Novel formulations comprising myeloid-derived growth factor

Abstract

This invention generally relates to pharmaceutical formulations that can be used to treat acute coronary syndromes. More particularly, this invention relates to a novel pharmaceutical formulation comprising myeloid-derived growth factor (MYDGF) protein, which can be used to treat myocardial infarction in subjects of need. Compared to commonly used formulations, the pharmaceutical formulation of this invention provides significantly improved long-term stability. Furthermore, the pharmaceutical formulation of this invention allows for easy lyophilization and reconstitution without any significant loss of therapeutically active protein. This invention also relates to a formulation for use in a method of treating myocardial infarction in subjects of need. The method includes intravenous administration of MYDGF protein to the subject in addition to standard care.

Description

[0001] This invention generally relates to pharmaceutical formulations that can be used to treat acute coronary syndromes. More particularly, this invention relates to a novel pharmaceutical formulation comprising myeloid-derived growth factor (MYDGF) protein, which can be used to treat myocardial infarction in subjects of need. Compared to commonly used formulations, the pharmaceutical formulation of this invention provides significantly improved long-term stability. Furthermore, the pharmaceutical formulation of this invention allows for easy lyophilization and reconstitution without any significant loss of therapeutically active protein. This invention also relates to a formulation for use in a method of treating myocardial infarction in subjects of need. The method includes intravenous administration of MYDGF protein to the subject in addition to standard care. Background Technology

[0002] Acute myocardial infarction (MI) remains a leading cause of morbidity and mortality worldwide, and a significant cause of disability. Acute MI is mediated by thrombotic occlusion of the coronary arteries, which leads to progressive cell death in non-perfused tissues. This triggers an inflammatory response, resulting in scarring and loss of viable tissue. Severe alterations in left ventricular histology can lead to ventricular dilation, systolic dysfunction, and heart failure.

[0003] ST-segment elevation myocardial infarction (STEMI) can be distinguished from non-ST-segment elevation myocardial infarction (NSTEMI) based on the presence or absence of persistent ST-segment elevation on an electrocardiogram (ECG). Both STEMI and NSTEMI are caused by the same pathophysiology and are typically due to acute thrombosis on atherosclerotic plaques in the coronary arteries. In the case of STEMI, this usually results in complete occlusion of the corresponding coronary artery. The angiographic morphology of NSTEMI may be more heterogeneous and usually indicates severe stenosis rather than complete occlusion of the vessel (Mitsis et al., 2021). ECG-based separation of these two patient subsets is currently used to select patients who will benefit most from immediate thrombolysis-reperfusion and direct percutaneous coronary intervention (PCI).

[0004] In emergency situations, PCI leading to rapid reperfusion is best practice for patients with acute STEMI, while antiplatelet therapy is used to prevent coronary re-occlusion. Reperfusion is undoubtedly beneficial, as evidenced by the decline in MI-related in-hospital morbidity and mortality over the past few decades. However, STEMI-related mortality has stabilized over the past decade, and despite PCI, up to 28% of patients still develop heart failure (HF) within 90 days after STEMI due to myocardial loss and scarring (Desta et al. 2015). Loss of cardiac tissue and subsequent cardiac remodeling, especially after larger myocardial infarctions (MI), can induce profound changes in the structure of the left ventricle (LV), leading to ventricular dilatation, systolic dysfunction, and ultimately heart failure.

[0005] Long-term prognosis remains poor, with over 50% of first-time STEMI patients over 45 years of age experiencing heart failure or death within a 5-year period (Desta et al., 2017). While PCI can reduce the degree of myocardial scarring in STEMI, it is also paradoxically associated with a process called "ischemia-reperfusion injury" (IRI), through which the myocardium can suffer additional damage at the reperfusion site due to a pro-inflammatory "burst," leading to apoptosis of cardiomyocytes downstream of the previously occluded tissue.

[0006] Therefore, even with successful PCI, STEMI patients may experience further myocardial injury and scarring. Furthermore, especially after extensive MI, natural repair mechanisms may be insufficient to prevent adverse remodeling and further scarring. Thus, STEMI remains a life-threatening emergency indication with a poor prognosis. To date, there are no treatments that can be combined with PCI to reduce ischemic injury and / or reperfusion-induced myocardial injury.

[0007] Myelin-derived growth factor (MYDGF) is a protein that has been shown to improve tissue repair and cardiac function in rodent models of myocardial infarction (WO 2014 / 111458). Treatment with recombinant MYDGF was found to protect cardiomyocytes from cell death and repair the heart after acute myocardial infarction. The development of protein-based therapies is a promising approach for cardiac repair and potentially for ischemic repair of other tissues (Ebenhoch et al., 2019; Polten et al., 2019; Botnov et al., 2018; Korf-Klingebiel et al., 2015).

[0008] Mouse MYDGF has been shown to block apoptosis in cardiomyocytes induced by simulated ischemia-reperfusion. MYDGF signaling is triggered via an unidentified molecular target / receptor on rat cardiomyocytes and is thought to involve a phosphatidylinositol 3-kinase (PI3K)-dependent pathway. This leads to phosphorylation of AKT1, which in turn phosphorylates its downstream targets Bad and Bax. Phosphorylation of Bad and Bax reduces cytochrome c release and caspase-9 cleavage. This reduces the activity of caspase-3 and caspase-7, well-known effector caspases that drive apoptosis (Korf-Klingebiel 2015). Furthermore, mouse MYDGF enhances in vitro angiogenesis by activating the mitogen-activated protein kinase (MAPK) pathway in human endothelial cells. This leads to phosphorylation of STAT3, which in turn increases cyclin D1 expression (Korf-Klingebiel 2015). MYDGF treatment in mice following myocardial infarction reduced infarct and scar size, increased recapillation at the infarct boundary, improved subacute and chronic cardiac function as assessed by echocardiography, and reduced heart failure-related mortality.

[0009] According to studies in mice (Korf-Klingebiel et al., 2015), the dosing regimen used to achieve the desired effect in a mouse model of myocardial infarction (ischemia / reperfusion) involved an intraventricular injection of 10 µg shortly before reperfusion, followed by subcutaneous infusion via a micropump for 7 days at a dose of 10 µg / day. To date, no formulations have been developed that allow for extended storage.

[0010] MYDGF is a large and complex molecule that undergoes various degradation processes, especially in the liquid state. The production and purification processes require careful control. Stabilizing the molecule through the development of appropriate formulations is a significant challenge. Because proteins exhibit various degradation pathways, this can lead to loss of therapeutic activity. Summary of the Invention

[0011] Our own research indicates that intravenous administration of myeloid-derived growth factor (MYDGF) protein to subjects (on a single dose) in addition to standard care is suitable for treating (acute) myocardial infarction in subjects, preferably human subjects.

[0012] The object of this invention is to provide formulations for intravenous injection, with or without further dilution. In the formulations provided by this invention, the stability of the MYDGF protein is increased due to the stabilizing excipients in the formulation. This stabilization enables MYDGF to be used for intravenous administration in a clinical setting.

[0013] The MYDGF formulations of this invention have a particular advantage: the liquid formulations can be lyophilized without any adjustment to the amount of excipients or the addition of any additional excipients. Compared to liquid formulations, the choice of lyophilization of the formulations of this invention provides extended shelf life. For example, lyophilized formulations can be stored at ambient temperature for extended periods without any significant degradation of the MYDGF protein.

[0014] When the formulation of the present invention is in liquid form, it should allow a concentration of MYDGF of 40-60 mg / ml and be suitable for intravenous administration. The liquid composition forming the basis of the formulation should be lyophilized, stored, and reconstituted before use.

[0015] Therefore, the present invention relates to a novel pharmaceutical formulation that has been optimized for maintaining the stability of MYDGF protein and reducing protein degradation during long-term storage of the pharmaceutical formulation.

[0016] In a first aspect, the present invention relates to a pharmaceutical preparation comprising:

[0017] (a) Myeloid-derived growth factor (MYDGF) protein at concentrations ranging from 0.5 mg / ml to 200 mg / ml;

[0018] (b) 10 mM to 100 mM buffer solution;

[0019] (c) Stabilizers ranging from 1 mM to 50 mM;

[0020] (d) Tension-inducing agents ranging from 20 mM to 250 mM; and

[0021] (e) 0.01% to 0.1% (w / v) of surfactant;

[0022] The pH of the composition is 5.0 to 7.0.

[0023] The pharmaceutical formulation of the present invention comprises five components: MYDGF protein, buffer, stabilizer, tensioning agent and surfactant.

[0024] As a first component, the formulation comprises a buffer. According to the invention, the buffer is an acetate buffer, citrate buffer, histidine buffer, succinate buffer, phosphate buffer, or tromethamine buffer.

[0025] The concentration of the buffer solution present in the pharmaceutical formulation of the present invention is from 10 mM to 100 mM. Preferably, the concentration of the buffer solution in the formulation is from 10 mM to 75 mM, from 10 mM to 50 mM, and more preferably from 10 mM to 30 mM. Most preferably, the concentration of the buffer solution in the formulation is 20 mM.

[0026] Preferably, the buffer solution in the pharmaceutical formulation of the present invention is a histidine buffer. The histidine buffer may be an L-histidine / HCl buffer, i.e., a buffer containing L-histidine or a mixture of L-histidine and L-histidine hydrochloride. The pH of the histidine buffer can be adjusted with hydrochloric acid.

[0027] Therefore, it is particularly preferred that the pharmaceutical formulation of the present invention comprises a histidine buffer solution, said histidine buffer solution being present in the formulation at a concentration of 10 mM to 100 mM. Preferably, the concentration of the histidine buffer solution in the formulation is 10 mM to 75 mM, 10 mM to 50 mM, and more preferably 10 mM to 30 mM. Most preferably, the concentration of the histidine buffer solution in the formulation is 20 mM.

