AU2015200603B2 - Compositions and methods for producing vascular occlusion using a solid-phase platelet binding agent - Google Patents

Abstract

The present invention relates generally to methods and compositions for targeting and delivering solid-phase platelet-dependent vascular occlusion agents. In particular, particles or coils or stents coated with platelet binding agents are directed to target vasculature, such as the vasculature of solid tumor masses or AV-malformations or aneurysms or endoleaks; the solid-phase agent then binds and activates platelets, which in turn bind and activate other platelets. This process results in the rapid formation of a platelet-mediated thrombus about the solid-phase agent causing vessel occlusion

Description

WO 2009/06779! PCX/C Α2β08Λ)02060 2015200603 06 Feb 2015

COMPOSITIONS AND METHODS FOR PRODUCING VASCULAR OCCLUSION USING A SOLID-PHASE PLATELET BINDING AGENT

[0001] This application is a continuing application of U.S. Serial Number 11/205,047 (now allowed); which is a continuation of U.S. Application Serial No.: 10/241,717 filed September 12, 2002, which is a non-provisional of U,S, Application Serial· No.: 60/318,339 filed September 12, 2001. These patent applications are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

[0002] The present invention is directed to compositions and methods for producing a therapeutic benefit by producing vascular occlusion using platelet activation as the initiating event. Compositions and methods of the invention involve delivering a soiid-phase platelet-binding agent to a target site, causing platelets to bind and activate, thereby forming a localized thrombus, in some embodiments of fee invention, fee thrombus may be further allowed to be replaced by connective tissue. Occlusion of the vasculature of the target tissue by the localized thrombus typically 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~endothe!ium, a complex series of events occurs to limit the loss of blood from the damaged vessel. Circulating platelets contacting components of fee 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 fee blood

-1—A WO 2009/067791 PCT/CA2<M18/M206&amp; 2015200603 06 Feb 2015 stream, and 5) form an efficient plug, clot, and/or thrombus stemming the flow of blood from the vessel.

[0004] In contrast to the coagulation cascade, a process defined in part by the conversion of fibrinogen to fibrin, platelets coalesce about the damaged area and are held together by bridging molecules that bind to specific receptors on the platelet surface. The initial bridging between platelets and the sub-endothelium is dependent on the interaction between the glycoprotein lb (GPIb) receptor on the surface of the platelet and von Willebrand Factor (VWF) in the subendothelium (i.e„ immobilized VWF). This interaction in itself is unique, since normal platelets circulating in the blood often contact soluble VWF, but are not activated, nor do they bind to the soluble VWF* In vitro experimentation has confirmed that immobilization of the soluble VWF to a surface facilitates binding and activation of platelets. Upon activation of the platelet, an additional receptor, glycoprotein llb/lila (GPIIb/llla), is altered enabling the binding of several plasma proteins, thereby promoting piatelet/plateiet binding. In addition to fibrinogen, soluble VWF binds to the activated GPIIb/llla receptor, in turn becoming immobilized and capable of binding other platelets via GPIb and GPIIb/lila.

[0005] Hyperactive platelets can induce thrombus formation at inopportune times resulting In reduced blood supply to various organs and tissues. A prime example is thrombus formation induced by blood flowing through a stenotic {narrowed} vessel supplying the heart. Reduction of the flow of blood to the heart muscle leads to infarction and eventually heart attack (cardiac cell death). Cerebral ischemia (transient ischemic attack (TIA); stroke) occurs when an embolus or thrombus occludes blood vessels feeding the brain.

[0006] Other pathological states exist that are caused by platelet activation as a result of an inappropriate antibody-mediated process. Heparin-induced thrombocytopenia (HIT) is characterized by a dramatic loss in platelet numbers and thrombus formation at sites of pre-existing pathology, From 1 % to 5% of all patients receiving unfractionated heparin as an anticoagulant to promote blood flow produce an antibody that binds to heparin in complex with a platelet granule protein. The binding of the antibody to the heparin/protein complex on the surface of the platelet induces rapid platelet activation and localized thrombus formation. This in turn leads to infarction of the affected area. ~2~ WO 2009/067791 PCT/CA2008/002060 2015200603 06 Feb 2015 [00071 Thrombosis is a well-described consequence of cancer. Controversy exists as to whether the presence of a hyper-coaguiable state is predictive of cancer. Many studies have been conducted demonstrating a prothrombdtic tendency with most neoplasia or neoplasms. It has been suggested that thrombosis is the most frequent complication with patients with overt malignant disease.

[0908] A key to the development of successful anti-tumor agents is the ability to design agents that will selectively kill tumor cells, while exerting relatively little, if any, untoward effects against norma! tissues. This goal has been elusive in that there are few qualitative differences between neoplastic and normal tissues. Because of this, much research over the years has focused on identifying tumor-specific “marker antigens" that can serve as immunological targets both for chemotherapy and diagnosis. Many tumor-specific or quasi-iumor-specific (tumor-associated) markers have been identified as tumor cell antigens that can be recognized by specific antibodies.

[0009J Unfortunately, it is generally the case that tumor-specific antibodies will not in and of themselves exert sufficient anti-tumor effects to make them useful in cancer therapy, in contrast with their efficacy in lymphomas, immunotoxins have proven to be relatively ineffective in the treatment of solid tumors such as carcinomas. The principal reason for this is that solid tumors are generally impermeable to antibody-sized molecules: specific uptake values of less than G.001% of the injected dose per gram of tumor are not uncommon in human studies. Furthermore, antibodies that enter the tumor mass do not distribute evenly for several reasons. Firstly, the dense packing of tumor cells and fibrous tumor stromas present a formidable physical barrier to macro-molecMlar transport and combined with the absence of lymphatic drainage create an elevated interstitial pressure in the tumor core which reduces extravasation and fluid convection. Secondly, the distribution of blood vessels in most tumors is disorganized and heterogeneous. As a result some tumor ceils are separated by large distances from capillaries so that the extravasating antibody must diffuse over a large volume in order to reach and bind to remote tumor ceils. Thirdly, all of the antibody entering the tumor may become absorbed in perivascular regions by the first tumor cells encountered, leaving none to reach tumor cells at more distant sites. -3- WO 2009/067791 PCT/CA2008/002060 2015200603 06 Feb 2015 [0010] One approach ίο 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.

