HK1143581B

HK1143581B - Novel anti-arrhythmic and heart failure drugs that target the leak in the ryanodine receptor and uses thereof

Info

Publication number: HK1143581B
Application number: HK10110024.7A
Authority: HK
Inventors: A‧R‧马克司; D‧W‧兰德瑞; S‧邓; Z‧Z‧程
Original assignee: 纽约市哥伦比亚大学理事会
Priority date: 2004-03-25
Filing date: 2010-10-25
Publication date: 2014-03-14

Description

Novel antiarrhythmic and heart failure drugs targeting ryanodine receptor leakage and uses thereof

The present application is a divisional application of a patent application having application number 200580015600.4, filed on 2005-3-22-days entitled "novel antiarrhythmic and heart failure drugs targeting the leakage of ryanodine receptor (RYR2) and uses thereof".

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of serial No. US10/809,089 from the continuation of the united states part filed 3, 25, 2004; claim the benefit of serial No. US 10/763,498 from the U.S. continuation application filed on 22/1/2004; the U.S. partial continuation application serial No. US 10/680,988, filed on 7/10/2003, is claimed; claim the benefit of U.S. partial continuation application serial No. US 10/608,723 filed on 26/6/2003; claim the benefit of U.S. partial continuation application serial No. US 10/288,606 filed on 5.11.2002; the U.S. division filed on month 5 and 10 of 2000 is asked to continue to claim the benefit of serial No. US 09/568,474, which is the current U.S. patent US 6,489,125B 1 granted on month 12 and 3 of 2002; the contents of these documents are incorporated herein by reference.

Statement of government interest

The invention is supported by the government under NIH Grant No. PO 1 HL 67849-01. As such, the united states government has certain rights in the invention.

Background

Despite advances in treatment, congestive heart failure remains a significant cause of mortality in western countries. Heart failure affects 5 million individuals in the united states alone and is characterized by 5-annual mortality rates-50% (Levy et al, "long-term trends in incidence and survival of heart failure," n.engl.j.med., 347: 1397-.

An important hallmark of heart failure is decreased myocardial contractility (Gwathmey et al, "abnormal intracellular calcium control in myocardium from patients with advanced heart failure, circ. Res., 61: 70-76, 1987). In healthy myocardium and other striated muscles (striated), calcium release channels on the Sarcoplasmic Reticulum (SR), including ryanodine receptors (RyRs), facilitate the coupling of action potentials to myocyte contraction (i.e., excitation-contraction (EC) coupling). Calcium (Ca)²⁺) Contraction begins when SR is released into the surrounding cytoplasm. In heart failure, contractile abnormalities are due, in part, to changes in the signaling cascade that cause the cardiac Action Potential (AP) to cause contraction. In particular, in damaged hearts, whole cell Ca²⁺Transient (transient) amplitude decreases (Beuckelmann et al, "intracellular calcium control in isolated ventricular myocytes from patients with advanced heart failure," Circ., 85: 1046-55, 1992; Gomez et al, "defective excitatory contraction coupling in experimental cardiac hypertrophy and heart failure," Science, 276: 800-06, 1997), and is prolonged (Beuckelmann et al, "intracellular calcium control in isolated ventricular myocytes from patients with advanced heart failure," Circ., 85: 1046-55, 1992).

Known as the common characteristic arrhythmia of heart failure and SR Ca in the structurally normal heart²⁺Leakage is relevant. In these cases, the most common mechanism for the induction and maintenance of ventricular tachycardia is autonomic abnormalities.

One form of autonomic abnormality called triggered arrhythmia and SR Ca²⁺Abnormal release is involved, which initiates delayed post-depolarization or DADs (Fozzard, H.A., "post-depolarization and trigger activity", Basic Res.Cardiol., 87: 105-13, 1992; Wit and Rosen, "pathophysiological mechanisms of arrhythmia", am. Heart.1., 106: 798-. DADs are abnormal depolarizations in cardiomyocytes that occur after repolarization of cardiac action potentials. The abnormal SR Ca responsible for DADs has not yet been fully elucidated²⁺Molecular basis of release. However, DADs are known to be blocked by lanolinidine, thereby providing RyR2 probably at this abnormal Ca²⁺Evidence for a key role in the pathogenesis of release (Marban et al, "mechanism of arrhythmogenic delayed post-and early post-depolarization in ventricular muscle in ferrets", J.Clin. invest., 78: 1185-92, 1986; Song and Belladinelli, "ATP promotes the development of post-depolarization and triggering events in cardiomyocytes", am.J.Physio., 267: H2005-11, 1994).

The most common arrhythmia in humans is Atrial Fibrillation (AF). It represents the major cause of morbidity and mortality (Chugh et al, "epidemiology and natural history of atrial fibrillation: clinical inclusion", j.am.coll.cardio., 37: 371-78, 2001; Falk, r.h., atrial fibrillation, n.engl.j.med., 344: 1067-78, 2001). However, despite its clinical importance, therapeutic options for AF remain limited, in part due to the fact that its underlying molecular mechanisms are poorly understood.

About 50% of all patients with heart disease die from fatal cardiac arrhythmias. Fatal arrhythmias are generally ventricular in nature. In some cases, a ventricular arrhythmia in the heart can be rapidly fatal-known as "sudden cardiac death" (SCD). Fatal cardiac arrhythmias (and SCD) may also occur among young people and, in addition, may also occur in healthy individuals not yet known to have structural heart disease. In fact, ventricular arrhythmias are sudden deaths, otherwise the most common cause of sudden death in healthy individuals.

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is a genetic disease in individuals with structurally abnormal hearts. It is characterized by stress-induced ventricular tachycardia-a fatal arrhythmia that can lead to SCD. In subjects with CPVT, physical exertion and/or stress induces bilateral and/or polymorphic ventricular tachycardia that leads to SCD in the absence of detectable structural heart disease (Laitinen et al, "mutation at the ryanodine receptor (RyR2) group in familial polymorphic ventricular tachycardia", Circulation, 103: 485-90, 2001; Leenhardt et al, "catecholaminergic polymorphic ventricular tachycardia in children: 7-year follow-up of 21 patients", Circulation, 91: 1512-19, 1995; Priori et al, "clinical and molecular characterization of patients with catecholaminergic polymorphic ventricular tachycardia", Circulation, 106: 69-74, 2002; Priori et al, "mutation at the noradrenaline receptor gene (hRyR2) in catecholaminergic polymorphic ventricular tachycardia", 103: 200, 196; Swan et al, "arrhythmic disease mapped by chromosome q42-q43 leads to malignant catecholaminergic polymorphic ventricular tachycardia in structurally abnormal hearts", j.am.coll.cardiol., 34: 2035-42, 1999).

CPVT is inherited predominantly in an autosomal dominant manner. Individuals with CPVT experience ventricular arrhythmias when exercising, but do not experience arrhythmias at rest. Association studies and direct sequencing have identified mutations in the human RyR2 gene on chromosome q42-q43 in individuals with CPVT (Laitinen et al, "mutation of the ryanodine receptor (RyR2) group in familial polymorphic ventricular tachycardia," Circulation, 103: 485-90, 2001; Priori et al, mutation of the ryanodine receptor gene (hRyR2) in based on catecholaminergic polymorphic ventricular tachycardia, "Circulation, 103: 196-200, 2001; Swan et al," arrhythmia diseases mapped on chromosome q42-q43 lead to malignant catecholaminergic polymorphic ventricular tachycardia "in structurally abnormal hearts," J.Am.Coll.Cardiol., 34: 2035-42, 1999).

There are three types of ryanodine receptors, all of which are highly related Ca²⁺A channel. RyR1 was found in skeletal muscle, RyR2 was found in the heart, and RyR3 was located in the brain. Lannogenin type 2 receptor (RyR2) is the major Ca required for EC coupling and muscle contraction in the striated muscle of the heart²⁺-a release channel.

RyR2 channel releases intracellular Ca²⁺The specific regions of the stored SR are packed into dense arrays and thereby cause muscle contraction (Marx et al, individual skeletal muscle Ca)²⁺Gating of coupling between release channels (ryanodine receptors), Science, 281: 818-21, 1998). Myocardial cell membrane depolarization in the AP null phase during EC coupling activates voltage controlCa of (2)²⁺A channel. Ca thereby passing through these channels²⁺The inward flux is at the name Ca²⁺-induced Ca²⁺Ca initiation during Release via RyR2²⁺Release from SR (Fabato, A., "calcium-induced release of calcium from the cardiac sarcoplasmic reticulum", am.J. Physio, 245: C1-C14, 1983; Nabauer et al, "modulation of calcium release by calcium current, rather than gated charge-gating, in cardiomyocytes", Science, 244: 800-03, 1989). RyR 2-mediated Ca²⁺-induced Ca²⁺Contractile proteins are released which subsequently activate the contraction of the heart muscle.

RyR2 is a protein complex comprising 4 565,000-dalton RyR2 polypeptides and 412,000-dalton FK506 binding proteins (FKBPs), particularly FKBP12.6 (calstabin). FKBPs are widely expressed cis-trans peptidyl-prolyl isomerases and are used for a variety of cellular functions (Marks, A.R., "inhibition of immune protein cellular functions-Physio. Rev., 76: 631-49, 1996). The FKBP12 protein binds tightly to and modulates the function of the following receptors: skeletal ryanodine receptor RyR1 (Brillants et al, "stabilization of calcium release channel (ryanodine receptor) function by FK 506-binding protein", Cell, 77: 513-23, 1994; Jayaraman et al, FK 506-binding protein associated with calcium release channel (ryanodine receptor), J.biol.chem., 267: 9474-77, 1992); cardiac ryanodine receptor RyR2(Kaftan et al, "action of rapamycin on ryanodine receptor/Ca (2+) -release channels from myocardium", circ. Res., 78: 990-97, 1996); related intracellular Ca²⁺A release channel, termed the inositol 1, 4, 5-triphosphate receptor type 1 (IP3R1) (Cameron et al, "FKBP 12 binds to the inositol 1, 4, 5-triphosphate receptor on leucine-proline (1400-1401) and anchors calcineurin to this FK 506-like domain", J.biol.chem., 272: 27582-88, 1997); and transforming growth factor beta (TGF β) receptor type I (T β RI) (Chen et al, the mechanism of TGF β receptor inhibition by FKBP 12-EMBO J., 16: 3866-76, 1997). FKBP12.6 binds to the RyR2 channel (one molecule per RyR2 subunit), stabilizes RyR 2-channel function (Brillants et al, "FK 506-binding protein stabilizes calcium release channels (Lannogen receptors)," Cell, 77: 513-23, 1994), and is advantageousCoupled gating between adjacent RyR2 channels (Marx et al, "Individual skeletal muscle Ca)²⁺Coupling gating between release channels (ryanodine receptors) — Science, 281: 818-21, 1998) to thereby prevent abnormal activation of the channels during the resting phase of the cardiac cycle.

It is clear that RyR2 channel leakage is associated with a number of pathological conditions in diseased and structurally abnormal hearts. Thus, methods of repairing leaks in RyR2 can treat or prevent heart failure, arrhythmias, and sudden cardiac death in millions of patients.

1, 4-benzothiazepinesClass of derivatives JTV-519 or 4- [3- (4-benzylpiperidin-1-yl) propanoyl]-7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepineMonohydrochloride-like salts (also known as k201 or ICP-Calstan 100) are novel modulators of calcium ion channels. Removing Ca from myocardial cells²⁺In addition to levels, JTV-519 also modulates Na in guinea pig ventricular cells⁺Current and inward rectification K⁺Current flow and inhibition of delayed rectifier K in guinea pig atrial cells⁺The current is applied. The study proves that JTV-519 has strong heart protection effect on catecholamine-induced myocardial injury, myocardial fiber over-contraction induced by myocardial injury and ischemia/reperfusion injury. JTV-519 exhibits greater than propranolol, verapamil and diltiazem in an experimental model of muscle fiber over-contractionGreater cardioprotective effect. The experimental data also suggest that JTV-519 reduces intracellular Ca in animal models²⁺Overload levels are effective in preventing ischemia/reperfusion.

Summary of The Invention

The present invention is based on the surprising discovery that RyR2 is a target for the treatment and prevention of heart failure and arrhythmias, including atrial fibrillation, ventricular arrhythmias, and exercise-induced arrhythmias. As described herein, the inventors made mutant RyR2 channels with 7 different CPVT mutations and studied their function. All 7 mutants have a functional defect of stimulating time-varying leakage (calcium leakage) during exercise. The inventors' study was to first identify the mechanism by which SR calcium leakage leads to DADs. It is clear that defects in mutant CPVT channels make these channels look like leaky channels in the heart of patients with advanced heart failure, an obstacle characterized by a high incidence of fatal arrhythmias. Thus, the inventors have demonstrated that the mechanism of VT in CPVT is the same as that of VT in heart failure.

The inventors also disclose herein JTV-519(k201 or ICP-Calstan 100) and other novel 1, 4-benzothiazepinesThe class derivatives can repair leaks in RyR2 channels. As the inventors have demonstrated, JTV-519 and related derivatives promote FKBP12.6 binding to PKA-phosphorylated RyR2 and to mutant RyR2s, which otherwise has reduced or no affinity for FKBP 12.6. This effect immobilizes RyR2 leakage causing fatal cardiac arrhythmias (sudden cardiac death (SCD)) and contributes to myocardial dysfunction in atrial/ventricular fibrillation and heart failure.

Accordingly, in one aspect, the invention provides a method for limiting or preventing a decrease in the level of RyR 2-bound FKBP12.6 in a subject having, or who is a candidate for, atrial fibrillation, comprising administering to the subject JTV-519 in an amount effective to limit or prevent a decrease in the level of RyR 2-bound FKBP12.6 in the subject, wherein RyR2 is atrial RyR 2. Also provided is the use of JTV-519 in a method of limiting or preventing a decrease in the level of RyR 2-bound FKBP12.6 in a subject who has, or is a candidate for, atrial fibrillation.

In another aspect, the invention provides a method for treating or preventing atrial fibrillation in a subject by administering JTV-519 to the subject in an amount effective to limit or prevent a decrease in the level of RyR 2-bound FKBP12.6 in the subject, thereby treating or preventing atrial fibrillation in the subject. In one embodiment, the amount of JTV-519 effective to limit or prevent a decrease in the level of RyR 2-bound FKBP12.6 in the subject is an amount of JTV-519 effective to treat or prevent atrial fibrillation in the subject.

In another aspect, the invention provides a method for limiting or preventing a decrease in the level of RyR 2-bound FKBP12.6 in a subject by administering to the subject an amount of 1, 4-benzothiazepine effective to limit or prevent a decrease in the level of RyR 2-bound FKBP12.6 in the subjectClass of derivatives in which 1, 4-benzothiazepineThe generic derivative is selected from the group consisting of:

wherein R is aryl, alkenyl, alkyl, - (CH)₂)_nNR′₂Or- (CH)₂)_nSR ', and n is 0, 1, 2 or 3, and R' is alkyl or cycloalkyl;

wherein R is aryl, alkyl, - (CH)₂)_nNR′₂Or- (CH)₂)_nSR ', and n is 0, 1, 2 or 3, and R' is alkyl or cycloalkyl;

wherein R ═ CO (CH)₂)_nXR′₂、SO₂(CH₂)_nXR′₂Or SO₂NH(CH₂)_nXR′₂And X ═ N or S, and N ═ 1, 2, or 3, and R' ═ alkyl or cycloalkyl; and wherein m is 1 or 2;

wherein R is aryl, alkyl, - (CH)₂)_nNR′₂、-(CH₂)_nSR ', and n is 0, 1, 2 or 3, and R' is alkyl or cycloalkyl; and wherein X ═ NH or O;

wherein R is₁OR ', SR', NR ', alkyl OR halide in the 2, 3,4, OR 5 position on the phenyl ring, and R' ═ alkyl, aryl OR H; wherein R is₂H, alkyl or aryl; and wherein R₃H, alkyl or aryl;

wherein R1 ═ H, OR ', SR ', NR ', alkyl, or halide at the 2, 3,4, or 5 positions on the phenyl ring, and R ═ alkyl, aryl, or acyl; wherein R is₂H, alkyl, alkenyl or aryl; wherein R is₃H, alkyl, alkenyl or aryl; wherein m is 0, 1 or 2; and wherein n is 0 or1；

Wherein R1 ═ H, OR ', SR', NR ', alkyl or halide in the 2, 3,4, or 5 position on the phenyl ring, and R' ═ alkyl, aryl or acyl; wherein R is₂H, alkyl, alkenyl or aryl; wherein R is₃H, alkyl, alkenyl or aryl; wherein R is₄H, halide, alkenyl, carboxylic acid or alkyl containing O, S or N; and wherein m is 0, 1 or 2; and

(h) any oxidized form thereof. Also provided are these 1, 4-benzothiazepinesUse of a class of derivatives in a method of limiting or preventing a decrease in the level of RyR 2-bound FKBP12.6 in a subject.

In another aspect, the invention provides a method for treating or preventing cardiac arrhythmia, heart failure and/or exercise-induced sudden cardiac death in a subject, comprising administering to the subject an amount of 1, 4-benzothiazepine effective to limit or prevent a decrease in the level of RyR 2-bound FKBP12.6 in the subjectClass of derivatives in which 1, 4-benzothiazepineThe generic derivative is selected from the group consisting of:

wherein R is₁OR ', SR', NR ', alkyl OR halide in the 2, 3,4, OR 5 position on the phenyl ring, and R' ═ alkyl, aryl OR H; wherein R is₂H, alkyl or aryl; and wherein R₃H, alkyl or aryl；

Wherein R1 ═ H, OR ', SR ', NR ', alkyl, or halide at the 2, 3,4, or 5 positions on the phenyl ring, and R ═ alkyl, aryl, or acyl; wherein R is₂H, alkyl, alkenyl or aryl; wherein R is₃H, alkyl, alkenyl or aryl; wherein m is 0, 1 or 2; and wherein n is 0 or 1;

(h) any oxidized form thereof.

In another aspect, the invention provides a method for treating or preventing cardiac arrhythmia, heart failure, and/or exercise-induced sudden cardiac death in a subject, comprising administering to the subject an amount of 1, 4-benzothiazepine effective to treat or prevent cardiac arrhythmia, heart failure, and/or exercise-induced sudden cardiac death in the subjectClass of derivatives in which 1, 4-benzothiazepineThe generic derivative is selected from the group consisting of:

(h) any oxidized form thereof. Also provided are these 1, 4-benzothiazepinesUse of a class of derivatives in a method of treatment or prophylaxis of cardiac arrhythmia, heart failure and/or exercise-induced sudden cardiac death in a subject.

In another aspect, the invention provides a method of identifying an agent for treating or preventing atrial fibrillation or heart failure by: (a) obtaining or producing a cell culture comprising RyR 2; (b) contacting the cell with a candidate agent; (c) contacting the cell with one or more conditions known to increase phosphorylation of RyR2 in the cell; and (d) determining whether the agent can limit or prevent a decrease in the level of RyR 2-bound FKBP12.6 in the cell. In one embodiment, the method further comprises the steps of: (e) determining whether the agent has an effect on an RyR 2-related biological event (event) in a cell. Also provided are agents identified by the methods and the use of the agents in methods of treating or preventing atrial fibrillation and heart failure.

In another aspect, the invention provides a method of identifying an agent for treating or preventing atrial fibrillation or heart failure, comprising the steps of: (a) obtaining or producing an animal containing RyR 2; (b) administering a candidate active agent to the animal; (c) contacting the animal with one or more conditions known to increase phosphorylation of RyR2 in the cell; and (d) determining whether the agent can limit or prevent a decrease in the level of RyR 2-bound FKBP12.6 in the animal. In one embodiment, the method further comprises the steps of: (e) determining whether the agent has an effect on an RyR 2-related biological event in an animal. Also provided are agents identified by the methods and the use of the agents in methods of treating or preventing atrial fibrillation and heart failure.

Other aspects of the invention will be apparent from the following description.

Brief Description of Drawings

FIG. 1 shows JTV-519 preventing FKBP12.6^+/-Exercise-induced ventricular arrhythmias in mice. (A) Untreated FKBP12.6^+/-Mouse, FKBP12.6 treated with JTV-519^+/-Mouse and FKBP12.6 treated with JTV-519^-/-Representative electrocardiogram of mice. There were no significant differences in heart rate or any measured ECG parameters. (B) Upper trace (tracing): in doing sportsUntreated FKBP12.6 tested and injected with 1.0mg/kg epinephrine^+/-Persistent polymorphic ventricular tachycardia recorded in mice. Middle trace: JTV-519-treated FKBP12.6 following the same protocol^+/-Electrograms of mice; no arrhythmia was detected. Lower trace: FKBP12.6 treated with JTV-519^-/-Exercise-induced ventricular arrhythmia (VT) in mice. The dashed line represents a VT of 16.31 seconds, not shown in the figure. 'P' denotes the P-wave, which indicates sinus rhythm after ventricular tachycardia. (C) Bar graph representation of FKBP12.6 with and without JTV-519 treatment, respectively^+/-And FKBP12.6^-/-Quantification of central sudden cardiac death (left), persistent ventricular tachycardia (> 10 beats, median) and non-persistent ventricular tachycardia (3-10 abnormal beats, right) in mice. Note 12.6 'with untreated FKBP'^+/-Mouse (n ═ 10) or JTV-519-treated FKBP12.6^-/-Treatment with JTV-519 completely prevented FKBP12.6 treatment with JTV-519 compared to mice (n ═ 5)^+/-Motor-and epinephrine-induced arrhythmias in mice (n ═ 9), suggesting that JTV-519 prevented FKBP12.6 by reassociation of FKBP12.6 with RyR2^+/-Arrhythmia and sudden death in mice.

