Chimeric C3-like Rho Antagonist Cardiac Therapeutic Cross References to Related Applications This application claims priority to U.S. Provisional Application No. 60/981,627 filed on

October 22, 2007.

Field of the Invention

This invention comprises a method of inhibiting Rho GTPase in a cardiac muscle cell by administering to the cardiac muscle cell an amount of a chimeric C3-like Rho antagonist effective to modulate Rho GTPase activity. This invention also comprises a method of treating cardiac disease in a subject in need of such treatment by administering to said subject a therapeutically effective dosage of a chimeric C3-like Rho antagonist such as SEQ ID NO: 1.

Background of the Invention

Heart failure (HF) is a complex pathophysiologic syndrome recognized as a significant cause of morbidity and mortality among the aging population. It is one of the most important public health problems in the United States. The incidence and prevalence of heart failure continues to rise, primarily owing to an increasing population, improved therapies prolonging survival after myocardial infarction, and enhanced diagnostic techniques that have increased the detection of heart failure. It has been estimated that heart failure affects 5 million patients and results in an economic burden of almost 30 billion dollars each year. Current therapies for American College of Cardiology- American Heart Association (ACC-AHA) Stage A-D patients include pharmacotherapeutic agents, coronary artery bypass grafting, mitral valve repair, and bi- ventricular pacemakers. None of these therapies specifically address cardiac remodeling, which is a fundamental pathophysiologic mechanism of heart failure.

Acute myocardial infarction (AMI) can lead to infarct expansion, chronic left ventricular (LV) dilatation, and, eventually, chronic heart failure. Postinfarction heart failure remains a major challenge for the clinical cardiologist.

It appears that experiments using inhibitors of Rho A, siRNA directed against Rho A expression, or Rho A knockout mice to learn about cardiac function, dysfunction, and repair have not been conducted. The lack of availability of a sufficiently potent Rho A inhibitor may explain the absence of direct experimental evidence, although one would expect in vitro attempts using the Clostridia botulinum C3 exotoxin that inhibits Rho A. The investigators in the field rely on ROCK inhibitors, siRNA directed against ROCK expression, or ROCK knockout mice to learn about cardiac function, dysfunction, and repair.

Rho family GTPases have been described as regulators of actin cytoskeletal organization. Some reports link RhoA and Racl signaling to serum response factor, NF-λB, and other transcription factors, myofilament proteins, ion channels, and reactive oxygen species generation. The utility of Rho kinase blockers in hypertrophy or heart failure has not been established. A recent review article is noted, Brown et al., "Rac and Rho Hall of Fame: A Decade of Hypertrophic Signaling Hits," Circulation Research. 98:730-742 (2006).

Further attention is drawn to Chang et al., "Activation of Rho-associated coiled-coil protein kinase 1 (ROCK-I) by caspase-3 cleavage plays an essential role in cardiac myocyte apoptosis," Proc Natl Acad Sci U S A.. 103(39): 14495-500 (2006); and Lin et al, "Acute Inhibition of Rho-kinase improves cardiac conctractile function in streptozocin-diabetic rats," Cardiovasc Res. JuI l;75(l):51-8. Epub (2007). Also noted is Zhang, et al., "Targeted deletion of ROCKl protects the heart against pressure overload by inhibiting reactive fibrosis," FASEB J., 20(7):916-25 (2006).

Notable RhoA antagonists are disclosed in US Patent No. 6,855,688 to McKerracher, "ADP-ribosyl transferase fusion proteins, pharmaceutical compositions, and methods of use." In this regard, particular mention is made of SEQ ID NO: 1, an engineered variant of a naturally occurring bacterial protein known as C3 exoenzyme, corresponding substantially to SEQ. ID NO.: 43 of US Patent No. 6,855,688 to McKerracher. After gaining access to the cytoplasm of mammalian cells, it interacts with and specifically inhibits Rho GTPases, which are major intracellular switching molecules that regulate a diversity of cellular functions including motility, growth, and differentiation. Whereas the naturally occurring C3 exoenzyme has a very limited capacity for cellular penetration, SEQ ID NO: 1 has been modified to include a transport motif that dramatically enhances its ability to enter the cytoplasm of mammalian cells.

Without being bound by any particular theory, it is believed that the Rho family GTPase transduction pathway is, alone or with co-factors, significant in cardiac remodeling. Cardiac remodeling is thought to be an important aspect of heart failure disease progression. Remodeling in failure is manifested clinically by pathological changes in cardiac size, shape, and function in response to cardiac injury or increased load. Other pathologies arise from pressure overload secondary to aortic stenosis or hypertension, and chemotherapy, e.g. anthracyclines such as doxorubicin. Also noted is inflammatory myocardial disease (myocarditis), with idiopathic dilated cardiomyopathy, or with volume overload including valvular regurgitation.

Germane to the present disclosure is the use of drug eluting stents in cardiac applications. Implantable medical devices capable of delivering medicinal agents have been described. In some embodiments this includes devices utilizing biodegradable or bioresorbable polymers as drug containing and releasing coatings, including U.S. Pat. No. 4,916,193 to Tang et al and U.S. Pat. No. 4,994,071 to MacGregor. Other patents are directed to the formation of a drug containing hydrogel on the surface of an implantable medical device. These include Amiden et al, U.S. Pat. No. 5,221,698 and Sahatjian, U.S. Pat. No. 5,304,121. Still other patents describe methods for preparing coated intravascular stents via application of polymer solutions containing dispersed therapeutic material to the stent surface followed by evaporation of the solvent. This method is described in Berg et al., U.S. Pat. No. 5,464,650. In drug eluting stents, coating thickness will typically be in the range of about 1 micrometer to about 500 micrometers. This level of coating thickness is generally required to provide an adequate density of drug and adequate activity under physiological conditions. Noted are stents having a drug-release coating comprising from about 10 and to about 80 weight percent and from about 20 to about 90 weight percent of therapeutic agent(s). Direct administration such as via intracardiac catheter is also noted as is depot administration. Above, and throughout this disclosure, a number of published materials are cited, all of which are incorporated herein by reference in their entirety.

Summary of the Invention

In one embodiment, a method for inhibiting Rho GTPase in a cardiac muscle cell is provided. The method comprises administering to the cardiac muscle cell a therapeutically

effective amount of a Rho inhibitor. With out being bound by any particular theory, it is believed that the Rho inhibitor modulates Rho GTPase activity.

In another embodiment, a method for preventing, delaying, attenuating, or ameliorating cardiac remodeling in a subject in need thereof is provided. The method comprises administering to the subject an amount of a Rho inhibitor effective to modulate cardiac remodeling.

In another embodiment, a method for the amelioration, attenuation, treatment or prevention of a condition associated with cardiac remodeling in a subject in need thereof is provided. The method comprises administering to a subject an amount of a Rho inhibitor effective to modulate cardiac remodeling, wherein the condition associated with cardiac remodeling is thereby improved, attenuated, or delayed.

