Methods and means for altering hemodynamics in infectious diseases

Abstract

To provide a method and a pharmaceutical preparation for reducing effects caused by infection, particularly hemodynamic effects.SOLUTION: A method of decreasing the permeability of an endothelial layer of a blood vessel in a subject, the method comprising providing to the endothelial layer a substance that decreases the ratio of Angiopoietin-2 to Angiopoietin-1 at a site of increased permeability as a result of infection. It is preferred that the substance comprises an AQGV peptide, an LQGV peptide or a functional analogue of either.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application relates to methods and means for alleviating certain effects, particularly hemodynamic effects, resulting from infection. More specifically, the present invention relates to peptide preparations for use in the treatment of viral infections that affect the permeability of the vascular system. [Background technology]

[0002] Recently, the world has been hit hard by a pandemic caused by a virus called SARS-Cov-2, the coronavirus that causes COVID-19. The new coronavirus appears to kill primarily by flooding and clogging the tiny air sacs in the lungs with fluid, cutting off the body's oxygen supply to the point of shutting down vital organs. This choking by the body's own fluids appears to be a model for the respiratory illnesses that many other coronaviruses can induce. Large accumulations of such viruses in various exotic animals, given the lack of pre-existing immunity, could trigger a similar pandemic with asphyxiation similar to that of SARS-Cov-2. Because virus-specific vaccines and / or antivirals typically become available only after the infection has already spread to a large portion of the population, other means and methods of treatment are desperately needed, as these types of viral infections are expected to increase further, typically through zoonotic outbreaks where vaccines or antivirals are not sufficiently specific or are developed too slowly. Summary of the Invention [Problem to be solved by the invention]

[0003] Therefore, there is a persistent and continuing need to be able to at least combat the common harmful effects of these infectious diseases, and the present invention provides the means and methods to do just that. [Means for solving the problem]

[0004] In a first embodiment, the present invention provides a method for reducing the permeability of the endothelial layer of blood vessels in a subject, the method comprising providing the endothelial layer with a substance that reduces the ratio of angiopoietin-2 to angiopoietin-1 at sites of increased permeability as a result of infection, e.g., viral infection. In a preferred embodiment, the present invention provides a method for reducing the gas diffusion distance between the alveoli and the vascular network surrounding the alveoli in a human subject suffering from a respiratory infection, thereby allowing for a reduction in intraalveolar fluid and / or improved oxygen delivery to the subject's body. In a preferred embodiment, the substance comprises an AQGV peptide, an LQGV peptide, or a functional analog of either. In a preferred embodiment, the present invention provides a method for reducing the gas diffusion distance between the alveoli and the vascular network surrounding the alveoli in a human subject suffering from a respiratory viral infection. Moreover, the viral infection is preferably caused by a virus that requires a specific receptor present on at least a portion of alveolar cells and a more ubiquitous binding partner. In a preferred embodiment, the specific receptor is preferably ACE-2. In another preferred embodiment, the more ubiquitous binding partner is preferably a glycoprotein containing sialic acid residues. It is particularly preferred that the ubiquitous binding partner binds to an fMLF-like amino acid sequence, for example, the sequence comprises at least a membrane-proximal-external-region (MPER, also referred to herein as a fusogenic sequence). The virus is preferably a coronavirus, particularly one having an MPER as identified in Figure 11, and more particularly comprising at least the fusogenic sequence as identified in Figure 12. It is most preferred that the MPER comprises at least the amino acid sequence KWPWIWL (amino acids identified herein by single-letter code).In a further preferred embodiment, the present invention provides a method for reducing the permeability of the endothelial layer of a blood vessel in a subject, comprising providing to the endothelial layer a substance that reduces the ratio of angiopoietin-2 to angiopoietin-1 at sites of increased permeability as a result of infection, e.g., infection by a virus, wherein the coronavirus is COVID-19 virus (SARS-COV-2) or a variant thereof.

[0005] The present invention also provides a method for reducing the permeability of the endothelial layer of a blood vessel in a subject, comprising providing the endothelial layer with a substance that reduces the ratio of angiopoietin-2 to angiopoietin-1 at sites of increased permeability as a result of infection, wherein the substance is administered intravenously to the subject, preferably at a rate of at least 75 mg / kg / hour, or more preferably at least 90 mg / kg / hour. Moreover, it is preferred that the substance be administered intermittently. It is preferred that the subject be monitored for hemodynamic stability during treatment. The present invention also provides a method for reducing the permeability of the endothelial layer of a blood vessel in a subject, comprising providing the endothelial layer with a substance that reduces the ratio of angiopoietin-2 to angiopoietin-1 at a site of increased permeability as a result of infection, wherein the substance is administered intravenously to the subject, and the method further comprises administering to the subject an antiviral agent, such as remdesivir (GS-5734), an inhibitor of viral RNA-dependent RNA polymerase.The present invention also provides a method for reducing the permeability of the endothelial layer of a blood vessel in a subject, comprising providing the endothelial layer with a substance that reduces the ratio of angiopoietin-2 to angiopoietin-1 at a site of increased permeability as a result of infection, wherein the substance is administered intravenously to the subject, and the method further comprises administering to the subject an anti-inflammatory agent, such as dexamethasone, or an interleukin-6 signaling inhibitor, such as tocilizumab.

[0006] The present invention also provides a pharmaceutical formulation for use in a method for reducing the permeability of the endothelial layer of a blood vessel in a subject, the method comprising providing the endothelial layer with a substance that reduces the ratio of angiopoietin-2 to angiopoietin-1 at a site of increased permeability as a result of infection, wherein the substance is administered intravenously, preferably at a rate of at least 75 mg / kg / hour, or more preferably at least 90 mg / kg / hour. Furthermore, it is preferred that the substance be administered intermittently. It is preferred that the subject be monitored for hemodynamic stability during treatment. The present invention also provides a pharmaceutical preparation for use in reducing the permeability of the endothelial layer of a blood vessel in a subject, the method comprising providing the endothelial layer with a substance that reduces the ratio of angiopoietin-2 to angiopoietin-1 at a site of increased permeability as a result of infection, wherein the substance is administered intravenously to the subject, and the method further comprises administering to the subject an antiviral agent, such as remdesivir (GS-5734), an inhibitor of viral RNA-dependent RNA polymerase.The present invention also provides a pharmaceutical preparation for use in reducing the permeability of the endothelial layer of a blood vessel in a subject, the method comprising providing the endothelial layer with a substance that reduces the ratio of angiopoietin-2 to angiopoietin-1 at a site of increased permeability as a result of infection, wherein the substance is administered intravenously to the subject, and the method further comprises administering to the subject an anti-inflammatory agent, such as dexamethasone, or an interleukin-6 signaling inhibitor, such as tocilizumab.

[0007] The present invention also provides pharmaceutical formulations for use in accordance with the present invention, comprising an AQGV peptide, an LQGV peptide, or a functional analogue of either, and an excipient suitable for parenteral administration.

[0008] When a human subject suffers from an infection, particularly a viral infection, more particularly a viral respiratory infection, there are many influences that affect hemodynamic stability.

[0009] One of the effects seen is increased permeability of blood vessels, leading to leakage of fluid from blood vessels into intercellular spaces and vice versa, resulting in exacerbated and traumatic damage to the lungs and other organs. Typical signs of such asphyxiation injury induced by vascular leakage include increased extracellular fluid in the lungs due to fluid overflow into the alveoli.

[0010] Thrombosis may also occur, particularly deep venous thrombosis (DVT) and pulmonary embolism (PE). Respiratory infections, especially those with pulmonary edema, can result in increased diffusion distances for gases, such as oxygen and carbon dioxide, across the distance between the alveoli and the blood, leading to hypoxemia. Both oxygen and carbon dioxide must pass through a thin layer in the lungs called the alveolar-capillary membrane. This is the thin layer between the tiny air sacs in the lungs (alveoli) and the smallest blood vessels (pulmonary capillaries) that pass through the lungs. How well oxygen can be inhaled and pass from the alveoli into the blood (diffusion), and how well carbon dioxide can be exhaled from the capillaries back to the alveoli, depends on the thickness (swelling) of this membrane and the amount of surface area available for transfer.

[0011] This problem is exacerbated in people who already suffer from limited oxygen availability through underlying disease. Diffusion capacity can be low when there is less surface area available for oxygen and carbon dioxide transfer, for example, in cases of emphysema, or when the lung or part of the lung has been removed due to lung cancer, PE, or pre-existing cardiovascular and metabolic problems and obesity.

[0012] Diffusion capacity may also be reduced in the presence of lung diseases that cause membrane thickening, such as chronic lung diseases, such as pulmonary fibrosis, such as that found in COPD and sarcoidosis. The present invention is particularly useful in such patients with only partial lung capacity.

[0013] Acute illness can also result in reduced diffusing capacity, such as in exacerbated viral respiratory infections accompanied by lung injury. This often leads to increased pulmonary capillary permeability, allowing fluid to flow from the capillaries into the thin layer of extracellular matrix separating the alveoli from the capillaries. This membrane thickens (expands) through fluid accumulation in the extracellular matrix (interstitium) that separates alveolar cells from vascular cells due to intercellular fluid accumulation. In patients with such lung injury and exacerbated infectious respiratory tract infections, plasma levels of biomarkers of endothelial activation, which can be measured by ELISA, often predict mortality and morbidity. In particular, the concentration of angiopoietin-2 relative to angiopoietin-1 (Ang-2 / Ang-1) can be a useful biological marker of mortality in patients with acute lung injury (ALI). Ang-2 / Ang-1 ratios were found to be significantly elevated in patients who died from lung injury [p=0.01; Crit Care Med. 2010 Sep;38(9):1845-1851.] In a multivariate analysis stratified by dead space fraction, Ang-2 / Ang-1 ratios were an independent predictor of mortality in patients with elevated dead space fraction, with an adjusted odds ratio of 4.3 (95% CI 1.3-13.5, p=0.01) (p=0.03 for the interaction between dead space fraction and Ang-2 / Ang-1 ratio).

[0014] Similarly, plasma levels of D-dimer can be used to track the health of patients with exacerbated viral respiratory infections accompanied by lung damage and endothelial activation. D-dimer, a lysate of cross-linked fibrin, indicates fibrinolysis in response to coagulation activation and fibrin formation (doi.org / 10.1111 / jth.12075). D-dimer levels are typically evident during the febrile and convalescent phases following viral infections that affect vascular endothelial cells and are associated with endothelial activation and plasma leakage. D-dimer assays can vary in sensitivity depending on the specific type of laboratory employed, and not all laboratories report the same units, providing a range of acceptable results. There are many factors that can cause elevated D-dimer levels beyond those associated with venous thromboembolism (VTE), such as age or pregnancy. The 8-hour half-life of D-dimer results in elevated levels for approximately three days after an inciting event. Quantitative D-dimer has a sensitivity of 94%-98% but a specificity of only 50%-60%, making it useful as a screening tool, but requires clinical evidence from history and physical examination, and preferably repeated testing at intervals to confirm the diagnosis or track the patient's health.

