Soluble extracellular matrix composition and method for intravascular delivery

Abstract

To provide a soluble extracellular matrix composition and a method for intravascular delivery.SOLUTION: Compositions and methods for manufacturing and using the same are provided. The compositions comprise a soluble extracellular matrix fraction for intravascular delivery which forms a gel or coating in situ for treatment of myocardial infarction and ischemia in a variety of tissues and endothelial injury / dysfunction.SELECTED DRAWING: Figure 7

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 62 / 750,303, filed Oct. 25, 2018, which is hereby incorporated by reference in its entirety.

[0002] Government Support This invention was made with government support under Grant No. HL113468 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention.

[0003] The present invention relates to injectable soluble extracellular matrix compositions and treatments for minimally invasive delivery to tissues / organs / cells including ischemic or damaged heart, brain, skeletal muscle, blood vessels, and endothelial cells.

Background Art

[0004] Extracellular matrix therapies contain natural tissue components that provide scaffolds for tissue regeneration. Current decellularized extracellular matrix therapies are limited to patches or direct injection. There is no extracellular matrix therapy that enables intravascular / injection delivery to the target tissue. ECM hydrogels made from decellularized tissues and digested tissues have been created, but they are not completely soluble or transparent colloids. They are translucent suspensions containing both soluble and insoluble components (Freytes et al, 2008, Singelyn et al, 2009), and the material passes through leaky vasculature (occurring in ischemic tissues such as acute myocardial infarction, stroke, cancer, etc.) (Nguyen et al, 2015, Dvorak et al, 1988, Yuan et al, 1995)) from the bloodstream into the tissue and prevents lining or filling the pores of the leaky vasculature.

[0005] Myocardial infarction (MI) is characterized by progressive ischemic necrosis of the heart muscle over time, leading to negative left ventricular (LV) remodeling and eventual heart failure. Current standard treatments do not address this ischemic injury. Minimally invasive tissue engineering treatments can repair the heart after MI. Many stem cell and growth factor therapies have reached clinical trials; however, these therapies have shown insufficient efficacy, possibly due to poor retention of unencapsulated therapeutic agents.

[0006] Extracellular matrix (ECM) hydrogels show great potential in the field of cardiac tissue engineering. In particular, tissue-specific hydrogels derived from decellularized porcine cardiomyocytes, referred to as cardiomyocyte matrix (MM), have shown increased cardiomyocytes, angiogenesis in infarcted areas, and improvements in local and global cardiac function in MI models [2-4]. Furthermore, investigation of repair mechanisms through whole transcriptome analysis of RNA isolated from infarcted areas of rat hearts injected with MM revealed upregulated pathways associated with cardiac repair (e.g., angiogenesis and cardiac development) and downregulated pathways associated with negative LV remodeling (e.g., hypertrophy, apoptosis, and fibrosis) [6]. This material was evaluated in a Phase I trial in post-MI patients using transendocardial injection 60 days to 3 years post-MI (ClinicalTrials.gov identifier: NCT02305602). However, cardiomyocyte death and negative LV remodeling are processes that begin within minutes to days post-MI [26, 27]. Current delivery of MM is limited to transendocardial catheter injection because it contains submicron particles that are too large to pass through the leaky coronary system and enter the infarct, making it unsuitable for intra-coronary injection. Furthermore, transendocardial injection is a specialized medical technique with several safety concerns, including ventricular rupture and arrhythmias within the first week after MI [7, 8]. This prevents the delivery of MM within a critical therapeutic range after acute MI.

[0007] Intra-coronary infusion is an alternative approach to transendocardial injection. Intra-coronary delivery is feasible in acute MI, as it may be accompanied by balloon angioplasty, typically performed shortly after the patient is admitted to the hospital. Such techniques are standard in interventional cardiology and do not require specialized training. Intra-coronary infusion utilizes the leaky vascular system after acute MI to allow biomaterials to pass through the coronary vascular system and enter the infarcted area.[5] Intra-coronary delivery of biomaterials demonstrated the feasibility of alginate hydrogel in a porcine MI model[9], which progressed to a phase II clinical trial (ClinicalTrials.gov identifier: NCT01226563). However, perhaps due to the limited bioactivity of alginate, the material did not show a significant improvement in cardiac function.

[10] [Overview of the project]

[0008] In embodiments, the present invention provides a method for preparing a soluble extracellular matrix (ECM) composition, comprising: enzymatically digesting ECM material with an acidic protease such as pepsin; neutralizing the digested ECM material in a liquid to pH 7.0-8.0; processing the liquid ECM to produce soluble and insoluble fractions; and separating at least a portion of the soluble fraction from the insoluble fraction to obtain a soluble ECM composition.

[0009] In embodiments, the present invention provides that the treatment of liquid ECM to produce soluble and insoluble fractions is achieved by centrifugation. In embodiments, the present invention provides that the soluble ECM composition is dialyzed and / or filtered to remove insoluble material. In embodiments, the present invention provides that the soluble ECM composition is further freeze-dried for storage and rehydrated for use.

[0010] In embodiments, the soluble ECM composition is substantially isolated from the ECM solid in the liquid ECM. In embodiments, the soluble ECM composition is clearer than the digested, unseparated ECM material. In embodiments, the soluble ECM composition comprises a clear ECM colloid that can pass through a 0.25 μm filter.

[0011] In embodiments, the present invention provides a method for treating a subject in need, comprising administering an effective amount of a soluble ECM composition to the subject to promote tissue repair or cell recruitment. In embodiments, the infusion is performed intravenously or intravascularly via a catheter. In embodiments, the present invention provides that, when delivered in vivo, the soluble fraction forms a gel in the tissue. In embodiments, the soluble fraction coats the inner lining of blood vessels. In embodiments, the soluble fraction fills pores, fenestrations, endothelial breakdown, open intercellular junctions, or gaps in leaky or damaged vascular systems.

[0012] In embodiments, the present invention provides that a soluble fraction of ECM is further crosslinked with glutaraldehyde, formaldehyde, bis-NHS molecules, or other crosslinking agents. In embodiments, the present invention provides that a soluble fraction of ECM is combined with and / or crosslinked with synthetic polymers or bio-derived materials. In embodiments, the present invention provides that a soluble fraction of ECM is combined with cells, peptides, proteins, DNA, drugs, nanoparticles, antibiotics, growth factors, nutrients, survival-promoting additives, proteoglycans, and / or glycosaminoglycans.

