term "aqueous formulation" is defined as a formulation comprising at least 50 %w/w water. Likewise, the term "aqueous solution" is defined as a solution comprising at least 50 %w/w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50 %w/w water.

In another embodiment the pharmaceutical formulation is a freeze-dried formulation, whereto the physician or the patient adds solvents and/or diluents prior to use. In another embodiment the pharmaceutical formulation is a dried formulation (e.g. freeze-dried or spray-dried) ready for use without any prior dissolution.

In a further aspect the invention relates to a pharmaceutical formulation comprising an aqueous solution of a peptide according to the present invention, and a buffer.

In a further embodiment of the invention the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethane, hepes, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each one of these specific buffers constitutes an alternative embodiment of the invention.

In a further embodiment of the invention the formulation further comprises a pharmaceutically acceptable preservative. In a further embodiment of the invention the preservative is selected from the group consisting of phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p- hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl p- hydroxybenzoate, benzethonium chloride, chlorphenesine (3p-chlorphenoxypropane-1 ,2-diol) or mixtures thereof. In a further embodiment of the invention the preservative is present in a concentration from 0.1 mg/ml to 20 mg/ml. In a further embodiment of the invention the preservative is present in a concentration from 0.1 mg/ml to 5 mg/ml. In a further embodiment of the invention the preservative is present in a concentration from 5 mg/ml to 10 mg/ml. In a further embodiment of the invention the preservative is present in a concentration from 10 mg/ml to 20 mg/ml. Each one of these specific preservatives constitutes an alternative embodiment of the invention. The use of a preservative in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19^th edition, 1995.

In a further embodiment of the invention the formulation further comprises an isotonic agent. In a further embodiment of the invention the isotonic agent is selected from the group consisting of a salt (e.g. sodium chloride), a sugar or sugar alcohol, an amino acid (e.g. glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), an alditol (e.g. glycerol (glycerine), 1 ,2-propanediol (propyleneglycol), 1 ,3-propanediol, 1 ,3- butanediol) polyethyleneglycol (e.g. PEG400), or mixtures thereof. Any sugar such as mono-, di-, or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na may be used. In one embodiment the sugar additive is sucrose. Sugar alcohol is defined as a sugar where the aldyhyde groups has been reduced to a hydroxyl group and includes, for example, mannitol, sorbitol, inositol, galacititol, dulcitol, xylitol, and arabitol. In one embodiment the sugar alcohol additive is mannitol. The sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to the amount used, as long as the sugar or sugar alcohol is soluble in the liquid preparation and does not adversely effect the stabilizing effects achieved using the methods of the invention. In one embodiment, the sugar or sugar alcohol concentration is between about 1 mg/ml and about 150 mg/ml. In a further embodiment of the invention the isotonic agent is present in a concentration from 1 mg/ml to 50 mg/ml. In a further embodiment of the invention the isotonic agent is present in a concentration from 1 mg/ml to 7 mg/ml. In a further embodiment of the invention the isotonic agent is present in a concentration from 8 mg/ml to 24 mg/ml. In a further embodiment of the invention the isotonic agent is present in a concentration from 25 mg/ml to 50 mg/ml. Each one of these specific isotonic agents constitutes an alternative embodiment of the invention. The use of an isotonic agent in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19^th edition, 1995.

In a further embodiment of the invention the formulation further comprises a chelating agent. In a further embodiment of the invention the chelating agent is selected from salts of ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic acid, and mixtures thereof. In a further embodiment of the invention the chelating agent is present in a concentration from 0.1 mg/ml to 5mg/ml. In a further embodiment of the invention the chelating agent is present in a concentration from 0.1 mg/ml to 2mg/ml. In a further embodiment of the invention the chelating agent is present in a concentration from 2mg/ml to 5mg/ml. Each one of these specific chelating agents constitutes an alternative embodiment of the invention. The use of a chelating agent in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19^th edition, 1995.