FIG. 2 shows JTV-519 increased FKBP12.6^+/-Affinity of FKBP12.6 for RyR2 in mice prevents exercise-induced Sudden Cardiac Death (SCD). (A-B) cardiac ryanodine receptor (RyR2) was immunoprecipitated using RyR2-5029 antibody. Immunoblots (A) and bar graphs (B) are shown, representing wild-type (FKBP12.6) in the absence or presence of JTV-519, respectively, under resting conditions and after exercise^+/+) Mouse, FKBP12.6^+/-Mouse and FKBP12.6^-/-Quantitative RyR2, PKA-phosphorylated RyR2(RyR 2-pSer) in mice²⁸⁰⁹Antibody) and FKBP 12.6. Under quiescent conditions, 70% of FKBP12.6 vs FKBP12.6^+/-RyR2 binding in mice. After exercise testing, the amount of FKBP12.6 bound to RyR2 complex was at FKBP12.6^+/-A significant increase in mice, and this condition can be recovered by treatment with JTV-519. (C) RyR2 was isolated from hearts obtained after exercise testing and epinephrine injection on a single channel. Shows from using andFKBP12.6 without pretreatment with JTV-519^+/-Mouse channel and FKBP12.6 from JTV-519 pretreatment^-/-Mouse channels. It should be noted that RyR 2-channel function is in motor FKBP12.6 using JTV-519 therapy^+/-Normalization was obtained in mice. FKBP12.6 from post-treatment exercise using JTV-519^-/-A representative single channel in mice shows that FKBP12.6 in the heart is required for JTV-519 action. The dashed line represents incomplete channel opening or're-conductance' opening and indicates an FKBP 12.6-deleted RyR2 channel. The left trace represents 5.0 seconds, while the right trace represents 500 milliseconds. In the figure, Po is open probability; to is the average open time; tc is the average closing time; and c is the closed state of the channel. (D) The summary bar graph indicates the open probability of a single RyR2 channel (see above). JTV-519 significantly reduced FKBP12.6 from the exercise test at diastolic calcium concentration (150nM)^+/-Open probability of mice.

FIG. 3 illustrates that JTV-519 calibrates RyR 2-channel gating by increasing the binding affinity of FKBP12.6 to PKA-phosphorylated RyR2 channels. (A, B) Canine cardiac SR membranes (A) and recombinantly expressed RyR2 channel (B) were prepared as described above (Kaftan et al, rapamycin for ryanodine receptor/Ca from myocardium⁽²⁺⁾-effect of release channels, circ.res., 78: 990-97, 1996). (A) PKI in the Presence or absence of PKA inhibitors_5-24Phosphorylation buffer in the Presence (8mM MgCl)₂10mM EGTA and 50mM Tris/PIPES; pH 6.8) using PKA catalytic subunits (40U; sigma Chemical co., st.louis, MO) phosphorylates ryanodine receptor (RyR 2). The samples were centrifuged at 100,000Xg for 10 minutes and washed 3 times with imidazole buffer (10mM imidazole; pH 7). Recombinantly expressed FKBP12.6 (final concentration 250nM) was added to the samples in the absence or presence of JTV-519 at various concentrations. After 60-minute incubation, the samples were centrifuged at 100,000Xg for 10 minutes and washed 2 times with imidazole buffer. The samples were heated to 95 ℃ and size fractionated using SDS-PAGE. anti-FKBP 12.6 antibody (1: 1,000) was used as described above (Jayaraman et al, "FK 506-binding protein binding to calcium Release channel (ryanodine receptor)", J.biol.chem., 267: 9474-77, 1992)) And anti-RyR 2-5029 antibody (1: 3,000) were subjected to immunoblotting of SR microsomes. The figure shows that JTV-519 is capable of binding FKBP12.6 to: (A) PKA-phosphorylated RyR2 (partially bound at 100 nM; fully bound at 1000nM) or (B) the RyR2-S2809D mutant channel, which is a constitutive PKA-phosphorylated RyR2 channel. (C-E) Single-channel Studies have shown that PKI is a specific inhibitor of PKA_5-24The open probability of RyR2 after PKA phosphorylation (D) is increased compared to PKA phosphorylation (C) in the presence. Single-channel function (E) was calibrated in PKA-phosphorylated RyR2 incubated with FKBP12.6 in the presence of JTV-519. The channel was open upwards, the level of complete opening (4pA) is indicated by the hatched line (dash), and the closed state is indicated by the letter 'c'. Channels are shown and recorded at 0mV at the compression (5 sec, upper trace) time scale and the expansion (500 msec, lower trace) time scale. The amplitude histogram (right) reveals increased activity and re-conductance opening in PKA-phosphorylated RyR2, but not after treatment with JTV-519 and FKBP 12.6. (F) Open probability as cytoplasm [ Ca ]²⁺]Normalized graph of function. Ca for incubation of PKA-phosphorylated RyR2 with FKBP12.6 in the presence of JTV-519 to activate RyR2²⁺Dependent rightward shift of Ca from unphosphorylated channels²⁺-dependency similarity.

FIG. 4 shows JTV-519 improves myocardial contractility in a rat model of heart failure. (A) The area of the cross section of the myocardial relaxation phase at the mastoid level was measured using echocardiography before treatment with JTV-519 or vehicle (control) and after 4 weeks of treatment. The relative increase in diastolic dysfunction is inhibited by JTV-519. (B) Although the contractile function deteriorated in untreated animals, JTV-519 significantly increased contractile function in post-myocardial infarction (post-MI) heart failure rats.

FIG. 5 shows that JTV-519 increases the affinity of calstabin2(FKBP12.6) for RyR2 in heart failure rats. Equal amounts of RyR2(a) were immunoprecipitated using anti-RyR 2 antibody. Representative immunoblots (a) and bar graphs (B) show the amount of PKA phosphorylation of RyR2 on Ser2809 (B, left) and the amount of calstabin2(FKBP12.6) bound to RyR2t (B, right) in different experimental groups. In heart failure, PKA significantly hyperphosphorylates RyR2 (B, left), resulting in dissociation of calstabin2(FKBP12.6) from the channel complex (B, right). Treatment with JTV-519 resulted in normalization of the PKA-phosphorylation state of RyR2 and FKBP12.6 binding to RyR 2. The number of experiments is shown in bar graph. P < 0.05, HF vs. simulated surgical group; # P < 0.05, HF + JTV-519 vs.

FIG. 6 illustrates JTV-519 calibrating RyR 2-channel gating in a defective heart. The RyR2 channel was isolated from either sham operated (sham operated) or Heart Failure (HF) rats. A representative single channel trace shows that RyR 2-channel open probability (Po) is significantly increased in the defective heart (middle) compared to the sham operated rats (upper). Treatment of heart failure rats with JTV-519 calibrates the channel opening probability to a level similar to that of the sham operated animals. For each condition, the upper scan represents 5000ms, while the lower scan represents 200 ms. The channel was open upward, the hatching indicated the complete open horizontal channel opening (4pA), and 'c' indicated the state of channel closure. The amplitude histogram (right) reveals an increase in Po and RyR2 channel re-conductance openings from the defective heart.

FIG. 7 shows that JTV-519 calibrates RyR2 channel gating by increasing FKBP12.6(calstabin2) binding to the RyR2 channel. (A) Wild type RyR2(RyR2-WT) channel in the absence or presence of specific PKA inhibitor PKI_5-24PKA was phosphorylated in the presence and then incubated with calstabin2(FKBP12.6) in the presence of JTV-519 at the indicated concentration. The RyR2 immunoblot indicates equal amounts of RyR2 in the sample; the calltabin 2 immunoblot indicates that JTV-519 is able to recombine calstabin2 with PKA-phosphorylated RyR2 either partially (100nM) or completely (1000 nM). (B) RyR2-S2809D, which mimics constitutive-PKA-phosphorylated RyR2, was incubated with calstabin2 in the presence of JTV-519 at the indicated concentration. The RyR2 immunoblot indicates equal amounts of RyR2 in the sample; a calstabin2 immunoblot indicates that JTV-519 is able to re-bind calstabin2 partially (100nM) or completely (1000nM) to RyR 2-S2809D. (C) 2 [ alpha ]³⁵S]Labeled calstabin2 binding curves indicate that TV-519 increased the binding affinity of alsstabin 2 to the PKA-phosphorylated RyR2 and RyR2-S2809D mutant channels to comparable levels to non-phosphorylated RyR 2-WT. (D-F) Single channel studyFruit JTV-519(1uM) by recombination with 150nM [ Ca ]²⁺]The lower calstabin2 reduced the open probability (Po) of PKA-phosphorylated RyR2-WT (D: n ═ 11; E: n ═ 12; F: n ═ 13). The channel was open upward, the hatching indicated the complete open horizontal channel opening (4pA), and 'c' indicated the state of channel closure. The amplitude histogram (right) reveals an increase in Po and re-conductance opening in PKA-phosphorylated RyR 2; this was not observed after treatment with JTV-519(1. mu.M) and calstabin2(FKBP 12.6).

FIG. 8 illustrates RyR2 macromolecular complexes in atrial tissue. (A) RyR2 was immunoprecipitated from atrial Sarcoplasmic Reticulum (SR) and phosphorylated using PKA or cyclic adenosine monophosphate (cAMP). The addition of PKA inhibitors (PKI) completely blocked the phosphorylation reaction. (B) The components of the RyR2 macromolecular complex are co-immunoprecipitated with RyR2 from atrial SRs. The positive control was atrial SR (using 50% Immunoprecipitation (IP) input). Negative control represents the blocking of the antigenic peptide by the antibody immunoprecipitated sample. (C) Calstabin2(FKBP12.6) was co-immunoprecipitated with RyR2 from atrial SR. The samples were phosphorylated with PKA in the presence and absence of PKI prior to size fractionation by SDS PAGE. PKA phosphorylation causes calstabin2(FKBP12.6) to dissociate from the channel complex in a PKI-inhibited manner. + Cont (CSR) ═ atrial SR; (FKBP) ═ recombinant FKBP; cont ═ IP using antibodies preabsorbed with antigenic peptides.

FIG. 9 shows PKA-hyperphosphorylation of RyR2 in Atrial Fibrillation (AF). (A) RyR2 Immunoprecipitated (IP) from control animals (control; n ═ 6) and dogs with atrial fibrillation (A Fib; n ═ 6) was phosphorylated with PKA. For the reverse phosphorylation experiments, immunoblots were performed in parallel on RyR2 to determine the amount of immunoprecipitated RyR2 protein in each sample. Bar graphs on the left represent quantification for the reverse phosphorylation study. Values represent the relative degree of PKA phosphorylation of RyR2 adjusted for the amount of immunoprecipitated protein. Dogs with AF showed a 130% increase in PKA phosphorylation compared to the control group (AF: n-6; control group: n-6; P-0.001). Calstabin2(FKBP12.6) was co-immunoprecipitated with RyR 2. For immunoprecipitation experiments, immunoblots were performed in parallel on RyR2 to determine the amount of RyR2 immunoprecipitated from each sample. Bars on the right represent quantification of co-immunoprecipitation experiments. Values represent the amount of calstabin2(FKBP12.6) co-immunoprecipitated with RyR2 adjusted for the amount of immunoprecipitated protein. Calstabin2(FKBP12.6) bound to RyR2 showed a 72% reduction in AF dogs compared to the control group (control group: n ═ 6; AF: n ═ 7; P < 0.0005). (B) The same series of experiments was performed using human atrial tissue (a Fib; n ═ 5) from patients with atrial fibrillation in a heart failure state and atrial tissue (control group; n ═ 3) from patients with normal hearts. Bar graphs on the left represent quantification for the reverse phosphorylation study. Persons with AF showed a 112% increase in PKA phosphorylation compared to the control group (a Fib: n-5; control group: n-3; P-0.002). The bar graph on the right represents the results of the calstabin2(FKBP12.6) co-immunoprecipitation experiment. Persons with AF showed a 70% decrease in the amount of calstabin2(FKBP12.6) that bound RyR2 (AFib: n ═ 5; control group: n ═ 3; P < 0.0001).

FIG. 10 illustrates the altered RyR 2-channel function in AF. (A) The upper trace (trace) shows a representative RyR2 channel from the left atrium of the control group; the lower trace is the AF channel. The right side of the trace is the corresponding current amplitude histogram. (B) Bar graphs show the quantification of the probability (Po) and frequency (Fo) of patency in control dogs (Cont.) and dogs with chronic atrial fibrillation (a Fib). 17 channels from 5 a Fib dogs and 11 channels from 5 control dogs were studied. The channels from the control dogs showed no increase in activity. In contrast, 15 (88%) of the 17 channels from A Fib dogs had open probability (AF: 0.39. + -. 0.07; control: 0.009. + -. 0.002; P < 0.001) and gating frequency (AF: 21.9. + -. 4.6 s)^-1(ii) a Control group: 1.6 +/-0.6 s^-1(ii) a P < 0.002) increased significantly.

FIG. 11 shows that treatment with JTV-519 restored normal RyR2 function in AF. (A) Cytoplasmic Ca at 150nM²⁺Representative traces of a single RyR2 channel from the dog heart at concentrations (as occurred during diastole) and in the presence of 0.25mM calstabin2(FKBP12.6)It was shown that the probability of patency (Po) and the frequency of gating increased significantly after PKA phosphorylation (control group: Po 0.3 ± 0.2%, n ═ 6; PKA: Po 14.8 ± 3.2%, n ═ 7; P < 0.001). As observed in the lower trace at higher time resolution and all point histograms, PKA phosphorylation of RyR2 resulted in partial opening (re-conductance state) that could be observed upon dissociation of calstabin2(FKBP12.6) from RyR 2. JTV-519(1.0mM) restored channel activity of PKA-phosphorylated RyR2 compared to PKA-treated RyR2 (Po ═ 0.8 ± 0.3%; n ═ 6; P < 0.001); JTV-519 also produced a discontinuous current amplitude distribution in the histogram, as can be observed in the unphosphorylated control channel. The upper and lower traces represent 5000 milliseconds and 200 milliseconds, respectively; the off state is represented by 'c'; complete channel opening as shown by upward deflection to a 4pA level, as indicated by the bar graph; the dashed line in the lower trace represents the partially open 1pA step. (B) In the presence or absence of 1, 4-benzothiazepinesRecombinant calstabin2(FKBP12.6) was incubated with PKA-phosphorylated RyR2 in the presence of the derivative JTV-519. Immunoblotting using an anti-calstabin 2 antibody revealed that JTV-519 allowed recombinant calstabin2(FKBP12.6) to bind PKA-phosphorylated RyR 2. Calstabin2 binding did not occur in the absence of JTV-519.

FIG. 12 shows novel 1, 4-benzothiazepinesClass of derivatives induces binding of calstabin2(FKBP12.6) to PKA-phosphorylated cardiolanidine receptor (RyR2) at 0.5 nM. Compounds of group 2.0 nM; compounds in the lower group were 0.5 nM.

FIG. 13 shows 1, 4-benzothiazepinesThe class derivative S36 (shown in figure 15) prevented arrhythmia in mice at 200 nM. Bar graphs illustrate FKBP12.6 +/mouse with and without drug treatment as shownIn the case of arrhythmia or sudden cardiac death during exercise testing. The left panel explains sudden cardiac death; the middle graph illustrates persistent VT; and the right hand graph illustrates non-persistent VT. The numbers refer to the total number of animals used in each group.

FIG. 14 shows that JTV-519 improves cardiac contractility in a rat model of heart failure.

FIG. 15 shows the structure of the derivatives.

Detailed Description

Phosphorylation of cardiac RyR2 by Protein Kinases (PKA) is an important component of the "fight or flight" response; it expands Ca released to specific trigger²⁺The amount of increases the cardiac EC-coupling gain (Marks, A.R., "intracellular calcium release channels of the heart: role in heart failure", circ.Res., 87: 8-11, 2000). This signaling pathway provides a mechanism by which activation of the sympathetic nervous system in response to stress results in an increase in cardiac output required to meet the metabolic requirements of the stress response. Upon binding catecholamines, beta 1-and beta 2-adrenergic receptors through stimulation of G-protein G.alpha._sActivating adenylate cyclase. Adenylate cyclase increases intracellular cyclic adenosine monophosphate (cAMP) levels, thereby activating cAMP-dependent PKA. PKA phosphorylation of RyR2 increases the probability of channel opening by dissociating calstabin2(FKBP12.6) from the channel complex. Thereby increasing RyR2 pairs of Ca²⁺Sensitivity to dependent activation (Hain et al, "phosphorylation regulates calcium release channel function from the sarcoplasmic reticulum of the myocardium", J.biol.chem., 270: 2074-81, 1995; Valldivia et al, Rapid Adaptation of cardiac Lanodide receptors: by Mg²⁺And phosphorylation regulation, Science, 267: 1997-2000, 1995; marx et al, "PKA phosphorylation dissociates KBP12.6 from calcium release channels (ryanodine receptors): defective regulation in defective heart ", Cell, 101: 365-76, 2000).

Defective hearts (e.g., in patients with heart failure and animal models of heart failure) are characterized by adaptive response dysfunction including chronic hyperadrenergic stimulation (Bristow et al, "catecholamine sensitivity and beta-adrenergic-receptor density decline in defective human hearts", n.engl.j.med., 307: 205-11, 1982). The pathogenic implications of this stimulus in heart failure are supported by therapeutic strategies that reduce beta-adrenergic stimulation and left ventricular myocardial wall pressure and effectively reverse ventricular remodeling (Barbone et al, "comparison of the response of right and left ventricles to left ventricular assist device support in patients with severe heart failure: the primary role in mechanical detachment based on reverse remodeling," Circulation, 104: 670-75, 2001; Eichhorn and Bristow, "medical therapy can improve the biological properties of chronically defective hearts. In heart failure, chronic β -adrenergic stimulation is associated with activation of β -adrenergic receptors in the heart, which activate adenylate cyclase by coupling to G-proteins and thereby increase intracellular cAMP concentrations. CAMP activates CAMP-dependent PKA, which has been shown to induce hyperphosphorylation of RyR 2. Thus, chronic heart failure is a long-term adrenergic excess state (Chidsey et al, "potentiation of plasma norepinephrine response in exercise in patients with congestive heart failure", N.Engl.J.Med., 267: 650, 1962), which can have several pathological consequences, including PKA hyperphosphorylation of RyR2 (Marx et al, "PKA phosphorylation dissociation of FKBP12.6 from calcium release channels (ryanodine receptor): defective regulation in defective heart", Cell, 101: 365-76, 2000).

PKA hyperphosphorylation of RyR2 has been suggested as a contributing factor to impaired contractile function and the development of arrhythmias in heart failure (Marks et al, "progression of heart failure: is a contributor to hyperphosphorylation of the lanodidine receptor. Consistent with this hypothesis, PKA hyperphosphorylation of RyR2 in defective hearts has been demonstrated in vivo in animal models and patients with heart failure following heart transplantation (Antos et al, "constitutive activation of protein kinase A leadsDilated cardiomyopathy and sudden death "circ. res., 89: 997-1004, 2001; marx et al, "PKA phosphorylation dissociates FKBP from calcium release channels (lanolinidine receptor) 12.6: defective regulation in defective heart ", Cell, 101: 365-76, 2000; ono et al, "FKBP 12.6 interaction with ryanodine receptor alterations as abnormal Ca in heart failure²⁺Cause of release ", cardiovasc. res, 48: 323-31, 2000; reiken et al, "beta-adrenergic receptor blockers restore cardiac calcium release channel (ryanodine receptor) structure and function in heart failure," Circulation, 104: 2843-48, 2001; semsarian et al, "L-type calcium channel inhibitor diltiazemPrevention of cardiomyopathy in mouse models ", j.clin.invest, 109: 1013-20, 2002; yano et al, "stoichiometry of FKBP12.6 and lanodidine receptor alterations in Heart failure as abnormal Ca via lanodidine receptor²⁺Cause of leakage ", Circulation, 102: 2131-36, 2000).

In defective hearts, hyperphosphorylation of RyR2 by PKA induces the dissociation of the regulatable FKBP12.6 subunit from the RyR2 channel (Marx et al, "PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in defective hearts", Cell, 101: 365-76, 2000). This result leads to a significant change in the biophysical properties of the RyR2 channel. Such changes are evidenced by the following: for Ca²⁺Increased probability of patency (Po) due to increased sensitivity to dependent activation (Brillants et al, "FK 506-binding protein stabilizes calcium release channel (ryanodine receptor) function", Cell, 77: 513-23, 1994; Kaftan et al, "rapamycin for ryanodine receptor/Ca from myocardium²⁺-effect of release channel ", circ.res., 78: 990-97, 1996); channel destabilization, leading to a re-conductance state; and impaired gating of channel coupling, leading to defective EC coupling and cardiac dysfunction (Marx et al, "individual skeletal muscle Ca)²⁺Coupling gating between release channels (ryanodine receptors) ", Science, 281: 818-21, 1998). Thus, PKA-hyperphosphorylated RyR2 is responsible for low levels of Ca²⁺The stimulus is extremely sensitive and this manifests itself as SR Ca²⁺Leakage through the hyperphosphorylation channel.

