Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/605—Glucagons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/06—Antihyperlipidemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/12—Antihypertensives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• 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).
• GLP-1 glucagon-like-peptide-1
• the invention further relates to methods of making and using such peptides.
• 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.
• GLP-1 glucagon-like peptide-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.
• GLP-1 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.
• [Gly 8 ]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.
• (N ⁇ 34 -tetradecanoyl)[Lys 34 ]GLP-1 (7-37) designates GLP-1 (7-37) wherein the ⁇ -amino group of the Lys residue in position 34 has been tetradecanoylated.
• 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.
• 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).
• 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:
• Xaa 7 is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, ⁇ -hydroxy-histidine, homohistidine, N ⁇ -acetyl-histidine, ⁇ -fluoromethyl-histidine, ⁇ -methyl-histidine, 3-pyridylalanine,
• Xaa 18 is Ser, Lys or Arg
• Xaa 2 o is Leu or Met
• Xaa 22 is GIy, GIu or Aib;
• Xaa 23 is GIn, GIu, Lys or Arg;
• Xaa 25 is Ala or VaI
• Xaa 26 is Lys, GIu or Arg;
• Xaa 27 is GIu or Leu
• Xaa 30 is Ala, GIu or Arg; Xaa 33 is VaI or Lys;
• Xaa 34 is Lys, GIu, Asn or Arg;
• Xaa 35 is GIy or Aib; Xaa 36 is Arg, Gly or Lys;
• Xaa 37 is GIy, Ala, GIu, Pro, Lys, amide or is absent;
• Xaa 38 is Lys, Ser, amide or is absent.
• Xaa 39 is Ser, Lys, amide or is absent;
• Xaa 40 is GIy, amide or is absent;
• Xaa 41 is Ala, amide or is absent;
• Xaa 42 is Pro, amide or is absent;
• Xaa 43 is Pro, amide or is absent;
• Xaa 44 is Pro, amide or is absent;
• Xaa 45 is Ser, amide or is absent;
• Xaa 46 is amide or is absent ; where if Xaa 38 , Xaa 39 , Xaa 40 , Xaa 41 , Xaa 42 , Xaa 43 , Xaa 44 , Xaa 45 or Xaa 46 is absent then each amino acid residue downstream is also absent and where the total number of basic residues
• the peptides of the invention may also comprise the amino acid sequence of the formula (IV)
• Xaa 7 is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, ⁇ -hydroxy-histidine, homohistidine, N ⁇ -acetyl-histidine, ⁇ -fluoromethyl-histidine, ⁇ -methyl-histidine, 3-pyridylalanine,
• Xaa 8 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;
• Xaa 9 is GIu or Asp
• Xaa 16 is VaI or Leu
• Xaa 17 is Ser or Thr;
• Xaa 18 is Ser, Lys or Arg;
• Xaa 21 is GIu or Asp;
• Xaa 22 is GIy, GIu or Aib;
• Xaa 23 is GIn, Lys or Arg
• Xaa 25 is Ala or VaI
• Xaa 26 is Lys, or Arg; Xaa 27 is GIu or Leu;
• Xaa 30 is Ala, GIu or Arg
• Xaa 33 is VaI or Lys
• Xaa ⁇ is Lys, GIu, Asn or Arg;
• Xaa 35 is GIy or Aib; Xaa 36 is Arg, GIy or Lys;
• Xaa 37 is GIy, Ala, GIu, Pro, Lys, amide or is absent;
• Xaa 38 is Lys, Ser, amide or is absent.
• Xaa 39 is Ser, Lys, amide or is absent;
• Xaa 40 is GIy, amide or is absent;
• Xaa 41 is Ala, amide or is absent;
• Xaa 42 is Pro, amide or is absent;
• Xaa 43 is Pro, amide or is absent;
• Xaa 44 is Pro, amide or is absent;
• Xaa 45 is Ser, amide or is absent; Xaa 46 is amide or is absent ; where if Xaa 38 , Xaa 39 , Xaa 40 , Xaa 41 , Xaa 42 , Xaa 43 , Xaa 44 , Xaa 45 or Xaa 46 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)
• Xaa 7 is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, ⁇ -hydroxy-histidine, homohistidine, N ⁇ -acetyl-histidine, ⁇ -fluoromethyl-histidine, ⁇ -methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;
• Xaa 8 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;
• Xaa 16 is VaI or Leu
• Xaa 18 is Ser, Lys or Arg
• Xaa 19 is Tyr or GIn
• Xaa 2 o is GIu or Asp;
• Xaa 22 is GIy, GIu or Aib or Lys;
• Xaa 23 is GIn, GIu, Lys or Arg;
• Xaa 25 is Ala or VaI
• Xaa 26 is Lys, GIu or Arg;
• Xaa 27 is GIu or Leu; Xaa 30 is Ala, GIu, Asp or Arg;
• Xaa 33 is VaI, Ne or Lys
• Xaa ⁇ is Lys, Asn or Arg
• Xaa 35 is GIy or Aib
• Xaa 36 is Arg, GIy or Lys
• Xaa 37 is GIy, Ala, GIu, Pro, Lys, amide or is absent;
• Xaa 38 is Lys, Ser, amide or is absent.