In a further embodiment of the invention the formulation further comprises a stabiliser. The use of a stabilizer in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19^th edition, 1995. More particularly, compositions of the invention are stabilized liquid pharmaceutical compositions whose therapeutically active components include a polypeptide that possibly exhibits aggregate formation during storage in liquid pharmaceutical formulations. By "aggregate formation" is intended a physical interaction between the polypeptide molecules that results in formation of oligomers, which may remain soluble, or large visible aggregates that precipitate from the solution. By "during storage" is intended a liquid pharmaceutical composition or formulation once prepared, is not immediately administered to a subject. Rather, fol- lowing preparation, it is packaged for storage, either in a liquid form, in a frozen state, or in a dried form for later reconstitution into a liquid form or other form suitable for administration to a subject. By "dried form" is intended the liquid pharmaceutical composition or formulation is dried either by freeze drying (i.e., lyophilization; see, for example, Williams and PoIIi (1984) J. Parenteral Sci. Technol. 38:48-59), spray drying (see Masters (1991 ) in Spray-Drying Handbook (5th ed; Longman Scientific and Technical, Essez, U.K.), pp. 491-676; Broadhead et al. (1992) Drug Devel. Ind. Pharm. 18:1 169-1206; and Mumenthaler et al. (1994) Pharm. Res. 11 : 12-20), or air drying (Carpenter and Crowe (1988) Cryobiology 25:459-470; and Roser (1991 ) Biopharm. 4:47-53). Aggregate formation by a polypeptide during storage of a liquid pharmaceutical composition can adversely affect biological activity of that polypeptide, resulting in loss of therapeutic efficacy of the pharmaceutical composition. Furthermore, aggregate formation may cause other problems such as blockage of tubing, membranes, or pumps when the polypeptide-containing pharmaceutical composition is administered using an infusion system. The pharmaceutical compositions of the invention may further comprise an amount of an amino acid base sufficient to decrease aggregate formation by the polypeptide during storage of the composition. By "amino acid base" is intended an amino acid or a combination of amino acids, where any given amino acid is present either in its free base form or in its salt form. Where a combination of amino acids is used, all of the amino acids may be present in their free base forms, all may be present in their salt forms, or some may be present in their free base forms while others are present in their salt forms. In one embodiment, amino acids to use in preparing the compositions of the invention are those carrying a charged side chain, such as arginine, lysine, aspartic acid, and glutamic acid. Glycine, imidazole and any stereoisomer of a particular amino acid (e.g. methionine, histidine, arginine, lysine, isoleucine, as- partic acid, tryptophan, threonine and mixtures thereof) or combinations of these stereoisomers, may be present in the pharmaceutical compositions of the invention so long as the particular amino acid is present either in its free base form or its salt form. In one embodiment the L-stereoisomer is used. Compositions of the invention may also be formulated with analogues of these amino acids. By "amino acid analogue" is intended a derivative of the natu- rally occurring amino acid that brings about the desired effect of decreasing aggregate formation by the polypeptide during storage of the liquid pharmaceutical compositions of the invention. Suitable arginine analogues include, for example, aminoguanidine, ornithine and N-monoethyl L-arginine, suitable methionine analogues include ethionine and buthionine and suitable cystein analogues include S-methyl-L cystein. As with the other amino acids, the amino acid analogues are incorporated into the compositions in either their free base form or their salt form. In a further embodiment of the invention the amino acids or amino acid ana- logues are used in a concentration, which is sufficient to prevent or delay aggregation of the protein.

In a further embodiment of the invention methionine (or other sulphuric amino acids or amino acid analogous) may be added to inhibit oxidation of methionine residues to me- thionine sulfoxide when the polypeptide acting as the therapeutic agent is a polypeptide comprising at least one methionine residue susceptible to such oxidation. By "inhibit" is intended minimal accumulation of methionine oxidized species over time. Inhibiting methionine oxidation results in greater retention of the polypeptide in its proper molecular form. Any stereoisomer of methionine or combinations thereof can be used. The amount to be added should be an amount sufficient to inhibit oxidation of the methionine residues such that the amount of methionine sulfoxide is acceptable to regulatory agencies. Typically, this means that the composition contains no more than about 10% to about 30% methionine sulfoxide. Generally, this can be achieved by adding methionine such that the ratio of methionine added to methionine residues ranges from about 1 :1 to about 1000:1 , such as 10:1 to about 100:1.

In a further embodiment of the invention the formulation further comprises a stabiliser selected from the group of high molecular weight polymers or low molecular compounds. In a further embodiment of the invention the stabilizer is selected from polyethylene glycol (e.g. PEG 3350), polyvinylalcohol (PVA), polyvinylpyrrolidone, carboxy-/hydroxycellulose or derivates thereof (e.g. HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, sulphur-containing substances as monothioglycerol, thioglycolic acid and 2-methylthioethanol, and different salts (e.g. sodium chloride). Each one of these specific stabilizers constitutes an alternative embodiment of the invention.

The pharmaceutical compositions may also comprise additional stabilizing agents, which further enhance stability of a therapeutically active polypeptide therein. Stabilizing agents of particular interest to the present invention include, but are not limited to, methionine and EDTA, which protect the polypeptide against methionine oxidation, and a nonionic surfactant, which protects the polypeptide against aggregation associated with freeze-th awing or mechanical shearing.

