U.S. patent application number 10/114998 was filed with the patent office on 2002-11-28 for tea catechins in sustained release formulations as cancer specific proliferation inhibitors.
This patent application is currently assigned to Purdue Research. Invention is credited to Chang, Michael N., Cooper, Raymond, Morre, D. James, Morre, Dorothy M..
Application Number | 20020176898 10/114998 |
Document ID | / |
Family ID | 27065413 |
Filed Date | 2002-11-28 |
United States Patent
Application |
20020176898 |
Kind Code |
A1 |
Morre, Dorothy M. ; et
al. |
November 28, 2002 |
Tea catechins in sustained release formulations as cancer specific
proliferation inhibitors
Abstract
The invention described herein encompasses a methods and
compositions of treating cancer or solid tumors comprising the
administration of a therapeutically effective amount of catechins,
a group of polyphenols found in green tea, to a mammal in need of
such therapy. Compositions of catechins include but not limited to,
epigallocatechin gallate (EGCg), epicatechin (EC), epicatechin
gallate (ECG), epigallocatechin (EGC). The unique compositions of
the invention contain various combinations of the catechins, alone
or in combination with each other or other therapeutic agents and
are used to treat primary and metastatic cancers in humans. The
invention also encompasses the varying modes of administration of
the therapeutic compounds, including a sustained release
formulation which may be used as a therapeutic compound for the
treatment of cancer or as a dietary supplement for the prevention
of cancer.
Inventors: |
Morre, Dorothy M.; (West
Lafayette, IN) ; Morre, D. James; (West Lafayette,
IN) ; Cooper, Raymond; (Mountain View, CA) ;
Chang, Michael N.; (Brisbane, CA) |
Correspondence
Address: |
PENNIE AND EDMONDS
1155 AVENUE OF THE AMERICAS
NEW YORK
NY
100362711
|
Assignee: |
Purdue Research
|
Family ID: |
27065413 |
Appl. No.: |
10/114998 |
Filed: |
April 3, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10114998 |
Apr 3, 2002 |
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09637840 |
Aug 10, 2000 |
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6410052 |
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09637840 |
Aug 10, 2000 |
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09537211 |
Mar 29, 2000 |
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6410061 |
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60126893 |
Mar 30, 1999 |
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60151109 |
Aug 27, 1999 |
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Current U.S.
Class: |
424/729 ;
514/27 |
Current CPC
Class: |
A61K 31/353 20130101;
A61K 36/82 20130101; A61K 2300/00 20130101; A61K 31/35 20130101;
A61K 9/2081 20130101; A61K 31/35 20130101; A61K 9/5047 20130101;
A61K 45/06 20130101; A23L 33/105 20160801 |
Class at
Publication: |
424/729 ;
514/27 |
International
Class: |
A61K 035/78; A61K
031/7048 |
Claims
What is claimed is:
1. A composition of a sustained release formulation of tea
catechins comprising tea catechins and at least one component which
controls the release of said catechins.
2. The composition of claim 1 wherein at least 30% of said
catechins is EGCg.
3. The composition of claim 2 wherein EGCg comprises about 35% to
about 45% of the catechins.
4. The composition of claim 2 wherein EGCg comprises about 40% of
the catechins.
5. The composition of claim 1 wherein at least 30% of said
catechins is EGCg and at least 5% of said catechins is ECG based on
the total amount of catechins present in said formulation.
6. The composition of claim 5 wherein EGCg comprises about 35% to
about 45% of the catechins and ECG comprises about 10% to about 20%
of the catechins.
7. The composition of claim 5 wherein EGCg comprises about 40% of
the catechins and ECG comprises about 15% of the catechins.
8. The composition of claim 1 wherein at least 30% of said
catechins is EGCg and at least 3% of said catechins is EC based on
the total amount of catechins present in said formulation.
9. The composition of claim 8 wherein EGCg comprises about 35% to
about 45% of the catechins and EC comprises about 3% to about 15%
of the catechins.
10. The composition of claim 8 wherein EGCg comprises about 40% of
the total catechins and EC comprises about 7% of the catechins.
11. The composition of claim 1 wherein EGCg and EC comprise the
catechins, EGCg comprises at least 0.01% of said catechins, and the
EC content is at least 10 fold greater than the EGCg content.
12. The composition of claim 11 wherein the EC content is at least
100 fold greater than the EGCg content.
13. The composition of claim 11 wherein the EC content is at least
1000 fold greater than the EGCg content.
14. The composition of claim 1 wherein at least 30% of said
catechins is EGCg and at least 1% of said catechins is EGC based on
the total amount of catechins present in said formulation.
15. The composition of claim 14 wherein EGCg comprises about 35% to
about 45% of the catechins and EGC comprises about 2% to about 5%
of the catechins.
16. The composition of claim 14 wherein EGCg comprises about 40% of
the total catechins and EGC comprises about 3% of the
catechins.
17. The composition of claim 1 wherein at least 30% of said
catechins is EGCg, at least 3% of said catechins is EC, and at
least 5% of said catechins is ECG based on the total amount of
catechins present in said formulation.
18. The composition of claim 17 wherein EGCg comprises about 35% to
about 45% of the catechins, EC comprises about 3% to about 15% of
the catechins, and ECG comprises about 10% to about 20% of the
catechins.
19. The composition of claim 17 wherein EGCg comprises about 45% of
the catechins, EC comprises about 7% of the catechins, and ECG
comprises about 15% of said catechins in said formulation.
20. The composition of claim 1 wherein at least 30% of said
catechins is EGCg, at least 3% of said catechins is EC, and at
least 1% of said catechins is EGC based on the total amount of
catechins present in said formulation.
21. The composition of claim 20 wherein EGCg comprises about 35% to
about 45% of the catechins, EC comprises about 3% to about 15% of
said catechins, and EGC comprises about 2% to about 5% of the
catechins.
22. The composition of claim 20 wherein EGCg comprises about 45% of
the catechins, EC comprises about 7% of the catechins, and EGC
comprises about 3% of the catechins.
23. The composition of claim 1 wherein at least 30% of said
catechins is EGCg, at least 3% of said catechins is EC, at least 5%
of said catechins is ECG, and at least 1% of said catechins is EGC
based on the total amount of catechins present in said
formulation.
24. The composition of claim 23 wherein EGCg comprises about 35% to
about 45% of the catechins, EC comprises about 3% to about 15% of
the catechins, ECG comprises about 10% to about 20% of the
catechins, and EGC comprises about 2% to about 5% of the
catechins.
25. The composition of claim 23 wherein EGCg comprises about 45% of
the catechins, EC comprises about 7% of the catechins, ECG
comprises about 15% of the catechins, and EGC comprises about 3% of
the catechins.
26. The composition of claim 1 wherein at least 30% of said
catechins is EGCg, at least 3% of said catechins is EC, at least 5%
of said catechins is ECG, at least 1% of said catechins is EGC, and
at least 5% of said catechins is C based on the total amount of
catechins present in said formulation.
27. The composition of claim 26 wherein EGCg comprises about 35% to
about 45% of the catechins, EC comprises about 3% to about 15% of
the catechins, ECG comprises about 10% to about 20% of the
catechins, EGC comprises about 2% to about 5% of the catechins, and
C comprises about 10% to about 20% of the catechins.
28. The composition of claim 26 wherein EGCg comprises about 45% of
the catechins, EC comprises about 7% of the catechins, ECG
comprises about 15% of the catechins, EGC comprises about 3% of the
catechins, and C comprises about 15% of the catechins.
29. The composition of claim 1, which is a dietary or nutritional
supplement, wherein the composition is formulated as an oral
preparation comprising tablets, capsules, gelcaps, or powders.
30. The composition of claim 1, which is a dietary or nutritional
supplement, wherein the composition is formulated as a sterile
preparation.
31. The composition of claim 1, which is a dietary or nutritional
supplement, wherein the composition is formulated as a parenteral
solution.
32. The composition of claim 1, which is a dietary or nutritional
supplement, wherein the component is a polymer matrix, gel,
permeable membrane, osmotic system, multilayer coating,
microparticle, liposome, or microsphere.
33. The composition of claim 32 wherein the polymer matrix is
cellulose.
34. The composition of claim 33 wherein the cellulose is
hydroprophylmethyl cellulose.
35. The composition of claim 1, which is a dietary or nutritional
supplement, wherein the composition is microencapsulated.
36. The composition of claim 35 wherein the composition is
microencapsulated with microcrystalline cellulose, maltodextrine,
ethylcellulose, and magnesium stearate.
37. The pharmaceutical composition of claim 1 wherein the
composition is formulated as an oral preparation comprising
tablets, capsules, gelcaps, or powders.
38. The pharmaceutical composition of claim 1 wherein the
composition is formulated as a sterile preparation.
39. The pharmaceutical composition of claim 1 wherein the
composition is formulated as a parenteral solution.
40. The pharmaceutical composition of claim 1 wherein the component
is a polymer matrix, gel, permeable membrane, osmotic system,
multilayer coating, microparticle, liposome, or microsphere.
41. The pharmaceutical composition of claim 40 wherein the polymer
matrix is cellulose.
42. The pharmaceutical composition of claim 41 wherein the
cellulose is hydroprophylmethyl cellulose.
43. The pharmaceutical composition of claim 1 wherein the
composition is microencapsulated.
44. The pharmaceutical composition of claim 43 wherein the
composition is microencapsulated with microcrystalline cellulose,
maltodextrine, ethylcellulose, and magnesium stearate.
45. A method of preventing cancer in a mammal, wherein said cancer
is a type having cancer cells which express tNOX, which comprises
administering to a mammal desirous of preventing cancer, an
effective dose of a sustained release formulation of tea catechins
so that the cancer cells which express tNOX do not accumulate to a
number which creates clinical symptoms associated with said
cancer.
46. The method of claim 45 wherein the reduction in the number of
cancer cells is a result of cell death.
47. The method of claim 45 wherein the reduction in the number of
cancer cells is a result of inhibition of cell growth.
48. The method of claim 45 wherein the reduction in the number of
cancer cells is a result of cell growth arrest.
49. The method of claim 45 wherein the mammal is a human.
50. The method of claim 49 wherein the cancer is selected from a
group comprising rectal carcinoma, colon carcinoma, breast
carcinoma, ovarian carcinoma, small cell lung carcinoma, colon
carcinoma, chronic lymphocytic carcinoma, hairy cell leukemia,
osophogeal carcinoma, prostate carcinoma, breast cancer, myeloma,
and lymphoma.
51. The method of claim 49 wherein the human is immunosuppressed by
reason of having undergone anti-cancer therapy prior to
administration of said formulation comprising catechins.
52. The method of claim 45 wherein at least 30% of said catechins
is EGCg.
53. The method of claim 52 wherein EGCg comprises about 35% to
about 45% of the catechins.
54. The method of claim 52 wherein EGCg comprises about 40% of the
catechins.
55. The method of claim 45 wherein at least 30% of said catechins
is EGCg and at least 5% of said catechins is ECG based upon the
total amount of catechins present in said formulation.
56. The method of claim 55 wherein EGCg comprises about 35% to
about 45% of the catechins and ECG comprises about 10% to about 20%
of the catechins.
57. The method of claim 55 wherein EGCg comprises about 40% of the
catechins and ECG comprises about 15% of the catechins.
58. The method of claim 45 wherein at least 30% of said catechins
is EGCg and at least 3% of said catechins is EC based upon the
total amount of catechins present in said formulation.
59. The method of claim 58 wherein EGCg comprises about 35% to
about 45% of the catechins and EC comprises about 3% to about 15%
of the catechins.
60. The method of claim 58 wherein EGCg comprises about 40% of the
total catechins and EC comprises about 7% of the catechins.
61. The method of claim 45 wherein EGCg and EC comprise the
catechins, EGCg comprises at least 0.01% of said catechins, and the
EC content is at least 10 fold greater than the EGCg content.
62. The method of claim 61 wherein the EC content is at least 100
fold greater than the EGCg content.
63. The method of claim 61 wherein the EC content is at least 1000
fold greater than the EGCg content.
64. The method of claim 45 wherein at least 30% of said catechins
is EGCg and at least 1% of said catechins is EGC based upon the
total amount of catechins present in said formulation.
65. The method of claim 64 wherein EGCg comprises about 35% to
about 45% of the catechins and EGC comprises about 2% to about 5%
of the catechins.
66. The method of claim 64 wherein EGCg comprises about 40% of the
total catechins and EGC comprises about 3% of the catechins.
67. The method of claim 45 wherein at least 30% of said catechins
is EGCg, at least 3% of said catechins is EC, and at least 5% of
said catechins is ECG based upon the total amount of catechins
present in said formulation.
68. The method of claim 67 wherein said formulation comprises EGCg,
EC, and ECG wherein EGCg comprises about 35% to about 45% of the
catechins, EC comprises about 3% to about 15% of the catechins, and
ECG comprises about 10% to about 20% of the catechins.
69. The method of claim 67 wherein said formulation comprises EGCg,
EC, and ECG wherein EGCg comprises about 45% of the catechins, EC
comprises about 7% of the catechins, and ECG comprises about 15% of
the catechins.
70. The method of claim 45 wherein at least 30% of said catechins
is EGCg, at least 3% of said catechins is EC, and at least 1% of
said catechins is EGC in said formulation.
71. The method of claim 70 wherein said formulation comprises EGCg,
EC, and EGC wherein EGCg comprises about 35% to about 45% of the
catechins, EC comprises about 3% to about 15% of the catechins, and
EGC comprises about 2% to about 5% of the catechins.
72. The method of claim 70 wherein said formulation comprises EGCg,
EC, and EGC wherein EGCg comprises about 45% of the catechins, EC
comprises about 7% of the catechins, and EGC comprises about 3% of
the catechins.
73. The method of claim 45 wherein at least 30% of said catechins
is EGCg, at least 3% of said catechins is EC, at least 5% of said
catechins is ECG, and least 1% of said catechins is EGC based upon
the total amount of catechins present in said formulation.
74. The method of claim 73 wherein said formulation comprises EGCg,
EC, and ECG wherein EGCg comprises about 35% to about 45% of the
catechins, EC comprises about 3% to about 15% of the catechins, ECG
comprises about 10% to about 20% of the catechins, and EGC
comprises about 2% to about 5% of the catechins.
75. The method of claim 73 wherein said formulation comprises EGCg,
EC, and ECG wherein EGCg comprises about 45% of said catechins, EC
comprises about 7% of said catechins, ECG comprises about 15% of
said catechins, and EGC comprises about 3% of said catechins in
said formulation.
76. The method of claim 45 wherein at least 30% of said catechins
is EGCg, at least 3% of said catechins is EC, at least 5% of said
catechins is ECG, at least 1% of said catechins is EGC, and at
least 5% of said catechins is C based upon the total amount of
catechins present in said formulation.
77. The method of claim 76 wherein said formulation comprises EGCg,
EC, and ECG wherein EGCg comprises about 35% to about 45% of the
catechins, EC comprises about 3% to about 15% of the catechins, ECG
comprises about 10% to about 20% of the catechins, EGC comprises
about 2% to about 5% of the catechins, and C comprises about 10% to
about 20% of the catechins.
78. The method of claim 76 wherein said formulation comprises EGCg,
EC, and ECG wherein EGCg comprises about 45% of the catechins, EC
comprises about 7% of the catechins, ECG comprises about 15% of the
catechins, EGC comprises about 3% of the catechins, and C comprises
about 15% of the catechins.
79. The method of claim 45 wherein said formulation is an ionic
aqueous solution of the composition comprising tea catechins.
80. The method of claim 45 wherein the total daily amount
administered is from about 10 to about 100,000 mg of the
composition comprising tea catechins.
81. The method of claim 45 wherein the formulation is provided at
constant levels in the sera for at least 48 hours.
82. The method of claim 45 wherein the formulation is provided at
constant levels in the sera for at least 72 hours.
83. A method for treating cancer in a mammal which comprises
administering to a mammal in need of cancer treatment, wherein said
cancer is a type having cancer cells which express tNOX, a
therapeutically effective dose of a sustained release formulation
of tea catechins so that there is a reduction in the number of
cancer cells in the body.
84. The method of claim 83 wherein the reduction in the number of
cancer cells is a result of cell death.
85. The method of claim 83 wherein the reduction in the number of
cancer cells is a result of inhibition of cell growth.
86. The method of claim 83 wherein the reduction in the number of
cancer cells is a result of cell growth arrest.
87. The method of claim 83 wherein the mammal is a human.
88. The method of claim 87 wherein the cancer is selected from a
group comprising rectal carcinoma, colon carcinoma, breast
carcinoma, ovarian carcinoma, small cell lung carcinoma, colon
carcinoma, chronic lymphocytic carcinoma, hairy cell leukemia,
osophogeal carcinoma, prostate carcinoma, breast cancer, myeloma,
and lymphoma.
89. The method of claim 87 wherein the human is immunosuppressed by
reason of having undergone anti-cancer therapy prior to
administration of said formulation comprising catechins.
90. A method for treating a solid tumor in a mammal, wherein the
solid tumor comprises cancer cells which express tNOX, which
comprises administering to a mammal, a therapeutically effective
dose of a sustained release formulation of tea catechins so that
there is a reduction in the number of cancer cells in the
tumor.
91. The method of claim 90 wherein the reduction in the number of
cancer cells is a result of cell death.
92. The method of claim 90 wherein the reduction in the number of
cancer cells is a result of inhibition of cell growth.
