Classifications

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  • C07K—PEPTIDES
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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • 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invention such as Glucagon-Like Peptide-1 (GLP-1 ), exendins and analogues thereof, the two N-terminal amino acids have been replaced by N-terminal mimetics.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 mimicing the N-terminus of the peptide.
• the new N-terminal is preferably a His-Ala, a
• His-Gly 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
• 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
• Chem. 4 represents a group of formula Chem. 4: , or Chem. 5:
• 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
• R1 1 , 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
• Chem. 6 *-(C(R15)(R16)) q -* , wherein 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 alkyi; 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.
• 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
• Chem. 4 represents a group of formula Chem. 4: , or Chem. 5:
• X 1 is N, O, or S
• X 2 , X3, X 4 , and X 5 independently represent C, or N, with the proviso that at least one of X 2 , X3, X 4 and X 5 is C
• R1 1 , 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
• Chem. 6 *-(C(R15)(R16)) q -* , wherein 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 alkyi; 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.
• W represents a group of formula Chem. 3: , wherein R1 and R2 independently represent (i) hydrogen, alkyl, aryl, heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy,
• X 2 , X3, 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;
• R1 1 , 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
• Chem. 6 *-(C(R15)(R16)) q -* , wherein 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 alkyi; 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.
• ⁇ alpha
• ⁇ beta
• ⁇ epsilon
• ⁇ gamma
• co omega
• 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-1 A(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
• sequences of the corresponding fragments lacking the two N-terminal amino acids viz. GLP-1 (9-37), exendin-4(3-39), and GLP-1 A(3-37), 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.
• 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, GLP1 R_HUMAN, Glucagon-like peptide 1 receptor, GLP-1 receptor, GLP-1 -R, or GLP-1 R.
• 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. Amino acids and peptides
• 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 (a-aminoisobutyric acid), ⁇ -alanine, and des-amino-histidine (desH, alternative name imidazopropionic acid, abbreviated Imp).
• D-isomers of the amino acids such as D-alanine and D-leucine, Aib (a-aminoisobutyric acid), ⁇ -alanine, and des-amino-histidine (desH, alternative name imidazopropionic acid, abbreviated Imp).
• GLP-1 receptor agonist peptides fragments, analogues, residue numbering, identity
• 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-1 A(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.
• SEQ ID NO: 2 which is also glutamic acid
• this glutamic acid residue is referred to as no. 3
• subsequent amino acid residues are numbered accordingly, ending with serine as no. 39.
• the first amino acid residue of GLP-1A(3-37) (SEQ ID NO: 3), which is aspartic acid, is assigned no. 1.
• this aspartic acid residue is referred to as no. 3, and subsequent amino acid residues are numbered accordingly, ending with serine as no. 37.
• 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 I D 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:
• [Lys 18 , Glu 22 , Gln 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;
• 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, 21 D, 22E, 23R, 26R, 30D, 33I, 34N, 36G, 37P, 38S, 39R, 40E, 41 1, 421, 43S, 44K) of GLP-1 (9-37) (SEQ ID NO: 1 ), having ⁇ 2-[2-(1 H- lmidazol-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; 31 H; 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, CD., (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.
• sequence no. 1 is
• SEQ ID NO: 1 SEQ ID NO: 1
• 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.
• 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
• 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 -0-(CH 2 ) 2 -0-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 examplified 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 S0 4 ⁇ (NH 4 ) 2 S0 4 .
• the salt may be a basic salt, an acid salt, or it may be neither nor (i.e. a neutral salt).
• Basic salts produce hydroxide ions and acid salts hydronium ions in water.
• the salts of the 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
• 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. In a still further particular embodiment, 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 R1 R2 PG 1 R O O wherein 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 PGi 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.
• the term half maximal effective concentration generally refers to the 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 AlphaS
• the medium has the following composition (final in-assay concentrations): ): 50 mM TRIS-HCI; 5 mM HEPES; 10 mM MgCl2, 6H2O; 150 mM NaCI; 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 2000pM, preferably below 1800pM, more preferably below 1700pM, even more preferably below 1600pM, or most preferably below 1500pM.