[0013] In embodiments, the present invention provides that a soluble fraction of ECM is used in combination with the above-mentioned components for endogenous intracellular proliferation, angiogenesis, and regeneration. In embodiments, the present invention provides that a soluble fraction of ECM is used in combination with the above-mentioned components as a matrix for altering the mechanical properties of tissue. In embodiments, the present invention provides that a soluble fraction of ECM is delivered with cells, either alone or in combination with the above-mentioned components, for the regeneration or repair of damaged tissue.

[0014] In embodiments, the present invention provides that, after concentration adjustment and / or sterile filtration, the soluble fraction of ECM can be freeze-dried and stored frozen for at least three months (e.g., 20°C, -80°C). The soluble ECM composition or fraction can then be resuspended and / or sterile-filtered before injection or infusion.

[0015] In embodiments, the present invention provides that, after concentration adjustment and / or sterile filtration, the soluble fraction of ECM can be freeze-dried and stored in a refrigerator (e.g., 4°C) for at least 3 months. The soluble ECM fraction can then be resuspended and / or sterile-filtered before injection or infusion.

[0016] In embodiments, the present invention provides that, after concentration adjustment and / or sterile filtration, the soluble fraction of ECM can be lyophilized and stored at room temperature for at least 3 months. The soluble ECM fraction can then be resuspended and / or sterile filtered before injection or infusion.

[0017] In embodiments, the present invention provides that a method for separating at least a portion of the soluble and insoluble fractions of a liquid extracellular matrix (pregel solution) can be performed by high-speed centrifugation, dialysis, filtration, or adjustment of pH or salinity. In embodiments, the separation of the soluble fraction is performed by removing at least a portion of the solid from the ECM material. In embodiments, the separation of the soluble fraction is performed using a filter having a size limit of 1 μm, 0.5 μm, 0.25 μm, 0.22 μm, or less than 0.2 μm. In embodiments, the present invention provides a soluble ECM composition derived from decellularized tissue and processed to isolate the soluble fraction before gelation in vivo. In embodiments, the present invention provides that a composition of soluble ECM is prepared for intravascular injection.

[0018] In embodiments, the present invention provides that the composition of the soluble extracellular matrix is ​​derived from human, animal, embryonic, and / or fetal tissue sources. In embodiments, the present invention provides that the composition of the soluble extracellular matrix is ​​derived from heart, brain, bladder, small intestine, skeletal muscle, kidney, liver, lung, blood vessels, and other tissue / organ tissue sources.

[0019] In embodiments, the present invention provides a method for treating acute myocardial infarction, comprising injecting or infusing an effective amount of a composition containing soluble decellularized extracellular matrix derived from muscle tissue into a subject in need having myocardial infarction.

[0020] In embodiments, the present invention provides that the soluble ECM composition is delivered intravascularly by injection. In embodiments, the present invention provides that the soluble ECM composition is delivered by intracoronary injection using a balloon-equipped injection catheter. In embodiments, the present invention provides that the soluble ECM composition transitions to a gel form in the tissue after delivery. In embodiments, the present invention provides that the soluble ECM composition transitions to form a coating on the endothelium of the damaged blood vessel after delivery. In embodiments, the present invention provides that the soluble ECM composition degrades within 1 to 14 days after injection or infusion.

[0021] In embodiments, the present invention provides that injection or infusion of the composition repairs sustained myocardial damage, such as myocardial infarction, in the subject. In embodiments, the present invention provides that injection or infusion of the composition is used to treat muscle or nerve damage caused by disease, trauma, stroke, and / or ischemia in the subject. In embodiments, the present invention provides that the effective amount is an amount that increases blood flow, increases the amount of viable tissue, or induces new angiogenesis in the area of ​​injection or infusion of the subject. In embodiments, the present invention provides that the effective amount is an amount that promotes cell survival, reduces inflammation, and repairs damaged vascular systems in the area of ​​injection or infusion of the subject. [Brief explanation of the drawing]

