U.S. patent application number 10/216026 was filed with the patent office on 2006-04-06 for detection and therapy of vulnerable plaque with fluorescent and/or radiolabeled compositions.
Invention is credited to Rox Anderson, David Elmaleh, Alan Fischman, Henry Gewirtz, Michael R. Hamblin, Tayyaba Hasan, James Muller, Ahmed Tawakol.
Application Number | 20060073100 10/216026 |
Document ID | / |
Family ID | 27388922 |
Filed Date | 2006-04-06 |
United States Patent
Application |
20060073100 |
Kind Code |
A1 |
Fischman; Alan ; et
al. |
April 6, 2006 |
Detection and therapy of vulnerable plaque with fluorescent and/or
radiolabeled compositions
Abstract
The present invention relates to methods for selectively
targeting Photodynamic Therapy ("PDT") to inflammatory components
of vulnerable plaques. As such, the present invention provides
methods for the identification of vulnerable plaques, using
fluorescent compositions, which include photosensitizer
compoisitions, and/or radiolabeled compounds, as well as methods to
treat vulnerable plaques by selectively targeting and/or
eliminating the inflammatory components of vulnerable plaques.
Inventors: |
Fischman; Alan; (Boston,
MA) ; Hamblin; Michael R.; (Boston, MA) ;
Tawakol; Ahmed; (Boston, MA) ; Hasan; Tayyaba;
(Boston, MA) ; Muller; James; (Boston, MA)
; Anderson; Rox; (Boston, MA) ; Elmaleh;
David; (Boston, MA) ; Gewirtz; Henry; (Boston,
MA) |
Correspondence
Address: |
EDWARDS & ANGELL, LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Family ID: |
27388922 |
Appl. No.: |
10/216026 |
Filed: |
August 9, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10163744 |
Jun 4, 2002 |
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10216026 |
Aug 9, 2002 |
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60295627 |
Jun 4, 2001 |
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60365673 |
Mar 15, 2002 |
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Current U.S.
Class: |
424/9.6 ;
424/178.1; 530/391.1 |
Current CPC
Class: |
A61N 5/0601 20130101;
A61K 47/643 20170801; A61B 5/0071 20130101; A61P 7/00 20180101;
A61K 41/0057 20130101; A61N 2005/0602 20130101; A61K 51/0474
20130101; A61B 18/245 20130101; A61B 5/0075 20130101; A61B 5/0086
20130101; A61B 2017/00057 20130101; A61B 2017/00079 20130101; A61K
41/0071 20130101; A61P 9/00 20180101; A61B 18/20 20130101; A61K
51/0491 20130101; A61K 41/0076 20130101; A61K 41/0061 20130101;
A61P 35/00 20180101; A61N 5/062 20130101; A61P 29/00 20180101; A61B
5/0084 20130101; A61B 2018/00904 20130101; A61B 6/4258
20130101 |
Class at
Publication: |
424/009.6 ;
424/178.1; 530/391.1 |
International
Class: |
A61K 49/00 20060101
A61K049/00; A61K 39/395 20060101 A61K039/395; C07K 16/46 20060101
C07K016/46 |
Goverment Interests
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0003] This work was supported by the government, in part, by a
grant from the United States Department of Defense, Grant No.
17-99-2-9001. The government may have certain rights to this
invention.
Claims
1. A method of stabilizing a vulnerable plaque in a subject
comprising the steps of: a) administering a therapeutically
effective amount of at least one photosensitizer composition,
wherein the photosensitizer composition is localized to the
vulnerable plaque; and b) light activating the photosensitizer
composition to produce a phototoxic species; and c) stabilizing the
vulnerable plaque against rupture.
2. The method of claim 1, wherein the vulnerable plaque comprises
inflammatory components, a large lipid pool, and a thin fibrous
cap.
3. The method of claim 2, wherein the thin fibrous cap is less than
about 150 microns thick.
4. The method of claim 2, wherein the thin fibrous cap is less than
about 100 microns thick.
5. The method of claim 2, wherein inflammatory components are
selected from the group consisting of inflammatory cells, lipids,
procoagulants and agents that promote inhibition of extracellular
matrix production or degradation of extracellular matrix.
6. The method of claim 5, wherein the inflammatory cells are
selected from the group consisting of smooth muscle cells,
leukocytes, lymphocytes, monocytes, macrophages, foam cells, mast
cells, endothelial cells, platelets, erythrocytes and
polymorphonuclear cells.
7. The method of claim 6, wherein the lymphocytes comprise
B-lymphocytes and T-lymophocytes.
8. The method of claim 6, wherein the polymorphonuclear cells
comprise granulocytes and neutrophils.
9. The method of claim 6, wherein the inflammatory cells comprise
greater than about 10% macrophages and/or monocytes.
10. The method of claim 6, wherein the inflammatory cells comprises
greater than about 25% macrophages and/or monocytes.
11. The method of claim 2, wherein the lipid content is greater
than about 10%.
12. The method of claim 2, wherein the lipid content is greater
than about 25%.
13. The method of claim 1, wherein the photosensitizer composition
comprises a photosensitizer coupled to a molecular carrier.
14. The method of claim 13, wherein the molecular carrier targets
inflammatory components selected from the group consisting of
inflammatory cells, lipids, procoagulants and agents that promote
inhibition of extracellular matrix production or degradation of
extracellular matrix.
15. The method of claim 14, wherein the inflammatory cells are
selected from the group consisting of smooth muscle cells,
leukocytes, lymphocytes, monocytes, macrophages, foam cells, mast
cells, endothelial cells, platelets, erythrocytes and
polymorphonuclear cells.
16. The method of claim 15, wherein the lymphocytes comprise
B-lymphocytes and T-lymophocytes.
17. The method of claim 15, wherein the polymorphonuclear cells
comprise granulocytes and neutrophils.
18. The method of claim 13, wherein the molecular carrier is
selected from the group consisting of serum proteins, receptor
ligands, microspheres, liposomes, antibodies, growth factors,
peptides, hormones and lipoproteins.
19. The method of claim 13, wherein the molecular carrier binds to
a scavenger receptor.
20. The method of claim 19, wherein the molecular carrier is
selected from the group consisting of maleylated albumin,
daunorubicin, doxorubicin, oxidized low density lipoprotein,
acetylated low density lipoprotein, oxidized high density
lipoprotein, malondialdehyde treated proteins, formaldehyde treated
albumin, glycated albumin, polyinosinic acid, glycated
lipoproteins, dextran sulfate, anionic phospholipids, fucoidin,
carrageenan, polyvinyl sulfate and monoclonal antibodies that
recognize CD11b, CD11c, CD13, CD14, CD16a, CD32 or CD68.
21. The method of claim 20, wherein the anionic phospholipid is
phosphatidyl serine.
22. The method of claim 13, where in the molecular carrier targets
the photosensitizer composition to a T cell.
23. The method of claim 22, where in the molecular carrier is
selected from the group consisting of monoclonal antibodies that
recognize CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD28, CD44
and CD71 and transferrin.
24. The method of claim 13, where in the molecular carrier targets
the photosensitizer composition to the lipids comprising the lipid
pool of the atheroma.
25. The method of claim 24, wherein the molecular carrier comprises
a hydrophobic vehicles selected from the group consisting of
liposomes, cremaphor EL, PEG/solvent mixtures, iodized castor oil,
nanoparticles and micellar preparations.
26. The method of claim 25, wherein the liposomes contain
cholesterol.
27. The method of claim 25, wherein the liposomes contain
cardiolipin.
28. The method of claim 13, wherein the molecular carrier targets
the photosensitizer composition to macrophages.
29. The method of claim 28, wherein the molecular carrier targets
the photosensitizer composition to a macrophage biomolecule
selected from the group consisting of For-Met-Leu-Phe, tenascin C,
tissue factor, tissue inhibitor of MMP 1, tissue inhibitor of MMP
2, oxidized LDL receptor, heme oxygenase-1, human cartilage gp-39,
IL-6, IL-6 receptor, IL-10, IL-10 receptor, lectin-like oxidized
LDL-receptor, monocyte inflammatory protein-1, monocyte
inflammatory protein-1 receptor and macrophage chemoattractant
protein-1 receptor.
30. The method of claim 22, wherein the molecular carrier targets
the photosensitizer composition to a T cell biomolecule selected
from the group consisting of IL-10, IL-10 receptor, monocyte
inflammatory protein-1, monocyte inflammatory protein-1 receptor
and transferrin.
31. The method of claim 13, wherein the molecular carrier targets
the photosensitizer composition to foam cells.
32. The method of claim 13, wherein the molecular carrier that
targets the photosensitizer composition is a protease that degrades
extracellular matrix.
33. The method of claim 32, wherein the protease is a
metalloproteinase.
34. The method of claim 32, wherein the molecular carrier is a
monoclonal antibody that binds to an epitope on a protease.
35. The method of claim 1, wherein the light activating the
photosensitizer composition to produce a phototoxic species is
administered in an amount sufficient to induce apoptosis and not
necrosis of the cells comprising the vulnerable plaque.
36. A method of stabilizing a vulnerable plaque in a subject
comprising the steps of: a) administering a therapeutically
effective amount of at least one photosensitizer composition
comprising a photosensitizer coupled to a molecular carrier; and b)
localizing the photosensitizer composition to a vulnerable plaque
consisting of inflammatory components, a large lipid pool, and a
thin fibrous cap that is less than about 150 microns thick; and b)
light activating the photosensitizer composition to produce a
phototoxic species; and c) stabilizing the vulnerable plaque
against rupture.
37. The method of claim 36, wherein the wherein the light
activating the photosensitizer composition to produce a phototoxic
species is administered in an amount sufficient to induce apoptosis
and not necrosis of the cells comprising the vulnerable plaque.
38. A method of stabilizing a vulnerable plaque in a subject
comprising the steps of: a) administering a therapeutically
effective amount of at least one photosensitizer composition
comprising a photosensitizer coupled to a molecular carrier; and b)
localizing the photosensitizer composition to a vulnerable plaque
consisting of inflammatory components, a large lipid pool, and a
thin fibrous cap that is less than about 150 microns thick; and c)
light activating the photosensitizer composition to produce a
phototoxic species, and wherein the light further produces cross
links in the fibrous cap; and d) stabilizing the vulnerable plaque
against rupture.
39. The method of claim 38, wherein the wherein the light
activating the photosensitizer composition to produce a phototoxic
species is administered in an amount sufficient to induce apoptosis
and not necrosis of the cells comprising the vulnerable plaque.
40. A method of detecting a vulnerable plaque in a subject
comprising the steps of: a) administering a fluorescent
composition; and b) localizing the composition to the vulnerable
plaque; and c) light activating the composition to illuminate the
vulnerable plaque; and d) identifying the vulnerable plaque.
41. The method of claim 40, wherein the fluorescent composition
comprises a photosensitizer coupled to a coupled to a molecular
carrier.
42. The method of claim 40, wherein the fluorescent composition
comprises a fluorescent dye coupled to a molecular carrier.
43. The method of claim 40, wherein the fluorescent composition
comprises a photoactive dye coupled to a molecular carrier.
44. A method of detecting a vulnerable plaque in a subject
comprising the steps of: a) administering a detectable amount of at
least one fluorescent composition, wherein the fluorescent
composition is localized to a vulnerable plaque; and b) light
activating the vulnerable plaque to produce a fluorescent species;
and c) identifying the vulnerable plaque.
45. The method of claim 44, wherein the fluorescent composition
comprises a photosensitizer coupled to a coupled to a molecular
carrier.
46. The method of claim 44, wherein the fluorescent composition
comprises a fluorescent dye coupled to a molecular carrier.
47. The method of claim 44, wherein the fluorescent composition
comprises a photoactive dye coupled to a molecular carrier.
48. A method of detecting and treating a vulnerable plaque in a
subject comprising the steps of: a) administering a detectable
amount of at least one fluorescent composition, wherein the
fluorescent composition is localized to a vulnerable plaque; and b)
administering a therapeutically effective amount of at least one
photosensitizer composition, wherein the photosensitizer
composition is localized to a vulnerable plaque; and c) light
activating the vulnerable plaque to produce a fluorescent species;
and d) identifying the vulnerable plaque; and e) light activating
the photosensitizer composition at the site of the vulnerable
plaque to produce a phototoxic species; and f) stabilizing the
vulnerable plaque against rupture.
49. The method of claim 41, further comprising the steps of: e)
light activating the photosensitizer at the site of the vulnerable
plaque to produce a phototoxic species; and f) stabilizing the
vulnerable plaque against rupture.
50. The method of claim 43, further comprising the steps of: e)
light activating the photoactive dye at the site of the vulnerable
plaque to produce a phototoxic species; and f) stabilizing the
vulnerable plaque against rupture.
51. The method of claim 45, further comprising the steps of: d)
light activating the photosensitizer at the site of the vulnerable
plaque to produce a phototoxic species; and e) stabilizing the
vulnerable plaque against rupture.
52. The method of claim 47, further comprising the steps of: d)
light activating the photoactive dye at the site of the vulnerable
plaque to produce a phototoxic species; and e) stabilizing the
vulnerable plaque against rupture.
53. The method of claim 1, wherein the photosensitizer is
chlorin.sub.e6.
54. The method of claim 53, wherein the light is administered in a
20-500 J/cm dose.
55. The method of claim 53, wherein the light is administered in a
50-300 J/cm dose.
56. The method of claim 53, wherein the light is administered in a
100-200 J/cm dose.
57. The method of claim 36, wherein the photosensitizer is
chlorin.sub.e6
58. The method of claim 57, wherein the light is administered in a
20-500 J/cm dose.
59. The method of claim 57, wherein the light is administered in a
50-300 J/cm dose.
60. The method of claim 57, wherein the light is administered in a
100-200 J/cm dose.
61. The method of claim 38, wherein the photosensitizer is
chlorin.sub.e6
62. The method of claim 61, wherein the light is administered in a
20-500 J/cm dose.
63. The method of claim 61, wherein the light is administered in a
50-300 J/cm dose.
64. The method of claim 61, wherein the light is administered in a
100-200 J/cm dose.
65. The method of claim 48, wherein the photosensitizer composition
comprises chlorin.sub.e6.
66. The method of claim 65, wherein the light activating the
photosensitizer composition is administered in a 20-500 J/cm
dose.
67. The method of claim 65, wherein the light activating the
photosensitizer composition is administered in a 50-300 J/cm
dose.
68. The method of claim 65, wherein the light activating the
photosensitizer composition is administered in a 100-200 J/cm
dose.
69. The method of claim 49, wherein the photosensitizer is
chlorin.sub.e6
70. The method of claim 69, wherein the light activating the
photosensitizer is administered in a 20-500 J/cm dose.
71. The method of claim 69, wherein the light activating the
photosensitizer is administered in a 50-300 J/cm dose.
72. The method of claim 69, wherein the light activating the
photosensitizer is administered in a 100-200 J/cm dose.
73. The method of claim 50, wherein the photoactive dye is
chlorin.sub.e6.
74. The method of claim 73, wherein the light activating the
photoactive dye is administered in a 20-500 J/cm dose.
75. The method of claim 73, wherein the light activating the
photoactive dye is administered in a 50-300 J/cm dose.
76. The method of claim 73, wherein the light activating the
photoactive dye is administered in a 100-200 J/cm dose.
77. The method of claim 51, wherein the photosensitizer is
chlorin.sub.e6.
78. The method of claim 77, wherein the light activating the
photosensitizer is administered in a 20-500 J/cm dose.
79. The method of claim 77, wherein the light activating the
photosensitizer is administered in a 50-300 J/cm dose.
80. The method of claim 77, wherein the light activating the
photosensitizer is administered in a 100-200 J/cm dose.
81. The method of claim 52, wherein the photoactive dye is
chlorin.sub.e6
82. The method of claim 81, wherein the light activating the
photoactive dye is administered in a 20-500 J/cm dose.
83. The method of claim 81, wherein the light activating the
photoactive dye is administered in a 50-300 J/cm dose.
84. The method of claim 81, wherein the light activating the
photosensitizer composition is administered in a 100-200 J/cm
dose.
85. The method of claim 41, wherein the photosensitizer is
chlorin.sub.e6
86. The method of claim 85, wherein the light is administered in a
dose that is less than about 10 J/cm.
87. The method of claim 85, wherein the light is administered in a
dose that is less than about 5 J/cm.
88. The method of claim 85, wherein the light is administered in a
dose that is less than about 1 J/cm.
89. The method of claim 43, wherein the photoactive dye is
chlorin.sub.e6
90. The method of claim 89, wherein the light is administered in a
dose that is less than about 10 J/cm.
91. The method of claim 89, wherein the light is administered in a
dose that is less than about 5 J/cm.
92. The method of claim 89, wherein the light is administered in a
dose that is less than about 1 J/cm.
93. The method of claim 45, wherein the photosensitizer is
chlorin.sub.e6
94. The method of claim 93, wherein the light is administered in a
dose that is less than about 10 J/cm.
95. The method of claim 93, wherein the light is administered in a
dose that is less than about 5 J/cm.
96. The method of claim 93, wherein the light is administered in a
dose that is less than about 1 J/cm.
97. The method of claim 47, wherein the photoactive dye is
chlorin.sub.e6
98. The method of claim 97, wherein the light is administered in a
dose that is less than about 10 J/cm.
99. The method of claim 97, wherein the light is administered in a
dose that is less than about 5 J/cm.
100. The method of claim 97, wherein the light is administered in a
dose that is less than about 1 J/cm.
101. The method of claim 48, wherein the fluorescent composition
comprises is chlorin.sub.e6
102. The method of claim 101, wherein the light activating the
fluorescent composition is administered in a dose that is less than
about 10 J/cm.
103. The method of claim 101, wherein the light activating the
fluorescent composition administered in a dose that is less than
about 5 J/cm.
104. The method of claim 101, wherein the light activating the
fluorescent composition is administered in a dose that is less than
about 1 J/cm.
105. A method of detecting and treating a vulnerable plaque in a
subject comprising the steps of: a) administering a detectable
amount of at least one fluorescent composition, wherein the
fluorescent composition is localized to a vulnerable plaque; and
administering a therapeutically effective amount of at least one
photosensitizer composition, wherein the photosensitizer
composition is localized to a vulnerable plaque; and b) light
activating the vulnerable plaque to produce a fluorescent species;
and c) identifying the vulnerable plaque; and d) light activating
the photosensitizer composition at the site of the vulnerable
plaque to produce a phototoxic species; e) and stabilizing the
vulnerable plaque against rupture.
106. A method of detecting and treating a vulnerable plaque in a
subject comprising the steps of: a) administering a composition
comprising a radiolabeled a molecular carrier; and b) localizing
the composition to the vulnerable plaque; and c) measuring
radioactive signal; and d) identifying the vulnerable plaque; and
e) administering a therapeutically effective amount of at least one
photosensitizer composition, wherein the photosensitizer
composition is localized to the vulnerable plaque; and f) light
activating the photosensitizer composition at the site of the
vulnerable plaque to produce a phototoxic species; and g)
stabilizing the vulnerable plaque against rupture.
107. A method of detecting a vulnerable plaque in a subject
comprising the steps of: a) administering a radiolabeled
composition; b) localizing the composition to the vulnerable
plaque; c) detecting a signal from the radiolabeled composition; d)
identifying the vulnerable plaque.
108. The method of claim 107, wherein the vulnerable plaque
comprises inflammatory components, a large lipid pool, and a thin
fibrous cap.
109. The method of claim 108, wherein the thin fibrous cap is less
than about 150 microns thick.
110. The method of claim 108, wherein the thin fibrous cap is less
than about 100 microns thick.
111. The method of claim 108, wherein inflammatory components are
selected from the group consisting of inflammatory cells, lipids,
procoagulants and agents that promote inhibition of extracellular
matrix production or degradation of extracellular matrix.
112. The method of claim 111, wherein the inflammatory cells are
selected from the group consisting of smooth muscle cells,
leukocytes, lymphocytes, monocytes, macrophages, foam cells, mast
cells, endothelial cells, platelets, erythrocytes and
polymorphonuclear cells.
113. The method of claim 112, wherein the lymphocytes comprise
B-lymphocytes and T-lymophocytes.
114. The method of claim 112, wherein the polymorphonuclear cells
comprise granulocytes and neutrophils.
115. The method of claim 112, wherein the inflammatory cells
comprise greater than about 10% macrophages and/or monocytes.
116. The method of claim 112, wherein the inflammatory cells
comprises greater than about 25% macrophages and/or monocytes.
117. The method of claim 108, wherein the lipid content is greater
than about 10%.
118. The method of claim 108, wherein the lipid content is greater
than about 25%.
119. The method of claim 107, wherein the radiolabeled composition
comprises a radioactive agent coupled to a molecular carrier.
120. The method of claim 119, wherein the molecular carrier targets
inflammatory components selected from the group consisting of
inflammatory cells, lipids, procoagulants and agents that promote
inhibition of extracellular matrix production or degradation of
extracellular matrix.
121. The method of claim 120, wherein the inflammatory cells are
selected from the group consisting of smooth muscle cells,
leukocytes, lymphocytes, monocytes, macrophages, foam cells, mast
cells, endothelial cells, platelets, erythrocytes and
polymorphonuclear cells.
122. The method of claim 121, wherein the lymphocytes comprise
B-lymphocytes and T-lymophocytes.
123. The method of claim 121, wherein the polymorphonuclear cells
comprise granulocytes and neutrophils.
124. The method of claim 119, wherein the molecular carrier is
selected from the group consisting of serum proteins, receptor
ligands, microspheres, liposomes, antibodies, growth factors,
peptides, hormones and lipoproteins.
125. The method of claim 119, wherein the molecular carrier binds
to a scavenger receptor.
126. The method of claim 125, wherein the molecular carrier is
selected from the group consisting of maleylated albumin,
daunorubicin, doxorubicin, oxidized low density lipoprotein,
acetylated low density lipoprotein, oxidized high density
lipoprotein, malondialdehyde treated proteins, formaldehyde treated
albumin, glycated albumin, polyinosinic acid, glycated
lipoproteins, dextran sulfate, anionic phospholipids, fucoidin,
carrageenan, polyvinyl sulfate and monoclonal antibodies that
recognize CD11b, CD11c, CD13, CD14, CD16a, CD32 or CD68.