[0015] Thus, in one aspect, the present invention provides a method for reducing the permeability of the endothelial layer of a blood vessel, comprising providing the endothelial layer with a substance that reduces the ratio of angiopoietin-2 to angiopoietin-1 at a site of increased permeability as a result of infection.

[0016] In one embodiment, the method is useful for reducing the gas diffusion distance (or at least preventing an increase in the diffusion distance) between the alveoli and the vascular network surrounding the alveoli in human subjects suffering from respiratory infections, particularly in patients with underlying diseases that result in limited oxygen availability. Reduced vascular permeability in patients suffering from vascular leakage is generally associated with reduced D-dimer levels. In one embodiment, the substance to be used in the method according to the present invention includes a peptide that affects hemodynamics, particularly a peptide that affects hemodynamics by affecting gap junctions between cells. Such peptides include AQGV and its functional analogs. A functional analog is defined as a substance that provides the same or similar function (in type, not necessarily quantity). Essentially, any substance that reduces the permeability of the vascular system can be used according to the present invention. For example, the tetrapeptide AQGV (also referred to herein as EA-230) has surprisingly been found to effectively regulate vascular permeability. In particular, EA-230 significantly improves hemodynamic stability in humans, even in the absence of inflammatory activity in the patient. Permeability governs the amount of fluid that leaks from blood vessels. The administration of fluid therapy generally increases leakage. Based on observations of patients in a Phase II study, the inventors found a significant reduction in harmful fluid retention (fluid leakage) in patients treated with EA-230 (p=0.03). Throughout the clinical trial, EA-230 has been shown to be safe and well tolerated. EA-230 shows a significant improvement in patient recovery compared to placebo patients. Patients treated with EA-230 are discharged earlier from the intensive care unit (p=0.0232) and hospital (p=0.0015).

[0017] EA-230 improved hemodynamic stability (p=0.006) and renal function (p=0.003). Long-term recovery of patients was significantly improved with EA-230. By improving vascular permeability, EA-230 reduces the harmful fluid buildup in the lungs associated with infection, reduces hypoxemia, reduces PE, and also reduces the use of mechanical ventilation with its deleterious systemic effects, particularly in viral respiratory infections, such as those caused by influenza viruses, particularly coronaviruses. Thus, methods according to the present invention are particularly provided, in which the active substance for controlling hemodynamic stability comprises the AQGV peptide. Functional and / or structural analogs of AQGV according to the present invention may be selected from the group consisting of peptides containing tetrapeptides selected from the group consisting of AQLP, PLQA, LQGV, LAGV, PQVG, PQVA, PQVR, VGQL, LQPL, RQGV, LQVG, LQGA, LQGR, AQGA, QPLA, PQVP, VGQA, QVGQ, and VGQG, or other permutations of peptides of 4 to 12 amino acids, particularly other permutations of peptides of 4 to 12 amino acids composed of the amino acids of the above tetrapeptides. The present invention further provides a method in which the viral infection is caused by a virus that requires a specific receptor present in at least a portion of alveolar cells and a more ubiquitous binding partner. In a preferred embodiment, the viral infection is caused by a coronavirus, wherein the specific receptor is ACE-2, and particularly, the coronavirus is SARS-CoV-2 or a variant or analog thereof. Other coronavirus infections that can be treated according to the present invention possess specific receptors DPP4 (e.g., in MERS coronavirus) or APN (aminopeptidase N). Also preferred are methods in which the more ubiquitous binding partner is a glycoprotein containing sialic acid residues. It is particularly preferred that the ubiquitous binding partner binds to fMLF-like amino acids, for example, where the sequence includes at least the membrane-proximal-external region (MPER, also known herein as the fusogenic sequence).Such a method according to the present invention is particularly provided when the virus is a coronavirus. It is particularly preferred that the virus is a coronavirus, particularly a coronavirus having an MPER as identified in Figure 11, and more particularly, at least comprising the fusion gene sequence as identified in Figure 12. Alternatively, the more ubiquitous binding partner is a glycoprotein containing sialic acid residues recognized by influenza viruses. The combination of specific and more ubiquitous binding / infective sites in cells is typical for coronaviruses, with their typical effects on hemodynamics as disclosed herein.

[0018] In a further preferred embodiment, the AQGV peptide or related substance is administered intravenously, preferably at a rate of at least 75 mg / kg / hour, more preferably at least 90 mg / kg / hour. Intermittent administration of the AQGV peptide or related substance is particularly useful. A preferred method of use involves administering at least 90 mg / kg / hour for 2-4 hours, then reducing to 30 mg / kg / hour for 2-4 hours, or until the patient's response to treatment is monitored by clinical or laboratory diagnostics, or suspending administration of the substance for 1-2 hours until a diagnostic study, e.g., point-of-care testing, is completed, and then resuming treatment for 2-4 hours at at least 90 mg / kg / hour. Preferably, the monitoring includes studying the subject for hemodynamic stability and / or fibrinolysis. Treatment with the AQGV peptide according to the present invention may further include administering an antiviral agent. The present invention also provides pharmaceutical formulations comprising an AQGV peptide or a related substance (preferably a functional analogue) for use in a method according to the invention, or a pharmaceutical formulation comprising an AQGV peptide or a functional analogue thereof and an excipient suitable for parenteral administration for use according to the invention.

[0019] Often, a human subject or patient experiencing reduced diffusion may be admitted to an intensive care unit (ICU) where their vital signs are monitored. If the patient receives medical treatment and recovers, and their vital signs are within acceptable limits, they may be released from the ICU and admitted to standard hospital care. If the patient is shown to be stable with standard treatment, they may be discharged and sent home.

[0020] Subsequently, if necessary, the patient can be readmitted to the hospital, for example, if the patient's condition or infection worsens. Improvements to the patient's health and recovery that impact the patient's length of stay in the ICU, length of stay with standard care in the hospital, and / or the patient's readmission will provide a significant benefit to the patient. Therefore, any means and methods according to the present invention that improve the patient's health, particularly the rate of recovery, through the use of the peptide compounds disclosed herein are of interest.

[0021] In clinical trials designed to evaluate the safety and tolerability of the AQGV peptide (also referred to herein as EA-230) and its immunomodulatory effects, the peptide was found to be safe, but unexpectedly, no immunomodulatory effects were observed in the test setting when treated patients were compared with control subjects. Instead of observing an immunomodulatory effect, the inventors surprisingly discovered that, upon analyzing the data obtained in the clinical trials, new and highly advantageous properties not previously observed could be attributed to the AQGV peptide. These properties are apparently unrelated to the known and observed immunomodulatory effects.

[0022] Thus, the present invention relates to methods of using the AQGV peptide and analogs thereof to improve clinical parameters of hospitalized human patients and / or clinical parameters of intensive care, such that the time from admission to discharge and / or the duration of intensive care can be shortened. In one embodiment, the use of the AQGV peptide and analogs thereof is for use in medical treatment to modify hemodynamics in a human subject. In a further embodiment, the use in a human subject to modify hemodynamics includes a reduction in undesirable fluid retention and / or a reduced use of vasopressor agents in the human subject. In another embodiment, the use of the AQGV peptide and analogs thereof is for use in a human subject with impaired pulmonary function.

[0023] In one embodiment, the AQGV peptide or a functional analog thereof is provided for use in a method of treating a human subject, wherein the use comprises altering hemodynamics in the human subject. Hemodynamics encompasses the dynamics of blood flow, i.e., the physical factors that govern blood flow through the human body. Hemodynamics in a human patient can be monitored, for example, by measuring blood pressure and / or fluid balance. If blood pressure is low and / or fluid balance is disturbed in a human patient, vasopressors or inotropes can be used and / or fluids can be administered intravenously. Inotropes and vasopressors are biologically and clinically important vasoactive medications that come from various pharmacological groups and act on some of the most fundamental receptors and signaling systems in the body. Although more than 20 such drugs are commonly used clinically, reviews of their pharmacology are rare outside of physiology and pharmacology textbooks. Despite their widespread use in critically ill patients, the clinical effects of these drugs in pathological conditions are poorly understood. The adverse effects of vasopressors and inotropes depend on the mechanism of action. For beta-stimulators, arrhythmias are one of the most common side effects that need to be alleviated.

[0024] The present inventors have found that by using the AQGV peptide or a functional analog thereof, hemodynamics in a human patient after trauma (e.g., viral infection) is significantly improved, as indicated, for example, by reduced use of vasopressors and / or improved fluid balance in the human patient. Thus, the use of the AQGV peptide or a functional analog thereof described herein improves hemodynamic stability in a human patient. Altering or optimizing hemodynamics in a human subject is important after injury, for example, when the human subject suffers from infection, trauma, and / or blood loss. Therefore, the AQGV peptide or an analog thereof can be advantageously used in hemodynamic therapy. Hemodynamic therapy includes optimizing the patient's hemodynamics in a goal-directed hemodynamic therapy. Such therapy can include therapeutic intervention, such as fluid management and / or the use of vasopressors in a patient.

[0025] As used herein, an AQGV functional analog is defined as a peptide that exerts a similar effect or function, not necessarily in kind but in quantity, to the AQGV peptide described herein. The AQGV peptide has a length of four amino acids. An AQGV functional analog may have sequence identity, i.e., may contain at least a portion or the entire AQGV peptide. Preferably, such an AQGV functional analog is a structural analog of the AQGV peptide. A preferred structural analog may be the LQGV peptide. AQGV peptide structural analogs may be selected from peptides consisting of amino acids selected from the group consisting of the following amino acids: alanine (in single-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), proline (P), and arginine (R). In a preferred embodiment, an AQGV structural analog is provided in which at least 50%, more preferably at least 75%, and most preferably at least 100% of the following autophagy-inhibiting amino acids are selected from the group consisting of alanine (single-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), proline (P), and arginine (R). Preferably, the AQGV structural analog has a length ranging from 4 to 12 amino acids. Preferably, such structural analogs are linear peptides. Suitable structural analogs of AQGV may have a length of less than 4, e.g., 3. However, such lengths may require higher doses of such peptides because the half-life of such peptides is shortened and therefore less preferred. Longer structural analogs, e.g., those longer than 12 residues, are less preferred due to the potential immunogenicity of such long peptides. Structural AQGV analogs according to the present invention may be selected from the group consisting of peptides including tetrapeptides selected from the group AQLP, PLQA, LQGV, LAGV, PQVG, PQVA, PQVR, VGQL, LQPL, RQGV, LQVG, LQGA, LQGR, AQGA, QPLA, PQVP, VGQA, QVGQ, VGQG.

