LU100526B1 - Ezetimibe and disulfiram for the treatment of cardiac diseases - Google Patents

Abstract

The present invention relates to the field of pharmaceutics and treatment of cardiac diseases. Specifically, the present invention provides activators of mitochondrial calcium uptake, in particular ezetimibe and disulfiram, for use in the treatment of various cardiac diseases. Further provided herein are pharmaceutical compositions and kits that are useful for treating cardiac diseases.

Description

New Luxemburgish patent application LU100526

Our ref.: LMU16091LU

Date: 4 December 2017 EZETIMIBE AND DISULFIRAM FOR THE TREATMENT OF______________CARDIAC DISEASES______________

BACKGROUND

[0001] Cardiovascular diseases (CVDs) are a group of disorders of the heart and bloodvessels that account for millions of deaths worldwide per year and cause significant costs tothe healthcare system. In Germany alone, cardiovascular diseases caused the death of350,000 people and lead to hospital costs in the amount of 37 billion Euros.

[0002] Although the death rates of cardiovascular diseases in general have decreased dueto better supply in recent years, morbidity and death rates due to cardiac arrhythmia andcongestive heart failure is still increasing. The new German Heart Report of the GermanSociety of Cardiology in 2016 estimated the death rate for cardiac arrhythmias to be 559.6per 100,000 people and for heart failure to be 541.5 per 100,000 people, whereinapproximately half of all heart failure patients die due to cardiac arrhythmia.

[0003] The main reason for this alarming state is that the drugs currently available for thetreatment of cardiac arrhythmia, i.e. antiarrhythmics, are extremely problematic in theirapplication due to significant side effects. With the exception of beta-blockers, allantiarrhythmics have pro-arrhythmic side-effects and therefore cannot be administered in thelong-term. Beta blockers are however ineffective for treatment of many types of arrhythmiaand limit the patient’s physical activity. For treatment of heart failure, medications to lowerblood pressure, such as ACE inhibitors and diuretics are commonly used in order to reducepreload and afterload on the damaged heart. A heart-specific treatment is only possible byusing cardiac glycosides. However, due to their strong pro-arrhythmic side effects inconjunction with a very narrow therapeutic index, these medicaments can only be usedunder observation in a hospital.

[0004] WO 2016/100379 A1 discloses agents, compositions, and methods for regulatingcardiac rhythmicity, such as efsevin. These agents, however, are only effective at higherconcentrations in the pm range, which limits their applicability as a drug for treating humandiseases.

[0005] There is thus a need in the art to explore new substances that overcome thedisadvantages set out above. SUMMARY OF THE INVENTION LU100526 [0006] The present invention provides a compound for use in a method of treatment of acardiac disease in a subject, wherein the cardiac disease can preferably be prevented,attenuated or alleviated by activating mitochondrial calcium uptake, wherein said compoundpreferably has a lower EC50 than kaempferol and/or efsevin for enhancing mitochondrialcalcium uptake, preferably determined as essentially described in Example 2. Saidcompound is preferably ezetimibe and/or disulfiram. The cardiac disease is preferablyselected from arrhythmia, in particular tachycardia such as atrial, supraventricular orventricular tachycardia, or heart failure, such as heart failure caused by systolic dysfuntion.

[0007] Further provided herein is a pharmaceutical composition comprising the compoundfor use in the treatment of a cardiac disease as described herein, wherein said compoundpreferably has a lower EC50 than kaempferol and/or efsevin for enhancing mitochondrialcalcium uptake, preferably determined as essentially described in Example 2. Saidcompound is preferably ezetimibe and/or disulfiram. A kit comprising said compound or apharmaceutical composition comprising the same; and at least one active agent useful fortreatment of said cardiac disease is also provided herein.

[0008] In a further aspect, the present invention also relates to a method for the treatment orprevention of a cardiac disease, wherein the cardiac disease can preferably be prevented,attenuated or alleviated by activating mitochondrial calcium uptake, comprising administeringa activator of mitochondrial calcium uptake to a subject in need thereof, said activator ofmitochondrial calcium uptake is a compound that preferably has a lower EC50 thankaempferol and/or efsevin for enhancing mitochondrial calcium uptake, preferablydetermined as essentially described in Example 2. Said compound is preferably ezetimibeand/or disulfiram.

BRIEF DESCRIPTION OF THE FIGURES

[0009] Figure 1: Ezetimbe and Disulfiram were identified as enhancers ofmitochondrial Ca2+ uptake in an assay on permeabilized HeLa cells. Left, mitochondrialCa2+ peak values (mM) relative to DMSO are fitted with a Hill equation (continuous lines) toextract the Michaelis constant (k0.5). Right, mitochondrial Ca2+ uptake rates (mM*s'1) relativeto DMSO are fitted with a Hill equation (continuous lines) to extract the Michaelis constant(k0.5). Data are represented as mean +- SEM (n=4) (*** p<0.001, t-test).

[0010] Figure 2: Ezetimib and Disulfiram enhance transfer of Ca2+ from thesarcoplasmic reticulum into mitochondria at significantly lower concentrationsrnmnarpri tn thp known Ca2+ uDtake enhancers efsevin and kaemDferol. Mitochondrial

Ca2+ uptake (expressed as change in fluorescence of the mitochondrial Ca2+ indicator rhod-2, LU100526AF/Fq) was measured after SR Ca2+ release with caffeine. Peak uptake was plotted againstdifferent concentrations of ezetimib and disulfiram. The dose response-curve for efsevin andkaempferol is shown for reference.

