U.S. patent application number 13/932609 was filed with the patent office on 2013-11-28 for method for treatment of aneurysms.
This patent application is currently assigned to LERS SURGICAL, LLC. The applicant listed for this patent is LERS SURGICAL, LLC. Invention is credited to Lionel C. SEVRAIN, Sylvie Y. VERDIER-SEVRAIN.
Application Number | 20130315908 13/932609 |
Document ID | / |
Family ID | 40824631 |
Filed Date | 2013-11-28 |
United States Patent
Application |
20130315908 |
Kind Code |
A1 |
SEVRAIN; Lionel C. ; et
al. |
November 28, 2013 |
METHOD FOR TREATMENT OF ANEURYSMS
Abstract
The present invention generally concerns the detection and/or
treatment of aneurysm in a non-invasive manner. In particular
cases, the invention concerns methods and compositions for
localizing a labeled composition to the site of an aneurysm for its
detection and, in further cases, treatment of the aneurysm. In
specific cases, the composition targets a subendothelial component
of the aneurysmal wall, such as a smooth muscle cell exposed at the
luminal surface of the vessel. In further specific cases, the
composition targets an integrin receptor or laminin.
Inventors: |
SEVRAIN; Lionel C.; (West
Palm Beach, FL) ; VERDIER-SEVRAIN; Sylvie Y.; (West
Palm Beach, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LERS SURGICAL, LLC |
WEST PALM BEACH |
FL |
US |
|
|
Assignee: |
LERS SURGICAL, LLC
West Palm Beach
FL
|
Family ID: |
40824631 |
Appl. No.: |
13/932609 |
Filed: |
July 1, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12809139 |
Jun 28, 2010 |
8491870 |
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PCT/US2008/084445 |
Nov 23, 2008 |
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13932609 |
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61016090 |
Dec 21, 2007 |
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Current U.S.
Class: |
424/134.1 ;
424/178.1 |
Current CPC
Class: |
A61P 9/14 20180101; A61P
9/10 20180101; A61P 43/00 20180101; A61K 31/70 20130101; A61K
38/363 20130101; A61K 38/39 20130101 |
Class at
Publication: |
424/134.1 ;
424/178.1 |
International
Class: |
A61K 47/48 20060101
A61K047/48 |
Claims
1-45. (canceled)
46. A method of treating an aneurysm in an individual, comprising
the step of delivering to the individual having or suspected of
having a cerebral aneurysm a composition comprising a) a labeled
antibody that specifically binds to at least one integrin expressed
on contractile SMCs, wherein the labeled antibody localizes to the
cerebral aneurysm site, b) a pharmaceutically acceptable excipient,
and c) a therapeutic agent, in an amount effective to treat the
aneurysm in the individual.
47. The method of claim 46, wherein the individual has an
unruptured cerebral aneurysm.
48. The method of claim 46, wherein the individual has a ruptured
cerebral aneurysm.
49. The method of claim 46, wherein the therapeutic agent is
selected from the group consisting of: a thrombogenic agent, a
polymerisable molecule, a protein, a cell growth factor, and a
protease inhibitor.
50. The method of claim 49, wherein the therapeutic moiety is
selected from the group consisting of: elastin, elastin degradation
fragment, fibronectin, fibrinogen, and a cell growth factor.
51. The method of claim 49, wherein the therapeutic agent is a
protease inhibitor.
52. The method of claim 51, wherein the protease inhibitor is an
elastase inhibitor or a matrix metalloproteinase inhibitor.
53. The method of claim 46, wherein the therapeutic agent comprises
a protease inhibitor and fibronectin or fibrinogen.
54. The method of claim 46, wherein the therapeutic moiety
comprises fibronectin and a cell growth factor.
55. The method of claim 46, wherein the composition is administered
to the individual intravascularly.
56. The method of claim 46, wherein administration of the
composition results in accumulation of the labeled antibody and
therapeutic agent at the aneurysm wall and thrombosis or aneurism
wall thickening.
57. The method of claim 46, wherein the at least one integrin is
one or more selected from the group consisting of: .alpha.1.beta.1,
.alpha.7.beta.1, .alpha.3.beta.1, and .alpha.8.beta.1.
58. The method of claim 46, wherein the composition is delivered in
situ via an arterial catheter.
59. The method of claim 46, wherein the labeled antibody is
conjugated to an intravascular targeting molecule.
60. The method of claim 59, wherein the intravascular targeting
molecule is a polymer.
61. The method of claim 60, wherein the polymer is albumin,
transferrin, a globulin, pectin, gelatin, dextran, or a cellulose
derivative.
62. The method of claim 46, further comprising administering to the
individual a drug or performing surgery or endovascular coiling on
the individual.
Description
[0001] The present application is a divisional application of U.S.
patent application Ser. No. 12/809,139, filed Jun. 18, 2010, which
is a national phase application filed under 35 USC .sctn.371 from
PCT/US2008/084445, filed Nov. 23, 2008, which claims priority to
U.S. Provisional Patent Application Ser. No. 61/016,090, filed Dec.
21, 2007, all of which applications are incorporated by reference
herein in their entirety.
TECHNICAL FIELD
[0002] The present invention generally concerns at least the fields
of medicine, cell biology, and molecular biology. In particular
aspects, the present invention concerns the detection and/or
treatment of cerebral aneurysms.
BACKGROUND OF THE INVENTION
[0003] Annually in the U.S., aneurysmal subarachnoid hemorrhage
affects greater than 30,000 people. Ten to 15 percent of these
individuals die before reaching the hospital and greater than 50
percent die within the first month following rupture. Of those
patients that survive, approximately half suffer some permanent
neurological deficit.
[0004] Intra-cranial saccular (berry) aneurysm is a balloon-like
distension of a major brain artery occurring at (or near) the apex
of arterial forks. It is frequently (90%) located on the anterior
part of the circle of Willis. Various hypothesis have been proposed
regarding the developmental mechanisms of Saccular Cerebral Artery
Aneurysms (SCAAs) such as the medial defect theory (Forbus, 1930),
the elastic lamellar theory (Glynn, 1940), degenerative theory
(Stehbens, 1963; Stehbens, 1972), congenital theories (Bremer,
1943; Agnoli, 1982), and others (Sekha et al., 1981). Recently the
development of an experimental animal model of the disease with
pathological features very similar to those of human SCCA has made
possible the study of the pathogenesis of human SCCAs (Hashimoto et
al., 1978). It has been shown that hemodynamic stress induces the
development of cerebral aneurysms causing degenerative changes of
the endothelium, the elastic lamina and the medial smooth muscle
cells at specific site on the arterial bifurcation (Kojima et al.,
1988).
Histological Features of SCAAs
[0005] The anterior cerebral artery/olfactory artery (ACA/OA)
junction is one of the most common sites of aneurysm development in
the animal model. Its normal structure and changes due to aneurysm
development have been widely studied.
[0006] 1. Normal ACA/OA Artery Junction
[0007] The apex of a normal ACA/OA junction consists of normal
arterial components (endothelial cells, internal elastic lamina,
medial smooth muscle cells, and thin adventitial fibrous connective
tissue). In the apical region, there is an intimal protrusion
called pad consistently located near the apex on the distal side of
the ACA. This pad is composed of spindle-shaped cells similar to
the medial smooth muscle cells, rich in interstitial tissue. Under
and just distal to the intimal pad on the side of the ACA, the
internal elastic lamina is thinned and fragmented.
[0008] 2. Aneurysm Formation at ACA/OA Artery Junction
[0009] The initial changes of aneurysm occur at the intimal pad and
the neighboring distal portion. In the histological structure of
early stage aneurysms, there are the following characteristics: 1)
the wall does not significantly protrude; 2) initial changes are
localized almost exclusively at the intimal pad and its neighboring
distal portion; and 3) there is fragmentation of internal elastic
lamina and slight thinning of the media (decrease of medial smooth
muscle cells in number). In the histological structure of advanced
stage aneurysms, there are the following characteristics: 1)
aneurismal wall consists only of a fibrous adventitia and a layer
of endothelial cells; 2) complete disappearance of the Internal
Elastic Lamina (I.E.L) at the level of the aneurismal neck; and 3)
media layer (smooth muscle cells (SMC)) ceases abruptly proximal to
the neck.
[0010] The histological features of SCAAs include degenerative
changes of endothelium, fragmentation and disappearance of I.E.L,
and thinning (then disappearance) of medial layer. In degenerative
changes of the endothelium, the following has been observed. Severe
changes in endothelium have been reported. Nagata et al. (1981)
examined by scanning electron microscopy the luminal surface of the
cerebral aneurysms. They noticed some variations in the shape of
the endothelial cells from fusiform to polygonal. Some of them
showed balloon-like protrusions. Crater-like depressions on the
endothelial surface and small holes and enlarged gaps at the
junction of the endothelial cells were frequently observed. Gap
formation at the junctions between the endothelial cells was one of
the most obvious changes on the luminal surface of the aneurysms.
Kojima et al. (1986) studying various stage of early aneurismal
changes reported alterations of the endothelium developing just
distal to intimal pad. Degenerated cells with balloons and craters
were observed intermingled with regenerated endothelial cells.
Interendothelial gaps were also seen. They concluded that some
hemodynamic stress, possibly turbulent flow or secondary flow, may
injure the endothelial cells located distal to the pad, and such
injured endothelial cells in turn develop saccular cerebral
aneurysms. Greenhill and Stehbens (1982) also described severe
alterations of the endothelium and subendothelial tissues caused by
hemodynamic stress. Kim et al. (1992) studied aneurismal changes in
experimental monkeys and found endothelial injury. They suggested
that aneurismal changes are initiated by degenerative changes in
the endothelium, which are followed by alterations in the
underlying elastic lamina and, in turn, in the medial layer.
[0011] Degenerative changes of the internal elastic lamina and the
medial smooth muscle cells are also known. Hazama et al. (1986)
showed that early aneurismal changes consist on degenerative
changes of the Internal Elastic Lamina (I.E.L) at the intimal pad
and the neighboring area distal to the pad associated to regressive
changes of medial smooth muscle layer. Kim et al. (1988) also
reported degenerative changes of the I.E.L and medial smooth muscle
layer. Morimoto et al. (2002) found that the characteristic of SCAA
formation in a mouse model was thinning of medial smooth muscle
layer and disappearance of the I.E.L. Kondo et al. (1998) found
that the histological features of aneurismal changes were thinning
of the medial layer accompanied by fragmentation or disappearance
of internal elastic lamina with wall dilatation. They noted a
decreased number of SMCs in the medial layer due to apoptosis. They
concluded that the death of medial SMCs through apoptosis plays an
important role in aneurysm formation.
Molecular Mechanisms of SCAAs Formation
[0012] While the pathological features of aneurismal lesions
described above are well documented, the precise molecular
mechanisms involved in the formation of cerebral aneurysms have not
yet been conclusively identified. Hemodynamic stress has been shown
in many investigations to be the major cause of various
degenerative changes in SCAA formation (Nakatani et al., 1991;
Stehbens, 1989). This hemodynamic stress might induce a complex,
multifactorial remodeling through a variety of mediators and
pathways. Recent studies have reported the role of nitric oxide in
the development of SCAA. Inducible NO synthase (iNOS) was induced
in response to hemodynamic stress and NO synthesized by iNOS serves
to damage the arterial wall and lead to aneurysm formation (Fukuda
et al., 2000). Other molecular mechanisms such as active
expressions of matrix metalloproteinases (Houghton et al., 2006),
apoptosis of medial smooth muscle cells (Cohen et al., 1991) have
been shown associated with SCAA. The role of elastase in the
degradation of I.E.L in early aneurismal lesions has also been
discussed. Nagata et al. (1981) reported that in experimental
aneurysms many leukocytes were present adhering to the inter
endothelial gaps, which may represent the participation of
leukocytes in degradation of the I.E.L. Cajander and Hassler (1976)
also found extracellular lysosome-like granules closely connected
to the disintegrated elastic lamella in the mouths of aneurysms and
hypothesized that discharged leukocyte granules containing elastase
help to destroy the elastic lamella. Enhanced activity of elastase
in the arterial wall may also participate in the degenerative
changes of the internal elastic lamina, as in the case of
hypertension (Yamada et al., 1983).
[0013] Two studies have brought significant insights into the
mechanism of formation of cerebral aneurysms.
[0014] 1. Futami et al. (1995) have demonstrated that fibronectin
(as well as collagen IV and I) normally expressed in the
subendothelium of artery, disappears in early aneurysmal lesions.
The absence of fibronectin in the aneurysm wall is a critical
feature in aneurysm formation considering the role of this
Extra-cellular Matrix (ECM) protein in wound repair and its role in
modulation of SMC phenotype (see below).
[0015] 2. Jamous et al. (2007) have demonstrated the sequence of
ultrastructural, morphological and pathological changes leading to
the formation of saccular intracranial aneurysms in vivo. They used
the current established animal model to induce cerebral aneurysm.
They studied the anterior cerebral artery-olfactory artery
bifurcation morphologically by using vascular corrosion casts and
immunohistochemically by using specific antibodies against
endothelial nitric oxide synthase (eNOS), .alpha.-smooth muscle
actin (.alpha.-SMA: marker of SMCs), macrophages, and matrix
metalloproteinase-9. They showed that the formation of intracranial
aneurysms starts with endothelial injury at the apical intimal pad
(evidenced by the loss of eNOS expression) (stage I); this leads to
the formation of an inflammatory zone. This inflammatory zone shows
subendothelial expression of .alpha.-SMA and a loss of eNOS. There
is no protusion on the vessel wall at this early inflammatory stage
(stage IIA). The progression of inflammation results in arterial
wall destruction and the development of a defect presenting as a
narrow slit; this is associated with protusion of the vessel wall
(Stage IIB). This defect is continuous with the lumen of the parent
artery, lacks eNOS expression, and contained .alpha.-SMA positive
SMCs and macrophages. Expansion of this defect results in the
formation of a saccular dilatation (stage III). The walls of the
cavity continued to lack eNOS expression, contained a layer of
.alpha.-SMA-positive SMCs and are positive for MMP-9 expression.
The authors suggested that endothelial injury and exposure of the
subendothelial matrix initiate platelet activation and adhesion.
Activated platelets secrete growth factors that contribute to the
recruitment of macrophages and promote migration of SMCs. These
processes result in the formation of the inflammatory zone. The
combined effects of hemodynamic changes and the destructive effects
of macrophages through their release of proteolytic enzymes may
lead to development of the defect.