[0028] As a second component, the formulation contains a stabilizer. According to the invention, the stabilizer is an amino acid, such as methionine, arginine, glycine, proline, lysine, or cysteine.

[0029] The concentration of the stabilizer in the pharmaceutical formulation of the present invention is from 1 mM to 50 mM. Preferably, the concentration of the stabilizer in the formulation is from 1 mM to 40 mM, 1 mM to 30 mM, 1 mM to 20 mM, or 1 mM to 10 mM. In some embodiments, the concentration of the stabilizer in the formulation is from 2 mM to 15 mM, 5 mM to 15 mM, and more preferably from 5 mM to 10 mM. Most preferably, the concentration of the stabilizer in the formulation is 10 mM.

[0030] Preferably, the stabilizer in the pharmaceutical formulation of the present invention is methionine.

[0031] Therefore, it is particularly preferred that the pharmaceutical formulation of the present invention comprises methionine, said methionine being present in the formulation at a concentration of 1 mM to 20 mM. Preferably, the concentration of methionine in the formulation is 2 mM to 15 mM, 5 mM to 15 mM, and more preferably 5 mM to 10 mM. Most preferably, the concentration of methionine in the formulation is 10 mM.

[0032] As a third component, the formulation contains a tensile agent. According to the invention, the tensile agent is a sugar or sugar alcohol, such as sucrose, trehalose, sorbitol, mannitol, or dextrose.

[0033] The concentration of the tensile agent present in the pharmaceutical formulation of the present invention is from 20 mM to 250 mM. Preferably, the concentration of the tensile agent in the formulation is from 50 mM to 250 mM, 100 mM to 250 mM, 150 mM to 250 mM, 175 mM to 250 mM, and more preferably from 200 mM to 250 mM, such as from 200 mM to 240 mM. Most preferably, the concentration of the tensile agent in the formulation is 220 mM.

[0034] Preferably, the tensile agent included in the pharmaceutical formulation of the present invention is sucrose.

[0035] Therefore, it is particularly preferred that the pharmaceutical formulation of the present invention comprises sucrose, said sucrose being present in the formulation at a concentration of 20 mM to 250 mM. Preferably, the concentration of sucrose in the formulation is 50 mM to 250 mM, 100 mM to 250 mM, 150 mM to 250 mM, 175 mM to 250 mM, and more preferably 200 mM to 250 mM, such as 200 mM to 240 mM. Most preferably, the concentration of sucrose in the formulation is 220 mM.

[0036] As a fourth component, the formulation contains a surfactant.

[0037] As used herein, the term "surfactant" refers to a surface-active agent. Examples of pharmaceutically acceptable surfactants include polyoxyethylene-sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (e.g., Brij), alkylphenyl polyoxyethylene ethers (e.g., Triton X), polyoxyethylene-polyoxypropylene copolymers (e.g., poloxamer, Pranic), and sodium dodecyl sulfate (SDS). The surfactant is preferably a polyoxyethylene-sorbitan fatty acid ester, i.e., a polysorbate, or a nonionic polyoxyethylene-polyoxypropylene copolymer, such as poloxamer. Preferred polysorbates for use in formulations of the present invention include polysorbate 20 and polysorbate 80. A preferred poloxamer for use in formulations of the present invention is Poloxamer 188™. Preferably, a nonionic surfactant is used.

[0038] The concentration of the surfactant present in the pharmaceutical formulation of the present invention is 0.01% to 0.1% (w / v). Preferably, the concentration of the surfactant in the formulation is 0.02% to 0.1% (w / v), 0.02% to 0.08% (w / v), 0.02% to 0.07% (w / v), 0.02% to 0.06% (w / v), 0.02% to 0.05% (w / v), and more preferably 0.02% to 0.04% (w / v). Most preferably, the concentration of the surfactant in the formulation is 0.03% (w / v) or 0.04% (w / v).

[0039] Preferably, the surfactant included in the pharmaceutical formulation of the present invention is polysorbate 20.

[0040] Therefore, it is particularly preferred that the pharmaceutical formulation of the present invention comprises polysorbate 20, said polysorbate 20 being present in the formulation at a concentration of 0.01% to 0.1% (w / v). Preferably, the concentration of polysorbate 20 in the formulation is 0.02% to 0.1% (w / v), 0.02% to 0.08% (w / v), 0.02% to 0.07% (w / v), 0.02% to 0.06% (w / v), 0.02% to 0.05% (w / v), and more preferably 0.02% to 0.04% (w / v). Most preferably, the concentration of polysorbate 20 in the formulation is 0.03% (w / v) or 0.04% (w / v).

[0041] In another embodiment, preferably, the surfactant included in the pharmaceutical formulation of the present invention is polysorbate 80.

[0042] Therefore, it is particularly preferred that the pharmaceutical formulation of the present invention comprises polysorbate 80, said polysorbate 80 being present in the formulation at a concentration of 0.01% to 0.1% (w / v). Preferably, the concentration of polysorbate 80 in the formulation is 0.02% to 0.1% (w / v), 0.02% to 0.08% (w / v), 0.02% to 0.07% (w / v), 0.02% to 0.06% (w / v), 0.02% to 0.05% (w / v), and more preferably 0.02% to 0.04% (w / v). Most preferably, the concentration of polysorbate 80 in the formulation is 0.03% (w / v) or 0.04% (w / v).

[0043] The MYDGF protein used in the pharmaceutical formulations of the present invention preferably comprises or consists of an amino acid sequence selected from or composed of the group of different MYDGF proteins shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 11 and SEQ ID NO: 13. All of these variants are derived from human MYDGF.

[0044] Because of the low levels of antidrug antibodies (ADAs) caused by this protein variant, the use of MYDGF protein comprising or consisting of the sequence of SEQ ID NO: 1 is particularly preferred for use in pharmaceutical formulations of the present invention. Furthermore, the MYDGF protein of SEQ ID NO: 1 can be produced in high amounts and with desired purity. According to the present invention, MYDGF protein is included in the formulations of the present invention at concentrations of 0.5 mg / ml to 200 mg / ml, and preferably at concentrations of 1 mg / ml to 200 mg / ml, 5 mg / ml to 200 mg / ml, 10 mg / ml to 200 mg / ml, or 25 mg / ml to 200 mg / ml. More preferably, MYDGF protein is included in the formulations of the present invention at concentrations of 1 mg / ml to 100 mg / ml, such as 10 mg / ml to 90 mg / ml, 20 mg / ml to 80 mg / ml, 30 mg / ml to 70 mg / ml, or 40 mg / ml to 60 mg / ml. A concentration of 50 mg / ml is particularly preferred.

[0045] The pharmaceutical formulations of the present invention have a pH of 5.0 to 7.0, and preferably a pH of 5.0 to 6.5, 5.2 to 6.5, 5.2 to 6.2, or 5.4 to 6.0. In some embodiments, the pharmaceutical formulations of the present invention have a pH of 5.5 to 6.5, 5.6 to 6.4, 5.7 to 6.3, 5.8 to 6.2, or 5.9 to 6.1. According to the present invention, a pH of 5.7 ± 0.3 is particularly preferred.

[0046] In one embodiment of the present invention, the pharmaceutical preparation of the present invention comprises or is composed of the following:

[0047] (a) Myeloid-derived growth factor (MYDGF) protein at concentrations ranging from 0.5 mg / ml to 200 mg / ml;

[0048] (b) 10 mM to 100 mM histidine buffer;

[0049] (c) 1 mM to 50 mM methionine;

[0050] (d) 20 mM to 250 mM sucrose; and

[0051] (e) 0.01% to 0.1% (w / v) polysorbate 20 or polysorbate 80;

[0052] The pH of the composition is 5.0 to 7.0.

[0053] In one embodiment of the present invention, the pharmaceutical preparation of the present invention comprises or is composed of the following:

[0054] (a) Myeloid-derived growth factor (MYDGF) protein at concentrations of 40 mg / ml to 60 mg / ml;

[0055] (b) 10 mM to 30 mM histidine buffer;

[0056] (c) 5 mM to 15 mM methionine;

[0057] (d) 100 mM to 250 mM sucrose; and

[0058] (e) 0.02% to 0.06% (w / v) polysorbate 20 or polysorbate 80;

[0059] The pH of the composition is 5.0 to 6.5.

[0060] In yet another embodiment of the invention, the pharmaceutical preparation of the invention comprises or is composed of the following:

[0061] (a) Myeloid-derived growth factor (MYDGF) protein at concentrations of 45 mg / ml to 55 mg / ml;

[0062] (b) 18 mM to 22 mM histidine buffer;

[0063] (c) 9 mM to 11 mM methionine;

[0064] (d) 200 mM to 240 mM sucrose; and

[0065] (e) 0.02% to 0.06% (w / v) polysorbate 20;

[0066] The pH of the composition is 5.7 ± 0.3.

[0067] In yet another embodiment of the invention, the pharmaceutical preparation of the invention comprises or is composed of the following:

[0068] (a) Myeloid-derived growth factor (MYDGF) protein at concentrations of 45 mg / ml to 55 mg / ml;

[0069] (b) 18 mM to 22 mM histidine buffer;

[0070] (c) 9 mM to 11 mM methionine;

[0071] (d) 200 mM to 240 mM sucrose; and

[0072] (e) 0.02% to 0.06% (w / v) polysorbate 80;

[0073] The pH of the composition is 5.7 ± 0.3.

[0074] In yet another embodiment of the invention, the pharmaceutical preparation of the invention comprises or is composed of the following:

[0075] (a) 50 mg / ml of myeloid-derived growth factor (MYDGF) protein;

[0076] (b) 20 mM histidine buffer;

[0077] (c) 10 mM methionine;

[0078] (d) 220 mM sucrose; and

[0079] (e) 0.03% (w / v) or 0.04% (w / v) polysorbate 20;

[0080] The pH of the composition is 5.7 ± 0.3.