In a further embodiment of the invention the formulation further comprises a surfactant. In a further embodiment of the invention the surfactant is selected from a detergent, ethoxylated castor oil, polyglycolyzed glycerides, acetylated monoglycerides, sorbitan fatty acid esters, polyoxypropylene-polyoxyethylene block polymers (eg. poloxamers such as Pluronic^® F68, poloxamer 188 and 407, Triton X-100 ), polyoxyethylene sorbitan fatty acid esters, polyoxyethylene and polyethylene derivatives such as alkylated and alkoxylated derivatives (tweens, e.g. Tween-20, Tween-40, Tween-80 and Brij-35), monoglycerides or ethoxylated derivatives thereof, diglycerides or polyoxyethylene derivatives thereof, alcohols, glycerol, lecitins and phospholipids (eg. phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, diphosphatidyl glycerol and sphingomyelin), derivates of phospholipids (eg. dipalmitoyl phosphatidic acid) and lysophospholipids (eg. palmitoyl lysophosphatidyl-L-serine and 1-acyl-sn-glycero-3- phosphate esters of ethanolamine, choline, serine or threonine) and alkyl, alkoxyl (alkyl ester), alkoxy (alkyl ether)- derivatives of lysophosphatidyl and phosphatidylcholines, e.g. lauroyl and myristoyl derivatives of lysophosphatidylcholine, dipalmitoylphosphatidylcholine, and modifications of the polar head group, that is cholines, ethanolamines, phosphatidic acid, serines, threonines, glycerol, inositol, and the positively charged DODAC, DOTMA, DCP, BISHOP, lysophosphatidylserine and lysophosphatidylthreonine, and glycerophospholipids (eg. cephalins), glyceroglycolipids (eg. galactopyransoide), sphingoglycolipids (eg. ceramides, gangliosides), dodecylphosphocholine, hen egg lysolecithin, fusidic acid derivatives- (e.g. sodium tauro-dihydrofusidate etc.), long-chain fatty acids and salts thereof C6-C12 (eg. oleic acid and caprylic acid), acylcarnitines and derivatives, N^α-acylated derivatives of lysine, arginine or histidine, or side-chain acylated derivatives of lysine or arginine, N^α-acylated derivatives of dipeptides comprising any combination of lysine, arginine or histidine and a neutral or acidic amino acid, N^α-acylated derivative of a tripeptide comprising any combination of a neutral amino acid and two charged amino acids, DSS

(docusate sodium, CAS registry no [577-11-7]), docusate calcium, CAS registry no [128-49- 4]), docusate potassium, CAS registry no [7491-09-0]), SDS (sodium dodecyl sulfate or sodium lauryl sulfate), sodium caprylate, cholic acid or derivatives thereof, bile acids and salts thereof and glycine or taurine conjugates, ursodeoxycholic acid, sodium cholate, sodium deoxycholate, sodium taurocholate, sodium glycocholate, N-Hexadecyl-N,N- dimethyl-3-ammonio-1-propanesulfonate, anionic (alkyl-aryl-sulphonates) monovalent surfactants, zwitterionic surfactants (e.g. N-alkyl-N,N-dimethylammonio-1-propanesulfonates, 3-cholamido-i-propyldimethylammonio-i-propanesulfonate, cationic surfactants (quarternary ammonium bases) (e.g. cetyl-trimethylammonium bromide, cetylpyridinium chloride), non-ionic surfactants (eg. Dodecyl β-D-glucopyranoside), poloxamines (eg.

Tetronic's), which are tetrafunctional block copolymers derived from sequential addition of propylene oxide and ethylene oxide to ethylenediamine, or the surfactant may be selected from the group of imidazoline derivatives, or mixtures thereof. Each one of these specific surfactants constitutes an alternative embodiment of the invention. The use of a surfactant in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19^th edition, 1995.

It is possible that other ingredients may be present in the peptide pharmaceutical formulation of the present invention. Such additional ingredients may include wetting agents, emulsifiers, antioxidants, bulking agents, tonicity modifiers, chelating agents, metal ions, oleaginous vehicles, proteins (e.g., human serum albumin, gelatin or proteins) and a zwitterion (e.g., an amino acid such as betaine, taurine, arginine, glycine, lysine and histidine). Such additional ingredients, of course, should not adversely affect the overall stability of the pharmaceutical formulation of the present invention.

Pharmaceutical compositions containing a peptide according to the present invention may be administered to a patient in need of such treatment at several sites, for example, at topical sites, for example, skin and mucosal sites, at sites which bypass absorption, for example, administration in an artery, in a vein, in the heart, and at sites which involve absorption, for example, administration in the skin, under the skin, in a muscle or in the abdomen.

Administration of pharmaceutical compositions according to the invention may be through several routes of administration, for example, lingual, sublingual, buccal, in the mouth, oral, in the stomach and intestine, nasal, pulmonary, for example, through the bronchioles and alveoli or a combination thereof, epidermal, dermal, transdermal, vaginal, rectal, ocular, for examples through the conjunctiva, uretal, and parenteral to patients in need of such a treatment. Compositions of the current invention may be administered in several dosage forms, for example, as solutions, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets, rinses, capsules, for example, hard gelatine capsules and soft gelatine capsules, suppositories, rectal capsules, drops, gels, sprays, powder, aerosols, inhalants, eye drops, ophthalmic ointments, ophthal- mic rinses, vaginal pessaries, vaginal rings, vaginal ointments, injection solution, in situ transforming solutions, for example in situ gelling, in situ setting, in situ precipitating, in situ crystallization, infusion solution, and implants.