[0011] The present inventors propose that this approach will provide several advantages over targeting tumor ceiis directly. Firstly, the target cells are directly accessible to vascuiariy 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.

[0012] The present invention is aiso directed to compositions and methods of treating abnormal tissue growth, abnormal bleeding (during or after surgery, postpartum), ectopic pregnancy, placenta previa, placenta accreta and uterine fibroids.

[0013] Under certain ciinical situations, inhibition of blood flow to a tissue through occlusion of its associated vasculature is desirable. Examples include hemorrhagic stroke, existence of saphenous vein side branches in saphenous bypass graft surgery, treatment of aortic aneurysm, correction of vascular malformations, and treatment of solid tumors, [0014] Vascular occlusion has been performed using a variety of techniques and materials including emboiotherapy. Examples of embolotherapy include the use of particles composed of a variety of materials Including polyvinyl alcohol (Boschetti, PGT WOQ023054), acrylamide (Boschetti et a!, US 5,635,215; Boschetti et ai, US 5,648,100), pptymeiby! methacrylate (Lemperie, US 5,344,452), physiea! piugs composed of collagen (Conston et ai, US 5,456,693) and coils (Variant, US 5,639,277). Emboiotherapy involves the delivery of these materials to the target vasculature by means Of a catheter. Since the vasculature in any given area proceeds from larger arteries to arterioles to metarterioles to capillaries, each with progressively smaller vessel diameters, the delivered material (embolus) continues to travel in the flowing blood until it becomes lodged in the smaller blood vessels thereby Impeding the flow of biood to the dependent tissue.

[0015] 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, coated with WO 2009/06779-1 PCT/CA2)K«/«02060 2015200603 06 Feb 2015 von Willebrand factor (VWF) of mammalian origin, in 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. SUMMARY OF THE INVENTION {0016] 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. The composition comprises an 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] The present invention provides methods for inducing thrombus formation by capturing platelets on the mrcrosphere, 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 collagen-containing microsphere (e.g., coated particles), the 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 (ike. {0018] Purposeful induction of thrombosis in a patient appears at first glance to be counter-intuitive, since thrombosis is well known to contribute significantly to patient morbidity and mortality. The present invention, solid-phase platelet-mediated occlusion, is based on the site-specific induction of thrombosis utilizing the body's natural capacity to produce a thrombus in response to immobilized von Wiliebrand factor (VWF) or other locally acting platelet activation agents. Although VWF circulates in the blood stream in soluble form, it is not until the molecule is exposed as part of the subendothelium or -5- WG 2009/067791 PCT/CA2(K)8/002060 2015200603 06 Feb 2015 binds to exposed collagen from the subendotheiium that it is capable of capturing platelets and inducing platelet activation* [0019] Contact of the solid-phase platelet-binding agent with the blbod 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 bloodvessels). Cells, including tumor ceils or hyperplastic tissue, diminish or die as a result of loss of localized blood flow. This approach avoids systemic platelet activation and thrombosis; relying on the fact that immobilized VWF (but not soluble WVF) binds to and activates circulating platelets. Thus, the methods and compositions of the present invention are an indirect means of treating a pathological condition, such as cancer, hyperplastic cells, excessive bleeding or arteriovenous (AV) malformations.

[0020] The present invention improves on existing methods for treating solid tumors* hyperplastic tissue, excessive bleeding and AV-malformations and any other disease or condition in which platelets (resting and/or activated) may play a therapeutic role.

[0021] In a manner similar to an existing pathological condition (Le. 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. Piatelet 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.

[0022] 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. GFlIb/HIa 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.

[0023] 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, -6- WG 2009/067791 ΡΓ17{;Α2008/002060 2015200603 06 Feb 2015 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-malformations, uterine fibroids, pelvic congestion, menorrhagia, varicoceles, hemoptysis, aneurysms, visceral artery aneurysms, pseudoaneurysms and endoleaks.

[0024] A preferred method of the invention includes preparing a coil or stent coated with VWF of recombinant or mammalian origin anti introducing the VWF-coated agent into the bloodstream of an animal, such as a human patient, an ariimaf 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 lengths of time.

[0025] 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 vascularfy 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.

[0026] The methods of the present invention are highly effective In inhibiting blood flow in a target vasculature. The methods and compositions may also be used in combination with other treatment protocols, e.g., chemotherapy, for the ultimate benefit of the patient.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention provides impositions and methods for capturing platelets at a predetermined site, activating the platelets, and harnessing the natural -7- PCT/CA2008/002060 WO 2009/067791 2015200603 06 Feb 2015 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 concentrated at or on 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 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.

[0028] The present invention provides a method for administering a biodegradable microsphere with a platelet binding agent, allowing the mtcrosphere to induce thrombus formation, and allowing the thrombus to be replaced by permanent connective tissue. [0029J The present invention also includes targeting platelets to a predetermined 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 predetermined 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.

[0030] 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 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-maiformations, neuro-embolism, varicoceles, hemoptysis, visceral artery aneurysms, arterial aneurysms, endoieaks, and the iike. Furthermore, the compositions and methods of the present invention provide a therapeutic benefit to the recipient of the composition. -8-- WO 2009/067791 PC'T/ΓA2008/002060 2015200603 06 Feb 2015 [0031] 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.

[0032] 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.

