U.S. patent application number 11/600613 was filed with the patent office on 2007-11-08 for methods and devices for detection and therapy of atheromatous plaque.
Invention is credited to Rox R. Anderson, David Elmaleh, Alan J. Fischman, Michael R. Hamblin, Tayyaba Hasan, James Muller, Ahmed Tawakol.
Application Number | 20070258906 11/600613 |
Document ID | / |
Family ID | 27388922 |
Filed Date | 2007-11-08 |
United States Patent
Application |
20070258906 |
Kind Code |
A1 |
Fischman; Alan J. ; et
al. |
November 8, 2007 |
Methods and devices for detection and therapy of atheromatous
plaque
Abstract
The present invention relates to devices for detection and
therapy of active atheromatous plaque and/or thin-capped
fibro-atheroma ("vulnerable plaque"), using selectively targeted
fluorescent, radiolabeled, or fluorescent and radiolabeled
compositions. The present invention further relates to methods and
devices for detection and therapy of active atheromatous plaques
and/or vulnerable plaques, using selectively targeted beta-emitting
compositions, optionally comprising fluorescent compositions.
Inventors: |
Fischman; Alan J.; (Boston,
MA) ; Hamblin; Michael R.; (Revere, MA) ;
Tawakol; Ahmed; (Boston, MA) ; Hasan; Tayyaba;
(Boston, MA) ; Muller; James; (Boston, MA)
; Anderson; Rox R.; (Boston, MA) ; Elmaleh;
David; (Boston, MA) |
Correspondence
Address: |
EDWARDS ANGELL PALMER & DODGE LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Family ID: |
27388922 |
Appl. No.: |
11/600613 |
Filed: |
November 16, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10215958 |
Aug 9, 2002 |
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11600613 |
Nov 16, 2006 |
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10163744 |
Jun 4, 2002 |
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10215958 |
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: |
424/9.71 |
Current CPC
Class: |
A61B 5/0086 20130101;
A61B 2018/00904 20130101; A61N 5/0601 20130101; A61B 5/0071
20130101; A61B 5/0075 20130101; A61K 41/0057 20130101; A61K 41/0071
20130101; A61P 9/00 20180101; A61P 35/00 20180101; A61B 6/4258
20130101; A61B 2017/00057 20130101; A61B 18/245 20130101; A61K
47/643 20170801; A61K 51/0491 20130101; A61P 7/00 20180101; A61P
29/00 20180101; A61N 5/062 20130101; A61B 2017/00079 20130101; A61K
41/0076 20130101; A61K 41/0061 20130101; A61B 5/0084 20130101; A61K
51/0474 20130101; A61B 18/20 20130101; A61N 2005/0602 20130101 |
Class at
Publication: |
424/009.71 |
International
Class: |
A61K 49/00 20060101
A61K049/00 |
Goverment Interests
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] 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
1-124. (canceled)
125. A method of detecting and treating a vulnerable plaque in a
subject comprising the steps of: a) administering a beta-emitting
composition; b) localizing the composition to 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) detecting a
signal from the beta-emitting composition; e) identifying the
vulnerable plaque; 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, thereby treating the vulnerable plaque in the
subject.
126. The method of claim 125, wherein the vulnerable plaque
comprises inflammatory components, a large lipid pool, and a thin
fibrous cap.
127. The method of claim 126, wherein the thin fibrous cap is less
than about 150 microns thick.
128. The method of claim 127, wherein the thin fibrous cap is less
than about 100 microns thick.
129. The method of claim 126, 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.
130. The method of claim 129, 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.
131. The method of claim 130, wherein the lymphocytes comprise
B-lymphocytes and T-lymophocytes.
132. The method of claim 130, wherein the polymorphonuclear cells
comprise granulocytes and neutrophils.
133. The method of claim 130, wherein the inflammatory cells
comprise greater than about 10% macrophages and/or monocytes.
134. The method of claim 130, wherein the inflammatory cells
comprise greater than about 25% macrophages and/or monocytes.
135. The method of claim 129, wherein the lipid content is greater
than about 10%.
136. The method of claim 129, wherein the lipid content is greater
than about 25%.
137. The method of claim 125, wherein the beta-emitting composition
comprises a beta-emitting agent selected from the group consisting
of .sup.18F-Fluorodeoxyglucose, I.sup.131, Re.sup.186 and
Re.sup.188 coupled to a molecular carrier.
138. The method of claim 137, 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.
139. The method of claim 138, 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.
140. The method of claim 139, wherein the lymphocytes comprise
B-lymphocytes and T-lymophocytes.
141. The method of claim 139, wherein the polymorphonuclear cells
comprise granulocytes and neutrophils.
142. The method of claim 137, wherein the molecular carrier is
selected from the group consisting of serum proteins, receptor
ligands, microspheres, liposomes, antibodies, growth factors,
peptides, hormones and lipoproteins.
143. The method of claim 137, wherein the molecular carrier binds
to a scavenger receptor.
144. The method of claim 143, 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.
145. The method of claim 144, wherein the anionic phospholipid is
phosphatidyl serine.
146. The method of claim 137, wherein the molecular carrier targets
the beta-emitting composition to a T lymphocyte.
147. The method of claim 146, wherein 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.
148. The method of claim 137, wherein the molecular carrier targets
the beta-emitting composition to lipids of the vulnerable
plaque.
149. The method of claim 148, 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.
150. The method of claim 149, wherein the liposomes contain
cholesterol.
151. The method of claim 150, wherein the liposomes contain
cardiolipin.
152. The method of claim 137, wherein the molecular carrier targets
the beta-emitting composition to macrophages.
153. The method of claim 152, wherein the molecular carrier targets
the beta-emitting 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.
154. The method of claim 146, wherein the molecular carrier targets
the beta-emitting 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.
155. The method of claim 137, wherein the molecular carrier targets
the beta-emitting composition to foam cells.
156. The method of claim 137, wherein the molecular carrier targets
the beta-emitting composition to a protease that degrades
extracellular matrix.
157. The method of claim 156, wherein the protease is a
metalloproteinase.
158. The method of claim 157, wherein the molecular carrier is a
monoclonal antibody that binds to an epitope on a protease.
159. The method of claim 125, wherein the photosensitizer is
chlorin.sub.e6.
160. The method of claim 125, wherein the light is administered in
a 20-500 J/cm dose.
161. The method of claim 125, wherein the light is administered in
a 50-300 J/cm dose.