In another embodiment, the Rho inhibitor compound prevents, attenuates or delays an increase in heart chamber dimension or wall thickness, increases E/ A ratio after myocardial infarction, decreases infarct size, increases exercise capacity; increases exercise efficiency, and/or normalizes cardiac output after myocardial infarction.

In another embodiment, a method for improving cardiac contractility in a subject in need thereof is provided. The method generally comprises administering to a subject an amount of a Rho inhibitor effective to improve cardiac contractility in the subject.

In another embodiment, a method for reducing or preventing atrial remodeling in a subject in need thereof is provided. In another embodiment, a method for reducing or preventing ventricular remodeling in a subject in need thereof is provided. The methods generally comprise administering to the subject an amount of a Rho inhibitor effective to reduce or prevent atrial or ventricular remodeling in the subject.

Also provided is the use of at least one Rho inhibitor, alone or in combination, to manufacture a medicament to mediate the effects or to treat the diseases or disorders disclosed herein.

Another embodiment of the present invention comprises the inhibition of Rho A by a cell- permeable fusion protein conjugate comprising a polypeptide comprising an amino acid sequence of a transport agent covalently linked to an amino acid sequence of an active agent,

said amino acid sequence of said active agent consisting of ADP-ribosyl transferase C3 or a fragment thereof retaining an ADP-ribosyl transferase activity, said amino acid sequence of said transport agent facilitating cellular uptake of the active agent, for example a fusion protein such as SEQ ID NO: 1. In one aspect, the functional analog of a Clostridium botulinum C3 exotransferase unit comprises a protein exhibiting an ADP-ribosyl transferase activity in the range of about 50% to about 500% of the ADP-ribosyl transferase activity of Clostridium botulinum C3 exotransferase.

A therapeutically effective amount or dosage of an active agent, e.g., SEQ ID NO: 1, may range from about 0.01 picograms(pg) to about 1 mg at the heart. While systemic administration to achieve such levels is contemplated, direct cardiac administration is noted. In one embodiment the active agent is administered to the coronary artery. In another embodiment, the active agent is administered by a coronary artery catheter.

In one embodiment, a non-limiting range for a therapeutically effective concentration is about 1 nM to about 1 M. In another embodiment, a range for a therapeutically effective concentration is about 1 μM to about 100 mM. In another embodiment, the therapeutically effective concentration is about 0.1 mM to about 10OmM. In another embodiment, the therapeutically effective concentration is about 0.1 mM to about 10 mM. In another embodiment, the therapeutically effective concentration is about 0.1 mM to about 2 mM.

In one embodiment, the therapeutically effective concentration is about 0.58 mM. In another embodiment, the therapeutically effective concentration is about 1.16 mM.

In one embodiment, the administered concentration is about 10 mM. In another embodiment, the administered concentration is about 50 mM. In another embodiment, the administered concentration is about 75 mM. In another embodiment, the administered concentration is about 100 mM. In another embodiment, the administered concentration is about 500 mM.

In one embodiment, SEQ ID NO: 1 is administered using a dose of about 0.01 pg/Kg to about 1 mg/Kg/. In another embodiment, SEQ ID NO: 1 is administered using a dose of about 0.1 ng/Kg/day to about 0.1 mg/Kg. In another embodiment, SEQ ID NO: 1 is administered using a dose of about 0.1 μg/Kg. In another embodiment, SEQ ID NO: 1 is administered using a dose of about 0.1 mg/Kg.

A skilled artisan will appreciate that the pharmaceutical compositions of the present invention may also be formulated as single-dose vials. For example, single-dose vials can be produced containing about 25, about 40, about 60, about 100, about 150, about 200, about 300, or about 500 micrograms of active agent. In a further example, single-dose vials can be produced containing a concentration of about 0.5 mM or about 1.0 mM active agent.

This invention comprises a method of treating cardiac disease in a subject in need of such treatment by administering to said subject a therapeutically effective dosage of a chimeric C3- like Rho antagonist, optionally wherein the chimeric C3-like Rho antagonist is SEQ ID NO: 1. Noted is administration by introduction into the coronary artery, and particularly wherein at least about 0.02 mg of SEQ ID NO: 1 is administered, and more particularly wherein at least about 0.2 mg of SEQ ID NO: 1 is administered. In some embodiments administration is daily for at least about 2 days and, optionally, wherein at least one introduction is at a site of cardiac damage. Also noted is administration by introduction into cardiac muscle at at least one site.

A further method comprises inhibiting Rho activity in a cardiac cell by the method of administering to the cell a therapeutically effective dosage of a chimeric C3-like Rho antagonist, optionally SEQ ID NO: 1.

In specific embodiments, administration comprises from about 0.1 picograms to about 0.1 mg/Kg body weight and particularly dosages from about 0.1 ng/Kg to about 0.01mg/Kg body weight. Further contemplated is administration of SEQ ID NO: 1 to heart muscle at a local concentration of at least about InM to about IM and particularly establishing a local concentration is at least about 1 μM-100mM. Durations of administration contemplated include the local concentration as maintained for at least about 4 hours, and optionally, at least about 8 hours and further for at least about 24 hours. Administration further includes administration directly to the heart by introduction by drug eluting cardiac stent, such as for example, a coronary artery stent.

Also included within the invention is a method of reducing damage to cardiac muscle arising from anthracycline therapy by administering a therapeutically effective dosage of SEQ ID NO: 1 from about 1 day prior to about concurrent with anthracycline dosing, and particularly administration from about 0.1 ng/Kg to about 0.01mg/Kg body weight.

Detailed Description of the Invention

There is a need to identify agents for inhibiting Rho GTP ase in cardiac muscle cells. There is also a need to identify agents for therapeutic cardiac remodeling which positively effect some or all aspects of cardiac physiology including cardiac size, shape, and function in response to cardiac insult or load. Rho antagonists are presented herein for such function.

The invention will be better understood with reference to the following definitions.

A. SEQ ID NO: 1 is a recombinant protein, believed to act as a Rho GTPase antagonist.

SEQ ID NO: 1 (C3-11) is composed of 232 amino acids with a theoretical molecular weight of 25,858 daltons and theoretical isoelectric point (pi) of 9.6. The extinction coefficient has been determined by amino acid analysis to be 0.72 units of absorbance at A280nm per mg of protein.

SEQ ID NO: 1 is described as SEQ ID NO.10 in US Patent Application Serial Nos.11/643,940 and 11/808,733, each of which are incorporated herein by reference in their entirety. It has been engineered by BioAxone Therapeutic (Montreal, Canada) in part through the addition of a proline rich peptidic transport sequence to a C3 exoenzyme sequence, (see Winton et al., J. Biol.

Chem., Vol.277, No.36, pp.32820-32829, (2002)). It is particularly understood that analogues and derivatives SEQ ID NO: 1 are also useful.

