Myeloid-derived growth factors for treatment of cardiac shock

Abstract

The present invention relates to a protein myeloid-derived growth factor (MYDGF) or a nucleic acid encoding said protein for use in the treatment and / or prevention of cardiac shock. The invention also relates to a vector comprising said nucleic acid, a host cell expressing said nucleic acid, and a pharmaceutical composition comprising said protein, nucleic acid, vector or host cell for use in the treatment and / or prevention of cardiac shock, as well as a method for the treatment and / or prevention of cardiac shock. The invention also relates to a method for preparing a cardiac shock animal model, and to the animals obtained by said method.

Description

[0001] Sequence Listing

[0002] This application contains a Sequence Listing submitted electronically in xml format and the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to a protein, myeloid-derived growth factor (MYDGF), for treating and / or preventing cardiogenic shock. The present invention also relates to a new animal model of cardiogenic shock. Background Art

[0004] Myeloid-derived growth factor (MYDGF), also known as factor 1, is a protein encoded in open reading frame 10 (C19Orf10) on human chromosome 19. In 2007, the protein was described as a new secreted factor in the synovium in a proteomic analysis of so-called fibroblast-like synoviocytes (FLS cells). In the absence of any experimental or statistical evidence, a correlation between the secretion of this protein and inflammatory diseases of the joints has been 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 protection of the protein as a therapeutic agent for the treatment of joints and for the diagnosis of tissues that have undergone growth changes and for monitoring changes in tissues (US2008 / 0004232A1, Characterization of c19orf10, a novel synovial protein). Another scientific publication describes the enhanced expression of this protein in hepatocellular carcinoma cells (Sunagozaka et al., International Journal of Cancer, 2010, identification of a secretariant protein c19orf10 activated in hepatocellular carcinoma). The recombinantly produced protein showed a proliferation enhancing effect on cultured hepatocellular carcinoma cells. It should be noted that C19Orf10 also refers to IL-25, IL-27 and IL-27W, as it was originally considered an interleukin. However, the terms "IL-25" and "IL-27" are not used consistently in the art and are used to refer to a variety of different proteins. For example, US2004 / 0185049 mentions a protein called IL-27 and discloses its use in regulating immune responses. This protein is structurally different from Factor 1 (the amino acid sequence of Factor 1 according to SEQ ID NO: 1 is compared with the amino acid sequence of "IL-27" according to UniProt: Q8NEV9). Similarly, EP 2 130 547 A1 mentions a protein called IL-25 and discloses its use in the treatment of inflammation. This protein is also known in the art as IL-17E and is structurally different from Factor 1 (comparing 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).

[0005] WO 2014 / 111458 discloses factor 1 for enhancing proliferation of non-transformed tissue or non-transformed cells and inhibiting apoptosis of non-transformed tissue or non-transformed cells, in particular for treating acute myocardial infarction. Also disclosed are inhibitors of factor 1 for medical use, in particular for treating or preventing diseases in which angiogenesis promotes disease development or progression.

[0006] Korf-Klingebiel et al. (Nature Medicine, 2015, Vol. 21(2): 140-149) reported that bone marrow cells secrete C19Orf10 after myocardial infarction, a protein that 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.

[0007] WO 2021 / 148411 discloses MYDGF for treating or preventing fibrosis, hypertrophy or heart failure. Heart failure is a clinical syndrome with a poor prognosis that can develop in response to persistent hemodynamic overload, myocardial damage or genetic mutations.

[0008] Cardiogenic shock (CS) is a life-threatening acute low cardiac output state caused by severe systolic and / or diastolic myocardial dysfunction, and leads to arterial hypotension, pulmonary congestion, severe end-organ hypoperfusion and impaired tissue oxygenation. CS is associated with insufficient blood flow to the limbs and vital organs including the heart itself, liver, kidneys and brain. Severe end-organ hypoperfusion and impaired tissue oxygenation lead to increased blood lactate concentration, which can be used for diagnosis and monitoring of CS patients. The clinical criteria for defining CS are summarized in a review article by, for example, Vahdatpour et al. (Journal of the American Heart Association, Vol 8 (8), 2019, e011991). Insufficient perfusion and impaired oxygenation of the heart itself lead to progressive deterioration of cardiac performance in CS, which triggers a spiral deterioration of increased hemodynamic instability, with a very high mortality rate.

[0009] Although CS may develop as a complication of myocardial infarction, the pathophysiology of uncomplicated myocardial infarction (without cardiogenic shock) and (typically massive) myocardial infarction with cardiogenic shock differs substantially. Patients with uncomplicated myocardial infarction do not have severe end-organ hypoperfusion and impaired tissue oxygenation, they do not experience the acute and progressive deterioration of cardiac function (deteriorative spiral), and they do not experience the very high acute mortality observed in patients with CS.

[0010] Likewise, patients with heart failure, cardiac fibrosis, and cardiac hypertrophy do not acutely develop severe end-organ hypoperfusion and impaired tissue oxygenation, they do not experience an acute progressive deterioration in cardiac function (a deteriorating spiral), and they do not experience the very high acute mortality observed in patients with CS.

[0011] As an example of the different pathophysiology of these conditions, medical treatments known to improve outcomes in patients with uncomplicated myocardial infarction and in patients with heart failure, cardiac fibrosis, and cardiac hypertrophy (such as angiotensin-converting enzyme inhibitors and beta-blockers) are ineffective or even contraindicated in CS. In fact, to date, no medical therapy has been found to improve survival in patients with CS.

[0012] Therefore, approaches and methods for treating and / or preventing cardiogenic shock are urgently needed. Suitable animal models of cardiogenic shock are also needed to further elucidate the underlying pathophysiology and identify new treatments for this condition. Summary of the invention

[0013] 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.

[0014] According to a preferred embodiment, the MYDGF protein comprises SEQ ID NO: 1. 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.

[0015] According to a particularly preferred embodiment, the MYDGF protein consists of SEQ ID NO:1 or SEQ ID NO:3.

[0016] According to other aspects, the present invention provides a nucleic acid encoding MYDGF or a fragment or variant thereof that exhibits the biological function of MYDGF, for use in treating and / or preventing cardiogenic shock.

[0017] According to one embodiment, the nucleic acid encodes an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:1.

[0018] According to a further aspect, the present invention provides a vector comprising the nucleic acid of the present invention for use in treating and / or preventing cardiogenic shock.

[0019] According to other aspects, the present invention provides a host cell comprising a nucleic acid according to the present invention or a vector according to the present invention for use in treating and / or preventing cardiogenic shock. Preferably, the host cell expresses the nucleic acid.

[0020] According to another aspect, the present invention provides a pharmaceutical composition comprising the MYDGF protein, nucleic acid, vector or host cell of the present invention and optionally a suitable pharmaceutical excipient and / or carrier for treating and / or preventing cardiogenic shock.

[0021] According to a preferred embodiment, the pharmaceutical composition used is administered orally, intravenously, subcutaneously, intramucosally, intraarterially, intramuscularly or intracoronary. Preferably, it is administered by one or more than one bolus injection and / or infusion.

[0022] According to other aspects, the present invention provides a method for treating and / or preventing cardiogenic shock, the method comprising administering a therapeutically effective amount of a myeloid-derived growth factor (MYDGF) protein to a patient in need thereof.

[0023] 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.

[0024] According to another embodiment, MYDGF or a fragment or variant thereof is administered by one or more than one bolus injection and / or infusion, preferably in a pharmaceutically acceptable carrier and / or excipient.

[0025] According to other aspects, the present invention provides a method for treating and / or preventing cardiogenic shock, the method 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.

[0026] According to a preferred embodiment, the pharmaceutical composition comprises a suitable pharmaceutical excipient.

[0027] According to another embodiment, the pharmaceutical composition is administered by one or more than one bolus injection and / or drip infusion.

[0028] According to other aspects, the present invention provides a method for generating a cardiogenic shock model in a non-human mammal, the method comprising (i) temporarily ligating a coronary artery of the mammal, (ii) establishing reperfusion, and (iii) ventilating the mammal with a fraction of inhaled oxygen at or below about 0.18.

[0029] According to a preferred embodiment, the non-human mammal is a rodent, more preferably a mouse.

[0030] According to a preferred embodiment, the coronary artery is ligated for about 30 minutes to about 90 minutes before reperfusion is established.

