BR112017012912B1 - USE OF A POPULATION OF STEM CELLS OR MESENCHYMAL LINEAGE PRECURSORS AND/OR PROGENIES THEREOF AND/OR SOLUBLE FACTORS DERIVED THEREOF IN THE TREATMENT OF PROGRESSIVE HEART FAILURE - Google Patents

Abstract

USE OF A POPULATION OF STEM CELLS OR MESENCHYMAL LINEAGE PRECURSORS AND/OR PROGENIES THEREOF AND/OR SOLUBLE FACTORS DERIVED THEREOF IN THE TREATMENT OF PROGRESSIVE HEART FAILURE. The present disclosure relates to methods for preventing progressive heart failure in subjects with persistent left ventricular (LV) dysfunction. Such methods can also be used to treat or prevent progressive heart failure in individuals with left proximal anterior descending (PAD) lesion and persistent left ventricular dysfunction.

Description

TECHNICAL FIELD

[001] The present disclosure relates to methods for preventing progressive heart failure in subjects with persistent left ventricular (LV) dysfunction. Such methods can also be used to treat or prevent progressive heart failure in individuals with left anterior descending (LAD) lesion and persistent left ventricular dysfunction.

BACKGROUND

[002] Myocardial infarction (MI) is still one of the leading causes of mortality and morbidity in developed countries. A recent update of US Medicare registries was published that evaluated data involving 350,509 acute MI hospitalizations in patients > 65 years, who were seen alive after their event (Schuster et al., 2004) Physiol Heart Circa Physiol., 287(2):525-532). In the first year after the indicator event, 25.9% of MI patients died with 50.5% rehospitalized. In the month after an MI, the odds of death were 21 times higher and the odds of hospitalization were 12 times higher than among the general Medicare-aged population.

[003] Patients who have larger infarcts after MI and more post-infarction LV dysfunction are at significantly increased risk of experiencing mid- to long-term cardiac events, and death. Specifically, individuals with anterior wall infarction, larger infarcts, and more LV dysfunction in the post-infarction period are at significantly increased risk of experiencing mid- to long-term cardiac events and death ( Eitel et al. (2010) J Am Coll Cardiol., 55:2470-2479 ).

[004] An infarction due to proximal PAD occlusion remains an important risk factor for progressive LV dilation, remodeling, and symptomatic progressive heart failure. In the post-angioplasty era, the 3-year mortality after MI is still the highest for proximal PAD lesions (10% versus 3% for distal PAD) (Elsman et al., 2006) Am J Cardiol., 97(8):1137 to 1141) and this is likely due to the 40% larger infarct size associated with this anatomical lesion (Elsman et al. (2006) Am J Cardiol ., 97(8):1137 to 1141). Since infarct size > 18.5% has been shown to likely result in a 30% incidence of heart failure-related major adverse cardiac events (HF-MACE, defined as hospitalization for heart failure or death) over 2 years (Wu et al. (2008) Heart, 94:730-736), this suggests that a population of patients with IM with proximal PAD lesions, low ejection fraction, and large infarcts is at greatest risk for HF- subsequent MACE.

[005] Clearly, there is a need in the art to treat or prevent progressive heart failure.

SUMMARY

[006] The present disclosure is based on the unexpected identification of a population of individuals with myocardial infarction (MI) who respond well to stem cell therapy. A large number of subjects with MI with elevated troponin or CK-MB (>4x the upper limit of normal (ULN)), a regional heart wall anomaly, and poor global left ventricular systolic function (less than or equal to 45% and greater than or equal to 20%), as determined on cardiac imaging screening performed within approximately 24 hours of MI, were given stem cell therapy or placebo shortly after MI.

[007] The present inventors found that global left ventricular systolic function had recovered to normal levels in most of these individuals approximately 5 days after MI. Administration of stem cell therapy did not provide therapeutic improvement over placebo therapy in these subjects.

[008] Surprisingly, the administration of stem cell therapy provided a pronounced therapeutic improvement over placebo therapy in subjects who had left anterior descending arterial (LAD) lesions. These results indicate that stem cell therapy may be useful for treating or preventing progressive heart failure in a subpopulation of individuals with IM, in particular, individuals with IM with proximal PAD lesions.

[009] Accordingly, in one example, the present disclosure provides a method of treating or preventing progressive heart failure in a subject with myocardial infarction, the method comprising administering to the subject a population of mesenchymal lineage stem or precursor cells and/or their progeny and/or soluble factors derived therefrom, wherein the subject has a left anterior descending (LAD) lesion.

[0010] In another example, the method comprises the steps of: i) selecting an individual with a left anterior descending lesion (LAD), and ii) administering to the individual a lineage population of mesenchymal cells or precursor cells and/or their progeny and/or soluble factors derived therefrom.

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

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

[0013] In one example, the subject also has an elevated left ventricular end-systolic volume (LVESV) greater than 70 mL. In one example, the LVESV is 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 LVESV is greater than 80 mL/m2, greater than 90 mL/m2, greater than 100 mL/m2, greater than 110 mL/m2, or greater than 120 mL/m2.

[0014] In one example, the individual 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, LVEF is measured using cardiovascular magnetic resonance (cMRI) imaging.

[0015] The present inventors have also identified that post IM administration time may also benefit individuals. Thus, in one example, mesenchymal lineage stem or precursor 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 lineage stem or precursor 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 lineage stem or precursor cells and/or progeny thereof and/or soluble factors derived therefrom are administered between about 3 and 5 days after myocardial infarction.

[0016] The present inventors have further characterized the population of individuals who may benefit from the methods of the present disclosure based on the level of serum biomarkers relative to the upper limit of normal (ULN). In one example, the subject has more than about 2x the upper limit of creatine kinase-MB and/or troponin. In another example, the subject has more than about 4x the upper limit of creatine kinase-MB and/or troponin and/or myoglobin.

[0017] The present inventors have further characterized the population of individuals who may benefit from the methods of the present disclosure based on infarct size. In one example, the individual has an infarct size between about 10 to 25% of the left ventricle. In another example, the subject has an infarct size greater than about 18.5% of the left ventricle. In one example, infarct size is measured using cMRI.

[0018] In another example, the methods of the present disclosure comprise administering a population of mesenchymal lineage stem or precursor cells enriched in STRO-1+ cells and/or progeny and/or soluble factors derived therefrom.

[0019] In another example, the methods of the present disclosure comprise administering a population of mesenchymal lineage stem or precursor cells enriched in STRO-1luminosa cells and/or progeny and/or soluble factors derived therefrom.

[0020] In one example, the stem cell population or mesenchymal lineage precursors express tissue non-specific alkaline phosphatase (TNAP) and/or the progeny cells and/or soluble factors derive from the stem cells or mesenchymal lineage precursors that express TNAP.

[0021] In one example, the mesenchymal lineage stem or precursor cell population expresses angiopoietin-1 (Ang1) in an amount of at least 0.1 μg/106 cells and/or the progeny cells and/or the soluble factors derive from mesenchymal lineage stem or precursor cells that express Ang1 in an amount of at least 0.1 μg/106 cells. In one example, the mesenchymal lineage stem or precursor cell population expresses Ang1 in an amount of at least 0.5 µg/106 cells. In one example, the mesenchymal lineage stem or precursor cell population expresses Ang1 in an amount of at least 0.7 µg/106 cells. In one example, the mesenchymal lineage stem or precursor cell population expresses Ang1 in an amount of at least 1 µg/106 cells.

[0022] In one example, the mesenchymal lineage stem or precursor cell population expresses vascular endothelial growth factor (VEGF) in an amount of less than about 0.05 µg/106 cells and/or the progeny cells and/or the soluble factors are derived from mesenchymal lineage stem or precursor cells that express VEGF in an amount of less than about 0.05 µg/106 cells and/or progeny cells. In one example, the mesenchymal lineage precursor or stem cell population expresses VEGF in an amount of less than about 0.03 µg/10 6 cells.

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

[0024] In one example, the mesenchymal lineage stem or precursor cell population and/or progeny thereof and/or the soluble factors derived therefrom are administered systemically. In one example, the mesenchymal lineage stem or precursor cell population and/or progeny thereof and/or the soluble factors derived therefrom are administered intravenously, intramuscularly or intranasally. For example, the mesenchymal lineage stem or precursor cell population and/or progeny thereof and/or soluble factors derived therefrom may be administered intravenously.

