Prevention of Progressive Heart Failure

Abstract

To provide methods for treating or preventing progressive heart failure.SOLUTION: A method for treating or preventing progressive heart failure in a myocardial infarction subject, comprises administering to the subject a population of mesenchymal lineage precursor or stem cells and / or progeny thereof and / or soluble factors derived therefrom, where the subject has a proximal left anterior descending (LAD) lesion.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to methods for preventing progressive heart failure in subjects having persistent left ventricular (LV) dysfunction. Such methods may be used to treat or prevent progressive heart failure in subjects having a proximal left anterior descending (LAD) lesion and persistent LV dysfunction.

Background Art

[0002] Myocardial infarction (MI) remains one of the leading causes of mortality and morbidity in developed countries. A recent update of US Medicare records evaluating data including 350,509 hospitalizations for acute MI in patients over 65 years old who survived and were discharged after the event was published (Schuster et al. (2004) Physiol Heart Circa Physiol., 287(2):525 - 32). Within the first year after the index event, 25.9% of MI patients died and 50.5% were readmitted. In the months after MI, the likelihood of death was 21 times higher and the likelihood of hospitalization was 12 times higher than in the general Medicare age population.

[0003] Patients with larger infarcts after MI and patients with more post - infarct LV dysfunction have a significantly increased risk of experiencing mid - to - long - term cardiac events and death. Specifically, subjects with an anterior wall infarct, larger infarcts, and more LV dysfunction during the post - infarct period have a significantly increased risk of experiencing mid - to - long - term cardiac events and death (Eitel et al. (2010) J Am Coll Cardiol., 55:2470 - 9).

[0004] The state where infarction due to proximal LAD occlusion continues to be a major risk factor for progressive LV dilation, remodeling, and symptomatic progressive heart failure. In the post-angioplasty stage, the 3-year mortality rate after MI remains higher for proximal LAD lesions (10% vs. 3% for distal LAD) (Elsman et al. (2006) Am J Cardiol., 97(8):1137-41), which is most likely due to the infarct size being 40% larger for this anatomical lesion (Elsman et al. (2006) Am J Cardiol., 97(8):1137-41). An infarct size exceeding 18.5% has been shown prospectively to result in a 30% incidence of major adverse cardiac events related to heart failure (HF-MACE, defined as hospitalization or death due to heart failure) over 2 years (Wu et al. (2008) Heart, 94:730-736), suggesting that the population of MI patients with proximal LAD lesions, low ejection fraction, and large infarcts is at the highest risk of subsequent HF-MACE.

[0005] Clearly, there is a need in the art to treat or prevent progressive heart failure. SUMMARY OF THE INVENTION

[0006] This disclosure is based on the unexpected identification of a population of myocardial infarction (MI) subjects in whom stem cell therapy is highly effective. A number of MI subjects with elevated troponin or CK-MB (exceeding 4 times the upper limit of normal (ULN)), regional cardiac wall abnormalities, and reduced overall left ventricular systolic function (45% or less and 20% or more), as determined by screening cardiac imaging performed within about 24 hours of MI, were administered stem cell therapy or placebo therapy immediately after MI.

[0007] The inventors found that at about 5 days after MI, overall left ventricular systolic function had recovered to normal levels in most of these subjects. The application of stem cell therapy did not provide a therapeutic improvement over placebo therapy in these subjects.

[0008] Surprisingly, the application of stem cell therapy provided a more significant therapeutic improvement in subjects with proximal left anterior descending (LAD) artery lesions than placebo treatment. These results indicate that stem cell therapy may be useful in treating or preventing progressive heart failure in a subset of MI subjects, particularly those with proximal LAD lesions.

[0009] Thus, in one example, the present disclosure provides a method of treating or preventing progressive heart failure in a subject having a myocardial infarction, the method comprising administering to the subject a population of mesenchymal progenitor cells or stem cells and / or their progeny and / or soluble factors derived therefrom, the subject having a proximal left anterior descending (LAD) artery lesion.

[0010] In another example, the method comprises: i) selecting a subject having a proximal left anterior descending (LAD) artery lesion; and ii) administering to the subject a population of mesenchymal stem cells or progenitor cells and / or their progeny and / or soluble factors derived therefrom.

[0011] The inventors also confirmed that subjects with proximal LAD lesions who responded well to stem cell therapy also had a persistent low ejection fraction approximately 5 days after MI. These results indicate that the methods of the present disclosure may also be useful in treating or preventing progressive heart failure in subjects with proximal LAD lesions and persistent left ventricular dysfunction.

[0012] Thus, in another example, the present disclosure provides a method of treating progressive heart failure in a subject having a myocardial infarction, the method comprising administering to the subject a population of mesenchymal progenitor cells or stem cells and / or their progeny and / or soluble factors derived therefrom, the subject having a proximal left anterior descending (LAD) artery lesion and having persistent left ventricular dysfunction.

[0013] In one example, the subject also has an elevated left ventricular end-systolic volume (LVESV) that exceeds 70 mL. In one example, the LVESV exceeds 80 mL, exceeds 90 mL, exceeds 100 mL, exceeds 110 mL, or exceeds 120 mL. In another example, the LVESV exceeds 80 mL / m 2 and exceeds 90 mL / m 2 and exceeds 100 mL / m 2 and exceeds 110 mL / m 2 and exceeds 120 mL / m 2 or exceeds 120 mL / m.

[0014] In one example, the subject has a left ventricular ejection fraction (LVEF) of less than about 55%. In another example, the subject has an LVEF of less than about 45%. In another example, the subject has an LVEF of less than about 40%. In one example, the LVEF is measured by cardiovascular magnetic resonance imaging (cMR).

[0015] The inventors have also confirmed that the timing of administration after MI may also be beneficial to the subject. Thus, in one example, mesenchymal progenitor cells or stem cells and / or their progeny and / or soluble factors derived therefrom are administered between about 1 and 7 days after myocardial infarction. In one example, mesenchymal progenitor cells or stem cells and / or their progeny and / or soluble factors derived therefrom are administered between about 2 and 7 days after myocardial infarction. In another example, mesenchymal progenitor cells or stem cells and / or their progeny and / or soluble factors derived therefrom are administered between about 3 and 5 days after myocardial infarction.

[0016] The inventors are further characterizing the population of subjects who may benefit from the methods of the present disclosure based on the levels of serum biomarkers compared to the upper limit of normal (ULM). In one example, the subject has creatine kinase-MB and / or troponin that exceeds about 2-fold the upper limit of normal. In another example, the subject has creatine kinase-MB and / or troponin and / or myoglobin that exceeds about 4-fold the upper limit of normal.

[0017] The inventors have further characterized a population of subjects who may benefit from the methods of the present disclosure based on infarct size. In some instances, the subject has an infarct size between about 10-25% of the left ventricle. In other instances, the subject has an infarct size greater than about 18.5% of the left ventricle. In some instances, the infarct size is measured by cMR.

[0018] In another example, the methods of the present disclosure include STRO-1 + The method comprises administering a population of mesenchymal precursor or stem cells enriched for cells and / or their progeny and / or soluble factors derived therefrom.

[0019] In another example, the methods of the present disclosure include STRO-1 bright The method comprises administering a population of mesenchymal precursor or stem cells enriched for cells and / or their progeny and / or soluble factors derived therefrom.

[0020] In some instances, the population of mesenchymal progenitor or stem cells express tissue-nonspecific alkaline phosphatase (TNAP), and / or the progeny cells and / or soluble factors are derived from mesenchymal progenitor or stem cells that express TNAP.

[0021] In some instances, the population of mesenchymal progenitor or stem cells is 6 express angiopoietin-1 (Ang1) in an amount of at least 0.1 μg per cell, and / or the progeny cells and / or soluble factors are 6 In one example, the population of mesenchymal progenitor or stem cells is derived from mesenchymal progenitor or stem cells that express Ang1 in an amount of at least 0.1 μg per cell. 6 In some instances, the population of mesenchymal progenitor or stem cells expresses an amount of Ang1 of at least 0.5 μg per cell. 6 In some instances, the population of mesenchymal progenitor or stem cells expresses an amount of Ang1 of at least 0.7 μg per cell. 6 Express Ang1 in amounts of at least 1 μg per cell.