93. The method of claim 90 wherein the reduction in the number of
cancer cells is a result of cell growth arrest.
94. The method of claim 90 wherein the mammal is a human.
95. The method of claim 94 wherein the tumor is a tumor of
epithelial tissue, lymphoid tissue, connective tissue, bone, or
central nervous system.
96. The method of claim 90 in which said administration is made
parenterally, orally, or directly into the tumor.
97. A method for treating metastases in a human which comprises
administering to a human having a primary cancer, wherein said
cancer is a type having cancer cells which express tNOX, a
therapeutically effective dose of a sustained release formulation
of tea catechins so that there is a reduction in the number of
cancer cells in the body.
98. The method of claim 97 wherein the reduction in the number of
cancer cells is a result of cell death.
99. The method of claim 97 wherein the reduction in the number of
cancer cells is a result of inhibition of cell growth.
100. The method of claim 97 wherein the reduction in the number of
cancer cells is a result of cell growth arrest.
101. A method for treating cancer in a mammal which comprises
administering to a mammal in need of cancer treatment, wherein said
cancer is a type having cancer cells which express tNOX, a
therapeutically effective dose of a sustained release formulation
of tea catechins, or a pharmaceutically acceptable salt thereof, in
combination with an effective amount of at least one other
chemotherapeutic agent so that there is a reduction in the number
of cancer cells in the body.
102. The method of claim 101 in which said other anti-cancer agent
is selected from the group consisting of adriamycin and adriamycin
conjugates, mechlorethamine, cyclophosphamide, ifosfamide,
melphalan, chlorambucil, hexamethylmelamine, thiotepa, busulfan,
carmustine, lomustine, semustine, streptozocin, dacarbazine,
methotrexate, fluorouacil, floxuridie, cytarabine, mercaptopurine,
thioguanine, pentostatin, vinblastine, vincristine, etoposide,
teniposide, actinomycin D, daunorubicin, doxorubicin, bleomycin,
plicamycin, mitomycin, L-asparaginase, interferon-alpha, cisplatin,
carboplatin, mitoxantrone, hydroxyurea, procarbazine, mitotane,
aminoglutethimide, prednisone, hydroxyprogesterone caproate,
medroxyprogesterone acetate, megestrol acetate, diethylstilbestrol,
ethinyl estradiol, tamoxifen, testosterone propionate,
fluoxymesterone, flutamide, leuprolide, acetogenins, e.g.,
bullatacin, and quassanoids, e.g. simalikalactone D and
glaucarubolone, and pharmaceutically acceptable derivatives
thereof.
103. The method of claim 89, 90, 97 or 101 in which said
administration is made via an implantation device.
104. The method of claim 89, 90, 97 or 101 wherein at least 30% of
said catechins is EGCg.
105. The method of claim 104 wherein EGCg comprises about 35% to
about 45% of the catechins.
106. The method of claim 104 wherein EGCg comprises about 40% of
the catechins.
107. The method of claim 89, 90, 97 or 101 wherein at least 30% of
said catechins is EGCg and at least 5% of said catechins is ECG
based upon the total amount of catechins present in said
formulation.
108. The method of claim 107 wherein EGCg comprises about 35% to
about 45% of the catechins and ECG comprises about 10% to about 20%
of the catechins.
109. The method of claim 107 wherein EGCg comprises about 40% of
the catechins and ECG comprises about 15% of the catechins.
110. The method of claim 89, 90, 97 or 101 wherein at least 30% of
said catechins is EGCg and at least 3% of said catechins is EC
based upon the total amount of catechins present in said
formulation.
111. The method of claim 110 wherein EGCg comprises about 35% to
about 45% of the catechins and EC comprises about 3% to about 15%
of the catechins.
112. The method of claim 110 wherein EGCg comprises about 40% of
the total catechins and EC comprises about 7% of the catechins.
113. The method of claim 89, 90, 97 or 101 wherein EGCg and EC
comprise the catechins, EGCg comprises at least 0.01% of said
catechins, and the EC content is at least 10 fold greater than the
EGCg content.
114. The method of claim 113 wherein the EC content is at least 100
fold greater than the EGCg content.
115. The method of claim 113 wherein the EC content is at least
1000 fold greater than the EGCg content.
116. The method of claim 89, 90, 97 or 101 wherein at least 30% of
said catechins is EGCg and at least 1% of said catechins is EGC
based upon the total amount of catechins present in said
formulation.
117. The method of claim 116 wherein EGCg comprises about 35% to
about 45% of the catechins and EGC comprises about 2% to about 5%
of the catechins.
118. The method of claim 116 wherein EGCg comprises about 40% of
the total catechins and EGC comprises about 3% of the
catechins.
119. The method of claim 89, 90, 97 or 101 wherein at least 30% of
said catechins is EGCg, at least 3% of said catechins is EC, and at
least 5% of said catechins is ECG based upon the total amount of
catechins present in said formulation.
120. The method of claim 119 wherein said formulation comprises
EGCg, EC, and ECG wherein EGCg comprises about 35% to about 45% of
the catechins, EC comprises about 3% to about 15% of the catechins,
and ECG comprises about 10% to about 20% of the catechins.
121. The method of claim 119 wherein said formulation comprises
EGCg, EC, and ECG wherein EGCg comprises about 45% of the
catechins, EC comprises about 7% of the catechins, and ECG
comprises about 15% of the catechins.
122. The method of claim 89, 90, 97 or 101 wherein at least 30% of
said catechins is EGCg, at least 3% of said catechins is EC, and at
least 1% of said catechins is EGC in said formulation.
123. The method of claim 122 wherein said formulation comprises
EGCg, EC, and EGC wherein EGCg comprises about 35% to about 45% of
the catechins, EC comprises about 3% to about 15% of the catechins,
and EGC comprises about 2% to about 5% of the catechins.
124. The method of claim 122 wherein said formulation comprises
EGCg, EC, and EGC wherein EGCg comprises about 45% of the
catechins, EC comprises about 7% of the catechins, and EGC
comprises about 3% of the catechins.
125. The method of claim 89, 90, 97 or 101 wherein at least 30% of
said catechins is EGCg, at least 3% of said catechins is EC, at
least 5% of said catechins is ECG, and least 1% of said catechins
is EGC based upon the total amount of catechins present in said
formulation.
126. The method of claim 125 wherein said formulation comprises
EGCg, EC, and ECG wherein EGCg comprises about 35% to about 45% of
the catechins, EC comprises about 3% to about 15% of the catechins,
ECG comprises about 10% to about 20% of the catechins, and EGC
comprises about 2% to about 5% of the catechins.
127. The method of claim 125 wherein said formulation comprises
EGCg, EC, and ECG wherein EGCg comprises about 45% of said
catechins, EC comprises about 7% of said catechins, ECG comprises
about 15% of said catechins, and EGC comprises about 3% of said
catechins in said formulation.
128. The method of claim 89, 90, 97 or 101 wherein at least 30% of
said catechins is EGCg, at least 3% of said catechins is EC, at
least 5% of said catechins is ECG, at least 1% of said catechins is
EGC, and at least 5% of said catechins is C based upon the total
amount of catechins present in said formulation.
129. The method of claim 128 wherein said formulation comprises
EGCg, EC, and ECG wherein EGCg comprises about 35% to about 45% of
the catechins, EC comprises about 3% to about 15% of the catechins,
ECG comprises about 10% to about 20% of the catechins, EGC
comprises about 2% to about 5% of the catechins, and C comprises
about 10% to about 20% of the catechins.
130. The method of claim 128 wherein said formulation comprises
EGCg, EC, and ECG wherein EGCg comprises about 45% of the
catechins, EC comprises about 7% of the catechins, ECG comprises
about 15% of the catechins, EGC comprises about 3% of the
catechins, and C comprises about 15% of the catechins.
131. The method of claim 89, 90, 97 or 101 wherein said formulation
is an ionic aqueous solution of the composition comprising tea
catechins.
132. The method of claim 89, 90, 97 or 101 wherein the total daily
amount administered is from about 10 to about 100,000 mg of the
composition comprising tea catechins.
133. The method of claim 89, 90, 97 or 101 wherein the formulation
is provided at constant levels in the sera for at least 48
hours.
134. The method of claim 89, 90, 97 or 101 wherein the formulation
is provided at constant levels in the sera for at least 72 hours.
Description
[0001] This is a continuation-in-part of U.S. patent application
Ser. No. 09/537,211, filed on Mar. 29, 2000, which is incorporated
herein, by reference, in its entirety, and which in turn claims
benefit to U.S. provisional application serial No. 60/126,893 filed
Mar. 30, 1999 and U.S. provisional application serial no.
60/151,109 filed Aug. 27, 1999.
1. INTRODUCTION
[0002] The present invention relates to novel methods and sustained
release compositions which utilize catechins, including but not
limited to, epigallocatechin gallate (EGCg), epicatechin (EC),
epicatechin gallate (ECG), and epigallocatechin (EGC), which are
found in varying levels in tea leaves. The unique sustained release
compositions of the invention contain various amounts of the
catechins, including combinations of catechins, or catechins and
other therapeutic agents. The invention also encompasses the
varying modes of administration of the therapeutic compounds, such
as a sustained release formulation which is used as a therapeutic
compound for the treatment of cancer or as a dietary supplement for
the prevention of cancer.
2. BACKGROUND OF THE INVENTION
[0003] Tea is generally in the form of black, oolong, and green
tea, all originating from the tea plant, Camellia sinensis. Tea is
cultivated in approximately thirty countries worldwide, and is
consumed globally. Although the level of tea consumption varies
around the world, it is believed that tea consumption is second
only to water (Ahmad et al., 1998, Nutrition and Chemical Toxicity,
John Wiley and Sons, Sussex, England, pp. 301-343). Black tea is
consumed predominantly in Western and some Asian countries and
green tea is consumed predominantly in China, Japan, India, and a
few countries in North Africa and the Middle East (Ahmad et al.,
1998, Nutrition and Chemical Toxicity, John Wiley and Sons, Sussex,
England, pp. 301-343).
[0004] Green tea has been prized as a traditional tonic and has
been widely consumed in East Asia. Recent studies have attempted to
link green tea to antioxidant benefits including protection against
the damage caused by cigarette smoke, pollution, stress, and other
toxins (for an overview, see e.g., Mitscher, 1998, The Green Tea
Book, Avery Publishing Group, Garden City Park, N.Y. and
Weisburger, 1997, Can. Lett. 114:315-317).
[0005] An empirical link between green tea and its cancer
prevention properties was made in the late 1980s (Khan et al.,
1988, Can. Lett. 42:7-12 and Wang et al., 1989, Carcinogenesis
10:411-415). Epidemiological studies show that cancer onset of
patients in Japan who had consumed ten cups of green tea per day
was 8.7 years later among females and 3 years later among males,
compared with patients who had consumed under three cups per day
(Fujiki et al., 1998, Mutation Res. 402:307-310). As such, a
possible relationship between high consumption of green tea and low
incidence of prostate and breast cancer in Asian countries where
green tea consumption is high has been postulated (Liao et al.,
1995, Can. Lett. 96:239-243 and Stoner and Mukhtar, 1995, J. Cell.
Biochem. 22:169-180). However, because of the many variables in
lifestyle inherent to such a study, a definitive link between green
tea and its cancer prevention effects could not be concluded.
[0006] Scientists have now identified many of the natural
substances in green tea that may provide the majority of its health
benefits. One class of chemicals that has attracted much study is
the polyphenols, also known as catechins.
2.1. Epigallocatechin Gallate (EGCg)
[0007] The polyphenols describe a class of substituted phenolic
compounds that are known as flavanols or catechins. The polyphenols
in green tea that have been identified are catechin (C),
epicatechin (EC), gallocatechin (GC), gallocatechin gallate (GCG),
epigallocatechin (EGC), epicatechin gallate (ECG), and
epigallocatechin gallate (EGCg) (FIG. 1). In addition, caffeine,
theobromine, theophylline, and phenolic acids, such as gallic acid,
are also present as constituents of green tea in smaller quantities
than the polyphenols (Ahmad et al., 1998, Nutrition and Chemical
Toxicity, John Wiley and Sons, Sussex, England, pp. 301-343).
[0008] Epigallocatechin gallate (EGCg), the major catechin in green
tea, has been the focus of many studies to determine if it is
responsible for the antioxidant and anti-carcinogenic effects of
green tea, as reviewed by Ahmad and Mukhtar, 1999, Nutr. Rev.
57:78-83. The administration of a pharmacologically effective
amount of EGCg has been alleged to reduce the incidence of lung
cancer in a mammal (U.S. Pat. No. 5,391,568). A bioavailability
study showed that frequent green tea consumption results in high
levels of EGCg in various body organs, suggesting that green tea
consumption may protect against cancers localized to different
sites of the body (Sugunama et al., 1998, Carcinogenesis
19:1771-1776).
[0009] EGCg has been implicated in blocking DNA transcription of a
number of genes in cancer cell lines. For example, in the human
epidermal carcinoma cell line A431, EGCg inhibits the DNA and
protein synthesis of the growth factor receptors epidermal growth
factor receptor (EGF-R), platelet-derived growth factor receptor
(PDGF-R), and fibroblast growth factor receptor (FGF-R) (Liang et
al., 1997, J. Cell. Biochem. 67:55-65). EGCg has also been
implicated in blocking transcription of nitric oxide (NO) synthase
by inhibiting the binding of transcription factor NF.kappa.B to the
NO synthase promotor (Lin and Lin, 1997, Mol. Pharmacol. 52:465-472
and Chan et al., 1997, Biochem. Pharmacol. 54:1281-1286). In the
tumor cell line JB6, EGCg inhibits AP-1 transcriptional activity
(Dong et al., 1997, Can. Res. 57:4414-4419). These results suggest
that EGCg may prevent cancer at the level of gene transcription,
i.e., by blocking the DNA synthesis of genes involved in signal
transduction pathways.
[0010] Further, the focus of many other studies has been the effect
of EGCg on apoptosis, or programmed cell death. Apoptosis differs
from necrosis, and is regarded as an ideal mechanism for the
elimination of cells. Studies have shown that several anti-cancer
preventative agents may induce apoptosis, and conversely, several
tumor-promoting agents inhibit apoptosis (Wright et al., 1994,
FASEB J 8:654-660 and Ahmad and Mukhtar, 1999, Nutr. Rev.
57:78-83).
[0011] Much of the prior work in the art has attempted to determine
what, if any, effect EGCg has on the growth inhibition and
apoptosis induction of cancer cells. A differential growth
inhibitory effect was reported in human colorectal cancer cells
CaCo-2, breast cancer cells Hs578T, and their non-cancer cell
counterparts (Ahmad and Mukhtar, 1999, Nutr. Rev. 57:78-83). EGCg
has been implicated in the growth arrest and subsequent induction
of apoptosis following cell growth inhibition has been shown in
virally transformed fibroblast cells WI138, human epidermal
carcinoma cells A431, lung cancer tumor cells H611, prostate cancer
cell lines LNCaP, PC-3, and DU145, human carcinoma keratinocytes
HaCaT, and mouse lymphoma cells LY-R (Chen et al., 1998, Can. Lett.
129:173-179; Ahmad et al., 1997, J. of the Nat. Can. Inst.
89:1881-1886; Yang et al., 1998, Carcinogenesis 19:611-616; Paschka
et al., 1998, Can. Lett. 130:1-7; and Ahmad and Mukhtar, 1999,
Nutr. Rev. 57:78-83). In studies where the apoptotic response was
studied in cancer cells versus their non-cancer counterparts, e.g.,
human carcinoma keratinocytes HaCaT versus normal human epidermal
keratinocytes, the apoptotic response to EGCg was reported to be
specific to the cancer cells (Ahmad et al., 1997, J. Nat. Can.
Inst. 89:1881-1886).
[0012] It has been suggested that EGCg induced apoptosis may result
from either cell cycle arrest and/or H.sub.2O.sub.2 production
(Ahmad et al., 1997, J. Nat. Can. Inst. 89:1881-1886; Fujiki et
al., 1998, Mutat. Res. 402:307-310; and Yang et al., 1998,
Carcinogenesis 19:611-616). EGCg may be involved in the growth
regulation of human epidermal carcinoma cells A431 by causing cell
cycle arrest of the G.sub.0 to G.sub.1 phase (Ahmad et al., 1997,
J. Nat. Can. Inst. 89:1881-1886). EGCg has also been implicated in
phase arrest between G.sub.2 to M phase of the cell cycle in human
lung cancer cells (Fujiki et al., 1998, Mutat. Res. 402:307-310).
In the EGCg induced inhibition of human lung cancer cells, it was
suggested that the tumor necrosis factor (TNF) .alpha. pathway that
is the mode of action of EGCg. Alternatively, the EGCg-induced
apoptosis of the lung cancer tumor cells H611 is inhibited by
catalase, suggesting the H.sub.2O.sub.2 production as a probable
cause of apoptosis (Yang et al., 1998, Carcinogenesis
19:611-616).
[0013] Despite the above studies, the efficacy of EGCg as a single
agent therapy for the prevention of cancer is still unclear.
Moreover, the efficacy of EGCg as a therapeutic drug to treat or
reverse cancer in a patient is unknown.