[0025] In one example, multiple doses of the mesenchymal lineage stem or precursor cell population and/or progeny thereof and/or soluble factors derived therefrom are administered.

[0026] In one example, the methods of the present disclosure comprise administering between 1 x 10 6 to 8 x 10 8 cells. In one example, the methods of the present disclosure comprise administering between 1.2 x 10 8 to 4 x 10 8 cells. In one example, the methods of the present disclosure comprise administering at least about 1.5 x 10 8 cells.

[0027] In one example, the cell population and/or progeny cells are autogenic or allogeneic and/or the soluble factors are derived from autogenic or allogeneic cells.

[0028] In one example, the cell population and/or its progeny was cultured expanded prior to administration and/or prior to obtaining the soluble factors.

[0029] In one example, the mesenchymal lineage stem or precursor cells and/or their progeny cells and/or the soluble factors derived therefrom are administered in the form of a composition comprising the mesenchymal lineage stem or precursor cells and/or their progeny cells and/or soluble factors derived therefrom and a vehicle and/or excipient. For example, the composition can comprise a cryopreservative.

[0030] Thus, by way of example, the present disclosure relates to a population of mesenchymal lineage stem or precursor cells and/or their progeny and/or soluble factors derived therefrom for use in treating or preventing progressive heart failure in a subject with myocardial infarction (MI), wherein the subject has a left anterior descending artery (LAD) lesion. In another example, the present disclosure relates to the use of a population of stem cells or mesenchymal lineage precursors and/or progeny thereof 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 left anterior descending (LAD) lesion. In these examples, the individual may have persistent left ventricular dysfunction. For example, the individual 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, mesenchymal lineage stem or precursor cells and/or progeny thereof and/or soluble factors derived therefrom can be administered between about 1 and 7 days after myocardial infarction. For example, mesenchymal lineage stem or precursor cells and/or progeny thereof and/or soluble factors derived therefrom are administered between about 3 and 5 days after myocardial infarction. In these examples, the individual may have more than about 2x the upper limit of creatine kinase-MB and/or troponin. In another example, the subject has more than about 4x the upper limit of creatine kinase-MB and/or troponin and/or myoglobin. In these examples, the individual may have an infarct size of between about 10 to 25% of the left ventricle. In another example, the individual may have an infarct size greater than about 18.5% of the left ventricle. In these examples, the size of the LVEF or infarct can be measured using cMRI.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1: Comparison of treatment groups with placebo and remestemcel-L - Reference.

[0032] Figure 2: Comparison of treatment groups with placebo and remestemcel-L - Month 6 change from baseline.

[0033] Figure 3: Comparison of placebo and remestemcel-L treatment groups - % of patients with change in left ventricular end-diastolic volume of less than -10ml after month 6. Change measured from baseline.

[0034] Figure 4: shows the change in the value of LVESV at 6 months in patients treated with placebo (control) and MPC (150 million cells) striated according to an LVESV greater than 70 mL.

[0035] Figure 5: shows the change in the value of LVESV at 6 months in patients treated with placebo (control) and MPC (150 million cells) striated according to an LVESV greater than 80 mL.

[0036] Figure 6: shows the change in the value of LVESV at 6 months in patients treated with placebo (control) and MPC (150 million cells) striated according to an LVESV greater than 90 mL.

[0037] Figure 7: shows the change in the value of LVESV at 6 months in patients treated with placebo (control) and MPC (150 million cells) striated according to an LVESV greater than 100 mL.

[0038] Figure 8: shows the change in the value of LVESV at 6 months in patients treated with placebo (control) and MPC (150 million cells) striated according to an LVESV greater than 110 mL.

[0039] Figure 9: shows the change in the value of LVESV at 6 months in patients treated with placebo (control) and MPC (150 million cells) striated according to an LVESV greater than 120 mL.

DETAILED DESCRIPTION General techniques and definitions

[0040] Unless specifically defined otherwise, all technical and scientific terms used herein should be understood to have the same meaning as understanding by a person skilled in the art (e.g., molecular genetics, molecular biology, cell culture, stem cell differentiation, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0041] Unless otherwise noted, the stem cells, cell culture, and surgical techniques used in the present disclosure are standard procedures, well known to those skilled 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 Harbor 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 to the present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988) and J.E. Coligan et al. (Editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0042] Throughout this specification, unless specifically indicated otherwise or the context requires otherwise, reference to a single stage, composition of matter, group of stages or group of compositions of matter shall be understood as encompassing one and a plurality (i.e., one or more) of these stages, compositions of matter, groups of stages or group of compositions of matter.

[0043] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for exemplary purposes only. Functionally equivalent products, compositions and methods are clearly within the scope of the disclosure as described herein.

[0044] Any example disclosed herein shall be taken to apply mutatis mutandis to any other example, unless specifically stated otherwise.

[0045] The term "and/or", eg "X and/or Y" should be understood to mean "X and Y" or "X or Y" and should be taken to provide explicit support for both meanings or for either meaning.

[0046] As used herein, the term about, unless otherwise noted, refers to +/- 10%, more preferably +/- 5%, of the designated value.

[0047] Throughout this descriptive report, the word "comprises", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a certain 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.

Mesenchymal lineage precursor cells

[0048] As used herein, the term "mesenchymal lineage stem or precursor cells" refers to undifferentiated multipotent cells that have the ability to self-renew while maintaining multipotency and the ability to differentiate into various cell types whether of mesenchymal origin, e.g., osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts, and tendons, or of non-mesodermal origin, e.g., hepatocytes, neural and epithelial cells. For the avoidance of doubt, a "mesenchymal lineage precursor cell" refers to a cell that can differentiate into a mesenchymal cell, such as bone, cartilage, muscle and fat cells, and fibrous connective tissue.

[0049] The term "mesenchymal lineage stem or precursor cells" includes both parental cells and their undifferentiated progeny. The term also includes mesenchymal precursor cells, multipotent stromal cells, mesenchymal stem cells (MSCs), perivascular mesenchymal precursor cells, and their undifferentiated progeny.

[0050] Stem cells or mesenchymal lineage precursors can be autologous, xenogeneic, syngeneic or isogenic. Autologous cells are isolated from the same individual to which they will be reimplanted. Allogeneic cells are isolated from a donor of the same species. Xenogeneic cells are isolated from a donor of another species. Syngeneic or isogenic cells are isolated from genetically identical organisms such as twins, clones or highly inbred research animal models.

[0051] Stem cells or mesenchymal lineage precursors reside primarily in the bone marrow, but have also been shown to be present in a variety of host tissues, including, for example, cord and umbilical cord blood, adult peripheral blood, adipose tissue, trabecular bone, and dental pulp.

[0052] In one example, the mesenchymal lineage stem or precursor cells are STRO-1+ mesenchymal precursor cells. As used herein, the phrase "STRO-1+ multipotential cells" should be understood to mean STRO-1+ and/or TNAP+ progenitor cells capable of forming colonies of multipotential cells.

[0053] STRO-1+ multipotential 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 node, thymus, bone, ligament, tendon, skeletal muscle, dermis, and periosteum; and are capable of differentiating into germ lineages, such as mesoderm and/or endoderm and/or ectoderm. Thus, STRO-1+ multipotential cells are capable of differentiating into a large number of cell types, including, but not limited to, adipose, bone, cartilaginous, elastic, muscle, and fibrous connective tissues. The specific pathway of differentiation and lineage commitment that these cells enter depends on various influences of mechanical influences and/or endogenous bioactive factors such as growth factors, cytokines and/or local microenvironmental conditions established by the host tissues.

[0054] Mesenchymal lineage precursor or stem cells can be isolated from host tissues and enriched by selection of STRO-1+ cells. For example, a bone marrow aspirate from an individual can be further treated with an antibody to STRO-1 or TNAP to allow selection of precursor mesenchymal lineage or stem cells. In one example, mesenchymal lineage precursor or stem cells can be enriched using the STRO-1 antibody described in (Simmons & Torok-Storb, 1991).

[0055] The terms "enriched", "enrichment" or variations thereof are used herein to describe a cell population in which the proportion of a particular cell type or the proportion of a number of particular cell types is increased when compared to an untreated population of cells (e.g., cells in their native environment). In one example, a population enriched for STRO-1+ cells comprises 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 in STRO-1+ cells" will be taken to provide explicit support for the term "population of cells comprising X% STRO-1+ cells", where X% is a percentage as cited herein. STRO-1+ cells can, in some instances, 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.

