WO2006097535A2

WO2006097535A2 - Peptide agonists of the glucagon family with secretin like activity

Info

Publication number: WO2006097535A2
Application number: PCT/EP2006/060853
Authority: WO
Inventors: Leif Christensen; Ingrid Pettersson; János Tiborg KODRA
Original assignee: Novo Nordisk A/S
Priority date: 2005-03-18
Filing date: 2006-03-20
Publication date: 2006-09-21
Also published as: WO2006097535A3

Abstract

Peptide agonists of the glucagon family with secretin like activity and their therapeutic use.

Description

PEPTIDE AGONISTS OF THE GLUCAGON FAMILY WITH SECRETIN LIKE ACTIVITY

The invention related to the filed of therapeutic peptides, i.e. to new peptides of the glucagon family.

BACKGROUND OF THE INVENTION

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. Since 1992 a number of peptides have been isolated from the venom of the GiIa monster lizards (Heloderma suspectum and Heloderma 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.

A range of different approaches have been used for modifying the structure of glucagon-like peptide 1 (GLP-1 ) compounds in order to provide a longer duration of action in vivo. WO 96/29342 discloses peptide hormone derivatives wherein the parent peptide hormone has been modified by introducing a lipophilic substituent in the C-terminal amino acid residue or in the N-terminal amino acid residue.

WO 98/08871 discloses GLP-1 derivatives wherein at least one amino acid residue of the parent peptide has a lipophilic substituent attached. WO 99/43708 discloses GLP-1 (7-35) and GLP-1 (7-36) derivatives which have a lipophilic substituent attached to the C-terminal amino acid residue. WO 00/34331 discloses acylated GLP-1 analogs.

WO 00/6991 1 discloses activated insulinotropic peptides to be injected into patients where they are supposed to react with blood components to form conjugates and thereby alledgedly providing longer duration of action in vivo.

WO 02/46227 discloses GLP-1 and exendin-4 analogs fused to human serum albumin in order to extend in vivo half-life.

Many diabetes patients particularly in the type 2 diabetes segment are subject to so-called "needle-phobia", i.e. a substantial fear of injecting themselves. In the type 2 diabetes segment most patients are treated with oral hypoglycaemic agents, and since GLP-1 compounds are expected to be the first injectable product these patients will be administered, the fear of injections may become a serious obstacle for the widespread use of the clinically very promising GLP-1 compounds. Thus, there is a need to develop new GLP-1 compounds which can be administered less than once daily, e.g. once every second or third day preferably once weekly, while retaining an acceptable clinical profile.

SUMMARY OF THE INVENTION

The present invention provides peptide analogs of the glucagon family having a non- conservative substitution in the N-terminal without loss of biological activity.

The present invention also provides a compound comprising the amino acid sequence of formula I :

XaarXaa₂-His-Gly- Xaa₅-Phe- Xaa₇- Xaa₈- Xaa₉-Xaai₀- Xaaii-Xaai₂-Xaai₃-Xaai4- Xaai₅- Xaa-|6-Xaa-|7" Xaa-i8-Xaa-ιg-Xaa2o-Xaa2i-r he- Xaa23-Xaa24- 1 rp- Xaa26-Xaa27-Xaa28-Xaa29- Xaa3o"Xaa3i -Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa3g-Xaa4o -Xaa4i — Xaa42 Formula (I) (SEQ. ID No: 1 )

wherein

Xaai is L-histidine, D-histidine, desamino-histidine, 2-amino-3-(2-aminoimidazol-4- yl)propionic acid, β-hydroxy-histidine, homohistidine, N^α-acetyl-histidine, α-fluoromethyl- histidine, α-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine; or L- tyrosine Xaa₂ is Ala, GIy, VaI, Leu, He, Lys, Aib, 1 -aminocyclopropanecarboxylic acid, 1 - aminocyclobutanecarboxylic acid, 1 -aminocyclopentanecarboxylic acid, 1 - aminocyclohexanecarboxylic acid, 1 -aminocycloheptanecarboxylic acid, or 1 - aminocyclooctanecarboxylic acid; Xaa₅ is Thr or Ser;

Xaa₇ is Thr or Ser;

Xaa₈ is Ser or Asp;

Xaa₉ is GIu or Asp;

Xaaio is VaI, Met, Leu or Tyr; Xaan is Ser, or Asn;

Xaa_i2 is Ser, Thr, Lys or He;

Xaai3 is Tyr, He, Ala or GIn;

Xaa_i4 is Leu or Met;

Xaais is Asp or GIu; Xaaie is GIy, Asn, GIu or Lys;

Xaaiy is Leu, GIn, GIu or He;

Xaais is Ala or His;

Xaaig is Ala, GIn or VaI;

Xaa₂₀ is Lys, Arg or GIn;

Xaa₂₃ is He or VaI;

Xaa₂₄ is Ala, Asn or GIu;

Xaa₂₆ is Leu or He;

Xaa₂₇ is VaI, He, Leu, Arg or Lys; Xaa₂₈ is Lys, GIn, Ala or Asn;

Xaa₂₉ is GIy, Thr or GIn;

Xaa₃₀ is Arg, Lys or GIy;

Xaa₃i is He, GIy, Pro, amide or is absent;

Xaa₃₂ is Thr, Lys, Ser, amide or is absent; Xaa₃₃ is Asp, Lys, Ser, amide or is absent;

Xaa₃₄ is Arg, Asn, GIy, amide or is absent;

Xaa₃₅ is Asp, Ala, amide or is absent ;

Xaa₃₆ is Trp, Pro, amide or is absent;

Xaa₃₇ is Lys, Pro, amide or is absent; Xaa₃₈ is His, Pro, amide or is absent;

Xaa₃₉ is Asn, Ser, amide or is absent; Xaa₄o is He, amide or is absent; Xaa₄i is Thr, amide or is absent; Xaa₄₂ is GIn, amide or is absent; provided that if Xaa₃i, Xaa₃₂, Xaa₃₃, Xaa₃₄, Xaa₃₅, Xaa₃₆, Xaa₃₇, Xaa₃₈, Xaa₃₉, Xaa₄₀, Xaa₄i, or Xaa₄₂ is absent then each amino acid residue downstream is also absent.

The present invention also provides pharmaceutical compositions comprising a compound according to the present invention and the use of compounds according to the present invention for preparing medicaments for treating disease.

DESCRIPTION OF THE INVENTION

In the present specification, the following terms have the indicated meaning :

The term "polypeptide" and "peptide" as used herein means a compound composed of at least five 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 they may 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, γ-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, anthranilic acid.

The term "analogue" as used herein referring to a polypeptide means a modified peptide wherein one or more amino acid residues of the peptide have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the peptide and/or wherein one or more amino acid residues have been deleted from the peptide and or wherein one or more amino acid residues have been added to the peptide. Such addition or deletion of amino acid residues can take place at the N-terminal of the peptide and/or at the C-terminal of the peptide. A simple system is often used to describe analogues : For example [Arg³⁴]GLP-1 (7-37)Lys designates a GLP-1 (7-37) analogue wherein the naturally occuring lysine at position 34 has been substituted with arginine and wherein a lysine has been added to the terminal amino acid residue, i.e. to the GIy³⁷. All amino acids for which the optical isomer is not stated is to be understood to mean the L-isomer.

The term "derivative" as used herein in relation to a peptide 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, esters and the like. An example of a derivative of GLP-1 (7-37) is N^ε26-((4S)-4-(hexadecanoylamino)- butanoyl)[Arg³⁴, Lys²⁶]GLP-1 -(7-37).

