Patent application title: FIBROBLAST GROWTH FACTOR-9 PROMOTES HAIR FOLLICLE REGENERATION AFTER WOUNDING
Inventors:
George Cotsarelis (Berwyn, PA, US)
Oh Sang Kwon (Seoul, KR)
IPC8 Class: AA61K3818FI
USPC Class:
Class name:
Publication date: 2015-08-06
Patent application number: 20150216934
Abstract:
The present invention provides methods for treating hair loss, treating,
inhibiting, or suppressing a degenerative skin disorder, treating
androgenetic alopecia (AGA), generating new hair follicles (HF), and
increasing the size of existing HF. The methods comprise epidermal
disruption or administration of wnt, and administration of a fibroblast
growth factor-9 polypeptide or another compound that upregulates sonic
hedgehog gene signaling.Claims:
1. A method of treating hair loss in a subject comprising the steps of
(a) disrupting the epidermis in the region of said hair loss in said
subject and (b) administering a composition comprising a fibroblast
growth factor-9 polypeptide to said subject.
2. The method of claim 1, wherein said hair loss is due to androgenetic alopecia (AGA).
3. The method of claim 2, wherein said AGA is male pattern baldness.
4. The method of claim 2, wherein said AGA is female pattern baldness.
5. The method of claim 1, wherein said hair loss is the result of a skin injury.
6. The method of claim 1, wherein said hair loss is in the scalp or eyebrow of said subject.
7. The method of claim 1, wherein said hair loss is in scarred skin tissue of said subject.
8. The method of claim 1, wherein said step of administering is performed 3-12 days after said step of disrupting.
9. The method of claim 1, wherein said step of disrupting is performed by exposing the region of said hair loss to a mechanical or chemical stimulus.
10. The method of claim 1, wherein said step of disrupting is performed by exposing the region of said hair loss to radiation.
11. The method of claim 1, wherein said administering step is via topical administration.
12. The method of claim 1, wherein said administering step is via subepidermal administration.
13. A method for generating a hair follicle in the dermis of a subject with hair loss comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a fibroblast growth factor-9 polypeptide to said subject.
14. The method of claim 13, wherein said subject has a reduced number of hair follicles as a result of androgenetic alopecia (AGA).
15. The method of claim 14, wherein said AGA is male pattern baldness.
16. The method of claim 14, wherein said AGA is female pattern baldness.
17. The method of claim 13, wherein said subject has a reduced number of hair follicles as a result of a skin injury.
18. The method of claim 13, wherein said subject has a reduced number of hair follicles in the scalp or eyebrow.
19. The method of claim 13, wherein said subject has a reduced number of hair follicles in scarred skin tissue.
20. The method of claim 13, wherein said step of administering is performed 3-12 days after said step of disrupting.
21. The method of claim 13, wherein said step of disrupting is performed by exposing said region of hair loss to a mechanical or chemical stimulus.
22. The method of claim 13, wherein said step of disrupting is performed by exposing the region of said hair loss to radiation.
23. The method of claim 13, wherein said administering step is via topical administration.
24. The method of claim 13, wherein said administering step is via subepidermal administration.
25. A method for increasing the size of a hair follicle in the dermis of a subject with hair loss comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a fibroblast growth factor-9 polypeptide to said subject.
26. The method of claim 25, wherein said subject has reduced hair follicle size as a result of androgenetic alopecia (AGA).
27. The method of claim 26, wherein said AGA is male pattern baldness.
28. The method of claim 26, wherein said AGA is female pattern baldness.
29. The method of claim 25, wherein said subject has reduced hair follicle size as a result of a skin injury.
30. The method of claim 25, wherein said subject has reduced hair follicle size in the scalp or eyebrow.
31. The method of claim 25, wherein said subject has reduced hair follicle size in scarred skin tissue.
32. The method of claim 25, wherein said step of administering is performed 3-12 days after said step of disrupting.
33. The method of claim 25 wherein said step of disrupting is performed by exposing said region of hair loss to a mechanical or chemical stimulus.
34. The method of claim 25, wherein said step of disrupting is performed by exposing the region of said hair loss to radiation.
35. The method of claim 25, wherein said administering step is via topical administration.
36. The method of claim 25, wherein said administering step is via subepidermal administration.
37. A method for increasing hair follicle formation in the skin of a subject with hair loss comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a fibroblast growth factor-9 polypeptide to said subject.
38. The method of claim 37, wherein said subject has decreased hair follicle numbers as a result of androgenetic alopecia (AGA).
39. The method of claim 37, wherein said subject has decreased hair follicle numbers as a result of a skin injury.
40. The method of claim 37, wherein said step of administering is performed 3-12 days after said step of disrupting.
41. The method of claim 37, wherein said step of disrupting is performed by exposing a region of said dermis of said subject comprising low hair follicle numbers to a mechanical or chemical stimulus or to radiation.
42. The method of claim 37, wherein said administering step is via topical administration.
43. The method of claim 37, wherein said administering step is via subepidermal administration.
44. A method for treating, inhibiting, or suppressing a degenerative skin disorder comprising the steps of (a) disrupting the epidermis in the region of said degenerative skin disorder in said subject and (b) administering a composition comprising a fibroblast growth factor-9 polypeptide to said subject.
45. The method of claim 44, wherein said degenerative skin disorder is granuloma annulare.
46. The method of claim 44, wherein said step of administering is performed 3-12 days after said step of disrupting.
47. The method of claim 44, wherein said step of disrupting is performed by exposing said region of said degenerative skin disorder to a mechanical or chemical stimulus or to radiation.
48. The method of claim 44, wherein said administering step is via topical administration.
49. The method of claim 44, wherein said administering step is via subepidermal administration.
50. A method for treating an androgenetic alopecia (AGA) in a scalp of a subject comprising the steps of (a) disrupting the epidermis in the region of said AGA in said subject and (b) administering a composition comprising a fibroblast growth factor-9 polypeptide to said subject.
51. The method of claim 50, wherein said step of administering is performed 3-12 days after said step of disrupting.
52. The method of claim 50, wherein said step of disrupting is performed by exposing the region of said AGA to a mechanical or chemical stimulus or to radiation.
53. The method of claim 50, wherein said administering step is via topical administration.
54. The method of claim 50, wherein said administering step is via subepidermal administration.
55. A method of treating hair loss in a subject comprising the step administering a composition comprising a fibroblast growth factor-9 polypeptide and a wnt polypeptide to said subject.
56. The method of claim 55, wherein said wnt polypeptide is a wnt7 polypeptide.
57. The method of claim 55, wherein said hair loss is due to androgenetic alopecia (AGA).
58. The method of claim 57, wherein said AGA is male pattern baldness.
59. The method of claim 57, wherein said AGA is female pattern baldness.
60. The method of claim 55, wherein said hair loss is the result of a skin injury.
61. The method of claim 55, wherein said hair loss is in the scalp or eyebrow of said subject.
62. The method of claim 55, wherein said hair loss is in scarred skin tissue of said subject.
63. The method of claim 55, wherein said administering step is via topical administration.
64. The method of claim 55, wherein said administering step is via subepidermal administration.
65. A method for generating a hair follicle in the dermis of a subject comprising the step of administering a composition comprising a fibroblast growth factor-9 polypeptide and a wnt polypeptide to said subject.
66. The method of claim 65, wherein said wnt polypeptide is a wnt7 polypeptide.
67. The method of claim 65, wherein said subject has a reduced number of hair follicles as a result of androgenetic alopecia (AGA).
68. The method of claim 67, wherein said AGA is male pattern baldness.
69. The method of claim 67, wherein said AGA is female pattern baldness.
70. The method of claim 65, wherein said subject has a reduced number of hair follicles as a result of a skin injury.
71. The method of claim 65, wherein said subject has a reduced number of hair follicles in the scalp or eyebrow.
72. The method of claim 65, wherein said subject has a reduced number of hair follicles in scarred skin tissue.
73. The method of claim 65, wherein said administering step is via topical administration.
74. The method of claim 65, wherein said administering step is via subepidermal administration.
75. A method for increasing the size of a hair follicle in the dermis of a subject comprising the step of administering a composition comprising a fibroblast growth factor-9 polypeptide and a wnt polypeptide to said subject.
76. The method of claim 75, wherein said wnt polypeptide is a wnt7 polypeptide.
77. The method of claim 75, wherein said subject has reduced hair follicle size as a result of androgenetic alopecia (AGA).
78. The method of claim 77, wherein said AGA is male pattern baldness.
79. The method of claim 77, wherein said AGA is female pattern baldness.
80. The method of claim 75, wherein said subject has reduced hair follicle size as a result of a skin injury.
81. The method of claim 75, wherein said subject has reduced hair follicle size in the scalp or eyebrow.
82. The method of claim 75, wherein said subject has reduced hair follicle size in scarred skin tissue.
83. The method of claim 75, wherein said administering step is via topical administration.
84. The method of claim 75, wherein said administering step is via subepidermal administration.
85. A method for increasing hair follicle formation in the skin of a subject comprising the step of administering a composition comprising a fibroblast growth factor-9 polypeptide and a wnt polypeptide to said subject.
86. The method of claim 85, wherein said wnt polypeptide is a wnt7 polypeptide.
87. The method of claim 85, wherein said subject has decreased hair follicle numbers as a result of androgenetic alopecia (AGA).
88. The method of claim 85, wherein said subject has decreased hair follicle numbers as a result of a skin injury.
89. The method of claim 85, wherein said administering step is via topical administration.
90. The method of claim 85, wherein said administering step is via subepidermal administration.
91. A method for treating, inhibiting, or suppressing a degenerative skin disorder comprising the step of administering a composition comprising a fibroblast growth factor-9 polypeptide and a wnt polypeptide to said subject.
92. The method of claim 91, wherein said degenerative skin disorder is granuloma annulare.
93. The method of claim 91, wherein said wnt polypeptide is a wnt7 polypeptide.
94. The method of claim 91, wherein said administering step is via topical administration.
95. The method of claim 91, wherein said administering step is via subepidermal administration.
96. A method for treating an androgenetic alopecia (AGA) in a scalp of a subject comprising the step of administering a composition comprising a fibroblast growth factor-9 polypeptide and a wnt polypeptide to said subject.
97. The method of claim 96, wherein said wnt polypeptide is a wnt7 polypeptide.
98. The method of claim 96, wherein said administering step is via topical administration.
99. The method of claim 96, wherein said administering step is via subepidermal administration.
100. A method of treating hair loss in a subject comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a compound or factor that upregulates sonic hedgehog (SHH) to said subject.
101. The method of claim 100, wherein said factor that upregulates SHH is a fibroblast growth factor-9 polypeptide.
102. The method of claim 100, wherein said hair loss is due to androgenetic alopecia (AGA).
103. The method of claim 102, wherein said AGA is male pattern baldness.
104. The method of claim 102, wherein said AGA is female pattern baldness.
105. The method of claim 100, wherein said hair loss is the result of a skin injury.
106. The method of claim 100, wherein said hair loss is in the scalp or eyebrow of said subject.
107. The method of claim 100, wherein said hair loss is in scarred skin tissue of said subject.
108. The method of claim 100, wherein said step of administering is performed 3-12 days after said step of disrupting.
109. The method of claim 100, wherein said step of disrupting is performed by exposing the region of said hair loss to a mechanical or chemical stimulus.
110. The method of claim 100, wherein said step of disrupting is performed by exposing the region of said hair loss to radiation.
111. The method of claim 100, wherein said administering step is via topical administration.
112. The method of claim 100, wherein said administering step is via subepidermal administration.
113. A method for generating a hair follicle in the dermis of a subject comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a compound or factor that upregulates sonic hedgehog (SHH) to said subject.
114. The method of claim 113, wherein said factor that upregulates SHH is a fibroblast growth factor-9 polypeptide.
115. The method of claim 113, wherein said subject has a reduced number of hair follicles as a result of androgenetic alopecia (AGA).
116. The method of claim 115, wherein said AGA is male pattern baldness.
117. The method of claim 115, wherein said AGA is female pattern baldness.
118. The method of claim 113, wherein said subject has a reduced number of hair follicles as a result of a skin injury.
119. The method of claim 113, wherein said subject has a reduced number of hair follicles in the scalp or eyebrow.
120. The method of claim 113, wherein said subject has a reduced number of hair follicles in scarred skin tissue.
121. The method of claim 113, wherein said step of administering is performed 3-12 days after said step of disrupting.
122. The method of claim 113, wherein said step of disrupting is performed by exposing said region of hair loss to a mechanical or chemical stimulus.
123. The method of claim 113, wherein said step of disrupting is performed by exposing the region of said hair loss to radiation.
124. The method of claim 113, wherein said administering step is via topical administration.
125. The method of claim 113, wherein said administering step is via subepidermal administration.
126. A method for increasing the size of a hair follicle in the dermis of a subject comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a compound or factor that upregulates sonic hedgehog (SHH) to said subject.
127. The method of claim 126, wherein said factor that upregulates SHH is a fibroblast growth factor-9 polypeptide.
128. The method of claim 126, wherein said subject has reduced hair follicle size as a result of androgenetic alopecia (AGA).
129. The method of claim 128, wherein said AGA is male pattern baldness.
130. The method of claim 128, wherein said AGA is female pattern baldness.
131. The method of claim 126, wherein said subject has reduced hair follicle size as a result of a skin injury.
132. The method of claim 126, wherein said subject has reduced hair follicle size in the scalp or eyebrow.
133. The method of claim 126, wherein said subject has reduced hair follicle size in scarred skin tissue.
134. The method of claim 126, wherein said step of administering is performed 3-12 days after said step of disrupting.
135. The method of claim 126 wherein said step of disrupting is performed by exposing said region of hair loss to a mechanical or chemical stimulus.
136. The method of claim 126, wherein said step of disrupting is performed by exposing the region of said hair loss to radiation.
137. The method of claim 126, wherein said administering step is via topical administration.
138. The method of claim 126, wherein said administering step is via subepidermal administration.
139. A method for increasing hair follicle formation in the skin of a subject comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a compound or factor that upregulates sonic hedgehog (SHH) to said subject.
140. The method of claim 139, wherein said factor that upregulates SHH is a fibroblast growth factor-9 polypeptide.
141. The method of claim 139, wherein said subject has decreased hair follicle numbers as a result of androgenetic alopecia (AGA).
142. The method of claim 139, wherein said subject has decreased hair follicle numbers as a result of a skin injury.
143. The method of claim 139, wherein said step of administering is performed 3-12 days after said step of disrupting.
144. The method of claim 139, wherein said step of disrupting is performed by exposing a region of said dermis of said subject comprising low hair follicle numbers to a mechanical or chemical stimulus or to radiation.
145. The method of claim 139, wherein said administering step is via topical administration.
146. The method of claim 139, wherein said administering step is via subepidermal administration.
147. A method for treating, inhibiting, or suppressing a degenerative skin disorder comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a compound or factor that upregulates sonic hedgehog (SHH) to said subject.
148. The method of claim 147, wherein said degenerative skin disorder is granuloma annulare.
149. The method of claim 147, wherein said factor that upregulates SHH is a fibroblast growth factor-9 polypeptide.
150. The method of claim 147, wherein said step of administering is performed 3-12 days after said step of disrupting.
151. The method of claim 147, wherein said step of disrupting is performed by exposing said region of said degenerative skin disorder to a mechanical or chemical stimulus or to radiation.
152. The method of claim 147, wherein said administering step is via topical administration.
153. The method of claim 147, wherein said administering step is via subepidermal administration.
154. A method for treating an androgenetic alopecia (AGA) in a scalp of a subject comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a compound or factor that upregulates sonic hedgehog (SHH) to said subject.
155. The method of claim 153, wherein said factor that upregulates SHH is a fibroblast growth factor-9 polypeptide.
156. The method of claim 153, wherein said step of administering is performed 3-12 days after said step of disrupting.
157. The method of claim 153, wherein said step of disrupting is performed by exposing the region of said AGA to a mechanical or chemical stimulus or to radiation.
158. The method of claim 153, wherein said administering step is via topical administration.
159. The method of claim 153, wherein said administering step is via subepidermal administration.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority from U.S. Provisional Application 61/114,028, filed Nov. 12, 2008, which is incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0003] The invention relates to pharmaceutical compositions and methods for treating hair loss and regenerating hair follicles. Specifically, the invention relates to fibroblast growth factor-9 polypeptides and administering a fibroblast growth factor-9 polypeptide for treating hair loss or regenerating hair follicles.
BACKGROUND OF THE INVENTION
[0004] Follicular neogenesis is defined as the generation of new hair follicles (HF) after birth. Humans are born with a full complement of HF, which can change in size and growth characteristics as in early baldness or can ultimately degenerate and disappear as in late stages of baldness or in permanent scarring (cicatricial) alopecias. Therefore, the generation of new HF is desirable in the treatment of common baldness as well as less common hair loss conditions, such as discoid lupus erythematosis, congenital hypotrichosis, lichen planopilaris and other scarring alopecias.
SUMMARY OF THE INVENTION
[0005] The present invention provides methods of treating hair loss, treating, inhibiting, or suppressing a degenerative skin disorder, and treating androgenetic alopecia (AGA) in a subject and generating new hair follicles (HF) and increasing the size of existing HF, comprising epidermal disruption or administration of wnt, and administration of a fibroblast growth factor-9 polypeptide or another compound that upregulates sonic hedgehog gene signaling.
[0006] Thus, in one embodiment, the present invention provides a method of treating hair loss in a subject comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a fibroblast growth factor-9 polypeptide to said subject.
[0007] In one embodiment, the hair loss is due to androgenetic alopecia (AGA). In one embodiment, the AGA is male pattern baldness. In another embodiment, the AGA is female pattern baldness. In one embodiment, the hair loss is the result of a skin injury. In one embodiment, the hair loss is in the scalp or eyebrow of said subject. In one embodiment, the hair loss is in scarred skin tissue of said subject. In one embodiment, the step of administering is performed 3-12 days after said step of disrupting. In one embodiment, the step of disrupting is performed by exposing the region of said hair loss to a mechanical, chemical, or optical stimulus. In one embodiment, the optical stimulus is radiation. In one embodiment, the administering step is via topical administration. In another embodiment, the administering step is via subepidermal administration.
[0008] In another embodiment, the present invention provides a method for generating a hair follicle in the dermis of a subject with hair loss comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a fibroblast growth factor-9 polypeptide to said subject.
[0009] In another embodiment, the present invention provides a method for increasing the size of a hair follicle in the dermis of a subject with hair loss comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a fibroblast growth factor-9 polypeptide to said subject.
[0010] In another embodiment, the present invention provides a method for increasing hair follicle formation in the skin of a subject with hair loss comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a fibroblast growth factor-9 polypeptide to said subject.
[0011] In another embodiment, the present invention provides a method for treating, inhibiting, or suppressing a degenerative skin disorder comprising the steps of (a) disrupting the epidermis in the region of said degenerative skin disorder in said subject and (b) administering a composition comprising a fibroblast growth factor-9 polypeptide to said subject.
[0012] In another embodiment, the present invention provides a method for treating an androgenetic alopecia (AGA) in a scalp of a subject comprising the steps of (a) disrupting the epidermis in the region of said AGA in said subject and (b) administering a composition comprising a fibroblast growth factor-9 polypeptide to said subject.
[0013] In another embodiment, the present invention provides a method of treating hair loss in a subject comprising the step administering a composition comprising a fibroblast growth factor-9 polypeptide and a wnt polypeptide to said subject.
[0014] In another embodiment, the present invention provides a method for generating a hair follicle in the dermis of a subject comprising the step of administering a composition comprising a fibroblast growth factor-9 polypeptide and a wnt polypeptide to said subject.
[0015] In another embodiment, the present invention provides a method for increasing the size of a hair follicle in the dermis of a subject comprising the step of administering a composition comprising a fibroblast growth factor-9 polypeptide and a wnt polypeptide to said subject.
[0016] In another embodiment, the present invention provides a method for increasing hair follicle formation in the skin of a subject comprising the step of administering a composition comprising a fibroblast growth factor-9 polypeptide and a wnt polypeptide to said subject.
[0017] In another embodiment, the present invention provides a method for treating, inhibiting, or suppressing a degenerative skin disorder comprising the step of administering a composition comprising a fibroblast growth factor-9 polypeptide and a wnt polypeptide to said subject.
[0018] In another embodiment, the present invention provides a method for treating an androgenetic alopecia (AGA) in a scalp of a subject comprising the step of administering a composition comprising a fibroblast growth factor-9 polypeptide and a wnt polypeptide to said subject.
[0019] In another embodiment, the present invention provides a method of treating hair loss in a subject comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a compound or factor that upregulates sonic hedgehog (SHH) to said subject.
[0020] In another embodiment, the present invention provides a method for generating a hair follicle in the dermis of a subject comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a compound or factor that upregulates sonic hedgehog (SHH) to said subject.
[0021] In another embodiment, the present invention provides a method for increasing the size of a hair follicle in the dermis of a subject comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a compound or factor that upregulates sonic hedgehog (SHH) to said subject.
[0022] In another embodiment, the present invention provides a method for increasing hair follicle formation in the skin of a subject comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a compound or factor that upregulates sonic hedgehog (SHH) to said subject.
[0023] In another embodiment, the present invention provides a method for treating, inhibiting, or suppressing a degenerative skin disorder comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a compound or factor that upregulates sonic hedgehog (SHH) to said subject.
[0024] In another embodiment, the present invention provides a method for treating an androgenetic alopecia (AGA) in a scalp of a subject comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a compound or factor that upregulates sonic hedgehog (SHH) to said subject.
[0025] Other features and advantages of the present invention will become apparent from the following detailed description examples and figures. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1. FGF9 is expressed during inductive period of hair follicle regeneration at Day 1 after scab detachment (SD). The ratio of FGF9 mRNA compared to control mRNA expression q-PCR of FGF9 mRNA expression in regenerated epidermis is presented.
[0027] FIG. 2. γδTCR immunostaining of regenerated epidermis (SD7, wholemount) (×200) and FGF9 immunostaining of SD1 sample (frozen section) (×400).
[0028] FIG. 3. γδTCR & FGF9 immunostaining of regenerated epidermis for SD1 sample.
[0029] FIG. 4. γδTCR & FGF9 immunostaining of E14 embryonic skin.
[0030] FIG. 5. Specificity of anti-FGF9 neutralization antibody for E14.5 mouse embryonal whole lysate (lanes 1 and 2) and for recombinant hFGF9 (+).
[0031] FIG. 6. Anti-FGF9 neutralization experiment in 3 week-old C57BL/6 mice. (A) Treatment schedule in which 50 μl of 10 μg/ml anti-FGF9 or IgG2a isotype control were injected subepidermally on scab detachment day (SD)1-SD4, and tissue was sampled at SD5. (B) Hair follicle numbers after anti-FGF9 or IgG2a control injections in mice using the treatment protocol described in (A). (C) Diagram showing injection site.
[0032] FIG. 7. Hair follicle number in anti-FGF9-treated mice vs controls at various stages of hair follicle development, as described in Paus R, et al. J Invest Dermatol 1999).
[0033] FIG. 8. Model showing how hair germ counting was conducted per mm2 at 3 different fields per each sample.
[0034] FIG. 9. rhFGF9 treatment for three days in embryonic skin explant culture (E13.5). Cultures were treated with 10, 20, or 40 ng/mL of rhFGF9 or control buffer for three days, and hair germ number/mm2 was evaluated as described in FIG. 8. Mean±SD. *: P<0.05, **: P<0.01, compared to control. EDA-A1 (50 ng/ml) was used as a positive control for hair germ number.
[0035] FIG. 10. Immunohistochemical staining showing alkaline phosphatase staining of the dermis in control and rhFGF9 (10, 20, 40 ng/ml)-treated embryonic skin explants.
[0036] FIG. 11. Anti-FGF9 neutralizing antibody treatment for three days in embryonic skin explant culture (E13.5). Cultures were treated with 10, 20, or 40 μg/mL of anti-FGF9 neutralizing antibody or control for three days, and hair germ number/mm2 was evaluated as described in FIG. 8.
[0037] FIG. 12. Immunohistochemical staining showing K17 staining of the epidermis in control and anti-FGF9 (10, 20, 40 μg/ml)-treated skin explants.
[0038] FIG. 13. Immunohistochemical staining showing alkaline phosphatase staining of the dermis in control and anti-FGF9 (10, 20, 40 μg/ml)-treated embryonic skin explants.
[0039] FIG. 14. Effect of 24 h treatment using rhFGF9 (10, 20, 40 ng/ml) on markers of embryonic hair follicle development sonic hedgehog (Shh), Ptch1, and Ptch2 by qPCR.
[0040] FIG. 15. Effect of 24 h treatment using rhFGF9 (10, 20, 40 ng/ml) on markers of embryonic hair follicle development Gli1 and Gli2 by qPCR.
[0041] FIG. 16. Fgf9 is expressed during HFN initiation and is important to HFN. (A) Fgf9 is highly expressed in regenerated epidermis prior to hair follicle formation at Day 1 and 3 after reepithelization with scab detachment (SD) and decreased to basal level at Day 5. The ratio of Fgf9 mRNA expression in regenerated epidermis was compared to the level of unwounded epidermis, Day 0. *: P<0.05, **: P<0.01, mean±standard deviation. (B) Effect of FGF9 neutralization on HFN after wounding in 3-week old mice. The number of regenerated hair follicles was significantly decreased in the mice treated with anti-FGF9 neutralizing antibody compared to controls. **: P<0.05 (B) Determination of developmental stages of hair follicles. Hair follicles in the anti-FGF9-treated mice showed delay in hair follicle maturation. (C-E) Wholemount hair follicle neogenesis assay stained for KRT17 protein and (F-H) alkaline phosphatase activity in separated epidermis and dermis at Day 5 after reepithelization, respectively. Overexpression of Fgf9 in K14rtTA; TRE-Fgf9-IRES-eGfp mice resulted in increased numbers of hair follicles at Day 17 after wounding. Scale bar, 1 mm.