2.2. Other Catechins and Combinations Thereof
[0014] Although the focus of much of the prior research has been on
EGCg, the putative biological functions of some of the other
catechins has been examined. For example, both epicatechin gallate
(ECG) and epigallocatechin (EGC) have been reported to be as
effective as EGCg in inducing apoptosis of human epidermal
carcinoma cells A431 at similar concentrations, whereas epicatechin
(EC) did not show a similar effect (Ahmad et al., 1997, J. of the
Nat. Can. Inst. 89:1881-1886). Growth inhibition in lung tumor cell
lines H661 and H1299 was also observed with EGCg and EGC, whereas
ECG and EC were less effective (Yang et al., 1998, Carcinogenesis
19:611-616).
[0015] Catechins have been implicated in growth inhibition of the
human lung cancer cell line PC-9, with the order of catechin
potency being reported as EGCg=ECG>EGCEC (Okabe et al., 1993,
Jpn. J. Clin. Oncol. 23:186-190). It has also been demonstrated
that catechin combinations of EGCg and EC, ECG and EC, and EGC and
EC induce apoptosis of the human lung cancer cell line PC-9 in
vitro (Suganuma et al., 1999, Can. Res. 59:44-47). EC is thought to
enhance incorporation of EGCg into the cells, which is thought to
inhibit TNF .alpha. release resulting in the induction of apoptosis
(Suganuma et al., 1999, Can. Res. 59:44-47).
[0016] Green tea extract, an important source of EGCg, has
previously been reported to enhance the effect of the anti-cancer
agents, e.g., adriamycin and doxorubicin (Sugiyama and Sadzuka,
1998, Can. Lett. 133:19-26 and Sadzuka et al., 1998, Clin. Can.
Res. 4:153-156). Green tea in combination with adriamycin inhibits
tumor growth in M5076 ovarian sarcoma cells, whereas adriamycin
alone does not inhibit tumor growth in M5076 ovarian sarcoma cells
(Sugiyama and Sadzuka, 1998, Can. Lett. 133:19-26). A similar
effect is observed with green tea extract and doxorubicin on the
same M5076 ovarian sarcoma cell line. Green tea extract, in
combination with doxorubicin, also enhances the inhibitory growth
effect on Ehrlich ascites carcinoma tumors in tumor-bearing mice,
presumably by increasing the concentration of doxorubicin
concentration in the tumor, but not in normal tissue (Sadzuka et
al., 1998, Clin. Can. Res. 4:153-156).
[0017] EGCg has also been shown to enhance the effect of cancer
prevention drugs in vitro. For example, EGCg has been shown to
enhance the apoptotic effect of sulindac and tamoxifin, presumably
by EGCg enhancing the intracellular concentration of the cancer
prevention drugs. (Suganuma et al., 1999, Can. Res. 59:44-47). Both
sulindac and tamoxifin induce apoptosis of human cancer cells and
inhibit TNF .alpha. release from BALB/c-3T3 cells (Piazza et al.,
1995, Can. Res. 55:3110-3116; Chen et al., 1996, J. Cell. Biochem.
61:9-17; and Sugunama et al., 1996, Can. Res. 56:3711-3715).
2.3. NADH Oxidase
[0018] A unique plasma membrane NADH oxidase (NOX), a unique cell
surface protein with hydroquinone (NADH) oxidase and protein
disulfide-thiol interchange activities that is responsive to
hormone and growth factors has been identified (Brightman et al.,
1992, Biochim. Biophys. Acta 1105:109-117; Morr, 1994, J. Bioenerg.
Biomemb. 26:421-433; and Morr, 1998, Plasma Membrane Redox Systems
and their Role in Biological Stress and Disease, Klewer Academic
Publishers, Dordrecht, The Netherlands, pp. 121-156). Further, a
hormone-insensitive and drug-responsive form of NOX designated tNOX
which is specific to cancer cells has been reported (Bruno et al.,
1992, Biochem. J. 284:625-628; Morr and Morr, 1995, Protoplasma
184:188-195; Morr et al., 1995, Proc. Natl. Acad. Sci. U.S.A.
92;1831-1835; Morr et al., 1995, Biochim. Biophys. Acta 1240:11-17;
Morr et al., 1996, Eur. J. Can. 32A:1995-2003; and Morr et al.,
1997, J. Biomemb. Bioenerg. 29:269-280).
[0019] Because the NOX protein is located at the external plasma
membrane surface and is not transmembrane, a functional role as an
NADH oxidase is not considered likely (Morr, 1994, J. Bioenerg.
Biomemb. 26:421-433; DeHaln et al., 1997, Biochim. Biophys. Acta
1328:99-108; and Morr, 1998, Plasma Membrane Redox Systems and
Their Role in Biological Stress and Disease, Klewer Academic
Publishers, Dordrecht, The Netherlands, pp. 121-156). While the
oxidation of NADH provides a basis for a convenient method to assay
the activity, the ultimate electron physiological donor is most
probably hydroquinones with specific activities for hydroquinone
oxidation greater than or equal to that of NADH oxidation and/or
protein thiol-disulfide interchange (Kishi et al., 1999, Biochim.
Biophys. Acta 1412:66-77).
[0020] CNOX was originally defined as a drug-indifferent
constitutive NADH oxidase activity associated with the plasma
membrane of non-transformed cells that was the normal counterpart
to tNOX (Morr, 1998, Plasma Membrane Redox Systems and Their Role
in Biological Stress and Disease, Kiewer Academic Publishers,
Dordrecht, The Netherlands, pp. 121-156). Indeed, a 36 kD protein
isolated from rat liver and from plants has NOX activity that is
unresponsive to tNOX inhibitors (Brightman et al., 1992, Biochim.
Biophys. Acta 1105: 109-117).
[0021] While cancer cells exhibit both drug-responsive and hormone
and growth factor-indifferent (tNOX) as well as drug inhibited and
hormone and growth factor dependent (CNOX) activities,
non-transformed cells exhibit only the drug indifferent hormone-
and drug-responsive CNOX. Among the first descriptions of so-called
constitutive or CNOX activity of non-transformed cells and tissues
was where the activity of rat liver plasma membranes was stimulated
by the growth factor, diferric transferrin (Sun et al., 1987, J.
Biol. Chem. 262:15915-15921). Subsequent work demonstrated that the
observed NADH oxidation was catalyzed by a unique enzyme exhibiting
responsiveness to several hormones and growth factors (Bruno et
al., 1992, Biochem J. 284:625-628). Unlike mitochondrial oxidases,
the hormone-stimulated NADH oxidase activity of rat liver plasma
membranes is not inhibited by cyanide (Morr, 1994, J. Bioenerg.
Biomemb. 26: 421-433). The enzyme also was distinguished from other
oxidase activities by its response to several common oxidoreductase
inhibitors, e.g., catalase, azide and chloroquine, as well as to
various detergents e.g., sodium cholate, Triton X-100 and CHAPS
(Morr and Brightman, 1991, J. Bioenerg. Biomemb. 23:469-489 and
Morr et al., 1997, J. Biomemb. Bioenerg. 29:269-280). Like tNOX of
cancer cells, CNOX is a unique membrane-associated protein that is
capable of oxidizing NADH but has an activity which is modulated by
hormones and growth factors.
2.4. Pathobiology of Cancer
[0022] Cancer is characterized primarily by an increase in the
number of abnormal cells derived from a given normal tissue,
invasion of adjacent tissues by these abnormal cells, and lymphatic
or blood-borne spread of malignant cells to regional lymph nodes
and to distant sites (metastasis). Clinical data and molecular
biologic studies indicate that cancer is a multistep process that
begins with minor preneoplastic changes, which may under certain
conditions progress to neoplasia.
[0023] Pre-malignant abnormal cell growth is exemplified by
hyperplasia, metaplasia, or most particularly, dysplasia (for
review of such abnormal growth conditions, see Robbins and Angell,
1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp.
68-79) Hyperplasia is a form of controlled cell proliferation
involving an increase in cell number in a tissue or organ, but
without significant alteration in structure or function. As but one
example, endometrial hyperplasia of ten precedes endometrial
cancer. Metaplasia is a form of controlled cell growth in which one
type of adult or fully differentiated cell substitutes for another
type of adult cell. Metaplasia can occur in epithelial or
connective tissue cells. Atypical metaplasia involves a somewhat
disorderly metaplastic epithelium. Dysplasia is frequently a
forerunner of cancer, and is found mainly in the epithelia; it is
the most disorderly form of non-neoplastic cell growth, involving a
loss in individual cell uniformity and in the architectural
orientation of cells. Dysplastic cells of ten have abnormally
large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia
characteristically occurs where there exists chronic irritation or
inflammation, and is of ten found in the cervix, respiratory
passages, oral cavity, and gall bladder.
[0024] The neoplastic lesion may evolve clonally and develop an
increasing capacity for invasion, growth, metastasis, and
heterogeneity, especially under conditions in which the neoplastic
cells escape the host's immune surveillance (Roitt, Brostoff, and
Kale, 1993, Immunology, 3rd ed., Mosby, St. Louis, pp.
17.1-17.12).
[0025] There remains a need for treatment of cancer that does not
have the adverse effects generally caused by non-selectivity, of
conventional chemotherapeutic agents. None of the above studies,
which are not to be construed as an admission that any of the above
studies is prior art, have suggested the present mechanism by which
the catechins are able to differentiate between cancer and
non-cancer cells. Moreover, none of the studies evaluated the
efficacy of varying levels of catechin combinations or compositions
of multiple catechins for the treatment of cancer. In contrast, the
Inventors have identified a cancer-specific isoform of a unique
plasma membrane NADH oxidase (tNOX) which is inhibited by the
catechins. Furthermore, the studies cited supra have hypothesized
that EGCg mediates its effects intracellularly, since EGCg
incorporation into the cell seems to be a prerequisite for the
inhibition of TNF .alpha. release. Inhibition of tNOX, an
extracellular membrane-associated protein) by EGCg, and
synergistically with other catechins and anti-cancer agents,
results in the selective inhibition of cancer cell growth and
ultimately, apoptosis. Further discussion of catechin-induced
apoptosis wherein tNOX is targeted is presented in Sections 6, 7,
and 8.
3. SUMMARY OF THE INVENTION
[0026] The invention encompasses sustained release formulations
comprising catechins, a group of polyphenols found in green tea,
which are used as therapeutic compounds for the treatment of cancer
or as a dietary supplement that offers white blood cell protection
and maintains healthy blood levels, all of which suggests that the
catechins play a role in the prevention of cancer. The sustained
release compositions optimally maintain circulating levels of said
composition in the body at a certain threshold level over an
extended time period. Specific therapeutic regimens, pharmaceutical
compositions, and kits are also provided by the invention.
[0027] In one embodiment, the invention described herein comprises
the administration of catechins in a sustained release formulation
to a mammal as a dietary supplement for the prevention of cancer.
In a preferred embodiment, the mammal is a human.
[0028] In one embodiment, the invention described herein comprises
the administration of a therapeutically effective amount of
catechins in a sustained release formulation to a mammal in need of
such therapy. In a preferred embodiment, the mammal is a human. In
another embodiment, the invention further encompasses the use of
combination therapy to treat cancer.
[0029] In a specific embodiment, the catechins comprise
epigallocatechin gallate (EGCg), epicatechin gallate (ECG),
epigallocatechin (EGC), and epicatechin (EC) or a combination
thereof , optionally in combination with other polyphenols.
[0030] The disclosure is based, in part, on the discovery that
epigallocatechin gallate (EGCg), alone and in combination with
other catechins and other anti-cancer therapeutic agents, inhibits
the activity of a cancer-specific protein, an isoform of NADH
oxidase specific to cancer cells (tNOX). The inhibition of tNOX
results in the inhibition of cell growth, and ultimately, apoptosis
of the cancer cell, whereas normal cells (which lack tNOX but
instead express the isoform CNOX) are less affected. Thus, the
invention provides a potent therapeutic effect without or while
reducing the adverse effects on normal, healthy cells.
[0031] Significantly the effect of the catechins such as EGCg is
reversible, i.e., if the EGCg is removed, cancer cells resume
normal rates of growth. Other discoveries include: (1) EGCg is
rapidly cleared from the blood and metabolized, (2) cancer cells
must be inhibited from growing for 48 to 72 hours before
EGCg-induced apoptosis occurs, and (3) when cancer cells are
challenged with 10.sup.-7 M EGCg every two hours during the day,
their growth is inhibited, but during the night normal cell growth
resumes in the absence of further EGCg addition. Thus, the
invention is directed to the administration of sustained release
formulations so that a constant level of the catechins is
maintained.
[0032] Particular compositions of the invention and their uses are
described in the sections and subsections which follow.
3.1. Definitions
[0033] As used herein, the term "cancer" describes a diseased state
in which a carcinogenic agent or agents causes the transformation
of a normal cell into an abnormal cell, the invasion of adjacent
tissues by these abnormal cells, and lymphatic or blood-borne
spread of malignant cells to regional lymph nodes and to distant
sites, i.e., metastasis.
[0034] As used herein, the terms "preventing cancer" and
"prevention of cancer" mean to inhibit the transformation of a
normal cell into an abnormal cell by a carcinogenic agent or agents
and/or to inhibit the accumulation of cells expressing
cancer-specific genes (e.g., tNOX) to a number which creates
clinical symptoms associated with cancer.
[0035] As used herein, the terms "treating cancer" and "treatment
of cancer" mean to inhibit the replication of cancer cells, to
inhibit the spread of cancer, to decrease tumor size, to lessen or
reduce the number of cancerous cells in the body, and to ameliorate
or alleviate the symptoms of the disease caused by the cancer. The
treatment is considered therapeutic if there is a decrease in
mortality and/or morbidity.
[0036] The term "synergistic" as used herein refers to a
combination which is more effective than the additive effects of
any two or more single agents. A determination of a synergistic
interaction between catechins, and another therapeutic agent may be
based on the results obtained from the NOX assays described in
Section 5.4 infra. The results of these assays are analyzed using
Chou and Talalay's combination method and Dose-Effect Analysis with
Microcomputers+ software in order to obtain a Combination Index
(Chou and Talalay, 1984, Adv. Enzyme Regul. 22:27-55 and Chou and
Chou, 1987, software and manual, Elsevier Biosoft, Cambridge, UK,
pp. 19-64). Combination Index values<1 indicates synergy,
values>1 indicate antagonism and values equal to 1 indicate
additive effects.
[0037] The term "pharmaceutically acceptable carrier" refers to a
carrier medium that does not interfere with the effectiveness of
the biological activity of the active ingredient, is chemically
inert and is not toxic to the patient to whom it is
administered.
[0038] The term "pharmaceutically acceptable salts" refers to salts
prepared from pharmaceutically acceptable non-toxic acids and
bases, including inorganic and organic acids and bases.
[0039] As used herein the term "pharmaceutically acceptable
derivative" refers to any homolog, analog, or fragment
corresponding to the catechin formulations as described in Section
5.1 infra which exhibits anti-cancer activity and is relatively
non-toxic to the subject.
[0040] The term "therapeutic agent" refers to any molecule,
compound or treatment that assists in the treatment of a cancer or
the diseases caused thereby.
[0041] As used herein, the term "sustained release formulation"
refers to any composition that provides slow, controlled, and/or
timed release of one or more active ingredients.
[0042] The catechins and target proteins defined herein are
abbreviated as follows:
1 (.+-.) - catechin C (-) - epicatechin EC gallocatechin GC
gallocatechin gallate GCG (-) - epigallocatechin EGC (-) -
epicatechin gallate ECG (-) - epigallocatechin gallate EGCg
nicotinamide adenine dinucleotide NADH cell surface hydroquinone
(NADH) oxidase with NOX protein disulfide - thiol isomerase
activity NOX present in both non-cancer and cancer cells CNOX NOX
specific to cancer cells tNOX
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0043] FIG. 1. Structures of six catechins from green tea. A.
(.+-.)-catechin (C). B. (-) epicatechin (EC). C.
(-)-epigallocatechin (EGC). D. (-)-epicatechin gallate (ECG). E.
(-)-epigallocatechin gallate (EGCg). F. (-)-gallocatechin gallate
(GCG)
[0044] FIG. 2. Dose-response of NADH oxidase of isolated plasma
membranes (A, B) and growth of attached cells (C, D) to
(-)-epigallocatechin gallate (EGCg). A, C. MCF-10A human mammary
epithelial (non-cancer) cells (.cndot.) and BT-20 human mammary
adenocarcinoma (cancer) cells (.largecircle.). B, D. HeLa (human
cervical carcinoma) cells. Values are averages of duplicate
determinations in each of three separate experiments
(n=6).+-.standard deviations among experiments (n=3).
[0045] FIG. 3. Dose-response of solubilized and partially purified
NADH oxidase to (-)-epigallocatechin gallate (EGCg). A. NADH
oxidase from MCF-10A and BT-20 cells. B. NADH oxidase from HeLa
cells. As with plasma membranes (FIG. 2), the preparations from
BT-20 and HeLa cells contained NOX activities both susceptible and
resistant to inhibition by EGCg whereas the preparations from
MCF10A cells was resistant to inhibition. Results are averages of
duplicate determinations in each of three separate experiments
(n=6).+-.standard deviations among experiments (n=3).
[0046] FIG. 4. Photomicrographs of MCF-10A mammary epithelial
(non-cancer), BT-20 mammary adenocarcinoma and HeLa cells treated
for 96 h with 10 .mu.M (-)-epigallocatechin gallate (EGCg) added at
t=0. The BT-20 and HeLa cells stopped growing and died whereas the
MCF-10 cells recovered fully.