127. The method of claim 126, wherein the anionic phospholipid is
phosphatidyl serine.
128. The method of claim 119, where in the molecular carrier
targets the radiolabeled composition to a T cell.
129. The method of claim 128, where in the molecular carrier is
selected from the group consisting of monoclonal antibodies that
recognize CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD28, CD44
and CD71 and transferrin.
130. The method of claim 119, where in the molecular carrier
targets the radiolabeled composition to lipids of the vulnerable
plaque.
131. The method of claim 130, wherein the molecular carrier
comprises a hydrophobic vehicles selected from the group consisting
of liposomes, cremaphor EL, PEG/solvent mixtures, iodized castor
oil, nanoparticles and micellar preparations.
132. The method of claim 131, wherein the liposomes contain
cholesterol.
133. The method of claim 131, wherein the liposomes contain
cardiolipin.
134. The method of claim 119, wherein the molecular carrier targets
the radiolabeled composition to macrophages.
135. The method of claim 134, wherein the molecular carrier targets
the radiolabeled composition to a macrophage biomolecule selected
from the group consisting of tenascin C, tissue factor, tissue
inhibitor of MMP 1, tissue inhibitor of MMP 2, oxidized LDL
receptor, heme oxygenase-1, human cartilage gp-39, IL-6, IL-6
receptor, IL-10, IL-10 receptor, lectin-like oxidized LDL-receptor,
monocyte inflammatory protein-1, monocyte inflammatory protein-1
receptor and macrophage chemoattractant protein-1 receptor.
136. The method of claim 128, wherein the molecular carrier targets
the radiolabeled composition to a T cell biomolecule selected from
the group consisting of IL-10, IL-10 receptor, monocyte
inflammatory protein-1, monocyte inflammatory protein-1 receptor
and transferrin.
137. The method of claim 119, wherein the molecular carrier targets
the radiolabeled composition to foam cells.
138. The method of claim 119, wherein the molecular carrier that
targets the radiolabeled composition a protease that degrades
extracellular matrix.
139. The method of claim 138, wherein the protease is a
metalloproteinase.
140. The method of claim 138, wherein the molecular carrier is a
monoclonal antibody that binds to an epitope on a protease.
141. A method of detecting a vulnerable plaque in a subject
comprising the steps of: a) administering a detectable amount of at
least one radiolabeled composition, wherein the radiolabeled
composition is localized to a vulnerable plaque; b) detecting a
signal from the radiolabeled composition; and c) identifying the
vulnerable plaque.
142. A method of detecting and treating a vulnerable plaque in a
subject comprising the steps of: a) administering a detectable
amount of at least one radiolabeled composition, wherein the
radiolabeled composition is localized to a vulnerable plaque; b)
administering a therapeutically effective amount of at least one
photosensitizer composition, wherein the photosensitizer
composition is localized to a vulnerable plaque; c) detecting a
signal from the radiolabeled composition; and d) identifying the
vulnerable plaque; e) light activating the photosensitizer
composition at the site of the vulnerable plaque to produce a
phototoxic species; f) stabilizing the vulnerable plaque against
rupture.
143. The method of claim 142, wherein the photosensitizer is
chlorin.sub.e6.
144. The method of claim 143, wherein the light is administered in
a 20-500 J/cm dose.
145. The method of claim 143, wherein the light is administered in
a 50-300 J/cm dose.
146. The method of claim 143, wherein the light is administered in
a 100-200 J/cm dose.
147. A method of detecting and treating a vulnerable plaque in a
subject comprising the steps of: a) administering a composition
comprising a radioactive agent and a photosensitizer coupled to a
molecular carrier; b) localizing the composition to the vulnerable
plaque; c) detecting a signal from the radiolabeled composition; d)
identifying the vulnerable plaque; e) light activating the
photosensitizer at the site of the vulnerable plaque to produce a
phototoxic species; and f) stabilizing the vulnerable plaque
against rupture.
148. In yet another embodiment, a method of detecting and treating
a vulnerable plaque in a subject comprising the steps of: a)
administering a radiolabeled composition wherein the radiolabeled
composition is localized to a vulnerable plaque; b) first detecting
a signal from the radiolabeled composition; and identifying the
vulnerable plaque; c) administering a therapeutically effective
amount of at least one photosensitizer composition, wherein the
photosensitizer composition is localized to the vulnerable plaque;
d) second light activating the photosensitizer composition at the
site of the vulnerable plaque to produce a phototoxic species; and
e) stabilizing the vulnerable plaque against rupture.
Description
RELATED APPLICATIONS/PATENTS & INCORPORATION BY REFERENCE
[0001] This application is a continuation-in-part application of
U.S. application Ser. No. 10/163,744, filed on Jun. 4, 2002, which
claims priority to U.S. Provisional Application No. 60/295,627,
filed Jun. 4, 2001, and U.S. Provisional Application No.
60/365,673, filed Mar. 15, 2002, the contents of which are
expressly incorporated herein by reference. Reference is also made
herein to PCT/US98/18685, published as WO 99/12579 on Mar. 18,
1999, the contents of which are expressly incorporated herein by
reference.
[0002] Each of the applications and patents cited in this text, as
well as each document or reference cited in each of the
applications and patents (including during the prosecution of each
issued patent; "application cited documents"), and each of the PCT
and foreign applications or patents corresponding to and/or
claiming priority from any of these applications and patents, and
each of the documents cited or referenced in each of the
application cited documents, are hereby expressly incorporated
herein by reference. More generally, documents or references are
cited in this text, either in a Reference List before the claims,
or in the text itself; and, each of these documents or references
("herein-cited references"), as well as each document or reference
cited in each of the herein-cited references (including any
manufacturer's specifications, instructions, etc.), is hereby
expressly incorporated herein by reference.
FIELD OF THE INVENTION
[0004] The present invention relates to methods for detection and
therapy of thin-capped fibro-atheroma ("vulnerable plaque") using
selectively targeted fluorescent compositions, which include
photosensitizer compositions, and/or radiolabeled compositions.
Other aspects of the invention are described in or are obvious from
the following disclosure (and within the ambit of the
invention).
BACKGROUND OF THE INVENTION
[0005] Cardiovascular disease remains the leading cause of
morbidity and mortality in the United States. A chief contributor
to the pathology of the disease is the formation of atherosclerotic
or "atheromatous" plaques in the coronary arteries (Farb et al.
(1995) Circulation 92:1701-1709). Yet, therapies designed to
ameliorate the occlusive effects of atheromatous plaques on
coronary blood flow, such as coronary artery bypass surgery and
percutaneous transluminal coronary angioplasty, do not prevent or
reduce the incidence of acute coronary syndrome. Acute coronary
syndrome covers a group of sudden-onset coronary diseases,
including unstable angina, acute myocardial infarction and sudden
cardiac death. The causative agent of acute coronary syndrome is
fissure, erosion or rupture of a specific kind of atheromatous
plaque known as a "vulnerable plaque." Vulnerable plaques are
responsible for the majority of heart attacks, strokes, and cases
of sudden death.
[0006] Post-mortem evidence suggests that vulnerable plaque rupture
occurs in areas of the coronary arteries that are less than about
50% stenosed. Thus, angioplasty and bypass procedures, which are
carried out on severely stenosed arteries, rarely remove vulnerable
plaques or reduce the incidence of acute coronary syndrome (Plutzky
(1999) Am J Cardiol 84:15J-20J). Even with currently available
therapeutic approaches, such as lipid lowering, angioplasty and
bypass, an unacceptably high incidence of acute coronary syndrome
remains (Sacks et al. (2000) Circulation 102:1893-1900).
[0007] Atheromatous plaques characteristically comprise a fibrous
cap surrounding a central core of extracellular lipids and debris
located in the central portion of the thickened vessel intima,
which is known as the "atheroma." On the luminal side of the lipid
core, the fibrous cap is comprised mainly of connective tissues,
typically a dense, fibrous, extracellular matrix made up of
collagens, elastins, proteoglycans and other extracellular matrix
materials. At the edges of the fibrous cap overlying the lipid core
is the shoulder region, enriched with macrophages. The macrophages
continually phagocytose oxidized LDL through scavenger receptors,
which have a high ligand specificity for oxidized LDL. Continuous
phagocytosis results in the formation of foam cells, a hallmark of
the atherosclerotic plaque (Parthasarathy et al. (1992) Annu Rev
Med 43:219-225). Foam cells, together with the binding of
extracellular lipids to collagen fibers and proteoglycans, play an
important role in the formation and growth of the lipid-rich
atheroma.
[0008] Histopathologic examination of atheromatous plaques has
revealed substantial variations in the thickness of fibrous caps,
the size of the atheromas, the extent of dystrophic calcification
and the relative contribution of major cell types (van der Wal et
al. (1994) Coron Artery Dis 5:463-469). Resident cells present in
atheromatous plaques include a significant population of
inflammatory cells, such as monocytes/macrophages and T
lymphocytes. The emigration of monocytes into the arterial wall,
and their subsequent differentiation into macrophages and
ultimately foam cells, remains one of the earliest steps in plaque
formation. Once there, these cells play a critical role in
secreting substances that further contribute to
atherosclerosis.
[0009] A vulnerable plaque is structurally and functionally
distinguishable from a stable atheromatous plaque. For example,
several histologic features distinguish a vulnerable plaque from a
stable atheromatous plaque. A vulnerable plaque is characterized by
an abundance of inflammatory cells (e.g., macrophages and/or T
cells), a large lipid pool, and a thin fibrous cap. An atheromatous
plaque refers to a wide range of coronary lesions, from subtle
collections of lipid, to obstructive coronary lesions that cause
angina.
[0010] In contrast to vulnerable plaques, the vast majority of
stable atheromatous plaques lay silent. Only the rare stable
atheromatous lesion causes heart attacks or strokes. Pathologic
studies have provided a further understanding of why vulnerable
plaques have a higher propensity for rupture than other
atheromatous plaques. The thickness and integrity of the fibrous
cap overlying the lipid-rich core is a principal factor in the
stability of the plaque. Vulnerable plaques prone to rupture can be
characterized as having thinner fibrous areas, increased numbers of
inflammatory cells (e.g., macrophages and T cells), and a relative
paucity of vascular smooth muscle cells. Vascular smooth muscle
cells are the major source of extra cellular matrix production, and
therefore, the absence of vascular smooth muscle cells from a
vulnerable plaque contributes to the lack of density in its fibrous
cap.
[0011] While the fibrous tissue within the cap provides structural
integrity to the plaque, the interior of the atheroma is soft, weak
and highly thrombogenic. It is rich in extracellular lipids and
substantially devoid of living cells, but bordered by a rim of
lipid-laden macrophages (van der Wal et al. (1999) Cardiovasc Res
41:334-344). The lipid core is a highly thrombogenic composition,
rich in tissue factor, which is one of the most potent
procoagulants known. The lesional macrophages and foam cells
produce a variety of procoagulant substances, including tissue
factor. The fibrous cap is the only barrier separating the
circulation from the lipid core and its powerful coagulation system
designed to generate thrombus. Essentially, the rapid release of
procoagulants into the blood stream at the site of rupture forms an
occlusive clot, inducing acute coronary syndrome. Thus, the thinner
the fibrous cap, the greater the instability of the thrombogenic
lipid core and the greater the propensity for rupture and
thrombosis.
[0012] Several factors can contribute to the weakened state of the
fibrous cap. In particular, inhibition of extracellular matrix
production or degradation of extracellular matrix components
adversely impacts the structural composition of the fibrous cap.
Macrophages and T lymphocytes have been identified as the dominant
cell types at the site of plaque rupture or superficial erosion,
and each of these inflammatory cells contributes to the inhibitory
and/or degradative pathways. Accelerated degradation of collagen
and other matrix components is carried out by macrophage proteases,
such as matrix metalloproteinases ("MMPs"), which are secreted at
the site of the plaque. MMPs constitute an extensive family of
enzymes, including interstitial collagenase (e.g., MMP-I),
gelatinases (e.g., MMP-2, MMP-9), and stromelysin (e.g., MMP-3).
Stromelysins can activate other members of the MMP family, causing
degradation among many matrix constituents. The presence of T cells
in the plaque can further contribute to weakening of the fibrous
cap. Activated T cells produce and secrete interferon-.gamma., a
potent inhibitor of collagen synthesis. Thus, the T lymphocytes
represent a potentially large source of, interferon-.gamma. that
can negatively regulate matrix production. Plaque rupture sites are
further characterized by expression of major histocompatibility
complex genes, (e.g., human lymphocyte antigen-DR on inflammatory
cells and adjacent smooth muscle cells), indicating an active
inflammatory reaction that also weakens the fibrous cap.
[0013] Present methods of plaque detection, several of which are
discussed herein, are inadequate for the identification of
vulnerable plaques. Common methods of plaque detection include
angiography and angioscopy. Except in rare circumstances
angiography gives almost no information about characteristics of
plaque components. Angiography is only sensitive enough to detect
hemodynamically significant lesions (>70% stenosis), which
account for approximately 33% of acute coronary syndrome cases.
Angioscopy is a technique based on fiber-optic transmission of
visible light that provides a small field of view with relatively
low resolution for visualization of interior surfaces of plaque and
thrombus. Because angioscopic visualization is limited to the
surface of the plaque, it is insufficient for use in detecting
vulnerable plaques.
[0014] Several methods are being investigated for their ability to
identify vulnerable plaques. However, none has proven to be
sufficiently sensitive. One such method, intravascular ultrasound
("IVUS") uses miniaturized crystals incorporated at catheter tips
and provides real-time, cross-sectional and longitudinal,
high-resolution images of the arterial wall with three-dimensional
reconstruction capabilities. IVUS can detect thin caps and
distinguish regions of intermediate density (e.g., intima that is
rich in smooth muscle cells and fibrous tissue) from echolucent
regions, but current technology does not determine which echolucent
regions are composed of cholesterol pools rather than thrombosis,
hemorrhage, or some combination thereof. Moreover, the spatial
resolution (i.e., approximately 2 cm) does not distinguish the
moderately thinned cap from the high risk cap (i.e., approximately
25-75 .mu.m) and large dense calcium deposits produce acoustic
echoes which "shadow" so that deeper plaque is not imaged.
[0015] Intravascular thermography is based on the premise that
plaques with dense macrophage infiltration give off more heat than
non-inflamed plaque (Casscells et al. (1996) Lancet.
347:1447-1451). The temperature of the plaque is inversely
correlated to cap thickness. However, thermography may not provide
information about eroded but non-inflamed vulnerable lesions.
[0016] Optical coherence tomography ("OCT") measures the intensity
of reflected near-infrared light from tissue. It provides images
with high resolution that is approximately 10 to 20 times higher
than that of IVUS resolution. OCT is primarily used for assessment
of atherosclerotic plaque morphology. However, long image
acquisition time, high costs, limited penetration and a lack of
physiologic data render this approach undesirable for detection of
vulnerable plaques.
[0017] Raman spectroscopy utilizes Raman effect: a basic principle
in photonic spectroscopy named after its inventor. Raman effect
arises when an incident light excites molecules in a sample, which
subsequently scatter the light. While most of this scattered light
is at the same wavelength as the incident light, some is scattered
at a different wavelength. This shift in the wavelength of the
scattered light is called Raman shift. The amount of the wavelength
shift and intensity depends on the size, shape, and strength of the
molecule. Each molecule has its own distinct "fingerprint" Raman
shift. Raman spectroscopy is a very sensitive technique and is
capable of reporting an accurate measurement of chemical compounds.
Conceivably, the ratio of lipid to proteins, such as collagen and
elastin, might help detect vulnerable plaques with large lipid
pools. However, it is unlikely that vulnerable plaques will be
reliably differentiated from stable plaques based solely on this
ratio.
[0018] Photodynamic therapy ("PDT") employs photoactivatable
compounds known as photosensitizers to selectively target and
destroy cells. Therapy involves delivering visible light of the
appropriate wavelength to excite the photosensitizer molecule to
the excited singlet state. This excited state can then undergo
intersystem crossing to the slightly lower energy triplet state,
which can then react further by one or both of two pathways, known
as Type I and Type II photoprocesses (Ochsner (1997) J Photochem
Photobiol B 39:1-18). The Type I pathway involves electron transfer
reactions from the photosensitizer triplet to produce radical ions
which can then react with oxygen to produce cytotoxic species such
as superoxide, hydroxyl and lipid derived radicals. The Type II
pathway involves energy transfer from the photosensitizer triplet
to ground state molecular oxygen (triplet) to produce the excited
state singlet oxygen, which can then oxidize many biological
molecules such as proteins, nucleic acids and lipids, and lead to
cytotoxicity.
[0019] Photodynamic therapy (PDT) has recently gained regulatory
approval in the United States for treatment of esophageal cancer
and in other countries for several other types of cancers
(Dougherty et al. (1998) J Natl Cancer Inst 90:889-905). Certain
photosensitizers accumulate preferentially in malignant tissues
(Hamblin & Newman (1994) J Photochem Photobiol B 23:3-8),
creating the advantage of dual selectivity: not only is the
photosensitizer ideally specific for the target tissue, but the
light can also be accurately delivered to the target tissue,
thereby limiting the area within which the toxic effects of the
photosensitizer are released.
[0020] Photodynamic therapy has been applied in cardiovascular
medicine for two broad indications: treatment of atherosclerosis
("photoangioplasty") and inhibition of restenosis due to intimal
hyperplasia after vascular interventions (Rockson et al. (2000)
Circulation 102:591-596, U.S. Pat. Nos. 5,116,864, 5,298,018,
5,308,861, 5,422,362, 5,834,503 and 6,054,449). Hematoporphyrin
derivative ("HpD") was the first of a number of photosensitizers
with demonstrable, selective accumulation within atheromatous
plaques (Litvack et al. (1985) Am J Cardiol 56:667-671). Subsequent
studies have underscored the affinity of porphyrin derivatives for
atheromatous plaques in rabbits and miniswine. There is maximal
photosensitizer accumulation within the arterial intimal surface
layers, which is diminished in comparison to the arterial media.
Both HpD and Photofrin, a more purified derivative of HpD, also
display in vitro preferential uptake by human atheromatous plaques.
However, there is generally a relative lack of selectivity of most
photosensitizers for atheromatous plaques and more particularly for
vulnerable plaques. Moreover, methods known in the art for
photodynamic destruction of atherosclerotic plaques generally fail
as a result of the inflammatory response that follows PDT.
[0021] Recently, interventional strategies leading to vulnerable
plaque stabilization have become an active area of research
(Rabbani & Topol (1999) Cardiovasc Res 41:402-417). A
photodynamic therapy designed to detect, stabilize and reduce or
eliminate vulnerable plaques without inducing an inflammatory
response would be highly desirable.
OBJECT AND SUMMARY OF THE INVENTION
[0022] The present invention provides methods for selectively
targeting fluorescent and/or radiolabeled compositions to
inflammatory components of vulnerable plaques, such as inflammatory
cells, proteases and lipids. As such, the present invention
provides methods for the identification of vulnerable plaques.
Detection methods of the present invention advantageously
differentiate stable atheromatous lesions from vulnerable plaques.
Furthermore, the present invention provides methods to treat
vulnerable plaques by selectively targeting and eliminating the
inflammatory components of vulnerable plaques. Once a vulnerable
plaque is identified by methods of the present invention, further
methods can be employed to stabilize the plaque against rupture
while additionally reducing specific populations of cells (e.g.,
inflammatory cells such as macrophages and T cells) or other
components (e.g., lipids and proteases) within or around the
plaque, thus reducing the overall size and severity of the
plaque.
[0023] In one aspect of the invention, fluorescent and/or
radiolabeled compositions, which include photodynamic compositions,
can be selectively targeted to inflammatory components within and
around the vulnerable plaque (e.g., macrophages, T cells, lipids
and proteases). In one embodiment, photodynamic compositions are
targeted to macrophages to reduce or eliminate secretion of
proteases. Reducing or eliminating protease activity greatly
enhances the stability of the fibrous cap and, thus, the vulnerable
plaque. In yet another embodiment, photodynamic compositions are
targeted to T cells to reduce or eliminate secretion of factors
that reduce or inhibit extracellular matrix production, such as
interferon-y. A carefully controlled application of PDT is
administered to induce apoptotic cell death in the target cells.
Advantageously, the parameters of PDT, including light dosimetry
and amount of photodynamic compound, can be controlled to induce
only apoptosis and not necrosis of the targeted cells. Inducing
apoptosis rather than necrosis reduces or eliminates the
inflammatory response following PDT and enhances the overall
therapeutic effect.
[0024] In yet another aspect of the invention, application of PDT
to the vulnerable plaque will induce cross-linking of extracellular
matrix proteins (e.g., collagen) to further stabilize the fibrous
cap against rupture. Advantageously, the parameters of PDT,
including the subcellular location of the photodynamic compounds,
can be controlled to optimize clustering of the photodynamic
compounds on the cell surface. Under these conditions, PDT induces
cell surface cross-linking and not cell necrosis, reducing or
eliminating the inflammatory response.
[0025] Other aspects of the invention are described in or are
obvious from the following disclosure (and within the ambit of the
invention).
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1A illustrates a detection/treatment system for
detecting and/or targeting and/or treating vulnerable plaque in
accordance with an embodiment of the invention. FIG. 1B is a
diagram illustrating a configuration of the control unit of FIG.
1A.
[0027] FIGS. 2A and 2B are diagrams showing a probe/catheter in
accordance with an embodiment with the present invention. FIGS. 2C
and 2D are diagrams showing alternative views of FIGS. 2A and 2B,
respectively. FIGS. 2E and 2F illustrate a probe/catheter in
accordance with an embodiment of the invention.
[0028] FIGS. 3A, 3B and 3C are diagrams showing a probe/catheter in
accordance with an embodiment of the invention.
[0029] FIGS. 4A and 4B show a probe/catheter in accordance with an
embodiment of the invention.
[0030] FIGS. 5A and 5B are diagrams illustrating a light delivery
element and a light deflection element in accordance with
respective embodiments of the invention.
[0031] FIGS. 6A, 6B and 6C illustrate a probe/catheter in
accordance with an embodiment of the present invention.
[0032] FIG. 7 shows the scheme for preparing maleylated
BSA-c.sub.e6 photosensitizer conjugates.
