NOVEL INHIBITORS OF MAMMALIAN TIGHT JUNCTION OPENING

Abstract

The present invention provides novel peptides that inhibit and/or reduce the opening of mammalian tight junctions, i.e. peptide tight junction antagonists. The present invention also provides methods for the treatment of excessive or undesirable permeability of a tissue by administering to a subject suffering from such a condition a composition comprising a peptide tight junction antagonist of the invention.

Claims

1. A peptide tight junction antagonist, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-69.

2. The peptide tight junction antagonist of claim 1, wherein the peptide is eight to ten amino acids in length.

3. The peptide tight junction antagonist of claim 1, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

4. The peptide tight junction antagonist of claim 1, wherein the peptide consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-69.

5. The peptide tight junction antagonist of claim 1, wherein the peptide consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

6. The peptide tight junction antagonist of claim 1, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-69.

7. The peptide tight junction antagonist of claim 1, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

8. A method of treating an excessive or undesirable permeability of a tissue containing tight junctions comprising: administering to a subject in need thereof a composition comprising a peptide tight junction antagonist, wherein the peptide comprises an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

9. The method of claim 8, wherein the subject is a human.

10. The method of claim 8, wherein the peptide is eight to ten amino acids in length.

11. The method of claim 8, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

12. The method of claim 8, wherein the peptide consists essentially of an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

13. The method of claim 8, wherein the peptide consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

14. The method of claim 8, wherein the peptide consists of an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

15. The method of claim 8, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

16. The method of claim 8, wherein the composition further comprises an additional therapeutic agent.

17. The method of claim 16, wherein the additional therapeutic agent is selected from the group consisting of aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics.

18. The method of claim 8, wherein the composition is formulated for intestinal delivery.

19. The method of claim 8, wherein the composition is formulated for pulmonary delivery.

20. The method of claim 8 wherein said excessive or undesirable permeability of a tissue containing tight junctions is associated with a disease selected form the group consisting of: celiac disease; inflammatory bowel disease; Crohn's disease; ulcerative colitis; acute respiratory distress syndrome; acute lung injury; chronic obstructive pulmonary disorder; type 1 diabetes; and asthma.

21. A method of treating inflammatory bowel disease comprising: administering to a subject in need thereof a composition comprising a peptide tight junction antagonist, wherein the peptide comprises an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

22. The method of claim 21, wherein the subject is a human.

23. The method of claim 21, wherein the peptide is eight to ten amino acids in length.

24. The method of claim 21, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

25. The method of claim 21, wherein the peptide consists essentially of an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

26. The method of claim 21, wherein the peptide consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

27. The method of claim 21, wherein the peptide consists of an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

28. The method of claim 21, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

29. The method of claim 21, wherein the composition further comprises an additional therapeutic agent.

30. The method of claim 29, wherein the additional therapeutic agent is selected from the group consisting of aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics.

31. The method of claim 21, wherein the composition is formulated for intestinal delivery.

32. A method of treating Crohn's disease comprising: administering to a subject in need thereof a composition comprising a peptide tight junction antagonist, wherein the peptide comprises an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

33. The method of claim 32, wherein the subject is a human.

34. The method of claim 32, wherein the peptide is eight to ten amino acids in length.

35. The method of claim 32, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

36. The method of claim 32, wherein the peptide consists essentially of an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

37. The method of claim 32, wherein the peptide consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

38. The method of claim 32, wherein the peptide consists of an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

39. The method of claim 32, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

40. The method of claim 32, wherein the composition further comprises an additional therapeutic agent.

41. The method of claim 40, wherein the additional therapeutic agent is selected from the group consisting of aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics.

42. The method of claim 32, wherein the composition is formulated for intestinal delivery.

43. A method of treating ulcerative colitis comprising: administering to a subject in need thereof a composition comprising a peptide tight junction antagonist, wherein the peptide comprises an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

44. The method of claim 43, wherein the subject is a human.

45. The method of claim 43, wherein the peptide is eight to ten amino acids in length.

46. The method of claim 43, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

47. The method of claim 43, wherein the peptide consists essentially of an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

48. The method of claim 43, wherein the peptide consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

49. The method of claim 43, wherein the peptide consists of an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

50. The method of claim 43, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69

51. The method of claim 43, wherein the composition further comprises a therapeutic agent.

52. The method of claim 51, wherein the therapeutic agent is selected from the group consisting of aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics.

