Novel platelet-derived growth factor peptide mimetics

Abstract

JPEG2025532522000027.jpg51170 Disclosed herein are compounds of formula (I), wherein X, L1, and the HSA conjugate are as defined herein, methods for their preparation, and methods for their use, particularly for improving cardiac repair after infarction.

Description

[Technical Field]

[0001] The present invention relates to peptides useful in cardiac repair. [Background technology]

[0002] Currently, no satisfactory treatment options are available to reverse damage and restore cardiac function after a large myocardial infarction resulting in severe cardiac dysfunction. While cardiac transplantation and ventricular assist devices are options, limited supply and the risks associated with these treatments mean that this is not a viable treatment option for most patients. For these reasons, considerable efforts have been directed toward research and development of cardiac regeneration. Several novel strategies have been developed with promising preclinical results, but none have yet been successfully translated into the clinic. These include gene therapy, cytokine therapy, cell-based therapy, and platelet-derived growth factor (PDGF)-based therapy.

[0003] PDGF is one of many growth factors that regulate cell growth and division. The main function of PDGF is in wound healing, and it has significant effects on mitogenesis, chemotaxis, and angiogenesis. The mechanism is activation of the PDGF receptor (PDGFr) by autophosphorylation. The loop III region of the PDGF-B chain is involved in the mitogenic and chemotactic functions of PDGF and is important for maintaining its receptor-binding function. Loop III is located within the cysteine ​​knot structure of PDGF and contains 12 amino acids, five of which play important roles in receptor binding. When loop III is removed from the chain or substituted within the chain, the result is loss of receptor-binding and mitogenic function.

[0004] Preclinical studies in both mouse and porcine models of myocardial infarction have shown that recombinant PDGF AB heterodimers increase cardiac repair by modulating cardiac scarring and improving vascularity in healing tissue.

[0005] In the field of PDGF-based therapy, researchers have also utilized PDGF-B chain loop III to develop peptides. Specifically, Lin et al. (2007) demonstrated that PDGF peptide mimics based on PDGF-B chain loop III have in vitro effects that mimic those of native PDGF. This peptide was conjugated to a heparin-binding domain (HBD).

[0006] However, PDGF is also well established as a potent mitogen, and upregulation of PDGF leads to increased proliferation. This upregulation has been linked to several disease states, including fibrosis. Chronic fibrosis is an essential component of wound healing, but in the cardiac context, it can lead to ventricular dysfunction, which can subsequently lead to the development of heart failure.

[0007] Fibrogenesis can occur as a result of increased proliferation of fibroblasts, which are often activated in wound healing by several growth factors, including PDGF. Given PDGF's known effects on the cell cycle and its subsequent role in fibrosis, research in this area has avoided prolonged exposure to PDGF-based therapies. Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, improved therapeutic options are needed to reverse damage and restore cardiac function after large myocardial infarction, which leads to severe cardiac dysfunction. PDGF-based therapies offer a potential approach, as long as the proliferation and fibrosis caused by PDGF are limited. [Means for solving the problem]

[0009] The present invention alleviates at least one drawback associated with current treatments for reversing damage and restoring cardiac function after large myocardial infarction.

[0010] Without being limited thereto, the present invention provides compounds for reversing damage and restoring cardiac function after myocardial infarction, methods for preparing such compounds, and methods for the prevention and / or treatment of various conditions or diseases by administering such compounds. [Brief explanation of the drawings]

[0011] [Figure 1] General scheme for solid phase peptide synthesis of compounds of formula (I). [Figure 2] Migration of C2C12 mouse fibroblasts treated with PDGF and peptides JC5 and JC5a. C2C12 mouse fibroblasts migrate across a scratch over 24 hours. Cells were treated with 10 ng / mL PDGF-AB (n = 6), PDGF-BB (n = 6), and 1 μg / mL of the novel PDGF mimetics JC5 (n = 6) and JC5a (n = 3). PDGF-BB (p < 0.0001) and the novel mimetic peptides JC5 (P < 0.0001) and JC5a (P < 0.0001) both showed significantly increased migration from untreated controls and AG1296, the negative control. There was no statistically significant difference between the PDGF mimetic peptides and the PDGF-AB and -BB positive controls. Error bars represent ±SD. [Figure 3] Collagen contraction in C2C12 mouse fibroblasts treated with PDGF and peptides JC5 and JC5a. C2C12 mouse fibroblasts significantly increased collagen contraction after treatment with 10 ng / mL PDGF-AB (p<0.0001), PDGF-BB (p>0.0001), and 1 μg / mL of the novel PDGF mimetic peptides JC5 (p>0.0001) and JC5a (p=0.0120) compared to the negative control, AG1296 + PDGF-AB. PDGF-AB (p=0.0073), PDGF-BB (p=0.0126), and the novel PDGF mimetic JC5 (p=0.0100) also significantly increased collagen contraction compared to the untreated control. Error bars represent ±SD, n=8 / 4. [Figure 4]Tube formation of human coronary artery endothelial cells treated with PDGF and the PDGF-mimetic peptide JC5. Human coronary artery endothelial cells treated with 50 ng / mL of PDGF-AB and PDGF-BB produced longer tubes than untreated cells and cells treated with the PDGF receptor inhibitor AG1296 + PDGF-AB. Cells treated with 5 μg / mL of JC5 also resulted in increased tube length when compared with untreated and AG1296 + PDGF-AB negative controls. Treatment with PDGF-AB, PDGF-BB, and JC5 resulted in significantly increased tube length compared with untreated controls (AB- p = 0.0062, BB- p = 0.0013, JC5- p ​​= 0.0025) and AG1296 + PDGF-AB negative controls (AB- p = 0.0068, BB- p = 0.0014, JC5- p ​​= 0.0027). Error bars represent ±SD, n=3 / group. [Figure 5] Phosphorylation of ERK after treatment of C2C12 cells with PDGF and JC5. Western blot for total (ERK) and phosphorylated ERK (pERK) at 42 and 44 kDa shows increased phosphorylation after treatment with 10 ng / mL PDGF-BB and 1 μg / mL JC5. [Figure 6] Phosphorylation of ERK after treatment of C2C12 cells with PDGF and JC5a. Western blots for total (ERK) and phosphorylated ERK (pERK) at 42 and 44 kDa show increased phosphorylation after 10 minutes of treatment with 10 ng / mL PDGF-BB and 1 μg / mL of the novel mimetic peptide JC5a. JC5a maintained this increased phosphorylation of ERK 30 minutes after treatment. [Figure 7] Annotated formula (I). [Figure 8]Proliferation of C3H10T1 / 2 cells after treatment with PDGF and novel PDGF-mimetic peptides. Cultured C3H10T1 / 2 mouse embryonic fibroblasts were treated for 24 hours with 20 ng / mL of recombinant PDGF-AB or PDGF-BB protein ligands and 2 μg / mL of the novel PDGF mimics JC5 or JC5a in the presence of 1% fetal bovine serum (FBS). After pulsing with the nucleotide analog EdU for 2 hours at the end of the incubation period, the percentage of proliferating cells was determined. PDGF-BB induced significantly greater proliferation compared to the 1% FBS control. However, PDGF-AB, JC5, and JC5a did not significantly increase proliferation relative to 1% FBS and were not significantly different from each other. Thus, unlike PDGF-BB, JC5 and JC5a do not exhibit significant proliferative activity. Cotreatment of the proteins / peptides with 10 μM of the PDGF receptor antagonist, AG1296, confirmed that PDGF-BB-stimulated proliferation occurred via the PDGF ligand / receptor pathway. AG1296 also significantly inhibited PDGF-AB and JC5 EdU incorporation, suggesting a weak PDGF receptor-mediated effect for these treatments. Error bars represent ±SD, n = 3. Statistical analysis was performed using one-way ANOVA: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. [Figure 9]Improved cardiac function 28 days after myocardial infarction. A) C57BL6 / J mice treated with PDGF-AB (p = 0.0073) and the novel mimetic peptide JC5 (p = 0.0129) had significantly increased ejection fractions at day 28 compared to the PBS vehicle control group. B) Mice treated with the positive control PDGF-AB (p = 0.0003) and the novel mimetic peptide JC5 (p = 0.0032) had significantly decreased end-systolic volumes compared to mice treated with the vehicle control PBS. C) Mice treated with PDGF-AB (p = 0.0016) and JC5 (p = 0.0185) also had decreased end-diastolic volumes compared to PBS. D) Both PDGF-AB (p = 0.0393) and JC5 (p = 0.0409) treated mice had significant delta ejection fractions from day 2 to day 28 compared to the PBS vehicle control. The novel mimetic peptide JC5a (p=0.0655) also had an increased delta ejection fraction (p=0.0082) from days 2 to 28 when compared to PBS. E) Timeline of mean EF by treatment group. Baseline represents pre-infarction EF per group. Days 2 and 28 represent days post-infarction. A-C) Error bars represent ±SD. D) Data expressed as median ± minimum and maximum. One-way ANOVA comparing all groups to the PBS vehicle control group (n=10 / 11). [Figure 10] Interobserver variability of ejection fraction at day 28. Interobserver variability at day 28 was within the normal range for rodent echocardiography with a bias of 14.06 (±7.346). Error bars (dotted lines) represent the 95% limits of agreement. [Figure 11] Decreased heart weight / tibia length in mice treated with JC5. C57BL6 / J mice treated with the positive control PDGF-AB (p=0.0066) (n=10) and the novel mimetic peptide JC5 (p=0.0366) (n=11) showed a significant decrease in heart weight normalized by tibia length compared to the vehicle control PBS (n=10). Error bars represent ±SD. DETAILED DESCRIPTION OF THE INVENTION

