Peptides for use in the treatment or prevention of myocardial injury - Patents.com

Abstract

The present invention relates to L-type Ca 2+ The present invention relates to a peptide that binds to a channel. The present invention also relates to a method for treating, preventing, or ameliorating the effects of myocardial injury. The present invention also relates to a pharmaceutical composition comprising said peptide. In a first aspect, the present invention provides a peptide comprising the amino acid sequence: QQX1EEDX2KGYLDWITQAE (SEQ ID NO: 2), wherein X1 is an amino acid selected from the group comprising Q, E, or R; and X2 is an amino acid selected from the group comprising L or E, or a variant thereof. Preferably, the peptide does not consist of SEQ ID NO: 1.

Description

[Technical Field]

[0001] Technical Field The present invention relates to L-type Ca 2+ The present invention relates to peptides that bind to the channel and their use for preventing and treating myocardial injury, including cardiac hypertrophy. The present invention provides methods for treating, preventing, or ameliorating the effects of myocardial injury by administering peptides and therapeutic compositions comprising the peptides. [Background technology]

[0002] Background technology The following discussion of the background art is intended solely to facilitate an understanding of the present invention and is not an acknowledgement or admission that any of the referenced material is or was part of the common general knowledge as of the priority date of this application.

[0003] Pathological cardiac hypertrophy (thickening of the heart muscle) can occur in humans or other animals as a response to stress; diseases such as hypertension; myocardial injury, including myocardial infarction; neurohormones; or pollution causing hypoxia due to atmospheric carbon monoxide.

[0004] Familial hypertrophic cardiomyopathy (HCM) is an inherited cardiac condition characterized by cardiac hypertrophy that affects an estimated 1 in 200 people worldwide (J. Am. Coll. Cardiol. 65 (12) (2015) 1249-1254). It is the leading cause of sudden cardiac death in people under the age of 40. Pharmacological therapy and surgical interventions, such as septal myectomy, are used to manage symptoms in patients with HCM.

[0005] Certain genetic variants are associated with the development of HCM. Cardiac troponin (cTn) is a sarcomeric protein complex consisting of three subunits (cTnT, cTnI, and cTnC) that plays a crucial role in regulating cardiac contraction and relaxation. The entire cTn complex is bound to tropomyosin via TnT. TnI regulates contraction in response to changes in intracellular calcium. In the relaxed state, TnI inhibits actin-myosin interaction. Upon calcium binding to TnC, TnI undergoes a conformational change, allowing actin-myosin interaction and, consequently, contraction.

[0006] Mutations in the cTnI gene TNNI3 account for approximately 3-5% of genotyped families with HCM. Human HCM, caused by the cTnI mutation Gly203Ser, is characterized by apical and ventricular hypertrophy and, in some cases, supraventricular and ventricular arrhythmias. Additionally, HCM is characterized by myocyte remodeling, myofibrillar disarray, and altered energy metabolism. Mutations in the MYH7 gene, specifically the alpha-MHCArg403Ser mutation, are also known to account for approximately 40% of HCM cases.

[0007] Medications used to manage symptoms in people with HCM may include calcium channel antagonists, beta-blockers, calcium channel blockers, amiodarone (Pacerone), or disopyramide (Norpace). The calcium channel blocker diltiazem is used to treat symptoms of HCM, such as angina, and cardiac arrhythmias. However, these drugs can cause negative inotropic (constrictive) effects and a drop in blood pressure, leading to heart failure.

[0008] Antiarrhythmic drug treatments include amiodarone, disopyramide, angiotensin receptor blockers, propafenone, angiotensin-converting enzyme (ACE) inhibitors, and perhexiline. These drugs are ineffective in preventing arrhythmias. Surgical insertion of an implantable cardioverter-defibrillator can prevent sudden death, but the socioeconomic and psychological costs to patients are substantial.

[0009] Although these treatments can help manage cardiac hypertrophy and the symptoms of HCM, there are currently no pharmacological treatments that can reverse or prevent the development of cardiac hypertrophy in patients.

[0010] Cardiac hypertrophy also exists in patients without HCM. Patients with cardiac hypertrophy as a result of complete or partial coronary artery blockage are typically treated with reperfusion therapy, for example, using thrombolytic drug therapy, percutaneous coronary intervention (PCI), or bypass surgery. However, reperfusion injury can occur after reperfusion therapy when blood supply to cardiac tissue is restored after an ischemic period. The absence of nutrients and oxygen from the blood during the ischemic period leads to a condition in which restoration of circulation leads to inflammation and oxidative damage. This occurs as a result of increased oxidative stress rather than restoration of normal function.

[0011] L-type calcium channels Long or L-type Ca 2+ L-type Ca channels are the major pathway for calcium entry into cardiomyocytes, generating the valuable muscle contraction of the beating heart. Increased intracellular calcium and oxidative stress are involved in the pathophysiology of cardiac hypertrophy, and L-type Ca channels are essential for the pathogenesis of cardiac hypertrophy. 2+ Increasing the influx through the channel or overexpressing the alpha subunit of the channel induces hypertrophy.

[0012] Pore-forming L-type Ca 2+The primary structure of the channel alpha-1 (α1) subunit is understood to consist of four homologous repeat motifs (I-IV), each of which consists of six putative transmembrane segments (S1-S6) (Figure 1). The cytoplasmic loops between the transmembrane segments are named according to the motifs they connect. The α2, δ, and γ subunits are extracellular subunits linked to the alpha subunit via disulfide bridges, and the L-type Ca channel is completely intracellular. 2+ The channel beta 2 (β2) subunit also exists. The structure of the β2 subunit can be expressed as one of four beta subunit isoforms (β1-β4). All isoforms are hydrophilic, non-glycosylated, and intracellular, lacking a transmembrane region. The β2 isoform binds tightly to a highly conserved motif in the cytoplasmic linker between repeats I and II of all cloned high-voltage activated α1 subunit isoforms, called the alpha interaction domain (AID).

[0013] L-type Ca 2+ L-type Ca channels also play an important role in regulating mitochondrial function, which involves both calcium-dependent and calcium-independent mechanisms. These channels are regulated by voltage clamping of the plasma membrane or by the DHP receptor agonist BayK(-). 2+ Activation of the L-type Ca channel is sufficient to increase intracellular and mitochondrial calcium, NADH production, superoxide production, and metabolic activity in a calcium-dependent manner in wt cardiomyocytes. 2+ Activation of the channel also causes a calcium-independent increase in mitochondrial membrane potential (Ψm). This response is attenuated in the presence of F-actin depolymerizing agents, suggesting that the response is due to F-actin-mediated L-type Ca(2+) transport. 2+ This indicates that the mechanism of action of L-type Ca channels is dependent in part on the interaction between the channels and mitochondria. 2+ The L-type Ca channel is a type of Ca signaling channel that occurs with each beat. 2+ After the conformational change of the channel, L-type Ca transport occurs via the cytoskeletal network. 2+It affects mitochondrial function through structure-function communication between the channel and mitochondria.

[0014] Patients with HCM have L-type Ca 2+ cTnI-G203S myocytes show altered communication between L-type Ca channels and mitochondria, as well as altered metabolic activity. 2+ Faster L-type Ca responses in response to channel activation 2+ These changes also occur in myocytes isolated from the hearts of cTnI-G203S mice that have not yet developed cardiomyopathy, suggesting that L-type Ca channels are involved in the regulation of mitochondrial metabolism. 2+ We show that alterations in channel kinetics and metabolic activity precede the development of cardiomyopathy.

[0015] The alpha subunit of the channel has been the target of many therapies aimed at protecting the myocardium during reperfusion and calcium overload. Examples of these therapies include monoclonal antibodies against the alpha subunit; Ca2+ channel antagonists, such as dihydropyridines, benzothiazepines, and phenylalkylamines. Ca2+ channel blockers specifically bind to the α1C subunit region of the L-type Ca2+ channel, but these drugs have found limited success because they can induce myocardial depression and heart failure.

[0016] AID peptide In WO / 2013 / 113060, the present inventors reported that peptides directed against AID inhibit L-type Ca2+ receptors. 2+We found that restricting the movement of the β2 subunit of the channel prevented the interaction of β2 with the α1 subunit. In WO / 2013 / 113060, we hypothesized that the β-interacting domain (BID) interacts with AID through a conserved hydrophobic cleft called the alpha-binding pocket (ABP). The I-II loop of the α1 subunit contains an endoplasmic reticulum retention signal that restricts cell surface expression. The β2 subunit reverses the inhibition conferred by the retention signal and binds to L-type Ca channels. 2+ It can modulate the biophysical properties of the channel α1 subunit, resulting in a leftward shift in the current-voltage relationship consistent with the involvement of the S4 domain in the voltage-sensing region of the α1 subunit. As described in WO / 2013 / 113060, the present inventors have demonstrated that L-type Ca2+ receptors in cardiomyocytes 2+ It interacts with BID of the β2 subunit of the channel, thereby connecting the β2 subunit with L-type Ca 2+ We developed a peptide with the sequence QQLEEDLKGYLDWITQAE (SEQ ID NO: 1) (AID peptide) that prevents the interaction of the channel α1 subunit with AID. The β2 subunit bound to the peptide cannot reverse the inhibition of the α1 subunit imposed by the endoplasmic reticulum retention signal that restricts cell surface expression of the α1 subunit. This is due to the fact that L-type Ca channels are highly susceptible to endoplasmic reticulum retention. 2+ This results in limited channel activation and reduced mitochondrial energy consumption. However, to effectively treat or prevent cardiac hypertrophy, the β2 subunit and L-type Ca 2+ There remains a need to develop additional peptides with improved potency in disrupting the channel's interaction with AID. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] International Application No. 2013 / 113060 [Non-patent literature]

[0018] [Non-Patent Document 1] J. Am. Coll. Cardiol. 65 (12) (2015) 1249-1254 Summary of the Invention [Means for solving the problem]

[0019] Summary of the Invention The present inventors have found that the activity of the AID peptide depends on the peptide composition. In particular, the present inventors have found that restricting substitution of the third and seventh amino acids in the AID peptide sequence is essential for the peptide to bind to the β2 subunit and L-type Ca2+. 2+ We identified an unexpected effect on the destructive potential of the β-amyloid β-glucanase (AID) produced by its interaction with AID.

[0020] Thus, in a first aspect, the present invention provides a method for producing a polypeptide having the amino acid sequence: QQX1EEDX2KGYLDWITQAE (SEQ ID NO: 2) wherein X1 is an amino acid selected from the group comprising Q, E, or R; X2 is an amino acid selected from the group including L or E. or a variant thereof. Preferably, the peptide does not consist of SEQ ID NO:1.

[0021] Preferably, the present invention relates to a method for producing a nucleotide sequence comprising the amino acid sequence: QQX1EEDX2KGYLDWITQAE wherein X1 is an amino acid selected from the group comprising Q, E, or R; X2 is an amino acid selected from the group including L or E. or a variant thereof, wherein the peptide does not consist of SEQ ID NO:1.

[0022] Preferably, the peptide is L-type Ca 2+More preferably, the peptide binds to BID of the β2 subunit of human L-type Ca channel. 2+ Binds to BID of the channel β2 subunit.

[0023] Preferably, the present invention provides a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 4 to 7 or a variant thereof, but not consisting of SEQ ID NO: 1. Preferably, the peptide is a peptide comprising an L-type Ca 2+ More preferably, the peptide binds to BID of the β2 subunit of human L-type Ca channel. 2+ Binds to BID of the channel β2 subunit.

[0024] In a second aspect, the present invention provides a peptide moiety comprising the amino acid sequence of SEQ ID NO: 2; and RKKRRQRRRZaa (SEQ ID NO: 3) wherein Zaa is 6-aminohexanoic acid. or a variant thereof. Preferably, the peptide is an L-type Ca 2+ More preferably, the peptide binds to BID of the β2 subunit of human L-type Ca channel. 2+ Binds to BID of the channel β2 subunit.

[0025] Preferably, the peptides of the present invention comprise an amino acid sequence selected from the group consisting of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity to a peptide comprising SEQ ID NO: 2. More preferably, the peptides of the present invention comprise an amino acid sequence comprising one or more conservative amino acid substitutions relative to SEQ ID NO: 2 selected from the suitable amino acid substitutions set forth in Table 3.

[0026] In a third aspect, the present invention provides a method for the treatment of cardiac myocarditis by inhibiting L-type Ca2+ receptors in cardiac cells of a subject. 2+A method for modulating the movement of the beta subunit of a channel is provided, comprising administering to a subject a peptide of the invention.

[0027] In a fourth aspect, the present invention provides a method for the treatment of cardiac myocarditis, including the administration of L-type Ca2+ in cardiac cells of a subject. 2+ A method for modulating the binding of beta subunits of a channel is provided, comprising administering to a subject a peptide of the invention.

[0028] Preferably, the peptide induces L-type Ca2+ in cardiac cells of the subject. 2+ Beta subunits of L-type Ca channels 2+ It prevents interaction between the alpha subunit of the channel and therefore prevents activation of the channel.

[0029] In a fifth aspect, the present invention provides a method for the treatment of cardiac myocytes with L-type Ca2+. 2+ A method for modulating a channel is provided, comprising administering to a subject a peptide of the invention.

[0030] In a sixth aspect, the present invention provides a method for treating, preventing or reducing myocardial damage and / or oxidative stress in the heart of a subject, the method comprising the step of administering to the subject a peptide of the present invention.

[0031] Preferably, the myocardial injury comprises cardiac hypertrophy. Preferably, cardiac hypertrophy and / or oxidative stress is reduced. More preferably, intracellular Ca in cardiac cells of the subject's heart is reduced. 2+ The level is slightly reduced or substantially maintained in cardiac cells of the subject's heart. The subject is preferably a mammal, more preferably a human. Most preferably, the subject suffers from hypertrophic cardiomyopathy.

[0032] In a seventh aspect, the present invention provides a method for treating or preventing cardiac hypertrophy in a subject, the method comprising administering a peptide of the present invention to the subject. Preferably, the subject is suffering from hypertrophic cardiomyopathy.

[0033] In an eighth aspect, the present invention provides a method for detecting L-type Ca in cardiac cells of the heart of a subject. 2+ There is provided the use of a peptide of the invention to modulate the movement of the beta subunit of a channel.