Compositions of the invention may further be compounded in, or attached to, for example through covalent, hydrophobic and electrostatic interactions, a drug carrier, drug de- livery system and advanced drug delivery system in order to further enhance stability of the compound, increase bioavailability, increase solubility, decrease adverse effects, achieve chronotherapy well known to those skilled in the art, and increase patient compliance or any combination thereof. Examples of carriers, drug delivery systems and advanced drug delivery systems include, but are not limited to, polymers, for example cellulose and derivatives, polysaccharides, for example dextran and derivatives, starch and derivatives, polyvinyl al- cohol), acrylate and methacrylate polymers, polylactic and polyglycolic acid and block copolymers thereof, polyethylene glycols, carrier proteins, for example albumin, gels, for example, thermogelling systems, for example block co-polymeric systems well known to those skilled in the art, micelles, liposomes, microspheres, nanoparticulates, liquid crystals and dispersions thereof, L2 phase and dispersions there of, well known to those skilled in the art of phase behaviour in lipid-water systems, polymeric micelles, multiple emulsions, self- emulsifying, self-microemulsifying, cyclodextrins and derivatives thereof, and dendrimers.

Compositions of the current invention are useful in the formulation of solids, semisolids, powder and solutions for pulmonary administration of the compound, using, for example a metered dose inhaler, dry powder inhaler and a nebulizer, all being devices well known to those skilled in the art. Pulmonary administration of GLP-1 peptides have been disclosed in WO 01/51071 and WO 00/12116, the contents of each of which are hereby inc- orporated by reference.

Compositions of the current invention are specifically useful in the formulation of controlled, sustained, protracting, retarded, and slow release drug delivery systems. More specifically, but not limited to, compositions are useful in formulation of parenteral controlled release and sustained release systems (both systems leading to a many-fold reduction in number of administrations), well known to those skilled in the art. Even more preferably, are controlled release and sustained release systems administered subcutaneous. Without limit- ing the scope of the invention, examples of useful controlled release system and compositions are hydrogels, oleaginous gels, liquid crystals, polymeric micelles, microspheres, nanoparticles,

Methods to produce controlled release systems useful for compositions of the current invention include, but are not limited to, crystallization, condensation, co-cystallization, precipitation, co-precipitation, emulsification, dispersion, high pressure homogenization, encapsulation, spray drying, microencapsulation, coacervation, phase separation, solvent evaporation to produce microspheres, extrusion and supercritical fluid processes. General reference is made to Handbook of Pharmaceutical Controlled Release (Wise, D. L., ed. Marcel Dekker, New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99: Protein Formulation and Delivery (MacNally, E.J., ed. Marcel Dekker, New York, 2000).

Pharmaceutical compositions containing a peptide according to the present invention may be administered parenterally to patients in need of such a treatment. Parenteral administra- tion may be performed by subcutaneous, intramuscular or intravenous injection by means of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump. A further option is a composition which may be a powder or a liquid for the administration of the peptide in the form of a nasal or pulmonal spray. As a still fur- ther option, the peptide of the invention can also be administered transdermal^, e.g. from a patch, optionally a iontophoretic patch, or transmucosally, e.g. bucally.

Thus, the compositions of the peptide of the invention can be prepared using the conventional techniques of the pharmaceutical industry which involves dissolving and mixing the ingredients as appropriate to give the desired end product.

In one embodiment, the peptide is dissolved in an amount of water which is somewhat less than the final volume of the composition to be prepared. An isotonic agent, a preservative and a buffer is added as required and the pH value of the solution is adjusted - if necessary - using an acid, e.g. hydrochloric acid, or a base, e.g. aqueous sodium hydroxide as needed. Finally, the volume of the solution is adjusted with water to give the desired concentration of the ingredients.

In one embodiment of the present invention, the peptide is provided in the form of a composition suitable for administration by injection. Such a composition can either be an injectable solution ready for use or it can be an amount of a solid composition, e.g. a lyophilised product, which has to be dissolved in a solvent before it can be injected.

The peptides of this invention can be used in the treatment of various diseases. The particular peptide to be used and the optimal dose level for any patient will depend on the disease to be treated and on a variety of factors including the efficacy of the specific peptide derivative employed, the age, body weight, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of the case. It is recommended that the dosage of the peptide of this invention be determined for each individual patient by those skilled in the art.

In one embodiment, it is envisaged that the peptide will be useful for the preparation of a medicament with a protracted profile of action for the treatment of non-insulin dependent diabetes mellitus and/or for the treatment of obesity.