[0012] definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0013] As used herein, the term "comprising" means "including." Variations of the word "comprising," such as "comprise" and "comprises," have correspondingly varied meanings.

[0014] When the term "about" is used herein in reference to a stated value, it will be understood to include the stated value and values ​​within ±10% of the stated value.

[0015] When the term "between" is used in referring to a range of numerical values, it will be understood that each of the endpoints of the range is included. For example, a temperature between 80°C and 150°C includes the temperatures of 80°C and 150°C.

[0016] Any statement in this specification of a document as prior art, or any statements made herein that are derived from or based on that document, is not an admission that the document or any statements made therefrom are part of the common general knowledge in the relevant art.

[0017] All materials referred to herein for illustrative purposes are incorporated herein by reference in their entirety unless otherwise stated.

[0018] (Detailed Description of the Invention) The present inventors have surprisingly found that linking a dimer of the loop III region of the PDGF-B chain of PDGF to a fatty acid conjugate of human serum albumin (HSA) results in compounds of formula (I), which mimic the useful properties of PDGF for cardiac repair while avoiding the undesirable side effects of PDGF-based therapy, such as increased cell proliferation and fibrosis.

[0019] [ka]

[0020] where X=NH2 or -L2-[HSA conjugate];

[0021] [ka]

[0022] and n, L1 and L2 are as described herein.

[0023] The HSA conjugate of the present invention is designed to extend the half-life of therapeutic peptides by binding them to the abundant carrier protein albumin, thereby extending the time until clearance by renal filtration. However, this particular HSA conjugate results in a shorter half-life extension than the complete albumin fusion protein due to its inability to utilize the FcRn recycling pathway. It is expected to extend the half-life of the PDGF-B chain loop III region dimer by up to 25-fold, but not by more than a few days.

[0024] The inventors theorize that this conjugation will allow for improved clinical use, providing more time for the peptide's mechanism of action to occur while ensuring that it is cleared before the onset of potentially problematic side effects such as fibrosis. Thus, the compounds of the present invention unexpectedly provide activity that mimics the beneficial properties of PDGF while avoiding excessive and harmful fibrosis.

[0025] compound In one aspect of the invention, there is provided a compound of formula (I) or a salt thereof:

[0026] [ka]

[0027] where X=NH2 or -L2-[HSA conjugate];

[0028] [ka]

[0029] and L1 and L2 are independently -HN(C2~C 60 Alkyl)C(O)-, -HN(C2-C 60 Alkaryl)C(O)-, -HN(C2-C 60 alkenyl)C(O)-, -HN(CH2) r (CH2CH2O) o (CH2) r C(O)-, -HN(CH2) r (O(CH2) m C(O)) o (CH2) r C(O)-, -HN(CH2) r ((NH(CH2) m C(O)) o )(CH2) r C(O)-, and

[0030] [ka]

[0031] wherein n=1 to 20, o=1 to 60, r=2 to 20, m=1 to 60, p=1 to 60, and q=1 to 60.

[0032] L1 and L2 may be the same, or L1 and L2 may be different.

[0033] In one embodiment, L1 and L2 are independently -HN(C2-C 20 Alkyl)C(O)-, -HN(C2-C 20 Alkaryl)C(O)-, -HN(C2-C 20 alkenyl)C(O)-, -HN(CH2) r(CH2CH2O) o (CH2) r C(O)-, -HN(CH2) r (O(CH2) m C(O)) o (CH2) r C(O)-, -HN(CH2) r ((NH(CH2) m C(O)) o )(CH2) r C(O)-, and

[0034] [ka]

[0035] wherein n=1 to 20, o=1 to 20, r=2 to 20, m=1 to 20, p=1 to 20, and q=1 to 20.

[0036] In each occurrence of the HSA conjugate in formula (I), n may be the same or n may be different.

[0037] -HN(C2~C 60 alkyl)C(O)- is -HN-(CH2) a -HN(C2-C3)-, where a is a branched or linear alkyl chain having 2 to 60 carbon atoms, such as -C(O)-, where a=2 to 60. In some embodiments, a=2 to 20. 60 Alkaryl)C(O)- is -HN-[(CH2) b -C6H4-(CH2) c ] d -HN(C2-C3)- refers to a straight chain containing one or more aryl groups, such as -C(O)-, where b=0-60, c=0-60, and d=1-60. In some embodiments, b=0-20, c=0-20, and d=1-20. 60 alkenyl)C(O)- is -HN-[(CH2) b -CH=CH-(CH2) c ] dmeans a straight chain containing one or more alkene groups, such as —C(O)—, where b=0-60, c=0-60, and d=1-60. In some embodiments, b=0-20, c=0-20, and d=1-20.

[0038] In one embodiment, n=15.

[0039] In one embodiment, r=2.

[0040] In some embodiments, L1 and L2 are

[0041] [ka]

[0042] In particular, L1 and L2 can be

[0043] [ka]

[0044] It could be.

[0045] In one embodiment where X is NH2, a dimer of the loop III region of the PDGF-B chain is linked to a single HSA conjugate. In this case, the compound of formula (I) is represented by compound Ia:

[0046] [ka]

[0047] It could be.

[0048] In another embodiment, where X is -L2-[HSA conjugate], a dimer of the loop III region of the PDGF-B chain is linked to two HSA conjugates. In this case, the compound of formula (I) is compound Ib:

[0049] [ka]

[0050] It could be.

[0051] In one embodiment, the compounds of formula (I) exist as their free base. In another embodiment, the compounds of formula (I) exist as pharmaceutically acceptable salts. The phrase "pharmaceutically acceptable salt" refers to any salt preparation suitable for use in pharmaceutical applications. A pharmaceutically acceptable salt means a salt that, within the scope of sound medical judgment, is suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reaction, etc., and is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are described, for example, in J. Pharmaceutical Sciences, 1977, 66:1-19. In one embodiment, the pharmaceutically acceptable salt is a hydrochloride salt. The compounds of formula (I) can also exist as any suitable salt, for example, a trifluoroacetate salt or a formate salt.

[0052] Method of preparation In another aspect, there is provided a method for preparing a compound of formula (I) or a salt thereof.

[0053] First, a method for preparing a compound of formula (I) comprises reacting a compound of the structure:

[0054] [ka]

[0055] The method includes preparing a PDGF loop III monomer (compound II) of

[0056] The PDGF loop III monomer is conveniently prepared by solid-phase peptide synthesis, e.g., Fmoc solid-phase peptide synthesis. Solid-phase peptide synthesis is a well-known standard technique for preparing polypeptides. Typically, a protected C-terminal amino acid residue is provided and covalently attached to a polystyrene resin via a functional group that can be cleaved under appropriate conditions. The terminal residue is deprotected, and the next amino acid is coupled via amide coupling to form a resin-linked dipeptide. This cycle is repeated until the entire polypeptide covalently attached to the resin is obtained. The polypeptide can then be cleaved from the resin and deprotected.