[0011] Figure 3: Ezetimibe reduces diastolic, arrhythmogenic Ca2+ waves. Incardiomyocytes from a murine CPVT model we quantified spontaneous Ca2+ waves duringdiastole. A. While only a minor portion of cells (3 out of 76) cells showed such events underbaseline, stimulation with the catecholamine isoproterenol (ISO) induced such Ca2+ waves inover 40% of cells, 1μΜ and 10μΜ ezetimibe successfully eliminated these events. B. Numberof Ca2+ waves per minute in the 4 groups described under A.

[0012] Figure 4: Ezetimibe enhances cytosolic Ca2+ transients and acceleratescytosolic Ca2+ removal. Systolic Ca2+ elevations (Ca2+ transients) in response toextracellular electrical pulsing was measured in isolated ventricular cardiomyocytes form amouse that underwent transverse aortic constriction to induce heart failure. Ezetimibeenhances the peak amplitude of the Ca2+ transient (i.e. the maximum amount of Ca2+ that isreleased during diastole) indicating positive inotropy and accelerated the removal of cytosolicCa2+ indicating positive lusitropy. Both effects are beneficial in heart failure.

DETAILED DESCRIPTION

[0013] The inventors of the present application have found that a compound that activatesmitochondrial calcium uptake is beneficial for the treatment of several cardiac diseases. Sucha compound preferably has a lower EC5o than kaempferol and/or efsevin for enhancingmitochondrial calcium uptake. Said EC50 is preferably determined as essentially described inExample 2. Such a compound according to the invention is preferably ezetimibe and/ordisulfiram. Without wishing to be bound to theory, it is believed that ezetimibe and/ordisulfiram target the mitochondrial calcium uniporter (MCU), which is an intracellular calciumchannel located in the inner membrane of the mitochondrion. It is further believed thattargeting a protein other than a receptor on the cellular membrane will at most marginallyinfluence the action potential of a heart muscle cell. It is thus assumed that targeting MCUwill only have a small or no impact on transmission of electric stimuli in the heart. Therefore,it is believed that pro-arrhythmic side effects can be significantly reduced when treating apatient with a compound that activates mitochondrial calcium uptake, e.g. by targeting MCU,as compared to using a conventional drug such as, for example, a class I or III antiarrhythmicagent.

[0014] A compound that activates mitochondrial calcium uptake is preferably a compound LU100526that facilitates or induces the passage of calcium ions from a cell’s cytosol into mitochondria.

[0015] The “mitochondrial calcium uniporter” or “MCU” as used herein is a transmembraneprotein that allows the passage of calcium ions from a cell's cytosol into mitochondria. It isone of the primary sources of mitochondria uptake of calcium, and is dependent onmembrane potential of the inner mitochondrial membrane and the concentration of calcium inthe cytosol relative to the concentration in the mitochondria. MCU is known to be present inseveral species, including, for example, human, mouse, rat, bovine, or zebrafish, just noname a few. A human MCU is the protein of the UniProt accession number Q8NE86 (version1 of 1 October 2002).

[0016] A compound that activates mitochondrial calcium uptake may for example be acompound that is an activator of MCU. An “activator of MCU” as used herein may be acompound that increases the biological activity of MCU, such as increasing the rate ofmitochondrial uptake of calcium by MCU, e.g. by binding to the mitochondrial calciumuniporter complex, such as by binding to MCU. In particular, such an activator ofmitochondrial calcium uptake may be ezetimibe and/or disulfiram.

[0017] The present invention thus envisions a compound described herein, preferablyezetimibe and/or disulfiram for use in a method of treatment of a cardiac disease in a subject.It is understood that such a cardiac disease can be alleviated by activating mitochondrialcalcium uptake.

[0018] The term “treatment” in all its grammatical forms includes therapeutic or prophylactictreatment of a subject in need thereof. A “therapeutic or prophylactic treatment” comprisesprophylactic treatments aimed at the complete prevention of clinical and/or pathologicalmanifestations or therapeutic treatment aimed at amelioration or remission of clinical and/orpathological manifestations. The term “treatment” thus also includes the amelioration orprevention of diseases.

[0019] A “cardiac disease” as used herein refers to a disease that affects the heart. The term“cardiac disease” generally includes heart arrhythmia, heart failure, coronary artery diseases(CAD) such as angina and myocardial, hypertensive heart disease, rheumatic heart disease,cardiomyopathy, congenital heart disease, valvular heart disease, or carditis.

[0020] In the context of the invention, the cardiac disease may preferably be arrhythmia. Asused herein, “arrhythmia”, “heart arrhythmia” or “cardiac arrhythmia”, which are usedsynonymously and interchangeably herein, refer to an irregular heartbeat, being either too fast or too slow. A heartbeat may be considered to be too fast if it is above 100 beats per LU100526minute in a human adult in resting state whereas a heartbeat may be considered to be tooslow if it is below 60 beats per minute in a human adult in resting state. Sick sinus syndrome(also referred to as sinus dysfunction (SND), or sinoatrial node disease), a group of abnormalheart rhythms (arrhythmias) caused by a malfunction of the sinus node, may however beexcluded from the scope of the invention.

[0021] The invention envisions that preferred arrhythmias to be treated include tachycardia.As used herein, the term "tachycardia" generally refers to any fast abnormal rhythm of theheart. In general, a resting heart rate over 100 beats per minute is accepted as tachycardiain human in adults. However, heart rates above the resting rate may be normal (such as withexercise) or abnormal (such as with electrical problems within the heart). However, heart rateshould be considered in the context of the prevailing clinical picture. For example: in sepsis>90 bp may be considered tachycardia. Tachycardia specifically includes sinus tachycardia,atrial tachycardia, ventricular tachycardia or supraventricular tachycardia. As used herein,“sinus tachycardia” refers to a sinus rhythm with an elevated rate of impulses. As usedherein, “atrial tachycardia” refers to a type of atrial arrhythmia in which the heart's electricalimpulse comes from ectopic atrial pacemaker, that is to say an abnormal site in the upperchambers of the heart or atria, rather than from the SA node which is the normal site of originof the heart's electrical activity. As used herein, “ventricular tachycardia” refers to a type oftachycardia, or a rapid heartbeat that arises from improper electrical activity of the heartpresenting as a rapid heart rhythm, that starts in the ventricles. As used herein,“supraventricular tachycardia” refers to an abnormally elevated heart rhythm arising fromimproper electrical activity of the heart, which originates at or above the atrioventricular node.