[0033] FIG. 8 shows BSA-c.sub.e6 purified from unreacted
c.sub.e6-NHS ester using a Sephadex G50 column and acetone
precipitation (8A: Thin Layer Chromatography; 8B: SDS-PAGE gel
visualized by fluorescence (left) and Coomassie stain (right)
before acetone precipitation; 8C: SDS-PAGE gel visualized by
fluorescence (left) and Coomassie stain (right) after acetone
precipitation)
[0034] FIG. 9 shows the UV-visible absorption spectra of the
purified mal-BSA-c.sub.e6 conjugates and free c.sub.e6.
[0035] FIG. 10 shows the selective targeting and phototoxicity of
maleylated BSA-c.sub.e6 conjugates.
[0036] FIG. 11 shows an optical multichannel analyzer used for
fluorescence localization within ex vivo aortas.
[0037] FIG. 12 shows an analysis of aortic sections from rabbits
injected with or without conjugates about 24 hours after injection
of the conjugate (Row 1: confocal fluorescence, Red=chlorin e6,
Green=elastic lamina auto-fluorescence; Row 2: fluorescence
emission spectra of intimal surface of aortic segments ex vivo; Row
3: Hematoxylin and eosin staining of formalin fixed paraffin
embedded aortic segments; Row 4: Verhoeff s elastic tissue stain).
Column 1 shows an atherosclerotic rabbit with no injection of
conjugate. Column 2 shows a normal non-atherosclerotic rabbit
injected with conjugate. Column 3 shows an atherosclerotic rabbit
injected with conjugate.
[0038] FIG. 13 shows a significant fluorescent signal from the
intimal surface (determined by Skin Scan) in all sections from
atherosclerotic rabbits compared to the corresponding sections of
aorta from normal rabbits injected with conjugate. (Top: 1=thoracic
aorta, 2=upper abdominal aorta below diaphragm, 3=mid abdominal
aorta, 4=lower abdominal aorta, 5=pelvic aorta just above
bifurcation; Middle: Measurement of intimal surface fluorescence
made by OMA-LIF system; Bottom: Data from extraction of gross
tissue samples).
[0039] FIG. 14 shows the contrast between a large aortic plaque and
an area of the abdominal aorta 5 mm beneath the plaque (14A),
between the balloon injured iliac artery and the contralateral
normal artery in the same rabbit (14B), and between the
plaque-laden aorta of an atherosclerotic rabbit and the same area
of the aorta in a normal rabbit (14C).
[0040] FIG. 15 shows a laparotomy and surgical exposure of the
aorta and surrounding tissues (15A) and a histological examination
of the arteries (15B: Top-histopathology of PDT treated
atherosclerotic aorta; Bottom-histopathology of atherosclerotic
aorta that received light but no conjugate).
[0041] FIG. 16 shows a transverse section of an atherosclerotic
aorta (16A) and a control aorta (16B).
[0042] FIG. 17 shows the accumulation of radiolabeled chemotactic
peptide within the aorta of control animals and atherosclerotic
animals.
[0043] FIG. 18 shows coronal and transverse cross-sectional SPECT
images of aortas from atherosclerotic and healthy animals. The red
arrows represent the abdominal aorta. No aortic uptake of tracer is
seen in the control animals, however, significant radiotracer
uptake is evident in the atherosclerotic rabbit's aorta.
DETAILED DESCRIPTION
Methods for Detecting and Treating Vulnerable Plaque
[0044] In one aspect, the present invention relates to methods for
the treatment of vulnerable plaques by selectively targeting and
destroying the inflammatory components of vulnerable plaques. In
one embodiment, a method of stabilizing a vulnerable plaque in a
subject comprises the steps of: [0045] a) administering a
therapeutically effective amount of at least one photosensitizer
composition, wherein the photosensitizer composition is localized
to a vulnerable plaque; [0046] b) light activating the
photosensitizer composition to produce a phototoxic species; and
[0047] c) stabilizing the vulnerable plaque against rupture.
[0048] A "vulnerable plaque" comprises an abundance of inflammatory
cells, a large lipid pool, and a thin fibrous cap. Preferably, a
vulnerable plaque comprises a fibrous cap that is less than about
150 microns thick. More preferably, a vulnerable plaque comprises a
fibrous cap that is less than about 100 microns thick (e.g.,
between about 60 and 100 microns thick). Preferably, a vulnerable
plaque comprises a macrophage and/or monocyte content that is
greater than about 10%. More preferably, a vulnerable plaque
comprises a macrophage and/or monocyte content that is greater than
about 25%. Preferably, a vulnerable plaque comprises a lipid
content that is greater than about 10%. More preferably, a
vulnerable plaque comprises a lipid content that is greater than
about 25%.
[0049] Methods of the present invention can be applied to vessels
throughout the body, and preferably, in the carotid artery.
Detection methods of the present invention can be practiced in an
invasive or non-invasive manner. In a preferred embodiment, methods
of the present invention are applied to patients who are undergoing
angiography (e.g., due to symptoms derived from stenotic plaques or
previously ruptured plaques) in order to identify and/or treat the
vulnerable plaque that would otherwise cause a coronary event.
[0050] "Inflammatory components" include inflammatory cells,
lipids, procoagulants (e.g., tissue factor) and enzymes or other
agents that promote inhibition of extracellular matrix production
or degradation of extracellular matrix components (e.g.,
proteases). "Inflammatory cells" include smooth muscle cells,
leukocytes, lymphocytes (B-lymphocytes and T-lymophocytes),
monocytes, macrophages, foam cells, mast cells, endothelial cells,
platelets, erythrocytes and polymorphonuclear cells (e.g.,
granulocytes and neutrophils). As used herein, the term, "thrombus"
refers to a clot of blood formed within a blood vessel from a
ruptured plaque and which remains attached to its place of
origin.
[0051] As used herein, a "photosensitizer" is a chemical compound,
or a biological precursor thereof, that produces a phototoxic or
other biological effect on biomolecules upon photoactivation. A
"phototoxic species" is an amount or variety of reactive species
that is sufficient to product a phototoxic effect on a cell,
cellular component or biomolecule. Preferably, the reactive species
is oxygen. As used herein, a "photosensitizer composition"
comprises a photosensitizer coupled to a molecular carrier.
Coupling to the carrier can be either direct or indirect (e.g.,
through a biotin/avidin or primary/secondary antibody association).
A "molecular carrier" refers to a biomolecule with targeting
specificity for one or more components comprising the vulnerable
plaque.
[0052] In yet another aspect, the present invention comprises
methods to detect and/or identify vulnerable plaques by targeting
fluorescent compositions, including photosensitizers, fluorescent
dyes, and photoactive dyes, to the inflammatory components
comprising vulnerable plaques. In one embodiment, a method of
detecting a vulnerable plaque in a subject comprises the steps of:
[0053] a) administering a fluorescent composition; [0054] b)
localizing the composition to the vulnerable plaque; [0055] c)
light activating the composition to illuminate the vulnerable
plaque; and identifying the vulnerable plaque.
[0056] As used herein, a "fluorescent composition" comprises a
photosensitizer, fluorescent dye or photoactive dye coupled to a
molecular carrier. Coupling to the carrier can be either direct or
indirect (e.g., through a biotin/avidin or primary/secondary
antibody association). As used herein, the term "fluorescent dye"
refers to dyes that are fluorescent when illuminated with light but
do not produce reactive species that are phototoxic or otherwise
capable of reacting with biomolecules. A photosensitizer will
fluoresce when illuminated with a certain wavelength and power of
light and also produce reactive species that is phototoxic under
the same or different wavelength and power of light. The term
"photoactive dye," as used herein, means that the illuminated
photosensitizer produces a fluorescent species, but not necessarily
a reactive species in phototoxic amounts (i.e., a phototoxic
species). Depending on the wavelength and power of light
administered, a photosensitizer can be activated to fluoresce and,
therefore, act as a photoactive dye, but not produce a phototoxic
species. The wavelength and power of light can be adapted by
methods known to those skilled in the art to bring about a
phototoxic effect where desired.
[0057] In yet another embodiment, a method of detecting a
vulnerable plaque in a subject comprises the steps of: [0058] a)
administering a detectable amount of at least one fluorescent
composition, wherein the fluorescent composition is localized to a
vulnerable plaque; [0059] b) light activating the vulnerable plaque
to produce a fluorescent species; and [0060] c) identifying the
vulnerable plaque.
[0061] In yet another aspect, methods of the present invention
comprise a combination of detection and treatment. In one
embodiment, a method of detecting and treating a vulnerable plaque
in a subject comprises the steps of: [0062] a) administering a
detectable amount of at least one fluorescent composition, wherein
the fluorescent composition is localized to a vulnerable plaque;
[0063] b) administering a therapeutically effective amount of at
least one photosensitizer composition, wherein the photosensitizer
composition is localized to a vulnerable plaque; [0064] c) light
activating the vulnerable plaque to produce a fluorescent species;
[0065] d) identifying the vulnerable plaque; [0066] e) light
activating the photosensitizer composition at the site of the
vulnerable plaque to produce a phototoxic species; and [0067] f)
stabilizing the vulnerable plaque against rupture.
[0068] In yet another embodiment, a method of detecting and
treating a vulnerable plaque in a subject comprises the steps of:
[0069] a) administering a fluorescent composition comprising a
photosensitizer coupled to a molecular carrier; [0070] b)
localizing the composition to the vulnerable plaque; [0071] c)
light activating the composition to illuminate the vulnerable
plaque; [0072] d) identifying the vulnerable plaque; [0073] e)
light activating the photosensitizer at the site of the vulnerable
plaque to produce a phototoxic species; and [0074] f) stabilizing
the vulnerable plaque against rupture.
[0075] In yet another embodiment, a method of detecting and
treating a vulnerable plaque in a subject comprises the steps of:
[0076] a) administering a fluorescent composition comprising a
photoactive dye coupled to a molecular carrier; [0077] b)
localizing the composition to the vulnerable plaque; [0078] c)
first light activating the composition to illuminate the vulnerable
plaque; and identifying the vulnerable plaque; [0079] d) second
light activating the photoactive dye at the site of the vulnerable
plaque to produce a phototoxic species; and [0080] e) stabilizing
the vulnerable plaque against rupture.
[0081] In yet another embodiment, a method of detecting and
treating a vulnerable plaque in a subject comprises the steps of:
[0082] a) administering a detectable amount of at least one
fluorescent composition comprising a photosensitizer coupled to a
molecular carrier, wherein the fluorescent composition is localized
to a vulnerable plaque; [0083] b) light activating the vulnerable
plaque to produce a fluorescent species; [0084] c) identifying the
vulnerable plaque; [0085] d) light activating the photosensitizer
at the site of the vulnerable plaque to produce a phototoxic
species; and [0086] e) stabilizing the vulnerable plaque against
rupture.
[0087] In yet another embodiment, a method of detecting and
treating a vulnerable plaque in a subject comprises the steps of:
[0088] a) administering a detectable amount of at least one
fluorescent composition comprising a photoactive dye coupled to a
molecular carrier, wherein the fluorescent composition is localized
to a vulnerable plaque; [0089] b) first light activating the
vulnerable plaque to produce a fluorescent species; [0090] c)
identifying the vulnerable plaque; [0091] d) second light
activating the photoactive dye at the site of the vulnerable plaque
to produce a phototoxic species; and [0092] e) stabilizing the
vulnerable plaque against rupture.
[0093] In yet another embodiment, a method of detecting and
treating a vulnerable plaque in a subject comprises the steps of:
[0094] a) administering a detectable amount of at least one
fluorescent composition, wherein the fluorescent composition is
localized to a vulnerable plaque; and administering a
therapeutically effective amount of at least one photosensitizer
composition, wherein the photosensitizer composition is localized
to a vulnerable plaque; [0095] b) light activating the vulnerable
plaque to produce a fluorescent species; and [0096] c) identifying
the vulnerable plaque; and [0097] d) light activating the
photosensitizer composition at the site of the vulnerable plaque to
produce a phototoxic species; and stabilizing the vulnerable plaque
against rupture.
[0098] In yet another aspect, the present invention comprises
methods to detect and/or identify vulnerable plaques by targeting
radiolabeled compositions to the inflammatory components comprising
vulnerable plaques.
[0099] In one embodiment, a method of detecting a vulnerable plaque
in a subject comprises the steps of: [0100] a) administering a
radiolabeled composition; [0101] b) localizing the composition to
the vulnerable plaque; [0102] c) detecting a signal from the
radiolabeled composition; [0103] d) identifying the vulnerable
plaque.
[0104] As used herein, a "radiolabeled composition" comprises a
radioactive agent, such as a radionuclide or paramagnetic contrast
agent, coupled to a molecular carrier. Coupling to the carrier can
be either direct or indirect (e.g., through a biotin/avidin or
primary/secondary antibody association).
[0105] In yet another embodiment, a method of detecting a
vulnerable plaque in a subject comprises the steps of: [0106] a)
administering a detectable amount of at least one radiolabeled
composition, wherein the radiolabeled composition is localized to a
vulnerable plaque; [0107] b) detecting a signal from the
radiolabeled composition; and [0108] c) identifying the vulnerable
plaque.
[0109] In yet another aspect, methods of the present invention
comprise a combination of detection and treatment. In one
embodiment, a method of detecting and treating a vulnerable plaque
in a subject comprises the steps of: [0110] a) administering a
detectable amount of at least one radiolabeled composition, wherein
the radiolabeled composition is localized to a vulnerable plaque;
[0111] b) administering a therapeutically effective amount of at
least one photosensitizer composition, wherein the photosensitizer
composition is localized to a vulnerable plaque; [0112] c)
detecting a signal from the radiolabeled composition; and [0113] d)
identifying the vulnerable plaque; [0114] e) light activating the
photosensitizer composition at the site of the vulnerable plaque to
produce a phototoxic species; [0115] f) stabilizing the vulnerable
plaque against rupture.
[0116] In yet another embodiment, a method of detecting and
treating a vulnerable plaque in a subject comprises the steps of:
[0117] a) administering a composition comprising a radioactive
agent and a photosensitizer coupled to a molecular carrier; [0118]
b) localizing the composition to the vulnerable plaque; [0119] c)
detecting a signal from the radiolabeled composition; [0120] d)
identifying the vulnerable plaque; [0121] e) light activating the
photosensitizer at the site of the vulnerable plaque to produce a
phototoxic species; and [0122] f) stabilizing the vulnerable plaque
against rupture.
[0123] In yet another embodiment, a method of detecting and
treating a vulnerable plaque in a subject comprises the steps of:
[0124] a) administering a radiolabeled composition wherein the
radiolabeled composition is localized to a vulnerable plaque;
[0125] b) first detecting a signal from the radiolabeled
composition; and identifying the vulnerable plaque; [0126] c)
administering a therapeutically effective amount of at least one
photosensitizer composition, wherein the photosensitizer
composition is localized to the vulnerable plaque; [0127] d) second
light activating the photosensitizer composition at the site of the
vulnerable plaque to produce a phototoxic species; and [0128] e)
stabilizing the vulnerable plaque against rupture.
Radiolabeled Compositions
[0129] Radiolabeled compositions of the present invention can
comprise any known radioactive agents in the art, including, but
not limited to radionuclide or paramagnetic contrast agents, which
are optionally coupled to molecular carriers. Examples of
appropriate radionuclides for use in radiolabeling include, but are
not limited to .sup.131I, .sup.125I, .sup.123I, .sup.99mTc,
including .sup.99mTc-sestamibi, .sup.99mTc-teboroxime,
.sup.99mTc-tetrofosmin, .sup.99mTc-furifosmin, .sup.99mTc-NOET,
.sup.18F, .sup.68Ga, .sup.67Ga, .sup.72As, .sup.89Zr, .sup.62Cu,
.sup.111Cu, .sup.203In, .sup.111In, .sup.198Pb, .sup.198Hg,
.sup.97Ru, .sup.11C, .sup.195mAu, .sup.82Rb, and .sup.201TI.
Suitable paramagnetic contrast agents include, but are not limited
to gadolinium, cobalt, nickel, manganese and iron.
Photosensitizer Compositions
[0130] Photosensitizers of the present invention can be any known
in the art, including, but not limited to, photofrin.RTM, synthetic
diporphyrins and dichlorins, phthalocyanines with or without metal
substituents, chloroaluminum phthalocyanine with or without varying
substituents, chloroaluminum sulfonated phthalocyanine,
O-substituted tetraphenyl porphyrins, 3,1-meso tetrakis
(o-propionamido phenyl) porphyrin, verdins, purpurins, tin and zinc
derivatives of octaethylpurpurin, etiopurpurin, hydroporphyrins,
bacteriochlorins of the tetra(hydroxyphenyl) porphyrin series,
chlorins, chlorin e.sub.6, mono-1-aspartyl derivative of chlorin
e.sub.6, di-1-aspartyl derivative of chlorin e.sub.6, tin(IV)
chlorin e.sub.6, meta-tetrahydroxphenylchlorin, benzoporphyrin
derivatives, benzoporphyrin monoacid derivatives,
tetracyanoethylene adducts of benzoporphyrin, dimethyl
acetylenedicarboxylate adducts of benzoporphyrin, Diels-Adler
adducts, monoacid ring "a" derivative of benzoporphyrin, sulfonated
aluminum PC, sulfonated AlPc, disulfonated, tetrasulfonated
derivative, sulfonated aluminum naphthalocyanines,
naphthalocyanines with or without metal substituents and with or
without varying substituents, zinc naphthalocyanine,
anthracenediones, anthrapyrazoles, aminoanthraquinone, phenoxazine
dyes, phenothiazine derivatives, chalcogenapyrylium dyes, cationic
selena and tellurapyrylium derivatives, ring-substituted cationic
PC, pheophorbide derivative, pheophorbide alpha and ether or ester
derivatives, pyropheophorbides and ether or ester derivatives,
naturally occurring porphyrins, hematoporphyrin, hematoporphyrin
derivatives, hematoporphyrin esters or ethers, protoporphyrin,
ALA-induced protoporphyrin IX, endogenous metabolic precursors,
5-aminolevulinic acid benzonaphthoporphyrazines, cationic imminium
salts, tetracyclines, lutetium texaphyrin, tin-etio-purpurin,
porphycenes, benzophenothiazinium, pentaphyrins, texaphyrins and
hexaphyrins, 5-amino levulinic acid, hypericin, pseudohypericin,
hypocrellin, terthiophenes, azaporphyrins, azachlorins, rose
bengal, phloxine B, erythrosine, iodinated or brominated
derivatives of fluorescein, merocyanines, nile blue derivatives,
pheophytin and chlorophyll derivatives, bacteriochlorin and
bacteriochlorophyll derivatives, porphocyanines, benzochlorins and
oxobenzochlorins, sapphyrins, oxasapphyrins, cercosporins and
related fungal metabolites and combinations thereof, as well as
cationic and/or lipophilic formulations thereof.
[0131] Several photosensitizers known in the art are FDA approved
and commercially available. In a preferred embodiment, the
photosensitizer is a benzoporphyrin derivative ("BPD"), such as
BPD-MA, also commercially known as BPD Verteporfin or "BPD"
(available from QLT). U.S. Pat. No. 4,883,790 describes BPD
compositions. BPD is a second-generation compound, which lacks the
prolonged cutaneous phototoxicity of Photofrin.RTM. (Levy (1994)
Semin Oncol 21: 4-10). BPD has been thoroughly characterized
(Richter et al., (1987) JNCI 79:1327-1331), (Aveline et al. (1994)
Photochem Photobiol 59:328-35), and it has been found to be a
highly potent photosensitizer for PDT. BPD tends to accumulate
within atheromatous plaques. Targeting BPD the inflammatory cells
comprising vulnerable plaques according to methods of the present
invention will increase the specificity of photoactivation.
[0132] Photosensitizers known as texaphyrins also tend to
accumulate within atherosclerotic plaques. Targeting texaphyrins to
the inflammatory cells comprising vulnerable plaques according to
methods of the present invention will increase the specificity of
photoactivation. In a preferred embodiment, the photosensitizer is
a texaphyrin photosensitizer, such as motexafin lutetium,
commercially known as Antrin (available from Pharmacyclics, Hayse
et al., (2001) Cardiovasc. Res., 2:449-55).
[0133] In a preferred embodiment, the photosensitizer is tin ethyl
etiopurpurin, commercially known as purlytin (available from
Miravant).
Fluorescent Compositions
[0134] Fluorescent compositions of the present invention can be any
known in the art, including photosensitizers, fluorescent dyes, and
photoactive dyes.
[0135] The photosensitizers used for detection of vulnerable
plaques can be any known in the art, as previously described. For
example, hematoporphyrin derivatives have been used as fluorescent
probes to investigate the development of human atherosclerotic
plaques (Spokojny (1986) J. Am. Coll. Cardiol. 8:1387-1392).
Hematoporphyrin derivatives can be used for the detection of
vulnerable plaques, particularly plaques with extensive
angiogenesis (i.e., new vasa vasorum are leaky, which will prompt
accumulation of the hematoporphyrin in the plaque in addition to
the selective targeting provided by the molecular carrier).