In another embodiment, the present invention relates to the use of a peptide accord- ing to the invention for the preparation of a medicament for the treatment of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syn- drome X, dyslipidemia, β-cell apoptosis, β-cell deficiency, myocardial infarction, inflammatory bowel syndrome, dyspepsia, cognitive disorders, e.g. cognitive enhancing, neuroprotection, atheroschlerosis, coronary heart disease and other cardiovascular disorders.

In another embodiment of the invention a peptide according to the invention is used for the preparation of a medicament for delaying or preventing disease progression in type 2 diabetes.

In another embodiment of the invention a peptide according to the is used for the preparation of a medicament for decreasing food intake, decreasing β-cell apoptosis, increasing β-cell funtion and β-cell mass, and/or for restoring glucose sensitivity to β-cells. The treatment with a peptide according to the present invention may also be combined with combined with a second or more pharmacologically active substances, e.g. selected from antidiabetic agents, antiobesity agents, appetite regulating agents, antihypertensive agents, agents for the treatment and/or prevention of complications resulting from or associated with diabetes and agents for the treatment and/or prevention of complications and disorders resulting from or associated with obesity. In the present context the expression

"antidiabetic agent" includes compounds for the treatment and/or prophylaxis of insulin resistance and diseases wherein insulin resistance is the pathophysiological mechanism.

Examples of these pharmacologically active substances are : Insulin, GLP-1 agonists, sulphonylureas (e.g. tolbutamide, glibenclamide, glipizide and gliclazide), biguanides e.g. metformin, meglitinides, glucosidase inhibitors (e.g. acorbose), glucagon antagonists, DPP- IV (dipeptidyl peptidase-IV) inhibitors, inhibitors of hepatic enzymes involved in stimulation of gluconeogenesis and/or glycogenosis, glucose uptake modulators, thiazolidinediones such as troglitazone and ciglitazone, compounds modifying the lipid metabolism such as anti- hyperlipidemic agents as HMG CoA inhibitors (statins), compounds lowering food intake, RXR agonists and agents acting on the ATP-dependent potassium channel of the β-cells, e.g. glibenclamide, glipizide, gliclazide and repaglinide; Cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol, dextrothyroxine, neteglinide, repaglinide; β-blockers such as alprenolol, atenolol, timolol, pindolol, propranolol and metoprolol, ACE (angiotensin converting enzyme) inhibitors such as benazepril, captopril, enalapril, fosinopril, lisinopril, alatriopril, quinapril and ramipril, calcium channel blockers such as nifedipine, felodipine, nicardipine, isradipine, nimodipine, diltiazem and verapamil, and α- blockers such as doxazosin, urapidil, prazosin and terazosin; CART (cocaine amphetamine regulated transcript) agonists, NPY (neuropeptide Y) antagonists, MC4 (melanocortin 4) agonists, orexin antagonists, TNF (tumor necrosis factor) agonists, CRF (corticotropin releas- ing factor) agonists, CRF BP (corticotropin releasing factor binding protein) antagonists, uro- cortin agonists, β3 agonists, MSH (melanocyte-stimulating hormone) agonists, MCH (melanocyte-concentrating hormone) antagonists, CCK (cholecystokinin) agonists, serotonin re-uptake inhibitors, serotonin and noradrenaline re-uptake inhibitors, mixed serotonin and noradrenergic compounds, 5HT (serotonin) agonists, bombesin agonists, galanin antagonists, growth hormone, growth hormone releasing compounds, TRH (thyreotropin releasing hormone) agonists, UCP 2 or 3 (uncoupling protein 2 or 3) modulators, leptin agonists, DA agonists (bromocriptin, doprexin), lipase/amylase inhibitors, RXR (retinoid X receptor) modulators, TR β agonists; histamine H3 antagonists.

It should be understood that any suitable combination of the peptides according to the invention with one or more of the above-mentioned compounds and optionally one or more further pharmacologically active substances are considered to be within the scope of the present invention.

The present invention is further illustrated by the following examples which, how- ever, are not to be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples may, both separately and in any combination thereof, be material for realising the invention in diverse forms thereof.

EXAMPLES

Example 1 Biological Potency and Chemical Stability of Peptides having the sequences shown in SEQ ID NOs 6-9.

The biological potency was measured in a cAMP formation assay for GLP-1 receptor agonist activity as described herein and the chemical stability was measured in the DPP-IV degradation assay described herein. For comparison, the same assays were run on exendin-4 and GLP-1 (7-37). The results were as follows:

The data on the two sequences shown as SEQ ID NOs: 8 and 9 are as follows.

