TW202421648A - Myeloid-derived growth factor for use in treating cardiogenic shock - Google Patents

Abstract

The present invention relates to the protein myeloid-derived growth factor (MYDGF) or nucleic acids encoding said protein for use in treating and/or preventing cardiogenic shock. The present invention also relates to vectors comprising the nucleic acid, host cells expressing the nucleic acid, pharmaceutical compositions comprising said protein, nucleic acid, vector or host cell for use in treating and/or preventing cardiogenic shock, and to methods for treating and/or preventing cardiogenic shock. The present invention further relates to a method of preparing an animal model of cardiogenic shock, and to animals obtained by said method.

Description

Myeloid-derived growth factor for the treatment of cardiogenic shock (I)

The present invention relates to a protein bone marrow-derived growth factor (MYDGF) for treating and/or preventing cardiogenic shock. The present invention also relates to a novel animal model of cardiogenic shock.

Myeloid-derived growth factor (MYDGF), also known as factor 1, is a protein encoded by open reading frame 10 (C19Orf10) on human chromosome 19. In 2007, the protein was described as a novel secreted factor in the synovium in a proteomic analysis of so-called fibroblast-like synoviocytes (FLS cells). Without any experimental or statistical evidence, a link between the secretion of this protein and inflammatory diseases of the joint was hypothesized (Weiler et al. , Arthritis Research and Therapy 2007, The identification and characterization of a novel protein, c19orf10, in the synovium). The corresponding patent application claims that the protein is used as a therapeutic agent for treating joints and for diagnosing tissues undergoing growth changes and monitoring changes in tissues (US 2008/0004232 A1, Characterization of c19orf10, a novel synovial protein). Another scientific publication describes the increased expression of the protein in hepatocellular carcinoma cells (Sunagozaka et al. , International Journal of Cancer, 2010, Identification of a secretory protein c19orf10 activated in hepatocellular carcinoma). The recombinantly produced protein showed a proliferation-enhancing effect on cultured hepatocellular carcinoma cells. It is worth noting that C19Orf10 is also known as IL-25, IL-27 and IL-27W, because it was originally considered to be an interleukin. However, the terms "IL-25" and "IL-27" are not used consistently in the art and have been used to refer to a variety of different proteins. For example, US 2004/0185049 refers to the protein as IL-27 and discloses its use in regulating immune responses. This protein is structurally different from Factor 1 (compare the Factor 1 amino acid sequence according to SEQ ID NO: 1 with the amino acid sequence of "IL-27" according to UniProt: Q8NEV9). Similarly, EP 2 130 547 A1 refers to the protein as IL-25 and discloses its use in treating inflammation. This protein is also known in the art as IL-17E and is structurally distinct from Factor 1 (compare the amino acid sequence of Factor 1 according to SEQ ID NO: 1 with the amino acid sequence of "IL-25" according to UniProt: Q9H293).

WO 2014/111458 discloses that factor 1 is used to increase proliferation and inhibit apoptosis of non-transformed tissues or cells, particularly for treating acute myocardial infarction. It further discloses that factor 1 inhibitors are used for medical purposes, particularly for treating or preventing diseases in which angiogenesis contributes to the occurrence or progression of the disease.

Korf-Klingebiel et al. (Nature Medicine, 2015, Vol. 21(2):140-149) describe C19Orf10 as secreted by bone marrow cells after myocardial infarction and that this protein promotes cardiomyocyte survival and angiogenesis. The authors demonstrated that bone marrow-derived monocytes and macrophages endogenously produce this protein to protect and repair the heart after myocardial infarction and proposed the name myeloid-derived growth factor (MYDGF). In particular, treatment with recombinant mouse Mydgf was reported to reduce scar size and contractile dysfunction after myocardial infarction.

WO 2021/148411 discloses that MYDGF is used to treat or prevent fibrosis, hypertrophy or heart failure. Heart failure is a clinical syndrome with a poor prognosis that may be caused by persistent hemodynamic overload, myocardial damage or genetic mutations.

Cardiogenic shock (CS) is a life-threatening, acute low cardiac output state caused by severe systolic and/or diastolic myocardial dysfunction, resulting in arterial hypotension, pulmonary congestion, severe end-organ hypoperfusion, and impaired tissue oxygenation. CS is associated with inadequate blood flow to the extremities and vital organs, including the heart itself, liver, kidneys, and brain. Severe end-organ hypoperfusion and impaired tissue oxygenation lead to increased blood lactate concentrations, which can be used to diagnose and monitor patients with CS. Clinical criteria defining CS are summarized, for example, in the review article by Vahdatpour et al. (Journal of the American Heart Association, Vol 8(8), 2019, e011991). Insufficient cardiac perfusion and impaired oxygenation can lead to a gradual deterioration of cardiac function in CS, which in turn triggers a downward spiral of hemodynamic instability, accompanied by an extremely high mortality rate.

Although CS may develop as a complication of myocardial infarction, the pathophysiology of uncomplicated myocardial infarction (without cardiogenic shock) and (usually massive) myocardial infarction with cardiogenic shock is very different. Patients with uncomplicated myocardial infarction do not have severe end-organ hypoperfusion and compromised tissue oxygenation, they do not have an acute progressive deterioration in cardiac function (a downward spiral), and they do not have the very high acute mortality observed in patients with CS.

Patients with heart failure, cardiac fibrosis, and hypertrophy similarly do not present with complete severe end-organ hypoperfusion and compromised tissue oxygenation, they do not experience an acute progressive deterioration in cardiac function (a downward spiral), and they do not experience the very high acute mortality observed in patients with CS.

As an example of the unique pathophysiology of these conditions, medical therapies known to improve outcomes in patients with uncomplicated myocardial infarction and those with heart failure, cardiac fibrosis, and hypertrophy (e.g., angiotensin-converting enzyme inhibitors and beta-blockers) are not useful or are even contraindicated in CS. In fact, to date, no medical therapy has been found to improve survival in patients with CS.

Therefore, there is an urgent need for means and methods for treating and/or preventing cardiogenic shock. Appropriate animal models of cardiogenic shock are also needed to further elucidate the underlying pathophysiology and identify new therapeutic approaches for this condition.

In a first aspect, the present invention provides myeloid-derived growth factor (MYDGF) or a fragment or variant thereof that exhibits the biological function of MYDGF, which is used for treating and/or preventing cardiogenic shock.

According to a preferred embodiment, the MYDGF protein comprises SEQ ID NO: 1. According to an embodiment, the MYDGF protein comprises a fragment or variant of SEQ ID NO: 1, which exhibits the biological function of MYDGF and comprises an amino acid sequence having at least 85% amino acid sequence identity with SEQ ID NO: 1.

According to a preferred embodiment, the MYDGF protein consists of SEQ ID NO: 1 or SEQ ID NO: 3.

According to another aspect, the present invention provides a nucleic acid encoding MYDGF or a fragment or variant thereof that exhibits the biological function of MYDGF, which is used for treating and/or preventing cardiogenic shock.

According to one embodiment, the nucleic acid encodes an amino acid sequence having at least 85% sequence identity to SEQ ID NO:1.

According to another aspect, the present invention provides a vector comprising the nucleic acid of the present invention, which is used for treating and/or preventing cardiogenic shock.

According to another aspect, the present invention provides a host cell comprising the nucleic acid of the present invention or the vector of the present invention, which is used for treating and/or preventing cardiogenic shock. Preferably, the host cell expresses the nucleic acid.

According to yet another aspect, the present invention provides a pharmaceutical composition comprising the MYDGF protein, nucleic acid, vector or host cell of the present invention and, as appropriate, a suitable pharmaceutical formulation and/or carrier, for treating and/or preventing cardiogenic shock.

According to a preferred embodiment, the pharmaceutical composition used is administered orally, intravenously, subcutaneously, intramucosally, intraarterially, intramuscularly or intracoronarily. Preferably, the administration is performed by one or more bolus injections and/or infusions.

According to another aspect, the present invention provides a method for treating and/or preventing cardiogenic shock, comprising administering a therapeutically effective amount of myeloid-derived growth factor (MYDGF) protein to a patient in need thereof.

According to one embodiment, the MYDGF protein comprises a fragment or variant of SEQ ID NO: 1, which exhibits the biological function of MYDGF and comprises an amino acid sequence having at least 85% amino acid sequence identity with SEQ ID NO: 1.

According to yet another embodiment, MYDGF or a fragment or variant thereof is administered by one or more bolus injections and/or infusions, preferably in a pharmaceutically acceptable carrier and/or formulation.

According to another aspect, the present invention provides a method for treating and/or preventing cardiogenic shock, comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a myeloid-derived growth factor (MYDGF) protein or a fragment or variant thereof. According to one embodiment, the MYDGF protein comprises a fragment or variant of SEQ ID NO: 1, which exhibits the biological function of MYDGF and comprises an amino acid sequence having at least 85% amino acid sequence identity with SEQ ID NO: 1.

According to a preferred embodiment, the pharmaceutical composition comprises a suitable pharmaceutical excipient.

According to another embodiment, the pharmaceutical composition is administered via one or more boluses and/or infusions.

According to yet another aspect, the present invention provides a method for producing a non-human mammalian model of cardiogenic shock, the method comprising (i) temporarily ligating a coronary artery of the mammal, (ii) establishing reperfusion, and (iii) ventilating the mammal at an inspired oxygen fraction of about 0.18 or less.

According to a preferred embodiment, the non-human mammal is a rodent, more preferably a mouse.

According to a preferred embodiment, the coronary artery is ligated for a period of about 30 minutes to about 90 minutes before reperfusion is established.

According to a preferred embodiment, mammals are ventilated at a fraction of inspired oxygen of approximately 0.16.

Sequence Listing

This application contains a sequence listing, which has been submitted electronically in XML format and is incorporated by reference in its entirety.

Before the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention, which is limited only by the scope of the attached patent application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art. Definitions

Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kölbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

Unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology and recombinant DNA technology will be used to practice the present invention, which are explained in the literature in this field (see, for example , Molecular Cloning: A Laboratory Manual , ^2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). In addition, conventional methods of clinical cardiology are used, which are also explained in the literature in this field (see, for example , Braunwald's Heart Disease. A Textbook of Cardiovascular Medicine , ^9th Edition, P. Libby et al. eds., Saunders Elsevier Philadelphia, 2011).