Dysfunction of the adaptive response to stress in heart failure leads to the evacuation of FKBP12.6 from the channel macromolecular complex. This results in RyR2 vs Ca²⁺-induced Ca²⁺Sensitivity of release shifts to the left, resulting in low-to-moderate [ Ca ]²⁺]The next more active channel (Marx et al, "PKA phosphorylation dissociating FKBP12.6 from calcium release channels (ryanodine receptors): defective regulation in defective heart", Cell, 101: 365-76, 2000; Yamamoto et al, "abnormal Ca in cardiac sarcoplasmic reticulum from tachycardia-induced heart failure²⁺Release ", cardiovasc. res, 44: 146-55, 1999; yano et al, "stoichiometry of FKBP12.6 and lanodidine receptor alterations in Heart failure as abnormal Ca via lanodidine receptor²⁺Cause of leakage ", Circulation, 102: 2131-36, 2000). Over time, increased "leakage" through RyR2 results in SR Ca²⁺Levels return to lower levels, thereby reducing EC coupling gain and contributing to impaired systolic contractility (Marx et al, "PKA phosphorylation disassociates FKBP12.6 from calcium release channels (ryanodine receptors): defective regulation in defective hearts", Cell, 101: 365-76, 2000).

In particular, the "leaky" subgroup of RyR2 channels releases SR Ca during the diastolic phase of the cardiac cycle²⁺. This leads to depolarization of the Cell membranes of the cardiac muscle cells known as delayed post-depolarization (DADs), which are known to cause fatal cardiac arrhythmias (Wehrens et al, "FKBP 12.6 deficiency and defective calcium release channel (ryanodine receptor) function associated with exercise-induced sudden cardiac death," Cell, 113: 829-40, 2003).

In a structurally normal heart, a similar phenomenon may be in operation. In particular, exercise and stress are known to induce the release of catecholamines which activate β -adrenergic receptors in the heart. Activation of the β -adrenergic receptor results in hyperphosphorylation of the RyR2 channel. Evidence also suggests that hyperphosphorylation of RyR2 due to β -adrenergic-receptor activation provides a mutated RyR2 channel to make opening of the diastolic phase of the cardiac cycle more likely, thereby increasing the likelihood of arrhythmia.

The inventors herein have demonstrated that JTV-519 prevents heart failure in the post-MI heart failure rat model. In this animal model, JTV-519 improved cardiac function in terms of reduced diastolic dysfunction and improved systolic function. Furthermore, the inventors have demonstrated that the skeletal muscle form of the RyR channel, RyR1, is also defective (or leaky) in heart failure skeletal muscle due to PKA-hyperphosphorylation (Reiken et al, "PKA phosphorylation dissociates FKBP12.6 from calcium release channels (ryanodine receptor): defective regulation in defective heart", J.cell biol., 160: 919-28, 2003). Therefore, it is expected that JTV-519 may also improve skeletal muscle function in patients with heart failure. As such, JTV-519 provides a new treatment for the two major symptoms in heart failure, early fatigue and shortness of breath, due to skeletal muscle weakness in the extremities (extremities) and septum, respectively.

As noted above, Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is an inherited disorder in individuals with structurally normal hearts. It is characterized by stress-induced ventricular tachycardia, a fatal arrhythmia (SCD) that can lead to sudden cardiac death. Mutations in the RyR2 channel located on the Sarcoplasmic Reticulum (SR) are associated with CPVT.

All individuals with CPVT have exercise-induced arrhythmias. The present inventors previously demonstrated that exercise-induced arrhythmias and sudden death (in patients with CPVT) result from a decrease in affinity of FKBP12.6 for RyR 2. The inventors herein have demonstrated that locomotor-activated RyR2 is a result of phosphorylation by adenosine-3 ', 5' -monophosphate-dependent Protein Kinase (PKA).

To determine the molecular mechanisms based on fatal arrhythmias in CPVT, the present inventors investigated CPVT-associated mutant RyR2 channels (e.g., S2246L, R2474S, N4104K, R4497C). Mutations with normal function in planar lipid bilayers under basal conditions compared to wild-type channelsThe bulk RyR2 channel is more sensitive to activation by PKA phosphorylation-exhibiting increased activity (open probability) and prolonged open states. In addition, the physiological inhibitor Mg for resisting RyR2 channel of PKA-phosphorylation mutant RyR2 channel²⁺And shows reduced binding to FKBP12.6 (stabilizes the channel in the closed state). These findings indicate that during exercise, when the yR2 channel PKA-is phosphorylated, the mutant CPVT channel is more likely to open during the diastolic phase (diastole) of the cardiac cycle, thereby increasing SR Ca²⁺The possibility of leak-induced arrhythmias. Since heart failure is a leading cause of death worldwide, methods of repairing leaks in RyR2 can prevent fatal arrhythmias in millions of patients.

The inventors herein have further demonstrated that the experimental drug JTV-519, 1, 4-benzothiazepineThe class of derivatives prevents fatal ventricular arrhythmias in mice heterozygous for the FKBP12.6 gene. JTV-519 has now been demonstrated to reduce diastolic SR Ca in animal models of heart failure²⁺Leakage (Yano et al, "FKBP 12.6-mediated calcium release channel (ryanodine receptor) stabilization as a new therapeutic strategy for heart failure," Circulation, 107: 477-84, 2003; Kohno et al, "New cardioprotectant JTV519 improves defective channel gating of ryanodine receptor in heart failure," am.J.Physiol.Heart Circuit. Physiol., 14: 14, 2002). In the current study, the inventors examined the efficacy and mechanism of action of JTV-519 in a arrhythmia model. For its in vivo experiments, the inventors required gram quantities of JTV-519(4- [3- (4-benzylpiperidin-1-yl) propanoyl) to be used]-7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepineA monohydrochloride-like salt).

To test for arrhythmias FKBP12.6 was administered^+/-And FKBP12.6^-/-Mice underwent the above exercise protocol (Wehrens et al "FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function associated with exercise-induced sudden cardiac death ", Cell, 113: 829-40, 2003; mohler et al, "ankyrin-B mutations cause type 4 long-QT arrhythmias and sudden cardiac death", Nature, 421: 634-39, 2003). Although FKBP12.6^+/-88% of the mice (7 out of 8) showed Ventricular Tachyarrhythmia (VT) or syncope during this protocol, but FKBP12.6 pre-treated with JTV-519^+/-None of the 1 mice (0 out of 6) developed arrhythmia or syncope events (figure 1). Furthermore, 90% of FKBP12.6^+/-Mice (9 out of 10) died during or after exercise, while JTV-519-treated FKBP12.6^+/-None of the mice (0 out of 9) died (fig. 1C). With pre-treated FKBP12.6^+/-In contrast, 100% of FKBP12.6 treated with JTV-519 despite treatment with JTV-519 (FIG. C)^-/-Mice (5 out of 5) developed VT and died during the stress regimen. Together, these data suggest that FKBP12.6 is required for JTV-519 anti-arrhythmic action.

To further characterize the anti-arrhythmic properties of JTV-519, the inventors performed work on FKBP12.6^+/+、FKBP12.6^+/-And FKBP12.6^-/-Mice were subjected to a programmed electrical stimulation protocol. After injection of 0.5mg/kg isoproterenol, the injection was stopped at 71% FKBP12.6^+/-Mice (5 out of 7), but not wild-type FKBP12.6^+/+Rapid overdrive pacing was performed to induce VTs in mice (P < 0.05, n ═ 5). FKBP12.6 pretreated with JTV-519(0.5mg/kg/h)^+/-Mice were significantly less sensitive to overdrive-induced VTs than untreated FKBP12.6^+/-Mice (1 of 7 vs.5 of 7; P < 0.05). In contrast, 67% FKBP12.6 pretreated with JTV-519^-/-VTs occurred in mice (4 out of 6) during overdrive pacing.

Can be at 71% FKBP12.6^+/-VTs were induced using premature beats in mice (5 out of 7). 7 FKBP12.6 pretreated with JTV-519^+/-No VTs were observed in mice. Using a double premature beat protocol (S1-S2-S3) in100% untreated FKBP12.6^+/-VTs were reproducibly induced in mice (7 out of 7). Treatment with JTV-519 completely abolished FKBP12.6^+/-Inducible VTs in mice (7 out of 7). JTV-519 treatment does not prevent FKBP12.6^-/-VTs in mice, supporting the concept that FKBP12.6 is required for the anti-arrhythmic effect of JTV-519.

The present inventors previously demonstrated that PKA phosphorylation of RyR2 on Ser2809 leads to dissociation of FKBP12.6 from the RyR2 channel (Marx et al, "PKA phosphorylation dissociates FKBP12.6 from calcium release channels (ryanodine receptor): defect regulation in defective hearts", Cell, 101: 365-76, 2000). In the current study, pre-treatment with JTV-519(0.5mg/kg/h) did not affect FKBP12.6^+/-And FKBP12.6^-/-The extent of RyR2 PKA phosphorylation in mice (figure 2). With FKBP12.6^+/+Mouse derived FKBP12.6^+/-The RyR2 complex in mice was significantly more deficient in FKBP12.6 (P < 0.05) after exercise. However, pretreatment with JTV-519 at FKBP12.6^+/-Loss of FKBP12.6 from the RyR2 macromolecular complex during locomotion was prevented in mice (fig. 2;. P < 0.05).

With FKBP12.6 from sports^+/+Mouse channel comparison, FKBP12.6 from locomotion^+/-The mice had a significant increase in the open probability (Po) of the RyR2 channel (+/-: 0.47 + -0.12, n-11; +/-: 0.04 + -0.01, n-13; P < 0.05). Treatment of motile FKBP12.6 with JTV-519(0.5mg/kg/h) compared to untreated motile mice^+/-Mice significantly reduced the channel Po (0.02 ± 0.01, n ═ 13) (fig. 2). This observation is consistent with an increase in the amount of KBP12.6 in the RyR2 complex (fig. 2). In contrast, JTV-519 treats motile FKBP12.6^-/-Mice do not produce channels with low Po.

150nM of low cis (cytosolic) [ Ca ] was used²⁺]Mixing Ca²⁺The RyR2 single channel was examined as a carrier. When RyR2 channel should have low probability of opening in order to prevent the possible arrhythmia induced diastolic SR Ca²⁺When leaky, these conditions stimulate those during diastole. Thus, exercise FKBP12.6 as treated in JTV-519^+/-A significant drop in RyR2 Po observed in mice suggests that RyR2 channels do not "leak" in the diastolic d observation, consistent with the absence of observed arrhythmias.

To further examine the mechanism of JTV-519 to prevent VTs, the inventors simulated locomotor conditions using PKA phosphorylation of wild-type RyR2(RyR2-WT) channels. The PKA-phosphorylated RyR2 channel was then incubated with FKBP12.6(250nM) in the presence of increasing concentrations of JTV-519. Incubation with 100nM or 1000nM JTV-519 induced binding of FKBP12.6 to PKA-phosphorylated RyR2 (FIG. 3). JTV-519 also induced FKBP12.6 binding to a mutant RyR2-S2809D channel that mimics the constitutive-PKA-phosphorylated RyR2 channel (FIG. 3).

The affinity of FKBP12.6 for PKA-phosphorylated RyR2 channels was significantly increased by addition of JTV-519. Dissociation constant (K) of FKBP12.6 binding channel_ds) is: RyR2-WT + PKA + PKI_5-24(PKA inhibitor): 148 +/-59.0 nM; RyR2-WT + PKA: 1972. + -. 39.9 nM; RyR2+ PKA + JTV-519: 158 ± 56.4nM (PKA-phosphorylation channel vs. PKA-phosphorylation channel using JTV-519: P < 0.05, n ═ 2) (fig. 3). Similar results were obtained using the RyR2-S2809D mutant channel (mimicking the constitutive-PKA-phosphorylation channel). FKBP12.6 bound K_ds is: RyR 2-S2809D: 2123 + -104 nM; and RyR2-S2809D + JTV-519: 428. + -.39 nM. PKA phosphorylation of RyR2 activates the channel (Po ═ 0.01 ± 0.002(PKA + PKI; n ═ 11) vs. Po ═ 0.40 ± 0.02 (PKA; n ═ 12; P < 0.05). addition of FKBP12.6(250nM) to the PKA-phosphorylated RyR2-WT channel did not decrease Po. whereas addition of 1 μ M JTV-519+ FKBP12.6 decreased Po to a comparable level to the non-PKA-phosphorylated channel (Po ═ 0.002 ± 0.001; n ═ 13; P < 0.05).

The results of the present inventors together show that by using 1, 4-benzothiazepineGeneric derivative JTV-519 treatment of RyR2 macromolecular complexation that can reverse FKBP12.6 from RyR2 open probability, ventricular tachycardia and sudden cardiac death associated with increased RyR2 open probability in FKBP12.6 +/-miceIs absent in the substance. Thus, the present inventors have identified a new molecular mechanism for the treatment of ventricular arrhythmias: the increased affinity of RyR2 for FKBP12.6 prevents diastolic SR calcium leakage that triggers arrhythmia. Since FKBP12.6 defect in RyR2 macromolecular complex (Marx et al, "PKA phosphorylation disassociates FKBP12.6 from calcium release channel (ryanodine receptor): defective regulation in defective heart", Cell, 101: 365-76, 2000) and hereditary exercise-induced ventricular arrhythmias (Wehrens et al, "FKBP 12.6 defect associated with exercise-induced sudden cardiac death and defective calcium release channel (ryanodine receptor) function", Cell, 113: 829-40, 2003) are common features in heart failure, TV-519 is expected to provide a new specific pathway for the treatment of molecular defects in RyR2 that cause sudden cardiac death.

As discussed above, atrial fibrillation is the most common form of human arrhythmia. To date, structural and electrical remodeling including atrial refractoriness shortening, loss of refractory heart rate (rate) related adaptation (Wijflips et al, "atrial fibrillation induced atrial fibrillation: Studies on Long-term Instrument Wake goats", Circulation, 92: 1954-68, 1995; Morillo et al, "Long-term Rapid atrial pacing": structural, functional and electrophysiological characteristics of a novel model of persistent atrial fibrillation ", Circulation, 91: 1588-95, 1995; Elvan et al," pace-induced chronic atrial fibrillation compromises sinus node function in dogs: electrophysiological remodeling ", Circulation, 94: 2953-60, 1996; Gaspo et al, functional mechanisms based on tachycardia-induced persistent atrial fibrillation in a chronic dog model, Circulation, 96: 4027-35, 1997) and reentry wave length shortening-associated persistent reentry (Rensma et al," Normal dogs in atrial fibrillation length and recurrent heart rhythm arrhythmia), circ.res., 62: 395-410, 1988). This remodeling may be important in the onset, maintenance, and progression of atrial fibrillation. It has also been suggested that calcium control may play a role in electrical remodeling in atrial fibrillation (Sun et al, "cellular mechanisms of atrial contractile dysfunction due to persistent atrial tachycardia", Circulation, 98: 719-27, 1998; Goette et al, electrical remodeling in atrial fibrillation: time course and mechanism, Circulation, 94: 2968-74, 1996; Daoud et al, "effects of verapamil and procainamide on atrial fibrillation-induced electrical remodeling in humans", Circulation, 96: 1542-50, 1997; Yu et al, "changes in atrial refractory period in humans induced by tachycardia: proportional dependence and effects of tachycardia", Circulation, 97: 2331-37, 1998; Leistal et al, "relieves atrial contractile dysfunction after verapamil short-term atrial fibrillation, whereas BAY K8644 increases atrial contractile dysfunction after atrial fibrillation", Circulation, 93: Tielan 54, 1996, verapamil reduces tachycardia-induced atrial electrical remodeling Circulation, 95: 1945-53, 1997).

Various mechanisms based on altered ion channel function have been proposed for AF. For example, studies have demonstrated that L-form Ca is present in a prolonged atrial tachycardia environment²⁺Current I_Ca，L)And instantaneous outward current (I)_to) (Yue et al, "ion remodeling based on action potential changes in atrial fibrillation canine models", circ. 512-25, 1997). Observed I_ca，LMay account, at least in part, for the shortening of AERP and the frequency-dependent loss of refractoriness-both of which are hallmarks of the electrical remodeling process that accompanies AF (Yue et al, "ion remodeling based on action potential changes in the canine model of atrial fibrillation," circ. res., 81: 512-25, 1997). Verapamil has been shown to inhibit electrical remodeling in experimental animal models of rapid atrial pacing and clinical studies in patients with AF, suggesting that Ca is involved²⁺Overload (Daoud et al, "effects of verapamil and procainamide on atrial fibrillation-induced electrical remodeling in humans," Circulation, 96: 1542-50, 1997; Leistad et al, "verapamil reduces atrial contractile dysfunction after short-term atrial fibrillation, while BAY K8644 increases atrial contractile dysfunction after short-term atrial fibrillation," Circulation, 93: 1747-54, 1996).

Although the sarcolemma ion channel apparently plays an important role in remodeling that accompanies atrial tachycardia and AF, intracellular Ca²⁺The contribution of control has not been fully studied. However, evidence exists suggesting abnormal cellsInternal Ca²⁺Control does play a role in the remodeling process. For example, previous studies have demonstrated that loss of frequency adaptation is entirely explained intracellularly as a sarcolemma ionic current, such as I_Ca，LAnd I_toChanges (Ramirez et al, "mathematical analysis of canine atrial action potentials: frequency, local factors and electrical remodeling," am.J.Physiol.Heart Circuit. Physiol., 279: H1767-85, 2000; Kneller et al, "Ca due to atrial tachycardia²⁺-controlled remodeling: evidence of a role in loss of frequency adaptation, "cardiovasc. res., 54: 416-26, 2002). Studies have also demonstrated that tachycardia-induced changes in this control within the cell also contribute significantly to the loss of frequency adaptation, which is believed to be critical to the pathogenesis of AF (Sun et al, "cellular mechanisms of atrial contractile dysfunction due to persistent atrial tachycardia", Circulation, 98: 719-27, 1998; Kneller et al, "Ca due to atrial tachycardia²⁺-controlled remodeling: evidence of a role in loss of frequency adaptation, "cardiovasc. res., 54: 416-26, 2002; hara et al, "Steady and astable action potentials in fibrotic Canine atria: abnormal frequency adaptability and its possible mechanism ", cardiovasc. res., 42: 455-69, 1999).

In previous studies, the atria from a canine model of pacing-induced AF showed AP-period-rate adaptation loss and changes in AP characteristics, which could be reversed by the presence of lanodidine. These observations suggest that the alteration is due, at least in part, to intracellular-Ca²⁺Dependent processes (Hara et al, "Steady and astable action potentials in fibrotic canine atria: abnormal frequency adaptation and its possible mechanisms", Cardiovasc. Res., 42: 455-69, 1999). In addition, Ca is present in canine atria with sustained pacing-induced atrial tachycardia²⁺A significant reduction in transients (Sun et al, "cellular mechanisms of atrial contractile dysfunction due to persistent atrial tachycardia", Circulation, 98: 719-27, 1998).

Because of Ca²⁺Transient cause of Ca²⁺-induced Ca²⁺Meat membrane Ca released from SR by RyR2²⁺Entering stationThus, previous studies suggest that changes in intracellular calcium control are accompanied by a tachycardia-induced remodeling process. Such abnormally reduced Ca²⁺The transient is associated with a suppressive shortening of isolated atrial myocytes, suggesting that calcium control contributes to atrial contractile dysfunction with AF (Sun et al, cellular mechanisms of atrial contractile dysfunction due to persistent atrial tachycardia, "Circulation, 98: 719-27, 1998).

As disclosed herein, the inventors provide that calcium balance plays an important role in electrical and contractile remodeling that accompanies persistent atrial tachycardia and AF. RyR2 Release SR Ca²⁺Storage as myocardial Ca²⁺The equilibrium of the integrated components remains stable. RyR2 modulation is well characterized in canine and human ventricular tissues, and RyR2 is involved in diseases of the ventricular muscle, including heart failure and sudden cardiac death (Marx et al, "PKA phosphorylation disassociates KBP12.6 from calcium release channel (Lannogen receptor): defective modulation in defective heart", Cell, 101: 365-76, 2000; Wehrens et al, "FKBP 12.6 defect and defective calcium release channel (Lannogen receptor) function associated with exercise-induced sudden cardiac death", Cell, 113: 829-40, 2003; Reiken et al, "beta-blockers restore calcium release channel function and improve myocardial performance in human heart failure", Circulation, 107: 2459-66, 2003). Despite the presence of Ca²⁺Evidence for a role in control of atrial arrhythmias, but the regulation and function of atrial ryanodine receptors has not been fully characterized in this setting. In particular, prior to the present invention, the role of such channels in AF was not known.

The inventors herein demonstrate that, just as in ventricular muscle, atrial intracellular-calcium-release channels exist as macromolecular complexes. The results of co-immunoprecipitation experiments by the inventors show that atrial RyR2 physically binds to the major regulatory subunit calstabin2(FKBP12.6), to phosphatases PP1 and PP2A and to the regulatory and catalytic subunits of PKA. Furthermore, the method is simple. The inventors have demonstrated that endogenous PKA specifically phosphorylates RyR2 in the atrial sarcoplasmic reticulum, resulting in the deletion of calstabin2(FKBP12.6) in the channel complex. These findings suggest that modulation of contractile function in the atria can be mediated by PKA phosphorylation of atrial RyR2 in a manner similar to that observed in ventricular muscle (Brillants, et al, "stabilization of calcium release channel (ryanodine receptor) function by FK-506 binding protein", Cell, 77: 513-523, 1994).