Affinity (nM) Potency (pM) Ty₂ ICjphigh IC50I0W ECso hours

H. sapiens GLP-I 0.4 0.9 36 (n>30) 1.6

H. suspectum Exendin-4 0.5 0.2 55 (n>20) >24

X. laevis GLP-IA 1.7 7.3 41±1 10 X. laevis GLP-IB 0.2 0.1 16±4 0.3

X /αev/s GLP-IC 0.2 0.3 24±7 0.3

SEQ ID NO: 8 0.2 0.3 11±7 2.2

SEQ ID NO: 9 02 02 31±2 L8

TABLE 1 : The receptor affinity, potency and half-life (T ¹A) in a DPP-IV stability assay of

the tested peptides are shown. Two or more measures have been made for the potency and the data is presented as the mean plus the standard deviation. T¹A is calculated from the average of two independent measures.

The data on SEQ ID NOs 6 and 7 are shown below.

TABLE 2: The receptor affinity, potency and half-life (T V₂) of the peptides in an in vitro DPP-IV stability are shown. Two or more measures have been made for the potency and the data is presented as the mean plus the standard deviation. TV₂ is calculated from the average of two or more independent measures. Affinity (nM) Potency (pM) VΛ IC^nhiqh [C₅₀I0W EC₅₀ hours

H. sapiens GLP-1 0.4 0.9 36 (n>30) 1.6

H. suspectum Exendin-4 0.5 0.2 55 (n>20) >24

X. laevis GLP-IA 1.7 7.3 41±1 10 SEQ ID NO: 6 0.3 0.2 40±18 10

SEQ ID NO: 7 05 L9 66±4 10

Example 2

Affinity and potency for the human GLP-I receptor for C. pyrrhogaster GLP-I and variations of hGLP-1 (7-37) based on the mutations observed in C. pyrrhogaster are shown. The potency is given as the average (± the standard deviation) of n independent measures.

Affinity (nM) Potency (pM)

ICsπhiεh IC₅₀I0W ECsn H. sapiens GLP-I 0.4 0.9 36 (n>30)

C. pyrrhogaster GLP-I 0.2 0.4 37±34

Ui 0.8±0.3 2.1±0.8 68±49 (n=3)

K₂₂ 0.1 0.2 43±24 (n=4)

Li₂, K₂₂ 0.8±0.1 1.7±0.2 67±41 (n=3)

Claims

1. A peptide comprising an amino acid sequence of formula I:

Xaa₇-Xaa₈-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Ala-

Xaa25-Xaa26-Xaa27-Phe-lle-Xaa30-Trp-Leu-Xaa33-Xaa34-Xaa35-Xaa36-Xaa₃7-Xaa38-Xaa39-Xaa40-

Xaa4i-Xaa42-Xaa43-Xaa44-Xaa45-Xaa46

Formula (I) (SEQ ID No: 1 ) wherein

2-pyridylalanine or 4-pyridylalanine;

Xaa₁₆ is VaI or Leu;

Xaa₁₈ is Ser, Lys or Arg;

Xaa₂o is Leu or Met;

Xaa₂₂ is GIy, GIu or Aib;

Xaa₂₃ is GIn, GIu, Lys or Arg;

Xaa₂₅ is Ala or VaI;

Xaa₂₆ is Lys, GIu or Arg; Xaa₂₇ is GIu or Leu;

Xaa₃₀ is Ala, GIu or Arg;

Xaa₃₃ is VaI or Lys;

Xaa₃₄ is Lys, GIu, Asn or Arg;

Xaa₃₅ is GIy or Aib; Xaa₃₆ is Arg, GIy or Lys;

Xaa₃₇ is GIy, Ala, GIu, Pro, Lys, amide or is absent;

Xaa₃₈ is Lys, Ser, amide or is absent.

Xaa₃₉ is Ser, Lys, amide or is absent;

Xaa₄₀ is GIy, amide or is absent; Xaa₄₁ is Ala, amide or is absent;

Xaa₄₂ is Pro, amide or is absent; Xaa₄₃ is Pro, amide or is absent; Xaa₄₄ is Pro, amide or is absent; Xaa₄₅ is Ser, amide or is absent; Xaa₄₆ is amide or is absent ; and provided that if Xaa₃₈, Xaa₃₉, Xaa₄₀, Xaa₄₁, Xaa₄₂, Xaa₄₃, XaB₄₄, Xaa₄₅ or Xaa₄₆ is absent then each amino acid residue downstream is also absent and where the total number of basic residues (Lys residues÷ Arg residues) in amino acids 7-37 is greater than the total number of acidic residues (Asp residues + GIu residues) in amino acids 7-37.