[0056] In one example, the cell population is enriched from a cell preparation comprising STRO-1+ cells in a selectable manner. In this sense, the term "selectable form" is understood to mean that the cells express a marker (for example, a cell surface marker) allowing the selection of STRO-1+ cells. The marker can be STRO-1, but it doesn't have to be. For example, cells (e.g., mesenchymal precursor cells) that express STRO-2 and/or STRO-3 (TNAP) and/or STRO-4 and/or VCAM-1 and/or CD146 and/or 3G5 also express STRO-1 (and may be luminous STRO-1). Consequently, an indication that the cells are STRO-1+ does not mean that the cells are selected for STRO-1 expression. In one example, cells are selected on the basis of at least STRO-3 expression, for example, they are STRO-3+ (TNAP+).

[0057] Reference to selection of a cell or its population does not necessarily require selection of a specific tissue source. As described herein, STRO-1+ cells can be selected, or isolated, or enriched from a wide variety of sources. That said, in some examples, these terms provide support for selecting any tissue that contains STRO-1+ cells (e.g., mesenchymal precursor cells) or vascularized tissue or tissue that comprises pericytes (e.g., STRO-1+ pericytes) or any one or more of the tissues cited herein.

[0058] In one example, the mesenchymal lineage precursor or stem cells used in the present disclosure express one or more markers individually or collectively selected from the group consisting of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90D), CD45+, CD146+, 3G5+ or any combination thereof.

[0059] By using the term "individually", it is understood that the disclosure separately encompasses the cited markers or groups of markers, and that, notwithstanding that the individual markers or groups of markers may not be separately listed herein, the appended claims may define such markers or groups of markers separately and divisibly from one another.

[0060] By use of the term "collectively" it is meant that the disclosure encompasses any number or combination of the foregoing markers or groups of markers, and that, notwithstanding that such numbers or combinations of markers or groups of markers may not be specifically listed herein, the appended claims may define such combinations or subcombinations separately and divisibly from any other combination of markers or groups of markers.

[0061] In one example, STRO-1+ cells are STRO-1luminous (similar, STRO-1bri). In another example, STRO-1bri cells are preferentially enriched over weak STRO-1 intermediate or intermediate STRO-1 cells. In another example, the STRO-1bri cells are additionally one or more of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90D) and/or CD146+. For example, cells are selected for one or more of the above markers and/or shown to express one or more of the above markers. In this sense, a cell 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 also be expressing the same marker.

[0062] In one example, mesenchymal precursor cells are perivascular mesenchymal precursor cell cells as defined in WO 2004/85630, characterized by the presence of the perivascular marker 3G5.

[0063] A cell that is referred to as being "positive" for a particular marker may express either a low (lo or weak) or a high (luminous, bri) level of that marker, depending on the degree to which the marker is present on the cell surface, where the terms relate to the intensity of the fluorescence or other marker used in the cell sorting process. The distinction of lo (or weak or dull) and bri will be understood in the context of the marker used in the particular cell population being classified. A cell that is said to be "negative" for a particular marker is not necessarily completely absent from that cell. This term means that the marker is expressed at a relatively very low level by that cell and that it generates a very low signal when detectably labeled or is undetectable above background levels, for example levels detected using an isotype control antibody.

[0064] The term "luminous" or "bri", as used herein, refers to a marker on a cell surface that generates a relatively high signal when detectably labeled. While not wishing to be bound by theory, it is proposed that "luminous" cells express more of the target marker protein (eg, the antigen recognized by STRO-1) than other cells in the sample. For example, STRO-1bri cells produce a greater fluorescent signal when labeled with a FITC-conjugated STRO-1 antibody as determined by fluorescence-activated cell sorting (FACS) analysis than non-luminous (dull/faint) cells. In one example, "luminous" cells constitute at least about 0.1% of the most luminously labeled bone marrow mononuclear cells contained in the initial sample. In other examples, the "luminous" cells constitute at least about 0.5%, at least about 1%, at least about 1.5%, or at least about 2% of the most luminously labeled bone marrow mononuclear cells contained in the starting sample. In one example, luminous STRO-1 cells have a magnitude 2 log greater surface expression of STRO-1 relative to "baseline", ie, cells that are STRO-1-. In comparison, weak STRO-1 and/or intermediate STRO-1 cells have less than greater than 2 log magnitude STRO-1 surface expression, typically about 1 log or less than "baseline".

[0065] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue non-specific alkaline phosphatase. For example, the term encompasses the liver isoform (LAP), the bone isoform (BAP), and the kidney isoform (KAP). In one example, the TNAP is BAP. In one example, TNAP as used herein refers to a molecule that can bind the STRO-3 antibody produced by the hybridoma cell line deposited with the ATCC on December 19, 2005 in accordance with the provisions of the Treaty of this Treaty under deposit accession number PTA-7282.

[0066] Furthermore, in one example, STRO-1+ cells are capable of giving rise to clonogenic CFU-F.

[0067] In one example, a significant proportion of STRO-1+ multipotential cells are capable of differentiating into at least two different germ lineages. Non-limiting examples of the lineages to which multipotential cells can be committed include bone precursor cells; hepatocyte progenitors, which are multipotent for bile duct epithelial cells and hepatocytes; restricted neural cells, which can generate glial cell precursors that progress to oligodendrocytes and astrocytes; neuronal precursors that progress to neurons; cardiac muscle precursors and cardiomyocytes, glucose-responsive insulin-secreting pancreatic beta cell lines. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells and chondrocytes, and precursor cells of the following: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal duct epithelial cells, smooth and skeletal muscle cells, testicular progenitors, vascular endothelial cells, tendon, ligament, cartilage, adipocyte, fibroblast , marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericyte, vascular, epithelial, glial, neuronal, astrocytes and oligodendrocytes.

[0068] In one aspect of the present disclosure, the presently described mesenchymal lineage precursor or stem cells are MSCs. The MSCs can be a homogeneous composition or they can be a mixed cell population enriched in MSCs. Homogeneous MSC cell compositions can be obtained by culturing adherent marrow or periosteal cells and MSCs can be identified by specific cell surface markers that are identified with unique monoclonal antibodies. A method for obtaining a cell population enriched in MSCs is described, for example, in US Patent No. 5,486,359. Alternative sources for MSCs include, but are not limited to, blood, skin, cord blood, muscle, fat, bone, and perichondrium.

[0069] In another example, the mesenchymal lineage stem or precursor cells are MSCs of CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ (eg, remestemcel-L).

[0070] Isolated or enriched mesenchymal lineage precursors or stem cells can be expanded in vitro by culture. Isolated or enriched mesenchymal lineage precursor or stem cells can be cryopreserved, thawed and further expanded in vitro by culture.

[0071] In one example, isolated or enriched mesenchymal lineage precursor or stem cells are seeded at 50,000 viable cells/cm2 in culture medium (serum-free or serum-supplemented), e.g., alpha minimal essential medium (aMEM) supplemented with 5% fetal bovine serum (FBS) and glutamine, and allowed to adhere to the culture vessel overnight at 37°C, 20% O2. The culture medium is subsequently replaced and/or changed as needed and the cells cultured for an additional 68 to 72 hours at 37°C, 5% O 2 .

[0072] As will be recognized by those skilled in the art, the cultured mesenchymal lineage precursor or stem cells are phenotypically different from cells in vivo. For example, in one embodiment they express one or more of the following markers, CD44, NG2, DC146 and CD140b. Cultured mesenchymal lineage precursor or stem cells are also biologically different from cells in vivo, with a higher rate of proliferation compared to largely non-cycling (quiescent) cells in vivo.

[0073] Stem cells or mesenchymal lineage precursors may also be cryopreserved prior to administration to an individual.

Expression of Ang1 and/or FCEV

[0074] The mesenchymal lineage stem or precursor cells of the present disclosure may be genetically modified or genetically unmodified and express high levels of Ang1. For example, mesenchymal lineage precursor or stem cells can express Ang1 in an amount of at least 0.1 µg/106 cells. In other examples, cells may express Ang1 in an amount of at least 0.2 μg/106 cells, 0.3 μg/106 cells, 0.4 μg/106 cells, 0.5 μg/106 cells, 0.6 μg/106 cells, 0.7 μg/106 cells 0.8 μg/106 cells, 1 μg/106 cells, 1.1 μg/106 cells, 1.2 μg/106 cells, 1.3 μg/106 cells, 1.4 μg/106 cells, 1.5 μg/106 cells.

