WO2024081245A1 - Polypeptides associés au facteur de différenciation tissulaire (tdfrp) pour le traitement d'une lésion myocardique - Google Patents

Polypeptides associés au facteur de différenciation tissulaire (tdfrp) pour le traitement d'une lésion myocardique Download PDF

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WO2024081245A1
WO2024081245A1 PCT/US2023/034834 US2023034834W WO2024081245A1 WO 2024081245 A1 WO2024081245 A1 WO 2024081245A1 US 2023034834 W US2023034834 W US 2023034834W WO 2024081245 A1 WO2024081245 A1 WO 2024081245A1
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seq
tdfrp
myocardial
injury
heart
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PCT/US2023/034834
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William Dean CARLSON
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Therapeutics By Design, LLC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • cardiomyopathy can develop in people of all ages, races, and ethnicities, and often medical conditions or lifestyle habits can raise the risk of cardiomyopathy. Some types of cardiomyopathy are more common in certain age groups; arrhythmogenic cardiomyopathy is more common in teens and young adults; dilated cardiomyopathy is more common in adults between 20 and 60 years old; hypertrophic cardiomyopathy is more common in people in their 30s; takotsubo cardiomyopathy is more common in women after menopause.
  • Some of the conditions that lead to cardiomyopathy include valvular heart disease, myocardial ischemia, pulmonary diseases, obstructive sleep apnea, inflammation (due to e.g., viral, bacterial, or fungal infection, or autoimmune disease), chemotherapy or radiation treatment for cancer, long term alcohol and drug abuse, heavy metal poisoning, nutritional issues, arrhythmias, constrictive pericarditis, diabetes, hemochromatosis, and complications of late-stage pregnancy.
  • cardiomyopathy Other medical conditions that may lead to cardiomyopathy include Duchenne muscular dystrophy, sarcoidosis or amyloidosis, heart inflammation from endocarditis, myocarditis, or pericarditis, infections, such as viral hepatitis and HIV, obesity, diabetes, or other problems with the metabolic system.
  • Myocardial infarction is a common presentation of coronary artery disease.
  • the World Health Organization estimated in 2004, that 12.2% of worldwide deaths were from ischemic heart disease; with it being the leading cause of death in high- or middle -income countries and second only to lower respiratory infections in lower-income countries.
  • STEMIs occur about twice as often in men as women.
  • IHD is becoming a more common cause of death in the developing world.
  • IHD had become the leading cause of death by 2004, accounting for 1.46 million deaths (14% of total deaths) and deaths due to IHD were expected to double during 1985- 2015.
  • DALYs disability adjusted life years
  • Cardiomyopathy is often characterized by a decrease in the pumping function of the heart.
  • Cardiomyopathy can be caused by a number of conditions including, including but not limited to, valvular abnormalities, hypertension, collagen vascular disease, myocardial ischemia, myocardial infarction, toxins, anticancer therapies, congenital abnormalities, arrhythmias, diabetes and other types of injury.
  • the underlying pathophysiology of cardiomyopathy is characterized by cardiomyocyte apoptosis, inflammation, and fibrosis (scarring). Loss of cardiac function results in increased stress on the heart and thus a worsening of the cardiomyopathic condition resulting in a spiral into congestive heart failure and death.
  • cardiomyopathy Treatments for cardiomyopathy generally address symptoms and causes: beta-blockers to relax stress by relaxing the heart, diuretics to remove excess fluid, spironolactone to reduce fluid and relax the heart, and pacemakers and implanted defibrillators to control arrhythmias.
  • beta-blockers to relax stress by relaxing the heart
  • diuretics to remove excess fluid
  • spironolactone to reduce fluid and relax the heart
  • pacemakers and implanted defibrillators to control arrhythmias.
  • cardiomyopathy is a condition of the heart muscle myocytes. If cardiomyocyte injury is allowed to progress, the result is apoptosis, necrosis (loss of heart muscle), inflammation and fibrosis (scarring and stiffening of the heart muscle), all of which result in loss of heart function resulting in increased mortality and morbidity.
  • cardiomyopathy encompasses a wide range of conditions, all resulting in loss of heart function, there exists a substantial need in the art for improved methods and compositions for treating a wide range of cardiac diseases and injuries characterized by cardiomyopathy.
  • Cardiac injury can be the result of myriad insults, but is mediated through a common pathophysiology including a apoptosis, inflammation, fibrosis, that leads to a syndrome called congestive heart failure or cardiomyopathy.
  • tissue differentiation factor related polypeptides can be used to block and reverse the intracellular pathways in cardiomyocytes that lead to inflammation and apoptosis, thereby preventing necrosis and fibrosis.
  • TDFRPs tissue differentiation factor related polypeptides
  • the present disclosure describes results showing that TDFRPs prevented apoptosis and inflammation in rat cardiomyocytes, and amelioratedthe effects of myocardial ischemia resulting from ischemia/reperfusion injury in vivo. Because the TDFRPs target intracellular pathways that lead to inflammation and apoptosis, the methods described herein are advantageously used to treat or prevent any condition that is associated with myocardial injury
  • the disclosure features a method for treating myocardial injury, or a condition associated with myocardial injury, in a mammal, wherein the method comprises administering at least one tissue differentiation factor related polypeptide (TDFRP) to the mammal, and wherein the administering is in an amount effective to treat myocardial injury in said mammal.
  • TDFRP tissue differentiation factor related polypeptide
  • the disclosure features a method for preventing myocardial injury, or a condition associated with myocardial injury, in a mammal, wherein the method comprises administering at least one tissue differentiation factor related polypeptide (TDFRP) to the mammal, and wherein the administering is in an amount effective to prevent myocardial injury in said mammal.
  • TDFRP tissue differentiation factor related polypeptide
  • the TDFRP is selected from an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.
  • the TDFRP is selected from an amino acid sequence at least 91%, 92%, 93%, 94% ,95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.
  • the TDFRP comprises SEQ ID NO: 1.
  • the TDFRP consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.
  • the TDFRP consists of SEQ ID NO: 1.
  • the mammal is a human.
  • the myocardial injury results from or is associated with an ischemia/reperfusion injury.
  • the ischemia/reperfusion injury results from myocardial infarction, severe trauma, renal insufficiency, pulmonary insufficiency, arterial stenosis, open heart surgery, or doxorubicin chemotherapy.
  • the myocardial injury results from or is associated with a surgical procedure; exposure to a cardiotoxic compound; hypertension; ischemic heart disease; dilated cardiac injury; myocarditis; thyroid disease; viral infection; gingivitis; drug abuse; alcohol abuse; periocarditis; atherosclerosis; vascular disease; hypertrophic cardiomyopathy; acute myocardial infarction; left ventricular systolic dysfunction; coronary bypass surgery; starvation; an eating disorder; or a genetic defect.
  • the cardiotoxic compound is an anthracycline, alcohol, or cocaine.
  • the anthracyline is doxorubicin, or daunomycin.
  • the TDFRP is administered prior to the diagnosis of the myocardial injury, or the condition associated with myocardial injury in the mammal. According to some embodiments of the aspects and embodiments herein, the TDFRP is administered after the diagnosis of the myocardial injury, or the condition associated with myocardial injury in the mammal. According to some embodiments of the aspects and embodiments herein, the mammal is at risk for myocardial injury.
  • the mammal is an individual that smokes, is obese, has been or will be exposed to a cardiotoxic compound, has or had high blood pressure, has or had ischemic heart disease, has or had a myocardial infarct, has a genetic defect that increases the risk of heart failure, has a family history of heart failure, has or had myocardial hypertrophy, has or had hypertrophic cardiomyopathy, has or had left ventricular systolic dysfunction, had coronary bypass surgery, has or had vascular disease, has or had atherosclerosis, has or had alcoholism, has or had pericarditis, has or had a viral infection, has or had gingivitis, has or had an eating disorder, has or had myocarditis, has or had a thyroid disease, or is a cocaine addict.
  • the mammal has or had a cancer.
  • administration of the TDFRP inhibits cardiomyocyte apoptosis.
  • administration of the TDFRP inhibits pericarditis inflammation levels.
  • the TDFRP is administered by a parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, oral, or topical route.
  • the method further comprises administration of an additional agent selected from the group consisting of: anti-neoplastic agents, antibiotics, vaccines, immunosuppressive agents, anti-hypertensive agents and mediators of the hedgehog signaling pathway.
  • an additional agent selected from the group consisting of: anti-neoplastic agents, antibiotics, vaccines, immunosuppressive agents, anti-hypertensive agents and mediators of the hedgehog signaling pathway.
  • FIG. 1 is a graph that shows anti-apopototic activity of THR-123 (SEQ ID NO: 1).
  • Phosphorylated Akt is a marker of apoptosis. Ischemic injury leads to apoptosis. As shown in FIG.l, cell starvation via serum withdrawal led to ischemia and the reduction of phosphorylated AKT in rat cardiomyocytes. Addition of PBS had no effect, whereas addition of THR-123 (SEQ ID NO:1) at 100 pM for 60 h reversed the effects of ischemia on AKT phosphorylation.
  • FIG. 2 is a graph that shows THR-123 (SEQ ID NO: 1) blocked adriamycin(doxorubicin) suppression of pAKT in human cardiomyocytes, thus demonstrating THR-123 anti-apoptotic activity.
