A dual-target peptide and its application in the treatment of heart failure

Abstract

The present invention discloses a dual-target polypeptide and its application in the treatment of heart failure. The dual-target polypeptide targets two targets, PDE9A and Neprilysin. The present invention has experimentally confirmed that the dual-target polypeptide can be used to effectively treat heart failure with preserved ejection fraction and heart failure without preserved ejection fraction. The present invention provides a new idea and strategy for the research and development of heart failure-related therapeutic and / or preventive drugs, and has good clinical application prospects and important translational significance.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a dual-target polypeptide having PDE9A and Neprilysin dual-target inhibitory activity, and more specifically relates to a dual-target polypeptide and its application in the treatment of heart failure. Background Art

[0002] Heart failure (HF) is a complex, severe, and life-threatening clinical syndrome. Based on the left ventricular ejection fraction (LVEF), HF can be categorized as either heart failure with preserved ejection fraction (HFpEF) or heart failure with no preserved ejection fraction (HFrEF). Patients with HFpEF typically have an LVEF ≥50% and present with symptoms and signs of heart failure, such as dyspnea, fatigue, and edema. Diagnosis requires a comprehensive assessment of the patient's symptoms, signs, cardiac structure and function, and natriuretic peptide levels. Echocardiography can reveal abnormalities such as left ventricular diastolic dysfunction and left atrial enlargement. Patients with HFrEF have an LVEF ≤40% and present with symptoms and signs of heart failure. Echocardiography can reveal findings such as left ventricular enlargement and weakened myocardial contractility. Although HFpEF and HFrEF are clinically common, to date, no single therapy consistently and reliably reduces their morbidity or mortality.

[0003] At present, the first-line drugs for the treatment of chronic heart failure in clinical practice (such as sacubitril / valsartan) mainly exert their antihypertensive and anti-heart failure effects by inhibiting neprilysin and the renin-angiotensin system. On the one hand, sacubitril / valsartan can inhibit the activation of the RASS system, dilate blood vessels, and improve myocardial remodeling. On the other hand, it can inhibit the degradation of BNP by neprilysin, thereby increasing endogenous BNP, dilating blood vessels, promoting diuresis, and preventing and reversing ventricular remodeling, thereby delaying the progression of heart failure and improving patients' cardiac function. However, based on current research results, the efficacy of sacubitril / valsartan in treating heart failure is limited. The treatment strategy represented by ARNI drugs (sacubitril / valsartan) has two major bottlenecks: (1) Target limitation: it cannot cover the myocardial metabolic abnormalities of heart failure and has no effect on metabolic disorders such as mitochondrial dysfunction; (2) Insufficient pathological coverage: clinical studies have shown that the efficiency of sacubitril / valsartan in inhibiting myocardial fibrosis is only 30-40%.

[0004] Neprilysin (NEP) is a neutral endopeptidase that catalyzes the degradation of various peptides, including ANP, BNP, and bradykinin. Blocking NEP prevents the degradation of endogenous natriuretic peptides. Therefore, theoretically, inhibiting neprilysin, which increases endogenous BNP, could lead to vasodilation, diuresis, and the prevention and reversal of ventricular remodeling, potentially treating heart failure. Recent studies have shown that abnormal expression of cyclic guanosine monophosphate-specific phosphodiesterase 9A (PDE9A) is closely associated with myocardial mitochondrial dysfunction and cardiac fibrosis. In cardiomyocytes, PDE9A is primarily localized in the cytoplasm and, by degrading cGMP, negatively regulates its signaling pathways, affecting myocardial hypertrophy, fibrosis, and contractility. Studies have shown that PDE9A expression is upregulated in heart failure, and its overactivation inhibits cGMP production, exacerbating myocardial dysfunction and pathological remodeling.

[0005] Therefore, developing a drug that can effectively treat heart failure has important practical significance and clinical needs. Summary of the Invention

[0006] In view of this, in order to overcome the above-mentioned technical problems existing in the art, the purpose of the present invention is to provide a dual-target polypeptide and its application in the treatment of heart failure.

[0007] The present invention creatively designs and synthesizes a novel dual-target inhibitory peptide for Neprilysin and PDE9A by targeting the enzymatically active regions of Neprilysin and PDE9A proteins. On the one hand, the dual-target peptide inhibits the enzyme activity of Neprilysin, blocking the degradation of natriuretic peptides in the blood, thereby dilating blood vessels and reducing cardiac preload through diuresis. On the other hand, by inhibiting the enzyme activity of PDE9A, it enhances cGMP in cardiomyocytes, activates the cGMP-PKG signaling pathway, improves myocardial metabolism, and inhibits myocardial dysfunction caused by myocardial fibrosis, thus filling the existing treatment gap for HFpEF and HFrEF. The present invention experimentally verifies for the first time that the dual-target peptide can improve cardiac diastolic or systolic dysfunction, cardiac hypertrophy, and myocardial fibrosis, increase the levels of plasma BNP and myocardial tissue cGMP, and has a significant effect in treating heart failure with preserved ejection fraction and heart failure without preserved ejection fraction. The dual-target peptide has the potential to be developed into an anti-heart failure drug.

[0008] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0009] The first aspect of the present invention provides a dual-target polypeptide targeting PDE9A and Neprilysin or a pharmaceutically acceptable salt thereof, wherein the sequence of the dual-target polypeptide is Ac-DArg-Arg-Gly-Trp-Gly-Pro-DCys-Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gly-Ala-Ser-DCys-NH2, wherein the disulfide bond position is Cys8-DCys19.

[0010] Furthermore, in the sequence of the dual-target polypeptide, DArg and DCys are D-type amino acids, and the rest are L-type amino acids.

[0011] In the present invention, sequences corresponding to polypeptides having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence homology to the sequence of the dual-target polypeptide of the present invention as described above all fall within the scope of protection of the present invention.

[0012] In some embodiments, pharmaceutically acceptable salts of the dual-targeting polypeptide refer to acidic salts formed with inorganic and / or organic acids and basic salts formed with inorganic and / or organic bases. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, but other salts are also useful. Pharmaceutically acceptable salts of the dual-targeting polypeptide can be formed, for example, by reacting the dual-targeting polypeptide with an amount of acid or base in a medium, such as a medium in which the salt precipitates or an aqueous medium (followed by lyophilization).

[0013] Specifically, pharmaceutically acceptable salts include those salts that are suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., within the scope of sound medical judgment, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts (pharmaceutically acceptable salts) are well known in the art. Pharmaceutically acceptable salts of the dual-target polypeptides of the present invention include salts derived from suitable inorganic and organic acids and inorganic and organic bases.

[0014] In some embodiments, suitable acids for preparing pharmaceutically acceptable salts of the dual-targeting polypeptide include, but are not limited to, acetic acid, oleic acid, orotic acid, 2,2-dichloroacetic acid, lauric acid, maleic acid, acylated amino acids, hexanoic acid, caprylic acid, cinnamic acid, adipic acid, alginic acid, ascorbic acid, benzenesulfonic acid, 4-acetamidobenzoic acid, boric acid, nicotinic acid, nitric acid, malonic acid, methanesulfonic acid, oxalic acid, palmitic acid, capric acid, citric acid, cyclamic acid, ethanesulfonic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, α-ketoglutaric acid, glycolic acid, hydroiodic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, and / or valeric acid.

[0015] In some embodiments, suitable bases for preparing pharmaceutically acceptable salts of the dual-target polypeptide include, but are not limited to, inorganic bases such as potassium hydroxide, magnesium hydroxide, calcium hydroxide, zinc hydroxide, and / or sodium hydroxide; organic bases such as primary, secondary, tertiary, butyl, aliphatic, and aromatic amines, including phenethylbenzylamine, dibenzylethylenediamine, choline, dimethylaminoethanol, diethanolamine, dimethylamine, propylamine, diethylamine, diisopropylamine, diethylaminoethanol, ethanolamine, ethylamine, ethylenediamine, isopropylamine, meglumine, hydrazine, methylamine, piperidine, piperazine, propylamine, pyrrolidine, quinuclidine, isoquinoline, secondary amines, triethanolamine, trimethylamine, triethylamine, tris(hydroxymethyl)aminomethane, and / or tromethamine.

[0016] In a specific embodiment of the present invention, the dual-target polypeptide can inhibit PDE9A enzyme activity and increase the cGMP content in myocardial tissue. The dual-target polypeptide can inhibit NEP enzyme activity and increase the BNP content in plasma. The dual-target polypeptide can improve the level of myocardial fibrosis in heart failure with preserved ejection fraction or heart failure without preserved ejection fraction and inhibit pathological remodeling.

[0017] The second aspect of the present invention provides a polynucleotide encoding the dual-target polypeptide described in the first aspect of the present invention.

[0018] In the present invention, variants of the polynucleotides described above are also encompassed within the scope of the present invention. A polynucleotide variant is a variant that differs from the polynucleotide described above by nucleotide substitution, deletion, or addition. The substitution, deletion, or addition may involve one or more nucleotides. The variant may be altered within the coding region, the non-coding region, or both.

