WO2007095256A2

WO2007095256A2 - Methods of promoting cardiac cell proliferation

Info

Publication number: WO2007095256A2
Application number: PCT/US2007/003842
Authority: WO
Inventors: C. M. Amy Chen; Lisa-Anne Whittemore
Original assignee: Hydra Biosciences, Inc.
Priority date: 2006-02-10
Filing date: 2007-02-12
Publication date: 2007-08-23
Also published as: WO2007095256A3

Abstract

The present invention provides compositions and methods for promoting cardiac cell proliferation.

Description

Methods of Promoting Cardiac Cell Proliferation

Background of the Invention

Injuries and diseases of the cardiovascular system exact a dramatic personal and financial toll both in this country and throughout the world. Scientific advances have resulted in a variety of medical and surgical therapies to decrease mortality following a serious cardiovascular incident, as well as to improve trie quality of life for survivors of such diseases and injuries. However, each of the available medical and surgical therapies has significant limitations. Most notably, since the term "cardiovascular disease and injury" encompasses a wide range of conditions, individual medical and/or surgical therapies must be developed to treat each indication. Accordingly, there exists a substantial need in the art for improved methods and compositions for treating a wide range of cardiac diseases and injuries.

Mammals typically heal an injury, whether induced from trauma or disease, by replacing the missing tissue with scar tissue. In the case of cardiac tissue, events such as a myocardial infarction result in substantial damage and even death to cardiomyocytes and other cardiac cells and tissues. However, instead of replacing the damaged cardiac muscle with functional cardiomyocytes, formation of scar tissue further strains and compromises the functional performance of the surviving cardiac tissue. This model, whereby diseased or damaged cardiomyocytes are replaced by scar tissue which further impedes the functional performance of the already compromised cardiovascular system, is recapitulated in a wide range of disease states including congenital cardiovascular disease states.

The loss of cardiac function resulting from injury or disease could be prevented if, as in other non-mammalian species, mammalian fetal, neonatal and adult cardiomyocytes and other cardiac cells regenerated following injury. In contrast to the tissue produced by scarring, regeneration would replace damaged or dead cardiac cells with functional cardiac cells, such as cardiomyocytes, thereby restoring functional performance following cardiac disease or injury. Furthermore, regeneration would replace cardiac cells, such as cardiomyocytes, damaged due to ischemia or other interruption of blood to cardiac tissue due to cardiovascular injury or disease. The present invention provides methods and compositions to promote cardiac cell proliferation, including mammalian fetal, neonatal and adult cardiac cell proliferation. The present invention further provides compositions and methods for promoting regeneration of cardiac cells, such as cardiomyocytes, following injury or disease. In contrast to currently available treatments designed for particular cardiac indications, the methods and compositions of the present invention can be used to treat a wide range of diseases and injuries characterized by damage to cardiac cells, including cardiomyocytes, and/or a decrease in cardiac function. Brief Summary of the Invention

The present invention is based on the finding that particular classes of GSK3 inhibitors promote cardiac cell proliferation. Such cardiac cell proliferation, for example cardiomyocyte proliferation, includes proliferation of mammalian fetal, neonatal and adult cardiac cells. Thus, particular classes of GSK3 inhibitors can be used in methods for promoting cardiac regeneration, as well as methods of treating a wide range of injuries and diseases characterized by injury to cardiomyocytes and/or a decrease in cardiac function.

Tn one aspect, the invention provides a method of promoting cardiac cell proliferation. The method comprises contacting a cardiac cell with an amount of a compound effective to promote cardiac cell proliferation. Compounds for use in this method selectively inhibit GSK3 kinase activity.

Most kinase inhibitors have significant cross reactivity against a range of kinases of different classes/families. This characteristic undermines their suitability for many in vitro and in vivo uses because the advantageous response promoted by inhibiting one kinase or class of kinases may be off-set by disadvantageous responses induced by simultaneously inhibiting a different kinase or class of kinases. The present invention is based on the appreciation that kinase inhibitors that have selective properties in inhibiting certain kinases over other kinases have useful applications.

The present invention is based on the observation that a certain class of selective kinase inhibitors can promote cardiac cell proliferation. Specifically, GSK3 kinase inhibitors that selectively inhibit the kinase activity of GSK3 in comparison to the kinase activity of CDKl can be used to promote cardiac cell proliferation. As shown in the Examples, structurally related compounds that are not selective for inhibiting the kinase activity of GSK3 over CDKl are significantly less effective in promoting cardiac cell proliferation. Without being bound by theory, compounds that inhibit CDKl may block cell cycle progress, thereby undermining any proliferative affect achieved by inhibiting GSK3 kinase activity. Accordingly, compounds that selectively inhibit GSK3 kinase activity in comparison to CDKl kinase activity can more effectively promote cardiac cell proliferation leading to cell division and an increase in total cell number.

In a first aspect, the invention provides a method of promoting cardiac cell proliferation. The method comprises contacting a cardiac cell with an amount of a compound effective to promote cardiac cell proliferation. Exemplary compounds selectively inhibit GSK3 kinase activity. For example, exemplary compounds inhibit GSK3 kinase activity with an IC50 at least 1 order of magnitude lower than its IC₅₀ for inhibiting CDKl kinase activity.

In a second aspect, the invention provides a method of promoting cardiac cell proliferation. The method comprises contacting a cardiac cell with an amount of a compound effective to promote cardiac cell proliferation. Exemplary compounds selectively inhibit GSK3 kinase activity. For example, exemplary compounds inhibit GSK3 kinase activity with an IC₅₀ at least 1 order of magnitude lower than its IC₅₀ for inhibiting the kinase activity of one or more of CDKl, CDK2, CDK4, CDK5, CDK6, or CDK7.

In a third aspect, the invention provides a method of promoting cardiac cell proliferation. The method comprises contacting a cardiac cell with an amount of a compound effective to promote cardiac cell proliferation. Exemplary compounds selectively inhibit GSK3 kinase activity with an IC₅₀ at least 1 order of magnitude lower than its IC50 for inhibiting CDKl kinase activity and at least 1 order of magnitude lower than its IC₅0 for inhibiting the kinase activity of one or more of CDK2, CDK4, CDK5, CDK6, or CDK7.

In a fourth aspect, the invention provides a method of treating an injury or disease of decreased cardiac function. The method comprises administering to a subject in need thereof an amount of a compound effective to promote cardiac cell proliferation. Exemplary compounds selectively inhibit GSK3 kinase activity. For example, exemplary compounds inhibit GSK3 kinase activity with an IC50 at least 1 order of magnitude lower than its IC5₀ for inhibiting the kinase activity of one or more of CDKl, CDK2, CDK4, CDK5, CDK6, or CDK7.

In a fifth aspect, the invention provides a method of treating an injury or disease of decreased cardiac function. The method comprises administering to a subject in need thereof an amount of a compound effective to promote cardiac cell proliferation. Exemplary compounds selectively inhibit GSK3 kinase activity. For example, exemplary compounds inhibit GSK3 kinase activity with an IC5₀ at least 1 order of magnitude lower than its IC₅₀ for inhibiting CDKl kinase activity and/or CDK2 kinase activity.

In a sixth aspect, the invention provides a method of treating an injury or disease of decreased cardiac function. The method comprises administering to a subject in need thereof an amount of a compound effective to promote cardiac cell proliferation. Exemplary compounds selectively inhibit GSK3 kinase activity with an IC₅₀ at least 1 order of magnitude lower than its IC5₀ for inhibiting CDKl kinase activity and at least 1 order of magnitude lower than its IC₅₀ for inhibiting the kinase activity of one or more of CDK2, CDK4, CDK5, CDK6, or CDK7.

In a seventh aspect, the invention provides the use of any of the compounds of the invention in the manufacture of a medicament for promoting cardiac cell proliferation.

In an eighth aspect, the invention provides the use of any of the compounds of the invention in the manufacture of a medicament for treating or preventing any of the injuries or diseases of cardiac tissue outlined herein.

In certain embodiments of any of the foregoing, the cardiac cell is a cardiomyocyte. In certain embodiments of any of the foregoing, the cardiomyocyte is selected from a neonatal, fetal, or adult cardiomyocyte.

In certain embodiments of any of the foregoing, the method is performed in vitro, for example using cells in culture. In certain embodiments of any of the foregoing, the method is performed in vivo.

In certain embodiments of any of the foregoing, the compound is a small organic molecule.

In certain embodiments of any of the foregoing, the cells are mammalian cells. Exemplary mammalian cells include but are not limited to mouse cells, rat cells, hamster cells, rabbit cells, dog cells, cat cells, goat cells, pig cells, sheep cells, non-human primate cells, and human cells.

In certain embodiments of any of the foregoing, the compound inhibits GSK3 kinase activity with an IC₅₀ of less than 250 nM. In other embodiments, the compound inhibits GSK3 kinase activity with an IC₅₀ less than or equal to any of the following: 200 nM, 150 nM, 100 nM, 50 nM, 25 nM, 20 nM, 15 nM, 10 nM, 7 nM, 5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, or even less than or equal to 1 nM.

In certain embodiments of any of the foregoing, the compound inhibits GSK3 kinase activity an IC₅₀ at least 1.5 orders of magnitude lower than its IC50 for inhibiting one or more of CDKl kinase activity or CDK2 kinase activity. In other embodiments, the compound inhibits GSK3 kinase activity an IC₅0 at least 2 orders of magnitude lower than its IC50 for inhibiting one or more of CDKl kinase activity or CDK2 kinase activity.

In certain embodiments of any of the foregoing, the compound inhibits GSK3 kinase activity with an IC₅₀ at least 25 times lower than its IC₅₀ for inhibiting CDKl kinase activity or CDK2 kinase activity. In other embodiments, the compound inhibits GSK3 kinase activity with an IC₅₀ at least 50, 60, 70, 80, 90, or 100 times lower than its IC₅₀ for inhibiting CDKl kinase activity or CDK2 kinase activity. In other embodiments, the compound inhibits GSK3 kinase activity with an IC₅₀ at least 200, 500, 1000, 1500, or even 2000 times lower than its IC₅₀ for inhibiting CDKl kinase activity or CDK2 kinase activity.

In certain embodiments of any of the foregoing, the compound inhibits GSK3 kinase activity with an IC₅₀ at least 5 times lower than its IC₅₀ for inhibiting the kinase activity of one or more of CDKl, CDK2, CDK4, CDK5, CDK6, or CDK7. In other embodiments, the compound inhibits GSK3 kinase activity with an IC₅₀ at least 10, 20, 25, 50, 100, 200, 500, 1000, 1500, or even 2000 times lower than its IC₅₀ for inhibiting the kinase activity of one or more of CDK2, CDK4, CDK5, CDK6, or CDK7.

In certain embodiments of any of the foregoing, the compound inhibits GSK3 kinase activity with an IC₅₀ at least 25 times lower than its IC₅₀ for inhibiting CDKl kinase activity and at least 5 times lower than its IC50 for inhibiting the kinase activity of one or more of CDK2, CDK4, CDK5, CDK6, or CDK7. In certain embodiments of any of the foregoing, the compound inhibits GSK3 kinase activity with an IC₅₀ at least 50, 60, 70, 80, 90, or 100 times lower than its ICso for inhibiting the kinase activity of one or more of the following: ERKl, ERK2, MAPKK, PD kinase, PKC (protein kinase C), casein kinase, insulin receptor tyrosine kinase, aktl, c-src, Fltl, bFGF receptor kinase, IGF receptor kinase, KDR, chkl or DNA protein kinase. In other embodiments, the compound inhibits GSK3 kinase activity with an IC₅₀ at least 200, 500, 1000, 1500, or even 2000 times lower than its IC50 for inhibiting the kinase activity of one or more of the following: ERKl, ERK2, MAPKK, PO kinase, PKC (protein kinase C), cAMP-dependent protein kinase, cGMP-dependent protein kinase, casein kinase, insulin receptor tyrosine kinase, aktl, c-src, Fltl, bFGF receptor tyrosine kinase, IGF receptor tyrosine kinase, KDR, chkl or DNA protein kinase.

