U.S. patent application number 09/942121 was filed with the patent office on 2002-10-10 for casein derived peptides and uses thereof in therapy.
Invention is credited to Sidelman, Zvi.
Application Number | 20020147144 09/942121 |
Document ID | / |
Family ID | 11073892 |
Filed Date | 2002-10-10 |
United States Patent
Application |
20020147144 |
Kind Code |
A1 |
Sidelman, Zvi |
October 10, 2002 |
Casein derived peptides and uses thereof in therapy
Abstract
Biologically active peptides that are derived from or are
similar to sequences identical with the N-terminus of the oS1
fraction of milk casein. These peptides are capable of stimulating
and enhancing immune response, protecting against viral infection,
normalizing serum cholesterol levels, and stimulating
hematopoiesis. The casein-derived peptides are non-toxic and can be
used to treat and prevent immune pathologies, hypercholesterolemia,
hematological disorders and viral-related diseases.
Inventors: |
Sidelman, Zvi; (Tel Aviv,
IL) |
Correspondence
Address: |
G.E. EHRLICH (1995) LTD.
c/o ANTHONY CASTORINA
SUITE 207
2001 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Family ID: |
11073892 |
Appl. No.: |
09/942121 |
Filed: |
August 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09942121 |
Aug 30, 2001 |
|
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PCT/IL01/00198 |
Mar 1, 2000 |
|
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Current U.S.
Class: |
514/3.8 ;
514/2.4; 514/3.7; 514/5.7; 514/7.8; 514/7.9 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 37/00 20180101; A61P 7/06 20180101; A61P 31/18 20180101; A61P
7/00 20180101; A61P 3/06 20180101; C07K 14/4732 20130101; A61P 7/04
20180101; C12P 21/06 20130101; A61P 3/10 20180101; A61K 38/08
20130101; A61P 3/00 20180101; A61P 37/02 20180101; A61K 38/1709
20130101; A61P 31/12 20180101; A61P 3/12 20180101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 038/17 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 1, 2000 |
IL |
134830 |
Claims
What is claimed is:
1. A method of preventing or treating an autoimmune disease, the
method comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
2. The method of claim 1, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
3. The method of claim 1, wherein said peptide is a synthetic
peptide.
4. The method of claim 1, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
5. A method of preventing or treating a viral disease, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
6. The method of claim 5, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
7. The method of claim 5, wherein said peptide is a synthetic
peptide.
8. The method of claim 5, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
9. A method of preventing viral infection, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
10. The method of claim 9, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
11. The method of claim 9, wherein said peptide is a synthetic
peptide.
12. The method of claim 9, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
13. A method of inducing hematopoiesis, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
14. The method of claim 13, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
15. The method of claim 13, wherein said peptide is a synthetic
peptide.
16. The method of claim 13, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
17. A method of inducing hematopoietic stem cells proliferation,
the method comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
18. The method of claim 17, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
19. The method of claim 17, wherein said peptide is a synthetic
peptide.
20. The method of claim 17, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
21. A method of inducing hematopoietic stem cells proliferation and
differentiation, the method comprising administering to a subject
in need thereof a therapeutically effective amount of a peptide
derived from an N terminus portion of oS1 casein.
22. The method of claim 21, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
23. The method of claim 21, wherein said peptide is a synthetic
peptide.
24. The method of claim 21, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs:1-25.
25. A method of inducing megakaryocytopoiesis, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
26. The method of claim 25, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
27. The method of claim 25, wherein said peptide is a synthetic
peptide.
28. The method of claim 25, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
29. A method of inducing erythropoiesis, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
30. The method of claim 29, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
31. The method of claim 29, wherein said peptide is a synthetic
peptide.
32. The method of claim 29, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
33. A method of inducing leukocytopoiesis, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
34. The method of claim 33, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
35. The method of claim 33, wherein said peptide is a synthetic
peptide.
36. The method of claim 33, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
37. A method of inducing thrombocytopoiesis, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
38. The method of claim 37, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
39. The method of claim 37, wherein said peptide is a synthetic
peptide.
40. The method of claim 37, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs:1-25.
41. A method of inducing plasma cell proliferation, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
42. The method of claim 41, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
43. The method of claim 41, wherein said peptide is a synthetic
peptide.
44. The method of claim 41, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs:1-25.
45. A method of inducing dendritic cell proliferation, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
46. The method of claim 45, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
47. The method of claim 45, wherein said peptide is a synthetic
peptide.
48. The method of claim 45, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs:1-25.
49. A method of inducing macrophage proliferation, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
50. The method of claim 49, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
51. The method of claim 49, wherein said peptide is a synthetic
peptide.
52. The method of claim 49, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
53. A method of preventing or treating thrombocytopenia, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
54. The method of claim 53, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
55. The method of claim 53, wherein said peptide is a synthetic
peptide.
56. The method of claim 53, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
57. A method of preventing or treating pancytopenia, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
58. The method of claim 57, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
59. The method of claim 57, wherein said peptide is a synthetic
peptide.
60. The method of claim 57, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
61. A method of preventing or treating granulocytopenia, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
62. The method of claim 61, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
63. The method of claim 61, wherein said peptide is a synthetic
peptide.
64. The method of claim 61, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
65. A method of preventing or treating hyperlipidemia, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
66. The method of claim 65, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
67. The method of claim 65, wherein said peptide is a synthetic
peptide.
68. The method of claim 65, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
69. A method of preventing or treating cholesteremia, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
70. The method of claim 69, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
71. The method of claim 69, wherein said peptide is a synthetic
peptide.
72. The method of claim 69, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
73. A method of preventing or treating glucosuria, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
74. The method of claim 73, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
75. The method of claim 73, wherein said peptide is a synthetic
peptide.
76. The method of claim 73, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
77. A method of preventing or treating diabetes, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
78. The method of claim 77, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
79. The method of claim 77, wherein said peptide is a synthetic
peptide.
80. The method of claim 77, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
81. A method of preventing or treating AIDS, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
82. The method of claim 81, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
83. The method of claim 81, wherein said peptide is a synthetic
peptide.
84. The method of claim 81, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
85. A method of preventing or treating infection by HIV, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
86. The method of claim 85, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
87. The method of claim 85, wherein said peptide is a synthetic
peptide.
88. The method of claim 85, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
89. A method of preventing or treating conditions associated with
myeloablative doses of chemoradiotherapy supported by autologous
bone marrow or peripheral blood stem cell transplantation (ASCT) or
allogeneic bone marrow transplantation (BMT), the method comprising
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
90. The method of claim 89, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
91. The method of claim 89, wherein said peptide is a synthetic
peptide.
92. The method of claim 89, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
93. A method of treating a thrombopoietin treatable condition, the
method comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
94. The method of claim 93, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
95. The method of claim 93, wherein said peptide is a synthetic
peptide.
96. The method of claim 93, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
97. A method of augmenting the effect of thrombopoietin, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
98. The method of claim 97, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
99. The method of claim 97, wherein said peptide is a synthetic
peptide.
100. The method of claim 1, wherein said peptide has a sequence as
set forth in one of SEQ ID NOs: 1-25.
101. A method of enhancing peripheral stem cell mobilization, the
method comprising administering to a subject in need thereof an
effective amount of a pharmaceutical composition comprising
effective amounts of thrombopoietin and a peptide derived from an N
terminus portion of .alpha. S1 casein.
102. The method of claim 101, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
103. The method of claim 101, wherein said peptide is a synthetic
peptide.
104. The method of claim 101, wherein said peptide has a sequence
as set forth in one of SEQ ID NOs: 1-25.
105. A pharmaceutical composition for preventing or treating an
autoimmune disease, the pharmaceutical composition comprising, as
an active ingredient, a peptide derived from an N terminus portion
of oS1 casein and a pharmaceutically acceptable carrier.
106. The pharmaceutical composition of claim 105, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
107. The pharmaceutical composition of claim 105, wherein said
peptide is a synthetic peptide.
108. The pharmaceutical composition of claim 105, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
109. A pharmaceutical composition for preventing or treating a
viral disease, the pharmaceutical composition comprising, as an
active ingredient, a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
110. The pharmaceutical composition of claim 109, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
111. The pharmaceutical composition of claim 109, wherein said
peptide is a synthetic peptide.
112. The pharmaceutical composition of claim 109, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
113. A pharmaceutical composition for preventing viral infection,
the pharmaceutical composition comprising, as an active ingredient,
a peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
114. The pharmaceutical composition of claim 113, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
115. The pharmaceutical composition of claim 113, wherein said
peptide is a synthetic peptide.
116. The pharmaceutical composition of claim 113, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
117. A pharmaceutical composition for inducing hematopoiesis, the
pharmaceutical composition comprising, as an active ingredient, a
peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
118. The pharmaceutical composition of claim 117, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
119. The pharmaceutical composition of claim 117, wherein said
peptide is a synthetic peptide.
120. The pharmaceutical composition of claim 117, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
121. A pharmaceutical composition for inducing hematopoietic stem
cells proliferation, the pharmaceutical composition comprising, as
an active ingredient, a peptide derived from an N terminus portion
of oS1 casein and a pharmaceutically acceptable carrier.
122. The pharmaceutical composition of claim 121, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
123. The pharmaceutical composition of claim 121, wherein said
peptide is a synthetic peptide.
124. The pharmaceutical composition of claim 121, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs:1-25.
125. A pharmaceutical composition for inducing hematopoietic stem
cells proliferation and differentiation, the pharmaceutical
composition comprising, as an active ingredient, a peptide derived
from an N terminus portion of oS1 casein and a pharmaceutically
acceptable carrier.
126. The pharmaceutical composition of claim 125, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
127. The pharmaceutical composition of claim 125, wherein said
peptide is a synthetic peptide.
128. The pharmaceutical composition of claim 125, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
129. A pharmaceutical composition for inducing
megakaryocytopoiesis, the pharmaceutical composition comprising, as
an active ingredient, a peptide derived from an N terminus portion
of oS1 casein and a pharmaceutically acceptable carrier.
130. The pharmaceutical composition of claim 129, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
131. The pharmaceutical composition of claim 129, wherein said
peptide is a synthetic peptide.
132. The pharmaceutical composition of claim 129, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
133. A pharmaceutical composition for inducing erythropoiesis, the
pharmaceutical composition comprising, as an active ingredient, a
peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
134. The pharmaceutical composition of claim 133, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
135. The pharmaceutical composition of claim 133, wherein said
peptide is a synthetic peptide.
136. The pharmaceutical composition of claim 133, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
137. A pharmaceutical composition for inducing leukocytopoiesis,
the pharmaceutical composition comprising, as an active ingredient,
a peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
138. The pharmaceutical composition of claim 137, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
139. The pharmaceutical composition of claim 137, wherein said
peptide is a synthetic peptide.
140. The pharmaceutical composition of claim 137, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
141. A pharmaceutical composition for inducing thrombocytopoiesis,
the pharmaceutical composition comprising, as an active ingredient,
a peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
142. The pharmaceutical composition of claim 141, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
143. The pharmaceutical composition of claim 141, wherein said
peptide is a synthetic peptide.
144. The pharmaceutical composition of claim 141, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
145. A pharmaceutical composition for inducing plasma cell
proliferation, the pharmaceutical composition comprising, as an
active ingredient a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
146. The pharmaceutical composition of claim 145, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
147. The pharmaceutical composition of claim 145, wherein said
peptide is a synthetic peptide.
148. The pharmaceutical composition of claim 1, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
149. A pharmaceutical composition for inducing dendritic cell
proliferation, the pharmaceutical composition comprising, as an
active ingredient a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
150. The pharmaceutical composition of claim 149, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
151. The pharmaceutical composition of claim 149, wherein said
peptide is a synthetic peptide.
152. The pharmaceutical composition of claim 149, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
153. A pharmaceutical composition for inducing macrophage
proliferation, the pharmaceutical composition comprising a peptide
derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
154. The pharmaceutical composition of claim 153, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
155. The pharmaceutical composition of claim 153, wherein said
peptide is a synthetic peptide.
156. The pharmaceutical composition of claim 153, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
157. A pharmaceutical composition for preventing or treating
thrombocytopenia, the pharmaceutical composition comprising, as an
active ingredient, a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
158. The pharmaceutical composition of claim 157, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
159. The pharmaceutical composition of claim 157, wherein said
peptide is a synthetic peptide.
160. The pharmaceutical composition of claim 157, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
161. A pharmaceutical composition for preventing or treating
pancytopenia, the pharmaceutical composition comprising, as an
active ingredient, a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
162. The pharmaceutical composition of claim 161, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
163. The pharmaceutical composition of claim 161, wherein said
peptide is a synthetic peptide.
164. The pharmaceutical composition of claim 161, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
165. A pharmaceutical composition for preventing or treating
granulocytopenia, the pharmaceutical composition comprising, as an
active ingredient, a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
166. The pharmaceutical composition of claim 165, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
167. The pharmaceutical composition of claim 165, wherein said
peptide is a synthetic peptide.
168. The pharmaceutical composition of claim 165, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
169. A pharmaceutical composition for preventing or treating
hyperlipidemia, the pharmaceutical composition comprising, as an
active ingredient, a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
170. The pharmaceutical composition of claim 169, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
171. The pharmaceutical composition of claim 169, wherein said
peptide is a synthetic peptide.
172. The pharmaceutical composition of claim 169, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
173. A pharmaceutical composition for preventing or treating
cholesteremia, the pharmaceutical composition comprising, as an
active ingredient, a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
174. The pharmaceutical composition of claim 173, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
175. The pharmaceutical composition of claim 173, wherein said
peptide is a synthetic peptide.
176. The pharmaceutical composition of claim 173, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
177. A pharmaceutical composition for preventing or treating
glucosuria, the pharmaceutical composition comprising, as an active
ingredient, a peptide derived from an N terminus portion of oS1
casein and a pharmaceutically acceptable carrier.
178. The pharmaceutical composition of claim 177, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
179. The pharmaceutical composition of claim 177, wherein said
peptide is a synthetic peptide.
180. The pharmaceutical composition of claim 177, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
181. A pharmaceutical composition for preventing or treating
diabetes, the pharmaceutical composition comprising, as an active
ingredient, a peptide derived from an N terminus portion of oS1
casein and a pharmaceutically acceptable carrier.
182. The pharmaceutical composition of claim 181, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
183. The pharmaceutical composition of claim 181, wherein said
peptide is a synthetic peptide.
184. The pharmaceutical composition of claim 181, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
185. A pharmaceutical composition for preventing or treating AIDS,
the pharmaceutical composition comprising, as an active ingredient,
a peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
186. The pharmaceutical composition of claim 185, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
187. The pharmaceutical composition of claim 185, wherein said
peptide is a synthetic peptide.
188. The pharmaceutical composition of claim 185, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
189. A pharmaceutical composition for preventing or treating
infection by HIV, the pharmaceutical composition comprising, as an
active ingredient, a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
190. The pharmaceutical composition of claim 189, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
191. The pharmaceutical composition of claim 189, wherein said
peptide is a synthetic peptide.
192. The pharmaceutical composition of claim 189, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
193. A pharmaceutical composition for preventing or treating
conditions associated with myeloablative doses of chemoradiotherapy
supported by autologous bone marrow or peripheral blood stem cell
transplantation (ASCT) or allogeneic bone marrow transplantation
(BMT), the pharmaceutical composition comprising, as an active
ingredient, a peptide derived from an N terminus portion of oS1
casein and a pharmaceutically acceptable carrier.
194. The pharmaceutical composition of claim 193, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
195. The pharmaceutical composition of claim 193, wherein said
peptide is a synthetic peptide.
196. The pharmaceutical composition of claim 193, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
197. A pharmaceutical composition for treating a thrombopoietin
treatable condition, the pharmaceutical composition comprising, as
an active ingredient a peptide derived from an N terminus portion
of oS1 casein and a pharmaceutically acceptable carrier.
198. The pharmaceutical composition of claim 197, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
199. The pharmaceutical composition of claim 197, wherein said
peptide is a synthetic peptide.
200. The pharmaceutical composition of claim 197, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
201. A pharmaceutical composition for augmenting the effect of
thrombopoietin, the pharmaceutical composition comprising, as an
active ingredient a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
202. The pharmaceutical composition of claim 201, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
203. The pharmaceutical composition of claim 201, wherein said
peptide is a synthetic peptide.
204. The pharmaceutical composition of claim 201, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
205. A pharmaceutical composition for enhancing peripheral stem
cell mobilization, the pharmaceutical composition comprising, as
active ingredients thrombopoietin and a peptide derived from an N
terminus portion of a S 1 casein and a pharmaceutically acceptable
carrier.
206. The pharmaceutical composition of claim 205, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
207. The pharmaceutical composition of claim 205, wherein said
peptide is a synthetic peptide.
208. The pharmaceutical composition of claim 205, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
209. A pharmaceutical composition for inducing hematopoiesis, the
pharmaceutical composition comprising, as active ingredients,
thrombopoietin and a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
210. The pharmaceutical composition of claim 209, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
211. The pharmaceutical composition of claim 209, wherein said
peptide is a synthetic peptide.
212. The pharmaceutical composition of claim 209, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
213. A pharmaceutical composition for inducing hematopoietic stem
cells proliferation, the pharmaceutical composition comprising, as
active ingredients, thrombopoietin and a peptide derived from an N
terminus portion of oS1 casein and a pharmaceutically acceptable
carrier.
214. The pharmaceutical composition of claim 213, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
215. The pharmaceutical composition of claim 213, wherein said
peptide is a synthetic peptide.
216. The pharmaceutical composition of claim 213, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
217. A pharmaceutical composition for inducing hematopoietic stem
cells proliferation and differentiation, the pharmaceutical
composition comprising, as active ingredients, thrombopoietin and a
peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
218. The pharmaceutical composition of claim 217, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
219. The pharmaceutical composition of claim 217, wherein said
peptide is a synthetic peptide.
220. The pharmaceutical composition of claim 217, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
221. A pharmaceutical composition for inducing
megakaryocytopoiesis, the pharmaceutical composition comprising, as
active ingredients, thrombopoietin and a peptide derived from an N
terminus portion of oS1 casein and a pharmaceutically acceptable
carrier.
222. The pharmaceutical composition of claim 221, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
223. The pharmaceutical composition of claim 221, wherein said
peptide is a synthetic peptide.
224. The pharmaceutical composition of claim 221, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
225. A pharmaceutical composition for inducing erythropoiesis, the
pharmaceutical composition comprising, as active ingredients,
thrombopoietin and a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
226. The pharmaceutical composition of claim 225, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
227. The pharmaceutical composition of claim 225, wherein said
peptide is a synthetic peptide.
228. The pharmaceutical composition of claim 225, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
229. A pharmaceutical composition for inducing leukocytopoiesis,
the pharmaceutical composition comprising, as active ingredients,
thrombopoietin and a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
230. The pharmaceutical composition of claim 229, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
231. The pharmaceutical composition of claim 229, wherein said
peptide is a synthetic peptide.
232. The pharmaceutical composition of claim 229, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
233. A pharmaceutical composition for inducing thrombocytopoiesis,
the pharmaceutical composition comprising, as active ingredients,
thrombopoietin and a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
234. The pharmaceutical composition of claim 233, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
235. The pharmaceutical composition of claim 233, wherein said
peptide is a synthetic peptide.
236. The pharmaceutical composition of claim 233, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
237. A pharmaceutical composition for preventing or treating
thrombocytopenia, the pharmaceutical composition comprising, as
active ingredients, thrombopoietin and a peptide derived from an N
terminus portion of oS1 casein and a pharmaceutically acceptable
carrier.
238. The pharmaceutical composition of claim 237, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
239. The pharmaceutical composition of claim 237, wherein said
peptide is a synthetic peptide.
240. The pharmaceutical composition of claim 237, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
241. A pharmaceutical composition for preventing or treating
pancytopenia, the pharmaceutical composition comprising, as active
ingredients, thrombopoietin and a peptide derived from an N
terminus portion of oS1 casein and a pharmaceutically acceptable
carrier.
242. The pharmaceutical composition of claim 241, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
243. The pharmaceutical composition of claim 241, wherein said
peptide is a synthetic peptide.
244. The pharmaceutical composition of claim 241, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
245. A pharmaceutical composition for preventing or treating
granulocytopenia, the pharmaceutical composition comprising, as
active ingredients, thrombopoietin and a peptide derived from an N
terminus portion of oS1 casein and a pharmaceutically acceptable
carrier.
246. The pharmaceutical composition of claim 245, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
247. The pharmaceutical composition of claim 245, wherein said
peptide is a synthetic peptide.
248. The pharmaceutical composition of claim 245, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
249. A pharmaceutical composition for treating or preventing an
indication selected from the group consisting of autoimmune disease
or condition, viral disease, viral infection, hematological
disease, hematological deficiencies, thrombocytopenia,
pancytopenia, granulocytopenia, hyperlipidemia,
hypercholesterolemia, glucosuria, hyperglycemia, diabetes, AIDS,
HIV-1, helper T-cell disorders, dendrite cell deficiencies,
macrophage deficiencies, hematopoietic stem cell disorders
including platelet, lymphocyte, plasma cell and neutrophil
disorders, pre-leukemic conditions, leukemic conditions, immune
system disorders resulting from chemotherapy or radiation therapy,
human immune system disorders resulting from treatment of diseases
of immune deficiency and bacterial infections, the pharmaceutical
composition comprising, as an active ingredient, a peptide derived
from an N terminus portion of oS1 casein and a pharmaceutically
acceptable carrier.
250. The pharmaceutical composition of claim 249, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
251. The pharmaceutical composition of claim 249, wherein said
peptide is a synthetic peptide.
252. The pharmaceutical composition of claim 249, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
253. A pharmaceutical composition for treating or preventing an
indication selected from the group consisting of hematological
disease, hematological deficiencies, thrombocytopenia,
pancytopenia, granulocytopenia, dendrite cell deficiencies,
macrophage deficiencies, hematopoietic stem cell disorders
including platelet, lymphocyte, plasma cell and neutrophil
disorders, pre-leukemic conditions, leukemic conditions,
myelodysplastic syndrome, aplastic anemia and bone marrow
insufficiency, the pharmaceutical composition comprising, as active
ingredients, thrombopoietin and a peptide derived from an N
terminus portion of oS1 casein and a pharmaceutically acceptable
carrier.
254. The pharmaceutical composition of claim 253, wherein said
peptide is a fragment derived by fragmentation of oS1 casein.