[0026] Vasopressors are a class of drugs that can increase hypotension. Some vasopressors act as vasoconstrictors, and another class of vasopressors sensitizes adrenergic receptors to catecholamine-glucocorticoids, while another class of vasopressors can increase cardiac output. Regardless of which vasopressor is used, the present invention allows for a reduction in vasopressor use. Reducing vasopressor use includes shortening the duration of vasopressor use and / or reducing the dosage of the vasopressor. Examples of vasopressors include epinephrine, noradrenaline, phenylephrine, dobutamine, dopamine, and vasopressin. Fluid management in patients includes, for example, monitoring oral, enteral, and / or intravenous fluid intake and fluid output (e.g., urine), and subsequently managing fluid intake if fluid retention is observed (i.e., fluid intake exceeds fluid output). Surprisingly, the use of the AQGV peptide or an analog thereof can reduce fluid retention. Thus, the AQGV peptide or an analog thereof can be used in addition to known interventions to improve hemodynamics in human patients, thereby resulting in a faster improvement in hemodynamics compared to without the use of the AQGV peptide or an analog thereof.

[0027] In another embodiment, the AQGV peptide or a functional analog thereof is provided for use in treating a human subject with impaired pulmonary function. In a further embodiment, the impaired pulmonary function is acute lung injury. In one embodiment, the AQGV peptide or a functional analog thereof is provided for use in treating a human subject to improve pulmonary function. Pulmonary function can also be assessed by measuring hypoxemia or by measuring the alveolar-arterial gradient (A-aO2, or Aa gradient). Assessing the Aa gradient to assess pulmonary function in humans is standard clinical practice (e.g., by determining the difference between the alveolar concentration of oxygen (A) and the arterial concentration of oxygen (a)). It is used in diagnosing the cause and degree of hypoxemia. The Aa gradient helps assess the integrity of the alveolar-capillary unit. Improvement in pulmonary function compared to not administering the AQGV peptide can include progression from a pulmonary function stage to a milder stage (e.g., a patient progressing from having lung injury to being at risk for lung injury or not having lung injury).

[0028] Regardless of how the assessment is performed, the use of the AQGV peptide or an analog thereof can improve lung function in humans with lung injury and / or impaired lung function in subjects without immunomodulatory effects.

[0029] The use of the AQGV peptide not only improves lung function, but also prevents lung function decline and / or damage. Thus, lung damage due to hypoxemia can be prevented. Thus, in one embodiment, the use of the AGQV peptide or an analog thereof allows for the maintenance of lung function in a human patient. Thus, the use of the AGQV peptide or an analog thereof allows for the protection of lung function in a human patient. In another embodiment, the use of the AQGV peptide or an analog thereof allows for the prevention of lung function decline and / or damage in a human patient. For example, a human patient who can be classified as having no lung damage or as being at risk of having lung damage (e.g., due to COVID-19) can be treated with the AQGV peptide. Thereby, such a patient can maintain their state of lung function instead of progressing to a (more serious) decline. Thus, a human patient at risk of developing lung damage, for example, due to (induced) trauma, such as infection, can be treated with the AQGV peptide or an analog thereof as a result of treatment with the AQGV peptide or an analog thereof, thereby maintaining the state of lung function of the patient.

[0030] In another embodiment, the AQGV peptide or a functional analog thereof is provided for use in treating a human subject with impaired pulmonary function, wherein the use comprises altering hemodynamics in the human subject. Because treatment of pulmonary function and treatment of hemodynamic stability can now be linked, the use of the AQGV peptide or a functional analog thereof according to the present invention can advantageously be used to protect and / or improve pulmonary function and alter hemodynamics. Such combined use can result, for example, in improved and / or maintained pulmonary function, as well as a reduction in the use of vasopressors and / or improved fluid management in the human subject.

[0031] In a further embodiment, the present invention provides for reduced use of vasoconstrictors.

[0032] The use of vasoconstrictors can be reduced by shortening the duration of use of the vasoconstrictor. The use of vasoconstrictors can be reduced by reducing the amount of the vasoconstrictor (e.g., reducing the amount per dose and / or increasing the time interval between doses). The use of vasoconstrictors can be reduced by reducing the amount of the vasoconstrictor and shortening the duration of use of the vasoconstrictor. By reducing the use of vasopressors, the human subject advantageously recovers more quickly compared to a human subject not administered AQGV or an analog thereof.

[0033] In another embodiment, the use of the AQGV peptide or a functional analog thereof reduces adverse fluid storage in a human subject. Fluid storage can occur in a human subject, and its symptoms include weight gain and edema. Fluid storage can be the result of decreased pulmonary function and / or impaired hemodynamics. Therefore, because the use of AQGV can affect pulmonary function and / or hemodynamic stability in a human subject, the use of AQGV can also affect fluid storage. Fluid storage can be the result of capillary leakage. Therefore, the use of AQGV and its analogs can affect capillary leakiness (permeability) and reduce plasma leakage from the blood into peripheral tissues and / or organs. Most preferably, edema can be reduced and / or avoided by the use of AQGV. Edema can also be referred to as adverse fluid storage because of its adverse effects on patients. Regardless of the cause of fluid retention, the use of the AAQGV peptide or a functional analog thereof can improve fluid storage dynamics in human subjects, thereby alleviating symptoms associated with fluid retention, such as weight gain and edema, and subsequently reducing the use of diuretics.

[0034] In another embodiment, the use of the AQGV peptide or its functional analog according to the present invention is not limited to patients with lung injury and / or patients requiring hemodynamic therapy. The use of the AQGV peptide or its functional analog according to the present invention includes the treatment of human patients considered to be at risk of lung injury and / or expected to require hemodynamic therapy. Such human patients include patients who are to be admitted to an intensive care unit or expected to be hospitalized. Accordingly, the use of the AQGV peptide or its functional analog includes use against trauma, such as infection, for example, as exemplified in the Examples. Use of the AQGV peptide against trauma, such as infection, may occur before infection, but is typically during and / or after infection. Use of the AQGV peptide or its analog during viral infection may be preferred. The AQGV peptides provided herein are particularly useful in patients undergoing prolonged mechanical ventilation, i.e., for 2.5 hours or more. Thus, in a further embodiment, the use of the AQGV peptide or analog thereof is during mechanical ventilation for longer than 2.5 hours, and the AQGV peptide or analog thereof is administered during mechanical ventilation. In another or further embodiment, the use of the AQGV peptide or functional analog thereof for use according to the present invention is for use in human subjects with COVID-19. It is well known that shortening the duration of mechanical ventilation is highly correlated with patient recovery and prevention of re-hospitalization.

[0035] Preferably, the use of the AQGV peptide or its functional analogue according to the present invention and as described above involves administration of the peptide into the bloodstream. Administration into the bloodstream is understood to include, for example, intravenous or intra-arterial administration. A constant supply of the AQGV peptide or its analogue is preferred, for example, via infusion, in which the peptide or its analogue is contained in a physiologically acceptable solution. Suitable physiologically acceptable solutions may include physiological salt solutions (e.g., 0.9% NaCl) or any other suitable solution for injection and / or infusion. Such physiological solutions may contain additional compounds (e.g., glucose) that may further benefit the human subject, and may also contain other medicinal compounds (e.g., vasopressors).

[0036] Preferably, the AQGV peptide is administered at a rate of at least 50 mg / kg patient body weight per hour (mg / kg / hr). Preferably, the administration rate is at least 60 mg, at least 70 mg, at least 80 mg, or most preferably at least 90 mg / kg / hr. Preferably, the AQGV peptide is administered for at least 1 hour, more preferably at least 1.5 hours, and most preferably at least 2 hours. Preferably, the administration of the AQGV peptide is at a rate of at least 70 mg / kg / hr and is administered for at least 1 hour, more preferably at least 1.5 hours, and most preferably at least 2 hours. Preferably, the administration is for the duration of the infection. More preferably, the administration is for essentially the entire duration of the illness resulting from the infection. Typically, treatment will begin after the level of severity warranting the treatment has been determined. Thus, the treatment can typically continue from its detection until there is no detectable infection or until sufficient recovery to allow for the termination of treatment.

[0037] As shown in the Examples section, the mean arterial maximum concentration (mean Cmax) measured in vivo in humans for EA-230 in a Phase II clinical trial was 30,500 ng / mL, with a range of 12,500 to 57,500 ng / mL. The mean venous Cmax found was 68,400 ng / mL, with a range of 19,600 to 113,000 ng / mL. Therefore, whatever means and methods are used for administering EA-230 (or AQGV), means and methods that preferably allow for an arterial Cmax in the range of 10,000 to 60,000 ng / mL and / or an venous Cmax in the range of 15,000 to 120,000 ng / mL may be contemplated. Therefore, the route of administration may not necessarily be limited to intravenous administration. However, other routes of administration that result in similar venous and / or arterial Cmax concentrations may be included.

[0038] In another embodiment, the AQGV peptide or a functional analog thereof is provided for any use according to the present invention as described above, wherein the human subject is admitted to an intensive care unit, and wherein the use improves a measured parameter of the human subject, which parameter of the human subject is typically determined to assess whether the patient needs to stay in the intensive care unit. As indicated above, parameters assessed when the human patient is in the intensive care unit include parameters related to pulmonary function and hemodynamics.

[0039] In any event, the use of the AQGV peptide or analog thereof improves such parameters, thereby shortening the length of stay in the intensive care unit. Not only does the use of the AQGV peptide or analog thereof shorten the length of stay in the intensive care unit, but the effect of using the AQGV peptide or analog thereof also shortens the length of hospital stay and reduces re-admissions.

[0040] In any case, the use of the AQGV peptide or its functional analog has a significant effect on pulmonary function and / or hemodynamics in a human subject, thereby advantageously benefiting the human subject, for example, when suffering from an induced trauma, for example, while undergoing mechanical ventilation. Thus, in one embodiment, the use of the AQGV peptide or its functional analog is for use in patients undergoing mechanical ventilation. In another embodiment, the use of the AQGV peptide or its functional analog is for use in human patients experiencing or suspected of experiencing COVID-19 or a similar infection.

[0041] The present invention relates to a distinct and novel class of drug: autophagy-inhibiting compounds, including peptides and / or amino acids, that target the nutrient-sensing system of mTOR, the mechanistic target of rapamycin, and inhibit autophagy. Upon testing the formyl peptide-associated signaling effects of the autophagy-inhibiting AQGV peptide, the peptide was found to unexpectedly attenuate the p38 / p38-MK2-HSP27 and / or PI3K / AKT / mTOR pathways, which govern signaling cytoskeletal contraction in regulating vascular permeability. Thus, the present invention relates to the use of autophagy-inhibiting peptides (also referred to herein as AQGV peptides) and analogs (functional equivalents) thereof to improve vascular permeability.

[0042] Without being bound by theory, the effect of the AQGV peptide or its functional analogue may have an effect on vasoconstriction. Vasoconstriction involves the narrowing of blood vessels due to contraction of the muscular walls of the blood vessels. Thus, in one embodiment, the use of the AQGV peptide or its functional analogue according to the present invention involves the induction of vasoconstriction.

