Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
  • A61K31/554—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one sulfur as ring hetero atoms, e.g. clothiapine, diltiazem
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/06—Antiarrhythmics
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/91—Transferases (2.)
  • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/32—Cardiovascular disorders
  • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/32—Cardiovascular disorders
  • G01N2800/326—Arrhythmias, e.g. ventricular fibrillation, tachycardia, atrioventricular block, torsade de pointes

Definitions

• Heart failure is a leading cause of mortality and morbidity, world wide. In the more severe cases of heart failure (New York Heart Association class IV), the 2-year mortality rate is over 50% (Braunwald, E.B., Heart Disease, 4 ed. (Philadelphia: W.B. Saunders Co., 1992)). Cardiac arrhythmia, a common feature of heart failure, results in many of the deaths associated with the disease. In particular, approximately 50% of all patients with heart disease die from fatal cardiac arrhythmias. Some ventricular arrhythmias in the heart are rapidly fatal - a phenomenon referred to as "sudden cardiac death" (SCD). However, fatal ventricular arrhythmias may also occur in young, otherwise-healthy individuals who are not known to have structural heart disease. In fact, ventricular arrhythmia is the most common cause of sudden death in otherwise-healthy individuals.
• SCD Sudden cardiac death
• Catecholaminergic polymorphic ventricular tachycardia is an inherited disorder in individuals with structurally-normal hearts. It is characterized by stress- induced ventricular tachycardia - a lethal arrhythmia that may cause sudden cardiac death.
• CPVT Catecholaminergic polymorphic ventricular tachycardia
• physical exertion and/or stress induce bidirectional and/or polymorphic ventricular tachycardias that lead to SCD in the absence of detectable structural heart disease (Laitinen et al, Mutations of the cardiac ryanodine receptor (RyR2) gene in familial polymorphic ventricular tachycardia. Circulation, 103:485-90, 2001; Leenhardt et al,
• hRyR2 cardiac ryanodine receptor gene
• CPVT is predominantly inherited in an autosomal-dominant fashion. Individuals with CPVT have ventricular arrhythmias when subjected to exercise, but do not develop arrhythmias at rest. Linkage studies and direct sequencing have identified mutations in the human RyR2 gene, on chromosome Iq42-q43, in individuals with CPVT (Laitinen et al, Mutations of the cardiac ryanodine receptor (RyR2) gene in familial polymorphic ventricular tachycardia.
• RyR2 cardiac ryanodine receptor
• Heart failure is characterized by a progressive decrease in the contractile function of cardiac muscle, which leads to hypoperfusion of critical organs.
• the contraction of heart muscle, and other striated muscle, is initiated when calcium (Ca 2+ ) is released from the sarcoplasmic reticulum (SR) into the surrounding cytoplasm.
• Calcium-release channels on the SR including ryanodine receptors (RyRs), are required for excitation-contraction (EC) coupling (i.e., coupling of an action potential to a muscle cell's contraction).
• ryanodine receptors There are three types of ryanodine receptors, all of which are highly-related Ca 2+ channels: RyRl, RyR2, and RyR3.
• RyRl is found in skeletal muscle, RyR2 is found in the heart, and RyR3 is located in the brain.
• the type 2 ryanodine receptor (RyR2) is the major Ca 2+ -release channel required for EC coupling and muscle contraction in cardiac striated muscle.
• RyR2 channels are packed into dense arrays in specialized regions of the SR that release intracellular stores of Ca 2+ , and thereby trigger muscle contraction (Marx et al, Coupled gating between individual skeletal muscle Ca2+ release channels (ryanodine receptors). Science, 281:818-21, 1998).
• depolarization of the cardiac- muscle cell membrane in phase zero of the action potential, activates voltage-gated Ca 2+ channels.
• Ca 2+ influx through these channels initiates Ca 2+ release from the SR via RyR2, in a process known as Ca 2+ -induced Ca 2+ release (Fabiato, A., Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am. J.
• RyR2 is a protein complex comprising four 565,000-dalton RyR2 polypeptides in association with four 12,000-dalton FK506 binding proteins (FKBPs), specifically FKBP12.6 proteins.
• FKBPs are cis-trans peptidyl-prolyl isomerases that are widely expressed, and serve a variety of cellular functions (Marks, A.R., Cellular functions of immunophilins. Physiol. Rev., 76:631-49, 1996).
• FKBP12 proteins are tightly bound to, and regulate the function of, the skeletal ryanodine receptor, RyRl (Brillantes et al.
• IP3R1 inositol 1,4,5-triphosphate receptor
• FKBP12 binds the inositol 1,4,5-trisphosphate receptor at leucine-proline (1400-1401) and anchors calcineurin to this FK506-like domain. J. Biol Chem. , 272:27582- 88, 1997
• TGF ⁇ transforming growth factor ⁇
• T ⁇ RI type I transforming growth factor ⁇
• FKBP12.6 binds to the RyR2 channel (one molecule per RyR2 subunit), stabilizes RyR2- channel function (Brillantes et al, Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein. Cell, 77:513-23, 1994), and facilitates coupled gating between neighboring RyR2 channels (Marx et al, Coupled gating between individual skeletal muscle Ca2+ release channels (ryanodine receptors). Science, 281:818-21, 1998), thereby preventing aberrant activation of the chamiel during the resting phase of the cardiac cycle.
• Failing hearts are characterized by a maladaptive response that includes chronic hyperadrenergic stimulation (Bristow et al, Decreased catecholamine sensitivity and beta-adrenergic-receptor density in failing human hearts. N Engl J. Med., 307:205-11, 1982).
• the pathogenic significance of this stimulation in heart failure is supported by therapeutic strategies that decrease ⁇ -adrenergic stimulation and left ventricular myocardial wall stress, and potently reverse ventricular remodeling (Barbone et al, Comparison of right and left ventricular responses to left ventricular assist device support in patients with severe heart failure: a primary role of mechanical unloading underlying reverse remodeling.
• chronic ⁇ -adrenergic stimulation is associated with the activation of ⁇ -adrenergic receptors in the heart, which, through coupling with G-proteins, activate adenylyl cyclase and thereby increase intracellular cAMP concentration.
• cAMP activates cAMP-dependent protein kinase (PKA), which has been shown to induce hyper- phosphorylation of RyR2.
• PKA cAMP-dependent protein kinase
• DADs delayed after-depolarizations
• JTV-519 (4-[3-(4-benzylpiperidin-l-yl)propionyl]-7-methoxy-2,3,4,5- tetrahydro-l,4-benzothiazepine monohydrochloride; also known as k201), a derivative of 1,4- benzothiazepine, is a new modulator of calcium-ion channels.
• JTV-519 In addition to regulating Ca 2+ levels in myocardial cells, JTV-519 also modulates the Na current and the inward-rectifier K + current in guinea pig ventricular cells, and inhibits the delayed-rectifier K + current in guinea pig atrial cells.
