WO2015009988A1 - Cardiac imaging methods and use in diagnosis and treatment of heart failure and related disease - Google Patents

Abstract

The present inventors have found that that the build-up of amyloid during a situation of increased, pathological, heart workload is an event which also occurs in the absence of any specific mutations of either systemic, or cardiac, proteins. The idea of an overarching mechanism of HF based on the toxicity of misfolded proteins is completely novel and could lead to revolutionary changes in the way HF is diagnosed and treated. In order to further this goal, the present inventors provide a novel, reliable, reproducible method to diagnose HF using positron emission tomography (PET) in mammals. The present invention also provides methods for screening compounds useful in treatment of HF in mammals using these methods.

Description

CARDIAC IMAGING METHODS AND USE IN DIAGNOSIS AND TREATMENT OF HEART FAILURE AND RELATED DISEASE

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61/856,287, filed on July 19, 2013, which is hereby incorporated by reference for all purposes as if fully set forth herein.

BACKGROUND OF THE INVENTION

[0002] Heart failure, presently affects 5 millions of individuals in the US alone. To date, the molecular mechanisms underlying this deadly disease have not been clarified and heart failure is diagnosed using functional parameters without a precise understanding of what is the molecular culprit for the decreased cardiac function.

[0003] Chronic heart failure is the final outcome of several diseases, including, but not limited to valvular defects, hypertension and myocardial infarction. The heart adapts to these conditions by hypertrophy, an increase in cell size rather than number. While myocardial hypertrophy is temporarily beneficial for cardiac function, sustained hypertrophy often becomes maladaptive and the heart decompensates and progresses to failure (Figure 1).

[0004] Typical features of the initial adaptation phase include: increased neurohumoral stimulation, fetal gene re-induction and cell hypertrophy. However, when hypertrophic stimulation is prolonged over time, myofibrillar and organelle disarrangement and malfunction, as well as impaired contractility, cell conductivity and energetics constitute the final outcomes. The present inventors suggest that the commonality of these features suggests an overarching mechanism of deterioration. To date the mechanism underlying the transition from hypertrophy to HF is not fully understood.

[0005] There still exists a need for non-invasive clinical indicators which can identify patients with HF before any outward clinical pathology is presented.

SUMMARY OF THE INVENTION

[0006] The present inventors have conceived that that the build-up of amyloid during a situation of increased, pathological, heart workload is an event which also occurs in the absence of any specific mutations of either systemic, or cardiac, proteins. The idea of an overarching mechanism of HF based on the toxicity of misfolded proteins is completely novel and could lead to revolutionary changes in the way HF is diagnosed and treated. In order to further this goal, the present inventors have devised a novel, reliable, reproducible method to diagnose HF using positron emission tomography (PET) in mammals.

[0007] In accordance with an embodiment, the present invention provides an in vivo method for diagnosing heart failure in a subject, comprising the steps of: (a) administering to the subject a detectable quantity of a pharmaceutical composition comprising the an amyloid binding compound having a detectable label, (b) determining whether there is binding of the compound to the cardiac tissue of the subject; (c) comparing the level of binding of the compound in the subject to the level of binding of the compound to normal cardiac tissue; and (d) determining that the subject is at a greater than normal risk for heart failure when the level of binding of the compound is greater than the level of binding of the compound in normal cardiac tissue.

[0008] In accordance with another embodiment, the present invention provides an in vivo method to monitor the progression to heart failure based on the detection of amyloid species in cardiac tissue comprising: (a) administering to the subject a detectable quantity of a pharmaceutical composition comprising the an amyloid binding compound having a detectable label, (b) determining whether there is binding of the compound to the cardiac tissue of the subject; (c) at one or more later time intervals, administering to the subject a detectable quantity of a pharmaceutical composition comprising the an amyloid binding compound having a detectable label, (d) determining whether there is binding of the compound to the cardiac tissue of the subject; (e) comparing the level of binding of the compound in cardiac tissue of the subject from a) to the level of binding of the compound in the cardiac tissue of the subject after (c); and (f) determining whether heart failure is progressing in the subject when the level of binding of the compound in the cardiac tissue of the subject is higher after (c) than the level of binding of the compound in the cardiac tissue of the subject after a).

[0009] In accordance with still another embodiment, the present invention provides an in vivo method for monitoring treatment of heart failure in a subject, comprising the steps of: (a) administering to the subject before commencement of a treatment regimen for heart failure a detectable quantity of a pharmaceutical composition comprising the an amyloid binding compound having a detectable label, (b) determining whether there is binding of the compound to the cardiac tissue of the subject; (c) commencing a treatment regimen for heart failure in the subject for a period of time; (d) administering to the subject a detectable quantity of a pharmaceutical composition comprising the an amyloid binding compound having a detectable label, (e) determining whether there is binding of the compound to the cardiac tissue of the subject; (f) comparing the level of binding of the compound in cardiac tissue of the subject before treatment to the level of binding of the compound in the cardiac tissue of the subject after (c); and (g) determining that the treatment regimen for heart failure in the subject is effective when the level of binding of the compound in the cardiac tissue of the subject is lower after (c) than the level of binding of the compound in the cardiac tissue of the subject before treatment.