[0022] The invention envisions that preferred arrhythmias to be treated include atrialarrhythmia, ventricular arrhythmia, or supraventricular arrhythmia. An “atrial arrhythmia” asused herein refers to an arrhythmia originating from the atria. It may include premature atrialcontractions, wandering atrial pacemaker, atrial tachycardia, atrial flutter and atrial fibrillation.A “ventricular arrhythmia” as used herein refers to an arrhythmia originating from theventricle. It may include premature ventricular contractions, accelerated idioventricularrhythms, ventricular tachycardia or ventricular fibrillation. A “supraventricular arrhythmia” asused herein refers to an arrhythmia originating from the areas above the ventricle. It mayinclude supraventricular tachycardia or paroxysmal supraventricular tachycardia, atrialfibrillation, Wolff-Parkinson-White syndrome, atrial flutter, or premature atrial contractions.

[0023] A preferred arrhythmia to be treated according to the invention is a calcium-inducedarrhythmia, such as catecholaminergic polymorphic ventricular tachycardia (CPVT), also called familial polymorphic ventricular tachycardia (FPVT) or catecholamine-induced LU100526polymorphic ventricular tachycardia. It is an inherited disorder in individuals with structurally-normal hearts. CPVT is due to a mutation in a gene encoding a calcium channel or proteinsrelated to this channel. All mutated proteins participate in the regulation of calcium ion flow inand out of the sarcoplasmatic reticulum of cardiac cells. CPVT is characterized by stress-induced ventricular tachycardia. In subjects with CPVT, physical exertion and/or stress mayinduce bidirectional and/or polymorphic ventricular tachycardia that may lead to suddencardiac death in the absence of detectable structural heart disease.

[0024] In the context of the invention, the cardiac disease may also preferably be heartfailure. “Heart failure”, as used herein refers to a state where the heart is unable to pumpsufficiently to maintain blood flow to meet the body’s needs. In the context of the invention,“heart failure” may refer to a state, in which the heart is still able to pump blood, which mayhowever be insufficient. A state in which the heart is not able to pump blood at all may beexcluded from the meaning of “heart failure” in the context of the invention.

[0025] Heart failure may include a systolic dysfunction or a diastolic dysfunction. A “diastolicdysfunction” as used herein generally refers to a backward failure of the ventricle toadequately relax and typically denotes a stiffer ventricular wall. This may cause inadequatefilling of the ventricle, and therefore may result in an inadequate stroke volume. A “systolicdysfunction” as used herein, generally refers to a failure of the pump function of the heart,such as an insufficient contraction. It may be characterized by a decreased ejection fraction.The strength of ventricular contraction may be attenuated and inadequate for creating anadequate stroke volume, which may result in inadequate cardiac output. In general, a systolicdysfunction may be caused by dysfunction or destruction of cardiac myocytes or theirmolecular components. It is envisioned that systolic dysfunction may be a preferred conditionto be treated according to the invention.

[0026] The state of heart failure may further be functionally classified. The New York HeartAssociation functional classification provides an established way of classifying the extent ofheart failure (The Criteria Committee of the New York Heart Association. (1994).Nomenclature and Criteria for Diagnosis of Diseases of the Heart and Great Vessels. (9thed.). Boston: Little, Brown &amp; Co. pp. 253-256.). It places patients in one of four categoriesbased on how much they are limited during physical activity; the limitations/symptoms are inregard to normal breathing and varying degrees in shortness of breath and/or angina pain.The classes (l-lV) are defined as follows: [0027] Class I: Cardiac disease, but no symptoms and no limitation in ordinary physicalactivity, e.g. no shortness of breath when walking, climbing stairs etc.

[0028] Class II: Mild symptoms (mild shortness of breath and/or angina) and slight limitation LU100526during ordinary activity.

[0029] Class III: Marked limitation in activity due to symptoms, even during less-than-ordinary activity, e.g. walking short distances (20-100 m). Comfortable only at rest.

[0030] Class IV: Severe limitations. Experiences symptoms even while at rest. Mostlybedbound patients.

[0031] It is envisioned by the present invention that heart failure to be treated is preferably aheart failure selected from the group consisting of class I, class II, class III, and class IVaccording to the New York Heart Association Functional Classification. Preferably, the heartfailure is selected from the group consisting of class I, class II, and class III heart failureaccording to the New York Heart Association Functional Classification. Preferably the heartfailure is class I or class II heart failure according to the New York Heart AssociationFunctional Classification. Preferably, the heart failure is class I heart failure according to theNew York Heart Association Functional Classification. It is further envisioned by the presentinvention that the heart failure to be treated may be selected from the group consisting of left-sided heart failure, including heart failure with reduced ejection fraction or systolic heartfailure and heart failure with preserved ejection fraction or diastolic heart failure, right-sidedheart failure, biventricular heart failure, congestive heart failure, acute heart failure, chronicheart failure, and acute decompensated heart failure.

[0032] It is envisioned by the invention that the “subject” or “patient”, which is usedsynonymously and interchangeably throughout the application, may be an animal, preferablya vertebrate, preferably a mammal. Preferred subjects include human, mouse, rat, rabbit,hamster, pig, dog, cat, cattle, sheep, goat, camel, monkey, or ape. It is envisioned by theinvention that a human may be most preferred.