Throughout this specification and the appended claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated number or step or group of steps but not the exclusion of any other number or step or group of steps. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.

When used in conjunction with a numerical value, the term "about" means that the numerical value is inclusive of a range having a lower limit that is 5% less than the indicated numerical value and an upper limit that is 5% greater than the indicated numerical value.

As used herein, the term "and/or" means that it refers to one or both/all of the options cited in the context of this term.

Nucleic acid molecules, also called nucleic acids, are understood to be polymeric macromolecules made of nucleotide monomers. Nucleotide monomers are composed of a nucleobase, a pentose (such as but not limited to ribose or 2'-deoxyribose), and one to three phosphate groups. Polynucleotides are usually formed through phosphodiester bonds between individual nucleotide monomers. In the context of the present invention, nucleic acid molecules referred to include but are not limited to ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

The term "open reading frame" (ORF) refers to a nucleotide sequence that can be translated into amino acids. Typically, such an ORF contains a start codon followed by a region that is usually a multiple of 3 nucleotides in length, but does not contain a stop codon (TAG, TAA, TGA, UAG, UAA or UGA) in a given reading frame. Typically, ORFs are naturally occurring or artificially constructed (i.e., by means of genetic technology). ORFs encode proteins in which the amino acids that can be translated form peptide-linked chains.

The terms "protein" and "polypeptide" are used interchangeably herein and refer to any peptide-bonded amino acid chain, regardless of length or post-translational modification. Proteins (including protein derivatives, protein variants, protein fragments, protein segments, protein epitopes and protein domains) that can be used in the present invention can be further modified by chemical modification. This means that the polypeptides so chemically modified contain other chemical groups in addition to the 20 naturally occurring amino acids. Examples of such other chemical groups include, but are not limited to, glycosylated amino acids and phosphorylated amino acids. Chemical modification of polypeptides can provide advantageous properties, such as one or more of increased stability, increased biological half-life or increased water solubility compared to the parent polypeptide. Chemical modifications suitable for use in the variants of the present invention include, but are not limited to, PEGylation, glycosylation of an unglycosylated parent polypeptide, covalent conjugation with a therapeutic small molecule (therapeutic small molecules such as glucagon-like peptide 1 agonists, including exenatide, albiglutide, taspoglutide, DPP4 inhibitors, secretin and liraglutide), or modification of the glycosylation pattern present in the parent polypeptide. Such chemical modifications suitable for use in the variants of the present invention may occur co-translationally or post-translationally.

The term "amino acid" includes naturally occurring amino acids and amino acid derivatives. In the context of the present invention, a hydrophobic non-aromatic amino acid is preferably any amino acid having a Kyte-Doolittle hydrophobicity index higher than 0.5, more preferably higher than 1.0, even more preferably higher than 1.5, and is not aromatic. Preferably, the hydrophobic non-aromatic amino acid in the context of the present invention is selected from the group consisting of the amino acids alanine (Kyte Doolittle hydrophobicity index 1.8), methionine (Kyte Doolittle hydrophobicity index 1.9), isoleucine (Kyte Doolittle hydrophobicity index 4.5), leucine (Kyte Doolittle hydrophobicity index 3.8), and valine (Kyte Doolittle hydrophobicity index 4.2), or a derivative thereof having a Kyte Doolittle hydrophobicity index as defined above.

The term "variant" is used herein to refer to a polypeptide that is different from the polypeptide or fragment thereof derived therefrom due to one or more changes in the amino acid sequence. The polypeptide derived from the protein variant is also referred to as the parent polypeptide. Similarly, the fragment derived from the protein fragment variant is referred to as the parent fragment. Typically, variants are artificially constructed, preferably constructed by genetic technology means. Typically, the parent polypeptide is a wild-type protein or a wild-type protein domain. In addition, variants that can be used in the present invention may also be derived from homologues, heterologous homologues or homologues of the parent polypeptide, or derived from artificially constructed variants, provided that the variant exhibits at least one biological activity of the parent polypeptide. The changes in the amino acid sequence may be amino acid exchanges, insertions, deletions, N-terminal truncations or C-terminal truncations, or any combination of these changes, which may occur at one or more sites. In preferred embodiments, variants useful in the present invention exhibit a total of up to 23 (up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23) changes (i.e., exchanges, insertions, deletions, N-terminal truncations and/or C-terminal truncations) in the amino acid sequence. A particularly preferred variant of SEQ ID NO: 1 is shown in SEQ ID NO: 3, which exhibits an additional amino acid (glycine) at its N-terminus (+G variant). Amino acid exchanges may be conservative, and/or semi-conservative, and/or non-conservative. In preferred embodiments, the variants useful in the present invention differ from the proteins or domains from which they are derived by at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 amino acid exchanges, preferably conservative amino acid changes.

Typical substitutions are between aliphatic amino acids, between amino acids with aliphatic hydroxyl side chains, between amino acids with acidic residues, between amide derivatives, between amino acids with basic residues, or between amino acids with aromatic residues. Typical semiconservative and conservative substitutions are:

A change from A, F, H, I, L, M, P, V, W or Y to C is semiconservative if the new cysteine is still a free thiol. Furthermore, the skilled artisan will appreciate that glycine should not be replaced by P at sterically demanding positions, and P should not be introduced into portions of the protein having alpha helical or beta sheet structures.

Alternatively or additionally, as used herein, a "variant" may be characterized by having a certain degree of sequence identity with the parent polypeptide or parent polynucleotide from which it is derived. More precisely, a protein variant in the context of the present invention exhibits at least 85% sequence identity with its parent polypeptide. Throughout the specification the term "at least 85% sequence identity" is used with respect to polypeptide and polynucleotide sequence comparisons. This expression preferably refers to at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a corresponding reference polypeptide or a corresponding reference polynucleotide.

Protein fragments include amino acid deletions, which may be N-terminal truncations, C-terminal truncations or internal deletions, or any combination of these. Such proteins including N-terminal truncations, C-terminal truncations and/or internal deletions are referred to as "fragments" in the context of this application. Fragments may be naturally occurring (e.g., splice variants) or may be artificially constructed (preferably constructed by genetic technology). Preferably, compared to the parent polypeptide, the fragment (or deletion variant) has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acid deletions at its N-terminus and/or at its C-terminus and/or internally, preferably at its N-terminus, at its N-terminus and C-terminus, or at its C-terminus.

If two sequences are compared and no reference sequence is specified to which the sequence identity percentage is calculated, the sequence identity is calculated with reference to the longer of the two sequences being compared, unless expressly stated otherwise.

Nucleotide and amino acid sequence similarity (ie, percent sequence identity) can be determined by sequence alignment. The alignment can be performed using any of several algorithms known in the art, preferably the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), using hmmalign (HMMER package, http://hmmer.wustl.edu/), or using the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80) or the CLUSTALW2 algorithm (Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG. (2007). Clustal W and Clustal X version 2.0. Bioinformatics , 23, 2947-2948.), which can be obtained, for example, at http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html or at http://www.ebi.ac.uk/Tools/clustalw2/index.html. Preferably, the CLUSTALW2 algorithm at http://www.ebi.ac.uk/Tools/clustalw2/index.html is used, wherein the parameters used are the default parameters as set at http://www.ebi.ac.uk/Tools/clustalw2/index.html: alignment type = slow, protein weight matrix = Gonnet, gap opening = 10, gap extension = 0,1 for the slow pairwise alignment option and protein weight matrix = Gonnet, gap opening = 10, gap extension = 0,20, gap distance = 5, no terminal gaps = none, output options: format = Aln with numbers, order = aligned.

The level of sequence identity (sequence match) can be calculated using, for example, BLAST, BLAT or BlastZ (or BlastX). Similar algorithms are incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215: 403-410. BLAST protein searches are performed using the BLASTP program, which is available, for example, at http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome. The algorithm parameters used are the default parameters as they are set at http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome: expectationthreshold=10, wordsize=3, maximummatchesofquery=0, matrix=BLOSUM62, gappenalty=presence:11 extension:1, componentadjustment=condition. The component score matrix was adjusted with the non-redundant protein sequence (nr) database to obtain amino acid sequences homologous to the factor 1 and factor 2 polypeptides.

For comparison purposes, Gapped BLAST was used to obtain gapped alignments as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs were used. Sequence matching analysis can be supplemented by established homology mapping techniques, such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:I54-I62) or Markov random fields. When percentages of sequence identity are mentioned in this application, these percentages are calculated relative to the full length of the longer sequence unless otherwise specifically stated.

As used herein, the term "host cell" refers to a cell that carries the nucleic acid of the invention, for example in the form of a plastid or virus. Such a host cell may be a prokaryotic cell, for example a bacterial cell, or a eukaryotic cell, for example a fungal, plant or animal cell. The cell may be transformed or non-transformed. The cell may be an isolated cell or part of a tissue, for example in a cell culture, which itself may be isolated or part of a more complex tissue structure, such as an organ or an individual.

The terms "myeloid derived growth factor", "MYDGF", "Factor 1", "MYDGF polypeptide or protein" or "Factor 1 polypeptide or protein" are used interchangeably and refer to the protein set forth in NCBI reference sequence NM_019107.3 (human homolog) and its mammalian homologs, particularly homologs from mouse or rat. The amino acid sequence of the human homolog is encoded in open reading frame 10 (C19Orf10) on human chromosome 19. Preferably, MYDGF and Factor 1 protein refer to a protein comprising, consisting essentially of or consisting of the core segment of human Factor 1 having an amino acid sequence according to SEQ ID NO: 1.

According to yet another preferred embodiment, MYDGF and Factor 1 protein refer to proteins comprising SEQ ID NO: 3, respectively. In yet another preferred embodiment, MYDGF refers to a protein consisting essentially of SEQ ID NO: 3. In yet another preferred embodiment, MYDGF refers to a protein consisting of SEQ ID NO: 3.