[0022] In one example, a population of mesenchymal progenitor cells or stem cells expresses less than about 0.05 μg of vascular endothelial growth factor (VEGF) per cell, and / or progeny cells and / or soluble factors are derived from mesenchymal progenitor cells or stem cells that express less than about 0.05 μg of VEGF and / or progeny cells per cell. In one example, a population of mesenchymal progenitor cells or stem cells expresses less than about 0.03 μg of VEGF per cell. 6 In one example, a population of mesenchymal progenitor cells or stem cells expresses less than about 0.05 μg of vascular endothelial growth factor (VEGF) per cell, and / or progeny cells and / or soluble factors are derived from mesenchymal progenitor cells or stem cells that express less than about 0.05 μg of VEGF and / or progeny cells per cell. In one example, a population of mesenchymal progenitor cells or stem cells expresses less than about 0.03 μg of VEGF per cell. 6 In one example, a population of mesenchymal progenitor cells or stem cells expresses less than about 0.05 μg of vascular endothelial growth factor (VEGF) per cell, and / or progeny cells and / or soluble factors are derived from mesenchymal progenitor cells or stem cells that express less than about 0.05 μg of VEGF and / or progeny cells per cell. In one example, a population of mesenchymal progenitor cells or stem cells expresses less than about 0.03 μg of VEGF per cell. 6 In one example, a population of mesenchymal progenitor cells or stem cells expresses less than about 0.03 μg of VEGF per cell.

[0023] In one example, a population of mesenchymal progenitor cells or stem cells expresses Ang1:VEGF in a ratio of at least about 2:1, and / or progeny cells and / or soluble factors are derived from mesenchymal progenitor cells or stem cells that express Ang1:VEGF in a ratio of at least about 2:1. In one example, a population of mesenchymal progenitor cells or stem cells expresses Ang1:VEGF in a ratio of at least about 10:1. In one example, a population of mesenchymal progenitor cells or stem cells expresses Ang1:VEGF in a ratio of at least about 20:1. In one example, a population of mesenchymal progenitor cells or stem cells expresses Ang1:VEGF in a ratio of at least about 30:1.

[0024] In one example, a population of mesenchymal progenitor cells or stem cells and / or their progeny and / or soluble factors derived therefrom are administered systemically. In one example, a population of mesenchymal progenitor cells or stem cells and / or their progeny and / or soluble factors derived therefrom are administered intravenously, intramuscularly, or intranasally. For example, a population of mesenchymal progenitor cells or stem cells and / or their progeny and / or soluble factors derived therefrom can be administered intravenously.

[0025] In one example, a population of mesenchymal progenitor cells or stem cells administered multiple times and / or their progeny and / or soluble factors derived therefrom are administered.

[0026] In one example, the method of the present disclosure involves administering between 1×10 6 and 8×10 8 cells. In one example, the method of the present disclosure involves administering between 1.2×10 8 and 4×10 8 cells. In one example, the method of the present disclosure involves administering at least about 1.5×10 8 cells.

[0027] In one example, the population of cells and / or progeny cells is autologous or allogeneic, and / or the soluble factor is derived from autologous or allogeneic cells.

[0028] In one example, the population of cells and / or their progeny is expanded in culture prior to administration and / or prior to obtaining the soluble factor.

[0029] In one example, mesenchymal progenitor cells or stem cells and / or their progeny cells and / or soluble factors derived therefrom are administered in the form of a composition comprising mesenchymal progenitor cells or stem cells and / or their progeny cells and / or soluble factors derived therefrom and a carrier and / or excipient. For example, the composition may contain a cryopreservative. Accordingly, in one example, the present disclosure relates to a population of mesenchymal progenitor cells or stem cells and / or their progeny and / or soluble factors derived therefrom that are used for the treatment or prevention of progressive heart failure in subjects with myocardial infarction (MI), wherein the subject has a proximal left anterior descending (LAD) artery lesion. In another example, the present disclosure relates to the use of a population of mesenchymal progenitor cells or stem cells and / or their progeny and / or soluble factors derived therefrom in the manufacture of a medicament for treating or preventing progressive heart failure in a subject with myocardial infarction (MI), wherein the subject has a proximal left anterior descending (LAD) artery lesion. In these examples, the subject may have persistent left ventricular dysfunction. For example, the subject may have an LVEF of less than about 55%. In another example, the subject has an LVEF of less than about 45%. In another example, the subject has an LVEF of less than about 40%. In these examples, the mesenchymal progenitor cells or stem cells and / or their progeny and / or soluble factors derived therefrom can be administered between about 1 and 7 days after myocardial infarction. For example, the mesenchymal progenitor cells or stem cells and / or their progeny and / or soluble factors derived therefrom are administered between about 3 and 5 days after myocardial infarction. In these examples, the subject may have creatine kinase-MB and / or troponin that exceeds about 2 times the upper limit of normal. In another example, the subject has creatine kinase-MB and / or troponin and / or myoglobin that exceeds about 4 times the upper limit of normal. In these examples, the subject may have an infarct size between about 10% and 25% of the left ventricle. In another example, the subject may have an infarct size that exceeds about 18.5% of the left ventricle. In these examples, the LVEF or infarct size may be measured by cMR. Brief Description of the Drawings

[0030]

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[0031] General techniques and definitions Unless specifically defined otherwise, all technical and scientific terms used in this specification shall be interpreted to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in molecular genetics, molecular biology, cell culture, stem cell differentiation, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0032] Unless otherwise noted, the stem cells, cell culture, and surgical techniques utilized in this disclosure are standard procedures well known to those of skill in the art. Such techniques are described and explained throughout the literature in sources such as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0033] Throughout this specification, unless otherwise specifically noted or the context otherwise requires, references to a single step, composition of matter, group of steps, or group of compositions of matter are to be construed to include one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps, or groups of compositions of matter.

[0034] The present disclosure should not be limited in scope by the specific embodiments described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure as described herein.

[0035] Any example disclosed herein should be construed as applicable to any other example unless otherwise specifically stated.

[0036] The term "and / or", e.g., "X and / or Y", should be understood to mean either "X and Y" or "X or Y", and should be construed as providing explicit support for both meanings or either meaning.

[0037] As used herein, the term "about" refers to ±10% of a given value, more preferably plus or minus 5%, unless otherwise stated.

[0038] Throughout this specification, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0039] Mesenchymal progenitor cells As used herein, the term "mesenchymal progenitor or stem cell" refers to an undifferentiated pluripotent cell that has the ability to self-renew while maintaining the ability to differentiate into a number of cell types, either mesenchymal in origin, such as osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts, and tendon, or non-mesodermal in origin, such as hepatocytes, neurons, and epithelial cells. To avoid misunderstanding, "mesenchymal progenitor cells" refers to mesenchymal cells such as osteocytes, chondrocytes, and adipocytes, as well as cells that can differentiate into fibrous connective tissue.

[0040] The term "mesenchymal progenitor or stem cell" includes both parental cells and their undifferentiated progeny. The term also includes mesenchymal progenitor cells, multipotent stromal cells, mesenchymal stem cells (MSCs), perivascular mesenchymal progenitor cells, and their undifferentiated progeny.

[0041] Mesenchymal progenitor or stem cells can be autologous, allogeneic, syngeneic, or isogeneic. Autologous cells are isolated from the same individual into which they are to be re-transplanted. Allogeneic cells are isolated from a donor of the same species. Xenogeneic cells are isolated from a donor of a different species. Syngeneic or isogeneic cells are isolated from a genetically identical organism, such as twins, clones, or highly inbred laboratory animal models.

[0042] Mesenchymal progenitor or stem cells are primarily present in the bone marrow, but have also been shown to be present in a variety of host tissues including, for example, umbilical cord blood and cord, adult peripheral blood, adipose tissue, trabecular bone, and dental pulp.

[0043] In one example, mesenchymal progenitor or stem cells are STRO-1+ mesenchymal progenitor cells. As used herein, the phrase "STRO-1+ pluripotent cells" should be interpreted to mean STRO-1+ and / or TNAP+ progenitor cells that can form pluripotent cell colonies.

[0044] STRO-1+ pluripotent cells are cells found in bone marrow, blood, dental pulp cells, adipose tissue, skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestine, lung, lymph nodes, thymus, bone, ligaments, tendons, skeletal muscle, dermis, and periosteum, and can differentiate into germ lines such as mesoderm and / or endoderm and / or ectoderm. Thus, STRO-1+ pluripotent cells can differentiate into a number of cell types including, but not limited to, adipose tissue, bone tissue, cartilage tissue, elastic tissue, muscle tissue, and fibrous connective tissue. The specific lineage commitment and differentiation pathways into which these cells enter depend on various influences from mechanical effects as well as / or endogenous bioactive factors such as growth factors, cytokines, and / or local microenvironmental conditions established by the host tissue.

[0045] Mesenchymal lineage progenitor cells or stem cells can be isolated from host tissue and enriched by selection of STRO-1+ cells. For example, bone marrow aspirates from a subject may be further treated with an antibody against STRO-1 or TNAP to enable selection of mesenchymal lineage progenitor cells or stem cells. In one example, mesenchymal lineage progenitor cells or stem cells can be enriched by using the STRO-1 antibody described in (Simmons and Torok-Storb, 1991).

