Incretin-insulin conjugates

• US 10,232,020 B2
• Filed: 09/23/2015
• Issued: 03/19/2019
• Est. Priority Date: 09/24/2014
• Status: Expired due to Fees

• Current Assignee: Indiana University Research & Technology Corporation (Indiana University)
• Original Assignee: Indiana University Research & Technology Corporation (Indiana University)
• Inventors: Dimarchi, Richard D., Mayer, John P., Smiley, David L., Liu, Fa
• Primary Examiner(s): Ha, Julie
• Assistant Examiner(s): Komatsu, Li N
• Application Number:
  US15/513,748
• Publication Number:
  US 20170281788A1
• Time in Patent Office: 1,273 Days
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 38/22 Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin A61K38/33, e.g. corticotropin A61K38/35
• CPC Class Codes: A61K 38/28 Insulins
• CPC Class Codes: A61K 47/02 Inorganic compounds
• CPC Class Codes: A61K 47/545 Heterocyclic compounds A61K47/558 takes precedence
• CPC Class Codes: A61K 47/64 Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent peptidic linkers A61K47/65
• CPC Class Codes: A61K 9/0019 Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner non-active ingredients are additionally classified in A61K47/00
• CPC Class Codes: A61K 9/0024 Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue compositions for intravenous administration, normal injectable solutions or dispersions for, e.g. subcutaneous administration A61K9/0019; brain implants A61K9/0085; (coated) prostheses, catheters or stents A61L
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/575 Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695
• CPC Class Codes: C07K 14/605 Glucagons
• CPC Class Codes: C07K 14/62 Insulins
• CPC Class Codes: C07K 2/00 Peptides of undefined number of amino acids; Derivatives thereof

Abstract

Disclosed herein are insulin agonist peptides conjugated to incretins wherein the incretin-insulin conjugate has agonist activity at the insulin receptor and the corresponding incretin receptor, and stimulates weight loss in an individual administered the compound.

Claims

1. An incretin-insulin conjugate comprising

an incretin peptide and

an insulin agonist peptide, wherein said conjugate has agonist activity at both the insulin receptor and an incretin receptor, and the incretin peptide is linked to the insulin agonist peptide via a linear chain spacer, wherein the incretin peptide is selected from the group consisting of:

(i) the amino acid sequence;

X1-X2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Z₁ (SEQ ID NO;

     839) with 1 to 3 amino acid modifications thereto, wherein;

X1 is selected from the group consisting of;

His, D-His, N-methyl-His, alpha-methyl-His, imidazole acetic acid, des-amino-His, hydroxyl-His, acetyl-His, homo-His, and alpha, alpha-dimethyl imidiazole acetic acid (DMIA);

X2 is selected from the group consisting of;

Ser, D-Ser, Ala, D-Ala, Gly, N-methyl-Ser, Val, and alpha-aminoisobutyric acid (Aib);

Z₁ is selected from the group consisting of Asn-Thr-COOH, and Y-COOH, wherein Y is 1 to 2 amino acids, and further wherein;

(1) a lactam bridge connects the side chains of an amino acid at position i and an amino acid at position i+4, wherein i is 12, 16, 20 or 24, or

(2) one, two, or three of the amino acids at positions 16, 20, 21, and 24 of the incretin peptide is substituted with an α

,α

-disubstituted amino acid;

and said incretin peptide has glucagon agonist activity;

(ii) the amino acid sequence of X₁X₂X₃GTFTSDX₁₀SX₁₂YLX₁₅X₁₆X₁₇X₁₈AX₂₀X₂₁FX₂₃X₂₄WLX₂₇X₂₈X₂₉ (SEQ ID NO;

     1926), wherein;

X₁ is selected from the group consisting of His, D-His, des-amino-His, hydroxyl-His, acetyl-His, homo-His or alpha, alpha-dimethyl imidiazole acetic acid (DMIA), N-methyl His, alpha-methyl His, and imidazole acetic acid;

X₂ is selected from the group consisting of Ser, D-Ser, Ala, D-Ala, Val, Gly, N-methyl Ser, aminoisobutyric acid (Aib) and N-methyl Ala;