As used herein, MYDGF and fragments and variants thereof as defined herein include pharmaceutically acceptable salts thereof.

Whether a protein, variant or fragment exhibits the biological function of MYDGF can be determined by any of the tests described in the following examples. According to the present invention, if the results obtained with such a peptide or protein demonstrate at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the efficacy described for MYDGF compared to the results obtained with the MYDGF protein of the present invention in at least one of the examples presented herein and outperform the control shown, then the peptide or protein exhibits the biological function of MYDGF.

As used herein, the terms "MYDGF" and "Mydgf" both refer to myeloid-derived growth factor.

The term "cardiogenic shock" describes a state of end-organ hypoperfusion due to heart failure and the inability of the cardiovascular system to provide adequate blood flow to the extremities and vital organs. Patients with cardiogenic shock who present with persistent hypotension (systolic blood pressure less than 80 to 90 mm Hg, or mean arterial pressure less than 30 mm Hg baseline, or SBP >90 mm Hg with support from vasopressors or inotropes, or mean arterial pressure <70 mm Hg, or systolic blood pressure <100 mm Hg despite adequate fluid resuscitation), evidence of end-organ damage (e.g., urine output <30 mL/h, or urine output <0.5 mL/kg for 1 hour, or cool extremities, or mottled skin, or serum lactate >2 mmol/L, or metabolic acidosis, or altered mental status), severely depressed cardiac indexes (less than 2.2 L/min/m ² ) in the presence of adequate or high filling pressures (left ventricular [LV] end-diastolic pressure greater than 15 mm Hg or right ventricular (RV) end-diastolic pressure greater than 10 to 15 mm Hg) (Vahdatpour et al., Journal of the American Heart Association, Vol 8(8), 2019, e011991).

As used herein, "treat," "treatment," or "treatment" of a disease or disorder refers to achieving one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing the development of symptoms characteristic of the disorder being treated; (c) inhibiting the worsening of symptoms characteristic of the disorder being treated; (d) limiting or preventing the recurrence of the disorder in an individual who previously had the disorder; (e) limiting or preventing the recurrence of symptoms in an individual who previously had symptoms of the disorder; (f) reducing the mortality rate after the onset of the disease or disorder; (g) curing; and (h) preventing the disease. The term "amelioration" is also encompassed by the term "treating." Consistent with this, as used herein, the term "treatment and/or prevention" of a condition or disease means that the condition or disease is treated or prevented, or both.

The term "stroke volume" describes the amount of blood ejected by the right or left ventricle during a single contraction. It is the difference between the end-diastolic volume (EDV) and the end-systolic volume (ESV).

The term "stroke work" describes the work done by the left or right ventricle to eject the stroke volume into the aorta or pulmonary artery, respectively.

The term "cardiac output" describes the amount of blood pumped by the heart's ventricles per unit time.

The terms "dP/dt _min " and "dP/dt _max " describe the minimum and maximum rates of pressure change in the ventricles, respectively. Peak dP/dt is used as an indicator of ventricular performance.

The term "isovolumic relaxation constant" or "Tau" describes the exponential decrease in ventricular pressure during isovolumic relaxation. It is also called the ventricular diastolic time constant.

The term "fraction of inspired oxygen" or "FiO ₂ " describes the molar or volume fraction of oxygen in the inspired air. Natural air contains 21% oxygen, which is equivalent to a FiO ₂ of 0.21.

The terms "subject," "individual," and "patient" are used interchangeably herein and refer to individuals such as humans, non-human primates (e.g., chimpanzees and other ape and monkey species); farm animals such as birds, fish, cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; and experimental animals, including rodents such as mice, rats, and guinea pigs. The term does not denote a particular age or sex. In a particular sense, an individual is a mammal. In a preferred sense, an individual is a human. An individual may be a healthy individual or an individual suffering from or suspected of suffering from one or more diseases. An individual suffering from or suspected of suffering from one or more diseases is also referred to as a patient.

Unless otherwise stated, these descriptions and definitions apply to the entire application.

sequence

The sequences used in the present invention are listed below.

SEQ ID NO: 1 (amino acid sequence of human factor 1 (MYDGF) lacking the 31-amino acid N-terminal signal peptide): VSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL

SEQ ID NO: 2 (amino acid sequence of MYDGF, including the N-terminal signal peptide (shown in bold and underlined); UniProtKB - Q969H8): MAAPSGGWNGVGASLWAALLLGAVALRPAEA VSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL

SEQ ID NO: 3 (amino acid sequence of a [+G] MYDGF variant, wherein the N-terminal V residue at position +1 of mature human MYDGF is preceded by a G residue): GVSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL

SEQ ID NO: 4 shows the nucleic acid sequence of human factor 1 encoding MYDGF of SEQ ID NO: 3 (NCBI Geene ID: 56005). Specific example

The elements of the present invention will be described below. These elements are listed together with specific embodiments, but it should be understood that they can be combined in any manner and in any quantity to create additional embodiments. The various described examples and preferred embodiments should not be interpreted as limiting the present invention to the explicitly described embodiments. This invention description should be understood to demonstrate and include embodiments that combine the explicitly described embodiments with any number of disclosed and/or preferred elements. In addition, unless the context indicates otherwise, any arrangement and combination of all described elements in this application should be considered to be disclosed by the invention description of this application.

The present disclosure demonstrates for the first time the anti-cardiogenic shock efficacy of MYDGF. In particular, the present disclosure demonstrates that administration of MYDGF in a mouse model of cardiogenic shock reduces symptoms associated with cardiogenic shock. Specifically, the present disclosure demonstrates that treatment of mice in an animal model of cardiogenic shock with MYDGF increases cardiac output, cardiac work, stroke volume, ventricular end-systolic pressure, and ventricular ejection fraction compared to saline-treated control mice. The present disclosure further demonstrates that MYDGF successfully treats cardiogenic shock and increases overall survival (see Examples below).

Therefore, in a first aspect, the present invention provides a protein bone marrow-derived growth factor (MYDGF) or a fragment or variant thereof for use in treating and/or preventing cardiogenic shock. According to a preferred embodiment, the fragment or variant of MYDGF exhibits the biological function of MYDGF.

According to a preferred embodiment, the cardiogenic shock to be treated or prevented is during (acute) myocardial infarction (AMI, MI), preferably ST-segment elevation myocardial infarction (STEMI), and even more preferably during MI or STEMI, during reperfusion after percutaneous coronary intervention (PCI).

In a particularly preferred embodiment of the present invention, the protein comprises the amino acid sequence SEQ ID NO: 1 or a fragment thereof. Preferably, the protein has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1.

In a preferred embodiment of this aspect of the invention, the protein comprises the amino acid sequence SEQ ID NO: 1, a fragment or variant thereof having at least 85% sequence identity with SEQ ID NO: 1. According to a preferred embodiment, the fragment or variant of MYDGF exhibits the biological function of MYDGF. Skilled artisans are able to decide, without undue burden, which positions in the parent polypeptide can be mutated to what extent and which positions must be maintained to retain the functionality of the polypeptide. Such information can be obtained, for example, from homologous sequences, which can be identified, aligned and analyzed by bioinformatics methods well known in the art. Such analysis is exemplarily described in Example 7 of WO 2014/111458 and in Figures 6 and 7. Preferably, mutations are introduced in those regions of the protein that are not completely conserved between species, preferably mammals. In a particularly preferred embodiment of the invention, the MYDGF protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 1 or 3 or a fragment or variant thereof. According to a preferred embodiment, the fragment or variant of MYDGF exhibits the biological function of MYDGF. Preferably, the protein has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.

Also included are N-terminal deletion variants which may, for example, lack one or more amino acids from amino acid positions 1 to 24 (based on SEQ ID NO: 1), ie, from the N-terminal conserved region.

Also contemplated are C-terminal deletion variants which may, for example, lack one or more amino acids from amino acid positions 114 to 142 (based on SEQ ID NO: 1).

On the other hand, amino acids can be added to the MYDGF protein. Such additions include additions at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The protein of the first aspect of the present invention may further comprise an additional amino acid sequence, for example, for stabilizing or purifying the resulting protein. Examples of such amino acids are 6xHis-tags, myc-tags, or FLAG-tags, which are well known in the art, and which may be present at any position in the protein, preferably at the N-terminus or the C-terminus. A particularly preferred additional sequence is a 6xHis-tag. Preferably, the 6xHis tag is present at the C-terminus of the MYDGF protein. Depending on the expression system used and the additional amino acids (such as the above-mentioned tags) (if present), one or more residual amino acids may be retained at the N-terminus and/or C-terminus of the protein. It should be emphasized that such artifacts may exist in the MYDGF protein and Mydgf protein according to the present invention, as shown, for example, in Ebenhoch R. et al., Nat Commun. 2019 Nov 26;10(1):5379, and Polten F. et al., Anal Chem. 2019 Jan 15;91(2):1302-1308.

In some cases, it is preferred to mutate the protease cleavage site in the MYDGF protein of the first aspect of the present invention to stabilize the protein (see Segers et al. Circulation 2007, 2011). The skilled artisan knows how to determine potential proteolytic cleavage sites in a protein. For example, the protein sequence can be submitted to a website that provides such analysis, such as, for example, http://web.expasy.org/peptide_cutter/ or http://pmap.burnham.org/proteases. If the protein sequence according to SEQ ID NO: 1 is submitted to http://web.expasy.org/peptide_cutter/, the following cleavage sites with low frequency (less than 10) are determined: Table 1 Protease Frequency Position (with reference to SEQ ID NO: 1) Arg-C protease 6 12 65 82 99 120 139 Asp-N endopeptidase 4 9 27 54 101 Clostripain 6 12 65 82 99 120 139 LysN 9 28 68 77 93 105 113 124 129 135 Proline-endopeptidase            3            13 66 121

These sites can be altered to remove the corresponding identified protease recognition/cleavage sequences, thereby increasing the serum half-life of the protein.