[0043] The present invention also provides a method for identifying a peptide capable of reducing p38 MAPK kinase activity, the method comprising: providing a cell with a peptide containing amino acids, wherein at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and more preferably 100% of the amino acids consist of amino acids selected from the group consisting of alanine (in single-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R); providing fMLP to the cell; and detecting phosphorylation of p38 MAPK in the absence and presence of the peptide, at an appropriate time interval after providing fMLP, preferably on the order of several minutes, most preferably about 0.5 to about 5 minutes, for example, 30 to 600 seconds; and comparing the results to determine the effect of the peptide on the phosphorylation. After testing the autophagy-inhibiting AQGV peptide, we used the prototype FPR ligand fMLP to detect FPR activation in FPR-expressing cells, which resulted in rapidly induced significant changes (p<0.05; p38 from 60-600 s, PKB (also known as AKT) at 600 s (Fig. 10a)) in the phosphorylation state of PKB and p38 MAPK kinase (Fig. 10c), but not in STAT3 kinase, JNK kinase (Fig. 10b), or P42 / p44 MAPK / ERK1,2 kinase (Fig. 10d).

[0044] In addition, the present invention also provides a method for identifying a peptide capable of reducing PI3K / AKT / mTOR activity, the method comprising: providing a cell with a peptide consisting of amino acids, wherein at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and more preferably 100% of the amino acids consist of amino acids selected from the group consisting of alanine (in single-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R); providing fMLP to the cell; and detecting phosphorylation of PKB (AKT) in the absence and presence of the peptide at an appropriate time interval after providing fMLP, preferably on the order of several minutes, most preferably about 0.5 to about 5 minutes, for example, 30 to 600 seconds. The effect of the AQGV peptide on p38 MAPK (Figure 10c) was already detected 30 s after FPR stimulation, followed by the effect of the AQGV peptide on PKB (AKT) (Figure 10a) at 300 s, in a biphasic pattern (Figure 10a). Both the effect of the AQGV peptide on p38 and PKB-mediated signaling persisted for the entire 600 s tested, while other kinases tested were unaffected throughout. This acute and specific response to treatment demonstrates the specific and rapid effect of the autophagy-inhibiting AQGV peptide on p38 signaling in the context of modulation of the PI3K / AKT / mTOR pathway, which influences vascular permeability and governs the balance between protein production and protein degradation, which regulates cytoskeletal changes. No such activity was detected in the STAT3, JNK (Figure 10b), and p42 / p44 MAPK / ERK1,2 (Figure 10d) kinases tested with the AQGV peptide. The AQGV peptide has been shown to reduce not only p38 MAPK kinase-activated changes but also PI3K / AKT / mTOR-activated induced changes in cytoskeletal rearrangements, affecting endothelial cell contraction and adverse vascular permeability.

[0045] The present invention also provides a method for identifying a peptide capable of reducing PI3K / AKT / mTOR activity, the method comprising: providing a cell with a peptide consisting of amino acids, wherein at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and more preferably 100% of the amino acids consist of amino acids selected from the group consisting of alanine (in single-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R); providing fMLP to the cell; and detecting phosphorylation of PKB (AKT) in the absence and presence of the peptide at an appropriate time interval after providing fMLP, preferably on the order of several minutes, most preferably about 0.5 to about 5 minutes, for example, 30 to 600 seconds; and comparing multiple results to determine the effect of the peptide on the phosphorylation. The identified AQGV peptides are useful and can address adverse vascular permeability, such as manifested by edema associated with vascular leakage, adverse leukocyte extravasation, and hypotension in human subjects.

[0046] The present invention also provides a method for identifying a peptide capable of reducing PI3K / AKT / mTOR activity, the method comprising: providing a cell with a peptide consisting of amino acids, wherein at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and more preferably 100% of the amino acids consist of amino acids selected from the group consisting of alanine (in single-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R); providing fMLP to the cell; and detecting phosphorylation of PKB (AKT) in the absence and presence of the peptide at an appropriate time interval after providing fMLP, preferably on the order of several minutes, most preferably about 0.5 to about 5 minutes, for example, 30 to 600 seconds; and comparing multiple results to determine the effect of the peptide on the phosphorylation. The present invention also provides a method for identifying a peptide capable of reducing cytoskeletal rearrangements, comprising providing a cell with a peptide consisting of amino acids, at least 50% of which are selected from the group consisting of alanine (in single-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R); providing fMLP to the cell; and detecting phosphorylation of PKB (AKT) in the absence and presence of the peptide at an appropriate time interval after providing fMLP, preferably on the order of several minutes, most preferably about 0.5 minutes to about 5 minutes, for example, 30 to 600 seconds; and comparing multiple results to determine the effect of the peptide on the phosphorylation.

[0047] Typically, the invention herein provides the molecular mechanism of action (MoA) of a group of autophagy-inhibitory peptides, so that their effect does not necessarily depend on their exact sequence.

[0048] Instead, their constituent amino acids provide a common household "no-danger or tissue-repair" signal to the mTOR nutrient-sensing system, resulting in the inhibition of autophagy and resulting in disease resolution. These tissue-repair signaling molecules shift the balance between protein production and protein degradation within the cell, resulting in disease resolution in three steps.

[0049] The administered peptide or amino acid fragment thereof is taken up by amino acid transport, PEPT1 / 2 transport, general endocytosis, and in the case of vascular cells, elastin receptor-mediated endocytosis or phagocytosis.

[0050] The internalized peptide is hydrolyzed and the amino acids are presented to the mTOR nutrient-sensing system.

[0051] Certain amino acids inhibit autophagy, thereby inhibiting protein degradation and resulting in resolution of protein synthesis and pharmacological effects.

[0052] Various peptides that meet one or more of the above-described characteristics, either derived from the degradation of peptide hormones or assembled as de novo synthetic peptides essentially containing amino acids selected from the group of autophagy-inhibiting amino acids, have been shown in various animal models in mice or rats to potently resolve local or systemic excessive or harmful vascular permeability through their effects on endothelial cells lining our vascular system. Some of them are in various stages of human clinical trials or are being rationally further developed. Exploiting the autophagy-inhibiting mechanisms involved through future clinical applications of these autophagy-inhibiting compounds and related peptide drugs offers exciting new avenues for the rational treatment of diseases. However, some autophagy-inhibiting peptide formulations for intravenous use have experienced peptide solubility issues, reducing the availability of autophagy-inhibiting amino acids and necessitating the provision of cumbersome storage solutions of large quantities of peptides to avoid peptide aggregation and loss of medicinal efficacy.

[0053] It is an object of the present disclosure to provide the autophagy-inhibiting amino acids to a subject likely to need them in the most convenient manner. The present invention also provides a tartrate or citrate salt of an autophagy-inhibiting peptide, preferably a recombinant or synthetic autophagy-inhibiting peptide, having an amino acid sequence containing at least 50%, more preferably at least 75%, and most preferably 100% of the autophagy-inhibiting amino acids selected from the group consisting of alanine (A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), and proline (P). More preferably, the present invention provides a storage solution, preferably an aqueous storage solution, comprising a peptide-tartrate or peptide-tartrate salt of an autophagy-inhibiting peptide tartrate or citrate, preferably a recombinant or synthetic autophagy-inhibiting peptide tartrate or citrate, wherein the peptide has an amino acid sequence comprising at least 50%, more preferably at least 75%, and most preferably 100% of autophagy-inhibiting amino acids selected from the group consisting of alanine (A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), and proline (P).

[0054] The present invention provides suitable solutions for several autophagy-inhibiting peptides to mitigate aggregation of the peptides and identifies tartrate (from tartaric acid, preferably (+)-tartaric acid) and more preferably citrate (from citric acid) as suitable counterions, pharmaceutical excipients, or pharmaceutical excipients or anions of choice for preparing salts of the autophagy-inhibiting peptides, which are neutral peptides as defined herein above. Various salts were screened herein to determine their effect on aggregation of the neutral peptides according to the present invention, and indeed demonstrated that the neutral peptides "salt out" from solution in an anion-specific and concentration-dependent manner.

[0055] At the aggregation point of such salts (the concentration point below which aggregated peptide salts tend to disintegrate), peptide sulfate, peptide maleate, peptide adenosine monophosphate, and peptide adenosine in aqueous solution were found to exhibit exacerbated aggregation relative to peptide acetate aggregation, whereas surprisingly, peptide-tartrate and, even more surprisingly, peptide-citrate exhibited (strongly) reduced aggregation in aqueous solution compared to peptide-acetate.

[0056] The autophagy inhibitor peptide salt according to the present invention preferably contains less than 25% charged residues selected from the group K, H, and R. More preferably, the autophagy inhibitor peptide contains less than 25% charged residues selected from the group D, K, R, H, and E. Most preferably, the autophagy inhibitor peptide salt does not contain any residues selected from the group D, K, R, H, and E. More preferably, the solution is an aqueous liquid. In a most preferred embodiment, the solution is a so-called storage solution, preferably an aqueous storage solution. A storage solution is generally a concentrated solution of an active substance, in this case an autophagy inhibitor peptide salt; this is a so-called working solution that will be diluted to a lower concentration for actual use of the substance.

[0057] Such low-concentration working solutions are, for example, infusion solutions for intravenous or intraperitoneal use, to which peptides are added from a stock solution for administering treatment to critically ill patients, such as those often found in hospital intensive care units or on the battlefield. Under such conditions, it is useful, and often considered necessary, to have a small amount (stock) of active (peptide) drug available for dilution into the infusion solution. So-called stock solutions are commonly provided and used to save solubilization and preparation time, conserve materials, reduce storage space, and improve accuracy when preparing solutions at low concentrations. Stock solutions of drugs are often prepared for imminent intravenous use, for example in critically ill patients, and then provided or stored. However, due to the high peptide concentration by default, stock solutions containing autophagy-inhibiting peptides always pose a higher risk of peptide drug aggregation than the final working solution. Stock solutions are typically prepared at concentrations (e.g., 40-50%) well below the aggregation concentration of the salt in question to prevent possible salting-out during long-term storage under various ambient conditions. The risk of peptide aggregation (salting-out) is a phenomenon that the present invention aims to avoid or mitigate using stock solutions according to the present invention. Such stock solutions are typically diluted 10- to 100-fold or more to provide suitable working solutions. However, it is also an object of the present invention to provide working solutions of peptide salts according to the present invention. In particular, since relatively high amounts / concentrations of peptide salts must be provided in the application of the peptides of the present invention, it is a prerequisite that the working solution is presented in a relatively small amount, despite being far from the salting-out point.

[0058] Various salts were screened to determine their effect on neutral peptide aggregation, revealing that the neutral peptide indeed "salted out" from solution in an anion-specific and concentration-dependent manner. Peptide-sulfate and peptide-maleate were found to exhibit exacerbated aggregation relative to that of peptide-acetate. Surprisingly, however, tartrate and, even more surprisingly, peptide-citrate exhibited (strongly) reduced aggregation compared to peptide-acetate.