[0019] The third aspect of the present invention provides a vector comprising the polynucleotide according to the second aspect of the present invention.

[0020] In some embodiments, the vector further comprises a transcriptional promoter, an enhancer, a translation signal, and a transcription and translation termination signal. Expression vectors for stable transformation generally have a selectable marker that allows selection and maintenance of transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vector in the cell. The vector can also include additional nucleotide sequences that are operably linked to the target nucleic acid molecule, such as an epitope tag for positioning, a tag for purification, a sequence for directing protein secretion and / or membrane association.

[0021] The present invention is not particularly limited to the type of vector, and may be a vector that can replicate and / or express a polynucleotide in eukaryotic or prokaryotic cells including mammalian cells (e.g., human, monkey, rabbit, rat, hamster or mouse cells), yeast cells, insect cells and bacteria (e.g., Escherichia coli). Preferably, the vector comprises at least one selective marker, operably linked to a suitable promoter, so that the polynucleotide can be expressed in the host cell. For example, the vector may include a bacteriophage, a plasmid, a cosmid, a minichromosome, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a poxvirus vector and / or a herpesvirus vector, which are conventionally used, for example, in genetic engineering.

[0022] The fourth aspect of the present invention provides a host cell, wherein the host cell comprises the vector according to the third aspect of the present invention.

[0023] In some embodiments, the host cell is a cell that is used to receive, maintain, replicate, and amplify the vector. The host cell can also be used to express the polypeptide sequence encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid.

[0024] In the present invention, there is no particular limitation on the host cells. For example, the host cells include, but are not limited to, CHO-K1, Pro-5, mouse myeloma cells NSO, mouse myeloma cells SP2 / 0-Ag14, CHO-S, rat myeloma cells YB2 / 0, mouse P3-X63-Ag8653 cells, dihydrofolate reductase gene-deficient CHO cells, lectin-resistant Lec13, α1,6-fucosyltransferase gene-deficient CHO cells, CHO-3E7 cells, immune cells, or any combination thereof.

[0025] The fifth aspect of the present invention provides a polypeptide derivative, which is a polypeptide derivative obtained by modifying the dual-target polypeptide or a pharmaceutically acceptable salt thereof described in the first aspect of the present invention, or a polypeptide derivative obtained by linking a conjugate to the dual-target polypeptide or a pharmaceutically acceptable salt thereof described in the first aspect of the present invention.

[0026] In some embodiments, the modification is esterification modification, amidation modification, hydrophobic group modification, carboxylation modification, carbonylation modification, alkylation modification, methylation modification, glycosylation modification, hydroxylation modification, phosphorylation modification, pegylation modification, sulfation modification and / or cyclization modification.

[0027] In some embodiments, the conjugate is a radioactive compound, a metal ion, a fluorescent dye, and / or an enzyme.

[0028] The sixth aspect of the present invention provides a pharmaceutical composition, comprising the dual-target polypeptide or a pharmaceutically acceptable salt thereof according to the first aspect of the present invention, the polynucleotide according to the second aspect of the present invention, the vector according to the third aspect of the present invention, the host cell according to the fourth aspect of the present invention, and / or the polypeptide derivative according to the fifth aspect of the present invention.

[0029] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0030] In the present invention, the pharmaceutically acceptable excipients and / or vehicles are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). These substances are used to improve the stability of the drug or to enhance the activity of the active ingredient (i.e., the dual-target polypeptide of the present invention as described above or its pharmaceutically acceptable salt, polynucleotide, vector, host cell). Such substances include, but are not limited to, diluents, surfactants, humectants, binders, fillers, disintegrants, adsorption carriers, lubricants, stabilizers, bactericides, buffers, isotonic agents, chelating agents, and pH control agents. The pharmaceutical composition thus formulated can be administered by any appropriate administration method known to those skilled in the art.

[0031] In some embodiments, the pharmaceutically acceptable adjuvants and / or excipients may additionally contain liquids such as water, saline, glycerol, and ethanol.

[0032] In some embodiments, the pharmaceutical composition may further comprise other anti-heart failure drugs. The present invention has no particular limitation on the other anti-heart failure drugs, and those skilled in the art may make conventional selections according to actual needs. For example, the other anti-heart failure drugs include, but are not limited to, angiotensin-converting enzyme inhibitors (e.g., enalapril, benazepril, etc.), angiotensin II receptor antagonists (e.g., losartan, valsartan, etc.), beta-blockers (metoprolol, bisoprolol, etc.), aldosterone receptor antagonists (e.g., spironolactone, etc.), diuretics (e.g., furosemide, hydrochlorothiazide, etc.), positive inotropic drugs (e.g., digoxin, dopamine, dobutamine, etc.), sodium-glucose cotransporter 2 (SGLT2) inhibitors (e.g., dapagliflozin, empagliflozin, etc.).

[0033] The seventh aspect of the present invention provides a biological preparation, which comprises the pharmaceutical composition described in the sixth aspect of the present invention.

[0034] In some embodiments, the dosage form of the biologic comprises injection, lyophilized powder, microspheres, powder, nasal spray, powder inhaler, capsule, tablet, pill, aerosol, enteric coating, microemulsion or multiple emulsion.

[0035] Specifically, the dosage form of the biological agent includes a gastrointestinal dosage form or a parenteral dosage form, wherein the gastrointestinal dosage form includes a solution, granules, tablets, capsules, suspensions, powders, sustained-release preparations, effervescent preparations, emulsions, syrups, drops and / or chewable preparations. The parenteral dosage form includes an injection dosage form, a respiratory tract dosage form, a cavity dosage form, a mucosal dosage form and / or a dermal dosage form.

[0036] In some embodiments, the biologic can be prepared according to any method known in the art. For this purpose, the dual-target polypeptide can be combined with one or more solid or liquid excipients, if necessary, to prepare a suitable administration or dosage form for human use.

[0037] The present invention does not particularly limit the specific dosage form of the biological preparation. In some embodiments, the pharmaceutical composition of the present invention as described above can be prepared into an injectable or oral biological preparation, including but not limited to: oral liquid preparations, granules, injections, tablets, capsules, pills, powders, sustained-release preparations, suppositories, aerosols, nanoformulations, tinctures, syrups, alcoholic preparations, and lotions. Those skilled in the art can select an appropriate dosage form known in the art based on actual needs (e.g., to improve drug bioavailability, improve patient medication compliance, etc.).

[0038] In some embodiments, the dual-target polypeptides provided herein, or pharmaceutically acceptable salts thereof, pharmaceutical compositions, or biological agents, can be administered in unit dosage forms, with routes of administration including, but not limited to, enteral or parenteral administration, such as oral, intramuscular, subcutaneous, nasal, oral mucosal, dermal, peritoneal, or rectal. Dosage forms include, but are not limited to, tablets, pills, powders, granules, solutions, capsules, pellets, aerosols, suspensions, emulsions, liposomes, transdermal formulations, buccal tablets, suppositories, and lyophilized powder injections. These formulations can include conventional preparations, sustained-release preparations, controlled-release preparations, and various microparticle delivery systems.

[0039] In some embodiments, to prepare a unit dosage form containing the dual-target polypeptide into an injectable preparation, such as a solution, emulsion, lyophilized powder injection, and suspension, all diluents commonly used in the art can be used, including but not limited to: water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. Furthermore, to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose, or glycerol can be added to the injectable preparation. Furthermore, conventional cosolvents, buffers, pH adjusters, etc. can also be added.

[0040] In some embodiments, colorants, preservatives, spices, flavorings, sweeteners or other materials may be added to the biological preparations as needed.

[0041] In the present invention, the effective therapeutic and / or preventive dose of the drug, pharmaceutical composition, or biological agent can be formulated in a variety of ways based on factors such as the actual formulation method, administration method, patient age, weight, sex, condition, diet, administration time, administration route, excretion rate, and reaction sensitivity. A skilled physician can generally easily determine a prescription and a dosage that is effective for the desired treatment and / or prevention. As long as such dosage can produce the desired therapeutic and / or preventive effect on the disease or related symptoms, such dosage falls within the scope of protection of the present invention.

