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United States Patent Application 20180133281
Kind Code A1
Tamiz; Amir May 17, 2018

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.


Inventors: Tamiz; Amir; (Silver Spring, MD)
Applicant:
Name City State Country Type

Alba Therapeutics Corporation

Baltimore

MD

US
Family ID: 1000003112971
Appl. No.: 15/860120
Filed: January 2, 2018


Related U.S. Patent Documents

Application NumberFiling DatePatent Number
15140850Apr 28, 2016
15860120
14305418Jun 16, 2014
15140850
12738815Dec 15, 20108785374
PCT/US08/80491Oct 20, 2008
14305418
60981399Oct 19, 2007

Current U.S. Class: 1/1
Current CPC Class: A61K 9/007 20130101; A61K 38/08 20130101; C07K 5/0819 20130101; C07K 5/0823 20130101; A61K 9/0053 20130101; C07K 5/0825 20130101; C07K 5/0806 20130101; C07K 5/0808 20130101; C07K 5/081 20130101; C07K 5/0812 20130101; C07K 5/0815 20130101; C07K 7/06 20130101; C07K 5/10 20130101; C07K 5/08 20130101; A61K 45/06 20130101; A61K 38/07 20130101; A61K 38/06 20130101; C07K 5/0817 20130101
International Class: A61K 38/06 20060101 A61K038/06; A61K 9/00 20060101 A61K009/00; A61K 38/08 20060101 A61K038/08; C07K 5/093 20060101 C07K005/093; C07K 5/097 20060101 C07K005/097; C07K 5/083 20060101 C07K005/083; C07K 5/087 20060101 C07K005/087; C07K 5/09 20060101 C07K005/09; C07K 7/06 20060101 C07K007/06; C07K 5/10 20060101 C07K005/10; C07K 5/08 20060101 C07K005/08; A61K 45/06 20060101 A61K045/06; A61K 38/07 20060101 A61K038/07

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



BACKGROUND

[0001] The tight junctions (tj) or zonula occludens (ZO) are one of the hallmarks of absorptive and secretory epithelia (Madara, J. Clin. Invest., 83:1089-1094 (1989); and Madara, Textbook of Secretory Diarrhea Eds. Lebenthal et al, Chapter 11, pages 125-138 (1990)). Tight junctions act as a barrier between apical and basolateral compartments, selectively regulating the passive diffusion of ions and water-soluble solutes through the paracellular (between cells) pathway (Gumbiner, Am. J. Physiol., 253 (Cell Physiol. 22):C749-C758 (1987)). This barrier maintains any gradient generated by the activity of pathways associated with the transcellular route (Diamond, Physiologist, 20:10-18 (1977)).

[0002] Variations in transepithelial conductance can usually be attributed to changes in the permeability of the paracellular pathway, since the resistances of enterocyte plasma membranes are relatively high (Madara, supra). The ZO represents the major barrier in this paracellular pathway, and the electrical resistance of epithelial tissues seems to depend on the number of transmembrane protein strands, and their complexity in the ZO, as observed by freeze-fracture electron microscopy (Madara et al, J. Cell Biol., 101:2124-2133 (1985)).

[0003] Tight Junction Dysfunctions

[0004] Tight junction dysfunction occurs in a variety of clinical conditions, including food allergies, infections of the gastrointestinal tract, autoimmune diseases, Celiac disease and inflammatory bowel diseases (Fasano, A., Pathological and therapeutical implications of macromolecule passage through the tight junction. In Tight Junctions, CRC Press, Inc., Boca Raton, Fla. 697-722 (2001)). Healthy, mature gut mucosa with its intact tight junction serves as the main barrier to the passage of macromolecules. During the healthy state, small quantities of immunologically active antigens cross the gut host barrier. These antigens are absorbed across the mucosa through at least two pathways. Up to 90% of the absorbed proteins cross the intestinal barrier via the transcellular pathway, followed by lysosomal degradation that converts proteins into smaller, non-immunogenic peptides. These residual peptides are transported as intact proteins through the paracellular pathway, which mediates a subtle, but sophisticated, regulation of intercellular tight junction that leads to antigen tolerance.