[0075] In another example, mesenchymal lineage stem or precursor cells express VEGF in an amount of less than about 0.05 µg/106 cells. In other examples, the cells express VEGF in an amount of less than about 0.05 µg/106 cells, 0.04 µg/106 cells, 0.03 µg/106 cells, 0.02 µg/106 cells, 0.01 µg/106 cells, 0.009 µg/106 cells, 0.008 µg/106 cells, 0.0 07 μg/106 cells, 0.006 μg/106 cells, 0.005 μg/106 cells, 0.004 μg/106 cells, 0.003 μg/106 cells, 0.002 μg/106 cells, 0.001 μg/106 cells.

[0076] In another example, mesenchymal lineage stem or precursor cells express Ang1:VEGF in a ratio of at least about 2:1. In another example, cells express Ang1:VEGF in a ratio of at least approximately 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.

[0077] In one example, the mesenchymal lineage precursor or stem cells are genetically unmodified and express the above mentioned levels of Ang-1 or VEGF or the above mentioned Ang-1:VEGF ratios. As used herein, the term "genetically unmodified" refers to cells that have not been modified by transfection with a nucleic acid. For the avoidance of doubt, in the context of the present disclosure, a mesenchymal lineage precursor or stem cell transfected with a nucleic acid encoding Ang1 would be considered genetically modified.

[0078] The amount of cellular Ang1 and/or VEGF that is expressed in a culture or present in a mesenchymal lineage stem or precursor cell composition can be determined by various methods known to those skilled in the art. Such methods include, but are not limited to, Western blotting, enzyme-linked immunosorbent assay (ELISA), fluorescence-linked immunosorbent assay (FLISA), competition assay, radioimmunoassay, lateral flow immunoassay, continuous flow immunoassay, electrochemiluminescent assay, nephelometry-based assays, turbidometry-based assays, fluorescence-activated cell sorting (FACS)-based assays for detection of Ang-1 or VEGF in culture medium used for culture of precursor cells of mesenchymal lineage or stem cells, and surface plasma resonance (SPR or Biacore).

[0079] In one example, the level of Ang1 and/or VEGF expressed by a culture or present in a mesenchymal lineage stem or precursor cell composition is determined by an ELISA assay. For example, a cell lysate from a culture of mesenchymal lineage stem or precursor cells is added to one well of an ELISA plate. The well can be coated with a primary antibody, either a monoclonal or polyclonal antibody(s), against Ang1 or VEGF. The well is washed and then contacted with a secondary antibody, either a monoclonal or polyclonal antibody(s), against the primary antibody. The secondary antibody is conjugated with an appropriate enzyme, such as horseradish peroxidase, for example. After an appropriate incubation period, the well is washed and then contacted with an appropriate substrate for the secondary antibody-conjugated enzyme, such as one or more chromogens. Chromogens that can be used include, but are not limited to, hydrogen peroxide and tetramethylbenzidine. After the substrate(s) is(are) added, the well is incubated for an appropriate amount of time. After the incubation is complete, a "stop" solution is added to the well to stop the enzyme from reacting with the substrate(s). The optical density (OD) of the sample is then measured. The optical density of the sample is correlated with the optical densities of samples containing known amounts of Ang1 or VEGF to determine the amount of Ang1 or VEGF expressed by the cultured mesenchymal stem or lineage precursor cells being tested. Methods for determining the ratio of Ang1:VEGF expression will also be recognized by those skilled in the art. For example, after quantifying Ang1 and VEGF levels, a ratio based on the quantified Ang1 and VEGF levels could be represented as: (Ang1 level/VEGF level) = Ang1:VEGF ratio.

Progressive heart failure treatment method

[0080] Heart failure occurs when the heart cannot pump enough to maintain blood flow to meet the body's needs. One cause of heart failure is systolic dysfunction after myocardial infarction (MI). An MI occurs when blood stops flowing properly to a part of the heart. The lack of blood supply results in a localized area of myocardial necrosis referred to as an infarction. The infarcted heart is unable to pump enough to maintain blood flow to meet the body's needs, leading to multiple pathophysiological responses and ultimately heart failure. After MI, a series of compensatory mechanisms are initiated that serve to cushion the drop in cardiac output and help maintain sufficient blood pressure to perfuse vital organs. As a result, heart failure patients may not progress for long periods of time. However, the compensatory mechanisms eventually fail to compensate for the damaged heart, resulting in 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, congestive heart failure, systolic dysfunction and advanced heart failure may be used interchangeably with "progressive heart failure".

[0081] The methods of the present disclosure relate to the treatment of the progressive decline in cardiac output characteristic of progressive heart failure. Accordingly, "treat" and "treatment" in the context of the present disclosure refer to both therapeutic treatment and prophylactic or preventative measures.

[0082] In one example, treatment reduces the chance or risk of major adverse cardiac events related to heart failure (HF-MACE) defined as a composite of cardiac death or resuscitated cardiac death or non-fatal decompensated heart failure events. In one example, the chance 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 chance or risk of all-cause mortality.

Individuals with myocardial infarction

[0083] The term "individuals with myocardial infarction (MI)" is used to define individuals who have had a myocardial infarction. The methods of the present disclosure can be used to treat progressive heart failure in a specific population of individuals with MI. Individuals in need of treatment include those who already have progressive heart failure, as well as those in whom progressive heart failure must be prevented, delayed, or stopped.

[0084] Subjects with MI treated with the methods of the present disclosure have a left anterior descending (LAD) lesion. As will be recognized by one skilled in the art, the PAD artery travels in the anterior interventricular sulcus that separates the right and left ventricle, in front of the heart. The diagonal branch (Dx) leaves the DAP and runs diagonally along the anterior wall towards its external or lateral portion. Thus, Dx supplies blood to the anterolateral portion of the left ventricle. An individual can have one or several Dx branches. The first Dx branch serves 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 proximal segment, the first main side of the branch. The distal segment of the DAP is the terminal third of the artery.

[0085] In the context of the present disclosure, the term "arterial lesion" encompasses an obstructive lesion occluding the PAD of the heart or an arterial lesion that previously occluded the PAD that was treated, for example, through percutaneous coronary intervention (PCI), also known as angioplasty.

[0086] In one example, a subject treated with the methods of the present disclosure received IPC within about 1 hour of ischemic symptoms. In other examples, the subject received PCI at about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 hours of ischemic symptoms. In one example, the subject received IPC within about 12 hours of ischemic symptoms. In other examples, the subject received IPC within about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or later. Individuals being treated with thrombolytic therapy with recurrent chest pain and/or ECG changes cannot be transferred to IPC until at least 24 hours after the onset of ischemic symptoms. Accordingly, in other examples, the subject received IPC at 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 of ischemic symptoms.

[0087] Individuals with MI may have elevated left ventricular end-systolic volume (LVESV). In one example, an individual with IM treated using the methods of the present disclosure has an elevated LVESV of 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/m2, greater than 90 mL/m2, greater than 100 mL/m2, greater than 110 mL/m2, or greater than 120 mL/m2.

[0088] MI can cause persistent left ventricular dysfunction. So, in another example, the individual with IM has a proximal PAD arterial lesion and persistent left ventricular dysfunction. Left ventricular dysfunction is characterized by a decrease in myocardial contractility. A reduction in left ventricular ejection fraction (LVEF) results when myocardial contractility decreases in the left ventricle. Thus, the LVEF provides a way to determine left ventricular dysfunction.

[0089] LVEF and LVESV can be measured by a number of methods known in the art, such as echocardiography, single photon emission computed tomography (SPECT) or cardiac magnetic resonance imaging (cMR).

[0090] In one example, an individual with an LVEF of less than about 60% has left ventricular dysfunction. In other examples, an individual with an LVEF of less than 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46% had left ventricular dysfunction. In another example, an individual with an LVEF of less than about 45% has left ventricular dysfunction. In other examples, an individual with an LVEF of less than about 44%, 43%, 42%, 41% has left ventricular dysfunction. In another example, an individual with an LVEF of less than about 40% has left ventricular dysfunction. In other examples, an individual with an LVEF of less than 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, and 30% has left ventricular dysfunction.