[0042] The eighth aspect of the present invention provides a method for preparing the dual-target polypeptide described in the first aspect of the present invention, the method comprising the following steps: culturing the host cell described in the fourth aspect of the present invention, and isolating the dual-target polypeptide described in the first aspect of the present invention from the cell culture obtained; or

[0043] Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gly-Ala-Ser-DCys-NH2 (disulfide bond position is Cys1-Dcys12), Ac-DArg(Pbf)-Arg(Pbf)-Gly-Trp(Boc)-Gly-Pro-DCys(Trt)-COOH were synthesized respectively, and the Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gl y-Ala-Ser-DCys-NH2 (disulfide bond position is Cys1-Dcys12) and Ac-DArg(Pbf)-Arg(Pbf)-Gly-Trp(Boc)-Gly-Pro-DCys(Trt)-COOH are mixed, EDCI, HOOBt, and NMM are added to react in a reaction solvent to obtain Ac-DArg(Pbf)-Arg(Pbf)-Gly-Trp(Boc)-Gly-Pro-DCys(Trt)-Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gly-Ala-Ser-DCys-NH2 (disulfide bond position is Cys1-Dcys12), and a cutting solution is added to cut the amino acid side chain protecting groups to obtain the dual-target polypeptide according to the first aspect of the present invention;

[0044] Optionally, the mixing ratio is 1:1; optionally, the amounts of EDCI, HOOBt, and NMM are 1-5 mmol, 1-5 mmol, and 0.1-3 mmol, respectively; optionally, the amounts of EDCI, HOOBt, and NMM are 3 mmol, 3 mmol, and 1 mmol, respectively; optionally, the reaction conditions for adding EDCI, HOOBt, and NMM to the reaction solvent are room temperature for 10-50 min; optionally, the reaction conditions for adding EDCI, HOOBt, and NMM to the reaction solvent are room temperature for 30 min; optionally, the cutting fluid is TFA:H2O=95%:5%;

[0045] Optionally, the synthesis of Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gly-Ala-Ser-DCys-NH2 (disulfide bond position is Cys1-Dcys12) comprises the following steps:

[0046] (1) Fmoc-DCys(Trt)-AM Resin was swelled, and then 20% Pip / DMF solution (3 times the volume of the resin) was added, nitrogen was blown, and the resin was dried to obtain H2N-DCys(Trt)-AM Resin;

[0047] (2) Fmoc-Ser(tBu)-OH amino acid, DIPEA, and HBTU were reacted with the obtained resin in a reaction solvent to obtain Fmoc-Ser(tBu)-DCys(Trt)-AM Resin, 20% Pip / DMF solution was added in an amount 3 times the volume of the resin, nitrogen was blown, and the mixture was dried to obtain H2N-Ser(tBu)-DCys(Trt)-AM Resin. After the resin was washed with the reaction solvent, Cys(Trt)-Gly-Gly-Gly-Gly-Ser(tBu)-Phe-Pro-Gly-Ala-Ser(tBu)-DCys(Trt)-AM Resin was obtained;

[0048] (3) After adding 6 times the volume of the resin-containing cutting solution, the resin was filtered, the filtrate was precipitated, and vacuum dried to obtain Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gly-Ala-Ser-DCys-NH2. The pH was adjusted to alkaline, and the mixture was stirred and naturally oxidized to obtain Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gly-Ala-Ser-DCys-NH2 (the disulfide bond position is Cys1-Dcys12);

[0049] Optionally, the amount of the Fmoc-DCys(Trt)-AM Resin is 0.1-5 mmol; Optionally, the Fmoc-DCys(Trt)-AM The amount of resin used is 1.8 mmol; optionally, the swelling condition is to use DCM to swell for 10-30 min; optionally, the swelling condition is to use DCM to swell for 20 min; optionally, the nitrogen bubbling time is 10-50 min; optionally, the nitrogen bubbling time is 30 min; optionally, the reaction solvent is DMF, DCM, NMP, HFIP and / or pyridine; optionally, the molar ratio of the Fmoc-Ser(tBu)-OH amino acid, DIPEA, HBTU, and the resulting resin is 1-5:3-9:1-5:0.1-5; optionally, the molar ratio of the Fmoc-Ser(tBu)-OH amino acid, DIPEA, HBTU, and the resulting resin is 3:6:2.85:1; optionally, the reaction condition is room temperature reaction for 10-50 min; optionally, the reaction condition is room temperature reaction for 30 min; optionally, the cutting fluid is 97.5% TFA+2.5% H2O; Optionally, the reaction time of adding the cutting fluid is 1-3h; Optionally, the reaction time of adding the cutting fluid is 2h;

[0050] Optionally, the synthesis of Ac-DArg(Pbf)-Arg(Pbf)-Gly-Trp(Boc)-Gly-Pro-DCys(Trt)-COOH comprises the following steps:

[0051] (1) Fmoc-DCys(Trt)-CTC Resin was swelled, and then 20% Pip / DMF solution (3 times the volume of the resin) was added, nitrogen was blown, and the resin was dried to obtain H2N-DCys(Trt)-CTC Resin;

[0052] (2) Fmoc-Pro-OH amino acid, DIPEA, and HBTU were reacted with the obtained resin in a reaction solvent to obtain Fmoc-Pro-DCys(Trt)-CTC Resin, 20% Pip / DMF solution was added in an amount three times the volume of the resin, nitrogen was blown, and the mixture was dried to obtain H2N-Pro-DCys(Trt)-CTC Resi n. After the resin was washed with the reaction solvent, H2N-DArg(Pbf)-Arg(Pbf)-Gly-Trp(Boc)-Gly-Pro-DCys(Trt)-CTC Resin was obtained;

[0053] (3) Adjusting the pH to alkaline, adding acetic anhydride, and acetylation modification to obtain Ac-DArg(Pbf)-Arg(Pbf)-Gly-Trp(Boc)-Gly-Pro-DCys(Trt)-CTC Resin, and then performing full protection cleavage to obtain Ac-DArg(Pbf)-Arg(Pbf)-Gly-Trp(Boc)-Gly-Pro-DCys(Trt)-COOH;

[0054] Optionally, the amount of the Fmoc-DCys(Trt)-CTC Resin is 0.1-5 mmol; Optionally, the Fmoc-DCys(Trt)-CTC The amount of resin used is 1.8 mmol; optionally, the swelling condition is to use DCM to swell for 10-30 min; optionally, the swelling condition is to use DCM to swell for 20 min; optionally, the nitrogen bubbling time is 10-50 min; optionally, the nitrogen bubbling time is 30 min; optionally, the reaction solvent is DMF, DCM, NMP, HFIP and / or pyridine; optionally, the molar ratio of the Fmoc-Pro-OH amino acid, DIPEA, HBTU, and the obtained resin is 1-5:3-9:1-5:0.1-5; optionally, the molar ratio of the Fmoc-Pro-OH amino acid, DIPEA, HBTU, and the obtained resin is 3:6:2.85:1; optionally, the reaction condition is room temperature reaction for 10-50 min; optionally, the reaction condition is room temperature reaction for 30 min; optionally, the acetic anhydride is 0.1-5 mmol; optionally, the acetic anhydride is 1.8 mmol.

[0055] The ninth aspect of the present invention provides the use of the dual-target polypeptide or pharmaceutically acceptable salt thereof described in the first aspect of the present invention, the polynucleotide described in the second aspect of the present invention, the vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention and / or the polypeptide derivative described in the fifth aspect of the present invention in the preparation of a medicament for treating and / or preventing heart failure with preserved ejection fraction, heart failure with no preserved ejection fraction, myocardial fibrosis caused by heart failure with preserved ejection fraction, myocardial fibrosis caused by heart failure with no preserved ejection fraction, cardiac hypertrophy caused by heart failure with preserved ejection fraction and / or cardiac hypertrophy caused by heart failure with no preserved ejection fraction.

[0056] Furthermore, the heart failure with preserved ejection fraction is diastolic dysfunction caused by diabetes, hypertension, hyperlipidemia, chronic renal failure, metabolic syndrome, obesity, coronary artery disease, hypertrophic cardiomyopathy, infiltrative cardiomyopathy, pulmonary hypertension, valvular heart disease and / or atrial fibrillation;

[0057] The non-ejection fraction preserved heart failure is a cardiac systolic dysfunction caused by coronary heart disease, hypertension, dilated cardiomyopathy, hypertrophic cardiomyopathy, valvular heart disease, myocarditis, congenital heart disease, ventricular tachycardia, atrial fibrillation and / or hyperthyroidism.

[0058] In the present invention, the heart failure with preserved ejection fraction or the heart failure with non-preserved ejection fraction is not limited to the heart failure with preserved ejection fraction or the heart failure with non-preserved ejection fraction caused by the diseases or causes as mentioned above. The heart failure with preserved ejection fraction or the heart failure with non-preserved ejection fraction and related diseases caused by any disease or cause fall within the scope of protection of the present invention.

[0059] In the present invention, the treatment and / or prevention refers to delaying the development of a disease, preventing the development of a disease, and / or reducing the severity of the symptoms that will develop or are expected to develop. Therefore, these terms include improving existing disease symptoms, preventing additional symptoms, improving or preventing potential metabolic causes of symptoms, inhibiting the disorder or disease, for example, preventing the development of the disorder or disease, alleviating the disorder or disease, regressing the disorder or disease, alleviating the symptoms caused by the disease or disorder, or stopping the symptoms of the disease or disorder. In a specific embodiment of the present invention, the disease is heart failure with preserved ejection fraction, heart failure without preserved ejection fraction, and related diseases.

[0060] It should be noted that the use of the dual-target polypeptide or pharmaceutically acceptable salt thereof described in the first aspect of the present invention, the polynucleotide described in the second aspect of the present invention, the vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention and / or the polypeptide derivative described in the fifth aspect of the present invention in increasing the BNP content in plasma or increasing the cGMP content in cardiac tissue also falls within the scope of protection of the present invention.