[0042] FIG. 17. FGF9 is expressed by activated DETC (A) Double immunostaining for FGF9 and γδ TCR. Fgf9 expression in repopulated γδ TCR-positive DETCs after reepithelization. Dot line, basement membrane. Scale bar, 50 μm. (B) Fgf9 gene expression is highly upregulated in the isolated DETCs after activation with mIL-2 and anti-CD3.
[0043] FIG. 18. Hair follicle neogenesis in TCRd-/- mice is severely impaired. (A-D) Wholemount epidermal and dermal samples treated to detect KRT17 protein and (E-H) alkaline phosphatase activity at Day 5 after reepithelization. Hair follicle formation was significantly impeded in 8 week and 40 week old mice. Scale bar, 1 mm *: P<0.05, mean±standard error.
[0044] FIG. 19. Developmental stages of HFs in control and anti-fgf9 antibody-treated wounds.
[0045] FIG. 20. FGF9 expression in K14rtTA; TRE-fgf9-IRES-eGfp mice compared to control mice during 2 days of doxycycline treatment.
[0046] FIG. 21. FGF9 expression in DETCs. (A) FGF9 is highly expressed in suprabasal dendritic cell. Wholemount double immunostaining of FGF9 and γδ TCR of ear epidermis from wild-type mouse without (B) or with IL-2 incubation (C).
[0047] FIG. 22. FGF9 expression in FGF9flox/flox; lck-cre mice compared to FGF9flox/flox control.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
[0048] The invention relates to pharmaceutical compositions and methods for treating hair loss and regenerating hair follicles. Specifically, the invention relates to fibroblast growth factor-9 polypeptides and administering a fibroblast growth factor-9 polypeptide for treating hair loss or regenerating hair follicles.
[0049] The present invention provides methods of treating hair loss, treating, inhibiting, or suppressing a degenerative skin disorder, and treating androgenetic alopecia (AGA) in a subject and generating new hair follicles (HF) and increasing the size of existing HF, comprising epidermal disruption or administration of wnt, and administration of a fibroblast growth factor-9 polypeptide or another compound that upregulates sonic hedgehog gene signaling.
[0050] In one embodiment, the present invention provides methods of treating hair loss, methods for generating a hair follicle, methods for increasing the size of a hair follicle, methods for treating an androgenetic alopecia (AGA), methods for arresting alopecia, methods of reversing alopecia, and methods of depilation comprising administering a composition comprising a neutralizing fibroblast growth factor-9 antibody to a subject.
[0051] In another embodiment, a composition or method of the present invention is utilized on human skin. In another embodiment, the composition or method is utilized on an area of unwanted hair growth. In another embodiment, the area is the face. In another embodiment, the area is the bikini area. In another embodiment, the area is the legs. In another embodiment, the area is the arms. In another embodiment, the area is the chest.
[0052] In one embodiment, the methods of the present invention include contacting a subject with an inhibitor of FGF9, SHH, WNT, or other compositions for use in the present invention. An "inhibitor" utilized in methods and compositions of the present invention is, in another embodiment, an antibody that binds the protein or biological factor that is the target of the inhibitor. In another embodiment, the inhibitor is a pharmacologic inhibitor. In another embodiment, the inhibitor is any other type of inhibitor known in the art. Each possibility represents a separate embodiment of the present invention.
[0053] In one embodiment, the present invention provides a method of treating hair loss comprising the step of administering a composition comprising a fibroblast growth factor-9 polypeptide to a subject.
[0054] In one embodiment, the present invention provides a method of treating hair loss in a subject comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a fibroblast growth factor-9 polypeptide to said subject.
[0055] In another embodiment, the present invention provides a method of treating hair loss in a subject comprising the step administering a composition comprising a fibroblast growth factor-9 polypeptide and a wnt polypeptide to said subject.
[0056] In another embodiment, the present invention provides a method of treating hair loss in a subject comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a compound or factor that upregulates sonic hedgehog (SHH) to said subject.
[0057] In another embodiment, the present invention provides a method for treating an androgenetic alopecia (AGA) in a scalp of a subject comprising the steps of (a) disrupting the epidermis in the region of said AGA in said subject and (b) administering a composition comprising a fibroblast growth factor-9 polypeptide to said subject.
[0058] In another embodiment, the present invention provides a method for treating an androgenetic alopecia (AGA) in a scalp of a subject comprising the step of administering a composition comprising a fibroblast growth factor-9 polypeptide and a wnt polypeptide to said subject.
[0059] In another embodiment, the present invention provides a method for treating an androgenetic alopecia (AGA) in a scalp of a subject comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a compound or factor that upregulates sonic hedgehog (SHH) to said subject.
[0060] In another embodiment, the present invention provides a method for generating a hair follicle in the dermis of a subject with hair loss comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a fibroblast growth factor-9 polypeptide to said subject.
[0061] In another embodiment, the present invention provides a method for generating a hair follicle in the dermis of a subject comprising the step of administering a composition comprising a fibroblast growth factor-9 polypeptide and a wnt polypeptide to said subject.
[0062] In another embodiment, the present invention provides a method for generating a hair follicle in the dermis of a subject comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a compound or factor that upregulates sonic hedgehog (SHH) to said subject.
[0063] In another embodiment, the present invention provides a method for increasing the size of a hair follicle in the dermis of a subject with hair loss comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a fibroblast growth factor-9 polypeptide to said subject.
[0064] In another embodiment, the present invention provides a method for increasing the size of a hair follicle in the dermis of a subject comprising the step of administering a composition comprising a fibroblast growth factor-9 polypeptide and a wnt polypeptide to said subject.
[0065] In another embodiment, the present invention provides a method for increasing the size of a hair follicle in the dermis of a subject comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a compound or factor that upregulates sonic hedgehog (SHH) to said subject.
[0066] In another embodiment, the present invention provides a method for increasing hair follicle formation in the skin of a subject with hair loss comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a fibroblast growth factor-9 polypeptide to said subject.
[0067] In another embodiment, the present invention provides a method for increasing hair follicle formation in the skin of a subject comprising the step of administering a composition comprising a fibroblast growth factor-9 polypeptide and a wnt polypeptide to said subject.
[0068] In another embodiment, the present invention provides a method for increasing hair follicle formation in the skin of a subject comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a compound or factor that upregulates sonic hedgehog (SHH) to said subject.
[0069] In another embodiment, the present invention provides a method for treating, inhibiting, or suppressing a degenerative skin disorder comprising the steps of (a) disrupting the epidermis in the region of said degenerative skin disorder in said subject and (b) administering a composition comprising a fibroblast growth factor-9 polypeptide to said subject.
[0070] In another embodiment, the present invention provides a method for treating, inhibiting, or suppressing a degenerative skin disorder comprising the step of administering a composition comprising a fibroblast growth factor-9 polypeptide and a wnt polypeptide to said subject.
[0071] In another embodiment, the present invention provides a method for treating, inhibiting, or suppressing a degenerative skin disorder comprising the steps of (a) disrupting the epidermis in the region of said hair loss in said subject and (b) administering a composition comprising a compound or factor that upregulates sonic hedgehog (SHH) to said subject.
[0072] In one embodiment, the methods of the present invention treat, inhibit or suppress a degenerative skin disorder. In one embodiment, a degenerative skin disorder is Hyperkeratosis, hyperpigmentation, depigmentation, atrophy, or a combination thereof. In one embodiment, a degenerative skin disorder is calcinosis; circumscripta; cutis; Colloid milium; skin degeneration; Senile dermatosis NOS; or Subcutaneous calcification.
[0073] In another embodiment, a degenerative skin disorder is granuloma annulare. In one embodiment, the degenerative skin disorder is localized granuloma annulare, which in one embodiment, is the most common form of granuloma annulare and in another embodiment, is characterized by the presence of small, firm red or yellow colored bumps (nodules or papules) that appear arranged in a ring on the skin. In one embodiment, the sizes of the lesions range from one to five centimeters. In one embodiment, the most commonly affected sites include the feet, hands, and fingers. In other embodiments, the degenerative skin disorder is generalized or disseminated, linear, perforating, or subcutaneous granuloma annulare. In one embodiment, the lesions associated with granuloma annulare may disappear without treatment (spontaneous remission) and reappear.
[0074] In another embodiment, the methods of the present invention are suitable for the prophylaxis and treatment of dryness, roughness of the skin, the formation of dry lines, reduced rehydration by sebaceous glands and an increased susceptibility to mechanical stress (tendency to crack), for the treatment of photodermatoses, the symptoms of senile xerosis, photoaging and other degenerative conditions which are associated with a decomposition of the connective tissue (collagen and elastin fibres and also glucosaminoglycans/hyaluronane) of the skin. "Photoaging" denotes the wrinkling, dryness and decreasing elasticity of the skin brought about by light and in particular UV radiation.
[0075] Further fields of application of the compositions according to the invention are the treatment and prevention of age- and/or UV-induced collagen degeneration and also the decomposition of elastin and glycosaminoglycans; of degenerative skin conditions such as loss of elasticity and also atrophy of the epidermal and dermal cell layers, of constituents of the connective tissue, of rete pegs and capillary vessels) and/or the skin adnexa; of environmentally-triggered negative changes in the skin and the skin adnexa, e.g. caused by ultraviolet radiation, smoking, smog, reactive oxygen species, free radicals and similar; of deficitary, sensitive or hypoactive skin conditions or deficitary, sensitive or hypoactive skin adnexa conditions; the reduction in skin thickness; of skin slackness and/or skin tiredness; of changes in the transepidermal water loss and normal moisture content of the skin; of a change in the energy metabolism of healthy skin; of deviations from the normal cell-cell communication in the skin which can manifest themselves e.g. in wrinkling; of changes in the normal fibroblast and keratinocyte proliferation; of changes in the normal fibroblast and keratinocyte differentiation; of polymorphic actinodermatosis, vitiligo; of wound healing disorders; disturbances to the normal collagen, hyaluronic acid, elastin and glycosaminoglycan homeostasis; of increased activation of proteolytic enzymes in the skin, such as e.g. metalloproteinases.
[0076] In another embodiment, the present invention provides a method of treating hair loss, generating a hair follicle, in creasing the size of a hair follicle, increasing hair follicle formation, treating, inhibiting or suppressing a degenerative skin disorder, treating androgenetic alopecia (AGA), comprising any combination of the following steps: (a) disrupting the epidermis in the region of said hair loss in said subject; (b) administering a fibroblast growth factor-9 polypeptide; (c) administering a wnt polypeptide; and (d) administering a compound or factor that upregulates Sonic Hedgehog (SHH), Patched-1 (Ptch1), Patched-2 (Ptch2), Gli1, Gli2, or a combination thereof to said subject.
[0077] In another embodiment, the present invention provides a method of depilation comprising the step of administering a composition comprising a neutralizing fibroblast growth factor-9 antibody to a subject. In one embodiment, the antibody is administered at a concentration of 10 μg/mL. In one embodiment, the depilation is in the legs, arms, underarms, pubic area, back, face, nose, or ears of said subject. In one embodiment, the method further comprises the step of disrupting the epidermis in the region of said depilation prior to said administering step. In one embodiment, the step of contacting is performed 3-12 days after said step of disrupting. In one embodiment, the step of disrupting is performed by exposing the region of said hair loss to a mechanical, chemical, or optical stimulus. In one embodiment, the optical stimulus is radiation. In one embodiment, the administering step is via topical administration. In another embodiment, the administering step is via subcutaneous administration.
[0078] In another embodiment, the present invention provides a method of reversing alopecia comprising the step of administering a composition comprising a fibroblast growth factor-9 polypeptide to a bald or balding subject. In another embodiment, the present invention provides a method of arresting alopecia comprising the step of administering a composition comprising a fibroblast growth factor-9 polypeptide to a bald or balding subject.
[0079] In another embodiment, the present invention provides a method of treating a wound in a subject comprising the step of administering a composition comprising a fibroblast growth factor-9 polypeptide to a bald or balding subject. In another embodiment, the present invention provides a method of treating an injury in a subject comprising the step of administering a composition comprising a fibroblast growth factor-9 polypeptide to a bald or balding subject.
FGF9
[0080] In one embodiment, the methods of the present invention comprise the step of administering a composition comprising a fibroblast growth factor-9 polypeptide, alone or in composition with one or more additional compounds. In one embodiment, FGF9 refers to Fgf-9, FGF-9, Fibroblast growth factor 9, GAF, glia activating factor, Glia-activating factor precursor, or HBGF-9. In one embodiment, the FGF9 protein of the methods of the present invention has the sequence: IFPNGTIQGTRKDHSRFGILEFISIAVGLVSIRGVDSGLYLGMNEKGELYGSEKLTQECV FREQFEENWYNTYSSNLYKHVDTGRRYYVALNKDGTPREGTRTKRHQKFTHFLPRP VDPDKVPELYKDILSQS (GenBank Accession No: BAA03572; SEQ ID No: 1). In another embodiment, the FGF9 protein has a sequence as set forth in GenBank Accession No. P31371, BAF83481, NP--002001, CAC17692, EAX08316, AAI03980, AAI03979, AAT74624 or AAH69692. In another embodiment, the FGF9 protein is encoded by a genomic nucleic acid molecule having a sequence as set forth in GenBank Accession No. AL139378.15, AY682094.1, or CH471075.1 or encoded by an mRNA molecule having a sequence as set forth in GenBank Accession No. AK290792.1, BC069692.1, BC103978.1, BC103979.1, CR746503.1, or D14838.1. In another embodiment, a biologically active fragment of an FGF9 protein is utilized in a method of the present invention. In another embodiment, a homolog of an FGF9 protein is utilized in a method of the present invention. Each possibility represents a separate embodiment of the present invention.
[0081] In one embodiment, administration of recombinant human FGF9 increased levels of sonic hedgehog (SHH) gene expression (FIG. 14).
Shh
[0082] In one embodiment, the methods of the present invention comprise the step of administering a composition comprising a sonic hedgehog (SHH) polypeptide, alone or in composition with one or more additional compounds. In another embodiment, the methods of the present invention comprise the step of administering a compound or factor that increases SHH expression. In one embodiment, SHH refers to TPT; HHG1; HLP3; HPE3; SMMCI; TPTPS; or MCOPCB5. In one embodiment, the SHH protein of the methods of the present invention has the sequence:
[0083] MLLLARCLLLVLVSSLLVCSGLACGPGRGFGKRRHPKKLTPLAYKQFIPNVAEK TLGASGRYEGKISRNSERFKELTPNYNPDBFKDEENTGADRLMTQRCKDKLNALAISV MNQWPGVKLRVTEGWDEDGHHSEESLHYEGRAVDITTSDRDRSKYGMLARLAVEAG DWVYYESKAHIFICSVKAENSVAAKSGGCFPGSATVHLEQGGTKLVKDLSPGDRVLA ADDQGRLLYSDFLTFLDRDDGAKKVFYVIETREPRERLLLTAAHLLFVAPHNDSATGEP EASSGSGPPSGGALGPRALFASRVRPGQRVYVVAERDGDRRLLPAAVHSVTLSEEAAG AYAPLTAQGTILINRVLASCYAVIEEHSWAHRAFAPIRLAHALLAALAPARTDRGGDS GGGDRGGGGGRVALTAPGAADAPGAGATAGIHWYSQLLYQIGTWLLDSEALHPLGM AVKSS (GenBank Accession No: Q15465.1; SEQ ID No: 2). In another embodiment, the SHH protein has a sequence as set forth in GenBank Accession No. BAA34689.1; AAB67604.1; AAS01990.1; AAQ87879.1; EAL23913.1; EAX04543.1; AAA62179.1; or AAI11926.1. In another embodiment, the SHH protein is encoded by a nucleic acid having a sequence as set forth in GenBank Accession No. AB020410.1; AC002484.1; AC078834.5; AY422195.1; CH236954.1; CH471149.1; AY927450.1; L38518.1; or BC111925.1. In another embodiment, a biologically active fragment of a SHH protein is utilized in a method of the present invention. In another embodiment, a homolog of a SHH protein is utilized in a method of the present invention. Each possibility represents a separate embodiment of the present invention.
[0084] In one embodiment, SHH binds to the patched (PTC) receptor, which functions in association with smoothened (SMO), to activate the transcription of target genes. In the absence of SHH, PTC represses the constitutive signaling activity of SMO. In another embodiment, SHH also regulates the gli oncogene. In another embodiment, SHH is an intercellular signal essential for a variety of patterning events during development: signal produced by the notochord that induces ventral cell fate in the neural tube and somites, and the polarizing signal for patterning of the anterior-posterior axis of the developing limb bud. In another embodiment, SHH displays both floor plate- and motor neuron-inducing activity.
[0085] In another embodiment, administration of recombinant human FGF9 increased levels of Patched homolog 1 (Drosophila), (PTCH1; FIG. 14), which in one embodiment, is a human gene. In one embodiment, Ptch1 encodes a member of the patched gene family. In one embodiment, Ptch1 is the receptor for sonic hedgehog (SHH), which in one embodiment, is a secreted molecule implicated in the formation of embryonic structures and in tumorigenesis. In one embodiment, Ptch1 functions as a tumor suppressor. In one embodiment, mutations of Ptch1 have been associated with nevoid basal cell carcinoma syndrome, esophageal squamous cell carcinoma, trichoepitheliomas, transitional cell carcinomas of the bladder, as well as holoprosencephaly. In one embodiment, alternative splicing of Ptch1 results in multiple transcript variants encoding different isoforms.
PTCH1
[0086] In one embodiment, the methods of the present invention comprise the step of administering a composition comprising a Patched-1 (PTCH1) polypeptide, alone or in composition with one or more additional compounds. In another embodiment, the methods of the present invention comprise the step of administering a compound or factor that increases PTCH1 expression. In one embodiment, PTCH1 refers to PTC; BCNS; HPE7; PTC1; PTCH; NBCCS; PTCH11; FLJ26746; or FLJ42602. In one embodiment, the PTCH1 protein of the methods of the present invention has the sequence:
[0087] MASAGNAAEPQDRGGGGSGCIGAPGRPAGGGRRRRTGGLRRAAAPDRDYLHR PSYCDAAFALEQISKGKATGRKAPLWLRAKFQRLLFKLGCYIQKNCGKFLVVGLLIFGA FAVGLKAANLETNVEELWVEVGGRVSRELNYTRQKIGEEAMFNPQLMIQTPKEEGAN VLTTEALLQHLDSALQASRVHVYMYNRQWKLEHLCYKSGELITETGYMDQUEYLYPC LITTPLDCFWEGAKLQSGTAYLLGKPPLRWTNFDPLEFLEELKKINYQVDSWEEMLNKA EVGHGYMDRPCLNPADPDCPATAPNKNSTKPLDMALVLNGGCHGLSRKYMHWQEEL IVGGTVKNSTGKLVSAHALQTMFQLMTPKQMYEHFKGYEYVSHINWNEDKAAAILEA WQRTYVEVVHQSVAQNSTQKVLSFTTTTLDDILKSFSDVSVIRVASGYLLMLAYACLT MLRWDCSKSQGAVGLAGVLLVALSVAAGLGLCSLIGISFNAATTQVLPFLALGVGVDD VFLLAHAFSETGQNKRIPFEDRTGECLKRTGASVALTSISNVTAFFMAALIPIPALRAFSL QAAVVVVFNFAMVLLIFPAILSMDLYRREDRRLDIFCCFTSPCVSRVIQVEPQAYTDTH DNTRYSPPPPYSSHSFAHETQITMQSTVQLRTEYDPHTHVYYTTAEPRSEISVQPVTVTQ DTLSCQSPESTSSTRDLLSQFSDSSLHCLEPPCTKWTLSSFAEKHYAPFLLKPKAKVVVIF LFLGLLGVSLYGTTRVRDGLDLTDIVPRETREYDFIAAQFKYFSFYNMYIVTQKADYPNI QHLLYDLHRSFSNVKYVMLEENKQLPKMWLHYFRDWLQGLQDAFDSDWETGKENIPN NYKNGSDDGVLAYKLLVQTGSRDKPIDISQLTKQRLVDADGENPSAFYIYLTAWVSND PVAYAASQANIRPHRPEWVHDKADYMPETRLRIPAAEPIEYAQFPFYLNGLRDTSDFVE AIEKVRTICSNYTSLGLSSYPNGYPFLFWEQYIGLRHWLLLFISVVLACTFLVCAVFLLNP WTAGIIVMVLALMTVELFGMMGLIGIKLSAVPVVILIASVGIGVEFTVHVALAFLTAIGD KNRRAVLALEHMFAPVLDGAVSTLLGVLMLAGSEFDFIVRYFFAVLAILTILGVLNGLV LLPVLLSFFGPYPEVSPANGLNRLPTPSPEPPPSVVRFAMPPGHTHSGSDSSDSEYSSQTT VSGLSEELRHYEAQQGAGGPAHQVIVEATENPVFAHSTVVHPESRHHPPSNPRQQPHL DSGSLPPGRQGQQPRRDPPREGLWPPPYRPRRDAFEISTEGHSGPSNRARWGPRGARSH NPRNPASTAMGSSVPGYCQPITTVTASASVTVAVHPPPVPGPGRNPRGGLCPGYPETDH GLFEDPHVPFHVRCERRDSKVEVIELQDVECEERPRGSSSN (GenBank Accession No: Q13635.2; SEQ ID No: 3). In another embodiment, the PTCH1 protein has a sequence as set forth in GenBank Accession No. CAH73817.1; CAH73818.1; CAH73819.1; AAR21238.1; AAR21239.1; AAR21240.1; EAW92631.1; EAW92632.1; BAD74184.1; BAD74185.1; BAD74186.1; BAD74187.1; BAD74188.1; BAD92732.1; BAF47711.1; BAE45300.1; BAE45302.1; BAE45304.1; BAF47712.1; BAC85893.1; AAH43542.1; AAC50496.1; AAC50550.1; or AAI52920.1. In another embodiment, the PTCH1 protein is encoded by a nucleic acid having a sequence as set forth in GenBank Accession No. AL161729.27; AY395758.1; AY395768.1; AY395772.1; CH471174.1; AB189436.1; AB189437.1; AB189438.1; AB189439.1; AB189440.1; AB209495.1; AB212827.1; AB212828.1; AB214500.1; AB233422.1; AB233424.1; AB239329.1; AI358880.1; AI494442.1; AK124593.1; AK130256.1; BC043542.1; BF195352.1; BM974119.1; BX117041.1; CR744004.1; DB093644.1; U43148.1; U59464.1; or BC152919.1. In another embodiment, a biologically active fragment of an PTCH1 protein is utilized in a method of the present invention. In another embodiment, a homolog of an PTCH1 protein is utilized in a method of the present invention. Each possibility represents a separate embodiment of the present invention.
[0088] In another embodiment, administration of recombinant human FGF9 increased levels of Patched homolog 2 (Drosophila), (PTCH; FIG. 14)
PTCH2
[0089] In one embodiment, the methods of the present invention comprise the step of administering a composition comprising a Patched-2 (PTCH2) polypeptide, alone or in composition with one or more additional compounds. In another embodiment, the methods of the present invention comprise the step of administering a compound or factor that increases PTCH2 expression. In one embodiment, ptch2 encodes a member of the patched gene family. In one embodiment, the patched protein is the receptor for sonic hedgehog, a secreted molecule implicated in the formation of embryonic structures and in tumorigenesis. In one embodiment, ptch2 is mutated in a medulloblastoma and in a basal cell carcinoma, suggesting that it plays a role in the development of some tumors. Alternative transcript variants have been described, but their biological function has not been determined. In one embodiment, the PTCH2 polypeptide for use in the methods of the present invention has the sequence:
[0090] FDFIVRYFTAALTVLTLLGLLHGLVLLPVLLSILGPPPEVIQMYKESPEILSPPAPQ GGGLRVGSLQVNISYWKELLWCQDLRPEEI (GenBank Accession No: Q5JR97; SEQ ID No: 4). In another embodiment, the PTCH2 protein has a sequence as set forth in GenBank Accession No. CAI23127.1; CAI13000.1; AAR05447.1; EAX07017.1; AAD25953.1; AAC79847.1; AAD17260.1; AAQ88919.1; AAQ89375.1; or AAI52912.1. In another embodiment, the PTCH2 protein is encoded by a nucleic acid having a sequence as set forth in GenBank Accession No. AL136380.22; AL592166.16; AY438664.1; CH471059.2; AF087651.1; AF091501.1; AF119569.1; AK307168.1; AY358555.1; AY359016.1; or BC152911.1. In another embodiment, a biologically active fragment of a PTCH2 protein is utilized in a method of the present invention. In another embodiment, a homolog of a PTCH2 protein is utilized in a method of the present invention. Each possibility represents a separate embodiment of the present invention.