[0010] In accordance with yet another embodiment, the present invention provides a method of screening for compounds useful in the treatment of heart failure comprising: (a) administering to the subject before commencement of a treatment regimen with a compound of interest for heart failure a detectable quantity of a pharmaceutical composition comprising the an amyloid binding compound having a detectable label, (b) determining whether there is binding of the amyloid binding compound to the cardiac tissue of the subject; (c)

commencing a treatment regimen for heart failure in the subject for a period of time with the compound of interest; (d) administering to the subject a detectable quantity of a

pharmaceutical composition comprising the an amyloid binding compound having a detectable label, (e) determining whether there is binding of the amyloid binding compound to the cardiac tissue of the subject; (f) comparing the level of binding of the amyloid binding compound in cardiac tissue of the subject before treatment to the level of binding of the amyloid binding compound in the cardiac tissue of the subject after (c); and (g) determining that the compound of interest for heart failure in the subject is effective when the level of binding of the amyloid binding compound in the cardiac tissue of the subject is lower after (c) than the level of binding of the amyloid binding compound in the cardiac tissue of the subject before treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is a schematic showing maladaptive transition from benign, adaptive hypertrophy, to heart failure.

[0012] Figure 2 depicts amyloid species (AS) from HF disrupted by treatment with epigallo catechin gallate (EGCG) and stained by Thioflavin T (ThT). Amyloid species were extracted for canine failing hearts and alternatively subjected to treatment with 20 μΜ EGCG (Sigma). EGCG selectively reduces a AS migrating at an apparent molecular size of 600 kDa in HF. Samples were separated using "not-so-native" or NSN-PAGE gels, stained with ThT for 10 minutes and rinsed in water overnight. Two filters (Cy2 and Cy5) were used to detect ThT signal specifically, before and after staining, using a Typhoon laser scanner (GE healthcare). The Cy2 filter is specific for ThT whereas the Cy5's was used as a reference filter.

[0013] Figure 3 shows ThT staining in R120G cryAB mice, a mouse model of cardiac amyloidosis. After separation of tissue homogenates by NSN-PAGE, gels were stained with ThT. Thioflavin T (Cy2, green, panel C) and coomassie (Cy5, red, panel B) signals were acquired by means of a Typhoon 9400 laser scanner (GE healthcare). Gel images were acquired before and after staining with Thioflavin T to prove specificity and rule out any contribution from auto fluorescence. A merged image providing the two colors/dyes is also provided (panel A) along with the results of the densitometric analysis (panel D). AS were increased 5-fold in cryAB R120G mouse hearts compared to non-transgenic controls (p=0.0002).

[0014] Figure 4 shows PiB staining in mice with heart failure. Mice were alternatively subjected to TAC for four weeks after which they were injected with PiB and imaged using PET. Specific PiB signal was only observed in the mouse with heart failure (left) whereas no signal was observed in healthy animals (right).

[0015] Figure 5 shows AMYVID™ (or Florbetapir F 18 or ¹⁸F-AV-45) staining in mice with heart failure. Mice were alternatively subjected to TAC for four weeks after which they were injected with AMYVID™ (Eli Lilly) and imaged using PET. Specific AMYVID™ AMYVID™ signal was only observed in the mice with heart failure (bottom, E and F) whereas no signal was observed in healthy animals (top, A and B). Magnified images of the heart highlighted in red are provided in the right panels for both control (C and D) and TAC animals (G and H)

DETAILED DESCRIPTION OF THE INVENTION

[0016] In accordance with some embodiments, the present invention provides methods for non-invasive, direct detection of amyloid deposition in the heart by means of a PET tracer that is based on a modified version of Thioflavin T (ThT) and other PET tracers (such as AMYVID (or Florbetapir F18 or F-AV-45) which have been already used in the diagnosis of Alzheimer's and other amyloid-related diseases in patients. Notably, PiB's structure is based on the same chemistry that allows the present inventors to monitor amyloid levels ex-vivo (Thioflavin T). Moreover, the same tracer has been recently utilized to detect amyloid signal in the heart of patients with frank cardiac amyloidosis proving its usefulness and applicability not only for the brain but also for the cardiac tissue (Antoni G et al, J Nucl Med 2013).

[0017] In accordance with an embodiment, the present invention provides an in vivo method for diagnosing heart failure in a subject, comprising the steps of: (a) administering to the subject a detectable quantity of a pharmaceutical composition comprising the an amyloid binding compound having a detectable label, (b) determining whether there is binding of the compound to the cardiac tissue of the subject; (c) comparing the level of binding of the compound in the subject to the level of binding of the compound to normal cardiac tissue; and (d) determining that the subject is at a greater than normal risk for heart failure when the level of binding of the compound is greater than the level of binding of the compound in normal cardiac tissue.

[0018] As used herein, the term "amyloid binding compound" includes any small molecule, compound or conjugate which binds with high affinity to amyloid species (AS), which includes but are not limited to pre-amyloid oligomers (PAO), amyloid oligomers (AO), amyloid prefibrils and fibrils as well as aggregates. Examples of such compounds include, but are not limited to ThT, Thioflavin S, PiB, Florbetapir F 18, Congo Red, and any analogs or derivatives thereof which bind AS with high affinity (Ki=10^{1 2} nM). Examples of ThT derivatives and analogs are known and can be found in U.S. Patent No. 7,270,800 and incorporated by reference herein.

[0019] In some embodiments, protein samples, such as from cardiac tissue, can be separated on gels, and stained using ThT for visualization of AS. Samples can be run using any gel protocol, including Blue Native to separate the proteins. In alternative embodiments, the samples can be treated before running the gel, with 0.1 to 2% w/v sodium dodecyl sulfate in a suitable buffer and then added to the gel, a protocol termed "Not-So-Native." Using this method can separate oligomers with an apparent molecular weights up to 1200 kDa. The gels can be run and then visualized with a laser scanner with filters for Cy2 and Cy5. This provides the feature of being able to visualize the ThT bound (using the Cy2 filter which fluoresces green) and the proteins (Cy5 filter which fluoresces red) to visualize the

Coomassie stain.