[0047] FIG. 5. Photomicrographs of MCF-10A, BT-20 and HeLa cells
stained with 4',6-diamidino-2-phenylindole (DAPI) (Wolvetang et
al., 1994, FEBS Lett. 339:40-44) to show condensed chromatin after
96 h in the presence of 10 or 50 .mu.M epigallocatechin gallate
(EGCg) characteristic of apoptosis for BT-20 and HeLa but not for
MCF-10A cells. Cells were grown on coverslips in the absence (upper
panel) or presence (lower 2 panels) of 10 or 50 .mu.M EGCg and
fixed. Nuclear DNA was stained with DAPI and analyzed with a
fluorescence microscope.
[0048] FIG. 6. Inhibition of partially purified tNOX from HeLa
cells by green tea infusions.
[0049] The EC.sub.50 for inhibition of the enzymatic activity was
at a tea dilution of about 1:1000. The preparations contained an
activity resistant to inhibition as well so that the inhibition by
the tea infusions was not complete and further inhibition by green
tea was not observed above a dilution of about 1:10. Results are
averages of duplicate determinations in each of three separate
experiments (n=6).+-.standard deviations among experiments
(n=3).
[0050] FIG. 7. Response of the NADH oxidase activity solubilized
and partially purified as described from plasma membrane vesicles
of HeLa cells to 1 nM (-)-epigallocatechin gallate (EGCg) alone and
in combination with (-)-epicatechin (EC) at 10, 50 and 100 .mu.M
(del Castillo et al, 1998, Arch. Biochem. Biophys. 358:125-140).
Values are from duplicate determinations from each of three
different experiments +standard deviations. HeLa cells contain NOX
activities containing both a drug-susceptible component (tNOX, 40
to 60% of the total) and a drug-resistant component (CNOX, 40 to
60%) of the total. The effect of EC in the presence of 1 nM EGCg
alone is to inhibit completely the tNOX component without an effect
on CNOX activity.
[0051] FIG. 8. Dose response of the growth of HeLa cells after 72 h
to (-)-epigallocatechin (EGCg) in the absence or presence of 100
.mu.M (-)-epicatechin (EC). Values are from duplicate
determinations from single experiments except for 10.sup.-7 M EGCg
which is the average of duplicate determinations from 3
experiments.+-.standard deviations.
[0052] FIG. 9. Response of the NADH oxidase of 4T1 mouse mammary
cells to (-)-epicatechin (EC) alone (upper curve, solid symbols) or
in the presence of 10.sup.-7 M (-)-epigallocatechin gallate (EGCg)
(lower curve, open symbols, dashed line). The tNOX activity (see
FIG. 12) was completely inhibited by 10.sup.-4 M EC in the presence
of 0.1 .mu.M EGCg without effect on CNOX activity. Values are
averages of 3 experiments.+-.standard deviations.
[0053] FIG. 10. Dose response of the growth of 4T1 cells after 72 h
to (-)-epigallocatechin gallate (EGCg) provided in combination with
other tea catechins as Tegreen.TM. in the absence or presence of
100 .mu.M (-)-epicatechin (EC). Values are from duplicate
determinations from single experiments except for 10.sup.-7 M EGCg
which is the average of duplicate determinations from 3
experiments.+-.standard deviations.
[0054] FIG. 11. Response of the NADH oxidase of 4T1 mouse mammary
cells to (-)-epicatechin (EC) in the presence of 10.sup.-7 M
Tegreen.TM. (upper curve, solid symbols) or 10.sup.-5 M Tegreen.TM.
(lower curve, open symbols, dashed line). The tNOX activity (see
FIG. 12) was completely inhibited by 10.sup.-4 EC in the presence
of 0.1 .mu.M EGCg without effect on CNOX activity. Values are
averages of 3 experiments.+-.standard deviations.
[0055] FIG. 12. Response of the NADH oxidase of HeLa S cells to
(-)-epigallocatechin gallate (EGCg) alone. The tNOX activity was
maximally inhibited by 0.1 .mu.M EGCg without effect on CNOX
activity. Values are averages of 3 experiments.+-.standard
deviations.
[0056] FIG. 13. Response of the NADH oxidase of HeLa S cells to
(-)-epicatechin (EC) alone (upper curve, solid symbols) or in the
presence of 10.sup.-7 M epigallocatechin gallate (EGCg) (lower
curve, open symbols, dashed line). The tNOX activity was completely
inhibited by 10.sup.-4 M EC in the presence of 0.1 .mu.M EGCg
without effect on CNOX activity. Values are averages of 3
experiments.+-.standard deviations.
[0057] FIG. 14. Response of the NADH oxidase of 4T1 mouse mammary
cells to varying concentrations of (-)-epicatechin gallate (ECG)
alone or in the presence of 10.sup.-7 M (-) -epigallocatechin
gallate (EGCg). The tNOX activity was completely inhibited by
10.sup.-6 M EC in the presence of 0.1 .mu.M EGCg without effect on
CNOX activity. Values are averages of duplicate determinations from
2 experiments.+-.mean average deviations between the two
experiments.
[0058] FIG. 15. Response of the NADH oxidase of 4T1 mouse mammary
cells to varying concentrations of (-)-epigallocatechin (EGC) in
the presence of 10.sup.-7 M (-)-epigallocatechin gallate (EGCg).
The tNOX activity was completely inhibited by 10.sup.-5 M EC in the
presence of 0.1 .mu.M EGCg without effect on CNOX activity. Values
are averages of duplicate determinations from 2 experiments.+-.mean
average deviations between the two experiments.
[0059] FIG. 16. Response of the NADH oxidase of 4T1 mouse mammary
cells to varying concentrations of (-)-epigallocatechin gallate
(GCG) in the presence of 10.sup.-7 M (-)-epigallocatechin gallate
(EGCg). The NOX activity was less affected by GCG in the presence
of 0.1 mM EGCg than for EC (Table 5), ECG (FIG. 14) or EGC (FIG.
15). Values are averages of duplicate determinations from 2
experiments.+-.standard deviations among the three experiments.
[0060] FIG. 17. Response of the NADH oxidase of 4T1 mouse mammary
cells to varying concentrations of.+-.catechin in the presence of
10.sup.-7 M (-)-epigallocatechin gallate (EGCg). The NOX activity
was little affected by.+-.catechin either in the presence or
absence (not shown) of 0.1 .mu.M EGCg. Values are averages of
duplicate determinations from 3 experiments.+-.standard deviations
among the three experiments.
[0061] FIG. 18. Response of the NADH oxidase of 4T1 mouse mammary
cells to varying concentrations of a mixture of equal parts of ECG,
EGC, EC and.+-.catechin in the presence of 10.sup.-7 M
(-)-epigallocatechin gallate (EGCg). The NOX activity was
completely inhibited by 10.sup.-5 to 10.sup.-6 M of the mixture in
the presence of 0.1 .mu.M EGCg without effect on CNOX activity.
Values are averages of duplicate determinations from 3
experiments.+-.standard deviations among the three experiments.
[0062] FIG. 19. Response of the NADH oxidase of 4T1 mouse mammary
cells to varying concentrations of Tegreen.TM. a concentration
equivalent to 10.sup.-7 EGCg treated with NaOH to cleave gallate
esters. The hydrolyzate was tested in the presence of 10.sup.-7 M
(-)-epigallocatechin gallate (EGCg). The base was neutralized to pH
7 with HCl and a control experiment with an equivalent amount of
NaCl was carried out. The tNOX activity was completely inhibited by
EGCg of Tegreen.TM. in the presence of 0.1 mM EGCg without effect
on CNOX activity. Values are averages of duplicate determinations
from 2 experiments.+-.mean average deviations between the two
experiments.
[0063] FIG. 20. Growth of HeLa cells (increase in cell number) with
time of culture. No addition (solid symbols, solid line). EGCg (100
nM) was added every 2 hours during the day (8:00 a.m. until 5:00
p.m.) (open symbols, dashed lines). Two different levels of
inoculum 10 .mu.l (circles) or 5 .mu.l (triangles) were
compared.
[0064] FIG. 21. Effect of sustained release formulation of
Tegreen.TM. on tumor growth in a Balb C mouse bearing a
transplantable 4T-1 mammary adenocarcinoma. A tumor mass lying on
an adhering sheet of mouse skin is shown in the center. The tumor
was treated with 100 .mu.l of a suspension of 2.3 mg/ml of the
sustained release formulation of Tegreen.TM.. On the left are the
lung, liver, and lymph nodes, as controls to indicate that
metastasis to these organs had not occured. Wherever a small
granule of the sustained release material was located in the tumor
mass, the cells were killed for several mm around the particle.
5. DETAILED DESCRIPTION OF THE INVENTION
[0065] The invention encompasses sustained release formulations
comprising catechins, a group of polyphenols found in green tea,
which are used as therapeutic compounds for the treatment of cancer
or as a dietary supplement that offers white blood cell protection
and maintains healthy blood levels, all of which suggests that the
catechins play a role in the prevention of cancer. The sustained
release compositions optimally maintain circulating levels of said
composition in the body at a certain threshold level over an
extended time period. Specific therapeutic regimens, pharmaceutical
compositions, and kits are also provided by the invention.
[0066] In one embodiment, the invention described herein comprises
the administration of a therapeutically effective amount of
catechins in a sustained release formulation to a mammal in need of
such therapy. In a preferred embodiment, the mammal is a human. In
another embodiment, the invention further encompasses the use of
combination therapy to treat cancer.
[0067] In a specific embodiment, the catechins comprise
epigallocatechin gallate (EGCg), epicatechin gallate (ECG),
epigallocatechin (EGC), and epicatechin (EC) or a combination
thereof , optionally in combination with other polyphenols.
[0068] The disclosure is based, in part, on the discovery that
epigallocatechin gallate (EGCg), alone and in combination with
other catechins and other anti-cancer therapeutic agents, inhibits
the activity of a cancer-specific protein, an isoform of NADH
oxidase specific to cancer cells (tNOX). The inhibition of tNOX
results in the inhibition of cell growth, and ultimately, apoptosis
of the cancer cell, whereas normal cells (which lack tNOX but
instead express the isoform CNOX) are less affected. Thus, the
invention provides a potent therapeutic effect without or while
reducing the adverse effects on normal, healthy cells.
[0069] Significantly the effect of the catechins such as EGCg is
reversible, i.e., if the EGCg is removed, cancer cells resume
normal rates of growth. Other discoveries include: (1) EGCg is
rapidly cleared from culture media and metabolized, (2) cancer
cells must be inhibited from growing for 48 to 72 hours before
EGCg-induced apoptosis occurs, and (3) when cancer cells are
challenged with 10.sup.-7 M EGCg every two hours during the day,
their growth is inhibited, but during the night normal cell growth
resumes in the absence of further EGCg addition. Thus, the
invention is directed to the administration of sustained release
formulations so that a constant level of the catechins is
maintained.
[0070] Particular compositions of the invention and their uses are
described in the sections and subsections which follow.
5.1. Catechin Formulations
5.1.1. Percentages of the Varying Polyphenols
[0071] The invention comprises formulations (e.g., specific
combination of catechins and specific levels) of green tea
polyphenols, in particular, catechins, for the prevention and/or
treatment of cancer. The typical percentage of the individual
catechins in green tea extracts is 10-15% EGCg, 2-3% ECG, 2% EC,
and 2-3% EGC (Suganuma et al., 1999, Can. Res. 59:44-47).
[0072] In contrast, in one embodiment of the present invention,
EGCg comprises at least 30% of the total catechins. In a preferred
embodiment, EGCg comprises about 35% to about 45% of the total
catechins. In a more preferred embodiment, EGCg comprises about 40%
of the total catechins.
[0073] Although the invention encompasses the use of a composition
containing certain levels of EGCg alone, it is preferred that EGCg
be used in combination with other catechins, more specifically,
those described infra.
[0074] In another embodiment, EGCg comprises at least 30% of the
total catechins and ECG comprises at least 5% of the total
catechins. In a preferred embodiment, EGCg comprises about 35% to
about 45% of the total catechins and ECG comprises about 10% to
about 20% of the total catechins. In a more preferred embodiment,
EGCg comprises about 40% of the total catechins and ECG comprises
about 15% of the total catechins.
[0075] In another embodiment, EGCg comprises at least 30% of the
total catechins and EC comprises at least 3% of the total
catechins. In a preferred embodiment, EGCg comprises about 35% to
about 45% of the total catechins and EC comprises about 3% to about
15% of the total catechins. In a more preferred embodiment, EGCg
comprises about 40% of the total catechins and EC comprises about
7% of the total catechins.
[0076] In an additional embodiment, EGCg comprises at least 0.01%
of the total catechins and EC comprises an amount which is at least
10 fold greater than the EGCg content of the total catechins. The
total catechins may or may not include the additional catechins
such as those described above, e.g., ECG, EGC, C, etc. In a
preferred embodiment, EC comprises an amount which is at least 100
fold greater than the EGCg content. In another preferred
embodiment, EC comprises an amount which is at least 1000 fold
greater than the EGCg content.
[0077] In another embodiment, EGCg comprises a negligible amount of
the catechin formulation.
[0078] In an additional embodiment, EGCg comprises at least 30% of
the total catechins and EGC comprises at least 1% of the total
catechins. In a preferred embodiment, EGCg comprises about 35% to
about 45% of the total catechins and EGC comprises about 2% to
about 5% of the total catechins. In a more preferred embodiment,
EGCg comprises about 40% of the total catechins and EGC comprises
about 3% of the total catechins.
[0079] In an additional embodiment, EGCg comprises at least 30% of
the total catechins, EC comprises at least 3% of the total
catechins, and ECG comprises at least 5% of the total catechins. In
a preferred embodiment, EGCg comprises about 35% to about 45% of
the total catechins, EC comprises about 3% to about 15% of the
total catechins, and ECG comprises about 10% to about 20% of the
total catechins. In a more preferred embodiment, EGCg comprises
about 40% of the total catechins, EC comprises about 7% of the
total catechins. and ECG comprises about 15% of the total
catechins.
[0080] In yet another embodiment, EGCg comprises at least 30% of
the total catechins, EC comprises at least 3% of the total
catechins, and EGC comprises at least 1% of the total catechins. In
a preferred embodiment, EGCg comprises about 35% to about 45% of
the total catechins, EC comprises about 3% to about 15% of the
total catechins, and EGC comprises about 2% to about 5% of the
total catechins. In a more preferred embodiment, EGCg comprises
about 40% of the total catechins, EC comprises about 7% of the
total catechins, and EGC comprises about 3% of the total
catechins.
[0081] In yet another embodiment, EGCg comprises at least 30% of
the total catechins, EC comprises at least 3% of the total
catechins, ECG comprises at least 5% of the total catechins, and
EGC comprises at least 1% of the total catechins. In a preferred
embodiment, EGCg comprises about 35% to about 45% of the total
catechins, EC comprises about 5% to about 15% of the total
catechins, ECG comprises about 10% to about 20% of the total
catechins, and EGC comprises 2% to about 5% of the total catechins.
In a more preferred embodiment, EGCg comprises about 40% of the
total catechins, EC comprises about 7% of the total catechins. ECG
comprises about 15% of the total catechins, and EGC comprises about
3% of the total catechins.
[0082] In yet another embodiment, EGCg comprises at least 30% of
the total catechins, EC comprises at least 3% of the total
catechins, ECG comprises at least 5% of the total catechins, EGC
comprises at least 1% of the total catechins, and C comprises at
least 5% of the total catechins. In a preferred embodiment, EGCg
comprises about 35% to about 45% of the total catechins, EC
comprises about 5% to about 15% of the total catechins, ECG
comprises about 10% to about 20% of the total catechins, EGC
comprises 2% to about 5% of the total catechins, and C comprises
about 10% to about 20% of the total catechins. In a more preferred
embodiment, EGCg comprises about 40% of the total catechins, EC
comprises about 7% of the total catechins. ECG comprises about 15%
of the total catechins, EGC comprises about 3% of the total
catechins, and C comprises about 15% of the total catechins.
[0083] The level of caffeine is generally less than about 5% and is
preferably less than 0.5% of the polyphenols.
[0084] The invention comprises all pharmaceutically acceptable
derivatives of the catechins listed supra, and their combinations
thereof.
5.1.2. Sustained Release Formulation
[0085] The invention comprises a mixture of catechins, including
but not limited to the percentages of the polyphenols described
supra, formulated as sustained release compositions. In a specific
embodiment, the invention comprises a mixture of catechins which
when administered to a human results in circulating levels of EGCg
is maintained between 10.sup.-9 and 10.sup.-4 M. In a preferred
embodiment for the prevention of cancer, the circulating levels of
all catechins in the catechin mixture is maintained up to 10.sup.-7
M. In a preferred embodiment for the treatment of cancer, the
circulating levels of all catechins in the catechin mixture is
maintained up to 10.sup.-7 M. The levels are either circulating in
the patient systemically, or in a preferred embodiment, localized
to the tumor, and in a most preferred embodiment, localized to the
cell surface of the cancer cells.
[0086] It is understood that the catechin levels are maintained
over a certain period of time as is desired and can be easily
determined by one of skill in the art using this disclosure and
available pharmaceutical compendia. In a preferred embodiment, the
invention includes a unique feature of administration comprising a
sustained release formulation so a constant level of EGCg is
maintained between 10.sup.-8 and 10.sup.-6 M between 48 to 96 hours
in the sera.