[0034] In a ninth aspect, the present invention provides a method for the treatment of cardiac myocytes with L-type Ca2+. 2+ The present invention provides the use of a peptide of the present invention for modulating a channel.

[0035] In a tenth aspect, the present invention provides use of a peptide of the present invention for treating, preventing or reducing myocardial damage and / or oxidative stress in the heart of a subject, preferably during and / or after reperfusion.

[0036] In an eleventh aspect, the present invention provides a polynucleotide encoding a peptide of the invention described herein.

[0037] In a twelfth aspect, the present invention provides use of a peptide of the present invention for the manufacture of a medicament for treating, preventing, or ameliorating myocardial damage and / or oxidative stress in the heart of a subject. Preferably, the myocardial damage is cardiac hypertrophy.

[0038] In a thirteenth aspect, the present invention provides a pharmaceutical, prophylactic or therapeutic composition comprising a peptide of the invention and one or more pharmaceutically acceptable carriers and / or diluents.

[0039] In a fourteenth aspect, the present invention provides a method for slowing the progression of myocardial fibrosis in a subject, the method comprising the step of administering to the subject a peptide of the present invention.

[0040] In a fifteenth aspect, the present invention provides the use of a peptide of the present invention for slowing the progression of myocardial fibrosis in a subject.

[0041] In a sixteenth aspect, the present invention provides a kit for treating, preventing or ameliorating the effects of myocardial damage and / or oxidative stress in the heart of a subject, the kit comprising at least a peptide of the present invention packaged in a suitable container together with instructions for its use.

[0042] In a seventeenth aspect, the present invention provides a method for the treatment of cardiac myocytes with L-type Ca2+. 2+ The present invention provides a use of a peptide of the invention for modulating binding to a channel alpha interaction domain.

[0043] In an eighteenth aspect, the present invention provides use of a peptide of the invention for the manufacture of a medicament for treating, preventing, or ameliorating reperfusion injury in the heart of a subject.

[0044] Further features of the present invention are described in more detail in the following description of certain non-limiting embodiments thereof. This description is included solely for the purpose of illustrating the present invention and should not be understood as a limitation on the broad summary, disclosure, or description of the invention set forth above. In an embodiment of the present invention, for example, the following items are provided: (Item 1) Amino acid sequence: QQX 1 EEDX 2 KGYLDWITQAE or a variant thereof, wherein X 1 is an amino acid selected from the group comprising Q, E, or R; X 2 is an amino acid selected from the group comprising L or E; The peptide does not consist of SEQ ID NO:1. (Item 2) A peptide comprising any one of the amino acid sequences of SEQ ID NOs: 4 to 7 or a variant thereof, wherein the peptide does not consist of SEQ ID NO: 1. (Item 3) A peptide moiety comprising the amino acid sequence according to item 1 or 2; and Amino acid sequence: RKKRRQRRRZaa or a variant thereof, wherein Zaa is 6-aminohexanoic acid. A peptide comprising: (Item 4) 3. The peptide according to items 1 to 2, comprising an amino acid sequence selected from the group consisting of at least 75%; at least 80%; at least 85%; at least 90%; at least 95%; at least 96%; at least 97%; at least 98%; and at least 99% sequence identity with a peptide comprising SEQ ID NO:2. (Item 5) 5. The peptide according to item 4, comprising an amino acid sequence comprising one or more conservative amino acid substitutions relative to SEQ ID NO: 2 selected from the suitable amino acid substitutions listed in Table 3. (Item 6) L-type Ca in cardiac cells of interest 2+ 6. A method for modulating the movement of a beta subunit of a channel, comprising administering to the subject a peptide according to any one of items 1 to 5. (Item 7) L-type Ca in cardiac cells of interest 2+ 6. A method for modulating the binding of beta subunits of a channel, comprising administering to the subject a peptide according to any one of items 1 to 5. (Item 8) L-type Ca in cardiac cells of interest 2+ 6. A method for modulating a channel, comprising administering to the subject a peptide according to any one of items 1 to 5. (Item 9) 10. A method for treating, preventing, or reducing myocardial damage and / or oxidative stress in the heart of a subject, the method comprising administering to the subject a peptide according to any one of items 1 to 5. (Item 10) 10. The method of item 9, wherein the myocardial damage comprises cardiac hypertrophy. (Item 11) Intracellular Ca 2+ 10. The method of claim 9, wherein the level is reduced or substantially maintained in cardiac cells of the heart of the subject. (Item 12) 6. A method for treating or preventing cardiac hypertrophy in a subject, the method comprising administering to the subject the peptide according to any one of items 1 to 5. (Item 13) 6. A method for slowing the progression of myocardial fibrosis in a subject, the method comprising administering to the subject the peptide according to any one of items 1 to 5. (Item 14) L-type Ca in cardiac cells of interest 2+ 6. Use of a peptide according to any one of items 1 to 5 for modulating the movement of the beta subunit of a channel. (Item 15) L-type Ca in cardiac cells of the target heart 2+ 6. Use of a peptide according to any one of items 1 to 5 for modulating binding to the channel alpha interaction domain. (Item 16) L-type Ca in cardiac cells of interest 2+ 6. Use of a peptide according to any one of items 1 to 5 for modulating a channel. (Item 17) 6. Use of the peptide according to any one of items 1 to 5 for treating, preventing or reducing myocardial damage and / or oxidative stress in the heart of a subject during and / or after reperfusion. (Item 18) 6. Use of the peptide according to any one of items 1 to 5 for slowing the progression of myocardial fibrosis in a subject. (Item 19) A polynucleotide encoding the peptide according to any one of items 1 to 5. (Item 20) 6. Use of the peptide according to any of items 1 to 5 in the manufacture of a medicament for the treatment of reperfusion injury in the heart of a subject. (Item 21) 6. Use of the peptide according to any of items 1 to 5 in the manufacture of a medicament for treating, preventing or ameliorating myocardial damage and / or oxidative stress in the heart of a subject. (Item 22) 6. A pharmaceutical, prophylactic or therapeutic composition comprising a peptide according to any one of items 1 to 5 and one or more pharmaceutically acceptable carriers and / or diluents. (Item 23) 10. A kit for treating, preventing or ameliorating the effects of myocardial damage and / or oxidative stress in the heart of a subject, comprising at least one peptide according to any one of items 1 to 5 packaged in a suitable container together with instructions for its use.

[0045] Reference will now be made to the accompanying drawings. [Brief explanation of the drawings]

[0046] [Figure 1] Figure 1 shows the primary structure of the pore-forming L-type Ca2+ channel alpha-1 (α1) subunit, which consists of four homologous repeat motifs (I-IV), each of which consists of six putative transmembrane segments (S1-S6). α2δ consists of a transmembrane protein (δ) and an extracellular α2 protein linked via a disulfide bond (SS). β2 is an intracellular protein bound to the linker between motifs 1 and 2 of α1C via an α-interacting domain (AID).

[0047] [Figure 2] FIG. 2 depicts the results of a competitive binding assay demonstrating the average binding affinity (K 0.5 ) of each mutant peptide, the full-length AID peptide, and diltiazem for the β subunit.

[0048] [Figure 3-1] FIG. 3 illustrates the effect of mutant peptides on the oxidative stress response in wild-type cardiomyocytes. [Figure 3-2] FIG. 3 illustrates the effect of the mutant peptides on the oxidative stress response in wild-type cardiomyocytes. [Figure 3-3] FIG. 3 illustrates the effect of the mutant peptides on the oxidative stress response in wild-type cardiomyocytes.

[0049] [Figure 4] FIG. 4 depicts the effect of mutant peptides on flavoprotein oxidation, a measure of metabolic activity and oxygen consumption, in wt myocytes.

[0050] [Figure 5] FIG. 5 shows the effect of mutant peptides on flavoprotein oxidation, a measure of metabolic activity and oxygen consumption, in myocytes isolated from hypertrophic cTnI-G203S mutant mouse hearts.

[0051] [Figure 6-1] Figure 6 shows the effect of the mutant peptides on CK and LDH release (indicators of necrosis) and oxidative stress, measured as the ratio of reduced to oxidized glutathione (GSH:GSSG), in hearts exposed ex vivo to 20 minutes of no-flow ischemia. [Figure 6-2] Figure 6 shows the effect of the mutant peptides on CK and LDH release (indicators of necrosis) and oxidative stress, measured as the ratio of reduced to oxidized glutathione (GSH:GSSG), in hearts exposed ex vivo to 20 minutes of no-flow ischemia. [Figure 6-3] Figure 6 shows the effect of the mutant peptides on CK and LDH release (indicators of necrosis) and oxidative stress, measured as the ratio of reduced to oxidized glutathione (GSH:GSSG), in hearts exposed ex vivo to 20 minutes of no-flow ischemia.

[0052] [Figure 7] FIG. 7 represents a table of sequences referenced in this application.

[0053] [Figure 8] FIG. 8 depicts echocardiographic parameters of cTnI-G203S mice administered 10 μM AID(S)-TAT or AID-TAT.

[0054] [Figure 9] Figure 9 presents the results of a competitive binding assay demonstrating the average binding affinity (K0.5) of each mutant peptide, full-length AID peptide, and diltiazem for the β subunit after exposure to 0, 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM of the peptides.

[0055] [Figure 10-1] Figure 10 illustrates that in vitro exposure of cTnI.2 (wt) and cTnI-G203S (mutant) cardiomyocytes to the variant peptide is more effective in restoring metabolic activity than the original AID-TAT, as assessed as changes in flavoprotein oxidation. [Figure 10-2] Figure 10 illustrates that in vitro exposure of cTnI.2 (wt) and cTnI-G203S (mutant) cardiomyocytes to the variant peptide is more effective in restoring metabolic activity than the original AID-TAT, as assessed as changes in flavoprotein oxidation. [Figure 10-3] Figure 10 illustrates that in vitro exposure of cTnI.2 (wt) and cTnI-G203S (mutant) cardiomyocytes to the variant peptide is more effective in restoring metabolic activity than the original AID-TAT, as assessed as changes in flavoprotein oxidation. [Figure 10-4] Figure 10 illustrates that in vitro exposure of cTnI.2 (wt) and cTnI-G203S (mutant) cardiomyocytes to the variant peptide is more effective in restoring metabolic activity than the original AID-TAT, as assessed as changes in flavoprotein oxidation.

[0056] [Figure 11-1]Figure 11 illustrates that in vitro exposure of cTnI.2 (wt) and cTnI-G203S (mutant) cardiomyocytes to the variant peptides is more effective in restoring metabolic activity than the original AID-TAT, as assessed as changes in JC-1 fluorescence. [Figure 11-2] Figure 11 illustrates that in vitro exposure of cTnI.2 (wt) and cTnI-G203S (mutant) cardiomyocytes to the variant peptides is more effective in restoring metabolic activity than the original AID-TAT, as assessed as changes in JC-1 fluorescence.

[0057] [Figure 12-1] FIG. 12 illustrates that in vivo treatment of cTnI-G203S (mutant) mice with the AID-TAT variant peptide does not alter blood pressure. [Figure 12-2] FIG. 12 illustrates that in vivo treatment of cTnI-G203S (mutant) mice with the AID-TAT variant peptide does not alter blood pressure.

[0058] [Figure 13-1] FIG. 13 illustrates that in vivo treatment of cTnI-G203S mice with the AID-TAT variant peptide is not toxic. [Figure 13-2] FIG. 13 illustrates that in vivo treatment of cTnI-G203S mice with the AID-TAT variant peptide is not toxic. [Figure 13-3] FIG. 13 illustrates that in vivo treatment of cTnI-G203S mice with the AID-TAT variant peptide is not toxic.

[0059] [Figure 14-1] FIG. 14 illustrates that in vivo treatment of cTnI-G203S mice with the AID-TAT variant peptide slows the progression of fibrosis. [Figure 14-2]FIG. 14 illustrates that in vivo treatment of cTnI-G203S mice with the AID-TAT variant peptide slows the progression of fibrosis.

[0060] [Figure 15-1] FIG. 15 illustrates echocardiographic parameters of mice exposed to 10 μM AID(S), 10 μM AID-TAT, 5 μM AIDP7-TAT, 5 μM AIDP14-TAT, 5 μM AIDP15-TAT, or 5 μM AIDP16-TAT. [Figure 15-2] FIG. 15 illustrates echocardiographic parameters of mice exposed to 10 μM AID(S), 10 μM AID-TAT, 5 μM AIDP7-TAT, 5 μM AIDP14-TAT, 5 μM AIDP15-TAT, or 5 μM AIDP16-TAT. [Figure 15-3] FIG. 15 illustrates echocardiographic parameters of mice exposed to 10 μM AID(S), 10 μM AID-TAT, 5 μM AIDP7-TAT, 5 μM AIDP14-TAT, 5 μM AIDP15-TAT, or 5 μM AIDP16-TAT. [Figure 15-4] FIG. 15 illustrates echocardiographic parameters of mice exposed to 10 μM AID(S), 10 μM AID-TAT, 5 μM AIDP7-TAT, 5 μM AIDP14-TAT, 5 μM AIDP15-TAT, or 5 μM AIDP16-TAT. [Figure 15-5] FIG. 15 illustrates echocardiographic parameters of mice exposed to 10 μM AID(S), 10 μM AID-TAT, 5 μM AIDP7-TAT, 5 μM AIDP14-TAT, 5 μM AIDP15-TAT, or 5 μM AIDP16-TAT. [Figure 15-6] FIG. 15 illustrates echocardiographic parameters of mice exposed to 10 μM AID(S), 10 μM AID-TAT, 5 μM AIDP7-TAT, 5 μM AIDP14-TAT, 5 μM AIDP15-TAT, or 5 μM AIDP16-TAT. [Figure 15-7]FIG. 15 illustrates echocardiographic parameters of mice exposed to 10 μM AID(S), 10 μM AID-TAT, 5 μM AIDP7-TAT, 5 μM AIDP14-TAT, 5 μM AIDP15-TAT, or 5 μM AIDP16-TAT. [Figure 15-8] FIG. 15 illustrates echocardiographic parameters of mice exposed to 10 μM AID(S), 10 μM AID-TAT, 5 μM AIDP7-TAT, 5 μM AIDP14-TAT, 5 μM AIDP15-TAT, or 5 μM AIDP16-TAT. [Figure 15-9] FIG. 15 illustrates echocardiographic parameters of mice exposed to 10 μM AID(S), 10 μM AID-TAT, 5 μM AIDP7-TAT, 5 μM AIDP14-TAT, 5 μM AIDP15-TAT, or 5 μM AIDP16-TAT. [Figure 15-10] FIG. 15 illustrates echocardiographic parameters of mice exposed to 10 μM AID(S), 10 μM AID-TAT, 5 μM AIDP7-TAT, 5 μM AIDP14-TAT, 5 μM AIDP15-TAT, or 5 μM AIDP16-TAT.