X₃ is selected from the group consisting of Gln, Glu, Orn and Nle;

X₁₀ is selected from the group consisting of Tyr, Val and Trp;

X₁₂ is selected from the group consisting of Ser, Lys and Arg;

X₁₅ is selected from the group consisting of Asp, Glu, cysteic acid, homoglutamic acid and homocysteic acid;

X₁₆ is selected from the group consisting of Ser, Gly, Glu, Gln, homoglutamic acid and homocysteic acid;

X₁₇ is selected from the group consisting of Arg, Gln, Lys, Cys, Orn, homocysteine and acetyl phenylalanine;

X₁₈ is selected from the group consisting of Arg, Ala, Lys, Cys, Orn, homocysteine and acetyl phenylalanine;

X₂₀ is selected from the group consisting of Gln, Lys, Arg, Orn and citrulline;

X₂₁ is selected from the group consisting of Gln, Glu, Asp, Lys, Cys, Orn, homocysteine and acetyl phenylalanine;

X₂₃ is selected from the group consisting of Val and Ile;

X₂₄ is selected from the group consisting of Ala, Gln, Glu, Lys, Cys, Orn, homocysteine and acetyl phenylalanine;

X₂₇ is selected from the group consisting of Met, Val, Leu and Nle;

X₂₈ is selected from the group consisting of Asn, Lys and Asp; and

X₂₉ is selected from the group consisting of Thr, Gly, Lys, Cys, Orn, homocysteine and acetyl phenylalanine;

or an analog of SEQ ID NO;

1926, wherein said analog differs from SEQ ID NO;

1926 by 1, 2 or 3 amino acid modifications;

(iii) an incretin peptide of SEQ ID NO;

701 or an analog thereof, wherein said analog is modified to comprise;

(a) an amino acid modification at position 1 that confers gastric inhibitory polypeptide (GIP) agonist activity,

(b) (1) a lactam bridge between the side chains of amino acids at positions i and i+4 or between the side chains of amino acids at positions j and j+3, wherein i is 12, 13, 16, 17, 20 or 24, and wherein j is 17, or

(2) one, two, three, or all of the amino acids at positions 16, 20, 21, and 24 of the incretin peptide of SEQ ID NO;

701 is substituted with an α

,α

-disubstituted amino acid,

(c) amino acid modifications at one, two or all of positions 27, 28 and 29, and

(d) 1-6 further amino acid modifications,

wherein the EC50 of the analog for GIP receptor activation is about 10 nM or less;

(iv) the sequence of SEQ ID NO;

701 with

(a) an amino acid at position 10 which is acylated with a C4 to C30 fatty acid, and

(b) an Aib at position 16;

(v) an amino acid sequence that differs from SEQ ID NO;

701 by no more than ten amino acid modifications, comprising one or more amino acid substitutions with Aib at positions 16, 20, 21, and/or 24, and an amino acid modification at position 1 and/or 2 that provides reduced susceptibility to cleavage by dipeptidyl peptidase IV, wherein said incretin peptide exhibits at least 20% of the activity of native glucagon like peptide-1 (GLP-1) at the GLP-1 receptor;

(vi) any of the incretin peptides (i)-(v), further comprising the amino acid sequence of GPSSGAPPPS (SEQ ID NO;

     1095) or XGPSSGAPPPS (SEQ ID NO;

     1096) attached to the C-terminus of the incretin peptide, wherein X is any amino acid;

(vii) the amino acid sequence of SEQ ID NO;

701 with the following amino acid modifications;

Tyr at position 1, an Aib at position 2, Lys at position 10, wherein the Lys is covalently bound to a C16 fatty acyl group via a γ

-Glu-γ

-Glu dipeptide spacer, Ile at position 12, Lys at position 16, Gln at position 17, Ala at position 18, Aib at position 20, Glu at position 21, Asn at position 24, Leu at position 27, Ala at position 28, Gly at position 29, followed by the amino acid sequence of SEQ ID NO;

1095 attached to the C-terminal amino acid at position 29, and a C-terminal amide in place of the C-terminal alpha carboxylate; and