[0020] Accordingly, included within the compounds and methods of the present invention are the tautomeric forms of the disclosed compounds, isomeric forms including enantiomers, stereoisomers, and diastereoisomers, and the pharmaceutically-acceptable salts thereof. The term "pharmaceutically acceptable salts" embraces salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases. Examples of acids which may be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, sulphuric acid and phosphoric acid, and such organic acids as maleic acid, succinic acid and citric acid. Other pharmaceutically acceptable salts include salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium and magnesium, or with organic bases, such as dicyclohexylamine. Suitable

pharmaceutically acceptable salts of the compounds of the present invention include, for example, acid addition salts which may, for example, be formed by mixing a solution of the compound according to the invention with a solution of a pharmaceutically acceptable acid, such as hydrochloric acid, sulphuric acid, methanesulphonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. All of these salts may be prepared by conventional means by reacting, for example, the appropriate acid or base with the corresponding compounds of the present invention.

[0021] Salts formed from free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0022] For use in medicines, the salts of the compounds used in the methods of the present invention should be pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of the compounds according to the invention or of their

pharmaceutically acceptable salts.

[0023] In addition, embodiments of the invention include hydrates of the compounds of the present invention. The term "hydrate" includes but is not limited to hemihydrate, monohydrate, dihydrate, trihydrate and the like. Hydrates of the compounds of the present invention may be prepared by contacting the compounds with water under suitable conditions to produce the hydrate of choice. [0024] By "detectable label(s) or moieties" is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens. Specific radioactive labels include most common commercially available isotopes including, for example, ³H, ⁿC, ¹³C, ¹⁵N, ¹⁸F, ¹⁹F, ¹²³1, ¹²⁴1, ¹²⁵I, ¹³¹1, ⁸⁶Y, ⁸⁹Zr, ^{U 1}ln, ^94mTc, ^99mTc, ⁶⁴Cu and ⁶⁸Ga. Suitable dyes include any commercially available dyes such as, for example, 5(6)-carboxyfluorescein, IRDye 680RD maleimide or IRDye 800CW, ruthenium polypyridyl dyes, and the like.

[0025] In accordance with another embodiment, the present invention provides an in vivo method for detecting the levels amyloid species in the cardiac tissue of a subject, comprising the steps of: (a) administering to the subject a detectable quantity of a pharmaceutical composition comprising the an amyloid binding compound having a detectable label, and (b) detecting the binding of the compound to amyloid species in the cardiac tissue of the subject.

[0026] In accordance with an embodiment, the present invention provides the use of a pharmaceutical composition comprising an amyloid binding compound having a detectable label, for detecting amyloid species in the cardiac tissue of a subject, characterized in that a detectable quantity of the pharmaceutical composition is administered to the subject for at least one interval, and determining whether there is binding of the compound to the cardiac tissue of the subject.

[0027] As used herein, the term "detecting amyloid species" means the amyloid binding compound having a detectable label is detected using known imaging methods. For example, when the detectable label is a radionuclide, the molecule can be detected by gamma counting, PET or SPECT imaging. The amyloid binding compounds can be chelated to an imaging agent. The most widely used agents include branched chelating agents such as di-ethylene tri-amine penta-acetic acid (DTPA), 1,4,7, 10-tetra-azacyclododecane-l,4,7, 10-tetraacetic acid (DOTA) and their analogs. Chelating agents, such as di-amine dithiols, activated

mercaptoacetyl-glycyl-glycyl-gylcine (MAG3), and hydrazidonicotinamide (HY IC), are able to chelate metals like ⁹⁹mTc and ¹⁸⁶Re. Instead of using chelating agents, a prosthetic group such as N-succinimidyl-4-¹⁸F-fluorobenzoate (18F-SFB) is necessary for labeling peptides with ¹⁸F. [0028] When the detectable label is a dye, the molecule is detected by fluorescence imaging. The dyes may be emitters in the visible or near-infrared (MR) spectrum. Known dyes useful in the present invention include carbocyanine, indocarbocyanine,

oxacarbocyanine, thuicarbocyanine and merocyanine, polymethine, coumarine, rhodamine, xanthene, fluorescein, boron-dipyrromethane (BODIPY), Cy5, Cy5.5, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S750, AlexaFluor660, AlexaFluor680, AlexaFluor700,

AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, and ADS832WS.

[0029] It is contemplated that any of the amyloid binding molecule embodiments of the present invention described above can also encompass a pharmaceutical composition comprising the molecules and a pharmaceutically acceptable carrier.

[0030] The carrier can be any of those conventionally used, and is limited only by physico-chemical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. The carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the carrier be one which is chemically inert to the active agent(s), and one which has little or no detrimental side effects or toxicity under the conditions of use. Examples of the carriers include soluble carriers such as known buffers which can be physiologically acceptable (e.g., phosphate buffer) as well as solid

compositions such as solid-state carriers or latex beads.

[0031] The carriers or diluents used herein may be solid carriers or diluents for solid formulations, liquid carriers or diluents for liquid formulations, or mixtures thereof.