53. The method of claim 43, wherein the composition is formulated for intestinal delivery.

54. A method of treating Celiac disease comprising: administering to a subject in need thereof a composition comprising a peptide tight junction antagonist, wherein the peptide comprises an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

55. The method of claim 54, wherein the subject is a human.

56. The method of claim 54, wherein the peptide is eight to ten amino acids in length.

57. The method of claim 54, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

58. The method of claim 54, wherein the peptide consists essentially of an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

59. The method of claim 54, wherein the peptide consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

60. The method of claim 54, wherein the peptide consists of an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

61. The method of claim 54, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

62. The method of claim 54, wherein the composition is formulated for intestinal delivery.

63. A method of treating acute respiratory distress syndrome comprising: administering to a subject in need thereof a composition comprising a peptide tight junction antagonist, wherein the peptide comprises an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

64. The method of claim 63, wherein the subject is a human.

65. The method of claim 63, wherein the peptide is eight to ten amino acids in length.

66. The method of claim 63, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

67. The method of claim 63, wherein the peptide consists essentially of an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

68. The method of claim 63, wherein the peptide consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

69. The method of claim 63, wherein the peptide consists of an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

70. The method of claim 63, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

71. The method of claim 63, wherein the composition is formulated for pulmonary delivery.

72. A method of treating acute lung injury comprising: administering to a subject in need thereof a composition comprising a peptide tight junction antagonist, wherein the peptide comprises an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

73. The method of claim 72, wherein the subject is a human.

74. The method of claim 72, wherein the peptide is eight to ten amino acids in length.

75. The method of claim 72, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

76. The method of claim 72, wherein the peptide consists essentially of an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

77. The method of claim 72, wherein the peptide consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

78. The method of claim 72, wherein the peptide consists of an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

79. The method of claim 72, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

80. The method of claim 72, wherein the composition is formulated for pulmonary delivery.

81. A method of treating chronic obstructive pulmonary disorder comprising: administering to a subject in need thereof a composition comprising a peptide tight junction antagonist, wherein the peptide comprises an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

82. The method of claim 81, wherein the subject is a human.

83. The method of claim 81, wherein the peptide is eight to ten amino acids in length.

84. The method of claim 81, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

85. The method of claim 81, wherein the peptide consists essentially of an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

86. The method of claim 81, wherein the peptide consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

87. The method of claim 81, wherein the peptide consists of an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

88. The method of claim 81, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

89. The method of claim 81, wherein the composition is formulated for pulmonary delivery.

90. A method of treating type 1 diabetes comprising: administering to a subject in need thereof a composition comprising a peptide tight junction antagonist, wherein the peptide comprises an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

91. The method of claim 90, wherein the subject is a human.

92. The method of claim 90, wherein the peptide is eight to ten amino acids in length.

93. The method of claim 90, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

94. The method of claim 90, wherein the peptide consists essentially of an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

95. The method of claim 90, wherein the peptide consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

96. The method of claim 90, wherein the peptide consists of an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

97. The method of claim 90, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

98. The method of claim 90, wherein the composition is formulated for intestinal delivery.

99. The method of claim 90, wherein the composition is formulated for pulmonary delivery.

100. A method of treating asthma comprising: administering to a subject in need thereof a composition comprising a peptide tight junction antagonist, wherein the peptide comprises an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

101. The method of claim 99, wherein the subject is a human.

102. The method of claim 99, wherein the peptide is eight to ten amino acids in length.

103. The method of claim 99, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

104. The method of claim 99, wherein the peptide consists essentially of an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

105. The method of claim 99, wherein the peptide consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

106. The method of claim 99, wherein the peptide consists of an amino acid sequence selected from the group of SEQ ID NOs: 2-69.

107. The method of claim 99, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11, 13, 17, 18, 20-32, 34, 35, 54, 57, and 67-69.

108. The method of claim 99, wherein the composition is formulated for pulmonary delivery.

109. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO3.

110. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:4.

111. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:5.

112. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:6.

113. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:7.

114. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:8.

115. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:9.

116. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:10.

117. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:11.

118. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:13.

119. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:17.

120. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:18.

121. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:20.

122. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:21.

123. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:22.

124. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:23.

125. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:24.

126. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:25.

127. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:26.

128. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:27.

129. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:28.

130. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:29.

131. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:30.

132. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:31.

133. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:32.

134. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:34.

135. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:35.

136. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:54.

137. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:57.

138. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:67.

139. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:68.

140. The peptide tight junction antagonist of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:69.

141. A method of treating an excessive or undesirable permeability of a tissue containing tight junctions comprising: administering to a subject in need thereof a composition comprising the peptide tight junction antagonist of any one of claims 109-140.

142. A method of treating inflammatory bowel disease comprising: administering to a subject in need thereof a composition comprising the peptide tight junction antagonist of any one of claims 109-140.

143. A method of treating Crohn's disease comprising: administering to a subject in need thereof a composition comprising the peptide tight junction antagonist of any one of claims 109-140.

144. A method of treating ulcerative colitis comprising: administering to a subject in need thereof a composition comprising the peptide tight junction antagonist of any one of claims 109-140.

145. A method of treating Celiac disease comprising: administering to a subject in need thereof a composition comprising the peptide tight junction antagonist of any one of claims 109-140.

146. A method of treating acute respiratory distress syndrome comprising: administering to a subject in need thereof a composition comprising the peptide tight junction antagonist of any one of claims 109-140.