[0005] When the integrity of the tight junction system is compromised, in premature infants or after exposure to radiation, chemotherapy, or toxins, a deleterious immune response to environmental antigens, resulting in autoimmune diseases and food allergies, may be elicited. In normal bowels, the immune reaction is regulated to maintain homeostasis of the gut.

[0006] Celiac disease (CD) is a chronic autoimmune disease that is HLA-DQ2/DQ8 haplotype restricted. Gluten, the major protein fraction of wheat, and related proteins in rye and barley are the triggering agents of the disease. Ingested gluten or its derivative fractions (gliadin and subunits) elicit a harmful T cell-mediated immune response after crossing the small bowel epithelial barrier, undergoing deamidation by tissue transglutaminase (tTG) and engaging class II MHC molecules. When the integrity of the tight junction system is compromised, as in CD, a paracellular leak ("leaky gut") and an inappropriate immune response to environmental antigens (i.e., gluten) may develop.

[0007] Inflammatory bowel disease (IBD) is a phrase used to describe an inappropriate immune response that occurs in the bowels of affected individuals. Two major types of IBD have been described: Crohn's disease and ulcerative colitis (UC). Both forms of IBD show abnormal profiles of T cell-mediated immunity. In the gut of a person with Crohn's disease a strong Th1 reaction is induced; the Th2 response is upregulated in the colon of a UC sufferer.

[0008] The barrier function of the intestines is impaired in IBD. For example, Crohn's disease is associated with increased permeability of the intestinal barrier even in quiescent patients (Oshitani, et al., Int. J. Mol. Med. 15(3):407-10 (2005)). A TNF-.alpha.-induced increase in intestinal epithelial tj permeability has been proposed to be an important proinflammatory mechanism contributing to intestinal inflammation in Crohn's disease and other inflammatory conditions (see Ye, et al., Am. J. Physiol.-Gastro. and Liver Physiol., 290(3):496-504 (2006)). Increased intestinal permeability during episodes of active disease correlates with destruction or rearrangement of the tight junction protein complex (Willemsen, et al., Clin. Exp. Immunol. 142(2): 275-284 (2005)).

[0009] Acute Respiratory Distress Syndrome (ARDS) presents in about 150,000 individuals in the US annually, with a mortality rate of 30-50%. ARDS occurs in response to diverse forms of severe injury, in which lung edema results in respiratory failure. The current standard of care for ARDS is limited to the management of the disease through supportive mechanical ventilation. The loss of endothelial barrier integrity is central to the pulmonary edema that occurs in ARDS.

[0010] Triggering causes for ALI (Acute Lung Injury) including ARDS can, for example, be diffuse pulmonary infections (e.g. due to viruses, bacteria, fungi), aspiration of liquids (e.g. gastric juice or water), inhalation of toxins or irritants (e.g. chlorine gas, nitrogen oxides, smoke), direct or indirect trauma (e.g. multiple fractures or pulmonary contusion), systemic reactions to inflammations outside the lung (e.g. hemorrhagic pancreatitis, gram-negative septicemia), transfusions of high blood volumes or alternatively after cardiopulmonary bypass.

[0011] The pulmonary vascular endothelium lines the intravascular space and presents a selective barrier that actively regulates paracellular movement of circulating fluid, macromolecules, and cells, into extravascular tissues and compartments. Loss of this endothelial barrier integrity is the central defect found in acute lung injury (ALI) and ARDS. The host response to a wide range of injurious stimuli includes the biosynthesis and release of endogenous mediators, some of which can open the paracellular pathway in lung microvascular endothelia. Several of these mediators have been identified, including tumor necrosis factor .alpha., interleukin-1, thrombospondin-1, and SPARC/osteonectin, and established as factors that disrupt endothelial barrier integrity. In recent preliminary studies, we found that .DELTA.G, the active domain of zonula occludens toxin (ZOT) of Vibrio cholerae, increases paracellular permeability across human lung microvascular endothelial cells (HMVEC-Ls).

