Note: Descriptions are shown in the official language in which they were submitted.
CA 02556287 2006-08-16
METHODS OF TREATMENT USING VASCULAR OCCLUSION IN COMBINATION
WITH ONE OR MORE THERAPIES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Application No.
10/241,717 filed 09/21 /2002 (now allowed), and U.S. Application No.
10/101,731 filed
3/21/2002, and the non-provisional of 60/708,757 filed 8/17/2005.
FEDERAL SPONSORSHIP
Not Applicable
BACKGROUND OF THE INVENTION
[0002] The present invention is directed to methods for producing a
therapeutic
benefit by producing vascular occlusion using platelet activation as the
initiating event in
combination with one or more therapies. Compositions and methods of; the
invention
involve delivering a solid-phase platelet-binding agent to a target site,
causing platelets
to bind and activate thereby forming a localized thrombus. Occlusion of the
vasculature
of the target tissue by the localized thrombus results in deprivation of
essential oxygen
and nutrients, in turn leading to tissue regression and ultimately tissue
death.
DESCRIPTION OF RELATED ART
[0003] Platelets function in the body to limit blood loss in the event of
vascular
damage. Normally, platelets circulate throughout the body with other cellular
components of blood, bathed in a mixture of various plasma proteins, many of
which
play key roles in the clotting process. Upon exposure of vascular sub-
endothelium, a
complex series of events occurs to limit the loss of blood from the damaged
vessel.
Circulating platelets contacting components of the exposed sub-endothelium: 1
) bind
and adhere, 2) spread across the exposed surface, 3) activate as evidenced by
release
of granule contents, 4) aggregate and recruit other circulating platelets from
the blood
stream, and 5) form an efficient plug, clot, and/or thrombus stemming the flow
of blood
from the vessel.
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[0004] One approach to overcoming the deficiencies of targeting tumors with
antibodies would be to target thrombus-inducing agents to the vasculature of
the tumor
rather than to the tumor.
[0005] The present inventors propose that this approach will provide several
advantages over targeting tumor cells directly. Firstly, the target cells are
directly
accessible to vascularly administered therapeutic~agents permitting rapid
localization of
a high percentage of the injected dose. Secondly, since each capillary
provides oxygen
and nutrients for thousands of cells in its surrounding cord of tumor, even
limited
damage to the tumor vasculature could produce an avalanche of tumor cell
death.
[0006] Under certain clinical situations, inhibition of blood flow to a tissue
through
occlusion of its associated vasculature is desirable. Examples include
treatment of:
hepatocellular carcinoma (HCC), renal cell carcinoma (RCC), hemorrhagic
stroke,
saphenous vein side branches in saphenous bypass graft surgery, aortic
aneurysm,
vascular malformations, and solid tumors. Embolization of tumor vasculature
prior to
organ transplantation, and embolization of vasculature prior to tissue or
organ resection
is also desirable. .-
[0007] HCC ranks among the most common malignancies worldwide, and the
prognosis for patients with HCC is typically poor. Hepatocellular tumors
derive their
blood supply nearly exclusively from the hepatic artery. As a result, an
arterial approach
to anti-tumor therapy is designed to spare the surrounding hepatic parenchyma,
including selective tumor necrosis. There are many treatments currently being
used and
tested (e.g. chemoembolization, resection, immunotherapy, and external
radiation).
Maintenance of arterial patency is important in some therapies.
[0008] Vascular occlusion has been performed using a variety of techniques and
materials including embolotherapy. Examples of embolotherapy include the use
of
particles composed of a variety of materials including polyvinyl alcohol
(Boschetti, PCT
W00023054), acrylamide (Boschetti et al, US 5,635,215; Boschetti et al, US
5,648,100),
polymethyl methacrylate (Lemperle, US 5,344,452), physical plugs composed of
collagen (Conston et al, US 5,456,693) and coils (Mariant, US 5,639,277).
Embolotherapy involves the delivery of these materials to the target
vasculature by
means of a catheter. Since the vasculature in most locations proceeds from
larger
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CA 02556287 2006-08-16
arteries to arterioles to metarterioles to capillaries, each with
progressively smaller
vessel diameters, the delivered material (embolus) continues to travel in the
flowing
blood~~ntil it becomes lodged in the smaller blood vessels thereby impeding
the flow of
blood to the dependent tissue.
[0009] Unfortunately, the suspension mediums currently used cause only
temporary or semi-permanent vascular occlusion. A number of the treatments,
such as
transcatheter arterial chemoembolization, which involves localized intra-
arterial infusion
of chemotherapy, emulsified oil, and an embolic material, provide better tumor
response
when repeated multiple times, so long-term arterial patency may be critical to
the
success of chemoembolization.
[0010] Other therapies have moderate success, but only resection or
transplantation is seen as curative treatments. For example, interferon has
been
reported to reduce the risk of HCC, but there is conflicting evidence in
patients who
have already developed cirrhosis. Further, the possibility that interferon has
anticarcinogenic effects unrelated to its antiviral efficacy is now widely
accepted, but
remains unproven. Internal radiation, e.g. 131--I-Lipiodol is used as a
vehicle for
chemotherapy, but does not achieve arterial occlusion. Percutaneous Ethanol
Injection
(PEI) is widely used to treat HCC. However, during the follow-up period after
PEI, local
recurrence was seen in approximately 10% of PEI-treated nodules.
[0011] Radiofrequency (RF) ablation is the most extensively used alternative
to
PEI. RF can be applied percutaneously, laparoscopically, or during laparotomy.
Yet,
robust survival advantages have not been proven.
[0012] As a result there is still a need for a method to enhance the
effectiveness
and success rates of current treatments. The present invention seeks to
address this
need by combining two or more therapies either, serially or in parallel to
achieve a
therapeutic benefit.
[0013] The present invention is novel and addresses unmet medical needs
through the use of a solid-phase material, such as microparticles or coils or
stents,
configured or adapted to activate platelets. In some embodiments of the
invention, the
solid-phase material itself binds and activates platelets; in other
embodiments, the solid-
phase material may be coated with von Willebrand factor (VWF) of mammalian
origin. In
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CA 02556287 2006-08-16
this way a therapeutic benefit may be achieved by delivering a solid-phase
platelet
binding agent to a target site and initiating efficient thrombus formation
leading to
occlusion of the associated vasculature in combination with one or more
therapies.
SUMMARY OF THE INVENTION
[0014] The present invention relates to therapeutic methods and compositions
for
targeting tissues and/or organs, and associated vasculature, which are
hyperplastic or
neoplastic in nature, or which have arterio-venous malformations, or which are
hemorrhaging, using solid-phase agents that induce thrombus formation via
localized
platelet activation.
[0015] The present invention relates to therapeutic methods for producing
vascular occlusion using a solid-phase agent in combination with one or more
therapies,
said therapy comprising any medical, pharmaceutical, or biological therapy or
agent.
Exemplary therapies or agents include, but are not limited to chemotherapy,
chemoembolization, radiofrequency ablation, microwave treatment, cryoablation,
percutaneous ethanol injection, resection, transplantation, angiogenesis
inhibitors,
immunotherapy, tyrosine kinase inhibitors, interferon, internal radiation,
external
radiation, cytostatic agents, gene therapy, embolic agents, hormone therapies,
and
growth factor receptor inhibitors. As used herein, in combination refers to
administering
two separate therapies whereby the overall health of the patient is improved.
The
therapies may be administered either serially or in parallel.
[0016] The composition comprises a solid phase agent for capturing platelets
on
a solid-phase agent such as a coil or a stent or a particle. In some
embodiments of the
invention, the solid-phase agent both captures and activates the platelets.
The method
utilizes localizing platelet collection and activating the platelets on the
solid-phase
particle to produce subsequent thrombus formation, thereby limiting the blood
supply to
the target area, without inducing a generalized or systemic pro-thrombotic
state.
[0017] Contact of the solid-phase platelet-binding agent with the blood from a
patient (ex vivo) or in the blood stream (in vivo) induces platelet binding
and localized
activation leading to accretion of platelets about the solid-phase agent
leading to
thrombosis and cessation of blood flow to the tissue supplied by the occluded
blood
4
CA 02556287 2006-08-16
vessel(s). Cells, including tumor cells or hyperplastic tissue, diminish or
die as a result
of loss of localized blood flow. This approach avoids systemic platelet
activation and
throm~b~osis. For example, immobilized VWF (but not soluble VWF) binds to and
activates circulating platelets. Thus, the methods and compositions of the
present
invention comprise both an indirect and direct means of treating any
pathological
condition where blood is involved or present, such as cancer, hyperplastic
cells,
excessive bleeding or arteriovenous (AV) malformations.