[0032] Solid carriers or diluents include, but are not limited to, gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, dextrose), cellulosic materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

[0033] For liquid formulations, pharmaceutically acceptable carriers may be, for example, aqueous or non-aqueous solutions, or suspensions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic/aqueous solutions, cyclodextrins, emulsions or suspensions, including saline and buffered media. [0034] Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Formulations suitable for parenteral administration include, for example, aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

[0035] Intravenous vehicles include, for example, fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.

[0036] The choice of carrier will be determined, in part, by the particular amyloid binding molecule, as well as by the particular method used to administer the composition.

Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention. The following formulations for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal and interperitoneal administration are exemplary, and are in no way limiting. More than one route can be used to administer the compositions of the present invention, and in certain instances, a particular route can provide a more immediate and more effective response than another route.

[0037] Injectable formulations are in accordance with the invention. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ^SHP Handbook on Injectable Drugs, Trissel, 15th ed., pages 622-630 (2009)).

[0038] By "disease" is meant any condition or disorder which damages or interferes with the normal function of a cell, tissue, or organ. Examples of such diseases include: heart failure secondary, but not limited to, coronary artery disease, hypertension, valvular defects, arrhythmias, conduction disturbances and idiopathic cardiomyopathies, as well as atherosclerosis, inflammatory diseases such as rheumatoid arthritis, infectious diseases, amyloidosis or Marfan's syndrome, collagen vascular disease, cancer therapies or other conditions, such as amyloidosis, which predispose the heart to failure.

[0039] In accordance with another embodiment, the present invention provides a method for the differential diagnosis of benign and maladaptive hypertrophy. It will be understood by those of ordinary skill that benign hypertrophy of muscle tissue occurs during strenuous exercise or training, for example, by athletes, whereas, maladaptive hypertrophy of muscle tissue occurs in subjects having certain genetic defects. Thus, the methods of the present invention can be used to differentiate these conditions.

[0040] In accordance with an embodiment, the present invention provides the use of a pharmaceutical composition comprising an amyloid binding compound having a detectable label, for diagnosing heart failure in a subject, characterized in that a detectable quantity of the pharmaceutical composition is administered to the subject at a first interval; determining whether there is binding of the compound to the cardiac tissue of the subject; comparing the level of binding of the compound in the subject to the level of binding of the compound to normal cardiac tissue; and determining that the subject is at a greater than normal risk for heart failure when the level of binding of the compound is greater than the level of binding of the compound in normal cardiac tissue.

[0041] By "an effective amount" is meant the amount required to identify, diagnose, image, or ameliorate the symptoms of a disease relative in an untreated or treated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a neurodegenerative disease varies depending upon the manner of

administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount. In a preferred embodiment, the disease is heart failure.

[0042] For purposes of the invention, the amount or dose of the amyloid binding molecules of the present invention that is administered should be sufficient to effectively target the cell, or population of cells in vivo, such that the amyloid binding molecules can be detected, in the subject over a reasonable time frame. The dose will be determined by the efficacy of the particular nanoparticle formulation and the location of the target population of cells in the subject, as well as the body weight of the subject to be treated.

[0043] The dose of the amyloid binding molecules of the present invention also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular amyloid binding molecule. Typically, an attending physician will decide the dosage of the amyloid binding molecules with which to treat each individual subject, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, compound to be administered, route of administration, and the severity of the condition being treated. By way of example, and not intending to limit the invention, the dose of the amyloid binding molecules of the present invention can be about 0.001 to about 1000 mg/kg body weight of the subject being treated, from about 0.01 to about 100 mg/kg body weight, from about 0.1 mg/kg to about 10 mg/kg, and from about 0.5 mg to about 5 mg/kg body weight. In another embodiment, the dose of the amyloid binding molecules of the present invention can be at a concentration from about 1 nM to about 10,000 nM, preferably from about 10 nM to about 5,000 nM, more preferably from about 100 nM to about 500 nM. In some embodiments, the amount of the radiolabeled amyloid binding molecule 100 MBq to about 2000 MBq, preferably between about 300 MBq to about 500 MBq.

[0044] In an embodiment, the term "administering" means that at least one or more amyloid binding molecules of the present invention are introduced into a subject, preferably a subject receiving treatment for a disease, and the at least one or more amyloid binding molecules are allowed to come in contact with AS containing cells or population of cells having AS in vivo.

[0045] As used herein, the term "subject" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.

[0046] As used herein, the term "test animal" refers to any mammal as described in the definition of the term "subject" but specifically excludes humans.

[0047] In a further embodiment, the amyloid binding molecules of the present invention can be used in combination with one or more additional therapeutically active agents which are known to be capable of treating conditions or diseases discussed above. For example, the described amyloid binding molecules of the present invention could be used in combination with one or more known therapeutically active agents, to treat a disease or condition. Non- limiting examples of other therapeutically active agents that can be readily combined in a pharmaceutical composition with the amyloid binding molecules of the present invention are enzymatic nucleic acid molecules, allosteric nucleic acid molecules, antisense, decoy, or aptamer nucleic acid molecules, antibodies such as monoclonal antibodies, small molecules, and other organic and/or inorganic compounds including metals, salts and ions.

[0048] It will be understood by those of ordinary skill in the art that one of the advantages of the compounds and methods of the present invention, is the ability to identify a subject which may have HF but be asymptomatic with regards to cardiac function. The compounds and methods of the present invention allow the diagnosis of a subject which has HF or is susceptible to develop HF, or has some other cardiac condition or damage which results in the induction and presence of AS in the cardiac tissue by the detection of AS in the cardiac tissue in vivo using imaging. The inventive compounds and methods can also be used to identify the extent of damage to the cardiac tissue and is useful in the clinic for staging HF and other cardiac diseases which impact on cardiac performance.