[0091] 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 series of measurements. For example, "persistent left ventricular dysfunction" can include left ventricular dysfunction that persists from about 1 to about 14 days after MI or longer. For example, persistent left ventricular dysfunction can include left ventricular dysfunction that persists from about 1 to about 10, from about 1 to about 9, from about 2 to about 8, from about 2 to about 7 days after MI. In another example, "persistent left ventricular dysfunction" can include left ventricular dysfunction that persists for approximately 1 to 10 measurements or more.

[0092] The size or amount of myocardial necrosis post MI is referred to clinically as the infarct size. The methods of the present disclosure relate to the treatment of MI subjects with large infarct size. For example, subjects treated using the methods of the present disclosure may have an infarct size greater than about 10 to 35% of the left ventricle. In other examples, subjects have an infarct size greater than about 11 to 34%, about 12 to 33%, about 13 to 32%, about 14 to 31%, about 15 to 30%, about 16 to 29%, about 17 to 28% of the left ventricle. In another example, subjects have an infarct size greater than about 18.5% of the left ventricle. In other examples, subjects have an infarct size greater than about 19 to 27%, about 20 to 26%, about 21 to 25%, about 22 to 24%, about 23% of the left ventricle.

[0093] Infarct size can be measured through a number of methods known in the art. Examples of such methods include the use of serum markers such as creatine kinase (CK), creatine kinase (CK-MB), troponin I and troponin brain natriuretic peptide.

[0094] In one example, subjects treated with the methods of the present disclosure have troponin levels at least about 2x the upper limits of normal (ULN). In another example, subjects have troponin levels at least about 3x, about 4x, about 5x, about 6x the ULN.

[0095] In one example, subjects treated with the methods of the present disclosure have creatine kinase-MB levels at least about 2x that of the ULN. In another example, subjects have creatine kinase-MB levels at least about 3x, about 4x, about 5x, about 6x of the ULN.

[0096] Other examples of infarct size measurement include Sestamibi single photon emission computed tomography (SPECT) imaging of myocardial perfusion, magnetic resonance imaging. In one example, infarct size is measured using MRI. Several MRI techniques can be used to diagnose infarct size. One of the most accurate and best validated techniques is delayed-enhanced cardiac magnetic resonance (DE-CMR). So, in one example, RMcI includes DE-RMC.

[0097] When the appropriate settings for DERMC are used, normal myocardium appears black or null, while non-viable regions appear bright or hyper-enhanced. Consequently, in one example, the size of the infarct can be determined by visually evaluating the bright and hyper-enhanced regions. Other examples of infarct size determination are known in the art ( Sievers et al. (2007), Circulation, 115, 236 to 244; Kim et al. (2000), N Engl J Med, 343, 1445 to 1453 ). In summary, hyperenhancement is scored on a 17-segment model with a 5-point scale for each segment (0 = no hyperenhancement, 1 = 1% to 25%, 2 = 26% to 50%, 3 = 51% to 75%, 4 = 76% to 100%). Entirely encompassed dark regions in the hyper-enhanced myocardium are interpreted as regions of microvascular damage (not reflux) and included as part of the infarction. Infarct size as a percentage of LV myocardium is calculated by summing the regional scores, each weighted by the midpoint of the hyper-enhancement range (i.e., 1 = 13%, 2 = 38%, 3 = 63%, 4 = 88%) and dividing by 17. In another example, infarct size can be quantified by planimetry of hyper-enhanced areas in the stack of short-axis images.

[0098] In one example, infarct size is measured between about 1 and 40 days after the MI. In other examples, infarct size is measured between about 1 and 40 days, between about 2 and 35 days, between about 3 and 30 days, between about 4 and 25 days, between about 5 and 20 days, between about 6 and 15 days after MI. For example, infarct size can be measured at about 30 days after MI.

[0099] In the context of the present disclosure, "infarct size" refers to the size of the infarct of the left ventricle. On the other hand, left ventricular infarct size refers to the amount of left ventricle that is infarcted.

[00100] The methods of the present disclosure can be used to treat progressive heart failure in an individual with MI with various stages or classifications of heart failure. For example, the individual may have stage A, B, C, or D heart failure. In one example, the individual has stage B or C heart failure. In these examples, testing for heart failure is based on the American College of Cardiology (ACC) and American Heart Association (AHA) stage determination criteria.

[00101] In another example, the individual may have Class I, II, III, or IV heart failure. In one example, the subject has Class II or III heart failure. In these examples, the heart failure classification is based on the New York Heart Association (NYHA) classification scale.

cellular compositions

[00102] In carrying out the methods of the present invention, mesenchymal lineage stem or precursor cells can be administered in the form of a composition. In one example, such a composition comprises a pharmaceutically acceptable carrier and/or excipient.

[00103] The terms "carrier" and "excipient" refer to compositions of matter that are conventionally used in the art to facilitate the storage, administration and/or biological activity of an active compound (see, for example, Remington's Pharmaceutical Sciences, 16th Ed., Mac Publishing Company (1980)). A carrier can also reduce any undesirable side effects of the active compound. A suitable vehicle is, for example, stable, for example incapable of reacting with other ingredients in the vehicle. In one example, the vehicle does not produce significant local or systemic adverse effects on recipients at the dosages and concentrations employed for the treatment.

[00104] Suitable vehicles for the present disclosure include those conventionally used, for example, water, saline, aqueous dextrose, lactose, Ringer's solution, a buffered solution, hyaluronan and glycols are exemplary liquid vehicles, particularly (when isotonic) for solutions. Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, and the like.

[00105] In another example, a vehicle is a composition of medium, for example, in which a cell is cultured or suspended. Such a medium composition does not induce any adverse effect in an individual to whom it is administered.

[00106] The exemplary vehicles and excipients do not adversely affect the viability of a cell and/or the ability of a cell to reduce, prevent or delay progressive heart failure.

[00107] In one example, the vehicle or excipient provides a buffering activity to keep the cells and/or soluble factors at a suitable pH to thereby exert a biological activity, for example, the vehicle or excipient is phosphate buffered saline (PBS). PBS represents an attractive vehicle or excipient because it minimally interacts with cells and factors and allows for rapid release of cells and factors, in such a case the disclosure composition can be produced as a liquid for direct application into the blood stream or into a tissue or a region surrounding or adjacent to a tissue, for example by injection.

[00108] Stem cells and/or their progeny cells can also be incorporated or integrated into scaffolds that are compatible with receptors and that degrade into products that are not harmful to the recipient. These scaffolds provide support and protection for cells that must be transplanted into recipient individuals. Natural and/or synthetic biodegradable scaffolds are examples of such scaffolds.

[00109] A variety of different scaffolds can be used successfully in the practice of disclosure. Exemplary scaffolds include, but are not limited to, biological and degradable scaffolds. Natural biodegradable scaffolds include collagen, fibronectin and laminated scaffolds. The synthetic material suitable for a cell transplant scaffold must be capable of supporting extensive cell growth and cell function. These scaffolds can also be resorbable. Suitable scaffolds include polyglycolic acid scaffolds (e.g., as described by Vacanti, et al., J. Ped. Surg. 23:3-9, 1988; Cima, et al. Biotechnol. Bioeng. 38:145, 1991; Vacanti et al. Plast. Reconstr. Surg. 88:753-759, 1 991); or synthetic polymers, such as polyanhydrides, polyorthoesters and polylactic acid.

[00110] In another example, the cells can be administered on a gel support (such as Gelfoam from the Upjohn Company).

[00111] The cellular compositions described herein can be administered alone or as mixtures with other cells. Cells of different types can be mixed with a composition of the disclosure immediately or shortly before administration, or they can be co-cultured together for a period of time before administration.

[00112] In one example, the composition comprises an effective amount or a therapeutically or prophylactically effective amount of cells. For example, the composition comprises about 1 x 10 5 cells to about 1 x 10 9 cells or about 1.25 x 10 3 cells to about 1.25 x 10 7 cells. The exact amount of cells to be administered depends on a variety of factors, including the individual's age, weight and gender, and the extent and severity of the disorder being treated.

