Inhibition of ZD17-JNK interaction as a therapy for acute myocardial infarction

Abstract

Disclosed herein are uses of polypeptides comprising NIMoEsh for treating diseases or conditions associated with acute myocardial infarction (AMI) in subjects in need thereof, uses of polypeptides comprising NIMoEsh for restoring cardiac function after AMI in subjects in need thereof, uses of polypeptides comprising NIMoEsh for reducing or preventing loss of cardiac function caused by AMI in subjects in need thereof, uses of polypeptides comprising NIMoEsh for reducing cardiac tissue infarction caused by AMI in subjects in need thereof, and uses of polypeptides comprising NIMoEsh for protecting cardiomyocytes from loss of function caused by AMI in subjects in need thereof. Also disclosed herein are methods for performing such treatment, restoration, reduction or prevention, reduction and / or protection, and polypeptides comprising NIMoEsh for such treatment, restoration, reduction or prevention, reduction and / or protection.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 899,440, filed on September 12, 2019. Technical Field

[0003] The present disclosure relates generally to cardioprotection, and more particularly to the use of polypeptides to treat acute myocardial infarction. Background Art

[0004] Acute myocardial infarction (AMI), commonly known as a heart attack, is a life-threatening cardiovascular disease. During an AMI, the blood supply to the heart is suddenly restricted due to a blockage in the coronary arteries, which can cause severe damage to the heart. AMI kills more than 2.4 million people in the United States and more than 4 million in Europe and North Asia each year, and is responsible for more than one-third of deaths in developed countries. (Reed, GW, Rossi, JE, and Cannon, CP. Acute myocardial infarction. Lancet 389, 197-210, doi:10.1016 / S0140-6736(16)30677-8(2017))

[0005] It is reported that the use of current drugs such as aspirin, nitroglycerin and statins helps reduce the risk of AMI (see, for example, Ferreira, JC and Mochly-Rosen, D. Circ J 76, 15-21 (2012); Dai, Y. and Ge, J. Thrombosis 2012, 245037, doi: 10.1155 / 2012 / 245037 (2012); Fung, V. et al., PLoS One 13, e0191817, doi: 10.1371 / journal.pone.0191817 (2018)), but cannot prevent the death of cardiomyocytes after AMI. Similarly, current surgical approaches such as percutaneous coronary intervention and coronary artery bypass surgery (Perrier, S. et al., Interact Cardiovasc Thorac Surg 17, 1015-1019, doi: 10.1093 / icvts / ivt381 (2013)) can help restore blood circulation to the heart, but have not been reported to have cardioprotective efficacy after AMI.

[0006] To date, the FDA has not approved any cardioprotective drugs for AMI. For example, cyclosporine is a small molecule that has shown potential cardioprotective efficacy in multiple animal AMI models, but failed in human clinical trials. (Rahman, FA et al., Efficacy and Safety of Cyclosporine in Acute Myocardial Infarction: A Systematic Review and Meta-Analysis. Front Pharmacol 9, 238, doi: 10.3389 / fphar.2018.00238 (2018))

[0007] Despite the progress achieved to date in the development of cardioprotective therapies, there is still room for improvement to address the above-mentioned problems and shortcomings of existing technologies. Summary of the invention

[0008] It is an object of the present invention to obviate or mitigate at least one of the above-mentioned disadvantages of the prior art.

[0009] Another object of the present invention is to provide a novel cardioprotective therapy.

[0010] Thus, in one aspect thereof, the present disclosure provides a method of treating a disease or condition associated with or as an acute myocardial infarction (AMI) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide comprising NIMoEsh.

[0011] In another aspect thereof, the present disclosure provides a method of restoring cardiac function after AMI in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide comprising NIMoEsh.

[0012] In another aspect thereof, the present disclosure provides a method of reducing or preventing AMI-induced loss of cardiac function in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide comprising NIMoEsh.

[0013] In another aspect thereof, the present disclosure provides a method of reducing AMI-induced cardiac tissue infarction in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide comprising NIMoEsh.

[0014] In another aspect thereof, the present disclosure provides a method of protecting cardiomyocytes from AMI-induced functional loss in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide comprising NIMoEsh.

[0015] In another aspect thereof, the present disclosure provides a use of a polypeptide comprising NIMoEsh for treating a disease or condition associated with AMI in a subject in need thereof.

[0016] In another aspect thereof, the present disclosure provides a use of a polypeptide comprising NIMoEsh to restore cardiac function after AMI in a subject in need thereof.

[0017] In another aspect thereof, the present disclosure provides a use of a polypeptide comprising NIMoEsh for reducing or preventing AMI-induced loss of cardiac function in a subject in need thereof.

[0018] In another aspect thereof, the present disclosure provides a use of a polypeptide comprising NIMoEsh for reducing cardiac tissue infarction caused by AMI in a subject in need thereof.

[0019] In another aspect thereof, the present disclosure provides a use of a polypeptide comprising NIMoEsh for protecting cardiomyocytes from loss of function caused by AMI in a subject in need thereof.

[0020] In another aspect thereof, the present disclosure provides a polypeptide comprising NIMoEsh for use in one or more of the above-mentioned uses.

[0021] Therefore, the inventors have developed a novel therapy for the cardioprotection of a subject. This cardioprotective therapy can be provided after AMI to reduce or prevent damage to cardiac tissue caused by AMI. In particular, by using this therapy, cardiomyocytes can be protected from cell death or loss of function caused by AMI. In addition, the use of the present therapy after AMI can at least partially restore cardiac function. The therapeutic use of the present invention can provide an alternative to existing drugs and non-drug treatment options, and is particularly a potential therapy for cardioprotection after AMI.