  • THR-123 SEQ ID NO: 1
  • doxorubicin adriamycin(doxorubicin) suppression of pAKT in human cardiomyocytes
  • FIG. 2 is a graph that shows THR-123 (SEQ ID NO: 1) blocked adriamycin(doxorubicin) suppression of pAKT in human cardiomyocytes, thus demonstrating THR-123 anti-apoptotic activity.
  • the addition of 333 nM doxorubicin to serum starved cardiomyocytes further reduces phosphorylation of AKT.
  • Addition of SEQ ID No. 1 at 100 pM and at 500 pM reversed the inhibition of AKT phosphorylation in a dose dependent manner.
  • FIG. 3 is a graph that shows THR-123 (SEQ ID NO: 1) and BMP-7 bloced adriamycin (doxorubicin) induction of caspase-3 in human cardiomyocytes, thus demonstrating THR-123 anti- apoptotic activity.
  • addition of 333 pM doxorubicin to cardiomyocytes stimulated caspase-3 activity.
  • Addition of 143 nM BMP-7 or 100 pM or 500 pM THR-123 (SEQ ID NO: 1) completely reversed the apoptotic effects of doxorubicin.
  • FIG. 4 is a graph that shows THR-123 (SEQ ID NO: 1) blocked LPS induction of caspase-3 release in human cardiomyocytes, thus demonstrating THR-123 anti-apoptotic activity.
  • LPS lipopolysaccharide;an endotoxin
  • FIG. 5 is a graph that shows BMP-7 and THR-123 (SEQ ID NO: 1) blocked LPS induction of IL-6 in human cardiomyocytes, thus demonstrating anti-inflammatory activity
  • FIG. 5 exposure of cardiomyocytes to 100 ng/ml LPS resulted in a significant release of inflammatory cytokine IL-6.
  • BMP-7 143 nM
  • FIG. 6 shows a schematic of the time course of the Left Anterior Descending Artery (LAD) model.
  • FIG. 7 shows a schematic of the histomorphometric analysis of the effects of LAD ligation.
  • the area of the necrotic area and the area of the whole area at risk are measured.
  • the areas are summed over all slices to obtain an estimate of the necrotic volume (NV) and the volume at risk (VR).
  • the ratio of NV/VR is a measure of the myocardial injury resulting from the period of ischemia and subsequent reperfusion injury.
  • FIG. 8 is a graph showing the results of the histomorphometric analysis. As shown in FIG.8, there was an 84% reduction in myocardial injury with THR-123 (SEQ ID NO: 1).
  • FIG. 9 is a graph that shows the effect of treatment on pericarditis on inflammation level.
  • tissue differentiation factor related polypeptides TDFRPs
  • analogs, homologs or variants thereof can be used to treat or prevent conditions associated with cardiomyopathy, particularly conditions where cardiomyocytes are stressed or injured leading to inflammation, fibrosis, apoptosis, and/or necrosis.
  • the term “about,” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 20% or ⁇ 10%, more preferably ⁇ 5%, even more preferably ⁇ 1%, and still more preferably ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • administering refers to introducing a composition or agent into a subject and includes concurrent and sequential introduction of a composition or agent.
  • administering can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods.
  • administering also encompasses in vitro and ex vivo treatments.
  • Administration includes self-administration and the administration by another. Administration can be carried out by any suitable route.
  • a suitable route of administration allows the composition or the agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject.
  • analog is meant to refer to a composition that differs from the compound of the present disclosure but retains essential properties thereof.
  • a non-limiting example of this is a polypeptide or peptide or peptide fragment that includes non-natural amino acids, peptidomimetics, unusual amino acids, amide bond isosteres.
  • cardiac remodeling is meant to refer to a group of molecular, cellular and interstitial changes that manifest clinically as changes in size, mass, geometry and function of the heart after injury. Often, the process results in poor prognosis because of its association with ventricular dysfunction and malignant arrhythmias.
  • cardiomyopathy is meant to refer to any disease or condition involving injured cardiomyocytes.
  • cardiovascular disease encompasses diseases and disorders of the muscle and/or blood vessels of the heart, diseases and disorders of the vascular system, and/or diseases and disorders of organs and anatomical systems caused by the diseased condition of the heart and/or vasculature.
  • Examples include, but are not limited to: inflammation of the heart and/or vasculature such as myocarditis, chronic autoimmune myocarditis, bacterial and viral myocarditis, as well as infective endocarditis; heart failure; congestive heart failure; chronic heart failure; cachexia of heart failure; cardiomyopathy, including non-ischemic (dilated cardiomyopathy; idiopathic dilated cardiomyopathy; cardiogenic shock, heart failure secondary to extracorporeal circulatory support (“post-pump syndrome”), heart failure following ischemia/reperfusion injury, brain death associated heart failure (as described in Owen et al., 1999 (Circulation.
  • cardiomyopathy 1999 May 18; 99(19):2565-70
  • hypertrophic cardiomyopathy restrictive cardiomyopathy; non-ischemic systemic hypertension; valvular disease; arythmogenic right ventricular cardiomyopathy) and ischemic (atherogenesis; atherosclerosis; arteriosclerosis; peripheral vascular disease; coronary artery disease; infarctions, including stroke, transient ischemic attacks and myocardial infarctions).
  • myocardial injury is meant to encompass all conditions causing cardiomyocyte death.
  • conditions associated with myocardial injury are meant to include any disease or disorder comprising injury of myocardial cells.
  • conditions associated with myocardial injury are those in which cardiomyocytes are stressed or injured leading to inflammation and fibrosis, apoptosis and/or necrosis.
  • myocardial contusion is a type of myocardial injury and refers to a bruise of the heart muscle. Myocardial contusions may be completely silent or cause an arrhythmia (supraventricular tachycardia or ventricular fibrillation) or hypotension secondary to reduced cardiac output.
  • myocardial fibrosis is meant to refer to a significant increase in the collagen volume of myocardial tissue. It is a complex process that involves all components of the myocardial tissue and can be triggered by tissue injury from myocardial ischemia (hypoxia), inflammation, and hypertensive overload. To reverse replacement fibrosis, resorption of fibrous tissue needs to be coupled with robust myocardial regeneration. The latter is currently not feasible in adult human hearts. Reversal of interstitial fibrosis poses several challenges (see text) and may require co-operation of several different cell types.
  • congestive heart failure is meant to refer to a condition of myocardial injury that is characterized by impaired cardiac function that renders the heart unable to maintain the normal blood output at rest or with exercise, or to maintain a normal cardiac output in the setting of normal cardiac filling pressure.
  • a left ventricular ejection fraction of about 40% or less is indicative of congestive heart failure (by way of comparison, an ejection fraction of about 60% percent is normal).
  • Patients in congestive heart failure display well-known clinical symptoms and signs, such as tachypnea, pleural effusions, fatigue at rest or with exercise, contractile dysfunction, and edema.
  • Congestive heart failure is readily diagnosed by well known methods (see, e.g., “Consensus recommendations for the management of chronic heart failure.” Am. J. Cardiol., 83(2A):lA-38-A, 1999).
  • ischemic heart disease is meant to refer to any disorder resulting from an imbalance between the myocardial need for oxygen and the adequacy of the oxygen supply. Most cases of ischemic heart disease result from narrowing of the coronary arteries, as occurs in atherosclerosis or other vascular disorders.
  • myocardial ischemia refers to when blood flow to the heart muscle (myocardium) is obstructed by a partial or complete blockage of a coronary artery by a buildup of plaques (atherosclerosis).
  • MI myocardial infarction
  • cardiotoxic is meant a compound that decreases heart function by directing or indirectly impairing or killing cardiomyocytes.
  • hypotension is meant to refer to blood pressure that is considered by a medical professional (e.g., a physician or a nurse) to be higher than normal and to carry an increased risk for developing congestive heart failure.
  • an "effective amount" of a compound is meant to refer to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, for example, an amount which results in the prevention of or a decrease in the symptoms associated with a disease that is being treated, e.g., myocardial injury.
  • the amount of compound administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • an effective amount of the compounds of the present disclosure sufficient for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day, to about 10,000 mg per kilogram body weight per day.
  • the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day.
  • the compounds of the present disclosure can also be administered in combination with each other, or with one or more additional therapeutic compounds.
  • reducing or preventing apoptosis is meant preventing apoptosis or reducing the levels of apoptosis in a cardiomocyte as compared with an equivalent untreated control; such reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique.
  • Standard techniques include for example DNA laddering, TUNEL assay, flow cytometry for DNA content, cell death ELISA, caspase activity, or detection of surrogate markers of apoptosis by immunohistochemistry, Western or Northern analysis.
  • treating, reducing, or preventing cardiac inflammation is meant preventing inflammation or decreasing the level of inflammation in a heart or cardiac tissue as compared with an equivalent untreated control; such reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique.
  • inflammation may be measured, for example, by the detection of infiltrating leucocytes (e.g., by immunohistochemistry), the release of pro-inflammatory molecules e.g., MCP-1), or the activation of inflammatory signaling pathway (e.g., activation of the NF-KB pathway).