162. The method of claim 125, wherein the light is administered in
a 100-200 J/cm dose.
163. The method of claim 125, wherein the photosensitizer induces
apoptosis and not necrosis of the cells comprising the vulnerable
plaque.
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.
[0003] 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 devices for detection and
therapy of active atheromatous plaque and/or thin-capped
fibro-atheroma ("vulnerable plaque"), using selectively targeted
fluorescent, radiolabeled, or fluorescent and radiolabeled
compositions. The present invention further relates to methods and
devices for detection and therapy of active atheromatous plaques
and/or vulnerable plaques, using selectively targeted beta-emitting
compositions, optionally comprising fluorescent 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; approximately 2600
deaths each day are the result of cardiovascular disease. In the
United States, 50-60% of heart attacks occur in people without
documented cardiovascular disease. 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). An atheromatous plaque refers to a wide
range of coronary lesions, from subtle collections of lipid, to
obstructive coronary lesions that cause angina.
[0006] Atheromatous plaques can be active, and prone to rupture, or
inactive and relatively stable. The progression of coronary
atherosclerotic disease can be divided into five phases (Fuster et
al. (1992) N Engl J Med 326:242-250). Phase I is represented by a
small plaque that is present in most people under the age of 30
years, regardless of their country of origin, and that usually
progresses slowly (i.e., type I-III lesions). Phase 2 is
represented by a plaque, not necessarily very stenotic, with a high
lipid content that is prone to rupture (i.e., type IV and Va
lesions). The plaque of phase 3 may have predisposition to change
in its geometry and to formation of mural thrombus, these processes
by definition represent phase 3 (i.e., type I lesion), with a
subsequent increase in stenosis, possibly resulting in angina, or
ischemic sudden death. The mural and occlusive thrombi from plaques
of phases 3 and 4, being organized by connective tissue, may
contribute to the progression of the atherosclerotic process
represented by severely stenotic or occlusive plaques of phase 5
(i.e., types Vb and Vc lesions). The severely stenotic plaques of
phase 5, by a phenomenon of stasis and/or de-endothelialization,
can become complicated by a thrombus and/or rapid myoproliferative
response, also leading to an occlusive plaque of phase 5. Of
interest is that about two thirds of coronary occlusions are the
result of this late stenotic-type of plaque and are unrelated to
plaque disruption. Unlike the rupture of less-stenotic lipid-rich
plaques, leading to occlusion and subsequent infarction or other
acute coronary syndromes, this process of occlusion from late
stenotic plaques tends to be silent because the preceding severe
stenosis and ischemia enhance protective collateral circulation
(Fuster et al., (1992) N Engl J Med 326:242-250; Chesebro et al.
(1992) Circulation 86 (suppl. 111)).
[0007] In general, 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.
[0008] At the edges of the fibrous cap overlying an active
atheromatous plaque, the lipid core comprises the shoulder region
and is 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.
[0009] 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
active 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.
[0010] New therapies designed to target the genesis of atheromatous
plaque and/or thrombus formation, or processes associated with
atheromatous plaque and/or thrombus formation, as well as internal
inflammation and infection are needed.
[0011] Current 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 always prevent the incidence of acute
coronary syndrome. Moreover, at least 50% of patients receiving
angioplasty must return for a further procedure between 6 months to
one year after the initial procedure. 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.
[0012] 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).
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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 mactophage 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-.gamma. R on
inflammatory cells and adjacent smooth muscle cells), indicating an
active inflammatory reaction that also weakens the fibrous cap.
[0017] Present methods of plaque detection, several of which are
discussed herein, are inadequate for detecting the genesis of
atheromatous plaque and/or thrombus formation, or processes
associated with atheromatous plaque and/or thrombus formation, as
well as internal inflammation and infection. Present methods of
plaque detection are also inadequate for detecting vulnerable
plaques.
[0018] Common methods of plaque detection include angiography and
angioscopy. Except in rare circumstances, angiography gives almost
no information about the 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 actively forming
atheromatous and/or vulnerable plaques.
[0019] Several methods are being investigated for their ability to
identify atheromatous plaques. However, none has proven to be
sufficiently sensitive to identify vulnerable plaques or monitor
the formation thereof. 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.
[0020] Intravascular thermography is based on the premise that
atheromatous 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 lesions, vulnerable or
otherwise, having a propensity to rupture.
[0021] 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
actively forming atheromatous and/or vulnerable plaques.
[0022] 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 actively-forming and/or
vulnerable plaques will be reliably differentiated from stable
plaques based solely on this ratio.
[0023] All of the existing technologies and methods used to date
are structural and therefore may be unable to detect actively
forming or vulnerable plaques. All vascular detection agents known
in the art involve the use of external imaging devices, such as
gamma or positron cameras. The usefulness of such agents is limited
and will not accurately detect plaque or thrombus due to the
background activity from the surrounding tissue. Although 3D
imaging via PET and SPECT is presently in use, the small size of
the arteries as compared to the scatter from the large surrounding
tissues lowers the utility of these imaging modalities as well.
[0024] Radiation-based methods for detection of diseased tissue are
known in the art (U.S. Pat. No. 4,995,396). The devices of U.S.
Pat. No. 4,995,396 are not designed to identify vulnerable plaques
and further, U.S. Pat. No. 4,995,396 does not disclose
intra-arterial beta probes. Use of beta-sensitive probes for the
detection of plaques has been reviewed (Daghighian et al. Med.
Phys. 21:153-7(1994); U.S. Pat. Nos. 5,008,546, 5,744,805,
5,932,879, 6,076,009 and 6,295,680). U.S. Pat. No. 5,744,805
relates to an ion-implanted silicon radiation detector located at
the tip of a probe with a preamplifier contained within the body of
the probe, connected to the detector as well as external
electronics for signal handling. U.S. Pat. Nos. 5,744,805 and
5,932,879 provide radio-pharmaceuticals for detecting diseased
tissue, such as a cancerous tumor, followed by the use of a probe
with one or more ion-implanted silicon detectors at its tip to
locate the radiolabeled diseased tissue; the detector is
preferentially responsive to beta emissions. U.S. Pat. Nos.