• JTV-519 has a strong cardioprotective effect against catecholamine-induced myocardial injury, myocardial-injury-induced myofibrillar overcontraction, and ischemia/reperfusion injury.
• JTV-519 demonstrated greater cardioprotective effects than propranolol, verapamil, and diltiazem.
• Experimental data also suggest that JTV-519 effectively prevents ventricular ischemia/reperfusion by reducing the level of intracellular Ca 2+ overload in animal models.
• the present invention is based upon the surprising discovery that RyR2 is a target for preventing cardiac arrhythmias that cause exercise-induced sudden cardiac death (SCD).
• SCD exercise-induced sudden cardiac death
• the inventors made mutant RyR2 channels with 7 different CPVT mutations, and studied their functions. All 7 mutants had functional defects that resulted in channels that became leaky (an SR calcium leak) when stimulated during exercise.
• the inventors' study is the first to identify a mechanism by which the SR calcium leak causes DADs.
• the defect in the mutant CPVT channels made the channels look like the leaky channels in the hearts of patients with end-stage heart failure - a disorder characterized by a high incidence of fatal cardiac arrhythmias. Therefore, the inventors demonstrate herein that the mechanism for the VT in CPVT is the same as the mechanism for VT in heart failure.
• JTV-519 a member of the 1,4 benzothiazepine family of compounds, repairs the leak in RyR2 channels.
• JTV-519 enhances binding of FKBP12.6 to PKA-phosphorylated RyR2, and to mutant RyR2s that otherwise have reduced affinity for, or do not bind to, FKBP12.6.
• This action of JTV-519 fixes the leak in RyR2 that triggers fatal cardiac arrhythmias (cardiac death) and contributes to heart muscle dysfunction in heart failure.
• the inventors have developed a novel synthesis for JTV-519, as well as a radio- labeled version of the drug.
• the present invention provides a method for limiting or preventing a decrease in the level of RyR2 -bound FKBP12.6 in a subject who is a candidate for exercise-induced cardiac arrhythmia, by administering to the subject an amount of JTV-519 effective to prevent a decrease in the level of RyR2 -bound FKBP12.6 in the subject. Also provided is a use of JTV-519 in a method for limiting or preventing a decrease in the level of RyR2 -bound FKBP12.6 in a subject who is a candidate for exercise-induced cardiac arrhythmia.
• the present invention provides a method for treating or preventing exercise-induced cardiac arrhythmia in a subject, by administering JTV-519 to the subject in an amount effective to treat or prevent the exercise-induced cardiac arrhythmia in the subject. Also provided is a use of JTV-519 in a method for treating or preventing exercise-induced cardiac arrhythmia in a subject.
• the present invention provides a method for preventing exercise-induced sudden cardiac death in a subject, by administering to the subject JTV-519 in an amount effective to prevent exercise-induced sudden cardiac death in the subject. Also provided is a use of JTV-519 in a method for preventing exercise-induced sudden cardiac death in a subject.
• the present invention provides a method for identifying an agent for use in preventing exercise-induced sudden cardiac death, by: (a) obtaining or generating a culture of cells containing RyR2; (b) contacting the cells with a candidate agent; (c) exposing the cells to one or more conditions known to increase phosphorylation of RyR2 in cells; and (d) determining if the agent prevents a decrease in the level of RyR2 -bound FKBP12.6 in the cells.
• the method may further comprise the step of: (e) determining if the agent has an effect on an RyR2-associated biological event in the cells.
• an agent identified by the method, and a method for preventing exercise-induced sudden cardiac death in a subject by administering the agent to the subject in an amount effective to prevent o exercise-induced sudden cardiac death in the subject.
• the present invention provides a method for identifying an agent for use in preventing exercise-induced sudden cardiac death, by: (a) obtaining or generating an animal containing RyR2; (b) administering a candidate agent to the animal; (c) exposing the animal to one or more conditions Icnown to increase phosphorylation of RyR2 in cells; and (d) determining if the agent increases binding between FKBP12.6 and RyR2 in the animal.
• The may further comprise the step of: (e) determining if the agent has an effect on an RyR2-associated biological event in the animal.
• an agent identified by the method, and a method for preventing exercise-induced sudden cardiac death in a subject by administering the agent to the subject in an amount effective to prevent exercise-induced sudden cardiac death in the subject.
• the present invention provides methods for synthesizing
• JTV-519 and 1,4-benzothiazepine intermediates and derivatives including the following:
• the present invention provides a method for synthesizing radio-labeled JTV-519.
• FIG. 1 demonstrates that JTV-519 prevents exercise-induced ventricular arrhythmias in FKBP12.6 4* ⁇ mice.
• A Representative ambulatory electrocardiograms of an untreated FKBP12.6 + " mouse, an FKBP12.6 +/” mouse treated with JTV-519, and an FKBP12.6 " " mouse treated with JTV-519. There were no significant differences in heart rate, or in any of the measured ECG parameters.
• middle tracing Electrocardiogram of a JTV-519-treated FKBP12.6 47" mouse following the same protocol; no arrhythmias were detected, bottom tracing: Exercise-induced ventricular tachycardia (VT) in an FKBP12.6 "7" mouse treated with JTV-519.
• the dotted line represents 16.31 seconds of VT that are not shown in the figure.
• 'P' indicates a P-wave, which is indicative of sinus rhythm following ventricular tachycardia.
• C Bar graph showing quantification of sudden cardiac death (left), sustained ventricular tachycardias (>10 beats, middle), and non-sustained ventricular tachycardias (3-10 abnormal beats, right) in FKBP12.6 +/" and FKBP12.6 7" mice, either treated or not treated with JTV-519, respectively.
• FIG. 2 shows that JTV-519 prevents exercise-induced sudden cardiac death
• the representative single channel from an exercised FKBP12.6 " " mouse after JTV-519 treatment shows that FKBP 12.6 in the heart is required for the action of JTV-519.
• the dotted lines represent incomplete channel openings, or 'subconductance' openings, and are indicative of FKBP12.6-depleted RyR2 channels. Tracings on the left represent 5.0 sec, while tracings on the right represent 500 msec.
• Po open probability
• To average open times
• Tc average closed times
• c closed state of the channel.
• D Summary bar graph showing average open probabilities of single RyR2 channels (see above). JTV-519 dramatically reduces the open probability of RyR2 from FKBP12.6 + " mice following exercise testing at diastolic calcium concentrations (150 nM).
• FIG. 3 illustrates JTV-519-normalized RyR2-channel gating by increased
• CPVT catecholaminergic polymorphic ventricular tachycardia
• SCD sudden cardiac death
• JTV-519 a benzothiazepine derivative, prevents lethal ventricular arrhythmias in mice heterozygous for the FKBP 12.6 gene.
• JTV-519 reduced the open probability of RyR2, isolated from FKBP12.6 +/" mice that died following exercise, by increasing the affinity of FKBP12.6 for PKA-phosphorylated RyR2.