147. A method of treating acute lung injury comprising: administering to a subject in need thereof a composition comprising the peptide tight junction antagonist of any one of claims 109-140.

148. A method of treating chronic obstructive pulmonary disorder comprising: administering to a subject in need thereof a composition comprising the peptide tight junction antagonist of any one of claims 109-140.

149. A method of treating type 1 diabetes comprising: administering to a subject in need thereof a composition comprising the peptide tight junction antagonist of any one of claims 109-140.

150. A method of treating asthma comprising: administering to a subject in need thereof a composition comprising the peptide tight junction antagonist of any one of claims 109-140.

151. A pharmaceutical composition comprising a peptide that reduces tight junction permeability and a pharmaceutically acceptable carrier and/or excipient, the peptide having from 3 to 5 amino acids.

152. The pharmaceutical composition of claim 151, wherein the peptide is defined by the formula: X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5; wherein: X.sub.1 is optional, and where present is a natural or non-natural amino acid; X.sub.2 is optional, and where present is a natural or non-natural amino acid; X.sub.3 is a natural or non-natural amino acid; X.sub.4 is selected from Pro or Ala; and X.sub.5 is Gly, Gln, or Ala.

153. The pharmaceutical composition of claim 152, wherein X.sub.1 and X.sub.2 are not present.

154. The pharmaceutical composition of claim 152 or 153, wherein X.sub.3 is a genetically-encoded amino acid.

155. The pharmaceutical composition of claim 152 or 153, wherein X.sub.3 is selected from Gln, (d)Gln, Ala, Gly, His, Arg, Phe, Glu, Lys, Leu, Met, Asn, Ser, Tyr, Ile, Trp, and Glp.

156. The pharmaceutical composition of any one of claims 152 to 155, wherein X.sub.4 is Pro.

157. The pharmaceutical composition of claim 156, wherein X.sub.3 is Gln.

158. The pharmaceutical composition of any one of claims 152 to 157, wherein X.sub.5 is Gly.

159. The pharmaceutical composition of claim 152 or 153, wherein X.sub.3 is Gln, X.sub.4 is Pro, and X.sub.5 is Gly.

160. The pharmaceutical composition of any one of claims 151 to 159, wherein the composition is formulated for pulmonary delivery.

161. The pharmaceutical composition of claim 160, wherein the composition is formulated as an aerosol.

162. A method for reducing tight junction permeability of lung tissue, comprising, administering the pharmaceutical composition of any one of claim 160 or 161 to a patient in need.

163. The method of claim 162, wherein the patient is afflicted with one or more of asthma, acute respiratory distress syndrome, acute lung injury, and chronic obstructive pulmonary disorder.

164. The method of claim 162 or 163, wherein the patient is human.

165. The pharmaceutical composition of any one of claims 151 to 159, wherein the composition is formulated for intestinal delivery.

166. A method for reducing tight junction permeability of intestinal tissue, comprising, administering the pharmaceutical composition of claim 165 to a patient in need.

167. The method of claim 166, wherein the patient is afflicted with one or more of celiac disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, and type 1 diabetes.

168. The method of claim 166 or 167, wherein the patient is human.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0067] FIG. 1 is a schematic showing the steps involved in solid phase synthesis of an exemplary tight junction antagonist of the invention.

[0068] FIG. 2 is a schematic showing the steps involved in solution phase synthesis of an exemplary tight junction antagonist of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0069] As used herein, about used to modify a numerical value means within 10% of the value.

[0070] Antagonists of Tight Junction Opening

[0071] As used herein, tight junction antagonists prevent, inhibit or reduce the opening of tight junctions, for example, the opening of tight junctions induced by a tight junction agonist. A tight junction antagonist may bind to a receptor that mediates tight junction agonist induced opening of tight junctions. For example, a tight junction antagonist may bind to the ZOT receptor and prevent, inhibit, reduce or reverse the tight junction opening triggered by the tight junction agonist ZOT.

[0072] As used herein a subject is any animal, e.g., mammal, upon which methods of the invention may be practiced and/or to which materials of the present invention may be administered. Subjects include, but are not limited to, humans.

[0073] Antagonists of the invention may comprise peptide antagonists. An example of a peptide tight junction antagonist is a peptide that consists of the amino acid sequence Gly Gly Val Leu Val Gln Pro Gly (SEQ ID NO: 1). Further examples of peptide tight junction antagonists are peptides that consist of the amino acid sequences Gly Val Leu Val Gln Pro Gly (SEQ ID NO:2), Val Leu Val Gln Pro Gly (SEQ ID NO:3), Leu Val Gln Pro Gly (SEQ ID NO:4), Val Gln Pro Gly (SEQ ID NO:5), or Gln Pro Gly (SEQ ID NO:6). Additional examples of peptide tight junction antagonists of the invention include, but are not limited to, peptides wherein one or more amino acids of the amino acid sequences Gly Val Leu Val Gln Pro Gly (SEQ ID NO:2), Val Leu Val Gln Pro Gly (SEQ ID NO:3), Leu Val Gln Pro Gly (SEQ ID NO:4), Val Gln Pro Gly (SEQ ID NO:5), or Gln Pro Gly (SEQ ID NO:6) have been substituted with a different amino acid. In some embodiments, only one position will be substituted. In some embodiments, two positions will be substituted. Substitutions may be made at any position of SEQ ID NO:1.

