WO2008145721A2 - N-terminal modification of polypeptides for protection against degradation by aminopeptidases - Google Patents

Abstract

N-terminally modified polypeptides comprising one or more N-terminal modifications consisting of an organic molecular substituent having a MW below 100 g per mol are ob- tained by the invention. Also compositions comprising such and the use thereof are com- prised by the invention.

Description

N-TERMINAL MODIFICATION OF POLYPEPTIDES FOR PROTECTION AGAINST DEGRADATION BY AMINOPEPTIDASES

FIELD OF THE INVENTION

An object of the invention is to provide N-terminally modified polypeptides compris- ing one or more N-terminal modifications consisting of an organic molecular substituent having a MW below 100 g pr. mol. Also compositions comprising such and the use thereof are comprised by the invention.

BACKGROUND OF THE INVENTION

Peptides and proteins are attractive drug candidates due to their generally high po- tency and selectivity towards relevant targets. Unfortunately, bioavailability of peptide and protein drugs is often low, due to enzymatic degradations in vivo, and due to poor up-take through relevant tissues. Enzymatic degradations of peptides are mediated by peptidases, such as exopeptidases and endopeptidases. Exopeptidases like aminopeptidases, dipeptidyl peptidases and carboxypeptidases cleave amino acids or dipeptide residues from the pep- tide termini. Endopeptidases cleave peptides at specific internal positions, e.g. chymotrypsin which cleave peptide bonds at amino acids with aromatic side-chains. Partial protection towards endopeptidases can often be obtained by careful mutation of specific residues, but the mutation strategy is often not effective for protection towards exopeptidases, which recognize the mere peptide backbone Pharmaceutical polypeptides such as insulins or GLP-1 analogues and derivatives are currently mainly delivered to patients by injection or via the pulmonary route to a small extend. Although modern injection devices/pens are quite convenient and virtually pain-free due to ultra-thin needles, many clinicians and diabetes patients are hesitant towards starting injection therapy. Administration of pharmaceutical polypeptides such as insulin and GLP-1 analogues via the oral route would be very convenient, but this route is not currently feasible, mainly due to very low bioavailability. Enzymatic degradation of polypeptides in the gut and during passage through stomach or intestinal tissues is a major factor in the low bioavailability.

N.N-di-C^ alkylation of the N-terminal of small peptides is known from structure ac- tivity relation (SAR) analyses (see e.g. WO 93/23424 and WO 2003/105677).

It is the object of the invention to provide a polypeptide which is stabilized against degradation by aminopeptidase. SUMMARY OF THE INVENTION

In one aspect of the invention an N-terminally modified polypeptide with enhanced proteolytic stability and retained biological activity of the wild type polypeptide is provided.

In another aspect an N-terminally modified polypeptide comprising one or more N- terminal modifications is obtained wherein an N-terminal modification is a conjugation of one or two substituents to an N-terminal of the parent polypeptide, and a substituent is an organic molecular substituent having a MW below 10O g pr. mol, wherein the size of the parent polypeptide is between 20 - 100 amino acids and wherein the N-terminal amine of the N- terminally modified polypeptide is at least partially positively charged at physiological pH.

In yet another aspect an N-terminally modified polypeptide comprising one or more N-terminal modifications is obtained wherein an N-terminal modification is a conjugation of one or two organic molecular substituents to an N-terminal of the parent polypeptide, and wherein the one or two organic molecular substituents are selected from the group consisting of C^ alkyl and amidinyl, wherein the size of the parent polypeptide is between 20 - 100 amino acids and wherein the N-terminal amine of the N-terminally modified polypeptide is at least partially positively charged at physiological pH.

In one aspect an N-terminally modified polypeptide is obtained wherein all free amino groups of the parent polypeptide have been conjugated to an organic molecular substituent having a MW below 100 g pr. mol.

In one aspect an N-terminally modified polypeptide is obtained which is at least par- tially resistant towards degradation by an aminopeptidase.

Also a pharmaceutical composition and methods for treating or preventing a disease or disorder are encompassed by the invention.

DESCRIPTION OF THE DRAWINGS

Fig 1. Stability of N-terminally protected insulin analogs evaluated by incubation with rat liver homogenates. Fig. 2. Biological activity of A1 (N,N-dimethyl), B1 (N,N-dimethyl), B29(N^ε,N^ε-dimethyl) A14E B25H desB30 human insulin and A14E B25H desB30 human insulin after administration into ileum of SPRD rats.

DESCRIPTION OF THE INVENTION It has by the inventors been found that derivatization of the terminal amino groups of a polypeptide wherein the derivatized amine of said polypeptide is at least partially positively charged at physiological pH can protect the polypeptide towards rapid degradation by amin- opeptidases while at least some activity of the parent polypeptide is retained.

In one aspect of the invention an N-terminally modified polypeptide is obtained comprising one or more N-terminal modifications, wherein an N-terminal modification is a conjugation of a substituent to the N-terminal of the parent polypeptide wherein the substituent is an organic molecular substituent having a MW below 100 g per mol. In a further aspect a polypeptide is obtained which comprises a di-C^ alkyl or an amidinyl substituent (i.e. a sub- stitutent of the form: R_nC(=NR)NR₂, where R_n is the polypeptide) conjugated to one of more of the N-terminals of the polypeptide. In yet a further aspect a polypeptide is obtained which comprises dimethyl substituents conjugated to one of more of the N-terminals of the polypeptide.

In one aspect of the invention an N-terminally modified polypeptide with enhanced proteolytic stability and retained biological activity relative to the wild type polypeptide is provided.

The following is a non-limiting list of aspects, which is further described elsewhere herein:

In aspect 1 of the invention an N-terminally modified polypeptide is obtained comprising one or more N-terminal modifications wherein an N-terminal modification is a conjugation of one or two substituents to an N-terminal of the parent polypeptide, and the substituent is an organic molecular substituent having a MW below 100 g per mol, wherein the size of the parent polypeptide is between 20 - 100 amino acids and wherein the N-terminal amine of the N-terminally modified polypeptide is at least partially positively charged at physiological pH. Aspect 2. The N-terminally modified polypeptide according to aspect 1 wherein the one or two organic molecular substituents are selected from the group consisting of C-ι_₇ alkyl and amidinyl.

Aspect 3. The N-terminally modified polypeptide according to any one of aspects 1 -2 wherein the N-terminal modification is a conjugation of two organic molecular subshiuerύs to one N-terminal.

Aspect 4. The polypeptide N-terminally modified polypeptide according to aspect 3 wherein the two organic molecular substituents are C^ alkyl substituents.

Aspect 5. The N-terminally modified polypeptide according to aspect 4 wherein the two organic molecular substituents are methyl substituents.

Aspect 6. The N-terminally modified polypeptide according to aspect 4 wherein the two organic molecular substituents are ethyl substituents.

Aspect 7. The N-terminally modified polypeptide according to any one of aspects 1 -6 which is further modified in one or more internal positions wherein the further modified posi- tions comprise the conjugation product of a free amino-group and an organic molecular sub- stituent having a MW below 10O g per mol.

Aspect 8. The N-terminally modified polypeptide according to any one of aspects 1 -7 which is further modified in one or more internal positions wherein the further modified positions comprise the conjugation product of one or more lysine(s) in the parent polypeptide and an organic molecular substituent having a MW below 100 g per mol.

Aspect 9. The N-terminally modified polypeptide according to aspect 7 wherein all free amino groups of the parent polypeptide have been conjugated to an organic molecular substituent having a MW below 100 g per mol.

Aspect 10. The N-terminally modified polypeptide according to aspect 9 wherein all free amino groups of the parent polypeptide have been alkylated and/or dialkylated with Ci_-7 alkyl.

Aspect 11. The N-terminally modified polypeptide according to any one of aspects 1- 10 which is at least partially resistant towards degradation by an aminopeptidase.

Aspect 12. The N-terminally modified polypeptide according to any one of aspects 1- 11 which possesses increased resistance towards degradation by an aminopeptidase compared to the wild-type polypeptide.

Aspect 13. The N-terminally modified polypeptide according to any one of aspects 1- 12 which possesses increased resistance towards degradation by an aminopeptidase compared to the parent polypeptide. Aspect 14. The N-terminally modified polypeptide according to any one of aspects 1- 13 wherein the parent polypeptide is a polypeptide analogue.

Aspect 15. The N-terminally modified polypeptide according to any one of aspects 1- 13 wherein the parent polypeptide is selected from the group consisting of: a) a pegylated polypeptide and b) an acylated polypeptide, wherein the acylation consists of a fatty acid or a difatty acid optionally attached via a linker.

Aspect 16. The N-terminally modified polypeptide according to any one of aspects 1-

15 wherein the parent polypeptide is selected from the group consisting of insulin, glucagon- like peptide, growth hormone, Factor VII, Factor VIII, Factor IX, Factor Xl and Factor XIII and analogues and derivatives thereof.

Aspect 17. The N-terminally modified polypeptide according to any one of aspects 1-

16 wherein the polypeptide is an insulin.

Aspect 18. The N-terminally modified polypeptide according to aspect 17 wherein the insulin is selected from the group consisting of human insulin, an insulin analogue and an insulin derivative.

Aspect 19. The N-terminally modified polypeptide according to any one of aspects 17-18 wherein the polypeptide is an insulin analogue.