[0016] Scanning electron microscopy studies of vascular corrosion
casts of the ACA-OA bifurcation and double immunostaining of the
vascular wall illustrates that normal endothelial cells are seen at
the apical intimal pad, and the endothelial cell markings are
elongated in the direction of the blood flow. The endothelial and
the smooth-muscle layer form two continuous layers. In stage I,
there are roughened apical intimal pad with irregularly shaped
imprints, and loss of eNOS expression at the apical intimal pad is
observed. In stage IIA, there is shallow elevation surrounded by an
area of depression of the apical intimal pad. Swelling of the
vessel wall at the apical intimal pad is shown, and part of this
swollen area lacks eNOS expression and shows subendothelial
expression of .alpha.-SMA-positive cells. In stage IIB, there is
pyramid-shaped elevation of the apical intimal pad, and the surface
of this elevation is covered by abnormal imprints. Thinning and
degradation of the smooth-muscle layer creates a defect in the
inflammatory zone (arrow) and produces vessel wall protrusion. In
stage III, there is saccular aneurysm covered with abnormal
imprints, expansion of the inflammatory zone defect, and
destruction and protrusion of the vessel wall representing the
nidus of the cerebral aneurysm.
[0017] Stage IIA has early inflammatory changes characterized by
SMC migration and macrophage infiltration. A sagittal cut of the
left ACA-OA bifurcation viewed at low and high magnification shows
swelling of the apical intimal pad. Double immunostaining of an
ACA-OA section with eNOS antibodies and .alpha.-SMA shows swelling
of the vessel wall at the apical intimal pad; part of this swollen
area lacks eNOS expression and shows migration of
.alpha.-SMA-positive cells into the neointima. In triple
immunostaining of an ACA-OA section with antibodies against eNOS,
.alpha.-SMA, and macrophages, macrophage expression confirms the
presence of an inflammatory zone.
BRIEF SUMMARY OF THE INVENTION
[0018] The present invention generally concerns a method for
detection of aneurysms, including cerebral aneurysm, using
non-invasive molecular imaging techniques, and in particular cases
further concerns treatment of the aneurysm.
[0019] The present invention can detect any aneurysm, regardless of
the stage or type of the aneurysm. In early stage aneurysms, the
initial changes are characterized by the alteration of the
endothelium and subendothelium and migration of medial smooth cells
in the intima. At the late stage of degeneration, there is a
disappearance of the media, and the aneurysmal wall just consists
in an endothelium and a few fibers of collagen (adventicia).
However, because these different stages of degeneration coexist in
the same aneurysm, the invention can detect any aneurysm.
[0020] In particular aspects of the invention, the methods and
compositions concern targeting a specific component of the
subendothelium at the aneurysm. In certain cases, the specific
component in the subendothelium that is targeted by compositions of
the invention for the detection of aneurysmal lesions are migrating
SMCs that are in a contractile phenotype, which is in contrast to
SMCs in neointima formation that are in a synthetic phenotype. That
is, specific markers of SMC phenotype will allow one to
differentiate SMCs in aneurysmal wall from SMCs in neointima. In
specific embodiments, particular proteins on the surface of SMCs
unique to contractile SMCs are the targets for detection/treatment
of the aneurysm. In specific embodiments, integrin receptors can
serve as markers of SMC phenotype. In further specific embodiments,
the differentiated contractile SMCs comprise .alpha.1.beta.1
integrin (which is a receptor for laminin, collagen I, and collagen
IV) and .alpha.7.beta.1 integrin, which is a receptor for
laminin-1. However, in the arterial wall, .alpha.1.beta.1 integrin
is expressed on SMCs but also on macrophages (which invade the
arterial wall in pathological conditions such as atherosclerosis
and aneurysm). Therefore, in some embodiments .alpha.7.beta.1 is
targeted, because in the arterial wall it is expressed exclusively
by SMCs. .alpha.7 integrin expression confers a gain of
function-motile phenotype to immobile cells and is responsible for
transduction of the laminin-induced cell motility, in certain
aspects of the invention. Laminin-1 is also useful in the invention
as a marker of aneurysmal lesion, because it is the specific ligand
of .alpha.7 integrin, and .alpha.7 integrin mediates adhesion and
migration of SMCs on laminin-1.
[0021] Thus, methods of the present invention are based at least in
part on two characteristic features of early aneurysmal lesions: 1)
the degeneration of endothelium and subendothelium that exposes
underlying components; and 2) the migration in the subendothelium
of SMCs of contractile, migrating phenotype (.alpha.7 integrin
positive cells that bind specifically laminin-1). In specific
cases, the method employs a labeled antibody directed against
.alpha.7 integrin or laminin-1, for example, and the labeled
antibody will have such characteristics that it binds exclusively
to SMCs exposed at the luminal surface of the vessel, as opposed to
being within the media. That is, such labeled antibody will not be
able to bind contractile SMCs in the medial layer of normal
arterial wall. In particular cases, the labeled antibody is coupled
to a macromolecular compound (such as Dextran, for example) that
keeps the compound in the intravascular compartment but yet allows
it to be cleared from the intravascular compartment. Such a
compound will bind exclusively SMCs exposed at the luminal surface
of the vessel due to endothelial and subendothelial degeneration
but will not be able to bind contractile SMCs in the medial layer
of normal arterial wall. In vivo detection of the aneurysm is
achieved, and the method may use different molecular imaging
techniques such as immunoscintigraphy using antibody radiolabeled
with 99mTc-dextran or MRI using antibody conjugated to
Gadolinium-DTPA-dextran, for example.
[0022] In one embodiment of the invention, there is an isolated
composition, comprising a cell targeting molecule, an intravascular
targeting molecule, and a label. In a specific embodiment, the
composition further comprises a therapeutic agent. In a further
specific embodiment, the cell targeting molecule is an antibody or
a peptide. In specific embodiments, the antibody immunologically
reacts with an integrin receptor on the cell surface of smooth
muscle cells or a laminin (such as laminin 1). In certain aspects,
the integrin receptor is .alpha.1.beta.1, .alpha.7.beta.1, or
.alpha.8.beta.1. In particular embodiments, the intravascular
targeting molecule is a polymer, such as one selected from the
group consisting of dextran, albumin, transferrin, globulins,
pectin, gelatin, and cellulose derivatives. In some cases, the
label is a radionuclide, a fluorophore, a lucigen, or a
paramagnetic chelator or microbubble contrast agent. In further
embodiments, the therapeutic agent is selected from the group
consisting of a thrombogenic molecule, a polymerisable molecule,
fibrinogen, a cell matrix protein (such as elastin, fibronectin, or
laminin), a synthetic peptide, a cell growth factor, an elastase
inhibitor, and a MMP inhibitor. In particular cases, the
composition is comprised in a pharmaceutically acceptable
excipient.
[0023] In another embodiment of the invention, there is a method of
detecting and/or treating a cerebral aneurysm in an individual,
comprising the step of delivering an effective amount of a compound
of the invention to the individual. The individual may be at risk
for developing an aneurysm, has a history of cerebral aneurysm, or
is asymptomatic with no history or known risk of cerebral aneurysm
(method may be used as a mass detection or routine screen for
individuals). In specific cases, the composition is delivered to
the individual once or more than once. In certain aspects, the
individual is provided an additional therapy for aneurysm, such as
one that comprises medication, surgery, endovascular coiling, or a
combination thereof.
[0024] In another embodiment of the invention, there is a kit for
detection and/or treatment of aneurysm, comprising the compound of
the invention, housed in a suitable container. In specific
embodiments, the kit further comprises a therapeutic agent.
[0025] In general embodiments of the invention, one may be able to
differentiate individuals in an "at-risk" aneurysm sub-group within
a cerebral aneurysm population.
[0026] In particular aspects of the invention, .alpha.1.beta.1,
.alpha.7.beta.1, .alpha.3.beta.1, .alpha.8.beta.1 integrins, and/or
laminin 1 are indicators that there is a risk for an aneurysm to
rupture; in specific embodiments,one can utilize one or more of
these targets to detect the cerebral aneurysms, that are prone to
rupture.
[0027] In other specific embodiments, the .alpha.7 expression, or
the ratio of expression of .alpha.7/.alpha.5 is a marker of
aneurysm that is "at risk" of rupture.
[0028] The foregoing has outlined rather broadly the features and
technical advantages of the present invention in order that the
detailed description of the invention that follows may be better
understood. Additional features and advantages of the invention
will be described hereinafter which form the subject of the claims
of the invention. It should be appreciated by those skilled in the
art that the conception and specific embodiment disclosed may be
readily utilized as a basis for modifying or designing other
structures for carrying out the same purposes of the present
invention. It should also be realized by those skilled in the art
that such equivalent constructions do not depart from the spirit
and scope of the invention as set forth in the appended claims. The
novel features which are believed to be characteristic of the
invention, both as to its organization and method of operation,
together with further objects and advantages will be better
understood from the following description when considered in
connection with the accompanying figures. It is to be expressly
understood, however, that each of the figures is provided for the
purpose of illustration and description only and is not intended as
a definition of the limits of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is an exemplary schema outlining embodiments of the
present invention for cerebral aneurysm formation versus neointima
formation.
[0030] FIG. 2 is an exemplary schema outlining embodiments of the
present invention regarding treatment of neo-intima formation vs.
cerebral aneurysm.
DETAILED DESCRIPTION OF THE INVENTION
[0031] This application incorporates WO 2007/092419 by reference
herein in its entirety.
[0032] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. For
purposes of the present invention, the following terms are defined
below.
[0033] As used herein, the use of the word "a" or "an" when used in
conjunction with the term "comprising" in the claims and/or the
specification may mean "one," but it is also consistent with the
meaning of "one or more," "at least one," and "one or more than
one." Some embodiments of the invention may consist of or consist
essentially of one or more elements, method steps, and/or methods
of the invention. It is contemplated that any method or composition
described herein can be implemented with respect to any other
method or composition described herein.
I. Definitions
[0034] The term "aneurysm" as used herein refers to an abnormal
bulge or "ballooning" in the wall of an artery.
[0035] The term "cerebral aneurysm" as used herein refers to a
cerebrovascular disorder wherein a localized weakness in the wall
of a cerebral blood vessel (artery) results in a localized dilation
or ballooning of the blood vessel. The majority of aneurysms are
saccular in shape. It may also be referred to as a Saccular
Cerebral Arterial Aneurysm (SCAA) or intracranial aneurysm.
[0036] The term "effective amount" or "therapeutically effective
amount" as used herein is defined as an amount of the agent that
will decrease, reduce, inhibit or otherwise abrogate an aneurysm.
Thus, an effective amount is an amount sufficient to detectably and
repeatedly ameliorate, reduce, minimize or limit the extent of the
disease or at least one of its symptoms.
[0037] As used herein, the term "tunica intima" (or just intima)
comprises a layer of endothelial cells supported by a basement
membrane and an internal elastic lamina. The connective tissue
between the endothelium and the internal elastic lamina is called
"subendothelium." The internal elastic lamina (I.E.L) is part of
the intima.
[0038] As used herein, the "subendothelium" is a definite zone
within the intima and corresponds to the connective tissue between
endothelial cell layer and inner elastic lamina.
[0039] As used herein, the "tunica media" refers to the main layer
of the artery wall and comprises SMCs.
[0040] The "tunica adventitia" as used herein refers to the
outermost layer and comprises mostly collagen. Cerebral arteries do
not have the external elastic lamina unlike systemic arteries.
II. General Embodiments of the Invention
[0041] Intracranial aneurysms are a major public health problem; it
is estimated that approximately 5% of the population harbors an
unruptured intracranial aneurysm (Iwamoto et al., 1999). The
consequences of rupture are catastrophic: approximately 50% of
patients die during the first post-rupture month, and 60% of deaths
occur within 2 days of the onset of aneurismal sub-arachnoid
hemorrhage (SAH) (Broderick et al., 1994). Half of the survivors
manifest physical or psychosocial deficits 1 year after SAH
(Hackett and Anderson, 2000). Clip placement and coil occlusion to
treat ruptured intracranial aneurysms, aimed at avoiding recurrent
bleeding, have no direct effect on the recovery from the initial
hemorrhage.
[0042] For surgical clipping, after a craniotomy, the surgeon then
spreads the brain tissue apart, opens the subarachnoid cisterns
under operative microscope and then places a tiny metal clip across
the neck to stop blood flow into the aneurysm and exclude it from
the blood stream. Endovascular treatment of brain aneurysms is a
minimally invasive procedure which requires insertion of a catheter
into the femoral artery and navigating it through the vascular
system into the aneurysm (referred to as Endovascular Coiling
(Guglielmi detachable coil)). Tiny platinum coils are threaded
through the catheter and deployed into the aneurysm, blocking blood
flow into the aneurysm and preventing rupture. However, these
treatments are responsible for 15% of the morbidity and mortality.
Recurrence is not insignificant: 2.2% at 10 years and 9.0% at 20
years (Molyneux et al., 2002). Given the poor prognosis of ruptured
intracranial aneurysms, cerebral aneurysms should be detected
before rupture, however current diagnosis means are invasive and
expensive (e.g., 4-axle digital subtraction angiography), and no
mass detection can be currently considered. Therefore, there is a
need for developing non-invasive diagnosis and safer treatment for
intracranial aneurysms.
[0043] Preliminary data from the literature have suggested that
degeneration of the endothelium and subendothelium at specific site
on the bifurcation of cerebral arteries is a characteristic feature
of cerebral aneurysm formation. In the present invention, these
degenerative changes render underlying components of the arterial
wall abnormally expressed at the luminal surface of the artery, and
these components are a useful a target for in vivo intravascular
immuno-detection of cerebral aneurysms. The diagnosis of aneurismal
lesions is made by coupling a specific antibody directed against a
subendothelial component of the arterial wall to a label moiety, in
certain cases. In specific embodiments, the labeled antibody is
coupled to a compound (such as dextran) that confines it into the
intravascular compartment, yet allows it to be rapidly cleared from
the intravascular space. This method allows the diagnosis of
cerebral aneurysm at an early stage, rendering possible a
biological treatment for repairing the wall before irreversible
damages. The treatment uses the same specific antibody coupled to a
therapeutic moiety. Several therapeutic agents have already been
considered such as the use of a thrombogenic or polymerisable
molecule (intended to clog the aneurysms) or proteins (elastin,
fibronectin, or fibrinogen, for example), or cell growth factors
(intended to reinforce or make the fundus thicker and stronger).
Thus, an antigenic component of the subendothelium of the arterial
wall is useful for in vivo immuno-detection of early-stage
aneurysm, although any stage of aneurysm may be detected and
treated with the present invention.
III. Cerebral Aneurysm
[0044] The present invention is useful for detection and treatment
of any cerebral aneurysm. Cerebral aneurysms are classified by size
and shape, with small aneurysms having a diameter of less than 15
mm and larger aneurysms are those classified as large (15 to 25
mm), giant (25 to 50 mm), and super giant (over 50 mm). Saccular
aneurysms, the most common form, have a saccular outpouching,
whereas berry aneurysms are particular saccular aneurysms having
necks or stems resembling a berry. Fusiform aneurysms lack
stems.