[0074] The peptide tight junction antagonists can be chemically synthesized and purified using well-known techniques, such as described in High Performance Liquid Chromatography of Peptides and Proteins: Separation Analysis and Conformation, Eds. Mant et al., C.R.C. Press (1991), and a peptide synthesizer, such as Symphony (Protein Technologies, Inc); or by using recombinant DNA techniques, i.e., where the nucleotide sequence encoding the peptide is inserted in an appropriate expression vector, e.g., an E. coli or yeast expression vector, expressed in the respective host cell, and purified therefrom using well-known techniques.

[0075] Compositions

[0076] Typically, compositions, such as pharmaceutical compositions, comprising a tight junction antagonist (e.g., peptide tight junction antagonist) comprise a pharmaceutically effective amount of that antagonist. The pharmaceutically effective amount of tight junction antagonist (e.g., peptide tight junction antagonist) employed in any given composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Generally, the amount of tight junction antagonist used for preventing, ameliorating and/or treating a disease in a subject will be in the range of about 1 g to 1 g, preferably from about 1 mg to about 1000 mg, or from about 10 mg to about 100 mg, or from about 10 mg to about 50 mg, or from about 10 mg to about 25 mg of antagonist.

[0077] Compositions of the invention may comprise one or more peptide tight junction antagonists at a level of from about 0.1 wt % to about 20 wt %, from about 0.1 wt % to about 18 wt %, from about 0.1 wt % to about 16 wt %, from about 0.1 wt % to about 14 wt %, from about 0.1 wt % to about 12 wt %, from about 0.1 wt % to about 10 wt %, from about 0.1 wt % to about 8 wt %, from about 0.1 wt % to about 6 wt %, from about 0.1 wt % to about 4 wt %, from about 0.1 wt % to about 2 wt %, from about 0.1 wt % to about 1 wt %, from about 0.1 wt % to about 0.9 wt %, from about 0.1 wt % to about 0.8 wt %, from about 0.1 wt % to about 0.7 wt %, from about 0.1 wt % to about 0.6 wt %, from about 0.1 wt % to about 0.5 wt %, from about 0.1 wt % to about 0.4 wt %, from about 0.1 wt % to about 0.3 wt %, or from about 0.1 wt % to about 0.2 wt % of the total weight of the composition. Compositions of the invention may comprise one or more peptide tight junction antagonists at a level of about 0.1 wt %, about 0.2 wt %, about 0.3 wt %, about 0.4 wt %, about 0.5 wt %, about 0.6 wt %, about 0.7 wt %, about 0.8 wt %, or about 0.9 wt % based on the total weight of the composition.

[0078] Compositions of the invention may comprise one or more peptide tight junction antagonists at a level of from about 1 wt % to about 20 wt %, from about 1 wt % to about 18 wt %, from about 1 wt % to about 16 wt %, from about 1 wt % to about 14 wt %, from about 1 wt % to about 12 wt %, from about 1 wt % to about 10 wt %, from about 1 wt % to about 9 wt %, from about 1 wt % to about 8 wt %, from about 1 wt % to about 7 wt %, from about 1 wt % to about 6 wt %, from about 1 wt % to about 5 wt %, from about 1 wt % to about 4 wt %, from about 1 wt % to about 3 wt %, or from about 1 wt % to about 2 wt % of the total weight of the composition. Compositions of the invention may comprise one or more peptide tight junction antagonists at a level of about 1 wt %, about 2 wt %, about 3 wt %, about 4 wt %, about 5 wt %, about 6 wt %, about 7 wt %, about 8 wt %, or about 9 wt % based on the total weight of the composition.

[0079] Compositions of the invention may formulated for pulmonary delivery (e.g., may be pulmonary dosage forms). Typically such compositions may be provided as pharmaceutical aerosols, e.g., solution aerosols or powder aerosols. Those of skill in the art are aware of many different methods and devices for the formation of pharmaceutical aerosols, for example, those disclosed by Sciarra and Sciarra, Aerosols, in Remington: The Science and Practice of Pharmacy, 20th Ed., Chapter 50, Gennaro et al. Eds., Lippincott, Williams and Wilkins Publishing Co., (2000).

[0080] In one embodiment, the dosage forms are in the form of a powder aerosol (i.e, comprise particles). These are particularly suitable for use in inhalation delivery systems. Powders may comprise particles of any size suitable for administration to the lung.