[0018] The present invention improves on existing methods for treating solid
hyperplastic tissue, excessive bleeding and AV-malformations and any other
tumors,
hyperplastic disease or condition in which platelets (resting and/or
activated) may play a
therapeutic role.
[0019] In a manner similar to an existing pathological condition (i.e. Heparin-
Induced Thrombocytopenia [HIT]), localized platelet activation can be enhanced
by
means of an Fc-mediated process by including or incorporating a human Fc
fragment
onto the solid-phase agent, or by directing select antibodies to the target
area. Platelet
activation in HIT syndrome results in localized-thrombosis and cessation of
blood flow to
the affected area. This leads to death of the affected tissue.
[0020] The extent or degree of site-specific thrombosis can be controlled in a
variety of ways. Inhibition of platelet activation through the use of anti-
platelet agents
(e.g. GPllb/llla inhibitors, aspirin, dipyridamole, etc.) decreases the
propensity to induce
a thrombus in a defined, titratable manner. Altering local blood flow, blood
pressure and
tissue temperature can also serve as means of controlling local platelet
activation to a
stimulus.
(0021] Typical vascularized tumors are the solid tumors, particularly
carcinomas,
which require a rich vascular blood supply. Exemplary solid tumors to which
the present
invention is directed include, but are not limited to, primary malignant
tumors of the lung,
breast, ovary, stomach, pancreas, larynx, esophagus, testes, liver, parotid,
biliary tract,
colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate,
thyroid, head and
neck, melanomas, gliomas, neuroblastomas, neuroendocrine tumors, and the like.
Other conditions to which the present invention is directed include, but are
not limited to,
secondary (metastatic) tumors of the above mentioned tumor types, cancer pain,
AV-
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CA 02556287 2006-08-16
malformations, uterine fibroids, pelvic congestion, menorrhagia, varicoceles,
hemoptysis, aneurysms, visceral artery aneurysms, pseudoaneurysms and
endoleaks.
[0022]:- A preferred method of the invention includes preparing a coil or
stent
coated with VWF of recombinant or mammalian origin and introducing the VWF-
coated
agent into the bloodstream of an animal, such as a human patient, an animal
patient, or
a test animal; the VWF is then delivered or collects at a desired target site.
The coils or
stents can be constructed of any suitable material capable of retaining VWF
either
within the coil or stent or on the surface of the coil or stent for an
indefinite or varying
length of time.
[0023] A solution to the problem of the unrestrained growth of solid tumors is
to
attack the blood vessels in the tumor. This approach offers several advantages
over
methods that directly target tumor cells. Firstly, the tumor vessels are
directly accessible
to vascularly administered therapeutic agents, thus permitting rapid
localization of high
percentage of the injected dose. Secondly, since each capillary provides
oxygen and
nutrients for thousands of cells in its surrounding cord of tumor even limited
damage to
the tumor vasculature could produce extensive tumor cell death. Finally, blood
vessels
are similar in different tumors, making it feasible to develop a single
reagent for treating
numerous types of cancer.
DESCRIPTION OF THE DRAWINGS
Not Applicable
DETAILED DESCRIPTION OF THE INVENTION
(0024] The present invention provides compositions and methods for capturing
platelets at a predetermined site, activating the platelets, and harnessing
the natural
function of platelets to achieve a beneficial therapeutic result. In
accordance with the
present invention, the platelets may be circulating platelets or may be
platelets obtained
from an external source. In accordance with the present invention, platelets
may be
targeted to a specific site, and then the natural ability of platelets to
induce thrombus
formation may be used to interrupt, disrupt, or reduce blood flow at the site.
Reduced
blood flow concomitantly reduces nutrient supply to a disease or condition
agent, such
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CA 02556287 2006-08-16
as a tumor, so the size of the disease agent is diminished. It is clear that
reducing the
size of a tumor is an obvious therapeutic benefit. In some instances reduction
of the
blood~supply to a target area alleviates pain.
[0025] The present invention relates to therapeutic methods for producing
vascular occlusion using a solid-phase agent in combination with one or more
therapies, as used herein combined refers to using one or more additional
protocols
together in any manner that results in a therapeutically beneficial result or
outcome for
the patient. For example, two or more therapies may be combined serially or in
conjunction with one another. It is intended that two or more therapies should
be
combined if it is determined that the patient's health will benefit, or that
it is believed that
the patient's health will benefit.
[0026] The present invention also includes targeting platelets to a pre-
determined
tissue capable of being selectively targeted, e.g., hyperplastic tissue, using
a solid-
phase agent capable of binding and activating the platelets. In these
embodiments of
the invention, targeting refers to the solid phase containing a targeting
moiety, e.g., a
ligand or the like, that specifically binds the p.re-determined site or
tissue. In other
embodiments of the invention, targeting may include delivering a composition
of the
present invention at or near a tumor site, e.g., by catheter, stent, or coil.
Activating the
platelets at the pre-selected site causes a therapeutic benefit by reducing
the nutrient
supply to the tissue or site. USP 6,960,532; 6,887,474; and USSN 11/205,047
(filed
August 17, 2005), each incorporated herein by reference
[0027] The present invention provides compositions and methods for inducing
thrombus formation by capturing platelets on the solid-phase agent, inducing
activation
of the platelets, and allowing a thrombus to form. Thrombus formation in the
target
vasculature reduces the blood supply to the downstream tissue. By capturing
platelets
on a VWF-containing, or collagen-containing solid phase (e.g., coated
particles), the
compositions and methods of the present invention may be used to treat cancer,
hyperplasia, uterine fibroids, pelvic congestion, menorrhagia, AV-
malformations, neuro-
embolism, varicoceles, hemoptysis, visceral artery aneurysms, arterial
aneurysms,
endoleaks, and the like. Furthermore, the compositions and methods of the
present
invention provide a therapeutic benefit to the recipient of the composition.
CA 02556287 2006-08-16
0028] In a preferred embodiment of the invention, the VWF is of mammalian
origin. In a most preferred embodiment of the invention, the VWF is of human
origin. In
a further most preferred embodiment of the invention, the VWF is of porcine
origin.
[0029] The VWF may be natural, synthetic, recombinant, or a peptide sequence
conforming to a biologically active portion of VWF. In a further most
preferred
embodiment of the invention, the VWF is of recombinant origin.
[0030] In a preferred embodiment of the invention, the collagen is of
mammalian
origin. In a most preferred embodiment of the invention, the collagen is of
human origin.
In a further most preferred embodiment of the invention, the collagen is of
bovine origin
or porcine origin. In a further most preferred embodiment of the invention,
the collagen
is of recombinant origin.
(0031] The present invention also provides compositions that bind a platelet-
binding agent (e.g. VWF or collagen) directly or indirectly through a spacer
to the solid
phase, so long as the ability of the platelet-binding agent to bind platelets
is not
impaired. Spacer, as used herein, refers to a group of inert or active
molecules that
physically separate the platelet binding agent-from the surface of the solid
phase agent.
Exemplary spacers are described below. The direct binding can occur either
covalently
or non-covalently. Indirect binding can occur through spacers, including but
not limited
to peptide spacer arms, spacers, antibody fragment spacers, fusion protein
spacers or
antibody carbohydrate spacers. These spacers normally act only as bridges
between
the particle and the platelet binding agent; however, the spacers could also
be used to
alter the degree of platelet activation. For example, an Fc component could be
used as
a spacer, thereby effecting enhanced platelet activation on and about the
solid-phase
agent. Coupling of platelet binding agent (e.g. VWF or collagen) to the solid
phase
agent can occur using methods known to those skilled in the art. Examples of
coupling
agents include but are not limited to glutaraldehyde, succinimide esters,
benzidine,
periodate and carbodiimide.
[0032] In a preferred embodiment of the invention, the positioning within the
vascular system of mammals of compositions without an active targeting agent
would
be selected by blood flow directed positioning following delivery by means of
a
superselective microcatheter.
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CA 02556287 2006-08-16
[0033] Compositions according to the present invention may also include a
targeting agent or moiety capable of binding a target antigen or site on the
vascular
endothelium or target tissue thereby enabling localization of the solid-phase
agent to a
selected site. Exemplary targeting agents or moieties are well known to those
skilled in
the art, and include, but are not limited to antibodies, ligands, receptors,
hormones,
lectins, and cadherins, or portions or fragments thereof. USP 6,960,532;
6,887,474;
and USSN 11/205,047 (filed August 17, 2005), each incorporated herein by
reference
[0034] In a preferred embodiment of the invention, the targeting agent would
include an antibody or antibody-like molecule with biotin, biotin mimetic
and/or a peptide
component. In a further preferred embodiment of the invention, the antibody or
antibody-like molecules would be directed toward a growth factor/receptor
complex.