[0045] Cerebral aneurysms commonly occur on the arteries at the
base of the brain, known as the Circle of Willis. About 85% of
cerebral aneurysms develop in the anterior part of the Circle of
Willis, thereby involving the internal carotid arteries and their
major branches that supply the anterior and middle sections of the
brain. Common locations are as follows: the anterior communicating
artery (30-35%), the bifurcation of the internal carotid and
posterior communicating artery (30-35%), the bifurcation of the
middle cerebral artery (20%), the bifurcation of the basilar
artery, and the remaining posterior circulation arteries (5%).
[0046] Cerebral aneurysms may occur at any age, although they are
more common in adults than children, and more common in women than
men.
[0047] Most of intracranial aneurysms are clinically quiescent
until they rupture. Onset of the aneurysm usually is sudden, with
no warning. Rupture of a cerebral aneurysm often results in
bleeding into the subarachnoid space or the brain itself, resulting
in a subarachnoid hemorrhage (SAH) or intracranial hematoma (ICH)
(Ruptured aneurysm). Although unruptured aneurysms are usually
asymptomatic, some may be known either because they are multiple
(The 4-axle digital subtraction angiography may reveal multiple
unruptured aneurysms), or symptomatic (third cranial nerve palsy,
headache, orbital pain) or incidental (brain imaging performed for
a neurological non-aneurysmal disease)
[0048] The histology of normal cerebral artery junction is
described as follows: the intimal pad is just distal to apex on
distal side of ACA, and the pad is composed of spindle-shaped cells
similar to the medial smooth muscle cells; the internal elastic
lamina is continuous along the curvature of the apex, but at the
proximal margin of the intimal pad, it is split into several layers
and considerably fragmented under the intimal pad, and just distal
to the intimal pad, it is thinned and fragmented for a short
distance; no medial defect is found. In early stage aneurysms,
initial changes are localized almost exclusively at the intimal pad
and its neighboring distal portion; the wall does not significantly
protrude; and there is fragmentation of internal elastic lamina and
slight thinning of the smooth muscles cells layer. In advanced
stage aneurysms, there is complete disappearance of the internal
elastic lamina at the level of the aneurysmal neck, the media layer
(SMC) ceases abruptly proximal to the neck, and the aneurysmal wall
consists only of a fibrous adventitia and a layer of endothelial
cells. Aneurysms form when hemodynamic stress plus pulsatile flow
patterns initiate degenerative changes in the endothelial layer
adjacent to the apex (distal side) of the arterial bifurcation, and
these endothelial injuries are followed by degenerative changes in
the internal elastic lamina, then in the medial layer (affecting
the SMCs).
IV. Risk Factors for Rupture of Aneurysm
[0049] After an aneurysmal subarachnoid hemorrhage, nearly half of
the patients die, with the remaining half who survive suffering
from irreversible cerebral damage. More unruptured cerebral
aneurysms are identified with increasing use of noninvasive
neuro-imaging techniques (for example, magnetic resonance and
computerized tomography angiography). The risk of rupture in
aneurysms smaller than 10 mm is a 0.5 to 2% annual risk, in
specific embodiments. Growing aneurysms and those larger than 10 mm
run a higher risk for rate of rupture.
[0050] Risk factors for aneurysm rupture include the size of
aneurysms in stable compared With Growing Lesions. In the ISUIA
(ISUIA=International Study of Unruptured Intracranial Aneurysms)
(1998) it has been pointed out that the size and location of
aneurysms were independent predictors of rupture. In Group 1,
aneurysms that were 25 mm or more in diameter had a rupture rate of
6% in the 1st year. It was observed that aneurysms that ruptured at
a later time had more often increased significantly in size (>
or =1 mm) than the largest aneurysms in patients without bleeding
(Juvela, 1993, 2002, and 2000).
[0051] Locations of aneurysms may also be a factor. For example,
aneurysms of the vertebrobasilar and middle cerebral arteries have
a statistically higher probability of subsequent bleeding. In ISUIA
Group 1, for example, the relative risk of rupture was 13.8 for
aneurysms that were located at the basilar tip, whereas the
relative risk was 13.6 for those in the vertebrobasilar or
posterior cerebral artery distribution, compared to other
locations. For posterior communicating artery aneurysms, the
relative risk of rupture was 8.0. The relative risk of rupture was
5.1 for aneurysms at the basilar tip in Group 2.
[0052] The shape of the aneurysms has an impact on rupture, in
specific examples. For example, multilobed lesions have a
significantly higher risk of hemorrhage than do single-lobed
unruptured aneurysms. In some embodiments, the age and sex of the
individual is a factor; females have a risk factor affecting both
aneurysm formation and growth, for example. In other cases,
cigarette smoking hastens the growth of preexisting aneurysms
(Juvela, 2002).
[0053] Families having intracranial aneurysms and rupture history
have a greater risk for rupture. In families with two or more
first-degree members, especially siblings and mother-daughter
pairs, or two first- and second-degree members with SAH, the risk
that other relatives will harbor unruptured intracranial aneurysms
is approximately 9 to 11%, which is higher than in the general
population (Raaymakers et al., 1998; Ronkainen et al., 1997)
[0054] Finally, genetic conditions in some cases have an impact on
risk factors for aneurysm rupture. For example, the presence of
ADPKD (ADPKD=autosomal-dominant polycystic kidney disease) is
associated with a 15% prevalence (Rinkel et al., 1998) of cerebral
aneurysms. Individuals having Type IV Ehlers-Danlos syndrome,
hereditary hemorrhagic telangiectasia, neurofibromatosis Type 1,
alpha-1-antitrypsin deficiency, Klinefelter syndrome, tuberous
sclerosis, Noonan syndrome, or alpha-glucosidase deficiency have a
propensity for intracranial aneurysms compared with the general
population, in certain embodiments of the invention.
V. Exemplary Molecular Basis of the Invention
[0055] Due to the degeneration of the endothelium and
subendothelium of the aneurysmal wall, SMCs that migrate into the
intima are abnormally exposed at the luminal surface of the
arterial wall and become available to react with labeled
antibodies. These cells can be labeled in vivo using a cell surface
marker (cytoskeletal antigen (such as .alpha.-SMA) are protected
from extracellular fluid by the cellular membrane and can not be
labeled in vivo).
[0056] The present invention exploits the pathogenesis of cerebral
aneurysm in which smooth muscle cells (SMCs) have a critical role.
Therein, one or more cell surface markers of SMCs, for example
specific integrin receptors, are antigenic components in the
arterial wall targeted for in vivo immuno-detection of
aneurysm.
[0057] A. Background on Smooth Muscle Cells (SMCs) and Their Role
in Arterial Wall Repair After Injury
[0058] 1. Smooth Muscle Cell (SMC)
[0059] SMC is the sole cell type normally found in the media of
mammalian arteries. In the adult, it is a terminally differentiated
cell that expresses cytoskeletal marker proteins like smooth muscle
alpha-actin (.alpha.-SM actin) and smooth muscle myosin heavy chain
(SMMHC), and contracts in response to chemical and mechanical
stimuli. They take part in the control of blood pressure and flow;
at this stage they are referred to as being in a contractile
phenotype. However, the smooth muscle cell is able to revert to a
proliferative and secretory active state equivalent to that seen
during vasculogenesis in the fetus; at this stage they are referred
to as being in a synthetic phenotype. SMCs in their synthetic
phenotype have a fibroblast-like appearance, a prominent
endoplasmic reticulum and Golgi complex, few filaments and only a
weak reactivity for .alpha.-SM actin. They secrete extracellular
matrix components: laminin, fibronectin, collagen and elastin
(Thyberg et al., 1997; Hultgardh-Nilsson et al., 1997). The
transition from a contractile to a synthetic phenotype occurs in
vascular diseases such as atherosclerosis and restenosis after
angioplasty. In these diseases, in response to endothelial injury,
smooth muscle cells migrate from the media to the intima, they
dedifferentiate into a synthetic phenotype, proliferate and secrete
components of the extracellular matrix and form what is called a
neointima or myo-intimal hyperplasia or intimal thickening
(Campbell and Campbell, 1985; Schwartz and Reidy, 1987). Neointima
formation is a common mechanism of arterial wall repair after
endothelial injury
[0060] 2. Factors that Control SMC Phenotype
[0061] Thyberg et al. (1997) have made extensive research on the
control of SMC phenotype. They demonstrated first that laminin
promotes the expression of a differentiated smooth muscle phenotype
in vitro and in vivo, whereas fibronectin stimulates the cells to
adopt a synthetic phenotype. Then, they demonstrated that after
being converted into a synthetic phenotype, the cells do not start
to proliferate without exogenous mitogen stimulation. They showed
that some growth factors and especially platelet-derived growth
factor (PDGF) stimulates SMC proliferation. After stimulation with
PDGF, converted SMCs divide and produce their own PDGF which
stimulates their growth in an autocrine and paracrine manner
(Sjolund et al., 1988). Therefore, it is concluded that at least
two requirements need to be fulfilled for inducing the synthetic,
proliferating phenotype of SMCs:
[0062] First, the cells must adhere to a substrate of fibronectin
and second, they must be stimulated with growth factors and
especially PDGF. These studies are in agreement with others
demonstrating that there is an accumulation of fibronectin at the
site of arterial injury, in association with neointima formation
(Bauters et al., 1995; Chemnitz and Collatz Christensen, 1983)
suggesting an important pathophysiological role of fibronectin in
neointima formation and vascular wall repair (Hedin and Johan,
1987; Boudreau et al., 1991; Molossi et al., 1995). To modulate the
phenotype of SMCs, laminin and fibronectin bind to integrin
receptors on the surface of SMCs.
[0063] 3. Integrin Receptors on SMCs
[0064] Integrins are a family of receptors involved in cell
interactions with extracellular matrix (ECM) components and with
other cells. Each integrin receptor is a heterodimer in which one
of several homologous .alpha. subunits associates noncovalently
with a .beta. subunit. Some integrin subunit combinations recognize
multiple ligands, while others are relatively specific. Although
some integrins are widely expressed by a variety of cell types,
others have a restricted distribution.
[0065] a. Integrin Receptors for Laminin
[0066] Several integrins (.alpha.1.beta.1, .alpha.2.beta.1,
.alpha.3.beta.1, .alpha.6.beta.1, .alpha.7.beta.1 and
.alpha.v.beta.3) bind laminin (Clyman et al., 1994). It has been
demonstrated that only .alpha.1.beta.1, .alpha.3.beta.1,
.alpha.7.beta.1 and .alpha.v.beta.3 are expressed on human SMCs in
vivo. .alpha.2.beta.1 is a receptor for collagen I to VI and
laminin. Despite the potentially significant in vitro functions of
.alpha.2.beta.1 in modulating SMC behavior, studies were unable to
detect this integrin complex in normal or atherosclerotic human
arteries (Glukhova et al., 1993). .alpha.2.beta.1 has not been
detected in SMCs in vivo (Thorsteinsdottir et al., 1995).
[0067] .alpha.1.beta.1: is a receptor for laminin, collagen-I,
collagen IV. Human medial SMCs (which in vivo are surrounded by a
basement membrane that contains laminin-1 and/or laminin 3 and
collagen IV) express high level of .alpha.1.beta.1. .alpha.1
subunit expression is an exceptional feature of SMCs. Other cell
types (fibroblasts, endothelial cells, keratinocytes, striated
muscles, and platelets) contained trace amounts of .alpha.1.beta.1
integrin (Belkin et al., 1990). Only activated T cells, monocytes
also express .alpha.1.beta.1 integrin. .alpha.1.beta.1 integrin
expression is characteristic of differentiated SMCs. It has been
demonstrated that SMCs from intimal thickening of human adult aorta
express less .alpha.1 subunit of .alpha.1.beta.1 integrin than SMCs
from adult aortic media (Belkin et al., 1990). In contrast, it has
been reported in a rat vascular injury model that .alpha.1.beta.1
is expressed by intimal SMC in response to vascular injury (46).
This discrepancy may be explained by the species-specificity of
integrins.
[0068] .alpha.3.beta.1: is able to bind a variety of ECM components
including laminin, nidogen/entactin, fibronectin, and collagen I.
It is expressed in vivo in medial SMC of human artery (Hillis et
al., 1998). It is also expressed on B-lymphocytes and cells of
kidney glomerulus.
[0069] .alpha.7.beta.1: is a specific receptor for laminin-1. This
integrin has a highly tissue-specific and limited expression
pattern. It is a muscle specific integrin being expressed by all
major types of muscle tissue, including skeletal, cardiac and
smooth muscle. Its presence in all muscle types suggests a role for
this integrin in transducing myofilament-generated forces to
anchoring sites in the surrounding laminin-rich basement membrane
during cellular contractile activity. No studies have documented
the expression of .alpha.7.beta.1 in human vascular SMCs. But, it
has been demonstrated that murine vascular SMCs express the
.alpha.7 integrin receptor. The expression of .alpha.7 integrin in
SMCs is associated with their differentiated phenotype and mediates
their interaction with laminin (Yao et al., 1997). Studies have
demonstrated that .alpha.7 integrin expression confers a gain of
function-motile phenotype to immobile cells and may be responsible
for transduction of the laminin-induced cell motility (Echtermeyer
et al., 1996). Therefore, in embodiments of the invention, there is
a role of .alpha.7 integrin in migration of SMCs to the intima
after vascular injury. It is likely that during neointima
formation, highly differentiated SMCs, which were originally
arranged in concentric layers and encircled by basement membranes,
become motile and migrate into the intima toward growth factor
signals using laminin-binding .alpha.7 integrin. In the intima,
growth factors (especially PDGF) and extracellular matrix
(fibronectin) modulate SMC phenotype and integrin expression
(switching from .alpha.7 to .alpha.5.beta.1 integrin expression)
leading to the formation of a neointima. Furthermore, a recent
study has demonstrated the importance of .alpha.7 integrin in
vascular remodeling (Welser et al., 2007). Using a carotid ligation
animal model, the authors have found a profound increase in
vascular remodeling and neointima formation in the carotid arteries
of .alpha.7 integrin-null mice subjected to ligation.
[0070] .alpha.v.beta.3: is able to bind a variety of ECM components
including laminin, vitronectin, von Willebrand factor,
thrombospondin, osteopontin, fibrinogen and fibronectin. It is
expressed on different cell types including platelets, endothelial
cells and SMCs. Studies have demonstrated that .alpha.v.beta.3 is
expressed on SMCs in the media of normal as well as atherosclerotic
coronary artery and on SMCs in the neointima. .alpha.a.beta.3 was
also strongly expressed by luminal endothelium (Hoshiga et al.,
1995). Clinical studies have shown that c7E3, an antibody directed
against .beta.3 integrin reduces SMC migration and neointima
formation and is useful in the prevention of the SMC response in
restenosis after angioplasty (Topol et al., 1997).