[0136] Fluorescent dyes of the present invention can be any known
in the art, including, but not limited to
6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimidyl
ester; 5-(and-6)-carboxyeosin; 5-carboxyfluorescein;
6-carboxyfluorescein; 5-(and-6)-carboxyfluorescein;
5-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl) ether,
-alanine-carboxamide, or succinimidyl ester; 5-carboxyfluorescein
succinimidyl ester; 6-carboxyfluorescein succinimidyl ester;
5-(and-6)-carboxyfluorescein succinimidyl ester;
5-(4,6-dichlorotriazinyl) aminofluorescein;
2',7'-difluorofluorescein; eosin-5-isothiocyanate;
erythrosin-5-isothiocyanate; 6-(fluorescein-5-carboxamido) hexanoic
acid or succinimidyl ester; 6-(fluorescein-5-(and-6)-carboxamido)
hexanoic acid or succinimidyl ester; fluorescein-5-EX succinimidyl
ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate;
Oregon Green.RTM. 488 carboxylic acid, or succinimidyl ester;
Oregon Green.RTM. 488 isothiocyanate; Oregon Green.RTM. 488-X
succinimidyl ester; Oregon Green.RTM. 500 carboxylic acid; Oregon
Green.RTM. 500 carboxylic acid, succinimidyl ester or
triethylammonium salt; Oregon Green.RTM. 514 carboxylic acid;
Oregon Green.RTM. 514 carboxylic acid or succinimidyl ester;
Rhodamine Green.TM. carboxylic acid, succinimidyl ester or
hydrochloride; Rhodamine Green.TM. carboxylic acid,
trifluoroacetamide or succinimidyl ester; Rhodamine Green.TM.-X
succinimidyl ester or hydrochloride; Rhodol Green.TM. carboxylic
acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester;
bis-(4-carboxypiperidinyl) sulfonerhodamine or di(succinimidyl
ester); 5-(and-6)-carboxynaphthofluorescein,
5-(and-6)-carboxynaphthofluorescein succinimidyl ester;
5-carboxyrhodamine 6G hydrochloride; 6-carboxyrhodamine 6G
hydrochloride, 5-carboxyrhodamine 6G succinimidyl ester;
6-carboxyrhodamine 6G succinimidyl ester;
5-(and-6)-carboxyrhodamine 6G succinimidyl ester;
5-carboxy-2',4',5',7'-tetrabromosulfonefluorescein succinimidyl
ester or bis-(diisopropylethylammonium) salt;
5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine;
5-(and-6)-carboxytetramethylrhodamine;
5-carboxytetramethylrhodamine succinimidyl ester;
6-carboxytetramethylrhodamine succinimidyl ester;
5-(and-6)-carboxytetramethylrhodamine succinimidyl ester;
6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimidyl ester;
6-carboxy-X-rhodamine succinimidyl ester;
5-(and-6)-carboxy-X-rhodamine succinimidyl ester;
5-carboxy-X-rhodamine triethylammonium salt; Lissamine.TM.
rhodamine B sulfonyl chloride; malachite green isothiocyanate;
NANOGOLD.RTM. mono(sulfosuccinimidyl ester); QSY.RTM. 21 carboxylic
acid or succinimidyl ester; QSY.RTM. 7 carboxylic acid or
succinimidyl ester; Rhodamine Red.TM.-X succinimidyl ester;
6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid
succinimidyl ester; tetramethylrhodamine-5-isothiocyanate;
tetramethylrhodamine-6-isothiocyanate;
tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Red.RTM.
sulfonyl; Texas Red.RTM. sulfonyl chloride; Texas Red.RTM.-X STP
ester or sodium salt; Texas Red.RTM.-X succinimidyl ester; Texas
Red.RTM.-X succinimidyl ester; and
X-rhodamine-5-(and-6)-isothiocyanate.
[0137] Fluorescent dyes of the present invention can be, for
example, bodipy dyes commercially available from Molecular Probes,
including, but not limited to BODIPY.RTM. FL; BODIPY.RTM. TMR STP
ester; BODIPY.RTM. TR-X STP ester; BODIPY.RTM. 630/650-X STP ester;
BODIPY.RTM. 650/665-X STP ester;
6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propi-
onic acid succinimidyl ester;
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid;
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic
acid;
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic
acid succinimidyl ester;
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid;
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid succinimidyl ester;
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid sulfosuccinimidyl ester or sodium salt;
6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)a-
mino)hexanoic acid;
6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)a-
mino)hexanoic acid or succinimidyl ester;
N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)cy-
steic acid, succinimidyl ester or triethylammonium salt;
6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a
4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid;
4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid succinimidyl ester;
4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid succinimidyl ester;
6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino-
)hexanoic acid or succinimidyl ester;
4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3--
propionic acid succinimidyl ester;
4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid succinimidyl ester;
6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styry-
loxy)acetyl)aminohexanoic acid or succinimidyl ester;
4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid;
4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid succinimidyl ester;
4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propioni-
c acid;
4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-p-
ropionic acid succinimidyl ester;
4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid succinimidyl ester;
6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza
s-indacene-3-yl)phenoxy)acetyl) amino)hexanoic acid or succinimidyl
ester; and
6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryl-
oxy)acetyl)aminohexanoic acid or succinimidyl ester.
[0138] Fluorescent dyes the present invention can be, for example,
alexa fluor dyes commercially available from Molecular Probes,
including but not limited to Alexa Fluor.RTM. 350 carboxylic acid;
Alexa Fluor.RTM. 430 carboxylic acid; Alexa Fluor.RTM. 488
carboxylic acid; Alexa Fluor.RTM. 532 carboxylic acid; Alexa
Fluor.RTM. 546 carboxylic acid; Alexa Fluor.RTM. 555 carboxylic
acid; Alexa Fluor.RTM. 568 carboxylic acid; Alexa Fluor.RTM. 594
carboxylic acid; Alexa Fluor.RTM. 633 carboxylic acid; Alexa
Fluor.RTM. 647 carboxylic acid; Alexa Fluor.RTM. 660 carboxylic
acid; and Alexa Fluor.RTM. 680 carboxylic acid.
[0139] Fluorescent dyes the present invention can be, for example,
cy dyes commercially available from Amersham-Pharmacia Biotech,
including, but not limited to Cy3 NHS ester; Cy 5 NHS ester; Cy5.5
NHS ester; and Cy 7 NHS ester.
[0140] Photoactive dyes of the present invention can be any
photosensitizer known in the art which will fluoresce but not
necessarily produce a reactive species in phototoxic amounts when
illuminated. Depending on the wavelength and power of light
administered, a photosensitizer can be activated to fluoresce and,
therefore, act as a photoactive dye, but not produce a phototoxic
effect unless, in some cases, the wavelength and power of light is
suitably adapted to induce a phototoxic effect.
Targeting Compositions
[0141] Selectivity for target tissues (i.e., vulnerable plaque) of
the present invention is achieved by using "targeting compositions"
comprising covalent conjugates or non-covalent complexes between
molecular carriers with targeting specificity for one or more
components of vulnerable plaque. Accordingly, targeting
compositions of the present invention comprise one or more
fluorescent dyes, photoactive dyes, photosensitizers, radiolabels,
and combinations thereof, "coupled" to molecular carriers. (Hasan,
T. (1992) In: B. Henderson and T. Dougherty (eds.), Photodynamic
Therapy: Basic Principles and Clinical Applications. pp. 187-200:
Marcel Dekker). Coupling to the carrier can be either direct or
indirect (e.g., through a biotin/avidin or primary/secondary
antibody association). Use of molecular carriers advantageously
allows, for example, the photosensitizer to be selected according
to optical and photophysical properties, without relying on the
molecular structure of the photosensitizer to provide a
tissue-targeting effect.
[0142] Generally, molecular targeting is based on two facets of
molecular structure. Firstly features of the molecular carriers,
such as size, charge, hydrophobicity and biodegradability, can be
manipulated to increase accumulation or retention in the plaque,
and, secondly, the molecular carrier can be designed to recognize
antigens, receptors or other cell type specific structures present
on inflammatory cells. In a preferred embodiment, the molecular
carrier is selected from the group consisting of serum proteins
including receptor ligands (Hamblin et al. (1994) J. Photochem.
Photobiol. 26:147-157; Hamblin and Newman (1994) J. Photochem.
Photobiol. 26:45-56), microspheres (Bachor et al. (1991) Proc.
Natl. Acad. Sci. U.S.A. 88:1580-1584), liposomes (Polo et al.
(1996) Cancer Lett. 109:57-61), polymers (Hamblin et al. (1999) Br.
J. Cancer 81:261-268), monoclonal antibodies (Hamblin et al. (2000)
Br. J. Cancer 83:1544-1551), growth factors (Gijsens and De Witte
(1998) Int. J. Oncol. 13:1171-1177), peptides (Krinick, (1994) J.
Biomater. Sci. Polym. Ed. 5: 303-324), hormones (Akhlynina et al.
(1995) Cancer Res. 55:1014-1019) and lipoproteins (Schmidt-Erfurth
et al. (1997) Br. J. Cancer 75:54-61).
[0143] In a preferred embodiment, targeting compositions of the
present invention are coupled to molecular carriers comprising
ligands that bind to "scavenger receptors." Scavenger receptors are
membrane proteins expressed on the surface of macrophages,
monocytes, endothelial cells and smooth muscle cells that recognize
a wide range of ligands, both naturally occurring and synthetic
(Freeman et al. (1997) Curr. Opin. Hematol. 4:41-47). Presently,
there are six members of the scavenger receptor family belonging to
three classes (e.g., class A, B or C). After initial binding to the
scavenger receptor, the ligands are rapidly internalized and are
routed to lysosomes for degradation by proteases and other
lysosomal enzymes. The wide and diverse range of structures
recognized by these receptors has led to them being termed
"molecular flypaper" (Krieger et al. (1992) Trends Biochem. Sci.
17:141-146, 1992). The ligands are all molecules with a pronounced
anionic charge that have some common conformational features
(Haberland and Fogelman (1985) Proc. Natl. Acad. Sci. U.S.A.
82:2693-2697; Takata (1989) Biochem. Biophys. Acta. 984:273-280).
Specific targeting of compositions to J774 and other
macrophage-like cells in vitro has been achieved with conjugates of
maleylated albumin, daunorubicin and doxorubicin (Mukhopadhyay et
al (1992) Biochem J. 284:237-241; Basu et al. (1994) FEBS Lett.
342:249-254; Hamblin et al. (2000) Photochem Photobiol.
4:533-540).
[0144] Numerous scavenger receptor ligands known in the art (either
with or without polyethyl glycolization) can be used to localize
targeting compositions of the present invention to vulnerable
plaques, including, but not limited to maleylated albumin, oxidized
low density lipoprotein, acetylated low density lipoprotein,
oxidized high density lipoprotein, malondialdehyde treated
proteins, lipotechoic acid, formaldehyde treated albumin, glycated
albumin, polyinosinic acid, glycated lipoproteins, dextran sulfate,
anionic phospholipids (phosphatidyl serine), fucoidin, carrageenan,
polyvinyl sulfate, monoclonal antibodies that recognize CD11b or c,
CD13, CD14, CD16a, CD32 or CD68.
[0145] In a preferred embodiment, targeting compositions of the
present invention are coupled to molecular carriers that target
macrophages and/or monocytes of vulnerable plaques. These molecular
carriers can be targeted to, for example, tenascin C, tissue
factor, tissue inhibitor of MMP 1 and 2, oxidized LDL receptor
(also known in the art as CD36), heme oxygenase-1, human cartilage
gp-39, IL-6, IL-6 receptor, IL-10, IL-10 receptor, lectin-like
oxidized LDL-receptor ("LOX-1"), bacterial chemotactic peptide
receptor agonists, preferably For-Met-Leu-Phe ("F-MLK"), macrophage
chemoattractant protein-1 receptor ("CCR-9") and monocyte
inflammatory protein-1 and receptors thereof (including "CCR-5").
Such molecular carriers can be, for example, antibodies against
these biomolecules, ligands binding the same or analogs
thereof.
[0146] In a preferred embodiment, targeting compositions of the
present invention are coupled to molecular carriers that target T
cells of vulnerable plaques. These molecular carriers can be
targeted to, for example, IL-10, IL-10 receptor, monocyte
inflammatory protein-1 and receptors thereof and transferrin. Such
molecular carriers can be, for example, antibodies against these
biomolecules, ligands binding the same or analogs thereof,
including, but not limited to monoclonal antibodies that recognize
CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD28, CD44, CD71 or
transferrin.
[0147] In a preferred embodiment, targeting compositions of the
present invention are delivered via molecular carriers that target
the lipid pool of the atheroma, including but not limited to
hydrophobic photosensitizers or photosensitizers delivered in
hydrophobic vehicles such as liposomes (with positive, neutral or
negatively charged and optionally containing cholesterol or
cardiolipin) cremaphor EL, PEG/solvent mixtures, iodized castor
oil, and various nanoparticles and micellar preparations.
[0148] In a preferred embodiment, targeting compositions of the
present invention are coupled to molecular carriers that target
proteases that degrade extracellular matrix (e.g.,
metalloproteinases), including but not limited to monoclonal
antibodies against the protease and proteinase substrates.
[0149] In a preferred embodiment, targeting compositions of the
present invention are coupled to molecular carriers that target the
endothelial cells of vulnerable plaques. These molecular carriers
can be targeted to, for example, endothelial adhesion molecules
including, but not limited to, ICAM (also known in the art as CD54)
and VCAM (also known in the art as CD106), angiotensin II,
angiotensin converting enzyme (also known in the art as CD143),
endothelial derived lipase, tissue factor, heme oxygenase-1, LOX-1,
low density lipoprotein ("LDL"), high density lipoprotein, ("HDL"),
P-selectin, L-selectin and E-selectin. Such molecular carriers can
be, for example, antibodies against these biomolecules, ligands
binding the same or analogs thereof.
[0150] In a preferred embodiment, targeting compositions of the
present invention are coupled to molecular carriers that target
neutrophils of vulnerable plaques. These molecular carriers can be
targeted to, for example, myeloperoxidase. Such molecular carriers
can be, for example, antibodies against these biomolecules, ligands
binding the same or analogs thereof.
[0151] In a preferred embodiment, targeting compositions of the
present invention are coupled to molecular carriers that target B
cells of vulnerable plaques. These molecular carriers can be
targeted to, for example, IL-6, IL-6 receptor, IL-10 and IL-10
receptor. Such molecular carriers can be, for example, antibodies
against these biomolecules, ligands binding the same or analogs
thereof.
[0152] In a preferred embodiment, targeting compositions of the
present invention are coupled to molecular carriers that target
smooth muscle cells of vulnerable plaques. These molecular carriers
can be targeted to, for example, LOX-1. Such molecular carriers can
be, for example, antibodies (e.g., Z2D3-monoclonal antibody)
against these biomolecules, ligands binding the same or analogs
thereof.
[0153] In a preferred embodiment, targeting compositions of the
present invention are coupled to molecular carriers that either
directly or indirectly associate with the target. For example,
indirect targeting can be achieved by first localizing a
biotinylated molecular carrier to a target, followed by
administration of a streptavidin-linked composition comprising a
photoactive dye, fluorescent dye, photosensitizer or radioactive
agent.
[0154] The features of a vulnerable plaque that are distinguishable
from other atheromatous plaques advantageously distinguish from
other atheromatous plaques according to methods of the present
invention.
[0155] Vulnerable plaques comprise an abundance of inflammatory
cells, a large lipid pool, and a thin fibrous cap. Preferably, a
vulnerable plaque comprises a fibrous cap that is less than about
150 microns thick. More preferably, a vulnerable plaque comprises a
fibrous cap that is less than about 100 microns thick (e.g.,
between about 60 and 100 microns thick). Preferably, a vulnerable
plaque comprises a macrophage and/or monocyte content that is
greater than about 10%. More preferably, a vulnerable plaque
comprises a macrophage and/or monocyte content that is greater than
about 25%. Preferably, a vulnerable plaque comprises a lipid
content that is greater than about 10%. More preferably, a
vulnerable plaque comprises a lipid content that is greater than
about 25%.
[0156] Thus, localizing a targeting composition to activated
macrophages or proteases that degrade extracellular matrix via a
molecular carrier, for example, confers a selective advantage on a
vulnerable plaque, such that uptake of the composition is far
greater than in a stable atheromatous plaque. Moreover, where the
targeting compositions comprise fluorescent compositions,
photodetection or photoactivation of the vulnerable plaque can be
carried out at a wavelength and power of light that has an
insubstantial or negligible effect on stable atheromatous plaques.
Thus, the methods and devices of the present invention are
advantageously suited for detection and therapy of vulnerable
plaques and not merely commonplace stable atheromatous plaques.
[0157] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature, such as,
"Molecular Cloning: A Laboratory Manual", second edition (Sambrook,
1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell
Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of
Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for
Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in
Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain
Reaction", (Mullis, 1994); "Current Protocols in Immunology"
(Coligan, 1991). These techniques are applicable to the production
of the polynucleotides and polypeptides of the invention, and, as
such, may be considered in making and practicing the invention.
Particularly useful techniques for particular embodiments will be
discussed in the sections that follow.
[0158] Compositions of the present invention that are useful for
detection of vulnerable plaques can comprise molecular carriers
that are radiolabeled. For example, photosensitizer compositions of
the present invention can comprise radiolabeled molecular carriers
coupled to photosensitizers. A number of radiolabeled molecular
carriers have been tested for their ability to bind to and permit
scintigraphic detection of atherothrombotic materials. These
include labeled antibodies to oxidized LDL, fibrinogen, autologous
platelets, fibrin fragment E1, plasminogen activators, and
.sup.99mTc-conjugated antibodies against modified LDL (Tsimikas et
al. (1999) J. Nucl. Cardiol. 6: 41-53).
[0159] Highly specific and sensitive labels are provided by
radionuclides, which can then be detected using positron emission
tomography (PET) or Single Photon Emission Computed Tomography
(SPECT) imaging. Such radiolabels can be coupled to the molecular
carrier by ionic association or covalent bonding directly to an
atom of the carrier. The radiolabel may be non-covalently or
covalently associated with the carrier through a chelating
structure. A "chelating structure" refers to any molecule or
complex of molecules which bind to both the label and targeting
moiety. Many such chelating structures are known in the art.
Chelating structures include, but are not limited to
--N.sub.2S.sub.2, --NS.sub.3, --N.sub.4, dota derivatives
[1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetrazacyclododecane],
an isonitrile, a hydrazine, a HYNIC (hydrazinonicotinic acid),
2-methylthiolnicotinic acid, phosphorus, or a carboxylate
containing group; or through an auxiliary molecule such as
mannitol, gluconate, glucoheptonate, tartrate, and the like. In
some cases, chelation can be achieved without including a separate
chelating structure, because the radionuclide chelates directly to
atom(s) in the molecular carrier, for example to oxygen atoms in
various moieties.
[0160] The chelating structure, auxiliary molecule, or radionuclide
may be placed in spatial proximity to any position of the molecular
carrier which does not interfere with the interaction of the
targeting molecule with its target site in cardiovascular tissue.
Accordingly, the chelating structure, auxiliary molecule, or
radionuclide can be covalently or non-covalently associated with
any moiety of the molecular carrier (except the receptor-binding
moiety where the molecular carrier is a receptor and the epitope
binding region where the molecular carrier is an antibody).
[0161] Radionuclides can be placed in spatial proximity to the
molecular carrier using known procedures that effect or optimize
chelation, association, or attachment of the specific radionuclide
to ligands. For example, when .sup.123I is the radionuclide, the
imaging agent can be labeled in accordance with the known
radioiodination procedures such as direct radioiodination with
chloramine T, radioiodination exchange for a halogen or an
organometallic group, and the like. When the radionuclide is
.sup.99mTc, the imaging agent can be labeled using any method
suitable for attaching .sup.99mTc to a ligand molecule. Preferably,
when the radionuclide is .sup.99mTc, an auxiliary molecule such as
mannitol, gluconate, glucoheptonate, or tartrate is included in the
labeling reaction mixture, with or without a chelating structure.
More preferably, .sup.99mTc is placed in spatial proximity to
carrier by reducing .sup.99mTcO.sub.4, with tin in the presence of
mannitol and the targeting molecule. Other reducing agents,
including tin tartrate or non-tin reductants such as sodium
dithionite, may also be used to make radiolabeled compositions of
the present invention.
[0162] In general, labeling methodologies vary with the choice of
radionuclide and the carrier to be labeled. Labeling methods are
described for example in Peters et al. (1986) Lancet 2:946-949;
Srivastava et al. (1984) Semin. Nucl. Med 14:68-82; Sinn et al.
(1984) J. Nucl. Med. 13:180; McAfee et al. (1976) J. Nucl. Med.
17:480-487; Welch et al., (1977) J. Nucl. Med. 18:558-562; Thakuret
et al. (1984) Semin. Nucl. Med. 14:107; Danpure et al. (1981) Br.
J. Radiol. 54:597-601; Danpure et al. (1982) Br. J. Radiol.
55:247-249; Peters et al. (1982) J. Nucl. Med. 24:39-44; Gunter et
al. (1983) Radiology 149:563-566 and Thakur et al. (1985) J. Nucl.
Med. 26:518-523.
[0163] After the labeling reaction is complete, the reaction
mixture may optionally be purified using one or more chromatography
steps such as Sep Pack or high performance liquid chromatography
(HPLC). Any suitable HPLC system may be used if a purification step
is performed, and the yield of cardiovascular imaging agent
obtained from the HPLC step may be optimized by varying the
parameters of the HPLC system, as is known in the art. Any HPLC
parameter may be varied to optimize the yield of the cardiovascular
imaging agent of the invention. For example, the pH may be varied,
e.g., raised to decrease the elution time of the peak corresponding
to the radiolabeled carrier.
[0164] The term "coupling agent" as used herein, refers to a
reagent capable of coupling a composition (e.g., photoactive dye,
fluorescent dye, photosensitizer or radioactive agent) to a
molecular carrier, or to a "backbone" or "bridge" moiety. Any bond
which is capable of linking the components such that they are
stable under physiological conditions for the time needed for
administration and treatment is suitable, but covalent linkages are
preferred. The link between two components may be direct, e.g.,
where a photosensitizer is linked directly to a molecular carrier,
or indirect, e.g., where a photosensitizer is linked to an
intermediate, e.g., linked to a backbone, and that intermediate
being linked to the molecular carrier. A coupling agent should
function under conditions of temperature, pH, salt, solvent system,
and other reactants that substantially retain the chemical
stability of the photosensitizer, the backbone (if present), and
the molecular carrier.
[0165] A coupling agent is not always required, for example, where
the fluorescent compound is in the form of a sulfonyl chloride,
isothiocyanate or succinimidyl ester, no coupling agent is
necessary.
[0166] A coupling agent can link components without the addition to
the linked components of elements of the coupling agent. Other
coupling agents result in the addition of elements of the coupling
agent to the linked components. For example, coupling agents can be
cross-linking agents that are homo- or hetero-bifunctional, and
wherein one or more atomic components of the agent can be retained
in the composition. A coupling agent that is not a cross-linking
agent can be removed entirely during the coupling reaction, so that
the molecular product can be composed entirely of the
photosensitizer, the targeting moiety, and a backbone moiety (if
present).