In certain embodiments, the compound does not substantially inhibit (e.g., inhibits with an IC50 of greater than or equal to 1 micromolar, greater than or equal to 5 micromolar, or greater than or equal to 10 micromolar) the kinase activity of one or more of the following: the kinase activity of one or more of the following: ERKl, ERK2, MAPKK, PI3 kinase, PKC (protein kinase C), cAMP-dependent protein kinase, cGMP-dependent protein kinase, casein kinase, insulin receptor tyrosine kinase, aktl, c-src, Fltl, bFGF receptor kinase, IGF receptor kinase, KDR, chkl or DNA protein kinase,

In certain embodiments of any of the foregoing, the compound binds to GSK3.

In certain embodiments of any of the foregoing, the compound is 6-bromo- indirubin-3 ' -monoxime .

In certain embodiments of any of the foregoing, the compound has the following structure:

In certain embodiments of any of the foregoing, the compound promotes cardiac cell proliferation with an EC₅₀ of less than or equal to 500 nM. In other embodiments, the compound promotes cardiac cell proliferation with an EC50 of less than or equal to 450 nM, 400 nM, 350 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, or 50 nM.

In certain embodiments of any of the foregoing, the compound is used to prevent, treat or alleviate a disease or injury of cardiac cells. In certain other embodiments, the compound is used to prevent, treat or alleviate a disease or injury characterized by decreased cardiac function.

In certain embodiments of any of the foregoing, the compound does not induce a hypertrophic response.

In certain embodiments of any of the foregoing, the compound is used to treat myocardial damage from myocardial infarction. In certain other embodiments, the compound is used to treat decreased cardiac function due to any of myocardial infarction; atherosclerosis; coronary artery disease; obstructive vascular disease; dilated cardiomyopathy; heart failure; myocardial necrosis; valvular heart disease; non-compaction of the ventricular myocardium; hypertrophic cardiomyopathy; cancer or cancer-related conditions such as structural defects resulting from cancer or cancer treatments.

In certain embodiments of any of the foregoing, the compound is administered systemically. In certain other embodiments, the compound is administered to the myocardium, pericardium, or endocardium via a syringe, catheter, stent, wire, or other intraluminal device.

The invention contemplates methods and preparations comprising any combination of any of the forgoing aspects and embodiments. By way of example, compounds having any of the foregoing potency and/or selectivity characteristics can be used to promote cardiac cell proliferation and/or can be used in the treatment of any of the diseases, injuries, or conditions described herein.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. Detailed Description of the Drawings

Figures la-b show the effect of a selective GSK3 kinase inhibitor on cardiomyocyte and fibroblast proliferation. Figure Ia shows that a selective GSK3 kinase inhibitor promotes cardiomyocyte proliferation in a rat neonatal cardiomyocyte assay. Figure Ib shows that the effect of this compound is specific to a subset of cardiac cell types, and that it does not increase proliferation of fibroblasts.

Figure 2 shows that a selective GSK3 kinase inhibitor increases both phases of cardiomyocyte proliferation: DNA synthesis and cell division (as reflected in an increase in the total number of cardiomyocytes).

Figure 3 shows that a selective GSK3 kinase inhibitor promotes proliferation of adult cardiomyocytes.

Figures 4a-c show that a selective GSK3 kinase inhibitor that promotes proliferation of neonatal cardiomyocytes does not induce hypertrophy. In contrast to treatment with fetal calf serum (FCS; Figure 4a and 4c), culture with a selective GSK3 kinase inhibitor did not induce hypertrophy (2 uM BIO; Figure 4b).

Figures 5a-e show that a selective GSK3 kinase inhibitor that promotes proliferation of adult cardiomyocytes does not induce hypertrophy. In contrast to treatment with fetal calf serum (FCS; Figure 5a and 5b), culture with a selective GSK3 kinase inhibitor did not induce hypertrophy (BIO; Figure 5d and 5e).

Figures 6a-c show that a selective GSK3 kinase inhibitor is more effective than a structurally related, non-selective GSK3 kinase inhibitor for increasing both phases of cell proliferation. The superior efficacy of the selective GSK3 inhibitor is most dramatically observed with respect to the cell division phase of proliferation. The selective GSK3 inhibitor significantly increases cardiomyocyte number.

Figure 7 summarizes experiments measuring plasma levels of a selective GSK3 kinase inhibitor following administration of a single intraperitoneal dose to male rats. Figures 8a-b show that a selective GSK3 kinase inhibitor has efficacy in a rat model of myocardial infarction.

Figures 9a-b show that a selective GSK3 kinase inhibitor has efficacy in a rat model of myocardial infarction.

Figure 10a-b summarize results showing that two GSK3 inhibitors (BIO and Compound A) promote cardiac cell proliferation in a neonatal cardiomyocyte assay. Figure 10c provides the structure of Compound A. Detailed Description of the Invention (i) Overview

The present invention provides methods and compositions with broad implications in the area of cardiovascular disease and treatment. By promoting proliferation and/or regeneration of cardiac cells, instead of the scarring that typically results following disease or injury, the present invention provides methods and compositions with a range of important applications including: promoting cardiomyocyte proliferation, promoting regeneration of cardiomyocytes, and treating a range of cardiovascular conditions. Additionally, the compositions of the present invention are particularly useful for promoting cardiomyocyte proliferation and/or regeneration without producing a hypertrophic response. This provides a substantial benefit over other agents that may increase proliferation, but also induce cardiomyocyte hypertrophy. More generally, the invention provides methods and compositions for promoting proliferation and/or regeneration of cardiac cells and tissues. (U) Definitions

Unless defined otherwise, all technical and scientific terms have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The terms "GSK3 antagonist" and "GSK3 inhibitor" are used interchangeably to refer to compounds that decreases or suppresses a biological activity of GSK3. Exemplary compounds for use in the methods of the present invention inhibit GSK3 kinase activity.

The terms "selective GSK3 inhibitor" or "compound that selectively inhibits GSK3 activity" or "compound that selectively inhibits GSK3 kinase activity" are used interchangeably throughout to refer to the compounds for use in the methods of the present invention. Specifically, and in contrast to many kinase inhibitors, the subject compounds do not inhibit the kinase activity of all classes of kinases. Instead, these compounds are selective for inhibiting the kinase activity of GSK3 in comparison to the kinase activity of CDKl . Optionally, the subject compounds may also be selective for inhibiting the kinase activity of GSK3 in comparison to the kinase activity of one or more of CDK2, CDK4, CDK5, CDK6, or CDK7. Selectivity does not require that the subject compounds have zero activity against non-GSK3 kinases. Selectively only requires that the subject compounds are more potent inhibitors of GSK3 kinase activity than one or more of CDKl, CDK2, CDK4, CDK5, CDK6, or CDK7 kinase activity by at least one order of magnitude.

A "marker" is used to determine the state of a cell. Markers are characteristics, whether morphological or biochemical (enzymatic), particular to a cell type, or molecules expressed by the cell type. A marker may be a protein marker, such as a protein marker possessing an epitope for antibodies or other binding molecules available in the art. A marker may also consist of any molecule found in a cell, including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids and steroids. Additionally, a marker may comprise a morphological or functional characteristic of a cell. Examples of morphological traits include, but are not limited to, shape, size, and nuclear to cytoplasmic ratio. Examples of functional traits include, but are not limited to, the ability to adhere to particular substrates, ability to incorporate or exclude particular dyes, ability to migrate under particular conditions, and the ability to differentiate along particular lineages.

Markers may be detected by any method available to one of skill in the art. In addition to antibodies (and all antibody derivatives) that recognize and bind at least one epitope on a marker molecule, markers may be detected using analytical techniques, such as by protein dot blots, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), or any other gel system that separates proteins, with subsequent visualization of the marker (such as Western blots), gel filtration, affinity column purification; morphologically, such as fluorescent-activated cell sorting (FACS), staining with dyes that have a specific reaction with a marker molecule (such as ruthenium red and extracellular matrix molecules), specific morphological characteristics (such as the presence of microvilli in epithelia, or the pseudopodia/fϊlopodia in migrating cells, such as fibroblasts and mesenchyme); and biochemically, such as assaying for an enzymatic product or intermediate, or the overall composition of a cell, such as the ratio of protein to lipid, or lipid to sugar, or even the ratio of two specific lipids to each other, or polysaccharides. In the case of nucleic acid markers, any known method may be used. If such a marker is a nucleic acid, PCR, RT-PCR, in situ hybridization, dot blot hybridization, Northern blots, Southern blots and the like may be used, coupled with suitable detection methods. If such a marker is a morphological and/or functional trait, suitable methods include visual inspection using, for example, the unaided eye, a stereomicroscope, a dissecting microscope, a confocal microscope, or an electron microscope.

"Differentiation" describes the acquisition or possession of one or more characteristics or functions different from that of the original cell type. A differentiated cell is one that has a different character or function from the surrounding structures or from the precursor of that cell (even the same cell). The process of differentiation gives rise from a limited set of cells (for example, in vertebrates, the three germ layers of the embryo: ectoderm, mesoderm and endoderm) to cellular diversity, creating all of the many specialized cell types that comprise an individual.

Differentiation is a developmental process whereby cells assume a specialized phenotype, e.g., acquire one or more characteristics or functions distinct from other cell types. In some cases, the differentiated phenotype refers to a cell phenotype that is at the mature endpoint in some developmental pathway. In many, but not all tissues, the process of differentiation is coupled with exit from the cell cycle. In these cases, the cells typically lose or greatly restrict their capacity to proliferate and such cells are commonly referred to as being "terminally differentiated. However, we note that the term "differentiation" or "differentiated" refers to cells that are more specialized in their fate or function than at a previous point in their development, and includes both cells that are terminally differentiated and cells that, although not terminally differentiated, are more specialized than at a previous point in their development.

"Muscle cells" are characterized by their principal role: contraction. Muscle cells are usually elongate and arranged in vivo in parallel arrays. The principal components of muscle cells, related to contraction, are the myofilaments. Two types of myofilaments can be distinguished: (1) those composed primarily of actin, and (2) those composed primarily of myosin. While actin and myosin can be found in many other cell types, enabling such cells, or portions, to be mobile, muscle cells have an enormous number of co-aligned contractile filaments that are used to perform mechanical work.

"Cardiac muscle" or "myocardium" consists of long fibers that, like skeletal muscle, are cross-striated. Cardiac muscle is composed of cells referred to as cardiomyocytes. In addition to the sanations, cardiac muscle also contains special cross bands, the intercalated discs, which are absent in skeletal muscle. Also unlike skeletal muscle in which the muscle fiber is a single multinucleated protoplasmic unit, in cardiac muscle the fiber consists of mononucleated (sometimes binucleated) cells aligned end-to-end. Usually, injured cardiac muscle is replaced with fibrous connective tissue, not cardiac muscle.

The term "cardiac cell" includes not only cardiomyocytes, but also other cell types that comprise functional cardiac tissue. Exemplary cardiac cells include, but are not limited to, endocardial cells, pericardial cells, cardiomyocytes, epicardial cells, and mesocardial cells. Further exemplary cardiac cells include cardiac stem and progenitor cells.

"Proliferation" refers to an increase in the number of cells in a population by means of cell division. Cell proliferation results from the coordinated activation of multiple signal transduction pathways, often in response to growth factors and other mitogens. Cell proliferation may also be promoted when cells are released from the actions of intra- or extracellular signals and mechanisms that block or down-regulate cell proliferation. Proliferation includes two distinct phases: (i) a DNA synthesis phase and (ii) a cell division phase. An increase in the DNA synthesis phase of cell proliferation can be assessed by, for example, examining an increase in BrdU incorporation. An increase in the cell division phase of proliferation can be assessed by, for example, observation of an increase in total cell number over time. Compounds that increase proliferation may increase the DNA synthesis phase of proliferation, the cell division phase of proliferation, or both.

"Cardiomyocyte proliferation" refers to an increase in proliferation in a population of cells, wherein the population of cells includes cardiomyocytes. The following are examples of cardiomyocyte proliferation within the meaning of the present application: (i) proliferation of a particular cardiomyocyte contacted with a selective GSK3 inhibitor; (ii) proliferation of a daughter cell (e.g., progeny) of a cardiomyocyte that was contacted with a selective GSK3 inhibitor; (iii) proliferation of a related cell adjacent to the cardiomyocyte contacted with a selective GSK3 inhibitor.

As used herein, "protein" is a polymer consisting essentially of any of the 20 amino acids. Although "polypeptide" is often used in reference to relatively large polypeptides, and "peptide" is often used in reference to small polypeptides, usage of these terms in the art overlaps and is varied.

The terms "peptide(s)", "protein(s)" and "polypeptide(s)" are used interchangeably herein.