Aspect 20. The N-terminally modified polypeptide according to any one of aspects 17-19 wherein the polypeptide is an insulin analogue selected from the group consisting of a) an insulin analogue wherein the amino acid residue in position B28 of insulin is

Pro, Asp, Lys, Leu, VaI, or Ala and the amino acid residue in position B29 is Lys or Pro and optionally the amino acid residue in position B30 is deleted; b) des(B28-B30) human insulin, des(B27) human insulin or des(B30) human insulin; c) an insulin analogue wherein the amino acid residue in position B3 is Lys and the amino acid residue in position B29 is GIu or Asp; and d) an insulin analogue wherein the amino acid in position A14 is selected from the group consisting of Lys, GIu, Arg, Asp, Pro and His, the amino acid in position B25 is His and which optionally further comprises one or more additional mutations; e) an insulin analogue wherein

• the amino acid in position A8 is His and/or the amino acid in position A12 is GIu or Asp and/or the amino acid in position A13 is His, Asn, GIu or Asp and/or the amino acid in position A14 is Asn, GIn, GIu, Arg, Asp, GIy or His and/or the amino acid in position A15 is GIu or Asp; and • the amino acid in position B1 is GIu and/or the amino acid in position B16 is GIu or His and or the amino acid in position B25 is His and/or the amino acid in posi-tion B26 is His, GIy, Asp or Thr and/or the amino acid in position B27 is His, GIu, Lys, GIy or Arg and/or the amino acid in position B28 is His, GIy or Asp; and which optionally further comprises one or more additional mutations; and f) an insulin analogue wherein the amino acid in position A14 is selected from the group consisting of Lys, GIu, Arg, Asp, Pro and His; and the B-chain of the insulin analogue comprises at least two mutations relative to the parent insulin, wherein two or more mutations are in the form of deletions of the amino acids in positions

B27, B28, B29 and B30, or a combination of a deletion of the amino acid in position B30 and a substitution of an amino acid selected from the amino acid substitutions in position: B25 to His, B26 to GIy or GIu, B27 to GIy or Lys and B28 to Asp, His, GIy, Lys or GIu. Aspect 21. The N-terminally modified polypeptide according to any one of aspects

17-20 wherein the one or more N-terminal modifications are one or more conjugations of an organic molecular substituent to the N-terminal of the A-chain of insulin and/or the N-terminal of the B-chain of insulin.

Aspect 22. The N-terminally modified polypeptide according to aspect 21 wherein the N-terminal modifications are conjugations of organic molecular substituents to the N- terminal of the A-chain of insulin and the N-terminal of the B-chain of insulin.

Aspect 23. The N-terminally modified polypeptide according to any one of aspects 17-22 wherein the N-terminal modifications are dialkylations to the N-terminal of the A-chain of insulin and the N-terminal of the B-chain of insulin. Aspect 24. The N-terminally modified polypeptide according to any one of aspects

17-23 wherein the N-terminal modifications are dimethylations to the N-terminal of the A- chain of insulin and the N-terminal of the B-chain of insulin.

Aspect 25. The N-terminally modified polypeptide according to any one of aspects 17-22 wherein the N-terminal modifications are guanidylations of the N-terminal of the A- chain of insulin and the N-terminal of the B-chain of insulin.

Aspect 26. The N-terminally modified polypeptide according to any one of aspects 1- 16 wherein the polypeptide is a glucagon-like peptide such as GLP-1.

Aspect 27. The N-terminally modified polypeptide according to aspect 26, which is an N-terminally modified derivative of a glucagon-like peptide which is acylated in another position than N-terminally with a lipophilic substituent. Aspect 28. The N-terminally modified polypeptide according to any one of aspects

26-27, which is an N-terminally modified derivative of a GLP-1 which is acylated in another position than N-terminally with a lipophilic substituent.

Aspect 29. The N-terminally modified polypeptide according to any one of aspects 26-28, which is an N-terminally modified derivative of a glucagon-like peptide GLP-1 which is acylated in another position than N-terminally with a lipophilic substituent, wherein the GLP-1 is selected from the group consisting of:

GLP-1 (7-37); Aib8,Arg34-GLP-1 (7-37); Aib8,Aib22,Arg34-GLP-1 (7-37); Arg34-GLP-

1 (7-37); [3-(4-lmidazolyl)propionyl7,Arg34]GLP-1 -(7-37); Gly8,Arg34-GLP-1 (7-37); Aib8,Arg34,Pro37-GLP-1 (7-37); Aib8,Aib22,Aib27,Aib30,Arg34,Aib35,Pro37- GLP-1 (7-

37)amide; Arg26,Lys38-GLP-1 (7-38), Arg26,Arg34,Lys38-GLP-1 (7-38);

Arg26,Arg34,Lys36,Lys38-GLP-1 (7-38); Gly8,Arg26,Lys38-GLP-1 (7-38);

Gly8,Arg26,Arg34,Lys36,Lys38-GLP-1 (7-38); DesaminoHis7,Glu22,Arg26,Arg34,Lys37-GLP-

1 (7-37); desaminoHis7,Glu22,Arg26,Arg34,Lys37-GLP-1 (7-37)amide; Aib8,Glu22,Arg26,Arg34,Lys37-GLP-1 -(7-37)amide; desaminoHis7,Glu22,Arg26,Arg

34,Phe(m-CF3)28-GLP-1-(7-37)amide; Aib8,Glu22,Arg26,Lys30-GLP-1-(7-37);

Aib8,Glu22,Arg26,Lys31 -GLP-1 -(7-37); Aib8,Glu22,Arg26,Lys31 ,Arg34-GLP-1-(7-37);

Aib8,Glu22,Arg26,Arg34,Lys37-GLP-1 -(7-37)amide; desamino-

His7,Glu22,Arg26,Arg34,Lys37-GLP-1 -(7-37)amide; Desamino- His7,Glu22,Arg26,Glu30,Arg34,Lys37]GLP-1-(7-37);

Aib8,Lys20,Glu22,Arg26,Glu30,Pro37]GLP-1-(7-37)amide;

Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide; Desamino-

His7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide; Aib8,Glu22,Arg26,Glu30,Pro37, Lys 38-

GLP-1-(7-38); DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1 (7-37) amide; Desamino- His7,Glu22,Arg26,Arg34,Lys37]GLP-1 (7-37)-amide; desaminoHis7,Glu22,Arg26,

Glu30,Arg34,Lys37] (GLP-1 -(7-37)amide; desaminoHis7,Glu22, Arg26,Arg34,Lys 37] (GLP-

1-(7-37)amide; desaminoHis7,Glu22,Arg26,Arg34,Lys37] GLP-1 (7-37)amide;

Aib8,Glu22,Arg26,Glu30,Lys36] GLP-1 -(7-37)Glu-amide; Desamino-

His7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide; Desamino- His7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37); Aib8,Glu22,Arg26,Lys31]GLP-1-(7-37);

Aib8,Lys20,Glu22,Arg26,Glu30,Pro37]GLP-1-(7-37)amide; and Desamino-

His7,Glu22,Arg26,Arg34,Lys37] GLP-1 -(7-37).

Aspect 30. The N-terminally modified polypeptide according to any one of aspects 1-

16 wherein the polypeptide is human growth hormone. Aspect 31. The N-terminally modified polypeptide according to any one of aspects 1- 16 wherein the polypeptide is selected from the group consisting of Factor VII, Factor VIII, Factor IX, Factor Xl and Factor Xl I.

Aspect 32. The N-terminally modified polypeptide according to any one of aspects 1- 25 which is selected from the group consisting of:

A1 (N,N-dimethyl), B1 (N,N-dimethyl), B29(N^ε,N^ε-dimethyl) human insulin;

A1 (N,N-diethyl), B1 (N,N-diethyl), B29(N^ε,N^ε-diethyl) human insulin;

A1 (N,N-dimethyl), B1 (N,N-dimethyl), B29(N^ε,N^ε-dimethyl) desB30 human insulin;

A1 (N,N-diethyl), B1 (N,N-diethyl), B29(N^ε,N^ε-diethyl) desB30 human insulin; A1 (N,N-dimethyl), B1 (N,N-dimethyl), B29(N^ε,N^ε-dimethyl) A14E B25H desB30 human insulin;

A1 (N,N-diethyl), B1 (N,N-diethyl), B29(N^ε,N^ε-diethyl) A14E B25H desB30 human insulin;

A1 (N,N-dimethyl), B1 (N,N-dimethyl), B29N^ε-hexadecandioyl-gamma-L-Glu desB30 human insulin;

A1 (N,N-diethyl), B1 (N,N-diethyl), B29N^ε-hexadecandioyl-gamma-L-Glu desB30 human insulin;

A1 (N,N-dimethyl), B1 (N,N-dimethyl), B29N^ε-myristoyl desB30 human insulin;

A1 (N,N-diethyl), B1 (N,N-diethyl), B29N^ε-myristoyl desB30 human insulin; A1 (N,N-dimethyl), B1 (N,N-dimethyl), B29N^ε-3-{2-[2-(2-methoxy-ethoxy)-ethoxy]- ethoxy}-propionyl A14E B25H desB30 human insulin;

A1 (N,N-diethyl), B1 (N,N-diethyl), B29N^ε-3-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}- propionyl A14E B25H desB30 human insulin;

A1 (N,N-dimethyl), B1 (N,N-dimethyl), B29(N^ε,N^ε-dimethyl) B28D human insulin; A1 (N,N-diethyl), B1 (N,N-diethyl), B29(N^ε,N^ε-dimethyl) B28D human insulin;

A1 N-amidinyl, B1 N-amidinyl, B29N^ε-amidinyl human insulin;

A1 N-amidinyl, B1 N-amidinyl, B29N^ε-amidinyl desB30 human insulin;

A1 N-amidinyl, B1 N-amidinyl, B29N^ε-amidinyl A14E B25H desB30 human insulin;

A1 N-amidinyl, B1 N-amidinyl, B29N^ε-hexadecandioyl-gamma-L-Glu desB30 human insulin;

A1 N-amidinyl, B1 N-amidinyl, B29N^ε-myristoyl desB30 human insulin;

A1 N-amidinyl, B1 N-amidinyl, B29N^ε-3-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}- propionyl A14E B25H desB30 human insulin; and

A1 N-amidinyl, B1 N-amidinyl, B29(N^ε,N^ε-dimethyl) B28D human insulin. Aspect 33. A pharmaceutical composition comprising one or more polypeptides according to any one of aspects 1-32 and a pharmaceutically acceptable excipient.

Aspect 34. The pharmaceutical composition according to aspect 33, which is suited for oral administration. Aspect 35. The pharmaceutical composition according to aspect 33, which is suited for pulmonary administration.

Aspect 36. A method for treating a disease or disorder selected from the group consisting of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive disorders, atheroschlerosis, myocardial in- farction, coronary heart disease and other cardiovascular disorders, CNS disorders such as Alzheimer's, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers, said method comprising administering to a subject in need of such treatment an effective amount of the pharmaceutical composition of any one of aspects 33-35.