[0167] Many coupling agents react with an amine and a carboxylate,
to form an amide, or an alcohol and a carboxylate to form an ester.
Coupling agents are known in the art, see, e.g., M. Bodansky,
"Principles of Peptide Synthesis", 2nd ed., referenced herein, and
T. Greene and P. Wuts, "Protective Groups in Organic Synthesis,"
2nd Ed, 1991, John Wiley, NY. Coupling agents should link component
moieties stably, but such that there is only minimal or no
denaturation or deactivation of the photosensitizer or the
molecular carrier.
[0168] The photosensitizer compositions of the invention can be
prepared by coupling the photosensitizer to molecular carriers
using methods described in the following Examples, or by methods
known in the art. A variety of coupling agents, including
cross-linking agents, can be used for covalent conjugation.
Examples of cross-linking agents include
N,N'-dicyclohexylcarbodiimide (DCC),
N-succinimidyl-S-acetylthioacetate (SATA),
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),
orthophenylenedimaleimide (o-PDM), and sulfosuccinimidyl
4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC)
(Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, MA et al.
(1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include
those described by Paulus and Behring (1985) Ins. Mitt.,
78:118-132; Brennan et al. (1985) Science 229:81-83 and Glennie et
al., (1987) J. Immunol, 139:2367-2375. A large number of coupling
agents for peptides and proteins, along with buffers, solvents, and
methods of use, are described in the Pierce Chemical Co. catalog,
pages T155-T-200, 1994 (3747 N. Meridian Rd., Rockford Ill., 61105,
U.S.A.; Pierce Europe B.V., P.O. Box 1512, 3260 BA Oud Beijerland,
The Netherlands), the contents of which are hereby incorporated by
reference.
[0169] DCC is a useful coupling agent (Pierce #20320; Rockland,
Ill.). It promotes coupling of the alcohol NHS to chlorin e6 in
DMSO (Pierce #20684), forming an activated ester which can be
cross-linked to polylysine. DCC (N,N'dicyclohexylcarbodiimide) is a
carboxy-reactive cross-linker commonly used as a coupling agent in
peptide synthesis, and has a molecular weight of 206.32. Another
useful cross-linking agent is SPDP (Pierce #21557), a
heterobifunctional cross-linker for use with primary amines and
sulfhydryl groups. SPDP has a molecular weight of 312.4, a spacer
arm length of 6.8 angstroms, is reactive to NHS-esters and
pyridyldithio groups, and produces cleavable cross-linking such
that, upon further reaction, the agent is eliminated so the
photosensitizer can be linked directly to a backbone or molecular
carrier. Other useful conjugating agents are SATA (Pierce #26102)
for introduction of blocked SH groups for two-step cross-linking,
which is deblocked with hydroxylamine-25-HCl (Pierce #26103), and
sulfo-SMCC (Pierce #22322), reactive towards amines and
sulfhydryls. Other cross-linking and coupling agents are also
available from Pierce Chemical Co. (Rockford, Ill.). Additional
compounds and processes, particularly those involving a Schiff base
as an intermediate, for conjugation of proteins to other proteins
or to other compositions, for example to reporter groups or to
chelators for metal ion labeling of a protein, are disclosed in EPO
243,929 A2 (published Nov. 4, 1987).
[0170] Photosensitizers which contain carboxyl groups can be joined
to lysine s-amino groups in the target polypeptides either by
preformed reactive esters (such as N-hydroxy succinimide ester) or
esters conjugated in situ by a carbodiimide-mediated reaction. The
same applies to photosensitizers that contain sulfonic acid groups,
which can be transformed to sulfonyl chlorides, which react with
amino groups. Photosensitizers that have carboxyl groups can be
joined to amino groups on the polypeptide by an in situ
carbodiimide method. Photosensitizers can also be attached to
hydroxyl groups, of serine or threonine residues or to sulfhydryl
groups, of serine or threonine residues or to sulfhydryl groups of
cysteine residues.
[0171] Methods of joining components of a composition, e.g.,
coupling polyamino acid chains bearing photosensitizers to
antibacterial polypeptides, can use heterobifunctional cross
linking reagents. These agents bind a functional group in one chain
and to a different functional group in the second chain. These
functional groups typically are amino, carboxyl, sulfhydryl, and
aldehyde. There are many permutations of appropriate moieties that
will react with these groups and with differently formulated
structures, to conjugate them together (described in the Pierce
Catalog and Merrifield et al. (1994) Ciba Found Symp.
186:5-20).
[0172] The production and purification of photosensitizers coupled
to molecular carriers can be practiced by methods known in the art.
Yield from coupling reactions can be assessed by spectroscopy of
product eluting from a chromatographic fractionation in the final
step of purification. The presence of uncoupled photosensitizer and
reaction products containing the photosensitizer can be followed by
the physical property that the photosensitizer moiety absorbs light
at a characteristic wavelength and extinction coefficient, so
incorporation into products can be monitored by absorbance at that
wavelength or a similar wavelength. Coupling of one or more
photosensitizer molecules to a molecular carrier or to a backbone
shifts the peak of absorbance in the elution profile in fractions
eluted using sizing gel chromatography, e.g., with the appropriate
choice of Sephadex G50, 6100, or 6200 or other such matrices
(Pharmacia-Biotech, Piscataway N.J.). Choice of appropriate sizing
gel, for example Sephadex gel, can be determined by that gel in
which the photosensitizer elutes in a fraction beyond the excluded
volume of material too large to interact with the bead, i.e., the
uncoupled starting photosensitizer composition interacts to some
extent with the fractionation bead and is concomitantly retarded to
some extent. The correct useful gel can be predicted from the
molecular weight of the uncoupled photosensitizer. The successful
reaction products of photosensitizer compositions coupled to
additional moieties generally have characteristic higher molecular
weights, causing them to interact with the chromatographic bead to
a lesser extent, and thus appear in fractions eluting earlier than
fractions containing the uncoupled photosensitizer substrate.
Unreacted photosensitizer substrate generally appears in fractions
characteristic of the starting material, and the yield from each
reaction can thus be assessed both from size of the peak of larger
molecular weight material, and the decrease in the peak of
characteristic starting material. The area under the peak of the
product fractions is converted to the size of the yield using the
molar extinction coefficient.
[0173] The product can be analyzed using NMR, integrating areas of
appropriate product peaks, to determine relative yields with
different coupling agents. A red shift in absorption of a
photosensitizer has often been observed following coupling to a
polyamino acid. Coupling to a larger carrier such as a protein
might produce a comparable shift, as coupling to an antibody
resulted in a shift of about 3-5 nm in that direction compared to
absorption of the free photosensitizer. Relevant absorption maxima
and extinction coefficients in O0.1M NaOH/1% SDS are, for
chlorin.sub.e6, 400 nm and 150,000 M.sup.-1, cm.sup.-1, and for
benzoporphyrin derivative, 430 nm and 61,000 M.sup.-1,
cm.sup.-1.
[0174] Photosensitizers compositions of the invention include those
in which a photosensitizer is coupled directly to a molecular
carrier, such as a scavenger receptor ligand. Other photosensitizer
compositions of the invention include a "backbone" or "bridge"
moiety, such as a polyamino acid, in which the backbone is coupled
both to a photosensitizer and to a molecular carrier.
[0175] Inclusion of a backbone in a composition with a
photosensitizer and a molecular carrier can provide a number of
advantages, including the provision of greater stoichiometric
ranges of photosensitizer and molecular carriers coupled per
backbone. If the backbone possesses intrinsic affinity for a target
organism, the affinity of the composition can be enhanced by
coupling to the backbone. The specific range of organisms that can
be targeted with one composition can be expanded by coupling two or
more different molecular carriers to a single
photosensitizer-backbone composition.
[0176] Peptides useful in the methods and compounds of the
invention for design and characterization of backbone moieties
include poly-amino acids which can be homo- and hetero-polymers of
L-, D-, racemic DL- or mixed L- and D-amino acid composition, and
which can be of defined or random mixed composition and sequence.
These peptides can be modeled after particular natural peptides,
and optimized by the technique of phage display and selection for
enhanced binding to a chosen target, so that the selected peptide
of highest affinity is characterized and then produced
synthetically. Further modifications of functional groups can be
introduced for purposes, for example, of increased solubility,
decreased aggregation, and altered extent of hydrophobicity.
Examples of nonpeptide backbones include nucleic acids and
derivatives of nucleic acids such as DNA, RNA and peptide nucleic
acids; polysaccharides and derivatives such as starch, pectin,
chitins, celluloses and hemimethylated celluloses; lipids such as
triglyceride derivatives and cerebrosides; synthetic polymers such
as polyethylene glycols (PEGS) and PEG star polymers; dextran
derivatives, polyvinyl alcohols, N-(2-hydroxypropyl)-methacrylamide
copolymers, poly (DL-glycolic acid-lactic acid); and compositions
containing elements of any of these classes of compounds.
[0177] The affinity of a photosensitizer composition can be refined
by modifying the charge of a component of the composition.
Conjugates such as poly-L-lysine chlorin e6 can be made in varying
sizes and charges (cationic, neutral, and anionic), for example,
free NH2 groups of the polylysine are capped with acetyl, succinyl,
or other R groups to alter the charge of the final composition. Net
charge of a composition of the present invention can be determined
by isoelectric focusing (IEF). This technique uses applied voltage
to generate a pH gradient in a non-sieving acrylamide or agarose
gel by the use of a system of ampholytes (synthetic buffering
components). When charged polypeptides are applied to the gel they
will migrate either to higher pH or to lower pH regions of the gel
according to the position at which they become non-charged and
hence unable to move further. This position can be determined by
reference to the positions of a series of known IEF marker
proteins.
[0178] Photosensitizer compositions of the present invention can
comprise photosensitizers coupled to antibodies, which are known in
the art as "photoimmunoconjugates." The antibody component of the
photoimmunoconjugate can bind with specificity to an epitope
present on the surface of a cell comprising the vulnerable plaque.
As used herein, the term "binding with specificity" means that
cells that do not express the epitope are only poorly recognized by
the antibody.
[0179] The term "antibody" as used in this invention includes
intact molecules as well as fragments thereof, such as Fab and
Fab', which are capable of binding the epitopic determinant. Fab
fragments retain an entire light chain, as well as one-half of a
heavy chain, with both chains covalently linked by the carboxy
terminal disulfide bond. Fab fragments are monovalent with respect
to the antigen-binding site. The antibodies of the invention
comprise whole native antibodies, bispecific antibodies; chimeric
antibodies; Fab, Fab', single chain variable region fragments
(scFv) and fusion polypeptides. Preferably, the antibodies of the
invention are monoclonal.
[0180] The antibodies of this invention can be prepared in several
ways. Methods of producing and isolating whole native antibodies,
bispecific antibodies; chimeric antibodies; Fab, Fab', single chain
V region fragments (scFv) and fusion polypeptides are known in the
art. See, for example, Harlow and Lane (1988) Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, New York (Harlow
and Lane, 1988).
[0181] Antibodies are most conveniently obtained from hybridoma
cells engineered to express an antibody. Methods of making
hybridomas are well known in the art. The hybridoma cells can be
cultured in a suitable medium, and spent medium can be used as an
antibody source. Polynucleotides encoding the antibody can in turn
be obtained from the hybridoma that produces the antibody, and then
the antibody may be produced synthetically or recombinantly from
these DNA sequences. For the production of large amounts of
antibody, it is generally more convenient to obtain an ascites
fluid. The method of raising ascites generally comprises injecting
hybridoma cells into an immunologically naive histocompatible or
immunotolerant mammal, especially a mouse. The mammal may be primed
for ascites production by prior administration of a suitable
composition, e.g., Pristane.
[0182] Another method of obtaining antibodies is to immunize
suitable host animals with an antigen and to follow standard
procedures for polyclonal or monoclonal production. Monoclonal
antibodies (Mabs) thus produced can be "humanized" by methods known
in the art. Examples of humanized antibodies are provided, for
instance, in U.S. Pat. Nos. 5,530,101 and 5,585,089.
[0183] "Humanized" antibodies are antibodies in which at least part
of the sequence has been altered from its initial form to render it
more like human immunoglobulins. In one version, the heavy chain
and light chain C regions are replaced with human sequence. In
another version, the CDR regions comprise amino acid sequences for
recognition of antigen of interest, while the variable framework
regions have also been converted to human sequences. See, for
example, EP 0329400. In a third version, variable regions are
humanized by designing consensus sequences of human and mouse
variable regions, and converting residues outside the CDRs that are
different between the consensus sequences. The invention
encompasses humanized Mabs.
[0184] The invention also encompasses hybrid antibodies, in which
one pair of heavy and light chains is obtained from a first
antibody, while the other pair of heavy and light chains is
obtained from a different second antibody. Such hybrids may also be
formed using humanized heavy and light chains.
[0185] Construction of phage display libraries for expression of
antibodies, particularly the Fab or scFv portion of antibodies, is
well known in the art (Heitner et al. (2001) J Immunol Methods
248:17-30). The phage display antibody libraries that express
antibodies can be prepared according to the methods described in
U.S. Pat. No. 5,223,409 incorporated herein by reference.
Procedures of the general methodology can be adapted using the
present disclosure to produce antibodies of the present invention.
The method for producing a human monoclonal antibody generally
involves (1) preparing separate heavy and light chain-encoding gene
libraries in cloning vectors using human immunoglobulin genes as a
source for the libraries, (2) combining the heavy and light chain
encoding gene libraries into a single dicistronic expression vector
capable of expressing and assembling a heterodimeric antibody
molecule, (3) expressing the assembled heterodimeric antibody
molecule on the surface of a filamentous phage particle, (4)
isolating the surface-expressed phage particle using immunoaffinity
techniques such as panning of phage particles against a preselected
antigen, thereby isolating one or more species of phagemid
containing particular heavy and light chain-encoding genes and
antibody molecules that immunoreact with the preselected
antigen.
[0186] Single chain variable region fragments are made by linking
light and heavy chain variable regions by using a short linking
peptide. Any peptide having sufficient flexibility and length can
be used as a linker in a scFv. Usually the linker is selected to
have little to no immunogenicity. An example of a linking peptide
is (GGGGS).sub.3, which bridges approximately 3.5 nm between the
carboxy terminus of one variable region and the amino terminus of
another variable region. Other linker sequences can also be used.
All or any portion of the heavy or light chain can be used in any
combination. Typically, the entire variable regions are included in
the scFv. For instance, the light chain variable region can be
linked to the heavy chain variable region. Alternatively, a portion
of the light chain variable region can be linked to the heavy chain
variable region, or a portion thereof. Also contemplated are
compositions comprising a biphasic scFv in which one component is a
polypeptide that recognizes an antigen and another component is a
different polypeptide that recognizes a different antigen, such as
a T cell epitope.
[0187] ScFvs can be produced either recombinantly or synthetically.
For synthetic production of scFv, an automated synthesizer can be
used. For recombinant production of scFv, a suitable plasmid
containing a polynucleotide that encodes the scFv can be introduced
into a suitable host cell, either eukaryotic, such as yeast, plant,
insect or mammalian cells, or prokaryotic, such as Escherichia
coli, and the protein expressed by the polynucleotide can be
isolated using standard protein purification techniques.
[0188] A particularly useful system for the production of scFvs is
plasmid pET-22b(+) (Novagen, Madison, Wis.) in E. coli. pET-22b(+)
contains a nickel ion binding domain consisting of 6 sequential
histidine residues, which allows the expressed protein to be
purified on a suitable affinity resin. Another example of a
suitable vector is pcDNA3 (Invitrogen, San Diego, Calif.),
described above.
[0189] Expression conditions should ensure that the scFv assumes
functional and, preferably, optimal tertiary structure. Depending
on the plasmid used (especially the activity of the promoter) and
the host cell, it may be necessary or useful to modulate the rate
of production. For instance, use of a weaker promoter, or
expression at lower temperatures, may be necessary or useful to
optimize production of properly folded scFv in prokaryotic systems;
or, it may be preferable to express scFv in eukaryotic cells.
Antibody purification methods may include salt precipitation (for
example, with ammonium sulfate), ion exchange chromatography (for
example, on a cationic or anionic exchange column run at neutral pH
and eluted with step gradients of increasing ionic strength), gel
filtration chromatography (including gel filtration HPLC), and
chromatography on affinity resins such as protein A, protein G,
hydroxyapatite, and anti-immunoglobulin.
[0190] Photosensitizers can be linked to antibodies according to
any method known in the art. For example, the antibody can be
directly linked to the photosensitizer through a polymer or a
polypeptide linkage. Polymers of interest include, but are not
limited to polyamines, polyethers, polyamine alcohols, derivitized
to components by means of ketones, acids, aldehydes, isocyanates or
a variety of other groups. Polypeptide linkages can comprise, for
example poly-L-lysine linkages (Del Govematore et al. (2000) Br. J.
Cancer 82:56-64; Hamblin et al. (2000) Br. J. Cancer 83:1544-41;
Molpus et al. (2000) Gynecol Oncol 76:397-404). The antibody can be
linked to a photosensitizer and at least one solubilizing agent
each of which are independently bound to the antibody through a
direct covalent linkage. The direct covalent linkage can be, for
example, an amide linkage to a lysine residue of the antibody, as
described in U.S. application Ser. No. 10/137,029, the contents of
which are herein incorporated by reference.
[0191] Photosensitizer compositions of the present invention can
comprise photosensitizers linked to molecular carriers comprising
the sequences of naturally occurring proteins and peptides, from
variants or fragments of these peptides, and from biologically or
chemically synthesized peptides or peptide fragments. Naturally
occurring peptides which have affinity for one or more target cells
can provide sequences from which additional peptides with desired
properties, e.g., increased affinity or specificity, can be
synthesized individually or as members of a library of related
peptides. Such peptides can be selected on the basis of affinity
for the target cell.
[0192] The term "or (a) fragment(s) thereof" as employed in the
present invention and in context with polypeptides of the
invention, comprises specific peptides, amino acid stretches of the
polypeptides as disclosed herein. It is preferred that said
"fragment(s) thereof" is/are functional fragment(s). The term
"functional fragment" denotes a part of the above identified
polypeptide of the invention which fulfills, at least in part,
physiologically and/or structurally related activities of the
polypeptide of the invention. The polypeptides of the present
invention can be recombinant polypeptides expressed in eukaryotic
cells, like mammalian cells.
[0193] Generally, recombinant DNA technology has enabled the
expression of foreign (heterologous) proteins in cell lines of
choice. In this process, a vector containing genetic material
directing a cell to produce a protein encoded by a portion of a
heterologous DNA sequence is introduced into the host, and the
transformed host cells can be fermented, cultured or otherwise
subjected to conditions which facilitate the expression of the
heterologous DNA, leading to the formation of large quantities of
the desired protein. Plasmids are extensively used as vectors to
clone DNA molecules. Most plasmid vectors are made by taking DNA
from a variety of replicons (plasmids, bacteriophage chromosomes
and bacterial chromosomes) and joining the DNA together (using
restriction enzymes and DNA ligase) to form a plasmid that has an
origin of replication, a selection marker (usually an
antibiotic-resistance gene) and a promoter for expressing genes of
interest in the required host cell. A vector can be, for example,
as in U.S. Pat. Nos. 5,990,091 and 6,004,777, and as in
PCT/US00/04203. Methods for generation and use of recombinant
vectors in vitro are well known in the art. See Sambrook, Fritsch
and Maniatis, Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold
Spring Harbor Laboratory Press, 1989 (e.g., procedures for
isolating DNA, constructing recombinant vectors, transfecting and
transforming cells and producing heterologous peptides).
[0194] Furthermore, the recombinant vector can, in addition to the
nucleic acid sequences of the invention (e.g., those encoding the
targeting peptide or functional fragments thereof), comprise
expression control elements, allowing proper expression of the
coding regions in suitable hosts. Such control elements are known
in the art and can include a promoter, a splice cassette,
translation initiation codon, translation and insertion site for
introducing an insert into the vector. Preferably, the nucleic acid
molecule is operatively linked to expression control sequences
allowing expression in eukaryotic or prokaryotic cells.
[0195] Control elements ensuring expression in eukaryotic and
prokaryotic cells are well known to those skilled in the art. As
mentioned herein above, they usually comprise regulatory sequences
ensuring initiation of transcription and optionally poly-A signals
ensuring termination of transcription and stabilization of the
transcript. Additional regulatory elements can include
transcriptional as well as translational enhancers, and/or
naturally-associated or heterologous promoter regions. Possible
regulatory elements permitting expression in for example mammalian
cells comprise the CMV-HSV thymikine kinase promoter, SV40,
RSV-promoter (Rous sarcoma virus), human elongation factor
1.alpha.-promoter, aPM-I promoter (Schaffer et al. (1999) Biochem.
Biophys. Res. Commun. 260:416-425), or inducible promoter(s), like,
metallothionein or tetracyclin, or enhancers, like CMV enhancer or
SV40-enhancer. For the expression in prokaryotic cells, a multitude
of promoters including, for example, the tac-lac-promoter or the
trp promoter, has been described. Besides elements that are
responsible for the initiation of transcription, such regulatory
elements can also comprise transcription termination signals, such
as SV40-poly-A site or the tk-poly-A site, downstream of the
polynucleotide. In this context, suitable expression vectors are
known in the art such as Okayama-Berg cDNA expression vector pcDV1
(Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), pSPORT1 (GIBCO
BRL), Casper, Casper-HS43, pUAST, or prokaryotic expression
vectors, such as lambda gt11.
[0196] Furthermore, depending on the expression system, leader
sequences capable of directing the polypeptide to a cellular
compartment can be added to the coding sequence of the nucleic acid
molecules of the invention and are well known in the art. The
leader sequence(s) is assembled in appropriate phase with
translation, initiation and termination sequences, and preferably,
a leader sequence capable of directing secretion of translated
protein, or a protein thereof, into the periplasmic space or
extracellular medium. Optionally, the heterologous sequence can
encode a fusion protein including an C- or N-terminal
identification peptide imparting desired characteristics, e.g.,
stabilization of expressed recombinant products. Once the vector
has been incorporated into the appropriate cell line, the cells are
maintained under conditions suitable for high level expression of
the nucleotide sequences.
[0197] A cell can be transfected or transformed with a recombinant
vector encoding the targeting peptide of the present invention.