The term "insulinotropic agent" as used herein means a compound which is an agonist of the human GLP-1 receptor, i.e. a compound which stimulates the formation of cAMP in a suitable medium containing the human GLP-1 receptor (one such medium disclosed below). The potency of an insulinotropic agent 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- 467-12A) were grown in DMEM media with the addition of 100 ILVmL penicillin, 100 μg/mL streptomycin, 5% fetal calf serum and 0.5 mg/mL Geneticin G-418 (Life Technologies). The cells were washed twice in phosphate buffered saline and harvested with Versene. Plasma membranes were prepared from the cells by homogenisation with an Ultraturrax in buffer 1 (20 mM HEPES-Na, 10 mM EDTA, pH 7.4). The homogenate was centrifuged at 48,000 x g for 15 min at 4⁰C. The pellet was suspended by homogenization in buffer 2 (20 mM HEPES-Na, 0.1 mM EDTA, pH 7.4), then centrifuged at 48,000 x g for 15 min at 4⁰C. The washing procedure was repeated one more time. The final pellet was suspended in buffer 2 and used immediately for assays or stored at -8O⁰C.

The functional receptor assay was carried out by measuring cyclic AMP (cAMP) as a response to stimulation by the insulinotropic agent. cAMP formed was quantified by the AlphaScreen™ cAMP Kit (Perkin Elmer Life Sciences). Incubations were carried out in half- area 96-well microtiter plates in a total volume of 50 μL buffer 3 (50 mM Tris-HCI, 5 mM HEPES, 10 mM MgCI₂, pH 7.4) and with the following addiditions: 1 mM ATP, 1 μM GTP, 0.5 mM 3-isobutyl-1 -methylxanthine (IBMX), 0.01 % Tween-20, 0.1 % BSA, 6 μg membrane preparation, 15 μg/mL acceptor beads, 20μg/mL donor beads preincubated with 6 nM biotinyl-cAMP. Compounds to be tested for agonist activity were dissolved and diluted in buffer 3. GTP was freshly prepared for each experiment. The plate was incubated in the dark with slow agitation for three hours at room temperature followed by counting in the Fusion™ instrument (Perkin Elmer Life Sciences). Concentration-response curves were plotted for the individual compounds and EC₅₀ values estimated using a four-parameter logistic model with

Prism v. 4.0 (GraphPad, Carlsbad, CA).

The term "GLP-1 peptide" as used herein means GLP-1 (7-37) (SEQ ID No 1 ), a

GLP-1 (7-37) analogue, a GLP-1 (7-37) derivative or a derivative of a GLP-1 (7-37) analogue. In one embodiment the GLP-1 peptide is an insulinotropic agent. The term "exendin-4 peptide" as used herein means exendin-4(1 -39) (SEQ ID No 2), an exendin-4(1 -39) analogue, an exendin-4(1 -39) derivative or a derivative of an exendin- 4(1 -39) analogue. In one embodiment the exendin-4 peptide is an insulinotropic agent. The term "DPP-IV protected" as used herein referring to a polypeptide means a polypeptide which has been chemically modified in order to render said compound resistant to the plasma peptidase dipeptidyl aminopeptidase-4 (DPP-IV). The DPP-IV enzyme in plasma is known to be involved in the degradation of several peptide hormones, e.g. GLP-1 , GLP-2, Exendin-4 etc. Thus, a considerable effort is being made to develop analogues and derivatives of the polypeptides susceptible to DPP-IV mediated hydrolysis in order to reduce the rate of degradation by DPP-IV. In one embodiment a DPP-IV protected peptide is more resistant to DPP-IV than GLP-1 (7-37) or Exendin-4(1 -39).

Resistance of a peptide to degradation by dipeptidyl aminopeptidase IV is determined by the following degradation assay : Aliquots of the peptide (5 nmol) are incubated at 37 ⁵C with 1 μl_ of purified dipeptidyl aminopeptidase IV corresponding to an enzymatic activity of 5 mU for 10-180 minutes in 100 μl_ of 0.1 M triethylamine-HCI buffer, pH 7.4. Enzymatic reactions are terminated by the addition of 5 μl_ of 10% trifluoroacetic acid, and the peptide degradation products are separated and quantified using HPLC analysis. One method for performing this analysis is : The mixtures are applied onto a Vydac C18 widepore (30 nm pores, 5 μm particles) 250 x 4.6 mm column and eluted at a flow rate of 1 ml/min with linear stepwise gradients of acetonitrile in 0.1 % trifluoroacetic acid (0% acetonitrile for 3 min, 0-24% acetonitrile for 17 min, 24-48% acetonitrile for 1 min) according to Siegel et al., Regul. Pept. 1999;79:93-102 and Mentlein et al. Eur. J. Biochem. 1993;214:829-35. Peptides and their degradation products may be monitored by their absorbance at 220 nm (peptide bonds) or 280 nm (aromatic amino acids), and are quantified by integration of their peak areas related to those of standards. The rate of hydrolysis of a peptide by dipeptidyl aminopeptidase IV is estimated at incubation times which result in less than 10% of the peptide being hydrolysed.

The term "Ci-₆-alkyl" as used herein means a saturated, branched, straight or cyclic hydrocarbon group having from 1 to 6 carbon atoms. Representative examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, terf-butyl, n- pentyl, isopentyl, neopentyl, terf-pentyl, n-hexyl, isohexyl, cyclohexane and the like. The term "pharmaceutically acceptable" as used herein means suited for normal pharmaceutical applications, i.e. giving rise to no adverse events in patients etc. The term "excipient" as used herein means the chemical compounds which are normally added to pharmaceutical compositions, e.g. buffers, tonicity agents, preservatives and the like.

The term "effective amount" as used herein means a dosage which is sufficient to be effective for the treatment of the patient compared with no treatment.

The term "pharmaceutical composition" as used herein means a product comprising an active compound or a salt thereof together with pharmaceutical excipients such as buffer, preservative, and optionally a tonicity modifier and/or a stabilizer. Thus a pharmaceutical composition is also known in the art as a pharmaceutical formulation. The term "treatment of a disease" as used herein means the management and care of a patient having developed the disease, condition or disorder. The purpose of treatment is to combat the disease, condition or disorder. Treatment includes the administration of the active compounds to eliminate or control the disease, condition or disorder as well as to alleviate the symptoms or complications associated with the disease, condition or disorder. In one aspect the present invention relates to a compound comprising the amino acid sequence of formula I :

XaarXaa₂-His-Gly- Xaa₅-Phe- Xaa₇- Xaa₈- Xaa₉-Xaai₀- Xaaii-Xaai₂-Xaai3-Xaai₄- Xaai₅- Xaa-|6-Xaa-|7" Xaa-i8-Xaa-ιg-Xaa₂o-Xaa₂i-Phe- Xaa₂3-Xaa₂₄-Trp- Xaa₂6-Xaa₂7-Xaa₂8-Xaa₂9- Xaa3o"Xaa3i -Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa3g-Xaa4o -Xaa4i — Xaa42 Formula (I) (SEQ. ID No: 1 )

wherein

Xaai is L-histidine, D-histidine, desamino-histidine, 2-amino-3-(2-aminoimidazol-4- yl)propionic acid, β-hydroxy-histidine, homohistidine, N^α-acetyl-histidine, α-fluoromethyl- histidine, α-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine; or L- tyrosine

Xaa₂ is Ala, GIy, VaI, Leu, He, Lys, Aib, 1 -aminocyclopropanecarboxylic acid, 1 - aminocyclobutanecarboxylic acid, 1 -aminocyclopentanecarboxylic acid, 1 - aminocyclohexanecarboxylic acid, 1 -aminocycloheptanecarboxylic acid, or 1 - aminocyclooctanecarboxylic acid;

