OF BIOLOGICAL TISSUE

FIELD OF THE INVENTION

[0001] The present invention relates to methods for treating biological tissue. More particularly, the present invention relates to methods and systems for treating damaged and diseased biological tissue; particularly, cardiovascular tissue.

BACKGROUND OF THE INVENTION

[0002] Myocardial infarction is a common presentation of ischemic heart

disease/coronary artery disease. The World Health Organization estimated in 2004 that 12.2% of worldwide deaths occurred as a result of ischemic heart disease. Ischemic heart disease was also deemed the leading cause of death in middle to high income countries and second only to respiratory infections in lower income countries. The Global Burden of

Disease: World Health Organization 2004 Update, Geneva (2008). Worldwide more than 3 million people present with a ST elevation myocardial infarction (STEMI) and 4 million people present with a non-ST elevation myocardial infarction (NSTEMI) a year. White, et al., Acute Myocardial Infarction, Lancet 372 (9638), pp. 570-84 (August 2008).

[0003] Rates of death from ischemic heart disease have slowed or declined in most high income countries, although cardiovascular disease still accounted for 1 in 3 of all deaths in the USA in 2008. Roger, et al., Executive summary: Heart Disease and Stroke Statistics— 2012 update: A report from the American Heart Association, Circulation 125 (1), pp. 188-97 (January 2012).

[0004] In contrast, ischemic heart disease is becoming a more common cause of death in the developing world. For example in India, ischemic heart disease had become the leading cause of death by 2004; accounting for 1.46 million deaths (14% of total deaths). Deaths in India due to ischemic heart disease were also expected to double during 1985-2015. Gupta, et al., Epidemiology and Causation of Coronary Heart Disease and Stroke in India, Heart 94 (1), pp. 16-26 (January 2008).

[0005] Globally, it is predicted that disability adjusted life years (DALYs) lost to ischemic heart disease will account for 5.5% of total DALYs in 2030, making it the second most important cause of disability (after unipolar depressive disorder), as well as the leading cause of death by this date.

[0006] A myocardial infarction (a common presentation of ischemic heart disease) often occurs when a coronary artery becomes occluded and can no longer supply blood to the myocardial tissue, thereby resulting in myocardial cell death. When a myocardial infarction occurs, the myocardial tissue that is no longer receiving adequate blood flow ultimately dies (without effective intervention) and is eventually replaced by scar tissue.

[0007] Within seconds of a myocardial infarction, the under-perfused myocardial cells no longer contract, leading to abnormal wall motion, high wall stresses within and surrounding the infarct, and depressed ventricular function. The high stresses at the junction between the infarcted tissue and the normal tissue lead to expansion of the infarcted area and remodeling, i.e. a cascading sequence of myocellular events, over time.

[0008] Various methods for treating a myocardial infarction are often employed. Such methods include stabilizing the hemodynamics associated with a myocardial infarction via systemic delivery of various pharmacological agents and restoring the patency of occluded vessels via thrombolytic therapy or angioplasty and stents.

[0009] Several additional methods for treating a myocardial infarction are directed to reestablishing blood flow to the ischemic area through stimulation of angiogenesis. Reestablishing blood flow at the ischemic area can, and in many instances will, reduce symptoms associated with a myocardial infarction and/or improve cardiac function.

[00010] Some methods for re-establishing blood flow and rehabilitating the heart involve invasive surgery, such as bypass surgery or angioplasty. Other methods employ lasers to bore holes through the infarctions and ischemic area(s) to promote blood flow. As one can readily appreciate, there are numerous incumbent risks associated with the noted methods.

[00011] A further method for treating a myocardial infarction is the direct or selective delivery of bioactive or pharmacological agents to the infarction and/or ischemic area (i.e. effected or damaged cardiovascular tissue). Direct delivery of a bioactive or pharmacological agent to the effected cardiovascular tissue is often preferred over the systemic delivery for several reasons. A primary reason is that a substantially greater concentration of such agents that can be delivered directly into the effected cardiovascular tissue, compared with the dilute concentrations possible through systemic delivery. Another reason is the risk of systemic toxicity which can, and in many instances will, occur with doses of pharmacological agents that are typically required to achieve desired drug concentrations in the effected

cardiovascular tissue.

[00012] One common method of delivering bioactive or pharmacological agents to effected cardiovascular tissue, e.g. damaged myocardial tissue, comprises advancing a catheter through the vasculature and into the heart to inject the agents directly into the effected cardiovascular tissue from within the heart.

[00013] Another method of delivering bioactive or pharmacological agents to effected cardiovascular tissue comprises epicardial, direct injection into the tissue during an open chest procedure. The bioactive agents that can be, and have been, administered to the effected cardiovascular tissue include various pharmacological agents, such as antithrombotic agents, e.g., heparin, hirudin, and ticlopidine, and cells that are capable of maturing into actively contracting cardiac muscle cells or regenerating cardiovascular tissue. Examples of such cells include myocytes, myoblasts, mesenchymal stem cells, and pluripotent cells.

[00014] However, to date, cell therapy of effected cardiovascular tissue has not reached its full potential, due, in part, to the failure of implanted cells to survive and regenerate the damaged tissue in ischemic area(s) or regions with inadequate vascularization.

[00015] It would thus be desirable to provide bioactive and pharmacological agents (and compositions) that promote tissue survival and induce neovascularization and regeneration of effected or damaged cardiovascular tissue, and improved methods for delivering same to effected cardiovascular tissue.