6,076,009, 5,568,532 and 5,864,141 relate to further designs for
probes containing scintillators and photomultiplier tubes connected
thereto. However, these techniques lack the precision of selective
targeting as first described herein.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] Recently, interventional strategies leading to vulnerable
plaque stabilization have become an active area of research
(Rabbani & Topol (1999) Cardiovasc Res 41:402-417). A therapy
designed to detect, stabilize and reduce or eliminate active
atheromatous and/or vulnerable plaques without inducing an
inflammatory response would be highly desirable.
OBJECT AND SUMMARY OF THE INVENTION
[0029] The present invention provides methods for selectively
targeting radiolabeled compositions, preferably comprising
beta-emitting radiolabels, and optionally photodynamic
compositions, to inflammatory components (e.g., inflammatory cells,
proteases and lipids) of active atheromatous and/or vulnerable
plaques as well as devices for the detection and therapy
thereof.
[0030] In one aspect, detection of active atheromatous and/or
vulnerable plaque can be carried out using a specially designed
intravascular device that detects a nuclear signal, preferably from
a radiolabeled composition, and even more preferably from a
beta-emitting composition, localized to the plaque.
[0031] It now been determined that intravascular beta-emitting
agents can be detected within atheromatous and/or vulnerable
plaques by beta detection devices of the present invention. The use
of a beta-emitting composition targeted to the active atheromatous
and/or vulnerable plaque should improve detection of the same due
to the improved sensitivity and reduced distance necessary to
accurately observe the area of the plaque. Beta-emission based
methods and devices of the present invention provide accurate
delineation of plaque areas, which will improve therapy and fosters
early detection.
[0032] In yet another aspect, detection and/or therapy can be
carried out using a specially designed intravascular device that
delivers excitation light to the surface of active atheromatous
and/or vulnerable plaques, to photoactivate fluorescent
compositions therein, and receives emitted fluorescence that is
transmitted to an analysis instrument. The same device can
optionally be used to deliver therapeutic light activating a
similarly located photosensitizer composition when a fluorescent or
nuclear signal, preferably from a beta-emitting composition, is
first detected.
[0033] Additionally, the present invention provides methods for the
identification of active atheromatous and/or vulnerable plaques.
Methods of the present invention can advantageously differentiate
stable atheromatous lesions from active atheromatous and/or
vulnerable plaques. Once such a 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.
[0034] In one aspect of the invention, photodynamic and/or
radiolabeled compositions can be selectively targeted to
inflammatory components (e.g., macrophages, T cells, lipids and
proteases) within and around the active atheromatous and/or
vulnerable plaque. 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. 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-.gamma.. 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.
[0035] In yet another aspect of the invention, application of PDT
to the active atheromatous and/or 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.
[0036] 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
[0037] 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.
[0038] 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.
[0039] FIGS. 3A, 3B and 3C are diagrams showing a probe/catheter in
accordance with an embodiment of the invention.
[0040] FIGS. 4A and 4B show a probe/catheter in accordance with an
embodiment of the invention.
[0041] FIGS. 5A and 5B are diagrams illustrating a light delivery
element and a light deflection element in accordance with
respective embodiments of the invention.
[0042] FIGS. 6A, 6B and 6C illustrate a probe/catheter in
accordance with an embodiment of the present invention.
[0043] FIG. 7 shows the scheme for preparing chlorin e6
photosensitizer conjugates.
[0044] 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)
[0045] FIG. 9 shows the UV-visible absorption spectra of the
purified mal-BSA-c.sub.e6 conjugates and free c.sub.e6.
[0046] FIG. 10 shows the selective targeting and phototoxicity of
maleylated BSA-c.sub.e6 conjugates.
[0047] FIG. 11 shows an optical multichannel analyzer used for
fluorescence localization within ex vivo aortas.
[0048] 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.
[0049] 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).
[0050] 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).
[0051] 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).
[0052] FIG. 18 depicts the chemical structure of Ap4A and
AppCHClppA.
[0053] FIG. 19 depicts blood clearance of .sup.99mTc-labeled
Ap.sub.4A and AppCHClppA in atherosclerotic and control rabbits.
The biexponential fits to the data are also indicated:
.sup.99mTc-Ap.sub.4A in atherosclerotic rabbits (solid line),
.sup.99mTc-Ap.sub.4A in control rabbits (dot-dashed line), and
.sup.99mTc-AppCHClppA in atherosclerotic rabbits (dashed line).
Each point is the mean .+-.SEM for three animals.
[0054] FIG. 20 depicts images of the aorta of a control rabbit. In
vivo gamma camera image acquired at 15 minutes (Left) and 2 hours
(Center) and after injection of .sup.99mTc-Ap.sub.4A-glucoheptonate
(Right). Corresponding ex vivo gamma camera image and sketch of the
lesioned areas are also shown.
[0055] FIG. 21 depicts images of the aorta of a rabbit with
experimental atherosclerosis. In vivo gamma camera images (i.e.,
lateral decubitus projection) acquired at 15 minutes (Left) and 2
hours (Center) after injection of .sup.99mTc-Ap.sub.4A (Right).
Corresponding ex vivo gamma camera image and sketch of the lesioned
areas are also shown.
[0056] FIG. 22 depicts a graph showing the correlation between
catheter-determined activity and counting measurements, (r-0.89,
P<0.001).
[0057] FIG. 23 depicts a bar graph showing the beta probe counts
(cps) in plaque and non-plaque.
DETAILED DESCRIPTION
Methods for Detecting and Treating Vulnerable Plaque
[0058] In one aspect, the present invention relates to devices for
the detection and/or therapy of active atheromatous and/or
vulnerable plaques by identifying and/or activating compositions
selectively targeted to the inflammatory components thereof.
[0059] An "active atheromatous plaque" comprises a plaque
accumulating aggregated platlets and monocytes, such that greater
than 50% stenosis is achieved. Preferably, 50% stenosis is achieved
within one month of the onset of growth, more preferably 50%
stenosis is achieved within six months of the onset of growth, and
even more preferably 50% stenosis is achieved within one year of
the onset of growth. In a preferred embodiment, the onset of active
atheromatous plaque growth follows a procedure to treat
atherosclerosis, such as a surgical procedure, e.g.,
angioplasty.
[0060] An "inactive or stable atheromatous plaque" comprises a
thick fibrous cap, preferably greater than 200 microns thick, a
small lipid pool or the absence thereof, which is only slowly
accumulating lipids, if at all, and less than 50% stenosis.
Preferably, less than 50% stenosis is maintained for one month,
more preferably less than 50% stenosis is maintained for six months
and even more preferably less than 50% stenosis is maintained for
one year.