[0035] Compositions according to the invention may also include one or more of
the following: one or more platelet binding modulators (e.g., inhibitors or
enhancers),
one or more thrombus formation controllers or modulators or one or more
complement
cascade components.
[0036] Methods according to the invention may also include administering a
solid-
phase agent capable of binding platelets at a pre-determined site; may also
include
inducing activation of the captured platelets; administering a bi-functional
binding agent
having an antigenic determinant and a platelet binding site; controlling
thrombus
generation by altering the temperature of one or more compositions of the
invention, or
by altering the temperature at the pre-selected site.
[0037] Methods according to the invention may further include one or more of
the
following: administering one or more platelet binding modulators,
administering one or
more thrombus formation modulators; administering one or more complement
cascade
components; administering one or more ligands and/or anti-ligands for binding
the solid
phase to a pre-determined site, and/or for binding a platelet binding moiety
or
component to the solid phase.
[0038] The present invention also includes a kit which may contain but is not
limited to any or all of the following components including a solid-phase
agent for
targeting platelets to an endothelial membrane component: a binding agent for
binding
platelets; a ligand for binding an endothelial membrane component; a ligand
conjugate;
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CA 02556287 2006-08-16
an anti-ligand for binding the ligand or the ligand conjugate; a platelet
binding modulator
(enhancer and/or inhibitor); a thrombus formation modulator; a complement
cascade
component; a complement cascade component inducer; and a binding agent for
binding
platelets that includes an anti-ligand. The kit may include a bi-functional
binding agent,
and/or a binding agent-ligand conjugate, and/or a platelet-binding agent -anti-
ligand
conjugate.
[0039] The compositions and methods of the present invention include any
mechanism of delivering a composition to the pre-selected site, including but
not limited
to systemically, locally, orally, or topically.
[0040] In accordance with some embodiments of the invention, binding agents
are used to capture platelets at a predetermined site.
Definitions:
[0041] As used herein, a solid-phase agent refers to any solid material
suitable
for binding, containing, or retaining a platelet-binding agent. The platelet-
binding agent
may be attached to the solid-phase agent sucks that platelet binding activity
is retained,
e.g., at or within a target site. The solid phase agent may be a coil, stent,
or particle,
e.g., a bead or the like, all of which are well known to those skilled in the
art. USP
6,960,532; 6,887,474; and USSN 11/205,047 (filed August 17, 2005), each
incorporated
herein by reference
[0042] As used herein, a particle refers to a discrete portion or part of a
solid-
phase material capable of containing or retaining a platelet-binding agent. A
preferred
method of the invention includes preparing a particle coated with VWF of
recombinant
or mammalian origin and introducing the VWF-coated particle into the
bloodstream of
an animal, such as a human patient, an animal patient, or a test animal.
[0043] A preferred method of the invention includes preparing a particle
coated
with collagen of recombinant or mammalian origin and introducing the collagen-
coated
particle into the bloodstream of an animal, such as a human patient, an animal
patient,
or a test animal.
[0044] As used herein, the term "particle" refers to any solid-phase material
capable of binding platelets, either directly or indirectly (e.g., through
ligands). The
to
CA 02556287 2006-08-16
particles can be homogenous or heterogeneous as related to size. Specifically,
the
particles can be of spherical (including ovoid) or irregular shape. The
particles can be
constructed of any suitable material capable of retaining VWF or collagen
either within
the particle or on the surface of the particle for an indefinite or varying
lengths of time.
Exemplary materials include polyvinyl alcohol (PVA), polystyrene,
polycarbonate,
polylactide, polyglycolide, lactide glycolide copolymers; polycaprolactone,
lactide-
caprolactone copolymers, polyhydroxybutyrate, polyalkylcyanoacrylates,
polyanhydrides, polyorthoesters, albumin, collagen, gelatin, polysaccharides,
dextrans,
starches, methyl methacrylate, methacrylic acid, hydroxylalkyl acrylates,
hydroxylalkyl
methacrylates, methylene glycol dimethacrylate, acrylamide, bisacrylamide,
cellulose-
based polymers, ethylene glycol polymers and copolymers, oxyethylene and
oxypropylene polymers, polyvinyl acetate, polyvinylpyrrolidone and
polyvinylpyridine,
magnetic particles, fluorescent particles, animal cells, plant cells, macro-
aggregated and
micro-aggregated albumin, denatured protein aggregates and liposomes, used
singly or
in combination. The solid phase materials suitable for use in the present
invention are
well known to those skilled in the art, and should not be limited to those
exemplary
materials recited above.
[0045] Exemplary materials for forming the stent or coil include, but are not
limited to: polyvinyl alcohol (PVA), polystyrene, polycarbonate, polylactide,
polyglycolide, lactide-glycolide copolymers, polycaprolactone, lactide-
caprolactone
copolymers, polyhydroxybutyrate, polyalkylcyanoacrylates, polyanhydrides,
polyorthoesters, polysaccharides, dextrans, starches, methyl methacrylate,
methacrylic
acid, hydroxylalkyl acrylates, hydroxylalkyl methacrylates, methylene glycol
dimethacrylate, acrylamide, bisacrylamide, cellulose-based polymers, ethylene
glycol
polymers and copolymers, oxyethylene and oxypropylene polymers, polyvinyl
acetate,
polyvinyl pyrrolidone and polyvinylpyridine; magnetic materials, fluorescent
materials;
gold, platinum, palladium, rhenium, rhodium, ruthenium, stainless steel,
tungsten,
titanium, nickel and alloys thereof; used singly or in combination.
[0046] The preferred size of the solid phase material depends on the type of
material being used. For example, those skilled in the art will recognize that
if the solid
phase is a stent or coil, the size is preferably of a diameter that fits
within a blood
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CA 02556287 2006-08-16
vessel, such as an artery. Typically the diameter will be up to about 15 mm or
greater. If
the solid phase is a particle, such as a bead, the diameter may be up to about
7 mm,
preferably from about 1 pm to about 5 mm, even more preferably from about 20
pm to
about 300 pm. The size of solid phase materials suitable for use in the
present invention
are well known to those skilled in the art, and should not be limited to the
exemplary
sizes recited above.
[0047] As used herein, a binding agent or targeting moiety refers to one or
more
solid phase chemical or biological molecules or structures for binding one
substance to
another. Specifically the binding agent, or solid phase agent, binds a ligand,
a receptor
or a ligand/receptor complex on a defined population of cells, typically
hyperplastic
tissue and/or associated vasculature, or a cancer cell and/or associated
vasculature. A
molecule's function as a binding agent should not be limited by the structural
mechanism of attachment. For example, a binding agent may bind a receptor, an
antigenic determinant or epitope, an enzymatic substrate, or other biological
structure
agent to a target cell or cell population. The binding agent may be a
conjugate, and
includes but is not limited to immunological conjugates, chemical conjugates
(covalent
or non-covalent), fusion proteins, and the like. USP 6,960,532; 6,887,474; and
USSN
11/205,047 (filed August 17, 2005), each incorporated herein by reference
[0048] As used herein, a ligand-binding agent refers to a complementary set of
molecules that demonstrate specific binding for each other. A ligand/anti-
ligand pair
generally binds with relatively high affinity, and for this reason, may be
highly desirable
for use with the present invention. A very well known ligand/anti-ligand pair
is biotin and
avidin. As used herein, avidin refers to avidin, streptavidin, neutravidin,
derivatives and
analogs thereof, and functional equivalents thereof. Avidin may bind biotin in
a
multivalent or univalent manner. Other exemplary ligand/anti-ligand pairs
include, but
are not limited to homophyllic peptides, heterophyllic peptides, "leucine
zippers", zinc
finger proteins/ds DNA fragment, enzyme/enzyme inhibitor, hapten/antibody,
ligand/ligand receptor, and growth factor/growth factor receptor.
[0049] As used herein, a selected site, a pre-determined site, targeting, and
pre-
targeting all refer to a site where the accumulation of platelets about a
solid-phase will
provide a therapeutically beneficial result. Typically, this involves target
site localization
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of a targeting moiety. Such sites include, but are not limited to, the
vasculature of solid
tumors, the vasculature of benign tumors, the vasculature of hyperplastic
tissue(s), the
vascu~lature of hyper-vascularized tissues, AV-malformations, vessel aneurysms
and
endoleaks. USP 6,960,532; 6,887,474; and USSN 11/205,047 (filed August 17,
2005),
each incorporated herein by reference
[0050] As used herein, delivery of the solid ~agerit comprising a platelet-
binding
agent can occur using a catheter, a microcatheter or by needle and syringe.