[00016] It is therefore an object of the present invention to provide bioactive and pharmacological agents (and compositions) that promote tissue survival, and induce neovascularization and regeneration of damaged cardiovascular tissue.

[00017] It is another object of the present invention to provide extracellular matrix (ECM) compositions, which, when delivered to damaged biological tissue; particularly,

cardiovascular tissue, induce neovascularization, host tissue proliferation, bioremodeling, and regeneration of cardiovascular tissue and associated structures with site-specific structural and functional properties. [00018] It is yet another object of the present invention to provide improved methods and systems for administering an ECM composition directly to damaged or diseased biological tissue; particularly, cardiovascular tissue.

SUMMARY OF THE INVENTION

[00019] The present invention is directed to methods and systems for treating damaged and diseased biological tissue; particularly, cardiovascular tissue. In some embodiments, the method comprises direct delivery or administration of at least one pharmacological composition of the invention to the damaged or diseased biological tissue.

[00020] In a preferred embodiment, the pharmacological compositions comprise extracellular matrix (ECM) compositions that include at least one ECM material.

[00021 ] According to the invention, the ECM material can be derived from various mammalian tissue sources, including the small intestine, large intestine, stomach, lung, liver, kidney, pancreas, placenta, heart, bladder, prostate, tissue surrounding growing enamel, tissue surrounding growing bone, and any fetal tissue from any mammalian organ, and methods for preparing same.

[00022] In a preferred embodiment, the ECM comprises cardiac tissue.

[00023] In some embodiments, the ECM compositions further include one or more additional biologically active components to facilitate the treatment of damaged tissue and/or the tissue regenerative process.

[00024] In some embodiments, the ECM compositions thus include at least one pharmacological agent or composition, which can comprise, without limitation, antibiotics or antifungal agents, anti-viral agents, anti-pain agents, anesthetics, analgesics, steroidal anti-inflammatories, non-steroidal anti-inflammatories, anti-neoplastics, anti-spasmodics, modulators of cell-extracellular matrix interactions, proteins, hormones, enzymes and enzyme inhibitors, anticoagulants and/or antithiOmbic agents, DNA, RNA, modified DNA and RNA, NSAIDs, inhibitors of DNA, RNA or protein synthesis, polypeptides,

oligonucleotides, polynucleotides, nucleoproteins, compounds modulating cell migration, compounds modulating proliferation and growth of tissue, and vasodilating agents.

[00025] In some embodiments of the invention, the biologically active component comprises a statin. According to the invention, suitable statins include, without limitation, atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.

[00026] In some embodiments of the invention, the biologically active component comprises a cell.

[00027] In some embodiments of the invention, the biologically active component comprises a protein.

[00028] In some embodiments of the invention, the EC compositions are formulated to facilitate injection of the ECM compositions to damaged or diseased tissue (i.e. injectable ECM compositions).

[00029] In some embodiments of the invention, one or more ECM compositions of the invention are directly administered to damaged cardiovascular tissue via a multi-needle injection system. According to the invention, the ECM compositions can be directly administered to the heart wall and/or the various cardiovascular structures associated therewith, including the epicardium, endocardium and myocardium.

BRIEF DESCRIPTION OF THE DRAWINGS

[00030] Further features and advantages will become apparent from the following and more particular description of the preferred embodiments of the invention, as illustrated in the accompanying drawings, and in which like referenced characters generally refer to the same parts or elements throughout the views, and in which:

[00031] FIGURE 1 is a depiction of a normal heart;

[00032] FIGURE 2 is a of a heart having an ischemic infracted region;

[00033] FIGURE 3 A is an exploded perspective view of one embodiment of a multi-needle injection apparatus that is suitable for direct administration of ECM compositions to biological tissue, e.g. cardiovascular tissue, in accordance with the invention; and

[00034] FIGURE 3B is an assembled perspective view of the multi-needle injection apparatus shown in FIGURE 3 A, in accordance with the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[00035] Before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified apparatus, systems, compositions or methods as such may, of course, vary. Thus, although a number of systems, compositions and methods similar or equivalent to those described herein can be used in the practice of the present invention, the preferred systems, compositions and methods are described herein.

[00036] It is also to be understood that, although the systems, pharmacological

compositions and methods of the invention are illustrated and described in connection with administration (or delivery) of pharmacological compositions (and bioactive and

pharmacological agents) to cardiovascular tissue, the systems, compositions and methods of the invention are not limited to such delivery. According to the invention, the systems and methods of the invention can be employed to administer pharmacological compositions (and bioactive and pharmacological agents) to numerous additional biological tissue, including, without limitation, gastrointestinal and respiratory organ tissue.

[00037] It is also to be understood that, although a preferred method of delivering a pharmacological composition of the invention to biological tissue comprises direct injection into the tissue. The delivery of the pharmacological composition is not limited to direct injection. According to the invention, a pharmacological composition of the invention can be delivered to biological tissue by other conventional means, including topical administration.

[00038] It is further to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.

[00039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one having ordinary skill in the art to which the invention pertains.

[00040] Further, all publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[00041] Finally, as used in this specification and the appended claims, the singular forms

"a, "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "an anti-inflammatory" includes two or more such agents and the like.