[0022] Other advantages of the present invention will become apparent to those skilled in the art after reading this specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Embodiments of the present invention will be described with reference to the accompanying drawings, wherein like reference numerals refer to like parts, and wherein:

[0024] Figure 1The protective effect of NIMoEsh-Tat peptide on primary cardiomyocyte culture against H2O2-induced cell injury was demonstrated. NIMoEsh-Tat peptide (30 min pretreatment + 12 h posttreatment) was bath-applied to primary cardiomyocyte cultures treated with H2O2 (300 μM for 4 h), and its protective efficacy was determined using multiple cell death assays 12 h after H2O2 treatment: (a) MTT assay (control: N = 3; H2O2: N = 6; H2O2 + NIMoEsh-Tat: N = 6; F(2,12) = 10.05, P < 0.01); (b) apoptosis assay (control: N = 3; H2O2: N = 4; H2O2 + NIMoEsh-Tat: N = 6; F(2,10) = 56.67, P < 0.001); (c) CK activity in the culture medium (control: N = 3; H2O2: N = 6; H2O2 + NIMoEsh-Tat: N = 6; F(2,10) = 56.67, P < 0.001). H2O2+NIMoEsh-Tat: N=6; F(2,12)=20.70, P<0.001); (d) MDA activity in cultured cells (control: N=3; H2O2: N=6; H2O2+NIMoEsh-Tat: N=6; F(2,12)=19.28, P<0.001); (e) LDH activity in culture medium (control: N=3; H2O2: N=6; H2O2+NIMoEsh-Tat: N=6; F(2,12)=16.10, P<0.001); (f) SOD activity in cultured cells (control: N=3; H2O2: N=6; H2O2+NIMoEsh-Tat: N=6; F(2,12)=13.42, P<0.01). Data are expressed as mean ± SEM. Statistical differences among groups were determined by one-way ANOVA followed by LSD post hoc test. *P<0.05, **P<0.01 and ***P<0.001 indicate significant differences. ns indicates not significant.

[0025] Figure 2The protective effect of NIMoEsh-Tat peptide on cardiac tissue infarction caused by AMI is illustrated. (a) TTC staining of fresh rat hearts and (b) quantitative bar graph showing that NIMoEsh-Tat peptide reduced the percentage of cardiac tissue infarction after AMI compared with the saline group (saline group: N=10; NIMoEsh-Tat group: N=11; t(19)=3.71, P<0.01). The cardiac tissue infarction area is highlighted by the dotted line in (a). For cardiac function analysis in (c)-(h): sham group: N=8; saline group: N=7; NIMoEsh-Tat peptide group: N=8. After sham or AMI surgery, the sham, saline, and NIMoEsh-Tat peptide groups showed no significant differences in (c) heart rate (F(2,20)=0.04, P=0.96) and (d) systolic blood pressure (F(2,20)=1.74, P=0.20). Compared with the saline group, the NIMoEsh-Tat peptide-treated group effectively rescued the loss of cardiac function caused by AMI, as shown by the following multiple measurements: (e) left ventricular systolic pressure (F(2,20)=5.09, P<0.05); (f) left ventricular end-diastolic pressure (F(2,20)=42.93, P<0.001); (g) +dp / dtmax (F(2,20)=27.98, P<0.001); (h) -dp / dtmax (F(2,20)=7.34, P<0.01). Data are expressed as mean ± SEM. Statistical differences between the groups in (b) were determined by unpaired t-test. Statistical differences between the groups in (c)-(h) were determined by one-way ANOVA followed by LSD post hoc test. **P<0.01 and ***P<0.001 indicate significant differences. ns indicates not significant.

[0026] Figure 3The protective effect of NIMoEsh-Tat peptide on AMI-induced cardiac injury and functional loss is illustrated. Compared with saline controls (N=5), NIMoEsh-Tat peptide (N=5) reduced AMI-induced deficits in (c) stroke volume (post-AMI: t(8)=-7.04, P<0.001) and (d) ejection fraction (post-AMI: t(4)=-4.31, P<0.05) in pigs, but not in (a) end-diastolic volume (post-AMI: t(8)=-1.71, P=0.13) and (b) end-systolic volume (AMI: t(8)=1.30, P=0.23). (e) Quantified bar graphs and (f) representative TTC staining images show that NIMoEsh-Tat peptide reduced AMI-induced myocardial infarction in pigs (t(8)=4.57, P<0.01) compared with saline. The infarcted area of ​​cardiac tissue is highlighted by the dotted line in (f). (g) Pig hearts treated with NIMoEsh-Tat peptide showed less fibrillation than pig hearts treated with saline. Scale bar in (g): 200 μm. Data are expressed as mean ± SEM. Statistical differences between groups were determined by unpaired t-test. *P<0.05, **P<0.01, and ***P<0.001 indicate significant differences. ns indicates not significant.

[0027] Figure 4 Figure 4 illustrates unbiased treatment and animal selection. Heart rate (a), electrocardiogram (QRS duration (b), QRS voltage (c), T voltage (d), and ST voltage (e)), and body weight (f) were monitored before arterial occlusion, after arterial occlusion, and after reperfusion. Pathological Q waves (b and c), increased T voltage (d), and increased ST voltage (e) indicate successful AMI surgery in pigs. The responses of the two groups of pigs to AMI surgery were almost the same, indicating unbiased treatment and animal selection. NIMoEsh-Tat group: N = 5; saline group: N = 5. Data are expressed as mean ± SEM. DETAILED DESCRIPTION

[0028] As used herein, the term "NIMoEsh" refers to the following amino acid sequence: WAAYRTHSVD [SEQ ID NO: 1].