[0057] Second, the PDGF loop III monomer is attached to an HSA conjugate and a linker to form a linked PDGF loop III HSA monomer (formula (II)):

[0058] [ka]

[0059] Form.

[0060] This step can also be carried out using solid-phase peptide synthesis, particularly since the linker and HSA conjugate are also linked by an amide bond. That is, the PDGF loop III monomer is prepared by solid-phase peptide synthesis, followed by coupling of the linked HSA conjugate. The linked PDGF loop III HSA monomer is then cleaved from the resin.

[0061] Third, the linked PDGF loop III HSA monomer of formula (II) can be linked to a PDGF loop III monomer (compound II) by disulfide coupling. This forms, for example, compound Ia. Alternatively, two linked PDGF loop III HSA monomers of formula (II) can be linked to each other by disulfide coupling. This forms, for example, compound Ib. Disulfide coupling can be achieved by using saturated NH4 + CO3- This can be carried out under any suitable conditions, such as in an aqueous solution.

[0062] In an alternative embodiment, the compounds of formula (I) or salts thereof may be prepared using native chemical ligation or a combination of solid phase peptide synthesis and native chemical ligation.

[0063] composition In another aspect of the present invention, there is provided a composition comprising a compound of formula (I) and a pharmaceutically acceptable excipient, carrier and / or diluent.

[0064] The compositions of the invention may contain a compound of formula (I) as the only therapeutic agent. Alternatively, the compositions of the invention may contain a compound of formula (I) in combination with an additional therapeutic agent.

[0065] The composition of the present invention can be formulated for oral administration or parenteral administration.The parenteral administration of the compound of the present invention can be, for example, by intravenous injection, intraarterial injection, intracoronary injection, intracoronary vein injection, subcutaneous injection, gastric autoinjector, implanted minipump, transdermal patch, implanted system that allows administration to local tissue by using bioengineered construct, gastrointestinal mucoadhesive patch system or microneedle system.The composition of the present invention can be formulated in any suitable manner to deliver effective amount of the compound of formula (I), for example, in nanoparticle formulation, polymer matrix formulation or lipid formulation.

[0066] Methods and Uses The compounds of formula (I) are useful in the methods and uses described below by mimicking the functions of PDGF, such as chemotaxis, collagen contraction, angiogenesis, and activation of the PDGF receptor via phosphorylation of Akt and ErK.

[0067] In the following embodiments of the methods and uses, the subject may have experienced a myocardial infarction, or the subject may not have experienced a myocardial infarction. In the following embodiments of the methods and uses, the chronic heart failure may be ischemic heart failure or non-ischemic heart failure. In some embodiments, if the subject suffers from non-ischemic heart failure, the subject may not have experienced a myocardial infarction.

[0068] In one aspect, there is provided a method of reversing cardiac damage caused by myocardial infarction in a subject in need thereof, comprising administering to the subject a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the invention.

[0069] In another aspect, there is provided a method for reversing cardiac damage in patients with chronic heart failure, the method comprising administering to a subject a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the invention.

[0070] In another aspect, there is provided a method of treating cardiac damage caused by myocardial infarction in a subject in need thereof, comprising administering to the subject a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the invention.

[0071] In another aspect, there is provided a method for treating cardiac damage in patients with chronic heart failure, the method comprising administering to a subject a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the invention.

[0072] In another aspect, there is provided a method for restoring or improving cardiac function after myocardial infarction in a subject in need thereof, comprising administering to the subject a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the invention.

[0073] In another aspect, there is provided a method for restoring or improving cardiac function in a patient suffering from chronic heart failure, the method comprising administering to a subject a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the present invention.

[0074] In another aspect, there is provided a method of enhancing cardiac repair after myocardial infarction in a subject in need thereof, comprising administering to the subject a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the invention.

[0075] In another aspect, there is provided a method for enhancing cardiac repair in a patient suffering from chronic heart failure, the method comprising administering to a subject a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the invention.

[0076] In another aspect, there is provided a method of treating post-myocardial infarction cardiac dysfunction in a subject in need thereof, comprising administering to the subject a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the invention.

[0077] In another aspect, there is provided a method for treating cardiac dysfunction in patients with chronic heart failure, the method comprising administering to a subject a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the invention.

[0078] In another aspect, there is provided a method of treating persistent post-myocardial infarction angina in a subject in need thereof, comprising administering to the subject a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the invention.

[0079] In another aspect, there is provided a method for treating persistent angina in a subject suffering from chronic heart failure, the method comprising administering to a subject a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the invention.

[0080] In another aspect, there is provided a method of treating persistent angina in a subject in need thereof, comprising administering to the subject a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the invention.

[0081] In another aspect, there is provided a method for improving survival after myocardial infarction, reducing arrhythmias, and / or increasing ventricular contractility and compliance in a subject in need thereof, comprising administering to the subject a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the invention.

[0082] In another aspect, there is provided a method for improving survival, reducing arrhythmias, and / or increasing ventricular contractility and compliance in a subject suffering from chronic heart failure, comprising administering to the subject a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the invention.

[0083] In another aspect, there is provided a method for prolonging survival of a subject after myocardial infarction, comprising administering to the subject a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the invention.

[0084] In another aspect, there is provided a method for prolonging survival of a subject suffering from chronic heart failure, the method comprising administering to the subject a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the invention.

[0085] In another aspect, there is provided a method for preventing the development of severe heart failure disease in a subject after myocardial infarction, the method comprising administering to the subject a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the invention.

[0086] In another aspect, there is provided a method for preventing the development of severe heart failure disease in a subject suffering from chronic heart failure, the method comprising administering to the subject a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the invention.

[0087] In another aspect, there is provided a method for treating chronic heart failure in a subject in need thereof, comprising administering to the subject a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the invention.

[0088] In another aspect, there is provided a compound of formula (I) or a salt thereof for use in reversing cardiac damage caused by myocardial infarction in a subject in need thereof.

[0089] In another aspect, there is provided a compound of formula (I) or a salt thereof for use in reversing cardiac damage in a subject suffering from chronic heart failure in a subject in need thereof.

[0090] In another aspect, there is provided a compound of formula (I) or a salt thereof for use in treating cardiac damage caused by myocardial infarction in a subject in need thereof.

[0091] In another aspect, there is provided a compound of formula (I) or a salt thereof for use in treating cardiac damage in a subject suffering from chronic heart failure.

[0092] In another aspect, there is provided a compound of formula (I) or a salt thereof for use in restoring or improving cardiac function after myocardial infarction in a subject in need thereof.

[0093] In another aspect, there is provided a compound of formula (I) or a salt thereof for use in restoring or improving cardiac function in a subject suffering from chronic heart failure.

[0094] In another aspect, there is provided a compound of formula (I) or a salt thereof for use in enhancing cardiac repair after myocardial infarction in a subject in need thereof.

[0095] In another aspect, there is provided a compound of formula (I) or a salt thereof for use in enhancing cardiac repair in a subject suffering from chronic heart failure.

[0096] In another aspect, there is provided a compound of formula (I) or a salt thereof for use in treating post-myocardial infarction cardiac dysfunction in a subject in need thereof.

[0097] In another aspect, there is provided a compound of formula (I) or a salt thereof for use in the treatment of cardiac dysfunction in a subject suffering from chronic heart failure.

[0098] In another aspect, there is provided a compound of formula (I) or a salt thereof for use in the treatment of persistent post-myocardial infarction angina in a subject in need thereof.

[0099] In another aspect, there is provided a compound of formula (I) or a salt thereof for use in the treatment of persistent angina in a subject suffering from chronic heart failure.

[0100] In another aspect, there is provided a compound of formula (I) or a salt thereof for use in the treatment of persistent angina.

[0101] In another aspect, there is provided a compound of formula (I) or a salt thereof for use in improving survival, reducing arrhythmias, and / or increasing ventricular contractility and compliance after myocardial infarction in a subject in need thereof.