[0046] The terms "enriched", "enrichment", or their inflections are used herein to describe a population of cells in which the population of a single specific cell type or populations of multiple specific cell types is increased when compared to an untreated population of cells (e.g., cells in their natural environment). In one example, a population enriched for STRO-1+ cells contains at least about 0.1% or 0.5% or 1% or 2% or 5% or 10% or 15% or 20% or 25% or 30% or 50% or 75% STRO-1+ cells. In this regard, the term "population of cells enriched for STRO-1+ cells" provides explicit support for the term "population of cells containing X% STRO-1+ cells", where X% is a percentage as listed herein. STRO-1+ cells, in some embodiments, can form clonogenic colonies, e.g., CFU-F (fibroblasts), or a subset thereof (e.g., 50% or 60% or 70% or 70% or 90% or 95%) can have this activity.

[0047] In one example, a population of cells is enriched from a cell preparation containing STRO-1+ cells in a selectable format. In this regard, the term "selectable format" will be understood to mean that the cells enabling the selection of STRO-1+ cells express a marker (e.g., a cell surface marker). The marker can be STRO-1, but need not be. For example, cells expressing STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 (e.g., mesenchymal progenitor cells) also express STRO-1 (and can be STRO-1). bright Thus, the designation that a cell is STRO-1+ does not mean that the cell is selected by STRO-1 expression. In one example, cells are selected based on at least STRO-3 expression, e.g., they are STRO-3+ (TNAP+).

[0048] References to selection of a cell or population thereof do not necessarily require selection from a specific tissue source. As described herein, STRO-1+ cells can be selected, isolated, or enriched from a wide variety of sources. In some embodiments, it is contemplated that these terms provide support for selection from any tissue containing STRO-1+ cells (e.g., mesenchymal progenitor cells), or angiogenic tissue, or tissue containing pericyte cells (e.g., STRO-1+ pericytes), or any one or more of the tissues recited herein.

[0049] In one example, the mesenchymal lineage progenitor cells or stem cells used in this disclosure express one or more markers selected individually or collectively from the group consisting of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β), CD45+, CD146+, 3G +, or any combination thereof.

[0050] The use of the term "individually" means that, although this disclosure encompasses the recited markers or groups of markers separately, and although such individual markers or groups of markers may not be described separately herein, the appended claims may define such markers or groups of markers separately and divisibly from one another.

[0051] The use of the term "collectively" means that, although this disclosure encompasses any number or combination of the recited markers or groups of markers, and although such numbers or combinations of markers or groups of markers may not be specifically described herein, the appended claims may define such combinations or sub - combinations separately and divisibly from any other combination of markers or groups of markers.

[0052] In one example, STRO-1+ cells are STRO-1 bright (synonym STRO-1 bri ) Another example, STRO-1 briThe cells are STRO-1 dim or STRO-1 intermediate cells are preferentially enriched compared to. In another example, STRO-1 bri cells are further one or more of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β) and / or CD146+. For example, the cells are selected for one or more of the above markers and / or are shown to express one or more of the above markers. In this regard, cells shown to express a marker need not be specifically tested; rather, previously enriched or isolated cells can be tested, and subsequently used, isolated, or enriched cells can reasonably be assumed to express the same marker.

[0053] In one example, the mesenchymal progenitor cells are perivascular mesenchymal progenitor cells as defined in WO2004 / 85630, characterized by the presence of the perivascular marker 3G5.

[0054] Cells referred to as "positive" for a given marker may express that marker at either a low (lo or dim) or high (bright, bri) level, depending on the degree to which the marker is present on the cell surface, and the term relates to the intensity of the fluorescent or other marker used in the cell sorting process. The distinction between lo (or dim or dull) and bri will be understood in the context of the marker used for the particular cell population being sorted. Cells referred to as "negative" for a given marker are not necessarily completely absent from the cell. This term means that the marker is expressed at a relatively very low level by the cell and that, when detected and labeled detectably or not detected above the background level, e.g., the level detected using an isotype control antibody, it gives a very low signal.

[0055] As used herein, the term "bright" or "bri" refers to a marker on the cell surface that produces a relatively large signal when detectably labeled. Without wishing to be bound by theory, it is proposed that "bright" cells express more of the target marker protein (e.g., an antibody recognized by STRO-1) than other cells in the sample. For example, STRO-1 bri cells are brighter when labeled with a FITC-conjugated STRO-1 antibody as measured by fluorescence-activated cell sorting (FACS) analysis than cells that are not (STRO-1 dull / dim ). In one example, "bright" cells constitute at least about 0.1% of the most brightly labeled bone marrow mononuclear cells contained in the starting sample. In other embodiments, "bright" cells constitute at least about 0.5%, at least about 1%, at least about 1.5%, or at least about 2% of the most brightly labeled bone marrow mononuclear cells contained in the starting sample. In one example, STRO-1 bright cells have an expression that is 2 logs (in magnitude) higher in STRO-1 surface expression compared to "background", i.e., cells that are STRO-1-negative. In comparison, STRO-1 dim and / or STRO-1 intermediate cells have an expression that is less than 2 logs (in magnitude), usually about 1 log or less than "background", higher in STRO-1 surface expression.

[0056] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue-nonspecific alkaline phosphatase. For example, the term encompasses the liver isoform (LAP), the bone isoform (BAP), and the kidney isoform (KAP). In one example, TNAP is BAP. In one example, TNAP as used herein refers to a molecule that can bind to an STRO-3 antibody produced by a hybridoma cell line deposited with the ATCC on December 19, 2005, under the terms of the Budapest Treaty with the deposit accession number PTA-7282.

[0057] Furthermore, in one example, STRO-1+ cells can give rise to clonogenic CFU-F.

[0058] In one example, a significant proportion of STRO-1+ pluripotent cells are capable of differentiating into at least two different germ cell lineages. Non-limiting examples of lineages into which pluripotent cells can be committed to differentiate include bone progenitor cells; stem cell progenitors with multipotency to bile duct epithelial cells and stem cells; neural restricted cells that can give rise to glial cell precursors that progress to oligodendrocytes and astrocytes; neural precursors that progress to neurons; precursors to myocardium and cardiomyocytes, glucose-responsive insulin-secreting pancreatic β cell lines. Other lineages include odontoblasts, dentin-producing cells, and chondrocytes, as well as the following progenitor cells: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal tubular epithelial cells, smooth muscle cells and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendon cells, ligament cells, cartilage tissue cells, adipocytes, fibroblasts, bone marrow stromal cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, pericytes, vascular cells, glial cells, nerve cells, astrocyte cells, as well as oligodendrocyte cells, but are not limited thereto.

[0059] In aspects of the present disclosure, the mesenchymal lineage progenitor cells or stem cells described herein are MSCs. The MSCs may be a homogeneous composition or a mixed cell population enriched in MSCs. The homogeneous MSC cell composition may be obtained by culturing adherent bone marrow cells or periosteal cells, and the MSCs may be identified by specific cell surface markers identified by unique monoclonal antibodies. Methods for obtaining a cell population enriched in MSCs are described, for example, in U.S. Patent No. 5,486,359. Alternative sources for MSCs include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium.

[0060] In another example, mesenchymal progenitor cells or stem cells are CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSCs (e.g., remestemcel-L).

[0061] Isolated or enriched mesenchymal progenitor cells or stem cells can be expanded in vitro by culture. Isolated or enriched mesenchymal progenitor cells or stem cells can be cryopreserved, thawed, and subsequently expanded in vitro by culture.

[0062] In one example, isolated or enriched mesenchymal progenitor cells or stem cells are seeded at 50,000 viable cells / cm in a medium (serum-free or serum-supplemented), e.g., α-minimum essential medium (αMEM) supplemented with 5% fetal bovine serum (FBS) and glutamine. 2 into a culture vessel and allowed to adhere overnight at 37 °C with 20% O2. Subsequently, the medium is exchanged and / or changed as needed, and the cells are cultured at 37 °C with 5% O2 for an additional 68 - 72 hours.

[0063] As will be understood by those skilled in the art, cultured mesenchymal progenitor cells or stem cells have a different phenotype from in vivo cells. For example, in one embodiment, they express one or more of the following markers: CD44, NG2, DC146, and CD140b. Cultured mesenchymal progenitor cells or stem cells are also biologically different from in vivo cells and have a high proliferation rate compared to mainly non-cycling (quiescent) cells in vivo.

[0064] Mesenchymal progenitor cells or stem cells may also be cryopreserved before administration to a subject.