[0087] Such sustained and/or timed release formulations may be made
by sustained release means or delivery devices that are well known
to those of ordinary skill in the art, such as those described in
U.S. Pat. Nos.: 3,845,770, 3,916,899, 3,536,809, 3,598,123,
4,008,719, 4,710,384, 5,674,533, 5,059,595, 5,591,767, 5,120,548,
5,073,543, 5,639,476, 5,354,556, and 5,733,566, the disclosures of
which are each incorporated herein by reference. These
pharmaceutical compositions can be used to provide slow or
sustained release of one or more of the active ingredients using,
for example, hydropropylmethyl cellulose, other polymer matrices,
gels, permeable membranes, osmotic systems, multilayer coatings,
microparticles, liposomes, microspheres, or the like, or a
combination thereof to provide the desired release profile in
varying proportions. Suitable sustained release formulations known
to those of ordinary skill in the art, including those described
herein, may be readily selected for use with the pharmaceutical
compositions of the invention. Thus, single unit dosage forms
suitable for oral administration, such as, but not limited to,
tablets, capsules, gelcaps, caplets, powders, and the like, that
are adapted for sustained release are encompassed by the present
invention.
[0088] In a highly preferred embodiment, the sustained release
formulation contains active ingredients such as, but not limited
to, microcrystalline cellulose, maltodextrine, ethylcellulose, and
magnesium stearate. In yet another highly preferred embodiment, the
formulation is synthesized with a CapsuDar.RTM. SR (Biodar, Yavne,
Israel) microencapsulation which consists of the active ingredients
microcrystalline cellulose, maltodextrine, ethylcellulose, and
magnesium stearate.
[0089] As described above, all known methods for encapsulation
which are compatible with the properties of tea catechins are
compassed by this invention. The sustained release formulation is
encapsulated by coating particles or granules of the pharmaceutical
composition of the invention with varying thicknesses of slowly
soluble polymers or by microencapsulation. In a preferred
embodiment, the sustained release formulation is encapsulated with
a coating material of varying thickness (e.g. about 1 micron to 200
microns) that allows the dissolution of the pharmaceutical
composition about 48 hours to about 72 hours after administration
to a mammal. In another embodiment, the coating material is a food
approved additive. In yet another embodiment, the coating material
is sold under the trademark Eudragit RS or RL (Rohm Pharma,
Germany).
[0090] In another embodiment, the sustained release formulation is
a matrix dissolution device, which is prepared by compressing the
drug with a slowly soluble polymer carrier into a tablet. In one
preferred embodiment, the coated particles have a size range
between about 0.1 to about 300 microns, as disclosed in U.S. Pat.
Nos. 4,710,384 and 5,354,556, which are incorporated herein by
reference in their entireties. Each of the particles is in the form
of a micromatrix, with the active ingredient uniformly distributed
throughout the polymer.
[0091] Sustained release formulations such as those described in
U.S. Pat. No. 4,710,384, which is incorporated herein by reference
in its entirety, have a relatively high percentage of plasticizer
in the coating in order to permit sufficient flexibility to prevent
substantial breakage during compression. The specific amount of
plasticizer varies depending on the nature of the coating and the
particular plasticizer used. The amount may be readily determined
empirically by testing the release characteristics of the tablets
formed. If the medicament is being released too quickly, then more
plasticizer is used. Release characteristics are also a function of
the thickness of the coating. When substantial amounts of
plasticizer are used, the sustained released capacity of the
coating diminishes. Thus, the thickness of the coating may be
increased slightly to make up for an increase in the amount of
plasticizer. Generally, the plasticizer in such an embodiment will
be present in an amount of about 15 to 30 percent of the sustained
release material in the coating, preferably 20 to 25 percent and
the amount of coating will be from 10 to 25 percent of the weight
of active material, preferably 15 to 20 percent. Any conventional
pharmaceutically acceptable plasticizer may be incorporated into
the coating.
5.2. Target Cancers
[0092] Cancers that can be treated by the methods of the present
invention include, but not limited to human sarcomas and
carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,
rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast
cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,
basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma,
Wilms.+-.tumor, cervical cancer, testicular tumor, lung carcinoma,
small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,
glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma,
retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and
acute myelocytic leukemia (myeloblastic, promyelocytic,
myclomonocytic, monocytic and erythroleukemia); chronic leukemia
(chronic myelocytic (granulocytic) leukemia and chronic lymphocytic
leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and
non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, and heavy chain disease.
[0093] In a preferred embodiment, the cancer is one where
circulating levels of tNOX are present in the sera of patients
suffering from said cancer, e.g., rectal carcinoma, colon
carcinoma, breast carcinoma, ovarian carcinoma, small cell lung
carcinoma, colon carcinoma, chronic lymphocytic carcinoma, hairy
cell leukemia, osophogeal carcinoma, prostate carcinoma, breast
cancer, myeloma, and lymphoma, see e.g. U.S. Pat. No. 5,605,810,
which is incorporated by reference in its entirety.
[0094] In a preferred embodiment, the patient already has cancer
and is undergoing treatment for said cancer. In a specific
embodiment, the patient already has cancer but no metastasis. i.e.,
secondary cancer. In another specific embodiment, the patient
already has cancer plus a metastatic cancer. In another specific
embodiment, the patient having a cancer is immunosuppressed by
reason of having undergone anti-cancer therapy (e.g., chemotherapy
or radiation) prior to administration of the catechin complexes of
the invention.
[0095] In another specific embodiment, the cancer is a tumor. In a
preferred embodiment, the tumor is a tumor of epithelial tissue,
lymphoid tissue, connective tissue, bone, or central nervous
system.
5.3. Combination Therapy
[0096] The invention encompasses the catechin formulations listed
in Section 5.1 administered in combination with other therapeutic
agents, such as anti-cancer drugs. The therapeutic agents include,
but are not limited to adriamycin and adriamycin conjugates,
mechlorethamine, cyclophosphamide, ifosfamide, melphalan,
chlorambucil, hexamethylmelamine, thiotepa, busulfan, carmustine,
lomustine, semustine, streptozocin, dacarbazine, methotrexate,
fluorouacil, floxuridie, cytarabine, mercaptopurine, thioguanine,
pentostatin, vinblastine, vincristine, etoposide, teniposide,
actinomycin D, daunorubicin, doxorubicin, bleomycin, plicamycin,
mitomycin, L-asparaginase, interferon-alpha, cisplatin,
carboplatin, mitoxantrone, hydroxyurea, procarbazine, mitotane,
aminoglutethimide, prednisone, hydroxyprogesterone caproate,
medroxyprogesterone acetate, megestrol acetate, diethylstilbestrol,
ethinyl estradiol, tamoxifen, testosterone propionate,
fluoxymesterone, flutamide, leuprolide, acetogenins, e.g.,
bullatacin, and quassanoids, e.g. simalikalactone D and
glaucarubolone, and pharmaceutically acceptable derivatives thereof
. The therapeutic agents which inhibit tNOX and cancer cell growth
include adriamycin, bullatacin, simalikalactone D, and
glaucarubolone has been demonstrated by the Inventors in U.S. Pat.
No. 5,605,810, which is incorporated by reference in its entirety
for all purposes.
[0097] The invention also embodies the catechin formulations,
anti-cancer agents, and combinations thereof for the treatment of
cancer patients undergoing chemotherapy and/or irradiation for a
primary cancer. In a preferred embodiment, the catechin
formulations, anti-cancer agents, and combinations thereof provides
a method for treating the metastasized, i.e. secondary cancer, in
said patients.
[0098] In another embodiment, the secondary agent administered, in
addition to the catechin formulations, includes a monoclonal
antibody directed against tNOX for combination therapy. A
monoclonal antibody to the human tNOX protein isolated from the
sera of cancer patients has already successfully been used in the
expression cloning of tNOX from HeLa cells (Chueh et al., 1997,
Arch. Biochem. Biophys. 342:38-44).
5.4. Pharmaceutical Compositions for Cancer Treatment
[0099] Catechin complexes of the invention may be formulated into
pharmaceutical preparations for administration to mammals for
treatment of cancer. In a preferred embodiment, the mammal is a
human.
[0100] Compositions comprising a compound of the invention
formulated in a compatible pharmaceutical carrier may be prepared,
packaged, and labelled for treatment of the indicated cancer, such
as human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms.+-.tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma,
retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and
acute myelocytic leukemia (myeloblastic, promyelocytic,
myelomonocytic, monocytic and erythroleukemia); chronic leukemia
(chronic myelocytic (granulocytic) leukemia and chronic lymphocytic
leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and
non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, and heavy chain disease.
5.5. Modes of Administration
5.5.1. Sustained Release Formulation
[0101] The catechins of the invention are formulated as a sustained
and/or timed release formulation. Applicants provide a description
of a response to the catechins which is reversible in cells (see
Example 6, infra). Furthermore, the levels of circulating catechin
compositions must be maintained above some minimum therapeutic dose
to reduce the number of cancer cells and/or prevent cancer. In one
embodiment, the reduction in the number of cancer cells is a result
of cell death or apoptosis. In another embodiment, the reduction in
the number of cancer cells is a result of inhibition of cell
growth. In yet another embodiment, the reduction in the number of
cancer cells is a result of cell growth arrest.
[0102] All sustained release pharmaceutical products have a common
goal of improving drug therapy over that achieved by their
non-sustained counterparts. Ideally, the use of an optimally
designed sustained release preparation in medical treatment is
characterized by a minimum of drug substance being employed to cure
or control the condition. Advantages of sustained release
formulations may include: (1) extended activity of the composition;
(2) reduced dosage frequency; and (3) increased patient compliance.
In addition, sustained release formulations can be used to affect
the time of onset of action or other characteristics, such as blood
levels of the composition, and thus can affect the occurrence of
side effects.
[0103] The sustained release formulations of the invention are
designed to initially release an amount of the therapeutic
composition that promptly produces the desired therapeutic effect,
and gradually and continually release of other amounts of
compositions to maintain this level of therapeutic effect over an
extended period of time. In order to maintain this constant level
in the body, the therapeutic composition must be released from the
dosage form at a rate that will replace the composition being
metabolized and excreted from the body.
[0104] The sustained release of an active ingredient may be
stimulated by various inducers, for example pH, temperature,
enzymes, water, or other physiological conditions or compounds. The
term "sustained release component" in the context of the present
invention is defined herein as a compound or compounds, including,
but not limited to, polymers, polymer matrices, gels, permeable
membranes, liposomes, microspheres, or the like, or a combination
thereof , that facilitates the sustained release of the active
ingredient.
[0105] Data showing the effectiveness of a sustained release
formulation on the inhibition of NADH oxidase activity in cancer
cells is presented in Section 9, infra.
5.5.2. Modes of Administration of Water-soluble Complexes
[0106] If the complex is water-soluble, then it may be formulated
in an appropriate buffer, for example, phosphate buffered saline or
other physiologically compatible solutions. Alternatively, if the
resulting complex has poor solubility in aqueous solvents, then it
may be formulated with a non-ionic surfactant such as Tween, or
polyethylene glycol. Thus, the compounds and their physiologically
acceptable solvates may be formulated for administration by
inhalation or insufflation (either through the mouth or the nose)
or oral, buccal, parenteral, rectal administration or, in the case
of tumors, directly injected into a solid tumor.
5.5.3. Oral Administration
[0107] For oral administration, the pharmaceutical preparation may
be in liquid form, (e.g., solutions, syrups or suspensions), or may
be presented as a drug product (e.g., capsule or powder) for
reconstitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, or fractionated vegetable
oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates
or sorbic acid). The pharmaceutical compositions may take the form
of , for example, tablets or capsules prepared by conventional
means with pharmaceutically acceptable excipients such as binding
agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well-known in the art. In a preferred embodiment,
the pharmaceutical composition may take the form of a capsule or
powder to be dissolved in a liquid for oral consumption.
[0108] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound. In a
preferred embodiment, the compounds of the present invention are
formulated as controlled release powders of discrete
micro-particles which can be readily formulated in liquid form. The
sustained release powder comprises particles containing an active
ingredient and optionally, an excipient with at least one non-toxic
polymer.
[0109] The powder can be dispersed or suspended in a liquid vehicle
and will maintain its sustained release characteristics for a
useful period of time. These dispersions or suspensions have both
chemical stability and stability in terms of dissolution rate. The
powder may contain an excipient comprising a polymer, which may be
soluble, insoluble, permeable, impermeable, or biodegradable. The
polymers may be polymers or copolymers. The polymer may be a
natural or synthetic polymer. Natural polymers include polypeptides
(e.g., zein), polysaccharides (e.g., cellulose), and alginic acid.
Representative synthetic polymers include those described, but not
limited to, those described in column 3, lines 33-45 of U.S. Pat.
No. 5,354,556 which is incorporated by reference in its entirety.
Particularly suitable polymers include those described, but not
limited to, those described in column 3, line 46-column 4, line 8
of U.S. Pat. No. 5,354,556 which is incorporated by reference in
its entirety.
5.5.4. Buccal Administration
[0110] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
5.5.5. Parenteral Administration
[0111] The sustained release compounds of the invention may be
formulated for parenteral administration, e.g., by intramuscular
injections or implants for subcutaneous tissues and various body
cavities and transdermal devices.
[0112] Formulations for injection may be presented in unit dosage
form, e.g., in ampules or in multi-dose containers, with an added
preservative. The compositions may take such forms as suspensions,
solutions or emulsions in oily or aqueous vehicles, and may contain
formulatory agents such as suspending, stabilizing and/or
dispersing agents. Alternatively, the active ingredient may be in
powder form for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
[0113] In a preferred embodiment, intramuscular injections are
formulated as aqueous or oil suspensions. In an aqueous suspension,
the sustained release effect is due to, in part, a reduction in
solubility of the active compound upon complexation or a decrease
in dissolution rate. A similar approach is taken with oil solutions
and suspensions, wherein the release rate of an active compound is
determined by partitioning of the active compound out of the oil
into the surrounding aqueous medium. Only active compounds which
are oil soluble and have the desired partition characteristics are
suitable. Oils that may be used for intramuscular injection
include, but are not limited to, sesame, olive, arachnis, maize,
almond, cottonseed, and castor oil.
[0114] A highly developed form of drug delivery that imparts
sustained release over periods of time ranging from days to years
is to implant a drug-bearing polymeric device subcutaneously or in
various body cavities. The polymer material used in an implant,
which must be biocompatible and nontoxic, include but are not
limited to hydrogels, silicones, polyethylenes, ethylene-vinyl
acetate copolymers, or biodegradable polymers.
5.5.6. Rectal Administration
[0115] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
5.5.7. Packs and Kits
[0116] The compositions may, if desired, be presented in a pack or
dispenser device 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.
[0117] The invention also provides kits for carrying out the
therapeutic regimens of the invention. Such kits comprise in one or
more containers having therapeutically or prophylactically
effective amounts of the catechin complexes in pharmaceutically
acceptable form. The catechin complex in a vial of a kit of the
invention may be in the form of a pharmaceutically acceptable
solution, e.g., in combination with sterile saline, dextrose
solution, or buffered solution, or other pharmaceutically
acceptable sterile fluid. Alternatively, the complex may be
lyophilized or desiccated; in this instance, the kit optionally
further comprises in a container a pharmaceutically acceptable
solution (e.g., saline, dextrose solution, etc.), preferably
sterile, to reconstitute the complex to form a solution for
injection purposes.
[0118] In another embodiment, a kit of the invention further
comprises a needle or syringe, preferably packaged in sterile form,
for injecting the complex, and/or a packaged alcohol pad.
Instructions are optionally included for administration of catechin
complexes by a clinician or by the patient.
5.6. Dosage
5.6.1. Catechins as a Dietary Supplement for Preventing Cancer
[0119] In one embodiment of this invention, a sustained release
formulation comprising catechins may be used as a dietary or
nutritional supplement for the prevention of cancer. In this
embodiment, the total daily dose ranges of the active catechins for
the conditions described herein are generally from about 10 mg to
about 800 mg administered in divided doses administered
parenterally or orally. A preferred total daily dose is from about
50 mg to about 400 mg of the active catechins.
[0120] In a another embodiment, a total daily dose of a sustained
release formulation may be used as a dietary supplement is about 10
mg to about 800 mg of active catechins administered twice daily
(e.g., in the morning and the evening) at a dose of about 5 mg to
about 400 mg. The dosage forms and compositions may comprise any of
the forms and compositions described supra. In a preferred
embodiment, the sustained release formulation comprising catechins
is a tablet, capsule, gel, or a liquid-soluble powder.
5.6.2. Catechins as a Therapeutic for Treating Cancer
[0121] In another embodiment of the invention, the magnitude of a
therapeutic dose of catechins in the acute or chronic management of
cancer will vary with the severity of the condition to be treated
and the route of administration. The dose, and dose frequency, will
also vary according to the age, body weight, condition and response
of the individual patient, and the particular catechin combination
used. All combinations described in the specification are
encompassed as therapeutic, active catechin mixtures and it is
understood that one of skill in the art would be able to determine
a proper dosage of particular catechin mixtures using the
parameters provided in the invention. In general, the total daily
dose ranges of the active catechins for the conditions described
herein are generally from about 10 mg to about 1000 mg administered
in divided doses administered parenterally or orally or topically.
A preferred total daily dose is from about 200 mg to about 600 mg
of the active catechins.
