US20130281512A1 - Agents for treating disorders involving modulation of ryanodine receptors - Google Patents

Agents for treating disorders involving modulation of ryanodine receptors Download PDF

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US20130281512A1
US20130281512A1 US13/865,359 US201313865359A US2013281512A1 US 20130281512 A1 US20130281512 A1 US 20130281512A1 US 201313865359 A US201313865359 A US 201313865359A US 2013281512 A1 US2013281512 A1 US 2013281512A1
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compound
diseases
disorders
disease
group
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Jiaming Yan
Sandro Belvedere
Yael Webb
Mark BERTRAND
Nicole Villeneuve
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Armgo Pharma Inc
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Laboratoires Servier SAS
Armgo Pharma Inc
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Assigned to LES LABORATOIRES SERVIER, ARMGO PHARMA, INC. reassignment LES LABORATOIRES SERVIER ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BERTRAND, MARK, VILLENEUVE, NICOLE, BELVEDERE, SANDRO, WEBB, YAEL, YAN, JIAMING
Publication of US20130281512A1 publication Critical patent/US20130281512A1/en
Priority to US14/076,474 priority patent/US8853198B2/en
Priority to US14/475,980 priority patent/US20140378437A1/en
Assigned to ARMGO PHARMA, INC. reassignment ARMGO PHARMA, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LES LABORATOIRES SERVIER
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Definitions

  • the present invention relates to 1,4-benzothiazepine derivatives and their use to treat disorders and diseases associated with ryanodine receptors (RyRs) that regulate calcium channel functioning in cells.
  • the invention also discloses pharmaceutical compositions comprising these compounds and uses thereof to treat diseases and conditions associated with RyRs, in particular cardiac, skeletal muscular and central nervous system (CNS) disorders.
  • RyRs ryanodine receptors
  • CNS central nervous system
  • the sarcoplasmic reticulum is a structure in cells that functions, among other things, as a specialized intracellular calcium (Ca 2+ ) store.
  • RyRs are channels in the SR, which open and close to regulate the release of Ca 2+ from the SR into the intracellular cytoplasm of the cell. Release of Ca 2+ into the cytoplasm from the SR increases cytoplasmic Ca 2+ concentration.
  • Open probability of RyRs refers to the likelihood that a RyR is open at any given moment, and therefore capable of releasing Ca 2+ into the cytoplasm from the SR.
  • RyR1 is found predominantly in skeletal muscle as well as other tissues
  • RyR2 is found predominantly in the heart as well as other tissues
  • RyR3 is found in the brain as well as other tissues.
  • the RyR is a tetramer.
  • Part of the RyR complex is formed by four RyR polypeptides in association with four FK506 binding proteins (FKBPs) (calstabins), specifically FKBP12 (calstabin1) and FKBP12.6 (calstabin2).
  • FKBPs FK506 binding proteins
  • Calstabin1 binds to RyR1 and RyR3 while calstabin2 binds to RyR2.
  • the calstabins bind to the RyR (one molecule per RyR subunit), stabilize the RyR function, facilitate coupled gating between neighboring RyRs and prevent abnormal activation (Ca 2+ leak) of the channel by stabilizing the channel's closed state.
  • RyR2 is the major Ca 2+ release channel required for excitation-contraction (EC) coupling and muscle contraction.
  • EC excitation-contraction
  • depolarization of the cardiac-muscle cell membrane during phase zero of the action potential activates voltage-gated Ca 2+ channels.
  • Ca 2+ influx through the open voltage-gated channels in turn initiates Ca 2+ release from the SR via RyR2.
  • This process is known as Ca 2+ -induced Ca 2+ release.
  • the RyR2-mediated Ca 2+ -induced Ca 2+ release then activates the contractile proteins in the cardiac cell, resulting in cardiac muscle contraction.
  • Phosphorylation of RyR2 by protein kinase A is an important part of the “fight or flight” response that increases cardiac EC coupling gain by augmenting the amount of Ca 2+ released for a given trigger.
  • This signaling pathway provides a mechanism by which activation of the sympathetic nervous system (SNS), in response to stress, results in increased cardiac output.
  • SNS sympathetic nervous system
  • Phosphorylation of RyR2 by PKA results in partial dissociation of calstabin2 from the channel, which in turn, leads to increased open probability, and increased Ca 2+ release from the SR into the intracellular cytoplasm.
  • Heart failure is characterized by a sustained hyperadrenergic state in which serum catecholamine levels are chronically elevated.
  • This chronic hyperadrenergic state is persistent PKA hyperphosphorylation of RyR2, such that 3-4 out of the four Ser2808 in each homotetrameric RyR2 channel are chronically phosphorylated (Marx S O, et al. Cell, 2000; 101(4):365-376).
  • chronic PKA hyperphosphorylation of RyR2 is associated with depletion of the channel-stabilization subunit calstabin2 from the RyR2 channel macromolecular complex.
  • mice engineered with RyR2 lacking the PKA phosphorylation site are protected from HF progression after myocardial infarction (MI) (Wehrens X H et al. Proc Natl Acad Sci USA. 2006; 103(3):511-518).
  • MI myocardial infarction
  • RyR2-S2808D+/+ (aspartic acid replacing serine 2808) knock-in mice, that mimic constitutive PKA hyperphosphorylation of RyR2, show depletion of calstabin2 and leaky RyR2.
  • RyR2-S2808D+/+ mice develop age-dependent cardiomyopathy, demonstrate elevated RyR2 oxidation and nitrosylation, a reduced SR Ca 2+ store content, and increased diastolic SR Ca 2+ leak. After myocardial infarction, RyR2-S2808D+/+ mice exhibit increased mortality compared with WT littermates.
  • RyR2 contains about 33 free thiol residues rendering it highly sensitive to the cellular redox state. Cysteine oxidation facilitates RyR opening and SR Ca 2+ leak. Shan et al, 2010, demonstrated that oxidation and nitrosylation of RyR2 and dissociation of the stabilizing subunit calstabin2 from RyR2 induces SR Ca 2+ leak.
  • CPVT Catecholaminergic polymorphic ventricular tachycardia
  • SCD syncope and sudden cardiac death
  • Individuals with CPVT have ventricular arrhythmias when subjected to exercise, but do not develop arrhythmias at rest.
  • CPVT-associated RyR2 mutations result in “leaky” RyR2 channels due to the decreased binding of the calstabin2 subunit (Lehnart et al., 2008).
  • Skeletal muscle contraction is activated by SR Ca 2+ release via RyR1.
  • Depolarization of the transverse (T)-tubule membrane activates the dihydropyridine receptor voltage sensor (Cav1.1) that in turn activates RyR1 channels via a direct protein-protein interaction causing the release of SR Ca 2+ stores.
  • Ca 2+ binds to troponin C allowing actin-myosin cross-bridging to occur and sarcomere shortening.
  • the RyR1 macromolecular complex consists of a tetramer of the 560-kDa RyR1 subunit that forms a scaffold for proteins that regulate channel function including PKA and the phosphodiesterase 4D3 (PDE4D3), protein phosphatase 1 (PP1) and calstabin1.
  • PKA PKA and the phosphodiesterase 4D3
  • PP1 protein phosphatase 1
  • mAKAP protein phosphatase 1
  • spinophilin targets PP1 to the channel (Marx et al. 2000; Brillantes et al., Cell, 1994, 77, 513-523; Bellinger et al. J. Clin. Invest. 2008, 118, 445-53).
  • Calstabin1 concentrations in skeletal muscle are reported to be approximately 200 nM and that PKA phosphorylation of RyR1 reduces the binding affinity of calstabin1 for RyR1 from approximately 100-200 nM to more than 600 nM.