[0031] According to a preferred embodiment, the mammal is ventilated at an inspired oxygen fraction of about 0.16. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : A: Schematic diagram of the process for preparing the animal model of cardiogenic shock. Myocardial infarction (MI) was induced in male C57BL / 6N mice aged 8 to 10 weeks by ligating the proximal left anterior descending coronary artery for 60 minutes. Animals were pretreated subcutaneously with 2 mg / kg butorphanol and 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 conductance catheter with a micromanometer tip was inserted through the right carotid artery, and left ventricular (LV) pressure-volume (PV) loops were continuously recorded. Subsequently, the heart was maintained at a concentration of 0.33 or 0.16 for a fraction of inspired oxygen (FiO 2 ) were ventilated for 120 min. As shown in preliminary experiments, an FiO of 0.33 2 Compared with normal blood oxygen [nx; arterial oxygen partial pressure (PaO 2 ), 144±16 mmHg; arterial oxygen saturation (SaO 2 ), 99 ± 1%], while 0.16 FiO 2 Causes mild hypoxemia (hx; PaO 2 , 75±16 mmHg; SaO 2, 89±3%) (4 to 6 mice per group). Subsequently, the infarcted mice and sham-operated mice were randomly subjected to the above two FiO 2 B: Left ventricular pressure-volume loops collected at 120 min in sham-operated mice (sham) (dashed lines) and mice with induced myocardial infarction (MI) (continuous lines) ventilated under normoxic (nx) and hypoxic (hx) conditions.

[0033] Figure 2 : Left ventricular end-systolic pressure (LVESP) in sham-operated mice (open circles) and mice with induced myocardial infarction (MI) (closed circles) ventilated under normoxic (nx) or hypoxic (hx) conditions at 120 min, in mmHg ( Figure 2 A), cardiac output, the unit is mL / min ( Figure 2 B), arterial lactate concentration, in mmol / L ( Figure 2 C). At an FiO of 0.16 2 Ventilated MI mice (MI-hx) progressively developed CS, which was defined by the clinical features of CS, namely hypotension (decreased LVESP), decreased cardiac output, and increased lactate concentration. 2 Ventilated MI mice (MI-nx) remained hemodynamically stable.

[0034] Figure 3 MI-hx mice were treated with an intravenous infusion of dobutamine (5 ng / g / min to 7.5 ng / g / min via the left jugular vein, titrated to LVESP of 70 mmHg) starting 60 min after the start of hypoxic ventilation (MI-hx, dobutamine). Saline-infused MI-hx mice were used as controls (MI-hx, saline). Dobutamine improved LVESP in MI-hx mice ( Figure 3 A) and cardiac output ( Figure 3 B) Similar hemodynamic improvements are commonly observed in CS patients treated with dobutamine.

[0035] Figure 4 : Phosphoproteome 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 distinct phosphoproteome signatures: Sham-nx = normoxemia in sham-operated mice; Sham-hx = hypoxemia in sham-operated mice; MI-nx = normoxemia in infarcted mice; MI-hx = hypoxemia in infarcted mice.

[0036] Figure 5 : Volcano plot showing differentially regulated phosphosites in the non-infarcted left ventricular myocardium of mice with myocardial infarction-induced CS (MI-hx) versus mice with myocardial infarction alone (MI-nx). The top 10 down- or up-regulated [P<0.05] phosphosites are shown in black. MI-nx = normoxemia in infarcted mice; MI-hx = hypoxemia in infarcted mice. Abbreviations indicate proteins, and amino acid positions in parentheses indicate the respective phosphorylated residues in each protein. RIPR1 = Rho family interacting cell polarization regulator 1. DDA1 = DET1 and DDB1-associated 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 precursor type A / C. XIRP1 = Xin actin-binding repeat-containing protein 1. TNNI3 = troponin I. TIF1B = transcription intermediary factor 1-beta.

[0037] Figure 6 : Kaplan-Meier survival curves showing mortality within 120 minutes in cardiogenic shock (MI-hx) mice treated with MYDGF or saline.

[0038] Figure 7 : Bar graph showing the results of PV loop recordings at the end of the 120-min observation period. Heart rate ( Figure 7 A) Left ventricular end-systolic volume (LVESV) Figure 7 B) Left ventricular end-diastolic volume (LVEDV) Figure 7 C) Cardiac output ( Figure 7 D) stroke volume ( Figure 7 E) and stroke work ( Figure 7 F). *P<0.05, **P<0.01, ***P<0.001.

[0039] Figure 8 : Bar graph showing the results of PV loop recordings at the end of the 120-min observation period. Left ventricular end-systolic pressure (LVESP) ( Figure 8A) Left ventricular end-diastolic pressure (LVEDP) Figure 8 B) Left ventricular ejection fraction (LVEF) Figure 8 C) Maximum rate of change of left ventricular pressure (dP / dt 最大 )( Figure 8 D) Minimum rate of change of left ventricular pressure (dP / dt 最小 )( Figure 8 E) and left ventricular diastolic time constant (Tau) ( Figure 8 F). **p<0.01, ***p<0.001.

[0040] Fig. 9 : Bar graph showing blood gas analysis results at the end of the 120-minute observation period. pH ( Fig. 9 A) Arterial oxygen partial pressure (PaO 2 , unit: mmHg)( Fig. 9 B) Arterial oxygen saturation (SaO 2 ,unit%)( Fig. 9 C) and lactate concentration (mmol / L) ( Fig. 9 D). *P<0.05.

[0041] Fig.10 : Results of continuous PV loop recordings during a 120-min observation period in the CS model. Cardiac output (mL / min) of saline-treated MI-hx mice (CS saline) versus MYDGF-treated MI-hx mice (CS MYDGF) ( Fig.10 A) and left ventricular end-systolic pressure (LVESP) (in mmHg) ( Fig.10 B).

[0042] Fig.11 Figure : Area at risk and infarct size of the left ventricle determined at the end of the 120-min observation period in the CS model. Saline-treated MI-hx mice (Saline) versus MYDGF-treated MI-hx mice (MYDGF). Area at risk / total LV area (%) ( Fig.11 A) and infarct size / risk area (unit %) ( Fig.11 B).

[0043] Fig.12 : Plasma concentrations of high-sensitivity cardiac troponin 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) in saline-treated MI-hx mice (saline) versus MYDGF-treated MI-hx mice (MYDGF) Fig.12 A) and alanine aminotransferase (Log 2 ALT, unit U / L)( Fig.12 B). Soluble nucleosome concentrations in the infarcted (I) region of the left ventricle relative to the non-infarcted (NI) region were measured by ELISA. The I / NI ratio was used as an indicator of cell death ( Fig.12 C).

[0044] Fig.13 :The aforementioned embodiment ( Figure 6 , Figure 7 , Figure 8 , Fig. 9 , Fig.10 , Fig.11 and Fig.12 Schematic diagram of the treatment protocol used in the animal model of cardiogenic shock in . Figure 1 The same definition as in .

[0045] Fig.14 : Kaplan-Meier survival curves showing the mortality within 120 minutes of wild-type mice (WT) and Mydgf gene-deficient (knockout) mice (MYDGFKO) in cardiogenic shock.

[0046] Fig.15 : Cardiogenic shock leads to aggravated left ventricular tissue damage. Fig.15 A shows the experimental setup. Fig.15 B shows experimental results indicating that cardiogenic shock further exacerbates the left ventricular tissue damage that persists during acute myocardial infarction.

[0047] Fig.16 : Delayed treatment of cardiogenic shock. Fig.16 A shows the experimental setup. Fig.16 B shows experimental results indicating that cardiogenic shock can be treated by administering MYDGF at a time point after reperfusion is established. DETAILED DESCRIPTION

[0048] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as they may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art.

[0049] definition

[0050] Preferably, the terms used herein have the same meaning as in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", HGW Leuenberger, B.Nagel and H. Definitions as described in Chimica Acta, ed., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0051] To practice the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology and recombinant DNA technology are used, which are explained in the literature of this field (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, edited by J. Sambrook et al., 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 of this field (see, for example, Braunwald's Heart Disease. A Textbook of Cardiovascular Medicine, 9th Edition, edited by P. Libby et al., Saunders Elsevier Philadelphia, 2011).

[0052] Throughout the specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprising" and "containing" will be understood to imply the inclusion of the stated integers or steps or groups of integers or steps but not the exclusion of any other integers or steps or groups of integers or steps. As used in this specification and the claims that follow, an element not preceded by a quantifier includes one or more than one unless the context clearly dictates otherwise.