[0061] [Figure 16] FIG. 16 illustrates that in vivo treatment of cTnI-G203S mice with the AID-TAT variant peptide slows the progression of hypertrophy. DETAILED DESCRIPTION OF THE INVENTION

[0062] Sequence Listing Table 1 below provides a list of the sequences referred to herein: [Table 1-1] [Table 1-2]

[0063] Description of the embodiment Those skilled in the art will recognize that the invention described herein is subject to variations and modifications other than those specifically described. It is understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions, and compounds individually or collectively referred to or shown in this specification, as well as any and all combinations or any two or more steps or features.

[0064] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the invention described herein.

[0065] The entire disclosures of all publications cited herein (including patents, patent applications, articles, laboratory manuals, books, or other documents) are hereby incorporated by reference. No admission is made that any of the references constitute prior art or are part of the common general knowledge of those working in the art to which this invention pertains.

[0066] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0067] Other definitions of selected terms used herein may be found in the detailed description of the invention and apply throughout. Unless defined otherwise, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0068] The inventions described herein may include one or more ranges of values ​​(e.g., size, displacement, field strength, etc.) A range of values ​​is understood to include all values ​​within the range, including the values ​​defining the range and values ​​immediately adjacent to that range that produce the same or substantially the same outcome as the values ​​immediately adjacent to that value defining the range boundary.

[0069] Features of the present invention will now be considered by reference to the following non-limiting descriptions and examples.

[0070] Variant AID peptides The present invention relates to a nucleic acid molecule having the amino acid sequence: QQX1EEDX2KGYLDWITQAE (SEQ ID NO: 2) wherein X1 is an amino acid selected from the group comprising Q, E, or R; X2 is an amino acid selected from the group including L or E. The present invention provides a peptide comprising:

[0071] Preferably, the peptide is L-type Ca 2+ More preferably, the peptide binds to BID of the β2 subunit of human L-type Ca channel. 2+ Most preferably, the peptide binds to BID of the L-type Ca channel β2 subunit with higher affinity compared to SEQ ID NO: 1. 2+ Binds to BID of the channel β2 subunit.

[0072] The present invention relates to human L-type Ca 2+ An isolated peptide is provided that includes a variant on the highly conserved AID motif of the channel α1 subunit (SEQ ID NO: 1).

[0073] Surprisingly, the present inventors found that L-type Ca 2+ A number of variants to specific amino acids in the original AID peptide were identified that improved the binding affinity of the peptide to BID of the channel β2 subunit.

[0074] In one embodiment, improved binding affinity results in improved functionality of the peptide in vitro and / or in vivo. In one embodiment, the peptides of the present invention bind to L-type Ca2+ receptors more efficiently than the original AID peptide. 2+ The rate of channel inactivation can be further slowed. In another embodiment, the peptides of the present invention can be administered to a subject at lower doses compared to the original AID peptide and produce the same effect in vitro or in vivo.

[0075] The inventors have identified that certain changes at positions X1 and X2 are effective in increasing the binding affinity of the peptide to BID compared to the original AID peptide. For example, the original AID peptide has the hydrophobic amino acid leucine at position X1, but in some embodiments, changing the X1 position to a negatively charged (e.g., glutamic acid), a positively charged (e.g., arginine), or a polar uncharged (e.g., glutamine) amino acid each increases the binding affinity of the peptide to BID compared to the original AID peptide.

[0076] The binding affinity of the peptides of the invention for BID can be measured by a number of techniques well known in the art, including SDS-PAGE assays.

[0077] Preferably, the peptide is provided in a pharmaceutically acceptable form.

[0078] Preferably, the peptide is not QQLEEDLKGYLDWITQAE (SEQ ID NO: 1).

[0079] Preferably, the peptide is not toxic. Most preferably, the peptide does not exhibit nephrotoxicity and / or hepatotoxicity. Nephrotoxicity can be measured by methods known in the art, including assessing urea and creatinine concentrations using a Quantichrom urea assay kit (BioAssay Systems, Hayward, CA) and a Quantichrom creatinine assay kit (BioAssay Systems, Hayward, CA), respectively. Hepatotoxicity can be measured by methods known in the art, including assessing alanine transaminase (ALT) and aspartate transaminase (AST) concentrations using alanine transaminase assay kit (BioAssay Systems, Hayward, CA) and an aspartate transaminase assay kit (BioAssay Systems, Hayward, CA), respectively.

[0080] The present invention further, and more preferably, provides peptides comprising the amino acid sequences of SEQ ID NOs: 4 to 7, as set forth in the table below: [Table 2]

[0081] The peptides of the present invention can be recombinant, natural, or synthetic. The peptides of the present invention can be mixed with diluents, adjuvants, or carriers (including nanoparticles) that do not interfere with the intended purpose of the peptide. The peptides of the present invention can also be in a substantially purified form, which generally includes peptides in preparations where at least 90%, 95%, 98%, or 99% of the protein in the preparation is a peptide of the present invention. As used herein, the term "peptide" can be used interchangeably with the term "polypeptide" when referring to a chain of at least two amino acid monomers.

[0082] In some embodiments, the peptides of the invention comprise one or more repeats of a peptide portion comprising SEQ ID NO: 2 or a variant thereof.

[0083] variant The present invention further includes variants of (1) the amino acid sequence of SEQ ID NO: 2; or (2) the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3. Preferably, the variants are L-type Ca 2+ More preferably, the peptide binds to BID of the β2 subunit of human L-type Ca channel. 2+ Most preferably, the variant binds to BID of the L-type Ca channel β2 subunit compared to SEQ ID NO: 1. 2+ It binds with high affinity to BID of the channel β2 subunit.

[0084] Preferably, the variant has an amino acid sequence homology selected from the group consisting of: (1) the amino acid sequence of SEQ ID NO: 2; or (2) at least 75% sequence identity; at least 80%; at least 85%; at least 90%; at least 95%; at least 96%; at least 97%; at least 98%; and at least 99% to the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3. Preferably, the variant is not QQLEEDLKGYLDWITQAE.

[0085] The term "% sequence identity" as used herein can be calculated, for example, as follows: The query sequence is aligned with the target sequence using the CLUSTAL W algorithm (Thompson et al., Nucleic Acids Research, 22: 4673-4680 (1994)). Comparison is performed, for example, over a window corresponding to one of the shortest aligned sequences. In some cases, the window can be defined by the target sequence. In other cases, the window can be defined by the query sequence. The amino acid residues at each position are compared, and the percentage of positions in the query sequence that have identical matches in the target sequence is reported as the % sequence identity.

[0086] Variants of (1) the amino acid sequence of SEQ ID NO:2; and (2) the amino acid sequences of SEQ ID NO:3 include polypeptides having an amino acid sequence that is substantially homologous to (1) the amino acid sequence of SEQ ID NO:2; or (2) the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:3, but that differs from (1) the amino acid sequence of SEQ ID NO:2; or (2) the amino acid sequence of SEQ ID NO:2 and SEQ ID NO:3 due to one or more amino acids being chemically modified or substituted with an amino acid analog. Preferably, any changes to the sequence to create a variant of (1) the amino acid sequence of SEQ ID NO:2; or (2) the amino acid sequence of SEQ ID NO:2 and SEQ ID NO:3 may also include amino acid deletions and / or amino acid additions in addition to amino acid substitutions.

[0087] Amino acid substitution is preferably conservative amino acid substitution known to those skilled in the art.For example, those skilled in the art may carry out amino acid substitution by selecting amino acids from the same class of amino acids that are shared with the specific amino acid identified for substitution.Examples of suitable amino acid substitution are shown in Table 3 below. [Table 3]

[0088] The peptide variants of the present invention also include fusions with additional peptides, in which additional peptide sequences are fused to the peptides of the present invention, for example to aid in extraction and purification. Examples of additional fusion peptide partners include glutathione-S-transferase (GST), hexahistidine, GAL4 (DNA binding and / or transcription activation domains), and β-galactosidase. Similarly, it may be convenient to include a proteolytic cleavage site between the additional peptide partner and the peptide of the present invention to allow removal of the additional peptide sequence. Preferably, the additional peptide is a peptide that binds to L-type Ca2+. 2+ It does not interfere with the binding of the peptides of the present invention to BID of the channel β2 subunit.

[0089] AID-TAT fusion peptide In a preferred form, the present invention further comprises: a peptide portion comprising the amino acid sequence of SEQ ID NO: 2; and Amino acid sequence: RKKRRQRRRZaa (SEQ ID NO: 3) wherein Zaa is 6-aminohexanoic acid. or a peptide moiety containing a variant thereof The present invention provides a peptide comprising:

[0090] The peptide portion of the peptide of the present invention comprising the amino acid sequence of SEQ ID NO:3 encodes the TAT peptide. The transactivating transcriptional activator from human immunodeficiency virus 1 (TAT) is a cell-penetrating peptide known in the art for delivering attached molecules, such as peptides, into cells. Therefore, without wishing to be bound by any particular mechanism, it is believed that the TAT peptide portion of the peptide of the present invention facilitates transport of the peptide into cardiac cells via endocytosis or by direct translocation across the cell membrane. A nuclear localization signal, GRKKR (SEQ ID NO:8), found within the domain mediates further translocation of TAT into the cell nucleus. The biological role of this domain and the exact mechanism of translocation are currently unknown. The amino acid sequence of the protein transduction domain is YGRKKRRQRRR (SEQ ID NO:9).

[0091] However, the peptides of the present invention may contain other or additional peptide moieties that aid or facilitate transport of the peptide into cardiac or other cells or that provide some other benefit, for example, that specifically localize the peptide of the present invention within the cell.

[0092] In some embodiments, the peptides of the invention comprise a peptide portion comprising SEQ ID NO:2 and one or more repeats of a peptide portion comprising SEQ ID NO:3 or a variant thereof.

[0093] Polynucleotides The present invention also provides isolated polynucleotides encoding the peptides of the present invention described herein, including peptides comprising SEQ ID NOs: 2-7. Due to the degeneracy of the amino acid code, it will be understood by those skilled in the art that multiple different polynucleotides may encode the same peptide as a result of the degeneracy of the genetic code. In addition, it will be understood that those skilled in the art may, using routine techniques, make nucleotide substitutions that do not affect the peptide sequence encoded by the polynucleotides of the present invention to reflect the codon usage of any particular host organism in which the polypeptides of the present invention will be expressed.

[0094] The polynucleotides of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including cDNA and genomic DNA, obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or the non-coding strand, also known as the antisense strand. They may also be polynucleotides containing synthetic or modified nucleotides therein. Many different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, and the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For purposes of the present invention, it is understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications may be made to enhance the in vivo activity or lifespan of the polynucleotides of the present invention.

[0095] Where a polynucleotide of the invention is double-stranded, both strands of the duplex, either individually or in combination, are encompassed by the invention. Where the polynucleotide is single-stranded, it is understood that the complementary sequence of that polynucleotide is also within the scope of the invention.

[0096] Reference to an "isolated" polynucleotide means a polynucleotide, DNA, or RNA that has been removed from its natural environment. For example, a recombinant DNA molecule contained in a vector is considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in a heterologous host cell or purified (partially or substantially) DNA molecules in solution.

[0097] Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated peptides of the present invention further include such molecules produced synthetically.

[0098] As noted above, polynucleotides of the present invention encoding peptides of the present invention include, but are not limited to, peptides encoded by the amino acid sequences of SEQ ID NOs: 2 and 4-7. Rather, polynucleotides of the present invention may include a coding sequence for a peptide and additional sequences, such as a sequence encoding a leader or secretory sequence, e.g., a pre-, pro-, or prepro-protein sequence; a coding sequence for a peptide with or without the additional coding sequences described above, along with additional non-coding sequences, such as, but not limited to, introns and non-coding 5' and 3' sequences, e.g., transcribed and untranslated sequences that play a role in mRNA processing, including transcription and splicing, and polyadenylation signals, e.g., for ribosome binding and stability of mRNA; and additional coding sequences encoding additional amino acids, e.g., amino acids that provide additional functionality. Preferably, the additional sequences include the peptide encoded by the amino acid sequence of SEQ ID NO: 3. Polynucleotides according to the present invention also include polynucleotides encoding peptides lacking an N-terminal methionine.

[0099] The present invention further relates to variants of the nucleic acid molecules of the present invention that encode variants of the peptides of the present invention.

[0100] Such variants include those produced by nucleotide substitution, deletion, or addition, which may involve one or more nucleotides.Non-naturally occurring variants may be produced using mutagenesis techniques known to those skilled in the art.Variants may be altered in coding regions, non-coding regions, or both.The alteration of coding regions may result in conservative or non-conservative amino acid substitution, deletion, or addition.Among these, silent substitutions, additions, and deletions that do not change the properties and activity of encoded peptides are particularly preferred.Similarly, conservative substitutions are particularly preferred in this regard.

[0101] For such nucleic acid molecules that are not degenerate variants, a reasonable number may be used, e.g., L-type Ca 2+ It is further recognized in the art that the peptides of the present invention also encode peptides having one or more properties of a full-length polypeptide capable of interacting with BID of a channel β2 subunit, because those skilled in the art are well aware of amino acid substitutions (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid) that are unlikely or unlikely to significantly affect protein function.