[0081] In yet another embodiment of the invention, the pharmaceutical preparation of the invention comprises or is composed of the following:

[0082] (a) 50 mg / ml of myeloid-derived growth factor (MYDGF) protein;

[0083] (b) 20 mM histidine buffer;

[0084] (c) 10 mM methionine;

[0085] (d) 220 mM sucrose; and

[0086] (e) 0.03% (w / v) or 0.04% (w / v) polysorbate 80;

[0087] The pH of the composition is 5.7 ± 0.3.

[0088] In yet another embodiment of the invention, the pharmaceutical preparation of the invention comprises or is composed of the following:

[0089] (a) 50 mg / ml of myeloid-derived growth factor (MYDGF) protein, wherein the myeloid-derived growth factor protein contains the amino acid sequence of SEQ ID NO: 4.

[0090] (b) 20 mM histidine buffer;

[0091] (c) 10 mM methionine;

[0092] (d) 220 mM sucrose; and

[0093] (e) 0.03% (w / v) or 0.04% (w / v) polysorbate 20 or polysorbate 80;

[0094] The pH of the composition is 5.7 ± 0.3.

[0095] In yet another embodiment of the invention, the pharmaceutical preparation of the invention comprises or is composed of the following:

[0096] (a) 50 mg / ml of myeloid-derived growth factor (MYDGF) protein, wherein the myeloid-derived growth factor protein comprises or is composed of the amino acid sequence of SEQ ID NO: 1.

[0097] (b) 20 mM histidine buffer;

[0098] (c) 10 mM methionine;

[0099] (d) 220 mM sucrose; and

[0100] (e) 0.03% (w / v) or 0.04% (w / v) polysorbate 20;

[0101] The pH of the composition is 5.7 ± 0.3.

[0102] In yet another embodiment of the invention, the pharmaceutical preparation of the invention comprises or is composed of the following:

[0103] (a) 50 mg / ml of myeloid-derived growth factor (MYDGF) protein, wherein the myeloid-derived growth factor protein comprises or is composed of the amino acid sequence of SEQ ID NO: 1.

[0104] (b) 20 mM histidine buffer;

[0105] (c) 10 mM methionine;

[0106] (d) 220 mM sucrose; and

[0107] (e) 0.03% (w / v) or 0.04% (w / v) polysorbate 80;

[0108] The pH of the composition is 5.7 ± 0.3.

[0109] The pharmaceutical formulations of the present invention can be provided in liquid or lyophilized form (i.e., as a dry powder). This powder can be reconstituted by adding a liquid (e.g., water for injection (WFI) or antibacterial water for injection (BWFI)). An advantage of the formulations of the present invention is that no additional excipients are required to make the formulation suitable for lyophilization. Images of the lyophilized formulations are included. Figure 9 In a preferred embodiment, the above-described formulation consists of components (a)-(e) and water, and contains no other components or excipients. Rapid reconstitution ensures rapid administration. Stability of the lyophilized formulation stored at 25ºC for up to 6 months showed no significant degradation of the MYDGF protein. Evaluation data included: monomer content determined by SEC-HPLC (e.g., Figure 7 (as shown) and the main peak determined by ion exchange chromatography (such as...) Figure 8 (As shown).

[0110] The pharmaceutical formulations of the present invention have particularly preferred storage stability, as described in the following examples and Figure 5 and Figure 6 As shown, the formulations of the present invention were found to be extremely stable when stored at 5ºC for 18 or 24 months. Preferably, when stored at 5ºC for 18 or 24 months, the pharmaceutical formulations of the present invention exhibited MYDGF protein degradation of less than 10.0%, less than 9.0%, less than 8.0%, less than 7.0%, less than 6.0%, or less than 5.0%, as determined by ion exchange chromatography or size exclusion chromatography.

[0111] In a second aspect, the present invention relates to pharmaceutical formulations according to the first aspect of the invention for use in medicine.

[0112] The drug formulation can be administered as an intravenous bolus or directly via infusion after dilution with an infusion medium. Standard infusion media are, for example, glucose solutions and sodium chloride solutions. Examples of dilution with sodium chloride solution without degrading MYDGF protein include... Figure 10 In this study, the monomer content was examined using size exclusion chromatography.

[0113] In a third aspect, the present invention relates to a pharmaceutical preparation according to a first aspect of the invention, said pharmaceutical preparation for use in a method of treating a subject with myocardial infarction. Preferably, the myocardial infarction to be treated by the preparation of the invention is ST-segment elevation myocardial infarction (STEMI). More preferably, the myocardial infarction to be treated is anterior wall STEMI, i.e., STEMI caused by occlusion of the left anterior descending artery (LAD) of the subject. The myocardial infarction to be treated may be STEMI associated with cardiogenic shock. The subject to be treated is preferably a mammal, and more preferably a human. The pharmaceutical preparation of the invention can be administered via various routes. However, it is preferred that the preparation of the invention be administered intravenously. Therefore, the preparation of the invention can be formulated for different routes of administration. Preferably, the preparation of the invention is formulated for intravenous administration, for example by intravenous infusion. The pharmaceutical preparation of the invention is preferably used in a method of treating a subject with myocardial infarction, said method comprising further percutaneous coronary intervention (PCI) to restore blood flow to the cardiac tissue.

[0114] In a fourth aspect, the present invention relates to a method for treating a subject with myocardial infarction, the method comprising administering a pharmaceutical preparation according to a first aspect of the invention. Preferably, the myocardial infarction to be treated by the method according to the fourth aspect of the invention is ST-segment elevation myocardial infarction (STEMI). More preferably, the myocardial infarction to be treated is anterior wall STEMI, i.e., STEMI caused by occlusion of the subject's left anterior descending artery (LAD). The myocardial infarction to be treated may be STEMI associated with cardiogenic shock. The subject to be treated is preferably a mammal, and more preferably a human. The method preferably comprises administering the pharmaceutical preparation according to the first aspect of the invention by intravenous infusion. The method may further comprise percutaneous coronary intervention (PCI) to restore blood flow to the cardiac tissue.

[0115] Any of the above treatment methods may also be applied to subjects with myocardial infarction for one or more of the following purposes:

[0116] • Reduce infarct size

[0117] • Reduce scar size

[0118] • Increase cardiac output

[0119] • To prevent or improve heart failure (HF).

[0120] • Reduce hospitalizations due to heart failure (HHF).

[0121] • Reduce mortality rate, and / or

[0122] • Reduce reperfusion injury.

[0123] In a particularly preferred embodiment, the above-described treatment method is applied to reduce infarct size, as measured by late gadolinium-enhanced cardiac magnetic resonance imaging (LGE-CMR) as reported by Ibanez et al. 2019. More preferably, the above-described treatment method is applied to reduce infarct size after PCI, as measured by LGE-CMR as reported by Ibanez et al. 2019.

[0124] In addition, any of the above treatments can be combined with commonly known interventions or pharmaceutical products for the treatment of MI.

[0125] In a fifth aspect, the present invention relates to a pharmaceutical preparation according to a first aspect of the invention, said pharmaceutical preparation being used in a method for treating or preventing cardiogenic shock in a subject.

[0126] In a sixth aspect, the present invention relates to a method for treating or preventing cardiogenic shock in a subject, the method comprising administering a pharmaceutical preparation according to a first aspect of the invention. For the use of MYDGF in the treatment or prevention of cardiogenic shock, see WO 2024 / 052563 A1.

[0127] As used in the fifth and sixth aspects of the invention, the term "cardiogenic shock" describes a state of inadequate end-organ perfusion due to heart failure and the cardiovascular system's inability to provide sufficient blood flow to the limbs and vital organs. Patients with cardiogenic shock present with persistent hypotension (systolic blood pressure less than 80 to 90 mm Hg, or mean arterial pressure less than 30 mm Hg from baseline, or requiring vasopressors or positive inotropic agents to maintain SBP > 90 mm Hg, or mean arterial blood pressure < 70 mm Hg, or systolic blood pressure < 100 mm Hg despite adequate fluid resuscitation), and evidence of end-organ damage (e.g., by urine output < 30 mL / h, or urine output < 0.5 mL / kg in 1 hour, or cold extremities, or mottled skin, or serum lactate > 2 mmol / L, or metabolic acidosis, or altered mental status), and with severely reduced cardiac index (less than 2.2 L / min / m²) in the presence of adequate or elevated filling pressure (left ventricular (LV) end-diastolic pressure greater than 15 mm Hg or right ventricular (RV) end-diastolic pressure greater than 10 to 15 mm Hg). 2 (Vahdatpour et al. 2019) Journal of the American Heart Association, Vol. 8 (8), 2019, pp. 1991.

[0128] According to a preferred embodiment related to the fifth or sixth aspect of the invention, the cardiogenic shock to be treated or prevented is cardiogenic shock occurring during myocardial infarction (MI), such as acute myocardial infarction (AMI), preferably ST-segment elevation myocardial infarction (STEMI), and even more preferably cardiogenic shock occurring during MI or STEMI during percutaneous coronary intervention (PCI) reperfusion. Attached Figure Description

[0129] Figure 1 The results (Y-axis) of the main peak measured by ion exchange chromatography (IEC-HPLC) after two weeks of storage at 40ºC are shown.

[0130] Figure 2 The initial turbidity results and those after 9 months of storage at 25ºC are shown (Y-axis).

[0131] Figure 3 The results (Y-axis) of the main peak measured by size exclusion chromatography (SEC-HPLC) during the preparation was stored at 25ºC for up to 9 months are shown.

[0132] Figure 4 The results (Y-axis) of the main peak measured by ion exchange chromatography (IEC-HPLC) during the preparation was stored at 25ºC for up to 9 months are shown.

[0133] Figure 5 The main peak (Y-axis) is shown as measured by size exclusion chromatography (SEC-HPLC) during the preparation's storage at 5ºC for up to 24 months.