[0053] When used in conjunction with a numerical value, the term "about" is intended to include a range of values ​​having a lower limit that is 5% less than the stated numerical value and an upper limit that is 5% greater than the stated numerical value.

[0054] As used herein, the term "and / or" means one or both / all of the options referenced in the context of the term.

[0055] Nucleic acid molecules are also referred to as nucleic acids, which are understood to be polymeric macromolecules consisting of nucleotide monomers. Nucleotide monomers are composed of bases, five-carbon sugars (such as but not limited to ribose or 2'-deoxyribose) and one to three phosphate groups. Typically, polynucleotides are formed by phosphodiester bonds between each nucleotide monomer. In the context of the present invention, the 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.

[0056] 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, a subsequent 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., constructed by genetic technology means. ORFs encode proteins, and the amino acids therein can be translated into peptide chains.

[0057] Regardless of length or post-translational modification, the terms "protein" and "polypeptide" are used interchangeably herein and refer to any peptide-bonded amino acid chain. Proteins (including protein derivatives, protein variants, protein fragments, protein segments, protein epitopes, and protein domains) that can be used in the present invention can also be modified by chemical modification. This means that such chemically modified polypeptides 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. Compared to the parent polypeptide, chemical modification of the polypeptide can provide advantageous properties, such as one or more of enhanced stability, increased biological half-life, or increased water solubility. Chemical modifications suitable for variants useful in the present invention include, but are not limited to, PEGylation, glycosylation of non-glycosylated parent polypeptides, covalent coupling with therapeutic small molecules such as glucagon-like peptide 1 agonists including exenatide, albiglutide, taseroglutide, DPP4 inhibitors, incretins, and liraglutide, or modification of the glycosylation pattern present in the parent polypeptide. Such chemical modifications applicable to variants useful in the present invention may occur during or after translation.

[0058] The term "amino acid" includes naturally occurring amino acids as well as 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 hydropathic index higher than 0.5, more preferably higher than 1.0, even more preferably higher than 1.5, and which is not aromatic. Preferably, in the context of the present invention, a hydrophobic non-aromatic amino acid is an amino acid selected from alanine (Kyte Doolittle hydropathic index 1.8), methionine (Kyte Doolittle hydropathic index 1.9), isoleucine (Kyte Doolittle hydropathic index 4.5), leucine (Kyte Doolittle hydropathic index 3.8) and valine (Kyte Doolittle hydropathic index 4.2), or a derivative thereof having a Kyte Doolittle hydropathic index as defined above.

[0059] The term "variant" as used herein refers to a polypeptide that has one or more than one change in the amino acid sequence compared to the polypeptide or fragment thereof from which it is derived. The polypeptide from which a protein variant is derived is also referred to as a parent polypeptide. Similarly, the fragment from which a protein fragment variant is derived is referred to as a parent fragment. Typically, variants are artificially constructed, preferably by genetic technology means. Typically, the parent polypeptide is a wild-type protein or a wild-type protein domain. In addition, the variants available in the present invention can also be derived from homologues, orthologues or paralogues of the parent polypeptide, or from artificially constructed variants, provided that the variant exhibits at least one biological activity of the parent polypeptide. The change in the amino acid sequence can be an amino acid exchange, an insertion, a deletion, an N-terminal truncation or a C-terminal truncation, or any combination of these changes, which can occur at one or several sites. In a preferred embodiment, the variants useful in the 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 invention have 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 changes, preferably conservative amino acid changes, from the protein or domain from which it is derived.

[0060] Typical substitutions occur in aliphatic amino acids, in amino acids with aliphatic hydroxyl side chains, in amino acids with acidic residues, in amide derivatives, in amino acids with basic residues, or in amino acids with aromatic residues. Typical semiconservative and conservative substitutions are:

[0061]

[0062] A change from A, F, H, I, L, M, P, V, W or Y to C is semiconservative if the new cysteine ​​is maintained as a free sulfhydryl group. Furthermore, the skilled artisan will appreciate that glycines at sterically demanding positions should not be substituted and that P should not be introduced into portions of the protein having alpha-helical or beta-sheet structures.

[0063] Alternatively or additionally, a "variant" as used herein is 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 has at least 85% sequence identity with its parent polypeptide. The term "at least 85% sequence identity" is used throughout the specification for comparisons of polypeptide sequences and polynucleotide sequences. This statement 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 to a corresponding reference polynucleotide.

[0064] Protein fragments include amino acid deletions, which may be N-terminal truncations, C-terminal truncations or internal deletions or any combination of these. In the context of the present application, such proteins comprising N-terminal truncations, C-terminal truncations and / or internal deletions are referred to as "fragments". Fragments may be naturally occurring (e.g., splice variants) or artificially constructed, preferably constructed by genetic technology means. Preferably, compared to the parent polypeptide, the fragment (or deletion variant) has a maximum of 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 its C-terminus and / or internally, preferably at its N-terminus, its N-terminus and C-terminus, or its C-terminus. Ebenhoch R. et al., 2019 proposed that receptor interaction is more likely to occur at the anterior / protruding portion of the MYDGF apical surface and suggested that Tyr73 may be a key residue for receptor interaction.

[0065] Where two sequences are aligned and no reference sequence is specified against which the percentage of sequence identity is to be calculated, the sequence identity is calculated with reference to the longer of the two sequences being aligned, unless otherwise specified.

[0066] The similarity of nucleotide and amino acid sequences, ie, the percentage of sequence identity, can be determined by sequence alignment. This alignment can be performed using several algorithms known in the art, preferably using 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 Xversion 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 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 set at http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html: Alignment type = Slow, protein weight matrix = Gonnet, gap open = 10, gap extension = 0,1 for slow pairwise alignment options and protein weight matrix = Gonnet, gap open = 10, gap extension = 0,20, gap distances = 5, No end gaps = no, Output options: format = Aln w / numbers, Order = aligned.

[0067] The rank of sequence identity (sequence match) can be calculated using, for example, BLAST, BLAT, or BlastZ (or BlastX). Similar algorithms are included in 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, available, for example, at

[0068] http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blastblas. The algorithm parameters used are preferably

[0069] The default parameters set on http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome were: Expect threshold=10, word size=3, max matches in a queryrange=0, matrix=BLOSUM62, gap costs=Existence:11Extension:1, compositionaladjustments=conditional compositional score matrix adjustment together with the non-redundant protein sequence (nr) database to obtain amino acid sequences homologous to Factor 1 and Factor 2 polypeptides.

[0070] To obtain gapped alignments for comparison purposes, Gapped BLAST was used 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 1:154-162) or Markov random fields. When the percentage of sequence identity is mentioned in this application, if not otherwise specified, these percentages are calculated relative to the full length of the longer sequence.

[0071] As used herein, the term "host cell" refers to a cell containing a nucleic acid of the invention (e.g., in the form of a plasmid or virus). Such a host cell may be a prokaryotic cell (e.g., a bacterial cell) or a eukaryotic cell (e.g., a fungal cell, a plant cell, or an animal cell). The cell may be transformed or non-transformed. The cell may be an isolated cell, such as an isolated cell in a cell culture or a portion of a tissue, which itself may be an isolated or more complex tissue structure such as a portion of an organ or an individual.

[0072] The terms "myeloid growth factor", "MYDGF", "factor 1", "MYDGF polypeptide or protein" or "factor 1 polypeptide or protein" are used interchangeably and refer to the protein shown in the NCBI reference sequence NM_019107.3 (human homolog) and its mammalian homologs, particularly proteins 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 the human factor having the amino acid sequence according to SEQ ID NO: 1.

[0073] According to other preferred embodiments, MYDGF and Factor 1 protein refer to proteins comprising SEQ ID NO: 3, respectively. In other preferred embodiments, MYDGF refers to a protein consisting essentially of SEQ ID NO: 3. In other preferred embodiments, MYDGF refers to a protein consisting of SEQ ID NO: 3.

[0074] As used herein, MYDGF and fragments and variants thereof as defined herein include pharmaceutically acceptable salts thereof.

[0075] Whether a protein, variant or fragment exhibits a biological function of MYDGF can be determined by any of the tests described in the examples below. According to the present invention, a peptide or protein exhibits a biological function of MYDGF if the results obtained with a certain peptide or protein are compared to the results obtained with the MYDGF protein of the present invention shown in at least one of the examples given below, achieving at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the reported effect of MYDGF over a specified control group.