The MYDGF protein may further comprise additional amino acid sequences, for example, for stabilizing or purifying the resulting protein. For example, a protease cleavage site within the MYDGF protein may be preferentially mutated to stabilize the protein. Appropriate proteolytic cleavage sites may be identified as described above.

The MYDGF protein or a composition comprising the protein can be administered in vivo, in vitro or in vitro, preferably in vivo.

Nucleic acid sequences can be optimized in an attempt to improve performance in a host cell. Parameters to consider include C:G content, preferred codons, and avoiding inhibitory secondary structures. These factors can be combined in different ways to try to obtain nucleic acid sequences with improved performance in a specific host (see, for example, Donnelly et al., International Publication No. WO 97/47358). The improvement of the performance of a specific sequence in a specific host involves some empirical experiments. Such experiments involve measuring the performance of a desired nucleic acid sequence and, if necessary, changing the sequence. Starting from a specific amino acid sequence and the known degeneracy of the genetic code, a large number of different coding nucleic acid sequences can be obtained. The degeneracy of the genetic code occurs because almost all amino acids are encoded by different combinations of nucleotide triplets or "codons". The translation of specific codons into specific amino acids is well known in the art (see, e.g., Lewin GENES IV, p. 119, Oxford University Press, 1990). Therefore, the present invention also provides a nucleic acid encoding MYDGF or a fragment or variant thereof for use in treating and/or preventing cardiogenic shock. According to a preferred embodiment, the fragment or variant of MYDGF exhibits the biological function of MYDGF. According to another preferred embodiment, the nucleic acid encodes an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1.

The nucleic acid used according to the present invention may further comprise a transcription control element or expression control sequence positioned to control protein expression. Such a nucleic acid together with the control element is generally referred to as an expression system. As used herein, the term "expression system" refers to a system designed to produce one or more gene products of interest. Typically, such a system is "artificially" designed, i.e., by means of gene technology that can be used to produce gene products of interest in vivo, in vitro or in vitro. The term "expression system" further encompasses gene products of interest, including transcription of polynucleotides, mRNA splicing, translation into polypeptides, co-translation and post-translational modification of polypeptides or proteins, and protein targeting to one or more compartments inside cells, secretion from cells or protein uptake into the same or another cell. This general description refers to expression systems for use in eukaryotic cells, tissues, or organisms. Expression systems for prokaryotic systems may differ, and how to construct expression systems for prokaryotic cells is well known in the art.

Regulatory elements present in a gene expression cassette typically include: (a) a promoter transcriptionally coupled to a nucleotide sequence encoding a polypeptide, (b) a 5' ribosomal binding site functionally coupled to the nucleotide sequence, (c) a terminator linked to the 3' end of the nucleotide sequence, and (d) a 3' polyadenylation signal functionally coupled to the nucleotide sequence. Additional regulatory elements that serve to increase or regulate gene expression or polypeptide processing may also be present. Promoters are genetic elements that are recognized by RNA polymerase and mediate transcription of downstream regions. Preferred promoters are strong promoters that provide increased levels of transcription. Examples of strong promoters are the immediate early human cytomegalovirus promoter (CMV) and CMV with intron A (Chapman et al, Nucl. Acids Res. 19:3979-3986, 1991). Additional examples of promoters include naturally occurring promoters, such as the EF1α promoter, the murine CMV promoter, the Rous sarcoma virus promoter, and the SV40 early/late promoter, and the [β]-actin promoter; and artificial promoters, such as a synthetic muscle-specific promoter and a chimeric muscle-specific/CMV promoter (Li et al., Nat. Biotechnol. 17:241-245, 1999, Hagstrom et al., Blood 95:2536-2542, 2000).

The ribosome binding site is located at or near the start codon. Examples of preferred ribosome binding sites include CCACCAUGG, CCGCCAUGG, and ACCAUGG, where AUG is the start codon (Kozak, Cell 44:283-292, 1986). The polyadenylation signal is responsible for cleaving the transcribed RNA and adding a poly(A) tail to the RNA. The polyadenylation signal of higher eukaryotes contains an AAUAAA sequence approximately 11-30 nucleotides away from the polyadenylation addition site. The AAUAAA sequence is involved in signaling RNA cleavage (Lewin, Genes IV, Oxford University Press, NY, 1990). The poly(A) tail is very important for mRNA processing, export from the cell nucleus, translation, and stability.

Polyadenylation signals that can be used as part of a gene expression cassette include the minimal rabbit [β]-globulin polyadenylation signal and bovine growth hormone polyadenylation (BGH) (Xu et al., Gene 272:149-156, 2001, Post et al., U.S. Patent No. 5,122,458).

Examples of additional regulatory elements that may be present to increase or regulate gene expression or polypeptide processing include enhancers, leader sequences, and operators. Enhancer regions increase transcription. Examples of enhancer regions include CMV enhancers and SV40 enhancers (Hitt et al., Methods in Molecular Genetics 7:13-30, 1995, Xu, et al., Gene 272:149-156, 2001). Enhancer regions can be combined with promoters.

The expression of the MYDGF protein or its variant according to the present invention can be regulated. Such regulation can be accomplished at many steps of gene expression. Possible regulatory steps are, for example, but not limited to, transcription initiation, promoter clearance, transcription elongation, splicing, nuclear export, mRNA stability, translation initiation, translation efficiency, translation elongation, and protein folding. Other regulatory steps that affect the concentration of MYDGF polypeptide in the cell will also affect the half-life of the protein. Such a regulatory step is, for example, regulated denaturation of the protein. Since the protein of the present invention comprises a secretory protein, the protein can be directed to the secretory pathway of the host cell. The secretion efficiency, together with the regulatory steps involving expression and protein stability, regulates the concentration of the corresponding protein outside the cell. Extracellular can refer to, for example but not limited to, culture medium, tissue, intracellular matrix or space, or body fluids (such as blood or lymph).

The control of the above-mentioned regulation step can be, for example, cell type or tissue type independent or cell type or tissue type specific. In a particularly preferred embodiment of the present invention, the control of the regulation step is cell type or tissue type specific. Such a cell type or tissue type specific regulation is preferably achieved through a regulation step involving nucleic acid transcription. This transcription regulation can be achieved by using a cell type or tissue type specific promoter sequence. The results of this cell type or tissue type specific regulation can have different degrees of specificity. This means that the expression of the corresponding polypeptide is increased in the corresponding cell or tissue compared to other cell or tissue types, or that the expression is restricted to the corresponding cell or tissue type. Cell or tissue type specific promoter sequences are well known in the art and can be used for a wide range of cell or tissue types.

The expression is not necessarily cell type or tissue type specific, but may depend on physiological conditions. Such conditions are, for example, inflammation or wounds. Such physiological condition-specific expression can also be achieved by regulation of all the above-mentioned regulatory steps. A preferred way to regulate physiological condition-specific expression is transcriptional regulation. For this purpose, wound or inflammation-specific promoters can be used. Corresponding promoters are, for example, naturally occurring sequences, which can, for example, be derived from genes that are specifically expressed during immune responses and/or regeneration of injured tissues. Another possibility is to use artificial promoter sequences, which are constructed, for example, by combining two or more naturally occurring sequences.

The regulation may be cell type or tissue type specific as well as physiological condition specific. In particular, the expression may be cardiac specific. Preferably, the expression is cardiac specific and/or wound specific.

Another possibility for regulating the expression of the MYDGF protein or its variant according to the present invention is the conditional regulation of gene expression. To achieve conditional regulation, an operator sequence can be used. For example, the Tet operator sequence can be used to inhibit gene expression. Conditional regulation of gene expression by the Tet operator together with the Tet repressor is well known in the art, and many corresponding systems have been established for a wide range of prokaryotic and eukaryotic organisms. The skilled artisan knows how to select a suitable system and transform it to the specific needs of the corresponding application.

In a particularly preferred embodiment, the use of the nucleic acid according to the present invention comprises administration to an individual or patient, preferably an individual or patient suffering from cardiogenic shock.

According to another aspect, the present invention provides a vector comprising the nucleic acid or expression system described herein for use in treating and/or preventing cardiogenic shock.

As used herein, the term "vector" refers to a protein or polynucleotide or a mixture thereof into which the protein and/or nucleic acid contained therein is to be introduced or introduced into a cell. In the context of the present invention, the gene of interest encoded by the introduced polynucleotide is preferably expressed in a host cell after introduction into one or more vectors. Examples of suitable vectors include, but are not limited to, plasmid vectors, cosmid vectors, phage vectors (such as lambda phage, filamentous phage vectors), viral vectors, virus-like particles, and bacterial spores.

In a preferred embodiment of the present invention, the vector is a viral vector. Suitable viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, alpha virus vectors, herpes virus vectors, measles virus vectors, pox virus vectors, vesicular stomatitis virus vectors, retrovirus vectors, and lentivirus vectors.

In a particularly preferred embodiment of the present invention, the vector is an adenovirus or adeno-associated virus (AAV) vector.

A nucleic acid encoding one or more MYDGF proteins or variants thereof according to the present invention can be introduced into a host cell, tissue or individual using a vector suitable for therapeutic administration. Suitable vectors can preferably deliver nucleic acids to target cells without causing unacceptable side effects.

In a particularly preferred embodiment, the use of the vector according to the present invention comprises administration to a subject in need thereof.

The vector comprising a nucleic acid encoding a MYDGF protein or a fragment or variant thereof that better exhibits the above-mentioned biological function of MYDGF is preferably used for treating and/or preventing cardiogenic shock.

According to another aspect, the present invention provides a host cell comprising a vector as described herein and expressing a nucleic acid encoding a MYDGF protein or a fragment or variant thereof, which is used to treat and/or prevent cardiogenic shock. According to a preferred embodiment, the fragment or variant of MYDGF exhibits the biological function of MYDGF.

According to another aspect, the present invention provides a pharmaceutical composition comprising MYDGF protein or its fragment or variant and a suitable pharmaceutical formulation as appropriate, for treating and/or preventing cardiogenic shock. According to a preferred embodiment, the fragment or variant of MYDGF exhibits the biological function of MYDGF.