[0061] In addition, the present invention also provides a method for treating and / or preventing heart failure with preserved ejection fraction or heart failure without preserved ejection fraction, the method comprising the following steps: administering to a subject in need thereof a therapeutically and / or preventive effective amount of the dual-target polypeptide or a pharmaceutically acceptable salt thereof described in the first aspect of the present invention, the polynucleotide described in the second aspect of the present invention, the vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, the polypeptide derivative described in the fifth aspect of the present invention, the pharmaceutical composition described in the sixth aspect of the present invention and / or the biological preparation described in the seventh aspect of the present invention.

[0062] In some embodiments, the subject includes mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds and fish. In one embodiment of the present invention, the subject is a human. The term subject includes confirmed patients.

[0063] In some embodiments, the routes of administration include, but are not limited to, oral, topical, intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal, intranasal, intrapulmonary, intrarectal, etc. In specific embodiments, a clinician may determine a dosage that is beneficial to the subject based on factors such as the type, age, weight, general disease condition, and mode of administration of the subject.

[0064] In some embodiments, the effective amount refers to the amount of the aforementioned drug, pharmaceutical composition, or biologic that effectively produces the desired preventive, allergic, or therapeutic effect. The amount of the drug, pharmaceutical composition, or biologic described herein that achieves an effective amount will vary depending on factors such as the active ingredient (in the present invention, particularly the aforementioned dual-target polypeptide or its pharmaceutically acceptable salt, pharmaceutical composition, or biologic), the symptoms and their severity, and the age of the treated mammal. However, the specific amount can be routinely determined by a person of ordinary skill in the art based on their knowledge in the art and the disclosure of this invention. Doses that can produce the aforementioned effects are within the scope of this invention.

[0065] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0066] (1) The present invention discloses for the first time a novel dual-target inhibitory peptide of Neprilysin and PDE9A, and experimentally confirms that the dual-target peptide can be used for the effective treatment of heart failure with preserved ejection fraction and heart failure without preserved ejection fraction, and has good pharmacological effects against heart failure with preserved ejection fraction and heart failure without preserved ejection fraction, as shown by the ability to significantly increase endogenous BNP levels, reduce cardiac preload, increase cGMP content in plasma and myocardial tissue, improve energy supply to myocardial cells, inhibit myocardial fibrosis, and improve cardiac diastolic or systolic dysfunction.

[0067] (2) This invention, for the first time, integrates polypeptides with neprilysin and PDE9A inhibitory effects into a single polypeptide molecule. By synergistically regulating plasma BNP and cardiac cGMP levels, it achieves comprehensive pathological coverage of both heart failure with preserved ejection fraction and heart failure without preserved ejection fraction, breaking through the technical bottlenecks of the existing technology, which are limited to a single drug target and limited indications. This invention provides a new approach and strategy for the research and development of drugs for the treatment and / or prevention of heart failure, and has promising clinical application prospects and important translational significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 This is a schematic diagram of the structure of the neprilysin and PDE9A dual-targeting polypeptide described in Example 1 of the present invention;

[0069] Figure 2This is the HPLC spectrum of the dual-target polypeptide in Example 1 of the present invention;

[0070] Figure 3 This is the MS spectrum of the dual-target peptide in Example 1 of the present invention;

[0071] Figure 4 These are the results of cardiac ultrasound and cardiac function evaluation of HFpEF model mice treated with the dual-target polypeptide in Example 2 of the present invention, wherein: Figure A: M-mode echocardiography for detecting cardiac contractile function; Figure B: Statistical graph of left ventricular ejection fraction by cardiac ultrasound; Figure C: Statistical graph of left ventricular fractional shortening by cardiac ultrasound; Figure D: diastolic function detected by tissue Doppler echocardiography; Figure E: Ratio of mitral early diastolic peak velocity (E) to late diastolic peak velocity (A); Figure F: Ratio of mitral early diastolic peak velocity (E) to mitral annular early diastolic peak velocity (E');

[0072] Figure 5 This is a graph showing the results of treating plasma BNP levels in HFpEF model mice with the dual-target peptide in Example 3 of the present invention;

[0073] Figure 6 This is a graph showing the results of treating cGMP content in myocardial tissue of HFpEF model mice with the dual-target peptide in Example 3 of the present invention;

[0074] Figure 7 This is a diagram showing the results of Masson and Sirius red staining of cardiac fibrosis in HFpEF model mice treated with the dual-target peptide in Example 4 of the present invention;

[0075] Figure 8 This is a graph showing the expression of cardiac fibrosis-related proteins in HFpEF model mice treated with the dual-target peptide in Example 5 of the present invention;

[0076] Figure 9 These are the results of cardiac ultrasound and cardiac function evaluation of HFrEF model mice treated with the dual-target polypeptide in Example 6 of the present invention, wherein: Figure A: M-mode echocardiography for detecting cardiac contractile function; Figure B: Statistical graph of left ventricular ejection fraction by cardiac ultrasound; Figure C: Statistical graph of left ventricular fractional shortening by cardiac ultrasound;

[0077] Figure 10 This is a graph showing the results of treating plasma BNP levels in HFrEF model mice with the dual-target peptide in Example 7 of the present invention;

[0078] Figure 11 This is a graph showing the results of treating cGMP content in myocardial tissue of HFrEF model mice with the dual-target peptide in Example 7 of the present invention;

[0079] Figure 12These are Masson staining results of cardiac fibrosis in HFrEF model mice treated with the dual-target polypeptide in Example 8 of the present invention, wherein: Figure A: Masson staining of mouse heart tissue; Figure B: Statistical graph of cardiac fibrosis area percentage;

[0080] Figure 13 This is a graph showing the expression of cardiac fibrosis-related proteins in HFrEF model mice treated with the dual-target peptide in Example 9 of the present invention. DETAILED DESCRIPTION

[0081] The present invention will be further described below in conjunction with specific examples. The following specific examples are only used to explain the present invention and are not to be construed as limiting the present invention. Those of ordinary skill in the art will appreciate that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental consumables, reagents and raw materials used in the present invention are readily available to those of ordinary skill in the art. Unless otherwise specified, they can be obtained commercially. The experimental methods for which specific conditions are not specified in the present invention are typically tested under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

[0082] Example 1 PDE9A / Neprilysin Dual-Target Peptide

[0083] The PDE9A / Neprilysin dual-target polypeptide is a dual-functional inhibitory polypeptide targeting both PDE9A and Neprilysin. The corresponding structural diagram is shown in FIG. Figure 1 The PDE9A / Neprilysin dual-target peptide sequence is Ac-DArg-Arg-Gly-Trp-Gly-Pro-DCys-Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gly-Ala-Ser-DCys-NH2 (SEQ ID NO: 1). The disulfide bond is located at Cys8-DCys19; DArg and DCys in the sequence are D-amino acids, while the rest are L-amino acids. Both ends of the sequence are capped: Ac represents acetylation (CH3CO-) and NH2 represents amino groups. Cys8-DCys19 refers to the disulfide bond formed between cysteine ​​at position 8 (Cys8) and D-cysteine ​​at position 19 (DCys19).

[0084] The specific synthesis steps of the PDE9A / Neprilysin dual-target polypeptide are as follows:

[0085] (1) Take 1.8 mmol of Fmoc-DCys(Trt)-AM Resin (Peptide Library Biotechnology, Product No.: TK6032) and swell it with DCM (dichloromethane) for 20 minutes.

[0086] (2) Add 3 times the resin volume of a 20% Pip / DMF solution (a 20% by mass solution of piperidine (Pip) dissolved in dimethylformamide (DMF)), purge with nitrogen for 30 minutes, and drain to obtain H2N-DCys(Trt)-AM Resin (Fmoc group removed).

[0087] (3) Wash the resin from the previous step five times with DMF twice the volume of the resin (to wash the resin and remove the residual solvent in preparation for the next reaction).

[0088] (4) Take 1.8 mmol of Fmoc-Ser(tBu)-OH amino acid (N-(9-fluorenylmethoxycarbonyl)-O-tert-butyl-L-serine), 3.6 mmol of DIPEA (N,N-diisopropylethylamine), and 1.71 mmol of HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate). React with 2 times the volume of resin in DMF (dimethylformamide) at room temperature for 30 minutes. Fmoc-Ser(tBu)-DCys(Trt)-AM Resin is obtained. Amino acid: DIPEA: HBTU:

[0089] Resin = 3:6:2.85:1 (molar ratio).

[0090] (5) Wash the resin from the previous step three times with DMF twice the volume of the resin (to wash the resin and remove the residual solvent and prepare for the next reaction).

[0091] (6) Add 20% Pip / DMF solution 3 times the volume of the resin, purge with nitrogen for 30 minutes, and drain to obtain H2N-Ser(tBu)-DCys(Trt)-AM Resin (Fmoc group removed).

[0092] (7) Detection: Drain the piperidine solution, take a dozen resin pellets, wash them three times with ethanol, add one drop each of ninhydrin, KCN, and phenol solution, heat at 105℃-110℃ for 5 minutes, and a dark blue color indicates a positive reaction.

[0093] (8) Wash the resin from the previous step five times with DMF twice the volume of the resin (to wash the resin and remove the residual solvent and prepare for the next reaction).

