WO2015193378A1 - Nouveaux agonistes des récepteurs au glp-1 à activité d'efflux de cholestérol - Google Patents

Nouveaux agonistes des récepteurs au glp-1 à activité d'efflux de cholestérol Download PDF

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Publication number
WO2015193378A1
WO2015193378A1 PCT/EP2015/063602 EP2015063602W WO2015193378A1 WO 2015193378 A1 WO2015193378 A1 WO 2015193378A1 EP 2015063602 W EP2015063602 W EP 2015063602W WO 2015193378 A1 WO2015193378 A1 WO 2015193378A1
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glu
glp
lys
gly
leu
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PCT/EP2015/063602
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Henning THØGERSEN
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Novo Nordisk A/S
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    • 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

Definitions

  • the present invention provides novel analogues of glucagon-like protein-1 (GLP-1 ) receptor agonist peptides that are capable of promoting cholesterol efflux.
  • the present invention also provides compositions comprising the novel glucagon-like protein-1 (GLP-1 ) receptor analogues, and relates to and to the use of said compounds in the manufacture of medicaments, the the use of said compounds in therapy, and to methods of treatment comprising administration of said compounds to patients.
  • CVD cardiovascular disease
  • Atherosclerotic plaque formation is initiated by the deposition of excess cholesterol, primarily derived from plasma low density lipoprotein (LDL), in the inner layer of the vascular wall (i.e. tunica intima).
  • LDL plasma low density lipoprotein
  • the cholesterol-containing LDL is oxidized or otherwise modified and taken up by resident macrophages, thus turning these into lipid-filled foam cells.
  • Cholesterol can be effluxed from the vascular wall, from macrophages and foam cells via specific transporters (ATP-binding casette (ABC) transporters; ABCA-1 and ABCG-1 ) to high density lipoprotein (HDL) particles and transported to the liver for excretion via the bile. This process is known as reverse cholesterol transport (RCT).
  • ABC ATP-binding casette
  • HDL high density lipoprotein
  • GLP-1 Glucagon-like protein-1 receptor agonist peptides
  • HbA1 c glycated hemoglobin A1 c
  • body weight with an overall improved lipid profile in type 2 diabetic patients.
  • GLP-1 receptor agonists There are currently three approved GLP-1 receptor agonists on the market Victoza ® , Byetta ® and Bydureon ® . These compounds are usually taken in combination with one or more blood glucose lowering agents.
  • many diabetes patients still suffer both from poor blood glucose control and elevated HbA1 c, and also have an increased risk of cardiovascular disease.
  • GLP-1 agonists do not possess cholesterol efflux activity, although, when bound to the N-terminal of the GLP-1 receptor (Underwood et al, J. Biol. Chem. 2010 285 723-730; and Runge et al, J. Biol. Chem. 2008 283 1 1340-1 1347), they do adopt an alpha helical conformation which in part is amphipathic.
  • One possible explanation for this lack of effect seems to be that the amphipathic part of the helix of GLP-1 agionist peptides
  • GLP-1 receptor agonist peptides that are capable of providing both good blood HbA1 c control and increased cholesterol efflux activity are of great benefit for diabetes patients, since they would address the unmet need for a treatment that can reduce the risk of cardiovascular diseases in patients with diabetes.
  • the present invention is devoted to the provision of novel, dual acting peptides, being capable of reducing blood glucose levels, while also confiing the risk of
  • the present invention therefore provides a novel therapeutic concept that addresses both reduction of blood glucose, and prevention or treatment of cardiovascular complications. This is an unmet need in diabetes care, as many diabetes patients have high risk of cardiovascular diseases.
  • the invention provides novel analogues of GLP-1 receptor agonist peptides, that show cholesterol efflux activity at physiological relevant concentrations (i.e. EC 50 is below 10 ⁇ ), which is in contrast to the native GLP-1 peptides, that do not show such cholesterol efflux activity.
  • the GLP-1 receptor agonist peptide of the invention may be regarded as an analogue of a GLP-1 (7-37) receptor agonist peptide holding a C-terminal extension.
  • analogue of a GLP-1 receptor agonist peptide of the invention may be characterised by
  • compositions comprising a therapeutically effective amount of the GLP-1 receptor analogue of the invention, optionally in combination with one or more pharmaceutically acceptable carriers or diluents.
  • the invention relates to use of the GLP-1 receptor analogue of the invention for use as a medicament.
  • the invention relates to a method for the treatment, prevention or alleviation of a disease or a disorder or a condition of a living animal body, which disease or a disorder or a condition is selected from a vascular disorder,
  • cardiovascular disease periheral vascular disease, restenosis, acute coronary syndrome, reperfusion myocardial injury, macrovascular disorder, microvascular disorder, endothelial dysfunction, atherosclerosis, stroke, ischemia, infarction, myocardial infarction, hemorrhage, intracranial hemorrhage, hypertension, dyslipidemia, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, hyperlipoproteinemia, HDL deficiency, apoA-l deficiency, coronary artery disease, or inflammation, and which method comprises the step of administering to such a living animal body in need thereof, a therapeutically effective amount of the GLP-1 receptor analogue of the invention.
  • GLP-1 glucagon-like protein-1
  • the GLP-1 receptor agonist peptide of the invention may be regarded as an analogue of a GLP-1 (7-37) receptor agonist peptide having a C-terminal extension.
  • analogue of a GLP-1 receptor agonist peptide of the invention may be characterised by
  • X 8 represents Ala or Aib
  • X 26 represents Lys or Arg, or a conservative substitution hereof;
  • X 34 represents Lys or Glu, or a conservative substitution hereof;
  • X 35 represents Gly, Arg or Lys, or a conservative substitution hereof;
  • X 36 represents Val, or a conservative substitution hereof
  • X 37 represents Gly, Arg or Lys, or a conservative substitution hereof; and ii) a C-terminal extension, having the amino acid sequence of Formula lb:
  • X 38 represents Glu, or a conservative substitution hereof
  • X 39 represents Gly, His or Phe, or a conservative substitution hereof;
  • X 40 represents His or Leu, or a conservative substitution hereof.
  • X 41 represents Glu or Gly, or a conservative substitution hereof; and X 42 represents Lys, or a conservative substitution hereof; or
  • X 41 represents Gly, or a conservative substitution hereof, or amide, or X 41 is absent;
  • X 42 is absent
  • GLP-1 receptor analogues of the invention may be further characterised by one of more of the following clauses:
  • the GLP-1 (7-37) receptor agonist peptide part of the compound of the invention holds the amino acid sequence of Formula la:
  • X 8 represents Ala or Aib
  • X 26 represents Lys or Arg
  • X 34 represents Lys or Glu
  • X 35 represents Gly, Arg or Lys
  • X 36 represents Val
  • X 37 represents Gly, Arg or Lys.
  • the amino acid sequence of the GLP-1 (7-37) receptor agonist peptide part of the compound of the invention holds 2-6 amino acid changes relative to the Homo sapiens GLP-1 (7-37) amino acid sequence.
  • the amino acid sequence of the GLP-1 (7-37) receptor agonist peptide part of the compound of the invention holds two amino acid changes relative to the Homo sapiens GLP-1 (7-37) amino acid sequence.
  • the amino acid sequence of the GLP-1 (7-37) receptor agonist peptide part of the compound of the invention holds three amino acid changes relative to the Homo sapiens GLP-1 (7-37) amino acid sequence.
  • the amino acid sequence of the GLP-1 (7-37) receptor agonist peptide part of the compound of the invention holds 4-6 amino acid changes relative to the Homo sapiens GLP-1 (7-37) amino acid sequence.