As used herein, the term "appropriate pharmaceutical excipient" refers to a pharmacologically inactive substance, such as, but not limited to, a diluent, excipient, surfactant, stabilizer, physiological buffer solution or vehicle administered with the therapeutically active ingredient. "Pharmaceutical excipient" is also called "pharmaceutical carrier" and can also be liquid or solid. Liquid carriers include, but are not limited to, sterile liquids, such as saline solutions in water and oils, including, but not limited to, those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions as well as aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. When the pharmaceutical composition is administered intravenously, saline solutions are preferred carriers. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. In a preferred embodiment of the present invention, the carrier is a suitable pharmaceutical excipient. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol and the like. These suitable pharmaceutical excipients are preferably pharmaceutically acceptable.

The terms "pharmaceutical," "medicament," and "drug" are used interchangeably herein to refer to a substance and/or combination of substances used to identify, prevent and/or treat a tissue condition or disease.

"Pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and more specifically in humans. The term "pharmaceutically acceptable salt" refers to a salt of a protein or peptide of the present invention. Suitable pharmaceutically acceptable salts include acid addition salts, which can be formed, for example, by mixing a solution of a peptide of the present invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. In addition, when the peptide carries an acidic moiety, its suitable pharmaceutically acceptable salt may include alkali metal salts (e.g., sodium salts or potassium salts); alkaline earth metal salts (e.g., calcium salts or magnesium salts); and salts formed with suitable organic ligands (e.g., ammonium, quaternary ammonium and amine cations using relative anion formation, the relative anion being such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyl sulfonates and aryl sulfonates). Illustrative examples of pharmaceutically acceptable salts include, but are not limited to, acetates, adipates, alginates, ascorbic acid, aspartates, benzenesulfonates, benzoates, bicarbonates, bisulfates, bitartrates, borates, bromides, butyrates, calcium edetate, camphorates, camphorsulfonates, Camsylate, carbonate, chloride, citrate, clavulanate, cyclopentane propionate, digluconate, dihydrochloride, dodecyl sulfate, edetate, edisylate, etopoate, benzenesulfonate, ethanesulfonate, formate, fumarate, glucoheptonate, glucoheptonate, gluconate, glutamine Acid salt, glycerophosphate, hydroxyacetylaminophenylarsine salt, hemisulfate, heptanoate, caproate, hexylisophthalate, hepamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, hydroxynaphthoate, iodide, isosulfate, lactate, lactobionate, laurate, dodecyl sulfate, apple acid, maleate, malonic acid Salt, mandelate, mesylate, methanesulfonate, methylsulfate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucosamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate/diphosphate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, theocyanate, toluenesulfonate, triethioidide, undecanoate, valerate, and the like (see, e.g., S. M. Berge et al., "Pharmaceutical Salts", J. Pharm. Sci., 66, pp. 1-19 (1977)).

The term "active ingredient" refers to a biologically active substance in a pharmaceutical composition or formulation, i.e., a substance that provides pharmaceutical value. A pharmaceutical composition may contain one or more active ingredients that may act in combination with one another or independently. The active ingredient may be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with free amine groups, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with free carboxyl groups, such as, but not limited to, those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

The terms "preparation" and "composition" are intended to include the formulation of the active compound with encapsulating materials as a carrier providing, for example, a capsule in which the active ingredient is surrounded by a carrier with or without other carriers which are in combination with the active ingredient.

As used herein, the term "carrier" refers to a pharmacologically inactive substance, such as, but not limited to, a diluent, excipient or vehicle administered with a therapeutically active ingredient. Such pharmaceutical carriers may be liquid or solid. Liquid carriers include, but are not limited to, sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions as well as aqueous dextrose and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions. Saline solutions are preferred carriers when the pharmaceutical composition is administered intravenously. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin.

Suitable pharmaceutical "formulations" include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol and the like. The term "composition" is preferably intended to include the formulation of the active compound with an encapsulating material such as a carrier providing a capsule in which the active ingredient, with or without other carriers, is surrounded by the carrier, which is in combination with the active compound.

According to one embodiment, the active ingredient is administered to a cell, tissue or individual in a therapeutically effective amount. A "therapeutically effective amount" is an amount of the active ingredient sufficient to achieve the intended purpose. The active ingredient may be a therapeutic agent. The effective amount of a given active ingredient will vary depending on parameters such as the nature of the ingredient, the route of administration, the size and species of the individual receiving the active ingredient, and the purpose of administration. The effective amount for each individual case can be determined empirically by those skilled in the art according to established methods in the art. As used in the context of the present invention, "administration" includes administration to an individual in vivo and direct administration to a cell or tissue in vitro or ex vivo.

In a preferred embodiment of the present invention, the pharmaceutical composition is customized for treating a disease and in particular for treating cardiogenic shock. In a further preferred embodiment of the present invention, the pharmaceutical composition is customized for preventing a disease and in particular for preventing cardiogenic shock. According to a particularly preferred embodiment, the pharmaceutical composition is customized for preventing and treating cardiogenic shock. In a particularly preferred embodiment of the present invention, treatment with the pharmaceutical composition according to the present invention comprises treating an individual in need of such treatment and/or preventing cardiogenic shock in an individual in need thereof.

The pharmaceutical compositions contemplated by the present invention can be formulated in a variety of ways well known to those skilled in the art. For example, the pharmaceutical compositions of the present invention can be in liquid form, such as in the form of solutions, emulsions or suspensions. Preferably, the pharmaceutical compositions of the present invention are formulated for parenteral administration, preferably intravenous, intraarterial, intramuscular, subcutaneous, transdermal, intrapulmonary, intraperitoneal, intracoronary, intracardiac administration, or administration via mucosa, preferably intravenous, subcutaneous, or intraperitoneal administration. Formulations for oral or anal administration are also feasible. Preferably, the pharmaceutical compositions of the present invention are in the form of sterile aqueous solutions, which may contain other substances, such as sufficient salts or glucose to make the solution isotonic with the blood. If necessary, the aqueous solution should be appropriately buffered (preferably pH 3 to 9, more preferably pH 5 to 7). The pharmaceutical composition is preferably in unit dosage form. In this form, the pharmaceutical composition is divided into unit doses containing appropriate amounts of the active ingredient. The unit dosage form can be a packaged preparation that contains discrete amounts of the pharmaceutical composition, such as a vial or an ampoule.

The pharmaceutical composition is preferably administered via intravenous, intraarterial, intramuscular, subcutaneous, transcutaneous, intrapulmonary, intraperitoneal, intracoronary or intracardiac routes, including other routes of administration known in the art.

If the pharmaceutical composition is used as a treatment for an individual or for the prevention of a disease or condition, the use of the pharmaceutical composition may replace the standard treatment or prevention of the corresponding disease or condition, or may be administered in addition to the standard treatment. In the case of additional use of the pharmaceutical composition, the pharmaceutical composition may be administered before, simultaneously with, or after the standard treatment and/or prevention.

It is further preferred that the pharmaceutical composition is administered once or more than once. This includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 times. There is no limit to the time span for administering the agent. Preferably, administration does not exceed 1, 2, 3, 4, 5, 6, 7 or 8 weeks.

A single dose of the pharmaceutical composition may independently form the total amount of the administered dose, or the corresponding time span of administration may include administration as one or more boluses and/or infusions.

According to another aspect, the present invention provides a method for treating and/or preventing cardiogenic shock, comprising administering a therapeutically effective amount of MYDGF or a fragment or variant thereof to a patient in need thereof. Suitable MYDGF proteins, fragments or variants thereof include those described in the first aspect. According to a preferred embodiment, a fragment or variant of MYDGF exhibits the biological function of MYDGF. In the method, MYDGF preferably comprises SEQ ID NO: 1, or a fragment or variant thereof that exhibits the biological function of MYDGF of SEQ ID NO: 1. In this aspect, the fragment or variant preferably comprises an amino acid sequence having at least 85% amino acid sequence identity with SEQ ID NO: 1.

For example, the administration can be carried out as described in the first aspect. According to a preferred embodiment of this method of the present invention, the MYDGF protein or its fragment or variant is administered via one or more bolus injections and/or infusions, preferably in a pharmaceutically acceptable carrier and/or formulation.

According to another aspect, the present invention provides a method for producing a non-human mammal model of cardiogenic shock, the method comprising (i) temporarily ligating a coronary artery of the mammal, (ii) establishing reperfusion, and (iii) ventilating the mammal at an inspired oxygen fraction of about 0.18 or less. Temporary ligation can be performed by any method known to those skilled in the art that is suitable for blocking or significantly reducing blood flow. According to a preferred embodiment, ligation is performed by placing and tightening a thread or wire around the coronary artery, thereby blocking or substantially blocking blood flow through the artery. Reperfusion is preferably established by reestablishing blood flow, such as by opening a thread or wire to allow blood to flow through the artery. Ventilation of the mammal is performed by standard devices known in the art, such as mechanical ventilation commonly used in mammalian surgery.

This approach does not specifically provide treatment to the animal, but rather induces a condition resembling cardiogenic shock. Cardiogenic shock is characterized by, for example, a decrease in ventricular end-systolic pressure (VESP) and cardiac output and an increase in arterial lactate concentration compared to healthy individuals or to individuals who have undergone the approach before. The advantage of the novel non-human mammalian model of cardiogenic shock is that small animals (such as rodents) that are established in laboratory practice can be used. There is no need to use large animals (such as pigs), which are more expensive to feed, house and husbandry and are more complicated to investigate. The novel model allows studies to be performed on genetically modified mice, for example to explore the molecular mechanisms of cardiogenic shock. Genetically modified mice can be easily generated, are already available in the research community, or can be obtained commercially from, for example, The Jackson Laboratory. In contrast, genetically modified large animals (such as pigs) are extremely difficult to generate, and there are very few genetically modified large animals available.

Therefore, according to a preferred embodiment, the non-human mammal is a rodent. According to a particularly preferred embodiment, the non-human animal is a mouse.

According to a preferred embodiment, the coronary artery is the proximal left anterior descending coronary artery. Alternatively, any other major coronary artery can be used, with temporary ligation ultimately resulting in a decrease in ventricular end-systolic pressure (VESP) and an increase in arterial lactate concentration.