Delivery by
a microcatheter is most often achieved by access through the arterial circuit,
however
delivery of the solid agent through the venous circuit is also desirable. As
an example,
the solid agent in the form of particles, coils or stents can be delivered by
catheter, to
target site using the arterial or venous circuits. Delivery of the solid agent
using arterial
circuit is advantageous since the capillary beds downstream of applied agent
in the
target tissue act as a means of trapping the agent, thereby preventing the
agent from
entering the systemic circulation. The solid agent can also be localized
within the
arterial circulation using a targeting agent associated with the solid agent.
Delivery of
the solid agent using the venous system is also desirable. Localized delivery
of the solid
agent in the venous system can be accomplished by binding the solid agent to
the
target site using a targeting agent associated with the solid agent. The solid
agent can
also be delivered to the target site during a surgical procedure. As an
example, the solid
agent in the form of particles can be delivered by syringe and needle to the
target site.
As a further example, the solid agent in the form of a coil or stent can be
placed
manually at the target site during the surgical procedure.
[0051] As used herein, thrombus refers to any semi-solid aggregate of blood
cells
enmeshed in fibrin and clumps of platelets originating from platelets actively
binding to
the solid-phase agent. In accordance with the invention, a thrombus is formed
as a
direct result of activated platelet accumulation at the pre-determined site.
Thrombosis
refers to the formation of a thrombus, typically within a blood vessel.
Thrombogenic
refers to substances that tend to cause thrombosis, or are thrombus forming.
[0052) As used herein, embolus refers to an intravascular mass, which travels
through the bloodstream, and through size constraints eventually becomes
lodged in a
blood vessel or capillary, distal from the site of origin of the intravascular
mass.
13
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Embolization does not imply an active process, but instead refers to a passive
process
whereby occlusion of blood vessels occurs by intravascular masses traveling
through
the blood stream where they become lodged in small blood vessels and
capillaries.
[0053] In contrast, the present invention involves the delivery of solid-phase
material to target vasculature whereupon platelets are actively recruited to
the solid-
phase surface through the use of a platelet-binding agent. In contrast to
embolizing
materials described in cited patents, included herein as reference, the agents
of the
present invention must be delivered in close proximity to the target
vasculature due to
rapid accumulation of platelets about the solid-phase material.
[0054] By way of example, macro-aggregated albumin (MAA), as supplied by
Draximage (Kirkland, Quebec, Canada), for example, is used as an embolizing
imaging
agent. The MAA consists of particles between 10 Nm and 70 pm in size, with a
maximum size of 150 pm that are radiolabeled with sodium pertechnetate Tc 99m
to
enable scintigraphy imaging. The MAA particles are injected intravenously, and
travel
through the blood stream as emboli where they become trapped in the,,pulmonary
alveolar capillary bed. Using the method of the present invention,
immobilization of VWF
on the MAA, with subsequent injection of the particles into the vascular
system, most
preferably upstream of the target tissue, causes immediate platelet binding to
the
particles and occlusion of the vasculature in close proximity to the site of
injection.
[0055] As used herein, combination therapy or similar phrases, refers to
administering a solid phase embodiment of the present invention in combination
with
any other therapy. The purpose of such combination treatment is to derive a
beneficial
affect for the patient.
[0056] The present invention improves upon existing methods of producing
vascular occlusion by securing platelets to the surface of a solid-phase
material through
the use of a platelet-binding agent, thereby increasing the effective size of
the solid-
phase material. For example, a particle coated with or containing VWF, which
is injected
into the blood stream, would rapidly accumulate platelets on its surface, in
effect
producing an 'onion-effect' of layered, activated platelets in close proximity
to the
injection site. Therefore the present invention enables delivery of a minimum
number of
small particles into the bloodstream, whereupon the particles rapidly grow in
size from
14
CA 02556287 2006-08-16
the accretion of platelets actively binding to the platelet-binding agent on
or within the
particle. Furthermore, the particle-bound platelets would interact with each
other thereby
forming aggregates of increasing size producing a tight matrix and effecting
occlusion of
the target vasculature.
[0057] The present invention further improves upon existing methods of
producing vascular occlusion by securing platelets to the surface of a solid-
phase
material by means of a platelet-binding agent. The agent of the invention
would
therefore have the following effects in vivo: a) molding to the contours of
the blood
vessel or capillary in which it resides, b) producing a solid, impermeable
three-
dimensional matrix; this in turn produces a tight, impermeable seal within the
vessel,
thereby maximally inhibiting the delivery of blood to downstream blood vessels
and
tissues.
(0058] For example, the introduction of a platelet-binding particle into the
blood
stream would proceed through the following sequence of events: a) a single
layer of
platelets would form on the surface of the particle thereby forming (i) a
,particle of
increased diameter and (ii) a particle coated v~ith activated platelets with
the propensity
to bind and activate nearby platelets in suspension, herein defined as 'single-
layered
surface activated platelets' particle (S-SAP particle), b) platelets flowing
in the blood
stream would interact with platelets bound to the S-SAP particle forming
'onion-like'
layers, herein defined as multi-layered surface activated platelet particle (M-
SAP
particle), c) M-SAP would interact with each other through platelet/platelet
interaction
forming larger aggregates, herein defined as the 'M-SAP matrix'.
[0059] As a further example, the introduction of an amorphous platelet binding
particle (e.g., MAA) containing or having a surface-bound platelet-binding
agent (e.g.,
VWF) into the blood stream would proceed through the following sequence of
events: a)
single platelets would bind on and within the matrix of the particle thereby
forming (i) a
particle with increased diameter and rigidity, (ii) a particle coated with and
containing
activated platelets with the propensity to bind and activate nearby platelets
in
suspension; b) platelets flowing in the blood stream would interact with the
platelets
bound to and/or bound within the particle thereby forming aggregates within
and/or on
Is
CA 02556287 2006-08-16
the particle, c) particles containing and/or having surface bound platelets
would interact
with each other to form large particle aggregates.
[0064] : As used herein, therapeutically beneficial, providing a therapeutic
benefit
or the like refers to a desirable change in the physiology of the recipient
animal. In a
preferred embodiment of the invention, the change is detectable. In accordance
with the
invention, any biological mechanism that involves~activated platelets or
platelet
modulation may be used or harnessed to achieve a beneficial therapeutic
result.
Exemplary therapeutic benefits produced in accordance with the present
invention
include, but are not limited to, forming a thrombus, forming a platelet-
mediated
occlusion, eliminating a hyperplastic tissue or cells, eliminating a tumor
and/or tumor
cells, diminishing the size of a hyperplastic tissue, diminishing the size of
a tumor,
causing the hyperplastic tissue or tumor to become susceptible to additional
therapies
such as chemotherapy and/or radiation therapy or the like, starving or
reducing the
nutrient supply to a hyperplastic tissue or cancer, repairing AV-
malformations, reducing
or preventing blood loss from endoleaks and repairing vessel aneurysms.
[0061] Another exemplary therapeutic benefits produced in accordance with the
present invention include, but are not limited to, forming a thrombus, forming
a platelet
mediated occlusion, eliminating a hyperplastic tissue or cells, eliminating a
tumor and/or
tumor cells, diminishing the size of a hyperplastic tissue, diminishing the
size of a tumor,
in combination with additional therapies either serially or alongside such as
surgery,
chemotherapy, chemobembolization, local ablation treatments, immunotherapy,
angiogenesis inhibition and/or radiation.
[0062] As used herein, "administering." refers to any action that results in
contacting or delivering a composition containing a solid-phase agent to a pre-
determined cell, cells, or tissue, typically mammalian. Administering may be
conducted
in vivo, in vitro, or ex vivo. For example, a composition may be administered
by injection
or through an endoscope or catheter. Administering also includes the direct
application
to cells of a composition according to the present invention. For example,
during the
course of surgery, the vasculature of tumor or hyperplastic tissue may be
exposed. In
accordance with an embodiment of the invention, the exposed cells or
vasculature may
16
CA 02556287 2006-08-16
be exposed directly to a composition of the present invention, e.g., by
washing or
irrigating the surgical site, vasculature, and/or the cells.