• Xaa 39 is Ser, Lys, amide or is absent;
• Xaa 40 is GIy, amide or is absent
• Xaa 41 is Ala, amide or is absent;
• Xaa 42 is Pro, amide or is absent;
• Xaa 43 is Pro, amide or is absent;
• Xaa 44 is Pro, amide or is absent;
• Xaa 45 is Ser, amide or is absent
• Xaa 46 is amide or is absent ; where if Xaa 38 , Xaa 39 , Xaa 40 , Xaa 41 , Xaa 42 , Xaa 43 , Xaa 44 , Xaa 45 or Xaa 46 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.
• 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 ( ⁇ -aminoisobutyric acid), Abu ( ⁇ -aminobutyric acid), Tie (tert-butylglycine), ⁇ -alanine, 3-aminomethyl benzoic acid, an- thranilic acid.
• agonist 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 EC 50 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 ⁇ l_/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.
• 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 ⁇ L and with the following final concentrations: 50 mM Tris-HCI, 1 mM EGTA, 1.5 mM MgSO 4 , 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 ⁇ g 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 ⁇ L 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 EC 50 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.
• enzyme 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).
• DPP-IV dipeptidyl aminopeptidase IV
• 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.
• 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).
• the peptides of the invention comprise an amino acid sequence of formula I:
• Xaa 7 -Xaa 8 -Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa 16 -Ser-Xaa 18 -Xaa 19 -Xaa 2 o-Glu-Xaa 22 -Xaa 23 -Ala- Xaa 2 5-Xaa 2 6-Xaa 27 -Phe-lle-Xaa30-Trp-Leu-Xaa33-Xaa3 4 -Xaa35-Xaa3 6 -Xaa3 7 -Xaa 38 -Xaa39-Xaa 4 0- Xaa4"i-Xaa42-Xaa43-Xaa44-Xaa45-Xaa4g Formula (I) (SEQ ID No: 1 ) wherein Xaa 7 is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, ⁇ -hydroxy-histidine, homoh
• Xaa 8 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;
• Xaa 16 is VaI or Leu
• Xaa 18 is Ser, Lys or Arg
• Xaa 19 is Tyr or GIn; Xaa 20 is Leu or Met;
• Xaa 22 is GIy, GIu or Aib;
• Xaa 23 is GIn, GIu, Lys or Arg;
• Xaa 25 is Ala or VaI
• Xaa 26 is Lys, GIu or Arg; Xaa 27 is GIu or Leu;
• Xaa 30 is Ala, GIu or Arg
• Xaa 33 is VaI or Lys
• Xaa 34 is Lys, GIu, Asn or Arg;
• Xaa 35 is GIy or Aib; Xaa 36 is Arg, GIy or Lys;
• Xaa 37 is GIy, Ala, GIu, Pro, Lys, amide or is absent;
• Xaa 38 is Lys, Ser, amide or is absent.
• Xaa 39 is Ser, Lys, amide or is absent;
• Xaa 40 is GIy, amide or is absent;
• Xaa 41 is Ala, amide or is absent;
• Xaa 42 is Pro, amide or is absent;
• Xaa 43 is Pro, amide or is absent;
• Xaa 44 is Pro, amide or is absent;
• Xaa 45 is Ser, amide or is absent; Xaa 46 is amide or is absent ; where if Xaa 38 , Xaa 39 , Xaa 40 , Xaa 41 , Xaa 42 , Xaa 43 , Xaa 44 , Xaa 45 or Xaa 46 is absent then each amino acid residue downstream is also absent.