In the present study, the inventors observed that PKA hyperphosphorylation of RyR2 was associated with a calstabin2(FKBP12.6) deletion in the atrium of canine AF. Similarly, the inventors observed that PKA hyperphosphorylation was associated with a calstabin (FKBP12.6) deletion in atrial tissue from humans with AF in a heart failure setting. This aberrant PKA hyperphosphorylation function of RyR2 has the consequence of increased rate of opening under conditions that stimulate diastole (low cytoplasmic Ca)²⁺). Such dysfunction is characteristic of a channel in which calstabin2(FKBP12.6) has been deleted (Brillants, et al, "stabilization of FK-506 binding protein for calcium release channel (ryanodine receptor) function", Cell, 77: 513-. This aberrant channel function of AF is consistent with previous studies, suggesting that the loss of calstabin2(FKBP12.6) from RyR2 under PKA hyperphosphorylation conditions creates "leaky channels" that are susceptible to Ca challenge²⁺-induced Ca²⁺Diastolic Ca followed by increased sensitivity to Release²⁺Leakage (Brillants et al, "stabilization of calcium release channel (ryanodine receptor) function by FK-506 binding protein", Cell, 77: 513-23, 1994; Kaftan et al, "Pasteur receptor/Ca on myocardial origin by Partamycin²⁺-effect of release channel ", circ.res., 78: 990-97, 1996). It is also known that channels open and close randomly (gates) in the absence of calstabin2(FKBP12.6), rather than harmoniously (coupled gates) (Marx et al, "individual skeletal muscle Ca)²⁺Coupling gating between release channels (ryanodine receptors) ", Science, 281: 818-21, 1998).

There is evidence that suggests that AF is generally initiated by premature atrial premature contractions (Bennett and Pentecost, "a pattern of onset and spontaneous cessation of atrial fibrillation in humans," Circulation, 41: 981-88, 1970), which are known to be caused by post-depolarization (Cranefield, P.F., "action potential, post-potential, and arrhythmia," Circuit. Res., 41: 415-23, 1977). It has been observed that atrial extra-systoles cause immediate reinitiation of AF after termination of the arrhythmia (Timmermans et al, "immediate reinitiation of atrial fibrillation after atrial defibrillation in vivo", J.Cardiovasc.Electrophysiol., 9: 122-28, 1998; Wellens et al, "Attrioverter: an implantable device for treating atrial fibrillation," Circulation, 98: 1651-56, 1998), and are particularly relevant to early posterior depolarization (Burashnikov and Antzevitch, "triggering activity induced by late 3 early post-depolarization mediates re-induction of atrial fibrillation immediately after termination of the arrhythmia," Circulation, 107: 2355-60, 2003). Extra-systoles are particularly likely to produce AF in the environment of a shortened effective refractory period of the atrium (Wang et al, "determining local and functional factors for atrial fibrillation induction and maintenance in dogs," am.J. Physio., 271: H148-58, 1996), similar to that which accompanies atrial electrical remodeling.

As described above, there is evidence that: abnormal diastolic Ca from "leaky" PKA-hyperphosphorylated RyR2²⁺Release results in delayed depolarization (DADs) sufficient to trigger fatal ventricular arrhythmias (Wehrens et al, FKBP 12.6-deficient and defective calcium release channel (ryanodine receptor) function associated with exercise-induced sudden cardiac death, Cell, 113: 829-40, 2003). It is believed that dysfunctional RyR2 calcium control may also be used to initiate AF in a similar manner. The abnormal channel function observed by the present inventors may contribute to the pathogenesis of AF by providing a source of DADs that is essential for initiating AF, and may also provide dysfunctional calcium control that is integral to arrhythmia-related remodeling. Given the evidence provided herein that PKA-hyperphosphorylation of the myocardium and the absence of calstabin2(FKBP12.6) from humans with AF in the heart failure setting, a functional portion of dysfunctional RyR2 could explain the frequency of atrial arrhythmias in patients with AF.

The ability of JTV-519 to repair specific molecule-level defects in the calcium control of RyR2 makes it an attractive candidate for new therapeutics. The potential of JTV-519 may be ascribed to the growing number of important cardiac diseases involving the modulation of RyR2 dysfunction, including heart failure and fatal ventricular arrhythmias (i.e., PKA hyperphosphorylation and calstabin2(FKBP12.6) loss from the channel complex may be important contributors to its pathogenesis).

Preliminary studies of JTV-519 have focused on their anti-ischemic properties (Aetas). However, recently, JTV-519 has been shown to inhibit the induction of AF in a canine sterile pericarditis model of atrial fibrillation (Kumagai et al, "antiarrhythmic effects of JTV-519, a novel cardioprotective drug, on atrial fibrillation/flutter in canine sterile pericarditis models", J.Cardiovasc. electrophysiol., 14: 880-84, 2003). However, this study does not identify a mechanism by which JTV-519 prevents AF inducibility and maintenance.

As demonstrated herein, the inventors have determined that treatment with JTV-519(1uM) results in the binding of recombinant calstabin2(FKBP12.6) to PKA-phosphorylated RyR2, which has been isolated from canine myocardium in vitro. Binding of calstabin2(FKBP12.6) to PKA-phosphorylated RyR2 did not occur using untreated channels. The re-association of calstabin2(FKBP12.6) with the channel complex restored normal function in the PKA-hyperphosphorylation channel. Thus, RyR 2-channel functional recovery may play a role in JTV-519 inhibition of AF inducibility and maintenance, as observed by Kumagai et al ("an antiarrhythmic effect of a novel cardioprotective drug JTV-519 on atrial fibrillation/flutter in the canine aseptic pericarditis model", J. Cardiovasc. electrophysiol., 14: 880-84, 2003).

Atrial fibrillation is a complex electrophysiological process; its molecular pathogenesis may be multifactorial. Abnormal myocardial Ca²⁺Control appears to significantly contribute to the course of the disease. The present inventors' studies suggest that intracellular-Ca is caused by PKA hyperphosphorylation of RyR2²⁺The release channel function may provide a remodeling process in AF and can act as a trigger for arrhythmia.

Novel methods of treatment and prevention using JTV-519

In accordance with the foregoing, the present invention provides methods for limiting or preventing a decrease in the level of RyR 2-bound FKBP12.6 in a subject. As used herein, "FKBP 12.6" includes "FKBP 12.6 protein" and "FKBP 12.6 analogue". Unless otherwise indicated, "protein" shall include proteins, protein domains, polypeptides or peptides and any fragments thereof. "FKBP 12.6 analogue" is a functional variant of FKBP12.6 protein having biological activity of FKBP12.6, which has more than 60% or 60% amino acid sequence homology with FKBP12.6 protein. The term "FKBP 12.6 biological activity" as further used herein refers to the activity of a protein or peptide that exhibits physical binding to or to unphosphorylated or unphosphorylated RyR2 under the assay conditions described herein (i.e., binding about 2-fold, or more preferably about 5-fold, above background binding of a negative control), although the affinity may be different from that of FKBP 12.6.

In addition, the term "RyR 2" as used herein includes "RyR 2 protein" (e.g., atrial RyR2 protein or ventricular RyR2 protein) and "RyR 2 analogs". An "RyR 2 analog" is a functional variant of RyR2 protein having the biological activity of RyR2, which has 60% or more amino acid sequence homology with RyR2 protein. As used herein, the term "RyR 2 analog" includes skeletal muscle isoforms of RyR1-RyR 2-and RyR 3. RyR2 of the invention may be unphosphorylated, phosphorylated (e.g., by PKA) or hyperphosphorylated (e.g., by PKA); preferably, RyR2 is phosphorylated or hyperphosphorylated. The term "RyR 2 biological activity" as further used herein refers to the activity of a protein or peptide that exhibits physical attachment to or binding to FKBP12.6 under the assay conditions described herein (i.e., about 2-fold, or more preferably about 5-fold, binding above background binding of a negative control), although the affinity may be different from that of RyR 2.

As described above, the cardiac ryanodine receptor RyR2 is a protein complex comprising 4 565,000-dalton RyR2 proteins bound to 412,000-dalton FKBP12.6 proteins. FK506 binding proteins (FKBPs) are widely expressed cis-trans peptidyl-prolyl isomerases and are used for a variety of cellular functions. The FKBP12.6 protein binds tightly to RyR2 and regulates its function. FKBP12.6 binds to RyR2 channels one molecule per RyR2 subunit, stabilizing RyR 2-channel function and facilitating coupled gating between adjacent RyR2 channels, thereby preventing abnormal activation of the channels during the resting phase of the cardiac cycle. Thus, the term "RyR 2-bound FKBP 12.6" as used herein includes molecules of FKBP12.6 protein that bind RyR2 protein subunits or tetramers of FKBP12.6 bound to RyR2 tetramers. The term "RyR 2-bound FKBP 12.6" also includes RyR2 protein subunits that bind FKBP12.6 protein molecules or RyR2 tetramers that bind FKBP12.6 tetramers. Thus, "a decrease in the level of RyR 2-bound FKBP12.6 in a subject" includes a decrease in the level of FKBP 12.6-bound RyR2 in a subject and a decrease in the level of FKBP12.6-RyR2 complex in a subject.

According to the methods of the invention, a "decreased" level of RyR 2-bound FKBP12.6 in a subject refers to a detectable decrease, or decrease in the level of RyR 2-bound FKBP12.6 in the subject. When this decrease is stopped, hindered, occluded, or reduced in any way by administration of JTV-519 (described below), such decrease is limited or prevented such that the level of RyR 2-bound FKBP12.6 in the subject is higher than it would be in the absence of JTV-519. "level" of RyR 2-bound FKBP12.6 in a subject refers to the total level in the subject, including the level of RyR 2-bound FKBP12.6 in blood (circulation), tissues and cells (e.g., cytoplasm or nucleus) in the subject. Then, as an example, a decrease in the total level of RyR 2-bound FKBP12.6 in a subject is accompanied by a decrease in the level of RyR 2-bound FKBP12.6 in the subject's blood, tissue and/or cells.

The level of RyR 2-bound FKBP12.6 in a subject can be detected by standard assays and techniques, including those readily determined by one skilled in the art (e.g., immunological techniques, hybridization assays, immunoprecipitation, western blot analysis, fluorescence imaging techniques, and/or radiation detection, etc.) as well as any of the assays and detection methods disclosed herein. For example, proteins can be isolated and purified from cells of a subject using standard methods well known in the art, including, but not limited to, cell: extracted from cells (e.g. the term solubilised egg)White matter detergents), if desired, followed by affinity chromatography using a column; chromatography (e.g., FTLC and HPLC); immunoprecipitation (using antibodies); and precipitation (e.g., using isopropanol and a reagent such as Trizol). Electrophoresis (e.g., using SDS-polyacrylamide gel) can be performed after separation and purification of the protein. Can be administered by administering a therapeutic/prophylactic agent (e.g., JTV-519 or another 1, 4-benzothiazepineClass of derivatives, as described below) to determine a decrease in, limit or prevent the level of RyR 2-bound FKBP12.6 in a subject, as compared to the amount detected at an appropriate time after administration of the therapeutic/prophylactic agent.

In the methods of the invention, for example, a decrease in the level of RyR 2-bound FKBP12.6 in a subject (e.g., in a cell of the subject) can be limited or prevented by: inhibiting dissociation of FKBP12.6 and RyR2 in a subject; increasing binding of FKBP12.6 to RyR2 in a subject; or to stabilize the RyR2-FKBP12.6 complex in a subject. As used herein, the term "inhibiting dissociation" includes blocking, reducing, inhibiting, limiting or preventing physical dissociation or separation of the FKBP12.6 subunit from the RyR2 molecule in a subject and blocking, reducing, inhibiting, limiting or preventing physical dissociation or separation of the RyR2 molecule from the FKBP12.6 subunit in a subject. As further used herein, the term "increase binding" includes increasing, increasing or improving the ability of phosphorylated RyR2 to physically bind to FKBP12.6 in a subject (e.g., about 2-fold, or more preferably about 5-fold, binding above background binding of a negative control) and increasing, increasing or improving the ability of phosphorylated FKBP12.6 to physically bind to phosphorylated RyR2 in a subject (e.g., about 2-fold, or more preferably about 5-fold, binding above background binding of a negative control).

Additionally, in the methods of the invention, the decrease in the level of RyR 2-bound FKBP12.6 in a subject (e.g., in a cell of the subject) can be limited or prevented by directly decreasing the level of phosphorylated RyR2 in the subject or by indirectly decreasing the level of phosphorylated RyR2 in the subject (e.g., by targeting an enzyme such as PKA) or another endogenous molecule that modulates or modulates the function or level of phosphorylated RyR2 in the cell). Preferably, the level of phosphorylated RyR2 in the subject is reduced by at least 10% in the methods of the invention. More preferably, the level of phosphorylated RyR2 is reduced by at least 20%.

According to the methods of the invention, a decrease in the level of RyR 2-bound FKBP12.6 in a subject (e.g., in a cell of the subject) is limited or prevented. The subject of the invention may be any animal including amphibians, birds, fish, mammals and marsupials, but is preferably a mammal (e.g. a human; domestic animals such as cats, dogs, monkeys, mice or rats; or commercial animals such as cows or pigs). In certain embodiments of the invention, the subject has or is a candidate for a cardiac disease. Examples of "heart diseases" include, but are not limited to, cardiac arrhythmias (e.g., tachycardia; tachycardia, including atrial tachyarrhythmia and atrial fibrillation (both sustained and non-sustained; ventricular arrhythmia, including ventricular fibrillation; and exercise-induced arrhythmia), heart failure, and exercise-induced sudden cardiac death.

Arrhythmias are disorders of electrical activity of the heart that manifest as abnormalities in heart rate or heart rhythm. Tachycardia (e.g., atrial, nodal, ventricular, and paroxysmal tachycardia) is a disease associated with excessive heart activity, particularly at heart rates above 100 beats per minute. Tachyarrhythmias are tachycardias associated with irregularities in the normal cardiac rhythm. Exercise-induced arrhythmias are heart diseases (e.g., ventricular fibrillation or ventricular tachycardia, including any condition that results in sudden cardiac death) that occur during/after a subject has undergone physical exercise.

Atrial fibrillation is an example of tachyarrhythmia. More specifically, atrial fibrillation is a disease associated with abnormal and irregular cardiac rhythms in which electrical signals are generated disorderly throughout the upper chamber or atrium of the heart. Common symptoms of atrial fibrillation include, but are not limited to, palpitations (discomfort of rapid and irregular beating of the heart). Atrial fibrillation can also cause blood clots to be transported from the heart to the brain, resulting in a stroke. Recent treatments for atrial fibrillation include controlling risk factors, administering drugs to slow the heart rate and/or convert the heart to a normal rhythm and prevent complications associated with coagulation.

Heart failure is a disease that manifests as a decrease in systolic function (contractility). Symptoms of heart failure include shortness of breath, decreased exercise endurance, and early muscle fatigue.

A "candidate" for a cardiac disease (e.g., arrhythmia or heart failure) is a subject that is known or believed or suspected to be at risk for developing a cardiac disease. Examples of cardiac candidates include, but are not limited to: animals/humans suspected of having cardiac arrhythmias (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmias) and/or heart failure; and animals/humans known or believed or suspected to be at risk of developing arrhythmias (e.g., tachycardia, atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained), ventricular arrhythmias including ventricular fibrillation, and exercise-induced arrhythmias), heart failure, and/or exercise-induced sudden cardiac death.

A "candidate" for exercise-induced arrhythmia is a subject that is known or believed or suspected to be at risk for developing arrhythmia during/after physical exercise. Examples of candidates for exercise-induced arrhythmias include, but are not limited to: animals/humans known to suffer from Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT); an animal/human suspected of having CPVT; and animals/humans known or believed or suspected to be at risk for arrhythmia during/after physical exercise and to be ready to exercise, currently exercising or just completing exercise. As noted above, CPVT is a genetic disease in individuals with structurally normal hearts. It is characterized by stress-induced ventricular tachycardia-a fatal arrhythmia that can lead to sudden cardiac death. In subjects with CPVT, physical exertion and/or stress can induce bidirectional and/or polymorphic ventricular tachycardia leading to Sudden Cardiac Death (SCD) in the absence of detectable structural heart disease. Individuals with CPVT experience ventricular arrhythmias when exercising, but do not develop arrhythmias at rest.

In the method of the present invention, the cell of the subject is preferably a striated muscle cell. Striated muscle is a repeating unit of contractile myofibrils (myofibril knots) arranged at a marker throughout the cell, creating striations or twills that can be observed under light microscopy scale. Examples of striated muscle cells include, but are not limited to, voluntary (skeletal) muscle cells and cardiac muscle cells. In a preferred embodiment, the cells used in the method of the invention are human cardiomyocytes. The term "cardiomyocytes" as used herein includes myocardial fibers, such as those found in the myocardium of the heart. The myocardial fibers consist of a continuous chain of cardiomyocytes or cardiomyocytes joined end to end on intercalated disks. These discs have two types of cell junctions: the expanded bridgework, which extends along its transverse portion, joins the gap, with its longest portion extending along its longitudinal portion.

In the methods of the invention, the level of RyR 2-bound FKBP12.6 in a subject (e.g., in a cell of the subject) is limited or induced to decrease by administering JTV-519 to the subject; this method then enables the subject cells to be contacted with JTV-519. JTV-519(4- [3- (4-benzylpiperidin-1-yl) propanoyl)]-7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepineMonohydrochloride-like salt), also known as k201 is 1, 4-benzothiazepineAre derivatives of (a) and are modulators of calcium ion channels. Removing Ca from myocardial cells²⁺In addition to levels, JTV-519 modulates Na in guinea pig ventricular cells⁺Current and inward-rectification K⁺Current and inhibition of delayed-rectifier K in guinea pig cells⁺The current is applied. FK506 and rapamycinAre drugs that may be used to design other compounds that stabilize the RyR2-FKBP12.6 complex in subjects of the invention. Both FKBP 506 and rapamycin can dissociate FKBP12.6 from RyR 2. Compounds that are structurally related to these drugs, but have an adverse effect, can be designed and/or screened.

In the methods of the invention, JTV-519 may be administered to a subject by treating a composition comprising JTV-519 and a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof. As used herein. The pharmaceutically acceptable carrier is selected from a variety of organic or inorganic substances which can be used as materials in pharmaceutical formulations and can be incorporated as analgesics, buffers, binders, disintegrants, diluents, emulsifiers, excipients, extenders, glidants, solubilizers, stabilizers, suspending agents, tonicity agents, vehicles and viscosity increasing agents. If necessary, pharmaceutical additives such as antioxidants, fragrances, colorants, flavors, preservatives and sweeteners may also be added. Examples of acceptable pharmaceutical carriers include carboxymethylcellulose, microcrystalline cellulose, glycerol, gum arabic, lactose, magnesium stearate, methylcellulose, powders, saline, sodium alginate, sucrose, starch, talc, water, and the like.

The pharmaceutical formulations of the present invention may be prepared by methods well known in the pharmaceutical arts. For example, JTV-519 may be mixed with a carrier or diluent as a suspension or solution. One or more adjuvants (e.g., buffers, flavoring agents, surfactants, etc.) may also optionally be added. The choice of carrier depends on the route of administration.

JTV-519 may be administered to a subject by contacting target cells (e.g., cardiomyocytes) with JTV-519 in the subject. JTV-519 may be contacted (e.g., introduced) with cells of a subject using well-known techniques for introducing and administering proteins, nucleic acids, and other drugs. Examples of methods for contacting cells with JTV-519 (i.e., treating cells therewith) include, but are not limited to, absorption, electroporation, immersion, injection, introduction, liposomal delivery, transfection, vectors, and other drug delivery vehicles and methods. Where the target cells are located in a particular part of the subject, it may be desirable to introduce JTV-519 directly into the cells by injection or by other means (e.g., by introducing JTV-519 into the blood or another body fluid). For example, target cells may be contained in cardiac tissue of a subject and may be detected in the cardiac tissue by standard detection methods readily determinable by well-known techniques, examples of which include, but are not limited to, immunological techniques (e.g., immunohistochemical staining), fluorescence imaging techniques, and microscopy techniques.

In addition, JTV-519 of the present invention may be administered to a human or animal subject by well known procedures, including, but not limited to, oral, parenteral, and transdermal administration. JTV-519 is preferably administered by suprafascial, intracapsular, intracranial, intradermal, intrathecal, intramuscular, intraorbital, intraperitoneal, intravenous, parenchymal (parenchymatus), subcutaneous or sublingual injection or parenterally by catheter. In one embodiment, the active agent is administered to the subject by targeted delivery to cardiomyocytes via a catheter inserted into the heart of the subject.

For oral administration, the JTV-519 formulation may be formulated as a capsule, tablet, powder, granule, or suspension. The formulation may contain conventional additives such as lactose, mannitol, corn starch or potato starch. The following components can also be used to prepare the formulation: binders such as microcrystalline cellulose, cellulose derivatives; acacia, corn starch or gelatin. In addition, the formulations may be prepared using disintegrating agents, such as corn starch, potato starch or sodium carboxymethyl cellulose. Formulations may also be prepared using dibasic calcium phosphate or sodium starch glycolate. Finally, the formulations can be prepared using lubricants, such as talc or magnesium stearate.