[0071] b. Integrin Receptors for Fibronectin
[0072] The integrin receptors for fibronectin are .alpha.3.beta.1,
.alpha.4.beta.1, .alpha.5.beta.1, .alpha.8.beta.1, .alpha.v.beta.1,
.alpha.v.beta.3, .alpha.-IIb/.beta.. .alpha.4.beta.1,
.alpha.5.beta.1, .alpha.8.beta.1, .alpha.v.beta.1 are specific
receptor for fibronectin (Topol et al., 1997).
[0073] .alpha.5.beta.1: is a specific receptor for fibronectin.
This integrin has been found to be expressed by SMCs in the media
of human aorta whereas this protein was absent in the destructive
media of aneurysmal aorta. The marked decrease in integrin
.alpha.5.beta.1 correlated to a decrease in density of SMCs (Cheuk
and Cheng, 2004). Studies in a rat model of vascular injury, have
shown that .alpha.5.beta.1 integrin was not expressed in medial
SMCs but highly expressed after vascular injury by the less
differentiated SMCs at the luminal surface of the neointima. This
subpopulation of .alpha.5.beta.1 positive SMCs repairs the arterial
wall by assembling the fibronectin matrix. Soluble fibronectin
protomers polymerize on the surface of these .alpha.5.beta.1
positive cells. This assembly process is of paramount importance
for wall repair because only insoluble fibrillar fibronectin can
act as an adhesive ligand and regulate cell function (Pickering et
al., 2000). In conclusion, these data indicate that .alpha.5.beta.1
integrin, a specific receptor for fibronectin is critical for
maintaining the integrity of the medial layer of normal artery and
for the processus of wall repair and neointima formation after
vascular injury. It is also expressed by T cells, monocytes,
platelets.
[0074] .alpha.8.beta.1: is a specific receptor for fibronectin.
.alpha.8 subunit has a restricted cellular distribution. It is
expressed in vascular and visceral smooth muscle cells. SMCs of
arteries show an intense staining. The endothelial cells of vessels
do not stain. The cells that expressed .alpha.8 subunit function as
contractile cells (Schnapp et al., 1995).
[0075] c. Integrin Receptors as Markers of Smooth Muscle Cell
Phenotype
[0076] According to the above data, .alpha.1.beta.1,
.alpha.7.beta.1, .alpha.3.beta.1, .alpha.8.beta.1 are useful
specific markers of differentiated contractile SMCs (these
integrins are highly specific of SMCs and are not expressed by
other cell types such as endothelial cells or platelets). Moreover,
.alpha.7 integrin-expression confers a gain of function-motile
phenotype to immobile cells and may be responsible for transduction
of the laminin-induced cell motility.
[0077] In contrast, .alpha.5.beta.1 a specific receptor or
fibronectin is expressed by less differentiated SMCs of the
neointima formation and is critical for fibronectin assembly and
wall repair after injury. .alpha.v.beta.3 is an integrin that is
widely expressed in the vessel wall (endothelial and SMCs cells)
and has multiple ECM ligands is overexpressed in neointima
formation.
[0078] d. Neointima Formation in Occlusive Arterial Disease.
[0079] Neointima formation is a common mechanism of arterial wall
repair after endothelial injury and occurs in vascular diseases
such as atherosclerosis and restenosis after angioplasty.
Endothelium injury induces platelet adhesion which induces the
release of growth factors and especially PDGF. PDGF stimulates
migration of SMCs for the media to the intima. Within the intima,
SMCs bind fibronectin (FN) of the basement membrane. Fibronectin
stimulates the cells to adopt a synthetic phenotype and then PDGF
stimulate the proliferation of the dedifferentiate SMCs. SMCs in
the neointima highly expressed .alpha.5.beta.1 and .alpha.v.beta.3
integrins.
[0080] B. Exemplary Model for Pathogenesis of Cerebral Aneurysm
[0081] It has been demonstrated that hemodynamic stress is
responsible for endothelium injury. But, in contrast to vascular
diseases such as atherosclerosis and restenosis after angioplasty,
neointima formation does not occur and there is no repair of the
arterial wall. During aneurysm formation, there is disappearance of
the medial layer. At an advanced stage, the aneurysm wall consists
only of a fibrous adventitia and a layer of endothelial cells.
Therefore, some factors that contribute to wound healing may be
missing in the course of cerebral aneurysm formation. As described
above, studies have demonstrated that fibronectin (as well as
collagen IV and I) normally expressed in the subendothelium of
artery, disappears in early aneurysmal lesions. This degeneration
of the endothelial basement membrane and the subendothelial
connective tissue may be due to endothelial dysfunction. Indeed,
degeneration of endothelial cells in aneurismal walls has been
demonstrated by scanning electron microscopy (described above) and
these degenerated cells may decrease the production of ECM. Also,
hemodynamic stress by itself may alter the subendothelium matrix.
The absence of fibronectin in aneurysm wall is a critical feature
in aneurysm formation considering the role of this ECM protein in
wound repair and its role in modulation of SMC phenotype (as
described above). In the invention, SMCs migrate from the media to
the intima toward growth factor (especially PDGF) secreted after
endothelial injury using laminin-binding .alpha.7 integrin. Because
of the absence of fibronectin, SMCs can not switch their phenotype
from a contractile to a synthetic phenotype. These cells can not
proliferate to form a neointima. Furthermore, since fibronectin is
known to facilitate SMC survival by providing integrin-mediated
inhibition of apoptose, in absence of fibronectin, SMCs may enter
apoptosis and disappear. The SMCs that migrate to the intima in the
early stage of aneurysm formation are SMCs of contractile phenotype
and expressed .alpha.1.beta.1, .alpha.3.beta.1, .alpha.7.beta.1 and
.alpha.8.beta.1 integrins in contrast to SMCs in the neointima
formation which are of synthetic phenotype and express
.alpha.5.beta.1 and .alpha.v.beta.3 integrins.
[0082] FIG. 1 illustrates an exemplary schema outlining embodiments
of the present invention for cerebral aneurysm formation versus
neointima formation. In certain embodiments of the invention,
integrin .alpha.7.beta.1 which is specific cell surface marker of
SMCs (they are not expressed by endothelial cells nor platelets)
and is characteristic of their contractile phenotype is the best
antigenic subendothelial components in the arterial wall useful for
in vivo immuno-detection of early-stage aneurysm. Data from the
literature suggest a prominent role for .beta.7 integrin in
vascular remodeling as described above, and in specific embodiments
.beta.7 integrin is a useful target antigen.
[0083] In vivo, human medial contractile SMCs are surrounded by a
basement membrane that contains laminin 1 and/or laminin 3 (Yao et
al., 1997) Laminin 1 is not expressed by the basement membrane of
endothelium (which is composed of laminin 8 and 10) (Halmann et
al., 2005; Falk et al., 1999; Viranen et al., 2000). It has been
demonstrated that .alpha.7 integrin binds to specific laminin
isoforms mediating adhesion and migration of SMCs (Yao et al.,
1996): .alpha.7 integrin binds to laminin 1 and a mixture of
laminin 2 and 4 but not to laminin 5. In contrast, it has been
demonstrated that Laminin 5, an ECM protein found predominantly in
epithelial tissues is expressed at low-level in the intima of
normal vessels but is overexpressed in the neointima of injured
vessels (Kingsley et al., 2002). In specific aspects, laminin 1 is
an antigenic subendothelial component of aneurismal lesions while
laminin 5 is a marker of neointima formation.
[0084] In certain aspects of the invention, immunohistochemistry is
utilized to characterize the in situ expression of .alpha.7
integrin and laminin-1 in the arterial wall of early aneurysmal
lesions in an established animal model of cerebral aneurysm. In
other aspects of the invention, the in vivo detection of cerebral
aneurysms is characterized by nuclear imaging or molecular MRI
using labeled antibodies directed against .alpha.7 integrin or
laminin-1 injected intravascularly.
VI. Compounds of the Invention
[0085] The compounds of the invention (which may also be referred
to as compositions of the invention) are prepared such that they
are suitable for detection and/or therapy of an aneurysm in an
individual. In general embodiments, the compounds comprise
different combinations of a targeting molecule, a label, an
intravascular targeting molecule, and a therapeutic agent.
[0086] A. Cell Targeting Molecule
[0087] The cell targeting molecule of the invention is any moiety
that is suitable to target a composition to a smooth muscle cell at
a cerebral aneurysm. In specific embodiments, the targeting
molecule is an antibody or a peptide, and in particular cases the
antibody or peptide targets proteins on the surface of the smooth
muscle cell, such as proteins that are receptors, for example.
[0088] 1. Antibodies
[0089] In specific embodiments of the invention, the cell targeting
molecule is an antibody. Although the antibody may immunologically
react with any target that identifies a smooth muscle cell at the
site of an aneurysm, in specific embodiments the antibody targets a
protein, such as a receptor, on the cell. In certain cases, the
antibody will bind exclusively smooth muscle cells exposed at the
luminal surface of the vessel, and in particular cases the antibody
does not bind contractile smooth muscle cells in the medial layer
of the normal arterial wall. In specific embodiment, the receptor
is an integrin or is a laminin. In further specific embodiments,
the antibody recognizes .alpha.7 integrin or laminin-1
[0090] 2. Peptides
[0091] In other embodiments of the invention, the cell targeting
molecule is a peptide. Although the peptide may bind with any
target on a smooth muscle cell at the site of an aneurysm, in
specific embodiments the peptide targets a protein, such as a
receptor, on the cell. In particular cases, the peptide will bind
exclusively smooth muscle cells exposed at the luminal surface of
the vessel, and in specific cases the peptide does not bind
contractile smooth muscle cells in the medial layer of the normal
arterial wall. In specific embodiment, the receptor is an integrin
or is a laminin. In further specific embodiments, the peptide
recognizes .alpha.7 integrin or laminin-1. In further specific
embodiments, the peptide also known as peptidomimetic (small
protein-like chain design to mimic a peptide) competes with
laminin-1 to bind .alpha.7 integrin. This peptidomimetic of
laminin-1 may be derived from the G domain of the .alpha.1 chain of
laminin-1 such as the .alpha.1 chain aminoacids 2179-2198 "SN
peptide" (Khan et al., 2002)
[0092] B. Labels
[0093] In particular aspects of the invention, one or more labels
are comprised on a composition of the invention. The label may be
attached to the cell targeting molecule, the intravascular
targeting molecule, and/or the therapeutic agent. Although any
labels suitable in the art may be employed, in specific embodiments
the label is a radionuclide, a fluorophore, a lucigen, or a
paramagnetic chelator or microbubble contrast agent.
[0094] A label moiety may be attached to the antibody moiety using
techniques readily available to the public. The label moiety may be
a radioactive label such as a gamma ray emitting radionuclide: 111
Indium, Technetium-99m, iodine 123 (123I), iodine 125 (125I). A
chelating agent such as dipropylaminetetraacetic acid (DPTA) may be
used to associate the radioactive label to the antibody. The label
moiety may be a positron emitting radionuclide such as Fluorin-18,
carbon-11, Gallium 68. The label moiety may be a near infrared
fluorophore (near infrared fluorescent dye) such as Cy7-NHS, Cy5.5
(Amersham Pharmacia). The label moiety may be a MRI contrast agent,
such as paramagnetic lanthanide Gadolinium or superparamagnetic
particles of iron oxide (SPIOs), for example. The label moiety may
be also a microbubble contrast agent.
[0095] C. Intravascular Targeting Molecule
[0096] In general embodiments of the invention, the composition
comprises a moiety that prevents the cell targeting molecule from
moving beyond the luminal surface of the aneurysmal wall to bind
smooth muscle cells in the medial layer of normal vascular wall
that harbors an intact endothelium. Any macromolecular agent with
intravascular retention may be coupled to the cell targeting
molecule is useful. The molecule may have a molecular weight that
confines it within the vessels, yet allows it to be cleared from
the intravascular compartment. In a specific embodiment, the
molecule is a macromolecule, and macromolecular species may include
any molecule, natural or synthetic, which has a molecular weight in
excess of 1 kilodalton such as but not limited to albumin,
transferrin, globulins, pectin, gelatin, dextran, and cellulose
derivatives, for example.
[0097] In specific embodiments, the molecule is a polymer. In
particular embodiments, the molecule is dextran. Dextran is a
hydrophilic molecule that does not pass the phospholipid bilayer of
endothelial membrane. Antibody (or any cell targeting molecule)
conjugated to dextran will not pass the endothelium. The conjugate
will bind specifically smooth muscle cells at the luminal surface
of the aneurysmal wall, such as .alpha.7 integrin or laminin-1
exposed at the luminal surface of the aneurysmal wall, and cannot
bind .alpha.7 integrin or laminin-1 on SMCs in the medial layer of
normal vascular wall that harbors an intact endothelium.
[0098] D. Therapeutic Agent
[0099] In particular aspects of the invention, the compounds for
delivery to the individual with the aneurysm or suspected of having
an aneurysm comprise one or more therapeutic agents. In specific
cases, the therapeutic agent is bound directly to the label, the
intravascular targeting molecule, and/or the cell targeting
molecule. Although in specific cases the therapeutic agent
comprises a thrombogenic or polymerisable molecule (intended to
clog the aneurysms) or proteins (elastin, fibronectin, or
fibrinogen, for example), or cell growth factors (intended to
reinforce or make the fundus thicker and stronger).
[0100] In a specific embodiment, the therapeutic agent is a
compound able to convert SMCs of contractile phenotype into SMCs of
synthetic phenotype. In a preferred embodiment, this compound
comprises an antibody directed against .alpha.7.beta.1 integrin
coupled to fibronectin or fragments of fibronectin (specific
fibronectin peptides). In an other embodiment, the
anti-.alpha.7.beta.1 integrin antibody is coupled to fibronectin or
fragments of fibronectin (specific fibronectin peptides) and Growth
Factors. In contrast to cerebral aneurysm formation, neo-intima
formation that occurs in vascular disease such as re-stenosis after
angioplasty, will be treated or prevented by using a compound able
to convert SMCs of synthetic phenotype into SMCs of contractile
phenotype. In this embodiment, the therapeutic agent is composed of
antibody directed against .alpha.5.beta.1 integrin coupled to
laminin, and especially laminin-1 (FIG. 2).