[0134] Figure 6 The main peak area (Y-axis) is shown as determined by ion exchange chromatography (IEC-HPLC) during the preparation's storage at 5ºC for up to 24 months.

[0135] Figure 7 The main peak (Y-axis) of the lyophilized formulation H was measured by size exclusion chromatography (SEC-HPLC) during storage at 25ºC for up to 6 months.

[0136] Figure 8 The main peak (Y-axis) is shown as measured by ion exchange chromatography (IEC-HPLC) during the storage of lyophilized formulation H at 25ºC for up to 6 months.

[0137] Figure 9 Images and µCT scans of the freeze-dried formulation are shown.

[0138] Figure 10The main peaks (Y-axis) of the undiluted and diluted solutions, measured by size exclusion chromatography (SEC-HPLC), are shown before and after storage at 25ºC.

[0139] Figure 11 The turbidity results (Y-axis) of the undiluted and diluted solutions before and after storage at 25ºC are shown. Example

[0140] The present invention will be illustrated by the following examples, given by way of illustration only. In particular, Examples 1-3 describe the generation of the production strain and the heterologous expression and purification of the MYDGF variant. Other examples describe studies on the stability of the MYDGF formulation.

[0141] Example 1 Production of recombinant human MYDGF protein

[0142] The preparation of MYDGF variants and the functional assays used to test their therapeutic efficacy are also described in WO 2023 / 233034A1. The following MYDGF proteins were prepared and used in the following studies and experiments:

[0143] 1.1 human [+G] MYDGF variant HEK (hereinafter “h[+G]-MYDGF-HEK”)

[0144] h[+G]-MYDGF-HEK has the sequence SEQ ID NO: 11 (the amino acid sequence of the [+G] MYDGF variant, wherein a G residue precedes the N-terminal V residue at the +1 position of mature human MYDGF):

[0145] h[+G]-MYDGF-HEK has the sequence SEQ ID NO: 11

[0146] GVSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL

[0147] The h[+G]-MYDGF-HEK precursor has the sequence SEQ ID NO: 12

[0148] MGWSLILLFLVAVATRVLSHHHHHHAGSENLYFQ↓GVSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL

[0149] Variants were manufactured as described in: Polten, F. et al. (2019), Anal Chem, 91, 1302–1308, pp. 1303, column 1 and Figure S1; and Ebenhoch, R. et al. (2019), Nat Commun 10, 5379, p. 8, left column. The precursor sequence of SEQ ID NO: 12 has a His tag integrated at the N-terminus, which is cleaved by the TEV protease to form the protein according to Seq ID NO: 11.

[0150] 1.2 Human MYDGF variants with HIS tags (hereinafter “hMYDGF-[HIS]-HEK”)

[0151] hMYDGF-[HIS]-HEK has the sequence SEQ ID NO: 13

[0152] VSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTELHHHHHH

[0153] The hMYDGF-[HIS]-HEK precursor has the sequence SEQ ID NO: 14

[0154] MGWSLILLFLVAVATRVLS↓VSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTELHHHHHH

[0155] It is produced using a stable HEK293f (human embryonic kidney) cell line.

[0156] Culture medium: F17 supplemented with 0.1% Pronic F-68 and 6 mM GlutaMAX

[0157] Culture Maintenance: Five vials of cells were previously cryopreserved for Project 1409 and stored in liquid nitrogen. For this project, one vial of cells was thawed and placed in 5 mL of pre-warmed medium. The cells were then centrifuged at 300 x g for 5 min, resuspended at a density of 5 x 10⁵ cells / mL, and transferred to a T-75 flask for static culture. The next day, the cells were transferred to shake flasks and incubated at 37ºC with shaking at 135 rpm in a humidified 5% CO₂ environment. MYDGF-His stabilized HEK293f cells were maintained in shake flasks at a density between 0.5–4 x 10⁶ cells / mL without antibiotics. The flasks were incubated at 37ºC with shaking at 135 rpm in a humidified 5% CO₂ environment.

[0158] Culture volume: 40 L (40 x 1 L in a 2 L shake flask); Initial density: 0.5 x 10⁶ cells / mL; method: 1 L of culture of MYDGF-His stable HEK293f cells was seeded in 2 L shake flasks (40 L total) at a density of 0.5 x 10⁶ cells / mL. Density and viability were monitored using a ViCell XR. Density, viability, and mean diameter were measured on the ViCell XR. Harvesting: The culture was harvested 10 days post-transfection by centrifugation at 1000 xg for 5 min at 4°C. The conditioned culture supernatant (CCS) was clarified by centrifugation at 9300 xg for 30 min at 4°C. For expression analysis, cells were harvested from 1 mL of culture by centrifugation at 1000 xg (25°C) for 5 min. Cell pellets were lysed by freezing / thawing and resuspended in a mixture of 1X TBS (pH 8.0), 0.1% BOG, 0.1% DDM, 1 mM β-ME, 10 U / mL Turbonuclease, and 1xComplete® protease inhibitor (10 μL / 1 x 10⁵ cells). Lysis was verified by optical microscopy.

[0159] 1.3 Human [+A] MYDGF variant Escherichia coli (hereinafter referred to as "h[+A]-MYDGF-Escherichia coli")

[0160] h[+A]-MYDGF-Escherichia coli corresponds to the [+A] variant described below and has the sequence shown in SEQ ID NO: 1.

[0161] This protein can be manufactured as follows:

[0162] 1.3.1 Preparation and transfection of the vector

[0163] To produce the cell bank, a derivative of the *E. coli* strain BL21(DE3), modified to prevent phage production, was used. The strain was transformed using one of the vectors shown in SEQ ID NO: 7-10 carrying genes encoding the corresponding MYDGF variants. The genes for each variant were codon-optimized to achieve high expression rates in *E. coli* and synthesized by ATUM (Newark, California, USA). Plasmids encoding the following MYDGF variants were produced:

[0164] • [+A] variant (h[+A]-MYDGF-E. coli), wherein the N-terminal V residue at the +1 position of mature human MYDGF is preceded by an A residue (as shown in the aa sequence of SEQ ID NO: 1:

[0165] AVSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL). The possible expression vector encoding this variant is shown in SEQ ID NO: 7.

[0166] • [+S] variant (h[+S]-MYDGF-E. coli), wherein the N-terminal V residue at the +1 position of mature human MYDGF is preceded by an S residue (as shown in the aa sequence of SEQ ID NO: 2).

[0167] SVSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL). The possible expression vector encoding this variant is shown in SEQ ID NO: 8.

[0168] • [+G] variant (h[+G]-MYDGF-E. coli), wherein the N-terminal V residue at the +1 position of mature human MYDGF is preceded by a G residue (as shown in the aa sequence of SEQ ID NO: 3:

[0169] GVSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL). The possible expression vector encoding this variant is shown in SEQ ID NO: 9.

[0170] • [-V] variant (h[-V]-MYDGF-E. coli), in which the N-terminal V residue at the +1 position of mature human MYDGF is deleted (as shown in the aa sequence of SEQ ID NO: 4:

[0171] SEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL). The possible expression vector encoding this variant is shown in SEQ ID NO: 10.

[0172] In the event of any discrepancy between the sequences listed above and those shown in the attached sequence list, the sequence list shall prevail. Hyphens appearing in the final sequence are the result of text processing truncation and must be ignored.

[0173] To prepare the expression strain, *E. coli* cells were transformed with the above-described vector plasmid via electroporation using the Gene Pulser Xcell™ electroporation system (BioRad). The protein was expressed in *E. coli* cells as inclusion bodies (IBs) that accumulate in the cytoplasm, as further described below.

[0174] 1.3.2 Expression of MYDGF variants

[0175] The following general protocol is used for the expression and purification of MYDGF variants:

[0176] 1. Fermentation

[0177] 2. IB Preparation

[0178] 3. Dissolving / Refolding

[0179] 4. Ultrafiltration / Percolation

[0180] 5. Anion exchange chromatography

[0181] 6. Preferably: Hydrophobic interaction chromatography

[0182] 7. Preferably: Concentration and formulation

[0183] Fermentation

[0184] One cell bank vial of the obtained production strain was thawed at room temperature. The preculture (PC) consisted of two 1 L shake flasks, each containing 300 mL of seed culture medium. The composition of the seed culture medium is depicted in Table 1 below. All buffers and media were prepared using reverse osmosis (RO) water and sterilized using a nanofiltration device or heat sterilization before use.

[0185] Table 1 Seed culture medium

[0186]

[0187] Add antifoaming agents and antibiotics as needed. Add kanamycin as an antibiotic (to a final concentration of 50 µg / mL). Inoculate each shake flask with 100 μL of the production strain. Cultures grow for approximately 9.4 h, targeting an OD of 1.75 ± 0.5 (optical density at 550 nm).

[0188] The main culture (MC) was carried out in a stainless steel bioreactor with a total volume of 20 L containing 10 L of batch culture medium. The composition of the batch culture medium is depicted in Table 2 below.

[0189] Table 2 Batch culture medium

[0190]

[0191] The batch culture medium was inoculated with 100 mL of cell broth from the preculture. Fermentation parameters were kept constant during both the batch and exponential feed phases: 33.5ºC, pH 6.8, 1.0 bar discharge pressure, and 20% DO setpoint. An exponential feed (600 g / L glucose; μ = 0.25 h⁻¹) was initiated after carbon source depletion was observed via the dissolved oxygen (DO) peak. -1 The composition of the feed medium is described in Table 3 below.

[0192] Table 3 Feed culture medium

[0193]

[0194] After an exponential feed rate of 9 h (60.48 to 573.78 g / h), the feed rate was maintained at 573.78 g / h for the remainder of fermentation (11.5 h). At 11.5 h after the start of exponential feed, a 60-min temperature ramp (from 33.5ºC to 30.0ºC) was initiated and completed immediately before IPTG induction. At 12.5 h after the start of feed, the culture was induced by swarming with IPTG. MC was terminated at 20.5 h after the start of feed. At the end of the culture, the culture broth was immediately cooled to <12ºC, diluted with reverse osmosis (RO) water to 15% of the target wet cell weight (WCW), and bacterial cell clumps were separated from the supernatant by centrifugation using a CEPA centrifuge. The biomass was harvested and transferred along with the supernatant to downstream processing.