• 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 WO09/030738.
• the derivatives of the invention are protracted.
• 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.
• HSA low albumin
• 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.
• 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.
• Such derivatives could be suitable candidates for oral administration, as long as their potency is generally satisfactory, and/or as long as their half-life is also generally satisfactory.
• 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 refers to the fraction of an administered dose of the compound that reaches the systemic circulation unchanged.
• bioavailability is 100%.
• 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
• 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,
• 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,
• 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.
• mannitol sorbitol
• inositol galactitol
• dulcitol xylitol
• arabitol 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),
• 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 benzamidineHCI.
• protease inhibitors such as, e.g., EDTA (ethylenediamine tetraacetic acid), and/or benzamidineHCI.
• 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;
• 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
• the term "physical stability” refers to the tendency of the polypeptide to form biologically inactive and/or insoluble aggregates as a result of exposure to thermo- mechanical stress, and/or interaction with destabilising interfaces and surfaces (such as hydrophobic surfaces).
• the physical stability of an aqueous polypeptide formulation may be evaluated by means of visual inspection, and/or by turbidity measurements after exposure to mechanical/physical stress (e.g. agitation) at different temperatures for various time periods.
• the physical stability may be evaluated using a spectroscopic agent or probe of the conformational status of the polypeptide such as e.g. Thioflavin T or "hydrophobic patch" probes.
• chemical stability refers to chemical (in particular covalent) changes in the polypeptide structure leading to formation of chemical degradation products potentially having a reduced biological potency, and/or increased immunogenic effect as compared to the intact polypeptide.
• the chemical stability can be evaluated by measuring the amount of chemical degradation products at various time-points after exposure to different
• the treatment with a 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
• 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 HbA1 C;
• diabetes such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes, MODY (maturity onset diabetes of the young), gestational diabetes, and/or for reduction of HbA1 C;
• diabetes delaying or preventing diabetic disease progression, such as progression in type 2 diabetes, delaying the progression of impaired glucose tolerance (IGT) to insulin requiring type 2 diabetes, and/or delaying the progression of non-insulin requiring type 2 diabetes to insulin requiring type 2 diabetes;
• ITT impaired glucose tolerance
• diabetes prevention and/or treatment of diabetic complications, such as neuropathy, including peripheral neuropathy; nephropathy; or retinopathy;
• 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
• W represents a group of formula Chem. 3:
• R1 and R2 independently represent
• R1 and R2 do not both represent hydrogen
• Y represents a group of formula Chem. 4 or Chem. 5:
• Xi is N, O, or S;
• X 2 , X 3 , X 4 , and X 5 independently represent C, or N,
• R1 1 , R12, R13, and R14 independently represent
• 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 alkyi;
• R1 and R2 independently represent hydrogen, alkyi, aryl, halogen, hydroxyl, hydroxylalkyi, amino, aminoalkyi, carboxyl, carboxylalkyi, alkoxy, aryloxy, carboxamide, alkyi ester, or aryl ester; wherein preferably alkyi, hydroxylalkyi, aminoalkyi, carboxylalkyi, alkoxy, and/or alkyi ester contains lower alkyi, straight or branched, more preferably having 1 -6 C-atoms.
• R1 and R2 independently represent hydrogen, alkyi having 1 -2 C-atoms (ethyl, or methyl), or alkoxy having 1 -2 C- atoms (ethoxy, methoxy).
• R1 and R2 independently represent alkyi, aryl, halogen, hydroxyl, hydroxylalkyi, amino, aminoalkyi, carboxyl, carboxylalkyi, alkoxy, aryloxy, carboxamide, alkyi ester, or aryl ester; wherein preferably alkyi, hydroxylalkyi, aminoalkyi, carboxylalkyi, alkoxy, and/or alkyi ester contains lower alkyi, straight or branched, more preferably having 1 -6 C-atoms.