[0065] Expression of Ang1 and / or VEGF The mesenchymal progenitor cells or stem cells of the present disclosure can be genetically modified or unmodified and express high levels of Ang1. For example, mesenchymal progenitor cells or stem cells are 10 6In other examples, the cells can express an amount of Ang1 of at least 0.1 μg per cell. 6 At least 0.2 μg per cell, 10 6 0.3 μg per cell, 10 6 0.4 μg per cell, 10 6 0.5 μg per cell, 10 6 0.6 μg per cell, 10 6 0.7 μg per cell, 10 6 0.8 μg per cell, 10 6 0.9 μg per cell, 10 6 1 μg per cell, 10 6 1.1 μg per cell, 10 6 1.2 μg per cell, 10 6 1.3 μg per cell, 10 6 1.4 μg per cell, 10 6 Ang1 may be expressed in amounts of 1.5 μg per cell.

[0066] In another example, a population of mesenchymal progenitor or stem cells is 6 In other examples, the cells express VEGF in an amount of less than about 0.05 μg per cell. 6 Approximately 0.05 μg per cell, 10 6 0.04 μg per cell, 10 6 0.03 μg per cell, 10 6 0.02 μg per cell, 10 6 0.01 μg per cell, 10 6 0.009 μg per cell, 10 6 0.008 μg per cell, 10 6 0.007 μg per cell, 10 6 0.006 μg per cell, 10 6 0.005 μg per cell, 10 6 0.004 μg per cell, 10 6 0.003 μg per cell, 10 6 0.002 μg per cell, 10 6 They express less than 0.001 μg of VEGF per cell.

[0067] In another example, mesenchymal progenitor cells or stem cells express Ang1:VEGF at a ratio of at least about 2:1. In other examples, the cells express Ang1:VEGF at ratios of at least about 10:1, 15:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1.

[0068] In one example, mesenchymal progenitor cells or stem cells are genetically unmodified and express levels of Ang-1 or VEGF as described above, or the above Ang-1:VEGF ratio. As used herein, the term "genetically unmodified" refers to cells that have not been modified by nucleic acid transfection. To avoid misunderstanding, in the context of the present disclosure, mesenchymal progenitor cells or stem cells transfected with a nucleic acid encoding Ang1 will be interpreted as being genetically modified.

[0069] The amount of Ang1 and / or VEGF expressed during culture or present in cells in a composition of mesenchymal progenitor cells or stem cells may be measured by various methods known to those of skill in the art. Such methods include, but are not limited to, Western blot, enzyme-linked immunosorbent assay (ELISA), fluorescence-linked immunosorbent assay (FLISA), competitive assay, radioimmunoassay, lateral flow immunoassay, flow-through immunoassay, electrochemiluminescence assay, assays based on nephelometry, assays based on turbidimetry, fluorescence-activated cell sorting (FACS)-based assays for detection of Ang-1 or VEGF in the medium used to culture mesenchymal progenitor cells or stem cells, and surface plasmon resonance (SPR or Biacore).

[0070] In one example, the levels of Ang1 and / or VEGF that are expressed by culture or present in a composition of mesenchymal progenitor cells or stem cells are measured by an ELISA assay. For example, cell lysates from cultures of mesenchymal progenitor cells or stem cells are added to the wells of an ELISA plate. The wells may be coated with either a primary antibody, monoclonal or polyclonal antibody(ies) against Ang1 or VEGF. The wells are washed and then contacted with either a secondary antibody, monoclonal or polyclonal antibody(ies) against the primary antibody. The secondary antibody is conjugated to a suitable enzyme such as, for example, horseradish peroxidase. After an appropriate incubation period, the wells are washed and then contacted with a suitable substrate for the enzyme conjugated to the secondary antibody, such as one or more chromogens. Chromogens that may be used include, but are not limited to, hydrogen peroxide and tetramethylbenzidine. After the substrate(s) are added, the wells are incubated for an appropriate period. After incubation is complete, a "stop" solution is added to the wells to stop the reaction of the enzyme with the substrate(s). Next, the optical density (OD) of the sample is measured. The optical density of the sample to determine the amount of Ang1 or VEGF expressed by the culture of mesenchymal progenitor cells or stem cells being tested correlates with the optical density of samples containing known amounts of Ang1 or VEGF. Methods for determining the Ang1:VEGF expression ratio will also be apparent to those skilled in the art. For example, after quantifying the levels of Ang1 and VEGF, the ratio based on the quantified levels of Ang1 and VEGF can be expressed as (level of Ang1 / level of VEGF) = Ang1:VEGF ratio.

[0071] Method for treating progressive heart failure Heart failure occurs when the heart is unable to pump sufficiently to maintain a blood flow rate that meets the body's needs. One cause of heart failure is systolic dysfunction after myocardial infarction (MI). MI occurs when blood flow to a part of the heart is inadequate. The lack of blood supply results in local myocardial necrosis, termed an infarct or infarction. The infarcted heart is unable to pump sufficiently to maintain the volume of blood required by the body, leading to multiple pathophysiological responses and ultimately heart failure. After MI, a series of compensatory mechanisms are initiated that help buffer the decline in cardiac output and maintain sufficient blood pressure to perfuse vital organs. As a result, patients with heart failure may not progress for a long time. However, the compensatory mechanisms eventually fail to adequately compensate for the damaged heart, leading to a progressive decline in cardiac output, termed "progressive heart failure". In the context of the present disclosure, the terms, chronic heart failure, congestive heart failure, systolic heart failure, and advanced heart failure can be used interchangeably with "progressive heart failure".

[0072] The methods of the present disclosure relate to treating the progressive decline in cardiac output characteristic of progressive heart failure. Accordingly, in the context of the present disclosure, "treating" and "treatment" refer to both therapeutic treatment and prophylactic or preventive measures.

[0073] In one example, treatment reduces the likelihood or risk of a heart failure-related adverse cardiac event (HF-MACE), defined as cardiac-related death or resuscitated cardiac death, or a non-fatal decompensated heart failure event. In one example, the likelihood or risk of HF-MACE is reduced over at least 6 months, at least 12 months, at least 24 months, at least 36 months. In one example, treatment reduces the likelihood or risk of all-cause mortality.

[0074] Myocardial infarction subject The term "myocardial infarction (MI) subject" is used to define a subject having myocardial infarction. The methods of the present disclosure can be used to treat progressive heart failure in a specific population of MI subjects. Subjects in need of treatment include subjects already having progressive heart failure and subjects in whom progressive heart failure should be prevented, delayed, or halted.

[0075] MI subjects treated by the methods of the present disclosure have a proximal left anterior descending (LAD) artery lesion. As will be understood by those skilled in the art, the LAD artery runs in the anterior interventricular groove that separates the right ventricle and the left ventricle in the front of the heart. The diagonal (Dx) branch exits from the LAD and runs across the anterior wall to the outer surface or lateral portion. Thus, the Dx supplies blood to the anterolateral portion of the left ventricle. A subject may have one or several Dx branches. The first Dx branch functions as the boundary between the proximal and middle portions of the LAD. Thus, the portion of the artery before the origin of the Dx is known as the "proximal LAD", while the segment is adjacent to the first major side of the branch. The distal segment of the LAD is the terminal third of the artery.

[0076] In the context of the present disclosure, the term "artery lesion" encompasses an occlusive lesion that occludes the LAD of the heart or an artery lesion that previously occluded the LAD that has been treated by percutaneous coronary intervention (PCI), also known as angioplasty, for example.

[0077] In one example, the subject treated by the method of the present disclosure received PCI within about 1 hour from the ischemic symptoms. In other examples, the subject received PCI within about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours from the ischemic symptoms. In one example, the subject received PCI within about 12 hours from the ischemic symptoms. In other examples, the subject received PCI within about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or more hours. A subject treated with thrombolytic therapy having recurrent chest pain and / or ECG changes may not be transferred to PCI until at least 24 hours after the onset of ischemic symptoms. Thus, in other examples, the subject received PCI within about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 35 hours, about 40 hours, about 48 hours from the ischemic symptoms.

[0078] MI subjects may have an increased left ventricular end-systolic volume (LVESV). In one example, an MI subject treated using the method of the present disclosure has an elevated LVESV greater than 70 mL. In one example, the subject has an elevated LVESV greater than 80 mL, greater than 90 mL, greater than 100 mL, greater than 110 mL, or greater than 120 mL. In another example, the subject has an elevated LVESV greater than 80 mL / m 2 greater than 90 mL / m 2 greater than 100 mL / m 2 greater than 110 mL / m 2 greater than 120 mL / m 2 or greater.

[0079] MI can cause persistent left ventricular dysfunction. Thus, in another example, an MI subject has a proximal LAD artery lesion and persistent left ventricular dysfunction. Left ventricular dysfunction is characterized by a decrease in myocardial contractility. When myocardial contractility within the left ventricle decreases, a reduction in left ventricular ejection fraction (LVEF) occurs. Thus, LVEF provides one means of determining left ventricular dysfunction.

[0080] LVEF and LVESV can be measured by a number of methods known in the art, such as echocardiogram, single photon emission computed tomography (SPECT), or cardiac magnetic resonance imaging (cMRI).