[0061] 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%.
[0062] "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).
[0063] 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 and "stenosis" refers to a
constriction or decrease in vascular diameter.
[0064] In one aspect, detection of active atheromatous and/or
vulnerable plaque can be carried out using a specially designed
intravascular device that detects a nuclear signal, preferably from
a radiolabeled composition and even more preferably from a
beta-emitting composition, localized to the plaque.
[0065] As used herein, a "beta-emitting composition" comprises a
beta-emitting agent, such as a radionuclide or a paramagnetic
contrast agent, that emits electron or positron rays ("beta rays")
and is 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). Preferably, the
beta-emitting agent is I.sup.131, .sup.18F-Fluorodeoxyglucose
("FDG"), Re.sup.186, which is electron-emitting, or Re.sup.188,
which is positron-emitting. Beta-detecting devices of the present
invention advantageously distinguish beta rays from gamma rays by a
ratio of about 100:1 (i.e., 100:1 beta to gamma), even more
preferably by a ratio of 1000:1 (i.e, 1000:1 beta to gamma).
Detection of beta-emitting compositions can comprise imaging or
standard means known in the art.
[0066] A "molecular carrier" refers to a biomolecule with targeting
specificity for one or more components comprising the active
atheromatous and/or vulnerable plaque.
[0067] In yet another aspect, detection and/or therapy can be
carried out using a specially designed intravascular device that
delivers excitation light to the surface of active atheromatous
and/or vulnerable plaques, to photoactivate fluorescent
compositions therein, and receives emitted fluorescence that is
transmitted to an analysis instrument. The same device can
optionally be used to deliver therapeutic light activating a
similarly located photosensitizer composition when a fluorescent or
nuclear signal, preferably from a beta-emitting composition, is
first detected.
[0068] 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 produce 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).
[0069] 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.
[0070] In yet another aspect, the present invention further
comprises methods to detect and/or identify active atheromatous
plaques by targeting beta-emitting compositions to the inflammatory
components comprising said plaques.
[0071] In one embodiment, a method of detecting an active
atheromatous plaque in a subject comprises the steps of: [0072] a)
administering a beta-emitting composition; [0073] b) localizing the
composition to the active atheromatous plaque; [0074] c) detecting
a signal from the beta-emitting composition; and [0075] d)
identifying the active atheromatous plaque.
[0076] In yet another embodiment, a method of detecting a
vulnerable plaque in a subject comprises the steps of: [0077] a)
administering a beta-emitting composition; [0078] b) localizing the
composition to the vulnerable plaque; [0079] c) detecting a signal
from the beta-emitting composition; and [0080] d) identifying the
vulnerable plaque.
[0081] In yet another aspect, methods of the present invention
comprise a combination of detection and treatment.
[0082] In one embodiment, a method of detecting and treating an
active atheromatous plaque in a subject comprises the steps of:
[0083] a) administering a beta-emitting composition; [0084] b)
localizing the composition to the active atheromatous plaque;
[0085] c) detecting a signal from the beta-emitting composition;
and [0086] d) identifying the active atheromatous plaque and
administering a suitable treatment thereto.
[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 beta-emitting composition; [0089] b)
localizing the composition to the vulnerable plaque; [0090] c)
detecting a signal from the beta-emitting composition; and [0091]
d) identifying the vulnerable plaque and administering a suitable
treatment thereto.
[0092] Suitable therapies comprise all known in the art for the
treatment of active atheromatous plaque and/or vulnerable plaque,
for example, treatment by statins (e.g., atorvastatin, or
pravastatin), cholesterol lowering drugs, aspirin,
anti-inflammatory agents, bisphosphonates, eicosapentaenoic acid,
docosahexaenoic acid, ACE inhibitors (e.g., ramipril), biomolecules
(e.g., thrombin-activatable fibrinolysis inhibitor, Angpt13, or
Apo-A1 mimetic peptide,) clot-reducing agents (e.g., TPA), or those
described in WO 01/04819 and U.S. Pat. No. 6,183,752.
[0093] In yet another aspect, methods of the present invention
comprise a combination of detection and treatment, wherein
treatment can comprise, for example, photodynamic therapy.
[0094] Accordingly, in one embodiment, a method of detecting and
treating an active atheromatous plaque in a subject comprises the
steps of: [0095] a) administering a detectable amount of at least
one beta-emitting composition, wherein the beta-emitting
composition is localized to an active atheromatous plaque; [0096]
b) administering a therapeutically effective amount of at least one
photosensitizer composition, wherein the photosensitizer
composition is localized to an active atheromatous plaque; [0097]
c) detecting a signal from the beta-emitting composition; [0098] d)
identifying the active atheromatous plaque; [0099] e) light
activating the photosensitizer composition at the site of the
active atheromatous plaque to produce a phototoxic species; and
[0100] f) stabilizing the active atheromatous plaque against
rupture.
[0101] In yet another embodiment, a method of detecting and
treating a vulnerable plaque in a subject comprises the steps of:
[0102] a) administering a detectable amount of at least one
beta-emitting composition, wherein the fluorescent composition is
localized to a vulnerable plaque; [0103] b) administering a
therapeutically effective amount of at least one photosensitizer
composition, wherein the photosensitizer composition is localized
to a vulnerable plaque; [0104] c) detecting a signal from the
beta-emitting composition; [0105] d) identifying the vulnerable
plaque; [0106] e) light activating the photosensitizer composition
at the site of the vulnerable plaque to produce a phototoxic
species; and [0107] f) stabilizing the vulnerable plaque against
rupture.
[0108] In yet another embodiment, a method of detecting and
treating an active atheromatous plaque in a subject comprises the
steps of: [0109] a) administering a beta-emitting composition
comprising a beta-emitting agent coupled to a molecular carrier;
wherein the beta-emitting composition is localized to an active
atheromatous plaque; [0110] b) administering a photosensitizer
composition comprising a photosensitizer coupled to a molecular
carrier; wherein the photosensitizer composition is localized to
the active atheromatous plaque; [0111] c) detecting a signal from
the beta-emitting composition; [0112] d) identifying the active
atheromatous plaque; [0113] e) light activating the photosensitizer
at the site of the active atheromatous plaque to produce a
phototoxic species; and [0114] f) stabilizing the active
atheromatous plaque against rupture.