[0195] Product quantification was performed using the LabChip GXII® system (Perkin Elmer), which provides an automated, high-throughput alternative to traditional SDS-PAGE and protein quantification. Sample preparation was performed using a liquid handling system (Tecan Freedom EVO150). For product quantification from fermentation samples, analytical cell lysis was promoted from the fermentation samples via enzymatic cell lysis. 90 µL of fermentation suspension was diluted at a 9:10 ratio (v / v) with cell lysis buffer (Lysonase™ in FastBreak™ Cell Lysis Reagent (Promega) (Merck), with 32 µL Lysonase / 1 mL FastBreak™ reagent). For total product assays (soluble and insoluble fractions), the sample was mixed prior to each pipetting step. Finally, the sample was diluted to the system's specific sample buffer. To minimize the required amount of sample buffer, all dilution steps were performed in PBS or another preparation buffer.

[0196] For the final dilution, dilute 8 µL of sample (from PBS diluent) or standard curve sample in 28 µL of non-reducing sample buffer in a 96-well plate (Eppendorf twin.tec PCR plate 95100401). For reducing conditions, use 28 µL of reducing sample buffer (with 35 mM DTT). Seal the plate with foil (Eppendorf PCR foil 0030127790), briefly centrifuge (25 g for 30 s) and denature at 70ºC for 10 min. After denaturation, centrifuge the plate at 2200 g for 5 min to centrifuge and settle any evaporated liquid. After centrifugation, remove the foil and dilute with 140 µL of DI water. Seal the 96-well plate (Eppendorf twin.tec PCR plate 95100401) with foil and centrifuge at 2200 g for 10 min to precipitate any potential aggregates that may cause LabChip analysis failures. After centrifugation, the samples were analyzed in a LabChip GXII plate set to "HT Protein Express 100 High Sensitivity". LabChip preparation was performed according to the manufacturing instructions. Standard curves were prepared by diluting the reference material. As reference materials, the [+G] variant generated in HEK 293-6E cells was used, as described below: Polten (2019), see page 1303, column 1 and Figure S1, and Ebenhoch (2019), see page 8, column 1.

[0197] Quantification was performed in the range of 1 mg / mL to 0.1 mg / mL via linear fitting. Reduction and non-reduction conditions did not change the integral area for quantification; however, changes in run time without affecting quantification were observed.

[0198] 1.3.3 Purification and Analysis of Inclusion Bodies

[0199] The frozen *E. coli* biomass obtained as described above was resuspended in IB preparation buffer 1 (1 M urea, 50 mM Tris, 0.1% (v / v) polysorbate 20, pH 7.5) at a 1:5 (w / v) ratio. After resuspending in Ultraturax for 15 min, *E. coli* cells were disrupted by three high-pressure homogenizations at 650–700 bar. Dense and heavy inclusion bodies (IBs) and large cell debris were separated by high-speed tubular centrifugation using a GLE rotor (CEPA). The feed flow rate was 55 mL / min, and the centrifugation speed was 24,500 g. The tubes had an inner diameter of 3.2 mm. The recovered precipitate was washed twice with HQ water. In all cases, the precipitate was diluted 1:5 (w / v) and resuspended in Ultraturax. After the HQ water step, the precipitate mainly contained IB.

[0200] 1.3.4 Protein solubility, refolding, and purification

[0201] The frozen IB obtained as described above was dissolved at room temperature in dissolution buffer (8 M urea, 0.14 M GuHCl, 6 mM DTT, 50 mM Tris, pH 8). The mixture was first stirred with an Ultraturax for 10 minutes, followed by stirring with a propeller mixer for 180 minutes. The target concentration during dissolution was 5 mg / mL, and the target volume was 100 mL. The dissolution cell was then filtered through a CUNO depth filter (filter E16E01A90ZB08A, 0.1–0.6 µm, 3M Deutschland GmbH, Neuschwanstein, Germany). The filter was pre-equilibrated with water for injection (WFI) and dissolution buffer. The dissolution cell was then loaded directly. The filtrate was collected using an ÄKTA system via UV monitoring. The inclusion body solution was diluted with a 1:5 refolding buffer (4 M urea, 0.3125 M Tris, 12.5 mM CaCl2, 3.75 mM cystamine, pH 7). The recovered refolding cell was stirred overnight. The next day, it was filtered using a CUNO deep filter (filter E16E01A60ZB05A, 3M Deutschland GmbH, CUNO).

[0202] After filtration using a depth filter, the filtrate was subjected to ultrafiltration / percolation (UFDF) for buffer exchange. UFDF was performed using a Pellicon 3 membrane (88 cm², Ultracell 3 kDa, C-type sieve) and percolation buffer (20 mM Tris, pH 9). Concentration factor 2 and percolation factor 5 were used.

[0203] Following UFDF, the filtrate was subjected to ion exchange chromatography (IEX). A YMC Biopro IEX (Q75) 75 µm column with a diameter of 1 cm, a bed height of 9 cm, and a column volume of 7.5 ml was used. The column was first equilibrated with 3 column volumes (CV) of equilibration buffer 1 (20 mM Hepes, 1 M NaCl, pH 7) for 5 min, followed by 5 CV of equilibration buffer 2 (20 mM Tris, pH 9) for 5 min. After loading the filtrate, it was washed with 5 CV of 20 mM Tris (pH 9) for 5 min. Proteins were eluted with 5 CV of elution buffer 1 (20 mM Hepes, 1 M NaCl, pH 7) for 5 min, followed by elution with 10 CV of elution buffer 2 (20 mM Hepes, pH 7) using a gradient (0% to 100% 20 mM Hepes, 1 M NaCl, pH 7) for 5 min. Finally, the column was stripped and washed with 5 CV (with 1 MHCl) for 5 min.

[0204] 1.3.5 Protein yield and product homogeneity

[0205] All four variants underwent at least one purification. Additionally, a second purification experiment was performed with the [+A] and [+S] variants. Purification resulted in high yields and high purity for each of the four variants. In particular, the overall process yield of the [+A] variant, after purification and refolding, was found to be 2.4 g / L for the first batch and 5.3 g / L for the second batch.

[0206] Analytical high-performance size exclusion chromatography (SMS) was used to test purity. The purified [+A] variant exhibited high purity with a main peak of 99.75%, low molecular weight impurities of 0.25%, and aggregate levels of 0.0%. The [-V] variant (99.64% main peak, 0.0% low molecular weight impurities, 0.04% aggregates) and the [+S] variant (99.73% main peak, 0.2% low molecular weight impurities, 0.05% aggregates) achieved similar high purity levels. In contrast, the [+G] variant showed poor homogeneity when examined with SMS, exhibiting a main peak purity of 60.36% and aggregate levels of 39.64%.

[0207] The table below summarizes the process yields for laboratory-scale purification runs of all N-terminal variants:

[0208] Table 4 Summary of laboratory-scale production of MYDGF N-terminal variants. Amounts of MYDGF at different process steps are provided in mg MYDGF.

[0209]

[0210] The batch fermentation process applied to all four variants resulted in very high cell densities at the end of fermentation (OD of 326–348 at 550 nm; wet cell mass of 310.42–337.58 g / L). Very high volumetric titers (23.1–27.1 g / L fermentation) were achieved for the recombinant MYDGF variants.

[0211] Example 2: Advanced MYDGF manufacturing process

[0212] Based on Examples 1.3.2-1.3.5, a manufacturing process for the h[+A]-MYDGF-Escherichia coli MYDGF variant was further developed. The MYDGF fermentation process was first developed at a 5 L scale using a research cell bank (RCB), then validated at a 20 L scale using a pooled run of a GMP working cell bank (WCB), and finally scaled up to 200 L. A typical 200 L fermentation batch yields 16-18 kg wet IB.

[0213] The downstream process for purifying MYDGF drugs from intracellular inclusion bodies was first developed at a laboratory scale, then validated using inclusion bodies from 10 L fermentation aliquots through a combined run at a pilot scale, and finally transferred to a cGMP facility, where one downstream batch started with 10 kg wet IB, representing approximately 110–125 L of fermentation aliquots.

[0214] Table 5 Description of the manufacturing process for CMC1a drugs (upstream process section)

[0215]

[0216] Table 6 Description of the CMC1a drug manufacturing process (downstream portion)

[0217]

[0218] Several batches were conducted under GMP conditions. Each batch yielded a high production rate of 330-355 g MYDGF, typically achieved from a 125 L fermentation aliquot. This reflects a total process yield of up to 2.84 g / L. The MYDGF produced by this process meets all quality requirements for use in toxicological and clinical studies.

[0219] The monomer content, measured by size exclusion chromatography (HP), is typically higher than 99%, with high molecular weight impurities (aggregates) below 1% and low molecular weight impurities (fragments) below 0.1%. Endotoxin levels are below 0.03 EU / mg MYDGF protein. Host cell DNA content is ≤ 3 pg / mg protein.

[0220] The excipients, pH, and concentration of MYDGF protein must be adjusted according to the specific formulation described herein.

[0221] Example 3: Molecular weight analysis by LCMS and advanced molecular weight analysis by LCMS after chemical modification, according to Tolonen, AC et al. (2011) (“aLCMS”).