  • the amino acid sequence of the GLP-1 (7-37) receptor agonist peptide part of the compound of the invention holds four amino acid changes relative to the Homo sapiens GLP-1 (7-37) amino acid sequence.
  • the amino acid sequence of the GLP-1 (7-37) receptor agonist peptide part of the compound of the invention holds five amino acid changes relative to the Homo sapiens GLP-1 (7-37) amino acid sequence.
  • the amino acid sequence of the GLP-1 (7-37) receptor agonist peptide part of the compound of the invention holds six amino acid changes relative to the Homo sapiens GLP-1 (7-37) amino acid sequence.
  • the GLP-1 (7-37) receptor agonist peptide part of the compound of the invention holds the amino acid sequence of Formula la, wherein X 8 represents Ala or Aib; X 26 represents Lys or Arg; X 34 represents Glu; X 35 represents Arg or Lys; X 36 represents Val; and X 37 represents Arg or Lys.
  • the GLP-1 (7-37) receptor agonist peptide part of the compound of the invention holds the amino acid sequence of Formula la, wherein X 8 represents Ala; X 26 represents Lys or Arg; X 34 represents Glu; X 35 represents Arg or Lys; X 36 represents Val; and X 37 represents Arg or Lys.
  • the GLP-1 (7-37) receptor agonist peptide part of the compound of the invention holds the amino acid sequence of Formula la, wherein X 8 represents Ala; X 26 represents Arg; X 34 represents Glu; X 35 represents Arg; X 36 represents Val; and X 37 represents Arg.
  • the GLP-1 (7-37) receptor agonist peptide part of the compound of the invention holds the amino acid sequence of Formula la, wherein X 8 represents Ala; X 26 represents Lys; X 34 represents Glu; X 35 represents Lys; X 36 represents Val; and X 37 represents Lys.
  • the GLP-1 (7-37) receptor agonist peptide part of the compound of the invention holds the amino acid sequence of Formula la, wherein X 8 represents Aib; X 26 represents Lys or Arg; X 34 represents Glu; X 35 represents Arg or Lys; X 36 represents Val; and X 37 represents Arg or Lys.
  • the GLP-1 (7-37) receptor agonist peptide part of the compound of the invention holds the amino acid sequence of Formula la, wherein X 8 represents Aib; X 26 represents Lys; X 34 represents Glu; X 35 represents Lys; X 36 represents Val; and X 37 represents Lys.
  • the GLP-1 (7-37) receptor agonist peptide part of the compound of the invention holds the amino acid sequence of Formula la, wherein X 8 represents Aib; X 26 represents Arg; X 34 represents Glu; X 35 represents Arg; X 36 represents Val; and X 37 represents Arg.
  • X 38 represent Glu
  • X 39 represents Gly, His or Phe
  • X 40 represents His or Leu; and X represents Glu or Gly; and X represents Lys; or
  • X 41 represents Gly or amide; and X 42 is absent.
  • the C-terminal extension of the compound of the invention holds the amino acid sequence of Formula lb, wherein X 38 represent Glu; X 39 represents Gly, His or Phe; X 40 represents His or Leu; and
  • X 41 represents Glu; and X 42 represents Lys; or
  • X 41 represents amide; and X 42 is absent.
  • the C-terminal extension of the compound of the invention holds the amino acid sequence of Formula lb, wherein X 38 represent Glu; X 39 represents Gly; X 40 represents Leu; and
  • X 41 represents Gly; and X 42 represents Lys; or
  • X 41 represents amide; and X 42 is absent.
  • the C-terminal extension of the compound of the invention holds the amino acid sequence of Formula lb, wherein X 38 represent Glu; X 39 represents Gly; X 40 represents Leu; X 41 represents Glu; and X 42 represents Lys.
  • the C-terminal extension of the compound of the invention holds the amino acid sequence of Formula lb, wherein X 38 represent Glu; X 39 represents Phe; X 40 represents His; and
  • X 41 represents Glu; and X 42 represents Lys; or
  • X 41 represents amide; and X 42 is absent.
  • the C-terminal extension of the compound of the invention holds the amino acid sequence of Formula lb, wherein X 38 represent Glu; X 39 represents His; X 40 represents Leu; and
  • X 41 represents Glu; and X 42 represents Lys; or
  • X 41 represents amide; and X 42 is absent.
  • the C-terminal extension of the compound of the invention holds the amino acid sequence of Formula lb, wherein X 38 represent Glu; X 39 represents Phe; X 40 represents Leu; and
  • X 41 represents Glu; and X 42 represents Lys; or X represents Gly or amide; and X is absent.
  • the C-terminal extension of the compound of the invention holds the amino acid sequence of Formula lb, wherein X 38 represent Glu; X 39 represents Phe; X 40 represents Leu; X 41 represents Glu; and X 42 represents Lys.
  • the GLP-1 receptor agonist analogue of the invention may be characterised by an amino acid sequence of Formula II:
  • X 8 represents Ala or Aib
  • X 26 represents Lys or Arg
  • X 34 represents Lys or Glu
  • X 35 represents Gly, Arg, or Lys
  • X 36 represents Val
  • X 37 represents Gly, Arg, or Lys
  • X 39 represents Gly, His or Phe
  • X 40 represents His or Leu
  • X 41 represents Glu or Gly
  • X 42 represents Lys
  • X 41 represents Gly or amide
  • X 42 is absent
  • the GLP-1 receptor agonist analogue of the invention may be characterised by an amino acid sequence of Formula II, wherein
  • X 8 represents Ala or Aib
  • X 26 represents Lys or Arg
  • X 34 represents Lys or Glu
  • X 35 represents Arg or Lys
  • X 36 represents Val
  • X 37 represents Arg or Lys
  • X 39 represents Gly, His or Phe
  • X 40 represents His or Leu; and X represents Glu, Gly or amide;
  • X 42 represents Lys
  • X 41 represents Gly or amide
  • X 42 is absent.
  • analogue of a GLP-1 receptor agonist peptide of the invention is selected from the group consisting of:
  • a receptor agonist is a peptide capable of binding to a receptor, and which elicits a response typical of the natural ligand.
  • GLP-1 receptor agonist or “GLP-1 receptor agonist peptide” is defined as a compound which is capable of binding to the GLP-1 receptor, and which is capable of activating it. This biological activity may be determined as described below.
  • GLP-1 refers to the human Glucagon-Like Peptide-1 (GLP-1 (7-37)), the sequence of which is included in the sequence listing as SEQ ID 1 , or an analogue thereof.
  • the peptide having the sequence of SEQ ID 1 may also be designated “native” GLP-1 .
  • GLP-1 analogue or “analogue of GLP-1" as used herein refers to a peptide, or a compound, which is a variant of GLP-1 (7-37).
  • the Homo sapiens GLP-1 (7-37) sequence is: HAEGTFTSDV SSYLEGQAAK EFIAWLVKGR G (SEQ ID 1 ).
  • the first amino acid residue (i.e. histidine) of SEQ ID 1 is assigned no. 1.
  • this histidine residue is usually referred to as residue no. 7, and subsequent amino acid residues are numbered accordingly, ending with glycine no. 37. Therefore, generally, any reference herein to an amino acid residue number or a position number of the GLP-1 (7-37) sequence is to the sequence starting with His at position 7 and ending with Gly at position 37.
  • GLP-1 analogues of the invention may be described by reference to i) the number of the amino acid residue in native GLP-1 (7-37), which corresponds to the amino acid residue which is changed (i.e., the corresponding position in native GLP-1 ), and to ii) the actual change.
  • a GLP-1 analogue according to the invention is a GLP-1 (7-37) peptide, in which a number of amino acid residues have been changed when compared to native GLP-1 (7-37).
  • 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 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 used to describe analogues. Formulae of peptide analogues and derivatives thereof are drawn using standard single letter or three letter abbreviations for amino acids used according to lUPAC-IUB nomenclature.