According to another preferred embodiment, the coronary artery is ligated for about 30 minutes to about 90 minutes before reperfusion is established. According to another preferred embodiment, the coronary artery is ligated for a time selected from a range having a lower value of about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or 85 minutes, and a higher value of about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90 minutes, or any combination thereof. More preferably, the coronary artery is ligated for about 45 to 70 minutes, and most preferably for about 60 minutes.

According to yet another preferred embodiment, the mammal is ventilated with a fraction of inspired oxygen (FiO ₂ ) between about 0.18 and about 0.12. According to yet another preferred embodiment, the mammal is ventilated with a fraction of inspired oxygen selected from a range having a lower value of about 0.12, 0.13, 0.14, 0.15, 0.16, or 0.17 and a higher value of about 0.13, 0.14, 0.15, 0.16, 0.17, or 0.18, and any combination thereof. More preferably, the mammal is ventilated with a fraction of inspired oxygen of about 0.17 to about 0.14, and most preferably about 0.16.

The present invention also provides a model animal obtained by implementing a method for producing a non-human mammal model of cardiogenic shock. Example

The examples are designed to further illustrate the present invention and facilitate a more complete understanding. They should not be interpreted as limiting the scope of the present invention in any way. Materials and methods used in the examples:

Unless otherwise stated, the following materials and methods were used in the examples. Recombinant MYDGF.

In the [+G] MYDGF variant (HEK) used in the examples, the N-terminal V residue at position +1 of mature human MYDGF is preceded by a G residue. The variant has a sequence as shown in SEQ ID NO: 3 and was produced as described in Polten, F. et al. (2019), Anal Chem, 91, 1302-1308, page 1303, column 1 and Figure S1, and Ebenhoch, R. et al. (2019), Nat Commun 10, 5379, page 8, left column. Mouse surgery and functional assessment.

All surgical procedures were approved by the authorities of Hannover, Germany (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit). All animal procedures complied with the guidelines of EU Directive 2010/63 on the protection of animals used for scientific purposes. Mice were housed in individually ventilated cages with a 12-h light/dark cycle at the Central Animal Facility of the Medical University of Hannover. Food and water were provided ad libitum. During surgery, mice were placed on a heating pad connected to a temperature controller (Föhr Medical Instruments) to maintain a rectal temperature of 37°C. Statistical analysis

Littermates were randomly assigned to different experimental groups. Data were normally distributed based on visual inspection, and the variance was similar between the different groups. Data are presented as mean ± sem. Two independent sample t tests were used to compare the two groups. For comparisons of more than two groups, one-way ANOVA was used if there was one independent variable and two-way ANOVA was used if there were two independent variables. Dunnett's post hoc test was used for multiple comparisons with a single control group. Tukey's post hoc test was used to adjust for multiple comparisons. Two-tailed P values less than 0.05 were considered statistically significant. KCW had full access to all data in the study and takes responsibility for the integrity of the data and data analysis. Example 1 :

Animal model of cardiogenic shock was established. Myocardial infarction (MI) was induced in 8-10 week old male C57BL/6N mice by temporary ligation of the proximal left anterior descending coronary artery for 60 minutes. Animals were pretreated with 2 mg/kg butorphanol and 0.02 mg/kg atropine subcutaneously to reduce bronchial secretions. Anesthesia was induced with 3% to 4% isoflurane. After intubation, mice were mechanically ventilated and anesthesia was maintained with 1% to 2% isoflurane. After left thoracotomy, the left anterior descending coronary artery (LAD) was ligated with a 7-0 prolene (Ethicon, catalog number EH7405) slipknot to induce ischemia and removed 1 h later to induce reperfusion. In sham-operated mice, the ligature around the LAD was not tied. After coronary reperfusion, a conductance catheter with a micromanometer tip was inserted through the right carotid artery to record left ventricular (LV) pressure-volume loops. At the end of the protocol, arterial blood samples were drawn from the left ventricle.

In preliminary experiments, coronary artery ligation alone only slightly reduced LV end-systolic pressure (LVESP) and did not increase arterial lactate concentration, a biomarker of peripheral hypoperfusion and tissue hypoxia in cardiogenic shock (CS). Based on the premise that MI combined with hypoxic ventilation may induce CS, mice were ventilated (medical O ₂ mixed with medical N ₂ ) after reperfusion with different inspired oxygen fractions (FiO ₂ ). During isoflurane anesthesia, infarcted and sham-operated mice were randomly ventilated with either an FiO ₂ of 0.33, which correlates with normoxemia (nx; arterial oxygen partial pressure [PaO ₂ ], 144 ± 16 mm Hg; arterial oxygen saturation [SaO ₂ ], 99 ± 1%), or an FiO ₂ of 0.16, which induces mild hypoxemia (hx; PaO ₂ , 75 ± 16 mmHg; SaO ₂ , 89 ± 3%; 4 to 6 mice per group) (Fig. 1A).

Exemplary pressure-volume loops recorded after 120 min are presented (Fig. 1B). During the 120-min period, LVESP (Fig. 2A) and cardiac output (Fig. 2B) progressively decreased in MI-hx mice (MI with CS) but remained stable in MI-nx mice (MI without CS). At 120 min, MI-hx mice developed arterial hyperlactatemia (Fig. 2C) and exhibited more severe systolic and diastolic dysfunction than MI-nx mice (LV ejection fraction, 18 ± 2 vs 33 ± 2%, P <0.001; LV end-diastolic pressure, 14 ± 1 vs 9 ± 1 mmHg, P = 0.005). Heart rate was unaffected (475 ± 16 vs 486 ± 8 min ^-1 ). The mortality rate of MI-hx mice (11 of 23 mice died; all deaths were related to CS) was higher than that of MI-nx mice (1 of 12 mice died, P = 0.031).

Intravenous infusion of dobutamine, an established symptomatic treatment for patients with CS (Vahdatpour et al. Journal of the American Heart Association, Vol 8(8), 2019, e011991), increased LVESP (Figure 3A) and cardiac output (Figure 3B) and decreased arterial lactate in MI-hx mice (5.6 ± 0.6 vs 11.1 ± 0.7 mmol/L, P < 0.001, 4-6 mice per group).

As a test case for this model, high-resolution mass spectrometry was used to define the phosphoproteomic signature of non-infarcted LV myocardium at 120 min. Among 1,264 different proteins, 9,004 phospho-sites were detected. Principal component analysis showed that the four groups (Sham-nx = normoxic blood in sham-operated mice; Sham-hx = hypoxic blood in sham-operated mice; MI-nx = normoxic blood in infarcted mice; MI-hx = hypoxic blood in infarcted mice) were associated with distinct phosphoproteomic signatures (Figure 4). The phosphoproteomic signatures of sham-hx and sham-nx mice overlapped (Figure 4), reflecting the observation that hypoxemia itself does not alter cardiac function (Figures 2A and 2B). In contrast, MI-hx and MI-nx mice displayed distinct phosphoproteomic signatures (Fig. 4), with 72 differentially regulated phosphosites (Fig. 5), suggesting potential therapeutic entry points.

Further details are provided in Wang Y, Polten F, Jäckle F, Korf-Klingebiel M, Kempf T, Bauersachs J, Freitag-Wolf S, Lichtinghagen R, Pich A, Wollert KC. A mouse model of cardiogenic shock. Cardiovasc Res. 2021;117:2414-2415, which is incorporated by reference in its entirety. The animal model of the present invention is the first of its kind and replicates the key features of CS in patients, including severe systolic and diastolic dysfunction, low cardiac output and hypotension, increased arterial lactate concentration, hemodynamic response to dopamine, and high mortality. The model provides a platform to explore the molecular pathophysiology of CS and develop much-needed therapies. Example 2 :

The MYDGF protein (human factor 1; C19orf10) was identified as described in detail in WO 2014/111458. The nucleic acid sequence encoding human factor 1 is available at NCBI Gene ID: 56005 (SEQ ID NO: 4). The amino acid sequence of human factor 1 including the N-terminal signal peptide is described in detail in SEQ ID NO: 2. In the example, the human [+G] MYDGF variant without a signal peptide according to SEQ ID NO: 3 is used, and as described in detail in Ebenhoch R. et al., Crystal structure and receptor-interacting residues of MYDGF - a protein mediating ischemic tissue repair (Nat Commun. 2019 Nov 26;10(1):5379 and Polten et al. Plasma Concentrations of Myeloid-Derived Growth Factor in Healthy Individuals and Patients with Acute Myocardial Infarction as Assessed by Multiple Reaction Monitoring-Mass Spectrometry. Anal Chem. 2019 Jan 15;91(2):1302-1308). Example 3 :

Acute myocardial infarction was induced in C57BL6/N mice by temporary ligation of the left anterior descending coronary artery for 60 minutes, pretreated with butorphanol (2 mg/kg, subcutaneous (sc)) and atropine (0.02 mg/kg, sc) to reduce bronchial secretions. Human MYDGF (10 µg in 100 µL) or saline (control) was bolus injected into the left ventricular cavity during reperfusion. After reperfusion, a conductance catheter with a micromanometer tip was inserted through the right carotid artery to continuously record left ventricular pressure-volume (PV) loops. Thereafter, cardiogenic shock (CS) was induced by hypoxic ventilation (FiO ₂ 0.16), and mice received a continuous infusion of MYDGF (5 µg/h, infusion rate: 2 µL/min) or saline (control) into the left jugular vein for 120 min. After 120 min, an arterial blood sample (anticoagulated with heparin) was drawn from the left ventricle and used for immediate blood gas and lactate analysis. Another blood sample was drawn, treated with EDTA, and centrifuged at 3,500 g for 10 min at 4°C to obtain plasma, which was stored at -80°C until further analysis. Figure 13 schematically illustrates this procedure (abbreviations in Figure 13 are as defined in Figure 1; sc = subcutaneous administration; iv = intravenous administration).

The results are shown in Figures 6 to 12.

MYDGF significantly improved the survival rate of mice with cardiogenic shock, as shown in Figure 6.