[0081] In one example, subjects with an LVEF of less than about 60% have left ventricular dysfunction. In other examples, subjects with an LVEF of less than about 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46% have left ventricular dysfunction. In another example, subjects with an LVEF of less than about 45% have left ventricular dysfunction. In other examples, subjects with an LVEF of less than about 44%, 43%, 42%, 41% have left ventricular dysfunction. In another example, subjects with an LVEF of less than about 40% have left ventricular dysfunction. In other examples, subjects with an LVEF of less than about 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30% have left ventricular dysfunction.

[0082] In the context of the present disclosure, the term "persistent left ventricular dysfunction" is used to define left ventricular dysfunction that persists over a period of time or a series of measurements. For example, "persistent left ventricular dysfunction" can include left ventricular dysfunction that persists for about 1 to about 14 days or longer after MI. For example, persistent left ventricular dysfunction can include left ventricular dysfunction that persists for about 1 to about 10 days, about 1 to about 9 days, about 2 to about 8 days, about 2 to about 7 days after MI. In another example, "persistent left ventricular dysfunction" can include left ventricular dysfunction that persists over about 1 to 10 or more measurements.

[0083] The size or amount of myocardial necrosis after MI is clinically referred to as the infarct size. The methods of the present disclosure relate to the treatment of MI subjects having a large infarct size. For example, a subject treated using the methods of the present disclosure can have an infarct size exceeding about 10-35% of the left ventricle. In other examples, the subject can have an infarct size exceeding about 11-34%, about 12-33%, about 13-32%, about 14-31%, about 15-30%, about 16-29%, about 17-28% of the left ventricle. In another example, the subject can have an infarct size exceeding about 18.5% of the left ventricle. In other examples, the subject can have an infarct size exceeding about 19-27%, about 20-26%, about 21-25%, about 22-24%, about 23% of the left ventricle.

[0084] The infarct size can be measured by a number of methods known in the art. Examples of such methods include the use of serum markers such as creatine kinase (CK), CK-MB, troponin I, and brain natriuretic peptide troponin.

[0085] In one example, a subject treated using the methods of the present disclosure has a troponin level of at least about 2 times the upper limit of normal (ULM). In another example, the subject has a troponin level of at least about 3 times, about 4 times, about 5 times, about 6 times the ULM.

[0086] In one example, a subject treated using the methods of the present disclosure has a creatine kinase-MB level of at least about 2 times the ULM. In another example, the subject has a creatine kinase-MB level of at least about 3 times, about 4 times, about 5 times, about 6 times the ULM.

[0087] Other examples of measuring infarct size include Sestamibi single photon emission computed tomography (SPECT), myocardial blood flow imaging, and magnetic resonance imaging. In one example, the infarct size is measured using cMRI. Several cMRI techniques can be used for the diagnosis of infarct size. One of the most accurate and well-validated techniques is delayed enhancement cardiac magnetic resonance imaging (DE-CMR). Thus, in one example, cMRI includes DE-CMR.

[0088] When appropriate settings for DE-CMR are used, normal myocardium appears black or as nothing, while areas that cannot survive appear bright or highly enhanced. Thus, in one example, infarct size can be measured by visual assessment of the bright, highly enhanced areas. Other examples of determining infarct size are known in the art (Sievers et al. (2007), Circulation, 115, 236-244; Kim et al. (2000), N Engl J Med, 343, 1445-1453). Briefly, hyperenhancement is scored in a 17-segment model using a 5-point scale for each segment (0 = no hyperenhancement, 1 = 1% - 25%, 2 = 26% - 50%, 3 = 51% - 75%, 4 = 76% - 100%). Dark areas completely enclosed within the highly enhanced myocardium are interpreted as areas of microvascular injury (no-reflow) and are included as part of the infarct. Infarct size as a percentage of LV myocardium is calculated by summing the segment scores, weighting each at the midpoint of the hyperenhancement range (i.e., 1 = 13%, 2 = 38%, 3 = 63%, 4 = 88%), and dividing by 17. In another example, infarct size can be quantified by measuring the area of the highly enhanced area on a stack of short-axis images.

[0089] In one example, infarct size is measured between about 1 and 40 days after MI. In other examples, infarct size is measured between about 1 and 40 days, about 2 and 35 days, about 3 and 30 days, about 4 and 25 days, about 5 and 20 days, about 6 and 15 days after MI. For example, infarct size can be measured about 30 days after MI.

[0090] In the context of the present disclosure, "infarct size" refers to left ventricular infarct size. In other words, left ventricular infarct size refers to the amount of the left ventricle that is infarcted.

[0091] The methods of the present disclosure can be used to treat progressive heart failure in various stages or classifications of heart failure with MI target. For example, the subject can have heart failure of stage A, B, C, or D. In one example, the subject has heart failure of stage B or C. In these examples, the staging of heart failure is based on the staging criteria of the American College of Cardiology (ACC) and the American Heart Association (AHA).

[0092] In another example, the subject can have heart failure of class I, II, III, or IV. In one example, the subject has heart failure of class II or III. In these examples, the heart failure classification is based on the classification scale of the New York Heart Association (NYHA).

[0093] Cell composition In practicing the methods of the present disclosure, mesenchymal progenitor cells or stem cells can be administered in the form of a composition. In one example, such a composition contains a pharmaceutically acceptable carrier and / or excipient.

[0094] The terms "carrier" and "excipient" refer to a composition of substances conventionally used in the art to facilitate storage, administration, and / or bioactivity of the active compound (see, e.g., Remington’s Pharmaceutical Sciences, 16th Ed., Mac Publishing Company (1980)). The carrier may also reduce any undesirable side effects of the active compound. Suitable carriers are, for example, stable and, for example, cannot react with other components in the carrier. In one example, the carrier does not cause local or systemic adverse reactions in the recipient at the dosage and concentration used for treatment.

[0095] Suitable carriers for the present disclosure include those conventionally used, for example, water, physiological saline, aqueous dextrose, lactose, Ringer's solution, buffer solutions, hyaluronic acid, and glycols are exemplary liquid carriers, especially in the case of solutions (isotonic cases). Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, and the like.

[0096] In another example, the carrier is, for example, a medium composition in which cells grow or are disrupted. Such a medium composition does not cause any harmful effects on the subject to which it is administered.

[0097] Exemplary monomers and excipients do not adversely affect the survival of cells and / or the ability of cells to reduce, prevent, or delay progressive heart failure.

[0098] In one example, the carrier or excipient provides soluble factors with buffering activity and / or a biologically active suitable pH for maintaining cells, whereby biological activity is exerted. For example, the carrier or excipient is phosphate buffered saline (PBS). PBS represents an attractive carrier or excipient. In this case, the composition of the present disclosure may be manufactured as a liquid for direct application, for example, by injection, into the bloodstream or into a tissue or an area surrounding or adjacent to the tissue because it minimally interacts with cells and factors and enables rapid release of cells and factors.

[0099] Those stem cells and / or progeny cells can also be incorporated into or embedded in a scaffold that degrades into a product that is compatible with the recipient and not harmful to the recipient. These scaffolds provide support and protection to the cells to be transplanted into the recipient subject. Examples of such scaffolds are natural and / or synthetic biodegradable scaffolds.

[0100] A variety of different scaffolds may be successfully used in the practice of the present disclosure. Exemplary scaffolds include, but are not limited to, biodegradable scaffolds. Natural biodegradable scaffolds include collagen, fibronectin, and laminin scaffolds. Synthetic materials suitable for cell transplantation scaffolds should be able to support extensive cell growth and cell function. Such scaffolds may also be resorbable. Suitable scaffolds include, for example, polyglycolic acid scaffolds (as described by, e.g., Vacanti, et al. J. Ped. Surg. 23:3-9 1988; Cima, et al. Biotechnol. Bioeng. 38:145 1991; Vacanti, et al. Plast. Reconstr. Surg. 88:753-9 1991); or synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acid.

[0101] In another example, the cells may be administered in a gel scaffold (e.g., Gelfoam from Upjohn Company).

[0102] The cell compositions described herein may be administered alone or as a mixture with other cells. Different types of cells may be mixed with the compositions of the present disclosure immediately or just prior to administration, or they may be co-cultured together for a period of time prior to administration.

[0103] In one example, the composition comprises an effective amount or a therapeutically or prophylactically effective amount of cells. For example, the composition may contain from about 1×10 5 cells to about 1×10 9 cells or from about 1.25×10 3 cells to about 1.25×10 7 cells. The exact amount of cells administered depends on various factors including the age, weight, and sex of the subject, as well as the degree and severity of the disorder being treated.