[00113] Exemplary doses include at least about 1.2 x 10 8 to about 8 x 10 8 cells, such as between about 1.3 x 10 8 to about 8 x 10 9 cells, about 1.4 x 10 8 to about 8 x 10 8 cells, about 1.5 x 10 8 to about 7.2 x 10 8 cells, about 1.6 x 10 8 to about 6.4 x 108 cells, about 1.7 x 108 to about 5.6 x 108 cells, about 1.8 x 108 to about 4.8 x 108 cells, about 1.9 x 108 to about 4.0 x 108 cells, about 2.0 x 108 to about 3.2 x 108 cells, about 2.1 x 108 to about 2.4 x 108 cells. For example, a dose can include at least about 1.5 x 10 8 cells. For example, a dose can include at least about 2.0 x 108 cells.

[00114] Stated another way, exemplary doses include at least about 1.5 x 10 6 cells/kg (80 kg subject). In one example, a dose can include at least about 2.5 x 10 6 cells/kg. In other examples, a dose may comprise between about 1.5 x 10 6 to about 1 x 10 9 cells/kg, about 1.6 x 10 6 to about 1 x 10 6 cells/kg, about 1.8 x 10 6 cells/kg, about 2.0 x 1 06 to about 8 x 106 cells/kg, about 2.1 x 106 to about 7 x 106 cells/kg, about 2.3 x 106 to about 6 x 106 cells/kg, about 2.4 x 106 to about 5 x 106 cells/kg, about 2.5 x 106 to about 4 x 106 cells/kg, about 2.6 x 10 6 to about 3 x 10 6 cells/kg.

[00115] In one example, the mesenchymal lineage stem or precursor cells comprise 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.

[00116] The compositions of the development can be cryopreserved. Cryopreservation of stem cells or mesenchymal lineage precursors can be performed using slow speed cooling methods or "fast" freezing protocols known in the art. Preferably, the cryopreservation method maintains similar phenotypes, cell surface markers and growth rates of cryopreserved cells compared to non-frozen cells.

[00117] The cryopreserved composition may comprise a cryopreservation solution. The pH of the cryopreservation solution is typically 6.5 to 8, preferably 7.4.

[00118] The cryopreservation solution may comprise a sterile, non-pyrogenic, isotonic solution such as, for example, PlasmaLyte ATM. 100 mL of PlasmaLyte ATM contains 526 mg of Sodium Chloride, USP (NaCl); 502 mg sodium gluconate (C6H11NaO7); 368 mg sodium acetate trihydrate, USP (C2H3NaO2 • 3H2O); Potassium chloride 37 mg, USP (KCl); and 30 mg of magnesium chloride, USP (MgCl2 • 6H2O). Does not contain antimicrobial agents. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 to 8.0).

[00119] The cryopreservation solution may comprise ProfreezeTM. The cryopreservation solution may additionally or alternatively comprise culture medium, for example DMEM.

[00120] To facilitate freezing, a cryoprotectant such as, for example, dimethylsulfoxide (DMSO), is usually added to the cryopreservation solution. Ideally, the cryoprotectant should be non-toxic to cells and patients, 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) can be used as a substitute or in combination with DMSO to reduce the cytotoxicity of the cryopreservation solution.

[00121] The cryopreservation solution may comprise one or more of DMSO, hydroxyethyl starch, human serum components and other protein bulking agents. In one example, the cryopreserved solution comprises about 5% human serum albumin (HSA) and about 10% DMSO. The cryopreservation solution may additionally comprise one or more of methylcellulose, polyvinylpyrrolidone (PVP) and trehalose.

[00122] In one embodiment, cells are suspended in 42.5% ProfreezeTM/50% aMEM/7.5% DMSO and cooled in a rate-controlled freezer.

[00123] The cryopreserved composition can be thawed and administered directly to the subject or added to another solution, for example comprising HA. Alternatively, the cryopreserved composition can be thawed and the stem cells or mesenchymal lineage precursors resuspended in an alternative vehicle prior to administration.

[00124] In one example, the cellular compositions described herein can be administered between about 1 and about 10 days after MI. In other examples, the cellular compositions described herein can be administered between about 1 and 9 days, between about 1 and 8 days, between about 2 and 7 days, between about 2 and 6 days, between about 3 and 5 days after MI. For example, the cellular compositions described herein can be administered about 5 days after MI.

[00125] In one example, the cellular compositions described herein can be administered between about 1 and about 10 days after PCI. In other examples, the cellular compositions described herein can be administered between about 1 and 9 days, between about 1 and 8 days, between about 2 and 7 days, between about 2 and 6 days, between about 3 and 5 days after PCI. For example, the cellular compositions described herein can be administered about 5 days after IPC.

[00126] In one example, the cellular compositions described herein can be administered as a single dose. In another example, cellular compositions are administered in 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 servings.

[00127] In one example, mesenchymal lineage stem or precursor cells may be expanded from culture prior to administration. Various methods of culturing mesenchymal lineage stem or precursor cells are known in the art. In one example, mesenchymal lineage stem or precursor cells are expanded in culture in a serum-free medium prior to administration.

[00128] The stem cells or precursors of mesenchymal lineage can be administered systemically, such as, for example, by intravenous, intraarterial or intraperitoneal administration. Stem cells or mesenchymal lineage precursors can also be administered by intranasal, intramuscular, or intracardiac administration. In one example, mesenchymal lineage stem or precursor cells are administered directly into the myocardium. For example, mesenchymal lineage stem or precursor cells can be administered directly into the left ventricular myocardium. In one example, mesenchymal lineage stem or precursor cells are administered through an endomyocardial catheter, such as the J&J Myostar™ injection catheter.

[00129] In one example, mesenchymal lineage stem or precursor cells are administered to viable myocardium. In one example, mesenchymal lineage stem or precursor cells are administered to the hibernating myocardium. A person skilled in the art would 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™ mapping catheter system can be used to identify viable and/or hibernating myocardium.

[00130] In another example, mesenchymal lineage stem or precursor cells are administered by intracoronary infusion. For example, mesenchymal lineage stem or precursor cells can be administered into the left descending artery (LAD). In one example, mesenchymal lineage stem or precursor cells are administered into the DAP artery immediately after revascularization of the DAP via PCI.

[00131] In one example, the cells are contained within a chamber that does not allow the cells to exit an individual's circulation, however it does allow factors secreted by the cells to enter the circulation. In this way, soluble factors can be administered to an individual, allowing the cells to secrete the factors into the individual's circulation. Such a chamber can also be implanted at a location in an individual to increase local levels of soluble factors, for example, implanted in or near the heart.

[00132] It will be recognized by persons skilled in the art that numerous variations and/or modifications can be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are therefore considered in all respects to be illustrative and not restrictive.

[00133] All publications discussed and/or mentioned herein are hereby incorporated in their entirety.

[00134] Any discussion of documents, acts, materials, devices, articles or the like that has been included in this descriptive report is solely for the purpose of providing a context for the present invention. It should not be taken as an admission that any or all of these individuals form part of the prior art basis or were common general knowledge in the field relevant to the present invention as it existed prior to the priority date of each claim of this application.

[00135] The present application claims priority from AU 2014905240 filed on December 23, 2014, the disclosures of which are incorporated herein by reference.

EXAMPLES Example 1 CLINICAL PHASE 2a/2b IV MSCs IN AMI

[00136] A randomized placebo-controlled phase 2a/2b trial was initiated to evaluate a single intravenous (IV) dose of 200 million mesenchymal stem cells (MSCs) administered over 2 to 7 days in patients with ST-elevation MI (STEMI) or non-ST-elevation myocardial infarction (NSTEMI) and a reduced cardiac ejection fraction.

[00137] A phase IIa/IIb, multicentre, randomized, double-blind, placebo-controlled study was designed to evaluate the safety and efficacy of intravenous infusion of remestemcel-L (adult human mesenchymal stem cells cultured ex vivo) after acute myocardial infarction.

[00138] The study was a phase II, multicentre, randomized, double-blind, placebo-controlled study of 220 subjects who had recently suffered an acute myocardial infarction (MI). Acute MI patients eligible for this study included those patients with any acute coronary syndrome resulting in: 1) positive biomarkers (troponin or CK-MB > 4x ULN); 2) a regional anomaly of wall motion; 3) an overall poor left ventricular systolic function < 45% and > 20% as determined on cardiac imaging screening that was performed within approximately 24 hours of the initial onset of the acute event.