Methods of transformation and transfection are well known in the
art. The transformed cells can be grown in fermentors and cultured
according to techniques known in the art to achieve optimal cell
growth. The resulting transformed or transfected cell lines are
genetically modified with a nucleic acid molecule according to the
invention or with a vector comprising such a nucleic acid molecule.
The term "genetically modified" means that the cell comprises in
addition to its natural genome a nucleic acid molecule or vector
according to the invention which was introduced into the cell or
host or into one of its predecessors/parents. The nucleic acid
molecule or vector can be present in the genetically modified cell
either as an independent molecule outside the genome, preferably as
a molecule that is capable of replication, or it can be stably
integrated into the genome of the cell.
[0198] The present invention can utilize any suitable prokaryotic
or eukaryotic cell. Suitable prokaryotic cells are those generally
used for cloning like Escherichia coli or Bacillus subtilis.
Eukaryotic cells comprise, for example, fungal or animal cells, and
are generally used for conducting the specificity assay. Animal
cells are preferably used for conducting the specificity assay.
Suitable animal cells are, for instance, insect cells, vertebrate
cells, preferably mammalian cells. Further suitable cell lines
known in the art are obtainable from cell line depositories, like
the American Type Culture Collection (ATCC) and the AIDS Research
and Reference Reagent Program Catalog. Derivation of primary cells
from an animal, preferably a mammal, and even more preferably a
human, can also be undertaken for the purposes of establishing a
suitable cell line.
Targeting Composition Administration
[0199] Targeting compositions of the invention can be administered
in a pharmaceutically acceptable excipient, such as water, saline,
aqueous dextrose, glycerol, or ethanol. The compositions can also
contain other medicinal agents, pharmaceutical agents, carriers,
and auxiliary substances such as wetting or emulsifying agents, and
pH buffering agents.
[0200] Standard texts, such as Remington: The Science and Practice
of Pharmacy, 17th edition, Mack Publishing Company, incorporated
herein by reference, can be consulted to prepare suitable
compositions and formulations for administration, without undue
experimentation. Suitable dosages can also be based upon the text
and documents cited herein. A determination of the appropriate
dosages is within the skill of one in the art given the parameters
herein.
[0201] A "therapeutically effective amount" is an amount sufficient
to effect a beneficial or desired clinical result. A
therapeutically effective amount can be administered in one or more
doses. In terms of treatment, an effective amount is an amount that
is sufficient to palliate, ameliorate, stabilize, reverse or slow
the progression of a cardiovascular disease characterized by the
presence of vulnerable plaques or otherwise reduce the pathological
consequences of the impending rupture. A therapeutically effective
amount can be provided in one or a series of administrations. The
effective amount is generally determined by the physician on a
case-by-case basis and is within the skill of one in the art.
[0202] As a rule, the dosage for in vivo therapeutics or
diagnostics will vary. Several factors are typically taken into
account when determining an appropriate dosage. These factors
include age, sex and weight of the patient, the condition being
treated, the severity of the condition and the form of the antibody
being administered.
[0203] Radiolabeled compositions of the present invention,
optionally coupled to molecular carriers or molecular carriers and
photosensitizers, can comprise, for example, from about 1 to about
30 mCi of the radionuclide in combination with a pharmaceutically
acceptable carrier. Such compositions may be provided in solution
or in lyophilized form. Suitable sterile and physiologically
acceptable reconstitution medium include water, saline, buffered
saline, and the like. Radionuclides can be combined with the
unlabeled molecular carrier/chelating agent and a reducing agent
for a sufficient period of time and at a temperature sufficient to
chelate the radionuclide to the molecular carrier prior to
injection into the patient.
[0204] Radiolabeled compositions of the invention can be used in
accordance with the methods of the invention by those of skill in
the art, e.g., by specialists in nuclear medicine, to image plaque
in the cardiovascular system of a subject. Images are generated by
virtue of differences in the spatial distribution of the
compositions which accumulate in the various tissues and organs of
the subject. The spatial distribution of the imaging agent
accumulated can be measured using devices of the present invention.
Stable atheromatous plaques are evident when a less intense signal
is detected, indicating the presence of tissue in which a lower
concentration of a radiolabeled composition accumulates relative to
the concentration of the same which accumulates in the vulnerable
plaque. Alternatively, a vulnerable plaque can be detected as a
more intense signal, indicating a region of enhanced concentration
of the radiolabeled composition at the site relative to the
concentration of the same which accumulates in stable atheromatous
plaques. The extent of accumulation of the radiolabeled composition
can be quantified using known methods for quantifying radioactive
emissions. A particularly useful imaging approach to employs more
than one imaging agent to perform simultaneous studies. For
example, simultaneous studies of perfusion and metabolic function
would allow study of coupling and uncoupling of flow of metabolism,
thus facilitating determinations of tissue viability after a
cardiac injury. Such determinations are useful in diagnosis of
cardiac ischemia, cardiomyopathy, tissue viability, hibernating
heart, and other heart abnormalities.
[0205] An effective amount of a radiolabeled composition comprising
at least one molecular carrier and a radiolabel (e.g. from about 1
to about 50 mCi of a radionuclide), or molecular carrier,
photosensitizer and radiolabel, can be combined with a
pharmaceutically acceptable carrier for use in detection and/or
therapeutic methods. In accordance with the invention, "an
effective amount of the radiolabeled composition" of the invention
is defined as an amount sufficient to yield an acceptable signal
using equipment which is available for clinical use. An effective
amount of the radiolabeled composition of the invention can be
administered in more than one dose. Effective amounts of the
radiolabeled composition of the invention will vary according to
factors such as the degree of susceptibility of the individual, the
age, sex, and weight of the individual, idiosyncratic responses of
the individual, and the dosimetry. Effective amounts of the imaging
agent of the invention will also vary according to instrument and
film-related factors.
[0206] Optimization of such factors is well within the level of
skill in the art. In general, the effective amount will be in the
range of from about 0.1 to about 10 mg by injection or from about 5
to about 100 mg orally.
[0207] The radiolabeled compositions, optionally comprising
molecular carriers or molecular carriers and photosensitizers, can
be administered to a subject in accordance with any means that
facilitates accumulation of the agent in a subject's cardiovascular
system. Preferably, the radiolabeled composition of the invention
is administered by arterial or venous injection, and has been
formulated as a sterile, pyrogen-free, parenterally acceptable
aqueous solution. The preparation of such parenterally acceptable
solutions, having due regard to pH, isotonicity, stability, and the
like, is within the skill in the art. A preferred formulation for
intravenous injection should contain an isotonic vehicle such as
Sodium Chloride Injection, Ringer's Injection, Dextrose Injection,
Dextrose and Sodium Chloride Injection, Lactated Ringer's
Injection, or other vehicle as known in the art.
[0208] The amount of radiolabeled composition used for diagnostic
purposes and the duration of the study will depend upon the nature
and severity of the condition being treated, on the nature of
therapeutic treatments which the patient has undergone, and on the
idiosyncratic responses of the patient. Ultimately, the attending
physician will decide the amount of radiolabeled composition to
administer to each individual patient and the duration of the
imaging study.
[0209] The dosage of fluorescent compositions, which include, for
example, photosensitizer compositions, can range from about 0.1 to
about 10 mg/kg. Methods for administering fluorescent compositions
are known in the art, and are described, for example, in U.S. Pat.
Nos. 5,952,329, 5,807,881, 5,798,349, 5,776,966, 5,789,433,
5,736,563, 5,484,803 and by (Sperduto et al. (1991) Int. J. Radiat.
Oncol. Biol. Phys. 21:441-6; Walther et al. (1997) Urology
50:199-206). Such dosages may vary, for example, depending on
whether multiple administrations are given, tissue type and route
of administration, the condition of the individual, the desired
objective and other factors known to those of skill in the art.
Where the fluorescent compositions comprises a photosensitizer
conjugated to an antibody, or a "photoimmunoconjugate," dosages can
vary from about 0.01 mg/m.sup.2 to about 500 mg/m.sup.2, preferably
about 0.1 mg/m.sup.2 to about 200 mg/m.sup.2, most preferably about
0.1 mg/m.sup.2 to about 10 mg/m.sup.2. Ascertaining dosage ranges
is well within the skill of one in the art. For instance, the
concentration of scFv typically need not be as high as that of
native antibodies in order to be therapeutically effective.
Administrations can be conducted infrequently, or on a regular
weekly basis until a desired, measurable parameter is detected,
such as diminution of disease symptoms. Administration can then be
diminished, such as to a biweekly or monthly basis, as
appropriate.
[0210] Compositions of the present invention are administered by a
mode appropriate for the form of composition. Available routes of
administration include subcutaneous, intramuscular,
intraperitoneal, intradermal, oral, intranasal, intrapulmonary
(i.e., by aerosol), intravenously, intramuscularly, subcutaneously,
intracavity, intrathecally or transdermally, alone or in
combination with other pharmaceutical agents. Therapeutic
compositions of photosensitizers are often administered by
injection or by gradual perfusion.
[0211] Compositions for oral, intranasal, or topical administration
can be supplied in solid, semi-solid or liquid forms, including
tablets, capsules, powders, liquids, and suspensions. Compositions
for injection can be supplied as liquid solutions or suspensions,
as emulsions, or as solid forms suitable for dissolution or
suspension in liquid prior to injection. For administration via the
respiratory tract, a preferred composition is one that provides a
solid, powder, or liquid aerosol when used with an appropriate
aerosolizer device. Although not required, compositions are
preferably supplied in unit dosage form suitable for administration
of a precise amount. Also contemplated by this invention are slow
release or sustained release forms, whereby a relatively consistent
level of the active compound are provided over an extended
period.
[0212] Another method of administration is intravascular, for
instance by direct injection into the blood vessel, plaque or
surrounding area.
[0213] Further, it may be desirable to administer the compositions
locally to the area in need of treatment; this can be achieved, for
example, by local infusion during surgery, by injection, by means
of a catheter, or by means of an implant, said implant being of a
porous, non-porous, or gelatinous material, including membranes,
such as silastic membranes, or fibers. A suitable such membrane is
Gliadel.RTM. provided by Guilford Pharmaceuticals Inc.
[0214] Following administration of the fluorescent composition, it
is necessary to wait for the fluorescent composition to reach an
effective tissue concentration at the site of the plaque before
light activation. Duration of the waiting step varies, depending on
factors such as route of administration, tumor location, and speed
of photosensitizer movement in the body. In addition, where
fluorescent composition target receptors or receptor binding
epitopes, the rate of photosensitizer uptake can vary, depending on
the level of receptor expression on the surface of the cells. For
example, where there is a high level of receptor expression, the
rate of binding and uptake is increased. Determining a useful range
of waiting step duration is within ordinary skill in the art and
may be optimized by utilizing fluorescence optical imaging
techniques.
Devices and Methods for Photosensitizer Composition Activation
[0215] Following the waiting step, the fluorescent composition is
activated by photoactivating light applied to the site of the
plaque. This is accomplished by applying light of a suitable
wavelength and intensity, for an effective length of time, at the
site of the plaque. As used herein, "photoactivation" means a
light-induced chemical reaction of a photosensitizer, which
produces a biological effect.
[0216] Target tissues are illuminated, preferably with red light.
Given that red and/or near infrared light best penetrates mammalian
tissues, photosensitizers with strong absorbances in the 600 nm to
900 nm range are optimal for PDT. The suitable wavelength, or range
of wavelengths, will depend on the particular photosensitizer(s)
used. Wavelength specificity for photoactivation depends on the
molecular structure of the photosensitizer. Photoactivation occurs
with sub-ablative light doses. Determination of suitable
wavelength, light intensity, and duration of illumination is within
ordinary skill in the art.
[0217] For photoactivation, the wavelength of light is matched to
the electronic absorption spectrum of the photosensitizer so that
photons are absorbed by the photosensitizer and the desired
photochemistry can occur. Except where the vessels being treated
are very superficial, the range of activating light is typically
between 600 and 900 nm. This is because endogenous molecules, in
particular hemoglobin, strongly absorb light below 600 nm and
therefore capture most of the incoming photons (Parrish et al.,
(1978) Optical properties of the skin and eyes. New York, N.Y.:
Plenum). The net effect would be the impairment of penetration of
the activating light through the tissue. The reason for the 900 nm
upper limit is that energetics at this wavelength may not be
sufficient to produce .sup.1O.sub.2, the activated state of oxygen
which, without wishing to necessarily be bound by any one theory,
is perhaps critical for successful PDT. In addition, water begins
to absorb at wavelengths greater than about 900 nm.
[0218] The effective penetration depth, .delta..sub.eff, of a given
wavelength of light is a function of the optical properties of the
tissue, such as absorption and scatter. The fluence (light dose) in
a tissue is related to the depth, d, as: e.sup.-d/.delta..sub.eff.
Typically, the effective penetration depth is about 2 to 3 mm at
630 nm and increases to about 5 to 6 nm at longer wavelengths
(700-800 nm) (Svaasand and Ellingsen, (1983) Photochem Photobiol.
38:293-299). These values can be altered by altering the biologic
interactions and physical characteristics of the photosensitizer.
In general, photosensitizers with longer absorbing wavelengths and
higher molar absorption coefficients at these wavelengths are more
effective photodynamic agents.
[0219] PDT dosage depends on various factors, including the amount
of the photosensitizer administered, the wavelength of the
photoactivating light, the intensity of the photoactivating light,
and the duration of illumination by the photoactivating light.
Thus, the dose of PDT can be adjusted to a therapeutically
effective dose by adjusting one or more of these factors. Such
adjustments are within ordinary skill in the art.
[0220] The light for photoactivation can be produced and delivered
to the plaque site by any suitable means known in the art.
Photoactivating light can be delivered to the plaque site from a
light source, such as a laser or optical fiber. Preferably, the
photoactivating light is delivered by optical fiber devices that
directly illuminate the plaque site. For example, the light can be
delivered by optical fibers threaded through small gauge hypodermic
needles. Light can be delivered by an appropriate intravascular
catheter, such as those described in U.S. Pat. Nos. 6,246,901 and
6,096,289, which can contain an optical fiber. Optical fibers can
also be passed through arthroscopes. In addition, light can be
transmitted by percutaneous instrumentation using optical fibers or
cannulated waveguides. For open surgical sites, suitable light
sources include broadband conventional light sources, broad arrays
of LEDs, and defocused laser beams.
[0221] Delivery can be by all methods known in the art, including
transillumination. Some photosensitizers can be activated by near
infrared light, which penetrates more deeply into biological tissue
than other wavelengths. Thus, near infrared light is advantageous
for transillumination. Transillumination can be performed using a
variety of devices. The devices can utilize laser or non-laser
sources, (e.g., lightboxes or convergent light beams).
[0222] Where treatment is desired, the dosage of photosensitizer
composition, and light activating the photosensitizer composition,
is administered in an amount sufficient to produce a phototoxic
species. For example, where the photosensitizer composition
includes chlorin.sub.e6, administration to humans is in a dosage
range of about 0.5-10 mg/kg, preferably about 1-5 mg/kg more
preferably about 2-4 mg/kg and the light delivery time is spaced in
intervals of about 30 minutes to 3 days, preferably about 12 hours
to 48 hours, and more preferably about 24 hours. The light dose
administered is in the range of about 20-500 J/cm, preferably about
50-300 J/cm and more preferably about 100-200 J/cm. The fluence
rate is in the range of about 20-500 mw/cm, preferably about 50-300
mw/cm and more preferably about 100-200 mw/cm. There is a
reciprocal relationship between photosensitizer compositions and
light dose, thus, determination of suitable wavelength, light
intensity, and duration of illumination is within ordinary skill in
the art.
[0223] Preferably, the phototoxic species induces apoptosis and not
necrosis of the cells comprising the vulnerable plaque. Lowering
the fluence rate will favor apoptosis (e.g., less than 100 mw/cm,
e.g., 10-60 mw/cm, for chlorin.sub.e6). Determination of a suitable
fluence rate for a photosensitizer composition is within ordinary
skill in the art.
[0224] Where the fluorescent composition comprises a photoactive
dye, the wavelength and power of light can be adjusted according to
standard methods known in the art to control the production of
phototoxic species. Thus, under certain conditions (e.g., low
power, low fluence rate, shorter wavelength of light or some
combination thereof), a fluorescent species is produced from the
photoactive dye and any reactive species produced has a negligible
effect. These conditions are easily adapted to bring about the
production of a phototoxic species. For example, where the
photoactive dye comprises chlorin.sub.e6, the light dose
administered to produce a fluorescent species and an insubstantial
reactive species is less than about 10 J/cm, preferably less than
about 5 J/cm and more preferably less than about 1 J/cm.
Determination of suitable wavelength, light intensity, and duration
of illumination is within ordinary skill in the art.
[0225] In a preferred embodiment, photoactivation can be carried
out using by a specially designed intravascular device that
delivers excitation light to the plaque surface inside the artery
and receives emitted fluorescence or other detectable signals
(e.g., heat or radioactivity) that are transmitted to an analysis
instrument. The same device can optionally be used to deliver
therapeutic light when a fluorescent signal, or other measurable
signal (e.g., heat or radioactivity) is detected.
[0226] FIG. 1 A illustrates a detection/treatment system 100 for
detecting and/or targeting and/or treating vulnerable plaque in
accordance with an embodiment of the invention. As shown in FIG.
1A, detection/treatment system 100 may include a control unit 105
and a detection/treatment unit 110, which may include a light
source/laser 113, and a detection/treatment device 115, which may
include a probe, a catheter, and so forth.
[0227] Control unit 105 may include a power supply, for example,
control unit may be coupled to a power source, for supplying power
to detection/treatment unit 110. Control unit 105 may also include
a computing device having control hardware and/or software for
controlling, based on inputted parameters and/or detected
properties, detection/treatment unit 110, light source/laser 113
and detection/treatment device 115.
[0228] FIG. 1B is a diagram illustrating a configuration of control
unit 105 in accordance with an embodiment of the invention. As
shown in FIG. 1B, control unit 105 may comprise a computing device
125, which may be a general purpose computer (such as a PC),
workstation, mainframe computer system, and so forth. Computing
device 125 may include a processor device (or central processing
unit "CPU") 130, a memory device 135, a storage device 140, a user
interface 145, a system bus 150, and a communication interface 155.
CPU 130 may be any type of processing device for carrying out
instructions, processing data, and so forth. Memory device 135 may
be any type of memory device including any one or more of random
access memory ("RAM"), read-only memory ("ROM"), Flash memory,
Electrically Erasable Programmable Read Only Memory ("EEPROM"), and
so forth. Storage device 140 may be any data storage device for
reading/writing from/to any removable and/or integrated optical,
magnetic, and/or optical-magneto storage medium, and the like
(e.g., a hard disk, a compact disc-read-only memory "CD-ROM",
CD-ReWritable "CD-RW", Digital Versatile Disc-ROM "DVD-ROM",
DVD-RW, and so forth). Storage device 140 may also include a
controller/interface (not shown) for connecting to system bus 150.
Thus, memory device 135 and storage device 140 are suitable for
storing data as well as instructions for programmed processes for
execution on CPU 130. User interface 145 may include a touch
screen, control panel, keyboard, keypad, display or any other type
of interface, which may be connected to system bus 150 through a
corresponding input/output device interface/adapter (not shown).
Communication interface 155 may be adapted to communicate with any
type of external device, including detection/treatment unit 110.
Communication interface 155 may further be adapted to communicate
with any system or network (not shown), such as one or more
computing devices on a local area network ("LAN"), wide area
network ("WAN"), the internet, and so forth. Interface 155 may be
connected directly to system bus 150, or may be connected through a
suitable interface (not shown). Control unit 105 may, thus, provide
for executing processes, by itself and/or in cooperation with one
or more additional devices, that may include algorithms for
controlling detection/treatment unit 110 in accordance with the
present invention. Control unit 105 may be programmed or instructed
to perform these processes according to any communication protocol,
or programming language on any platform. Thus, the processes may be
embodied in data as well as instructions stored in memory device
135 and/or storage device 140 or received at interface 155 and/or
user interface 145 for execution on CPU 130.
[0229] Referring back to FIG. 1A, detection/treatment unit 110 may
be a handheld device, an automated apparatus, and the like. As
shown in FIG. 1A, detection/treatment device 115 may be inserted
and extended into a blood vessel 120, such as an artery, in tissue
125. Detection/treatment device 115 may be a handheld device, an
automated apparatus, and the like. It is further noted that the
elements of detection/treatment system 100 may be integrated into a
single physical unit or may comprise any number of discrete units,
such that any number of these elements or the functionality
thereof, may be incorporated into a physical device. As will be
described in further detail below, detection/treatment device 115
may include a number of light delivery elements for delivering
detected light from targeted plaque, delivering therapeutic light,
and/or delivering detection/excitation light.
[0230] In accordance with an embodiment of the invention, light
source 113 may include a pulse blue laser for delivering detection
or excitation light via detection/treatment device 115. Depending
on the dye and/or excitation effect on target plaque as described
above, reflected and/or emitted light from the target plaque may
include light with a particular wavelength and/or frequency, which
may then be detected through detection/treatment device 115. A
large number of fluorescent probes (e.g., photosensitizers,
fluorescent dyes or photoactive dyes) and methods of use thereof
(e.g., excitation and emission wavelengths), are described in the
Molecular Probes, Inc. catalog, (Handbook of Fluorescent Probes and
Research Chemicals, 6.sup.th Edition by Richard Haugland), the
contents of which are hereby incorporated by reference.
[0231] In accordance with an embodiment of the invention where in
the fluorescent composition or photosensitizer composition includes
chlorine6, detection/excitation light may include a wavelength of
337 nm (for example, nitrogen laser), therapeutic light may include
a wavelength of 405 nm (for example, pump dye laser), and light or
fluorescence emitted from target plaque as a result of excitation
by detection/excitation light may include a wavelength of 666-668
nm. The power of detection/excitation light may, for example, be
adjusted in accordance with the specific excitation or emission
wavelength of the particular fluorescent or photosensitizer
composition used. The power of detection/excitation light may, for
example, be adjusted in accordance with a size and/or dimension of
blood vessel 120. The power of therapeutic light may, for example,
be adjusted in accordance with a size and/or dimension of blood
vessel 120, and/or the level of light detected from target
plaque.