Aspect 37. A method for decreasing food intake, decreasing β-cell apoptosis, increas- ing β-cell function and β-cell mass, and/or for restoring glucose sensitivity to β-cells, said method comprising administering to said subject an effective amount of the pharmaceutical composition of any one of aspects 33-35.

Aspect 38. A method of treating or preventing a glucagon-mediated disease, disorder or condition in a mammal comprising administering to said mammal an effective amount of one or more polypeptides according to any one of aspects 1-32.

Aspect 39. The N-terminally modified polypeptide according to any one of aspects 1- 32 for use as a medicament.

Aspect 40. The N-terminally modified polypeptide according to any one of aspects 1- 32 for use as a medicament for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive disorders, atheroschlerosis, myocardial infarction, coronary heart disease and other cardiovascular disorders, CNS disorders such as Alzheimer's, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers.

Aspect 41. The N-terminally modified polypeptide according to any one of aspects 1- 32 for use as a medicament for delaying or preventing disease progression in type 2 diabetes.

When used herein, the term "conjugate" is intended to indicate the process of bonding a substituent to a polypeptide to modify the properties of said polypeptide. "Conjugation" or a "conjugation product" of a molecule and a polypeptide is thus a term for said substituent bonded to an amino acid of the polypeptide and a "substituent" as described herein thus means the substituent which is attached to the polypeptide.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to mean a compound composed of at least five constituent amino acids connected by peptide bonds.

The term "parent polypeptide" as used herein is intended to mean the polypeptide before any N-terminal modification according to the invention has been applied thereto. A parent polypeptide may e.g. be a wild-type polypeptide, an analogue of a wild-type polypeptide or a derivative of a wild-type or an analogue polypeptide such as a polypeptide that has been PEGylated or acylated.

An N-terminal modification of a polypeptide according to the invention is a conjuga- tion of a molecule to the N-terminal of a parent polypeptide wherein the substituent is an organic molecular substituent having a MW below 100 g per mol. In one aspect the N-terminal modification is in the form of a monoalkylation, dialkylation or guadinylation for protection towards aminopeptidases. In one aspect of the invention the N-terminal modification is in the form of a dialkylation. In a further aspect the N-terminal modification is in the form of a di- methylation of polypeptides for protection towards aminopeptidases. In another aspect of the invention the N-terminal modification is in the form of a guadinylation.

"Monoalkylation" is herein to be understood as conjugation of one alkyl substituent to a free amino group of a polypeptide and "dialkylation" is to be understood as conjugation of two alkyl substituents to a free amino group of a polypeptide as illustrated below, where a "free amino group" is to be understood as a primary amine, R-NH2, or a secondary amine, R1-NH-R2, where R, R1 and R2 represents a substituent.

"Guadinylation" is herein to be understood as conjugation of an amidinyl substituent (which may also be referred to as carboxamidine, i.e. a substitutent of the form: R_nC(=NR)NR₂, where R_n is the polypeptide) to a free amino group of the polypeptide resulting in transformation of the amino group to a guadinyl group as illustrated below. monoalkylation

R-NH, dialkylation primary amine

NhL guadinylation

An "organic molecular substituent" or "substituent", as used interchangeably herein, according to the invention is an organic molecular substituent having a MW below 10O g per mol. In one aspect of the invention the MW of the organic molecular substituent is below 90 g per mol, 80 g per mol, 70 g per mol or 60 g per mol. In a further aspect of the invention the MW of the organic molecular substituent is below 55 g per mol.

In a yet further aspect an organic molecular substituent of the invention is selected from the group consisting of C_1-7 alkyl and amidinyl. In a yet further aspect of the invention an organic molecular substituent of the invention is selected from the group consisting of C_1-6 alkyl and amidinyl, C_1-5 alkyl and amidinyl, C_1-4 alkyl and amidinyl and C_1-3 alkyl and amidinyl. The term C_1-x alkyl as used herein represents a saturated, branched or unbranched hydro- carbon group having from 1 to X carbon atoms, for example the term C_1-7 alkyl represents a saturated, branched or straight hydrocarbon group having from 1 to 7 carbon atoms. Representative examples of C_1-7 alkyl are methyl, ethyl, propyl (e.g. prop-1-yl, prop-2-yl (or iso- propyl)), butyl (e.g. 2-methylprop-2-yl (or terf-butyl), but-1-yl, but-2-yl), pentyl (e.g. pent-1-yl, pent-2-yl, pent-3-yl), 2-methylbut-1-yl, 3-methylbut-1 -yl, hexyl (e.g. hex-1-yl) and heptyl (e.g. hept-1-yl).

The size of the parent polypeptide before N-terminal modification according to the invention is between 20 - 100 amino acids. In one aspect of the invention the size of the parent polypetide is between 20 - 90 amino acids, 20 - 80 amino acids or 20 - 70 amino ac- ids. In a further aspect the size of the parent polypeptide is between 25 - 70 amino acids, 25 - 65 amino, 25 - 60 amino acids, or 25 - 55 amino acids. In a yet further aspect the size of the parent polypeptide is between 30 - 70 amino acids, 30 - 65 amino acids, 30 - 60 amino acids or 30 - 55 amino acids. The described alkylations, dialkylations or guanidylations alter the involved amines from primary or secondary amines to secondary or tertiary amines or guadinyl groups with the basic properties of the functional group retained, i.e. little change of pKa occurs. In one aspect of the invention an amine of the N-terminally modified polypeptide is at least partially positively charged at physiological pH.

By "at least partially positively charged" amine at physiological pH as herein described is meant, that in a solution comprising the N-terminally polypeptide at least 10 % of the amines have a charge of +1 at physiological pH. In one aspect at least 30 % of the amines in a solution of the N-terminally polypeptide have a charge of +1 at physiological pH. In a further aspect at least 50 % of the amines in a solution of the N-terminally polypeptide have a charge of +1 at physiological pH. In yet a further aspect at least 70 % of the amines in a solution of the N-terminally polypeptide have a charge of +1 at physiological pH. In still a further aspect at least 90 % of the amines in a solution of the N-terminally polypeptide have a charge of +1 at physiological pH.

In one aspect of the invention an N-terminally modified polypeptide is obtained which possesses increased resistance towards degradation from aminopeptidases relative to the wild-type polypeptide, while exhibiting substantially the same or improved biological activity relative to the wild-type polypeptide. In a further aspect of the invention an N-terminally modified polypeptide is obtained which possesses increased resistance towards degradation from aminopeptidases relative to the parent polypeptide, while exhibiting substantially the same or improved biological activity relative to the parent polypeptide. In a yet further aspect of the invention an N-terminally modified polypeptide is obtained which possesses increased resistance relative to the wild-type polypeptide towards degradation from one or more aminopeptidases selected from the group consisting of DPP-I, DPP-II, DPP-III and DPP-IV, while exhibiting substantially the same or improved biological activity relative to the wild-type polypeptide. In a still further aspect of the invention an N- terminally modified polypeptide is obtained which possesses increased resistance relative to the parent polypeptide towards degradation from one or more aminopeptidases selected from the group consisting of DPP-I, DPP-II, DPP-III and DPP-IV, while exhibiting substantially the same or improved biological activity relative to the parent polypeptide. The biological activity of a polypeptide or a polypeptide derivative may be measured in an assay as known by a person skilled in the art as e.g. described in WO 2005/012347.

The production of polypeptides is well known in the art. Polypeptides may for in- stance be produced by classical peptide synthesis, e.g. solid phase peptide synthesis using t-Boc or Fmoc chemistry or other well established techniques, see e.g. Greene and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999. The polypeptides may also be produced by a method which comprises culturing a host cell containing a DNA sequence encoding the polypeptide and capable of expressing the polypeptide in a suitable nu- trient medium under conditions permitting the expression of the peptide. For polypeptides comprising non-natural amino acid residues, the recombinant cell should be modified such that the non-natural amino acids are incorporated into the polypeptide, for instance by use of tRNA mutants.

The term "analogue" as used herein referring to a polypeptide means a modified polypeptide wherein one or more amino acid residues of the polypeptide have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the polypeptide and or wherein one or more amino acid residues have been added to the polypeptide. Such addition or deletion of amino acid residues can take place at the N-terminal of the polypeptide and/or at the C-terminal of the polypeptide.

A simple system is often used to describe analogues: For example Arg34-GLP-1 (7- 37)Lys designates a GLP-1 (7-37) analogue wherein the naturally occurring lysine at position 34 has been substituted with arginine and wherein a lysine has been added to the terminal amino acid residue, i.e. to the glycine in position 37 (Gly37). Another example is

AspB28,DesB30 human insulin, which designates an insulin analogue wherein the naturally occurring proline at position 28 of the B-chain has been substituted with asparagine and the naturally occurring threonine at position 30 of the B-chain has been deleted.

In one embodiment an analogue according to the invention comprises less than 17 modifications (substitutions, deletions, additions) relative to the parent polypeptide. In one embodiment an analogue comprises less than 16 modifications (substitutions, deletions, additions) relative to the parent polypeptide. In one embodiment an analogue comprises less than 15 modifications (substitutions, deletions, additions) relative to the parent polypeptide. In another embodiment an analogue comprises less than 14 modifications (substitutions, de- letions, additions) relative to the parent polypeptide. In another embodiment an analogue comprises less than 13 modifications (substitutions, deletions, additions) relative to the parent polypeptide. In another embodiment an analogue comprises less than 12 modifications (substitutions, deletions, additions) relative to the parent polypeptide. In another embodiment an analogue comprises less than 1 1 modifications (substitutions, deletions, additions) relative to the parent polypeptide. In another embodiment an analogue comprises less than 10 modifications (substitutions, deletions, additions) relative to the parent polypeptide. In another embodiment an analogue comprises less than 9 modifications (substitutions, deletions, additions) relative to the parent polypeptide. In another embodiment an analogue comprises less than 8 modifications (substitutions, deletions, additions) relative to the parent polypeptide. In one embodiment an analogue comprises less than 7 modifications (substitutions, deletions, additions) relative to the parent polypeptide. In one embodiment an analogue comprises less than 6 modifications (substitutions, deletions, additions) relative to the parent polypeptide. In another embodiment an analogue comprises less than 5 modifications (sub- stitutions, deletions, additions) relative to the parent polypeptide. In another embodiment an analogue comprises less than 4 modifications (substitutions, deletions, additions) relative to the parent polypeptide. In another embodiment an analogue comprises less than 3 modifications (substitutions, deletions, additions) relative to the parent polypeptide. In another embodiment an analogue comprises less than 2 modifications (substitutions, deletions, addi- tions) relative to the parent polypeptide.