Ezetimibe and disulfiram [0033] As set out previously herein, the present inventors surprisingly identified ezetimibeand disulfiram as a promising agents for treatment of various cardiac diseases.

[0034] Ezetimibe is commonly known for capacity to lower plasma cholesterol levels. Itinhibits the absorption of cholesterol from the small intestine and decreases the amount ofcholesterol normally available to liver cells, leading them to absorb more from circulation andthus lowering levels of circulating cholesterol.

[0035] Disulfiram is a drug used to support the treatment of chronic alcoholism by producingan acute sensitivity to ethanol. Disulfiram works by inhibiting the enzyme acetaldehyde dehydrogenase. Disulfiram is also being studied as a treatment for cocain dependence, LU100526cancer, and HIV.

[0036] However, the beneficial effect of ezetimibe or disulfiram in treatment of variouscardiac diseases that can be alleviated by activating mitochondrial calcium uptake was notknown before and was surprisingly found out by the inventors of the present application.

[0037] As used herein, “Ezetimibe” also known as “Ezetimib”, commonly refers to thecompound with the IUPAC name: 1-(4-fluorophenyl)-(3R)-(3-(4-fluorophenyl)-(3S)-hydroxypropyl)-(4S)-(4-hydroxyphenyl)-2-azetidinone; PubChem CID 3117 (Modify date:2017-11-11)). The compound has the following formula (I):

The substance is commercially available from various suppliers or can be prepared usingmethods well-known in the art. It is marketed under the trade names Zetia and Ezetrol.

[0038] “Disulfiram”, also known as “Bis(diethylthiocarbamoyl) Disulfide”, “TetraethylthiuramDisulfide”, or “Tetraethylthioperoxydicarbonic Diamide”, commonly refers to the compoundwith the IUPAC name: diethylcarbamothioylsulfanyl Ν,Ν-diethylcarbamodithioate; PubChem CID 150311 (Modify date: 2017-11-11)). is The compound has the following formula (II):

The substance is commercially available from various suppliers or can be prepared usingmethods well-known in the art. It is marketed under the trade names Antabuse.

[0039] As used herein the terms “ezetimibe” and “disulfiram” may also refer to derivatives ofezetimibe or disulfiram, which are also envisaged for the uses of the present invention. Suchderivatives are compounds that are structurally related to, and derivable from, ezetimibe or

disulfiram, such as salts, esters, and the like. Ezetimibe derivatives or disulfiram derivatives LU100526include chemical modifications, for instance different or additional side groups. Suchderivatives may for example be prodrugs of ezetimibe or disulfiram. The derivatives intendedfor the use according to the present invention are preferably pharmaceutically acceptable,i.e. capable of eliciting the desired therapeutic effect without causing any undesirable local orsystemic effects in the recipient.

[0040] The skilled person will readily be able to select suitable ezetimibe derivatives ordisulfiram derivatives based on their ability to activate mitochondrial calcium uptake asascertainable using routine experiments as shown in the appended examples. The selectedezetimibe derivative or disulfiram derivative will further preferably be pharmaceuticallyacceptable and elicit the desired therapeutic effect described elsewhere herein.

Pharmaceutical composition [0041] Compounds described herein, such as ezetimibe and/or disulfiram, used fortreatment of cardiac diseases and conditions as described herein will typically beadministered in the form of a pharmaceutical composition. The present invention thus furtherprovides a pharmaceutical composition comprising, as an active agent, a compounddescribed herein, preferably ezetimibe and/or disulfiram, and, optionally, one or morepharmaceutically excipient(s). Accordingly, the use of a compound described herein,preferably ezetimibe and/or disulfiram, for the manufacture of a pharmaceutical compositionor medicament is also envisaged herein. The pharmaceutical composition or medicament ispreferably for the treatment of the cardiac diseases described herein.

[0042] The term "pharmaceutical composition" particularly refers to a composition suitablefor administering to a human. However, compositions suitable for administration to non-human animals are generally also encompassed by the term.

[0043] The pharmaceutical composition and its components (i.e. active agents and optionalexcipients) are preferably pharmaceutically acceptable, i.e. capable of eliciting the desiredtherapeutic effect without causing any undesirable local or systemic effects in the recipient.Pharmaceutically acceptable compositions of the invention may for instance be sterile ornon-sterile. Specifically, the term "pharmaceutically acceptable" may mean approved by aregulatory agency or other generally recognized pharmacopoeia for use in animals, andmore particularly in humans.

[0044] The compound, e.g. ezetimibe and/or disulfiram, is preferably present in thepharmaceutical composition in a therapeutically effective amount. By "therapeuticallyeffective amount" is meant an amount of the active agent that elicits the desired therapeutic effect. Therapeutic efficacy and toxicity can be determined by standard procedures, e.g. in LU100526cell culture or in test animals, e.g., ED50 (the dose therapeutically effective in 50% of thepopulation) and LD50 (the dose lethal to 50% of the population). The dose ratio betweentherapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio,ED5O/LD5o. Pharmaceutical compositions that exhibit large therapeutic indices are preferred.

Excipients [0045] The term “excipient” includes fillers, binders, disintegrants, coatings, sorbents,antiadherents, glidants, preservatives, antioxidants, flavoring, coloring, sweeting agents,solvents, co-solvents, buffering agents, chelating agents, viscosity imparting agents, surfaceactive agents, diluents, humectants, carriers, diluents, preservatives, emulsifiers, stabilizersand tonicity modifiers. It is within the knowledge of the skilled person to select suitableexcipients for preparing the desired pharmaceutical composition of the invention. Exemplarycarriers for use in the pharmaceutical composition of the invention include saline, bufferedsaline, dextrose, and water. Typically, choice of suitable excipients will inter alia depend onthe specific active agent used, the disease to be treated, and the desired formulation of thepharmaceutical composition.