[0081] Powder formulations may optionally contain at least one particulate pharmaceutically acceptable carrier known to those of skill in the art. Examples of suitable pharmaceutical carriers include, but are not limited to, saccharides, including monosaccharides, disaccharides, polysaccharides and sugar alcohols such as arabinose, glucose, fructose, ribose, mannose, sucrose, trehalose, lactose, maltose, starches, dextran, mannitol or sorbitol. In one embodiment, a powder formulation may comprise lactose as a carrier.

[0082] Powder formulations may be contained in any container known to those in the art. Containers may be capsules of, for example, gelatin or plastic, or in blisters (e.g. of aluminum or plastic), for use in a dry powder inhalation device. In some embodiments, the total weight of the formulation in the container may be from about 5 mg to about 50 mg. In other embodiments, powder formulations may be contained in a reservoir in a multi-dose dry powder inhalation device adapted to deliver a suitable amount per actuation.

[0083] Powder formulations typically comprise small particles. Suitable particles can be prepared using any means known in the art, for example, by grinding in an airjet mill, ball mill or vibrator mill, sieving, microprecipitation, spray-drying, lyophilisation or controlled crystallisation. Typically, particles will be about 10 microns or less in diameter. Particles for use in the compositions of the invention may have a diameter of from about 0.1 microns to about 10 microns, from about 0.1 microns to about 9 microns, from about 0.1 microns to about 8 microns, from about 0.1 microns to about 7 microns, from about 0.1 microns to about 6 microns, from about 0.1 microns to about 5 microns, from about 0.1 microns to about 4 microns, from about 0.1 microns to about 3 microns, from about 0.1 microns to about 2 microns, from about 0.1 microns to about 1 micron, from about 0.1 microns to about 0.5 microns, from about 1 micron to about 10 microns, from about 1 micron to about 9 microns, from about 1 micron to about 8 microns, from about 1 micron to about 7 microns, from about 1 micron to about 6 microns, from about 1 micron to about 5 microns, from about 1 micron to about 4 microns, from about 1 micron to about 3 microns, from about 1 micron to about 2 microns, from about 2 microns to about 10 microns, from about 2 microns to about 9 microns, from about 2 microns to about 8 microns, from about 2 microns to about 7 microns, from about 2 microns to about 6 microns, from about 2 microns to about 5 microns, from about 2 microns to about 4 microns, or from about 2 microns to about 3 microns. In some embodiments, particles for use in the invention may be about 1 micron, about 2 microns, about 3 microns, about 4 microns, about 5 microns, about 6 microns, about 7 microns, about 8 microns, about 9 microns, or about 10 microns in diameter.

[0084] In one embodiment, the dosage forms are in the form of a solution aerosol (i.e., comprise droplets). Typically, droplets will be about 10 microns or less in diameter. Droplets for use in the compositions of the invention may have a diameter of from about 0.1 microns to about 10 microns, from about 0.1 microns to about 9 microns, from about 0.1 microns to about 8 microns, from about 0.1 microns to about 7 microns, from about 0.1 microns to about 6 microns, from about 0.1 microns to about 5 microns, from about 0.1 microns to about 4 microns, from about 0.1 microns to about 3 microns, from about 0.1 microns to about 2 microns, from about 0.1 microns to about 1 micron, from about 0.1 microns to about 0.5 microns, from about 1 micron to about 10 microns, from about 1 micron to about 9 microns, from about 1 micron to about 8 microns, from about 1 micron to about 7 microns, from about 1 micron to about 6 microns, from about 1 micron to about 5 microns, from about 1 micron to about 4 microns, from about 1 micron to about 3 microns, from about 1 micron to about 2 microns, from about 2 microns to about 10 microns, from about 2 microns to about 9 microns, from about 2 microns to about 8 microns, from about 2 microns to about 7 microns, from about 2 microns to about 6 microns, from about 2 microns to about 5 microns, from about 2 microns to about 4 microns, or from about 2 microns to about 3 microns. In some embodiments, particles and/or droplets for use in the invention may be about 1 micron, about 2 microns, about 3 microns, about 4 microns, about 5 microns, about 6 microns, about 7 microns, about 8 microns, about 9 microns, or about 10 microns in diameter.

[0085] The compositions of the invention may be formulated for enteric delivery, for example, may comprise one or more coatings that may include, for example, a delayed-release coating containing one or more enteric agents. A delayed-release coating is typically substantially stable in gastric fluid and substantially unstable (e.g., dissolves rapidly or is physically unstable) in intestinal fluid, thus providing for substantial release of the peptide tight junction antagonist from the composition in the duodenum or the jejunum.

[0086] The terms stable in gastric fluid or stable in acidic environments refers to a composition that releases 30% or less by weight of the total peptide tight junction antagonist in the composition in gastric fluid with a pH of 5 or less, or simulated gastric fluid with a pH of 5 or less, in approximately sixty minutes. Examples of simulated gastric fluid and simulated intestinal fluid include, but are not limited to, those disclosed in the 2005 Pharmacopeia 23NF/28USP in Test Solutions at page 2858 and/or other simulated gastric fluids and simulated intestinal fluids known to those of skill in the art, for example, simulated gastric fluid and/or intestinal fluid prepared without enzymes.