[0102] In another aspect, there is provided a compound of formula (I) or a salt thereof for use in improving survival, reducing arrhythmias, and / or increasing ventricular contractility and compliance in a subject suffering from chronic heart failure.

[0103] In another aspect, there is provided a compound of formula (I) or a salt thereof for use in prolonging survival of a subject after a myocardial infarction.

[0104] In another aspect, there is provided a compound of formula (I) or a salt thereof for use in prolonging survival of a subject suffering from chronic heart failure.

[0105] In another aspect, there is provided a compound of formula (I) or a salt thereof for use in preventing the development of severe heart failure disease in a subject following a myocardial infarction.

[0106] In another aspect, there is provided a compound of formula (I) or a salt thereof for use in preventing the development of severe heart failure disease in a subject suffering from chronic heart failure.

[0107] In another aspect, there is provided a compound of formula (I) or a salt thereof for use in the treatment of chronic heart failure in a subject in need thereof.

[0108] In another aspect, there is provided the use of a compound of formula (I) or a salt thereof in the manufacture of a medicament for reversing cardiac damage caused by myocardial infarction in a subject in need thereof.

[0109] In another aspect, there is provided the use of a compound of formula (I) or a salt thereof in the manufacture of a medicament for reversing cardiac damage in a subject suffering from chronic heart failure.

[0110] In another aspect, there is provided the use of a compound of formula (I) or a salt thereof in the manufacture of a medicament for treating cardiac damage caused by myocardial infarction in a subject in need thereof.

[0111] In another aspect, there is provided the use of a compound of formula (I) or a salt thereof in the manufacture of a medicament for treating the heart in a subject suffering from chronic heart failure.

[0112] In another aspect, there is provided the use of a compound of formula (I) or a salt thereof in the manufacture of a medicament for restoring or improving cardiac function after myocardial infarction in a subject in need thereof.

[0113] In another aspect, there is provided the use of a compound of formula (I) or a salt thereof in the manufacture of a medicament for restoring or improving cardiac function in a subject suffering from chronic heart failure.

[0114] In another aspect, there is provided the use of a compound of formula (I) or a salt thereof in the manufacture of a medicament for enhancing cardiac repair after myocardial infarction in a subject in need thereof.

[0115] In another aspect, there is provided the use of a compound of formula (I) or a salt thereof in the manufacture of a medicament for enhancing cardiac repair in a subject suffering from chronic heart failure.

[0116] In another aspect, there is provided the use of a compound of formula (I) or a salt thereof in the manufacture of a medicament for the treatment of post-myocardial infarction cardiac dysfunction in a subject in need thereof.

[0117] In another aspect, there is provided the use of a compound of formula (I) or a salt thereof in the manufacture of a medicament for the treatment of cardiac dysfunction in a subject suffering from chronic heart failure.

[0118] In another aspect, there is provided the use of a compound of formula (I) or a salt thereof in the manufacture of a medicament for the treatment of persistent post-myocardial infarction angina in a subject in need thereof.

[0119] In another aspect, there is provided the use of a compound of formula (I) or a salt thereof in the manufacture of a medicament for the treatment of persistent angina in a subject suffering from chronic heart failure.

[0120] In another aspect, there is provided the use of a compound of formula (I) or a salt thereof in the manufacture of a medicament for the treatment of persistent angina in a subject in need thereof.

[0121] In another aspect, there is provided a use of a compound of formula (I) or a salt thereof in the manufacture of a medicament for improving survival, reducing arrhythmias, and / or increasing ventricular contractility and compliance after myocardial infarction in a subject in need thereof.

[0122] In another aspect, there is provided the use of a compound of formula (I) or a salt thereof in the manufacture of a medicament for improving survival, reducing arrhythmias, and / or increasing ventricular contractility and compliance in a subject suffering from chronic heart failure.

[0123] In another aspect, there is provided the use of a compound of formula (I) or a salt thereof in the manufacture of a medicament for prolonging survival of a subject following a myocardial infarction.

[0124] In another aspect, there is provided the use of a compound of formula (I) or a salt thereof in the manufacture of a medicament for prolonging survival of a subject suffering from chronic heart failure.

[0125] In another aspect, there is provided the use of a compound of formula (I) or a salt thereof in the manufacture of a medicament for preventing the development of severe heart failure disease in a subject following a myocardial infarction.

[0126] In another aspect, there is provided the use of a compound of formula (I) or a salt thereof in the manufacture of a medicament for preventing the development of severe heart failure disease in a subject suffering from chronic heart failure.

[0127] In another aspect, there is provided the use of a compound of formula (I) or a salt thereof in the manufacture of a medicament for the treatment of chronic heart failure in a subject in need thereof.

[0128] In the above-mentioned methods and uses of the present invention, the compound of formula (I) or a salt thereof is administered in an amount of about 0.1 to 100 mg / kg, or about 0.1 to 0.5, 0.1 to 1, 0.1 to 2, 0.1 to 3, 0.1 to 4, 0.1 to 5, 0.1 to 6, 0.1 to 7, 0.1 to 8, 0.1 to 10, 0.5 to 1, 0.5 to 2, 0.5 to 3, 0.5 to 4, 0.5 to 5, 0.5 to 6, 0.5 to 7, 0.5 to 8, 0.5 to 9, 0.5 to 10, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 ... 3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, 9-1 0, 5-10, 5-20, 5-30, 5-40, 5-50, 5-60, 5-70, 5-80, 5-90, 5-100, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 30-40, 30-50, 30-60, 30-70 , 30-80, 30-90, 30-100, 40-50, 40-60, 40-70, 40-80, 40-90, 40-100, 50-60, 50-70, 50-80, 50-90, 50-100, 60-70, 60-80, 60-90, 60-100, 70-80, 70-90, 70-100, 80-90, 80-100, or 90-100 mg / kg. In the above methods and uses of the present invention, the compound of formula (I) may be administered to a subject in need thereof in an amount of 0.1 mg / kg, or about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / kg.

[0129] In the above-mentioned methods and uses of the present invention, the compound of formula (I) or a salt thereof can be administered to a subject in need thereof twice a day, once a day, twice a week, once a week, once every two weeks, monthly, once every two months, or once every six months. In the above-mentioned methods and uses of the present invention, the treatment period with the compound of formula (I) or a salt thereof can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. Any of the above-mentioned administration frequencies can be combined with any of the above-mentioned treatment periods. For example, the compound of formula (I) can be administered once a day for 5 days.

[0130] As discussed above, the compounds of the present invention unexpectedly provide activity that mimics that of PDGF while avoiding excessive and harmful fibrosis.

[0131] For example, administering a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the present invention to a subject does not significantly change the fibrotic area of ​​the heart after infarction. That is, after administering a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the present invention, the fibrotic area of ​​the heart after infarction does not increase by more than 5%, or by more than 10, 15, 20, 30, 40, or 50%, compared to the size of cardiac fibrosis after infarction but before administration of a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the present invention. In some embodiments, the fibrotic area of ​​the heart after infarction can be reduced compared to the size of cardiac fibrosis after infarction but without administration of a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the present invention. The fibrotic area of ​​the heart after infarction can be measured by Gomori's trichrome staining of the infarcted heart.

[0132] Furthermore, administration of a compound of Formula (I) or a pharmaceutical composition of the present invention to a subject does not significantly increase the size of cardiac fibrosis after infarction compared to administration of a corresponding dose of a platelet-derived growth factor (PDGF)-B chain homodimer not conjugated to human serum albumin (HSA). The term "corresponding dose" refers to a dose of a compound of Formula (I) determined to have the same efficacy as PDGF-AB or PDGF-BB in an in vitro assay. The term "corresponding dose" may also refer to the dose of a compound of Formula (I) required to achieve a plasma concentration that provides a therapeutic effect in vivo. The term "does not significantly increase" means that administration of a compound of Formula (I) or a salt thereof, or a pharmaceutical composition of the present invention does not increase the size of cardiac fibrosis by more than 5%, or by more than 10, 15, 20, 30, 40, or 50%, compared to the size of cardiac fibrosis after infarction without administration of a compound of Formula (I) or a salt thereof, or a pharmaceutical composition of the present invention. In some embodiments, the size of cardiac fibrosis may be reduced compared to the size of cardiac fibrosis following infarction but without administration of a compound of formula (I) or a salt thereof, or a pharmaceutical composition of the present invention. [Example]

[0133] Preparation of compounds Reagents and Solvents Peptide-grade N,N-dimethylformamide (DMF) and dichloromethane (DCM) were purchased from RCI Labscan and Merck, respectively. Chromatographic acetonitrile (MeCN) was purchased as "gradient grade" from Sigma-Aldrich, and ultrapure water was purchased from a Merck Millipore Direct-Q 5 water purification system. All solvents for chromatography were supplemented with formic acid (FA) purchased from Sigma-Aldrich. All standard Fmoc-protected amino acids were purchased from Mimotopes. Rink amide resin for peptide synthesis was purchased from Mimotopes. Automated SPPS was performed on a Biotage Syro1.