255. The pharmaceutical composition of claim 253, wherein said
peptide is a synthetic peptide.
256. The pharmaceutical composition of claim 253, wherein said
peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
257. A purified peptide having an amino acid sequence selected from
the group consisting of SEQ ID NOs: 1-25.
258. A pharmaceutical composition comprising a purified peptide
having an amino acid sequence selected from the group consisting of
SEQ ID NOs: 1-25 and a pharmaceutically acceptable carrier.
259. A pharmaceutical composition comprising thrombopoietin and a
purified peptide having an amino acid sequence selected from the
group consisting of SEQ ID NOs: 1-25 and a pharmaceutically
acceptable carrier.
260. A method of enhancing colonization of donated blood stem cells
in a mycloablated recipient, the method comprising treating a donor
of said donated blood stem cells with a peptide derived from an N
terminus portion of oS1 casein prior to donation and implanting the
donated blood stem cells in the recipient.
261. The method of claim 260, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
262. The method of claim 260, wherein said peptide is a synthetic
peptide.
263. The method of claim 260, wherein said peptide has a sequence
as set forth in one of SEQ ID NOs: 1-25.
264. A method of enhancing colonization of donated blood stem cells
in a myeloablated recipient, the method comprising treating said
donated blood stem cells with a peptide derived from an N terminus
portion of oS1 casein prior to implanting the donated blood stem
cells in the recipient.
265. The method of claim 264, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
266. The method of claim 264, wherein said peptide is a synthetic
peptide.
267. The method of claim 264, wherein said peptide has a sequence
as set forth in one of SEQ ID NOs: 1-25.
268. A method of enhancing colonization of blood stem cells in a
myeloablated recipient, the method comprising treating said blood
stem cells with a peptide derived from an N terminus portion of oS1
casein prior to implanting the blood stem cells in the
recipient.
269. The method of claim 268, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
270. The method of claim 268, wherein said peptide is a synthetic
peptide.
271. The method of claim 268, wherein said peptide has a sequence
as set forth in one of SEQ ID NOs: 1-25.
272. A method of enhancing colonization of donated blood stem cells
in a myeloablated recipient, the method comprising treating a donor
of said donated blood stem cells with a peptide derived from an N
terminus portion of oS1 casein and thrombopoietin prior to donation
and implanting the donated blood stem cells in the recipient.
273. The method of claim 272, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
274. The method of claim 272, wherein said peptide is a synthetic
peptide.
275. The method of claim 272, wherein said peptide has a sequence
as set forth in one of SEQ ID NOs: 1-25.
276. A method of enhancing colonization of donated blood stem cells
in a myeloablated recipient, the method comprising treating said
donated blood stem cells with a peptide derived from an N terminus
portion of oS1 casein and thrombopoietin prior to implanting the
donated blood stem cells in the recipient.
277. The method of claim 276, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
278. The method of claim 276, wherein said peptide is a synthetic
peptide.
279. The method of claim 276, wherein said peptide has a sequence
as set forth in one of SEQ ID NOs: 1-25.
280. A method of enhancing colonization of blood stem cells in a
myeloablated recipient, the method comprising treating said blood
stem cells with a peptide derived from an N terminus portion of oS1
casein and thrombopoietin prior to implanting the blood stem cells
in the recipient.
281. The method of claim 280, wherein said peptide is a fragment
derived by fragmentation of oS1 casein.
282. The method of claim 280, wherein said peptide is a synthetic
peptide.
283. The method of claim 280, wherein said peptide has a sequence
as set forth in one of SEQ ID NOs: 1-25.
Description
[0001] This is a continuation-in-part of PCT/IL01/00198, filed
March 1, 2001, which claims the benefit of priority from IL 134830,
filed March 1, 2000.
FIELD OF THE INVENTION
[0002] The present invention relates to biologically active
peptides that are derived from or are similar to sequences
identical with the N-terminus of the oS1 fraction of milk casein.
These peptides are capable of stimulating and enhancing immune
response, protecting against viral infection, normalizing serum
cholesterol levels, and stimulating hematopoiesis. The
casein-derived peptides are non-toxic and can be used to treat and
prevent immune pathologies, hypercholesterolemia, hematological
disorders and viral-related diseases.
BACKGROUND OF THE INVENTION
[0003] Bioactive Molecules from Nutrients:
[0004] In addition to the nutritional value of many foods, certain
fractions and products of digestive pathways possess the ability to
influence physiological processes. Some of these "extranutritional"
constituents are present in their active form in the whole
nutriment, such as the immunoglobulins in mother's milk and
colostrums, phytoestrogens found in soy-based foods, polyphenolic
antioxidants from fruits and vitamins. Others are encrypted within
nutrient molecules, and are released in an active form during
digestion or food processing, for example antihypertensive peptides
from lactoglobin [Kitts, D. D. (1999), Can. J. Physiol. Pharmacol.
72:4; 423-434].
[0005] Biological Activity in Milk Proteins:
[0006] Casein, the predominant milk protein, has been traditionally
defined as composed of three fractions, o, .beta., and .gamma.,
according to their electrophoretic mobility [N. J. Hipp, et al.
(1952), Dairy Sci., 35:272]. Today casein is defined according to
the amino acid sequences of each of the subgroups oS1, oS2, .beta.
and .kappa. [W. N. Engel et al. (1984), J. Dairy Sci. 67:
1599].
[0007] In the course of digestion, the casein proteins are
subjected to proteolytic cleavage by acid proteases such as
chymosin (rennin), trypsin and pepsin, producing shorter peptides
and causing curdling and calcium sequestration by the resultant
protein fragments. A few studies with milk compounds demonstrated
casein-related bacteriocidal activity. U.S. Pat. No. 3,764,670
discloses proteolytic casein digests possessing antibiotic
properties against microorganisms. Israel Patent No. 42863
describes a casein-derived peptide consisting of 23 amino acids of
the N-terminus of casein, possessing anti-bacterial activity. In
addition, other physiologically active properties, such as opioid
and growth factor-like activities have been proposed for casein or
its derivatives [Kitts, D. D., (1999), ibid.].
[0008] Immune modulating activity has also been observed in casein
peptides. Coste et al. (1992, Immun. Lett. 33: 41-46) observed
enhancement of rat lymphocyte proliferation following treatment
with a peptide derived from the C-terminus of .beta. casein.
However, none of these studies have determined the specific
sequences in these casein peptides which confer their
"extranutritional" properties.
[0009] Hematopoiesis in Cancer Therapy:
[0010] Following high-dose chemotherapy, especially following
myeloablative doses of chemoradiotherapy supported by autologous
bone marrow or peripheral blood stem cell transplantation (ASCT) or
allogeneic bone marrow transplantation (BMT), patients are at high
risk due to pancytopenia. Granulocytopenia may lead to development
of serious, occasionally fatal infectious complications from common
bacterial, viral, fungal and parasitic agents in the immediate post
transplant period. Similarly, thrombocytopenia frequently results
in bleeding tendency and occasionally, in long lasting platelet
dependence. Whenever resistance to platelets develops, bleeding
episodes can be life threatening and hemorrhagic complications are
frequently lethal. The risk due to granulocytopenia can be
partially overcome by supportive measures and most effectively by
administration of recombinant human cytokines that can enhance
reconstitution of granulocytes, particularly granulocyte colony
stimulation factor (G-CSF) and granulocyte macrophage colony
stimulating factor (GM-CSF). These agents are extremely expensive
(approximately $200-400/day/patient) and infrequently cause side
effects due to hypersensitivity reactions, fever, bone pain and
occasionally vascular leak syndromes, including pericarditis and
pleuritis. Some of the side effects may be due to other cytokines
that may be intrinsically released by these hematopoietic growth
factors. Moreover, the use of these hematopoietic growth factors
may be prohibitive in patients with tumor cells bearing G-CSF or
GM-CSF receptors such as in acute and chronic myeloid leukemias and
in myelodysplastic syndromes. Whereas major progress in treating
patients at risk of pancytopenia has been achieved from the use of
hematopoietic cytokines, no progress has been made in the treatment
of thrombocytopenia. Following high dose chemotherapy and
especially following ASCT, patients are at risk for
thrombocytopenia which may last for many months even up to 3 years
and some thromboctyopenic patients may never recover. Many patients
previously treated with multiple blood products become platelet
resistant and hence thrombocytopenia may be impossible to overcome,
even transiently, despite intensive and frequent platelet
transfusions from a single donor. Resistance to platelets and
protracted thrombocytopenia represent a common cause of death at
ASCT centers worldwide.
[0011] Currently, several new recombinant cytokines such as
recombinant human interleukin-3 (rhIL3) and recombinant human
interleukin-6 (rhIL6) are being investigated as potential agents
for enhancing megakaryocytopoiesis and platelet reconstitution.
Unfortunately, preliminary clinical trials showed that although
rhIL3 and rhIL6 may enhance platelet reconstitution, such effects
are by no means dramatic and may take considerable time.
[0012] Clearly, protracted thrombocytopenia represents a major
problem in clinical Bone Marrow Transplant centers today, for which
no satisfactory solution has yet been found.
[0013] There is thus a widely recognized need for, and it would be
highly advantageous to have a safe, inexpensive, rapidly effective
and well-defined stimulator of hematopoiesis, and specifically
megakaryocytopoiesis, devoid of the above limitations.
[0014] Thrombopoetin (TPO) in Regulation of Hematopoiesis and
Platelet Function:
[0015] TPO appears to be the major regulator of platelet production
in vivo, although increase in the kidney- and liver-derived growth
factor in platelet deficiencies is not caused by adaptation of TPO
biosynthesis in these organs. Rather, a "feed-back loop" seems to
exist in which the number of circulating platelets determines how
much of the circulating TPO is available to the bone marrow for
platelet production. In addition, it has been demonstrated that TPO
is an early acting cytokine with important multilineage effects:
TPO alone, or in combination with other early acting cytokines, can
(i) promote viability and suppress apoptosis in progenitor cells;
(ii) regulate hematopoietic stem cell production and function;
(iii) trigger cell division of dormant multipotent cells; (iv)
induce multilineage differentiation and (v) enhance formation of
multilineage colonies containing granulocytes, erythrocytes,
macrophages, and megakaryocytes (MK, CFU-GEMM). Moreover, TPO
stimulates the production of more limited progenitors for
granulocyte/monocyte, megakaryocyte and erythroid colonies, and
stimulates adhesion of primitive human bone marrow and
megakaryocytic cells to fibronectin and fibrinogen. Thus, TPO is an
important cytokine for clinical hematologists/transplanters: for
the mobilization, amplification and ex vivo expansion of stem cells
and committed precursor cells for autologous and allogeneic
transplantation (von dem Borne, A.E.G.Kr., et al., (1998)
Thrombopoietin: it's role in platelet disorders and as a new drug
in clinical medicine. In Bailliers Clin. Hematol. June:11 (2),
427-45.
[0016] In addition to TPO effects in hematopoiesis, this potent
growth factor primes platelets for various agonists and modulates
platelet-extracellular matrix interactions. Although it does not
itself cause platelet aggregation, TPO upregulates ADP-induced
aggregation, especially on the second wave of aggregation,
upregulates granule (ADP, ATP, serotonin, etc.) release and
production of thromboxane B2, increases platelet attachment to
collagen and potentiates shear-induced platelet aggregation. TPO
also stimulates PMN activation, inducing IL-8 release and priming
oxygen metabolite production, likely enhancing antimicrobial
defense.
[0017] Clinical studies suggest TPO's value in understanding and
treating a variety of hematological conditions. In patients with
idiopathic aplastic anemia (AA), elevated TPO levels persist even
in remission following immunosupressive therapy, indicating a
hematopoietic defect. TPO is elevated in other forms of aplastic
thrombocytopenia as well, but not in conditions of increased
platelet destruction. Apparently, the reactive increase in TPO
production is insufficient in cases of destructive
thrombocytopenia. Thus, TPO is not only a therapeutic option for
aplastic, but also for destructive thrombocytopenia.
[0018] Thrombopoietic agents are of great clinical interest, for
prevention and/or treatment of pathological or treatment-induced
thrombocytopenia, and as a substitute for platelet transfusions. Of
the cytokines evaluated, all but the marginally potent IL-11 have
been deemed unacceptable for clinical use. TPO is widely believed
to become the cytokine of choice for throbocytopenia treatment.
Recombinant human TPO (Genentech) has recently become available,
enabling accurate pharmacokinetic determinations and clinical
trials. Thus, TPO's potential applications encompass the realms of
supportive care (post chemo/radio-therapy, bone marrow and stem
cell transplantation), hematological disease (AA, myelodysplasia,
congenital and acquired thrombocytopenia), liver diseases,
transfusion (expansion, harvest, mobilization and storage of
platelets) and surgery (including liver transplantation). Of
particular interest is the potential use of TPO/EPO/G-CSF cocktail
for myelodysplasia, G-CSF and TPO combination for peripheral stem
cell mobilization and TPO in harvesting CD 34+cells and ex vivo
expansion of megakaryocytes for superior platelet reconstitution.
However, similar to other hematopoietic agents under consideration
for clinical application, TPO is costly and potentially antigenic
at therapeutically effective levels. Thus, it would be advantageous
to have a safe, inexpensive and readily available stimulator of
thrombopoiesis capable of augmenting TPO activity.
[0019] The oS1 Fraction of Casein:
[0020] The oS1 fraction of casein can be obtained from milk
proteins by various methods [D. G. Schmidth and T. A. J. Paynes
(1963), Biochim., Biophys. Acta, 78:492; M. P. Thompson and C. A.
Kiddy (1964), J. Dairy Sci., 47:626; J. C. Mercier, et al. (1968),
Bull. Soc. Chim. Biol. 50:521], and the complete amino acid
sequence of the oS1 fraction of casein was determined by J. C.
Mercier et al. (1971) (Eur. J. Biochem. 23:41). The genomic and
coding sequences of bovine oS1 fraction of casein have also been
cloned and sequenced employing recombinant DNA techniques [D.
Koczan, et al. (1991), Nucl. Acids Res. 19(20): 5591; McKnight, R.
A., et a. (1989), J. Dairy Sci. 72:2464-73]. Proteolytic cleavage
and identification of N-terminal fragments from the oS1 fraction of
casein has been documented [J. C. Mercier, et al. (1970), Eur. J.
Biochem. 16:439; P. L. H. McSweeney et al. (1993), J. Dairy Res.,
60:401], as has the intestinal absorption and appearance of this
fragment in mammalian plasma following ingestion of whole milk
proteins [Fiat, A.M., et al. (1998) Biochimie, 80(2):2155-65].
Meisel, H. and Bockelmann, W. [(1999), Antonie Van Leeuwenhoek,
76:207-15] detected amino acid sequences of immunopeptides,
casokinins and casomorphins in peptides liberated by lactic acid
bacteria digests of o and p casein fractions. Of particular
interest is the anti-aggregating and thrombolytic activity
demonstrated for C-terminal portions of the o- and .kappa.-casein
fractions [Chabance, B. et al. (1997), Biochem. Mol. Biol. Int.
42(1) 77-84; Caen J. et al. (1993), J. Dairy Sci. 76(1): 301-310].
Previous studies documented potential bioactive peptides encrypted
in the N-terminal oS1 amino acid sequence, but no mention was made
of use of these protein fragments, specific sequences or defined
synthetic peptides to enhance hematopoiesis, prevent viral
infection or modulate the development of autoimmune diseases.
SUMMARY OF THE INVENTION
[0021] According to the present invention there is provided a
method of preventing or treating an autoimmune disease, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0022] Further according to the present invention there is provided
a method of preventing or treating a viral disease, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0023] Further according to the present invention there is provided
a method of preventing viral infection, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
[0024] Further according to the present invention there is provided
a method of inducing hematopoiesis, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
[0025] Further according to the present invention there is provided
a method of inducing hematopoietic stem cells proliferation, the
method comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0026] Further according to the present invention there is provided
a method of inducing hematopoietic stem cells proliferation and
differentiation, the method comprising administering to a subject
in need thereof a therapeutically effective amount of a peptide
derived from an N terminus portion of oS1 casein.
[0027] Further according to the present invention there is provided
a method of inducing megakaryocytopoiesis, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
[0028] Further according to the present invention there is provided
a method of inducing erythropoiesis, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
[0029] Further according to the present invention there is provided
a method of inducing leukocytopoiesis, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
[0030] Further according to the present invention there is provided
a method of inducing plasma cell proliferation, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0031] Further according to the present invention there is provided
a method of inducing dendritic cell proliferation, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0032] Further according to the present invention there is provided
a method of inducing macrophage cell proliferation, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0033] Further according to the present invention there is provided
a method of preventing or treating thrombocytopenia, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0034] Further according to the present invention there is provided
a method of preventing or treating pancytopenia, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0035] Further according to the present invention there is provided
a method of preventing or treating granulocytopenia, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0036] Further according to the present invention there is provided
a method of preventing or treating hyperlipidemia, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0037] Further according to the present invention there is provided
a method of preventing or treating hypercholesterolemia, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0038] Further according to the present invention there is provided
a method of preventing or treating glucosuria, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0039] Further according to the present invention there is provided
a method of preventing or treating diabetes, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
[0040] Further according to the present invention there is provided
a method of preventing or treating AIDS, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
[0041] Further according to the present invention there is provided
a method of preventing or treating infection by HIV, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0042] Further according to the present invention there is provided
a method of preventing or treating conditions associated with
myeloablative doses of chemoradiotherapy supported by autologous
bone marrow or peripheral blood stem cell transplantation (ASCT) or
allogeneic bone marrow transplantation (BMT), the method comprising
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
[0043] Further according to the present invention there is provided
a method of treating a thrombopoietin treatable condition, the
method comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0044] Further according to the present invention there is provided
a method of augmenting the effect of thrombopoietin, the method
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0045] Further according to the present invention there is provided
a method of enhancing peripheral stem cell mobilization, the method
comprising administering to a subject in need thereof an effective
amount of a pharmaceutical composition comprising effective amounts
of thrombopoietin and a peptide derived from an N terminus portion
of a SI casein.
[0046] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating an
autoimmune disease, the pharmaceutical composition comprising, as
an active ingredient, a peptide derived from an N terminus portion
of oS1 casein and a pharmaceutically acceptable carrier.
[0047] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating a viral
disease, the pharmaceutical composition comprising, as an active
ingredient, a peptide derived from an N terminus portion of oS1
casein and a pharmaceutically acceptable carrier.
[0048] Further according to the present invention there is provided
a pharmaceutical composition for preventing viral infection, the
pharmaceutical composition comprising, as an active ingredient, a
peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0049] Further according to the present invention there is provided
a pharmaceutical composition for inducing hematopoiesis, the
pharmaceutical composition comprising, as an active ingredient, a
peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0050] Further according to the present invention there is provided
a pharmaceutical composition for inducing hematopoietic stem cells
proliferation, the pharmaceutical composition comprising, as an
active ingredient, a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0051] Further according to the present invention there is provided
a pharmaceutical composition for inducing hematopoietic stem cells
proliferation and differentiation, the pharmaceutical composition
comprising, as an active ingredient, a peptide derived from an N
terminus portion of oS1 casein and a pharmaceutically acceptable
carrier.
[0052] Further according to the present invention there is provided
a pharmaceutical composition for inducing megakaryocytopoiesis, the
pharmaceutical composition comprising, as an active ingredient, a
peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0053] Further according to the present invention there is provided
a pharmaceutical composition for inducing erythropoiesis, the
pharmaceutical composition comprising, as an active ingredient, a
peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0054] Further according to the present invention there is provided
a pharmaceutical composition for inducing leukocytopoiesis, the
pharmaceutical composition comprising, as an active ingredient, a
peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0055] Further according to the present invention there is provided
a pharmaceutical composition for inducing thrombocytopoiesis, the
pharmaceutical composition comprising, as an active ingredient, a
peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0056] Further according to the present invention there is provided
a pharmaceutical composition for inducing plasma cell
proliferation, the pharmaceutical composition comprising, as an
active ingredient a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0057] Further according to the present invention there is provided
a pharmaceutical composition for inducing dendritic cell
proliferation, the pharmaceutical composition comprising, as an
active ingredient a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0058] Further according to the present invention there is provided
a pharmaceutical composition for inducing macrophage proliferation,
the pharmaceutical composition comprising, as an active ingredient
a peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0059] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating
thrombocytopenia, the pharmaceutical composition comprising, as an
active ingredient, a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0060] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating
pancytopenia, the pharmaceutical composition comprising, as an
active ingredient, a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0061] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating
granulocytopenia, the pharmaceutical composition comprising, as an
active ingredient, a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0062] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating
hyperlipidemia, the pharmaceutical composition comprising, as an
active ingredient, a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0063] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating
hypercholesterolemia, the pharmaceutical composition comprising, as
an active ingredient, a peptide derived from an N terminus portion
of oS1 casein and a pharmaceutically acceptable carrier.
[0064] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating glucosuria,
the pharmaceutical composition comprising, as an active ingredient,
a peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0065] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating diabetes,
the pharmaceutical composition comprising, as an active ingredient,
a peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0066] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating AIDS, the
pharmaceutical composition comprising, as an active ingredient, a
peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0067] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating infection
by HIV, the pharmaceutical composition comprising, as an active
ingredient, a peptide derived from an N terminus portion of oS1
casein and a pharmaceutically acceptable carrier.
[0068] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating conditions
associated with myeloablative doses of chemoradiotherapy supported
by autologous bone marrow or peripheral blood stem cell
transplantation (ASCT) or allogeneic bone marrow transplantation
(BMT), the pharmaceutical composition comprising, as an active
ingredient, a peptide derived from an N terminus portion of oS1
casein and a pharmaceutically acceptable carrier.
[0069] Further according to the present invention there is provided
a pharmaceutical composition for treating a thrombopoietin
treatable condition, the pharmaceutical composition comprising, as
an active ingredient a peptide derived from an N terminus portion
of oS1 casein and a pharmaceutically acceptable carrier.
[0070] Further according to the present invention there is provided
a pharmaceutical composition for augmenting the effect of
thrombopoietin, the pharmaceutical composition comprising, as an
active ingredient a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0071] Further according to the present invention there is provided
a pharmaceutical composition for enhancing peripheral stem cell
mobilization, the pharmaceutical composition comprising, as active
ingredients thrombopoietin and a peptide derived from an N terminus
portion of oS1 casein and a pharmaceutically acceptable
carrier.
