US20120329711A1 - Glp-1 receptor agonist compounds with a modified n-terminus - Google Patents

Glp-1 receptor agonist compounds with a modified n-terminus Download PDF

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US20120329711A1
US20120329711A1 US13/516,311 US201013516311A US2012329711A1 US 20120329711 A1 US20120329711 A1 US 20120329711A1 US 201013516311 A US201013516311 A US 201013516311A US 2012329711 A1 US2012329711 A1 US 2012329711A1
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chem
glp
peptide
derivative
ester
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Janos Tibor Kodra
Johnny Madsen
Patrick William Garibay
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Nordisk AS
Novo Nordisk AS
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Nordisk AS
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Assigned to NOVO NORDISK A/S reassignment NOVO NORDISK A/S ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KODRA, JANOS TIBOR, MADSEN, JOHNNY, GARIBAY, PATRICK WILLIAM
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    • C07K—PEPTIDES
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    • C07—ORGANIC CHEMISTRY
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    • G01N2333/575—Hormones
    • G01N2333/605—Glucagons

Definitions

  • the present invention relates to analogues and derivatives of GLP-1 receptor agonist peptides, and their pharmaceutical use.
  • GLP-1 receptor agonist peptides of the invention such as Glucagon-Like Peptide-1 (GLP-1), exendins and analogues thereof, the two N-terminal amino acids have been replaced by N-terminal mimetics.
  • WO 2004/067548 A2 relates to chemically modified metabolites of regulatory peptides and methods of producing and using same.
  • Liraglutide a GLP-1 derivative for once daily administration which is marketed by Novo Nordisk A/S, is disclosed in Example 37 of WO 98/08871.
  • Semaglutide a GLP-1 derivative for once weekly administration which is under development by Novo Nordisk A/S, is disclosed in Example 4 of WO 06/097537.
  • the invention relates to GLP-1 receptor agonist compounds comprising a modified N-terminus.
  • Preferred compounds have the formula Chem. 1: Y—Z—P, wherein P represents a fragment of a GLP-1 receptor agonist peptide lacking the N-terminus; and Y—Z represents a group mimicking the N-terminus of the peptide.
  • the new N-terminal is preferably a His-Ala, a His-Gly, and/or a His-Ser mimetic.
  • the invention relates to a GLP-1 receptor agonist peptide having the formula Chem. 1: Y—Z—P, wherein P represents a fragment of a GLP-1 receptor agonist peptide lacking the two N-terminal amino acid residues; Z represents a group of the formula Chem. 2:
  • R1 and R2 independently represent (i) hydrogen, alkyl, aryl, heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy, carboxamide, substituted carboxamide, alkyl ester, aryl ester, alkyl sulfonyl, or aryl sulfonyl, or R1 and R2 together form (ii) cyclo alkyl, heterocyclyl, or heteroaryl; and Y represents a group of formula Chem. 4:
  • X 1 is N, O, or S
  • X 2 , X 3 , X 4 , and X 5 independently represent C, or N, with the proviso that at least one of X 2 , X 3 , X 4 and X 5 is C
  • R11, R12, R13, and R14 independently represent hydrogen, alkyl, aryl, heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy, carboxamide, substituted carboxamide, alkyl ester, aryl ester, alkyl sulfonyl, or aryl sulfonyl
  • Q represents a bond, or a group of formula
  • q is 1-6, and R15 and R16 independently of each other and independently for each value of q represent hydrogen, alkyl, carboxyl, or hydroxyl; and R represents hydrogen, or alkyl; or a pharmaceutically acceptable salt, amide, or ester thereof.
  • the invention also relates to a derivative of this peptide, and a pharmaceutically acceptable salt, amide, or ester thereof.
  • the invention also relates to the pharmaceutical use of these compounds, preferably for the treatment and/or prevention of all forms of diabetes and related diseases, such as eating disorders, cardiovascular diseases, gastrointestinal diseases, diabetic complications, critical illness, and/or polycystic ovary syndrome; and/or for improving lipid parameters, improving ⁇ -cell function, and/or for delaying or preventing diabetic disease progression.
  • diabetes and related diseases such as eating disorders, cardiovascular diseases, gastrointestinal diseases, diabetic complications, critical illness, and/or polycystic ovary syndrome
  • lipid parameters improving ⁇ -cell function, and/or for delaying or preventing diabetic disease progression.
  • the invention relates to intermediate products corresponding to the new N-terminus, as well as to the peptide fragments, i.e. before attachment of the new N-terminus, both relevant for the preparation of the peptides of the invention.
  • the peptides and derivatives of the invention are biologically active, preferably of a high potency. Also, or alternatively, they have a protracted pharmacokinetic profile. Also, or alternatively, they are stable against degradation by gastro intestinal enzymes. Also, or alternatively, they have a high oral bioavailability. These properties are of importance in the development of next generation GLP-1 compounds for subcutaneous, intravenous, and/or in particular oral administration.
  • the invention relates to a GLP-1 receptor agonist peptide having the formula Chem. 1: Y—Z—P, wherein P represents a fragment of a GLP-1 receptor agonist peptide lacking the two N-terminal amino acid residues; Z represents a group of the formula Chem. 2:
  • R1 and R2 independently represent (i) hydrogen, alkyl, aryl, heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy, carboxamide, substituted carboxamide, alkyl ester, aryl ester, alkyl sulfonyl, or aryl sulfonyl, or R1 and R2 together form (ii) cyclo alkyl, heterocyclyl, or heteroaryl; and Y represents a group of formula Chem. 4:
  • X 1 is N, O, or S
  • X 2 , X 3 , X 4 , and X 5 independently represent C, or N, with the proviso that at least one of X 2 , X 3 , X 4 and X 5 is C
  • R11, R12, R13, and R14 independently represent hydrogen, alkyl, aryl, heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy, carboxamide, substituted carboxamide, alkyl ester, aryl ester, alkyl sulfonyl, or aryl sulfonyl
  • Q represents a bond, or a group of formula
  • q is 1-6, and R15 and R16 independently of each other and independently for each value of q represent hydrogen, alkyl, carboxyl, or hydroxyl; and R represents hydrogen, or alkyl; or a pharmaceutically acceptable salt, amide, or ester thereof.
  • R1 and R2 do not both represent hydrogen, and the invention accordingly relates to a GLP-1 receptor agonist peptide having the formula Chem. 1: Y—Z—P, wherein P represents a fragment of a GLP-1 receptor agonist peptide lacking the two N-terminal amino acid residues; Z represents a group of the formula Chem. 2:
  • R1 and R2 independently represent (i) hydrogen, alkyl, aryl, heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy, carboxamide, substituted carboxamide, alkyl ester, aryl ester, alkyl sulfonyl, or aryl sulfonyl, or R1 and R2 together form (ii) cyclo alkyl, heterocyclyl, or heteroaryl, with the proviso that (iii) R1 and R2 do not both represent hydrogen; and Y represents a group of formula Chem. 4:
  • X 1 is N, O, or S
  • X 2 , X 3 , X 4 , and X 5 independently represent C, or N, with the proviso that at least one of X 2 , X 3 , X 4 and X 5 is C
  • R11, R12, R13, and R14 independently represent hydrogen, alkyl, aryl, heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy, carboxamide, substituted carboxamide, alkyl ester, aryl ester, alkyl sulfonyl, or aryl sulfonyl
  • Q represents a bond, or a group of formula
  • q is 1-6, and R15 and R16 independently of each other and independently for each value of q represent hydrogen, alkyl, carboxyl, or hydroxyl; and R represents hydrogen, or alkyl; or a pharmaceutically acceptable salt, amide, or ester thereof.
  • R1 and R2 may both represent hydrogen, and Q-NR—* is not attached to a nitrogen atom of Chem. 4.
  • R1 and R2 may both represent hydrogen, and Q-NR—* is attached to a carbon atom of Chem. 4.
  • the invention also relates to a derivative of each of these peptides, and to pharmaceutically acceptable salts, amides, or esters thereof.
  • the invention also relates to the pharmaceutical use of these compounds, preferably for the treatment and/or prevention of all forms of diabetes and related diseases, such as eating disorders, cardiovascular diseases, gastrointestinal diseases, diabetic complications, critical illness, and/or polycystic ovary syndrome; and/or for improving lipid parameters, improving ⁇ -cell function, and/or for delaying or preventing diabetic disease progression.
  • diabetes and related diseases such as eating disorders, cardiovascular diseases, gastrointestinal diseases, diabetic complications, critical illness, and/or polycystic ovary syndrome
  • lipid parameters improving ⁇ -cell function, and/or for delaying or preventing diabetic disease progression.
  • the invention relates to intermediate products corresponding to the new N-terminus, as well as to the peptide fragments, i.e. before attachment of the new N-terminus, both relevant for the preparation of the peptides of the invention.
  • An asterisk (*) in a chemical formula designates i) a point of attachment, ii) a radical, and/or iii) an unshared electron.
  • the GLP-1 receptor agonist compounds of the invention may be derived, or are derivable, from human GLP-1(7-37), exendin-4(1-39), and/or GLP-1A(1-37).
  • the amino acid sequences of these peptides may be found in the UniProt Knowledgebase (UniProtKB)—SwissProt section (www.uniprot.org) with the following accession numbers, sequence identifiers, and sequence names: UNIPROT:P01275 — 8, GLUC_HUMAN, Glucagon-like peptide 1(7-37); UNIPROT:P26349 — 3, EXE4_HELSU, Exendin-4, or exenatide; and UNIPROT:042143 — 5, GLUC1_XENLA, Glucagon-like peptide 1A; respectively.
  • sequences of the corresponding fragments lacking the two N-terminal amino acids are included in the appended sequence listing as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.
  • GLP-1 receptor agonist fragment from which the compounds of the invention may be derived, or are derivable, is the peptide designated exendin-3(3-39) which is the D3 analogue of SEQ ID NO: 2, i.e. identical to SEQ ID NO: 2 except for having aspartic acid (D, Asp) at position 3, the first amino acid residue.
  • GLP-1 receptor The sequence of the GLP-1 receptor may be found in the UniprotKB database referred to above with the following accession number, identifier, and name: UNIPROT:P43220, GLP1R_HUMAN, Glucagon-like peptide 1 receptor, GLP-1 receptor, GLP-1-R, or GLP-1R.
  • GLP-1 receptor agonist refers to a compound which is an agonist of the human GLP-1 receptor, i.e. a compound that stimulates the formation of cAMP in a medium containing the human GLP-1 receptor.
  • GLP-1 receptor agonism, or potency is determined as described below, in the section headed “Potency”, see also Example 13 herein.
  • peptide refers to a compound which comprises a series of amino acids intereconnected by amide (or peptide) bonds.
  • the peptide is to a large extent, or predominantly, composed of amino acids interconnected by amide bonds (e.g., at least 50%, 60%, 70%, 80%, or at least 90%, by molar mass). In another particular embodiment the peptide consists of amino acids interconnected by peptide bonds.
  • the peptides of the invention comprise at least five constituent amino acids connected by peptide bonds.
  • the peptide comprises at least 10, preferably at least 15, more preferably at least 20, even more preferably at least 25, or most preferably at least 28 amino acids.
  • the peptide is composed of at least five constituent amino acids, preferably composed of at least 10, at least 15, at least 20, at least 25, or most preferably composed of at least 28 amino acids.
  • the peptide is a) composed of, or b) consists of, i) 29, ii) 30, iii) 31, or iv) 32 amino acids.
  • the peptide is a) composed of, or b) consists of, i) 33, ii) 34, iii) 35, or iv) 36 amino acids.
  • the peptide consists of amino acids interconnected by peptide bonds.
  • Amino acids are molecules containing an amine group and a carboxylic acid group, and, optionally, one or more additional groups, often referred to as a side chain.
  • amino acid includes proteogenic amino acids (encoded by the genetic code, including natural amino acids, and standard amino acids), as well as non-proteogenic (not found in proteins, and/or not coded for in the standard genetic code), and synthetic amino acids.
  • the amino acids may be selected from the group of proteinogenic amino acids, non-proteinogenic amino acids, and/or synthetic amino acids.
  • Non-limiting examples of amino acids which are not encoded by the genetic code are gamma-carboxyglutamate, ornithine, and phosphoserine.
  • Non-limiting examples of synthetic amino acids are the D-isomers of the amino acids such as D-alanine and D-leucine, Aib ( ⁇ -aminoisobutyric acid), ⁇ -alanine, and des-amino-histidine (desH, alternative name imidazopropionic acid, abbreviated Imp).
  • GLP-1 Receptor Agonist Peptides Fragments, Analogues, Residue Numbering, Identity
  • a “GLP-1 receptor agonist peptide” is a peptide as defined above, and also a GLP-1 receptor agonist as defined above.
  • the peptides of the invention are GLP-1 receptor agonist peptides.
  • GLP-1 receptor agonist peptides are the following known compounds: Human GLP-1(7-37), exendin-4(1-39), exendin-3(1-39), and GLP-1A(1-37).
  • the GLP-1 receptor agonist compound of the invention may be derived, or is derivable, from any one or more of these known GLP-1 receptor agonist peptides.
  • fragment as it refers to a GLP-1 receptor agonist peptide means a peptide which is shorter than the peptide referred to.
  • the fragment lacks the two N-terminal amino acids as compared to the corresponding full-length peptide being a GLP-1 receptor agonist.
  • this particular fragment is not in itself a GLP-1 receptor agonist, due to a i) substantial, ii) preferably almost complete, or iii) more preferably for all practical purposes complete, loss of biological activity (i.e., GLP-1 receptor agonism).
  • P fragments of a GLP-1 receptor agonist peptide lacking the two N-terminal amino acid residues
  • GLP-1(9-37), exendin-4(3-39), and GLP-1A(3-37) are the following: GLP-1(9-37), exendin-4(3-39), and GLP-1A(3-37), which are included in the appended sequence listing as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.
  • Another example of P is exendin-3(3-39) which is variant D3 of SEQ ID NO: 2.
  • any reference herein to an amino acid residue number or a position number in the context of the peptides of SEQ ID NO: 1, 2, or 3 or analogues thereof is to the sequence starting with Glu at position 9, Glu at position 3, or Asp at position 3, respectively; and ending with Gly at position 37, Ser at position 39, or Ser at pos. 37, respectively.
  • P is analogues of SEQ ID NO: 1, SEQ ID NO: 2, and/or SEQ ID NO: 3.
  • an “analogue” as used herein in the context of SEQ ID NO: 1, 2, or 3 refers to a peptide, or a compound, which is a variant of any one or more of SEQ ID NO: 1, 2, or 3.
  • the analogue of SEQ ID NO: 1 refers to a modification of SEQ ID NO: 1 in which a number of amino acid residues have been exchanged as compared to SEQ ID NO: 1.
  • These exchanges, or modifications may represent, independently, one or more amino acid substitutions, additions, and/or deletions. Additions at the N-terminus are, however, preferably excluded.
  • Analogues of SEQ ID NO: 2, and SEQ ID NO: 3 are defined similarly, by analogy to the definition of analogues of SEQ ID NO: 1.
  • Analogues may be described by reference to a reference sequence, the number of the amino acid residue in the reference sequence corresponding to the one which is modified, i.e., its position, and to the actual modification.
  • the reference sequence is i) GLP-1(9-37) (SEQ ID NO: 1); ii) exendin-4(3-39) (SEQ ID NO: 2); or iii) GLP-1A(3-37) (SEQ ID NO: 3).
  • GLP-1(9-37) peptide is a GLP-1 receptor agonist peptide of the invention derivable from GLP-1(9-37) (SEQ ID NO: 1), i.e. P in Chem. 1 is an analogue of SEQ ID NO: 1, viz. the analogue in which the serine at position 18 has been substituted with lysine, the glycine at position 22 has been substituted with glutamic acid, and the lysine at position 34 has been substituted with glutamine;
  • N 9 - ⁇ 2-[2-(1H-Imidazol-4-yl)propylcarbamoyl]-2-methyl-propionyl ⁇ -[Glu 30 ,Lys 36 ]GLP-1(9-37)Glu 38 -peptide is a GLP-1 receptor agonist peptide of the invention derivable from GLP-1(9-37) (SEQ ID NO: 1), i.e. P in Chem. 1 is an analogue of SEQ ID NO: 1, viz. the analogue in which the alanine at position 30 has been substituted with glutamic acid, the arginine at position 36 has been substituted with lysine, and a glutamic acid has been added at the C-terminus, viz. at position 38; and
  • GLP-1A(3-37)-peptide is a GLP-1 receptor agonist peptide of the invention derivable from GLP-1A(3-37) (SEQ ID NO: 3), i.e. P in Chem. 1 is an analogue of SEQ ID NO: 3, viz. the analogue in which the lysines at position 17, 20, and 33 have been substituted with arginine, and a lysine has been added at the C-terminus, viz. at position 38.
  • This peptide by the way, is also derivable from SEQ ID NO: 1, and it can therefore also be designated as analogue (17T, 18Q, 19Q, 21D, 22E, 23R, 26R, 30D, 33I, 34N, 36G, 37P, 38S, 39R, 40E, 41I, 42I, 43S, 44K) of GLP-1(9-37) (SEQ ID NO: 1), having ⁇ 2-[2-(1H-Imidazol-4-yl)-methylcarbamoyl]-2-methyl-propionyl ⁇ - attached to the N-terminus, N 9 .
  • a GLP-1 receptor agonist peptide of the invention which “comprises at least one of the following substitutions as compared to GLP-1(9-37) (SEQ ID NO: 1): 18K; 22E; 30E; 31H; 34Q,R; 36K; 37K; and/or 38E” refers to a GLP-1 receptor agonist peptide in which P of Chem.
  • SEQ ID NO: 1 is considered an analogue of SEQ ID NO: 1, which analogue has a lysine at position 18, a glutamic acid at position 22, a glutamic acid at position 30, a histidine at position 31, a glutamine at position 34, a lysine at position 36, a lysine at position 37, and/or a glutamic acid at position 38, and which analogue may comprise further modifications as compared to SEQ ID NO: 1.
  • amino acid residues may be identified by their full name, their one-letter code, and/or their three-letter code. These three ways are fully equivalent.
  • a position equivalent to or “corresponding position” may be used to characterise the site of modification in a modified GLP-1 receptor agonist peptide sequence by reference to any one or more of SEQ ID NO: 1, 2, or 3. Equivalent or corresponding positions, as well as the number of modifications, are easily deduced, e.g. by simple handwriting and eyeballing; and/or a standard protein or peptide alignment program may be used, such as “align” which is a Needleman-Wunsch alignment. The algorithm is described in Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48: 443-453, and the align program by Myers and W.
  • the default scoring matrix BLOSUM50 and the default identity matrix may be used, and the penalty for the first residue in a gap may be set at ⁇ 12, or preferably at ⁇ 10, and the penalties for additional residues in a gap at ⁇ 2, or preferably at ⁇ 0.5.
  • This algorithm may also suitably be used for determining the degree of identity of the P-group of a GLP-1 receptor agonist peptide of the invention to each of SEQ ID NO: 1, 2, and 3, e.g. with a view to determining which of these three sequences has the highest percentage of identity to the P-group in question, and thus for determining the number of amino acid residues that have been exchanged as compared to the closest related sequence of SEQ ID NOs: 1-3 (the one with the highest percentage of identity). If the percentages of identity of a given P group of a GLP-1 receptor agonist of the invention to SEQ ID NO: 1, 2, and 3, respectively, should happen to be the same, any of those having the same highest percentage of identity may be used for the determination.
  • An example of such alignment is inserted hereinbelow, in which sequence no. 1 is SEQ ID NO: 1, and sequence no. 2 is SEQ ID NO: 3:
  • analogues comprising non-natural amino acids such as Imp, and/or Aib being included in the sequence, these may, for alignment purposes, be replaced with X. If desired, X can later be manually corrected.
  • derivative as used herein in the context of the GLP-1 receptor agonist peptides of the invention means a chemically modified peptide or analogue, in which one or more substituents have been covalently attached to the peptide.
  • the substituent(s) may also be referred to as side chain(s).
  • the derivative of the invention has one side chain. In another particular embodiment it has two side chains.
  • the group Y—Z— of formula I is preferably not considered a substituent/side chain.
  • the side chain has at least 10 carbon atoms, or at least 15, 20, 25, 30, 35, 40, or at least 43 carbon atoms.
  • the side chain may further include at least 5 hetero atoms, in particular O and N, for example at least 7, 9, 10, 12, 15, 17, or at least 20 hetero atoms, such as at least 1, 2, 3, or 4 N-atoms, and/or at least 3, 4, 6, 9, 12, 13, or 15 O-atoms.
  • GLP-1 receptor agonist derivatives include heterologous fusion proteins or conjugates of the GLP-1 receptor agonist peptides of the invention, with e.g. the Fc portion of an immunoglobulin such as IgG, with human albumin, with antibodies such as a glucagon binding antibody heavy chain variable region, or with fragments or analogues of any of these (see, e.g., US 2007/0161087, WO 2005/058958, and WO 2007/124463 A2).
  • PEGylated peptides see, e.g., WO 2005/058954, WO 2004/093823, and WO 2006/124529
  • acylated peptides see, e.g., WO 98/08871, WO2005/027978, WO 2006/097537, and WO 2009/030771).
  • the side chain is capable of forming non-covalent aggregates with albumin, thereby promoting the circulation of the derivative with the blood stream, and also having the effect of protracting the time of action of the derivative, due to the fact that the aggregate of the derivative and albumin is only slowly disintegrated to release the active pharmaceutical ingredient.
  • a preferred substituent, or side chain, as a whole may be referred to as an albumin binding moiety.
  • the albumin binding moiety comprises a portion which is particularly relevant for the albumin binding and thereby the protraction, which portion may accordingly be referred to as a protracting moiety.
  • the protracting moiety may be at, or near, the opposite end of the albumin binding moiety, relative to its point of attachment to the peptide.
  • the albumin binding moiety comprises a portion in-between the protracting moiety and the point of attachment to the peptide, which portion may be referred to as a linker, linker moiety, spacer, or the like.
  • a linker is optional; hence if no linker is present the albumin binding moiety may be identical to the protracting moiety.
  • the albumin binding moiety and/or the protracting moiety is lipophilic, and/or negatively charged at physiological pH (7.4).
  • the albumin binding moiety, the protracting moiety, or the linker may be covalently attached to a lysine residue of the GLP-1 receptor agonist peptide by conjugation chemistry such as by alkylation, acylation, ester formation, or amide formation; or to a cysteine residue, such as by maleimide or haloacetamide (such as bromo-/fluoro-/iodo-) coupling.
  • conjugation chemistry such as by alkylation, acylation, ester formation, or amide formation
  • cysteine residue such as by maleimide or haloacetamide (such as bromo-/fluoro-/iodo-) coupling.
  • an active ester of the albumin binding moiety and/or the protracting moiety is covalently linked to an amino group of a lysine residue, preferably the epsilon amino group thereof, under formation of an amide bond (this process being referred to as acylation).
  • the invention relates to a derivative of a GLP-1 receptor agonist peptide which comprises, preferably has, an albumin binding moiety attached to one or more of 18K, 26K, 36K, and/or 37K, wherein reference may be had to the sequence of GLP-1(9-37) (SEQ ID NO: 1).
  • each residue number refers to the corresponding position in GLP-1(9-37) (SEQ ID NO: 1).
  • ordinary script may be used instead of superscript to designate the position number. E.g., “K 18 ” is fully equivalent to “18K”.
  • Corresponding position numbers are preferably identified by handwriting and eyeballing, or by using a suitable alignment program, as explained above.
  • albumin binding moiety include the un-reacted as well as the reacted forms of these molecules. Whether or not one or the other form is meant is clear from the context in which the term is used.
  • albumin binding moiety comprises, or consists of, a protracting moiety selected from
  • x is an integer in the range of 6-18
  • y is an integer in the range of 3-17
  • z is an integer in the range of 1-5
  • R 18 is a group having a molar mass not higher than 150 Da.
  • *—(CH 2 ) x —* refers to straight or branched, preferably straight, alkylene in which x is an integer in the range of 6-18.
  • *—(CH 2 ) y —* refers to straight or branched, preferably straight, alkylene in which y is an integer in the range of 3-17.
  • *—(CH 2 ) z —* refers to straight or branched, preferably straight, alkylene in which z is an integer in the range of 1-5.
  • the molar mass (M) of a chemical substance is the mass of one mole of the substance.
  • Molar mass may be calculated from standard atomic weights, and is often listed in chemical catalogues.
  • the molar mass of a compound is given by the sum of the standard atomic weights of the atoms which form the compound multiplied by the molar mass constant, M u which equals 1 g/mol.
  • M u which equals 1 g/mol.
  • the acid group of the fatty acid, or one of the acid groups of the fatty diacid forms an amide bond with the epsilon amino group of a lysine residue in the GLP-1 receptor agonist peptide.
  • fatty acid refers to aliphatic monocarboxylic acids having from 4 to 28 carbon atoms, it is preferably unbranched, and/or even numbered, and it may be saturated or unsaturated.
  • fatty diacid refers to fatty acids as defined above but with an additional carboxylic acid group in the omega position.
  • fatty diacids are dicarboxylic acids.
  • the aromatics such as the phenoxy and the phenylene radicals, may be, independently, ortho, meta, or para.
  • the linker moiety if present, has from 5 to 30 C-atoms. In additional preferred embodiments, the linker moiety, if present, has from 4 to 20 hetero atoms. H-atoms are not hetero atoms.
  • the linker comprises at least one OEG molecule, at least one glutamic acid residue, and/or at least one piperidine molecule, optionally substituted, or rather the corresponding radicals
  • OEG designates 8-amino-3,6-dioxaoctanic acid, i.e. this di-radical: *—NH—(CH 2 ) 2 —O—(CH 2 ) 2 —O—CH 2 —CO—*).
  • the amino acid glutamic acid comprises two carboxylic acid groups. Its gamma-carboxy group is preferably used for forming an amide bond with the epsilon-amino group of lysine, or with an amino group of an OEG molecule, if present, or with the amino group of another Glu residue, if present.
  • the amino group of Glu in turn forms an amide bond with the carboxy group of the protracting moiety, or with the carboxy group of an OEG molecule, if present, or with the gamma-carboxy group of another Glu, if present. This way of inclusion of Glu is occasionally briefly referred to as “gamma-Glu”.
  • the derivatives of the invention may exist in different stereoisomeric forms having the same molecular formula and sequence of bonded atoms, but differing only in the three-dimensional orientation of their atoms in space.
  • the stereoisomerism of the exemplified derivatives of the invention is indicated in the experimental section, in the names as well as the structures, using standard nomenclature. Unless otherwise stated the invention relates to all stereoisomeric forms of the claimed derivative.
  • the concentration in plasma of the GLP-1 receptor agonist peptides and derivatives of the invention may be determined using any suitable method.
  • LC-MS Liquid Chromatography Mass Spectroscopy
  • immunoassays such as RIA (Radio Immuno Assay), ELISA (Enzyme-Linked Immuno Sorbent Assay), and LOCI (Luminescence Oxygen Channeling Immunoasssay).
  • RIA Radio Immuno Assay
  • ELISA Enzyme-Linked Immuno Sorbent Assay
  • LOCI Luminescence Oxygen Channeling Immunoasssay
  • a preferred assay is the LOCI assay in which the plasma concentrations of the compounds are determined using a Luminescence Oxygen Channeling Immunoasssay (LOCI), generally as described for the determination of insulin by Poulsen and Jensen in Journal of Biomolecular Screening 2007, vol. 12, p. 240-247.
  • the donor beads are coated with streptavidin, while acceptor beads are conjugated with a monoclonal antibody recognising a mid-/C-terminal epitope of the peptide.
  • Another monoclonal antibody, specific for the N-terminus is biotinylated.
  • the three reactants are combined with the analyte and form a two-sited immuno-complex. Illumination of the complex releases singlet oxygen atoms from the donor beads, which are channeled into the acceptor beads and trigger chemiluminescence which is measured in an Envision plate reader. The amount of light is proportional to the concentration of the compound.
  • the GLP-1 receptor agonist peptides, derivatives, 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.: NH 3 +H 2 SO 4 ⁇ (NH 4 ) 2 SO 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 peptides and derivatives of the invention may be formed with added cations or anions that react with anionic or cationic groups, respectively. These groups may be situated in the peptide moiety, and/or in the side chain of the compounds of the invention.
  • Non-limiting examples of anionic groups of the compounds 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.
  • ester of the peptides and derivatives 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 peptides and derivatives 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 or derivative is in the form of a pharmaceutically acceptable salt.
  • the peptide or derivative is in the form of a pharmaceutically acceptable amide, preferably with an amide group at the C-terminus of the peptide.
  • the peptide or derivative is in the form a pharmaceutically acceptable ester.
  • the invention also relates to an intermediate product of the formula Chem. 50 or Chem. 51:
  • Q, R, R1, and R2 are as defined for the GLP-1 receptor agonist peptide of the invention, having the formula Chem. 1, and each of PG 1 and PG 2 represents a protection group.
  • Non-limiting examples of PG1 groups are Boc, Trt, Mtt, Mmt, and Fmoc.
  • Non-limiting examples of PG2 groups are —OH, or groups functionalised as an activated ester, for example, without limitation, OPfp, OPnp, and OSuc.
  • activated esters may be selected, e.g., according to the teaching of M. Bodanszky, “Principles of Peptide Synthesis”, 2nd ed., Springer Verlag, 1993.
  • the GLP-1 receptor agonist peptides and/or derivatives of the invention have a good potency. Also, or alternatively, in a second functional aspect, they have a protracted pharmacokinetic profile. Also, or alternatively, in a third functional aspect, they are stable against degradation by gastro intestinal enzymes. Also, or alternatively, in a fourth functional aspect, they have a high oral bioavailability.
  • the GLP-1 receptor agonist derivatives are biologically active, or have a good potency.
  • the derivatives of the invention have a high binding affinity to the GLP-1 receptor at low albumin concentration (0.005%), i.e. a low IC 50 value, which is discussed further below under the heading of receptor binding.
  • EC 50 half maximal effective concentration
  • concentration which induces a response halfway between the baseline and maximum by reference to the dose response curve.
  • EC 50 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 derivatives of the invention may be determined as described hereinbelow, and the EC 50 of the derivative in question determined. The lower the EC 50 , the better the potency.
  • the derivatives of the invention are at least 3 times more potent than Chem. 40; preferably at least 4 times more potent; even more preferably at least 5 times more potent; or most preferably at least 6 times more potent than Chem. 40.
  • the derivatives of the invention are at least 7 times more potent than Chem. 40; preferably at least 8 times more potent; even more preferably at least 9 times more potent; or most preferably at least 10 times more potent than Chem. 40.
  • the derivatives of the invention are at least 20 times more potent than Chem. 40; preferably at least 50 times more potent; even more preferably at least 100 times more potent; still more preferably at least 200 times more potent; or most preferably at least 400 times more potent than Chem. 40.
  • Potency is preferably determined as described below, and it is noted that a, e.g., three times more potent compound has an EC 50 which is three times lower.
  • 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 stimulating cAMP formation in a cell line expressing the cloned human GLP-1 receptor.
  • the stimulation of the formation of cAMP in a medium containing the human GLP-1 receptor may preferably be determined using a stable transfected cell-line such as BHK467-12A (tk-ts13), and/or using for the determination of cAMP a functional receptor assay, e.g. based on competition between endogenously formed cAMP and exogenously added biotin-labelled cAMP, in which assay cAMP is more preferably captured using a specific antibody, and/or wherein an even more preferred assay is the AlphaScreen cAMP Assay, most preferably the one described in Example 13.
  • a stable transfected cell-line such as BHK467-12A (tk-ts13)
  • a functional receptor assay e.g. based on competition between endogenously formed cAMP and exogenously added biotin-labelled cAMP, in which assay cAMP is more preferably captured using a specific antibody, and/or wherein an even more preferred assay is the AlphaScreen c
  • the medium has the following composition (final in-assay concentrations):): 50 mM TRIS-HCl; 5 mM HEPES; 10 mM MgCl 2 , 6H 2 O; 150 mM NaCl; 0.01% Tween; 0.1% BSA; 0.5 mM IBMX; 1 mM ATP; 1 uM GTP; pH 7.4.
  • the GLP-1 receptor agonist has anEC 50 below 2000 pM, preferably below 1800 pM, more preferably below 1700 pM, even more preferably below 1600 pM, or most preferably below 1500 pM.
  • the derivatives of the invention 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, e.g. as described in Example 43 of WO09/030,738.
  • the derivatives of the invention are protracted.
  • Example 14 A suitable assay for determining receptor binding of the peptides and derivatives of the invention at high and low albumin concentration is disclosed in Example 14 herein.
  • the binding to the GLP-1 receptor at low albumin concentration should be as good as possible, corresponding to a low IC 50 value.
  • the IC 50 value at high albumin concentration is a measure of the influence of albumin on the binding of the compound to the GLP-1 receptor.
  • the peptides GLP-1 receptor agonist peptide derivatives of the invention also bind to albumin. This is a generally desirable effect, which extends their lifetime in plasma. Therefore, the IC 50 value at high albumin will generally be higher than the IC 50 value at low albumin, corresponding to a reduced binding to the GLP-1 receptor, caused by albumin binding competing with the binding to the GLP-1 receptor.
  • a high ratio (IC 50 value (high albumin)/IC 50 value (low albumin)) may therefore be taken as an indication that the derivative in question binds well to albumin (may have a long half-life), and also per se binds well to the GLP-1 receptor (the IC 50 value (high albumin) is high, and the IC 50 value (low albumin) is low).
  • albumin binding may not always be desirable, or the binding to albumin may become too strong. Therefore, the desirable ranges for IC 50 (low albumin), IC 50 (high albumin)/, and the ratio high/low may vary from compound to compound, depending on the intended use and the circumstances surrounding such use, and on other compound properties of potential interest.
  • the peptides and derivatives of the invention have a high binding affinity to the GLP-1 receptor at low albumin concentration (0.005%), i.e. a low IC 50 value.
  • the GLP-1 receptor binding affinity (IC 50 ) in the presence of 0.005% HSA (low albumin) is below 600.00 nM, preferably below 500.00 nM, more preferably below 200.00 nM, even more preferably below 100.00 nM, or most preferably below 45.00 nM.
  • the GLP-1 receptor agonist peptides and/or derivatives of the invention are stable, or stabilised, against degradation by one or more gastro intestinal enzymes.
  • Gastro intestinal enzymes include, without limitation, exo and endo peptidases, such as pepsin, trypsin, chymotrypsin, elastases, and carboxypeptidases.
  • exo and endo peptidases such as pepsin, trypsin, chymotrypsin, elastases, and carboxypeptidases.
  • the stability may be tested against these gastro intestinal enzymes in the form of purified enzymes, or in the form of extracts from the gastrointestinal system.
  • the derivative of the invention has an in vitro half-life (T 1/2 ), in an extract of rat small intestines, divided by the corresponding half-life (T 1/2 ) of GLP-1(7-37), of above 1.0, preferably above 2.0, more preferably above 3.0, even more preferably above 4.0, or most preferably above 5.0.
  • a ratio (SI) may be defined for each derivative, viz. as the in vitro half-life (T 1/2 ) of the derivative in question, in an extract of rat small intestines, divided by the corresponding half-life (T 1/2 ) of GLP-1(7-37).
  • the derivatives of the invention are protracted.
  • protraction may be determined as half-life (T 1/2 ) in vivo in rats after i.v. administration.
  • the half-life is at least 4 hours, preferably at least 5 hours, even more preferably at least 6 hours, or most preferably at least 8 hours.
  • the derivatives of the invention are protracted.
  • protraction may be determined as half-life (T 1/2 ) in vivo in minipigs after i.v. administration.
  • the half-life is at least 12 hours, preferably at least 24 hours, more preferably at least 36 hours, even more preferably at least 48 hours, or most preferably at least 60 hours.
  • Example 16 A suitable assay for determining half-life in vivo in minipigs after i.v. administration is disclosed in Example 16 herein.
  • the derivatives of the invention have a high oral bioavailability.
  • the oral bioavailability of commercial GLP-1 receptor agonist peptide derivatives is very low.
  • the oral bioavailability of such derivatives under development for i.v. or s.c. administration is also low.
  • the present inventors identified a novel class of GLP-1 receptor agonist peptide derivatives, which have a high oral bioavailability, and at the same time a satisfactory potency, and/or half-life.
  • these derivatives have a high oral bioavailability, and at the same time a high binding affinity (i.e. a low IC 50 value) to the GLP-1 receptor at a low concentration of albumin.
  • bioavailability of a GLP-1 receptor agonist compound of the invention refers to the fraction of an administered dose of the compound that reaches the systemic circulation unchanged.
  • bioavailability when a medication is administered intravenously, its bioavailability is 100%.
  • a medication is administered via other routes (such as orally), its bioavailability decreases (due to incomplete absorption and first-pass metabolism).
  • Knowledge about bioavailability is essential when calculating dosages for non-intravenous routes of administration.
  • Absolute oral bioavailability compares the bioavailability (estimated as the area under the curve, or AUC) of the active drug in systemic circulation following oral administration, with the bioavailability of the same drug following intravenous administration. It is the fraction of the drug absorbed through non-intravenous administration compared with the corresponding intravenous administration of the same drug. The comparison must be dose normalised if different doses are used; consequently, each AUC is corrected by dividing the corresponding dose administered.
  • a plasma drug concentration vs time plot is made after both oral and intravenous administration.
  • the absolute bioavailability (F) is the dose-corrected AUC-oral divided by AUC-intravenous.
  • the GLP-1 receptor agonist compounds of the invention have an absolute oral bioavailability which is higher than that of a) liraglutide, and/or b) semaglutide; preferably at least 10% higher, more preferably at least 20% higher, even more preferably at least 30% higher, or most preferably at least 40% higher.
  • the GLP-1 receptor agonist compounds of the invention may suitably be formulated as is known in the art of oral formulations of insulinotropic compounds, e.g. using any one or more of the formulations described in WO 2008/145728.
  • the fragment P of the peptides of formula I 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, “Protective Groups in Organic Synthesis”, John Wiley & Sons, 1999, Florencio Zaragoza Dörwald, “Organic Synthesis on solid Phase”, Wiley-VCH Verlag GmbH, 2000, and “Fmoc Solid Phase Peptide Synthesis”, Edited by W. C. Chan and P. D. White, Oxford University Press, 2000.
  • a host cell containing a DNA sequence encoding the fragment and capable of expressing the peptide in a suitable nutrient medium under conditions permitting the expression of the peptide.
  • 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.
  • the complete GLP-1 receptor agonist peptides of the invention incorporating, viz. adding Y—Z to P of formula I, may e.g. be produced as described in the experimental part. Or see Hodgson et al: “The synthesis of peptides and proteins containing non-natural amino acids”, Chemical Society Reviews, vol. 33, no. 7 (2004), p. 422-430; and in WO 2009/083549 A1 entitled “Semi-recombinant preparation of GLP-1 analogues”.
  • Derivatives of the invention may be prepared as is known in the art, and specific examples of methods of preparing a number of derivatives of the invention are included in the experimental part herein.
  • compositions comprising a peptide or a derivative 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, tablet aid, 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.
  • a pharmaceutical composition may be a solid formulation, e.g. a freeze-dried or spray-dried composition, which may be used as is, or whereto the physician or the patient adds solvents, and/or diluents prior to use.
  • 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 7.0.
  • a pharmaceutical composition may comprise a buffer.
  • the buffer may e.g. be selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid, and mixtures thereof.
  • a pharmaceutical composition may comprise a preservative.
  • the preservative may e.g.
  • 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), and mixtures thereof.
  • a pharmaceutical composition may comprise an isotonic agent.
  • the isotonic agent may e.g. be selected from the group consisting of a salt (e.g. sodium chloride), a sugar or sugar alcohol, an amino acid (e.g. glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), an alditol (e.g.
  • glycerol glycerine
  • 1,2-propanediol propyleneglycol
  • 1,3-propanediol 1,3-butanediol
  • polyethyleneglycol e.g. PEG400
  • 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, alfa and beta HPCD, soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na may be used.
  • Sugar alcohol is defined as a C4-C8 hydrocarbon having at least one —OH group and includes, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol.
  • the sugar alcohol additive is mannitol.
  • a pharmaceutical composition may comprise a chelating agent.
  • the chelating agent may e.g. be selected from salts of ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic acid, and mixtures thereof.
  • 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 peptide molecules resulting in formation of oligomers, which may remain soluble, or large visible aggregates that precipitate from the solution. Aggregate formation by a peptide 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 the pharmaceutical composition may comprise a stabiliser selected from the group of high molecular weight polymers or low molecular compounds.
  • the stabiliser may e.g. be selected from polyethylene glycol (e.g. PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, carboxy-/hydroxycellulose or derivates thereof (e.g. HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, sulphur-containing substances as monothioglycerol, thioglycolic acid and 2-methylthioethanol, and different salts (e.g. sodium chloride).
  • PEG 3350 polyethylene glycol
  • PVA polyvinyl alcohol
  • PVpyrrolidone polyvinylpyrrolidone
  • carboxy-/hydroxycellulose or derivates thereof e.g. HPC, HPC-SL, HPC-L and HPMC
  • cyclodextrins e.g. sulphur-containing substances as mono
  • a pharmaceutical 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, preferably a surfactant, at least one surfactant, or two different surfactants.
  • surfactant refers to any molecules or ions that are comprised of a water-soluble (hydrophilic) part, and a fat-soluble (lipophilic) part.
  • 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, such as, e.g., EDTA (ethylenediamine tetraacetic acid), and/or benzamidineHCl.
  • protease inhibitors such as, e.g., EDTA (ethylenediamine tetraacetic acid), and/or benzamidineHCl.
  • 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.
  • a pharmaceutical composition may be formulated as is known in the art of oral formulations of insulinotropic compounds, e.g. using any one or more of the formulations described in WO 2008/145728.
  • An administered dose may contain from 0.01 mg-100 mg of the GLP-1 receptor agonist derivative, or from 0.01-50 mg, or from 0.01-20 mg, or from 0.01-10 mg of the GLP-1 receptor agonist derivative.
  • the derivative 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.
  • 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
  • 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; conjunctival; uretal; vaginal; rectal; and/or ocular.
  • a composition may be an oral composition, and the route of administration is per oral.
  • 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 environmental conditions, e.g. by SEC-HPLC, and/or RP-HPLC.
  • the treatment with a derivative 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.
  • Examples of these pharmacologically active substances are: Insulin, sulphonylureas, biguanides, meglitinides, glucosidase inhibitors, glucagon antagonists, DPP-IV (dipeptidyl peptidase-IV) inhibitors, inhibitors of hepatic enzymes involved in stimulation of gluconeogenesis and/or glycogenolysis, glucose uptake modulators, compounds modifying the lipid metabolism such as antihyperlipidemic agents as HMG CoA inhibitors (statins), Gastric Inhibitory Polypeptides (GIP analogs), compounds lowering food intake, RXR agonists and agents acting on the ATP-dependent potassium channel of the ⁇ -cells; Cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol, dextrothyroxine, neteglinide, repaglinide; ⁇ -blockers such as
  • the treatment with a derivative 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 a GLP-1 receptor agonist peptide of the invention, and a derivative thereof, for use as a medicament.
  • these compounds may be used for the following medical treatments, all preferably relating one way or the other to diabetes:
  • diabetes 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 HbA1C;
  • 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 HbA1C;
  • 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
  • eating disorders such as obesity, e.g. by decreasing food intake, reducing body weight, suppressing appetite, inducing satiety; treating or preventing binge eating disorder, bulimia nervosa, and/or obesity induced by administration of an antipsychotic or a steroid; reduction of gastric motility; and/or delaying gastric emptying;
  • diabetes prevention and/or treatment of diabetic complications, such as neuropathy, including peripheral neuropathy; nephropathy; or retinopathy;
  • lipid parameters such as prevention and/or treatment of 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)) in vitro and/or in vivo;
  • cardiovascular diseases such as syndrome X; atherosclerosis; myocardial infarction; coronary heart disease; stroke, cerebral ischemia; an early cardiac or early cardiovascular disease, such as left ventricular hypertrophy; coronary artery disease; essential hypertension; acute hypertensive emergency; cardiomyopathy; heart insufficiency; exercise tolerance; chronic heart failure; arrhythmia; cardiac dysrhythmia; syncopy; atheroschlerosis; mild chronic heart failure; angina pectoris; cardiac bypass reocclusion; intermittent claudication (atheroschlerosis oblitterens); diastolic dysfunction; and/or systolic dysfunction;
  • cardiovascular diseases such as syndrome X; atherosclerosis; myocardial infarction; coronary heart disease; stroke, cerebral ischemia; an early cardiac or early cardiovascular disease, such as left ventricular hypertrophy; coronary artery disease; essential hypertension; acute hypertensive emergency; cardiomyopathy; heart insufficiency; exercise tolerance; chronic heart failure; arrhythm
  • 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, septicaemia, and/or septic shock during hospitalisation; and/or
  • CIPNP critical illness poly-nephropathy
  • SIRS systemic inflammatory response syndrome
  • the indication is selected from the group consisting of (i)-(iii) and (v)-(iix), such as indications (i), (ii), and/or (iii); or indication (v), indication (vi), indication (vii), and/or indication (iix).
  • the indication is (i). In a further particular embodiment the indication is (v). In a still further particular embodiment the indication is (iix).
  • Type 2 diabetes and/or obesity.
  • P represents a fragment of a GLP-1 receptor agonist peptide lacking the two N-terminal amino acid residues;
  • Z represents a group of the formula Chem. 2:
  • R11 and R12 independently represent hydrogen, alkyl, aryl, halogen, hydroxyl, hydroxylalkyl, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy, carboxamide, alkyl ester, or aryl ester; wherein preferably alkyl, hydroxylalkyl, aminoalkyl, carboxylalkyl, alkoxy, and/or alkyl ester contains lower alkyl, straight or branched, more preferably having 1-6 C-atoms. 16.
  • the peptide of any one of embodiment 1-15 wherein R11 and R12 independently represent hydrogen, lower alkyl, or lower alkoxy, wherein the lower alkyl and lower alkoxy, independently, have 1-5 C-atoms, preferably 1-4 C-atoms, or most preferably 1-3 C-atoms. 17.
  • R11 and R12 are methyl or hydrogen, preferably hydrogen. 19.
  • Y is a derivative of 1H-imidazole, preferably 1H-imidazol-4-yl, optionally substituted at one or two of positions 2, 3, and/or 5, wherein the position numbering of imidazole is according to IUPAC, and/or as shown for imidazole on wikipedia on 3 Dec. 2010 at 18:00 DK time.
  • Q is attached to i) X 2 , ii) X 3 , X 4 , or X 5 , preferably to X 2 or X 4 , most preferably to X 4 . 21.
  • Y is a derivative of an imidazole, such as 1H-imidazol, being substituted with a group of formula Chem. 7:
  • R17 represents alkylene, straight or branched, having 1-6 C-atoms, preferably 1-5 C-atoms, more preferably 1-4 C-atoms, or most preferably 1-3 C-atoms. 26. The peptide of any one of embodiments 1-10, wherein Y is Chem. 5.
  • Y is a derivative of pyridine, preferably pyridin-2-yl, optionally substituted at one or two of positions 3, 4, 5, and/or 6, where the position numbering of pyridine is according to IUPAC, and/or as shown for pyridine on wikipedia on 3 Dec. 2010 at 18:00 DK time.
  • Q is attached to position 2, 3, 4, 5, or 6 of the pyridine ring, preferably to position 2, where the position numbering of pyridine is as defined in embodiment 31.
  • 33 The peptide of any one of embodiments 1-32, wherein q is as defined in embodiment 21. 34.
  • the medium is a suitable medium, such as a medium containing the human GLP-1 receptor and having the following composition (final in-assay concentrations): 50 mM Tris-HCl, 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; more preferably the following composition (final in-assay concentrations): 50 mM TRIS-HCl; 5 mM HEPES; 10 mM MgCl 2 , 6H 2 O; 150 mM NaCl; 0.01% Tween; 0.1% BSA; 0.5 mM IBMX; 1 mM ATP; 1 uM GTP; pH 7.4.
  • a suitable medium such as a medium containing the human GLP-1 receptor and having the following composition (final in-assay concentrations): 50 mM Tris-HCl
  • the peptide of any one of embodiments 1-39, wherein the GLP-1 receptor agonist peptide of which P is a fragment lacking the two N-terminal amino acid residues is selected from His-Ala-“P” and His-Gly-“P”. 41.
  • P is selected from i) GLP-1(9-37) (SEQ ID NO: 1), ii) exendin-4(3-39) (SEQ ID NO: 2), iii) GLP-1A(3-37) (SEQ ID NO: 3), and iv) analogues of i), ii), or iii) having a maximum of eight amino acid residues exchanged as compared to the respective sequence i), ii), or iii) with which the analogue has the highest similarity, or, preferably, percentage of identity. 42.
  • the peptide of any one of embodiments 1-44 which has a C-terminal amide. 46.
  • the peptide of any one of embodiments 1-45 which has a C-terminal —COOH group. 47.
  • the peptide of any one of embodiments 1-46 which comprises at least one of the following substitutions as compared to GLP-1(9-37) (SEQ ID NO: 1): 18K; 22E; 30E; 31H; 34Q,R; 36K; 37K; and/or 38E. 48.
  • the peptide of any one of embodiments 1-47 which comprises 18K.
  • the peptide of any one of embodiments 1-48 which comprises 22E. 50.
  • the peptide of any one of embodiments 1-49 which comprises 30E. 51.
  • the peptide of any one of embodiments 1-55 which comprises 34R and 37K. 57.
  • the peptide of any one of embodiments 1-59 which comprises the following substitutions: (i) 18K, 22E, 34Q; (ii) 31H, 34Q; (iii) 30E, 36K; (iv) 30E, 36K, 38E; (v) 34R; (vi) 34R, 37K; or (vii) 34R, 37K, 38E. 61.
  • the peptide of any one of embodiments 1-60 which has the following substitutions as compared to GLP-1(9-37) (SEQ ID NO: 1), all other amino acid residues being as in SEQ ID NO: 1: (i) 18K, 22E, 34Q; (ii) 31H, 34Q; (iii) 30E, 36K; (iv) 30E, 36K, 38E; (v) 34R; (vi) 34R, 37K; of (vii) 34R, 37K, 38E. 62.
  • the peptide of any one of embodiments 1-46 which comprises at least one of the following substitutions as compared to GLP-1A(3-37) (SEQ ID NO: 3): 17Q,R; 20R; 33R; and/or 38K.
  • the peptide of any one of embodiments 1-46, and 62 which has the following substitutions, as compared to GLP-1A(3-37) (SEQ ID NO: 3), all other amino acid residues being as in SEQ ID NO: 3: 17R, 20R, 33R, and 38K.
  • a GLP-1 receptor agonist peptide selected from the following:
  • a residue number preferably any residue number, be it in superscript after an amino acid residue, or in ordinary script before or after the amino acid residue in question, refers to the corresponding position in one of the sequences of i) GLP-1(9-37) (SEQ ID NO: 1), ii) exendin-4(3-39) (SEQ ID NO: 2), and iii) GLP-1A(3-37) (SEQ ID NO: 3).
  • the derivative of embodiment 74 which comprises, preferably has, an albumin binding moiety attached to a lysine residue, more preferably to the epsilon-amino group thereof, via an amide bond.
  • the derivative of embodiment 75 which comprises, preferably has, an albumin binding moiety attached to one or more of 18K, 26K, 36K, and/or 37K; preferably one albumin binding moiety attached to 18K, 26K, or 36K; or two albumin binding moieties attached to 26K and 37K; wherein reference may be had to the sequence of GLP-1(9-37) (SEQ ID NO: 1).
  • SEQ ID NO: 1 sequence of GLP-1(9-37)
  • the derivative of embodiment 75 which has an albumin binding moiety attached to one or more of 17K, 20K, 33K, and/or 38K; preferably one albumin binding moiety attached to 38K; or two albumin binding moieties attached to 20K and 33K; wherein reference may be had to the sequence of GLP-1A(3-37) (SEQ ID NO: 3).
  • 78. The derivative of any one of embodiments 75-77, in which the albumin binding moiety comprises a protracting moiety.
  • the protracting moiety is selected from Chem. 8, Chem. 9, and Chem. 10:
  • x is an integer in the range of 6-18
  • y is an integer in the range of 3-17
  • z is an integer in the range of 1-5
  • R 18 is a group having a molar mass not higher than 150 Da.
  • k is an integer in the range of 1-5, and n is an integer in the range of 1-5; and wherein Chem. 12 and Chem. 13 are di-radicals of Glu. 91.
  • the derivative of any one of embodiments 90-92, wherein n is 1. 94.
  • Chem. 11 is included m times, wherein m is an integer in the range of 1-10. 95.
  • k and n are as defined in any one of embodiments 90-97. 99.
  • the derivative of any one of embodiments 90-99, wherein Chem. 12 and Chem. 13, independently, may be represented by Chem. 12a and Chem. 13a, respectively:
  • Chem. 12a 101.
  • the derivative of any one of embodiments 90-102, wherein the Glu di-radical is a radical of L-Glu or D-Glu, preferably of L-Glu. 104.
  • the derivative of any one of embodiments 90-103, wherein the linker consists of a Glu di-radical, preferably Chem. 12, more preferably Chem. 12a.
  • the derivative of any one of embodiments 90-104, wherein the linker comprises Chem. 14. 106.
  • the derivative of any one of embodiments 90-105, where Chem. 14 is represented by Chem. 14a:
  • any one of embodiments 90-106 wherein the linker consists of one time Chem. 14, one time Chem. 12, and two times Chem. 11, interconnected via amide bonds and in the sequence indicated, the linker being connected at its *—NH end to the *—CO end of the protracting moiety, and at its *—CO end to the epsilon amino group of a lysine residue of the peptide. 110.
  • the peptide or derivative of embodiment 115, wherein GLP-1 activity refers to the capability of activating the human GLP-1 receptor.
  • 117. The peptide or derivative of embodiment 116, wherein activation of the human GLP-1 receptor is measured in an in vitro assay, as the potency of cAMP production.
  • 118. The peptide or derivative of any one of embodiments 1-117, which has a potency corresponding to an EC 50 at or below 4500 pM, preferably below 4500 pM, more preferably below 4000 pM, even more preferably below 3500 pM, or most preferably below 3000 pM. 119.
  • the peptide or derivative of any one of embodiments 1-118 which has a potency corresponding to an EC 50 below 2500 pM, preferably below 2000 pM, more preferably below 1500 pM, even more preferably below 1000 pM, or most preferably below 800 pM. 120.
  • the peptide or derivative of any one of embodiments 1-119 which has a potency corresponding to an EC 50 below 600 pM, preferably below 500 pM, more preferably below 400 pM, even more preferably below 300 pM, or most preferably below 200 pM. 121.
  • the peptide or derivative of any one of embodiments 1-120 which has a potency corresponding to an EC 50 below 180 pM, preferably below 160 pM, more preferably below 140 pM, even more preferably below 120 pM, or most preferably below 100 pM. 122.
  • the peptide or derivative of any one of embodiments 1-121 which has a potency corresponding to an EC 50 below 80 pM, preferably below 60 pM, more preferably below 50 pM, even more preferably below 40 pM, or most preferably below 30 pM. 123.
  • a functional receptor assay e.g. based on competition between endogenously formed cAMP and exogenously added biotin-labelled cAMP, in which assay cAMP is more preferably captured using a specific antibody, and/or wherein an even more preferred assay is the AlphaScreen cAMP Assay, most preferably the one described in Example 13. 124.
  • the derivative of any one of embodiments 74-127, for which the ratio [GLP-1 receptor binding affinity (IC 50 ) in the presence of 2.0% HSA (high albumin), divided by GLP-1 receptor binding affinity (IC 50 ) in the presence of 0.005% HSA (low albumin)] is: a) at least 0.5, preferably at least 1.0, more preferably at least 10, even more preferably at least 20, or most preferably at least 30; b) at least 40, preferably at least 50, more preferably at least 60, even more preferably at least 70, or most preferably at least 80; c) at least 90, preferably at least 100, more preferably at least 110, or most preferably at least 120; d) at least 20% of the ratio of semaglutide, preferably at least 50% of the ratio of semaglutide, more preferably at least 75% of the ratio of semaglutide, even more preferably at least equal to the ratio of semaglutide, or most preferably at least twice the ratio of semaglutide; or e) at least
  • any one of embodiments 74-128, for which the GLP-1 receptor binding affinity (IC 50 ) in the presence of 0.005% HSA (low albumin) is a) below 600.00 nM, preferably below 500.00 nM, more preferably below 200.00 nM, even more preferably below 100.00 nM, or most preferably below 45.00 nM; or b) below 20.00 nM, preferably below 10.00 nM, more preferably below 5.00 nM, even more preferably below 2.00 nM, or most preferably below 1.00 nM. 130.
  • the derivative of any one of embodiments 1-129, for which the GLP-1 receptor binding affinity (IC 50 ) in the presence of 2.0% HSA (high albumin) is a) below 900 nM, more preferably below 800 nM, even more preferably below 700 nM, or most preferably below 600 nM; or b) below 400.00 nM, preferably below 300.00 nM, more preferably below 200.00 nM, even more preferably below 100.00 nM, or most preferably below 50.00 nM.
  • the derivative of any one of embodiments 74-133 which has an oral bioavailability, preferably an absolute oral bioavailability, which is higher than that of liraglutide; and/or higher than that of semaglutide. 135.
  • the derivative of embodiment 134 wherein oral bioavailability is measured in vivo in rats, as exposure in plasma after direct injection into the intestinal lumen.
  • the derivative of any one of embodiments 74-136, for which the plasma concentration (pM) of the derivative, determined 30 minutes after injection of a solution of the derivative in the jejunum of rat, divided by the concentration (pM) of the injected solution (dose-corrected exposure at 30 min) is at least 30, preferably at least 40, more preferably at least 50, still more preferably at least 60, even more preferably at least 70, or most preferably at least 80.
  • the derivative of any one of embodiments 74-142 wherein 100 ⁇ l of the derivative is injected into the jejunal lumen through a catheter with a 1 ml syringe, and subsequently 200 ⁇ l of air is pushed into the jejunal lumen with another syringe, which is then left connected to the catheter to prevent flow back into the catheter.
  • 144 The derivative of any one of embodiments 74-143, wherein blood samples (200 ul) are collected into EDTA tubes from the tail vein at desired intervals, such as at times 0, 10, 30, 60, 120 and 240 min, and centrifuged 5 minutes, 10000G, at 4° C. within 20 minutes. 145.
  • protraction means half-life in vivo in a relevant animal species, such as db/db mice, rat, pig, and/or, preferably, minipig; wherein the derivative is administered i) s.c., and/or, preferably, ii) s.c. 149.
  • administration in minipigs is a) at least 12 hours, preferably at least 24 hours, more preferably at least 36 hours, even more preferably at least 48 hours, or most preferably at least 60 hours; or b) at least 0.2 times the half-life of semaglutide, preferably at least 0.4 times the half-life of semaglutide, more preferably at least 0.6 times the half-life of semaglutide, even more preferably at least 0.8 times the half-life of semaglutide, or most preferably at least the same as the half-life of semaglutide.
  • the derivative of embodiment 149, wherein the minipigs are male Göttingen minipigs. 151.
  • 154. The derivative of any one of embodiments 149-153, wherein the animals are fasted for approximately 18 h before dosing and for at least 4 h after dosing, and have ad libitum access to water during the whole period. 155.
  • a peptide according to any one of embodiments 1-73 and 115-127 for use as a medicament.
  • a peptide according to any one of embodiments 1-73 and 115-127 in the manufacture of a medicament for treatment and/or prevention of all forms of diabetes and related diseases, such as eating disorders, cardiovascular diseases, gastrointestinal diseases, diabetic complications, critical illness, and/or polycystic ovary syndrome; and/or for improving lipid parameters, improving ⁇ -cell function, and/or for delaying or preventing diabetic disease progression. 163.
  • a derivative according to any one of embodiments 74-157 in the manufacture of a medicament for treatment and/or prevention of all forms of diabetes and related diseases, such as eating disorders, cardiovascular diseases, gastrointestinal diseases, diabetic complications, critical illness, and/or polycystic ovary syndrome; and/or for improving lipid parameters, improving ⁇ -cell function, and/or for delaying or preventing diabetic disease progression.
  • diabetes and related diseases such as eating disorders, cardiovascular diseases, gastrointestinal diseases, diabetic complications, critical illness, and/or polycystic ovary syndrome
  • lipid parameters improving ⁇ -cell function
  • delaying or preventing diabetic disease progression 164.
  • a method of treating or preventing all forms of diabetes and related diseases such as eating disorders, cardiovascular diseases, gastrointestinal diseases, diabetic complications, critical illness, and/or polycystic ovary syndrome; and/or for improving lipid parameters, improving ⁇ -cell function, and/or for delaying or preventing diabetic disease progression, by administering a pharmaceutically active amount of a peptide according to any one of embodiments 1-73 and 115-127. 165.
  • a method of treating or preventing all forms of diabetes and related diseases such as eating disorders, cardiovascular diseases, gastrointestinal diseases, diabetic complications, critical illness, and/or polycystic ovary syndrome; and/or for improving lipid parameters, improving ⁇ -cell function, and/or for delaying or preventing diabetic disease progression, by administering a pharmaceutically active amount of a derivative according to any one of embodiments 74-157. 166.
  • q is 1-6, and R 15 and R16 independently of each other and independently for each value of q represent hydrogen, alkyl, carboxyl, or hydroxyl;
  • R represents hydrogen, or alkyl;
  • R1 and R2 independently represent (i) hydrogen, alkyl, aryl, heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy, carboxamide, substituted carboxamide, alkyl ester, aryl ester, alkyl sulfonyl, or aryl sulfonyl, or (ii) R1 and R2 together form cyclo alkyl, heterocyclyl, or heteroaryl; and each of PG 1 and PG 2 represents a protection group; with the optional proviso (iii) that R1 and R2 do not both represent hydrogen; or a pharmaceutically acceptable salt, ester, or
  • the activated ester is an ester of p-nitrophenol; 2,4,5-trichlorophenol; N-hydroxysuccinimide; N-hydroxysulfosuccinimide; 3,4-dihydro-3-hydroxy-1,2,3-benzotriazine-4-one; 5-chloro-8-hydroxyquinoline; N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide; pentafluorophenol; p-sulfotetrafluorophenol; N-hydroxyphthalimide; 1-hydroxybenzotriazole; 1-hydroxy-7-azabenzotriazole; N-hydroxymaleimide; 4-hydroxy-3-nitrobenzene sulfonic acid; or any other activated ester known in the art.
  • R1 and R2 independently represent hydrogen, alkyl, aryl, halogen, hydroxyl, hydroxylalkyl, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy, carboxamide, alkyl ester, or aryl ester; wherein preferably alkyl, hydroxylalkyl, aminoalkyl, carboxylalkyl, alkoxy, and/or alkyl ester contains lower alkyl, straight or branched, more preferably having 1-6 C-atoms. 180.
  • q is 1-6, and R15 and R16 independently of each other and independently for each value of q represent hydrogen, alkyl, carboxyl, or hydroxyl;
  • R represents hydrogen, or alkyl;
  • R1 and R2 independently represent (i) alkyl, aryl, heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy, carboxamide, substituted carboxamide, alkyl ester, aryl ester, alkyl sulfonyl, or aryl sulfonyl, or (ii) R1 and R2 together form cyclo alkyl, heterocyclyl, or heteroaryl; and each of PG 1 and PG 2 represents a protection group, preferably as defined in any one of embodiments 167-173; or a pharmaceutically acceptable salt, amide, or ester thereof.
  • a peptide intermediate product which is selected from the following analogues of GLP-1(9-37) (SEQ ID NO: 1): (i) (18K, 22E, 34Q); (ii) (30E, 36K, 38E); (iii) (31H, 34Q); (iv) 34R; (v) (34R, 37K); and (vi) (34R, 37K, 38E); or a pharmaceutically acceptable salt, amide, or ester thereof. 191.
  • a peptide intermediate product which is the following analogue of GLP-1A(3-37) (SEQ ID NO: 3): (17R, 20R, 33R, 38K); or a pharmaceutically acceptable salt, amide, or ester thereof.
  • 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.
  • pseudoproline di-peptides may be used (available from, e.g., Novabiochem, see also W. R. Sampson (1999), J. Pep. Sci. 5, 403).
  • the protected amino acid derivatives used were standard Fmoc-amino acids (supplied from e.g. Anaspec, IRIS, or Novabiochem).
  • the 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 H is at the N-terminus).
  • the epsilon amino group of lysines in the sequence were either protected with Mtt, Mmt, Dde, ivDde, or Boc, depending on the route for attachment of the albumin binding moiety and spacer.
  • the albumin binding moiety and/or linker can be attached to the peptide either by acylation of the resin bound peptide or by acylation in solution of the unprotected peptide.
  • the attachment can be modular using SPPS and suitably protected building blocks such as but not limited to Fmoc-OEG-OH (Fmoc-8-amino-3,6-dioxaoctanoic acid), Fmoc-Trx-OH (Fmoc-tranexamic acid), Fmoc-Glu-OtBu, octadecanedioic acid mono-tert-butyl ester, nonadecanedioic acid mono-tert-butyl ester, or 4-(9-carboxynonyloxy)benzoic acid tert-butyl ester.
  • Fmoc-OEG-OH Fmoc-8-amino-3,6-dioxaoctanoic acid
  • Fmoc-Trx-OH Fmoc-tranexamic acid
  • Fmoc-Glu-OtBu octadecanedioic acid mono-tert-butyl este
  • SPPS method A refers to the synthesis of a protected peptidyl resin using Fmoc chemistry on an Applied Biosystems 433 peptide synthesiser (also designated ABI433A synthesiser) in 0.25 mmol or 1.0 mmol scale using the manufacturer's FastMoc UV protocols which employ HBTU or HATU mediated couplings in NMP, and UV monitoring of the de-protection of the Fmoc protection group.
  • the starting resin used for the synthesis of peptide amides was a suitable Rink-Amide resin (for peptide amides), or (for peptides with a carboxy C-terminus) either a suitable Wang resin or a suitable chlorotrityl resin.
  • Suitable resins are commercially available from, e.g., Novabiochem.
  • SPPS method B refers to the synthesis of a protected peptidyl resin using Fmoc chemistry on a microwave-based Liberty peptide synthesiser (CEM Corp., North Carolina).
  • a suitable resin is a pre-loaded, low-load Wang resin available from Novabiochem (e.g. low load Fmoc-Lys(Mtt)-Wang resin, 0.35 mmol/g).
  • Fmoc-deprotection was with 5% piperidine in NMP at up to 70 or 75° C.
  • the coupling chemistry was DIC/HOAt in NMP.
  • Amino acid/HOAt solutions (0.3 M in NMP at a molar excess of 3-10 fold) were added to the resin followed by the same molar equivalent of DIC (0.75M in NMP).
  • DIC molar equivalent of NMP
  • the following amounts of 0.3M amino acid/HOAt solution were used per coupling for the following scale reactions: Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml, 1 mmol/15 ml.
  • Coupling times and temperatures were generally 5 minutes at up to 70 or 75° C. Longer coupling times were used for larger scale reactions, for example 10 min.
  • Histidine amino acids were double coupled at 50° C., or quadruple coupled if the previous amino acid was sterically hindered (e.g.
  • Arginine amino acids were coupled at RT for 25 min then heated to 70 or 75° C. for 5 min. Some amino acids such as but not limited to Aib, were “double coupled”, meaning that after the first coupling (e.g. 5 min at 75° C.), the resin is drained and more reagents are added (amino acid, HOAt and DIC), and the mixture in heated again (e.g. 5 min at 75° C.). When a chemical modification of a lysine side chain was desired, the lysine was incorporated as Lys(Mtt).
  • the Mtt group was removed by washing the resin with DCM and suspending the resin in neat (undiluted) hexafluoroisopropanol for 20 minutes followed by washing with DCM and NMP.
  • the chemical modification of the lysine was performed either by manual synthesis (see SPPS method D) or by one or more automated steps on the Liberty peptide synthesiser as described above, using suitably protected building blocks (see General methods), optionally including a manual coupling.
  • SPPS method D refers to synthesis of the protected peptidyl resin using manual Fmoc chemistry. This was typically used for the attachment of the linkers and side chains to the peptide backbone. The following conditions were employed at 0.25 mmol synthesis scale. The coupling chemistry was DIC/HOAt/collidine in NMP at a 4-10 fold molar excess. Coupling conditions were 1-6 h at room temperature. Fmoc-deprotection was performed with 20-25% piperidine in NMP (3 ⁇ 20 ml, each 10 min) followed by NMP washings (4 ⁇ 20 mL).
  • Dde- or ivDde-deprotection was performed with 2% hydrazine in NMP (2 ⁇ 20 ml, each 10 min) followed by NMP washings (4 ⁇ 20 ml).
  • Mtt- or Mmt-deprotection was performed with 2% TFA and 2-3% TIS in DCM (5 ⁇ 20 ml, each 10 min) followed by DCM (2 ⁇ 20 ml), 10% MeOH and 5% DIPEA in DCM (2 ⁇ 20 ml) and NMP (4 ⁇ 20 ml) washings, or by treatment with neat hexafluoroisopropanol (5 ⁇ 20 ml, each 10 min) followed by washings as above.
  • the albumin binding moiety and/or linker can be attached to the peptide either by acylation of the resin bound peptide or acylation in solution of the unprotected peptide (see the routes described below).
  • the attachment can be modular using SPPS and suitably protected building blocks (see General methods).
  • Activated (active ester or symmetric anhydride) albumin binding moiety or linker such as octadecanedioic acid mono-(2,5-dioxo-pyrrolidin-1-yl)ester (Ebashi et al. EP511600, 4 molar equivalents relative to resin bound peptide) was dissolved in NMP (25 mL), added to the resin and shaken overnight at room temperature. The reaction mixture was filtered and the resin was washed extensively with NMP, DCM, 2-propanol, methanol and diethyl ether.
  • NMP octadecanedioic acid mono-(2,5-dioxo-pyrrolidin-1-yl)ester
  • the albumin binding moiety was dissolved in NMP/DCM (1:1, 10 ml).
  • the activating reagent such as HOBt (4 molar equivalents relative to resin) and DIC (4 molar equivalents relative to resin) was added and the solution was stirred for 15 min.
  • the solution was added to the resin and DIPEA (4 molar equivalents relative to resin) was added.
  • the resin was shaken 2 to 24 hours at room temperature.
  • the resin was washed with NMP (2 ⁇ 20 ml), NMP/DCM (1:1, 2 ⁇ 20 ml) and DCM (2 ⁇ 20 ml).
  • Activated (active ester or symmetric anhydride) albumin binding moiety or linker such as octadecanedioic acid mono-(2,5-dioxo-pyrrolidin-1-yl)ester (Ebashi et al. EP511600) 1-1.5 molar equivalents relative to the peptide was dissolved in an organic solvent such as acetonitrile, THF, DMF, DMSO or in a mixture of water/organic solvent (1-2 ml) and added to a solution of the peptide in water (10-20 ml) together with 10 molar equivalents of DIPEA.
  • organic solvent such as acetonitrile, THF, DMF, DMSO
  • the reaction mixture was lyophilised overnight and the isolated crude peptide deprotected afterwards.
  • the deprotection was performed by dissolving the peptide in a mixture of trifluoroacetic acid, water and triisopropylsilane (90:5:5). After for 30 min the mixture was evaporated in vacuo and the crude peptide purified by preparative HPLC as described later.
  • SPPS method E refers to peptide synthesis by Fmoc chemistry on a Prelude Solid Phase Peptide Synthesiser from Protein Technologies (Tucson, Ariz. 85714 U.S.A.).
  • a suitable resin is a pre-loaded, low-load Wang resin available from Novabiochem (e.g. low load fmoc-Lys(Mtt)-Wang resin, 0.35 mmol/g).
  • Fmoc-deprotection was with 25% piperidine in NMP for 2 ⁇ 10 min.
  • the coupling chemistry was DIC/HOAt/collidine in NMP.
  • Amino acid/HOAt solutions (0.3 M in NMP at a molar excess of 3-10 fold) were added to the resin followed by the same molar equivalent of DIC (3 M in NMP) and collidine (3 M in NMP).
  • DIC 3 M in NMP
  • collidine 3 M in NMP
  • the following amounts of 0.3M amino acid/HOAt solution were used per coupling for the following scale reactions: Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml. Coupling times were generally 60 minutes.
  • Some amino acids including, but not limited to arginine, Aib or histidine were “double coupled”, meaning that after the first coupling (e.g.
  • amino acids and fatty acid derivatives including but not limited to Fmoc-Oeg-OH, Fmoc-Trx-OH, Fmoc-Glu-OtBu, octadecanedioic acid mono-tert-butyl ester, nonadecanedioic acid mono-tert-butyl ester, or 4-(9-carboxynonyloxy)benzoic acid tert-butyl ester were coupled for prolonged time, for example 6 hours.
  • the lysine was incorporated as Lys(Mtt).
  • the Mtt group was removed by washing the resin with DCM and suspending the resin in hexafluoroisopropanol/DCM (75:25) for 3 ⁇ 10 minutes followed by washings with DCM, 20% piperidine and NMP.
  • the chemical modification of the lysine was performed either by manual synthesis (see SPPS method D) or by one or more automated steps on the Prelude peptide synthesiser as described above using suitably protected building blocks (see General methods).
  • the resin was washed with DCM, and the peptide was cleaved from the resin by a 2-3 hour treatment with TFA/TIS/water (95/2.5/2.5 or 92.5/5/2.5) followed by precipitation with diethylether.
  • the peptide was dissolved in a suitable solvent (such as, e.g., 30% acetic acid) and purified by standard RP-HPLC on a C18, 5 ⁇ M column, using acetonitrile/water/TFA.
  • the fractions were analysed by a combination of UPLC, MALDI and LCMS methods, and the appropriate fractions were pooled and lyophilised.
  • a Perkin Elmer Sciex API 3000 mass spectrometer was used to identify the mass of the sample after elution from a Perkin Elmer Series 200 HPLC system.
  • a Waters Micromass ZQ mass spectrometer was used to identify the mass of the sample after elution from a Waters Alliance HT HPLC system.
  • LCMS4 was performed on a setup consisting of Waters Acquity UPLC system and LCT Premier XE mass spectrometer from Micromass.
  • the UPLC pump was connected to two eluent reservoirs containing:
  • A 0.1% Formic acid in water
  • B 0.1% Formic acid in acetonitrile
  • the analysis was performed at RT by injecting an appropriate volume of the sample (preferably 2-10 ⁇ l) onto the column which was eluted with a gradient of A and B.
  • the UPLC conditions, detector settings and mass spectrometer settings were: Column: Waters Acquity HPLC BEH, C-18, 1.7 ⁇ m, 2.1 mm ⁇ 50 mm Gradient: Linear 5%-95% acetonitrile during 4.0 min (alternatively 8.0 min) at 0.4 ml/min Detection: 214 nm (analogue output from TUV (Tunable UV detector)) MS ionisation mode: API-ES Scan: 100-2000 amu (alternatively 500-2000 amu), step 0.1 amu
  • UPLC (method 05_B5 — 1): 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 UBEH130, C18, 130 ⁇ , 1.7 um, 2.1 mm ⁇ 150 mm column, 40° C.
  • the UPLC system was connected to two eluent reservoirs containing:
  • UPLC (method 05_B7 — 1): 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 ⁇ , 1.7 um, 2.1 mm ⁇ 150 mm column, 40° C.
  • the UPLC system was connected to two eluent reservoirs containing:
  • UPLC (method 04_A2 — 1): 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 ⁇ , 1.7 um, 2.1 mm ⁇ 150 mm column, 40° C.
  • the UPLC system was connected to two eluent reservoirs containing: A: 90% H 2 O, 10% CH 3 CN, 0.25 M ammonium bicarbonate
  • UPLC (method 04_A3 — 1): 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 ⁇ , 1.7 um, 2.1 mm ⁇ 150 mm column, 40° C.
  • the UPLC system was connected to two eluent reservoirs containing: A: 90% H 2 O, 10% CH 3 CN, 0.25 M ammonium bicarbonate
  • UPLC (method 04_A4 — 1): 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 ⁇ , 1.7 um, 2.1 mm ⁇ 150 mm column, 40° C.
  • the UPLC system was connected to two eluent reservoirs containing: A: 90% H 2 O, 10% CH 3 CN, 0.25 M ammonium bicarbonate
  • UPLC (method 08_B2 — 1): 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 ⁇ , 1.7 um, 2.1 mm ⁇ 150 mm column, 40° C.
  • the UPLC system was connected to two eluent reservoirs containing:
  • UPLC (method 08_B4 — 1): 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 ⁇ , 1.7 um, 2.1 mm ⁇ 150 mm column, 40° C.
  • the UPLC system was connected to two eluent reservoirs containing:
  • UPLC (Method 05_B10 — 1): The RP-analyses 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 ⁇ , 1.7 um, 2.1 mm ⁇ 150 mm column, 40° C.
  • the UPLC system was connected to two eluent reservoirs containing:
  • UPLC Method 02_B4 — 4
  • the RP-analysis was performed using a Alliance Waters 2695 system fitted with a Waters 2487 dualband detector. UV detections at 214 nm and 254 nm were collected using a Symmetry300 C18, 5 um, 3.9 mm ⁇ 150 mm column, 42° C. Eluted with a linear gradient of 5-95% acetonitrile, 90-0% water, and 5% trifluoroacetic acid (1.0%) in water over 15 minutes at a flow-rate of 1.0 ml/min.
  • HPLC Method 01_B4 — 1: The RP-analysis was performed using a Waters 600S system fitted with a Waters 996 diode array detector. UV detections were collected using a Waters 3 mm ⁇ 150 mm 3.5 um C-18 Symmetry column. The column was heated to 42° C. and eluted with a linear gradient of 5-95% acetonitrile, 90-0% water, and 5% trifluoroacetic acid (1.0%) in water over 15 minutes at a flow-rate of 1 ml/min.
  • Histamine dihydrochloride (20.47 g; 0.111 mol) and triethylamine (48 mL; 0.345 mol) in absolute methanol (400 mL) were stirred at room temperature for 10 min.
  • Trifluoroacetic acid ethyl ester (14.6 mL; 0.122 mol) in methanol (30 mL) was added dropwise over 30 min at 0° C. Reaction mixture was stirred for 3.5 hrs at room temperature and then it was evaporated to dryness in vacuo. The residue was dissolved in dichlormethane (450 mL) and triethylamine (31 mL; 0.222 mol) was added.
  • R F SiO 2 , chloroform/ethyl acetate, 98:2): 0.60.
  • Aluminum chloride powder (80.0 g, 600 mmol) was added in portions to a stirred mixture of tert-butylbenzene (40.0 g, 300 mmol) and succinic anhydride (26.7 g, 267 mmol) and 1,1,2,2-tetrachloroethane (100 mL). After all the aluminum chloride had been added, the mixture was poured into a mixture of ice (500 mL) and concentrated hydrochloric acid (100 mL). The organic layer was separated, washed with water (500 mL) and the solvent distilled off.
  • Acetyl chloride (51.0 mL, 718 mmol) was added dropwise to methanol (670 mL) at 0° C. under argon. After 30 min, the cooling bath was removed and the above oxime (16.0 g, 144 mmol) was added, followed by palladium on carbon (5 wt %, 6.1 g). The mixture was hydrogenated at atmospheric pressure for 17 hrs, then it was filtered through Celite and the solvent evaporated to give pure 4-(aminomethyl)-imidazole dihydrochloride as colorless crystals.
  • the aqueous phase was extracted with chloroform (100 mL); the chloroform phases were combined, washed with water (150 mL) dried over anhydrous magnesium sulfate and solvent removed in vacuo.
  • the residue was purified by flash column chromatography (silica gel Fluke 60, dichloromethane/methanol 98:2 to 9:1) and crystallised from chloroform/hexanes mixture to give the title product as beige crystals.
  • Methanesulfonyl chloride (8 mL, 104 mmol) was added dropwise to a solution of the above alcohol (32.0 g, 86.8 mmol) in dichloromethane (400 mL) and triethyl amine (15.5 mL) at 0° C. during 1 hr. The mixture was stirred without cooling for an additional 1 hr; then it was washed with 5% sodium bicarbonate and dried over anhydrous magnesium sulfate. Dichloromethane was evaporated at 30° C. in vacuo and the residual oily mesylate was used directly in the next step.
  • 2-Chlorotrityl chloride resin (2.3 g, 3.0 mmol) was swelled in DCM for 20 mins and filtrered.
  • Dimethylmalonic acid (2 eq; 6.0 mmol; 793 mg) was dissolved i DCM:DMF 1:1 (10 mL) and added to the resin followed by DIPEA (6 eq; 18.0 mmol; 3.14 mL) and DCM (10 mL).
  • DIPEA 6 eq; 18.0 mmol; 3.14 mL
  • DCM 10 mL
  • the resin was shaken overnight at RT.
  • the resin was filtered and washed with DCM:MeOH:DIPEA (17:2:1), DCM, NMP og DCM (2 ⁇ 25 mL of each).
  • the resin was swelled in DMF for 20 mins and filtered.
  • Chlorotrityl chloride resin (2.3 g, 3.0 mmol) was swelled in DCM for 20 mins and filtered.
  • Dimethylmalonic acid (2 eq; 6.0 mmol; 793 mg) was dissolved i DCM:NMP 1:1 (10 mL) and added to the resin followed by DIPEA (6 eq; 18.0 mmol; 3.14 mL) and DCM (10 mL).
  • DIPEA 6 eq; 18.0 mmol; 3.14 mL
  • DCM 10 mL
  • the resin was shaken overnight at RT.
  • the resin was filtered and washed with DCM:MeOH:DIPEA (17:2:1), DCM, NMP og DCM (2 ⁇ 25 mL of each).
  • the resin was swelled in NMP for 20 mins and filtered.
  • SPPS method E 2,2-Dimethyl-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-malonamic acid was coupled using the same coupling condition as an Aib amino acid
  • SPPS method B 2,2-Dimethyl-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-malonamic acid, Fmoc-Oeg-OH, Fmoc-Glu-OtBu, and octadecanedioic acid mono-tert-butyl ester were coupled using the same coupling condition as an Aib amino acid.
  • SPPS method B 2,2-Dimethyl-N-[3-(1-trityl-1H-imidazol-4-yl)-propyl]-malonamic acid, Fmoc-Oeg-OH, and octadecanedioic acid mono-tert-butyl ester were coupled using the same coupling condition as an Aib amino acid
  • SPPS method B 2,2-Dimethyl-N-(1-trityl-1H-imidazol-4-ylmethyl)-malonamic acid, Fmoc-Oeg-OH, and octadecanedioic acid mono-tert-butyl ester were coupled using the same coupling condition as an Aib amino acid
  • SPPS method B 2,2-Dimethyl-N-(1-trityl-1H-imidazol-4-ylmethyl)-malonamic acid, Fmoc-Oeg-OH, Fmoc-Glu-OtBu, and octadecanedioic acid mono-tert-butyl ester were coupled using the same coupling condition as an Aib amino acid
  • SPPS method B 2,2-Dimethyl-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-malonamic acid was coupled using the same coupling condition as an Aib amino acid. 8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid (commercially available from Iris Biotech), Fmoc-Glu-OtBu and 4-(9-carboxy-nonyloxy)-benzoic acid tert-butyl ester (prepared as described in Example 25, step 2 of WO 2006/082204) were coupled using SPPS method D.
  • SPPS method B 2,2-Dimethyl-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-malonamic acid was coupled using the same coupling condition as an Aib amino acid. 8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid (commercially available from Iris Biotech), Fmoc-Glu-OtBu, and 4-(9-carboxy-nonyloxy)-benzoic acid tert-butyl ester (prepared as described in Example 25, step 2 of WO 2006/082204) were coupled using SPPS method D.
  • SPPS method B 2,2-Dimethyl-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-malonamic acid was coupled using the same coupling condition as Fmoc-Aib amino acid. 8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid (commercially available from Iris Biotech), Fmoc-Glu-OtBu and 4-(4-t-butylphenyl)butyric acid were coupled using SPPS method D.
  • SPPS method E 2,2-Dimethyl-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-malonamic acid was coupled using the same coupling condition as an Aib amino acid
  • the purpose of this example is to test the activity, or potency, of the GLP-1 receptor agonist derivatives in vitro.
  • the potencies of the GLP-1 receptor agonist derivatives of Examples 1-12 were determined as described below, i.e. as the stimulation of the formation of cyclic AMP (cAMP) in a medium containing membranes expressing the human GLP-1 receptor.
  • cAMP cyclic AMP
  • Example 11 is a comparative compound based on compound 215 (p. 24) of WO 2004/067548, which according to FIG. 1 of this WO publication is one of the most potent compounds of this publication
  • a stable transfected cell line and a high expressing clone were selected for screening.
  • the cells were grown at 5% CO 2 in DMEM, 5% FCS, 1% Pen/Strep (Penicillin/Streptomycin) and 0.5 mg/ml of the selection marker G418.
  • Cells at approximate 80% confluence were washed 2 ⁇ with PBS and harvested with Versene (aqueous solution of the tetrasodium salt of ethylenediaminetetraacetic acid), centrifuged 5 min at 1000 rpm and the supernatant removed. The additional steps were all made on ice.
  • the suspension was homogenised for 20-30 sec and centrifuged 15 min at 20,000 rpm. Suspension in Buffer 2, homogenisation and centrifugation was repeated once and the membranes were resuspended in Buffer 2. The protein concentration was determined and the membranes stored at ⁇ 80° C. until use.
  • the assay was performed in 1 ⁇ 2-area 96-well plates, flat bottom (Costar cat. no: 3693). The final volume per well was 50 ⁇ l.
  • AlphaScreen cAMP Assay Kit from Perkin Elmer Life Sciences (cat. No: 6760625M); containing Anti-cAMP Acceptor beads (10 U/ ⁇ l), Streptavidin Donor beads (10 U/ ⁇ l) and Biotinylated-cAMP (133 U/ ⁇ l).
  • AlphaScreen Buffer, pH 7.4: 50 mM TRIS-HCl (Sigma, cat.no: T3253); 5 mM HEPES (Sigma, cat.no: H3375); 10 mM MgCl 2 , 6H 2 O (Merck, cat.no: 5833); 150 mM NaCl (Sigma, cat.no: S9625); 0.01% Tween (Merck, cat.no: 822184).
  • BSA Sigma, cat. no. A7906
  • IBMX Sigma, cat. no. 15879): 0.5 mM
  • ATP Sigma, cat. no. A7699
  • GTP Sigma, cat. no. G8877
  • Suitable dilution series in AlphaScreen Buffer were prepared of the cAMP standard as well as the GLP-1 analogue or derivative to be tested, e.g. the following eight concentrations of the GLP-1 compound: 10 ⁇ 7 , 10 ⁇ 8 , 10 ⁇ 9 , 10 ⁇ 10 , 10 ⁇ 11 , 10 ⁇ 12 , 10 ⁇ 13 and 10 ⁇ 14 M, and a series from, e.g., 10 ⁇ 6 to 3 ⁇ 10 ⁇ 11 of cAMP.
  • any handling was in the dark (as dark as possible), or in green light. All dilutions were made on ice.
  • the fold variation in relation to GLP-1 may be calculated as EC 50 (GLP-1)/EC 50 (analogue)—3693.2.
  • All tested derivatives of the invention had a good in vitro potency corresponding to an EC 50 of 2100 pM or below; eight derivatives were even more potent having and EC 50 at 1000 pM or below; five derivatives had a still further improved potency corresponding to an EC 50 at 500 pM or below; four derivatives were very potent corresponding to an EC 50 at 300 pM or below; and one derivative had a very good potency corresponding to an EC 50 at 100 pM or below.
  • the comparative compound of Example 11 was much less potent, namely with an EC 50 of above 8000 pM.
  • the purpose of this experiment is to investigate the binding to the GLP-1 receptor of the GLP-1 agonist derivatives, and how the binding is potentially influenced by the presence of albumin. This is done in an in vitro experiment as described below.
  • the binding affinity of the GLP-1 receptor agonist derivatives of Examples 1-12 to the human GLP-1 receptor was measured by way of their ability to displace of 125 I-GLP-1 from the receptor.
  • Example 11 is a comparative compound based on compound 215 (p. 24) of WO 2004/067548, which according to FIG. 1 of this WO publication is one of the most potent compounds of this publication.
  • the assay was performed with a low concentration of albumin (0.005%—corresponding to the residual amount thereof in the tracer), as well as with a high concentration of albumin (2.0% added).
  • a shift in the binding affinity, IC 50 is an indication that the peptide in question binds to albumin, and thereby a prediction of a potential protracted pharmacokinetic profile of the peptide in question in animal models.
  • GLP-1 receptor GLP-1 receptor
  • a stable transfected cell line and a high expressing clone were selected for screening.
  • the cells were grown at 5% CO 2 in DMEM, 10% FCS, 1% Pen/Strep (Penicillin/Streptomycin) and 1.0 mg/ml of the selection marker G418.
  • the cells (approx. 80% confluence) were washed twice in PBS and harvested with Versene (aqueous solution of the tetrasodium salt of ethylenediaminetetraacetic acid), following which they were separated by centrifugation at 1000 rpm for 5 min.
  • the cells/cell pellet must be kept on ice to the extent possible in the subsequent steps.
  • the cell pellet was homogenised with Ultrathurrax for 20-30 seconds in a suitable amount of Buffer 1 (depending on the amount of cells, but e.g. 10 ml). The homogenate was centrifuged at 20000 rpm for 15 minutes. The pellet was resuspended (homogenised) in 10 ml Buffer 2 and re-centrifuged. This step was repeated once more. The resulting pellet was resuspended in Buffer 2, and the protein concentration was determined.
  • the membranes were stored at minus 80° C.
  • Buffer 1 20 mM Na-HEPES+10 mM EDTA, pH 7.4
  • Buffer 2 20 mM Na-HEPES+0.1 mM EDTA, pH 7.4
  • Test compounds, membranes, SPA-particles and [ 125 I]-GLP-1(7-36)NH 2 were diluted in assay buffer. 25 ul (micro liter) of test compounds were added to Optiplate. HSA (“high albumin” experiment containing 2% HSA), or buffer (“low albumin” experiment containing 0.005% HSA), was added (50 ul). 5-10 ug protein/sample was added (50 ul) corresponding to 0.1-0.2 mg protein/ml (to be preferably optimised for each membrane preparation).
  • SPA-particles (Wheatgerm agglutinin SPA beads, Perkin Elmer, #RPNQ0001) were added in an amount of 0.5 mg/well (50 ul).
  • HSA was SIGMA A1653
  • the IC 50 value was read from the curve as the concentration which displaces 50% of 125 I-GLP-1 from the receptor, and the ratio of [(IC 50 /nM) high HSA]/[(IC 50 /nM) low HSA] was determined.
  • the binding to the GLP-1 receptor at low albumin concentration should be as good as possible, corresponding to a low IC 50 value.
  • the IC 50 value at high albumin concentration is a measure of the influence of albumin on the binding of the derivative to the GLP-1 receptor.
  • the GLP-1 receptor agonist derivatives also bind to albumin. This is a generally desirable effect, which extends their lifetime in plasma. Therefore, the IC 50 value at high albumin will generally be higher than the IC 50 value at low albumin, corresponding to a reduced binding to the GLP-1 receptor, caused by albumin binding competing with the binding to the GLP-1 receptor.
  • a high ratio (IC 50 value (high albumin)/IC 50 value (low albumin)) may therefore be taken as an indication that the derivative in question binds well to albumin (may have a long half-life), and also per se binds well to the GLP-1 receptor (the IC 50 value (high albumin) is high, and the IC 50 value (low albumin) is low).
  • ratio refers to [(IC 50 /nM) high HSA]/[(IC 50 /nM) low HSA]):
  • IC 50 low albumin
  • all derivatives, except the comparative compound of Example 11 had an IC 50 (low albumin) below 40 nM; all but one below 20 nM; all but four were below 10.0 nM; five were below 5.00 nM; and three derivatives were below 1.00 nM.
  • IC 50 high albumin
  • all derivatives of the invention had an IC 50 (high albumin) below 900.00 nM; nine were below 500.00 nM; four were below 100.00 nM; and two derivatives were below 50.00 nM.
  • the IC 50 (high albumin) for the comparative compound of Example 11 was above 800.00 nM.
  • the purpose of this experiment is to estimate the oral bioavailability of the GLP-1 receptor agonist derivatives.
  • the compounds are tested in a concentration of 1000 uM in a solution of 55 mg/ml sodium caprate.
  • the compounds are administered in the jejunum either in the proximal part (10 cm distal for the duodenum) or in the mid-intestine (50 cm proximal for the cecum).
  • a PE50-catheter 10 cm long is inserted into the jejunum, forwarded at least 1.5 cm into the jejunum, and secured before dosing by ligature around the gut and the catheter with 3/0 suture distal to tip to prevent leak or catheter displacement.
  • Catheter is placed without syringe and needle and 2 ml saline is administered into abdomen before closing the incision with wound clips.
  • 100 ⁇ l of the respective compound is injected into the jejunal lumen through the catheter with a 1 ml syringe. Subsequently, 200 ⁇ l of air is pushed into the jejunal lumen with another syringe to “flush” the catheter. This syringe is leaved connected to the catheter to prevent flow back into the catheter.
  • Plasma samples are collected at desired intervals (usually at times 0, 10, 30, 60, 120 and 240 min) into EDTA tubes from the tail vein and centrifuged 5 minutes, 10000G, at 4° C. within 20 minutes.
  • Plasma 75 ul is separated to Micronic tubes, immediately frozen, and kept at ⁇ 20° C. until analyzed for plasma concentration of the respective GLP-1 receptor agonist derivative with LOCI (Luminescent Oxygen Channeling Immunoassay), generally as described for the determination of insulin by Poulsen and Jensen in Journal of Biomolecular Screening 2007, vol. 12, p. 240-247.
  • LOCI Luminescent Oxygen Channeling Immunoassay
  • the donor beads are coated with streptavidin, while acceptor beads are conjugated with a monoclonal antibody recognising a mid-/C-terminal epitope of the peptide.
  • Another monoclonal antibody, specific for the N-terminus is biotinylated.
  • the three reactants are combined with the analyte and formed a two-sited immuno-complex. Illumination of the complex released singlet oxygen atoms from the donor beads, which are channeled into the acceptor beads and triggered chemiluminescence which is measured in an Envision plate reader. The amount of light is proportional to the concentration of the compound.
  • the rats are sacrificed under anaesthesia and the abdomen is opened to verify correct catheter placement.
  • plasma concentrations are determined as a function of time.
  • the dose-corrected exposure has been shown to correlate significantly with the actual bioavailability.
  • the results may be given as dose-corrected exposure at 30 min which refers to (the plasma concentration 30 minutes after injection of the compound in the jejunum (pM)), divided by (the concentration of the compound in the dosing solution (pM)).
  • the purpose of this study is to determine the protraction in vivo of the GLP-1 receptor agonist derivatives after i.v. administration to minipigs, i.e. the prolongation of their time of action. This is done in a pharmacokinetic (PK) study, where the terminal half-life of the derivative in question is determined.
  • terminal half-life is generally meant the period of time it takes to halve a certain plasma concentration, measured after the initial distribution phase.
  • the animals are fasted for approximately 18 h before dosing and for at least 4 h after dosing, but had ad libitum access to water during the whole period.
  • the compounds are dissolved in 50 mM sodium phosphate, 145 mM sodium chloride, 0.05% tween 80, pH 7.4 to a concentration of usually from 20-60 nmol/ml.
  • Intravenous injections (the volume corresponding to usually 1-2 nmol/kg, for example 0.033 ml/kg) of the compounds are given through a catheter, and blood is sampled at predefined time points for up till 13 days post dosing (preferably through the other catheter). Blood samples (for example 0.8 ml) are collected in EDTA buffer (8 mM) and then centrifuged at 4° C. and 1942G for 10 minutes. Plasma is pippetted into Micronic tubes on dry ice, and kept at ⁇ 20° C.
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