Additional active agents [0046] The present invention further provides pharmaceutical compositions comprising oneor more of the active agents specified above and one or more additional active agents thatare suitable for treatment of the disease to be treated. Examples of active ingredientssuitable for combinations include Na+ channel blockers such as Quinidine, Ajmaline,Procainamide, Disopyramide, Lidocaine, Phenytoin, Mexiletine, Tocainide, Encainide,Flecainide, Propafenone, Moricizine, beta blockers such as Carvedilol, Propranolol, Esmolol,Timolol, Metoprolol, Atenolol, Bisoprolol; K+ channel blockers such as Amiodarone, Sotalol,Ibutilide, Dofetilide, Dronedarone; Ca+ channel blockers such as Verapamil, Diltiazem; orother agents such as Adenosine, Digoxin, Efsevin, Kaempferol, or Magnesium Sulfate.

Dosage [0047] The exact dosage of the compound described herein, such as ezetimibe and/ordisulfiram (or the pharmaceutical composition comprising the same), will be ascertainable byone skilled in the art using known techniques. Suitable dosages provide sufficient amounts ofthe compound, such as ezetimibe and/or disulfiram, and are preferably therapeuticallyeffective.

[0048] As is known in the art, adjustments for purpose of the treatment (e.g. remissionmaintenance vs. acute flare of disease), route, time and frequency of administration, time and frequency of administration formulation, age, body weight, general health, sex, diet, LU100526severity of the disease state, drug combination(s), reaction sensitivities, andtolerance/response to therapy may be required. Suitable dosage ranges for ezetimibe and/ordisulfiram or any other compound described herein can be determined using data obtainedfrom cell culture assays and animal studies and may include the ED50. Typically, dosageamounts may vary from 0.1 to 100000 micrograms, up to a total dose of about 2 g,depending upon the route of administration. Exemplary dosages are in the range from about0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about 1 mg/kgto about 10 mg/kg, from about 1 mg/kg to about 5 mg/kg, from about 0.01 mg/kg to about 1mg/kg, or from about 0.1 mg/kg to about 1 mg/kg. Guidance as to particular dosages andmethods of delivery is provided in the literature. It is recognized that treatment may require asingle administration of a therapeutically effective dose or multiple administrations of atherapeutically effective dose of the compound described herein, such as ezetimibe and/ordisulfiram. As an illustrative example, the compound described herein, such as ezetimibeand/or disulfiram, (or a pharmaceutical composition comprising the same) might beadministered daily, every 3 to 4 days, every week, or once every two weeks, or once within amonth depending on formulation, half-life and clearance rate of the particular compound orcomposition.

Administration [0049] A variety of routes are applicable for administration of the pharmaceutical compositionaccording to the present invention. Typically, administration may be accomplished enterally.Methods of enteral delivery include oral application, for example as a tablet or capsule or inthe form of powder or granulate. However, administration can also be accomplishedparenterally. Methods of parenteral delivery include topical, intra-arterial, intramuscular,subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal,intrauterine, intravaginal, sublingual or intranasal administration.

Formulation [0050] The pharmaceutical compositions of the invention can be formulated in various forms,e.g. in solid, liquid, gaseous or lyophilized form and may be, for instance, in the form of atablet, a pill, a capsule, a powder, a granule, a solution, an ointment, a cream, transdermalpatches, a gel, an aerosol, suspensions, emulsions, syrups, liquids, elixirs, extracts, tinctureor fluid extracts or in a form which is particularly suitable for the desired method ofadministration. Processes known per se for producing medicaments are indicated in 22ndedition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa.,2012) and may include, for instance conventional mixing, dissolving, granulating, dragee- making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. After LU100526pharmaceutical compositions of the invention have been prepared, they can be placed in anappropriate container and labeled for treatment of an indicated condition. Such labelingwould for instance include amount, frequency and method of administration.

Kit [0051] The invention further provides a kit comprising a compound described herein, such asezetimibe and/or disulfiram, and at least one further active agent suitable for treatment of acardiac disease as described herein as exemplified in the context of the pharmaceuticalcomposition. The components are preferably provided in one or more containers or vialswhich may be associated with a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biological products, reflectingapproval by the agency of the manufacture, use or sale of the product for humanadministration.

[0052] The present invention further envisions a compound described herein, such as amitochondrial calcium uptake activator, such as a MCU activator, preferably ezetimibe and/ordisulfiram for use in a method of activating mitochondrial calcium uptake in a patient sufferingfrom a cardiac disease. Said cardiac disease may be any cardiac disease defined herein.Preferred cardiac diseases are also described herein. The preferred cardiac diseases mayinclude cardiac arrhythmia, preferably tachydardia, or preferably atrial arrhythmia, ventriculararrhythmia, or supraventricular arrhythmia, preferably CPVT. These preferred cardiacdiseases may also include heart failure, preferably caused by systolic dysfunction. The heartfailure may be one selected from the group consisting of class I, class II, class III, and classIV according to the New York Heart Association Functional Classification, preferably class I,class II, or class III, preferably class I or class II, preferably class I. ***** [0053] It must be noted that as used herein, the singular forms “a”, “an”, and “the”, includeplural references unless the context clearly indicates otherwise. Thus, for example, referenceto “a reagent” includes one or more of such different reagents and reference to “the method”includes reference to equivalent steps and methods known to those of ordinary skill in the artthat could be modified or substituted for the methods described herein.

[0054] Unless otherwise indicated, the term "at least" preceding a series of elements is to beunderstood to refer to every element in the series. Those skilled in the art will recognize, orbe able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be LU100526encompassed by the present invention.

