Novel peptide agonists of GLP-1 activity
AssigneeZealand Pharma A/S
InventorsBjarne Due Larsen, Jens Damsgaard Mikkelsen, Soren Neve
Life of this patent
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Abstract
Novel peptide agonists of GLP-1 activity useful for lowering blood glucose levels. The novel peptides comprise variants of the GLP-1 or the exendin-4 polypeptide sequence and are pharmacologically active and stable. These peptides are useful in the treatment of diseases that benefit from regulation of excess levels of blood glucose and/or regulation of gastric emptying, such as diabetes and eating disorders.
Claims48
A peptide conjugate comprising a peptide X selected from the group consisting of
- (a) an exendin having at least 90% homology to exendin-4;
- (b) a variant of said exendin wherein said variant comprises a modification selected from the group consisting of between one and five deletions at positions 34-39 and contains a Lys at position 40 having a lipophilic substituent;
- or
- (c) GLP-1 (7-36) (SEQ ID NO:
- 114) or GLP-1 (7-37) (SEQ ID NO:
- 123) having at least one modification selected from the group consisting of:
- (i) substitution of D-alanine, glycine or alpha-amino isobutyric acid for alanine at position 8 and
- (ii) a lipophilic substituent;
- and Z, a peptide sequence of 4-20.amino acid units covalently bound to said variant, wherein each amino acid unit in said peptide sequence, Z is selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Asn, Gin, Asp, Glu, Lys, Arg, His, Met, Orn, and amino acid units of the general formula I
- —NH—C(R1)(R2)—C(═O)—(1)
- wherein R1 and R2 are selected from the group consisting of hydrogen, C1-6-alkyl, phenyl, and phenyl-methyl, wherein C1-6-alkyl is optionally substituted with from one to three substituents selected from halogen, hydroxy, amino, cyano, nitro, sulfono, and carboxy, and phenyl and phenyl-methyl is optionally substituted with from one to three substituents selected from C1-6-alkyl, C2-6-alkenyl, halogen, hydroxy, amino, cyano, nitro, sulfono, and carboxy, or R1 and R2 together with the carbon atom to which they are bound form a cyclopentyl, cyclohexyl, or cycloheptyl ring, e.g. 2,4-diaminobutanoic acid and 2,3-diaminopropanoic acid;
- and a pharmaceutically acceptable salt or the c-terminal amide of said peptide conjugate, with the proviso that x is not exendin-4 or exendin-3.
A peptide conjugate according to any one of the preceding claims, characterised in that the ratio between the minimum effective dose of said peptide conjugate and the minimum effective oral dose of the peptide, X is at least 1:5.
The peptide conjugate according to any one of the preceding claims, wherein the lipophilic substituent is a palmitoyl attached to the epsilon amino group of a Lys in peptide X.
The peptide conjugate according to any one of the preceding claims, wherein X is selected from the group consisting of Gly8-GLP-1-(7-36)(Human)-NH2(SEQ ID NO:
- 87), Gly8-GLP-1-(7-36)(Human) (SEQ ID NO:
- 87), Gly8Lys37(palmitoyl)-GLP-1-(7-36)(Human) (SEQ ID NO:
- 111), Gly8Lys34(palmitoyl-GLP-1-(7-36)(Human) (SEQ ID NO:
- 90), des Ser39-exendin-4(1-39) (SEQ ID NO:
- 129), exendin-4(1-39) (SEQ ID NO:
- 102), des Pro-exendin-4(1-39) (SEQ ID NO:
- 101), des Ala35-exendin-4(1-39) (SEQ ID NO:
- 105), des Gly34-exendin-4(1-39) (SEQ ID NO:
- 106), des Ser39-(Lys40 (palmitoyl))exendin-4(1-39) (SEQ ID NO:
- 107), des Gly34-(Lys (palmitoyl))exendin-4(1-39) (SEQ ID NO:
- 108), des Ala35-(Lys40 (palmitoyl))exendin-4(1-39) (SEQ ID NO:
- 109), des Pro36-(Lys40(palmitoyl))exendin-4(1-39) (SEQ ID NO:
- 110) and Lys40 (palmitoyl)exendin-4(1-39) (SEQ ID NO:
- 151), and the free acids or C-terminal primary amides and pharmaceutically acceptable salts thereof.
The peptide conjugate according to the preceding claim, wherein the lipophilic substituent is attached to the epsilon amino group of a Lys in said GLP-1.