  • Apolipoproteins or "apo” or “Apo” refers to any of the several water soluble proteins that combine with lipid to form lipoproteins. These lipoproteins can be separated by size or by flotation densities and generally classified as chylomicrons, VLDL, LDL and HDL. Apolipoproteins include non-exchangeable protein Apo B and exchangeable proteins e.g. Apo A-l, Apo A-ll, Apo A-IV, Apo C-l, Apo C-ll, Apo C-lll, Apo E, and serum amyloid proteins such as serum amyloid A.
  • apolipoprotein A-l refers to a polypeptide comprising 243 amino acids forming N and C-terminal domains. Residues 44-243 of ApoA-l contain the necessary structural determinants for mediating cholesterol efflux via ABCA1 or other ABC transporters. This region of ApoA-l (aa44-243) is comprised of a series of ten amphipathic alpha-helices of 1 1- or 22-amino acids. The individual alpha-helical segments of ApoA-l are defined, in part, by the relative distribution of positively charged residues and are designated as Class A or Y. Class A helices possess positively charged amino acid toward the middle of the polar surface in addition to interfacial cationic residues. Additional definitions
  • conservative substitution refers to substitution of one peptide amino acid residue with another amino acid residue with similar characteristics such as charge, size, hydrophobicity, hydrophilicity, presence of identical functional group (eg. hydroxyl group) and/or aromaticity, or when both residues are classified as lipophilic amino acid residues (non-limiting examples are Ser with Thr, Lys with Arg, Phe with Trp and Asp with Glu), and includes exchanges within the following four groups:
  • ABC ATP Binding Casette
  • lipids e.g. cholesterol and phospholipids
  • ABC transporters are trans-membrane proteins that utilize the energy of adenosine triphosphate (ATP) hydrolysis to carry out certain biological processes including translocation of various substrates across membranes. They transport a wide variety of substrates across extra- and intracellular membranes, including metabolic products, lipids and sterols, and drugs. Proteins are classified as ABC
  • ABS ATP-binding cassette
  • ABC transporters There are 48 known ABC transporters present in humans, which are classified into seven families by the Human Genome Organization.
  • the ABCA family contains some of the largest transporters (over 2, 100 amino acids long). Five of them are located in a cluster in the 17q24 chromosome. These transporters are responsible for the transportation of cholesterol and lipids, among other things. Examples are ABCA1 and ABCA12.
  • the ABCG family also transports lipids, diverse drug substrates, bile, cholesterol, and other steroids. Examples are ABCG1 and ABCG2.
  • ABC1 refers to the ATP-binding cassette transporter ABCA1 (member 1 of human transporter sub-family ABCA), also known as the cholesterol efflux regulatory protein (CERP) is a protein which in humans is encoded by the ABCA1 gene. This transporter is a major regulator of cellular cholesterol and phospholipid homeostasis.
  • amino acid abbreviations used in the present context have the following meanings:
  • the GLP-1 receptor agonist analogues of the invention have GLP-1 activity. This term refers to the ability to bind to the GLP-1 receptor and initiate a signal transduction pathway resulting in an insulinotropic action or other physiological effects as is known in the art.
  • the analogues of the invention can be tested for GLP-1 activity using the assay described in Example 2 (in vitro). Cholesterol efflux
  • Macrophage or foam cells in the artery wall release or export cholesterol to acceptors, such as apolipoproteins and/or HDL or the peptides of the current invention.
  • acceptors such as apolipoproteins and/or HDL or the peptides of the current invention.
  • a compound that mediates cholesterol efflux enhances the release of cholesterol out of the cell and into the extracellular compartment.
  • Cholesterol efflux is often accompanied by the efflux of phospholipids from the cell.
  • the coordinated release of both cholesterol and phospholipids produces HDL in the presence of a suitable lipid acceptor, eg. apolipoprotein or peptide. Therefore, the processes of cholesterol-and phospholipid efflux are linked and synonymous with one another.
  • ABCA1 -dependent lipid efflux refers to a process whereby apolipoproteins or peptides that facilitate cholesterol efflux, interact with a cell or vesicle and efflux lipid from the cell by a process that is facilitated by the ABCA1 transporter.
  • the current invention relates to analogues of GLP-1 receptor agonists that promote cholesterol efflux.
  • the term "cholesterol efflux” or “cholesterol efflux activity” indicates the capability to induce efflux of cholesterol from a macrophage cell line as described in Example 3.
  • Preferred compounds of the invention show an efficacy measured as E max of at least 65%, or at least 70%, or at least 75%, or at least 80%, of that of apolipoprotein A-l mimetic peptide L-4F, and a potency measured as EC 50 better than the potency of L-4F measured as described in Example 3.
  • L-4F means an ApoA-l mimetic, i.e. L-4F, also referred to as 4F or L4F, which is a synthetic mimetic containing four phenylalanine amino acids (see e.g. Datta G et al; J. Lipid Res. 2001 42 (7) 1096-1 104).
  • the cholesterol efflux potency can be expressed as the EC 50 value, defined as the half maximal effective concentration, and refers to the concentration of a drug, antibody or toxicant, which induces a response halfway between the baseline and maximum after a specified exposure time. It is commonly used as a measure of the potency of a drug.
  • Y Bottom + exercise program—— -— where Y is the observed value, Bottom is the lowest observed value, Top is the highest observed value (which equals E max ), and the Hill coefficient gives the largest absolute value of the slope of the curve.
  • RCT refers to the mediation of cholesterol efflux from cells of the arterial wall to the liver or other steroidogenic organs.
  • the reverse cholesterol transport pathway has three main steps, i) cholesterol efflux, i.e. the initial removal of cholesterol from various pools of peripheral cells; ii) cholesterol esterification by the action of lechitin cholesterol acyltransferase (LCAT), thereby preventing re-entry of effluxed cholesterol into cells; iii) uptake of the cholesteryl ester by HDL and deloivery of the cholesteryl ester complex to liver cells. Enhancement of cholesterol efflux and of reverse cholesterol transport (RCT) is considered an important target for anti-atherosclerotic drug therapy.
  • LCAT lechitin cholesterol acyltransferase
  • the analogues and intermediate products of the invention may be in the form of a pharmaceutically acceptable salt, amide, or ester.
  • Salts are e.g. formed by a chemical reaction between a base and an acid, e.g. 2NH 3 + H 2 S0 4 ⁇ (NH 4 ) 2 S0 4 .
  • the salt may be a basic salt, an acid salt, or it may be neither nor (i.e. a neutral salt).
  • Basic salts produce hydroxide ions and acid salts hydronium ions in water.
  • the salts of the analogues of the invention may be formed with added cations or anions between anionic or cationic groups, respectively. These groups may be situated in the peptide moiety, and/or in the side chain of the analogues of the invention.
  • Non-limiting examples of anionic groups of the analogues of the invention include free carboxylic groups in the side chain, if any, as well as in the peptide moiety.
  • the peptide moiety often includes a free carboxylic acid group at the C-terminus, and it may also include free carboxylic groups at internal acid amino acid residues such as Asp and Glu.
  • Non-limiting examples of cationic groups in the peptide moiety include the free amino group at the N-terminus, if present, as well as any free amino group of internal basic amino acid residues such as His, Arg, and Lys.
  • the ester of the analogues of the invention may, e.g., be formed by the reaction of a free carboxylic acid group with an alcohol or a phenol, which leads to replacement of at least one hydroxyl group by an alkoxy or aryloxy group.
  • the ester formation may involve the free carboxylic group at the C-terminus of the peptide, and/or any free carboxylic group in the side chain.