[0055] The term "and/or" wherever used herein includes the meaning of "and", "or" and "allor any other combination of the elements connected by said term". Accordingly, “ezetimibeand/or disulfiram” includes the meaning of ezetimibe, disulfiram, or a combination ofezetimibe and disulfiram.

[0056] The term "about" or "approximately" as used herein means within 20%, preferablywithin 10%, and more preferably within 5% of a given value or range. It includes, however,also the concrete number, e.g., about 20 includes 20.

[0057] The term “less than” or “greater than” includes the concrete number. For example,less than 20 means less than or equal to. Similarly, more than or greater than means morethan or equal to, or greater than or equal to, respectively.

[0058] Throughout this specification and the claims which follow, unless the context requiresotherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will beunderstood to imply the inclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integer or step. When used hereinthe term “comprising” can be substituted with the term “containing” or “including” orsometimes when used herein with the term “having”.

[0059] When used herein “consisting of excludes any element, step, or ingredient notspecified in the claim element. When used herein, "consisting essentially of does notexclude materials or steps that do not materially affect the basic and novel characteristics ofthe claim.

[0060] In each instance herein any of the terms "comprising", "consisting essentially of' and"consisting of may be replaced with either of the other two terms.

[0061] It should be understood that this invention is not limited to the particular methodology,protocols, material, reagents, and substances, etc., described herein and as such can vary.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 theclaims.

[0062] All publications cited throughout the text of this specification (including all patents,patent applications, scientific publications, manufacturer’s specifications, instructions, etc.),whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such LU100526disclosure by virtue of prior invention. To the extent the material incorporated by referencecontradicts or is inconsistent with this specification, the specification will supersede any suchmaterial.

[0063] A better understanding of the present invention and of its advantages will be obtainedfrom the following example, offered for illustrative purposes only. The example is notintended to limit the scope of the present invention in any way.

EXAMPLES

Example 1: Identification of Ezetimibe and Disulfiram as enhancers or mitochondrialCa2+ uptake [0064] A library of small-molecule compounds, which includes about 700 compounds wasscreened in biological duplicates. The drug screen was performed in human epitheloid cervixcarcinoma (HeLa) cells stably expressing a mitochondrial matrix-targeted GFP-aequorin (mt-AEQ). The mitochondrial-targeting sequence consisted of the first 31 residues of the subunitVIII of the human cytochome c oxidase fused in frame to GFP-aequorin. The screeningassay consists in measuring mt-Ca2+ uptake in permeabilized HeLa mt-AEQ cells. First, theendoplasmic reticulum (ER) was depleted of Ca2+ by pre-treatment with the SERCA inhibitorthapsigargin, and the plasma membrane was selectively permeabilized with digitonin. Then,the mitochondria of permeabilized cells were energized in a potassium-based medium withsuccinate and pyruvate as respiratory substrates, resulting in the full activation of the TCAcycle and the electron transport chain. Last, an exogenous Ca2+ bolus is delivered tomitochondria, which causes a rapid enhancement of mt-AEQ luminescence.

[0065] Permeabilized HeLa cell-based drug discovery screen was performed as it follows:HeLa cells stably expressing mt-AEQ were harvested at a density of 500,000 cells/mL ingrowth medium supplemented with 20 mM HEPES (pH 7.4/NaOH), and the photoproteinaequorin was reconstituted by incubation with 3 μΜ coelenterazine derivative n for 3 hr atroom temperature. Cells were centrifuged at 300 g for 3 min and the pellet was re-suspendedin an extracellular-like buffer containing 145mM NaCI, 5mM KCI, 1mM MgCI2, 10mM glucose,10mM HEPES and 500 mM EGTA (pH 7.4/NaOH), supplemented with 200 nM thapsigargin.After 20 min at room temperature, cells were collected by centrifugation at 300 g for 3 minand the pellet was resuspended in an intracellular-like buffer containing 140mM KCI, 1mMKH2PO4/K2HPO4, 1mM MgCI2, 20mM HEPES, 100 mM EGTA (pH 7.2/KOH), supplementedwith 1 mM Na+-pyruvate, 1 mM ATP/MgCI2 and 2 mM Na+-succinate. Cells werepermeabilized with 60 μΜ digitonin for 5 min, collected by centrifugation at 300 g for 3 min,resuspended in intracellular-like buffer at a density of -800 cells/mL, and 90 mL were then dispensed in a white 96-well compound plate (PerkinElmer) using a MultiDrop Combi LU100526(Thermo Fisher Scientific). The first and last column of each plate contained 10 mL of1%DMSO (negative control) and 100 μΜ Ru360 (positive control), respectively. Cells wereincubated for 5 min at room temperature and Ca2+-stimulated light signal was recorded at469 nm every 0.1 s with a luminescence counter (MicroBeta2 LumiJET Microplate Counter,PerkinElmer).

[0066] Drug screening in permeabilized HeLa cells was robust, with a Z'-factor of 0.47 and0.46 for calcium peak and uptake rate, suggesting that it is amenable for high-throughputscreening (HTS). The screen was also reproducible (Rpeak(R1 vsR2)2 =0.87; Ruptake rate(R1 vs r2)2=0.78). To systematically quantify the effect of each drug on mt-Ca2+ uptake kinetics, themaximum amplitude of the luminescence signal (peak) and the rising phase of the bell-shaped kinetic trace (uptake rate) were extracted after automated fitting of raw light signals.Each drug was scored based on its inhibitory effect on either peak amplitude or uptake ratewhen compared to positive (Ru360) and negative (DMSO) controls. The compoundsezetimib and disulfiram that increased mt-Ca2+ uptake kinetics (ldrug <-0.5) were selected ashits.