Experiments further demonstrated that administration of MYDGF improved left ventricular systolic and diastolic function (PV loop recordings in Figures 7A to 7F and 8A to 8F).

MYDGF further reversed acidemia and reduced lactate concentration, two defining features of cardiogenic shock (blood gas analysis in Figure 9).

MYDGF has also been shown to prevent the decline in cardiac output and LVESP, two further defining features of cardiogenic shock (serial PV loop recordings in Figures 10A and 10B).

The experiment also demonstrated that treatment with MYDGF reduced infarct size (Figures 11A and 11B). To this end, the left ventricle was removed at the end of the observation period, and the area at risk and infarct size were measured by staining with Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) (as described in Korf-Klingebiel et al. Nat Med. 2015;21:140-149).

The effect of MYDGF treatment on plasma sarcoma and alanine aminotransferase (ALT) concentrations measured at the end of the 120-minute observation period are shown in Figures 12A and 12B. Plasma sarcoma is an indicator of cardiac damage, while ALT reflects end-organ (liver) damage. MYDGF treatment of CS mice reduced plasma sarcoma and alanine aminotransferase levels. At the end of the 120-minute observation period, the effect of MYDGF on cell death in the infarct area was determined by measuring soluble nucleosomes released due to cell death. Measurements were performed using the commercially available Cell Death Detection ELISA from Roche. FIG12C shows the ratio of soluble nucleosomes in the infarct area (I) to soluble nucleosomes in the non-infarct area (NI) as an indicator of cell death, and demonstrates that MYDGF significantly reduces the amount of soluble nucleosomes, thereby reducing cell death in the infarct area. Example 4 :

MYDGF knockout mice were obtained as described by Korf-Klingebiel et al. (Nature Medicine, 2015, Vol. 21(2):140-149). As described in Example 1, cardiogenic shock (CS) was induced in wild-type (WT) and MYDGF knockout (KO) mice. The mortality rates of WT and MYDGF knockout mice were compared during the 120-minute observation period. As shown in FIG14 , the mortality rate of KO mice was significantly higher, demonstrating that endogenous MYDGF has a protective effect against cardiogenic shock. Example 5 :

As shown, for example, in FIG10 , cardiac function continues to deteriorate during cardiogenic shock. This deterioration is thought to be due to continued aggravation of left ventricular tissue damage during cardiogenic shock. To test this hypothesis, acute MI was induced by temporary ligation of the left anterior descending coronary artery for 60 min. After reperfusion, CS was induced with hypoxic ventilation (FiO ₂ 0.16; MI/with shock). Infarct control mice were ventilated with normoxic ventilation (FiO ₂ 0.33; MI/without shock). The experimental setup is shown in FIG15A .

60 minutes after reperfusion and at the end of the experiment, the left ventricles were removed (5 MI/non-shock mice and 5 MI/shock mice at each time point) and cell death (soluble nucleosomes) in the infarct area (I) and the non-infarct distal area (NI) was measured using Roche's cell death detection ELISA. The I/NI ratio was recorded as a measure of cell death. The results are shown in Figure 15B. During the observation period, the concentration of soluble nucleosomes in the infarct area increased in both groups ([apoptotic] cell death takes time to develop gradually). The increase in soluble nucleosome concentrations in MI/shock mice was significantly more obvious than in MI/non-shock mice, indicating that CS further exacerbates the persistent damage of left ventricular tissue during acute MI. Example 6 :

As shown in Example 5, cardiogenic shock further exacerbates left ventricular persistent tissue damage during acute myocardial infarction. Therefore, it was tested whether delayed MYDGF therapy could alleviate the symptoms of cardiogenic shock and improve cardiac function. To this end, acute myocardial infarction was induced in mice by temporary ligation of the left anterior descending coronary artery for 60 min. After reperfusion, a conductance catheter with a micromanometer at the tip was inserted through the right carotid artery to continuously record left ventricular pressure-volume (PV) loops. Thereafter, cardiogenic shock was induced with hypoxic ventilation (FiO ₂ 0.16). Starting 60 min after reperfusion, mice received a continuous left jugular intravenous infusion of (i) human MYDGF (5 µg/h, infusion rate: 2 µL/min) or (ii) saline (control) until the end of the infusion. Blood was drawn from the left ventricle (anticoagulated with heparin) and used for immediate blood gas and lactate analysis. Three MI/shock mice received delayed MYDGF therapy, while three MI/shock mice received delayed saline infusion. The experimental set-up is shown in Figure 16A.

The results are shown in Figure 16B. One saline-treated control mouse died. In the surviving animals, cardiac function (e.g., left ventricular ejection fraction, LVEF) and peripheral tissue perfusion (e.g., lactate concentration) were better in MYDGF-treated mice than in saline-treated control mice. These data suggest that delayed MYDGF therapy can still promote beneficial effects in cardiogenic shock. Project

The projects of the present invention relate to the following: Project 1.   A myeloid-derived growth factor (MYDGF) or a fragment or variant thereof that exhibits the biological function of MYDGF, which is used for treating and/or preventing cardiogenic shock. Project 2.   The MYDGF as in Project 1, wherein the MYDGF comprises: (i) SEQ ID NO: 1; or (ii) a fragment or variant of SEQ ID NO: 1 that exhibits the biological function of MYDGF, wherein the variant comprises an amino acid sequence having at least 85% amino acid sequence identity with SEQ ID NO: 1. Project 3.   The MYDGF as in Project 1, wherein the MYDGF protein consists of SEQ ID NO: 1 or SEQ ID NO: 3. Project 4.   A nucleic acid encoding MYDGF or a fragment or variant thereof that exhibits the biological function of MYDGF, which is used for treating cardiogenic shock. Item 5.   A nucleic acid as in Item 4, wherein the nucleic acid encodes an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1. Item 6.   A vector comprising the nucleic acid as in Item 5, for use in treating and/or preventing cardiogenic shock. Item 7.   A host cell comprising the nucleic acid as in Item 5 or the vector as in Item 6 and preferably expressing the nucleic acid, for use in treating and/or preventing cardiogenic shock. Item 8.   A pharmaceutical composition comprising the MYDGF protein as in any one of Items 1 to 3 or a fragment or variant thereof exhibiting the biological function of MYDGF, the nucleic acid as in any one of Items 4 or 5, the vector as in Item 6, or the host cell as in Item 7, for use in treating and/or preventing cardiogenic shock. Item 9.   The pharmaceutical composition of Item 8, wherein the pharmaceutical composition is administered orally, intravenously, subcutaneously, intramucosally, intraarterially, intramuscularly or intracoronarily. Item 10. The pharmaceutical composition of Item 9, wherein the administration is by one or more boluses and/or infusions. Item 11. A method for treating and/or preventing cardiogenic shock, comprising administering to a patient in need thereof a therapeutically effective amount of MYDGF or a fragment or variant thereof that exhibits the biological function of MYDGF. Item 12. A method for treating and/or preventing cardiogenic shock, comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a myeloid-derived growth factor (MYDGF) protein. Item 13. A method as in item 11 or 12, wherein the MYDGF comprises: (i) SEQ ID NO: 1; or (ii) a fragment or variant of SEQ ID NO: 1 that exhibits the biological function of MYDGF, wherein the variant comprises an amino acid sequence having at least 85% amino acid sequence identity with SEQ ID NO: 1. Item 14. A method as in any one of items 11 to 13, wherein the MYDGF or its fragment or variant is administered by one or more bolus injections and/or infusions, preferably in a pharmaceutically acceptable carrier and/or formulation. Item 15. A method for producing a non-human mammal model of cardiogenic shock, the method comprising: (i) temporarily ligating a coronary artery of a mammal, (ii) establishing reperfusion, and (iii) ventilating the mammal at a fraction of inspired oxygen of about 0.18 or less. Item 16. The method of Item 15, wherein the non-human mammal is a rodent, preferably wherein the non-human mammal is a mouse. Item 17. The method of Item 15 or 16, wherein the coronary artery is ligated for about 30 minutes to about 90 minutes before establishing reperfusion. Item 18. The method of any one of Items 15 to 17, wherein the mammal is ventilated at a fraction of inspired oxygen of about 0.16.

Figure 1: A: Schematic diagram of the procedure used to prepare the animal model of cardiogenic shock. Myocardial infarction (MI) was induced in 8- to 10-week-old male C57BL/6N mice by ligation of the proximal left anterior descending coronary artery for 60 min. Animals were pretreated subcutaneously with 2 mg/kg butorphanol and with 0.02 mg/kg atropine to reduce bronchial secretions. Anesthesia was induced with 3% to 4% isoflurane. After intubation, mice were mechanically ventilated and anesthesia was maintained with 1% to 2% isoflurane. After reperfusion, a conductive catheter with a micromanometer tip was inserted through the right carotid artery to record left ventricular (LV) pressure-volume (PV) loops. Subsequently, mice were ventilated for 120 min at a fraction of inspired oxygen (FiO ₂ ) concentration of 0.33 or 0.16. As shown in the pilot experiment, FiO ₂ of 0.33 was associated with normoxemia (nx; arterial oxygen partial pressure [PaO ₂ ], 144 ± 16 mm Hg; arterial oxygen saturation [SaO ₂ ], 99 ± 1%), whereas FiO ₂ of 0.16 resulted in mild hypoxemia (hx; PaO ₂ , 75 ± 16 mmHg; SaO ₂ , 89 ± 3%) (4 to 6 mice per group). Subsequently, infarcted and sham-operated mice were randomly ventilated with FiO ₂ and followed for 120 min as described in Wang et al. Cardiovasc Res. 2021;117:2414-2415. B: Left ventricular pressure-volume loops sampled at 120 min in sham-operated mice (sham) (dashed lines) and mice with induced myocardial infarction (MI) (unbroken lines) ventilated under normoxic (nx) and hypoxic (hx) conditions.