[0029] The present disclosure relates to a method of treating a disease or condition that is or is associated with acute myocardial infarction (AMI) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide comprising NIMoEsh. In one aspect of the present disclosure, the disease or condition is AMI.

[0030] The present disclosure also relates to a method of restoring cardiac function after AMI in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide comprising NIMoEsh.

[0031] The present disclosure also relates to a method of reducing or preventing AMI-induced loss of cardiac function in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide comprising NIMoEsh.

[0032] The present disclosure also relates to a method of reducing AMI-induced cardiac tissue infarction in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide comprising NIMoEsh.

[0033] The present disclosure also relates to a method of protecting cardiomyocytes from loss of function caused by AMI in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide comprising NIMoEsh.

[0034] The present disclosure also relates to the use of a polypeptide comprising NIMoEsh to treat a disease or condition associated with AMI in a subject in need thereof.

[0035] The present disclosure also relates to the use of polypeptides comprising NIMoEsh to restore cardiac function after AMI in a subject in need thereof.

[0036] The present disclosure also relates to the use of a polypeptide comprising NIMoEsh to reduce or prevent AMI-induced loss of cardiac function in a subject in need thereof.

[0037] The present disclosure also relates to the use of a polypeptide comprising NIMoEsh to reduce cardiac tissue infarction caused by AMI in a subject in need thereof.

[0038] The present disclosure also relates to the use of a polypeptide comprising NIMoEsh to protect cardiomyocytes from loss of function caused by AMI in a subject in need thereof.

[0039] Implementations of these methods and uses may include any one or any combination of two or more of the following features:

[0040] The subject is a human;

[0041] The polypeptide is conjugated to a dat moiety;

[0042] The dat portion is a protein transduction domain;

[0043] The protein transduction domain is HIV-1 Tat;

[0044] The polypeptide and the dat portion together have at least about 90%, or at least about 95%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 3, or the polypeptide and the dat portion together have the amino acid sequence of SEQ ID NO: 3;

[0045] The polypeptide is co-administered to the subject with one or more other active therapeutic ingredients, or the polypeptide is the only active therapeutic ingredient administered to the subject, or the subject is already receiving other cardiovascular drugs;

[0046] The polypeptide is administered in a pharmaceutical composition comprising one or more excipients;

[0047] The pharmaceutical composition is for systemic administration;

[0048] The pharmaceutical composition is for intravenous administration.

[0049] The present disclosure also relates to a polypeptide comprising NIMoEsh for use in any of the above uses.

[0050] As used herein, "peptide" or "polypeptide" can be used interchangeably and generally refer to a compound consisting of at least two amino acid residues covalently linked by a peptide bond or a modified peptide bond. However, when used specifically with reference to a particular SEQ ID NO, it is intended to include an amino acid sequence, such as a sequence represented by SEQ ID NO: 1 or 3, wherein the peptide has cardioprotective activity. Modified peptide bonds can include, for example, peptide isosteres (modified peptide bonds), which can provide additional desired properties to the peptide, such as an increased half-life. The amino acids that make up the peptides or polypeptides described herein can also be modified by natural processes such as post-translational processing or by chemical modification techniques well known in the art. Modifications can occur anywhere in the peptide, including the peptide backbone, amino acid side chains, and the amino or carboxyl termini. It should be understood that the same type of modification can be present in several sites of a given peptide to the same or varying degrees.

[0051] Amino acids are molecules containing an amine group, a carboxylic acid group, and a side chain that varies between different amino acids. Amino acids can be in their natural form, or they can be synthetic amino acids. Amino acids can be described as, for example, polar, nonpolar, acidic, basic, aromatic, or neutral. Polar amino acids are amino acids that can interact with water through hydrogen bonding at biological or near-neutral pH values. The polarity of an amino acid is an indicator of the degree of hydrogen bonding at biological or near-neutral pH values. Examples of polar amino acids include serine, proline, threonine, cysteine, asparagine, glutamine, lysine, histidine, arginine, aspartic acid, tyrosine, and glutamic acid. Examples of nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, and tryptophan. Acidic amino acids have a net negative charge at neutral pH values. Examples of acidic amino acids include aspartic acid and glutamic acid. Basic amino acids have a net positive charge at neutral pH values. Examples of basic amino acids include arginine, lysine and histidine. Aromatic amino acids are generally non-polar and may participate in hydrophobic interactions. Examples of aromatic amino acids include phenylalanine, tyrosine and tryptophan. Tyrosine may also participate in hydrogen bonding through the hydroxyl group on the aromatic side chain. Neutral aliphatic amino acids are generally non-polar and hydrophobic. Examples of neutral amino acids include alanine, valine, leucine, isoleucine and methionine. An amino acid may be described by more than one description category. Amino acids with a common description category may be substituted for each other in a peptide. According to Table A below, amino acid residues may generally be represented by single-letter or three-letter names, corresponding to the common names of amino acids. The amino acids constituting the peptides described herein will be understood to be in L- or D- configurations. The amino acids described herein may be modified by methylation, amidation, acetylation or substitution with other chemical groups that may change the circulation half-life of the peptide without adversely affecting its biological activity.