The terms "polynucleotide sequence" and "nucleotide sequence" are also used interchangeably herein.

The term "treating" includes prophylactic and/or therapeutic treatments. The term "prophylactic or therapeutic" treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, (i.e., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic, (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

The terms "compound" and "agent" are used interchangeably to refer to the inhibitors/antagonists of the invention. In certain embodiments, the compounds are small organic or inorganic molecules, e.g., with molecular weights less than 7500 amu, preferably less than 5000 amu, and even more preferably less than 2000, 1500, 1000, or 500 amu. One class of small organic or inorganic molecules are non- peptidyl, e.g., containing 2, 1, or no peptide and/or saccharide linkages. In certain other embodiments, the compounds are peptidyl agents such as polypeptides or antibodies.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrastemal injection and infusion.

The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the animal's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

The phrase "effective amount" as used herein means that the amount of one or more agent, material, or composition comprising one or more agents as described herein which is effective for producing some desired effect in a subject.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings. In certain preferred embodiments, "pharmaceutically acceptable" refers to those agents which can be used in animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation. (Ui) Exemplary Compositions and Methods

Most kinase inhibitors have significant cross reactivity against a range of kinases of different classes/families. This characteristic undermines their suitability for many in vitro and in vivo uses because the advantageous response promoted by inhibiting one kinase or class of kinases may be off-set by disadvantageous responses induced by simultaneously inhibiting a different kinase or class of kinases. The present invention is based on the appreciation that kinase inhibitors that are somewhat selective in inhibiting certain kinases over other kinases have useful applications.

The present invention is based on the observation that a certain class of selective kinase inhibitors can promote cardiac cell proliferation. Specifically, GSK3 kinase inhibitors that selectively inhibit the kinase activity of GSK3 in comparison to the kinase activity of CDKl and/or CDK2 can be used to promote cardiac cell proliferation. As shown in the Examples, structurally related compounds that are not selective for inhibiting the kinase activity of GSK3 over CDKl are significantly less effective in promoting cardiac cell proliferation. Specifically, such compounds are significantly less effective in promoting the cell division phase of proliferation. Without being bound by theory, compounds that inhibit the kinase activity of CDKl may block cell cycle progress, thereby undermining any proliferative effect achieved by inhibiting GSK3 kinase activity. Accordingly, compounds that selectively inhibit GSK3 kinase activity in comparison to CDKl kinase activity can more effectively promote cardiac cell proliferation. Such cardiac cell proliferation includes, but is not limited to, cell division. In one aspect, the invention provides a method of promoting cardiac cell proliferation. The method comprises contacting a cardiac cell with an amount of a compound effective to promote cardiac cell proliferation. Compounds for use in this method selectively inhibit GSK3 kinase activity.

In certain embodiments, such compounds inhibit GSK3 kinase activity with an IC₅₀ at least 1 order of magnitude lower than its IC₅₀ for inhibiting CDKl kinase activity. In certain other embodiments, such compounds inhibit GSK3 kinase activity with an IC₅₀ at least 1 order of magnitude lower than its IC₅0 for inhibiting the kinase activity of one or more of CDK2, CDK4, CDK5, CDK6, or CDK7. In certain other embodiments, such compounds inhibit GSK3 kinase activity with an ICsoat least 1 order of magnitude lower than its IC₅₀ for inhibiting CDKl kinase activity and at least 1 order of magnitude lower than its IC₅₀ for inhibiting the kinase activity of one or more of CDK2, CDK4, CDK5, CDK6, or CDK7.

In certain embodiments of any of the foregoing, the compounds promote regeneration.

In certain embodiments, the cardiac cells are cardiomyocytes. Such cardiac cells, including cardiomyocytes, may be neonatal, fetal, or adult cells. Such cardiac cells may be in, isolated from, or derived from any human or non-human species. Exemplary cells are mammalian cells including, but not limited to, mouse, rat, rabbit, cat, dog, pig, cow, non-human primate, or human.

In certain embodiments, the cardiac cells include one or more of pericardial cells, endocardial cells, mesocardial cells, or epicardial cells. In certain other embodiments, the cardiac cells include cardiac stem or progenitor cells, or other stem or progenitor cell populations resident in or transiting through the heart. Exemplary stem or progenitor cell populations resident in or transiting through the heart include, but are not limited to, mesenchymal stem cells and hematopoietic stem cells.

In certain embodiments, the compound is a small organic or inorganic molecule.

In certain embodiments, the method is conducted in an animal and said compound is formulated as a pharmaceutical preparation. In certain embodiments, exemplary compounds inhibit GSK3 kinase activity with an IC₅₀ of less than 500 nM, preferably less than 250 nM, 200 nM, or 100 nM. In certain other embodiments, exemplary compounds inhibit GSK3 kinase activity with an IC₅₀ of less than: 75 nM, 50 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM.

In certain embodiments, exemplary compounds inhibit GSK3 kinase activity with an IC₅₀ at least 1.5 orders of magnitude lower than its IC₅₀ for inhibiting CDKl kinase activity. Alternatively or additionally, in certain embodiments, exemplary compounds inhibit GSK3 kinase activity with an IC50 at least 1.5 orders of magnitude lower than its IC₅₀ for inhibiting one or more of CDK2, CDK4, CDK5, CDK6, or CDK7.

Certain exemplary compounds are chosen because they inhibit GSK3 kinase activity with an ICsoat least 10, 20, 25, 30, or 50 times lower than its IC5₀ for inhibiting CDKl kinase activity. Certain exemplary compounds are chosen because they inhibit GSK3 kinase activity with an IC₅₀ at least 2.5, 5, 10, 15, 20, 25, 30, or 50 times lower than its IC₅₀ for inhibiting the kinase activity of one or more of CDK2, CDK4, CDK5, CDK6, or CDK7. It is understood that exemplary compounds that inhibit or do not inhibit one or more of the aforementioned kinases may do so with the same or different IC50.

In any of the foregoing, the methods and compounds can be used to prevent, treat, or alleviate a disease or injury of cardiac cells. Furthermore, in any of the foregoing, the methods and compounds of the invention can be used to prevent, treat or alleviate a disease or injury characterized by decreased cardiac function.

Exemplary conditions and injuries that may be treated or alleviated include, but are not limited to, myocardial damage from myocardial infarction; myocardial infarction; atherosclerosis; coronary artery disease; obstructive vascular disease; dilated cardiomyopathy; heart failure; myocardial necrosis; valvular heart disease; non-compaction of the ventricular myocardium; hypertrophic cardiomyopathy; cancer or cancer-related conditions such as structural defects resulting from cancer or cancer treatments. Further exemplary conditions and injuries that may be treated or alleviated include, but are not limited to, myocarditis, exposure to a toxin, exposure to an infectious agent, or from a mineral deficiency. In certain embodiments, compounds may be administered systemically. In certain other embodiments, compounds may be administered to the myocardium, pericardium, or endocardium via a syringe, catheter, stent, wire, or other intraluminal device.

In certain embodiments, the compound is a small organic or inorganic molecule.

In certain embodiments, the compound binds to GSK3. In certain embodiments, the compound binds to GSK3α, GSK3β, or both GSK3α and GSK3β.

In certain embodiments, the compound promotes proliferation and/or regeneration without inducing a hypertrophic response. For example, the compound promotes cardiac cell proliferation without inducing cardiac hypertrophy.

Compounds, for example 6-bromo-indirubin-3'-monoxime, may be used in vitro or in vivo to promote cardiac cell proliferation and/or regeneration.

Compounds, for example 6-bromo-indirubin-3'-monoxime, may be used in the manufacture of medicaments for the treatment of any diseases disclosed herein.

Compounds, for example 6-bromo-indirubin-3'-monoxime, may be used to inhibit a function of a GSK3. In certain embodiments, the function inhibited is the kinase activity of GSK3.

Compounds according to the present invention may be used in in vitro or in vivo methods of promoting cardiac cell proliferation or cardiac cell regeneration. Furthermore, such compounds may be used in vitro or in vivo to inhibit the kinase activity of GSK3.

Particularly preferred compounds for use in any of the methods of the present invention promote both the DNA synthesis phase of proliferation and the cell division phase of proliferation leading to an increase in total cell number.

In particular embodiments, the small molecule is chosen for use because it is more selective for GSK3 than for certain other kinases. In a preferred embodiment, the small molecule is at least: 10-fold, 25-fold, 50-fold, 100-fold, or even 1000-fold more selective or 2000-fold more selective for GSK3 than for CDKl and/or CDK2. Alternatively or additionally, the small molecule is at least: 10-fold, 25-fold, 50-fold, 100-fold, or even 1000-fold more selective for inhibiting the kinase activity of GSK3 than for inhibiting the kinase activity of one or more of CDK2, CDK4, CDK5, CDK6, or CDK7. Such comparisons may be made, for example, by comparing IC₅₀ values.

In certain embodiments, a compound which inhibits GSK3 does so more strongly (e.g., inhibits kinase activity with a lower IC₅₀ value) than CDKl, preferably at least one and a half orders of magnitude more strongly, more preferably at least two orders of magnitude more strongly, even more preferably at least three orders of magnitude more strongly. Alternatively or additionally, the small molecule inhibits GSK3 kinase activity more strongly (e.g., with a lower IC50 value) than the kinase activity of one or more of CDK2, CDK4, CDK5, CDK6, or CDK7, preferably at least one order of magnitude more strongly, one and a half orders of magnitude more strongly, more preferably at least two orders of magnitude more strongly, even more preferably at least three orders of magnitude more strongly. Such comparisons may be made, for example, by comparing IC₅₀ values.

Similarly, in particular embodiments, the small molecule is chosen for use because it lacks significant activity against one or more targets other than GSK3. For example, the compound may have an IC₅₀ above 500 nM, above 1 μM, or even above 10 μM or 100 μM for inhibiting the kinase activity of one or more of CDKl, CDK2, CDK4, CDK5, CDK6, or CDK7. Additionally or alternatively, the compound may have an IC₅₀ above 500 nM, above 1 μM, or even above 10 μM or 100 μM for inhibiting the kinase activity of one or more of ERKl, ERK2, PKC (protein kinase C), casein kinase, insulin receptor tyrosine kinase, aktl, PI3 kinase, c-src, Fltl, bFGF receptor kinase, or IGF receptor kinase.

For any of the foregoing, in addition to inhibiting GSK3 kinase activity, the compound also promotes proliferation and/or regeneration of cardiac cells. In certain embodiments, the compound is chosen because it promotes cardiac cell proliferation and/or regeneration with an EC50 value of less than or equal to 500 nM. Tn certain other embodiments, the compound is chosen because it promotes cardiac cell proliferation and/or regeneration with an EC50 value of less than or equal to 400 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, 50 nM, or less than 50 nM. Such EC₅₀ value can be measured using a cardiomyocyte assay like that described in the Examples. In certain embodiments of any of the foregoing, the small molecule may be chosen because it inhibits a GSK function, for example, GSK kinase activity, with an IC₅O less than 250 nM, 200 nM, 100 nM, or even less than 50 nM, 25, 20, 10, 5, or 1 nM.

In certain embodiments of any of the foregoing, inhibition of a GSK3 function means that a function, for example kinase activity, is decreased by at least 50%, 60%, 70%, 75%, 80%, 85%, or 90% in the presence of an effective amount of a compound in comparison to in the absence of the compound. In still other embodiments, the inhibition of a GSK3 function means that a function, for example kinase activity, is decreased by at least 92%, 95%, 97%, 98%, 98%, 99%, or 100% in the presence of an effective amount of a compound in comparison to in the absence of the compound.

In any of the foregoing embodiments, IC₅₀ values for inhibiting kinase activity are measured in vitro in a cell-based or cell-free system using, for example, standard in vitro kinase assays described in the examples and the references cited therein. EC₅₀ values for promoting cardiac cell proliferation can be measured in vitro or in vivo in, for example, a neonatal or adult cardiomyocyte assay, as described in the Examples.

Without being bound by theory, a compound may inhibit a function of GSK3 by binding covalently or non-covalently to a portion of GSK3. Alternatively, a compound may inhibit a function of GSK3 indirectly, for example, by associating with a protein or non-protein cofactor necessary for a function of GSK3. One of skill in the art will readily appreciate that an inhibitory compound may associate reversibly or irreversibly with GSK3 or a cofactor thereof. Compounds that reversibly associate with GSK3 or a cofactor thereof may continue to inhibit a function of GSK3 even after dissociation.