2. A peptide comprising amino acid sequence of formula (IV)

Xaa₇-Xaa₈- Xaa₉-Gly-Thr- Phe-Thr-Ser-Asp-Xaa₁₆- Xaa₁₇-Xaa₁₈-Gln-Leu- Xaa₂i-Xaa₂₂-Xaa₂₃-Ala- Xaa25-Xaa26-Xaa2₇-Phe-lle-Xaa3o-Trp-Leu-Xaa33-Asn-Xaa35-Xaa36-Xaa₃₇-Xaa38-Xaa39-Xaa₄o- Xaa₄-|-Xaa₄₂-Xaa₄₃-Xaa₄₄-Xaa₄₅~Xaa₄Q

Formula (IV) (SEQ ID No: 4)

Xaa₇ is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, N^α-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;

Xaa₉ is GIu or Asp; Xaa₁₆ is VaI or Leu;

Xaa₁₇ is Ser or Thr;

Xaa₁₈ is Ser, Lys or Arg;

Xaa₂₂ is GIy, GIu or Aib; Xaa₂₃ is GIn, Lys or Arg;

Xaa₂₅ is Ala or VaI;

Xaa₂₆ is Lys, or Arg;

Xaa₂₇ is GIu or Leu;

Xaa₃₀ is Ala, GIu or Arg; Xaa₃₃ is VaI or Lys;

XaB₃₄ is Lys, GIu, Asn or Arg; Xaa₃₅ is GIy or Aib;

Xaa₃₆ is Arg, GIy or Lys;

Xaa₃₇ is GIy, Ala, GIu, Pro, Lys, amide or is absent;

Xaa₃₈ is Lys, Ser, amide or is absent. Xaa₃₉ is Ser, Lys, amide or is absent;

Xaa₄₀ is GIy, amide or is absent;

Xaa₄₁ is Ala, amide or is absent;

Xaa₄₂ is Pro, amide or is absent;

Xaa₄₃ is Pro, amide or is absent; Xaa₄₄ is Pro, amide or is absent;

Xaa₄₅ is Ser, amide or is absent;

Xaa₄₆ is amide or is absent ; where if Xaa₃₈, Xaa₃₉, Xaa₄₀, Xaa₄₁, Xaa₄₂, Xaa₄₃, Xaa₄₄, Xaa₄₅ or Xaa₄₆ is absent then each amino acid residue downstream is also absent.

3. A peptide comprising an amino acid sequence of formula (V)

Xaa₇-Xaa₈-Glu-Gly-Thr- Leu-Thr-Ser-Asp-Xaa₁₆- Xaa₁₇-Xaa₁₈-Xaa₁₉-Xaa2o- Xaa₂i-Xaa22-Xaa₂₃- Ala-Xaa₂₅-Xaa₂₆-Xaa₂₇-Phe-lle-Xaa₃o-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉- Xa a₄o~Xaa₄ -i -Xa a₄₂-Xaa₄₃-Xa 3₄₄-Xaa₄₅-Xa a₄g

Formula (V) (SEQ ID No: 5)

2-pyridylalanine or 4-pyridylalanine;

Xaa₁₇ is Ser or Thr;

Xaa₁₈ is Ser, Lys or Arg;

Xaa₁₉ is Tyr or GIn;

Xaa₂o is GIu or Asp;

Xaa₂₂ is GIy, GIu or Aib or Lys; Xaa₂₃ is GIn, GIu, Lys or Arg;

Xaa₂₅ is Ala or VaI;

Xaa₂₆ is Lys, GIu or Arg;

Xaa₂₇ is GIu or Leu; Xaa₃₀ is Ala, GIu, Asp or Arg;

Xaa₃₃ is VaI, Ne or Lys;

Xaa₃₄ is Lys, Asn or Arg;

Xaa₃₅ is GIy or Aib;

Xaa₃₈ is Lys, Ser, amide or is absent.

Xaa₃₉ is Ser, Lys, amide or is absent;

Xaa₄₀ is GIy, amide or is absent;

Xaa₄₁ is Ala, amide or is absent; Xaa₄₂ is Pro, amide or is absent;

Xaa₄₃ is Pro, amide or is absent;

Xaa₄₄ is Pro, amide or is absent;

Xaa₄₅ is Ser, amide or is absent;

4. A peptide according to claim 3, where Xaa₂₂ is Lys.

5. A peptide according to claim 1 , said peptide comprising an amino acid sequence of formula

(II):

Xaa₇-Xaa₈-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Xaa₁₈-Tyr-Leu-Glu-Xaa₂₂-Xaa₂₃-Ala-Ala-Xaa₂₆-

Glu-Phe-lle-Xaa₃o-Trp-Leu-Val-Xaa₃₄-Xaa₃5-Xaa₃6-Xaa₃₇-Xaa₃8

Formula (II) (SEQ ID No: 2) wherein

2-pyridylalanine or 4-pyridylalanine;

Xaa₈ is Ala, GIy, VaI, Leu, lie, Lys, Aib, (1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl) carboxylic acid, (1- aminocycloheptyl) carboxylic acid, or (1-aminocyclooctyl) carboxylic acid; Xaa₁₈ is Ser, Lys or Arg; Xaa₂₂ is GIy, GIu or Aib; Xaa₂₃ is GIn, GIu, Lys or Arg; Xaa₂₆ is Lys, GIu or Arg; Xaa₃₀ is Ala, GIu or Arg; Xaa₃₄ is Lys, GIu or Arg; Xaa₃₅ is GIy or Aib; Xaa₃₆ is Arg or Lys; Xaa₃₇ is GIy, Ala, GIu or Lys; and Xaa₃₈ is Lys, amide or is absent.