[0063]-~ The solid-phase platelet-binding agent can be localized to a specific
target
site using a binding or targeting agent. Exemplary binding or targeting agents
include,
but are not limited to: monoclonal antibodies; polyclonal antibodies; chimeric
monoclonal antibodies; humanized antibodies; genetically engineered
antibodies;
fragments of antibodies, selected from the group consisting of F(ab)2,
F(ab')2, Fab,
F(ab') , Dab, Fv, sFv, scFv, Fc, and minimal recognition unit; single chains
representing
the reactive portion of monoclonal antibodies (SC-MAb); tumor-binding
peptides; a
protein, including receptor proteins; peptide; polypeptide; glycoprotein;
lipoprotein, or
the like, e.g., growth factors; lymphokines and cytokines; enzymes, immune
modulators;
hormones, for example, somatostatin; a ligand (paired with its complementary
anti-
ligand); oligonucleotides; any of the above joined to a molecule that mediates
an-
effector function; and mimics or fragments of any of the above. Analogs of the
above-
listed targeting moieties that retain the capacity to bind to a defined target
cell
population may also be used within the claimed invention. In addition,
synthetic
targeting moieties may be designed.
[0064] Monoclonal antibodies useful in the practice of the present invention
include whole antibodies and fragments thereof. Such monoclonal antibodies and
fragments are producible in accordance with conventional techniques, such as
hybridoma synthesis, recombinant DNA techniques and protein synthesis. Useful
monoclonal antibodies and fragments may be derived from any species (including
humans) or may be formed as chimeric proteins, which employ sequences from
more
than one species. See, generally, Kohler and Milstein, Nature, 256:495-97,
1975; Eur. J.
Immunol., 6:511-19, 1976. The preferred binding and/or targeting agent capable
of
localizing the solid-phase agent to a target site is an antibody or antibody-
like molecule,
preferably a monoclonal antibody. A more preferred binding agent is an
antibody that
binds a ligand/receptor complex on hyperplastic tissue or cells (e.g., tumor)
or the
vasculature associated with hyperplastic tissue or cells. The most preferred
binding
agent is an antibody or antibody-like molecule that binds a growth
factor/growth factor
receptor complex either on or in the vicinity of the tumor mass such as the
tumor
m
CA 02556287 2006-08-16
vasculature. In a preferred embodiment of the invention, the binding agent
(i.e.,
antibody or antibody-like molecules) would bind to the VEGFNEGF receptor
complex. In
a further preferred embodiment of the invention, the antibody or antibody-like
molecule
binding would recognize a neo-epitope (cryptic or previously unavailable
epitope)
formed due to ligand/receptor (i.e., growth factor/growth factor receptor)
interaction.. In
a further preferred embodiment of the invention, the binding of the antibody
or antibody-
like molecules to the growth factor/growth factor receptor complex would not
affect the
function of either the growth factor or the growth factflr receptor.
[0065] As used herein, VEGF receptor refers to all members of the Vascular
Endothelial Growth Factor Receptor family, including but not limited to FLT1/
VEGFR,
FLK1/KDR/VEGFR2, and FLT4/VEGFR3.
[0066] As used herein "causing a tissue or tumor to become susceptible to
additional therapies" refers to inducing a condition of low nutrient and/or
oxygen supply
to the tissue or tumor, through the method of the present invention including,
but not
limited to, forming a thrombus in the tumor vasculature and/or causing .a
reduced blood
supply to the tumor. ~--
[0067] Exemplary proteins useful in the practice of this invention include but
are
not limited to proteins corresponding to known cell surface receptors
(including low
density lipoproteins, transferrin and insulin), fibrinolytic enzymes, anti-
HER2, platelet
binding proteins such as annexins, and biological response modifiers
(including
interleukin, interferon, erythropoietin and colony-stimulating factor).
Oligonucleotides,
e.g., anti-sense oligonucleotides that are complementary to portions of target
cell
nucleic acids (DNA or RNA), are also useful as targeting moieties in the
practice of the
present invention. Oligonucleotides binding to cell surfaces are also useful.
[0068] Any growth factor may be used for such a targeting purpose so long as
it
binds to a tumor or tumor-associated endothelial cell. Suitable growth factors
for
targeting include but are not limited to VEGF/VPF (vascular endothelial growth
factor/vascular permeability factor), FGF (which, as used herein refers to the
fibroblast
growth factor family of proteins), TGFb (transforming growth factor b), EGF
and
pleitotropin. Preferably the growth factor receptor to which the targeting
factor binds
should be present at a higher concentration on the surface of tumor-associated
Is
CA 02556287 2006-08-16
endothelial cells than on non-tumor associated endothelial cells. Most
preferably, the
growth factor receptor to which the targeting growth factor binds should
further be
present at a higher concentration on the surface of tumor-associated
endothelial cell
than on any non-tumor-associated cell type.
[0069] Functional equivalents of the aforementioned molecules are also useful
as
targeting moieties of the present invention. One targeting moiety functional
equivalent is
a "mimetic" compound, an organic chemical construct designed to mimic the
proper
configuration and/or orientation for targeting moiety-target cell binding.
Another
targeting moiety functional equivalent is a short polypeptide designated as a
"minimal"
polypeptide, constructed using computer-assisted molecular modeling and
mutants
having altered binding affinity, such minimal polypeptides exhibiting the
binding affinity
of the targeting moiety.
[0070] The Fv fragments of immunoglobulins have many significant advantages
over whole immunoglobulins for the purpose of targeted tumor therapy,
including better
lesion penetration on solid tumor tissue and more rapid blood clearance, as
well as
potentially lower Fc-mediated immunogenicity: An exemplary single-chain Fv
(scFv)
binding agent may be engineered from the genes isolated from the variable
regions of
antibodies recognizing a ligand/receptor complex.
[0071] An embodiment of the invention involves a targeting agent having a
binding affinity for a marker found, expressed, accessible to binding, or
otherwise
localized on the cell surfaces of tumor-associated vascular endothelial cells
as
compared to normal non-tumor-associated vasculature. Further, certain markers
for
which a targeting agent has a binding affinity may be associated with
components of the
tumor-associated vasculature rather than on the tumor-associated endothelial
cells,
themselves. For example, such markers may be located on basement membranes or
tumor-associated connective tissue.
[0072] It may be desirable to prepare and employ an antibody or other binding
agent or moiety having a relatively high degree of selectivity for tumor
vasculature,
together with little or no reactivity with the cell surface of normal
endothelial cells as
assessed by immunostaining of tissue sections. It may also be desirable to
prepare and
19
CA 02556287 2006-08-16
employ an antibody or other binding agent or moiety capable of binding an
epitope
common to all vasculature.
(0073] ~~ Any composition that includes a solid-phase platelet-binding agent
with or
without a targeting agent according to the invention may be used to initiate
an in vivo
therapeutic benefit, thrombus formation, and/or cell killing or regression.
The
composition may include one or more adjuvants, one or more carriers, one or
more
excipients, one or more stabilizers, one or more permeating agents (e.g.,
agents that
modulated movement across a cell membrane), one or more imaging reagents, one
or
more effectors; and/or physiologically-acceptable saline and buffers.
Generally,
adjuvants are substances mixed with an immunogen in order to elicit a more
marked
immune response. The composition may also include pharmaceutically acceptable
carriers. Pharmaceutically acceptable carriers include, but are not limited
to, saline,
sterile water, phosphate buffered saline, and the like. Other buffering
agents, dispersing
agents, and inert non-toxic substances suitable for delivery to a patient may
be included
in the compositions of the present invention. The compositions may
be,~solutions
suitable for administration, and are typically sterile, non-pyrogenic and free
of
undesirable particulate matter. The compositions may be sterilized by
conventional
sterilization techniques.
[0074] In a preferred embodiment of the invention, a suitable composition
includes a binding or targeting agent that binds to ligand/receptor complex.
Exemplary
antigens useful as targets in accordance with the present invention include,
but are not
limited to, antigens associated with cancer, including, lung, colon, rectum,
breast, ovary,
prostate gland, head, neck, bone, immune system, blood, or any other
anatomical
location. Exemplary antigens and/or pre-determined sites include but are not
limited to
VEGF/VEGF receptor complex, FGF/FGF receptor complex, or TGF beta/TGF beta
receptor complex, p-selectin, sialyl-lewis X, endothelin, endothelin receptor,
endothelin/endothelin receptor complex, alpha-fetoprotein, platelet-
endothelial cell
adhesion molecule (PECAM), CD31, CD34, CD36, glycoprotein Ib (GPIb), endoglin,
thrombomodulin, endothelial leukocyte adhesion molecule (ELAM), intercellular
adhesion molecule 1 (ICAM-1 ), MHC-I, and MHC-II. The subject may be a human
or
animal subject.