[0033] The present invention also provides compositions that bind a platelet-binding agent (e.g., VWF) directly or indirectly through a spacer to the solid phase, so long as the ability of the piatelet-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-covalentiy. Indirect binding can occur through spacers, including but not limited to peptide spacer arms, antibody spacers, antibody fragment spacers, fusion protein spacers or carbohydrate spacers. These spacers normally act only as bridges between the particle and the VWF; 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 VWF 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 giutaraidehyde and carbodiimtde.

[0034] In an embodiment of the invention, the platelet binding agent may comprise collagen.

[0035] in a further 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, ovine, or porcine origin. In a further most preferred embodiment of the invention, the collagen is of recombinant origin.

[0036] In a preferred embodiment of the invention, the positioning within the vascular system of mammals of compositions without an active targeting agent would be 9- WO 2009/067791 PCT/CA2008/002060 2015200603 06 Feb 2015 selected by blood flow directed positioning following delivery by means of a superselective microcatheter.

[0037] 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 [0038] in a preferred embodiment of the invention, the targeting agent would include an antibody or antibody-tike 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.

[0039] Compositions according to the Invention may aiso 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.

[0040] Methods according to the invention may also include administering a solid-phase agent capable of binding platelets at a pfe-determined site; may also include inducing activation of the captured platelets; administering a bifunctionai binding agent having an antigenic determinant and a platelet binding site; controlling thrombus generation by altering the temperature of one or more com positions of the invention, or by altering the temperature at the pre-selected site.

[0041] 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.

[0042] The present invention also includes a kit which may contain but is not limited to any or all of the following components including a soiid-phase agent for targeting platelets to an endothelial membrane component: a binding agent for binding platelets; -10- WO 2009/067791 PCT/CA2(H)8/00206() 2015200603 06 Feb 2015 a ligand for binding an endothelial membrane component; a ligand conjugate; 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 bifunctional binding agent, and/or a binding agent-ligand conjugate, and/or a platelet-binding agent -anti-ligand conjugate, £0043] 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 systemicaily, locally, orally, or topically.

[0044] In accordance with some embodiments of the invention, binding agents are used to capture platelets at a predetermined site.

Definitions; [0045] As used herein, a solid-phase agent refers to any solid materiai suitable for binding , containing, or retaining a platelet-binding agent. The platelet-binding agent may be attached to the solid-phase agent such 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, 0,0., a bead or the like, al! of which are well known to those skilled in the art.

[0046] As used herein, a particle refers to a d iscrete portion or part of a solid-phase materiai 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. 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 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 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, poiylactide, polyglycolide, lactide- -11- PCT/CA2008/002060 WO 2009/067791 2015200603 06 Feb 2015 glycolide copolymers, polycaprolactone, ladide-caproiactone copolymers, polyhydroxybutyrate, polyalkylcyanoacrylates, poiyanhydrides, polyorthoesters, albumin, collagen, gelatin, polysaccharides, dextrans, starches, methyl methacrylate, methacrylsc add, hydroxylalkyl acrylates, hydroxylalkyl methacrylates, methylene glycol dimethacrylate, acrylamide, bisacrylamide, cellulose-based polymers, ethylene glycol polymers and copolymers, oxyethyiene and oxypropylene polymers, polyvinyi 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.

[0047] In preferred embodiments, the polymer particle can include a non-biological polymer. Sn some embodiments, the polymer particles are selected from polylactide, polygfycolide, and po!y(!actic-co-glycolic acid) (PLGA).

[0048] in an embodiment of the invention, the platelet binding agent may comprise collagen. £0049] in a further 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.

[0050] Exemplary materials for forming the stent or coil include, but are not limited to: polyvinyl alcohol (PVA), polystyrene, polycarbonate, polylactide, potyglycolide, lactide-giycolide copolymers, polycaprolactone, ladide-caproiactone copolymers, polyhydroxybutyrate, polyalkylcyanoacrylates, poiyanhydrides, 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, oxyethyiene and oxypropylene polymers, polyvinyl acetate, polyvinylpyrrolidone and polyvinylpyridine; magnetic materials, fluorescent materials; -12- PCT/CA2008/002060 WO 2009/067791 2015200603 06 Feb 2015 gold, platinum, palladium, rhenium, riiodium, ruthenium, stainless steel, tungsten, titanium, nickel and alloys thererof; used singly or in combination.

[0051] 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 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. {00521 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 iigand/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 linking the binding 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. 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; and USP 6,960,532; 6,887,474; and USSN 11/205,047 (filed August 17,2005), each incorporated herein by reference. 10053] 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/antMigand pair is biotin and avidin, As used herein, avidin refers to avid in, sireptavidln, neutravidih, derivatives and -13- WO 2009/067791 PO'/CA2008/002fl6« 2015200603 06 Feb 2015 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, heterophyliic peptides, 'leucine zippers", zinc finger proteins/ds DMA fragment, enzyme/enzyme Inhibitor, hapten/antibody, ligand/ligand receptor, and growth factor/growth factor receptor.

[0054] As used herein, a selected site, a pre-determined site, targeting, and pretargeting all refer to a site where the accumulation of platelets about a solid-phase agent will provide a therapeutically beneficial result. Typically, this involves target site localization 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), AV-matformations, vessel aneurysms and endoteaks, [00553 As used herein, delivery of the solid agent comprising a platelet-binding agent can occur using a catheter, a microcatheter or by needle and syringe. Delivery by catheter or 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 the target site using the arterial or venous circuits. Delivery of the solid agent using the arterial circuit is advantageous since the capillary beds downstream of the 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, [0056] 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 --14-- WO 2()1)9/067791 PCT/CA2008/002060 2015200603 06 Feb 2015 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.

[00573 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. 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, [0058] 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 embotizing 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.

[0059] 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 pm and 70 pm in size, with a maximum size of 150 pm that are radiOiabelled 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 causes immediate platelet binding to the particles and occlusion of the vasculature in close proximity to the site of injection.