For parenteral administration (i.e., administration by injection via a route other than the digestive tract), JTV-519 may be combined with a sterile aqueous solution that is preferably isotonic with the blood of the subject. Such formulations may be prepared by the following steps: the solid active ingredient is dissolved in water containing a physiologically compatible substance, such as sodium chloride, glycine and the like, and has a buffered pH compatible with physiological conditions so as to produce an aqueous solution, which is then rendered sterile. The formulations may be presented in unit-or multi-dose containers, such as sealed ampoules or vials. The formulation may be delivered by any injection means, including, but not limited to, suprafascially, intravesicularly, intracranially, intradermally, intrathecally, intramuscularly, intraorbitally, intraperitoneally, intraspinally, intrasternally, intravascularly, intravenously, parenchymally, subcutaneously, or sublingually, or by a catheter inserted into the heart of the subject.

For transdermal administration, JTV-519 may be combined with a skin penetration enhancer, such as propylene glycol, polyethylene glycol, isopropyl alcohol, ethanol, oleic acid, N-methylpyrrolidone, or the like, which may increase the permeability of the skin to JTV-519 and allow JTV-519 to penetrate into the skin and enter the bloodstream. The JTV-519/accelerator composition may also be further combined with a polymer, such as ethyl cellulose, hydroxypropyl cellulose, ethylene/vinyl acetate, polyvinylpyrrolidone, and the like, to produce a composition in the form of a gel, which may be dissolved in a solvent, such as methylene chloride, evaporated to a desired viscosity and then spread on a backing material to make a patch.

According to the methods of the invention, JTV-519 can be administered to a subject (and JTV-519 can be contacted with cells of the subject) in an amount effective to limit or prevent a decrease in the level of RyR 2-bound FKBP12.6 in the subject, particularly in cells of the subject. Such amounts are readily determined by one skilled in the art based on well known procedures, including established in vivo titration curve analysis and the methods and assays disclosed herein. A suitable amount of JTV-519 that is effective to limit or prevent a decrease in the level of RyR 2-bound FKBP12.6 in a subject may range from about 5 mg/kg/day to about 20 mg/kg/day, and/or may be an amount sufficient to achieve a plasma level of about 300ng/ml to about 1000 ng/ml. Preferably, JTV-519 is used in an amount of about 10 mg/kg/day to about 20 mg/kg/day.

In one embodiment of the invention, the subject has not developed a heart disease, such as a cardiac arrhythmia (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmias), heart failure, or exercise-induced sudden cardiac death. In this case, the amount of JTV-519 effective to limit or prevent a decrease in the level of RyR 2-bound FKBP12.6 in the subject may be an amount of JTV-519 effective to prevent a cardiac disease (e.g., arrhythmia, heart failure, or exercise-induced sudden cardiac death) in the subject.

As used herein, an amount of a drug (e.g., JTV-519) that is "effective in preventing heart disease" includes an amount of a drug (e.g., JTV-519) that is effective in preventing clinical damage or symptoms of heart disease. For example, if the cardiac disease is atrial fibrillation, the amount of JTV-519 effective to prevent atrial fibrillation may be an amount of JTV-519 effective to prevent palpitations and/or blood clots in the subject. Similarly, if the heart disease is exercise-induced arrhythmia, then the amount of JTV-519 effective to prevent exercise-induced arrhythmia may be an amount of JTV-519 effective to prevent exercise-induced palpitations, syncope, ventricular fibrillation, ventricular tachycardia, and sudden cardiac death in the subject. In addition, if the heart disease is heart failure, the amount of JTV-519 effective to prevent heart failure may be an amount of JTV-519 effective to prevent shortness of breath, decreased exercise endurance, and early muscle fatigue in the subject.

The amount of drug (e.g., JTV-519) effective to prevent heart disease in a subject will vary with the particular factors of each case, including the type of heart disease, the weight of the subject, the severity of the disease in the subject, and the mode of administration of the drug (e.g., JTV-519). Such amounts are readily determined by those skilled in the art based upon well known procedures, including the clinical trials and methods disclosed herein. In one embodiment of the invention, the amount of the agent (e.g., JTV-519) effective to prevent exercise-induced arrhythmia is an amount of the agent (e.g., JTV-519) effective to prevent exercise-induced sudden cardiac death in the subject. In another embodiment, the medicament (e.g., JTV-519) prevents at least one heart disease (e.g., a cardiac arrhythmia (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmias), heart failure, or exercise-induced sudden cardiac death) in the subject.

JTV-519 can also be used to treat subjects who have already begun to develop clinical symptoms of heart disease due to the ability to stabilize RyR 2-bound FKBP12.6 and to maintain and restore the dynamic PKA phosphorylation and dephosphorylation equilibrium of RyR 2. JTV-519 may be effective in limiting or preventing further decreases in RyR 2-bound FKBP12.6 levels in subjects if symptoms of heart disease in the subject are well observed at an early stage.

Thus, in another embodiment of the invention, the subject has developed a heart disease. For example, the subject has performed exercise or is currently exercising and has experienced exercise-induced arrhythmia. In such cases, the amount of JTV-519 effective to limit or prevent a decrease in the level of RyR 2-bound FKBP12.6 in the subject may be an amount of JTV-519 effective to treat a cardiac disease (e.g., cardiac arrhythmia (e.g., tachycardia; atrial arrhythmia, including atrial tachyarrhythmia and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmia, including ventricular fibrillation; and exercise-induced arrhythmia) or heart failure) in the subject.

As used herein, an amount of a drug (e.g., JTV-519) that is "effective in treating a cardiac disease" includes an amount of a drug (e.g., JTV-519) that is effective in ameliorating or ameliorating a clinical lesion or symptom of a cardiac disease. For example, if the cardiac disease is atrial fibrillation, the amount of JTV-519 effective to treat atrial fibrillation may be an amount of JTV-519 effective to relieve or ameliorate palpitations and/or blood clots in the subject. Similarly, if the heart disease is exercise-induced arrhythmia, the amount of JTV-519 effective to treat the exercise-induced arrhythmia may be an amount of JTV-519 effective to alleviate or ameliorate exercise-induced palpitations, syncope, ventricular fibrillation, and ventricular tachycardia in the subject. Additionally, if the heart disease is heart failure, then the amount of JTV-519 effective to treat heart failure may be an amount of JTV-519 effective to alleviate or improve shortness of breath, decreased exercise endurance, and early muscle fatigue in the subject.

The amount of drug (e.g., JTV-519) effective to treat a cardiac disorder in a subject will vary with the particular factors of each case, including the type of cardiac disorder, the weight of the subject, the severity of the disorder in the subject, and the mode of administration of the drug (e.g., JTV-519). Such amounts are readily determined by those skilled in the art based upon well known procedures, including the clinical trials and methods disclosed herein. In a preferred embodiment, the medicament (e.g., JTV-519) treats at least one heart disease in a subject.

The invention further provides methods of treating at least one heart disease (e.g., cardiac arrhythmia (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmias) or heart failure) in a subject. The method includes administering to the subject JTV-519 in an amount effective to treat at least one heart disease in the subject. An appropriate amount of JTV-519 that is effective in treating heart disease (e.g., cardiac arrhythmias (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmias) or heart failure) in a subject may range from about 5 mg/kg/day to about 20 mg/kg/day, and/or may be an amount sufficient to achieve a plasma level of about 300ng/ml to about 1000 ng/ml.

The invention also provides methods of preventing at least one heart disease (e.g., arrhythmia (e.g., tachycardia; atrial arrhythmia, including atrial tachyarrhythmia and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmia, including ventricular fibrillation; and exercise-induced arrhythmia), heart failure, or exercise-induced sudden cardiac death) in a subject. The method includes administering to the subject JTV-519 in an amount effective to treat at least one heart disease in the subject. An appropriate amount of JTV-519 effective to treat heart disease (e.g., cardiac arrhythmias (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmias), heart failure, or exercise-induced sudden cardiac death) in a subject may be in the range of about 5 mg/kg/day to about 20 mg/kg/day, and/or may be an amount sufficient to achieve a plasma level of about 300ng/ml to about 1000 ng/ml.

In various embodiments of the above methods, the exercise-induced arrhythmia in the subject is associated with VT. In a preferred embodiment, VT is CPVT. In other embodiments of these methods, the subject is a candidate for exercise-induced arrhythmia, including a candidate for exercise-induced sudden cardiac death.

In addition, according to the above method, the present invention also provides JTV-519 for use in a method of limiting or preventing a decrease in the level of RyR 2-bound FKBP12.6 in a subject suffering from or being a candidate for at least one cardiac disease (e.g., cardiac arrhythmias (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmias), heart failure, or exercise-induced sudden cardiac death). The invention also provides for the use of JTV-519 in a method of treating or inducing at least one heart disease (e.g., cardiac arrhythmia (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmia), heart failure, or induction-induced sudden cardiac death) in a subject.

Novel screening method

As described above and provided herein, the present inventors' data demonstrate that protein kinase a (pka) phosphorylation of the cardiac ryanodine receptor RyR2 at serine 2809 activates the channel by releasing the FK506 binding protein FKBP 12.6. In defective hearts (including human hearts and animal models of heart failure), RyR2 is PKA-hyperphosphorylated, yielding a defective channel with reduced amounts of bound FKBP12.6 and with increased sensitivity to calcium-induced activation. The net result of these changes is that the RyR2 channel "leaks". These channel leakages can result in the loss of intracellular storage of calcium to the point where sufficient calcium is no longer present in the Sarcoplasmic Reticulum (SR), thereby providing a strong stimulus for muscle contraction. This may result in myocardial contraction. As a second consequence of channel leakage, RyR2 channels release calcium during the resting phase of the cardiac cycle, called the "diastolic phase". This calcium release during diastole can trigger cardiac fatal arrhythmias (e.g., ventricular tachycardia and ventricular fibrillation) that lead to Sudden Cardiac Death (SCD).

The present inventors have also demonstrated that the treatment of heart failure using a mechanical pumping device called a Left Ventricular Assist Device (LVAD) that puts the heart in a resting and recovering state is associated with a decrease in PKA hyperphosphorylation of RyR2 and can calibrate channel function. Furthermore, the present inventors have demonstrated that treatment of dogs (with pulsatile induced heart failure) with β adrenergic blockers (β blockers) can reverse PKA hyperphosphorylation of RyR 2. Beta blockers inhibit the pathway for activating PKA. The conclusion that can be drawn from the working results of the present inventors is that PKA phosphorylation of RyR2 increases the activity of the channel, resulting in more calcium release into cells that specify the channel trigger (activator).

As further disclosed herein, the present inventors have established that cardiac diseases (e.g., cardiac arrhythmias (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmias), heart failure, or exercise-induced sudden cardiac death) are associated with increased phosphorylation of RyR2 protein (particularly CPVT-related RyR2 mutant protein) and decreased levels of RyR 2-bound FKBP 12.6. This mechanism can be used to design effective drugs for the treatment and prevention of such heart diseases. Candidate agents having the ability to limit or prevent a decrease in the level of RyR 2-bound FKBP12.6 have an effect on RyR 2-related biological events as a result of such limiting or preventing activity, thereby treating or preventing such heart diseases.

Accordingly, the present invention further provides methods of identifying an agent for treating or preventing at least one heart disease (e.g., cardiac arrhythmia (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmias), heart failure, or exercise-induced sudden cardiac death). The method comprises the following steps: (a) obtaining or producing a cell culture comprising RyR 2; (b) contacting the cell with a candidate agent; (c) contacting the cell with one or more conditions known to increase phosphorylation of RyR2 in the cell; and (d) determining whether the agent can limit or prevent a decrease in the level of RyR 2-bound FKBP12.6 in the cell.

As used herein, "active agent" shall include proteins, polypeptides, peptides, nucleic acids (including DNA or RNA), antibodies, Fab fragments, F (ab')₂Fragments, molecules, compounds, antibiotics, drugs, and any combination thereof. The agent that limits or prevents the reduction in the level of RyR 2-bound FKBP12.6 may be natural or synthetic and may be an agent that reacts with (i.e., has an affinity for, binds to, or is directed to) RyR2 and/or FKBP 12.6. As further used herein, a cell "containing RyR 2" is a cell (preferably a cardiomyocyte) in which RyR2 or a derivative or homologue thereof is naturally expressed or naturally occurring. Conditions known to increase phosphorylation of RyR2 in cells include, but are not limited to, PKA.

In the methods of the invention, any standard method of contacting a drug/active agent with a cell can be provided for contacting the cell with a candidate active agent, including any mode of introduction and administration disclosed herein. The level of RyR 2-bound FKBP12.6 in a cell can be determined or detected by well-known procedures, including any of the methods, molecular procedures, and assays well known to those of skill in the art or described herein. In one embodiment of the invention, the agent limits or prevents a decrease in the level of RyR 2-bound FKBP12.6 in the cell.

As disclosed herein, RyR2 is involved in many biological events (events) in striated muscle cells. For example, the RyR2 channel has been shown to play an important role in EC coupling and contractility in cardiomyocytes. Thus, it is apparent that therapeutic or prophylactic agents designed to limit or prevent a decrease in the level of RyR 2-bound FKBP12.6 in a cell may be used to modulate a number of RyR 2-related biological events, including EC coupling and contractility. Thus, once candidate agents of the invention have been screened and determined to have a suitable limiting or preventing effect on the reduction in the level of RyR 2-bound FKBP12.6, their effect on EC coupling and contractility in cells can be assessed. It is contemplated that the therapeutic/prophylactic agents of the present invention may be used to treat or prevent cardiac disease, including cardiac arrhythmias (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmias), heart failure, and induction-induced sudden cardiac death.

Thus, the method of the present invention may further comprise the steps of: (e) contacting a candidate agent with a cell culture comprising RyR 2; and (f) determining whether the agent has an effect on an RyR 2-related biological event in the cell. As used herein, "RyR 2-related biological events" include biochemical or physiological processes involving RyR2 levels or activity. As disclosed herein, examples of RyR 2-related biological events include, but are not limited to, EC coupling and contractility in cardiomyocytes. According to the methods of the invention, candidate agents may be contacted with one or more cells (preferably cardiomyocytes) in vitro. For example, a cell culture can be incubated with a preparation containing a candidate active agent. The effect of the candidate agent on RyR 2-related biological events can then be evaluated by any biological assay or method known in the art, including immunoblotting, single channel recording, and any other method disclosed herein.

The invention further relates to an active agent identified by the above-described identification method and a pharmaceutical composition comprising the active agent and a pharmaceutically acceptable carrier. The agents can be used to prevent exercise-induced sudden cardiac death in a subject and to treat or prevent other RyR 2-related conditions. As used herein, "RyR 2-related conditions" are conditions, diseases or disorders that involve levels or activity of RyR2 and include RyR 2-related biological events and heart disease (e.g., cardiac arrhythmias (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmias), heart failure, or exercise-induced sudden cardiac death).

RyR 2-related conditions in a subject can be treated or prevented by administering to the subject an amount of the active agent effective to treat or prevent RyR 2-related conditions in the subject. This amount is readily determined by one skilled in the art. In one embodiment, the invention provides a method for treating or preventing at least one heart disease (e.g., cardiac arrhythmias (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both persistent and non-persistent); ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmias), heart failure, or exercise-induced sudden cardiac death) in a subject by administering to the subject an amount of the active agent effective to treat or prevent the at least one heart disease in the subject.

The invention also provides in vivo methods of identifying an agent for treating or preventing a cardiac disease (e.g., cardiac arrhythmia (e.g., tachycardia; atrial arrhythmia, including atrial tachyarrhythmia and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmia, including ventricular fibrillation; and exercise-induced arrhythmia), heart failure, or exercise-induced sudden cardiac death). The method comprises the following steps: (a) obtaining or producing an animal containing RyR 2; (b) administering a candidate active agent to the animal; (c) contacting the animal with one or more conditions known to increase phosphorylation of RyR2 in the cell; and (d) determining whether the agent can limit or prevent a decrease in the level of RyR 2-bound FKBP12.6 in the animal. The method further comprises the steps of: (e) administering said agent to an animal containing RyR 2; and (f) determining whether the agent has an effect on an RyR 2-related biological event in the animal. Also provided are active agents identified by the method; pharmaceutical compositions comprising the active agent; and methods for treating or preventing at least one heart disease (e.g., cardiac arrhythmias (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmias), heart failure, or exercise-induced sudden cardiac death) in a subject by administering to the subject an amount of the active agent effective to treat or prevent the at least one heart disease in the subject.

Work by the present inventors has demonstrated that compounds that block activation of PKA activation can be expected to reduce activation of the RyR2 channel, resulting in this lesser release into cells. Compounds that are expected to bind to the RyR2 channel at the FKBP12.6 binding site, but do not leave the channel when phosphorylated by PKA, can reduce channel activity in response to PKA activation or other triggers that activate the RyR2 channel. Such compounds may also result in less release into cells. Based on these findings, the present invention further provides additional tests to identify compounds that may be used to treat or prevent cardiac disease (e.g., arrhythmias (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmias), heart failure, or exercise-induced sudden cardiac death) because they may block or inhibit RyR2 activation.

As an example, the diagnostic assays of the invention can use calcium sensitive fluorescent dyes (e.g., Fluo-3, Fura-2, etc.) to screen for the release of calcium into cells via the RyR2 channel. Cells can be loaded with a selected fluorescent dye and then stimulated with RyR2 activator to determine whether compounds added to the cells can attenuate calcium-dependent fluorescence signals (Brillants et al, "FK 506-binding protein stabilizes calcium release channel (ryanodine receptor) function" Cell, 77: 513-23, 1994; Gillo et al, "calcium entry during differentiation induced in murine erythroleukemia cells," Blood, 81: 783-92, 1993; Jayaraman et al, "modulation of inositol 1, 4, 5-triphosphate receptor by tyrosine phosphorylation," Science, 272: 1492-94, 1996). The calcium-dependent fluorescence signal can be monitored using a photomultiplier tube as described above and analyzed using appropriate software (Brillants et al, "FK 506-binding protein stabilizes calcium release channel (ryanodine receptor) function", Cell, 77: 513-23, 1994; Gillo et al, "calcium entry during differentiation induced in murine erythroleukemia cells", Blood, 81: 783-92, 1993; Jayaraman et al, "Regulation of inositol 1, 4, 5-triphosphate receptor by tyrosine phosphorylation" -Science, 272: 1492-94, 1996). The assay is easily automated to screen large numbers of compounds using multi-well dishes.

To identify compounds that inhibit PKA-dependent activation of RyR 2-mediated intracellular calcium release, assays may include expression of recombinant RyR2 channels in heterologous expression systems, such as Sf9, HEK293, or CHO cells (Brillants et al, "FK 506-binding protein stabilizes calcium release channel (ryanodine receptor) function", Cell, 77: 513-23, 1994). RyR2 may also be co-expressed with the β -adrenergic receptor. This allows the evaluation of the effect of compounds on RyR2 activation as a response to the addition of β -adrenergic receptor agonists.

The level of RyR2 PKA phosphorylation associated with heart failure extent can also be measured and then used to determine the efficacy of compounds designed to block RyR2 channel PKA phosphorylation. Such assays may be based on the use of antibodies specific for the RyR2 protein. For example, RyR 2-channel proteins can be co-immunoprecipitated and then PKA and [ γ ] can be used³²P]-ATP reverse-phosphorylation. The radioactivity transferred to the RyR2 protein can then be determined using a photoimager³²P]The amount of label (Marx et al, "PKA phosphorylation dissociating FKBP12.6 from calcium release channels (ryanodine receptors): defective regulation in defective heart", Cell, 101: 365-76, 2000).

Another assay of the invention involves the use of a phosphorylated epitope-specific antibody to detect PKA phosphorylated RyR2 on Ser 2809. Immunoblotting using such antibodies can be used to evaluate the efficacy of therapy in heart failure and arrhythmias. In addition, RyR2S 2809A and RyR2S 2809D knockout mice can be used to evaluate the efficacy of therapy in heart failure and arrhythmias. Such mice further provide evidence that PKA hyperphosphorylation of RyR2 contributes to heart failure and arrhythmia by exhibiting that the RyR2S 2809A mutation suppresses heart failure and arrhythmia and that the RyR2S 2809D mutation exacerbates heart failure and arrhythmia.

1, 4-benzothiazepinesClass of derivatives and synthesis method thereof

1, 4-benzothiazepinesDerivatives of the class, in particular 2, 3,4, 5-tetrahydro-1, 4-benzothiazepinesThe derivatives are important building blocks for preparing bioactive molecules, including JTV-519. The inventors have developed a process for the preparation of 1, 4-benzothiazepinesIntermediate compounds, such as 7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepineAnd (3) a new method of class. The process of the present inventors uses key 1, 4-benzothiazepines which are available and inexpensive as starting materials and provide high yieldsAnd (c) a class of intermediates.

In the early 90 s of the last century, Kaneko et al (U.S. Pat. No. 5, 5,416,066; WO 92/12148; JP4230681) disclosed that 7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepine can be prepared by reactingClass (1, 4-benzothiazepines)Intermediate) with acryloyl chloride and then reacting the resulting product with 4-benzylpiperidine to prepare JTV-519.