[0102] Vectors and host cells Nucleic acid molecules encoding the amino acid sequence of the peptides of the present invention can be inserted into an appropriate expression vector using standard ligation techniques. The vector is typically selected to be functional in the particular host cell to be used (i.e., the vector is compatible with the host cell machinery so that amplification of the nucleic acid molecule and / or expression of the nucleic acid molecule can occur). Nucleic acid molecules encoding the amino acid sequence of the peptides of the present invention can be amplified / expressed in prokaryotic, yeast, insect (baculovirus system), and / or eukaryotic host cells. The choice of host cell depends in part on whether the peptide is to be post-translationally modified (e.g., glycosylated and / or phosphorylated). In such cases, yeast, insect, or mammalian host cells are preferred.

[0103] Typically, expression vectors used in any host cell contain sequences for maintaining the plasmid and for cloning and expressing exogenous nucleotide sequences. Such sequences, collectively referred to in certain embodiments as "flanking sequences," typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for secreting the peptide, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding the peptide to be expressed, and a selectable marker element.

[0104] Preferred vectors for practicing the present invention are those compatible with bacterial, insect, and mammalian host cells, including, among others, pCRII, pCR3, and pcDNA3.1 (Invitrogen Company, Carlsbad, CA), pBSII (Stratagene Company, La Jolla, CA), pET15 (Novagen, Madison, WI), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFP-N2 (Clontech, Palo Alto, CA), pETL (BlueBacII; Invitrogen), pDSR-alpha (PCT Publication No. WO90 / 14363), and pFastBacDual (Gibco / BRL, Grand Island, NY).

[0105] Additional suitable vectors include, but are not limited to, cosmids, plasmids, or modified viruses, although it is recognized that the vector system must be compatible with the selected host cell. Such vectors include, but are not limited to, plasmids such as Bluescript® plasmid derivatives (high copy number ColE1-based phagemids, Stratagene Cloning Systems Inc., La Jolla, CA), PCR cloning plasmids designed for cloning Taq-Taq amplified PCR products (e.g., TOPO™ TA Cloning® Kit, PCR2.1® plasmid derivatives, Invitrogen, Carlsbad, CA), and mammalian, yeast, or viral vectors, such as the baculovirus expression system (pBacPAK plasmid derivatives, Clontech, Palo Alto, CA).

[0106] After constructing the vector and inserting the polynucleotide molecule encoding the peptide of the present invention into the appropriate site of the vector, the completed vector may be inserted into a suitable host cell for amplification and / or fusion protein expression. Transformation of the expression vector of the peptide of the present invention into the selected host cell can be accomplished by well-known methods, including transfection, infection, calcium chloride, electroporation, microinjection, lipofection, or DEAE-dextran methods, or other known techniques. The method selected will depend in part on the type of host cell used. These and other suitable methods are well known to those of skill in the art and are described, for example, in Sambrook et al., supra.

[0107] Host cells can be prokaryotic host cells (e.g., E. coli) or eukaryotic host cells (e.g., yeast cells, insect cells, or vertebrate cells). When cultured under appropriate conditions, the host cells synthesize the peptide, which can then be recovered from the culture medium (if the host cells secrete it into the medium) or directly from the host cells that produce it (if it is not secreted). The selection of an appropriate host cell depends on various factors, such as the desired expression level, peptide modifications desired or necessary for activity, such activity (e.g., glycosylation or phosphorylation), and the ease of folding into a biologically active molecule.

[0108] Many suitable host cells are known in the art, and many can be obtained from the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209. Examples include, but are not limited to, mammalian cells, such as Chinese hamster ovary cells (CHO) (ATCC No. CCL61); CHO DHFR cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 97:4216-4220 (1980)); human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573); or 3T3 cells (ATCC No. CCL92). The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening, and screening, product production, and purification are known in the art. Other suitable mammalian cell lines are the monkey COS-1 (ATCC No. CRL1650) and COS-7 (ATCC No. CRL1651) cell lines, and the CV-1 cell line (ATCC No. CCL70). Further exemplary mammalian host cells include primate and rodent cell lines, including transformed cell lines. Cell lines derived from in vitro cultures of normal diploid cells, primary tissues, and primary explants are also suitable. Candidate cells may be genotypically deficient in the selection gene or may contain a dominantly acting selection gene. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse LL-929 cells, 3T3 cell lines derived from Swiss, Balb-c, or NIH mice, and BHK or HaK hamster cell lines, all of which are available from the ATCC. Each of these cell lines is known and available to those skilled in the art of protein expression.

[0109] Similarly, bacterial cells are also useful as host cells suitable for the present invention. For example, various strains of E. coli (e.g., HB101 (ATCC No. 33694), DH5α, DH10, and MC1061 (ATCC No. 53338)) are well known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas spp., other Bacillus spp., Streptomyces spp., etc. can be used in this method.

[0110] Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the peptides of the invention. Preferred yeast cells include, for example, Saccharomyces cerivisae and Pichia pastoris.

[0111] Additionally, if desired, insect cell systems may be utilized in the methods of the present invention. Such systems are described, for example, in Kitts et al., Biotechniques, 14:810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4:564-572 (1993); and Lucklow et al. (J. al., J. Virol., 67:4566-4579 (1993). Preferred insect cells are Sf-9 and Hi5 (Invitrogen, Carlsbad, CA).

[0112] Transgenic animals may be used to express the glycosylated peptides of the present invention. For example, transgenic milk-producing animals (e.g., cows or goats) may be used, and the glycosylated peptides of the present invention may be obtained in the animal's milk. Similarly, plants may be used to produce the peptides of the present invention. However, the glycosylation generally present in plants may differ from that produced in mammalian cells and may result in glycosylated products that are not suitable for use in human therapy.

[0113] therapeutic composition Therapeutic compositions are within the scope of the present invention. The peptides of the present invention can be combined with various components to produce compositions of the present invention. Such compositions may contain a therapeutically effective amount of a peptide or nucleotide of the present invention mixed with a pharmaceutically or physiologically acceptable formulation agent selected to be appropriate for the mode of administration. Pharmaceutical compositions may also contain a therapeutically effective amount of one or more peptides of the present invention mixed with a pharmaceutically or physiologically acceptable formulation agent selected to be appropriate for the mode of administration. Preferably, the composition is combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition (which may be for human or animal use). Suitable carriers and diluents include isotonic saline solutions, such as phosphate-buffered saline. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. See, e.g., Remington's Pharmaceutical Sciences, 19th Ed. (1995, Mack Publishing Co., Easton, Pa.), incorporated herein by reference.

[0114] Pharmaceutical compositions may contain formulation materials to alter, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeation of the composition. Suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., Pluronic, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancers (sucrose or sorbitol); tonicity enhancers (e.g., alkali metal halides, preferably sodium or potassium chloride), delivery vehicles, diluents, excipients, and / or pharmaceutical adjuvants.

[0115] The optimal pharmaceutical composition will be determined by one skilled in the art depending on, for example, the intended route of administration, delivery format, and desired dosage. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the peptides of the present invention. The preferred form of the pharmaceutical composition depends on the intended mode of administration and therapeutic application. The pharmaceutical composition prepared according to the present invention can be administered by any means that brings the peptides of the present invention into contact with the causative agent of the diseases or disorders described herein, including cardiac hypertrophy or oxidative stress.

[0116] The primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier can be water for injection, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other ingredients common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer at about pH 7.0-8.5 or acetate buffer at about pH 4.0-5.5, which may further contain sorbitol or a suitable substitute. In one embodiment of the present invention, pharmaceutical compositions can be prepared for storage in the form of a lyophilized cake or aqueous solution by mixing a selected composition having the desired degree of purity with optional formulating agents. Additionally, peptide products can be formulated as lyophilizates using appropriate excipients, such as sucrose.

[0117] The pharmaceutical composition may be capable of parenteral delivery. Alternatively, the composition may be capable of delivery through the digestive tract, for example, orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.

[0118] The formulation components are present in concentrations that are acceptable to the site of administration, e.g., buffers are used to maintain the composition at physiological pH or a slightly lower pH, typically within a pH range of about 5 to about 8.

[0119] When parenteral administration is intended, the therapeutic composition for use in the present invention can be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired peptide of the present invention in a pharmaceutically acceptable vehicle.A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the active agent is formulated as a sterile, isotonic solution, properly preserved.Yet another preparation can include a formulation of the desired molecule with an agent that provides controlled or sustained release of the product, such as injectable microspheres, biodegradable particles, polymeric compounds (e.g., polylactic acid, acid, or polyglycolic acid), or beads or liposomes, which can then be delivered as a depot injection.Hyaluronic acid can also be used, which can have the effect of promoting sustained duration in the circulation.Other suitable means for introducing the desired molecule include implantable drug delivery devices.

[0120] It is also contemplated that certain formulations may be administered orally. In one embodiment of the present invention, the peptides of the present invention administered in this manner can be formulated with or without carriers typically used in compounding solid dosage forms, such as tablets and capsules. For example, capsules can be designed to release the active portion of the formulation at a location in the gastrointestinal tract that maximizes bioavailability and minimizes pre-systemic degradation. Additional agents can be included to facilitate absorption of the active agent. Diluents, flavoring agents, low-melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be used.

[0121] Another pharmaceutical composition can contain an effective amount of the peptide of the present invention in a mixture with non-toxic excipients suitable for tablet manufacture.By dissolving tablets in sterile water or other suitable vehicle, a solution can be prepared in unit dose form.Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate, or sodium bicarbonate, lactose, or calcium phosphate; or binders such as starch, gelatin, or acacia; or lubricants such as magnesium stearate, stearic acid, or talc.

[0122] Additional pharmaceutical compositions, including formulations containing the peptides of the present invention in sustained- or controlled-delivery formulations, will be apparent to those skilled in the art. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, biodegradable microparticles or porous beads, and depot injections, are also known to those skilled in the art. See, for example, PCT Application No. PCT / US93 / 00829, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. Additional examples of sustained-sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. Sustained-release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyrate. Sustained-release compositions may also include liposomes, which can be prepared by any of several methods known in the art.

[0123] The pharmaceutical composition used for in vivo administration must typically be sterile.This can be achieved by filtration through sterilizing membrane.When lyophilizing composition, sterilization using these methods can be carried out either before or after lyophilization and reconstitution.The composition for parenteral administration can be stored in lyophilized form or in solution.In addition, parenteral composition is generally placed in a container with sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0124] Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.

[0125] The effective amount of active agent in a pharmaceutical composition used therapeutically will depend, for example, on the therapeutic situation and purpose. Those skilled in the art will recognize that the appropriate dosage level for such treatment will vary, in part, depending on the molecule being delivered, the indication for which the active agent is being used, the route of administration, and the patient's size (weight, body surface, or organ size) and condition (age and general health). Thus, a physician may titrate the dosage and modify the route of administration to achieve optimal therapeutic effect. Typical dosages may range from about 0.1 μg / kg up to about 100 mg / kg or more, depending on the factors mentioned above. In other embodiments, dosages may range from 0.1 μg / kg up to about 100 mg / kg; or 1 μg / kg up to about 100 mg / kg; or 5 μg / kg up to about 100 mg / kg.

[0126] The number of administrations depends on the pharmacokinetic parameters of the active agent and formulation used.Typically, physicians administer the composition until the dosage reaches the desired effect.Therefore, the composition can be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as continuous infusion via implanted device or catheter.Further refinement of the appropriate dosage is routinely performed by those skilled in the art and is within the scope of the tasks routinely performed by those skilled in the art.The appropriate dosage can be confirmed through the use of appropriate dose-response data.

[0127] Peptide or pharmaceutical compositions comprising peptide can be administered to subject in a range of treatment regimens.For example, peptide or pharmaceutical compositions can be administered hourly, 3 times a day, twice a day, once a day, once every 2 days, once every 3 days, once a week, once every 2 weeks, once a month, once every 2 months, once every 6 months, and once a year.Appropriate regimen can be determined by those skilled in the art based on the nature of the condition to be treated.For example, peptide or pharmaceutical compositions comprising peptide can be administered 3 times a week.

[0128] The pharmaceutical composition can be administered by known methods, for example, orally, by injection via intravenous, intracoronary, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release system or implant. If desired, the composition can be administered by bolus injection or continuously by infusion, or by implantable device.

[0129] Alternatively or additionally, the composition may be administered locally via implantation of a membrane, sponge, or another suitable material onto which the desired molecule has been absorbed or encapsulated. When an implantable device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.

[0130] In some cases, it may be desirable to use the pharmaceutical compositions herein in an ex vivo manner, in which cells, tissues, or organs that have been removed from a patient are exposed to the pharmaceutical composition, after which the cells, tissues, and / or organs are then implanted back into the patient.

[0131] In another embodiment, nanoparticles can be used as a carrier for delivering the peptide of the present invention.For example, nanoparticles can be spherical polymer nanoparticles.Nanoparticles have been shown to overcome some of the limitations of traditional therapeutic delivery, such as non-specific biodistribution and targeting, and especially the lack of aqueous solubility.In this way, nanoparticles can be used to deliver the peptide of the present invention to cardiac cells to treat patients with peptides.

[0132] In one embodiment, the peptides of the present invention are delivered through the use of dendronized polymers. Preferably, the peptides of the present invention are delivered through the use of dendronized linear polymers (denpol). Most preferably, the peptides of the present invention are complexed with the dendronized linear polymer to form polymeric peptide nanoparticles. The polymeric peptide nanoparticles can be delivered intracellularly.

[0133] The routes of administration described herein are intended as a guide only, as a person of ordinary skill in the art can readily determine the optimum route of administration and dosage for any particular patient.

[0134] Uses and Methods of the Peptides of the Invention The present invention relates to L-type Ca 2+ The present invention provides the use of a peptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof, or the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3 or variants thereof, for modulating binding to a channel alpha interaction domain, which preferably occurs intracellularly in cardiac cells.

[0135] The present invention relates to the treatment of cardiac myocytes, such as cardiac myocytes, with L-type Ca2+. 2+The present invention provides the use of a peptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof; or the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3 or variants thereof, for modulating the movement of a channel β2 subunit.

[0136] In this regard, the present invention also provides a method for the production of L-type Ca2+ in cardiac cells, such as cardiac myocytes, of a subject. 2+ Also provided is a method for modulating the movement of a channel β2 subunit, the method comprising administering to a subject a peptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof; or SEQ ID NO: 2 and SEQ ID NO: 3 or a variant thereof.