[0022] [Figure 1] This shows the formation of a soluble myocardial matrix. Figure 1A shows the fragmentation of isolated left ventricular myocardium. Figure 1B shows decellularization after continuous stirring in 1% sodium dodecyl sulfate. Figure 1C shows the lyophilized and pulverized material. Figure 1D shows the partially digested myocardial matrix. (E) Fractionated myocardial matrix after centrifugation, (1) SolMM fraction in the supernatant, and (2) insoluble pellet. (F) SolMM hydrogel after subcutaneous injection. Images (A-D) were obtained from [3]. [Figure 2]PAGE showing the protein distribution comparison of ladder (Full-Range RPN800E, lane 1), collagen (lane 2), myocardial matrix (lane 3), and soluble myocardial matrix (lane 4). [Figure 3] Distribution and retention of SolMM (red grayscale) 12 hours after intracoronary injection of 200 μL of 10 mg / mL SolMM in an ischemia-reperfusion rat model. (Left) Short-axis image of an infarcted heart stained with hematoxylin and eosin, with the infarct spreading to the lower half of the heart, scale bar 3 mm. (Right) Insert of infarcted myocardium showing SolMM microscale gel across the infarcted myocardium, scale bar 200 μm. [Figure 4] Distribution and retention of SolMM (red grayscale) 1 hour after intracoronary injection in a porcine ischemia-reperfusion model. (Left) Gross short-axis tissue image of an infarcted porcine heart. The infarct is outlined in blue grayscale. (Right) Infarcted myocardium showing SolMM microgel across the infarcted myocardium. [Figure 5] Showing alleviated negative left ventricular remodeling (preserved EDV and ESV) after intracoronary injection of SolMM in an ischemia-reperfusion model at 24 hours and 5 weeks after injection. EDV - end-diastolic volume, ESV - end-systolic volume, EF - ejection fraction, SolMM (square in blue grayscale), saline (circle in red grayscale). N = 10 - 11 per group. [Figure 6] Showing an increase in infarct arteriole density in SolMM-injected rats 5 weeks after injection and ischemia-reperfusion. Arterioles were identified by co-staining with α-smooth muscle actin and isolectin and manually traced in ImageJ. N = 10 - 11 per group. [Figure 7] Showing a decrease in cardiomyocyte apoptosis in the infarct border zone of SolMM-injected rats 3 days after injection and ischemia-reperfusion. Tissues were stained with α-actinin for cardiomyocytes and cleaved caspase 3 for apoptosis. Apoptotic cardiomyocytes were manually counted in ImageJ. N = 5 - 6 per group. [Figure 8]Shows relative gene expression changes in SolMM-injected rats 1 day after injection and ischemia-reperfusion. Gene expression was measured by RT-qPCR using RNA isolated from the LV free wall. Gene expression on day 1 suggests an increase in the angiogenesis pathway and the metabolic pathway of reactive oxygen species. N = 5 - 6 per group. [Figure 9] Shows relative gene expression changes in SolMM-injected rats 3 days after injection and ischemia-reperfusion. Gene expression was measured by RT-qPCR using RNA isolated from the LV free wall. Gene expression on day 3 suggests a decrease in the apoptosis / necrosis and fibrosis pathways. LRG1 is suggested in the angiogenesis pathway, and down-regulation of LRG1 is related to fibrosis. N = 5 - 6 per group. [Figure 10] Shows confocal images of soluble matrix (red grayscale) and lectin (green grayscale) of endothelial cells after injecting soluble matrix into the ischemia-reperfusion rat model. The panels are consecutive images from a z-stack. The soluble matrix coats the inside of small (about 5 μm in diameter) capillaries, but the soluble matrix does not completely occlude the lumen. [Figure 11] Shows confocal images of soluble matrix (red grayscale) and lectin (green grayscale) of endothelial cells after injecting soluble matrix into the ischemia-reperfusion rat model. The soluble matrix overlaps with endothelial cells and does not occlude the lumen of blood vessels. [Figure 12] Shows the retention of soluble matrix in the soluble matrix-injected heart 24 hours after injection and ischemia-reperfusion. From left to right, 1) saline, 2) 10 mg / ml soluble matrix conjugated with Vivo Tag750, 3) 10 mg / ml trilysin conjugated with Vivo Tag750, 4) 10 mg / ml soluble matrix conjugated with Vivo Tag750 was injected into the heart. Trilysin was used as a small peptide control and showed minimal heart retention. [Figure 13]Scanning electron microscope images of a soluble matrix hydrogel are shown. The left image has a scale bar of 20 μm, and the right image has a scale bar of 5 μm. [Figure 14] The dynamic light scattering data for soluble matrix (SolMM) and complete matrix (MM) at 1:50 dilutions (1.0 mg / ml and 0.6 mg / ml, respectively) are shown, indicating that soluble matrix particles have a diameter of less than 100 nm, while the complete matrix has larger particles. [Figure 15] The dynamic light scattering data of the soluble matrix (SolMM) at 1:10 and 1:100 dilutions (1.0 mg / ml and 0.1 mg / ml, respectively) is shown, representing particles with a diameter of less than 100 nm. [Figure 16] The absorbance (left) and transmittance (right) of physiological saline, soluble matrix (SolMM), and complete matrix (MM) are shown. [Figure 17] The relative absorbance (left) and transmittance (right) of soluble matrix (SolMM) and complete matrix (MM) are shown. [Modes for carrying out the invention]

[0023] All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.

[0024] Unless otherwise defined, all technical and scientific terms, as well as any acronyms, used herein have the same meaning as commonly understood by those skilled in the art of the present invention. Any methods and materials similar or equivalent to those described herein may be used in carrying out the present invention, but exemplary methods, apparatus, and materials are described herein.

[0025] The implementation of this invention, unless otherwise specified, utilizes conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the skill of those skilled in the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, 2 nd (Sambrook et al., 1989), Oligonucleotide Synthesis (MJGait, ed., 1984), Animal Cell Culture (RIFreshney, ed., 1987), Methods in Enzymology (Academic Press, Inc.), Current Protocols in Molecular Biology (FMAusubel et al., eds., 1987, and periodic updates), PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994), Remington, The Science and Practice of Pharmacy, 20 th ed., (Lippincott, Williams & Wilkins 2003), and Remington, The Science and Practice of Pharmacy, 22 th This is fully explained in publications such as (Pharmaceutical Press and Philadelphia College of Pharmacy at University of the Sciences 2012).

[0026] In embodiments, the present invention relates to a method for preparing one or more biologically active soluble fractions of the extracellular matrix (ECM) for therapeutic delivery, a. Partial or complete digestion of decellularized ECM prepared from tissue using acidic proteases such as pepsin, b. Neutralizing the digested ECM material to pH 7.0-8.0, c. Treating with liquid ECM (Pregel solution) to produce soluble and insoluble fractions, d. A method is provided which includes separating at least a portion of the insoluble fraction from the soluble fraction in order to obtain a soluble ECM composition.

[0027] In embodiments, the present invention provides that the soluble ECM composition is further freeze-dried, dialyzed, and / or filtered. In embodiments, the present invention provides that the soluble ECM composition is rehydrated after freeze-drying.

[0028] In embodiments, the present invention provides a method for treating a subject in need of such treatment, comprising administering an effective amount of a soluble ECM composition intravascularly to the subject to promote the repair or mobilization of organs, tissues, or cells. In embodiments, the infusion is performed intravenously or intravascularly via a catheter. In embodiments, the present invention provides that, when delivered in vivo, the soluble ECM composition forms a gel within and / or around the microvessel system of the tissue.

[0029] In embodiments, the present invention provides a method for treating acute myocardial infarction, comprising injecting or infusing an effective amount of a composition containing soluble decellularized extracellular matrix derived from muscle tissue into a subject in need having myocardial infarction.

[0030] In embodiments, the present invention provides that the composition is delivered intravascularly. In embodiments, the present invention provides that the composition is delivered by a balloon-equipped infusion catheter. In embodiments, the present invention provides that the composition transitions to a gel form in tissue after delivery. In embodiments, the present invention provides that the composition degrades within 1 to 14 days after injection or infusion. In embodiments, the present invention provides that injection or infusion of the composition repairs myocardial damage sustained by the subject. In embodiments, the present invention provides that injection or infusion of the composition repairs non-cardiac tissue damage caused by trauma or ischemia in the subject.

[0031] In embodiments, the present invention provides that an effective amount is an amount that increases blood flow, increases the amount of viable tissue, or induces new angiogenesis in the area of ​​the injection or infusion of interest.

[0032] For human treatment, there are many sources of extracellular matrix: for example, human, pig, cattle, goat, mouse, rat, rabbit, chicken, and other animal sources. Furthermore, there are many tissue sources: for example, heart, brain, bladder, small intestine, skeletal muscle, kidney, liver, lung, blood vessels, and other tissues and organs.