  • PKA phosphorylation of RyR1 reduces the binding affinity of calstabin1 for RyR1 from approximately 100-200 nM to more than 600 nM.
  • RyR1 regulation of RyR1 by posttranslational modifications other than phosphorylation, such as by nitrosylation of free sulfhydryl groups on cysteine residues (S-nitrosylation), as well as channel oxidation, have been reported to increase RyR1 channel activity. S-nitrosylation and oxidation of RyR1 have each been shown to reduce calstabin1 binding to RyR1.
  • DMD Duchenne muscular dystrophy
  • X-linked Duchenne muscular dystrophy
  • Mutations in dystrophin associated with DMD lead to a complete loss of the dystrophin protein, thereby disrupting the link between the subsarcolemma cytoskeleton and the extracellular matrix. This link is essential for protecting and stabilizing the muscle against contraction induced injury.
  • Emerging interventions in Phase I/II clinical trials are exon skipping, myostatin inhibition, and up-regulation of utrophin.
  • PCT International patent publication WO 2008/060332 relates to the use of 1,4-benzothiazepine derivatives for treating muscle fatigue in subjects suffering from pathologies such as muscular dystrophy, or in subjects suffering from muscle fatigue as a result of sustained, prolonged and/or strenuous exercise, or chronic stress.
  • PCT International patent publication WO 2008/021432 relates to the use of 1,4-benzothiazepine derivatives for the treatment and/or prevention of diseases, disorders and conditions affecting the nervous system.
  • PCT International patent publication WO 2012/019076 relates to the use of 1,4-benzothiazepine derivatives for the treatment and/or prevention of cardiac ischemia/reperfusion injury. Fauconnier et al., Proc Natl Acad Sci USA, 2011, 108(32): 13258-63 reported that RyR leak mediated by caspase-8 activation leads to left ventricular injury after myocardial ischemia-reperfusion, and that treatment with 5107 inhibited the SR Ca 2+ leak, reduced ventricular arrhythmias, infarct size, and left ventricular remodeling at 15 days after reperfusion.
  • PCT International patent publication WO 2012/019071 relates to the use of 1,4-benzothiazepine derivatives for the treatment and/or prevention of sarcopenia.
  • PCT International patent publication WO 2012/037105 relates to the use of 1,4-benzothiazepine derivatives for the treatment and/or prevention of stress-induced neuronal disorders and diseases.
  • the present invention provides novel 1,4-benzothiazepine derivatives, and their pharmaceutically acceptable salts.
  • the compounds of the present invention are ryanodine receptor (RyR) calcium channel stabilizers, sometimes referred to as “Ryca1sTM”
  • the present invention further provides methods of using these compounds for treating disorders and diseases associated with RyRs.
  • the compounds of the present invention are a selection from the 1,4-benzothiazepine derivatives described in WO 2007/024717.
  • WO 2007/024717 describes structurally similar compounds, however, as further described herein, these compounds have been found to be highly unstable and thus their therapeutic utility as drugs is limited.
  • the problem underlying the present application is thus to provide alternative 1,4-benzothiazepine derivatives that are not only pharmacologically active—but also have favorable properties such as high metabolic stability, and thus are suitable as drugs in treating diseases and conditions associated with the RyR, for example cardiac, skeletal muscular and central nervous system (CNS) disorders.
  • CNS central nervous system
  • R is COOH
  • the compounds of Formula (I) may be present in the form of a salt with a pharmaceutically acceptable acid or base.
  • Such salts are preferably selected from the group consisting of sodium, potassium, magnesium, hemifumarate, hydrochloride and hydrobromide salts, with each possibility representing a separate embodiment of the present invention.
  • One currently preferred salt is the sodium salt.
  • Another currently preferred salt is the hemifumarate salt.
  • the compound is selected from the group consisting of compound 1, compound 4 and compound 6, and pharmaceutically acceptable salts thereof.
  • the structures of these compounds are described hereinbelow.
  • the compound is represented by the structure of compound (1):
  • compound 1 is provided as the parent compound. In other embodiments, however, compound 1 is provided in the form of a salt with a pharmaceutically acceptable acid or base.
  • a salt is selected from the group consisting of sodium, potassium, magnesium, hemifumarate, hydrochloride and hydrobromide salts, with each possibility representing a separate embodiment of the present invention.
  • One currently preferred salt is the sodium salt.
  • Another currently preferred salt is the hemifumarate salt.
  • the present invention also provides methods for the synthesis of compounds of the invention, and salts thereof.
  • the present invention also provides pharmaceutical compositions comprising one or more of the compounds of the invention, and at least one additive or excipient, e.g., fillers, diluents, binders, disintegrants, buffers, colorants, emulsifiers, flavor-improving agents, gellants, glidants, preservatives, solubilizers, stabilizers, suspending agents, sweeteners, tonicity agents, wetting agents, emulsifiers, dispersing agents, swelling agents, retardants, lubricants, absorbents, and viscosity-increasing agents.
  • the compositions may be presented in capsules, granules, powders, solutions, sachets, suspensions, or tablet dosage form.
  • the present invention further provides methods of treating or preventing various disorders, diseases and conditions associated with RyRs, such as cardiac, musculoskeletal, cognitive, CNS and neuromuscular disorders and diseases, comprising administering to a subject in need of such treatment an amount of a compound of Formula (I) or a salt thereof, effective to prevent or treat a disorder or disease associated with an RyR.
  • the present invention also provides a method of preventing or treating a leak in RyR (including RyR1, RyR2 and RyR3) in a subject, including administering to the subject an amount of a compound of Formula (I) or a salt thereof, effective to prevent or treat a leak in RyR.
  • the present invention provides a method of modulating the binding of RyRs and calstabins in a subject, including administering to the subject an amount of a compound of Formula (I) or a salt thereof, effective to modulate the amount of RyR-bound calstabin.
  • the present invention further relates to the use of a compound of Formula (I) for the manufacture of a medicament for the treatment and/or prevention of disorders, diseases and conditions associated with RyRs, such as cardiac, musculoskeletal and cognitive/CNS disorders and diseases.
  • the present invention relates to the use of a compound of Formula (I) for the manufacture of a medicament for preventing or treating a leak in RyR.
  • the present invention relates to the use of a compound of Formula (I) for the manufacture of a medicament for modulating the amount of RyR-bound calstabins.
  • the methods of the invention can be practiced on an in vitro system (e.g., cultured cells or tissues) or in vivo (e.g., in a non-human animal or a human).
  • in vitro system e.g., cultured cells or tissues
  • in vivo e.g., in a non-human animal or a human.
  • the compounds of the invention are provided in combination with exon skipping therapy, e.g., antisense oligonucleotides (AOs) so as to enhance exon skipping in an mRNA of interest, e.g., the DMD gene, as further described herein.
  • exon skipping therapy e.g., antisense oligonucleotides (AOs) so as to enhance exon skipping in an mRNA of interest, e.g., the DMD gene, as further described herein.
  • FIG. 1A Immunoblot with calstabin2 antibody showing binding of calstabin2 to PKA-phosphorylated RyR2 in the absence ( ⁇ ) or presence of 100 nM compound 1. (+): calstabin binding to non-PKA phosphorylated RyR2. S36 (U.S. Pat. No. 7,544,678), is used as a positive control.
  • FIG. 1B Immunoblot with calstabin2 antibody showing binding of calstabin2 to PKA-phosphorylated RyR2 in the absence ( ⁇ ) or presence of 100 nM compound 2, compound 3 or compound 4. (+): calstabin binding to non-PKA phosphorylated RyR2. S36 is used as a positive control.