Xaa₅ is Thr or Ser;

Xaa₇ is Thr or Ser;

Xaa₈ is Ser or Asp;

Xaa₉ is GIu or Asp; Xaaio is VaI, Met, Leu or Tyr;

Xaan is Ser, or Asn; Xaai₂ is Ser, Thr, Lys or He;

Xaai3 is Tyr, He, Ala or GIn;

Xaais is Asp or GIu; Xaaie is GIy, Asn, GIu or Lys;

Xaaiy is Leu, GIn, GIu or He;

Xaais is Ala or His;

Xaaig is Ala, GIn or VaI;

Xaa₂₀ is Lys, Arg or GIn;

Xaa₂₃ is He or VaI;

Xaa₂₄ is Ala, Asn or GIu;

Xaa₂₆ is Leu or He;

Xaa₂₇ is VaI, He, Leu, Arg or Lys; Xaa₂₈ is Lys, GIn, Ala or Asn;

Xaa₂₉ is GIy, Thr or GIn;

Xaa₃₀ is Arg, Lys or GIy;

Xaa₃i is He, GIy, Pro, amide or is absent;

Xaa₃₄ is Arg, Asn, GIy, amide or is absent;

Xaa₃₅ is Asp, Ala, amide or is absent ;

Xaa₃₆ is Trp, Pro, amide or is absent;

Xaa₃₉ is Asn, Ser, amide or is absent;

Xaa₄₀ is He, amide or is absent;

Xaa₄i is Thr, amide or is absent;

Xaa₄₂ is GIn, amide or is absent; provided that if Xaa₃i, Xaa₃₂, Xaa₃₃, Xaa₃₄, Xaa₃₅, Xaa₃₆, Xaa₃₇, Xaa₃₈, Xaa₃₉, Xaa₄₀, Xaa_4i, or

Xaa₄₂ is absent then each amino acid residue downstream is also absent.

In another aspect the present invention relates to a compound wherein the amino acid sequence is according to formula 2 : His-Xaa₂-His-Gly- Xaa₅-Phe- Xaa₇- Xaa₈- Xaa₉-Xaai₀- Xaaii-Xaai₂-Xaai₃-Xaai4- Xaai₅-

Xaai6-Xaai7-Ala-Xaai9-Xaa₂o-Xaa₂i-Phe-lle-Xaa₂₄-Trp- Leu-Xaa₂7-Xaa₂8-Xaa₂9-Xaa₃o-Xaa3i-

Xaa3₂-Xaa33"Xaa3₄-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39

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

Xaa₂ is Ala, GIy, VaI, Leu, He, Lys, Aib, 1 -aminocyclopropanecarboxylic acid, 1 - aminocyclobutanecarboxylic acid, 1 -aminocyclopentanecarboxylic acid, 1 - aminocyclohexanecarboxylic acid, 1 -aminocycloheptanecarboxylic acid, or 1 - aminocyclooctanecarboxylic acid; Xaa₅ is Thr or Ser;

Xaa₇ is Thr or Ser;

Xaa₈ is Ser or Asp;

Xaa₉ is GIu or Asp;

Xaaio is VaI, Met, or Leu; Xaan is Ser or Asn;

Xaais is Asp or GIu;

Xaaiy is Leu, GIn or GIu;

Xaa₁₉ is Ala or VaI;

Xaa₂o is Lys or Arg;

Xaa₂₄ is Ala, Asn or GIu;

Xaa₂₇ is VaI, He or Lys;

Xaa₂s is Lys, GIn or Asn;

Xaa₂₉ is GIy or Thr;

Xaa₃₀ is Arg, Lys or GIy; Xaa₃i is He, Pro, amide or is absent;

Xaa₃₂ is Thr, Ser, amide or is absent;

Xaa₃₃ is Asp, Ser, amide or is absent;

Xaa₃₄ is Arg, GIy, amide or is absent;

Xaa₃₅ is Ala, amide or is absent ; Xaa₃₆ is Pro, amide or is absent;

Xaa₃₇ is Pro, amide or is absent; Xaa₃s is Pro, amide or is absent;

Xaa₃₉ is Ser, amide or is absent; provided that if Xaa₃i, Xaa₃₂, Xaa₃₃, Xaa₃₄, Xaa₃₅, Xaa₃₆, Xaa₃₇, Xaa₃₈, or Xaa₃₉ is absent then each amino acid residue downstream is also absent.

In antoher aspect the present invention relates to a compound wherein the amino acid sequence is according to formula 3 :

His-Xaa₂-His-Gly-Thr-Phe-Thr-Ser-Asp-Xaaio-Ser-Xaai₂-Xaai₃-Xaai₄-Glu-Xaai6-Xaai₇-Ala-

Xaai ₉-Xaa₂o-Xaa₂i - P he- 1 Ie-Xaa₂₄-Trp- Leu -Xaa₂7-Xaa₂8-G ly-Xaa₃₀-Xaa₃i -Xaa₃₂-Xaa₃₃-Xaa₃₄- Xaa₃5-Xaa₃6-Xaa₃7-Xaa₃8"Xaa₃g

Formula (3) (SEQ. ID. No : 3)

Xaaio is VaI or Leu;

Xaa_i2 is Ser or Lys;

Xaa_i4 is Leu or Met;

Xaaiy is GIn or GIu;

Xaa₁₉ is Ala or VaI;

Xaa₂₀ is Lys or Arg;

Xaa₂₄ is Ala or GIu;

Xaa₂₇ is VaI or Lys;

Xaa₂₈ is Lys or Asn;

Xaa₃₀ is Arg, Lys or GIy;

Xaa₃i is Pro, amide or is absent; Xaa₃₂ is Ser, amide or is absent;

Xaa₃₃ is Ser, amide or is absent;

Xaa₃₄ is GIy, amide or is absent;

Xaa₃₅ is Ala, amide or is absent ;

Xaa₃₆ is Pro, amide or is absent; Xaa₃₇ is Pro, amide or is absent;

Xaa₃₈ is Pro, amide or is absent; Xaa₃g is Ser, amide or is absent; provided that if Xaa₃i, Xaa₃₂, Xaa₃₃, Xaa₃₄, Xaa₃₅, Xaa₃₆, Xaa₃₇, Xaa₃₈, or Xaa₃₉ is absent then each amino acid residue downstream is also absent.

In one embodiment one or more of the amino acids residues have been modified by acylation.

In another embodiment an acyl group of a straight-chain, branched fatty acid or branched alkane α,ω-dicarboxylic acid is attached to one or more amino acid residues. In another embodiment the acyl group is selected from the group comprising CH₃(CH₂)_nCO-, wherein n is 4 to 38, such as CH₃(CH₂)₆CO-, CH₃(CH₂)₈CO-, CH₃(CH₂)₁₀CO-, CH₃(CH₂)₁₂CO- , CH₃(CH₂)₁₄CO-, CH₃(CH₂)₁₆CO-, CH₃(CH₂)₁₈CO-, CH₃(CH₂)₂₀CO- and CH₃(CH₂)₂₂CO- acid is attached to one or more amino acid residues.

In another embodiment the acyl group is selected from the group comprising HOOC(CH₂)_mCO-, wherein m is 4 to 38, such as HOOC(CH₂)₁₄CO-, HOOC(CH₂)₁₆CO-,

HOOC(CH₂)₁₈CO-, HOOC(CH₂)₂₀CO- and HOOC(CH₂)₂₂CO- acid is attached to one or more amino acid residues.