  • the treatment, reduction, or prevention of cardiac inflammation may also be measured by the ability to reduce the activation of inflammatory signaling pathways in a cardiomyocyte as measured by any standard technique.
  • reducing or preventing pericarditis is meant preventing pericarditis or reducing the levels of pericarditis in a cardiomocyte as compared with an equivalent untreated control; such reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique.
  • treating, reducing, or preventing ischemic-reperfusion injury or by “treating, reducing or preventing a cardiac disorder” is meant treating, or ameliorating such injury or cardiac disorder, respectively, before or after it has occurred.
  • reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique known in the art.
  • ST elevation refers to the ST segment of an electrocardiogram where the myocardial cells have gone through depolarization, but not repolarization. This segment should be on the isoelectric line because the there should be no voltage difference across the cardiac muscle cell membrane at this point.
  • STMI refers to a ST elevation myocardial infarction
  • NSTEMI refers to a non-ST elevation myocardial infarction. NSTEMI is a type of heart attack that happens when the heart’s need for oxygen cannot be met.
  • iatrogenically-induced is meant a condition that is of longer duration than acute, and is planned, or is a consequence of a medical treatment (for example, open heart surgery or chemotherapy).
  • an “isolated” or “purified” polypeptide or polypeptide or biologically-active portion thereof is substantially free of cellular material or other contaminating polypeptides from the cell or tissue source from which the tissue differentiation factor-related polypeptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • polypeptide As used herein, the terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a natural or synthetic peptide containing two or more amino acids linked typically via the carboxy group of one amino acid and the amino group of another amino acid. As will be appreciated by those having skill in the art, the above definition is not absolute and polypeptides or peptides can include other examples where one or more amide bonds could be replaced by other bonds, for example, isosteric amide bonds. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • polypeptide The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids.
  • polypeptide The terms “polypeptide”, “peptide” and “protein” also are inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gammacarboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. It will be appreciated, as is well known and as noted above, that polypeptides may not be entirely linear.
  • polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslational events, including natural processing event and events brought about by human manipulation which do not occur naturally.
  • Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well.
  • the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil, and various types of wetting agents.
  • the term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans, as well as any carrier or diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered compound.
  • small molecule is meant to refer to a composition that has a molecular weight of less than about 5 kDa and more preferably less than about 2 kDa.
  • Small molecules can be, e.g., nucleic acids, peptides, polypeptides, glycopeptides, peptidomimetics, carbohydrates, lipids, lipopolysaccharides, combinations of these, or other organic or inorganic molecules.
  • the terms “subject,” “individual,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. The methods described herein are applicable to both human therapy and veterinary applications.
  • the subject is a mammal, and in other embodiments the subject is a human.
  • a “subject in need” is meant to refer to a subject that (i) will be administered a TDFRP.
  • the terms “therapeutic amount”, “therapeutically effective amount”, an “amount effective”, or “pharmaceutically effective amount” of an active agent e.g. a TDFRP), as described herein, are used interchangeably to refer to an amount that is sufficient to provide the intended benefit of treatment.
  • dosage levels are based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular active agent employed. Thus the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods.
  • the terms “therapeutic amount”, “therapeutically effective amounts” and “pharmaceutically effective amounts” include prophylactic or preventative amounts of the compositions of the disclosure.
  • compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of, a disease, disorder or condition in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease, disorder or condition, including biochemical, histologic and/or behavioral symptoms of the disease, disorder or condition, its complications, and intermediate pathological phenotypes presenting during development of the disease, disorder or condition. It is generally preferred that a maximum dose be used, that is, the highest safe dose according to some medical judgment.
  • dose and “dosage” are used interchangeably herein.
  • TGF-[3) superfamily of polypeptides is meant to refer to a superfamily of polypeptide factors with pleiotropic functions that is composed of many multifunctional cytokines which includes, but is not limited to, TGF-Bs, activins, inhibins, anti-mullerian hormone (AMH), mullerian inhibiting substance (MIS), bone morphogenetic proteins (BMPs), and myostatin.
  • TGF-Bs Transforming Growth Factor- beta
  • AMH anti-mullerian hormone
  • MIS mullerian inhibiting substance
  • BMPs bone morphogenetic proteins
  • myostatin myostatin.
  • TGF-B1 TGF- B2 and TGF-B3 potently inhibit cellular proliferation of many cell types, including those from epithelial origin. Most mesenchymal cells, however, are stimulated in their growth by TGF-B.
  • TGF-Bs strongly induce extracellular matrix synthesis and integrin expression and modulate immune responses.
  • BMPs also known as osteogenic proteins (OPs)
  • OPs osteogenic proteins
  • Activins named after their initial identification as activators of follicle-stimulating hormone (FSH) secretion from pituitary glands, are also known to promote erythropoiesis, mediate dorsal mesoderm induction, and contribute to survival of nerve cells.
  • FSH follicle-stimulating hormone
  • tissue differentiation factor includes, but is not limited to, all members of the TGF- beta superfamily of polypeptides.
  • TGF-beta superfamily polypeptides can be antagonists or agonists of TGF-beta superfamily receptors.
  • TGF-beta Transforming Growth Factor- beta superfamily receptors
  • TGF-beta Transforming Growth Factor- beta
  • TGF-B Transforming Growth Factor- beta
  • TGF-B tissue differentiation factor receptor
  • treating is meant that administration of a TDFRP slows or inhibits the progression of myocardial injury and/or conditions associated with myocardial injury during the treatment, relative to the disease progression that would occur in the absence of treatment, in a statistically significant manner.
  • Well known indicia such as left ventricular ejection fraction, exercise performance, and other clinical tests as enumerated above, as well as survival rates and hospitalization rates may be used to assess disease progression.
  • Whether or not a treatment slows or inhibits disease progression in a statistically significant manner may be determined by methods that are well known in the art (see, e.g., SOLVD Investigators, N. Engl. J. Med. 327:685-691, 1992 and Cohn et al., N. Engl. J. Med. 339:1810-1816, 1998).
  • preventing is meant minimizing or partially or completely inhibiting the development of myocardial injury and/or conditions associated with myocardial injury in a mammal at risk for developing congestive heart failure (as defined in “Consensus recommendations for the management of chronic heart failure.” Am. J. Cardiol., 83(2A):lA-38-A, 1999). Determination of whether myocardial injury and conditions associated with myocardial injury is minimized or prevented by administration of a TDFRP is made by known methods, such as those described in SOLVD Investigators, supra, and Cohn et al., supra.
  • being “at risk for myocardial injury” is meant to refer to an individual who smokes, is obese (i.e., 20% or more over their ideal weight), has been or will be exposed to a cardiotoxic compound (such as a chemotherapeutic or an anthracycline antibiotic), or has (or had) high blood pressure, ischemic heart disease, a myocardial infarct, a genetic defect known to increase the risk of heart failure, a family history of heart failure, myocardial hypertrophy, hypertrophic cardiomyopathy, left ventricular systolic dysfunction, coronary bypass surgery, vascular disease, atherosclerosis, alcoholism, periocarditis, a viral infection, gingivitis, or an eating disorder (e.g., anorexia nervosa or bulimia), or is an alcoholic or cocaine addict.
  • a cardiotoxic compound such as a chemotherapeutic or an anthracycline antibiotic
  • Beneficial or desired clinical results include, but are not limited to, preventing the disease, disorder or condition from occurring in a subject that may be predisposed to the disease, disorder or condition but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), alleviation of symptoms of the disease, disorder or condition, diminishment of extent of the disease, disorder or condition, stabilization i.e., not worsening) of the disease, disorder or condition, preventing spread of the disease, disorder or condition, delaying or slowing of the disease, disorder or condition progression, amelioration or palliation of the disease, disorder or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.
  • proliferative treatment preventing the disease, disorder or condition from occurring in a subject that may be predisposed to the disease, disorder or condition but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), alleviation of symptoms of the disease, disorder or condition, diminishment of extent of the disease, disorder or condition, stabilization i.e., not worsening) of
  • therapeutic effect refers to a consequence of treatment, the results of which are judged to be desirable and beneficial.
  • a therapeutic effect can include, directly or indirectly, the arrest, reduction, or elimination of a disease manifestation.
  • a therapeutic effect can also include, directly or indirectly, the arrest reduction or elimination of the progression of a disease manifestation.
  • the therapeutically effective amount may be initially determined from preliminary in vitro studies and/or animal models.
  • a therapeutically effective dose may also be determined from human data.
  • the applied dose may be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other well-known methods is within the capabilities of the ordinarily skilled artisan.
  • General principles for determining therapeutic effectiveness which may be found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001), incorporated herein by reference, are summarized below.
  • Pharmacokinetic principles provide a basis for modifying a dosage regimen to obtain a desired degree of therapeutic efficacy with a minimum of unacceptable adverse effects. In situations where the drug's plasma concentration can be measured and related to the therapeutic window, additional guidance for dosage modification can be obtained.
  • Drug products are considered to be pharmaceutical equivalents if they contain the same active ingredients and are identical in strength or concentration, dosage form, and route of administration. Two pharmaceutically equivalent drug products are considered to be bioequivalent when the rates and extents of bioavailability of the active ingredient in the two products are not significantly different under suitable test conditions.
  • variant is meant to refer to a compound that differs from the compound of the present disclosure but retains essential properties thereof.