[0104] Exemplary dosages are at least about 1.2×10 8 to about 8×10 10Cells, for example, about 1.3×10 8 ~ about 8×10 9 cells, about 1.4×10 8 ~ about 8×10 8 cells, about 1.5×10 8 ~ about 7.2×10 8 cells, about 1.6×10 8 ~ about 6.4×10 8 cells, about 1.7×10 8 ~ about 5.6×10 8 cells, about 1.8×10 8 ~ about 4.8×10 8 cells, about 1.9×10 8 ~ about 4.0×10 8 cells, about 2.0×10 8 ~ about 3.2×10 8 cells, about 2.1×10 8 ~ about 2.4×10 8 includes between cells. For example, the dosage can include at least about 1.5×10 8 cells. For example, the dosage can include at least about 2.0×10 8 cells.

[0105] In other words, an exemplary dosage (for an 80 kg subject) includes at least about 1.5×10 6 cells per kg. In one example, the dosage can include at least about 2.5×10 6 cells per kg. In other examples, the dosage is about 1.5×10 6 ~ about 1×10 9 cells per kg, about 1.6×10 6 ~ about 1×10 8 cells per kg, about 1.8×10 6 ~ about 1×10 7 cells per kg, about 1.9×10 6 ~ about 9×10 6 cells per kg, about 2.0×10 6 ~ about 8×10 6 cells per kg, about 2.1×10 6 ~ about 7×10 6 cells per kg, about 2.3×10 6 ~ about 6×10 6Cells, about 2.4×10 per kg 6 ~ about 5×10 6 Cells, about 2.5×10 per kg 6 ~ about 4×10 6 Cells, about 2.6×10 per kg 6 ~ about 3×10 6 It can include between cells.

[0106] In one example, mesenchymal progenitor cells or stem cells account for at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% of the cell population of the composition.

[0107] The compositions of the present disclosure may be cryopreserved. Cryopreservation of mesenchymal progenitor cells or stem cells can be carried out using slow cooling methods or "rapid" freezing protocols known in the art. Preferably, the cryopreservation method maintains a similar phenotype, cell surface markers, and growth rate of the cryopreserved cells compared to the non-frozen cells.

[0108] The cryopreserved composition may contain a cryopreservation solution. The pH of the cryopreservation solution is usually 6.5 - 8, preferably 7.4.

[0109] The cryopreservation solution may contain, for example, a sterile non-pyrogenic isotonic solution such as PlasmaLyte A™. 100 mL of PlasmaLyte A™ contains 526 mg of sodium chloride, USP (NaCl); 502 mg of sodium gluconate (C6H 11It contains 368 mg of sodium acetate trihydrate, USP (C2H3NaO2·3H2O); 37 mg of potassium chloride, USP (KCl); and 30 mg of magnesium chloride, USP (MgCl2·6H2O). It does not contain antibacterial agents. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 - 8.0).

[0110] The cryopreservation solution may contain Profreeze™. The cryopreservation solution may additionally or alternatively contain a medium, such as αMEM.

[0111] To facilitate freezing, a cryoprotectant, such as dimethyl sulfoxide (DMSO), is usually added to the cryopreservation solution. Ideally, the cryoprotectant should be non-toxic to cells and the subject, non-antigenic, chemically inert, provide a high survival rate after thawing, and allow transplantation without washing. However, the most commonly used cryoprotectant, DMSO, shows some cytotoxicity. Hydroxyethyl starch (HES) may be used as a substitute or in combination with DMSO to reduce the cytotoxicity of the cryopreservation solution.

[0112] The cryopreservation solution may contain one or more of DMSO, hydroxyethyl starch, human serum components, and other protein fillers. In one example, the cryopreserved solution contains about 5% human serum albumin (HSA) and about 10% DMSO. The cryopreservation solution may further contain one or more of methylcellulose, pyrrolidone (PVP), and trehalose.

[0113] In one embodiment, the cells are suspended in 42.5% Profreeze™ / 50% αMEM / 7.5% DMSO and cooled in a freezer at a controlled rate.

[0114] The cryopreserved composition may be thawed and administered directly to the subject or added to another solution containing, for example, HA. Alternatively, the cryopreserved composition may be thawed, and the mesenchymal progenitor cells or stem cells may be resuspended in an alternative carrier prior to administration.

[0115] In one example, the cell compositions described herein may be administered between about 1 and about 10 days after MI. In other examples, the cell compositions described herein may be administered between about 1 and 9 days after MI, between about 1 and 8 days after MI, between about 2 and 7 days after MI, between about 2 and 6 days after MI, between about 3 and 5 days after MI. For example, the cell compositions described herein may be administered about 5 days after MI.

[0116] In one example, the cell compositions described herein may be administered between about 1 and about 10 days after PCI. In other examples, the cell compositions described herein may be administered between about 1 and 9 days after PCI, between about 1 and 8 days after PCI, between about 2 and 7 days after PCI, between about 2 and 6 days after PCI, between about 3 and 5 days after PCI. For example, the cell compositions described herein may be administered about 5 days after PCI.

[0117] In one example, the cell compositions described herein may be administered as a single dose. In another example, the cell composition is administered over multiple doses. For example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 doses.

[0118] In one example, the mesenchymal progenitor cells or stem cells can be expanded in culture prior to administration. Various methods of mesenchymal progenitor cell or stem cell culture are known in the art. In one example, the mesenchymal progenitor cells or cells are expanded in serum-free medium prior to administration.

[0119] Mesenchymal progenitor cells or stem cells may be systemically administered, for example, by intravenous administration, intra-arterial administration, or intraperitoneal administration. Mesenchymal progenitor cells or stem cells may also be administered by nasal administration, intramuscular administration, or intracardiac administration. In one example, mesenchymal progenitor cells or stem cells are administered directly to the myocardium. For example, mesenchymal progenitor cells or stem cells can be directly administered to the myocardium of the left ventricle. In one example, mesenchymal progenitor cells or stem cells are administered by an endocardial myocardial catheter such as the J&J Myostar (trademark) injection catheter.

[0120] In one example, mesenchymal progenitor cells or stem cells are administered to viable myocardium. In one example, mesenchymal progenitor cells or stem cells are administered to hibernating myocardium. One of ordinary skill in the art will be able to identify viable and / or hibernating myocardium using methods known in the art. For example, a mapping catheter system such as the NOGASTAR (trademark) mapping catheter system can be used to identify viable and / or hibernating myocardium.

[0121] In another example, mesenchymal progenitor cells or stem cells are administered by intracoronary injection. For example, mesenchymal progenitor cells or stem cells can be administered to the left anterior descending (LAD) artery. In one example, mesenchymal progenitor cells or stem cells are administered to the LAD artery immediately after PCI for LAD revascularization.

[0122] In one example, the cells are contained within a chamber that does not allow the cells to exit into the circulation of the subject, but allows factors secreted by the cells to enter the circulation. Thus, the soluble factors may be administered to the subject by allowing the cells to secrete the factors into the circulation of the subject. Such a chamber may be implanted equally at the site of the subject to increase the local level of the soluble factor, for example, implanted in or near the heart.

[0123] It will be understood by those skilled in the art that numerous modifications and / or variations can be made to the present invention without departing from the spirit or scope of the invention as broadly described. Accordingly, the embodiments are to be construed in all respects as illustrative and not restrictive.

[0124] All publications considered and / or referenced herein are hereby incorporated by reference in their entirety.

[0125] Any discussion of the documents, acts, materials, devices, articles, etc. included herein is intended solely to provide a context for the present invention. It is not to be construed as an admission that any or all of these constitute prior art or common general knowledge in the relevant field as it existed before the priority date of each claim of the present application.

[0126] This application claims priority from AU 2014905240, filed on 23 December 2014, the disclosure of which is incorporated herein by reference.

Examples

[0127] Example 1 MSC in Clinical Phase 2a / 2b IV AMI A randomized placebo-controlled Phase 2a / 2b trial was initiated to evaluate the single intravenous (IV) administration of 200 million mesenchymal stem cells (MSC) administered between 2 and 7 days in patients with ST-elevation myocardial infarction (STEMI) or non-ST-elevation myocardial infarction (NSTEMI) and reduced cardiac ejection fraction.

[0128] A Phase IIa / IIb multicenter, randomized, double-blind, placebo-controlled study was designed to evaluate the safety and efficacy of the intravenous infusion of remestemcel-L (adult human mesenchymal stem cells cultured ex vivo) after acute myocardial infarction.

[0129] The study was a phase II, multi-center, randomized, double-blind, placebo-controlled study of 220 subjects who had recently experienced acute myocardial infarction (MI). Eligible acute MI patients for this study were 1) positive biomarker (troponin or CK-MB exceeding 4 times the ULN); 2) regional wall motion abnormality; 3) any acute coronary syndrome resulting in a reduction in overall left ventricular systolic function of 45% or less and 20% or more as determined by screening cardiac imaging performed within approximately 24 hours after the initial onset of the acute event.