[0072] Further according to the present invention there is provided
a pharmaceutical composition for enhancing hematopoiesis, the
pharmaceutical composition comprising, as active ingredients
thrombopoietin and a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0073] Further according to the present invention there is provided
a pharmaceutical composition for enhancing hematopoietic stem cell
proliferation, the pharmaceutical composition comprising, as active
ingredients thrombopoietin and a peptide derived from an N terminus
portion of oS1 casein and a pharmaceutically acceptable
carrier.
[0074] Further according to the present invention there is provided
a pharmaceutical composition for enhancing hematopoietic stem cell
proliferation and differentiation, the pharmaceutical composition
comprising, as active ingredients thrombopoietin and a peptide
derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0075] Further according to the present invention there is provided
a pharmaceutical composition for enhancing megakaryocytopoiesis,
the pharmaceutical composition comprising, as active ingredients
thrombopoietin and a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0076] Further according to the present invention there is provided
a pharmaceutical composition for enhancing erythropoiesis, the
pharmaceutical composition comprising, as active ingredients
thrombopoietin and a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0077] Further according to the present invention there is provided
a pharmaceutical composition for enhancing leukocytopoiesis, the
pharmaceutical composition comprising, as active ingredients
thrombopoietin and a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0078] Further according to the present invention there is provided
a pharmaceutical composition for enhancing thrombocytopoiesis, the
pharmaceutical composition comprising, as active ingredients
thrombopoietin and a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0079] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating
thrombocytopenia, the pharmaceutical composition comprising, as
active ingredients thrombopoietin and a peptide derived from an N
terminus portion of oS1 casein and a pharmaceutically acceptable
carrier.
[0080] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating
pancytopenia, the pharmaceutical composition comprising, as active
ingredients thrombopoietin and a peptide derived from an N terminus
portion of oS1 casein and a pharmaceutically acceptable
carrier.
[0081] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating
granulocytopenia, the pharmaceutical composition comprising, as
active ingredients thrombopoietin and a peptide derived from an N
terminus portion of oS1 casein and a pharmaceutically acceptable
carrier.
[0082] Further according to the present invention there is provided
a pharmaceutical composition for treating or preventing an
indication selected from the group consisting of autoimmune disease
or condition, viral disease, viral infection, hematological
disease, hematological deficiencies, thrombocytopenia,
pancytopenia, granulocytopenia, hyperlipidemia,
hypercholesterolemia, glucosuria, hyperglycemia, diabetes, A/DS,
infection with HIV-1, helper T-cell disorders, dendrite cell
deficiencies, macrophage deficiencies, hematopoietic stem cell
disorders including platelet, lymphocyte, plasma cell and
neutrophil disorders, pre-leukemic conditions, leukemic conditions,
immune system disorders resulting from chemotherapy or radiation
therapy, human immune system disorders resulting from treatment of
diseases of immune deficiency and bacterial infections, the
pharmaceutical composition comprising, as an active ingredient, a
peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0083] Further according to the present invention there is provided
a pharmaceutical composition for treating or preventing an
indication selected from the group consisting of hematological
disease, hematological deficiencies, thrombocytopenia,
pancytopenia, granulocytopenia, dendrite cell deficiencies,
macrophage deficiencies, hematopoietic stem cell disorders
including platelet, lymphocyte, plasma cell and neutrophil
disorders, pre-leukemic conditions, leukemic conditions,
myelodysplastic syndrome, aplastic anemia and bone marrow
insufficiency, the pharmaceutical composition comprising, as active
ingredients, thrombopoietin and a peptide derived from an N
terminus portion of oS1 casein and a pharmaceutically acceptable
carrier.
[0084] Further according to the present invention there is provided
a method of enhancing colonization of donated blood stem cells in a
myeloablated recipient, the method comprising treating a donor of
the donated blood stem cells with a peptide derived from an N
terminus portion of oS1 casein prior to donation and implanting the
donated blood stem cells in the recipient.
[0085] Further according to the present invention there is provided
a method of enhancing colonization of donated blood stem cells in a
myeloablated recipient, the method comprising treating the donated
blood stem cells with a peptide derived from an N terminus portion
of oS1 casein prior to implanting the donated blood stem cells in
the recipient.
[0086] Further according to the present invention there is provided
a method of enhancing colonization of blood stem cells in a
myeloablated recipient, the method comprising treating the blood
stem cells with a peptide derived from an N terminus portion of oS1
casein prior to implanting the blood stem cells in the
recipient.
[0087] Further according to the present invention there is provided
a method of enhancing colonization of donated blood stem cells in a
myeloablated recipient, the method comprising treating a donor of
the donated blood stem cells with a peptide derived from an N
terminus portion of oS1 casein and thrombopoietin prior to donation
and implanting the donated blood stem cells in the recipient.
[0088] Further according to the present invention there is provided
a method of enhancing colonization of donated blood stem cells in a
myeloablated recipient, the method comprising treating the donated
blood stem cells with a peptide derived from an N terminus portion
of oS1 casein and thrombopoietin prior to implanting the donated
blood stem cells in the recipient.
[0089] Further according to the present invention there is provided
a method of enhancing colonization of blood stem cells in a
myeloablated recipient, the method comprising treating the blood
stem cells with a peptide derived from an N terminus portion of oS1
casein and thrombopoietin prior to implanting the blood stem cells
in the recipient.
[0090] According to further features in preferred embodiments of
the invention described below, the peptide is a fragment derived by
fragmentation of oS1 casein.
[0091] According to still further features in the described
preferred embodiments the peptide is a synthetic peptide.
[0092] According to still further features in the described
preferred embodiments the peptide has a sequence as set forth in
one of SEQ ID NOs:1-25.
[0093] Further according to the present invention there is provided
a purified peptide having an amino acid sequence selected from the
group consisting of SEQ ID NOs:1-25.
[0094] Further according to the present invention there is provided
a pharmaceutical composition comprising a purified peptide having
an amino acid sequence selected from the group consisting of SEQ ID
NOs:1-25 and a pharmaceutically acceptable carrier.
[0095] Further according to the present invention there is provided
a pharmaceutical composition comprising thrombopoietin and a
purified peptide having an amino acid sequence selected from the
group consisting of SEQ ID NOs:1-25 and a pharmaceutically
acceptable carrier.
[0096] The present invention successfully addresses the
shortcomings of the presently known configurations by providing
peptides for the treatment of human disease, which peptides are
derived from the N terminus portion of o S1 casein and posses no
detectable toxicity and high therapeutic efficacy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0097] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the
invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0098] In the drawings:
[0099] FIG. 1 depicts the stimulation of Natural Killer (NK) cell
activity in cultured murine bone marrow cells by peptides derived
from natural casein. Lysis of .sup.35S labeled YAC target cells by
cultured murine bone marrow cells incubated in the presence or
absence of 100 .mu.g per ml peptides derived from natural casein is
expressed as the fraction of total radioactivity released from the
YAC cells into the culture supernatant (% Release .sup.35S). FIG. 1
represents NK activity at an effector:target cell ratio of 25:1 and
50:1.
[0100] FIGS. 2a and 2b depict the stimulation of Natural Killer
(NK) cell activity in cultured human Peripheral Blood Stem Cells
(PBSC) by peptides derived from natural casein. Lysis of .sup.35S
labeled K562 target cells by cultured human PBSC from Granulocyte
Colony Stimulating Factor (G-CSF) treated donors incubated without
(0 .mu.g) or with increasing concentrations (5-500 .mu.g per ml) of
peptides derived from natural casein is expressed as the fraction
of total radioactivity released from the K562 cells into the
culture supernatant (% Release .sup.35S). FIG. 2a represents NK
activity of two blood samples from the same patient, incubated at
different effector:target cell ratios (1:25 and 1:50). FIG. 2b
represents NK activity of blood samples from normal and affected
donors incubated at the same effector:target cell ratio. Squares
represent an effector:target cell ratio of 100:1, diamonds
represent an effector:target cell ratio of 50:1.
[0101] FIGS. 3a-c depict the stimulation of proliferation of
Natural Killer (NK) and T-lymphocyte (T) cells from cultured human
Peripheral Blood Stem Cells (PBSC) by peptides derived from natural
casein. NK and T cell proliferation in cultured PBSC from
Granulocyte Colony Stimulating Factor treated donors incubated with
or without peptides derived from natural casein is expressed as the
percentage (%) of cells binding the anti-CD.sub.3/FITC fluorescent
anti-T cell antibody UCHT.sub.1, or the anti CD.sub.56/RPE
fluorescent anti-NK cell antibody MOC-1 (DAKO A/S Denmark).
Controls are FITC and RPE-conjugated anti-mouse IgG antibody. FIG.
3a represents the percentage of cultured human PBSC binding
fluorescent antibody CD.sub.56 (5 independent samples) after 10
days incubation with (peptides) or without (control) 100 .mu.g per
ml peptides derived from natural casein. FIG. 3b represents the
percentage of cultured human PBSC cells binding fluorescent
anti-CD.sub.3 (T cell) antibody, following 14 days of incubation
with (peptides) or without (control) 100 .mu.g per ml peptides
derived from natural casein. FIG. 3c represents the percentage of
cultured human PBSC cells binding fluorescent anti-CD.sub.3 (T
cell) antibody and cells binding both CD.sub.3 and CD.sub.56 (T and
NK-like cells) antibodies after 28 days incubation with (peptides)
or without (control) 100 .mu.g per ml peptides derived from natural
casein.
[0102] FIG. 4 depicts the stimulation of Natural Killer (NK) cell
activity in cultured human Peripheral Blood Stem Cells (PBSC) by
synthetic peptides derived from casein. Lysis of .sup.35S labeled
K562 target cells by cultured human PBSC (from a breast cancer
patient) incubated without (0 .mu.g) or with increasing
concentrations (10-500 .mu.g per ml) of synthetic peptides derived
from casein is expressed as the fraction of total radioactivity
released from the K562 cells into the culture supernatant (%
Release). Peptides represent N-terminal sequences of 1-10 (1a,
diamonds), 1-11 (2a, squares) and 1-12 (3a, triangles) first amino
acids of the N terminus portion of oS1 casein (see Table 3 below
for sequences of synthetic peptides).
[0103] FIGS. 5a-c depict the stimulation of proliferation of
cultured human cells of diverse origin by peptides derived from
natural casein. Proliferation of the cultured human cells after
14-21 days incubation with increasing concentrations of the
peptides derived from natural casein is expressed as the amount of
[.sup.3H]-thymidine incorporated into each sample. FIG. 5a
represents the incorporation of label into two samples (PBSC 1,
squares, 15 days incubation; and PBSC 2, diamonds, 20 days
incubation) of human Peripheral Blood Stem Cells incubated with or
without (ctrl) 50-600 .mu.g per ml peptides derived from natural
casein. FIG. 5b represents the incorporation of [.sup.3H]-thymidine
into cultured human bone marrow cells after 21 days incubation with
or without (ctrl) 50-600 .mu.g per ml peptides derived from natural
casein. Bone marrow was donated by cancer patients in remission (BM
Auto, squares, BM 1, triangles, and BM 2,-?-) or healthy volunteers
(BM normal, diamons). FIG. 5c represents incorporation of
[.sup.3H]-thymidine into cultured human Cord Blood cells after 14
days incubation with or without (ctrl) 50-1000 .mu.g per ml
peptides derived from natural casein. Cord Blood cells were donated
by two separate donors (C. B. 1, triangles, C. B. 2, squares).
[0104] FIG. 6 shows a Table depicting the proliferation of blood
cell progenitors from human bone marrow and cord blood in response
to incubation with peptides derived from natural casein. The
relative cell number.times.10.sup.4 per ml, reflecting the
proliferation of cultured cells, was determined by counting cells
as described in the Examples section that follows. Bone marrow from
healthy volunteers (Bone Marrow) and Cord Blood from normal births
(Cord Blood) was incubated for 13 (Cord Blood) or 14 (Bone Marrow)
days in the presence of growth factors and AB serum, with or
without increasing concentrations of peptides derived from natural
casein (25-500 .mu.g).
[0105] FIG. 7 shows a table depicting the effect of in-vitro
incubation with Synthetic peptides derived from casein on the
relative distribution of Megakaryocyte, Erythroid, Plasma and
Dendritic cells (differential count) in CFU-GEMM colonies from
murine bone marrow progenitor cells. Cells were scored in the
macroscopic colonies grown from murine bone marrow cells prepared
similarly to the CFU-GEMM colonies previously described. Cells were
incubated with hematopoietic factors, and 25 .mu.g or more of
Synthetic peptides derived from casein for 14 days. The
differential count is expressed as the percentage of total cells
represented by individual cell types.
[0106] FIG. 8 depicts the stimulation of peripheral white blood
cell reconstitution in myeloablated, bone marrow transplanted mice
in response to treatment with peptides derived from natural casein.
Cell counts represent the number of white blood cells
(.times.10.sup.4 per ml, as counted in a haemocytometer). The mice
(n=6 per group) received lethal irradiation and syngeneic bone
marrow transplantation (10.sup.6 cells per mouse) on the following
day, and intravenous administration of 1 mg per recipient peptides
derived from natural casein (peptides: squares) or 1 mg per
recipient human serum albumin (CONTROL: diamonds) one day
later.
[0107] FIG. 9 depicts the stimulation of platelet reconstitution in
myeloablated, bone marrow transplanted mice in response to
treatment with peptides derived from natural casein. Platelet (PLT)
counts represent the number of thrombocytes (.times.10.sup.3 per
ml, as counted in a haemocytometer). The mice (n=60 per group)
received lethal irradiation and syngeneic bone marrow
transplantation (10.sup.6 cells per mouse) on day 1, and
intravenous administration of 1 mg per recipient peptides derived
from natural casein (Peptides, diamonds) or 1 mg per recipient
human serum albumin (control, squares).
[0108] FIGS. 10a-f depict the penetration and nuclear uptake of
FITC-conjugated peptides derived from natural casein in cultured
human T-lymphocyte cells, as recorded by fluorescent microscopy. F1
and F2 are identical fractions of the FITC-conjugated peptides
derived from natural casein. Sup-T.sub.1 cells were incubated with
100 .mu.g per ml FITC-conjugated peptides derived from natural
casein as described in the Examples section that follows. At the
indicated times, the cells were washed of free label, fixed in
formalin and prepared for viewing and recording by Laser Scanning
Confocal Microscopy. FIGS. 10a through 10f are selected images of
cells from consecutive incubation times, demonstrating
FITC-conjugated peptides derived from natural casein penetrating
the Sup-T.sub.1 cell membrane (FIGS. 10a, 10b) and concentrating in
the nucleus (FIGS. 10c-10f).
[0109] FIG. 11 shows a Table depicting the stimulation of Sup-T1
Lymphocyte cell proliferation in response to incubation with
peptides derived from natural casein. Sup-T1 cells (5000 per well)
were incubated with increasing concentrations (50-1000 .mu.g per
ml) of peptides derived from natural casein, counted in their wells
at the indicated times post culture and pulsed with
[.sup.3H]-thymidine for 18 hours. Proliferation index is the ratio
of the average of the incorporation of [.sup.3 H]-thymidine into
triplicate samples divided by the incorporation into cells cultured
without peptides derived from natural casein (control).
[0110] FIG. 12 shows a Table depicting inhibition of HIV-1
infection of CEM lymphocytes by peptides derived from natural
casein. CEM cells were preincubated with increasing concentrations
(50-1000 .mu.g per ml) of peptides derived from natural casein for
the indicated number of hours (3, 24 and 48 hours) before contact
with HIV-1 virus, as described in the Examples section that
follows. On day 15 post infection, cells were counted for cell
numbers and assayed for severity of HIV-1 infection by the P34
antigen assay, as described in the Examples section that follows.
Control cultures were IF: CEM cells contacted with HIV-1 virus
without pretreatment with peptides derived from natural casein, and
UIF: CEM cells cultured under identical conditions without peptides
derived from natural casein and without contact with HIV-1
virus.
[0111] FIG. 13 shows a Table depicting inhibition of HIV-1
infection of CEM lymphocytes by Synthetic peptides derived from
casein. CEM cells were preincubated with various concentrations
(10-250 .mu.g per ml) of peptides derived from natural casein (1P,
3P and 4P) for 3 hours before contact with HIV-1 virus, as
described in the Examples section that follows. On day 7 post
infection, cells were counted for cell numbers and assayed for
severity of HIV-1 infection by the P34 antigen assay, as described
in the Examples section that follows. Control cultures (IF) were
CEM cells contacted with HIV-1 virus without pretreatment with
peptides derived from natural casein.
[0112] FIG. 14 depicts the prevention by peptides derived from
natural casein of Juvenile (Type I, IDDM) Diabetes in female Non
Obese Diabetic mice.
[0113] Glucosuria was monitored at intervals during 365 days post
treatment in female NOD mice receiving a once (triangles) or twice
(squares) weekly injection of 100 .mu.g peptides derived from
natural casein for 5 weeks (6 or 11 injections total) and untreated
controls. All the controls developed glucosuria and subsequently
died.
[0114] FIG. 15 depicts the reduction by Synthetic peptides derived
from casein of diet-induced hypercholesterol/hyperlipidemia in
female C57 Black/6j mice. Total cholesterol (TC), High Density
(HDL) and Low Density Lipoproteins (LDL) were assayed in pooled
blood of two (2) mice per sample from
hypercholesterol/hyperlipidemic mice receiving (IP) casein-derived
peptides B, C, 2a or 3P, or no treatment (control). "Normal"
samples represent control mice not fed the atherogenic diet.
[0115] FIG. 16 shows a Table depicting the stimulation of
hematopoiesis in cancer patients in response to injections of
peptides derived from natural casein. Peripheral blood from five
female cancer patients either receiving or having received
chemotherapy, as described above, was counted for total White Blood
Cells (WBC, .times.10.sup.3), Platelets (PLT, .times.10.sup.3),
Erythrocytes (RBC, .times.10.sup.3) and Hemoglobin (gm per dl)
before (n) and after (n +. . . ) intramuscular injections with
peptides derived from natural casein, as described above. Patient 1
relates to G.T.; patient 2 relates to E.C.; patient 3 relates to
E.S.; patient 4 relates to J.R. and patient 5 relates to D.M.
[0116] FIG. 17 depicts the stimulation by peptides derived from
natural casein of thrombocytopoiesis in a platelet-resistant
patient with Acute Myeloid Leukemia (M-1). Thrombocyte
reconstitution was expressed as the change in platelet content of
peripheral blood (PLA, .times.10.sup.6 per ml), counted as
described above at the indicated intervals following intramuscular
injection (as described in the Examples section that follows) of
100 .mu.g peptides derived from natural casein.
[0117] FIG. 18 depicts the stimulation by peptides derived from
natural casein of thrombocytopoiesis in a platelet-resistant
patient with Acute Myeloid Leukemia (M-2). Thrombocyte
reconstitution was expressed as the change in platelet content of
peripheral blood (PLA, .times.10.sup.6per ml), counted as described
above at the indicated intervals following intramuscular injection
(as described in the Examples section that follows) of 100 .mu.g
peptides derived from natural casein.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0118] The present invention is of biologically active peptides
that are derived from or are similar to sequences identical with
the N-terminus of the oS1 fraction of milk casein, compositions
containing same and methods of utilizing same in, for example,
stimulating and enhancing immune response, protecting against viral
infection, normalizing serum cholesterol levels, and stimulating
hematopoiesis. The casein-derived peptides are non-toxic and can be
used to treat and prevent, for example, immune pathologies,
hypercholesterolemia, hematological disorders and viral-related
diseases.
[0119] The principles and operation of the present invention may be
better understood with reference to the drawings and accompanying
descriptions.
[0120] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details set forth in the following
description or exemplified by the Examples. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0121] As used herein, the term "treating" includes substantially
inhibiting, slowing or reversing the progression of a disease,
substantially ameliorating clinical symptoms of a disease.
[0122] As used herein, the term "preventing" includes substantially
preventing the appearance of clinical symptoms of a disease.
[0123] As used herein the term "peptide" includes native peptides
(either degradation products, synthetically synthesized peptides or
recombinant peptides) and peptido-mimetics (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. Such modifications include, but are not limited to,
cyclization, N terminus modification, C terminus modification,
peptide bond modification, including, but not limited to,
CH.sub.2--NH, CH.sub.2--S, CH.sub.2--S.dbd.O, O.dbd.C--NH,
CH.sub.2--O, CH.sub.2--CH.sub.2, S.dbd.C--NH, CH.dbd.CH or
CF.dbd.CH, backbone modification and residue modification. Methods
for preparing peptido-mimetic 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. Further
detail in this respect are provided hereinunder.
[0124] Thus, a peptide according to the present invention can be a
cyclic peptide. Cyclization can be obtained, for example, through
amide bond formation, e.g., by incorporating Glu, Asp, Lys, Orn,
di-amino butyric (Dab) acid, di-aminopropionic (Dap) acid at
various positions in the chain (--CO--NH or --NH--CO bonds).
Backbone to backbone cyclization can also be obtained through
incorporation of modified amino acids of the formulas
H--N((CH.sub.2).sub.n--COOH)--C(R)H--COOH or
H--N((CH.sub.2).sub.n--COOH)--C(R)H--NH.sub.2, wherein n 1=4, and
further wherein R is any natural or non-natural side chain of an
amino acid.
[0125] Cyclization via formation of S--S bonds through
incorporation of two Cys residues is also possible. Additional
side-chain to side chain cyclization can be obtained via formation
of an interaction bond of the formula
--(--CH.sub.2--).sub.n--S--CH.sub.2--C--, wherein n=1 or 2, which
is possible, for example, through incorporation of Cys or homoCys
and reaction of its free SH group with, e.g., bromoacetylated Lys,
Orn, Dab or Dap.
[0126] Peptide bonds (--CO--NH--) within the peptide may be
substituted, for example, by N-methylated bonds
(--N(CH.sub.3)--CO--), ester bonds (--C(R)H--C--O--O--C(R)--N--),
ketomethylen bonds (--CO--CH.sub.2--), o-aza bonds
(--NH--N(R)--CO--), wherein R is any alkyl, e.g., methyl, carba
bonds (--CH.sub.2--NH--), hydroxyethylene bonds
(--CH(OH)--CH.sub.2--), thioamide bonds (--CS--NH--), olefinic
double bonds (--CH.dbd.CH--), retro amide bonds (--NH--CO--),
peptide derivatives (--N(R)--CH.sub.2--CO--), wherein R is the
"normal" side chain, naturally presented on the carbon atom.