• JTV-519 normalized gating of CPVT-associated mutant RyR2 channels by increasing FKBP 12.6 binding affinity.
• the present invention provides a method for limiting or preventing a decrease in the level of RyR2-bound FKBP12.6 in cells of a subject.
• FKBP12.6 includes both an "FKBP12.6 protein” and an "FKBP12.6 analogue”.
• protein shall include a protein, protein domain, polypeptide, or peptide, and any fragment thereof.
• An "FKBP 12.6 analogue” is a functional variant of the FKBP12.6 protein, having FKBP12.6 biological activity, that has 60% or greater (preferably, 70% or greater) amino-acid-sequence homology with the FKBP 12.6 protein.
• FKBP 12.6 biological activity refers to the activity of a protein or peptide that demonstrates an ability to associate physically with, or bind with, unphosphorylated or non-hyperphosphorylated RyR2 (i.e., binding of approximately two fold, or, more preferably, approximately five fold, above the background binding of a negative control), under the conditions of the assays described herein, although affinity may be different from that of FKBP 12.6.
• RyR2 includes both an “RyR2 protein” and an
• RyR2 analogue is a functional variant of the RyR2 protein, having RyR2 biological activity, that has 60% or greater (preferably, 70% or greater) amino-acid- sequence homology with the RyR2 protein.
• the RyR2 of the present invention may be unphosphorylated, phosphorylated, or hyperphosphorylated.
• RyR2 biological activity refers to the activity of a protein or peptide that demonstrates an ability to associate physically with, or bind with, FKBP 12.6 (i.e., binding of approximately two fold, or, more preferably, approximately five fold, above the background binding of a negative control), under the conditions of the assays described herein, although affinity may be different from that of RyR2.
• the cardiac ryanodine receptor, RyR2 is a protein complex comprising four 565,000-dalton RyR2 proteins in association with four 12,000- dalton FKBP12.6 proteins.
• FK506 binding proteins FKBPs
• FKBP 12.6 protein is tightly bound to, and regulates the function of, RyR2.
• FKBP12.6 binds to the RyR2 channel, one molecule per RyR2 subunit, stabilizes RyR2-channel function, and facilitates coupled gating between neighboring RyR2 channels, thereby preventing aberrant activation of the channel during the resting phase of the cardiac cycle.
• RyR2-bound FKBP12.6 includes a molecule of an FKBP12.6 protein that is bound to an RyR2 protein subunit or a tetramer of FKBP 12.6 that is in association with a tetramer of RyR2.
• a "decrease" in the level of RyR2-bound FKBP12.6 in cells of a subject refers to a detectable decrease, diminution, or reduction in the level of RyR2-bound FKBP12.6 in cells of the subject.
• Such a decrease is limited or prevented in cells of a subject when the decrease is in any way halted, hindered, impeded, obstructed, or reduced by the administration of JTV-519 (as described below), such that the level of RyR2-bound FKBP 12.6 in cells of the subject is higher than it would otherwise be in the absence of JTV-519.
• the level of RyR2-bound FKBP12.6 in a subject may be detected by standard assays and techniques, including those readily determined from the known art (e.g., immunological techniques, hybridization analysis, immunoprecipitation, Western-blot analysis, fluorescence imaging techniques, and/or radiation detection, etc.), as well as any assays and detection methods disclosed herein.
• standard assays and techniques including those readily determined from the known art (e.g., immunological techniques, hybridization analysis, immunoprecipitation, Western-blot analysis, fluorescence imaging techniques, and/or radiation detection, etc.), as well as any assays and detection methods disclosed herein.
• protein may be isolated and purified from cells of a subject using standard methods known in the art, including, without limitation, extraction from the cells (e.g., with a detergent that solubilizes the protein) where necessary, followed by affinity purification on a column, chromatography (e.g., FTLC and HPLC), immunoprecipitation (with an antibody), and precipitation (e.g., with isopropanol and a reagent such as Trizol). Isolation and purification of the protein may be followed by electrophoresis (e.g., on an SDS-polyacrylamide gel).
• a decrease in the level of RyR2-bound FKBP12.6 in a subject, or the limiting or prevention thereof, may be determined by comparing the amount of RyR2 -bound FKBP 12.6 detected prior to the administration of JTV-519 (in accordance with methods described below) with the amount detected a suitable time after administration of JTV-519. [0036] In the method of the present invention, a decrease in the level of RyR2-bound FKBP12.6 in a subject, or the limiting or prevention thereof, may be determined by comparing the amount of RyR2 -bound FKBP 12.6 detected prior to the administration of JTV-519 (in accordance with methods described below) with the amount detected a suitable time after administration of JTV-519. [0036] In the method of the present invention, a decrease in the level of RyR2-bound
• FKBP 12.6 in cells of a subject maybe limited or prevented, for example, by inhibiting dissociation of FKBP12.6 and RyR2 in cells of the subject; by increasing binding between FKBP12.6 and RyR2 in cells of the subject; or by stabilizing the RyR2-FKBP12.6 complex in cells of a subject.
• inhibiting dissociation includes blocking, decreasing, inhibiting, limiting, or preventing the physical dissociation or separation of an FKBP12.6 subunit from an RyR2 molecule in cells of the subject, and blocking, decreasing, inhibiting, limiting, or preventing the physical dissociation or separation of an RyR2 molecule from an FKBP 12.6 subunit in cells of the subject.
• the term "increasing binding” includes enhancing, increasing, or improving the ability of phosphorylated RyR2 to associate physically with FKBP 12.6 (e.g. , binding of approximately two fold, or, more preferably, approximately five fold, above the background binding of a negative control) in cells of the subject, and enhancing, increasing, or improving the ability of FKBP12.6 to associate physically with phosphorylated RyR2 (e.g., binding of approximately two fold, or, more preferably, approximately five fold, above the background binding of a negative control) in cells of the subject.
• a decrease in the level of RyR2 -bound FKBP12.6 in cells of a subject may be limited or prevented by directly decreasing the level of phosphorylated RyR2 in cells of the subject, or by indirectly decreasing the level of phosphorylated RyR2 in the cells (e.g., by targeting an enzyme (such as PKA) or another endogenous molecule that regulates or modulates the functions or levels of phosphorylated RyR2 in the cells).
• the level of phosphorylated RyR2 in the cells is decreased by at least 10% in the method of the present invention. More preferably, the level of phosphorylated RyR2 is decreased by at least 20%.
• a decrease in the level of RyR2 -bound FKBP 12.6 is limited or prevented in a subject, particularly in cells of a subject.
• the subject of the present invention may be any animal, including amphibians, birds, fish, mammals, and marsupials, but is preferably a mammal (e.g., a human; a domestic animal, such as a cat, dog, monkey, mouse, or rat; or a commercial animal, such as a cow or pig).
• the subject of the present invention is a candidate for exercise-induced cardiac arrhythmia.