In certain embodiments of any of the foregoing, the compound that selectively inhibits a function of GSK3 is a small organic molecule or a small inorganic molecule. Exemplary small molecules include, but are not limited to, small molecules that bind to GSK3 and inhibit one or more function of GSK3. In certain embodiments the compound binds to and inhibits GSK3-alpha and GSK3- beta with approximately equal IC₅₀ values. In certain other embodiments, the compound binds to and inhibits GSK3-alpha and GSK3-beta with IC50 values that differ by less than or equal to 2-fold, 3-fold, 4-fold, or 5-fold.

The subject selective GSK3 inhibitors can be used alone or in combination with other pharmaceutically active agents. Examples of such other pharmaceutically active agents include, but are not limited to, anti-fungal agents, anti-viral agents, anti-septic agents (e.g., antibacterials), local anaesthetics, anti-coagulents, beta- blockers, or other agents suitable for use as part of a therapeutic regimen appropriate in the treatment of a particular cardiovascular disease or condition.

The subject selective GSK3 inhibitors can be used alone or in combination with one or more other therapeutic agents or treatment regimens appropriate for the particular disease or condition being treated. Such combinatorial therapies may be administered simultaneously, concurrently, etc. Furthermore, combinatorial approaches include administering one selective GSK3 inhibitor or more than one (2, 3, or 4) selective GSK3 kinase inhibitors. When more than one selective GSK3 kinase inhibitors are used, the inhibitors may have the same or differing IC50 values for inhibiting GSK3, CDKl, CDK2, CDK4, CDK5, CDK6, and/or CDK7.

An exemplary selective GSK3 kinase inhibitor for use in the methods of the present invention is 6-brorno-indirubin-3'-monoxime (also known as BIO). BIO (6- bromo-indirubin-3'-oxime) has previously been shown to inhibit GSK3 kinase activity (See, Meijer et al. (2003) Chem Biol 10: 1255). BIO is a potent inhibitor of GSK3 kinase activity with an IC₅₀ in in vitro kinase assays of approximately 5 nM. Furthermore, unlike many potent kinase inhibitors that tend to potently inhibit the activity of multiple different families of kinases, BIO is somewhat selective for GSK3 kinases in comparison to cyclin dependent kinases. For example, its IC₅₀ in vitro for CDKl is approximately 320 nM and its IC₅₀ in vitro for CDK5 is approximately 83 nM. In other words, BIO inhibits the kinase activity of GSK greater than 50-times more (e.g., greater than 1¹A orders of magnitude more potently) than the kinase activity of CDKl. Furthermore, BIO inhibits the kinase activity of GSK3 greater than 15-times more (e.g., greater than 1 order of magnitude more potently) than the kinase activity of CDK5.

In addition to selectivity over CDK family kinases, BIO also selectively inhibits GSK kinase activity in comparison to numerous other kinases. For example, BIO only weakly (IC₅0 greater than 10 micromolar) inhibits the activity of the following kinases: ERKl, ERK2, MAPKK, PKCα, PKCβl, PKCβ2, PKCγ, cAMP- dependent protein kinase, cGMP-dependent protein kinase, casein kinase, and insulin receptor tyrosine kinase. Thus, BIO inhibits the kinase activity of GSK3 greater than 1000 times more potently than that of any of the foregoing kinases.

As detailed in the Examples, BIO promotes proliferation of neonatal and adult cardiomyocytes. The increased proliferation promoted by BIO is accompanied by cell division, and thus exposing cells to this compound also increases cell number over time. Furthermore, the effect of BIO on cardiac cells is independent of cardiac hypertrophy (e.g., contacting cardiac cell with BIO does not induce a hypertrophic response).

Similar results were not observed with the structurally related compound 5- iodo-indirubin-3-monoxime (ITO). Although structurally related, these two GSK3 kinase inhibitors differ in their potency against CDKl and CDK5. HO inhibits GSK3 with an IC₅₀ similar to that of BIO. However, HO inhibits CDKl with similar potency while BIO does not. Specifically, HO inhibits CDKl kinase activity with an IC₅₀ of 25 nM making it approximately 12 times more potent (in an absolute sense) against CDKl than BIO is.

Another exemplary selective GSK3 kinase inhibitor for use in the methods of the present invention is depicted in Figure 10c (Compound A) and has the following structure:

Compound A, which is structurally distinct from BIO, is a potent inhibitor of GSK3 kinase activity with an IC50 in in vitro kinase assays of less than 1 nM (Diabetes, 2003, 52: 588-595). Unlike many potent kinase inhibitors that tend to potently inhibit the activity of multiple different families of kinases, Compound A is somewhat selective for GSK3 kinases in comparison to cyclin dependent kinases. For example, its IC50 in vitro for CDK2 is approximately 3700 nM. In other words, Compound A inhibits the kinase activity of GSK3 greater than 3000-times more potently (e.g., greater than 3 orders of magnitude more potently) than the kinase activity of CDK2.

In addition to selectivity over CDK family kinases, Compound A also selectively inhibits GSK kinase activity in comparison to numerous other kinases. For example, Compound A only weakly (IC₅₀ greater than 1 micromolar) inhibits the activity of the following kinases: ERK2 (IC₅0 greater than 10 micromolar), PKCα (IC₅₀ greater than 10 micromolar), insulin receptor tyrosine kinase, aktl (IC50 greater than 5 micromolar), p70 S6K, p90 RSK2 (IC₅₀ greater than 10 micromolar), c-src, tie2 (IC₅₀ greater than 5 micromolar), Fltl (IC₅₀ greater than 5 micromolar), KDR, bFGF receptor tyrosine kinase, IGFl receptor tyrosine kinase, chkl (IC50 greater than 10 micromolar), DNA protein kinase (IC5₀ greater than 10 micromolar), and PI3 kinase. Thus, Compound A inhibits the kinase activity of GSK3 greater than 1000 times more potently than that of any of the foregoing kinases.

As detailed in the Examples, Compound A promotes proliferation of neonatal cardiomyocytes. (iv) Exemplary Injuries and Conditions

The methods and compositions of the present invention provide a treatment for any of a wide range of injuries and diseases that compromise the functional performance of cardiac tissue. Because the methods and compositions of the present invention promote, for example, cardiomyocyte or other cardiac cell proliferation, regeneration, and/or survival, and thus overcome the typical scarring response of cardiac tissue to myocardial damage, these methods and compositions help restore cardiac function independent of the cause of the original injury. Accordingly, the present invention has broad applicability to a wide range of conditions, including developmental disorders and congenital defects.

As outlined in detail throughout the application, the invention contemplates administration of one or more selective GSK3 inhibitors. Compounds of the invention can be administered alone, in combination with other related or unrelated agents, or as part of a therapeutic regimen. Compounds of the invention can be administered as pharmaceutical compositions formulated in a pharmaceutically acceptable carrier or excipient.

By way of non-limiting example, we provide a brief description of exemplary conditions that diminish the functional performance of cardiac tissue. The invention contemplates methods of treating any of these diseases, as well as other diseases that result in myocardial injury that diminishes cardiac function. Myocardial infarction: Myocardial infarction is defined as myocardial cell death due to prolonged ischemia. Cell death is categorized pathologically as either coagulation or contraction band necrosis, or both, which usually evolves through necrosis, but can result to a lesser degree from apoptosis.

After the onset of myocardial ischemia, cell death is not immediate but takes a finite period to develop (as little as 15 min in some animal models, but even this may be an overestimate). It takes 6 hours before myocardial necrosis can be identified by standard macroscopic or microscopic postmortem examination. Complete necrosis of all myocardial cells at risk requires at least 4-6 hours or longer, depending on the presence of collateral blood flow into the ischemic zone, persistent or intermittent coronary artery occlusion and the sensitivity of the myocytes.

Infarcts are usually classified by size — microscopic (focal necrosis), small (<10% of the left ventricle), medium (10% to 30% of the left ventricle) or large (>30% of the left ventricle) — as well as by location (anterior, lateral, inferior, posterior or septal or a combination of locations). The pathologic identification of myocardial necrosis is made without reference to morphologic changes in the epicardial coronary artery tree or to the clinical history.

The term MI in a pathologic context may be preceded by the words "acute, healing or healed." An acute or evolving infarction is characterized by the presence of polymorphonuclear leukocytes. If the interval between the onset of infarction and death is brief (e.g., 6 h), minimal or no polymorphonuclear leukocytes may be seen. The presence of mononuclear cells and fibroblasts and the absence of polymorphonuclear leukocytes characterize a healing infarction. A healed infarction is manifested as scar tissue without cellular infiltration. The entire process leading to a healed infarction usually requires five to six weeks or more. Furthermore, reperfiision alters the gross and microscopic appearance of the necrotic zone by producing myocytes with contraction bands and large quantities of extravasated erythrocytes.

Infarcts are classified temporally according to the pathologic appearance as follows: acute (6 h to 7 days); healing (7 to 28 days), healed (29 days or more). It should be emphasized that the clinical and ECG timing of an acute ischemic event may not be the same as the pathologic timing of an acute infarction. For example, the ECG may still demonstrate evolving ST-T segment changes, and cardiac troponin may still be elevated (implying a recent infarct) at a time when, pathologically, the infarct is in the healing phase.

Myocardial necrosis results in and can be recognized by the appearance in the blood of different proteins released into the circulation due to the damaged myocytes: myoglobin, cardiac troponins T and I, creatine kinase, lactate dehydrogenase, as well as many others. Myocardial infarction is diagnosed when blood levels of sensitive and specific biomarkers, such as cardiac troponin and the MB fraction of creatine kinase (CK-MB), are increased in the clinical setting of acute ischemia. These biomarkers reflect myocardial damage but do not indicate its mechanism. Thus, an elevated value in the absence of clinical evidence of ischemia should prompt a search for other causes of cardiac damage, such as myocarditis.

The presence, absence, and amount of myocardial damage resulting from prolonged ischemia can be assessed by a number of different means, including pathologic examination, measurement of myocardial proteins in the blood, ECG recordings (ST-T segment wave changes, Q waves), imaging modalities such as myocardial perfusion imaging, echocardiography and contrast ventriculography. For each of these techniques, a gradient can be distinguished from minimal to small to large amounts of myocardial necrosis. Some clinicians classify myocardial necrosis as microscopic, small, moderate and large on the basis of the peak level of a particular biomarker. The sensitivity and specificity of each of these techniques used to detect myocardial cell loss, quantitate this loss and recognize the sequence of events over time, differ markedly. We note that the term myocardial necrosis refers to any myocardial cell death regardless of its cause. Although myocardial infarction is one cause of myocardial necrosis, many other conditions result in necrosis. The methods and compositions of the invention can be used to promote cardiomyocyte proliferation and/or regeneration, and thus improve cardiac function following myocardial infarction, as well as myocardial necrosis caused by any injury or condition.

Noncompaction of the ventricular myocardium: This rare condition, also known as "spongy myocardium," is a congenital cardiomyopathy of children and adults resulting from arrested myocardial development during embryogenesis. Prior to formation of the epicardial coronary circulation at about 8 weeks of life, the myocardium is a meshwork of interwoven myocardial fibers that form trabeculae and deep trabecular recesses. The increased surface area permits perfusion of the myocardium by direct communication with the left ventricular cavity. Normally, as the myocardium undergoes gradual compaction, the epicardial coronary vessels form.

In this developmental disorder, echocardiography demonstrates a thin epicardium with extremely hypertrophied endocardium and prominent trabeculations with deep recesses. These features tend to be apically localized since compaction would normally proceed from base to apex, and from epicardium to endocardium.

Clinical presentation consists of congestive heart failure with depressed left ventricular systolic function, ventricular arrhythmias, arterial thromboemboli from thrombus formation within the inter-trabecular recesses, as well as restrictive physiology from endocardial fibrosis. The diagnosis can be made echocardiographically, and the entity may be associated with problems of cardiac rhythm. The methods and compositions of the present invention can be used to improve the impairments of the ventricular myocardium, and thus to help restore some of the diminished cardiac function.

The severity of noncompaction of the ventricular myocardium varies among patients, and patients with less severe disease may not present until later in life. In addition to patient populations presenting with only noncompaction of the ventricular myocardium, this disorder is also associated with more complex, multisystem syndromes. For example, noncompaction of the ventricular myocardium is also observed in Wolf-Parkinson- White syndrome and Roifman syndrome. Accordingly, the methods and compositions of the present invention may also be useful in ameliorating the noncompaction of the ventricular myocardium-related effects in these multi-system syndromes.