Definitions

[00042] The terms "cardiac tissue damage", "cardiac tissue injury" and "cardiovascular tissue damage" are used interchangeably herein, and mean and include any area of abnormal tissue in the cardiovascular system or heart caused by a disease, disorder, injury or damage, including damage to the epicardium, endocardium and/or myocardium. Non-limiting examples of causes of cardiovascular tissue damage include acute or chronic stress (systemic hypertension, pulmonary hypertension, valve dysfunction, etc.), coronary artery disease, ischemia or infarction, inflammatory disease and cardiomyopathies.

[00043] As is well known in the art, cardiovascular tissue damage most often involves damage or injury to the myocardium and, therefore, for the purposes of this disclosure, myocardial damage or injury is equivalent to cardiovascular tissue damage.

[00044] The term "damaged tissue", as used herein, means and includes biological tissue; particularly, cardiovascular tissue damaged or injured by trauma, ischemic tissue, infarcted tissue or tissue damaged by any means which results in interruption of normal blood flow to the tissue.

[00045] The terms "prevent" and "preventing" are used interchangeably herein, and mean and include reducing the frequency or severity of a disease, condition or disorder. The term does not require an absolute preclusion of the disease, condition or disorder. Rather, this tenn includes decreasing the chance for disease occurrence.

[00046] The terms "treat" and "treatment" are used interchangeably herein, and mean and include medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition or disorder. The terms include "active treatment", i.e. treatment directed specifically toward the improvement of a disease, pathological condition or disorder, and "causal treatment", i.e. treatment directed toward removal of the cause of the associated disease, pathological condition or disorder.

[00047] The terms "treat" and "treatment" further include "palliative treatment", i.e. treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition or disorder, "preventative treatment", i.e. treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition or disorder, and "supportive treatment", i.e. treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition or disorder.

[00048] The tenn "chamber remodeling", as used herein, means and includes a series of events (which may include changes in gene expression, molecular, cellular and interstitial changes) that result in changes in size, shape and function of cardiac tissue following stress or injury. As is well known in the art, remodeling can occur after a myocardial infarction, pressure overload (e.g., aortic stenosis, hypertension), volume overload (e.g., valvular regurgitation), inflammatory heart disease (e.g., myocarditis), or in idiopathic cases (e.g., idiopathic dilated cardiomyopathy).

[00049] The term "angiogenesis", as used herein, means a physiologic process involving the growth of new blood vessels from pre-existing blood vessels.

[00050] The term "neovascularization", as used herein, means and includes the formation of functional vascular networks that can be perfused by blood or blood components.

Neovascularization includes angiogenesis, budding angiogenesis, intussuceptive angiogenesis, sprouting angiogenesis, therapeutic angiogenesis and vasculogenesis.

[00051] The terms "extracellular matrix", "extracellular matrix material" and "ECM material" are used interchangeably herein, and mean a collagen-rich substance that is found in between cells in animal tissue and serves as a structural element in tissues. It typically comprises a complex mixture of polysaccharides and proteins secreted by cells. The extracellular matrix can be isolated and treated in a variety of ways. Extracellular matrix material (ECM) can be isolated from small intestine submucosa, stomach submucosa, urinary bladder submucosa, tissue mucosa, dura mater, liver basement membrane, pericardium or other tissues. Following isolation and treatment, it is commonly referred to as extracellular matrix or ECM material.

[00052] The terms "pharmacological agent", "pharmaceutical agent", "agent", "active agent", "drug" and "active agent formulation" are used interchangeably herein, and mean and include an agent, drug, compound, composition of matter or mixture thereof, including its formulation, which provides some therapeutic, often beneficial, effect. This includes any physiologically or pharmacologically active substance that produces a localized or systemic effect or effects in animals, including warm blooded mammals, humans and primates;

avians; domestic household or farm animals, such as cats, dogs, sheep, goats, cattle, horses and pigs; laboratory animals, such as mice, rats and guinea pigs; fish; reptiles; zoo and wild animals; and the like. [00053] The terms "pharmacological agent", "pharmaceutical agent", "agent", "active agent", "drug" and "active agent formulation" thus mean and include, without limitation, antibiotics, anti-viral agents, analgesics, steroidal anti-inflammatories, non-steroidal antiinflammatories, anti -neoplastics, anti-spasmodics, modulators of cell-extracellular matrix interactions, proteins, hormones, enzymes and enzyme inhibitors, anticoagulants and/or antithrombic agents, DNA, RNA, modified DNA and RNA, NSAIDs, inhibitors of DNA, RNA or protein synthesis, polypeptides, oligonucleotides, polynucleotides, nucleoproteins, compounds modulating cell migration, compounds modulating proliferation and growth of tissue, and vasodilating agents.