[0094] (9) Repeat steps (4), (5), (6), (7), and (8) to obtain Cys(Trt)-Gly-Gly-Gly-Gly-Ser(tBu)-Phe-Pro-Gly-Ala-Ser(tBu)-DCys(Trt)-AM Resin.

[0095] (10) Wash the resin with methanol three times and drain the resin (in preparation for cleavage).

[0096] (11) Cutting: Add 6 times the volume of resin to the cutting solution (97.5% TFA + 2.5% H2O) and shake on a shaker for 2 hours. Filter out the resin, precipitate the filtrate with anhydrous ether, and wash the precipitate three times with anhydrous ether. Finally, place the precipitate in a vacuum drying kettle and dry it at room temperature for 24 hours to obtain the crude product Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gly-Ala-Ser-DCys-NH2.

[0097] (12) Sodium bicarbonate (NaHCO3) was added to adjust the pH to alkaline, and the mixture was stirred and naturally oxidized to form a ring to obtain Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gly-Ala-Ser-DCys-NH2 (Disulfide Bridge: C1-c12, i.e., a disulfide bond was formed between the cysteine ​​at positions 1 and 12).

[0098] (13) Purify the crude product to obtain Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gly-Ala-Ser-DCys-NH2 (Disul fide Bridge: C1-c12) of higher purity for use.

[0099] (14) Take Fmoc-DCys(Trt)-CTC Resin (Peptide Library Biotechnology, catalog number: TK5479) and swell it with DCM for 20 minutes.

[0100] (15) Add 20% Pip / DMF solution 3 times the volume of the resin, purge with nitrogen for 30 minutes, and drain to obtain H2N-DCys(Trt)-CTC Resin (Fmoc group removed).

[0101] (16) Wash the resin from the previous step five times with DMF twice the volume of the resin (to wash the resin and remove the residual solvent and prepare for the next reaction).

[0102] (17) 1.8 mmol of Fmoc-Pro-OH amino acid, 3.6 mmol of DIPEA, and 1.71 mmol of HBTU were reacted with 2 times the resin volume of DMF for 30 minutes to obtain Fmoc-Pro-DCys(Trt)-CTC resin. The molar ratio of amino acid: DIPEA: HBTU: resin was 3:6:2.85:1.

[0103] (18) Wash the resin from the previous step three times with DMF twice the volume of the resin (to wash the resin and remove the residual solvent and prepare for the next reaction).

[0104] (19) Add 20% Pip / DMF solution 3 times the volume of the resin, purge with nitrogen for 30 minutes, and drain to obtain H2N-Pro-DCys(Trt)-CTC Resin (Fmoc group removed).

[0105] (20) Detection: Drain the piperidine solution, take a dozen resin pellets, wash them three times with ethanol, add one drop each of ninhydrin, KCN, and phenol solution, heat at 105-110°C for 5 min, and a dark blue color indicates a positive reaction.

[0106] (21) Wash the resin from the previous step five times with DMF twice the volume of the resin (to wash the resin and remove the residual solvent and prepare for the next reaction).

[0107] (22) Repeat steps (17)-(21) to obtain H2N-DArg(Pbf)-Arg(Pbf)-Gly-Trp(Boc)-Gly-Pro-DCys(Trt)-CTC Resin.

[0108] (23) DIPEA was added to adjust the pH to alkaline, and 1.8 mmol of acetic anhydride was added. The mixture was reacted at room temperature for 30 min to obtain Ac-DArg(Pbf)-Arg(Pbf)-Gly-Trp(Boc)-Gly-Pro-DCys(Trt)-CTC resin through acetylation.

[0109] (24) Perform full protection cleavage and prepare a 0.5% TFA / DCM solution with a volume of about 3 times the volume of the resin. Wash the resin twice with DCM (to remove residual DMF), then pour the prepared solution into the reactor and react for 30 minutes. Filter the resin and collect the filtrate (the polypeptide has been separated from the resin and is present in the filtrate). Add DIEPA to the filtrate and adjust the pH to 7-8. Wash the filtrate with saturated NaHCO3 to separate the DCM layer solution. The DCM layer solution can be properly rotary evaporated to reduce the organic solvent. Add 1 or 2 times the volume of ethyl acetate again, adjust the pH to slightly acidic with dilute HCl solution, and extract the polypeptide from the DCM layer to the ethyl acetate layer. Wash the ethyl acetate layer twice with saturated NaCl. Absorb the moisture of the ethyl acetate layer with anhydrous magnesium sulfate. Use vacuum rotary evaporation to completely evaporate the ethyl acetate to obtain a crystalline solid polypeptide for the next C-terminal reaction. Alternatively, retain an appropriate volume of ethyl acetate solution by vacuum rotary evaporation, add ice ether to precipitate the polypeptide, and then dry the polypeptide for the next C-terminal reaction. Ac-DArg(Pbf)-Arg(Pbf)-Gly-Trp(Boc)-Gly-Pro-DCys(Trt)-COOH was obtained.

[0110] (25) Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gly-Ala-Ser-DCys-NH2 (DisulfideBridge: C1-c12) and Ac-DArg(Pbf)-Arg(Pbf)-Gly-Trp(Boc)-Gly-Pro-DCys(Trt)-COOH were directly mixed in a 1:1 ratio, and 3 mmol EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride), 3 mmol HOOBt (3-hydroxy-1,2,3-benzotriazine-4(3H)-one), and 1 mmol NMM (N-methylmorpholine) was reacted in DMF at room temperature for 30 minutes to obtain Ac-DArg(Pbf)-Arg(Pbf)-Gly-Trp(Boc)-Gly-Pro-DCys(Trt)-Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gly-Ala-Ser-DCys-NH2 (Disulfide Bridge: C1-c12).

[0111] (26) After adding a cutting solution (TFA:H2O=95%:5%) to cut the amino acid side chain protecting group, the target product was obtained: Ac-DArg-Arg-Gly-Trp-Gly-Pro-DCys-Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gly-Ala-Ser-DCys-NH2(Cys8-19) crude product.

[0112] (27) The target product with a purity greater than 95% was obtained by purification, and the molecular weight was verified by mass spectrometry. After freeze-drying, 1 mg was taken for re-dissolution, and the HPLC and MS tests were both correct and qualified. Figure 2 、 Figure 3 ).

[0113] Example 2 Effects of PDE9A / Neprilysin Dual-Target Peptide on Cardiac Diastolic Function in Mice with Heart Failure with Preserved Ejection Fraction (HFpEF)

[0114] 1. Experimental Materials

[0115] Experimental animals: Six-week-old male Lepr-WT and Lepr-KO mice were purchased from Jiangsu Jicui Pharmaceutical. Experimental instruments included a Vevo 3100 small animal ultrasound system and a small animal gas anesthesia machine. Experimental consumables included a 60% high-fat diet (Research Diet, D12492) and isoflurane (Rayward, R510-22-10).

[0116] 2. Experimental methods

[0117] (1) HFpEF model: 6-week-old male Lepr-WT mice served as the normal control group, and 6-week-old male Lepr-KO mice were divided into a model group, a dual-target peptide 1 mg / kg group, and a dual-target peptide 3 mg / kg group. The model group was modeled using the following literature: Myeloid Cell Derived IL1βContributes to Pulmonary Hypertensionin HFpEF. Circ Res. 2023; 133(11): 885-898. doi: 10.1161 / CIRCRESAHA.123.323119. Specifically, a high-fat diet (the feed product used in animal experiments was from Research Diet, product number D12492) was used to induce heart failure with preserved ejection fraction to obtain a HFpEF animal model. The systolic function parameter LVEF (left ventricular ejection fraction) of the mice was detected 8 weeks after model induction, and no changes were found. However, the diastolic function parameter (E / E') increased significantly at the fifth week of model induction, indicating that the heart failure model with preserved ejection fraction described in the aforementioned literature had been successfully obtained. At the same time, there was no significant difference in the diastolic function parameter (E / E') between the model group and the treatment groups at 8 weeks of induction. Subsequent drug administration was carried out under the same baseline conditions, and the cardiac systolic and diastolic functions were evaluated 4 weeks after administration.

[0118] (2) Cardiac ultrasound: In this experiment, transthoracic echocardiography was performed using a VisualVevo 3100 system equipped with an MS400 transducer (Visual Sonics). Anesthesia was induced using 2.5% isoflurane, and the anesthetic effect was determined by the lack of response to light pressure on the mouse's hind paw. After anesthesia, a short-axis M-mode scan was performed at the mid-ventricular level marked by the location of the papillary muscle to obtain the left ventricular ejection fraction (LVEF) and other related indicators of contractile function. For anesthetized mice, an apical four-chamber view was obtained, and diastolic function was measured at the level of the mitral valve using pulse wave and tissue Doppler imaging techniques. During the acquisition of echocardiographic data (maintaining constant body temperature), the isoflurane concentration was reduced to 1.0-1.5% and adjusted according to actual conditions to maintain the mouse heart rate at around 500 beats per minute. After the experiment, all mice successfully recovered from anesthesia without any abnormalities. Each parameter was measured at least 3 times, and the final data was averaged. This ultrasound test covers both systolic function and diastolic function testing.