[0127] These modifications can occur at any of the bonds along the
peptide chain and even at several (2-3) at the same time.
[0128] Natural aromatic amino acids, Trp, Tyr and Phe, may be
substituted for synthetic non-natural acid such as TIC,
naphthylelanine (Nol), ring-methylated derivatives of Phe,
halogenated derivatives of Phe or o-methyl-Tyr.
[0129] Tables 1-2 below list all the naturally occurring amino
acids (Table 1) and non-conventional or modified amino acids (Table
2).
1 TABLE 1 Three-Letter One-Letter Amino Acid Abbreviation Symbol
Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D
Cysteine Cys C Glutamine Gln Q Glutamic Acid Glu E Glycine Gly G
Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K
Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S
Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Any
amino acid as above Xaa X
[0130]
2TABLE 2 Non-conventional amino acid Code Non-conventional amino
acid Code .alpha.-aminobutyric acid Abu L-N-methylalanine Nmala
.alpha.-amino-.alpha.-methylbutyrate Mgabu L-N-methylarginine Nmarg
aminocyclopropane- Cpro L-N-methylasparagine Nmasn Carboxylate
L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib
L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine
Nmgin carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine
Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen
L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine Nmleu
D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp
L-N-methylmethionineNmmet D-cysteine Dcys L-N-methylnorleucine
Nmnle D-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid
Dglu L-N-methylornithine Nmorn D-histidine Dhis
L-N-methylphenylalanine Nmphe D-isoleucine Dile L-N-methylproline
Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys
L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophan
Nmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine
Dphe L-N-methylvaline Nmval D-proline Dpro L-N-merhylethylglycine
Nmetg D-serine Dser L-N-methyl-t-butylglyci- ne Nmtbug D-threonine
Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine
Dtyr .alpha.-methyl-aminoisobutyra- te Maib D-valine Dval
.alpha.-methyl-.gamma.-aminobutyrate Mgabu D-.alpha.-methylalanine
Dmala .alpha.-methylcyclohexylalanine Mchexa
D-.alpha.-methylarginine Dmarg .alpha.-methylcyclopentylalanine
Mcpen D-.alpha.-methylasparagine Dmasn .alpha.-methyl-.alpha.-napt-
hylalanine Manap D-.alpha.-methylaspartate Dmasp
.alpha.-methylpenicillamine Mpen D-.alpha.-methylcysteine Dmcys
N-(4-aminobutyl)glycine Nglu D-.alpha.-methylglutamine Dmgln
N-(2-aminoethyl)glycine Naeg D-.alpha.-methylhistidine Dmhis
N-(3-aminopropyl)glycine Norn D-.alpha.-methylisoleucine Dmile
N-amino-.alpha.-methylbutyrate Nmaabu D-.alpha.-methylleucine Dmleu
.alpha.-napthylalanine Anap D-.alpha.-methyllysine Dmlys
N-benzylglycine Nphe D-.alpha.-methyhnethionine Dmmet
N-(2-carbamylethyl)glycine Ngln D-.alpha.-methylornithine Dmorn
N-(carbamylmethyl)glycine Nasn D-.alpha.-methylphenylalanine Dmphe
N-(2-carboxyethyl)glycine Nglu D-.alpha.-methylproline Dmpro
N-(carboxymethyl)glycine Nasp D-.alpha.-methylserine Dmser
N-cyclobutylglycine Ncbut D-.alpha.-methylthreonine Dmthr
N-cycloheptylglycine Nchep D-.alpha.-methyltryptophan Dmtrp
N-cyclohexylglycine Nchex D-.alpha.-methyltyrosine Dmty
N-cyclodecylglycine Ncdec D-.alpha.-methylvaline Dmval
N-cyclododeclglycine Ncdod D-.alpha.-methylalnine Dnmala
N-cyclooctylglycine Ncoct D-.alpha.-methylarginine Dnmarg
N-cyclopropylglycine Ncpro D-.alpha.-methylasparagine Dnmasn
N-cycloundecylglycine Ncund D-.alpha.-methylasparatate Dnmasp
N-(2,2-diphenylethyl)glycine Nbhm D-.alpha.-methylcysteine Dnmcys
N-(3,3-diphenylpropyl)glycine Nbhe D-N-methylleucine Dnmleu
N-(3-indolylyethyl) glycine Nhtrp D-N-methyllysine Dnmlys
N-methyl-.gamma.-aminobutyrate Nmgabu N-methylcyclohexylalanine
Nmchexa D-N-methylmethionine Dnmmet D-N-methylornithine Dnmorn
N-methylcyclopentylalanine Nmcpen N-methylglycine Nala
D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib
D-N-methylproline Dnmpro N-(1-methylpropyl)glycine Nile
D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nile
D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nleu
D-N-methylthreonine Dnmthr D-N-methyltryptophan Dnmtrp
N-(1-methylethyl)glycine Nva D-N-methyltyrosine Dnmtyr
N-methyla-napthylalanine Nmanap D-N-methylvaline Dnmval
N-methylpenicillamine Nmpen .gamma.-aminobutyric acid Gabu
N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine Tbug
N-(thiomethyl)glycine Ncys L-ethylglycine Etg Penicillamine Pen
L-homophenylalanine Hphe L-.alpha.-methylalanine Mala
L-.alpha.-methylarginine Marg L-.alpha.-methylasparagine Masn
L-.alpha.-methylaspartate Masp L-.alpha.-methyl-t-butylglycine
Mtbug L-.alpha.-methylcysteine Mcys L-methylethylglycine Metg
L-.alpha.-methylglutamine Mgln L-.alpha.-methylglutamate Mglu
L-.alpha.-methylhistidine Mhis L-.alpha.-methylhomo phenylalanine
Mhphe L-.alpha.-methylisoleucine Mile N-(2-methylthioethyl)glycine
Nmet D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg
D-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine Nthr
D-N-methylhistidine Dnmhis N-(hydroxyethyl)glycine Nser
D-N-methylisoleucine Dnmile N-(imidazolylethyl)glycine Nhis
D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp
D-N-methyllysine Dnmlys N-methyl-.gamma.-aminobutyrate Nmgabu
N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet
D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen
N-methylglycine Nala D-N-methylphenylalanine Dnmphe
N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro
N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr
D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nval
D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen
.gamma.-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr
L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg
Penicillamine Pen L-homophenylalanine Hphe L-.alpha.-methylalanine
Mala L-.alpha.-methylarginine Marg L-.alpha.-methylasparagine Masn
L-.alpha.-methylaspartate Masp L-.alpha.-methyl-t-butylglycine
Mtbug L-.alpha.-methylcysteine Mcys L-methylethylglycine Metg
L-.alpha.-methylglutamine Mgln L-.alpha.-methylglutamate Mglu
L-.alpha.-methylhistidine Mhis L-.alpha.-methylhomophenylalanine
Mhphe L-.alpha.-methylisoleucine Mile N-(2-methylthioethyl)glycine
Nmet L-.alpha.-methylleucine Mleu L-.alpha.-methyllysine Mlys
L-.alpha.-methylmethionine Mmmet L-.alpha.-methylnorleucine Mnle
L-.alpha.-methylnorvaline Mnva L-.alpha.-methylornithine Morn
L-.alpha.-methylphenylalanine Mphe L-.alpha.-methylproline Mpro
L-.alpha.-methylserine Mser L-.alpha.-methylthreonine Mthr
L-.alpha.-methylvaline Mtrp L-.alpha.-methyltyrosine Mtyr
L-.alpha.-methylleucine Mval L-N-methylhomophenylalanine Nmhphe
Nnbhm N-(N-(2,2-diphenylethyl) N-(N-(3,3-diphenylpropyl)
carbamylmethyl-glycine Nnbhm carbamylmethyl(1)glycine Nnbhe
1-carboxy-1-(2,2-diphenyl Nmbc ethylamino)cyclopropane
[0131] A peptide according to the present invention can be used in
a self standing form or be a part of moieties such as proteins and
display moieties such as display bacteria and phages. The peptides
of the invention can also be chemically modified to give active
dimers or multimers, in one polypeptide chain or covalently
crosslinked chains.
[0132] Additionally, a peptide according to the present invention
includes at least two, optionally at least three, optionally at
least four, optionally at least five, optionally at least six,
optionally at least seven, optionally at least eight, optionally at
least nine, optionally at least ten, optionally at least eleven,
optionally at least twelve, optionally at least thirteen,
optionally at least fourteen, optionally at least fifteen,
optionally at least sixteen, optionally at least seventeen,
optionally at least eighteen, optionally at least nineteen,
optionally at least twenty, optionally at least twenty-one,
optionally at least twenty-two, optionally at least twenty-three,
optionally at least twenty-four, optionally at least twenty-five,
optionally at least twenty-six, optionally between twenty-seven and
sixty, or more amino acid residues (also referred to herein
interchangeably as amino acids).
[0133] Accordingly, as used herein 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.
[0134] As used herein the phrase "derived from an N terminus
portion of oS1 casein" refers to peptides as this term is defined
herein, e.g., cleavage products of oS1 casein (referred to herein
as peptides derived from natural casein), synthetic peptides
chemically synthesized to correspond to the amino acid sequence of
an N terminus portion of oS1 casein (referred to herein as
synthetic peptides derived from casein), peptides similar
(homologous) to an N terminus portion of oS1 casein, for example,
peptides characterized by one or more amino acid substitutions,
such as, but not limited to, permissible substitutions, provided
that at least 70%, preferably at least 80%, more preferably at
least 90% similarity is maintained, and functional homologues
thereof. The terms "homologues" and "functional homologues" as used
herein mean peptides with any insertions, deletions and
substitutions which do not affect the biological activity of the
peptide.
[0135] As used herein the term "oS1 casein" refers to oS1 casein of
a mammal, including, but not limited to, livestock mammals (e.g.,
cow, sheep, goat, mare, camel, deer and buffalo) human beings and
marine mammals. The following provides a list of oS1 caseins having
a known amino acid sequence, identified by their GenBank (NCBI)
Accession Nos. and source: CAA26982 (Ovis aries (sheep)), CAA51022
(Capra hircus (goat)), CAA42516 (Bos taurus (bovine)), CAA55185
(Homo sapiens), CAA38717 (Sus scrofa (pig)), P09115 (rabbit) and
097943 (Camelus dromedurius (camel)).
[0136] As used herein the term "N terminus portion" refers to M
amino acids of oS1 casein derived from the first 60 amino acids of
oS1 casein, wherein M is any of the integers between 2 and 60
(including the integers 2 and 60). Preferably, the term refers to
the first M amino acids of oS1 casein.
[0137] The peptides of the invention can be obtained by extraction
from milk as previously described, or by solid phase peptide
synthesis, which is a standard method known to the man skilled in
the art. Purification of the peptides of the invention is performed
by standard techniques, known to the man skilled in the art, such
as high performance liquid chromatography (HPLC). Milk casein
fragmentation to obtain the peptides of the invention may be
effected using various enzymatic and/or chemical means.
[0138] As is further detailed hereinunder and exemplified in the
Examples section that follows, the peptides of the present
invention have a variety of therapeutic effects. In the Examples
section there are provided numerous assays with which one of
ordinary skills in the art can test a specific peptide designed in
accordance with the teachings of the present invention for a
specific therapeutic effect.
[0139] Any of the peptides described herein can be administered per
se or be formulated into a pharmaceutical composition which can be
used for treating or preventing a disease. Such a composition
includes as an active ingredient any of the peptides described
herein and a pharmaceutically acceptable carrier.
[0140] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the peptides described herein, with
other chemical components such as pharmaceutically suitable
carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration of a compound to an
organism.
[0141] Hereinafter, the term "pharmaceutically acceptable carrier"
refers to a carrier or a diluent that does not cause significant
irritation to an organism and does not abrogate the biological
activity and properties of the administered compound. Examples,
without limitations, of carriers are:
[0142] propylene glycol, saline, emulsions and mixtures of organic
solvents with water. Herein the term "excipient" refers to an inert
substance added to a pharmaceutical composition to further
facilitate administration of a compound. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0143] Techniques for formulation and administration of drugs may
be found in "Remington's Pharmaceutical Sciences," Mack Publishing
Co., Easton, Pa., latest edition.
[0144] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, transdermal, intestinal or parenteral
delivery, including intramuscular, subcutaneous and intramedullary
injections as well as intrathecal, direct intraventricular,
intravenous, intraperitoneal, intranasal, or intraocular
injections.
[0145] Pharmaceutical compositions of the present invention may be
manufactured by processes well known in the art, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0146] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in conventional manner
using one or more pharmaceutically acceptable carriers comprising
excipients and auxiliaries, which facilitate processing of the
active peptides into preparations which, can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0147] For injection, the peptides of the invention may be
formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hank's solution, Ringer's solution, or
physiological saline buffer with or without organic solvents such
as propylene glycol, polyethylene glycol. For transmucosal
administration, penetrants are used in the formulation. Such
penetrants are generally known in the art.
[0148] For oral administration, the peptides can be formulated
readily by combining the active peptides with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
peptides of the invention to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions,
and the like, for oral ingestion by a patient. Pharmacological
preparations for oral use can be made using a solid excipient,
optionally grinding the resulting mixture, and processing the
mixture of granules, after adding suitable auxiliaries if desired,
to obtain tablets or dragee cores. Suitable excipients are, in
particular, fillers such as sugars, including lactose, sucrose,
mannitol, or sorbitol; cellulose preparations such as, for example,
maize starch, wheat starch, rice starch, potato starch, gelatin,
gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose,
sodium carbomethylcellulose; and/or physiologically acceptable
polymers such as polyvinylpyrrolidone (PVP). If desired,
disintegrating agents may be added, such as cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate.
[0149] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, titanium dioxide, lacquer
solutions and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active ingredient doses.
[0150] Pharmaceutical compositions, which can be used orally,
include push-fit capsules made of gelatin as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. The push-fit capsules may contain the active ingredients
in admixture with filler such as lactose, binders such as starches,
lubricants such as talc or magnesium stearate and, optionally,
stabilizers. In soft capsules, the active peptides may be dissolved
or suspended in suitable liquids, such as fatty oils, liquid
paraffin, or liquid polyethylene glycols. In addition, stabilizers
may be added. All formulations for oral administration should be in
dosages suitable for the chosen route of administration.
[0151] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0152] For administration by inhalation, the peptides according to
the present invention are conveniently delivered in the form of an
aerosol spray presentation from a pressurized pack or a nebulizer
with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichloro-tetrafluoroethane or carbon dioxide. In the case of a
pressurized aerosol, the dosage unit may be determined by providing
a valve to deliver a metered amount. Capsules and cartridges of,
e.g., gelatin for use in an inhaler or insufflator may be
formulated containing a powder mix of the compound and a suitable
powder base such as lactose or starch.
[0153] The peptides described herein may be formulated for
parenteral administration, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multidose containers with
optionally, an added preservative. The compositions may be
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0154] Pharmaceutical compositions for parenteral administration
include aqueous solutions of the active preparation in
water-soluble form. Additionally, suspensions of the active
peptides may be prepared as appropriate oily injection suspensions.
Suitable lipophilic solvents or vehicles include fatty oils such as
sesame oil, or synthetic fatty acids esters such as ethyl oleate,
triglycerides or liposomes. Aqueous injection suspensions may
contain substances, which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the peptides to allow for
the preparation of highly concentrated solutions.
[0155] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water, before use.
[0156] The peptides of the present invention may also be formulated
in rectal compositions such as suppositories or retention enemas,
using, e.g., conventional suppository bases such as cocoa butter or
other glycerides.
[0157] The pharmaceutical compositions herein described may also
comprise suitable solid of gel phase carriers or excipients.
Examples of such carriers or excipients include, but are not
limited to, calcium carbonate, calcium phosphate, various sugars,
starches, cellulose derivatives, gelatin and polymers such as
polyethylene glycols.
[0158] Persons ordinarily skilled in the art can easily determine
optimum dosages and dosing methodology for any of the peptides of
the invention.
[0159] For any peptide used in accordance with the teachings of the
present invention, a therapeutically effective amount, also
referred to as a therapeutically effective dose, which can be
estimated initially from cell culture assays or in vivo animal
assays. For example, a dose can be formulated in animal models to
achieve a circulating concentration range that includes the
IC.sub.50 or the IC.sub.100 as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Initial dosages can also be estimated from in vivo data.
Using these initial guidelines one having ordinary skill in the art
could determine an effective dosage in humans.
[0160] Moreover, toxicity and therapeutic efficacy of the peptides
described herein can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., by
determining the LD.sub.50 and the ED.sub.50. The dose ratio between
toxic and therapeutic effect is the therapeutic index and can be
expressed as the ratio between LD.sub.50 and ED.sub.50. Peptides
which exhibit high therapeutic indices are preferred. The data
obtained from these cell cultures assays and animal studies can be
used in formulating a dosage range that is not toxic for use in
human. The dosage of such peptides lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. The exact formulation, route of
administration and dosage can be chosen by the individual physician
in view of the patient's condition (see, e.g., Fingl et al., 1975,
In: The Pharmacological Basis of Therapeutics, chapter 1, page
1).
[0161] Dosage amount and interval may be adjusted individually to
provide plasma levels of the active ingredient which are sufficient
to maintain therapeutic effect. Usual patient dosages for oral
administration range from about 50-2000 mg/kg/administration,
commonly from about 100-1000 mg/kg/administration, preferably from
about 150-700 mg/kg/administration and most preferably from about
250-500 mg/kg/administration. In some cases, therapeutically
effective serum levels will be achieved by administering multiple
doses each day. In cases of local administration or selective
uptake, the effective local concentration of the drug may not be
related to plasma concentration. One having skill in the art will
be able to optimize therapeutically effective local dosages without
undue experimentation.
[0162] Depending on the severity and responsiveness of the
condition to be treated, dosing can also be a single administration
of a slow release composition, with course of treatment lasting
from several days to several weeks or until cure is effected or
diminution of the disease state is achieved.
[0163] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc. Compositions of the present invention
may, if desired, be presented in a pack or dispenser device, such
as FDA approved kit, which may contain one or more unit dosage
forms containing the active ingredient. The pack may, for example,
comprise metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration. The pack or dispenser may also be accompanied by a
notice associated with the container in a form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals, which notice is reflective of approval by the
agency of the form of the compositions or human or veterinary
administration. Such notice, for example, may be of labeling
approved by the U.S. Food and Drug Administration for prescription
drugs or of an approved product insert. Compositions comprising a
peptide of the invention formulated in a compatible pharmaceutical
carrier may also be prepared, placed in an appropriate container,
and labeled for treatment or prevention of an indicated condition
or induction of a desired event. Suitable indica on the label may
include treatment and/or prevention of an autoimmune disease or
condition, viral disease, viral infection, bacterial infection,
hematological disease, hematological deficiencies,
thrombocytopenia, pancytopenia, granulocytopenia, hyperlipidemia,
hypercholesterolemia, glucosuria, hyperglycemia, diabetes, AIDS,
infection with HIV-1, helper T-cell disorders, dendrite cell
deficiencies, macrophage deficiencies, hematopoietic stem cell
disorders including platelet, lymphocyte, plasma cell and
neutrophil disorders, hematopoietic stem cell proliferation,
hematopoietic stem cell proliferation and differentiation,
pre-leukemic conditions, leukemic conditions, immune system
disorders resulting from chemotherapy or radiation therapy, and
human immune system disorders resulting from treatment of diseases
of immune deficiency.
[0164] The pharmaceutical compositions according to the invention
may be useful in maintaining and/or restoring blood system
constituents, in balancing blood cell counts, in balancing levels
of metabolites in the blood including sugar, cholesterol, calcium,
uric acid, urea and enzymes such as alkaline phosphatase. Further,
the pharmaceutical compositions of the invention may be useful in
inducing blood cell proliferation, modulating white and/or red
blood cell counts, particularly increasing white and/or red blood
cell counts, elevating haemoglobin blood level and in modulating
platelet counts.
[0165] The term "balancing" as used herein with relation to levels
of certain physiological parameters, means changing the levels of
referred parameters and bringing them closer to normal values.
[0166] The term "normal values" as used herein with relation to
physiological parameters, means values which are in the range of
values of healthy humans or animals.
[0167] In specifically preferred embodiments, the peptides of the
invention balance counts of red blood cells, white blood cells,
platelets and haemoglobin level. The pharmaceutical compositions of
the invention may be used for activating blood cell
proliferation.
[0168] In addition, the pharmaceutical compositions may be used for
the treatment and/or prevention of hemopoietic stem cell disorders,
including platelet, lymphocyte, plasma cell and neutrophil
disorders, as well as deficiency and malfunction in pre-leukemic
and leukemic conditions and thrombocytopenia.
[0169] Further, the pharmaceutical compositions may be used for the
treatment and/or prevention of cell proliferative diseases. In this
connection, it is worth noting that the pharmaceutical compositions
of the invention are advantageous in the stimulation of the immune
response during chemotherapy or radiation treatments, in
alleviating the negative effects, reducing chemotherapy and
irradiation-induced vomiting and promoting a faster recovery.
[0170] Still further, the pharmaceutical compositions of the
invention may be used for the stimulation of human immune response
during treatment of diseases associated with immune deficiency, for
example HIV and autoimmune diseases.
[0171] The compositions of the invention may also be intended for
veterinary use.
[0172] The pharmaceutical compositions of the invention may be used
in the treatment and/or prevention of, for example, disorders
involves abnormal levels of blood cells, disorders involving
hemopoietic stem cells production and differentiation, treatment of
platelet, lymphocyte and/or neutrophil disorders, for the treatment
of pre-leukemic and leukemic conditions and for the treatment of
thrombocytopenia. The pharmaceutical compositions of the invention
may also be used in the treatment of cell proliferative diseases
and diseases involving immune deficiency, such as HIV, and of
autoimmune diseases. Further, the pharmaceutical compositions of
the invention may be used for modulating the immune response during
chemotherapy or radiation treatments, for example for reducing
chemotherapy-associated vomiting.