• the peptide of the invention comprises an amino acid sequence of formula (II): Xaa7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Xaa 18 -Tyr-Leu-Glu-Xaa22-Xaa23-Ala-Ala-Xaa26-
• Formula (II) (SEQ ID No: 2) wherein Xaa 7 is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, ⁇ -hydroxy-histidine, homohistidine, N ⁇ -acetyl-histidine, ⁇ -fluoromethyl-histidine, ⁇ -methyl-histidine, 3-pyridylalanine,
• Xaa 8 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;
• Xaa 18 is Ser, Lys or Arg
• Xaa 22 is GIy, GIu or Aib;
• Xaa 23 is GIn, GIu, Lys or Arg;
• Xaa 26 is Lys, GIu or Arg; Xaa 30 is Ala, GIu or Arg;
• Xaa 34 is Lys, GIu or Arg;
• Xaa 35 is GIy or Aib
• Xaa 36 is Arg or Lys
• Xaa 37 is GIy, Ala, GIu or Lys; Xaa 38 is Lys, amide or is absent.
• the peptide of the invention comprises an amino acid sequence of formula (III):
• Xaa 18 is Lys or Arg
• Xaa 2 o is Leu or Met
• Xaa 23 is GIn or Asn
• Xaa 34 is Lys or Arg
• Xaa 36 is Arg or Lys
• Xaa 38 is Lys, amide or is absent.
• the peptide of the invention comprises the amino acid sequence of the formula (IV) (SEQ ID NO:4)
• Xaa 7 is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, ⁇ -hydroxy-histidine, homohistidine, N ⁇ -acetyl-histidine, ⁇ -fluoromethyl-histidine, ⁇ -methyl-histidine, 3-pyridylalanine,
• Xaa 8 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;
• Xaa 9 is GIu or Asp
• Xaa 16 is VaI or Leu
• Xaa 18 is Ser, Lys or Arg
• Xaa 22 is GIy, GIu or Aib
• Xaa 23 is GIn, Lys or Arg
• Xaa 25 is Ala or VaI
• Xaa 26 is Lys, or Arg
• Xaa 27 is GIu or Leu; Xaa 30 is Ala, GIu or Arg;
• Xaa 33 is VaI or Lys
• Xaa 34 is Lys, GIu, Asn or Arg;
• Xaa 35 is GIy or Aib
• Xaa 36 is Arg, GIy or Lys
• Xaa 37 is GIy, Ala, GIu, Pro, Lys, amide or is absent;
• Xaa 38 is Lys, Ser, amide or is absent.
• Xaa 39 is Ser, Lys, amide or is absent;
• Xaa 40 is GIy, amide or is absent
• Xaa 41 is Ala, amide or is absent;
• Xaa 42 is Pro, amide or is absent;
• Xaa 43 is Pro, amide or is absent;
• Xaa 44 is Pro, amide or is absent;
• Xaa 45 is Ser, amide or is absent;
• Xaa 46 is amide or is absent ; where if Xaa 38 , Xaa 39 , Xaa 40 , Xaa 41 , Xaa 42 , Xaa 43 , Xaa 44 , Xaa 45 or Xaa 46 is absent then each amino acid residue downstream is also absent.
• peptides of the invention may comprise the amino acid sequence of the formula (V)
• Xaa 7 -Xaa 8 -Glu-Gly-Thr- Leu-Thr-Ser-Asp-Xaa 16 - Xaa 17 -Xaa 18 -Xaa 19 -Xaa2o- Xaa 2 i-Xaa22-Xaa 2 3- Ala-Xaa 25 -Xaa 26 -Xaa 27 -Phe-lle-Xaa 30 -Trp-Leu-Xaa 33 -Xaa 34 -Xaa 35 -Xaa 36 -Xaa 37 -Xaa 38 -Xaa 39 - Xaa 4 o-Xaa 4 -
• Xaa 7 is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, ⁇ -hydroxy-histidine, homohistidine, N ⁇ -acetyl-histidine, ⁇ -fluoromethyl-histidine, ⁇ -methyl-histidine, 3-pyridylalanine,
• Xaa 8 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;
• Xaa 16 is VaI or Leu
• Xaa 17 is Ser or Thr
• Xaa 18 is Ser, Lys or Arg; Xaa 19 is Tyr or GIn;
• Xaa 2 o is GIu or Asp
• Xaa 22 is GIy, GIu or Aib or Lys;
• Xaa 23 is GIn, GIu, Lys or Arg; Xaa 25 is Ala or VaI;
• Xaa 26 is Lys, GIu or Arg;
• Xaa 27 is GIu or Leu
• Xaa 30 is Ala, GIu, Asp or Arg;
• Xaa 33 is VaI, Ne or Lys;
• Xaa 34 is Lys, Asn or Arg;
• Xaa 35 is GIy or Aib; Xaa 36 is Arg, Gly or Lys;
• Xaa 37 is GIy, Ala, GIu, Pro, Lys, amide or is absent; Xaa 38 is Lys, Ser, amide or is absent.