• Exercise-induced cardiac arrhythmia is a heart condition (e.g.
• a ventricular fibrillation or ventricular tachycardia including any that leads to sudden cardiac death
• a "candidate" for exercise-induced cardiac arrhythmia is a subject who is known to be, or is believed to be, or is suspected of being, at risk for developing cardiac arrhythmia during/after physical exercise.
• Examples of candidates for exercise-induced cardiac arrhythmia include, without limitation, an animal/person known to have catecholaminergic polymorphic ventricular tachycardia (CPVT); an animal/person suspected of having CPVT; and an animal/person who is known to be, or is believed to be, or is suspected of being, at risk for developing cardiac arrhythmia during/after physical exercise, and who is about to exercise, is currently exercising, or has just completed exercise.
• CPVT catecholaminergic polymorphic ventricular tachycardia
• CPVT catecholaminergic polymorphic ventricular tachycardia
• an animal/person suspected of having CPVT and an animal/person who is known to be, or is believed to be, or is suspected of being, at risk for developing cardiac arrhythmia during/after physical exercise, and who is about to exercise, is currently exercising, or has just completed exercise.
• CPVT is an inherited disorder in individuals with structurally-normal hearts. It is characterized by stress-induced ventricular tachycardia -
• the cells of a subject are preferably striated muscle cells.
• a striated muscle is a muscle in which the repeating units (sarcomeres) of the contractile myo fibrils are arranged in registry throughout the cell, resulting in transverse or oblique striations that may be observed at the level of a light microscope.
• striated muscle cells include, without limitation, voluntary (skeletal) muscle cells and cardiac muscle cells.
• the cell used in the method of the present invention is a human cardiac muscle cell.
• cardiac muscle cell includes cardiac muscle fibers, such as those found in the myocardium of the heart. Cardiac muscle fibers are composed of chains of contiguous heart-muscle cells, or cardiomyocytes, joined end to end at intercalated disks. These disks possess two kinds of cell junctions: expanded desmosomes extending along their transverse portions, and gap junctions, the largest of which lie along their longitudinal portions. [0039] In the method of the present invention, a decrease in the level of RyR2 -bound
• FKBP12.6 is limited or prevented in cells of a subject by administering JTV-519 to the subject; this would also permit contact between cells of the subject and JTV-519.
• JTV-519 (4-[3-(4-benzylpiperidin-l-yl) ⁇ ropionyl]-7-methoxy-2,3,4,5-tetrahydro-l,4-benzothiazepine monohydrochloride), also known as k201, is a derivative of 1,4-benzothiazepine, and a modulator of calcium-ion channels.
• JTV-519 modulates the Na + current and the inward-rectifier K + current in guinea pig ventricular cells, and inhibits the delayed-rectifier K current in guinea pig atrial cells.
• FK506 and rapamycin are drugs that may be used to design other compounds that stabilize RyR2-FKBP12.6 binding in cells of a subject who is a candidate for exercise-induced cardiac arrhythmia.
• FK506 and rapamycin both dissociate FKBP12.6 from RyR2. It is possible to design and/or screen for compounds that are structurally related to these drugs, but have the opposite effects.
• JTV-519 may be administered to a subject by way of a therapeutic composition, comprising JTV-519 and a pharmaceutically- acceptable carrier.
• a therapeutic composition comprising JTV-519 and a pharmaceutically- acceptable carrier.
• the pha ⁇ naceutically-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.
• the pharmaceutically-acceptable carrier employed herein is selected from various organic or inorganic materials that are used as materials for pharmaceutical formulations, and which may be incorporated as analgesic agents, buffers, binders, disintegrants, diluents, emulsifiers, excipients, extenders, glidants, solubilizers, stabilizers, suspending agents, tonicity agents, vehicles, and viscosity-increasing agents.
• pharmaceutical additives such as antioxidants, aromatics, colorants, flavor-improving agents, preservatives, and sweeteners, may also be added.
• acceptable pharmaceutical carriers include carboxymethyl cellulose, crystalline cellulose, glycerin, gum arabic, lactose, magnesium stearate, methyl cellulose, powders, saline, sodium alginate, sucrose, starch, talc, and water, among others.
• the pharmaceutical formulations of the present invention may be prepared by methods well-known in the pharmaceutical arts.
• the JTV-519 may be brought into association with a carrier or diluent, as a suspension or solution.
• a carrier or diluent as a suspension or solution.
• one or more accessory ingredients e.g., buffers, flavoring agents, surface active agents, and the like
• the choice of carrier will depend upon the route of administration.
• JTV-519 may be administered to a subj ect by contacting target cells (e.g. , cardiac muscle cells) in vivo in the subject with the JTV-519.
• JTV-519 may be contacted with (e.g. , introduced into) cells of the subject using known techniques utilized for the introduction and administration of proteins, nucleic acids, and other drugs.
• methods for contacting the cells with (i.e., treating the cells with) JTV-519 include, without limitation, absorption, electroporation, immersion, injection, introduction, liposome delivery, transfection, transfusion, vectors, and other drug-delivery vehicles and methods.
• the target cells When the target cells are localized to a particular portion of a subject, it may be desirable to introduce the JTV-519 directly to the cells, by injection or by some other means (e.g., by introducing the JTV-519 into the blood or another body fluid).
• the target cells may be contained in heart tissue of a subject, and may be detected in heart tissue of the subject by standard detection methods readily determined from the known art, examples of which include, without limitation, immunological techniques (e.g., immunohistochemical staining), fluorescence imaging techniques, and microscopic techniques.
• the JTV-519 of the present invention may be administered to a ' human or animal subject by known procedures, including, without limitation, oral administration, parenteral administration, and transdermal administration.
• the JTV-519 is administered parenterally, by epifascial, intracapsular, intracranial, intracutaneous, intrathecal, intramuscular, intraorbital, intraperitoneal, intraspinal, intrasternal, intravascular, intravenous, parenchymatous, subcutaneous, or sublingual injection, or by way of catheter.
• the agent is administered to the subject by way of targeted delivery to cardiac muscle cells via a catheter inserted into the subject's heart.
• a JTV-519 formulation may be presented as capsules, tablets, powders, granules, or as a suspension.
• the formulation may have conventional additives, such as lactose, mannitol, corn starch, or potato starch.
• the formulation also may be presented with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatins.
• the formulation may be presented with disintegrators, such as corn starch, potato starch, or sodium carboxymethylcellulose.
• the formulation also may be presented with dibasic calcium phosphate anhydrous or sodium starch glycolate.
• the formulation may be presented with lubricants, such as talc or magnesium stearate.
• JTV-519 may be combined with a sterile aqueous solution that is preferably isotonic with the blood of the subject.
• a sterile aqueous solution that is preferably isotonic with the blood of the subject.