[0222] Samples of the folded and purified product obtained from Example 2 were subjected to liquid chromatography-mass spectrometry (LC-MS) analysis. Complete (non-reducing) molecular weight analysis of the MYGDF construct was performed using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) to (1) validate the sequences by the consistency of the observed molecular weights with the predicted values ​​for each sequence, and (2) capture an overall profile of net post-translational modifications (PTMs) on each protein. The sample (0.5 µg / injection) was desalted using an Agilent 1290 UPLC with a 1.0 mm × 30 mm C3 POROS reversed-phase column and introduced into the mass spectrometer. A three-minute binary gradient consisting of mobile phase A (98.9% water, 1% acetonitrile, 0.1% formic acid, and 2 mM ammonium acetate) and mobile phase B (70% isopropanol, 20% acetonitrile, 9.9% water, and 0.1% formic acid) was used to capture, desalt, and elute proteins from the column at a rate of 150 µl / min, increasing from 5% to 80% of mobile phase B. Mass spectrometric data of the eluted material were acquired using an Agilent 6224 Time-of-Flight (TOF) MS and then processed (deconvolutioned) using the maximum entropy algorithm within the MassHunter analysis software (Agilent). The data obtained by this method are referred to herein as “full MW LCMS data” or “data measured by liquid chromatography-mass spectrometry (LCMS)”.

[0223] For peptide-level sequence confirmation and site-specific post-transcriptional modification (PTM) analysis, aliquots of each sample were digested with trypsin and chymotrypsin, respectively, to achieve complete sequence coverage. 100 µg of each sample was desalted and concentrated via acetone precipitation and centrifugation of the precipitate. Each protein precipitate was redissolved, denatured, and reduced in 10 µl of denaturation / reduction buffer (5% w / v sodium deoxycholate (SDC), 10 mM dithiothreitol (DTT), 20 mM ammonium bicarbonate) and incubated at 70ºC for 2 min, followed by a 10-fold dilution with 20 mM ammonium bicarbonate and 2 mM methionine. The reduced / denatured molecules were then aliquoted into two vials (50 µg each), and trypsin and chymotrypsin were added to each vial at a 1:10 enzyme-to-substrate ratio, and the samples were incubated at 37ºC for 10 min. The reaction was quenched by adding 10% v / v trifluoroacetic acid to obtain a final reagent concentration of 1% v / v. The short (10 min) digestion step avoids the need for the alkylation step commonly used in peptide mapping. The precipitate of sodium deoxycholate was removed by centrifugation at 16,000 x g, and the peptide-containing supernatant was recovered and transferred to an autosampler vial, which was immediately stored at -80ºC until analysis. The data obtained by this method are referred to herein as “peptide mapping LCMS data”.

[0224] aLCMS: Additionally, since the first four N-terminal residues of various MYGDF constructs have been shown to undergo fragmentation during electrospraying (at both the intact and peptide levels), reductive dimethylation (also known as stable isotopic dimethyl labeling (SIDL)) was performed on aliquots of peptide digests according to Tolonen et al. (2019) to distinguish sample-derived N-terminal truncation relative to electrospray-derived N-terminus. The data obtained by this method are referred to herein as “LCMS after reductive dimethylation (stable isotopic dimethyl labeling, SIDL)”. Briefly, 50 µg of peptide from each digest was immobilized into individual Waters Oasis SPE columns using a vacuum manifold. The SPE medium and peptides were adjusted to pH 5.5 with citrate buffer (90 mM citrate, 230 mM sodium divalent phosphate), and then 10 ml of 0.8% v / v formaldehyde (in citrate buffer) and 120 mM sodium cyanoborohydride was passed through the bound peptides for 10 minutes. The reactants were then removed by washing with 10 column volumes of 0.1% formic acid aqueous solution and eluting with 10 volumes of 50% acetonitrile and 0.1% formic acid. The labeled peptides were collected in low-retention microcentrifuge tubes and dried in a vacuum centrifuge. The dried peptides were reconstituted in 50 µl of 0.1% TFA and transferred to an autosampler vial for LC-MS / MS analysis.

[0225] LC-MS / MS (tandem mass spectrometry) analysis was performed using a Vanquish UHPLC system connected to a Lumos FusionOrbitrap (ThermoFisher) and operated under Xcalibur 4.1.31.9 software (ThermoFisher). 0.5 µg of each peptide digest was loaded onto a 2.1 mm x 150 mm C18 CSH Acquity UPLC reversed-phase column (1.7 µm particles, Waters Corp.) and separation was performed using a binary gradient: (mobile phase A = 0.1% difluoroacetic acid (DFA) in water) 0.5% to 40% mobile phase B (99.9% acetonitrile, 0.1% DFA), at a flow rate of 200 µl / min and a column temperature of 50ºC. The LC eluent was analyzed using a top-4 data-dependent acquisition (DDA) MS workflow. Full-scan MS spectra were acquired at 120,000 resolution (FWHM) at 200 m / z, and HCD (high-energy collisional dissociation) and EThcD (electron transfer dissociation with complementary HCD energy) MS / MS spectra were acquired at 15,000 resolution in a charge-state dependent manner in an Orbitrap analyzer. The .RAW files from each LC-MS / MS analysis were further processed using Protein Metrics Inc. (PMI) Byonic and Byos software to identify and quantify PTMs. Manual analysis of various spectra was performed using the QualBroswer function in Xcalibur software.

[0226] Table 7: MYDGF variants examined by aLCMS or LCMS:

[0227]

[0228] Table 8: Complete LCMS MW data for +S MYDGF variant

[0229]

[0230] (PTM = Post-translation Modification). Na in the complete analysis + Adducts are a common artifact, not a molecular property. The term "N-terminus" in a peptide map refers to the N-terminal amino group. Sequence coverage is 100%. ND = PTM < Detection limit.

[0231] Table 9: LCMS data of peptide mapping for +S MYDGF variants

[0232]

[0233] *: Determined by complete Mw analysis. Na + Add the adduct to the original value

[0234] Table 10: Complete LCMS MW data for the -V MYDGF variant

[0235]

[0236] Table 11: LCMS data of peptide mapping for -V MYDGF variants

[0237]

[0238] *Determined by full MW analysis; Na+ adduct added to the original value.

[0239] Table 12: +A Complete MW LCMS Data for MYDGF Variants

[0240]

[0241] Table 13: +A Peptide Plotting LCMS Data of MYDGF Variants

[0242]

[0243] *As determined by complete mass analysis, the Na+ adduct was added to the original value.

[0244] **No N-terminal methionine was observed.

[0245] Table 14: Complete MW LCMS data for the +G MYDGF variant

[0246]

[0247] Table 15: aLCMS data (combined with intact MW LCMS and dimethyl-capped peptide level LCMS after trypsin digestion as described*):

[0248]

[0249] * No N-terminal methionine was observed in the +A variant; however, 2.5% N-terminal methionine was observed in the +S variant.

[0250] Example 4 Studies on conformational stability

[0251] Thermal stability was evaluated in the pH range of 5.75 to 8.00 by monitoring the thermal unfolding temperature (Tm). Conformational stability at different pH values ​​was assessed by differential light scattering. Samples were prepared using different buffers containing the protein (h[+A]-MYDGF-E. coli) at concentrations of 20 mM and 50 mg / mL. Thermal stability data indicated a slight increase in conformational stability with increasing pH. Table 16 shows the evaluated buffers and their corresponding Tm values.

[0252] Table 16: pH / Buffer Screening: Determined T m value

[0253]

[0254] Ion exchange chromatography (IEC) data after storage at 40ºC for two weeks are depicted in Figure 1 The values ​​of the main peak before storage for all samples were 96.7% to 96.9%. Based on expectations of conformational stability, the lower pH after two weeks of storage at 40ºC resulted in less protein degradation detected by IEC.

[0255] Example 5 pH study

[0256] The stability of the h[+A]-MYDGF-E. coli protein of the present invention was evaluated as a function of pH / buffer. Samples were prepared at 50 mg / mL protein at low pH of approximately 6 and high pH of pH 8.0. Details of the formulation composition are summarized in Table 17. The solutions were filled into 6 mL glass vials (2.6 mL / vial).

[0257] Table 17: Formulations evaluated in pH studies

[0258]

[0259] Surprisingly, the results showed that formulations containing h[+A]-MYDGF-E. coli protein exhibited the most promising properties at pH 5.7, 6.0, and 6.2 (20 mM histidine). After 9 months of storage at 25ºC, the turbidity of all histidine formulations (formulations: B, C, D, E) did not exceed 4 NTU. For the tromethamine formulation (formulation A), the turbidity increased significantly to 6.5 NTU after storage. Figure 2 ).

[0260] The results of size exclusion chromatography (SEC-HPLC) are evidenced by the main peak representing the content of monomers of h[+A]-MYDGF-Escherichia coli. Figure 3In samples stored at 25ºC for up to 9 months, all histidine formulations showed only minimal reduction in the main peak. Although only minor changes were observed in histidine-containing formulations, the main peak of the tromethamine formulation (formulation A) showed a continuous reduction of more than 3% during the 9-month storage period at 25ºC.

[0261] The ion-exchange chromatography (IEC-HPLC) results were consistent with the SEC-HPLC and turbidity data, as only minor changes were detected in all histidine formulations after storage at 25ºC for up to 9 months. The main peak represents the purity of h[+A]-MYDGF-E. coli protein. The main peak of the tromethamine formulation (formulation A) decreased, and the decrease was significantly greater compared to the histidine formulation (9.2% decrease vs. 1.9% decrease), as shown in the results. Figure 4 middle.

[0262] Example 6 Surfactant Research

[0263] This working example was conducted to optimize the concentration of polysorbate in four different formulations containing 20 mM histidine (pH 6) and 50 mg / mL h[+A]-MYDGF-E. coli protein (Table 18). The formulations were filled into 6 mL glass vials (3 mL / vial).

[0264] Table 18: Formulations evaluated in surfactant optimization studies

[0265]

[0266] The following experiments were conducted for surfactant research: freeze-thaw stability and stirring stability. Turbidity results were evaluated.

[0267] Freeze-thaw stability study

[0268] The preparations listed in Table 19 were frozen in glass vials and exposed to five freeze-thaw cycles (-65ºC to 25ºC). The results are summarized in Table 19.