  • FIG. 1C Immunoblot with calstabin1 antibody showing binding of calstabin1 to PKA-phosphorylated RyR1 in the absence (Neg) or presence of the indicated concentrations of compound 1 or compound 4.
  • FIG. 2 FIG. 2A : Immunoblot with calstabin1 antibody showing the levels of calstabin1 in immunoprecipitated RyR1 complexes from tibialis lysates in mice administered vehicle (50:50 DMSO/PEG), isoproterenol alone (ISO) or isoproterenol together with the indicated concentrations of compound 1 in osmotic pumps. S36 is used as control at 3.6 mM.
  • FIG. 2B quantification of % calstabin1 rebinding to RyR1.
  • FIG. 3 Rat chronic heart failure model induced by ischemia-reperfusion (I/R) injury.
  • I/R protocol the left anterior descending (LAD) coronary artery was occluded for 1 h.
  • FIG. 4 Left ventricular (LV) volumes and ejection fraction (EF) in rats treated with compound 1 at 5 mg/kg/d (5 MK) or 10 mg/kg/d (10 MK) in drinking water vs. vehicle (H 2 O)-treated and sham-operated animals.
  • Chronic heart failure was induced by ischemia-reperfusion (I/R) injury.
  • LAD artery was occluded for 1 h; treatment started 1 week after reperfusion and continued for 3 months. Echocardiographic parameters were obtained after 1, 2 or 3 months of treatment.
  • FIG. 4A LV End Diastolic Volume
  • FIG. 4B LV End Systolic Volume
  • FIG. 4C EF.
  • FIG. 4A and 4B ⁇ P ⁇ 0.001 vs. sham; * P ⁇ 0.05 vs. vehicle; ⁇ P ⁇ 0.001 vs. vehicle.
  • FIG. 4C ⁇ P ⁇ 0.001 vs. sham, ⁇ P ⁇ 0.001 vs. vehicle.
  • FIG. 5 FIGS. 5A-C depict body weight (BW) ( 5 A), Infarct size ( 5 B), and LV weight ( 5 C), and FIG. 5D depicts collagen content in rats treated with compound 1 at 5 mg/kg/d (5 MK) and 10 mg/kg/day (10 MK) in drinking water vs. vehicle (H 2 O)-treated and sham-operated animals.
  • Chronic heart failure was induced by ischemia-reperfusion (I/R) injury.
  • LAD artery was occluded for 1 h; treatment started 1 week after reperfusion and continued for 3 months. Parameters were measured after 3 months of treatment.
  • FIGS. 5A-C not significant.
  • FIG. 5D ⁇ 0.001 vs. sham; * P ⁇ 0.05 vs. vehicle.
  • FIG. 6 Invasive hemodynamics: Left ventricular systolic pressure (LV SP) ( 6 A), dP/dtmax ( 6 B); and dP/dtmin ( 6 C) in rats treated with compound 1 at 5 mg/kg/d (5 MK) or 10 mg/kg/day (10 MK) in drinking water vs. vehicle (H 2 O)-treated and sham-operated animals.
  • Chronic heart failure was induced by ischemia-reperfusion (I/R) injury.
  • LAD artery was occluded for 1 h; treatment started 1 week after reperfusion and continued for 3 months. Hemodynamic parameters were measured after 3 months of treatment.
  • FIG. 6A not significant.
  • FIG. 6B ⁇ P ⁇ 0.05 vs. sham; * P ⁇ 0.05 vs. vehicle.
  • FIG. 6C ⁇ P ⁇ 0.01 vs. sham; *P ⁇ 0.05 vs. vehicle.
  • FIG. 7 Compound 1 plasma concentrations ( ⁇ M) vs. time of day.
  • FIG. 8 EF in rats treated with compound 1 or compound A at 5 mg/kg/d (5 MK) in drinking water vs. vehicle (H 2 O)-treated and sham-operated animals.
  • LAD artery was occluded for 1 h; treatment started 1 week after reperfusion and continued for 3 months. Echocardiographic parameters were obtained after 1, 2 or 3 months of treatment.
  • FIG. 9 Effect of compound 1 on spontaneous physical activity of mdx and WT mice as compared with vehicle (H 2 O)-treated controls. P ⁇ 0.001 for days 1-19 activity in mdx mice dosed with 10 and 50 mg/kg/day (target dose) administered in drinking water, compared to vehicle control.
  • FIG. 10 Specific force-frequency relationship of EDL muscle.
  • FIG. 11 Average body weight ( 11 A) and average water consumption ( 11 B) of mdx and WT mice treated with vehicle (H 2 O) or compound 1 administered in drinking water.
  • Raceca1sTM refers to ryanodine receptor calcium channel stabilizers, represented by compounds of the general Formula (I) or (IA) as provided by the invention, as well as the specific compounds designated by numerical numbers as provided by the invention, and herein collectively referred to as “compound(s) of the invention”.
  • the compounds of the present invention are represented by the structure of Formula (IA):
  • R is COOH or a bioisostere thereof, COOR 1 or CN;
  • R 1 is a C 1 -C 4 alkyl
  • R in Formula (IA) is a carboxylic acid (COOH).
  • R in Formula (IA) is a carboxylic acid bioisostere, for example tetrazole.
  • the carboxylic acid bioisostere may be an acidic heterocycle such as 1,2,4-oxadiazol-5(4H)-one, 1,2,4-thiadiazol-5(4H)-one, 1,2,4-oxadiazole-5(4H)-thione, 1,3,4-oxadiazole-2(3H)-thione, 4-methyl-1H-1,2,4-triazole-5(4H)-thione, 5-fluoroorotic acid, and the like.
  • the compounds of the present invention are represented by the structure of Formula (IA) wherein R is COOH and pharmaceutically acceptable salts thereof (i.e., a compound of formula (I)).
  • R in Formula (IA) is at position 4 of the phenyl ring (i.e., position 7 of the benzothiazepine ring).
  • the compounds of Formula (IA) or (I) may be present in the form of a salt with a pharmaceutically acceptable acid or base.
  • Such salts are preferably selected from the group consisting of sodium, potassium, magnesium, hemifumarate, hydrochloride and hydrobromide salts, with each possibility representing a separate embodiment of the present invention.
  • One currently preferred salt is the sodium salt.
  • Another currently preferred salt is the hemifumarate salt.
  • the compound is selected from the group consisting of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, compound 9, compound 10, compound 11, and compound 12, and pharmaceutically acceptable salts thereof.
  • These compounds are represented by the following structures:
  • alkyl refers to a linear or branched, saturated hydrocarbon having from 1 to 4 carbon atoms (“C 1 -C 4 alkyl”).
  • Representative alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, and tert-butyl.
  • the alkyl group may be unsubstituted or substituted by one or more groups selected from halogen, haloalkyl, hydroxy, alkoxy, haloalkoxy, cycloalkyl, aryl, heterocyclyl, heteroaryl, amido, alkylamido, dialkylamido, nitro, amino, cyano, N 3 , oxo, alkylamino, dialkylamino, carboxyl, thio, thioalkyl and thioaryl.
  • All stereoisomers of the compounds of the present invention are contemplated within the scope of this invention.
  • Individual stereoisomers of the compounds of the invention may, for example, be substantially free of other isomers (e.g., as a pure or substantially pure optical isomer having a specified activity), or may be admixed, for example, as racemates, or as mixtures enriched by one stereoisomer.
  • the chiral centers of the present invention may have the S or R configuration as defined by the IUPAC 1974 Recommendations.
  • racemic forms can be resolved by physical methods, such as, for example, fractional crystallization, separation or crystallization of diastereomeric derivatives or separation by chiral column chromatography.