• Such a formulation may be prepared by dissolving a solid active ingredient in water containing physiologically-compatible substances, such as sodium chloride, glycine, and the like, and having a buffered pH compatible with physiological conditions, so as to produce an aqueous solution, then rendering said solution sterile.
• the formulation may be presented in unit or multi-dose containers, such as sealed ampoules or vials.
• the formulation may be delivered by any mode of injection, including, without limitation, epifascial, intracapsular, intracranial, intracutaneous, intrathecal, intramuscular, intraorbital, intraperitoneal, intraspinal, intrasternal, intravascular, intravenous, parenchymatous, subcutaneous, or sublingual, or by way of catheter into the subject's heart.
• JTV-519 may be combined with skin penetration enhancers, such as propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone, and the like, which increase the permeability of the skin to the JTN-519, and permit the JTN-519 to penetrate through the skin and into the bloodstream.
• the JTV-519/enhancer composition also may be further combined with a polymeric substance, such as ethylcellulose, hydroxypropyl cellulose, ethylene/vinylacetate, polyvmyl pyrrolidone, and the like, to provide the composition in gel form, which may be dissolved in a solvent, such as methylene chloride, evaporated to the desired viscosity, and then applied to backing material to provide a patch.
• a polymeric substance such as ethylcellulose, hydroxypropyl cellulose, ethylene/vinylacetate, polyvmyl pyrrolidone, and the like
• JTV-519 may be administered to the subject (and JTV-519 may be contacted with cells of the subject) in an amount effective to limit or prevent a decrease in the level of RyR2 -bound FKBP 12.6 in the subject, particularly in cells of the subject.
• This amount may be readily determined by the skilled artisan, based upon known procedures, including analysis of titration curves established in vivo, and methods and assays disclosed herein.
• a suitable amount of JTV-519 effective to limit or prevent a decrease in the level of RyR2-bound FKBP12.6 in the subject may range from about 5 mg/kg/day to about 20 mg/kg/day, and/or may be an amount sufficient to achieve plasma levels ranging from about 300 ng/ml to about 1000 ng/ml.
• the amount of JTV-519 ranges from about 10 mg/kg/day to about 20 mg/kg/day.
• the subject has not yet developed exercise-induced cardiac arrhythmia.
• the amount of JTV-519 effective to limit or prevent a decrease in the level of RyR2 -bound FKBP12.6 in the subject may be an amount of JTV-519 effective to prevent exercise-induced cardiac arrhythmia in the subject.
• Cardiac arrhythmia is a disturbance of the electrical activity of the heart that manifests as an abnormality in heart rate or heart rhythm.
• an amount of JTV-519 "effective to prevent exercise-induced cardiac arrhythmia” includes an amount of JTV-519 effective to prevent the development of the clinical impairment or symptoms of the exercise-induced cardiac arrhythmia (e.g., palpitations, fainting, ventricular fibrillation, ventricular tachycardia, and sudden cardiac death).
• the amount of JTV-519 effective to prevent exercise-induced cardiac arrhythmia in a subject will vary depending upon the particular factors of each case, including the type of exercise-induced cardiac arrhythmia, the subject's weight, the severity of the subject's condition, and the mode of administration of the JTV- 519. This amount may be readily determined by the skilled artisan, based upon known procedures, including clinical trials, and methods disclosed herein.
• the amount of JTV-519 effective to prevent the exercise-mduced cardiac arrhythmia is an amount of JTV-519 effective to prevent exercise-induced sudden cardiac death in the subject.
• the JTV- 19 prevents exercise- induced cardiac arrhythmia and exercise-induced sudden cardiac death in the subject.
• JTV-519 may also be useful in treating a subject who has already started to experience clinical symptoms of exercise-induced cardiac arrhythmia. If the symptoms of arrhythmia are observed in the subject early enough, JTV-519 might be effective in limiting or preventing a further decrease in the level of RyR2 -bound FKBP12.6 in the subject.
• the subject has been exercising, or is currently exercising, and has developed exercise-induced cardiac arrhythmia.
• the amount of JTV-519 effective to limit or prevent a decrease in the level of RyR2 -bound FKBP12.6 in the subject may be an amount of JTV-519 effective to treat exercise-induced cardiac arrhythmia in the subject.
• an amount of JTV- 51,9 "effective to treat exercise-induced cardiac arrhythmia" includes an amount of JTV-519 effective to alleviate or ameliorate the clinical impairment or symptoms of the exercise- induced cardiac arrhythmia (e.g. , palpitations, fainting, ventricular fibrillation, ventricular tachycardia, and sudden cardiac death).
• the amount of JTV-519 effective to treat exercise- induced cardiac arrhythmia in a subject will vary depending upon the particular factors of each case, including the type of exercise-induced cardiac arrhythmia, the subject's weight, the severity of the subject's condition, and the mode of administration of the JTV-519. This amount may be readily determined by the skilled artisan, based upon known procedures, including clinical trials, and methods disclosed herein, hi a preferred embodiment, the JTV- 519 treats exercise-induced cardiac arrhythmia in the subject.
• the present invention further provides a method for treating exercise-induced cardiac arrhythmia in a subject.
• the method comprises administering JTV-519 to the subject in an amount effective to treat exercise-induced cardiac arrhythmia in the subject.
• a suitable amount of JTV-519 effective to treat exercise-induced cardiac arrhythmia in the subject may range from about 5 mg/kg/day to about 20 mg/kg/day, and/or may be an amount sufficient to achieve plasma levels ranging from about 300 ng/ml to about 1000 ng/ml.
• the present invention also provides a method for preventing exercise-induced cardiac arrhythmia in a subject. The method comprises administering JTV-519 to the subject in an amount effective to prevent exercise-induced cardiac arrhythmia in the subject.
• a suitable amount of JTV-519 effective to prevent exercise-induced cardiac arrhythmia in the subject may range from about 5 mg/kg/day to about 20 mg/kg/day, and/or may be an amount sufficient to achieve plasma levels ranging from about 300 ng/ml to about 1000 ng/ml.
• the present invention provides a method for preventing exercise-induced sudden cardiac death in a subject. The method comprises administering JTV-519 to the subject in an amount effective to prevent exercise-induced sudden cardiac death in the subject.
• a suitable amount of JTV- 519 effective to prevent exercise-induced sudden cardiac death in the subject may range from about 5 mg/kg/day to about 20 mg/kg/day, and/or may be an amount sufficient to achieve plasma levels ranging from about 300 ng/ml to about 1000 ng/ml.
• the exercise-induced cardiac arrhythmia in the subject is associated with VT.
• the VT is CPVT.
• the subject is a candidate for exercise- induced cardiac arrhythmia, including candidates for exercise-induced sudden cardiac death.
• the present invention also provides use of
• JTV-519 in a method for limiting or preventing a decrease in the level of RyR2-bound FKBP12.6 in a subject who is a candidate for exercise-induced cardiac arrhythmia.
• the present invention also provides use of JTV-519 in a method for treating or preventing exercise-induced cardiac arrhythmia in a subject.