[0119] 3. Experimental results

[0120] The results are as follows Figure 4 As shown in Figures AC, the systolic function (LVEF, LVFS) of the mice in each group did not change significantly after modeling. In the model control group mice that were not treated with the PDE9A / NEP dual-targeting peptide, an increase in the E / E' ratio and a deterioration in the diastolic function were observed. After treatment with the PDE9A / NEP dual-targeting peptide, the diastolic function of HFpEF mice was significantly improved ( Figure 4 The high-dose treatment group showed a better therapeutic effect, indicating that the PDE9A / NEP dual-targeting peptide can have both preventive and therapeutic effects on HFpEF in the mouse HFpEF model.

[0121] Example 3 Effects of PDE9A / Neprilysin Dual-Target Peptide on Plasma BNP and Cardiac Tissue cGMP Levels in HFpEF Mice

[0122] 1. Experimental Materials

[0123] Experimental instruments: Multifunctional microplate reader (Thermo Fisher, K3). Reagents and consumables: Mouse BNP Elisa detection kit (Wuhan Clone Cloud, SEA541Mu), Mouse cGMP Elisa detection kit (Wuhan Clone Cloud, MEA577Ge).

[0124] 2. Experimental methods

[0125] After anesthesia, mice in each group were placed in a supine position, their chest hair was removed and disinfected, the apex of the heart was exposed, and a needle was inserted into the heart at an angle of 25-30°. 0.5-1 mL of blood was collected and placed in an EDTA anticoagulant tube. After centrifugation at 3500 rpm for 10 minutes, plasma was separated and used for plasma BNP content detection. After blood collection, the mice were sacrificed by cervical dislocation and quickly fixed on the operating table. The chest skin was cut open with surgical scissors to fully expose the heart and lungs. The heart was cut from the aortic root and removed. According to the cGMP test kit requirements, 30 mg of tissue blocks were taken and washed in pre-cooled PBS (0.01 mol / L, pH = 7.0-7.2) to remove blood. The heart tissue was cut into small pieces and evenly placed in a glass homogenizer placed on ice with fresh lysis buffer. The resulting suspension was sonicated until clarified. Finally, the prepared homogenate was centrifuged at 10,000 × g for 5 minutes, the pellet was discarded, and the supernatant was used to detect cGMP content in myocardial tissue.

[0126] (1) The BNP content detection method is as follows: add samples: set up standard wells, test sample wells, and blank wells respectively. Set up 7 standard wells, and add 100 μL of standard samples of different concentrations in sequence. Add 100 μL of standard dilution solution to the blank well, and add 100 μL of the test sample to the remaining wells. Cover the ELISA plate with a film and incubate at 37°C for 1 hour. Discard the liquid, shake dry, and do not wash. Add 100 μL of detection solution A working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 1 hour. Discard the liquid in the well, wash each well with 350 μL of washing solution, soak, and tap the ELISA plate on absorbent paper to remove all liquid in the well. Repeat the plate washing 3 times. After the last wash, aspirate or pour out the remaining washing buffer, turn the ELISA plate upside down on absorbent paper, and absorb all the liquid remaining in the well. Add 100 μL of detection solution B working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 30 minutes. Discard the liquid in the wells, spin dry, and wash the plate. Add 90 μL of TMB substrate solution to each well, cover the plate with film, and develop at 37°C in the dark. Add 50 μL of stop solution to each well to terminate the reaction; the blue color will immediately turn yellow. The stop solution should be added in the same order as the substrate solution. After ensuring that there are no water droplets on the bottom of the plate and no bubbles in the wells, immediately measure the optical density (OD) of each well at a wavelength of 450 nm using a microplate reader.

[0127] (2) The cGMP content detection method is as follows: add samples: set up standard wells, test sample wells, and blank wells respectively. Set up 5 standard wells, add 50μL of standard samples of different concentrations in sequence (blank wells add 50μL of standard diluent), add 50μL of test sample to the remaining wells, and then immediately add 50μL of detection solution A working solution to each well, cover the ELISA plate with a film, and incubate at 37℃ for 1 hour. Discard the liquid in the well, wash each well with 350μL of washing solution, soak, and tap the ELISA plate on absorbent paper to remove all liquid in the well. Repeat the plate wash 3 times. After the last wash, absorb or pour out the remaining washing buffer, turn the ELISA plate upside down on absorbent paper, and absorb all the liquid remaining in the well. Add 100μL of detection solution B working solution to each well, cover with a film, and incubate at 37℃ for 30 minutes. Discard the liquid in the well, spin dry, and wash the plate. Add 90 μL of substrate solution to each well, cover the plate with film, and develop at 37°C in the dark. Terminate the reaction by adding 50 μL of stop solution to each well, which will immediately turn the blue color to yellow. Try to add the stop solution in the same order as the substrate solution. After ensuring there are no water droplets on the bottom of the plate and no bubbles in the wells, immediately measure the optical density (OD) of each well using a microplate reader at a wavelength of 450 nm.

[0128] 3. Experimental results

[0129] The results are as follows Figure 5-6 As shown in Figure 2, after treatment with PDE9A / NEP dual-targeting peptide, the plasma BNP level of HFpEF mice ( Figure 7 ) and cardiac tissue cGMP( Figure 8 This indicates that the PDE9A / NEP dual-targeting polypeptide described in Example 1 can improve HFpEF by increasing the levels of BNP in mouse plasma and cGMP in cardiac tissue.

[0130] Example 4 Effects of PDE9A / Neprilysin Dual-Targeting Peptide on Myocardial Fibrosis in HFpEF Mice

[0131] 1. Experimental Materials

[0132] Experimental instruments: embedding machine (JB-P5, Junjie Electronics Co., Ltd., Wuhan), pathological slicer (RM2016, Leica Instrument Co., Ltd., Shanghai), tissue slide spreader (KD-P, Kedi Instrument Equipment Co., Ltd., Jinhua City, Zhejiang Province), upright optical microscope (NIKON ECLIPSE E100, Japan). Experimental reagents: anhydrous ethanol, xylene (Sinopharm Group), universal tissue fixative (G1128, Wuhan Sevier), hematoxylin and eosin (H&E) high-definition constant staining kit (G1076, Wuhan Sevier), Sirius red staining kit (G1078, Wuhan Sevier), Masson trichrome staining solution (G1006, Wuhan Sevier).

[0133] 2. Experimental methods

[0134] Mice from each group were sacrificed by cervical dislocation after cardiac blood collection. The mice were quickly secured on an operating table. The chest skin was cut open with surgical scissors to fully expose the heart and lungs. The heart was then removed by scissoring at the root of the aorta. The heart was rinsed three times and weighed after rinsing for subsequent Sirius Red and Masson staining.

[0135] (1) Sirius red staining: The heart was fixed with tissue fixative, treated with different concentrations of gradient alcohol, and embedded in paraffin. The embedded paraffin blocks were frozen at 4°C for final shape. The paraffin blocks were cut into 4-5 μm paraffin sections using a blade cutter. The sections were dewaxed in xylene I for 5 minutes, then in xylene II for 5 minutes, then in xylene III for 5 minutes, then in anhydrous ethanol for 1 minute, then in 95% ethanol for 1 minute, then in 75% ethanol for 1 minute, and finally washed with distilled water for 5 minutes. Add iron hematoxylin staining solution to the sections for 5-10 minutes, then wash with distilled water for 10-20 seconds to remove excess staining solution. Wash the sections with tap water for 5 minutes. Add Sirius red staining solution to the sections for 15-30 minutes. The sections were dehydrated in 75% ethanol for 1 minute, then in 95% ethanol for 1 minute, then in anhydrous ethanol for 1 minute, and then in xylene for 3 times, each time for 1-2 minutes, and finally sealed with neutral gum.

[0136] (2) Masson staining: Prepare sections from the paraffin blocks, stain them in a hematoxylin aqueous solution for 15 minutes, differentiate them in hydrochloric acid and ethanol for a few seconds, reverse blue with ammonia water for 2 minutes, rinse with water and stain with Masson's fuchsin solution for 10 minutes, soak in a 2% glacial acetic acid aqueous solution, differentiate them in a 1% phosphomolybdic acid aqueous solution for 5 minutes, stain with aniline blue for 5 minutes, rinse with 0.2% glacial acetic acid, dehydrate with 95% ethanol and anhydrous ethanol, make them transparent with xylene, add neutral gum to the slides and seal them.

[0137] 3. Experimental results

[0138] The results are as follows Figure 7 As shown, significant fibrotic pathological changes occurred in the hearts of HFpEF mice. After treatment with PDE9A / NEP dual-target peptides, the cardiac fibrosis of HFpEF mice was significantly improved, and the percentage of fibrosis area was reduced, which was more significant in the high-dose treatment group.