[0137] Binding of peptides to the alpha-interacting domain inhibits L-type Ca 2+ It can prevent the movement of the β2 subunit during channel activation and inactivation. The β2 subunit is proposed to facilitate the inactivation of the alpha subunit, which may result in a delay in the inactivation of the current.

[0138] Subjects that can be treated with the peptides of the present invention include humans and other mammals and animals.

[0139] In one aspect, the present invention provides a method for the treatment of cardiac myocytes, such as cardiac myocytes, with L-type Ca2+. 2+ There is provided the use of a peptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof; or the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3 or variants thereof, for modulating a channel.

[0140] In this regard, the present invention also provides a method for the production of L-type Ca2+ in cardiac cells, such as cardiac myocytes, of a subject. 2+ Also provided is a method for modulating a channel, the method comprising administering to a subject a peptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof; or the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3 or variants thereof.

[0141] In another aspect, the present invention provides the use of a peptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof; or the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3 or variants thereof, for preventing or reducing myocardial damage and / or oxidative stress in the heart of a subject.

[0142] In this regard, the present invention also provides a method for preventing or reducing myocardial damage and / or oxidative stress in the heart of a subject, the method comprising the step of administering to the subject a peptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof; or the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3 or variants thereof.

[0143] Myocardial damage may include cardiac hypertrophy. Preferably, cardiac hypertrophy is reduced or prevented, but intracellular Ca 2+ The Ca levels are reduced or substantially maintained in the subject's heart. Substantially maintained means that the Ca levels are the same as or near levels normally observed in the subject, such as before cardiac hypertrophy. 2+ It means the level.

[0144] L-type Ca 2+ Binding of the peptides of the present invention to the channel results in a decrease in mitochondrial oxygen consumption or metabolism. This can occur without substantially altering calcium influx because the peptides anchor the β2 subunit but not the pore-forming α1C subunit. While any agent that reduces calcium influx can decrease contractility, this can occur at concentrations (1 μM) that do not alter calcium influx, making them preferred agents for use in treating or preventing cardiac hypertrophy.

[0145] Thus, the present invention provides L-type Ca 2+ Provided are methods and uses of the peptides of the invention as treatments for preventing or reducing damage and / or oxidative stress in cardiac muscle cells of a subject by modulating channel activity.

[0146] The peptide of the present invention can be administered to treat or prevent cardiac hypertrophy in patients who have developed or are at risk of developing cardiac hypertrophy.The peptide of the present invention can also be administered to reduce the damage and / or oxidative stress to ischemic cardiomyocytes after myocardial infarction in subjects during and after reperfusion treatment.The peptide can be administered before, after, or during reperfusion.

[0147] In this regard, the present invention also provides the use of a peptide comprising the amino acid sequence of SEQ ID NO: 2; or the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3, in the preparation of a medicament for treating, preventing, or ameliorating myocardial damage in a patient.

[0148] The present invention also provides the use of a peptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof; or the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3 or variants thereof, in the manufacture of a medicament for treating, preventing, or ameliorating myocardial damage and / or oxidative stress in cardiomyocytes in a patient.

[0149] It may be preferable to administer the peptide of the present invention in combination with other therapeutic agents that are useful for treating cardiac hypertrophy or HCM in subjects, or other agents that help reduce myocardial damage and / or oxidative stress.Such combinations can use conjugates that contain peptides, or treatment can be simultaneous or involve the sequential administration of agents.Examples of such therapeutic agents can include, but are not limited to, antioxidants, such as N-acetylcysteine, reduced glutathione, TAT-conjugated catalase, or TAT-conjugated superoxide dismutase.

[0150] The peptides may be administered via a variety of methods, eg, as a therapeutic agent depending on the particular situation and as deemed appropriate by a medical practitioner.

[0151] In one non-limiting example, the peptides of the present invention may be administered via the coronary arteries by a cardiologist / physician during an in-hospital angiogram or angioplasty after hospitalization for chest pain and diagnosis of coronary artery blockage (myocardial infarction).

[0152] In another non-limiting example, the peptides of the present invention may be administered to HCM patients before the onset of cardiomyopathy. The peptides of the present invention may be administered to patients at risk of developing cardiomyopathy via intraperitoneal injection or via the coronary artery. Prevention of the onset of cardiomyopathy may be measured by a decrease in ventricular septal or posterior wall thickness and an increase in left ventricular end-diastolic dimension on echocardiography. The peptides of the present invention may be administered approximately three times a week, based on the half-life of the peptides of the present invention in the body, which may be approximately 3 to 4 days, consistent with the turnover rate of the Cav1.2 channel protein (as described in Catalucci et al., The Journal of Cell Biology 184, 923-933 (2009)).

[0153] The effectiveness of the administered therapeutic composition can be monitored by standard diagnostic procedures.

[0154] For example, in one example, the effectiveness of the peptides may be monitored by echocardiography (ultrasound analysis of cardiac function). The size of the lesion can be assessed by the release of muscle enzymes into the blood and by changes in the electrocardiogram (ECG).

[0155] Treatment method In yet another aspect, the present invention provides a method for the treatment of cardiac muscle ulcers, including the administration of L-type Ca2+ in cardiac cells of a subject's heart. 2+ A method for modulating the movement of a beta subunit of a channel is provided, comprising administering to a subject a peptide comprising the amino acid sequence of SEQ ID NO:2; or the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:3.

[0156] The present invention further provides a method for the treatment of cardiac myocytes in a subject's heart with L-type Ca2+. 2+The present invention provides a method for modulating the binding of beta subunits of L-type Ca2+ channels, the method comprising administering to a subject a peptide comprising the amino acid sequence of SEQ ID NO: 2; or the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3. Preferably, the peptide prevents interaction between beta subunits of L-type Ca2+ channels and alpha subunits of L-type Ca2+ channels in cardiac cells of the subject.

[0157] The present invention further provides a method for the treatment of cardiac myocytes in a subject's heart with L-type Ca2+. 2+ A method for modulating a channel is provided, comprising administering to a subject a peptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof; or the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3 or variants thereof.

[0158] The present invention further provides a method for reducing myocardial damage and / or oxidative stress in the heart of a subject, the method comprising the step of administering to the subject a peptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof; or a peptide comprising the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3 or variants thereof.

[0159] The methods of the present invention may reduce cardiac hypertrophy in a subject's heart, but may also reduce intracellular Ca 2+ The level can be substantially maintained.

[0160] Patients suffering from HCM exhibit elevated Ψm and mitochondrial metabolic activity, as well as more rapid L-type Ca uptake, even before the onset of cardiomyopathy. 2+ Therefore, L-type Ca channels exhibit a high inactivation rate. 2+ Administration of the peptides of the present invention, which can increase the rate of channel inactivation and decrease mitochondrial metabolic activity, can be used to prevent the development of cardiac hypertrophy in HCM patients.

[0161] Preferably, the method of treatment or use does not cause vasodilatory or negative inotropic effects, as can occur with treatment with calcium channel antagonists.

[0162] In one aspect, the present invention provides a method of preventing cardiac hypertrophy in a subject's heart, comprising administering to the subject a peptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof; or a peptide comprising the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3 or variants thereof. Preferably, the subject is suffering from HCM.

[0163] In a further aspect, the present invention provides a method or use for slowing the progression of myocardial fibrosis in a subject, the method or use comprising the step of administering to the subject a peptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof; or a peptide comprising the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3 or variants thereof.

[0164] In a further aspect, the present invention provides a method for detecting L-type Ca in cardiac cells of a subject. 2+ A method or use for modulating the binding of beta subunits of L-type Ca channels, comprising administering a peptide of the present invention to a subject, wherein the method reduces the binding of L-type Ca channels in cardiac cells of the subject compared to a method of administering QQLEEDLKGYLDWITQAE (SEQ ID NO: 1). 2+ The present invention provides a method or use which is more effective in modulating the binding of the beta subunit of the channel.

[0165] In a further aspect, the present invention provides a method for detecting L-type Ca in cardiac cells of a subject. 2+ A method or use for modulating L-type Ca channels in cardiac cells of a subject, the method comprising administering a peptide of the present invention to a subject, the method comprising administering a peptide of the present invention to a subject, the method comprising administering a peptide of the present invention to a subject, the method comprising administering a peptide of the present invention to a subject 2+ The present invention provides a method or use which is more effective in modulating the channel.

[0166] In a further aspect, the present invention provides a method or use for reducing myocardial damage and / or oxidative stress in the heart of a subject, comprising a step of administering a peptide of the present invention to the subject, wherein the method is more effective in reducing myocardial damage and / or oxidative stress in the heart of the subject compared to a method of administering QQLEEDLKGYLDWITQAE (SEQ ID NO: 1).

[0167] As used herein, the term "subject" generally refers to mammals, such as humans; farm animals, such as sheep, goats, pigs, cows, horses, and llamas; companion animals, such as dogs and cats; primates; birds, such as chickens, geese, and ducks; fish; and reptiles. The subject is preferably a human.

[0168] The effective dose of the composition of the present invention for treating the above conditions varies depending on many different factors, including the means of administration, the target site, the physiological state of the patient, whether the patient is human or animal, and other medications administered. Treatment dosages need to be titrated to optimize safety and efficacy.

[0169] The following examples are illustrative only, and should not be construed as limiting in any way the remainder of the disclosure. These examples are included solely for the purpose of illustrating the present invention. They are not to be construed as limitations on the broad summary, disclosure, or description of the invention above. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. In the foregoing and following examples, all temperatures are set forth unmodified in degrees Celsius; all parts and percentages are by weight unless otherwise indicated. [Example]

[0170] Example 1 Synthesis of AID peptide A peptide corresponding to the α1C-β2a interaction domain (AID) within the cytoplasmic I-II linker of the cardiac α1C subunit was synthesized using the amino acid sequence QQLEEDLKGYLDWITQAE (SEQ ID NO: 1). A scrambled (inactive) control peptide (AID[S]) was also synthesized using the sequence QKILGEWDLAQYTDQELE (SEQ ID NO: 10). If necessary, a cell-permeable TAT sequence was attached to AID or AID(S) via 6-aminohexanoic acid (6-Ahx) (RKKRRQRRR) (SEQ ID NO: 3), resulting in the AID-TAT and AID(S)-TAT peptides.

[0171] Variant AID peptides were also synthesized, as described in Table 4. Based on the original AID-TAT sequence, variant sequences were generated by truncating the amino and / or carboxyl termini of the original sequence or by introducing amino acid point mutations. [Table 4]

[0172] Example 2 Binding assay - NHS magnetic beads The affinity of each of the variant peptides for the β2 subunit was examined through a binding competition assay.

[0173] PureProteome™ NHS FlexiBind magnetic beads (catalog number LSKMAGN04, Merck) were used according to the manufacturer's recommendations. Aminohexanoic acid-linked AID peptide was coupled to NHS FlexiBind magnetic beads at concentrations greater than 2 mg / mL to obtain bead saturation with peptide. The beads were first primed with equilibration buffer (1 mM HCl) to prepare them for binding. The Ahx-AID peptide was dissolved in wash / coupling buffer (PBS, pH 7.4) and added to the beads. The bead-AID peptide mixture was mixed uniformly at 300 rpm on a multiplate shaker at room temperature for 2 hours. To reduce nonspecific binding, the beads were incubated in Quench Buffer (100 mM Tris-HCl, 150 mM NaCl, pH 8.0) at room temperature for 1 hour. After washing, cardiac lysates were added to increasing concentrations of variant peptides for competitive binding to the AID-bound beads at the concentrations shown in Table 5. Table 5: Sample preparation for pre-binding of AID / variant peptides to the β2 subunit in whole heart lysates at various concentrations [Table 5]

[0174] The AID peptide-conjugated beads were divided into five aliquots for the five samples prepared in Table 5. Whole heart lysate was added to each of the peptide-conjugated beads and allowed to competitively bind for 2 hours at room temperature.

[0175] Upon completion, the conjugate was washed in coupling buffer to remove unbound fractions. SDS sample buffer was added to the beads, and the tubes were heated to 95°C for 5 minutes. Eluted samples were stored at -20°C and run through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine the binding affinity of each of the variants.

[0176] Example 3 Binding assay - streptavidin beads The use of streptavidin Dynabeads and biotinylated peptides was developed to improve binding affinity assays. The peptides were first biotinylated using EZ-Link Amine-PEG11-biotin (spacer arm: 53.2 Å, Thermofisher Scientific, catalog no. 26136) dissolved at 1 mM in 0.1 M MES buffer ((2-N-morpholino)-ethanesulfonic acid). Just prior to the reaction, 0.1 M EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) was dissolved in the MES buffer. To achieve complete biotinylation of the AID peptide, a 50x molar excess of biotin relative to the peptide was required; the total volumes of each component required are listed in Table 6. Table 6 - Volume of each component required to achieve sufficient biotinylation of the AID peptide for one complete assay [Table 6]

[0177] Each 78 μL aliquot of biotinylated peptide shown in Table 4 was sufficient for six reactions, covering a range of concentrations in which cardiac lysates were again prepared with various concentrations of variant AID peptide according to Table 7. Table 7. Sample preparation for pre-binding of AID / variant peptides to the β2 subunit in whole heart lysates at various concentrations. [Table 7]

[0178] A 78 μL aliquot of biotinylated AID peptide was mixed with streptavidin Dynabeads (1 mg Dynabeads per 200 pmol biotinylated peptide) for 30 minutes at room temperature. Heart lysate (100 μg) was incubated with the variant peptide for 2 hours at room temperature. After removing all unbound peptide from the beads, they were mixed with the prebound heart lysate for 2 hours at room temperature. The unbound fraction was removed. The conjugate was heated at 95°C for 5 minutes, then eluted with sample buffer and stored at -20°C for SDS-PAGE analysis.