• R1 and R2 independently represent lower alkyi, or lower alkoxy, wherein the lower alkyi and lower alkoxy, independently, have 1 -5 C-atoms, preferably 1 -4 C-atoms, or most preferably 1 -3 C- atoms.
• alkyi having 1 -2 C-atoms ethyl, or methyl
• alkoxy having 1 -2 C- atoms ethoxy, methoxy
• R1 1 and R12 independently represent hydrogen, alkyi, aryl, halogen, hydroxyl, hydroxylalkyl, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy, carboxamide, alkyi ester, or aryl ester; wherein preferably alkyi, hydroxylalkyl, aminoalkyl, carboxylalkyl, alkoxy, and/or alkyi ester contains lower alkyi, straight or branched, more preferably having 1 -6 C-atoms.
• R1 1 and R12 independently represent hydrogen, lower alkyi, or lower alkoxy, wherein the lower alkyi and lower alkoxy, independently, have 1 -5 C-atoms, preferably 1 -4 C-atoms, or most preferably 1 -3 C-atoms.
• R1 1 and R12 independently represent hydrogen, alkyi having 1 -2 C-atoms (ethyl, or methyl), or alkoxy having 1 -2 C- atoms (ethoxy, methoxy).
• Y is a derivative of 1 H- imidazole, preferably 1 H-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 lUPAC, and/or as shown for imidazole on wikipedia on 03-DEC-2010 at 18:00 DK time.
• 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.
• 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 lUPAC, and/or as shown for pyridine on wikipedia on 03-DEC-2010 at 18:00 DK time.
• 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-HCI, 1 mM EGTA, 1 .5 mM MgS0 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-HCI; 5 mM HEPES; 10 mM MgCI 2 , 6H 2 0; 150 mM NaCI; 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):
• 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.
• 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 ).
• 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).
• 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
• N-radical is represented by a first *-NR 19 R 20 group, where R 19 and R 20 may,
• the CO radical is represented by a first *-CO group
• the first *-NR 19 R 20 group is capable of forming an amide bond with a second *-CO group
• the first *-CO group is capable of forming an amide bond with a second *-NR 19 R 20 group
• the second *-NR 19 R 20 group and the second *-CO group are defined as the first *-NR 19 R 20 group and the first *-CO group, respectively, and form part, independently, of the structure of i) the analogue, ii) the protracting moiety, and/or iii) another linker.
• 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.
• k and n are as defined in any one of embodiments 90-97.
• Glu di-radical is a radical of L-Glu or D-Glu, preferably of L-Glu.
• 1 17 The peptide or derivative of embodiment 1 16, wherein activation of the human GLP- 1 receptor is measured in an in vitro assay, as the potency of cAMP production. 1 18.
• 1500pM even more preferably below 1000pM, or most preferably below 800pM.
• peptide or derivative of any one of embodiments 1 -120 which has a potency corresponding to an EC 50 below 180pM, preferably below 160pM, more preferably below 140pM, even more preferably below 120pM, or most preferably below 100pM.
• peptide or derivative of any one of embodiments 1 -121 which has a potency corresponding to an EC 50 below 80pM, preferably below 60pM, more preferably below 50pM, even more preferably below 40pM, or most preferably below 30pM.
• At least 40 preferably at least 50, more preferably at least 60, even more preferably at least 70, or most preferably at least 80;
• At least 90 preferably at least 100, more preferably at least 1 10, or most preferably at least 120;
• nM 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
• below 900 nM more preferably below 800 nM, even more preferably below 700 nM, or most prefererably 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.
• any one of embodiments 1 -131 , wherein the GLP-1 receptor is prepared using a stable, transfected cell line, preferably a hamster cell line, more preferably a baby hamster kidney cell line, such as BHK tk-ts13.
• 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.
• 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 ( ⁇ ) of the injected solution (dose-corrected exposure at 30 min) is at least 15, preferably at least 30, more preferably at least 48, still more preferably at least 62, even more preferably at least 80, or most preferably at least 100.
• 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 ( ⁇ ) 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.
• 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.
• a peptide according to any one of embodiments 1 -73 and 1 15-127 for use in 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.