[0134] General procedure for solid phase peptide synthesis: The resin (164 mg, 100 μmol, 0.61 mmol g, 1 equiv.) was treated with 40% (v / v) piperidine in DMF (1.6 mL) for 3 min, drained, then treated with 20% (v / v) piperidine in DMF (1.6 mL) for 10 min, drained, and washed with DMF (4 × 1.6 mL). The resin was then treated with a solution of Fmoc-Xaa-OH (400 μmol, 4 equiv.) and Oxyma (57 mg, 400 μmol, 4 equiv.) in DMF (800 μL), followed by a solution of DIC (63 μL, 400 μmol, 4 equiv.) in DMF (800 μL) and shaken at room temperature for 1 h. The resin was then drained, washed with DMF (4 × 1.6 mL), and then treated with a solution of 5% (v / v) AcO and 10% (v / v) iPrNEt in DMF (1.6 mL) at room temperature for 5 min, drained, washed with DMF (4 × 1.6 mL), and drained. The general scheme is shown in Figure 1.

[0135] Peptide cleavage from resin: The resin-bound peptide was shaken in a cleavage solution of TFA / TIS / HO (90:5:5 v / v / v) at room temperature for 2 hours. The crude product was drained, and the resin was rinsed with cleavage cocktail (approximately 2 mL). These solutions were combined and concentrated to <1 mL under a stream of nitrogen. To precipitate the free peptide, diethyl ether (14 mL) was added to the crude concentrate. The resulting suspension was centrifuged at 7000 rcf for 4 minutes to pellet the free peptide. The supernatant was then decanted, and the precipitation process was repeated once more. The crude peptide was dried under a stream of nitrogen and then redissolved in 50% v / v aq. MeCN (~6 mL) for cyclization.

[0136] Disulfide formation The crude or purified peptide fragment was dissolved in saturated aqueous ammonium carbonate and stirred under air for 24 hours. Upon completion, as determined by UHPLC, the solution was concentrated under a stream of nitrogen. The residue was then dissolved in DMF for HPLC purification.

[0137] Preparative Chromatography Reversed-phase high-performance liquid chromatography (HPLC) was performed on a Waters 600E multisolvent delivery system equipped with a Rheodyne 7725i injection valve (5 mL loading loop), a Waters 500 pump, and a Waters 490E programmable wavelength detector operating at 214 nm and 230 nm. Preparative reversed-phase HPLC was performed using a Waters semi-preparative Sunfire OED C18 column (5 μm, 19 × 150 mm) with 15 mL min. -1 All preparative HPLC runs used mobile phases of ultrapure (type 1) water (solvent A) and MeCN (solvent B) supplemented with 0.1% by volume formic acid or trifluoroacetic acid (TFA) with the gradients specified.

[0138] Analytical chromatography Liquid chromatography-mass spectrometry (UPLC) was performed on a Shimadzu 2020 UPLC instrument equipped with a Nexera X2 LC-30AD pump, a Nexera X2 SPD-M30A UV / Vis diode array detector, and a Shimadzu 2020 (ESI) mass spectrometer operated in either positive or negative mode. Separations were performed on a Waters Acquity BEH300 1.7 μm, 2.1 × 50 mm (C18) column at a flow rate of 0.6 mL min . -1 All separations were performed using a mobile phase of 0.1% by volume formic acid in water (solvent A) and 0.1% by volume formic acid in MeCN (solvent B) with the specified gradient. Analytical reversed-phase HPLC was performed on a Waters Acquity UPLC system equipped with a PDA λ detector (λ = 210-400 nm). Separations were performed on a Waters Acquity BEH300 1.7 μm, 2.1 × 50 mm (C18) column at a flow rate of 0.6 mL min -1 All separations were performed using a mobile phase of 0.1% by volume TFA in water (solvent A) and 0.1% by volume TFA in MeCN (solvent B) using the gradients specified.

[0139] Matrix-assisted laser desorption / ionization (MALDI) mass spectrometry Matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectra were recorded on a Bruker Autoflex™ Speed ​​MALDI-TOF instrument operated in linear positive mode using a matrix of 10 mg / mL sinapinic acid in TFA-free HO:MeCN (1:1 v / v).

[0140] compound

[0141] [ka]

[0142] PDGF loop III monomer was synthesized via Fmoc-strategy SPPS as specified in the general method. The linear sequence N'-CVRKIEIVRKK-C' was generated by automated SPPS on Rink amide resin (204 mg, 100 μmol, capacity: 0.49 mmol g -1 ). A 50 μmol portion was cleaved from the resin as described in General Methods. The crude linear peptide was purified by semi-preparative RP-HPLC (0-30% B + 0.1% formic acid over 30 min). The appropriate fractions were combined and lyophilized to give the PDGF loop III monomer as a white solid (22.6 mg, 32%). HPLC t = 16.64 min (1 to 40% B + 0.1% TFA over 30 min). LRMS (ESI+): m / z = 1371.5 [M+H] + .

[0143] [ka]

[0144] The PDGF loop III HSA monomer was synthesized via Fmoc-strategy SPPS as specified in the general method. The linear sequence N'-CVRKIEIVRKK(Peg3)EYEK(Palm)EYE-C' was generated by automated SPPS on Rink amide resin (204 mg, 100 μmol, capacity: 0.49 mmol g). -1). A 50 μmol portion was cleaved from the resin as described in General Methods. The crude linear peptide was used without further purification. PDGF Loop III HSA Monomer (53 mg, 36%) as a white solid. HPLC t = 34.61 min (1 to 50% B + 0.1% TFA over 40 min). LRMS (ESI+): m / z = 1494.2 [M+2H] 2+ .

[0145] [ka]

[0146] The PDGF loop III dimer HSA(CVRKIEIVRKK)2(PEG3)2EYEK(Palm)EYE-C' was synthesized via disulfide bond formation as specified in the general method. The linear sequence N'-CVRKIEIVRKK-C' (12.2 mg (0.009 mmol)) and N'-(CVRKIEIVRKK)2(PEG3)2EYEK(Palm)EYE-C' (27 mg (0.009 mmol)) were dissolved in DMF (1 mL), and the solution was saturated with NH4. + CO3 - Aqueous solution was added and the reaction mixture was stirred for 48 h. The crude linear peptide was purified by semi-preparative RP-HPLC (0-30% B + 0.1% formic acid over 30 min). The appropriate fractions were combined and lyophilized to give PDGF loop III dimer HSA as a white solid (3.2 mg, 8%). HPLC t = 21.77 min (1 to 80% B + 0.1% TFA over 40 min). LRMS (ESI+): m / z = 1452.7 [M+3H] 3+ . MALDI-TOF(ESI+):m / z=4357.1[M+H] + .