[0076] As used herein, the terms "MYDGF" and "Mydgf" both refer to myeloid-derived growth factor.

[0077] 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 present with persistent hypotension (systolic blood pressure less than 80 mmHg to 90 mmHg, or mean arterial pressure less than 30 mmHg below baseline, or need for vasopressor or inotropic support to maintain SBP > 90 mmHg, or mean arterial blood pressure < 70 mmHg, or systolic blood pressure < 100 mmHg despite adequate fluid resuscitation) with evidence of end-organ damage (eg, urine output < 30 mL / hour, or urine output < 0.5 mL / kg per hour, or cool extremities, or mottled skin, or serum lactate > 2 mmol / L, or metabolic acidosis, or altered mental status) and a severely reduced cardiac index (less than 2.2 L / min / m2) in the presence of adequate or elevated filling pressures (left ventricular [LV] end-diastolic pressure greater than 15 mmHg or right ventricular [RV] end-diastolic pressure greater than 10 mmHg to 15 mmHg) 2 )(Vahdatpour et al., Journal of the American Heart Association, Vol 8(8), 2019, e011991).

[0078] As used herein, "treatment" or "treating" of a disease or condition refers to accomplishing one or more of the following: (a) reducing the severity of the condition; (b) limiting or preventing the development of symptoms characteristic of the one or more conditions being treated; (c) inhibiting the worsening of symptoms characteristic of the one or more conditions being treated; (d) limiting or preventing the recurrence of one or more conditions in an individual previously suffering from the one or more conditions; (e) limiting or preventing the recurrence of symptoms of one or more conditions in an individual previously suffering from the symptoms; (f) reducing mortality after the onset of the disease or condition; (g) healing; and (h) prevention of the disease. The term "amelioration" is also encompassed by the term "treatment". Accordingly, the term "treating and / or preventing" a condition or disease as described herein refers to treating or preventing the condition or disease, or treating and preventing the condition or disease.

[0079] The term "stroke volume" describes the volume of blood expelled by the right / left ventricle in a single contraction. It is the difference between the end-diastolic volume (EDV) and the end-systolic volume (ESV).

[0080] 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.

[0081] The term "cardiac output" describes the amount of blood pumped by the ventricles per unit time.

[0082] The term "dP / dt 最小 ” and “dP / dt 最大 " describe the minimum and maximum rates of pressure change in the ventricle, respectively. Peak dP / dt is used as an indicator of ventricular performance.

[0083] The term "isovolumetric relaxation constant" or "Tau" describes the exponential decay of intraventricular pressure during isovolumetric relaxation. It is also called the ventricular relaxation time constant.

[0084] The term "fraction of inspired oxygen" or "FiO 2 ” describes the mole fraction or volume fraction of oxygen in the inspired gas. Natural air contains 21% oxygen, which corresponds to an FiO of 0.21 2 .

[0085] 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 laboratory animals including rodents such as mice, rats and guinea pigs. The term does not indicate a specific age or sex. In particular, the subject is a mammal. Preferably, the subject is a human. The subject may be a healthy subject or a subject suffering from or suspected of suffering from one or more than one disease. A subject suffering from or suspected of suffering from one or more than one disease is also referred to as a patient.

[0086] Unless stated otherwise, these descriptions and definitions are valid for the entire application.

[0087] sequence

[0088] The sequences used in the present invention are listed below.

[0089] SEQ ID NO: 1 (amino acid sequence of human factor 1 (MYDGF) lacking the 31 amino acid N-terminal signal peptide):

[0090]

[0091] SEQ ID NO:2 (amino acid sequence of MYDGF, including the N-terminal signal peptide (shown in bold and underlined); UniProtKB-Q969H8):

[0092]

[0093] SEQ ID NO:3 (amino acid sequence of [+G] MYDGF variant, wherein the V residue at position +1 of the N-terminus of mature human MYDGF is preceded by a G residue):

[0094]

[0095] SEQ ID NO: 4 shows the nucleic acid sequence encoding human factor 1 of MYDGF of SEQ ID NO: 3 (NCBI gene ID: 56005).

[0096] Implementation

[0097] Hereinafter, the elements of the present invention will be described. These elements are listed in specific embodiments, but it should be understood that they can be combined in any manner and in any number to create additional embodiments. The different described embodiments and preferred embodiments should not be construed as limiting the present invention to only the embodiments clearly described. This description should be understood to support and encompass the embodiments that combine the clearly described embodiments with any number of disclosed and / or preferred elements. In addition, any arrangement and combination of all described elements in this application should be considered to be disclosed by the description of the application, unless otherwise indicated by the context.

[0098] The present disclosure demonstrates for the first time the anti-cardiogenic shock effect of MYDGF. The present disclosure particularly demonstrates that administration of MYDGF in a mouse model of cardiogenic shock alleviates 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, stroke work, stroke volume, ventricular end-systolic pressure, and ventricular ejection fraction compared to control mice treated with saline. The present disclosure also demonstrates that MYDGF successfully treats cardiogenic shock and increases overall survival (see Examples below).

[0099] Therefore, in a first aspect, the present invention provides protein myeloid-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.

[0100] According to a preferred embodiment, the cardiogenic shock to be treated or prevented occurs during (acute) myocardial infarction (AMI, MI), preferably ST-segment elevation myocardial infarction (STEMI), and even more preferably cardiogenic shock occurs during reperfusion after percutaneous coronary intervention (PCI) of MI or STEMI.

[0101] In a particularly preferred embodiment of the invention, the protein comprises the amino acid sequence of SEQ ID NO: 1 or a fragment thereof. Preferably, the protein is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1.

[0102] 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, said variant 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. A person skilled in the art is able to decide, without undue burden, which positions in the parent polypeptide can be mutated to what extent, and which positions must be maintained in order to maintain 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 known in the art. Examples 7 and 8 of WO 2014 / 111458 Figure 6 and Figure 7 Such analysis is exemplarily described in . Preferably, mutations are introduced into those regions of the protein that are not completely conserved between species, preferably between mammals. In particularly preferred embodiments of the present invention, the MYDGF protein comprises, consists essentially of, or consists of the amino acid sequence SEQ ID NO:1 or SEQ ID NO: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 with SEQ ID NO:1.

[0103] N-terminal deletion variants are also included, which may, for example, delete one or more amino acids from amino acid position 1 to position 24 (based on SEQ ID NO: 1), ie, from the N-terminal conserved region.

[0104] C-terminal deletion variants are also included, which may, for example, delete one or more than one amino acid from amino acid position 114 to position 142 (based on SEQ ID NO: 1).

[0105] On the other hand, amino acids can be added to the MYDGF protein. This addition includes addition at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof. Therefore, the protein of the first aspect of the present invention may also include additional amino acid sequences, such as additional amino acid sequences 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 may be present at any position in the protein, preferably at the N-terminus or C-terminus. A particularly preferred additional sequence is the 6xHis tag. Preferably, the 6xHis tag is present at the C-terminus of the MYDGF protein. Depending on the expression system used and, if present, additional amino acids such as the above-mentioned tags, one or more than one residual amino acids may be retained at the N-terminus and / or C-terminus of the protein. It should be emphasized that in the MYDGF protein and Mydgf protein according to the present invention, such artifacts may exist, 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.

[0106] In some cases, it is preferred to mutate the protease cleavage site in the MYDGF protein of the first aspect of the invention to stabilize the protein (see Segers et al., Circulation 2007, 2011). The skilled person 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 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 lower frequencies (less than 10) are determined:

[0107] Table 1

[0108]

[0109] These sites can be altered to remove the recognition / cleavage sequences of the respectively identified proteases, thereby increasing the serum half-life of the protein.

[0110] The MYDGF protein may also contain additional amino acid sequences, for example, additional amino acid sequences for stabilizing or purifying the resulting protein. For example, preferably the protease cleavage site in the MYDGF protein is mutated to stabilize the protein. As described above, suitable proteolytic cleavage sites can be identified.

[0111] The MYDGF protein or the composition comprising the protein can be administered in vivo, ex vivo or in vitro, preferably in vivo.