  • the amide of the analogues of the invention may, e.g., be formed by the reaction of a free carboxylic acid group with an amine or a substituted amine, or by reaction of a free or substituted amino group with a carboxylic acid.
  • the amide formation may involve the free carboxylic group at the C-terminus of the peptide, any free carboxylic group in the side chain, the free amino group at the N-terminus of the peptide, and/or any free or substituted amino group of the peptide in the peptide and/or the side chain.
  • the peptide is in the form of a pharmaceutically acceptable salt. In another particular embodiment, the peptide is in the form of a
  • the peptide is in the form a pharmaceutically acceptable ester.
  • pharmaceutical composition means a product comprising an active compound, or a salt thereof, together with pharmaceutically acceptable excipients, such as buffers, preservatives, and optionally a tonicity modifier and/or a stabilizer.
  • pharmaceutically acceptable as used herein means suited for normal pharmaceutical applications, i.e. giving rise to no adverse events in patients etc.
  • excipient means a chemical that is normally added to pharmaceutical compositions, e.g. buffers, tonicity agents, preservatives and the like.
  • an effective amount means a dosage which is sufficient to be effective for the treatment of the patient compared with no treatment.
  • treatment of a disease means the management and care of a patient having developed the disease, condition or disorder and includes treatment, prevention or alleviation of the disease.
  • the purpose of a treatment is to combat a 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, and prevention of the disease, condition or disorder.
  • diabetes or "diabetes mellitus” includes type 1 diabetes, type 2 diabetes, gestational diabetes (during pregnancy) and other states that cause
  • hyperglycaemia hyperglycaemia.
  • the term is used for a metabolic disorder in which the pancreas produces insufficient amounts of insulin, or in which the cells of the body fail to respond appropriately to insulin thus preventing cells from absorbing glucose. As a result, glucose builds up in the blood.
  • Type 1 diabetes also called insulin-dependent diabetes mellitus (IDDM) and juvenile-onset diabetes, is caused by beta-cell destruction, usually leading to absolute insulin deficiency.
  • IDDM insulin-dependent diabetes mellitus
  • juvenile-onset diabetes is caused by beta-cell destruction, usually leading to absolute insulin deficiency.
  • Type 2 diabetes also known as non-insulin-dependent diabetes mellitus (NIDDM) and adult-onset diabetes, is associated with predominant insulin resistance and thus relative insulin deficiency and/or a predominantly insulin secretory defect with insulin resistance.
  • NIDDM non-insulin-dependent diabetes mellitus
  • adult-onset diabetes is associated with predominant insulin resistance and thus relative insulin deficiency and/or a predominantly insulin secretory defect with insulin resistance.
  • cardiovascular disease refers to a class of diseases that involve the heart or blood vessels (arteries, capillaries and veins). Cardiovascular disease refers to any disease that affects the cardiovascular system, principally cardiac disease, vascular diseases of the brain and kidney, and peripheral arterial disease. The causes of tha cardiovascular disease may be diverse, but atherosclerosis and/or hypertension are the most common.
  • Types of CVD include, coronary heart disease (also ischaemic heart disease or coronary artery disease), cardiomyopathy (diseases of cardiac muscle), hypertensive heart disease (diseases of the heart secondary to high blood pressure), heart failure, coronary heart disease, pulmonale (a failure of the right side of the heart), cardiac dysrhythmias (abnormalities of heart rhythm), inflammatory heart disease (such as endocarditis (inflammation of the inner layer of the heart, the endocardium), inflammatory cardiomegaly and myocarditis (inflammation of the myocardium, the muscular part of the heart), valvular heart disease, stroke and cerebrovascular disease; and peripheral arterial disease.
  • coronary heart disease also ischaemic heart disease or coronary artery disease
  • cardiomyopathy diseases of cardiac muscle
  • hypertensive heart disease diseases of the heart secondary to high blood pressure
  • heart failure coronary heart disease
  • pulmonale a failure of the right side of the heart
  • the peptides of the present invention may be administered to an animal or human suffering from a dyslipidemic or vascular disorder, such as hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, hyperlipoproteinemia, HDL deficiency, apoA-l deficiency, coronary artery disease, atherosclerosis, stroke, ischemia, infarction, myocardial infarction, hemorrhage, periferal vascular disease, restenosis, acute coronary syndrome, or reperfusion myocardial injury, in an amount sufficient to inhibit or treat the dyslipidemic or vascular disorder. Amounts effective for this use will depend upon the severity of the disorder and the general state of the subject's health. A therapeutically effective amount of the peptide is that which provides either subjective relief of a symptom(s) or an objective identifiable improvement as noted by the clinician or other qualified observer.
  • a dyslipidemic or vascular disorder such as hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, hyperlip
  • Dyslipidemia refers to a disorder associated with any altered amount of any or all of the lipids or lipoproteins in the blood.
  • Dyslipidemic disordes include hyperlipidemia, hyperlipoproteinemia, hypercholesterolemia, hypertriglyceridemia, HDL deficiency, apoA-l deficiency, and cardiovascular disease (e.g. coronary artery disease, atherosclerosis and restenosis).
  • cardiovascular disease e.g. coronary artery disease, atherosclerosis and restenosis.
  • the peptides of the present invention can be used in a combination therapy together with statins (HMG-CoA reductase inhibitors) e.g. atorvastatin (Lipitor and Torvast), fluvastatin (Lescol), lovastatin (Mevacor, Altocor, Altoprev), pitavastatin (Livalo, Pitava), pravastatin (Pravachol, Selektine, Lipostat), rosuvastatin (Crestor) or simvastatin, or fibrates Bezafibrate (e.g. Bezalip), Ciprofibrate (e.g. Modalim), Gemfibrozil (e.g. Lopid), Fenofibrate (e.g. TriCor) to treat hyperlipidemia,
  • statins HMG-CoA reductase inhibitors
  • statins HMG-CoA reductase inhibitors
  • statins HMG-CoA reductase inhibitors
  • hypertriglyceridemia hypercholesterolemia
  • cardiovascular disease such as atherosclerosis
  • the peptides of the present invention can be used in combination with anti-microbial and/or anti-inflammatory agents such as, for example, but not limited to, aspirin.
  • anti-microbial and/or anti-inflammatory agents such as, for example, but not limited to, aspirin.
  • the peptides of the present invention can be used in combination with anti-hypertensive medicines known to one of ordinary skill in the art. It is to be understood that more than one additional therapy may be combined with administration of the peptides of the present invention.
  • potency and/or activity refers to in vitro potency, i.e. performance in a functional GLP-1 receptor assay, more in particular to the capability of activating the human GLP-1 receptor.
  • the response of the human GLP-1 receptor may be measured in a reporter gene assay, e.g. in a stably transfected BHK cell line that expresses the human GLP-1 receptor and contains the DNA for the cAMP response element (CRE) coupled to a promoter and the gene for firefly luciferase (CRE luciferase).
  • CRE cAMP response element
  • Luciferase may be determined by adding luciferin, which by the enzyme is converted to oxyluciferin and produces bioluminescence, which is measured and is a measure of the in vitro potency.
  • luciferin which by the enzyme is converted to oxyluciferin and produces bioluminescence, which is measured and is a measure of the in vitro potency.
  • bioluminescence which is measured and is a measure of the in vitro potency.
  • EC 50 half maximal effective concentration
  • EC 5 o is used as a measure of the potency of a compound and represents the concentration where 50% of its maximal effect is observed.
  • the in vitro potency of the peptides of the invention may be determined as described above, and the EC 5 o of the peptide in question determined. The lower the EC 5 o value, the better the potency.
  • the peptide of the invention has an in vitro potency determined using the method of Example 2, corresponding to an EC 50 at or below 10000 pM, more preferably below 5000 pM, even more preferably below 1000 pM, or most preferably below 500 pM.