[0147] [ka]

[0148] The PDGF loop III dimer HSA dimer (CVRKIEIVRKK)2(Peg3)2(EYEK(Palm)EYE)2-C' was synthesized via disulfide bond formation as specified in the general method. The linear sequence (CVRKIEIVRKK)2(Peg3)2EYEK(Palm)EYE-C' (10 mg, 0.0032 mmol) was dissolved in DMF (1 mL) and the solution was saturated with NH4. + CO3 - Aqueous solution (1 mL) was added and the reaction mixture was stirred for 48 h. The crude linear peptide was purified by semi-preparative RP-HPLC (0-30% B + 0.1% formic acid over 30 min). The appropriate fractions were combined and lyophilized to give the PDGF loop III dimer HSA dimer as a white solid (3.6 mg, 36%). HPLC R t =25.33 minutes (1-80% B+0.1% TFA over 40 minutes). LRMS(ESI+):m / z=1493.7[M+4H] 4+ . MALDI-TOF(ESI+):m / z=5973.2[M+H] + 。

[0149] In vitro testing The functions of compounds Ia (also referred to herein as JC5) and Ib (also referred to herein as JC5a) were tested in vitro on the continuous cell line C2C12 mouse myoblasts. These in vitro assays used recombinant PDGF-AB and -BB as positive controls, along with a serum-starved untreated control and the PDGF receptor inhibitor AG1296 co-treated with PDGF-AB as negative controls. The in vitro assays were designed to evaluate the primary known functions of PDGF while also considering functions relevant to its in vivo application. These functions were chemotaxis, collagen contraction, angiogenesis, and activation of the PDGF receptor via phosphorylation of Akt and Erk.

[0150] Chemotaxis was assessed by creating scratches through 2D cultures of C2C12 cells after treatment with four controls and peptides JC5 and JC5a. These were then incubated at 37°C for 24 hours. Cell migration across the scratch was measured at the end of the incubation period and compared with the relevant controls. PDGF is a well-established mediator of chemotaxis and is expected to increase cell migration across the scratch over the 24-hour period following treatment.

[0151] Figure 2 shows a significant increase in migration of C2C12 cells treated with recombinant PDGF-BB and peptides JC5 and JC5a. Migration of the positive control PDGF-BB-treated cells was 75.02% (±18.14%), which was not significantly higher than the migration of JC5-treated cells (68.28% (±17.80%) and lower than the migration of JC5a (79.88% (±9.096%)). The untreated control group migrated 20.32% (±12.08%), as did the negative control AG1296 + PDGF-AB-treated cells (16.57% (±9.60%)). PDGF-BB-treated cells had a 54.70% (mean difference) increase compared to the untreated control (p<0.0001) and a 58.45% (mean difference) increase compared to the negative control (p<0.0001). Cells treated with the novel mimetic peptide JC5 showed a 47.96% (mean difference) increase compared to the untreated control group (p<0.0001) and a 51.71% (mean difference) increase compared to the negative control (p<0.0001). Cells treated with the novel mimetic peptide JC5a showed a 59.56% (mean difference) increase compared to the untreated control group (p<0.0001) and a 62.71% (mean difference) increase compared to the negative control (p<0.0001). In summary, our novel peptides exhibited chemotactic function similar to that of recombinant human PDGF-AB and PDGF-BB.

[0152] Collagen contraction is a key function during wound healing in many tissues, including the heart. This can be simulated in vitro using a collagen gel contraction assay. This was evaluated using C2C12 cells treated with 10 ng / mL recombinant PDGF-BB and PDGF-AB as positive controls, 10 μM AG1296 cells co-treated with 10 ng / mL recombinant PDGF-AB as negative controls, a serum-starved untreated control, and 1 μg / mL peptides JC5 and JC5a. C2C12 mouse myoblasts were seeded into 3D collagen gels cross-linked with NaOH and treated with the four controls and peptides JC5 and JC5a, followed by incubation at 37°C for 24 hours.

[0153] Increased collagen contraction is known to occur after treatment with PDGF and was observed in this assay after treatment with PDGF-BB, PDGF-AB, and peptides JC5 and JC5a (Figure 3). The increase in collagen contraction was statistically significant when compared to our negative control, AG1296 + PDGF-AB, whereas PDGF-AB, PDGF-BB, and JC5 were statistically significant when compared to the untreated control. These results are consistent with other reported data on PDGF in collagen contraction assays. After 24 h of incubation, C2C12 cells treated with PDGF-AB contracted collagen gels to 22.46% (±7.081) of their initial surface area, whereas PDGF-BB contracted them to 23.42% (±7.522), JC5 contracted them to 23.01% (±6.040), and JC5a contracted them to 33.28% (±6.979). The negative control AG1296 cells co-treated with PDGF-AB contracted to 55.25% (±15.61%), which was 32.79% (mean difference) less than PDGF-AB (p>0.0001), 31.83% (mean difference) less than PDGF-BB (p>0.0001), 32.23% (mean difference) less than our novel mimetic peptide JC5 (p>0.0001), and 21.96% (mean difference) less than our novel mimetic peptide JC5a (p=0.0120). The untreated control contracted to 41.27% (±7.675), which was 18.82% (mean difference) less than PDGF-AB (p=0.0073), 17.86% (mean difference) less than PDGF-BB (p=0.0126), and 18.26% (mean difference) less than our novel mimetic peptide JC5 (p=0.0100). In summary, our novel mimetic peptide has similar collagen contractile ability to recombinant human PDGF-AB and PDGF-BB.

[0154] PDGF has a well-established role in in vivo angiogenesis. To simulate this function in vitro, we performed an angiogenesis assay on primary human coronary artery endothelial cells (HCAECs). Cells were seeded on 3D growth factor-reduced Geltrex basement membrane extract and treated with PDGF-AB, PDGF-BB, untreated control, AG1296 + PDGF-AB negative control, and peptides JC5 and JC5a, followed by 24-hour incubation. Over the course of 24 hours, cells formed tubes throughout the gel. PDGF has been reported to promote endothelial cell tube formation in in vitro 3D assays.

[0155] Cells treated with 50 ng / mL PDGF-AB, PDGF-BB, and 5 μg / mL JC5 produced longer, more uniform tubes than untreated cells or cells treated with the negative control 10 μM PDGF receptor inhibitor AG1296 co-treated with 50 ng / mL PDGF-AB (Figure 4). The positive controls PDGF-AB and PDGF-BB produced tubes that were 560.7 μm (±53.93) and 616.3 μm (±39.07), respectively. These were not significantly different from JC5, which produced tubes that were 591.3 μm (±73.23). The untreated control and the AG1296 + PDGF-AB negative control produced tubes that were 305 μm and 307.7 μm, respectively. The PDGF-AB-treated group had an increased tube length of 255.7 μm (mean difference) compared to untreated controls (p=0.0062) and 253 μm (mean difference) compared to negative controls (p=0.0068). The PDGF-BB-treated group had an increased tube length of 311.3 μm (mean difference) compared to untreated controls (p=0.0013) and 308.7 μm (mean difference) compared to negative controls (p=0.0014). The JC5-treated group had an increased tube length of 286.3 μm (mean difference) compared to untreated controls (p=0.0025) and 283.7 μm (mean difference) compared to negative controls (p=0.0027). In summary, JC5 has angiogenic potential similar to that of recombinant human PDGF-AB and PDGF-BB.

[0156] PDGF receptor activation occurs via autophosphorylation of tyrosine and threonine residues at key sites throughout the intracellular domain. Autophosphorylation is involved in different signaling pathways, including ERK (MAPK), which is involved in proliferation, cell survival, angiogenesis, and chemotactic activity of cells. ERK phosphorylation indicates successful activation of the PDGF receptor. In Figure 5, phosphorylation of ERK (pERK) can be seen in the PDGF-BB-treated and JC5-treated groups compared to the untreated control, the AG1296 + PDGF-AB negative control, and the PDGF-AB group. In Figure 6, phosphorylation of ERK (pERK) can be seen in the PDGF-BB-treated C2C12 cells and JC5a-treated cells compared to the untreated control, the AG1296 + PDGF-AB negative control, and the PDGF-AB cells. We also observed conserved phosphorylation of ERK (pERK) after 30 minutes of treatment with our PDGF-mimetic peptide, JC5a. These results demonstrate successful activation of the PDGF receptor after treatment with JC5 and JC5a. Furthermore, it should be noted that the AB ligand requires the presence of PDGFRα, which is expressed at very low levels in C2C12 cells (Contreras et al.), whereas PDGFRβ is expressed at high levels; therefore, pERK is induced by PDGF-BB but not PDGF-AB.