[0101] E. Assembly of the Compounds
[0102] The compounds of the invention may be assembled in any
suitable manner. In specific embodiments, however, Dextran is the
molecule used as the intravascular targeting molecule. For nuclear
imaging Technetium-99m-Dextran is prepared according to method
known in the art (Line et al., 2000) and then coupled to the cell
targeting molecule (antibody or peptide). 99mTc-Dextran is
restricted to the blood pool and is broken down by the liver and
cleared through the kidneys. These properties allow low background
activity in the extra vascular space and rapid clearance from the
intra vascular space. Radiolabeling through 99mTc-Dextran will
avoid the diffusion of the compound to the extra vascular
compartment. 99mTc-Dextran coupled to the cell targeting molecule
will bind specifically SMCs of the luminal surface of the
aneurysmal lesion and will not bind SMCs in the medial layer of
normal artery. For molecular MRI, gadolinium-DPTA-Dextran will be
prepared according to method known in the art (Sirlin C B et al.,
2004) and coupled to the cell targeting molecule.
VII. Detection of the Compounds
[0103] Detection of a compound accumulated at the aneurysm site/or
in the aneurysm vicinity may be performed according to techniques
known in the art and which may vary depending on the characteristic
of the label moiety of the conjugate. For example, when the
conjugate comprises a gamma-ray emitting radionuclide, the aneurysm
may be detected by scintigraphy using a gamma-camera or by single
photon emission computed tomography (SPECT). When the conjugate
comprises a positron emitting radionuclide, the aneurysm may be
detected by positron emission tomography (PET). Combined positron
emission tomography (PET) and computerized tomography (CT) or
magnetic resonance (MR) can be used (PET/CT or PET/MR). This
combined technique allows accurate detection and localization for
aneurysmal lesions. When the conjugate comprises a MRI agent
contrast, the aneurysm may be detected by MRI. When the conjugate
comprises a positron emitting radionuclide, the aneurysm may be
detected by positron emission tomography (PET). When the conjugate
is a near infrared fluorophore (near infrared fluorescent dye), the
aneurysm may be detected by near-infrared imager. When the
conjugate is a microbubble contrast agent, the aneurysm may be
detected by ultrasound imagery.
[0104] Thus, different molecular imaging techniques could be used
depending on the nature of the labeling agent such as
immunoscintigraphy and SPECT using antibody radiolabeled with
99mTc-dextran or MRI using antibody conjugated to
Gadolinium-DTPA-dextran. Ultrasound may also be employed. Other
techniques could use a positron emitting radionuclide and the
aneurysm will be detected by positron emission tomography
(PET).
VIII. Methods of Using the Compounds
[0105] In general embodiments of the invention, the compounds are
employed to detect an aneurysm, and in further embodiments the
compounds are used to treat an aneurysm. In certain cases the same
compound is employed to detect the aneurysm as to treat the
aneurysm, although in other cases a different compound is employed
to detect the aneurysm as to treat the aneurysm. The methods may be
employed in an individual suspected of having an aneurysm
(symptomatic aneurysm, for example), an individual at risk for
having an aneurysm (such as one with head trauma, high blood
pressure, cigarette smoking, or having certain disease states known
to be associated with an increased prevalence of aneurysm such as,
but not limited to familial intracranial aneurysm, autosomal
dominant polycystic kidney disease, fibrous dysplasia or
coarctation of the aorta, for example), an individual that has a
history of aneurysms, or an individual known to harbor aneurysm(s)
(multiple aneurysms, for example).
[0106] A. Diagnosis
[0107] The present invention utilizes particular compounds to
detect one or more aneurysms in an individual. In certain cases,
the compound is delivered to the individual, the compound localizes
to the aneurysm site, and the brain or a part thereof is monitored
for detection of the compound. When the compound is detected at the
localized site, the individual may be given therapy for the
aneurysm, using a compound of the present invention having a
therapeutic agent, endovascular coil embolization or surgical clip
occlusion.
[0108] The detection of a signal at the major brain artery site and
more particularly, at or near the apex of arterial forks may be
indicative of a cerebral aneurysm. A positive detection of a signal
may be followed by a digital subtraction angiography in patients in
need thereof. Follow-up may be performed using the same method of
diagnosis to ensure the disappearance or reduction of any
intracranial labeling after treatment.
[0109] Currently, ruptured aneurysms are diagnosed with the aid of
computerized tomography scanning followed by a 4-axle digital
subtraction angiography.
[0110] Unruptured aneurysms are mainly found by serendipity during
evaluation of neurological non-aneurysmal disease (incidental
aneurysm), part of a multiple aneurysm constellation (multiple
aneurysms), or those that are symptomatic (symptomatic
aneurysm).
[0111] Both computerized tomography scanning and magnetic resonance
angiography are poorer methods than digital subtraction angiography
for detection of aneurysms smaller than 5 mm. Such invasive method
cannot be used for mass detection.
[0112] Methods and compositions of the present invention that can
non-invasively detect and treat ruptured as well as unruptured
aneurysms is a more desirable embodiment and allows mass detection
and preventive treatment.
[0113] B. Therapy
[0114] In particular aspects, the present invention employs a
therapeutic agent on the compound, wherein the compound targets
subendothelial components of an aneurysmal wall. In specific
embodiments, the aneurysm is suspected of being in the individual,
although in other cases the aneurysm has already been detected,
including by a compound of the present invention, for example. The
therapy may be delivered to the individual once or multiple
times.
[0115] An exemplary embodiment of a therapeutic molecule may
include for example, but not limited to a compound for inducing
thrombosis, a compound for promoting aneurismal wall thickening
and/or a compound for promoting cell growth. The therapeutic
molecule may, more particularly be selected from the group
consisting of a thrombogenic molecule, a polymerisable molecule
(intended to clog the aneurysms), a protein (e.g., elastin,
fibronectin, or fibrinogen etc), and a cell growth factor (intended
to reinforce or make the fundus thicker and stronger). Another
exemplary embodiment of a therapeutic molecule may include for
example a protease inhibitor, such as an elastase inhibitor or a
matrix metallo-proteinase inhibitor. Elastase inhibitors may
include, without limitation, alpha-1 antitrypsin, alpha-2
macroglobulin which are the main elastase inhibitors in the serum.
Elafin is also a potent inhibitor of elastase and proteinase 3
which is encompassed herewith. Matrix metallo-proteinase inhibitors
may include, for example and without limitation tissue inhibitors
of metallo-proteinase (TIMPs) or synthetic inhibitors known in the
art, such as tetracyclines and tetracycline derivatives such as
doxycycline.
IX. Combination Therapy
[0116] In order to increase the effectiveness of a compound of the
invention, it may be desirable to combine these compositions with
other therapies effective in the treatment of aneurysms. More
generally, these other compositions would be provided in a combined
amount effective to treat the aneurysm. This process may involve
treating the individual with the compound of the invention and the
additional therapy (drugs and/or surgery and/or endovascular
coiling) at the same time. In cases wherein the additional therapy
is a compound, as opposed to surgery and/or endovascular coiling,
this may be achieved by providing the individual with a single
composition or pharmacological formulation that includes both
agents, or by delivering to the individual with the two distinct
compositions or formulations, at the same time, wherein one
composition includes the therapy of the invention and the other
includes the second agent(s).
[0117] In the context of the present invention, it is contemplated
that the composition of the invention could be used in conjunction
with surgery and/or endovascular coiling or drug(s). Alternatively,
delivery of the compound of the present invention may precede or
follow the other agent treatment by intervals ranging from minutes
to weeks. In embodiments where the other agent (or surgery and/or
endovascular coiling) and the compound of the present invention are
delivered separately to the individual, one would generally ensure
that a significant period of time did not expire between the time
of each delivery, such that the other agent and compound of the
invention would still be able to exert an advantageously combined
effect on the cell. In such instances, it is contemplated that one
may contact the cell with both modalities within about 12-24 h of
each other and, more preferably, within about 6-12 h of each other.
In some situations, it may be desirable to extend the time period
for treatment significantly, however, where several d (2, 3, 4, 5,
6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the
respective administrations.
[0118] In some cases, the treatments are repeated as necessary.
X. Antibodies
[0119] In certain aspects of the invention, one or more antibodies
are employed in the methods and compositions of the invention.
These antibodies may be used in various diagnostic or therapeutic
applications, described herein below. As used herein the term
"antibody" means a polyclonal antibody, a monoclonal antibody, a
chimeric antibody, a humanized antibody, a deimmunized antibody, an
antigen-binding fragment, an Fab fragment; an F(ab')2 fragment, and
Fv fragment, or a synthetic molecule comprising an antigen-binding
fragment.
[0120] In some cases, the term "antibody" is intended to refer
broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD
and IgE. In certain cases, IgG and/or IgM may be utilized, because
they are the most common antibodies in the physiological situation
and because they are most easily made in a laboratory setting.
[0121] The term "antibody" is used to refer to any antibody-like
molecule that has an antigen binding region, and includes antibody
fragments such as Fab', Fab, F(ab')2, single domain antibodies
(DABs), Fv, scFv (single chain Fv), and the like. The techniques
for preparing and using various antibody-based constructs and
fragments are well known in the art. Means for preparing and
characterizing antibodies are also well known in the art (See,
e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, 1988; incorporated herein by reference).
[0122] "Mini-antibodies" or "minibodies" are also contemplated for
use with the present invention. Minibodies are sFv polypeptide
chains which include oligomerization domains at their C-termini,
separated from the sFv by a hinge region. Pack et al. (1992)
Biochem 31:1579-1584. The oligomerization domain comprises
self-associating .alpha.-helices, e.g., leucine zippers, that can
be further stabilized by additional disulfide bonds. The
oligomerization domain is designed to be compatible with vectorial
folding across a membrane, a process thought to facilitate in vivo
folding of the polypeptide into a functional binding protein.
Generally, minibodies are produced using recombinant methods well
known in the art. See, e.g., Pack et al. (1992) Biochem
31:1579-1584; Cumber et al. (1992) J Immunology 149B:120-126.
[0123] Antibody-like binding peptidomimetics are also contemplated
in the present invention. Liu et al. Cell Mol Biol
(Noisy-le-grand). 2003 March; 49(2):209-16 describe "antibody like
binding peptidomimetics" (ABiPs), which are peptides that act as
pared-down antibodies and have certain advantages of longer serum
half-life as well as less cumbersome synthesis methods.
[0124] Monoclonal antibodies (MAbs) are recognized to have certain
advantages, e.g., reproducibility and large-scale production, and
their use is generally preferred. The invention thus provides
monoclonal antibodies of the human, murine, monkey, rat, hamster,
rabbit and even chicken origin. Due to the ease of preparation and
ready availability of reagents, murine monoclonal antibodies will
often be preferred.
[0125] However, "humanized" antibodies are also contemplated, as
are chimeric antibodies from mouse, rat, or other species, bearing
human constant and/or variable region domains, bispecific
antibodies, recombinant and engineered antibodies and fragments
thereof. As used herein, the term "humanized" immunoglobulin refers
to an immunoglobulin comprising a human framework region and one or
more CDR's from a non-human (usually a mouse or rat)
immunoglobulin. The non-human immunoglobulin providing the CDR's is
called the "donor" and the human immunoglobulin providing the
framework is called the "acceptor". A "humanized antibody" is an
antibody comprising a humanized light chain and a humanized heavy
chain immunoglobulin.
[0126] A. Methods for Generating Monoclonal Antibodies
[0127] The methods for generating monoclonal antibodies (MAbs)
generally begin along the same lines as those for preparing
polyclonal antibodies. Briefly, a polyclonal antibody is prepared
by immunizing an animal with a LEE or CEE composition in accordance
with the present invention and collecting antisera from that
immunized animal.
[0128] A wide range of animal species can be used for the
production of antisera. Typically the animal used for production of
antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a
goat. The choice of animal may be decided upon the ease of
manipulation, costs or the desired amount of sera, as would be
known to one of skill in the art. Antibodies of the invention can
also be produced transgenically through the generation of a mammal
or plant that is transgenic for the immunoglobulin heavy and light
chain sequences of interest and production of the antibody in a
recoverable form therefrom. In connection with the transgenic
production in mammals, antibodies can be produced in, and recovered
from, the milk of goats, cows, or other mammals. See, e.g., U.S.
Pat. Nos. 5,827,690, 5,756,687, 5,750,172, and 5,741,957.
[0129] As is also well known in the art, the immunogenicity of a
particular immunogen composition can be enhanced by the use of
non-specific stimulators of the immune response, known as
adjuvants. Suitable adjuvants include all acceptable
immunostimulatory compounds, such as cytokines, chemokines,
cofactors, toxins, plasmodia, synthetic compositions or LEEs or
CEEs encoding such adjuvants.
[0130] Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7,
IL-12, .gamma.-interferon, GMCSP, BCG, aluminum hydroxide, MDP
compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and
monophosphoryl lipid A (MPL). RIBI, which contains three components
extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell
wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion is also
contemplated. MHC antigens may even be used. Exemplary, often
preferred adjuvants include complete Freund's adjuvant (a
non-specific stimulator of the immune response containing killed
Mycobacterium tuberculosis), incomplete Freund's adjuvants and
aluminum hydroxide adjuvant.
[0131] In addition to adjuvants, it may be desirable to
coadminister biologic response modifiers (BRM), which have been
shown to upregulate T cell immunity or downregulate suppressor cell
activity. Such BRMs include, but are not limited to, Cimetidine
(CIM; 1200 mg/d) (Smith/Kline, PA); low-dose Cyclophosphamide (CYP;
300 mg/m2) (Johnson/Mead, NJ), cytokines such as
.gamma.-interferon, IL-2, or IL-12 or genes encoding proteins
involved in immune helper functions, such as B-7.
[0132] The amount of immunogen composition used in the production
of polyclonal antibodies varies upon the nature of the immunogen as
well as the animal used for immunization. A variety of routes can
be used to administer the immunogen including but not limited to
subcutaneous, intramuscular, intradermal, intraepidermal,
intravenous and intraperitoneal. The production of polyclonal
antibodies may be monitored by sampling blood of the immunized
animal at various points following immunization.
[0133] A second, booster dose (e.g., provided in an injection), may
also be given. The process of boosting and titering is repeated
until a suitable titer is achieved. When a desired level of
immunogenicity is obtained, the immunized animal can be bled and
the serum isolated and stored, and/or the animal can be used to
generate MAbs.
[0134] For production of rabbit polyclonal antibodies, the animal
can be bled through an ear vein or alternatively by cardiac
puncture. The removed blood is allowed to coagulate and then
centrifuged to separate serum components from whole cells and blood
clots. The serum may be used as is for various applications or else
the desired antibody fraction may be purified by well-known
methods, such as affinity chromatography using another antibody, a
peptide bound to a solid matrix, or by using, e.g., protein A or
protein G chromatography.
[0135] MAbs may be readily prepared through use of well-known
techniques, such as those exemplified in U.S. Pat. No. 4,196,265,
incorporated herein by reference. Typically, this technique
involves immunizing a suitable animal with a selected immunogen
composition, e.g., a purified or partially purified protein,
polypeptide, peptide or domain, be it a wild-type or mutant
composition. The immunizing composition is administered in a manner
effective to stimulate antibody producing cells.