[0033] In embodiments, the tissue is first decellularized, leaving only the extracellular matrix, for example, as disclosed in U.S. Patent Publication US2013 / 0251687, which incorporates the entire matrix by reference. The matrix is ​​then lyophilized, pulverized, or micronized, solubilized with pepsin or other enzymes, and subsequently neutralized and buffered as previously reported. After neutralization, the digest (Pregel solution) is fractionated to separate the soluble and insoluble fractions. The separation of the soluble and insoluble fractions can be achieved by centrifugation, dialysis, filtration, or adjustment of pH or salinity. The soluble fraction can be dialyzed to remove salt, lyophilized, and resuspended to adjust the ECM concentration. The ECM can be sterile filtered, lyophilized, and stored in a sterile container. The ECM can be resuspended to an appropriate / physiological concentration for injection.

[0034] A soluble ECM composition refers to an extracellular matrix material that has been decellularized, freeze-dried, pulverized, and digested to remove at least a portion of its solid components. In embodiments, the soluble ECM composition is obtained from the supernatant of centrifugation. In embodiments, the soluble ECM composition can pass through filter sizes of 1 μm, 500 nm, 250 nm, 220 nm, or less than 200 nm. A soluble ECM composition from which at least a portion of the naturally occurring solid ECM components have been removed is a clearer material than before the removal of the ECM solids. However, it should be understood that some insoluble small particulate matter, such as ECM colloids, may still be present in the soluble ECM composition. A soluble ECM composition is substantially isolated when at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% by volume of the naturally occurring ECM solids has been removed.

[0035] After adjusting the concentration and / or sterile filtration, the soluble ECM composition can be lyophilized and stored for at least 3 months (e.g., -20°C, -80°C). The soluble ECM composition can then be rehydrated with sterile water before injection or infusion.

[0036] Soluble ECM compositions can be injected via catheter, delivered intravenously, or injected intravascularly with or without a balloon. Soluble ECM compositions can pass through damaged, leaky vascular systems, such as those found in acute myocardial infarction, stroke, other ischemic tissues, and tumors. Once inside the tissue, soluble ECM compositions form a gel.

[0037] Soluble ECM compositions can be injected via catheter, delivered intravenously, or injected intravascularly with or without a balloon. Soluble ECM compositions can assemble to build a coating of leakage vascular lining, such as in acute myocardial infarction, stroke, other ischemic tissues, tumors, and traumatized tissues, or to fill the pores thereof.

[0038] Soluble ECM composition gels can be crosslinked with glutaraldehyde, formaldehyde, bis-NHS molecules, or other crosslinking agents. Soluble ECM compositions can be combined with cells, peptides, proteins, DNA, drugs, nutrients, survival-promoting additives, proteoglycans, and / or glycosaminoglycans. Soluble ECM compositions can be combined with and / or crosslinked with synthetic polymers. Soluble ECM compositions can be used alone or in combination with the above-mentioned components for endogenous intracellular proliferation, angiogenesis, and regeneration. Soluble ECM compositions can be used alone or in combination with the above-mentioned components as a matrix for altering the mechanical properties of tissue. Soluble ECM compositions can be delivered with cells, alone or in combination with the above-mentioned components, for the regeneration of damaged tissue.

[0039] Soluble ECM compositions can be used for tissue repair after tissue damage caused by conditions such as myocardial infarction, stroke, traumatic brain injury, peripheral artery disease, hepatic chiralism, cancerous tumors, or kidney injury. Soluble ECM compositions can be used alone or act as a therapeutic drug delivery vehicle.

[0040] The present invention provides soluble ECM compositions and methods for treating conditions involving endothelial cell damage or dysfunction, leaky vascular systems, disruption of endothelial cell junctions, inhibition of vasodilation, and inflammation.

[0041] The present invention provides soluble ECM compositions and methods for treating conditions involving potential reperfusion injury, including myocardial infarction, stroke, and peripheral artery and vascular diseases. The soluble ECM compositions can act as tissue engineering scaffolds to reduce reperfusion injury, reduce apoptosis, and promote tissue repair.

[0042] The present invention provides soluble ECM compositions and methods for treating the production / signaling of excessive or persistent reactive oxygen species (ROS) that lead to endothelial cell activation and inflammation. The soluble ECM compositions can protect cells and tissues from ROS damage and inflammation through physical shielding and / or ROS sequestration.

[0043] This invention provides soluble ECM compositions and methods for treating heart disease, ischemia, and perfusion. The soluble ECM compositions can promote angiogenesis and increase tissue perfusion.

[0044] This invention provides soluble ECM compositions and methods for treating diabetes and insulin resistance. The soluble ECM compositions can treat endothelial cells and restore endothelium-dependent vasodilation.

[0045] This invention provides soluble ECM compositions and methods for treating cancer, including tumor growth and metastasis. The soluble ECM compositions can treat leaky vascularization and endothelial dysfunction present in cancer. ECM degradation products have been shown to inhibit tumor growth and formation.

[0046] This invention provides a soluble ECM composition and method for treating lung diseases such as chronic obstructive pulmonary disease, asthma, and pulmonary arterial hypertension. The soluble ECM composition can be injected to treat damaged lung tissue and / or endothelial cells.

[0047] This invention provides a soluble ECM composition and method for treating chronic renal failure. The soluble ECM composition can treat blood vessels and restore vasodilation and vasoconstriction.

[0048] This invention provides a soluble ECM composition and method for the treatment of venous thrombosis. Injection of the soluble ECM composition can coat blood vessels to prevent thrombosis and platelet aggregation.

[0049] The present invention provides a soluble ECM composition and method for treating severe infectious diseases, particularly diseases involving disruption of the endothelial barrier, such as hemorrhagic fever and hantavirus pulmonary syndrome, which are caused by hemorrhagic fever viruses. Injection of the soluble matrix can treat and restore the endothelial barrier.

[0050] This invention provides a soluble ECM composition and method for treating atherosclerosis. Injection of the soluble ECM composition can prevent plaque rupture by coating and stabilizing atherosclerotic plaques, or it can reduce inflammation by adhering to endothelial cells.

[0051] This invention provides a soluble ECM composition and method for treating cirrhosis and acute liver failure. The soluble ECM composition can treat endothelial dysfunction in cirrhosis. The soluble ECM composition can reduce inflammation and oxidative stress.

[0052] The present invention provides soluble ECM compositions and methods for treating tissue bleeding and edema. The soluble matrix can coat endothelial cells, fill the gaps in the endothelial cell layer, and increase tissue perfusion or decrease fluid entering the tissue through a vascular stimulating effect.