[0139] Example 5 Effect of PDE9A / Neprilysin Dual-Target Peptide on Expression of Myocardial Fibrosis-Related Proteins in HFpEF Mice

[0140] 1. Experimental Materials

[0141] Experimental instruments: Bio-Rad electrophoresis instrument (165-8001), Bio-Red protein gel imaging analyzer (GelDoc XR+). Experimental reagents: 10% Tris SwePAGE precast gel (Wuhan Seville, G2302-10), protein marker (Wuhan Seville, G26616), 5× Tris-glycine SDS-PAGE running buffer (Wuhan Seville, G2162), 10× transfer buffer (Wuhan Seville, G2028), blocking buffer (Wuhan Seville, G2052), β-actin antibody (Proteintech, 66009-1-Ig), α-SMA (Proteintech, 14395-1-AP), Collagen I (Proteintech, 67288-1-Ig), Collagen III (Proteintech, 22734-1-AP).

[0142] 2. Experimental methods

[0143] Wash twice with pre-chilled PBS, transfer to a 1.5 mL centrifuge tube, and add 200 μL of the above protein extraction and lysis buffer. Lyse on ice for 30 minutes, then centrifuge at 12,000 rpm at 4°C for 30 minutes. Aspirate the supernatant, quantify protein using the BCA assay, and adjust the protein concentration to the same level. Add 5× SDS gel loading buffer, mix well, and denature at 98°C for 10 minutes. Perform SDS-polyacrylamide gel electrophoresis on a portion of the protein sample.

[0144] 3. Experimental results

[0145] The results are as follows Figure 8 As shown, the expression of fibrosis-related proteins Collagen I, Collagen III, and α-SMA in the heart of HFpEF mice was significantly upregulated. After treatment with the PDE9A / NEP dual-targeting peptide, the expression of fibrosis-related proteins decreased. This indicates that the PDE9A / NEP dual-targeting peptide can inhibit cardiac fibrosis in HFpEF mice.

[0146] Example 6 Effects of PDE9A / Neprilysin Dual-Target Peptide on Cardiac Contractile Function in Mice with Heart Failure with Preserved Ejection Fraction (HFrEF)

[0147] 1. Experimental Materials

[0148] Experimental animals: 8-week-old male C57BL / 6J mice, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Experimental instruments: Vevo 3100 small animal ultrasound system, small animal gas anesthesia machine. Experimental consumables: isoflurane (Rayward, R510-22-10).

[0149] 2. Experimental methods

[0150] (1) HFrEF model: 8-week-old male C57BL / 6J mice were divided into a control group, a HFrEF model group, a dual-target peptide 1 mg / kg treatment group, and a dual-target peptide 3 mg / kg treatment group. Specifically, 8-week-old male C57BL / 6J mice were used to induce heart failure with reduced ejection fraction by ligating the left anterior descending artery of the heart. After modeling, cardiac ultrasound showed that LVEF was <60%, indicating that the HFrEF animal model had been successfully obtained. The control group mice underwent sham surgery. Subsequent dual-target peptide administration was performed under the same baseline conditions, and cardiac contractile function was evaluated 4 weeks after administration. The specific modeling method is as follows: After the anesthesia of the mouse takes effect, the mouse is placed in a supine position on a fixed board, and the upper incisor is fixed to the edge of the mouse board. The four limbs of the mouse are punctured with pins to connect to the ECG leads (right upper limb: green; left lower limb: red; right lower limb: black) for ECG monitoring. After the ECG stabilizes for 5-10 minutes, a normal ECG is recorded as a control. After shaving and disinfecting the midline of the mouse neck, a small 10 mm midline incision was made along the anatomical direction of the trachea. The fascia was bluntly dissected, the thyroid gland was freed, and the anterior cervical muscles were separated. The trachea was isolated and exposed. An inverted T-shaped incision was made in the trachea using ophthalmic scissors. A suture was threaded under the trachea, and a catheter was carefully inserted along the trachea and secured with a ligature. The tracheal catheter was connected to an animal ventilator for positive pressure ventilation (respiratory rate: 75 breaths / min, ventilation volume 100-120 mL, expiratory:inspiratory ratio 3:1). After observing that the mouse's breathing and electrocardiogram were stable, the left anterior chest was shaved and disinfected. A longitudinal incision was made 2 mm to the left of the sternum or one finger width from the axilla. The fascia and muscles were separated, and the ribs were exposed. The intercostal muscles were dissected downwards in the fourth intercostal space using a blunt dissector. A suture was threaded under the ribs for later use. The ends of the ribs were ligated, and the fourth rib was severed. The ligatures were secured with hemostats, and the thorax was opened to expose the thoracic cavity. The pericardium was removed to expose the heart. The left atrial appendage and pulmonary artery cone can be used as landmarks to determine the position of the left anterior descending coronary artery. The insertion point of the 5-0 suture is 3-4 mm below the lower edge of the left atrial appendage, and the exit point is obliquely to the upper right pulmonary artery cone. The insertion depth is about 1.5 mm, the needle distance is about 3-4 mm, and the left anterior descending branch is ligated with a cannula 2-3 mm away from the aortic root. Observation of the electrocardiogram shows ST segment elevation and a large and widened QRS wave, indicating successful ligation. In the control group, the suture was passed under the left anterior descending branch but no ligation was performed.

[0151] (2) Cardiac ultrasound: as in the aforementioned embodiment 2.

[0152] 3. Experimental results

[0153] The results are as follows Figure 9As shown in AC, in the model group mice that were not treated with the PDE9A / NEP dual-targeting peptide, a significant decrease in contractile function was observed. After treatment with the PDE9A / NEP dual-targeting peptide, the contractile function of HFpEF mice was significantly improved and the left ventricular ejection fraction was increased, indicating that the PDE9A / NEP dual-targeting peptide can play a therapeutic role in the HFrEF mouse model.

[0154] Example 7 Effects of PDE9A / Neprilysin Dual-Target Peptide on Plasma BNP and Cardiac Tissue cGMP Levels in HFrEF Mice

[0155] 1. Experimental Materials

[0156] The experimental materials are the same as those in Example 3 above.

[0157] 2. Experimental methods

[0158] The detection method is the same as that in Example 3 above.

[0159] 3. Experimental results

[0160] The results are as follows Figure 10-11 As shown in Figure 2, after treatment with PDE9A / NEP dual-targeting peptide, the plasma BNP level of HFrEF mice ( Figure 10 ) and cardiac tissue cGMP( Figure 11 This indicates that the PDE9A / NEP dual-target peptide can increase the plasma BNP level and cardiac tissue cGMP level in HFrEF mice.

[0161] Example 8 Effects of PDE9A / Neprilysin Dual-Targeting Peptide on Myocardial Fibrosis in HFrEF Mice

[0162] 1. Experimental Materials

[0163] The experimental materials are the same as those in Example 4 above.

[0164] 2. Experimental methods

[0165] The same detection method as in Example 4 above.

[0166] 3. Experimental results

[0167] The results are as follows Figure 12 As shown in Figures AB, after treatment with the PDE9A / NEP dual-targeting peptide, the area of ​​fibrosis in HFrEF mice was reduced, and cardiac fibrosis was significantly improved. This indicates that the PDE9A / NEP dual-targeting peptide can inhibit myocardial fibrosis in HFrEF mice.

[0168] Example 9 Effect of PDE9A / Neprilysin Dual-Target Peptide on Expression of Myocardial Fibrosis-Related Proteins in HFrEF Mice

[0169] 1. Experimental Materials

[0170] The experimental materials are the same as those in Example 5 above.

[0171] 2. Experimental methods

[0172] The detection method is the same as that in Example 5 above.

[0173] 3. Experimental results

[0174] The results are as follows Figure 13 As shown, the expression of fibrosis-related proteins Collagen I, Collagen III, and α-SMA in the heart of HFrEF mice was significantly upregulated. After treatment with the PDE9A / NEP dual-targeting peptide, the expression of fibrosis-related proteins decreased. This indicates that the PDE9A / NEP dual-targeting peptide can inhibit cardiac fibrosis in HFrEF mice.

Claims

1. A dual-target polypeptide targeting PDE9A and Neprilysin or a pharmaceutically acceptable salt thereof, characterized in that: The sequence of the dual-target polypeptide is Ac-DArg-Arg-Gly-Trp-Gly-Pro-DCys-Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gly-Ala-Ser-DCys-NH2, wherein the disulfide bond position is Cys8-DCys19.

2. A polynucleotide, characterized in that The polynucleotide encodes the dual-target polypeptide according to claim 1.

3. A carrier, characterized in that The vector comprises the polynucleotide according to claim 2.

4. A host cell, characterized in that The host cell comprises the vector according to claim 3.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the dual-target polypeptide or a pharmaceutically acceptable salt thereof according to claim 1, the polynucleotide according to claim 2, the vector according to claim 3 and / or the host cell according to claim 4.

6. A biological agent, characterized in that The biological preparation comprises the pharmaceutical composition according to claim 5.