CA 02556287 2006-08-16
[0075] As noted above, a composition or method of the present invention
includes a platelet binding agent or component. Exemplary platelet binding
agents or
components include but are not limited to von Willebrand factor (VWF),
osteopontin,
fibrinogen, fibrin, fibronectin, vitronectin, collagen, thrombospondin,
laminin, heparin,
heparan sulfate, chondroitin sulfate, phospholipase A2 (PLA2), matrix metal
lop
proteinases (MMPs), thrombin, glass, sialyl-lewis X, fibulin-1, platelet-
endothelial cell
adhesion molecule (PECAM), intercellular adhesion molecule 1 (ICAM-1 ),
intercellular
adhesion molecule 2 (ICAM-2), CD11 b/CD18 (MAC-1 ), CD11 a/CD18 (LFA-1 ), p-
selectin glycoprotein ligand 1 (PSGL-1 ), either singly or in combination.
[0076] As noted above, a composition or method of the present invention may
include a platelet-mediated occlusion enhancer. The platelet-mediated
occlusion
enhancer may be a moiety that forms a portion of a bi-functional molecule as
noted
above, may be an ingredient in a composition according to the invention,
and/or may be
administered separately from a composition according to the invention.
[0077] Those skilled in the art will recognize that it may be desirable to
include or
use an occlusion enhancer when the individual receiving the therapy is in a
state of
compromised haemostasis. Under such conditions, the individual receiving the
therapy
has a propensity to bleed due to a pathological process that may have been
acquired or
is congenital in nature. Since the utility of the present invention is reliant
upon the
formation of a thrombus in the tissue or tumor vasculature after targeting
platelets to the
area, use of methods that augment platelet activation and/or the coagulation
process
could compensate for the individuals hemorrhagic tendencies. Examples of such
conditions include, but are not limited to, haemophilia, von Willebrand's
disease,
coagulation factor deficiencies, Glanzmann's thrombasthenia, and Bernard
Soulier
Syndrome.
[0078] Exemplary platelet-mediated occlusion enhancers include but are not
limited to ristocetin, thrombin, heparin-induced thrombocytopenia (HIT)
antibodies or
portions thereof, antiphospholipid antibodies (APA) or portions thereof, whole
antibody
molecules via an Fc-mediated mechanism, anti-LIES antibodies, anti-CD9
antibodies,
epinephrine, thrombin receptor activating peptide (TRAP), proteinase-activated
receptor
(also known as protease activated receptor, PAR) agonists, cathepsin G,
elastase,
21
CA 02556287 2006-08-16
arachidonate, platelet activating factor (PAF), thromboxane A2 (TxA2), TxA2
mimetics,
phospholipase A2 (PLA2), activators of protein kinase C (PKC), adenosine
diphosphate
(ADP),:inducers of cyclo-oxygenase 1 (COX-1 ), inducers of cyclo-oxygenase 2
(COX-
2), collagen, von Willebrand factor (VWF), matrix metalloproteinases (MMPs),
heparin,
heparan sulfate, chondroitin sulfate, ionophores, complement cascade
components
(e.g., C5b-9) platelet microparticles, platelet membrane fractions.
[0079] As noted above, a composition or method of the present invention may
include a platelet-mediated occlusion retarder or the like. The platelet-
mediated
occlusion retarder may be a moiety that forms a portion of a bi-functional
molecule as
noted above, may be an ingredient in a composition according to the invention,
and/or
may be administered separately from a composition according to the invention.
Those
skilled in the art will recognize that it may be desirable to include or use a
platelet-
mediated occlusion retarder when the individual receiving therapy based on the
method
of the present invention has an underlying propensity to thrombose (i.e. form
clots too
rapidly and/or in inappropriate locations in the body). Although the metf?od
of the
present invention is directed to the formation.of a thrombus in the tumor
vasculature,
individuals with a propensity to thrombose may form thrombi in inappropriate
locations
during the course of the therapy described by the present invention. Use of
agents to
reduce the rapidity and/or extent of thrombosis could be used to minimize the
risk of
forming thrombi in inappropriate locations in the body. Examples of conditions
whereby
the individual receiving therapy encompassed by the present invention may
require the
use of occlusion retarders are, but are not limited to, coronary artery
disease, acute
myocardial infarction, transient ischemic attack, stroke, high blood pressure,
ATIII
deficiency, Protein C deficiency, Protein S deficiency, heparin-induced
thrombocytopenia, deep vein thrombosis, peripheral vascular disease and/or
Factor V
Leiden deficiency.
[0080] Exemplary platelet-mediated occlusion retarders include but are not
limited to aspirin, ibuprofen, acetaminophen, ketoprofen, ticlopidine,
clopidogrel,
indomethacin, dipyridamole, omega-3 fatty acids, prostacyclin, nitric oxide,
inducers of
nitric oxide, inducers of nitric oxide synthase, matrix metalloproteinase
inhibitors
(MMPIs, TIMPs), anti-GPllb/Illa agents, anti-ovo3 agents, anti-o2o1 agents,
anti-CD36
22
CA 02556287 2006-08-16
agents, anti-GPVI agents, aurintricarboxylic acid, thrombin receptor
antagonists,
thromboxane receptor antagonists, streptokinase, urokinase, tissue plasminogen
activator (WA).
[0081] In addition, it is known that platelets that have been cooled below
their
membrane phase transition temperature (i.e., < 15 degrees C) become
irreversibly
activated. Although the platelets function normally if transfused into a
patient, the
platelets are rapidly cleared from the body (i.e., in approximately 24 hours,
in contrast to
normal circulating platelet life span of 7 to 10 days). Although these
platelets are
cleared rapidly, they bind with high avidity to immobilized VWF. Therefore,
transfusion
of cooled platelets provides an additional means to enhance thrombus formation
at the
target site. Therefore, one embodiment of the invention includes controlling
platelet-
mediated occlusion by administering platelets cooled as noted above.
[0082] As noted above, the targeting moiety may be, or may be bound to, one
member of a binding pair. Methods according to the invention may require a
time period
sufficient for accumulation of the targeting moiety at the site of
localization, for optimal
target to non-target accumulation, for accum~,+lation and binding of the
second member
of the binding pair, and/or for clearance of unbound substances.
[0083] In accordance with the invention, two, three or more step targeting or
localization steps may be used. Many of these protocols are well known in the
art (see,
for example, U.S. patent 5,578,287 using a biotin/avidin protocol). Exemplary
multiple
step protocols include, but are not limited to, administering a binding agent-
ligand,
administering an anti-ligand to clear unbound binding agent and to localize
bound
binding agent-ligand, and administering an active agent-ligand. As used
herein, active
agent refers to any therapeutic agent that is active or becomes active and
leads to a
therapeutic benefit.
[0084] In accordance with a method of the invention, the binding agent must be
capable of binding a ligand/receptor complex, and may be administered to the
patient
by any immunologically suitable route. The immune perspective of cancer
differs
somewhat from the perspective centered on the cancer cell itself. The presence
of
tumor in a host logically demonstrates that the immune system and its ability
to acquire
new, useful immunity either is generally damaged or has become specifically
tolerant.
23
CA 02556287 2006-08-16
Insofar as the primary tumor bulk has a negative effect on the immune system,
its
removal can be considered to have immunotherapeutic potential.
[0085] < The present invention in combination with immunotherapy may enhance
immunologic memory, targeting immunogenic proteins involved in malignant
transformation, we may be able to prevent relapse, which is one of the major
problems
in the long term survival of cancer patients.
[0086] For example, the binding agent may be introduced into the patient by an
intravenous, intra-arterial, subcutaneous, intraperitoneal, intrathecal,
intravesical,
intradermal, intramuscular, or intralymphatic route. The composition may be in
solid,
solution, tablet, aerosol, or multi-phase formulation forms. Liposomes, long-
circulating
liposomes, immunoliposomes, biodegradable microspheres, micelles, or the like
may
also be used as a carrier, vehicle, or delivery system. Further more, using ex
vivo
procedures well known in the art, blood, plasma or serum may be removed from
the
patient; optionally, it may be desirable to purify the antigen in the
patient's blood; the
blood or serum may then be mixed with a composition that includes a binding
agent or
the solid-phase agent according to the invention; and the treated blood or
serum is
returned to the patient. The clinician may compare the responses associated
with these
different routes in determining the most effective route of administration.
The invention
should not be limited to any particular method of introducing the binding
agent into the
patient.
[0087] Administration may be once, more than once, or over a prolonged period.