[0112] Nucleic acid sequences can be optimized to enhance expression in host cells. Parameters to be considered include C: G content, preferred codons, and avoidance of inhibitory secondary structures. These factors can be combined in different ways to try to obtain nucleic acid sequences that enhance expression in specific hosts (see, e.g., Donnelly et al., International Publication No. WO 97 / 47358). The ability of a specific sequence to enhance expression in a specific host involves some empirical experiments. Such experiments include detecting the expression of the expected 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 encoding 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". It is well known in the art that specific codons are translated into specific amino acids (see, e.g., Lewin GENES IV, p.119, Oxford University Press, 1990). Therefore, the present invention also provides nucleic acids encoding MYDGF or fragments or variants thereof for treating and / or preventing cardiogenic shock. According to a preferred embodiment, a fragment or variant of MYDGF exhibits the biological function of MYDGF. According to other preferred embodiments, the nucleic acid encodes an amino acid sequence having at least 85% sequence identity to SEQ ID NO:1.

[0113] The nucleic acid of the use according to the present invention can also include transcription control elements or expression control sequences placed in order to control protein expression. Such nucleic acid together with the control elements are generally referred to as expression systems. As used herein, the term "expression system" refers to a system designed to produce one or more than one target gene product. Typically, such a system is "artificially" designed, i.e., designed by means of genetic technology, which can be used to produce a target gene product in vivo, in vitro or in vitro. The term "expression system" also includes the expression of a target gene product, including transcription of polynucleotides, mRNA splicing, translation into polypeptides, co-translational modifications and post-translational modifications of polypeptides or proteins, and protein targeting to one or more compartments within a cell, secretion of a cell, and protein uptake by the same cell or another cell. This overview relates to expression systems for eukaryotic cells, tissues, or organisms. The expression system of a prokaryotic system can be different, and how to construct an expression system of a prokaryotic cell is well known in the art.

[0114] The regulatory elements present in the gene expression assembly generally include: (a) a promoter coupled to the transcription of the nucleotide sequence encoding the polypeptide, (b) a 5' ribosome binding site functionally coupled to the nucleotide sequence, (c) a terminator connected to the 3' end of the nucleotide sequence, and (d) a 3' polyadenylation signal functionally coupled to the nucleotide sequence. Other regulatory elements for enhancing or regulating gene expression or polypeptide processing may also be present. A promoter is a genetic element that is recognized by RNA polymerase and mediates transcription in the downstream region. A preferred promoter is a strong promoter that provides an increased level 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). Other examples of promoters include naturally occurring promoters, such as the EF1α promoter, the mouse 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).

[0115] 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 cutting the transcribed RNA and adding poly (A) tails to the RNA. The polyadenylation signal in higher eukaryotes comprises an AAUAAA sequence of about 11 to 30 nucleotides from the polyadenylation addition site. The AAUAAA sequence is involved in signal RNA cutting (Lewin, Genes IV, Oxford University Press, NY, 1990). Poly (A) tails are very important for the processing of mRNA, export from the nucleus, translation and stability.

[0116] Polyadenylation signals that can be used as part of the gene expression assembly include the minimal rabbit β-globin polyadenylation signal and bovine growth hormone polyadenylation (BGH) (Xu et al., Gene 272:149-156, 2001, Post et al., US Pat. No. 5,122,458).

[0117] Examples of other regulatory elements that may be present for enhancing or regulating 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 may be associated with promoters.

[0118] The expression of the MYDGF protein or its variant according to the present invention can be regulated. This regulation can be accomplished in many steps of gene expression. Possible regulatory steps are, for example, but not limited to, transcription initiation, promoter clearance, transcription elongation, splicing, export from the nucleus, mRNA stability, translation initiation, translation efficiency, translation elongation and protein folding. Other regulatory steps that affect the concentration of the intracellular MYDGF polypeptide are those that affect the half-life of the protein. The regulatory step is, for example, regulated protein denaturation. Since the protein of the present invention includes secretory proteins, 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.

[0119] The control of the above-mentioned regulating step can be, for example, independent of cell type or tissue type or cell type or tissue type specific. In a particularly preferred embodiment of the present invention, the control of the regulating step is cell type or tissue type specific. This cell type or tissue type specific regulation is preferably completed by a regulating step involving nucleic acid transcription. This transcriptional regulation can be completed by using a cell type or tissue type specific promoter sequence. The result of this cell type or tissue type specific regulation can have different levels of specificity. This means that, compared with other cell types or tissue types, the expression of the corresponding polypeptide in the corresponding cell or tissue is enhanced, or expression is confined to the corresponding cell type or tissue type. Cell type or tissue type specific promoter sequences are well known in the art and are all accessible for a wide range of cell types or tissue types.

[0120] The expression is not necessarily cell type or tissue type specific, but can depend on physiological conditions. Such conditions are, for example, inflammation or trauma. Such physiological condition-specific expression can also be achieved by regulation in all of the above-mentioned regulation steps. The preferred mode of regulating physiological condition-specific expression is transcriptional regulation. For this reason, a promoter specific for trauma or inflammation can be used. The corresponding promoter is, for example, a naturally occurring sequence, which can, for example, be derived from a gene specifically expressed during an immune response and / or wound tissue regeneration process. Another possibility is to use an artificial promoter sequence, which is, for example, constructed by a combination of two or more naturally occurring sequences.

[0121] The regulation may be cell type or tissue type specific and physiological condition specific. In particular, the expression may be cardiac specific. Preferably, the expression is cardiac specific and / or wound specific.

[0122] According to the present invention, another possibility of regulating the expression of MYDGF protein or its variants is the conditional regulation of gene expression. In order to achieve conditional regulation, an operon sequence can be used. For example, the Tet operon sequence can be used to inhibit gene expression. Conditional regulation of gene expression by the Tet operon and the Tet repressor is well known in the art, and many corresponding systems have been established for a wide range of prokaryotes and eukaryotes. Those skilled in the art know how to select a suitable system and adapt it to the special needs of the corresponding application.

[0123] In a particularly preferred embodiment, the use of the nucleic acid according to the invention comprises application to an individual or patient, preferably an individual or patient suffering from cardiogenic shock.

[0124] According to other aspects, the present invention provides a vector comprising the nucleic acid or expression system described herein for use in treating and / or preventing cardiogenic shock.

[0125] As used herein, the term "vector" refers to a protein or polynucleotide or a mixture thereof that can be introduced into or can introduce the protein and / or nucleic acid contained therein into a cell. In the context of the present invention, it is preferred that upon introduction of one or more than one vector, the gene of interest encoded by the introduced polynucleotide is expressed in the host cell. 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.

[0126] 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, alphavirus vectors, herpes virus vectors, measles virus vectors, poxvirus vectors, vesicular stomatitis virus vectors, retroviral vectors, and lentiviral vectors.

[0127] In a particularly preferred embodiment of the invention, the vector is an adenoviral vector or an adeno-associated virus (AAV) vector.

[0128] Nucleic acids encoding one or more than one MYDGF protein or variant thereof according to the present invention can be introduced into host cells, tissues or individuals using vectors suitable for therapeutic administration. Suitable vectors can preferably deliver nucleic acids into target cells without causing unacceptable side effects.

[0129] In a particularly preferred embodiment, the use of the vector according to the invention comprises application to an individual in need thereof.

[0130] The vector comprising a nucleic acid encoding a MYDGF protein or a fragment or variant thereof preferably exhibits the above-mentioned biological function of MYDGF, and is preferably used for treating and / or preventing cardiogenic shock.

[0131] According to other aspects, 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 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.

[0132] According to other aspects, the present invention provides a pharmaceutical composition comprising MYDGF protein or a fragment or variant thereof and optionally a suitable pharmaceutical excipient for treating and / or preventing cardiogenic shock. According to a preferred embodiment, the fragment or variant of MYDGF exhibits the biological function of MYDGF.

[0133] As used herein, the term "suitable pharmaceutical excipient" refers to a pharmaceutically inactive substance, such as, but not limited to, a diluent, an excipient, a surfactant, a stabilizer, a physiological buffer solution or a carrier, which is administered together with a therapeutically active ingredient."Pharmaceutical excipient" is also referred to as "pharmaceutical carrier" and can be liquid or solid. Liquid carriers include, but are not limited to, sterile liquids, such as saline solutions and oils in water, which include, but are not limited to, oils of petroleum origin, animal origin, plant origin or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Saline solutions and aqueous dextrose solutions and aqueous glycerol solutions can also be used as liquid pharmaceutical 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 EWMartin. 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, glycol, water, ethanol, etc. Such suitable pharmaceutical excipients are preferably pharmaceutically acceptable.

[0134] The terms "drug," "medicament," and "pharmaceutical" 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.