[0052] It is understood by those skilled in the art that aspects of individual amino acids in the polypeptides described herein can be substituted. In addition, it is understood by those skilled in the art that certain substitutions are more likely to result in activity retention. For example, amino acids can be described as, for example, polar, non-polar, acidic, basic, aromatic or neutral. Polar amino acids are amino acids that can interact with water through hydrogen bonding at biological or near neutral pH values. The polarity of an amino acid is an indicator of the degree of hydrogen bonding at biological or near neutral pH values. Examples of polar amino acids include serine, proline, threonine, cysteine, asparagine, glutamine, lysine, histidine, arginine, aspartic acid, tyrosine and glutamic acid. Examples of non-polar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine and tryptophan. Acidic amino acids have a net negative charge at neutral pH values. Examples of acidic amino acids include aspartic acid and glutamic acid. Basic amino acids have a net positive charge at neutral pH values. Examples of basic amino acids include arginine, lysine and histidine. Aromatic amino acids are generally nonpolar and may participate in hydrophobic interactions. Examples of aromatic amino acids include phenylalanine, tyrosine, and tryptophan. Tyrosine may also participate in hydrogen bonding via the hydroxyl group on the aromatic side chain. Neutral aliphatic amino acids are generally nonpolar and hydrophobic. Examples of neutral amino acids include alanine, valine, leucine, isoleucine, and methionine. An amino acid may be described by more than one description class. Amino acids with a common description class may substitute for each other in a peptide.

[0053] As used herein, the term "identity" refers to a measure of sequence identity between two peptides. Identity can be determined by comparing the position in each sequence that is compared for the purpose of comparison. For example, identity can be determined by the currently used BLAST algorithm, and the algorithm was originally described in Altschul et al., (1990) J.Mol.Biol.215:403-410. The BLAST algorithm can be used with the published default settings. When a position in the comparison sequence is occupied by the same amino acid, the molecule is considered to have shared identity at that position. The degree of identity between sequences is a function of the number of matching positions shared by the sequences and the degree of overlap between the sequences. In addition, when considering the degree of identity with SEQ ID NO:1 or 3, it is expected that an equal number of amino acids will be compared with SEQ ID NO:1 or 3, respectively. When determining the degree of identity with SEQ ID NO:1 or 3, it is not intended to consider additional sequences (i.e., except for those corresponding to 10 or 21 amino acids of SEQ ID NO:1 or 3, respectively). The sequence identity of a given sequence can be calculated over the length of a reference sequence (ie, SEQ ID NO: 1 or 3).

[0054] The nomenclature used to describe peptides or polypeptides can follow conventional practice, wherein the amino group is presented on the left side of each amino acid residue and the carboxyl group is presented on the right side of each amino acid residue. In the sequences representing selected embodiments of the present invention, the amino terminal and carboxyl terminal groups, although not specifically shown, will be understood as the form they present at physiological pH, unless otherwise indicated. According to Table A below, in the amino acid structural formula, each residue can generally be represented by a single letter or three letter name, corresponding to the name of the amino acid.

[0055] Table A. Nomenclature and abbreviations of the 20 standard L-amino acids commonly found in naturally occurring peptides

[0056] Full name Three letter abbreviation Single letter abbreviation Alanine Ala A Cysteine Cys C Aspartic acid Asp D Glutamate Glu E Phenylalanine Phe F Glycine Gly G Histidine His H Isoleucine Ile I Lysine Lys K Leucine Leu L Methionine Met M Asparagine Asp N Proline Pro P Glutamine Gln Q Arginine Arg R Serine Ser S Threonine Thr T Valine Val V Tryptophan Trp W Tyrosine Tyr Y

[0057] One or both ends of the peptide, but usually one end, can be substituted by a lipophilic group, which is usually an aliphatic or aralkyl group, which can include heteroatoms. The chain can be saturated or unsaturated. Conveniently, commercially available aliphatic fatty acids, alcohols and amines can be used, such as caprylic acid, capric acid, lauric acid, myristic acid and myristyl alcohol, palmitic acid, palmitoleic acid, stearic acid and stearylamine, oleic acid, linoleic acid, docosahexaenoic acid, etc. are preferred. Unbranched, naturally occurring fatty acids with a length between 14-22 carbon atoms are preferred. Other lipophilic molecules include glycerolipids and sterols, such as cholesterol. The lipophilic group can react with the appropriate functional group on the oligopeptide according to conventional methods, usually during synthesis on a support, depending on the attachment site of the oligopeptide to the support. Lipid attachment is useful when the oligopeptide can be introduced into the lumen of the liposome together with other therapeutic agents, for administering the peptide and the agent to the host.

[0058] Depending on their intended use, particularly administration to a mammalian host, the subject peptides may also be modified by linkage to other compounds for the purpose of incorporating carrier molecules, altering the bioavailability of the peptide, extending or shortening the half-life, controlling distribution to various tissues or the bloodstream, reducing or enhancing binding to blood components, etc.

[0059] The exemplary peptides may also include a delivery and targeting (dat) moiety to help the exemplary peptides transport across the cell membrane. As used herein, the term delivery and targeting (dat) moiety is intended to cover any moiety that helps deliver and / or target the peptides described herein to a target cell or tissue or within a target cell or within a cell of a target tissue. In addition, the dat moiety can "help" delivery and / or targeting by promoting the biological efficacy of the peptides described herein. Moieties that can deliver or target biologically active molecules to cells in a suitable manner to provide an effective amount (e.g., a pharmacologically effective amount) are known in the art. Optionally, the delivery and targeting (dat) moiety can be selected from one or more of the following: a receptor ligand, a protein transduction domain, a micelle, a liposome, a lipid particle, a viral vector, a peptide carrier, a protein fragment, or an antibody. Optionally, the protein transduction domain can be a cell membrane transduction domain of HIV-1 Tat (Demarchi et al., (1996) J Virol. 70: 4427-4437). Other examples and related details of such protein transduction domains have been described and are known to those skilled in the art. HIV-1 Tat cell membrane transduction domain can form a fusion protein with the exemplary peptides described herein (eg, as in SEQ ID NO: 3). These proteins can be produced by chemical synthesis, recombinant DNA, genetic and molecular engineering techniques known in the art.