[0087] Compositions of the of the invention may release from about 0% to about 30%, from about 0% to about 25%, from about 0% to about 20%, from about 0% to about 15%, from about 0% to about 10%, from about 0% to about 5%, from about 5% to about 30%, from about 5% to about 25%, from about 5% to about 20%, from about 5% to about 15%, or from about 5% to about 10% by weight of the total peptide tight junction antagonist in the composition in gastric fluid with a pH of 5 or less or simulated gastric fluid with a pH of 5 or less, in approximately sixty minutes. Compositions of the invention may release about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% by weight of the total peptide tight junction antagonist in the composition in gastric fluid with a pH of 5 or less, or simulated gastric fluid with a pH of 5 or less, in approximately sixty minutes.

[0088] The term unstable in intestinal fluid refers to a composition that releases 70% or more by weight of the total peptide tight junction antagonist in the composition in intestinal fluid or simulated intestinal fluid in approximately sixty minutes. The term unstable in near neutral to alkaline environments refers to a composition that releases 70% or more by weight of the total amount of tight junction antagonist in the composition in intestinal fluid with a pH of 5 or greater, or simulated intestinal fluid with a pH of 5 or greater, in approximately ninety minutes. For example, a composition that is unstable in near neutral or alkaline environments may release 70% or more by weight of the total peptide tight junction antagonist in the composition in a fluid having a pH greater than about 5 (e.g., a fluid having a pH of from about 5 to about 14, from about 6 to about 14, from about 7 to about 14, from about 8 to about 14, from about 9 to about 14, from about 10 to about 14, or from about 11 to about 14) in from about 5 minutes to about 90 minutes, or from about 10 minutes to about 90 minutes, or from about 15 minutes to about 90 minutes, or from about 20 minutes to about 90 minutes, or from about 25 minutes to about 90 minutes, or from about 30 minutes to about 90 minutes, or from about 5 minutes to about 60 minutes, or from about 10 minutes to about 60 minutes, or from about 15 minutes to about 60 minutes, or from about 20 minutes to about 60 minutes, or from about 25 minutes to about 60 minutes, or from about 30 minutes to about 60 minutes.

[0089] In addition to one or more peptide tight junction antagonists, compositions of the invention may further comprise one or more additional therapeutic agents. Additional therapeutic agents include, but are not limited to, steroids and other anti-inflammatory compounds. Suitable additional therapeutic agents may include one or more of aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapies. Examples of suitable therapeutic agents that may be included in the compositions of the invention to treat IBD (e.g., Crohn's disease and/or ulcerative colitis) include, but are not limited to: [0090] 5-ASA agents (e.g., Sulfasalazine), Azulfidine, Asacol, Dipentum, Pentasa, and Rowasa; [0091] Antibiotics, for example, metronidazole (Flagyl) and ciprofloxacin (Cipro), although there are many others that may be effective in certain individuals; [0092] Steroids, e.g., corticosteroids. Suitable steroids include, but are not limited to, prednisone, hydrocortisone, Medrol, and budesonide multiple-release capsule MRC (EntocortREC). [0093] 6-mercaptopurine (6-MP, Purinethol) and azathioprine (Imuran); and antibodies against inflammatory cytokines, e.g., Infliximab (Remicade).

[0094] Compositions of the invention may also comprise one or more pharmaceutically acceptable excipients. Suitable excipients include, but are not limited to, buffers, buffer salts, bulking agents, salts, surface active agents, acids, bases, sugars, binders, and the like.

[0095] Methods of Use

[0096] The compositions of the invention can be used for preventing, slowing the onset of, ameliorating and/or treating any disease associated with an excessive or undesirable permeability of tissues containing tight junctions. Specific examples of diseases of this type include, but are not limited to, Celiac Disease, Inflammatory Bowel Disease, Crohn's disease, ulcerative colitis, Irritable Bowel Syndrome, Type 1 Diabetes, Asthma, Acute Respiratory Distress Syndrome, Acute Lung Injury, and Chronic Obstructive Pulmonary Disease. In one embodiment, the present invention provides a method of treating Celiac disease comprising administering to a subject in need thereof a composition comprising a peptide tight junction antagonist. In one embodiment, the present invention provides a method of treating Crohn's disease comprising administering to a subject in need thereof a composition comprising a peptide tight junction antagonist. In one embodiment, the present invention provides a method of treating ulcerative colitis comprising administering to a subject in need thereof a composition comprising a peptide tight junction antagonist. In one embodiment, the present invention provides a method of treating irritable bowel syndrome comprising administering to a subject in need thereof a composition comprising a peptide tight junction antagonist. In one embodiment, the present invention provides a method of treating type 1 diabetes comprising administering to a subject in need thereof a composition comprising a peptide tight junction antagonist. In one embodiment, the present invention provides a method of treating asthma comprising administering to a subject in need thereof a composition comprising a peptide tight junction antagonist. In one embodiment, the present invention provides a method of treating acute respiratory distress syndrome comprising administering to a subject in need thereof a composition comprising a peptide tight junction antagonist. In one embodiment, the present invention provides a method of treating acute lung injury comprising administering to a subject in need thereof a composition comprising a peptide tight junction antagonist. In one embodiment, the present invention provides a method of treating chronic obstructive pulmonary disease comprising administering to a subject in need thereof a composition comprising a peptide tight junction antagonist.