[0179] Example 4 Binding affinity analysis using SDS-PAGE All samples obtained from the binding affinity assay were analyzed using SDS-PAGE. 25 μL of each sample was loaded onto a 10-well Mini-Protean TGX Stain-Free Gel (Biorad). A molecular weight ladder (Precision Protein Western C) was also loaded alongside the samples to identify the presence of the β2 subunit (68 kDa). The gels were run for 35 minutes at a constant current of 30 mA in a Biorad electrophoresis tank powered by a PowerPac3000. Upon completion, the gels were placed onto nitrocellulose membranes for 3 minutes in a TransBlot Turbo. The membranes were placed in blocking buffer (5% BSA) for 1 hour and then incubated overnight at 4°C with rabbit anti-Cavβ2 polyclonal primary antibody (Alomone Labs #ACC-105). The membrane was washed three times with TBST for 10 min each and then incubated with a goat anti-rabbit IgG(H+L)-HRP conjugated secondary antibody (Abcam #ab97080, pre-absorbed) in 5% BSA for 1 h at room temperature. Protein bands were visualized using Luminata Crescendo, Western HRP substrate (Merck Millipore), and a ChemiDoc imager (Biorad) equipped with ImageLab software. Densitometric analysis was performed using ImageJ software to quantify the intensity of the band on the membrane corresponding to Cavβ2.

[0180] average K 0.5 was calculated for each variant. These values ​​are listed in Figure 2 and in the table below. Variants P7, P14, P15, and P16 have higher binding affinity (lower K) for the target β2 subunit than the original AID peptide. 0.5 ) was demonstrated.

[0181] Example 5 Isolation of adult mouse cardiomyocytes Adult male C57BL / 6J mice were anesthetized via intraperitoneal injection of pentobarbitone sodium (240 mg / kg) and then their hearts were excised. Ventricular myocytes were isolated. Mouse hearts were intubated via the aorta into a Langendorff apparatus and retrogradely perfused for 4 minutes at 37°C with Krebs-Henseleit Buffer (KHB) (pH = 7.35, containing O2 / CO2 at 37°C) containing 120 mM NaCl, 25 mM NaHCO3, 4.8 mM KCl, 2.2 mM MgSO4, 1.2 mM NaH2PO4, and 11 mM glucose. The hearts were then perfused for 3 minutes with KHB supplemented with 2.4 mg / ml collagenase B, followed by 8 minutes in the presence of 40 μM calcium. After perfusion, the aorta and atria were removed, and the ventricles were gently dissected and triturated to dissociate myocytes into a suspension. The myocyte suspension was centrifuged at 500 RPM for 3 minutes, the supernatant discarded, and the myocytes resuspended in calcium-free HEPES-buffered solution (HBS) containing 140 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl, 5.5 mM HEPES, and 11 mM glucose (pH = 7.4 at 37 °C) in the presence or absence of 3 mM EGTA (for JC-1 experiments with 0 mM calcium). For calcium-containing (uptake) experiments, calcium was again titrated to achieve a final extracellular concentration of 2.0 mM.

[0182] Example 6 In vitro assessment of cardiomyocyte function All in vitro studies were performed at 37°C in freshly isolated myocytes. Fluorescence was recorded using a Hamamatsu Orca ER digital camera attached to a Nikon TE2000-U inverted microscope. After 20 min of incubation with 0.5–1 μM AID(S)-TAT, AID-TAT, peptide 6, or peptide 7, the fluorescence signal of individual myocytes was quantified using Metamorph 6.3, measuring the signal intensity of manually traced cell regions. An equivalent region containing no cells was used for background subtraction.

[0183] Assessment of intracellular calcium levels Intracellular calcium was monitored in cardiomyocytes using the fluorescent indicator Fura-2 AM (Fura-2, 1 μM, excitation 340 / 380 nm, emission 510 nm, Molecular Probes). A non-necrotic / non-apoptotic concentration of HO (30 μM) was applied to myocytes for 5 min, followed by oxidative stress induction by the application of 10 U / ml catalase. Fluorescence at excitation 340 / 380 nm and emission 510 nm was measured at 1-min intervals with 50 ms exposure before and after exposure to 30 μM HO (5 min) and 10 U / ml catalase (5 min). Ratiometric 340 / 380 nm signals were quantified and reported as a percentage of the baseline pretreatment mean.

[0184] The results are shown in Figure 3(a). Figure 3(a) demonstrates the effect of peptides on the increase in intracellular calcium induced by hydrogen peroxide injury (H2O2). Only P7 altered the increase in Fura2 after H2O2. Mutant P7 increased intracellular Ca2+ more than AID-TAT. 2+ It was more effective in reducing H2O2-induced increases in (Fura2) and superoxide production (DHE) and had similar effects as AID-TAT on mitochondrial membrane potential (JC-1).

[0185] Assessment of intracellular superoxide levels Superoxide generation was assessed in cardiomyocytes using the fluorescent indicator dihydroethidium (DHE, 5 μM, 515-560 nm excitation filter, 590 long-pass emission, Merck). Fluorescence images were acquired at 1-min intervals with 200 ms exposure before and after exposure to 30 μM HO (5 min) and 10 U / ml catalase (5 min). Fluorescence was reported as the percentage change in the slope of the signal measured at 20-40 min (treatment) compared to 1-10 min (pretreatment).

[0186] The results are shown in Figure 3(b). Peptide 7 statistically significantly reduced superoxide production compared to peptide 6, AID-TAT, or AID(S)-TAT.

[0187] In vitro assessment of mitochondrial membrane potential Mitochondrial membrane potential (Ψ) was monitored by assessing changes in 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide fluorescence (JC-1: 200 nM, excitation 480 nm, emission 580 / 535 nm, Molecular Probes) in cardiomyocytes. Myocytes were incubated in calcium-free HBS (additionally supplemented with 0 mM calcium and 3 mM EGTA) for at least 3 h before measuring Ψ. m Changes in Ψ were measured. Fluorescence images were acquired every 2 min (exposure = 50 ms) before and after exposure to 30 μM H2O2 (5 min) and 10 U / ml catalase (5 min). Ratiometric 580 / 535 nm fluorescence signals were quantified and reported as a percentage of the baseline pretreatment mean value. 40 mM NaCN was added at the end of each experiment to reduce Ψ m and the JC-1 signal decays to Ψ m It was confirmed that it shows.

[0188] The results are shown in Figure 3(c). Only AID-TAT and peptide 7 reduced mitochondrial membrane potential.

[0189] In vitro assessment of mitochondrial flavoprotein oxidation Autofluorescence was used to measure flavoprotein oxidation in cardiomyocytes as previously described (excitation 480 nm, emission 535 nm). Fluorescence images were acquired at 1-minute intervals with 1-second exposures before and after exposure to the L-type Ca2+ channel agonist BayK(-). Fluorescence was reported as a percentage of the baseline pretreatment mean value.

[0190] Results from mutant peptides (P6-TAT, P7-TAT, P14-TAT, and P15-TAT) and the AID-TAT peptide are presented in Figure 4 for wild-type cardiomyocytes and in Figure 5 for myocytes from HCM hearts. The results show that P7, P14, and P15 exhibited a statistically significant reduction in flavoprotein oxidation compared to the AID-TAT peptide in HCM hearts, and P7 exhibited a statistically significant reduction in flavoprotein oxidation compared to the AID-TAT peptide in wild-type hearts.

[0191] Example 7 Assessment of myocardial ischemia-reperfusion injury Adult male guinea pigs were anesthetized via intraperitoneal injection of sodium pentobarbitone (240 mg / kg) before heart excision. The guinea pig hearts were intubated via the aorta into a Langendorff apparatus and placed in a CaCl2-containing solution containing 120 mM NaCl, 25 mM NaHCO3, 4.8 mM KCl, 2.2 mM MgSO4, 1.2 mM NaH2PO4, 11 mM glucose, and 1.5 mM CaCl2 as previously described. 2+ The rats were retrogradely perfused with KHB (pH = 7.35, containing O2 / CO2, at 37°C) at a rate of 7 mL / min for 30 min at 37°C, followed by 30 min of no-flow ischemia and 30 min of reperfusion.

[0192] Perfusate was collected 20 and 25 min before ischemia and 20 and 30 min after reperfusion. A single dose of 0.5–10 μM AID(S)-TAT, AID-TAT, peptide 6, or peptide 7 was administered immediately before reperfusion using CaCl2. 2+Creatine kinase (CK) and lactate dehydrogenase (LDH) activities were measured in perfusates collected before and after ischemia, and postischemic values ​​were normalized to preischemic values.

[0193] For each sample, 10 μL of perfusate was added to 100 μL of CK enzyme reagent (CK NAC Activation Diagnostic Kit, Randox Laboratories), and the rate of absorbance increase was recorded using a spectrophotometer (PowerWave XS, BioTek, 340 nm) over 15 min at 30° C. CK activity was calculated according to the following equation:

number

[0194] The results are shown in Figure 6(a). CK activity was reduced compared to the AID(S)-TAT peptide in the AID-TAT, P7-TAT (1 μM), and P7-TAT (0.5 μM) samples. CK activity was reduced in both P7-TAT (1 μM) and P7-TAT (0.5 μM) compared to the P6-TAT peptide. P7-TAT (0.5 μM) was at least as effective as AID-TAT (1 μM).

[0195] To determine LDH activity, 150 μL of each sample was mixed with 50 μL of LDH reagent (50 mM imidazole buffer, 375 μM NADH, pyruvate, and 0.05% BSA, pH=7), and the rate of absorbance decrease was recorded over 15 minutes at 25° C. using a spectrophotometer (PowerWave XS, BioTek, 340 nm). LDH activity was calculated according to the following equation:

number

[0196] To determine GSH:GSSG, whole heart tissue was homogenized immediately after the ischemia-reperfusion protocol, and GSH and GSSG levels were analyzed using a GSH / GSSG ratio detection assay kit according to the manufacturer's instructions (abcam, ab205811). 8 Total glutathione and GSH were measured using a FLUOstar OPTIMA (BMG Labtech, excitation 485-12 nm, emission 520 nm). GSSG was calculated by subtracting GSH from total glutathione.

[0197] The results are presented in Figure 6(c). The AIDS-TAT sample did not show a statistically significant increase in the GSH:GSSG ratio at 1 μM, while the AID-TAT sample showed a statistically significant increase in the GSH:GSSG ratio at 10 μM. The P7-TAT sample produced a statistically significant increase in the GSH:GSSG ratio at 1 μM and 0.5 μM that was comparable to the AID-TAT sample at 10 μM.

[0198] The reduction in CK activity, LDH activity, and the increase in the GSH:GSSG ratio each represent a reduction in oxidative stress. The results demonstrate that administration of P7-TAT reduced oxidative stress with reference to each of these markers. Furthermore, P7-TAT was more effective than AID-TAT in reducing oxidative stress, producing superior results at the same concentration or similar results at lower dosages of P7-TAT compared to AID-TAT.

[0199] Example 8 Prophylactic administration of AID-TAT to cTnIG203S mice 10 μM AID-TAT was administered intraperitoneally to cTnIG203S mutant mice three times a week for five weeks prior to the onset of hypertrophic cardiomyopathy. The peptide was dissolved in phosphate-buffered saline (PBS), and the total amount of 10 μM AID-TAT administered over five weeks was 0.9 mg. Treatment prevented the onset of hypertrophy, as evidenced by a decrease in interventricular septal thickness and an increase in left ventricular end-diastolic dimension on echocardiography. Fractional shortening was also improved. The results are shown in Figure 8. The AID-TAT peptide was administered three times a week because it was hypothesized that the bound AID-TAT peptide would persist for 3–4 days, consistent with the turnover rate of the Cav1.2 channel protein.

[0200] Example 9 I Ca-L In vitro binding to the β2 subunit of AID P7 , A.I.D. P14 , A.I.D. P15 , and A.I.D. P16 demonstrate increased binding affinity of In this experiment, a binding affinity assay was utilized to examine the binding potency of the AID peptide variants presented in Table 8 compared to the original AID peptide. [Table 8]

[0201] Biotinylation of original AID (without TAT): For each reaction (a dose-response curve consisted of six reactions), 0.02 nM AID-TAT was added to the biotinylation solution (1 nM amine-PEG 11The beads were incubated in 0.1 M MES buffer containing 0.1 nM EDC and biotin for 2 hours at room temperature with agitation (400 rpm). Affinity beads (Dynabeads M-280 Streptavidin, ThermoFisher Scientific) were then incubated with biotinylated AID peptide for 30 minutes at room temperature with agitation (400 rpm). The biotinylated AID-coated affinity beads were then washed first with 0.1% BSA in PBS, followed by three 3-minute washes in PBS.

[0202] Prebinding of the AID (without TAT) mutant peptide of interest to the CaVβ2 subunit: The AID mutant peptide was serially diluted (0, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM) and assayed for binding as previously described (Haase H, Podzuweit T, Lutsch G, Hohaus A, Kostka S, Lindschau C, et al. Signaling from beta-adrenoceptor to L-type calcium channels: identification of a novel cardiac protein kinase A target possessing similarities to AHNAK. FASEB J. 1999;13(15):2161-72; Hohaus A, Poteser M, Romanin C, Klugbauer N, Hofmann F, Morano I, et al. Modulation of the smooth-muscle L-type Ca2+ channel alpha1 subunit (alpha1C-b) by the beta2a subunit: a peptide which inhibits binding of beta to the I-II linker of alpha1 induces functional uncoupling. Biochem J. 2000;348 Pt 3:657-65; Haase H, Striessnig J, Holtzhauer M, Vetter R, Glossmann H. A rapid procedure for the purification of cardiac 1,4-dihydropyridine receptors from porcine heart. Eur J Pharmacol. 1991;207(1):51-9), were incubated with 100 μg of Cavβ2 subunit from cardiac homogenates and cytosolic protein preparations at room temperature for 2 hours with agitation (400 rpm).

[0203] Competitive binding of AID-affinity beads and Cavβ2 subunit: Next, the diluted Cavβ2-AID mutant peptide was incubated with biotinylated AID-coated affinity beads for 2 hours at room temperature with gentle agitation (400 rpm). The sample was placed on a magnetic stand to allow the affinity beads to migrate toward the magnet. The supernatant was removed and treated with 1x sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 100 mM DTT, 0.002% bromophenol blue) at 95°C for 5 minutes. The resulting proteins were analyzed by immunoblotting.

[0204] Using dose-response curves, decreased K values ​​were observed for AID compared to the AID peptide. P7 -TAT, AID P14、 AID P15 , and A.I.D. P16 These four peptides were found to bind to the AID domain, indicating the strong binding affinity of these variants to the AID region (Figure 9). In Figure 9, n indicates the number of technical replicates. P values ​​compared to AID(S)-TAT as determined by Mann-Whitney t-test are shown. Further in vitro and in vivo experiments focused on these four peptides.