Protein Sequence of SEQ ID NO: 1 (C3-11):

M S A Y S N T Y Q E F T N I D Q A K A W G N A Q Y K K Y G L S K S E K E A I V S

Y T K S A S E I N G K L R Q N K G V I N

G F P S N L I K Q V E L L D K S F N K M

K T P E N I M L F R G D D P A Y L G T E

F Q N T L L N S N G T I N K T A F E K A K A K F L N K D R L E Y G Y I S T S L M

N V S Q F A G R P I I T K F K V A K G S

K A G Y I D P I S A F A G Q L E M L L P R H S T Y H I D D M R L S S D G K Q I I I T A T M M G T A I N P K E F V M N P A N A Q G R H T P G T R L (SEQ ID NO: 1)

B. Cardiothoracic ratio shall mean the transverse cardiac diameter (the horizontal distance between the most rightward and leftward borders of the heart seen on a postero-anterior (PA) chest radiograph) divided by the transverse chest diameter (measured from the inside rib margin at the widest point above the costophrenic angles on a PA chest film). A cardiothoracic ratio of more than 50% is considered abnormal in an adult; more than 66% in a neonate.

C. The term "unfavorable left ventricular remodeling" refers to alterations in chamber size, wall thickness, and other dimensional changes of the left ventricle and to any other changes to the left ventricle which occur in response to myocardial or cardiac damage that may be evidenced by decreased diastolic and/or systolic performance. D. The term "Rho antagonists" as used herein includes, but is not restricted to, C3 proteins, including C3-like proteins.

E. The term "C3 protein" refers to ADP-ribosyl transferase C3 of the composition isolated from Clostridium botulinum, Bacillus cereus or Staphylococcus aureus or a recombinant ADP-ribosyl transferase. F. The terms "C3-like protein", "ADP-ribosyl transferase C3-like protein", "ADP- ribosyl transferase C3 analogue", "C3-like transferase" or "C3 chimeric proteins" as used herein refers to any protein or polypeptide having a biological activity similar (e.g., the same, substantially similar) to ADP-ribosyl transferase C3. Examples of C3-like proteins include, but are not limited to those found in U.S. Patent Application Serial No. 11/808,733, which is incorporated herein by reference in its entirety. In one embodiment, the C3-like protein is SEQ ID NO: 1.

G. The term "subject" means a patient in need of a treatment. In one embodiment, a subject is a mammal. In another embodiment, a subject is a human.

Conditions associated with cardiac remodeling include, for example, myocardial infarction, inflammation, ischemial reperfusion, oxidative stress, cor pulmonale, advanced glycation endproducts, abnormal cardiac wall tension, sympathetic stimulation, myocarditis, hypertension, viral cardiomyopathy, idiopathic cardiomyopathy, heart transplantation, and surgical procedures of the heart.

Provided herein are methods of delaying or preventing conditions that result from cardiac remodeling. Conditions associated with or resulting from cardiac remodeling that can benefit from the methods provided herein include left ventricular hypertrophy, coronary artery disease, essential hypertension, acute hypertensive emergency, cardiomyopathy, heart insufficiency, exercise tolerance, chronic heart failure, arrhythmia, cardiac dysrhythmia, sudden death, syncopy, atherosclerosis, mild chronic heart failure, angina pectoris, cardiac bypass reocclusion, intermittent claudication, diastolic dysfunction, and/or systolic dysfunction.

The pharmacologically active compositions of this invention can be processed in accordance with conventional methods of Galenic pharmacy to produce medicinal agents for administration to patients, e.g., mammals including humans.

The compositions of this invention individually or in combination are employed in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral (e.g., oral or inhalation) or topical application which do not deleteriously react with the active compositions. Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatine, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc. The pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifϊers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compositions. They can also be combined where desired with other active agents, e.g., vitamins.

In some embodiments of the present invention, dosage forms include instructions for the use of such compositions. For parenteral application, particularly suitable are injectable, sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories. Ampules are convenient unit dosages.

Also for parenteral application, particularly suitable are tablets, dragees, troches, liquids, drops, suppositories, or capsules. A syrup, elixir, or the like can be used wherein a sweetened vehicle is employed. Sublingual and buccal forms are also noted.

Sustained or directed release compositions can be formulated, e.g., liposomes or those wherein the active component is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc. It is also possible to freeze-dry the new compositions and use the lyophilizates obtained, for example, for the preparation of products for injection. It will be appreciated that the actual preferred amounts of active compositions in a specific case will vary according to the specific compositions being utilized, the particular compositions formulated, the mode of application, and the particular situs and organism being treated. Dosages for a given host can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the subject compositions and of a known agent, e.g., by means of an appropriate, conventional pharmacological protocol.

In addition, other therapeutic cardiovascular compounds that may be concurrently administered for use in methods of the invention, including but not limited to an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist, a calcium channel blocker, an antithrombolytic agent, a β-adrenergic receptor antagonist, a vasodilator, a diuretic, an α- adrenergic receptor antagonist, an antioxidant, and a mixture thereof. A compound suitable for use in methods of the invention also may be concurrently administered with pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid.

Cardiomyopathy refers to diseases of the heart muscle. These diseases have a variety of causes, symptoms, and treatments. In cardiomyopathy, the heart muscle becomes enlarged or abnormally thick or rigid. In rare cases, the muscle tissue in the heart is replaced with scar tissue. As cardiomyopathy progresses, the heart becomes weaker and less able to pump blood through the body, leading to heart failure, arrhythmias, fluid buildup in the lungs or legs, or endocarditis. The weakening of the heart also can lead to other severe complications. Cardiomyopathy can affect people of all ages, from newborns to older adults. However, certain age groups are more likely to have certain types of cardiomyopathy. Treatment may involve medicines, surgery, non-surgical procedures, and lifestyle changes.

The four main types of cardiomyopathy include Dilated Cardiomyopathy, Hypertrophic Cardiomyopathy, Restrictive Cardiomyopathy, and Arrhythmogenic Right Ventricular Dysplasia.

According to the National Heart, Lung and Blood institute, in some manifestations, Cardiomyopathy has a specific cause, such as damage to the heart from a heart attack, high blood pressure, or a viral infection. Some types of cardiomyopathy are caused by a gene mutation and may run in families. In many cases, the cause is unknown. When this happens, the disease is called idiopathic (or primary) cardiomyopathy. The majority of cardiomyopathies in children are idiopathic.

Sometimes, cardiomyopathy is inherited (passed down in the genes from parent to child) or caused by another disease or condition. Dilated cardiomyopathy can be inherited. It also can be caused by certain diseases, conditions, and substances, including: Coronary artery disease and heart attacks (ischemic cardiomyopathy); infections, especially viral infections that cause the heart muscle to become inflamed (myocarditis); Alcohol, especially when a person has a poor diet (alcoholic cardiomyopathy); Complications during the last month of pregnancy or within 5 months of birth (peripartum cardiomyopathy); Certain toxins, such as cobalt; Certain drugs, such as cocaine, amphetamines, and two medicines used to treat cancer (doxorubicin and daunorubicin); and Diseases such as diabetes and thyroid disease.