  • a non-limiting example of this is a polynucleotide or polypeptide compound having conservative substitutions with respect to the reference compound, commonly known as degenerate variants.
  • Another non-limiting example of a variant is a compound that is structurally different but retains the same active domain of the compounds of the present disclosure.
  • Variants include N-terminal or C-terminal extensions, capped amino acids, modifications of reactive amino acid side chain functional groups, e.g., branching from lysine residues, pegylation, and/or truncations of a polypeptide compound.
  • variants are overall closely similar, and in many regions, identical to the compounds of the present disclosure. Accordingly, the variants may contain alterations in the coding regions, non-coding regions, or both.
  • compositions comprising at least one TDFRP.
  • TDFRPs Tissue Differentiation Factor Related Polypeptides
  • the present disclosure provides compounds that are functional analogs of tissue differentiation factors, i.e., compounds that functionally mimic TGF-beta superfamily proteins, for example by acting as TGF-beta superfamily receptor agonists, and preferentially bind to select ALK receptor(s).
  • the present compounds are called TDFRPs, and include small molecules, more particularly polypeptides.
  • the TDFRP compound has the general structure identified as SEQ ID NOs: 1-208, disclosed in International Publication No. WG/2003/106656, incorporated by reference in its entirety herein.
  • a TDFRP compound includes an analog or homolog of SEQ ID NOs: 1-208.
  • Compounds of the present disclosure include those with homology to SEQ ID Nos: 1-208, for example, preferably 50% or greater amino acid identity, more preferably 75% or greater amino acid identity, and even more preferably 90% or greater amino acid identity.
  • the compounds of the present disclosure also include one or more polynucleotides encoding SEQ ID Nos: 1-208, including degenerate variants thereof. Accordingly, nucleic acid sequences capable of hybridizing at low stringency with any nucleic acid sequences encoding SEQ ID Nos:l- 208 are considered to be within the scope of the disclosure.
  • the TDFRP compound has the general structure identified as SEQ ID NOs: 1-347, disclosed in International Publication No. WO/2007/035872, incorporated by reference in its entirety herein.
  • a TDFRP compound includes an analog or homolog of SEQ ID NOs: 1-347.
  • Compounds of the present disclosure include those with homology to SEQ ID Nos: 1-347, for example, preferably 50% or greater amino acid identity, more preferably 75% or greater amino acid identity, and even more preferably 90% or greater amino acid identity.
  • the compounds of the present disclosure also include one or more polynucleotides encoding SEQ ID Nos: 1-347, including degenerate variants thereof. Accordingly, nucleic acid sequences capable of hybridizing at low stringency with any nucleic acid sequences encoding SEQ ID Nos:l- 347 are considered to be within the scope of the disclosure.
  • the TDFRP compound has the general structure identified as SEQ ID NOs: 1-314, disclosed in International Publication No. WO/2006/009836, incorporated by reference in its entirety herein.
  • a TDFRP compound includes an analog or homolog of SEQ ID NOs:l-314.
  • Compounds of the present disclosure include those with homology to SEQ ID Nos: 1-314, for example, preferably 50% or greater amino acid identity, more preferably 75% or greater amino acid identity, and even more preferably 90% or greater amino acid identity.
  • the compounds of the present disclosure also include one or more polynucleotides encoding SEQ ID Nos: 1-314, including degenerate variants thereof.
  • the TDFRP compound has the general structure set forth as SEQ ID NOs:l-77, disclosed in International Publication No. WO/2013/013085 incorporated by reference in its entirety herein.
  • a TDFRP compound includes an analog or homolog of SEQ ID NOs:l-77.
  • Compounds of the present disclosure include those with homology to SEQ ID Nos: 1-77, for example, preferably 50% or greater amino acid identity, more preferably 75% or greater amino acid identity, and even more preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid identity.
  • the compounds of the present disclosure also include one or more polynucleotides encoding one or more of SEQ ID Nos: 1-77, including degenerate variants thereof. Accordingly, nucleic acid sequences capable of hybridizing at low stringency with any nucleic acid sequences encoding SEQ ID Nos: 1-77 are considered to be within the scope of the disclosure.
  • Sequence identity can be measured using sequence analysis software (Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705), with the default parameters therein.
  • non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence.
  • Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
  • peptides having mutated sequences such that they remain homologous, e.g., in sequence, in structure, in function, and in antigenic character or other function, with a polypeptide having the corresponding parent sequence.
  • Such mutations can, for example, be mutations involving conservative amino acid changes, e.g., changes between amino acids of broadly similar molecular properties. For example, interchanges within the aliphatic group alanine, valine, leucine and isoleucine can be considered as conservative. Sometimes substitution of glycine for one of these can also be considered conservative.
  • conservative interchanges include those within the aliphatic group aspartate and glutamate; within the amide group asparagine and glutamine; within the hydroxyl group serine and threonine; within the aromatic group phenylalanine, tyrosine, and tryptophan; within the basic group lysine, arginine, and histidine; and within the sulfur-containing group methionine and cysteine. Sometimes substitution within the group methionine and leucine can also be considered conservative.
  • Preferred conservative substitution groups are aspartate-glutamate; asparagine-glutamine; valine -leucine -isoleucine; alanine-valine; phenylalanine- tyrosine; and lysinearginine.
  • altered sequences including insertions such that the overall amino acid sequence is lengthened, while the compound still retains the appropriate TDF agonist or antagonist properties.
  • altered sequences may include random or designed internal deletions that truncate the overall amino acid sequence of the compound, however the compound still retains its TDF-like functional properties.
  • one or more amino acid residues within the sequence are replaced with other amino acid residues having physical and/or chemical properties similar to the residues they are replacing.
  • conservative amino acid substitutions are those wherein an amino acid is replaced with another amino acid encompassed within the same designated class, as will be described more thoroughly below. Insertions, deletions, and substitutions are appropriate where they do not abrogate the functional properties of the compound. Functionality of the altered compound can be assayed according to the in vitro and in vivo assays described below that are designed to assess the TDF-like properties of the altered compound.
  • particularly preferred peptides include, but are not limited to, the following:
  • the peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 10. According to some embodiments of the embodiments and aspects herein, the peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 11. According to some embodiments of the embodiments and aspects herein, the peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 12.
  • the peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 13. According to some embodiments of the embodiments and aspects herein, the peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 14. According to some embodiments of the embodiments and aspects herein, the peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 15.
  • the peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 16. According to some embodiments of the embodiments and aspects herein, the peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 17. According to some embodiments of the embodiments and aspects herein, the peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 18. According to some embodiments of the embodiments and aspects herein, the peptide consists of SEQ ID NO: 10.
  • the peptide consists of SEQ ID NO: 11. According to some embodiments of the embodiments and aspects herein, the peptide consists of SEQ ID NO: 12. According to some embodiments of the embodiments and aspects herein, the peptide consists of SEQ ID NO: 13. According to some embodiments of the embodiments and aspects herein, the peptide consists of SEQ ID NO: 14. According to some embodiments of the embodiments and aspects herein, the peptide consists of SEQ ID NO: 15. According to some embodiments of the embodiments and aspects herein, the peptide consists of SEQ ID NO: 16.
  • the peptide consists of SEQ ID NO: 17. According to some embodiments of the embodiments and aspects herein, the peptide consists of SEQ ID NO: 18.
  • SEQ ID NOs 10-18 set forth above further comprise a N- terminal (H) and a C-terminal (OH):
  • the peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 1.
  • the peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 2.
  • the peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 3.
  • the peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 4. According to some embodiments of the embodiments and aspects herein, the peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 5. According to some embodiments of the embodiments and aspects herein, the peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 6.
  • the peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 7. According to some embodiments of the embodiments and aspects herein, the peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 8. According to some embodiments of the embodiments and aspects herein, the peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 9. According to some embodiments of the embodiments and aspects herein, the peptide consists of SEQ ID NO: 1.
  • the peptide consists of SEQ ID NO: 2. According to some embodiments of the embodiments and aspects herein, the peptide consists of SEQ ID NO: 3. According to some embodiments of the embodiments and aspects herein, the peptide consists of SEQ ID NO: 4. According to some embodiments of the embodiments and aspects herein, the peptide consists of SEQ ID NO: 5. According to some embodiments of the embodiments and aspects herein, the peptide consists of SEQ ID NO: 6. According to some embodiments of the embodiments and aspects herein, the peptide consists of SEQ ID NO: 7.
  • the peptide consists of SEQ ID NO: 8. According to some embodiments of the embodiments and aspects herein, the peptide consists of SEQ ID NO: 9.
  • the disclosure includes vectors containing one or more nucleic acid sequences encoding a TDFRP compound.
  • the nucleic acid containing all or a portion of the nucleotide sequence encoding the polypeptide is inserted into an appropriate cloning vector, or an expression vector (i.e., a vector that contains the necessary elements for the transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA techniques well known in the art and as detailed below.
  • expression vectors useful in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the disclosure is intended to include such other forms of expression vectors that are not technically plasmids, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • the recombinant expression vectors of the disclosure comprise a nucleic acid encoding a compound with TDF-like properties in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression that is operatively-linked to the nucleic acid sequence to be expressed.
  • “operably-linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
  • the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc.