Congenital heart defects: Congenital heart defects are heart problems present at birth. They happen when the heart does not develop normally before birth. About 8 out of every 1,000 infants are born with one or more heart or circulatory problems. Doctors usually do not know the cause of congenital heart defects, but they do know of some conditions that increase a child's risk of being bom with a heart defect. Such conditions include the following: (i) congenital heart disease in the mother or father; (ii) congenital heart disease in a sibling; (iii) diabetes in the mother; (iv) German measles, toxoplasmosis, or HIV infection in the mother; (v) mother's use of alcohol during pregnancy; (vi) mother's use of cocaine or other drugs during pregnancy; (vii) mother's use of certain over-the-counter and prescription medicines during pregnancy.

Congenital heart defects are often detected at birth, however certain defects are not diagnosed until later in life. In still other cases, the heart defect can be detected in utero — prior to birth. Given the broad range of congenital heart defects, as well as the variability in their onset and severity, effective methods of treatment previously needed to be designed for each particular condition. The present methods and compositions provide effective treatment option for this diverse class of disorders that decrease myocardial function. By way of example, congenital heart defects include atrial septal defects (ASD); ventricular septal defects (VSD); atrioventricular canal defects; patent ductus arteriosus; aortic Stenosis; pulmonary stenosis; Ebstein's anomaly; coarctation of the aorta; Tetralogy of Fallot; transposition of the great arteries; persistent truncus arteriosus; tricuspid atresia; pulmonary atresia; total anomalous pulmonary venous connection; and hypoplastic left heart syndrome.

Hypoplastic left heart syndrome: HLHS is an underdevelopment of the left side of the heart characterized by aortic valve atresia, hypoplastic ascending aorta, hypoplastic/atretic mitral valve, and endocardial fibroelastosis. Hypoplastic left heart syndrome is the most common cause of congenital heart failure in newborns, and is responsible for 25% of cardiac deaths occurring during the first week of life. If left untreated, this disorder has a 100% fatality rate. The PDA usually closes a few days after birth, and separates the left and right sides of the heart. It is at this time that babies with undetected HLHS will exhibit problems as they experience a lack of blood flow to the body. They may look blue, have trouble eating, and breathe rapidly. If left untreated, this heart defect is fatal - usually within the first few days or weeks of life.

Currently, treatment for hypoplastic left heart syndrome requires one of two surgical procedures, and the patient must remain on the drug prostaglandin until surgery is performed. The present invention provides a novel, less invasive treatment option for this otherwise fatal disorder, and can be used alone or in combination with currently available surgical procedures. Dilated Cardiomyopathy: DCM is an acquired disease characterized by the progressive loss of cardiac contractility. Although the causes of many forms of DCM are unknown, the causes of particular forms of DCM have been identified and include taurine deficiency, adriamycin, and parvovirus. As cardiac contractile function is progressively lost, there is a decrease in cardiac output. Increased blood volume and pressure within the chambers causes them to dilate, most dramatically evident in the left atrium and left ventricle. In response to the poor contractility and decreased cardiac output, the sympathetic nervous system and the renin-angiotensin- aldosterone axis are activated. As with degenerative valve disease, these compensatory mechanisms are initially beneficial, however their chronic activation becomes deleterious. Constant stimulation of the heart by the sympathetic nervous system causes ventricular arrhythmias and myocyte death, while constant activation of the renin-angiotensin-aldosterone axis causes excessive vasoconstriction and retention of sodium and water. The majority of cases exhibit signs of left-sided congestive heart failure, although right-sided signs (ascites) can also occur. Infection and toxicity: The myocardium is affected by a variety of disease processes including the primary muscle disorders such as dilated cardiomyopathy and hypertrophic cardiomyopathy, degenerative and inflammatory diseases, neoplasia, and infarction. The myocardium is also sensitive to toxin exposure, including adriamycin, oleander, and fluoroacetate.

Myocarditis occurs in all species and may be caused by viral, bacterial, parasitic, and protozoal infection. Canine parvovirus, encephalomyocarditis virus, and equine infectious anemia are viruses with a propensity to cause myocarditis. Myocardial degeneration occurs in lambs, calves, and foals with white muscle disease, and in pigs with mulberry heart disease or hepatosis dietetica. Mineral deficiencies can also result in myocardial degeneration, including iron, selenium, and copper.

Common causes of myocarditis include the following: streptococcus, Salmonella, Clostridium, viral Equine influenza, Borrelia burgdorferi, and Strongylosis. Furthermore, vitamin E and selenium deficiency are known to cause myocardial necrosis.

Cardiac toxins include ionophore antibiotics such as monensin and salinomycin, cantharidin (blister beetle toxicosis), Cryptostegia grandiflora (rubber vine poisoning), and Eupatorium rugosum (white snake root poisoning). These diseases cause typical signs of congestive heart failure - exercise intolerance, tachycardia, and tachypnea.

Current treatment for toxicity and infection aim to stabilize the cardiac symptoms, while addressing the underlying infection or poisoning. However, this approach does not address the actual myocardial damage or necrosis that may result from infection or exposure to toxins. The present invention addresses such myocardial damage resulting from infection and toxicity.

DiGeorge syndrome: DiGeorge syndrome is a multi-system disorder characterized by a few specific cardiac malformations, a sub-set of facial attributes, and certain endocrine and immune anomalies. The cause of DiGeorge syndrome has been identified as a submicroscopic deletion of chromosome 22 in the DiGeorge chromosomal region. It is classified along with velo-cardio-facial syndrome (Shprintzen syndrome) and conotruncal anomaly face syndrome as a 22ql 1 microdeletion and is sometimes referred to by the simple name 22ql 1 syndrome.

People with DiGeorge syndrome may have the following congenital heart lesions: tetralogy of Fallot, interrupted aortic arch type B, truncus arteriosus, aberrant left subclavian artery, right infundibular stenosis, or ventricular septal defect. 74% of patients with 22ql 1 syndrome have conotruncal malformations. 69% of patients are found to have palatal abnormalities including velopharyngeal incompetence (VPI), submucosal cleft palate, and cleft palate. Given the large percentage of DiGeorge syndrome patients with significant cardiac malformation, the methods and compositions of the present invention may be used to help augment, improve, or restore diminished cardiac function.

The foregoing examples are merely illustrative of the broad range of diseases and injuries of vastly different mechanisms that can be treated using the methods and compositions of the present invention. Additionally, we note that although some of the foregoing conditions effect the vasculature, any condition that alters blood flow to or from the heart can damage cardiac tissue. Accordingly, the methods and compositions of the present invention can be used to treat diseases and injuries that primarily affect cardiac tissue, as well as diseases and injuries that affect cardiac tissue secondarily to a defect in the vasculature that alters blood flow or oxygenation of cardiac tissue. (v) Pharmaceutical Compositions and Methods of Administration

The present invention provides compounds that selectively inhibit the activity of GSK3. Such agents can be used alone or in combination with other agents or with other therapeutic regimens appropriate for the particular application of the invention.

The invention further contemplates pharmaceutical compositions comprising one or more compounds that selectively inhibit the activity of GSK3. Further exemplary pharmaceutical compositions include pharmaceutical compositions comprising one or more of the above referenced compositions formulated or delivered with one or more other unrelated agents.

The pharmaceutical compositions of the present invention are formulated according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA). Pharmaceutical formulations of the invention can contain the active polypeptide and/or agent, or a pharmaceutically acceptable salt thereof. These compositions can include, in addition to an active polypeptide and/or agent, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other material well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active agent. Preferable pharmaceutical compositions are non-pyrogenic. The carrier may take a wide variety of forms depending on the route of administration, e.g., intravenous, intravascular, oral, intrathecal, epineural or parenteral, transdermal, etc. Furthermore, the carrier may take a wide variety of forms depending on whether the pharmaceutical composition is administered systemically or administered locally, as for example, via a biocompatible device such as a catheter, stent, wire, or other intraluminal device. Additional methods of local administration include local administration that is not via a biocompatible device.

Illustrative examples of suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin. The carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like.

In one embodiment, the pharmaceutical composition is formulated for sustained-release. An exemplary sustained-release composition has a semi permeable matrix of a solid biocompatible polymer to which the composition is attached or in which the composition is encapsulated. Examples of suitable polymers include a polyester, a hydrogel, a polylactide, a copolymer of L-glutamic acid and ethyl-L-glutamase, non-degradable ethylene-vinyl acetate, a degradable lactic acid-glycolic acid copolymer, and poly-D+- hydroxybutyric acid.

Polymer matrices can be produced in any desired form, such as a film, or microcapsules.

Other sustained-release compositions include liposomally entrapped modified compositions. Liposomes suitable for this purpose can be composed of various types of lipids, phospholipids, and/or surfactants. These components are typically arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposomes containing the selective GSK3 inhibitors of the present invention are prepared by known methods (see, for example, Epstein, et al. (1985) PNAS USA 82:3688-92, and Hwang, et al., (1980) PNAS USA, 77:4030).

Pharmaceutical compositions according to the invention include implants, i.e., compositions or device that are delivered directly to a site within the body and are, preferably, maintained at that site to provide localized delivery. For example, a preferred use for the methods and compositions of the present invention is to promote cardiac cell (e.g., cardiomyocyte) proliferation and/or regeneration. The compositions, including the pharmaceutical compositions described in the present application can be administered systemically, or locally. Locally administered compositions can be delivered, for example, to the pericardial sac, to the pericardium, to the endocardium, to the great vessels surrounding the heart (e.g., intravascularly to the heart), via the coronary arteries, or directly to the myocardium. When delivering to the myocardium to promote proliferation and repair damaged myocardium, the invention contemplates delivering directly to the site of damage or delivery to another site at some distance from the site of damage. Exemplary methods of administering compositions systemically or locally will be described in more detail herein.

The compounds, compositions, and pharmaceutical compositions thereof, of the invention also include implants comprising one or more of the compounds of the invention attached to a biocompatible support. Preferable biocompatible supports include, without limitation, stents, wires, catheters, and other intraluminal devices. In one embodiment, the biocompatible support can be delivered intravascularly or intravenously.

The support can be made from any biologically compatible material, including polymers, such as polytetrafluorethylene (PFTE), polyethylene terphthalate, Dacronftpolypropylene, polyurethane, polydimethyl siloxame, fluorinated ethylene propylene (FEP), polyvinyl alcohol, poly(organo)phosphazene (POP), poly-1-lactic acid (PLLA), polyglycolic/polylactic acid copolymer, methacrylphosphorylcholine and laurylmethacrylate copolymer, phosphorylcholine, polycaprolactone, silicone carbide, cellulose ester, polyacrylic acid, and the like, as well as combinations of these materials. Metals, such as stainless steel, nitinol, titanium, tantalum, and the like, can also be employed as or in the support. The compounds may be cross-linked or covalently attached to the biocompatible support. Alternatively, the compounds may be formulated on, dissolved in, or otherwise noncovalently associated with the biocompatible support. In certain embodiments, the support is sufficiently porous to permit diffusion of compounds or products thereof across or out of the support. In other embodiments, the compound remains substantially associated with or attached to the support. Supports can provide pharmaceutical compositions of the invention with desired mechanical properties. Those skilled in the art will recognize that minimum mechanical integrity requirements exist for implants that are to be maintained at a given target site.

Preferred intravascular implants, for example, should resist the hoop stress induced by blood pressure without rupture or aneurysm formation.

The size and shape of the support is dictated by the particular application. If the support is to be maintained at a vascular site, a tubular support is conveniently employed.

In other embodiments, the one or more compounds are delivered via a biocompatible, intraluminal device, however, the compound is not crosslinked or otherwise dissolved in the device. For example, the invention contemplates use of a catheter or other device to deliver a bolus of a compound, composition, or pharmaceutical composition. In such embodiments, the compound may not necessarily be associated with the catheter. The use of a catheter, or other functionally similar intraluminal device, allows localized delivery via the vasculature. For example, an intraluminal device can be used to deliver a bolus of compound directly to the myocardium, endocardium, or pericardium/pericardial space. Alternatively, an intraluminal device can be used to locally deliver a bolus of compound in the vasculature adjacent to cardiac tissue.