[0269] Table 19: Turbidity Results in Freeze-Thaw Study

[0270]

[0271] Formulations without polysorbate (0%) showed increased turbidity, while all formulations containing polysorbate did not show any changes in turbidity. Adding 0.02% to 0.04% polysorbate stabilizes proteins during freezing and thawing.

[0272] Stirring stability

[0273] Formulations 1 through 4 were subjected to agitation studies on a shaker in filled vials at 25ºC. Surprisingly, no change in turbidity was detected in all formulations containing polysorbate (0.02% to 0.04%), even after five days of shaking. All turbidity values ​​in formulations containing polysorbate remained at 2 NTU (turbidimetric turbidity units). In formulations without polysorbate, turbidity increased from an initial 3 NTU to an unmeasurable value exceeding 400 NTU. The addition of polysorbate stabilized the MYDGF protein during freezing and thawing and protected it during shaking stress.

[0274] In addition, further studies were conducted at pH 5.7 using formulations containing 50 mg / mL protein MYDGF, 20 mM histidine, 10 mM methionine, 220 mM sucrose, and varying amounts of polysorbate 80 (0%, 0.02%, 0.04%, and 0.06% (w / v)). The formulations were filled into 20 mL glass vials with a filling volume of 6.2 mL and agitated at 25ºC for up to seven days. Even after seven days of agitation, none of the vials containing polysorbate showed any increase in turbidity. All turbidity values ​​of the solutions containing polysorbate were initially equal to or less than 3 NTU and after seven days. Only after one day of agitation, the turbidity value of the formulations without polysorbate increased from an initial 3 NTU to an unmeasurable value exceeding 400 NTU.

[0275] Adding 0.02% to 0.06% (w / v) of polysorbate protects the MYDGF protein in the formulation from molecular degradation caused by shaking stress.

[0276] Example 7 Long-term stability study

[0277] Three formulations (Table 20) were selected for testing in a long-term stability study. The formulations containing 50 mg / mL of h[+A]-MYDGF-E. coli protein were stored in Type I 6 mL glass vials at 5ºC for 24 months.

[0278] Table 20: Formulation compositions used in long-term stability studies

[0279]

[0280] The stability of each formulation was monitored for 0, 1, 2, 3, 6, 9, 12, 18, and 24 months. Samples were analyzed by the following determinations: turbidity, subvisible particles, size exclusion chromatography (SE-HPLC), ion exchange chromatography (IEC), and binding.

[0281] Turbidity

[0282] The samples were analyzed by measuring turbidity. Surprisingly, throughout the 24-month study, all three formulations exhibited very low turbidity relative to the initial sampling points. All results at each sampling point were within an extremely narrow range, from 1.8 to 2.7 NTU.

[0283] Subvisible particles

[0284] Subvisible particles in the formulation were monitored using light obscuration. Surprisingly, a very low number of subvisible particles were observed in samples stored at 5ºC for up to 24 months. Only a maximum of 20 particles / mL with diameters ≥ 10 µm and ≥ 25 µm were detected (data not shown).

[0285] Size exclusion chromatography

[0286] The stability of the formulations was assessed by evaluating the monomer content of the main peak using SEC-HPLC. Surprisingly, the main peak changes in all three formulations were negligible over a period of 24 months at 5ºC. Figure 5 ).

[0287] Ion exchange chromatography

[0288] Charge distribution was examined by measuring acidity, basicity, and main peak groups using ion-exchange chromatography. Over 24 months, the charge distribution of all three formulations showed only very minor changes. Main peak data are shown below. Figure 6 middle.

[0289] Combination

[0290] The binding of h[+A]-MYDGF-E. coli protein to anti-MYDGF antibody was assessed using a binding assay. Notably, no loss of binding was observed in any of the three formulations during storage at 5ºC for up to 9 months (data not shown).

[0291] Summary of long-term stability studies

[0292] Evaluation of data from a 24-month stability study showed that formulations C, D, and E performed equally well when evaluated by various assays. Notably, all three formulations showed almost no change in their main peaks over time, as indicated by size exclusion and ion exchange chromatography.

[0293] Example 8 Freeze-drying research

[0294] Formulation E (Table 21), containing 50 mg / mL h[+A]-MYDGF-E. coli protein, was stored in a Type I 6 mL glass vial at 5ºC for 18 months. The stability of this liquid formulation is described in detail in Example 7.

[0295] Table 21: Composition of Preparation E

[0296]

[0297] A formulation E with unique properties was developed, namely that the solution can be lyophilized without any adjustment to the excipient dosage or addition of excipients. The formulation was lyophilized in glass vials using standard lyophilization methods. Although the drug is provided in lyophilized form, rapid administration is ensured by rapid reconstitution with water for injection. The reconstitution time of this formulation is extremely fast, less than 60 seconds. Compared to standard liquid formulations, the choice of lyophilized solutions provides opportunities for more extreme shelf-life conditions. For example, lyophilized drugs can have extended shelf lives and provide long-term storage at ambient temperatures.

[0298] Preparation H (Table 22) containing 50 mg / mL h[+A]-MYDGF-E. coli protein was prepared and filled into 20 mL glass vials with a filling volume of 11 mL. The vials were then freeze-dried using standard drying methods.

[0299] Table 22: Composition of Preparation H

[0300]

[0301] The lyophilized formulation H was stored at 25ºC for up to 6 months. Stability was assessed by analyzing monomer content (main peak) using SEC-HPLC and ion-exchange chromatography. Results are shown in [Figures to be inserted]. Figure 7 and Figure 8 As can be seen, no significant degradation of MYDGF protein was detected over a period of up to 6 months. Therefore, long-term storage at room temperature is possible.

[0302] Example 9: Feasibility Study of Freeze-drying

[0303] Two different formulations were freeze-dried, resulting in formulations F and G. Formulation F contained MYDGF protein and excipients: 20 mM histidine, 220 mM sucrose, 10 mM methionine, and 0.04% polysorbate 80, with a pH of 5.7. Formulation G differed from formulation F in that it contained 440 mM sucrose (the amounts of methionine, polysorbate 80, and pH remained unchanged).

[0304] Both solutions were filled into 20 mL glass vials, with a filling volume of 12 mL, and lyophilized using the same method. After lyophilization, both formulations were visually examined and subjected to microcomputed tomography (µCT) to evaluate the structure of the lyophilized drugs. µCT images and 3D structures were depicted on... Figure 9Significant differences in pore size and even collapsed structure were found in formulation G. Collapse in formulation G... Figure 9 The foam-like structure at the bottom of the vial is an indication of this. In µCT scanning, collapse of formulation G was detected through larger pores. Surprisingly, formulation F showed a complete structure and a more uniform pore distribution. This demonstrates that liquid formulation F can be easily freeze-dried.

[0305] Example 10: Compatibility Study of Dilution Media

[0306] The stability of the drug solution containing MYDGF protein was investigated in both undiluted and sodium chloride-based solutions. Diluting the drug with 0.9% NaCl solution is the standard procedure for parenteral administration. To examine the stability of the drug solution, two formulations were diluted in 0.9% NaCl solution to 18 mg / mL and 3 mg / mL MYDGF protein, respectively. The two formulations used were: Formulation F (pH 5.7) containing 50 mg / mL MYDGF protein, 20 mM histidine, 220 mM sucrose, 10 mM methionine, and 0.04% polysorbate 80, and Formulation H (pH 5.7) containing 50 mg / mL MYDGF protein, 20 mM histidine, 220 mM sucrose, 10 mM methionine, and 0.04% polysorbate 20. After storage at 25ºC for at least 24 hours, no significant changes were observed in turbidity and SEC-HPLC data for both diluted and undiluted formulations. Data are shown in [Table data would be inserted here]. Figure 10 and Figure 11 middle.

[0307] literature

[0308] 1.Botnov, V. et al. (2018), J Biol Chem, 293(34), 13166–13175.

[0309] 2.Ebenhoch, R. et al. (2019), Nat Commun 10, 5379.

[0310] 3.Desta L. et al. (2015), JACC Heart Fail, 3(3), 234-242.

[0311] 4.Desta L, et al. (2017), Int J Cardiol, 248, 221-226.

[0312] 5.Korf-Klingebiel, M. et al. (2015), Nat Med 21(2): 140-9.

[0313] 6.Korf-Klingebiel, M. et al. (2021), Circulation.144(15): 1227-40.

[0314] 7.Polten, F. et al. (2019), Anal Chem, 91, 1302−1308.

[0315] 8.Tolonen, AC et al. (2011), Mol Systems Biol 7(1).

[0316] 9.Zhao, L. et al. (2020), J Cell Mol Med, 24(2):1189–1199.

[0317] 10.Frottin, F. et al. (2006), The Proteomics of N-terminal MethionineCleavage, Molecular & Cellular Proteomics, Volume 5, Issue 12, 2336-2349.

[0318] 11.Bodenhausen G. & Ruben DJ (1980), Chem. Phys. Lett. 69 , 185 .

[0319] 12.Piotto, M. et al. (1992), J Biomol NMR, 2, 661-666.

[0320] 13.Sklenar, V. et al. (1993), J Magn Reson, Series A 102, 241-245.

[0321] 14.Mori, S. et al. (1995), J Magn Reson B 108, 94-98.

[0322] 15.Wishart, DS et al. (1995), J Biomol NMR, 5, 67-81.

[0323] 16.Dyson, H. J. & Wright, P. E. (1998), Nat Struct Biol, 5, 499-503.

[0324] 17.Shen Y. & Bax A. (2015), Methods Mol Biol, 1260:17-32.

[0325] 18.CN 111544572 A

[0326] 19.EP 2 918 676 B1

[0327] 20.US 5,744,328 B

[0328] 21.US 7,851,433 B

[0329] 22.US 2015 / 0291683 A1

[0330] 23.WO 2011 / 154349 A2

[0331] 24.Peternel, S. & Komel, R. (2010), Microbial Cell Factories, 9:66.