(viii) HSQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQID NO;

     2042), HSQGTFTSDYSKYLDERRAQDFVQWLMNT (SEQ ID NO;

     2041), Y(aib)EGTFISDYSIYLDRQAA(aib)EFVNWLLAGGPSSGAPPPS (SEQ ID NO;

     2044), Y(aib)EGTFTSDX₁₀SIYLDKQAA(aib)EFVNWLLAGGPSSGAPPPS (SEQ ID NO;

     1987), X₁X₂EGTFTSDX₁₀SIYLDKQAAX₂₀EFVNWLLAGGPSSGAPPPS (SEQ ID NO;

     2037) or X₁X₂EGTFTSDVSIYLDKQAAX₂₀EFVNWLLAGGPSSGAPPPSX₄₀ (SEQ ID NO;

     2038), wherein;

X₁ is His or Tyr;

X₂ is alpha-aminoisobutyric acid (Aib);

X₁₀ is Lys acylated with a C16 to C20 fatty acid group via a gamma Glu linker;

X₂₀ is alpha-aminoisobutyric acid (Aib); and

X₄₀ is Lys acylated with a C16 to C20 fatty acid group via a gamma Glu linker; and

(ix) X ₁X₂X₃GTFX₇SDX₁₀SX₁₂YLX₁₅X₁₆X₁₇AAX₂₀X₂₁FVX₂₄WLLX₂₈X₂₉ (SEQ ID NO;

     2039), wherein;

X₁ is Tyr or His;

X₂ is Ser, D-serine, Ala, Val, glycine, N-methyl serine, aminoisobutyric acid (Aib), N-methyl alanine or D-alanine;

X₃ is Glu or Gln;

X₇ is Thr or Ile;

X₁₀ is Lys, Tyr or Val;

X₁₂ is Ser, Lys, Arg or Ile;

X₁₅ is Glu or Asp;

X₁₆ is Glu or Lys;

X₁₇ is Gln or Arg;

X₂₀ is Gln or Aib;

X₂₁ is Glu or Asp;

X₂₄ is Asn, Gln or Ala;

X₂₈ is Ala, Glu, or Asp;

X₂₉ is Ala or Gly,

wherein said insulin agonist peptide comprises an insulin A chain and an insulin B chain, wherein said insulin A chain comprises the amino acid sequence GIVX₄X₅CCX₈X₉X₁₀CX₁₂LX₁₄X₁₅LX₁₇X₁₈YCX₂₁-R₁₃ (SEQ ID NO;

     19), and said B chain comprises the amino acid sequence R₂₂-X₂₅LCGX₂₉X₃₀LVX₃₃X₃₄LYLVCGX₄₁X₄₂GFX₄₅ (SEQ ID NO;

     20), wherein;

X₄ is glutamic acid or aspartic acid;

X₅ is glutamine or glutamic acid;

X₈ is histidine, threonine or phenylalanine;

X₉ is serine, arginine, lysine, ornithine or alanine;

X₁₀ is isoleucine or serine;

X₁₂ is serine or aspartic acid;

X₁₄ is tyrosine, arginine, lysine, ornithine or alanine;

X₁₅ is glutamine, glutamic acid, arginine, alanine, lysine, ornithine or leucine;

X₁₇ is glutamic acid, aspartic acid, asparagine, lysine, ornithine or glutamine;

X₁₈ is methionine, asparagine, glutamine, aspartic acid, glutamic acid or threonine;

X₂₁ is selected from the group consisting of alanine, glycine, serine, valine, threonine, isoleucine, leucine, glutamine, glutamic acid, asparagine, aspartic acid, histidine, tryptophan, tyrosine, and methionine;

X₂₅ is histidine or threonine;

X₂₉ is selected from the group consisting of alanine, glycine and serine;

X₃₀ is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid;

X₃₃ is selected from the group consisting of aspartic acid and glutamic acid;

X₃₄ is selected from the group consisting of alanine and threonine;

X₄₁ is selected from the group consisting of glutamic acid, aspartic acid and asparagine;

X₄₂ is selected from the group consisting of alanine, ornithine, lysine and arginine;