By way of illustration, intracardial injection catheters can be used to deliver the compositions of the invention directly to, for example, the myocardium or endocardium. Such catheters can be used, for example, in combination with imaging technology to deliver compositions directly into the myocardium. By way of specific example, the Stiletto™ injection system (Boston Scientific) includes two concentric fixed guide catheters and a spring loaded needle component. This and other similar injection catheters can be used for localized delivery to, for example, the myocardium or endocardium. Furthermore, such injection catheters can be used for delivery of agents into the pericardial sac. (Karmarkar et al. (2004) Magnetic Resonance in Medicine 51: 1163-1172; Naimark et al. (2003) Human Gene Therapy 14: 161-166; Bao et al. (2001) Catheter Cardiovasc Interv. 53: 429-434). As outlined above, biocompatible devices for use in the various methods of delivery contemplated herein can be composed of any of a number of materials. The biocompatible devices include wires, stents, catheters, balloon catheters, and other intraluminal devices. Such devices can be of varying sizes and shapes depending on the intended vessel, duration of implantation, particular condition to be treated, and overall health of the patient. A skilled physician or cardiovascular surgeon can readily select from among available devices based on the particular application.

By way of further illustration, exemplary biocompatible, intraluminal devices are currently produced by several companies including Cordis, Boston Scientific, Guidant, and Medtronic (Detailed description of currently available catheters, stents, wires, etc., are available at www.cordis.com; www.medtronic.com: www.bostonscientific.comy One of skill in the art can readily select from amongst currently available or later designed devices to select a device appropriate for a particular application of the methods and compositions of the present invention.

The invention also provides articles of manufacture including pharmaceutical compositions of the invention and related kits. The invention encompasses any type of article including a pharmaceutical composition of the invention, but the article of manufacture is typically a container, preferably bearing a label identifying the composition contained therein.

The container can be formed from any material that does not react with the contained composition and can have any shape or other feature that facilitates use of the composition for the intended application. A container for a pharmaceutical composition of the invention intended for parental administration generally has a sterile access port, such as, for example, an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle.

Kits of the invention generally include one or more such articles of manufacture and preferably include instructions for use. Preferred kits include one or more devices that facilitate delivery of a pharmaceutical composition of the invention to a target site.

Compounds for use in the methods of the present invention may be conveniently formulated for administration with a biologically acceptable medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.

Optimal concentrations of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists. As used herein, "biologically acceptable medium" includes solvents, dispersion media, and the like which may be appropriate for the desired route of administration of the one or more agents. The use of media for pharmaceutically active substances is known in the art. Except insofar as a conventional media or agent is incompatible with the activity of a particular agent or combination of agents, its use in the pharmaceutical preparation of the invention is contemplated. Suitable vehicles and their formulation inclusive of other proteins are described, for example, in the book Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA 1985). These vehicles include injectable "deposit formulations".

Methods of introduction may also be provided by delivery via a biocompatible, device. Biocompatible devices suitable for delivery of the subject agents include intraluminal devices such as stents, wires, catheters, sheaths, and the like. However, administration is not limited to delivery via a biocompatible device. As detailed herein, the present invention contemplates any of number of routes of administration and methods of delivery. Furthermore, when an agent is delivered via a biocompatible device, the invention contemplates that the agent may be covalently linked, crosslinked to or otherwise associated with or dissolved in the device, or may not be so associated.

The agents identified using the methods of the present invention may be given orally, parenterally, or topically. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, ointment, controlled release device or patch, or infusion.

The effective amount or dosage level will depend upon a variety of factors including the activity of the particular one or more agents employed, the route of administration, the time of administration, the rate of excretion of the particular agents being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular agents employed, the age, sex, weight, condition, general health and prior medical history of the animal, and like factors well known in the medical arts.

The one or more agents can be administered as such or in admixtures with pharmaceutically acceptable and/or sterile carriers and can also be administered in conjunction with other compounds. These additional compounds may be administered sequentially to or simultaneously with the agents for use in the methods of the present invention.

Agents can be administered alone, or can be administered as a pharmaceutical formulation (composition). Said agents may be formulated for administration in any convenient way for use in human or veterinary medicine. In certain embodiments, the agents included in the pharmaceutical preparation may be active themselves, or may be a prodrug, e.g., capable of being converted to an active compound in a physiological setting.

Thus, another aspect of the present invention provides pharmaceutically acceptable compositions comprising an effective amount of one or more agents, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) delivery via a stent or other biocompatible, intraluminal device; (2) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (3) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (4) topical application, for example, as a cream, ointment or spray applied to the skin; or (5) ophthalmic administration, for example, for administration following injury or damage to the retina; (6) intramyocardial, intrapericardial, or intraendocardial administration; (7) intravascularly, intravenously, or via the coronary artiers. However, in certain embodiments the subject agents may be simply dissolved or suspended in sterile water. In certain embodiments, the pharmaceutical preparation is non-pyrogenic, i.e., does not elevate the body temperature of a patient. Some examples of the pharmaceutically acceptable carrier materials that may be used include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safϊlower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

In certain embodiments, one or more agents may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term "pharmaceutically acceptable salts" in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of agent of the present invention. These salts can be prepared in situ during the final isolation and purification of the agents of the invention, or by separately reacting a purified agent of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. ScL 66:1-19)

The pharmaceutically acceptable salts of the agents include the conventional nontoxic salts or quaternary ammonium salts of the agents, e.g., from non-toxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.

In other cases, the one or more agents may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of agents of the present invention. These salts can likewise be prepared in situ during the final isolation and purification of the agents, or by separately reacting the purified agent in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (See, for example, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Formulations of the present invention may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the agent which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.

Methods of preparing these formulations or compositions include the step of bringing into association an agent with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association an agent of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a agent of the present invention as an active ingredient. An agent of the present invention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

Liquid dosage forms for oral administration of the agents of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active agents, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Transdermal patches have the added advantage of providing controlled delivery of an agent of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the agents in the proper medium. Absorption enhancers can also be used to increase the flux of the agents across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the agent in a polymer matrix or gel.

Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more agents of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of an agent, it is desirable to slow the absorption of the agent from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the agent then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered agent form is accomplished by dissolving or suspending the agent in an oil vehicle.

For any of the foregoing, the invention contemplates administration to neonatal, adolescent, and adult patients, and one of skill in the art can readily adapt the methods of administration and dosage described herein based on the age, health, size, and particular disease status of the patient. Furthermore, the invention contemplates administration in utero to treat conditions in an affected fetus. Exemplifications

The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention. Example 1 : A Selective GSK3 Inhibitor Promotes Cardiomyocvte Proliferation

BIO (6-bromo-indirubin-3'-oxime) has previously been shown to inhibit GSK3 kinase activity (See, Meijer et al. (2003) Chem Biol 10: 1255). BIO is a potent inhibitor of GSK3 kinase activity with an IC₅₀ in in vitro kinase assays of approximately 5nM. Furthermore, unlike many potent kinase inhibitors that tend to potently inhibit the activity of multiple different families of kinases, BIO is somewhat selective for GSK3 kinases in comparison to cyclin dependent kinases. For example, its IC50 in vitro for CDKl is approximately 320 nM and its IC50 in vitro for CDK5 is approximately 83 nM. In other words, BIO inhibits the kinase activity of GSK greater than 50-times more (e.g., greater than 114 orders of magnitude more potently) than the kinase activity of CDKl. Furthermore, BIO inhibits the kinase activity of GSK3 greater than 15-times more (e.g., greater than 1 order of magnitude more potently) than the kinase activity of CDK5.

We assessed the ability of this selective GSK3 inhibitor to promote cardiomyocvte proliferation in a rat, neonatal cardiomyocvte assay. As summarized in Figure Ia, BIO promotes cardiomyocvte proliferation, as assessed by incorporation of BrdU (e.g., it promotes the DNA synthesis phase of proliferation). The EC₅₀ of BIO in this in vitro neonatal cardiomyocvte assay was approximately 200-30OnM. Across various experiments and preparations of neonatal cardiomyocytes, the EC₅0 has varied between 100-30OnM. In contrast and as summarized in Figure Ib, BIO does not promote proliferation of cardiac fibroblasts. Without being bound by theory, this indicates that the proliferative affects of BIO are, at least, somewhat selective to subsets of cardiac cell types including cardiomyocytes. METHODS:

(a) Preparation of Neonatal Rat Cardiomyocytes: Neonatal rat cardiomyocytes were isolated from postnatal day 2 Wistar rat pups. Rat pups were anesthetized by hypothermia on ice for 10 min and euthanized by decapitation. Hearts were collected and placed in cold ADS buffer (NaCl 6.8g/L, HEPES 4.76g/L, NaH₂PO₄ 0.12g/L, Glucose lg/L, KCl 0.4g/L, MgSO₄ O.lg/L, pH 7.4). The atria were removed and ventricles were washed in ADS buffer and cut into pieces smaller than 2 millimeters. Ventricles were dissociated in ADS buffer containing 0.4mg/ml collagenase type IT and 0.2mg/ml pancreatin in a spinner flask at 37 ⁰C. Cell suspension from the first 15 minutes dissociation was discarded. Cell suspension was then collected every 20 minutes, for up to 6 times. To each collection, ¹A volume of newborn calf serum (NCS) was added to inactivate the collagenase/pancreatin; the cells were centrifuged at 1200 rpm for 6 minutes and resuspended in the same amount of NCS. The cell collection was incubated at 37 ⁰C, 10% CO₂. Cells from all the collections were pooled together and centrifuged at 1200 rpm for 6 min at room temperature. Cell pellets were resuspended in ADS buffer (4ml/ 10- 12 pups). 2 ml of cell suspension was applied to each Percoll gradient set up in the following manner. The top Percoll gradient (density 1.059 g/ml) was generated by mixing Percoll stock (9 parts Percoll: 1 part 1Ox ADS buffer) with Ix ADS buffer in a 9:1 1 ratio, and the bottom Percoll gradient (density 1.082 g/ml) was made by mixing Percoll stock and Ix ADS buffer in a 13:7 ratio. 4 ml of the top Percoll was added to a 15 ml conical tube, and three ml of the bottom Percoll was laid below it by placing a pipette tip at the bottom of the tube, then carefully withdrawing it as the solution was delivered. The gradients were centrifuged at 3000 rpm for 30 minutes at room temperature without brake. Cardiac fibroblast were located above the top percoll buffer while cardiac myocytes were located at the interface of the top and bottom percoll layers. Blood and nondissociated tissue were found at the bottom of the tube. Cardiac myocytes were carefully collected and washed in 50 ml Ix ADS buffer after the top percoll was aspirated. After centrifugation at 1200 rpm for 6 minutes, myocyte pellets were resuspended in plating medium [DMEM (Gibco cat# 11965-092) plus 5% horse serum, 1% penicillin/streptomycin] at the density of 100 K/ml. 100 ul were added to each well of 96 half-area plates, which had previously been coated with 0.1% gelatin/12.5 ug/ml fibronectin. Plated cells were grown for 48 hours at 37 ⁰C , 10% CO₂ prior to use in further experiments (b) Neonatal cardiomyocyte (NCM) Assay: Neonatal rat cardiomyocytes were prepared and cultured as outlined in above. Cells were grown for 48 hours at 37 ⁰C, then washed 4 times with neonatal base medium containing DMEM, 25 mM HEPES, 4mM glutamine, penicillin and streptomycin (neonatal base medium). Care was taken to leave 40 ul in the well with each wash to avoid drying the cells. Cells were left in 40 ul of neonatal base medium. 40 ul base medium containing 2x of the desired stimuli was added to each well. 24 hours after the addition of stimulation, 10 ul of base medium containing lOOuM 5-bromo-2'-deoxyuridine (BrdU) was added to each well. After an additional 24 hours the cells were fixed in 3.7% formaldehyde.

Following fixation, immunocytochemistry to detect incorporation of BrdU was performed. Cells were washed 2X with PBS and treated with 4M HCl/1% Triton X-100 in H_∑O for 8 minutes to denature the nuclear DNA. The acid was washed away with 4 washes of PBS. Cells were blocked with 5% goat serum in PBS for one hour. Primary antibody mixture containing rat anti-BrdU antibody (Oxford Biotechnology Limited, 250 fold dilution) and mouse anti-tropomyosin CHl antibody (Developmental Studies Hybridoma Bank, 100 dilution) was applied at 37 ⁰C for 2 hours. Primary antibodies were washed away with 4 washes of PBS. Then secondary antibody solution containing Goat anti-mouse Alexa 488 and Goat anti-rat Alexa 594 (Invitrogen, 250 fold dilution) was applied for 2 hours. DAPI (400 ug/ml) was included in the secondary antibody solution to counterstain cell nuclei.