[0059] The present invention thereby contributes to improving the solubility of this emerging, distinct, and novel class of drugs. Here, small autophagy-inhibiting peptides contain amino acids that preferentially inhibit autophagy and target the mTOR nutrient-sensing system, a mechanistic target of rapamycin. Typically, peptides are defined as having 50 amino acids or less, and for purposes of this disclosure, proteins are defined as having more than 50 amino acids. An autophagy-inhibiting peptide is defined herein as a linear, branched, or cyclic string of 50 amino acids or less, comprising a peptide sequence in which at least 50%, more preferably at least 70%, and most preferably 100% of the amino acids are selected from the group of autophagy-inhibiting amino acids: alanine (in single-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), proline (P), isoleucine (I), and arginine (R).

[0060] The molecular mode-of-action (MoA) of this group of peptides does not depend on their exact sequence. Instead, their constituent amino acids provide a common household "no-danger or tissue-repair" signal to the mTOR nutrient-sensing system; resulting in the inhibition of autophagy and resulting in disease resolution.

[0061] In another embodiment, the present invention provides a peptide, preferably an autophagy-inhibiting peptide, preferably a recombinant or synthetic autophagy-inhibiting peptide, in the form of an organic acid salt, such as maleate, more preferably acetate, more preferably tartrate, and most preferably citrate, wherein the peptide has an amino acid sequence containing at least 50%, more preferably at least 75%, and most preferably 100% autophagy-inhibiting amino acids selected from the group consisting of alanine (A), glutamine (Q), leucine (L), valine (V), glycine (G), and proline (P). More preferably, the present invention provides a peptide-tartrate salt or a storage solution, preferably an aqueous solution, containing the peptide-tartrate salt of an autophagy-inhibiting peptide, preferably a recombinant or synthetic autophagy-inhibiting peptide, wherein the autophagy-inhibiting peptide has an amino acid sequence containing at least 50%, more preferably at least 75%, and most preferably 100% of autophagy-inhibiting amino acids selected from the group consisting of alanine (A), glutamine (Q), glycine (G), valine (V), leucine (L), and proline (P).

[0062] Mindful of the risk of aggregation, to date, vials containing stock solutions of the AQGV peptide as defined above for use in clinical trials have contained less than (0.8 mol / L) of active substrate in solution.

[0063] According to the present invention, there is provided a stock solution of an AQGV organic acid salt, in particular an AQGV peptide-maleate, an AQGV peptide-acetate, an AQGV peptide-tartrate or an AQGV peptide-citrate (but not adenosine or adenosine monophosphate) having an amino acid sequence, wherein the amino acid sequence contains at least 50% of the autophagy-inhibiting amino acids selected from the group of alanine (A), glutamine (Q), glycine (G), valine (V), leucine (L) and proline (P), more preferably or at least 75%, most preferably 100%, of the AQGV peptide-acetate, AQGV peptide-tartrate, or AQGV peptide-citrate, wherein the storage solution contains at least 0.85 mol / L, more preferably at least 0.9 mol / L, more preferably at least 1 mol / L, more preferably at least 1.2 mol / L, more preferably at least 1.4 mol / L, more preferably at least 1.6 mol / L, and most preferably at least 1.8 mol / L of the AQGV peptide-acetate, AQGV peptide-tartrate, or AQGV peptide-citrate. In a more preferred embodiment, the present invention provides a storage solution of the AQGV peptide-tartrate or AQGV peptide-citrate, wherein the concentration of the AQGV peptide is 2 mol / L to 2.5 mol / L. In a more preferred embodiment, the present invention provides a storage solution of the AQGV peptide-citrate, wherein the concentration of the peptide-citrate is 2.5 mol / L to 3 mol / L. In a more preferred embodiment, the present invention provides a stock solution of the peptide-citrate, wherein the concentration of the peptide-citrate is 3 mol / L to 3.5 mol / L. In a more preferred embodiment, the present invention provides a stock solution of the peptide-citrate, wherein the concentration of the peptide-citrate is 3.5 mol / L to 4.5 mol / L. In a more preferred embodiment, the present invention provides a stock solution of the peptide-citrate, wherein the concentration of the peptide-citrate is 4.5 mol / L to 5.5 mol / L.In a more preferred embodiment, the present invention provides a stock solution of the peptide-citrate, wherein the concentration of the peptide-citrate is 5.5 mol / L or more. Preferably, the stock solution is an aqueous solution.

[0064] The stock solution contains the following dipeptides: AQ, QQ, LQ, GQ, PQ, VQ, AL, LL, QL, GL, PL, VL, QA, QL, QG, QP, QV, LA, LG, LP, LV, and the following tripeptides: AQG, QQG, LQG, GQG, PQG, VQG, ALG, LLG, QLG, GLG, PLG, VLG, QAG, QLG, QGG, QPG, QVG, LAG, LGG. , LPG, LVG, or the following tetrapeptides: AQGV, QQGV, LQGV, GQGV, PQGV, VQGV, ALGV, LLGV, QLGV, GLGV, PLGV, VLGV, QAGV, QLGV, QGGV, QPGV, QVGV, LAGV, LGGV, LPGV, LVGV, or a mixture thereof.

[0065] The peptides according to the invention preferably have a peptide sequence length of 2 to 40 amino acids, preferably 3 to 30 amino acids, and preferably 4 to 20 amino acids. Most preferably, the peptides according to the invention have a peptide sequence containing at least 6 amino acids, especially when at least 4 of them inhibit autophagy. The maximum length of the peptide-tartrate or peptide-citrate according to the invention preferably contains up to 50 amino acids, more preferably up to 40 amino acids, more preferably up to 30 amino acids, more preferably up to 20 amino acids, more preferably up to 15 amino acids, more preferably up to 12 amino acids, and most preferably up to 9 amino acids.

[0066] The present invention provides a method for reducing p38 MAPK kinase activity that results in cytoskeletal rearrangements, comprising providing to cells, preferably cells having formyl peptide receptors associated with their surface, a source of autophagy-inhibiting amino acids, preferably the AQGV peptide provided herein, wherein at least 50% of the amino acids consist of amino acids selected from the group consisting of alanine (in single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R).

[0067] The present invention provides a method for reducing formyl-peptide receptor (FPR)-mediated p38 MAPK kinase activity, comprising providing to cells, preferably cells having formyl-peptide receptors associated with their surface, a source of autophagy-inhibiting amino acids, preferably wherein the source is the AQGV peptide provided herein, wherein at least 50% of the amino acids consist of amino acids selected from the group consisting of alanine (in single-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R).

[0068] The present invention provides a method for reducing PI3K / AKT / mTOR activity, resulting in cytoskeletal reorganization, comprising providing to cells, preferably cells having formyl peptide receptors associated with their surface, a source of autophagy-inhibiting amino acids, preferably wherein the source is the AQGV peptide provided herein, wherein at least 50% of the amino acids consist of amino acids selected from the group consisting of alanine (in single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R).

[0069] The present invention provides a method for reducing formyl peptide receptor (FPR)-mediated PI3K / AKT / mTOR activity, comprising providing to cells, preferably cells having formyl peptide receptors associated with their surface, a source of autophagy-inhibiting amino acids, preferably wherein the source is the AQGV peptide provided herein, wherein at least 50% of the amino acids consist of amino acids selected from the group consisting of alanine (in single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R).

[0070] The present invention provides a method for reducing cytoskeletal rearrangements, comprising providing to cells, preferably cells having formyl peptide receptors associated with their surface, a source of autophagy-inhibiting amino acids, preferably wherein the source is the AQGV peptide provided herein, and wherein at least 50% of the amino acids consist of amino acids selected from the group consisting of alanine (in single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R).

[0071] The present invention provides a method for reducing formyl peptide receptor (FPR)-mediated cytoskeletal rearrangements, comprising providing to cells, preferably cells having formyl peptide receptors associated with their surface, a source of autophagy-inhibiting amino acids, preferably the AQGV peptide provided herein, wherein at least 50% of the amino acids consist of amino acids selected from the group consisting of alanine (in single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R).

[0072] The present invention provides a method of altering vascular permeability, comprising providing to cells, preferably cells having formyl peptide receptors associated with their surface, a source of an autophagy-inhibiting amino acid, preferably wherein the source is the AQGV peptide provided herein, and the amino acid is selected from the group of alanine (in single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R).

[0073] The present invention provides a method of improving tissue repair, comprising providing to cells, preferably cells having formyl peptide receptors associated with their surface, a source of an autophagy-inhibiting amino acid, preferably wherein the source is the AQGV peptide provided herein, and the amino acid is selected from the group of alanine (in single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R).

[0074] The invention provides a method according to the invention, wherein the peptide comprising an autophagy-inhibiting amino acid comprises the following dipeptides: AQ, QQ, LQ, GQ, PQ, VQ, AL, LL, QL, GL, PL, VL, QA, QL, QG, QP, QV, LA, LG, LP, LV, tripeptides. AQG, QQG, LQG, GQG, PQG, VQG, ALG, LLG, QLG, GLG, PLG, VLG, QAG, QLG, QGG, QPG, QVG, LAG, LGG, LPG, LVG, or the tetrapeptides AQGV, QQGV, LQGV, GQGV, PQGV, VQGV, ALGV, LLGV, QLGV, GLGV, PLGV, VLGV, QAGV, QLGV, QGGV, QPGV, QVGV, LAGV, LGGV, LPGV, LVGV, or mixtures thereof.

[0075] In another embodiment, the present application finds the SARS-COV-2 spike protein to carry a distinct peptide motif sequence KWPWYIWL or variant KWPWYVWL in the membrane proximal external region (MPER) capable of binding to one of the receptors of the FPR family of receptors (see Figures 11-13).

[0076] COVID-19 activates FPR-mediated pathways, particularly through interactions with vascular cells and their spike proteins, leading to vascular leakage, thrombotic events, and modulation of the Ang1 / Ang2 ratio. The MPER region may also be involved in the sparsely reported and occasional thrombotic events following vaccination with different coronavirus-based vaccines that express unfixed spike proteins in the pre-fusion state. The AQGV-peptide inhibits formyl peptide-activated FPR-mediated pathways (p38-MK2-HSP27 and PI3K-AKT-mTOR, see Figure 10) involved in the disruption of vascular integrity. Therefore, after an event resulting in the expression of at least the fusogenic region bearing the motif KWPWYIWL or variant KWPWYVWL in a subject, the AQGV-peptide ameliorates vascular leakage and thrombotic events by inhibiting thrombus formation and modulating the Ang1 / Ang2 ratio.