X₄₅ is tyrosine or phenylalanine;

R₂₂ is selected from the group consisting of AYRPSE (SEQ ID NO;

     14), FVNQ (SEQ ID NO;

     12), FVKQ (SEQ ID NO;

     8), PGPE (SEQ ID NO;

     11), a tripeptide glycine-proline-glutamic acid, a tripeptide valine-asparagine-glutamine, a dipeptide proline-glutamic acid, a dipeptide asparagine-glutamine, glutamine, glutamic acid and an N-terminal amine; and

R₁₃ is COOH or CONH₂;

wherein said linear chain spacer comprises the general structure of;

2. The conjugate of claim 1, wherein the linear chain spacer comprises the general structure of:

3. The conjugate of claim 2, wherein

R₁, R₂, R₃ and R₄ are each H;

and

m is 1 or 2.

4. The conjugate of claim 1, wherein said A chain comprises the amino acid sequence GIVEQCCX₈X₉ICSLYQLENYCX₂₁-R₁₃ (SEQ ID NO:

2040), said B chain comprises the amino acid sequence R₂₂-X₂₅LCGX₂₉X₃₀LVX₃₃X₃₄LYLVCGX₄₁X₄₂GFX₄₅ (SEQ ID NO;

     20), wherein;

X₈ is histidine or threonine;

X₉ is serine, lysine, or alanine;

X₂₁ is alanine, glycine or asparagine;

X₂₅ is histidine or threonine;

X₂₉ is selected from the group consisting of alanine, glycine and serine;

X₃₀ is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid;

X₃₃ is selected from the group consisting of aspartic acid and glutamic acid;

X₃₄ is selected from the group consisting of alanine and threonine;

X₄₁ is selected from the group consisting of glutamic acid, aspartic acid and asparagine;

X₄₂ is selected from the group consisting of alanine, ornithine, lysine and arginine;

X₄₅ is tyrosine or phenylalanine;

R₂₂ is selected from the group consisting of FVNQ (SEQ ID NO;

     12), FVKQ (SEQ ID NO;

     8), a tripeptide valine-asparagine-glutamine, a dipeptide asparagine-glutamine, glutamine and an N-terminal amine; and

R₁₃ is COOH or CONH₂.

5. The conjugate of claim 4, wherein

said incretin peptide is selected from the group consisting of:

Y(aib)EGTFTSDYSIYLDKQAA(aib)EFVNWLLAGGPSSGAPPPS (SEQ ID NO;

1930), H(aib)QGTFTSDYSKYLDERAAQDFVQWLLDGGPSSGAPPPS (SEQ ID NO;

     1931), H(aib)EGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPS (SEQ ID NO;

     1932), HSQGTFTSDYSKYLDSRRAQDFVQWLMNTGPSSGAPPPS (SEQ ID NO;

     2043), Y(aib)EGTFISDYSIYLDRQAA(aib)EFVNWLLAGGPSSGAPPPS (SEQ ID NO;

     2044), and Y(aib)EGTFTSDX₁₀SIYLDKQAA(aib)EFVNWLLAGGPSSGAPPPS (SEQ ID NO;

     1987);

wherein X₁₀ is Lys acylated with a C16 fatty acid group via a gamma Glu linker;

said linear chain spacer joining the incretin to the insulin agonist peptide comprises the general structure of;

6. The conjugate of claim 5, wherein

i) the side chain of the amino acid at position 10 of the incretin peptide further comprises a non-native alkyl or acyl group;

or

ii) the side chain of the amino acid at position 24, or an added C-terminal amino acid at position 40 of the incretin peptide, or the side chain of the amino acid at position B28 or B29 of the insulin agonist peptide is linked to a hydrophilic moiety;

or

iii) a combination of i) and ii).