[0115] In yet another embodiment, a method of detecting and
treating a vulnerable plaque in a subject comprises the steps of:
[0116] a) administering a beta-emitting composition comprising a
beta-emitting agent coupled to a molecular carrier; wherein the
beta-emitting composition is localized to a vulnerable plaque;
[0117] b) administering a photosensitizer composition comprising a
photosensitizer agent coupled to a molecular carrier; wherein the
photosensitizer is localized to the vulnerable plaque [0118] c)
detecting a signal from the beta-emitting composition; [0119] d)
identifying the vulnerable plaque; [0120] e) light activating the
photosensitizer at the site of the vulnerable plaque to produce a
phototoxic species; and [0121] f) stabilizing the vulnerable plaque
against rupture.
[0122] In yet another embodiment, a method of detecting and
treating an active atheromatous plaque in a subject comprises the
steps of: [0123] a) administering a composition comprising a
beta-emitting agent and a photosensitizer coupled to a molecular
carrier; wherein the composition is localized to an active
atheromatous plaque; [0124] b) detecting a signal from the
beta-emitting composition; [0125] c) identifying the active
atheromatous plaque; [0126] d) light activating the photosensitizer
at the site of the active atheromatous plaque to produce a
phototoxic species; and [0127] e) stabilizing the active
atheromatous plaque against rupture.
[0128] In yet another embodiment, a method of detecting and
treating a vulnerable plaque in a subject comprises the steps of:
[0129] a) administering a composition comprising a beta-emitting
agent and a photosensitizer coupled to a molecular carrier; wherein
the composition is localized to an active vulnerable plaque; [0130]
b) detecting a signal from the beta-emitting composition; [0131] c)
identifying the vulnerable plaque; [0132] d) light activating the
photosensitizer at the site of the vulnerable plaque to produce a
phototoxic species; and [0133] e) stabilizing the vulnerable plaque
against rupture.
[0134] In yet another embodiment, the present invention further
comprises methods to identify active atheromatous and/or vulnerable
plaques by targeting beta-emitting compositions to the inflammatory
components comprising said plaques and employing one or more
additional means to identify said plaques, including, but not
limited to thermal detection, OCT, MRI or other detection
modalities known in the art.
[0135] Accordingly, in one embodiment, a method of detecting an
active atheromatous plaque in a subject comprises the steps of:
[0136] a) administering a beta-emitting composition; [0137] b)
localizing the composition to the active atheromatous plaque;
[0138] c) detecting a signal from the beta-emitting composition;
[0139] d) employing one or more additional means to identify said
plaque; and [0140] e) identifying the active atheromatous
plaque.
[0141] In yet another embodiment, a method of detecting a
vulnerable plaque in a subject comprises the steps of: [0142] a)
administering a beta-emitting composition; [0143] b) localizing the
composition to the vulnerable plaque; [0144] c) detecting a signal
from the beta-emitting composition; [0145] d) employing one or more
additional means to identify said plaque; and [0146] e) identifying
the vulnerable plaque.
Radiolabeled Compositions
[0147] Radiolabeled compositions of the present invention can
comprise any known radioactive agents in the art, including, but
not limited to radionuclide or a paramagnetic contrast agents,
preferably beta-emitting 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, .sup.18F, .sup.68Ga, .sup.67Ga,
.sup.72As, .sup.89Zr, .sup.62Cu, .sup.111Cu, .sup.203In,
.sup.198Pb, .sup.198Hg, .sup.97Ru, .sup.11C, Re.sup.188 and
.sup.201TI. Suitable paramagnetic contrast agents include, but are
not limited to gadolinium, cobalt, nickel, manganese and iron.
Preferred radionuclides or paramagnetic contrast agents are
detected by gamma detecting devices of the present invention.
Detection of radiolabeled compositions can comprise imaging or
standard means known in the art.
[0148] Preferably, the radiolabeled composition is a beta-emitting
composition, wherein the radionuclide is
.sup.18F-Fluorodeoxyglucose ("FDG"). Other beta-emitting
compositions include, but are not limited to I.sup.131, Re.sup.186
and Re.sup.188.
Photosensitizer Compositions
[0149] 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. Several
photosensitizers known in the art are FDA approved and commercially
available.
[0150] 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.
[0151] 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).
[0152] In a preferred embodiment, the photosensitizer is tin ethyl
etiopurpurin, commercially known as purlytin (available from
Miravant).
Fluorescent Compositions
[0153] Fluorescent compositions of the present invention can be any
known in the art, including photosensitizers, fluorescent dyes, and
photoactive dyes.
[0154] 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).
[0155] 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.
[0156] 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)st-
yryloxy)acetyl)aminohexanoic acid or succinimidyl ester.
[0157] 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.
[0158] 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.
[0159] 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
[0160] Selectivity for target tissues of the present invention is
achieved by using covalent conjugates or non-covalent complexes
between molecular carriers with targeting specificity for one or
more components comprising the active atheromatous and/or
vulnerable plaque. Accordingly, targeting compositions of the
present invention comprise one or more 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). 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.
[0161] 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).
[0162] 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).
[0163] 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 active
atheromatous and/or 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.
[0164] In a preferred embodiment, targeting compositions of the
present invention are coupled to molecular carriers that target
macrophages and/or monocytes of active atheromatous and/or
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.
[0165] In a preferred embodiment, targeting compositions of the
present invention are coupled to molecular carriers that target T
cells of active atheromatous and/or 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.
[0166] 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.
[0167] 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.
[0168] In a preferred embodiment, targeting compositions of the
present invention are coupled to molecular carriers that target the
endothelial cells of active atheromatous and/or 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. Targeting compositions of the present invention can be
coupled to molecular carriers that target the subendothelial matrix
of active atheromatous and/or vulnerable plaques.
[0169] In a preferred embodiment, targeting compositions of the
present invention are coupled to molecular carriers that target
neutrophils of active atheromatous and/or 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.
[0170] In a preferred embodiment, targeting compositions of the
present invention are coupled to molecular carriers that target B
cells of active atheromatous and/or 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.
[0171] In a preferred embodiment, targeting compositions of the
present invention are coupled to molecular carriers that target
smooth muscle cells of active atheromatous and/or vulnerable
plaques. These molecular carriers can be targeted to, for example,
LOX-1. Such molecular carriers can be, for example, antibodies
against these biomolecules, ligands binding the same or analogs
thereof.
[0172] 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.