[0060] In therapeutic applications, the compositions described herein can be applied to subjects suffering from one or more symptoms of a disease or condition (e.g., a disease or condition associated with acute myocardial infarction (AMI)). The compositions described herein can be applied to subjects in an amount sufficient to cure or at least partially prevent or prevent a disease or condition and / or its complications or to help alleviate the symptoms associated therewith. An amount sufficient to achieve treatment, cure, or preventive treatment is defined as a "therapeutically effective dose" or "therapeutically effective amount". The effective amount for this purpose will depend on the severity of the disease or condition, the intended use (treatment, cure, prevention, relief of symptoms, etc.), and the general state of health of the subject. Single or multiple administrations of the composition can be applied according to the dosage and frequency required and tolerated by the patient. The composition will typically provide a sufficient amount of active peptide or peptides described herein in the subject to effectively treat (e.g., at least improve one or more symptoms).

[0061] The concentration of the peptides described herein can vary widely and can be selected based primarily on fluid volume, viscosity, body weight, etc., depending on the particular mode of administration selected and the needs of the subject. However, the concentration is generally selected to provide a dosage ranging from about 0.01 or 1 mg / kg / day to about 50 mg / kg / day and sometimes higher. It should be understood that such dosages can be varied to optimize the treatment regimen in a particular subject or subject group.

[0062] Additional active therapeutic ingredients can be administered to a subject together with a primary active agent (e.g., an exemplary peptide described herein) or prior to the primary active agent. The exemplary peptide can be co-administered with other therapeutically active agents to enhance the therapeutic effect on target cells or tissues by delivering a second compound with similar or complementary activity. In one embodiment, such agents include, but are not limited to, agents that reduce the risk of acute myocardial infarction (AMI) and / or its complications. Such agents include, but are not limited to, anticoagulants (e.g., Acenocoumarol, Coumatetralyl, Dicoumarol, Ethyl Coumarate, Phenprocoumon, Warfarin, Clorindione, Diphenadione, Phenindione, Tioclomarol, Bemiparin, Certoparin, Dalteparin, Enoxaparin, Nadroparin, Parnaparin, Reviparin, Tinzaparin, Fondaparinux, Idroparinux, Idraparinux, Danaparoid, Sulodexide, Dermatan sulfate, Apixaban, Betrixaban, Edoxaban, Otamixaban, Rivaroxaban, Hirudin, Bivalirudin, Lepirudin, Desirudin, Argatroban, Dabigatran, Melagatran, Ximelagatran, REG1, Defibrotide, Ramatroban, Antithrombin III, and Drotrecogin alfa), antiplatelet drugs (e.g.,Abciximab, Eptifibatide, Tirofiban, Clopidogrel, Prasugrel, Ticlopidine, Ticagrelor, Beraprost, Prostacyclin, Lloprost, Treprostinil, Acetylsalicylic Acid / Aspirin, Aloxiprin, Carbasalate Calcium calcium), Indobufen, Triflusal, Dipyridamole, Picotamide, Terutroban, Cilostazol, Dipyridamole, Triflusal, Cloricromen, Ditazole), and thrombolytic and fibrinolytic drugs (e.g., tissue plasminogen activator (tPA) or recombinant tissue plasminogen activator (rtPA), such as Alteplase, Reteplase, Tenecteplase, Urokinase, Saruplase, Streptokinase, Anistreplase, Monteplase, Ancrod, Fibrinolysin and Brinase), etc. or in combination with other cardioprotective agents. ,

[0063] Peptides can be prepared in a variety of ways. Chemical synthesis of peptides is well known in the art. Solid phase synthesis is commonly used, and various commercial synthesis devices can be used, such as automatic synthesizers from Applied Biosystems Inc., Foster City, Calif.; Beckman, etc. Solution phase synthesis methods can also be used, especially for large-scale production.

[0064] The peptides may also exist in the form of salts, usually in the form of pharmaceutically acceptable salts. These include inorganic salts of sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, etc. Various organic salts of the peptides may also be made, including but not limited to acetic acid, propionic acid, pyruvic acid, maleic acid, succinic acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, salicylic acid, etc. The previous examples are used as examples and are non-limiting.

[0065] Experimental example

[0066] Embodiments of the present invention will be described with reference to the following exemplary information, which should not be used to limit or interpret the teachings described herein.

[0067] animal

[0068] Juvenile Sprague Dawley rats (P1-2) and adult male Sprague Dawley rats (180-220 g) were purchased from B&K Universal Ltd, China. Adult rats were housed in plastic cages with free access to food and water and maintained in a temperature-controlled room (22-25° C.) with a 12 / 12 h light / dark cycle. All experimental protocols were approved by the Second Military Medical University, and methods were performed in accordance with approved guidelines and regulations. Every effort was made to minimize animal suffering and reduce the number of animals used.