GLI1
[0091] In one embodiment, the methods of the present invention comprise the step of administering a composition comprising a glioma-associated oncogene homolog 1 (zinc finger protein) (GLI1) polypeptide, alone or in a composition with one or more additional compounds. In one embodiment, gli1 encodes a protein which is a member of the Kruppel family of zinc finger proteins. In another embodiment, the methods of the present invention comprise the step of administering a compound or factor that increases Gill expression. In one embodiment, the GLI1 polypeptide for use in the methods of the present invention has the sequence:
[0092] MFNSMTPPPISSYGEPCCLRPLPSQGAPSVGTEGLSGPPFCHQANLMSGPHSYGP ARETNSCTEGPLFSSPRSAVKLTKKRALSISPLSDASLDLQTVIRTSPSSLVAFINSRCTSP GGSYGHLSIGTMSPSLGFPAQMNHQKGPSPSFGVQPCGPHDSARGGMIPHPQSRGPFPT CQLKSELDMLVGKCREEPLEGDMSSPNSTGIQDPLLGMLDGREDLEREEKREPESVYET DCRWDGCSQEFDSQEQLVHHINSEHIFIGERKEFVCHWGGCSRELRPFKAQYMLVVHM RRHTGEKPHKCTFEGCRKSYSRLENLKTHLRSHTGEKPYMCEHEGCSKAFSNASDRAK HQNRTHSNEKPYVCKLPGCTKRYTDPSSLRKHVKTVHGPDAHVTKRHRGDGPLPRAP SISTVEPKREREGGPIREESRLTVPEGAMKPQPSPGAQSSCSSDHSPAGSAANTDSGVEM TGNAGGSTEDLSSLDEGPCIAGTGLSTLRRLENLRLDQLHQLRPIGTRGLKLPSLSHTGT TVSRRVGPPVSLERRSSSSSSISSAYTVSRRSSLASPFPPGSPPENGASSLPGLMPAQHYL LRARYASARGGGTSPTAASSLDRIGGLPMPPWRSRAEYPGYNPNAGVTRRASDPAQA ADRPAPARVQRFKSLGCVHTPPTVAGGGQNFDPYLPTSVYSPQPPSITENAAMDARGL QEEPEVGTSMVGSGLNPYMDFPPTDTLGYGGPEGAAAEPYGARGPGSLPLGPGPPTNY GPNPCPQQASYPDPTQETWGEFPSHSGLYPGPKALGGTYSQCPRLEHYGQVQVKPEQG CPVGSDSTGLAPCLNAHPSEGPPHPQPLFSHYPQPSPPQYLQSGPYTQPPPDYLPSEPRP CLDFDSPTHSTGQLKAQLVCNYVQSQQELLWEGGGREDAPAQEPSYQSPKFLGGSQVS PSRAKAPVNTYGPGFGPNLPNHKSGSYPTPSPCHENFVVGANRASHRAAAPPRLLPPLP TCYGPLKVGGTNPSCGHPEVGRLGGGPALYPPPEGQVCNPLDSLDLDNTQLDFVAILD EPQGLSPPPSHDQRGSSGHTPPPSGPPNMAVGNMSVLLRSLPGETEFLNSSA (GenBank Accession No: P08151; SEQ ID No: 5). In another embodiment, the GLI1 protein has a sequence as set forth in GenBank Accession No. AAM13391.1; EAW97013.1; BAG60219.1; AAH13000.1; or CAA30297.1. In another embodiment, the GLI1 protein is encoded by a nucleic acid having a sequence as set forth in GenBank Accession No. ACO22506.38; AF316573.1; CH471054.1; AK297899.1; BC013000.2; or X07384.1. In another embodiment, a biologically active fragment of a GLI1 protein is utilized in a method of the present invention. In another embodiment, a homolog of a GLI1 protein is utilized in a method of the present invention. Each possibility represents a separate embodiment of the present invention.
GLI2
[0093] In one embodiment, the methods of the present invention comprise the step of administering a composition comprising a glioma-associated oncogene homolog 2 (zinc finger protein) (GLI2) polypeptide, alone or in a composition with one or more additional compounds. In another embodiment, the methods of the present invention comprise the step of administering a compound or factor that increases GLI2 expression. In one embodiment, GLI2 may be referred to as HPE9; THP1; or THP2. In one embodiment, gli2 encodes a protein which belongs to the C2H2-type zinc finger protein subclass of the Gli family. Members of this subclass are characterized as transcription factors which bind DNA through zinc finger motifs. These motifs contain conserved H--C links. Gli family zinc finger proteins are mediators of Sonic hedgehog (Shh) signaling and they are implicated as potent oncogenes in the embryonal carcinoma cell. The protein encoded by this gene localizes to the cytoplasm and activates patched Drosophila homolog (PTCH) gene expression. It is also thought to play a role during embryogenesis. The encoded protein is associated with several phenotypes--Greig cephalopolysyndactyly syndrome, Pallister-Hall syndrome, preaxial polydactyly type IV, postaxial polydactyly types A1 and B. In one embodiment, the GLI2 polypeptide for use in the methods of the present invention has the sequence: MALTSINATPTQLSSSSNCLSDTNQNKQSSESAVSSTVNPVAITIKRSKVKTEPEGLRPAS PLALTQGQVLDTAHVGVPFPSPQEQLADLKEDLDRDDCKQEAEVVIYETNCHWEDCT KEYDTQEQLVHHINNEHITIGEKKEFVCRWQACTREQKPFKAQYMLVVHMRRHTGEK PHKCTFEGCSKAYSRLENLKTHLRSHTGEKPYVCEHEGCNKAFSNASDRAKHQNRTHS NEKPYICKIPGCTKRYTDPSSLRKHVKTVHGPDAHVTKKQRNDVHLRTPLLKENGDSE AGTEPGGPESTEASSTSQAVEDCLHVRAIKTESSGLCQSSPGAQSSCSSEPSPLGSAPNN DSGVEMPGTGPGSLGDLTALDDTPPGADTSALAAPSAGGLQLRKHMTTMHRIEQLKK EKLKSLKDSCSWAGPTPHTRNTKLPPLPGSGSILENFSGSGGGGPAGLLPNPRLSELSAS EVTMLSQLQERRDSSTSTVSSAYTVSRRSSGISPYFSSRRSSEASPLGAGRPHNASSADS YDPISTDASRRSSEASQCSGGSGLLNLTPAQQYSLRAKYAAATGGPPPTPLPGLERMSL RTRLALLDAAEGTLPAGCPRPLGPRRGSDGPTYGHGHAGAAPAFPHEAPGGGTRRASD PVRRPDALSLPRVQRFHSTHNVNPGPLPPCADRRGLRLQSHPSTDGGLARGAYSPRPPSI SENVAMEAVAAGVDGAGPEADLGLPEDDLVLPDDVVQYIKAHASGALDEGTGQVYP TESTGFSDNPRLPSPGLHGQRRMVAADSNVGPSAPMLGGCQLGFGAPSSLNKNNMPV QWNEVSSGTVDSLASQVKPPPFPQGNLAVVQQKPAFGQYPGYSPQGLQASPGGLDST QPHLQPRSGAPSQGIPRVNYMQQLRQPVAGSQCPGMTTTMSPHACYGQVHPQLSPSTI SGALNQFPQSCSNMPAKPGHLGHPQQTEVAPDPTTMGNRHRELGVPNSALAGVPPPHP VQSYPQQSHHLAASMSQEGYHQVPSLLPARQPGFMEPQTGPMGVATAGFGLVQPRPP LEPSPTGRHRGVRAVQQQLAYARATGHAMAAMPSSQETAEAVPKGAMGNMGSVPPQ PPPQDAGGAPDHSMLYYYGQIIIMYEQDGGLENLGSCQVMRSQPPQPQACQDSIQPQP LPSPGVNQVSSTVDSQLLEAPQIDIDAINIDDGDHSSLFSGALSPSLLHSLSQNSSRLTTPR NSLTLPSIPAGISNMAVGDMSSMLTSLAEESKFLNMMT (GenBank Accession No: P10070; SEQ ID No: 6). In another embodiment, the GLI2 protein has a sequence as set forth in GenBank Accession No. AAA35898.1; BAA25665.1; BAA25666.1; BAA25667.1; BAA25668.1; BAD92591.1; BAG61875.1; AAS72889.1; AAS72890.1; AAS72891.1; AAI11411.1; BAA03568.1; BAA03569.1; AAY58315.1; AAY58316.1; AAY58317.1; or AAY87165.1. In another embodiment, the GLI2 protein is encoded by a nucleic acid having a sequence as set forth in GenBank Accession No. AC016764.8; AC017033.5 (60664..181887); M20672.1; M20673.1; AB007295.1; AB007296.1; AB007297.1; AB007298.1; AB209354.1; AJ707583.1; AK300071.1; AY493737.1; AY493738.1; AY493739.1; BC111410.1; D14827.1; D14828.1; DQ004396.1; DQ004397.1; DQ004398.1; or DQ086814.1. In another embodiment, a biologically active fragment of a GLI2 protein is utilized in a method of the present invention. In another embodiment, a homolog of a GLI2 protein is utilized in a method of the present invention. Each possibility represents a separate embodiment of the present invention.
WNT
[0094] In one embodiment, the methods of the present invention comprise the step of administering a composition comprising a Wnt polypeptide. The Wnt polypeptide of methods and compositions of the present invention has, in another embodiment, the sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAII VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAH AITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIK QNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGS VGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCS ERTEMYTCK (GenBank Accession No: BC008811; SEQ ID No: 7). In another embodiment, the Wnt polypeptide has a sequence selected from the sequences set forth in GenBank entries NM--004625, D83175, U53476, and NP--004616. In another embodiment, the Wnt polypeptide is a Wnt7 protein. In another embodiment, the Wnt polypeptide is a Wnt7a polypeptide. In another embodiment, the Wnt polypeptide is Wnt1 protein. In another embodiment, the Wnt polypeptide is a Wnt3 polypeptide. In another embodiment, the Wnt polypeptide is a Wnt3a polypeptide. In another embodiment, the Wnt polypeptide is a Wnt10 polypeptide. In another embodiment, the Wnt polypeptide is a Wnt10a protein. In another embodiment, the Wnt polypeptide is a Wnt10b polypeptide. In another embodiment, the Wnt polypeptide is encoded by a nucleic acid molecule having a sequence set forth in the one of the above GenBank entries. In another embodiment, a biologically active fragment of a Wnt polypeptide is utilized in a method of the present invention. In another embodiment, a biologically active fragment of a Wnt7 protein is utilized in a method of the present invention. In another embodiment, a biologically active fragment of a Wnt polypeptide is utilized in a method of the present invention. In another embodiment, a biologically active fragment of a Wnt7a polypeptide is utilized in a method of the present invention. Each possibility represents a separate embodiment of the present invention.
[0095] In another embodiment, methods of the present invention stimulate one or more members of the SHH signaling pathway, which in one embodiment is N-Shh (cleavage product), N-Shh-Cho1, which in one embodiment, inhibits Patched-1 and Patched-2, which in one embodiment, inhibit Smoothened, which in one embodiment, stimulates GLI-1, which in one embodiment, stimulates transcription of other genes (in one embodiment, GLI-1, PTC1, HNF3β) and GLI-2, and GLI-3, which in one embodiment inhibit transcription of other genes. Thus, in one embodiment, FGF9 stimulation and the resulting increase in SHH will relieve the tonic inhibition of Patched proteins on the Smoothened protein and increase levels of GLI-1, leading to enhancement of gene transcription.
[0096] In another embodiment, methods of the present invention stimulate one or more members of the WNT signaling pathway, which in one embodiment is Frizzled, SFRP, Dishevelled (Dsh), TCF, LRP, APC, β-catenin, Axin, Dickkopf, GSK3, Naked, Porcupine, or FRAT/GBP.
[0097] In another embodiment, the wnt pathway is stimulated before the hedgehog pathway. In another embodiment, the two pathways are stimulated in an overlapping fashion. In another embodiment, the two pathways are stimulated simultaneously. Each possibility represents a separate embodiment of the present invention.
[0098] In another embodiment, homologues and variants of transcripts and proteins of the present invention are administered in methods of the present invention. In another embodiment, homologues and variants of transcripts and proteins of the present invention are targeted in methods of the present invention. Each possibility represents a separate embodiment of the present invention.
[0099] The terms "homology," "homologous," etc, when in reference to any protein or peptide, refer in one embodiment, to a percentage of amino acid residues in the candidate sequence that are identical with the residues of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Methods and computer programs for the alignment are well known in the art.
[0100] In another embodiment, the term "homology," when in reference to any nucleic acid sequence similarly indicates a percentage of nucleotides in a candidate sequence that are identical with the nucleotides of a corresponding native nucleic acid sequence.
[0101] In another embodiment, "homology" refers to identity of greater than 70%. In another embodiment, "homology" refers to identity of greater than 75%. In another embodiment, "homology" refers to identity of greater than 80%. In another embodiment, "homology" refers to identity of greater than 82%. In another embodiment, "homology" refers to identity of greater than 85%. In another embodiment, "homology" refers to identity of greater than 87%. In another embodiment, "homology" refers to identity of greater than 90%. In another embodiment, "homology" refers to identity of greater than 92%. In another embodiment, "homology" refers to identity of greater than 95%. In another embodiment, "homology" refers to identity of greater than 97%. In another embodiment, "homology" refers to identity of greater than 98%. In another embodiment, "homology" refers to identity of greater than 99%. In another embodiment, "homology" refers to identity of 100%.
[0102] Protein and/or peptide homology for any amino acid sequence listed herein is determined, in one embodiment, by methods well described in the art, including immunoblot analysis, or via computer algorithm analysis of amino acid sequences, utilizing any of a number of software packages available, via established methods. Some of these packages may include the FASTA, BLAST, MPsrch or Scanps packages, and may employ the use of the Smith and Waterman algorithms, and/or global/local or BLOCKS alignments for analysis, for example. Each method of determining homology represents a separate embodiment of the present invention.
[0103] In one embodiment, the term "peptide" includes native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), such as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into bacterial cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S═O, O═C--NH, CH2-O, CH2-CH2, S═C--NH, CH═CH or CF═CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein.
[0104] Peptide bonds (--CO--NH--) within the peptide may be substituted, for example, by N-methylated bonds (--N(CH3)-CO--), ester bonds (--C(R)H--C--O--O--C(R)--N--), ketomethylen bonds (--CO--CH2-), α-aza bonds (--NH--N(R)--CO--), wherein R is any alkyl, e.g., methyl, carba bonds (--CH2-NH--), hydroxyethylene bonds (--CH(OH)--CH2-), thioamide bonds (--CS--NH--), olefinic double bonds (--CH═CH--), retro amide bonds (--NH--CO--), peptide derivatives (--N(R)--CH2-CO--), wherein R is the "normal" side chain, naturally presented on the carbon atom.
[0105] These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time.
[0106] Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted for synthetic non-natural acid such as TIC, naphthylelanine (No1), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
[0107] In addition to the above, the peptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
[0108] As used herein in the specification and in the claims section below the term "amino acid" or "amino acids" is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term "amino acid" includes both D- and L-amino acids.
[0109] In another embodiment, naturally occurring amino acids and non-conventional or modified amino acids as are known in the art can be used with the present invention.
[0110] In another embodiment, the present invention provides a kit, comprising a tools and/or a compound suitable for performing a method of the present invention.
[0111] In another embodiment, the present invention provides a device, comprising a tool suitable for epidermal disruption and a means of delivering a compound or factor that upregulates expression of SHH.
[0112] It is to be understood that included in the present invention are methods comprising the step of administering an isolated nucleic acid, in one embodiment, a vector or plasmid, encoding a polypeptide of the present invention, which in one embodiment, is a fibroblast growth factor-9 polypeptide, shh, wnt, ptch1, ptch, gli1, or gli2, or a composition comprising such a vector.
[0113] In one embodiment, an isolated nucleic acid that encodes a polypeptide of the present invention for use in the methods of the present invention is provided.
[0114] In one embodiment, an "isolated nucleic acid" refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
[0115] In one embodiment, the present invention provides a cell comprising an isolated nucleic acid or vector of the present invention.
[0116] In one embodiment, two polynucleotides of the present invention are operably linked. For example, in one embodiment, polynucleotides encoding FGF9 and WNT may be operably linked. In one embodiment, "operably linked" indicates that a single-stranded or double-stranded nucleic acid moiety comprises the two polynucleotides arranged within the nucleic acid moiety in such a manner that they are expressed together. By way of example, a promoter operably linked to the coding region of a gene is able to promote transcription of the coding region.
[0117] In one embodiment, a polynucleotide of the present invention comprises a promoter/regulatory sequence, which in one embodiment, the promoter/regulatory is positioned at the 5' end of the desired protein coding sequence such that it drives expression of the desired protein in a cell. Together, the nucleic acid encoding the desired protein and its promoter/regulatory sequence comprise a "transgene."
[0118] In one embodiment, the term "promoter/regulatory sequence" refers to a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner. In one embodiment, a promoter used in the present invention may be constitutive or inducible. In another embodiment, a promoter for use in the methods of the present invention may be tissue-specific. Such promoters are well known in the art.
[0119] In another embodiment, the present invention provides a delivery vehicle for administration of a polypeptide of the present invention. Examples of such delivery vehicles are known in the art and may include recombinant viruses or bacteria engineered to express said polypeptide. In one embodiment, said viruses or bacteria are attenuated. In one embodiment, viruses for use in the methods of the present invention may include retroviruses, adenoviruses, adeno-associated viruses, etc. In one embodiment, the virus may be of any known serotype or subgroup.
[0120] In one embodiment, any one of a number of different vectors can be used in the methods of the present invention, such as viral vectors, plasmid vectors, linear DNA, etc., as known in the art, to introduce an exogenous nucleic acid fragment encoding a therapeutic agent into target cells and/or tissue. These vectors can be inserted, for example, using infection, transduction, transfection, calcium-phosphate mediated transfection, DEAE-dextran mediated transfection, electroporation, liposome-mediated transfection, biolistic gene delivery, liposomal gene delivery using fusogenic and anionic liposomes (which are an alternative to the use of cationic liposomes), direct injection, receptor-mediated uptake, magnetoporation, ultrasound, or any combination thereof, as well as other techniques known in the art (for further detail see, for example, "Methods in Enzymology" Vol. 1-317, Academic Press, Current Protocols in Molecular Biology, Ausubel F. M. et al. (eds.) Greene Publishing Associates, (1989) and in Molecular Cloning: A Laboratory Manual, 2nd Edition, Sambrook et al. Cold Spring Harbor Laboratory Press, (1989), or other standard laboratory manuals). The polynucleotide segments encoding sequences of interest can be ligated into an expression vector system suitable for transducing mammalian cells and for directing the expression of recombinant products within the transduced cells. The introduction of the exogenous nucleic acid fragment is accomplished by introducing the vector into the vicinity of the micro-organ. Once the exogenous nucleic acid fragment has been incorporated into the cells using any of the techniques described above or known in the art, the production and/or the secretion rate of the therapeutic agent encoded by the nucleic acid fragment can be quantified. In one embodiment, the term "exogenous" refers to a substance that originated outside, for example a nucleic acid that originated outside of a cell or tissue.
[0121] In one embodiment, a vector for use in the methods of the present invention is a non-immunogenic gene transfer agent such as a nonviral vector (e.g. DNA plasmids or minicircle DNA), a "gutless" viral vector i.e. without endogenous genes (which in one embodiment, is due to a deletion, while in another embodiment, due to an insertion, substitution or deletion in a gene that prevents gene expression), a helper-dependent adenovirus (HDAd) vector, or adeno associated virus AAV (which in one embodiment is single stranded and in another embodiment, double stranded). In another embodiment, said formulation is so chosen such that recombinant gene expression results in lack of toxicity or immune-mediated rejection of the gene product by the tissue. In one embodiment, the vector is virally derived, and in another embodiment, the vector is a plasmid. In one embodiment, the virally-derived vector is derived from adenovirus, which in one embodiment, is helper-dependent adenovirus, while in another embodiment, the virally-derived vector is derived from adenovirus-associated vector.
[0122] In one embodiment, the term "vector" or "expression vector" refers to a carrier molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. In one embodiment, the nucleic acid molecules are transcribed into RNA, which in some cases are then translated into a protein, polypeptide, or peptide. In other cases, RNA sequences are not translated, for example, in the production of antisense molecules or ribozymes. In one embodiment, expression vectors can contain a variety of "control sequences" which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In another embodiment, a vector further includes an origin of replication. In one embodiment the vector may be a shuttle vector, which in one embodiment can propagate both in prokaryotic and eukaryotic cells, or in another embodiment, the vector may be constructed to facilitate its integration within the genome of an organism of choice. The vector, in other embodiments may be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome. In one embodiment, the vector is a viral vector, which in one embodiment may be a bacteriophage, mammalian virus, or plant virus.
[0123] In other embodiments, the viral vector is derived from a virus such as vaccinia virus, lentivirus, polio virus, hepatitis virus, papilloma virus, cytomegalovirus, simian virus, or herpes simplex virus.
[0124] In certain embodiments of the invention, the vector comprising a nucleic acid sequence may comprise naked recombinant DNA or plasmids. Transfer of the construct may be performed by any method which physically or chemically permeabilizes the cell membrane. In one embodiment, the vector is a mini-circle DNA, which in one embodiment, is a supercoiled DNA molecule for non-viral gene transfer, which has neither a bacterial origin of replication nor an antibiotic resistance marker.
[0125] Construction of vectors using standard recombinant techniques is well known in the art (see, for example, Maniatis, et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor, 1990) and Ausubel, et al., 1994, Current Protocols in Molecular Biology (John Wiley & Sons, 1996), both incorporated herein by reference).
[0126] In one embodiment, compositions of the present invention comprise the indicated agent, while in another embodiment, compositions of the present invention consist essentially of the indicated agent, while in another embodiment, compositions of the present invention consist of the indicated agent. In some embodiments, the term "comprise" refers to the inclusion of the indicated active agent, such as human fibroblast growth factor-9 polypeptide, shh, wnt, etc, as well as inclusion of other active agents, and pharmaceutically acceptable carriers, excipients, emollients, stabilizers, etc., as are known in the pharmaceutical industry. In some embodiments, the term "consisting essentially of" refers to a composition, whose only active ingredient is the indicated active ingredient, however, other compounds may be included which are for stabilizing, preserving, etc. the formulation, but are not involved directly in the therapeutic effect of the indicated active ingredient. In some embodiments, the term "consisting essentially of" may refer to components which facilitate the release of the active ingredient. In some embodiments, the term "consisting" refers to a composition, which contains the active ingredient and a pharmaceutically acceptable carrier or excipient.
[0127] In one embodiment, methods of the present invention treat, inhibit or suppress hair loss. In one embodiment, methods of the present invention generate hair follicles or increase hair follicle size, in one embodiment, in a subject with hair loss. In one embodiment, hair loss is due to androgenetic alopecia (AGA). In another embodiment, hair loss is due to male pattern baldness. In another embodiment, hair loss is due to female pattern baldness. In another embodiment, hair loss is the result of a skin injury.
[0128] In another embodiment, the methods of the present invention treat, inhibit, or suppress a disease or disorder in a subject. In one embodiment, the subject has a disease or disorder comprising balding. In another embodiment, the subject does not have a disease or disorder comprising balding. In another embodiment, the disease or disorder is androgenetic alopecia (AGA). In another embodiment, the disease or disorder is male pattern baldness. In another embodiment, the disease or disorder is female pattern baldness. In another embodiment, the disease or disorder is a discoid lupus erythematosis. In another embodiment, the disease or disorder is a congenital hypotrichosis. In another embodiment, the disease or disorder is a lichen planopilaris.
[0129] In another embodiment, the disease or disorder is a scarring (or, in another embodiment, cicatricial) alopecia, which in one embodiment, is hair loss due to scarring of the scalp area. In one embodiment, scarring alopecia typically involves the top of the scalp and occurs predominantly in women. The condition frequently occurs in African-American women and is believed to be associated with persistent tight braiding or "corn-rowing" of scalp hair. A form of scarring alopecia also may occur in post-menopausal women, associated with inflammation of hair follicles and subsequent scarring. In another embodiment, the disease or disorder is any other disease or disorder comprising balding known in the art.
[0130] In another embodiment, the present invention provides methods for treating Alopecia areata, which in one embodiment, is an autoimmune disorder that causes patchy hair loss that can range from diffuse thinning to extensive areas of baldness with "islands" of retained hair.
[0131] In another embodiment, the present invention provides methods for treating Trichotillomania, which in one embodiment, compulsive hair pulling. Hair loss due to trichotillomania is typically patchy, as compulsive hair pullers tend to concentrate the pulling in selected areas.
[0132] In another embodiment, the present invention provides methods for treating Triangular alopecia, which in one embodiment, is a loss of hair in the temporal areas that sometimes begins in childhood. Hair loss may be complete, or a few fine, thin-diameter hairs may remain.
[0133] In another embodiment, the present invention provides methods for treating Telogen effluvium, which in one embodiment, is a common type of hair loss caused when a large percentage of scalp hairs are shifted into "shedding" phase. The causes of telogen effluvium may be hormonal, nutritional, drug-associated, or stress-associated.
[0134] In another embodiment, the present invention provides methods for treating Loose-anagen syndrome, which in one embodiment, is a condition occurring primarily in fair-haired persons in which scalp hair sits loosely in hair follicles and is easily extracted by combing or pulling. In one embodiment, the condition may appear in childhood.
[0135] In another embodiment, the present invention provides methods for treating Tinea Capitis (Scalp Ringworm), which in one embodiment, is caused by a fungal infection, and in one embodiment, is characterized by patches of scaling that can spread and result in broken hair, redness, swelling, and oozing on the scalp.
[0136] In another embodiment, the present invention provides methods for treating hair loss associated with particular conditions, which in one embodiment, is cancer, thyroid disease, inadequate protein in diet, low serum iron levels, or associated with particular environmental stimuli, which in one embodiment, is chemotherapy, or, in another embodiment, radiotherapy.
[0137] In other embodiments, the present invention provides a method of treating any disease, disorder, or symptom associated with balding. In other embodiments, the present invention provides a method of treating any disease, disorder, or symptom associated with degenerative skin disorder. In other embodiments, the present invention provides a method of treating any disease, disorder, or symptom associated with alopecia. Each disease, disorder, or symptom represents a separate embodiment of the present invention.