[0173] The features of an active atheromatous and/or vulnerable
plaque that are distinguishable from stable atheromatous plaques
advantageously distinguish active atheromatous and/or vulnerable
plaques from stable atheromatous plaques according to methods of
the present invention.
[0174] An "active atheromatous plaque" comprises a plaque
accumulating aggregated platelets and monocytes, such that greater
than 50% stenosis is achieved. Preferably, 50% stenosis is achieved
within one month of the onset of growth, more preferably 50%
stenosis is achieved within six months of the onset of growth, and
even more preferably 50% stenosis is achieved within one year of
the onset of growth. In a preferred embodiment, the onset of active
atheromatous plaque growth follows a surgical procedure, such as
angioplasty.
[0175] An "inactive or stable atheromatous plaque" comprises a
thick fibrous cap, preferably greater than 200 microns thick, a
small lipid pool or the absence thereof, which is only slowly
accumulating lipids, if at all, and less than 50% stenosis.
Preferably, less than 50% stenosis is maintained for one month,
more preferably less than 50% stenosis is maintained for six months
and even more preferably less than 50% stenosis is maintained for
one year.
[0176] 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%.
[0177] 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 an
active atheromatous and/or 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
active atheromatous and/or vulnerable plaques and not merely
commonplace stable atheromatous plaques.
[0178] 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.
[0179] Compositions of the present invention that are useful for
detection of active atheromatous and/or vulnerable plaques of 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 99mTc-conjugated antibodies against
modified LDL (Tsimikas et al. (1999) J. Nucl. Cardiol. 6:
41-53).
[0180] 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. Alternatively, devices of the present invention
can be employed for intravascular detection of beta waves.
[0181] Such radiolabels may be associated with 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.
[0182] 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 may 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).
[0183] Radionuclides may 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 may 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 may 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.
[0184] 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.
[0185] 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.
[0186] 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.
[0187] 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.
[0188] 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).
[0189] 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.
[0190] 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-5-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, M A 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.
[0191] 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).
[0192] 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.
[0193] 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 join them together (described in the Pierce Catalog
and Merrifield et al. (1994) Ciba Found Symp. 186:5-20).
[0194] 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.
[0195] 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 0.1M 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.
[0196] 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.
[0197] 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.
[0198] 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.
[0199] 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.sub.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.
[0200] 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.
[0201] 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.
[0202] 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).
[0203] 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.
[0204] 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.
[0205] "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.
[0206] 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.
[0207] 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.
[0208] 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.
[0209] 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.
[0210] 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.
[0211] 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.
[0212] 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 Governatore 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.
[0213] 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.
[0214] 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.
[0215] 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).
[0216] 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.
[0217] 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.
[0218] 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.
[0219] 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.
[0220] 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
[0221] 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.
[0222] 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.
[0223] 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.
[0224] 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.
[0225] 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.
[0226] 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 active
atheromatous plaque and/or vulnerable plaque. Alternatively, an
active atheromatous plaque and/or 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.
[0227] 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.
[0228] 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.
[0229] 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.
[0230] 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.
[0231] 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.
[0232] 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.
[0233] 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.
[0234] Another method of administration is intravascular, for
instance by direct injection into the blood vessel, plaque or
surrounding area.
[0235] 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.
[0236] 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
[0237] 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.
[0238] 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.
[0239] 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.
[0240] 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.
[0241] 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.
[0242] 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.
[0243] 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).
[0244] 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 about 3 days, preferably about 12
hours to about 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.
[0245] 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.
[0246] 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.
[0247] 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.
[0248] FIG. 1A 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.
[0249] 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.
[0250] 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.
[0251] 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.
[0252] 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.
[0253] In accordance with an embodiment of the invention where in
the fluorescent composition or photosensitizer composition includes
chlorin.sub.e6, 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.
[0254] 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.
[0255] 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.
[0256] 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.
[0257] 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.
[0258] 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.
[0259] 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.
[0260] 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.
[0261] 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.
[0262] 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.
[0263] 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.
[0264] 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.
[0265] 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.
[0266] 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.
[0267] 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.
[0268] 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.
[0269] FIG. 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.
[0270] 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.
[0271] FIGS. 16A and 16B are diagrams illustrating a probe/catheter
1600 in accordance with an embodiment of the invention.
Probe/catheter 1600 may include a cylindrical structure with an
open, or hollow, center region 1605, so that when probe/catheter
1600 is deploy in blood vessel 120, blood can flow through center
region 1605, as shown in FIG. 16B. One or more light delivery
and/or deflection elements 1610 may be disposed along the outer
surface of the cylindrical structure. Light delivery/deflection
elements 1610 may deliver detection/excitation light and/or
therapeutic light from light source 113 and shine the
detection/excitation light and/or therapeutic light outward from
the circumference of the cylindrical structure towards the
surrounding wall of blood vessel 120. Probe/catheter 1600 may
include a corresponding black seal mash 1615 for each light
delivery/deflection element 1610. Black seal mash 1615 may be
placed around an inner portion of the cylindrical structure of
probe/catheter 1600 from light delivery/deflection elements 1610.
Probe/catheter 1600 may also include a pair of black seal rings
1620 on either end of the cylindrical structure. Preferably, as
shown in FIG. 16B, black seal rings 1620 engage the wall 1630 of
blood vessel 120. Black seal rings 1620 and black seal mash 1615
may include light absorbing and/or outward reflecting material. As
a result, black seal rings 1620 and black seal mash 1615 may form a
light seal around a region on vessel wall 1635 where
detection/excitation light and/or therapeutic light would be
targeted. Thus, black seal rings 1620 and black seal mash 1615 may
absorb or deflect outward any stray detection/excitation light
and/or therapeutic light. Advantageously, blood flowing through
center region 1605 would be protected from any stray
detection/excitation light and/or therapeutic light. Probe/catheter
1600 may further include a vessel (or "balloon") 1625 that may be
expanded by filling it with a fluid. The fluid may be any non-toxic
fluid but is preferably a transparent fluid. Vessel 1625 may
include any elastic material, such as rubber or latex, and the
like, and includes preferably a transparent material. Control unit
105 and/or detection/treatment unit 110 may control fluid flow to
and from vessel 1625 so that fluid is delivered thereto when, as
shown in FIG. 16B, vessel 1625 is filled with fluid and expanded,
engaging the surrounding wall 1635 of blood vessel 120.