Figure 2: Left ventricular end-systolic pressure (LVESP) (in mmHg) (Fig. 2A), cardiac output (in mL/min) (Fig. 2B), and arterial lactate concentration (in mmol/L) (Fig. 2C) at 120 min in sham-operated mice (Sham) (open circles) and mice with induced myocardial infarction (MI) (closed circles), all ventilated under normoxic (nx) or hypoxic (hx) conditions, respectively. MI mice ventilated with an FiO ₂ of 0.16 (MI-hx) progressively developed CS, as defined by the clinical features of CS, i.e., hypotension (decreased LVESP), decreased cardiac output, and increased lactate concentration. MI mice ventilated with an FiO ₂ of 0.33 (MI-nx) maintained hemodynamic stability.

Figure 3: MI-hx mice were treated with intravenous dopamine infusion (5 to 7.5 ng/g/min via the left jugular vein, titrated to LVESP of 70 mmHg) starting 60 minutes after the start of hypoxic ventilation (MI-hx, dopamine). MI-hx mice infused with saline served as controls (MI-hx, saline). Dopamine improved LVESP (Fig. 3A) and cardiac output (Fig. 3B) in MI-hx mice. Similar hemodynamic improvements are commonly observed in CS patients treated with dopamine.

Figure 4: Phosphoproteomic analysis of non-infarcted left ventricular myocardium using high-resolution mass spectrometry and unsupervised principal component analysis (n=6 mice per group). Four experimental groups were associated with unique phosphoproteomic signatures: Sham-nx = normoxic blood in sham-operated mice; Sham-hx = hypoxic blood in sham-operated mice; MI-nx = normoxic blood in infarcted mice; MI-hx = hypoxic blood in infarcted mice.

Figure 5: Volcano plot showing phosphosites differentially regulated in non-infarcted left ventricular myocardium of mice with myocardial infarction-induced CS (MI-hx) versus mice with myocardial infarction only (MI-nx). The top 10 down- or up-regulated [P<0.05] phosphosites are shown in black. MI-nx = normoxic infarcted mice; MI-hx = hypoxic infarcted mice. Abbreviations indicate proteins, and the amino acid positions shown in parentheses indicate the corresponding phosphorylated residues in each protein. RIPR1 = Rho family interacting cell polarization regulator 1. DDA1 = DET1 and DDB1 associating protein 1. OSB11 = oxysterol-binding protein-associated protein 11. ODPA = pyruvate dehydrogenase E1 component subunit alpha. SCRIB = protein Scribble homolog. SRF=serum response factor. KCNH2=potassium voltage-gated channel subfamily H member 2. CSRP1=cysteine- and glycine-rich protein 1. MYH6=myosin-6. DP13A=DCC-interacting protein 13-alpha. PDLI5=PDZ and LIM domain-containing protein 5. TITIN=titin. HSPB1=heat shock protein beta-1. DESMIN=desmin. LMNA=lamin-A/C. XIRP1=Xin actin-binding repeat-containing protein 1. TNNI3=actin I. TIF1B=transcriptional intermediary factor 1-beta.

Figure 6: Kaplan-Meier survival curves showing the mortality rate within 120 minutes in MYDGF- or saline-treated cardiogenic shock (MI-hx) mice.

Figure 7: Bar graphs showing the results of PV loop recordings at the end of the 120-minute observation period. Heart rate (Figure 7A), left ventricular end-systolic volume (LVESV) (Figure 7B), left ventricular end-diastolic volume (LVEDV) (Figure 7C), cardiac output (Figure 7D), stroke volume (Figure 7E), and cardiac work (Figure 7F) in MYDGF- or saline-treated cardiogenic shock (MI-hx) mice. *P<0.05, **P<0.01, ***P<0.001.

Fig. 8: Bar graphs showing the results of PV loop recordings at the end of the 120-minute observation period. Left ventricular end-systolic pressure (LVESP) (Fig. 8A), left ventricular end-diastolic pressure (LVEDP) (Fig. 8B), left ventricular ejection fraction (LVEF) (Fig. 8C), left ventricular maximum pressure change rate (dP/dt _max ) (Fig. 8D), left ventricular minimum pressure change rate (dP/dt _min ) (Fig. 8E), and left ventricular diastolic time constant (Tau) (Fig. 8F) in MYDGF- or saline-treated cardiogenic shock (MI-hx) mice. **P<0.01, ***P<0.001.

Figure 9: Bar graphs showing the results of blood gas analysis at the end of the 120-minute observation period. pH (Figure 9A), arterial oxygen partial pressure ( _PaO2 , in mmHg) (Figure 9B), arterial oxygen saturation ( _SaO2 , in %) (Figure 9C), and lactate concentration (in mmol/L) (Figure 9D) in MYDGF- or saline-treated cardiogenic shock (MI-hx) _mice . *P<0.05.

Figure 10: Results of continuous PV loop recordings during the 120-minute observation period in the CS model. Cardiac output (in mL/min) (Figure 10A) and left ventricular end-systolic pressure (LVESP) (in mmHg) (Figure 10B) in saline-treated MI-hx mice (CS saline) and MYDGF-treated MI-hx mice (CS MYDGF).

Figure 11: Area at risk and infarct size of the left ventricle were determined at the end of the 120-minute observation period in the CS model. Saline-treated MI-hx mice (Saline) vs. MYDGF-treated MI-hx mice (MYDGF). Area at risk/total LV area (in %) (Figure 11A) and infarct size/area at risk (in %) (Figure 11B).

Figure 12: Plasma concentrations of high-sensitive cardiac calcitonin T (cTnT) and alanine aminotransferase (ALT), a marker of liver damage, at the end of the 120-minute observation period in the CS model. Soluble nucleosome concentrations in the left ventricle at the end of the 120-minute observation period in the CS model. cTnT (in ng/mL) (Figure 12A) and alanine aminotransferase (in U/L) in saline-treated MI-hx mice (Saline) and MYDGF-treated MI _- hx mice (MYDGF). Soluble nucleosome concentrations measured in the infarcted (I) and non-infarcted (NI) regions of the left ventricle were measured by ELISA. The I/NI ratio was used as an indicator of cell death (Figure 12C).

Figure 13: Schematic representation of the treatment regimen used in the animal model of cardiogenic shock in the previous examples (Figures 6, 7, 8, 9, 10, 11 and 12). The abbreviations shown in the figure are as defined in Figure 1.

Figure 14: Kaplan-Meier survival curves showing the mortality of wild-type mice (WT) and Mydgf gene-deficient (knockout) mice (MYDGF KO) within 120 minutes of cardiogenic shock.

Figure 15: Cardiogenic shock exacerbates left ventricular tissue damage. Figure 15A shows the experimental set-up. Figure 15B shows the experimental results, demonstrating that cardiogenic shock further exacerbates the persistent left ventricular tissue damage during acute myocardial infarction.

Figure 16: Delayed treatment of cardiogenic shock. Figure 16A shows the experimental set up. Figure 16B shows the experimental results, indicating that cardiogenic shock can be treated by administering MYDGF at a time point after reperfusion has been established.

TW202421648A_112134385_SEQL.xml

Claims (18)

A myeloid-derived growth factor (MYDGF) or a fragment or variant thereof that exhibits the biological function of MYDGF, which is used for treating and/or preventing cardiogenic shock. MYDGF as claimed in claim 1, wherein the MYDGF comprises: (i) SEQ ID NO: 1; or (ii) a fragment or variant of SEQ ID NO: 1 that exhibits the biological function of MYDGF, wherein the variant comprises an amino acid sequence having at least 85% amino acid sequence identity with SEQ ID NO: 1. The MYDGF of claim 1, wherein the MYDGF protein consists of SEQ ID NO: 1 or SEQ ID NO: 3. A nucleic acid encoding MYDGF or a fragment or variant thereof that exhibits the biological function of MYDGF, which is used for treating and/or preventing cardiogenic shock. The nucleic acid of claim 4, wherein the nucleic acid encodes an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1. A vector comprising the nucleic acid of claim 5, which is used for treating and/or preventing cardiogenic shock. A host cell comprising the nucleic acid of claim 5 or the vector of claim 6 and preferably expressing the nucleic acid, which is used for treating and/or preventing cardiogenic shock. A pharmaceutical composition comprising a MYDGF protein as described in any one of claims 1 to 3 or a fragment or variant thereof that exhibits the biological function of MYDGF, a nucleic acid as described in any one of claims 4 or 5, a vector as described in claim 6, or a host cell as described in claim 7, for use in treating and/or preventing cardiogenic shock. The pharmaceutical composition for use in claim 8, wherein the pharmaceutical composition is administered orally, intravenously, subcutaneously, intramucosally, intraarterially, intramuscularly or intracoronarily. A pharmaceutical composition for use as claimed in claim 9, wherein administration is by one or more boluses and/or infusions. A method for treating and/or preventing cardiogenic shock comprises administering to a patient in need thereof a therapeutically effective amount of MYDGF or a fragment or variant thereof that exhibits the biological function of MYDGF. A method for treating and/or preventing cardiogenic shock comprises administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a myeloid-derived growth factor (MYDGF) protein or a fragment or variant thereof. The method of claim 11 or 12, wherein the MYDGF comprises: (i) SEQ ID NO: 1; or (ii) wherein the fragment or variant is a fragment or variant of SEQ ID NO: 1 and exhibits the biological function of MYDGF, wherein the variant comprises an amino acid sequence having at least 85% amino acid sequence identity with SEQ ID NO: 1. A method as claimed in any one of claims 11 to 13, wherein the MYDGF or a fragment or variant thereof is administered by one or more bolus injections and/or infusions, preferably in a pharmaceutically acceptable carrier and/or formulation. A method for producing a non-human mammalian model of cardiogenic shock, the method comprising: (i) temporarily ligating a coronary artery of the mammal, (ii) establishing reperfusion, and (iii) ventilating the mammal at a fraction of inspired oxygen of about 0.18 or less. The method of claim 15, wherein the non-human mammal is a rodent, preferably wherein the non-human mammal is a mouse. The method of claim 15 or 16, wherein the coronary artery is ligated for a period of about 30 minutes to about 90 minutes before reperfusion is established. The method of any of claims 15 to 17, wherein the mammal is ventilated at a fraction of inspired oxygen of about 0.16.

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