  • the individual optical isomers can be obtained from the racemates by any suitable method, including without limitation, conventional methods, such as, for example, salt formation with an optically active acid or base, followed by crystallization.
  • Compounds of the present invention are, subsequent to their preparation, preferably isolated and purified to obtain a composition containing an amount by weight equal to or greater than about 90% of the compound, about 95% of the compound, and even more preferably greater than about 99% of the compound (“substantially pure” compound), which is then used or formulated as described herein. Such “substantially pure” compounds of the present invention are also contemplated herein as part of the present invention.
  • the present invention provides compounds that are capable of treating conditions, disorders and diseases associated with RyRs. More particularly, the present invention provides compounds that are capable of fixing a leak in RyR channels, which may be RyR1, RyR2 and/or RyR3 channels. In one embodiment, the compounds of the invention enhance association and/or inhibit dissociation of RyR and calstabin (e.g., RyR1 and calstabin1; RyR2 and calstabin2; and RyR3 and calstabin1).
  • RyR and calstabin e.g., RyR1 and calstabin1; RyR2 and calstabin2; and RyR3 and calstabin1
  • “Conditions, disorders and diseases associated with RyRs” means disorders and diseases that can be treated and/or prevented by modulating RyRs and include, without limitation, cardiac disorders and diseases, muscle fatigue, musculoskeletal disorders and diseases, CNS disorders and diseases, cognitive dysfunction, neuromuscular diseases and disorders, cognitive function improvement, bone disorders and diseases, cancer cachexia, malignant hyperthermia, diabetes, sudden cardiac death, and sudden infant death syndrome.
  • the present invention relates to a method of treating or preventing a condition selected from the group consisting of cardiac disorders and diseases, muscle fatigue, musculoskeletal disorders and diseases, CNS disorders and diseases, cognitive dysfunction, neuromuscular diseases and disorders, bone disorders and diseases, cancer cachexia, malignant hyperthermia, diabetes, sudden cardiac death, and sudden infant death syndrome, or for improving cognitive function, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound of Formula (I) or (IA) as described herein, or a salt thereof, to effectuate such treatment.
  • a currently preferred compound is a compound of Formula (1).
  • the present invention relates to the use of an effective amount of compound of Formula (I) or (IA), as described herein, or a salt thereof, for the manufacture of a medicament for treating or preventing a condition selected from the group consisting of cardiac disorders and diseases, muscle fatigue, skeletal muscular disorders and diseases, CNS disorders and diseases, neuromuscular disorder and diseases, cognitive dysfunction, bone disorders and diseases, cancer cachexia, malignant hyperthermia, diabetes, sudden cardiac death, and sudden infant death syndrome, or for improving cognitive function.
  • a currently preferred compound is a compound of Formula (1).
  • the present invention relates to a compound of Formula (I) or (IA) as described herein, or a salt thereof, for use in the manufacture of a medicament for treating or preventing a condition selected from the group consisting of cardiac disorders and diseases, muscle fatigue, skeletal muscular disorders and diseases, CNS disorders and diseases, cognitive dysfunction, neuromuscular diseases and disorders, bone disorders and diseases, cancer cachexia, malignant hyperthermia, diabetes, sudden cardiac death, and sudden infant death syndrome, or for improving cognitive function.
  • a currently preferred compound is a compound of Formula (1).
  • condition, disorder or disease is associated with an abnormal function of RyR1. In another embodiment, the condition, disorder or disease is associated with an abnormal function of RyR2. In another embodiment, the condition, disorder or disease is associated with an abnormal function of RyR3.
  • RyR1 In another embodiment, the condition, disorder or disease is associated with an abnormal function of RyR2.
  • Cardiac disorders and diseases include, but are not limited to, irregular heartbeat disorders and diseases, exercise-induced irregular heartbeat disorders and diseases, heart failure, congestive heart failure, chronic heart failure, acute heart failure, systolic heart failure, diastolic heart failure, acute decompensated heart failure, cardiac ischemia/reperfusion (I/R) injury (including I/R injury following coronary angioplasty or following thrombolysis during myocardial infarction (MI)), chronic obstructive pulmonary disease, and high blood pressure.
  • I/R cardiac ischemia/reperfusion
  • MI myocardial infarction
  • Irregular heartbeat disorders and diseases include, but are not limited to atrial and ventricular arrhythmia, atrial and ventricular fibrillation, atrial and ventricular tachyarrhythmia, atrial and ventricular tachycardia, catecholaminergic polymorphic ventricular tachycardia (CPVT), and exercise-induced variants thereof.
  • atrial and ventricular arrhythmia atrial and ventricular fibrillation
  • atrial and ventricular tachyarrhythmia atrial and ventricular tachycardia
  • atrial and ventricular tachycardia atrial and ventricular tachycardia
  • CPVT catecholaminergic polymorphic ventricular tachycardia
  • the compounds of the invention are also useful in treating muscle fatigue, which may be due to prolonged exercise or high-intensity exercise, or may be caused by musculoskeletal diseases.
  • muscular disorders and diseases include, but are not limited to, skeletal muscle fatigue, central core diseases, exercise-induced skeletal muscle fatigue, bladder disorders, incontinence, age-associated muscle fatigue, sarcopenia, congenital myopathies, skeletal muscle myopathies and/or atrophies, cancer cachexia, myopathy with cores and rods, mitochondrial myopathies [e.g., Kearns-Sayre syndrome, MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke) syndrome, and MERRF (myoclonus epilepsy with ragged-red fibers) syndrome], endocrine myopathies, muscular glycogen storage diseases [e.g., Pompe's disease, Andersen's disease, and Cori's diseases], myoglobinurias [e.g., McAr
  • Preferred skeletal muscular disorders include, but are not limited to exercise-induced skeletal muscle fatigue, a congenital myopathy, muscular dystrophy, age-related muscle fatigue, sarcopenia, central core disease, cancer cachexia, bladder disorders, and incontinence.
  • muscular dystrophy examples include, but are not limited to, Duchenne Muscular Dystrophy (DMD), Becker's Muscular Dystrophy (BMD), Limb Girdle Muscular Dystrophy (LGMD), Congenital Muscular Dystrophy (CMD), distal muscular dystrophy, facioscapulohumeral dystrophy, myotonic muscular dystrophy, Emery-Dreifuss muscular dystrophy, and oculopharyngeal muscular dystrophy, with DMD being currently preferred.
  • DMD Duchenne Muscular Dystrophy
  • BMD Becker's Muscular Dystrophy
  • LGMD Limb Girdle Muscular Dystrophy
  • CMD Congenital Muscular Dystrophy
  • distal muscular dystrophy facioscapulohumeral dystrophy
  • myotonic muscular dystrophy myotonic muscular dystrophy
  • Emery-Dreifuss muscular dystrophy Emery-Dreifuss muscular dystrophy
  • Congenital muscular dystrophy refers to muscular dystrophy that is present at birth.
  • CMD is classified based on genetic mutations: 1) genes encoding for structural proteins of the basal membrane or extracellular matrix of the skeletal muscle fibres; 2) genes encoding for putative or demonstrated glycosyltransferases, that in turn affect the glycosylation of dystroglycan, an external membrane protein of the basal membrane; and 3) other.
  • CMD examples include, but are not limited to Laminin- ⁇ 2-deficient CMD (MDC1A), Ullrich CMG (UCMDs 1, 2 and 3), Walker-Warburg syndrome (WWS), Muscle-eye-brain disease (MEB), Fukuyama CMD (FCMD), CMD plus secondary laminin deficiency 1 (MDC1B), CMD plus secondary laminin deficiency 2 (MDC1C), CMD with mental retardation and pachygyria (MDC1D), and Rigid spine with muscular dystrophy Type 1 (RSMD1).