[0130] Whichever occurred first, the subjects were evaluated for safety and efficacy up to 60 months after death, discontinuation, or infusion of the investigational agent (IA). The treatment window was 2 to 7 days after the initial onset of acute MI.

[0131] The subjects were randomized to placebo or treatment group (200 × 10 6 cells of remestemcel-L per injection). Approximately equal numbers of subjects in each cohort were assigned to each of the two groups (1:1 randomization).

[0132] All study data over 24 months All intravenous mesenchymal stem cell (MSC) treatments were well tolerated, and the study demonstrated that the use of IV MSC during acute myocardial infarction was safe. There were no significant differences between remestemcel-L and placebo in all adverse events (90.9% vs 90.9%) and serious adverse events (32.7% vs 33.6%) in acute MI subjects. There were 5 deaths during the study: 2 in the remestemcel-L group and 3 in the placebo group. Only 1 subject's death was considered possibly related to the study treatment, and that subject received placebo.

[0133] In the original study design, the primary inclusion criterion for study participants was an LVEF of less than 45% as determined by cardiac imaging approximately 24 hours after the acute event, and more than 70% of the eligible subjects at screening were found to have an LVEF of more than 45% by cMR immediately prior to the intravenous administration of the study product administered at a mean time of 5.4 days after MI. This is most likely due to the natural course of myocardial recovery after MI and angioplasty procedures. Therefore, less than 30% of the enrolled patients actually met the study design criteria for persistent left ventricular (LV) dysfunction at the time of remestemcel-L infusion, and thus most patients in this study actually had normal LV function at the time of treatment.

[0134] Sixty-six patients had left anterior descending (LAD) lesions with low ejection fraction at screening. For the primary efficacy variable of the change obtained from cMR of left ventricular end-systolic volume at 3 months, subjects in the remestemcel-L group showed numerical, non-statistically significant changes (+3.31 mL vs -0.35, p = 0.17). Similar non-significant numerical changes were also evident at 6 months in ESV and mean infarct size and ejection fraction.

[0135] Next, the effect of remestemcel-L therapy was evaluated in a subset of patients with persistent LV dysfunction and proximal LAD lesions as measured by cMR immediately prior to infusion.

[0136] Post-hoc efficacy analysis The central theoretical basis for this study was that remestemcel-L would be effective in patients after MI with persistent LV dysfunction. However, in the overall patient population of more than 70%, ejection fraction normalized between the selection of trial inclusion 24 hours after MI and treatment on days 2 - 7 (mean treatment application was day 5.4). This reflects the early recovery of stunned myocardium within days after reperfusion by PCI, and improvement in LV systolic function may occur prior to treatment application. The differences in LVEF data between study enrollment and treatment application strongly negated the performance of the original trial in properly testing the hypothesis underlying the study.

[0137] Therefore, a post hoc analysis was designed to further investigate the potential effect of remestemcel-L in a subset of patients who would be expected to have the most extensive disease after MI, specifically, those with a proximal LAD culprit lesion and a cMR LVEF of 45% or less at the time of treatment application.

[0138] In patients with the highest risk of progressive heart failure, a post hoc analysis was designed to answer whether a single intravenous administration (200 × 10 6 cells) of MSCs was more effective than placebo in reversing left ventricular heart failure and preventing progressive deleterious LV remodeling 6 months after index AMI. The highest risk patients were selected for study participation as follows. First anterior wall acute myocardial infarction due to a proximal LAD culprit lesion; The culprit coronary artery lesion was successfully treated with PCI within 12 hours of the onset of ischemic symptoms. Persistent LV systolic dysfunction (i.e., post-PCI LVEF of less than 45% by cMR) was present between 2 and 7 days after index AMI.

[0139] Of the 220 patients randomized in the overall trial, a total of 25 subjects met the criteria consisting of MI localized to the proximal LAD, ischemic time of less than 12 hours, successful percutaneous coronary intervention (PCI), baseline cMR LVEF of less than 45%, and treatment with the investigational product between 2 and 7 days after index AMI.

[0140] These 25 subjects constituted the evaluation group for the post hoc analysis and were assigned such that 10 subjects were in the remestemcel-L group and 15 subjects were in the placebo group. In the post hoc analysis conducted for the phase 2 AMI trial of remestemcel-L, the responder index approach was used to evaluate the effect of cell therapy in adverse LV remodeling. The primary endpoint of the post hoc study, which evaluated the change from baseline in LVESV and determined whether there was a clinically significant difference between the patients treated with remestemcel-L and placebo 6 months after AMI, was evaluated using this approach.

[0141] Of the commonly evaluated measurements of LV systolic function, LVESV has previously been shown to be a strong predictor of long-term survival after recovery from acute myocardial infarction (White et al. (1987) Circulation, 76:44-51).

[0142] Indeed, LVESV is now well recognized as a useful surrogate efficacy endpoint for biologically and clinically meaningful changes, as it is associated with the development of adverse LV remodeling and related MACE in patients at risk for the development / progression of heart failure related to LV systolic function. This analytical model has been shown to be highly correlated with MACE outcomes in phase 2 gene therapy trials (Hajjar et al. (2008) J Card Fail., 14(5):355-67; Jaski et al. (2009) Card Fail., 15(3):171-81; Jessup et al. (2011) Circulation, 124:304-313).

[0143] In the current analysis of the primary and secondary endpoints, treatment responders were determined using pre-specified threshold boundaries for success. Using these values calculated as data subtracted from the baseline at 6 months, it was confirmed that the change in the interval was outside the range of normal measurement error and both potentially clinically significant.

[0144]

Table 1

[0145] Results of the effectiveness of the post hoc group At baseline, there were no significant differences between treatment groups with respect to LVESV, LVEDV, LVEF, or LV infarct volume. However, an interesting trend was evident within the changes from baseline data for the 6-month follow-up visit.

[0146] LV infarct volume; At baseline, the mean infarct volumes between the placebo group and the remestemcel-L group were similar and very high, 40.9 + 13.75 g (mean + standard deviation) and 40.76 + 17.82 g (mean + standard deviation), respectively. Assuming a total mean myocardial mass of approximately 130 g in these patients (Stone et al. (2012) JAMA., 307:17, 1817 - 26), these values represent a very high-risk population with an infarct size of approximately 30% at the time of therapeutic intervention. Since infarct sizes greater than 18.5% have been shown prospectively to result in an incidence of 30% of major adverse cardiac events (HF-MACE, defined as heart failure hospitalization or death) associated with failure beyond 2 years, this indicates that the population of AMI patients evaluated in this post hoc analysis was at the highest risk of subsequent HF-MACE.

[0147] At the end of 6 months, subjects in the remestemcel-L group demonstrated a decrease in LV infarct volume from a baseline value of 40.76 + 17.82 g to 26.6 + 12.94 g (mean + standard deviation). This represented a change of -14.14 + 13.94 g. On the other hand, placebo subjects demonstrated a nominally smaller decrease in LV infarct volume from a baseline value of 40.9 + 13.75 g (mean + standard deviation) to 31.59 + 12.70 g. This represented a change of -7.74 + 10.84 g. The placebo-corrected difference was -6.40 g (p = 0.187).

[0148] These results indicate that remestemcel-L enhanced the natural endogenous healing process, with a two-fold reduction in infarct volume over six months, from approximately 30% of infarct size at baseline (assuming an average LV mass of 130 g) to approximately 20% of infarct size at six months, compared to placebo.

[0149] LVESV: At the end of six months, subjects in the remestemcel-L group showed a decrease in LVESV from 85.0 + 15.89 mL at baseline to 73.0 + 24.24 mL (mean ± standard deviation), representing a change of -12.0 + 16.57 mL. In contrast, placebo subjects showed an increase in LVESV from 90.5 + 23.54 mL (mean ± standard deviation) at baseline to 92.8 + 35.40 mL, representing a change of 2.2 + 28.53 mL. The placebo-corrected difference was -14.2 mL (p = 0.174).

[0150]

Table 2

[0151] In particular, the change in infarct size from approximately 30% at baseline (assuming an average LV mass of 130 g) to approximately 20% at six months, as seen in the remestemcel-L group, would be expected to reduce LVESV by at least 10 mL over this period (Wu et al. (2007) Stem Cells, 25:26, 48 - 59). The inventors' results are consistent and confirm the concordance of the inventors' dataset.

[0152] LVEDV: At the end of 6 months, subjects in the Remestemcel-L group showed an increase in LVEDV from 142.9 + 24.01 mL at baseline to 154.4 + 37.52 mL (mean + standard deviation). This represented a change of 11.5 + 27.91 mL. On the other hand, placebo subjects showed an increase in LVEDV from 151.2 + 35.98 mL (mean + standard deviation) at baseline to 167.8 + 41.66 mL. This represented a change of 16.6 + 27.30 mL. The placebo-corrected difference was -5.1 mL (p = 0.618).