[0077] Surprisingly, the fusion region having the motif KWPWYIWL or the variant KWPWYVWL also contains an FPR-binding site involved in inducing vascular leakage in a subject. The present application also provides an alternative treatment or use: a method for treating a subject suspected of expressing a peptide or protein comprising a fusion region derived from a virus, the method comprising adoptive cell therapy using at least one cell equipped with a receptor that recognizes the fusion region. Preferably, the fusion region comprises at least the peptide motif KWPWYIWL or at least the peptide motif KWPWYVWL, wherein the cell is a transformed T cell, such as a CAR-T cell or a TCR-T cell, and preferably the cell is directed against a T cell epitope (preferably CD8+) that comprises or overlaps with the fusion region. Such adoptive cell therapy methods provided herein (see, e.g., June et al., Adoptive cellular therapy: A race to the finish line Science Translational Medicine 25 Mar 2015: Vol. 7, Issue 280, pp. 280 ps7) involve the use of at least one cell equipped with a receptor that recognizes a fusion region from a virus in a method of treating a subject deemed to express a peptide or protein comprising the fusion region, wherein the fusion region comprises at least the peptide motif KWPWYIWL or comprises at least the peptide motif KWPWYVWL, and wherein the cell is preferably a transformed T cell, e.g., a CAR-T cell or a TCR-T cell, directed against a (preferably CD8+) T cell epitope comprising or overlapping with the fusion region.

[0078] Further embodiments

[0079] Item 1. An AQGV peptide or a functional analog thereof for use in treating a human subject, wherein the use comprises altering hemodynamics in the human subject.

[0080] Item 2. An AQGV peptide or a functional analog thereof for use in treating a human subject, wherein the human subject is suffering from a viral infection, and the use comprises altering hemodynamics in the human subject. The AQGV peptide or a functional analog thereof.

[0081] Item 3. An AQGV peptide or a functional analog thereof for use in treating a human subject having impaired pulmonary function, wherein the use includes altering hemodynamics and improving hypoxemia in the human subject. The AQGV peptide or a functional analog thereof.

[0082] Item 4. An AQGV peptide or a functional analogue thereof for use according to any one of the further aspects of items 1 to 3 above, wherein said use reduces fluid retention in said human subject.

[0083] Item 5. An AQGV peptide or a functional analogue thereof for use according to any one of the further aspects of items 1 to 3 above, wherein the use comprises reduced use of a vasoconstrictor.

[0084] Item 6. An AQGV peptide or a functional analogue thereof for use according to any one of the further aspects of items 1 to 3 above, wherein the use comprises reduced fluid uptake.

[0085] Item 7. An AQGV peptide or a functional analog thereof for use according to a further aspect of item 5 above, wherein the reduced use of vasoconstrictors comprises a reduced duration of use of vasopressors.

[0086] Item 8. An AQGV peptide or a functional analogue thereof for use according to any one of the further aspects of items 3 to 6 above, wherein the subject is suffering from a respiratory viral infection.

[0087] Item 9. The AQGV peptide or a functional analogue thereof for use according to any one of the further aspects of items 3 to 8 above, wherein the use improves lung function in the human subject.

[0088] Item 10. An AQGV peptide or a functional analogue thereof for use according to a further aspect of item 9 above, wherein the improved lung function is accompanied by improved oxygen saturation of the blood.

[0089] Item 11. The AQGV peptide or a functional analog thereof for use according to any one of the further aspects of items 3 to 10 above, wherein the human subject has a pulmonary dysfunction, and the pulmonary dysfunction is ARDS.

[0090] Item 12. An AQGV peptide or a functional analogue thereof for use according to any one of the further aspects of items 1 to 11 above, wherein the use is for reducing plasma leakage from the blood into peripheral tissues and / or organs.

[0091] Clause 13. The AQGV peptide or a functional analogue thereof for use according to any one of the further aspects of clauses 1 to 12 above, wherein the use is in a human subject suffering from or at risk of the harmful effects of mechanical ventilation.

[0092] Item 14. The AQGV peptide or a functional analog thereof for use according to any one of the further aspects of items 1 to 13 above, wherein the use is in a human subject at risk of having edema.

[0093] Clause 15. An AQGV peptide or a functional analogue thereof for use according to any one of the further aspects of clauses 2 to 14 above, wherein the human subject is suffering from or is suspected to be suffering from a coronavirus infection.

[0094] Clause 16. An AQGV peptide or a functional analogue thereof for use according to a further aspect of clause 15 above, wherein the infection is a SARS-Cov-2 infection.

[0095] Item 17. An AQGV peptide or a functional analogue thereof for use according to any one of the further aspects of items 1 to 16 above, wherein the peptide is administered into the bloodstream.

[0096] Item 18. The AQGV peptide or a functional analogue thereof for use according to a further aspect of Item 17 above, wherein the peptide is administered at a rate of at least 70 mg / kg body weight / hour.

[0097] Item 19. An AQGV peptide or a functional analogue thereof for use according to a further aspect of item 15 above, wherein the peptide is administered for at least 1 hour.

[0098] Item 20. An AQGV peptide or a functional analogue thereof for use according to any one of the further aspects of items 15 to 19 above, wherein the administration is intermittent.

[0099] Clause 21. The AQGV peptide or a functional analogue thereof for use according to any one of the further aspects of clauses 1 to 20 above, wherein the human subject is admitted to an intensive care unit, and the use improves a parameter measured in the human subject, and the parameter of the human subject is determined to assess whether the human subject remains in the intensive care unit.

[0100] Clause 22. An AQGV peptide or a functional analogue thereof for use according to a further aspect of clause 21 above, wherein said improvement in a parameter results in a shortened length of stay in an intensive care unit.

[0101] Item 23. An AQGV peptide or a functional analogue thereof for use according to any one of the further aspects of items 1 to 22 above, wherein the use induces vasoconstriction.

[0102] Clause 24. An AQGV peptide or a functional analogue thereof for use according to any one of the further aspects of clauses 1 to 22 above, wherein the subject is considered to be at risk for VALI or VILI.

[0103] Paragraph 25. An AQGV peptide or a functional analogue thereof for use according to any one of the further aspects of paragraphs 1 to 23 above, wherein the subject is considered to express a peptide or protein comprising a fusogenic region derived from a virus. The AQGV peptide or a functional analogue thereof.

[0104] Item 26. An AQGV peptide or a functional analogue thereof for use according to a further aspect of item 25 above, wherein the fusion region comprises at least the peptide motif KWPWYIWL or the variant KWPWYVWL.

[0105] Item 27. An AQGV peptide or a functional analogue thereof for use according to a further embodiment of item 25 or 26 above, wherein the fusion region comprises at least an FPR binding site.

[0106] Item 28. A method of treatment comprising administering an AQGV peptide or a functional analogue thereof to a human subject, wherein the human subject is in need of maintaining hemodynamic stability.

[0107] Item 29. A method of treatment comprising administering an AQGV peptide or a functional analogue thereof to a human subject, wherein the human subject is in need of improved hemodynamic stability.

[0108] Item 30. A method of treatment comprising administering an AQGV peptide or a functional analog thereof to a human subject, wherein the human subject has impaired pulmonary function, and wherein administering the AQGV peptide comprises maintaining or improving hemodynamic stability in the human subject.

[0109] Item 31. A method of treatment comprising intermittently administering an AQGV peptide or a functional analog thereof to a human subject, wherein the human subject has impaired pulmonary function, and wherein administering the AQGV peptide comprises maintaining or improving hemodynamic stability in the human subject.

[0110] Item 31-2. A method for treating a subject that is considered to express a peptide or protein containing a fusion region derived from a virus, the method comprising adoptive cell therapy using at least one cell equipped with a receptor that recognizes the fusion site.

[0111] Item 32. A method according to item 31 or 31-2, which is a further embodiment, wherein the fusion region comprises at least the peptide motif KWPWYIWL.

[0112] Item 33. A method according to item 31 or 31-2, which is a further embodiment, wherein the fusion region comprises at least the peptide motif KWPWYVWL.

[0113] Item 33-2. The method according to any one of the further embodiments of any one of claims 31 to 33, wherein the cell is a transformed T cell, such as a CAR-T cell or a TCR-T cell.

[0114] Clause 34. The method according to the embodiment of claim 33, wherein the cells are directed against a T cell epitope that includes the fusion region or that overlaps with the fusion region.

[0115] Item 35. A method for using at least one cell having a receptor that recognizes a fusion domain derived from a virus, wherein the subject treated is considered to express a peptide or protein containing the fusion domain.

[0116] Clause 36. A further embodiment of the use according to clause 35, wherein the fusion domain comprises at least the peptide motif KWPWYIWL.

[0117] Clause 37. A further embodiment of the use according to clause 35, wherein the fusion domain comprises at least the peptide motif KWPWYVWL.

[0118] Item 38. A further embodiment of the use according to any one of items 35 to 37, wherein the cell is a transformed T cell, such as a CAR-T cell or a TCR-T cell.

[0119] Clause 39. The use of embodiment clause 38, wherein the cells are directed against a T cell epitope comprising the fusion region or a T cell epitope that overlaps with the fusion region. [Brief explanation of the drawings]