[0135] "Pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, particularly 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 the 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 portion, its suitable pharmaceutically acceptable salts may include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); and salts formed with suitable organic ligands (e.g., ammonium, quaternary ammonium and amine cations, and formed using counteranions 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, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cypionate, digluconate, dihydrochloride, dodecyl sulfate, edetate, edisylate, ylate, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolyllarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methosulfate, mucate, 2-naphthalenesulfonate, naphthalenesulfonate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (spicate) , embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate / diphosphate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, toluenesulfonate, triethiodide, undecanoate, valerate, and the like (see, e.g., SM Berge et al., "Pharmaceutical Salts", J. Pharm. Sci., 66, pp. 1-19 (1977)).

[0136] 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 than one active ingredient, which may act in combination with one another or independently of one another. The active ingredient may be formulated in a neutral form or in the form of a salt. Pharmaceutically acceptable salts include those formed with free amino 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, etc.

[0137] The terms "preparation" and "composition" are intended to include the formulation of an active compound with encapsulating material as a carrier, thereby providing a capsule in which the active component, with or without other carriers, is surrounded by a carrier, thereby bringing the carrier into association with the active compound.

[0138] As used herein, the term "carrier" refers to a pharmacologically inactive substance, such as, but not limited to, a diluent, excipient or carrier that is administered with a therapeutically active ingredient. These pharmaceutical carriers can be liquid or solid. Liquid carriers include, but are not limited to, sterile liquids, such as saline solutions and oils in water, including oils derived from petroleum, animals, plants or synthesis, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Saline solutions and aqueous dextrose solutions and aqueous 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 EWMartin.

[0139] 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, glycol, water, ethanol, etc. The term "composition" is preferably intended to include the formulation of an active compound with an encapsulating material, such as a carrier, thereby providing a capsule in which the active component and or without other carriers are surrounded by a carrier, thereby bringing the carrier into association with the active compound.

[0140] According to one embodiment, the active ingredient is applied to cells, tissues or individuals in a therapeutically effective amount. "Therapeutically effective amount" refers to the amount of the active ingredient that is sufficient to achieve the desired purpose. The active ingredient can be a therapeutic agent. The effective amount of a given active ingredient will vary with 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 in each individual case can be determined empirically by the technician according to the established methods in the art. As used in the context of the present invention, "application" includes administration in vivo to an individual and direct in vitro administration or ex vivo administration to a cell or tissue.

[0141] In a preferred embodiment of the present invention, the pharmaceutical composition is tailored for treating a condition, in particular for treating cardiogenic shock. In other preferred embodiments of the present invention, the pharmaceutical composition is tailored for preventing a condition, in particular for preventing cardiogenic shock. According to a particularly preferred embodiment, the pharmaceutical composition is tailored 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.

[0142] The pharmaceutical composition contemplated by the present invention can be formulated in various ways well known to those skilled in the art. For example, the pharmaceutical composition of the present invention can be in liquid form, such as in the form of a solution, emulsion or suspension. Preferably, the pharmaceutical composition of the present invention is formulated for parenteral administration, preferably for intravenous, intraarterial, intramuscular, subcutaneous, percutaneous, intrapulmonary, intraperitoneal, intracoronary, intracardiac administration, or by mucosal administration, preferably for intravenous, subcutaneous or intraperitoneal administration. Oral or anal administration preparations are also possible. Preferably, the pharmaceutical composition of the present invention is in the form of a sterile aqueous solution, which may contain other substances, such as, enough salt or glucose to make the solution isotonic with 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 subdivided into unit doses containing an appropriate amount of active ingredient. The unit dosage form can be a packaged preparation, which contains a discrete amount of pharmaceutical composition, such as a vial or an ampoule.

[0143] The pharmaceutical composition is preferably administered by intravenous, intraarterial, intramuscular, subcutaneous, transdermal, intrapulmonary, intraperitoneal, intracoronary or intracardiac routes, which also include other routes of administration known in the art.

[0144] If the pharmaceutical composition is used as an individual treatment or for the prevention of a disease or condition, the use of the pharmaceutical composition can replace the standard treatment or standard prevention of the corresponding disease or condition, or can be applied additionally outside the standard treatment. In the case of additional use of the pharmaceutical composition, the pharmaceutical composition can be applied before, simultaneously or after the standard treatment and / or prevention.

[0145] It is also 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. The time span of drug administration is not limited. Preferably, the administration does not exceed 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks.

[0146] Single doses of the pharmaceutical composition may independently form the total amount of the administered dose, or the respective administration time span may include one or more than one administration in the form of a bolus and / or drip.

[0147] According to other aspects, 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, which exhibits the biological function of MYDGF of SEQ ID NO: 1. In this regard, the fragment or variant preferably comprises an amino acid sequence having at least 85% amino acid sequence identity with SEQ ID NO: 1.

[0148] For example, the administration may be performed as described in the first aspect. According to a preferred embodiment of the method of the present invention, the MYDGF protein or fragment or variant thereof is administered by one or more than one bolus injection and / or drip infusion, preferably in a pharmaceutically acceptable carrier and / or excipient.

[0149] According to other aspects, the present invention provides a method for generating a cardiogenic shock model in a non-human mammal, the method comprising (i) temporarily ligating a coronary artery of the mammal, (ii) establishing reperfusion, and (iii) administering a cardiogenic shock model at a fraction of inspired oxygen (FiO2) of about 0.18 or less. 2) Ventilate the mammal. The transient ligation may be performed by any method known to the skilled person that is suitable for blocking or significantly reducing blood flow. According to a preferred embodiment, the ligation is performed by placing and tightening a surgical thread or surgical wire around the coronary artery, thereby blocking or substantially blocking blood flow through the artery. Reperfusion is preferably established by reestablishing blood flow, for example by opening the surgical thread or surgical wire, allowing blood to flow through the artery. Ventilation of the mammal is performed by standard methods known in the art, such as mechanical ventilation commonly used in mammalian surgery.

[0150] This method does not specifically provide treatment to the animal, but rather induces a state similar to cardiogenic shock. Cardiogenic shock is characterized by, for example, a decrease in ventricular end-systolic pressure (VESP), a decrease in cardiac output, and an increase in arterial lactate concentration compared to healthy subjects or to subjects before the method is implemented. The advantage of the new non-human mammalian cardiogenic shock model is that small animals such as rodents that are mature in laboratory practice can be used. There is no need to use larger animals, such as pigs, which involve higher costs for food, shelter, and animal husbandry, and are more complicated to study. The new model allows for studies such as genetically modified mice, such as exploring the molecular mechanisms of cardiogenic shock. Genetically modified mice can be easily generated, or can already be obtained from research groups, or can be commercially purchased, such as from the Jackson Laboratory. In contrast, genetically modified large animals, such as pigs, are extremely difficult to generate, and only a few genetically modified large animals are available.

[0151] Thus, 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.

[0152] According to a preferred embodiment, the coronary artery is the proximal left anterior descending coronary artery. Alternatively, any other major coronary heart artery, whose transient ligation ultimately results in a decrease in ventricular end-systolic pressure (VESP) and an increase in arterial lactate concentration, may be used.

[0153] According to other preferred embodiments, before reperfusion is established, the coronary artery is ligated for about 30 minutes to about 90 minutes. According to other preferred embodiments, the duration of coronary artery ligation is selected from a range having a lower limit of about 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes or 85 minutes and an upper limit of about 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes or 90 minutes, or any combination thereof. More preferably, the coronary artery is ligated for about 45 minutes to 70 minutes, most preferably about 60 minutes.

[0154] According to other preferred embodiments, the oxygen fraction (FiO) is about 0.18 to about 0.12. 2 ) The mammal is ventilated. According to other preferred embodiments, the mammal is ventilated with an inspired oxygen fraction selected from a range having a lower limit of about 0.12, 0.13, 0.14, 0.15, 0.16 or 0.17 and an upper limit 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 an inspired oxygen fraction of about 0.17 to about 0.14, most preferably about 0.16.

[0155] The present invention also provides a model animal obtained by implementing the method for generating a cardiogenic shock model in a non-human mammal.

[0156] Example

[0157] These examples are intended to further illustrate the present invention and contribute to a better understanding. They should not be interpreted as limiting the scope of the present invention in any way.

[0158] Materials and methods in the examples:

[0159] Unless otherwise stated, the following materials and methods were used in the examples.

[0160] Recombinant MYDGF.