[0232] In accordance with an embodiment of the invention,
detection/treatment system 100 may include a number of
configurations and instruments. Algorithms that are designed for
different types of procedures, configurations and/or instruments
may be included for control unit 105.
[0233] It is noted that detection/treatment system 100 may be
controlled remotely. For example, the link between control unit 105
and detection/treatment unit 110 may be a remote link (wired or
wireless) providing control unit 105 remote control over light
source 113 and detection/treatment device 115.
[0234] While the above exemplary detection/treatment system 100 is
illustrative of the basic components of a system suitable for use
with the present invention, the architecture shown should not be
considered limiting since many variations of the hardware
configuration are possible without departing from the present
invention.
[0235] The present invention is additionally described by way of
the following illustrative, non-limiting Examples, that provide a
better understanding of the present invention and of its many
advantages.
[0236] As described before, target plaque may accumulate on the
wall of blood vessels, e.g. arteries, and the like. Thus,
detection/treatment device 115 embodying the present invention may
include a probe/catheter and the like, as described below, which
may include a number of elements for detecting the target plaque on
the wall of these blood vessels, distinguishing the target plaque
from non-target plaque and/or treating the target plaque without
obstructing the blood flow through these vessels.
[0237] FIGS. 2A, 2B, 2C, 2D, 2E and 2F are diagrams showing a
probe/catheter 200 in accordance with an embodiment with the
present invention. As shown in FIG. 2A, probe/catheter 200 may
include an external unit 202 and an extendible internal unit, which
may include a number of light delivery element(s) 205 and light
deflection element(s) 210 and a tip 215. As an example, external
unit 202 may include any plastic and/or metallic material (e.g.,
nitinol alloy) and the like. FIG. 2A illustrates probe/catheter 200
with its internal unit retracted within and extended from external
unit 202, and FIG. 2B illustrates probe/catheter 200 with its
internal unit extended and deployed. In accordance with an
embodiment of the invention, the internal unit may be extended and
deployed to detect target plaque, then retracted to move
probe/catheter 200 to a different area within, say, blood vessel
120. For example, probe/catheter 200 may be used to scan blood
vessel 120 where probe/catheter 200 is moved along blood vessel 120
and the internal unit is extended every one to six millimeters to
make a detection. A guidewire 223 may be used to guide
probe/catheter 200 along blood vessel 120 and/or extend/retract the
internal unit (e.g., light delivery element(s) 205 and light
deflection elements 210, and so forth) from/into external unit 202.
As an example, guidewire 223 may include any plastic and/or
metallic material (e.g., nitinol alloy) and the like. Light
deflection element(s) 210 may include a smooth surface for
contacting the wall of blood vessel 120, thus allowing detection
while probe/catheter 200 is being moved. Detection may be made
without contacting the wall or probe/catheter 200 may also be
stopped to make such a detection. Probe/catheter 200 may include
four light delivery elements 205, each including a light deflection
element 210. Each of the four light delivery elements 205 may be
disposed such that the corresponding light deflection elements 210
form a circumference separated by 90 degrees, as shown by the
cross-sectional views in FIGS. 2C and 2D. It is noted that
probe/catheter 200 may include any number of light delivery
element(s) 205 (and light deflection element(s) 210) separated by a
corresponding angle around a circumference for covering a divided
area of the surrounding wall of blood vessel 120. Probe/catheter
200 may also be rotatable to cover the circumference of blood
vessel 120. In accordance with a preferred embodiment of the
invention, probe/catheter 200 may include three to six light
delivery elements 205 (and light deflection elements 210). It is
noted, of course, that light delivery elements 205 may be split
from a single element connected to detection/treatment unit 110 or
they may be separately connected to detection/treatment unit
110.
[0238] As will be described in further detail below, light
deflection element(s) 210 may deflect external light received from
blood vessel 120 into light delivery element(s) 205, which may then
deliver the received light to detection/treatment unit 110 and/or
control unit 105 for analysis. Light deflection element(s) 210 may
also deflect detection/excitation light, which may be delivered
from detection/treatment unit 110 through light delivery element(s)
205, and shine the detection/excitation light onto a target area in
blood vessel 120. And so, reflected light and/or light emitted from
excited target plaque may be received as described above. Depending
on the dye and/or excitation effect on target plaque as described
before, the target plaque may reflect and/or emit light having a
particular wavelength and/or frequency. Thus, target plaque may be
identified and located by detecting and identifying light having
such a particular wavelength and/or frequency from the light
received from blood vessel 120.
[0239] Light delivery element(s) 205 may include an optical fiber
for delivering light received at its corresponding light deflection
element(s) 210 to treatment unit 110 and/or control unit 105. Light
delivery element(s) 205 may also deliver detection/excitation light
from light source 113 to its corresponding light deflection
element(s) 210 where it is deflected and shone onto blood vessel
120. As shown in FIG. 2A, light delivery element(s) 205 may extend
to and joined at a tip 215.
[0240] As shown in FIG. 2B, light delivery element(s) 205 may move
outward so that light deflection element(s) 210 are moved towards
the surrounding wall of blood vessel 120, thus allowing better
plaque detection. In accordance with an embodiment of the
invention, light delivery element(s) 205 may include a rigid and/or
spring-like structure, for example, a plastic structure, such that
the structure expands when extended, as shown in FIG. 2B, and may
be compressed within external unit 202 when retracted, as shown in
FIG. 2A. The rigid structure may include any elastic material so
that the structure expands to substantially the same size and shape
every time it is extended as shown in FIG. 2B.
[0241] In accordance with an embodiment of the invention,
probe/catheter 200 may include a vessel (or "balloon") 220 that may
be expanded by filling it with a fluid. Thus, when extended as
shown in FIG. 2B, vessel 220 may be filled with fluid and expanded,
pushing light deflection element(s) 210 towards the surrounding
wall of blood vessel 120. The fluid may be any non-toxic fluid,
such as saline and so forth. As an example, vessel 220 may include
any elastic material, such as rubber or latex, and the like.
Control unit 105 and/or detection/treatment unit 110 may control
fluid flow to and from vessel 220 so that fluid is delivered
thereto when probe/catheter 200 is extended, and drained when
probe/catheter 200 is retracted. Advantageously, the amount of
fluid may be controlled so as to fit the size of the surrounding
blood vessel 120. In other words, less fluid may be delivered if
blood vessel 120 is relatively small and more fluid may be
delivered if blood vessel 120 is relatively large. Thus, light
deflection element(s) 210 may be moved towards the wall of a blood
vessel 120 of any size, while preventing light deflection
element(s) 210 from being pressed against the wall of a smaller
blood vessel 120.
[0242] FIGS. 2C and 2D are diagrams showing cross-sectional views
of FIGS. 2A and 2B, respectively. When expanding vessel 220 or
otherwise moving light deflection element(s) 210 towards the wall
of blood vessel 120, it is important that blood flow through blood
vessel 120 be unhindered. Therefore, in accordance with an
embodiment of the invention, vessel 220 may include a number of
rigid element(s) 225 so that only a particular portion of vessel
220 expands when filled with fluid. As an example, rigid element(s)
225 may include a rigid material, for instance any plastic and/or
metallic material (e.g., nitinol alloy), and the like. As shown in
FIGS. 2C and 2D, vessel 220 may include four rigid element(s) 225,
such as plastic ribbings, and the like. As shown in FIG. 2D, rigid
element(s) 225 may hold vessel 220 in place where only regions of
vessel 220 that are adjacent light delivery element(s) 205 and
light deflection element(s) 210 may expand outward. Therefore,
vessel 220 does not substantially block blood vessel 120 when it is
expanded. Light deflection element(s) 210 may, thus, be moved
outward to the wall of blood vessel 120 without obstructing blood
flow.
[0243] FIGS. 2E and 2F illustrate cross-sectional views of
probe/catheter 200 in accordance with an embodiment of the
invention. As shown in FIGS. 2E and 2F, vessel 220 may include an
isolated chamber corresponding to a particular light deflection
element 210. Therefore, each of any number of particular light
deflection element(s) 210 may correspond to such a chamber in
vessel 220 so that element(s) 210 can be individually moved towards
and away from the wall of blood vessel 120, by individually
inflating and deflating each chamber. For example, as shown in FIG.
2F, a chamber 230 may be individually deflated (i.e., drained of
fluid), in the event that therapeutic light may be directed to the
corresponding region on blood vessel 120, say, from tip 215, in the
event that the corresponding region need not be detected or
monitored for any reason, or to fit to a particular dimension of a
blood vessel.
[0244] FIGS. 3A, 3B and 3C are diagrams illustrating a
probe/catheter 300 in accordance with an embodiment of the
invention. Probe/catheter 300 as shown in FIGS. 3A and 3B is
similar to probe/catheter 200 shown in FIGS. 2A and 2B,
respectively, except that probe/catheter 300 may include only one
light delivery element 205 and corresponding light deflection
element 210. Advantageously, the cross-sectional area of
probe/catheter 300, when extended and deployed, may be further
reduced. For example, as shown in FIG. 3C, probe/catheter 300 may
include only one prong compared to the four prongs shown in FIG. 2D
for probe/catheter 200. As a result, blood flow obstruction may be
further reduced. Probe/catheter 200 may include a platform 305 for
supporting, say, vessel 220. As an example, platform 305 may
include a rigid material, for instance any plastic and/or metallic
material (e.g., nitinol alloy), and the like, so that it is held in
place while vessel 220 expands and pushes light deflection element
210 outward. As mentioned before, light delivery element 205 may
include a rigid structure that pushes outward when extended from
external unit 202. Platform 305 may support such a structure.
[0245] FIGS. 4A and 4B show a probe/catheter 400 in accordance with
an embodiment of the invention. As shown in FIGS. 4A and 4B,
probe/catheter 400 may include light delivery elements 205 disposed
on a rigid structure that is compressed when enclosed in external
unit 202, as shown in FIG. 4A, and expands when extended, as shown
in FIG. 4B. As described before, the rigid structure may include
any elastic material so that the structure expands to substantially
the same size and shape every time it is extended as shown in FIG.
4B. As an example, the rigid structure may include any plastic
and/or metallic material (e.g., nitinol alloy) and the like.
[0246] FIGS. 5A and 5B are diagrams illustrating light delivery
element 205 and light deflection element 210 in accordance with
respective embodiments of the invention. As shown in FIG. 5A, light
deflection element 210 may include a reflective surface 505 and/or
a refractive element 510 for deflecting light from a target area
back to detection/treatment unit 110 through light delivery element
205, and/or deflecting detection/excitation light from light source
113 to the target area. In accordance with an embodiment of the
invention, light source 113 may include a light source for
therapeutic light having a difference wavelength and/or frequency.
Thus, light deflection element 210 may deflect only
detection/excitation light, while allowing therapeutic light to
pass through. Referring back to FIGS. 2A and 2B, the passed through
therapeutic light may be deflected out at tip 215 for effecting
treatment on the surrounding wall of blood vessel 120.
Probe/catheter 200 may further be extended and/or retracted
partially when effecting treatment so as to ensure that therapeutic
light from tip 215 reaches the areas covered by light deflection
element(s) 210.
[0247] FIGS. 5B illustrates light deflection element 210 that may
be used in probe/catheter 400, as shown in FIGS. 4A and 4B, in
accordance an embodiment of the invention. As shown in FIG. 5B, a
therapeutic light deflection unit 515 may be placed adjacent light
deflection element 210. Since it is advantageous to target
therapeutic light more broadly to cover tissue surrounding the
detected plaque, therapeutic light deflection unit 515 may include
a refractive material for spreading or diffusing the therapeutic
light in all directions to cover the surrounding wall of blood
vessel 120. In accordance with an embodiment of the invention,
therapeutic light deflection unit 515 may also include a reflective
element 520 for targeting the therapeutic light to a general
direction or a particular area. Thus, referring back to FIGS. 4A
and 4B, a therapeutic light deflection unit 515 may be disposed at
the end, or tip, of each light deflection element 210. In
accordance with an embodiment of the invention,
detection/excitation light and therapeutic light may be carried on
separate light delivery elements.
[0248] FIGS. 6A, 6B, and 6C illustrate a probe/catheter 600 in
accordance with an embodiment of the present invention. As shown in
FIG. 6A, probe/catheter 600 may include a detector 605, such as a
scintillation detector, and the like, for detecting emitted and/or
reflected light, radioactive signals (e.g., gamma rays, beta rays,
and so forth), nuclear isotopes, radio frequency/microwave signals,
magnetic fields, electric fields, temperature (e.g., heat),
vibration, and so forth. By detecting any one or more of the
foregoing, target plaque may be identified and/or located from
surrounding plaque/tissue. As further shown in FIG. 6A,
probe/catheter 600 may also include a therapeutic light deflector
610, such as a diffusing fiber, and the like, for diffusing
therapeutic light to surrounding plaque/tissue. As shown in FIG.
6B, detector 605 may be independently retracted so that therapeutic
light may be directed to the general direction or particular area
where target plaque/tissue is detected. Furthermore, as shown in
FIG. 6C, therapeutic light deflector 610 may include a reflective
element 615, such as a shield, and the like, to block therapeutic
light from diffusing to a non-target direction. For example, after
detector 605 detects target plaque/tissue, it may be retracted and
therapeutic light deflector 610 and reflective element 615 may
diffuse therapeutic light only to the general direction and/or
target area covered by detector 605. In accordance with an
embodiment of the invention, probe/catheter 600 may be rotatable
in, say, blood vessel 120 so that detector 605 and therapeutic
light may be directed in any direction therewithin.
[0249] The present invention is additionally described by way of
the following illustrative, non-limiting Examples, that provide a
better understanding of the present invention and of its many
advantages.
EXAMPLES
Example 1
Preparation and Purification of Photosensitizer Compositions
[0250] A photosensitizer composition comprising chlorin.sub.e6
("c.sub.e6") coupled to maleylated-albumin) was prepared for
optimal targeting to macrophages of a vulnerable plaque animal
model system.
Results
[0251] Four photosensitizer compositions were studied (i.e., two
BSA-c.sub.e6 conjugates and their maleylated counterparts). The
N-hydroxy succinimide (NHS) ester of c.sub.e6was prepared by
reacting approximately 1.5 equivalents of dicyclohexylcarbodiimide
and approximately 1.5 equivalents of NHS with approximately 1
equivalent of c.sub.e6 (Porphyrin Products, Logan, Utah) in dry
DMSO. After standing in the dark at room temperature for
approximately 24 hours, the NHS ester was frozen in aliquots for
further use. BSA (Sigma Chemical Co, St Louis, Mo.) (approximately
2.times.50 mg) was dissolved in NaHCO.sub.3 buffer (0.1 M, pH 9.3,
approximately 3 ml), and approximately 30 .mu.l and approximately
120 .mu.l of c.sub.e6-NHS ester added to respective tubes with
vortex mixing. After standing in the dark at room temperature for
approximately 6 hours, the crude conjugate preparations were each
divided into two approximately equal parts. One portion of each of
the conjugate preparations was maleylated by adding solid maleic
anhydride (approximately 20 mg) to the protein preparation in
portions and with vortex mixing, and by adding saturated
NaHCO.sub.3 solution as needed to keep the pH above approximately
7.0 (Takata et al. (1989) Biochim. Biophys. Acta 984:273). The
reaction mixture was allowed to stand at room temp in the dark for
approximately 3 hours (FIG. 7). Unmodified BSA was also maleylated
to act as a control and as a competitor for the cellular uptake of
conjugates.
[0252] Crude conjugate preparations (approximately 5 mg/ml) were
added to approximately 10.times. volume of acetone (ACS grade)
slowly at approximately 4.degree. C., and were kept at
approximately 4.degree. C. for approximately 6 hours, followed by
centrifugation at about 4000.times.g for approximately 15 minutes
at about 4.degree. C. The supernatant was removed and the pellet
again suspended in approximately the same volume of acetone and the
centrifugation repeated. After each precipitation step the
preparation was monitored by thin layer chromatography (TLC).
Approximately five precipitation steps were necessary to completely
remove non-covalently bound chlorin species. Finally, the pellet
was dissolved in approximately 2 ml PBS and dialyzed approximately
twice against 20 L PBS overnight to remove traces of acetone.
[0253] Sephadex G50 column chromatography was carried out by
applying the reaction mixture from conjugation of approximately 50
mg BSA with approximately 5 mg c.sub.e6-NHS ester to a 50.times.1
cm Sephadex column that was eluted with PBS at about 4.degree. C.
The absorbance of the eluted fractions was monitored at 400 nm and
at 280 nm.
[0254] A problem that can be encountered in the preparation of
covalent conjugates of tetrapyrrole photosensitizer (PS) with
proteins is the tendency of the dye to form tightly bound
non-covalent complexes, as well as conjugates. These mixtures can
be difficult to separate into pure conjugate and non-bound dye.
This is illustrated by the attempted use of a Sephadex G50 column
to separate the BSA-c.sub.e6 conjugate from unreacted c.sub.e6-NHS
ester and its subsequent reaction products. Monitoring of the
eluted fractions at 400 nm and at 280 nm showed a single peak that
contained both c.sub.e6 and protein. However, when the material
obtained from combining the fractions was examined by TLC, as shown
in FIG. 8A, it was apparent that there was a considerable amount of
unbound dye present. Lane 1 on the TLC shows the single peak
isolated from the size exclusion column and demonstrates that there
was still considerable unbound c.sub.e6 present as a fast running
spot. When this material was used in cell-uptake experiments, it
was difficult to distinguish receptor targeting between J774 and
EMT-6 cell due to indiscriminate uptake of unbound c.sub.e6 by both
receptor-positive and receptor-negative cells. Likewise, lane 3
shows the crude mixture after maleylation and that there was
unbound c.sub.e6 present.
[0255] Therefore, the conjugates were purified using an acetone
precipitation that allowed the lipophilic c.sub.e6 species to be
retained in the acetone supernatant and the precipitated conjugates
to be redissolved in a purified form. The sodium dodecyl sulfate
polyacrylamide (SDS-PAGE) gels were viewed by fluorescence imaging
to localize the c.sub.e6 after staining with Coomassie Blue. FIG.
8B shows the corresponding fluorescence and Coomassie images of
BSA, BSA mixed with free c.sub.e6 and conjugates (BSA-c.sub.e6 1
and mal-BSA-c.sub.e61) after Sepahadex column chromatography, but
before acetone precipitation. The mixture of BSA and c.sub.e6
(lanes 2a and 2b) showed that no fluorescence is retained by the
protein band on the gel, thus demonstrating that a fluorescent band
localizing with the protein is evidence of covalent conjugation.
The lanes of the conjugates (3a and 3b, 4a and 4b) show that a
fluorescent band running at the gel front remained after Sephadex
chromatography.
[0256] The efficiency of the purification by acetone precipitation
of the conjugates was confirmed by the gel electrophoresis images
shown in FIG. 8C. It can be seen that the fast running fluorescent
band disappeared from both the BSA-c.sub.e6 and the
mal-BSA-c.sub.e6 (lanes 2c and 2d, 3c and 3d), while the TLC also
showed the disappearance of the fast running spot (FIG. 8A, lanes 2
and 4)
[0257] The concentrations of the constituents in the conjugates
and, hence the substitution ratios, were measured by absorbance
spectroscopy. An aliquot of the conjugate was diluted in
approximately 0.1 M NaOH/1% SDS and absorbance between 240 nm and
700 nm scanned. The extinction coefficient of BSA at 280 nm is
approximately 47000 cm.sup.-M.sup.-1 (Markwell et al. (1978) Anal
Biochem 87:206) while the extinction coefficient of c.sub.e6 at 400
nm is approximately 150000 cm.sup.-1M.sup.-1. Thin layer
chromatography was performed on silica gel plates (Polygram SIL
G/UV254, Macherey Nagel, Duren, Germany). The chromatograms were
developed with an approximately 1:1 mixture of approximately 10%
aqueous ammonium chloride and methanol, and spots were observed
with fluorescence and absorbance imaging. SDS-PAGE was carried out
essentially according to the methods known in the art (Laemmli
(1970) Nature 227:680). Gradients of 4-10% acrylamide were used in
a non-reducing gel and c.sub.e6 was localized on the gel by a
fluorometer (excitation at 400-440 nm bandpass filter, emission
scanned from 580-720 nm longpass filter (Chemilmager 4000, Alpha
Innotech Corp, San Leandro, Calif.). Proteins were localized by
Coomassie blue staining.
[0258] The UV-visible absorption spectra of the purified
mal-BSA-c.sub.e6 conjugates with the two substitution ratios
measured at approximately equal protein concentrations are shown in
FIG. 9, together with free c.sub.e6 at approximately the same
concentration as was present in mal-BSA-c.sub.e6 2. Similar spectra
were obtained for BSA-c.sub.e6 1 and 2. Using the values for molar
extinction coefficients of BSA at 280 nm of approximately 47000
cm.sup.-1M.sup.-1 (Markwell et al (1978) Anal Biochem 87:206) and
c.sub.e6 at 400 nm of approximately 150000 cm.sup.-1M.sup.-1, and
correcting for the small absorbance of c.sub.e6 at 280 nm, then the
substitution ratios can be calculated to be mal-BSA-c.sub.e6 1
ratio equals approximately 1 protein to approximately 1 dye, and
mal-BSA- c.sub.e6 2, ratio equals approximately 1 protein to
approximately 3 dye.
Example 2
Macrophage-Targeting of Photosensitizers
[0259] The photosensitizer composition comprising chlorin.sub.e6
coupled to maleylated-albumin described in Example 1 was shown to
accumulate in the macrophage-rich plaques of an animal model system
that are analogous to vulnerable plaques in humans. Thus, methods
of the present invention provide highly specific intravascular
detection and therapy of vulnerable plaques.
Cell Culture
[0260] J774.A1 (J774) and RAW 264.7 mouse macrophage-like cell
lines, together with EMT-6 mouse mammary fibrosarcoma cells, were
obtained from ATCC (Rockville, Md.). Cells were grown in RPMI 1640
media containing HEPES, glutamine, 10% fetal calf serum (FCS), 100
U/ml penicillin and 100 .mu.g/ml streptomycin. They were passaged
by washing with phosphate buffered saline (PBS) without Ca.sup.2+
and Mg.sup.2+ and by adding trypsin-EDTA to the plate for 10
minutes at 37.degree. C.