[0136] The methods for generating monoclonal antibodies (MAbs)
generally begin along the same lines as those for preparing
polyclonal antibodies. Rodents such as mice and rats are preferred
animals, however, the use of rabbit, sheep or frog cells is also
possible. The use of rats may provide certain advantages (Goding,
1986, pp. 60 61), but mice are preferred, with the BALB/c mouse
being most preferred as this is most routinely used and generally
gives a higher percentage of stable fusions.
[0137] The animals are injected with antigen, generally as
described above. The antigen may be mixed with adjuvant, such as
Freund's complete or incomplete adjuvant. Booster administrations
with the same antigen or DNA encoding the antigen would occur at
approximately two-week intervals.
[0138] Following immunization, somatic cells with the potential for
producing antibodies, specifically B lymphocytes (B cells), are
selected for use in the MAb generating protocol. These cells may be
obtained from biopsied spleens, tonsils or lymph nodes, or from a
peripheral blood sample. Spleen cells and peripheral blood cells
are preferred, the former because they are a rich source of
antibody-producing cells that are in the dividing plasmablast
stage, and the latter because peripheral blood is easily
accessible.
[0139] Often, a panel of animals will have been immunized and the
spleen of an animal with the highest antibody titer will be removed
and the spleen lymphocytes obtained by homogenizing the spleen with
a syringe. Typically, a spleen from an immunized mouse contains
approximately 5.times.10.sup.7 to 2.times.10.sup.8 lymphocytes.
[0140] The antibody producing B lymphocytes from the immunized
animal are then fused with cells of an immortal myeloma cell,
generally one of the same species as the animal that was immunized.
Myeloma cell lines suited for use in hybridoma producing fusion
procedures preferably are non antibody producing, have high fusion
efficiency, and enzyme deficiencies that render then incapable of
growing in certain selective media which support the growth of only
the desired fused cells (hybridomas).
[0141] Any one of a number of myeloma cells may be used, as are
known to those of skill in the art (Goding, pp. 65 66, 1986;
Campbell, pp. 75 83, 1984). cites). For example, where the
immunized animal is a mouse, one may use P3 X63/Ag8, X63 Ag8.653,
NS1/1.Ag 4 1, Sp210 Ag14, FO, NSO/U, MPC 11, MPC11 X45 GTG 1.7 and
S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3 Ag 1.2.3, IR983F
and 4B210; and U 266, GM1500 GRG2, LICR LON HMy2 and UC729 6 are
all useful in connection with human cell fusions. See Yoo et al., J
Immunol Methods. 2002 Mar. 1; 261(1-2):1-20, for a discussion of
myeloma expression systems.
[0142] One preferred murine myeloma cell is the NS-1 myeloma cell
line (also termed P3-NS-1-Ag4-1), which is readily available from
the NIGMS Human Genetic Mutant Cell Repository by requesting cell
line repository number GM3573. Another mouse myeloma cell line that
may be used is the 8 azaguanine resistant mouse murine myeloma
SP2/0 non producer cell line.
[0143] Methods for generating hybrids of antibody producing spleen
or lymph node cells and myeloma cells usually comprise mixing
somatic cells with myeloma cells in a 2:1 proportion, though the
proportion may vary from about 20:1 to about 1:1, respectively, in
the presence of an agent or agents (chemical or electrical) that
promote the fusion of cell membranes. Fusion methods using Sendai
virus have been described by Kohler and Milstein (1975; 1976), and
those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by
Gefter et al., (1977). The use of electrically induced fusion
methods is also appropriate (Goding pp. 71 74, 1986).
[0144] Fusion procedures usually produce viable hybrids at low
frequencies, about 1.times.10.sup.-6 to 1.times.10.sup.-8. However,
this does not pose a problem, as the viable, fused hybrids are
differentiated from the parental, unfused cells (particularly the
unfused myeloma cells that would normally continue to divide
indefinitely) by culturing in a selective medium. The selective
medium is generally one that contains an agent that blocks the de
novo synthesis of nucleotides in the tissue culture media.
Exemplary and preferred agents are aminopterin, methotrexate, and
azaserine. Aminopterin and methotrexate block de novo synthesis of
both purines and pyrimidines, whereas azaserine blocks only purine
synthesis. Where aminopterin or methotrexate is used, the media is
supplemented with hypoxanthine and thymidine as a source of
nucleotides (HAT medium). Where azaserine is used, the media is
supplemented with hypoxanthine.
[0145] The preferred selection medium is HAT. Only cells capable of
operating nucleotide salvage pathways are able to survive in HAT
medium. The myeloma cells are defective in key enzymes of the
salvage pathway, e.g., hypoxanthine phosphoribosyl transferase
(HPRT), and they cannot survive. The B cells can operate this
pathway, but they have a limited life span in culture and generally
die within about two weeks. Therefore, the only cells that can
survive in the selective media are those hybrids formed from
myeloma and B cells.
[0146] This culturing provides a population of hybridomas from
which specific hybridomas are selected. Typically, selection of
hybridomas is performed by culturing the cells by single-clone
dilution in microtiter plates, followed by testing the individual
clonal supernatants (after about two to three weeks) for the
desired reactivity. The assay should be sensitive, simple and
rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity
assays, plaque assays, dot immunobinding assays, and the like.
[0147] The selected hybridomas would then be serially diluted and
cloned into individual antibody producing cell lines, which clones
can then be propagated indefinitely to provide MAbs. The cell lines
may be exploited for MAb production in two basic ways. First, a
sample of the hybridoma can be injected (often into the peritoneal
cavity) into a histocompatible animal of the type that was used to
provide the somatic and myeloma cells for the original fusion
(e.g., a syngeneic mouse). Optionally, the animals are primed with
a hydrocarbon, especially oils such as pristane
(tetramethylpentadecane) prior to injection. The injected animal
develops tumors secreting the specific monoclonal antibody produced
by the fused cell hybrid. The body fluids of the animal, such as
serum or ascites fluid, can then be tapped to provide MAbs in high
concentration. Second, the individual cell lines could be cultured
in vitro, where the MAbs are naturally secreted into the culture
medium from which they can be readily obtained in high
concentrations.
[0148] Further, expression of antibodies of the invention (or other
moieties therefrom) from production cell lines can be enhanced
using a number of known techniques. For example, the glutamine
sythetase and DHFR gene expression systems are common approaches
for enhancing expression under certain conditions. High expressing
cell clones can be identified using conventional techniques, such
as limited dilution cloning and Microdrop technology. The GS system
is discussed in whole or part in connection with European Patent
Nos. 0 216 846, 0 256 055, and 0 323 997 and European Patent
Application No. 89303964.4.
[0149] MAbs produced by either means may be further purified, if
desired, using filtration, centrifugation and various
chromatographic methods such as HPLC or affinity chromatography.
Fragments of the monoclonal antibodies of the invention can be
obtained from the monoclonal antibodies so produced by methods
which include digestion with enzymes, such as pepsin or papain,
and/or by cleavage of disulfide bonds by chemical reduction.
Alternatively, monoclonal antibody fragments encompassed by the
present invention can be synthesized using an automated peptide
synthesizer.
[0150] It is also contemplated that a molecular cloning approach
may be used to generate monoclonals. In one embodiment,
combinatorial immunoglobulin phagemid libraries are prepared from
RNA isolated from the spleen of the immunized animal, and phagemids
expressing appropriate antibodies are selected by panning using
cells expressing the antigen and control cells. The advantages of
this approach over conventional hybridoma techniques are that
approximately 10.sup.4 times as many antibodies can be produced and
screened in a single round, and that new specificities are
generated by H and L chain combination which further increases the
chance of finding appropriate antibodies. In another example, LEEs
or CEEs can be used to produce antigens in vitro with a cell free
system. These can be used as targets for scanning single chain
antibody libraries. This would enable many different antibodies to
be identified very quickly without the use of animals.
[0151] Another embodiment of the invention for producing antibodies
according to the present invention is found in U.S. Pat. No.
6,091,001, which describes methods to produce a cell expressing an
antibody from a genomic sequence of the cell comprising a modified
immunoglobulin locus using Cre-mediated site-specific recombination
is disclosed. The method involves first transfecting an
antibody-producing cell with a homology-targeting vector comprising
a lox site and a targeting sequence homologous to a first DNA
sequence adjacent to the region of the immunoglobulin loci of the
genomic sequence which is to be converted to a modified region, so
the first lox site is inserted into the genomic sequence via
site-specific homologous recombination. Then the cell is
transfected with a lox-targeting vector comprising a second lox
site suitable for Cre-mediated recombination with the integrated
lox site and a modifying sequence to convert the region of the
immunoglobulin loci to the modified region. This conversion is
performed by interacting the lox sites with Cre in vivo, so that
the modifying sequence inserts into the genomic sequence via
Cre-mediated site-specific recombination of the lox sites.
[0152] Alternatively, monoclonal antibody fragments encompassed by
the present invention can be synthesized using an automated peptide
synthesizer, or by expression of full-length gene or of gene
fragments in E. coli.
[0153] B. Antibody Conjugates
[0154] The present invention further provides antibodies against
contractile SMC surface proteins, polypeptides and peptides that
are linked to at least one agent to form an antibody conjugate. In
order to increase the efficacy of antibody molecules as diagnostic
or therapeutic agents, it is conventional to link or covalently
bind or complex at least one desired molecule or moiety. Such a
molecule or moiety may be, but is not limited to, at least one
effector or reporter molecule. Effector molecules comprise
molecules having a desired activity, e.g., cytotoxic activity.
Non-limiting examples of effector molecules which have been
attached to antibodies include toxins, anti-tumor agents,
therapeutic enzymes, radio-labeled nucleotides, antiviral agents,
chelating agents, cytokines, growth factors, and oligo- or
poly-nucleotides. By contrast, a reporter molecule is defined as
any moiety which may be detected using an assay. Non-limiting
examples of reporter molecules that have been conjugated to
antibodies include enzymes, radiolabels, radionuclides, haptens,
fluorescent labels, phosphorescent molecules, chemiluminescent
molecules, chromophores, luminescent molecules, photoaffinity
molecules, colored particles or ligands, such as biotin.
[0155] Any antibody of sufficient selectivity, specificity or
affinity may be employed as the basis for an antibody conjugate.
Such properties may be evaluated using conventional immunological
screening methodology known to those of skill in the art. Sites for
binding to biological active molecules in the antibody molecule, in
addition to the canonical antigen binding sites, include sites that
reside in the variable domain that can bind pathogens, B-cell
superantigens, the T cell co-receptor CD4 and the HIV-1 envelope
(Sasso et al., 1989; Shorki et al., 1991; Silvermann et al., 1995;
Cleary et al., 1994; Lenert et al., 1990; Berberian et al., 1993;
Kreier et al., 1991). In addition, the variable domain is involved
in antibody self-binding (Kang et al., 1988), and contains epitopes
(idiotopes) recognized by anti-antibodies (Kohler et al.,
1989).
[0156] Certain examples of antibody conjugates are those conjugates
in which the antibody is linked to a detectable label. "Detectable
labels" are compounds and/or elements that can be detected due to
their specific functional properties, and/or chemical
characteristics, the use of which allows the antibody to which they
are attached to be detected, and/or further quantified if desired.
Another such example is the formation of a conjugate comprising an
antibody linked to a cytotoxic or anti cellular agent, and may be
termed "immunotoxins".
[0157] Antibody conjugates are generally preferred for use as
diagnostic agents. Antibody diagnostics generally fall within two
classes, those for use in in vitro diagnostics, such as in a
variety of immunoassays, and/or those for use in vivo diagnostic
protocols, generally known as "antibody directed imaging".
[0158] Many appropriate imaging agents are known in the art, as are
methods for their attachment to antibodies (see, for e.g., U.S.
Pat. Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporated
herein by reference). The imaging moieties used can be paramagnetic
ions; radioactive isotopes; fluorochromes; NMR-detectable
substances; X-ray imaging.
[0159] In the case of paramagnetic ions, one might mention by way
of example ions such as chromium (III), manganese (II), iron (III),
iron (II), cobalt (II), nickel (II), copper (II), neodymium (III),
samarium (III), ytterbium (III), gadolinium (III), vanadium (II),
terbium (III), dysprosium (III), holmium (III) and/or erbium (III),
with gadolinium being particularly preferred. Ions useful in other
contexts, such as X-ray imaging, include but are not limited to
lanthanum (III), gold (III), lead (II), and especially bismuth
(III).
[0160] In the case of radioactive isotopes for therapeutic and/or
diagnostic application, one might mention astatine211, 14carbon,
51chromium, 36chlorine, 57cobalt, 58cobalt, copper67, 152Eu,
gallium67, 3hydrogen, iodine123, iodine125, iodine131, indium111,
59iron, 32phosphorus, rhenium186, rhenium188, 75selenium,
35sulphur, technicium99m and/or yttrium90. .sup.125I is often being
preferred for use in certain embodiments, and technicium99m and/or
indium111 are also often preferred due to their low energy and
suitability for long range detection. Radioactively labeled
monoclonal antibodies of the present invention may be produced
according to well-known methods in the art. For instance,
monoclonal antibodies can be iodinated by contact with sodium
and/or potassium iodide and a chemical oxidizing agent such as
sodium hypochlorite, or an enzymatic oxidizing agent, such as
lactoperoxidase. Monoclonal antibodies according to the invention
may be labeled with technetium99m by ligand exchange process, for
example, by reducing pertechnate with stannous solution, chelating
the reduced technetium onto a Sephadex column and applying the
antibody to this column. Alternatively, direct labeling techniques
may be used, e.g., by incubating pertechnate, a reducing agent such
as SNCl2, a buffer solution such as sodium-potassium phthalate
solution, and the antibody. Intermediary functional groups which
are often used to bind radioisotopes which exist as metallic ions
to antibody are diethylenetriaminepentaacetic acid (DTPA) or
ethylene diaminetetracetic acid (EDTA).
[0161] Among the fluorescent labels contemplated for use as
conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650,
BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX,
Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX,
6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514,
Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin,
ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.
[0162] Another type of antibody conjugates contemplated in the
present invention are those intended primarily for use in vitro,
where the antibody is linked to a secondary binding ligand and/or
to an enzyme (an enzyme tag) that will generate a colored product
upon contact with a chromogenic substrate. Examples of suitable
enzymes include urease, alkaline phosphatase, (horseradish)
hydrogen peroxidase or glucose oxidase. Preferred secondary binding
ligands are biotin and/or avidin and streptavidin compounds. The
use of such labels is well known to those of skill in the art and
are described, for example, in U.S. Pat. Nos. 3,817,837; 3,850,752;
3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each
incorporated herein by reference.