  • Cholesterol efflux activity may be assessed in vitro by measuring the capacity of compounds to efflux cholesterol from macrophage cell line, primarily transported via the ABCA1 transporter.
  • the cholesterol efflux activity is determined in vitro as described in Example 3.
  • ABCA1 -mediated efflux may be obtained from the difference between induced efflux and non-induced efflux.
  • EC 50 values which were calculated by the software and reported in ⁇ are shown in Table 1 , as well as E max values expressed in %.
  • potency and/or activity refers to in vitro potency, i.e. performance in an assay determining cholesterol efflux, more in particular in a cell or tissue based assay measuring the efflux of cholesterol out of the cells.
  • cells like mouse monocyte/macrophage cell line, RAW 264.7 or other cells like but not limited to THP-1 , BHK cells transfected with the ABCA1 (and/or ABCG1 ) transporter or other monocyte or macrophage primary cells or cell lines can be used for establishment of a cholesterol efflux assay.
  • cAMP can be used to up-regulate the ABCA1 transporter allowing the measurement of cholesterol efflux mediated specifically by the ABCA1 transporter.
  • the cholesterol efflux may be measured by incubating the cells with 3H-Cholesterol and subsequently measuring the amount of cholesterol effluxed to the media by measuring the radioactivity of the labelled cholesterol effluxed into the media.
  • Non-specific cholesterol efflux can be measured in non-induced cells (i.e. cells not induced by cAMP).
  • ABCA1 -mediated efflux can be obtained from the difference between induced efflux and non-induced efflux.
  • the peptide of the invention has an in vitro potency determined using the method of Example 3 corresponding to an EC 50 potency at or below 2 ⁇ , even more preferably at or below 1 ⁇ , or most preferably below 0.8 ⁇ .
  • the peptide of the invention has an in vitro E max , as determined by the method of Example 3, at or above 65% of the E max of L-4F, or most preferably at or above 75% of the E max of L-4F.
  • the cholesterol efflux potency of the compounds of the present invention of the present invention can also be expressed relative to that of L-4F.
  • the cholesterol efflux potency of the compounds of the present invention has an EC 50 value at or below that of L-4F.
  • the peptides of the invention or analogues thereof are potent in vivo, which may be determined as is known in the art in any suitable animal model, as well as in clinical trials.
  • the diabetic db/db mouse is one example of a suitable animal model, and the blood glucose lowering effect may be determined in such mice in vivo.
  • the peptides of the invention have improved pharmacokinetic properties compared to hGLP-1 .
  • the peptides of the invention have pharmacokinetic properties suitable for once daily administration.
  • the pharmacokinetic properties may be determined as terminal half-life (T 1 ⁇ 2 ) in vivo in mice after i.v. and s.c. administration.
  • the terminal half-life is at least 1 hour, preferably at least 3 hours, preferably at least 4 hours, even more preferably at least 5 hours, or most preferably at least 6 hours.
  • the peptides of the invention have improved pharmacokinetic properties compared to hGLP-1 and preferably suitable for once daily administration.
  • the pharmacokinetic properties may be determined as terminal half-life (T 1 ⁇ 2 ) in vivo in mini-pigs after i.v. administration.
  • the terminal half-life in mini- pigs is at least 5 hours, preferably at least 10 hours, even more preferably at least 15 hours, or most preferably at least 20 hours.
  • the GLP-1 receptor agonist peptide of the invention may be obtained by conventional methods for the preparation of peptides and peptide derivatives, and in particular according to the methods described in the working examples.
  • the GLP-1 moiety of the invention may for instance be produced by classical peptide synthesis, e.g., solid phase peptide synthesis using t-Boc or Fmoc chemistry or other well established techniques, see, e.g., Greene and Wuts,
  • telomeres may be produced by recombinant methods, viz. by culturing a host cell containing a DNA sequence encoding the analogue and capable of expressing the peptide in a suitable nutrient medium under conditions permitting the expression of the peptide.
  • host cells suitable for expression of these peptides are: Escherichia coli, Saccharomyces cerevisiae, as well as mammalian BHK or CHO cell lines.
  • peptides of the invention which include non-natural amino acids and/or a covalently attached N-terminal mono- or dipeptide mimetic may e.g. be produced as described in the experimental part, or as described by Hodgson et al: The synthesis of peptides and proteins containing non-natural amino acids; Chemical Society Reviews 2004 33 (7) 422-430; or as described in WO 2009/083549 A1 entitled "Semi-recombinant preparation of GLP-1 analogues".
  • compositions comprising a peptide of the invention or a
  • pharmaceutically acceptable salt, amide, or ester thereof, and a pharmaceutically acceptable excipient may be prepared as is known in the art.
  • excipient broadly refers to any component other than the active therapeutic ingredient(s).
  • the excipient may be an inert substance, an inactive substance, and/or a not medicinally active substance.
  • the excipient may serve various purposes, e.g. as a carrier, vehicle, diluent, and/or to improve administration, and/or absorption of the active substance.
  • Non-limiting examples of excipients are: Solvents, diluents, buffers, preservatives, tonicity regulating agents, chelating agents, and stabilisers.
  • formulations include liquid formulations, i.e. aqueous formulations comprising water.
  • a liquid formulation may be a solution, or a suspension.
  • An aqueous formulation typically comprises at least 50% w/w water, or at least 60%, 70%, 80%, or even at least 90% w/w of water.
  • the pH in an aqueous formulation may be anything between pH 3 and pH 10, for example from about 7.0 to about 9.5; or from about 3.0 to about 9.0.
  • a pharmaceutical composition may comprise a buffer.
  • a pharmaceutical composition may comprise a preservative.
  • a pharmaceutical composition may comprise a chelating agent.
  • the chelating agent may e.g. be selected from salts of
  • EDTA ethylenediaminetetraacetic acid
  • citric acid citric acid
  • aspartic acid citric acid
  • a pharmaceutical composition may comprise a stabiliser.
  • the stabiliser may e.g. be one or more oxidation inhibitors, aggregation inhibitors, surfactants, and/or one or more protease inhibitors.
  • Non-limiting examples of these various kinds of stabilisers are disclosed in the following.
  • aggregate formation refers to a physical interaction between the polypeptide molecules resulting in formation of oligomers, which may remain soluble, or large visible aggregates that precipitate from the solution. 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.
  • a pharmaceutical composition may comprise an amount of an amino acid base sufficient to decrease aggregate formation of the polypeptide during storage of the composition.
  • amino acid base refers to one or more amino acids (such as methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), or analogues thereof. Any amino acid may be present either in its free base form or in its salt form. Any stereoisomer (i.e., L, D, or a mixture thereof) of the amino acid base may be present.
  • 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. Any stereoisomer of methionine (L or D) or combinations thereof can be used.
  • a pharmaceutical composition may comprise a stabiliser selected from the group of high molecular weight polymers or low molecular compounds.
  • composition may comprise additional stabilising agents such as, but 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.
  • additional stabilising agents such as, but 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.
  • a pharmaceutical composition may comprise one or more surfactants.
  • surfactant refers to any molecules or ions that are comprised of a water-soluble surfactant.
  • the surfactant may e.g. be selected from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants, and/or zwitterionic surfactants.
  • a pharmaceutical composition may comprise one or more protease inhibitors. Additional, optional, ingredients of a pharmaceutical composition include, e.g., wetting agents, emulsifiers, antioxidants, bulking agents, metal ions, oily vehicles, proteins (e.g., human serum albumin, gelatine), and/or a zwitterion (e.g., an amino acid such as betaine, taurine, arginine, glycine, lysine and histidine).
  • a zwitterion e.g., an amino acid such as betaine, taurine, arginine, glycine, lysine and histidine.