The use for the preparation of 7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepines has previously been reported in the literatureTwo methods for analogous and analogous compounds. The first method disclosed by Kaneko et al (U.S. Pat. No. 5,416,066) involves a 6-step synthetic route using 2, 5-dihydroxybenzoic acid as starting material. In this process, dimethyl sulfate is used to selectively methylate 2, 5-dihydroxybenzoic acid. The resulting compound was then reacted with dimethylthiocarbamoyl chloride for 20 hours and then subjected to high temperature (270 ℃) for 9 hours. The product of this step was refluxed with sodium methoxide in methanol for 20 hours. The product of the reflux step is then reacted with 2-chloroethylamine under basic conditions and at elevated temperature to give the cyclic amide. By LiAlH₄Reduction of the cyclized amide to give 7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepineClass (1, 4-benzothiazepines)A class of intermediates).

Hitoshi in Japanese patent (JP 10045706) discloses the preparation of 7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepineClass second method. The method takes 2-bromine-5-methoxybenzaldehyde as a raw material. The bromide was replaced with NaSMe and the resulting product was oxidized with chlorine, followed by reflux in water to give the disulfide dialdehyde. Treating the dialdehyde with 2-chloroethylamine and reducing agent such as NaBH₄Reducing the obtained product. Cyclizing the resulting compound to obtain7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepineAnd (4) class.

Initially, the present inventors attempted to prepare 1, 4-benzothiazepines using the above-described methodThe intermediate 7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepineAnd (4) class. However, they found that the first method described by Kaneko et al (U.S. Pat. No. 5,416,066) involves a synthesis step at high temperature and long reaction time. In addition, the inventors have found that the thio group in the third thiolated intermediate is readily air-oxidized to a disulfide compound, making synthesis of subsequent cyclization products impossible. The method of the present inventors also substituted Hitoshi (JP 10045706) includes Cl₂And another proprietary method of preparing the first intermediate had to be used to avoid the use of NaSMe instead of bromide.

In order to overcome the above problems, the present inventors have developed a method for preparing 7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepine from easily available and inexpensive raw materialsAnd (3) a new method of class. The inventors' method simplifies the separation and purification steps and can be used to prepare a variety of 1, 4-benzothiazepinesIntermediates, including 7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepinesClasses and other compounds having the general structure shown in the following general formula:

r1 ═ n-MeO, n-MeS, n-alkyl, n ═ 6, 7, 8, 9

R2 ═ alkyl

R3 ═ alkyl

The process may also be used to prepare JTV-519.

Thus, in light of the above, the present invention provides a process for the synthesis of a compound having the general formula:

wherein R ═ OR ', SR ', NR ', alkyl OR halide and R ═ alkyl, aryl OR H, and wherein R may be in the 2, 3,4 OR 5 position, the process comprising the steps of:

(a) treating a compound having the formula:

wherein R is as defined above, to produce a compound having the formula:

wherein R is as defined above;

(b) treating the compound formed in step (a) with a diazotizing agent and a disulfide to produce a compound having the general formula:

wherein R is as defined above;

(c) treating the compound formed in step (b) with an activating agent and chloroethylamine to produce a compound having the general formula:

wherein R is as defined above;

(d) treating the compound formed in step (c) with a reducing agent and a base to form a compound having the general formula:

wherein R is as defined above; and

(e) treating the compound formed in step (d) with a reducing agent to form a compound having the general formula:

wherein R is as defined above.

According to the process of the present invention, the reducing agent in step (a) may be H₂. In addition, the diazotizing agent in step (b) may be NaNO₂And the disulfide in step (b) may be Na₂S₂. Further, the chloride in step (c)May be SOCl₂. The reducing agent in step (d) may be trimethophosphine (Pme)₃) And the base in step (d) is triethylamine. In another embodiment, the reducing agent in step (e) is LiAlH₄。

The invention further provides a process for the synthesis of a compound having the general formula:

(a) treating a compound having the formula:

wherein R is as defined above, said one compound having the formula:

thereby forming a compound having the general formula:

wherein R is as defined above.

As an example, a compound having the following general formula may be synthesized as follows:

wherein R ═ OR ', SR ', NR ', alkyl OR halide and R ═ alkyl, aryl OR H, and wherein R may be located in the 2, 3,4 OR 5 positions:

r ═ OR ', SR ', NR ', alkyl, halide; r' ═ alkyl, aryl, HR can be in position2. 3,4 or 5 position

By way of example and as shown in example 9 and scheme 1 below, 7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepine may be prepared from 2-nitro-5-methoxybenzoic acid as followsAnd (4) class. Using H₂And Pd/C as a catalyst to reduce the nitro group of 2-nitro-5-methoxybenzoic acid to give 2-amino-5-methoxybenzoic acid. NaNO can be used₂Diazotising 2-amino-5-methoxybenzoic acid and then reacting with Na₂S₂The treatment results in stable di-sulphide compounds. Without further purification, SOCl may be used₂Treating the stabilized disulfide compound and then reacting it with 2-chloroethylamine in the presence of Et₃In the presence of N to give an amide. The amide compound can then be converted to the cyclized compound by the following one-pot procedure. A reducing agent such as trimethophosphine or triphenylphosphine and a base such as triethylamine may be added to the amide compound in THF (tetrahydrofuran)In the solution of (1). The resulting reaction mixture was then refluxed for 3 hours. The reducing agent (trimetaphosphine or triphenylphosphine) cleaves the disulfide (S-S) to the monosulfide (-S) and intramolecular cyclization is carried out in situ using the chloride to give the cyclized amide. LiAlH can then be used₄Reduction of the cyclized amide to give the 1, 4-benzothiazepineThe intermediate 7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepineAnd (4) class. This can then be done by reacting 7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepineBy reaction of a compound with 3-bromopropionyl chloride and reaction of the resulting compound with benzylpiperidine from 7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepinePreparation of JTV-519.

The invention further provides a composition comprising radiolabeled JTV-519. JTV-519 may be labeled using a variety of different radiolabels known in the art. For example, the radiolabel of the invention may be a radioisotope. The radioisotope can be any isotope that emits detectable radiation, including but not limited to³⁵S、¹²⁵I、³H or¹⁴C. The radioactivity emitted by the radioisotope can be detected by techniques well known in the art. For example, gamma emission from a radioisotope can be detected using gamma imaging techniques, particularly scintigraphic imaging.

By way of example and as shown in example 10 and scheme 2 below, radiolabeled JTV-519 may be prepared as follows. BBr can be used₃JTV-519 is demethylated on the phenyl ring. Then using a radiolabeled methylating agent (such as³H-dimethyl sulfate)Obtained phenol compound is remethylated in the presence of a base (such as NaH) to obtain³H-labeled JTV-519.

The invention further provides novel 1, 4-benzothiazepinesIntermediates and derivatives, including 2, 3,4, 5-tetrahydro-1, 4-benzothiazepines, analogous to JTV-519And (4) class. By way of example, the present invention provides a compound having the general formula:

wherein R is aryl, alkenyl, alkyl, (CH)₂)_nNR′₂Or- (CH)₂)_nSR ', and n is 0, 1, 2 or 3, and R' is alkyl or cycloalkyl;

wherein R ═ CO (CH)₂)_nXR′₂、SO₂(CH₂)_nXR′₂Or SO₂NH(CH₂)_nXR’₂And X ═ N or S, and N ═ 1, 2 or 3, andr ═ alkyl or cycloalkyl; and wherein m is 1 or 2; and

wherein R is aryl, alkyl, - (CH)₂)_nNR′₂、-(CH₂)_nSR ', and n is 0, 1, 2 or 3, and R' is alkyl or cycloalkyl; and wherein X ═ NH or O. Also provided are additional 2, 3,4, 5-tetrahydro-1, 4-benzothiazepines having the formulaClass (c):

wherein R is₁OR ', SR', NR ', alkyl OR halide at the 2, 3,4 OR 5 position on the phenyl ring, and R' ═ alkyl, aryl OR H; wherein R is₂H, alkyl or aryl; and wherein R₃H, alkyl or aryl;

wherein R is₁H, OR ', SR', NR ', alkyl or halide at the 2, 3,4 or 5 position on the phenyl ring, and R' ═ alkyl, aryl or acyl; wherein R is₂H, alkyl, alkenyl or aryl;

wherein R is₃H, alkyl, alkenyl or aryl; wherein m is 0, 1 or 2; and wherein n is 0 or 1; and

wherein R1 ═ H, OR ', SR ', NR ', alkyl or halide at the 2, 3,4 or 5 position on the phenyl ring, and R ═ alkyl, aryl or acyl; wherein R is₂H, alkyl, alkenyl or aryl;

wherein R is₃H, alkyl, alkenyl or aryl; wherein R is₄H, halide, alkenyl, carboxylic acid or alkyl containing O, S or N; and wherein m is 0, 1 or 2.

The novel 1, 4-benzothiazepines of the present inventorsExamples of such compounds include, but are not limited to, S7, S-20, S-25, S-27 and S36. Preferably the compound is S36.

The structures of S7, S-20, S-25, S-27, and S36 can be found in FIG. 15 of the present application. These and any other novel compounds of the present invention may be combined with a pharmaceutically acceptable carrier as described above to form a pharmaceutical composition.

The invention further provides for the synthesis of the novel 1, 4-benzothiazepines disclosed hereinA method for preparing the compound. For example, the invention provides a process for the synthesis of a compound having the general formula:

wherein R is aryl, alkenyl, alkyl, - (CH)₂)_nNR′₂Or- (CH)₂)_nSR ', and n is 0, 1, 2 or 3, and R' is alkyl or cycloalkyl, comprising the following steps:

(a) treating a compound having the formula:

thereby producing a compound having the general formula:

(b) optionally treating the compound formed in step (a) with a primary or secondary amine to produce a compound having the general formula:

wherein R is as defined above. In one embodiment, the sulfonyl chloride compound in step (a) is selected from the group consisting of alkyl sulfonyl chlorides and aryl sulfonyl chlorides. In another embodiment, the base in step (a) is Et₃And N is added. In another embodiment, the primary or secondary amine in step (b) is 4-benzylpiperidine.

The method of the present invention may further comprise the step of oxidizing a compound having the general formula:

wherein R is aryl, alkenyl, alkyl, - (CH)₂)_nNR′₂Or- (CH)₂)_nSR ', and n is 0, 1, 2 or 3, and R' is alkyl or cycloalkyl, to give a compound having the general formula:

wherein R is as defined above, and wherein m ═ 1 or 2. In one embodiment of the invention, the oxidizing agent is hydrogen peroxide.

As an example and as shown in example 11 and scheme 3, the inventors have developed a method by which compounds having the following structure can be synthesized:

wherein R is aryl, alkenyl, alkyl, - (CH)₂)_nNR′₂Or- (CH)₂)_nSR ', and n is 0, 1, 2 or 3, and R' is alkyl or cycloalkyl. Can be prepared by reacting 7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepineWith alkylsulphonyl or arylsulphonyl chlorides in the presence of bases such as Et₃Reacting in the presence of N to prepare the novel compound with the general structure. Additional reactions (e.g., addition of 4-benzylpiperidine) may then be performed to extend the side chain as desired. As shown in scheme 3, 2-chloroethanesulfonyl chloride (e.g. 180 mg; 1.1mM) and Et can be reacted at 0 deg.C₃N (e.g. 140 mg; 1.1mM) is added to CH₂Cl₂7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepine (e.g. 20ml)Class (1) (e.g., 194 mg; 1 mM). The mixture is then stirred (e.g. at 0 ℃ for 2 hours) and washed (e.g. with H)₂O and saturated NaHCO₃A solution). The solvent is removed to give a crude product, which can be purified by silica gel chromatography. The structure was confirmed by NMR. Scheme 3 further shows that this compound can be prepared by reacting(e.g., 28 mg; 0.1mM) with a base in CH₂Cl₂The 4-benzylpiperidine (e.g., 21 mg; 0.13mM) in (A) is reacted to extend the side chain of the resulting compound. After the reaction is complete, excess amine can be removed with a base scavenger (e.g., 3- (2-succinic anhydride) propyl-functionalized silica gel, 0.5 g).

The present invention also provides a process for the synthesis of a compound having the general formula:

wherein R is aryl, alkyl, - (CH)₂)_nNR₂Or- (CH)₂) SR ', and n is 0, 1, 2 or 3, and R' is alkyl or cycloalkyl, which process comprises treating a compound having the formula:

thereby producing a compound having the formula

Wherein R is as defined above. In one embodiment of the invention, the base is Et₃And N is added. In another embodiment, the primary or secondary amine is 1-piperonyl piperidine.

wherein R is aryl, alkyl, - (CH)₂)_nNR′₂Or- (CH)₂)_nSR ', and n is 0, 1, 2 or 3, and R' is alkyl or cycloalkyl, to give a compound having the general formula:

wherein R is as defined above, and wherein m ═ 1 or 2. In one embodiment, the oxidizing agent is hydrogen peroxide.

As an example and as shown in example 11 and scheme 4, the present inventors developed a method of synthesizing a compound having the general structure:

wherein R is aryl, alkyl, - (CH)₂)_nNR′₂Or- (CH)₂)_nSR ', and n is 0, 1, 2 or 3, and R' is alkyl or cycloalkyl. Can be prepared by reaction with a base (e.g., Et)₃N) in the presence of 7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepineThe novel compounds of this general structure are prepared by one-pot reaction of the compounds with sulfonyl chlorides, followed by a primary or secondary amine. As shown in scheme 4, sulfonyl chloride (e.g., 15.0 mg; 0.111mM) and Et can be reacted at 0 deg.C₃N (e.g. 28.0 mg; 0.22mM) is added to the solution in CH₂Cl₂7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepine (e.g. 20ml)Class (e.g., 19.4 mg; 0.1 mM). In stirring theAfter the mixture (e.g., at 0 ℃ for 2 hours), 1-piperonylpiperazine (e.g., 27 mg; 0.12mM) can be added. The compound is stirred for a further 2 hours and then washed (for example with H)₂O and saturated NaHCO₃A solution). Excess amine can be removed by addition of a base scavenger (e.g., 3- (2-succinic anhydride) propyl functional silica gel, 0.2 g).

wherein R ═ CO (CH)₂)_nXR′₂、SO₂(CH₂)_nXR′₂Or SO₂NH(CH₂)_nXR′₂And X ═ N or S, and N ═ 1, 2, or 3, and R' ═ alkyl or cycloalkyl; and wherein m is 1 or 2, comprising the step of treating a compound having the formula:

wherein R is as defined above, to produce a compound having the formula:

wherein R and m are as defined above. In one embodiment, the oxidizing agent is hydrogen peroxide. The process may also be used to oxidize JTV-519.

thereby producing a compound having the general formula:

As an example and as shown in example 11 and scheme 5, the present inventors developed a method of synthesizing a compound having the general structure:

wherein R ═ CO (CH)₂)_nXR′₂、SO₂(CH₂)_nXR′₂Or SO₂NH(CH₂)_nXR′₂And X ═ N or S, and N ═ 1, 2, or 3, and R' ═ alkyl or cycloalkyl; and wherein m is 1 or 2. Can be made by usingOxidation of JTV-519 by hydrogen peroxide or the novel 1, 4-benzothiazepines disclosed hereinOne of the derivatives prepares the novel compound with the general structure. As shown in scheme 5, the 1, 4-benzothiazepine of interest may be dissolved in MeOH (e.g., 5ml)Such compounds (e.g. 21 mg; 0.05mM) are added to H₂O₂(e.g., 0.1ml, excess). The mixture can be stirred (e.g., for 2 days) and chromatographed on silica gel (e.g., CH)₂Cl₂OH 10: 1) purifying the obtained product.

In addition, the present invention provides a method for synthesizing a compound having the general formula:

wherein R is aryl, alkyl, - (CH)₂)_nNR′₂Or- (CH)₂)_nSR ', and n is 0, 1, 2 or 3, and R' is alkyl or cycloalkyl; and wherein X ═ NH or O, comprising the step of treating a compound having the general formula:

thereby producing a compound having the general formula:

wherein R andx is as defined above. In one embodiment, the carbonyl chloride compound is triphosgene. In another embodiment, the base is Et₃And N is added. In another embodiment, the primary or secondary amine is 4-benzylpiperidine.

As an example and as shown in example 11 and scheme 6, the inventors developed a method of synthesizing a compound having the general structure:

wherein R is aryl, alkyl, - (CH)₂)_nNR′₂、-(CH₂)_nSR ', and n is 0, 1, 2 or 3, and R' is alkyl or cycloalkyl; and wherein X ═ NH or O. Can be prepared by reacting 7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepineWith triphosgene in the presence of a base (e.g. Et)₃N) in the presence of a primary or secondary amine or an alcohol, followed by addition of a primary or secondary amine or an alcohol.

The invention further provides processes for the synthesis of 2, 3,4, 5-tetrahydro-1, 4-benzothiazepines having the general formulaThe method for preparing the compound comprises the following steps:

wherein R is₁OR ', SR', NR ', alkyl OR halide in the 2, 3,4 OR 5 position on the phenyl ring, and R' ═ alkyl, aryl OR H; wherein R is₂H, alkyl or aryl; and wherein R₃H, alkyl or aryl, the process comprising the steps of:

(a) treating a compound having the formula:

wherein R is₁As defined above, to produce a compound having the general formula:

wherein R is₁As defined above;

wherein R is₁、R₂And R₃As defined above;

and

wherein R is₁、R₂And R₃As defined above.

Using novel 1, 4-benzothiazepines Therapeutic and prophylactic methods for the class of derivatives

The novel 1, 4-benzothiazepines of the present inventorsThe compounds share functional characteristics with JTV-519. For example, compound S36(mwt ═ 267) modulates calcium channels such as JTV-519(mwt ═ 423). In fact, S36 (a carboxylic acid) was about 910 times more potent in modulating calcium channels than JTV-51910 (data not shown). However, unlike JTV-519, the novel compounds of the present inventors exhibit weak hERGs blocking activity.

Fast delay rectification (i (kr)) channel-potassium channel-is important for repolarization of cardiac action potentials. HERG is the pore-forming subunit of the I (Kr) channel. I (kr) functional inhibition-as a result of adverse drug action and/or genetic defects in hERG-can produce long-term-qt (lqt) syndrome, which carries an increased risk of fatal cardiac arrhythmias. HERGs are then potassium channel subunits, which when blocked can lead to arrhythmias and sudden cardiac death.

The blocking effect of our compounds on the hERG (I (Kr)) channel is significantly reduced when compared to JTV-519. For example, as shown in FIGS. 4-7, one of the compounds of the present inventors, S36, has approximately 5 to 10 times less hERG blocking activity than JTV-519. Since the compounds of the present inventors have weak hERG blocking activity, their toxicity is expected to be lower than JTV-519.

Based on the above, the present inventors have found that 1, 4-benzothiazepinesThe compounds have higher efficacy than JTV-519 and reduced toxicity. Accordingly, the novel compounds of the present inventors are believed to be particularly useful in any of the above-described methods of limiting or preventing a decrease in the level of RyR 2-bound FKBP12.6 in a subject, including subjects having or being a candidate for at least one cardiac disease, including, but not limited to, cardiac arrhythmias (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmias), heart failure, and exercise-induced sudden cardiac death. The compounds of the present inventors are also considered to be particularly useful in methods of treating or preventing such heart diseases in a subject.

Thus, the invention further provides a method for reducing the level of RyR 2-bound FKBP12.6 in a subject by administering to the subject an amount of an active agent effective to limit or prevent a reduction in the level of RyR 2-bound FKBP12.6 in the subject. The active agent of the invention may be any 1, 4-benzothiazepineDerivatives of the class include the following compounds:

wherein R1 ═ H, OR ', SR ', NR ', alkyl, or halide at the 2, 3,4, or 5 positions on the phenyl ring, and R ═ alkyl, aryl, or acyl; wherein R2 ═ H, alkyl, alkenyl, or aryl; wherein R3 ═ H, alkyl, alkenyl, or aryl; wherein m is 0, 1 or 2; and wherein n is 0 or 1;

wherein R1 ═ H, OR ', SR', NR ', alkyl or halide in the 2, 3,4, or 5 position on the phenyl ring, and R' ═ alkyl, aryl or acyl; wherein R2 ═ H, alkyl, alkenyl, or aryl; wherein R3 ═ H, alkyl, alkenyl, or aryl; wherein R4 ═ H, halide, alkenyl, carboxylic acid, or alkyl containing O, S or N; and wherein m is 0, 1 or 2; and

(h) an oxidized form of any of (a) - (g) above. Also provided are uses of these agents in methods of limiting or preventing a decrease in the level of RyR 2-bound FKBP12.6 in a subject. In one embodiment of the invention, the active agent is selected from S4, S7, S-20, S-24, S-25, S-26, S-27 and S36. The structures of these active agents can be found in figure 15. Preferably, the active agent is S36.

As mentioned above, the subject may be any animal, but is preferably a human. In one embodiment, the subject has Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). In another embodiment, the subject has or is a candidate for at least one cardiac disease including, but not limited to, cardiac arrhythmias (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both persistent and non-persistent ventricular arrhythmias), ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmias), heart failure, and exercise-induced sudden cardiac death.