7. The conjugate of claim 1, wherein the insulin agonist peptide comprises:

an A chain sequence of GIVEQCCTSICSLYQLENYCN-R₁₃ (SEQ ID NO;

     1); and

a B chain sequence selected from the group consisting of;

FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO;

     2), FVNQHLCGSHLVEALYLVCGERGFFYTKPT (SEQ ID NO;

     9), FVNQHLCGSHLVEALYLVCGERGFFYTDKT (SEQ ID NO;

     5) and FVKQHLCGSHLVEALYLVCGERGFFYTEKT (SEQ ID NO;

     6), wherein R₁₃ is COOH or CONH₂; and

said linear chain spacer joining the incretin to the insulin agonist peptide comprises the general structure of;

8. The conjugate of claim 7, wherein the insulin agonist peptide comprises:

an A chain sequence of GIVEQCCTSICSLYQLENYCN (SEQ ID NO;

     1); and

a B chain sequence of FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO;

     2);

said incretin peptide comprises the amino acid sequence Y(aib)EGTFTSDX₁₀SIYLDKQAA(aib)EFVNWLLAGGPSSGAPPPS (SEQ ID NO;

     1987);

wherein X₁₀ is Lys acylated with a C16 fatty acid group via a gamma Glu linker; and

said linear chain spacer joining the incretin to the insulin agonist peptide comprises the general structure of;

9. The conjugate of claim 1, wherein

said incretin peptide comprises the amino acid sequence Y(aib)EGTFTSDX₁₀SIYLDKQAA(aib)EFVNWLLAGGPSSGAPPPS (SEQ ID NO:

1987), Y(aib)EGTFTSDYSIYLDKQAA(aib)EFVNWLLAGGPSSGAPPPS (SEQ ID NO;

     1930), or H(aib)QGTFTSDYSKYLDERAAQDFVQWLLDGGPSSGAPPPS (SEQ ID NO;

     1931);

wherein X₁₀ is Lys acylated with a C16 fatty acid group via a gamma Glu linker; and

said insulin agonist peptide comprises an A chain sequence of GIVEQCCTSICSLYQLENYCN (SEQ ID NO;

     1), and a B chain sequence selected from the group consisting of;

FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO;

     2), FVNQHLCGSHLVEALYLVCGERGFFYTKPT (SEQ ID NO;

     9), FVNQHLCGSHLVEALYLVCGERGFFYTDKT (SEQ ID NO;

     5), and FVKQHLCGSHLVEALYLVCGERGFFYTEKT (SEQ ID NO;

     6), and wherein the linear chain spacer comprises the general structure of;

10. The conjugate of claim 1, wherein the insulin agonist peptide is a single chain insulin and the peptide linker joining the B and A chains is selected from the group consisting of SSSSKAPPPSLPSPSRLPGPSDTPILPQR (SEQ ID NO:

1922), SSSSRAPPPSLPSPSRLPGPSDTPILPQK (SEQ ID NO;

     1923), GAGSSSX₅₇X₅₈ (SEQ ID NO;

     76), GYGSSSRR (SEQ ID NO;

     61), GAGSSSRR (SEQ ID NO;

     1925) and GYGSSSX₅₇X₅₈APQT (SEQ ID NO;

     77), wherein

X₅₇ and X₅₈ are independently arginine, lysine or ornithine.

11. The conjugate of claim 1, wherein the incretin peptide comprises the amino acid sequence X₁X₂EGTFTSDX₆SSYLEEQAAKEFIAWLVK-R₄ (SEQ ID NO:

1936), or X₁X₂QGTFTSDYSKYLDERX₅AKDFVX₃WLMN-R₄ (SEQ ID NO;

     1937);

wherein;

X₁ is selected from the group consisting of His, D-histidine, desaminohistidine, hydroxyl-histidine, acetyl-histidine, homo-histidine, alpha, alpha-dimethyl imidiazole acetic acid (DMIA), N-methyl histidine, alpha-methyl histidine, and imidazole acetic acid;

X₂ is selected from the group consisting of Ser, D-serine, Ala, Val, glycine, N-methyl serine, aminoisobutyric acid (Aib), N-methyl alanine and D-alanine;

X₃ is selected from the group consisting of Ala, Gln and Cys-PEG;

R₄ is selected from the group consisting of Thr-CONH₂, Cys-PEG, GGPSSGAPPPS (SEQ ID NO;

     515), GGPSSGAPPPSC-PEG (SEQ ID NO;

     516) and GGPSSGAPPPSCK(rEC16)C (SEQ ID NO;

     1935);

X₅ is Ala or Arg and

X₆ is selected from the group consisting of valine, tyrosine and K(rEC16)C; and

wherein when X₃ is Cys-PEG, R₄ is not Cys-PEG or GGPSSGAPPPSC-PEG (SEQ ID NO;

     516), and when X₂ is Ser, X₁ is not His.