[0053] This invention provides soluble ECM compositions and methods for treating traumatic brain and other neurological injuries. The soluble matrix can treat endothelial cells to repair leaky vessels, restore endothelium-dependent dilation and nitric oxide production, and reduce inflammation and oxidative stress.

[0054] Intra-coronary injection is an alternative approach to transendocardial injection. Intra-coronary delivery may be accompanied by balloon angioplasty during the typical treatment course of myocardial infarction (MI). Intra-coronary injection utilizes the leaky vascular system after acute MI, allowing the biomaterial to enter the infarcted area [5]. In conventional formulations, the matrix material (MM) consists of a soluble fraction and insoluble submicron particles (>800 nm), which are too large to pass through or adhere to the leaky vascular system. As a result, methods have been provided to isolate the soluble fraction, referred to as soluble MM (SolMM), which can pass through and / or coat the leaky vascular system and still form a hydrogel in vivo. Since SolMM is derived from MM, SolMM has similar therapeutic effects, including reduced cardiomyocyte apoptosis, angiogenesis, and reduced negative LV remodeling.

[0055] To facilitate understanding of the present invention, some terms and abbreviations used herein are defined as follows:

[0056] When describing elements of the present invention or its preferred embodiments, the articles "a," "an," "the," and "said" are intended to indicate that one or more of the elements exist. The terms "comprising," "including," and "having" are intended to indicate comprehensiveness and that additional elements other than those listed may exist.

[0057] When the term "and / or" is used in an enumeration of two or more items, it means that any one of the enumerated items may be used alone or in combination with any one or more of the enumerated items. For example, the expression "A and / or B" is intended to mean either A or B, or both, i.e., A only, B only, or a combination of A and B. The expression "A, B and / or C" is intended to mean A only, B only, C only, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C.

[0058] The aspects and embodiments of the present invention described herein are understood to include aspects and embodiments that "consist of ~" and / or "essentially consist of ~".

[0059] It should be understood that descriptions in range form are for convenience and brevity only and should not be interpreted as inflexible limitations on the scope of the invention. Therefore, descriptions of ranges should be considered to specifically disclose all possible subranges and the individual numerical values ​​within those ranges. For example, a description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and the individual numerical values ​​within those ranges, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range. Also, in this specification, values ​​or ranges may be expressed as “approximately,” “approximately” one particular value, and / or “approximately” another particular value. Where such values ​​or ranges are expressed, other embodiments disclosed will include the enumerated specific values, from one particular value to and / or other specific values. Similarly, where values ​​are expressed as approximations by using the antecedent “approximately,” it will be understood that a particular value forms another embodiment. There are several values ​​disclosed herein, and it will be further understood that each value is disclosed herein not only as the value itself, but also “about” that particular value. In embodiments, “about” may be used to mean, for example, within 10% of the enumerated value, within 5% of the enumerated value, or within 2% of the enumerated value.

[0060] As used herein, the term “pharmaceutical composition” means a pharmaceutically acceptable composition which comprises a pharmaceutically active agent and, in some embodiments, further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may be a combination of a pharmaceutically active agent and a carrier.

[0061] The term “combination” refers to either a fixed combination in a single drug dosing unit or a parts kit for combination administration, in which one or more active compounds and combination partners (e.g., another drug described below, also referred to as “therapeutic agent” or “adjunct agent”) may be administered simultaneously, independently, or separately within a time interval. In some situations, combination partners exhibit a synergistic effect. As used herein, terms such as “co-administration” or “combined administration” encompass the administration of selected combination partners to a single subject (e.g., a patient) that requires them, and are intended to include treatment regimens in which the drugs are not necessarily administered via the same route of administration or at the same time. As used herein, the term “combination of pharmaceuticals” means a product obtained by mixing or combining two or more active ingredients, which may include both fixed and unfixed combinations of active ingredients. The term “fixed combination” means that both the active ingredients, e.g., compounds and combination partners, are administered to the patient simultaneously in the form of a single entity or dose. The term "non-fixed combination" means that both the active ingredients, e.g., compounds and their combination partners, are administered to the patient simultaneously, concurrently, or sequentially as separate entities without any specific temporal constraints, thereby providing the patient with therapeutically effective concentrations of the two compounds. The latter also applies to cocktail therapies, e.g., the administration of three or more active ingredients.

[0062] As used herein, the term “pharmaceutically acceptable” means that, in addition to other formulations that are safe for use in animals, more specifically in humans and / or non-human mammals, it is approved by a federal or state regulatory authority or listed in the United States Pharmacopeia or any other commonly accepted pharmacopoeia.

[0063] As used herein, the term “pharmaceutically acceptable carrier” refers to excipients, diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or vehicles administered with a demethylene compound(s). Such carriers may be sterile liquids such as water and oil, and oils may be petroleum, animal, plant, or synthetic oils, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.; polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents. Antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for adjusting tonicity such as sodium chloride or dextrose can also be carriers. Methods for preparing compositions in combination with carriers are known to those skilled in the art. In some embodiments, the term “pharmaceutically acceptable carrier” is intended to include any solvent, dispersion medium, coating, isotonic agent, and absorption retarder, etc., suitable for pharmacopoeia administration. The use of such media and agents for pharmaceutically active substances is well known in the art. See, for example, Remington, The Science and Practice of Pharmacy, 20th ed., (Lippincott, Williams & Wilkins 2003). Such use in this composition is intended unless conventional media or agents are incompatible with the active compound.

[0064] As used herein, “therapeutically effective” means a quantity of a compound(s) that is pharmaceutically active enough to treat, improve, or alleviate in any way the symptoms associated with a disease or condition. When used in reference to a method, the method is sufficiently effective to treat, improve, or alleviate in any way the symptoms associated with a disease or condition. For example, with respect to age-related eye disease, an effective dose is sufficient to prevent or inhibit the onset of the disease, or, if the disease has already begun, to mitigate, improve, stabilize, recover from, or delay the progression of the disease, or to reduce the pathological outcomes of the disease. In either case, the effective dose may be administered as a single dose or in divided doses.

[0065] As used herein, the terms “to treat,” “to cure,” or “to treat” encompass at least improvement of the symptoms associated with a disease in a patient, in which case improvement is used in a broad sense to mean at least a reduction in the degree of a parameter such as the symptoms associated with the disease or condition being treated. Thus, “treatment” includes situations in which a disease, disorder, or pathological condition, or at least the symptoms associated therewith, are completely inhibited (e.g., prevented from occurring) or stopped (e.g., terminated) so that the patient no longer suffers from that condition, or at least the symptoms that characterize that condition.