[0012] Disruption of lung tight junction function has been implicated in the development of allergic sensitization and asthma. In order for an allergen to reach antigen-presenting cells and induce an immune response, such as in asthma, the allergen must cross the lung epithelium. It has been shown that dust mite allergen Der p 1 causes disruption of lung tight junction structure and an increase in the permeability of lung epithelia. Wan, et al. J. Clinical Investigation 104(1): 123-133 (1999). It was suggested that the transepithelial movement of Der p1 may have been facilitated by the inherent proteolytic activity of Der p 1.

[0013] Antagonism and/or inhibition of intestinal tight junction function has been demonstrated to slow, delay and/or reverse the progression toward diabetes. (see U.S. Pat. No. 7,026,294 and US Application Publication No. 20060287233). Thus, the compositions and methods of the present invention are believed to be useful to prevent and or delay the onset of diabetes, and to mitigate the long-term complications of diabetes. Further, the permeability changes associated with autoimmune diseases are long standing, and early intervention using the compositions and methods of the present invention is believed to have untold benefits to the diabetic patient.

[0014] Zonula occludens toxin (ZOT), which is produced by Vibrio cholerae, has been characterized by Fasano et al., (Proc. Natl. Acad. Sci., USA, 8:5242-5246 (1991)) and the sequence has been determined (GenBank accession no. A43864). ZOT increases the intestinal permeability of rabbit ileal mucosa by modulating the structure of intercellular tight junctions. Mammalian proteins that are immunologically and functionally related to ZOT have been identified. Sec U.S. Pat. No. 5,945,510. These proteins, referred to as "zonulin," function as the physiological effector of mammalian tight junctions. These proteins are useful for enhancing absorption of therapeutic agents across tight junctions of intestinal and nasal mucosa, as well as across tight junctions of the blood brain barrier.

[0015] ZOT and .DELTA.G have been previously identified as tight junction agonists, i.e., compounds that mediate or facilitate or augment the physiological, transient opening of tight junctions that form a barrier between adjacent epithelial cells. The ability of ZOT and .DELTA.G to open tight junctions has been used to facilitate the transfer of macromolecule across epithelial barriers (see U.S. Pat. No. 5,665,389 and Salama et al. J. Pharmacology and Experimental Therapeutics 312(1):199-205, 2005). ZOT has been shown to act as a tight junction agonist that allows opening of tight junctions between adjacent mucosal epithelial cells. Compounds that antagonize the opening of tight junctions have been identified (see U.S. Pat. Nos. 6,458,925, 6,670,448, 6,936,689 and 7,189,696). One such antagonist, AT-1001, is currently in Phase II clinical trials for the treatment of celiac disease where it protects against loss of gut mucosal barrier function.

[0016] Peptide antagonists of tight junction opening were described in U.S. Pat. No. 6,458,925 (corresponds to WO 00/07609), which is incorporated by reference herein in its entirety. Peptide antagonists of tight junction opening may bind to the receptor utilized by the zonula occludens toxin expressed by Vibrio cholerae, and not function to physiologically modulate the opening of mammalian tight junctions. The peptide antagonists may competitively inhibit the binding of ZOT and/or zonulin to the ZOT receptor, thereby inhibiting the ability of ZOT and/or zonulin to physiologically modulate the opening of mammalian tight junctions.

[0017] There remains a need in the art for compositions and methods to treat such diseases characterized by excessive or undesirable permeability of tissues containing tight junctions. This need and others are met by the present invention.

SUMMARY OF THE INVENTION

[0018] The present invention provides antagonists of tight junctions. Tight junction antagonists of the invention may be peptide tight junction antagonists. Peptide tight junction antagonists of the invention may be of any length. In some embodiments, peptide tight junction antagonists according to the invention may be three amino acids in length. In some embodiments, peptide tight junction antagonists according to the invention may be four amino acids in length. In some embodiments, peptide tight junction antagonists according to the invention may be five amino acids in length. In some embodiments, peptide tight junction antagonists according to the invention may be six amino acids in length. In some embodiments, peptide tight junction antagonists according to the invention may be seven amino acids in length. In some embodiments, a peptide tight junction antagonist of the invention may comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-69. In some embodiments, a peptide tight junction antagonist of the invention may comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of or 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.