[0138] In one embodiment, "treating" refers to either therapeutic treatment or prophylactic or preventative measures, wherein the object is to prevent or lessen the targeted pathologic condition or disorder as described hereinabove. Thus, in one embodiment, treating may include directly affecting or curing, suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, reducing symptoms associated with the disease, disorder or condition, or a combination thereof. Thus, in one embodiment, "treating" refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof. In one embodiment, "preventing" refers, inter alia, to delaying the onset of symptoms, preventing relapse, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, or a combination thereof. In one embodiment, "suppressing" or "inhibiting", refers inter alia to reducing the severity of symptoms, reducing the severity of the current episode, reducing the number of symptoms, reducing the incidence of symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, or a combination thereof.
[0139] In one embodiment, symptoms are primary, while in another embodiment, symptoms are secondary. In one embodiment, "primary" refers to a symptom that is a direct result of the alopecia, while in one embodiment, "secondary" refers to a symptom that is derived from or consequent to a primary cause. In one embodiment, the compositions and strains for use in the present invention treat primary or secondary symptoms or secondary complications related to alopecia, in one embodiment, seborrheic dermatitis.
[0140] In another embodiment, "symptoms" may be any manifestation of alopecia, comprising hair loss, balding, temporary hair loss, patchy hair loss, degenerative skin disorders or a combination thereof.
[0141] Methods of determining the presence and severity of alopecia and/or degenerative skin disorders such as those described herein are well known in the art. Each method represents a separate embodiment of the present invention.
[0142] In one embodiment, the methods of the present invention are for treating a subject with hair loss. In one embodiment, the hair loss is in the scalp of the subject. In another embodiment, the hair loss is in the eyebrow of the subject. In another embodiment, the hair loss is in scarred skin tissue of the subject, which in one embodiment, may be scalp, eyebrow, arm, or leg of a subject. In another embodiment, any other hair-bearing area or region of the skin is treated by a method of the present invention. Each possibility represents a separate embodiment of the present invention.
[0143] In one embodiment, methods of the present invention comprise the step of disrupting the epidermis in the region of said hair loss prior to said administering step. In another embodiment, the epithelium is disrupted.
[0144] In another embodiment of methods and compositions of the present invention, the first step (e.g. epidermal disruption) is performed 3-12 days prior to the second step (e.g. addition of an active compound, factor, cell, etc). In another embodiment, the interval is 4-12 days. In another embodiment, the interval is 5-12 days. In another embodiment, the interval is 4-11 days. In another embodiment, the interval is 6-11 days. In another embodiment, the interval is 6-10 days. In another embodiment, the interval is 6-9 days. In another embodiment, the interval is 6-8 days. In another embodiment, the interval is 7-8 days. In another embodiment, the interval is 5-11 days. In another embodiment, the interval is 5-10 days. In another embodiment, the interval is 7-10 days. In another embodiment, the interval is about 1 week. In another embodiment, the compositions for use in the methods of the present invention are applied as the scabbing starts to heal, which in one embodiment is 3-12 days after epidermal disruption. In one embodiment, the compositions for use in the methods of the present invention are applied one day after scab detachment, in another embodiment, two days after scab detachment, in another embodiment, three days after scab detachment, in another embodiment, four days after scab detachment, in another embodiment, five days after scab detachment, in another embodiment, six days after scab detachment, in another embodiment, seven days or more after scab detachment. In another embodiment, the compositions for use in the present invention are administered on days 1-4 after scab detachment. Each possibility represents a separate embodiment of the present invention.
[0145] In one embodiment, the step of disrupting is performed by exposing the region of said hair loss to a mechanical, chemical, or optical stimulus. In one embodiment, the optical stimulus is radiation.
[0146] The step of disrupting the epidermis in methods of the present invention is performed, in another embodiment, by abrading the skin region of interest. In another embodiment, the term "abrading" refers to an act of creating an abrasion. In another embodiment, "abrading" refers to rubbing. In another embodiment, "abrading" refers to wearing away by friction. In one embodiment, epidermal abrasion causes, under the conditions utilized herein, de novo HF neo-genesis. In another embodiment, the epidermal layer is disrupted.
[0147] In one embodiment, "abrasion" refers to a wound consisting of superficial damage to the skin. In another embodiment, "abrasion" refers to an area of the scalp or skin from which the epidermis is removed. In another embodiment, "abrasion" refers to an area of the scalp or skin from which the epidermis and dermis are removed. Each definition of "abrading" and "abrasion" represents a separate embodiment of the present invention.
[0148] In one embodiment, epidermal disruption by a method of the present invention converts the skin region of interest back to an embryonic-like state, in which the follicle regenerates. In another embodiment, a subsequent window of opportunity is created, during which the number and size of new HF in the skin region of interest can be manipulated. In another embodiment, the administration of a compound or factor that promotes a differentiation of an uncommitted epidermal cell into a HF cell during this window causes regeneration of larger and more numerous HF. In one embodiment, the morphology of HF in abraded skin is similar to that of embryonic HF, and the markers expressed are similar as well.
[0149] In another embodiment, the excisional wounds of methods of the present invention are created using a surgical tool. In one embodiment, the surgical tool is a dermal biopsy punch. In another embodiment, the excisional wounds are induced by freezing or cryoinjury. The use of freezing or cryoinjury is well known in the art, and is used, for example by dermatologists to injure skin. In one embodiment, the freezing or cryoinjury results in a blister. In another embodiment, the blister is used as a "chamber" to introduce drugs and or cells into the reepithelialized area. Each possibility represents a separate embodiment of the present invention.
[0150] In another embodiment, the epidermal disruption in methods of the present invention further removes dermal tissue from the skin region of interest. In another embodiment, the epidermal disruption does not remove dermal tissue from the skin region of interest. Each possibility represents a separate embodiment of the present invention.
[0151] "Disrupting" an epidermis or epidermal layer refers, in another embodiment, to removing part of the epidermis or epidermal layer. In another embodiment, the term refers to disturbing the intactness of the epidermis or epidermal layer. In another embodiment, the term refers to perforating the epidermis or epidermal layer. In another embodiment, only part of the epidermal layer need be removed. In another embodiment, the entire epidermal layer is removed. In another embodiment, the term refers to abrading the epidermis or epidermal layer. In another embodiment, the term refers to wounding the epidermis or epidermal layer. Each possibility represents a separate embodiment of the present invention.
[0152] In another embodiment, the epidermal disruption is performed with a tool that comprises sandpaper. In another embodiment, the epidermal disruption is performed with a laser. In another embodiment, the laser is a Fraxel laser. In another embodiment, the laser is a CO2 laser. In another embodiment, the laser is an excimer laser. In another embodiment, the laser is any other type of laser capable of inducing trans-epithelial injury. In another embodiment, the epidermal disruption is performed with a felt wheel. In another embodiment, the epidermal disruption is performed with a surgical tool. In another embodiment, the epidermal disruption is performed with any other tool known in the art that is capable of epidermal disruption. In another embodiment, the epidermal disruption comprises use of a micro-dermabrasion device. In another embodiment, the epidermal disruption comprises a burn treatment.
[0153] In another embodiment, the epidermal disruption comprises a disruption of a follicle of said epidermis and a disruption of an interfollicular region of said epidermis. In another embodiment, the epidermal disruption comprises a disruption of a follicle of said epidermis and does not comprise a disruption of an interfollicular region of said epidermis. Each possibility represents a separate embodiment of the present invention.
[0154] In another embodiment, the epidermal disruption comprises a light-based method. In another embodiment, the epidermal disruption comprises irradiation with visible light. In another embodiment, the epidermal disruption comprises irradiation with infrared light. In another embodiment, the epidermal disruption comprises irradiation with ultraviolet radiation. In another embodiment, the epidermal disruption comprises orthovoltage irradiation. In another embodiment, the epidermal disruption comprises X-ray irradiation. In another embodiment, the epidermal disruption comprises any other type of irradiation known in the art.
[0155] In another embodiment, the epidermal disruption is performed by mechanical means. In another embodiment, "mechanical means" refers to abrading. In another embodiment, the term refers to wounding. In another embodiment, the term refers to ultrasound. In another embodiment, the term refers to radio-frequency. In another embodiment, the term refers to an electrical process or the use of an electrical current. In another embodiment, the term refers to electoporation. In another embodiment, the term refers to excision. In another embodiment, the term refers to tape-stripping. In another embodiment, the term refers to microdermabrasion. In another embodiment, the term refers to the use of peels. In another embodiment, the term refers to any other type of mechanical means known in the art. Each possibility represents a separate embodiment of the present invention.
[0156] In another embodiment, the epidermal disruption comprises chemical treatment. In another embodiment, the chemical is phenol. In another embodiment, the chemical is trichloracetic acid. In another embodiment, the chemical is ascorbic acid. In another embodiment, the chemical is any other chemical capable of epidermal disruption that is known in the art.
[0157] In another embodiment, epidermal trauma is utilized in a method of the present invention.
[0158] Each method or type of epidermal disruption, abrasion, and trauma represents a separate embodiment of the present invention.
[0159] In one embodiment, "WIHN" refers to HF neogenesis induced by disruption of the epithelial layer. In another embodiment, the term refers to HF neogenesis induced by abrasion. In another embodiment, the term refers to HF neogenesis induced by wounding. In another embodiment, the term refers to HF neogenesis induced by disruption of the epithelial layer, followed by administration of a compound or factor that promotes a differentiation of an uncommitted epidermal cell into a HF cell. Each possibility represents a separate embodiment of the present invention.
[0160] In another embodiment, the epidermal disruption of methods of the present invention creates an abrasion at least about 1-1.5 centimeters (cm) in width. In another embodiment, the abrasion is at least about 1 cm in width. In another embodiment, the abrasion is at least about 1.5 cm in width. In another embodiment, the abrasion is at least about 2 cm in width. Each type of abrasion represents a separate embodiment of the present invention.
[0161] In another embodiment, the excisional wounds of methods of the present invention are not surgically closed. In another embodiment, the excisional wounds are allowed to heal by secondary intention. In another embodiment, the skin region of interest is not contacted with a bandage or dressing following the epidermal disruption. In another embodiment, the skin region of interest is not contacted with an ointment following the epidermal disruption. In another embodiment, the skin region of interest is allowed to heal for a period of time without being contacted by any substance, device, ointment, etc., that is ordinarily administered to an abrasion or wound to facilitate healing. In another embodiment, the skin region of interest is allowed to heal for a period of time without being contacted by any substance, device, ointment, etc., that is ordinarily administered to an abrasion or wound to prevent infection. In another embodiment, the "period of time" is the time it takes the epidermal disruption to heal. In another embodiment, the period of time is any time or range of times between 2 days and 3 weeks. Each possibility represents a separate embodiment of the present invention.
[0162] In one embodiment, "following" refers to a period of time of about 2 days. In another embodiment, "following" refers to a period of time of about 3 days. In another embodiment, "following" refers to a period of time of about 4 days. In another embodiment, "following" refers to a period of time of about 5 days. In another embodiment, "following" refers to a period of time of about 7 days. In another embodiment, "following" refers to a period of time of about 10 days. In another embodiment, "following" refers to a period of time of about 2 weeks. In another embodiment, "following" refers to a period of time of about 3 weeks. Each possibility represents a separate embodiment of the present invention.
[0163] In another embodiment, a method of the present invention further comprises the step of depilating the skin in the region in which hair growth or follicle formation is desired. In one embodiment, said step of depilating is performed prior to said step of epidermal disruption.
[0164] In another embodiment, the depilation is epilation. In another embodiment, the depilation comprises the step of waxing. In another embodiment, the depilation comprises the step of plucking. In another embodiment, the depilation comprises the use of an abrasive material. In another embodiment, the depilation comprises the use of a laser. In another embodiment, the depilation comprises the use of electrolysis. In another embodiment, the depilation comprises the use of a mechanical device. In another embodiment, the depilation comprises the use of thioglycolic acid. In another embodiment, the depilation comprises the use of any other method of depilation or epilation known in the art. Each possibility represents a separate embodiment of the present invention.
[0165] In another embodiment, the additional step (depilation or administration of a retinoid) is performed prior to the step of disrupting the epidermis. In another embodiment, the additional step is performed following the step of disrupting the epidermis, but prior to the addition of the compound or factor that of the present invention. In another embodiment, the additional step is performed concurrently with the addition of the differentiation-promoting compound or factor. In another embodiment, the additional step is performed following the addition of the differentiation-promoting compound or factor. Each possibility represents a separate embodiment of the present invention.
[0166] In another embodiment, a method of the present invention further comprises the step of administering a topical retinoid to the skin region of interest. In one embodiment, the topical retinoid induces resting (telogen) HF in the skin region of interest to enter anagen. Each possibility represents a separate embodiment of the present invention.
[0167] In another embodiment, the additional step is performed between about two days and about three weeks before the step of abrading. In another embodiment, the additional step is performed about two days before the step of abrading. In another embodiment, the additional step is performed about three days before the step of abrading. In another embodiment, the additional step is performed about four days before the step of abrading. In another embodiment, the additional step is performed about one week before the step of abrading. In another embodiment, the additional step is performed about ten days before the step of abrading. In another embodiment, the additional step is performed about two weeks before the step of abrading. In another embodiment, the additional step is performed about three weeks before the step of abrading. Each possibility represents a separate embodiment of the present invention.
[0168] In one embodiment, the methods of the present invention further comprise the step of administering an antagonist of an androgen or an antagonist of an androgen receptor. In another embodiment, the methods of the present invention further comprise the step of administering a 5 alpha-reductase type 2 inhibitor.
[0169] In another embodiment, a method of the present invention further comprises the step of contacting the skin region of interest with an anti-androgen compound. In one embodiment, the anti-androgen compound is finasteride. In another embodiment, the anti-androgen compound is Fluridil®. In another embodiment, the anti-androgen compound is dutasteride. In another embodiment, the anti-androgen compound is spironolactone. In another embodiment, the anti-androgen compound is cyproterone acetate. In another embodiment, the anti-androgen compound is bicalutamide. In another embodiment, the anti-androgen compound is flutamide. In another embodiment, the anti-androgen compound is nilutamide. In another embodiment, the anti-androgen compound is an inhibitor of an androgen receptor. In another embodiment, the anti-androgen compound is any other anti-androgen compound known in the art. Each possibility represents a separate embodiment of the present invention.
[0170] In another embodiment, a method of the present invention further comprises the step of contacting the skin region of interest with an estrogen compound. In another embodiment, a method of the present invention further comprises the step of contacting the skin region of interest with an estrogen receptor agonist. In another embodiment, a method of the present invention further comprises the step of contacting the skin region of interest with an estrogen analogue. In one embodiment, the estrogen analogue is estradiol. In another embodiment, the estrogen analogue is 17 beta-estradiol. In another embodiment, the estrogen analogue is 17 alpha-estradiol. In another embodiment, the estrogen analogue is ZYC3. In another embodiment, the estrogen compound, estrogen receptor agonist, or estrogen analogue is any other estrogen compound, estrogen receptor agonist, or estrogen analogue known in the art. Each possibility represents a separate embodiment of the present invention.
[0171] In another embodiment, a method of the present invention further comprises the step of contacting the skin region of interest with an inhibitor of an EGF protein. In another embodiment, a method of the present invention further comprises the step of contacting the skin region of interest with an inhibitor of an EGFR. In another embodiment, a method of the present invention further comprises the step of contacting the skin region of interest with a compound that reduces an expression of an EGF protein or an EGFR. Each possibility represents a separate embodiment of the present invention.
[0172] In another embodiment, the inhibitor of an EGF or an EGF receptor is panitumumab. In another embodiment, the inhibitor is AG1478. In another embodiment, the inhibitor is nimotuzumab. In another embodiment, the inhibitor is an antibody that binds EGF or EGFR. In another embodiment, the inhibitor is HuMax-EGFR® (Genmab, Copenhagen, Denmark). In another embodiment, the inhibitor is cetuximab. In another embodiment, the inhibitor is IMC 11F8. In another embodiment, the inhibitor is matuzumab. In another embodiment, the inhibitor is SC 100. In another embodiment, the inhibitor is ALT 110. In another embodiment, the inhibitor is PX 1032. In another embodiment, the inhibitor is BMS 599626. In another embodiment, the inhibitor is MDX 214. In another embodiment, the inhibitor is PX 1041. In another embodiment, the inhibitor is any other inhibitor of an EGF or an EGF receptor known in the art. Each possibility represents a separate embodiment of the present invention.
[0173] In another embodiment, a method of the present invention further comprises the step of contacting the skin region of interest with an inhibitor of a tyrosine kinase activity of an EGF receptor. In another embodiment, the inhibitor is gefitinib. In another embodiment, the inhibitor is erlotinib. In another embodiment, the inhibitor is canertinib. In another embodiment, the inhibitor is leflunomide. In another embodiment, the inhibitor is A77 1726. In another embodiment, the inhibitor is pelitinib. In another embodiment, the inhibitor is ZD 1839. In another embodiment, the inhibitor is CL 387785. In another embodiment, the inhibitor is EKI 785. In another embodiment, the inhibitor is vandetanib. In another embodiment, the inhibitor is any other inhibitor of a tyrosine kinase activity of an EGF receptor known in the art. Each possibility represents a separate embodiment of the present invention.
[0174] In another embodiment, a method of the present invention further comprises the step of contacting the skin region of interest with an EGF or EGFR antagonist. In another embodiment, the EGF or EGFR antagonist is a carboxypeptidase inhibitor from potato (PCI) protein or a homologue, fragment or mimetic thereof. In another embodiment, the EGF or EGFR antagonist is a sprouty protein or a homologue, fragment or mimetic thereof. In another embodiment, the EGF or EGFR antagonist is an Argos protein or a homologue, fragment or mimetic thereof. In another embodiment, the EGF or EGFR antagonist is a lefty protein or a homologue, fragment or mimetic thereof. In another embodiment, the EGF or EGFR antagonist is an antibody that recognizes EGF or EGFR, or a fragment or mimetic thereof. In another embodiment, the EGF or EGFR antagonist is small molecule inhibitor that binds and reduces the activity of EGF or EGFR. In another embodiment, the EGF or EGFR antagonist is CRM197. In another embodiment, the EGF or EGFR antagonist is IMC-C225 (ImClone Systems, New York, N.Y.). In another embodiment, the EGF or EGFR antagonist is any other antagonist of EGF or EGFR known in the art. Each possibility represents a separate embodiment of the present invention.
[0175] In another embodiment, the EGF or EGFR antagonist is a carboxypeptidase inhibitor from potato (PCI) protein or a homologue, fragment or mimetic thereof. In another embodiment, the EGF or EGFR antagonist is a sprouty protein or a homologue, fragment or mimetic thereof. In another embodiment, the EGF or EGFR antagonist is an Argos protein or a homologue, fragment or mimetic thereof. In another embodiment, the EGF or EGFR antagonist is a lefty protein or a homologue, fragment or mimetic thereof. In another embodiment, the EGF or EGFR antagonist is an antibody that recognizes EGF or EGFR, or a fragment or mimetic thereof. In another embodiment, the EGF or EGFR antagonist is small molecule inhibitor that binds and reduces the activity of EGF or EGFR. In another embodiment, the EGF or EGFR antagonist is CRM197. In another embodiment, the EGF or EGFR antagonist is IMC-C225 (ImClone Systems, New York, N.Y.). In another embodiment, the EGF or EGFR antagonist is any other antagonist of EGF or EGFR known in the art. Each possibility represents a separate embodiment of the present invention.
[0176] The EGFR of methods and compositions of the present invention has, in another embodiment, the sequence: MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNN CEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENS YALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLS NMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSD CCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYS FGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGI GEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITG FLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIIS GNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPR DCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDN CIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGL EGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPL TPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREAT SPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNI GSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAE EKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASE ISSILEKGERLPQPPICTIDVYMENIVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQ GDERMHLPSPTDSNEYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSA TSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKR PAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAH WAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA (GenBank Accession No: NM--005228; SEQ ID No: 8). In another embodiment, the EGFR has a sequence selected from the sequences set forth in GenBank entries NM--201282, NM--201283, NM--201284, BC094761, AF288738, AY588246, AY573061, X17054, AF125253, U48722, K03193, and AY698024. In another embodiment, the EGFR is encoded by a nucleic acid molecule having a sequence set forth in the one of the above GenBank entries. In another embodiment, a biologically active fragment of an EGFR is utilized in a method of the present invention. Each possibility represents a separate embodiment of the present invention.
[0177] The EGF of methods and compositions of the present invention has, in another embodiment, the sequence: MLLTLIILLPVVSKFSFVSLSAPQHWSCPEGTLAGNGNSTCVGPAPFLIFSHGNSIFRIDT EGTNYEQLVVDAGVSVIMDFHYNEKRIYWVDLERQLLQRVFLNGSRQERVCNIEKNV SGMAINWINEEVIWSNQQEGIITVTDMKGNNSHILLSALKYPANVAVDPVERFIFWSSE VAGSLYRADLDGVGVKALLETSEKITAVSLDVLDKRLFWIQYNREGSNSLICSCDYDG GSVHISKHPTQHNLFAMSLFGDRIFYSTWKMKTIWIANKHTGKDMVRINLHSSFVPLG ELKVVHPLAQPKAEDDTWEPEQKLCKLRKGNCSSTVCGQDLQSHLCMCAEGYALSR DRKYCEDVNECAFWNHGCTLGCKNTPGSYYCTCPVGFVLLPDGKRCHQLVSCPRNV SECSHDCVLTSEGPLCFCPEGSVLERDGKTCSGCSSPDNGGCSQLCVPLSPVSWECDCF PGYDLQLDEKSCAASGPQPFLLFANSQDIRHMHFDGTDYGTLLSQQMGMVYALDHDP VENKIYFAHTALKWIERANMDGSQRERLIEEGVDVPEGLAVDWIGRRFYWTDRGKSLI GRSDLNGKRSKIITKENISQPRGIAVHPMAKRLFWTDTGINPRIESSSLQGLGRLVIASSD LIWPSGITIDFLTDKLYWCDAKQSVIEMANLDGSKRRRLTQNDVGHPFAVAVFEDYV WFSDWAMPSVIRVNKRTGKDRVRLQGSMLKPSSLVVVHPLAKPGADPCLYQNGGCE HICKKRLGTAWCSCREGFMKASDGKTCLALDGHQLLAGGEVDLKNQVTPLDILSKTR VSEDNITESQHMLVAEIMVSDQDDCAPVGCSMYARCISEGEDATCQCLKGFAGDGKL CSDIDECEMGVPVCPPASSKCINTEGGYVCRCSEGYQGDGIHCLDIDECQLGVHSCGE NASCTNTEGGYTCMCAGRLSEPGLICPDSTPPPHLREDDHHYSVRNSDSECPLSHDGY CLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELRHAGHGQQQKVIVVAV CVVVLVMLLLLSLWGAHYYRTQKLLSKNPKNPYEESSRDVRSRRPADTEDGMSSCPQ PWFVVIKEHQDLKNGGQPVAGEDGQAADGSMQPTSWRQEPQLCGMGTEQGCWIPVS SDKGSCPQVMERSFHMPSYGTQTLEGGVEKPHSLLSANPLWQQRALDPPHQMELTQ (GenBank Accession No: NM--001963; SEQ ID No: 9). In another embodiment, the EGF has a sequence selected from the sequences set forth in GenBank entries BC093731, AY548762, and X04571. In another embodiment, the EGF is encoded by a nucleic acid molecule having a sequence set forth in the one of the above GenBank entries. In another embodiment, a biologically active fragment of an EGF is utilized in a method of the present invention. Each possibility represents a separate embodiment of the present invention.
[0178] In another embodiment, a method of the present invention further comprises the step of contacting the skin region of interest with a Hedgehog protein. In another embodiment, a method of the present invention further comprises the step of contacting the skin region of interest with a nucleotide encoding a Hedgehog protein. In another embodiment, a method of the present invention further comprises the step of contacting the skin region of interest with an activator of a Hedgehog protein. Each possibility represents a separate embodiment of the present invention.
[0179] In one embodiment, the methods of the present invention further comprise the step of administering a 5 alpha-reductase type 2 inhibitor, which in one embodiment, is Finasteride or, in another embodiment, turosteride.
[0180] In another embodiment, the methods of the present invention further comprise the step of administering a reductase inhibitor, which in one embodiment is an FCE-dual inhibitor, which in one embodiment is FCE 28260; FCE 28175, or FCE 27837; in another embodiment is an MK inhibitor, which in one embodiment, is MK 0434, MK 0963, or MK 386; in another embodiment, is an FK Nonsteroidal Inhibitor, which in one embodiment is FK 143; and in another embodiment is a LY Nonsteroidal Inhibitor, which in one embodiment, is LY 191704; and in another embodiment, is a SK&F Inhibitor, which in one embodiment, is SK&F 105657.
[0181] In another embodiment, the methods of the present invention further comprise the step of administering an additional composition. In one embodiment, the composition is Dutasteride (Avodart®, GI198745), Finasteride (Propecia®, Proscar®), Turosteride, Azelaic acid, Zinc sulphate, CS 891, or a combination thereof.
[0182] In another embodiment, the methods of the present invention further comprise the step of administering an antiandrogen, which in one embodiment is Spironolactone (Aldactone®), Flutamide (Euflex®, Eulexin®), Casodex, Inocoterone, an RU Antiandrogen, TZP-4238, Win 49596, Fluridil (Eucapil®), or a combination thereof.
[0183] In another embodiment, the methods of the present invention further comprise the step of administering a K+ Channel Opener, which in one embodiment, is minoxidil (Rogaine®), Diazoxide, Cromakalim, Pinacidil, or a combination thereof.
[0184] In another embodiment, the methods of the present invention further comprise the step of administering a vasodilator, which in one embodiment, is minoxidil (Rogaine®).