[0332] 25.Eggenreich, B. et al. (2020), Journal of Biotechnology 324S,100022.

[0333] 26.Brinson, R. G. et al. (2019), mAbs, 11, 94-105.

[0334] 27.WO 2021 / 148411

[0335] 28.WO 2014111458

[0336] 29.McAlindon E, et al. (2015); Heart; 101; 155-160.

[0337] 30.Kramer CM, et al. (2020); J Cardiovasc Magn Reson; 22; 17

[0338] 31.Ware JE (1993), SF-36 Health Survey: Manual and InterpretationGuide.

[0339] 32.Ware JE (2000), SF-36 health survey update. Spine; 25(24); 3130-3139.

[0340] 33.Bernklev T et al. (2005), Inflamm Bowel Dis; 11(10); 909-918

[0341] 34.Feagan BG et al. (2007), Am J Gastroenterol; 102; 794-802

[0342] 35.Feagan BG et al. (2017), Aliment Pharmacol Ther; 45; 264-275

[0343] 36.Shinde AV et al. (2015), Circ Res;117:10-12

[0344] 37.Zhao et al. (2020), Cell Mol Med.;24:1189–1199

[0345] 38.Korf-Klingebiel, M et al. (2021), Circulation, 144(15), 1227-1240

[0346] 39.Ibanez, B et al. (2019), J Am Coll Cardiol, 74(2):238-256.

[0347] 40.Gibson and Schömig (2004), Circulation, 109:3096-3105.

[0348] 41.Martin and Rankin, Heart 1999;82:163–16

[0349] 42.WO 2023 / 233034 A1

[0350] 43.Vahdatpour et al. (2019), Journal of the American HeartAssociation, 8(8), eOl 1991

[0351] 44.WO 2024 / 052563 A1

Claims

1. A pharmaceutical preparation comprising: (a) Myeloid-derived growth factor (MYDGF) protein at concentrations ranging from 0.5 mg / ml to 200 mg / ml; (b) 10 mM to 100 mM buffer solution; (c) Stabilizers ranging from 1 mM to 50 mM; (d) Tension-inducing agents ranging from 20 mM to 250 mM; and (e) 0.01% to 0.1% (w / v) of surfactant; The pH of the composition is 5.0 to 7.

0.

2. The pharmaceutical preparation according to claim 1, wherein the buffer solution is selected from acetate buffer, citrate buffer, histidine buffer, succinate buffer, phosphate buffer, and tromethamine buffer.

3. The pharmaceutical formulation according to claim 2, wherein the buffer solution is a histidine buffer solution.

4. The pharmaceutical formulation according to claim 3, wherein the histidine buffer is present in the composition at a concentration of 10 mM to 30 mM.

5. The pharmaceutical formulation according to any one of claims 1-4, wherein the stabilizer is selected from methionine, arginine, glycine, proline, lysine, and cysteine.

6. The pharmaceutical formulation according to claim 5, wherein the stabilizer is methionine.

7. The pharmaceutical formulation according to claim 6, wherein the methionine is present in the composition at a concentration of 5 mM to 15 mM.

8. The pharmaceutical preparation according to any one of claims 1-7, wherein the tonic agent is selected from sucrose, trehalose, sorbitol, mannitol and dextrose.

9. The pharmaceutical formulation according to claim 8, wherein the tonic agent is sucrose.

10. The pharmaceutical formulation according to claim 3, wherein the sucrose is present in the composition at a concentration of 100 mM to 250 mM.

11. The pharmaceutical formulation according to any one of claims 1-10, wherein the surfactant is selected from polysorbate 20, polysorbate 80 and poloxamer 188.

12. The pharmaceutical formulation according to claim 11, wherein the surfactant is polysorbate 80 or polysorbate 20.

13. The pharmaceutical formulation according to claim 12, wherein the polysorbate 20 is present in the composition at 0.02% to 0.06% (w / v).

14. The pharmaceutical preparation according to any one of claims 1-13, wherein the MYDGF protein comprises or is composed of an amino acid sequence selected from or consisting of SEQ ID NO: 1, 2, 3, 6, 11 or 13.

15. The pharmaceutical formulation according to claim 14, wherein the MYDGF protein comprises or is composed of the amino acid sequence of SEQ ID NO:

1.

16. The pharmaceutical formulation according to any one of claims 1-15, wherein the MYDGF protein is present in the composition at a concentration of 1 mg / ml to 100 mg / ml.

17. The pharmaceutical formulation according to claim 16, wherein the MYDGF protein is present in the composition at a concentration of 40 mg / ml to 60 mg / ml, and preferably 50 mg / ml.

18. The pharmaceutical preparation according to any one of claims 1-17, wherein the pH of the preparation is 5.2 to 6.

2.

19. The pharmaceutical preparation according to claim 18, wherein the pH of the preparation is 5.4 to 6.

0.

20. The pharmaceutical preparation according to any one of claims 1-19, wherein the pharmaceutical preparation comprises or is composed of the following: (a) Myeloid-derived growth factor (MYDGF) protein at concentrations ranging from 0.5 mg / ml to 200 mg / ml; (b) 10 mM to 100 mM histidine buffer; (c) 1 mM to 50 mM methionine; (d) 20 mM to 250 mM sucrose; and (e) 0.01% to 0.1% (w / v) polysorbate 20 or polysorbate 80; The pH of the composition is 5.0 to 7.

0.

21. The pharmaceutical preparation according to any one of claims 1-20, wherein the pharmaceutical preparation comprises or is composed of the following: (a) Myeloid-derived growth factor (MYDGF) protein at concentrations of 40 mg / ml to 60 mg / ml; (b) 10 mM to 30 mM histidine buffer; (c) 5 mM to 15 mM methionine; (d) 100 mM to 250 mM sucrose; and (e) 0.02% to 0.06% (w / v) polysorbate 20 or polysorbate 80; The pH of the composition is 5.0 to 6.

5.

22. The pharmaceutical preparation according to any one of claims 1-21, wherein the pharmaceutical preparation comprises or is composed of the following: (a) Myeloid-derived growth factor (MYDGF) protein at concentrations of 45 mg / ml to 55 mg / ml; (b) 18 mM to 22 mM histidine buffer; (c) 9 mM to 11 mM methionine; (d) 200 mM to 240 mM sucrose; and (e) 0.02% to 0.06% (w / v) polysorbate 20 or polysorbate 80; The pH of the composition is 5.7 ± 0.

3.

23. The pharmaceutical preparation according to any one of claims 1-22, wherein the pharmaceutical preparation comprises or is composed of the following: (a) 50 mg / ml of myeloid-derived growth factor (MYDGF) protein; (b) 20 mM histidine buffer; (c) 10 mM methionine; (d) 220 mM sucrose; and (e) 0.04% (w / v) polysorbate 20 or polysorbate 80; The pH of the composition is 5.7 ± 0.

3.

24. The pharmaceutical preparation according to any one of claims 1-23, wherein the pharmaceutical preparation comprises or is composed of the following: (a) 50 mg / ml of myeloid-derived growth factor (MYDGF) protein, wherein the myeloid-derived growth factor protein comprises the amino acid sequence of SEQ ID NO: 4; (b) 20 mM histidine buffer; (c) 10 mM methionine; (d) 220 mM sucrose; and (e) 0.04% (w / v) polysorbate 20 or polysorbate 80; The pH of the composition is 5.7 ± 0.

3.

25. The pharmaceutical preparation according to any one of claims 1-24, wherein the pharmaceutical preparation comprises or is composed of the following: (a) 50 mg / ml of myeloid-derived growth factor (MYDGF) protein, wherein the myeloid-derived growth factor protein comprises or is composed of the amino acid sequence of SEQ ID NO: 1; (b) 20 mM histidine buffer; (c) 10 mM methionine; (d) 220 mM sucrose; and (e) 0.04% (w / v) Polysorbate 20; The pH of the composition is 5.7 ± 0.

3.

26. The pharmaceutical preparation according to any one of claims 1-24, wherein the pharmaceutical preparation comprises or is composed of the following: (a) 50 mg / ml of myeloid-derived growth factor (MYDGF) protein, wherein the myeloid-derived growth factor protein comprises or is composed of the amino acid sequence of SEQ ID NO: 1; (b) 20 mM histidine buffer; (c) 10 mM methionine; (d) 220 mM sucrose; and (e) 0.04% (w / v) Polysorbate 80; The pH of the composition is 5.7 ± 0.

3.

27. The pharmaceutical preparation according to any one of claims 1-26, wherein the preparation is in liquid form or in lyophilized form.

28. The pharmaceutical preparation according to claim 27, wherein the preparation is in liquid form.

29. The pharmaceutical preparation according to any one of claims 1-28, wherein the pharmaceutical preparation is used in a medicament.

30. The pharmaceutical preparation according to any one of claims 1-28, wherein the pharmaceutical preparation is used in a method of treating a subject with myocardial infarction.

31. A pharmaceutical formulation for use in the method of claim 30, wherein the myocardial infarction is ST-segment elevation myocardial infarction (STEMI).

32. A pharmaceutical formulation for use in the method according to claim 30 or 31, wherein the method comprises administering the formulation by intravenous infusion.

33. A pharmaceutical formulation for use in the method of claim 32, wherein the method further comprises percutaneous coronary intervention (PCI) to restore blood flow to cardiac tissue.

34. The pharmaceutical preparation according to any one of claims 1-28, wherein the pharmaceutical preparation is used in a method for treating or preventing cardiogenic shock.

35. A pharmaceutical formulation for use in the method of claim 34, wherein the cardiogenic shock occurs during myocardial infarction.

36. A pharmaceutical formulation for use in the method of claim 35, wherein the myocardial infarction is ST-segment elevation myocardial infarction (STEMI).

Images