[0050] The present invention improves upon existing methods of producing vascular occlusion by securing platelets to the surface of a solid-phase materia! 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. -15-- PCT/CA2008/002060 WO 2009/067791 2015200603 06 Feb 2015

Therefore the present invention enables delivery of a minimum number of small particles into the bloodstream, whereupon the particles rapidly grow in size from 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, [0061] 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 threendimensional 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.

[0062] For example, the introduction of a pSatelet-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 (S) a particle of increased diameter and (ii) a particle coated with 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-iayered 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', [0063] As a further example, the introduction of an amorphous platelet binding particle {e,g„ MAA) containing or having a surface-bound piatelet-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 the particle, c) -16-- WO 2009/067791 PCT/CA2008/002060 2015200603 06 Feb 2015 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 piateiets 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 ceils, 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-maiformations, reducing or preventing blood loss from endoleaks and repairing vessel aneurysms, [0065] As used herein, “administering" refers to any action that results in delivering a composition containing a solid-phase agent to a pre-detentiined 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 be exposed directly to a composition of the present Invention, e.g., by washing or irrigating the surgical site, vasculature, and/or the cells.

[0066] 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, sFy, scFv, Fc, and minimal recognition unit; single chains representing the reactive 17- PCT/CA2008/002060 WO 2009/067791 2015200603 06 Feb 2015 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.

[00871 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 ashybridoma 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 totalizing 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 Iigand/receptor complex on hyperplastic tissue or cells (e.g., tumor) orthe vasculature associated with hyperplastic tissue or cells. The most preferred Ending 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 vasculature, in a preferred embodiment of the invention, the binding agent (i.e., antibody or antibodylike molecules) would bind to the VEGF/VEGF 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 Iigand/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 factor receptor. -18” WO 2009/067791 PCT/CA2008/002060 2015200603 06 Feb 2015 [0068] Oligonucleotides, e.g., anti-sense oligonucleotides that are complementary to portions of target ceil nucleic acids (DNA or RNA), are also useful as targeting moieties in the practice of the present invention. Oligonucleotides binding to ceil surfaces are also useful [0069] Functional equivalents of the aforementioned moiecuies are also useful as targeting moieties of the present invention. One targeting moiety functional equivalent is a “mimetic’1 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 “minima!" 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 iigand/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 father than on the tumor-associated endothelial ceils, 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 tittle 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 employ an 19- PCT/C A2908/002060 WO 201)9/(167791 2015200603 06 Feb 2015 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 ceil 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 cefi membrane), one or more imaging reagents, one or more effectors; and/or physioiogicaliy-acceptabie 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 pharmaceuticaliy 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 indude a binding or targeting agent that binds to iigand/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, coion, 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-seiectin, siaiyl-lewis X, endotheiin, endothelin receptor, endothelin/endothelin receptor complex, alpha-fetoprotein, platelet-endothelial cell adhesion molecule (PECAM), CD31, CD34, CD36, glycoprotein lb (GPIb), endogiin, thrombomodulin, endothelial leukocyte adhesion molecule (ELAM), intercellular adhesion molecule 1 (IGAM-1), MHC-i, and MHC-fl. The subject may be a human or animal subject. -20- 2015200603 06 Feb 2015 WO 2009/067791 PCT/CA2008/(M)2«60 [0O75J 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, fibranectin, vitronectin, collagen, thrombospondin, laminin, heparin, heparan sulfate, chondroitin sulfate, phospholipase A2 (PLA2), matrix metailoproteinases (MMPs), thrombin, glass, stalyl-lewis X, fsbulin-1, platelet-endothelial cel! adhesion molecule (FECAM), intercellular adhesion molecule 1 (ICAM-1), intercellular adhesion molecule 2 (iCAy-2), CD11b/CDl8 (MAC-1), CD1la/CD18 (LFA-1), p-seiectin glycoprotein ligand i (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} 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-LiBS antibodies, anti-CD9 antibodies, epinephrine, thrombin receptor activating peptide (TRAP), proteinase-activated receptor (also known as protease activated receptor, PAR) agonists, cathepsin G, eiastase, arachidonate, platelet activating factor (PAF), thromboxane A2 (TxA2), TxA2 rnimeites, phospholipase A2 (PLA2), activators of protein kinase C (PKC), adenosine diphosphate (ADP), inducers of cycto-oxygenase.-1 (COX-1), inducers of cyclo-oxygenase 2 (COX-2), collagen, von Willebrand factor (VWF), matrix metailoproteinases (MMPs), heparin, heparan sulfate, chondroitin sulfate, ionophores, complement cascade components (e.g., C5b-9) platelet microparticles, platelet membrane fractions.

[00783 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. PCT/CA2008/Q03060 WO 2009/067791 2015200603 06 Feb 2015 [0079] Exemplary platelet-mediated occlusion retarders include but are not limited to aspirin, ibuprofen, acetaminophen, ketoprofen, ticlopidine, clopidogrei, indomethacln, dipyridamole, omega-3 fatty acids, prostacyclin, nitric oxide, inducers of nitric oxide, inducers of nitric oxide synthase, matrix metalloproteinase inhibitors (MMPIs, TiMPs), anti-GPlib/llla agents, anti-avp3 agents, anti-a2pl agents, anti-CD36 agents, anti-GPVi agents, aurlntricarboxyiic acid, thrombin receptor antagonists, thromboxane receptor antagonists, streptokinase, urokinase, tissue plasminogen activator (tPA).

[0080] 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 cooied as noted above, [0081] 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 accumulation and binding of the second member of the binding pair, and/or fbr clearance of unbound substances.

[0082] In accordance with the invention, two, three or more step targeting or localization steps may be used. Many of these protocols are weli 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.

