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Patent Searching and Data


Title:
PHARMACEUTICAL CARRIERS AND COMPOSITIONS FOR TRANSDERMAL DRUG DELIVERY
Document Type and Number:
WIPO Patent Application WO/2002/011784
Kind Code:
A2
Abstract:
A pharmaceutical carrier for enhancing transdermal delivery of a therapeutic agent is disclosed. The pharmaceutical carrier comprises at least one skin permeation enhancing agent; and at least one at least one surface adhesion molecule modulating agent; the at least one skin permeation enhancing agent and the at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the therapeutic agent.

Inventors:
HERZBERG MAX (IL)
MESSIKA ERIC (IL)
GHOZI MICHAL (IL)
Application Number:
PCT/IL2001/000729
Publication Date:
February 14, 2002
Filing Date:
August 07, 2001
Export Citation:
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Assignee:
TRANSDERMICS LTD (IL)
HERZBERG MAX (IL)
MESSIKA ERIC (IL)
GHOZI MICHAL (IL)
International Classes:
A61K47/42; A61K47/08; A61K47/10; A61K47/12; A61K47/14; A61K47/18; A61K47/20; A61K47/22; A61K47/24; A61K47/28; (IPC1-7): A61M/
Foreign References:
US6019988A2000-02-01
Attorney, Agent or Firm:
G. E. EHRLICH (1995) LTD. (Ramat Gan, IL)
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Claims:
WHAT IS CLAIMED IS:
1. A pharmaceutical carrier for enhancing transdermal delivery of a therapeutic agent, the pharmaceutical carrier comprising: at least one skin permeation enhancing agent; and at least one surface adhesion molecule modulating agent; said at least one skin permeation enhancing agent and said at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the therapeutic agent.
2. The pharmaceutical carrier of claim 1, wherein said at least one skin permeation enhancing agent is selected from the group consisting of an alcohol, a fatty alcohol, a fatty acid ester, an alkyl ester, a polyol, an amid, a surfactant, a sulfoxide and a terpene and an alkanone.
3. The pharmaceutical carrier of claim 2, wherein : (i) said alcohol is selected from the group consisting of ethanol, propanol and nonanol; (ii) said fatty alcohol is lauryl alcohol; (iii) said fatty acid is selected from the group consisting of valeric acid, caproic acid and capric acid; (iv) said fatty acid ester is selected from the group consisting of isopropyl myristate and isopropyl nhexanoate; (v) said alkyl ester is selected from the group consisting of ethyl acetate and butyl acetate; (vi) said polyol is selected from the group consisting of propylene glycol, propanedione and hexanetriol ; (vii) said sulfoxide is selected from the group consisting of dimethylsulfoxide and decylmethylsulfoxide ; (viii) said amide is selected from the group consisting of urea, dimethylacetamide and pyrrolidone derivatives; (ix) said surfactant is selected from the group consisting of sodium lauryl sulfate, cetyltrimethylammonium bromide, polaxamers, spans, tweens, bile salts and lecithin ; (x) said terpene is selected from the group consisting of dlimonene, alphaterpeneol, 1,8cineole and menthone; and (xi) said alkanone is selected from the group consisting of Nheptane and Nnonane.
4. The pharmaceutical carrier of claim 1, wherein said at least one skin permeation enhancing agent includes at least one biodegradable skin permeation enhancer.
5. The pharmaceutical carrier of claim 4, wherein said at least one biodegradable skin permeation enhancer is selected from the group consisting of dodecylN, Ndimethylamino acetate and N, Ndimethylamino isopropionate.
6. The pharmaceutical carrier of claim 1, wherein said at least one surface adhesion molecule modulating agent is selected from the group consisting of a cadherin antagonist, a selectin antagonist, and an integerin antagonist.
7. The pharmaceutical carrier of claim 6, wherein said cadherin antagonist is a peptide which includes a HisAlaVal amino acid sequence.
8. The pharmaceutical carrier of claim 7, wherein said peptide is a cyclic peptide containing 415 amino acid residues.
9. The pharmaceutical carrier of claim 8, wherein said cyclic peptide is of a general formula: (Zi) (Yi) (Xi)HisAlaVal (X2) (Y2) (Z2) wherein, (i) Xi and X2, are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations of amino acid residues in which the residues are linked by peptide bonds, and wherein Xi and X2 independently range in size from 0 to 10 residues, such that the sum of residues contained within Xi and X2 ranges from 1 to 12 ; (ii) Y1 and Y2 are independently selected from the group consisting of amino acid residues, and wherein a covalent bond is formed between residues Y1 and Y2 ; and (iii) Zl and Z2 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations of amino acid residues in which the residues are linked by peptide bonds.
10. The pharmaceutical carrier of claim 9, wherein Z1 is not present and Yl comprises an Nacetyl group in said cyclic peptide.
11. The pharmaceutical carrier of claim 9, wherein Z2 is not present and Y2 comprises a Cterminal amide group in said cyclic peptide.
12. The pharmaceutical carrier of claim 9, wherein Y1 and Y2 in said cyclic peptide are covalently linked via a disulfide bond.
13. The pharmaceutical carrier of claim 12, wherein Y1 and Y2 are each independently selected from the group consisting of penicillamine, P, Ptetramethylene cysteine, P, ppentamethylene cysteine, Pmercaptopropionic acid, (3, ßpentamethyleneßmercaptopropionic acid, 2mercaptobenzene, 2mercaptoaniline, 2mercaptoproline and derivatives of cysteine residues containing side chain modifications.
14. The pharmaceutical carrier of claim 12, wherein Y1 and Y2, in said cyclic peptide, are cysteine residues or derivatives of cysteine residues containing side chain modifications.
15. The pharmaceutical carrier of claim 14, wherein said cyclic peptide comprises the sequence CysHisAlaValCys (SEQ ID NO : 4).
16. The pharmaceutical carrier of claim 15, wherein said cyclic peptide further comprising an Nacetyl group.
17. The pharmaceutical carrier of claim 15, wherein said cyclic peptide further comprising a Cterminal amide group.
18. The pharmaceutical carrier of claim 14, wherein said cyclic peptide comprises a sequence selected from the group consisting of CysAlaHisAlaValAspIleCys (SEQ ID NO : 5), CysSerHisAlaValCys (SEQ ID NO : 6), CysHisAlaValSerCys (SEQ ID NO : 7), CysAlaHisAlaValAspCys (SEQ ID NO : 8) and CysSerHisAlaValSerSerCys (SEQ ID NO : 9).
19. The pharmaceutical carrier of claim 9, wherein Y1 and Y2, in said cyclic peptide, are covalently linked via an amide bond.
20. The pharmaceutical carrier of claim 19, wherein said amide bond is formed between terminal functional groups.
21. The pharmaceutical carrier of claim 19, wherein said amide bond is formed between residue sidechains.
22. The pharmaceutical carrier of claim 19, wherein said amide bond is formed between one terminal functional group and one residue side chain.
23. The pharmaceutical carrier of claim 19, wherein (i) Y1 is selected from the group consisting of lysine, ornithine, and derivatives of lysine or ornithine containing side chain modifications and Y2 is selected from the group consisting of aspartate, glutamate and derivatives of aspartate or glutamate containing side chain modifications; or (ii) Y2 is selected from the group consisting of lysine, ornithine and derivative of lysine or ornithine containing side chain modifications and Y1 is selected from the group consisting of aspartate, glutamate and derivatives of aspartate or glutamate containing side chain modifications.
24. The pharmaceutical carrier of claim 19, wherein said cyclic peptide comprises the sequence LysHisAlaValAsp (SEQ ID NO : 10) or AlaHisAlaValAspIle (SEQ ID NO : 11).
25. The pharmaceutical carrier of claim 9, wherein Yl and Y2, in said cyclic peptide, are covalently linked via a thioether bond.
26. The pharmaceutical carrier of claim 9, wherein Y1 and Y2 in said cyclic peptide, are each tryptophan or a derivative of tryptophan containing side chain modifications, such that said covalent bond generates a 61 81ditryptophan, or a derivative of 81 81tryptophan containing side chain modifications.
27. A pharmaceutical composition comprising: a therapeutic effective amount of at least one at least one therapeutic agent; and a pharmaceutical carrier including: (i) at least one skin permeation enhancing agent; and (ii) at least one surface adhesion molecule modulating agent; said at least one skin permeation enhancing agent and said at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the at least one therapeutic agent.
28. The pharmaceutical composition of claim 27, wherein said at least one therapeutic agent is selected from the group consisiting of a drug, a nucleic acid construct, a vaccine, a hormon, an enzyme, an antibody and cells.
29. The pharmaceutical composition of claim 27, wherein said at least one skin permeation enhancing agent is selected from the group consisting of an alcohol, a fatty alcohol, a fatty acid ester, an alkyl ester, a polyol, an amid, a surfactant, a sulfoxide, a terpene and an alkanone.
30. The pharmaceutical composition of claim 29, wherein: (i) said alcohol is selected from the group consisting of ethanol, propanol and nonanol; (ii) said fatty alcohol is lauryl alcohol; (iii) said fatty acid is selected from the group consisting of valeric acid, caproic acid and capric acid ; (iv) said fatty acid ester is selected from the group consisting of isopropyl myristate and isopropyl nhexanoate; (v) said alkyl ester is selected from the group consisting of ethyl acetate and butyl acetate; (vi) said polyol is selected from the group consisting of propylene glycol, propanedione and hexanetriol ; (vii) said sulfoxide is selected from the group consisting of dimethylsulfoxide and decylmethylsulfoxide ; (viii) said amide is selected from the group consisting of urea, dimethylacetamide and pyrrolidone derivatives; (ix) said surfactant is selected from the group consisting of sodium lauryl sulfate, cetyltrimethylammonium bromide, polaxamers, spans, tweens, bile salts and lecithin; (x) said terpene is selected from the group consisting of dlimonene, alphaterpeneol, 1, 8cineole and menthone; and (xi) said alkanone is selected from the group consisting of Nheptane and Nnonane.
31. The pharmaceutical composition of claim 27, wherein said at least one skin permeation enhancing agent includes at least one biodegradable skin permeation enhancer.
32. The pharmaceutical composition of claim 31, wherein said at least one biodegradable skin permeation enhancer is selected from the group consisting of dodecylN, Ndimethylamino acetate and N, Ndimethylamino isopropionate.
33. The pharmaceutical composition of claim 27, wherein said at least one surface adhesion molecule modulating agent is selected from the group consisting of a cadherin antagonist, a selectin antagonist, and an integerin antagonist.
34. The pharmaceutical composition of claim 33, wherein said cadherin antagonist is a peptide which includes a HisAlaVal amino acid sequence.
35. The pharmaceutical composition of claim 34, wherein said peptide is a cyclic peptide containing 415 amino acid residues.
36. The pharmaceutical composition of claim 34, wherein said cyclic peptide is of a general formula : (Z1) (Y1) (X1)HisAlaVal(X2) (Y2) (Z2) wherein, (i) X1 and X2, are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations of amino acid residues in which the residues are linked by peptide bonds, and wherein Xi and X2 independently range in size from 0 to 10 residues, such that the sum of residues contained within Xi and X2 ranges from 1 to 12; (ii) Y1 and Y2 are independently selected from the group consisting of amino acid residues, and wherein a covalent bond is formed between residues Y1 and Y2; and (iii) Z1 and Z2 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations of amino acid residues in which the residues are linked by peptide bonds.
37. The pharmaceutical composition of claim 36, wherein Z I is not present and Y1 comprises an Nacetyl group in said cyclic peptide.
38. The pharmaceutical composition of claim 36, wherein Z2 is not present and Y2 comprises a Cterminal amide group in said cyclic peptide.
39. The pharmaceutical composition of claim 36, wherein Y1 and Y2 in said cyclic peptide are covalently linked via a disulfide bond.
40. The pharmaceutical composition of claim 39, wherein Y1 and Y2 are each independently selected from the group consisting of penicillamine, P, Ptetramethylene cysteine, p. ßpentamethylene cysteine, Pmercaptopropionic acid, P, ßpentamethyleneßmercaptopropionic acid, 2mercaptobenzene, 2mercaptoaniline, 2mercaptoproline and derivatives of cysteine residues containing side chain modifications.
41. The pharmaceutical composition of claim 39, wherein Y1 and Y2, in said cyclic peptide, are cysteine residues or derivatives of cysteine residues containing side chain modifications.
42. The pharmaceutical composition of claim 41, wherein said cyclic peptide comprises the sequence CysHisAlaValCys (SEQ ID NO : 4).
43. The pharmaceutical composition of claim 42, wherein said cyclic peptide further comprising an Nacetyl group.
44. The pharmaceutical composition of claim 42, wherein said cyclic peptide further comprising a Cterminal amide group.
45. The pharmaceutical composition of claim 41, wherein said cyclic peptide comprises a sequence selected from the group consisting of CysAlaHisAlaValAspIleCys (SEQ ID NO : 5), CysSerHisAlaValCys (SEQ ID NO : 6), CysHisAlaValSerCys (SEQ ID NO : 7), CysAlaHisAlaValAspCys (SEQ ID NO : 8) and CysSerHisAlaValSerSerCys (SEQ ID NO : 9).
46. The pharmaceutical composition of claim 36, wherein Y1 and Y2, in said cyclic peptide, are covalently linked via an amide bond.
47. The pharmaceutical composition of claim 46, wherein said amide bond is formed between terminal functional groups.
48. The pharmaceutical composition of claim 46, wherein said amide bond is formed between residue sidechains.
49. The pharmaceutical composition of claim 46, wherein said amide bond is formed between one terminal functional group and one residue side chain.
50. The pharmaceutical composition of claim 46, wherein (i) YI is selected from the group consisting of lysine, ornithine ; and derivatives of lysine or ornithine containing side chain modifications and Y2 is selected from the group consisting of aspartate, glutamate and derivatives of aspartate or glutamate containing side chain modifications ; or (ii) Y2 is selected from the group consisting of lysine, ornithine and derivative of lysine or ornithine containing side chain modifications and Y1 is selected from the group consisting of aspartate, glutamate and derivatives of aspartate or glutamate containing side chain modifications.
51. The pharmaceutical composition of claim 46, wherein said cyclic peptide comprises the sequence LysHisAlaValAsp (SEQ ID NO : 10) or AlaHisAlaValAspIle (SEQ ID NO : 11).
52. The pharmaceutical composition of claim 36, wherein Y1 and Y2, in said cyclic peptide, are covalently linked via a thioether bond.
53. The pharmaceutical composition of claim 36, wherein Yl and Y2 in said cyclic peptide, are each tryptophan or a derivative of tryptophan containing side chain modifications, such that said covalent bond generates a 81 §iditryptophan, or a derivative of 61 S 1tryptophan containing side chain modifications.
54. A method of transdermal delivery of at least one therapeutic agent, the method comprising the step of topically administering the at least one therapeutic agent in a presence of a pharmaceutical carrier including: at least one skin permeation enhancing agent; and at least one surface adhesion molecule modulating agent; said at least one skin permeation enhancing agent and said at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdennal delivery of the at least one therapeutic agent.
55. The method of claim 54, wherein said at least one skin permeation enhancing agent is selected from the group consisting of an alcohol, a fatty alcohol, a fatty acid ester, an alkyl ester, a polyol, an amid, a surfactant, a sulfoxide, a terpene and an alkanone.
56. The method of claim 55, wherein: (i) said alcohol is selected from the group consisting of ethanol, propanol and nonanol ; (ii) said fatty alcohol is lauryl alcohol; (iii) said fatty acid is selected from the group consisting of valeric acid, caproic acid and capric acid; (iv) said fatty acid ester is selected from the group consisting of isopropyl myristate and isopropyl nhexanoate; (v) said alkyl ester is selected from the group consisting of ethyl acetate and butyl acetate; (vi) said polyol is selected from the group consisting of propylene glycol, propanedione and hexanetriol ; (vii) said sulfoxide is selected from the group consisting of dimethylsulfoxide and decylmethylsulfoxide ; (viii) said amide is selected from the group consisting of urea, dimethylacetamide and pyrrolidone derivatives; (ix) said surfactant is selected from the group consisting of sodium lauryl sulfate, cetyltrimethylammonium bromide, polaxamers, spans, tweens, bile salts and lecithin; (x) said terpene is selected from the group consisting of dlimonene, alphaterpeneol, 1, 8cineol and menthone; and (xi) said alkanone is selected from the group consisting of Nheptane and Nnonane.
57. The method of claim 54, wherein said at least one skin permeation enhancing agent includes at least one biodegradable skin permeation enhancer.
58. The method of claim 57, wherein said at least one biodegradable skin permeation enhancer is selected from the group consisting of dodecylN, Ndimethylamino acetate and N, Ndimethylamino isopropionate.
59. The method of claim 54, wherein said at least one surface adhesion molecule modulating agent is selected from the group consisting of a cadherin antagonist, a selectin antagonist, and an integerin antagonist.
60. The method of claim 59, wherein said cadherin antagonist is a peptide which includes a HisAlaVal amino acid sequence.
61. The method of claim 60, wherein said peptide is a cyclic peptide containing 415 amino acid residues.
62. The method of claim 61, wherein said cyclic peptide is of a general formula: (Zi) (Yi) (Xi)HisAlaVal (X2) (Y2) (Z2) wherein, (i) Xi and X2, are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations of amino acid residues in which the residues are linked by peptide bonds, and wherein Xi and X2 independently range in size from 0 to 10 residues, such that the sum of residues contained within Xi and X2 ranges from 1 to 12; (ii) Yi and Y2 are independently selected from the group consisting of amino acid residues, and wherein a covalent bond is formed between residues Y1 and Y2 ; and (iii) Zl and Z2 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations of amino acid residues in which the residues are linked by peptide bonds.
63. The method of claim 62, wherein Zl is not present and Y comprises an Nacetyl group in said cyclic peptide.
64. The method of claim 62, wherein Z2 is not present and Y2 comprises a Cterminal amide group in said cyclic peptide.
65. The method of claim 62, wherein Y1 and Y2 in said cyclic peptide are covalently linked via a disulfide bond.
66. The method of claim 65, wherein Y1 and Y2 are each independently selected from the group consisting of penicillamine, ß, ßtetramethylene cysteine, ß, ppentamethylene cysteine, Pmercaptopropionic acid, ß, ßpentamethyleneßmercaptopropionic acid, 2mercaptobenzene, 2mercaptoaniline, 2mercaptoproline and derivatives of cysteine residues containing side chain modifications.
67. The method of claim 65, wherein Y1 and Y2, in said cyclic peptide, are cysteine residues or derivatives of cysteine residues containing side chain modifications.
68. The method of claim 67, wherein said cyclic peptide comprises the sequence CysHisAlaValCys (SEQ ID NO : 4).
69. The method of claim 68, wherein said cyclic peptide further comprising an Nacetyl group.
70. The method of claim 68, wherein said cyclic peptide further comprising a Cterminal amide group.
71. The method of claim 67, wherein said cyclic peptide comprises a sequence selected from the group consisting of CysAlaHisAlaValAspIleCys (SEQ ID NO : 5), CysSerHisAlaValCys (SEQ ID NO : 6), CysHisAlaValSerCys (SEQ ID NO : 7), CysAlaHisAlaValAspCys (SEQ ID NO : 8) and CysSerHisAlaValSerSerCys (SEQ ID NO : 9).
72. The method of claim 62,wherein Y1 and Y2, in said cyclic peptide, are covalently linked via an amide bond.
73. The method of claim 72, wherein said amide bond is formed between terminal functional groups.
74. The method of claim 72, wherein said amide bond is formed between residue sidechains.
75. The method of claim 72, wherein said amide bond is formed between one terminal functional group and one residue side chain.
76. The method of claim 72, wherein (i) Y1 is selected from the group consisting of lysine, ornithine, and derivatives of lysine or ornithine containing side chain modifications and Y2 is selected from the group consisting of aspartate, glutamate and derivatives of aspartate or glutamate containing side chain modifications ; or (ii) Y2 is selected from the group consisting of lysine, ornithine and derivative of lysine or ornithine containing side chain modifications and Y1 is selected from the group consisting of aspartate, glutamate and derivatives of aspartate or glutamate containing side chain modifications.
77. The method of claim 72, wherein said cyclic peptide comprises the sequence LysHisAlaValAsp (SEQ ID NO : 10) or AlaHisAlaValAspIle (SEQ ID NO : 11).
78. The method of claim 62, wherein Y1 and Y2, in said cyclic peptide, are covalently linked via a thioether bond.
79. The method of claim 62, wherein Y1 and Y2 in said cyclic peptide, are each tryptophan or a derivative of tryptophan containing side chain modifications, such that said covalent bond generates a 61 blditryptophan, or a derivative of 81 61tryptophan containing side chain modifications.
80. A device for transdermal application of at least one therapeutic agent, the device comprising a solid support having on a skincontacting side thereof a pharmaceutical carrier including: at least one skin permeation enhancing agent; and at least one surface adhesion molecule modulating agent; said at least one skin permeation enhancing agent and said at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the at least one therapeutic agent.
81. The device of claim 80, wherein said solid support is selected from the group consisting of a patch, a foil, a plaster and a film.
82. The device of claim 80, wherein said at least one skin permeation enhancing agent is selected from the group consisting of an alcohol, a fatty alcohol, a fatty acid ester, an alkyl ester, a polyol, an amid, a surfactant, a sulfoxide, a terpene and an alkanone.
83. The device of claim 82, wherein: (i) said alcohol is selected from the group consisting of ethanol, propanol and nonanol; (ii) said fatty alcohol is lauryl alcohol; (iii) said fatty acid is selected from the group consisting of valeric acid, caproic acid and capric acid; (iv) said fatty acid ester is selected from the group consisting of isopropyl myristate and isopropyl nhexanoate; (v) said alkyl ester is selected from the group consisting of ethyl acetate and butyl acetate; (vi) said polyol is selected from the group consisting of propylene glycol, propanedione and hexanetriol; (vii) said sulfoxide is selected from the group consisting of dimethylsulfoxide and decylmethylsulfoxide ; (viii) said amide is selected from the group consisting of urea, dimethylacetamide and pyrrolidone derivatives; (ix) said surfactant is selected from the group consisting of sodium lauryl sulfate, cetyltrimethylammonium bromide, polaxamers, spans, tweens, bile salts and lecithin; (x) said terpene is selected from the group consisting of dlimonene, alphaterpeneol, 1, 8cineole and menthone; and (xi) said alkanone is selected from the group consisting of Nheptane and Nnonane.
84. The device of claim 80, wherein said at least one skin permeation enhancing agent includes at least one biodegradable skin permeation enhancer.
85. The device of claim 84, wherein said at least one biodegradable skin permeation enhancer is selected from the group consisting of dodecylN, Ndimethylamino acetate and N, Ndimethylamino isopropionate.
86. The device of claim 82, wherein said at least one surface adhesion molecule modulating agent is selected from the group consisting of a cadherin antagonist, a selectin antagonist, and an integerin antagonist.
87. The device of claim 86, wherein said cadherin antagonist is a peptide which includes a HisAlaVal amino acid sequence.
88. The device of claim 87, wherein said peptide is a cyclic peptide containing 415 amino acid residues.