[0066] As used herein, unless otherwise specified, the terms “prevent,” “prevent,” and “prevention” refer to the prevention of the onset, recurrence, or spread of a disease or disorder, or one or more of its symptoms. In certain embodiments, these terms refer to treatment or administration of a compound or dosage form provided herein, with or without one or more additional activators, to a subject at risk of the disease or disorder provided herein, particularly before the onset of symptoms. These terms encompass the inhibition or reduction of symptoms of a particular disease. In certain embodiments, a subject with a family history of the disease is a potential candidate for a preventive regimen. In certain embodiments, a subject with a history of recurrent symptoms is also a potential candidate for prevention. In this regard, the term “prevention” may be used interchangeably with the term “preventive treatment.”

[0067] As used herein, unless otherwise specified, the “prophylactic effective dose” of a compound is an amount sufficient to prevent a disease or disorder, or to prevent its recurrence. A prophylactic effective dose of a compound means the amount of a therapeutic agent, either alone or in combination with one or more other agents, that provides a prophylactic benefit in the prevention of a disease. The term “prophylactic effective dose” may include an amount that improves overall prevention or enhances the prophylactic efficacy of another prophylactic agent.

[0068] Where used herein, unless otherwise specified, the term “subject” is defined herein to include animals such as mammals, including but not limited to primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, and mice. In certain embodiments, the subject is human. The terms “subject” and “patient” are used herein interchangeably with respect to mammalian subjects such as humans. [Examples]

[0069] Experiment 1 - Production and characterization of SolMM The myocardial matrix (MM) formulation can be prepared based on a previously described protocol (Figure 1) [3]. In short, a fresh heart is taken from a pig (approximately 30-45 kg) and the LV myocardium is isolated. Major blood vessels and connective tissue are removed, and the remaining tissue is divided into 5 mm sections. 3 Cut into pieces smaller than 1 mm (Figure 1A). Decellularize the tissue in 1% (w / v) sodium dodecyl sulfate (SDS) for 4-5 days until the tissue is completely white, then rinse with water for another day to remove residual SDS (Figure 1B). Freeze-dry the material and grind it into a fine powder (Figure 1C), followed by partial enzymatic digestion for 48 hours. Then neutralize the material and buffer it to match in vivo conditions to obtain a MM capable of thermally induced gelation (Figure 1D).

[0070] Next, MM is centrifuged at 15,000 RCF and 4°C to separate the soluble and insoluble fractions (Figure 1E). The supernatant is isolated from the insoluble pellet and is referred to as the soluble MM fraction (SolMM). SolMM is then dialyzed and freeze-dried to adjust the salt concentration and ratio to maintain the physiological conditions of SolMM. SolMM is then resuspended at a high concentration (16 mg / mL), passed through a 0.22 μm filter, placed in a sterile container, freeze-dried, weighed, and stored at -80°C until needed. Approximately 30 minutes before injection, SolMM is resuspended in sterile water to the appropriate concentration. This suspension can then be subcutaneously injected into rats and gelled within 5 minutes (Figure 1F, 10 mg / mL, 500 μL). The consistency of the material can be assessed by polyacrylamide gel electrophoresis for protein distribution, PicoGreen assay for DNA content, dimethylmethylene blue assay for sulfated glycosaminoglycan (sGAG) content, and methylene blue assay for SDS content. Due to the digestion process that produces MM and the resulting SolMM, accurate data cannot be obtained from mass spectrometry. However, PAGE shows an overlapping distribution of proteins between MM and SolMM, except for high molecular weight proteins in SolMM (Figure 2, lane 4).

[0071] Experiment 2 - Blood compatibility between SolMM and human blood The interaction between SolMM and human blood samples (n=4) is evaluated using SolMM at different dilutions (1:1, 1:2, 1:10) to human whole blood or platelet-rich plasma. 1:1 represents the highest possible ratio between blood and SolMM, and 1:10 represents a physiologically appropriate dilution based on the volumetric flow rate of the coronary system and the intended infusion rate (1 mL / min). Blood compatibility is evaluated as previously described for MM [4]. Red blood cell agglutination is performed within 4 hours of sampling using a Myrenne agglutinator (Myrenne GmbH) after adjusting the hematocrit value to 45% in autologous plasma. Agglutination is evaluated after stasis (M0) or low shear rate (3 Hz; M1), and absorbance (800 nm) is measured for 5 seconds. Similarly, platelet agglutination is measured using platelet-rich plasma isolated with a Lumi agglutinator (Chrono-log). High concentrations of coagulation cascade agonists (adenosine diphosphate, epinephrine, and collagen) are added to platelet-rich plasma samples using the same dilution ratios as above (dilution 1:200 to 1:1000), and platelet aggregation is measured by absorbance (600 to 620 nm).

[0072] The results for 1:1 and 1:10 dilutions (material versus human blood) show that all values ​​are within the normal physiological range, suggesting that SolMM is blood-compatible (Table 1). [Table 1]

[0073] Experiment 3 - Distribution, retention, and effectiveness in a small animal ischemia-reperfusion model. Using a myocardial ischemia-reperfusion model of MI Sprague Dawley rats (225–250 g), the left coronary artery was occluded for 45 minutes and then reperfused. Within 5 minutes after reperfusion, the aorta was clamped for approximately 15 seconds to simulate intra-coronary injection, and 200 μl of SolMM was injected into the LV lumen at concentrations of 6, 10, or 14 mg / mL. This forced the material into the coronary artery and distributed it to the infarcted myocardium

[12] . Since the non-gelling material is removed from the heart within 1 hour, the heart (n=2 per concentration) was isolated 60 minutes after injection to determine whether the material was initially distributed and then retained in the heart [5]. SolMM was conjugated with Alexa Fluor® 568 N-hydroxysuccinimidyl ester (Invitrogen) to enable fluorescence detection and analysis. At 6, 12, and 24 hours, 2, 3, 4, and 5 days, and 1 week after injection (n=2-3 per time point), the retention of the material at optimal concentrations was examined to evaluate degradation. Physiological saline (n=3 per time point) was mixed with unbound Alexa Fluor® 568 to serve as a control. The heart was fresh-frozen in OCT Tissue-Tek compound and thinned axially into 16 equally spaced regions (approximately 300 μm apart), creating two sets of four slides per region, each section being 10 μm thick. One slide per region was used for H&E to confirm infarction, and one slide per region was used for SolMM fluorescence analysis.