[0205] Example 10 Exposure of cTnI-G203S cardiomyocytes to the AID-TAT variant in vitro demonstrated that Ψ m and more effective in restoring flavoprotein oxidation In this experiment, wt myocytes were pre-exposed to 0.1, 0.5, or 1 μM for 20 min to induce flavoprotein oxidation and Ψ m The changes were evaluated.

[0206] Measurement of mitochondrial membrane potential: The fluorescent indicator 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was used to measure mitochondrial membrane potential (Ψ) as previously described (Viola HM, Arthur PG, Hool LC. Transient exposure to hydrogen peroxide causes an increase in mitochondria-derived superoxide as a result of sustained alteration in L-type Ca2+ channel function in the absence of apoptosis in ventricular myocytes. Circ Res. 2007;100(7):1036-44). m ) was measured. Fluorescence signals were measured on a Hamamatsu Orca ER digital camera attached to a Nikon TE2000-U inverted microscope. Images were acquired at 2-minute intervals with 50 ms exposure. Signals were quantified by manually tracking myocytes using Metamorph 6.3 (version 7.10.3). An equivalent area containing no cells was used as background and subtracted. 580 nm / 535 nm ratiometric fluorescence values ​​recorded over a period of 6 minutes before and 4 minutes after drug addition were averaged, and the change in the fluorescence ratio was reported as a percentage increase from the basal mean value. When the signal was Ψ m To confirm that Ψ m In addition, the individual 580 and 535 signals were evaluated in each experiment, and the fluorescent indicator was m In calcium-free experiments, cells were exposed to calcium-free HBS (additionally supplemented with 3 mM EGTA and 200 nM JC-1) for at least 3 hours, followed by Ψ m The change in was measured.

[0207] Measurement of mitochondrial flavoprotein oxidation: Flavoprotein autofluorescence was used to measure flavoprotein oxidation based on a previously described method (Yaniv Y, Juhaszova M, Lyashkov AE, Spurgeon HA, Sollott SJ, Lakatta EG. Ca2+-regulated-cAMP / PKA signaling in cardiac pacemaker cells links ATP supply to demand. J Mol Cell Cardiol. 2011;51(5):740-8). Fluorescence at excitation 480 nm and emission 535 nm was measured on a Hamamatsu Orca ER digital camera attached to a Nikon TE2000-U inverted microscope. Fluorescence images were acquired at 1-min intervals with 200-ms exposure. Signal was quantified by manually tracking myocytes using Metamorph 6.3 (version 7.10.3). Equivalent areas containing no cells were used as background and subtracted. Fluorescence values ​​recorded over 5 min before and 5 min after drug addition were averaged, and the change in fluorescence ratio was reported as a percentage increase from the basal mean value. FCCP (50 μM) and NaCN (40 mM) were added at the end of each experiment to achieve maximum and minimum fluorescence values, indicating maximum and minimum flavoprotein oxidation, respectively.

[0208] I ca-LThe agonist BayK(-) was used to activate the channel and assess mitochondrial function. Consistent with previous findings, in the presence of 10 μM inactive scrambled peptide (AID(S)-TAT), BayK(-) induced an increase in flavoprotein oxidation that was comparable to the response elicited by application of BayK(-) alone (Figure 10A-C) (Viola HM, Shah AA, Johnstone VPA, Cserne Szappanos H, Hodson MP, Hool LC. Characterization and validation of a preventative therapy for hypertrophic cardiomyopathy in a murine model of the disease. Proc Natl Acad Sci U S A. 2020;117(37):23113-24). In addition, exposure of wt myocytes to 1 μM AID-TAT peptide was found to significantly attenuate the BayK(-) response (Figure 10A). At 1 μM, the peptide variant AID P7 -TAT, AID P14 -TAT, and AID P15 AID-TAT was as effective as the original AID-TAT in attenuating responses in wt myocytes (Fig. 10A). At a concentration of 0.5 μM, the original AID-TAT did not attenuate responses to BayK(-), but AID P7 -TAT, AID P14 , and A.I.D. P15 At a concentration of 0.1 μM, neither the original AID-TAT peptide nor any of the AID-TAT peptide variants was effective (P<0.05). In cTnI-G203S myocytes, 0.5 μM AID-TAT did not attenuate the response, whereas AID P7 -TAT and AID P14AID(S)-TAT significantly attenuated the increase in flavoprotein oxidation caused by BayK(-) exposure (P<0.01 and P<0.05, respectively) (Figure 10D). Figure 10 shows the mean ± SEM of flavoprotein fluorescence from cTnI.2 wt and cTnI-G203S mutant myocytes before and after exposure to 10 μM BayK(-) in the presence of 1 μM AID(S)-TAT and increasing concentrations of variant peptides as indicated. N = number of animals, n = number of cardiomyocytes. P values ​​compared to AID(S)-TAT as determined by Kruskal-Wallis test are shown.

[0209] Based on the results obtained from the in vitro assessment of flavoprotein oxidation, 0.5 μM AID-TAT variants inhibited mitochondrial membrane potential (Ψ m We further investigated the effect of JC-1 fluorescence under calcium-free conditions as previously described (Viola H, Johnstone V, Cserne Szappanos H, Richman T, Tsoutsman T, Filipovska A, et al. The L-type Ca(2+) channel facilitates abnormal metabolic activity in the cTnI-G203S mouse model of hypertrophic cardiomyopathy. J Physiol. 2016;594(14):4051-70; Viola HM, Shah AA, Johnstone VPA, Cserne Szappanos H, Hodson MP, Hool LC. Characterization and validation of a preventative therapy for hypertrophic cardiomyopathy in a murine model of the disease. Proc Natl Acad Sci U S A. 2020;117(37):23113-24). Consistent with previous studies, application of BayK(-) reduced Ψ in the presence of 10 μM AID(S)-TAT. mIn both wt and cTnI-G203S myocytes, exposure to 0.5 μM AID-TAT was found to be ineffective in attenuating the response to BayK(-), whereas exposure to 0.5 μM AID-TAT was not effective in attenuating the response to BayK(-). P7 -TAT, AID P14 -TAT, and AID P15 These data suggest that, at the in vitro level, AID P7 -TAT and AID P14 Figure 11 shows that AID(S)-TAT is most effective at normalizing mitochondrial metabolic activity in cTnI-G203S myocytes. Figure 11 shows the mean ± SE of JC-1 fluorescence from cTnI.2 wt and cTnI-G203S mutant myocytes before and after exposure to 10 μM BayK(-) in the presence of 1 μM AID(S)-TAT and 0.5 μM of the indicated variant peptide. N = number of animals, n = number of cardiomyocytes. P values ​​compared to AID(S)-TAT as determined by Kruskal-Wallis test are shown.

[0210] Example 11 In vivo treatment of cTnI-G203S mice with the AID-TAT variant does not alter blood pressure method Male mice aged 20-30 weeks expressing the human cTnI gene encoding human disease-causing cTnI-G203S were generated and used for in vivo studies. Depending on the background, mice develop hallmarks of HCM between 20 and 25 weeks of age. (Viola H, Johnstone V, Cserne Szappanos H, Richman T, Tsoutsman T, Filipovska A, et al. The L-type Ca(2+) channel facilitates abnormal metabolic activity in the cTnI-G203S mouse model of hypertrophic cardiomyopathy. J Physiol. 2016;594(14):4051-70; Viola HM, Shah AA, Johnstone VPA, Cserne Szappanos H, Hodson MP, Hool LC. Characterization and validation of a preventative therapy for hypertrophic cardiomyopathy in a murine model of the disease. Proc Natl Acad Sci U S A. 2020;117(37):23113-24; Tsoutsman T, Chung J, Doolan A, Nguyen L, Williams IA, Tu E, et al. Molecular insights from a novel cardiac troponin I mouse model of familial hypertrophic cardiomyopathy. J Mol Cell Cardiol. 2006;41(4):623-32). Age-matched male mice expressing normal human troponin I gene were used as controls and designated wild-type (wt). For in vivo studies, 20-week-old cTnI-G203S mice were treated with 10 μM AID(S)-TAT (2 mg / kg) or 5 μM AID-TAT mutant peptide via intraperitoneal injection three times a week for a total period of 5 weeks.The peptide was dissolved in phosphate-buffered saline (PBS), and the total amount of 10 μM AID-TAT mutant peptide administered over 5 weeks was 0.9 mg. The total amount of 5 μM AID-TAT mutant peptide administered over 5 weeks was 0.45 mg. Male mice were utilized to eliminate potential differences in response due to gender. The number of mice used is indicated by N.

[0211] Blood pressure was assessed before the start of the treatment regimen, 1 hour after the first injection (acute), and after the completion of the 5-week treatment regimen. Blood pressure measurements of mice were obtained via a CODA non-invasive blood pressure system (Tail-Cuff method, Kent Scientific Corporation). Conscious mice were placed in a restrainer of appropriate size based on body weight, and pre-set parameters for assessing blood pressure (software details provided by Henrietta) were used for a total of 15 cycles (the first 5 cycles were used for acclimatization). The average of 10 measurements was used.

[0212] result Systolic and diastolic blood pressures were recorded at three time points: 1 hour before the first dose, 1 hour after the first dose (acute response), and at the end of the 5-week treatment regimen (chronic response). No significant changes in systolic or diastolic blood pressure were observed in mice treated with any of the AID-TAT variants at any time point (Figure 12). These data indicate that the peptides do not cause vasodilatory or negative inotropic effects that can occur with treatment with calcium channel antagonists (e.g., diltiazem). Figure 12 shows (A-B) 10 μM AID(S)-TAT, 5 μM AID P7 -TAT, 5 μM AID P14 -TAT, 5 μM AID P15 -TAT, or 5 μM AID P16Figures represent the mean ± SEM of blood pressure measurements from wt and cTnI-G203S mice treated with -TAT (three times a week for 5 weeks). Measurements were taken before treatment initiation (Pre), 1 hour after the first injection (1 hour post-injection), or after the 5-week treatment regimen (After), as indicated. N = number of mice as indicated. P = ns as determined by Kruskal-Wallis test.

[0213] Example 12 in vitro toxicity The fluorescent indicator propidium iodide (P4170; Sigma-Aldrich) was used to assess the toxicity of the AID-TAT mutant peptide in vitro in cardiomyocytes. Fluorescence at excitation 480 nm and emission 580 nm was measured on a Hamamatsu Orca ER digital camera attached to a Nikon TE2000-U inverted microscope. Fluorescent images were captured using 1 μM AID. P7 -TAT(P7-TAT), AID P14 -TAT(P14-TAT), AID P15 -TAT (P15-TAT), or AID P16 Images were acquired using a 50 ms exposure after an initial 20 minute preincubation with -TAT (P16-TAT) and after a 5 minute incubation with the dye. No evidence of toxicity was observed.

[0214] Example 13 In vivo treatment of cTnI-G203S mice with the AID-TAT variant is not toxic In vivo toxicity parameters Mice were treated with 5 μM AID(S)-TAT, AID P7 -TAT(P7-TAT), AID P14 -TAT (P14-TAT), or AID P15 -TAT (P15-TAT), or AID P16Mice were treated with AID-TAT three times a week for 5 weeks (equivalent to 15 doses). The peptide was dissolved in phosphate-buffered saline (PBS), and the total amount of 5 μM AID-TAT mutant peptide administered over the 5 weeks was 0.45 mg. Body weights were recorded before administration of each peptide dose. A 5-10% reduction in body weight may indicate toxicity and warrants additional monitoring as recommended by the University of Western Australia Animal Ethics Committee in accordance with the Guidelines to Promote the Wellbeing of Animals Used for Scientific Purposes (NHMRC 2008). After completion of the treatment regimen, mice were anesthetized and a terminal blood sample was drawn. Blood was allowed to clot in lithium-heparin tubes at room temperature for 20 minutes. Plasma was separated by centrifugation at 3000 g for 10 minutes at 4°C. To measure nephrotoxicity, urea and creatinine concentrations were assessed using the Quantichrom Urea Assay Kit (BioAssay Systems, Hayward, CA) and Quantichrom Creatinine Assay Kit (BioAssay Systems, Hayward, CA), respectively. To measure hepatotoxicity, alanine transaminase (ALT) and aspartate transaminase (AST) concentrations were measured using the Alanine Transaminase Assay Kit (BioAssay Systems, Hayward, CA) and Aspartate Transaminase Assay Kit (BioAssay Systems, Hayward, CA), respectively. Assays were performed using a spectrophotometer (CLARIOstar, BMG Labtech) according to the manufacturer's instructions.

[0215] result After completing the 5-week in vivo treatment regimen, final plasma samples were collected and evaluated for nephrotoxicity and hepatotoxicity. No significant alterations in urea or alanine transaminase (ALT) were observed compared to plasma evaluated from AID(S)-TAT-treated wild-type and cTnI-G203S mice, indicating that treatment with the AID-TAT variants does not cause nephrotoxicity or hepatotoxicity (Figure 13A-B). Additionally, no significant reduction in body weight was observed over the duration of the entire treatment protocol (Figure 13C). Overall, these data demonstrate that in vivo treatment of cTnI-G203S mice with AID-TAT variants is not toxic. Figure 13 shows the effects of 10 μM AID(S)-TAT, 5 μM AID, and 10 μM AID-TAT, as indicated. P7 -TAT, or 5 μM AID P14 -TAT, or AID P15 -TAT, or AID P16 Figures represent mean ± SEM values ​​of urea (A) and ALT (B) concentrations from plasma of wt and cTnI-G203S mice treated with 5 μM AID(S)-TAT (three times a week for 5 weeks). N = number of mice. P = ns (A-B) as determined by ANOVA and Kruskal-Wallis test. (C) 5 μM AID(S)-TAT, 5 μM AID(S)-TAT, and 5 μM AID(S)-TAT, as indicated. P7 -TAT, 5 μM AID P14 -TAT, 5 μM AID P15 -TAT, or 5 μM AID P16 Mean ± SEM of body weights recorded from cTnI-G203S mice treated with -TAT (3 times per week for 5 weeks, equivalent to 15 doses in total). N = number of mice as indicated. The red line indicates the threshold for 5% weight loss.