A derivatized polypeptide or a "derivative" as used herein means a polypeptide in which one or more of the amino acid residues of the polypeptide have been chemically modified (e.g. by alkylation, acylation, ester formation, or amide formation) or associated with one or more non-amino acid organic and/or inorganic atomic or molecular substituents.

The N-terminal protection can be combined with polypeptide acylations or pegyla- tions at for example lysine side chains, in order to e.g. gain prolonged action or better uptake in vivo. In one aspect of the invention an N-terminally modified polypeptide according to the invention is a pegylated or acylated polypeptide which is monoalkylated, dialkylated or gua- nidylated in the N-terminal.

Pegylation is the conjugation to a polypeptide of a polyethylene glycol (PEG) chain, which is thus based on the repeating unit -CH₂CH₂O-. Non-limiting examples of pegylated polypeptides may e.g. be found in WO 02/094200, WO 94/20069 and WO 02/092147092147 which are hereby incorporated by reference. Acylated polypeptides are derivatives of wild- type polypeptides or polypeptide analogues, which have a fatty acid or difatty acid substitu- ent attached optionally via a linker to one or more amino acids. Non-limiting examples of acylated polypeptides may e.g. be found in WO2005012347A3, WO 95/07931 , WO 98/08871 and WO 2006/097537which are hereby incorporated by reference.

A polypeptide suitable for N-terminal modification according to the invention is any polypeptide, polypeptide analogue or polypeptide derivative possessing one or more amino terminal(s) suitable for N-terminal modification. Non-limiting examples of polypeptides suit- able for N-terminal modification are insulin, glucagon-like peptide (GLP), growth hormone, Factor VII, Factor VIII, Factor IX, Factor Xl, Factor XIII and analogues and derivatives thereof.

In one aspect of the invention an N-terminally modified polypeptide according to the invention is an N-terminally modified insulin. Insulin is a polypeptide hormone produced in the beta cells of the islets of Langerhans. The active insulin molecule is a two-chain molecule consisting of a B- and an A-chain connected by two disulphide bridges.

In one aspect the N-terminally modified polypeptide is selected from the group con- sisting of an N-terminally modified human insulin, an N-terminally modified insulin analogue and a further derivatized N-terminally modified insulin.

An insulin analogue according to the invention is a polypeptide which has a molecular structure which formally can be derived from the structure of a naturally occurring insulin, for example that of human insulin, by deleting and/or substituting at least one amino acid residue occurring in the natural insulin and/or by adding at least one amino acid residue.

In one aspect an insulin analogue according to the invention is such wherein the amino acid residue in position B28 of insulin is Pro, Asp, Lys, Leu, VaI, or Ala and the amino acid residue in position B29 is Lys or Pro and optionally the amino acid residue in position B30 is deleted. In another aspect an insulin analogue according to the invention is des(B28- B30) human insulin, des(B27) human insulin or des(B30) human insulin. In yet another aspect an insulin analogue according to the invention is an insulin analogue wherein the amino acid residue in position B3 is Lys and the amino acid residue in position B29 is GIu or Asp. In a further aspect an insulin analogue according to the invention is an insulin which is stabilized towards endopeptidases by internal mutations, e.g. an insulin analogue wherein the amino acid in position A14 is GIu or His, the amino acid in position B25 is His and which optionally further comprises one or more additional mutations; or an insulin analogue wherein

• the amino acid in position A8 is His and/or the amino acid in position A12 is GIu or Asp and/or the amino acid in position A13 is His, Asn, GIu or Asp and/or the amino acid in position A14 is Asn, GIn, GIu, Arg, Asp, GIy or His and/or the amino acid in position A15 is GIu or Asp; and • the amino acid in position B1 is GIu and/or the amino acid in position B16 is GIu or His and or the amino acid in position B25 is His and/or the amino acid in position B26 is His, GIy, Asp or Thr and/or the amino acid in position B27 is His, GIu, Lys, GIy or Arg and/or the amino acid in position B28 is His, GIy or Asp; and which optionally further comprises one or more additional mutations; or an insulin analogue wherein the amino acid in position A14 is selected from the group consisting of Lys, GIu, Arg, Asp, Pro and His; and the B-chain of the insulin analogue comprises at least two mutations relative to the parent insulin, wherein two or more mutations are in the form of deletions of the amino acids in positions B27, B28, B29 and B30, or a combination of a deletion of the amino acid in position B30 and a substitution of an amino acid selected from the amino acid substitutions in position: B25 to His, B26 to GIy or GIu, B27 to GIy or Lys and B28 to Asp, His, GIy, Lys or GIu.

In a yet further aspect an insulin analogue according to the invention is selected from the group consisting of: human insulin; DesB30 human insulin; AspB28 human insulin; AspB28,DesB30 human insulin; LysB3,GluB29 human insulin; LysB28,ProB29 human insulin; GluA14,HisB25 human insulin; HisA14,HisB25 human insulin; GluA14,HisB25,DesB30 human insulin; HisA14, HisB25,DesB30 human insulin; GluA14,HisB25,desB27,desB28,desB29,desB30 human insulin; GluA14,HisB25,GluB27,desB30 human insulin; GluA14,HisB16,HisB25,desB30 human insu- Nn; HisA14,HisB16,HisB25,desB30 human insulin; HisA8,GluA14,HisB25,GluB27,desB30 human insulin; HisA8,GluA14, GIuBI , GluB16,HisB25,GluB27,desB30 human insulin; and HisA8,GluA14,GluB16,HisB25,desB30 human insulin.

Also, derivatives of precursors or intermediates are covered by the invention. An ex- ample of such a derivative is a single-chain insulin which comprises the B- and the A-chain of human insulin or analogues or derivatives thereof connected by a connecting peptide. Non- limiting examples of singlechain insulins according to the invention are described in WO 2005/054291 which is hereby incorporated by reference.

An insulin derivative according to the invention is a naturally occurring insulin or an insulin analogue which has been chemically modified, e.g. by introducing a side chain in one or more positions of the insulin backbone or by oxidizing or reducing groups of the amino acid residues in the insulin or by converting a free carboxylic group to an ester group or to an amide group. Other derivatives are obtained by acylating a free amino group or a hydroxy group, such as in the B29 position of human insulin or desB30 human insulin. A non-limiting example of acylated polypeptides may e.g. be found in WO 95/07931 which is are hereby incorporated by reference.

According to the invention one or both of the N-terminal amino groups of the two chains of insulin may be derivatized.

In one aspect of the invention an N-terminal modification of an insulin is in the form of dialkylation of an insulin A- or B-chain for protection towards aminopeptidases. In another aspect of the invention an N-terminal modification of an insulin is in the form of dialkylation of both the insulin A- and B-chain for protection towards aminopeptidases. In a further aspect of the invention an N-terminal modification of an insulin is in the form of dimethylation of an insulin A- or B-chain for protection towards aminopeptidases. In a yet further aspect of the invention an N-terminal modification of an insulin is in the form of dimethylation of both the insulin A- and B-chain for protection towards aminopeptidases. In a further aspect of the inven- tion an N-terminal modification of an insulin is in the form of diethylation of an insulin A- or B- chain for protection towards aminopeptidases. In a yet further aspect of the invention an N- terminal modification of an insulin is in the form of diethylation of both the insulin A- and B- chain for protection towards aminopeptidases. In another aspect of the invention an N- terminal modification of an insulin is in the form of guanylidation of an insulin A- or B-chain for protection towards aminopeptidases. In a further aspect of the invention an N-terminal modification of an insulin is in the form of guanylidation of both the insulin A- and B-chains for protection towards aminopeptidases.

In one aspect of the invention an N-terminally modified insulin according to the in- vention is selected from the group consisting of: A1 (N,N-dimethyl), B1 (N,N-dimethyl), B29(N^ε,N^ε-dimethyl) human insulin, A1 (N,N-diethyl), B1 (N,N-diethyl), B29(N^ε,N^ε-diethyl) human insulin, A1 (N,N-dimethyl), B1 (N,N-dimethyl), B29(N^ε,N^ε-dimethyl) desB30 human insulin, A1 (N,N-diethyl), B1 (N,N-diethyl), B29(N^ε,N^ε-diethyl) desB30 human insulin, A1 (N,N-dimethyl), B1 (N,N-dimethyl), B29(N^ε,N^ε-dimethyl) A14E B25H desB30 human insulin,

A1 (N,N-diethyl), B1 (N,N-diethyl), B29(N^ε,N^ε-diethyl) A14E B25H desB30 human insulin

A1 (N,N-dimethyl), B1 (N,N-dimethyl), B29N^ε-hexadecandioyl-gamma-L-Glu desB30 human insulin

A1 (N,N-diethyl), B1 (N,N-diethyl), B29N^ε-hexadecandioyl-gamma-L-Glu desB30 human insulin

A1 (N,N-dimethyl), B1 (N,N-dimethyl), B29N^ε-myristoyl desB30 human insulin A1 (N,N-diethyl), B1 (N,N-diethyl), B29N^ε-myristoyl desB30 human insulin A1 (N,N-dimethyl), B1 (N,N-dimethyl), B29N^ε-3-{2-[2-(2-methoxy-ethoxy)-ethoxy]- ethoxy}-propionyl A14E B25H desB30 human insulin

A1 (N,N-diethyl), B1 (N,N-diethyl), B29N^ε-3-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}- propionyl A14E B25H desB30 human insulin

A1 (N,N-dimethyl), B1 (N,N-dimethyl), B29(N^ε,N^ε-dimethyl) B28D human insulin A1 (N,N-diethyl), B1 (N,N-diethyl), B29(N^ε,N^ε-dimethyl) B28D human insulin