Detection and quantification of DNA synthesis in cardiomyocytes was performed as follows. Immunocytochemistry was visualized using Molecular Devices ImageXpress automated image analyzer and software. An Axon ImageXpress software script written by Molecular Devices for the purpose of detecting overlapping red and blue nuclei surrounded by green cytoplasmic staining was applied to the acquired images. This software separately identified nuclei (blue, stained with DAPI) that were or were not BrdU positive (red, rat anti-BrdU antibody-Alexa 594 goat anti-rat antibody pair). Furthermore, the software separately identified each class of nucleus based on whether it was surrounded by tropomyosin stain, indicative of a cardiomyocytes (green, mouse anti-tropomyosin CHl-Alexa 488 goat anti-mouse antibody pair), within a 5 uM ring drawn around the red nuclei. Thresholds were set appropriately for each plate such that overall background for each stain was not counted as positive.

Data from the ImageXpress script was imported to Microsoft Excel, and percentage of total cardiomyocytes that were BrdU positive was plotted for each condition. Images were exported from ImageXpress files into Adobe Photoshop. Adjustments of color and contrast were made simultaneously on all images shown in each figure.

(c) Neonatal cardiac fibroblasts were isolated the same way as NCM with the following exceptions. (1) The fibroblast layer was collected from the Percol gradient instead of the NCM layer. (2) NCFs were cultured in 10%FCS/DMEM on regular tissue culture plates without additional coating. (3) NCFs were passaged three times before using for assay in order to select against contaminating NCM. The proliferation assay on NCF was conducted similarly to the NCM proliferation assay except that the NCM were serum starved for 24 hours before sample addition in order to decrease background. Example 2: A Selective GSK3 Inhibitor Promotes Cardiomyocvte Proliferation

The selective GSK3 inhibitor BIO, described in detail above, increased both phases of cell proliferation (eg., as assessed by BrdU incorporation and total cardiomyocvte number) in the neonatal cardiomyocyte assay. These results are summarized in Figure 2. Briefly, neonatal cardiomyocytes were prepared as outlined in detail above. Cells were treated for 5 days with the indicated concentrations of BIO (concentrations ranging from 0.25 uM to 2 uM). Control cultures were treated with DMSO. The total number of cardiomyocytes was scored each day. BrdU incorporation was scored on day 2 — which is about 24 hours after exposure to BrdU. (See, Example 1 for detailed description of methods). As depicted in Figure 2, the selective GSK3 inhibitor BIO increased the DNA synthesis phase of cell proliferation, as assessed by BrdU incorporation. The selective GSK3 inhibitor BIO also increased the cell division phase of cell proliferation, as evidenced by the increase in the total number of cardiomyocytes. Example 3: A Selective GSK3 Inhibitor Promotes Adult Cardiomyocyte Proliferation

We assessed the ability of this selective GSK3 inhibitor to promote cardiomyocyte proliferation in adult cardiomyocyte assay. As summarized in Figure 3, BIO (indicated with the abbreviation CRF3) promoted cardiomyocyte proliferation, as assessed by incorporation of BrdU, in adult cardiomyocytes. Methods: Adult rat cardiomyocytes were isolated from 8-week-old (weight 250- 30Og) male Wistar rat hearts and cultured in ACCT medium (DMEM supplemented with 0.2% BSA, 0.4mg/ml L-carnitine, 0.66mg/ml Creatine, 0.62mg/ml Taurine, 1% Penicillin/Streptomycin). To isolate the cardiomyocytes, the heart was first isolated from a rat that was anesthetized with 2 ml Isoflurane in a 10-liter chamber. The isolated heart was placed in 50ml cold Ca²⁺-free buffer (NaCl, 6.895mg/ml; KCl, 0.35mg/ml; MgSO₄, 0.1.44mg/ml; KH₂PO₄ 0.1635mg/ml; NaHCO₃, 2.1mg/ml; Glucose, 2mg/ml) before perfusion apparatus through aorta. The heart was first perfused with Ca²⁺-free buffer for 2 to 3 minutes to remove blood, then with 30 ml enzyme solution I ( Ca²⁺- free buffer plus 100 unit/ml of collagenase type II and 150unit/ml of Hyaluronidase) for 20 minutes. The perfused heart was removed from perfusion apparatus and placed in 15ml enzyme solution II (enzyme solution I plus 1 mM CaCl₂, 0.3 mg/ml Trypsin and 0.3 mg/ml DNAase). The heart was cut into 8 to 10 small pieces and incubated for another 18 minutes at 37 ⁰C. 15 ml of wash media (1 to 1 mixture of ACCT medium and Ca²⁺ free buffer) was added and the heat tissue was sheared by pipetting gently. The cell suspension was filtered through a 250 micron nylon mesh filter and centrifuged at 50 g for 3 minutes. The isolated cells were washed three times with wash media by settling the cells for 5-8 min by gravity. After the last wash, the cell suspension (7ml) was slowly layered on top of a BSA solution (ACCT plus 6.45%BSA). After settling for 6-8 minutes, the cell pellet was resupended in ACCT medium at 100,000 cells/ml density, and plated at 50ul/welI on 96 well plates which had been precoated with lOug/ml laminin in ACCT. Cells were incubated at 37 ⁰C, 10% CO₂ for 1 hour, and medium was changed to lOOul/well of ACCT medium containing 10% FCS and 2OuM araC. After culturing for 3 days, medium was changed again to serum-free ACCT medium, and the cells were cultured for another 4 days. On day 7, medium was changed to ATTC medium containing stimuli and BrdU. The stimuli and BrdU steps were repeated on day 10. Cells were fixed on day 14, immunostained, and analyzed with the ImageXpress as described in neonatal cardiomyocyte culture and assay. Example 4: A Selective GSK3 Inhibitor Promotes Neonatal Cardiomyocyte Proliferation Without Inducing Hypertrophy

One limitation of some agents that promote cardiomyocyte proliferation is that they also promote hypertrophy. For example, 10% fetal calf serum (FCS) robustly promotes cardiomyocyte proliferation. However, 10% FCS also induces a hypertrophic response. Induction of hypertrophy may not always be desirable, and thus hypertrophic cells may not be suitable for some uses.

Induction of a hypertrophic response was a particular possibility when administering GSK3 inhibitors. Previous studies implicated inactivation of GSK3 in AKT induced hypertrophy (Hardt and Sadoshima, 2002, Circulation Res. 90: 1055- 1063; Molkentin et al., 2000, J. Cell Biol 151: 117-130). Thus, we assessed the selective GSK3 inhibitor BIO to ascertain whether this compound that promotes cardiomyocyte proliferation also induces hypertrophy, or whether it was able to promote cardiomyocyte proliferation without inducing hypertrophy.

Figure 4 shows exemplary results of these experiments. Briefly, cultures of neonatal cardiomyocytes were prepared, as outlined above. Cells were cultured in the presence of 0% FCS (panel a), BIO (panel b), or 10% FCS (panel c). Cells treated with any of the foregoing were assayed for BrdU incorporation and hypertrophy to identify whether compounds that promoted cardiomyocyte proliferation did so in the presence or absence of hypertrophy.

As shown in Figure 4, culturing cells in 0% FCS promoted neither proliferation nor hypertrophy (See, panel a). Culturing cells in 10% FCS promoted proliferation, but also induced hypertrophy (See, panel c). Culturing cells in 2uM BIO promoted proliferation of neonatal cardiomyocytes, and did not induce hypertrophy (See, panel b). Thus, despite some suggestion in the literature linking inhibitor of GSK3 activity to cardiac hypertrophy, hypertrophy was not observed following administration of a selective GSK3 kinase inhibitor to cardiomyocytes.

In the forgoing experiments, hypertrophy was assayed by examining an increase in cardiomyocyte cell size. Immuno staining was performed on neonatal cardiomyocyte cultures treated with FCS or BIO, at the indicated concentrations. Cardiomyocyte morphology was observed using tropomyosin staining. Cell nuclei were stained with DAPI. BrdU incorporation was determined, as described below. Example 5: A Selective GSK3 Inhibitor Promotes Adult Cardiomvocvte Proliferation Without Inducing Hypertrophy

Using similar methods as outlined above, we assessed the selective GSK3 inhibitor BIO to ascertain whether this compound that promotes cardiomyocyte proliferation without inducing hypertrophy in neonatal cardiomyocytes also does so in adult cardiomyocytes. Figure 5 shows exemplary results of these experiments. Briefly, cultures of adult cardiomyocytes were prepared, as outlined above. Cells were cultured in the presence of 0% FCS (panel a), 10% FCS (panel b), 100 ng/ml Wnt3a (panel c), 2 uM BIO (panel d), or 0.5 uM BIO (panel e). Cells treated with any of the foregoing were assayed for BrdU incorporation and hypertrophy to identify whether compounds that promoted cardiomyocyte proliferation in adult cardiomyocytes did so in the presence or absence of hypertrophy.

As shown in Figure 5, culturing adult cells in 0% FCS promoted neither proliferation nor hypertrophic (See, panel a). Culturing adult cells in 10% FCS promoted proliferation, but also induced hypertrophy (See, panel b). As has been previously shown, culturing adult cells in Wnt3a protein promoted proliferation without inducing hypertrophy. Culturing cells in either 2uM BIO or 0.5 uM BIO promoted proliferation of adult cardiomyocytes, and did not induce hypertrophy (See, panel d and e).

Example 6: A Class of Selective GSK3 Inhibitors More Potently Promotes Cardiomvocvte Cell Proliferation

BIO is an example of a selective GSK3 inhibitor that inhibits GSK3 kinase activity with an IC₅₀ more than 50 times lower than its IC₅0 for CDKl . As detailed herein, BIO also promotes proliferation of neonatal and adult cardiomyocytes. BIO promoted both the DNA synthesis and the cell division phases of proliferation, and resulted in an increase in cardiomyocyte number over time.

We tested a structurally related compound, HO [5-Iodo-indirubin-3- monoxime], to ascertain whether HO also promoted cardiomyocyte proliferation. HO inhibits GSK3 with an IC50 similar to that of BIO. However, HO inhibits CDKl with similar potency while BIO does not. Specifically, HO inhibits CDKl kinase activity with an IC₅0 of 25 nM making it approximately 12 times more potent against CDKl than BIO is.

Figure 6 summarizes the results of these experiments performed in neonatal cardiomyocytes. Figure 6a indicates the Vmax and EC₅0 based on BrdU incorporation. BIO and HO promote proliferation with a similar Vmax of 60%. However, BIO promoted the DNA synthesis phase of cardiomyocyte proliferation with a EC50 approximately 4 times less than IIO. In the particular experiment summarized in Figure 6, the EC50 for BIO was 100 nM while the EC50 for IIO was 400 nM. In other experiments, the EC50 for BIO has ranged from 100-300 nM while that for IIO has typically been approximately 500 nM.

In addition to the difference in potency of BIO, Figure 6b shows that BIO and IIO differ significantly in their ability to stimulate cardiomyocyte division. In other words, BIO and HO differ significantly in their ability to promote the cell division phase of proliferation. Specifically, BIO treated cultures contained a greater number of cardiomyocytes in comparison to IIO treated cultures. Thus, BIO more potently stimulated not only DNA synthesis, as measured by BrdU incorporation, but also cell division as evinced by increased cell numbers over time.

Figure 6c shows the increase in cardiomyocyte cell number relative to the increase in BrdU incorporation for BIO and IIO. Doses of BIO and HO that stimulate equivalent BrdU incorporation differ in their ability to stimulate cell division. Thus the difference between BIO and IIO in the ability to increase cardiomyocyte cell number (Fig. 6b) is not simply the result of the difference in the ability to induce BrdU incorporation (fig. 6a), but can be explained by the difference in selectivity over CDKs. Example 7: A Selective GSK3 Inhibitor Can Be Formulated for In Vivo Delivery

The selective GSK3 inhibitor BIO was formulated in Captisol or Solutol. Pharmacokinetic properties were evaluated via HPLC/MS following a single intraperitoneal dose of 30 and/or 100 mg/kg in male Sprague-Dawley rats. Briefly, BIO was administered at a dose volume of 10 mL/kg IP to conscious rats. Blood was obtained from the animals via the jugular vein. For the last collection point, animals were anesthetized and blood was taken via cardiac puncture. The results of these experiments are summarized in Figure 7. Briefly, when administered IP at a dose of 30 mg/kg in Captisol [40% Captisol, 0.6% DMSO, 20 mM citrate buffer, pH 6], the following properties were observed: Plasma level > ECso: 4-5 hours; Plasma level > EC₉₀: 2 hours.