Loss of some blood-pumping ability in the hearts is expected as people age. but significant loss is generally known as heart failure, in some embodiments, heart failure results from the added stress of health conditions that either damage the heart or make it work too hard, subjects at risk of heart failure may have or have had coronary artery disease, past heart attacks (myocardial infarction), high blood pressure (hypertension), abnormal heart valves, heart muscle disease (cardiomyopathy), heart defects present at birth (congenital heart disease), severe lung disease, diabetes, low red blood cell count (severe anemia), an overactive thyroid gland (hyperthyroidism), and abnormal heart rhythm (arrhythmia or dysrhythmia).

In the practice of this invention, any pharmacologically suitable Rho antagonist may be used in treatment. In one embodiment, the Rho antagonist is a chimeric C3-like Rho antagonists. In another embodiment, the Rho antagonists are polypeptides corresponding to chimeric (i.e. conjugate) C3-like Rho antagonists, as well as compositions comprising the polypeptides. Examples of polypeptides corresponding to chimeric C3-like Rho antagonists are found in U.S. Patent Application Serial No. 11/808,733, which is incorporated herein by reference in its

entirety. In another embodiment, the Rho antagonist is SEQ ID NO: 1, the active pharmaceutical ingredient in Cethrin® (BioAxone Therapeutic, Inc Montreal).

The invention also relates to pharmaceutical compositions comprising the polypeptides of the invention. In an aspect, the pharmaceutical compositions include a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical composition is sterile, sterilizable or sterilized. In a further aspect, the pharmaceutical composition is in a vial in a unit dosage amount or in an integral multiple of a unit dosage amount. In another aspect, the pharmaceutical composition is dried or lyophilized or comprises a dehydrated matrix, or comprises a fusion protein in a lyophilized matrix. In another aspect the pharmaceutical composition of the invention may include a tissue adhesive, tissue sealant, polymer and/or fibrin, which may be a fibrin sealant such as, for example, Tisseel®.

In another aspect the pharmaceutical composition is a coating for a drug-eluting stent.

The invention also relates to therapeutic methods comprising administering to a subject the polypeptides or compositions of the invention. In one aspect, a therapeutically effective amount of polypeptide or composition is administered. In another aspect, the subject is in need of such treatment. The subject may be a mammal, particularly a human, in certain aspects of the invention. In an aspect, polypeptides and compositions of the invention may be administered by injection. Administration may be by injection, by topical application, or by implantation. In one aspect, administration is intravenous, intraarterial, intraperitoneal, intramuscular, or subcutaneous. In one embodiment, administration is local. In another embodiment, the administration is systemic.

In one aspect, provided herein are methods for using a Rho inhibitor for the treatment of conditions associated with cardiac remodeling. In another aspect, provided herein are methods of delaying or preventing conditions that result from cardiac remodeling.

Conditions associated with or resulting from cardiac remodeling that can benefit from the methods provided herein include, cardiomyopathy, heart failure, chronic heart failure, an acute myocardial infarction, left ventricular hypertrophy, right ventricular hypertrophy, coronary artery

disease, essential hypertension, acute hypertensive emergency, heart insufficiency, exercise tolerance, arrhythmia, cardiac dysrhythmia, sudden death, syncope, atherosclerosis, angina pectoris, cardiac bypass reocclusion, intermittent claudication, diastolic dysfunction, and/or systolic dysfunction. A review of Cardiac disorders is found, for example in Braunwald's Heart Disease: A

Textbook of Cardiovascular Medicine, Eugene Braunwald, Saunders; 7th Ed. (2004).

The pharmaceutical compositions containing one or more active agents may be formulated according to conventional pharmaceutical practice (see, for example, Remington: The Science and Practice of Pharmacy, (21st ed.) Ed. A.R. Gennaro (2006), Lippincott Williams & Wilkins, Philadelphia, PA. and Encyclopedia of Pharmaceutical Technology, eds., J. Swarbrick and J. C. Boylan, 1988-2002, Marcel Dekker, New York).

Formulations include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. A variety of aqueous carriers can be used, e.g., water, buffered water, saline, and the like. Non limiting examples of other suitable vehicles include DMSO, polypropylene glycol, polyethylene glycol, vegetable oils, gelatin, hydrogels, hydrogenated naphthalenes, and injectable organic esters, such as ethyl oleate. Such formulations may also contain auxiliary substances, such as preserving, wetting, buffering, emulsifying, and/or dispersing agents. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene- polyoxypropylene copolymers may be used to control the release of the active ingredients. The terms "pharmaceutically acceptable carrier" and "adjuvant" and "physiologically acceptable vehicle" and the like, are to be understood as referring to an acceptable carrier or adjuvant that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof. Further, as used herein "pharmaceutically acceptable carrier" or "pharmaceutical carrier" are known in the art and include, but are not limited to, about 0.01 to about 0.1 M and preferably about 0.05 M phosphate buffer or about 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include

sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, collating agents, inert gases and the like.

As used herein, "pharmaceutical composition" means therapeutically effective amounts (dose) of the agent together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifϊers, adjuvants and/or carriers. A "therapeutically effective amount" as used herein refers to that amount which provides a therapeutic effect for a given condition and administration regimen.

Pharmaceutical composition may include liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCL, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, and detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts). The pharmaceutical composition of the present invention can comprise pharmaceutically acceptable solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance.

Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended by the invention are medical devices or particulate compositions coated with polymers. In one embodiment the pharmaceutical composition is administered locally, systemically, parenterally, intramuscularly, intravenously, subcutaneously, intraperitoneally, intracoronaraly, or directly at the heart.

In addition, the term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount (dose) effective in treating a patient, having a cardiac disorder. It is

also to be understood herein that a "pharmaceutically effective amount" may be interpreted as an amount giving a desired therapeutic effect, either taken in one dose or in any dosage or route, taken alone or in combination with other therapeutic agents. In one embodiment, a "pharmaceutically effective amount" may be understood as an amount of ADP-ribosyl transferase C3 or ADP-ribosyl transferase C3 analogues (e.g., fusion proteins such as SEQ ID NO: 1) that may for example, suppress Rho activity, prevent cell apoptosis, reduce actin polymerization, or suppress (e.g., totally or partially) blunt hypertrophy.

A therapeutically effective amount or dosage of an active agent, e.g., SEQ ID NO: 1, may range from about 0.01 picograms(pg)/Kg to about 0.1 mg/Kg body weight. In one embodiment, the intravenous therapeutically effective amount or dosage of SEQ

ID NO: 1 ranges from about 0.1 pg/Kg to 0.1 mg/Kg body weight, from about 0.1 ng/Kg to 0.1 mg/Kg body weight, or from about 0.1 μg/Kg to 0.1 mg/Kg body weight.

In one embodiment, the intraarterial therapeutically effective amount or dosage of SEQ ID NO: 1 ranges from about 0.01 pg/Kg to 0.01 mg/Kg body weight, from about 0.01 ng/Kg to 0.01 mg/Kg body weight, from about 0.01 μg/Kg to 0.01 mg/Kg body weight.