[0069] Chinese Bama miniature pigs (2-3 months; 7.81-10.43 Kg) were purchased from Wujiang Tianyu Biotech (Suzhou, China). The pigs were housed in stainless steel cages with free access to food and water and kept in a temperature-controlled room (18-26°C) with a 12 / 12 hour light / dark cycle. All experimental protocols were approved by JOINN Laboratories (Suzhou, China, http: / / www.joinn-lab.com / ), a contract research organization, and the methods were performed in accordance with the approved guidelines and regulations. Every effort was made to minimize the suffering of the animals and reduce the number of animals used.

[0070] Chemicals and reagents

[0071] The following chemicals and reagents were used: ketamine hydrochloride (Fujian Gutian Pharmaceutical, China, H35020148), diazepam (Henan Anyang Yikang Pharmaceutical, China, H41021491), normal saline (Shandong Hualu Pharmaceutical, China, H37022749), 2,3,5-triphenyl-tetrazolyl chloride (TTC) (Sangon Biotech, China, CA25BA0012), urethane (Sinopharm Chemical Reagent, China, CA25BA0013), and 1,2-dihydro-2-nitropropene (DHT) (DHT-10001). Reagent, China, 20150908), DMEM (Hyclone, AB10155403), FBS (SAFC Biosciences, 8J0157), trypsin (Gibco, 1766146), collagenase type II (Sigma, 234155), 5'-BrdU (Bio Basic Inc, MCO325B2011Z), MTT (AMRESCO, LJ0628A5010J), Annexin V-FITC cytotoxicity kit (Beyotime Biotechnology, C1063), creatine kinase detection kit (Nanjing Jiancheng Bioengineering Institute Institute, 20161130), lactate dehydrogenase detection kit (Nanjing Jiancheng Bioengineering Institute, 20161206), malondialdehyde detection kit (Nanjing Jiancheng Bioengineering Institute, 20161205), superoxide dismutase detection kit (Nanjing Jiancheng Bioengineering Institute, 20161205).

[0072] As used herein, the term "NIMoEsh" refers to the following amino acid sequence: WAAYRTHSVD [SEQ ID NO: 1]. In the examples described herein, the NIMoEsh peptide (WAAYRTHSVD) was conjugated to the TAT protein transduction domain (YGRKKRRQRRR; SEQ ID NO: 2). The NIMoEsh-Tat peptide (WAAYRTHSVD-YGRKKRRQRRR; SEQ ID NO: 3) was chemically synthesized using a Prelude peptide synthesizer (Protein Technologies Inc.) at the Peptide Facility at the Center for Brain Health at the University of British Columbia.

[0073] Buffers and media

[0074] The cell digestion buffer (without EDTA) contained 0.05% trypsin and 0.5 mg / ml collagenase type II. The cell culture medium contained 15% FBS, 1% penicillin-streptomycin, 1% 0.1 mM Brdu and 83% DMEM.

[0075] Peptide preparation and handling

[0076] Prepare peptide solutions freshly before each use by dissolving dry peptide powder into sterile water or saline. For cell culture experiments, the peptide stock solution was dissolved in culture medium to the desired concentration. For animal experiments, the peptide solution was injected intravenously.

[0077] Primary cardiomyocyte culture

[0078] Hearts were removed from P1-2 Sprague Dawley rats and cut into 1 mm 3 cubes. The heart tissue was then digested in cell digestion buffer at 37°C for 4 minutes and centrifuged at 200 rpm for 1 minute to remove the supernatant. The pellet was digested again in cell digestion buffer at 37°C until it was completely digested and then centrifuged at 200 rpm for 1 minute to remove debris. The sample was mixed with FBS to neutralize trypsin and collagenase and then centrifuged at 1000 rpm for 5 minutes to remove the supernatant. Cardiomyocytes were isolated from the sample by differential adhesion and then cultured in cell culture medium in a plate. Primary cardiomyocyte cultures were maintained in a 37°C incubator at 95% O2 and 5% CO2 for 5 days before being used in experiments.

[0079] Cell death assay

[0080] Use multiple determinations to measure the cell death of primary cardiomyocyte cultures. Use commercial kits and measure the activity of MTT and creatine kinase, lactate dehydrogenase, malondialdehyde and superoxide dismutase according to the instructions of manufacturers. By processing primary cardiomyocyte cultures with Annexin V-FITC cytotoxicity kit, apoptosis determination is carried out, then apoptosis percentage is determined by flow cytometry (BD, FACS Alibur).

[0081] AMI rat model

[0082] As previously described, rats were subjected to AMI by ligating the left anterior descending coronary artery (Yu, JG et al., ActaPharmacol Sin 34, 1508-1514, doi: 10.1038 / aps.2013.147 (2013)). In brief, rats were anesthetized with 100 mg / kg ketamine hydrochloride and 10 mg / kg diazepam by intraperitoneal injection, and then artificially ventilated using a ventilator (Shanghai Alcott Biotech, China, ALC-V8) at a tidal volume of 20 mL and a respiratory rate of 60 times / minute. The heart was externalized through the 4th intercostal space, and the left anterior descending coronary artery was ligated using a 6-0 suture. After the incision was closed, the rats were returned to their home cages. Sham-operated rats received the same protocol except that the coronary artery was not ligated.

[0083] Transient AMI pig model

[0084] Pigs were subjected to transient AMI by temporarily occluding the left circumflex coronary artery as described above (lchimura, K. et al., PLoS One 11, e0162425, doi: 10.1371 / journal.pone.0162425 (2016)). In short, pigs were anesthetized with ketamine hydrochloride (10 mg / kg, intramuscularly), and a ventilator (SN23402, Hallowell engineering and manufacturing corporation, the United States) was used to maintain anesthesia with isoflurane (tidal volume: 80 mL; respiratory rate: 20 times / min). Left thoracotomy was performed, and a pneumatic cuff occluder was placed at the proximal end of the left circumflex coronary artery. After the incision was closed, the pig was returned to its home cage. On the day of the experiment, AMI was induced by occluding the left circumflex coronary artery for 60 minutes by inflating the cuff. The cuff was then deflated and blood reperfusion was started. The ECG signals of pigs were monitored using jacketed external telemetry (DataScience International Inc., USA) before arterial occlusion and until 10 hours after blood reperfusion.