[0122] For example, in one embodiment, the daily dose ranges of
EGCg and EC for the conditions described herein are generally from
about 0.15 to about 15 mg per kg body weight of EGCg and 10 to
about 100 mg per kg weight of body EC. Preferably the catechin
formulation of the invention is given daily until remission,
followed by two to ten additional cycles, each lasting about 60
days in duration. When the dose is administered orally, a sustained
release formulation is preferred so that a fairly constant level of
catechins is provided over the course of treatment, which is
generally at least 48 hours and preferably at least 96 hours per
cycle. As the catechins are not particularly toxic, the formulation
may be administered for as long as necessary to achieve the desired
therapeutic effect.
[0123] In the case where an intravenous injection or infusion
composition is employed, a suitable dosage range for use is, e.g.,
from about 0.01 to about 1.5 mg per kg body weight of EGCg and
about 1 to about 10 mg per kg body weight of EC total daily.
[0124] For treatment of solid tumors, a preferred dosing regimen
involves intravenous infusion of about 0.01 to about 1.5 mg per kg
body weight of EGCg and about 1 to about 10 mg per kg body weight
of EC per day. This daily treatment protocol is repeated once per
month until the tumor growth tumor is inhibited or when the tumor
shows signs of regression.
[0125] As stated in Section 5.1, EGCg and EC are present in varying
percentages in the formulation. Thus, the formulation will be
adjusted to reflect the concentrations of EGCg and EC, i.e., in one
preferred embodiment, EGCg is 40% and EC is 7% of the total
catechins in the formulation. So, in one non-limiting example, 15
to 1500 mg of the total formulation will be required for a dose of
6 to 600 mg of EGCg and 1 to 105 mg of EC.
[0126] In another preferred embodiment, EGCg is 0.1% of the total
catechins and EC is 100 fold greater than the EGCg content of the
total catechins in the formulation. So, in this non-limiting
example, 15 to 1500 mg of the total formulation will be required
for a dose of 0.15 to 1.5 mg of EGCg and 1.5 to 150 mg of EC.
[0127] In an alternative embodiment of the invention, the effect of
the therapy with EGCg and EC on cancer treatment can be monitored
by any methods known in the art, including but not limited to
monitoring circulating tNOX activity in patient sera, as well as
more traditional approaches such as determining levels of tumor
specific antigens and putative biomarkers, e.g., carcinoembryonic
antigens (CEA), alpha-fetoprotein; and changes in morphology and/or
size using computed tomographic scan and/or sonogram.
[0128] Desirable blood levels may be maintained by a continuous
infusion of EGCg and EC as ascertained by plasma levels. It should
be noted that the attending physician would also know how to and
when to adjust treatment to higher levels if the clinical response
is not adequate (precluding toxic side effects, if any).
[0129] Again, any suitable route of administration may be employed
for providing the patient with an effective dosage of EGCg and EC
or another catechin combination of this invention. Dosage forms
include tablets, troches, cachet, dispersions, suspensions,
solutions, capsules, gel caps, caplets, compressed tablets,
sustained release devices, patches, and the like.
[0130] The pharmaceutical compositions of the present invention
comprise catechins as the active ingredients, as well as
pharmaceutically acceptable salts thereof , and may also contain a
pharmaceutically acceptable carrier, and optionally, other
therapeutic ingredients. The term "pharmaceutically acceptable
salts" refers to salts prepared from pharmaceutically acceptable
non-toxic acids and bases, including inorganic and organic acids
and bases.
[0131] The pharmaceutical compositions include compositions
suitable for oral and parenteral (including subcutaneous,
intramuscular, intrathecal, intravenous, and other injectables)
routes, although the most suitable route in any given case will
depend on the nature and severity of the condition being
treated.
[0132] In addition, the catechin carrier could be delivered via
charged and uncharged matrices used as drug delivery devices such
as cellulose acetate membranes, also through targeted delivery
systems such as fusogenic liposomes attached to antibodies or
specific antigens.
[0133] In practical use, catechins can be combined as the active
ingredient(s) in intimate admixture with a pharmaceutical carrier
according to conventional pharmaceutical compounding techniques.
The carrier may take a wide variety of forms depending on the form
of preparation desired for administration, e.g., oral or parenteral
(including tablets, capsules, powders, intravenous injections or
infusions). In preparing the compositions for oral dosage form any
of the usual pharmaceutical media may be employed, e.g., water,
glycols, oils, alcohols, flavoring agents, preservatives, coloring
agents, and the like; in the case of oral liquid preparations,
e.g., suspensions, solutions, elixirs, liposomes and aerosols;
starches, sugars, micro-crystalline cellulose, diluents,
granulating agents, lubricants, binders, disintegrating agents, and
the like in the case of oral solid preparations e.g., powders,
capsules, and tablets. In preparing the compositions for parenteral
dosage form, such as intravenous injection or infusion, similar
pharmaceutical media may be employed, e.g., water, glycols, oils,
buffers, sugar, preservatives and the like know to those skilled in
the art. Examples of such parenteral compositions include, but are
not limited to Dextrose 5% (w/v), normal saline or other solutions.
The total dose of the catechins may be administered in a vial of
intravenous fluid, e.g., ranging from about 0.01 to about 1000 mg
per kg body weight of catechins. The volume of dilution fluid will
vary according to the total dose administered and over the length
of the period of time of administration.
[0134] An exemplary course of treatment of a patient with cancer or
solid cancer can involve daily administration by intravenous
infusion of catechins in an aqueous solution at a daily dose of
about 0.01 to about 1.5 mg of the EGCg and about 1 to about 10 mg
of the EC compositions per kg of body weight of the patient. The
course of treatment may be repeated for up to ten times over
approximately 10 months with a break of about three to six weeks in
between courses. The post-remission course of treatment involves
infusion of EGCg and EC at a daily dose of about 0.01 to about 1 mg
per kg of body weight of the patient on a daily or weekdays-only
basis for a cumulative total of 25 days.
[0135] In another embodiment, the invention encompasses the daily
dose ranges of EGCg and ECG for the conditions described herein are
generally from about 0.1 to about 15 mg per kg body weight
administered in divided doses administered orally. Preferably the
catechin formulation of the invention is given daily, or until
remission, followed by two to ten additional cycles, each lasting
about 60 days in duration. When the dose is administered orally, a
sustained release formulation is preferred so that a fairly
constant level of catechins is provided over the course of
treatment, which is generally at least 48 hours and preferably at
least 96 hours per cycle. As the catechins are not particularly
toxic, the formulation may be administered for as long as necessary
to achieve the desired therapeutic effect. In the case where an
intravenous injection or infusion composition is employed, a
suitable dosage range for use is, e.g., from about 0.01 to about
1.5 mg per kg body weight of EGCg and ECG total daily.
[0136] For treatment of solid tumors, a preferred dosing regimen
involves intravenous infusion of the active catechins of the
invention, as described above, in the amount of about 0.01 to about
10 mg per kg body weight per day. This daily treatment protocol is
repeated once per month until the tumor growth tumor is inhibited
or when the tumor shows signs of regression.
[0137] As stated in Section 5.1, EGCg and ECG are present in
varying percentages in the formulation. Thus, the formulation will
be adjusted to reflect the concentrations of EGCg and ECG, i.e., in
a preferred embodiment, EGCg is 40% and ECG is 15% of the total
catechins in the formulation. Thus, in one non-limiting example, 15
to 1500 mg of the total formulation will be required for a dose of
6 to 600 mg of EGCg and 2.25 to 225 mg of ECG.
[0138] The effect of the therapy with EGCg and ECG on cancer
treatment can be monitored by methods stated supra in the example
of EGCg and EC. Similarly, pharmaceutical compositions and routes
of administration are similar as those described supra for EGCg and
EC.
[0139] For the purposes described above, the invention also
encompasses methods for monitoring patient response to tea
catechins. By monitoring circulating tNOX activity in patient sera,
it will be possible to determine therapeutic dosages and to monitor
therapeutic benefit from tea catechins. The response of neoplastic
cells to the subject compositions may be monitored by assaying the
blood or urine of the patient for the NOX activity that is
responsive to the catechin compositions, i.e., tNOX. Various assays
may be used to monitor activity, such as a NOX assay for neoplasia
determination see e.g., U.S. Pat. No. 5,605,810. By following the
above monitoring procedures, an effective dosage of the subject
compositions may be administered in accordance with the requirement
of an individual patient.
6. EXAMPLE
Epigallocatechin Gallate Inhibits Preferentially the NADH Oxidase
and Growth of Transformed Cells in Culture
6.1. Materials and Methods
6.1.1. Growth of Cells
[0140] HeLa (ATCC CCL2) cells were grown in 175 cm.sup.2 flasks in
Minimal Essential Medium (Gibco), pH 7.4, at 37.degree. C. with 10%
bovine calf serum (heat-inactivated), plus 50 mg/l gentamycin
sulfate (Sigma). Cells were harvested by scraping and taken up in
140 mM NaCl, 5 mM KCl, 0.7 mM Na.sub.2HPO.sub.4 and 25 mM Tris, pH
7.4 to a final cell concentration of 0.1 g wet weight (gww) per
ml.
[0141] MCF-10 A human mammary epithelial cells were cultured in a
1:1 mixture of Ham's F12 medium and Dulbecco's Modified Eagle's
medium containing cholera enterotoxin (100 ng/ml), insulin (10
.mu.g/ml), hydrocortisone (0.5 .mu.g/ml), epidermal growth factor
(EGF, 20 mg/ml), and 5% horse serum. Media were renewed every 2-3
days.
[0142] BT-20 human breast adenocarcinoma cells were cultured in
Eagle's minimal essential medium nonessential amino acids and
Earle's balanced salts supplement with 10% fetal bovine serum.
Media were renewed as for MCF-10A cells.
[0143] Cell lines were from the American Type Culture Collection
(Rockville, Md.).
6.1.2. Purification of Plasma Membranes from Cultured Cells
[0144] Cultured cells were collected by centrifugation for 6-15 min
at 175-1000.times. g. The cell pellets were resuspended in 0.2 mM
EDTA in 1 mM NaHCO.sub.3 in an approximate ratio of 1 ml per
10.sup.8 cells and incubated on ice for 10-30 min to swell the
cells. Homogenization was achieved in 7- to 8-ml aliquots with a
Polytron homogenizer (Brinkmann) for 30-40 sec at 10,500 rpm, using
a PT-PA 3012/23 or ST-10 probe. To estimate breakage, the cells
were monitored by light microscopy before and after homogenization.
At least 90% cell breakage without breakage of nuclei was achieved
routinely.
[0145] The homogenates were centrifuged for 10 min at 175.times. g
to remove unbroken cells and nuclei and the supernatant was
centrifuged a second time at 1.4.times.10.sup.6 g.multidot.min
(e.g., 1 h at 23,500.times.g) to prepare a plasma membrane-enriched
microsome fraction. The supernatant was discarded and the pellets
were resuspended in 0.2 M potassium phosphate buffer in a ratio of
.about.1 ml per pellet from 5.times.10.sup.8 cells. The resuspended
membranes were then loaded onto the two-phase system constituted on
a weight basis consisting of 6.6% (w/w) Dextran T-500 (Pharmacia)
and 6.6% (w/w) Polyethylene Glycol 3350 (Fisher) in a 5 mM
potassium phosphate buffer (pH 7.2) for aqueous two-phase
separation as described (Morr1971, Methods Enzymol. 22:130-148, and
Morr and Morr, 1989, BioTechniques 7:946-958). The upper phase,
enriched in plasma membranes, was diluted 5-fold with 1 mM sodium
bicarbonate and the membranes were collected by centrifugation. The
purity of the plasma membrane was determined to be >90% by
electron microscope morphometry. The yield was 20 mg plasma
membrane protein from 10.sup.10 cells.
6.1.3. Preparation of HeLa Cells and Cell-free Extracts
[0146] HeLa S cells were collected by centrifugation and shipped
frozen in 0.1 M sodium acetate, pH 5 in a ratio of 1 ml packed cell
volume to 1 ml of acetate (Cellex Biosciences, Minneapolis, Minn.).
The cells were thawed at room temperature, resuspended and
incubated at 37.degree. C. for 1 h to release the protein (del
Castillo-Oliveras et al.,1998, Arch. Biochem. Biophys.
358:125-140). The cells were removed by centrifugation at 37,000 g
for 60 min and the cell-free supernatants were refrozen and stored
in 1 ml aliquots at -70.degree. C.
[0147] For heat treatment, 1 ml aliquots of the above supernatant
material were thawed at room temperature and heated to 50.degree.
C. for 10 min. The denatured proteins were removed by
centrifugation (1,500 g, 5 min). Full activity was retained from
this step (del Castillo-Oliveras et al., 1998, Arch. Biochem.
Biophys. 358:125-140).
[0148] For protease treatment, the pH of the heat-stable
supernatant was adjusted to 7.8 by addition of 0.1 M sodium
hydroxide. Tritirachium album proteinase K (Calbiochem) was added
(4 ng/ml) and incubated at 37.degree. C. for 1 h with full
retention of enzymatic activity and drug response (del
Castillo-Oliveras et al., 1998, Arch. Biochem. Biophys.
358:125-140). The reaction was stopped either by freezing for
determination of enzymatic activity or by addition of 0.1 M
phenylmethylsulfonyl fluoride (PMSF) in ethanol to yield a final
concentration of 10 mM PMSF.
6.1.4. Spectrophotometric Assay of NADH Oxidase
[0149] NADH oxidase activity was determined as the disappearance of
NADH measured at 340 nm in a reaction mixture containing 25 mM
Tris-Mes buffer (pH 7.2), 1 mM KCN to inhibit low levels of
mitochondrial oxidase activity, and 150 mM NADH at 37.degree. C.
with stirring. Activity was measured using a Hitachi U3210 or SLM
Aminco DW2000 spectrophotometer with continuous recording over two
intervals of 5 min each. A millimolar extinction coefficient of
6.22 was used to determine specific activity. EGCg was added at the
final concentrations indicated at the beginning of the assay and
was present during the assay period.
[0150] Proteins were estimated by the bicinchonic acid method
(Smith et al., 1985, Anal. Biochem. 150:76-85) with bovine serum
albumin as standard.
6.1.5. Fluorescence Microscopy
[0151] Cells were grown for 72 h on glass coverslips placed in
small culture dishes with media containing 100 .mu.M EGCg in
ethanol or an equivalent amount of ethanol alone. The coverslips
were rinsed and the cells fixed in methanol followed by addition of
fluorescent dye 4',6-diamidino-2-phenylindole (DAPI) as described
(Wolvetang et al., 1994, FEBS Lett. 339:40-44). Cells were observed
and photographed at a primary magnification of 400.times..
6.1.6. Determination of EGCg
[0152] EGCg was determined with the hot water extracts using the
standardized chromatographic procedure described by Katiyar et al.
(Katiyar et al., 1992, Nutr. Can. 18:73-83). Authentic EGCg (Sigma)
was used as the standard.
6.1.7. Chemicals
[0153] All chemicals were from Sigma (St. Louis, Mo.) unless
otherwise specified. EGF was from mouse, culture grade, from
Upstate Biotechnology Inc. (Lake Placid, N.Y.). Tea infusions were
prepared by sequential steeping of ca. 2-g bags of tea (Lipton) in
10 ml of water for 10 min each. At the end of the infusion, bags
were pressed to remove liquid.
6.2. Results
6.2.1. NADH Oxidase Activity in Plasma Membrane Vesicles
[0154] Epigallocatechin gallate (EGCg) was without effect on the
NADH oxidase activity of plasma membrane vesicles (FIG. 2) or NADH
oxidase solubilized and partially purified from the cell surface
(FIG. 3) of human mammary epithelia (MCF-10A). However, with plasma
membranes from human mammary adenocarcinoma (BT-20) or HeLa (human
cervical carcinoma) cells, NADH oxidase activities were inhibited
by 30 to 40% with an ED.sub.50 of about 1 nM (FIG. 2). BT-20 and
HeLa cells contain a drug-responsive component of NADH oxidase
activity inhibited by capsaicin or the antitumor sulfonylurea as
well as NADH oxidase activities resistant to inhibition. The
responses to EGCg were comparable to those for capsaicin and the
sulfonylurea.
[0155] With plasma membrane vesicles from the BT-20 mammary
adenocarcinoma cell line, the NADH oxidase specific activity was
approximately 1.5 that of the MCF-10A cell line (FIG. 2A). Upon
addition of EGCg, the specific activity of the MCF-10A cells was
unchanged, whereas, that of the BT-20 was reduced to approximately
the same level as that of the MCF-10A cells (FIG. 2A). Also
inhibited by EGCg in a similar fashion was the NADH oxidase
activity from plasma membranes of HeLa cells (FIG. 2B). Thus, in
the plasma membrane vesicles from the BT-20 and HeLa cells, there
were both EGCg-resistant and EGCg-susceptible components whereas in
the plasma membrane vesicles from the MCF 10A cells only an
EGCg-resistant activity was observed (FIG. 2A).
6.2.2. NADH Oxidase Activity Released from Cultured Cells
[0156] Results similar to those observed with isolated plasma
membrane vesicles were obtained as well with solubilized NADH
oxidase preparations of NADH oxidase released from cultured cells
by low pH treatment (FIG. 3). With BT-20 (FIG. 3A) and HeLa (FIG.
3B) preparations, activity was strongly inhibited by EGCg with an
EC.sub.50 of between 1 and 10 nM. The released and solubilized NADH
oxidase for the MCF-10A cells was much less affected by the EGCg
(FIG. 3A). As with isolated plasma membrane vesicles, the specific
activity of the released NADH oxidase preparations from BT-20 cells
was greater (approximately twice) than that of the released
preparations from MCF-10A cells. Following treatment with EGCg, the
specific activity of the preparations from BT-20 cells was reduced
to a level comparable to the specific activity of the preparations
from MCF-10A cells. Thus, the EGCg appears to inhibit specifically
the drug-responsive NADH oxidase component of the tumorigenically
transformed cell lines but not that of the constitutive NADH
oxidase activity of the MCF-10A mammary epithelial line.