Consequently, black seal rings 1620 and vessel 1625 may form a seal
between light delivery/deflection elements 1610 and the surrounding
wall 1635 of blood vessel 120. In other words, this seal prevents
blood in vessel 120 from flowing between light delivery/deflection
elements 1610 and the surrounding wall 1635 of blood vessel 120.
Advantageously, detection/excitation light and/or therapeutic light
may be delivered/deflected from light delivery/deflection elements
1610 to the surrounding wall 1635 without any interference from the
blood flowing through blood vessel 120.
[0272] FIGS. 17A and 17B show a probe/catheter 1700 in accordance
with an embodiment of the present invention. As shown in FIGS. 17A
and 17B, probe/catheter 1700 may include an external unit 1702, a
therapeutic structure 1705, a detector 1710 and a vessel 1715.
External unit 1702, detector 1710 and vessel 1715 may operate in a
similar manner to external unit 202, detector 605 and vessel 220,
respectively, as described above. Description of such operations
will not be repeated here.
[0273] Probe/catheter 1700 advantageously includes a therapy
release system allowing more accurate medication delivery to an
artherosclerotic injury. The device and methods allow deposition of
a therapy regiment in the immediate area of a plaque-diagnosed
region. Detector 1710 may include a fluorescence, temperature, or
beta detection probe and therapeutic structure 1705 may include a
medicated stent.
[0274] As shown in FIGS. 17A and 17B, detector 1710 and stent 1705
may be mounted on the catheter type used for balloon
catheterisation. Probe/catheter 1700 may be inserted into the
femoral or carotid artery as used by catheterisation physicians.
Probe/catheter may be moved through the artery until vulnerable
plaque or plaque zone is detected by their elevated amount of beta
radiation, temperature, or fluorescence emission on the arterial
wall. As describe above, the detection may be determined by an
increased beta emitting signal or fluorescence color of a
preinjected plaque-targeted diagnostic agent, or increased
temperature due to inflammatory process. After the location of the
plaque formation region, plaque or vulnerable plaque, is detected,
stent 1705 may be pushed forward out from external unit 1702, as
shown in FIG. 17B. Stent 1705 may thus be move to the exact
location on or near the arterial wall by inflating vessel 1715.
Stent 1705 may be attached to a lower part of probe/catheter 1700
and after the correct location of plaque has been determined, stent
1705 may be deposited by an action, which resembles that of a
sleeve. In accordance with another embodiment, stent 1705 may be a
cover of probe/catheter 1700 and be deployed by inflating vessel
1715 at the area of the diagnosed plaque. Probe/catheter 1700 may
then be disconnected from the inflated stent 1705.
[0275] The therapeutic compounds that heal or prevent the formation
of the internal hyperplasia or vulnerable plaque are incorporated
in the coating of stent 1705, or as integral part of a porous
support on stent 1705, may be released in the exact location where
vulnerable plaque or restinosis may occur. The medication or
radiation treatment may be assembled or absorbed on tiny porous
cavities on stent 1705 and slowly released directly or via the
delivery of a second drug to the body of the patient. Furthermore,
the drugs, peptides, glycopeptides protein glycoproteins,
antisense, DNA or their modifications may be attached or fixed on
stent 1705. A biodegradable polymer or a specifically modified
delivery polymer, and drugs may be enclosed in an organic or
inorganic chemical matrix and be fixed on stent 1705. Stent 1705
may be coated or attached by other forms to antibody or ligand or
compound which may attract and/or bind the medication, chosen from
anti-infective or other plaque-preventing drugs which are in
combination with stent 1705 given systemically from time to time to
control, prevent or treat plaque formation or thrombus. Stent 1705
may have a trapped enzyme in forms such as sol gel, fulrenes, or
other inert inorganic or organic matrix.
[0276] In the case where detector 1710 is a beta emitting
radioactive detector, stent 1705 may be pulled back a distance that
is far enough from detector 1710 while it is working to find the
location of the plaque. This may prevent false readings from the
radioactive accumulation in the vulnerable plaque. Since beta rays
have short ranges in the order of a few millimeters, any affect on
the readings of detector 1710 may be prevented by retracting stent
1705 such distances.
[0277] Examples of treatment compounds include: for brachytherapy,
an appropriate treatment radionuclide may be enclosed in the matrix
without the tissue being physically exposed to the radionuclide
that is able to emit the radiation necessary to treat the tissue;
and an enzyme such as NTPase like 6CD39 or similar structure in the
matrix, without the enzyme or the polymeric derived enzyme being in
contact with the tissue. The chemical precursors may move freely
through the matrix and be transformed to the plaque prevention
form. In the case of NTPase the degradation of ADP may prevent the
plaque internal hyperplasia formation.
[0278] 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
[0279] 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
[0280] 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.e6 was 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.
[0281] 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.
[0282] 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.
[0283] 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.
[0284] 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.e6 1) 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.
[0285] 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)
[0286] 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.-1M.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 (ChemiImager 4000, Alpha
Innotech Corp, San Leandro, Calif.). Proteins were localized by
Coomassie blue staining.
[0287] 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 Photo Sensitizers
[0288] 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
[0289] 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
[0290] 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
[0291] 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).
[0292] 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 mm. Data
points were the average of 3 wells of the 24 well plate (15 wells
of 96 well plate).
[0293] 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.
[0294] 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).
[0295] 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.
[0296] 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).
[0297] 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).
[0298] 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.
[0299] 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.
[0300] 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 macrophages
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).
[0301] 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.
[0302] 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.
[0303] 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
[0304] 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
[0305] 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
[0306] 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.
[0307] 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.
[0308] 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.
[0309] 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
In Vivo Detection of Radionuclide Emitting Signals
[0310] Methods and devices of the present invention are readily
employed by the skilled artisan.
Synthesis of .sup.99mTc-labeled Ap4A Derivatives
[0311] .sup.99mTc-labeled Ap4A derivatives (FIG. 18) were
synthesized and HPLC-characterized using Tc0.sub.4.sup.- reduction
with stannous chloride and mannitol as the coligand. The process
was optimized for greater yield and consistency. The conveniently
synthesized .sup.99mTc precursor
[.sup.99mTc(CO).sub.3(H.sub.2O.sub.3)].sup.+ was also employed. By
utilizing the precursor
[.sup.99mTc(CO).sub.3(H.sub.2O.sub.3)].sup.+, additional Tc-Ap4A
complexes were isolated from the pool of derivatives. The precursor
was prepared from .sup.99mTcO.sub.4 in saline and CO at normal
pressure. The novel one-pot synthesis of the organometellic
precursor was designed for use in radiopharmacy (Schubiger et al.