[0216] Example 14 In vivo treatment of precardiomyopathic cTnI-G203S mice with the AID-TAT variant slows the progression of fibrosis method After the treatment regimen was completed, mouse hearts were excised and prepared for Masson's Trichrome staining based on a previously described method with minor modifications (Van De Vlekkert D, Machado E, d'Azzo A. Analysis of Generalized Fibrosis in Mouse Tissue Sections with Masson's Trichrome Staining. Bio Protoc. 2020;10(10):e3629). Hearts were washed in PBS and fixed overnight in 10% buffered formalin saline (FSAL-5L; Hurst Scientific, AUS). After fixation, hearts were washed three times for 10 minutes in PBS and incubated overnight in 30% sucrose solution (in PBS). After overnight incubation, hearts were then coated in Tissue-Tek OCT compound (IA018; ProSciTech, AUS) and embedded in a 4 cm deep cylindrical aluminum foil mold containing approximately 2 ml of OCT. The mold was submerged in an isopentane-filled tube in a canister containing liquid nitrogen to slowly freeze the cardiac tissue. The tissue samples were stored at -80°C until cryosectioning.

[0217] Hearts were cut into 7 μm sections, collected onto warm (room temperature) subbed Superfrost slides and stored at −80° C. until staining.

[0218] To achieve thermal equilibrium before Masson's trichrome staining, slides were transferred from -80°C to -20°C overnight. After overnight incubation, slides were transferred from -20°C to room temperature for 1 hour, then washed once in running water for 2 minutes to remove the OCT and secondary fixed overnight in Bouin's solution. To completely remove the Bouin's solution, slides were washed 1 to 3 times in running water for 5 minutes each. Slides were stained using the following steps: Celestine blue (5 minutes), running water (2 minutes), Harris hematoxylin (5 minutes), running water (2 minutes), distilled water (2 minutes), Ponceau-fuchsin (10 minutes), distilled water (20 seconds), phosphomolybdic acid (4 minutes), aniline blue (1 minute), and 1% acetic acid (1 minute). Slides were then blotted dry with filter paper and dehydrated, and washing and mounting steps were performed as previously outlined (Van De Vlekkert D, Machado E, d'Azzo A. Analysis of Generalized Fibrosis in Mouse Tissue Sections with Masson's Trichrome Staining. Bio Protoc. 2020;10(10):e3629).

[0219] To observe the level of cardiac fibrosis in mice, at least 15 sections of the microscopic field were randomly selected for histological analysis for each animal. Images of cardiac sections were obtained at 20x magnification using a Nikon upright light microscope (DS-Qi2, Japan) and analyzed using ImageJ (NIH). Collagen accumulation was analyzed using the ratio of blue area to total myocardial area in Masson's trichrome-stained sections. Two researchers performed the analysis of histological features blindly.

[0220] result It is well established that myocardial fibrosis is a characteristic feature of HCM (O'Connell TD, Rodrigo MC, Simpson PC. Isolation and culture of adult mouse cardiac myocytes. Methods Mol Biol. 2007;357:271-96). Myocardial fibrosis is a collagen-rich extracellular matrix that is significantly increased in the hypertrophied heart (Haase H, Striessnig J, Holtzhauer M, Vetter R, Glossmann H. A rapid procedure for the purification of cardiac 1,4-dihydropyridine receptors from porcine heart. Eur J Pharmacol. 1991;207(1):51-9). Increased collagen accumulation and fibrosis are maladaptive and associated with impaired cardiac relaxation, thereby increasing the risk of heart failure (Tang H, Viola HM, Filipovska A, Hool LC. Ca(v)1.2 calcium channel is glutathionylated during oxidative stress in guinea pig and ischemic human heart. Free Radic Biol Med. 2011;51(8):1501-11).

[0221] Herein, we evaluated the efficacy of treating 20-week-old prehypertrophic cTnI-G203S mice with 5 μM AID-TAT variant peptide (three times a week for 5 weeks) on the development and progression of fibrosis. The peptide was dissolved in phosphate-buffered saline (PBS), and the total amount of 5 μM AID-TAT variant peptide administered over 5 weeks was 0.45 mg. After the 5-week treatment regimen, mouse hearts were excised, fixed, and embedded in OCT for cryosectioning. Collagen deposition in cardiac tissue sections was visualized using Masson's trichrome staining, which shows myocardium (stained red), nuclei (stained purple / black), and collagen (stained blue). Images were acquired using a Nikon upright microscope, and collagen volume fraction (%) was determined using color thresholding in ImageJ. Consistent with the development of HCM, we found that the left ventricular posterior wall collagen volume fraction (CVF, %) was significantly increased in cTnI-G203S mice treated with 10 μM AID(S)-TAT compared to wt myocytes (Figure 14). P7 The vasopressin-induced vasoconstriction was significantly reduced in cTnI-G203S mice treated with 10 μM AID(S)-TAT (P7-TAT) (Figure 14). Figure 14 shows (A) a representative section of left ventricular cardiac tissue. Scale bar = 50 μM. (B) vasopressin-induced vasoconstriction was significantly reduced in cTnI-G203S mice treated with 10 μM AID(S)-TAT, 5 μM AID(S)-TAT, or 5 μM AID(S)-TAT. P7 -TAT, or 5 μM AID P14 LV collagen volume fraction (%) from cardiac sections of 25-week-old wt and 25-week-old cTnI-G203S mice after treatment with AID(S)-TAT (3 times per week for 5 weeks). N = number of mice indicated. Values ​​reported as mean ± SEM; P values ​​compared to cTnI-G203S AID(S)-TAT as determined by Kruskal-Wallis test.

[0222] Example 15 In vivo treatment of precardiopathic cTnI-G203S mice with AID peptide variants prevents the development of hypertrophic cardiomyopathy method Echocardiographic studies to measure left ventricular function were performed on mice under light methoxyflurane anesthesia using an i13L probe on a Vivid™ 7 IQ ultrasound system (GE Healthcare, Little Chalfont, UK) as previously described (Viola H, Johnstone V, Cserne Szappanos H, Richman T, Tsoutsman T, Filipovska A, et al. The L-type Ca(2+) channel facilitates abnormal metabolic activity in the cTnI-G203S mouse model of hypertrophic cardiomyopathy. J Physiol. 2016;594(14):4051-70; Viola HM, Shah AA, Johnstone VPA, Cserne Szappanos H, Hodson MP, Hool LC. Characterization and validation of a preventative therapy for hypertrophic cardiomyopathy in a murine model of the disease. Proc Natl Acad Sci US A. 2020;117(37):23113-24; Viola HM, Johnstone VPA, Cserne Szappanos H, Richman TR, Tsoutsman T, Filipovska A, et al. The Role of the L-Type Ca(2+) Channel in Altered Metabolic Activity in a Murine Model of Hypertrophic Cardiomyopathy. JACC Basic Transl Sci. 2016;1(1-2):61-72). Each N represents the mean of quantitative measurements from wt or cTnI-G203S mice in each treatment group.

[0223] result Echocardiographic evaluation of cTnI-G203S mice reveals that HCM features develop from approximately 21 weeks of age (Viola H, Johnstone V, Cserne Szappanos H, Richman T, Tsoutsman T, Filipovska A, et al. The L-type Ca(2+) channel facilitates abnormal metabolic activity in the cTnI-G203S mouse model of hypertrophic cardiomyopathy. J Physiol. 2016;594(14):4051-70; Viola HM, Shah AA, Johnstone VPA, Cserne Szappanos H, Hodson MP, Hool LC. Characterization and validation of a preventative therapy for hypertrophic cardiomyopathy in a murine model of the disease. Proc Natl Acad Sci U S A. 2020;117(37):23113-24). Consistent with previous findings, treatment of 20-week-old cTnI-G203S mice with 10 μM AID(S)-TAT (the peptide was dissolved in phosphate-buffered saline (PBS) and the total amount of 10 μM AID-TAT mutant peptide administered over 5 weeks was 0.9 mg) until 25 weeks of age (10 μM, 3 times per week for 5 weeks) resulted in a significant decrease in left-ventricular end diameter (LVEDd and LVEDs) and a significant increase in fractional shortening (FS), ejection fraction (EF), left ventricular posterior wall thickness (LVPWd and LVPWs), and interventricular septum (IVDs) compared with age-matched wt mice treated with AID(S)-TAT (Figure 15). In addition, age-matched cTnI-G203S mice treated with 10 μM AID-TAT demonstrated restoration of FS, EF, IVSd, LVEDd, LVEDs, and LVPWd compared to AID(S)-TAT-treated cTnI-G203S mice (Figure 15).Figure 15 represents: LVEDd, left ventricular end-diastolic diameter; LVEDs, left ventricular end-systolic diameter; FS, fractional shortening; EF, ejection fraction; LVPWd, left ventricular posterior wall diastole; LVPWs, left ventricular posterior wall systole; IVDd, interventricular septum diastole; IVDs, interventricular septum systole; HR, heart rate. N = number of mice. Values ​​are reported as mean ± SEM; P values ​​compared to cTnI-G203S AID(S)-TAT as determined by Kruskal-Wallis test.

[0224] The efficacy of treating 20-week-old prehypertrophic cTnI-G203S mice with 5 μM AID-TAT variant was investigated using the same treatment protocol (three times a week for 5 weeks). We performed echocardiography on the mice before and after 5 weeks of treatment to evaluate cardiac morphology and function. P7 Treatment of cTnI-G203S mice with 5 μM AID-TAT (P7-TAT) effectively restored FS, EF, IVSs, LVPWd, and LVPWs compared to AID(S)-TAT, similar to 10 μM AID-TAT (Figure 15). A trend toward increased LVEDd and LVEDs, as well as decreased IVDS, was observed (Figure 15). Interestingly, 5 μM AID P7 Twenty-five week old cTnI-G203S mice treated with 5 μM AID-TAT (SEQ ID NO: 4-TAT) demonstrate a significant reduction in LVPWs and IVSs that was not achieved by 10 μM AID-TAT (FIG. 15). P14 Treatment of cTnI-G203S mice with 10 μM AID-TAT (P14-TAT) effectively restored all echocardiographic parameters (except LVEDd), including some that were unaltered by 10 μM AID-TAT (FS, EF, IVSd, IVSs, LVEDs, LVPWd, and LVPWs). A trend toward increased LVEDd was observed. In other cohorts, treatment with 5 μM AID-TAT effectively restored all echocardiographic parameters (except LVEDd). P15 -TAT (P15-TAT) effectively restored FS, EF, IVSd, IVSs, and LVPWs, but 5 μM AID P16 Treatment with -TAT (P16-TAT) demonstrated recovery of only IVSd and IVSs.

[0225] The effect of treating mice with the peptide on the development of hypertrophy, assessed as the change in the ratio of heart weight to body weight, was also examined. P7 -TAT (P7-TAT) or 5 μM AID P14 We found that treatment with 10 μM AID(S)-TAT (P14-TAT) significantly reduced heart weight / body weight, indicating a regression of hypertrophy (Figure 16). P7 -TAT, 5 μM AID P14 -TAT, 5 μM AID P15 -TAT, or 5 μM AID P16 Figures represent the mean ± SEM of heart weight:body weight ratio measurements for wt and cTnI-G203S mice treated with -TAT, n = number of mice indicated. * P<0.05, ** P<0.01 compared with cTnI-G203S AID(S)-TAT as determined by Kruskal-Wallis test.

[0226] Overall, this data demonstrates that treatment with the peptides of the invention prior to the onset of hypertrophy prevents the onset of hypertrophy, reverses fibrosis, and improves cardiac function in cTnI-G203S mice, with the variant peptide being more effective than the original AID-TAT in preventing hypertrophy.

Claims

1. Amino acid sequence: QQX １ EEDX ２ KGYLDWITQAE A peptide comprising: X １ is an amino acid selected from the group comprising Q, E, or R; X ２ is an amino acid selected from the group comprising L or E; The peptide does not consist of SEQ ID NO:

1.

2. A peptide comprising any one of the amino acid sequences of SEQ ID NO: 4 to 7, wherein the peptide does not consist of SEQ ID NO:

1.

3. A peptide moiety comprising the amino acid sequence of claim 1 or claim 2; and Amino acid sequence: RKKRRQRRRZaa wherein Zaa is 6-aminohexanoic acid. A peptide comprising:

4. A method for enhancing L-type Ca in cardiac cells of a subject, comprising administering the peptide according to any one of claims 1 to 3. ２＋ A composition for modulating the movement of the beta subunit of a channel.

5. A method for enhancing L-type Ca in cardiac cells of a subject, comprising administering the peptide according to any one of claims 1 to 3. ２＋ A composition for modulating the binding of beta subunits of a channel.

6. A method for enhancing L-type Ca in cardiac cells of a subject, comprising administering the peptide according to any one of claims 1 to 3. ２＋ Compositions for modulating channels.

7. A composition for treating, preventing or reducing myocardial damage and / or oxidative stress in the heart of a subject, comprising a peptide according to any one of claims 1 to 3.

8. The composition of claim 7 , wherein the myocardial damage comprises cardiac hypertrophy.

9. Intracellular Ca ２＋ 8. The composition of claim 7, wherein the level is reduced or substantially maintained in cardiac cells of the heart of the subject.

10. A composition for treating or preventing cardiac hypertrophy in a subject, comprising the peptide of any one of claims 1 to 3.

11. A composition for slowing the progression of myocardial fibrosis in a subject, comprising the peptide of any one of claims 1 to 3.

12. A polynucleotide encoding the peptide according to any one of claims 1 to 3.

13. 10. Use of a peptide according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment of reperfusion injury in the heart of a subject.

14. Use of a peptide according to any one of claims 1 to 3 in the manufacture of a medicament for treating, preventing or ameliorating myocardial damage and / or oxidative stress in the heart of a subject.

15. A pharmaceutical, prophylactic or therapeutic composition comprising a peptide according to any one of claims 1 to 3 and one or more pharmaceutically acceptable carriers and / or diluents.

16. 10. A kit for treating, preventing or ameliorating the effects of myocardial damage and / or oxidative stress in the heart of a subject, comprising at least one peptide according to any one of claims 1 to 3 packaged in a suitable container together with instructions for its use.

17. A composition for treating reperfusion injury in the heart of a subject, comprising the peptide of any one of claims 1 to 3.

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