[0049] Thus, in accordance with still another embodiment, the present invention provides an in vivo method for monitoring treatment of heart failure in a subject, comprising the steps of: (a) administering to the subject before commencement of a treatment regimen for heart failure a detectable quantity of a pharmaceutical composition comprising the an amyloid binding compound having a detectable label, (b) determining whether there is binding of the compound to the cardiac tissue of the subject; (c) commencing a treatment regimen for heart failure in the subject for a period of time; (d) administering to the subject a detectable quantity of a pharmaceutical composition comprising the an amyloid binding compound having a detectable label, (e) determining whether there is binding of the compound to the cardiac tissue of the subject; (f) comparing the level of binding of the compound in cardiac tissue of the subject before treatment to the level of binding of the compound in the cardiac tissue of the subject after (c); and (g) determining that the treatment regimen for heart failure in the subject is effective when the level of binding of the compound in the cardiac tissue of the subject is lower after (c) than the level of binding of the compound in the cardiac tissue of the subject before treatment.

[0050] In accordance with still another embodiment, the present invention provides the use of a pharmaceutical composition comprising an amyloid binding compound having a detectable label, to monitor the progression to heart failure in a subject based on the detection of amyloid species in cardiac tissue, characterized in that a detectable quantity of the pharmaceutical composition is administered to the subject at a first interval; determining whether there is binding of the compound to the cardiac tissue of the subject; at one or more later time intervals, administering to the subject a detectable quantity of the pharmaceutical composition, making subsequent determinations of whether there is binding of the compound to the cardiac tissue of the subject; comparing the level of binding of the compound in cardiac tissue of the subject from the first interval, to the level of binding of the compound in the cardiac tissue of the subject after one or more subsequent intervals; and determining whether heart failure is progressing in the subject when the level of binding of the compound in the cardiac tissue of the subject is higher after subsequent intervals than the level of binding of the compound in the cardiac tissue of the subject after the first interval.

[0051] Dr. Jeffrey Robbins and colleagues first investigated the formation of amyloid in mice expressing a mutated form of the chaperone protein alpha-B-crystallin (R120G CryAB). In this animal model the impaired chaperone function makes the animal more susceptible to protein misfolding and pre-amyloid oligomers deposition leading to cardiac dilation and HF. Thus, in some examples of the inventive embodiments, the compositions and methods involve the use of R120G CryAB mice.

[0052] It is also contemplated that the compounds and methods of the present invention can be useful in the screening of novel compounds of interest for their effect on the increase or decrease in AS in cardiac tissue of a subject.

[0053] In some embodiments, the present invention provides a screening assay comprises a plurality of R120G CryAB mice that are treated with a compound of interest are compared to a plurality of R120G CryAB mice that are treated with a control composition, to examine the effect on AS formation in the cardiac tissue. Both groups of mice are then subjected to in vivo imaging using the compounds of the present invention.

[0054] In some embodiments, the present invention provides a screening assay comprising a plurality of mice which have heart failure that was induced by transverse aortic constriction (TAC), simulating aortic stenosis that are treated with a compound of interest are compared to a plurality of mice which have heart failure that was induced by TAC that are treated with a control composition, to examine the effect on AS formation in the cardiac tissue. Both groups of mice are then subjected to in vivo imaging using the compounds of the present invention. [0055] The compounds tested novel compounds of interest for their effect on the increase or decrease in AS can be any small chemical compound, or a biological entity, such as, without limitation, include small organic molecules, peptides, proteins, oligonucleotides, aptamers, antibodies, and siR As, saccharides, nucleic acids, lipids, enzymes, receptor antagonists or agonists, hormones, growth factors, antibiotics, antimicrobial agents, and antibodies. Typically, test compounds will be small synthetic and natural molecules and peptides or antibodies.

[0056] In some embodiments, the agents have a molecular weight of less than 1,500 daltons, and in some cases less than 1,000, 800, 600, 500, or 400 daltons. The relatively small size of the agents can be desirable because smaller molecules have a higher likelihood of having physiochemical properties compatible with good pharmacokinetic characteristics, including oral absorption than agents with higher molecular weight. For example, agents less likely to be successful as drugs based on permeability and solubility were described by Lipinski et al. as follows: having more than 5 H-bond donors (expressed as the sum of OHs and NHs); having a molecular weight over 500; having a Log P over 5 (or M Log P over 4.15); and/or having more than 10 H-bond acceptors (expressed as the sum of Ns and Os). See, e.g., Lipinski et al. Adv Drug Delivery Res 23 :3-25 (1997). Compound classes that are substrates for biological transporters are typically exceptions to the rule.

[0057] Essentially any chemical compound can be used as a novel compound of interest for their effect on the increase or decrease in AS in the assays of the present invention. Most often, compounds can be dissolved in aqueous or organic (especially DMSO-based) solutions. The assays are designed to screen large chemical libraries by automating the assay steps, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and the like.