It will be appreciated that calibration of SEQ ID NO: 1 dosage in high blood flow areas such as the heart presents difficulties in dosage computation. Injured or scarred tissue will, by definition, exhibit altered behavior. The amounts stated here are those to be achieved at the site of introduction and are presumed to be transient and are termed "local concentration." In one embodiment, a non-limiting range for a therapeutically effective concentration is about 1 nM to about 1 M. In another embodiment, a range for a therapeutically effective concentration is about 1 μM to about 100 mM. In another embodiment, the therapeutically effective concentration at the heart is about 0.1 mM to about 10OmM. In another embodiment, the therapeutically effective concentration is about 0.1 mM to about 10 mM. In another embodiment, the therapeutically effective concentration is about 0.1 mM to about 2 mM.

In one embodiment, the therapeutically effective concentration is about 0.5 mM. In another embodiment, the therapeutically effective concentration is about 1.0 mM.

In one embodiment, the administered concentration is about 0.1 mM. In one embodiment, the administered concentration is about 0.5 mM. In one embodiment, the

administered concentration is about 1.0 rnM. In one embodiment, the administered concentration is about 10 mM. In another embodiment, the administered concentration is about 50 mM. In another embodiment, the administered concentration is about 75 mM. In another embodiment, the administered concentration is about 100 mM. In another embodiment, the administered concentration is about 500 mM.

In one embodiment, SEQ ID NO: 1 is administered using a dose of about 0.01 pg/Kg to about 1 mg/Kg. In another embodiment, SEQ ID NO: 1 is administered using a dose of about 0.1 ng/Kg to about 0.1 mg/Kg. In another embodiment, SEQ ID NO: 1 is administered using a dose of about 0.1 u_g/Kg . In another embodiment, SEQ ID NO: 1 is administered using a dose of about 0.1 mg/Kg.

A skilled artisan will appreciate that the pharmaceutical compositions of the present invention may also be formulated as single-dose vials. For example, single-dose vials can be produced containing about 25, about 40, about 60, about 100, about 150, about 200, about 300, or about 500 micrograms of active agent. In a further example, single-dose vials can be produced containing a concentration of about 0.5 mM or about 1.0 mM active agent.

The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.

Treatment of a subject with a therapeutically effective amount of an active compound can a single treatment, continuous treatment, or a series of treatments divided into multiple doses.

Also contemplated is treatment as a single administration, continuous administration, or periodic administration over one or more years. Chronic, long-term administration may be indicated in many cases.

In one embodiment a subject is treated for up to 1 year. In one embodiment a subject is treated for up to 6 months. In one embodiment a subject is treated for up to 100 days. In one example, a subject is treated with an active compound in a time frame of one time per week for between about 1 to about 10 weeks, alternatively between 2 to about 8 weeks, between about 3 to about 7 weeks, or for about 4, 5, or 6 weeks.

In one embodiment a subject is treated substantially continuously. In another embodiment a subject is treated once per day. In another embodiment a subject is treated twice per day. In another embodiment a subject is treated once per week. In another embodiment a subject is treated once per month. It will also be appreciated that the effective dosage of an active compound used for treatment may increase or decrease over the course of a particular treatment.

Generally, each formulation is administered in an amount sufficient to suppress or reduce or eliminate a deleterious effect or a symptom of a cardiac disorder. The amount of an active ingredient that is combined with the carrier materials to produce a single dosage will vary depending upon the subject being treated and the particular mode of administration.

The dosage of each formulation depends on several factors including the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the patient to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular patient may affect dosage used. Furthermore, one skilled in the art will appreciate that the exact individual dosages may be adjusted somewhat depending on a variety of factors, including the specific composition being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disorder being treated, the severity of the disorder, and the anatomical location of the disorder. Wide variations in the needed dosage are to be expected in view of the differing efficiencies of the various routes of administration. For instance, intravenous administration generally would be expected to require higher dosage levels than administration by intracoronary injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, which are well- known in the art. The precise therapeutically effective dosage levels and patterns are typically determined by the physician such as a cardiologist or cardiothoracic surgeon or interventional radiologist in consideration of the above-identified factors.

In addition to treating pre-existing cardiac disorders, the therapies of the present invention can be administered in order to prevent or slow the onset of such disorders. In one embodiment, the therapies of the present invention can be administered for prophylactic applications. In another embodiment, the therapies are administered to a patient susceptible to or

otherwise at risk of a cardiac disorder. In another embodiment, the therapies are administered to a patient who has a pre-existing cardiac disorder and is susceptible to or otherwise at risk of a further cardiac disorder. In another embodiment the therapy is administered to a patient with cardiomyopathy in order to prevent heart failure. Suppression of a cardiac disorder is evaluated by any accepted method of measuring whether cardiac remodeling is slowed or diminished. This includes direct observation and indirect evaluation such as by evaluating subjective symptoms or objective physiological indicators. Treatment efficacy, for example, may be evaluated based on the prevention or reversal of ventricular dysfunction, cardiac chamber size, abnormality or dysmorphology, regional or global wall motion abnormalities or any combination thereof. In a non-limiting example, treatment efficacy for evaluating suppression of heart failure, may also be defined in terms of stabilizing or improving ventricular systolic and diastolic function.

The following examples serve to illustrate certain useful embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof. Alternative materials and methods can be utilized to obtain similar results.

EXAMPLES

Example 1. Effect of SEQ ID NO: 1 on cell size and myofibrillar organization

The following example adapted from Kuwahara et al., FEBS Letters, 1999, 452, 314-318, incorporated herein in its entirety, studies the effect of SEQ ID NO: 1 on an increase in myocyte cell size, protein synthesis and myofibrillar organization. SEQ ID NO: 1 attenuates ET-I -induced increases in protein synthesis and size of cardiac myocytes. In addition, striated sarcomeric actin fibers appear thinner and less dense in cells treated with SEQ ID NO: 1.

Cultured ventricular myocytes are pre-incubated with or without 0.1 μM SEQ ID NO: 1 for 1 h prior to the addition of either a vehicle or 1 nM ET-I for 48 h. The area of cell attachment

is measured by digital image analysis. Values are means +/-S.E.M. (μm ² ) (sizes of 40 cells for each group are measured).

The radioactivity of the incorporated [3H] leucine in cells pretreated with 0.1 μM SEQ ID NO: 1 for 1 h prior to the addition of 1 nM ET-I is measured. The radioactivity in cells treated with a vehicle as a control is arbitrarily assigned a value of 100(%). Values obtained from two independent cultures in quadruplicate are shown as means +/- S.E.M.; P<0.05 vs. control;

P<0.05 vs. ET-I without SEQ ID NO: 1. The effect of SEQ ID NO: 1 on ET-1-induces sarcomeric actin organization. Cardiac myocytes grown on a gelatin-coated slide glass are incubated with a vehicle or 1 nM ET-I in the presence or absence of 0.1 μM SEQ ID NO: 1 for 48 h. Cells are stained with FITC-phalloidin. Results indicate more robust myocytes with SEQ ID NO: 1 administration.