89. The device of claim 88, wherein said cyclic peptide is of a general formula: (xi) (Yi) (Xi)HisAlaVal (X2) (Y2) (Z2) wherein, (i) Xi and X2, are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations of amino acid residues in which the residues are linked by peptide bonds, and wherein Xi and X2 independently range in size from 0 to 10 residues, such that the sum of residues contained within X1 and X2 ranges from 1 to 12; (ii) Y1 and Y2 are independently selected from the group consisting of amino acid residues, and wherein a covalent bond is formed between residues Y1 and Y2; and (iii) Z1 and Z2 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations of amino acid residues in which the residues are linked by peptide bonds.
90. The device of claim 89, wherein Zl is not present and Y comprises an Nacetyl group in said cyclic peptide.
91. The device of claim 89, wherein Z2 is not present and Y2 comprises a Cterminal amide group in said cyclic peptide.
92. The device of claim 89, wherein Yl and Y2 in said cyclic peptide are covalently linked via a disulfide bond.
93. The device of claim 92, wherein Y1 and Y2 are each independently selected from the group consisting of penicillamine, P, Ptetramethylene cysteine, ß, ppentamethylene cysteine, Pmercaptopropionic acid, P, ßpentamethyleneßmercaptopropionic acid, 2mercaptobenzene, 2mercaptoaniline, 2mercaptoproline and derivatives of cysteine residues containing side chain modifications.
94. The device of claim 92, wherein Yi and Y2, in said cyclic peptide, are cysteine residues or derivatives of cysteine residues containing side chain modifications.
95. The device of claim 94, wherein said cyclic peptide comprises the sequence CysHisAlaValCys (SEQ ID NO : 4).
96. The device of claim 95, wherein said cyclic peptide further comprising an Nacetyl group.
97. The device of claim 95, wherein said cyclic peptide further comprising a Cterminal amide group.
98. The device of claim 94, wherein said cyclic peptide comprises a sequence selected from the group consisting of CysAlaHisAlaValAspIleCys (SEQ ID NO : 5), CysSerHisAlaValCys (SEQ ID NO : 6), CysHisAlaValSerCys (SEQ ID NO : 7), CysAlaHisAlaValAspCys (SEQ ID NO : 8) and CysSerHisAlaValSerSerCys (SEQ ID NO : 9).
99. The device of claim 89, ~wherein Y1 and Y2, in said cyclic peptide, are covalently linked via an amide bond.
100. The device of claim 99, wherein said amide bond is formed between terminal functional groups.
101. The device of claim 99, wherein said amide bond is formed between residue sidechains.
102. The device of claim 99, wherein said amide bond is formed between one terminal functional group and one residue side chain.
103. The device of claim 99, wherein (i) Y1 is selected from the group consisting of lysine, ornithine, and derivatives of lysine or ornithine containing side chain modifications and Y2 is selected from the group consisting of aspartate, glutamate and derivatives of aspartate or glutamate containing side chain modifications; or (ii) Y2 is selected from the group consisting of lysine, ornithine and derivative of lysine or ornithine containing side chain modifications and Y1 is selected from the group consisting of aspartate, glutamate and derivatives of aspartate or glutamate containing side chain modifications.
104. The device of claim 99, wherein said cyclic peptide comprises the sequence LysHisAlaValAsp (SEQ ID NO : 10) or AlaHisAlaValAspIle (SEQ ID NO : 11).
105. The device of claim 89, wherein Yl and Y2, in said cyclic peptide, are covalently linked via a thioether bond.
106. The device of claim 89, wherein Y1 and Y2 in said cyclic peptide, are each tryptophan or a derivative of tryptophan containing side chain modifications, such that said covalent bond generates a 6ditryptophan, or a derivative of 61 81tryptophan containing side chain modifications.
107. A device for transdermal application of at least one therapeutic agent, the device comprising a solid support having on skincontacting side thereof a pharmaceutical composition including: a therapeutic effective amount of at least one therapeutic agent; and a pharmaceutical carrier including: (i) at least one skin permeation enhancing agent; and (ii) at least one surface adhesion molecule modulating agent; said at least one skin permeation enhancing agent and said at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the at least one therapeutic agent.
108. The device of claim 107, wherein said solid support is selected from the group consisting of a patch, a foil, a plaster and a film.
109. The device of claim 107, wherein said at least one skin permeation enhancing agent is selected from the group consisting of an alcohol, a fatty alcohol, a fatty acid ester, an alkyl ester, a polyol, an amid, a surfactant, a sulfoxide, a terpene and an alkanone.
110. The device of claim 109, wherein: (i) said alcohol is selected from the group consisting of ethanol, propanol and nonanol; (ii) said fatty alcohol is lauryl alcohol ; (iii) said fatty acid is selected from the group consisting of valeric acid, caproic acid and capric acid; (iv) said fatty acid ester is selected from the group consisting of isopropyl myristate and isopropyl nhexanoate; (v) said alkyl ester is selected from the group consisting of ethyl acetate and butyl acetate; (vi) said polyol is selected from the group consisting of propylene glycol, propanedione and hexanetriol; (vii) said sulfoxide is selected from the group consisting of dimethylsulfoxide and decylmethylsulfoxide ; (viii) said amide is selected from the group consisting of urea, dimethylacetamide and pyrrolidone derivatives ; (ix) said surfactant is selected from the group consisting of sodium lauryl sulfate, cetyltrimethylammonium bromide, polaxamers, spans, tweens, bile salts and lecithin; (x) said terpene is selected from the group consisting of dlimonene, alphaterpeneol, 1,8cineole and menthone; and (xi) said alkanone is selected from the group consisting of Nhep and Nnonane.
111. The device of claim 107, wherein said at least one skin permeation enhancing agent includes at least one biodegradable skin permeation enhancer.
112. The device of claim 111, wherein said at least one biodegradable skin permeation enhancer is selected from the group consisting of dodecylN, Ndimethylamino acetate and N, Ndimethylamino isopropionate.
113. The device of claim 107, wherein said at least one surface adhesion molecule modulating agent is selected from the group consisting of a cadherin antagonist, a selectin antagonist, and an integerin antagonist.
114. The device of claim 113, wherein said cadherin antagonist is a peptide which includes a HisAlaVal amino acid sequence.
115. The device of claim 114, wherein said peptide is a cyclic peptide containing 415 amino acid residues.
116. The device of claim 115, wherein said cyclic peptide is of a general formula: (Zi) (Yi) (Xi)HisAlaVal (X2) (Y2) (Z2) wherein, (i) X1 and X2, are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations of amino acid residues in which the residues are linked by peptide bonds, and wherein XI and X2 independently range in size from 0 to 10 residues, such that the sum of residues contained within Xi and X2 ranges from 1 to 12 ; (ii) Y1 and Y2 are independently selected from the group consisting of amino acid residues, and wherein a covalent bond is formed between residues Y1 and Y2 ; and (iii) Z1 and Z2 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations of amino acid residues in which the residues are linked by peptide bonds.
117. The device of claim 116, wherein Z1 is not present and Il comprises an Nacetyl group in said cyclic peptide.
118. The device of claim 116, wherein Z2 is not present and Y2 comprises a Cterminal amide group in said cyclic peptide.
119. The device of claim 116, wherein Yl and Y2 in said cyclic peptide are covalently linked via a disulfide bond.
120. The device of claim 119, wherein Y1 and Y2 are each independently selected from the group consisting of penicillamine, ß, ptetramethylene cysteine, ß, ppentamethylene cysteine, ßmercaptopropionic acid, ß, ßpentamethyleneßmercaptopropionic acid, 2mercaptobenzene, 2mercaptoaniline, 2mercaptoproline and derivatives of cysteine residues containing side chain modifications.
121. The device of claim 119, wherein Yl and Y2, in said cyclic peptide, are cysteine residues or derivatives of cysteine residues containing side chain modifications.
122. The device of claim 121, wherein said cyclic peptide comprises the sequence CysHisAlaValCys (SEQ ID NO : 4).
123. The device of claim 122, wherein said cyclic peptide further comprising an Nacetyl group.
124. The device of claim 122, wherein said cyclic peptide further comprising a Cterminal amide group.
125. The device of claim 121, wherein said cyclic peptide comprises a sequence selected from the group consisting of CysAlaHisAlaValAspIleCys (SEQ ID NO : 5), CysSerHisAlaValCys (SEQ ID NO : 6), CysHisAlaValSerCys (SEQ ID N0 : 7), CysAlaHisAlaValAspCys (SEQ ID NO : 8) and CysSerHisAlaVaISerSerCys (SEQ ID NO : 9).
126. The device of claim 116, wherein Yl and Y2, in said cyclic peptide, are covalently linked via an amide bond.
127. The device of claim 126, wherein said amide bond is formed between terminal functional groups.
128. The device of claim 126, wherein said amide bond is formed between residue sidechains.
129. The device of claim 126, wherein said amide bond is formed between one terminal functional group and one residue side chain.
130. The device of claim 126, wherein (i) Y1 is selected from the group consisting of lysine, ornithine, and derivatives of lysine or ornithine containing side chain modifications and Y2 is selected from the group consisting of aspartate, glutamate and derivatives of aspartate or glutamate containing side chain modifications; or (ii) Y2 is selected from the group consisting of lysine, ornithine and derivative of lysine or ornithine containing side chain modifications and Y1 is selected from the group consisting of aspartate, glutamate and derivatives of aspartate or glutamate containing side chain modifications.
131. The device of claim 126, wherein said cyclic peptide comprises the sequence LysHisAlaValAsp (SEQ ID NO : 10) or AlaHisAlaValAspIle (SEQ ID NO : 11).
132. The device of claim 126, wherein Y1 and Y2, in said cyclic peptide, are covalently linked via a thioether bond.
133. The device of claim 126, wherein Yi and Y2 in said cyclic peptide, are each tryptophan or a derivative of tryptophan containing side chain modifications, such that said covalent bond generates a 61 Alditryptophan, or a derivative of 81 81tryptophan containing side chain modifications.
134. A method of transdermal delivery of at least one therapeutic agent, the method comprising the step of placing a device for transdermal application of the at least one therapeutic agent over a skin region to which the at least one therapeutic agent has been previously applied, the device including a solid support having on a skincontacting side thereof a pharmaceutical carrier including: at least one skin permeation enhancing agent; and at least one surface adhesion molecule modulating agent ; said at least one skin permeation enhancing agent and said at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the at least one therapeutic agent.
135. The method of claim 134, wherein said at least one skin permeation enhancing agent is selected from the group consisting of an alcohol, a fatty alcohol, a fatty acid ester, an alkyl ester, a polyol, an amid, a surfactant, a sulfoxide, a terpene and an alkanone.
136. The method of claim 135, wherein: (i) said alcohol is selected from the group consisting of ethanol, propanol and nonanol; (ii) said fatty alcohol is lauryl alcohol; (iii) said fatty acid is selected from the group consisting of valeric acid, caproic acid and capric acid; (iv) said fatty acid ester is selected from the group consisting of isopropyl myristate and isopropyl nhexanoate; (v) said alkyl ester is selected from the group consisting of ethyl acetate and butyl acetate; (vi) said polyol is selected from the group consisting of propylene glycol, propanedione and hexanetriol; (vii) said sulfoxide is selected from the group consisting of dimethylsulfoxide and decylmethylsulfoxide ; (viii) said amide is selected from the group consisting of urea, dimethylacetamide and pyrrolidone derivatives; (ix) said surfactant is selected from the group consisting of sodium lauryl sulfate, cetyltrimethylammonium bromide, polaxamers, spans, tweens, bile salts and lecithin; (x) said terpene is selected from the group consisting of dlimonene, alphaterpeneol, 1,8cineole and menthone; and (xi) said alkanone is selected from the group consisting of Nheptane and Nnonane.
137. The method of claim 134, wherein said at least one skin permeation enhancing agent includes at least one biodegradable skin permeation enhancer.
138. The method of claim 137, wherein said at least one biodegradable skin permeation enhancer is selected from the group consisting of dodecylN, Ndimethylamino acetate and N, Ndimethylamino isopropionate.
139. The method of claim 134, wherein said at least one surface adhesion molecule modulating agent is selected from the group consisting of a cadherin antagonist, a selectin antagonist, and an integerin antagonist.
140. The method of claim 139, wherein said cadherin antagonist is a peptide which includes a HisAlaVal amino acid sequence.
141. The method of claim 140, wherein said peptide is a cyclic peptide containing 415 amino acid residues.
142. The method of claim 140, wherein said cyclic peptide is of a general formula: (Zi) (Yi) (Xi)HisAlaVaI (X2) (Y2) (Z2) wherein, (i) Xi and X2, are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations of amino acid residues in which the residues are linked by peptide bonds, and wherein Xi and X2 independently range in size from 0 to 10 residues, such that the sum of residues contained within X1 and X2 ranges from 1 to 12; (ii) Y1 and Y2 are independently selected from the group consisting of amino acid residues, and wherein a covalent bond is formed between residues Y1 and Y2 ; and (iii) Zl and Z2 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations of amino acid residues in which the residues are linked by peptide bonds.
143. The method of claim 142, wherein Z1 is not present and Y comprises an Nacetyl group in said cyclic peptide.
144. The method of claim 142, wherein Z2 is not present and Y2 comprises a Cterminal amide group in said cyclic peptide.
145. The method of claim 142, wherein Y1 and Y2 in said cyclic peptide are covalently linked via a disulfide bond.
146. The method of claim 142, wherein Yi and Y2 are each independently selected from the group consisting of penicillamine, P. ßtetramethylene cysteine, ß, ppentamethylene cysteine, pmercaptopropionic acid, P, ßpentamethyleneßmercaptopropionic acid, 2mercaptobenzene, 2mercaptoaniline, 2mercaptoproline and derivatives of cysteine residues containing side chain modifications.
147. The method of claim 142, wherein Y1 and Y2, in said cyclic peptide, are cysteine residues or derivatives of cysteine residues containing side chain modifications.
148. The method of claim 147, wherein said cyclic peptide comprises the sequence CysHisAlaValCys (SEQ ID NO : 4).
149. The method of claim 148, wherein said cyclic peptide further comprising an Nacetyl group.
150. The method of claim 148, wherein said cyclic peptide further comprising a Cterminal amide group.
151. The method of claim 147, wherein said cyclic peptide comprises a sequence selected from the group consisting of CysAlaHisAlaValAspIleCys (SEQ ID NO : 5), CysSerHisAlaValCys (SEQ ID NO : 6), CysHisAlaValSerCys (SEQ ID NO : 7), CysAlaHisAlaValAspCys (SEQ ID NO : 8) and CysSerHisAlaValSerSerCys (SEQ ID NO : 9).
152. The method of claim 142, wherein Y1 and Y2, in said cyclic peptide, are covalently linked via an amide bond.
153. The method of claim 152, wherein said amide bond is formed between terminal functional groups.
154. The method of claim 152, wherein said amide bond is formed between residue sidechains.
155. The method of claim 152, wherein said amide bond is formed between one terminal functional group and one residue side chain.
156. The method of claim 152, wherein (i) Y1 is selected from the group consisting of lysine, ornithine, and derivatives of lysine or ornithine containing side chain modifications and Y2 is selected from the group consisting of aspartate, glutamate and derivatives of aspartate or glutamate containing side chain modifications; or (ii) Y2 is selected from the group consisting of lysine, ornithine and derivative of lysine or ornithine containing side chain modifications and Y1 is selected from the group consisting of aspartate, glutamate and derivatives of aspartate or glutamate containing side chain modifications.
157. The method of claim 142, wherein said cyclic peptide comprises the sequence LysHisAlaValAsp (SEQ ID NO : 10) or AlaHisAlaValAspIle (SEQ ID NO : 11).
158. The method of claim 142, wherein Y1 and Y2, in said cyclic peptide, are covalently linked via a thioether bond.
159. The method of claim 142, wherein Y1 and Y2 in said cyclic peptide, are each tryptophan or a derivative of tryptophan containing side chain modifications, such that said covalent bond generates a 61 blditryptophan, or a derivative of 5151tryptophan containing side chain modifications.
160. A method of transdermal delivery of at least one therapeutic agent, the method comprising the step of placing a device for transdermal application of the at least one therapeutic agent over a skin region, the device including a solid support having on a skincontacting side thereof a pharmaceutical composition including: a therapeutic effective amount of at least one therapeutic agent; and a pharmaceutical carrier including: (i) at least one skin permeation enhancing agent; and (ii) at least one surface adhesion molecule modulating agent; said at least one skin permeation enhancing agent and said at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the at least one therapeutic agent.
161. The method of claim 160, wherein said solid support is selected from the group consisting of a patch, a foil, a plaster and a film.
162. The method of claim 160, wherein said at least one skin permeation enhancing agent is selected from the group consisting of an alcohol, a fatty alcohol, a fatty acid ester, an alkyl ester, a polyol, an amid, a surfactant, a sulfoxide, a terpene and an alkanone.
163. The method of claim 162, wherein: (i) said alcohol is selected from the group consisting of ethanol, propanol and nonanol; (ii) said fatty alcohol is lauryl alcohol; (iii) said fatty acid is selected from the group consisting of valeric acid, caproic acid and capric acid; (iv) said fatty acid ester is selected from the group consisting of isopropyl myristate and isopropyl nhexanoate ; (v) said alkyl ester is selected from the group consisting of ethyl acetate and butyl acetate; (vi) said polyol is selected from the group consisting of propylene glycol, propanedione and hexanetriol; (vii) said sulfoxide is selected from the group consisting of dimethylsulfoxide and decylmethylsulfoxide ; (viii) said amide is selected from the group consisting of urea, dimethylacetamide and pyrrolidone derivatives; (ix) said surfactant is selected from the group consisting of sodium lauryl sulfate, cetyltrimethylammonium bromide, polaxamers, spans, tweens, bile salts and lecithin ; (x) said terpene is selected from the group consisting of dlimonene, alphaterpeneol, 1, 8cineole and menthone; and (xi) said alkanone is selected from the group consisting of Nheptane and Nnonane.
164. The method of claim 160, wherein said at least one skin permeation enhancing agent includes at least one biodegradable skin permeation enhancer.
165. The method of claim 164, wherein said at least one biodegradable skin permeation enhancer is selected from the group consisting of dodecylN, Ndimethylamino acetate and N, Ndimethylamino isopropionate.
166. The method of claim 160, wherein said at least one surface adhesion molecule modulating agent is selected from the group consisting of a cadherin antagonist, a selectin antagonist, and an integerin antagonist.
167. The method of claim 166, wherein said cadherin antagonist is a peptide which includes a HisAlaVal amino acid sequence.
168. The method of claim 167, wherein said peptide is a cyclic peptide containing 415 amino acid residues.
169. The method of claim 168, wherein said cyclic peptide is of a general formula: (xi) (Yi) (Xi)HisAlaVal (X2) (Y2) (Z2) wherein, (i) Xi and X2, are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations of amino acid residues in which the residues are linked by peptide bonds, and wherein Xi and X2 independently range in size from 0 to 10 residues, such that the sum of residues contained within Xi and X2 ranges from 1 to 12; (ii) Y1 and Y2 are independently selected from the group consisting of amino acid residues, and wherein a covalent bond is formed between residues YI and Y2 ; and (iii) Zl and Z2 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations of amino acid residues in which the residues are linked by peptide bonds.
170. The method of claim 169, wherein Zl is not present and Yl comprises an Nacetyl group in said cyclic peptide.
171. The method of claim 169, wherein Z2 is not present and Y2 comprises a Cterminal amide group in said cyclic peptide.
172. The method of claim 169, wherein Y1 and Y2 in said cyclic peptide are covalently linked via a disulfide bond.
173. The method of claim 172, wherein Y1 and Y2 are each independently selected from the group consisting of penicillamine, P. ßtetramethylene cysteine, P, Ppentamethylene cysteine, Pmercaptopropionic acid, ß, Ppentamethylenepmercaptopropionic acid, 2mercaptobenzene, 2mercaptoaniline, 2mercaptoproline and derivatives of cysteine residues containing side chain modifications.
174. The method of claim 172, wherein Y1 and Y2, in said cyclic peptide, are cysteine residues or derivatives of cysteine residues containing side chain modifications.
175. The method of claim 174, wherein said cyclic peptide comprises the sequence CysHisAlaValCys (SEQ ID NO : 4).
176. The method of claim 175, wherein said cyclic peptide further comprising an Nacetyl group.
177. The method of claim 175, wherein said cyclic peptide further comprising a Cterminal amide group.
178. The method of claim 174, wherein said cyclic peptide comprises a sequence selected from the group consisting of CysAlaHisAlaValAspIleCys (SEQ ID NO : 5), CysSerHisAlaValCys (SEQ ID NO : 6), CysHisAlaValSerCys (SEQ ID NO : 7), CysAlaHisAlaValAspCys (SEQ ID NO : 8) and CysSerHisAlaValSerSerCys (SEQ ID NO : 9).
179. The method of claim 169, wherein Y1 and Y2, in said cyclic peptide, are covalently linked via an amide bond.
180. The method of claim 179, wherein said amide bond is formed between terminal functional groups.
181. The method of claim 179, wherein said amide bond is formed between residue sidechains.
182. The method of claim 179, wherein said amide bond is formed between one terminal functional group and one residue side chain.
183. The method of claim 179, wherein (i) Yl is selected from the group consisting of lysine, ornithine, and derivatives of lysine or ornithine containing side chain modifications and Y2 is selected from the group consisting of aspartate, glutamate and derivatives of aspartate or glutamate containing side chain modifications; or (ii) Y2 is selected from the group consisting of lysine, ornithine and derivative of lysine or ornithine containing side chain modifications and Yl is selected from the group consisting of aspartate, glutamate and derivatives of aspartate or glutamate containing side chain modifications.
184. The method of claim 179, wherein said cyclic peptide comprises the sequence LysHisAlaValAsp (SEQ ID NO : 10) or AlaHisAlaValAspIle (SEQ ID NO : 11).
185. The method of claim 169, wherein Yi and YS, in said cyclic peptide, are covalently linked via a thioether bond.
186. The method of claim 169, wherein Y1 and Y2 in said cyclic peptide, are each tryptophan or a derivative of tryptophan containing side chain modifications, such that said covalent bond generates a 81 Slditryptophan, or a derivative of 61 61tryptophan containing side chain modifications.
Description:
PHARMACEUTICAL CARRIERS AND COMPOSITIONS FOR TRANSDERMAL DRUG DELIVERY FIELD AND BACKGROUND OF THE INVENTION The present invention relates to pharmaceutical carriers and further to pharmaceutical compositions and skin patches containing same for transdermal delivery of a therapeutic agent into the body of a treated subject. More particularly, the present invention relates to the use of at least one skin permeation enhancer agent combined with at least one surface adhesion molecule modulating agent, so as to in synergy facilitate and thereby accelerate the delivery of the therapeutic agent into the blood stream and into targeted organs of the treated subject.

MetAtods of drug delivery Therapeutic agents such as drugs are a mainstay of modern medicine and are used for the prevention diagnosis, alleviation, treatment, or cure of diseases.

Delivery of therapeutic agents to target tissue is often limited by biological, biochemical and/or physical barriers. For example, the skin is a physical barrier which must be traversed by a topically administered drug targeted at internal tissues. Orally administered drugs must be resistant to the low pH conditions and digestive enzymes present in the gastrointestinal (GI) tract. Moreover, there are many clinical situations where it is difficult to maintain compliance, for example, patients with mental problems (e. g., Alzheimer or psychosis). Children and infants will also benefit from other than oral routes of administration.

To traverse the skin barrier, drugs targeted at internal tissues are often administered via a transepidermal injection, using a syringe and a needle or other mechanical devices. A transepidermal injection delivers drugs into the subcutaneous space and not into the epidermis-dermis layers, which are the major barriers for efficient drug delivery.

Anatomically, the skin of the human body is subdivided into three compartments: an epidermis, a dermis and an endodermis, of which the

epidermis plays a key role in blocking drug delivery via the skin. The epidermis is 0.1 mm or more in thickness and consists mainly of about 20 percent lipid and about 40 percent protein. Each segment of protein is surrounded by lipid, thus rendering the epidermis highly hydrophobic.

Although the syringe and needle is an effective delivery device, it is sensitive to contamination, while use thereof is often accompanied by pain and/or bruising. In addition, the use of such a device is accompanied by risk of accidental needle injury to a health care provider.

Mechanical injection devices based on compressed gasses have been developed to overcome the above mentioned limitations of syringe and needle devices. Mechanical injection devices typically utilize compressed gas (such as, helium or carbon dioxide) to deliver medications at high velocity through a narrow aperture.

Although such devices traverse some of the limitations mentioned above, their efficiency is medication dependent, and their use can lead to pain, bruising and lacerations.

Transdermal delivery is a controlled drug delivery system. It controls the release of drug continuously to the surface of skin, then the drug penetrates the skin and enters the capillary blood circulation system. Blood circulation later brings the drug to the target organ wherein the drug exerts its action. The advantage of transdermal drug delivery is its convenience and ease of removing away from skin, thus the chance of dose dumping is minimized. In general, the surface area of an adult is 2 square meters, and capillary blood flowing throughout body surface area accounts for one third of the whole blood circulation, this offers an unique advantage for transdermal drug delivery system. In addition, transdermal drug delivery system not only avoids some side effects of traditional preparations, but also controls the release of drug.

For these reasons, transdermal drug delivery system is practical in clinical use.

For several years, transdermal drug delivery systems have been employed to effectively introduce certain drugs into the bloodstream through unbroken skin. Aside from comfort and convenience, transdermal systems

avoid the barriers, delivery rate control problems and potential toxicity concerns associated with traditional administration techniques, such as oral, intramuscular or intravenous delivery. Such systems have proven particularly effective in the delivery of melatonin and other natural hormones to the body, since transdermal delivery mimics the body's own system of secretion.

Transdermal delivery has traditionally involved the transport of a drug or drugs across the stratum corneum, the layer of the skin responsible for preventing water loss and the transport of substances through the skin, and into the bloodstream.

Less common drug delivery methods utilize a pulsed Yag laser to punctuate the stratum corneum in order to deliver medication via diffusion and enhancement of ionic compound flux across the skin by the application of an electric current. Such methods are effective in delivering small charged molecules over a long range of time although with an inherent danger of inflicting skin burns.

U. S. Pat. No. 4,767,402 (Kost et al.) discloses the use of ultrasound for enhancing transdermal drug delivery. However, relatively few drugs are known to be deliverable transdermally via ultrasound, insofar as the majority of drugs will not penetrate the skin at rates sufficiently high for therapeutic efficacy. It will be appreciated that the use of laser and ultrasound requires skills and cannot be practiced by the patient himself.

Most transdermal drug delivery systems include at least one substance which serves as a skin permeation enhancing agent. The following paragraphs provide some examples in which skin permeation enhancing agents are used in skin patches to enhance permeation of a variety of drugs through the skin.

For example, U. S. Pat. No. 4,638,043 (Szycher et al.) teaches a polyurethane matrix for dispensing drugs dispersed therein, primarily for incorporation in a medical patch comprised of successive layers of a substrate, a pressure sensitive adhesive, the drug dispensing matrix and optionally a second layer of adhesive. The matrix may also include, skin permeation enhancing agents, polypropylene glycol, polyethylene glycol or glycerine, to

soften layer softer and to aid the transport of the drug out of the matrix and into the skin.

U. S. Pat. No. 4,792,450 (Kydonieus et al.) discloses a transdermal drug delivery device which comprises a vinyl gel layer comprising PVC and a drug uniformly dispersed therein, the vinyl gel layer comprising a primary plasticizer for the PVC and an organic nonvolatile gel forming additive in an amount sufficient to form a gel and which serve as skin permeation enhancing agent. Examples of such additives are isopropyl palmitate, isopropyl myristate, soybean oil, castor oil, linseed oil, olive oil, mineral oil, petrolatum, caprylic/capric triglyceride and non-ionic surfactants.

In U. S. Pat. No. 4,818,540 (Chien et al.), there is disclosed essentially a transdermal fertility-controlling polymer matrix dosage unit comprising an impervious backing layer, a polymer matrix disc layer adhered thereto containing microdispersed fertility-controlling estrogen and progestin hormones, and an adhesive layer for securing the dosage unit to the subject.

The device may contain, preferably in the adhesive layer, but alternatively or additionally in the matrix layer, a skin permeation enhancing agent, in particular a fatty acid CH3 (CH2) nCOOH, where n is 2-16, isopropyl myristate or decyl methyl sulfoxide.

U. S. Pat. No. 4,820,525 (Leonard et al.) discloses the use of a foamed polyethylene having specified properties, as a drug reservoir in a transdermal/transmucosal pharmaceutical delivery system. Thus, fertility hormones and albuterol were applied transdermally from such reservoirs attached to adhesive tape across nude mouse skin or cadaver skin, using menthol as penetration enhancer.

In U. S. Pat. No. 4,822,617 (Panoz), there is disclosed a device for the transdermal administration of skin-permeable drugs (e. g., nitroglycerin, clonidine, methadone and scopolamine) in an ointment, cream or jelly-like carrier, comprising a laminar applicator adapted to receive a predetermined quantity of the drug on a skin-contacting surface thereof, the latter being overlaid by a drug-impervious layer to ensure a unidirectional transfer of the

drug to the skin surface. In an exemplified embodiment, the applicator is loaded with a predetermined amount of ointment containing 2% nitroglycerin and lactose in an absorptive lanolin and white petrolatum base formulated to provide controlled release of the active ingredient and serve to enhance skin permeation.

In U. S. Ser. No. 07/876, 153 as originally filed April 30,1992, there is described a pharmaceutical composition for use in the transdermal administration of a medicament intended to be detectable in the blood stream within two hours after administration, comprising the medicament and a pharmaceutically acceptable carrier including esters of C8-24 fatty acids with at least one aliphatic C2 12 hydroxy compound containing 2-3 hydroxy groups which serve as skin permeation enhancing agents.

U. S. Pat. No. 5,707,641 discloses a pharmaceutical formulation which is adapted particularly for transdermal administration, and which comprises an aqueous emulsion or dispersion including, in addition to the aqueous phase, (a) at least one therapeutically active protein or polypeptide; (b) at least one pharmaceutically acceptable emulsifier ; and (c) an oil phase comprising at least one ester of an aliphatic hydroxy compound containing 1-12 carbon atoms and 1-4 alcoholic hydroxy groups with an aliphatic carboxylic acid containing 8-24 carbon atoms and 1-3 carboxylic acid groups, which serve as skin permeation enhancing agents.

A different approach for transdermal drug delivery of therapeutic agents into the blood stream involves using antagonists or inhibitors of recognition sites of cell adhesion molecules.

Cell adliesion molecules Cell adhesion is a complex process that is important for maintaining tissue integrity and generating physical and permeability barriers within the body. All tissues are divided into discrete compartments, each of which is composed of a specific cell type that adheres to similar cell types. Such adhesion triggers the formation of intercellular junctions (i. e., readily definable contact sites on the surfaces of adjacent cells that are adhering to one another),

also known as tight junctions, gap junctions and belt desmosomes. The formation of such junctions gives rise to physical and permeability barriers that restrict the free passage of cells and other biological substances from one tissue compartment to another. For example, the blood vessels of all tissues are composed of endothelial cells. In order for components in the blood to enter a given tissue compartment, they must first pass from the lumen of a blood vessel through the barrier formed by the endothelial cells of that vessel. Similarly, in order for substances to enter the body via the gut, the substances must first pass through a barrier formed by the epithelial cells of that tissue. To enter the blood via the skin, both epithelial and endothelial cell layers must be crossed.

Cell adhesion is mediated by specific cell surface adhesion molecules (CAMs). There are many different families of CAMs, including the immunoglobulin, integrin, selectin and cadherin superfamilies, and each cell type expresses a unique combination of these molecules.

Cadherins : Cadherins are calcium dependant cell adhesion molecules that have both adhesion and calcium binding sites. These molecules are homophilic. Intracellularly, cadherins attach to a group of molecules known as catenins which link the cytoplasmic domain of the cadherin molecule to intermediate filaments of the cytoskeleton. Cadherins have been shown to regulate epithelial, endothelial, neural and cancer cell adhesion, with different cadherins expressed in different cell types. N (neural)-cadherin is predominantly expressed by neural cells, endothelial cells and a variety of cancer cell types. E (epithelial)-cadherin is predominantly expressed by epithelial cells. Other cadherins include P (placental)-cadherin, which is found in human skin and R (retinal)-cadherin. A detailed discussion of the classical cadherins is provided in Munro S B et al., 1996, In: Cell Adhesion and Invasion in Cancer Metastasis, P. Brodt, ed., pp. 17-34 (RG Landes Company, Austin Tex.). E cadherin, is the best characterized molecule in this group. Its expression is regulated by the ErbB2 proto-oncogene. Both E-cadherin and P-cadherin are related to skin diseases such as Paget's disease, carcinoma,

melanoma and psoriasis and the intracellular binding site of the two molecules is catenin or alpha actinin, whereas the cytoplasmatic filament is actin.

Integrins : Integrins are cell-cell and cell-matrix adhesion molecules.

They are heterodimers consisting of one a and one ß chain, wherein both chains are required for binding. The presence of divalent cations such as Ca and Mg is necessary for binding. Integrins are sub-classified according to which ß sub-unit is involved in the complex. There are three main classes of integrins, (31, ß2 and (33. The pi and ß3 subfamilies predominantly mediate cell matrix interactions, while the members of the ß2 class are cell-cell adhesion molecules. (31 integrins are connected to fibronectin, laminin and collagens, whilst the ß3 integrins bind to vascular ligands such as fibrinogen, von Willebrand factor, thrombospondin and vitronectin. PI and ß3 integrins are co-expressed on most cell types whereas ß2 integrins are restricted to leucocytes.

Selectins : Selectins have lectin-like (carbohydrate binding) domains on the extracellular component of the molecule. There are three major groups of selectins; the L selectins (the main example of which was once known as LCAM or MEL-14), which are receptors for specific adhesion of lymphocytes to endothelial cells of peripheral lymph nodes ; the E selectins (endothelial leucocyte adhesion molecules) which are important mediators of inflammatory reactions and P selectin which is contained in the bodies of endothelial cells and in the a granules of platelets and is released during clotting and at times of platelet activation, mediating adhesion between leucocytes and platelets.

The role that cell adhesion molecules play in body function, their implications in diseases and pathologic conditions and the recognition of the molecular structure of these molecules have led to the search for possible antagonists for these molecules.

Examples for cell adhesion molecule antagonists are provided in the following paragraph.

Cadherin antagonists : U. S. Pat. No. 6,031,072 discloses cyclic peptides which comprise a cadherin cell adhesion recognition sequence His-Ala-Val (SEQ ID NO : 1). This sequence is involved in the"homophylic" biniding site. Methods for using such peptides and compositions for modulating cadherin-mediated cell adhesion in a variety of contexts are also disclosed, wherein one of the preferred embodiments is the use of these peptides for transdermal transport of therapeutic agents.

INP, a novel N-cadherin antagonist targeted to the amino acids that flank the HAV motif was published by Wiliams et al. (Mol Cell Neurosci 2000 May; 15 (5): 456-64). The authors demostrated that a linear peptide mimetic of a short sequence in ECD1 of N-cadherin (INPISGQ, (SEQ ID NO : 3)) functions as a highly specific and potent antagonist of N-cadherin function with an ICso value of approximately 15 microM.

Integrin antagonists : Non-peptide integrin antagonists were designed after the adhesion recognition sequence RGD (Arg-Gly-Asp, (SEQ ID NO : 2)) was recognized. Combinatorial organic synthesis of chemical mini-libraries have facilitated non-peptide lead optimization of integrin antagonists and these were used as antithrombptic agents and for the treatment of cancer and osteoporosis (Curr Med, Chem. 1998 3 : 195-204).

BIRT 377 (J ; of Immunology, 1999 163 : 5173-5177) is a small molecule antagonist of LFA-1 (an integrin in the white blood cells), which is orally bioavailable compound and inhibits lymphocyte activity Selectin antagonists : Inhibition of adhesion of human neutrophils and eosinophils to P-selectin by the sialyl Lewis antagonist TBC1269 showed preferential activity against neutrophil adhesion in vitro (J Allergy Clifa Immunol2000 Apr; 105 (4) 769-75).

Each of the approaches described herein with respect to transdermal drug delivery suffers at least one a major limitation: either transdermal delivery is too slow so as to establish a therapeutic concentration of the delivered drug, and/or transdermal delivery is limited to relatively small molecules. None of

these approaches yielded satisfactory results with respect to effective transdermal delivery of high molecular weight proteins.

There is thus a widely recognized need for, and it would be highly advantageous to have, a transdermal drug delivery carrier devoid of the above limitations.

SUMMARY OF THE INVENTION While conceiving the present invention, it was hypothesized that the combination of a skin permeation enhancing agent with at least one surface adhesion molecule modulating agent may have a synergetic effect on transdermal delivery of a therapeutic agent because, and without being bound to any theory, the penetration of the at least one surface adhesion molecule modulating agent should be facilitated by the skin permeation enhancing agent, so as to result in superior transdermal delivery of the therapeutic agent.

As is further detailed and exemplified hereinunder, this hypothesis was tested and such synergy detected both with respect to acceleration and efficiency of transdermal delivery of therapeutic agents of a given size, and with respect to the size of therapeutic agents, which may be transdermally delivered.

According to the present invention there is provided a pharmaceutical carrier for enhancing transdermal delivery of a therapeutic agent, the pharmaceutical carrier comprising: (a) at least one skin permeation enhancing agent; and (b) at least one at least one surface adhesion molecule modulating agent; the at least one skin permeation enhancing agent and the at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the therapeutic agent.

According to another aspect of the present invention there is provided a pharmaceutical composition comprising: (a) a therapeutic effective amount of at least one at least one therapeutic agent; and (b) a pharmaceutical carrier including:

(i) at least one skin permeation enhancing agent; and (ii) at least one surface adhesion molecule modulating agent ; the at least one skin permeation enhancing agent and the at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the at least one therapeutic agent.

According to yet another aspect of the present invention there is provided a method of transdermal delivery of at least one therapeutic agent, the method comprising the step of topically administering the at least one therapeutic agent in a presence of a pharmaceutical carrier including: (a) at least one skin permeation enhancing agent; and (b) at least one surface adhesion molecule modulating agent; The at least one skin permeation enhancing agent and the at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the at least one therapeutic agent.

According to still another aspect of the present invention there is provided a device for transdermal application of at least one therapeutic agent, the device comprising a solid support having on a skin-contacting side thereof a pharmaceutical carrier including: (a) at least one skin permeation enhancing agent; and (b) at least one surface adhesion molecule modulating agent; the at least one skin permeation enhancing agent and the at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the at least one therapeutic agent According to an additional aspect of the present invention there is provided a device for transdermal application of at least one therapeutic agent, the device comprising a solid support having on skin-contacting side thereof a pharmaceutical composition including: (a) a therapeutic effective amount of at least one therapeutic agent; and (b) a pharmaceutical carrier including: (i) at least one skin permeation enhancing agent; and (ii) at least one surface adhesion molecule modulating agent; the at least one skin permeation enhancing agent and the at least one surface adhesion molecule modulating

agent acting in synergy for enhancing the transdermal delivery of the at least one therapeutic agent.

According to yet an additional aspect of the present invention there is provided a method of transdermal delivery of at least one therapeutic agent, the method comprising the step of placing a device for transdermal application of the at least one therapeutic agent over a skin region to which the at least one therapeutic agent has been previously applied, the device including a solid support having on a skin-contacting side thereof a pharmaceutical carrier including: (a) at least one skin permeation enhancing agent; and (b) at least one surface adhesion molecule modulating agent; the at least one skin permeation enhancing agent and the at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the at least one therapeutic agent.

According to still an additional aspect of the present invention there is provided a method of transdermal delivery of at least one therapeutic agent, the method comprising the step of placing a device for transdermal application of the at least one therapeutic agent over a skin region, the device including a solid support having on a skin-contacting side thereof a pharmaceutical composition including: (a) a therapeutic effective amount of at least one therapeutic agent; and (b) a pharmaceutical carrier including: (i) at least one skin permeation enhancing agent; and (ii) at least one surface adhesion molecule modulating agent; the at least one skin permeation enhancing agent and the at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the at least one therapeutic agent According to further features in preferred embodiments of the invention described below, the at least one skin permeation enhancing agent is selected from the group consisting of an alcohol, a fatty alcohol, a fatty acid ester, an alkyl ester, a polyol, an amid, a surfactant, a sulfoxide, a terpene and an alkanone.

According to still further features in the described preferred embodiments the alcohol is selected from the group consisting of ethanol, propanol and nonanol; (ii) the fatty alcohol is lauryl alcohol; (iii) the fatty acid is selected from the group consisting of valeric acid, caproic acid and capric acid; (iv) the fatty acid ester is selected from the group consisting of isopropyl myristate and isopropyl n-hexanoate; (v) the alkyl ester is selected from the group consisting of ethyl acetate and butyl acetate; (vi) the polyol is selected from the group consisting of propylene glycol, propanedione and hexanetriol; (vii) the sulfoxide is selected from the group consisting of dimethylsulfoxide and decylmethylsulfoxide ; (viii) the amide is selected from the group consisting of urea, dimethylacetamide and pyrrolidone derivatives; (ix) the surfactant is selected from the group consisting of sodium lauryl sulfate, cetyltrimethylammonium bromide, polaxamers, spans, tweens, bile salts and lecithin; (x) the terpene is selected from the group consisting of d-limonene, alpha-terpeneol, 1, 8-cineole and menthone; and (xi) the alkanone is selected from the group consisting of N-heptane and N-nonane.

According to still further features in the described preferred embodiments the at least one skin permeation enhancing agent includes at least one biodegradable skin permeation enhancer.

According to still further features in the described preferred embodiments the at least one biodegradable skin permeation enhancer is selected from the group consisting of dodecyl-N, N-dimethylamino acetate and N, N-dimethylamino isopropionate.

According to still further features in the described preferred embodiments the at least one surface adhesion molecule modulating agent is selected from the group consisting of a cadherin antagonist, a selectin antagonist, and an integerin antagonist.

According to still further features in the described preferred embodiments the cadherin antagonist is a peptide which includes a His-Ala-Val amino acid sequence.

According to still further features in the described preferred embodiments the peptide is a cyclic peptide containing 4-15 amino acid residues.

According to still further features in the described preferred embodiments the cyclic peptide is of a general formula: (Z1)----(Y1)----(X1)-His-Ala-Val-(X2)----(Y2)----(Z2) wherein, (i) X1 and X2, are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations of amino acid residues in which the residues are linked by peptide bonds, and wherein X1 and X2 independently range in size from 0 to 10 residues, such that the sum of residues contained within Xi and X2 ranges from 1 to 12; (ii) Y1 and Y2 are independently selected from the group consisting of amino acid residues, and wherein a covalent bond is formed between residues Y1 and Y2; and (iii) Zl and Z2 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations of amino acid residues in which the residues are linked by peptide bonds.

According to still further features in the described preferred embodiments the Z I is not present and Y1 comprises an N-acetyl group in the cyclic peptide.

According to still further features in the described preferred embodiments the Z2 is not present and Y2 comprises a C-terminal amide group in the cyclic peptide.

According to still further features in the described preferred embodiments the Y1 and Y2 in the cyclic peptide are covalently linked via a disulfide bond.

According to still further features in the described preferred embodiments the Y1 and Y2 are each independently selected from the group consisting of penicillamine, P, ß-tetramethylene cysteine, ß, ß-pentamethylene cysteine, ß-mercaptopropionic acid, P, ß-pentamethylene-ß-mercaptopropionic

acid, 2-mercaptobenzene, 2-mercaptoaniline, 2-mercaptoproline and derivatives of cysteine residues containing side chain modifications.

According to still further features in the described preferred embodiments the Y1 and Y2, in the cyclic peptide, are cysteine residues or derivatives of cysteine residues containing side chain modifications.

According to still further features in the described preferred embodiments the cyclic peptide comprises the sequence Cys-His-Ala-Val-Cys (SEQ ID NO : 4).

According to still further features in the described preferred embodiments the cyclic peptide further comprising an N-acetyl group.

According to still further features in the described preferred embodiments the cyclic peptide further comprising a C-terminal amide group.

According to still further features in the described preferred embodiments the cyclic peptide comprises a sequence selected from the group consisting of Cys-Ala-His-Ala-Val-Asp-Ile-Cys (SEQ ID NO : 5), Cys-Ser-His-Ala-Val-Cys (SEQ ID NO : 6), Cys-His-Ala-Val-Ser-Cys (SEQ ID NO : 7), Cys-Ala-His-Ala-Val-Asp-Cys (SEQ ID NO : 8) and Cys-Ser-His-Ala-Val-Ser-Ser-Cys (SEQ ID NO : 9).

According to still further features in the described preferred embodiments Y1 and Y2, in the cyclic peptide, are covalently linked via an amide bond.

According to still further features in the described preferred embodiments the amide bond is formed between terminal functional groups.

According to still further features in the described preferred embodiments the amide bond is formed between residue side-chains.

According to still further features in the described preferred embodiments the amide bond is formed between one terminal functional group and one residue side chain.

According to still further features in the described preferred embodiments (i) Y1 is selected from the group consisting of lysine, ornithine, and derivatives of lysine or ornithine containing side chain modifications and

Y2 is selected from the group consisting of aspartate, glutamate and derivatives of aspartate or glutamate containing side chain modifications; or (ii) Y2 is selected from the group consisting of lysine, ornithine and derivative of lysine or ornithine containing side chain modifications and Y1 is selected from the group consisting of aspartate, glutamate and derivatives of aspartate or glutamate containing side chain modifications.

According to still further features in the described preferred embodiments the cyclic peptide comprises the sequence Lys-His-Ala-Val-Asp (SEQ ID NO : 10) or Ala-His-Ala-Val-Asp-Ile (SEQ ID NO : 11).

According to still further features in the described preferred embodiments the Y1 and Y2, in the cyclic peptide, are covalently linked via a thioether bond.

According to still further features in the described preferred embodiments the Y1 and Y2 in the cyclic peptide, are each tryptophan or a derivative of tryptophan containing side chain modifications, such that the covalent bond generates a 61 61-ditryptophan, or a derivative of 6 tryptophan containing side chain modifications.

According to still further features in the described preferred embodiments the at least one therapeutic agent is selected from the group consisiting of a drug, a nucleic acid construct, a vaccine, a hormon, an enzyme, an antibody and cells.

According to still further features in the described preferred embodiments the solid support is selected from the group consisting of a patch, a foil, a plaster and a film.

The present invention successfully addresses the shortcomings of the presently known configurations by providing an efficient drug delivery system including at least one skin permeation enhancing agent and at least one surface cell adhesion molecule which by acting in synergy enable rapid and efficient penetration of large therapeutic agents.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

In the drawings: FIGs. la-b are a photograph and a scheme, respectively, of six station Franz cells.

FIG. 2 demonstrates the combined, synergistic effect of E-cadherin antagonist (100 ug) as described in U. S. Pat. No. 6,031,072 and skin permeation enhancing emulsion as described in U. S. Pat. No. 5,707,641 on the penetration of biotinylated a-lactalbumin through nude mice skin, in vitro, using the Franz cells of Figures la-b.

FIG. 3 demonstrates the combined, synergistic effect of E-cadherin antagonist (10 pg) as described in U. S. Pat. No. 6,031,072 and skin permeation enhancing emulsion as described in U. S. Pat. No. 5,707,641 on the penetration of 100 pg oc-lactalbumin, in vitro, using the Franz cells of Figures la-b.

FIG. 4 demonstrates the combined, synergistic effect of E-cadherin antagonist (0.10 u. g) as described in U. S. Pat. No. 6,031,072 and skin permeation enhancing emulsion as described in U. S. Pat. No. 5,707,641, on the penetration of 100, ug ot-lactalbumin, in vitro, using the Franz cells of Figures la-b.

FIG. 5 demonstrates the effect of pretreatment with E-cadherin antagonist (1 mg/ml) as described in U. S. Pat. No. 6,031,072 followed by

administration of a skin patch containing insulin (6.4 IU) and skin permeation enhancing emulsion as described in U. S. Pat. No. 5,707,641, on glucose level of nude mice, in vivo.

FIG. 6 demonstrates the effect of the presence of E-cadherin antagonist (0.15 Lg/CM2) as described in U. S. Pat. No. 6,031,072, in a skin patch containing insulin (6.4 IU and 25 IU/cm2) and skin permeation enhancing emulsion as described in U. S. Pat. No. 5,707,641, on glucose level of nude mice, in vivo.

FIG. 7 demonstrates the efficacy of the skin permeation enhancing emulsion as described in U. S. Pat. No. 5,707,641, for transdermal delivery of fluorescent molecular weight markers through nude mice skin, in vitro, using the Franz cells of Figures 1 a-b.

FIG. 8 demonstrates the effect of the presence of E-cadherin antagonist as described in U. S. Pat. No. 6,031,072, in a skin permeation enhancing emulsion as described in U. S. Pat. No. 5,707,641, on permeation of Sulforhodamine B (581 Da), in vitro, using the franz cells of Figures la-b.

FIGs. 9 and 10 demonstrate the effect of the presence of E-cadherin antagonist as described in U. S. Pat. No. 6,031,072, in a skin permeation enhancing emulsion as described in U. S. Pat. No. 5,707,641, on permeation of FITC-Dextran (4 kDa), in vitro, using the franz cells of Figures la-b.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is of pharmaceutical carriers, pharmaceutical compositions and pharmaceutical devices for transdermal application of therapeutic agents. The pharmaceutical carriers, pharmaceutical compositions and pharmaceutical devices for transdermal application of therapeutic agents according to the present invention include at least one surface adhesion molecule modulating agent and at least one skin permeation enhancing agent, which by acting in synergy, enable transfer of pharmaceutically active agents into a skin region. The present invention can be used to deliver drugs, nucleic acid constructs, vaccines, hormons, enzymes, antibodies and cells into a tissue

region of a subject in need. Moreover, the present invention overcomes the currently used methods, by enabling the efficient transdermal passage of large molecules, through, for example, the skin into the blood stream of a treated subject.

The principles and operation of the described drug delivery system according to the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

According to the present invention there is provided a pharmaceutical carrier for enhancing transdermal delivery of a therapeutic agent, the pharmaceutical carrier comprising: (a) at least one skin permeation enhancing agent; and (b) at least one at least one surface adhesion molecule modulating agent; the at least one skin permeation enhancing agent and the at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the therapeutic agent.

According to another aspect of the present invention there is provided a pharmaceutical composition comprising: (a) a therapeutic effective amount of at least one at least one therapeutic agent; and (b) a pharmaceutical carrier including: (i) at least one skin permeation enhancing agent; and (ii) at least one surface adhesion molecule modulating agent ; the at least one skin permeation enhancing agent and the at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the at least one therapeutic agent.

According to yet another aspect of the present invention there is provided a method of transdermal delivery of at least one therapeutic agent, the

method comprising the step of topically administering the at least one therapeutic agent in a presence of a pharmaceutical carrier including: (a) at least one skin permeation enhancing agent; and (b) at least one surface adhesion molecule modulating agent; The at least one skin permeation enhancing agent and the at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the at least one therapeutic agent.

According to still another aspect of the present invention there is provided a device for transdermal application of at least one therapeutic agent, the device comprising a solid support having on a skin-contacting side thereof a pharmaceutical carrier including: (a) at least one skin permeation enhancing agent; and (b) at least one surface adhesion molecule modulating agent; the at least one skin permeation enhancing agent and the at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the at least one therapeutic agent According to an additional aspect of the present invention there is provided a device for transdermal application of at least one therapeutic agent, the device comprising a solid support having on skin-contacting side thereof a pharmaceutical composition including: (a) a therapeutic effective amount of at least one therapeutic agent; and (b) a pharmaceutical carrier including: (i) at least one skin permeation enhancing agent; and (ii) at least one surface adhesion molecule modulating agent; the at least one skin permeation enhancing agent and the at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the at least one therapeutic agent.

According to yet an additional aspect of the present invention there is provided a method of transdermal delivery of at least one therapeutic agent, the method comprising the step of placing a device for transdermal application of the at least one therapeutic agent over a skin region to which the at least one therapeutic agent has been previously applied, the device including a solid support having on a skin-contacting side thereof a pharmaceutical carrier

including: (a) at least one skin permeation enhancing agent; and (b) at least one surface adhesion molecule modulating agent; the at least one skin permeation enhancing agent and the at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the at least one therapeutic agent.

According to still an additional aspect of the present invention there is provided a method of transdermal delivery of at least one therapeutic agent, the method comprising the step of placing a device for transdermal application of the at least one therapeutic agent over a skin region, the device including a solid support having on a skin-contacting side thereof a pharmaceutical composition including : (a) a therapeutic effective amount of at least one therapeutic agent; and (b) a pharmaceutical carrier including: (i) at least one skin permeation enhancing agent; and (ii) at least one surface adhesion molecule modulating agent; the at least one skin permeation enhancing agent and the at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the at least one therapeutic agent The term"acting in synergy"refers to a transdermal delivery effect which is superior over the effect achieved when using each agent individually and/or the summ of effects achieved when using each agent individually. The effect can be, for example, improved efficiency in transdermal delivery of a molecule of a given size. Alternatively, the effect can be improved efficiency in transdermal delivery of molecules having a higher molecular weight.

The examples below demonstrate the synergistic effect of the combination a surface adhesion molecule antagonist and a skin permeation enhancing agent on transdermal delivery of a variety of proteins through the skin. The synergy is evident both in terms of efficiency of delivery and in terms of molecular weight efficiently deliverable.

The surface adhesion molecule antagonist can be administered either together, before or after the skin permeation enhancing agent. In every experimental design, the results clearly show that the combination of the

surface adhesion molecule modulating agent and the skin permeation enhancing agent act in synergy to enhance the permeation of proteins through skin.

This synergistic effect is the gist of the present invention. Without being bound to any theory in particular, it is hypothesized that the different modes of action of the surface adhesion molecule modulating agent and the skin permeation enhancing agent in enhancing skin penetration enhance the penetration of the agents themselves, which thereby act in synergy to facilitate the penetration of a therapeutic agent through the skin.

The term"antagonist"is used herein in the broadest sense to include any molecule, which blocks, prevents, inhibits, or neutralizes a process.

Thus, the phrase"surface adhesion molecule modulating agent"refers to substances having high binding affinity, high specificity and agonistic effect towards a specified surface adhesion molecule. In general, these agents have the ability to prevent interaction of any of the surface adhesion molecules with their target proteins. For example, cadherins are calcium dependant cell adhesion molecules that have both adhesion and calcium binding sites.

Intracellularly, cadherins attach to a group of molecules known as catenins, which link the cytoplasmic domain of the cadherin molecule to intermediate filaments of the cytoskeleton. A carherin modulating agent according to the present invention shall inhibit one or more of these interactions.

The phrase"skin permeation enhancing agent"refers to substances characterized by a less specific mode of action, as is further exemplified hereinbelow.

According to a preferred embodiment of the present inventoion the at least one surface adhesion molecule modulating agent is selected from the group consisting of cadherin antagonists, such as peptides with a cadherin adhesion recognition sequence which include a His-Ala-Val amino acid sequence.

According to a preferred embodiment the cadherin antagonist peptide is a cyclic peptide containing 4-15 amino acid residues of a general formula:

(Zi)-- (Yi)-- (Xi)-His-Ala-Val- (X2)-- (Y2)-- (Z2) wherein, (i) X1 and X2, are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations of amino acid residues in which the residues are linked by peptide bonds, and wherein X1 and X2 independently range in size from 0 to 10 residues, such that the sum of residues contained within Xi and X2 ranges from 1 to 12; (ii) Y1 and Y2 are independently selected from the group consisting of amino acid residues, and wherein a covalent bond is formed between residues Y1 and Y2 ; and (iii) ZI and Z2 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations of amino acid residues in which the residues are linked by peptide bonds.

According to a preferred embodiment the Zl is not present and Y1 comprises an N-acetyl group in the cyclic peptide. According to a preferred embodiment the Z2 is not present and Y2 comprises a C-terminal amide group in the cyclic peptide. According to a preferred embodiment the Y1 and Y2 in the cyclic peptide are covalently linked via a disulfide bond. According to a preferred embodiment Y1 and Y2 are each independently selected from the group consisting of penicillamine, ß, ß-tetramethylene cysteine, ß, P-pentamethylene cysteine, ß-mercaptopropionic acid, P, ß-pentamethylene-ß-mercaptopropionic acid, 2-mercaptobenzene, 2-mercaptoaniline, 2-mercaptoproline and derivatives of cysteine residues containing side chain modifications. According to a preferred embodiment the Y1 and Y2, in the cyclic peptide, are cysteine residues or derivatives of cysteine residues containing side chain modifications.

According to a preferred embodiment the cyclic peptide comprises the sequence Cys-His-Ala-Val-Cys (SEQ ID NO : 4). According to a preferred embodiment the cyclic peptide further comprising an N-acetyl group.

According to a preferred embodiment the cyclic peptide further comprising a C-terminal amide group. According to a preferred embodiment the cyclic peptide comprises a sequence selected from the group consisting of Cys-Ala-His-Ala-Val-Asp-Ile-Cys (SEQ ID NO : 5), Cys-Ser-His-Ala-Val-Cys

(SEQ ID NO : 6), Cys-His-Ala-Val-Ser-Cys (SEQ ID NO : 7), Cys-Ala-His-Ala-Val-Asp-Cys (SEQ ID NO : 8) and Cys-Ser-His-Ala-Val-Ser-Ser-Cys (SEQ ID NO : 9). According to a preferred embodiment Y1 and Y2, in the cyclic peptide, are covalently linked via an amide bond. According to a preferred embodiment the amide bond is formed between terminal functional groups. According to a preferred embodiment the amide bond is formed between residue side-chains. According to a preferred embodiment the amide bond is formed between one terminal functional group and one residue side chain. According to a preferred embodiment (i) Y1 is selected from the group consisting of lysine, ornithine, and derivatives of lysine or ornithine containing side chain modifications and Y2 is selected from the group consisting of aspartate, glutamate and derivatives of aspartate or glutamate containing side chain modifications; or (ii) Y2 is selected from the group consisting of lysine, ornithine and derivative of lysine or ornithine containing side chain modifications and Y1 is selected from the group consisting of aspartate, glutamate and derivatives of aspartate or glutamate containing side chain modifications. According to a preferred embodiment the cyclic peptide comprises the sequence Lys-His-Ala-Val-Asp (SEQ ID NO : 10) or Ala-His-Ala-Val-Asp-Ile (SEQ ID NO : 11). According to a preferred embodiment the Y1 and Y2, in the cyclic peptide, are covalently linked via a thioether bond. According to a preferred embodiment the Y1 and Y2 in the cyclic peptide, are each tryptophan or a derivative of tryptophan containing side chain modifications, such that the covalent bond generates a 61 81-ditryptophan, or a derivative of 81 81-tryptophan containing side chain modifications.

The term"cyclic peptide,"as used herein, refers to a peptide or salt thereof that comprises an intramolecular covalent bond between two non-adjacent residues and at least one cadherin cell adhesion recognition (CAR) sequence. The intramolecular bond may be a backbone to backbone, side-chain to backbone or side-chain to side-chain bond (i. e., terminal

functional groups of a linear peptide and/or side chain functional groups of a terminal or interior residue may be linked to achieve cyclization). Preferred intramolecular bonds include, but are not limited to, disulfide, amide and thioether bonds. At least one CAR sequence generally comprises HAV (His-Ala-Val). Cyclic peptides may contain only one CAR sequence, or may additionally contain one or more other adhesion molecule binding sites, which may or may not be CARs. Such additional sequences may be separated by a linker (i. e., one or more peptides not derived from a CAR sequence or other adhesion molecule binding site). Within one such embodiment, the cyclic peptide contains two HAV sequences. Within another embodiment, the cyclic peptide contains one HAV and one CAR sequence recognized by a different CAM. In a preferred embodiment, the second CAR sequence is derived from fibronectin and is recognized by an integrin (i. e., Arg-Gly-Asp; see Cardarelli etal., J. Biol. Chem. 267: 23159-23164,1992).

In addition to the CAR sequence (s), cyclic peptides generally comprise at least one additional residue, such that the size of the cyclic peptide ring ranges from 4 to about 15 residues, preferably from 5 to 10 residues. Such additional residue (s) may be present on the N-terminal and/or C-terminal side of a CAR sequence, and may be derived from sequences that flank the HAV sequence within one or more naturally occurring cadherins (e. g., N-cadherin, E-cadherin, P-cadherin, R-cadherin or other cadherins containing the HAV sequence) with or without amino acid substitutions and/or other modifications.

Database accession numbers for representative naturally occurring cadherins are as follows: human N-cadherin M34064, mouse N-cadherin M31131 and M22556, cow N-cadherin X53615, human P-cadherin X63629, mouse P-cadherin X06340, human E-cadherin Z13009, mouse E-cadherin X06115.

Alternatively, additional residues present on one or both sides of the CAR sequence (s) may be unrelated to an endogenous sequence (e. g., residues that facilitate cyclization).

Within certain preferred embodiments, as discussed below, relatively small cyclic peptides that do not contain significant sequences flanking the

HAV sequence are preferred for modulating N-cadherin and E-cadherin mediated cell adhesion. Such peptides may contain, for example, an N-acetyl group and a C-amide group. The finding, within the present invention, that such relatively small cyclic peptides may be effective and all-purpose inhibitors of cell adhesion represents a unexpected discovery. Such cyclic peptides can be thought of as"master keys"that fit into peptide binding sites of each of the different classical cadherins, and are capable of disrupting cell adhesion of neural cells, endothelial cells, epithelial cells and/or certain cancer cells. Small cyclic peptides may generally be used to specifically modulate cell adhesion of neural and/or other cell types by topical administration or by systemic administration, with or without linking a targeting agent to the peptide.

Within other preferred embodiments, a cyclic peptide may contain sequences that flank the HAV sequence on one or both sides that are designed to confer specificity for cell adhesion mediated by one or more specific cadherins, resulting in tissue and/or cell-type specificity. Suitable flanking sequences for conferring specificity include, but are not limited to, endogenous sequences present in one or more naturally occurring cadherins, and cyclic peptides having specificity may be identified using the representative screens provided herein. For example, it has been found, within the context of the present invention, that cyclic peptides that contain additional residues derived from the native E-cadherin sequence on the C-terminal side of the CAR sequence are specific for epithelial cells (i. e., such peptides disrupt E-cadherin mediated cell adhesion to a greater extent than they disrupt N-cadherin expression). The addition of appropriate endogenous sequences may similarly result in peptides that disrupt N-cadherin mediated cell adhesion.

To facilitate the preparation of cyclic peptides having a desired specificity, nuclear magnetic resonance (NMR) and computational techniques may be used to determine the conformation of a peptide that confers a known specificity. NMR is widely used for structural analysis of molecules.

Cross-peak intensities in nuclear Overhauser enhancement (NOE) spectra, coupling constants and chemical shifts depend on the conformation of a

compound. NOE data provide the interproton distance between protons through space and across the ring of the cyclic peptide. This information may be used to facilitate calculation of the lowest energy conformation for the HAV sequence. Conformation may then be correlated with tissue specificity to permit the identification of peptides that are similarly tissue specific or have enhanced tissue specificity.

Cyclic peptides as described herein may comprise residues of L-amino acids, D-amino acids, or any combination thereof. Amino acids may be from natural or non-natural sources, provided that at least one amino group and at least one carboxyl group are present in the molecule; a-and (3-amino acids are generally preferred. The 20 L-amino acids commonly found in proteins are identified herein by the conventional three-letter or one-letter abbreviations, and the corresponding D-amino acids are designated by a lower case one letter symbol. A cyclic peptide may also contain one or more rare amino acids (such as 4-hydroxyproline or hydroxylysine), organic acids or amides and/or derivatives of common amino acids, such as amino acids having the C-terminal carboxylate esterified (e. g., benzyl, methyl or ethyl ester) or amidated and/or having modifications of the N-terminal amino group (e. g., acetylation or alkoxycarbonylation), with or without any of a wide variety of side-chain modifications and/or substitutions (e. g., methylation, benzylation, t-butylation, tosylation, alkoxycarbonylation, and the like). Preferred derivatives include amino acids having an N-acetyl group (such that the amino group that represents the N-terminus of the linear peptide prior to cyclization is acetylated) and/or a C-terminal amide group (i. e., the carboxy terminus of the linear peptide prior to cyclization is amidated). Residues other than common amino acids that may be present with a cyclic peptide include, but are not limited to, penicillamine, ß, p-tetramethylene cysteine, P, p-pentamethylene cysteine, ß-mercaptopropionic acid, ß, P-pentamethylene-p-mercaptopropionic acid, 2-mercaptobenzene, 2-mercaptoaniline, 2-mercaptoproline, ornithine, diaminobutyric acid, a-aminoadipic acid, m-aminomethylbenzoic acid and a, ß-diaminopropionic acid.

Cyclic peptides as described herein may be synthesized by methods well known in the art, including recombinant DNA methods and chemical synthesis.

Chemical synthesis may generally be performed using standard solution phase or solid phase peptide synthesis techniques, in which a peptide linkage occurs through the direct condensation of the a-amino group of one amino acid with the a-carboxy group of the other amino acid with the elimination of a water molecule. Peptide bond synthesis by direct condensation, as formulated above, requires suppression of the reactive character of the amino group of the first and of the carboxyl group of the second amino acid. The masking substituents must permit their ready removal, without inducing breakdown of the labile peptide molecule.

In solution phase synthesis, a wide variety of coupling methods and protecting groups may be used (see Gross and Meienhofer, eds.,"The Peptides: Analysis, Synthesis, Biology,"Vol. 1-4 (Academic Press, 1979); Bodansky and Bodansky,"The Practice of Peptide Synthesis,"2d ed. (Springer Verlag, 1994)). In addition, intermediate purification and linear scale up are possible.

Those of ordinary skill in the art will appreciate that solution synthesis requires consideration of main chain and side chain protecting groups and activation method. In addition, careful segment selection is necessary to minimize racemization during segment condensation. Solubility considerations are also a factor.

Solid phase peptide synthesis uses an insoluble polymer for support during organic synthesis. The polymer-supported peptide chain permits the use of simple washing and filtration steps instead of laborious purifications at intermediate steps. Solid-phase peptide synthesis may generally be performed according to the method of Merrifield et al., J. Am. Chem. Soc. 85: 2149, 1963, which involves assembling a linear peptide chain on a resin support using protected amino acids. Solid phase peptide synthesis typically utilizes either the Boc or Fmoc strategy. The Boc strategy uses a 1% cross-linked polystyrene resin. The standard protecting group for a-amino functions is the tert-butyloxycarbonyl (Boc) group. This group can be removed with dilute

solutions of strong acids such as 25% trifluoroacetic acid (TFA). The next Boc-amino acid is typically coupled to the amino acyl resin using dicyclohexylcarbodiimide (DCC). Following completion of the assembly, the peptide-resin is treated with anhydrous HF to cleave the benzyl ester link and liberate the free peptide. Side-chain functional groups are usually blocked during synthesis by benzyl-derived blocking groups, which are also cleaved by HF. The free peptide is then extracted from the resin with a suitable solvent, purified and characterized. Newly synthesized peptides can be purified, for example, by gel filtration, HPLC, partition chromatography and/or ion-exchange chromatography, and may be characterized by, for example, mass spectrometry or amino acid sequence analysis. In the Boc strategy, C-terminal amidated peptides can be obtained using benzhydrylamine or methylbenzhydrylamine resins, which yield peptide amides directly upon cleavage with HF.

In the procedures discussed above, the selectivity of the side-chain blocking groups and of the peptide-resin link depends upon the differences in the rate of acidolytic cleavage. Orthoganol systems have been introduced in which the side-chain blocking groups and the peptide-resin link are completely stable to the reagent used to remove the a-protecting group at each step of the synthesis. The most common of these methods involves the 9-fluorenylmethyloxycarbonyl (Fmoc) approach. Within this method, the side-chain protecting groups and the peptide-resin link are completely stable to the secondary amines used for cleaving the N-a-Fmoc group. The side-chain protection and the peptide-resin link are cleaved by mild acidolysis. The repeated contact with base makes the Merrifield resin unsuitable for Fmoc chemistry, and p-alkoxybenzyl esters linked to the resin are generally used.

Deprotection and cleavage are generally accomplished using TFA.

Those of ordinary skill in the art will recognize that, in solid phase synthesis, deprotection and coupling reactions must go to completion and the side-chain blocking groups must be stable throughout the entire synthesis. In

addition, solid phase synthesis is generally most suitable when peptides are to be made on a small scale.

Acetylation of the N-terminal can be accomplished by reacting the final peptide with acetic anhydride before cleavage from the resin. C-amidation is accomplished using an appropriate resin such as methylbenzhydrylamine resin using the Boc technology.

Following synthesis of a linear peptide, with or without N-acetylation and/or C-amidation, cyclization may be achieved by any of a variety of techniques well known in the art. Within one embodiment, a bond may be generated between reactive amino acid side chains. For example, a disulfide bridge may be formed from a linear peptide comprising two thiol-containing residues by oxidizing the peptide using any of a variety of methods. Within one such method, air oxidation of thiols can generate disulfide linkages over a period of several days using either basic or neutral aqueous media. The peptide is used in high dilution to minimize aggregation and intermolecular side reactions. This method suffers from the disadvantage of being slow but has the advantage of only producing H20 as a side product. Alternatively, strong oxidizing agents such as I2 and K3 Fe (CN) 6 can be used to form disulfide linkages. Those of ordinary skill in the art will recognize that care must be taken not to oxidize the sensitive side chains of Met, Tyr, Trp or His. Cyclic peptides produced by this method require purification using standard techniques, but this oxidation is applicable at acid pHs.

Suitable thiol-containing residues for use in such oxidation methods include, but are not limited to, cysteine, P, ß-dimethyl cysteine (penicillamine or Pen), ß, ß-tetramethylene cysteine (Tmc), ß, ß-pentamethylene cysteine (Pmc), p-mercaptopropionic acid (Mpr), P, ß-pentamethylene-ß-mercaptopropionic acid (Pmp), 2-mercaptobenzene, 2-mercaptoaniline and 2-mercaptoproline (For specific examples, refer to U. S.

Pat. No. 6031072). It will be readily apparent to those of ordinary skill in the art that, within each of these representative formulas, any of the above

thiol-containing residues may be employed in place of one or both of the third-containing residues recited.

In another embodiment, cyclization may be achieved by amide bond formation. For example, a peptide bond may be formed between terminal functional groups. Within another such embodiment, the linear peptide comprises a D-amino acid. Alternatively, cyclization may be accomplished by linking one terminus and a residue side chain or using two side chains, as in.

Residues capable of forming a lactam bond include lysine, ornithine (Orn), a-amino adipic acid, m-aminomethylbenzoic acid, (x,-diaminopropionic acid, glutamate or aspartate.

Methods for forming amide bonds are well known in the art and are based on well established principles of chemical reactivity. Within one such method, carbodiimide-mediated lactam formation can be accomplished by reaction of the carboxylic acid with DCC, DIC, EDAC or DCCI, resulting in the formation of an O-acylurea that can be reacted immediately with the free amino group to complete the cyclization. The formation of the inactive N-acylurea, resulting from O. fwdarw. N migration, can be circumvented by converting the O-acylurea to an active ester by reaction with an N-hydroxy compound such as 1-hydroxybenzotriazole, 1-hydroxysuccinimide, 1-hydroxynorbornene carboxamide or ethyl 2-hydroximino-2-cyanoacetate. In addition to minimizing O. fwdarw. N migration, these additives also serve as catalysts during cyclization and assist in lowering racemization. Alternatively, cyclization can be performed using the azide method, in which a reactive azide intermediate is generated from an alkyl ester via a hydrazide. Hydrazinolysis of the terminal ester necessitates the use of a t-butyl group for the protection of side chain carboxyl functions in the acylating component. This limitation can be overcome by using diphenylphosphoryl acid (DPPA), which furnishes an azide directly upon reaction with a carboxyl group. The slow reactivity of azides and the formation of isocyanates by their disproportionation restrict the usefulness of this method. The mixed anhydride method of lactam formation is widely used because of the facile removal of reaction by-products. The

anhydride is formed upon reaction of the carboxylate anion with an alkyl chloroformate or pivaloyl chloride. The attack of the amino component is then guided to the carbonyl carbon of the acylating component by the electron donating effect of the alkoxy group or by the steric bulk of the pivaloyl chloride t-butyl group, which obstructs attack on the wrong carbonyl group.

Mixed anhydrides with phosphoric acid derivatives have also been successfully used. Alternatively, cyclization can be accomplished using activated esters.

The presence of electron withdrawing substituents on the alkoxy carbon of esters increases their susceptibility to aminolysis. The high reactivity of esters of p-nitrophenol, N-hydroxy compounds and polyhalogenated phenols has made these"active esters"useful in the synthesis of amide bonds. The last few years have witnessed the development of benzotriazolyloxytris- (dimethylamino) phosphonium hexafluorophosphonate (BOP) and its congeners as advantageous coupling reagents. Their performance is generally superior to that of the well established carbodimide amide bond formation reactions.

In a further embodiment, a thioether linkage may be formed between the side chain of a thiol-containing residue and an appropriately derivatized a-amino acid. By way of example, a lysine side chain can be coupled to bromoacetic acid through the carbodiimide coupling method (DCC, EDAC) and then reacted with the side chain of any of the thiol containing residues mentioned above to form a thioether linkage. In order to form dithioethers, any two thiol containing side-chains can be reacted with dibromoethane and diisopropylamine in DMF.

Representative structures of cyclic peptides are provided in FIG. 1 (which is taken from U. S. Pat. No. 6031072). Within FIG. 1, certain cyclic peptides having the ability to modulate cell adhesion (shown on the left) are paired with similar inactive structures (on the right). The structures and formulas recited herein are provided solely for the purpose of illustration, and are not intended to limit the scope of the cyclic peptides described herein.

As was mentioned above, the effect of surface adhesion molecule antagonists on the delivery of drug through the skin is amplified by coadministration with an emulsion of known one or more skin permeation enhancing agents.

The term"Emulsion"refers to a mixture of two or more generally immiscible liquids, and is generally in the form of a colloid. The mixture may be of lipids, for example, which may be homogeneously or heterogeneously dispersed throughout the emulsion. Alternatively, the lipids may be aggregated in the form of, for example, clusters or layers, including monolayers or bilayers According to further features in preferred embodiments of the invention described below, the at least one skin permeation enhancing agent can be from any the following groups: alcohol, fatty alcohol, fatty acid ester, alkyl ester, polyol, amid, surfactant, sulfoxide, terpene and alkanone or combinations thereof.

According to still further features in the described preferred embodiments the alcohol is selected from the group consisting of ethanol, propanol and nonanol; (ii) the fatty alcohol is lauryl alcohol; (iii) the fatty acid is selected from the group consisting of valeric acid, caproic acid and capric acid; (iv) the fatty acid ester is selected from the group consisting of isopropyl myristate and isopropyl n-hexanoate; (v) the alkyl ester is selected from the group consisting of ethyl acetate and butyl acetate; (vi) the polyol is selected from the group consisting of propylene glycol, propanedione and hexanetriol; (vii) the sulfoxide is selected from the group consisting of dimethylsulfoxide and decylmethylsulfoxide ; (viii) the amide is selected from the group consisting of urea, dimethylacetamide and pyrrolidone derivatives; (ix) the surfactant is selected from the group consisting of sodium lauryl sulfate, cetyltrimethylammonium bromide, polaxamers, spans, tweens, bile salts and lecithin; (x) the terpene is selected from the group consisting of d-limonene, alpha-terpeneol, 1,8-cineole and menthone; and (xi) the alkanone is selected from the group consisting of N-heptane and N-nonane.

According to a preferred embodiment the at least one skin permeation enhancing agent includes at least one biodegradable skin permeation enhancer.

Examples for biodegradable skin permeation enhancer are dodecyl-N, N-dimethylamino acetate and N, N-dimethylamino isopropionate.

According to a preferred embodiment of the present invention the therapeutic agent (s) can be drugs, genetic materials, cells, proteins such as for example hormones, enzymes, vaccines and antibodies.

"Pharmaceutical agent"or"drug"refers to any therapeutic or prophylactic agent which may be used in the treatment (including the prevention, alleviation, or cure) of a malady, affliction, disease or injury in a patient. Therapeutically useful peptides, polypeptides and polynucleotides may be included within the meaning of the term pharmaceutical or drug.

Virtually any drug can be applied in the present invention, due to the ability of the carrier to transfer high molecular weight agents. Examples include, but are not limited to antibiotic agents, free radical generating agents, anti fungal agents, anti-viral agents, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, non-steroidal anti inflammatory drugs, immunosuppressants, anti-histamine agents, retinoid agents, tar agents, anti-pruritic agents, antidepressants, anti cancer, antiphsychotics, contarceptives, toxins, antihypertensives, cerebral or peripheral vasodilators, anaesthetics and scabicide agents.

Hereinafter, the phrase"pharmaceutically acceptable carrier"refers to a carrier, which does not cause significant irritation to the individual treated and does not abrogate the biological activity and properties of the active ingredient.

The therapeutic agent can also be a prodrug, which is activatable prior to, during, or following penetration of the therapeutic agent into a tissue region.

As used herein in the specification and in the claims section, which follows, the term"prodrug"refers to an agent which is inactive but which is convertable into an active form via enzymatic, chemical or physical activators.

An example, without limitation, of a prodrug would be a compound, which is administered as an inactive ester to facilitate transmittal across a cell

membrane where water solubility is not beneficial, but which is then hydrolyzed to the carboxylic acid and active form once inside the cell where water solubility is beneficial.

A prodrug (for example an enzyme) can be activated just prior to stinging cell discharge by providing an activator compound (for example an ion), which can be diffused or pumped (during discharge) into the capsule.

Alternatively, specific enzymes, molecules or pH conditions present in the target tissues, can activate the prodrug.

"Protein"refers to molecules comprising, and preferably consisting essentially of, a-amino acids in peptide linkages. Included within the term "protein"are globular proteins such as albumins, globulins and histones, and fibrous proteins such as collagens, elastins and keratins. Also included within the term"protein"are"compound proteins,"wherein a protein molecule is united with a nonprotein molecule, such as nucleoproteins, mucoproteins, lipoproteins and metalloproteins. The proteins may be naturally-occurring, synthetic or semi-synthetic. Thus, the synergistic effect of the skin permeation enhancing agent and the surface adhesion molecule antagonist enable the passage of medium and even large polypeptides and hormones, such as insulin, growth factor, TSH, FSH, LH and male and female sex hormones.

The compositions described herein may be administered as part of a sustained release formulation (i. e., a formulation such as a capsule or sponge that effects a slow release of cyclic peptide following administration). Such formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations may contain a cyclic peptide and one or more skin permeation enhancers dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane (see, e. g., European Patent Application 710,491 A).

Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of the cyclic peptide and the skin permeation enhancer release. The amount of

each the cell adhesion modulating agent and the skin permeation enhancer, independently, and the ratio between them within a sustained release formulation depends upon the site of implantation, the rate, the duration of release and the nature of the condition to be treated or prevented.

The combination of surface adhesion molecule modulating agent and skin permeation enhancing agent of the present invention can also be utilized for vaccination. Vaccine antigens can be delivered to specialized immune cells underlying the skin or into blood circulation (as described above).

Absorption into the blood stream following transdermal delivery will most likely result in transport of the antigen to the phagocytic cells of the liver, spleen, and bone marrow. Since such cells serve as antigen presenting cells, a strong immunogenic response will be elicited leading to effective immunization.

Further advantages of the present invention is that it is not necessary that the administered medicament is completely soluble in the carrier, preparation of the compositions is uncomplicated, and the same or similar carriers are effective for different sort of drugs.

The combination of medicament and carrier will preferably be in the liquid state at ambient temperatures, although the invention also extends to such combinations, which are semi-solid or semi-liquid.

The combination of the surface adhesion molecule modulating agent and skin permeation enhancing agent may be linked to a targeting agent to further increase the local concentration of these agent. This is one of the main advantages of the present invention. For example, a targeting agent may be an antibody that binds to a fibrin degradation product or a cell enzyme such as a peroxidase that is released by granulocytes or other cells in necrotic or inflamed tissues. These methods, which are beneficial in theory, are not feasible with the currently used methods. The ability of the two agents to act in synergy enable the passage of high molecular weight agents, thus, antibodies will easily penetrate into the skin, to the blood stream and into the target organ.

Monoclonal antibodies specific for the surface cell adhesion molecules of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6: 511-519,1976, and improvements thereto.

Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity from spleen cells obtained from an animal immunized as described above. The spleen cells are immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. Single colonies are selected and their culture supernatants tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies, with or without the use of various techniques known in the art to enhance the yield. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. Antibodies having the desired activity may generally be identified using immunofluorescence analyses of tissue sections, cell or other samples where the target cadherin is localized.

Within certain embodiments, instead of surface adhesion molecule modulating antagonists, antibodies to surface adhesion molecules (or, preferably, antigen-binding fragments thereof) may be used in combination with the skin permeation enhancing agents. Such antibodies or fragments may be used, for example, for treatment of demyelinating diseases, such as MS, or to inhibit interactions between tumor cells, as described above. The use of Fab fragments is generally preferred.

There are a variety of assay formats known to those of ordinary skill in the art for using an antibody to detect a target molecule in a sample. See, e. g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For example, the assay may be performed in a Western blot format, wherein a protein preparation from the biological sample is submitted to gel electrophoresis, transferred to a suitable membrane and allowed to react

with the antibody. The presence of the antibody on the membrane may then be detected using a suitable detection reagent, as described below.

"Genetic material"refers generally to nucleotides and polynucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The genetic material may be made by synthetic chemical methodology known to one of ordinary skill in the art, or by the use of recombinant technology, or by a combination thereof. The DNA and RNA may optionally comprise unnatural nucleotides and may be single or double stranded."Genetic material"also refers to sense and anti-sense DNA and RNA, that is, a nucleotide sequence which is complementary to a specific sequence of nucleotides in DNA and/or RNA.

The administration of cells which was mentioned above is related to gene therapy approach which have evolved ex vivo; and in vivo gene therapy.

In ex vivo gene therapy cells are removed from a patient or are derived from another source, and while being cultured are treated in vitro. Generally, a functional replacement gene is introduced into the cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient. These genetically reimplanted cells have been shown to express the transfected genetic material in situ. Here again, the synergistic effect of surface adhesion molecule modulating agent and skin permeaton enhancing agent on the penetration of large agents, will enable passage of large amounts of modificated cells, in a constant rate and without being painful to the subjects.

Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented).

Appropriate dosages and the duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient's disease and the method of administration. In general, an appropriate dosage and treatment regimen provides the cell adhesion modulating antagonist (s) and the skin permeation enhancing agent (s) in an

amount sufficient to provide therapeutic and/or prophylactic benefit.

Appropriate dosages may generally be determined using experimental models and/or clinical trials. In general, the use of the minimum dosage that is sufficient to provide effective therapy is preferred. Patients may generally be monitored for therapeutic effectiveness using assays suitable for the condition being treated or prevented, which will be familiar to those of ordinary skill in the art.

According to yet another aspect of the present invention there is provided a method of transdermal or mucosal delivery of at least one therapeutic agent, the method comprising the step of topically administering the at least one therapeutic agent in the presence of a pharmaceutical carrier includes at least one skin permeation enhancing agent and at least one surface adhesion molecule modulating agent; The at least one skin permeation enhancing agent and the at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal or the mucosal delivery of the at least one therapeutic agent.

Compositions of the present invention may be formulated for any appropriate manner of administration onto a tissue region, including for example, topical, oral, nasal, intracranial, administration. For certain topical applications, formulation as a cream or lotion, using well known components, is preferred.

The present invention can be served also for increasing vasopermeability in a mammal by administering one or more surface cell adhesion molecule antagonists in combination with at least one skin permeation enhancing agent and at least one therapeutic agent. Within blood vessels, endothelial cell adhesion (mediated by N-cadherin) results in decreased vascular permeability.

Accordingly, surface cell adhesion molecule antagonists and skin permeation enhancing agents, such as, those described herein may be used to increase vascular permeability. In certain embodiments, preferred surface cell adhesion molecule antagonists for use within such methods include peptides capable of decreasing both endothelial and tumor cell adhesion. Such peptides may be

used to facilitate the penetration of anti-tumor therapeutic agents (e. g., monoclonal antibodies) through endothelial cell permeability barriers and tumor barriers. The combination of the surface adhesion molecule modulating agent and skin permeation enhancing agent will enable better penetration of the therapeutic agent in comparison to each of the agents individually.

According to still another aspect of the present invention there is provided a device for transdermal application of at least one therapeutic agent, the device comprising a solid support having on a tissue region-contacting side thereof a pharmaceutical carrier including at least one skin permeation enhancing agent and at least one surface adhesion molecule modulating agent; the at least one skin permeation enhancing agent and the at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal delivery of the at least one therapeutic agent. The solid support may be any material known to those of ordinary skill in the art such as for example microtiter plate, a nitrocellulose filter or another suitable membrane.

Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic such as polystyrene or polyvinylchloride. The surface adhesion molecule and the skin permeation enhancing agent may be attached on the solid support using a variety of techniques known to those in the art, which are amply described in the patent and scientific literature According to an additional aspect of the present invention there is provided a device for transdermal or mucosal application of at least one therapeutic agent, the device comprising a solid support having on tissue-contacting side thereof a pharmaceutical composition including: a therapeutic effective amount of at least one therapeutic agent; and a pharmaceutical carrier including: at least one skin permeation enhancing agent; and at least one surface adhesion molecule modulating agent; the at least one skin permeation enhancing agent and the at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal or mucosal delivery of the at least one therapeutic agent.

Preferably the solid support is selected from the group consisting of a patch, a foil, a plaster and a film or other support which are known in the art.

According to yet an additional aspect of the present invention there is provided a method of transdermal or mucosal delivery of at least one therapeutic agent, the method comprising the step of placing a device for transdermal application of the at least one therapeutic agent over a tissue region to which the at least one therapeutic agent has been previously applied, the device including a solid support having on a tissue region-contacting side thereof a pharmaceutical carrier including: at least one skin permeation enhancing agent and at least one surface adhesion molecule modulating agent; the at least one skin permeation enhancing agent and the at least one surface adhesion molecule modulating agent acting in synergy for enhancing the transdermal or thee mucosal delivery of the at least one therapeutic agent.

"Tissue region"refers generally to specialized cells, which may perform a particular function. The term"tissue"may refer to an individual cell or a plurality or aggregate of cells, for example, membranes, blood skin or organs.

The term"tissue"also includes reference to an abnormal cell or a plurality of abnormal cells. Exemplary tissues include myocardial tissue, including myocardial cells and cardiomyocites, membranous tissues, including endothelium and epithelium, laminae, connective tissue, including interstitial tissue, and tumors According to still an additional aspect of the present invention there is provided a method of transdermal or mucosal delivery of at least one therapeutic agent, the method comprising the step of placing a device for transdermal or mucosal application of the at least one therapeutic agent over a tissue region, the device including a solid support having on a skin-contacting side thereof a pharmaceutical composition including: a therapeutic effective amount of at least one therapeutic agent; and a pharmaceutical carrier which include at least one skin permeation enhancing agent; and at least one surface adhesion molecule modulating agent; the at least one skin permeation enhancing agent and the at least one surface adhesion molecule modulating

agent acting in synergy for enhancing the transdermal delivery of the at least one therapeutic agent Excipiefzts and additives for the described phartnaceutical compositions : "Excipients", which refer hereinunder to inert substances added to a pharmaceutical composition to further facilitate processes and administration of the active ingredients will be added to the present invention pharmaceutical composition. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of active ingredients may be found in"Remington's Pharmaceutical Sciences,"Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

While various routes for the administration of active ingredients are possible, and were previously described, for the purpose of the present invention, the topical route is preferred, and is assisted by a topical carrier. The topical carrier is one, which is generally suited for topical active ingredients administration and includes any such materials known in the art. The topical carrier is selected so as to provide the composition in the desired form, e. g., as a liquid or non-liquid carrier, lotion, cream, paste, gel, powder, ointment, solvent, liquid diluent, drops and the like, and may be comprised of a material of either naturally occurring or synthetic origin. It is essential, clearly, that the selected carrier does not adversely affect the active agent or other components of the topical formulation, and which is stable with respect to all components of the topical formulation. Examples of suitable topical carriers for use herein include water, alcohols and other nontoxic organic solvents, glycerin, mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetable oils, parabens, waxes, and the like. Preferred formulations herein are colorless, odorless ointments, liquids, lotions, creams and gels.

Ointments are semisolid preparations, which are typically based on petrolatum or other petroleum derivatives. The specific ointment base to be

used, as will be appreciated by those skilled in the art, is one that will provide for optimum active ingredients delivery, and, preferably, will provide for other desired characteristics as well, e. g., emolliency or the like. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and nonsensitizing. As explained in Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa.: Mack Publishing Co., 1995), at pages 1399-1404, ointment bases may be grouped in four classes: oleaginous bases; emulsifiable bases; emulsion bases; and water-soluble bases. Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum. Emulsifiable ointment bases, also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin and hydrophilic petrolatum. Emulsion ointment bases are either water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, and include, for example, cetyl alcohol, glyceryl monostearate, lanolin and stearic acid. Preferred water-soluble ointment bases are prepared from polyethylene glycols of varying molecular weight; again, reference may be made to Remington: The Science and Practice of Pharmacy for further information.

Lotions are preparations to be applied to the skin surface without friction, and are typically liquid or semiliquid preparations, in which solid particles, including the active agent, are present in a water or alcohol base.

Lotions are usually suspensions of solids, and may comprise a liquid oily emulsion of the oil-in-water type. Lotions are preferred formulations herein for treating large body areas, because of the ease of applying a more fluid composition. It is generally necessary that the insoluble matter in a lotion be finely divided. Lotions will typically contain suspending agents to produce better dispersions as well as compounds useful for localizing and holding the active agent in contact with the skin, e. g., methylcellulose, sodium carboxymethylcellulose, or the like.

Creams containing the selected active ingredients are, as known in the art, viscous liquid or semisolid emulsions, either oil-in-water or water-in-oil.

Cream bases are water-washable, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase, also sometimes called the"internal"phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation, as explained in Remington, supra, is generally a nonionic, anionic, cationic or amphoteric surfactant.

Gel formulations are preferred for application to the scalp. As will be appreciated by those working in the field of topical active ingredients formulation, gels are semisolid, suspension-type systems. Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous, but also, preferably, contain an alcohol and, optionally, an oil.

Carriers for nucleic acids include, but are not limited to, liposomes including targeted liposomes, nucleic acid complexing agents, viral coats and the like. However, transformation with naked nucleic acids may also be used.

Various additives, known to those skilled in the art, may be included in the topical formulations of the invention. For example, solvents may be used to solubilize certain active ingredients substances. Other optional additives include skin permeation enhance, opacifiers, anti-oxidants, gelling agents, thickening agents, stabilizers, and the like.

The topical compositions of the present invention may also be delivered to the skin using conventional dermal-type patches or articles, wherein the active ingredients composition is contained within a laminated structure, that serves as a drug delivery device to be affixed to the skin. In such a structure, the active ingredients composition is contained in a layer, or"reservoir", underlying an upper backing layer. The laminated structure may contain a single reservoir, or it may contain multiple reservoirs. In one embodiment, the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during active ingredients delivery. Examples of suitable skin contact adhesive

materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like. The particular polymeric adhesive selected will depend on the particular active ingredients, vehicle, etc., i. e., the adhesive must be compatible with all components of the active ingredients-containing composition. Alternatively, the active ingredients-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form.

The backing layer in these laminates, which serves as the upper surface of the device, functions as the primary structural element of the laminated structure and provides the device with much of its flexibility. The material selected for the backing material should be selected so that it is substantially impermeable to the active ingredients and to any other components of the active ingredients-containing composition, thus preventing loss of any components through the upper surface of the device. The backing layer may be either occlusive or nonocclusive, depending on whether it is desired that the skin become hydrated during active ingredients delivery. The backing is preferably made of a sheet or film of a preferably flexible elastomeric material.

Examples of polymers that are suitable for the backing layer include polyethylene, polypropylene, and polyesters.

During storage and prior to use, the laminated structure includes a release liner. Immediately prior to use, this layer is removed from the device to expose the basal surface thereof, either the active ingredients reservoir or a separate contact adhesive layer, so that the system may be affixed to the skin.

The release liner should be made from a active ingredients/vehicle impermeable material.

Such devices may be fabricated using conventional techniques, known in the art, for example by casting a fluid admixture of adhesive, active ingredients and vehicle onto the backing layer, followed by lamination of the release liner. Similarly, the adhesive mixture may be cast onto the release

liner, followed by lamination of the backing layer. Alternatively, the active ingredients reservoir may be prepared in the absence of active ingredients or excipient, and then loaded by"soaking"in a active ingredients/vehicle mixture.

As with the topical formulations of the invention, the active ingredients composition contained within the active ingredients reservoirs of these laminated system may contain a number of components. In some cases, the active ingredients may be delivered"neat,"i. e., in the absence of additional liquid. In most cases, however, the active ingredients will be dissolved, dispersed or suspended in a suitable pharmaceutically acceptable vehicle, typically a solvent or gel. Other components, which may be present, include preservatives, stabilizers, surfactants, and the like.

The pharmaceutical compositions herein described may also comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycols.

Dosing is dependent on the type, the severity and manifestation of the affliction and on the responsiveness of the subject to the active ingredients, as well as the dosage form employed the potency of the particular conjugate and the route of administration utilized. Persons of ordinary skill in the art can easily determine optimum dosages, dosing methodologies and repetition rates.

The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e. g., Fingl, et al., 1975, in"The Pharmacological Basis of Therapeutics", Ch. 1 p. l).

Thus, depending on the severity and responsiveness of the condition to be treated, dosing can be a single or repetitive administration, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the skin lesion is achieved.

In some aspects the present invention utilizes in vivo and ex vivo (cellular) gene therapy techniques, which involve cell transformation and gene knock-in type transformation. Gene therapy as used herein refers to the

transfer of genetic material (e. g. DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition or phenotype. The genetic material of interest encodes a product (e. g., a protein, polypeptide, peptide, functional RNA, antisense RNA) whose production in vivo is desired. For example, the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide or peptide of therapeutic value. For review see, in general, the text"Gene Therapy" (Advanced in Pharmacology 40, Academic Press, 1997).

As was mentioned before two basic approaches to gene therapy have evolved (1) ex vivo; and (ii) in vivo gene therapy. In ex vivo gene therapy cells are removed from a patient or are derived from another source, and while being cultured are treated in vitro. Generally, a functional replacement gene is introduced into the cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient. These genetically reimplanted cells have been shown to express the transfected genetic material in situ.