[0058] In accordance with a further embodiment, the present invention provides the use of a pharmaceutical composition comprising an amyloid binding compound having a detectable label, to monitor the treatment of heart failure in a subject based on the detection of amyloid species in cardiac tissue, characterized in that a detectable quantity of the pharmaceutical composition is administered to the subject before commencement of a treatment regimen for heart failure; determining whether there is binding of the compound to the cardiac tissue of the subject; commencing a treatment regimen for heart failure in the subject for a period of time; subsequently administering to the subject a detectable quantity of the pharmaceutical composition; determining whether there is binding of the compound to the cardiac tissue of the subject; comparing the level of binding of the compound in cardiac tissue of the subject before treatment to the level of binding of the compound in the cardiac tissue of the subject after treatment; and determining that the treatment regimen for heart failure in the subject is effective when the level of binding of the compound in the cardiac tissue of the subject is lower after treatment than the level of binding of the compound in the cardiac tissue of the subject before treatment.

[0059] In accordance with another embodiment, the present invention provides a composition comprising an amyloid binding compound having a detectable label, for use in detection of amyloid proteins in vitro. For example, in some embodiments, the amyloid binding compound can comprise Thioflavin T. In some embodiments, the compositions can comprise Thioflavin T having one or more detectable labels, as discussed herein.

[0060] In accordance with a further embodiment, the present invention provides a method for detection of amyloid proteins in cardiac tissue comprising obtaining a sample of cardiac tissue from a subject, isolating and purifying the proteins in the sample, applying the proteins in the sample to a protein separation means, contacting the proteins in the sample with Thioflavin T for a sufficient amount of time to allow the Thioflavin T to bind to any amyloid proteins in the sample, and detecting the presence of Thioflavin T.

[0061] The protein separation means can use any known means for separating and isolating proteins, including, for example, gels, capilliary gels, PAGE and others known in the art. In some embodiments, the cardiac proteins can be separated by PAGE or other gel electrophoresis and then contacted for a time period of between about 5 minutes to 30 minutes with a solution comprising Thioflavin T at a concentration of between about 0.05% w/v to about 1% w/v, preferably between about 0.1% w/v to about 0.3% w/v.

[0062] The detection of Thioflavin T can include any known detection means, such as fluorescence, light, or radioisotopic detection means. In some embodiments detection of Thioflavin T is accomplished by observing fluorescence through the use of various filters. In some embodiments, the filter wavelengths used are in the range for Cy2 and Cy5 fluorescent dyes.

EXAMPLE 1 [0063] Amyloid Species (AS) from Heart Failure (HF) are disrupted by treatment with EGCG and stained by Thioflavin T. Amyloid species defined by their reactivity to ThT stain and EGCG treatment were extracted from canine failing hearts using the IN-sequence protocol which we developed (see Kane LA et al, Methods Mol Biol 2007) and alternatively subjected to treatment with 20 μΜ epigallocatechin gallate (EGCG, Sigma) for 10 min at RT or vehicle alone (DMSO). EGCG selectively reduces AS in HF. Samples were separated on "not-so-native" (blue-native in the presence of 1% SDS, which we optimized, unpublished data) and stained with the Thioflavin T (ThT) in-gel staining of the present invention.

Thioflavin T is a natural molecule around which chemical structure PiB is based (Figure 2). Briefly, gels were stained with 0.1% (w/v) ThT for 10 minutes and rinsed in water overnight. Two filters (Cy2 and Cy5) were used to detect ThT signal specifically, before and after staining. The Cy2 filter is specific for ThT whereas the Cy5's was used as a reference filter. These findings support the existence of amyloid in heart failure, its susceptibility to pharmacological treatment in vitro and the affinity for a PiB analogue (Thioflavin T).

EXAMPLE 2

[0064] Other lines of evidence obtained by the present inventors were aimed at validating the specificity of Thioflavin T (ThT) staining in a well-established mouse model of cardiac amyloidosis (R120G cryAB mice, Proc. Natl. Acad. Sci. USA, 2004; 101(27): 10132-6). In these mice, the most abundant small heat shock protein in the heart, alpha-B-crystallin or cryAB is mutated (R120G) causing loss of function. CryAB is the chaperone protein that maintains the correct desmin protein folding, and AS found in these hearts contains desmin. Therefore, R120G cryAB and non-trans genic frozen mice hearts were processed according to the ΓΝ-sequence method. Protein extracts were separated using NSN-PAGE gels followed by in-gel staining with ThT as described. Regardless of the composition of the AS, ThT staining was able to highlight the increased accumulation of AS in the cryAB R120G tissue (Figure 3).

[0065] ThT staining in cryAB mice. After separation of tissue homogenates on NSN- PAGE AS were increased in cryAB compared to non-trans genic mice (by

561.7%±54.38SEM, p=0.0005).

EXAMPLE 3 [0066] The effectiveness of PiB staining was tested in mice with experimental heart failure. In these mice, heart failure was induced by transverse aortic constriction (TAC), simulating aortic stenosis. Mice were monitored by echocardiogram for signs of heart failure and subjected to PET imaging after PiB injection as well as CT scan. Briefly, after 4 weeks after TAC surgery mice were echoed for signs of overt decompensation (fractional shortening <40%). Mice with heart failure as well as healthy controls were transferred in the imaging suite and anesthesized using 2.5% isofluorane in oxygen. Mice were subsequently injected with PiB via a tail vein catheter and imaged using a PET scanner after which they were injected with a lethal dose of pentobarbital and subjected to CT scan. The heart failure induced a significant increase in the uptake of PiB compared to controls (Figure 4). These results are proof of principle that PiB could be used to detect AS, as defined by their reactivity to PiB stain, in the failing heart, independently of the presence of genetic mutations.