12. The conjugate of claim 1, wherein the incretin peptide comprises an analog of glucagon (SEQ ID NO:

701) having GIP agonist activity, said analog comprising one or more of the following modifications;

(a) an amino acid modification at position 1 that confers GIP agonist activity, wherein the amino acid at position 1 is an amino acid lacking an imidazole side chain;

(b) an amino acid substitution of Ser at position 16 with an amino acid of Formula IV;

13. The conjugate of claim 12, wherein the incretin peptide comprises the following modifications:

(a) the amino acid at position 1 is a Tyr, and (b) wherein (i) the Met at position 27 is substituted with Leu, (ii) the Asn at position 28 is substituted with Ala, or (iii) the Thr at position 29 is substituted with Gly, or wherein the analog comprises a combination of (i), (ii), and (iii).

14. The conjugate of claim 13, wherein the incretin peptide further comprises one or more of the following modifications:

(a) Ser at position 2 substituted with D-Ser, Ala, D-Ala, Gly, N-methyl-Ser, Aib, Val, or α

-amino-N-butyric acid;

(b) Gln at position 3 substituted with Glu;

(c) substitution of the amino acid Tyr at position 10 with an amino acid of Formula I;

15. The conjugate of claim 1, wherein the incretin peptide comprises the amino acid sequence of:

Y(aib)EGTFTSDX₁₀SIYLDKQAA(aib)EFVNWLLAGGPSSGAPPPS (SEQ ID NO;

     1987), HAEGTFTSDVSSYLEEQAAREFIAWLVRGRG (SEQ ID NO;

     700), HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO;

     703), HAEGTFTSDVSSYLEGQAAKEFICWLVKGR (SEQ ID NO;

     717), HSQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO;

     2042), HSQGTFTSDYSKYLDERRAQDFVQWLMNT (SEQ ID NO;

     2041), or an analog of SEQ ID NO;

703, SEQ ID NO;

717, SEQID NO;

2042 or SEQ ID NO;

2041, wherein the incretin peptide or the analog further comprises a terminal extension of the amino acid sequence of GPSSGAPPPS (SEQ ID NO;

     820) or GGPSSGAPPPS (SEQ ID NO;

     515), wherein X₁₀ is Lys acylated with a C16 fatty acid group via a gamma Glu linker.

16. The conjugate of claim 1, wherein

said incretin peptide comprises an amino acid sequence selected from the group consisting of:

Y(aib)EGTFTSDX₁₀SIYLDKQAAXEFVNWLLAGGPSSGAPPPS (SEQ ID NO;

     1987), Y(aib)EGTFTSDYSIYLDKQAA(aib)EFVNWLLAGGPSSGAPPPS (SEQ ID NO;

     1930), H(aib)QGTFTSDYSKYLDERAAQDFVQWLLDGGPSSGAPPPS (SEQ ID NO;

     1931), H(aib)EGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPS (SEQ ID NO;

     1932), HSQGTFTSDYSKYLDSRRAQDFVQWLMNTGGPSSGAPPPS (SEQ ID NO;

     2043), and Y(aib)EGTFISDYSIYLDRQAA(aib)EFVNWLLAGGPSSGAPPPS (SEQ ID NO;

     2044), wherein X₁₀ is Lys acylated with a C16 fatty acid group via a gamma Glu linker;

said insulin peptide comprises

an A chain sequence consisting of GIVEQCCTSICSLYQLENYCN (SEQ ID NO;

     1); and

a B chain sequence consisting of FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO;

     2); and

said linear chain spacer joining the incretin to the insulin peptide comprises the general structure of;