[00054] The terms "anti-inflammatory" and "anti-inflammatory agent" are also used interchangeably herein, and mean and include a "pharmacological agent" and/or "active agent formulation", which, when a therapeutically effective amount is administered to a subject, prevents or treats bodily tissue inflammation i.e. the protective tissue response to injury or destruction of tissues, which serves to destroy, dilute, or wall off both the injurious agent and the injured tissues. Anti-inflammatory agents thus include, without limitation, alclofenac, alclometasone dipropionate, algestone acetonide, alpha amylase, amcinafal, amcinafide, amfenac sodium, amiprilose hydrochloride, anakinra, anirolac, anitrazafen, apazone, balsalazide disodium, bendazac, benoxaprofen, benzydamine hydrochloride, bromelains, broperamole, budesonide, carprofen, cicloprofen, cintazone, cliprofen, clobetasol propionate, clobetasone butyrate, clopirac, cloticasone propionate, coraiethasone acetate, cortodoxone, decanoate, deflazacort, delatestryl, depo-testosterone, desonide, desoximetasone, dexamethasone dipropionate, diclofenac potassium, diclofenac sodium, diflorasone diacetate, diflumidone sodium, diflunisal, difluprednate, diftalone, dimethyl sulfoxide, drocinonide, endrysone, enlimomab, enolicam sodium, epirizole, etodolac, etofenamate, felbinac, fenamole, fenbufen, fenclofenac, fenclorac, fendosal, fenpipalone, fentiazac, flazalone, fluazacort, flufenamic acid, flumizole, fiunisolide acetate, flunixin, flunixin meglumine, fluocortin butyl, fluorometholone acetate, fluquazone, flurbiprofen, fluretofen, fluticasone propionate, furaprofen, furobufen, halcinonide, halobetasol propionate, halopredone acetate, ibufenac, ibuprofen, ibuprofen aluminum, ibuprofen piconol, ilonidap, indomethacin, indomethacin sodium, indoprofen, indoxole, intrazole, isoflupredone acetate, isoxepac, isoxicam, ketoprofen, lofemizole hydrochloride,

lomoxicam, loteprednol etabonate, meclofenamate sodium, meclofenamic acid, meclorisone dibutyrate, mefenamic acid, mesalamine, meseclazone, mesterolone, methandrostenolone, methenolone, methenolone acetate, methylprednisolone suleptanate, momiflumate, nabumetone, nandrolone, naproxen, naproxen sodium, naproxol, nimazone, olsalazine sodium, orgotein, orpanoxin, oxandrolane, oxaprozin, oxyphenbutazone, oxymetholone, paranyline hydrochloride, pentosan polysulfate sodium, phenbutazone sodium glycerate, pirfenidone, piroxicam, piroxicam cinnamate, piroxicam olamine, pirprofen, prednazate, prifelone, prodolic acid, proquazone, proxazolc, proxazole citrate, rimexolone, romazarit, salcolex, salnacedin, salsalate, sanguinarium chloride, seclazone, sermetacin, stanozolol, sudoxicam, sulindac, suprofen, talmetacin, talniflumate, talosalate, tebufelone, tenidap, tenidap sodium, tenoxicam, tesicam, tesimide, testosterone, testosterone blends,

tetrydamine, tiopinac, tixocortol pivalate, tolmetin, tolmetin sodium, triclonide, triflumidate, zidometacin, and zomepirac sodium.

[00055] The term "chitosan", as used herein, means and includes the family of linear polysaccharides consisting of varying amounts of β (1→4) linked residues of N-acetyl-2 amino-2-deoxy-D-glucose and 2-amino-2-deoxy-Dglucose residues, and all derivatives thereof.

[00056] The terms "active agent formulation", "pharmacological agent formulation" and "agent formulation", are also used interchangeably herein, and mean and include an active agent (and chitosan) optionally in combination with one or more pharmaceutically acceptable carriers and/or additional inert ingredients. According to the invention, the formulations can be either in solution or in suspension in the carrier.

[00057] The term "pharmacological composition", as used herein, means and includes a composition comprising a "pharmacological agent" and/or an "extracellular matrix material" and/or a "pharmacological agent formulation" and/or any additional agent or component identified herein.

[00058] The term "therapeutically effective", as used herein, means that the amount of the "pharmacological composition" and/or "pharmacological agent" and/or "active agent formulation" administered is of sufficient quantity to ameliorate one or more causes, symptoms, or sequelae of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination, of the cause, symptom, or sequelae of a disease or disorder.

[00059] The terms " delivery" and "administration ^" are used interchangeably herein, and mean and include providing a "pharmacological composition" or "pharmacological agent ^" or "active agent formulation" to a treatment site, e.g., damaged tissue, through any method appropriate to deliver the functional agent or formulation or composition to the treatment site. Non-limiting examples of delivery methods include direct injection, percutaneous delivery and topical application at the treatment site.

[00060] The term "percutaneous' ^* , as used herein, means and includes any penetration through the skin of a patient or subject, whether in the form of a small cut, incision, hole, cannula, tubular access sleeve or port or the like.

[00061] The terms "patient" and "subject" are used interchangeably herein, and mean and include warm blooded mammals, humans and primates; avians; domestic household or farm animals, such as cats, dogs, sheep, goats, cattle, horses and pigs; laboratory animals, such as mice, rats and guinea pigs; fish; reptiles; zoo and wild animals; and the like.

[00062] The term "comprise" and variations of the term, such as "comprising" and

"comprises," means "including, but not limited to" and is not intended to exclude, for example, other additives, components, integers or steps.

[00063] The following disclosure is provided to further explain in an enabling fashion the best modes of performing one or more embodiments of the present invention. The disclosure is further offered to enhance an understanding and appreciation for the inventive principles and advantages thereof, rather than to limit in any manner the invention. The invention is defined solely by the appended claims including any amendments made during the pendency of this application and all equivalents of those claims as issued.

[00064] As will readily be appreciated by one having ordinary skill in the art, the present invention substantially reduces or eliminates the disadvantages and drawbacks associated with prior art methods of treating damaged or diseased biological tissue.