A1 N-amidinyl, B1 N-amidinyl, B29N^ε-amidinylhuman insulin A1 N-amidinyl, B1 N-amidinyl, B29N^ε-amidinyldesB30 human insulin A1 N-amidinyl, B1 N-amidinyl, B29N^ε-amidinylA14E B25H desB30 human insulin A1 N-amidinyl, B1 N-amidinyl, B29N^ε-hexadecandioyl-gamma-L-Glu desB30 human insulin

A1 N-amidinyl, B1 N-amidinyl, B29N^ε-myristoyl desB30 human insulin A1 N-amidinyl, B1 N-amidinyl, B29N^ε-3-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}- propionyl A14E B25H desB30 human insulin

A1 N-amidinyl, B1 N-amidinyl, B29(N^ε,N^ε-dimethyl) B28D human insulin A1 (N,N-dimethyl), B1 (N,N-dimethyl), B29N^ε-hexadecandioyl-gamma-amino-butanoyl desB30 human insulin

A1 (N,N-diethyl), B1 (N,N-diethyl), B29N^ε-hexadecandioyl-gamma-amino-butanoyl desB30 human insulin A1 N-amidinyl, B1 N-amidinyl, B29N^ε-hexadecandioyl-gamma-amino-butanoyl desB30 human insulin

A1 (N,N-dimethyl), B1 (N,N-dimethyl), B29N^ε-hexadecandioyl-gamma-L-Glu-amide desB30 human insulin A1 (N,N-diethyl), B1 (N,N-diethyl), B29N^ε-hexadecandioyl-gamma-L-Glu-amide desB30 human insulin

A1 N-amidinyl, B1 N-amidinyl, B29N^ε-hexadecandioyl-gamma-L-Glu-amide desB30 human insulin

In one aspect of the invention the N-terminally modified polypeptide is an N- terminally modified glucagon-like peptide. In a further aspect the N-terminally modified polypeptide is selected from the group consisting of N-terminally modified GLP-1 , N-terminally modified GLP-2, N-terminally modified exendin-4 and analogues and derivatives thereof. In one aspect the glucagon-like peptide is an insulinotropic agent.

The terms GLP-1 , GLP-2 and exendin-4 are known to a person skilled in the art. For example "GLP-1 polypeptide" as used herein means GLP-1 (7-37), a GLP-1 (7-37) analogue, a GLP-1 (7-37) derivative or a derivative of a GLP-1 (7-37) analogue and the term "ex- endin-4 polypeptide" as used herein means exendin-4(1-39), an exendin-4(1-39) analogue, an exendin-4(1 -39) derivative or a derivative of an exendin-4(1 -39) analogue.

The term "insulinotropic agent" as used herein means a compound which is an agonist of the human GLP-1 receptor, i.e. a compound which stimulates the formation of cAMP in a suitable medium containing the human GLP-1 receptor. The potency of an insulinotropic agent is determined by calculating the EC₅₀ value from the dose-response curve. Methods for measuring if a compound is insulinotropic are well known to a person skilled in the art, see e.g. WO 2005/058954

In one aspect a glucagon-like peptide derivative according to the invention is obtained which possesses resistance towards dipeptidyl aminopeptidase-IV (DPP-IV), while exhibiting substantially the same or improved biological activity as insulinotropic agent relative to the wild-type glucagon-like peptide. In another aspect a glucagon-like peptide derivative according to the invention is obtained which possesses resistance towards dipeptidyl aminopeptidase-IV (DPP-IV), while exhibiting substantially the same or improved biological activity as insulinotropic agent relative to the parent glucagon-like peptide.

The peptidase DPP-IV in plasma is known to be involved in the degradation of sev- eral polypeptide hormones, for example, GLP-1 , GLP-2, Exendin-4 etc. In one aspect a glucagon-like peptide according to the invention is more resistant to DPP-IV than GLP-1 (7-37) or Exendin-4(1 -39).

Resistance of a polypeptide to degradation by dipeptidyl aminopeptidase IV can be determined by a degradation assay known to the person skilled in the art as e.g. described in the examples

In one aspect a polypeptide of the invention is an analogue of a glucagon-like peptide. A simple system is often used to describe analogues: Human GLP-1 is hydrolysed to GLP-1 (7-37) and GLP-1 (7-36)-amide which are both insulinotropic polypeptides. Thus, for example, [Gly⁸]GLP-1 (7-37) designates an analogue of GLP-1 (7-37) formally derived from GLP-1 (7-37) by substituting the naturally occurring amino acid residue in position 8 (Ala) by GIy. Similarly, (N^ε34-tetradecanoyl)[Lys³⁴]GLP-1 (7-37) designates GLP-1 (7-37) wherein the ε- amino group of the Lys residue in position 34 has been tetradecanoylated.

In one aspect of the invention an N-terminally modified polypeptide of the invention is an N-terminally modified derivative of a glucagon-like peptide. In a further aspect an N- terminally modified polypeptide of the invention is an N-terminally modified derivative of a glucagon-like peptide which is acylated in another position than N-terminally with a moiety comprising a hydrophilic linker. In a yet further aspect an N-terminally modified polypeptide of the invention is an N-terminally modified derivative of a GLP-1 which is acylated in another position than N-terminally with a moiety comprising a hydrophilic linker.

In a still further aspect an N-terminally modified polypeptide of the invention is an N- terminally modified derivative of a glucagon-like peptide GLP-1 acylated with a lipophilic sub- stituent, wherein the GLP-1 is selected from the group consisting of: GLP-1 (7-37); Arg34-GLP-1 (7-37); Aib8,Arg34-GLP-1 (7-37); Aib8,Aib22,Arg34-GLP-

1 (7-37); Arg34-GLP-1 (7-37); [3-(4-lmidazolyl)propionyl7,Arg34]GLP-1-(7-37); Gly8,Arg34- GLP-1 (7-37); Aib8,Arg34,Pro37-GLP-1 (7-37); Aib8,Aib22,Aib27,Aib30,Arg34,Aib35,Pro37- GLP-1 (7-37)amide; Arg26,Lys38-GLP-1 (7-38); Arg26,Arg34,Lys38-GLP-1(7-38); Arg26,Arg34,Lys36,Lys38-GLP-1 (7-38); Gly8,Arg26,Lys38-GLP-1 (7-38); Gly8,Arg26,Arg34,Lys36,Lys38-GLP-1 (7-38); DesaminoHis7Glu22Arg26Arg34Lys37-GLP-1(7- 37); desaminoHis7,Glu22,Arg26,Arg34,Lys37-GLP-1 (7.37)_amjde; Aib8,Glu22,Arg26,Arg34,Lys37-GLP-1 -(7-37)amide; desaminoHis7,Glu22,Arg26,Arg 34,Phe(m-CF3)28-GLP-1-(7-37)amide; Aib8,Glu22,Arg26,Lys30-GLP-1-(7-37);

Aib8,Glu22,Arg26,Lys31 -GLP-1 -(7-37); Aib8,Glu22,Arg26,Lys31 ,Arg34-GLP-1-(7-37); Aib8,Glu22,Arg26,Arg34,Lys37-GLP-1 -(7-37)amide; d_esamjno.

His7,Glu22,Arg26,Arg34,Lys37-GLP-1 -(7-37)amide; Desamino- His7,Glu22,Arg26,Glu30,Arg34,Lys37]GLP-1-(7-37); Aib8,Lys20,Glu22,Arg26,Glu30,Pro37]GLP-1-(7-37)amide; Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide; Desamino- His7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide; Aib8,Glu22,Arg26,Glu30,Pro37, Lys 38- GLP-1-(7-38); DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1 (7-37) amide; Desamino- His7,Glu22,Arg26,Arg34,Lys37]GLP-1 (7-37)-amide; desaminoHis7,Glu22,Arg26, Glu30,Arg34,Lys37] (GLP-1 -(7-37)amide; desaminoHis7,Glu22, Arg26,Arg34,Lys 37] (GLP- 1-(7-37)amide; desaminoHis7,Glu22,Arg26,Arg34,Lys37] GLP-1 (7-37)amide; Aib8,Glu22,Arg26,Glu30,Lys36] GLP-1 -(7-37)Glu-amide; Desamino- His7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide; Desamino- His7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37); Aib8,Glu22,Arg26,Lys31]GLP-1-(7-37); Aib8,Lys20,Glu22,Arg26,Glu30,Pro37]GLP-1-(7-37)amide; and Desamino- His7,Glu22,Arg26,Arg34,Lys37] GLP-1 -(7-37).

In one aspect of the invention an N-terminally modified polypeptide of the invention is an N- terminally modified derivative of a glucagon-like peptide which is acylated in another position than N-terminally and which is selected from the group consisting of:

• N-epsilon26-(17-carboxyheptadecanoyl)-[Aib8,Arg34]GLP-1 -(7-37)-peptide, • N-epsilon26-(19-carboxynonadecanoyl)-[Aib8,Arg34]GLP-1 -(7-37)-peptide,

• N-epsilon26- (4-{[N-(2-carboxyethyl)-N-(15- carboxypentadecanoyl)amino]methyl} benzoyl)[Arg34]GLP-1-(7-37),

• N-epsilon26-[2-(2-[2-(2-[2-(2-[4-(17-Carboxyheptadecanoylamino)-4(S)- carboxybutyrylamino] eth- oxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Aib8,Arg34]GLP-1-(7-37)peptide,

• N-epsilon37{2-[2-(2-{2-[2-((R)-3-carboxy-3-{[1 -(19- carboxynonadecanoyl)piperidine-4- carbonyljaminojpropionylaminojethoxylethoxyjacetylaminojethoxylethoxyjacetyl [desaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1 (7-37)amide, • N-epsilon37{2-[2-(2-{2-[2-((S)-3-carboxy-3-{[1 -(19- carboxynonadecanoyl)piperidine-4- carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy} acetyl Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide, • N-epsilon37-[2-(2-[2-(2-[2-(2-((R)-3-[1 -(17-Carboxyheptadecanoyl)piperidin-4- ylcarbonylamino]3- carboxypropionylamino)ethoxy)ethoxy]acetylamino) eth- oxy]ethoxy)acetyl][DesaminoHis7, Glu22 Arg26, Arg 34, Phe(m-CF3)28]GLP-1- (7-37)amide,