EXAMPLE 4

[0067] Another commercially available tracer which is FDA-approved and fluorinated (AMYVID™ or Florbetapir F 18 or ¹⁸F-AV-45, see Yang L et al, NEJM 2012) was tested in the same mouse model for heart failure (TAC) for its capacity to distinguish heart failure and control mice. Notably, AMYVID is based of a different chemical structure then PiB or ThT. Briefly, mice were echoed and transferred to the imaging suite. Heart failure (TAC) and control mice were anesthetized using 2.5% isofluorane in oxygen. After 10 minute AMYVID signal was dynamically acquired for 20 minutes in gated mode. After PET mice were euthanized using a lethal dose of pentobarbital after which a CT scan was acquired for 512 slices.

[0068] After reconstruction AMYVID signal was present on the myocardium of TAC, heart failure mice but absent in healthy controls (Figure 5). Figure 5 shows the signal of AMYVID overlapped to the CT scan for 2 controls (panels A and B, and relative magnified AMVID signal with the heart shape outlined in red for clarity in C and D) and 2 TAC mice (panels E and F and magnified and heart shape-outlined in G and H)

EXAMPLE 5 [0069] Non-invasive detection of amyloid protein deposition in patients with heart failure (HF). The present inventions suggest that patients with heart failure, including non-ischemic cardiomyopathy, proven by biopsy not to be amyloidosis, do in fact have myocardial amyloid protein deposition that can be detected by PET imaging using [ⁿC]PiB (Pittsburg Compound B), ¹⁸F- florbetapir and other PET tracers which bind to AS.

[0070] Without being bound to any particular theory, the present inventors conceived that that the build-up of amyloid during a situation of increased, pathological, cardiac workload is an event which also occurs in the absence of any specific mutations of either systemic, or cardiac, proteins.

[0071] Human subjects which have been clinically diagnosed with HF at JHU, in which familiar systemic, or cardiac-specific amyloidosis, have alternatively been excluded by genotyping, will be subjected to cardiac PET imaging with, N-methyl-[ⁿC]2-(4- methylaminophenyl)-6-hydroxybenzothiazole, (PiB) or ¹⁸F- florbetapir. A total of 10 patients will be enrolled cardiomyopathy service at Johns Hopkins University. An equal number of healthy individuals with a similar age/sex distribution will be also employed in the study as controls.

[0072] Patient Population - Eligibility and Exclusion Criteria - This present invention will leverage access to the Johns Hopkins Hospital Cardiovascular Diagnostic Laboratory in the Division of Cardiology and the Division of Nuclear Medicine. The study will enroll three groups of patients: 1) test group, (HF, n=10) 2) positive control group (n=6), and 3) negative control group (n=10). The test group will include men and women with an idiopathic nonischemic cardiomyopathy (EF < 35%) and a clinical indication for myocardial biopsy. The positive control group will include men and women with proven genetic amyloidosis. All subjects must be capable of providing informed consent. The negative control group will include men and women with normal left ventricular systolic (EF >55%) and diastolic function that are matched with the test group in regards to age and gender. Patients will be excluded for inability to lie flat for 1 hour, cardiac transplant, and patients in the test group will also be excluded if they have proven amyloidosis.

[0073] Study Procedures. Following signed informed consent, all women will have a urine pregnancy test. Subjects with a positive test will be excluded from the study. Eligible subjects will have a 20-22 gauge IV placed in a peripheral vein. [^uC]PiB and/or or t- florbetapir and ¹³N- Ammonia will be prepared in the Department of Radiology, Division of Nuclear Medicine at Johns Hopkins Hospital as described previously (J Med Chem 2003;46:2740-2754). N-Ammonia is a quantitative PET tracer that is used to quantify myocardial blood flow in absolute terms (ml/g/min) and will be used to determine whether [^uC]PiB and/or or ¹⁸F- florbetapir uptake is independent of myocardial blood flow. PET imaging will be performed on the Discovery VCT (General Electric) CT/PET scanner. A low dose attenuation scan will be performed for attenuation correction. 300 MBq of [ⁿC]PiB and/or or ¹⁸F- florbetapir will be infused intravenously and PET acquisition will be performed in listmode for 1 hour after injection. Following [^uC]PiB and/or or ¹⁸F- florbetapir imaging, 400-600 MBq of ¹³N-ammonia will be infused and a 20 minute PET acquisition will be performed in listmode 2 minutes after injection.

[0074] Image Analysis. Using custom software (MunichHeart, Technical University, Munich Germany) previously validated by our lab and others (Eur JNucl Med 1998;25: 1313- 1321), volumetric sampling of the ¹³N-ammonia perfusion study's myocardial radioactivity will be defined in 460 left ventricular sectors and depicted in a polar map. Absolute myocardial blood flow will be calculated as previously described and expressed in ml/g of tissue/min. Using the same software we will calculate the standardized uptake value (SUV) over time by normalizing tissue concentration (nCi/ml) by injected dose (nCi) and body mass (in units of ml, making the approximation that 1 gram equals 1 ml). We will compare

11 18 11 18 myocardial blood flow and [ C]PiB or F- florbetapir uptake to determine if [ C]PiB or F- florbetapir uptake is independent of blood flow. We will calculate the mean SUV time curve for each group and compare each time point, as well as, the area under the curve to determine differences between the groups as described by Sojkova and colleagues (Arch Neurol 201 1;68:232-240). Statistical comparison of standardized uptake values will be performed by two-sample, unequal variance, two-tailed Student's t test, with statistical significance set at p=0.05. Correlation will be calculated by linear regression using the Pearson correlation coefficient, r.