[0185] In another embodiment, the methods of the present invention further comprise the step of administering an estrogen blocker, which in one embodiment, is an ICI Estrogen Blocker.
[0186] The subject of methods of the present invention, is, in another embodiment, a human. In another embodiment, the subject is a rodent, in one embodiment, a mouse, in another embodiment, a rat. In another embodiment, the subject is a mammal. In another embodiment, the subject is a vertebrate. In another embodiment, the subject is feline, canine, ovine, or bovine. In another embodiment, the subject is a male. In another embodiment, the subject is a female. In another embodiment, the subject is any other subject known in the art. Each possibility represents a separate embodiment of the present invention.
[0187] In another embodiment, the subject is an adult. In one embodiment, "adult" refers to an age greater than about 18 years. In another embodiment, "adult" refers to an age greater than about 20 years. In another embodiment, "adult" refers to an age greater than about 25 years. In another embodiment, "adult" refers to an age greater than about 30 years. In another embodiment, "adult" refers to an age greater than about 35 years. In another embodiment, "adult" refers to an age greater than about 40 years. In another embodiment, "adult" refers to an age greater than about 45 years.
[0188] In another embodiment, the subject is elderly. In one embodiment, "elderly" refers to an age greater than about 45 years. In another embodiment, "elderly" refers to an age greater than about 50 years. In another embodiment, "elderly" refers to an age greater than about 55 years. In another embodiment, "elderly" refers to an age greater than about 60 years. In another embodiment, "elderly" refers to an age greater than about 65 years. In another embodiment, "elderly" refers to an age greater than about 70 years.
[0189] In another embodiment, the first subject, or, where applicable, both the first subject and the second subject, is a laboratory animal. In another embodiment, the subject(s) is/are mice. In another embodiment, the subject(s) is/are rats. In another embodiment, the subject(s) is/are gerbils. In another embodiment, the subject(s) is/are hamsters. In another embodiment, the subject(s) is/are guinea pigs. In another embodiment, the subject(s) is/are rabbits. In another embodiment, the subject(s) is/are pigs. In another embodiment, the subject(s) is/are dogs. In another embodiment, the subject(s) is/are cats. In another embodiment, the subject(s) is/are primates. In another embodiment, the subject(s) is/are any other laboratory animal known in the art. Each possibility represents a separate embodiment of the present invention.
[0190] In one embodiment, the subject is contacted with FGF9, or in another embodiment, with a composition comprising FGF9. In another embodiment, FGF9 or a composition comprising FGF9 is administered to a subject.
[0191] "Contacting" as used herein refers, in another embodiment, to bringing skin, in one embodiment, scalp, eyebrow, etc, into to contact with a compound, factor, cell, etc. In another embodiment, the term refers to embedding the compound, factor, cell, etc into the skin region of interest. In another embodiment, the term refers to injecting the compound, factor, cell, etc into the skin region of interest. In another embodiment, term refers to any other type of contacting known in the art. Each possibility represents a separate embodiment of the present invention.
[0192] In another embodiment, the step of contacting in methods of the present invention comprises directly contacting the skin region of interest with the compound, RNA, protein, etc. In another embodiment, the step of contacting comprises indirectly contacting the skin region of interest via contacting another site or tissue of the subject, after which the compound, RNA, or protein is transported to the skin region of interest by a biological process; e.g, diffusion, active transport, or circulation in a fluid such as the blood, lymph, interstitial fluid, etc. Each possibility represents a separate embodiment of the present invention.
[0193] In one embodiment, other fibroblast growth factors may be used in the methods of the present invention. In one embodiment, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, or a combination thereof may be used in the methods of the present invention. In one embodiment, FGF1 through FGF10 all bind fibroblast growth factor receptors (FGFRs). In one embodiment, FGF1 is known as acidic fibroblast growth factor, and FGF2 is also known as basic fibroblast growth factor.
[0194] In another embodiment, FGF11, FGF12, FGF13, or FGF14, may be used in the methods of the present invention. In one embodiment, FGF11, FGF12, FGF13, and FGF14 are known as FGF homologous factors 1-4 (FHF1-FHF4), and in another embodiment, have distinct functional differences compared to the FGFs. In one embodiment, these factors possess remarkably similar sequence homology, they do not in one embodiment, bind FGFRs and are involved in intracellular processes unrelated to the FGFs. In one embodiment, this group is also known as "iFGF".
[0195] In another embodiment, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, or FGF23 may be used in the methods of the present invention. In one embodiment, FGF15/FGF19, FGF21 and FGF23 have systemic rather than local effects.
Pharmaceutical Compositions
[0196] In another embodiment, methods of the present invention comprise administering a pharmaceutical composition comprising FGF9 or an up-regulator of SHH and/or its analog, derivative, isomer, metabolite, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, or any combination thereof; and a pharmaceutically acceptable carrier. Each possibility represents a separate embodiment of the present invention.
[0197] The pharmaceutical compositions containing FGF9 or an up-regulator of SHH can, in another embodiment, be administered to a subject by any method known to a person skilled in the art, such as topically, parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, subepidermally, intraperitonealy, intraventricularly, intra-arteriolly, intravascularly, intracranially, intravaginally, intrarectally, or intratumorally. Each possibility represents a separate embodiment of the present invention. In one embodiment, the dosage regimen will be determined by skilled clinicians, based on factors such as exact nature of the condition being treated, the severity of the condition, the age and general physical condition of the patient, body weight, and response of the individual patient, etc.
[0198] In another embodiment, the pharmaceutical compositions are administered orally, and are thus formulated in a form suitable for oral administration, i.e. as a solid or a liquid preparation. Suitable solid oral formulations include tablets, capsules, pills, granules, pellets and the like. Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In one embodiment of the present invention, the FGF9 or other polypeptide as provided herein composition is formulated in a capsule. In another embodiment, the compositions of the present invention comprise, in addition to FGF9 or other polypeptide as provided herein an inert carrier or diluent, or a hard gelating capsule.
[0199] In another embodiment, the pharmaceutical compositions are administered topically to body surfaces and are thus formulated in a form suitable for topical administration. Suitable topical formulations include gels, ointments, creams, lotions, drops, gels; pastes; powders; aerosol sprays; syrups or ointments on sponges or cotton applicators; and solutions or suspensions in an aqueous liquid, non-aqueous liquid, oil-in-water emulsion, or water-in-oil liquid emulsion, and the like. Because of its ease of administration, a cream, lotion, or ointment represents the most advantageous topical dosage unit form, in which case liquid pharmaceutical carriers may be employed in the composition. These creams, lotions, or ointments, may be prepared as rinse-off or leave-on products, as well as two stage treatment products for use with other skin cleansing or managing compositions. In a preferred embodiment, the compositions are administered as a rinse-off product in a higher concentration form, such as a gel, and then a leave-on product in a lower concentration to avoid irritation of the skin. Each of these forms is well understood by those of ordinary skill in the art, such that dosages may be easily prepared to incorporate the pharmaceutical composition of the invention. In one embodiment, a delayed release patch may be used for administration of FGF9. For topical administration, the FGF9 composition or its physiologically tolerated derivatives such as salts, esters, N-oxides, and the like are prepared and applied as solutions, suspensions, or emulsions in a physiologically acceptable diluent with or without a pharmaceutical carrier.
[0200] Ointment preparations may be roughly classified into fat/oil type ointments, emulsified ointments, water-soluble ointments and suspended ointments according to the type of the base (vehicle) used therefor. An ointment may comprise, for example, fats, fatty oils, lanolin, vaseline, paraffins, waxes, resins, plastics, glycols, higher alcohols, glycerol, water, emulsifiers, suspending agents or other appropriate additives as a diluent, carrier or as a vehicle. Manufacture of an ointment comprises, for example, adding the compound of the present invention to the appropriate additives, diluents, carriers or vehicles followed by mixing to make the mixture homogeneous.
[0201] For parenteral application, particularly suitable are injectable, sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories and enemas. Ampoules are convenient unit dosages. Such a suppository may comprise any agent described herein.
[0202] For application by inhalation, solutions or suspensions of the compounds mixed and aerosolized or nebulized in the presence of the appropriate carrier suitable. Such an aerosol may comprise any agent described herein.
[0203] For enteral application, particularly suitable are tablets, dragees, liquids, drops, or capsules. In one embodiment, a sweetened vehicle is employed when a syrup, elixir, or the like is used for enteral application.
[0204] For liquid formulations, pharmaceutically acceptable carriers are, in another embodiment, aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, in another embodiment, water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil.
[0205] In another embodiment, the pharmaceutical compositions are administered by subcutaneous implantation of a pellet. In another embodiment, the pellet provides for controlled release of the FGF9 over a period of time.
[0206] In one embodiment, the pharmaceutical compositions are controlled-release compositions, i.e. compositions in which the FGF9 composition is released over a period of time after administration. Controlled- or sustained-release compositions include, in another embodiment, formulation in lipophilic depots (e.g. fatty acids, waxes, oils). In another embodiment, the composition is an immediate-release composition, i.e. a composition in which all the FGF9 composition is released immediately after administration. Sustained or directed release compositions can be formulated, e.g., liposomes or those wherein the active compound is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc. It is also possible to freeze-dry the new compounds and use the lyophilisates obtained, for example, for the preparation of products for injection.
[0207] In one embodiment, compositions of this invention are pharmaceutically acceptable. In one embodiment, the term "pharmaceutically acceptable" refers to any formulation which is safe, and provides the appropriate delivery for the desired route of administration of an effective amount of at least one compound for use in the present invention. This term refers to the use of buffered formulations as well, wherein the pH is maintained at a particular desired value, ranging from pH 4.0 to pH 9.0, in accordance with the stability of the compounds and route of administration.
[0208] In one embodiment, FGF9 or upregulators of SHH used in the methods of this invention may be administered alone or within a composition. In another embodiment, compositions comprising FGF9 or upregulators of SHH in admixture with conventional excipients, i.e. pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral (e.g., oral) or topical application which do not deleteriously react with the active compounds may be used. In one embodiment, suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatine, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, white paraffin, glycerol, alginates, hyaluronic acid, collagen, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc. In another embodiment, the pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds. In another embodiment, they can also be combined where desired with other active agents, e.g., vitamins.
[0209] In one embodiment, the therapeutic compositions of the present invention comprise an FGF9 composition and one or more additional compounds effective in preventing or treating dermatologic conditions such as alopecia. In one embodiment, the additional compound is a moisturizer or an emollient, which in one embodiment is petrolatum, white petrolatum, hydrogenated vegetable oil, hydrophilic petrolatum, panthenol, primrose oil, omega-3 fish oils, omega-6 fish oils, linoleic acid, flax seed oil, ceramide, borage oil (linoleic acid), tocopherol (Vitamin E), tocopherol linoleate, dimethicone, glycerine or a combination thereof. In one embodiment, moisturizers improve the ability of the skin to absorb other administered compounds, including inter alia, the compounds for use in the present invention. In another embodiment, moisturizing agents minimize or prevent the skin from drying and cracking, thereby decreasing susceptibility of skin to environmental factors that generate free radicals, thereby preventing additional damage to the skin.
[0210] In another embodiment, the additional compound is a topical steroid, which in one embodiment is hydrocortisone, in one embodiment 1% hydrocortisone, triamcinolone, fluocinolone acetonide, halcinonide, halobetasol propionate, clobetasol propionate, betamethasone dipropionate, betamethasone valerate, and triamcinolone acetonide or a combination thereof; oral steroids; topical immunomodulators including, inter alia, tacrolimus, pimecrolimus, Ascomycin, cyclosporine, or a combination thereof; antihistamines, which in one embodiment is hydroxyzine or diphenhydramine hydrochloride, Ketotifen, Doxepin; biologics, which in one embodiment comprises Amevive (alefacept), Enbrel, Humira, Raptiva, Remicade, or a combination thereof; or a combination thereof. In another embodiment, the additional compound is an antibiotic, which in one embodiment comprise tetracycline, doxycline, minocycline, cloxacillin, cephalexin, penicillin, clindamycin or a combination thereof. In another embodiment, the additional compound is methotrexate, tar, coal tar, anthralin, dovonex, salicyclic acid, tazorac, moisturizers, aloe vera, soriatane, accutane, hydrea, mycophenolate mofetil, sulfasalazine, 6-thioguanine, or a combination thereof. In another embodiment, additional compounds comprise acyclovir, which in one embodiment is particularly effective in patients with eczema herpeticum. In one embodiment, additional compounds to treat seborrheic dermatitis comprise zinc pyrithione, selenium sulfide, sulfur, tar shampoo, flucinolone acetonide solution, triamcinolone acetonide lotion, ketoconazole cream, other imidazoles, or a combination thereof.
[0211] In another embodiment, the additional compound is an anti-inflammatory agent, which in one embodiment comprises aspirin, ibuprofen, ketoprofen, naproxen, or a combination thereof. In another embodiment, the additional compound is a prostaglandin or prostaglandin inhibitor, which in one embodiment is an inhibitor of PGD2.
[0212] In another embodiment, the additional compound is an exfoliant, which in one embodiment comprises an enzymatic exfoliant or a mono- or -poly-hydroxy acid. In one embodiment, the exfoliant is an alpha-hydroxy acid, beta-hydroxy acid, tannic acid, glycolic acid, lactic acid, citric acid, salicylic acid, or a combination thereof. In another embodiment, the additional compound is an analgesic, or anesthetic, while in another embodiment it is aloe vera gel, aloe vera, licorice extract, pilewort, Canadian willow root, zinc, allantoin, or a combination thereof. In another embodiment, the additional compound is an anti-oxidant.
[0213] In one embodiment, fibroblast growth factor-9 protein is administered at a concentration of 10 ng/mL. In another embodiment, fibroblast growth factor-9 protein is administered at a concentration of 20 ng/mL. In another embodiment, fibroblast growth factor-9 protein is administered at a concentration of 40 ng/mL. In another embodiment, fibroblast growth factor-9 protein is administered at a concentration of 80 ng/mL. In another embodiment, fibroblast growth factor-9 protein is administered at a concentration of 5 ng/mL. In another embodiment, fibroblast growth factor-9 protein is administered at a concentration of 3 ng/mL. In another embodiment, fibroblast growth factor-9 protein is administered at a concentration of 1 ng/mL. In another embodiment, fibroblast growth factor-9 protein is administered at a concentration of between 1 and 50 ng/mL. In another embodiment, fibroblast growth factor-9 protein is administered at a concentration of between 1 and 15 ng/mL. Each dose represents a separate embodiment.
[0214] In general, the doses utilized for the above described purposes will vary, but will be in an effective amount to exert the desired effect. As used herein, the term "pharmaceutically effective amount" refers to an amount of a FGF9 or other composition for use in the present invention, which will produce the desired alleviation in symptoms or other desired phenotype in a patient. The doses utilized for any of the above-described purposes will generally be from 1 to about 1000 milligrams per kilogram of body weight (mg/kg), administered one to four times per day, or by continuous IV infusion. In one embodiment, a topical daily dose range, in single or divided doses, for the conditions described herein is from about 1 mg to 20,000 mg, more preferably about 2,000 mg to 16,000 mg, and most preferably about 6,000 mg to 10,000 mg of the active components (i.e., excluding excipients and carriers). When the compositions are dosed topically or intraocularly, they will generally be in a concentration range of from 0.1 to about 10% w/v, administered 1-4 times per day. In one embodiment, the compositions for use in the methods of the present invention are administered topically two times a day.
[0215] In one embodiment of the invention, the concentrations of the compounds will depend on various factors, including the nature of the condition to be treated, the condition of the patient, the route of administration and the individual tolerability of the compositions.
[0216] In one embodiment, the administering step is via topical administration. In another embodiment, the administering step is via subcutaneous administration.
[0217] In one embodiment, the compound administered as part of methods of the present invention is administered systemically. In another embodiment, the compound is administered topically. In another embodiment, the compound is administered subepidermally. In another embodiment, the compound is administered subcutaneously. In another embodiment, the compound is administered transdermally. In another embodiment, the compound is administered to the site of the abrasion. In another embodiment, the compound is administered to the site of the wound induction. In another embodiment, the compound is administered to the site of the depilation. In another embodiment, the compound is administered during wound healing. In another embodiment, the compound is administered prior to HF neo-genesis. In another embodiment, the compound is administered during HF neo-genesis. Each possibility represents a separate embodiment of the present invention.
[0218] In one embodiment, the route of administration may be directed to an organ or system that is affected by alopecia. For example, compounds may be administered topically to treat dermatologic conditions such as alopecia. In another embodiment, the route of administration may be directed to a different organ or system than the one that is affected by dermatologic conditions such as alopecia. For example, compounds may be administered parenterally to treat dermatologic conditions such as alopecia. Thus, the present invention provides for the use of FGF9 or other composition for use in the present invention in various dosage forms suitable for administration using any of the routes listed hereinabove.
[0219] In one embodiment, the methods of the present invention of testing a compound are repeated using a plurality of subjects, until a statistically significant sample has been tested.
[0220] In one embodiment, FGF9 augments hair germ formation in embryonic tissue, but is not essential for hair germ formation. In one embodiment, FGF9 is necessary for hair follicle formation and/or size in adult tissue after epidermal disruption.
[0221] In one embodiment, the signaling pathway for embryonic hair germ formation and for wound-induced hair follicle neogenesis share one or more components. In another embodiment, the signaling pathway for embryonic hair germ formation and for wound-induced hair follicle neogenesis is not identical. Thus, in one embodiment, FGF9 is essential for hair follicle formation in WIHF but not during ED13.5.
[0222] In another embodiment, the signaling pathway for hair growth signals differs in different parts of the body. Thus, in one embodiment, embryonic hair germ formation and wound-induced hair follicle neogenesis differ in their dependence on FGF9 due to the differences in their respective developmental stages and differences in location in the body.
[0223] In one embodiment, the combination of FGF9 and wound healing increases its efficacy as a hair growth promoter. In one embodiment, FGF9 application alone causes epidermal thickening.
[0224] In another embodiment, the invention provides a method of treating hair loss or regenerating hair follicles in a subject comprising the step of disrupting the epidermis in the region of said hair loss in said subject. In some embodiments, the method further comprises the step of recruiting the gamma-delta T cells to the wound epidermis. In an exemplary embodiment, the method further comprises the step of recruiting the gamma-delta T cells to the wound epidermis through cytokines.
EXAMPLES
Experimental Details
Depilation and Epidermal Abrasion
[0225] Mice were anesthetized with an injection of sodium pentobarbital before the hair on the back was clipped and depilated with Nair (Carter-Wallace, New York, N.Y.), then epidermis was removed using a rotating felt wheel as described by Argyris T, J Invest Dermatol, 75: 360-362, 1980). After scrubbing with 70% ethanol and drying under an incandescent lamp, the basal and supra-basal layers in an area of (1.5 cm)2 cm of the inter-follicular epidermis were removed by careful abrasion with a felt wheel mounted on a Dremel Moto-tool (Racine, Wis.). After abrasion, the skin was shiny and smooth, and there was no blood. One day later, the abraded area was covered by a fibrin crust, which fell off after 3-7 days, exposing the newly regenerated epidermis. A group of control mice was sacrificed immediately after abrasion to confirm microscopically the complete removal of the interfollicular epidermis.
Punch Wound and Excisional Wound Induction
[0226] The backs of 21-day-old mice were depilated as described for Example 1 and sterilized with alcohol, followed by 1% iodine solution. Punch wounds, 4 mm in diameter, were induced using a dermal biopsy punch, down to, but not through, the muscle fascia. Excisional wounds were full thickness and 1 cm in diameter; skin and panniculus carnosus was excised using fine surgical scissors.
Immunohistochemistry
[0227] Skin samples were fixed in PBS-buffered 10% formalin. Six-micron thick paraffin sections were cut and stained, where applicable, with antibodies.
Whole Mounting and Immunofluorescence
[0228] HF whole mounts were obtained by incubating fresh skin with EDTA (20 mM in PBS) at 37° C. overnight, then separating the epidermis and dermis. Epidermis was then fixed in 10% formalin for 10 min, room temperature (RT). Dermis was fixed in acetone overnight, RT. After rinsing with PBS, whole mounts were stained with antibodies for immunohistochemistry (schematically depicted in FIG. 12) and were imaged using a Leica confocal microscope.
Statistics
[0229] Hair follicle numbers are expressed as mean±s.d. The student's two-tailed t-test function in Excel was used to calculate P values.
Embryonic Mouse Skin Culture Protocol
[0230] The following materials were used: Center well dishes (Fisher 08-772-12); Metal grids (Goodfellow 688-485-21); Nitrocellulose filters (Millipore AABP04700); Media: DMEM+5% FBS+1× Pen/Strep.
[0231] Gestational day 13.5 timed pregnant mothers (Charles River) were ordered. Center well dishes were set up with 2 ml media/dish. Metal grid was placed in center well. Dishes were stored in incubator so the media warmed to 37° C. Nitrocellulose filter were cut into rectangles and placed in a beaker of dH2O on a hot plate. Water was allowed to boil and then filters were boiled for 10 min.
[0232] Two petri dishes with sterile PBS were prepared. Mothers were euthanized and embryos were dissected out in the sac. Embryos were placed into one petri dish. The embryos were dissected out of the sac and placed in a second clean petri dish with sterile PBS. The dish of embryos were placed on ice. Dorsal skin was dissected from the embryo under a dissecting scope, in a clean petri dish containing sterile PBS.
[0233] The crown-rump length of the embryo was checked with a ruler to ensure it is E13.5 stage. (˜10-10.3 mm). The head of the embryo was removed with micro dissecting scissors. A smaller pair of micro dissecting scissors were used to make incisions along both sides of the back, above the limbs. A third incision was made across the back, anterior to the tail. Using fine tipped Dumont tweezers, the skin was peeled from the tail towards the head. The skin was laid onto the black side of nitrocellulose filter, as flat as possible. The nitrocellulose filter was placed onto metal grid so that the skin is at the liquid-air interface. The dish was incubated at 37° C. When all skins were dissected, compounds were added to culture media (if necessary), and returned to the incubator. Skins were cultured for up to 3 days. Placodes started to develop on E14.5.
Example 1
FGF9 Expressed in Early Period of Hair Germ Formation
[0234] FGF9 mRNA expression was evaluated in regenerated epidermis by quantitative real time-PCR. FGF9 was expressed at higher levels prior to the earliest stages of hair follicle regeneration at Day 1 after scab detachment (SD; which occurs at reepithelialization) compared to Day 5 after scab detachment when follicles have formed (FIG. 1). Skin γδ T-cells (detected by immunostaining using antibodies against γδTCreceptor) repopulate the reepithelialized epidermis by SD7 (FIG. 2, left panel) and these cells express FGF9 protein at SD1 (red dendritic cell in epidermis, FIG. 2, right panel, and FIG. 3).
[0235] Thus, FGF9 was selectively expressed prior to hair germ formation (during the undifferentiated period) rather than during differentiation. Skin γδ T cells appeared to be the source of FGF9, which suggests inflammatory cells may have a role in Wound-induced hair follicle neogenesis (WIHN).
Example 2
FGF9 Expressed in Embryonic Day 14 (E14) Skin
[0236] FGF9 (red staining) is expressed by γδTC (green staining) in embryonic day 14 (E14) skin (FIG. 4).
Example 3
FGF9 Plays a Role in Wound-Induced Hair Follicle Neogenesis (WIHN)
Anti-FGF9 Neutralization Experiment in Adult Mice
[0237] 3 week-old (adult) C57BL/6 mice were subjected to the wounding model as described hereinabove. Mice then received subepidermal injections of 50 μl of 10 μg/ml anti-FGF9 or IgG2a isotype control on days SD1-SD4. Tissue samples were taken and analyzed at SD5.
[0238] Immunoblots were used to verify the specificity of the anti-FGF9 neutralization antibody. Mouse FGF9 has 198 bp and greater than 99% homology with human FGF9 (with only one amino acid difference). FGF9 exists in both monomer (25-27 kd) and dimer forms. Immunoblots demonstrated the presence of both the 26 Kd monomer and the 52 Kd dimmer forms in E14.5 mouse embryonal whole cell lysates, as well as in control samples containing recombinant hFGF9 (FIG. 5).
[0239] Mice receiving anti-FGF9 antibody had significantly lower hair follicle numbers on SD5 than IgG2a controls (FIG. 6). Thus, FGF9 plays a role in wound-induced hair follicle neogenesis.
[0240] The developmental stages of the hair follicles were quantitated as described in Paus R et al., J Invest Dermatol 1999. There was a decrease in mature hair follicles and an increase in immature hair follicles in the anti-FGF9 treated group (FIG. 7).
Example 4
FGF9 Plays a Role in Embryonic Skin Development
[0241] Scheduled pregnant C57BL/6 mice were sacrificed at E13.5, and embryonic whole back skin was dissected. E13.5 skin was cultured for three days floated on filter paper with metal grid.
[0242] To determine the role of FGF-9 in hair follicle neogenesis in embryonic skin, embryonic skin explant cultures were treated for three days with recombinant human (rh)FGF9 (control, 10, 20, or 40 ng/mL) or with an anti-FGF9 neutralizing antibody (control, 10, 20, or 40 μg/mL) or IgG2a isotype control (10, 20, 40 μg/mL) Alkaline phosphatase (AP) for dermis immunostaining (FIG. 10) and K17 was used for epidermis immunostaining (FIG. 12).
[0243] Hair germ counting was performed at three separate fields per sample and was evaluated per mm2 (FIG. 8). q-PCR for Shh, Ptch1, Ptch2, Gli1, and Gli2 was performed after 24 h of rhFGF9 treatment.