A peptide conjugate according to the preceding claim, wherein X is selected from the group consisting of
- des Pro36-exendin-4(1-39)-NH2(SEQ ID NO:
- 101),
- des Pro36, Pro37-exendin-4(1-39)-NH2(SEQ ID NO:
- 130), and
- des Pro36, Pro37, Pro38-exendin-4(1-39)-NH2(SEQ ID NO:
- 132).
A peptide conjugate according to any one of the preceding claims, characterised in that X is bound to Z via a peptide bond.
The peptide conjugate according to any one of the preceding claims, wherein Z consists of 4-15, preferably 4-10, more preferably 4-7, most preferably 6-7 amino acid units.
A peptide conjugate according to any one of the preceding claims, wherein the overall charge of the peptide sequence (Z) is in the range from 0 to +15 at pH 7, preferably in the range from 0 to +10, such as in the range from 0 to +8, more preferably in the range from 0 to +7 or 0 to +6.
The peptide conjugate according to any one of the preceding claims, wherein each amino acid unit in Z is independently selected from the group consisting of Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Orn, 2,4-diaminobutanoic acid, 2,3-diaminopropanoic acid and Met.
A peptide conjugate according to any one of the preceding claims, wherein Z is (Lys)n, wherein n is an integer in the range from 4 to 15, preferably in the range from 4 to 10, such as in the range from 4 to 8, e.g. in the range from 4 to 7.
A peptide conjugate according to the preceding claim wherein Z is Lys6 (SEQ ID NO:
- 9) or Lys7(SEQ ID NO:
- 30).
A peptide conjugate according to any one of the preceding claims wherein said amino acid residues have an L-configuration.
A novel peptide conjugate comprising X, a peptide agonist of GLP-1 activity and/or exendin-4 activity, wherein X is selected from the group consisting of:
- des Pro36-exendin-4(1-39)-NH2 (SEQ ID NO:101),
- des Pro36-des Pro37-exendin-4(1-39)-NH2(SEQ ID NO:
- 130),
- des Pro36-des Pro37-des Pro38-exendin-4(1-39)-NH2(SEQ ID NO:
- 132),
- des Ala35-exendin-4(1-39)-NH2 (SEQ ID NO:105),
- des Gly34-exendin-4(1-39)-NH2 (SEQ ID NO:
- 106),
- des Gly34-(Lys40 (palmitoyl))exendin-4(1-39)-NH2 (SEQ ID NO:108),
- des Ala35-(Lys40 (palmitoyl))exendin-4(1-39)-NH2 (SEQ ID NO:109),
- des Pro36-(Lys40 (palmitoyl))exendin-4(1-39)-NH2 (SEQ ID NO:110),
- Gly8-Glp-1(7-36)-NH2(SEQ ID NO:
- 87), and Gly8-Glp-1(7-36) (SEQ ID NO:
- 87),
- and wherein X is C-terminally bound via a peptide bond to a peptide sequence Z selected from the group consisting of (Lys)n where n is an integer from 4 to 8, preferably n is 6, and the free acid thereof and a pharmaceutically acceptable salt thereof.
The peptide conjugate which is exendin-4(1-39)-Lys6-NH2 (SEQ ID NO:92) and the free acid thereof and a pharmaceutically acceptable salt thereof.
A variant of a parent exendin, wherein said parent exendin has an amino acid sequence having at least an 90% homology to exendin-4 and wherein said variant lowers the blood glucose level and binds to a GLP-1 receptor in a mammal and has at least one modification selected from the group consisting of:
- (a) between one and five deletions at positions 34-38, and
- (b) contains a Lys at position 40 having a lipophilic substituent bound to the epsilon-amino group of Lys via an amide bond.
The variant according to the preceding claim wherein said parent exendin is exendin-4.
The variant according to the preceding claim wherein said exendin-4 is exendin-4(1-39).
A pharmaceutical composition according to the preceding claim wherein the variant is selected from the group consisting of des Pro36-exendin-4(1-39)-NH2 (SEQ ID NO:
- 101) and the free acid thereof and a pharmaceutically acceptable salt thereof.
The composition of the preceding claim for use in therapy.
A polypeptide sequence encoding the peptide conjugates of any one of the preceding claims having a natural polypeptide sequence.
A method of increasing the plasma half life of exendin 4 and exendin 4 variants comprising linking a polypeptide sequence of preferably 6 lysine residues via a peptide bond to the C-terminal of said exendin 4 or exendin 4 variant.
Dependencies are inferred from the claim text.