In the process of the invention, 1, 4-benzothiazepines may be reacted as described aboveThe class of derivatives is administered to a subject as part of a therapeutic composition comprising the derivative and a pharmaceutically acceptable carrier. The derivative or pharmaceutical composition can be administered to a subject by any technique known in the art and/or disclosed herein.

According to the process of the invention, 1, 4-benzothiazepinesThe class of derivatives is administered to a subject (and can result in 1, 4-benzothiazepine) in an amount effective to limit or prevent a decrease in the level of RyR 2-bound FKBP12.6 in a subjectThe class derivative is contacted with the subject cell). Such amounts are readily determined by one skilled in the art based on well known procedures, including established in vivo titration curve analysis and the methods and assays disclosed herein. Effective in limiting or preventing a decrease in the level of RyR 2-bound FKBP12.6 in a subjectSuitable amounts of the derivative may range from about 5 mg/kg/day to about 20 mg/kg/day, and/or may be sufficientAmounts to achieve plasma levels of about 300ng/ml to about 1000 ng/ml. Preference is given to 1, 4-benzothiazepinesThe amount of the derivative is about 10 mg/kg/day to about 20 mg/kg/day.

According to the methods of the invention, a decrease in the level of RyR 2-bound FKBP12.6 in a subject can be limited or prevented by decreasing the level of phosphorylated RyR2 in the subject. In one embodiment of the invention, the subject has not developed a cardiac disease (cardiac condition), such as an arrhythmia (e.g., tachycardia; atrial arrhythmia, including atrial tachyarrhythmia and atrial fibrillation (both persistent and non-persistent); ventricular arrhythmia, including ventricular fibrillation; and exercise-induced arrhythmia), heart failure, or exercise-induced sudden cardiac death. In this case, 1, 4-benzothiazepine, which is effective to limit or prevent a decrease in the level of RyR 2-bound FKBP12.6 in a subjectThe amount of the class of derivatives can be 1, 4-benzothiazepine effective to prevent heart disease (e.g., arrhythmia, heart failure, or exercise-induced sudden cardiac death) in the subjectThe amount of the derivative is used. In one embodiment, the 1, 4-benzothiazepineThe class of derivatives prevents at least one heart disease (e.g., cardiac arrhythmia (e.g., tachycardia; atrial arrhythmia, including atrial tachyarrhythmia and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmia, including ventricular fibrillation; and exercise-induced arrhythmia), heart failure, or exercise-induced sudden cardiac death) in a subject.

In another embodiment of the invention, the subject has developed a heart disease. In this case, effectively limit orPrevention of decreased levels of RyR 2-bound FKBP12.6 in a subject 1, 4-benzothiazepineThe amount of the class of derivatives can be an amount of 1, 4-benzothiazepine effective to treat a cardiac disease (e.g., cardiac arrhythmia (e.g., tachycardia; atrial arrhythmia, including atrial tachyarrhythmia and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmia, including ventricular fibrillation; and exercise-induced arrhythmia) or heart failure) in a subjectAmount of the derivative of the class. In a preferred embodiment, JTV-519 treats at least one heart disease in a subject.

The invention further provides a method for treating or preventing a cardiac disease (e.g., cardiac arrhythmia (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmias), heart failure, or exercise-induced sudden cardiac death) in a subject comprising administering to the subject an amount of an active agent effective to limit or prevent a decrease in the level of RyR 2-bound FKBP12.6 in the subject. The active agent of the invention may be any 1, 4-benzothiazepineDerivatives of the class include the following compounds:

(h) an oxidized form of any of (a) - (g) above.

The invention further provides methods of treating at least one heart disease (e.g., cardiac arrhythmia (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmias) or heart failure) in a subject. The method comprises administering to the subject an amount of 1, 4-benzothiazepine effective to treat at least one heart disease in the subjectA derivative of the class. 1, 4-benzothiazepines effective in treating heart disease (e.g., cardiac arrhythmias (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmias) or heart failure) in a subjectSuitable amounts of the class of derivatives may range from about 5 mg/kg/day to about 20 mg/kg/day, and/or may be in amounts sufficient to achieve plasma levels in the range from about 300ng/ml to about 1000 ng/ml.

The invention also provides methods for preventing at least one heart disease (e.g., arrhythmia (e.g., tachycardia; atrial arrhythmias including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmias including ventricular fibrillation; and exercise-induced arrhythmias), heart failure, or exercise-induced sudden cardiac death) in a subject. The method comprises administering to the subject an amount of 1, 4-benzothiazepine effective to prevent at least one heart disease in the subjectA derivative of the class. 1, 4-benzothiazepine effective to prevent at least one heart disease (e.g., cardiac arrhythmia (e.g., tachycardia; atrial arrhythmia, including atrial tachyarrhythmia and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmia, including ventricular fibrillation; and exercise-induced arrhythmia) or heart failure) in a subjectSuitable amounts of the class of derivatives may range from about 5 mg/kg/day to about 20 mg/kg/day, and/or may be in amounts sufficient to achieve plasma levels in the range from about 300ng/ml to about 1000 ng/ml.

1, 4-Benzothiazepine according to the above processExamples of generic derivatives include, but are not limited to:

wherein R ═Aryl, alkenyl, alkyl, - (CH)₂)_nNR′₂Or- (CH)₂)_nSR ', and n is 0, 1, 2 or 3, and R' is alkyl or cycloalkyl;

wherein R is₁OR ', SR', NR ', alkyl OR halide in the 2, 3,4, OR 5 position on the phenyl ring, and R' ═ alkyl, aryl ORH; wherein R is₂H, alkyl or aryl; and wherein R₃H, alkyl or aryl;

(h) an oxidized form of any of (a) - (g) above. In one embodiment of the invention, 1, 4-benzothiazepinesThe derivative is selected from S4, S7, S-20, S-24, S-25, S-26, S-27 and S36. Preference is given to 1, 4-benzothiazepinesThe derivative is S36. The invention also provides these 1, 4-benzothiazepinesClass of derivatives in the treatment or prevention of at least one heart disease (e.g., arrhythmia (e.g., heart)Moving and over-speeding; atrial arrhythmias, including atrial tachyarrhythmias and atrial fibrillation (both sustained and non-sustained); ventricular arrhythmias, including ventricular fibrillation; and exercise-induced cardiac arrhythmia), heart failure, or exercise-induced sudden cardiac death).

The present invention also provides novel assays for facilitating the ordered or high throughput screening of other biologically active small molecules that bind FKBP12.6 to RyR 2. In particular, the invention provides a method for identifying an agent that promotes binding of RyR2 to FKBP12.6, comprising the steps of: (a) a source to obtain or produce FKBP 12.6; (b) contacting RyR2 with KBP12.6 in the presence of a candidate active agent; and (c) determining whether the agent can promote binding of RyR2 to FKBP 12.6. In one embodiment, RyR2 is PKA-phosphorylated. In another embodiment, RyR2 is PKA-hyperphosphorylated. In another embodiment, RyR2 is not phosphorylated.

In the method of the invention, RyR2 is immobilized on a solid phase, such as a plate or bead. To facilitate detection of RyR2-FKBP12.6 binding, FKBP12.6 may be radiolabeled (e.g., with³²S). In addition, increased binding of RyR2 to FKBP12.6 can be detected using FKBP 12.6-binding agents. In one embodiment, the FKBP 12.6-binding agent is an anti-FKBP 12.6 antibody. The invention also provides an agent identified by the method and the use of the agent in a method for limiting or preventing a decrease in the level of RyR 2-bound FKBP12.6 in a subject; use in a method for treating or preventing heart failure, atrial fibrillation, or exercise-induced arrhythmia in a subject; and in a method for preventing exercise-induced sudden cardiac death in a subject.

In addition, the present invention provides a method of identifying an agent for promoting binding of RyR2 to FKBP12.6, comprising the steps of: (a) a source to obtain or produce FKBP 12.6; (b) contacting FKBP12.6 with RyR2 in the presence of a candidate active agent; and (c) determining whether the agent can promote binding of RyR2 to FKBP 12.6. In one embodiment, RyR2 is PKA-phosphorylated. In another embodiment, RyR2 is PKA-hyperphosphorylated. In another embodiment, RyR2 is not phosphorylated.

In the method of the present invention, FKBP12.6 is immobilized on a solid phase, such as a plate or bead. To facilitate detection of RyR2-FKBP12.6 binding, RyR2 may be radiolabeled (e.g., with³²P). In addition, improved binding of RyR2 to FKBP12.6 can be detected using RyR 2-binding agents. In one embodiment, the RyR 2-binding agent is an anti-RyR 2 antibody. The invention also provides an agent identified by the method and the use of the agent in a method for limiting or preventing a decrease in the level of RyR 2-bound FKBP12.6 in a subject; use in a method for treating or preventing heart failure, atrial fibrillation, or exercise-induced arrhythmia in a subject; and in a method for preventing exercise-induced sudden cardiac death in a subject.

As an example and as shown in example 12 below, a high efficiency assay for high throughput screening of small molecules can be developed by immobilizing FKBP12.6 (e.g., wild-type FKBP12.6 or a fusion protein such as GST-FKBP12.6) on glutathione coated 96-well plates using standard procedures. PKA-phosphorylated ryanodine (ryanodine) receptor type 2(RyR 2) can be loaded onto FKBP 12.6-coated plates and incubated with JTV-519 analogs and other 1, 4-benzothiazepines at various concentrations (10-100nM)The derivatives were incubated together for 30 minutes. Thereafter, the plate is washed to remove unbound RyR2, and then incubated with anti-RyR 2 antibody (e.g., 30 minutes). The plate was washed again to remove unbound anti-RyR 2 antibody and then treated with a fluorescently labeled secondary antibody. Binding activity in the plate was read by an automated fluorescent plate reader.

Or, can be at³²RyR2 PKA-is phosphorylated in the presence of P-ATP. Radioactive PKA-phosphorylated RyR2 can be loaded onto KBP 12.6-coated JTV-519 analogs and other 1, 4-benzothiazepines at various concentrations (10-100nM)Class derivatives were present in 96-well plates for 30 min. The plate may be washed to remove unbound radiolabeled RyR2 and then read with an automatic plate reader. The plates were also coated with PKA-phosphorylated RyR2 and reacted with the analogs and derivatives in the presence of³²S-labeled FKBP12.6 was incubated together.

The following examples illustrate the invention and are presented to aid in the understanding of the invention but are not intended to limit the scope of the invention in any way as defined in the claims which follow.

Examples

Example 1-FKBP 12.6-deficient mice

FKBP 12.6-deficient mice were generated as described above (Wehrens et al, "FKBP 12.6 deficient and defective calcium release channel (ryanodine receptor) function associated with exercise-induced sudden cardiac death," Cell, 113: 829-40, 2003). Briefly, mouse genomic lambda-phage clones for the murine ortholog (ortholog) of human FK506 binding protein 12.6(FKBP12.6) were isolated from the DBA/11acJ library using full-length murine cDNA probes. Targeting vectors were designed to delete exons 3 and 4 containing the full coding sequence of murine FKBP12.6 by replacing the 3.5kb murine genomic DNA with the PGK-neo selectable marker (Bennett et al, "identification and characterization of the murine FK506 binding protein (FKBP)12.6 gene," Mamm. genome, 9: 1069-71, 1998). The 5.0-kb 5 'fragment and the 1.9-kb 3' fragment were cloned into pJNS2, a backbone vector containing the PGK-neo and PGK-TK clips. DBA/lacJ Embryonic Stem (ES) cells were grown and transfected using established protocols. Targeted ES cells were first screened by southern blot analysis and 5 positive ES cell lines were analyzed by PCR to confirm homologous recombination. The male chimeras were mated with DBA/11acJ females and germ line progeny were identified based on brown coat color. Germline progeny genotypes were generated using 5' southern blot analysis. Make positive FKBP12.6^+/-Male and female crossed and gave BP12.6 with a frequency of about 25%^-/-A mouse. FKBP12.6^-/-The mice were fertile.

Using FKBP12.6^-/-All studies performed in mice used age and sex matched FKBP12.6^+/+Mice served as control group. FKBP12.6 produced in the context of^-/-No difference was observed between mice: DBA/C57BL6 mixed; pure DBA; and pure C57BL 6.

Example 2 telemetry recording and exercise testing in mice

Maintenance and study of FKBP12.6 according to the Institutional Animal Care and Use Committee of Columbia university approved protocol^+/+And FKBP12.6^-/-A mouse. Mice were anesthetized using 2.5% isoflurane inhalation anesthesia. Ambulatory animal ECG radiotelemetry recordings obtained after intraperitoneal implantation for > 7 days (Data Sciences International, St. Paul, MN) (Wehrens et al, "FKBP 12.6 deficiency and defective calcium release channel (ryanodine receptor) function associated with exercise-induced sudden cardiac death," Cell, 113: 829-40, 2003). For stress testing, mice were allowed to move on an inclined treadmill to failure and then were injected intraperitoneally with epinephrine (0.5-2.0mg/kg) (Wehrens et al, "FKBP 12.6 deficiency and defective calcium release channel (ryanodine receptor) function associated with exercise-induced sudden cardiac death," Cell, 113: 829-40, 2003). The resting heart rate of the pedestrian animals was averaged over 4 hours.

Example 3 expression of wild type and RyR2-S2809D mutant

RyR2(RyR2-S2809D) was subjected to mutagenesis of PKA target sites as described above (Wehrens et al, "FKBP 12.6 deficient and defective calcium release channel (ryanodine receptor) function associated with exercise-induced sudden cardiac death," Cell, 113: 829-40, 2003). Using Ca²⁺Phosphate precipitation HEK293 cells were co-transfected with 20. mu.g RyR2 Wild Type (WT) or mutant cDNA and 5. mu.g FKBP12.6cDNA. Preparation of a composition containing RyR2 as described aboveVesicles of the channel (Wehrens et al, "FKBP 12.6 deficiency and defective calcium release channel (ryanodine receptor) function associated with exercise-induced sudden cardiac death," Cell, 113: 829-40, 2003).

Example 4-RyR2 PKA phosphorylation and FKBP12.6 binding

Cardiac SR membranes were prepared as described above (Marx et al, "PKA phosphorylation dissociating FKBP12.6 from calcium release channels (ryanodine receptor): defective regulation in defective heart" -Cell, 101: 365-76, 2000; Kaftan et al, "rapamycin to ryanodine receptor/Ca from myocardium)²⁺-effect of release channel ", circ.res., 78: 990-97, 1996). TNT from Promega (Madison, Wis.) was used^TMQuick coupled transcription/translation system generation³⁵S-labeled FKBP 12.6. [³H]Lannogidine binding was used to quantify RyR2 levels. Mu.g of microsomes were diluted in 100ul of 10-mM imidazole buffer solution (pH 6.8) at 37 ℃ with 250-nM (final concentration) ([ solution ]³⁵S]FKBP12.6 was incubated together for 60 min and then quenched with 500. mu.l ice cold imidazole buffer. The samples were centrifuged at 100,000g for 10 min and washed 3 times in imidazole buffer. Measuring the bound [2 ] by liquid scintillation counting of the precipitate³⁵S]-amount of FKBP 12.6.

Example 5 immunoblotting

Microsomes (50 μ g) were immunoblotted for 1 hour at room temperature as described using anti-FKBP 12/12.6 (1: 1,000), anti-RyR-5029 (1: 3,000) (Jayaraman et al, "FK 506 binding protein binding to calcium release channel (ryanodine receptor)", J.biol. chem., 267: 9474-77, 1992) or anti-phosphoRyR 2-P2809 (1: 5,000) (IkReen et al, "beta-receptor blockers restored calcium release channel function in human heart failure and improved myocardial performance" -Circulation, 107: 2459-66, 2003). P²⁸⁰⁹Phosphorylated epitope-specific anti-RyR 2 antibody was used by Zymed Laboratories (San Francisco, Calif.) using the equivalent of Ser²⁸⁰⁹The upper RyR2 PKA-phosphorylated peptide CRTRRI- (pS) -QTSQ was a custom affinity purified polyclonal rabbit antibody. In the presence of HRP-labeled anti-Homeotic antibodiesAfter incubation of rabbit IgG (1: 5,000 dilution; Transduction Laboratories, Lexington, KY), the blots were developed using ECL (Amersham Pharmacia, Piscataway, N.J.). These antibodies can also be used in the following ratios: 1: 4,000 (anti-rabbit IgG); and 1: 5,000 (anti-RyR 2-5029 and anti-FKBP 12.6).

Example 6 Single-channel recording

Single channel recordings of native RyR2 or recombinant RyR2 from rodent (mouse or rat) hearts were obtained under 0mV and potential clamp conditions as described above (Marx et al, "PKA phosphorylation disassociates FKBP12.6 from calcium release channels (ryanodine receptor): defective regulation in defective hearts" Cell, 101: 365-76, 2000). The homogeneous solutions used for channel recording were: trans compartment-HEPES, 250 mmol/L; ba (OH)₂53mmol/L (in some experiments, with Ca (OH))₂Alternative Ba (OH)₂) (ii) a pH 7.35; and cis compartment-HEPES, 250 mmol/L; tris-base, 125 mmol/L; EGTA, 1.0 mmol/L; and CaCl₂0.5 mmol/L; the pH was 7.35. Unless otherwise stated, there is 150-nM [ Ca ] in the cis compartment²⁺]And 1.0-mM [ Mg²⁺]There is a single channel recorded. Lannogen (5mM) was applied in the cis compartment in order to confirm the identity of all channels. Data were analyzed from digitized current recordings using fetcan software (Axon Instruments, Union City, CA). All data are expressed as mean ± SE. Unpaired student's t-test was used to statistically compare the means between experiments. Values of p < 0.05 were considered statistically significant.

The effect of JTV-519 on the RyR2 channel is shown in FIGS. 1-3 and Table 1 (below). As shown in FIG. 3, the single channel study demonstrated that PKI is a specific inhibitor of PKA_5-24(C) In the presence of PKA phosphorylation, there is an increased chance of opening RyR2 following PKA phosphorylation (D). Single channel function was calibrated in PKA-phosphorylated RyR2 incubated with FKBP12.6 in the presence of JTV-519 (E). The amplitude histogram (right) reveals an increase in activity and re-conductance opening in PKA-phosphorylated RyR2, but not after treatment with JTV-519 and FKBP 12.6. FIG. 3F shows a liquid crystal display panelCa for RyR2 activation by incubation of PKA-phosphorylated RyR2 with FKBP12.6 in the presence of JTV-519²⁺Dependent rightward shift, making it interact with Ca of unphosphorylated channels²⁺-dependency similarity.

TABLE 1 Steps before, during and after exercise and with adrenaline injection Horizontal ECG data

FKBP12.6 in treatment with JTV-519^+/-Mice (n-8) or controls (n-6) and FKBP12.6 treated with JTV-519^-/-Walking ECG data in mice (n-5) were summarized. SCL ═ sinus cycle length; HR-heart rate; ms is millisecond; bpm is the number of heart beats per minute; FKBP12.6^+/-FKBP12.6 heterozygous mouse; FKBP12.6^-/-FKBP12.6 deficient mice

Example 7 rat model of Heart failure

As described above, 25 Sprague-Dawley (300-400g) were left coronary artery ligated by left thoracotomy for myocardial infarction (Alvarez et al, "tetrodotoxin-resistant Na in rat cardiomyocytes induced after late myocardial infarction)⁺Current "-j.mol.cellCardiol, 32: 1169-79, 2000). Briefly, rats were anesthetized with a mixture of 150mg/kg intraperitoneal ketamine and 15mg/kg chlorpromazine. The breath was maintained using a ventilation aid and an endotracheal tube (3ml air/60 strokes/min). Following a medial-left thoracotomy and pericardial patency, the left main trunk of the coronary artery was occluded at the proximal point below the left atrial appendage using a 7-0 # suture. Rats (n ═ 5) simulating surgical procedures were treated in the same manner, but without coronary artery ligation.

Heart failure was confirmed using echocardiography 6 weeks after myocardial infarction. JTV-519 or vehicle (DMSO) (0.5mg/kg/h) was continuously infused via an implantable osmotic infusion pump (Alzet mini-osmotic pump; Durect Corporation, Cupertino, Calif.). After 4 weeks of continuous treatment, echocardiography and hemodynamic measurements were performed. Animals were sacrificed and tissue samples were collected.

Heart failure was induced in rats by ligation of the left anterior descending coronary artery according to the above technique. This results in myocardial infarction, which can develop into dilated cardiomyopathy with reduced cardiac function within 4 weeks. 3 groups of animals were studied, with 25 animals in each group: simulated surgical operation group (control group); heart failure + therapy group (JTV-519); and heart failure group without therapy (vehicle). 4-week treatment with JTV-519 significantly reduced diastolic and systolic dysfunction as determined from echocardiography (FIG. 4). Thus, therapy with JTV-519 significantly improved cardiac function and slowed progression of heart failure in the rat model of ischemia-induced heart failure.

It was determined that JTV-519 can increase the affinity of FKBP12.6 for the RyR2 channel using a mutant RyR2-S2809D channel that mimics the constitutive-PKA-phosphorylated RyR2 channel (FIG. 5). In particular, treatment with JTV-519 enables FKBP12.6 to bind to mutant channels, thereby revealing a mechanism by which JTV-519 prevents heart failure. Treatment with JTV-519 also prevented leakage in RyR2 channels in the defective heart (fig. 6). Furthermore, JTV-519 restored binding of FKBP12.6 to PKA-phosphorylated RyR2 and mutant RyR2-S2809D that mimics constitutive mutant-PKA-phosphorylated RyR2 in a dose-dependent manner (FIG. 7).