• Xaa 39 is Ser, Lys, amide or is absent;
• Xaa 40 is GIy, amide or is absent;
• Xaa 41 is Ala, amide or is absent;
• Xaa 42 is Pro, amide or is absent;
• Xaa 43 is Pro, amide or is absent;
• Xaa 44 is Pro, amide or is absent;
• Xaa 45 is Ser, amide or is absent;
• Xaa 46 is amide or is absent ; where if Xaa 38 , Xaa 39 , Xaa 40 , Xaa 41 , Xaa 42 , Xaa 43 , Xaa 44 , Xaa 45 or Xaa 46 is absent then each amino acid residue downstream is also absent.
• 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.
• 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).
• 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).
• 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).
• 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).
• the peptides of the invention comprise no more than 4 amino acid residues which are not encoded by the genetic code.
• 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.
• 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.
• the peptides of the invention comprise two lysine residues.
• the peptides of the invention comprise only one lysine residue.
• the peptides of the invention have an isoelectric point of greater than 7.
• 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.
• the peptides of the invention have an isoelectric point of greater than 10.
• the peptide of the invention comprises an amino acid sequence selected from the group consisting of: formula (Vl) (SEQ ID No: 6)
• 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-Xaa 39 Where Xaa 39 is amide or is absent and
• 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 peptid
• GLP- 1 (7-37) Arg 34 , Lys 26 (N ⁇ -( ⁇ -Glu(N ⁇ -hexadecanoyl)))-GLP-1 (7-37).
• 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.
• 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.
• a salt e.g. ammonium sulphate
• 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 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.
• 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.
• 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.
• 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.
• 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 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.
• suitable host cells well known and used in the art are, without limitation, E. coli, Saccharomyces cerevisiae, or mammalian BHK or CHO cell lines.
• 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.
• the peptide is present in the formulation at a concentration of from about 0.1 mg/ml to about 25 mg/ml.
• the peptide is present in the formulation at a concentration of from about 1 mg/ml to about 10 mg/ml.
• the formulation has a pH from 4.0 to 10.0.
• 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.
• the formulation has a pH from 4.0 to 7.0.
• the formulation has a pH from 7.0 to 10.0.
• 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.
• the pharmaceutical formulation is an aqueous formulation, i.e. formulation comprising water. Such formulation is typically a solution or a suspension.
• the pharmaceutical formulation is an aqueous solution.
• aqueous formulation is defined as a formulation comprising at least 50 %w/w water.
• aqueous solution is defined as a solution comprising at least 50 %w/w water
• aqueous suspension is defined as a suspension comprising at least 50 %w/w water.
• the pharmaceutical formulation is a freeze-dried formulation, whereto the physician or the patient adds solvents and/or diluents prior to use.
• the pharmaceutical formulation is a dried formulation (e.g. freeze-dried or spray-dried) ready for use without any prior dissolution.
• the invention relates to a pharmaceutical formulation
• a pharmaceutical formulation comprising an aqueous solution of a peptide according to the present invention, and a buffer.
• 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.
• the formulation further comprises a pharmaceutically acceptable preservative.
• 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.
• 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.
• the formulation further comprises an isotonic agent.
• 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.
• 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.
• 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.
• 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.
• the sugar or sugar alcohol concentration is between about 1 mg/ml and about 150 mg/ml.
• 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.
• the formulation further comprises a chelating agent.
• the chelating agent is selected from salts of ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic acid, and mixtures thereof.
• the chelating agent is present in a concentration from 0.1 mg/ml to 5mg/ml.
• the chelating agent is present in a concentration from 0.1 mg/ml to 2mg/ml.
• 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.
• 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.
• 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.
• during storage is intended a liquid pharmaceutical composition or formulation once prepared, is not immediately administered to a subject.
• 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.
• dried form 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.
• 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.
• 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.
• 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.
• compositions of the invention 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.
• the L-stereoisomer is used.
• Compositions of the invention may also be formulated with analogues of these amino acids.
• 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.
• the amino acid analogues are incorporated into the compositions in either their free base form or their salt form.