[0157] Cultured C3H10T 1 The mitogenic effects of the PDGF mimetic peptides JC5 and JC5a were tested using 1 / 2 mouse embryonic fibroblasts. Proliferation was assayed after pulsing with the nucleotide analog 5-ethynyl-2'-deoxyuridine (EdU) and fluorescence detection using flow cytometry. Cells were treated with 20 ng / mL PDGF-BB or PDGF-AB and 2 μg / mL peptide JC5 or JC5a for 24 hours in the presence of 1% fetal bovine serum (FBS) and DMEM. 10% FBS was used as a positive control, and cotreatment with the specific PDGF receptor antagonist, AG1296, was used to confirm whether proliferation occurred via the PDGF ligand / receptor pathway. Assays were performed in biological triplicate.

[0158] Both 10% FBS (positive control) and PDGF-BB stimulated EdU incorporation >2-fold (p<0.001), the latter of which was significantly inhibited by AG1296, confirming stimulation via the PDGF ligand / receptor pathway. However, PDGF-AB, JC5, and JC5a did not significantly stimulate proliferation compared to 1% FBS. Thus, even though the PDGF peptide moiety of JC5 and JC5a is based on the loop III sequence of PDGF-BB, they did not exhibit significant proliferative activity in this assay. The inhibition of EdU levels shown by PDGF-AB and JC5 after cotreatment with AG1296 (p<0.01 and p<0.05, respectively) suggests weak stimulation of the PDGF ligand / receptor pathway by PDGF-AB and JC5 in the presence of 1% FBS, although at a much lower level than that seen with PDGF-BB. In summary, the novel mimetic peptides JC5 and JC5a exhibit significantly lower effects on proliferation in C3H10T1 / 2 cells than PDGF-BB. This contrasts with results seen in cell migration, collagen gel contraction, and angiogenesis assays, where PDGF-AB, PDGF-BB, JC5, and JC5a exhibit comparable activity (Figures 2–4). These data suggest novel biological activities for JC5 and JC5a compared to native recombinant PDGF-BB.

[0159] In vivo testing The in vivo efficacy of the peptides was evaluated using a mouse model of MI established using surgically induced acute myocardial infarction (MI) via permanent occlusion of the left anterior descending (LAD) artery, along with non-infarcted sham control mice. Cardiac function was monitored at baseline post-MI on days 2 and 28, and at endpoints. Subsequently, tissues were harvested and heart weight divided by tibia length was calculated. Mice were randomly assigned to treatment groups and, at the time of infarction, received either 60 μg / kg of the positive control recombinant human PDGF-AB, 32.50 mg / kg of the novel mimetic peptide JC5, 32.50 mg / kg of the novel mimetic peptide JC5a, or phosphate-buffered saline (PBS) as a vehicle control via an implanted minipump. In addition to the sham control, non-infarcted healthy mice received either 32.50 mg / kg of the novel mimetic peptide JC5 or 32.50 mg / kg of the novel mimetic peptide JC5a.

[0160] At day 28 post-MI, mice treated with PDGF-AB(MI) and the novel mimetic peptide JC5(MI) had significantly increased ejection fractions when compared with mice receiving the PBS(MI) vehicle control (Figure 9A). Mice treated with PDGF-AB(MI) had a day 28 ejection fraction of 43.44% (±11.90%), similar to mice treated with JC5(MI) that had a day 28 ejection fraction of 42.06% (±13.41%) compared with mice receiving PBS(MI) that had a day 28 ejection fraction of 26.89% (±6.966%) (PDGF-AB p=0.0073, JC5 p=0.0129). The novel mimetic peptide JC5a(MI) showed a trend toward increased ejection fraction, but this was not significant when compared with PBS(MI) at day 28 (p=0.0655), resulting in an ejection fraction of 39.24% (±13.51%). The validity of the ejection fraction results at day 28 was confirmed using Bland-Altman interobserver variability analysis (Figure 10). The bias for this analysis was 14.06 (±7.346), consistent across all replicates when comparing observer 1 with observer 2. This falls within the range reported for murine echocardiography of injured hearts (Grune, Blumrich et al. 2018; Hume, Kanagalingam et al. 2023). Both observers were blinded during the analysis.

[0161] Left ventricular ejection fraction (LVEF) is directly proportional to the difference between left ventricular end-diastolic volume and left ventricular end-systolic volume (LVEDV-LVESV) and inversely proportional to left ventricular end-diastolic volume (LVEDV). This value is calculated using the following formula: LVEF = (LVEDV-LVESV) / LVEDV. An increase in end-diastolic volume (EDV) indicates potential ventricular dilation, while an increase in end-systolic volume (ESV) indicates potential decreased contractile function. Mice treated with the positive control PDGF-AB (MI) and the novel mimetic peptide JC5 (MI) showed significant decreases in both ESV and EDV at day 28 when compared to the vehicle control, PBS (MI), cohort. The reduced ESV of the PDGF-AB(MI) cohort on day 28 was consistent with that expected for the positive control, with a mean ESV of 55.04 μL (±23.66) and 61.54 μL (mean difference) lower than the PBS(MI) cohort (p=0.0003). The PDGF-AB(MI) cohort had a mean EDV of 93.75 μL (±23.17), while the JC5(MI) cohort had a mean EDV of 108.6 μL (±53.37). Both were significant decreases from the PBS(MI) cohort, with a mean EDV of 155.9 μL (±47.15) (JC5 p=0.0185, PDGF-AB p=0.0016). The JC5(MI) mouse cohort had a mean ESV of 67.01 μL (±42.94), which was 49.58 μL (mean difference) lower than the PBS(MI) cohort, which had a mean ESV of 116.6 μL (±45.00) (p=0.0032).

[0162] Although JC5a(MI) did not have a significantly increased ejection fraction on day 28 compared with PBS(MI), mice treated with JC5a(MI) had the greatest increase from their day 2 ejection fraction. After treatment with 32.50 mg / kg of the novel mimetic peptide JC5a(MI), mice had a mean change in ejection fraction of 10.82% from day 2 to day 28. This was 18.02% (mean difference) greater than that of mice receiving the vehicle control, PBS(MI) (p=0.0082). JC5a(MI) mice maintained the greatest decrease in ejection fraction from baseline to day 2 (Figure 9E), potentially contributing to their lower ejection fraction on day 28 when compared with PDGF-AB(MI) and JC5(MI). Mice treated with the positive controls PDGF-AB(MI) and JC5(MI) also had a significant increase in ejection fraction from day 2 to day 28 when compared to PBS(MI). Mice treated with PDGF-AB(MI) had a mean Δ from day 2 to day 28 of 7.104% (±13.19) (PDGF-AB p=0.0393), while mice treated with JC5(MI) had a mean Δ from day 2 to day 28 of 6.686% (±12.22) (JC5 p=0.0409).

[0163] These results demonstrate a significant improvement in cardiac function in mice treated with the novel mimetic peptides JC5 and JC5a, both of which function similarly to the positive control, human recombinant PDGF-AB. The significant reductions in ESV and EDV in PDGF-AB and JC5-treated mice indicate that these treatments have effects on left ventricular dilatation (EDV) and contractility (ESV), leading to increased ejection fraction and improved cardiac function. While both are significant, the greater significance seen in ESV suggests that the effects of these peptides are likely driven more by effects on left ventricular contractility than dilatation. From this, we can conclude that the comparable effects of the mimetic peptides in vitro (Figures 2-6 and 8) translate into maintained biological functions of PDGF-AB in vivo (Figures 9-11), in agreement with previous PDGF-AB studies conducted by our group (Asli, Xaymardan et al. 2019, Sujitha Thavapalachandran 2020, Hume, Deshmukh et al. 2023).

[0164] Supporting the improved cardiac function identified in Figure 9, mice treated with both the PDGF-AB(MI) positive control and the novel mimetic peptide JC5(MI) had reduced heart weights, normalized by tibia length, compared to mice receiving the vehicle control PBS(MI). JC5(MI) mice had a mean heart weight / tibia length of 0.01462 (±0.005002), as did mice treated with the positive control PDGF-AB(MI), which had a mean heart weight / tibia length of 0.01344 (±0.003075). Both were statistically significant when compared to PBS(MI) mice, whose mean heart weight / tibia length was 0.01913 (±0.004728) (JC5 p=0.0366, PDGF-AB p=0.0066). This indicates that there is a reduction in hypertrophy and inflammation in mice treated with the novel mimetic peptide JC5(MI) and the positive control PDGF-AB(MI) when compared to PBS vehicle control (MI) mice.