In another embodiment the acyl group is selected from the group comprising

CH₃(CH₂)_p((CH₂)_qCOOH)CHNH-CO(CH₂)₂CO-, wherein p and q are integers and p+q is an integer of from 8 to 40, such as from 12 to 35 acid is attached to one or more amino acid residues.

In another embodiment the acyl group is selected from CH₃(CH₂)_rCO-

NHCH(COOH)(CH₂)₂CO-, wherein r is an integer of from 10 to 24 acid is attached to one or more amino acid residues. In another embodiment an acyl group of the formula CH₃(CH₂)_SCO-NHCH((CH₂)₂COOH)CO-

, wherein s is an integer of from 8 to 24 acid is attached to one or more amino acid residues.

In another embodiment the acyl group is attached to an amino acid with a nuclophilic atom in the side chain, i.e. GLP-1 residue number 18, 22, 26, 34, 36, 37 or 38, GLP-2 residue number 10 or 17, Exendin-4 residue number 12, 16, 20, 32, 33 or 34.

In another embodiment the acyl group is attached to amino acid residue number 18 relative to GLP-1 (7-37) (SEQ ID No. 1 ). In another embodiment the acyl group is attached to amino acid residue number 22 relative to GLP-1 (7-37) (SEQ ID No. 1 ). In another embodiment the acyl group is attached to amino acid residue number 26 relative to GLP-1 (7-37) (SEQ ID No. 1 ). In another embodiment the acyl group is attached to amino acid residue number 34 relative to GLP-1 (7-37) (SEQ ID No. 1 ). In another embodiment the acyl group is attached to amino acid residue number 36 relative to GLP-1 (7-37) (SEQ ID No. 1 ). In another embodiment the acyl group is attached to amino acid residue number 37 relative to GLP-1 (7- 37) (SEQ ID No. 1 ). In another embodiment the acyl group is attached to amino acid residue number 38 relative to GLP-1 (7-37) (SEQ ID No. 1 ).

In another embodiment one or more of the amino acids residues have been modified by pegylation.

Another object of the present invention is to provide a pharmaceutical formulation comprising a compound according to the present invention which is present in a concentration from 0.1 mg/ml to 25 mg/ml, and wherein said formulation has a pH from 3.0 to 9.0. The formulation may further comprise a buffer system, preservative(s), tonicity 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 compound according to the present invention, and a buffer, wherein said compound is present in a concentration from 0.1 mg/ml or above, and wherein said formulation has a pH from about 3.0 to about 9.0.

In another embodiment of the invention the pH of the formulation is from about 7.0 to about 9.5. In another embodiment of the invention the pH of the formulation is from about 3.0 to about 7.0. In another embodiment of the invention the pH of the formulation is from about 5.0 to about 7.5. In another embodiment of the invention the pH of the formulation is from about 7.5 to about 9.0. In another embodiment of the invention the pH of the formulation is from about 7.5 to about 8.5. In another embodiment of the invention the pH of the formulation is from about 6.0 to about 7.5. In another embodiment of the invention the pH of the formulation is from about 6.0 to about 7.0. 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)-aminomethan, 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 C4-C8 hydrocarbon having at least one -OH group and includes, for example, mannitol, sorbitol, inositol, galactitol, 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 stabilizer. 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, following 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. 1 1 :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. Any stereoisomer (i.e., L, D, or a mixture thereof) of a particular amino acid (e.g. methionine, histidine,arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine and mixtures thereof) or combinations of these stereoisomers or imidazole, 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 naturally 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 cysteine analogues include S- methyl-L cysteine. 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 analogues 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 methionine 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 (L or D) 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 stabilizer 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), polyvinyl alcohol (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-thawing 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, lectins 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 sulphate or sodium lauryl sulphate), 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-1 -propyldimethylammonio-1 -propanesulfonate, cationic surfactants (quaternary 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.

In a further embodiment of the invention the formulation further comprises protease inhibitors such as EDTA (ethylenediamine tetraacetic acid) and benzamidineHCI, but other commercially available protease inhibitors may also be used. The use of a protease inhibitor is particular useful in pharmaceutical compositions comprising zymogens of proteases in order to inhibit autocatalysis.

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, gelatine 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 compound 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, ophthalmic 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 delivery system and advanced drug delivery system in order to further enhance stability of the compound of the present invention, 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 alcohol), acrylate and methacrylate polymers, polylactic and polyglycolic acid and block co-polymers 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 compounds of the present invention, using, for example a metered dose inhaler, dry powder inhaler and a nebulizer, all being devices well known to those skilled in the art. 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 limiting 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-crystallization, precipitation, co-precipitation, emulsification, dispersion, high pressure homogenisation, encapsulation, spray drying, microencapsulating, 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). Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal 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 solution or suspension for the administration of the compound of the present invention in the form of a nasal or pulmonal spray. As a still further option, the pharmaceutical compositions containing the compound of the invention can also be adapted to transdermal administration, e.g. by needle-free injection or from a patch, optionally an iontophoretic patch, or transmucosal, e.g. buccal, administration.

The compounds of the present invention can be administered via the pulmonary route in a vehicle, as a solution, suspension or dry powder using any of known types of devices suitable for pulmonary drug delivery. Examples of these comprise, but are not limited to, the three general types of aerosol-generating for pulmonary drug delivery, and may include jet or ultrasonic nebulizers, metered-dose inhalers, or dry powder inhalers (Cf. Yu J, Chien YW. Pulmonary drug delivery: Physiologic and mechanistic aspects. Crit Rev Ther Drug Carr Sys 14(4) (1997) 395-453). Based on standardised testing methodology, the aerodynamic diameter (d_a) of a particle is defined as the geometric equivalent diameter of a reference standard spherical particle of unit density (1 g/cm³). In the simplest case, for spherical particles, d_a is related to a reference diameter (d) as a function of the square root of the density ratio as described by:

Modifications to this relationship occur for non-spherical particles (cf. Edwards DA, Ben- Jebria A, Langer R. Recent advances in pulmonary drug delivery using large, porous inhaled particles. J Appl Physiol 84(2) (1998) 379-385). The terms "MMAD" and "MMEAD" are well- described and known to the art (cf . Edwards DA, Ben-Jebria A, Langer R and represents a measure of the median value of an aerodynamic particle size distribution. Recent advances in pulmonary drug delivery using large, porous inhaled particles. J Appl Physiol 84(2) (1998) 379-385). Mass median aerodynamic diameter (MMAD) and mass median effective aerodynamic diameter (MMEAD) are used inter-changeably, are statistical parameters, and empirically describe the size of aerosol particles in relation to their potential to deposit in the lungs, independent of actual shape, size, or density (cf. Edwards DA, Ben-Jebria A, Langer R. Recent advances in pulmonary drug delivery using large, porous inhaled particles. J Appl Physiol 84(2) (1998) 379-385). MMAD is normally calculated from the measurement made with impactors, an instrument that measures the particle inertial behaviour in air. In a further embodiment, the formulation could be aerosolized by any known aerosolisation technology, such as nebulisation, to achieve a MMAD of aerosol particles less than 10 μm, more preferably between 1 -5 μm, and most preferably between 1 -3 μm. The preferred particle size is based on the most effective size for delivery of drug to the deep lung, where protein is optimally absorbed (cf . Edwards DA, Ben-Jebria A, Langer A, Recent advances in pulmonary drug delivery using large, porous inhaled particles. J Appl Physiol 84(2) (1998) 379-385).

Deep lung deposition of the pulmonal formulations comprising the compound of the present invention may optional be further optimized by using modifications of the inhalation techniques, for example, but not limited to: slow inhalation flow (eg. 30 L/min), breath holding and timing of actuation.