[0074] Using concentrations of 6, 10, and 14 mg / mL (n=2 per group), the distribution of the infarct area increases with concentration, as indicated by the larger distribution of the pre-labeled gel and increased infarct area intensity (10 mg / mL shown in Figure 2) 1 hour after injection; however, based on the yield of SolMM generation, 10 mg / mL will be used for future experiments. Resuspending SolMM at 16 mg / mL and subsequently filtering yields a concentration of approximately 10 mg / mL, thus the yield is particularly limited based on the filtration step described in Experiment 1. Resuspend concentrations above 16 mg / mL typically do not pass through the filter.

[0075] Based on histological analysis over time, the material was observed in infarcted hearts for approximately 3 days after injection.

[0076] In a rat ischemia-reperfusion model, the left coronary artery was occluded for 35 minutes to simulate myocardial infarction. The heart was then reperfused, and a soluble matrix was injected through the coronary arteries using an aortic cross-clamp model. Rats were imaged using magnetic resonance imaging 24 hours and 5 weeks after injection. Left ventricular (LV) volume and ejection fraction are shown in Figure 5. 24 hours after injection, significantly preserved LV volume (end-systolic and end-diastolic) was observed in controls injected with saline. Ejection fraction showed an increasing trend compared to the saline controls. At 5 weeks, the LV volume in the matrix-injected rats was also significantly reduced compared to the saline-injected controls, demonstrating that matrix injection mitigates negative left ventricular remodeling.

[0077] Experiment 4: Injectable extracellular matrix using a balloon-tipped infusion catheter to repair the heart after myocardial infarction. Following myocardial infarction, extracellular matrix was injected through cardiac vessels (e.g., left anterior descending artery or left main artery) for targeted delivery using a balloon-assisted infusion catheter. Figure 4 shows the distribution and retention of soluble myocardial matrix (SolMM) one hour after intracoronary infusion in a porcine ischemia-reperfusion model using a balloon-assisted infusion catheter. Figure 4 left shows a macroscopic short-axis histological view of the infarcted porcine heart. The infarct is outlined in blue grayscale. Figure 4 right shows the infarcted myocardium with SolMM microgel displayed throughout the entire myocardium, indicated by a red grayscale channel.

[0078] Associated organs (brain, kidneys, liver, lungs, and spleen) were evaluated by blinded histopathologists, and no abnormal signs of ischemia or inflammation were observed one hour after matrix injection (Table 2). The absence of any observed soluble matrix gel in the associated organs suggests the ability of the injectable matrix to target ischemic tissue. [Table 2]

[0079] Experiment 5: The injectable matrix can be used as a scaffold to promote angiogenesis in ischemic or damaged tissue. Figure 6 shows the increase in arteriole density after matrix injection in a myocardial infarction model. Infarctions were imaged 5 weeks after injection in a rat ischemia-reperfusion model. Arterioles were identified by co-staining with α-smooth muscle actin and isolectin and manually tracked in ImageJ. Upregulation of the angiogenic pathway is shown in Figure 6.

[0080] Experiment 6: The injectable matrix can be used as a scaffold to reduce cellular apoptosis or necrosis in ischemic or damaged tissue. Figure 7 shows the reduction in cardiomyocyte apoptosis after matrix injection in a myocardial infarction model. Infarcts and the infarct boundary zone were stained with α-actinin for cardiomyocytes and with cleaved caspase 3 for apoptosis. Apoptotic cardiomyocytes were manually counted in ImageJ. The reduction in apoptosis may extend to other cell types, but is not limited to endothelial cells, immune cells, fibroblasts, neurons, and (cardiac) myocytes. The reduction in the apoptosis / necrosis pathway is shown in Figure 9. The reduction in apoptosis may be explained by increased ROS metabolism, as the upregulated reactive oxygen species (ROS) metabolic pathway is shown in Figure 8.

[0081] Figures 8 and 9 show differential gene expression suggestive of repair pathways for injectable extracellular matrix therapeutics. RNA was isolated from left ventricular free wall tissue 1 and 3 days after matrix injection and ischemia-reperfusion injury. On day 1, angiogenesis and reactive oxygen species metabolic pathways were upregulated. On day 3, a decrease in apoptosis / necrosis and a decrease in fibrous pathways were observed. Downregulation of LRG1 was associated with myocardial fibrosis, and a trend in the opposite direction was observed. Saline injection was used as a control.

[0082] Experiment 7: Matrix injection can treat endothelial cell damage / dysfunction. Soluble matrix can coat endothelial cells to reduce damage from reactive oxygen species, increase endothelial cell survival, and / or fill gaps in the leaky vascular system after ischemic injury. Following ischemic injury and matrix injection, soluble matrix was observed to coat the lumen of small vessels (capillaries / endothelial cells). Figure 10 shows the lumen of endothelial cells (green grayscale) coated with soluble matrix (red grayscale). Note that the matrix does not occlude the lumen. Additionally, Figure 11 shows that the soluble matrix overlaps with endothelial cells of large vessels, but does not occlude the lumen. The heart was imaged using a confocal microscope up to 24 hours post-injection to simulate myocardial infarction.

[0083] Experiment 8: The injectable matrix can be delivered co-delivered with drugs, growth factors, microRNAs, or other therapeutic agents. The soluble extracellular matrix composition has potential binding domains for growth factors, microRNAs, and other potential drugs or therapeutic agents. Since the injectable matrix can gel within the tissue after injection, it can be used for sustained release of therapeutic agents.

[0084] Figure 12 shows the retention of soluble ECM in infarct tissue 24 hours after matrix infusion in an ischemia-reperfusion model. From left to right, saline, VivoTag750-bound matrix, VivoTag750-bound trilysine, and VivoTag750-bound matrix were infused into the heart. 24 hours after infusion, the heart was harvested and imaged with Licor Odyssey. In contrast to hearts infused with saline and trilysine, the heart infused with matrix showed greater signal intensity. When trilysine containing VivoTag750 was used as a small peptide control, no perceptible retention was observed.

[0085] Figure 13 shows the nanofiber structure of the soluble matrix hydrogel. A 10 mg / ml pregel solution was subcutaneously injected into the backs of rats, and after the gel formed, it was collected for scanning electron microscopy imaging. The gel structure is reminiscent of the natural extracellular matrix.