Real-Time PCR Protocol
[0244] The following materials were used: RNeasy® fibrous tissue mini kit (Qiagen, 74704); High capacity cDNA reverse transcription kit (Applied Biosystems, P/N 4368814); Taqman® Fast universal PCR master mix (2×) (Applied Biosystems, P/N 4352042); Applied Biosystems StepOne® real-time PCR system; (Applied Biosystems, P/N 4376373); MicroAmp® 48-well optical adhesive film (Applied Biosystems, P/N 4375928); MicroAmp® 48-well reaction plate (Applied Biosystems, P/N 4375816).
[0245] The following PCR primers were used (Taqman® gene expression assay, Applied Biosystems):
TABLE-US-00001 TABLE 1 Target Reference gene Gene name Assay primer ID sequence Fgf9 fibroblast Mm00442795_m1 NM_013518.3 growth factor 9 Shh Sonic Mm00436527_m1 NM_009170.3 hedgehog Ptch1 patched Mm00436026_m1 NM_008957.2 homolog 1 Ptch2 patched Mm00436047_m1 NM_008958.2 homolog 2 Gli1 GLI-Kruppel Mm00494645_m1 NM_010296.2 family member GLI1 Gli2 GLI-Kruppel Mm01293116_m1 NM_001081125.1 family member GLI2 ACTB actin, beta P/N 4352933E NM_007393.1 (endogenous control)
Protocol for q-PCR with Cultured Embryonic Skin Samples
[0246] Embryonic skin culture: E13.5 timed pregnant B57BL/6 female mice (Charles-River) were euthanized in CO2 chamber. Embryos were dissected and placed in sterile cold PBS on ice.
[0247] Preparation: Millipore nitrocellulose membrane (0.5×1.0 cm2); Autoclave metal grids; Culture dishes (Falcon center-well organ culture dish, 35-3037); 5% FBS-DMEM (lx penicillin/streptomycin, not necessary to inactivate FBS).
[0248] 2.5 mL of culture media was added and metal mesh and nitrocellulose membrane were set on the individual culture dishes. Embryonic back skin was dissected. Head & buttock area were cut. Dissection was through flank in a caudo-cranial direction. Dissected back skin was loaded on Millipore membrane (dermal side down). The samples were prepared in triplicate per needed for each concentration. Skin samples were cultured for 24 hr at 37° C. in 5% CO2.
RNA Isolation & cDNA Preparation
[0249] Skin samples were incubated in 20 mM EDTA for 10 min Epidermis and dermis were separated with fine-tipped tweezers under a dissecting microscope, respectively.
[0250] Samples were disrupted with a homogenizer and total RNA extracted with RNeasy® fibrous tissue mini kit (Qiagen, 74704) following manufacturer's information.
[0251] RNA concentration was measured by spectrophotometer and then converted to lag of total RNA to cDNA using High capacity cDNA reverse transcription kit (Applied Biosystems, P/N 4368814) with program in thermal cycler.
Real-Time PCR
[0252] PCR running program was set up and arranged the reaction plate layout with provided StepOne software in comparative CT (ΔΔCT) method. The reaction mixture of target gene and β-actin endogenous control were prepared together in triplicate. cDNA template was diluted from stock to final total cDNA amount of 30-50 ng in 2 μl.
[0253] Reaction Mix Components
TABLE-US-00002 Volume (μl) Component for 1 reaction Taqman ® Fast universal PCR master mix (2X) 10.0 PCR primers (Taqman ® gene expression assay) 1.0 H2O 7.0 cDNA template 2.0 Total volume 20.0
[0254] 2. Prepare the reaction plate: A reaction volume of 20 μl/well is added on 48-well reaction plate. The plate is sealed tightly with optical adhesive film.
[0255] 3. Load the plate into StepOne instrument and start the programmed reaction.
[0256] 4. Analyze the results with the StepOne software and obtain relative quantitation data of gene expression.
[0257] The effect of rhFGF9 treatment for three days in the dermis of embryonic skin explant culture (E13.5) was dose-dependent, with 10 ng/mL and 20 ng/ml resulting in an increase in hair germ number/mm2, while a 40 ng/mL dose resulted in decreased hair germ number/mm2 (FIGS. 9-10). On the other hand, there was no discernable effect of anti-FGF9 neutralizing antibody treatment for three days in the epidermis or dermis of embryonic skin explant culture (E13.5; FIGS. 11-13).
[0258] 24-hr treatment of E13.5 embryonic skin explant culture with 10 ng/mL of rhFGF9 resulted in increases in markers of embryonic hair follicle development including sonic hedgehog (Shh), Ptch1, Ptch2, and Gli1, particularly in the epidermis (FIGS. 14-15).
[0259] Fibroblast growth factor 9 increases hair follicle formation when injected into the wound after healing. This is just prior to and during the time when new hair follicles are forming FGF9 also increases hair follicle formation during hair follicle development by using embryonic mouse skin explanted in culture. These findings support the notion that wounding converts the epidermis to a "receptive" state in which it responds to exogenous factors.
Example 5
Overexpression of FGF9 in Basal Keratinocytes
[0260] The gain of function mutant TRE-fgf9-IRES-eGfp; K5-rtTA (×Ptch1-LacZ reporter) (White et al., Development 133, 1507-1517, 2006; Diamond, et al. (2000) J. Invest. Dermatol. 115,788-794, both incorporated herein by reference) is used to validate that early FGF9 expression in hair neogenesis stage would enhance hair follicle development and to confirm that FGF9 is upstream of Shh signaling.
Example 6
Deletion of FGF9 Expression in γδ T Cells
[0261] Deletion of FGF9 expression in γδT cells is accomplished using the loss of function mutant FGF9flox/flox; lck-cre to selectively delete FGF9 using T-cell targeting lck-cre promoter. The Lck-Cre uses the proximal promoter of the Lck (lymphocyte protein tyrosine kinase) gene, which is first expressed early in thymocyte development at the double negative stage. After T cells fully mature, the level of expression of this transgene decreases by approximately 10 fold. This particular mouse gene shows a high degree of expression of the transgene in the thymus and has been found to bring about the selective deletion of genes flanked by loxP targeting sequences in almost all early thymocytes. It thus is used to delete a specific gene in the T cell lineage starting at the double negative stage. Since the homozygous Lck-Cre mice strains are crossed to a strain containing a floxed FGF9 and offspring with deleted FGF9 in the T cell lineage are obtained. Control animals are obtained in the same litter by typing for the presence or absence of the floxed gene in genomic DNA tail samples. This system is described in more detail in Lee et al., Immunity November 2001:15(5) 763-74, which is incorporated herein by reference.
Example 7
K17-eGFP Reporter Mice
[0262] K17-eGFP reporter mice (Bianchi et al., Mol Cell Biol., 2005 August; 25(16): 7249-7259, incorporated herein by reference) are used to confirm the accumulation of FGF9-producing γδ T-cells around newly developing hair germs.
Example 8
FGF9 Mediates Hair Follicle Neogenesis Through Epidermal γδ T Cells
[0263] Understanding molecular mechanisms responsible for hair follicle regeneration during wound healing raises the opportunity to develop new treatments for hair loss and other skin disorders. Here, it is clearly shown that Fibroblast Growth Factor 9 (Fgf9) modulates hair follicle formation following wounding of adult mice. Forced overexpression of Fgf9 in the newly formed wound epidermis results in a 2-3-fold increase in the number of neogenic hair follicles. Remarkably, during wound healing in normal mice, γδ T cells, which reside in the epidermis, serve as the primary source for Fgf9. Specific deletion of the Fgf9 gene in T cells using Lck-Cre; floxed fgf9 transgenic mice results in a marked reduction of hair follicle neogenesis following wounding. Similarly, mice lacking γδ T cells demonstrate severe impairment of follicular neogenesis. Overall, these findings explain the robustness of hair follicle regeneration in mouse compared to human and highlight the important relationship between the immune system and tissue regeneration.
Materials and Methods:
[0264] Mice and Wounding.
[0265] Full thickness excision (FTE) of skin was performed on the back of C57BL/6J mice (Jackson laboratory) under ketamine/zylazine anesthesia as previously described (1). Three-week-old mice were used for all experiments with a 1×1 cm2 FTE, except as indicated. Timed pregnant C57BL/6 female mice of gestational day 13.5 (Charles River) were utilized for embryonic skin explant culture. K14-rtTA mice harboring the doxycycline-sensitive transactivator were mated to TRE-Fgf9-IRES-eGfp mice. Both K14-rtTA and K14rtTA/TRE-Fgf9 mice were fed Dox-containing food (Bio-SERV) for 4 days after complete reepithelization. Deletion of FGF9 expression in γδ T cells was accomplished using Fgf9 flox/flox mated to lck-cre mice (Jackson Laboratory) with T-cell targeting proximal promoter of the lymphocyte protein tyrosine kinase (lck). γδ T cell null mice (Tcrd.sup.-/-) were purchased from Jackson Laboratory. All animal protocols were approved by the University of Pennsylvania IACUC.
[0266] Whole-Mount Hair Follicle Neogenesis Assay.
[0267] Healed skin was taken at day 5 after reepithelization. Whole-mount hair follicle neogenesis assays for epidermal KRT17 immunostaining (1:5000, from P. Coulombe) and dermal NBT/BCIP incubation were performed to identify new hair germs and follicular dermal papillae in wound area as previously described.
[0268] Real-Time PCR.
[0269] Dorsal skins were as day 0 samples or the wounded skin at day 1, 3 and 5 after scab detachment after reepithelization (SD), respectively. The epidermis was separated from dermis by incubation with 4° C. dispase overnight or 20 mM EDTA for 30 mM at 37° C. RNA was isolated using RNeasy minikit (Qiagen) and then 1 μg of total RNA was converted to cDNA with a High capacity cDNA kit (Applied Biosystems). All primer sets including fgf9 of Taqman gene expression assay were purchased from Applied Biosystems. Reactions were performed in triplicate and relative expression levels were standardized using β-actin as an internal control. The results were analyzed using StepOne program.
[0270] Immunostaining.
[0271] Reepithelialized skin after wounding was placed either frozen in OCT (Tissue-Tek). Staining for FGF9 (1:200; R&D systems) and γδTCR (1:100; GL3, BD Bioscience) were performed on 8 μm frozen section Immunohistochemisty with antibodies against BrdU (1:500; Harlan-Seralab) was done as previously described. For pulse-chase experiments, BrdU (Sigma) was administered 2 hr before sample preparation.
[0272] Isolation of DETCs and Activation of the Cells.
[0273] Epidermal cell suspension was prepared from C57BL/6 mice and was incubated overnight at 37° C. in complete DMEM containing 20 U/ml of recombinant mouse IL-2 (mIL-2) to allow surface receptor re-expression as described. DETCs were isolated by FACS sorting with PE-γδTCR (GL3, Abcam) and allophycocyanin-Thy1.2 (BD Bioscience) staining. The isolated DETCs were cultured in RPMI-1640 medium supplemented with 10% FCS, 25 mM HEPES, 100 U penicillin, 100 μg streptomycin, 2 mM glutamine, 100 μM nonessential amino acids, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol and 20 U/ml mIL-2 For cell stimulation, the cells were harvested for 4 h in the growth factor-free media excluding FCS and mIL-2 and then incubated in the media described above supplemented with anti-CD3ε (10 μg/ml, eBioscience) at 37° C. for 4, 24 and 48 h. Stimulation was arrested by the addition of ice-cold PBS and samples were placed on ice. Supernatants were removed and cells were collected with lysis buffer. RNA isolation and subsequent cDNA generation were performed with Gene expression cells-to CT kit (Applied Biosystems).
[0274] Wholemount Epidermal γδ T Cell Staining.
[0275] Ears were cut from 8-week old C57BL/6J mice and epidermal sheets were separated as previously described. Epidermal sheets were incubated overnight in the growth factor-free media described above or complete media with 20 U/ml mIL-2 at 37° C. Epidermis was then washed in PBS and fixed in ice-cold acetone for 20 mM at -20° C. Primary antibodies of FGF9 and γδTCR mentioned above were incubated overnight at 4° C. The following morning, sheets were incubated with secondary antibodies for 1 hr and mounted on silane-coated slides.
[0276] In Vitro Embryonic Skin Culture.
[0277] Embryos at E13.5 were dissected out of the sac and the crown-lump length was checked to ensure exact developmental age. Dorsal skin was dissected and then cultured for up to 3 days as previously described. Recombinant human FGF-9 (0-20 ng/ml, R&D systems) or EDA1 (50 ng/ml, R&D systems) as a positive control were added into culture media. In addition, FGF9 neutralization experiment was paralleled with anti-FGF9 antibody incubation (0-40 μg/ml; MAB273, R&D systems). Epidermal-dermal separations were performed by incubating skin samples in 20 mM EDTA at 37° C. for 5 min Tissues were homogenized to isolated RNA or harvested for wholemount assay as described above. The number of hair follicles was counted per mm2 at 3 different fields of each sample and the mean value was calculated.
[0278] Neutralization Experiment in Adult Mice.
[0279] Reepithelization of epidermis, indicated by scab detachment, was complete 10-12 days after FTE. One day after complete reepithelization, 50 μl of anti-FGF9 neutralization antibody or IgG isotype control (MAB 003) at 10 μg/ml were daily injected just beneath epidermis for 4 consecutive days. After then, tissues were harvested at day 5, epidermis and dermis were separated using 20 mM EDTA solution and processed for KRT17 immunostaining and detecting alkaline phosphatase activity, respectively. The number of regenerated hair follicles was characterized with respect to their density inside the epidermis. The developmental stages of the hair follicles were quantified as previously described.
[0280] In Vivo Confocal Microscopy.
[0281] To chase dynamic process of hair follicle neogenesis, the changes of newly formed hair follicle number was quantified using in vivo confocal microscope (Vivascope 1500, Lucid). Briefly, surrounding area of healed skin was clipped and adhesive window (Lucid) and ultrasonic transmission gel (Parker laboratory) were applied under ketamine/zylazine anesthesia. New hair follicles could be visualized and counted at the level beneath epidermal-dermal junction. The number was measured at day 2 after reepithelization and then every 3 days for 2 weeks.
Results
[0282] Fgf9 Expression Significantly Increases after Wounding Prior to Hair Follicle Neogenesis:
[0283] To define molecular events responsible for hair follicle neogenesis following wounding, we compared gene expression in wounded epidermis soon after reepithelialization (1 and 3 days after scab detachment "SD") to the initiation of hair follicle neogenesis (SD5). Microarray analyses showed that Fibroblast growth factor 9 (Fgf9) was significantly upregulated (4.2 fold) prior to hair follicle germ formation. We further analyzed Fgf9 gene expression changes in reepithelialized epidermis around the time of hair follicle neogenesis by quantitative RTPCR (FIG. 16A). Fgf9 gene expression increased significantly after reepithelialization until the initial stages of hair follicle neogenesis when expression decreased dramatically. These results show that Fgf9 is upregulated in the newly formed epidermis just prior to hair follicle neogenesis presumably at a time when cells are committing to the hair follicle lineage.
[0284] Inhibition of Fgf9 Decreases Hair Follicle Neogenesis.
[0285] Fgf9 is a secreted ligand with a known role in lung, kidney and gonad development, but it has not been previously implicated in hair follicle development or regeneration. Nevertheless, the main receptor for Fgf9 in the skin, Fgr3b, is expressed in epidermis and is upregulated in regenerated skin after wounding. To address the importance of Fgf9 in hair follicle neogenesis following wounding, we injected Fgf9 neutralizing antibody into the reepithelialized skin daily for four days (FIG. 16b, Table 2). Wounds treated with anti-Fgf9 antibody showed a significant reduction of new hair follicle formation when compared with controls. The hair follicles that did form in anti-Fgf9-treated wounds were in immature stages of development (FIG. 19).
[0286] Forced Overexpression of Fgf9 in the New Epidermis Increases Hair Follicle Formation.
[0287] Since blocking Fgf9 inhibited hair follicle neogenesis, we asked whether increasing levels of Fgf9 in the wound would promote hair follicle neogenesisfollowing wounding. We used a doxycycline-inducible transgenic mouse (K14rtTAx TRE-Fgf9-IRES-eGfp) to inducibly target Fgf9 expression to the epidermis following wound re-epithelialization. Administration of doxcycline from SD1 to SD4 increased Fgf9 expression 150-fold (FIG. 20) compared to doxycycline treated control mice. This targeted overexpression of Fgf9 to the epidermis for four days after reepithelialization led to a marked increase in the number of hair follicles compared to controls (FIG. 16c, Table2).
TABLE-US-00003 TABLE 2 Hair follicle neogenesis assay. The number of new hair follicles were counted at Day 5 after reepithelization. NS: not significant. Hair follicle No. Mice Experiment mice (mean ± SD) No. Range P-value Deletion of lck-cre; Fgf9flox/flox 9.1 ± 16.7 11 0-49 <0.05 FGF9 Fgf9flox/flox, Fgf9flox/+ 30.7 ± 34.0 15 1-131 in T cells (Control) FGF9 Double transgenic 168.2 ± 117.1 12 2-189 <0.05 overexpression K14rtTA; TRE-Fgf9- IRES-eGfp Single transgenic 64.8 ± 50.3 21 26-431 K14rtTA, TRE-Fgf9- IRES-eGfp (Control) Absence of 8-week old wild-type 43.4 ± 31.7 8 1-87 NS γδ T cells (1.5 × 1.5 cm2 wounding) 24-40 week old wild-type 36.7 ± 24.5 6 1-76 (1.5 × 1.5 cm2 wounding) 8-week old γδ T cell null 9.8 ± 10.1 13 0-27 <0.01 mice (1.5 × 1.5 cm2 wounding) 24-40 week old 7.8 ± 13.7 8 0-39 γδ T cell null mice (1.5 × 1.5 cm2 wounding)
[0288] Fgf9 Expression Localizes to γδT Cells.
[0289] To identify the source of Fgf9 in re-epithelialized skin of normal mice, we immunostained tissue sections of healed skin prior to HFN. Surprisingly, we discovered that γδ T cell receptor-bearing epidermal T cells (DETC), which repopulate the epidermis, express Fgf9. DETCs appear to be the primary source of Fgf9 in epidermis, with little or no contribution from keratinocytes or other epithelial residents (FIG. 17A). Previous gene expression data from basal keratinocytes (Alpha-6-integrin-positive) isolated by FACS showed an absence of Fgf9 expression (NAT BIOTech paper). To further confirm the origin of the Fgf9, we treated unwounded ear epidermis with mIL-2 and analysed wholemount preparations for Fgf9 expression by immunofluorescence. IL2-induced DETCs stained strongly with anti-FGF9 antibodies whereas adjacent keratinocytes exhibited background staining (FIG. 21).
[0290] To determine if FGF9 is constitutively expressed by DETCs in skin or upregulated following stimulation, DETCs were isolated from skin by cell sorting and were cultured in vitro with anti-CD3 and IL2 as previously described (havren ref). Fgf9 mRNA levels increased by greater than 10 fold within 4 hours, followed by diminution to baseline levels within 24 hours. (FIG. 17B). This rapid upregulation contrasts with the much longer 48 hour induction period required for expression of FGF7 and FGF10, two factors known to be secreted by DETCs during wound repair and indicates distinct transcriptional regulatory mechanisms.
[0291] DETCs are Essential for Hair Follicle Neogenesis.
[0292] Since Fgf9 mediates hair follicle neogenesis and DETCs appear to be the primary source of Fgf9 in re-epithelialized epidermis, we hypothesized that activated DETCs repopulate the wound during reepithelialization and secrete FGF9 to induce hair follicle neogenesis. To better define the role of DETCs in hair follicle neogenesis, we studied Tcrd-/- mice that fail to develop these cells.
[0293] We wounded age-matched wild-type and Tcrd-/- mice at 8 or 24-40 weeks of age and quantified hair follicle neogenesis. As previously reported, TCRd-/- mice showed slight delays in wound closure (data not shown), but hair follicle neogenesis was markedly decreased. Quantitation of Fgf9 levels indicated that Fgf9 was consistently negligible in the Tcrd-/- mice.
[0294] As shown in FIG. 18, 8 wk and 40 wk -/- mice exhibited profound defects in HFN, with reductions of >80% in HF numbers compared with wt mice (18A,B, Table 2). Thus, reduced numbers of HFs in -/- mice reflect a true defect in hair follicle neogenesis rather than delayed kinetics of response.
[0295] The above findings supported the hypothesis that activation of DETCs following wounding leads to FGF9 production and subsequent hair follicle neogenesis. Nevertheless, to address the concern that DETCs may have a role in hair follicle neogenesis other than the production of FGF9, mutant (lck-cre×Fgf9flox/flox) mice carrying a deletion of the FGF9 gene specifically in T cells, including DETCs, were analysed for hair follicle neogenesisfollowing wounding. Quantitative rtPCR analyses showed that these mice express low constitutive levels of FGF9 in skin. (FIG. 22). Wounding studies showed that these mutant (lck-cre×Fgf9flox/flox) mice exhibited a dramatic reduction in post-wound hair follicle numbers comparable to that observed in TCRd-/- animals (FIG. 18E, Table 2).
[0296] Taken together, the above described results show that DETCs are essential immunologic contributors to HF neogenesis through the production of FGF9.
[0297] In summary, we discovered that DETCs are the source of FGF9. Further, the findings indicate that more divergent cellular & molecular events could be implicated in HFN after wounding, not exactly the recapitulation of embryonic development, and provide additional evidence that acquired immune system including DETCs would have a role in tissue regeneration.
[0298] FGF9 and DETCs are critical for HFN after wounding. Overexpression of FGF9 in reepithelized epidermis resulted in increase of hair follicle formation. These results show that manipulation of FGF9 expression during wound healing or after reepithelization could be a useful approach to develop a new treatment for hair loss.
[0299] Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.