(1998) J Am Chem 120:7987-7988).
Generation of Experimental Atherosclerotic Lesions
[0312] Male New Zealand White rabbits weighing 2.5-3.0 kg (Charles
River Breeding Laboratories) were maintained on a 2% cholesterol-6%
peanut oil diet (ICN) for 3 months. After 1 week of the
hyperlipidemic diet, the abdominal aorta was denuded of endothelium
by a modified Baumgartener technique (Narula, J. et al. (1995)
Circulation 92:474-484). Briefly, each animal was anesthetized with
a mixture of ketamine and xylazine (100 mg/ml, 10:1 vol/vol;
1.5-2.5 ml sc), and the right femoral artery was isolated. A 4F
Fogarty embolectomy catheter (Catalog Number 12-040-4F; Edwards
Laboratories, Santa Ana, Calif.) was introduced through an
arteriotomy and advanced under fluoroscopic guidance to the level
of the diaphragm. The catheter was inflated to a pressure of 3 psi
above the balloon inflation pressure with radiographic contrast
media (Conray, Mallinckrodt), and three passes were made down the
abdominal aorta with the inflated catheter. The femoral artery was
then ligated, and the wound closed. The animals were allowed to
recover from anesthesia and then returned to their cages.
Gamma Camera Imaging of Atherosclerotic and Normal Rabbits
[0313] Two to four millicuries of the
.sup.99mTc-Ap.sub.4A-glucoheptonate,
.sup.99mTc-AppCHClppA-glucoheptonate, or .sup.99mTc-glucoheptonate
(control) was injected into marginal ear veins of groups of three
rabbits with experimental atherosclerotic lesions. As a control,
three unlesioned animals were injected with 2-4 mCi of
.sup.99mTc-Ap.sub.4A-glucoheptonate. After radiopharmaceutical
administration, serial gamma images were collected every minute for
the first 5 minutes, every 2 minutes for the next 25 minutes, and
every 5 minutes for the next 2.5 hours. In all of the rabbits,
images were acquired in the anterior and lateral decubitus
projections, including the heart and aorta, by using a standard
field-of-view gamma camera (Series 100, Ohio Nuclear, Solon, Ohio)
equipped with a high-resolution parallel-hole collimator and
interfaced with a dedicated computer system (Technicare 560, Solon,
Ohio). The pulse height analyzer was adjusted to record the 140 KeV
photopeak of .sup.99mTc, and all images were recorded in a
256.times.256 matrix.
[0314] After acquiring the final images, the animals were
sacrificed with an overdose of sodium pentobarbital. The aortas
were removed, opened along the ventral surface, and mounted on
styrofoam blocks. The aortas then were placed on the face of the
gamma camera and ex vivo images were recorded for 10 minutes.
Blood Clearance of .sup.99mTc-Ap.sub.4A
[0315] Blood clearance of the .sup.99mTc-Ap.sub.4A-glucoheptonate
was rapid. In the control rabbits, the concentration of
radioactivity (% ID/g) in the circulation averaged 0.25% at 2
minutes, after injection, decreased to 0.08% ID/g at 60 minutes and
only slightly thereafter (i.e., up to 180 minutes). For all groups
of rabbits, blood clearance was well described by bi-exponential
functions with fast and slow components (t.sub.1/2s) of .apprxeq.4
and .apprxeq.250 min, respectively (FIG. 19). Blood clearance was
not significantly different between rabbits with artherosclerotic
lesions and controls.
Results
[0316] All of the rabbits with experimental atherosclerosis showed
rapid accumulation of radioactivity in the lesioned areas;
representative images are shown in FIG. 20. The lesions were
clearly visible within 20 minutes after injection, and
radioactivity was retained in the lesions for the full 3 hours of
the imaging session. When the aortas were imaged ex vivo, the
pattern of radioactivity distribution closely paralleled the
imaging results (FIG. 20). Inspection of the excised aortas
revealed lesion patterns that were virtually identical to the
results of in vivo and ex vivo imaging (FIG. 20). In contrast, both
in vivo and ex vivo gamma camera imaging failed to demonstrate
evidence of focal tracer accumulation in aortas of unlesioned
rabbits; representative images are shown in FIG. 21. Inspection of
the aortic specimens showed no evidence of vessel damage. Imaging
atherosclerotic rabbits with .sup.99mTc-labeled glucoheptonate
showed no evidence of specific accumulation in the aortic lesions,
and the images were indistinguishable from those obtained in
control rabbits imaged with .sup.99mTc-labeled Ap.sub.4A or
AppCHClppA (data not shown). With this tracer, radioactivity
cleared rapidly from all organs (t.sub.1/2s: 5-10 min) and
accumulated in the kidneys and bladder.
Characterization of Vulnerable Plaque Using a .sup.18F-FDG Beta
Probe
[0317] Additionally, FDG was administered intravenously to two
additional rabbits. FDG selectively accumulates in vulnerable
plaques. Thereafter, a 1.6 mm thin, flexible beta probe, was
inserted into the aorta. This probe is selectively sensitive to
positron emissions, and was built by optically coupling a 1 mm
diameter, 2 mm-long plastic scintillator to a PMT via a 40 cm long
optical fiber. Measurements of FDG activity were made in
triplicate, at 2 seconds per measurement, at grossly visible sites
of plaque within areas of balloon injury; at non-injured sites in
the cholesterol fed rabbits; and in corresponding areas in control
aorta. Aortic segments at previously assessed sites of plaque and
control areas were excised and examined for uptake of FDG by
standard well counting.
[0318] Sensitivity of the probe was 850.+-.11 cps/microCi (mean
.+-.SD). Catheter-determined activity correlated well with well
counting measurements, (r-0.89, P<0.001, FIG. 22). Moreover,
atherosclerotic regions were readily distinguished from control by
catheter mounted beta probe, (11.9.+-.2.1 [n=9, range 9.7.+-.15.3]
vs. 4.8.+-.1.9[n=14, range 1.3.+-.7.3], cps in atherosclerotic vs.
control regions, respectively, P<0.001).
[0319] The intravascular detection of positron emissions was
achieved with sensitivity and specificity in an in vivo system.
* * * * *