[0173] While reducing the present invention to practice, it was
surprisingly observed that the peptides of the invention exert a
synergistic effect on human hematopoietic stem cell proliferation
and differentiation with addition of other hematopoietic growth
factors. Of notable significance was the dose-dependent enhancement
of thrombopoietin (TPO) induction of megakaryocyte proliferation by
peptides of the present invention. Thrombopoietin is an early
acting cytokine with important multilineage effects: TPO alone, or
in combination with other early acting cytokines, can (i) promote
viability and suppress apoptosis in progenitor cells; (ii) regulate
hematopoietic stem cell production and function; (iii) trigger cell
division of dormant multipotent cells; (iv) induce multilineage
differentiation and (v) enhance formation of multilineage colonies
containing granulocytes, erythrocytes, macrophages, and
megakaryocytes (MK, CFU-GEMM). Moreover, TPO stimulates the
production of more limited progenitors for granulocyte/monocyte,
megakaryocyte and erythroid colonies, stimulates adhesion of
primitive human bone marrow and megakaryocytic cells to fibronectin
and fibrinogen. Thus, TPO is an important cytokine for clinical
hematologists/transplante- rs: for the mobilization, amplification
and ex vivo expansion of stem cells and committed precursor cells
for autologous and allogeneic transplantation. In addition,
administration of TPO to healthy platelet donors has been employed
to enhance pheresis yields. However, clinical application of TPO
therapy is complicated by, among other considerations, relatively
high costs of the recombinant human cytokine rhTPO, and the
potential antigenicity of TPO with repeated administration.
[0174] Combined treatment with TPO and the peptide of the present
invention, either together in a pharmaceutical composition
comprising both, or separately, can provide inexpensive, proven
non-toxic augmentation of TPOs effects on target cell proliferation
and function. In such a combination, the peptide of the present
invention may be applied to the treatment of, in addition to the
abovementioned conditions, disorders such as myelodysplastic
syndrome (MDS), aplastic anemia and complications of liver failure.
Pre-treatment of platelet donors with the peptide of the present
invention, alone or in combination with TPO, may even further
enhance the efficiency of pheresis yields.
[0175] Thus, according to the present invention there is provided a
method of treating a thrombopoietin treatable condition, the method
effected by administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0176] Further according to the present invention there is provided
a method of augmenting the effect of thrombopoietin, the method
effected by administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0177] Further according to the present invention there is provided
a method of enhancing peripheral stem cell mobilization, the method
effected by administering to a subject in need thereof an effective
amount of a pharmaceutical composition comprising effective amounts
of thrombopoietin and a peptide derived from an N terminus portion
of a SI casein.
[0178] Further according to the present invention there is provided
a pharmaceutical composition for treating a thrombopoietin
treatable condition, the pharmaceutical composition comprising, as
an active ingredient a peptide derived from an N terminus portion
of oS1 casein and a pharmaceutically acceptable carrier.
[0179] Further according to the present invention there is provided
a pharmaceutical composition for augmenting the effect of
thrombopoietin, the pharmaceutical composition comprising, as an
active ingredient a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0180] Further according to the present invention there is provided
a pharmaceutical composition for enhancing peripheral stem cell
mobilization, the pharmaceutical composition comprising, as active
ingredients thrombopoietin and a peptide derived from an N terminus
portion of a SI casein and a pharmaceutically acceptable
carrier.
[0181] Further according to the present invention there is provided
a pharmaceutical composition for inducing hematopoiesis, the
pharmaceutical composition comprising, as active ingredients,
thrombopoietin and a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0182] Further according to the present invention there is provided
a pharmaceutical composition for inducing hematopoietic stem cells
proliferation, the pharmaceutical composition comprising, as active
ingredients, thrombopoietin and a peptide derived from an N
terminus portion of oS1 casein and a pharmaceutically acceptable
carrier.
[0183] Further according to the present invention there is provided
a pharmaceutical composition for inducing hematopoietic stem cells
proliferation and differentiation, the pharmaceutical composition
comprising, as active ingredients, thrombopoietin and a peptide
derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0184] Further according to the present invention there is provided
a pharmaceutical composition for inducing megakaryocytopoiesis, the
pharmaceutical composition comprising, as active ingredients,
thrombopoietin and a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0185] Further according to the present invention there is provided
a pharmaceutical composition for inducing erythropoiesis, the
pharmaceutical composition comprising, as active ingredients,
thrombopoietin and a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0186] Further according to the present invention there is provided
a pharmaceutical composition for inducing leukocytopoiesis, the
pharmaceutical composition comprising, as active ingredients,
thrombopoietin and a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0187] Further according to the present invention there is provided
a pharmaceutical composition for inducing thrombocytopoiesis, the
pharmaceutical composition comprising, as active ingredients,
thrombopoietin and a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0188] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating
thrombocytopenia, the pharmaceutical composition comprising, as
active ingredients, thrombopoietin and a peptide derived from an N
terminus portion of oS1 casein and a pharmaceutically acceptable
carrier.
[0189] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating
pancytopenia, the pharmaceutical composition comprising, as active
ingredients, tbrombopoietin and a peptide derived from an N
terminus portion of oS1 casein and a pharmaceutically acceptable
carrier.
[0190] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating
granulocytopenia, the pharmaceutical composition comprising, as
active ingredients, thrombopoietin and a peptide derived from an N
terminus portion of oS1 casein and a pharmaceutically acceptable
carrier.
[0191] Further according to the present invention there is provided
a pharmaceutical composition for treating or preventing an
indication selected from the group consisting of hematological
disease, hematological deficiencies, thrombocytopenia,
pancytopenia, granulocytopenia, dendrite cell deficiencies,
macrophage deficiencies, hematopoietic stem cell disorders
including platelet, lymphocyte, plasma cell and neutrophil
disorders, pre-leukemic conditions, leukemic conditions,
myelodysplastic syndrome, aplastic anemia and bone marrow
insufficiency, the pharmaceutical composition comprising, as active
ingredients, thrombopoietin and a peptide derived from an N
terminus portion of oS1 casein and a pharmaceutically acceptable
carrier.
[0192] Further according to the present invention there is provided
a pharmaceutical composition comprising thrombopoietin and a
purified peptide having an amino acid sequence selected from the
group consisting of SEQ ID NOs: 1-25 and a pharmaceutically
acceptable carrier.
[0193] Further according to the present invention there is provided
a method of enhancing colonization of donated blood stem cells in a
myeloablated recipient, the method effected by treating a donor of
the donated blood stem cells with a peptide derived from an N
terminus portion of oS1 casein and thrombopoietin prior to donation
and implanting the donated blood stem cells in the recipient.
[0194] Further according to the present invention there is provided
a method of enhancing colonization of donated blood stem cells in a
myeloablated recipient, the method effected by treating the donated
blood stem cells with a peptide derived from an N terminus portion
of oS1 casein and thrombopoietin prior to implanting the donated
blood stem cells in the recipient.
[0195] Further according to the present invention there is provided
a method of enhancing colonization of blood stem cells in a
myeloablated recipient, the method comprising treating the blood
stem cells with a peptide derived from an N terminus portion of oS1
casein and thrombopoietin prior to implanting the blood stem cells
in the recipient.
[0196] The invention further relates to anti-bacterial
pharmaceutical compositions comprising as active ingredient at
least one peptide of the invention and to the use of the peptides
of the invention as anti-bacterial agents.
[0197] Thus, according to the present invention there is provided a
method of preventing or treating an autoimmune disease, the method
is effected by administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0198] Further according to the present invention there is provided
a method of preventing or treating a viral disease, the method is
effected by administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0199] Further according to the present invention there is provided
a method of preventing viral infection, the method is effected by
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
[0200] Further according to the present invention there is provided
a method of inducing hematopoiesis, the method is effected by
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
[0201] Further according to the present invention there is provided
a method of inducing hematopoietic stem cells proliferation, the
method is effected by administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0202] Further according to the present invention there is provided
a method of inducing hematopoietic stem cells proliferation and
differentiation, the method is effected by administering to a
subject in need thereof a therapeutically effective amount of a
peptide derived from an N terminus portion of oS1 casein.
[0203] Further according to the present invention there is provided
a method of inducing megakaryocytopoiesis, the method is effected
by administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
[0204] Further according to the present invention there is provided
a method of inducing erythropoiesis, the method is effected by
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
[0205] Further according to the present invention there is provided
a method of inducing leukocytopoiesis, the method is effected by
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
[0206] Further according to the present invention there is provided
a method of inducing thrombocytopoiesis, the method is effected by
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
[0207] Further according to the present invention there is provided
a method of inducing plasma cell proliferation, the method is
effected by administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0208] Further according to the present invention there is provided
a method of inducing dendritic cell proliferation, the method is
effected by administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0209] Further according to the present invention there is provided
a method of inducing macrophage cell proliferation, the method is
effected by administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0210] Further according to the present invention there is provided
a method of preventing or treating thrombocytopenia, the method is
effected by administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0211] Further according to the present invention there is provided
a method of preventing or treating pancytopenia, the method is
effected by administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0212] Further according to the present invention there is provided
a method of preventing or treating granulocytopenia, the method is
effected by administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0213] Further according to the present invention there is provided
a method of preventing or treating hyperlipidemia, the method is
effected by administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0214] Further according to the present invention there is provided
a method of preventing or treating hypercholesterolemia, the method
is effected by administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0215] Further according to the present invention there is provided
a method of preventing or treating glucosuria, the method is
effected by administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0216] Further according to the present invention there is provided
a method of preventing or treating diabetes, the method is effected
by administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
[0217] Further according to the present invention there is provided
a method of preventing or treating AIDS, the method is effected by
administering to a subject in need thereof a therapeutically
effective amount of a peptide derived from an N terminus portion of
oS1 casein.
[0218] Further according to the present invention there is provided
a method of preventing or treating infection by HIV, the method is
effected by administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0219] Further according to the present invention there is provided
a method of preventing or treating conditions associated with
myeloablative doses of chemoradiotherapy supported by autologous
bone marrow or peripheral blood stem cell transplantation (ASCT) or
allogeneic bone marrow transplantation (BMT), the method is
effected by administering to a subject in need thereof a
therapeutically effective amount of a peptide derived from an N
terminus portion of oS1 casein.
[0220] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating an
autoimmune disease, the pharmaceutical composition comprising, as
an active ingredient, a peptide derived from an N terminus portion
of oS1 casein and a pharmaceutically acceptable carrier.
[0221] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating a viral
disease, the pharmaceutical composition comprising, as an active
ingredient, a peptide derived from an N terminus portion of oS1
casein and a pharmaceutically acceptable carrier.
[0222] Further according to the present invention there is provided
a pharmaceutical composition for preventing viral infection, the
pharmaceutical composition comprising, as an active ingredient, a
peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0223] Further according to the present invention there is provided
a pharmaceutical composition for inducing hematopoiesis, the
pharmaceutical composition comprising, as an active ingredient, a
peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0224] Further according to the present invention there is provided
a pharmaceutical composition for inducing hematopoietic stem cells
proliferation, the pharmaceutical composition comprising, as an
active ingredient, a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0225] Further according to the present invention there is provided
a pharmaceutical composition for inducing hematopoietic stem cells
proliferation and differentiation, the pharmaceutical composition
comprising, as an active ingredient, a peptide derived from an N
terminus portion of oS1 casein and a pharmaceutically acceptable
carrier.
[0226] Further according to the present invention there is provided
a pharmaceutical composition for inducing megakaryocytopoiesis, the
pharmaceutical composition comprising, as an active ingredient, a
peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0227] Further according to the present invention there is provided
a pharmaceutical composition for inducing erythropoiesis, the
pharmaceutical composition comprising, as an active ingredient, a
peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0228] Further according to the present invention there is provided
a pharmaceutical composition for inducing leukocytopoiesis, the
pharmaceutical composition comprising, as an active ingredient, a
peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0229] Further according to the present invention there is provided
a pharmaceutical composition for inducing thrombocytopoiesis, the
pharmaceutical composition comprising, as an active ingredient, a
peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0230] Further according to the present invention there is provided
a pharmaceutical composition for inducing plasma cell
proliferation, the pharmaceutical composition comprising, as an
active ingredient a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0231] Further according to the present invention there is provided
a pharmaceutical composition for inducing dendritic cell
proliferation, the pharmaceutical composition comprising, as an
active ingredient a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0232] Further according to the present invention there is provided
a pharmaceutical composition for inducing macrophage proliferation,
the pharmaceutical composition comprising, as an active ingredient
a peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0233] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating
thrombocytopenia, the pharmaceutical composition comprising, as an
active ingredient, a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0234] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating
pancytopenia, the pharmaceutical composition comprising, as an
active ingredient, a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0235] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating
granulocytopenia, the pharmaceutical composition comprising, as an
active ingredient, a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0236] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating
hyperlipidemia, the pharmaceutical composition comprising, as an
active ingredient, a peptide derived from an N terminus portion of
oS1 casein and a pharmaceutically acceptable carrier.
[0237] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating
hypercholesterolemia, the pharmaceutical composition comprising, as
an active ingredient, a peptide derived from an N terminus portion
of oS1 casein and a pharmaceutically acceptable carrier.
[0238] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating glucosuria,
the pharmaceutical composition comprising, as an active ingredient,
a peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0239] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating diabetes,
the pharmaceutical composition comprising, as an active ingredient,
a peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0240] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating AIDS, the
pharmaceutical composition comprising, as an active ingredient, a
peptide derived from an N terminus portion of oS1 casein and a
pharmaceutically acceptable carrier.
[0241] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating infection
by HIV, the pharmaceutical composition comprising, as an active
ingredient, a peptide derived from an N terminus portion of oS1
casein and a pharmaceutically acceptable carrier.
[0242] Further according to the present invention there is provided
a pharmaceutical composition for preventing or treating conditions
associated with myeloablative doses of chemoradiotherapy supported
by autologous bone marrow or peripheral blood stem cell
transplantation (ASCT) or allogeneic bone marrow transplantation
(BMT), the pharmaceutical composition comprising, as an active
ingredient, a peptide derived from an N terminus portion of oS1
casein and a pharmaceutically acceptable carrier.
[0243] Further according to the present invention there is provided
a purified peptide having an amino acid sequence selected from the
group consisting of SEQ ID NOs:1-25.
[0244] Further according to the present invention there is provided
a pharmaceutical composition comprising a purified peptide having
an amino acid sequence selected from the group consisting of SEQ ID
NOs:1-25 and a pharmaceutically acceptable carrier.
[0245] The present invention successfully addresses the
shortcomings of the presently known configurations by providing
peptides for the treatment of human disease, which peptides are
derived from the N terminus portion of o S1 casein and posses no
detectable toxicity and high therapeutic efficacy.
[0246] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0247] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
MATERIALS AND EXPERIMENTAL METHODS
[0248] Preparation of Peptides Derived from Natural Casein:
[0249] The casein fraction of cow's milk was isolated as described
by Hipp et al. (1952), ibid., and subjected to exhaustive
proteolytic digestion with chymosin (also known as rennin) (20 ng
per ml) at 30.degree. C. Upon completion of the reaction, the
solution was heated to inactivate the enzyme, and the digest was
precipitated as paracaseinate by acidification with an organic
acid, acetic or trichloracetic acid. Paracaseinate was separated by
centrifugation, and the supernatant fraction, containing the
peptide fragments of interest, was dialyzed and re-precipitated as
caseicidin by higher acid concentrations. The resulting caseicidin,
following re-suspension, dialysis and neutralization was
lyophilized. The resulting powdered preparation was assayed for
biological activity as described below, and separated by HPLC for
peptide analysis.
[0250] HPLC Analysis of Peptides Derived from Natural Casein:
[0251] Peptides derived from natural casein as described above were
analyzed by HPLC in two stages. Initially, the lyophilized casein
digests were separated using a C 18 reversed phase with a 0.1%
water triflouroacetic acid (w/w)--acetonitrile gradient. Detection
was according to UV absorption at 214 nm. Following this the
samples were analyzed by HPLC-Mass Spectroscopy (MS) equipped with
an electrospray source. Mass calculations represent the mass of the
ionized peptide samples, as derived from the retention times.
Following separation, the amino acid composition of the peptides
was determined with a gas-phase microsequencer (Applied Biosystems
470A).
[0252] The following data is representative: Eight peptide peaks
were typically observed of which 3 were major peaks having Rt
values of 17.79, 19.7, 23.02 and 5 were minor peaks having Rt
values of 12.68, 14.96, 16.50, 21.9 and 25.1, which Rt values
represent molecular mass of 2764, 1697, 1880, 2616, 3217, 2333,
1677 and 1669 Da, respectively. At Rt of 17.79 (corresponding to
2,764 Da) a major peak of a peptide of 23 amino acids representing
amino acids 1-23 of oS1 casein, having the sequence
RPKHPIKHQGLPQEVLNENLLRF (SEQ ID NO:15, see McSweeny et al., 1993,
ibid., for the complete sequence of oS1 casein). Other peptides
were from positions 208-224 of .beta. casein, positions 16-37 of
oS1 casein and positions 197-222 of oS2 like casein precursor.
Other peptides were also present.
[0253] Synthetic Peptides Derived from Casein:
[0254] Peptides of increasing lengths corresponding to the
N-terminal 1-25 amino acids of oS1 casein were synthesized by
NoVetide Ltd., Haifa, Israel, with purity of >95% (HPLC).
Quality Control included: HPLC, Mass Spectrometry (EI), Amino acid
analysis and Peptide Content. Table 3 below provides the sequence
of these peptides:
3TABLE 3 Sequence No. of Identification (N terminus - C terminus)
amino acids SEQ ID NO: 74 RP 2 1 1P RPK 3 2 2P RPKH 4 3 3P RPKHP 5
4 4P RPKHPI 6 5 5P RPKHPIK 7 6 Y RPKHPIKH 8 7 X RPKHPIKHQ 9 8 1a
RPKHPIKHQG 10 9 2a RPKHPIKHQGL 11 10 3a RPKHPIKHQGLP 12 11 A
RPKHPIKHQGLPQ 13 12 B RPKHPIKHQGLPQE 14 13 C RPKHPIKHQGLPQEV 15 14
D RPKHPIKHQGLPQEVL 16 15 E RPKHPIKHQGLPQEVLN 17 16 F
RPKHPIKHQGLPQEVLNE 18 17 G RPKHPIKHQGLPQEVLNEN 19 18 H
RPKHPIKHQGLPQEVLNENL 20 19 I RPKHPIKHQGLPQEVLNENLL 21 20 J
RPKHPIKHQGLPQEVLNENLLR 22 21 K RPKHPIKHQGLPQEVLNENLLRF 23 22 L
RPKHPIKHQGLPQEVLNENLLRFF 24 23 M RPKHPIKHQGLPQEVLNENLLRFFV 25 24 N
RPKHPIKHQGLPQEVLNENLLRFFVA 26 25
[0255] Juvenile (Type I, IDDM) Diabetes in Non-Obese Diabetic (NOD)
Mice:
[0256] Peptides Derived from Natural Casein:
[0257] NOD mice are a commonly used model for research of
autoimmune disease and human Juvenile Diabetes. Six week old female
NOD mice received either one or two injections per week of 100
.mu.g of peptides derived from natural casein, for a total of 5 or
11 treatments. Control mice received no treatment. The severity of
disease was determined according to glucosuria, which was measured
using Combi test sticks [Gross, D. J. et al. (1994), Diabetology,
37:1195]. Results were expressed as the percent of glucosuria-free
mice in each sample over a 365-day period.
[0258] Synthetic Peptides Derived from Casein:
[0259] In another experiment, 6 week old female NOD mice received
one injection per week of 100 .mu.g of Synthetic peptides derived
from casein for a total of 5 treatments. Control mice received no
treatment. Results were expressed as the number of healthy mice in
the various treated groups.
[0260] Intraperitoneal Glucose Tolerance Test (IPGTT):
[0261] The glucose tolerance test is the definitive method for
investigating glucose metabolism and diabetic tendencies in
mammals. Twenty five (25) weeks after receiving Synthetic peptides
derived from casein, response to a glucose load was assessed with
an intraperitoneal glucose tolerance test. Glucose injection
consisted of 1 g/kg body weight. Glycemic values were determined
from blood drawn prior to test (0 minutes) and 60 minutes after
loading. Plasma glucose levels were determined with a Glucose
Analyzer 2 (Beckman Instruments, Fullerton, Calif.) and expressed
as mmol/L. Normal values do not exceed 140 mmol/L.
[0262] Stimulation of Proliferation of Natural Killer (NK)
Cells:
[0263] From Human Peripheral Blood Stem Cells (PBSC):
[0264] PBSC of G-CSF treated subjects were separated on a FICOLL
gradient, washed twice with RPMI-1640 medium and seeded into 1.5 ml
wells with or without peptides derived from natural casein or
synthetic peptides derived from casein, as indicated, (0-500 .mu.g
per ml). Following two days incubation the cells were assayed for
Natural Killer activity by measuring radioactivity released from
.sup.35S-labeled K562 target cells (NEG-709A, 185.00 MBq, 2.00 mCi
EASYTAGth Methionine, L-[.sup.35S] 43.48 TBq per mmol, 1175.0 Ci
per mmol, 0.488 ml, Boston USA). Two concentrations of effector
cells (2.5.times.10.sup.4 and 5.times.10.sup.4 cells per well) were
incubated with 5.times.10.sup.3 target cells per well
(effector:target cell ratios of 50:1 and 100:1, respectively) in
U-bottomed 96 well tissue culture plates. The cells were incubated
for 5 hours at 37.degree. C. in 5% CO.sub.2, 95% air and
precipitated by 5 minutes centrifugation at 1000 rpm. .sup.35S
release was measured in 50 .mu.l samples of the supernatant
liquid.
[0265] From murine Bone Marrow (BM) Cells:
[0266] Bone marrow was collected from 4 untreated BALB/c and
C57B1/6 mice. Bone marrow was harvested from the long bones of
front and hind limbs of the mice by injection of medium using a 25
Gauge needle. Aspirated cells were washed with RPMI 1640, counted
in a haemocytometer and vital-stained (20 .mu.of cells in 380 .mu.l
acetic acid-trypan blue), then seeded in culture bottles at
2-5.times.106 cells per ml in RPMI-1640 containing 10% Fetal Calf
Serum, antibiotics and glutamine with or without 100 .mu.g per ml
peptides derived from natural casein. The cell cultures were
incubated in 5% CO.sub.2, 95% air for 12-15 days at 37.degree. C.,
harvested by 10 minutes centrifugation at 1500 rpm, counted, and
seeded in U-bottom wells with .sup.51Cr (Chromium-51, 740 MBq, 2.00
mCi activity) or .sup.35S (NEG-709A, 185.00 MBq, 2.00 mCi EASYTAGth
Methionine, L-[.sup.35S] 43.48 TBq per mmol, 1175.0 Ci per mmol,
0.488 ml, Boston USA) labeled murine lymphoma (YAC) cells at either
25:1 or 50:1 effector:target cell ratio. NK activity is expressed
as the percent radioactivity in the cell-free supernatants.