Example 2. Left ventricular (LV) dysfunction and LV chamber remodeling in dogs

The following example examines the effects of SEQ ID NO: 1 on the progression of left ventricular (LV) dysfunction and LV chamber remodeling in dogs with chronic heart failure produced by multiple sequential intracoronary microembolizations. (See Blackburn and Sabbah, U.S. 2006/0111361) Animal Preparation

Chronic LV dysfunction and failure in dogs is produced by multiple sequential intracoronary embolizations with polystyrene Latex microspheres (77-109 μm in diameter) as previously described by Sabbah et al. (1991) Am. J. Physiol. 260:H1379-H1384. Coronary microembolizations are performed during coronary artery catheterization under general anesthesia and sterile conditions. Anesthesia is induced using a combination of intravenous injections of hydromorphone (0.22 mg/Kg), diazepam (0.2-0.6 mg/Kg) and sodium pentobarbital 50-100 mg to effect. Plane of anesthesia is maintained throughout the study using 1% to 2% isoflurane. Left and right heart catheterization is performed via a femoral arteriotomy and venotomy. After each catheterization, the vessels are repaired using 6-0 silk and the skin closed

with 4-0 suture. Microembolizations are discontinued when LV ejection fraction, determined angiographically, is between 30% and 40%. A period of 2 weeks is allowed after the last embolization to ensure that infarctions produced by the last microembolizations have completely healed and heart failure is established. The study protocol is then performed. Study Animals

Healthy, conditioned, purpose-bred mongrel dogs weighing between 19 and 25 Kg are used.

Study Protocol

A randomized, blinded, placebo controlled study design is used. A total of 28 dogs underwent multiple sequential intracoronary microembolizations as described above to produce chronic heart failure. Two weeks after the last embolization, dogs are randomized into 4 study groups (treatment arms). Dogs are randomized to 3 months therapy with SEQ ID NO: 1 or placebo (vehicle). Hemodynamic, angiographic, echocardiographic, Doppler and neurohumoral measurements are made prior to randomization (2 weeks after the last embolization) and after completion of therapy (3 months after initiating therapy). After completing the final hemodynamic and angiographic study, dogs are euthanized and the hearts removed and tissue prepared and saved for future histological and biochemical evaluations. The study primary and secondary end-points are as follows:

Primary Endpoints o Prevention or attenuation of progressive LV dysfunction based on an assessment of LV ejection fraction determined angiographically. o Prevention or attenuation of progressive LV remodeling based on measurements of LV end-diastolic volume and LV end-systolic volume determined angiographically.

Secondary Endpoints o Prevention or attenuation of progressive LV diastolic dysfunction based on assessments of 1) LV peak -dP/dt, 2) LV time constant of early relaxation (T au),

3) mitral valve velocity PE/PA, and 4) LV end-diastolic circumferential wall stress. o Extent of attenuation of cardiomyocyte hypertrophy, volume fraction of replacement fibrosis, volume fraction of interstitial fibrosis, capillary density and oxygen diffusion distance. o Changes in circulating levels of plasma neurohormones (plasma norepinephrine (PNE), plasma renin activity (PRA) and plasma atrial natriuretic factor (ANF) as well as changes of transmyocardial plasma norepinephrine (arterial to coronary sinus difference). Hemodynamic Measurements

All hemodynamic measurements are made during left and right heart catheterizations in anesthetized dogs. Baseline measurements, prior to any microembolizations, are be made to ensure that all hemodynamic parameters are within normal limits. Abnormal dogs are excluded from the study. The following parameters are measured in all dogs at all three study time periods: heart rate, mean aortic pressure, peak rate of change of LV pressure during isovolumic contraction (peak +dP/dt) and relaxation (peak -dP/dt), and LV end-diastolic pressure.

Ventriculographic Measurements

Left ventriculograms are performed during coronary artery catheterization after completion of the hemodynamic measurements. Ventriculograms are performed with the dog placed on its right side and are recorded on 35 mm cine at 30 frames per second during a power injection of 20 mL of contrast material (RENO-M-60, Squibb Diagnostics). Correction for image magnification are made using a radiopaque grid placed at the level of the LV. LV end- systolic and end-diastolic volumes are calculated from angiographic silhouettes using the area length method (4). Premature beats and postextrasystolic beats are excluded from any analysis. LV ejection fraction is calculated as the ratio of the difference of end-diastolic and end- systolic volumes to end-diastolic volume times 100. Stroke volume is calculated as the difference between LV end-diastolic and end-systolic volumes. Cardiac output is calculated as the stroke volume times heart rate and cardiac index as the cardiac output divided by body surface area.

Echocardiography and Doppler Measurements

Echocardiographic and Doppler studies are performed in all dogs at all specified study time points using a 77030A ultrasound system (Hewlett-Packard) with a 3.5 MHZ transducer. All echocardiographic measurements are made with the dog placed in the right lateral decubitus position and recorded on a Panasonic 6300 VHS recorder for subsequent off-line analysis.

Transverse 2-dimensional echocardiograms are obtained at the level of the LV papillary muscle and are used to calculate LV fractional area of shortening. The latter are calculated as the end- diastolic LV cavity area minus the end-systolic cavity area divided by the end-diastolic cavity area times 100. Two chamber view 2-dimensional echocardiograms are also obtained to ascertain LV major and minor semiaxes to be used for calculation of LV end-diastolic circumferential wall stress. Wall stress is calculated as follows: Stress=Pb/h(l-h/2b)(l-hb/2a2), where P is LV end- diastolic pressure, a is LV major semiaxis, b is LV minor semiaxis, and h is LV wall thickness.

Mitral inflow velocity is measured by pulsed-wave Doppler echocardiography. The velocity waveforms are used to calculate peak mitral flow velocity in early diastole (PE), peak mitral inflow velocity during LA contraction (PA), the ratio of PE to PA and early mitral inflow deceleration time. The presence or absence of functional mitral regurgitation (MR) is determined with Doppler color flow mapping (Hewlett-Packard model 77020A Ultrasound System) using both an apical two-chamber and an apical four-chamber views. When present, the severity of functional MR is quantified based on the ratio of the regurgitant jet area to the area of the left atrium times 100. The ratios calculated from both views are then averaged to obtain single representative measure of the severity of functional MR.

Blood Neurohumoral and Electrolyte Measurements

Evaluation of plasma concentrations of several neurohormones is made to complement the hemodynamic assessments. Measurements are made at each of the study time periods described for hemodynamic and angiographic assessments. Transmyocardial PNE concentration is estimated by obtaining blood samples from the ascending aorta and coronary sinus during coronary artery catheterization. Transmyocardial PNE is calculated as the difference between the two samples. Venous blood samples are obtained in duplicate from conscious dogs prior to coronary artery catheterizations for measurement of plasma concentration of norepinephrine, plasma renin activity and plasma atrial natriuretic factor using radioimmunoassay. In addition,

blood samples are obtained at the same time intervals for determination of serum electrolytes (Na+, K+, creatinine and BUN).