Example 8 Canine model of atrial fibrillation

a) Animal protocol

Pacemakers were implanted in female adult crossbred dogs weighing 24-26kg using the technique described above (Dun et al, "chronic atrial fibrillation did not further reduce outward currents". Am.J. Physiol. Heart. Physiol., 285: H1378-84, 2003) can increase these outward currents. Animals were anesthetized with thiopentone sodium (17mg/kg, i.v.) andintroducing isoflurane (1.5-2%) and O₂(2 l/min). Active fixation leads (lead) were implanted in the right atrial appendage and right atrial free wall, passed subcutaneously and connected to an Itrel pulse generator and Thera8962 pacemaker (Medtronics, Minneapolis, MN), respectively. 40% formaldehyde (0.1-0.3ml) was injected into the bundle of his in order to obtain complete AV block. The ventricular pacemaker was programmed to operate at a rate of 60bpm and this rate was maintained throughout the pacing protocol. After recovery, atrial pacing was initiated at a rate of 600-.

The animals were then anesthetized with pentobarbital (30mg/kg) and their hearts removed. Atrial tissue was dissected, snap frozen immediately in liquid nitrogen and stored at-80 ℃.

b) Heart acquisition

The human data provided in this study was derived from 5 human hearts from patients with atrial fibrillation in the context of advanced heart failure following orthotopic heart transplantation according to the Institutional Review Board of the New York Presbyterians Hospital approved protocol. In addition, data were also obtained from samples of 3 normal hearts that were not suitable for transplantation. At the time of transplantation, the heart was preserved on explants with cold (4 ℃), hypocalcemic (hypocalcemic), hyperkalemic (hyperkalemic) cardioplegic solutions.

c) Immunoprecipitation and reverse-phosphorylation of RyR2

Heart membranes (100. mu.g) prepared from Left Atrial (LA) tissue as described above (Marx et al, "PKA phosphorylation to dissociate FKBP12.6 from calcium Release channels (ryanodine receptor) -Defect Regulation in defective hearts" -Cell, 101: 365-]0.9% NaCl, 0.25% Triton 100x, 5mM NaF and protease inhibitor) and then incubated with rabbit anti-RyR 2 antibody overnight at 4 ℃. Protein A agarose beads were added and incubated at 4 ℃ for 1 hour. Followed byUsing 1 Xkinase buffer (50mM Tris-HCl, 50mM piperazine-N, N' -bis [ 2-ethanesulfonic acid)]8mM MgCl and 10mM EGTA [ pH 6.8]) Protein a beads were washed and then resuspended in 1.5x kinase buffer. Using PKA (5 units), 100nM MgATP, and [ gamma. ]³²P]ATP (NEN Life Sciences, Boston) initiates the reaction; the system was incubated at room temperature for 8 minutes and then stopped using 5. mu.l of 6 XLoading buffer (4% SDS and 0.25M DTT). The samples were heated to 95 ℃ and then size fractionated on 6% SDS-PAGE. The RyR2 radioactivity was quantified using Molecular dynamics spectroradiometer, and Imagequant software (amersham pharmacia Biotech, Pescataway, NJ). The value was divided by the amount of immunoprecipitated RyR2 (determined by immunoblotting and densitometry) and expressed as [ gamma ]³²P]The reciprocal of the ATP signal.

d)Combined with Calstabin2 of JTV-519 (FKBP12.6)

As described above, RyR2 was immunoprecipitated from atrial SR (100 μ g) and washed with 1 × kinase buffer. Immunoprecipitated RyR2 was phosphorylated using PKA (5 units) and 100 μ M MgATP at room temperature and the reaction was terminated after 8 min by washing with ice cold RIPA buffer. Followed by reaction at room temperature with or without 1, 4-benzothiazepineClass of derivatives, JTV-519(1. mu.M), recombinant calstabin2(FKBP 12.6; 200nM) was incubated with phosphorylated RyR 2. After washing, the reaction was performed with RIPA buffer, the proteins were size fractionated by 15% SDS PAGE and calstabin2(FKBP12.6) was immunoblotted.

Summarized below are the results obtained by the inventors in conjunction with the experiment of example 8:

modulation of atrial RyR2

Atrial RyR2 PKA was phosphorylated according to the technique described above (fig. 8A). PKA phosphorylation of atrial RyR2 reduced the amount of calstabin2(FKBP12.6) in the RyR2 macromolecular complex, as determined by co-immunoprecipitation (fig. 8C). Atrial RyR2 macromolecular complex includes calstabin2(FKBP12.6), the catalytic subunit of PKA, the PKA regulatory subunit (RII), PP2A, PP1 and mAKAP (FIG. 8B), as reported above for ventricular RyR2 (Marx et al, "PKA phosphorylation causes dissociation of FKBP12.6 from calcium release channels (ryanodine receptor): defective regulation in defective heart" -Cell, 101: 365-76, 2000).

PKA hyperphosphorylation of ryanodine receptors in atrial fibrillation

PKA phosphorylation of immunoprecipitated RyR2 in atrial tissue from dogs with persistent Atrial Fibrillation (AF) increased 130% over the control (AF: n ═ 6; control: n ═ 6; P < 0.001; fig. 9A). Calstabin2(FKBP12.6) binding to RyR2 was reduced by 72% in atrial tissue from dogs with persistent AF compared to the control (AF: n ═ 6; control: n ═ 6; P < 0.0005; FIG. 9A).

Similarly, PKA phosphorylation of immunoprecipitated RyR2 in atrial tissue from persons with chronic atrial fibrillation in a heart failure setting increased 112% over that in the control (AF: n-5; control: n-3; P < 0.002; fig. 9B). The reduction in Calstabin2(FKBP12.6) bound to RyR2 was 70% (AF: n ═ 5; control: n ═ 6; P < 0.0001; fig. 9B).

For all reverse-phosphorylation and co-immunoprecipitation experiments, the total amount of loaded RyR2 was confirmed by parallel immunoblotting of immunoprecipitated ryrs with anti-RyR 2-5029 antibody (fig. 9A and 9B).

Cardiac ryanodine receptor channel function

To determine the physiological significance of the RyR2 PKA hyper-phosphorylation observed in AF dogs, RyR2 single channel measurements were taken on planar lipid bilayers using symmetric ion conditions at 0mV and potential clamp conditions. Atrial RyR2 single channel characteristics were studied in 17 channels from 5 AF dogs and 11 channels from 5 control dogs. None of the channels from the control dogs showed increased activity, while from AF dogs15 of the 17 channels (88%) showed a significant increase in open probability (Po; AF: 0.412 + -0.07; control: 0.008 + -0.002; P < 0.001) and gating frequency (Fo; AF: 21.9 + -4.6; control: 1.6 + -0.6 s)^-1(ii) a P < 0.002 (FIGS. 10A and 10B).

Recombination of Calstabin2(FKBP12.6) in the Presence of JTV-519

Treatment with TV-519(1mM) allowed recombinant calstabin2(FKBP12.6) to bind to PKA-phosphorylated RyR2 that had been isolated from canine myocardium. In these experiments, calstabin2(FKBP12.6) was unable to bind to PKA-phosphorylated RyR2 in the absence of JTV-519 (FIG. 11B).

The physiological significance of FKBP-12.6 rebinding in the presence of JTV-519 was demonstrated by performing a RyR2 single channel assay in planar lipid bilayers. In the presence of recombinant calstabin2(FKBP12.6) alone, the isolated channels exhibited significant aberrant behavior, with an increase in the open probability (Po) and the state of repolarization. These abnormalities in channel function were not observed upon addition of specific PKA inhibitors (PKI), indicating that the abnormalities observed in channel function are specific for PKA-induced phosphorylation of RyR 2. When the PKA-phosphorylation channel was treated with JTV-519 in the presence of recombinant calstabin2(FKBP12.6), single-channel measurements were similar to those observed in the presence of PKI (FIG. 11A).

Example 9-1, 4-Benzothiazepine Synthesis of intermediate and JTV-519

To perform in vivo experiments, the inventors required JTV-519 in gram amounts. However, the 1, 4-benzothiazepines originally reportedThe intermediate 7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepineClass (compound in scheme 1 below) attempts to prepare this compound were unsuccessful. The thio group in this intermediate is readily oxidized by air to disulfide compounds, making synthesis of the cyclized product impossible. To overcome this problem, the present inventors have developed a novel method using 2-nitro-5-methoxybenzoic acid (1) which is easily available and inexpensive as a raw material. This method is described in scheme 1 below.

Using H₂Reduction of the nitro group of compound (1) with Pd/C as a catalyst gave 2-amino-5-methoxybenzoic acid (2) in quantitative yield. Using NaNO₂Diazotizing compound (2) and then using Na₂S₂The work-up gave the stable disulfide compound (3) in 80% yield. Without further purification, with SOCl₂The stabilized disulfide (3) is disposed of and then in the presence of Et₃Reaction with 2-chloroethylamine in the presence of N gave the amide (4) in 90% yield. By a one-pot procedure, via reaction with triphosphine and Et₃N is refluxed together in THF to convert compound (4) to cyclized compound (5). Then using LiAlH₄Reduction of the cyclized amide (5) to 7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepineClass (6).

Scheme 1

JTV-519 is prepared by reacting compound (6) with 3-bromopropionyl chloride and then reacting the resulting product with 4-benzylpiperidine. According to¹H NMR established the structure of JTV-519.

EXAMPLE 10 Synthesis of radiolabeled JTV-519

The novel method used by the present inventors for the synthesis of radiolabeled JTV-51 is described in scheme 2 below. For the preparation of radiolabeled JTV-519, BBr is used₃Demethylation of JTV-519 on a benzene ring gives phenol compound (21). Using a radiolabelled methylating agent in the presence of a base (NaH) ((³H-dimethyl sulfate) to remethylate the phenol compound (21) to obtain³H-labelled JTV-519 (scheme 2).

Scheme 2

Example 11 novel 1, 4-Benzothiazepine Class of derivatives and synthesis method thereof

The inventors have also developed novel 1, 4-benzothiazepines for the treatment and prevention of cardiac arrhythmiasA derivative of the class. In particular, the inventors have prepared compounds having the following general structure:

wherein R is aryl, alkenyl, alkyl, - (CH)₂)_nNR′₂Or- (CH)₂)_nSR', and n is 0, 1, 2 or 3; and wherein R' is alkyl or cycloalkyl. By reacting 7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepineWith alkylsulfonyl or arylsulfonyl chloridesChlorine in the presence of a base, such as Et₃The novel compounds of this general structure are prepared by reaction in the presence of N. Additional reactions (e.g., addition of 4-benzylpiperidine) may then be performed to extend the side chain as desired. A representative synthesis of this general approach is described in scheme 3 below.

As shown in scheme 3, 2-chloroethanesulfonyl chloride (180 mg; 1.1mM) and Et are reacted at 0 ℃ with₃N (140 mg; 1.1mM) was added to the solution in CH₂Cl₂7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepine in (20ml)Class (1) (194 mg; 1 mM). The mixture was stirred at 0 ℃ for 2 hours and with H₂O and saturated NaHCO₃And (4) washing the solution. The solvent was removed to give the crude product (Ia) which was purified by silica gel chromatography (petroleum ether: ethyl acetate 3: 1). The yield from this synthesis was 280mg or 95%. The structure was confirmed by NMR.

Scheme 3 further shows the treatment of a disease caused by contacting Compound (Ia) (28 mg; 0.1mM) with 4-benzylpiperidine (21 mg; 0.13mM) in CH₂Cl₂The side chain of the compound (Ia) is extended by the reaction of (A) and (B). After the reaction is complete (by TLC), excess amine can be removed with a base scavenger (e.g., 3- (2-succinic anhydride) propyl functional silica gel, 0.5 g).¹HNMR and HPLC showed the product (Ib) to be > 98% pure.

Scheme 3

In addition, the present inventors have produced compounds having the following general structure:

wherein R is aryl, alkenyl, alkyl, - (CH)₂)_nNR′₂Or- (CH)₂)_nSR', and n is 0, 1, 2 or 3; and wherein R' is alkyl or cycloalkyl. By reacting 7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepineIn the presence of a base (Et) of class (1)₃N) with sulphuryl chloride in the presence of a primary or secondary amine followed by addition of a primary or secondary amine. After the mixture was stirred at 0 ℃ for 2 hours, 1-piperonylpiperazine (27 mg; 0.12mM) was added. The mixture was stirred for a further 2 hours and then with H₂O and saturated NaHCO₃And (4) washing the solution. Excess amine was removed by addition of a base scavenger (e.g., 3- (2-succinic anhydride) propyl functional silica gel, 0.2 g). The yield from this synthesis was 36mg or 77%.

Scheme 4

The present inventors have also produced compounds having the general structure:

wherein R ″ ═ CO (CH)₂)_nXR′″₂、SO₂(CH₂)_nXR′″₂Or SO₂NH(CH₂)_nXR′″₂And X ═ N or S, and N ═ 1, 2, or 3. By oxidation of JTV-519 or the above-mentioned novel 1, 4-benzothiazepines using hydrogen peroxideOne of the derivatives prepares the novel compound with the general structure. In this general methodA representative synthesis is described in scheme 5 below.

As shown in scheme 5, Compound (Ib) (21 mg; 0.05mM) in MeOH (5ml) was added to H₂O₂(0.1ml, excess). The mixture was stirred for 2 days and the product III (CH) was purified by silica gel chromatography₂Cl₂MeOH 10: 1). The yield from this synthesis was 19mg or 91%.

Scheme 5

Finally, the inventors have prepared compounds having the general structure:

wherein R is aryl, alkyl, - (CH)₂)_nNR′₂、-(CH₂)_nSR ', and n is 0, 1, 2 or 3, and R' is alkyl, cycloalkyl; and wherein X ═ NH or O. By reacting 7-methoxy-2, 3,4, 5-tetrahydro-1, 4-benzothiazepineIn the presence of a base (Et) of class (1)₃N) with triphosgene in the presence of a primary or secondary amine or an alcohol followed by addition of a primary or secondary amine. A representative synthesis of this general approach is described in scheme 6 below.

Scheme 6

Example 12 highThroughput screening assay

The present inventors have developed assays for screening biologically active small molecules. These assays are based on the re-binding of FKBP12 protein to RyR.

A high-performance assay for high-throughput screening of small molecules can be developed by immobilizing FKBP12.6 (GST-fusion protein) on glutathione-coated 96-well plates. PKA-phosphorylated ryanodine receptor type 2(RyR 2) can be loaded onto FKBP 12.6-coated plates and incubated with JTV-519 analogs at different concentrations (10-100nM) for 30 min. Thereafter, the plate was washed to remove unbound RyR2, and then incubated with anti-RyR 2 antibody for 30 minutes. The plate was washed again to remove unbound anti-RyR 2 antibody and then treated with a fluorescently labeled secondary antibody. Binding activity in the plate was read by an automated fluorescent plate reader.

In an alternative test, there may be³²RyR2 PKA-is phosphorylated in the presence of P-ATP. Radioactive PKA-phosphorylated RyR2 can be loaded into KBP 12.6-coated 96-well plates in the presence of JTV-519 analogs at various concentrations (10-100nM) for 30 min. The plate was washed to remove unbound radiolabeled RyR2 and then read with an automatic plate reader.

Example 13 novel Re-binding of FKBP12.6 to RYR2 1, 4-benzothiazepines Class of derivatives

Phosphorylation of cardiac Sarcoplasmic Reticulum (SR) with PKA for 30 min at room temperature resulted in complete dissociation of FKBP12.6 from the RyR2 complex. SR (50mg) was then incubated with 250nmfkbp12.6 and test compound for 30 minutes at room temperature. The sample was centrifuged at 100,000g for 10 min and the pellet was washed 3 times with 10mM imidazole buffer. After washing, proteins were separated by 15% PAGE. Immunoblots were developed using anti-FKBP antibodies. The results of this study are shown in figure 12.

Example 14 telemetry recording and exercise/EKG testing in mice

Using FKBP12.6^+/-Mice (intervention group), age-and sex-matched FKBP12.6^-/-Mouse (positive control) and wild-type FKBP12.6^+/+Mice (negative control) were studied. Absence of FKBP12.6 protein was confirmed by immunoblotting in cardiac tissue (FKBP12.6)^-/-) Or decrease (FKBP12.6)^+/-)。

The drug JTV-519 (plasma target concentration 1.0. mu.M) or derivative S36 (plasma target concentration 1.0. mu.M or 0.02. mu.M) was continuously injected subcutaneously via a mini-osmotic pump at a rate of 1.0. mu.l/hr into FKBP12.6^+/-Or FKBP12.6^-/-Mice were subjected to exercise testing 7 days after which (AlzetDurect co., Cupertino, CA). Mice were anesthetized with intraperitoneal injections of ketamine (50 μ g/kg) and xylazine (10 μ g/kg) and implanted with a radioactive EKG transmitter. ECG radiotelemetry recordings of walking animals were obtained 1 week after intraperitoneal implantation (Data sciences international, st. paul, MN). Standard dimensions were used to determine ECG parameters.

For stress testing, mice were run in a stepped fashion on an inclined treadmill to failure and then injected intraperitoneally with epinephrine (2.0mg/kg) for maximum sympathetic stimulation. The resting heart rate of the pedestrian animals was then averaged over 4 hours. Recovery was performed after exercise testing and conditions after exercise were monitored overnight. Sinus Cycle Length (SCL) and PR, QRS and QT intervals were determined and the rate-corrected QT interval (QTc) was calculated using the Mitchell formula. Plasma drug levels were confirmed by HPLC. The results are summarized in fig. 13.

Example 15-JTV-519 improvement of cardiac contractility in rat model of Heart failure

Rats were subjected to Myocardial Infarction (MI) by ligation of the left coronary artery. Treatment with JTV-519(n ═ x) or vehicle (n ═ x) using an implantable osmotic pump (Alzet, Durect Corporation, Cupertino, CA) started directly after MI. Myocardial systolic (Ds) and diastolic (Dd) diameters were measured at mid-papillary (mid-papillary) level 24 hours and 6 weeks post-MI using echocardiography; a foreshortening score (fractional shortening) is then calculated. As shown in figure 14, treatment with JTV-519 significantly improved cardiac function in rats with heart failure.

Although the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this specification that various changes in form and detail can be made without departing from the true scope of the invention as defined in the appended claims.

Claims

1. A compound represented by the following formula,

2. a pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier, additive or diluent.

3. A pharmaceutical composition according to claim 2, comprising one or more of the following: antioxidants, colorants, flavors, preservatives, sweeteners, binders, lubricants, carboxymethylcellulose, crystalline cellulose, glycerin, gum arabic, lactose, magnesium stearate, methylcellulose, saline, sodium alginate, sucrose, starch, talc, or water; the pharmaceutical composition is in the form of a capsule, tablet, powder, granule or suspension.

4. A pharmaceutical composition according to claim 2 or 3 for use in the preparation of a medicament for limiting or preventing a decrease in the level of RyR 2-bound FKBP12.6 in a subject having a heart condition.

5. A pharmaceutical composition according to claim 2 or 3 for use in the preparation of a medicament for the treatment or prevention of a cardiac condition in a subject.

6. The pharmaceutical composition according to claim 4 or 5, wherein said cardiac condition is arrhythmia, tachycardia, atrial fibrillation, ventricular fibrillation, heart failure or sudden cardiac death.

7. The pharmaceutical composition according to claim 6, wherein said arrhythmia is an atrial arrhythmia or a ventricular arrhythmia; the atrial fibrillation is persistent atrial fibrillation or non-persistent atrial fibrillation; the tachycardia is ventricular tachycardia; and said sudden cardiac death is exercise-induced sudden cardiac death.

8. The pharmaceutical composition according to claim 7, wherein said atrial arrhythmia is an atrial tachyarrhythmia; and said ventricular tachycardia is persistent ventricular tachycardia, non-persistent ventricular tachycardia or catecholamine polymorphic ventricular tachycardia.

9. The pharmaceutical composition according to claim 6, wherein said cardiac condition is heart failure.

10. The pharmaceutical composition according to claim 6, wherein said cardiac condition is catecholaminergic polymorphic ventricular tachycardia.

11. Use of a compound of claim 1 in the manufacture of a medicament for limiting or preventing a decrease in the level of RyR 2-bound FKBP12.6 in a subject having a heart condition, or for treating or preventing a heart condition in a subject.

12. The use according to claim 11, wherein the cardiac condition is arrhythmia, tachycardia, atrial fibrillation, ventricular fibrillation, heart failure or sudden cardiac death.

13. Use according to claim 12, wherein the arrhythmia is an atrial arrhythmia or a ventricular arrhythmia; the atrial fibrillation is persistent atrial fibrillation or non-persistent atrial fibrillation; the tachycardia is ventricular tachycardia; and said sudden cardiac death is exercise-induced sudden cardiac death.

14. The use according to claim 13, wherein the atrial arrhythmia is an atrial tachyarrhythmia; and said ventricular tachycardia is persistent ventricular tachycardia, non-persistent ventricular tachycardia or catecholamine polymorphic ventricular tachycardia.

15. The use according to claim 12, wherein said cardiac condition is heart failure.

16. The use according to claim 12, wherein said cardiac condition is catecholaminergic polymorphic ventricular tachycardia.