  • An administered dose may contain from 0.01 mg - 100 mg of the peptide or from 0.1-50 mg, or from 1 -25 mg of the peptide.
  • the GLP-1 receptor agonist peptide of the present invention may be administered in the form of a pharmaceutical composition. It may be administered to a patient in need thereof at several sites, for example, at topical sites such as skin or mucosal sites; at sites which bypass absorption such as in an artery, in a vein, or in the heart; and at sites which involve absorption, such as in the skin, under the skin, in a muscle, or in the abdomen.
  • the route of administration may be, for example, lingual; sublingual; buccal; in the mouth; oral; in the stomach; in the intestine; nasal; pulmonary, such as through the bronchioles, the alveoli, or a combination thereof; parenteral, epidermal; dermal;
  • transdermal transdermal; conjunctival; uretal; vaginal; rectal; and/or ocular.
  • a composition may be administered in several dosage forms, for example as a solution; a suspension; an emulsion; a microemulsion; multiple emulsions; a foam; a salve; a paste; a plaster; an ointment; a tablet; a coated tablet; a chewing gum; a rinse; a capsule such as hard or soft gelatine capsules; a suppositorium; a rectal capsule; drops; a gel; a spray; a powder; an aerosol; an inhalant; eye drops; an ophthalmic ointment; an ophthalmic rinse; a vaginal pessary; a vaginal ring; a vaginal ointment; an injection solution; an in situ transforming solution such as in situ gelling, setting, precipitating, and in situ crystallisation; an infusion solution; or as an implant.
  • a composition may be a tablet, optionally coated, a capsule, or a chewing gum.
  • a composition may further be compounded in a drug carrier or drug delivery system, e.g. in order to improve stability, bioavailability, and/or solubility.
  • a composition may be attached to such system through covalent, hydrophobic, and/or electrostatic interactions.
  • the purpose of such compounding may be, e.g., to decrease adverse effects, achieve chronotherapy, and/or increase patient compliance.
  • a composition may also be used in the formulation of controlled, sustained, protracting, retarded, and/or slow release drug delivery systems.
  • Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal, or intravenous injection by means of a syringe, optionally a pen-like syringe, or by means of an infusion pump.
  • a composition may be administered nasally in the form of a solution, a suspension, or a powder; or it may be administered pulmonally in the form of a liquid or powder spray.
  • Transdermal administration is a still further option, e.g. by needle-free injection, from a patch such as an iontophoretic patch, or via a transmucosal route, e.g. buccally.
  • a composition may be a stabilised formulation.
  • stabilized formulation refers to a formulation with increased physical and/or chemical stability, preferably both. 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.
  • the term "physical stability” refers to the tendency of the polypeptide to form biologically inactive and/or insoluble aggregates as a result of exposure to thermo- mechanical stress, and/or interaction with destabilising interfaces and surfaces (such as hydrophobic surfaces).
  • the physical stability of an aqueous polypeptide formulation may be evaluated by means of visual inspection, and/or by turbidity measurements after exposure to mechanical/physical stress (e.g. agitation) at different temperatures for various time periods.
  • the physical stability may be evaluated using a spectroscopic agent or probe of the conformational status of the polypeptide such as e.g. Thioflavin T or "hydrophobic patch" probes.
  • chemical stability refers to chemical (in particular covalent) changes in the polypeptide structure leading to formation of chemical degradation products potentially having a reduced biological potency, and/or increased immunogenic effect as compared to the intact polypeptide.
  • the chemical stability can be evaluated by measuring the amount of chemical degradation products at various time-points after exposure to different
  • the treatment with a peptide according to the present invention may also be combined with one or more additional 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.
  • additional 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.
  • Insulins and insulin analogues such as but not limited to Lantus also known as insulin glargine, sulphonylureas, biguanides, meglitinides, glucosidase inhibitors, glucagon antagonists, DPP-IV (dipeptidyl peptidase-IV) inhibitors, inhibitors of hepatic enzymes involved in stimulation of
  • gluconeogenesis and/or glycogenolysis glucose uptake modulators, compounds modifying the lipid metabolism such as antihyperlipidemic agents as HMG CoA inhibitors (statins), compounds lowering food intake, RXR agonists and agents acting on the ATP-dependent potassium channel of the ⁇ -cells; 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) inhibitors such as benazepril, captopril, enalapril, fosinopril, lisinopril, alatriopril, quinapri
  • lipase/amylase inhibitors examples include RXR (retinoid X receptor) modulators, TR ⁇ agonists; histamine H3 antagonists.
  • the treatment with a peptide according to this invention may also be combined with a surgery that influences the glucose levels, and/or lipid homeostasis such as gastric banding or gastric bypass.
  • the present invention also relates to analogues of GLP-1 receptor agonist peptides for use as medicaments.
  • the GLP-1 receptor analogues of the invention may in particular find use in the treatment of diseases or disorders relating to diabetes or to cardiovascular diseases, or to a combination of diabetes and cardiovascular diseases, and in particular (i) prevention and/or treatment of all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes, MODY (maturity onset diabetes of the young), gestational diabetes, and/or for reduction of HbA1 C;
  • diabetes delaying or preventing diabetic disease progression, such as progression in type 2 diabetes, delaying the progression of impaired glucose tolerance (IGT) to insulin requiring type 2 diabetes, and/or delaying the progression of non-insulin requiring type 2 diabetes to insulin requiring type 2 diabetes;
  • ITT impaired glucose tolerance
  • diabetes prevention and/or treatment of diabetic complications, such as neuropathy, including peripheral neuropathy; nephropathy; or retinopathy;
  • dyslipidemia lowering total serum lipids; lowering HDL; lowering small, dense LDL; lowering VLDL: lowering triglycerides; lowering cholesterol; increasing HDL; lowering plasma levels of lipoprotein a (Lp(a)) in a human; inhibiting generation of apolipoprotein a (apo(a));
  • dysrhythmia syncopy; atheroschlerosis; mild chronic heart failure; angina pectoris; cardiac bypass reocclusion; intermittent claudication (atheroschlerosis oblitterens); diastolic dysfunction; and/or systolic dysfunction; (ix) prevention and/or treatment of gastrointestinal diseases, such as inflammatory bowel syndrome; small bowel syndrome, or Crohn's disease; dyspepsia; and/or gastric ulcers;
  • x prevention and/or treatment of critical illness, such as treatment of a critically ill patient, a critical illness poly-nephropathy (CIPNP) patient, and/or a potential CIPNP patient; prevention of critical illness or development of CIPNP; prevention, treatment and/or cure of systemic inflammatory response syndrome (SIRS) in a patient; and/or for the prevention or reduction of the likelihood of a patient suffering from bacteraemia,
  • critical illness such as treatment of a critically ill patient, a critical illness poly-nephropathy (CIPNP) patient, and/or a potential CIPNP patient
  • SIRS systemic inflammatory response syndrome
  • the indication is selected from the group consisting of
  • the indication is (i). In a further particular embodiment the indication is (v). In a still further particular embodiment the indication is
  • Type 2 diabetes and/or obesity and/or cardiovascular disease, especially atherosclerosis.
  • EMBODIMENTS are particularly preferred: Type 2 diabetes, and/or obesity and/or cardiovascular disease, especially atherosclerosis.
  • the GLP-1 receptor agonist analogue of the invention showing a cholesterol efflux in vitro EC 50 potency of less than 3 ⁇ , less than 2 ⁇ , less than 1 ⁇ or less than
  • the GLP-1 receptor agonist analogue of the invention showing a cholesterol efflux in vitro EC 50 potency of less than 2 ⁇ .
  • the GLP-1 receptor agonist analogue according of the invention showing a cholesterol efflux in vitro EC 50 potency of less than 1 ⁇ .