[0019] The present invention also provides compositions, e.g., pharmaceutical compositions, comprising one or more peptide tight junction antagonists of the invention. Compositions of the invention may comprise one or more peptide tight junction antagonists. Peptide tight junction antagonists for use in compositions of the invention may be of any length. In some embodiments, such peptide antagonists of tight junctions are three amino acids in length. Suitable peptide tight junction antagonists for use in the compositions of the invention include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-69. In some embodiments, peptide tight junction antagonists for use in the compositions of the invention include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or 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. Compositions of the invention may further comprise one or more additional therapeutic agents. Suitable additional therapeutic agents include, but are not limited to, aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics. Compositions of the invention may comprise one or more pharmaceutically acceptable excipients, for example, buffers, buffer salts, bulking agents, salts, surface active agents, acids, bases, sugars, binders and the like.

[0020] Compositions of the invention may be formulated for any type of delivery. For example, compositions of the invention may be formulated for intestinal delivery, e.g., may be delayed release compositions. Compositions of the invention may be formulated for pulmonary delivery.

[0021] In some embodiments, the invention provides methods 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 of the invention as described above. As used herein, a "subject" may be any mammal, for example, a human, dog, cat, horse, cow, etc. In some embodiments, a subject may be a human. In other embodiments, a subject may be a dog.

[0022] Compositions for treating an excessive or undesirable permeability of a tissue containing tight junctions may comprise one or more peptide tight junction antagonists as described above. Peptide tight junction antagonists for use in compositions for treating an excessive or undesirable permeability of a tissue containing tight junctions may be of any length. In some embodiments, such peptide tight junction antagonists of are three amino acids in length.

[0023] Suitable peptide tight junction antagonists for use in compositions for treating an excessive or undesirable permeability of a tissue containing tight junctions include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-69. In some embodiments, peptide tight junction antagonists for use in compositions for treating an excessive or undesirable permeability of a tissue containing tight junctions include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or 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. Compositions for treating an excessive or undesirable permeability of a tissue containing tight junctions may further comprise one or more additional therapeutic agents. Suitable additional therapeutic agents include, but are not limited to, aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics. Compositions for use in treating an excessive or undesirable permeability of a tissue containing tight junctions may comprise one or more pharmaceutically acceptable excipients, for example, buffers, buffer salts, bulking agents, salts, surface active agents, acids, bases, sugars, binders and the like.

[0024] Compositions for treating an excessive or undesirable permeability of a tissue containing tight junctions may be formulated for any type of delivery. For example, compositions for treating an excessive or undesirable permeability of a tissue containing tight junctions may be formulated for intestinal delivery, e.g., may be delayed release compositions. Compositions for treating an excessive or undesirable permeability of a tissue containing tight junctions may be formulated for pulmonary delivery.

[0025] In some embodiments, the invention provides methods of treating Celiac disease comprising administering to a subject in need thereof a composition comprising a peptide tight junction antagonist of the invention as described above. Compositions for treating Celiac disease may comprise one or more peptide tight junction antagonists as described above. Peptide tight junction antagonists for use in compositions for treating Celiac disease may be of any length. In some embodiments, such peptide tight junction antagonists are three amino acids in length.

[0026] Suitable peptides for use in the compositions for treating Celiac disease include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-69. In some embodiments, peptide tight junction antagonists for use in compositions for treating Celiac disease include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or 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. Compositions for use in treating Celiac disease may comprise one or more pharmaceutically acceptable excipients, for example, buffers, buffer salts, bulking agents, salts, surface active agents, acids, bases, sugars, binders and the like.

[0027] Compositions for treating Celiac disease may be formulated for any type of delivery. For example, such compositions may be formulated for intestinal delivery, e.g., may be delayed release compositions.

[0028] In some embodiments, the invention provides methods of treating inflammatory bowel disease comprising administering to a subject in need thereof a composition comprising a peptide tight junction antagonist as described above. Compositions for treating inflammatory bowel disease may comprise one or more peptide tight junction antagonists as described above. Peptide tight junction antagonists for use in compositions for treating inflammatory bowel disease may be of any length. In some embodiments, such peptide tight junction antagonists are three amino acids in length.