[0075] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0076] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0077] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as 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 elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Claims:

1. Use of a pharmaceutical composition comprising an amyloid binding compound having a detectable label, for detecting amyloid species in the cardiac tissue of a subject, characterized in that a detectable quantity of the pharmaceutical composition is administered to the subject for at least one interval, and determining whether there is binding of the compound to the cardiac tissue of the subject.

2. Use of a pharmaceutical composition comprising an amyloid binding compound having a detectable label, for diagnosing heart failure in a subject, characterized in that a detectable quantity of the pharmaceutical composition is administered to the subject at a first interval; determining whether there is binding of the compound to the cardiac tissue of the subject; comparing the level of binding of the compound in the subject to the level of binding of the compound to normal cardiac tissue; and determining that the subject is at a greater than normal risk for heart failure when the level of binding of the compound is greater than the level of binding of the compound in normal cardiac tissue.

3. Use of a pharmaceutical composition comprising an amyloid binding compound having a detectable label, to monitor the progression to heart failure in a subject based on the detection of amyloid species in cardiac tissue, characterized in that a detectable quantity of the pharmaceutical composition is administered to the subject at a first interval; determining whether there is binding of the compound to the cardiac tissue of the subject; at one or more later time intervals, administering to the subject a detectable quantity of the pharmaceutical composition, making subsequent determinations of whether there is binding of the compound to the cardiac tissue of the subject; comparing the level of binding of the compound in cardiac tissue of the subject from the first interval, to the level of binding of the compound in the cardiac tissue of the subject after one or more subsequent intervals; and determining whether heart failure is progressing in the subject when the level of binding of the compound in the cardiac tissue of the subject is higher after subsequent intervals than the level of binding of the compound in the cardiac tissue of the subject after the first interval.

4. Use of a pharmaceutical composition comprising an amyloid binding compound having a detectable label, to monitor the treatment of heart failure in a subject based on the detection of amyloid species in cardiac tissue, characterized in that a detectable quantity of the pharmaceutical composition is administered to the subject before commencement of a treatment regimen for heart failure; determining whether there is binding of the compound to the cardiac tissue of the subject; commencing a treatment regimen for heart failure in the subject for a period of time; subsequently administering to the subject a detectable quantity of the pharmaceutical composition; determining whether there is binding of the compound to the cardiac tissue of the subject; comparing the level of binding of the compound in cardiac tissue of the subject before treatment to the level of binding of the compound in the cardiac tissue of the subject after treatment; and determining that the treatment regimen for heart failure in the subject is effective when the level of binding of the compound in the cardiac tissue of the subject is lower after treatment than the level of binding of the compound in the cardiac tissue of the subject before treatment.

5. A method of screening for compounds useful in the treatment of heart failure comprising:

(a) administering to a test animal before commencement of a treatment

regimen with a compound of interest for heart failure a detectable quantity of a pharmaceutical composition comprising the an amyloid binding compound having a detectable label,

(b) determining whether there is binding of the amyloid binding compound to the cardiac tissue of the test animal;

(c) commencing a treatment regimen for heart failure in the test animal for a period of time with the compound of interest;

(d) administering to the test animal a detectable quantity of a pharmaceutical composition comprising the an amyloid binding compound having a detectable label,

(e) determining whether there is binding of the amyloid binding compound to the cardiac tissue of the test animal;

(f) comparing the level of binding of the amyloid binding compound in cardiac tissue of the test animal before treatment to the level of binding of the amyloid binding compound in the cardiac tissue of the test animal after (c); and

(g) determining that the compound of interest for heart failure in the test

animal is effective when the level of binding of the amyloid binding compound in the cardiac tissue of the test animal is lower after (c) than the level of binding of the amyloid binding compound in the cardiac tissue of the test animal before treatment.

6. The method of any of claims 1 to 5, wherein the amyloid binding compound having a detectable label is selected from the group consisting of Thioflavin T analogs such as Pittsburg compound B, and florbetapir and derivatives.

7. The amyloid binding compound of claim 6, wherein the detectable label is selected from the group consisting of ^UC, ¹³¹1, ¹²⁵1, ¹²³1, ⁷⁶Br, ⁷⁵Br, ¹⁸F, or ¹⁹F.

8. The method of claim 7, wherein the detecting is selected from the group consisting of gamma imaging, magnetic resonance imaging, and magnetic resonance spectroscopy.

9. The method of claim 8, wherein the detecting is gamma imaging, and the gamma imaging is either PET or SPECT.

10. The method of any of claims 1 to 4, wherein the pharmaceutical composition is administered by intravenous injection.

1 1. The method of claim 5, wherein the test animal is a R120G cryAB mutant or a TAC mouse, as well as other established experimental models of heart failure.

12. The method of any of claims 1 to 4, wherein the subject is a human.

13. A method for detection of amyloid proteins in a sample of cardiac tissue from a subject comprising:

a) obtaining a sample of cardiac tissue from a subject;

b) isolating the proteins from the sample of a);

c) applying the proteins of b) to a protein separating gel and allowing the proteins to separate by molecular weight;

d) contacting the proteins from c) with a solution comprising between about

0.05% w/v to about 1% w/v of Thioflavin T for a time sufficient for the Thioflavin T to bind to the amyloid proteins in c);

e) washing the gel to remove all Thioflavin T; and

e) detecting the Thioflavin T bound to the gel.