[0165] References: Asli,N.S.,et al.(2019).「PDGFRα signaling in cardiac fibroblasts modulates quiescence,metabolism and self-renewal, and promotes anatomical and functional repair.」BioRxiv. Contreras et al,Cellular Signalling 202184:110036. Grune,J.,et al.(2018).「Evaluation of a commercial multi-dimensional echocardiography technique for ventricular volumetry in small animals.」Cardiovasc Ultrasound 16(1):10. Hume,R.D.,et al.(2023).「PDGF-AB Reduces Myofibroblast Differentiation Without Increasing Proliferation After Myocardial Infarction.」JACC:Basic to Translational Science. Hume,R.D.,et al.(2023).「Tropoelastin Improves Post-Infarct Cardiac Function.」Circ Res 132(1):72-86. Lin et al(2007)Growth Factors 25(2):87-93). Sujitha Thavapalachandran,S.M.G.,Robert D.Hume,Thi Yen Loan Le,Kalyan Raguram,James E.Hudson,Jim Pouliopoulos,Gemma A.Figtree,Rafael P.Dye,Anthony M.Barry,Paula Brown,Juntang Lu,Sean Coffey,Scott H.Kesteven,Richard J.Mills,Fairooj N.Rashid,Elena Taran,Pramesh Kovoor,Liza Thomas,Alan Robert Denniss,Eddy Kizana, Naisana S.Asli,Munira Xaymardan,Michael P.Feneley,Robert M.Graham,Richard P.Harvey,James J.H.Chong(2020).「Platelet-derived growth factor-AB improves scar mechanics and vascularity after myocardial infarction.」Science Translational Medicine 12.

Claims

1. A compound of formula (I) or a salt thereof: 【Chemical 1】 In the formula, X=NH ２ or -L2-[HSA conjugate], 【Chemistry 2】 and L1 and L2 are independently -HN(C ２ ~C ６０ alkyl)C(O)-, -HN(C ２ ~C ６０ Alkaryl)C(O)-, -HN(C ２ ~C ６０ alkenyl)C(O)-, -HN(CH ２ ) ｒ (CH ２ CH ２ O) ｏ (CH ２ ) ｒ C(O)-, -HN(CH ２ ) ｒ (O(CH ２ )) ｍ C(O)) ｏ (CH ２ ) ｒ C(O)-, -HN(CH ２ ) ｒ ((NH(CH ２ )) ｍ C(O)) ｏ (CH ２ ) ｒ C(O)-, and 【Chemistry 3】 wherein n=1-20, o=1-60, r=2-20, m=1-60, p=1-60, and q=1-60.

2. L1 and L2 are independently -HN(C ２ ~C ２０ alkyl)C(O)-, -HN(C ２ ~C ２０ Alkaryl)C(O)-, -HN(C ２ ~C ２０ alkenyl)C(O)-, -HN(CH ２ )(CH ｒ CH ２ O)(CH ２ C(O)-、-HN(CH ｏ )(O(CH ２ C(O))(CH ｒ C(O)-、-HN(CH ２ )(NH(CH ｒ )(CH ２ C(O))(CH ｍ C(O)-、and ｏ [[ID=来的文本]] ２ )(CH ２ C(O)-、and ｒ -HN(CH ２ )(NH(CH ｒ )(CH ２ C(O))(CH ｍ C(O)-、and ｏ )(CH ２ C(O)-、and ｒ C(O)-、and It should be noted that the original text contains some unclear or potentially incorrect chemical structure expressions, which may lead to some difficulties in accurate translation. The above translation is based on the best understanding of the original text. If possible, it is recommended to double-check and clarify the original chemical content for a more accurate translation. 【Chemistry 4】 2. The compound of claim 1, wherein n=1-20, o=1-20, r=2-20, m=1-20, p=1-20, and q=1-20.

3. L1 and L2 are 【Chemistry 5】 2. The compound of claim 1, wherein:

4. The compound according to any one of claims 1 to 3, wherein n=15.

5. The compound according to any one of claims 1 to 4, wherein r=2.

6. L1 and L2 are 【Chemistry 6】 The compound according to any one of claims 1 to 5, 【Request 7】 【Chemical 7】 The compound according to any one of claims 1 to 6, selected from the group consisting of:

8. A process for preparing a compound of formula (I) or a salt thereof according to any one of claims 1 to 4, wherein X = H, (i) Structure: 【Chemistry 8】 preparing a PDGF loop III monomer (compound II) of the formula: (ii) From the PDGF loop III monomer (compound II), the structure: 【Chemistry 9】 preparing a linked PDGF Loop III HSA monomer of Formula II: (iii) preparing a compound of formula (I) by disulfide coupling said compound of formula (II) with said PDGF loop III HSA monomer (compound (II)) or with a second compound of formula (II).

9. 9. The method of claim 8, wherein the PDGF Loop III monomer (Compound II) and / or the PDGF Loop III HSA monomer (Formula II) are prepared by solid phase peptide synthesis.

10. 10. A compound of formula (I) prepared by the method of claim 8 or claim 9.

11. A pharmaceutical composition comprising a compound of formula (I) or a salt thereof according to any one of claims 1 to 7 or claim 10, and a pharmaceutically acceptable excipient, carrier and / or diluent.

12. 12. The pharmaceutical composition of claim 11, wherein the composition is formulated for parenteral administration.

13. Reversing cardiac damage caused by myocardial infarction in a subject in need thereof; Reversing cardiac damage in a subject suffering from chronic heart failure; Treating cardiac damage caused by myocardial infarction in a subject in need thereof; Treating cardiac damage in a subject suffering from chronic heart failure; Restoring or improving cardiac function after myocardial infarction in a subject in need thereof; Restoring or improving cardiac function in a subject suffering from chronic heart failure; Enhancing cardiac repair after myocardial infarction in a subject in need thereof; Enhancing cardiac repair in a subject suffering from chronic heart failure; Treating cardiac dysfunction after myocardial infarction in a subject in need thereof; Treating cardiac dysfunction in a subject suffering from chronic heart failure; Treating persistent angina after myocardial infarction in a subject in need thereof; Persistent angina in a subject suffering from chronic heart failure treating persistent angina in a subject in need thereof; improving survival, reducing arrhythmias and / or increasing ventricular contractility and compliance after myocardial infarction in a subject in need thereof; improving survival, reducing arrhythmias and / or increasing ventricular contractility and compliance in a subject suffering from chronic heart failure; prolonging survival of a subject after myocardial infarction; prolonging survival of a subject suffering from chronic heart failure; preventing the onset of severe heart failure in a subject after myocardial infarction; preventing the onset of severe heart failure in a subject suffering from chronic heart failure; or a method of treating chronic heart failure in a subject in need thereof, said method comprising administering to said subject a compound of formula (I) or a salt thereof according to any one of claims 1 to 7 or claim 10, or a pharmaceutical composition according to claim 11 or claim 12.

14. 14. The method according to claim 13, wherein the compound of formula (I) or a salt thereof is administered in an amount of about 0.1 to 100 mg / kg; and / or the compound of formula (I) is administered twice daily, once daily, twice weekly, once weekly, once every two weeks, once monthly, once every two months, or once every six months; and / or the treatment period is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months; and / or wherein administering to the subject a compound of formula (I) according to any one of claims 1 to 7 or claim 10, or a pharmaceutical composition according to claim 11 or claim 12, does not significantly change the size of cardiac fibrosis after infarction.

15. 15. The method of claim 13 or claim 14, wherein administration to the subject of a compound of formula (I) according to any one of claims 1 to 7 or claim 10, or a pharmaceutical composition according to claim 11 or claim 12, does not significantly increase post-infarction cardiac fibrosis compared to administration of a corresponding dose of a platelet-derived growth factor (PDGF)-B chain homodimer that is not conjugated to human serum albumin (HSA).

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