GLUCAGON ANALOGS EXHIBITING ENHANCED SOLUBILITY AND STABILITY IN PHYSIOLOGICAL pH BUFFERS

• US 20110190200A1
• Filed: 06/16/2009
• Published: 08/04/2011
• Est. Priority Date: 06/17/2008
• Status: Active Grant

• Current Assignee: David L. Smiley, Jonathan Day, Joseph Chabenne, Maria Dimarchi, Richard D. DiMarchi
• Original Assignee: Indiana University Research & Technology Corporation (Indiana University)
• Inventors: DiMarchi, Richard D., Dimarchi, Maria, Smiley, David L., Day, Jonathan, Chabenne, Joseph
• Granted Patent: US 8,450,270 B2
• Time in Patent Office: Days
• US Class Current: 514/6.8
• 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: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 23/02 Local anaesthetics
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/08 for glucose homeostasis pancreatic hormones A61P5/48
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: C07K 14/605 Glucagons

Abstract

Modified glucagon peptides are disclosed having improved solubility and/or half-life while retaining glucagon agonist activity. The glycogen peptides have been modified by substitution of native amino acids with, and/or addition of, charged amino acids to the carboxy terminus of the peptide. The modified glucagon agonists can be further modified by pegylation, or the addition of a carboxy terminal peptide selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, or both to further enhance the solubility of the glucagon agonist analogs.

Claims

1. A glucagon peptide with glucagon agonist activity, comprising (i) the amino acid sequence of SEQ ID NO:

1 with one, two, three, or all of the amino acids at positions 16, 20, 21, and 24 substituted with an α

,α

-disubstituted amino acid, and (ii) a C-terminal alpha carboxylate.

2-24. -24. (canceled)

25. A glucagon peptide with glucagon agonist activity, comprising the amino acid sequence of SEQ ID NO:

77, wherein the Xaa at position 3 is an amino acid comprising a side chain of Structure I, II, or III;

26. The glucagon peptide of claim 25, wherein:

(a) X is NH or Y is NHR⁴;

(b) R¹ is C_0-2 alkyl or C₁ heteroalkyl;

(c) R² is NHR⁴ or C₁ alkyl;

(d) R⁴ is H or C¹ alkyl;

(e) a combination thereof.

30. The glucagon peptide of claim 25, wherein the Xaa at position 3 is an amino acid comprising a side chain of:

(i) Structure I and R¹ is CH₂—

S, X is NH, and R² is CH₃;

(ii) Structure I and R¹ is CH₂, X is NH, R² is CH₃,

(iii) Structure I and R1 is C0 alkyl, X is NH, R2 is NHR4, and R4 is H;

(iv) Structure II and R1 is CH2, Y is NHR4, and R4 is CH3;

(v) Structure III and R1 is CH2 and R4 is H;

or

(vi) Structure I and R¹ is CH₂—

CH₂, X is NH, and R² is CH₃.

32. The analog of claim 25, wherein the α

,α

-disubstituted amino acid is AIB.

33. The analog of claim 32, wherein the AIB is at position 16 of the glucagon peptide.

34. The analog of claim 25, wherein the glucagon peptide comprises an amino acid modification selected from the group consisting of:

(a) substitution of His at position 1 with a non-native amino acid that reduces susceptibility of the glucagon peptide to cleavage by dipeptidyl peptidase IV (DPP-IV),

(b) substitution of Ser at position 2 with a non-native amino acid that reduces susceptibility of the glucagon peptide to cleavage by dipeptidyl peptidase IV (DPP-IV),

(c) substitution of Thr at position 7 with Abu or Ile;

(d) substitution of Tyr at position 10 with Phe or Val;

(e) substitution of Lys at position 12 with Arg;

(f) substitution of Asp at position 15 with Glu,

(g) substitution of Ser at position 16 with Thr or AIB;

(h) substitution of Gln at position 20 with Ala or AIB;

(i) substitution of Asp at position 21 with Glu;

(j) substitution of Gln at position 24 with Ala or AIB;

(k) substitution of Met at position 27 with Leu or Nle;

(l) deletion of amino acids at positions 27-29;

(m) deletion of amino acids at positions 28-29;

(n) deletion of the amino acid at positions 29;

(o) addition of the amino acid sequence of SEQ ID NO;

20 to the C-terminus, wherein the amino acid at position 29 is Thr or Gly,

(p) substitution or addition of an amino acid comprising a side chain covalently attached to an acyl or alkyl group which is non-native to a naturally-occurring amino acid;

(q) a substitution of Asn at position 28 with a charged amino acid;

(r) a substitution of Thr at position 29 with a charged amino acid;

(s) an insertion of 1-3 charged amino acids after position 29;

(t) and a combination thereof.

35. The analog of claim 34, wherein the glucagon peptide comprises the amino acid sequence of any of SEQ ID NOs:

62-67 and 69-74.

36. The analog of claim 25, wherein:

(a) a hydrophilic moiety is covalently linked to the amino acid at any of positions 16, 17, 20, 21, 24, and 29 or at the C-terminal amino acid;

(b) an acyl group or alkyl group is covalently attached to the side chain of the amino acid at position 10 or the C-terminal amino acid;

(c) a combination thereof.