[0163] Yet another known method of site-specific attachment of
molecules to antibodies comprises the reaction of antibodies with
hapten-based affinity labels. Essentially, hapten-based affinity
labels react with amino acids in the antigen binding site, thereby
destroying this site and blocking specific antigen reaction.
However, this may not be advantageous since it results in loss of
antigen binding by the antibody conjugate.
[0164] Molecules containing azido groups may also be used to form
covalent bonds to proteins through reactive nitrene intermediates
that are generated by low intensity ultraviolet light (Potter &
Haley, 1983). In particular, 2- and 8-azido analogues of purine
nucleotides have been used as site-directed photoprobes to identify
nucleotide binding proteins in crude cell extracts (Owens &
Haley, 1987; Atherton et al., 1985). The 2- and 8-azido nucleotides
have also been used to map nucleotide binding domains of purified
proteins (Khatoon et al., 1989; King et al., 1989; and Dholakia et
al., 1989) and may be used as antibody binding agents.
[0165] Several methods are known in the art for the attachment or
conjugation of an antibody to its conjugate moiety. Some attachment
methods involve the use of a metal chelate complex employing, for
example, an organic chelating agent such a
diethylenetriaminepentaacetic acid anhydride (DTPA);
ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;
and/or tetrachloro-3.alpha.-6.alpha.-diphenylglycouril-3 attached
to the antibody (U.S. Pat. Nos. 4,472,509 and 4,938,948, each
incorporated herein by reference). Monoclonal antibodies may also
be reacted with an enzyme in the presence of a coupling agent such
as glutaraldehyde or periodate. Conjugates with fluorescein markers
are prepared in the presence of these coupling agents or by
reaction with an isothiocyanate. In U.S. Pat. No. 4,938,948,
imaging of breast tumors is achieved using monoclonal antibodies
and the detectable imaging moieties are bound to the antibody using
linkers such as methyl-p-hydroxybenzimidate or
N-succinimidyl-3-(4-hydroxyphenyl)propionate.
[0166] In other embodiments, derivatization of immunoglobulins by
selectively introducing sulfhydryl groups in the Fc region of an
immunoglobulin, using reaction conditions that do not alter the
antibody combining site are contemplated. Antibody conjugates
produced according to this methodology are disclosed to exhibit
improved longevity, specificity and sensitivity (U.S. Pat. No.
5,196,066, incorporated herein by reference). Site-specific
attachment of effector or reporter molecules, wherein the reporter
or effector molecule is conjugated to a carbohydrate residue in the
Fc region have also been disclosed in the literature (O'Shannessy
et al., 1987). This approach has been reported to produce
diagnostically and therapeutically promising antibodies which are
currently in clinical evaluation.
[0167] In another embodiment of the invention, the antibodies are
linked to semiconductor nanocrystals such as those described in
U.S. Pat. Nos. 6,048,616; 5,990,479; 5,690,807; 5,505,928;
5,262,357 (all of which are incorporated herein in their
entireties); as well as PCT Publication No. 99/26299 (published May
27, 1999). In particular, exemplary materials for use as
semiconductor nanocrystals in the biological and chemical assays of
the present invention include, but are not limited to those
described above, including group II-VI, III-V and group IV
semiconductors such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, MgS, MgSe,
MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, GaN, GaP,
GaAs, GaSb, InP, InAs, InSb, AlS, AlP, AlSb, PbS, PbSe, Ge and Si
and ternary and quaternary mixtures thereof. Methods for linking
semiconductor nanocrystals to antibodies are described in U.S. Pat.
Nos. 6,630,307 and 6,274,323.
XI. Pharmaceutical Preparations and Delivery
[0168] Pharmaceutical compositions of the present invention
comprise an effective amount of one or more compositions of the
invention or additional agent dissolved or dispersed in a
pharmaceutically acceptable carrier. The phrases "pharmaceutical or
pharmacologically acceptable" refers to molecular entities and
compositions that do not produce an adverse, allergic or other
untoward reaction when administered to an animal, such as, for
example, a human, as appropriate. The preparation of an
pharmaceutical composition that contains at least one composition
of the invention or additional active ingredient will be known to
those of skill in the art in light of the present disclosure, as
exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack
Printing Company, 1990, incorporated herein by reference. Moreover,
for animal (e.g., human) administration, it will be understood that
preparations should meet sterility, pyrogenicity, general safety
and purity standards as required by FDA Office of Biological
Standards.
[0169] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
surfactants, antioxidants, preservatives (e.g., antibacterial
agents, antifungal agents), isotonic agents, absorption delaying
agents, salts, preservatives, drugs, drug stabilizers, gels,
binders, excipients, disintegration agents, lubricants, sweetening
agents, flavoring agents, dyes, such like materials and
combinations thereof, as would be known to one of ordinary skill in
the art (see, for example, Remington's Pharmaceutical Sciences,
18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated
herein by reference). Except insofar as any conventional carrier is
incompatible with the active ingredient, its use in the
pharmaceutical compositions is contemplated.
[0170] The composition of the invention may comprise different
types of carriers depending on whether it is to be administered in
solid, liquid or aerosol form, and whether it need to be sterile
for such routes of administration as injection. The present
invention can be administered intravenously, intradermally,
transdermally, intrathecally, intraarterially, intraperitoneally,
intranasally, intravaginally, intrarectally, topically,
intramuscularly, subcutaneously, mucosally, orally, topically,
locally, inhalation (e.g., aerosol inhalation), injection,
infusion, continuous infusion, localized perfusion bathing target
cells directly, via a catheter, via a lavage, in cremes, in lipid
compositions (e.g., liposomes), or by other method or any
combination of the forgoing as would be known to one of ordinary
skill in the art (see, for example, Remington's Pharmaceutical
Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein
by reference).
[0171] The present invention is generally intravascularly
(intra-arterially or intravenously) administered, or may be
delivered in situ via an arterial catheter. The administration can
be injection, infusion, or continuous infusion.
[0172] In some embodiments of the invention, systemic injection is
employed for delivery, whereas in other embodiments of the
invention there is local or in situ administration using a catheter
(same technique as coiling, for example) in order to increase the
local concentration of the antibody while decreasing the blood
concentration in the systemic bloodstream/circulation.
[0173] In an other embodiment, a coil coated with the therapeutic
compound is brought to the aneurysm via an arterial catheter using
a standardized endovascular coiling technique.
[0174] This coil behaves as a drug delivery system and therefore
realizes an in situ continuous infusion of the composition.
[0175] Once released within the aneurysm, the coated coil deploys
blocking or decreasing the blood flow into the aneurysm. These
conditions allow the topical delivery of the therapeutic agent and
promote its assimilation within the aneurysmal wall.
[0176] This dual mechanism combines a short-term effect of clogging
the aneurysmal lumen and the long-term effect of repairing the
aneurysmal wall.
[0177] Both combined actions are aimed to preclude a new hemorrhage
when a re-permeabilization occurs after coiling. The simultaneous
delivery of the therapeutic agent to the aneurysm will repair the
aneurysmal wall and prevent any hemorrhagic recurrence.
[0178] The composition of the invention may be formulated into a
composition in a free base, neutral or salt form. Pharmaceutically
acceptable salts, include the acid addition salts, e.g., those
formed with the free amino groups of a proteinaceous composition,
or which are formed with inorganic acids such as for example,
hydrochloric or phosphoric acids, or such organic acids as acetic,
oxalic, tartaric or mandelic acid. Salts formed with the free
carboxyl groups can also be derived from inorganic bases such as
for example, sodium, potassium, ammonium, calcium or ferric
hydroxides; or such organic bases as isopropylamine,
trimethylamine, histidine or procaine. Upon formulation, solutions
will be administered in a manner compatible with the dosage
formulation and in such amount as is therapeutically effective. The
formulations are easily administered in a variety of dosage forms
such as formulated for parenteral administrations such as
injectable solutions, or aerosols for delivery to the lungs, or
formulated for alimentary administrations such as drug release
capsules and the like.
[0179] Further in accordance with the present invention, the
composition of the present invention suitable for administration is
provided in a pharmaceutically acceptable carrier with or without
an inert diluent. The carrier should be assimilable and includes
liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar
as any conventional media, agent, diluent or carrier is detrimental
to the recipient or to the therapeutic effectiveness of a the
composition contained therein, its use in administrable composition
for use in practicing the methods of the present invention is
appropriate. Examples of carriers or diluents include fats, oils,
water, saline solutions, lipids, liposomes, resins, binders,
fillers and the like, or combinations thereof. The composition may
also comprise various antioxidants to retard oxidation of one or
more component. Additionally, the prevention of the action of
microorganisms can be brought about by preservatives such as
various antibacterial and antifungal agents, including but not
limited to parabens (e.g., methylparabens, propylparabens),
chlorobutanol, phenol, sorbic acid, thimerosal or combinations
thereof.
[0180] In accordance with the present invention, the composition is
combined with the carrier in any convenient and practical manner,
i.e., by solution, suspension, emulsification, admixture,
encapsulation, absorption and the like. Such procedures are routine
for those skilled in the art.
[0181] In a specific embodiment of the present invention, the
composition is combined or mixed thoroughly with a semi-solid or
solid carrier. The mixing can be carried out in any convenient
manner such as grinding. Stabilizing agents can be also added in
the mixing process in order to protect the composition from loss of
therapeutic activity, i.e., denaturation in the stomach. Examples
of stabilizers for use in an the composition include buffers, amino
acids such as glycine and lysine, carbohydrates such as dextrose,
mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol,
mannitol, etc.
[0182] In further embodiments, the present invention may concern
the use of a pharmaceutical lipid vehicle compositions that include
composition of the invention, one or more lipids, and an aqueous
solvent. As used herein, the term "lipid" will be defined to
include any of a broad range of substances that is
characteristically insoluble in water and extractable with an
organic solvent. This broad class of compounds are well known to
those of skill in the art, and as the term "lipid" is used herein,
it is not limited to any particular structure. Examples include
compounds which contain long-chain aliphatic hydrocarbons and their
derivatives. A lipid may be naturally occurring or synthetic (i.e.,
designed or produced by man). However, a lipid is usually a
biological substance. Biological lipids are well known in the art,
and include for example, neutral fats, phospholipids,
phosphoglycerides, steroids, terpenes, lysolipids,
glycosphingolipids, glycolipids, sulphatides, lipids with ether and
ester-linked fatty acids and polymerizable lipids, and combinations
thereof. Of course, compounds other than those specifically
described herein that are understood by one of skill in the art as
lipids are also encompassed by the compositions and methods of the
present invention.
[0183] One of ordinary skill in the art would be familiar with the
range of techniques that can be employed for dispersing a
composition in a lipid vehicle. For example, the composition may be
dispersed in a solution containing a lipid, dissolved with a lipid,
emulsified with a lipid, mixed with a lipid, combined with a lipid,
covalently bonded to a lipid, contained as a suspension in a lipid,
contained or complexed with a micelle or liposome, or otherwise
associated with a lipid or lipid structure by any means known to
those of ordinary skill in the art. The dispersion may or may not
result in the formation of liposomes.
[0184] The actual dosage amount of a composition of the present
invention administered to an animal patient can be determined by
physical and physiological factors such as body weight, severity of
condition, the type of disease being treated, previous or
concurrent therapeutic interventions, idiopathy of the patient and
on the route of administration. Depending upon the dosage and the
route of administration, the number of administrations of a
preferred dosage and/or an effective amount may vary according to
the response of the subject. The practitioner responsible for
administration will, in any event, determine the concentration of
active ingredient(s) in a composition and appropriate dose(s) for
the individual subject.
[0185] In certain embodiments, pharmaceutical compositions may
comprise, for example, at least about 0.1% of an active compound.
In other embodiments, the an active compound may comprise between
about 2% to about 75% of the weight of the unit, or between about
25% to about 60%, for example, and any range derivable therein.
Naturally, the amount of active compound(s) in each therapeutically
useful composition may be prepared is such a way that a suitable
dosage will be obtained in any given unit dose of the compound.
Factors such as solubility, bioavailability, biological half-life,
route of administration, product shelf life, as well as other
pharmacological considerations will be contemplated by one skilled
in the art of preparing such pharmaceutical formulations, and as
such, a variety of dosages and treatment regimens may be
desirable.
[0186] In other non-limiting examples, a dose may also comprise
from about 1 microgram/kg/body weight, about 5 microgram/kg/body
weight, about 10 microgram/kg/body weight, about 50
microgram/kg/body weight, about 100 microgram/kg/body weight, about
200 microgram/kg/body weight, about 350 microgram/kg/body weight,
about 500 microgram/kg/body weight, about 1 milligram/kg/body
weight, about 5 milligram/kg/body weight, about 10
milligram/kg/body weight, about 50 milligram/kg/body weight, about
100 milligram/kg/body weight, about 200 milligram/kg/body weight,
about 350 milligram/kg/body weight, about 500 milligram/kg/body
weight, to about 1000 mg/kg/body weight or more per administration,
and any range derivable therein. In non-limiting examples of a
derivable range from the numbers listed herein, a range of about 5
mg/kg/body weight to about 100 mg/kg/body weight, about 5
microgram/kg/body weight to about 500 milligram/kg/body weight,
etc., can be administered, based on the numbers described
above.
[0187] A. Alimentary Compositions and Formulations
[0188] In preferred embodiments of the present invention, the
composition of the invention are formulated to be administered via
an alimentary route. Alimentary routes include all possible routes
of administration in which the composition is in direct contact
with the alimentary tract. Specifically, the pharmaceutical
compositions disclosed herein may be administered orally, buccally,
rectally, or sublingually. As such, these compositions may be
formulated with an inert diluent or with an assimilable edible
carrier, or they may be enclosed in hard- or soft-shell gelatin
capsule, or they may be compressed into tablets, or they may be
incorporated directly with the food of the diet.
[0189] In certain embodiments, the active compounds may be
incorporated with excipients and used in the form of ingestible
tablets, buccal tables, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like (Mathiowitz et al., 1997; Hwang et
al., 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792,451, each
specifically incorporated herein by reference in its entirety). The
tablets, troches, pills, capsules and the like may also contain the
following: a binder, such as, for example, gum tragacanth, acacia,
cornstarch, gelatin or combinations thereof; an excipient, such as,
for example, dicalcium phosphate, mannitol, lactose, starch,
magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate or combinations thereof; a disintegrating agent, such as,
for example, corn starch, potato starch, alginic acid or
combinations thereof; a lubricant, such as, for example, magnesium
stearate; a sweetening agent, such as, for example, sucrose,
lactose, saccharin or combinations thereof; a flavoring agent, such
as, for example peppermint, oil of wintergreen, cherry flavoring,
orange flavoring, etc. When the dosage unit form is a capsule, it
may contain, in addition to materials of the above type, a liquid
carrier. Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. For
instance, tablets, pills, or capsules may be coated with shellac,
sugar, or both. When the dosage form is a capsule, it may contain,
in addition to materials of the above type, carriers such as a
liquid carrier. Gelatin capsules, tablets, or pills may be
enterically coated. Enteric coatings prevent denaturation of the
composition in the stomach or upper bowel where the pH is acidic.