Histomorphometric Evaluations

On the day of sacrifice, after completion of all hemodynamic and angiographic studies, the dog's chest is opened through a left thoracotomy, the pericardium is opened and the heart rapidly removed and placed in ice-cold, Tris buffer (pH 7.4). Three 2 mm thick transverse slices are obtained from the LV; one slice from the basal third, one from the middle third and one from the apical third and placed in 10% formalin. Transmural blocks are also obtained and rapidly frozen in isopentane cooled to -16O ⁰ C by liquid nitrogen and stored at -7O ⁰ C. until needed. LV tissue samples are also obtained and stored in gluteraldehyde for future scanning and transmission electron microscopic studies.

From each heart, 3 transverse slices one from the basal third, middle third and apical third of the LV, each approximately 3 mm thick, are obtained. For comparison, tissue samples from normal dogs are obtained and prepared in an identical manner. From each transverse slice, transmural tissue blocks are obtained and embedded in paraffin blocks. From each block, 6 μm thick sections are prepared and stained with Gomori trichrome to identify fibrous tissue. The volume fraction of replacement fibrosis namely, the proportion of scar tissue to viable tissue in all three transverse LV slices, is calculated as the percent total surface area occupied by fibrous tissue using computer-based video densitometry (MOCHA, Jandel Scientific, Corte Madera, Calif). LV free wall tissue blocks are obtained from a second mid-ventricular transverse slice, are mounted on cork using Tissue-Tek embedding medium (Sakura, Torrance, Calif.) and rapidly frozen in isopentane pre-cooled in liquid nitrogen and stored at -7O ⁰ C until used. Cryostat sections, approximately 8 μm thick, are prepared from each block and stained with fluorescein- labeled peanut agglutinin (Vector Laboratories Inc., Burlingame, Calif.) after pretreatment with 3.3 U/ml neuroaminidase type V (Sigma Chemical Co., St. Louis. Mo.) to delineate the myocyte border and the interstitial space including capillaries (5). Sections are double stained with rhodamine-labeled Griffonia simplicifolia lectin I (GSL-I) to identify capillaries. Ten radially oriented microscopic fields (magnification XlOO, objective X40, and ocular 2.5) are selected at random from each section for analysis. Fields containing scar tissue (infarcts) are excluded. An average myocyte cross-sectional area is calculated for each dog using computer-assisted

planimetry. The total surface area occupied by interstitial space and the total surface occupied by capillaries are measured from each randomly selected field using computer-based video densitometry (MOCHA, Jandel Scientific, Corte Madera, Calif). The volume fraction of interstitial collagen is calculated as the percent total surface area occupied by interstitial space minus the percent total area occupied by capillaries (5). Capillary density is calculated as the number of capillaries per mm . Oxygen diffusion distance is calculated as half the distance between two adjoining capillaries. For comparison, identical measurements are made using LV tissue obtained from 7 normal dogs.

Statistical Analysis To ensure that all study measures are similar at baseline, comparisons are made between all 4 study groups before any embolizations and at the time of randomization before initiation of therapy. For these comparisons, a one way analysis of variance (ANOVA) is used with a set at 0.05. If significance is attained, then group wise comparisons are made using the Student- Newman-Kuels test with significance set at p%0.05. Within group comparisons between pre- treatment and post-treatment measures are made using a Students paired t-test with p<0.05 considered significant. To assess treatment effect, the change (δ) in each measure from pre- treatment to post-treatment is calculated for each of the 4 study arms. To determine whether significant differences in δ are present between groups, ANOVA is used with a set at 0.05. If significance is attained, then groupwise comparisons are made using the Student-Newman-Kuels test with significance set at p%0.05. All data are stated as a mean ±SEM.

Baseline Measures

Baseline hemodynamic, ventriculographic, echocardiographic, Doppler and plasma neurohormones and electrolytes measures are obtained prior to any microembolizations.

Pre-Treatment Measures Hemodynamic, ventriculographic, echocardiographic, Doppler and plasma neurohormones and electrolytes measures are obtained at the time of randomization.

Intra-Group Comparisons:

In dogs randomized to placebo, there are no differences in heart rate, mean aortic pressure and LV end-diastolic pressure between pre-treatment and post-treatment. However,

both LV peak +dP/dt and -dP/dt decreased. In this study group, LV end-diastolic volume and end- systolic volume increases significantly at the end of 3 months of treatment while LV ejection fraction and stroke volume decreases significantly. Cardiac output and cardiac index tends to also decrease but the reduction did not reach statistical difference. Echocardiographic and Doppler results show significant reduction in LV fractional area of shortening, mitral inflow PE/PA ratio and deceleration time with significant increases in the severity of function mitral regurgitation and LV end-diastolic circumferential wall stress. Plasma neurohormones and electrolytes remain substantially unchanged.

Intra-Group Comparisons: SEQ ID NO: 1 In dogs randomized to therapy with SEQ ID NO: 1 , there are minor differences in heart rate, mean aortic pressure, LV peak +dP/dt and peak -dP/dt but LV end-diastolic pressure decrease significantly. In this study group, LV end-diastolic volume and end-systolic volume are substantially unchanged at the end of 3 months of treatment while LV ejection fraction, stroke volume and cardiac index increase. Cardiac output tends to also increase but the increase. Echocardiographic and Doppler results show significant no change in LV fractional area of shortening, mitral inflow PE/PA ratio, deceleration time and severity of function mitral regurgitation. LV end-diastolic circumferential wall stress, however, decrease. Plasma neurohormones and electrolytes remain substantially unchanged.

Histomorphometric Findings Compared to normal dogs, dogs treated with placebo show an increase in myocyte cross- sectional area, volume fraction of replacement and interstitial fibrosis and oxygen diffusion distance along with a decrease in capillary density. Treatment with SEQ ID NO: 1 improves histomorphometric measures compared to placebo. The extent of improvement is greater in dogs treated with combination therapy that those treated with SEQ ID NO: 1 alone. The results of this study performed in dogs with moderate heart failure indicate that therapy with SEQ ID NO: 1 prevents the progression of heart failure as evidenced by preservation of LV function and attenuation of LV remodeling. SEQ ID NO: 1 as a therapy for treatment of chronic heart failure is useful.

Example 3

Heart failure (HF)

SEQ ID NO: 1 is indicated for use in adult patients who have been diagnosed with heart failure and are symptomatic despite treatment with optimal heart failure medical management. Without being bound by any particular theory it is believed that in patients with HF and reduced ejection fraction (EF), the left ventricle (LV) progressively dilates from LV remodeling and this process continues long after the time of the initial myocardial injury. It has been suggested that prevention or reversal of remodeling is associated with more favorable natural history outcomes. Again, without being bound by any particular theory, it is believed that the Rho family GTPase transduction pathway is, alone or with co-factors, significant in cardiac remodeling.