• the present invention provides use of JTV-519 in a method for preventing exercise-induced sudden cardiac death in a subject.
• PKA protein kinase A
• RyR2 In failing hearts (including human hearts and animal models of heart failure), RyR2 is PKA- hyperphosphorylated, resulting in defective channels that have decreased amounts of bound FKBP 12.6, and have increased sensitivity to calcium-induced activation. The net result of these changes is that the RyR2 channels are "leaky". These channel leaks can result in a depletion of intracellular stores of calcium to such an extent that there is no longer enough calcium in the sarcoplasmic reticulum (SR) to provide a strong stimulus for muscle contraction. This results in weak contraction of heart muscle. As a second consequence of the channel leaks, RyR2 channels release calcium during the resting phase of the heart cycle known as "diastole". This release of calcium during diastole can trigger the fatal arrhythmias of the hearts (e.g., ventricular tachycardia and ventricular fibrillation) that cause sudden cardiac death (SCD).
• SCD sudden cardiac death
• LVAD left ventricular assist device
• a mechanical pumping device referred to as a left ventricular assist device (LVAD)
• LVAD left ventricular assist device
• the inventors have shown that treatment of dogs (who have pacing-induced heart failure) with beta-adrenergic blockers (beta blockers) reverses the PKA hyperphosphorylation of RyR2.
• Beta blockers inhibit the pathway that activates PKA.
• PKA phosphorylation of RyR2 increases the activity of the channel, resulting in the release of more calcium into the cell for a given trigger (activator) of the channel.
• the inventors have established that exercise- induced sudden cardiac death is associated with an increase in phosphorylation of RyR2 proteins (particularly CPVT-associated RyR2 mutant proteins) and a decrease in the level of RyR2 -bound FKBP 12.6. It is possible to use this mechanism to design effective drugs for preventing exercise-induced sudden cardiac death.
• a candidate agent having the ability to limit or prevent a decrease in the level of RyR2 -bound FKBP 12.6 may, as a consequence of this limiting or preventive activity, have an effect on an RyR2-associated biological event, thereby preventing exercise-induced sudden cardiac death.
• the present invention further provides a method for identifying an agent for use in preventing exercise-induced sudden cardiac death.
• the method comprises the steps of: (a) obtaining or generating a culture of cells containing RyR2; (b) contacting the cells with a candidate agent; (c) exposing the cells to one or more conditions known to increase phosphorylation of RyR2 in cells; and (d) determining if the agent limits or prevents i a decrease in the level of RyR2 -bound FKBP12.6 in the cells.
• an "agent” shall include a protein, polypeptide, peptide, nucleic acid (including DNA or RNA), antibody, Fab fragment, F(ab') 2 fragment, molecule, compound, antibiotic, drug, and any combination(s) thereof.
• An agent that limits or prevents a decrease in the level of RyR2- bound FKBP12.6 may be either natural or synthetic, and may be an agent reactive with (i.e., an agent that has affinity for, binds to, or is directed against) RyR2 and/or FKBP12.6.
• a cell "containing RyR2" is a cell (preferably, a cardiac muscle cell) in which RyR2, or a derivative or homologue thereof, is naturally expressed or naturally occurs. Conditions Icnown to increase phosphorylation of RyR2 in cells include, without limitation, PKA.
• cells may be contacted with a candidate agent by any of the standard methods of effecting contact between drugs/agents and cells, including any modes of introduction and administration described herein.
• the level of RyR2-bound FKBP12.6 in the cell may be measured or detected by known procedures, including any of the methods, molecular procedures, and assays Icnown to one of slcill in the art or described herein.
• the agent limits or prevents a decrease in the level of RyR2-bound FKBP12.6 in the cells.
• RyR2 has been implicated in a number of biological events in striated muscle cells. For example, it has been shown that RyR2 channels play an important role in EC coupling and contractility in cardiac muscle cells. Therefore, it is clear that preventive drugs designed to limit or prevent a decrease in the level of RyR2 -bound FKBP12.6 in cells, particularly cardiac muscle cells, may be useful in the regulation of a number of RyR2-associated biological events, including EC coupling and contractility. Thus, once the candidate agent of the present invention has been screened, and has been determined to have a suitable limiting or preventive effect on decreasing levels of RyR2-bound FKBP 12.6, it may be evaluated for its effect on EC coupling and contractility in cells, particularly cardiac muscle cells. It is expected that the preventive agent of the present invention will be useful for preventing exercise-induced sudden cardiac death.
• the method of the present invention may further comprise the steps of: (e) contacting the candidate agent with a culture of cells containing RyR2; and (f) determining if the agent has an effect on an RyR2-associated biological event in the cells.
• an "RyR2-associated biological event” includes a biochemical or physiological process in which RyR2 levels or activity have been implicated.
• examples of RyR2-associated biological events include, without limitation, EC coupling and contractility in cardiac muscle cells.
• a candidate agent may be contacted with one or more cells (preferably, cardiac muscle cells) in vitro.
• a culture of the cells may be incubated with a preparation containing the candidate agent.
• the candidate agent's effect on an RyR2-associated biological event then may be assessed by any biological assays or methods known in the art, including immunoblotting, single-channel recordings and any others disclosed herein.
• the present invention is further directed to an agent identified by the above- described identification method, as well as a pharmaceutical composition comprising the agent and a pharmaceutically-acceptable carrier.
• the agent may be useful for preventing exercise-induced sudden cardiac death in a subject, and for treating or preventing other RyR2-associated conditions.
• an "RyR2-associated condition" is a condition, disease, or disorder in which RyR2 level or activity has been implicated, and includes an RyR2-associated biological event.
• the RyR2-associated condition may be treated or prevented in the subject by administering to the subject an amount of the agent effective to treat or prevent the RyR2-associated condition in the subject. This amount may be readily determined by one skilled in the art.
• the present invention provides a method for preventing exercise-induced sudden cardiac death in a subject, by administering the agent to the subject in an amount effective to prevent the exercise-induced sudden cardiac death in the subject.
• the present invention also provides an in vivo method for identifying an agent for use in preventing exercise-induced sudden cardiac death.
• the method comprises the steps of: (a) obtaining or generating an animal containing RyR2; (b) administering a candidate agent to the animal; (c) exposing the animal to one or more conditions known to increase phosphorylation of RyR2 in cells; and (d) determining if the agent limits or prevents a decrease in the level of RyR2-bound FKBP12.6 in the animal.
• the method may further comprise the steps of: (e) administering the agent to an animal containing RyR2; and (f) determining if the agent has an effect on an RyR2-associated biological event in the animal.
• an agent identified by this method a pharmaceutical composition comprising this agent; and a method for preventing exercise-induced sudden cardiac death in a subject, by administering this agent to the subject in an amount effective to prevent the exercise-induced sudden cardiac death in the subject.