[0161] In the [+G] MYDGF variant (HEK) used in the examples, the V residue at the +1 position of the N-terminus of mature human MYDGF is preceded by a G residue. The variant has the sequence shown in SEQ ID NO: 3 and is made 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.

[0162] Mouse surgery and functional assessment.

[0163] All surgical procedures were approved by the authorities in Hannover, Germany ( 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 in the central animal facility of the Hannover Medical School. Food and water were provided ad libitum. During surgery, mice were housed in a room with a temperature controller ( Rectal temperature was maintained at 37°C on a heating pad connected to a 400-well Medical Instruments.

[0164] Statistical analysis.

[0165] Mice from the same litter were randomly assigned to different experimental groups. Based on visual inspection, the data were normally distributed with similar variance in different groups. Data are presented as mean ± sem. Two independent sample t tests were used to compare 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 of 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 the data in the study and takes responsibility for the integrity of the data and data analysis.

[0166] Embodiment 1:

[0167] Establishment of the animal model of cardiogenic shock. Myocardial infarction (MI) was induced in male C57BL / 6N mice aged 8 to 10 weeks by transient ligation of the proximal left anterior descending coronary artery for 60 minutes. Animals were pretreated subcutaneously with 2 mg / kg butorphanol and 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 left thoracotomy, the left anterior descending coronary artery (LAD) was ligated with a 7-0prolene (Ethicon, Cat. No. EH7405) slipknot to induce local ischemia, and the slipknot was 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.

[0168] In preliminary studies, isolated coronary artery ligation 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 hypothesis that hypoxic ventilation combined with MI can induce CS, we compared the effects of ventilation (medical O2) and reperfusion in mice. 2 With medical N 2 Mixture) of different inspired oxygen fractions (FiO 2 During isoflurane anesthesia, infarcted mice and sham-operated mice were randomly assigned to receive anesthesia with normoxia (nx; arterial oxygen partial pressure [PaO 2 ], 144±16 mmHg; arterial oxygen saturation [SaO 2 ], 99 ± 1%) with a FiO of 0.33 2 , or mild hypoxemia (hx; arterial oxygen partial pressure 75±16 mmHg; SaO 2 , 89±3%; 4 to 6 mice per group) with an FiO of 0.16 2 Ventilation ( Figure 1 A).

[0169] An exemplary pressure volume loop recorded after 120 minutes is presented ( Figure 1 B) During a 120-minute period, LVESP ( Figure 2 A) and cardiac output ( Figure 2 B) gradually decreased in MI-hx mice (MI with CS), but remained stable in MI-nx mice (MI without CS). At 120 minutes, MI-hx mice already showed arterial hyperlactatemia ( Figure 2 C), and showed 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 mmHg vs. 9±1 mmHg, P=0.005). Heart rate was not affected (475±16 min -1 Compared with 486±8min -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).

[0170] Intravenous dobutamine, a well-established symptomatic treatment for CS patients (Vahdatpour et al., Journal of the American Heart Association, Vol 8(8), 2019, e011991), increased LVESP in MI-hx mice ( Figure 3 A) and cardiac output ( Figure 3 B), and decreased arterial lactate (5.6±0.6mmol / L vs. 11.1±0.7mmol / L, P<0.001, 4 to 6 mice per group).

[0171] As an example of the use of this model, high-resolution mass spectrometry was used to define the phosphoproteome signature in the non-infarcted LV myocardium at 120 minutes. 9004 phosphorylation sites were detected among 1264 different proteins. Principal component analysis showed that the four groups (Sham-nx = normoxic sham-operated mice; Sham-hx = hypoxic sham-operated mice; MI-nx = normoxic MI-infarcted mice; MI-hx = hypoxic MI-infarcted mice) were associated with different phosphoproteome signatures ( Figure 4 ). This reflects the observation that hypoxemia itself does not alter cardiac performance ( Figure 2 A and Figure 2 B), Overlapping phosphorylation proteome signatures between sham-hx mice and sham-nx mice ( Figure 4 In contrast, MI-hx mice and MI-nx mice displayed very different phosphoproteome signatures ( Figure 4 ), with 72 differentially regulated phosphorylation sites ( Figure 5 ), thus indicating a potential therapeutic entry point.

[0172] In Wang Y, Polten F, 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 provides a more detailed description, the contents of which are incorporated herein by reference in their 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 dobutamine, and high mortality. This model provides a platform to explore the molecular pathophysiology of CS and develop urgently needed therapies for CS.

[0173] Embodiment 2:

[0174] As described in detail in WO 2014 / 111458, the MYDGF protein (human factor 1; C19orf10) is identified. The nucleic acid sequence encoding human factor 1 can be obtained from 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 examples, the human [+G]MYDGF variant without a signal peptide according to SEQ ID NO: 3 is used and is 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 AcuteMyocardial Infarction as Assessed by Multiple Reaction Monitoring-MassSpectrometry. Anal Chem. 2019 Jan 15; 91(2): 1302-1308).

[0175] Embodiment 3:

[0176] Acute myocardial infarction was induced in C57BL6 / N mice pretreated with butorphanol (2 mg / kg, subcutaneous injection (sc)) and atropine (0.02 mg / kg, sc) to reduce bronchial secretions by transient ligation of the left anterior descending coronary artery for 60 minutes. At the time of reperfusion, human MYDGF (10 μg / 100 μL) or saline (control) was bolus injected into the left ventricular cavity. After reperfusion, a conductivity catheter with a micromanometer tip was inserted through the right carotid artery to continuously record the left ventricular pressure-volume (PV) loop. Thereafter, hypoxic ventilation (FiO 20.16) induced cardiogenic shock (CS), and mice received 120 minutes of continuous left jugular vein infusion of MYDGF (5 μg / h, infusion rate: 2 μL / min) or saline (control). After 120 minutes, arterial blood samples (anticoagulated with heparin) were drawn from the left ventricle and used for immediate blood gas analysis and lactate analysis. Another blood sample was drawn, treated with EDTA, and centrifuged at 3500g for 10 minutes at 4°C to obtain plasma, which was stored at -80°C until further analysis. Fig.13 The process is schematically shown ( Fig.13 The abbreviations in Figure 1 as defined; sc = subcutaneous administration; iv = intravenous administration).

[0177] The results are as follows Figures 6 to 12 shown.

[0178] like Figure 6 As shown, MYDGF significantly improved the survival rate of mice with cardiogenic shock.

[0179] The experiment also showed that the administration of MYDGF improved left ventricular systolic and diastolic function ( Figure 7 A to Figure 7 F and Figure 8 A to Figure 8 PV loop recording in F).

[0180] MYDGF also reversed acidosis and reduced lactate concentrations, two defining features of cardiogenic shock ( Fig. 9 blood gas analysis in ).

[0181] It was also shown that MYDGF prevented the decline in cardiac output and LVESP, two other defining features of cardiogenic shock ( Fig.10 A and Fig.10 Continuous PV loop recording in B).

[0182] The experiments also showed that MYDGF treatment reduced infarct size ( Fig.11 A and Fig.11 B). To this end, at the end of the observation period, the left ventricle was removed and the area at risk and infarct size were measured by staining with Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) (method as described in Korf-Klingebiel et al., Nat Med. 2015; 21: 140-149).

[0183] Fig.12 A and Fig.12B shows the effect of MYDGF treatment on the plasma troponin level and alanine aminotransferase (ALT) concentration measured at the end of the 120-minute observation period. Plasma troponin is an indicator of cardiac damage, and ALT reflects end-organ (liver) damage. MYDGF treatment of CS mice reduces plasma troponin and alanine aminotransferase levels. At the end of the 120-minute observation period, the effect of MYDGF on cell death in the infarct zone was determined by measuring the soluble nucleosomes released due to cell death. Measured using a cell death ELISA detection kit commercially available from Roche. Fig.12 C shows the ratio of soluble nucleosomes in the infarct area (I) to the non-infarct area (NI), which is an indicator of cell death, and indicates that MYDGF significantly reduces the amount of soluble nucleosomes, thereby reducing cell death in the infarct area.

[0184] Embodiment 4:

[0185] MYDGF knockout mice were obtained as described by Korf-Klingebiel et al. (Nature Medicine, 2015, Vol. 21(2): 140-149). Cardiogenic shock (CS) was induced in wild-type (WT) mice and MYDGF knockout (KO) mice as described in Example 1. The mortality rates of WT mice and MYDGF knockout mice were compared during an observation period of 120 minutes. Fig.14 As shown, the mortality rate of KO mice was significantly higher, indicating that endogenous MYDGF has a protective role in cardiogenic shock.