  • the expression vectors of the disclosure can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides, encoded by nucleic acids as described herein (e.g., TDFRP compounds and TDFRP-derived fusion polypeptides, etc.).
  • the present disclosure pertains to TDFRP-expressing host cells, which contain a nucleic acid encoding one or more TDFRP compounds.
  • the recombinant expression vectors of the disclosure can be designed for expression of TDFRP compounds in prokaryotic or eukaryotic cells.
  • TDFRP compounds can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), fungal cells, e.g., yeast, yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, CA. (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide.
  • Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant polypeptide; (ii) to increase the solubility of the recombinant polypeptide; and (iii) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion polypeptide.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988.
  • GST glutathione S-transferase
  • suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET l id (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
  • One strategy to maximize recombinant polypeptide expression in E. coli is to express the polypeptide in host bacteria with an impaired capacity to proteolytically cleave the recombinant polypeptide. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, CA. (1990) 119-128.
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the expression host, e.g., E. coli (see, e.g., Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences of the disclosure can be carried out by standard DNA synthesis techniques.
  • the TDFRP expression vector is a yeast expression vector.
  • yeast Saccharomyces cerivisae examples include pYepSecl (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, CA.), and picZ (Invitrogen Corp, San Diego, CA.).
  • TDFRP can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of polypeptides in cultured insect cells include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
  • a nucleic acid of the disclosure is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195).
  • the expression vector When used in mammalian cells, the expression vector’s control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liverspecific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J.
  • promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the a-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
  • the disclosure further provides a recombinant expression vector comprising a DNA molecule of the disclosure cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to a TDRFP mRNA.
  • Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • TDFRP can be expressed in bacterial cells such as E. coll, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2 nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding TDFRP or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a host cell that includes a compound of the disclosure can be used to produce (i.e., express) recombinant TDFRP.
  • the method comprises culturing the host cell of disclosure (into which a recombinant expression vector encoding TDFRP has been introduced) in a suitable medium such that TDFRP is produced.
  • the method further comprises the step of isolating TDFRP from the medium or the host cell. Purification of recombinant polypeptides is well-known in the art and include ion-exchange purification techniques, or affinity purification techniques, for example with an antibody to the compound.
  • a TDFRP-derived “chimeric polypeptide” or “fusion polypeptide” comprises a TDFRP operatively-linked to a polypeptide having an amino acid sequence corresponding to a polypeptide that is not substantially homologous to the TDFRP, e.g., a polypeptide that is different from the TDFRP and that is derived from the same or a different organism (i.e., non- TDFRP).
  • the TDFRP can correspond to all or a portion of a TDFRP.
  • a TDFRP-derived fusion polypeptide comprises at least one biologically-active portion of a TDFRP, for example a fragment of SEQ ID Nos: 1-347.
  • a TDFRP-derived fusion polypeptide comprises at least two biologically active portions of a TDFRP.
  • a TDFRP-derived fusion polypeptide comprises at least three biologically active portions of a TDFRP polypeptide.
  • the term “operatively linked” is intended to indicate that the TDFRP polypeptide and the non-TDFRP polypeptide are fused in-frame with one another.
  • the non-TDFRP polypeptide can be fused to the N-terminus or C-terminus of the TDFRP.
  • the fusion polypeptide is a GST-TDFRP fusion polypeptide in which the TDFRP sequences are fused to the N-or C-terminus of the GST (glutathione S -transferase) sequences.
  • Such fusion polypeptides can facilitate the purification of recombinant TDFRP by affinity means.
  • the fusion polypeptide is a TDFRP polypeptide containing a heterologous signal sequence at its N-terminus.
  • TDFRP polypeptide containing a heterologous signal sequence at its N-terminus.
  • expression and/or secretion of TDFRP can be increased through use of a heterologous signal sequence.
  • the fusion polypeptide is a TDFRP-immunoglobulin fusion polypeptide in which the TDFRP sequences are fused to sequences derived from a member of the immunoglobulin superfamily.
  • the TDFRP-immunoglobulin fusion polypeptides of the disclosure can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a TDF and a TDF receptor polypeptide on the surface of a cell, to thereby suppress TDF-mediated signal transduction in vivo.
  • the TDFRP-immunoglobulin fusion polypeptides can be used to affect the bioavailability of a TDFRP, for example to target the compound to a particular cell or tissue having the requisite antigen. Inhibition of the TDF/TDF receptor interaction can be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g., promoting or inhibiting) cell survival.
  • TDFRP multiple TDF-related polypeptides (i.e., multiple domain TDF-related polypeptide compounds, hereinafter “TDFRP”) with the general structure shown below:
  • TDFRP 1 TDF-related polypeptide 1
  • TDF-related polypeptide 1 TDF-related polypeptide 1
  • TDF-related polypeptide 1 TDF-related polypeptide 1
  • TDF-related polypeptide 2 TDF-related polypeptide 2
  • the TDRFP domains are compounds that include small molecules. Variants, analogs, homologs, or fragments of these compounds, such as species homologs, are also included in the present disclosure, as well as degenerate forms thereof.
  • a first domain is linked to a second domain through a linker.
  • linker refers to an element capable of providing appropriate spacing or structural rigidity, or structural orientation, alone, or in combination, to a first and a second domain, e.g., TDFRP 1 and TDFRP2, such that the biological activity of the TDFRP is preserved.
  • linkers may include, but are not limited to, a diamino alkane, a dicarboxylic acid, an amino carboxylic acid alkane, an amino acid sequence, e.g., glycine polypeptide, a disulfide linkage, a helical or sheet-like structural element or an alkyl chain.
  • the linker is not inert, e.g., chemically or enzymatically cleavable in vivo or in vitro.
  • the linker is inert, i.e., substantially unreactive in vivo or in vitro, e.g., is not chemically or enzymatically degraded.
  • inert groups which can serve as linking groups include aliphatic chains such as alkyl, alkenyl and alkynyl groups (e.g., C1-C20), cycloalkyl rings e.g., C3-C10), aryl groups (carbocyclic aryl groups such as 1- naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl and heteroaryl group such as /V-imidazolyl, 2- imidazole, 2-thienyl, 3-thienyl, 2-furanyl, 3-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidy, 4- pyrimidyl, 2-pyranyl, 3-pyranyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 2-pyrazinyl, 2-thiazole, 4- thiazole, 5-thiazole, 2-oxazolyl, 4-oxazolyl,
  • the TDFRP compounds include small molecules, more particularly TDFRP compound domains, with the general structure identified herein, as detailed below.
  • the TDFRP compound domains disclosed herein may be present in an TDFRP compound in any combination or orientation. Variants, analogs, homologs, or fragments of these TDFRP compound domains, such as species homologs, are also included in the present disclosure, as well as degenerate forms thereof.
  • the TDFRP compound domains of the present disclosure may be capped on the N-terminus, or the C- terminus, or on both the N-terminus and the C-terminus.
  • the TDFRP compounds may be pegylated, or modified, e.g., branching, at any amino acid residue containing a reactive side chain, e.g., lysine residue, or chemically reactive group on the linker.
  • the TDFRP compound of the present disclosure may be linear or cyclized.
  • the tail sequence of the TDFRP or TDFRP domains may vary in length.
  • the TDFRP compounds of the disclosure are prodrugs, i.e., the biological activity of the TDFRP compound is altered, e.g., increased, upon contacting a biological system in vivo or in vitro.
  • the TDFRP compounds can contain natural amino acids, non-natural amino acids, d-amino acids and 1-amino acids, and any combinations thereof.
  • the compounds of the disclosure can include commonly encountered amino acids, which are not genetically encoded.
  • These non-genetically encoded amino acids include, but are not limited to, - alanine ( -Ala) and other omega-amino acids such as 3-aminopropionic acid (Dap), 2,3- diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth; a-aminoisobutyric acid (Aib); 8- aminohexanoic acid (Aha); 5-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly); ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (M
  • the biological activity namely the agonist or antagonist properties of TDF polypeptides or TDFRP compounds can be characterized using any conventional in vivo and in vitro assays that have been developed to measure the biological activity of the TDFRP compound, a TDF polypeptide or a TDF signaling pathway component.
  • TGF-b/BMPs Superfamily members are associated with a number of cellular activities involved in injury responses and regeneration.
  • TDFRP compounds can be used as agonists of BMPs or antagonists of TGF-b molecules to mediate activities that can prevent, repair or alleviate injurious responses in cells, tissues or organs. Key activities involved in mediating these effects would be antiinflammatory, anti-apoptotic and anti-fibrotic properties.
  • Several in vitro models for inflammation can be used to assess cytokine, chemokine and cell adhesion responses, which are well-documented markers of inflammation.
  • TNF-a tumor necrosis factor-alpha
  • LPS lipopolysaccharide
  • pro-inflammatory molecules for example, IL-1, IL-6, IL-8, NF-kappaB
  • adhesion molecules for example, intercellular adhesion molecule-1 or ICAM-1.
  • these agents induce chemokines (IL-6, IL-8, monocyte chemoattractant protein- 1 or MCP-1 and RANTES), which cause immune cells to infiltrate tissues resulting in organ damage.
  • chemokines IL-6, IL-8, monocyte chemoattractant protein- 1 or MCP-1 and RANTES
  • Apoptosis or programmed cell death is initiated through either a mitochondrial pathway, in response to stress factors or through a receptor-mediated pathway, triggered by the binding of ligands, such as TNF-a.
  • ligands such as TNF-a.
  • Multiple factors contribute to the complex apoptotic process, including the infiltration neutrophils and other inflammatory cells that activate a class of enzymes known as caspases.
  • Other useful markers of apoptosis are Bax and the human vascular anticoagulant, Annexin V, which binds to a protein that gets translocated from the inner to the outer plasma membrane in apoptotic cells.
  • the anti-apoptotic activity of TDFRP compounds can be assessed using in vitro models of apoptosis in cultured cells (for example, heart muscle cells or cardiomyocytes) .
  • cultured cells for example, heart muscle cells or cardiomyocytes
  • Cardiomyocytes rarely proliferate in adult cardiac muscles and the loss of cardiac muscle cells can lead to permanent loss of cardiac function.
  • Myocardial apoptosis caused by injury to cardiomyoctes, contributes to or aggravates the development of myocardial dysfunction in various cardiac diseases.
  • the chemotherapeutic agent, doxorubicin causes heart failure, a reduced number of functioning cardiac muscle cells, activation of caspase 3 and apoptosis.
  • the anti-apoptotic activity of TDFRP compounds can be demonstrated by showing the inhibition of Bax and caspase-3 expression that was induced by doxorubicin, LPS or ischemia, as well as by showing an increase in the levels of phosphorylated Akt, a sensitive indicator of cardiomyocyte health.
  • MI Myocardial injury
  • MI Myocardial ischemia
  • TPA administered soon after a myocardial infarction, limits damage (by reopening the blood vessel) but has no effect on the cellular processes of apoptosis, inflammation, and fibrosis.
  • TDFRP compounds can be used to demonstrate anti-inflammatory and anti-apoptotic activities in cultured cardiomyoctes.
  • TDFRP compounds can be used to reduce the size of infarct, maintain coronary artery endothelial function and inhibit neutrophil adherence to vascular endothelium (reduced reperfusion injury) in rat models of MI.
  • the MI rat model (called the Left Anterior Descending Artery (LAD) occlusion model) involves the transient ligation of the left anterior descending artery to create ischemia.
  • LAD Left Anterior Descending Artery
  • TDFRP compounds can be administered before and after ischemia induced by ligation of the heart ventricle. Efficacy can be determined by morphology and by assessing Creatine Kinase - Myocardioband (CK- MB) levels between infarct and non-infarct regions of the ventricle following reperfusion.
  • CK- MB Creatine Kinase - Myocardioband
  • TDFRPs to block and reverse the intracellular pathways in cardiomyocytes that lead to inflammation and apoptosis, thereby preventing necrosis and fibrosis.
  • the disclosure features a method of treating a condition that is associated with myocardial injury, the method comprising administering to a subject in need of treatment at least one tissue differentiation factor related polypeptide (TDFRP).
  • TDFRP tissue differentiation factor related polypeptide
  • the disclosure features a method of preventing a condition that is associated with myocardial injury, the method comprising administering to a subject in need of prevention at least one TDFRP.
  • cardiomyocytes can be injured. Some of those injuries are immediate, and include, but are not limited to: ischemia, myocardial infarction, chronic arterial insufficiency, atherosclerosis, mechanical stress (e.g., over work of cardiomyocytes), pressure overload, due to valvular obstruction, hypertension, volume overload and arrhythmias.
  • Congenital disorders include Coarctation, where section of the aorta developed narrow; atrial septal defect, a hole between two upper chambers of the heart; ventricular septal defect, a hole between two lower chambers of the heart; noncompaction, a developmental defect in which the lower left ventricle failed to develop; and pulmonary hypertension, narrowed arteries in lung and heart.
  • Infectious diseases can also lead to myocardial injury, and include, but are not limited to, viral, bacterial, fungal infections and Ricksttsia.
  • Autoimmune diseases can also lead to myocardial injury, and include, but are not limited to, Systemic Lupus Erythematosis and Scleroderma.
  • TDFRPs represent a possible chronic treatment or preventative treatment for myocardial injury associated with the autoimmune diseases.
  • Cardiotoxic compound e.g., cocaine, alcohol, an anti-ErbB2 antibody or anti-HER2 antibody, such as HERCEPTIN, or an anthracycline antibiotic, such as doxorubicin or daunomycin
  • TDFRPs represent a possible chronic treatment or preventative treatment for myocardial injury associated withe ardiotoxic compounds.
  • TDFRP administration to cancer patients prior to and during anthracycline chemotherapy or anthracycline/anti-ErbB2 (anti- HER2) antibody (e.g., HERCEPTIN) combination therapy may prevent the patients' cardiomyocytes from undergoing apoptosis, thereby preserving cardiac function.
  • toxins include, but are not limited to, toxins, heavy metals, doxorubicin, gingivitis, drug abuse, alcohol abuse, starvation, amphetamines, cancer treatments, genetic or congenital conditions, missing, unusual, or abnormal protein expression (extracellular - interacting with cardiomyocytes or intracellular - internal structural or signaling).
  • CI Cardiac Injury
  • Cardiac injury refers to a group of diseases that affect the heart muscle. Early on there may be few or no symptoms of cardiac injury. However, as the disease worsens, shortness of breath, feeling tired, and swelling of the legs may occur, due to the onset of heart failure. An irregular heart beat and fainting may occur. Those affected are at an increased risk of sudden cardiac death.
  • Types of cardiac injury include: ischemic cardiomyopathy, hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular dysplasia, and Takotsubo cardiomyopathy (broken heart syndrome) (CI-3).
  • hypertrophic cardiomyopathy the heart muscle enlarges and thickens.
  • dilated cardiomyopathy the ventricles enlarge and weaken.
  • restrictive cardiomyopathy the ventricle stiffens.
  • Hypertrophic cardiomyopathy (HCM, or HOCM when obstructive) is a condition in which the heart becomes thickened without an obvious cause.
  • the parts of the heart most commonly affected are the interventricular septum and the ventricles. This results in the heart being less able to pump blood effectively and also may cause electrical conduction problems.
  • hypertrophic cardiomyopathy the heart muscle enlarges and thickens.
  • dilated cardiomyopathy the ventricles enlarge and weaken.
  • People who have HCM may have a range of symptoms. People may be asymptomatic, or may have fatigue, leg swelling, and shortness of breath. It may also result in chest pain or fainting. Symptoms may be worse when the person is dehydrated. Complications may include heart failure, an irregular heartbeat, and sudden cardiac death.
  • HCM is most commonly inherited from a person's parents in an autosomal dominant pattern. It is often due to mutations in certain genes involved with making heart muscle proteins. Other inherited causes of left ventricular hypertrophy may include Fabry disease, Friedreich's ataxia, and certain medications such as tacrolimus. Other considerations for causes of enlarged heart are athlete's heart and hypertension (high blood pressure). Making the diagnosis of HCM often involves a family history or pedigree, an electrocardiogram, echocardiogram, and stress testing. Genetic testing may also be done. HCM can be distinguished from other inherited causes of cardiomyopathy by its autosomal dominant pattern, whereas Fabry disease is X-linked and Friedreich's Ataxia is inherited in an autosomal recessive pattern.
  • DCM Dilated Cardiomyopathy
  • DCM Dilated cardiomyopathy
  • Coronary artery disease and high blood pressure may play a role, but are not the primary cause. In many cases the cause remains unclear. It is a type of cardiomyopathy, a group of diseases that primarily affects the heart muscle. The diagnosis may be supported by an electrocardiogram, chest X-ray, or echocardiogram.
  • treatment may include medications in the ACE inhibitor, beta blocker, and diuretic families.
  • a low salt diet may also be helpful.
  • blood thinners or an implantable cardioverter defibrillator may be recommended.
  • Cardiac resynchronization therapy (CRT) may be necessary. If other measures are not effective a heart transplant may be an option in some.
  • Restrictive cardiomyopathy is a form of cardiomyopathy in which the walls of the heart are rigid (but not thickened). Thus the heart is restricted from stretching and filling with blood properly. It is the least common of the three original subtypes of cardiomyopathy: hypertrophic, dilated, and restrictive. In restrictive cardiomyopathy the ventricle stiffens. It should not be confused with constrictive pericarditis, a disease which presents similarly but is very different in treatment and prognosis.
  • Arrhythmogenic cardiomyopathy ACM
  • arrhythmogenic right ventricular dysplasia AMVD
  • arrhythmogenic right ventricular cardiomyopathy AR VC
  • ACM is caused by genetic defects of the parts of heart muscle (also called myocardium or cardiac muscle) known as desmosomes, areas on the surface of heart muscle cells which link the cells together.
  • the desmosomes are composed of several proteins, and many of those proteins can have harmful mutations.
  • ARVC can also develop in intense endurance athletes in the absence of desmosomal abnormalities. Exercise-induced ARVC cause possibly is a result of excessive right ventricular wall stress during high intensity exercise.
  • the disease is a type of non-ischemic cardiomyopathy that primarily involves the right ventricle, though cases of exclusive left ventricular disease have been reported. It is characterized by hypokinetic areas involving the free wall of the ventricle, with fibrofatty replacement of the myocardium, with associated arrhythmias often originating in the right ventricle.
  • the nomenclature ARVD is currently thought to be inappropriate and misleading as ACM does not involve dysplasia of the ventricular wall. Cases of ACM originating from the left ventricle led to the abandonment of the name AR VC.
  • ACM can be found in association with diffuse palmoplantar keratoderma, and woolly hair, in an autosomal recessive condition called Naxos disease, because this genetic abnormality can also affect the integrity of the superficial layers of the skin most exposed to pressure stress.
  • ACM is an important cause of ventricular arrhythmias in children and young adults. It is seen predominantly in males, and 30-50% of cases have a familial distribution.
  • Takotsubo cardiomyopathy or Takotsubo syndrome also known as stress cardiomyopathy, is a type of non-ischemic cardiomyopathy in which there is a sudden temporary weakening of the muscular portion of the heart. It usually appears after a significant stressor, either physical or emotional; when caused by the latter, the condition is sometimes called broken heart syndrome. Examples of physical stressors that can cause TTS are sepsis, shock, and pheochromocytoma, and emotional stressors include bereavement, divorce, or the loss of a job. Reviews suggest that of patients diagnosed with the condition, about 70-80% recently experienced a major stressor, including 41-50% with a physical stressor and 26-30% with an emotional stressor. TTS can also appear in patients who have not experienced major stressors.
  • catecholamines such as adrenaline and norepinephrine from extreme stress or a tumor secreting these chemicals
  • catecholamines when released directly by nerves that stimulate cardiac muscle cells, have a toxic effect and can lead to decreased cardiac muscular function or "stunning".
  • this adrenaline surge triggers the arteries to tighten, thereby raising blood pressure and placing more stress on the heart, and may lead to spasm of the coronary arteries that supply blood to the heart muscle. This impairs the arteries from delivering adequate blood flow and oxygen to the heart muscle. Together, these events can lead to congestive heart failure and decrease the heart's output of blood with each squeeze.
  • Takotsubo cardiomyopathy occurs worldwide. The condition is thought to be responsible for 2% of all acute coronary syndrome cases presenting to hospitals. Although TTS has generally been considered a self-limiting disease, spontaneously resolving over the course of days to weeks, contemporary observations show that "a subset of TTS patients may present with symptoms arising from its complications, e.g. heart failure, pulmonary oedema, stroke, cardiogenic shock, or cardiac arrest". This does not imply that rates of shock/death of TTS are comparable to those of acute coronary syndrome (ACS), but that patients with acute complications may co-occur with TTS.
  • ACS acute coronary syndrome
  • ICM Coronary infarct
  • Ischemic cardiomyopathy is a type of cardiomyopathy caused by a narrowing of the coronary arteries which supply blood to the heart.
  • patients with ischemic cardiomyopathy have a history of acute myocardial infarction, however, it may occur in patients with coronary artery disease, but without a past history of acute myocardial infarction.
  • This cardiomyopathy is one of the leading causes of sudden cardiac death.
  • the adjective ischemic means characteristic of, or accompanied by, ischemia — local anemia due to mechanical obstruction of the blood supply.
  • the TDFRPs described herein are used to treat likely or existing myocardial injury due to an unanticipated occurrence, such as a myocardial infarct, or to prevent further damage from an ongoing condition associated with myocardial injury such as certain congenital conditions or in the wake of an infection.
  • therapeutic uses include, but are no limited to, untreatable chronic conditions that result in cardiomyopathy.
  • untreatment conditions include, but are not limited to, such as autoimmune disease, pulmonary insufficiency, haemochromatosis, Anderson-Fabry disease, glycogen storage disease, medications for treating chronic conditions for which a side effect is cardiomyopathy, Gaucher’s disease, Hurler’s disease, Hunter’s disease, diabetes mellitus, hyper and hypothyroidism, hyperparathyroidism, and pheochromocytoma.
  • the disclosure features a method of preventing a condition that is associated with myocardial injury, the method comprising administering to a subject in need of treatment at least one tissue differentiation factor related polypeptide (TDFRP).
  • TDFRP tissue differentiation factor related polypeptide
  • TDFPRs are used prophylactically, for example to prevent damage from a planned event such as the use of doxorubicin for chemotherapy or prior to a major operative procedure where either a long period under anesthesia is anticipated and/or open-heart surgery where a cardio-pulmonary pump may or will be used.
  • a planned event such as the use of doxorubicin for chemotherapy or prior to a major operative procedure where either a long period under anesthesia is anticipated and/or open-heart surgery where a cardio-pulmonary pump may or will be used.
  • the disclosure features a method of providing protection for an ischemic event in a subject, wherein the subject is undergoing a chemotherapy regimen, or will undergo chemotherapy regimen where a cardiotoxic agent is used, the method comprising administering to a subject in need of treatment at least one tissue differentiation factor related polypeptide (TDFRP).
  • TDFRP tissue differentiation factor related polypeptide
  • the disclosure features a method of providing protection for an ischemic event in a subject, wherein the subject will undergo open-heart surgery, the method comprising administering to a subject in need of treatment at least one tissue differentiation factor related polypeptide (TDFRP).
  • TDFRP tissue differentiation factor related polypeptide
  • Isolated cardiomyocyte cultures or cardiomyocytes present within cardiac tissue or the heart organ may be treated with the TDFRPs described herein.
  • a cardiomyocyte according to the methods of the present disclosure may be derived from any mammal, including for example, a pig, mouse, or non-human primate monkey. Cardiomyocytes amenable to treatment may be of any maturity state, and thus include neonatal cardiomyocytes, stem cells, cells committed to differentiate to cardiomyocytes, a myocyte derived from a non-heart muscle, a myoblast, or adult cardiomyocytes.
  • Mammals having a cardiac disorder may be directly administered a TDFRP.
  • cardiomyocytes, or cardiac tissue may be isolated from a mammal, treated with a TDFRP ex vivo and transplanted back into the patient.
  • Such cardiomyocytes, cardiac tissue, or heart may also derive from a donor mammal for transplantation into a recipient mammal.
  • the donor and recipient may or may not be from the same species.
  • a pig heart treated according to the methods of this disclosure may be transplanted into a human recipient.
  • suitable in vitro or in vivo assays are performed to determine the effect of a specific TDFRP-based therapeutic.
  • in vitro assays can be performed with representative cells of the type(s) involved in the patient's disorder, to determine if a given TDFRP-based therapeutic exerts the desired effect upon the cell type(s).
  • Compounds for use in therapy can be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects.
  • suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects.
  • any of the animal model system known in the art can be used prior to administration to human subjects.
  • a more complex in vitro system that better mimics the in vivo setting may be used for testing the efficacy of a compound or analog, e.g., TDFRP compound
  • a number of assays may be used to measure cardiomyocyte apoptosis in vitro, including for example DNA laddering, cell death ELISA, flow cytometry for DNA content, TUNEL assay, the measurement of caspase activity, or the detection of surrogate markers of apoptosis by Western or Northern analysis, or alternatively by RT-PCR.
  • DNA laddering For determination of DNA laddering, cellular DNA is extracted with phenol: chloroform, treated with RNase, 32P-labeled, and then visualized by electrophoresis in 1.8% agarose gel.
  • terminal deoxynucleotidyl transferase is used to incorporate digoxigenin-labeled dUTP into 3'-OH DNA ends generated by DNA fragmentation and detected by counterstaining with peroxidase labeled anti-digoxigenin mAh (ApoTag, Intergen).
  • Flow cytometry may also be used to measure DNA content in cardiomyocytes.
  • Cells are fixed with 80% ethanol and stained with propidium iodide after RNase treatment. Apoptotic cells register as containing less than the diploid DNA quantity (2N).
  • Histone-associated DNA fragments are quantified by cell death ELISA as described in the manufacturer's protocol. DNA fragmentation data are corrected for background and normalized to the result with normoxic cardiomyocytes.
  • apoptosis may also be measured by the protein or gene expression, or alternatively, by the activity of surrogate markers of apoptosis including, for example, caspase activity.
  • Caspase-3, -8, and -9 activity may be examined by using the caspase colorimetric assay kit from R&D Systems according to the manufacturer's protocol. Briefly, cells are scraped, collected, washed with cold PBS, and lysed in cold lysis buffer. Lysates are incubated on ice for 10 min and centrifuged (10,000xg, 1 min). The supernatants are removed and assayed for caspase activity.
  • the specific peptide substrates used for each individual caspase are DEAD-pNA, LEHD-pNA, and IETD- pNA for caspase-3, -9, and -8, respectively. Release of the pNA cleavage product is quantitated in a microplate reader (Bio-Rad) at a wavelength of 405 nm.
  • overlapping assays may be performed to ensure that cell death is apoptotic (nuclear morphology and DNA laddering), that dying cells are in fact CM (TUNEL/double staining/confocal), and that quantitative comparisons of different populations can be made (ELISA for histone-associated DNA fragments/FACS for DNA content).
  • ELISA histone-associated DNA fragments/FACS for DNA content.
  • General assays of cell viability e.g., trypan blue exclusion, MTT, etc.