[0083] 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 --22-- WO 2009/067791 FCT/€A2«08/0ft206fl 2015200603 06 Feb 2015 by any immunoiogicaliy suitable route. For example, the binding agent may be introduced into the patient by an intravenous, intra-arterial, subcutaneous, intraperitoneal, intrathecal, intravesical, intradennai, intramuscular, or intraiymphatic route. The composition may be in solid, solution, tablet, aerosol, or multi-phase formulation forms. Liposomes, long-circulating liposomes, immunoiiposomes, 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.

[0084] Administration may be 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. , iife-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).

[0085} 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 Geifoam or polyvinyl alcohol (PVA) particles or the like.

[0086] 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 i—23— WO 2009/067791 PCT/CA2008/00206I) 2015200603 06 Feb 2015 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.

[0087] In accordance with a preferred embodiment of the invention, therefore, unactivated platelets accumulate or are induced to accumulate at a pre-deterrnined site through binding to the solid-phase agent, and then the properly localized platelets are selectively activated.

[0088] 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.

[0089] 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, biood 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.

[0090] 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 dampen a method 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 contro lled administration of a composition according to the invention under norma! circumstances (i.e., normal hemostasis).

[0091] 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 VLeiden deficiency, antiphospholipid syndrome (APS), Protein C and/or Protein S and/or Antithrombin HI deficiency, deep -24- WO 2009/067791 Ρ€Τ/€Α2008/0020όϋ 2015200603 06 Feb 2015 vein thrombosis (DVT), pseudo-von Willebrands disease, Type ilb von Willebrands 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 defidendes, hemophilia, thrombocytopenia, and anticoaguiatlon therapy, among others. Controlling thrombus generation includes at 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 predetermined site.

[0092] 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-GPilb/llla agents, anti-avp3 agents, anti-a201 agents, anti-CD36 agents, aurintricarboxylic acid, thrombin receptor antagonists, thromboxane receptor antagonists, streptokinase, urokinase, tissue plasminogen activator (tPA).

[9093] An exemplary process in which it may be desirable to enhance or augment platelet occlusion process includes thrombocytopenic (low piatelet 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 skilied 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, -25- WO 2009/067791 PCT/CA2008/(8)2060 2015200603 06 Feb 2015 cooled platelets, frozen platelets, particles containing or expressing platelet membrane components, platelet substitutes and whole blood.

[0084] 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 degranuiation and/or platelet/plateiet binding and/or platelet accretion about an existing thrombus. These agents include but are not limited to: ristocetin, thrombin, heparin-induced thrombocytopenia (HIT) antibodies or portions thereof, antiphosphoiipid antibodies (APA) or portions thereof, whole antibody molecules via an Fc-mediated mechanism, ants-ligand-induced binding site (anti-LIBS) antibodies or portions thereof, anti~CD9 antibodies or portions thereof, epinephrine, thrombin receptor activating peptide (TRAP), PAR agonists, cathepsih G, elastase, arachidonate, thromboxane A2 (TxA2) mimetics, TxA2, phospholipase A2 (PLA2), activators of protein kinase C (PKG), adenosine diphosphate (ADP), collagen, von Wiliebrand factor (VWF), matrix metalloproteinases {MMPs), heparin, heparan sulfate, chondroitin sulfate, ionophones, platelet microparticles, platelet membrane fractions.

[0085] 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 enhancing occlusion of the target vasculature.

Example 1— Acute Effects of Particie-Immobilized VWF in a Porcine Model [0100] The study was designed to evaluate the effectiveness of particles coated with human VWF in inducing thrombus formation in the vasculature of the pig kidney. In this procedure, the renal artery was catheterized using a 20-22 gauge angiocatheter. The -26-- WO 2009/067791 PCT/CA2008/002060 2015200603 06 Feb 2015 renai vessels were exposed by surgery and a Doppler flow probe attached to the renal vessels to monitor blood flowing into and out of the target organ. Mo noticeable difference was seen between flow readings taken from the renal artery and vein; therefore, all readings were further taken from the renal vein to be indicative of blood flow through the target kidney. Human VWF was covalently bound to human MAA by overnight incubation with giutaraldehyde. Previous titration studies varying the amount of VWF bound to MAA on a per weight protein basis had established that ratios of 1:5 through 1:80 (MAArVWF) provided sufficient immobilized VWF to induce platelet binding and activation. On this basis, a ratio of 1:20 (MAA:VWF) was chosen for in vivo studies. Particle analysts by phase contrast microscopy demonstrated that the particle size in the final preparation ranged from 30 pm through to 250 pm (as estimated by light microscopy using a micrometer/hemacytometer).

[0101} Injection of MAA/VWF (500 p! or 1 mi) followed by human PRP (500 pi containing approximately 200 - 400 x 106 platelets) into the renal artery of the pig caused a dramatic decrease in blood flow. Human platelets were injected after the test agent to approximate conditions within a human as closely as possible, In addition, binding of porcine platelets to human VWF was noted to be very weak. Within 10 minutes of injection of the test agent, the blood flow had dropped to less than half of baseline levels. Within 15 minutes of injection of the test agent, the blood flow had dropped to less than 20% of the baseline values. Blood flow to the organ did not increase during the 0Q-minute monitoring period, [0102} Conversely, control agent (MAA alone; identical protein concentration) injected into the renai artery of the contra lateral kidney showed a transient decrease in blood flow. The blood flow decreased by 50% initially, but rapidly returned to near baseline levels, i.e,, >80% of original blood flow within 15 minutes and > 90% of original blood flow within 30 minutes.