A study is designed to examine the effects of SEQ ID NO: 1 on the progression of heart failure (clarified to refer to hospitalization for heart failure) and changes in left ventricular (LV) volume over time (reduction of LV dilation [six months versus baseline LV end -diastolic volume index by 2D-echocardiogram], LV end systolic and diastolic volume [LVESV, LVEDV], and LV-ejection fraction).

Study Design

Mulitcenter, randomized, double-blind, placebo-controlled study of SEQ ID NO: 1 in patients with HF on optimal medical treatment and considered stable for specialized intervention. One objective is to evaluate the effect of SEQ ID NO: 1 at a dose of 0.01 pg to 0.01 mg/Kg body weight on left ventricular end-diastolic volume (LVEDV) compared with placebo in such patients. Patients undergo quantitative radionuclide ventriculography (RVG) at baseline and at 6 months. (See "Current Guidelines for Treatment of Heart Failure": 2006 Update, Nabil Uddin et al, Pharmacotherapy. 2007; 27(4):12S-17S "Society of Nuclear Medicine Procedure Guideline for Gated Equilibrium Radionuclide Ventriculography," version 3.0, approved June 15, 2002)

Patient Population

Patients with American College of Cardiology- American Heart Association (ACC-AHA) stage C and D HF who are at least 18 years of age and who have a left ventricular ejection

fraction less than 40% within 1 year are eligible for screening. Patients are on standard background therapy for HF, including beta-blocker therapy, ACE inhibitor, or angiotension receptor blocker therapy if they are ACE inhibitor intolerant. They must receive therapy for 3 months before enrollment and on stable doses for 2 weeks before enrollment. Exclusion criteria comprise but are not limited to the following: women of child-bearing potential not using acceptable double-barrier contraceptive methods, cardiac surgery within 90 days, biventricular pacing device implanted within 2 months, percutaneous coronary interventions or implantable cardioverter-defibrillator implant within 2 months of potential study enrollment, history of myocardial infarction (documented by electrocardiogram or enzymes) within 3 months, systolic arterial blood pressure <90 mm Hg at screening, and serum creatinine >3.0 mg/dl or blood urea nitrogen (BUN) >60 mg/dl.

Study Drug Administration

Patients are randomized to receive either SEQ ID NO: 1 or saline (matching placebo) in a double-blinded fashion. Patients remain on concomitant medications during the course of the study; however, all cardiac medications, with the exception of short-acting nitrates if needed, are withheld for at least 6 hours before RVG acquisitions.

RVG

Gated equilibrium radionuclide ventriculography (RVG) is a procedure in which the patient's red blood cells (RBCs) are radiolabeled and an electrocardiograph (ECG)-gated cardiac scintigraphy is obtained. Single or multiple measurements of left and/or right ventricular function are obtained. Alternative terminologies for this technique include gated cardiac blood- pool imaging, multigated acquisition (MUGA), and gated equilibrium radionuclide angiography (RNA).

Data are collected from several hundred cardiac cycles to generate an image set of the beating heart that is presented as single, composite cardiac cycle. The method is used to assess (a) regional and global wall motion: (b) cardiac chamber size and morphology; and (c) ventricular systolic and diastolic function, including left and right ventricular ejection fractions (LVEF and RVEF, respectively). An RVG may be acquired at rest, during exercise, or after either pharmacologic or mechanical interventions.

Assessment of Symptom Changes

The overall treatment effect scale is used to determine if there are any changes in the way each subject is feeling over the duration of the study treatment. The Minnesota Living with Heart Failure questionnaire was designed to measure the effects of HF and treatments for HF on an individual's quality of life. Subjects complete the Minnesota Living with Heart Failure Questionnaire on day 1 and at 6 months.

Statistical Analysis and Sample Size Calculations

The primary outcome variable is defined as the change from baseline in LV end diastolic volume index (adjusted for body surface) at 6 months. For the Minnesota Living with Heart Failure Questionnaire, comparisons between SEQ ID NO: 1 and placebo are performed.

Current Practice Guidelines

The Heart Failure Society of America (HFSA), the European Society of Cardiology (ESC), and the American College of Cardiology- American Heart Association (ACC-AHA) have published guidelines for the management of heart failure. The guidelines are relatively consistent in their recommendations; however, one key difference is the staging system for classifying the extent of disease progression. Whereas the HFSA and ESC guidelines use the familiar New York Heart Association (NYHA) functional class system (classes I-IV) based pm signs and symptoms of dyspnea, the ACC-AHA has identified four separate stages of heart failure, paraphrased as follows: • Stage A: High risk for heart failure with no structural damage or heart failure symptoms

• Stage B: Structural damage without heart failure symptoms

• Stage C: Structural damage with previous or current heart failure symptoms

• Stage D: Refractory heart failure, specialized intervention. Stage C is roughly equivalent to NYHA classes II and III.

Example 4

Acute myocardial infarction (AMI)

SEQ ID NO: 1 is indicated for use in adult AMI patients who have undergone successful reperfusion therapy. Study Design

A randomized, double-blind, placebo-controlled, multicenter trial is designed to investigate whether intracoronary infusion of SEQ ID NO: 1 improves global LV function in AMI patients who have undergone successful reperfusion therapy.

Patients are randomized to receive intracoronary infusion of either cells SEQ ID NO: 1 or placebo 3 to 6 days after the infarction. All patients undergo follow-up left ventriculography at 4 months.

Primary Endpoint

Improvement of global LV function by quantitative LV angiography after 4 months.

Secondary Endpoints • Interaction of primary endpoint with baseline ejection fraction (EF) and time to intracoronary infusion

• Changes in LV end-diastolic (EDV) and end-systolic volumes (ESV)

• Major adverse cardiac events (death, MI, coronary revascularization, and rehospitalization due to heart failure Inclusion Criteria

• Acute ST-segment elevation MI

• Successful revascularization by either primary percutaneous coronary intervention (PCI) or thrombolysis (followed by PCI)

• Significant regional wall motion abnormality (EF < 45%) on visual estimate at the time of acute PCI

Exclusion Criteria

• Regional wall motion abnormality outside index infarction areas

• Necessity for additional revascularization

• Active infection or fever Methods

Following randomization, study subjects undergo intracoronary injection of either SEQ ID NO: 1 or the placebo into the infarct-related artery during stop flow (3 x 3 minutes infusion of 3.3 mL each).

Dose of SEQ ID NO: 1 is 0.1 mg/Kg Clinical Events at 4-Month Follow-up Clinical Event Placebo SEQ ID NO: 1 HF = heart failure; MI = myocardial infarction

Change in EF From Baseline to 4-Month Follow-up

Placebo SEQ ID NO: 1

EF at baseline (%) EF at 4 months (%)

P value (within group) Absolute change in EF (%) EF = ejection fraction

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

What is claimed is: