THERAPEUTIC USES OF CARDIAC MYOSIN INHIBITORS

RELATED APPLICATIONS

[0001] The application claims priority to U.S. provisional patent application number 64/403,547 filed on September 2, 2022. The contents of the aforementioned application are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention relates generally to small molecule therapeutics, and more specifically, it relates to cardiac myosin inhibitors and methods of treating heart disease.

BACKGROUND

[0003] Cardiovascular disease (CVD) is a class of diseases that involve the heart or blood vessels. CVD includes coronary artery diseases (CAD) such as angina and myocardial infarction (commonly known as a heart attack). Other CVDs include stroke, heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, abnormal heart rhythms, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, thromboembolic disease and venous thrombosis.

[0004] Cardiovascular disease (CVD) is a major cause of disability and premature death throughout the world. CVD is a major factor in healthcare costs, mortality and morbidity. Moreover, CVD is the leading cause of death in the United States. About 659,000 people in the United States die from heart disease every year.

[0005] The underlying mechanisms of CVD vary depending on the disease. It is estimated that dietary risk factors are associated with 53% of CVD deaths. Coronary artery disease, stroke, and peripheral artery disease involve atherosclerosis. This may be caused by high blood pressure, smoking, diabetes mellitus, lack of exercise, obesity, high blood cholesterol, poor diet, excessive alcohol consumption and poor sleep.

Based on these risk factors, it is estimated that up to 90% of CVD is preventable. Prevention of CVD involves improving risk factors through, for example, healthy eating, exercise, limiting alcohol intake and not smoking. Treating risk factors, such as high blood pressure, blood lipids and diabetes can also be beneficial.

[0006] The five most common heart diseases are heart attack, stroke, heart failure, arrhythmia, and heart valve complications. Arrhythmias are irregular heartbeats (heart can beat quickly or too slowly or have an erratic pattern). This can cause severe chest pain, dangerous blood clots and the heart to abruptly stop pumping blood to the body. Without immediate treatment, the arrhythmia can cause death within minutes. Current anti-arrhythmic drugs are far from being optimal and are limited by their low to moderate efficacy and by their QT-prolonging/pro-arrhythmic effects. Also, no anti-arrhythmic drug has been approved by the Food and Drug Administration for the last decade. Furthermore, heart failure can be systolic (heart muscle can become weak and enlarged, and it cannot pump enough blood forward when the ventricles contract) and diastolic (heart muscle becomes stiff and it doesn’t relax normally between contractions which keeps the ventricles from filling with blood).

[0007] Consequently, patients with heart failure need extra support to make necessary lifestyle changes and need treatment to maintain the function of their hearts. Some medications for the treatment of heart failure are available, although they have been found not to reduce hospitalization and mortality. Additionally, hypertrophic cardiomyopathy which is characterized with excessive contraction and reduced ability to fill the heart with blood can lead to cardiac dysfunction and has been associated with increased risk of heart failure and sudden cardiac death. In obstructive or nonobstructive hypertrophic cardiomyopathy, exertion can result in excessive myocardial contraction, leading to fatigue or shortness of breath and inability to participate in activities of daily life.

[0008] Heart arrhythmia are conditions affecting the electrical system of the heart, causing irregular heart rhythms. Arrhythmias can occur when the electrical impulses that coordinate the heart rhythm do not travel normally. Arrhythmia can originate in the atria, or the ventricles of the heart and they can induce tachycardia (i. e. , fast heart rate) or bradycardia (i.e. , slow heart rate). This causes the heart to beat too fast, too slow, or with an irregular rhythm. Such irregular rhythm affects the heart’s ability to pump enough blood to the body. Lack of blood flow can damage the brain, heart, and other organs. Without treatment, this can lead to death within minutes. Arrhythmia can be atrial (e.g., atrial fibrillation) or ventricular (e.g., ventricular fibrillation, ventricular tachycardia, long QT syndrome/Torsades de Pointes (polymorphic ventricular tachycardia), catecholaminergic polymorphic ventricular tachycardia, etc.).

[0009] Several medications can be used to treat arrhythmia. However, conventional anti-arrhythmic drugs often lack efficacy and are associated with side effects. For example, p-blockers are the first-line treatment for patients with ventricular tachycardia. However, [3-blockers can cause fatigue, stomach or sleep problems and sexual dysfunction. Moreover, they can make some heart disorders worse. Other evidence suggests that in patients with long QT syndrome, [3 -blockers are not effective. Other anti-arrhythmic drugs such as blockers of calcium, sodium and potassium channels have also been linked to side effects including those affecting the heart itself. Calcium channel blockers often cause digestive trouble, swollen feet, and low blood pressure. Similarly, potassium channel blockers can cause low blood pressure, problems with thyroid levels, lung conditions, or QT prolongation/Torsades de Pointes arrhythmia.

Further, sodium channel blockers can reduce myocardial contraction and raise the risk of sudden cardiac arrest in people who have heart disease. Because of these limitations, there is a need for improved anti-arrhythmic drugs. The present studies demonstrate that the inhibition of the cardiac myosin can be considered as an innovative approach to anti-arrhythmic drug therapy.

[0010] In summary, improved treatments are needed for arrhythmias and heart failure/cardiomyopathies. Aspects of the present invention fulfill these needs and provide further related advantages as described in the following summary.

SUMMARY OF THE INVENTION

[0011] Aspects of the present disclosure teach certain benefits in construction and use which give rise to the exemplary advantages described below. [0012] Embodiments include compositions and methods that target the cardiac sarcomere (e.g., by modulating cardiac myosin) for laboratory and therapeutic use.

[0013] Embodiments also include compositions for preserving the viability of cardiomyocytes during cold storage in artificial media or buffer. The compositions can include an effective amount of a cardiac myosin inhibitor (e.g., blebbistatin, mavacamten or aficamten).

[0014] Embodiments also include methods of preserving the viability of cardiomyocytes during cold storage in artificial media or buffer. The methods can include administering an effective amount of a cardiac myosin inhibitor to the cells. Preservation can be measured by, for example, cellular appearance, biomarker levels or excitationcontraction coupling.

[0015] The present disclosure also provides pharmaceutical compositions for treating heart disease and ailments related to heart function. The methods can be used clinically to treat heart conditions such as arrhythmia. The methods can include administering a myosin inhibitor such as blebbistatin, mavacamten or aficamten.

[0016] Additional embodiments include methods of treating heart disease. The heart disease can be, for example, obstructive hypertrophic cardiomyopathy. The methods can include administering an inhibitor of cardiac myosin.

[0017] Embodiments also include methods of treating arrhythmia. The arrhythmia can be atrial fibrillation, ventricular fibrillation, ventricular tachycardias or supraventricular tachycardias or long QT syndrome. The methods can include administering an inhibitor of cardiac myosin.

[0018] Embodiments also include methods of reducing or ameliorating sympathetic response to cardiac muscle. The methods can include administering a myosin inhibitor.

[0019] The methods described herein can be used as alternatives to the use of conventional medications that have unwanted side-effects (e.g., [3-blockers, blockers of calcium channels, blockers of sodium channels and blockers of potassium channels). [0020] Other features and advantages of the present invention will become apparent from the following more detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings illustrate aspects of the present invention. In such drawings:

[0022] FIG. 1 A is an image of freshly isolated cardiomyocytes in Tyrode solution.

[0023] FIG. 1 B is an image of isolated cardiomyocytes that were stored for ten days in Tyrode solution.

[0024] FIG. 1 C is an image of isolated cardiomyocytes that were stored for ten days in Tyrode solution + inhibition of cardiac myosin (sample 1 ).

[0025] FIG. 1 D is an image of isolated cardiomyocytes that were stored for ten days in Tyrode solution + inhibition of cardiac myosin (sample 2).

[0026] FIG. 1 E is an image of isolated cardiomyocytes that were stored for ten days in Tyrode solution + inhibition of cardiac myosin (sample 3).

[0027] FIG. 2A is a bar graph showing the effects of 0.3pM mavacamten (n=4 cells) on isoproterenol-induced contractility increase in human cardiac cells at 1 Hz pacing frequency.

[0028] FIG. 2B is a bar graph showing the effects of 1 pM mavacamten (n=7 cells) on isoproterenol-induced contractility increase in human cardiac cells at 1 Hz pacing frequency.

[0029] FIG. 2C is a bar graph showing the effects of 0.3pM mavacamten (n=7 cells) on contractility in the presence of vehicle and after exposure to 0.03pM isoproterenol. Isoproterenol increased contractility in human cardiac cells at 1 Hz pacing frequency which was inhibited with mavacamten.

[0030] FIG. 2D is a bar graph showing the effects of 1 pM mavacamten (n=11 cells, 2 donors) on contractility in the presence of vehicle and after exposure to 0.03pM isoproterenol. Isoproterenol increased contractility in human cardiac cells at 1 Hz pacing frequency which was inhibited with mavacamten.

[0031] FIG. 2E is a bar graph showing the effects of blebbistatin (n=12 cells, 1 donor) on isoproterenol-induced contractility increase in human cardiac cells at 1 Hz pacing frequency.

[0032] FIG. 2F is a bar graph showing the effects of 1 pM mavacamten on isoproterenol- induced aftercontraction incidence, a biomarker of arrhythmia

[0033] FIG. 2G is a bar graph showing the effects of 0.3pM mavacamten on isoproterenol-induced aftercontraction incidence, a biomarker of arrhythmia

[0034] FIG. 3A shows typical contractility transients that were recorded from a ventricular cell at a pacing frequency of 1 Hz in the presence of vehicle control.

[0035] FIG. 3B shows the effects of ATX-II on human ventricular contractility. Typical contractility transients were recorded from the same ventricular cell as in FIG. 3A at a pacing frequency of 1 Hz after exposure to 0.03pM ATX-II.

[0036] FIG. 3C shows the effects of mavacamten on ATX-ll-induced changes in human ventricular contractility. Typical contractility transients were recorded from the same ventricular cell as in FIG. 3A and FIG. 3B at a pacing frequency of 1 Hz in the presence of 0.03pM ATX-II and 0.3pM mavacamten during the transition to the steady-state effect.

[0037] FIG. 3D shows the effects of mavacamten on ATX-ll-induced changes in human ventricular contractility. Typical contractility transients were recorded from the same ventricular cell as in FIG. 3A, FIG. 3B and FIG. 3C at a pacing frequency of 1 Hz in the presence of 0.03pM ATX-II alone and 0.3pM mavacamten at steady-state effect. [0038] FIG. 4A is a bar graph that shows the mean percentage change in contractility when human cardiomyocytes were treated with 0.03pM ATX alone or in combination with either 0.3pM and 1 pM mavacamten (n=8 cells, 2 donors at 1 Hz pacing frequency). Differences were tested for statistical significance using one-way ANOVA. *p < 0.05, **p

< 0.01 , ****p < 0.0001. ATX: ATX-II; Mava: mavacamten; BBS, blebbistatin

[0039] FIG. 4B is a bar graph that shows the mean percentage change in contractility, when human cardiomyocytes were treated with 0.03pM ATX alone or in combination with 0.3pM blebbistatin (n=10 cells, 1 donor) at 1 Hz pacing frequency.

[0040] FIG. 4C is a bar graph that shows the mean percentage incidence of aftercontraction when human cardiomyocytes were treated with 0.03pM ATX alone or in combination with either 0.3pM and 1 pM mavacamten (n=8 cells, 2 donors).

[0041] FIG. 4D is a bar graph that shows the mean percentage incidence of aftercontraction when human cardiomyocytes were treated with 0.03pM ATX alone or in combination with 0.3pM blebbistatin (n=10 cells, 1 donor) at 1 Hz pacing frequency.

[0042] FIG. 5A shows the change in sarcomere length (pm) over time (seconds) in the presence of a vehicle control.

[0043] FIG. 5B shows the change in sarcomere length (pm) over time (seconds) after exposure to 0.2pM dofetilide.

[0044] FIG. 5C shows the change in sarcomere length (pm) over time (seconds) after exposure to 1 pM mavacamten and dofetilide before steady state effect.

[0045] FIG. 5D shows the change in sarcomere length (pm) over time (seconds) after exposure to 1 pM Mavacamten and dofetilide after steady state effect.

[0046] FIG. 5E is bar graph that shows the change in contractility when cells were treated with 0.2pM dofetilide or dofetilide + 1 pM mavacamten. [0047] FIG. 5F shows the mean percentage incidence of after contraction when human cardiomyocytes were treated with 0.2pM dofetilide alone or in combination with 1 pM mavacamten.

[0048] FIG. 6A shows the change in sarcomere length (pm) over time (seconds) in the presence of a vehicle control.

[0049] FIG. 6B shows the change in sarcomere length (pm) over time (seconds) after exposure to 0.2pM dofetilide and 1 pM HMR-1556.

[0050] FIG. 6C shows the change in sarcomere length (pm) over time (seconds) after exposure to 1 pM mavacamten in the presence of dofetilide and HMR-1556.

[0051] FIG. 6D shows the results in Poincare plot of contractility amplitude after exposure to the vehicle control, and in the presence of 0.2pM dofetilide and 1 pM HMR- 1556 alone or in combination with 1 pM mavacamten.

[0052] FIG. 7A shows the change in voltage (mV) over time (seconds) in the presence of a vehicle control.

[0053] FIG. 7B shows the change in voltage (mV) over time (seconds) after exposure to 0.03pM ATX.

[0054] FIG. 7C shows the change in voltage (mV) over time (seconds) after exposure to 0.1 pM ATX.

[0055] FIG. 7D shows the effects of mavacamten on human ventricular trabeculae action potential from normal donors.

[0056] FIG. 8A shows the change in force (grams) over time (seconds). Typical contraction/relaxation cycles were recorded from one ventricular trabecula of a donor with heart failure with preserved ejection fraction (HFpEF) at a pacing of 1 Hz in the presence of vehicle control. [0057] FIG. 8B shows typical contraction/relaxation cycles that were recorded from the sameventricular trabecula as in FIG. 5A of a donor with HFpEF at a pacing of 1 Hz after exposure to 0.03 pM ATX.

[0058] FIG. 8C shows typical contraction/relaxation cycles that were recorded from the same ventricular trabecula of a donor with HFpEF as in FIG. 5A and FIG. 5B at a pacing of 1 Hz after exposure to 0.03 pM ATX in the presence of 1 pM mavacamten.

[0059] FIG. 8D is a bar graph showing the mean percentage control in maximum amplitude of contraction (Max. Ampl. Contraction) when HFpEF ventricular trabeculae were treated with vehicle or after exposure to 0.03pM ATX alone or in combination with mavacamten (n=4 trabeculae, 1 donor) at 1 Hz.

[0060] FIG. 8E is a bar graph showing the mean percentage incidence of aftercontraction when HFpEF ventricular trabeculae were treated with 0.03pM ATX alone or in combination with 1 pM mavacamten (n=4 trabeculae, 1 donor) at 1 Hz

Definitions

[0061] Reference in this specification to "one embodiment/aspect" or "an embodiment/aspect" means that a particular feature, structure, or characteristic described in connection with the embodiment/aspect is included in at least one embodiment/aspect of the disclosure. The use of the phrase "in one embodiment/aspect" or "in another embodiment/aspect" in various places in the specification are not necessarily all referring to the same embodiment/aspect, nor are separate or alternative embodiments/aspects mutually exclusive of other embodiments/aspects. Moreover, various features are described which may be exhibited by some embodiments/aspects and not by others. Similarly, various requirements are described which may be requirements for some embodiments/aspects but not other embodiments/aspects. Embodiment and aspect can in certain instances be used interchangeably.

[0062] The terms used in this specification generally have their ordinary meanings in the art, within the context of the disclosure, and in the specific context where each term is used. Certain terms that are used to describe the disclosure are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the disclosure. It will be appreciated that the same thing can be said in more than one way.

[0063] Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein. Nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only and is not intended to further limit the scope and meaning of the disclosure or of any exemplified term. Likewise, the disclosure is not limited to various embodiments given in this specification.

[0064] Without intent to further limit the scope of the disclosure, examples of instruments, apparatus, methods and their related results according to the embodiments of the present disclosure are given below. Note that titles or subtitles may be used in the examples for convenience of a reader, which in no way should limit the scope of the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In the case of conflict, the present document, including definitions, will control.

[0065] The term “cardiomyocyte” refers to a cell responsible for generating contractile force in the intact heart. Specialized cardiomyocytes form the cardiac conduction system, responsible for control of rhythmic beating of the heart.

[0066] The term “cardiovascular disease” or “CVD” refers to a class of diseases that involve the heart or blood vessels. There are many cardiovascular diseases involving the blood vessels including, coronary artery disease (also known as coronary heart disease and ischemic heart disease), peripheral arterial disease (i.e. , disease of blood vessels that supply blood to the arms and legs), cerebrovascular disease (i.e., disease of blood vessels that supply blood to the brain, including stroke), renal artery stenosis and aortic aneurysm. There are also many cardiovascular diseases that involve the heart. These include cardiomyopathy, hypertensive heart disease (i.e. , diseases of the heart secondary to high blood pressure or hypertension), heart failure (i.e., a clinical syndrome caused by the inability of the heart to supply sufficient blood to the tissues to meet their metabolic requirements), pulmonary heart disease, cardiac dysrhythmias, inflammatory heart disease (including endocarditis, inflammatory cardiomegaly, myocarditis and eosinophilic myocarditis), valvular heart disease, congenital heart disease and rheumatic heart disease.

[0067] Acute myocardial infarction (AMI) is the leading cause of death in the US. AMI is caused by a sudden and sustained lack of blood flow to an area of the heart, commonly caused by narrowing of a coronary artery. Without adequate blood supply, the tissue becomes ischemic, leading to the death of myocytes and vascular structures. This area of necrotic tissue is referred to as the infarct site and will eventually become scar tissue. The remaining cardiomyocytes are unable to reconstitute the necrotic tissue, and the heart deteriorates with time. The deterioration may be in the form of a loss of function of the heart muscle associated with remodeling of the damaged myocardium.

[0068] The term “arrhythmia” refers to improper beating of the heart, whether irregular, too fast or too slow. Cardiac arrhythmia can occur when electrical impulses in the heart do not work properly. Symptoms can include a fluttering in the chest, chest pain, fainting or dizziness. However, a person with arrhythmia can be asymptomatic. Treatments can include anti-arrhythmic drugs, medical procedures, implantable devices and surgery. Conventional anti-arrhythmic drugs are limited by their low to moderate efficacy and by their QT-prolonging/pro-arrhythmic effects.

[0069] The term “hypertrophic cardiomyopathy” or “HCM” refers to a disease in which the heart muscle becomes thickened (i.e., hypertrophied). The thickened heart muscle can make it harder for the heart to pump blood. Hypertrophic cardiomyopathy often goes undiagnosed because many people with the disease have few, if any, symptoms. Mavacamten, sold under the brand name Camzyos™, is a medication used to treat obstructive hypertrophic cardiomyopathy. Mavacamten is a cardiac myosin inhibitor.

Mavacamten was approved for medical use in the United States in April 2022.

[0070] The term “long QT syndrome” or “LQTS” refers to an abnormal feature of the heart's electrical system that can lead to a potentially life-threatening arrhythmia called torsades de pointes. Torsades de pointes may result in syncope (fainting) or sudden cardiac death.

[0071] The sympathetic nervous system (SANS or SNS) is one of the three divisions of the autonomic nervous system, the others being the parasympathetic nervous system and the enteric nervous system. In general, the SNS increases heart rate, blood pressure, breathing rate, and pupil size. It also causes blood vessels to narrow and decreases digestive juices. The SNS provides inotropic support to the failing heart increasing stroke volume, and peripheral vasoconstriction to maintain mean arterial perfusion pressure, but eventually accelerates disease progression affecting survival.

[0072] The term “myosin” refers to a superfamily of motor proteins best known for their roles in muscle contraction and in a wide range of other motility processes in eukaryotes. They are ATP-dependent and responsible for actin-based motility. Most myosin molecules are composed of a head, neck, and tail domain. The head domain binds the filamentous actin and uses ATP hydrolysis to generate force and to "walk" along the filament towards the barbed (+) end (with the exception of myosin VI, which moves towards the pointed (-) end). The neck domain acts as a linker and as a lever arm for transducing force generated by the catalytic motor domain. The neck domain can also serve as a binding site for myosin light chains which are distinct proteins that form part of a macromolecular complex and generally have regulatory functions. The tail domain generally mediates interaction with cargo molecules and/or other myosin subunits. In some cases, the tail domain may play a role in regulating motor activity. Skeletal muscle myosin, the most conspicuous of the myosin superfamily due to its abundance in muscle fibers, was the first to be discovered. This protein makes up part of the sarcomere and forms macromolecular filaments composed of multiple myosin subunits. Similar filament-forming myosin proteins were found in cardiac muscle, smooth muscle and non-muscle cells.

[0073] The term “Tyrode's solution” refers to a solution that is roughly isotonic with interstitial fluid and used in physiological experiments and tissue culture. It resembles lactated Ringer's solution, but contains magnesium, a sugar (usually glucose) as an energy source and uses bicarbonate and phosphate as a buffer instead of lactate. As used herein, the solution includes 110 mM Sucrose, 0.005mM CaCl2, 3 mM MgCl2, 70 mM KOH, 60 mM lactobionic acid, 10 mM KH2PO4, 20 mM Taurine, 20 mM L-Histidine, 20 mM HEPES, 2 mM L-Glutamic acid and 2 mM L-(-)-Malic acid (pH adjusted to 7.4 with KOH).

[0074] The term “blebbistatin” refers to (±)~1 ,2,3,3a-Tetrahydro~3a~hydroxy-6-methyl~1 - phenyl-4H-pyrrolo[2,3-£)]quinolin-4-one. It is a small molecule myosin inhibitor mostly specific for myosin II. It is widely used in research to inhibit heart muscle myosin, nonmuscle myosin II, and skeletal muscle myosin.

[0075] The term “mavacamten” sold under the brand name Camzyos, is a medication used to treat obstructive hypertrophic cardiomyopathy. Mavacamten is a cardiac myosin inhibitor, with chemical name 6-[[(1 S)-1-phenylethyl]amino]-3-propan-2-yl-1 H- pyrimidine-2, 4-dione.

[0076] The term “aficamten” refers to N-[(1 R)-5-(5-ethyl-1 ,2,4-oxadiazol-3-yl)~2,3- dihydro~1 H~inden-1 ~yl]-1 ~methylpyrazole~4-carboxamide a small molecule cardiac myosin inhibitor. It is currently being studied for therapeutic use.

[0077] The term “beta blocker,” “P-blocker” or “beta-adrenergic blocking agent” refers to a medication that reduces blood pressure. [3-blockers work by blocking the effects of the hormone epinephrine (i.e. , adrenaline). [3-blockers cause the heart to beat more slowly and with less force, which lowers blood pressure. [3-blockers also help widen veins and arteries to improve blood flow. Examples of [3-blockers include acebutolol, atenolol (Tenormin), bisoprolol (Zebeta), metoprolol (Lopressor, Toprol XL), nadolol (Corgard), nebivolol (Bystolic) and propranolol (Inderal, InnoPran XL). [0078] The term “ATX-II,” “neurotoxin 2” or “6-AITX-Avd1c” refers to a neurotoxin derived from the venom of the sea anemone Anemonia sulcata. ATX-II slows down the inactivation of different voltage-gated sodium channels, including Na _v 1.5, Na _v 1.1 and Na _v 1 .2, thus prolonging action potentials.

[0079] The term “cold storage,” as used herein, refers to storage of isolated cardiac cells at a temperature between -196°C and 8°C and ideally between 0°Cand 4°C, for a period of time (e.g., more than 24 hours).

[0080] The term “formulation” as used herein refers to the antibodies disclosed herein and excipients combined together which can be administered and has the ability to bind to the corresponding receptors and initiate a signal transduction pathway resulting in the desired activity. The formulation can optionally comprise other agents.

[0081] The term "administration" refers to the introduction of an amount of a predetermined substance into a patient by a certain suitable method. The composition disclosed herein may be administered via any of the common routes, as long as it is able to reach a desired tissue, for example, but is not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, or intrarectal administration. However, since peptides are digested upon oral administration, active ingredients of a composition for oral administration should be coated or formulated for protection against degradation in the stomach.

[0082] As applicable, the terms "about" or "generally", as used herein in the specification and appended claims, and unless otherwise indicated, means a margin of +/- 20%. Also, as applicable, the term "substantially" as used herein in the specification and appended claims, unless otherwise indicated, means a margin of +/- 10%. It is to be appreciated that not all uses of the above terms are quantifiable such that the referenced ranges can be applied.

[0083] The term “medicament,” “active agent” or “active ingredient” refers to a substance, compound, or molecule, which is biologically active or otherwise, induces a biological or physiological effect on a subject to which it is administered to. In other words, “active agent” or “active ingredient” refers to a component or components of a composition to which the whole or part of the effect of the composition is attributed. An active agent can be a primary active agent, or in other words, the component(s) of a composition to which the whole or part of the effect of the composition is attributed. An active agent can be a secondary agent, or in other words, the component(s) of a composition to which an additional part and/or other effect of the composition is attributed.

[0084] The term “bioavailability” refers to the fraction of an administered dose of unchanged drug that reaches the systemic circulation. For example, when a medication is administered intravenously, its bioavailability is 100%. However, when a medication is administered via other routes (such as orally), its bioavailability generally decreases due to incomplete absorption and first-pass metabolism. Bioavailability is one of the essential tools in pharmacokinetics, as bioavailability must be considered when calculating dosages for non-intravenous routes of administration.

[0085] The term “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, in a sterile composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo. In one aspect, the pharmaceutical composition is substantially free of endotoxins or is non-toxic to recipients at the dosage or concentration employed.

[0086] In an embodiment, a “subject” of diagnosis or treatment is, without limitation, a prokaryotic or a eukaryotic cell, a tissue culture, a tissue or an animal, e.g. a mammal, including a human. Non-human animals subject to diagnosis or treatment include, for example, without limitation, a simian, a murine, a canine, a leporid, such as a rabbit, livestock, sport animals, and pets.

[0087] The terms “treating,” “treatment” and the like are used herein, without limitation, to mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disorder or sign or symptom thereof, and/or may be therapeutic in terms of amelioration of the symptoms of the disease or infection, or a partial or complete cure for a disorder and/or adverse effect attributable to the disorder.

[0088] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are to be understood as approximations in accordance with common practice in the art. When used herein, the term “about” may connote variation (+) or (-) 1 %, 5% or 10% of the stated amount, as appropriate given the context. It is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0089] Many known and useful compounds and the like can be found in Remington’s Pharmaceutical Sciences (13 ^th Ed), Mack Publishing Company, Easton, PA — a standard reference for various types of administration. As used herein, the term “formulation(s)” means a combination of at least one active ingredient with one or more other ingredient, also commonly referred to as excipients, which may be independently active or inactive. The term “formulation” may or may not refer to a pharmaceutically acceptable composition for administration to humans or animals and may include compositions that are useful intermediates for storage or research purposes.

[0090] As the patients and subjects of the invention method are, in addition to humans, veterinary subjects, formulations suitable for these subjects are also appropriate. Such subjects include livestock and pets as well as sports animals such as horses, greyhounds, and the like.

[0091] For purposes herein, a formulation, a formulation for transdermal delivery and a transdermal delivery formulation are each a formulation for transdermal delivery, including, the transdermal delivery of an active ingredient for the treatment of a syndrome and or a disease in an individual.

DETAILED DESCRIPTION

[0092] The disclosure relates to treatment methods that can include administering a therapeutically effective amount of a myosin modulator or a pharmaceutically acceptable salt thereof to a subject in need. Also included are methods of preserving cardiac cells in culture using myosin modulators.

Myosin inhibitors

[0093] The contractile portion of the heart muscle (what makes the heart relax and contract) is called the sarcomere. The interaction between two parts of the sarcomere, actin and myosin, is responsible for how much the heart contracts. In hypertrophic cardiomyopathy (HCM), the heart squeezes too hard and fails to relax enough to fill well. Myosin inhibitors can reduce the interaction between actin and myosin so that the heart does not squeeze as hard.

[0094] Myosin inhibitors (e.g., mavacamten) are a new class of medication that can be used to treat patients with HCM. Mavacamten is an allosteric and reversible inhibitor of cardiac myosin ATPase. It reduces actin-myosin cross-bridge formation in muscle tissue, helping the heart muscle to relax, reducing contractility and improving myocardial energetics.

[0095] The U.S. Food and Drug Administration approved mavacamten (CAMZYOS™) for medical use in April 2022. Efficacy of mavacamten is mediated via its ability to reduce cardiac muscle contractility by inhibiting excessive myosin-actin cross-bridge formation that results in hypercontractility, left ventricular hypertrophy and reduced compliance. In clinical and preclinical studies, mavacamten reduced biomarkers of cardiac wall stress, lessened excessive cardiac contractility. In separate studies, another myosin inhibitor (i.e., aficamten) is being studies for the treatment of obstructive hypertrophic cardiomyopathy. Like mavacamten, aficamten is also a selective, small molecule cardiac myosin inhibitor.

[0096] Blebbistatin (“BBS”) is another myosin inhibitor mostly specific for myosin II. It is widely used in research to inhibit heart muscle myosin, non-muscle myosin II, and skeletal muscle myosin.

Cardiac Arrhythmia [0097] Cardiac arrhythmia (also referred to as dysrhythmia) is a term for any of a large and heterogeneous group of conditions in which there is abnormal electrical activity in the heart. The heartbeat can be too fast or too slow and can be regular or irregular. Some arrhythmias are life-threatening medical emergencies that can result in cardiac arrest and sudden death. Others cause symptoms such as an abnormal awareness of heartbeat (i.e. , palpitations) which usually do not present a danger. Others may not be associated with any symptoms but can pre-dispose one to potentially life-threatening stroke or embolus.

[0098] There are many classes of antiarrhythmic medications with different mechanisms of action and many different individual drugs within these classes. The methods described herein can also include administering to the subject one or more antiarrhythmic medications. Anticoagulant medications such as warfarin and heparin, and anti-platelet drugs such as aspirin can reduce the risk of clotting. Thus, the methods described herein can include administering an anticoagulant.

[0099] Arrhythmias can also be treated electrically, by applying a shock across the heart — either externally to the chest wall, or internally to the heart via implanted electrodes or intra-operatively. Cardioversion can be achieved either pharmacologically or via the application of a shock synchronized to the underlying heartbeat. It is used for treatment of supraventricular tachycardias. In elective cardioversion, the recipient is usually sedated or lightly anesthetized for the procedure. For example, atrial flutter can be treated by cardioversion. Thus, the methods described herein can also include treating the subject with cardioversion.

[00100] With synchronized cardioversion, a reversion shock is delivered by way of pads or paddles of a selected amount of electric current over a predefined number of milliseconds at the optimal moment in the cardiac cycle which corresponds to the R wave of the QRS complex on the ECG. Timing the shock to the R wave prevents the delivery of the shock during the vulnerable period (or relative refractory period) of the cardiac cycle, which could induce ventricular fibrillation. [00101 ] Defibrillation differs from cardioversion in that the shock is not synchronized to a cardiac cycle. It is needed for the chaotic rhythm of ventricular fibrillation and is also used for pulseless ventricular tachycardia. Often, more electricity is required for defibrillation than for cardioversion. Because most subjects with ventricular fibrillation are unconscious, there is generally no need for sedation. Thus, the methods described herein can also include treating the subject with defibrillation.

Sympathetic Response

[00102] The autonomic nervous system (ANS) has a significant influence on the structural integrity and electrical conductivity of the atria. The sympathetic nervous system (“SNS”) releases the hormones (i.e. , the catecholamines epinephrine and norepinephrine) to accelerate the heart rate. Aberrant activation of the sympathetic nervous system can induce heterogeneous changes with arrhythmogenic potential which can result in atrial tachycardia, atrial tachyarrhythmias and atrial fibrillation (AF).

[00103] Heart failure is a syndrome characterized initially by left ventricular dysfunction that triggers countermeasures aimed to restore cardiac output. These responses are compensatory at first but eventually become part of the disease process itself leading to further worsening cardiac function. Among these responses is the activation of the sympathetic nervous system (SNS) that provides inotropic support to the failing heart increasing stroke volume, and peripheral vasoconstriction to maintain mean arterial perfusion pressure, but eventually accelerates disease progression affecting survival.

[00104] Conventional efforts of modulating the sympathetic response (i.e., sympathetic inhibition) are generally limited to drugs such as clonidine and moxonidine. These drugs, classified as sedatives and antihypertensives, have non-specific modes of action. Applicants have discovered that a myosin inhibitor can be administered to a patient to reduce modulation by the sympathetic nervous system.

Administration of Cardiac Myosin Inhibitors and Dosing

[00105] Methods for treating, preventing or ameliorating a disease, disorder, a condition, or a symptom thereof or a condition related thereto are provided herein. Preferred, but non-limiting embodiments are directed to methods for treating, preventing, inhibiting or ameliorating a disease, disorder, a condition, or a symptom described below.

[00106] The present invention provides methods of treating an ailment such as heart disease in a subject comprising administering to the subject one or more compounds described herein or a salt thereof, or a pharmaceutical composition of the same. In some embodiments, the subject is a subject in need of such treatment. In some embodiments, the compound is administered as a pro-drug.

[00107] In another aspect, certain embodiments are directed to a sustained release drug delivery platform releases a therapeutic compound or compounds disclosed and made as a formulation described herein over a period of, without limitation, about 3 days after administration, about 7 days after administration, about 10 days after administration, about 15 days after administration, about 20 days after administration, about 25 days after administration, about 30 days after administration, about 45 days after administration, about 60 days after administration, about 75 days after administration, or about 90 days after administration. In other aspects of this embodiment, a sustained release drug delivery platform releases a therapeutic compound or compounds disclosed herein with substantially first order release kinetics over a period of, without limitation, at least 3 days after administration, at least 7 days after administration, at least 10 days after administration, at least 15 days after administration, at least 20 days after administration, at least 25 days after administration, at least 30 days after administration, at least 45 days after administration, at least 60 days after administration, at least 75 days after administration, or at least 90 days after administration.

[00108] In embodiment, the ailment is heart disease. Heart disease can include (a) coronary artery and vascular disease, (b) heart rhythm disorders (arrhythmias), (c) structural heart disease and (d) heart failure.

[00109] Any suitable route or mode of administration can be employed for providing the patient with a therapeutically or prophylactically effective dose of the agents. Exemplary routes or modes of administration include parenteral {e.g., intravenous, intraarterial, intramuscular, subcutaneous, intratumoral), oral, topical (nasal, transdermal, intradermal or intraocular), mucosal {e.g., nasal, sublingual, buccal, rectal, vaginal), inhalation, intralymphatic, intraspinal, intracranial, intraperitoneal, intratracheal, intravesical, intrathecal, enteral, intrapulmonary, intralymphatic, intracavital, intraorbital, intracapsular and transurethral, as well as local delivery by catheter or stent.

[00110] A pharmaceutical composition comprising an agent in accordance with the present disclosure can be formulated in any pharmaceutically acceptable carrier(s) or excipient(s). As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Pharmaceutical compositions can include suitable solid or gel phase carriers or excipients. Exemplary carriers or excipients include calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Exemplary pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers can further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the therapeutic agents.

[00111 ] The therapeutic agents in the pharmaceutical compositions may be formulated in a "therapeutically effective amount" or a "prophylactically effective amount". A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the recombinant vector may vary depending on the condition to be treated, the severity and course of the condition, the mode of administration, whether the antibody or agent is administered for preventive or therapeutic purposes, the bioavailability of the particular agent(s), the ability of the agent to elicit a desired response in the individual, previous therapy, the age, weight and sex of the patient, the patient's clinical history and response to the antibody, the type of the agent used, discretion of the attending physician, etc. A therapeutically effective amount is also one in which any toxic or detrimental effects of the recombinant vector is outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.

[00112] As a general proposition, a therapeutically effective amount or prophylactically effective amount of the agent will be administered in a range from about 1 ng/kg body weight/day to about 100 mg/kg body weight/day whether by one or more administrations. In a particular embodiment, each agent is administered in the range of from about 1 ng/kg body weight/day to about 10 mg/kg body weight/day, about 1 ng/kg body weight/day to about 1 mg/kg body weight/day, about 1 ng/kg body weight/day to about 100 g/kg body weight/day, about 1 ng/kg body weight/day to about 10 g/kg body weight/day, about 1 ng/kg body weight/day to about 1 g/kg body weight/day, about 1 ng/kg body weight/day to about 100 ng/kg body weight/day, about 1 ng/kg body weight/day to about 10 ng/kg body weight/day, about 10 ng/kg body weight/day to about 100 mg/kg body weight/day, about 10 ng/kg body weight/day to about 10 mg/kg body weight/day, about 10 ng/kg body weight/day to about 1 mg/kg body weight/day, about 10 ng/kg body weight/day to about 100 g/kg body weight/day, about 10 ng/kg body weight/day to about 10 mg/kg body weight/day, about 10 ng/kg body weight/day to about 1 mg/kg body weight/day, 10 ng/kg body weight/day to about 100 ng/kg body weight/day, about 100 ng/kg body weight/day to about 100 mg/kg body weight/day, about 100 ng/kg body weight/day to about 10 mg/kg body weight/day, about 100 ng/kg body weight/day to about 1 mg/kg body weight/day, about 100 ng/kg body weight/day to about 100 mg/kg body weight/day, about 100 ng/kg body weight/day to about 10 mg/kg body weight/day, about 100 ng/kg body weight/day to about 1 mg/kg body weight/day, about 1 mg/kg body weight/day to about 100 mg/kg body weight/day, about 1 mg /kg body weight/day to about 10 mg/kg body weight/day, about 1 mg /kg body weight/day to about 1 mg/kg body weight/day, about 1 mg /kg body weight/day to about 100 mg/kg body weight/day, about 1 mg /kg body weight/day to about 10 mg/kg body weight/day, about 10 mg/kg body weight/day to about 100 mg/kg body weight/day, about 10 mg /kg body weight/day to about 10 mg/kg body weight/day, about 10 mg /kg body weight/day to about 1 mg/kg body weight/day, about 10 mg /kg body weight/day to about 100 mg/kg body weight/day, about 100 mg/kg body weight/day to about 100 mg/kg body weight/day, about 100 mg /kg body weight/day to about 10 mg/kg body weight/day, about 100 mg /kg body weight/day to about 1 mg/kg body weight/day, about 1 mg/kg body weight/day to about 100 mg/kg body weight/day, about 1 mg/kg body weight/day to about 10 mg/kg body weight/day, about 10 mg/kg body weight/day to about 100 mg/kg body weight/day.

[00113] In other embodiments, each agent is administered in the range of about 10 ng to about 100 ng per individual administration, about 10 ng to about 1 g per individual administration, about 10 ng to about 10 g per individual administration, about 10 ng to about 100 mg per individual administration, about 10 ng to about 1 mg per individual administration, about 10 ng to about 10 mg per individual administration, about 10 ng to about 100 mg per individual administration, about 10 ng to about 1000 mg per injection, about 10 ng to about 10,000 mg per individual administration, about 100 ng to about 1 mg per individual administration, about 100 ng to about 10 mg per individual administration, about 100 ng to about 100 mg per individual administration, about 100 ng to about 1 mg per individual administration, about 100 ng to about 10 mg per individual administration, about 100 ng to about 100 mg per individual administration, about 100 ng to about 1000 mg per injection, about 100 ng to about 10,000 mg per individual administration, about 1 mg to about 10 mg per individual administration, about 1 mg to about 100 mg per individual administration, about 1 mg to about 1 mg per individual administration, about 1 mg to about 10 mg per individual administration, about 1 mg to about 100 mg per individual administration, about 1 mg to about 1000 mg per injection, about 1 mg to about 10,000 mg per individual administration, about 10 mg to about 100 mg per individual administration, about 10 mg to about 1 mg per individual administration, about 10 mg to about 10 mg per individual administration, about 10 mg to about 100 mg per individual administration, about 10 mg to about 1000 mg per injection, about 10 mg to about 10,000 mg per individual administration, about 100 mg to about 1 mg per individual administration, about 100 mg to about 10 mg per individual administration, about 100 mg to about 100 mg per individual administration, about 100 mg to about 1000 mg per injection, about 100 mg to about 10,000 mg per individual administration, about 1 mg to about 10 mg per individual administration, about 1 mg to about 100 mg per individual administration, about 1 mg to about 1000 mg per injection, about 1 mg to about 10,000 mg per individual administration, about 10 mg to about 100 mg per individual administration, about 10 mg to about 1000 mg per injection, about 10 mg to about 10,000 mg per individual administration, about 100 mg to about 1000 mg per injection, about 100 mg to about 10,000 mg per individual administration and about 1000 mg to about 10,000 mg per individual administration. The agent may be administered daily, every 2, 3, 4, 5, 6 or 7 days, or every 1 , 2, 3 or 4 weeks.

[00114] In other particular embodiments, the amount of the agent may be administered at a dose of about 0.0006 mg/day, 0.001 mg/day, 0.003 mg/day, 0.006 mg/day, 0.01 mg/day, 0.03 mg/day, 0.06 mg/day, 0.1 mg/day, 0.3 mg/day, 0.6 mg/day, 1 mg/day, 3 mg/day, 6 mg/day, 10 mg/day, 30 mg/day, 60 mg/day, 100 mg/day, 300 mg/day, 600 mg/day, 1000 mg/day, 2000 mg/day, 5000 mg/day or 10,000 mg/day. As expected, the dosage will be dependent on the condition, size, age and condition of the patient.

[00115] In other aspects of this embodiment, a pharmaceutical composition compound disclosed herein reduces a sign/symptom of heart disease by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%. In yet other aspects of this embodiment, a pharmaceutical composition disclosed herein reduces a sign/symptom of heart disease from, e.g., about 5% to about 100%, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%. [00116] A pharmaceutical composition disclosed herein may comprise a solvent, emulsion or other diluent in an amount sufficient to dissolve a pharmaceutical composition disclosed herein. In other aspects of this embodiment, a pharmaceutical composition disclosed herein may comprise a solvent, emulsion or a diluent in an amount of, e.g., less than about 90% (v/v), less than about 80% (v/v), less than about 70% (v/v), less than about 65% (v/v), less than about 60% (v/v), less than about 55% (v/v), less than about 50% (v/v), less than about 45% (v/v), less than about 40% (v/v), less than about 35% (v/v), less than about 30% (v/v), less than about 25% (v/v), less than about 20% (v/v), less than about 15% (v/v), less than about 10% (v/v), less than about 5% (v/v), or less than about 1 % (v/v). In other aspects of this embodiment, a pharmaceutical composition disclosed herein may comprise a solvent, emulsion or other diluent in an amount in a range of, e.g., about 1 % (v/v) to 90% (v/v), about 1 % (v/v) to 70% (v/v), about 1 % (v/v) to 60% (v/v), about 1 % (v/v) to 50% (v/v), about 1 % (v/v) to

40% (v/v), about 1 % (v/v) to 30% (v/v), about 1 % (v/v) to 20% (v/v), about 1 % (v/v) to

10% (v/v), about 2% (v/v) to 50% (v/v), about 2% (v/v) to 40% (v/v), about 2% (v/v) to

30% (v/v), about 2% (v/v) to 20% (v/v), about 2% (v/v) to 10% (v/v), about 4% (v/v) to

50% (v/v), about 4% (v/v) to 40% (v/v), about 4% (v/v) to 30% (v/v), about 4% (v/v) to

20% (v/v), about 4% (v/v) to 10% (v/v), about 6% (v/v) to 50% (v/v), about 6% (v/v) to

40% (v/v), about 6% (v/v) to 30% (v/v), about 6% (v/v) to 20% (v/v), about 6% (v/v) to

10% (v/v), about 8% (v/v) to 50% (v/v), about 8% (v/v) to 40% (v/v), about 8% (v/v) to

30% (v/v), about 8% (v/v) to 20% (v/v), about 8% (v/v) to 15% (v/v), or about 8% (v/v) to 12% (v/v).

[00117] The final concentration of a pharmaceutical composition disclosed herein may be of any concentration desired. In an aspect of this embodiment, the final concentration of a pharmaceutical composition may be a therapeutically effective amount. In other aspects of this embodiment, the final concentration of a pharmaceutical composition (total plasma concentration) may be, e.g., at least 0.00001 mg/mL, at least 0.0001 mg/mL, at least 0.001 mg/mL, at least 0.01 mg/mL, at least 0.1 mg/mL, at least 1 mg/mL, at least 10 mg/mL, at least 25 mg/mL, at least 50 mg/mL, at least 100 mg/mL, at least 200 mg/mL, at least 500 mg/mL, at least 700 mg/mL, at least 1 ,000 mg/mL, or at least 1 ,200 mg/mL. In other aspects of this embodiment, the concentration of a pharmaceutical composition disclosed herein in the solution may be, e.g., at most 1 ,000 mg/mL, at most 1 ,100 mg/mL, at most 1 ,200 mg/mL, at most 1 ,300 mg/mL, at most 1 ,400 mg/mL, at most 1 ,500 mg/mL, at most 2,000 mg/mL, at most 2,000 mg/mL, or at most 3,000 mg/mL. In other aspects of this embodiment, the final concentration of a pharmaceutical composition in a pharmaceutical composition may be in a range of, e.g., about 0.00001 mg/mL to about 3,000 mg/mL, about 0.0001 mg/mL to about 3,000 mg/mL, about 0.01 mg/mL to about 3,000 mg/mL, about 0.1 mg/mL to about 3,000 mg/mL, about 1 mg/mL to about 3,000 mg/mL, about 250 mg/mL to about 3,000 mg/mL, about 500 mg/mL to about 3,000 mg/mL, about 750 mg/mL to about 3,000 mg/mL, about 1 ,000 mg/mL to about 3,000 mg/mL, about 100 mg/mL to about 2,000 mg/mL, about 250 mg/mL to about 2,000 mg/mL, about 500 mg/mL to about 2,000 mg/mL, about 750 mg/mL to about 2,000 mg/mL, about 1 ,000 mg/mL to about 2,000 mg/mL, about 100 mg/mL to about 1 ,500 mg/mL, about 250 mg/mL to about 1 ,500 mg/mL, about 500 mg/mL to about 1 ,500 mg/mL, about 750 mg/mL to about 1 ,500 mg/mL, about 1 ,000 mg/mL to about 1 ,500 mg/mL, about 100 mg/mL to about

1 ,200 mg/mL, about 250 mg/mL to about 1 ,200 mg/mL, about 500 mg/mL to about

1 ,200 mg/mL, about 750 mg/mL to about 1 ,200 mg/mL, about 1 ,000 mg/mL to about

1 ,200 mg/mL, about 100 mg/mL to about 1 ,000 mg/mL, about 250 mg/mL to about 1 ,000 mg/mL, about 500 mg/mL to about 1 ,000 mg/mL, about 750 mg/mL to about 1 ,000 mg/mL, about 100 mg/mL to about 750 mg/mL, about 250 mg/mL to about 750 mg/mL, about 500 mg/mL to about 750 mg/mL, about 100 mg/mL to about 500 mg/mL, about 250 mg/mL to about 500 mg/mL, about 0.00001 mg/mL to about 0.0001 mg/mL, about 0.00001 mg/mL to about 0.001 mg/mL, about 0.00001 mg/mL to about 0.01 mg/mL, about 0.00001 mg/mL to about 0.1 mg/mL, about 0.00001 mg/mL to about 1 mg/mL, about 0.001 mg/mL to about 0.01 mg/mL, about 0.001 mg/mL to about 0.1 mg/mL, about 0.001 mg/mL to about 1 mg/mL, about 0.001 mg/mL to about 10 mg/mL, or about 0.001 mg/mL to about 100 mg/mL.

[00118] Aspects of the present specification disclose, in part, treating an individual suffering from heart disease. As used herein, the term "treating," refers to reducing or eliminating in an individual a clinical symptom of heart disease; or delaying or preventing in an individual the onset of a clinical symptom of heart disease. For example, the term "treating" can mean reducing a symptom of a condition characterized by a heart disease, including, but not limited to, arrhythmias, by, e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, or at least 100%. The actual symptoms associated with heart disease are well known and can be determined by a person of ordinary skill in the art by taking into account factors, including, without limitation, the type of heart disease, the cause of the heart disease, the severity of the heart disease, and/or the affected tissues. Those of skill in the art will know the appropriate symptoms or indicators associated with a specific type of heart disease and will know how to determine if an individual is a candidate for treatment as disclosed herein.

[00119] In another aspect, a pharmaceutical composition disclosed herein reduces the severity of a symptom of a disorder associated with heart disease. In aspects of this embodiment, a pharmaceutical composition disclosed herein reduces the severity of a symptom of a disorder associated with heart disease by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%. In other aspects of this embodiment, a pharmaceutical composition disclosed herein reduces the severity of a symptom of a disorder associated with heart disease by, e.g., about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

[00120] In aspects of this embodiment, a therapeutically effective amount of a pharmaceutical composition disclosed herein reduces a symptom associated with heart disease by, e g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100%. In other aspects of this embodiment, a therapeutically effective amount of a pharmaceutical composition disclosed herein reduces a symptom associated with heart disease by, e.g., at most 10%, at most 15%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 95% or at most 100%. In yet other aspects of this embodiment, a therapeutically effective amount of a pharmaceutical composition disclosed herein reduces a symptom associated with heart disease by, e.g., about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[00121 ] In yet other aspects of this embodiment, a therapeutically effective amount of a pharmaceutical composition disclosed herein generally is in the range of about 0.001 mg/kg/day to about 100 mg/kg/day. In aspects of this embodiment, an effective amount of a pharmaceutical composition disclosed herein may be, e.g., at least 0.001 mg/kg/day, at least 0.01 mg/kg/day, at least 0.1 mg/kg/day, at least 1.0 mg/kg/day, at least 5.0 mg/kg/day, at least 10 mg/kg/day, at least 15 mg/kg/day, at least 20 mg/kg/day, at least 25 mg/kg/day, at least 30 mg/kg/day, at least 35 mg/kg/day, at least 40 mg/kg/day, at least 45 mg/kg/day, or at least 50 mg/kg/day. In other aspects of this embodiment, an effective amount of a pharmaceutical composition disclosed herein may be in the range of, e.g., about 0.001 mg/kg/day to about 10 mg/kg/day, about 0.001 mg/kg/day to about 15 mg/kg/day, about 0.001 mg/kg/day to about 20 mg/kg/day, about 0.001 mg/kg/day to about 25 mg/kg/day, about 0.001 mg/kg/day to about 30 mg/kg/day, about 0.001 mg/kg/day to about 35 mg/kg/day, about 0.001 mg/kg/day to about 40 mg/kg/day, about 0.001 mg/kg/day to about 45 mg/kg/day, about 0.001 mg/kg/day to about 50 mg/kg/day, about 0.001 mg/kg/day to about 75 mg/kg/day, or about 0.001 mg/kg/day to about 100 mg/kg/day. In yet other aspects of this embodiment, an effective amount of a pharmaceutical composition disclosed herein may be in the range of, e.g., about 0.01 mg/kg/day to about 10 mg/kg/day, about 0.01 mg/kg/day to about 15 mg/kg/day, about 0.01 mg/kg/day to about 20 mg/kg/day, about 0.01 mg/kg/day to about 25 mg/kg/day, about 0.01 mg/kg/day to about 30 mg/kg/day, about 0.01 mg/kg/day to about 35 mg/kg/day, about 0.01 mg/kg/day to about 40 mg/kg/day, about 0.01 mg/kg/day to about 45 mg/kg/day, about 0.01 mg/kg/day to about 50 mg/kg/day, about 0.01 mg/kg/day to about 75 mg/kg/day, or about 0.01 mg/kg/day to about 100 mg/kg/day. In still other aspects of this embodiment, an effective amount of a pharmaceutical composition disclosed herein may be in the range of, e.g., about 0.1 mg/kg/day to about 10 mg/kg/day, about 0.1 mg/kg/day to about 15 mg/kg/day, about 0.1 mg/kg/day to about 20 mg/kg/day, about 0.1 mg/kg/day to about 25 mg/kg/day, about 0.1 mg/kg/day to about 30 mg/kg/day, about 0.1 mg/kg/day to about 35 mg/kg/day, about 0.1 mg/kg/day to about 40 mg/kg/day, about 0.1 mg/kg/day to about 45 mg/kg/day, about 0.1 mg/kg/day to about 50 mg/kg/day, about 0.1 mg/kg/day to about 75 mg/kg/day, or about 0.1 mg/kg/day to about 100 mg/kg/day.

[00122] Dosing can be single dosage or cumulative (serial dosing), and can be readily determined by one skilled in the art. For instance, treatment of heart disease may comprise a one-time administration of an effective dose of a pharmaceutical composition disclosed herein. Alternatively, treatment of heart disease may comprise multiple administrations of an effective dose of a pharmaceutical composition carried out over a range of time periods, such as, e.g., once daily, twice daily, trice daily, once every few days, or once weekly. The timing of administration can vary from individual to individual, depending upon such factors as the severity of an individual's symptoms. For example, an effective dose of a pharmaceutical composition disclosed herein can be administered to an individual once daily for an indefinite period of time, or until the individual no longer requires therapy. A person of ordinary skill in the art will recognize that the condition of the individual can be monitored throughout the course of treatment and that the effective amount of a pharmaceutical composition disclosed herein that is administered can be adjusted accordingly. [00123] In one embodiment, a therapeutic disclosed herein is capable of reducing the extent of arrhythmia in an individual suffering from arrhythmia by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% as compared to a patient not receiving the same treatment.

[00124] In an embodiment, the period of administration of a therapeutic disclosed herein is for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more. In a further embodiment, a period of during which administration is stopped is for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more.

[00125] In another aspect, a pharmaceutical composition disclosed herein reduces the frequency of a symptom of a disorder associated with heart disease incurred over a given time period. In aspects of this embodiment, a pharmaceutical composition disclosed herein reduces the frequency of a symptom of a disorder associated with heart disease incurred over a given time period by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%. In other aspects of this embodiment, a pharmaceutical composition disclosed herein reduces the frequency of a symptom of a disorder associated with a heart disease incurred over a given time period by, e.g., about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

[00126] As described above, if desired, other therapeutic agents can be employed in conjunction with those provided in the above-described compositions. The amount of active ingredients that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated, the nature of the disease, disorder, or condition, and the nature of the active ingredients.

[00127] It is understood that a specific dose level for any particular patient will vary depending upon a variety of factors, including the activity of the specific active agent; the age, body weight, general health, sex and diet of the patient; the time of administration; the rate of excretion; possible drug combinations; the severity of the particular condition being treated; the area to be treated and the form of administration. One of ordinary skill in the art would appreciate the variability of such factors and would be able to establish specific dose levels using no more than routine experimentation.

[00128] Pharmacokinetic parameters such as bioavailability, absorption rate constant, apparent volume of distribution, unbound fraction, total clearance, fraction excreted unchanged, first-pass metabolism, elimination rate constant, half-life, and mean residence time can be determined by methods well known in the art.

[00129] If desired, other therapeutic agents can be employed in conjunction with those provided in the above-described compositions. The amount of active ingredients that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated, the nature of the disease, disorder, or condition, and the nature of the active ingredients.

[00130] It is understood that a specific dose level for any particular patient will vary depending upon a variety of factors, including the activity of the specific active agent; the age, body weight, general health, sex and diet of the patient; the time of administration; the rate of excretion; possible drug combinations; the severity of the particular condition being treated; the area to be treated and the form of administration. One of ordinary skill in the art would appreciate the variability of such factors and would be able to establish specific dose levels using no more than routine experimentation.

[00131 ] Pharmacokinetic parameters such as bioavailability, absorption rate constant, apparent volume of distribution, unbound fraction, total clearance, fraction excreted unchanged, first-pass metabolism, elimination rate constant, half-life, and mean residence time can be determined by methods well known in the art.

EXAMPLES

[00132] The following non-limiting examples are provided for illustrative purposes only in order to facilitate a more complete understanding of representative embodiments now contemplated. These examples are intended to be a mere subset of all possible contexts in which the components of the formulation may be combined. Thus, these examples should not be construed to limit any of the embodiments described in the present specification, including those pertaining to the type and amounts of components of the formulation and/or methods and uses thereof.

Example 1

Inhibition of cardiac myosin preserves viability of human heart cells.

[00133] Primary culture of cardiomyocytes can be a valuable tool for pharmacological and toxicological studies. The availability of differentiated cells for research is a challenge in the cardiac field as postnatal mammalian cardiomyocytes do not proliferate, and primary human cells are generally unavailable in significant quantities. In this example, cardiac cells were grown in culture untreated and in the presence of 3p.M - 10p.M myosin inhibitor.

[00134] Studies demonstrate that death occurs within a few hours after isolation if cells are stored in refrigerated Tyrode solution (or similar solution). FIG. 1 A is an image of healthy cardiomiocytes that were isolated and immediately added to Tyrode solution. Rod-shaped morphology and cross-striations represent important markers of the health of freshly isolated human cardiac cells. FIG. 1 B is an image of cardiomiocytes that were stored for ten days in the solution. The change is cell shape is indicative of cell death. The short lifespan limits the usage of these cells to run translational research by scientists in the pharmaceutical industry and academia. This has forced scientists to rely on non-predictive cardiac cells from animal hearts.

[00135] Applicants proposed that cardiac myosin inhibitors could extend the lifespan of cardiomyocytes in culture. FIG. 1 C, FIG 1 D and FIG. 1 E are images of cardiomiocytes that were stored for ten days in Tyrode solution with a cardiac myosin inhibitor. When cells were stored in the Tyrode solution containing inhibitors of the cardiac myosin, blebbistatin (“BBS”) or mavacamten, cells maintained their rod-shaped morphology with clear cross-striations for ten to fourteen days. The preserved cells showed a normal excitation-contraction coupling and responded well to pharmacological agents and drugs. Additionally, data aligns with the 3Rs (Replacement, Reduction, Refinement) concept in animal welfare.

[00136] NT-proBNP and Troponin I are known biomarkers that are released during the injury/death of cardiac cells. Improvement in cardiac biomarkers of wall stress and myocardial injury has been reported in patients treated with mavacamten and aficamten (data not shown). Taken together the clinical observations with mavacamten and aficamten, and data showing preservation of cardiac cell health with inhibition of the cardiac myosin indicate that improvements in the heart function of patients treated with mavacamten and aficamten can be explained by the role of cardiac myosin in cell’s preservation. Because it is not available publicly, Aficamten was not included in the studies.

Example 2

Inhibition of the cardiac myosin reduces the modulation of the heart by the sympathetic nervous system.

[00137] Exertion (e.g., high level of activity of the sympathetic nervous system) in patients with obstructive hypertrophic cardiomyopathy can cause excessive myocardial contraction, result in fatigue or shortness of breath, etc. Conventional treatment typically includes administration of p-blockers. The aim of the p-blockers is to block/reduce the effect of the sympathetic nervous system on the force/contraction of the heart and consequently reduce exertion-induced excessive myocardial contraction. Since its approval by the FDA, Mavacamten, is often administered in combination with conventional regimens (e.g., p-blockers, calcium channel blockers).

[00138] The present study with cardiac myosin inhibitors and isoproterenol indicate that P-blockers are not necessary when treating patients with obstructive hypertrophic cardiomyopathy with mavacamten. Thus, the present findings may impact clinical practice and ensure patients do not experience side effects caused by p-blockers. The sympathetic nervous system releases hormones (e.g., catecholamines: epinephrine and norepinephrine) to accelerate the heart rate and increase its myocardial force. To mimic sympathetic nervous system-induced increase in the myocardial force, isoproterenol (an agonist of the cardiac p-adrenoceptors) was used at a concentration of 0.03pM. The results showed that 0.03pM isoproterenol increased the contractility of human cardiac cells. The contractility was inhibited with mavacamten at concentrations of 0.3pM and 1 pM. The results are shows in the FIG 2A - FIG. 2G.

[00139] FIG. 2A is a bar graph showing the contractility amplitude using four cells (n=4). A vehicle control (a) was compared with cells treated with (b) isoproterenol, (c) isoproterenol + mavacamten and (d) mavacamten. The concentrations of isoproterenol and mavacamten were 0.03pM and 0.3pM, respectively. The results demonstrate isoproterenol increased the contractility of human cardiac cells. Treatment with mavacamten significantly reduced the contractility.

[00140] Similarly, FIG. 2B shows the effects of mavacamten using seven cells (n=7). A vehicle control (a) was compared with cells treated with (b) isoproterenol, (c) isoproterenol + mavacamten and (d) mavacamten. In this study, the concentration of isoproterenol was 0.03pM; the concentration of mavacamten was 1 pM. The results demonstrate isoproterenol increased the contractility of human cardiac cells. Treatment with mavacamten significantly reduced the contractility such that it was not detectable. [00141 ] FIG. 2C is a bar graph showing the effects of mavacamten using seven cells (n=7). As above (FIG. 2A), the concentrations of isoproterenol and mavacamten were 0.03pM and 0.3pM, respectively. Isoproterenol increased the contractility of the cells. Treatment with mavacamten significantly reduced contractility.

[00142] Similarly, FIG. 2D shows the effects of mavacamten using eleven cells from two donors (n=11 ). As above (FIG. 2B), the concentration of isoproterenol was 0.03pM and the concentration of mavacamten was 1 pM. Isoproterenol increased the contractility of human cardiac cells. Treatment with mavacamten significantly reduced the contractility.

[00143] Blebbistatin, another cardiac myosin inhibitor was also tested. FIG. 2E shows the effects of isoproterenol and blebbistatin using twelve cells (n=12) on isoproterenol- induced contractility increase in human cardiac cells at 1 Hz pacing frequency. Isoproterenol increased the contractility of human cardiac cells. Blebbistatin significantly reduced the contractility.

[00144] It is notable that the clinically effective concentrations of mavacamten were ranged between 0.33 M and 0.5 M. Like mavacamten, blebbistatin (BBS), at a concentration of 0.3 pM, also inhibited the isoproterenol-induced increase in contractility.

Example 3

Inhibitors of the cardiac myosin are effective for anti-arrhythmic therapy.

[00145] Heart arrhythmia are conditions affecting the electrical system of the heart, causing irregular heart rhythms. Arrhythmia can originate in the atria or the ventricles of the heart, and they can induce tachycardia (i.e. , fast heart rate) or bradycardia (i.e. , slow heart rate). The irregular heart rhythm experienced during atrial fibrillation or other heart arrhythmia may reduce the volume of blood pumped by the heart, and/or may put a patient at an elevated risk for stroke.

[00146] Several medications can be used to treat arrhythmia. However, conventional anti-arrhythmic drugs often lack efficacy and are associated with side effects. The present studies show that the inhibition of the cardiac myosin can be considered as an innovative approach to anti-arrhythmic drug therapy. Here, isoproterenol was used at a concentration of 0.03pM to mimic the occurrence of catecholaminergic polymorphic ventricular tachycardia in human cardiac cells. FIG. 2F and FIG. 2G show the effects of mavacamten on isoproterenol-induced aftercontraction incidence, a biomarker of arrhythmia. FIG. 2F shows the percent incidence (after contraction incidence) cells treated with isoproterenol, isoproterenol + mavacamten and mavacamten. Similarly, FIG. 2G shows percent incidence for cells treated with mavacamten, isoproterenol + mavacamten and isoproterenol.

[00147] The results show that mavacamten, at both concentrations of 0.3pM and 1 M, blocked isoproterenol-induced aftercontractions, a biomarker of arrhythmia. It was also assessed whether the inhibition of the cardiac myosin could inhibit the long QT syndrome/Torsades de Pointes-like arrhythmia. To this purpose, ATX-II was used at a concentration of 0.03pM to mimic the occurrence of long QT syndrome type 3 arrhythmia in human cardiac cells. The effects of ATX-II on human ventricular contractility are shows in FIG. 3 and FIG. 4. Dofetilide was used at a concentration of 0.2pM to mimic the occurrence of long QT syndrome type 2 arrhythmia in human cardiac cells. The effects of dofetilide on human ventricular contractility are shown in FIG. 5. Dofetilide and HMR-1556 were used at concentrations of 0.2pM and 1 pM, respectively, to mimic the occurrence of decreased relaxation reserve arrhythmia. The effects of dofetilide and HMR-1556 on human ventricular contractility are shown in FIG.

6. Finally, ATX-II was used at concentration of 0.03pM and 0.1 pM to mimic the occurrence of long QT syndrome type 3 action potential arrhythmia in human ventricular trabeculae. The effects of ATX-II on human ventricular action potential are shown in FIG. 7.

[00148] FIG. 3A - FIG. 3D show the change in sarcomere length (pm) over time (seconds). FIG. 3A was conducted in the presence of a vehicle control. FIG. 3B shows the results after exposure to 0.03pM ATX-II (ATX). ATX slows down the inactivation of cardiac voltage-gated sodium channels, thus increasing the contractility and causing aftercontraction incidence. FIG. 3C shows the results after exposure to 0.3pM mavacamten and ATX before steady state effect. FIG. 3D shows the results after exposure to 0.3pM Mava and ATX after steady state effect. Differences were tested for statistical significance using one-way ANOVA. *p < 0.05, **p < 0.01 , ****p < 0.0001 .

[00149] FIG. 4A - FIG 4D are bar graphs that show the change in contractility. FIG. 4A shows the mean percentage control in contractility when cells were treated with 0.03pM ATX, ATX + 0. 3pM Mava, ATX + 1 pM Mava (n=8 cells, 2 donors) at 1 Hz pacing frequency. FIG. 4B shows the mean percent change in contractility when cells were treated with 0.03pM ATX alone or in combination with 0.3 pM blebbistatin (n=10 cells, 1 donor) at 1 Hz pacing frequency. Differences were tested for statistical significance using one-way ANOVA. **p < 0.01 , ****p < 0.0001 .

[00150] FIG. 4C and FIG. 4D show the mean % incidence of aftercontraction when human cardiomyocytes were treated with 0.03pM ATX alone or in combination with either 0.3pM and 1 pM mavacamten (n=8 cells, 2 donors) or 0.3pM blebbistatin (n=10 cells, 1 donor) at 1 Hz pacing frequency.

[00151 ] FIG. 5A - FIG. 5D show the change in sarcomere length (pm) over time (seconds). FIG. 5A was conducted in the presence of a vehicle control. FIG. 5B shows the results after exposure to 0.2pM dofetilide. Dofetilide inhibits the hERG channel I IKr current, thus increasing the contractility and causing aftercontraction incidence. FIG.

5C shows the results after exposure to 1 pM mavacamten and dofetilide before steady state effect. FIG. 5D shows the results after exposure to 1 pM Mavacamten and dofetilide after steady state effect. Differences were tested for statistical significance using one-way ANOVA. *p < 0.05, **p < 0.01 .

[00152] FIG. 5E is bar graph that show the change in contractility. FIG. 5E shows the mean percentage control in contractility when cells were treated with 0.2pM dofetilide, dofetilide + 1 pM mavacamten (n=8 cells, 2 donors) at 1 Hz pacing frequency.

Differences were tested for statistical significance using one-way ANOVA. *p < 0.05, **p < 0.01. [00153] FIG. 5F shows the mean % incidence of after contraction when human cardiomyocytes were treated with 0.2pM dofetilide alone or in combination with 1 pM mavacamten (n=8 cells, 2 donors) at 1 Hz pacing frequency.

[00154] FIG. 6A - FIG. 6D show the change in sarcomere length (pm) over time (seconds). FIG. 6A was conducted in the presence of a vehicle control. FIG. 6B and FIG. 6D shows the results after exposure to 0.2pM dofetilide and 1 M HMR-1556. Dofetilide inhibits the hERG channel / IKr current and HMR-1556 inhibits the KvLQTI channel / IKs current, thus causing beat-to-beat variability in contractility amplitude (FIG. 6B) and causing alternans incidence (FIG. 6D). FIG. 6C shows the results after exposure to 1 pM mavacamten in the presence of dofetilide and HMR-1556. FIG. 6D shows the results in Poincare plot of contractility amplitude after exposure to the vehicle control, and in the presence of 0.2pM dofetilide and 1 pM HMR-1556 alone or in combination with 1 pM mavacamten.

[00155] FIG. 7A - FIG. 7D show the change in voltage (mV) over time (seconds). FIG. 7A was conducted in the presence of a vehicle control. FIG. 7B and FIG. 70 shows the results after exposure to 0.03pM and 0.1 pM ATX, respectively. ATX slows down the inactivation of cardiac voltage-gated sodium channels, thus increasing the action potential duration (FIG. 7B) and causing incidence of arrhythmic events: triggered action potential (Triggered AP), early afterdepolarization (EAD) and action potential escape (FIG. 7C).

[00156] FIG. 7D shows the effects of mavacamten on human ventricular trabeculae action potential from normal donors. 1 pM mavacamten completely blocked 0.1 pM ATX- induced arrhythmic events.

[00157] FIG 8A - FIG 8E show the effects of mavacamten on human ventricular trabeculae contraction from donors with heart failure with preserved ejection fraction (HFpEF). FIG. 8A - FIG. 8C show the change in force (grams) over time (seconds). The contraction/relaxation cycles were recorded from one ventricular trabecula of a donor with HFpEF at a pacing of 1 Hz in the presence of vehicle control (FIG. 8A) and after exposure to 0.03pM ATX (FIG. 8B). FIG. 80 shows the results with treatment of ATX and 1 pM mavacamten (Mava).

[00158] FIG. 8D shows the mean percentage control in maximum amplitude of contraction (Max. Ampl. Contraction) with vehicle or after exposure to ATX alone or in combination with mavacamten (n=4 trabeculae, 1 donor) at 1 Hz. Differences were tested for statistical significance using one-way ANOVA. *p < 0.05 and nsP > 0.05. FIG. 8E shows the mean percentage incidence of aftercontraction when HFpEF ventricular trabeculae were treated with ATX and ATX + mavacamten (n=4 trabeculae, 1 donor) at 1 Hz.

[00159] The results demonstrate that ATX increased contractility amplitude (FIG. 3B, 4A and 4B) and caused manifestation of aftercontraction events (FIG. 3B, 4C and 4D). These ATX-induced increases in contractility and incidence of aftercontraction were completely blocked with mavacamten (FIG. 30, 3D and 4C) and blebbistatin (FIG. 4D).

[00160] The results also demonstrate that dofetilide increased contractility amplitude (FIG. 5B and 5E) and caused manifestation of aftercontraction events (FIG. 5B and 5F). These dofetilide-induced increases in contractility and incidence of aftercontraction were completely blocked with mavacamten (FIG. 5C, 5D and 5F).

[00161 ] The results also demonstrate that dofetilide and HMR-1556 caused manifestation of beat-to-beat variability in contractility amplitude and alternans events (FIG. 6B and 6D). These dofetilide+HMR-1556-induced events were completely blocked with mavacamten (FIG. 6C and 6D).

[00162] The results also demonstrate that ATX-induced action potential prolongation (FIG. 7B and FIG. 70) caused manifestation of arrhythmic events (Triggered AP, EAD and action potential escape) (FIG. 7C). These ATX-induced events were completely blocked with mavacamten (FIG. 7D).

[00163] Next, Applicants assessed the potential of cardiac myosin inhibitors in blocking arrhythmias in hearts from donors affected by heart failure with preserved ejection fraction (HFpEF). The data demonstrate that ATX increased contractility amplitude (FIG. 8B and 8D) and caused manifestation of aftercontraction events (FIG. 8B and 8E) in ventricular trabeculae from HFpEF donors. These findings correlate with clinical data reporting ventricular tachycardia, atrial fibrillation, and sudden cardiac death among HFpEF patients. Again, these ATX-induced increase in contractility and incidence of aftercontraction were completely blocked with 1 pM mavacamten (FIG. 80, 8D and 8E). Taken together, the ATX/cardiac myosin inhibition data from normal and diseased human hearts, and the well-tolerated treatments with mavacamten and aficamten in patients suggest that cardiac myosin inhibitors can provide an effective and safe anti- arrhythmic therapy.

[00164] Embodiments also include methods of preserving the viability and function of cardiac cells during cold storage in artificial media or buffer. In embodiments, the cardiac cells are treated with a therapeutic amount of a myosin inhibitor. The therapeutic amount can be, for example, about 0.10 pM, about 0.20 pM, about 0.30 pM, about 0.40 pM, about 0.50 pM, about 0.60 pM, about 0.70 pM, about 0.80 pM, about 0.90 pM, about 1.0 pM, about 1.2 pM, about 1.4 pM, about 1.6 pM, about 1.8 pM or about 2.0 pM. In aspects, the myosin inhibitor is one or more of blebbistatin, mavacamten and aficamten.

[00165] In embodiments, cardiac cells are treated with a therapeutic amount of a myosin inhibitor. The therapeutic amount can be, for example, at least 0.10 pM, at least 0.20 pM, at least 0.30 pM, at least 0.40 pM, at least 0.50 pM, at least 0.60 pM, at least 0.70 pM, at least 0.80 pM, at least 0.90 pM, at least 1.0 pM, at least 1 .2 pM, at least 1 .4 pM, at least 1.6 pM, at least 1 .8 pM or at least 2.0 pM. In aspects, the myosin inhibitor is one or more of blebbistatin, mavacamten and aficamten.

[00166] In embodiments, treatment of cardiac cells with a myosin inhibitor preserves the viability of the cells (e.g., maintains their viability and/or functionality). Preservation can be determined by, for example, cellular appearance, biomarker levels and/or excitationcontraction coupling. In aspects, the treatment preserves cardiac cells by at least 100% compared to untreated cells (e.g., as measured in days). In aspects, the treatment preserves the cardiac cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 200% (i.e. , two-fold), at least 300% (i.e., three-fold), at least 400% (i.e., four-fold), at least 500% (i.e., fivefold) or more compared to untreated cells.

[00167] In other aspects, the treatment preserves cardiac cells (e.g., maintains their functionality) by at least 10 days compared to untreated cells. In aspects, the treatment preserves the cardiac cells by at least 5 days, at least 15 days, at least 20 days, at least 30 days, at least 50 days or more compared to untreated cells.

[00168] In embodiments, cardiac cells are maintained in culture, artificial media or buffer for greater than twice the lifespan of untreated cardiac cells. The cells can be maintained in cold storage. In aspects, the treated cells are maintained three times, four times, five times, six times, seven times, eight times, nine time or ten times the lifespan of untreated cells. In aspects, treated cells are maintained for 10 days, for 12 days, for 14 days, for 16 days, for 18 days, for 20 days, for 24 days, for 30 days or longer than untreated cells. In aspects, lifespan of cells (treated and untreated) is measured by cellular appearance (e.g., cell shape). In aspects, one or more biomarkers (e.g., NT-proBNP or troponin I) are used to measure the lifespan. In aspects, excitation-contraction coupling is used to measure lifespan.

[00169] In embodiments, the methods of treatment with a myosin modulator as described herein results in an improvement in one or more clinical endpoints (e.g., one or more functional endpoints or one or more outcome endpoints). In some embodiments, the improved clinical endpoint is a symptom such as, for example, shortness of breath (e.g., as measured by a change in dyspnea index), fatigue (e.g., as measured by a change in peak VO2 or NYHA class), palpitations (e.g., as measured by a change in atrial fibrillation), chest discomfort, edema, and premature mortality, or any combination thereof. In some embodiments, the improved clinical endpoint is a functional endpoint selected from one or more of peak VO2, VE/VCO2, VENCO2 slope, six-minute walk test, KCCQ subscores, Canadian Cardiovascular Society chest pain score, and Seattle angina score or any combination thereof. In some embodiments, the improved clinical endpoint is an outcome endpoint from one or more of reduction in mortality, reduction in hospitalization or rehospitalization, reduction in major adverse cardiovascular events (MACE), reduction in atrial fibrillation, and reduction in atrial fibrillation embolic phenomenon, or a combination thereof. In some embodiments, the improvement is a change (e.g., increase or decrease) from baseline, either in percentage or in amount.

[00170] In embodiments, a pharmaceutical composition described herein (e.g., a composition containing a myosin inhibitor) is administered to a patient with heart disease. A pharmaceutical composition disclosed herein can include a therapeutic compound in an amount sufficient to allow customary administration to an individual. In certain embodiments, a pharmaceutical composition disclosed herein may comprise, e.g., at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mg of a therapeutic compound. In certain embodiments, a pharmaceutical composition disclosed herein may comprise, e.g., at least 5 mg, at least 10 mg, at least 20 mg, at least 25 mg, at least 50 mg, at least 75 mg, at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, at least 500 mg, at least 600 mg, at least 700 mg, at least 800 mg, at least 900 mg, at least 1 ,000 mg, at least 1 ,100 mg, at least 1 ,200 mg, at least 1 ,300 mg, at least 1 ,400 mg, or at least 1 ,500 mg of a therapeutic compound. In yet other aspects of this embodiment, a pharmaceutical composition disclosed herein may comprise in the range of, e.g., about 5 mg to about 100 mg, about 10 mg to about 100 mg, about 50 mg to about 150 mg, about 100 mg to about 250 mg, about 150 mg to about 350 mg, about 250 mg to about 500 mg, about 350 mg to about 600 mg, about 500 mg to about 750 mg, about 600 mg to about 900 mg, about 750 mg to about 1 ,000 mg, about 850 mg to about 1 ,200 mg, or about 1 ,000 mg to about 1 ,500 mg. In still certain embodiments, a pharmaceutical composition disclosed herein may comprise in the range of, e.g., about 10 mg to about 250 mg, about 10 mg to about 500 mg, about 10 mg to about 750 mg, about 10 mg to about 1 ,000 mg, about 10 mg to about 1 ,500 mg, about 50 mg to about 250 mg, about 50 mg to about 500 mg, about 50 mg to about 750 mg, about 50 mg to about 1 ,000 mg, about 50 mg to about 1 ,500 mg, about 100 mg to about 250 mg, about 100 mg to about 500 mg, about 100 mg to about 750 mg, about 100 mg to about 1 ,000 mg, about 100 mg to about 1 ,500 mg, about 200 mg to about 500 mg, about 200 mg to about 750 mg, about 200 mg to about 1 ,000 mg, about 200 mg to about 1 ,500 mg, about 5 mg to about 1 ,500 mg, about 5 mg to about 1 ,000 mg, or about 5 mg to about 250 mg.

[00171 ] In one embodiment, the dose of the composition may be administered daily, semi-weekly, weekly, bi-weekly, or monthly. The period of treatment may be for a week, two weeks, a month, two months, four months, six months, eight months, a year, or longer. The initial dose may be larger than a sustaining dose. In one embodiment, the dose ranges from a weekly dose of at least 0.01 mg/kg, at least 0.25 mg/kg, at least 0.3 mg/kg, at least 0.5 mg/kg, at least 0.75 mg/kg, at least 1 mg/kg, at least 2 mg/kg, at least 3 mg/kg, at least 4 mg/kg, at least 5 mg/kg, at least 6 mg/kg, at least 7 mg/kg, at least 8 mg/kg, at least 9 mg/kg, at least 10 mg/kg, at least 15 mg/kg, at least 20 mg/kg, at least 25 mg/kg, or at least 30 mg/kg In one embodiment, a weekly dose may be at most 1 .5 mg/kg, at most 2 mg/kg, at most 2.5 mg/kg, at most 3 mg/kg, at most 4 mg/kg, at most 5 mg/kg, at most 6 mg/kg, at most 7 mg/kg, at most 8 mg/kg, at most 9 mg/kg, at most 10 mg/kg, at most 15 mg/kg, at most 20 mg/kg, at most 25 mg/kg, or at most 30 mg/kg. In a particular aspect, the weekly dose may range from 5 mg/kg to 20 mg/kg. In an alternative aspect, the weekly dose may range from 10 mg/kg to 15 mg/kg.

[00172] The present specification also provides a pharmaceutical composition for the administration to a subject. The pharmaceutical composition disclosed herein may further include a pharmaceutically acceptable carrier, excipient, or diluent. As used herein, the term “pharmaceutically acceptable” means that the composition is sufficient to achieve the therapeutic effects without deleterious side effects, and may be readily determined depending on the type of the diseases, the patient’s age, body weight, health conditions, gender, and drug sensitivity, administration route, administration mode, administration frequency, duration of treatment, drugs used in combination or coincident with the composition disclosed herein, and other factors known in medicine. [00173] The pharmaceutical composition including the pro-drug disclosed herein may further include a pharmaceutically acceptable carrier. For oral administration, the carrier may include, but is not limited to, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersing agent, a stabilizer, a suspending agent, a colorant, and a flavorant. For injectable preparations, the carrier may include a buffering agent, a preserving agent, an analgesic, a solubilizer, an isotonic agent, and a stabilizer. For preparations for topical administration, the carrier may include a base, an excipient, a lubricant, and a preserving agent.

[00174] The disclosed compositions may be formulated into a variety of dosage forms in combination with the aforementioned pharmaceutically acceptable carriers. For example, for oral administration, the pharmaceutical composition may be formulated into tablets, troches, capsules, elixirs, suspensions, syrups or wafers. For injectable preparations, the pharmaceutical composition may be formulated into an ampule as a single dosage form or a multidose container. The pharmaceutical composition may also be formulated into solutions, suspensions, tablets, pills, capsules and long-acting preparations.

[00175] On the other hand, examples of the carrier, the excipient, and the diluent suitable for the pharmaceutical formulations include, without limitation, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils. In addition, the pharmaceutical formulations may further include fillers, anti-coagulating agents, lubricants, humectants, flavorants, and antiseptics.

[00176] Further, the pharmaceutical composition disclosed herein may have any formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquids for internal use, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized formulations and suppositories. [00177] The composition may be formulated into a single dosage form suitable for the patient’s body, and preferably is formulated into a preparation useful for peptide drugs according to the typical method in the pharmaceutical field so as to be administered by an oral or parenteral route such as through skin, intravenous, intramuscular, intraarterial, intramedullary, intramedullary, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intracolonic, topical, sublingual, vaginal, or rectal administration, but is not limited thereto.

[00178] The composition may be used by blending with a variety of pharmaceutically acceptable carriers such as physiological saline or organic solvents. In order to increase the stability or absorptivity, carbohydrates such as glucose, sucrose or dextrans, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers may be used.

[00179] The administration dose and frequency of the pharmaceutical composition disclosed herein are determined by the type of active ingredient, together with various factors such as the disease to be treated, administration route, patient’s age, gender, and body weight, and disease severity.

[00180] The total effective dose of the compositions disclosed herein may be administered to a patient in a single dose, or may be administered for a long period of time in multiple doses according to a fractionated treatment protocol. In the pharmaceutical composition disclosed herein, the content of active ingredient may vary depending on the disease severity. Preferably, the total daily dose of the peptide disclosed herein may be approximately 0.0001 pg to 500 mg per 1 kg of body weight of a patient. However, the effective dose of the peptide is determined considering various factors including patient’s age, body weight, health conditions, gender, disease severity, diet, and secretion rate, in addition to administration route and treatment frequency of the pharmaceutical composition. In view of this, those skilled in the art may easily determine an effective dose suitable for the particular use of the pharmaceutical composition disclosed herein. The pharmaceutical composition disclosed herein is not particularly limited to the formulation, and administration route and mode, as long as it shows suitable effects.

[00181 ] Moreover, the pharmaceutical composition may be administered alone or in combination or coincident with other pharmaceutical formulations showing prophylactic or therapeutic efficacy.

[00182] Given the teachings and guidance provided herein, those skilled in the art will understand that a formulation described herein can be equally applicable to many types of biopharmaceuticals, including those exemplified, as well as others known in the art. Given the teachings and guidance provided herein, those skilled in the art also will understand that the selection of, for example, type(s) or and/or amount(s) of one or more excipients, surfactants and/or optional components can be made based on the chemical and functional compatibility with the biopharmaceutical to be formulated and/or the mode of administration as well as other chemical, functional, physiological and/or medical factors well known in the art. For example, non-reducing sugars exhibit favorable excipient properties when used with polypeptide biopharmaceuticals compared to reducing sugars. Accordingly, exemplary formulations are exemplified further herein with reference to polypeptide biopharmaceuticals. However, the range of applicability, chemical and physical properties, considerations and methodology applied to polypeptide biopharmaceutical can be similarly applicable to biopharmaceuticals other than polypeptide biopharmaceuticals.

[00183] In various embodiments, a formulation can include, without limitation, combinations of bioactive agents (such as viruses, proteins, antibodies, peptides and the like as described herein) in the formulation. For example, a formulation as described herein can include a single bioactive agent for treatment of one or more conditions, including without limitation, disease. A formulation as described herein also can include, in an embodiment, without limitation, two or more different bioactive agents for a single or multiple conditions. Use of multiple bioactive agents in a formulation can be directed to, for example, the same or different indications. Similarly, in another embodiment, multiple bioactive agents can be used in a formulation to treat, for example, both a pathological condition and one or more side effects caused by the primary treatment. In a further embodiment, multiple bioactive agents also can be included, without limitation, in a formulation as described herein to accomplish different medical purposes including, for example, simultaneous treatment and monitoring of the progression of the pathological condition. In an additional embodiment, multiple, concurrent therapies such as those exemplified herein as well as other combinations well known in the art are particularly useful for patient compliance because a single formulation can be sufficient for some or all suggested treatments and/or diagnosis. Those skilled in the art will know those bioactive agents that can be admixed for a wide range of combination therapies. Similarly, in various embodiments, a formulation can be used with a small molecule drug and combinations of one or more bioactive agents together with one or more small molecule pharmaceuticals. Therefore, in various embodiments a formulation is provided containing 1 , 2, 3, 4, 5 or 6 or more different bioactive agents, as well as, for one or more bioactive agents combined with one or more small molecule pharmaceuticals.

[00184] In various embodiments, a formulation can include, one or more preservatives and/or additives known in the art. Similarly, a formulation can further be formulated, without limitation, into any of various known delivery formulations. For example, in an embodiment, a formulation can include, surfactants, adjuvant, biodegradable polymers, hydrogels, etc., such optional components, their chemical and functional characteristics are known in the art. Similarly known in the art are formulations that facilitate rapid, sustained or delayed release of the bioactive agents after administration. A formulation as described can be produced to include these or other formulation components known in the art.

[00185] The composition can therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages may be ascertained through use of appropriate dose-response data. In various embodiments, the bioactive agents in formulations described herein can, without limitation, be administered to patients throughout an extended time period, such as chronic administration for a chronic condition. The composition can be a solid, a semisolid or an aerosol and a pharmaceutical compositions is formulated as a tablet, geltab, lozenge, orally dissolved strip, capsule, syrup, oral suspension, emulsion, granule, sprinkle or pellet.

[00186] In an embodiment, for oral, rectal, vaginal, parenteral, pulmonary, sublingual and/or intranasal delivery formulations, tablets can be made by compression or molding, optionally with one or more accessory ingredients or additives. In an embodiment, compressed tablets are prepared, for example, by compressing in a suitable tableting machine, the active ingredients in a free-flowing form such as a powder or granules, optionally mixed with a binder (for example, without limitation, povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, without limitation, sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) and/or surface-active or dispersing agent.

[00187] In an embodiment, molded tablets are made, for example, by molding in a suitable tableting machine, a mixture of powdered compounds moistened with an inert liquid diluent. In an embodiment, the tablets may optionally be coated or scored, and may be formulated so as to provide slow or controlled release of the active ingredients, using, for example, without limitation, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. In an embodiment, tablets may optionally be provided with a coating, without limitation, such as a thin film, sugar coating, or an enteric coating to provide release in parts of the gut other than the stomach. In an embodiment, processes, equipment, and toll manufacturers for tablet and capsule making are well-known in the art.

[00188] In an embodiment, capsule formulations can utilize either hard or soft capsules, including, without limitation, gelatin capsules or vegetarian capsules such as those made out of hydroxymethylpropylcellulose (HMPC). In an embodiment, a type of capsule is a gelatin capsule. In an embodiment, capsules may be filled using a capsule filling machine such as, without limitation, those available from commercial suppliers such as Miranda International or employing capsule manufacturing techniques well- known in the industry, as described in detail in Pharmaceutical Capules, 2. sup. nd Ed., F. Podczeck and B. Jones, 2004. In an embodiment, capsule formulations may be prepared, without limitation, using a toll manufacturing center such as the Chao Center for Industrial Pharmacy & Contract Manufacturing, located at Purdue Research Park.

[00189] Packaging and instruments for administration may be determined by a variety of considerations, such as, without limitation, the volume of material to be administered, the conditions for storage, whether skilled healthcare practitioners will administer or patient self-compliance, the dosage regime, the geopolitical environment (e.g., exposure to extreme conditions of temperature for developing nations), and other practical considerations.

[00190] Also described herein, are methods for treating a subject in need of therapy, comprising administering to the subject an effective amount of a formulation as described herein. The therapeutically effective amount or dose of a formulation will depend on the disease or condition of the subject and actual clinical setting.

[00191 ] In an embodiment, a formulation as described herein can be administered by any suitable route, specifically by parental (including subcutaneous, intramuscular, intravenous and intradermal) administration. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary, without limitation, with the composition used for therapy, the purpose of the therapy, and the subject being treated. Single or multiple administrations can be carried out, without limitation, the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art.

[00192] The formulations as described herein can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures. [00193] Compositions in accordance with embodiments described herein have desirable properties, such as desirable solubility, viscosity, syringeability and stability. Lyophilates in accordance with embodiments described herein have desirable properties, as well, such as desirable recovery, stability and reconstitution.

[00194] In an embodiment, the pH of the pharmaceutical formulation is at least about 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, or 9.

[00195] In an embodiment, the pH of the pharmaceutical formulation is from about 3 to about 9, about 4 to about 19, about 5 to about 9, about 6 to about 8, about 6 to about 7, about 6 to about 9, about 5 to about 6, about 5 to about 7, about 5 to about 8, about 4 to about 9, about 4 to about 8, about 4 to about 7, about 4 to about 6, about 4 to about 5, about 3 to about 8, about 3 to about 7, about 3 to about 6, about 3 to about 5, about 3 to about 4, about 7 to about 8, about 7 to about 9, about 7 to about 10.

[00196] Certain embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the present invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described embodiments in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[00197] Groupings of alternative embodiments, elements, or steps of the present invention are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[00198] Unless otherwise indicated, all numbers expressing a characteristic, item, quantity, parameter, property, term, and so forth used in the present specification and claims are to be understood as being modified in all instances by the term “about.” As used herein, the term “about” means that the characteristic, item, quantity, parameter, property, or term so qualified encompasses a range of plus or minus ten percent above and below the value of the stated characteristic, item, quantity, parameter, property, or term. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical indication should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and values setting forth the broad scope of the invention are approximations, the numerical ranges and values set forth in the specific examples are reported as precisely as possible. Any numerical range or value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Recitation of numerical ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate numerical value falling within the range. Unless otherwise indicated herein, each individual value of a numerical range is incorporated into the present specification as if it were individually recited herein.

[00199] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the present invention so claimed are inherently or expressly described and enabled herein.

[00200] Groupings of alternative embodiments, elements, or steps of the present invention are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[00201 ] All patents, patent publications, and other publications referenced and identified in the present specification are individually and expressly incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[00202] In closing, it is to be understood that although aspects of the present specification are highlighted by referring to specific embodiments, one skilled in the art will readily appreciate that these disclosed embodiments are only illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is in no way limited to a particular methodology, protocol, and/or reagent, etc., described herein. As such, various modifications or changes to or alternative configurations of the disclosed subject matter can be made in accordance with the teachings herein without departing from the spirit of the present specification. Lastly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Accordingly, the present invention is not limited to that precisely as shown and described.