[0067] Ezetimbe and Disulfiram were identified as enhancers of mitochondrial Ca2+ uptake inan assay on permeabilized HeLa cells (Figure 1). Left, mitochondrial Ca2+ peak values (mM)relative to DMSO are fitted with a Hill equation (continuous lines) to extract the Michaelisconstant (k0.5). Right, mitochondrial Ca2+ uptake rates (mM*s-1) relative to DMSO are fittedwith a Hill equation (continuous lines) to extract the Michaelis constant (k0.5). Data arerepresented as mean +- SEM (n=4) (*** p<0.001, t-test).

Example 2: Ezetimib and Disulfiram enhance transfer of Ca2+ from the sarcoplasmicreticulum into mitochondria [0068] Ca2+ transfer from the SR into mitochondria was measured in cultured HL-1cardiomyocytes. For the measurement of SR-mitochondria Ca2+ transfer cells were plated ina 96-well plate. Cells were loaded with 6 μΜ Rhod-2 AM (Life technologies) and 0.12% (w/v)pluronic® F-127 for 30 min at 37°C. Thereafter cells were washed with external solutioncontaining (in mM) 140 NaCI, 6 KCI, 1 MgCI2, 10 Glucose, 20 HEPES, 1 EGTA (pH=7.4 withNaOH) and incubated at 37°C for 20 min to allow complete intracellular de-esterification ofthe dye. An additional washing step with Ca2+-free external solution was performed beforepermeabilizing the cells with 100 mM digitonin in internal solution containing (in mM) 1BAPTA, 20 HEPES, 100 L-Aspartic acid potassium salt, 40 KCI, 0.5 MgCI2, 2 maleic acid, 2glutamic acid, 5 pyruvic acid, 0.5 KH2PO4, 5 MgATP, 0.46 CaCI2 (pH=7.2 with KOH) for 1min. Cells were then washed 3 times for 1.5 min with internal solution. The internal solution was supplemented with different concentrations of either ezetimibe, disulfiram or DMSO as a LU100526vehicle control. Measurements were performed with the Infinite® 200 PRO multimode reader(Tecan, Maennedorf, Switzerland) at excitation wavelength 540±9 nm and emissionwavelength 580±20 nm. The measurement period was 1.5 min with a sampling rate of 650ms. After 30 s, 10 mM caffeine was added to open RyRs and release Ca2+ from the SR.

[0069] Ezetimib and Disulfiram enhance transfer of Ca2+ from the sarcoplasmic reticulum intomitochondria at significantly lower concentrations compared to the known Ca2+ uptakeenhancers efsevin and kaempferol (Figure 2). Mitochondrial Ca2+ uptake (expressed aschange in fluorescence of the mitochondrial Ca2+ indicator rhod-2, AF/F0) was measuredafter SR Ca2+ release with caffeine. Peak uptake was plotted against different concentrationsof ezetimib and disulfiram. The dose response-curve for efsevin and kaempferol is shown forreference.

Example 3: Ezetimibe reduces diastolic, arrhythmogenic Ca2+ waves [0070] Cardiomyocytes were loaded with 3 μΜ Fluo-4 AM (Thermo Fisher, Germany) and0.06% (w/v) pluronic® F-127 (Sigma-Aldrich, Germany) under protection from light for 40minutes at room temperature (RT) in external solution containing (in mM) 140 NaCI, 4 KCI, 1MgCI2, 1 CaCI2, 10 glucose, 5 HEPES, 10 BDM (pH=7.4 with NaOH), followed by incubationin external solution for 20 minutes to allow de-esterification of the dye. Calcium transients inFluo-4 AM loaded cardiomyocytes were elicited by electric field stimulation using a S48square pulse Stimulator (Grass Technologies, Warwick, Rl, USA) to apply 5 ms test pulsesat 0.5 Hz with 30 V/cm electrode distance and visualized on an inverted confocal microscopeZeiss LSM 880 (Zeiss, Germany). Line scan series were generated along the long axis of amyocyte at 400 Hz. After steady-state was reached pulsing was stopped and cells werefurther recorded for 90s to detect spontaneous, diastolic Ca2+ waves.

[0071] The results show that ezetimibe reduces diastolic, arrhythmogenic Ca2+ waves(Figure 3). In cardiomyocytes from a murine CPVT model we quantified spontaneous Ca2+waves during diastole. Figure 3A shows that while only a minor portion of cells (3 out of 76)cells showed such events under baseline, stimulation with the catecholamine isoproterenol(ISO) induced such Ca2+ waves in over 40% of cells, 1μΜ and 10μΜ ezetimibe successfullyeliminated these events. Figure 3B shows the number of Ca2+ waves per minute in the 4groups described under A.

Example 4: Ezetimibe enhances cytosolic Ca2+ transients and accelerates cytosolic LU100526Ca2+ removal [0072] Cardiomyocytes were loaded with 3 μΜ Fluo-4 AM (Thermo Fisher, Germany) and0.06% (w/v) pluronic® F-127 (Sigma-Aldrich, Germany) under protection from light for 40minutes at room temperature (RT) in external solution containing (in mM) 140 NaCI, 4 KCI, 1MgCl2, 1 CaCl2, 10 glucose, 5 HEPES, 10 BDM (pH=7.4 with NaOH), followed by incubationin external solution for 20 minutes to allow de-esterification of the dye. Calcium transients inFluo-4 AM loaded cardiomyocytes were elicited by electric field stimulation using a S48square pulse Stimulator (Grass Technologies, Warwick, Rl, USA) to apply 5 ms test pulsesat 0.5 Hz with 30 V/cm electrode distance and visualized on an inverted confocal microscopeZeiss LSM 880 (Zeiss, Germany). Line scan series were generated along the long axis of amyocyte at 400 Hz. Ca2+ transient kinetics were further analyzed using pCIamp 10.4 software(Molecular Devices LLC, CA, USA). The amplitude of the Ca2+ transient is expressed asAF/F0 and Ca2+ removal from the cytosol was analyzed by calculating the inactivationconstant Tau of a monoexponential fit of the decay phase of the Ca2+ transient.