[0029] Suitable peptides for use in the compositions for treating inflammatory bowel disease include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-69. In some embodiments, peptide tight junction antagonists for use in compositions for treating inflammatory bowel disease include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of; or consist of a peptide that comprises, consists essentially of, or 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.

[0030] Compositions for treating inflammatory bowel disease may further comprise one or more additional therapeutic agents. Suitable additional therapeutic agents include, but are not limited to, aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics. Compositions for use in treating inflammatory bowel disease may comprise one or more pharmaceutically acceptable excipients, for example, buffers, buffer salts, bulking agents, salts, surface active agents, acids, bases, sugars, binders and the like.

[0031] Compositions for treating inflammatory bowel disease may be formulated for any type of delivery. For example, such compositions may be formulated for intestinal delivery, e.g., may be delayed release compositions.

[0032] The present invention provides methods and materials for treating Crohn's disease. In some embodiments, the invention provides methods of treating Crohn's disease comprising administering to a subject in need thereof a composition comprising a peptide tight junction antagonist as described above.

[0033] Compositions for treating Crohn's disease may comprise one or more peptide tight junction antagonists as described above. Peptide tight junction antagonists for use in compositions for treating Crohn's disease may be of any length. In some embodiments, such peptide tight junction antagonists are three amino acids in length.

[0034] Suitable peptides for use in the compositions for treating Crohn's disease include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-69. In some embodiments, peptide tight junction antagonists for use in compositions for treating Crohn's disease include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or 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.

[0035] Compositions for treating Crohn's disease may further comprise one or more additional therapeutic agents. Suitable additional therapeutic agents include, but are not limited to, aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics. Compositions for use in treating Crohn's disease may comprise one or more pharmaceutically acceptable excipients, for example, buffers, buffer salts, bulking agents, salts, surface active agents, acids, bases, sugars, binders and the like.

[0036] Compositions for treating Crohn's disease may be formulated for any type of delivery. For example, such compositions may be formulated for intestinal delivery, e.g., may be delayed release compositions.

[0037] The present invention provides methods and materials for treating ulcerative colitis. In some embodiments, the invention provides methods of treating ulcerative colitis comprising administering to a subject in need thereof a composition comprising a peptide tight junction antagonist as described above.

[0038] Compositions for treating ulcerative colitis may comprise one or more peptide tight junction antagonists as described above. Peptide tight junction antagonists for use in compositions for treating ulcerative colitis may be of any length. In some embodiments, such peptide tight junction antagonists are three amino acids in length.

[0039] Suitable peptides for use in the compositions for treating ulcerative colitis include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-69. In some embodiments, peptide tight junction antagonists for use in compositions for treating ulcerative colitis include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or 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.

[0040] Compositions for treating ulcerative colitis may further comprise one or more therapeutic agents. Suitable therapeutic agents include, but are not limited to, aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics. Compositions for use in treating ulcerative colitis may comprise one or more pharmaceutically acceptable excipients, for example, buffers, buffer salts, bulking agents, salts, surface active agents, acids, bases, sugars, binders and the like.

[0041] Compositions for treating ulcerative colitis may be formulated for any type of delivery. For example, such compositions may be formulated for intestinal delivery, e.g., may be delayed release compositions.

[0042] The present invention provides methods and materials for treating acute respiratory distress syndrome. In some embodiments, the invention provides methods of treating acute respiratory distress syndrome comprising administering to a subject in need thereof a composition comprising a peptide tight junction antagonist as described above.

[0043] Compositions for treating acute respiratory distress syndrome may comprise one or more peptide tight junction antagonists as described above. Peptide tight junction antagonists for use in compositions for treating acute respiratory distress syndrome may be of any length. In some embodiments, such peptide tight junction antagonists are three amino acids in length.

[0044] Suitable peptides for use in the compositions for treating acute respiratory distress syndrome include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-69. In some embodiments, peptide tight junction antagonists for use in compositions for treating acute respiratory distress syndrome include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or 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.

[0045] Compositions for treating acute respiratory distress syndrome may further comprise one or more therapeutic agents. Suitable therapeutic agents include, but are not limited to, aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics. Compositions for use in treating acute respiratory distress syndrome may comprise one or more pharmaceutically acceptable excipients, for example, buffers, buffer salts, bulking agents, salts, surface active agents, acids, bases, sugars, binders and the like.