[0267] Proliferation of Human Cells in Culture:
[0268] Peripheral blood (PB) was collected from healthy or affected
patients. Affected patients received no treatment other than G-CSF
supplementation prior to plasmapheresis. Bone marrow (BM) cells
were collected from consenting healthy patients or affected
patients in remission following chemotherapy by medium injection
into the medullary space and aspiration. Umbilical cord blood was
collected during normal births. Human cells of the various origins
were separated on a FICOLL gradient, washed twice with RPMI-1640
medium, and seeded into 0.2 ml flat bottom tissue culture wells at
the indicated concentrations with or without peptides derived from
natural casein or with or without synthetic peptides derived from
casein, as indicated. All treatments, including controls, were
repeated in triplicate. Cell proliferation was measured by addition
of radioactive thymidine [thymidine (methyl-[.sup.3H]) Specific
activity 6.7 Ci per ml 37 MBq per ml, ICN Corp.] following
incubation for the indicated number of days. Cells were then
incubated 16-20 hours with the label, harvested and washed twice
with medium. Incorporated radioactivity was measured in a .beta.
scintillation counter.
[0269] Proliferation of K562 leukemia and colon cancer cell lines:
Colon and K526 are established lines of cancer cells grown in
culture. Both cell lines were grown in culture bottles in 5%
CO.sub.2, 95% air at 37.degree. C., harvested and washed with
medium before seeding in tissue culture wells at 4.times.10.sup.5
cells (K562) or 3.times.10.sup.3 cells (Colon) per well. Peptides
derived from natural casein were added to the wells, at the
indicated concentrations, and after 9 (K562) or 3 (Colon) days of
incubation labeled thymidine was added as described above.
Harvesting and measurement of radioactive uptake was as described
above.
[0270] Fluorescent Antibody Detection of NK and T Cell
Proliferation in Human Peripheral Blood Stem Cells (PBSC):
[0271] Peripheral Blood Stem cells (PBSC) from human subjects
receiving G-CSF treatment were collected by plasmapheresis,
separated on a FICOLL gradient, washed twice with RPMI-1640 medium
containing 10% Fetal Calf Serum and incubated in culture bottles at
37.degree. C. in 5% CO.sub.2, 95% air with or without peptides
derived from natural casein at the indicated concentrations.
Following 10, 14 or 28 days incubation with peptides derived from
natural casein, the presence of T cells (CD.sub.3 surface antigen)
and NK cells (CD.sub.56 surface antigen) was detected by direct
immunofluorescence using anti-CD.sub.3 fluorescent antibody
(CD.sub.3/FITC clone UHCT.sub.1), anti-CD.sub.56 fluorescent
antibody (CD.sub.56/RPE clone MOC-1) (DAKO A/S, Denmark) and mouse
IgG1/RPE and IgG1/FITC antibodies as a control. Detection of
fluorescently tagged cells was performed using fluorescence
activated cell sorting (FACS) and fluorescent microscopy.
[0272] Stimulation of Hematopoiesis from Bone Marrow (BM) Cells in
Culture:
[0273] Proliferation of Megakaryocytes in Multipotential Colonies
(CFU-GEMM) from Murine Bone Marrow Cells:
[0274] Primary bone marrow cells (1.times.10.sup.5 per ml) from
8-12 week-old C3H/HeJ mice were grown in serum-free methyl
cellulose-IMDM medium for 8-9 days at 5% CO.sub.2, 95% air, at
37.degree. C. The medium, appropriate for the growth of
multipotential colonies (CFU-GEMM), contained 1% BSA (Sigma),
10.sup.-4 M thioglycerol (Sigma), 2.8.times.10.sup.-4 M human
transferrin (TF, Biological industries, Israel), 10% WEHI-CM as a
source of IL-3 and 2 units per ml erythropoietin (rhEPO, R & D
Systems, Minneapolis). Colonies were scored after 8-9 days using an
Olympus dark field microscope. They were picked with a
micropipette, cytocentrifuged and stained with May-Grunwald-Giemsa
for differential counts. At least 700 cells were counted for each
preparation.
[0275] Proliferation of Dendritic Cells in CFU-GEMM: Multipotent
(CFU-GEMM) colonies grown from primary bone marrow cells as
described for the assay of megakaryocyte proliferation above were
collected, stained and counted for dendritic cells. At least 700
cells were counted for each preparation.
[0276] Proliferation of Plasma Cells in CFU-GEMM:
[0277] Multipotent (CFU-GEMM) colonies grown from primary bone
marrow cells as described for the assay of megakaryocyte
proliferation above were collected, stained and counted for plasma
cells. At least 700 cells were counted for each preparation.
[0278] Proliferation of Macrophage Cells in CFU-GEMM:
[0279] Multipotent (CFU-GEMM) colonies grown from primary bone
marrow cells as described for the assay of megakaryocyte
proliferation above were collected, stained and counted for
macrophage cells. At least 700 cells were counted for each
preparation.
[0280] Proliferation of Red Blood Cells in CFU-GEMM:
[0281] Multipotent (CFU-GEMM) colonies grown from primary bone
marrow cells as described for the assay of megakaryocyte
proliferation above were collected, stained and counted for red
blood cells. At least 700 cells were counted for each
preparation.
[0282] Proliferation of Polymorphonuclear Cells (PMN) in
CFU-GEMM:
[0283] Multipotent (CFU-GEMM) colonies grown from primary bone
marrow cells as described for the assay of megakaryocyte
proliferation above were collected, stained and counted for
polymorphonuclear cells. At least 700 cells were counted for each
preparation.
[0284] Proliferation of Megakaryocyte- and Erythroid Forming Cells
from Human Bone Marrow and Cord Blood Cells:
[0285] A sample of bone marrow from an apparently healthy human
being was processed by density gradient separation using
Histopaque-107 (Sigma Diagnostics) to obtain a purified population
of mononuclear cells (MNC). Colony assays were performed in a
plating medium containing final concentrations of 0.92% methyl
cellulose (4000 centripase powder, Sigma Diagnostic), rehydrated in
Iscoves modified Dulbecco's medium containing 36 mM sodium
bicarbonate (Gibco), 30% fetal bovine serum (FBS) (Hyclone), 0.292
mg/ml glutamine, 100 units per ml penicillin and 0.01 mg per ml
streptomycin (Biological Industries, Beit Haemek). Cord blood from
normal births was collected and prepared as mentioned above.
[0286] Colony assay medium containing 105 MNC per ml was plated in
triplicate wells within a 24 well tissue culture plate (Greiner),
0.33 ml per well. The cultures were incubated at 37.degree. C. in
5% C0.sub.2, 95% air and 55% relative humidity with or without
peptides derived from natural casein or synthetic peptides derived
from casein, at the indicated concentrations. Plates were scored
after 14 days for colonies containing more than 50 cells.
Megakaryocytes were identified by indirect immunofluorescence using
a highly specific rabbit antibody recognizing human platelet
glycoproteins, and an FITC-conjugated goat anti-rabbit IgG. Added
growth factors included 15 ng per ml leucomax (GM-CSF) (Sandoz
Pharma), and 5% vol. per vol. human phyto-hemagglutinin-m (Difco
Lab)-induced conditioned medium (CM) to induce development of
granulocyte-monocyte colonies (CFU-GM). Erythropoietin (EPO) 2
units/ml was used to induce formation of erythroid colonies.
[0287] Alternatively, human bone marrow cells from consenting
volunteer donors or patients undergoing autologous bone marrow
transplantation were precultured in medium containing 10-1000 .mu.g
per ml peptides derived from natural casein, grown in semi-solid
agar, and scored for granulocyte-macrophage hematopoietic colonies
(GM-CFU) at 7 or 14 days post treatment.
[0288] Megakaryocytopoiesis was measured in normal bone marrow
cells from healthy consenting human donors by either scoring of the
number of megakaryocytes in samples of liquid culture (RPMI-1640
plus 10% human AB serum, glutamine and antibiotics) with or without
100 .mu.g per ml peptides derived from natural casein, or in a
methylcellulose assay for assessing colony formation.
2.times.10.sup.5 bone marrow cells were seeded in the presence of a
standard growth factor combination with or without peptides derived
from natural casein. In the methylcellulose assay megakaryocytes
were counted with an inverted microscope on days 12-14 after
seeding.
[0289] Clinical Trials Using Peptides Derived from Natural
Casein:
[0290] In one series of trials, a single dose containing 50 mg
peptides derived from natural casein was administered
intra-muscular to human subjects in 3 depots, over a period of 2
hours. Clinical parameters were monitored at the indicated
intervals. In other trials, patients at various stages of treatment
for and/or remission from cancer and metastatic disease received
peptides derived from natural casein once or twice, and were
monitored for changes in the cell count of peripheral blood.
[0291] Inhibition of in vitro HIV Infection of Human Lymphocyte
Cells:
[0292] Peptides: Peptides (either peptides derived from natural
casein or s synthetic peptides derived from casein (1-25 amino
acids in length, see table 3) supplied as lyophilized powder were
resuspended in RPMI complete medium and added to cell cultures at a
final concentrations of 50 to 1000 .mu.g per ml.
[0293] Cells: Several types of freshly isolated human cells
(primary cells) and cell lines are known to be susceptible to in
vitro HIV-1 infection, although essentially any cell displaying
even low surface levels of the CD.sub.4 molecule can be considered
a potential target for HIV-1 infection. Two commonly used human
cell lines which are highly sensitive for HIV-1 infection were
chosen, CEM and Sup-T1.
[0294] CEM is a human T4-lymphoblastoid cell line initially derived
by G. E. Foley et al. [(1965), Cancer 18:522] from peripheral blood
buffy coat of a 4-year old caucasian female with acute
lymphoblastic leukemia. These cells were continuously maintained in
suspension in medium, and have been used widely for analysis of
infectivity, antiviral agents and neutralizing antibodies.
[0295] Sup-T1 is a human T-lymphoblastoid cell line isolated from a
pleural effusion of an 8-year old male with Non-Hodgkin's T-cell
lymphoma [Smith, S. D. et al. [(1984) Cancer Research 44:5657].
This cell expresses high levels of surface CD.sub.4 and is useful
in studies of cell fusion, cytopathic effect and infectivity of
HIV-1. Sup-TI cells are grown in suspension in enriched medium.
[0296] Medium: Cells were grown in RPMI-1640 complete medium
enriched with 10% Fetal bovine serum, 2 mM glutamine and 2 mM
penicillin- streptomycin (GIBCO).
[0297] Virus: The HIV virus strain employed was HIV-1IIIB,
originally designated HTLV-IIIB. Concentrated culture fluids of
peripheral blood from several patients with AIDS or related
diseases were used to establish a permanent productive infection in
H-9 cells. This subtype B virus has high capacity to replicate in
human T-cell lines. Viral titer was 5.38 ng per ml in stock
solution.
[0298] FITC-labeled peptides: FITC F-1300 (Fluorescein
isothiocyanate, isomer I, Sigma (F25o-2) St. Louis, MI, USA) having
excitation/emission maxima of about 494/520 nm, respectively, was
employed. The amine-reactive fluorescein derivative is probably the
most common fluorescent derivatization reagent for covalently
labeling proteins. FITC-conjugated peptides derived from natural
casein were prepared by covalent binding of FITC to the amine
groups of lysine.
[0299] HIV-1 P24 antigen capture assay: An HIV-1 P24 Antigen
capture assay kit employed was designed to quantitate the HIV-1 P24
core antigen, which is proportionally related to the degree of
viral production in cells. This kit was purchased from the AIDS
Vaccine program of the SAIC-NCI-Frederick Cancer Research
Institute, P.O. Box B, Frederick, M.D. 21702, USA and included 96
well plates coated with monoclonal antibody to HIV-1 P24, primary
antibody-rabbit anti-HIV P24 serum, secondary antibody-Goat
anti-rabbit-IgG (H+L) peroxidase conjugated antibody, TMB
peroxidase substrate system and lysed HIV-1 P24 standard. The HIV-1
P24 antigen capture assay was analyzed by Organon-Technica ELISA
reader at 450 nm with a reference at 650 nm.
[0300] HIV-1 P24 antigen capture ELISA: HIV infection was measured
with an indirect enzyme immunoassay which detects HIV-1 P24 core
antigens in tissue culture media. Tissue culture supernatant was
reacted with primary rabbit anti-HIV-1 P24 antigen and visualized
by peroxidase conjugated goat anti rabbit IgG. The reaction was
terminated by adding 4N H.sub.2SO.sub.4, wherein the intensity of
the color developed is proportional to the amount of HIV-1 antigen
present in the tissue culture supernatant.
[0301] Biological hazard level (BL-3) laboratory: All virus
production isolation and infection, tissue culture of HIV-1
infected cells, P24 antigen containing supernatant harvesting and
P24 antigen capture ELISA, were performed in BL-3 facility of the
Hebrew University, Hadassah Medical School and were in accordance
with the bio safety practices set by the NIH and CDC (USA).
[0302] Flow cytometry: A FACSort cell sorter (Becton &
Dickinson, San Jose, Calif. USA) was used to (i) determine the
percentage of CD4 positive CEM and sup-T1 cells batches before
infection with HIV-1 in order to assure the same degree of
infection in each experiment; and (ii) detect T cells that harbor
FITC conjugated peptides derived from natural casein in their
cytoplasm and nuclei.
[0303] CO.sub.2 incubator: For viral culture production cells with
HIV-1, cells and virus pretreated with peptides derived from
natural casein and cells which were further incubated with HIV-1,
were all kept in humidified C0.sub.2 incubator for the duration of
the experiment.
[0304] HIV infection of human cultured CD.sub.4+cells: The cells
(CEM, Sup-TI) were preincubated with several increasing
concentrations of peptides derived from natural casein (50-1000
.mu.g per ml) or synthetic peptides derived from casein (10-500
.mu.g per ml) for 3, 24 (for synthetic and natural peptides) and 48
(only for natural peptides) hours and HIV-1IIIB (45 .mu.g per ml
final concentration) was added to each well thereafter. HIV-1IIIB
was preincubated with the peptides for 3 hours and then added to
cells (5000 cells/well) in tissue culture plates. Controls were IF
(Infected, cells cultured with HIV-1 and without peptides), UIF
(Uninfected, cells cultured without HIV-1 and without peptides) and
UI +Ch (Uninfected +peptides derived from natural casein, cells
cultured in the presence of peptides derived from natural casein
{50-1000 .mu.g per ml}) to test the effect of peptides derived from
natural casein and synthetic peptides derived from casein on cell
viability and growth. Cells were counted for viability and
proliferation rate on day 7, 10 and day 14 post infection (the day
of P24 antigen culture supernatant harvest). Cells and tissue
culture supernatants (media) were harvested and lysed immediately
in {fraction (1/10)}volume of 10% Triton X-100. These samples were
farther incubated at 37.degree. C. for 1 hour and kept at
-80.degree. C. until tested for p24 antigen.
[0305] Confocal microscopy: A Zeiss LSM 410 confocal laser scanning
system attached to TW Zeiss Axiovert 135M inverted microscope,
employing the laser scanning confocal microscopy technique, was
used to detect penetration of FITC conjugated peptides into cells.
T cells were incubated with FITC conjugated peptides derived from
natural casein in a 5% CO.sub.2, 95% air, 37.degree. C. incubator,
after which the cells were washed 3 times with phosphate buffer
saline (PBS) to remove unbound FITC-peptides. Cells were fixed with
3.8% formalin for 10 minutes, washed twice with PBS and resuspended
in 50-100 .mu.l PBS before viewing the cells under the microscope.
Selected images of cells from different time points of incubation
(15 minutes, 30 minutes, 1 hour, 1.5 hour and 3 hours) displaying
various amounts of FITC-peptides derived from natural casein in
their cytoplasms and nuclei were stored on 3.5" Zip derive (230 MB)
and processed for pictures using Photoshop software.
[0306] [.sup.3H]-thymidine incorporation test: In order to test the
effect of peptides derived from natural casein on T cell
proliferation, several concentrations of peptides derived from
natural casein (1 .mu.g/ml stock in RPMI) were added to Sup-T1 cell
cultures in 96 flat bottom microwell plate (5000 cells/well), as
described for HIV-1 infection in Sup-TI cells. Cells were counted
and their viability was determined by trypan blue dye exclusion.
They were pulsed with [.sup.3H]-thymidine at each time point (3, 7,
10 and 14 days) for 18 hours (over night) and harvested on glass
fiber filters for radioactivity reading (Incorporation of
[.sup.3H]-thymidine into cellular DNA is proportional to degree of
cell proliferation).
[0307] Toxicity of Peptides Derived from Natural Casein in Normal,
myeloablated and Transplant Recipient Mice and Guinea Pigs:
[0308] Intramuscular, or intravenous injections of up to 5,000 mg
peptides derived from natural casein per kg animal were
administered in a single dose, or in three doses to normal. A
variety of strains were employed, including BALB/c, C3H/HeJ and
Non-Obese Diabetic (NOD) mice. The mice were either monitored for
10 months before sacrifice and post-mortem examination (toxicity
assay) or observed for 200 days (survival rate). Guinea pigs
received a single intramuscular injection of 20 mg peptides derived
from natural casein per animal. Fifteen days later they were
sacrificed and examined for pathology.
[0309] Leukocyte and Platelet Reconstitution in Bone Marrow
Transplant Recipient Mice:
[0310] BALB/c mice were lethally irradiated at a source to skin
distance of 70 cm, dosage of 50 cGy per minute, for a total of 600
cGy. The irradiated mice were reconstituted with syngeneic bone
marrow as described above and injected intravenously 24 hours later
with 1 mg per animal peptides derived from natural casein,
synthetic peptides derived from casein (13-26 amino acids, see
Table 3 above), or human serum albumin (controls), following a
double-blinded protocol. Leukocyte reconstitution was determined
according to cell count in peripheral blood collected at indicated
intervals from 6 to 12 days post treatment. Platelet reconstitution
was determined by cell count in blood collected from the retro
orbital plexus, into heparinized capillaries, at indicated
intervals from day 6 to day 15 post treatment.
[0311] In an additional series of experiments, CBA mice were
lethally irradiated (900 cGy), reconstituted and treated with
peptides derived from natural casein or human serum albumin as
described above. Platelet reconstitution was assayed as mentioned
above.
[0312] In a third series of experiments, the mice were irradiated
(800 cGy), reconstituted and injected intraperitoneally with 100
.mu.g synthetic peptides derived from casein (peptides 3a and 4P,
representing the first 6 and 12 amino acids of the N terminus of
.alpha.S1 casein, respectively--see Table 3 above) daily, on days
4, 5, 6 and 7 post-transplantation. Platelet reconstitution was
assayed at 10 and 12 days post-transplantation.
[0313] Reconstitution of Bone Marrow Transplant Recipient Mice:
[0314] C57/Black/6 mice were lethally irradiated at a source to
skin distance of 70 cm, dosage of 50 cGy per minute, for a total of
600 cGy. The irradiated mice were reconstituted with syngeneic bone
marrow from mice which were either treated a day prior to bone
marrow aspiration with 1 mg per animal peptides derived from
natural casein or not treated, following a double-blinded protocol.
In one experiment mice survival was monitored for 18 days. In
another experiment mice were sacrificed after 10 days and spleen
colonization monitored.
[0315] Synthetic Peptides Derived from Casein Significantly Reduce
Cholestrol Levels:
[0316] The ability of synthetic caesin derived peptides to reduce
cholesterol levels in 7-week old female C57B1/6j mice was assessed
after feeding an atherogenic diet. The mice were divided into
groups of 8. One control group was fed a normal diet. A second
control group was fed the modified Thomas Hartroft diet containing
cholate (#TD 88051: Teklad, Madison, Wis.) [Gerber, D. W. et
al.(2000), Journal of Lipid Research. 42, 2001]. The remaining
experimental groups were all fed the modified Thomas Hartroft diet.
After one week on the diet, serum cholesterol values increased
significantly and the synthetic peptides derived from casein were
injected intraperitoneally, 1 mg per mouse, followed by a second
injection of 0.1 mg one week later.
[0317] Cholestrol blood levels were determined according to Roche
Cholesterol Assay based on Roeschlou & Allin enzymatic method
(Roche, Inc., Germany).
EXPERIMENTAL RESULTS
[0318] Peptides Derived from Natural Casein:
[0319] Originating from the observation that curdled milk
occasionally failed to support bacterial growth, a casein fragment
possessing bacteriocidal properties was isolated from milk proteins
(U.S. Pat. No. 3,764,670 to Katzirkatchalsky, et al.). Crude
peptides derived by proteolysis of natural casein were prepared by
acid precipitation of the soluble fraction of the casein
proteolytic digest, dialysis and lyophilization. When tested for
biological activity after extended storage, it was noted that this
crude preparation, when lyophilized and stored at 4.degree. C.,
remained active (in vitro and in vivo) for at least 24 months.
[0320] In order to identify the active peptides contained in the
peptides derived from natural casein the lyophilized crude
preparation was fractionated using high performance liquid
chromatography (HPLC), as described hereinabove. All of the
lyophilized samples analyzed demonstrated similar retention time
profiles, with contents as described above.
[0321] Thus, a major component of the crude peptides derived from
natural casein preparation is the N-terminal fragment of oS1
casein.
[0322] Peptides Derived from Natural Casein are Non-Toxic in
Rodents and Humans:
[0323] Extensive investigation of the short and long term effects
of high doses of peptides derived from natural casein on mice,
guinea pigs and human volunteers confirmed the absence of toxicity,
teratogenicity or adverse side effects of the preparation. In one
series of tests, single doses representing 7,000 times the
estimated effective dose of peptides derived from natural casein
were administered intra muscularly to mice. Standard post-mortem
pathology examination of the mice at 14 days post treatment
revealed no toxic effects on internal organs or other
abnormalities. Similar toxicity tests in guinea pigs revealed no
abnormalities two weeks after single 20 mg intra-muscular doses of
peptides derived from natural casein. In another series of
experiments, high doses of peptides derived from natural casein
administered to healthy mice had no effect on several hematological
parameters measured two weeks later, including white blood cells
(WBC), red blood cells (RBC), hemoglobin (HGB), electrolytes,
glucose and others. A third series of experiments tested repeated
high doses of 100 mg per kg body weight in mice and rats for two
weeks, revealing no allergic, delayed cutaneous or anaphylactic
responses and no pathological effects upon post-mortem examination.