[00139] Subjects were evaluated for safety and efficacy until death, withdrawal, or 60 months after investigational agent (AI) infusion, whichever occurred first. The treatment window was 2 to 7 days after the initial onset of acute MI.

[00140] Subjects were randomized to either the placebo or the treatment group (rememtemcel-L 200 x 106 cells per infusion). Approximately equal numbers of subjects in each cohort were assigned to each of the two groups (1:1 randomization).

GLOBAL STUDY DATA THROUGH MONTH 24

[00141] In general, intravenous mesenchymal stem cell (MSC) therapy was well tolerated and the study demonstrated that the use of IV MSC at the time of an acute myocardial infarction was safe. There were no apparent differences between remestecel-L and placebo in overall adverse events (90.9% versus 90.9%) and serious adverse events (32.7% versus 33.6%) in subjects with acute MI. There were 5 deaths in the study: 2 in the remestemcel-L group, 3 in the placebo group. Only 1 subject death was considered possibly related to study treatment, and that subject received placebo.

[00142] Although in the original study design the main inclusion criterion for entry into the study was an LVEF < 45% determined by cardiac imaging approximately 24 hours after the acute event, more than 70% of subjects who qualified at separation were actually found to have LVEF > 45% by cMRI shortly before the intravenous administration of the study product, which was administered at the median time of 5.4 days after the MI. This is likely due to the natural course of myocardial recovery after an MI and angioplasty procedure. Therefore, less than 30% of patients enrolled met the study design criteria of persistent left ventricular (LV) dysfunction at the time of remestemcel-L infusion, and consequently, the majority of patients in this study actually had normal LV function at the time they received treatment.

[00143] 66 patients had left anterior descending (LAD) lesions with low ejection fraction on selection. For the primary efficacy variable of NMR-derived change in left ventricular end-systolic volume at 3 months, subjects in the remestemcel-L group had numerical but not statistically significant changes (+3.31 mL versus -0.35, p = 0.17). Similar non-significant numerical changes were also evident at 6 months in ESV, as well as in mean infarct size and ejection fraction.

[00144] The effects of therapy with remestemcel-L were then evaluated in the subset of patients with persistent LV dysfunction and proximal DAP lesions, as measured by cMR immediately before infusion.

POST HOC EFFECTIVENESS ANALYSIS

[00145] The central rationale for this study was that rememstemcel-L would be effective in those post-MI patients with persistent LV dysfunction. However, in the general patient population, more than 70% normalized their ejection fraction between selection for trial enrollment 24 hours post MI and treatment infusion on day 2 through 7 (mean treatment administration on day 5.4). This may reflect early recovery of the damaged myocardium in the days after reperfusion with PCI, resulting in improved LV systolic function prior to administration of treatment. The disparity in the LVEF data between study entry and treatment administration significantly negated the ability of the original trial to adequately test the study's underlying hypothesis.

[00146] Consequently, a post hoc analysis was designed and intended to further investigate the potential effect of remestemcel-L in the subset of patients who are expected to have the most extensive disease after MI, specifically individuals with proximal DAP culprit lesions and cMR LVEF < 45% at the time of treatment administration.

[00147] The post hoc analysis was designed to answer whether, in patients at the highest risk of progressive heart failure, a single dose (200 x 106 cells) of MSC administered intravenously would be more effective than placebo for reversing left ventricular cardiac dysfunction and preventing progressive adverse LV remodeling at 6 months after the AMI index. Higher-risk patients were selected for study entry as follows: • a first anterior wall acute myocardial infarction due to a LAD proximal culprit lesion; • the culprit coronary lesion was successfully treated with a PCI procedure within 12 hours of the onset of ischemic symptoms; • Persistent LV systolic dysfunction (ie, post CPI LVEF <45% by cMRI) presents 2 to 7 days after the AMI index.

[00148] Of the 220 patients randomized to the overall study, a total of 25 subjects met the criteria consisting of an MI located at the proximal DAP, ischemic time < 12 hours, a successful percutaneous coronary intervention (PCI), a baseline LVEF cMR < 45%, and investigational product treatment between 2 and 7 days after the AMI index.

[00149] These 25 subjects constituted the evaluation population for the post hoc analysis and were allocated as 10 subjects in the remestemcel-L group and 15 subjects in the placebo group. In the post hoc analysis performed for the phase 2 trial of remestemcel-L AMI, a response rate approach was used to assess the effects of cell therapy on adverse LV remodeling. This approach was used to assess the primary endpoint of the post hoc study in which LVESV change assessments from baseline were conducted to determine whether a clinically significant difference was present at 6 months post AMI between patients treated with remestemcel-L compared to placebo.

[00150] It has previously been shown that of the commonly evaluated measures of LV systolic function, LVESV is a strong predictor of long-term survival after recovery from an acute myocardial infarction (White et al. (1987) Circulation, 76:44-51).

[00151] Indeed, it is now well accepted that LVESV is a useful surrogate efficacy endpoint of biologically and clinically significant changes as it relates to the development of adverse LV remodeling and associated MACE in patients at risk of developing/progressing heart failure related to LV systolic function. This analysis model has been shown to correlate well with MACE results in a Phase 2 gene therapy trial (Hajjar et al. (2008) J Card Fail., 14(5):355 to 367; Jaski et al. (2009) Card Fail., 15(3):171 to 181; Jessup et al. (2011) Circulation, 124:304 to 3 13).

[00152] In the current analysis of primary and secondary endpoints, a treatment responder was defined using pre-specified threshold limits for success. These values calculated at 6 months minus baseline data were used to ensure that the range change was both outside the usual measurement error range and potentially clinically significant.

POST HOC POPULATION EFFECTIVENESS RESULTS

[00153] At baseline, there were no significant differences between treatment groups for LVESV, LVEDV, LVEF, or LV infarct volume. However, interesting developments were evident in the change from baseline data to the 6-month follow-up visit.

LV infarct volume:

[00154] At baseline, the mean infarct volumes between the placebo and remestemcel-L group were similar and very high, 40.9 + 13.75 g (mean + SD) and 40.76 + 17.82 g (mean + SD), respectively. Assuming a mean total myocardial mass of approximately 130 g in these patients (Stone et al. (2012) JAMA., 307:17, 1817 to 1826), these values represent a high-risk population with approximately 30% infarct size at the time of therapeutic intervention. Since infarct size > 18.5% was prospectively shown to result in a 30% incidence of major adverse cardiac events related to heart failure (HF-MACE, defined as hospitalization for heart failure or death) over 2 years, this confirms that the population of patients with AMI evaluated in this post hoc analysis were at increased risk for subsequent HF-MACE.

[00155] At the end of 6 months, subjects in the remestemcel-L group showed a decrease in LV infarct volume from a baseline value of 40.76 + 17.82 g to 26.6 + 12.94 g (mean + SD). This represented a -14.14 + 13.94 g change. In contrast, placebo subjects showed a smaller symbolic decrease in LV infarct volume from a baseline value of 40.9 ± 13.75 g (mean + SD) 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).

[00156] These results indicate that remestemcel-L enhanced the natural endogenous healing process of infarct volume reduction two-fold over 6 months relative to placebo, from approximately 30% infarct size at baseline to approximately 20% infarct size (assuming a mean LV mass of 130 g) at 6 months.

LVESV:

[00157] At the end of 6 months, subjects in the remestemcel-L group showed decreases in LVESV from a baseline value of 85.0 + 15.89 mL to 73.0 + 24.24 mL (mean + SD). This represented a -12.0 + 16.57 mL change. In contrast, subjects on placebo showed an increase in LVESV from a baseline value of 90.5 ± 23.54 mL (mean + SD) to 92.8 ± 35.40 mL. This represented a change of 2.2 + 28.53 mL. The placebo-corrected difference was -14.2 mL (p = 0.154). Summary of LV end-systolic volume at baseline and 6 months post-treatment (LOCF analysis)

[00158] In particular, a change in infarct size from approximately 30% at baseline to approximately 20% at 6 months (assuming a mean LV mass of 130 g), as was observed in the rememstemcel-L group, would be expected to reduce LVESV by at least 10 mL (Wu et al. (2007) Stem Cells, 25:26, 48-59) during this period; the results of the present invention are consistent and confirm the agreement of the dataset of the present invention.