  • the GLP-1 receptor agonist analogue of the invention showing a cholesterol efflux in vitro EC 50 potency of less than 0.5 ⁇ .
  • the GLP-1 receptor agonist analogue of the invention showing a GLP-1 in vitro potency of at least 25%, between 10% and 25%, or between 1 % and 10% of that of native GLP-1 .
  • the GLP-1 receptor agonist analogue of the invention showing a GLP-1 in vitro potency of at least 25%of that of native GLP-1.
  • the GLP-1 receptor agonist analogue of the invention showing a GLP-1 in vitro potency of between 10% and 25% of that of native GLP-1 .
  • the GLP-1 receptor agonist analogue of the invention showing a GLP-1 in vitro potency of between 1 % and 10% of that of native GLP-1.
  • the GLP-1 receptor agonist analogue of the invention showing a cholesterol efflux in vitro EC 50 potency of less than 3 ⁇ and a GLP-1 in vitro potency of at least 25%, between 10% and 25% or between 1 % and 10% of that of GLP-1 .
  • the GLP-1 receptor agonist analogue of the invention showing a cholesterol efflux in vitro EC 50 potency of less than 2 ⁇ and a GLP-1 in vitro potency of at least 25%, between 10% and 25% or between 1 % and 10% of that of GLP-1 .
  • the GLP-1 receptor agonist analogue of the invention showing a cholesterol efflux in vitro EC 50 potency of less than 1 ⁇ and a GLP-1 in vitro potency of at least 25%, between 10% and 25% or between 1 % and 10% of that of GLP-1 .
  • the GLP-1 receptor agonist analogue of the invention showing a cholesterol efflux in vitro EC 50 potency of less than 0.5 ⁇ and a GLP-1 in vitro potency of at least 25%, between 10% and 25% or between 1 % and 10% of that of GLP-1 .
  • the GLP-1 receptor agonist analogue of the invention showing shows an in vitro E max , as determined by the method of Example 3, at or above 65% of the E max of L-4F; or at or above 75% of the E max of L-4F.
  • GLP-1 receptor agonist analogue of the invention wherein said GLP-1 receptor agonist peptide is for use as an anti-inflammatory agent.
  • the GLP-1 receptor agonist analogue of the invention which GLP-1 receptor agonist peptide reduces insulin resistance in diabetic patients.
  • the GLP-1 receptor agonist analogue of the invention which GLP-1 receptor agonist peptide improves HbA1 C levels in diabetic patients.
  • the GLP-1 receptor agonist analogue of the invention which GLP-1 receptor agonist peptide improves HbA1 C levels in diabetic patients, with 1 %.
  • the GLP-1 receptor agonist analogue of the invention which lowers HbA1 c in diabetes patients with at least 0.5%.
  • the GLP-1 receptor agonist analogue of the invention wherein the terminal half-life of said peptide is prolonged.
  • the GLP-1 receptor agonist analogue of the invention, wherein the terminal half-life of said peptide in mini-pigs is at least 5 hours, at least 10 hours, at least 15 hours or at least 20 hours.
  • a method for treating and/or preventing diseases or states associated with dyslipidemia, inflammation and vascular disorder such as cardiovascular disease, endothelial dysfunction, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, hyperlipoproteinemia, HDL deficiency, apoA-l deficiency, coronary artery disease, atherosclerosis, hypertension, stroke, ischemia, infarction, myocardial infarction, hemorrhage, periheralperiferal vascular disease, restenosis, acute coronary syndrome, or reperfusion myocardial injury, macrovascular disorder and microvascular disorder; or treating, in an diabetes patient, a disease or state selected from cardiovascular disease, endothelial dysfunction, a macrovascular disorder, microvascular disorder, atherosclerosis and hypertension - by administering a pharmaceutically active amount of the GLP-1 receptor agonist analogue of the invention.
  • diseases or states associated with dyslipidemia, inflammation and vascular disorder such as cardiovascular disease, endothelial dysfunction, hyperlipidemia, hypert
  • a method of treating or preventing a disease or state associated with dyslipidemia, hypercholesterolemia and inflammation comprising administering to a patient in need thereof an effective amount of the GLP-1 receptor agonist analogue of the invention, optionally in combination with one or more additional therapeutically active compounds.
  • a method according to any one of the embodiments 25-27 comprising administering to a patient in need thereof an effective amount of the GLP-1 receptor agonist analogue of the invention, or a pharmaceutical composition according to the present invention, optionally in combination with one or more additional therapeutically active compounds.
  • a method of treating, in a diabetes patient, a disease or state selected from cardiovascular disease, endothelial dysfunction, a macrovascular disorder, microvascular disorder, atherosclerosis and hypertension comprising administering to a diabetes patient in need thereof an effective amount of the GLP-1 receptor agonist analogue of the invention, optionally in combination with one or more additional therapeutically active compounds.
  • a pharmaceutical composition comprising the GLP-1 receptor agonist analogue of the invention.
  • composition according to embodiment 26 which further comprises a pharmaceutical acceptable carrier and/or excipient.
  • the GLP-1 receptor agonist analogue of the invention for use in therapy.
  • the GLP-1 receptor agonist analogue of the invention for use in the treatment of diseases or states associated with dyslipidemia, hypercholesterolemia and inflammation, such as cardiovascular disease, endothelial dysfunction, macrovascular disorder, microvascular disorder, atherosclerosis and hypertension; or treating, in an diabetes patient, a disease or state selected from cardiovascular disease, endothelial dysfunction, a macrovascular disorder, microvascular disorder, atherosclerosis and hypertension.
  • diseases or states associated with dyslipidemia, hypercholesterolemia and inflammation such as cardiovascular disease, endothelial dysfunction, macrovascular disorder, microvascular disorder, atherosclerosis and hypertension
  • a disease or state selected from cardiovascular disease, endothelial dysfunction, a macrovascular disorder, microvascular disorder, atherosclerosis and hypertension such as a
  • cardiovascular disease endothelial dysfunction
  • a macrovascular disorder a macrovascular disorder
  • microvascular disorder a macrovascular disorder
  • atherosclerosis a macrovascular disorder
  • hypertension a vascular hypertension
  • the GLP-1 receptor agonist analogue of the invention for use as a
  • GLP-1 receptor agonist analogue of the invention in the manufacture of a medicament for treating and/or preventing diseases or states associated with
  • dyslipidemia, inflammation and vascular disorder such as cardiovascular disease, endothelial dysfunction, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, hyperlipoproteinemia, HDL deficiency, apoA-l deficiency, coronary artery disease, atherosclerosis, hypertension, stroke, ischemia, infarction, myocardial infarction, hemorrhage, periheralperiferal vascular disease, restenosis, acute coronary syndrome, or reperfusion myocardial injury, macrovascular disorder and microvascular disorder; or treating, in an diabetes patient, a disease or state selected from cardiovascular disease, endothelial dysfunction, a macrovascular disorder, microvascular disorder, atherosclerosis and hypertension.
  • Aib oaminoisobutyric acid (2-aminoisobutyric API: Active Pharmaceutical Ingredient
  • BSA Bovine serum albumin
  • DesH des-amino histidine (may also be referred to as imidazopropionic acid, Imp)
  • DIPEA diisopropylethylamine
  • DMEM Dulbecco's Modified Eagle's Medium
  • EDTA ethylenediaminetetraacetic acid
  • EGTA ethylene glycol tetraacetic acid
  • HSA Human Serum Albumin
  • Imp Imidazopropionic acid (also referred to as des-amino histidine,
  • ivDde 1 -(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl
  • MALDI-MS See MALDI-TOF MS
  • MALDI-TOF MS Matrix-Assisted Laser Desorption/lonisation Time of Flight Mass
  • NMP N-methyl pyrrolidone
  • OEG 8-amino-3,6-dioxaoctanic acid
  • PBS Phosphate Buffered Saline
  • Pen/Strep Penicillin/Streptomycin
  • TIS triisopropylsilane
  • Tris tris(hydroxymethyl)aminomethane or 2-amino-2-hydroxymethyl- propane-1 ,3-diol
  • This section relates to methods for solid phase peptide synthesis (SPPS methods, including methods for de-protection of amino acids, methods for cleaving the peptide from the resin, and for its purification), as well as methods for detecting and characterising the resulting peptide (LCMS, MALDI, and UPLC methods).