[0086] Experiment 9: Dynamic light scattering analysis will demonstrate the differences between MM and SolMM. Figure 14 shows the dynamic light scattering data for the soluble matrix (SolMM) and complete matrix (MM) at a 1:50 dilution (1.0 mg / ml and 0.6 mg / ml, respectively), indicating that the soluble matrix particles are less than 100 nm in diameter, while the complete matrix has larger particles.

[0087] Figure 15 shows the dynamic light scattering data of the soluble matrix (SolMM) at 1:10 and 1:100 dilutions (1.0 mg / ml and 0.1 mg / ml, respectively), showing particles with a diameter of less than 100 nm.

[0088] Figure 16 shows the absorbance (left) and transmittance (right) of physiological saline, soluble matrix (SolMM), and complete matrix (MM).

[0089] Figure 17 shows the relative absorbance (left) and transmittance (right) of the soluble matrix (SolMM) and the complete matrix (MM).

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Claims

1. A method for preparing a soluble extracellular matrix (ECM) composition, a. Digestion of decellularized ECM material with acidic protease, b. Neutralizing the digested ECM material in a liquid to a pH of 7.0 to 8.0, c. The process involves treating the Pregel liquid ECM by centrifugation to produce soluble and insoluble fractions, wherein the soluble fraction includes the supernatant obtained from the centrifugation. d. A method for obtaining a soluble ECM composition containing soluble matrix particles, comprising separating at least a portion of the soluble fraction from the insoluble fraction, wherein the soluble matrix particles have a diameter of less than 100 nm and the soluble extracellular matrix is ​​soluble cardiomyocyte matrix (SolMM).

2. The method according to claim 1, further comprising dialysis of the soluble fraction.

3. The method according to claim 1, wherein the separation is performed using an exclusion filter with a size of 250 nm or less.

4. The method according to claim 1, wherein the soluble ECM composition is further freeze-dried and rehydrated.

5. A soluble ECM composition comprising decellularized, digested and neutralized tissue from which at least a portion of the solid ECM material has been removed, wherein the soluble ECM composition passes through a 250 nm size exclusion filter, the soluble ECM composition contains soluble matrix particles with a diameter of less than 100 nm, and the soluble ECM composition is a soluble cardiac matrix (SolMM).

6. The soluble ECM composition according to claim 5, wherein the composition is formulated for intravascular injection.

7. The soluble ECM composition according to claim 5, wherein the composition is liquid at room temperature and forms a gel in tissue after in vivo injection.

8. The soluble ECM composition according to claim 5, wherein the composition is liquid at room temperature and forms a coating that lines damaged blood vessels after in vivo injection or delivery.

9. The soluble ECM composition according to claim 5, wherein the composition is liquid at room temperature and fills the pores between endothelial cells after in vivo injection or delivery.

10. The soluble ECM composition according to claim 5, derived from human, animal, embryo, or fetal tissue.

11. The soluble ECM composition according to claim 5, for use in a method comprising administering an effective amount of an injectable solution of the soluble ECM composition to a subject in need to promote tissue repair.

12. The soluble ECM composition according to claim 11, wherein the injection solution is delivered intravenously or intravascularly via a catheter.

13. The soluble ECM composition according to claim 11, wherein when delivered in vivo, the soluble ECM composition forms a gel in the tissue.

14. The soluble ECM composition according to claim 11, wherein the soluble ECM composition is crosslinked with glutaraldehyde, formaldehyde, a bis-NHS molecule, or another crosslinking agent before administration.

15. The soluble ECM composition according to claim 11, wherein the soluble ECM composition is combined with cells, peptides, proteins, DNA, drugs, nanoparticles, nutrients, survival-promoting additives, proteoglycans, and / or glycosaminoglycans before administration.

16. The soluble ECM composition according to claim 11, wherein the soluble ECM composition is combined with and / or crosslinked with a synthetic polymer or a bio-derived material before administration.

17. The soluble ECM composition according to claim 11, wherein the soluble ECM composition induces endogenous intracellular proliferation, angiogenesis, and regeneration in the subject.

18. The soluble ECM composition according to claim 11, wherein the soluble ECM composition promotes cell survival and reduces inflammation in the subject.

19. A pharmaceutical composition for treating acute myocardial infarction, comprising an effective amount of a soluble ECM composition containing decellularized, digested and neutralized tissue from which at least a portion of a solid ECM material has been removed, for injection or infusion into a subject, wherein the soluble ECM composition comprises soluble matrix particles with a diameter of less than 100 nm, and the soluble ECM composition is a soluble myocardial matrix (SolMM).

20. The pharmaceutical composition according to claim 19, wherein the composition is intended for delivery into blood vessels.

21. The pharmaceutical composition according to claim 19, wherein the composition is intended for delivery using a balloon-equipped infusion catheter.

22. The pharmaceutical composition according to claim 19, wherein the composition transitions to a gel form within the tissue after delivery.

23. The pharmaceutical composition according to claim 19, wherein the composition is liquid at room temperature and forms a coating that lines infarcted blood vessels after delivery.

24. The pharmaceutical composition according to claim 19, wherein the composition is liquid at room temperature and fills the pores between infarcted endothelial cells after delivery.

25. The pharmaceutical composition according to claim 19, wherein the composition decomposes within 1 to 14 days after injection or infusion.

26. The pharmaceutical composition according to claim 19, wherein injection or infusion of the composition repairs damage to the myocardium sustained by the subject.

27. The pharmaceutical composition according to claim 19, wherein injection or infusion of the composition repairs damage caused by ischemia in the subject.

28. The pharmaceutical composition according to claim 19, wherein the effective amount is an amount that increases blood flow, increases the amount of viable tissue, or induces new angiogenesis in the area of ​​the injection or infusion of the target.

29. A pharmaceutical composition for treating endothelial cell damage and / or dysfunction by injection or infusion into a target of interest, comprising an effective amount of a soluble ECM composition containing decellularized, digested and neutralized tissue from which at least a portion of a solid ECM material has been removed, wherein the soluble ECM composition comprises soluble matrix particles less than 100 nm in diameter, and the soluble ECM composition is a soluble cardiac matrix (SolMM).

30. The pharmaceutical composition according to claim 29, wherein the effective amount promotes the survival, proliferation, or vasodilation of endothelial cells and / or reduces inflammation, apoptosis, damage from reactive oxygen species, or leakage of the vascular system.

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