[00065] In overview, the present disclosure is directed to methods and systems for treating damaged and diseased biological tissue; particularly, cardiovascular tissue, via the "direct" delivery of a pharmacological composition (and/or pharmacological agent and/or formulation) to the damaged or diseased tissue. According to the invention, the delivery of a

therapeutically effective amount of a pharmacological composition of the invention to damaged or diseased tissue induces neovascularization, host tissue proliferation,

bioremodeling and regeneration of new tissue.

[00066] According to the invention, the pharmacological compositions can comprise mixed liquids, mixed emulsions, mixed gels, mixed pastes, or mixed solid particulates.

[00067] In some embodiments, one or more pharmacological compositions of the invention are directly administered to the damaged or diseased tissue via a multi-needle injection system, such as disclosed in Co-pending Application No. 61/704,634, filed September 24, 2012 and illustrated in FIGS. 3 A and 3B.

[00068] In a preferred embodiment, the pharmacological compositions comprise extracellular matrix (ECM) compositions that include at least one extracellular matrix (hereinafter "ECM material").

[00069] According to the invention, the ECM material can be derived from various mammalian tissue sources and methods for preparing same, such as disclosed in U.S. Pat. Nos. 7,550,004, 7,244,444, 6,379,710, 6,358,284, 6,206,931 , 5,733,337 and 4,902,508 and U.S. Application No. 12/707,427; which are incorporated by reference herein in their entirety. The mammalian tissue sources include, without limitation, the small intestine, large intestine, stomach, lung, liver, kidney, pancreas, placenta, heart, bladder, prostate, tissue surrounding growing enamel, tissue surrounding growing bone, and any fetal tissue from any mammalian organ.

[00070] In a preferred embodiment of the invention, the ECM material comprises mesothelium, i.e. mesothelial tissue.

[00071] According to the invention, the ECM material can be formed into a particulate and fluidized, as described in U.S. Pat. Nos. 5,275,826, 6,579,538 and 6,933,326, to form an ECM composition of the invention.

[00072] According to the invention, various conventional means can be employed to form a particulate ECM material. In some embodiments, the ECM material is formed into a sheet, fluidized (or hydrated), if necessary, frozen and ground. [00073] In some embodiments of the invention, the ground ECM material is subsequently filtered to achieve a desired particulate size. Thus, in some embodiments, the ECM material has a particulate size no greater than 2000 microns. In some embodiments, the ECM material preferably has a particulate size no greater than 500 microns. In a preferred embodiment, the ECM material has a particulate size in the range of about 20 microns to about 300 microns.

[00074] According to the invention, fluidized or emulsified compositions (the liquid or semi-solid forms) can comprise various certain concentrations of ECM material. In some embodiments of the invention, the concentration of the ECM material is greater than about 5%, more preferably, greater than about 20%, even more preferably, greater than about 70%.

[00075] According to the invention, the particulate ECM material can be fluidized or hydrated by various conventional buffer materials. Suitable buffer materials include, without limitation, water and saline.

[00076] According to the invention, the liquid or semi-solid components of the ECM compositions (i.e. liquids, gels, emulsions or pastes) can comprise various concentrations. Preferably, the concentration of the liquid or semi-solid components of the ECM

compositions are in the range of about 0.001 mg/ml to about 200 mg/ml. Suitable concentration ranges thus include, without limitation: about 5 mg/ml to about 150 mg/ml, about 10 mg/ml to about 125 mg/ml, about 25 mg/ml to about 100 mg/ml, about 20 mg/ml to about 75 mg/ml, about 25 mg/ml to about 60 mg/ml, about 30 mg/ml to about 50 mg/ml, and about 35 mg/ml to about 45 mg/ml and about 40 mg/ml. to about 42 mg/ml.

[00077] The noted concentration ranges are, however, merely exemplary and not intended to be exhaustive or limiting. It is understood that any value within any of the listed ranges is deemed a reasonable and useful value for a concentration of a liquid or semi-solid component of an ECM composition.

[00078] According to the invention, the dry particulate or reconstituted particulate that forms a gel emulsion or paste of the two ECM materials can also be mixed together in various proportions. For example, the particulates can comprise 50% of small intestine submucosa mixed with 50% of pancreatic basement membrane. The mixture can then similarly be fluidized by hydrating in a suitable buffer, such as saline.

[00079] As indicated above, in some embodiments of the invention, the ECM

compositions are formulated to be injected into damaged or cardiovascular tissue, i.e.

injectable ECM compositions. In some embodiments of the invention, the injectable ECM compositions thus comprise approximately 70% particulate ECM material and

approximately 30% fully hydrolyzed ECM gel.

[00080] According to the invention, the pharmacological compositions of the invention can further include one or more additional bioactive agents or components to aid in the treatment of damaged tissue and/or facilitate the tissue regenerative process.

[00081 ] In some embodiments, the pharmacological compositions of the invention thus include at least one pharmacological agent or composition, which can comprise, without limitation, antibiotics or antifungal agents, anti-viral agents, anti-pain agents, anesthetics, analgesics, steroidal anti-inflammatories, non-steroidal anti-inflammatories, antineoplastics, anti-spasmodics, modulators of cell-extracellular matrix interactions, proteins, hormones, enzymes and enzyme inhibitors, anticoagulants and/or antithrombic agents, DNA, RNA, modified DNA and RNA, NSAIDs, inhibitors of DNA, RNA or protein synthesis, polypeptides, oligonucleotides, polynucleotides, nucleoproteins, compounds modulating cell migration, compounds modulating proliferation and growth of tissue, and vasodilating agents.