[0103} Histological examination of the target vasculature revealed a large thrombus in the immediate branches of the renai artery in the test organ with minor thrombosis of the renal vasculature in the control kidney. No thrombi were noted in the liver, lungs., spleen, brain, heart and eyes of the control or test animals. -27- PCT/CA2008/002060 WO 2009/067791 2015200603 06 Feb 2015

Example 2 - Chronic Effects of Particle-Immobilized VWF in a Porcine Model [01043 In a manner similar to the procedure outlined in Example 1, the renal arteries of two test and two control pigs were catheterized using 20-22 gauge angiocatheters, A Doppler flow probe was placed surgically on the renal vein of each animal and the lead wires exposed on the outer skin of the animal after closing the initial incision. After establishing a baseline for blood flowing through the affected kidney, 1 ml of MAA:VWF particles (11 mg total protein) followed by 1 mL human platelet rich plasma (approximately 460 x 106 platelets (platelet count 458 x 109/ liter) were injected into the renal arteries of the test animals, and an identical amount of MAA was injected into the control animals. The animals were transferred to metabolic crates and monitored at varying time intervals over a 7-day period. The following table presents bipod flow results from these studies. PORCINE DOPPLER BLOOD FLOW (milliliters per minute)

Day Test 1 | Control 1 | Test 2 Control 2 0 28 1 70 Γ 4..................................f 35 35 1 17 | 15 11 103 2 32 ) 24 | ! 15 71 3 3 ( 100 i 16 85 4 3 ! 101 ! 17 78 5 3 i 140 S 3 65 6 1 [ 140 I ..j ..... | 0 55 7 3 I 148 I 2 30 [0105] The major organs and tissues of the control and test animals were examined histologically for evidence of thrombosis and other treatment-associated damage. No , thrombosis was observed except in the renal vasculature of the target kidney.

Example 3 - Fluoroscopy Studies of MAA/VWF Thrombosis of Porcine Renal Vasculature -28- WG 2009/067791 P€T7CA20O8/O«i'O60 2015200603 06 Feb 2015 [0106] MAA/VWF was delivered to porcine renal vasculature using a femoral artery approach. A 5-Fr sheath catheter was inserted Into the femoral artery of 18 to 20 kg piglets after general anesthesia (halothane) and guided by fluoroscopy to the left renal artery. The catheter was positioned such that upon delivery of contrast dye, only the lower pole of the kidney’s vasculature was visualized. At time zero 1 ml of MAA/VWF particles (size range 30 - 250 micron) were delivered slowly (over 10 seconds) to the renal vasculature. Ten (10) seconds later 1 ml human PRP (420 x 109 per liter) was delivered and the catheter flushed with 1 mi saline. The catheter was kept in place and after a twenty (20) minute waiting period contrast agent was again delivered to the target vasculature. The contrast agent was observed to pool at the end of the catheter then move rapidly into the vasculature of the upper pole of the kidney. The vasculature of the lower pole of the kidney siowly accumulated contrast agent that did not dissipate (greater than 20 minutes), while the upper pole vasculature rapidly lost the contrast agent (less than 5 seconds). With the animal under anesthetic and the catheter still in place, the affected kidney was surgically exposed, the renal vasculature damped just proximal to the catheter and the affected kidney removed. The renal artery Was opened through a longitudinal incision and dissected toward the kidney. A large thrombus was noted distal to the tip of the catheter extending deep within the vasculature of the lower pole of the kidney. No dotting was noted in the vasculature of the upper pole of the kidney. In addition, the lower pole of the affected kidney was noticeably blanched indicating a lack of blood flow, while the upper pole of the kidney exhibited normal red coloration.

[0107] In a separate experiment following the above outlined procedure the catheter was directed to the vasculature of the lower pole of the target kidney and used to deliver 1 ml of MAA/VWF particles followed by 1 ml of human PRP. After 20 minutes blood flow to the tower pole of the kidney was notably impeded as determined by fluoroscopy. As before, the contrast dye moved rapidly into the upper pole vasculature after pooling at the tip of the catheter. The catheter was then repositioned and MAA/VWF followed by human PRP was delivered to the upper pole vasculature. After a 20 minute waiting period contrast dye was again injected. Fluoroscopy revealed that blood flow to the upper pole vasculature was blocked. The contrast dye did not enter the lower pole --29- WO 2009/067791 PCT/CA2008/002060 2015200603 06 Feb 2015 vasculature. The target kidney with associated renal vessels was then removed surgically prior to sacrificing the animal. Immediate dissection of the kidney revealed extensive thrombosis of the blood vessels in both the upper and lower poles of the

Example A- Study of the Safety and Efficacy of an Artificial Embolization Device in Sheep [0108] Introduction: This preciinical animal study evaluated safety, effectiveness, and biocompatibility of biodegradable poly lactic-co-giycoiic acid (RIGA) microspheres coated with bovine fibrillar collagen type 1 (100-300 pm particle size), as compared with Embosphere® Microspheres (Biosphere Medical Inc.}. This study also assessed changes to the test and control microspheres in animals over time. 101093 Materials and Method: Unilateral uterine artery occlusion was carried out in 32 adult ewes. Animals were randomly assigned the RIGA microspheres (100-300 pm particle size) or Embospheres (300-500 pm) as the embolic agent. Both groups were treated in a similar manner with standard angiographic equipment including coaxial microcatheters and the vessels emboiized to stasis. Embolization procedures were carried out under genera! anesthesia with femoral artery cannulation. Procedural data and follow up clinical and hematological data were recorded. Groups of 8 animals (4 Embospheres, 4 PLGA) were scheduled to be sacrificed at 1 , :3, 6 or 12 months. Target and non-target tissues were examined postmortem.

[01103 Results: Uterine arteries (UA) were clearly Identified in 31/32 animals. In one case the occluded vessel was identified post mortem as the vaginal artery (VA). Embolization to effective stasis was achieved in ai! 32 cases. Post procedure course was uneventful. There were no procedural related complications. One ewe developed a spinal abscess secondary to jugular venous line placement and was euthanized 7 weeks post procedure.

[01113 Results: Particles were dearly identified in all occluded arteries one month post procedure. At 3 months post procedure there was evidence of particle remodeling and potential recannaiization of the distal vasculature in the PLGA animals. Particles were identified in the VA in 6/8 Embosphere animals and in 0/9 of the PLGA animals. -30- WO 2009/067791 PCT/CA2008/002060 2015200603 06 Feb 2015

No other evidence of non target embolization was seen in any animal. AH uteri were viable without evidence of chronic ischemia.