82. The glucagon peptide or analog of claim 25, wherein, when the glucagon peptide, or analog thereof, lacks a hydrophilic moiety, the glucagon peptide, or analog thereof, exhibits at least about 20% of the activity of native glucagon at the glucagon receptor.

87. A conjugate, a fusion peptide, or a dimer comprising a glucagon peptide of claim 25.

89. A pharmaceutical composition comprising (i) a glucagon peptide or an analog of claims 25, (ii) a dimer, a conjugate, or a fusion peptide comprising the glucagon peptide or the analog, or (iii) a combination thereof, and a pharmaceutically acceptable carrier.

90. A kit for administering a glucagon agonist to a patient in need thereof, said kit comprising a pharmaceutical composition of claim 89 and a device for administering said pharmaceutical composition to the patient.

94. A method of treating or preventing hypoglycemia in a patient in need thereof, comprising administering to the patient a pharmaceutical composition of claim 89 in an amount effective to treat or prevent hypoglycemia in the patient.

95. A method of stabilizing a blood glucose level in a patient in need thereof, wherein the patient is on a treatment regimen comprising administration of insulin, comprising administering to the patient a pharmaceutical composition of claim 89 in an amount effective to stabilize the blood glucose level of the patient.

97. The analog of claim 34, wherein the charged amino acid is selected from the group consisting of Lys, Arg, His, Asp, Glu, cysteic acid, and homocysteic acid.

98. The analog of claim 35, comprising the amino acid sequence of SEQ ID NO:

71.

27-29. -29. (canceled)

31. (canceled)

37-42. -42. (canceled)

43. A glucagon peptide with glucagon agonist activity, comprising 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-Z (SEQ ID NO;

     39) with 1 to 3 amino acid modifications thereto,

wherein X1 and/or X2 is a non-native amino acid that reduces susceptibility of the glucagon peptide to cleavage by dipeptidyl peptidase IV (DPP-IV),

wherein Z is selected from the group consisting of —

COOH, -Asn-COOH, Asn-Thr-COOH, and Y—

COOH, wherein Y is 1 to 2 amino acids, and

wherein 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.

44-57. -57. (canceled)

58. A glucagon peptide with glucagon agonist activity, comprising the amino acid sequence:

59-73. -73. (canceled)

74. An analog of the glucagon peptide of SEQ ID NO:

1 with up to 10 modifications relative to native glucagon (SEQ ID NO;

     1) that has glucagon agonist activity and comprises

(a) at least one amino acid modification selected from the group consisting of;

substitution of Asn at position 28 with a charged amino acid;

(ii) substitution of Asn at position 28 with a charged amino acid selected from the group consisting of Lys, Arg, His, Asp, Glu, cysteic acid, and homocysteic acid;

(iii) substitution at position 28 with Asn, Asp, or Glu;

(iv) substitution at position 28 with Asp;

(v) substitution at position 28 with Glu;

(vi) substitution of Thr at position 29 with a charged amino acid;

(vii) substitution of Thr at position 29 with a charged amino acid selected from the group consisting of Lys, Arg, His, Asp, Glu, cysteic acid, and homocysteic acid;

(viii) substitution at position 29 with Asp, Glu, or Lys;

(ix) substitution at position 29 with Glu;

(x) insertion of 1-3 charged amino acids after position 29;

(xi) insertion after position 29 of Glu or Lys;

(xii) insertion after position 29 of Gly-Lys or Lys-Lys;

or a combination thereof;

(b) and at least one amino acid modification selected from Group A or Group B, or a combination thereof;

wherein Group A is an amino acid modification selected from the group consisting of substitution of Asp at position 15 with Glu, and substitution of Ser at position 16 with Thr or AIB; and

wherein Group B is an amino acid modification selected from the group consisting of;

(i) substitution of His at position 1 with a non-native amino acid that reduces susceptibility of the glucagon peptide to cleavage by dipeptidyl peptidase IV (DPP-IV),

(ii) substitution of Ser at position 2 with a non-native amino acid that reduces susceptibility of the glucagon peptide to cleavage by dipeptidyl peptidase IV (DPP-IV),

(iii) substitution of Tyr at position 10 with Phe or Val;

(iv) substitution of Lys at position 12 with Arg;

(v) substitution of Gln at position 20 with Ala or AIB;

(vi) substitution of Asp at position 21 with Glu;

(vii) substitution of Gln at position 24 with Ala or AIB;

(viii) substitution of Met at position 27 with Leu or Nle;

(ix) deletion of amino acids at positions 27-29;

(x) deletion of amino acids at positions 28-29;

(xi) deletion of the amino acid at positions 29;

(xii) or a combination thereof.

75. The analog of claim 74, further comprising one or a combination of the following amino acid modifications:

(a) substitution of Gln at position 3 with an amino acid comprising a side chain of Structure I, II, or III;

78. The analog of claim 75, wherein the α

,α

-disubstituted amino acid is AIB.

76. (canceled)

77. (canceled)

79. (canceled)

80. A glucagon peptide comprising the amino acid sequence of any of SEQ ID NOs:

40-56.

81. (canceled)

83-86. -86. (canceled)

88. (canceled)

91-93. -93. (canceled)

96. (canceled)