Sequence CWU
1
1
91133PRTHomo sapiens 1Ile Phe Pro Asn Gly Thr Ile Gln Gly Thr Arg Lys Asp
His Ser Arg 1 5 10 15
Phe Gly Ile Leu Glu Phe Ile Ser Ile Ala Val Gly Leu Val Ser Ile
20 25 30 Arg Gly Val Asp
Ser Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu 35
40 45 Leu Tyr Gly Ser Glu Lys Leu Thr Gln
Glu Cys Val Phe Arg Glu Gln 50 55
60 Phe Glu Glu Asn Trp Tyr Asn Thr Tyr Ser Ser Asn Leu
Tyr Lys His 65 70 75
80 Val Asp Thr Gly Arg Arg Tyr Tyr Val Ala Leu Asn Lys Asp Gly Thr
85 90 95 Pro Arg Glu Gly
Thr Arg Thr Lys Arg His Gln Lys Phe Thr His Phe 100
105 110 Leu Pro Arg Pro Val Asp Pro Asp Lys
Val Pro Glu Leu Tyr Lys Asp 115 120
125 Ile Leu Ser Gln Ser 130 2462PRTHomo
sapiens 2Met Leu Leu Leu Ala Arg Cys Leu Leu Leu Val Leu Val Ser Ser Leu
1 5 10 15 Leu Val
Cys Ser Gly Leu Ala Cys Gly Pro Gly Arg Gly Phe Gly Lys 20
25 30 Arg Arg His Pro Lys Lys Leu
Thr Pro Leu Ala Tyr Lys Gln Phe Ile 35 40
45 Pro Asn Val Ala Glu Lys Thr Leu Gly Ala Ser Gly
Arg Tyr Glu Gly 50 55 60
Lys Ile Ser Arg Asn Ser Glu Arg Phe Lys Glu Leu Thr Pro Asn Tyr 65
70 75 80 Asn Pro Asp
Ile Ile Phe Lys Asp Glu Glu Asn Thr Gly Ala Asp Arg 85
90 95 Leu Met Thr Gln Arg Cys Lys Asp
Lys Leu Asn Ala Leu Ala Ile Ser 100 105
110 Val Met Asn Gln Trp Pro Gly Val Lys Leu Arg Val Thr
Glu Gly Trp 115 120 125
Asp Glu Asp Gly His His Ser Glu Glu Ser Leu His Tyr Glu Gly Arg 130
135 140 Ala Val Asp Ile
Thr Thr Ser Asp Arg Asp Arg Ser Lys Tyr Gly Met 145 150
155 160 Leu Ala Arg Leu Ala Val Glu Ala Gly
Phe Asp Trp Val Tyr Tyr Glu 165 170
175 Ser Lys Ala His Ile His Cys Ser Val Lys Ala Glu Asn Ser
Val Ala 180 185 190
Ala Lys Ser Gly Gly Cys Phe Pro Gly Ser Ala Thr Val His Leu Glu
195 200 205 Gln Gly Gly Thr
Lys Leu Val Lys Asp Leu Ser Pro Gly Asp Arg Val 210
215 220 Leu Ala Ala Asp Asp Gln Gly Arg
Leu Leu Tyr Ser Asp Phe Leu Thr 225 230
235 240 Phe Leu Asp Arg Asp Asp Gly Ala Lys Lys Val Phe
Tyr Val Ile Glu 245 250
255 Thr Arg Glu Pro Arg Glu Arg Leu Leu Leu Thr Ala Ala His Leu Leu
260 265 270 Phe Val Ala
Pro His Asn Asp Ser Ala Thr Gly Glu Pro Glu Ala Ser 275
280 285 Ser Gly Ser Gly Pro Pro Ser Gly
Gly Ala Leu Gly Pro Arg Ala Leu 290 295
300 Phe Ala Ser Arg Val Arg Pro Gly Gln Arg Val Tyr Val
Val Ala Glu 305 310 315
320 Arg Asp Gly Asp Arg Arg Leu Leu Pro Ala Ala Val His Ser Val Thr
325 330 335 Leu Ser Glu Glu
Ala Ala Gly Ala Tyr Ala Pro Leu Thr Ala Gln Gly 340
345 350 Thr Ile Leu Ile Asn Arg Val Leu Ala
Ser Cys Tyr Ala Val Ile Glu 355 360
365 Glu His Ser Trp Ala His Arg Ala Phe Ala Pro Phe Arg Leu
Ala His 370 375 380
Ala Leu Leu Ala Ala Leu Ala Pro Ala Arg Thr Asp Arg Gly Gly Asp 385
390 395 400 Ser Gly Gly Gly Asp
Arg Gly Gly Gly Gly Gly Arg Val Ala Leu Thr 405
410 415 Ala Pro Gly Ala Ala Asp Ala Pro Gly Ala
Gly Ala Thr Ala Gly Ile 420 425
430 His Trp Tyr Ser Gln Leu Leu Tyr Gln Ile Gly Thr Trp Leu Leu
Asp 435 440 445 Ser
Glu Ala Leu His Pro Leu Gly Met Ala Val Lys Ser Ser 450
455 460 31447PRTHomo sapiens 3Met Ala Ser Ala
Gly Asn Ala Ala Glu Pro Gln Asp Arg Gly Gly Gly 1 5
10 15 Gly Ser Gly Cys Ile Gly Ala Pro Gly
Arg Pro Ala Gly Gly Gly Arg 20 25
30 Arg Arg Arg Thr Gly Gly Leu Arg Arg Ala Ala Ala Pro Asp
Arg Asp 35 40 45
Tyr Leu His Arg Pro Ser Tyr Cys Asp Ala Ala Phe Ala Leu Glu Gln 50
55 60 Ile Ser Lys Gly Lys
Ala Thr Gly Arg Lys Ala Pro Leu Trp Leu Arg 65 70
75 80 Ala Lys Phe Gln Arg Leu Leu Phe Lys Leu
Gly Cys Tyr Ile Gln Lys 85 90
95 Asn Cys Gly Lys Phe Leu Val Val Gly Leu Leu Ile Phe Gly Ala
Phe 100 105 110 Ala
Val Gly Leu Lys Ala Ala Asn Leu Glu Thr Asn Val Glu Glu Leu 115
120 125 Trp Val Glu Val Gly Gly
Arg Val Ser Arg Glu Leu Asn Tyr Thr Arg 130 135
140 Gln Lys Ile Gly Glu Glu Ala Met Phe Asn Pro
Gln Leu Met Ile Gln 145 150 155
160 Thr Pro Lys Glu Glu Gly Ala Asn Val Leu Thr Thr Glu Ala Leu Leu
165 170 175 Gln His
Leu Asp Ser Ala Leu Gln Ala Ser Arg Val His Val Tyr Met 180
185 190 Tyr Asn Arg Gln Trp Lys Leu
Glu His Leu Cys Tyr Lys Ser Gly Glu 195 200
205 Leu Ile Thr Glu Thr Gly Tyr Met Asp Gln Ile Ile
Glu Tyr Leu Tyr 210 215 220
Pro Cys Leu Ile Ile Thr Pro Leu Asp Cys Phe Trp Glu Gly Ala Lys 225
230 235 240 Leu Gln Ser
Gly Thr Ala Tyr Leu Leu Gly Lys Pro Pro Leu Arg Trp 245
250 255 Thr Asn Phe Asp Pro Leu Glu Phe
Leu Glu Glu Leu Lys Lys Ile Asn 260 265
270 Tyr Gln Val Asp Ser Trp Glu Glu Met Leu Asn Lys Ala
Glu Val Gly 275 280 285
His Gly Tyr Met Asp Arg Pro Cys Leu Asn Pro Ala Asp Pro Asp Cys 290
295 300 Pro Ala Thr Ala
Pro Asn Lys Asn Ser Thr Lys Pro Leu Asp Met Ala 305 310
315 320 Leu Val Leu Asn Gly Gly Cys His Gly
Leu Ser Arg Lys Tyr Met His 325 330
335 Trp Gln Glu Glu Leu Ile Val Gly Gly Thr Val Lys Asn Ser
Thr Gly 340 345 350
Lys Leu Val Ser Ala His Ala Leu Gln Thr Met Phe Gln Leu Met Thr
355 360 365 Pro Lys Gln Met
Tyr Glu His Phe Lys Gly Tyr Glu Tyr Val Ser His 370
375 380 Ile Asn Trp Asn Glu Asp Lys Ala
Ala Ala Ile Leu Glu Ala Trp Gln 385 390
395 400 Arg Thr Tyr Val Glu Val Val His Gln Ser Val Ala
Gln Asn Ser Thr 405 410
415 Gln Lys Val Leu Ser Phe Thr Thr Thr Thr Leu Asp Asp Ile Leu Lys
420 425 430 Ser Phe Ser
Asp Val Ser Val Ile Arg Val Ala Ser Gly Tyr Leu Leu 435
440 445 Met Leu Ala Tyr Ala Cys Leu Thr
Met Leu Arg Trp Asp Cys Ser Lys 450 455
460 Ser Gln Gly Ala Val Gly Leu Ala Gly Val Leu Leu Val
Ala Leu Ser 465 470 475
480 Val Ala Ala Gly Leu Gly Leu Cys Ser Leu Ile Gly Ile Ser Phe Asn
485 490 495 Ala Ala Thr Thr
Gln Val Leu Pro Phe Leu Ala Leu Gly Val Gly Val 500
505 510 Asp Asp Val Phe Leu Leu Ala His Ala
Phe Ser Glu Thr Gly Gln Asn 515 520
525 Lys Arg Ile Pro Phe Glu Asp Arg Thr Gly Glu Cys Leu Lys
Arg Thr 530 535 540
Gly Ala Ser Val Ala Leu Thr Ser Ile Ser Asn Val Thr Ala Phe Phe 545
550 555 560 Met Ala Ala Leu Ile
Pro Ile Pro Ala Leu Arg Ala Phe Ser Leu Gln 565
570 575 Ala Ala Val Val Val Val Phe Asn Phe Ala
Met Val Leu Leu Ile Phe 580 585
590 Pro Ala Ile Leu Ser Met Asp Leu Tyr Arg Arg Glu Asp Arg Arg
Leu 595 600 605 Asp
Ile Phe Cys Cys Phe Thr Ser Pro Cys Val Ser Arg Val Ile Gln 610
615 620 Val Glu Pro Gln Ala Tyr
Thr Asp Thr His Asp Asn Thr Arg Tyr Ser 625 630
635 640 Pro Pro Pro Pro Tyr Ser Ser His Ser Phe Ala
His Glu Thr Gln Ile 645 650
655 Thr Met Gln Ser Thr Val Gln Leu Arg Thr Glu Tyr Asp Pro His Thr
660 665 670 His Val
Tyr Tyr Thr Thr Ala Glu Pro Arg Ser Glu Ile Ser Val Gln 675
680 685 Pro Val Thr Val Thr Gln Asp
Thr Leu Ser Cys Gln Ser Pro Glu Ser 690 695
700 Thr Ser Ser Thr Arg Asp Leu Leu Ser Gln Phe Ser
Asp Ser Ser Leu 705 710 715
720 His Cys Leu Glu Pro Pro Cys Thr Lys Trp Thr Leu Ser Ser Phe Ala
725 730 735 Glu Lys His
Tyr Ala Pro Phe Leu Leu Lys Pro Lys Ala Lys Val Val 740
745 750 Val Ile Phe Leu Phe Leu Gly Leu
Leu Gly Val Ser Leu Tyr Gly Thr 755 760
765 Thr Arg Val Arg Asp Gly Leu Asp Leu Thr Asp Ile Val
Pro Arg Glu 770 775 780
Thr Arg Glu Tyr Asp Phe Ile Ala Ala Gln Phe Lys Tyr Phe Ser Phe 785
790 795 800 Tyr Asn Met Tyr
Ile Val Thr Gln Lys Ala Asp Tyr Pro Asn Ile Gln 805
810 815 His Leu Leu Tyr Asp Leu His Arg Ser
Phe Ser Asn Val Lys Tyr Val 820 825
830 Met Leu Glu Glu Asn Lys Gln Leu Pro Lys Met Trp Leu His
Tyr Phe 835 840 845
Arg Asp Trp Leu Gln Gly Leu Gln Asp Ala Phe Asp Ser Asp Trp Glu 850
855 860 Thr Gly Lys Ile Met
Pro Asn Asn Tyr Lys Asn Gly Ser Asp Asp Gly 865 870
875 880 Val Leu Ala Tyr Lys Leu Leu Val Gln Thr
Gly Ser Arg Asp Lys Pro 885 890
895 Ile Asp Ile Ser Gln Leu Thr Lys Gln Arg Leu Val Asp Ala Asp
Gly 900 905 910 Ile
Ile Asn Pro Ser Ala Phe Tyr Ile Tyr Leu Thr Ala Trp Val Ser 915
920 925 Asn Asp Pro Val Ala Tyr
Ala Ala Ser Gln Ala Asn Ile Arg Pro His 930 935
940 Arg Pro Glu Trp Val His Asp Lys Ala Asp Tyr
Met Pro Glu Thr Arg 945 950 955
960 Leu Arg Ile Pro Ala Ala Glu Pro Ile Glu Tyr Ala Gln Phe Pro Phe
965 970 975 Tyr Leu
Asn Gly Leu Arg Asp Thr Ser Asp Phe Val Glu Ala Ile Glu 980
985 990 Lys Val Arg Thr Ile Cys Ser
Asn Tyr Thr Ser Leu Gly Leu Ser Ser 995 1000
1005 Tyr Pro Asn Gly Tyr Pro Phe Leu Phe Trp
Glu Gln Tyr Ile Gly 1010 1015 1020
Leu Arg His Trp Leu Leu Leu Phe Ile Ser Val Val Leu Ala Cys
1025 1030 1035 Thr Phe
Leu Val Cys Ala Val Phe Leu Leu Asn Pro Trp Thr Ala 1040
1045 1050 Gly Ile Ile Val Met Val Leu
Ala Leu Met Thr Val Glu Leu Phe 1055 1060
1065 Gly Met Met Gly Leu Ile Gly Ile Lys Leu Ser Ala
Val Pro Val 1070 1075 1080
Val Ile Leu Ile Ala Ser Val Gly Ile Gly Val Glu Phe Thr Val 1085
1090 1095 His Val Ala Leu Ala
Phe Leu Thr Ala Ile Gly Asp Lys Asn Arg 1100 1105
1110 Arg Ala Val Leu Ala Leu Glu His Met Phe
Ala Pro Val Leu Asp 1115 1120 1125
Gly Ala Val Ser Thr Leu Leu Gly Val Leu Met Leu Ala Gly Ser
1130 1135 1140 Glu Phe
Asp Phe Ile Val Arg Tyr Phe Phe Ala Val Leu Ala Ile 1145
1150 1155 Leu Thr Ile Leu Gly Val Leu
Asn Gly Leu Val Leu Leu Pro Val 1160 1165
1170 Leu Leu Ser Phe Phe Gly Pro Tyr Pro Glu Val Ser
Pro Ala Asn 1175 1180 1185
Gly Leu Asn Arg Leu Pro Thr Pro Ser Pro Glu Pro Pro Pro Ser 1190
1195 1200 Val Val Arg Phe Ala
Met Pro Pro Gly His Thr His Ser Gly Ser 1205 1210
1215 Asp Ser Ser Asp Ser Glu Tyr Ser Ser Gln
Thr Thr Val Ser Gly 1220 1225 1230
Leu Ser Glu Glu Leu Arg His Tyr Glu Ala Gln Gln Gly Ala Gly
1235 1240 1245 Gly Pro
Ala His Gln Val Ile Val Glu Ala Thr Glu Asn Pro Val 1250
1255 1260 Phe Ala His Ser Thr Val Val
His Pro Glu Ser Arg His His Pro 1265 1270
1275 Pro Ser Asn Pro Arg Gln Gln Pro His Leu Asp Ser
Gly Ser Leu 1280 1285 1290
Pro Pro Gly Arg Gln Gly Gln Gln Pro Arg Arg Asp Pro Pro Arg 1295
1300 1305 Glu Gly Leu Trp Pro
Pro Pro Tyr Arg Pro Arg Arg Asp Ala Phe 1310 1315
1320 Glu Ile Ser Thr Glu Gly His Ser Gly Pro
Ser Asn Arg Ala Arg 1325 1330 1335
Trp Gly Pro Arg Gly Ala Arg Ser His Asn Pro Arg Asn Pro Ala
1340 1345 1350 Ser Thr
Ala Met Gly Ser Ser Val Pro Gly Tyr Cys Gln Pro Ile 1355
1360 1365 Thr Thr Val Thr Ala Ser Ala
Ser Val Thr Val Ala Val His Pro 1370 1375
1380 Pro Pro Val Pro Gly Pro Gly Arg Asn Pro Arg Gly
Gly Leu Cys 1385 1390 1395
Pro Gly Tyr Pro Glu Thr Asp His Gly Leu Phe Glu Asp Pro His 1400
1405 1410 Val Pro Phe His Val
Arg Cys Glu Arg Arg Asp Ser Lys Val Glu 1415 1420
1425 Val Ile Glu Leu Gln Asp Val Glu Cys Glu
Glu Arg Pro Arg Gly 1430 1435 1440
Ser Ser Ser Asn 1445 487PRTHomo sapiens 4Phe Asp
Phe Ile Val Arg Tyr Phe Phe Ala Ala Leu Thr Val Leu Thr 1 5
10 15 Leu Leu Gly Leu Leu His Gly
Leu Val Leu Leu Pro Val Leu Leu Ser 20 25
30 Ile Leu Gly Pro Pro Pro Glu Val Ile Gln Met Tyr
Lys Glu Ser Pro 35 40 45
Glu Ile Leu Ser Pro Pro Ala Pro Gln Gly Gly Gly Leu Arg Val Gly
50 55 60 Ser Leu Gln
Val Asn Ile Ser Tyr Trp Lys Glu Leu Leu Trp Cys Gln 65
70 75 80 Asp Leu Arg Pro Glu Glu Ile
85 51106PRTHomo sapiens 5Met Phe Asn Ser Met Thr
Pro Pro Pro Ile Ser Ser Tyr Gly Glu Pro 1 5
10 15 Cys Cys Leu Arg Pro Leu Pro Ser Gln Gly Ala
Pro Ser Val Gly Thr 20 25
30 Glu Gly Leu Ser Gly Pro Pro Phe Cys His Gln Ala Asn Leu Met
Ser 35 40 45 Gly
Pro His Ser Tyr Gly Pro Ala Arg Glu Thr Asn Ser Cys Thr Glu 50
55 60 Gly Pro Leu Phe Ser Ser
Pro Arg Ser Ala Val Lys Leu Thr Lys Lys 65 70
75 80 Arg Ala Leu Ser Ile Ser Pro Leu Ser Asp Ala
Ser Leu Asp Leu Gln 85 90
95 Thr Val Ile Arg Thr Ser Pro Ser Ser Leu Val Ala Phe Ile Asn Ser
100 105 110 Arg Cys
Thr Ser Pro Gly Gly Ser Tyr Gly His Leu Ser Ile Gly Thr 115
120 125 Met Ser Pro Ser Leu Gly Phe
Pro Ala Gln Met Asn His Gln Lys Gly 130 135
140 Pro Ser Pro Ser Phe Gly Val Gln Pro Cys Gly Pro
His Asp Ser Ala 145 150 155
160 Arg Gly Gly Met Ile Pro His Pro Gln Ser Arg Gly Pro Phe Pro Thr
165 170 175 Cys Gln Leu
Lys Ser Glu Leu Asp Met Leu Val Gly Lys Cys Arg Glu 180
185 190 Glu Pro Leu Glu Gly Asp Met Ser
Ser Pro Asn Ser Thr Gly Ile Gln 195 200
205 Asp Pro Leu Leu Gly Met Leu Asp Gly Arg Glu Asp Leu
Glu Arg Glu 210 215 220
Glu Lys Arg Glu Pro Glu Ser Val Tyr Glu Thr Asp Cys Arg Trp Asp 225
230 235 240 Gly Cys Ser Gln
Glu Phe Asp Ser Gln Glu Gln Leu Val His His Ile 245
250 255 Asn Ser Glu His Ile His Gly Glu Arg
Lys Glu Phe Val Cys His Trp 260 265
270 Gly Gly Cys Ser Arg Glu Leu Arg Pro Phe Lys Ala Gln Tyr
Met Leu 275 280 285
Val Val His Met Arg Arg His Thr Gly Glu Lys Pro His Lys Cys Thr 290
295 300 Phe Glu Gly Cys Arg
Lys Ser Tyr Ser Arg Leu Glu Asn Leu Lys Thr 305 310
315 320 His Leu Arg Ser His Thr Gly Glu Lys Pro
Tyr Met Cys Glu His Glu 325 330
335 Gly Cys Ser Lys Ala Phe Ser Asn Ala Ser Asp Arg Ala Lys His
Gln 340 345 350 Asn
Arg Thr His Ser Asn Glu Lys Pro Tyr Val Cys Lys Leu Pro Gly 355
360 365 Cys Thr Lys Arg Tyr Thr
Asp Pro Ser Ser Leu Arg Lys His Val Lys 370 375
380 Thr Val His Gly Pro Asp Ala His Val Thr Lys
Arg His Arg Gly Asp 385 390 395
400 Gly Pro Leu Pro Arg Ala Pro Ser Ile Ser Thr Val Glu Pro Lys Arg
405 410 415 Glu Arg
Glu Gly Gly Pro Ile Arg Glu Glu Ser Arg Leu Thr Val Pro 420
425 430 Glu Gly Ala Met Lys Pro Gln
Pro Ser Pro Gly Ala Gln Ser Ser Cys 435 440
445 Ser Ser Asp His Ser Pro Ala Gly Ser Ala Ala Asn
Thr Asp Ser Gly 450 455 460
Val Glu Met Thr Gly Asn Ala Gly Gly Ser Thr Glu Asp Leu Ser Ser 465
470 475 480 Leu Asp Glu
Gly Pro Cys Ile Ala Gly Thr Gly Leu Ser Thr Leu Arg 485
490 495 Arg Leu Glu Asn Leu Arg Leu Asp
Gln Leu His Gln Leu Arg Pro Ile 500 505
510 Gly Thr Arg Gly Leu Lys Leu Pro Ser Leu Ser His Thr
Gly Thr Thr 515 520 525
Val Ser Arg Arg Val Gly Pro Pro Val Ser Leu Glu Arg Arg Ser Ser 530
535 540 Ser Ser Ser Ser
Ile Ser Ser Ala Tyr Thr Val Ser Arg Arg Ser Ser 545 550
555 560 Leu Ala Ser Pro Phe Pro Pro Gly Ser
Pro Pro Glu Asn Gly Ala Ser 565 570
575 Ser Leu Pro Gly Leu Met Pro Ala Gln His Tyr Leu Leu Arg
Ala Arg 580 585 590
Tyr Ala Ser Ala Arg Gly Gly Gly Thr Ser Pro Thr Ala Ala Ser Ser
595 600 605 Leu Asp Arg Ile
Gly Gly Leu Pro Met Pro Pro Trp Arg Ser Arg Ala 610
615 620 Glu Tyr Pro Gly Tyr Asn Pro Asn
Ala Gly Val Thr Arg Arg Ala Ser 625 630
635 640 Asp Pro Ala Gln Ala Ala Asp Arg Pro Ala Pro Ala
Arg Val Gln Arg 645 650
655 Phe Lys Ser Leu Gly Cys Val His Thr Pro Pro Thr Val Ala Gly Gly
660 665 670 Gly Gln Asn
Phe Asp Pro Tyr Leu Pro Thr Ser Val Tyr Ser Pro Gln 675
680 685 Pro Pro Ser Ile Thr Glu Asn Ala
Ala Met Asp Ala Arg Gly Leu Gln 690 695
700 Glu Glu Pro Glu Val Gly Thr Ser Met Val Gly Ser Gly
Leu Asn Pro 705 710 715
720 Tyr Met Asp Phe Pro Pro Thr Asp Thr Leu Gly Tyr Gly Gly Pro Glu
725 730 735 Gly Ala Ala Ala
Glu Pro Tyr Gly Ala Arg Gly Pro Gly Ser Leu Pro 740
745 750 Leu Gly Pro Gly Pro Pro Thr Asn Tyr
Gly Pro Asn Pro Cys Pro Gln 755 760
765 Gln Ala Ser Tyr Pro Asp Pro Thr Gln Glu Thr Trp Gly Glu
Phe Pro 770 775 780
Ser His Ser Gly Leu Tyr Pro Gly Pro Lys Ala Leu Gly Gly Thr Tyr 785
790 795 800 Ser Gln Cys Pro Arg
Leu Glu His Tyr Gly Gln Val Gln Val Lys Pro 805
810 815 Glu Gln Gly Cys Pro Val Gly Ser Asp Ser
Thr Gly Leu Ala Pro Cys 820 825
830 Leu Asn Ala His Pro Ser Glu Gly Pro Pro His Pro Gln Pro Leu
Phe 835 840 845 Ser
His Tyr Pro Gln Pro Ser Pro Pro Gln Tyr Leu Gln Ser Gly Pro 850
855 860 Tyr Thr Gln Pro Pro Pro
Asp Tyr Leu Pro Ser Glu Pro Arg Pro Cys 865 870
875 880 Leu Asp Phe Asp Ser Pro Thr His Ser Thr Gly
Gln Leu Lys Ala Gln 885 890
895 Leu Val Cys Asn Tyr Val Gln Ser Gln Gln Glu Leu Leu Trp Glu Gly
900 905 910 Gly Gly
Arg Glu Asp Ala Pro Ala Gln Glu Pro Ser Tyr Gln Ser Pro 915
920 925 Lys Phe Leu Gly Gly Ser Gln
Val Ser Pro Ser Arg Ala Lys Ala Pro 930 935
940 Val Asn Thr Tyr Gly Pro Gly Phe Gly Pro Asn Leu
Pro Asn His Lys 945 950 955
960 Ser Gly Ser Tyr Pro Thr Pro Ser Pro Cys His Glu Asn Phe Val Val
965 970 975 Gly Ala Asn
Arg Ala Ser His Arg Ala Ala Ala Pro Pro Arg Leu Leu 980
985 990 Pro Pro Leu Pro Thr Cys Tyr Gly
Pro Leu Lys Val Gly Gly Thr Asn 995 1000
1005 Pro Ser Cys Gly His Pro Glu Val Gly Arg Leu
Gly Gly Gly Pro 1010 1015 1020
Ala Leu Tyr Pro Pro Pro Glu Gly Gln Val Cys Asn Pro Leu Asp
1025 1030 1035 Ser Leu Asp
Leu Asp Asn Thr Gln Leu Asp Phe Val Ala Ile Leu 1040
1045 1050 Asp Glu Pro Gln Gly Leu Ser Pro
Pro Pro Ser His Asp Gln Arg 1055 1060
1065 Gly Ser Ser Gly His Thr Pro Pro Pro Ser Gly Pro Pro
Asn Met 1070 1075 1080
Ala Val Gly Asn Met Ser Val Leu Leu Arg Ser Leu Pro Gly Glu 1085
1090 1095 Thr Glu Phe Leu Asn
Ser Ser Ala 1100 1105 61258PRTHomo sapiens 6Met
Ala Leu Thr Ser Ile Asn Ala Thr Pro Thr Gln Leu Ser Ser Ser 1
5 10 15 Ser Asn Cys Leu Ser Asp
Thr Asn Gln Asn Lys Gln Ser Ser Glu Ser 20
25 30 Ala Val Ser Ser Thr Val Asn Pro Val Ala
Ile His Lys Arg Ser Lys 35 40
45 Val Lys Thr Glu Pro Glu Gly Leu Arg Pro Ala Ser Pro Leu
Ala Leu 50 55 60
Thr Gln Gly Gln Val Leu Asp Thr Ala His Val Gly Val Pro Phe Pro 65
70 75 80 Ser Pro Gln Glu Gln
Leu Ala Asp Leu Lys Glu Asp Leu Asp Arg Asp 85
90 95 Asp Cys Lys Gln Glu Ala Glu Val Val Ile
Tyr Glu Thr Asn Cys His 100 105
110 Trp Glu Asp Cys Thr Lys Glu Tyr Asp Thr Gln Glu Gln Leu Val
His 115 120 125 His
Ile Asn Asn Glu His Ile His Gly Glu Lys Lys Glu Phe Val Cys 130
135 140 Arg Trp Gln Ala Cys Thr
Arg Glu Gln Lys Pro Phe Lys Ala Gln Tyr 145 150
155 160 Met Leu Val Val His Met Arg Arg His Thr Gly
Glu Lys Pro His Lys 165 170
175 Cys Thr Phe Glu Gly Cys Ser Lys Ala Tyr Ser Arg Leu Glu Asn Leu
180 185 190 Lys Thr
His Leu Arg Ser His Thr Gly Glu Lys Pro Tyr Val Cys Glu 195
200 205 His Glu Gly Cys Asn Lys Ala
Phe Ser Asn Ala Ser Asp Arg Ala Lys 210 215
220 His Gln Asn Arg Thr His Ser Asn Glu Lys Pro Tyr
Ile Cys Lys Ile 225 230 235
240 Pro Gly Cys Thr Lys Arg Tyr Thr Asp Pro Ser Ser Leu Arg Lys His
245 250 255 Val Lys Thr
Val His Gly Pro Asp Ala His Val Thr Lys Lys Gln Arg 260
265 270 Asn Asp Val His Leu Arg Thr Pro
Leu Leu Lys Glu Asn Gly Asp Ser 275 280
285 Glu Ala Gly Thr Glu Pro Gly Gly Pro Glu Ser Thr Glu
Ala Ser Ser 290 295 300
Thr Ser Gln Ala Val Glu Asp Cys Leu His Val Arg Ala Ile Lys Thr 305
310 315 320 Glu Ser Ser Gly
Leu Cys Gln Ser Ser Pro Gly Ala Gln Ser Ser Cys 325
330 335 Ser Ser Glu Pro Ser Pro Leu Gly Ser
Ala Pro Asn Asn Asp Ser Gly 340 345
350 Val Glu Met Pro Gly Thr Gly Pro Gly Ser Leu Gly Asp Leu
Thr Ala 355 360 365
Leu Asp Asp Thr Pro Pro Gly Ala Asp Thr Ser Ala Leu Ala Ala Pro 370
375 380 Ser Ala Gly Gly Leu
Gln Leu Arg Lys His Met Thr Thr Met His Arg 385 390
395 400 Phe Glu Gln Leu Lys Lys Glu Lys Leu Lys