[0112] Autopsy and histology data are available for the 24 animals sacrificed by 6 months. Treated uterine arteries were effectively occluded at all time points. Embosphere® Microspheres were identified in ihe uterine (11/12)., contralateral uterine ¢4/12), vaginal (7/12) or ovarian (1/12) arteries. OCL 500 microspheres were only identified in the uterine (12/12) and contralateral uterine (1/12) arteries. No other evidence of non target embolization was seen. Embosphere® Microspheres were structurally unchanged through 6 months, whereas the OCL 500 microspheres degraded with time.

[0113] Embosphere® Microspheres, a permanent implant, appeared identical at one, three and six months after implantation, it should be noted that Embosphere® Microspheres are made of polyacrylamide, a biocompatibie material that does not biodegrade. Therefore, the continued presence of Embosphere® Microspheres is anticipated and supported by the literature. In contrast, the Occiusin® 500 AED microspheres showed significant surface changes one month after implantation, No intact Occiusin® 500 AED microspheres Were observed three and six months after implantation, although there was residual material present in many, but not all, tissue sections at three months after implantation.

[0114] Three months after implantation into the sheep uterine artery, there are no identifiable intact Occiusin® 503 AED microspheres (100 - 300 pm). The residua! material is highly deformed and compressed in the center of the artery as it continues to degrade. In other sections, there is even (ess visible material. In contrast, the Embosphere® Microspheres (300 — 500 pm) retain their round shapes and the artery remains highly distended. Both arteries remain fully occluded.

[0115] Although the Occiusin® 500 AED microspheres were highly degraded after six months in the body, the inner luminal wall of the treated artery appears to have been replaced by fibrous connective tissue and the treated arteries remained completely occluded. This morphological change suggests that the treated arteries may not recannalize. Residual Occiusin® 500 AED mierosphere material may be present at six months after implantation. No residual debris from the impfanted Occiusin® 503 AED -~31— WO 2009/067791 PCT/CA2008/002060 2015200603 06 Feb 2015 mierospheres was visible. In contrast, the Embosphere® Microspheres (300 - 500 pm) retained their round shapes and the artery remained highly distended. Connective tissue is present between the Embosphere® Microspheres. Both arteries remain fully occluded. All uteri were viable without evidence of chronic ischemia. {0116] Conclusion: PLGA microspheres coated with collagen are an effective emboiic agent when used to occlude uterine arteries in sheep. No device related issues or adverse events were recorded. The PLGA microspheres were at least as effective as Embospheres in this context with potential advantages.

Example 5 - Study of the Safety and Efficacy of an Artificial Embolization Device in Sheep, 12 Month Data [01.17] This example provides the 12-month results of the study conducted in Example 4.

[0118] This study demonstrated that Occlusin™ 503 AED microspheres are as effective as Embosphere® Mierospheres in embolizing a treated artery for at least 6 months post implantation. Although the Embosphere® Microspheres were unchanged throughout the study, Occlusin™ 503 AED mierospheres biodegrade and allow the artery to re-open by one year after treatment, allowing normal blood flow through the treated vasculature. Occlusin™ 503 AED microspheres showed significant surface changes one month after implantation. Only residual material of Occlusin™ microspheres were observed three months after implantation in many, but not all, tissue sections. Although the Occlusin™ 503 AED mierospheres were fully degraded after six months in the body, the inner luminal wali of the treated artery appears to have been replaced by fibrous connective tissue and the treated arteries remained completely occluded. By 12 months post embolization, uterine arteries treated with Occlusin™ mierospheres are fully recanaiized in three of the four treated animals. The fourth animal in the group was in the process of recannalization. In contrast, the Embosphere® Mierospheres, a permanent implant, remained unchanged throughout the study. -32· PGT/C A2008/062060 WO 2009/06779! 2015200603 06 Feb 2015 £0119} Occiusin™ 503 AED microspheres were localized to the uterine vasculature after implantation, in contrast, Embosphere® Microspheres were detected in the uterine, ovarian and urinary bladder arteries after implantation. Embosphere® Microspheres were also detected in the untreated contralateral uterine vasculature in a much higher percentage of treated animals.

Conclusions: [0120] Occiusin™ 503 AED is readily and consistently suspended to neutral buoyancy for administration. The resuspended microspheres are easily delivered through standard microcatheters.

[0121} Occiusin™ 503 AED can be administered stte-specifically and remains localized at the site following injection.

[0122] Occiusin™ 503 AED is as effective as EmbospheFe™ Microspheres at occluding a treated artery. Occiusin™ 503 AED-treated arteries remain fully occluded more than six months post treatment/ [0123] Occiusin™ 503 AED is fully biodegraded between three and six months post implantation. This is in sharp contrast with Embosphere® Microspheres, which remained unchanged throughout the duration of the study.

[0124] Fibrous connective tissue replaces the Occiusin™ 503 microspheres as they degrade and continues to completely block blood flow for more than six months post implantation.

[0125J Vasculature treated with Occiusin™ 503 AED is fully recanaiized (or in the process of recanalizing) one year post embolization, allowing normal blood flow to the organ.

[0126] Occiusin™ 503 AED is safe and biocompatible in all pre-clinica! studies conducted to date. No systemic or local toxicity was observed following implantation of Occiusin ™ 503 AED. Implantation did not cause any detectable infarction to the uterus .33« WO 2009/067791 PCT/CA2008/002060 2015200603 06 Feb 2015 or surrounding tissues. Fibrous connective tissue formed following implantation with Occlusin™ 503 AED is fully resolved by one year post implantation.