• the inventors' work has demonstrated that compounds which block PKA activation would be expected to reduce the activation of the RyR2 channel, resulting in less release of calcium into the cell.
• Compounds that bind to the RyR2 channel at the FKBP12.6 binding site, but do not come off the channel when the channel is phosphorylated by PKA, would also be expected to decrease the activity of the channel in response to PKA activation or other triggers that activate the RyR2 channel. Such compounds would also result in less calcium release into the cell.
• the present invention further provides additional assays for identifying agents that may be useful in preventing exercise-induced sudden cardiac death, in that they block or inhibit activation of RyR2.
• the diagnostic assays of the present invention may screen for the release of calcium into cells via the RyR2 channel, using calcium-sensitive fluorescent dyes (e.g., Fluo-3, Fura-2, and the like).
• Cells may be loaded with the fluorescent dye of choice, then stimulated with RyR2 activators to determine whether or not compounds added to the cell reduce the calcium-dependent fluorescent signal (Brillantes et al, Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein. Cell, 77:513-23, 1994; Gillo et al, Calcium entry during induced differentiation in murine erythroleukemia cells.
• an assay may involve the expression of recombinant RyR2 channels in a heterologous expression system, such as Sf9, HEK293, or CHO cells (Brillantes et al. , Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein. Cell, 77:513-23, 1994).
• RyR2 could also be co-expressed with beta- adrenergic receptors. This would permit assessment of the effect of compounds on RyR2 activation, in response to addition of beta-adrenergic receptor agonists.
• the level of PKA phosphorylation of RyR2 which correlates with the degree of heart failure may also be assayed, and then used to determine the efficacy of compounds designed to block the PKA phosphorylation of the RyR2 channel.
• Such an assay may be based on the use of antibodies that are specific for the RyR2 protein.
• the RyR2- channel protein may be immunoprecipitated, and then back-phosphorylated with PKA and
• [ ⁇ 32 P]-ATP The amount of radioactive [ 32 P] label that is transferred to the RyR2 protein may be then measured using a phosphorimager (Marx et al, PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell, 101:365-76, 2000).
• Another assay of the present invention involves use of a phosphoepitope- specific antibody that detects RyR2 that is PKA phosphorylated on Ser 2809. Immunob lotting with such an antibody can be used to assess efficacy of therapy for heart failure and cardiac arrhythmias.
• RyR2 S2809A and RyR2 S2809D knock-in mice may be used to assess efficacy of therapy for heart failure and cardiac arrhythmias. Such mice further provide evidence that PKA hyperphosphorylation of RyR2 is a contributing factor in heart failure and cardiac arrhythmias, by showing that the RyR2
• 1,4-benzothiazepine derivatives are important building blocks in the preparation of biologically- active molecules, including JTV-519.
• the inventors have developed a novel process for preparing 1,4-benzothiazepine intermediate compounds, such as 7-methoxy-2,3,4,5-tetrahydro-l,4-benzothiazepine.
• the inventors' process utilizes readily- available and inexpensive starting materials, and provides high yields of key 1,4- benzothiazepine intermediates.
• Kaneko et al. US Patent 5,416,066; WO 92/12148;
• JP4230681 disclosed that JTV-519 could be prepared by reacting 7-methoxy-2,3,4,5- tetrahydro- 1,4-benzothiazepine (a 1,4-benzothiazepine intermediate) with acryloyl chloride, and then reacting the resulting product with 4-benzyl piperidine.
• the product of this step was refluxed with sodium methoxide in methanol for 20 h.
• the product of the reflux step was then reacted with 2-chloroethylamine, under basic conditions and at a high temperature, to produce a cyclized amide.
• the cyclized amide was reduced with LiAlH 4 to yield 7-methoxy-2,3,4,5-tetrahydro- 1,4-benzothiazepine (a 1,4-benzothiazepine intermediate).
• the second process for the preparation of 7-methoxy-2,3,4,5-tetrahydro-l ,4- benzothiazepine was disclosed by Hitoshi in a Japanese patent (JP 10045706).
• This process started with 2-bromo-5-methoxy benzaldehyde.
• the bromide was substituted with NaSMe, and the resulting product was oxidized with chlorine, followed by reflux in water, to yield disulfide dialdehyde.
• the dialdehyde was treated with 2-chloroethylamine, and the resulting product was reduced with a reducing agent, such as NaBH 4 .
• the resulting compound was cyclized to give 7-methoxy-2,3,4,5-tetrahydro-l,4-benzothiazepine.
• the inventors also determined that the process described by Hitoshi (JP 10045706) involved Cl , and that another patented method for the preparation of the first intermediate, apart from the substitution of bromide with NaSMe, had to be used. [0073] To overcome the foregoing problems, the inventors developed a novel process for making 7-methoxy-2,3,4,5-tetrahydro-l,4-benzothiazepine from readily-available and inexpensive starting materials. The inventors' process simplifies isolation and purification steps, and can be used to prepare various 1,4-benzothiazepine intermediates, including 7- methoxy-2,3,4,5-tetrahydro-l,4-benzothiazepine and other compounds having the general structure shown in formula:
• R2 alkyl
• R3 alkyl
• This process may also be used to prepare JTV-519.
• the present invention provides a method for the synthesis of a compound of a compound having formula:
• R is as defined above;
• step (b) treating the compound formed in step (a) with a diazotizing agent and a disulfide, to form a compound having formula:
• R is as defined above;
• step (c) treating the compound formed in step (b) with a chloride and a chloroethylamine, to form a compound having formula:
• R is as defined above;
• step (d) treating the compound formed in step (c) with a reducing agent and a base, in the presence of tetrahydrolate, to form a compound having fonnula:
• step (e) treating the compound formed in step (d) with a reducing agent, to form a compound having formula:
• the reducing agent in step (a) may be H 2 .
• the diazotizing agent in step (b) may be NaNO 2
• the disulfide in step (b) may be Na S 2 .
• the chloride in step (c) may be SOCl 2 .
• the reducing agent in step (d) may be trimethylphosphine (PMe 3 ), while the base in step (d) is triethyl amine.
• the reducing agent in step (e) is LiAlH .
• the present invention further provides a method for the synthesis of a compound of having formula:
• the method of the present invention further provides a method for the synthesis of a compound having formula: X7
• said method comprising the steps of:
• step (b) treating the compound formed in step (a) with a diazotizing agent and a disulfide, to fonn a compound having formula:
• step (c) treating the compound formed in step (b) with a chloride and a chloroethylamme, to form a compound having formula:
• step (d) treating the compound formed in step (c) with a reducing agent and a base, in the presence of tetrahydrolate, to form a compound having formula:
• step (e) treating the compound formed in step (d) with a reducing agent, to form a compound having formula: 5
• the present invention also provides a method for the synthesis of a compound having formula:
• said method comprising the step of:
• 7- methoxy-2,3,4,5-tetrahydro-l,4-benzothiazepine may be prepared from 2-nitro-5- methoxybenzoic acid as follows.