[0120] [Figure 1] Figure 1 shows that initial infection with the SARS-CoV-2 virus is almost always mild or even uneventful. Stage I disease is seen in over 80% of infected individuals. The majority of patients experience an upper respiratory tract infection of the nose and throat, with a dry cough that usually resolves within 2–12 days. In the remaining <20% of cases, two distinct pathological stages may occur, often beginning in mild cases as viral infection diminishes due to the emergence of an immune response directed against the virus. Stage II is viral pneumonia (pulmonary stage with lung damage), characterized by loss of alveolar-capillary membrane permeability (Stage IIA), diffuse and massive involvement of the deeper airways and alveoli of both lungs (Stage IIB), and reduced oxygen uptake due to respiratory failure. This bilateral pneumonia may be followed by Stage III disease, characterized by general fatigue, high fever, and ultimately by total organ (kidney, liver, and heart) failure. [Figure 2] Figure 2 shows that acute disease in stage II increases vascular permeability and results in fluid leakage from pulmonary capillaries into lung tissue (see also lung injury in Figure 3). This angiopoietin-regulated permeability is depicted in Figure 2. Angiopoietin 1 (ANG1) is constitutively secreted by perivascular mural cells. When gaps form between cells, ANG1 is released into the vascular lumen. Ligand binding of ANG1 to TIE2 induces sequestration of the tyrosine kinase Src, thus establishing stable expression of VE-cadherin on the surface of endothelial cells and allowing gap closure. ANG2 is stored in Weibel-Palade (WBP) bodies and is rapidly released in response to triggering signals. Its binding to TIE2 abolishes ANG1-induced sequestration of Src, resulting in the internalization of VE-cadherin. [Figure 3]Figure 3 shows that acute disease in stage II results in lung injury characterized by edematous lung tissue, resulting in a reduced gas (oxygen and carbon dioxide) diffusion capacity. SARS-CoV-2 infection begins in type II cells. Pulmonary capillary permeability is increased by increased vascular cell gap formation, as shown in Figure 2. This process generates fluid flow from capillaries into the thin layer of extracellular matrix that separates alveoli from capillaries, where intercellular fluid storage occurs. This, along with the accumulation of fluid within the extracellular matrix, thickens (expands) the membrane (resulting in an enlarged, edematous interstitium), separating alveolar cells from vascular cells, and entering the alveoli. This typically triggers local inflammatory activity of leukocytes that migrate into the lung tissue. Overall, gas diffusion is significantly impaired, resulting in respiratory distress. In such patients with stage II lung injury, plasma levels of biomarkers of endothelial activation, which can be measured by ELISA, often predict mortality. In particular, the concentration of angiopoietin-2 relative to angiopoietin-1 (Ang-2 / Ang-1) may be a useful biological marker of mortality in patients with acute lung injury (ALI). [Figure 4]Figure 4 shows a schematic diagram of the intermittent administration of AQGV peptides during various stages of COVID-19 infection, based on the infection phases shown in Figure 1. The shaded bars indicate time slots during which the AQGV peptide or related substance is administered intravenously, preferably at a rate of at least 75 mg / kg / hour, more preferably at least 90 mg / kg / hour. Intermittent administration of AQGV peptides or related substances is particularly useful. A preferred use is a 2-4 hour dosing regimen of at least 75 mg / kg / hour, more preferably at least 90 mg / kg / hour, followed by optional reduction to 30 mg / kg / hour for 2-4 hours (between the shaded bars) or as long as it takes to monitor the patient's response to treatment with clinical or laboratory diagnostics, or cessation of substance administration for 1-2 hours until diagnostic studies are completed, and then resuming treatment at at least 75 mg / kg / hour, more preferably at least 90 mg / kg / hour, for 2-4 hours. Depending on the stage of the disease, the therapeutic effect of EA-230 can be monitored by measuring hypoxia, plasma Ang2 / Ang1 ratio and plasma levels of D-dimer in stage II and stage III. [Figure 5] Figure 5 shows that the need for treatment of hemodynamic instability with the use of vasopressors (left) and fluid therapy was significantly improved in patients receiving EA-230 peptide during the first 24 hours in the intensive care unit (ICU), with EA-230 significantly improving hemodynamic recovery and hemodynamic stability (reducing the composite measure of fluid therapy and blood pressure medication requirements; 2-way ANOVA; p=0.006). [Figure 6] Figure 6 shows that patients treated with EA-230 (AQGV) had a shorter length of stay compared to the placebo group, both in the ICU (p=0.02) and hospital (p=0.001). The AQGV peptide EA-230g reduced the number of patients in the ICU by 48% over 24 hours; and reduced the length of hospital stay by 20%. [Figure 7] Figure 7 shows an overview of the solubility experiments, along with the results shown in Table 1. [Figure 8]Figure 8 shows that, based on the results shown in Figure 7, the concentration below which the aggregated peptide salts of the screened neutral peptide salts tend to disintegrate (aggregation point) was determined. It can be concluded that changing the anion significantly affects the solubility characteristics of AQGV. Higher solubility (solubility in 0.9% NaCl) and correspondingly higher aggregation point were observed for AQGV-citrate (AQGV-citrate) and AQGV-tartrate (AQGV-tartrate), while maleic acid and KHSO4 salts showed lower solubility compared to AQGV-Ac. Solubility was not achieved using adenosine monophosphate or adenosine. Citric acid appears to be a special case. Highly concentrated solutions did not crystallize or aggregate, but tended to form highly viscous solutions. [Figure 9]Figure 9 shows that formyl peptide receptors mediate vascular permeability after cell and tissue trauma. Human formyl peptide receptors (FPRs) are N-glycosylated and activate cells through G(i) proteins. Site-directed mutagenesis of extracellular Asn residues prevented FPR glycosylation but not FPR expression in the plasma membrane. However, non-glycosylated FPRs are significantly less active than native FPRs in terms of high-affinity agonist binding, GTP gamma S binding kinetics, number of activated G(i) proteins, and constitutive activity. Mitochondrial N-formyl peptides (F-MITs) released from trauma / cell injury activate the formyl peptide receptor (FPR), leading to changes in the endothelial cell cytoskeleton, subsequently inducing endothelial cell contraction and vascular permeability, leukocyte extravasation, and hypotension. N-formyl peptides are a common molecular signature of bacteria and mitochondria that activate the formyl peptide receptor (FPR). FPR activation by mitochondrial N-formyl peptide (F-MIT) induces changes in cytoskeletal regulatory proteins in endothelial cells, resulting in increased endothelial cell contractility accompanied by increased vascular leakage and leukocyte extravasation. FPR activation via mitochondrial N-formyl peptide (F-MIT) resulting from tissue damage, such as trauma, is a major cause of barrier dysfunction following cell and tissue damage or injury, resulting in deleterious effects on the blood vessels, such as adverse vascular permeability with edema, vascular leakage, adverse leukocyte extravasation, and hypotension. [Figure 10A]Figure 10A shows formyl peptide receptor-mediated peptide effects. FPR activation of FPR-expressing cells with the prototypic FPR ligand fMLP rapidly induced and significant (p<0.05; p38 at 60-600 s, PKB at 600 s) changes in the phosphorylation status of PKB (also known as AKT) (Figure 10A) and p38 MAPK kinase (Figure 10C), but did not induce (or detect) changes in STAT3, JNK (Figure 10B), and p42 / p44 MAPK / ERK1,2 (Figure 10D) kinases. The AQGV peptide effect on p38 MAPK (Figure 10C) was detected as early as 30 s after FPR stimulation, whereas the AQGV peptide effect on PKB (AKT) continued in a biphasic pattern at 300 s (Figure 10A). Both the AQGV peptide effects on p38 and PKB-mediated signaling persisted throughout the 600-second time period tested, while other kinases tested were generally unaffected. This acute and specific response to treatment demonstrates the specific and rapid effect of the autophagy-inhibiting AQGV peptide on p38 signaling in the context of regulating the PI3K / AKT / mTOR pathway. This pathway governs the balance between protein degradation and protein production, which regulates cytoskeletal changes that affect vascular permeability. The AQGV peptide has been shown to reduce p38 MAPK kinase activation changes and PI3K / AKT / mTOR activation-induced changes in cytoskeletal reorganization that affect endothelial cell contraction and vascular permeability. The AQGV peptide is useful and can address adverse vascular permeability, such as those manifested by edema associated with vascular leakage, harmful leukocyte extravasation, and hypotension in human subjects. [Figure 10B]Figure 10B shows formyl peptide receptor-mediated peptide effects. FPR activation of FPR-expressing cells with the prototypic FPR ligand fMLP rapidly induced and significant (p<0.05; p38 at 60-600 s, PKB at 600 s) changes in the phosphorylation status of PKB (also known as AKT) (Figure 10A) and p38 MAPK kinase (Figure 10C), but did not induce (or detect) changes in STAT3, JNK (Figure 10B), and p42 / p44 MAPK / ERK1,2 (Figure 10D) kinases. The AQGV peptide effect on p38 MAPK (Figure 10C) was already detected 30 s after FPR stimulation, whereas the AQGV peptide effect on PKB (AKT) continued in a biphasic pattern at 300 s (Figure 10A). Both the AQGV peptide effects on p38 and PKB-mediated signaling persisted throughout the 600-second time period tested, while other kinases tested were generally unaffected. This acute and specific response to treatment demonstrates the specific and rapid effect of the autophagy-inhibiting AQGV peptide on p38 signaling in the context of regulating the PI3K / AKT / mTOR pathway. This pathway governs the balance between protein degradation and protein production, which regulates cytoskeletal changes that affect vascular permeability. The AQGV peptide has been shown to reduce p38 MAPK kinase activation changes and PI3K / AKT / mTOR activation-induced changes in cytoskeletal reorganization that affect endothelial cell contraction and vascular permeability. The AQGV peptide is useful and can address adverse vascular permeability, such as those manifested by edema associated with vascular leakage, harmful leukocyte extravasation, and hypotension in human subjects. [Figure 10C]Figure 10C shows formyl peptide receptor-mediated peptide effects. FPR activation of FPR-expressing cells with the prototypic FPR ligand fMLP rapidly induced and significant (p<0.05; p38 at 60-600 s, PKB at 600 s) changes in the phosphorylation status of PKB (also known as AKT) (Figure 10A) and p38 MAPK kinase (Figure 10C), but did not induce (or detect) changes in STAT3, JNK (Figure 10B), and p42 / p44 MAPK / ERK1,2 (Figure 10D) kinases. The AQGV peptide effect on p38 MAPK (Figure 10C) was already detected 30 s after FPR stimulation, whereas the AQGV peptide effect on PKB (AKT) continued in a biphasic pattern at 300 s (Figure 10A). Both the AQGV peptide effects on p38 and PKB-mediated signaling persisted throughout the 600-second time period tested, while other kinases tested were generally unaffected. This acute and specific response to treatment demonstrates the specific and rapid effect of the autophagy-inhibiting AQGV peptide on p38 signaling in the context of regulating the PI3K / AKT / mTOR pathway. This pathway governs the balance between protein degradation and protein production, which regulates cytoskeletal changes that affect vascular permeability. The AQGV peptide has been shown to reduce p38 MAPK kinase activation changes and PI3K / AKT / mTOR activation-induced changes in cytoskeletal reorganization that affect endothelial cell contraction and vascular permeability. The AQGV peptide is useful and can address adverse vascular permeability, such as those manifested by edema associated with vascular leakage, harmful leukocyte extravasation, and hypotension in human subjects. [Figure 10D]Figure 10D shows formyl peptide receptor-mediated peptide effects. FPR activation of FPR-expressing cells with the prototypic FPR ligand fMLP rapidly induced and significant (p<0.05; p38 at 60-600 s, PKB at 600 s) changes in the phosphorylation status of PKB (also known as AKT) (Figure 10A) and p38 MAPK kinase (Figure 10C), but did not induce (or detect) changes in STAT3, JNK (Figure 10B), and p42 / p44 MAPK / ERK1,2 (Figure 10D) kinases. The AQGV peptide effect on p38 MAPK (Figure 10C) was already detected 30 s after FPR stimulation, whereas the AQGV peptide effect on PKB (AKT) continued in a biphasic pattern at 300 s (Figure 10A). Both the AQGV peptide effects on p38 and PKB-mediated signaling persisted throughout the 600-second time period tested, while other kinases tested were generally unaffected. This acute and specific response to treatment demonstrates the specific and rapid effect of the autophagy-inhibiting AQGV peptide on p38 signaling in the context of regulating the PI3K / AKT / mTOR pathway. This pathway governs the balance between protein degradation and protein production, which regulates cytoskeletal changes that affect vascular permeability. The AQGV peptide has been shown to reduce p38 MAPK kinase activation changes and PI3K / AKT / mTOR activation-induced changes in cytoskeletal reorganization that affect endothelial cell contraction and vascular permeability. The AQGV peptide is useful and can address adverse vascular permeability, such as those manifested by edema associated with vascular leakage, harmful leukocyte extravasation, and hypotension in human subjects. [Figure 11]Figure 11 shows AQGV peptides targeting viral spike protein-induced pulmonary and vascular leakage in coronavirus infections, such as those seen in SARS, MERS, and COVID-19. The SARS-CoV-2 spike (S) glycoprotein is a class I viral fusion protein that facilitates viral entry into cells and is the primary target of antibodies (White et al., Critical reviews in biochemistry and molecular biology. 2008 Jan 1;43(3):189-219). The C-terminus of the spike protein contains a heptad repeat (HR2), a short linker region (membrane proximal external region, or MPER), a transmembrane helix domain (TMD), and a C-terminal cytoplasmic or internal domain (CTD / IC).After the ACE2 receptor in the target cell binds to the receptor binding domain (RBD) of the S protein, the heptad repeat 1 (HR1) and heptad repeat 2 (HR2) domains form a six-helix bundle fusion core (6HB), bringing the fusogenic MPER domain and the cell membrane together for virus fusion and cell entry (Walls et al., Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion. Proceedings of the National Academy of Sciences. 2017 Oct 17;114(42):11157-62.; Xia et al., Fusion mechanism of 2019-nCoV and fusion inhibitors targeting the HR1 domain in spike protein. Cellular & molecular immunology. 2020 Feb 11:1-3.). The MPER is essential for viral entry into cells, as identified in Figure 12. Note that at least one T cell epitope that allows for the generation of CD8+ T cell cross-reactivity against SARS-CoV-2 and other coronavirus strains (Lee et al, Front. Immunol., 05 November 2020 | https: / / doi.org / 10.3389 / fimmu.2020.579480) overlaps with the fusion site identified in Figure 12. In patients, virus-specific CD4+ and CD8+ T cell responses are associated with mild disease, suggesting the involvement of this fusion region in protective immunity against COVID-19.Typically, the fusion site, and thereby the T cell epitope, is highly conserved in SARS-CoV-2 (Guo E, Guo H (2020) CD8 T cell epitope generation toward the continually mutating SARS-CoV-2 spike protein in genetically diverse human population: Implications for disease control and prevention. PLOS ONE 15(12):e0239566), and is provided herein for developing adoptive cellular therapy (ACT) directed against the fusogenic region that can be used in virus- or vaccine-based infections, such as those with coronaviruses or vaccines. [Figure 12]Figure 12 shows that the short membrane proximal external region (MPER) connects HR2 and the transmembrane domain and contains an aromatic amino acid-rich fusion peptide sequence that destabilizes the membrane during fusion (Mahajan M, Bhattacharjya S. NMR structures and localization of the potential fusion peptides and the pre-transmembrane region of SARS-CoV: Implications in membrane fusion. Biochimica et Biophysica Acta (BBA)-Biomembranes. 2015 Feb 1;1848(2):721-30.; Guillen J, Kinnunen PK, Villalain J. Membrane insertion of the three main membranotropic sequences from SARS-CoV S2 glycoprotein. Biochimica et Biophysica Acta (BBA)-Biomembranes. 2008 Dec 1;1778(12):2765-74.). This fusogenic region is sometimes referred to as the "membrane proximal ectodomain region" or "pre-transmembrane region" (PTM). The MPER peptides 1185-LGKYEQYIKWPWYVWLGF-1202 and 1193-KWPWYVWLGFIAGLIAIV-1210 from SARS-CoV-1 have been shown to intercalate into lipid membranes and to be highly surface active; the corresponding fusion gene sequences in SARS-CoV-2 and MERS-CoV are identical except for a V to I substitution at position 1216. [Figure 13]Figure 13 shows that the present invention finds that the SARS-CoV-2 spike protein carries a distinct and conserved fusogenic motif in its MPER domain (KWPWYIWL) that can bind to FPR. This motif is highly homologous to a related coronavirus spike protein motif that has been demonstrated to bind to FPR (Mills, Biochim Biophys Acta Mol Basis Dis. 2006 Jul;1762(7):693-704). Vascular leakage in COVID-19 is at least partially mediated by binding and / or fusion of this spike protein, containing at least the minimal essential fusogenic sequence KWPWYIWL or variant KWPWYVWL, to pulmonary vascular cells that carry formyl peptide receptors, which may result in thrombotic events during coronavirus infection or vaccination against COVID-19 with a spike protein vaccine, e.g., ChAdOx1-S, especially if such vaccines are not engineered to express the spike protein only in the pre-fusion state. The FPR-mediated pathway is known to be activated in thrombotic events (Salamah et al., The formyl peptide fMLF primes platelet activation and augments thrombus formation. J Thromb Haemost. 2019;17:1120-1133.) and in acute lung injury and acute respiratory disease syndromes (ALI / ARDS), with pulmonary vascular leak as the primary clinical manifestation (Thorax. 2017;72:928-936). COVID-19 infection with SARS-COV-2, particularly severe, typically increases the risk of ALI / ARDS with pulmonary vascular leak, leading to significant mortality. DETAILED DESCRIPTION OF THE INVENTION