The term "stabilized formulation" refers to a formulation with increased physical stability, increased chemical stability or increased physical and chemical stability.

The term "physical stability" of the protein formulation as used herein refers to the tendency of the protein to form biologically inactive and/or insoluble aggregates of the protein as a result of exposure of the protein to thermo-mechanical stresses and/or interaction with interfaces and surfaces that are destabilizing, such as hydrophobic surfaces and interfaces. Physical stability of the aqueous protein formulations is evaluated by means of visual inspection and/or turbidity measurements after exposing the formulation filled in suitable containers (e.g. cartridges or vials) to mechanical/physical stress (e.g. agitation) at different temperatures for various time periods. Visual inspection of the formulations is performed in a sharp focused light with a dark background. The turbidity of the formulation is characterized by a visual score ranking the degree of turbidity for instance on a scale from 0 to 3 (a formulation showing no turbidity corresponds to a visual score 0, and a formulation showing visual turbidity in daylight corresponds to visual score 3). A formulation is classified physical unstable with respect to protein aggregation, when it shows visual turbidity in daylight. Alternatively, the turbidity of the formulation can be evaluated by simple turbidity measurements well-known to the skilled person. Physical stability of the aqueous protein formulations can also be evaluated by using a spectroscopic agent or probe of the conformational status of the protein. The probe is preferably a small molecule that preferentially binds to a non-native conformer of the protein. One example of a small molecular spectroscopic probe of protein structure is Thioflavin T. Thioflavin T is a fluorescent dye that has been widely used for the detection of amyloid fibrils. In the presence of fibrils, and perhaps other protein configurations as well, Thioflavin T gives rise to a new excitation maximum at about 450 nm and enhanced emission at about 482 nm when bound to a fibril protein form. Unbound Thioflavin T is essentially non-fluorescent at the wavelengths.

Other small molecules can be used as probes of the changes in protein structure from native to non-native states. For instance the "hydrophobic patch" probes that bind preferentially to exposed hydrophobic patches of a protein. The hydrophobic patches are generally buried within the tertiary structure of a protein in its native state, but become exposed as a protein begins to unfold or denature. Examples of these small molecular, spectroscopic probes are aromatic, hydrophobic dyes, such as antrhacene, acridine, phenanthroline or the like. Other spectroscopic probes are metal-amino acid complexes, such as cobalt metal complexes of hydrophobic amino acids, such as phenylalanine, leucine, isoleucine, methionine, and valine, or the like.

The term "chemical stability" of the protein formulation as used herein refers to chemical covalent changes in the protein structure leading to formation of chemical degradation products with potential less biological potency and/or potential increased immunogenic properties compared to the native protein structure. Various chemical degradation products can be formed depending on the type and nature of the native protein and the environment to which the protein is exposed. Elimination of chemical degradation can most probably not be completely avoided and increasing amounts of chemical degradation products is often seen during storage and use of the protein formulation as well-known by the person skilled in the art. Most proteins are prone to deamidation, a process in which the side chain amide group in glutaminyl or asparaginyl residues is hydrolysed to form a free carboxylic acid. Other degradations pathways involves formation of high molecular weight transformation products where two or more protein molecules are covalently bound to each other through transamidation and/or disulfide interactions leading to formation of covalently bound dimer, oligomer and polymer degradation products (Stability of Protein Pharmaceuticals, Ahem. T.J. & Manning M. C, Plenum Press, New York 1992). Oxidation (of for instance methionine residues) can be mentioned as another variant of chemical degradation. The chemical stability of the protein formulation can be evaluated by measuring the amount of the chemical degradation products at various time-points after exposure to different environmental conditions (the formation of degradation products can often be accelerated by for instance increasing temperature). The amount of each individual degradation product is often determined by separation of the degradation products depending on molecule size and/or charge using various chromatography techniques (e.g. SEC-HPLC and/or RP-HPLC).

Hence, as outlined above, a "stabilized formulation" refers to a formulation with increased physical stability, increased chemical stability or increased physical and chemical stability. In general, a formulation must be stable during use and storage (in compliance with recommended use and storage conditions) until the expiration date is reached.

In one embodiment of the invention the pharmaceutical formulation comprising the compound of the present invention is stable for more than 6 weeks of usage and for more than 3 years of storage.

In another embodiment of the invention the pharmaceutical formulation comprising the compound of the present invention is stable for more than 4 weeks of usage and for more than 3 years of storage. In a further embodiment of the invention the pharmaceutical formulation comprising the compound of the present invention is stable for more than 4 weeks of usage and for more than two years of storage.

In an even further embodiment of the invention the pharmaceutical formulation comprising the compound of the present invention is stable for more than 2 weeks of usage and for more than two years of storage.

In another aspect the present invention relates to the use of a compound according to the invention for the preparation of a medicament.

In one embodiment a compound according to the invention is used for the preparation of a medicament for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive disorders, atheroschlerosis, myocardial infarction, stroke, coronary heart disease and other cardiovascular disorders, inflammatory bowel syndrome, dyspepsia and gastric ulcers.

In another embodiment a compound 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 a compound according to the invention is used for the preparation of a medicament 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.

The treatment with a compound according to the present invention may also be 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. Examples of these pharmacologically active substances are : Insulin, sulphonylureas, biguanides, meglitinides, glucosidase inhibitors, glucagon antagonists, DPP-IV (dipeptidyl peptidase-IV) inhibitors, inhibitors of hepatic enzymes involved in stimulation of gluconeogenesis and/or glycogenosis, glucose uptake modulators, compounds modifying the lipid metabolism such as antihyperlipidemic agents as HMG CoA inhibitors (statins), Gastric Inhibitory Polypeptides (GIP analogs), compounds lowering food intake, RXR agonists and agents acting on the ATP-dependent potassium channel of the β-cells; 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, PYY agonist, PYY2 agonists, PYY4 agonits, mixed PPY2/PYY4 agonists, MC4 (melanocortin 4) agonists, orexin antagonists, TNF (tumor necrosis factor) agonists, CRF (corticotropin releasing factor) agonists, CRF BP (corticotropin releasing factor binding protein) antagonists, urocortin 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, Gastric Inhibitory Polypeptide agonists or antagonists (GIP analogs), gastrin and gastrin analogs.

It should be understood that any suitable combination of the compounds 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, however, are not to be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples may, both separately and in any combination thereof, be material for realising the invention in diverse forms thereof.

EXAMPLES

In the present context the three-letter or one-letter indications of the amino acids have been used in their conventional meaning. Unless indicated explicitly, the amino acids mentioned herein are L-amino acids. Further, the left and right ends of an amino acid sequence of a peptide are, respectively, the N- and C-termini unless otherwise specified.

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

The following abbreviations are used: amu: atomic mass units.

DIPEA: diisopropylethylamine. DMF: N,N-Dimethylformamide.

HBTU: 2-(1 H-Benzotriazole-1 -yl)-1 ,1 ,3,3-tetramethyluronium hexafluoro phosphate.

HOAt: N-hydroxy-9-azabenzotriazole.

HPLC: High Performance Liquid Chromatography.

NMP: N-Methyl-2-pyrrolidone. PyBop: Benzotriazol-1 -yl-oxytripyrrolidinophosphonium hexafluorophosphate

TFA: Trifluoroacetic acid.

THF: Tetrahydrofuran.