17. A pharmaceutical composition comprising a conjugate of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

18. An incretin-insulin conjugate comprising

an incretin peptide and

an insulin agonist peptide, wherein said conjugate has agonist activity at both the insulin receptor and an incretin receptor, and the incretin peptide is linked to the insulin agonist peptide via a linear chain spacer, wherein

said insulin agonist peptide comprises:

an A chain sequence of GIVEQCCX₈SICSLYQLENYCX₂₁ (SEQ ID NO;

     3); and

a B chain sequence of R₂₂-HLCGSHLVEALYLVCGERGFX₄₅ (SEQ ID NO;

     15), wherein the B chain is linked to the A chain through disulfide linkages; and

R₂₂ is selected from the group consisting of FVNQ (SEQ ID NO;

     12), FVKQ (SEQ ID NO;

     8), VNQ, NQ and Q;

X₈ is selected from the group consisting of threonine and histidine;

X₂₁ is selected from the group consisting of asparagine, lysine, glycine, and alanine; and

X₄₅ is histidine, tyrosine or phenylalanine; and

said incretin peptide comprises the sequence X₈₀X₈₁X₈₂GTFTSDX₇₉SX₈₃YLX₈₄X₈₅X₈₆X₈₇AX₈₈X₈₉FX₉₀X₉₁WLX₉₂X₉₃X₉₄ (SEQ ID NO;

     1928) or X₁X₂X₃GTFX₇SDX₁₀SX₁₂YLX₁₅X₁₆X₁₇AAX₂₀X₂₁FVX₂₄WLLX₂₈X₂₉ (SEQ ID NO;

     2039), wherein

the peptide of SEQ ID NO;

1928 further comprises a C-terminal extension Z₁;

wherein;

X₁ is Tyr or His;

X₂ is Ser, D-serine, Ala, Val, glycine, N-methyl serine, aminoisobutyric acid (Aib), N-methyl alanine or D-alanine;

X₃ is Glu or Gln;

X₇ is Thr or Ile;

X₁₀ is Lys, Tyr or Val;

X₁₂ is Ser, Lys, Arg or Ile;

X₁₅ is Glu or Asp;

X₁₆ is Glu or Lys;

X₁₇ is Gln or Arg;

X₂₀ is Gln or Aib;

X₂₁ is Glu or Asp;

X₂₄ is Asn, Gln or Ala;

X₂₈ is Ala, Glu, or Asp;

X₂₉ is Ala or Gly;

X₇₉ is an amino acid covalently attached to a C12 to C18 acyl or alkyl group;

X₈₀ is His, Tyr, D-histidine, desaminohistidine, hydroxyl-histidine, acetyl-histidine, homo-histidine or alpha, alpha-dimethyl imidiazole acetic acid (DMIA) N-methyl histidine, alpha-methyl histidine, or imidazole acetic acid;

X₈₁ is Ser, D-serine, Ala, Val, glycine, N-methyl serine or alpha-aminoisobutyric acid (Aib), N-methyl alanine or D-alanine;

X₈₂ is Gln or Glu;

X₈₃ is Lys or Ile;

X₈₄ is Asp or Glu;

X₈₅ is Lys, Arg, Ser or Glu;

X₈₆ is Arg or Gln;

X₈₇ is Ala or Arg;

X₈₈ is alpha-aminoisobutyric acid (Aib), Gln or Lys;

X₈₉ is Asp or Glu;

X₉₀ is Val or Be;

X₉₁ is Asn, Gln or Ala;

X₉₂ is Leu, Val or Met;

X₉₃ is Ala, Asp, Lys Asn or Ala;

X₉₄ is Gly or Thr; and

Z₁ is selected from the group consisting of —

COOH, GPSSGAPPPS (SEQ ID NO;

     820), KRNRNNIA (SEQ ID NO;

     821) and KRNR (SEQ ID NO;

     822); and

said linear chain spacer comprises the general structure of;

19. The conjugate of claim 18, wherein

X₈₀ is His or Tyr;

X₈₈ is aminoisobutyric acid (Aib); and

Z₁ is GPSSGAPPPS (SEQ ID NO;

     820).