[00082] Suitable pharmacological agents and/or compositions thus include, without limitation, atropine, tropicamide, dexamethasone, dexamethasone phosphate, betamethasone, betamethasone phosphate, prednisolone, triamcinolone, triamcinolone acetonide, fluocinolone acetonide, anecortave acetate, budesonide, cyclosporine, FK-506, rapamycin, ruboxistaurin, midostaurin, flurbiprofen, suprofen, ketoprofen, diclofenac, ketorolac, nepafenac, lidocaine, neomycin, polymyxin b, bacitracin, gramicidin, gentamicin, oyxtetracycline, ciprofloxacin, ofloxacin, tobramycin, amikacin, vancomycin, cefazolin, ticarcillin, chloramphenicol, miconazole, itraconazole, trifluridine, vidarabine, ganciclovir, acyclovir, cidofovir, ara-amp, foscarnet, idoxuridine, adefovir dipivoxil, methotrexate, carboplatin, phenylephrine, epinephrine, dipivefrin, timolol, 6-hydroxydopamine, betaxolol, pilocaipine, carbachol, physostigmine, demecarium, dorzolamide, brinzolamide, latanoprost, sodium hyaluronate, insulin, verteporfin, pegaptanib, ranibizumab, and other antibodies, antineoplastics, Anti VGEFs, ciliary neurotrophic factor, brain-derived neurotrophic factor, bFGF, Caspase-1 inhibitors, Caspase-3 inhibitors, a-Adrenoceptors agonists, NMDA antagonists, Glial cell line-derived neurotrophic factors (GDNF), pigment epithelium-derived factor (PEDF), and NT-3, NT-4, NGF, IGF-2.

[00083] In a preferred embodiment of the invention, pharmacological agent comprises an antibiotic selected from the group comprising, without limitation, aminoglycosides, cephalosporins, chloramphenicol, clindamycin, erythromycins, fluoroquinolones, macrolides, azolides, metronidazole, penicillins, tetracyclines, trimethoprim-sulfamethoxazole and vancomycin.

[00084] In some embodiments of the invention, the pharmacological agent comprises a statin, i.e. a HMG-CoA reductase inhibitor, selected from the group comprising, without limitation, atorvastatin (LIPITOR®), cerivastatin, fluvastatin (Lescol®), lovastatin

(Mevacor®, Altocor®, Altoprev®), mevastatin, pitavastatin (Livalo ®, Pitava®), pravastatin (Pravachol®, Selektine®, Lipostat®), rosuvastatin (Crestor®), and simvastatin (Zocor®, Lipex®).

[00085] Applicant has found that statins exhibit numerous beneficial properties that provide several beneficial biochemical actions or activities. The properties and beneficial actions resulting therefrom are set forth in U.S. Application No. 13/573,569.

[00086] According to the invention, the amount of a pharmacological agent added to an ECM composition of the invention will, of course, vary from agent to agent. For example, in one embodiment, wherein the pharmacological agent comprises dicloflenac (Voltaren ^® ), the amount of dicloflenac included in the ECM composition is preferably in the range of 10 μg - 75 mg.

[00087] In some embodiments of the invention, the bioactive agent comprises chitin or a derivative thereof, e.g. chitosan.

[00088] Chitosan similarly exhibits a wide range of favorable biochemical properties that make it an outstanding agent for use in the medical field. The biochemical properties of chitosan include biocompatibility, biodegradability and non-toxicity. Additional properties, such as analgesic, hemostatic, antimicrobial, and antioxidant have also been reported. See Aranaz, et al., Functional Characterization of Chitin and Chitosan, Current Chemical Biology, vol. 3, pp. 203-230 (2009); and Kumar MNVR, A Review ofChitin and Chitosan Applications, React. Funct. Polm., vol. 46, pp. 1-27 (2000).

[00089] In some embodiments of the invention, the bioactive agent comprises a cell. According to the invention, the cell can comprise, without limitation, a stem cell, such as, for example, a human embryonic stem cell, fetal cell, fetal cardiomyocyte, myofibroblast, mesenchymal stem cell, auto transplanted expanded cardiomyocyte, adipocyte, totipotent cell, pluripotent cell, blood stem cell, myoblast, adult stem cell, bone marrow cell, mesenchymal cell, embryonic stem cell, parenchymal cell, epithelial cell, endothelial cell, mesothelial cell, fibroblast, myofibroblast, osteoblast, chondrocyte, exogenous cell, endogenous cell, stem cell, hematopoetic stem cell, pluripotent stem cell, bone marrow- derived progenitor cell, progenitor cell, myocardial cell, skeletal cell, undifferentiated cell, multi-potent progenitor cell, unipotent progenitor cell, monocyte, cardiomyocyte, cardiac myoblast, skeletal myoblast, macrophage, capillary endothelial cell, xenogenic cell, and allogenic cell.