[0046] Compositions for treating acute respiratory distress syndrome may be formulated for any type of delivery. For example, such compositions may be formulated for pulmonary delivery, e.g., may be solution aerosol or powder aerosol compositions.

[0047] The present invention provides methods and materials for treating acute lung injury. In some embodiments, the invention provides methods of treating acute lung injury comprising administering to a subject in need thereof a composition comprising a peptide tight junction antagonist as described above.

[0048] Compositions for treating acute lung injury may comprise one or more peptide tight junction antagonists as described above. Peptide tight junction antagonists for use in compositions for treating ac acute lung injury may be of any length. In some embodiments, such peptide tight junction antagonists are three amino acids in length.

[0049] Suitable peptides for use in the compositions for treating acute lung injury include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-69. In some embodiments, peptide tight junction antagonists for use in compositions for treating acute lung injury include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or 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.

[0050] Compositions for treating acute lung injury may further comprise one or more therapeutic agents. Suitable therapeutic agents include, but are not limited to, aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics. Compositions for use in treating acute lung injury may comprise one or more pharmaceutically acceptable excipients, for example, buffers, buffer salts, bulking agents, salts, surface active agents, acids, bases, sugars, binders and the like.

[0051] Compositions for treating acute lung injury may be formulated for any type of delivery. For example, such compositions may be formulated for pulmonary delivery, e.g., may be solution aerosol or powder aerosol compositions.

[0052] The present invention provides methods and materials for treating chronic obstructive pulmonary disorder. Chronic obstructive pulmonary disorder may be emphysema or bronchitis. In some embodiments, the invention provides methods of treating chronic obstructive pulmonary disorder comprising administering to a subject in need thereof a composition comprising a peptide tight junction antagonist as described above.

[0053] Compositions for treating chronic obstructive pulmonary disorder may comprise one or more peptide tight junction antagonists as described above. Peptide tight junction antagonists for use in compositions for treating ac chronic obstructive pulmonary disorder may be of any length. In some embodiments, such peptide tight junction antagonists are three amino acids in length.

[0054] Suitable peptides for use in the compositions for treating chronic obstructive pulmonary disorder include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-69. In some embodiments, peptide tight junction antagonists for use in compositions for treating chronic obstructive pulmonary disorder include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or 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.

[0055] Compositions for treating chronic obstructive pulmonary disorder may further comprise one or more therapeutic agents. Suitable therapeutic agents include, but are not limited to, aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics. Compositions for use in treating chronic obstructive pulmonary disorder may comprise one or more pharmaceutically acceptable excipients, for example, buffers, buffer salts, bulking agents, salts, surface active agents, acids, bases, sugars, binders and the like.

[0056] Compositions for treating chronic obstructive pulmonary disorder may be formulated for any type of delivery. For example, such compositions may be formulated for pulmonary delivery, e.g., may be solution aerosol or powder aerosol compositions.

[0057] The present invention provides methods and materials for treating asthma. In some embodiments, the invention provides methods of treating asthma comprising administering to a subject in need thereof a composition comprising a peptide tight junction antagonist as described above.

[0058] Compositions for treating asthma may comprise one or more peptide tight junction antagonists as described above. Peptide tight junction antagonists for use in compositions for treating ac asthma may be of any length. In some embodiments, such peptide tight junction antagonists are three amino acids in length.

[0059] Suitable peptides for use in the compositions for treating asthma include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-69. In some embodiments, peptide tight junction antagonists for use in compositions for treating asthma include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or 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.

[0060] Compositions for treating asthma may further comprise one or more therapeutic agents. Suitable therapeutic agents include, but are not limited to, bronchodilators, anti-inflammatories, aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics. Compositions for use in treating asthma may comprise one or more pharmaceutically acceptable excipients, for example, buffers, buffer salts, bulking agents, salts, surface active agents, acids, bases, sugars, binders and the like.

[0061] Compositions for treating asthma may be formulated for any type of delivery. For example, such compositions may be formulated for pulmonary delivery, e.g., may be solution aerosol or powder aerosol compositions.