[0153] LVEF: At the end of 6 months, subjects in the Remestemcel-L group showed an increase in LVEF from 40.6 + 4.23% at baseline to 53.1 + 8.71% (mean + standard deviation). This represented a change of 12.5 + 8.88 LVEF units. On the other hand, placebo subjects showed an increase in LVEF from 40.3 + 3.47% (mean + standard deviation) at baseline to 45.8 + 9.08%. This represented a change of 5.6 + 9.48 LVEF units. The placebo-corrected difference was 6.9 LVEF units (p = 0.066) (see Figures 1 and 2).

[0154]

Table 3

[0155] Efficacy analysis There was a significant trend towards statistical superiority (p = 0.095) regarding the difference between the percentages of Remestemcel-L patients and placebo patients who were treatment responders 6 months after index AMI. Specifically, 60% of patients treated with Remestemcel-L were treatment responders compared to 27% of placebo patients. This represented a 2.2-fold increase in the responder rate for the Remestemcel-L group compared to the placebo group (Figure 3).

[0156] Cohort analysis of the "responder" vs. "non-responder" subgroups The responder rate for the reduction in LVESV was 2.2 times higher in the remestemcel-L group than in the placebo group, but the mean change over 6 months in LV remodeling and parameters of overall LV systolic function was similar for remestemcel-L and placebo responders (decrease in LVESV, no change in LVEDV, and increase in LVEF) compared to remestemcel-L and placebo non-responders (nominal increase in LVESV, large increase in LVEDV, and minimal increase in LVEF).

[0157] On the other hand, the remestemcel-L treatment responders demonstrated a greater reduction in LV infarct volume from baseline to 6 months (-18.8 g) compared to any other subgroup (-7.1 g for remestemcel-L non-responders, -4.8 g for placebo responders, and -9.1 g for placebo non-responders). The mean change in LV infarct volume for the 17 patients included in the latter three groups was -7.6 g. This difference in LV infarct volume for remestemcel-L treatment responders was strikingly contrasted with the data generated for the LVESV, LVEDV, and LVEF analyses, and the responder data were generally similar for remestemcel-L and placebo subjects.

[0158] Assuming an average left ventricular mass of 130 g in patients with large occlusions after proximal LAD occlusion (Stone et al. (2012) JAMA., 307:17, 1817 - 26), this represents a change in infarct size from approximately 30% at baseline to approximately 17% at 6 months in remestemcel-L responders. This reduction in infarct volume may have a major impact on the 2-year HF-MACE event rate in this group (Wu et al. (2007) Stem Cells, 25:26, 48 - 59).

[0159] Therefore, remestemcel-L responders had 1) An achievement rate 2.2 times higher for the primary efficacy endpoint related to LVESV (p = 0.095); 2) Higher levels of concordance between improvement in LV remodeling, improvement in overall LV systolic function, and reduction in LV infarct volume; 3) That is, reduction in infarct volume demonstrated a unique mechanism by which LVESV and adverse LV remodeling were improved.

[0160]

Table 4

[0161] For all Remestemcel-L groups (n = 10) versus placebo groups (n = 15), there were no significant differences with respect to baseline patient demographics, time from onset of ischemic MI symptoms to PCI, or incidence of TIMI perfusion grade 3 after PCI. There was a tendency for the time from PCI to infusion of the investigational product and the time from the first ischemic MI symptoms to infusion of the investigational product to be shorter for the Remestemcel-L groups compared to the placebo groups.

[0162] In conclusion, intravenous administration of Remestemcel-L 2 - 7 days after AMI helped to reduce LV infarct size, attenuate adverse LV remodeling, and improve overall LV systolic function 6 months after the index event. The beneficial effects of Remestemcel-L compared to placebo were evident in both the infarcted area (reduction in infarct volume) and the remote myocardial area (reduction in LVESV leading to an increase in LVEF). These findings are expected to ultimately be equivalent to a reduction in the incidence of heart failure development in patients at high risk for this condition after AMI.

[0163] Example 2 Correlation between disease severity and therapeutic effect of MPC in LVESV Figures 4 - 9 show placebo (control) or MPC (1.5×10 8Shows the change in LVESV in subjects evaluated 6 months after administration of (mesenchymal progenitor cells). The reduction in LVESV correlated with the level of heart failure (as determined by measurement of baseline LVESV). Subjects were stratified by LVESV as shown below.

[0164]

Table 5

[0165] These data demonstrate that the greater the magnitude of baseline left ventricular systolic abnormality in subjects with chronic heart failure due to left ventricular systolic dysfunction, the more beneficial the MPC-related cardioprotective effect observed over a 6-month follow-up period. The data further demonstrate that the progressive deleterious natural history associated with progressive chronic heart failure can be beneficially altered by treatment with MPCs. While not wishing to be bound by theory, the findings support the paracrine cross-talk hypothesis that tissue-level biochemical / physiological impairments create a local environment that promotes the release of beneficial paracrine mediators from MPCs. Thereby, the optimal effect achieved by administration of MPCs in heart failure subjects is seen in subjects at highest risk of disease worsening, i.e., those with a baseline LVESV greater than 70 mL.

Claims

1. A pharmaceutical composition for use in the treatment of progressive heart failure in a human subject having a left ventricular (LV) myocardial infarction, wherein the subject is susceptible to HF-MACE, has a proximal left anterior descending (LAD) artery lesion, has an LVESV of more than 70 ml, has persistent left ventricular dysfunction, and has an LVEF of less than 45%, a pharmaceutical composition comprising a population of mesenchymal progenitor cells or mesenchymal stem cells in an amount effective to improve the LV systolic function of the patient.

2. The pharmaceutical composition according to claim 1, administered in an amount of 150 million mesenchymal progenitor cells or mesenchymal stem cells.

3. The pharmaceutical composition according to claim 1 or 2, wherein the subject has an LVEF of less than 40%.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the mesenchymal progenitor cells or mesenchymal stem cells are present in a composition for administration between 1 and 7 days after myocardial infarction.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the mesenchymal progenitor cells or mesenchymal stem cells are present in a composition for administration between 3 and 5 days after myocardial infarction.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the subject has creatine kinase-MB and / or troponin and / or myoglobin in excess of four times the upper normal limit.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the subject has an infarct size of more than 18.5% of the left ventricle.

8. The pharmaceutical composition according to any one of claims 1 to 7, comprising a population of mesenchymal progenitor cells or mesenchymal stem cells in an amount effective to reduce the size of the LV infarct in the subject.

9. (1) Improve the LV systolic function of the patient, (2) reduce the size of the LV infarct in the subject, thereby reducing the risk of HF-MACE in the subject for a period of at least 6 months, a pharmaceutical composition comprising a population of mesenchymal progenitor cells or mesenchymal stem cells in an amount effective to do so, according to any one of claims 1 to 8.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the population of mesenchymal progenitor cells or mesenchymal stem cells is present in a composition for systemic administration.

11. The pharmaceutical composition according to any one of claims 1 to 9, wherein the population of mesenchymal progenitor cells or mesenchymal stem cells is present in a composition for intravenous administration.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the population of mesenchymal progenitor cells or mesenchymal stem cells is present in a composition administered over multiple administrations.

13. the population of mesenchymal progenitor cells or mesenchymal stem cells is 1.2 x 10 ８ ~4 x 10 ８ The pharmaceutical composition according to any one of claims 1 to 12, which is present in a composition comprising cells.

14. Use of a population of mesenchymal progenitor cells or mesenchymal stem cells in the manufacture of a medicament for treating progressive heart failure in a human subject having left ventricular (LV) myocardial infarction, wherein the subject is susceptible to the effects of HF-MACE, has a proximal left anterior descending (LAD) artery lesion, has an LVESV of more than 70 ml, has persistent left ventricular dysfunction, and has an LVEF of less than 45%, wherein the medicament comprises a population of mesenchymal progenitor cells or mesenchymal stem cells in an amount effective to improve the LV systolic function of the patient and thereby reduce the risk of HF-MACE in the subject.

15. The use according to claim 14, wherein the medicament comprises 150 million mesenchymal progenitor cells or mesenchymal stem cells.

16. The use according to claim 14 or 15, wherein the subject has an LVEF of less than 40%.

17. The use according to any one of claims 14 to 16, wherein the subject has an infarct size of more than 18.5% of the left ventricle.

18. The use according to any one of claims 14 to 17, wherein the medicament comprises a population of mesenchymal progenitor cells or mesenchymal stem cells in an amount effective to reduce the size of the LV infarct in the subject.

19. The use according to any one of claims 14 to 18, wherein the medicament comprises a population of mesenchymal progenitor cells or mesenchymal stem cells in an amount effective to (1) improve the LV contractility of the patient, (2) reduce the size of the LV infarct in the subject, and thereby reduce the risk of HF-MACE in the subject for a period of at least 6 months.

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