[0097] In some embodiments, compositions of the invention may be administered repeatedly over a protracted period, i.e., may be chronically administered. Typically, compositions may be administered one or more times each day in an amount suitable to prevent an attack of, reduce the likelihood of an attack of, or reduce the severity of an attack of the underlying disease condition (e.g., Celiac disease, IBD etc.). Such compositions may be administered chronically, for example, one or more times daily over a plurality of days.

[0098] In some embodiments, compositions of the invention may be used to treat acute attacks of the underlying disease (e.g., Celiac disease, IBD (e.g., Crohn's disease and/or ulcerative colitis)). Typically, embodiments of this type will require administration of the compositions of the invention to a subject undergoing an attack in an amount suitable to reduce the severity of the attack. One or more administrations may be used.

[0099] The following examples are provided for illustrative purposes only, and are in no way intended to limit the scope of the present invention.

EXAMPLES

Example 1

[0100] Measurement of the Inhibition of the Decrease in Trans Epithelial Electric Resistance (TEER) and Epithelial Flux of a Fluorescent Marker Lucifer Yellow

[0101] CaCo2 cells form monolayers that exhibit tight junctions between adjacent cells. Treatment of CaCo2 monolayers with peptide FCIGRL (SEQ ID NO: 70) enhanced 51-fold Lucifer Yellow permeability through CaCo2 monolayers compared to vehicle alone. Peptide FCIGRL decreased TEER 16-fold in CaCo2 monolayers compared to vehicle alone. Antagonists of tight junctions can be identified by their ability to prevent or decrease the enhancement of the flux of compounds (e.g. Lucifer Yellow) through the monolayer induced by agonists of tight junctions (e.g., SEQ ID NO: 70). Antagonists of tight junctions can also be identified by their ability to prevent the decrease in TEER induced by agonists of tight junctions (e.g., SEQ ID NO: 70).

[0102] Tight junction antagonists can be identified using the following method:

[0103] Prepare Modified Hank's Balanced Salt Solution (MHBSS) by obtaining IL bottle of HBSS removing 10 ml of HBSS and replacing it with 10 ml HEPES buffer pH 7.0. Adjust pH to 7.40.1 using concentrated NaOH (10N).

[0104] Remove Caco-2 cells from incubator, grown on 12-well, 3.0 M, polycarbonate Transwell filters (Corning) and record passage number, date cells seeded and age in days.

[0105] Aspirate cell culture medium from both the apical (AP) and basolateral (BL) compartments, replacing with 0.5 ml and 1.5 ml of MHBSS, respectively. Incubate cells at 37 C. for 30 minutes.

[0106] Using the MilliCell.circle-solid.-ERS instrument (Millipore), measure and record the transepithelial electrical resistance (TEER) across each filter and record.

[0107] Prepare a stock solution of the Antagonist Pretreatment Solution by dissolving the appropriate amount of antagonist in MHBSS. Vortex or sonicate the solution until it is clear then adjust pH to 7.40.1 using 1N NaOH. Preincubate each filter for 30 min with these solutions at 37 C. and 50 RPM.

[0108] Prepare stock solutions of Antagonist Treatment Solution by dissolving appropriate amounts of antagonist and agonist (for example, a peptide agonist such as a peptide having the sequence FCIGRL (SEQ ID NO: 70)) in 7.5 mM Lucifer Yellow solution in MHBSS. Vortex or sonicate the solution until it is clear then adjust pH to 7.40.1 using 1N NaOH.

[0109] Aspirate Antagonist Pretreatment Solution from the apical compartment of each filter (n=3 per condition) and replace with 0.5 ml of control and test solutions.

[0110] Place all plates into incubator set at 37 C. (0.2), 50 RPM (5) for a total of 180 minutes.

[0111] At t=30, 60, 120 and 180 minutes, measure and record the transepithelial electrical resistance (TEER) across each filter using the MilliCell-ERS instrument.

[0112] At t=60, 120 and 180 minutes remove 100 l from each basolateral compartment and place it in a 96-well plate for Lucifer Yellow analysis, replace with 100 l of HBSS.