[00090] In some embodiments of the invention, the bioactive agent comprises a protein. According to the invention, the protein can comprise, without limitation, a growth factor, collagen, proteoglycan, glycosaminoglycan (GAG) chain, glycoprotein, cytokine, cell- surface associated protein, cell adhesion molecule (CAM), angiogenic growth factor, endothelial ligand, matrikine, matrix metalloprotease, cadherin, immunoglobin, fibril collagen, non-fibrillar collagen, basement membrane collagen, multiplexin, small-leucine rich proteoglycan, decorin, biglycan, fibromodulin, keratocan, lumican, epiphycan, heparan sulfate proteoglycan, perlecan, agrin, testican, syndecan, glypican, serglycin, selectin, lectican, aggrecan, versican, nuerocan, brevican, cytoplasmic domain-44 (CD44), macrophage stimulating factor, amyloid precursor protein, heparin, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate A, heparan sulfate, hyaluronic acid, fibronectin (Fn), tenascin, elastin, fibrillin, laminin, nidogen/entactin, fibulin I, fibulin II, integrin, a transmembrane molecule, platelet derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), transforming growth factor beta (TGF-beta), fibroblast growth factor-2 (FGF-2) (also called basic fibroblast growth factor (bFGF)), thrombospondin, osteopontin, angiotensin converting enzyme (ACE), and vascular epithelial growth factor (VEGF).

[00091] According to the invention, the bioactive agents referenced above can comprise any form. In some embodiments of the invention, the bioactive component or components, e.g. simvastatin and/or chitosan, comprise microcapsules that provide delayed delivery of the agent contained therein.

[00092] Additional suitable pharmacological compositions that can be delivered within the scope of the invention are disclosed in Pat. Pub. Nos. 20070014874, 20070014873, 20070014872, 20070014871, 20070014870, 20070014869, and 20070014868; which are expressly incorporated by reference herein in its entirety.

[00093] As indicated above, in some embodiments of the invention, one or more pharmacological compositions of the invention are directly administered or delivered to damaged or diseased cardiovascular tissue via a unique multi-needle injection system. As will readily be appreciated by one having ordinary skill in the art, the pharmacological compositions of the invention can also be delivered to damaged tissue via one or more conventional injection apparatus and systems, e.g., syringe. According to the invention, the pharmacological compositions can be directly administered to the heart wall and/or the various cardiovascular structures associated therewith.

[00094] Referring now to Fig. 1 there is shown a depiction of a normal heart 100. The heart wall 102 consists of an inner layer of simple squamous epithelium, referred to as the endocardium. The endocardium overlays the myocardium (a variably thick heart muscle) and is enveloped within a multi-layer tissue structure referred to as the pericardium. The innermost layer of the pericardium, referred to as the visceral pericardium or epicardium, covers the myocardium. An outermost layer of the pericardium, referred to as the fibrous pericardium, attaches the parietal pericardium to the sternum, the great vessels and the diaphragm.

[00095] According to the invention, a pharmacological composition can be delivered to each of the noted structures; particularly, the myocardium, whereby neovascularization, host tissue proliferation, and bioremodeling is induced. [00096] Referring now to Fig. 2, there is shown a depiction of a heart 200 having an ischemic infracted region 202, and a peri-infarcted region 204 that is surrounded by healthy non-ischemic myocardium tissue 206.

[00097] As indicated above, a myocardial infarction, i.e. irreversible myocardial injury resulting in necrosis of a significant portion of myocardium, can result in an acute depression in ventricular function and expansion of the infarcted tissue under stress. This triggers a cascading sequence of myocellular events. In many cases, this progressive myocardial infarct expansion and remodeling leads to deterioration in ventricular function and heart failure.

[00098] When a myocardial infarction occurs, the myocardial tissue that is no longer receiving adequate blood flow dies and is replaced with scar tissue. This infarcted tissue cannot contract during systole, and may actually undergo lengthening in systole and leads to an immediate depression in ventricular function. This abnormal motion of the infarcted tissue can cause delayed or abnormal conduction of electrical activity to the still surviving peri- infarct tissue (tissue at the junction between the normal tissue and the infarcted tissue) and also places extra structural stress on the peri-infarct tissue.

[00099] In addition to immediate hemodynamic effects, the infarcted heart tissue and undergoes three major processes: infarct expansion, infarct extension, and chamber remodeling. These factors individually and in combination contribute to the eventual dysfunction observed in the cardiac tissue remote from the site of the infarction.

[000100] Infarct expansion is a fixed, permanent, disproportionate regional thinning and dilatation of tissue within the infarct zone. Infarct extension is additional myocardial necrosis following myocardial infarction. Infarct extension results in an increase in total mass of infarcted tissue.

[000101 ] However, as indicated above, the noted effects of a myocardial infarction can be ameliorated or eliminated by administering a pharmacological composition of the invention directly to the infarcted cardiovascular tissue. As also indicated herein, the pharmacological compositions of the invention will specifically induce neovascularization, host tissue proliferation, bioremodeling, and regeneration of new cardiac tissue structures with site- specific structural and functional properties. A preferred means of administering the pharmacological compositions to infracted cardiovascular tissue comprises direct injection via the multi-needle injection apparatus 300 illustrated in Figs. 3A and 3B and the associated control system described in Co-Pending Application No. 61/704,634.

EXAMPLES

[000102] The following examples are provided to enable those skilled in the art to more clearly understand and practice the present invention. They should not be considered as limiting the scope of the invention, but merely as being illustrated as representative thereof.

Example 1

[000103] Five (5) porcine hearts were obtained from young calves. After removal, the hearts were stored in a saline bath.