Administration may be made in combination with, serially or alongside, two or
more
therapies. Administration may be made in combination with two or more
therapies once,
more than once, or over a prolonged period. As the compositions of this
invention may
be used for patients in a serious disease state, i.e., life-threatening or
potentially life-
threatening, excesses of the solid-phase agent may be administered if
desirable. Actual
methods and protocols for administering pharmaceutical compositions, including
dilution
techniques for injections of the present compositions, are well known or will
be apparent
to one skilled in the art. Some of these methods and protocols are described
in
Remington"s Pharmaceutical Science, Mack Publishing Co. (1982).
24
CA 02556287 2006-08-16
[0088] A solid-phase agent may be administered in combination with other
binding agents, or may be administered in combination with other treatment
protocols or
agents; e.g., chemotherapeutic agents, embolizing agents such as Gelfoam or
polyvinyl
alcohol (PVA) particles or the like.
[0089] As is well known in the art, a disadvantage associated with
administering
treatment agents or treatment agent conjugates in vivo includes non-target or
undesirable target binding. It is therefore a desirable attribute of any
administered
composition to minimize non-target binding, to minimize non-target exposure to
the
treatment agent or active agent, and/or to maximize clearance of non-bound
binding
agent, ligand, or active agent. Moreover, optimizing these attributes
typically permits
administering a higher dose of active agent, a therapeutic agent, or an
element of the
process that activates a previously un-activated agent. Those skilled in the
art are well
versed in selecting the optimal parameters for administering the highest
possible dose
while remaining safely below a toxic threshold.
[0090] In accordance with a preferred embodiment of the invention, therefore,
un-
activated platelets accumulate or are induced-to accumulate at a pre-
determined site
through binding to the solid-phase agent, and then the properly localized
platelets are
selectively activated.
[0091] In accordance with a preferred embodiment of the invention, activated
platelets accumulate or are induced to accumulate at a pre-determined site
through
binding to the solid-phase agent or through platelets bound to the solid-phase
agent.
[0092] The effectiveness of the present invention may be monitored by
conventional assays that determine thrombus formation, morphometric studies of
thrombus formation, tumor necrosis, tumor size, tumor morphology, and/or
thrombus
formation that results in tumor necrosis, blood flow studies (e.g.,
angiography, Doppler
ultrasound, radiography, CT scan, MRI), or reduction in pain symptoms. One
skilled in
the art will recognize that other tests may be performed to assess or monitor
therapeutic
benefit.
[0093] Since some binding agents such as proteins are by themselves poor
immunogens, their immunogenicity may be augmented by administration in
immunological adjuvants and antigen delivery systems. The immunogenicity of a
CA 02556287 2006-08-16
specific composition may also be increased or optimized by choice of delivery
route. For
example, the immunogenicity of compositions produced in accordance with the
present
invention that include a monoclonal antibody may be increased by choosing a
mode of
delivery that increases the direct contact between the binding agent and the
antigen.
The referred route is intravenous. Those skilled in the art are conversant
with the
various choices available, and why one route might be chosen over another
route for a
particular binding agent.
(0094] One skilled in the art will also recognize that liposomes, nanospheres,
micelles, or microspheres may be used to administer a composition, and that
such
administration may result in a therapeutically desirable benefit.
[0095] It will be recognized by those skilled in the art that for certain
congenital
and pathological conditions, some of which are listed below, it is desirable
to modify a
composition or method of the present invention to compensate for a
predisposition of
the patient to bleed excessively or to thrombose. Under these circumstances,
use of
modifying agents, which either enhance or Under these circumstances,, use of
modifying
agents, which either enhance or dampen a m$thod or composition of the
invention, can
be employed. The use of these modifying agents is predicted to minimize
bleeding or
clotting episodes. Moreover, the use of modifying agents enables controlled
administration of a composition according to the invention under normal
circumstances
(i.e., normal hemostasis).
[0096] Exemplary pro-thrombotic or pro-coagulant conditions that may warrant
the using of controllers, retarders, or agents that diminish a method or
composition of
the invention include, but are not limited to, Factor V~eiden deficiency,
antiphospholipid
syndrome (APS), Protein C and/or Protein S and/or Antithrombin III deficiency,
deep
vein thrombosis (DVT), pseudo-von Willebrand's disease, Type Ilb von
Willebrand's
disease, peripheral vascular disease (PVD), and high blood pressure, among
others.
Exemplary conditions that include a risk of hemorrhage that may warrant using
enhancers or agents that augment a method or composition of the invention
include but
are not limited to, any condition that includes a risk of hemorrhage,
including but not
limited to coagulation factor deficiencies, hemophilia, thrombocytopenia, and
anticoagulation therapy, among others. Controlling thrombus generation
includes at
26
CA 02556287 2006-08-16
least one of altering the temperature at the pre-determined site, altering the
rate of
blood flow at the pre-determined site, and altering the blood pressure at the
pre-
determfned site.
[0097] As an example of the foregoing, it will be recognized by those skilled
in the
art that upon initiation of the vascular occlusion process, reversal or
dampening of the
associated prothrombotic condition may be necessary: In such cases,
administration of
agents that reduce platelet reactivity will, in turn, reduce response to the
vascular
occlusion initiators. Such agents are readily known by those skilled in the
art and
include, but are not limited to: aspirin or aspirin-like compounds, ibuprofen,
acetaminophen, ketoprofen, ticlopidine, clopidogrel, indomethacin, omega-3
fatty acids,
prostacyclin, nitric oxide, inducers of nitric oxide, inducers of nitric oxide
synthase,
matrix metalloproteinase inhibitors (MMPIs, TIMPs), anti-GPIb agents, anti-
GPllb/Illa
agents, anti-av~33 agents, anti-a2[i1 agents, anti-CD36 agents,
aurintricarboxylic acid,
thrombin receptor antagonists, thromboxane receptor antagonists,
streptokinase,
urokinase, tissue plasminogen activator (tPA).
[0098] An exemplary process in which~it may be desirable to enhance or
augment platelet occlusion process includes thrombocytopenic (low platelet
count)
patients. These individuals would benefit from concomitant or pre-
administration
(transfusion) of platelet products to provide an adequate resource of
platelets to
accomplish platelet occlusion. It will be recognized by those skilled in the
art that all
transfusable products mimicking or approximating normal platelet function can
be used
under such circumstances. Such agents include but are not limited to: random
donor
platelets, apheresis platelets, autologous platelets, washed platelets,
platelet membrane
fractions, cooled platelets, frozen platelets, particles containing or
expressing platelet
membrane components, platelet substitutes and whole blood.
[0099] As a further example, specific platelet-function enhancing agents can
be
employed to boost or enhance initial platelet reactivity once targeted to the
site of
therapy. Agents known to those skilled in the art have been demonstrated to
enhance
existing platelet reactivity and/or lower the threshold limiting sufficient
platelet reactivity
to facilitate irreversible platelet adhesion and/or platelet degranulation
and/or
platelet/platelet binding and/or platelet accretion about an existing
thrombus. These
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CA 02556287 2006-08-16
agents include but are not limited to: ristocetin, thrombin, heparin-induced
thrombocytopenia (HIT) antibodies or portions thereof, antiphospholipid
antibodies
(APA) or portions thereof, whole antibody molecules via an Fc-mediated
mechanism,
anti-ligand-induced binding site (anti-LIBS) antibodies or portions thereof,
anti-CD9
antibodies or portions thereof, epinephrine, thrombin receptor activating
peptide
(TRAP), PAR agonists, cathepsin G, elastase, arachidonate, thromboxane A2
(TxA2)
mimetics, TxA2, phospholipase A2 (PLA2), activators of protein kinase C (PKC),
adenosine diphosphate (ADP), collagen, von Willebrand factor (VWF), matrix
metalloproteinases (MMPs), heparin, heparan sulfate, chondroitin sulfate,
ionophores,
platelet microparticles, platelet membrane fractions.
[0100] Once introduced into the bloodstream of an animal bearing a tumor,
hyperplastic tissue, AV-malformation, aneurysm or endoleak, the solid-phase
agent will
localize in the target vasculature; bind or immobilize platelets, whereby
immobilization
activates the platelets; and the activated platelets in turn bind and activate
other
platelets until an occlusion is formed. Platelet activation and binding
facilitates leukocyte
binding to the activated platelets further enha+acing occlusion of the target
vasculature.
Example 1.
[0101] Each vial of VWF-coated macroaggregated albumin particles (MAA/VWF)
contains 5.0 mL of the particulate suspension, which consists of 4.5 to 7
million particles
of the MAA/VWF conjugate in an isotonic vehicle. The human serum protein
content per
vial ranges from 5 to 15 mg. By light microscopy, more than 90% of the MAA/VWF
conjugate particles are 10-150 pm in diameter and less than 10% are less than
10 pm
in diameter.