TES: Triethylsilane

The compounds of the present can be synthesized by those skilled in the art using standard coupling and deprotection steps. A description of all necessary tools and synthetic methods can be found in "The Fine Art Of Solid Phase Synthesis", 2002/3 Catalog, Novabiochem. A typical example that include a cyclization step is as follows:

Radioligand binding to plasma membranes expressing the human GLP-1 receptor The binding assay was performed with purified plasma membranes containing the human GLP-1 receptor. The plasma membranes containing the receptors were purified from stably expressing BHK tk-ts 13 cells. The membranes were diluted in Assay Buffer (50 mM HEPES, 5 mM EGTA, 5 mM MgCI₂, 0.005% Tween 20, pH=7.4) to a final concentration of 0.2 mg/ml of protein and destributed to 96-well microtiter plates precoated with 0.3 % PEI. Membranes in the presence of 0.05 nM [¹²⁵I]GLP-I , unlabelled ligands in increasing concentrations and different HSA concentrations (0.005%, 0.05%, and 2%) were incubated 2 hr at 3O⁰C. After incubation, unbound ligands were separated from bound ligands by filtration through a vacuum-manifold followed by 2X100 μl washing with ice cold assaybuffer. The filters were dried overnight at RT, punched out and quantified in a γ-counter.

Example 1

[His3, LΘU10]-GLP-2(1 -33).

S F S D E L N T I L D N L A A R D F I N W L I Q T K I T D—_™

1 .a Synthesis of the peptidyl resin:

The protected peptidyl resin was synthesized according to the Fmoc strategy on an MultiSyntech Syro Il synthesizer in 53 micromol scale using the manufacturer supplied protocols which employ HBTU ( 2-(1 H-Benzotriazol-1 -yl-)-1 ,1 ,3,3 tetramethyluronium hexafluorophosphate) mediated couplings in NMP (N-methyl pyrrolidone). The starting resin (100 mg) used for the synthesis was (4-((2', 4'-dimethoxyphenyl)-(Fmoc-Asp(OBut)-O-p- Benzyloxybenzyl resin (Wang resin) with a substitution capacity of 0.53 mmol / g. The protected aminoacid derivatives used were Fmoc-Ala-OH, Fmoc-Arg(Pmc)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-His(Trt)-OH, Boc-His(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OBut)-OH, Fmoc-Gly-OH, Fmoc-lle-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Dde), Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Ser(But)-OH, Fmoc-Thr(But)-OH, Fmoc-Trp(Boc)-OH.

1 .b Cleavage of the peptide from the resin: The peptide was cleaved from the protected peptidyl resin by stirring with a mixture of TFA (trifluoro acetic acid) (2 ml), triisopropylsilane (50 ml) and water (50 ml) for 60 min at room temperature. The cleavage mixture was filtered and the filtrate is concentrated to approximately 1 ml by a stream of nitrogen. The crude peptide was precipitated from this oil with diethyl ether (49.5 ml), washed 3 times with diethyl ether (3 times 50 ml) and dried to a white powder.

1 ,c Purification of the peptide;

The crude peptide was dissolved in water/acβtonitrjle (65:35) (100ml) adjusted to pH 7.5 with

NH₄OH and purified by semipreparative HPLC on a 25 mm x 250 mm column packed with 7μ C-13 silica. The column is eluted with a gradient of 50 to 70% acetonitrile against 0.1% TFAΛivater at 10 ml/min at a temperature of 40 ^PC for 47mn, The peptide containing fractions were collected, diluted with 3 volumes of water and lyophiliz;ed.

The final product obtained was characterized by RP-HPLC / ion spray mass spectrometry (LC-MS)

(retention time and molecular mass) and by analytical RP-HPLC (retention time and peptide amount). The peptide amount was calculated by comparing the UV detector response with that of a standard where the amount had been determined by amino acid analysis.The RP-HPLC analysis is performed on a Vydac 218TP544.6mm x 250mm 5m C-18 silica column (Hie Separations Group,

Hesperia) with UV detection at 214 nm . The column is equilibrated with 0,1% TFA/ H_∑Q and eluted by a gradient of 0 to 90% CHgCN against 0.1 %TFA/water for 50 min at 42⁰C, with a flow of

0.5ml/min.

The LC-MS analysis was performed using a Symmetry 3.0 mm x 150 mm 5m C-18 silica column

(Waters, Milford MA-, USA) which is eluted at 1 ml/miπ at room temperature. It was equilibrated with 5 % CH₃CN / 0.1 % TFA / H₂O and βluted by a gradient of 5% CH₃CN / 0.1 % TFA / H₂O to

90% CH₃CN / 0.1 % TFA / H₂O during 15 min. Besides the UV detection at 214nm, a fraction of the column eluate was introduced into the ionspray interface of a PE-Scjex AP1 100 mass spectrometer. The mass range 300 - 3000 amu was scanned every 2 seconds during the run.

Using these conditions, the retention time of the product as determined from the UV trace was found to be 10.80 min, and the molecular mass was found to be 1262.5 (M+3) amu, which is in agreement with the expected structure within the experimental error of the method (± 1 snπu).

Example 2

[His3]N-epsilon32-(17-carboxyheptadecanoyl)[Lys32]Exendin[1-39]amide

'2,

2. a Synthesis of the protected peptidyl resin:

The protected peptidyl resin was synthesized according to the Fmoc strategy on an Applied Biosystems 431 A peptide synthesizer in 0.25 mmol scale using the manufacturer supplied FastMoc UV protocols which employ HBTU ( 2-(1 H-Benzotriazol-1 -yl-)-1 ,1 ,3,3 tetramethyluronium hexafluorophosphate) or HATU (O-(7-azabenzotriazol-1 -yl)-1 ,1 ,3,3- tetramethyluronium hexafluorophosphate) mediated couplings in NMP (N-methyl pyrrolidone), and UV monitoring of the deprotection of the Fmoc protection group. The starting resin (416 mg) used for the synthesis was Rink-Amide with a substitution capacity of 0.60 mmol / g. The protected aminoacid derivatives used were Fmoc-Ala-OH, Fmoc-Arg(Pmc)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-His(Trt)-OH, Boc-His(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OBut)-OH, Fmoc-Gly-OH, Fmoc-lle-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Dde), Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Ser(But)-OH, Fmoc-Thr(But)-OH, Fmoc-Trp(Boc)-OH.

2.b Dde removal and acylation

To the protected peptidyl resin resulting from (1 .a) (300 mg, 75μmol) was added a freshly prepared solution of hydrazine hydrate 2% in NMP (12ml). The reaction mixture was shaken for 3 minuntes at room temperature, and then filtered. More hydrazine solution (20ml) was added on the reaction mixture was shaken for 15 minutes and then filtered. The resin was then washed extensively with NMP (5 x 20 ml_). Octadecanedioic acid mono-(2,5-dioxo-pyrrolidin-1 -yl) ester (Ebashi et al. EP51 1600)

(123,46 mg, 0.3 mmol) was dissolved in NMP (25 ml_), added to the resin and shaken overnight at room temperature. The reaction mixture was filtered and the resin was washed extensively with NMP, dichloromethane, 2-propanol, methanol and diethyl ether.

2. c Cleavage of the acylated peptide from the resin:

The peptide was cleaved from the protected peptidyl resin by stirring with a mixture of TFA

(trifluoro acetic acid) (20 ml), triisopropylsilane (500 ml) and water (500 ml) for 3 hours at room temperature. The cleavage mixture was filtered and the filtrate was concentrated to approximately 5 ml by a stream of nitrogen. The crude peptide was precipitated from diethyl ether (30 ml), washed 3 times with diethyl ether (3 times 10 ml) and dried to a white powder.