[0186] Embodiment 5:

[0187] For example Fig.10 As shown, cardiac function deteriorates continuously during cardiogenic shock. It is hypothesized that this deterioration is caused by the continued worsening of left ventricular tissue damage during cardiogenic shock. To test this hypothesis, acute MI was induced by transient ligation of the left anterior descending coronary artery for 60 min. After reperfusion, CS (FiO 2 0.16; MI / shock). Infarction control mice were ventilated with normoxia (FiO 2 0.33; MI / no shock). Schematic diagram of the experimental setup is shown in Fig.15 As shown in A.

[0188] At 60 min after reperfusion and at the end of the experiment, the left ventricles were removed (5 MI / non-shocked mice and 5 MI / shocked mice at each time point) and cell death (soluble nucleosomes) in the infarcted (I) and distal non-infarcted (NI) regions was measured using a cell death ELISA kit from Roche. The I / NI ratio is reported as a measure of cell death. Fig.15 B. During the observation period, the concentration of soluble nucleosomes increased in the infarcted area in both groups ([apoptotic] ​​cell death takes time to develop). The increase in soluble nucleosome concentration was significantly more pronounced in MI / shocked mice than in MI / non-shocked mice, indicating that CS also exacerbates the left ventricular tissue damage that persists during acute MI.

[0189] Embodiment 6:

[0190] As shown in Example 5, cardiogenic shock also exacerbates the left ventricular tissue damage that persists during acute myocardial infarction. Therefore, it was tested whether delayed MYDGF treatment can alleviate the symptoms of cardiogenic shock and improve cardiac function. To this end, acute myocardial infarction was induced in mice by transient ligation of the left anterior descending coronary artery for 60 minutes. After reperfusion, a conductivity catheter with a micromanometer tip was inserted through the right carotid artery to continuously record the left ventricular pressure-volume (PV) loop. Thereafter, hypoxic ventilation (FiO 2 Cardiogenic shock was induced by 1:1 ratio (0.16). Starting 60 min after reperfusion, mice received a continuous left jugular vein infusion of (i) human MYDGF (5 μg / h, infusion rate: 2 μL / min) or (ii) saline (control) until the end of the experiment, when 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 treatment, and three MI / shock mice received delayed saline infusion. Fig.16 A shows a schematic diagram of the experimental setup.

[0191] The results are shown in Fig.16 B. One saline-treated control mouse died. In 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 treatment can also promote beneficial effects in cardiogenic shock.

[0192] Terms

[0193] The invention relates to the following:

[0194] Item 1: Myeloid-derived growth factor (MYDGF) or a fragment or variant thereof that exhibits the biological function of MYDGF for use in treating and / or preventing cardiogenic shock.

[0195] Clause 2: MYDGF for use according to clause 1, wherein the MYDGF comprises:

[0196] (i) SEQ ID NO: 1; or

[0197] (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.

[0198] Item 3: The MYDGF for use according to Item 1, wherein the MYDGF protein consists of SEQ ID NO: 1 or SEQ ID NO: 3.

[0199] Item 4: A nucleic acid encoding MYDGF or a fragment or variant thereof that exhibits the biological function of MYDGF, for use in treating cardiogenic shock.

[0200] Item 5: The nucleic acid for use according to Item 4, wherein the nucleic acid encodes an amino acid sequence having at least 85% sequence identity with SEQ ID NO:1.

[0201] Item 6: A vector comprising the nucleic acid according to Item 5, for use in treating and / or preventing cardiogenic shock.

[0202] Item 7: A host cell comprising and preferably expressing the nucleic acid according to Item 5 or the vector according to Item 6, for use in the treatment and / or prevention of cardiogenic shock.

[0203] Item 8: A pharmaceutical composition comprising the MYDGF protein according to any one of items 1 to 3 or a fragment or variant thereof that exhibits the biological function of MYDGF, the nucleic acid according to any one of items 4 or 5, the vector according to item 6, or the host cell according to item 7, which is used for treating and / or preventing cardiogenic shock.

[0204] Clause 9: The pharmaceutical composition for use according to clause 8, wherein the pharmaceutical composition is administered orally, intravenously, subcutaneously, intramucosally, intraarterially, intramuscularly or intracoronary.

[0205] Clause 10: The pharmaceutical composition for use according to clause 9, wherein the administration is performed by one or more than one bolus injection and / or infusion.

[0206] Item 11: A method for treating and / or preventing cardiogenic shock, the method 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.

[0207] Item 12: A method for treating and / or preventing cardiogenic shock, the method comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a myeloid-derived growth factor (MYDGF) protein.

[0208] Clause 13: The method according to clause 11 or 12, wherein the MYDGF comprises:

[0209] (i) SEQ ID NO: 1; or

[0210] (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.

[0211] Clause 14: The method according to any one of clauses 11 to 13, wherein the MYDGF or fragment or variant thereof is administered by one or more than one bolus injection and / or infusion, preferably in a pharmaceutically acceptable carrier and / or excipient.

[0212] Clause 15: A method of generating a cardiogenic shock model in a non-human mammal, the method comprising:

[0213] (i) temporarily ligating the coronary arteries of a mammal,

[0214] (ii) establish reperfusion, and

[0215] (iii) ventilating the mammal at a fraction of inspired oxygen at or below about 0.18.

[0216] Clause 16: The method according to Clause 15, wherein the non-human mammal is a rodent, preferably wherein the non-human mammal is a mouse.

[0217] Clause 17: The method of clause 15 or 16, wherein the coronary artery is ligated for about 30 minutes to about 90 minutes before reperfusion is established.

[0218] Clause 18: The method of any one of clauses 15 to 17, wherein the mammal is ventilated at a fraction of inspired oxygen of about 0.16.

Claims

1. A myeloid-derived growth factor (MYDGF) or a fragment or variant thereof that exhibits the biological function of MYDGF, for use in treating and / or preventing cardiogenic shock.

2. The MYDGF for use according to 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.

3. The MYDGF for use according to claim 1, wherein the MYDGF protein consists of SEQ ID NO: 1 or SEQ ID NO:

3.

4. A nucleic acid encoding MYDGF or a fragment or variant thereof that exhibits the biological function of MYDGF, for use in treating and / or preventing cardiogenic shock.

5. The nucleic acid for use according to claim 4, wherein the nucleic acid encodes an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

1.

6. A vector comprising the nucleic acid according to claim 5, for use in the treatment and / or prevention of cardiogenic shock.

7. A host cell comprising the nucleic acid according to claim 5 or the vector according to claim 6 and preferably expressing said nucleic acid for use in the treatment and / or prevention of cardiogenic shock.

8. A pharmaceutical composition comprising the MYDGF protein according to any one of claims 1 to 3 or a fragment or variant thereof that exhibits the biological function of MYDGF, the nucleic acid according to any one of claims 4 or 5, the vector according to claim 6, or the host cell according to claim 7, for treating and / or preventing cardiogenic shock.

9. The pharmaceutical composition for use according to claim 8, wherein the pharmaceutical composition is administered orally, intravenously, subcutaneously, intramucosally, intraarterially, intramuscularly or intracoronary.

10. The pharmaceutical composition for use according to claim 9, wherein the administration is performed by one or more than one bolus injection and / or drip infusion.

11. A method for treating and / or preventing cardiogenic shock, the method 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.

12. A method for treating and / or preventing cardiogenic shock, the method 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.

13. The method according to 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.

14. The method according to any one of claims 11 to 13, wherein the MYDGF or fragment or variant thereof is administered by one or more than one bolus injection and / or drip infusion, preferably in a pharmaceutically acceptable carrier and / or excipient.

15. A method for generating a cardiogenic shock model in a non-human mammal, the method include: (i) temporarily ligating the coronary arteries of a mammal, (ii) establish reperfusion, and (iii) ventilating the mammal at a fraction of inspired oxygen at or below about 0.

18.

16. The method of claim 15, wherein the non-human mammal is a rodent, preferably wherein the non-human mammal is a mouse.

17. The method of claim 15 or 16, wherein the coronary artery is ligated for about 30 minutes to about 90 minutes before reperfusion is established.

18. The method of any one of claims 15 to 17, wherein the mammal is ventilated at an inspired oxygen fraction of about 0.16.

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