Example 6 - Study of the Safety and Efficacy of an Artificial Embolization Device in Pigs [0127] Introduction: This preclinica! animal study in pigs evaluated safety, effectiveness, and biocompatibifity of biodegradable poly (factic-co-glycolic acid) (PLGA) microspheres coated with bovine fibrillar collagen type l.

[0128] Materials and Methods: Occlusion of file target artery was earned out in 12 adult male pigs. Animals were administered coated PLGA mierospheres (300-500 pm) as the embolic agent. Animals were randomly assigned to have either a hepatic artery (6 pigs) ora renal artery (6 pigs) embolized. All animals were treated in a similar manner with standard angiographic equipment including coaxial catheters and the vessels embolized to effective stasis. Procedural data and follow up clinical and hematological data were recorded. Four animals (2 renal and 2 hepatic) were sacrificed immediately following embolization and 8 animals (4 renal and 4 hepatic) were sacrificed 1 month following embolization. Target and non target tissues were examined postmortem.

[0129] Results: Embolization procedures were carried out under general anesthesia with femoral artery cannuiation. Target arteries were clearly identified in 12/12 animals. Embolization to effective stasis was achieved in all 12 cases. Post procedure course was uneventful. There were no procedural related complications. The coated PLGA (300-500 pm) particles were clearly identified in all occluded arteries, the particles were spherical in animals sacrificed immediately after embolization. At one month post procedure there was evidence of particle remodeling consistent with the particles biodegrading. No evidence of non target embolization was seen in any animal. Occlusion of the renal artery caused significant chronic ischemia in the occluded kidneys, Contralateral kidneys showed signs of hypertrophy to compensate for the loss of function by the occluded kidney. In contrast, all livers were viable without evidence of chronic ischemia. 2015200603 06 Feb 2015 WO 2009/067791 PCT/C A2008/002060 [0130] Conclusion: RIGA microspheres coated with collagen are an effective embolic agent when used to occlude the hepatic and renal arteries in pigs. No device related issues or adverse were recorded,

Example 7 - Effectiveness of implantation and location of device

Animal Number Implanted Device Result of implantation at Sacrifice Microspheres Observed in Vasculature Treated Uterine Artery Vasculature Contralateral Uterine Vasculature Vagina! Artery Ovarian Artery Bladder Artery Y26 OCL 503 Occluded Yes No No No No Y30 OCL 503 Occluded Yes Yes No No No Y187 OCL 503 Occluded Yes No No No No Y790 OCL 503 Occluded Yes No No No No G57 OCL 503 Occluded Yes No No No No G186 OCL 503 Occluded Yes No No No No G194 OCL 503 Occluded Yes No No No No G261 OCL 503 Occluded Yes No No No No R56 OCL 503 Occluded Yes No No No No R179 OCL 503 Occluded — — — — R193 OCL 503 Occluded — — .- — R198 OCL 503 Occluded — — -- — — BS3 OCL 503 Recanalized — — — — --: B182 OCL 503 Recanalized — — — — B183 OCL 503 Recanalizing — — — — B346 OCL 503 Recanalized — — — Number of Sheep : 9/9 9© 1/9 0/9 0/9 0/9 Percent: 100 100% 11% 0% 0% 0% Partly Y28 Embospheres® Occluded Yes ONE’ No No No Y33 Embospheres® Occluded Yes No No No No 'VUG Embospbetes© Occluded Yes No Yes No No ΥΪ842 Smbeephwes® Occluded Few Few Yes No No G188 Embospheres® Occluded Yes Few No No No G196 Embospheres® Occluded Yes No Yes No No G2S3 Embospheres® Occluded Yes Few Yes No No G41Q Embaspheres© Occluded Yes No Few No No R27 Embospheres® Occluded Yes No Yes No No R32 Embospheres® Occluded Yes Yes Yes No No PCT/GA2008/002060 WO 2009/067791 2015200603 06 Feb 2015 10131] Although the present invention has been described in terms of particular preferred embodiments, it is not limited to those 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. 36-

Claims (14)

1. Claims:

   1. Use of a biodegradable solid-phase agent comprising a polylactic-co-glycolic acid and bovine fibrillar collagen type 1 in producing formation of intravascular connective tissue, wherein the intravascular connective tissue replaces the solid-phase agent as it degrades.
2. 2. Use according to claim 1, wherein the connective tissue replaces the biodegradable solid-phase agent as it degrades.
3. 3. Use according to claim 2, wherein the connective tissue degrades after the solid-phase agent degrades allowing recanalization.
4. 4. Use according to any of claims 1 to 3, wherein the solid-phase agent is a particle, coil or stent.
5. 5. Use according to claim 4, wherein the particle is a microsphere.
6. 6. Use according to claim 5, wherein the microsphere is from about 1 micron to about 7000 microns.
7. 7. A biodegradable solid-phase agent comprising polylactic-co-glycolic acid and bovine fibrillar collagen type 1 when used in forming an occlusion in a target vessel, wherein thrombus formation is induced and the solid-phase agent is degraded and replaced by connective tissue.
8. 8. The biodegradable solid-phase agent of claim 7, wherein the occlusion is temporary.
9. 9. The biodegradable solid-phase agent of claim 7, wherein the occlusion is permanent.
10. 10. The biodegradable solid-phase agent of any one of claims 7 to 9, wherein the solid-phase agent is a particle, coil or stent.
11. 11. The biodegradable solid-phase agent of claim 10, wherein the particle is a microsphere.
12. 12. The biodegradable solid-phase agent of claim 11, wherein the microsphere is from about 1 micron to about 7000 microns.
13. 13. The biodegradable solid-phase agent of any one of claims 7 to 12, wherein the connective tissue is intravascular connective tissue.
14. 14. A kit when used in producing connective tissue, the kit comprising: a) polylactic-co-glycolic acid; b) bovine fibrillar collagen type 1; and c) instructions for combining the polylactic-coglycolic acid and bovine fibrillar collagen type 1 and replacing it, as it degrades, with connective tissue.