When peptides derived from natural casein were tested for their
effect on the long-term survival of irradiated, bone marrow
reconstituted BALB/c and C3H/HeJ mice, survival of the treated mice
(18 of 27 BALB/c and C3H/HeJ; 66%) clearly exceeded the survival
rates of the albumin-treated controls (4 of 26 BALB/c and C3H/HeJ;
15%). Standard teratogenicity tests [for details see, for example,
Drug Safety in Pregnancy, Folb and Dakes, p. 336, Elsevier;
Amsterdam, N.Y., Oxford (1990)] in mice treated with peptides
derived from natural casein revealed no effect of the peptides on
any developmental parameters.
[0324] Similar to its lack of toxicity or side effects when tested
in rodents, peptides derived from natural casein were safe when
administered to humans as well. Comparison of blood and urine
samples from seven healthy human volunteers before, during and 7
days after intramuscular injection of peptides derived from natural
casein revealed no changes in any of the clinical parameters. No
other negative effects were observed.
[0325] Thus, high dose and extended treatment of rodents with
peptides derived from natural casein revealed no apparent toxic,
pathological, hypersensitivity, teratogenic, serological or any
other negative effects. Moreover, peptides derived from natural
casein administration to irradiated mice, at risk for short-and
long-term complications, conferred a significant survival advantage
over 200-300 days. These, and the absence of any undesirable
effects in healthy human volunteers receiving peptides derived from
natural casein via injections clearly demonstrate the peptide's
safety in parenteral administration.
[0326] Reconstitution of Bone Marrow in Transplant Recipient
Mice:
[0327] When C57/Black/6 mice were lethally irradiated and
reconstituted with syngeneic bone marrow from mice that were either
treated a day prior to bone marrow aspiration with 1 mg per animal
peptides derived from natural casein or not so treated, survival of
irradiated mice that received bone marrow from treated mice far
exceeded that of irradiated mice that received bone marrow from non
treated mice (survival of irradiated mice that received bone marrow
from treated mice was 15 out of 18, 10 days post irradiation;
whereas survival of irradiated mice that received bone marrow from
non treated mice was 4 out of 17, 10 days post irradiation).
Spleens derived from irradiated mice that received bone marrow from
treated mice included about twice to three times as many colonies
per spleen, as compared to spleens of irradiated mice that received
bone marrow from non treated mice (1-5 colonies as compared to 0-3
colonies).
[0328] Peptides Derived from Natural Casein Stimulate the
Proliferation of Lymphocytes:
[0329] Natural killer (NK) and cytotoxic T cells are crucial to the
immune system's ability to protect against invasion by both
infectious pathogens and cancer cells, by both active cytotoxicity
and the secretion of immunoregulatory lymphokines. Immune
compromise, such as in AIDS or following chemotherapy, results in
abnormal, weakened T or NK cell activity. When normal murine bone
marrow cells from BALB/c and C57B1/6 mice were cultured in the
presence of 100 .mu.g per ml peptides derived from natural casein,
a clear increase in NK activity was observed in both
effector:target cell ratio groups. Moreover, comparison between the
two groups revealed a clear dose response relationship. At the 1:25
effector:target cell ratio the average NK activity was elevated
from 13.93% to 30.77% and at the 1:50 effector:target cell ratio
the average NK activity was elevated from 13.68% to 44.05% (FIG.
1). Similar experiments using human Peripheral Blood Stem Cells
from Granulocyte Colony Stimulating Factor-treated donors
demonstrated an even more significant, concentration-dependent
stimulation of target cell lysis by peptides derived from natural
casein In the first set of experiments (FIG. 2a), NK activity was
measured in blood samples taken from one patient and incubated at
two effector:target cell ratios with increasing peptides derived
from natural casein concentration. Only 4% .sup.35S release was
measured in the control, untreated PBSC culture. Almost the same
percent radioactivity (4%) was found at the lowest peptide
concentration (5.mu.g per ml). However, at higher peptide
concentrations, in the range of 10.mu.g per ml up to 100.mu.g per
ml, a release of 10.8-14.9% .sup.35S was measured for
effector:target cell ratios of 100:1 and 8.3-14.5% .sup.35S for
effector target cell ratios of 50:1 (FIG. 2a).
[0330] When PBS cells from normal (patient 1) and affected
(patients 2-6) human donors were incubated with increasing
concentrations of the peptides derived from natural casein, a
significant enhancement of affected patients' NK cell activity
could be measured. Thus, while the peptides derived from natural
casein had a minimal effect on the normal patient's NK activity
(increased from 13-15% .sup.35S release, patient 1), PBS cells from
both breast cancer and Non-Hodgkins Lymphoma patients (patients 3
and 4, for example) exhibited dramatic, dose-dependent increases in
NK activity (3.5-10.8% .sup.35S; 12.2-19.1% .sup.35S, respectively)
(FIG. 2b).
[0331] Peptides Derived from Natural Casein Stimulate the
Proliferation of CD56 Surface Antigen Positive (NK) Cells:
[0332] In another series of experiments Peripheral Blood Stem Cells
(PBSC) from 5 human donors receiving GCSF treatment were incubated
with peptides derived from natural casein for 10, 14, or 28 days,
then assayed for proliferation of the CD 56 antigen. A sometimes
dramatic increase in CD 56 antigen detection was observed in the
peptide-treated cells from all the donors but one (patient 1). A
representative response is depicted in FIG. 3a: Following 10 days
of incubation with or without peptides derived from natural casein,
the presence of CD56 surface antigen-positive (NK) cells was
detected by direct immunofluorescent staining. % Overall,
incubation with peptides derived from natural casein increased the
mean percentage of the cells positively stained for CD56 from 0.64%
in the control group to 2.0% following treatment (FIG. 3a).
[0333] Peptides Derived from Natural Casein Stimulate the
Proliferation of CD3 Surface Antigen-Positive (T) Cells:
[0334] The effect of peptides derived from natural casein on the
proliferation of CD3 surface antigen-positive (T) cells in PBS
cells from 5 subjects was assayed by direct immunofluorescence. In
all but one patient (patient 4), 14 days incubation with peptides
derived from natural casein significantly increased T-cell
proliferation, up to more than 5 fold in some. Taken together, the
mean percentage of the cells positively stained for CD3 increased
from 19.45% in the control group to 35.54% in the treated group
(FIG. 3b).
[0335] Peptides Derived from Natural Casein Stimulate the
Proliferation of--CD56 and CD3 (NK/T-cells) Positive Cells:
[0336] In an additional experiment PBSCs from 7 patients were
incubated with peptides derived from natural casein for 28 days,
and the effect on proliferation of NK/T cells (CD56 and CD3 surface
antigen-positive) was detected by direct immunofluoresence.
Incubation with peptides derived from natural casein stimulated
proliferation of T-cell up greater than 5 fold in some cases
(patient 6), while the mean percentage of the CD3- positive (T-)
cells increased from 2.08% in the control group to 6.49% in the
treated group The number of both CD56 and CD3 surface
antigen-positive (NK/T) cells was increased from 1.1% in the
control to 4.3% in the treated group (FIG. 3c). Thus, peptides
derived from natural casein stimulate the proliferation of both
T-lymphocytes and Natural Killer cells from normal murine and human
blood cell progenitors. Significantly, the greatest
immune-stimulatory effect of the peptides derived from natural
casein was noted in human donors having initially low T- and NK
cell levels (FIGS. 3a-c).
[0337] Synthetic Peptides Derived from Casein Stimulate human
Lymphocyte Proliferation in vitro:
[0338] When synthetic peptides derived from casein representing the
first 3 to 26 residues of oS1 casein were incubated with human PBSC
cells from healthy and cancer patients (see below), a significant
increase in NK cell activity was observed. Target cell lysis was
greatest (from 3 to greater than 5 fold that of controls) in
Non-Hodgkin's Lymphoma and Breast Cancer patient's PBSC cultures
after two days incubation with as little as 10 .mu.g per ml of
peptides containing the first 9 or more residues of o SI casein
(FIG. 4). Under identical conditions, none of the peptides tested
had a significant effect on NK activity in PBSC cultures from
healthy human donors. Thus, even low concentrations of peptides
containing the first 10 residues of the N-terminal sequence of oS1
casein are capable of selectively stimulating in vitro lymphocyte
proliferation in cells from cancer patients. Similar stimulation of
NK cell activity was observed when PBS cells from human donors with
hematopoietic disease were incubated with Synthetic peptides
derived from casein representing the first 3 amino acid residues of
o S1 casein. Incubation of the PBS cells with the peptides
increased target cell lysis from 2- to greater then 8-fold that of
the untreated controls. Of the 5 patients tested, three (3)
responded to 25 .mu.g/ml peptide concentration, one (1) responded
to 100 .mu.g/ml peptide concentration and one (1) to 250 .mu.g/ml.
Three out of the five (5) patients responded at 25 .mu.g/ml. No
significant effect on NK activity in PBSC cultures from healthy
human donors treated with the synthetic peptide representing the
first 3 amino acids of oS1 casein, was observed, confirming the
selective nature of the human lymphocyte-stimulating properties of
casein-derived peptides.
[0339] Stimulation of Hematopoiesis in Human Blood Cell
Progenitors:
[0340] Blood cell progenitors differentiate into a variety of blood
cells: macrophages, monocytes, granulocytes, lymphocytes,
erythrocytes and megakaryocytes. Progenitor cells are abundant in
bone marrow, but are also found in peripheral blood after
Granulocyte Colony Stimulating Factor treatment (PBSC cells), in
fresh Cord Blood. When increasing concentrations (50-600 .mu.g per
ml) of peptides derived from natural casein were added to cultures
of human Bone Marrow, PBSC and Cord Blood, an increase in cell
proliferation, as measured by [.sup.3H]-thymidine incorporation was
noted (FIGS. 5a-5c). Human PBSC proliferation was most greatly
effected by 300 .mu.g per ml (FIG. 5a) after 15 days in culture. An
even greater effect was noted for Cord Blood cells in culture (3 to
4 fold increase in [.sup.3H]-thymidine incorporation) after 14 days
incubation (but not after 7 days) with peptides derived from
natural casein (600 .mu.g per ml, FIG. 5c). Cultured human bone
marrow cells from three out of four donors also reacted strongly (3
to 5 fold increase in incorporation) to peptides derived from
natural casein (300 .mu.g per ml) after 21 days incubation (FIG.
5b). Thus, peptides derived from natural casein stimulate
proliferation of human blood cell progenitors from bone marrow as
well as other sources. Interestingly, incubation of cultured human
K562 (Chronic Myeloid Leukemia) and Colon (Colon cancer) cell lines
with high concentrations (up to 500 .mu.g per ml) of peptides
derived from natural casein under similar conditions had no effect
on [.sup.3H]-thymidine incorporation. Thus, peptides derived from
natural casein stimulate proliferation of human blood cell
progenitors but not growth of cancerous cells.
[0341] Stimulation of Megakaryocytopoiesis by Peptides Derived from
Casein:
[0342] Peptides Derived from Natural Casein Stimulate Megakaryocyte
Progenitor Proliferation in Cultured Murine Bone Marrow Cells:
[0343] Multinucleated megakaryocytes develop in the bone marrow
from primitive stem cells, mature to giant cells and give rise to
thousands of thrombocytes per megakaryocyte. Thrombocytes are
crucial for clot formation and thrombocytopenia is a major concern
in myeloablative conditions (following chemotherapy or
radiotherapy).
[0344] Primary bone marrow cell cultures can be induced to form
CFU-GM (Granulocyte and Megakaryocyte) colonies, giving rise to
megakaryocytes, and CFU-GEMM (Granulocyte, Erythroid, Macrophage
and Megakaryocyte) colonies, containing additional blood cell
types. Colony counts reflect expansion of specific progenitors,
cell numbers reflect proliferation rates and differential cell
counts reflect which specific cell lineages have developed
[Patenkin, D. et al. (1990), Mol. Cel. Biol. 10, 6046-50]. In
cultured murine bone marrow cells incubated with erythropoietin and
IL-3, addition of 25 .mu.g per ml peptides derived from natural
casein for 8 days increased the number of CFU-GEMM two and one half
fold over controls, stimulating a three fold increase in relative
cell numbers per colony in the CFU-GEMM. In a similar series of
experiments, addition of peptides derived from natural casein to
bone marrow cells incubated with erythropoietin and conditioned
medium (see Materials and Experimental Methods) stimulated a
concentration-dependent increase in the percentage of early and
late megakaryocytes (15% megakaryocytes without peptides, to 50%
with 500 p g per ml peptides derived from natural casein). Thus, 8
days treatment with peptides derived from natural casein stimulated
a significant increase in megakaryocyte formation and development
in primary murine bone marrow cultures.
[0345] Synthetic Peptides Derived from Casein Stimulate
Megakaryocyte Progenitor Proliferation in Cultured Murine Bone
Marrow Cells:
[0346] Similar to the above and under similar experimental
conditions, synthetic peptides derived from casein representing the
first 5 to 24 amino acids of oS1 casein increase the percentage of
early and late megakaryocytes from 15% without the synthetic
peptide to more than 40% with 25 .mu.g per ml of synthetic peptides
(FIG. 7). Thus, 8 days treatment with synthetic casein derived
peptides representing the first 5, 6, 11, 12, 17, 18, 19, 20, 21
and 24 amino acids stimulated a significant increase in
megakaryocyte formation and development in primary murine bone
marrow culture. Somewhat milder, yet appreciable, stimulation was
observed with the other synthetic peptides derived from casein.
[0347] Peptides derived From Natural Casein Stimulate
Megakaryocytopoiesis in Cultured Human Bone Marrow Cells:
[0348] When 100 .mu.g per ml peptides derived from natural casein
were added under similar conditions to human bone marrow cell
cultures from healthy donors, CFU-GM colony formation was increased
with or without additional stimulating factors (GM-CSF, CM).
Peptides derived from natural casein also stimulated erythroid cell
forming colonies in the presence of erythropoietin. Treatment of
the human bone marrow cells with thrombopoietin (TPO) stimulates
megakaryocyte (MK) colony formation. Addition of 300 .mu.g per ml
peptides derived from natural casein to TPO-treated cells
stimulates a more than twofold increase (16 colonies per 2 x 105
cells without peptides, 35 colonies per 2.times.105 with peptides
derived from natural casein) in MK colony proliferation.
[0349] In the presence of additional hematopoietic factors, such as
erythropoietin, human IL-3, hSCF and AB serum, 14 days incubation
with peptides derived from natural casein stimulated a nearly three
fold increase in CFU-GEMM colonies from human bone marrow cells
(158 colonies with 500 .mu.g per ml peptides derived from natural
casein, 68 colonies with the factors alone), but had a smaller (one
and one half fold) effect on cultured cord blood CFU-GEMM
formation. The relative cell number counts in the cultured human
bone marrow and cord blood colonies reflect megakaryocyte cell
proliferation in response to addition of 25 .mu.g per ml peptides
derived from natural casein (see Table shown in FIG. 6). Thus,
incubation of cultured human primary bone marrow and cord blood
cells with peptides derived from natural casein stimulates the
development and proliferation of both committed megakaryocyte and
erythroid cell colonies. Significantly, the synergy observed
between TPO and peptides derived from natural casein in stimulating
megakaryocytopoiesis indicates a probable role for this potent
hematopoietic growth factor in the mechanism of peptides derived
from casein's stimulatory properties, and further suggests the
likelihood of similar augmentation of a wide range of TPO-mediated
effects by peptides derived from natural casein.
[0350] Synthetic Peptides Derived from Casein Stimulate Dendritic
Cells Proliferation in Murine CFU-GEMM:
[0351] The effect of Synthetic peptides derived from casein on
dendritic cell proliferation in murine primary bone marrow cells
was assessed under the same conditions outlined for the stimulation
of megakaryocytes. Synthetic peptides derived from casein
representing the first: 2, 3, 5, 6, 7, 9, 11, 12, 16, 23, 24 and 26
amino acids of oS1 casein stimulated the proliferation of dendritic
cells, from 2.2% and up 23% of total cells compared with 0.1-0.2%
dendritic cells in the cell samples incubated without Synthetic
peptides derived from casein (FIG. 7).
[0352] Synthetic Peptides Derived from Casein Stimulate Plasma Cell
Proliferation in Murine CFU-GEMM:
[0353] The effect of Synthetic peptides derived from casein on
plasma cell proliferation in murine primary bone marrow cells was
demonstrated under the same conditions outlined for the stimulation
of megakaryocytes. Synthetic peptides derived from casein
representing the first: 2, 3, 5, 7, 11, 16, 17, 18, 19, 20, 21, 22,
23 and 24 and 26 amino acids of oS1 casein, significantly
stimulated the proliferation of plasma cells, from 1.5% and up
12.3% of total cell count, compared with 0.3% of total without
Synthetic peptides derived from casein (FIG. 7).
[0354] Synthetic Peptides Derived from Casein Stimulate Macrophage
Proliferation in CFU-GEMM:
[0355] The effect of Synthetic peptides derived from casein on
macrophage proliferation in murine primary bone marrow cells was
demonstrated under the same conditions outlined for the stimulation
of megakaryocytes. Incubation of cells with synthetic peptides
derived from casein representing the first: 7, 9, 16, and 23 amino
acids of oS1casein significantly stimulated the proliferation of
macrophages, from approximately 17% of total cell count in
unincubated controls, to nearly 30% of total in cells incubated
with Synthetic peptides derived from casein (FIG. 7).
[0356] Synthetic Peptides Derived from Casein Stimulate Red Blood
Cells Proliferation in CFU-GEMM:
[0357] The effect of Synthetic peptides derived from casein on red
blood cell proliferation in murine primary bone marrow cells was
demonstrated under the same conditions outlined for the stimulation
of megakaryocytes. Incubation of cells with Synthetic peptides
derived from casein representing the first 4 amino acids from the N
terminus of oS1 casein significantly stimulated the proliferation
of red blood cells, from 53% of total cell count in unincubated
controls, to 71% of total in cells incubated with the synthetic
peptide derived from casein (FIG. 7).
[0358] Synthetic Peptides Derived from Casein Stimulate
Polymorphonuclear (PMN) Cell Proliferation in CFU-GEMM:
[0359] The effect of Synthetic peptides derived from casein on the
proliferation of polymorphonuclear (PMN) cells in murine primary
bone marrow cells was demonstrated under the same conditions
outlined for the stimulation of megakaryocytes. Incubation of cells
with Synthetic peptides derived from casein representing the first:
3, 6, 7, 9, 16 and more, up to and including 26 amino acids of oS1
casein significantly stimulated the proliferation of Pmns, from
1.6% of total cell count in unincubated controls, to between 2.9%
and 14.9% of total in cells incubated with Synthetic peptides
derived from casein (FIG. 7).
[0360] Peptides Derived from Natural Casein Stimulate Hematopoiesis
in vivo Following Irradiation and Bone Marrow Transplant:
[0361] Myeloablative therapy may lead to life-threatening reduction
in thrombocytes and leukocytes, which may persist despite
administration of blood cells and growth factors. The following
demonstrates the effect of peptides derived from natural casein
following irradiation and bone marrow transplantation.
[0362] Peptides Derived from Natural Casein Enhance Leukocyte and
Platelet Reconstitution Following Syngeneic Bone Marrow
Transplantation in Mice:
[0363] When lethally irradiated (600 cGy), minimally bone
marrow-reconstituted, BALB/c mice (n=12) received 1 mg per mouse
peptides derived from natural casein via intravenous injection
along with the bone marrow cells, significant increases in
peripheral white blood cell counts on days 4, 6 and post-treatment
were noted, compared to controls receiving human serum albumin
(FIG. 8). Platelet counts in the peripheral blood of both the
treated and control irradiated, bone marrow transplanted mice were
equally depressed up to 8 days post treatment. However, by the
thirteenth day a clear advantage was noted for the mice treated
with the peptides derived from natural casein, demonstrating a
significant increase over the human serum albumin-treated controls
which became even more pronounced by day 15 (FIG. 9). Thus,
peptides derived from natural casein enhance platelet and leukocyte
reconstitution following transplantation with limiting numbers of
bone marrow cells. It is expected that this effect will be further
increased in reconstitution with optimal, rather than limiting
numbers of bone marrow cells.
[0364] Synthetic Peptides Derived from Casein Enhance Leukocyte
Reconstitution Following Syngeneic Bone Marrow Transplantation in
Mice:
[0365] When lethally irradiated (600 cGy), minimally bone
marrow-reconstituted, BALB/c mice (n=5 per synthetic peptide, n=10
in the control group) received 1 mg per mouse synthetic peptides
(15-26 amino acids in length, see Table 3) derived from casein via
an intraperitoneal injection along with the bone marrow cells, a
clear enhancement of leukocyte reconstitution was observed.
Significant increases in peripheral white blood cell counts over a
10 to 14 day period were noted with peptides having 13 (day 10:
17.2.times.10.sup.6 cells per dl; day 12: 65.4.times.10.sup.6 cells
per dl) and 17 (day 10: 27.4 cells.times.10.sup.6 per dl; day 12:
52.0.times.10.sup.6 cells per dl) amino acids (see Table 3),
compared to controls receiving human serum albumin (day 10:
16.7.times.10.sup.6 cells per dl; day 12: 46.4.times.10.sup.6 cells
per dl). Thus, synthetic peptides derived from casein enhance
leukocyte reconstitution following transplantation with limiting
numbers of bone marrow cells.