• N-epsilon30{2-[2-(2-{2-[2-((S)-3-carboxy-3-{[1 -(19- carboxynonadecanoyl)piperidine-4- carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy} acetyl [Aib8,Glu22,Arg26,Lys30]GLP-1 -(7-37),

• N-epsilon31 {2-[2-(2-{2-[2-((S)-3-carboxy-3-{[1 -(19- carboxynonadecanoyl)piperidine-4- carbonyllaminojpropionylaminojethoxylethoxyjacetylaminojethoxylethoxy} acetyl [Aibδ, Glu22, Arg26,Lys 31 ]GLP-1-(7-37),

• N-epsilon31 -(2-{2-[2-(2-{2-[2-((S)-3-Carboxy-3-{[1 -(19-carboxy- nonadecanoyl)piperidine-4- carbonyllaminojpropionylaminojethoxylethoxyjacetylaminojethoxylethoxyjacetyl ) [Aibδ, Glu22,Arg26,Lys31 ,Arg34]GLP-1 -(7-37),

• N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxy- nonadecanoyl- amino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylam ino] ethoxy}ethoxy)acetyl][Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide, • N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxy- nonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}et hoxy) acetylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GL

P-1 -(7-37)amide, • N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxy- nonadecanoyl- amino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylam ino] ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-

37), • N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxy- nonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino] ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg26,GI u30,Arg34, Lys37]GLP-1-(7-37), • N-epsilon20-[2-(2-{2-[(S)-4-Carboxy-4-((S)-4-carboxy-4-{12-[4-(16-(1 H-tetrazol-

5- yl)hexadecanoylsulfamoyl)butyrylamino]dodecanoylamino}butyrylamino)butyryla mino]ethoxy}ethoxy)acetyl][Aib8,Lys20,Glu22_lAi'g26,Glu30,Pro37]GLP-1-(7- 37)amide, • N-epsilon37-[2-(2-{2-[(S)-4-Carboxy-4-((S)-4-carboxy-4-{12-[4-(16-(1 H-tetrazol-

5- yl)hexadecanoylsulfamoyl)butyrylamino]dodecanoylamino}butyrylamino)butyryla mino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Arg26,Ar"g34,Lys37]GLP-1 -(7-37)amide,

• N-epsilon37-[2-(2-{2-[(S)-4-Carboxy-4-((S)-4-carboxy-4-{12-[4-(16-(1 H-tetrazol- 5- yl)hexadecanoylsulfamoyl)butyrylamino]dodecanoylamino}butyrylamino)butyryla mino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Ai'g26,Ai'g34,Lys37]GLP-1 -(7- 37)amide,

• [Aib8,Glu22,Arg26,Glu30,Pro37]GLP-1-(7-37)Lys [2-(2-{2-[4-Carboxy-4-(4- carboxy-4-{4-[4-(16-1 H-tetrazol-5-yl- hexadecanoylsulfamoyl)butyrylamino]butyrylamino} butyrylamino)butyrylamino]ethoxy}ethoxy)acetyl],

• N-epsilon37 (Polyethyleneglycol2000)[DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1 (7- 37) amide,

• N-epsilon37 (3-((2-(2-(2-(2-(2-

Hexadecyloxyethoxy)ethoxy)ethoxy)ethoxy)ethoxy)) propionyl) [DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1 (7-37)-amide,

• N-epsilon37-{2-(2-(2-(2-[2-(2-(4-(hexadecanoylamino)-4- carboxybutyrylamino)ethoxy) ethoxy]acetyl)ethoxy)ethoxy)acetyl)}-

[desaminoHis7,Glu22,Arg26, Glu30,Arg34,Lys37] (GLP-1-(7-37)amide,

• N-epsilon37-{2-(2-(2-(2-[2-(2-(4-(hexadecanoylamino)-4- carboxybutyrylamino)ethoxy) ethoxy]acetyl)ethoxy)ethoxy)acetyl)}- [desaminoHis7,Glu22, Arg26,Arg34,Lys 37] (GLP-1-(7-37)amide, • N-epsilon37-(2-(2-(2-(2-(2-(2-(2-(2-(2- Octadecanoylamino)ethoxy)ethoxy)acetylamino) ethoxy)ethoxy) acetylamino)ethoxy)ethoxy)acetyl) [desaminoHis7,Glu22,Arg26,Arg34,Lys37] GLP-1 (7-37)amide, • N-epsilon36-(2-(2-(2-((2-[2-(2-(17-carboxyheptadecanoylamino)ethoxy)ethoxy] acetylamino)ethoxy)ethoxy)acetyl) [Aib8,Glu22,Arg26,Glu30,Lys36] GLP-1 -(7- 37)Glu-amide,

• N-epsilon37-[4-(16-(1 H-Tetrazol-5-yl)hexadecanoylsulfamoyl)butyryl] [DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide, • N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(19-carboxynonadecanoyl aminoJbutyrylaminojethoxyJethoxyJacetylaminojethoxyJethoxyJacetylHDesamino His7,Glu22,Arg26,Arg34,Lys37]GLP-1 -(7-37), and

• N-epsilon31 -[2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino) butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Arg2 6,Lys31 ]GLP-1-(7-37).

In one aspect an N-terminally modified polypeptide according to the invention is selected from the group consisting of N-terminally modified GLP-1 , an N-terminally modified GLP-1 analogue or an N-terminally modified GLP-1 derivative for protection towards amin- opeptidases.

In one aspect of the invention an N-terminally modified polypeptide according to the invention is selected from the group consisting of N-terminally modified Factor VII, N- terminally modified Factor VIII, N-terminally modified Factor IX, N-terminally modified Factor Xl and N-terminally modified Factor XIII. As used herein, the terms "Factor VII polypeptide " or "FVII polypeptide" means any protein comprising the amino acid sequence 1 -406 of wild- type human Factor Vila, variants thereof as well as Factor Vl l-related polypeptides, Factor VII derivatives and Factor VII conjugates. This includes FVII variants, Factor Vll-related polypeptides, Factor VII derivatives and Factor VII conjugates exhibiting substantially the same or improved biological activity relative to wild-type human Factor Vila. Similar principles are used in applying similar terms related to Factor VIII, Factor IX, Factor Xl and Factor XIII.

In one aspect, the growth hormone derivative is a derivatized human growth hormone (hGH). The growth hormone from man and from the common domestic animals are proteins of approximately 191 amino acids, synthesized and secreted from the anterior lope of the pituitary gland.

In one aspect, the growth hormone is an analogue of hGH, wherein an analogue is understood to be the compound obtained by substituting one or more amino acid residues in the hGH sequence with another natural or unnatural amino acid; and/or by adding one or more natural or unnatural amino acids to the hGH sequence; and/or by deleting one or more amino acid residue from the hGH sequence, wherein any of these steps may optionally be followed by further derivatization of one or more amino acid residue. In particular, such substitutions are conservative in the sense that one amino acid residue is substituted by another amino acid residue from the same group, i.e. by another amino acid residue with similar properties.

PHARMACEUTICAL COMPOSITIONS

Another object of the present invention is to provide a pharmaceutical composition comprising an N-terminally modified polypeptide according to the present invention which is present in a concentration from 0.1 mg/ml to 500 mg/ml, and wherein said composition has a pH from 2.0 to 10.0. The composition may further comprise protease inhibitor(s) known to the person skilled in the art, a buffer system, preservative(s), tonicity agent(s), chelating agent(s), stabilizers and surfactants.

Administration of pharmaceutical compositions according to the invention may be through several routes of administration, for example, lingual, sublingual, buccal, in the mouth, oral, in the stomach and intestine, nasal, pulmonary, for example, through the bronchioles and alveoli or a combination thereof, epidermal, dermal, transdermal, vaginal, rectal, ocular, for examples through the conjunctiva, uretal, and parenteral to patients in need of such a treatment.

Compositions of the current invention may be administered in several dosage forms, for example, as solutions, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets, rinses, capsules, for ex- ample, hard gelatine capsules and soft gelatine capsules, suppositories, rectal capsules, drops, gels, sprays, powder, aerosols, inhalants, eye drops, ophthalmic ointments, ophthalmic rinses, vaginal pessaries, vaginal rings, vaginal ointments, injection solution, in situ transforming solutions, for example in situ gelling, in situ setting, in situ precipitating, in situ crystallization, infusion solution, and implants. Compositions of the invention may further be compounded in, or attached to, for example through covalent, hydrophobic and electrostatic interactions, a drug carrier, drug delivery system and advanced drug delivery system in order to further enhance stability of the insulin analogue compound, increase bioavailability, increase solubility, decrease adverse effects, achieve chronotherapy well known to those skilled in the art, and increase patient compliance or any combination thereof.

In one aspect of the invention the pharmaceutical composition is an aqueous composition, i.e. composition comprising water. Such composition is typically a solution or a sus- pension. In a further aspect of the invention the pharmaceutical composition is an aqueous solution. The term "aqueous composition" is defined as a composition comprising at least 50 % w/w water. Likewise, the term "aqueous solution" is defined as a solution comprising at least 50 % w/w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50 % w/w water. In another aspect the pharmaceutical composition is a freeze-dried composition, whereto the physician or the patient adds solvents and/or diluents prior to use.

In another aspect the pharmaceutical composition is a dried composition (e.g. freeze-dried or spray-dried) ready for use without any prior dissolution.

Aqueous suspensions may contain the active compounds in admixture with excipi- ents suitable for the manufacture of aqueous suspensions.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil or in a mineral oil. The oily suspensions may contain a thickening agent, sweetening agents, and flavouring agents. These compositions may be preserved by the addition of an anti-oxidant.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active compound in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.

The pharmaceutical compositions comprising a compound for use according to the present invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, a mineral oil or a mixture thereof. Suitable emulsifying agents may be naturally- occurring gums, naturally-occurring phosphatides, or partial esters derived from fatty acids and hexitol anhydrides and condensation products of said partial esters with ethylene oxide. The emulsions may also contain sweetening and flavouring agents.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such compositions may also contain a demulcent, preservative and flavouring and colouring agent.