2.d Purification of the peptide:

The crude peptide was dissolved in water adjusted to pH 8.5 with NH₄OH and purified by preparative HPLC (Waters, Prep LC2000) on a 20 mm x 250 mm column packed with C-18 silica. The column was eluted with a gradient of 30 to 50% acetonitrile against 0.1 % TFA/water at 10 ml/min at room temperature for 40minutes. The peptide containing fractions were collected, diluted with 3 volumes of water and lyophilized, yield determined by UV- spectrophotometer to be 10.25 mg.

The final product obtained was characterized by RP-HPLC / ion spray mass spectrometry (LC-MS) (retention time and molecular mass).

The LC-MS analysis was performed using the following instrumentation:

Hewlett Packard series 1100 G1312A Bin Pump

Hewlett Packard series 1100 Column compartment

Hewlett Packard series 1100 G1315A DAD diode array detector Hewlett Packard series 1100 MSD

Sedere 75 Evaporative Light Scattering detector

The instrument was controlled by HP Chemstation software.

The HPLC pump was connected to two eluent reservoirs whicj contained.

A: 0.01 % TFA in water B: 0.01 % TFA in acetonitrile

The analysis was performed at 4O⁰C by injecting an appropriate volume of the sample (preferably 1 ml) onto the column which was eluted with a gradient of acetonitrile. The HPLC conditions, detector settings and mass spectrometer settings used are giving in the following table.

Column Waters Xterra MS C-18 X 3 mm id 5 mm

Gradient 5% - 100% acetonitrile linear during 7.5 min at 1 .5ml/min

Detection 210 nm (analogue output from DAD) ELS (analogue output from ELS)

MS ionisation mode API-ES

Scan 100-1000 amu step 0.1 amu

After the DAD the flow was divided yielding approx 1 ml/min to the ELS and 0.5 ml/min to the

MS. Using these conditions, the retention time of the product as determined from the UV trace was found to be 3.54 min, and the molecular masspeaks identified were 1133 (m/4) and

151 1 (m/3) which is in agreement with the expected structure within the experimental error of the method (± 1 amu).

Example 3

[His9, Arg34, Lys26]-{Λ/-epsilon-(gamma-L-glutamyl(Λ/-alfa-hexadecanoyl))}-GLP-1 (7-37) VR«3 R G—COOH

3.a Synthesis of the protected peptidyl rβsiπ:

The protected peptidyl resin was synthesized similar as described aboveThe starting resjn (385 mg) used for the synthesis was Fmoc-G(y^»Wang resin with a substitution capacity of 0,65 mmol / g. The protected aminoacid derivatives used were Fmoc-Ala-OH, Fmoc-Arg(Pmc>OH, Fmoc- AsnOTrtyOH, Fmoc-Asp(OBut)-OH, Fmoc-His(Trt)-OH, Boc-His(BocK>H, Fmoc-Gln(Trt)-OH, FmooGlu(OBut)"OH, Fmoc-<3ly-OH, Fmoc-lle-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc- Lys(Dde), Fmoc-Met-OH, Fmoc-Phe-OH, Frτioc-Ser(But)-OH, Fmoc-Thr(But)-OH, Fmoc-Tφ(Boc)- OH.

3.b Dde removal and acylatioπ

This step was carried out similar as described above using 2-HeκadecanoylaminθHpentanedioic acid 5'fert-butyl ester 1-(2,5-dioxo-pyrro1idin-1-yl) ester (Knudsen era/. J. Med. Chem 43, 1664-

1$69 (200O)) as acylatlng reagent

3.c Cleavage of the gcylated peptide from the resin:

The step carried out similar as described above.

3.d Purification of the peptide:

The crude peptide was dissolved in water adjusted to pH 8.5 with NH₄OH and purified by preparative HPLC (Waters, Prep LC2000) on a 20 mm x 250 mm column packed with C- 18 silica.

The column was eluted with a gradient of 30 to 60% acetonrtrile against 0.1% TFA/water at 10 ml/min at room temperature for 40minutes, The peptide containing fractions were collected, diluted with 3 volumes of water and lyophilized, yield determined by UV-spectrophotometerto be 17 mg.

Using the same analysis method as described above, the retention time of the product as determined from the UV trace was found to be 4.6 miπ, and the molecular mass peaks identified were 1253 (rn/3) which is in agreement with the expected structure within the experimental error of the method (± 1 smu).

Example 4.

[His3] Eχeπ.diπ-4 amid NHp-H G H G T F T S D L S K Q M E E E A V R L F I E W L K N G G P S S G A P P PP--NN - NNH₂ H C^ 1— '

O

Synthesized according to procedure described above.

Using the same analysis method as described above, the retention time of the product as determined from the UV trace was found to be 10.44 min, and the molecular mass peak identified was m/z:1399.3 (M+3/3) which is in agreement with the expected structure within the experimental error of the method (± 1 amu).

Example 5:

[His9] GLP-1 (7-37) amid

NHp-H A H G T F T S D V S S Y L E G Q A A K E F I A W L V K G R—N"~~g— NH₂

Synthesized according to procedure described above.

Using the same analysis method as described above, the retention time of the product as determined from the UV trace was found to be 9.94 min, and the molecular mass peaks identified were m/z:1121.6 (M+3/3) and 841.5 (M+4/4) which is in agreement with the expected structure within the experimental error of the method (± 1 amu).

Claims

1 . A compound comprising the amino acid sequence of formula I :

XaarXaa₂-His-Gly- Xaa₅-Phe- Xaa₇- Xaa₈- Xaa_g-Xaa₁₀- Xaan-Xaa^-Xaa^-Xaa^- Xaa₁₅- Xaai₆-Xaai₇- Xaa-is-Xcicii_Ei-Xcic^o-Xcic^i-Phe- Xaa₂₃-Xaa₂₄-Trp- Xaa₂₆-Xaa₂₇-Xaa₂₈-Xaa₂₉- Xaa3o"Xaa3i -Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39-Xaa4o -Xaa4i — Xaa42 Formula (I) (SEQ. ID No: 1 )

wherein

Xaai is L-histidine, D-histidine, desamino-histidine, 2-amino-3-(2-aminoimidazol-4- yl)propionic acid, β-hydroxy-histidine, homohistidine, N^α-acetyl-histidine, α-fluoromethyl- histidine, α-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine; or L- tyrosine Xaa₂ is Ala, GIy, VaI, Leu, He, Lys, Aib, 1 -aminocyclopropanecarboxylic acid, 1 - aminocyclobutanecarboxylic acid, 1 -aminocyclopentanecarboxylic acid, 1 - aminocyclohexanecarboxylic acid, 1 -aminocycloheptanecarboxylic acid, or 1 - aminocyclooctanecarboxylic acid;

Xaa₅ is Thr or Ser; Xaa₇ is Thr or Ser;

Xaa₈ is Ser or Asp;

Xaa₉ is GIu or Asp;

Xaaio is VaI, Met, Leu or Tyr;

Xaan is Ser, or Asn; Xaa_i2 is Ser, Thr, Lys or He;

Xaai3 is Tyr, He, Ala or GIn;

Xaais is Asp or GIu;

Xaaie is GIy, Asn, GIu or Lys; Xaaiy is Leu, GIn, GIu or He;

Xaais is Ala or His;

Xaaig is Ala, GIn or VaI;

Xaa₂o is Lys, Arg or GIn;

Xaa₂₄ is Ala, Asn or GIu; Xaa₂e is Leu or He;

Xaa₂₇ is VaI, He, Leu, Arg or Lys;

Xaa₂s is Lys, GIn, Ala or Asn;

Xaa₂₉ is GIy, Thr or Gln; Xaa₃₀ is Arg, Lys or GIy;

Xaa₃i is He, GIy, Pro, amide or is absent;

Xaa₃₂ is Thr, Lys, Ser, amide or is absent;

Xaa₃₃ is Asp, Lys, Ser, amide or is absent;

Xaa₃₄ is Arg, Asn, GIy, amide or is absent; Xaa₃₅ is Asp, Ala, amide or is absent ;

Xaa₃₆ is Trp, Pro, amide or is absent;

Xaa₃₇ is Lys, Pro, amide or is absent;

Xaa₃₈ is His, Pro, amide or is absent;

Xaa₃₉ is Asn, Ser, amide or is absent; Xaa₄₀ is He, amide or is absent;

Xaa₄i is Thr, amide or is absent;

Xaa₄₂ is GIn, amide or is absent; provided that if Xaa₃i , Xaa₃₂, Xaa₃₃, Xaa₃₄, Xaa₃₅, Xaa₃₆, Xaa₃₇, Xaa₃₈, Xaa₃₉, Xaa₄₀, Xaa₄i , or

Xaa₄₂ is absent then each amino acid residue downstream is also absent.