In in vivo gene therapy, target cells are not removed from the subject rather the genetic material to be transferred is introduced into the cells of the recipient organism in situ, that is within the recipient. In an alternative embodiment, if the host gene is defective, the gene is repaired in situ (Culver, 1998. (Abstract) Antisense DNA & RNA based therapeutics, February 1998, Coronado, CA). These genetically altered cells have been shown to express the transfected genetic material in situ.

The gene expression vehicle is capable of delivery/transfer of heterologous nucleic acid into a host cell. The expression vehicle may include elements to control targeting, expression and transcription of the nucleic acid in a cell selective manner as is known in the art. It should be noted that often the 5'UTR and/or 3'UTR of the gene may be replaced by the 5'UTR and/or 3'UTR of the expression vehicle. Therefore, as used herein the expression vehicle

may, as needed, not include the 5'UTR and/or 3'UTR of the actual gene to be transferred and only include the specific amino acid coding region.

The expression vehicle can include a promoter for controlling transcription of the heterologous material and can be either a constitutive or inducible promoter to allow selective transcription. Enhancers that may be required to obtain necessary transcription levels can optionally be included.

Enhancers are generally any nontranslated DNA sequence, which works contiguously with the coding sequence (in cis) to change the basal transcription level dictated by the promoter. The expression vehicle can also include a selection gene as described herein below.

Vectors can be introduced into cells or tissues by any one of a variety of known methods within the art. Such methods can be found generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York 1989,1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland 1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, MI 1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor MI (995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston MA 1988) and Gilboa et al. (Biotechniques 4 (6): 504-512,1986) and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see United States patent 4,866,042 for vectors involving the central nervous system and also United States patents 5,464,764 and 5,487,992 for positive-negative selection methods.

Introduction of nucleic acids by infection offers several advantages over the other listed methods. Higher efficiency can be obtained due to their infectious nature. Moreover, viruses are very specialized and typically infect and propagate in specific cell types. Thus, their natural specificity can be used to target the vectors to specific cell types in vivo or within a tissue or mixed culture of cells. Viral vectors can also be modified with specific receptors or ligands to alter target specificity through receptor mediated events.

A specific example of DNA viral vector introducing and expressing recombination sequences is the adenovirus-derived vector Adenop53TK. This vector expresses a herpes virus thymidine kinase (TK) gene for either positive or negative selection and an expression cassette for desired recombinant sequences. This vector can be used to infect cells that have an adenovirus receptor which includes most tissues of epithelial origin as well as others. This vector as well as others that exhibit similar desired functions can be used to treat a mixed population of cells and can include, for example, in vitro or ex vivo culture of cells, a tissue or a human subject.

Features that limit expression to particular cell types can also be included. Such features include, for example, promoter and regulatory elements that are specific for the desired cell type.

In addition, recombinant viral vectors are useful for in vivo expression of a desired nucleic acid because they offer advantages such as lateral infection and targeting specificity. Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny.

Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.

Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting.

Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

The following experiments were aimed to assess the effects of an E-cadherin antagonist administered together with a skin permeation enhancing agent on the permeability of low and high molecular weight proteins in vitro and in vivo. The results clearly demonstrate that the administration of the skin permeation enhancing agent together with the E-cadherin antagonist has a dramatic synergistic effect on the permeability of the assessed proteins through the skin.

MATERIALSAND EXPERIMENTAL PROCEDURES Marker proteins : a-Lactalbumin (14 kDa) was biotinylated using the EZ-Link Sulfo-NHS-LC biotinylation kit (Pierce, Rockford, IL). Biotinylated cc-Lactalbumin was purified using a size fractionation column chromatography provided with the kit (D-Salt-Dextran). Skin permeation preparations : (i) a skin permeation enhancing emulsion was prepared containing final concentration of 33.75 % Fractionated Coconut Oil (FCO) together with 0.003 % tocopheryl Acetate, 0.006 % nipagine and 3.75 % oleic acid, 10 % lecithin and 3 % Tween 80; and (ii) an E-Cadherin antagonist was prepared as described in U. S. Pat. No. 6,031,072.

Preparation of ski portions : Nude mouse skin was used since it is considered one of the best substitutes for human skin for transdermal delivery assays (Methods Find Exp Clin Pharmacol 1997, Jun; 19 (5): 335-41). Ten to twelve weeks nude mice were sacrificed, an incision was made on the dorsal part of the animal and a skin sheet including the dorsal and ventral areas was removed and placed in a buffered environment. After removing the subdermal fat, the skin was divided into 6-8 portions and placed in the Franz cell system.

In vitro assays : For the performance of the in vitro assays, four horizontal Franz cell systems (6 cells each, Figure 1) were employed. The lower cells of the Franz cells were blocked overnight, prior to the assay, with a

blocking solution containing 0.001 % tryptone, 1 % Penicilin-Streptomycin, in a PBS buffer, pH 7.0. For each experiment, skin from a single mouse was divided into six substantially equal pieces and placed on a Franz cells system, where the inner part of the skin is facing the lower cell. The lower cells were filled with 5 ml buffer containing 0.001 % tryptone, 1 % Penicilin-Streptomycin, in a PBS buffer, pH 7.0. Air trapped in the interphase between the buffer and the skin was removed in order to insure full contact of the skin with the buffer. In some of the cases the skin portions were pre-treated with an E-cadherin antagonist solution, prior to the application of the marker-containing emulsions as is further described hereinunder. Two hundred microliters of a marker-containing emulsion were then placed in the upper cell, in contact with the skin surface. Duplicates of one hundred microliter samples were then collected at fixed time intervals from the lower Franz cell and transferred to an ELISA plate. The missing buffer volume from the lower Franz cell was then replaced with fresh buffer.

ELISA assay : 100 p1 samples were placed into wells of a Nunc-Immunoplate Maxisorp microtiterplate and were incubated overnight at 4'C. In Parallel, a known amount of biotinylated protein was added to separate wells for the generation of a standard curve. Thereafter, the wells were washed three times with a washing buffer (PBS + 0.1 % Tryptone + 0.05% Tween 20).

The wells were then blocked with 100 ul of PBS + 1 % Tryptone for one hour at room temperature, washed three times with 200 pl of washing buffer, and filled with 100 p, l of Streptavidin-HRP solution (Jackson Immunoresearch Labs). After incubation of 30 minutes at room temperature, the wells were washed three times with washing buffer as above and filled with 100 ul of TMB (3,3,5,5,'-Tetramethylbenzidine) (Savyon Diagnostics, Ashdod, Israel).

Following the development of color, 100 ul of fixing solution was added and the results were read in an Anthos HT LL ELISA reader at 450 nm.

Fluoreseftce-based Assays : The following fluorescent reagents were used as molecular weight markers in a Franz cells in vitro diffusion assays: Fluorescein sodium (376 Da, Merck, Cat. No. 1.03992.0050), Sulforhodamine

B monosodium (581 Da, Fluka, Cat. No. 86183), FITC-Dextran (4,000 Da, Sigma, FD-4) and FITC-Dextran (10,000 Da, Sigma, Cat. No. FD-IOS). The sulforhodamine B was monitored at the emission wavelength of 590 nm, following excitation at 544 nm, while the other three fluorescent markers were monitored at 538 nm, following excitation at 485 nm. A standard curve for each fluorophore was prepared so as to allow for the calculation of the marker's concentration.

EXPERIMENTAL RESULTS EXAMPLE I Effect of E-Cadherin inAlibitor given after tAle skin pernzeation enhancing emulsion on traiisdermalpermeation of biotinylated a-Lactalbumin. (a-Lac) Experimental Design : In order to further prove the concept of the combined effect of E-Cadherin antagonist together with a skin permeation enhancing emulsion to intensify the transfer of peptides through the skin, another protein, a-Lactalbumin, was assessed using the same methods. Since a-lactalbumin was not available commercially as a biotinylated protein, the protein was biotinylated in the laboratory, purified and was concentrated to a working concentration of three fold the original suspension.

As before, Franz cells were covered with nude mice skin taken from two mice. Three cells were treated for one hour with 200 ul of 0.5 mg/ml E-cadherin antagonist (the final amount was 100 gag). The other three cells were treated with 200 pl of lower cell buffer (0.1 % BSA in PBS). At the end of the incubation time the E-cadherin antagonist was removed from the upper cells and an emulsion of biotinylated a-lactalbumin and skin permeation enhancing emulsion was added to all six cells. Samples were taken at intervals of 30 minutes up to 5 hours. The result of this experiment are depicted in Figure 2.

Experimental Results : Pre-treatment of skin portions for one hour with 100 pg of E-cadherin antagonist created pores in the skin which were large enough to allow the transfer of a-lactalbumin in a skin permeation enhancer for three hours. The kinetics of the two experiments was similar, wherein the maximal effect obtained after three hours was followed by a decline in the protein amount.

The main difference between the experiments was in the amounts of the transferred proteins. This experiment shows that the combination of an E-cadherin antagonist with a skin permeation enhancer has a dramatic effect on the permeation of the two proteins, iii vitro, through the skin.

EXAMPLE 2 Effect of E-cadElerin antagonist mixed witA2 skin permeation enhanci7lg emulsion on a-lactalbumin permeation Experimental design : The combined effect of an E-cadherin antagonist mixed with a skin permeation enhancing emulsion on a-lactalbumin permeation was assessed. A skin permeation enhancing emulsion containing 10 u. g E-cadherin antagonist and 100 pg a-lactalbumin was administered onto nude mice skin portions placed in a Franz cells system. A second emulsion containing only 100 jig a-lactalbumin was applied as control. The design of this experiment was similar to that described under Example 1 above with one exception: the E-cadherin antagonist was not used as a part of a pre-treatment of the skin, rather it was dissolved in the emulsion itself.

Experimental Results : The result of this experiment show that the modification made in the detection system increased its sensitivity by almost 15 fold (Figure 3 and Figure 4). In addition, as is demonstrated in Figure 4, E-cadherin antagonist in a concentration of 100 ng was as effective as in a concentration of 100 pLg in its

ability to increase the permeation, in the presence of skin permeation enhancing emulsion.

EXAMPLE 3 EnAIancement of the in vivo pe7meation of monomeric Insulin I Experimental Design : The combined effect of an E-cadherin antagonist and a skin permeation enhancing emulsion, in vivo, on monomeric insulin was assessed in nude mice.

A concentration of 1 mg/ml insulin was chosen for this assay. The in vivo experiment was divided into two stages: a pre-treatment stage with E-cadherin antagonist and a treatment stage with insulin. This design was used to avoid potential interference between the E-cadherin antagonist and the compounds of the insulin emulsion.

Two patches were prepared: E-cadherin antagonist patch: 1 mg/ml E-cadherin antagonist solution reinforced with lecithin was prepared the day before the experiment. 0.7 ml of this solution was applied on a 2.3 mm2 patch.

Insulin patch: 0.7 ml of insulin (54 IU total), with a skin permeation enhancer emulsion were applied on another set of patches.

On the day of the experiment, 15 NMRI (5 in each group) male nude mice were starved for 3 hours. Blood samples were taken by vein puncture from the tail and the blood glucose level was measured using a Glucometer (g) Gx and Glucostixs@. In the pretreatment stage, groups of 5 mice were treated with either placebo patches or E-cadherin antagonist patches (n=5) for one hour and a third group was maintained on placebo treatment through the entire experiment. The patches were hold on the back of the mice by a tailor-make elastic socket that ensured a proper function of the patch without harming the animals. At the next stage, E-cadherin antagonist patch and the placebo patch were removed and replaced with insulin patches. Mice were then tested for their blood glucose level at 30 minutes, 1 hour, 2, hours, 3 hours, and 5 hours after the application of the insulin patch.

Experinzental esults : As can be clearly seen from Figure 5, pretreatment with E-cadherin antagonist prior to the application of the insulin patch resulted in a significance increase in the permeation of the skin to insulin. The maximal effect was observed two hours after the application of the patch (Figure 5). Although the experimental results varied due to intensive bleeding of the animals, the statistical analysis demonstrated strong effect of the skin permeation enhancing emulsion contained insulin (T = 0.02435) on the glucose level in comparison to the placebo, whereas the effect of E-cadherin antagonist pretreatment was much stronger. These results provide the first evidence that E-cadherin antagonist together with skin permeation enhancing emulsion has an in vivo effect on the permeation of insulin through skin.

EXAMPLE 4 Enhancement of tAle in vivo permeation of monomeric Insulin II Experimental Design : The combined effect of a skin permeation enhancing emulsion together with an E-cadherin antagonist on insulin permeation through skin was further assessed on NMRI mice. The mice were deprived from food for 3-4 hours and were treated as follows: placebo patch, insulin 25 IU/cm2 with or without 0.15 llg/cm2 E-cadherin antagonist in skin permeation enhancing emulsion; and insulin 6.4 IU/cm2 with or without 0.15 Fg/cm2 E-cadherin antagonist in skin permeation enhancing emulsion. Blood samples were taken at 2 hour intervals and assessed using a Glucometer.

Experimental results : As is demonstrated in Figure 6 the effect of E-cadherin antagonist and skin permeation enhancing emulsion permeation of both low and high dose of insulin can be clearly seen. At high insulin concentration the presence of E-cadherin antagonist mainly affect the kinetics at which insulin lower glucose concentration in the blood. At low insulin concentration, the addition of E-cadherin antagonist is much more significant. By assuming a linear

correlation between insulin permeation and glucose level, and by summarizing the above data, it can be estimated that of E-cadherin antagonist at a surface concentration of 0.15 llg/cm2 increases by four fold the permeation of 6.4 IU/cm2 insulin.

EXAMPLE 5 Figure 7 represents a typical Franz type diffusion chambers experiment using fluorescent dyes as molecular weight markers. Fluorescein (376 Da), Sulforhodamine B (581 Da) and FITC-Dextran (4 kDa) were incorporated at 0.5 mg/ml in a skin permeation enhancing emulsion. 200 p1 of each sample were loaded onto four separate chambers and the results reported are the average of these replicates. The cumulating amount in nanograms of fluorophore permeating through nude mice skin into the bottom reservoir of a Franz cell is plotted against time. This experiment shows that the emulsion is an efficient vehicle for the transdermal delivery of molecules up to at least 10 kDa. The delivery is efficient for at least 7 hours since saturation was not reached at the end of the experiment.

EXAMPLE 6 Figure 8 represents a Franz type diffusion chambers experiment in which the impact of an E-cadherin antagonist on transdermal permeation of Sulforhodamine B (581 Da) was studied. Sulforhodamine B was incorporated at 0.5 mg/ml in an emulsion as described above together with 3 concentrations of E-cadherin antagonist (0,1 and 10 pg/ml). 200 zip of each sample were loaded onto four separate chambers and the results shown are the average of these replicates. The cumulating amount in nanograms of fluorophore permeating through nude mice skin into the bottom reservoir is plotted against time. This experiment shows that the addition of 10 llg/ml E-cadherin antagonist to the emulsion further increases permeation of Sulforhodamine B.

The results are statistically significant between 4 to 5 hours (p<0.05). The

delivery is efficient for at least 6 hours since saturation was not reached at the end of the experiment.

EXAMPLE 7 Figure 9 represents a Franz type diffusion chambers experiment in which the impact of E-cadherin antagonist on transdermal permeation of FITC-Dextran 4 kDa (about 4,000 Da) was studied. FITC-Dextran 4 kDa was incorporated at 0.5 mg/ml in the emulsion together with 4 concentrations of E-cadherin antagonist (0,0.01,0.1 and 1 pg/ml). 200 u. l of each sample were loaded onto four separate Franz chambers and the results reported are the average of these replicates. The cumulating amount in nanograms of fluorophore permeating through nude mice skin into the bottom reservoir is plotted against time. This experiment shows that the addition of E-cadherin antagonist to the emulsion further increases permeation of FITC-Dextran (4 kDa) in a dose dependent manner. The results are statistically significant for the highest dose of E-cadherin antagonist (1 u. g/ml) from 4 hours up to the end of the experiment (p<0.05). The delivery is efficient for at least 7 hours since saturation was not reached at the end of the experiment.

EXAMPLE 8 Figure 10 represents a Franz type diffusion chambers experiment in which the impact of E-cadherin antagonist on transdermal permeation of FITC-Dextran 4 kDa (about 4,000 Da) was studied. FITC-Dextran (4 kDa) was incorporated at 2 mg/ml in the emulsion together with 4 concentrations of E-cadherin antagonist (0,0.1,1 and 10 llg/ml). 200, ul of each sample were loaded onto four separate chambers and the results shown are the average of these replicates. The cumulating amount in nanograms of fluorophore permeating through nude mice skin into the bottom reservoir is plotted against time. This experiment confirms that the addition of E-cadherin antagonist to the emulsion further increases the permeation of FITC-Dextran 4 kDa in a dose dependent manner. The delivery is efficient for at least 6 hours since saturation was not reached at the end of the experiment.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference in the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.