[0085] TTC staining and measurement of cardiac infarction

[0086] Rats were sacrificed, their hearts were removed and weighed on an electronic scale. After rapid freezing in a -20°C freezer for 20 minutes, the heart was cut into 5 small pieces of equal size and placed in a 0.5% TTC solution at 37°C for 5 minutes. The infarcted tissue identified as white tissue was separated from the healthy tissue identified as red tissue. The infarcted tissue was then weighed on an electronic scale, and the percentage of cardiac infarction was calculated by dividing the weight of the infarcted tissue by the weight of the whole heart.

[0087] The pig was killed and the heart was removed. After rinsing with normal saline, the heart was perfused with 37°C 1% TTC and then cut into 6 slices (5 mm thickness). The heart slices were placed in 37°C 1% TTC solution for 5-10 minutes and then fixed with 10% formalin solution. Images of the heart slices were taken and analyzed using NIH Image J software. The volume of myocardial infarction in each slice = (infarct area on one side of the slice + infarct area on the other side of the slice) / 2 × thickness (5 mm). The percentage of myocardial infarction was calculated by dividing the volume of the infarcted tissue by the volume of the left ventricle.

[0088] Cardiac function measurement

[0089] Hemodynamic assessment of rats was performed as described by Yu et al. (Acta Pharmacol Sin 34, 1508-1514, doi: 10.1038 / aps.2013.147 (2013)). Briefly, rats were anesthetized with 1.25 g / kg 25% urethane, and a polyethylene catheter connected to a pressure transducer (Powerlab 30, ADInstruments) was inserted into the right carotid artery and then into the left ventricular cavity. Mean heart rate, systolic blood pressure, left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), and maximum pressure rise rate (+dp / dtmax) and fall rate (-dp / dtmax) were recorded for 30 minutes and then analyzed.

[0090] After pigs were anesthetized with telazol (10 mg / kg, intramuscularly) and isoflurane, transthoracic echocardiography was performed using a portable color Doppler (S8 Exp, Shenzhen SonoScape Medical Corp, China). End-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), and ejection fraction (EF) were recorded.

[0091] Histological analysis of porcine heart

[0092] The second section from each pig heart was processed for H&E staining and then histological analysis. The level of cardiac pathology (atrial fibrillation, necrosis, hemorrhage, inflammation, granulomas, and pericarditis) was divided into 4 categories for comparison: mild (+; barely visible), mild (++; visible but very small), moderate (+++; visible and quantifiable), and severe (++++; extensive damage).

[0093] Statistical analysis

[0094] Cell cultures and animals were randomly assigned to experimental treatment groups, and data were analyzed using SPSS software and expressed as mean ± SEM. Data were analyzed by one-way ANOVA followed by LSD post hoc test or unpaired t-test. *P<0.05, **P<0.01 and ***P<0.001 were considered significant differences.

[0095] result

[0096] Cardioprotective efficacy of NIMoEsh-Tat peptide in an in vitro model of AMI

[0097] Primary cardiomyocyte cultures were treated with 300uM H2O2 for 4 hours to induce oxidative stress. Cardiomyocytes were then returned to normal culture medium for 12 hours before cell death analysis. Some cardiomyocytes were treated with 10uM NIMoEsh-Tat peptide 30 minutes before H2O2 treatment and during the 12-hour recovery period. H2O2 treatment induced cell death compared to untreated controls, as measured by MTT assay ( Figure 1 a) and cell apoptosis assay ( Figure 1 b) showed that the activity of myocardial cell injury markers, such as creatine kinase (CK, Figure 1 c), malondialdehyde (MDA, Figure 1 d) and lactate dehydrogenase (LDH, Figure 1 e) and reduced the activity of the pro-survival protein marker superoxide dismutase (SOD) ( Figure 1 f). Compared with the H2O2 group, NIMoEsh-Tat peptide effectively reduced H2O2-induced cell death, e.g. Figure 1 ab and Figure 1 Although NIMoEsh-Tat peptide could not completely rescue the increase in CK activity induced by H2O2, there was a trend of decreased CK activity in the peptide-treated group compared with the H2O2 group ( Figure 1 c) These results suggest that NIMoEsh-Tat peptide has cardioprotective effects against oxidative stress-induced cardiomyocyte death.

[0098] NIMoEsh-Tat peptide reduces cardiac damage and restores cardiac function in rats after AMI

[0099] The cardioprotective efficacy of NIMoEsh-Tat peptide was studied in a well-established AMI rat model (Yu, JG et al., Acta Pharmacol Sin 34, 1508-1514, doi: 10.1038 / aps.2013.147 (2013)). 20 mg / kg NIMoEsh-Tat or saline control was injected intravenously (iv) into rats immediately after ligation of the left anterior descending coronary artery. Four hours later, the hearts were removed for TTC staining and infarct percentage measurement. Figure 2 As shown in ab, AMI surgery induced severe tissue infarction in the hearts of saline-treated rats, and this surgery-induced cardiac damage was significantly reduced by NIMoEsh-Tat treatment, indicating the cardioprotective efficacy of NIMoEsh-Tat peptide.