• the nitro group of 2-nitro-5-methoxybenzoic acid is reduced, using H 2 with Pd/C as a catalyst, to give 2-amino-5-methoxybenzoic acid.
• 2-amino- 5-methoxybenzoic acid may be diazotized with NaNO 2 , and then treated with Na S 2 , to provide a stable disulfide compound.
• the stable disulfide compound may be treated with SOCl 2 , and then reacted with 2-chloroethylamine, in the presence of Et 3 N, to' give an amide.
• the amide compound may then be converted to a cyclized compound via a one-pot procedure, as follows.
• a reducing reagent such as trimethylphosphine or triphenylphosphine
• a base such as triethylamine
• the reducing agent (trimethylphosphine or triphenylphine) cleaves the disulfide (S-S) to its monosulfide (-S), which, in situ, undergoes intramolecular cyclization with the chloride to yield a cyclized amide.
• the cyclized amide may then be reduced with LiAlH 4 to yield the 1,4-benzothiazepine intermediate, 7-methoxy- 2,3,4,5-tetrahydro-l,4-benzothiazepine.
• JTV-519 may then be prepared from 7-methoxy- 2,3, 4,5-tetrahydro- 1,4-benzothiazepine by reacting the 7-methoxy-2,3,4,5-tetrahydro-l,4- benzothiazepine with 3-bromopropionic chloride, and then reacting the resulting compound with 4-benzyl piperidine.
• radio- labeled JTV-519 may be prepared as follows. JTV-519 may be demethylated at the phenyl ring using BBr . The resulting phenol compound may then be re-methylated with a radio- labeled methylating agent (such as 3 H-dimethyl sulfate) in the presence of a base (such as NaH) to provide 3 H-labeled JTV-519.
• a radio- labeled methylating agent such as 3 H-dimethyl sulfate
• a base such as NaH
• the present invention further provides a composition, comprising radio- labeled JTV-519.
• Labeling of JTV-519 may be accomplished using one of a variety of different radioactive labels known in the art.
• the radioactive label of the present invention may be, for example, a radioisotope.
• the radioisotope may be any isotope that emits detectable radiation, including, without limitation, S, P, I, H, or C. Radioactivity emitted by the radioisotope can be detected by techniques well known in the art. For example, gamma emission from the radioisotope may be detected using gamma imaging techniques, particularly scintigraphic imaging.
• mouse genomic ⁇ -phage clones for the murine orthologue of the human FK506 binding protein 12.6 were isolated from a DBA/llacJ library, using a full-length murine cDNA probe.
• the targeting vector was designed to delete exons 3 and 4, which contain the entire coding sequences for murine FKBP 12.6 (Bennett et al, Identification and characterization of the murine FK506 binding protein (FKBP) 12.6 gene. Mamm. Genome, 9:1069-71, 1998), by replacing 3.5 kb of murine genomic DNA with a PGK-neo selectable marker.
• a 5.0-lcb 5' fragment and a 1.9-kb 3' fragment were cloned into pJNS2, a backbone vector with PGK-neo and PGK-TK cassettes.
• the DBA/lacJ embryonic stem (ES) cells were grown and transfected, using established protocols. Targeted ES cells were first screened by Southern analysis, and 5 positive ES cell lines were analyzed by PCR to confirm homologous recombination. Male chimeras were bred to DBA/llacJ females, and germline offspring identified by brown coat color. Germline offspring were genotyped using 5' Southern analysis.
• FKBP12.6 + + mice as controls. No differences were observed between FKBP12.6 " " mice raised on the following backgrounds: DBA C57BL6 mixed, pure DBA, and pure C57BL6.
• mice were exercised on an inclined treadmill until exhaustion, and then intraperitoneally injected with epinephrine (0.5-2.0 mg/kg) (Wehrens et al, FKBP 12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death. Cell, 113:829-40, 2003). Resting heart rates of ambulatory animals were averaged over 4 h.
• EXAMPLE 3 EXPRESSION OF WILD-TYPE AND RvR2-S2809D MUTANTS
• Vesicles containing RyR2 channels were prepared, as previously described (Wehrens et al, FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death. Cell, 113:829-40, 2003).
• microsomes 100 ⁇ g of microsomes were diluted in 100 ⁇ l of 10-mM imidazole buffer (pH 6.8), incubated with 250-nM (final concentration) [ 35 S]-FKBP12.6 at 37°C for 60 min, then quenched with 500 ⁇ l of ice-cold imidazole buffer. Samples were centrifuged at 100,000 g for 10 min, and washed three times in imidazole buffer. The amount of bound [ 35 S]-FKBP12.6 was detennined by liquid scintillation counting of the pellet.
• EXAMPLE 5 IMMUNOBLOTS [0089] Immunoblotting of microsomes (50 ⁇ g) was performed as described, with anti-FKBP12/12.6 (1:1,000), anti-RyR (5029; 1:3,000) (Jayaraman et al, FK506 binding protein associated with the calcium release channel (ryanodine receptor). J. Biol. Chem., 267:9474-77, 1992), or anti-phosphoRyR2 (P2809; 1 :5,000) for 1 h at room temperature (Reilcen et al, Beta-b lockers restore calcium release channel function and improve cardiac muscle performance in human heart failure. Circulation, 107:2459-66, 2003).
• the P2809- phosphoepitope-specific anti-RyR2 antibody is an affinity-purified polyclonal rabbit antibody, custom-made by Zymed Laboratories (San Francisco, CA) using the peptide, CRTRRI-(pS)-QTSQ, which conesponds to RyR2 PKA-phosphorylated at Ser 2809 . After incubation with HRP-labeled anti-rabbit IgG (1 :5,000 dilution; Transduction Laboratories, Lexington, KY), the blots were developed using ECL (Amersham Pharmacia, Piscataway, NJ).
• Symmetric solutions used for channel recordings were: trans compartment - HEPES, 250 mmol/L; Ba(OH) , 53 mmol/L (in some experiments, Ba(OH) 2 was replaced by Ca(OH) 2 ); pH 7.35; and cis compartment - HEPES, 250 mmol/L; Tris-base, 125 mmol/L; EGTA, 1.0 mmol/L; and CaCl , 0.5 mmol/L; pH 7.35. Unless otherwise indicated, single-channels recordings were made in the presence of 150-nM [Ca 2+ ] and 1.0-mM [Mg 2+ ] in the cis compartment.
• 3F shows that incubation of PKA-phosphorylated RyR2 with FKBP12.6, in the presence of JTV-519, shifted the Ca 2+ -dependence of RyR2 activation towards the right, making it similar to the Ca 2+ -dependence of unphosphorylated channels.
• JTV-519 was prepared by reacting compound (6) with 3-bromopropionic chloride, and then reacting the resulting product with 4-benzyl piperidine.
• the structure of JTV-519 was established by 1H NMR.

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