  • MDC1A Laminin- ⁇ 2-deficient CMD
  • Ullrich CMG Ullrich CMG
  • WWS Walker-Warburg syndrome
  • MB Muscle-eye-brain disease
  • FCMD Fukuyama CMD
  • CMD plus secondary laminin deficiency 1 MDC1B
  • CMD plus secondary laminin deficiency 2 CMD with mental retardation and pachygyria
  • MDC1D CMD
  • Cognitive dysfunction may be associated with or includes, but is not limited to memory loss, age-dependent memory loss, post-traumatic stress disorder (PTSD), attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), generalized anxiety disorder (GAD), obsessive compulsive disorder (OCD), Schizophrenia, Bipolar disorder, or major depression
  • CNS disorders and diseases include, but are not limited to Alzheimer's Disease (AD), neuropathy, seizures, Parkinson's Disease (PD), and Huntington's Disease (HD).
  • AD Alzheimer's Disease
  • PD Parkinson's Disease
  • HD Huntington's Disease
  • Neuromuscular disorders and diseases include, but are not limited to Spinocerebellar ataxia (SCA), and Amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease).
  • SCA Spinocerebellar ataxia
  • ALS Amyotrophic lateral sclerosis
  • the compounds of the present invention improve cognitive function, which may be selected from short term memory, long term memory, attention, learning, and any combination thereof.
  • the compounds of the present invention are useful in the treatment of cancer cachexia, i.e., muscle weakness which is associated with cancer in general, and preferably muscle weakness in metastatic cancer, such as bone metastases.
  • Muscle weakness and muscle atrophy are common paraneoplastic symptoms in cancer patients. These conditions cause significant fatigue and dramatically reduce patients' quality of life.
  • the present invention provides a method for treating and preventing muscle weakness in a cancer patient, based, in part, on the discovery that, in certain types of cancers, e.g., prostate and breast cancer with bone metastases, RyR1 is oxidized which induces it to become “leaky”. It has further been found that prevention of the leak by administration of Ryca1 compounds improves muscle function.
  • Exemplary cancers include, but are not limited to, breast cancer, prostate cancer, bone cancer, pancreatic cancer, lung cancer, colon cancer, and gastrointestinal cancer.
  • the compounds of the present invention modulate (e.g., enhance) mRNA splicing by enhancing antisense-mediated exon skipping. This modulation of splicing is accomplished in the presence of antisense oligonucleotides (AOs) that are specific for splicing sequences of interest.
  • AOs antisense oligonucleotides
  • the compound of formula (I) or (IA) and the AO can act synergistically wherein the compound of formula (I) or (IA) enhances AO mediated exon skipping.
  • the present invention relates to a pharmaceutical composition for use in the treatment or prevention of any of the conditions described herein that are associated with Leaky RyR, further comprising the use of an antisense AO which is specific for a splicing sequence in an mRNA sequence, for enhancing exon skipping in the mRNA of interest.
  • DMD Duchenne Muscular Dystrophy
  • DMD is a lethal X-linked recessive disease characterized by progressive muscle weakness over a patient's lifetime.
  • DMD is primarily caused by out of frame multi-exon deletions in the DMD gene that ablate dystrophin protein production. Loss of dystrophin expression alone does not explain DMD pathophysiology.
  • DGC dystrophin-glycoprotein complex
  • the present invention relates to a method for treating DMD, by administering to a subject in need thereof a compound of formula (I) or (IA) according to the present invention, in combination with an antisense oligonucleotide (AO) which is specific for a splicing sequence of one or more exons of the DMD gene, for example exon 23, 45, 44, 50, 51, 52 and/or 53 of the DMD gene.
  • AOs include, but are not limited to, AOs targeting DMD exon 23, 50 and/or 51 of the DMD gene, such as 2′-O-methyl (2′OMe) phosphorothioate or phosphorodiamidate morpholino (PMO) AOs.
  • Examples of such AOs include, but not limited to, Pro051/GSK2402968, AVI4658/Eteplirsen, and PMO E23 morpholino (5′-GGCCAAACCTCGGCTTACCTGAAAT-3′).
  • an “effective amount,” “sufficient amount” or “therapeutically effective amount” of an agent as used herein interchangeably, is that amount sufficient to effectuate beneficial or desired results, including clinical results and, as such, an “effective amount” or its variants depends upon the context in which it is being applied. The response is in some embodiments preventative, in others therapeutic, and in others a combination thereof.
  • the term “effective amount” also includes the amount of a compound of the invention, which is “therapeutically effective” and which avoids or substantially attenuates undesirable side effects.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • the compounds of the invention are formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo.
  • the present invention provides a pharmaceutical composition comprising compounds of the invention in admixture with a pharmaceutically acceptable diluent and/or carrier.
  • the pharmaceutically-acceptable carrier is preferably “acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.
  • the compound may be administered alone, but is preferably administered with one or more pharmaceutically acceptable carriers.
  • the pharmaceutically-acceptable carrier employed herein may be selected from various organic or inorganic materials that are used as materials for pharmaceutical formulations and which are incorporated as any one or more of fillers, diluents, binders, disintegrants, buffers, colorants, emulsifiers, flavor-improving agents, gellants, glidants, preservatives, solubilizers, stabilizers, suspending agents, sweeteners, tonicity agents, wetting agents, emulsifiers, dispersing agents, swelling agents, retardants, lubricants, absorbents, and viscosity-increasing agents.
  • the compounds of the present invention are administered to a human or animal subject by known procedures including, without limitation, oral, sublingual, buccal, parenteral (intravenous, intramuscular or subcutaneous), transdermal, per- or trans-cutaneous, intranasal, intra-vaginal, rectal, ocular, and respiratory (via inhalation administration).
  • the compounds of the invention may also be administered to the subject by way of delivery to the subject's muscles including, but not limited to, the subject's cardiac or skeletal muscles.
  • the compound is administered to the subject by way of targeted delivery to cardiac muscle cells via a catheter inserted into the subject's heart.
  • the compounds may be administered directly into the CNS, for example by intralumbar injection or intreventricular infusion of the compounds directly into the cerebrospinal-fluid (CSF), or by intraventricular, intrathecal or interstitial administration. Oral administration is currently preferred.
  • CSF cerebrospinal-fluid
  • compositions according to the invention for solid oral administration include especially tablets or dragées, sublingual tablets, sachets, capsules including gelatin capsules, powders, and granules, and those for liquid oral, nasal, buccal or ocular administration include especially emulsions, solutions, suspensions, drops, syrups and aerosols.
  • the compounds may also be administered as a suspension or solution via drinking water or with food.
  • acceptable pharmaceutical carriers include, but are not limited to, cellulose derivatives including carboxymethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, ethyl cellulose and microcrystalline cellulose; sugars such as mannitol, sucrose, or lactose; glycerin, gum arabic, magnesium stearate, sodium stearyl fumarate, saline, sodium alginate, starch, talc and water, among others.
  • compositions according to the invention for parenteral injections include especially sterile solutions, which may be aqueous or non-aqueous, dispersions, suspensions or emulsions and also sterile powders for the reconstitution of injectable solutions or dispersions.
  • sterile solutions which may be aqueous or non-aqueous, dispersions, suspensions or emulsions and also sterile powders for the reconstitution of injectable solutions or dispersions.
  • the compounds of the invention may be combined with a sterile aqueous solution that is isotonic with the blood of the subject.