6.2.3. Effect of EGCg on Intact Cells in Culture
[0157] EGCg also inhibited the growth of the BT-20 mammary
adenocarcinoma and HeLa cells in culture (FIG. 2C, D). While not as
striking as for the inhibition of NADH oxidase, EGCg did
preferentially restrict the growth of the HeLa and BT-20 cells
compared to MCF-10A (FIG. 2C, D). Growth of the MCF-10A mammary
epithelial cells was unaffected by EGCg except at very high doses
of 10.sup.-4 (FIG. 2C), whereas that of the tumorigenically
transformed BT-20 and HeLa cells was 50% inhibited at about
5.times.10.sup.-3 M (FIG. 2C, D).
[0158] Despite early growth inhibition of MCF-10A cells by EGCg,
the cells quickly recovered and eventually grew normally (FIG. 4).
This is in contrast to HeLa and BT-20 cells where the cells did not
recover and died (FIG. 4).
[0159] Measurements of the diameters of treated HeLa and BT-20
cells taken directly from printed micrographs revealed that, on
average, the cells treated with 5.times.10.sup.-6 to
5.times.10.sup.-5 M EGCG exhibited volumes.about.50% those of
untreated cells. At 10.sup.-6 M EGCg, there was no response of any
of the cell lines at 72 h despite the fact that this EGCg
concentration inhibited the tNOX activity of isolated plasma
membranes. The possibility was considered that the combination of a
reversible inhibition and rapid metabolism of EGCg might result in
an overall lack of growth inhibition at 10.sup.-6 M EGCg after 3
days. To test this possibility, cells were treated with 10.sup.-6 M
EGCg twice daily for 96 h after which time the cells were
photographed, measured and counted. Cell diameters were reduced on
average by about 25% and cell volume by 50% by the twice daily
10.sup.-6 M EGCg dosage. Cell number also was reduced by about 25%
with both HeLa and BT-cells by the 10.sup.-6 M EGCg provided twice
daily whereas with the non-cancer MCF-10A cells, growth rate and
cell diameters were unaffected or slightly increased. When the
cells treated with 10.sup.-5 or 5.times.10.sup.-5 EGCg were stained
to reveal DAPI fluorescence, a very large percentage of the treated
cells showed nuclear DNA with the condensed and fragmented
appearance characteristic of apoptotic cells (FIG. 5).
6.2.4. Green Tea Inhibits NADH Oxidase
[0160] Since EGCg is considered as one of the major compounds
contributing to the cancer preventative actions attributed to green
tea, green tea infusions were examined as well for their ability to
inhibit the NADH oxidase (Weisburger, 1997, Can. Lttr. 114:315-317;
Chen et al., 1998, Can. Lttr. 129:173-179; Fujiki et al., 1998,
Mutation Res. 402:307-310; Liao et al., 1995; Can. Lttr.
96:239-243; Stoner and Mukhtar, 1995, J. Cell. Biochem. 22:169-180;
and Ahmad et al., 1997, J. Nat. Can. Inst. 89:1881-1886). Both the
solubilized and partially purified NADH oxidase released from cells
by low pH treatment (FIG. 6) and the NADH oxidase of sera pooled
from cancer patients (Table 1) were inhibited by green tea
infusions. Infusions of green tea (Lipton) were approximately ten
times more effective than those of black tea (Lipton) and
correlated approximately with the content of EGCg with an EC.sub.50
of 2.times.10.sup.6 M EGCg equivalent to 1 .mu.g/ml.
2TABLE 1 Inhibition of tNOX activity by tea infusions and by
epigallocatechin gallate (EGCg), the major tea polyphenol
(catechin) of green tea, for sera pooled from patients with cancer.
The EGCg content was determined as described (Katiyar et al., 1992,
Nutr. Can. 18:73-83). Results were repeated 3 to 5 times with
different sources and preparations of both black and green tea and
with consistent findings. Source EC.sub.50 EGCg (.mu.g/ml) Black
tea (Lipton) 1:10 to 1:100 1 Green tea (Lipton) 1:1000 1
Epigallocatechin gallate (EGCg) 2 .times. 10.sup.-6 M 1
6.2.5. EGCg Inhibits Cancer Cell Growth
[0161] Not only did EGCg inhibit the NADH oxidase of plasma
membrane vesicles from cancer cells and not that of normal cells,
the substance exerted a parallel response on growth. Growth of HeLa
cells was almost completely inhibited by EGCg whereas growth of CHO
cells and mammary epithelial cells was much less affected by EGCg.
With treated HeLa cells, nuclei exhibited patterns of fluorescence
characteristic of apoptosis (Smith et al., 1985, Anal. Biochem.
150:76-85). Thus, the cyanide-resistant NADH oxidase of the plasma
membrane appears to represent an enzymatic activity whose
inhibition by EGCg correlates with an inhibition of growth and
subsequent apoptosis in susceptible cancer cell lines.
7. EXAMPLE
Synergistic Effects of (-)- Epigallocatechin Gallate with (-)-
Epicatechin on Inhibition of Cell Surface NADH Oxidase (NOX)
Activity and Growth if 4T1 Mouse Mammary and Hela Cells in
Culture
[0162] 7.1. Materials and Methods
7.1.1. Chemicals
[0163] EGCg and EC were purchased from Sigma (St. Louis, Mo.) or
purified from leaves of green tea and supplied by Pharmanex
(Brisbane, Calif.). The stability and purity (>98%) of the EGCg
and EC were confirmed by high performance liquid chromatographic
analysis.
7.1.2. Growth of Cells
[0164] HeLa (ATCC CCL2) cells were grown in 150 cm.sup.2 flasks in
Minimal Essential Medium (Gibco), pH 7.4, at 37.degree. C. with 10%
bovine calf serum (heat-inactivated), plus 50 mg/l gentamicin
sulfate (Sigma). Cells were trypsinized with Sigma IX trypsin for 1
to 2 min and harvested by scraping and taken up in 140 mM NaCl, 5
mM KCl, 0.7 mM Na.sub.2HPO.sub.4 and 25 mM Tris, pH 7.4, to a final
cell concentration of 0.1 g wet weight (gww) per ml.
[0165] A mouse mammary tumor subpopulation line 4T1 arising from a
BALB/cf C3H mouse was grown in DME-10, Dulbecco's modified Eagle's
medium supplemented with 5% fetal calf serum, 5% newborn calf
serum, 1 mM mixed non-essential amino acids, 2 mM L-glutamine,
penicillin (100 units/ml), and streptomycin (100 .mu.g/ml) (Miller
et al., 1987, Brit. J. Can. 56:561-569 and Miller et al., 1990,
Invasion Metastasis 10:101-112).
7.1.3. Purification of Plasma Membranes from Cultured Cells
[0166] Cultured cells were collected by centrifugation for 6 to 15
min at 1,000 to 3,000 rpm. The cell pellets were resuspended in 0.2
mM EDTA in 1 mM NaHCO.sub.3 in an approximate ratio of 1 ml per
10.sup.8 cells and incubated on ice for 10 to 30 min to swell the
cells. Homogenization was with a Polytron Homogenizer for 30 to 40
sec at 10,500 rpm using a PT-PA 3012/23 or ST-probe in 7 to 8 ml
aliquots. To estimate breakage, the cells were monitored by light
microscopy before and after homogenization. At least 90% cell
breakage without breakage of nuclei was achieved routinely.
[0167] The homogenates were centrifuged for 10 min at 175 g to
remove unbroken cells and nuclei and the supernatant was
centrifuged a second time at 1.4.times.10.sup.6 g min (e.g., 1 h at
23,500 g) to prepare a plasma membrane-enriched microsome fraction.
The supernatant was discarded and the pellets were resuspended in
0.2 M potassium phosphate buffer in a ration of approximately 1 ml
per pellet from 5.times.10.sup.8 cells. The resuspended membranes
were then loaded onto the two-phase system constituted on a weight
basis. The two-phase system contained 6.4% (w/w) Dextran T-500
(Pharmacia), 6.4% (w/w) Polyethylene Glycol 3350 (Fisher), and 5 mM
potassium phosphate, pH 7.2 (Morr and Morr, 1989, BioTechniques
7:946-958). The homogenate (1 g) was added to the two-phase system
and the weight of the system was brought to 8 g with distilled
water. The tubes were inverted vigorously for 40 times in the cold
(4.degree. C.). The phases were separated by centrifugation at 750
rpm (150.times.g) in a Sorvall HB 4 rotor for 5 min. The upper
phases were withdrawn carefully with a Pasteur pipette, divided in
half and transferred into 40 ml plastic centrifuge tubes. The tube
contents were diluted with cold 1 mM NaHCO.sub.3 and collected by
centrifugation at 10,000.times.g in a HB rotor for 30 min. Plasma
membrane pellets were resuspended in 50 mM Tris-Mes buffer (pH 7.2)
and stored at -70.degree. C. Proteins were determined using the
bicinchoninic acid (BCA) assay (Smith et al., 1985, Anal. Biochem.
100:76-85) with bovine serum albumin as standard. The upper phase,
enriched in plasma membranes, was diluted 5-fold with 1 mM sodium
bicarbonate and the membranes are collected by centrifugation. The
purity of the plasma membrane was determined to be >90% by
electron microscope morphometry. The yield was 20 mg plasma
membrane protein from 10.sup.10 cells.
7.1.4. Spectrophotometric Assay of NADH Oxidase
[0168] NADH oxidase activity was determined as the disappearance of
NADH measured at 340 nm in a reaction mixture containing 25 mM
Tris-Mes buffer (pH 7.2), 1 mM KCN, and 150 .mu.M NADH at
37.degree. C. Activity was measured using a Hitachi U3210
spectrophotometer with stirring and continuous recording over two
intervals of 5 min each. A millimolar extinction coefficient of
6.22 was used to determine specific activity.
7.2. Results
7.2.1. Effect of EGCg and EC on Solubilized NOX
[0169] Mixtures of EGCg with EC were tested first with a NOX
preparation solubilized from HeLa cells (FIG. 7) and subsequently
with cells. With the solubilized NOX protein, maximum inhibition
was achieved by a mixture of 10.sup.-9 M EGCg plus
5.times.10.sup.-6 M EC. Neither EC alone (up to and including
10.sup.-4 M) nor 10.sup.-9 M EGCg were effective in inhibiting the
activity of the solubilized plasma membrane NADH oxidase
protein.
[0170] Apoptosis was considerably enhanced by the combination of
EGCg and EC (FIG. 8). In the absence of EC, 50% growth arrest by
EGCg was observed at 10.sup.-5 M. However, in the presence of
10.sup.-4 M EC, the concentration of EGCg for 50% growth arrest was
lowered to 10.sup.-7 M and, in one experiment, the cells were
totally killed by the combination of 10.sup.-7 M EGCg plus
10.sup.-4 M EC.
7.2.2. Effect of EGCg and EC on NOX in Intact Cells
[0171] A similar response was seen with the NADH oxidase activity
of intact 4T1 cells (FIG. 9). With 10.sup.-4 M EC, 10.sup.-7 M
EGCg, or no addition, the response was minimal. However, in the
presence of 10.sup.-7 M EGCg, a substantial dose response to EC was
observed.
[0172] Mouse 4T1 mammary carcinoma cells are particularly
refractory to drug-induced growth inhibition and cell killing.
However in the presence of the combination of 10.sup.-7 M EGCg and
10.sup.-4 EC, the cells were killed (Table 2). This remarkable drug
response was reflected in the inhibition of the oxidation of NADH
by the intact 4T1 cells (Table 3, FIG. 9). The activity was
completely inhibited back to basal levels by 10.sup.-7 M EGCg plus
10.sup.-4 M EC. The EC.sub.50 for inhibition of the drug-responsive
component of the plasma membrane NADH oxidase was 2.times.10.sup.-9
M in the presence of 10.sup.-4 M EC alone, 10.sup.-7 M EGCg alone
or 10.sup.31 4 M EC+10.sup.-7 M EGCg, the EC.sub.50 for inhibition
by EC was between 2.times.10.sup.-9 M and 5.times.10.sup.-8 M
(Table 3).
3TABLE 2 Killing of 4T1 metastatic mouse mammary cancer cells in
culture. Addition Increase in cell number cm.sup.-2 over 72 h
.multidot. 10.sup.2 None 550 EGCg 10.sup.-7 M 520 BC 10.sup.-4 M
560 EGCg 10.sup.-7 M + EC 10.sup.-4 M -40* *100% Dead
[0173]
4TABLE 3 Preliminary Animal Study. Balb/c mice, 4T1 mouse mammary
cancer. Treated for 5 days. Metastases to axillary nodes Tumor wt
(Number Lung Treatment Amount/animal (g) of mice) Mets Control ---
2.3 .+-. 0.3 +++ + (water only) Glaucarubolone 1 mg 1.5 .+-. 0.1
+++ + (4 .times. 10.sup.-6 M) EGCg 10.sup.-7 M + 0.2 + 1.2 mg 0.75
+ 0.35 + --- EC 10.sup.-4 M Glaucarubolone + 1 + 0.2 + 1.2 mg 1.2
.+-. 0.4 +++ +* EGCg + EC Each animal received 100 .mu.l/day *One
animal with liver metastases
[0174] Epicatechin alone was largely without effect on the cell
surface NADH oxidase of 4T-1 cells (FIG. 9, no addition) over the
range 10.sup.-7 M to 10.sup.-4 M. However, in the presence of
10.sup.-7 M EGCg, the drug responsive component of the cell surface
NADH oxidase was inhibited maximally at about 10.sup.-4 with an
EC.sub.50 of about 2.times.10.sup.-7 M. The effect of EGCg was
approximately the same as the concentration is increased up to
10.sup.-4 (Table 4). The EC.sub.50 was increased slightly at
10.sup.-5 and 10.sup.-4 M EGCg although the difference is not
significant. The forms of the dose response curves including
maximum inhibition were unchanged from that with 10.sup.-7 M EGCg
and only a function of the concentration of EC (FIG. 10).
5TABLE 4 EC.sub.50 for (-)-epicatechin in the presence of varying
concentrations of EGCg alone or supplied as Tegreen .TM. on the
inhibition of tNOX activity of intact 4T1 mouse mammary cells in
culture. EC.sub.50 for tNOX inhibition by (-)-epicatechin,
10.sup.-6 M EGCg concentration, M EGCg Tegreen .TM. 0.sup. No
effect No effect 10.sup.-8 No effect No effect 10.sup.-7 0.2 .+-.
0.1 No effect 10.sup.-6 0.15 .+-. 0.05 0.5 .+-. 0.4 10.sup.-5 0.7
.+-. 0.3 0.4 .+-. 0.1 10.sup.-4 0.5 .+-. 0.4 0.3 .+-. 0.2
7.2.3. Effect of Tegreen.TM. on NOX in Intact Cells
[0175] With a commercially supplied tea concentrate (Tegreen.TM.,
Pharmanex, Brisbane, Calif.), results were similar except that on
an EGCg basis a higher concentration of Tegreen.TM. was required to
achieve the same response (Table 4). With Tegreen.TM., an EGCg
equivalent concentration of 10.sup.-6 M was required to elicit the
response and 10.sup.-7 M was largely without effect or slightly
stimulatory (FIG. 11, upper curve). Tegreen.TM. alone tended to
stimulate the surface NADH oxidase activity of the intact 4T1 cells
but the dose response with respect to EC was similar to that when
EGCg in the absence of other tea constituents was added (FIG. 11,
lower curve). The EC.sub.50 for inhibition of activity by EC was
0.4.+-.0.1.times.10.sup.-7 M comparing 10.sup.-6, 10.sup.-5 and
10.sup.-4 EGCg supplied as Tegreen.TM. (Table 4).
[0176] With intact HeLa cells, the tNOX activity was maximally
inhibited at 10.sup.-7 M to 10.sup.-6 M (FIG. 12). At 10.sup.-5 M
EGCg or higher, NOX activity was stimulated. (-)-Epicatechin (EC)
alone is without effect on NOX activity of HeLa cells (FIG. 13,
upper curve). However, in the presence of 10.sup.-7 M EGCg, a
further inhibitory response to EC was noted.
[0177] It should be noted that the formulation of Tegreen.TM. is an
old formulation which is not encompassed within the scope of this
invention. It should also be noted that the data suggested by the
Tegreen.TM. experiments indicates that combinations of catechins
are therapeutically more effective than EGCg alone on tNOX
inhibition.
8. EXAMPLE
Synergistic Interaction of Different Tea Catechins with (-)-
Epigallocatechin Gallate on Inhibition of Cell Surface NADH Oxidase
(NOX) Activity and Growth of 4T1 Mouse Mammary Cells in Culture
[0178] 8.1. Materials and Methods
8.1.1. Chemicals
[0179] The (-)-epigallocatechin gallate (EGCg), (-)-epicatechin
(EC), gallocatechin gallate (GCG) and .+-.catechin were purchased
from Sigma (St. Louis, Mo.) or purified from leaves of green tea
and supplied by Pharmanex (Brisbane, Calif.). The
(-)-epigallocatechin (EGC) and (-)-epicatechin gallate (ECG) were
purified from leaves of green tea and supplied by Pharmanex
(Brisbane, Calif.). The stability and purity (>90%) of the
catechins were confirmed by high performance liquid chromatographic
analysis.