[000104] A first heart was removed from the bath. The thickness of the heart wall was determined to range from 4 mm to greater than 2 cm with an A scan ultrasound sensor.

[000105] A multi-needle injection system of the invention, such as illustrated in Figs. 2 and 3 was provided and prepared for the injection procedure.

[000106] A pharmacological composition of the invention was also provided. The pharmacological composition comprised two components: an ECM (i.e. SIS) particulate derived from porcine intestines and a SIS gel. The SIS particulate comprised SIS material, which was cryogenically ground to a characterized particle size, and subsequently thawed and loaded into a syringe for delivery. The particulate size was in the range of 50 - 350 microns.

[000107] The SIS gel comprised SIS material that was cryogenically ground, subject to enzymatic digestion in acid, lyophihzed, and reconstituted to a predetermined concentration. The SIS gel was also subjected to a subsequent disinfection and neutralization process. The SIS gel was also loaded into a syringe.

[000108] The materials were maintained in refrigerated conditions throughout processing.

[000109] Approximately 4 cc of SIS gel was mixed with 6 cc of particulate SIS to derive an injectable ECM composition.

[0001 10] The injectable pharmacological composition was then transferred into the reservoirs of the injector apparatus.

[0001 1 1] The injector control system was then set to provide the following delivery parameters: two (2) equal pulses at 20 and 30 milliseconds and at pressures ranging from approximately 60 - 120 psi. The noted parameters provided an ECM composition delivery in the range of approximately 0.5 - 1.0 ml per pulse.

[0001 12] The pharmacological composition was then delivered into the wall of a first heart. The injected portion of the heart wall was then observed visually and with a B scan ultrasound (i.e. echo) sensor to assess the ECM composition delivery pattern. Substantially uniform delivery (i.e. amount and spread) at each needle injection site was observed.

[0001 13] The injected portion of the heart wall was also sectioned to observe the delivery pattern. The procedure confirmed that delivery was uniform and at the prescribed needle depth at each needle injection site, with a good safety margin from the ventricular cavity.

[0001 14] The above noted test procedures were similarly employed with the remaining four (4) porcine hearts. The only parameter that varied was the proportion of SIS gel in the ECM composition.

[0001 15] In the second heart, the pharmacological composition was similar to the composition employed for the first heart, i.e. approximately 4 cc of SIS gel and 6 cc of particulate SIS.

[0001 16] In the third and fourth hearts, the pharmacological composition comprised approximately 2 cc of SIS gel and 8 cc of particulate SIS.

[0001 17] In the fifth heart, no SIS gel was employed. The pharmacological composition thus comprised approximately 10 cc of particulate SIS.

[0001 18] In each instance, the delivery was similarly uniform and at the prescribed needle depth at each needle injection site, with a good safety margin from the ventricular cavity.

Example 2

[0001 19] A young porcine was provided in which CHF had been induced via serial microsphere injections down the coronary arteries.

[000120] A multi-needle injection system of the invention, such as illustrated in Figs. 2 and 3, was prepared for injection of a pharmacological composition of the invention.

[000121] An ECM composition, such as described in Example 1 , was also provided. The composition mixture comprised approximately 4 cc of SIS gel was mixed with 6 cc of particulate SIS to derive an injectable composition. [000122] The injectable pharmacological composition was then transferred into the reservoirs of the injector apparatus.

[000123] The injector control system was similarly set to provide the following delivery parameters: two (2) equal pulses at 20 and 30 milliseconds and at pressures ranging from approximately 60 - 120 psi. The noted parameters provided an ECM composition delivery in the range of approximately 0.5 - 1.0 ml per pulse.

[000124] The heart of the porcine was then exposed. A B scan ultrasound sensor was then employed to assess the depth of the infarcted region.

[000125] The pharmacological composition was then delivered into the infarcted region. The injected portion of the heart wall was then observed visually and with a B scan ultrasound (i.e. echo) sensor to assess the ECM composition delivery pattern. Substantially uniform delivery (i.e. amount and spread) at each needle injection site was observed. The ECM composition also stayed within the infarcted region without coming out of the ventricle wall.

[000126] Further, no extravisation or emboli was observed.

[000127] A ventricular assist device was subsequently placed into the apex and the animal recovered without incident.

[000128] In accord with the invention, within 2 - 4 weeks, neovascularization, host tissue proliferation, bioremodeling and regeneration of new tissue proximate the infracted region will be observed.

[000129] As will readily be appreciated by one having ordinary skill in the art, the present invention provides numerous advantages compared to prior art methods and systems for treating damaged cardiac tissue. Among the advantages are the following:

« The provision of pharmacological compositions which, when delivered to damaged biological tissue; particularly, cardiovascular tissue, induce neovascularization, and promote survival and regeneration of damaged cardiovascular tissue.

• The provision of extracellular matrix (ECM) compositions which, when delivered to damaged biological tissue; particularly, cardiovascular tissue, induce host tissue proliferation, bioremodeling, and regeneration of cardiovascular tissue structures with site-specific structural and functional properties. • The provision of improved methods and systems for administering pharmacological compositions; particularly, ECM compositions directly to damaged or diseased biological tissue.

[000130] Without departing from the spirit and scope of this invention, one of ordinary skill can make various changes and modifications to the invention to adapt it to various usages and conditions. As such, these changes and modifications are properly, equitably, and intended to be, within the full range of equivalence of the following claims.