2. The compound according to claim 1 , wherein the amino acid sequence is according to formula 2 :

His-Xaa₂-His-Gly- Xaa₅-Phe- Xaa₇- Xaa₈- Xaa₉-Xaai₀- Xaan-Xaai₂-Xaai₃-Xaai₄- Xaai₅-

Xaai₆-Xaai₇-Ala-Xaai₉-Xaa₂₀-Xaa₂i-Phe-lle-Xaa₂₄-Trp- Leu-Xaa₂₇-Xaa₂₈-Xaa₂₉-Xaa₃₀-Xaa₃r Xaa₃₂-Xaa₃₃-Xaa₃₄-Xaa₃₅"Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉

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

Xaa₅ is Thr or Ser;

Xaa₇ is Thr or Ser;

Xaa₈ is Ser or Asp; Xaa₉ is GIu or Asp;

Xaaio is VaI, Met, or Leu; Xaan is Ser or Asn;

Xaa_i2 is Ser, Thr or Lys;

Xaais is Asp or GIu;

Xaaie is GIy, Asn or GIu;

Xaaiy is Leu, GIn or GIu;

Xaaig is Ala or VaI;

Xaa₂₀ is Lys or Arg;

Xaa₂₄ is Ala, Asn or GIu;

Xaa₂₈ is Lys, GIn or Asn;

Xaa₂₉ is GIy or Thr; Xaa₃₀ is Arg, Lys or GIy;

Xaa₃i is He, Pro, amide or is absent;

Xaa₃₂ is Thr, Ser, amide or is absent;

Xaa₃₃ is Asp, Ser, amide or is absent;

Xaa₃₄ is Arg, GIy, amide or is absent; Xaa₃₅ is Ala, amide or is absent ;

Xaa₃₆ is Pro, amide or is absent;

Xaa₃₇ is Pro, amide or is absent;

Xaa₃₈ is Pro, amide or is absent;

3. The compound according to any one of claims 1 -2, wherein the amino acid sequence is according to formula 3 : His-Xaa^His-Gly-Thr-Phe-Thr-Ser-Asp-Xaaio-Ser-Xaa^-Xaais-Xaa_M-Glu-Xaaie-Xaaiy-Ala-

Xaai ₉-Xaa₂₀-Xaa₂i - P he- 1 Ie-Xaa₂₄-Trp- Leu -Xaa₂₇-Xaa₂₈-G ly-Xaa₃₀-Xaa₃i -Xaa₃₂-Xaa₃₃-Xaa₃₄-

Xaa₃₅"Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃g

Formula (3) (SEQ. ID. No : 3)

Xaaio is VaI or Leu;

Xaaie is GIy or GIu;

Xaaiy is GIn or GIu;

Xaaig is Ala or VaI; Xaa₂o is Lys or Arg;

Xaa₂₇ is VaI or Lys;

Xaa₂₈ is Lys or Asn; Xaa₃₀ is Arg, Lys or GIy;

Xaa₃i is Pro, amide or is absent;

Xaa₃₂ is Ser, amide or is absent;

Xaa₃₃ is Ser, amide or is absent;

Xaa₃₄ is GIy, amide or is absent; Xaa₃₅ is Ala, amide or is absent ;

Xaa₃₆ is Pro, amide or is absent;

Xaa₃₇ is Pro, amide or is absent;

Xaa₃₈ is Pro, amide or is absent;

4. A compound which is a peptide according to any one of claims 1 -3 wherein one or more of the amino acids residues have been modified by acylation.

5. A compound according to claim 4, wherein an acyl group of a straight-chain, branched fatty acid or branched alkane α,ω-dicarboxylic acid is attached to one or more amino acid residues.

6. A compound according to any one of claims 4-5, wherein the acyl group is selected from the group comprising CH₃(CH₂)_nCO-, wherein n is 4 to 38, such as CH₃(CH₂)₆CO-, CH₃(CH₂)₈CO-, CH₃(CH₂)₁₀CO-, CH₃(CH₂)₁₂CO-, CH₃(CH₂)₁₄CO-, CH₃(CH₂)₁₆CO-, CH₃(CH₂)₁₈CO-, CH₃(CH₂)₂₀CO- and CH₃(CH₂)₂₂CO- acid is attached to one or more amino acid residues.

7. A compound according to any one of claims 4-5, wherein the acyl group is selected from the group comprising HOOC(CH₂)_mCO-, wherein m is 4 to 38, such as HOOC(CH₂)₁₄CO-,

HOOC(CH₂)₁₆CO-, HOOC(CH₂)₁₈CO-, HOOC(CH₂)₂₀CO- and HOOC(CH₂)₂₂CO- acid is attached to one or more amino acid residues.

8. A compound according to any one of claims 4-5, wherein the acyl group is selected from the group comprising CH₃(CH₂)_p((CH₂)_qCOOH)CHNH-CO(CH₂)₂CO-, wherein p and q are integers and p+q is an integer of from 8 to 40, such as from 12 to 35 acid is attached to one or more amino acid residues.

9. A compound according to any one of claims 4-5, wherein the acyl group is selected from CH₃(CH₂)_rCO-NHCH(COOH)(CH₂)₂CO-, wherein r is an integer of from 10 to 24 acid is attached to one or more amino acid residues.

10. A compound according to any one of claims 4-5, wherein an acyl group of the formula CH₃(CH₂)_SCO-NHCH((CH₂)₂COOH)CO-, wherein s is an integer of from 8 to 24 acid is attached to one or more amino acid residues.

1 1 . A compound according to any one of claims 4-10, wherein the acyl group is attached to an amino acid with a nuclophilic atom in the side chain, i.e. GLP-1 residue number 18, 22, 26, 34, 36, 37 or 38, GLP-2 residue number 10 or 17, Exendin-4 residue number 12, 16, 20, 32, 33 or 34.

12. A compound which is a peptide according to any one of claims 1 -3 wherein one or more of the amino acids residues have been modified by pegylation.

13. A pharmaceutical composition comprising a compound according to any one of claims 1 -12, and a pharmaceutically acceptable excipient.

14. The pharmaceutical composition according to claim 13 which is suited for parenteral administration.

15. Use of a compound according to any one of the claims 1 -12 for the preparation of a medicament.

16. Use of a compound according to any one of the claims 1 -12 for the preparation of a medicament for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive disorders, atheroschlerosis, myocardial infarction, stroke, coronary heart disease and other cardiovascular disorders, inflammatory bowel syndrome, dyspepsia and gastric ulcers.

17. Use of a compound according to any one of the claims 1 -12 for the preparation of a medicament for delaying or preventing disease progression in type 2 diabetes.

18. Use of a compound according to any one of the claims 1 -12 for the preparation of a medicament 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.