Rabbits
[0261] Male New Zealand white rabbits weight 2.5-3.0 kg (Charles
River Breeding Lab) were maintained on a 2% cholesterol-6% peanut
oil diet (ICN) for 6 weeks.
Results
[0262] For cellular uptake studies, cells were grown to
approximately 90% confluency in twenty-four well plates and the
conjugate or photosensitizer was added in about 1 ml medium
containing approximately 10% serum to each well. The concentration
range for the conjugates and free c.sub.e6 was between
approximately 0.5 and 4 .mu.M c.sub.e6 equivalent and the
incubation time was approximately 3 hours. After incubation at
37.degree. C., the medium was removed and cells were washed about
three times with approximately 1 ml sterile PBS and incubated with
approximately 1 ml trypsin-EDTA for about 20 minutes (OVCAR-5) or
60 minutes (J774). The cell suspension was then removed and
centrifuged (about 5 minutes at approximately 250.times.g). The
trypsin supernatant was aspirated and retained and the pellets
(frequently visibly fluorescent under long wave UV) were dissolved
in about 1.5 ml of approximately 0.1M NaOH/1% SDS for at least
about 24 hours to give a homogenous solution. The trypsin
supernatant was checked for the presence of fluorescence to
quantify any surface binding which might easily be removed by
trypsin. The fluorescence was measured using an excitation
wavelength of 400 nm and the emission scanned from 580 to 700 nm in
order to calculate the peak area (.lamda..sub.max=664 nm). A series
of dilutions in approximately 1.5 ml 0.1M NaOH/1% SDS of known
concentrations of each separate conjugate and photosensitizer was
scanned for fluorescence as above in order to prepare calibration
curves to allow for quantitation of the c.sub.e6 by conversion of
the measured peak areas into mol c.sub.e6 equivalent. The protein
content of the entire cell extract was then determined by a
modified Lowry method (Marwell et al (1978) Anal Biochem 87:296)
using BSA dissolved in approximately 0.1M NaOH/1% SDS to construct
calibration curves. Results were expressed as mol of c.sub.e6 per
mg cell protein. For measuring the cellular uptake at 4.degree. C.,
pre-cooled growth media was used and the plates with cells were
cooled to about 4.degree. C. in an ice-bath for approximately 20
minutes before the addition of photosensitizer solutions as well as
after the addition. The incubation was carried out in the normal
atmosphere in the dark (e.g., plates wrapped in aluminum foil).
[0263] Cells were seeded in 24 well plates, at densities of
approximately 100,000 cells in about 1 ml medium. After about 24
hours, the cells were given about 1 ml fresh medium containing 10%
serum and a specific conjugate or free c.sub.e6 (c.sub.e6
equivalent concentration of approximately 4 nmoles per well) and
incubated for about 3 hours at 37.degree. C. Immediately prior to
illumination, the cells were washed about 3 times with PBS with
Mg.sup.2+/Ca.sup.2+ and the wells were replenished with
approximately 1 ml medium containing HEPES and about 10% FCS. Light
(660 nm) was delivered from beneath the wells from a diode laser at
a fluence rate of about 50 mW/cm.sup.2 via a fiber optic coupled
microscope objective. Wells were illuminated in blocks of four
defined by a black mask placed beneath the 24 well plate. Fluences
were about 2, 5, and 10 J/cm2. After completion of illumination,
the dishes were returned to the incubator for a further
approximately 24 hour incubation. Cell survival was determined by
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay, which measures mitochondrial dehydrogenase activity.
It has been extensively used for measuring viability of cell
cultures after PDT and has been shown to have close correlation
with colony forming assays (McHale et al (1988) Cancer Letters
41:315). Approximately twenty-four hours post illumination, the
cells were given fresh media and about 100 .mu.L MTT (5 mg/ml)
solution was added to each well and cells were incubated at
37.degree. C. After approximately 1 hour incubation, the
supernatant medium was gently aspirated and about 1 ml of DMSO was
added to lyse the cells and dissolve the deep blue formazan. Plates
were gently shaken on an orbital shaker in the dark for
approximately 15 min to complete the dissolution of any formazan
crystals and the blue DMSO solution was transferred to 96 well
plates (about 200 .mu.l per well, 5 wells per well of 24-well
plate). Absorbance was read on an automated plate reader (Model
2550 EIA, Bio-Rad Laboratories, Hercules, Calif.) at 570 nm. Data
points were the average of 3 wells of the 24 well plate (15 wells
of 96 well plate).
[0264] The role of scavenger receptors in the uptake of these
conjugates was tested by measuring the reduction in the cellular
content of photosensitizer produced by competing the uptake with a
ligand known to be recognized by the scavenger receptor. The
reduction in cellular uptake was then related to protection of the
cells from phototoxicity. Increasing amounts of unlabeled mal-BSA
were added simultaneously with the conjugates to J774 and OVCAR-5
cells and incubated for about 3 hours. Approximately 0, 50, 100,
and 200 .mu.g/ml mal-BSA were used, representing a range of about
0.25 to 3 fold molar excess of the BSA contained in approximately 4
.mu.M BSA-c.sub.e6 or mal-BSA-c.sub.e6. The cellular uptakes and
phototoxicities were measured as described above.
[0265] Mouse-macrophage cells (J774 or RAW264.7) took up more than
ten times as much dye as non-target EMT-6 cells and, upon
illumination with modest levels of red light, were killed
approximately 1000 times as much. The maleylated conjugates had
greater macrophage selectivity and, therefore, higher phototoxicity
than their non-maleylated counterparts (FIG. 10).
[0266] After 1 week on the peanut oil diet, the abdominal aorta was
denuded of endothelium by a modified Baumgartener technique.
Briefly, each animal was anesthetized with a mixture of ketamine
and xylazine and the right femoral artery was isolated.
Subsequently, a 4F Fogarty embolectomy catheter was introduced via
arteriotomy and advanced under fluoroscopic guidance to the level
of the diaphragm. The balloon was then inflated to 3 psi above
balloon inflation pressure and three passes were made down the
abdominal aorta with the inflated catheter. The femoral artery was
subsequently ligated and the wound closed.
[0267] For fluorescence localization within ex vivo aortas, aortic
segments were cut open and flattened and the luminal side examined
by spectrofluorometry using either a fiber-bundle based double
monochromator spectrofluorimeter (Skin Scan, Spex Figure), where
emission spectra (excitation 400 nm, emission 580-720 nm) was
collected about every 3 mm across the entire area of the exposed
intimal surface, or an optical multichannel analyzer (FIG. 11).
[0268] For confocal fluorescence microscopy, selected parts of the
aortas were snap frozen in liquid nitrogen and approximately 10-20
.mu.m frozen sections were prepared. These sections underwent laser
scanning confocal fluorescence microscopy to detect the tissue
distribution of the c.sub.e6. The red intracellular fluorescence
from c.sub.e6 together with green tissue auto-fluorescence was
imaged in the cells in 10 .mu.m frozen sections. Sections were
examined with a laser scanning confocal fluorescence microscope. A
Leica DMR confocal laser fluorescence microscope (Leica Mikroskopie
und Systeme GmbH, Wetzler, Germany) (excitation 488 nm argon laser)
and 4.times.-40.times. air immersion lens or a 100.times. oil
immersion objective was used to image at a resolution of
1024.times.1024 pixels. Two channels collected fluorescence signals
in either the green range (580 nm dichroic mirror plus 530 nm
(+/-10 nm) bandpass filter) or the red range (580 nm dichroic
mirror plus 590 nm longpass filter) and were displayed as false
color images. These channels were overlaid using TCS NT software
(Version 1.6.551, Leica Lasertechnik, Heidelberg, Germany) to allow
visualization of overlap of red and green fluorescence. These
sections were also stained by immunohistochemistry using macrophage
specific monoclonal antibodies and conventional H&E staining.
Other parts of normal and atherosclerotic aorta were cut into small
pieces, weighed and dissolved in sodium hydroxide/SDS and the
tissue content of c.sub.e6 was determined by spectrofluorimetry as
previously described (Hamblin et al (2000) Br. J. Cancer
83:1544).
[0269] FIG. 12 shows an analysis of aortic sections from rabbits
injected with or without conjugate (approximately 2 mg/kg in PBS)
about 24 hours after injection of the conjugate. Row 1 shows
confocal fluorescence micrographs of frozen aortic sections
(Red=chlorin.sub.e6, Green=elastic lamina auto-fluorescence). Row 2
shows fluorescence emission spectra (excitation=400 nm) of initmal
surface of aortic segments ex vivo. Row 3 shows Hematoxylin and
eosin staining of formalin fixed paraffin embedded aortic segments.
Row 4 shows Verhoeff's elastic tissue stain. The confocal
micrographs showed red fluorescence from the PS (c.sub.e6) and
green auto-fluorescence principally from the elastic lamina of the
arteries. Column 1 shows an atherosclerotic rabbit with no
injection of conjugate. There was no red c.sub.e6 fluorescence in
the tissue section, nor any fluorescence signal from the intimal
surface. Column 2 shows a normal non-atherosclerotic rabbit
injected with conjugate. There is a small amount of red
fluorescence visible in the adventitia rather than the intima in
the fluorescence micrographs, and a small fluorescence emission
signal from the intimal surface. Column 3 shows an atherosclerotic
rabbit injected with conjugate. There was a large amount of red
fluorescence visible in the plaque and this gave a corresponding
large fluorescence emission signal from the intimal surface.
[0270] The intimal fluorescence signal was measured from different
sections of aortas from atherosclerotic and normal rabbits. The
areas of the abdominal aorta that received balloon injury developed
greater amounts of plaque than the neighboring thoracic and lower
abdominal aortas. The results from the intimal fluorescence
measurements were confirmed by extracting sections of the aortas
and measuring fluorescence with a spectrofluorimeter that gives a
measure of the number of c.sub.e6 molecules in the tissue
sections.
[0271] FIG. 13 shows a significant fluorescent signal from the
intimal surface (determined by Skin Scan) in all sections from
atherosclerotic rabbits compared to the corresponding sections of
aorta from normal rabbits injected with conjugate, but particularly
higher in the sections from the balloon-injured areas. The section
1 depicts thoracic aorta, section 2 depicts upper abdominal aorta
below the diaphragm, section 3 depicts mid-abdominal aorta, section
4 depicts lower abdominal aorta and section 5 depicts pelvic aorta
just above bifurcation. At least 6 separate measurements were taken
from each artery segment. By the nature of the balloon injury,
sections 3 and 4 generally sustained a more severe endothelial
injury than other sections and hence developed more severe
atherosclerosis. These plaques are extremely rich in marcophages
and therefore, are most analogous to vulnerable plaques in humans.
Such lesions represent the animal model system used by those of
skill in the art to study the features of vulnerable plaques. The
signal from atherosclerotic rabbit section 3 was greater than
normal control section 3 (p<0.0005) and the signal from
atherosclerotic section 4 was greater than normal control section 4
(p<0.005).
[0272] The second measurement of intimal surface fluorescence was
made by the OMA-LIF system described above. At least 15 separate
fluorescence measurements were taken from each artery segment. In
addition, the iliac artery through which the balloon was passed
also sustained an injury due to its relatively small diameter
compared to aortic section 5 and, therefore, developed
atherosclerosis compared to the uninjured iliac artery. FIG. 13
shows a similar pattern to the Skin Scan measurements that can be
seen with highly significant increases in fluorescence in the
arteries with inflamed plaque (i.e., balloon injured aorta and
iliac). Sections 3, 4 and injured iliac of atherosclerotic compared
to normal control had p values <0.0001, while section 5 and
uninjured iliacs had p values <0.0005. Accordingly, the less
severe plaques of section 5 are distinguishable from the
macrophage-rich plaques of sections 3 and 4. Sections 1 and 2 were
not significantly different in atherosclerotic and normal
rabbits.
[0273] To corroborate the selectivity of the macrophage targeted
conjugate for inflamed plaque, the dye molecules were extracted out
of the pre-weighed tissue sections by dissolving the tissue in a
solvent (1M NaOH/0.2% SDS) designed to preserve c.sub.e6
fluorescence. These dissolved tissue sections were then measured on
the spectrofluorimeter and the fluorescent signal was divided by
the tissue weight to give a value per gram tissue. At least four
pieces of tissue were dissolved for each data point. The
differences between atherosclerotic and normal rabbits were
significant (p<0.05) for sections 1, 2, and 4. The lower level
of significance in this assay was probably due to the inability to
sample as many points as was possible with the surface fluorescence
measurement. In addition, it is possible that surface measurement
of fluorescence was more sensitive than bulk extraction for
detecting macrophage population because macrophages are more likely
to be concentrated in the inflamed surface of the plaque.
[0274] In FIG. 14a, a marked contrast was seen between a large
aortic plaque and an area of the abdominal aorta 5 mm beneath the
plaque. In FIG. 14b, another marked contrast was seen between the
balloon injured iliac artery and the contralateral normal artery in
the same rabbit. Similarly, FIG. 14c shows a contrast between the
plaque-laden aorta of an atherosclerotic rabbit and the same area
of the aorta in a normal rabbit. These spectra were obtained in a
rabbit that had received an overdose of anesthesia. The rabbit
received a laparotomy that exposed the abdominal aorta and iliac
arteries. The rabbit also had an arterotomy in the right leg to
expose the femoral artery. The fiber-optic catheter of the OMA-LIF
apparatus was advanced through the femoral and iliac arteries, to
the abdominal aorta, up to the thoracic aorta. Spectra were
obtained and the fiber optic catheter pulled back about 5 mm each
time successive spectra were obtained. By palpation of the outside
of the artery, the position of the catheter in relation to plaques
was determined
[0275] Thus, a novel method has been developed for targeting a
photosensitizer composition to the activated macrophages of a
vulnerable plaque with high specificity.
Example 3
In Vivo Photodynamic Therapy
[0276] An intravascular fluorescence catheter that efficiently
localized a fluorescence signal from a vulnerable plaque in the
rabbit coronary (although not limited to rabbit) through flowing
blood was developed. In addition, a therapeutic intravascular light
delivery system was developed that illuminated the vulnerable
plaques through flowing blood with the appropriate wavelength,
fluence and fluence rate of light, achieving the desired
therapeutic effect.
Results
[0277] PDT in rabbit aorta was demonstrated to be possible in vivo
in living rabbits through flowing blood without undue harm to the
rabbits and with no short-term toxicity. The same parameters were
used as above (photosensitizer composition, dose and time interval)
in order to be able to correlate treatment effects with previously
determined dye localization in plaque lesions. Animals (one
atherosclerotic and one normal rabbit, each injected with
Mal-BSA-c.sub.e6 24 hours previously; and one atherosclerotic
rabbit that received no injection) were anesthetized as before and
a cylindrical diffusing tipped fiber optic (length of tip=2 cm,
diameter=1 mm) was advanced to a position midway along the
balloon-injured abdominal aorta. The fiber had a SMA connector at
the proximal end that can be connected to a diode laser emitting
light at approximately 665 nm for Mal-BSA-c.sub.e6. Light was
delivered at a fluence rate of approximately 100 mW/cm of diffusing
tip and a total fluence of approximately 100 J/cm was delivered. At
the conclusion of the illumination, the fiber was withdrawn and the
arteriotomy and overlying wound were closed. Animals were
sacrificed 48 hours later. They received a laparotomy and surgical
exposure of the aorta and surrounding tissues (FIG. 15A). The top
panel of FIG. 15A shows light delivery into the abdominal aorta via
a diffusing tip catheter inserted into the femoral artery,
demonstrating the feasibility of intra-arterial illumination. The
middle panel of FIG. 15A shows atherosclerotic aorta that is thick
such that light did not penetrate to extra-aortic tissue. The
bottom panel of FIG. 15A shows normal aorta that is thin such that
light penetrates to give a slight but definite damage to psoas
muscle. Complete aortas and iliac arteries were removed from the
PDT treated normal and atherosclerotic rabbits and control (no
Mal-BSA-c.sub.e6 injection) atherosclerotic rabbit and were
examined by histology using H&E, Masson Trichrome and Verhoeffs
stain.
[0278] The two rabbits that received both the photosensitizer
composition and light showed no ill effects of the treatment during
the two days they lived before sacrifice. At necropsy, the
atherosclerotic rabbit had no gross damage visible in the
illuminated aortic section or surrounding tissue. By contrast, the
normal rabbit had some minor damage visible in the para-aortic
muscle, consisting of hemorrhage and purpura. Without being bound
by theory, it is hypothesized that this damage was caused because
the thickness of the normal artery was much less than the
atherosclerotic aorta, and consequently, much of the light
penetrated the artery and illuminated the surrounding tissue. The
atherosclerotic rabbit that received light, but no conjugate was
associated with any change to artery or surrounding tissue.
[0279] Histological examination of the arteries (FIG. 15B. Top
panel: histopathology of PDT treated atherosclerotic aorta; Bottom
panel: histopathology of atherosclerotic aorta that received light
but no conjugate) showed changes in the illuminated section of the
atherosclerotic rabbit that received both conjugate and light,
consistent with PDT effects in the targeted tissue. There was
evidence of apoptosis (pyknotic nuclei) and an inflammatory
infiltrate in the plaque (FIG. 15B, left panel), together with some
coagulative necrosis (FIG. 15B, middle panel), and extravasated
erythrocytes that may have come from the vasa vasorum and visible
damage in the plaque (FIG. 15B, right panel). Together, these
histological data indicate that the treatment produced favorable
modifications of plaque histology and reduced vulnerability.
Histological changes were not observed in the normal rabbit that
received photosensitizer composition and light, nor were any
changes observed in the atherosclerotic rabbit that received light
but no conjugate.
[0280] This technology satisfies the clear need for a new therapy
that allows localized stabilization of vulnerable plaques in
coronary arteries with the consequent reduced risk of rupture.
Example 4
Detection of Atherosclerotic Lesions
[0281] Chemotactic peptide receptor agonists are bacterial products
that induce chemotaxis by binding to specific receptors on
inflammatory cells. Because vulnerable plaque contains an abundance
of inflammatory cells, atherosclerotic lesions can be detected
non-invasively using radiolabeled chemotactic peptide receptor
agonists.
Peptide Synthesis and Characterization
[0282] The chemotactic peptide receptor agonist For-Met-Leu-Phe
(F-MLK) was used as a ligand to selectively target inflammatory
cells within plaques.
[0283] N-Formyl-Methionyl-Leucyl-Phenylalanyl-Lysine
(N-For-Met-Leu-Phe-Lys) and the nicotinyl hydrazine-derivatized
chemotactic peptide analog, N-Formyl-Met-Leu-Phe-Lys-HYNIC were
synthesized and purified by standard solid-phase techniques
(Fischman et al., 1995, J. Trauma, Injury, Infection and Critical
Care, 38(2):223-227).
Radiolabeling with .sup.99mTc
[0284] A .sup.99mTc generator was eluted five hours after a
previous elution to yield a total activity of approximately 500
mCi. .sup.99mTc-glucoheptonate, prepared from stannous
glucoheptonate, was used to provide the Tc(v) oxo species for
radiolabeling the hydrazinonicotinamide-conjugated peptide. Peptide
labeling was monitored by ITLC-sg using three solvent systems:
acetone, saline, and acetone:water (9:1). Radiolabeled peptide was
purified by reversed-phase HPLC methods as described in Fischman et
al. A similar method was used for .sup.125I.
SPECT Imaging Technique
[0285] In all SPECT acquisitions, a large field-of-view gamma
camera and a high resolution collimator were used to obtain 64
projections at 30 seconds per projection over a semicircular 180
degree arc. A 15% window centered on the 140-keV peak was used. All
projection images were stored on a magnetic disk with the use of a
64.times.64 16-bit matrix. Preprocessing was performed using a
Butterworth filter, order 5, with a cutoff frequency of 66%
Nyquist. Short-axis, as well as vertical and horizontal long-axis
tomograms were constructed. No attenuation or scatter correction
was used.
Experimental Atherosclerotic Model
[0286] A balloon-injured, cholesterol-fed rabbit model of
atherosclerosis was used (FIG. 16). Lesions were produced in the
aortas of seven New Zealand rabbits by de-endothelialization of the
infradiaphragmatic aorta followed by a 6% peanut oil-2% cholesterol
diet. Seven untreated rabbits fed standard chow were used as
controls. After one week on diet, the abdominal aorta was denuded
of endothelium by a modified Baumgartener technique (Elmaleh, et
al., 1998, PNAS, 99:691-695). Briefly, each animal was anesthetized
with a mixture of ketamine and xylazine and the right femoral
artery was isolated. Subsequently, a 4F Fogarty embolectomy
catheter was introduced via arteriotomy and advanced under
fluoroscopic guidance to the level of the diaphragm. The balloon
was then inflated to 3 psi above balloon inflation pressure and
three passes were made down the abdominal aorta with the inflated
catheter. The femoral artery was subsequently ligated and the wound
was closed.
[0287] After eight-ten weeks, .sup.99mTc- and
.sup.125I-radiolabeled F-MLK was administered by injection to the
control and experimental groups. At 12 hours after administration
of the radiolabel, the rabbits' aortas were imaged using single
photon emission tomography (SPECT). After 16 hours, Evan's blue
stain was injected intravenously, the animals were sacrificed and
the aortas were examined for uptake of the radiolabeled F-MLK.
Histology
[0288] The rabbit aortas were excised and fixed with neutral
buffered formalin. Histological assessment was performed using
H&E and eleastic tissue stains. Autoradiography was performed
using previously described methods (Elmaleh, et al.).
Statistical Analysis
[0289] Results are presented as mean i standard error of the mean.
A 5% probability of type I experimental error (p<0.05) is
considered to be of statistical significance.
Results
[0290] Examination of excised aortas revealed that radiotracer
uptake is selective for atherosclerotic lesions. Uptake within the
atherosclerotic aortas was 72-fold higher than in the control
aortas (FIG. 17). Moreover, in living rabbits, atherosclerotic
aortas were readily imaged with SPECT, whereas healthy aortas were
not (FIG. 18). These results demonstrate that selective targeting
of radionuclides via molecular carriers is an effective method for
the detection of vulnerable plaque.
* * * * *