• a derivative according to any one of embodiments 74-157 for use in 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.
• a peptide according to any one of embodiments 1 -73 and 1 15-127 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.
• 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.
• a method of treating or preventing all forms of diabetes and related diseases such as eating disorders, cardiovascular diseases, gastrointestinal diseases, diabetic
• a method of treating or preventing all forms of diabetes and related diseases such as eating disorders, cardiovascular diseases, gastrointestinal diseases, diabetic
• R represents hydrogen, or alkyi
• R1 and R2 independently represent (i) hydrogen, alkyi, aryl, heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy, carboxamide, substituted carboxamide, alkyi ester, aryl ester, alkyi sulfonyl, or aryl sulfonyl, or (ii) R1 and R2 together form cyclo alkyi, heterocyclyl, or heteroaryl; and each of PG-i and PG 2 represents a protection group;
• R1 and R2 independently represent hydrogen, alkyi, aryl, halogen, hydroxyl, hydroxylalkyl, amino, aminoalkyl, carboxyl, carboxylalkyi, alkoxy, aryloxy, carboxamide, alkyi ester, or aryl ester; wherein preferably alkyi, hydroxylalkyl, aminoalkyl, carboxylalkyi, alkoxy, and/or alkyi ester contains lower alkyi, straight or branched, more preferably having 1 -6 C-atoms.
• R15 and R16 independently of each other and independently for each value of q represent hydrogen, alkyi, carboxyl, or hydroxyl;
• R represents hydrogen, or alkyi;
• R1 and R2 independently represent (i) alkyi, aryl, heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano, amino, aminoalkyl, carboxyl,
• each of PGi and PG 2 represents a protection group, preferably as defined in any one of embodiments 167-173;
• 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) (31 H, 34Q); (iv)
• a peptide intermediate product which is the following analogue of GLP-1 A(3-37) (SEQ ID NO: 3): (17R, 20R, 33R, 38K); or a pharmaceutically acceptable salt, amide, or ester thereof.
• Aib aminoisobutyric acid (a-aminoisobutyric acid)
• API Active Pharmaceutical Ingredient
• DIPEA diisopropylethylamine
• DMEM Dulbecco's Modified Eagle's Medium
• EDTA ethylenediaminetetraacetic acid
• EGTA ethylene glycol tetraacetic acid
• FCS Fetal Calf Serum
• HATU (0-(7-azabenzotriazol-1 -yl)-1 , 1 ,3,3-tetramethyluronium hexafluoro- phosphate)
• HBTU (2-(1 H-benzotriazol-1 -yl-)-1 , 1 ,3,3 tetramethyluronium hexafluorophosphate)
• HSA Human Serum Albumin
• IBMX 3- isobutyl-1 -methylxanthine
• Imp Imidazopropionic acid (also referred to as des-amino histidine, DesH) i.v. intravenously
• ivDde 1 -(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl
• IVGTT Intravenous Glucose Tolerance Test
• MALDI-TOF MS Matrix-Assisted Laser Desorption/lonisation Time of Flight Mass Spectroscopy
• NMP N-methyl pyrrolidone
• OEG 8-amino-3,6-dioxaoctanic acid
• PBS Phosphate Buffered Saline
• Pen/Strep Pencillin/Streptomycin
• TIS triisopropylsilane
• Tris tris(hydroxymethyl)aminomethane or 2-amino-2-hydroxymethyl-propane-
• Trt triphenylmethyl, or trityl
• Trx tranexamic acid
• 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 solid phase peptide synthesis
• LCMS, MALDI, and UPLC methods methods for detecting and characterising the resulting peptide.
• 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 His 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.
• 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. Aib). 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).
• 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 x 20 ml, each 10 min) followed by NMP washings (4 x 20 mL). Dde- or ivDde-deprotection was performed with 2% hydrazine in NMP (2 x 20 ml, each 10 min) followed by NMP washings (4 x 20 ml).