  • Such a formulation is 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 is presented in unit or multi-dose containers, such as sealed ampoules or vials.
  • the formulation is 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.
  • compositions for rectal or vaginal administration are preferably suppositories, and those for per- or trans-cutaneous administration include especially powders, aerosols, creams, ointments, gels and patches.
  • the compounds of the invention are 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 compounds of the invention and permit the compounds to penetrate through the skin and into the bloodstream.
  • 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 compounds of the invention and permit the compounds to penetrate through the skin and into the bloodstream.
  • the compound/enhancer compositions also may be further combined with a polymeric substance, such as ethylcellulose, hydroxypropyl cellulose, ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to provide the composition in gel form, which is dissolved in a solvent, evaporated to the desired viscosity and then applied
  • the pharmaceutical formulations of the present invention are prepared by methods well-known in the pharmaceutical arts, including but not limited to wet and dry granulation methods, or by direct compression.
  • the choice of carrier is determined by the solubility and chemical nature of the compounds, chosen route of administration and standard pharmaceutical practice.
  • compositions mentioned above illustrate the invention but do not limit it in any way.
  • any of these compounds may be administered to the subject (or are contacted with cells of the subject) in an amount effective to limit or prevent a decrease in the level of RyR-bound calstabin in the subject, particularly in cells of the subject.
  • This amount is 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 the compounds of the invention effective to limit or prevent a decrease in the level of RyR-bound calstabin in the subject ranges from about 0.01 mg/kg/day to about 100 mg/kg/day (e.g., 1, 2, 5, 10, 20, 25, 50 or 100 mg/kg/day), and/or is an amount sufficient to achieve plasma levels ranging from about 300 ng/ml to about 5,000 ng/ml.
  • the amount of compounds from the invention ranges from about 1 mg/kg/day to about 50 mg/kg/day.
  • the amount of compounds from the invention ranges from about 10 mg/kg/day to about 20 mg/kg/day. Also included are amounts of from about 0.01 mg/kg/day or 0.05 mg/kg/day to about 5 mg/kg/day or about 10 mg/kg/day which can be administered.
  • the present invention provides, in a further aspect, processes for the preparation of a compound of the invention, and salts thereof. More particularly, the present invention provides processes for the preparation of compounds of Formula (I) or (IA), e.g., compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, compound 9, compound 10, compound 11, and compound 12, or salts thereof.
  • compounds of Formula (I) or (IA) e.g., compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, compound 9, compound 10, compound 11, and compound 12, or salts thereof.
  • the various synthetic routes to the compounds are described in the examples.
  • the general route of synthesis (ROS) is set forth in Scheme 1 below:
  • R a COOR 1 or CN R 1 is a C 1 -C 4 alkyl
  • L is a leaving group, which is, by way of example, a halogen, a sulfonate (OSO 2 R′ wherein R′ is alkyl or aryl, e.g., OMs (mesylate), OTs (tosylate)), and the like.
  • the amine starting material is reacted with the alkylating agent (benzyl derivative shown above), preferably in the presence of a base, to yield the desired product or a precursor thereof (R ⁇ R a ).
  • such precursor may further be reacted to convert the group R a to the group R as exemplified in the experimental section hereinbelow, or by any other method known to a person of skill in the art.
  • R 1 is a C 1 -C 4 alkyl
  • R ⁇ COOH carboxylic acid
  • a tetrazole a carboxylic acid isostere
  • the amine starting material may be prepared in accordance with the methods described in WO 2009/111463 or WO 2007/024717, or by any other method known to a person of skill in the art.
  • the contents of all of the aforementioned references are incorporated by reference herein.
  • the nature of the base is not particularly limiting.
  • Preferred bases include, but are not limited to, hydrides (e.g., sodium or potassium hydride) and N,N-diisopropylethylamine.
  • Suitable bases include, but are not limited to an organic base such as a tertiary amine, selected from the group consisting of acyclic amines (e.g., trimethylamine, triethylamine, dimethylphenylamine diisopropylethylamine and tributylamine), cyclic amines (e.g., N-methylmorpholine) and aromatic amines (dimethylaniline, dimethylaminopyridine and pyridine).
  • acyclic amines e.g., trimethylamine, triethylamine, dimethylphenylamine diisopropylethylamine and tributylamine
  • cyclic amines e.g., N-methylmorpholine
  • aromatic amines dimethylaniline, dimethylaminopyridine and pyridine
  • the reaction may be conducted in the presence or absence of a solvent.
  • a solvent such as an ester (e.g., ethyl acetate), an ether (e.g., THF), a chlorinated solvent (e.g., dichloromethane or chloroform), dimethylformamide (DMF), and other solvents such as acetonitrile or toluene or mixtures of these solvents with each other or with water.
  • Salts of compounds of formula (I) wherein R ⁇ COOH may be prepared by reacting the parent molecule with a suitable base, e.g., NaOH or KOH to yield the corresponding alkali metal salts, e.g., the sodium or potassium salts.
  • a suitable base e.g., NaOH or KOH
  • esters (R ⁇ COOR 1 ) may be directly converted to salts by reactions with suitable bases.
  • Salts of compounds of formula (I) may also be prepared by reacting the parent molecule with a suitable acid, e.g., HCl, fumaric acid, or para-toluenesulfonic acid to yield the corresponding salts, e.g., hydrochloride, tosylate or hemi-fumarate.
  • a suitable acid e.g., HCl, fumaric acid, or para-toluenesulfonic acid
  • hydrochloride tosylate or hemi-fumarate.
  • LC/MS Waters Delta 600 equipped with Autosampler 717Plus, Photo Diode Array Detector 2996, and Mass Detector 3100, or Shimadzu 210
  • Methyl ester (3 mmol) was dissolved in 30 ml of THF/methanol/1 M NaOH (1:1:1, v/v). The mixture was stirred for 8 hours and TLC showed complete disappearance of the ester. 1 ml Conc. HCl was added to adjust to acidic pH. The organic solvent was removed and the formed solid was collected by filtration. The solid was dried in the air.
  • the sodium salt of compound 1 was prepared from the parent molecule using 1 equivalent of NaOH in EtOH (m.p. of the salt: >290° C.).
  • Nitrile precursor (3.22 mmol), sodium azide (830 mg, 12.9 mmol) and triethylamine hydrochloride (1.72 g, 12.9 mmol) were stirred in 40 ml anhydrous DMF at 100° C. for 5 days.
  • the DMF was removed under high vacuum and the residue was mixed with water.
  • the water solution was extracted with dichloromethane (3 ⁇ 100 ml), The pure compound was purified by column chromatography (EtOAc/methanol).
  • Cardiac SR membranes were prepared as previously described (Marx et al., 2000; Kaftan et al., Circ. Res., 1996, 78:990-97). Immunoblotting of microsomes (50 ⁇ g) was performed as described, with anti-calstabin antibody (1:1,000) (Jayaraman et al., J. Biol. Chem., 1992, 267:9474-77) for 1 hr at room temperature (Reiken et al., Circulation, 107:2459-66, 2003).
  • CSR cardiac sarcoplasmic reticulum
  • Reaction mixture was set up in 1.5 ml microfuge tube. 200 ⁇ g of cardiac SR were added to a reaction mix of kinase buffer, PKA and ATP to a final volume of 100 (Reaction mix below). ATP was added last to initiate the reaction.
  • FIG. 1A depicts an immunoblot with calstabin2 antibody showing binding of calstabin2 to PKA-phosphorylated RyR2 in the absence ( ⁇ ) or presence of 100 nM compound 1.
  • (+) calstabin binding to non-PKA phosphorylated RyR2.