  • SPPS methods including methods for de-protection of amino acids, methods for cleaving the peptide from the resin, and for its purification), as well as methods for detecting and characterising the resulting peptide (LCMS, MALDI, and UPLC methods).
  • the solid phase synthesis of peptides may in some cases be improved by the use of di-peptides protected on the di-peptide amide bond with a group that can be cleaved under acidic conditions such as, but not limited to, 2-Fmoc-oxy-4-methoxybenzyl, or 2,4,6- trimethoxybenzyl.
  • a serine or a threonine is present in the peptide
  • pseudoproline di-peptides may be used (available from, e.g., Novabiochem, see also W.R. Sampson et al, J. Pep. Sci. 1999 5 403-409).
  • Fmoc-protected amino acid derivatives used were the standard recommended: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)- OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-lle-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc- Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc- Trp(Boc)-OH, Fm
  • N-terminal amino acid was Boc protected at the alpha amino group (e.g. Boc-His(Boc)-OH, or Boc-His(Trt)-OH for peptides with His at the N-terminus).
  • the following suitably protected building blocks such as but not limited to Fmoc-8-amino-3,6-dioxaoctanoic acid, Fmoc-tranexamic acid, Fmoc- Glu-OtBu, octadecanedioic acid mono-tert-butyl ester, nonadecanedioic acid mono-tert- butyl ester, tetradecanedioic acid mono-tert-butyl ester, or 4-(9-carboxynonyloxy) benzoic acid tert-butyl ester were used. All operations stated below were performed at 250- ⁇ synthesis scale.
  • SPPS_P was performed on a Prelude Solid Phase Peptide Synthesizer from
  • Fmoc-amino acids 300 mM in NMP with 300 mM HOAt or Oxyma Pure®
  • resin loading e.g. Rinkamide-Chematrix (0.5 mmol/g) or low load Fmoc-Gly-Wang (0.35 mmol/g).
  • Fmoc-deprotection was performed using 20% piperidine in NMP.
  • Coupling was performed using 3:3:3:4 amino acid/(HOAt or Oxyma Pure®)/DIC/collidine in NMP.
  • NMP and DCM top washes (7 ml, 0.5 min, 2 x 2 each) were performed between deprotection and coupling steps. Coupling times were generally 60 minutes.
  • Some amino acids including, but not limited to Fmoc-Arg(Pbf)-OH, Fmoc-Aib-OH or Boc-His(Trt)-OH were "double coupled", meaning that after the first coupling (e.g. 60 min), the resin is drained and more reagents are added (amino acid, (HOAt or Oxyma Pure®), DIC, and collidine), and the mixture allowed to react again (e.g. 60 min).
  • LCMS_AP was performed using a Micromass Quatro micro API mass spectrometer to identify the mass of the sample after elution from a HPLC system composed of Waters
  • the RP-analysis was performed using a Waters UPLC system fitted with a dual band detector. UV detections at 214 nm and 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130 A, 1.7 urn, 2.1 mm x 150 mm column, 30°C.
  • the UPLC system was connected to two eluent reservoirs containing: A: 99.95% H20, 0.05% TFA; B: 99.95%
  • In vitro potency (CRE luciferase; whole cells)
  • the purpose of this example is to test the activity, or potency, of the GLP-1 receptor agonist peptides in vitro.
  • the in vitro potency is the measure of human GLP-1 receptor activation in a whole cell assay.
  • In vitro potency was determined by measuring the response of the human GLP-1 receptor in a reporter gene assay.
  • the assay was performed in a stably transfected BHK cell line that expresses the human GLP-1 receptor and contains the DNA for the cAMP response element (CRE) coupled to a promoter and the gene for firefly luciferase (CRE luciferase).
  • CRE cAMP response element
  • CRE luciferase firefly luciferase
  • the cells used in this assay were BHK cells with BHKTS13 as a parent cell line.
  • the cells were derived from a clone (FCW467-12A) that expresses the human GLP-1 receptor and were established by further transfection with CRE luciferase to obtain the current clone.
  • the cells were cultured at 5% C0 2 in Cell Culture Medium. They were aliquoted and stored in liquid nitrogen. Before each assay an aliquot is taken up and washed twice in PBS before being suspended at the desired concentration in the assay specific buffer. For 96-well plates the suspension was made to give a final concentration of 5x10 3 cells/well. Materials
  • Pluronic F-68 (10%) (Gibco 2404), human serum albumin (HSA) (Sigma A951 1 ), ovalbumin (Sigma A5503), DMEM w/o phenol red (Gibco 1 1880-028), 1 M Hepes (Gibco 15630), Glutamax 100x (Gibco 35050) and steadylite plus (PerkinElmer 6016757).
  • Cell Culture Medium consisted of DMEM medium with 10% FBS (Fetal Bovine Serum), 1 mg/ml G418, 240 nM MTX (methotrexate) and 1 % pen/strep
  • Assay Medium consisted of DMEM w/o phenol red, 10mM Hepes and 1x Glutamax.
  • the 1 % Assay Buffer consisted of 2% ovalbumin, 0.2% Pluronic F-68 and 2% HSA in assay medium.
  • the 0% Assay Buffer consisted of 2% ovalbumin and 0.2% Pluronic F-68 in Assay Medium.
  • the assay plate was incubated for 3 h in a 5% C0 2 incubator at 37°C.
  • Cholesterol efflux was assessed in vitro by measuring the capacity of compounds to efflux cholesterol from macrophage cell line, primarily transported via the ABCA1 transporter.
  • 8-(4-Chlorophenyl-thio) adenosine 3 ' , 5 ' -cyclic monophosphate sodium salt (CPT-cAMP) was used to up-regulate the ABCA1 transporter.
  • Penicillin/Streptomycin (GIBCO, Cat. #15140).

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Abstract

La présente invention concerne de nouveaux analogues des peptides agonistes des récepteurs à la protéine-1 semblable au glucagon (GLP-1) qui sont capables de favoriser l'efflux de cholestérol. La présente invention concerne également des compositions comprenant les nouveaux analogues des récepteurs à la protéine-1 semblable au glucagon (GLP-1), et l'utilisation desdits composés dans la fabrication de médicaments, l'utilisation desdits composés en thérapie, et des procédés de traitement comprenant l'administration desdits composés à des patients.
PCT/EP2015/063602 2014-06-18 2015-06-17 Nouveaux agonistes des récepteurs au glp-1 à activité d'efflux de cholestérol WO2015193378A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10660939B2 (en) 2015-04-14 2020-05-26 Palatin Technologies, Inc. Therapies for obesity, diabetes and related indications
WO2023124847A1 (fr) * 2021-12-28 2023-07-06 北京惠之衡生物科技有限公司 Dérivé de glp-1 à action prolongée

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10660939B2 (en) 2015-04-14 2020-05-26 Palatin Technologies, Inc. Therapies for obesity, diabetes and related indications
WO2023124847A1 (fr) * 2021-12-28 2023-07-06 北京惠之衡生物科技有限公司 Dérivé de glp-1 à action prolongée

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