Compositions intended for oral use may be prepared according to any known method, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents, and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets may contain the active ingredient in a mixture with non-toxic pharmaceutically-acceptable excipients which are suitable for the manufacture of tablets. The tablets may be uncoated or they may be coated by known techniques to delay disintegration or release of the therapeutically active polypeptide.

The orally administerable compositions of the present invention may be prepared and administered according to methods well known in pharmaceutical chemistry, see Remington's Pharmaceutical Sciences, 17^th ed. (A. Osol ed., 1985).

In one aspect of the invention, the pharmaceutical compositions of the present invention may be administered by means of solid dosage forms such as tablets and capsules. The tablets may be prepared by wet granulation, by dry granulation, by direct compression or melt granulation. Tablets for this invention may be prepared utilizing conventional tabletting techniques.

Compositions for oral use may also be presented as hard or soft gelatine capsules where the active ingredient is mixed with an inert solid diluent, for example, such as manni- tol, maltodextrin, calcium carbonate, sodium carbonate, lactose, kaolin, calcium phosphate or sodium phosphate, or a soft gelatine capsule wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

Entrapment and encapsulation is a technique used in drug delivery systems for therapeutically active polypeptides to optimize delivery properties including protection against enzymatic degradation. Entrapment or encapsulation could be in the form of polymeric drug delivery systems such as hydrogels and nanocapsules/microspheres, and lipid drug delivery systems such as liposomes and micro emulsions.

A composition of the invention may further comprise one or more protease inhibitors such as EDTA (ethylenediamine tetraacetic acid) and benzamidine hydrochloride, but other commercially available protease inhibitors such as protease inhibitors of serine protease, as- partic proteases, cysteine proteases and metalloproteases may also be used.

In another aspect of the invention, the composition comprises a further inhibitor of (a) proteolytic enzyme(s) such as aminopeptidase inhibitor, amastatin, bestatin, boroleucine and puromycin. Examples of protease inhibitors are sodium glycolate, camostat mesilate, bacitracin, soybean trypsin inhibitor and aprotinin.

In a further aspect of the invention, the composition further comprises a permeation enhancer. Bile salts and fatty acids are most often considered to increase the oral permeability of the lipid bi-layer membranes of the epithelial cell lining of the Gl tract. In general, permeation enhancers increase paracellular and trancellular transport of macromolecules by reversible altering the membrane integrity.

In a further aspect of the invention, the composition further comprises a mucoadhe- sive polymer. An intimate contact of the drug delivery system to the mucosa of the gastrointestinal tract can be obtained by use of such a mucoadhesive polymer. An intimate contact of the dosage form to the membrane seems advantageous as an enzymatic degradation of the therapeutically active polypeptide on the way between the delivery system and the absorp- tion membrane can be avoided. Moreover, a steep concentration gradient on the absorption membrane representing the driving force for passive drug uptake can be provided.

Compositions of the current invention are useful in the formulation of solids, semisolids, powder and solutions for pulmonary administration of N-terminally modified polypeptides according to the invention, using, for example a metered dose inhaler, dry powder inhaler and a nebulizer, all being devices well known to those skilled in the art.

The N-terminally modified polypeptide according to the invention can be administered via the pulmonary route in a vehicle, as a solution, suspension or dry powder using any of known types of devices suitable for pulmonary drug delivery. Examples of these comprise, but are not limited to, the three general types of aerosol-generating for pulmonary drug delivery, and may include jet or ultrasonic nebulizers, metered-dose inhalers, or dry powder inhalers (Cf. Yu J, Chien YW. Pulmonary drug delivery: Physiologic and mechanistic aspects. Crit Rev Ther Drug Carr Sys 14(4) (1997) 395-453).

In a further aspect, the composition could be aerosolized by any known aerosolisa- tion technology, such as nebulisation, to achieve a MMAD of aerosol particles less than 10 μm, more preferably between 1 -5 μm, and most preferably between 1 -3 μm. The preferred particle size is based on the most effective size for delivery of drug to the deep lung, where protein is optimally absorbed (cf. Edwards DA, Ben-Jebria A, Langer A, Recent advances in pulmonary drug delivery using large, porous inhaled particles. J Appl Physiol 84(2) (1998) 379-385).

Deep lung deposition of the pulmonal compositions comprising the insulin analogue compound may optional be further optimized by using modifications of the inhalation tech- niques, for example, but not limited to: slow inhalation flow (eg. 30 L/min), breath holding and timing of actuation.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each refer- ence were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law).

All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents.

This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. EXAMPLES

General procedure for reductive N-methylation of peptides and proteins

The polypeptide (0.022 mmol) is dissolved in a mixture of Λ/-methylformamide (3.8 ml) and 0.2 M citrate buffer pH 4.5 (2.2 ml_, 0.44 mmol; preparation of the buffer: citric acid 0.2 M + NaOH 0.35 M) and the mixture is carefully stirred.

37 % aqueous formaldehyde solution (0.063 ml, appr. 0.82 mmol) is added, followed by addition of a freshly prepared solution of sodium cyanoborohydride (21 mg, 0.33 mmol) in methanol (0.3 ml_). The mixture is carefully stirred for 90 min.

After completed reaction, the mixture is carefully acidified by adding 37 % hydrochloric acid drop by drop until pH 2-3. The product is isolated by preparative HPLC.

Example 1 : A1 (Λ/,Λ/-dimethyl), B1 (Λ/,Λ/-dimethyl), B29(Λ/^ε,Λ/^ε-dimethyl) desB30 human insulin

Reductive methylation of desB30 human insulin (0.022 mmol) was performed ac- cording to the general procedure described above. After completed reaction and acidification approximately to pH 2.5, the crude mixture was diluted with acetonitrile (2 ml) and water (12 ml) to give a total volume of about 20 ml.

This solution was divided into two equal portions. Each portion was diluted with ace- tonitrile (2.5 ml) and water (7.5 ml) and then purified using a Gilson preparative HPLC device. Elution was performed with water/acetonitrile containing 0.1 % TFA with a gradient from 29 % to 41% acetonitrile. The eluate was collected as fractions of 5 ml (peak fractions) or 12 ml (non-peak fractions), respectively. Relevant fractions were checked by analytical HPLC. Fractions containing the pure target product were mixed and concentrated under reduced pressure to give a colourless aqueous solution (45 ml). The solution was cooled with ice- water and then carefully treated with 1 M aqueous sodium hydroxide solution drop by drop until pH 5.

A Waters "Sep-Pak® C18 12cc (2 g)" reversed phase cartridge (Waters Corporation, Milford, Massachusetts, USA; catalogue no. WAT036915) was washed first with isopro- panol/water 9:1 (30 ml) and then with water (20 ml). The aqueous product solution was loaded onto the cartridge and the eluate was collected (fractions 1 -4). The cartridge was then washed with water (50 ml, eluate fractions 4-6). The product was eluted with methanol/water 7:3 (50 ml) and the eluate was collected as 5 ml fractions (fractions 7-16).

According to analytical HPLC, eluate fractions 8-14 contained the pure product. Fractions 8-14 were pooled and concentrated under reduced pressure to give a suspension. Freeze-drying for three days afforded the target compound as a white solid (60 mg by weight; yield 47 %).

LCMS (Sciex AP 1-100 Quad ru pole MS, electrospray, m/z = 300 to m/z = 5000; column: Waters XTerra® MS C₁₈ 5μm 3. OxSOmm; water/acetonitrile containing 0.05 % TFA; gradient: 5 % → 90 % acetonitrile from 0 to 7.5 m in; flow 1.5 ml/min): m/z = 1449.1 [(M+4)/4] at t_R = 2.95 min.

HPLC (Waters Symmetry300 C18, 5 μm, 3.9 x 150 mm; 42 ⁰C; water/acetonitrile containing 0.05 % TFA; gradient: 0 % → 60 % acetonitrile from 0 to 15 min; flow 1 ml/min): t_R = 1 1.64 min (100 % purity by UV 214 nm).

The affinity of the analog for the human insulin receptor was 68 % relative to human insulin.

Example 2: A1 (Λ/,Λ/-dimethyl), B1 (Λ/,Λ/-dimethyl), B29(Λ/^ε,Λ/^ε-dimethyl) A14E B25H desB30 human insulin

A1 (Λ/,Λ/-dimethyl), B1 (Λ/,Λ/-dimethyl), B29(Λ/^ε,Λ/^ε-dimethyl) A14E B25H desB30 human insulin was prepared from A14E B25H desB30 human insulin, formaldehyde and sodium cyanoborohydride following the general procedure above. LCMS of the purified product showed m/z = 1484.9 [(M+4)/4].

The affinity of the analog for the human insulin receptor was 17 % relative to human insulin (71 % relative to A14E B25H desB30 human insulin).

Example 3: A1 (Λ/,Λ/-dimethyl), B1 (Λ/,Λ/-dimethyl), B29Λ/^ε-hexadecandioyl-gamma-L-Glu desB30 human insulin

A1 (Λ/,Λ/-dimethyl), B1 (Λ/,Λ/-dimethyl), B29Λ/^ε-hexadecandioyl-gamma-L-Glu desB30 human insulin was prepared from B29N^ε-hexadecandioyl-gamma-L-Glu desB30 human insulin (WO2005012347), formaldehyde and sodium cyanoborohydride following the general procedure above. LCMS of the purified product showed m/z = 1540.6 [(M+4)/4].

The affinity of the analog for the human insulin receptor was 16 % relative to human insulin (64 % relative to B29 Λf-hexadecandioyl-gamma-L-Glu desB30 human insulin). Example 4: A1 (Λ/,Λ/-dimethyl), B1 (Λ/,Λ/-dimethyl), B29Λ/^ε-myristoyl desB30 human insulin

A1 (Λ/,Λ/-dimethyl), B1 (Λ/,Λ/-dimethyl), B29Λ/^e-hexadecandioyl-gamma-L-Glu desB30 human insulin was prepared from B29Af-myristoyl desB30 human insulin (insulin detemir), formaldehyde and sodium cyanoborohydride following the general procedure above. LCMS of the purified product showed m/z = 1493.8 [(M+4)/4].