  • MTT histone-associated DNA fragments/FACS for DNA content
  • TUNEL staining can be performed using Apoptag (Intergen) according to the manufacturer's instructions, with Hoechst 33258 (Sigma) nuclear counter-staining.
  • DNA laddering can also be used to detect apoptosis of cardiomyocytes.
  • Fresh tissues (without TTC staining) are microdissected under UV light into ischemic and non-ischemic regions and processed simultaneously. Tissues from each region are lysed and DNA is prepared, labeled with [a-32P] dCTP, and subjected to electrophoresis and autoradiography as described by Vazquez- Jimenez et al (J. Am. Coll. Cardiol., 2001).
  • apoptosis of cardiomyocytes may further be determined by the detection of surrogate markers of apoptosis, such caspase activation, for example.
  • Cardiac inflammation may be determined by the detection of pro-inflammatory markers, the release of pro-inflammatory molecules (MCP-1), or by the activation of pro-inflammatory signaling in cardiomyocytes.
  • NF-KB activation correlates with cardiac inflammation and may be determined both in vitro (by Western or Northern analysis for example) or in vivo (as measured by immunohistochemical methods).
  • NF-KB activation is typically demonstrated by phosphorylation and degradation of IKB, nuclear translocation of the p65 NF-KB subunit, or increased mRNA for the NF- KB -dependent transcripts, VCAM-1 and ICAM-1.
  • compositions of the disclosure typically contain a therapeutically effective amount of a compound described herein.
  • a pharmaceutical composition may contain more than a therapeutically effective amount, such as in bulk compositions, or less than a therapeutically effective amount, that is, individual unit doses designed for multiple administration to achieve a therapeutically effective amount.
  • the composition will contain from about 0.01-95 wt % of active agent, including, from about 0.01-30 wt %, such as from about 0.01-10 wt %, with the actual amount depending upon the formulation itself, the route of administration, the frequency of dosing, and so forth.
  • a composition suitable for an oral dosage form may contain about 5-70 wt %, or from about 10-60 wt % of active agent.
  • TDFRPs may be administered to patients or experimental animals with a pharmaceutically- acceptable diluent, carrier, or excipient, in unit dosage form.
  • a pharmaceutically- acceptable diluent, carrier, or excipient in unit dosage form.
  • Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer such compositions to patients or experimental animals.
  • intravenous administration is preferred, any appropriate route of administration may be employed, for example, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, oral, or topical (e.g., by applying an adhesive patch carrying a formulation capable of crossing the dermis and entering the bloodstream) administration.
  • Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols. Any of the above formulations may be a sustained-release formulation.
  • Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Sustained-release, biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • Other potentially useful parenteral delivery systems for administering molecules of the disclosure include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the polypeptide may be administered by administering an expression vector encoding the polypeptide to the mammal.
  • TDFRP polypeptides described herein may also be administered by somatic gene therapy.
  • Expression vectors for gene therapy e.g., plasmids, artificial chromosomes, or viral vectors, such as those derived from adenovirus, retrovirus, poxvirus, or herpesvirus
  • the promoter may be any non-tissue- specific promoter known in the art (for example, an SV-40 or cytomegalovirus promoter).
  • the promoter may be a tissue-specific promoter, such as a striated muscle-specific, an atrial or ventricular cardiomyocyte-specific (e.g., as described in Franz et al., Cardiovasc. Res.
  • the promoter may be an inducible promoter, such as the ischemia-inducible promoter described in Prentice et al. (Cardiovasc. Res. 35:567-574, 1997).
  • the expression vector may be administered as naked DNA mixed with or conjugated to an agent to enhance the entry of the DNA into cells, e.g., a cationic lipid such as LIPOFECTIN, LIPOFECT AMINE (Gibco/BRL, Bethesda, Md.), DOTAPTM (Boeringer-Mannheim, Indianapolis, Ind.) or analogous compounds, liposomes, or an antibody that targets the DNA to a particular type of cell, e.g., a cardiomyocyte or an endothelial cell.
  • a cationic lipid such as LIPOFECTIN, LIPOFECT AMINE (Gibco/BRL, Bethesda, Md.), DOTAPTM (Boeringer-Mannheim, Indianapolis, Ind.) or analogous compounds, liposomes, or an antibody that targets the DNA to a particular type of cell, e.g., a cardiomyocyte or an endothelial cell.
  • the method of administration may be any
  • DNA for somatic gene therapy has been successfully delivered to the heart by intravenous injection, cardiac perfusion, and direct injection into the myocardium e.g., see Losordo et al., Circulation 98:2800-2804, 1998; Lin et al., Hypertension 33:219-224, 1999; Labhasetwar et al., J. Pharm. Sci. 87:1347-1350, 1998; Yayama et al., Hypertension 31:1104-1110, 1998).
  • Example 1 Cell starvation via serum withdrawal led to ischemia and the reduction of phosphorylated AKT
  • Cell Applications Primary neonatal rat cardiomyocytes (Cell Applications) were cultured in medium with 10% growth supplement (Cell Applications) for 24 hours at 37° C., 5% CO2. Cells were then starved of growth supplement for 12 hours to create ischemic injury. Controls received medium alone. Cells were incubated with culture medium alone, BMP- 7 (143 nM) or SEQ ID No. 1 (100 pM) for 60 h.
  • BMP- 7 143 nM
  • SEQ ID No. 1 100 pM
  • Example 2 The effect of TDFRPs on the depression of pAKT due to doxorubicin
  • Neonatal rat cardiomyocytes were pre -treated with 0.33 pM doxorubicin for 24h, followed by treatment with BMP-7 (143 nM) or THR-123 (SEQ ID NO: 1) at 100 and 500 pM for 60h.
  • the TDFRP peptide significantly increased cellular Akt phosphorylation (p ⁇ 0.01) compared to doxorubicin alone (FIG. 2), and inhibited doxorubicin induced Caspase-3 activity in a dose dependent manner (p ⁇ 0.05) (FIG. 2).
  • Example 3 The chemotherapeutic agent doxorubicin stimulated Caspase-3.
  • Neonatal rat cardiomyocytes were pre -treated with 0.33 pM doxorubicin for 24h, followed by treatment with BMP-7 (143 nM) or SEQ ID No. 1 at 100 and 500 pM for 60h. Both BMP-7 and THR-123 (SEQ ID NO: 1) significantly inhibited doxorubicin induced Caspase-3 activity (p ⁇ 0.05) (FIG. 3). Caspase-3 activation is a step common to both apoptotic pathways and leads to apoptosis.
  • Example 4 The effect of TDFRP peptide SEQ ID NO: 1 on LPS induced Caspase-3
  • LPS Lipopolysaccharide
  • TDFRP peptide SEQ ID NO: 1 reduces inflammation in cardiomyocytes
  • LPS induces the production of cytokine IL-6, a marker of inflammation.
  • cytokine IL-6 a marker of inflammation.
  • BMP-7 143 nM
  • THR-123 SEQ ID NO: 1
  • Example 6 An in vivo study of the effect of TDFRP peptide THR-123 (SEQ ID NO: 1) in ameliorating the effects of myocardial ischemia The efficacy of BMP mimetic THR-123 in ameliorating the effects of myocardial ischemia was investigated in the rat after ligation of the coronary artery.
  • the rat LAD model is used for studying the effect of treatments for ameliorating the effect of a heart attack (coronary infarct).
  • the Left Anterior Descending artery (LAD) supplies blood to the left ventricle. Blocking blood flow through the LAD creates ischemia in a region of the left ventricle muscle (region at risk). Releasing the blockage leads to reperfusion injury when immune cells in the recirculating blood attack injured cardiomyocytes.
  • Myocardial ischemia was created by ligating the left anterior descending artery (LAD), which supplies blood to the left ventricle of the heart.
  • LAD left anterior descending artery
  • the chest and the pericardium are opened to expose the LAD.
  • a suture is placed around the LAD and loops are added to the two ends, then the suture is tightened with an over hand knot (ligation).
  • the ligation blockage to the LAD was maintained for 20 min to simulate a coronary infarct, then released using the two loops.
  • the animals were divided into three groups that were dosed with PBS, BMP-7, or THR-123 (SEQ ID NO: 1). Administrations were at 2 hr prior to ligation and at 24, 72 and 120 hr post ligation. On the seventh day post ligation the suture was tightened again and Methyl Blue was injected into the blood stream to stain region of the heart that the ligation did not affect (the unaffected region).
  • Treatment with BMP-7 and THR-123 also reduced paracardial inflammation relative to PBS.
  • the predominance of the hearts from the PBS treated group had more severe inflammation than hearts from the BMP-7 treated and the THR-123 (SEQ ID NO: 1) treated groups (FIG. 9).

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Abstract

La présente invention concerne de manière générale des méthodes de traitement ou de prévention d'une lésion myocardique, ou d'un état pathologique associé à une lésion myocardique, chez un mammifère, la méthode comprenant l'administration d'au moins un polypeptide associé au facteur de différenciation tissulaire (TDFRP) au mammifère, et l'administration étant effectuée en une quantité efficace pour traiter ou prévenir une lésion myocardique chez ledit mammifère.
PCT/US2023/034834 2022-10-10 2023-10-10 Polypeptides associés au facteur de différenciation tissulaire (tdfrp) pour le traitement d'une lésion myocardique WO2024081245A1 (fr)

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