• Mtt- or Mmt-deprotection was performed with 2% TFA and 2-3% TIS in DCM (5 x 20 ml, each 10 min) followed by DCM (2 x 20 ml), 10% MeOH and 5% DIPEA in DCM (2 x 20 ml) and NMP (4 x 20 ml) washings, or by treatment with neat hexafluroisopropanol (5 x 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). In case of attachment of the albumin binding moiety and/or linker to the protected peptidyl resin the attachment can be modular using SPPS and suitably protected building blocks (see General methods).
• Attachment to resin bound peptide - Route I 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. EP51 1600, 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.
• active ester or symmetric anhydride such as octadecanedioic acid mono-(2,5-dioxo- pyrrolidin-1 -yl) ester
• Attachment to peptide in solution - Route III 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. EP51 1600) 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 (10-20ml) together with 10 molar equivalents of DIPEA.
• an 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 30min 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, AZ 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 x 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 x 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). 2. Cleavage of peptide from the resin and purification
• 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 ⁇ 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:
• the analysis was performed at RT by injecting an appropriate volume of the sample
• the UPLC conditions, detector settings and mass spectrometer settings were: Column: Waters Acq uity UPLC BEH, C-18, 1 .7 ⁇ , 2.1 mm x 50mm
• 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 214nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130A, 1.7um, 2.1 mm x 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 214nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130A, 1.7um, 2.1 mm x 150 mm column, 40°C.
• the UPLC system was connected to two eluent reservoirs containing:
• the following linear gradient was used: 80 % A, 20 % B to 40 % A, 60 % B over 8 minutes at a flow-rate of 0.40 ml/min.
• UPLC (method 04_A2_1 ): The RP-analysis was performed using a Waters UPLC system fitted with a dual band detector. UV detections at 214nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130A, 1.7um, 2.1 mm x 150 mm column, 40°C. The UPLC system was connected to two eluent reservoirs containing:
• UPLC (method 04_A3_1 ): The RP-analysis was performed using a Waters UPLC system fitted with a dual band detector. UV detections at 214nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130A, 1.7um, 2.1 mm x 150 mm column, 40°C. The UPLC system was connected to two eluent reservoirs containing:
• UPLC (method 04_A4_1 ): The RP-analysis was performed using a Waters UPLC system fitted with a dual band detector. UV detections at 214nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130A, 1.7um, 2.1 mm x 150 mm column, 40 °C. The UPLC system was connected to two eluent reservoirs containing:
• UPLC (method 08_B2_1 ): The RP-analysis was performed using a Waters UPLC system fitted with a dual band detector. UV detections at 214nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130A, 1.7um, 2.1 mm x 150 mm column, 40°C. The UPLC system was connected to two eluent reservoirs containing:
• Method 08 B4 1 UPLC (method 08_B4_1 ): The RP-analysis was performed using a Waters UPLC system fitted with a dual band detector. UV detections at 214nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130A, 1.7um, 2.1 mm x 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 214nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130A, 1.7um, 2.1 mm x 150 mm column, 40°C. The UPLC system was connected to two eluent reservoirs containing:
• 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 x 150 mm 3.5um 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.
• MALDI-MS method Molecular weights were determined using matrix-assisted laser desorption and ionisation time-of-flight mass spectroscopy, recorded on a Microflex or Autoflex (Bruker). A matrix of alpha-cyano-4-hydroxy cinnamic acid was used. NMR method
• Aluminum chloride powder (80.0 g, 600 mmol) was added in portions to a stirred mixture of ferf-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.
• 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 filtered.
• 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). The resin was shaken overnight at RT. The resin was filtered and washed with
• the resin was filtrered and 3-(1 -Trityl-1 H-imidazol-4-yl)-propyl amine (1 .8 eq; 5.40 mmol; 1.84 g), DIPEA (4 eq; 6.0 mmol; 2.09 mL), and DMF (10 mL) was added. The resin was shaken for 2 days. The resin was filtered and washed with NMP (5 x 20 mL) and DCM (10 x 20 mL). 2,2,2-Trifluoroethanol/dichlormethan 1 : 1 (20 mL) was added to the resin and it was shaked for 2 hrs. The resin was washed with 2,2,2-Trifluoroethanol/dichlormethan 1 : 1 (10 mL) and the combined filtrates were collected and concentrated in vacuo to yield the title compound. Yield: 600 mg (41 %).
• 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). The resin was shaken overnight at RT. The resin was filtered and washed with
• SPPS method B 2-Dimethyl-A/-[2-(1 -trityl-1 H-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-A/-[3-(1 -trityl-1 /-/-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-A/-(1 -trityl-1 /-/-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
• Example 6 A/ 3 - ⁇ 2-[2-(1 H-lmidazol-4-yl)-methylcarbamoyl]-2-methyl-propionyl ⁇ - [2-(2- ⁇ 2-[2-(2- ⁇ 2-[(S)-4- Carboxy-4-(17-carboxyheptadecanoylamino)-butyrylamino]ethoxy ⁇ ethoxy)- acetylamino]ethoxy ⁇ -ethoxy)acetyl][Arg 17 ,Arg 20 ,Arg 33 ,Lys 38 ]GLP-1A(3-37)-peptide
• SPPS method B 2,2-Dimethyl-A/-(1 -trityl-1 /-/-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-A/-[2-(1 -trityl-1 /-/-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 ferf-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-A/-[2-(1 -trityl-1 /-/-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 ferf-butyl ester
• SPPS method B 2,2-Dimethyl-A -[2-(1 -trityl-1 H-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-f-butylphenyl)butyric acid were coupled using SPPS method D.
• Preparation method SSPS method B. 2,2-Dimethyl-N-pyridin-2-ylmethyl-malonamic acid was coupled using the same coupling condition as used for 2,2-Dimethyl-A/-[2-(1 -trityl-1 /-/- imidazol-4-yl)-ethyl]-malonamic acid in the previous examples.
• Fmoc-Glu-OtBu and 4-(9- carboxy-nonyloxy)-benzoic acid ferf-butyl ester (prepared as described in Example 25, step 2 of WO 2006/082204) were coupled using SPPS method D
• 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 1 1 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% C0 2 in DMEM, 5% FCS, 1 % Pen/Strep
• Cells at approximate 80% confluence were washed 2X 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 assay was performed in 1 ⁇ 2-area 96-well plates, flat bottom (Costar cat.
• AlphaScreen cAMP Assay Kit from Perkin Elmer Life Sciences (cat. No: 6760625M); containing Anti-cAMP Acceptor beads (10 U/ ⁇ ), Streptavidin Donor beads (10 U/ ⁇ ) and Biotinylated-cAMP (133 ⁇ / ⁇ ).
• HEPES (Sigma, cat.no: H3375); 10 mM MgCI 2 , 6H 2 0 (Merck, cat.no: 5833); 150 mM NaCI (Sigma, cat.no: S9625); 0.01 % Tween (Merck, cat.no: 822184).
• BSA Sigma, cat. no. A7906
• IBMX Sigma, cat. no. I5879
• 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 , 10-1 2 , 10-1 3 and 10" 14M, and a series from, e.g., 10" 6 to 3x10-1 1 0 f 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.
• Example 1 1 The comparative compound of Example 1 1 was much less potent, namely with an
• 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 1 1 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% C0 2 in DMEM, 10% FCS, 1 % Pen/Strep
• 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
• 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 + 10mM 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
• SPA- particles Wheatgerm agglutinin SPA beads, Perkin Elmer, #RPNQ0001 ) were added in an amount of 0.5 mg/well (50ul). The incubation was started with [ 125 l]-GLP-1 ]-(7-36)NH 2 (final concentration 0.06 nM corresponding to 49.880 DPM, 25ul). The plates were sealed with PlateSealer and incubated for 120 minutes at 30°C while shaking. The plates were centrifuged (1500 rpm, 10min) and counted in Topcounter.
• 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] / [(ICso/nM) low HSA]):
• the comparative compound of Example 1 1 had a ratio above 300.
• IC 50 low albumin
• all derivatives except the comparative compound of Example 1 1 , 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.

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