  • S36 a benzothiazepine described in U.S. Pat. No. 7,544,678, is used as a control.
  • compound 1, at a concentration of 100 nM prevented the dissociation of calstabin2 from PKA-phosphorylated RyR2 and/or enhanced the (re)binding of calstabin2 to PKA-phosphorylated RyR.
  • SR membranes from skeletal muscle were prepared in a manner similar to Example 2, and as further described in US patent application publication No. 2004/0224368, the contents of which are incorporated by reference herein. Immunoblotting of microsomes (50 ⁇ g) was performed as described, with anti-calstabin1 antibody (Zymed) (1:1,000). The blots were developed and quantified as described in Example 2.
  • FIG. 1C depicts an immunoblot with calstabin1 antibody showing binding of calstabin1 to PKA phosphorylated RyR1 in the absence (Neg) or presence of the indicated concentrations of compound 1 or compound 4.
  • (Pos) calstabin binding to non-PKA phosphorylated RyR1. S36 is used as a control.
  • compound 1 and compound 4 prevented the dissociation of calstabin1 from PKA phosphorylated RyR1 and/or enhanced the (re)binding of calstabin1 to PKA-phosphorylated RyR1 in a dose-dependent manner, with an estimated EC50 of about 100 nM and 150 nM, respectively.
  • Isoproterenol a beta adrenergic receptor agonist, induces heart failure in mice via overstimulation of the beta adrenergic receptor. Concurrent with this is the activation of PKA, phosphorylation of the RyR2 on the SR, and decreased interaction of calstabin2 (FKBP12.6) to RyR2. A similar cascade of events occurs in skeletal muscle, wherein RyR1 is phosphorylated, leading to decreased binding of calstabin1 (FKBP12) to RyR1.
  • mice C57Bl/6 mice were maintained and studied according to approved protocols.
  • the synthetic beta-adrenergic agonist, isoproterenol (ISO) was obtained from Sigma (165627) and prepared as a 100 mg/ml stock in water. Lysis buffer was made by adding sucrose (1 mM), dithiothreitol (320 mM), and 1 protease inhibitor tablet (10 ⁇ ) to 10 ml stock solution (10 mM HEPES, 1 mM EDTA, 20 mM NaF, 2 mM Na 3 VO 4 ).
  • mice were continually infused for five days with 10 mg/ml isoproterenol (1 ⁇ l/hr) by means of a subcutaneously implanted osmotic infusion pump (Alzet MiniOsmotic pump, Model 2001, Durect Corporation, Cupertino, Calif.).
  • osmotic infusion pump Alzet MiniOsmotic pump, Model 2001, Durect Corporation, Cupertino, Calif.
  • the osmotic pump was held vertically and 200 ⁇ l drug solution was injected into the pump via a 1 ml syringe (attached to a cannula) that contained an excess of drug solution ( ⁇ 250-300 ⁇ l).
  • the drug solution was injected slowly downward, while the syringe was slowly lifted, until the pump was overfilled. Overflow of displaced fluid upon capping the pump confirmed that the pump was properly filled.
  • the loaded osmotic pumps were implanted subcutaneously by the following steps.
  • the recipient mouse was anesthetized with 1.5-2% isoflurane in O 2 administered at 0.6 L/min, and its weight was then measured and recorded.
  • the mouse was then placed chest-down on styrofoam, its face in the nose cone. The fur was clipped on the back of the neck, extending behind the ears to the top of the head. The area was wiped gently with 70% alcohol, and a small incision was made at the midline on the nape of head/neck.
  • a suture holder was swabbed with alcohol, inserted into the cut, and opened to release the skin from the underlying tissue. To accommodate the pump, this opening was extended back to the hindquarters.
  • the loaded pump was inserted into the opening, with its release site positioned away from the incision, and was allowed to settle underneath the skin with minimal tension.
  • the incision was closed with 5.0 nylon suture, requiring about 5-6 sutures, and the area was wiped gently with 70% alcohol. Following surgery, mice were placed in individual cages to minimize injury and possible activation of the sympathetic nervous system.
  • Mouse skeletal muscle tissue was isolated as follows. The leg muscles were exposed by cutting the skin at the ankle and pulling upward. The tissue was kept moistened with Tyrode's buffer (10 mM HEPES, 140 mM NaCl, 2.68 mM KCl, 0.42 mM Na 2 HPO 4 , 1.7 mM MgCl 2 , 11.9 mM NaHCO 3 , 5 mM glucose, 1.8 mM CaCl 2 , prepared by adding 20 mg CaCl 2 to 100 ml 1 ⁇ buffer made from a 10 ⁇ solution without CaCl 2 ). The following muscles were isolated and frozen in liquid nitrogen.
  • Tyrode's buffer (10 mM HEPES, 140 mM NaCl, 2.68 mM KCl, 0.42 mM Na 2 HPO 4 , 1.7 mM MgCl 2 , 11.9 mM NaHCO 3 , 5 mM glucose, 1.8 mM CaCl 2 , prepared by adding 20 mg CaCl 2
  • extensor digitorum longus was isolated by inserting scissors between lateral tendon and the X formed by the EDL and Tibialis tendons, cutting upward toward the knee; cutting the fibularis muscle to expose the fan-shaped tendon of gastrocnemius; inserting forceps under X and under the muscle to loosen the EDL tendon; cutting the EDL tendon and pulling up the muscle; and finally cutting loose the EDL.
  • the soleus was isolated by removing the fibularis muscle from top of gastrocnemius; exposing the soleus on the underside of the gastrocnemius by cutting and lifting up the Achilles tendon; cutting the soleus at the top of the muscle behind the knee; and finally pulling the soleus and cutting it away from the gastrocnemius muscle.
  • the tibialis was isolated by cutting the tibialis tendon from the front of ankle, pulling the tendon upwards, and cutting it away from the tibia.
  • the vastus (thigh muscle) was isolated from both legs, by cutting the muscle just above the knee and removing the muscle bundle. The samples were frozen in liquid nitrogen.
  • RyR1 was immunoprecipitated from samples by incubating 200-500 ⁇ g of homogenate with 2 ⁇ l anti-RyR1 antibody (Zymed) in 0.5 ml of a modified RIPA buffer (50 mM Tris-HCl (pH 7.4), 0.9% NaCl, 5.0 mM NaF, 1.0 mM Na 3 VO 4 , 0.5% Triton-X100, and protease inhibitors) at 4° C. for 1.5 hr. The samples were then incubated with Protein A sepharose beads (Amersham Pharmacia Biotech, Piscatawy, N.J.) at 4° C. for 1 hour, after which the beads were washed three times with ice cold RIPA.
  • a modified RIPA buffer 50 mM Tris-HCl (pH 7.4), 0.9% NaCl, 5.0 mM NaF, 1.0 mM Na 3 VO 4 , 0.5% Triton-X100, and protease inhibitors
  • Osmotic pumps containing isoproterenol with or without test compound were implanted in mice as described above.
  • the mice were osmotically perfused for five days with either vehicle alone (DMSO/PEG), isoproterenol alone (ISO) (0.5 mg/kg/hr), or a combination of isoproterenol (0.5 mg/kg/hr) and compound 1 at the indicated concentrations.
  • vehicle alone DMSO/PEG
  • ISO isoproterenol alone
  • compound 1 0.5 mg/kg/hr
  • compound 1 0.5 mg/kg/hr
  • each mouse was sacrificed, and skeletal muscle tissue was isolated and used to analyze calstabin1 binding in RyR1 immunoprecipates.