8.1.2. Growth of Cells
[0180] A mouse mammary tumor subpopulation line 4T1 arising from a
BALB/cf C3H mouse was grown in DME-10, Dulbecco's modified Eagle's
medium supplemented with 5% fetal calf serum, 5% newborn calf
serum, 1 mM mixed non-essential amino acids, 2 mM L-glutamine,
penicillin (100 units/ml), and streptomycin (100 .mu.g/ml) (Miller
et al., 1987, Brit. J. Can. 56:561-569 and Miller et al., 1990,
Invasion Metastasis 10:101-112).
8.1.2. Spectrophotometric Assay of NADH Oxidase
[0181] NADH oxidase activity was determined as the disappearance of
NADH measured at 340 nm in a reaction mixture containing 25 mM
Tris-Mes buffer (pH 7.2), 1 mM KCN, and 150 .mu.M NADH at
37.degree. C. Activity was measured at 340 nm with reference at 430
nm using an SLM Aminco DW-2000 spectrophotometer (Milton Roy,
Rochester, N.Y.) in the dual beam mode of operation with stirring
and continuous recording over two intervals of 5 min each. A
millimolar extinction coefficient of 6.22 was used to determine
specific activity.
8.2. Results
8.2.1. Effect of Catechin Combinations on NOX in Intact Cells
[0182] Mixtures of EGCg with other catechins and mixtures of other
catechins were tested for inhibition of tNOX activity intact with
4T1 mouse mammary carcinoma cells. Previously, maximum inhibition
of the tNOX activity component was achieved by a mixture of
10.sup.-7 M EGCg plus 10.sup.-5 to 10.sup.-4 EC. Neither EC alone
(up to and including 10.sup.-4) nor EGCg (up to and including
10.sup.-6 M) were effective in inhibiting the residual CNOX
activity of the cells.
8.2.2. Effect of EGCg and EC on NOX in Intact Cells
[0183] These findings were extended to a more detailed comparison
of different concentrations of EGCg in the presence of 0,
10.sup.-8, 10.sup.-6 and 10.sup.-4 EC on the NOX activity of the
4T1 cells (Table 5). In the absence of EC, the EC.sub.50 for tNOX
inhibition by EGCg was about 10.sup.-8 M with >90% inhibition at
10.sup.-7 M. In the presence of 10.sup.-8 EC EGCg inhibition was
little enhanced. However with both 10.sup.-6 and 10.sup.-4 M EC,
the efficacy of EGCg inhibition was enhanced 10-fold or greater
(Table 5). EC alone was largely without effect on tNOX activity of
the 4T1 cells.
6TABLE 5 EC.sub.50 and EC.sub.>90 for (-)-epigallocatechin
gallate (EGCg) in the presence of varying concentrations of
(-)-epicatechin (EC) on the inhibition of tNOX activity of intact
cultured 4T1 mouse mammary carcinoma cells. tNOX inhibition by
(-)-epigallocatechin gallate (EGCg), M EC concentration, M
EC.sub.50 EC.sub.>90 0.sup. 10.sup.-8 10.sup.-7 10.sup.-8
10.sup.-8 10.sup.-7 10.sup.-6 10.sup.-9 10.sup.-8 10.sup.-4 5
.times. 10.sup.-10 10.sup.-8
8.2.3. Effect of Other Catechins and EGCg on NOX in Intact
Cells
[0184] Several catechins and catechin mixtures were tested for
their ability to replace the EC in the combination with 10.sup.-7 M
EGCg. ECG (FIG. 14) and EGC (FIG. 15) both were effective in
enhancing the inhibition by EGCg of tNOX activity of cultured 4T1
cells. The residual tNOX activity remaining after 10.sup.-7 M EGCg
was inhibited 50% by 5.times.10.sup.-7 and 10.sup.-6 M of ECG and
EGC respectively (Table 6).
[0185] Gallocatechin gallate (GCG) (FIG. 16) was less effective due
to a propensity of the GCG to stimulate activity as the
concentrations of GCG exceeded 10.sup.-7 M in the mixture.
[0186] Catechin was largely without effect on the cell surface NADH
oxidase of 4T1 cells over the range 10.sup.-7 M to 10.sup.-4 M both
in the presence (FIG. 17) or absence (not shown) of 10.sup.-7 M
EGCg. Unlike GCG, catechin did not stimulate NOX activity and
therefore, may represent an activity-neutral catechin component. A
mixture of equal parts of ECG, EGC, EC and catechin (FIG. 18) was
approximately equivalent in effectiveness to EC, ECG or EGC alone.
There appeared to be no marked enhancement of inhibition by the
mixture compared to the individual components tested singly (Table
6).
7TABLE 6 Estimated EC.sub.50 and EC.sub.>90 for different
catechins and catechin mixtures in the presence of
(-)-epigallocatechin gallate (EGCg) on the inhibition of the
residual tNOX activity of intact cultured 4T1 mouse mammary
carcinoma cells remaining after addition of 0.1 mM EGCg. Inhibition
of residual tNOX remaining in the presence of 10.sup.-6 M EGCg
Catechin EC.sub.50 EC.sub.>90 Epicatechin gallate (ECG) 5
.times. 10.sup.-7 M 10.sup.-6 M Epigallocatechin (EGC) 10.sup.-7 M
10.sup.-5 M Gallocatechin gallate (GCG) Not reached due to
stimulation Catechin (C) Not reached due to lack of inhibition ECG
+ EGC + EC + C 5 .times. 10.sup.-7 M 10.sup.-5 M Base-cleaved
Tegreen .TM. <10.sup.-7 M 10.sup.-7 M
15 8.2.4. Effect of Tegreen.TM. on NOX in Intact Cells
[0187] When a commercially supplied tea concentrate (Tegreen.TM.,
Pharmanex, Brisbane, Calif.), was treated with NADH to cleave the
gallate esters, results were similar (FIG. 19) except that on an
EGCg basis less catechin was required to achieve the same response
as compared to individual catechins (Table 6). With the hydrolyzed
Tegreen.TM., >90% inhibition was achieved at an EGCg equivalent
concentration of 10.sup.-7 M and with an EC.sub.50 of less than
10.sup.-7 M. The hydrolyzate was largely without effect on CNOX. A
control preparation containing an amount of NaCl equivalent to the
salt concentration of the tNOX hydrolyzate was without effect on
activity (not shown).
[0188] In this Example, the synergy in inhibition of tNOX activity
of cultured 4T1 mouse mammary carcinoma cells between the most
potent tea catechin EGCg and less potent tea catechins such as EC
was confirmed. Additionally, an equivalency among the catechins
(EC, EGC, ECG) in eliciting the synergistic response has been
shown, which is of considerable importance in efforts to optimize
tea catechin mixtures for use in cancer therapy.
9. EXAMPLE
Effect of a Sustained Release Formulation of Tegreen on Inhibition
of Cell Surface NADH Oxidase (NOX) Activity, Growth of 4T1 Mouse
Mammary and Hela Cells in Culture, and Transplanted Adenocarcinoma
Tumors
[0189] 9.1. Materials and Methods
9.1.1. Chemicals
[0190] A commercially supplied tea concentrate, Tegreen.TM., was
supplied by Pharmanex (Brisbane, Calif.). Microencapsulated
formulations of Tegreen.TM. were synthesized by Biodar (Yavne,
Israel), and designated as P-3039, P-3041, and a sustained release
formulation, known as P-3069.
[0191] The sustained release formulation (P-3069), was synthesized
with a CapsuDar.RTM. SR microencapsulation to achieve a slow, or
sustained release effect. In addition to the green tea extract, the
sustained release formulation also contains the active ingredients
microcrystalline cellulose, maltodextrine, ethylcellulose, and
magnesium stearate. The EGCg content of this sustained release
formulation is 28.5%.
9.1.2. Growth of Cells
[0192] HeLa (ATCC CCL2) cells were grown in 175 cm.sup.2 flasks in
Minimal Essential Medium (Gibco), pH 7.4, at 37.degree. C. with 10%
bovine calf serum (heat-inactivated), plus 50 mg/l gentamycin
sulfate (Sigma). Cells were harvested by scraping and taken up in
140 mM NaCl, 5 mM KCl, 0.7 mM Na.sub.2HPO.sub.4 and 25 mM Tris, pH
7.4 to a final cell concentration of 0.1 g wet weight (gww) per
ml.
[0193] A mouse mammary tumor subpopulation line 4T1 arising from a
BALB/cf C3H mouse was grown in DME-10, Dulbecco's modified Eagle's
medium supplemented with 5% fetal calf serum, 5% newborn calf
serum, 1 mM mixed non-essential amino acids, 2 mM L-glutamine,
penicillin (100 units/ml), and streptomycin (100 .mu.g/ml) (Miller
et al., 1987, Brit. J. Can. 56:561-569 and Miller et al., 1990,
Invasion Metastasis 10: 101-112).
9.1.3. Spectrophotometric Assay of NADH Oxidase
[0194] NADH oxidase activity was determined as the disappearance of
NADH measured at 340 nm in a reaction mixture containing 25 mM
Tris-Mes buffer (pH 7.2), 1 mM KCN, and 150 .mu.M NADH at
37.degree. C. Activity was measured using a Hitachi U3210
spectrophotometer with stirring and continuous recording over two
intervals of 5 min each. A millimolar extinction coefficient of
6.22 was used to determine specific activity.
9.1.4. Experimental Metastasis in Mice
[0195] Female BALB/c mice (8 to 12 weeks old) were injected with
cells from the tumor subpopulation line 4TO7 arising from a BALB/c
C3H mouse (Miller et al., 1987, Brit. J. Can. 56:561-569). Cells
from a monolayer culture were suspended in Hank's buffered salt
solution and 1.times.10.sup.5 cells were injected subcutaneously
into the mice in a volume of 0.1 ml. Primary tumors were measured
twice a week in two perpendicular dimensions using a vernier
caliper. Catechin mixtures were administered intratumoral on
alternate days beginning after palpable tumor masses are
discernible. A5 15 days post tumor implantation, the mice were
sacrificed and major organs (e.g., lung, liver, and lymph nodes)
were examined for evidence of metastases.
9.2. Results
9.2.1. Need for a Sustained Release Formulation
[0196] As described supra, the effect of the catechins on the
inhibition of cell growth and tNOX inhibition is reversible, i.e.,
if EGCg is removed, cancer cells resume normal rates of growth. As
shown in FIG. 20, with repeat additions of 100 nM EGCg four times a
day, growth was inhibited during the day but recovered during the
night (16 hours). As a result, there was no apoptosis. However,
when the dose of EGCg was increased to 1 .mu.M provided twice daily
to cells in culture, growth was inhibited and the resultant cells
were smaller (data not shown).
[0197] Indications are that NOX activity and cell growth must be
inhibited for approximately 72 hours or more to induce apoptosis.
Even a modest 30% decrease in cell diameter might exert little or
no effect in slowing of cell multiplication. It would appear that a
nearly complete inhibition of the cell volume increase following
division would be required to block cell proliferation. Thus, there
exists a need for a sustained release formulation of catechins in
order for apoptosis to occur.
9.2.2. Effect of Sustained Release Formulation on Cell Growth
[0198] The effect of the microencapsulated formulations of
Tegreen.TM. on the growth of cancer cells is presented in Table 7.
Dosages were administered every two hours four times a day over
eight hours. The sustained release formulation inhibited the growth
of both HeLa cells and 4T-1 mouse mammary tumor cells. The
inhibition of HeLa cell growth by the sustained release formulation
was similar to Tegreen.TM. and the other microencapsulated
formulations tested. In contrast, two of the microencapsulated
formulations of Tegreen.TM. were less effective at inhibiting 4T-1
cell growth than the Tegreen.TM. starting material. However, the
sustained release formulation inhibited 4T-1 cell growth at a
concentration similar to that of the Tegreen.TM. starting
material.
8TABLE 7 Effect of Tegreen .TM. and microencapsulated Tegreen .TM.
formulations on HeLa cell and 4T-1 mouse mammary tumor cell growth.
Cells/cm.sup.2 .times. 10.sup.2 after 72 h HeLa cells 4T-1 cells
(10.sup.-5 M of Tegreen .TM. (5 .times. 10.sup.-5 M of Tegreen .TM.
Addition formulation added) formulation added) None 260 892 Tegreen
.TM. starting 132 16 material P-3039 135 492 P-3041 191 720
Sustained release 164 15 (10.sup.-4 M of Tegreen .TM. formulation
added)
9.2.3. Effect of Sustained Release Formulation on NOX
[0199] The effect of the Tegreen.TM. starting material and the
microencapsulated Tegreen.TM. formulations on cell surface NOX in
HeLa and 4T-1 cells is presented in Table 8. All of the
microencapsulated formulations of Tegreen.TM., including the
sustained release formulation, inhibited NOX activity in both HeLa
and 4T-1 cells. All of the microencapsulated formulations were
effective at concentrations less than or equal to the Tegreen.TM.
starting material. Significantly, the sustained release formulation
inhibited NOX activity in both HeLa and 4T 1 cells and was
effective at a concentration less than Tegreen.TM.. Furthermore,
the overall concentrations of active catechins in the
microencapsulated formulations are less than the concentrations of
active catechins, e.g., EGCg, in the data presented supra in
Examples 6 to 8. In other words, the microencapsulated formulations
contain approximately 70% active catechins, thus the actual
concentration of active catechins will be less than the
concentration of the microencapsulated formulation that is
administered.
9TABLE 8 Effect of microencapsulated Tegreens on NOX activity 24
hours post addition. The number of experiments (n) is indicated.
The range of EC.sub.50 values (if n > 1) is noted below.
EC.sub.50 4T-1 mouse mammary HeLa cells tumor cells Tegreen .TM.
starting material 10.sup.-7 M.sup.(a) (n = 7) 10.sup.-7(b) M (n =
5) P-3039 10.sup.-7(c) M (n = 2) 10.sup.-8 M (n = 1) P-3041
10.sup.-7 M (n = 1) 5 .times. 10.sup.-8 M (n = 1) Sustained release
10.sup.-8 M (n = 1) 10.sup.-8 M.sup.(d) (n = 4) .sup.(a)10.sup.-8
to 5 .times. 10.sup.-6 M .sup.(b)5 .times. 10.sup.-8 to 5 .times.
10.sup.-7 M .sup.(c)10.sup.-7 to 5 .times. 10.sup.-7 M
.sup.(d)10.sup.-8 to 2 .times. 10.sup.-8 M
9.2.4. Effect of Sustained Release Formulation on a Tumor
[0200] The effect of the microencapsulated sustained release
Tegreen.TM. formulation on a transplantable 4T-1 mammary
adenocarcinoma in a BALB/c mouse is shown in FIG. 21. The tumor
resulted from a subcutaneous injection of a tumor subpopulation
line. The tumor was treated with 100 .mu.l of a 2.3 mg/ml
suspension of the sustained release formulation. Whenever a small
granule of the sustained release formulation was located within the
tumor mass, cells that were several mm around the particle were
killed. In between the particles, the tumor continued to grow. As
shown in FIG. 21, this gave rise to the distinctive "lumpy"
appearance of the tumor mass with growing areas not receiving
Tegreen.TM. surrounded by areas receiving Tegreen.TM. that were
killed. Thus, the sustained release formulation of Tegreen.TM. is
effective for eradicating tumor cell growth.
[0201] In this example, a microencapsulated sustained release
formulation of Tegreen.TM. has been shown to be effective over time
(>72 h) for the inhibition of both in vitro and in vivo cancer
cell growth. Furthermore, this sustained release formulation is
effective at a concentration less than an equivalent amount of
Tegreen.TM. at inhibiting NOX in the cancer cells tested.
10. EXAMPLE
Method for the Encapsulation of Epigallocatechin Gallate
[0202] As described in Section 5.1.2, all known methods for
encapsulation which are compatible with the properties of tea
catechins are encompassed by this invention.
[0203] For example, a sustained release formulation is described in
U.S. Pat. No. 4,710,384, which is incorporated herein by reference
in its entirety. Using U.S. Pat. No. 4,710,384 as an example, a
sustained release formulation of EGCg can be prepared in the
following manner:
[0204] One kg of EGCg is coated in a modified Uni-Glatt powder
coater with ethyl cellulose. The ethyl cellulose is type 10 ethyl
cellulose obtained from Dow Chemical Company. The spraying solution
comprises an 8 percent solution of the ethyl cellulose in 90
percent acetone to 10 percent ethanol. Castor oil is added as
plasticizer in an amount equal to 20 percent of the ethyl cellulose
present.
[0205] The spraying conditions are as follows:
[0206] (i) Speed: 1 liter/hour
[0207] (ii) Flap: 10-15 percent
[0208] (iii) Inlet Temperature: 50 C.
[0209] (iv) Outlet temp.: 30 C.
[0210] (v) Percent of Coating: 17 percent
[0211] The coated EGCg is sieved to particle sizes between 74-210
microns. Attention is paid to ensure a good mix of particles of
different sizes within that range. 400 mg of the coated particles
are mixed with 100 mg of starch and the mixture is compressed in a
hand press to 1.5 tons to produce 500 mg tablets.
[0212] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described will
become apparent to those skilled in the art from the foregoing
description and accompanying figures. Such modifications are
intended to fall within the scope of the appended claims.
[0213] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
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