Ser Leu Lys Asp Ser Cys 405 410
415 Ser Trp Ala Gly Pro Thr Pro His Thr Arg Asn Thr Lys Leu Pro
Pro 420 425 430 Leu
Pro Gly Ser Gly Ser Ile Leu Glu Asn Phe Ser Gly Ser Gly Gly 435
440 445 Gly Gly Pro Ala Gly Leu
Leu Pro Asn Pro Arg Leu Ser Glu Leu Ser 450 455
460 Ala Ser Glu Val Thr Met Leu Ser Gln Leu Gln
Glu Arg Arg Asp Ser 465 470 475
480 Ser Thr Ser Thr Val Ser Ser Ala Tyr Thr Val Ser Arg Arg Ser Ser
485 490 495 Gly Ile
Ser Pro Tyr Phe Ser Ser Arg Arg Ser Ser Glu Ala Ser Pro 500
505 510 Leu Gly Ala Gly Arg Pro His
Asn Ala Ser Ser Ala Asp Ser Tyr Asp 515 520
525 Pro Ile Ser Thr Asp Ala Ser Arg Arg Ser Ser Glu
Ala Ser Gln Cys 530 535 540
Ser Gly Gly Ser Gly Leu Leu Asn Leu Thr Pro Ala Gln Gln Tyr Ser 545
550 555 560 Leu Arg Ala
Lys Tyr Ala Ala Ala Thr Gly Gly Pro Pro Pro Thr Pro 565
570 575 Leu Pro Gly Leu Glu Arg Met Ser
Leu Arg Thr Arg Leu Ala Leu Leu 580 585
590 Asp Ala Ala Glu Gly Thr Leu Pro Ala Gly Cys Pro Arg
Pro Leu Gly 595 600 605
Pro Arg Arg Gly Ser Asp Gly Pro Thr Tyr Gly His Gly His Ala Gly 610
615 620 Ala Ala Pro Ala
Phe Pro His Glu Ala Pro Gly Gly Gly Thr Arg Arg 625 630
635 640 Ala Ser Asp Pro Val Arg Arg Pro Asp
Ala Leu Ser Leu Pro Arg Val 645 650
655 Gln Arg Phe His Ser Thr His Asn Val Asn Pro Gly Pro Leu
Pro Pro 660 665 670
Cys Ala Asp Arg Arg Gly Leu Arg Leu Gln Ser His Pro Ser Thr Asp
675 680 685 Gly Gly Leu Ala
Arg Gly Ala Tyr Ser Pro Arg Pro Pro Ser Ile Ser 690
695 700 Glu Asn Val Ala Met Glu Ala Val
Ala Ala Gly Val Asp Gly Ala Gly 705 710
715 720 Pro Glu Ala Asp Leu Gly Leu Pro Glu Asp Asp Leu
Val Leu Pro Asp 725 730
735 Asp Val Val Gln Tyr Ile Lys Ala His Ala Ser Gly Ala Leu Asp Glu
740 745 750 Gly Thr Gly
Gln Val Tyr Pro Thr Glu Ser Thr Gly Phe Ser Asp Asn 755
760 765 Pro Arg Leu Pro Ser Pro Gly Leu
His Gly Gln Arg Arg Met Val Ala 770 775
780 Ala Asp Ser Asn Val Gly Pro Ser Ala Pro Met Leu Gly
Gly Cys Gln 785 790 795
800 Leu Gly Phe Gly Ala Pro Ser Ser Leu Asn Lys Asn Asn Met Pro Val
805 810 815 Gln Trp Asn Glu
Val Ser Ser Gly Thr Val Asp Ser Leu Ala Ser Gln 820
825 830 Val Lys Pro Pro Pro Phe Pro Gln Gly
Asn Leu Ala Val Val Gln Gln 835 840
845 Lys Pro Ala Phe Gly Gln Tyr Pro Gly Tyr Ser Pro Gln Gly
Leu Gln 850 855 860
Ala Ser Pro Gly Gly Leu Asp Ser Thr Gln Pro His Leu Gln Pro Arg 865
870 875 880 Ser Gly Ala Pro Ser
Gln Gly Ile Pro Arg Val Asn Tyr Met Gln Gln 885
890 895 Leu Arg Gln Pro Val Ala Gly Ser Gln Cys
Pro Gly Met Thr Thr Thr 900 905
910 Met Ser Pro His Ala Cys Tyr Gly Gln Val His Pro Gln Leu Ser
Pro 915 920 925 Ser
Thr Ile Ser Gly Ala Leu Asn Gln Phe Pro Gln Ser Cys Ser Asn 930
935 940 Met Pro Ala Lys Pro Gly
His Leu Gly His Pro Gln Gln Thr Glu Val 945 950
955 960 Ala Pro Asp Pro Thr Thr Met Gly Asn Arg His
Arg Glu Leu Gly Val 965 970
975 Pro Asn Ser Ala Leu Ala Gly Val Pro Pro Pro His Pro Val Gln Ser
980 985 990 Tyr Pro
Gln Gln Ser His His Leu Ala Ala Ser Met Ser Gln Glu Gly 995
1000 1005 Tyr His Gln Val Pro
Ser Leu Leu Pro Ala Arg Gln Pro Gly Phe 1010 1015
1020 Met Glu Pro Gln Thr Gly Pro Met Gly Val
Ala Thr Ala Gly Phe 1025 1030 1035
Gly Leu Val Gln Pro Arg Pro Pro Leu Glu Pro Ser Pro Thr Gly
1040 1045 1050 Arg His
Arg Gly Val Arg Ala Val Gln Gln Gln Leu Ala Tyr Ala 1055
1060 1065 Arg Ala Thr Gly His Ala Met
Ala Ala Met Pro Ser Ser Gln Glu 1070 1075
1080 Thr Ala Glu Ala Val Pro Lys Gly Ala Met Gly Asn
Met Gly Ser 1085 1090 1095
Val Pro Pro Gln Pro Pro Pro Gln Asp Ala Gly Gly Ala Pro Asp 1100
1105 1110 His Ser Met Leu Tyr
Tyr Tyr Gly Gln Ile His Met Tyr Glu Gln 1115 1120
1125 Asp Gly Gly Leu Glu Asn Leu Gly Ser Cys
Gln Val Met Arg Ser 1130 1135 1140
Gln Pro Pro Gln Pro Gln Ala Cys Gln Asp Ser Ile Gln Pro Gln
1145 1150 1155 Pro Leu
Pro Ser Pro Gly Val Asn Gln Val Ser Ser Thr Val Asp 1160
1165 1170 Ser Gln Leu Leu Glu Ala Pro
Gln Ile Asp Phe Asp Ala Ile Met 1175 1180
1185 Asp Asp Gly Asp His Ser Ser Leu Phe Ser Gly Ala
Leu Ser Pro 1190 1195 1200
Ser Leu Leu His Ser Leu Ser Gln Asn Ser Ser Arg Leu Thr Thr 1205
1210 1215 Pro Arg Asn Ser Leu
Thr Leu Pro Ser Ile Pro Ala Gly Ile Ser 1220 1225
1230 Asn Met Ala Val Gly Asp Met Ser Ser Met
Leu Thr Ser Leu Ala 1235 1240 1245
Glu Glu Ser Lys Phe Leu Asn Met Met Thr 1250
1255 7349PRTHomo sapiens 7Met Asn Arg Lys Ala Arg Arg Cys
Leu Gly His Leu Phe Leu Ser Leu 1 5 10
15 Gly Met Val Tyr Leu Arg Ile Gly Gly Phe Ser Ser Val
Val Ala Leu 20 25 30
Gly Ala Ser Ile Ile Cys Asn Lys Ile Pro Gly Leu Ala Pro Arg Gln
35 40 45 Arg Ala Ile Cys
Gln Ser Arg Pro Asp Ala Ile Ile Val Ile Gly Glu 50
55 60 Gly Ser Gln Met Gly Leu Asp Glu
Cys Gln Phe Gln Phe Arg Asn Gly 65 70
75 80 Arg Trp Asn Cys Ser Ala Leu Gly Glu Arg Thr Val
Phe Gly Lys Glu 85 90
95 Leu Lys Val Gly Ser Arg Glu Ala Ala Phe Thr Tyr Ala Ile Ile Ala
100 105 110 Ala Gly Val
Ala His Ala Ile Thr Ala Ala Cys Thr Gln Gly Asn Leu 115
120 125 Ser Asp Cys Gly Cys Asp Lys Glu
Lys Gln Gly Gln Tyr His Arg Asp 130 135
140 Glu Gly Trp Lys Trp Gly Gly Cys Ser Ala Asp Ile Arg
Tyr Gly Ile 145 150 155
160 Gly Phe Ala Lys Val Phe Val Asp Ala Arg Glu Ile Lys Gln Asn Ala
165 170 175 Arg Thr Leu Met
Asn Leu His Asn Asn Glu Ala Gly Arg Lys Ile Leu 180
185 190 Glu Glu Asn Met Lys Leu Glu Cys Lys
Cys His Gly Val Ser Gly Ser 195 200
205 Cys Thr Thr Lys Thr Cys Trp Thr Thr Leu Pro Gln Phe Arg
Glu Leu 210 215 220
Gly Tyr Val Leu Lys Asp Lys Tyr Asn Glu Ala Val His Val Glu Pro 225
230 235 240 Val Arg Ala Ser Arg
Asn Lys Arg Pro Thr Phe Leu Lys Ile Lys Lys 245
250 255 Pro Leu Ser Tyr Arg Lys Pro Met Asp Thr
Asp Leu Val Tyr Ile Glu 260 265
270 Lys Ser Pro Asn Tyr Cys Glu Glu Asp Pro Val Thr Gly Ser Val
Gly 275 280 285 Thr
Gln Gly Arg Ala Cys Asn Lys Thr Ala Pro Gln Ala Ser Gly Cys 290
295 300 Asp Leu Met Cys Cys Gly
Arg Gly Tyr Asn Thr His Gln Tyr Ala Arg 305 310
315 320 Val Trp Gln Cys Asn Cys Lys Phe His Trp Cys
Cys Tyr Val Lys Cys 325 330
335 Asn Thr Cys Ser Glu Arg Thr Glu Met Tyr Thr Cys Lys
340 345 81210PRTHomo sapiens 8Met Arg Pro
Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala 1 5
10 15 Ala Leu Cys Pro Ala Ser Arg Ala
Leu Glu Glu Lys Lys Val Cys Gln 20 25
30 Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu
Asp His Phe 35 40 45
Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn 50
55 60 Leu Glu Ile Thr
Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys 65 70
75 80 Thr Ile Gln Glu Val Ala Gly Tyr Val
Leu Ile Ala Leu Asn Thr Val 85 90
95 Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn
Met Tyr 100 105 110
Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn
115 120 125 Lys Thr Gly Leu
Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130
135 140 His Gly Ala Val Arg Phe Ser Asn
Asn Pro Ala Leu Cys Asn Val Glu 145 150
155 160 Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe
Leu Ser Asn Met 165 170
175 Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro
180 185 190 Ser Cys Pro
Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln 195
200 205 Lys Leu Thr Lys Ile Ile Cys Ala
Gln Gln Cys Ser Gly Arg Cys Arg 210 215
220 Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala
Ala Gly Cys 225 230 235
240 Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp
245 250 255 Glu Ala Thr Cys
Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 260
265 270 Thr Thr Tyr Gln Met Asp Val Asn Pro
Glu Gly Lys Tyr Ser Phe Gly 275 280
285 Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr
Asp His 290 295 300
Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu 305
310 315 320 Asp Gly Val Arg Lys
Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val 325
330 335 Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys
Asp Ser Leu Ser Ile Asn 340 345
350 Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly
Asp 355 360 365 Leu
His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr 370
375 380 Pro Pro Leu Asp Pro Gln
Glu Leu Asp Ile Leu Lys Thr Val Lys Glu 385 390
395 400 Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro
Glu Asn Arg Thr Asp 405 410
415 Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
420 425 430 His Gly
Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu 435
440 445 Gly Leu Arg Ser Leu Lys Glu
Ile Ser Asp Gly Asp Val Ile Ile Ser 450 455
460 Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn
Trp Lys Lys Leu 465 470 475
480 Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu
485 490 495 Asn Ser Cys
Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro 500
505 510 Glu Gly Cys Trp Gly Pro Glu Pro
Arg Asp Cys Val Ser Cys Arg Asn 515 520
525 Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu
Leu Glu Gly 530 535 540
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro 545
550 555 560 Glu Cys Leu Pro
Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565
570 575 Asp Asn Cys Ile Gln Cys Ala His Tyr
Ile Asp Gly Pro His Cys Val 580 585
590 Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu
Val Trp 595 600 605
Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys 610
615 620 Thr Tyr Gly Cys Thr
Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly 625 630
635 640 Pro Lys Ile Pro Ser Ile Ala Thr Gly Met
Val Gly Ala Leu Leu Leu 645 650
655 Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg
His 660 665 670 Ile
Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu 675
680 685 Val Glu Pro Leu Thr Pro
Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu 690 695
700 Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile
Lys Val Leu Gly Ser 705 710 715
720 Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu
725 730 735 Lys Val
Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser 740
745 750 Pro Lys Ala Asn Lys Glu Ile
Leu Asp Glu Ala Tyr Val Met Ala Ser 755 760
765 Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile
Cys Leu Thr Ser 770 775 780
Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp 785
790 795 800 Tyr Val Arg
Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn 805
810 815 Trp Cys Val Gln Ile Ala Lys Gly
Met Asn Tyr Leu Glu Asp Arg Arg 820 825
830 Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val
Lys Thr Pro 835 840 845
Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala 850
855 860 Glu Glu Lys Glu
Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp 865 870
875 880 Met Ala Leu Glu Ser Ile Leu His Arg
Ile Tyr Thr His Gln Ser Asp 885 890
895 Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe
Gly Ser 900 905 910
Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu
915 920 925 Lys Gly Glu Arg
Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr 930
935 940 Met Ile Met Val Lys Cys Trp Met
Ile Asp Ala Asp Ser Arg Pro Lys 945 950
955 960 Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala
Arg Asp Pro Gln 965 970
975 Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro
980 985 990 Thr Asp Ser
Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp 995
1000 1005 Asp Val Val Asp Ala Asp
Glu Tyr Leu Ile Pro Gln Gln Gly Phe 1010 1015
1020 Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu
Leu Ser Ser Leu 1025 1030 1035
Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn
1040 1045 1050 Gly Leu Gln
Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg 1055
1060 1065 Tyr Ser Ser Asp Pro Thr Gly Ala
Leu Thr Glu Asp Ser Ile Asp 1070 1075
1080 Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser
Val Pro 1085 1090 1095
Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln 1100
1105 1110 Pro Leu Asn Pro Ala
Pro Ser Arg Asp Pro His Tyr Gln Asp Pro 1115 1120
1125 His Ser Thr Ala Val Gly Asn Pro Glu Tyr
Leu Asn Thr Val Gln 1130 1135 1140
Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala
1145 1150 1155 Gln Lys
Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln 1160
1165 1170 Gln Asp Phe Phe Pro Lys Glu
Ala Lys Pro Asn Gly Ile Phe Lys 1175 1180
1185 Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val
Ala Pro Gln 1190 1195 1200
Ser Ser Glu Phe Ile Gly Ala 1205 1210 91207PRTHomo
sapiens 9Met Leu Leu Thr Leu Ile Ile Leu Leu Pro Val Val Ser Lys Phe Ser
1 5 10 15 Phe Val
Ser Leu Ser Ala Pro Gln His Trp Ser Cys Pro Glu Gly Thr 20
25 30 Leu Ala Gly Asn Gly Asn Ser
Thr Cys Val Gly Pro Ala Pro Phe Leu 35 40
45 Ile Phe Ser His Gly Asn Ser Ile Phe Arg Ile Asp
Thr Glu Gly Thr 50 55 60
Asn Tyr Glu Gln Leu Val Val Asp Ala Gly Val Ser Val Ile Met Asp 65
70 75 80 Phe His Tyr
Asn Glu Lys Arg Ile Tyr Trp Val Asp Leu Glu Arg Gln 85
90 95 Leu Leu Gln Arg Val Phe Leu Asn
Gly Ser Arg Gln Glu Arg Val Cys 100 105
110 Asn Ile Glu Lys Asn Val Ser Gly Met Ala Ile Asn Trp
Ile Asn Glu 115 120 125
Glu Val Ile Trp Ser Asn Gln Gln Glu Gly Ile Ile Thr Val Thr Asp 130
135 140 Met Lys Gly Asn
Asn Ser His Ile Leu Leu Ser Ala Leu Lys Tyr Pro 145 150
155 160 Ala Asn Val Ala Val Asp Pro Val Glu
Arg Phe Ile Phe Trp Ser Ser 165 170
175 Glu Val Ala Gly Ser Leu Tyr Arg Ala Asp Leu Asp Gly Val
Gly Val 180 185 190
Lys Ala Leu Leu Glu Thr Ser Glu Lys Ile Thr Ala Val Ser Leu Asp
195 200 205 Val Leu Asp Lys
Arg Leu Phe Trp Ile Gln Tyr Asn Arg Glu Gly Ser 210
215 220 Asn Ser Leu Ile Cys Ser Cys Asp
Tyr Asp Gly Gly Ser Val His Ile 225 230
235 240 Ser Lys His Pro Thr Gln His Asn Leu Phe Ala Met
Ser Leu Phe Gly 245 250
255 Asp Arg Ile Phe Tyr Ser Thr Trp Lys Met Lys Thr Ile Trp Ile Ala
260 265 270 Asn Lys His
Thr Gly Lys Asp Met Val Arg Ile Asn Leu His Ser Ser 275
280 285 Phe Val Pro Leu Gly Glu Leu Lys
Val Val His Pro Leu Ala Gln Pro 290 295
300 Lys Ala Glu Asp Asp Thr Trp Glu Pro Glu Gln Lys Leu
Cys Lys Leu 305 310 315
320 Arg Lys Gly Asn Cys Ser Ser Thr Val Cys Gly Gln Asp Leu Gln Ser
325 330 335 His Leu Cys Met
Cys Ala Glu Gly Tyr Ala Leu Ser Arg Asp Arg Lys 340
345 350 Tyr Cys Glu Asp Val Asn Glu Cys Ala
Phe Trp Asn His Gly Cys Thr 355 360
365 Leu Gly Cys Lys Asn Thr Pro Gly Ser Tyr Tyr Cys Thr Cys
Pro Val 370 375 380
Gly Phe Val Leu Leu Pro Asp Gly Lys Arg Cys His Gln Leu Val Ser 385
390 395 400 Cys Pro Arg Asn Val
Ser Glu Cys Ser His Asp Cys Val Leu Thr Ser 405
410 415 Glu Gly Pro Leu Cys Phe Cys Pro Glu Gly
Ser Val Leu Glu Arg Asp 420 425
430 Gly Lys Thr Cys Ser Gly Cys Ser Ser Pro Asp Asn Gly Gly Cys
Ser 435 440 445 Gln
Leu Cys Val Pro Leu Ser Pro Val Ser Trp Glu Cys Asp Cys Phe 450
455 460 Pro Gly Tyr Asp Leu Gln
Leu Asp Glu Lys Ser Cys Ala Ala Ser Gly 465 470
475 480 Pro Gln Pro Phe Leu Leu Phe Ala Asn Ser Gln
Asp Ile Arg His Met 485 490
495 His Phe Asp Gly Thr Asp Tyr Gly Thr Leu Leu Ser Gln Gln Met Gly
500 505 510 Met Val
Tyr Ala Leu Asp His Asp Pro Val Glu Asn Lys Ile Tyr Phe 515
520 525 Ala His Thr Ala Leu Lys Trp
Ile Glu Arg Ala Asn Met Asp Gly Ser 530 535
540 Gln Arg Glu Arg Leu Ile Glu Glu Gly Val Asp Val
Pro Glu Gly Leu 545 550 555
560 Ala Val Asp Trp Ile Gly Arg Arg Phe Tyr Trp Thr Asp Arg Gly Lys
565 570 575 Ser Leu Ile
Gly Arg Ser Asp Leu Asn Gly Lys Arg Ser Lys Ile Ile 580
585 590 Thr Lys Glu Asn Ile Ser Gln Pro
Arg Gly Ile Ala Val His Pro Met 595 600
605 Ala Lys Arg Leu Phe Trp Thr Asp Thr Gly Ile Asn Pro
Arg Ile Glu 610 615 620
Ser Ser Ser Leu Gln Gly Leu Gly Arg Leu Val Ile Ala Ser Ser Asp 625
630 635 640 Leu Ile Trp Pro
Ser Gly Ile Thr Ile Asp Phe Leu Thr Asp Lys Leu 645
650 655 Tyr Trp Cys Asp Ala Lys Gln Ser Val
Ile Glu Met Ala Asn Leu Asp 660 665
670 Gly Ser Lys Arg Arg Arg Leu Thr Gln Asn Asp Val Gly His
Pro Phe 675 680 685
Ala Val Ala Val Phe Glu Asp Tyr Val Trp Phe Ser Asp Trp Ala Met 690
695 700 Pro Ser Val Ile Arg
Val Asn Lys Arg Thr Gly Lys Asp Arg Val Arg 705 710
715 720 Leu Gln Gly Ser Met Leu Lys Pro Ser Ser
Leu Val Val Val His Pro 725 730
735 Leu Ala Lys Pro Gly Ala Asp Pro Cys Leu Tyr Gln Asn Gly Gly
Cys 740 745 750 Glu
His Ile Cys Lys Lys Arg Leu Gly Thr Ala Trp Cys Ser Cys Arg 755
760 765 Glu Gly Phe Met Lys Ala
Ser Asp Gly Lys Thr Cys Leu Ala Leu Asp 770 775
780 Gly His Gln Leu Leu Ala Gly Gly Glu Val Asp
Leu Lys Asn Gln Val 785 790 795
800 Thr Pro Leu Asp Ile Leu Ser Lys Thr Arg Val Ser Glu Asp Asn Ile
805 810 815 Thr Glu
Ser Gln His Met Leu Val Ala Glu Ile Met Val Ser Asp Gln 820
825 830 Asp Asp Cys Ala Pro Val Gly
Cys Ser Met Tyr Ala Arg Cys Ile Ser 835 840
845 Glu Gly Glu Asp Ala Thr Cys Gln Cys Leu Lys Gly
Phe Ala Gly Asp 850 855 860
Gly Lys Leu Cys Ser Asp Ile Asp Glu Cys Glu Met Gly Val Pro Val 865
870 875 880 Cys Pro Pro
Ala Ser Ser Lys Cys Ile Asn Thr Glu Gly Gly Tyr Val 885
890 895 Cys Arg Cys Ser Glu Gly Tyr Gln
Gly Asp Gly Ile His Cys Leu Asp 900 905
910 Ile Asp Glu Cys Gln Leu Gly Val His Ser Cys Gly Glu
Asn Ala Ser 915 920 925
Cys Thr Asn Thr Glu Gly Gly Tyr Thr Cys Met Cys Ala Gly Arg Leu 930
935 940 Ser Glu Pro Gly
Leu Ile Cys Pro Asp Ser Thr Pro Pro Pro His Leu 945 950
955 960 Arg Glu Asp Asp His His Tyr Ser Val
Arg Asn Ser Asp Ser Glu Cys 965 970
975 Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly Val Cys
Met Tyr 980 985 990
Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile
995 1000 1005 Gly Glu Arg
Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg 1010
1015 1020 His Ala Gly His Gly Gln Gln Gln
Lys Val Ile Val Val Ala Val 1025 1030
1035 Cys Val Val Val Leu Val Met Leu Leu Leu Leu Ser Leu
Trp Gly 1040 1045 1050
Ala His Tyr Tyr Arg Thr Gln Lys Leu Leu Ser Lys Asn Pro Lys 1055
1060 1065 Asn Pro Tyr Glu Glu
Ser Ser Arg Asp Val Arg Ser Arg Arg Pro 1070 1075
1080 Ala Asp Thr Glu Asp Gly Met Ser Ser Cys
Pro Gln Pro Trp Phe 1085 1090 1095
Val Val Ile Lys Glu His Gln Asp Leu Lys Asn Gly Gly Gln Pro
1100 1105 1110 Val Ala
Gly Glu Asp Gly Gln Ala Ala Asp Gly Ser Met Gln Pro 1115
1120 1125 Thr Ser Trp Arg Gln Glu Pro
Gln Leu Cys Gly Met Gly Thr Glu 1130 1135
1140 Gln Gly Cys Trp Ile Pro Val Ser Ser Asp Lys Gly
Ser Cys Pro 1145 1150 1155
Gln Val Met Glu Arg Ser Phe His Met Pro Ser Tyr Gly Thr Gln 1160
1165 1170 Thr Leu Glu Gly Gly
Val Glu Lys Pro His Ser Leu Leu Ser Ala 1175 1180
1185 Asn Pro Leu Trp Gln Gln Arg Ala Leu Asp
Pro Pro His Gln Met 1190 1195 1200
Glu Leu Thr Gln 1205
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