[0100] The long-term protective effect of NIMoEsh-Tat peptide against AMI was also studied. Rats with ligation of the left anterior descending coronary artery received three iv injections of NIMoEsh-Tat peptide (20 mg / kg) or saline at 0 hours, 24 hours and 48 hours after surgery. Rats undergoing sham surgery were used as controls. Four weeks after the last peptide injection, cardiac function was measured using several well-characterized parameters (Yu, JG et al., Acta Pharmacol Sin 34, 1508-1514, doi: 10.1038 / aps.2013.147 (2013)). Compared with the sham group, neither saline nor NIMoEsh-Tat peptide changed the heart rate of rats after AMI ( Figure 2 c) and systolic blood pressure ( Figure 2 d). After AMI, saline-treated rats showed reduced left ventricular systolic pressure ( Figure 2 e) and maximum pressure rise rate (+dp / dtmax) ( Figure 2 g) and descent rate (-dp / dtmax) ( Figure 2 h), and an increase in left ventricular end-diastolic pressure ( Figure 2 f), indicating impaired cardiac function after AMI. Compared with the saline group, NIMoEsh-Tat peptide treatment rescued cardiac function after AMI ( Figure 2 eh).

[0101] NIMoEsh-Tat peptide reduces myocardial infarction and restores cardiac function after transient AMI in minipigs

[0102] The cardioprotective efficacy of the NIMoEsh-Tat peptide was also studied in Chinese Bama miniature pigs. Transient AMI was induced in these pigs by occluding the left circumflex coronary artery for 1 hour followed by reperfusion of blood. The pigs received one iv injection of either NIMoEsh-Tat peptide (2 mg / kg) or saline 40 minutes after occlusion and another iv injection 24 hours after reperfusion. Cardiac function in these pigs was assessed 30 days before the pigs were sacrificed for histological analysis. Both groups of pigs had the same level of arterial occlusion during AMI surgery and also showed the same level of post-AMI weight recovery ( Figure 4 ).like Figure 3 As shown, NIMoEsh-Tat peptide rescued the stroke volume (SV, Figure 3 c) and ejection fraction (EF, Figure 3 TTC staining of pig heart sections also revealed that NIMoEsh-Tat peptide reduced the volume of AMI-induced myocardial infarction compared with saline controls ( Figure 3 e and Figure 3 f). This is consistent with histological studies of the heart ( Figure 3 g and Table 1), indicating that NIMoEsh-Tat peptide reduced AMI-induced cardiac pathology, such as atrial fibrillation and pericarditis, compared with controls.

[0103] Table 1

[0104]

[0105] A summary of the pathology observed under the microscope is provided in Table 1. NIMoEsh-Tat peptide treated pigs showed much less AMI-induced pathology compared to saline controls.

[0106] discuss

[0107] NIMoEsh-Tat not only protected cardiomyocytes from cell death in primary cultures of cardiomyocytes, but also protected rats and pigs from myocardial infarction and functional loss caused by AMI. While not wishing to be bound by any particular theory or mode of action, the results described herein suggest that the interaction between zD17 and JNK may play a role, at least in part, in inducing cardiac cell death, and thus the cardioprotective effects of NIMoEsh-Tat described herein may be at least in part attributable to the blockade of the interaction between zD17 and JNK during cellular stress.

[0108] One potential problem with peptide therapeutics may be their short half-life. Once inside the body, chemically unmodified peptides are easily digested by enzymes. This can be a problem for the treatment of chronic diseases such as Alzheimer's disease, Parkinson's disease, and hypertension, where patients may need multiple injections per day. However, for acute indications, a small number of injections may be sufficient for disease treatment, so the short half-life of the peptide is no longer an issue. The results described in this article support this. Figure 2 As shown, a single iv injection of NIMoEsh-Tat peptide was sufficient to protect rats from AMI-induced cardiac tissue damage, and daily injections for 3 days were sufficient to effectively protect the heart from AMI-induced functional loss. Figure 3 As shown in Table 1, two iv injections of NIMoEsh-Tat peptide are sufficient to induce cardioprotection in pigs after AMI.

[0109] Although the present invention has been described with reference to illustrative embodiments and examples, the description is not intended to be interpreted in a limiting sense. Therefore, various modifications to the illustrative embodiments and other embodiments of the present invention will be apparent to those skilled in the art upon reference to this description. It is therefore contemplated that the claims cover any such modifications or embodiments.

[0110] All publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0111] Sequence Listing

[0112] SEQ ID NO:1(NIMoEsh):WAAYRTHSVD

[0113] SEQ ID NO:2 (HIV-1 Tat protein transduction domain): YGRKKRRQRRR

[0114] SEQ ID NO: 3 (NIMoEsh-Tat peptide): WAAYRTHSVDYGRKKRRQRRR

Claims

1. A use of a polypeptide conjugated to a delivery and targeting (dat) moiety in the preparation of a medicament for treating acute myocardial infarction (AMI) in a subject in need thereof, wherein the polypeptide and the dat moiety together consist of the amino acid sequence of SEQ ID NO:

3.

2. The use according to claim 1, wherein the subject is a human.

3. The use according to claim 1 or 2, wherein the medicament comprises one or more other active therapeutic ingredients.

4. The use according to claim 1 or 2, wherein the polypeptide is the only active therapeutic ingredient in the medicament.

5. The use according to claim 1 or 2, wherein the medicament comprises one or more excipients.

6. The use according to claim 1 or 2, wherein the medicament is for systemic administration.

7. The use according to claim 1 or 2, wherein the medicament is for intravenous administration.

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