LVEDV:

[00159] At the end of 6 months, subjects in the remestemcel-L group exhibited an increase in LVEDV from a baseline value of 142.9 +24.01 mL to 154.4 + 37.52 mL (mean + SD). This represented a change of 11.5 + 27.91 mL. In contrast, subjects on placebo showed an increase in LVEDV from a baseline value of 151.2 + 35.98 mL (mean + SD) 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).

LVEF:

[00160] At the end of 6 months, subjects in the remestemcel-L group exhibited an increase in LVEF from a baseline value of 40.6 + 4.23% to 53.1 + 8.71% (mean + SD). This represented a 12.5 + 8.88 unit change of LVEF. In contrast, subjects on placebo showed a nominally smaller increase in LVEF from a baseline value of 40.3 ± 3.47% (mean + SD) to 45.8 ± 9.08%. This represented a unit change of 5.6 + 9.48 of LVEF. The placebo-corrected difference was 6.9 LVEF units (p = 0.066) (see Figures 1 and 2). Summary of LV ejection fraction at baseline and 6 months post-treatment (LOCF analysis) * Comparison of the reference treatment using ANOVA. Other treatment comparisons using ANCOVA with treatment as a fixed effect and reference as a covariate Source: Table 14.2.19.S.1

L versus PLACEBO

[00161] There was an important trend towards statistical significance (p = 0.095) for the difference between the percentage of patients on remestemcel-L versus PLACEBO who responded to treatment at 6 months after the AMI index. Specifically, 60% of patients treated with remestemcel-L were treatment responders, compared with 27% of patients treated with placebo. This represented a 2.2-fold increase in response rate for the rememstemcel-L group compared to the placebo group (Figure 3).

COHORT ANALYSIS FOR "RESPONDER" VERSUS "NON-RESPONDER" SUBGROUPS

[00162] Although the response rate for the decrease in LVESV was 2.2 times greater in the remestemcel-L group than in the placebo group, mean changes at 6 months for LV remodeling parameters and general LV systolic function were similar for remestemcel-L and placebo responders (decrease in LVESV, no change in LVEDV, and increase in LVEF) compared with non-responders with remestemcel-L and with placebo (nominal increase in LVESV, large increase in LVEDV, and minimal increase in LVEF).

[00163] In contrast, responders to remestemcel-L treatment demonstrated a greater decrease in LV infarct volume from baseline to month 6 (-18.8 g) than was observed for any other subgroups (remestemcel-L non-responders = -7.1 g, PLACEBO responders = -4.8 g, and placebo non-responders = -9.1 g). The mean change in LV infarct volume for the 17 patients included in the last three groups was -7.6 g. This differentiation in LV infarct volume for remestemcel-L treatment responders is in marked contrast to the data generated for the LVESV, LVEDV, and LVEF analyses, where treatment responder data were generally similar for remestemcel-L and placebo subjects.

[00164] Assuming a mean left ventricular mass of 130 g in patients with large infarcts after a proximal PAD occlusion (Stone et al. (2012) JAMA., 307: 17, 1817 to 1826), this represents a change in infarct size from approximately 30% at baseline to approximately 17% at 6 months in the remestemcel-L responders. This reduction in infarct volume is likely to have a large impact on the HF-MACE event rate over two years in this group (Wu et al. (2007) Stem Cells, 25:26, 48-59).

[00165] Thus, remestemcel-L responders demonstrated: 1) a 2.2-fold higher achievement rate of the primary endpoint relative to LVESV (p = 0.095); 2) a higher level of concordance between improvement in LV remodeling, improvement in overall LV systolic function, and reduction in LV infarct volume. 3) a unique mechanism by which LVESV and adverse LV remodeling were improved, namely by reduction in infarct volume.

[00166] For the overall remestemcel-L group (n = 10) versus the placebo group (n = 15), there were no significant differences for baseline demographics, time from onset of MI ischemic symptoms to time of PCI, or incidence of TIMI grade 3 perfusion flow post PCI. There was a trend towards shorter IPC time to investigational product infusion and investigational time from first ischemic MI symptoms to investigational product infusion for the remestemcel-L group compared to the placebo group.

[00167] In conclusion, intravenous administration of remestemcel-L 2 to 7 days after AMI helped to decrease LV infarct size, attenuated adverse LV remodeling, and improved overall LV systolic function at 6 months after the index event. It appears that the beneficial effects of remestemcel-L compared to placebo were evident both in the infarct region (decreased infarct volume) and in remote myocardial areas (decreased LVESV leading to increased LVEF). These findings are predicted to ultimately equate to a reduction in the incidence of developing heart failure in post-AMI patients at high risk for this condition.

Example 2 CORRELATION BETWEEN DISEASE SEVERITY AND THE THERAPEUTIC BENEFIT OF MPC IN LVESV

[00168] Figures 4 to 9 show the change in LVESV in subjects evaluated at 6 months after administration of placebo (control) or MPC (1.5 x 108 mesenchymal precursor cells). The reduction in LVESV was correlated with the level of heart failure (as determined by measuring baseline LVESV). Subjects were streaked according to LVESV as shown below.

[00169] These data demonstrate that the greater the magnitude of left ventricular contractile abnormality at baseline in individuals with chronic heart failure due to left ventricular systolic dysfunction, the more beneficial the CPM-related cardioprotective effect observed during a 6-month follow-up period. The data further show that the progressive adverse natural history associated with advanced chronic heart failure can be beneficially altered by treatment with MPCs. Without wishing to be bound by theory, the findings support the conversational paracrine hypothesis that biochemical/physiological disturbances of tissue levels create a local environment that facilitates MPC release of beneficial paracrine mediators. Thus, the optimal benefit obtained by the administration of MPC in individuals with heart failure is seen in individuals with a higher risk of disease progression, namely, individuals with baseline LVESV > 70 mL.

Claims (12)

1. Use of a population of stem cells or mesenchymal lineage precursors and/or progeny thereof and/or soluble factors derived therefrom characterized in that it is for the manufacture of a medicament for the treatment of progressive heart failure in a subject with myocardial infarction, wherein the subject has a left proximal anterior descending artery (PAD) lesion, has a left ventricular end systolic volume (LVESV) greater than 70 mL, has persistent left ventricular dysfunction and has a left ventricular ejection fraction (LVEF) less than 45%. 2. Use, according to claim 1, characterized by the fact that the individual has an LVEF of less than 40%. 3. Use according to any one of claims 1 or 2, characterized by the fact that the individual has more than 4x the upper limit of creatine kinase-MB and/or troponin and/or myoglobin. 4. Use according to any one of claims 1 to 3, characterized in that the individual has an infarct size between 10 and 25% of the left ventricle. 5. Use according to any one of claims 1 to 3, characterized in that the individual has an infarct size greater than 18.5% of the left ventricle. 6. Use according to claim 5, characterized in that the size of the infarction is measured using cardiovascular magnetic resonance imaging (cMR). 7. Use according to any one of claims 1 to 6, characterized by the fact that the population of stem cells or mesenchymal lineage precursors is enriched by STRO-1+ cells and/or STRO-1luminosa cells and/or their progeny and/or soluble factors derived therefrom. 8. Use according to any one of claims 1 to 7, characterized in that the medicine comprises between 1.2 x 108 and 4 x 108 cells. 9. Use according to any one of claims 1 to 8, characterized by the fact that the population of stem cells or precursors of mesenchymal lineage and/or progeny cells is autogenic or allogeneic and/or the soluble factors are derived from such autogenic or allogeneic cells. 10. Use according to any one of claims 1 to 9, characterized by the fact that the population of stem cells or mesenchymal lineage precursors and/or their progeny was expanded in culture before administration and/or before obtaining soluble factors. 11. Use according to any one of claims 1 to 10, characterized by the fact that the population of stem cells or mesenchymal lineage precursors express tissue non-specific alkaline phosphatase (TNAP) and/or the progeny cells and/or the soluble factors are derived from such stem cells or mesenchymal lineage precursors that express TNAP. 12. Use according to any one of claims 1 to 11, characterized in that the population of stem cells or precursors of mesenchymal lineage expresses: - Ang1:VEGF in a ratio of at least 2:1 and/or the progeny cells and/or soluble factors are derived from such a population of stem cells or precursors of mesenchymal lineage that expresses Ang1:VEGF in a ratio of at least 2:1.