The affinity of the analog for the human insulin receptor was 22 % relative to human insulin (100 % relative to B29Af-myristoyl desB30 human insulin).

Example 5: A1 (Λ/,Λ/-dimethyl), B1 (Λ/,Λ/-dimethyl), B29Af-3-{2-[2-(2-methoxy-ethoxy)-ethoxy]- ethoxy}-propionyl A14E B25H desB30 human insulin

A1 (Λ/,Λ/-dimethyl), B1 (Λ/,Λ/-dimethyl), B29Af-3-{2-[2-(2-methoxy-ethoxy)-ethoxy]- ethoxy}-propionyl A14E B25H desB30 human insulin was prepared from A14E B25H desB30 human insulin by derivatization with succinimidyl 3-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}- propionate (as described in US2006019874), followed by treatment with formaldehyde and sodium cyanoborohydride following the general procedure above. LCMS of the purified product showed m/z = 1484.9 [(M+4)/4].

The affinity of the analog for the human insulin receptor was 1 1 % relative to human insulin (48 % relative to A14E B25H desB30 human insulin).

Example 6: A1 (Λ/,Λ/-dimethyl), B1 (Λ/,Λ/-dimethyl), B29(Λ/^ε,Λ/^ε-dimethyl) B28D human insulin

A1 (Λ/,Λ/-dimethyl), B1 (Λ/,Λ/-dimethyl), B29(Λ/^ε,Λ/^ε-dimethyl) B28D human insulin was prepared from B28D human insulin (insulin aspart), formaldehyde and sodium cyanoboro- hydride following the general procedure above. LCMS of the purified product showed m/z = 1478.2 [(M+4)/4].

Example 7: A1 (Λ/,Λ/-diethyl), B1 (Λ/,Λ/-diethyl), 629(^/^6^1) A14E B25H desB30 human insulin

A1 (Λ/,Λ/-diethyl), B1 (Λ/,Λ/-diethyl), B29(N^e,N^e-diethyl) A14E B25H desB30 human insulin was prepared from A14E B25H desB30 human insulin, acetaldehyde and sodium cyanoborohydride following the general procedure above. LCMS of the purified product showed m/z = 1458.4 [(M+4)/4].

The affinity of the analog for the human insulin receptor was 23 % relative to human insulin (100 % relative to A14E B25H desB30 human insulin).

Example 8: A1Λ/-amidinyl, B1 Λ/-amidinyl, B29Λ/^ε-amidinyl human insulin

200 mg human insulin was dissolved in 12 ml dimethylacetamide and 60 μl diiso- propylethylamine. 50 mg 1 H-pyrazol-1-carboxamidine,HCI was added and the mixture shaken at room temperature. After 4 weeks RP-HPLC and MALDI showed a mixture of di- and triguanylated product which were difficult to separate by RP-HPLC. The solvent was evaporated under vacuum at 60⁰C and 20 ml acetonitrile was added to the resulting oil. A white solid formed which was washed three times with acetonitrile. The precipitate was dis- solved in 50 ml glacial acetic acid and 15 ml acetonitrile. 2 ml acetic anhydride was added and the mixture left to react overnight at room temperature to block any free amino groups. The reaction was stopped by addition of 5 ml of water and the solution was evaporated to dryness, redissolved in water and lyophilized. The desired compound was isolated in a yield of 15 mg by RP-HPLC in acetonitrile/TFA. Mass by MALDI-MS 5934 (5933.8 expected). The affinity of the analog for the human insulin receptor was 91% relative to human insulin. Example 9: His7-ΛΛΛΛdimethyl, Lys26-Λ/^ε,Λ/^ε-dimethyl, Lys34-Λ/^ε,Λ/^ε-dimethyl, GLP-1-(7-37)

His7-ΛΛΛΛdimethyl, Lys26-Λ/^ε,Λ/^ε-dimethyl, Lys34-Λ/^ε,Λ/^ε-dimethyl, GLP-1 -(7-37) was prepared from recombinant GLP-1 (7-37), formaldehyde and sodium cyanoborohydride following the general procedure above. LCMS of the purified product showed m/z = 1721 [(M+2)/2].

DPP-IV stability was examined according to a literature procedure (Journal of Mo- lecular Biology. Volume 363, Issue 5, 10 November 2006, Pages 977-988). The compound of example 9 showed no sign of DPP-IV degradation after 4 hours incubation with the DPP- IV enzyme, versus unmodified hGLP-1 which was 30 % degraded after 1 hours treatment under the same in vitro conditions.

Example 10: Af26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoyl- amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Λ/,Λ/-dimethylHis7, Aib8,Arg34]GLP-1-(7-37)

This compound was prepared according to the procedure as described above starting with 100 mg of Af26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17 carboxyheptadecanoylamino) bu- tyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Arg34]GLP-1 -(7-37) (prepared as described in WO2006097537) to yield the title compound (30 mg). HPLC-MS: m/z = 1381 (m/3), 1036 (m/4), EC50 = 12 nM Example 11 : Stability of insulins towards rat liver homogenate

Stability of N-terminally protected insulin analogs was evaluated by incubation of the analogs with rat liver homogenates. Human insulin and A14E, B25H, desB30 human insulin were rapidly degraded by aminopeptidases present in the homogenate. In contrast, the ^-terminally protected analogs of example 1 (Λ/-hexamethyl human insulin) and example 2 (Λ/-hexamethyl A14E, B25H, desB30 human insulin) were highly resistant to degradation by liver homogenates, see figure 1. In further experiments, A1 (Λ/,Λ/-dimethyl), B1 (Λ/,Λ/-dimethyl), B29 Λf-3- {2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-propionyl A14E B25H desB30 human insulin and A1 (Λ/,Λ/-diethyl), B1 (Λ/,Λ/-diethyl), B29(N^e,Λf-diethyl) A14E B25H desB30 human insulin were also found to have superior stability towards rat liver homogenates (data not shown).

Example 12: Insulin receptor binding of the insulin derivatives of the invention

The affinity of the insulin analogues of the invention for the human insulin receptor was determined by a SPA assay (Scintillation Proximity Assay) microtiterplate antibody capture assay. SPA-PVT antibody-binding beads, anti-mouse reagent (Amersham Biosciences, Cat No. PRNQ0017) were mixed with 25 ml of binding buffer (100 mM HEPES pH 7.8; 100 mM sodium chloride, 10 mM MgSO₄, 0.025% Tween-20). Reagent mix for a single Packard Optiplate (Packard No. 6005190) is composed of 2.4 μl of a 1 :5000 diluted purified recombinant human insulin receptor - exon 11 , an amount of a stock solution of A14 Tyr[¹²⁵l]-human insulin corresponding to 5000 cpm per 100 μl of reagent mix, 12 μl of a 1 :1000 dilution of F12 antibody, 3 ml of SPA-beads and binding buffer to a total of 12 ml. A total of 100 μl was then added and a dilution series is made from appropriate samples. To the dilution series was then added 100 μl of reagent mix and the samples were incubated for 16 hours while gently shaken. The phases were the then separated by centrifugation for 1 min and the plates counted in a Topcounter. The binding data were fitted using the nonlinear regression algorithm in the GraphPad Prism 2.01 (GraphPad Software, San Diego, CA).

Example 13: Biological activity of A1 (Λ/,Λ/-dimethyl), B1 (Λ/,Λ/-dimethyl), B29(Λ/^ε,Λ/^ε-dimethyl) A14E B25H desB30 human insulin

Biological activity of A1 (Λ/,Λ/-dimethyl), B1 (Λ/,Λ/-dimethyl), B29(Λ/^ε,Λ/^ε-dimethyl) A14E B25H desB30 human insulin and A14E B25H desB30 human insulin were compared after administration into ileum of SPRD rats. Both insulin analogues were active in lowering blood glucose (figure 2).

Claims

1. An N-terminally modified polypeptide comprising one or more N-terminal modifications wherein an N-terminal modification is a conjugation of one or two substituents to an N- terminal of the parent polypeptide, and a substituent is an organic molecular substituent having a MW below 10O g pr. mol, wherein the size of the parent polypeptide is between 20 - 100 amino acids and wherein the N-terminal amine of the N-terminally modified polypeptide is at least partially positively charged at physiological pH.

2. The N-terminally modified polypeptide according to claim 1 wherein the one or two organic molecular substituents are selected from the group consisting of C^ alkyl and amidinyl.

3. The N-terminally modified polypeptide according to claims 1 or 2 wherein the organic molecular substituents are methyl substituents.

4. The N-terminally modified polypeptide according to anyone of claims 1-3 which is further modified in one or more internal positions wherein the further modified positions comprise the conjugation product of a free amino-group and an organic molecular substituent having a MW below 100 g pr. mol.

5. The N-terminally modified polypeptide according to any one of claims 1-4 which is at least partially resistant towards degradation by an aminopeptidase.

6. The N-terminally modified polypeptide according to any one of claims 1-5 wherein the polypeptide is selected from the group consisting of insulin, glucagon-like peptide, growth hormone, Factor VII, Factor VIII, Factor IX, Factor Xl and Factor XIII and analogues and derivatives thereof.

7. The N-terminally modified polypeptide according to any one of claims 1 -6 wherein the polypeptide is an insulin.

8. The N-terminally modified polypeptide according to any one of claims 1 -6 wherein the polypeptide is GLP-1.

9. A pharmaceutical composition comprising one or more polypeptides according to any one of claims 1-8 and a pharmaceutically acceptable excipient.

10. A method for treating or preventing a disease or disorder selected from the group consisting of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive disorders, atheroschlerosis, myocardial infarction, coronary heart disease and other cardiovascular disorders, CNS disorders such as Alzheimer's, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers, said method comprising administering to a subject in need of such treatment an effective amount of the pharmaceutical composition of claim 9.

11. An N-terminally modified polypeptide according to any one of claims 1 -8 for for use as a medicament for delaying or preventing disease progression in type 2 diabetes.