See, e.g., U.S. Pat. No. 5,629,001. Upon reaching the small
intestines, the basic pH therein dissolves the coating and permits
the composition to be released and absorbed by specialized cells,
e.g., epithelial enterocytes and Peyer's patch M cells. A syrup of
elixir may contain the active compound sucrose as a sweetening
agent methyl and propylparabens as preservatives, a dye and
flavoring, such as cherry or orange flavor. Of course, any material
used in preparing any dosage unit form should be pharmaceutically
pure and substantially non-toxic in the amounts employed. In
addition, the active compounds may be incorporated into
sustained-release preparation and formulations.
[0190] For oral administration the compositions of the present
invention may alternatively be incorporated with one or more
excipients in the form of a mouthwash, dentifrice, buccal tablet,
oral spray, or sublingual orally-administered formulation. For
example, a mouthwash may be prepared incorporating the active
ingredient in the required amount in an appropriate solvent, such
as a sodium borate solution (Dobell's Solution). Alternatively, the
active ingredient may be incorporated into an oral solution such as
one containing sodium borate, glycerin and potassium bicarbonate,
or dispersed in a dentifrice, or added in a
therapeutically-effective amount to a composition that may include
water, binders, abrasives, flavoring agents, foaming agents, and
humectants. Alternatively the compositions may be fashioned into a
tablet or solution form that may be placed under the tongue or
otherwise dissolved in the mouth.
[0191] Additional formulations which are suitable for other modes
of alimentary administration include suppositories. Suppositories
are solid dosage forms of various weights and shapes, usually
medicated, for insertion into the rectum. After insertion,
suppositories soften, melt or dissolve in the cavity fluids. In
general, for suppositories, traditional carriers may include, for
example, polyalkylene glycols, triglycerides or combinations
thereof. In certain embodiments, suppositories may be formed from
mixtures containing, for example, the active ingredient in the
range of about 0.5% to about 10%, and preferably about 1% to about
2%.
[0192] B. Parenteral Compositions and Formulations
[0193] In further embodiments, composition of the invention may be
administered via a parenteral route. As used herein, the term
"parenteral" includes routes that bypass the alimentary tract.
Specifically, the pharmaceutical compositions disclosed herein may
be administered for example, but not limited to intravenously,
intradermally, intramuscularly, intraarterially, intrathecally,
subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,613,308,
5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specifically
incorporated herein by reference in its entirety).
[0194] Solutions of the active compounds as free base or
pharmacologically acceptable salts may be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions may also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms. The
pharmaceutical forms suitable for injectable use include sterile
aqueous solutions or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable solutions or
dispersions (U.S. Pat. No. 5,466,468, specifically incorporated
herein by reference in its entirety). In all cases the form must be
sterile and must be fluid to the extent that easy injectability
exists. It must be stable under the conditions of manufacture and
storage and must be preserved against the contaminating action of
microorganisms, such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (i.e., glycerol, propylene glycol, and liquid
polyethylene glycol, and the like), suitable mixtures thereof,
and/or vegetable oils. Proper fluidity may be maintained, for
example, by the use of a coating, such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. The prevention of the action of
microorganisms can be brought about by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or
sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminum monostearate and
gelatin.
[0195] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous, and
intraperitoneal administration. In this connection, sterile aqueous
media that can be employed will be known to those of skill in the
art in light of the present disclosure. For example, one dosage may
be dissolved in isotonic NaCl solution and either added
hypodermoclysis fluid or injected at the proposed site of infusion,
(see for example, "Remington's Pharmaceutical Sciences" 15th
Edition, pages 1035-1038 and 1570-1580). Some variation in dosage
will necessarily occur depending on the condition of the subject
being treated. The person responsible for administration will, in
any event, determine the appropriate dose for the individual
subject. Moreover, for human administration, preparations should
meet sterility, pyrogenicity, general safety and purity standards
as required by FDA Office of Biologics standards.
[0196] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof. A
powdered composition is combined with a liquid carrier such as,
e.g., water or a saline solution, with or without a stabilizing
agent.
[0197] C. Miscellaneous Pharmaceutical Compositions and
Formulations
[0198] In other preferred embodiments of the invention, the active
compound may be formulated for administration via various
miscellaneous routes, for example, topical (i.e., transdermal)
administration, mucosal administration (intranasal, vaginal, etc.)
and/or inhalation.
[0199] Pharmaceutical compositions for topical administration may
include the active compound formulated for a medicated application
such as an ointment, paste, cream or powder. Ointments include all
oleaginous, adsorption, emulsion and water-solubly based
compositions for topical application, while creams and lotions are
those compositions that include an emulsion base only. Topically
administered medications may contain a penetration enhancer to
facilitate adsorption of the active ingredients through the skin.
Suitable penetration enhancers include glycerin, alcohols, alkyl
methyl sulfoxides, pyrrolidones and luarocapram. Possible bases for
compositions for topical application include polyethylene glycol,
lanolin, cold cream and petrolatum as well as any other suitable
absorption, emulsion or water-soluble ointment base. Topical
preparations may also include emulsifiers, gelling agents, and
antimicrobial preservatives as necessary to preserve the active
ingredient and provide for a homogenous mixture. Transdermal
administration of the present invention may also comprise the use
of a "patch". For example, the patch may supply one or more active
substances at a predetermined rate and in a continuous manner over
a fixed period of time.
[0200] In certain embodiments, the pharmaceutical compositions may
be delivered by eye drops, intranasal sprays, inhalation, and/or
other aerosol delivery vehicles. Methods for delivering
compositions directly to the lungs via nasal aerosol sprays has
been described e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212
(each specifically incorporated herein by reference in its
entirety). Likewise, the delivery of drugs using intranasal
microparticle resins (Takenaga et al., 1998) and
lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,
specifically incorporated herein by reference in its entirety) are
also well-known in the pharmaceutical arts. Likewise, transmucosal
drug delivery in the form of a polytetrafluoroetheylene support
matrix is described in U.S. Pat. No. 5,780,045 (specifically
incorporated herein by reference in its entirety).
[0201] The term aerosol refers to a colloidal system of finely
divided solid of liquid particles dispersed in a liquefied or
pressurized gas propellant. The typical aerosol of the present
invention for inhalation will consist of a suspension of active
ingredients in liquid propellant or a mixture of liquid propellant
and a suitable solvent. Suitable propellants include hydrocarbons
and hydrocarbon ethers. Suitable containers will vary according to
the pressure requirements of the propellant. Administration of the
aerosol will vary according to subject's age, weight and the
severity and response of the symptoms.
XII. Kits
[0202] Any of the compositions described herein may be comprised in
a kit for the detection and/or treatment of aneurysm. The kit may
include a cell targeting molecule, a label, an intravascular
targeting molecule, and/or a therapeutic agent. These components
may or may not be assembled into one composition. In a non-limiting
example, there may be an additional agent provided in the kit. The
kits may thus comprise, in suitable container means, a composition
of the invention and, optionally, an additional agent of the
present invention. Kits of the present invention will generally
contain, in suitable container means, a pharmaceutically acceptable
formulation of the composition of the invention.
[0203] The component(s) of the kits may be packaged either in
aqueous media or in lyophilized form. When reagents and/or
components are provided as a dry powder, the powder can be
reconstituted by the addition of a suitable solvent. It is
envisioned that the solvent may also be provided in another
container means. The container means of the kits will generally
include at least one vial, test tube, flask, bottle, syringe or
other container means, into which a component may be placed, and
preferably, suitably aliquoted. Where there are more than one
component in the kit, the kit also will generally contain a second,
third or other additional container into which the additional
components may be separately placed. However, various combinations
of components may be comprised in a vial. The kits of the present
invention also will typically include a means for containing the
composition of the invention and any other reagent containers in
close confinement for commercial sale. Such containers may include
injection or blow molded plastic containers into which the desired
vials are retained.
EXAMPLES
[0204] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Exemplary Experimental Designs and Methods
[0205] The present example provides exemplary materials and methods
for the invention, although one of skill in the art recognizes that
they are merely exemplary in nature and may be modified within
routine standards in the art.
[0206] 1. In Situ Immunohistochemistry
Animal Preparation
[0207] Intracranial aneurysms are induced in 20 male Sprague-Dawley
rats (age range, 6 to 7 weeks) according to the method of Hashimoto
et al (Hashimoto et al., 1978). Ligation of the left common carotid
artery and the posterior branches of both renal arteries will be
performed under anesthesia with the use of an intraperitoneal
injection of chloral hydrate (3%, 0.01 mL/g body wt). One week
after the operation, 1% saline will be substituted for drinking
water. The previous literature reported this method of
preferentially induced experimental cerebral aneurysms at the right
anterior cerebral artery (ACA)-olfactory artery (OA) bifurcations,
where hemodynamic stress is assumed to increase by the ligation of
the opposite common carotid artery (Kojima et al., 1986). An
additional 5 age-matched rats will served as controls.
[0208] Three months after the aneurysm induction procedure, the
rats will be cannulated into the ascending aorta through the left
cardiac ventricle under general anesthesia and perfused at a
pressure of 80 mmHg with 4% paraformaldehyde in PBS. After the
perfusion fixations, the major arteries at the base of the brain
will be carefully dissected under a surgical microscope. The
specimens will be further immersed in 4% paraformaldehyde in PBS
for 24 hours. The specimens will be rinsed with PBS, embedded in
OCT compound (Tissue-Tek, Inc) and 7 .mu.m-thick serial sections
from ACA/OlfA bifurcation will be cut with a cryotome.
Light Microscopic Examination
[0209] Using elastica-van Gieson stain and a light microscope, we
will examine the bifurcation of the ACA and OA on both sides and
examine aneurismal changes on the nonligated side.
Definition of Aneurysmal Changes
[0210] It is established that fragmentation and disappearance of
internal elastic lamina is a characteristic histological feature of
aneurysmal lesions. Therefore, aneurysmal changes will be defined
as lesions representing the outward dilatation of the wall that are
accompanied by discontinuity of the internal elastic lamina in more
than half the length of the dilated wall (evidenced by elastica-van
Gieson staining). The lesions will be classified into two stages:
(1) a stage of early aneurismal lesion preserving the smooth cell
layer in the whole area of the wall and (2): saccular aneurysm
lacking the smooth muscle layer even in part of the whole area of
the lesion. Moreover, to detect endothelial injury at the apical
intimal pad which is characteristic of early aneurismal lesion, we
will performed immunohistochemical study using antibody against
eNOS (endothelial injury is evidenced by the loss of eNOS
expression).
Immunohistochemical Studies
[0211] Immunohistochemical studies will be performed on early
aneurismal lesions and saccular aneurysm to study .alpha.7 integrin
and laminin-1 expression. Our goal is to demonstrate that early
aneurismal lesions show a lack of eNOS expression and a
subendothelial staining for .alpha.7 integrin and laminin-1 in
contrast to control sections that show a positive expression of
eNOS and a medial without subendothelial staining for .alpha.7
integrin and laminin-1. Sections will be fixed in ice-cold acetone
(10 minutes), air-dried (30 minutes), and incubated in 5% skim milk
(30 minutes) before overnight incubation at 4.degree. C. with
primary antibodies. The primary antibodies will be mouse anti-eNOS
antibody (BD Biosciences), mouse anti-rat .alpha.7 integrin
antibody (H36 provide by SJ Kaufman) rabbit polyclonal anti-laminin
.alpha.-1 (H-300) (sc-5582, Santa Cruz Biotechnology, Inc).
Sections will be washed with PBS, then incubated 30 minutes with
fluorescein-conjugated secondary antibody (Alexa Fluor 594, 488 and
647 goat anti-mouse, anti-rabbit or anti-rat immunoglobulin G;
Molecular Probes). For double immunofluorescence staining (eNOS and
.alpha.7, eNOs and laminin-1) the same procedure will be repeated.
After washing with PBS, the specimens will be mounted with
Vectashield (Vector Laboratories).
[0212] Slides will be inspected under a fluorescent microscope
combined with a laser confocal system. Image files will be
digitally processed using Adobe Photoshop (Adobe Systems).
[0213] 2. In Vivo Immunodetection of Aneurysmal Lesions
[0214] Molecular Imaging techniques well suitable for intravascular
applications will be used such as immunoscintigraphy using antibody
radiolabeled with 99mTc-dextran and/or MRI using antibody
conjugated to Gadolinium-DTPA-dextran.
[0215] Dextran is a hydrophilic molecule that does not pass the
phospholipid bilayer of endothelial membrane. Antibody conjugated
to dextran will not pass the endothelium. The conjugate will bind
specifically .alpha.7 integrin or laminin-1 exposed at the luminal
surface of the aneurysmal wall and can not bind .alpha.7 integrin
or laminin-1 on SMCs in the medial layer of normal vascular wall
that harbors an intact endothelium. Dextran is useful in the
invention, but any macromolecular agent with intravascular
retention may be coupled to the antibody.
REFERENCES
[0216] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by
reference.
PATENTS AND PATENT APPLICATIONS
[0217] U.S. Pat. No. 3,817,837
[0218] U.S. Pat. No. 3,850,752
[0219] U.S. Pat. No. 3,939,350
[0220] U.S. Pat. No. 3,996,345
[0221] U.S. Pat. No. 4,196,265
[0222] U.S. Pat. No. 4,275,149
[0223] U.S. Pat. No. 4,277,437
[0224] U.S. Pat. No. 4,366,241
[0225] U.S. Pat. No. 4,472,509
[0226] U.S. Pat. No. 4,938,948
[0227] U.S. Pat. No. 5,021,236
[0228] U.S. Pat. No. 5,741,957
[0229] U.S. Pat. No. 5,750,172
[0230] U.S. Pat. No. 5,756,687
[0231] U.S. Pat. No. 5,827,690
[0232] U.S. Pat. No. 6,091,001
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[0304] Although the present invention and its advantages have been
described in detail, it should be understood that various changes,
substitutions and alterations can be made herein without departing
from the spirit and scope of the invention as defined by the
appended claims. Moreover, the scope of the present application is
not intended to be limited to the particular embodiments of the
process, machine, manufacture, composition of matter, means,
methods and steps described in the specification. As one of
ordinary skill in the art will readily appreciate from the
disclosure of the present invention, processes, machines,
manufacture, compositions of matter, means, methods, or steps,
presently existing or later to be developed that perform
substantially the same function or achieve substantially the same
result as the corresponding embodiments described herein may be
utilized according to the present invention. Accordingly, the
appended claims are intended to include within their scope such
processes, machines, manufacture, compositions of matter, means,
methods, or steps.
* * * * *