[0073] Ezetimibe enhances cytosolic Ca2+ transients and accelerates cytosolic Ca2+ removal(Figure 4). Systolic Ca2+ elevations (Ca2+ transients) in response to extracellular electricalpulsing was measured in isolated ventricular cardiomyocytes form a mouse that underwenttransverse aortic constriction to induce heart failure. Ezetimibe enhances the peak amplitudeof the Ca2+ transient (i.e. the maximum amount of Ca2+ that is released during diastole)indicating positive inotropy and accelerated the removal of cytosolic Ca2+ indicating positivelusitropy. Both effects are beneficial in heart failure.

Claims (32)

An ezetimibe for use in a method of treating a heart disease in a subject, wherein the heart disease can be alleviated by activating mitochondrial calcium uptake. 2. Ezetimibe for use according to claim 1, wherein the disease is arrhythmia. 3. Ezetimibe for use according to claim 2, wherein the disease is tachycardia. 4. Ezetimibe for use according to claim 2 or 3, wherein the disease is atrial arrhythmia, ventricular arrhythmia or supraventricular arrhythmia. 5. Ezetimibe for use according to any one of claims 2 to 4, wherein the disease is Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). 6. Ezetimibe for use according to claim 1, wherein the disease is cardiac failure. 7. Ezetimibe for use according to claim 6, wherein the heart failure is caused by systolic dysfunction. 8. Ezetimibe for use according to claim 6 or 7, wherein the cardiac insufficiency is one selected from the group consisting of class I, class II, class III and class IV according to the New York Heart Association Functional Classification, preferably class I, class II or Class III, preferably Class I or Class II, preferably Class I. The ezetimibe for use according to any one of claims 6 to 8, wherein the heart failure is selected from the group consisting of left ventricular failure including cardiac failure with restricted ejection fraction or systolic heart failure and heart failure with unrestricted ejection fraction or diastolic heart failure, right heart failure, biventricular heart failure, congestive heart failure, acute heart failure, chronic heart failure and acute decompensated heart failure. 1 10. Ezetimibe for use according to any one of the preceding claims, wherein the LHH 00526 subject is a human. A pharmaceutical composition comprising ezetimibe for use in the treatment of heart disease in a subject, wherein the heart disease can be alleviated by activating mitochondrial calcium uptake. The pharmaceutical composition for use according to claim 12, wherein the heart disease is a disease as defined in any one of claims 2 to 9. 13. Ezetimibe for use in a method of activating mitochondrial calcium uptake in a patient suffering from heart disease. 14. A kit comprising ezetimibe and at least one further active substance suitable for the treatment of heart disease, wherein the heart disease can be alleviated by activating mitochondrial calcium uptake. A method of treating or preventing heart disease, wherein the heart disease can be prevented or alleviated by activating mitochondrial calcium uptake, comprising administering ezetimibe to a subject in need of it. 16. Use of ezetimibe for the manufacture of a medicament for the treatment or prevention of heart disease, wherein the heart disease can be alleviated by activating mitochondrial calcium uptake. 17. Disulfiram for use in a method of treating heart disease in a subject, wherein the heart disease can be alleviated by activating mitochondrial calcium uptake. 18. Disulfiram for use according to claim 17, wherein the disease is arrhythmia. 19. Disulfiram for use according to claim 18, wherein the disease is tachycardia. 20. Disulfiram for use according to claim 18 or 19, wherein the disease is atrial arrhythmia, ventricular arrhythmia or supraventricular arrhythmia. LU100526 The disulfiram for use according to any one of claims 18 to 20, wherein the disease is catecholaminergic polymorphic ventricular tachycardia (CPVT). 22. Disulfiram for use according to claim 17, wherein the disease is heart failure. 23. Disulfiram for use according to claim 22, wherein the heart failure is caused by systolic dysfunction. 24. Disulfiram for use according to claim 22 or 23, wherein the heart failure is one selected from the group consisting of class I, class II, class III and class IV according to the New York Heart Association Functional Classification, preferably class I, class II or Class III, preferably Class I or Class II, preferably Class I. 25. A disulfiram for use according to any one of claims 22 to 24, wherein the heart failure is selected from the group consisting of left ventricular failure including cardiac insufficiency with restricted ejection fraction or systolic heart failure and heart failure with unrestricted ejection fraction or diastolic heart failure, right heart failure, biventricular heart failure, congestive heart failure, acute heart failure, chronic heart failure and acute decompensated heart failure. 26. Disulfiram for use according to any one of claims 17 to 25, wherein the subject is a human. A pharmaceutical composition comprising disulfiram for use in the treatment of heart disease in a subject, wherein the heart disease can be alleviated by activating mitochondrial calcium uptake. A pharmaceutical composition for use according to claim 27, wherein the heart disease is a disease as defined in any one of claims 18 to 25. 29. Disulfiram for use in a method of activating mitochondrial calcium uptake in a patient suffering from heart disease. A kit comprising disulfiram and at least one further active substance suitable for the treatment of heart disease with LU100526, wherein the heart disease can be alleviated by activating mitochondrial calcium uptake. A method of treating or preventing heart disease, wherein the heart disease can be prevented or alleviated by activating mitochondrial calcium uptake, comprising administering disulfiram to a subject in need of it. Use of disulfiram for the manufacture of a medicament for the treatment or prevention of heart disease, wherein the heart disease can be alleviated by activating mitochondrial calcium uptake.