Summary of the MAA/VWF Particle In Vitro Studies
[0102] Mixing MAA/VWF particles with either platelet rich plasma or whole
blood
in vitro resulted in platelet activation and subsequently platelet
aggregation. MAA/VWF
particles manufactured with a wide range of ratios of VWF to MAA (by weight)
was
studied for its ability to cause platelet activation and was found effective
over the entire
range of ratios studied (1:1 to 1:80). The distribution of VWF on MAA
particles was also
28
CA 02556287 2006-08-16
investigated over the same range of ratios of VWF and MAA, and von Willebrand
Factor
was found to be uniformly distributed over the MAA particulate surfaces.
Summary of the MAA/VWF Particle In Vivo Studies
[0103] The effects of MAA/VWF particles on renal blood flow were evaluated in
vivo in a porcine renal infarct model. Administration of'MAA/VWF particles by
transcatheter arterial injection into the arterial network of the porcine
kidney had the
following effects:
1. In an acute study in pigs, blood flowing through the kidney decreased over
a 20
minute period to less than 20% of the baseline rate (20 mL/minute versus 100
mL/minute).
2. In a chronic study of pigs monitored over a seven day period after
treatment with
MAA/VWF particles:
a. The blood flow rates to the target kidneys were maintained at less than 10%
of
the baseline rate (3-5 mL/min);
b. The animals treated with MAA/VWF particles did not develop any adverse
events
during the observation period.
3. Inspection of the renal vasculature grossly and histologically after
surgical
removal of the affected organ (acute treatment) demonstrated thrombus
formation distal
to the angiocatheter down to the level of the capillary beds.
4. Histological examination of the target kidney seven days (chronic study)
after
treatment showed no evidence of necrosis.
(0104] Histological examination of normal tissue from acute (90 minutes) and
chronic studies (7 days) showed no sign of thrombosis, indicating that the
MAA/VWF
particles were completely retained in the capillary beds of the porcine
kidneys.
[0105] In further studies of MAA/VWF particles to evaluate the agent in acute
porcine infarct models, MAA/VWF particles were used to block the blood flow to
the
porcine spleen, kidneys or lobes of the liver. Sequential small doses of
MAA/VWF
particles (0.1 mL) were administered by transcatheter arterial injection into
the major
29
CA 02556287 2006-08-16
artery supplying each organ. (Each dose of MAA/VWF particles was followed by
0.1 mL
of human platelet rich plasma.) The MAA/VWF particles induced a rapid and
sustained
reduction in blood flow to each of the targeted organs. No evidence was found
of
induction of thrombosis in any other organ or tissue of the test animals. No
adverse
effects of treatment were noted by observation of the living animals.
Summary of the MAA/VWF Particles Biodistribution Studies
[0106] Following transcatheter arterial injection of technetium-99m labelled
MAA/VWF particles into the arterial network of the porcine kidney or liver,
the agent was
virtually completely retained within target, organ. The data clearly
demonstrate that
negligible amounts of radioactivity reached the systemic circulation, and the
near
background levels of radioactivity in other organs may be attributed to the
presence of
free pertechnetate and to the metabolic products of the radiolabelled MAA/VWF
particles in the blood.
[0107] Although the present invention has been described in terms of
particular
preferred embodiments, it is not limited to tha~se embodiments. Alternative
embodiments, examples, and modifications, which would still be encompassed by
the
invention, may be made by those skilled in the art, particularly in light of
the foregoing
teachings.
Example 2
[0108] Advanced renal cancers including renal cell carcinoma and
angiomyolipoma can be treated by embolization prior to surgical resection.
MAA/VWF
particles or particles coated with a similar platelet capture agent can be
used as embolic
agents to induce a thromboembolus in the arterial supply of advanced renal
tumors. A
standard embolization procedure would be performed prior to surgical removal
of the
affected kidney. Prior to the embolic procedures, patients would undergo renal
imaging
by computerized tomography (CT), magnetic resonance imaging (MRI) or
ultrasound to
delineate the extent of disease and to measure the tumor masses. Scintigraphic
imaging of the metabolic status of the patient's tumor masses would also be
performed
with F-18 fluorodeoxy-glucose (FDG), and at the discretion of the physician, a
baseline
CA 02556287 2006-08-16
study of hypoxic imaging of the abdomen could be performed by means of F-18
misonidazole.
[0109] ~ Following selective and global angiography to delineate the renal
vasculature, thromboembolization of the renal tumor arterial network would be
performed with MAA/VWF particles or particles coated with a similar platelet
capture
agent such as collagen-coated particles. Treatment with MAA/VWF particles
would
begin with a starting dose of approximately 1.0 mL (1 x 106 particles)
administered intra-
arterially; additional 1.0 mL doses of MAA/VWF particles would be administered
as
required to achieve blockage of the blood supply to the tumor mass.
Obstruction of
target arteries would be assessed by injection of contrast agent after
injection of the
dose of MAA/VWF particles. Following the procedure, supportive therapy would
be
given to the patient as required to ameliorate the effects of the post-
embolization
syndrome (pain, nausea, fever).
[0110] In the interval between the embolization procedure and the complete or
partial nephrectomy, scintigraphic imaging of the chest may be repeated with
FDG. At
the discretion of the physician, scintigraphy with F-18 misonidazole may also
be
repeated.
[0111] Safety laboratory studies could be performed pre-treatment, daily for
the
first week after the embolization procedure (Days 2-7), every other day for
the second
week (Day 8, 10, and 12), weekly for the remainder of the first month (Days
15, 22, and
[0112] 29), and biweekly for the second month (Days 43 and 57).
During the second and fifth weeks post-operatively (Study Days 8-12 and Days
29-33),
the patient could have a CT examination of the abdomen performed to evaluate
the
patient's status post-surgery and to evaluate the patency of the residual
renal arterial
vasculature (if any). At this time and at the discretion of the physician,
scintigraphic
imaging may be repeated with FDG and/or F-18 misonidazole.
Example 3
[0113] Patients with primary hepatocellular carcinoma (HCC) who are candidates
for standard embolotherapeutic procedures were treated with MAA/VWF particles
to
induce embolization of target vasculature feeding the tumors. Patients were
imaged by
31
CA 02556287 2006-08-16
triphasic CT to delineate the extent of disease and to measure the size of
index tumor
lesions.
[0114] <' Following selective catheterization of the right or left hepatic
artery using a
percutaneous femoral artery approach, a suitable dose of Technetium Tc-99m
Albumin
Aggregated Injection (Tc-99m MAA) was injected through the catheter.
Scintigraphic
imaging confirmed the absence of arterial shunts that might result in untoward
arterial
embolization.
[0115] Following selective and global angiography to delineate the liver
vasculature, the patients were treated by means of the standard protocol for
transcatheter arterial chemoembolization (TACE). Doxorubicin (75 mg/m2) was
emulsified in 10 mL of Lipiodol~ and infused in accordance with standard
procedures,
while the subsequent embolization of the arteries feeding the tumors was
performed
with MAA/VWF particles.
[0116] Embolization of the target hepatic artery branches) with MAA/VWF
particles began with a starting dose of approximately 1.0 mL (approxim,ately 1
x 106
VWF-MAA particles), which was injected by t~anscatheter arterial injection.
Additional
1.0 mL doses of MAA/VWF particles were administered as required until blockage
of the
target arteries was achieved. Obstruction of target arteries was assessed by
injection of
contrast agent 2 minutes after injection of the dose of MAA/VWF particles.
[0117] Following the chemoembolization procedure, symptomatic therapy was
given as required to ameliorate the effects of the post-embolization syndrome
(pain,
nausea, fever).
[0118] The patients were re-imaged at one and four weeks after the procedure
by
CT, and then in accordance with standard medical care, every 3 months by CT
and/or
ultrasound. Long term follow up involved standard patterns of care with time
to disease
progression and patient survival data obtained by chart review.
Example 4
[0119] HCC patients treated by TACE as outlined in Example.3 using MAA/VWF
as the embolic agent were assessed by CT to determine the affect of therapy on
index
tumor lesions. Five patients were treated with MAA/VWF in combination with
32
CA 02556287 2006-08-16
doxorubicin and Lipiodol~. No serious adverse events were attributed to the
administration of MAA/VWF particles. Four of the five patients treated with
MAA/VWF
particles showed tumor regression ranging from approximately 8% to 27%
decrease in
tumor volume (response measured 5 weeks after treatment). The remaining
patient
showed an increase in tumor size amounting to 2% (by volume). All responses
fell into
the "stable disease" category. One patient received a (fiver transplant after
TACE using
MAA/VWF particles as an embolic agent.
33