7. A method for preparing the dual-target polypeptide according to claim 1, characterized in that: The method comprises the following steps: culturing the host cell according to claim 4, isolating the dual-target polypeptide according to claim 1 from the cell culture obtained by culturing; or respectively synthesizing Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gly-Ala-Ser-DCys-NH2 (the disulfide bond position is Cys1-Dcys12), Ac-DArg(Pbf)-Arg(Pbf)-Gly-Trp(Boc)-Gly-Pro-DCys(Trt)-COOH, and isolating the Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gly-Ala-Ser-DCys-NH2 (the disulfide bond position is Cys1-Dcys12) and Ac-DArg(Pbf)-Arg(Pbf)-Gly-Trp(Boc)-Gly-Pro-DCys(Trt)-COOH. 1-Dcys12) and Ac-DArg(Pbf)-Arg(Pbf)-Gly-Trp(Boc)-Gly-Pro-DCys(Trt)-COOH are mixed, EDCI, HOOBt, and NMM are added to react in a reaction solvent to obtain Ac-DArg(Pbf)-Arg(Pbf)-Gly-Trp(Boc)-Gly-Pro-DCys(Trt)-Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gly-Ala-Ser-DCys-NH2 (disulfide bond position is Cys1-Dcys12), and a cutting solution is added to cut the amino acid side chain protecting groups to obtain the dual-target polypeptide as claimed in claim 1.

8. The method according to claim 7, characterized in that The mixing ratio is 1:

1.

9. The method according to claim 7, characterized in that The amounts of EDCI, HOOBt and NMM used are 1-5 mmol, 1-5 mmol and 0.1-3 mmol respectively.

10. The method according to claim 9, characterized in that The amounts of EDCI, HOOBt and NMM used are 3 mmol, 3 mmol and 1 mmol respectively.

11. The method according to claim 7, characterized in that The reaction conditions for adding EDCI, HOOBt, and NMM in the reaction solvent are room temperature for 10-50 min.

12. The method according to claim 11, characterized in that The reaction conditions for adding EDCI, HOOBt, and NMM in the reaction solvent are room temperature and 30 min.

13. The method according to claim 7, characterized in that The cutting fluid is TFA:H2O=95%:5%.

14. The method according to claim 7, wherein: The synthesis of Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gly-Ala-Ser-DCys-NH2 (disulfide bond position is Cys1-Dcys12) comprises the following steps: (1) Take Fmoc-DCys(Trt)-AM Resin, swell it, add 20% Pip / DMF solution 3 times the volume of the resin, blow nitrogen, and dry it to obtain the resin H2N-DCys(Trt)-AM Resin; (2) Fmoc-Ser(tBu)-OH amino acid, DIPEA, and HBTU were reacted with the obtained resin in a reaction solvent to obtain Fmoc-Ser(tBu)-DCys(Trt)-AM Resin, 20% Pip / DMF solution was added in an amount 3 times the volume of the resin, nitrogen was blown, and the mixture was dried to obtain H2N-Ser(tBu)-DCys(Trt)-AM Resin. After the resin was washed with the reaction solvent, Cys(Trt)-Gly-Gly-Gly-Gly-Ser(tBu)-Phe-Pro-Gly-Ala-Ser(tBu)-DCys(Trt)-AM Resin was obtained; (3) After adding 6 times the volume of the resin-containing cutting solution, the resin was filtered, the filtrate was precipitated, and vacuum dried to obtain Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gly-Ala-Ser-DCys-NH2. The pH was adjusted to alkaline and stirred for natural oxidation to obtain Cys-Gly-Gly-Gly-Gly-Ser-Phe-Pro-Gly-Ala-Ser-DCys-NH2 (the disulfide bond position is Cys1-Dcys12).

15. The method according to claim 14, characterized in that The amount of the Fmoc-DCys(Trt)-AM Resin used is 0.1-5 mmol.

16. The method according to claim 15, characterized in that The amount of the Fmoc-DCys(Trt)-AM Resin used is 1.8 mmol.

17. The method according to claim 14, characterized in that The swelling condition is to use DCM to swell for 10-30 minutes.

18. The method according to claim 17, characterized in that The swelling condition is to use DCM to swell for 20 min.

19. The method according to claim 14, wherein The nitrogen bubbling time is 10-50 min.

20. The method according to claim 19, characterized in that The nitrogen bubbling time is 30 min.

21. The method according to claim 14, wherein The reaction solvent is DMF, DCM, NMP, HFIP and / or pyridine.

22. The method according to claim 14, wherein The molar ratio of the Fmoc-Ser(tBu)-OH amino acid, DIPEA, HBTU and the obtained resin is 1-5:3-9:1-5:0.1-5.

23. The method according to claim 22, characterized in that The molar ratio of the Fmoc-Ser(tBu)-OH amino acid, DIPEA, HBTU, and the obtained resin is 3:6:2.85:

1.

24. The method according to claim 14, wherein The reaction conditions are room temperature for 10-50 min.

25. The method according to claim 24, characterized in that The reaction conditions are room temperature for 30 min.

26. The method according to claim 14, wherein The cutting fluid is 97.5% TFA+2.5% H2O.

27. The method according to claim 14, wherein The reaction time of adding the cutting solution is 1-3 h.

28. The method according to claim 27, characterized in that The reaction time after adding the cutting solution was 2 h.

29. The method according to claim 7, characterized in that The synthesis of Ac-DArg(Pbf)-Arg(Pbf)-Gly-Trp(Boc)-Gly-Pro-DCys(Trt)-COOH comprises the following steps: (1) Take Fmoc-DCys(Trt)-CTC Resin, swell it, add 20% Pip / DMF solution 3 times the volume of the resin, blow nitrogen, and dry it to obtain the resin H2N-DCys(Trt)-CTC Resin; (2) Fmoc-Pro-OH amino acid, DIPEA, HBTU and the obtained resin were reacted in a reaction solvent to obtain Fmoc-Pro-DCys(Trt)-CTC Resin, 20% Pip / DMF solution was added in an amount 3 times the volume of the resin, nitrogen was blown, and the resin was dried to obtain H2N-Pro-DCys(Trt)-CTC Resin. After the resin was washed with the reaction solvent, H2 N-DArg(Pbf)-Arg(Pbf)-Gly-Trp(Boc)-Gly-Pro-DCys(Trt)-CTC Resin; (3) Adjust the pH to alkaline, add acetic anhydride, and obtain Ac-DArg(Pbf)-Arg(Pbf)-Gly-Trp(Boc)-Gly-Pro-DCys(Trt)-CTC Resin by acetylation. After full protection cleavage, obtain Ac-DArg(Pbf)-Arg(Pbf)-Gly-Trp(Boc)-Gly-Pro-DCys(Trt)-COOH.

30. The method according to claim 29, wherein The amount of the Fmoc-DCys(Trt)-CTC Resin used is 0.1-5 mmol.

31. The method according to claim 30, characterized in that The amount of the Fmoc-DCys(Trt)-CTC Resin used is 1.8 mmol.

32. The method according to claim 29, wherein The swelling condition is to use DCM to swell for 10-30 minutes.

33. The method according to claim 32, characterized in that The swelling condition is to use DCM to swell for 20 min.

34. The method according to claim 29, wherein The nitrogen bubbling time is 10-50 min.

35. The method according to claim 34, wherein The nitrogen bubbling time is 30 min.

36. The method according to claim 29, wherein The reaction solvent is DMF, DCM, NMP, HFIP and / or pyridine.

37. The method according to claim 29, wherein The molar ratio of the Fmoc-Pro-OH amino acid, DIPEA, HBTU and the obtained resin is 1-5:3-9:1-5:0.1-5.

38. The method according to claim 37, wherein The molar ratio of the Fmoc-Pro-OH amino acid, DIPEA, HBTU and the obtained resin is 3:6:2.85:

1.

39. The method according to claim 29, wherein The reaction is carried out at room temperature for 10-50 min.

40. The method according to claim 39, wherein The reaction conditions are room temperature for 30 min.

41. The method according to claim 29, wherein The acetic anhydride is 0.1-5 mmol.

42. The method according to claim 41, wherein The amount of acetic anhydride was 1.8 mmol.

43. Use of the dual-target polypeptide or pharmaceutically acceptable salt thereof according to claim 1, the polynucleotide according to claim 2, the vector according to claim 3, and / or the host cell according to claim 4 in the preparation of a medicament for treating and / or preventing heart failure with preserved ejection fraction, heart failure without preserved ejection fraction, myocardial fibrosis caused by heart failure with preserved ejection fraction, myocardial fibrosis caused by heart failure without preserved ejection fraction, cardiac hypertrophy caused by heart failure with preserved ejection fraction, and / or cardiac hypertrophy caused by heart failure without preserved ejection fraction.

44. The use according to claim 43, characterized in that The heart failure with preserved ejection fraction is a cardiac diastolic dysfunction caused by diabetes, hypertension, hyperlipidemia, chronic renal failure, metabolic syndrome, obesity, coronary artery disease, hypertrophic cardiomyopathy, infiltrative cardiomyopathy, pulmonary hypertension, valvular heart disease and / or atrial fibrillation.

45. The use according to claim 43, characterized in that The non-ejection fraction preserved heart failure is a cardiac systolic dysfunction caused by coronary heart disease, hypertension, dilated cardiomyopathy, hypertrophic cardiomyopathy, valvular heart disease, myocarditis, congenital heart disease, ventricular tachycardia, atrial fibrillation and / or hyperthyroidism.

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