[0062] The present invention provides methods and materials for treating type 1 diabetes. In some embodiments, the invention provides methods of treating type 1 diabetes comprising administering to a subject in need thereof a composition comprising a peptide tight junction antagonist as described above.

[0063] Compositions for treating type 1 diabetes may comprise one or more peptide tight junction antagonists as described above. Peptide tight junction antagonists for use in compositions for treating ac type 1 diabetes may be of any length. In some embodiments, such peptide tight junction antagonists are three amino acids in length.

[0064] Suitable peptides for use in the compositions for treating type 1 diabetes include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-69. In some embodiments, peptide tight junction antagonists for use in compositions for treating type 1 diabetes include, but are not limited to, peptide tight junction antagonists that comprise, consist essentially of, or consist of a peptide that comprises, consists essentially of, or 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.

[0065] Compositions for treating type 1 diabetes may further comprise one or more therapeutic agents. Suitable therapeutic agents include, but are not limited to, insulin, insulin mimetics, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics. Compositions for use in treating type 1 diabetes may comprise one or more pharmaceutically acceptable excipients, for example, buffers, buffer salts, bulking agents, salts, surface active agents, acids, bases, sugars, binders and the like.

[0066] Compositions for treating type 1 diabetes may be formulated for any type of delivery. For example, such compositions may be formulated for intestinal delivery, e.g., may be delayed release compositions.

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 .mu.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.RTM., Asacol,.RTM. Dipentum,.RTM. Pentasa,.RTM. and Rowasa.RTM.; [0091] Antibiotics, for example, metronidazole (Flagyl.RTM.) and ciprofloxacin (Cipro.RTM.), 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.RTM., and budesonide multiple-release capsule MRC (EntocortREC.RTM.). [0093] 6-mercaptopurine (6-MP, Purinethol.RTM.) and azathioprine (Imuran.RTM.); and antibodies against inflammatory cytokines, e.g., Infliximab (Remicade.TM.).

[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 7.4.+-.0.1 using concentrated NaOH (10N).

[0104] Remove Caco-2 cells from incubator, grown on 12-well, 3.0 .mu.M, polycarbonate Transwell.RTM. 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.degree. 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 7.4.+-.0.1 using 1N NaOH. Preincubate each filter for 30 min with these solutions at 37.degree. 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 7.4.+-.0.1 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.degree. 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 .mu.l from each basolateral compartment and place it in a 96-well plate for Lucifer Yellow analysis, replace with 100 .mu.l of HBSS.

[0113] Make a Lucifer Yellow standard curve with the following dilutions (7500 .mu.M, 3750 .mu.M, 750 .mu.M, 375 .mu.M, 75 .mu.M, 37.5 .mu.M, 7.5 .mu.M, 3.75 .mu.M, 0.75 .mu.M) and pipette 100 .mu.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 .mu.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 .mu.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.degree. 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.RTM. 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: 100.times.20 mm Tissue culture dish Falcon.

[0123] Plates: 12 well polycarbonate Transwell.RTM. 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 TABLE 1 Tight junction antagonism by SEQ ID NOs: 1-69 SEQ Prevented ID Reduced LY 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.