[0113] Make a Lucifer Yellow standard curve with the following dilutions (7500 M, 3750 M, 750 M, 375 M, 75 M, 37.5 M, 7.5 M, 3.75 M, 0.75 M) and pipette 100 L of each into a 96-well plate except for the first three standards mentioned above which require a 1:10 dilution prior to transferring to the 96-well plate.

[0114] At t=0 make 1:10 dilutions of each starting Antagonist Treatment Solution mentioned above and pipette 100 L of each into a 96-well plate.

[0115] At t=180 make 1:10 dilutions of Antagonist Treatment Solution from the apical compartment of each filter and pipette 100 L of each into the 96-well plate.

[0116] Harvest the remaining start solutions and what is left in each apical compartment into 1.5 ml vials. Freeze at 20 C. for future analysis.

[0117] Analyze each 96-well plate in a Tecan Spectra Fluor Plus using Magellan at 485 and 535 nm.

[0118] Materials:

[0119] Cells: Caco-2 cells passage 40-60 grown on Transwell plates for 21-28 days

[0120] Culture Medium: DMEM supplemented with 10% fetal bovine serum, 1% NEAA, 1% Penn/Strep

[0121] Buffers: Hank's Balanced Salt Solution (HBSS) without calcium and magnesium

[0122] Flasks: 10020 mm Tissue culture dish Falcon.

[0123] Plates: 12 well polycarbonate Transwell filters; 0.3 uM pore size

[0124] The results of these assays are provided in the following table. The first column of the table provides SEQ ID NO: of the peptide, the second column provides the sequence of the peptides tested, the third column provides the results of an assay of inhibition of the increase in Lucifer Yellow permeability induced by SEQ ID NO:70, and the fourth column provides the results of an assay of the inhibition of the reduction in TEER induced by SEQ ID NO: 70. SEQ ID NO: 70 is a 6-mer peptide tight junction agonist having the sequence FCIGRL. See US patent publication US 2005/0059593 A1. In the following tables + indicates attenuation of the effects of the tight junction agonist were observed and indicates no attenuation of the effects of the tight junction agonist were observed.

TABLE-US-00001 TABLE1 TightjunctionantagonismbySEQIDNOs:1-69 SEQ Prevented ID ReducedLY TEER NO: Sequence Permeability Reduction 1 Gly-Gly-Val-Leu-Val- + + Gln-Pro-Gly 2 Gly-Val-Leu-Val-Gln- Pro-Gly 3 Val-Leu-Val-Gln-Pro-Gly + + 4 Leu-Val-Gln-Pro-Gly + + 5 Val-Gln-Pro-Gly + + 6 Gln-Pro-Gly + + 7 Ala-Pro-Gly + + 8 Gln-Ala-Gly + + 9 Gln-Pro-Ala + + 10 (d)Gln-Pro-Gly + + 11 Gln-(d)Pro-Gly + + 12 (d)G1n-(d)Pro-Gly 13 Gly-Pro-Gln + + 14 Gly-(d)Pro-Gln 15 Gly-Pro-(d)Gln 16 Gly-(d)Pro-(d)Gln 17 Ala-Pro-Gly + + 18 His-Pro-Gly + + 19 Asp-Pro-Gly 20 Arg-Pro-Gly + + 21 Phc-Pro-Gly + + 22 Gly-Pro-Gly + + 23 Glu-Pro-Gly + + 24 Lys-Pro-Gly + + 25 Leu-Pro-Gly + + 26 Met-Pro-Gly + + 27 Asn-Pro-Gly + + 28 Ser-Pro-Gly + + 29 Tyr-Pro-Gly + + 30 Thr-Pro-Gly + 31 Ile-Pro-Gly + + 32 Trp-Pro-Gly + + 33 Pro-Pro-Gly 34 Val-Pro-Gly + 35 Glp-Pro-Gly + + 36 Glp-Val-Gly 37 Glp-Gln-Gly 38 Glp-Ser-Gly 39 Glp-Lys-Gly 40 Glp-Phe-Gly 41 Glp-Glu-Gly 42 Glp-Thr-Gly 43 Glp-Ile-Gly 44 Glp-Tyr-Gly 45 Glp-His-Gly 46 Glp-Asn-Gly 47 Glp-Arg-Gly 48 Glp-Gly-Gly 49 Glp-Trp-Gly 50 Glp-Asp-Gly 51 Glp-Met-Gly 52 Glp-Leu-Gly 53 Glp-Pro-Gln 54 Glp-Pro-Asn + 55 Glp-Pro-Gln 56 Glp-Pro-Ser 57 Glp-Pro-Pro + 58 Glp-Pro-Trp 59 Glp-Pro-Asp 60 Glp-Pro-His 61 Glp-Pro-Leu 62 Glp-Pro-Arg 63 Glp-Pro-Val 64 Glp-Pro-Lys 65 Glp-Pro-Glu 66 Glp-Pro-Phe 67 Glp-Pro-Ile + 68 Glp-Pro-Met + 69 Glp-Pro-Tyr + Glp - Pyroglutamic acid

[0125] All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.