6. A peptide according to claim 1 wherein said peptide has an amino acid sequence of formula (III): His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Xaa₁₈-Tyr-Xaa₂₀-Glu-Gly-Xaa₂₃-Ala-Val-Xaa₂₆-

Leu-Phe-lle-Ala-Trp-Leu-Xaass-Xaa^-Gly-Xaase-Gly-Xaass

Formula (III) (SEQ ID No: 3) where

Xaa₁₈ is Lys or Arg;

Xaa₂₀ is Leu or Met; Xaa₂₃ is GIn or Asn;

Xaa₂₆ is Lys or Arg;

Xaa₃₃ is Lys or Arg

Xaa₃₄ is Lys or Arg;

Xaa₃₆ is Arg or Lys; Xaa₃₈ is Lys, amide or is absent.

7. A peptide according to claim 6, wherein said peptide comprises an amino acid sequence of formula (IV): His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Tyr-Met-Glu-Gly-Gln-Ala-Val-Arg-Leu-Phe- Ile-Ala-Trp-Leu-Lys-Lys-Gly-Arg-Gly-Xaa₃₈

Formula (Vl) (SEQ ID No: 6) where Xaa₃₈ is amide or is absent.

8. A peptide comprising an amino acid sequence of formula (VII) (SEQ ID NO:7):

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Gln-Leu-Glu-Glu-Gln-Ala-Ala- Lys-Glu-Phe-lle-Glu-Trp-Leu-Val-Asn-Gly-Gly-Pro-Ser-Xaa₃₉

where Xaa₃₉ is amide or is absent.

9. A peptide according to claims 2-3, wherein the total number of basic residues (Lys residues÷ Arg residues) in amino acids 7-37 is greater than the total number of acidic residues (Asp residues + GIu residues) in amino acids 7-37.

10. A peptide according to claims 1-3, wherein said peptide has an isoelectric point of greater than 7.

11. A peptide according to claims 1-3, wherein said peptide has an isoelectric point of greater than 8

12. A peptide according to claims 1-3, wherein said peptide has an isoelectric point of greater than 9

13. A peptide according to claims 1-3, wherein said peptide has an isoelectric point of greater than 10

14. A peptide according to claims 1-3, wherein the C terminal amino acid of said peptide is Xaa₃₇ or Xaa₃₈.

15. A peptide according to claims 1-3, wherein the amino acid sequence of said peptide comprises no more than eight amino acid residues which have been exchanged, added or deleted as compared to GLP-1 (7-37).

16. A peptide according to claims 1-3, wherein the amino acid sequence of said peptide comprises no more than six amino acid residues which have been exchanged, added or deleted as compared to GLP-1 (7-37).

17. A peptide according to claims 1-3, wherein the amino acid sequence of said peptide com- prises no more than four amino acid residues which have been exchanged, added or deleted as compared to GLP-1 (7-37).

18. A peptide according to claims 1-3, wherein the amino acid sequence of said peptide comprises no more than 4 amino acid residues which are not encoded by the genetic code.

19. A peptide comprising an amino acid sequence of formula VIII (SEQ ID NO: 8)

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Thr-GIn-Tyr-Leu-Glu-Glu-Lys-Ala-Ala- Lys-Glu-Phe-lle-Asp-Trp-Leu-Val-Asn-Gly-Arg-Pro-Lys-Xaa₃₉

where Xaa₃₉ is amide or is absent.

20. A peptide comprising an amino acid sequence of formula IX (SEQ ID NO: 9) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Thr-Ser-Tyr-Leu-Glu-Glu-Lys-Ala-Ala- Lys-Glu-Phe-lle-Asp-Trp-Leu-lle-Lys-Gly-Arg-Pro-Lys-Xaa₃₉

where Xaa₃₉ is amide or is absent.

21. A pharmaceutical composition comprising a peptide according to claims 1-3, 8, 19 and 20 and a pharmaceutically acceptable excipient.

22. The pharmaceutical composition according to claim 21 , which is suited for oral administration.

23. The pharmaceutical composition according to claim 21 , which is suited for pulmonary administration.

24. A method for treating a disease or disorder selected from the group consisting of hypergly- cemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive disorders, atheroschlerosis, myocardial infarction, and coronary heart disease, said method comprising administering to a subject in need of such treatment an effective amount of the pharmaceutical composition of claim 21.

25. A method for decreasing food intake, decreasing β-cell apoptosis, increasing β-cell function and β-cell mass, and/or for restoring glucose sensitivity to β-cells, said method comprising administering to said subject an effective amount of the pharmaceutical composition of claim 21.