Example 8: A Selective GSK3 Inhibitor Has Efficacy in a Rat Model of Myocardial Infarction

We tested the selective GSK3 inhibitor BIO in an established rat model of myocardial infarction (Fishbein et al., 1978, American Journal of Pathol 90: 57-70). Briefly, under general anesthesia, the left anterior descending coronary artery of male Sprague-Dawley rats was ligated using 5.0 mm silk sutures to create a permanent occlusion and induce myocardial infarction. Animals were allowed to recover from anesthesia, and heart function was assessed at various time points post ligation by echocardiography. Amongst the measures evaluated was fractional shortening indicative of cardiac damage post-infarction. Routinely, rats were assessed on day 2, 23, and 35 post infarction.

Occluded rats were administered BIO or a vehicle control daily from day 2- 23 post infarction. Drug or vehicle was administered IP. Animals were compared to rats on which a sham procedure was performed.

Figures 8a and 8b summarize experiments in which occluded rats were administered BIO formulated in solutol (30 mg/kg/day — daily — IP) or a solutol vehicle control. Administration of BIO resulted in a statistically significant improvement in cardiac symptoms following infarction in comparison to daily administration of vehicle alone.

Figures 9a and 9b summarize experiments in which occluded rats were administered BIO formulated in captisol (30 mg/kg/day - daily - IP) or a captisol vehicle control. Administration of BIO resulted in a statistically significant improvement in cardiac symptoms following infarction in comparison to daily administration of vehicle alone.

In addition to monitoring cardiac function, the rats were weighed and the ratio of heart to body weight was calculated. No cardiac hypertrophy relative to control animals was observed following administration of BIO. This indicated that administration of BIO improved cardiac function following infarction without inducing a hypertrophic response.

Example 9: Promotion of Neonatal Cardiac Cell Proliferation by Selective GSK3 Kinase Inhibitors

Figures lOa-c summarize the results of experiments performed in the neonatal cardiomyocyte assay described in detail above. As detailed in Figures 10a and 10b, two selective GSK3 inhibitors promoted cardiac cell proliferation in this in vitro model. The selectivity profile of BIO is detailed above. BIO is selective for inhibiting, at least, GSK3 activity preferentially over CDKl activity. BIO may also be selective for inhibiting GSK3 activity preferentially over the activity of other kinases. BIO promoted cardiac cell proliferation in the neonatal cardiomyocyte assay.

A second selective inhibitor [Compound A] was also examined. Compound A is selective for inhibiting, at least, GSK3 activity preferentially over CDK2 activity. Compound A may also be selective for inhibiting GSK3 activity preferentially over the activity of other kinases. Compound A promoted cardiac cell proliferation in the neonatal cardiomyocyte assay. The structure of Compound A is provided in Figure 10c.

AU publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS:

1. A method of promoting cardiac cell proliferation, comprising contacting a cardiac cell with an amount of a compound effective to promote cardiac cell proliferation, which compound selectively inhibits GSK3 kinase activity, wherein said compound inhibits GSK3 kinase activity with an IC₅₀ at least 1 order of magnitude lower than its IC50 for inhibiting CDKl kinase activity.

2. A method of promoting cardiac cell proliferation, comprising contacting a cardiac cell with an amount of a compound effective to promote cardiac cell proliferation, which compound selectively inhibits GSK3 kinase activity, wherein said compound inhibits GSK3 kinase activity with an IC50 at least 1 order of magnitude lower than its IC₅0 for inhibiting the kinase activity of one or more of CDKl, CDK2, CDK4, CDK5, CDK6, or CDK7.

3. A method of promoting cardiac cell proliferation, comprising contacting a cardiac cell with an amount of a compound effective to promote cardiac cell proliferation, which compound selectively inhibits GSK3 kinase activity, wherein said compound inhibits GSK3 kinase activity with an IC50 at least 1 order of magnitude lower than its IC50 for inhibiting CDKl kinase activity and at least 1 order of magnitude lower than its IC50 for inhibiting the kinase activity of one or more of CDK2, CDK4, CDK5, CDK6, or CDK7.

4. The method of any of claims 1-3, wherein the cardiac cell is a cardiomyocyte.

5. The method of claim 4, wherein the cardiomyocyte is selected from a neonatal, fetal, or adult cardiomyocyte.

6. The method of claim 4, wherein the cardiomyocyte is an adult cardiomyocyte.

7. The method of any of claims 1-3, wherein the compound is a small organic molecule. ^"

8. The method of any of claims 1-3, wherein said method is conducted in an animal and said compound is formulated as a pharmaceutical preparation.

9. The method of any of claims 1-3, wherein said method is conducted in vitro and said cardiac cells are in culture.

10. The method of any of claims 1-3, wherein said compound inhibits GSK3 kinase activity with an IC50 of less than 250 nM.

11. The method of claim 10, wherein said compound inhibits GSK3 kinase activity with an IC50 of less than 100 nM.

12. The method of claim 1, wherein said compound inhibits GSK3 kinase activity with an IC₅₀ at least 1.5 orders of magnitude lower than its IC50 for inhibiting CDKl kinase activity.

13. A method of promoting cardiac cell proliferation, comprising contacting a cardiac cell with an amount of a compound effective to promote cardiac cell proliferation, which compound selectively inhibits GSK3 kinase activity, wherein said compound inhibits GSK3 kinase activity with an IC50 at least 25 times lower than its IC₅₀ for inhibiting CDKl kinase activity.

14. A method of promoting cardiac cell proliferation, comprising contacting a cardiac cell with an amount of a compound effective to promote cardiac cell proliferation, which compound selectively inhibits GSK3 kinase activity, wherein said compound inhibits GSK3 kinase activity with an IC50 at least 5 times lower than its IC₅0 for inhibiting the kinase activity of one or more of CDK2, CDK4, CDK5, CDK6, or CDK7.

15. A method of promoting cardiac cell proliferation, comprising contacting a cardiac cell with an amount of a compound effective to promote cardiac cell proliferation, which compound selectively inhibits GSK3 kinase activity, wherein said compound inhibits GSK3 kinase activity with an IC₅₀ at least 25 times lower than its IC₅₀ for inhibiting CDKl kinase activity and at least 5 times lower than its IC₅₀ for inhibiting the kinase activity of one or more of CDK2, CDK4, CDK5, CDK6, or CDK7.

16. The method of any of claims 13-15, wherein the cardiac cell is a cardiomyocyte.

17. The method of claim 16, wherein the cardiomyocyte is selected from a neonatal, fetal, or adult cardiomyocyte.

18. The method of claim 17, wherein the cardiomyocyte is an adult cardiomyocyte.

19. The method of any of claims 13-18, wherein the compound is a small organic molecule.

20. The method of any of claims 13-19, wherein said method is conducted in an animal and said compound is formulated as a pharmaceutical preparation.

21. The method of any of claim 13-19, wherein said method is conducted in vitro and said cardiac cells are in culture.

22. The method of claim 13, wherein said compound inhibits GSK3 kinase activity with an IC5₀ at least 50 times lower than its IC₅₀ for inhibiting CDKl kinase activity.

23. The method of any of claims 13-15, wherein said compound inhibits GSK3 kinase activity with an IC5₀ of less than 25OnM.

24. The method of claim 23, wherein said compound inhibits GSK3 kinase activity with an IC5₀ of less than 100 nM.

25. The method of any of claims 1-24, wherein the compound binds to GSK3.

26. The method of any of claims 1-25, wherein the compound is 6-bromo- indirubin-3 '-monoxime.

27. The method of any of claims 1-25, wherein the compound has the following structure represented in Figure 1 Oc:

28. The method of any of claims 1-27, wherein the compound promotes cardiac cell proliferation with an EC₅₀ of less than or equal to 500 nM.

29. The method of any of claims 1-28, used to prevent, treat or alleviate a disease or injury of cardiac cells.

30. The method of any of claims 1-29, used to prevent, treat or alleviate a disease or injury characterized by decreased cardiac function.

31. The method of any of claims 1-30, wherein said compound does not induce a hypertrophic response.

32. A method of treating an injury or disease of decreased cardiac function, comprising administering to a subject in need thereof an amount of a compound effective to promote cardiac cell proliferation, which compound selectively inhibits GSK3 kinase activity, wherein said compound inhibits GSK3 kinase activity with an IC₅₀ at least 1 order of magnitude lower than its IC₅₀ for inhibiting CDKl kinase activity.

33. The method of claim 32, wherein said injury or disease of decreased cardiac function is myocardial damage from myocardial infarction, and wherein said compound is administered in an amount effective to treat said myocardial damage.

34. The method of claim 32, wherein said injury or disease of decreased cardiac function is selected from any of myocardial infarction; atherosclerosis; coronary artery disease; obstructive vascular disease; dilated cardiomyopathy; heart failure; myocardial necrosis; valvular heart disease; non-compaction of the ventricular myocardium; hypertrophic cardiomyopathy; cancer or cancer-related conditions such as structural defects resulting from cancer or cancer treatments.

35. The method of claim 34, wherein said injury results from myocarditis, exposure to a toxin, exposure to an infectious agent, or from a mineral deficiency.

36. The method of any of claims 32-35, wherein said compound is administered systemically.

37. The method of any of claims 32-35, wherein said compound is administered to the myocardium, pericardium, or endocardium^" via a syringe, catheter, stent, wire, or other intraluminal device.

38. The method of any of claims 32-37, wherein said compound is 6-bromo- indirubin-3 '-monoxime.

39. The method of any of claims 32-38, wherein said compound does not induce a hypertrophic response.

40. A method of treating an injury or disease of decreased cardiac function, comprising administering to a subject in need thereof an amount of a compound effective to promote cardiac cell proliferation, which compound selectively inhibits GSK3 kinase activity, wherein said compound inhibits GSK3 kinase activity with an IC_5O at least 1 order of magnitude lower than its IC₅₀ for inhibiting CDK2 kinase activity.

41. The method of claim 40, wherein said injury or disease of decreased cardiac function is myocardial damage from myocardial infarction, and wherein said compound is administered in an amount effective to treat said myocardial damage.

42. The method of claim 40, wherein said injury or disease of decreased cardiac function is selected from any of myocardial infarction; atherosclerosis; coronary artery disease; obstructive vascular disease; dilated cardiomyopathy; heart failure; myocardial necrosis; valvular heart disease; non-compaction of the ventricular myocardium; hypertrophic cardiomyopathy; cancer or cancer-related conditions such as structural defects resulting from cancer or cancer treatments.

43. The method of claim 42, wherein said injury results from myocarditis, exposure to a toxin, exposure to an infectious agent, or from a mineral deficiency.

44. The method of any of claims 40-43, wherein said compound is administered systemically.

45. The method of any of claims 40-43, wherein said compound is administered to the myocardium, pericardium, or endocardium via a syringe, catheter, stent, wire, or other intraluminal device.

46. The method of any of claims 40-45, wherein said compound does not induce a hypertrophic response.

47. Use of a small molecule agent that inhibits GSK3 kinase activity with an IC50 at least one order of magnitude lower than its IC₅₀ for inhibiting CDKl kinase activity in the manufacture of a medicament for treating an injury or disease of decreased cardiac function.

48. Use of a small molecule agent that inhibits GSK3 kinase activity with an IC₅₀ at least one order of magnitude lower than its IC₅₀ for inhibiting CDK2 kinase activity in the manufacture of a medicament for treating an injury or disease of decreased cardiac function.

49. Use of 6-bromo-indirubin-3'-rnonoxirne in the manufacture of a medicament for treating an injury or disease of decreased cardiac function.

50. A method of promoting cardiac cell proliferation, comprising contacting a cardiac cell with an amount of a composition comprising 6-bromo-indirubin- 3'-monoxime effective to promote cardiac cell proliferation.

51. The method of claim 50, wherein the composition comprising 6-bromo- indirubin-3'-monoxime is a pharmaceutical composition.