[0366] Synthetic Peptides Derived from Casein Enhance Platelet
Reconstitution Following Syngeneic Bone Marrow Transplantation in
Mice:
[0367] In order to confirm the observed ability of synthetic
peptides derived from casein to enhance megakaryocyte proliferation
in hematopoietic stem cell cultures (see FIGS. 6 and 7), the
peptides' effects on platelet reconstitution in vivo was
investigated. When lethally irradiated (800 cGy), minimally bone
marrow-reconstituted, mice (n=5 per group) received 100 .mu.g per
mouse synthetic peptides 4P and 3a (6 and 12 amino acids in length,
respectively--see Table 3) in 4 daily intraperitoneal injections
(4-7 days post-transplantation), a clear enhancement of platelet
reconstitution over untreated controls was observed. Significant
increases in platelet counts at 10 and 12 days post transplantation
were noted for both peptides. Treatment with peptide 4P increased
counts by 29% (872.times.10.sup.3/ml compared with 676
.times.10.sup.3/ml in the control group) at 12 days post
transplantation while treatment with peptide 3a increased counts by
up to 35.5% (229.times.10.sup.3/ml compared with
169.times.10.sup.3/ml in the control group) at 10 days, and up to
13.5% (622.times.10.sup.3/ml compared with 461.times.10.sup.3/ml in
the control group) at 12 days post transplantation. Thus, the same
synthetic peptides derived from casein enhance megakaryocyte
proliferation in vitro and platelet reconstitution following bone
marrow transplantation in vivo.
[0368] Peptides Derived from Natural Casein inhibit in vitro
Infection of Lymphocytic T Cell Lines by HIV-1 Virus:
[0369] In order to investigate the mechanisms of immune stimulatory
and anti-viral effects of peptides derived from natural casein,
susceptible Sup-T1 and CEM cultured human T-cells were treated with
peptides derived from natural casein prior to in vitro infection
with HIV-1 virus. Fluorescent microscopy revealed that
FITC-conjugated peptides derived from natural casein (100 .mu.g per
ml) penetrated the Sup-T1 cells when incubated therewith as
described above (FIGS. 10a-f). A small amount of label was observed
in the cytoplasm of the cells after 15 minutes (FIGS. 10a-b). At 30
minutes (FIGS. 10c-d) more label was observed in the cytoplasm,
with limited nuclear uptake. From 1 hour incubation and on (FIGS.
10e-f), FITC-labeled peptides derived from natural casein were
observed in the cytoplasm, but mostly they were concentrated in the
cell nucleus. Analysis of the Sup-T1 cells by flow cytometry
confirmed increasing uptake of the labeled peptides derived from
natural casein from 5 minutes post incubation.
[0370] Peptides Derived from Natural Casein Enhance Human
Lymphocyte Proliferation:
[0371] The presence of peptides derived from natural casein in the
culture medium resulted in increased Sup-T1 cell counts over a
period of 14 days. The greatest increases in cell number at 7 days
was observed for 50 .mu.g per ml peptides derived from natural
casein (42%), for 1000 .mu.g at 10 days (30%) and for 600 .mu.g
(32%) at 14 days incubation (data not shown).
[0372] Measurement of [.sup.3H]-thymidine incorporation by the
cultured cells, providing a proliferation index, reflected the
increase in cell number, with the most significant effect noted for
600 .mu.g per ml peptides derived from natural casein on day 10 and
50 .mu.g per ml on day 14 (FIG. 11). The reduced proliferation
indices at 14 days probably reflect cell overgrowth and nutrient
depletion.
[0373] Synthetic Peptides Derived from Casein Enhance Human
Lymphocyte Proliferation:
[0374] The presence of synthetic peptides derived from casein (all
peptides listed in Table 3) in the culture medium resulted in
increased Sup-T1 cell counts over a period of 10 days. The increase
was similar for all synthetic peptides. The greatest increases in
lymphocyte cell number in infected cells were observed for 250
.mu.g and 500 .mu.g per ml of peptide representing the first 9
amino acids (80% and 33%, respectively) (data not shown).
[0375] Peptides Derived from Natural Casein Inhibit HIV-1 Infection
in Human Lymphocyte Cells:
[0376] Susceptible CEM lymphocyte cells pretreated with peptides
derived from natural casein (50-1000 .mu.g per ml) 3, 24 or 48
hours prior to incubation with HIV-1 exhibited enhanced cell
proliferation and reduced levels of viral infection compared to
untreated controls. Cell counts and HIV-1 P24 antigen assay at 15
days post infection revealed 100% inhibition of viral infection
after 3 hours incubation with 600-1000 .mu.g per ml peptides
derived from natural casein and 98% and 99% inhibition after 24
hours incubation with 50 and 600 .mu.g per ml peptides,
respectively (comparing cell numbers with uninfected controls UIF).
Longer incubation times were not found to be more effective (FIG.
12). Although increasing concentrations of peptides derived from
natural casein enhanced cell proliferation at 3 and 24 hours post
infection, viral infection is most significantly inhibited in these
fastest growing cultures. An even more dramatic enhancement of cell
proliferation and inhibition of HIV-1 infection was observed in
Sup-T1 cells pretreated with peptides derived from natural casein
before HIV-1 infection (average inhibition of viral infection of
96.7%, 88.7% and 95.7% for 3 hours, 24 hours and 48 hours
pretreatment, respectively) (not shown). Thus, peptides derived
from natural casein penetrate human cultured lymphocyte cells and
their nuclei, enhance cell growth, and significantly reduce the
susceptibility of CD.sub.4+cells to HIV-1 infection. As such,
peptides derived from natural casein are expected to be useful both
at preventing HIV infection and for post infection treatment of HIV
infected and AIDS patients.
[0377] Synthetic Peptides Derived from Casein Inhibit HIV-1
Infection in Human Lymphocyte Cells:
[0378] The ability of Synthetic peptides derived from casein to
inhibit HIV-1 infection in human lymphocyte cells was demonstrated
using CEM-lymphocyte cells under the same conditions outlined
above. Three hours pretreatment of CEM lymphocytes with the
synthetic peptide derived from casein representing the first 3
amino acids of oS1 casein conferred a significant degree of
resistance to infection following incubation with HIV-1. Lymphocyte
cell numbers were 1.29.times.10.sup.6 (100 .mu.g per ml) and
2.01.times.10.sup.6 (500.mu.g per ml) in the treated cells as
compared to the infected HIV-1 control of 1.06.times.10.sup.6 (FIG.
13). HIV-1 infection levels in the same cells, measured by the
HIV-P.sup.24 antigen assay at 7 days post infection, was
significantly reduced in the peptide treated cells (0.17 and 0.14ng
P24 Antigen/ml with 100 .mu.g/ml and 500.mu.g/ml respectively), as
compared to the untreated controls(0.52 ng P Ag/ml).
[0379] Likewise, significant inhibition of HIV-1 infection was
observed in the CEM cells pre-treated (3 hours) with the synthetic
casein derived peptide representing the first 5 amino acids of oS1
casein.
[0380] Cell counts in the cultures incubated with 10 and 25.mu.g
peptide 3P per ml were 1.17.times.10.sup.6 and 1.26.times.10.sup.6
respectively, as compared to the infected HIV-1 control of
1.06.times.10.sup.6.
[0381] HIV-P24 antigen assay at 7 days post infection, revealed
significant reduction in HIV-1 infection levels in treated cultures
(0.26 and 0.18ng P24 Ag per ml for 10 and 25.mu.g per ml
respectively, as compared to the control of s15 0.52 ng P24 Ag per
ml).
[0382] Likewise, 3 hours preincubation with the synthetic peptide
derived from casein4P, representing the first 6 amino acids of
oS1casein had a significant effect on the susceptibility of CEM
lymphocyte cells to infection with HIV-1. Cell numbers were most
affected at concentrations of 25 and 250.mu.g per ml
(1.26.times.10.sup.6 , and 1.59.times.10.sup.6 respectively, as
compared to the infected control value of 1.06.times.10.sup.6).
[0383] Assay of HIV-P.sup.24 antigen at 7 days post infection,
revealed a dose dependent reduction in viral particles as compared
to the untreated, infected control cultures (FIG. 13).Thus, the
protection from HIV-1 infection afforded lymphocyte cells by the
peptides derived from natural casein is retained in Synthetic
peptides derived from casein representing as few as the first five
N-terminal amino acids of .alpha.S-1 casein.
[0384] Peptides Derived from Natural Casein Prevent Development of
Glucosuria in Non-Obese Diabetic (NOD) mice: Non-Obese Diabetic
(NOD) mice spontaneously develop Juvenile (Type I, IDDM) Diabetes,
an autoimmune condition causing inflammation of the pancreatic
.beta. cells and ending in disease and death. Female NOD mice are
extremely susceptible, demonstrating evidence of macrophage
invasion of the pancreatic islet interstitial matrix as early as 5
weeks old. A once or twice weekly injection of 100 .mu.g peptides
derived from natural casein for 5 weeks (6 or 11 injections total)
were completely effective in preventing the glucosuria associated
with the onset and course of the disease. By 200 days 100% of the
untreated control mice (n=5) had become diabetic, and subsequently
died, while the treated mice (n=5) remained 100% euglycemic, all
still surviving at 365 days (FIG. 14). Thus, peptides derived from
natural casein effectively protected genetically susceptible mice
against the onset of this autoimmune inflammatory condition.
[0385] Synthetic Peptides Derived from Casein Prevent Development
of Glucosuria in Non-Obese Diabetic (NOD) Mice:
[0386] The preventative effect of Synthetic peptides derived from
casein on the development of glucosuria in NOD mice was
demonstrated under the same conditions outlined above, except that
the mice were injected only once weekly for five (5) weeks with 100
.mu.g of Synthetic peptides derived from casein. The results of
these experiments are presented in Table 4 below:
4TABLE 4 The effect of synthetic peptides on IDDM in NOD mice
Peptide IPGT TEST Derivative Urine 0 min. 60 min. code
Healthy/Total* Sugar (pre-load) post load Y 1/5 Negative 121 138 X
3/5 Negative 94 114 Negative 104 119 Negative 141 114 1a 1/5
Negative 88 106 2a 4/5 Negative 215 183 Negative 112 119 Negative
95 107 Negative 159 204 3a 3/5 Negative 135 137 Negative 205 197
Negative 201 211 A 2/5 Negative 134 164 Negative 105 107 B 2/5
Negative 130 117 Negative 130 97 D 2/5 Negative 99 108 Negative 130
136 I 2/5 Negative 324 not tested Negative 124 138 J 3/5 Negative
166 not tested Negative 193 not tested Negative 186 not tested K
2/5 Negative 116 143 Negative 443 not tested Chay-13 2/5 Negative
123 130 Negative 111 111 Chay-13 2/5 Negative 128 116 Negative 113
125 Control 0/5 Blood was drawn from the paraorbital plexus at 0
min and 60 min after the intraperitoneal injection of glucose 1
g/kg body weight. Plasma glucose levels were determined with a
Glucose Analyzer 2 (Beckman lnstruments, Fullerton, CA) and
expressed as mmol/L. *Healthy and well = Sugar not detected in
urine. Glucosuria = >1000 mg/dL. IPGTT performed with 6 healthy
female control mice: 0 min- 110 mmol/L; 60 min- 106 mmol/L blood
glucose.
[0387] The synthetic peptides derived from casein representing the
first 9 (X), 11 (2a) and 12 (3a) amino acids and higher chain
length of oS1 casein, were highly effective in preventing the
glucosuria associated with the onset and course of the disease.
[0388] Effect of treatment with synthetic peptides derived from
casein was evaluated after 25 weeks. At that time, all 5 mice in
the untreated control group (n=5) had become diabetic, as indicated
by the presence of frank (>1000 mg/dl) glucosuria (Table 4).
[0389] No glucosuria was detected in three of the five (3/5) NOD
mice treated with the synthetic peptide representing the first nine
(9) amino acids from the N terminal of oS1 casein. Of the group
injected with the synthetic peptide of eleven (11) amino acids from
the N terminal of oS1 casein, no glucosuria was detected in four
out of five (4/5) of the NOD mice In the groups of peptide treated
mice in which glucosuria was detected, the onset was generally
significantly delayed (by 3-5 weeks) relative to the onset of
glucosuria in untreated controls (data not shown), indicating a
clearly protective effect of the peptides even when incomplete.
[0390] The protective effects of shorter Synthetic peptides derived
from casein have also been studied in NOD mice. In an additional
series of experiments similar to the abovementioned, administration
of peptides representing the first 3 (1P) and 4 (2P) N-terminal
amino acids of oS1 casein effectively prevented the onset of
glucosuria in the treated mice (assayed at week 16), while the
untreated controls had all become diabetic (100% glucosuria) (data
not shown).
[0391] The glucose tolerance (IPGT) test performed after 25 weeks
with the healthy and well NOD mice, of the group injected with the
synthetic casein derived peptide of the first 9 amino acids, showed
no evidence of abnormal glucose metabolism (normal glycemic values
pre- and 60 minutes post- glucose loading).
[0392] In the group treated with the synthetic peptide derived from
casein representing the first 11 amino acids of the N-terminal of
oS1 casein (2a), resting plasma glucose levels were somewhat
elevated in two of the five mice (215 and 159 mmol/L), and remained
mildly elevated at (183 and 204 mmol/L) 60 minutes post load,
indicating mild diabetic tendencies. The other two mice remained
within normal glycemic range throughout the test (Table 4). In
general, the normal results of the IPGTT reflected the absence of
glucosuria in the healthy, surviving peptide-treated mice (Table
4). Thus, synthetic peptides representing only a few amino acids
from the N-terminal of .alpha.S 1 casein, as well as peptides
derived from native casein dramatically reduce the susceptibility
of genetically predisposed NOD mice to onset of autoimmune diabetic
disease.
[0393] Synthetic Casein-Derived Peptides Significantly Reduce Total
Cholestrol Blood Levels (TC), Low Density Lipoprotein (LDL) and
High Density Lipoprotein (HDL):
[0394] Intraperitoneal administration of Synthetic peptides derived
from casein caused a significant reduction in the blood lipid (HDL,
LDL and TC) values in experimentally hypercholesterolemic mice.
After one week of the atherogenic Thomas Hartroft diet, the blood
cholesterol levels of the mice had risen to the levels of 31
8mg/dl.
[0395] One week post treatment with 1 mg synthetic peptides derived
from casein per mouse, the group treated with the Synthetic
peptides derived from casein representing the first 5 (3P) and 11
(2a) amino acids of oS1 casein, had significantly reduced TC, HDL
and LDL values, compared to those of the control group [TC: 308 and
279mg/dl respectively; HDL: 41mg/dl and 40mg/dl respectively and
LDL: 247mg/dl and 221mg/dl respectively as compared to 393mg/dl
(TC), 52 mg/dl (HDL) and 326 mg/dl (LDL) in the diet-induced
hypercholesterol-/hyperlipidemic control group] (FIG. 15). Thus,
synthetic peptides representing the first few N-terminal amino
acids of .alpha.S 1 casein effectively reduced experimentally
induced hyperlipidemia and hypercholesterolemia within 1 week after
a single, intraperitoneal administration.
[0396] Clinical Trials with Peptides Derived from Natural
Casein:
[0397] Patients received intramuscular injections of 50 mg peptides
derived from natural casein each, in one or three depots, as
indicated.
[0398] Peptides derived from natural casein stimulates
hematopoiesis in cancer patients: The hematology profiles of six
cancer patients who had received or were receiving chemotherapy
were examined before and following administration of peptides
derived from natural casein, as indicated. Special attention was
paid to changes in the Platelet (PLT), Leukocyte (WBC), Erythrocyte
(RBC) and Hemoglobin (HGB) values, representing thrombocytopoiesis,
leukocytopoiesis, and erythrocytopoiesis, respectively.
[0399] G.T., (Female Patient, Patient 1):
[0400] Patient had ovarian cancer, undergone a hysterectomy
followed by chemotherapy. She received two intramuscular injections
of peptides derived from natural casein at two and then two and one
half months post operation. No chemotherapy was administered
between the first and second administrations of peptides derived
from natural casein. Blood tests from 6 days post first injection,
7, and 13 days post second injection reflect a considerable
increase in platelet and WBC components, as well as increased RBC
(FIG. 16).
[0401] E.C., (Female Patient, Patient 2):
[0402] Patient underwent a radical mastectomy for lobular carcinoma
in 1983, and six years later suffered from gastric metastases.
Three days prior to commencement of chemotherapy, she received one
intramuscular peptides derived from natural casein by injection,
and a second 10 days after the chemotherapy. Although the blood
counts from 10 and 16 days post chemotherapy indicated an
attenuation of the depressed hematological profile usually
encountered following chemotherapy, the most significant effects of
peptides derived from natural casein were noted 3 days after the
first injection, prior to the chemotherapy (FIG. 16).
[0403] E.S., (Female Patient, Patient 3):
[0404] Patient was suffering from widespread metastatic
dissemination of a breast carcinoma first discovered in 1987. Two
years later, she received a first intramuscular injection of
peptides derived from natural casein, and a second 23 days later.
No additional therapy was administered during this period. Blood
tests indicate a strong enhancement of PLT seven days after the
first treatment and a significant increase in RBC and WBC seven
days after the second treatment (FIG. 16).
[0405] JR., (Female Patient, Patient 4):
[0406] Patient's diagnosis is breast cancer with bone metastases.
She received one intramuscular injection of peptides derived from
natural casein 8 days before commencing chemotherapy, and another,
14 days later. The most significant effect is clearly seen in the
rapid return of WBC levels following chemotherapy-induced
depression (FIG. 16).
[0407] D.M., (Female Patient, Patient 5):
[0408] Patient suffering from hepatic cancer with widespread
metastatic dissemination. She received three intramuscular
injections of peptides derived from natural casein at 10, 8 and 6
days before receiving chemotherapy. A second series of injections
was initiated 10, 12 and 14 days following the chemotherapy
treatment. Although a significant effect on the hematological
profile is noted following the first series of injections and prior
to the chemotherapy, the most dramatic improvements are seen in the
rapid return of depressed post-chemotherapy values to normalized
cell counts following the second series of peptides derived from
natural casein injections (FIG. 16).
[0409] Thus, administration of peptides derived from natural casein
to cancer patients results in improved hematological profiles,
specifically enhanced erythropoiesis, leukocytopoiesis and
thrombocytopoiesis, and is capable of moderating and shortening the
duration of chemotherapy-induced depression of blood
components.
[0410] Peptides Derived from Natural Casein Stimulates
Thrombocytopoiesis in Transplant Recipients With Resistant
Thrombocytopenia:
[0411] Prolonged transfusion-resistant thrombocytopenia with
episodes of severe bleeding, may be a life threatening complication
of bone marrow transfusion, especially where traditional therapies
are ineffective. Two patients with severe resistant
thrombocytopenia were treated with peptides derived from natural
casein.
[0412] M-1 (Female Patient):
[0413] 32 year old patient suffering from Acute Myeloid Leukemia in
complete remission, following autologous stem cell transfusion. She
had experienced two life-threatening bleeding episodes, involving
pulmonary hemorrhage and a large obstructive hematoma in the soft
palate. At more than 114 days post transfusion, platelet counts
were refractive to rhIL-3, rhIL-6, intravenous gamma globulin, and
recombinant erythropoietin. Following a single intra muscular
treatment with 50 mg peptides derived from natural casein, divided
into three depots, her condition improved immediately. Along with
the rapid return of normal platelet counts (FIG. 17), her distal
limb bleeding with exertion and patechyae subsided, she was able to
resume walking, and returned to her home overseas with no
complications or side effects.
[0414] M-2 (Male Patient):
[0415] 30 year old patient suffering from Acute Myeloid Leukemia in
a second complete remission following autologous stem cell
transfusion, exhibiting totally resistant platelet counts and
massive gastrointestinal bleeding episodes. He required daily
transfusions of packed cells, had developed hypoalbuminia, and
failed to respond to extensive therapy with rhIL-3, rhIL-6 and
gamma globulin. Following one intramuscular administration of 50 mg
peptides derived from natural casein in three depots 86 days post
transfusion, rapid platelet reconstitution (FIG. 18) and gradual
discontinuation of the bleeding was observed. No further treatment
was required, and the patient is presently completely asymptomatic
with normal platelet count.
[0416] Thus, one course of intramuscular administration of peptides
derived from natural casein at 1 mg per kg body weight, divided
into three depots was effective in rapidly reconstituting platelet
counts and diminishing associated clinical symptoms in patients
suffering from prolonged, transfusion resistant thrombocytopenia
with life-threatening bleeding episodes.
[0417] Peptides Derived from Natural Casein Decreases Triglycerides
and LDL-cholesterol in Familial Hyperlipidemia:
[0418] M.S. (Female Patient):
[0419] Patient is a 38 year old female with family history of
hyperlipidemia. Before treatment with peptides derived from natural
casein, blood chemistry profile revealed elevated total cholesterol
(321 mg per dl), triglycerides (213 mg per dl; normal range 45-185
mg per dl) and elevated LDL-cholesterol (236.4 mg per dl; normal
range 75-174 mg per dl). One month after administration of 50 mg
peptides derived from natural casein in three intra muscular depots
the hyperlipidemia was stabilized: total cholesterol was reduced to
270 mg per dl, triglycerides were 165 mg per dl and LDL-cholesterol
was 201 mg per dl, still higher than normal range but significantly
reduced from the pretreatment value. No additional treatment was
administered. Thus, treatment with peptides derived from natural
casein is effective in rapidly bringing about a significant
reduction in otherwise untreated hyperlipidemia in humans.
[0420] Peptides Derived from Natural casein Stimulate
Normoglobinemia in a Case of Occult Bleeding:
[0421] D. G. (Male Patient):
[0422] Patient is a 75 year old male suffering from anemia and
hypoglobinemia (depressed RBC, HGB, HCT, MCH and MCHC) associated
with extensive occult bleeding. One month after receiving one
intramuscular injection of 50 mg peptides derived from natural
casein in three depots, a significant reduction of the anemia was
observed. After two months, RBC approached normal values (4.32
instead of 3.44 M per .mu.l), HGB increased (11.3 instead of 8.9 g
per dl) and HCT, MCH and MCHC all improved to nearly normal values,
despite the persistence of occult bleeding. Thus, one injection of
peptides derived from natural casein seemed capable of stimulating
erythropoiesis and reducing anemia associated with blood loss in
humans.
[0423] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable sub
combination.
[0424] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims. All
publications, patents, patent applications and sequences identified
by an accession number, mentioned in this specification are herein
incorporated in their entirety by reference into the specification,
to the same extent as if each individual publication, patent,
patent application or sequence was specifically and individually
indicated to be incorporated herein by reference. In addition,
citation or identification of any reference in this application
shall not be construed as an admission that such reference is
available as prior art to the present invention.
* * * * *