Sequence CWU 1

1

7018PRTArtificial SequenceTight junction antagonist 1Gly Gly Val Leu Val Gln Pro Gly 1 5 27PRTArtificial SequenceTight junction antagonist 2Gly Val Leu Val Gln Pro Gly 1 5 36PRTArtificial SequenceTight junction antagonist 3Val Leu Val Gln Pro Gly 1 5 45PRTArtificial SequenceTight junction antagonist 4Leu Val Gln Pro Gly 1 5 54PRTArtificial SequenceTight junction antagonist 5Val Gln Pro Gly 1 63PRTArtificial SequenceTight junction antagonist 6Gln Pro Gly 1 73PRTArtificial SequenceTight junction antagonist 7Ala Pro Gly 1 83PRTArtificial SequenceTight junction antagonist 8Gln Ala Gly 1 93PRTArtificial SequenceTight junction antagonist 9Gln Pro Ala 1 103PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Gln may be D-Gln 10Gln Pro Gly 1 113PRTArtificial SequenceTight junction antagonistmisc_feature(2)..(2)Pro may be D-Pro 11Gln Pro Gly1 123PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Gln may be D-Glnmisc_feature(2)..(2)Pro may be D-Pro 12Gln Pro Gly1 133PRTArtificial SequenceTight junction antagonist 13Gly Pro Gln1 143PRTArtificial SequenceTight junction antagonistmisc_feature(2)..(2)Pro may be D-Pro 14Gly Pro Gln 1 153PRTArtificial SequenceTight junction antagonistmisc_feature(3)..(3)Gln may be D-Gln 15Gly Pro Gln1 163PRTArtificial SequenceTight junction antagonistmisc_feature(2)..(2)Pro may be D-Promisc_feature(3)..(3)Gln may be D-Gln 16Gly Pro Gln1 173PRTArtificial SequenceTight junction antagonist 17Ala Pro Gly1 183PRTArtificial SequenceTight junction antagonist 18His Pro Gly 1 193PRTArtificial SequenceTight junction antagonist 19Asp Pro Gly 1 203PRTArtificial SequenceTight junction antagonist 20Arg Pro Gly 1 213PRTArtificial SequenceTight junction antagonist 21Phe Pro Gly 1 223PRTArtificial SequenceTight junction antagonist 22Gly Pro Gly 1 233PRTArtificial SequenceTight junction antagonist 23Glu Pro Gly 1 243PRTArtificial SequenceTight junction antagonist 24Lys Pro Gly1 253PRTArtificial SequenceTight junction antagonist 25Leu Pro Gly1 263PRTArtificial SequenceTight junction antagonist 26Met Pro Gly 1 273PRTArtificial SequenceTight junction antagonist 27Asn Pro Gly 1 283PRTArtificial SequenceTight junction antagonist 28Ser Pro Gly 1 293PRTArtificial SequenceTight junction antagonist 29Tyr Pro Gly 1 303PRTArtificial SequenceTight junction antagonist 30Thr Pro Gly1 313PRTArtificial SequenceTight junction antagonist 31Ile Pro Gly 1 323PRTArtificial SequenceTight junction antagonist 32Trp Pro Gly1 333PRTArtificial SequenceTight junction antagonist 33Pro Pro Gly 1 343PRTArtificial SequenceTight junction antagonist 34Val Pro Gly 1 353PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 35Xaa Pro Gly 1 363PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 36Xaa Val Gly1 373PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 37Xaa Gln Gly1 383PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 38Xaa Ser Gly 1 393PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 39Xaa Lys Gly 1 403PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 40Xaa Phe Gly1 413PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 41Xaa Glu Gly1 423PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 42Xaa Thr Gly1 433PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 43Xaa Ile Gly1 443PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 44Xaa Tyr Gly 1 453PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 45Xaa His Gly 1 463PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 46Xaa Asn Gly1 473PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 47Xaa Arg Gly 1 483PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 48Xaa Gly Gly 1 493PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 49Xaa Trp Gly1 503PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 50Xaa Asp Gly1 513PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 51Xaa Met Gly1 523PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 52Xaa Leu Gly 1 533PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 53Xaa Pro Gln 1 543PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 54Xaa Pro Asn1 553PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 55Xaa Pro Gln 1 563PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 56Xaa Pro Ser 1 573PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 57Xaa Pro Pro 1 583PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 58Xaa Pro Trp1 593PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 59Xaa Pro Asp1 603PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 60Xaa Pro His 1 613PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 61Xaa Pro Leu1 623PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 62Xaa Pro Arg 1 633PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 63Xaa Pro Val 1 643PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 64Xaa Pro Lys1 653PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 65Xaa Pro Glu 1 663PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 66Xaa Pro Phe1 673PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 67Xaa Pro Ile 1 683PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 68Xaa Pro Met1 693PRTArtificial SequenceTight junction antagonistmisc_feature(1)..(1)Xaa may be pyroglutamic acid 69Xaa Pro Tyr1 706PRTArtificial SequenceTight junction antagonist 70Phe Cys Ile Gly Arg Leu1 5

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