  • FIGS. 2A and 2 B The effect of compound 1 on enhancing calstabin1 binding to RyR1 in skeletal muscle isolated from isoproterenol treated mice is depicted in FIGS. 2A (immunoblot) and 2 B (graphical quantification). As shown, compound 1 enhanced levels of calstabin1 bound to RyR1 in skeletal muscle membranes to a level similar to that observed by administration of 3.6 mM S36, another benzothiazepine derivative used as a positive control (WO2008/064264). Similar results were obtained for compound 4 (data not shown).
  • the objective of this study was to test the ability of compound 1 to reduce cardiac dysfunction and attenuate ventricular remodelling in a model of ischemia-reperfusion induced heart failure.
  • I/R ischemia-reperfusion
  • LAD left anterior descending coronary artery
  • Drug treatment (5 mg/kg/d or 10 mg/kg/d in drinking water) was initiated 1 week after reperfusion and was maintained for a 3 month study period.
  • the efficacy of compound 1 was assessed by echocardiography at one, two and three months after treatment began, and by invasive hemodynamics at 3 months in comparison with vehicle-treated and sham-operated animals. Cardiac specimens were also analyzed to assess hypertrophy and collagen content. Blood was collected from each rat on the final study day to assess drug plasma concentrations as shown in FIG. 3 . The study design is depicted in FIG. 3 . Experiments were performed in a blinded manner.
  • LV ESV left ventricular end systolic
  • LV EDV end diastolic
  • EF Ejection Fraction
  • FIG. 5D increased interstitial collagen content
  • compound 1, administered at 5 and 10 mg/kg/d significantly increased EF, as well as decreased both LVESV and LVEDV compared to vehicle, from one to three months ( FIGS. 4A-C ), as well as reduced interstitial collagen content ( FIG. 5D ).
  • FIGS. 5A-C No effects on body weight (BW), infarct size or hypertrophy (LV weight) were observed upon treatment ( FIGS. 5A-C ).
  • Drug plasma concentrations are depicted in FIG. 7 .
  • Compound 1 was significantly and surprisingly more active in comparison with compound A, a structurally related benzothiazepine derivative described in WO 2007/024717. As shown in FIG. 8 , compound A, administered at a concentration of 5 mg/kg/d for 3 months, failed to improve systolic and diastolic cardiac function when compared with compound 1 in the chronic post-ischemic heart failure rat model at the end of the study. Thus, beneficial effects of compound 1, but not compound A, were observed at a dose of 5 mg/kg/d after 3 months of treatment in the rat CHF model.
  • the objective of this study was to test whether treatment with compound 1 improves muscle function in a dystrophin-deficient mouse model (mdx).
  • vehicle H 2 O
  • target doses 5 mg/kg/d, 10 mg/kg/d, or 50 mg/kg/d (actual doses: 7.9 mg/kg/d; 12.8 mg/kg/d;
  • vehicle H 2 O
  • target dose 50 mg/kg/d (actual dose: 67.7 mg/kg/d) of the sodium salt of compound 1.
  • Voluntary activity on wheel, body weight, and average water consumption were measured in the first 3 weeks. Specific muscle force was measured after 4 weeks of treatment, at the end of the study.
  • Extensor digitorum longus (EDL) muscle was isolated for muscle force analysis as further described hereinbelow. Blood was collected from each mouse by retro-orbital bleeds at the end of the study (after end of dark cycle—about 7 AM) to assess drug plasma concentrations. Experiments were blinded.
  • EDL muscle was dissected from hind limbs for isometric force analysis using the 407A Muscle Test System from Aurora Scientific (Aurora, Ontario, Canada).
  • a 6-0 suture were tied to each tendon and the entire EDL muscle, tendon to tendon, was transferred to a Ragnoti bath of O 2 /CO 2 (95%/5%) bubbled Tyrode solution (in mM: NaCl 121, KCl 5.0, CaCl 2 1.8, MgCl 2 , NaH 2 PO 4 , NaHCO 3 24, and glucose 5.5).
  • the EDL muscle was then removed from the system and weighed after clipping the end tendons and sutures off. The EDL muscle was then frozen in liquid nitrogen.
  • the cross-sectional area (mm 2 ) of the EDL muscle was calculated by dividing the EDL muscle weight by the EDL muscle length and the mammalian muscle density constant of 1.056 mg/m 3 (Yamada, T., et al. Arthritis and rheumatism 60:3280-3289).
  • EDL specific force kN/m 2
  • the absolute tetanic force was divided by the EDL muscle cross-sectional area.
  • compound 1 treatment increased specific force in mdx muscle dose-dependently.
  • stimulation frequencies of 150 Hz and above the 50 mg/kg/d-treated mdx mice showed statistically significant increase in specific muscle force (P ⁇ 0.05). No effect of compound 1 treatment on specific muscle force was observed in WT mice.
  • compound 1 treatment did not affect body weight. No dose-dependent effects on water consumption were observed.
  • Morning blood exposure of compound 1 was (average ⁇ SEM) 3.3 ⁇ 0.4 ⁇ M for the 5 mg/kg/d-dosed mdx mice, 10.7 ⁇ 0.9 ⁇ M for the 10 mg/kg/d-dosed mdx mice, 52.8 ⁇ 1.7 ⁇ M for the 50 mg/kg/d-dosed mdx mice and 72.8 ⁇ 7.0 ⁇ M for the 50 mg/kg/d-dosed WT mice.
  • Metabolic bioavailability predictions were based on in vitro metabolic stability measurements with hepatic microsomes assuming total absorption. Briefly, unchanged drugs were quantified by LC-MS-MS following incubation (10 ⁇ 7 M) with rat and human hepatic microsomes (0.33 mg protein/ml) after 0, 5, 15, 30 and 60 min of incubation in presence of NADPH (1 mM). Enzymatic reaction was stopped with methanol (v/v) and proteins were precipitated by centrifugation. The in vitro intrinsic clearances (Clint_mic) expressed as ml/min/g protein were the slope (after LN linearization) of the unchanged drug remaining concentration versus incubation time.
  • Compound solubilization Stock solutions were made in DMSO, and working solutions in William medium containing 1/10 rat plasma or 1/4 human plasma.
  • Metabolic stability determination Compounds were incubated at 10 ⁇ 7 M with isolated hepatocytes (6E+5 cells/ml for rat hepatocytes and 4E+5 cells/ml for human hepatocytes) at 37° C. in plasma from the same species diluted in Wiliams medium (1/10 dilution for rat and 1/4 dilution for human). Sampling times were performed at 0, 10, 20, 30, 60 and 120 min and enzymatic reaction stopped with methanol (v/v). Proteins were precipitated by centrifugation and the supernatant was analyzed by LC/MS/MS.
  • Clint expressed as ml/min/g protein were calculated as for hepatic microsomes using a ratio of 0.134 mg protein/ml for 4E+5 cells/ml for human and 0.201 mg protein/ml for 6E+5 cells/ml for rat.
  • the presence of the reference drug and the potential metabolite was checked by LC/MS/MS during the assay in each sample. The results are presented in Table 2:
  • a 50 mL falcon tube containing 3.32 mL of dilution buffer was prewarmed at 37° C. for 15 min. (at least 10 min.) 0.178 mL of microsome (24.6 mg/mL) were added to the prewarmed dilution buffer.
  • the protein concentration of this microsome preparation was 1.25 mg/mL.
  • a 1 mg/mL (0.5 mg/mL was used for compound 1) solution of the test compound in methanol was prepared.
  • 100 ⁇ M intermediate solution of the test compound from the original stock solution were prepared using the dilution buffer.
  • a 5 ⁇ M solution was prepared by diluting the 100 ⁇ M intermediate solution using dilution buffer.
  • the MS area used is an average of duplicate injections.

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