• the amino acids or amino acid ana- logues are used in a concentration, which is sufficient to prevent or delay aggregation of the protein.
• 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.
• inhibitor 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.
• the formulation further comprises a stabiliser selected from the group of high molecular weight polymers or low molecular compounds.
• 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).
• PEG 3350 polyethylene glycol
• PVA polyvinylalcohol
• PVpyrrolidone polyvinylpyrrolidone
• carboxy-/hydroxycellulose or derivates thereof e.g. HPC, HPC-SL, HPC-L and HPMC
• cyclodextrins e.g. sulphur-containing substances as monothiogly
• 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.
• the formulation further comprises a surfactant.
• 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.
• phospholipids eg. dipalmitoyl phosphatidic acid
• lysophospholipids eg.
• ceramides e.g. sodium tauro-dihydrofusidate etc.
• C6-C12 e.g.
• acylcarnitines and derivatives N ⁇ -acylated derivatives of lysine, arginine or histidine, or side-chain acylated derivatives of lysine or arginine, N ⁇ -acylated derivatives of dipeptides comprising any combination of lysine, arginine or histidine and a neutral or acidic amino acid, N ⁇ -acylated derivative of a tripeptide comprising any combination of a neutral amino acid and two charged amino acids, DSS
• N-alkyl-N,N-dimethylammonio-1-propanesulfonates 3-cholamido-i-propyldimethylammonio-i-propanesulfonate
• cationic surfactants quarternary ammonium bases
• cetyl-trimethylammonium bromide cetylpyridinium chloride
• non-ionic surfactants eg. Dodecyl ⁇ -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.
• 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).
• additional ingredients should not adversely affect the overall stability of the pharmaceutical formulation of the present invention.
• 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.
• topical sites for example, skin and mucosal sites
• sites which bypass absorption for example, administration in an artery, in a vein, in the heart
• 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.
• 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.
• solutions for example, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets, rinses
• 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.
• 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.
• polymers for example cellulose and derivatives, polysaccharides, for example dextran
• 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.
• parenteral controlled release and sustained release systems both systems leading to a many-fold reduction in number of administrations
• 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).
• 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.
• 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.
• the peptide of the invention can also be administered transdermal ⁇ , e.g. from a patch, optionally a iontophoretic patch, or transmucosally, e.g. bucally.
• 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.
• 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.
• the volume of the solution is adjusted with water to give the desired concentration of the ingredients.
• the peptide is provided in the form of a composition suitable for administration by injection.
• 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.
• 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.
• 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, ⁇ -cell apoptosis, ⁇ -cell deficiency, myocardial infarction, inflammatory bowel syndrome, dyspepsia, cognitive disorders, e.g. cognitive enhancing, neuroprotection, atheroschlerosis, coronary heart disease and other cardiovascular disorders.
• cognitive disorders e.g. cognitive enhancing, neuroprotection, atheroschlerosis, coronary heart disease and other cardiovascular disorders.
• a peptide according to the invention is used for the preparation of a medicament for delaying or preventing disease progression in type 2 diabetes.
• a peptide according to the is used for the preparation of a medicament for decreasing food intake, decreasing ⁇ -cell apoptosis, increasing ⁇ -cell funtion and ⁇ -cell mass, and/or for restoring glucose sensitivity to ⁇ -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.
• 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
• insulin therapy 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 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 ⁇ -cells, e.g.
• glibenclamide glipizide, gliclazide and repaglinide
• Cholestyramine colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol, dextrothyroxine, neteglinide, repaglinide
• ⁇ -blockers such as alprenolol, atenolol, timolol, pindolol, propranolol and metoprolol
• ACE angiotensin converting enzyme
• benazepril captopril, enalapril, fosinopril, lisinopril, alatriopril, quinapril and ramipril
• calcium channel blockers such as nifedipine, felodipine, nicardipine, isradipine, nimodipine, diltiazem and
• Example 1 Biological Potency and Chemical Stability of Peptides having the sequences shown in SEQ ID NOs 6-9.
• TABLE 1 The receptor affinity, potency and half-life (T 1 A) in a DPP-IV stability assay of
• T 1 A is calculated from the average of two independent measures.
• T V 2 The receptor affinity, potency and half-life (T V 2 ) 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. TV 2 is calculated from the average of two or more independent measures. Affinity (nM) Potency (pM) V ⁇ IC ⁇ nhiqh [C 50 I0W EC 50 hours
• 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.

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