[0121] Example

[0122] Autophagy-inhibiting peptides One-character code In the description of the composition, structure, and function of proteins or peptides herein, reference is made to amino acids. In the present specification, amino acid residues are represented by the following abbreviations. Furthermore, unless otherwise specified, the amino acid sequences of peptides and proteins are shown from the N-terminus to the C-terminus and from the left to the right, with the N-terminus as the first residue. Ala: alanine residue; Asp: aspartic acid residue; Glu: glutamic acid residue; Phe: phenylalanine residue; Gly: glycine residue; His: histidine residue; Ile: isoleucine residue; Lys: lysine residue; Leu: leucine residue; Met: methionine residue; Asn: asparagine residue; Pro: proline residue; Gln: glutamine residue; Arg: arginine residue; Ser: serine residue; Thr: threonine residue; Val: valine residue; Trp: tryptophan residue; Tyr: tyrosine residue; Cys: cysteine ​​residue. Amino acids may also be represented by the following abbreviations of their conventional one-letter code: A=Ala; T=Thr; V=Val; C=Cys; L=Leu; Y=Tyr; I=Ile; N=Asn; P=Pro; Q=Gln; F=Phe; D=Asp; W=Trp; E=Glu; M=Met; K=Lys; G=Gly; R=Arg; S=Ser; and H=His.

[0123] peptide As used herein, peptide refers to a short chain of amino acid monomers, either natural, biological, or synthetic, linked by peptide (amide) bonds. Glutamine peptide refers to a short chain of amino acid monomers, either natural, biological, or synthetic, linked by peptide (amide) bonds, one of which is glutamine. Chemically synthesized peptides generally have free N- and C-termini. N-terminal acetylation and C-terminal amidation can reduce the overall charge of the peptide and therefore its overall solubility. However, terminal acetylation / amidation can also improve peptide stability by creating a closer mimic of a natural protein. These modifications can increase the biological activity of the peptide and are also provided herein.

[0124] Peptide synthesis Herein, peptides are synthesized by classically known chemical synthesis on a solid support (Ansynth BV, Roosendaal, The Netherlands) or in solution (Syncom BV, Groningen, The Netherlands and Diosynth BV, Oss, The Netherlands). Pharmaceutical peptide compositions may be synthesized using trifluoroacetate as a counterion or salt, which is then exchanged for a counterion such as maleate (from maleic acid), acetate (from acetic acid), tartrate (from tartaric acid), or citrate (from citric acid). The drug substance of AQGV (EA-230) for use in preclinical and clinical human studies is manufactured by Organon NV (formerly Diosynth BV), Oss, The Netherlands, while the filling and finishing of the final product is carried out by Octoplus Development in Leiden, The Netherlands. The molecular weight of EA-230 (AQGV) is 373 g / mol.

[0125] FPR-mediated vascular permeability and hypotension The concept that active endothelial cell contraction regulates vascular permeability was first proposed by Majno in 1961 (J Biochem Ytol (1961) 11:571.10.1083 / jcb.11.3.571). However, the intracellular events regulating endothelial contractile activity are currently relatively unknown. N-formyl peptides are a common molecular signature of bacteria and mitochondria that activate formyl peptide receptors (FPRs). FPR activation by mitochondrial N-formyl peptides (F-MIT) or bacterial N-formyl peptides (F-MLPs), such as N-formyl-methionyl-leucyl-phenylalanine, induces changes in cytoskeletal regulatory proteins in endothelial cells, leading to increased endothelial cell contractility accompanied by increased vascular leakage and increased leukocyte extravasation. FPR activation is a major cause of barrier dysfunction after trauma. It has been proposed that mitochondrial components from damaged tissues may initiate the development of vascular leakage in patients (Wenceslau et al., Front Immunol. 2016;7:297). For evolutionary reasons, mitochondria share several characteristics with bacteria, and when mitochondrial fragments are released into the circulation, they are recognized by cells carrying the formyl peptide receptor (FPR). Because protein translation is initiated by formyl-methionine in both bacteria and mitochondria, N-formyl peptides are a common molecular signature of bacteria and mitochondria and are known to play a role in initiating vascular leakage by activating the formyl peptide receptor (FPR).

[0126] The vascular system, composed of blood vessels of various forms and functions, distributes blood to all tissues and maintains physiological tissue homeostasis. Specifically, to explain its central role in maintaining homeostasis, the vascular system not only serves as the primary carrier of gas exchange from the lungs to tissues (e.g., oxygen (and vice versa, e.g., carbon dioxide)), but also transports nutrients from the intestine to the liver and from the liver to tissues, and metabolizes toxic by-products from tissues to the kidneys and from the kidneys to the urine for excretion.

[0127] In a variety of pathological conditions, the vasculature is often affected by and involved in the disease process.

[0128] This can result in, among other things, adverse vascular permeability with edema, adverse vascular leakage, adverse leukocyte extravasation, and hypotension, and also in the excessive formation of new, unstable, and highly permeable blood vessels with poor blood flow, which further promotes hypoxia and disease propagation. Chronic adverse vascular permeability can also promote cancer metastasis. Thus, there is a strong motivation to learn more about (and be able to modulate) the regulation of vascular permeability, an important aspect of vascular biology in health and disease.

[0129] Endothelial cells in different blood vessels and organs have different functions and morphologies (Aird WC. Molecular heterogeneity of tumor endothelium. Cell Tissue Res. 2009;335:271-81.), but generally serve to provide a barrier between blood and tissue. In certain organs, such as the brain and endocrine organs, endothelial cells exhibit certain morphological characteristics that reflect the need for communication between the organ and the circulation. In the brain, the vasculature forms a particularly strong barrier, the blood-brain barrier (BBB), protecting the brain parenchyma from harmful edema. In hormone-producing organs, such as the endocrine pancreas, endothelial cells display specialized fenestrae on their surface.

[0130] These are diaphragm-covered "holes" in the plasma membrane that allow very rapid exocytosis of hormones. In most organs, endothelial cells form a dynamic barrier between the blood and tissues. Under resting conditions, the vasculature continuously leaks solutes and small molecules but restricts the extravasation of larger molecules and cells. In many diseases, including cancer, the vascular barrier breaks down and leakage can increase and become chronic. Leakage of larger molecules and cells can result in edema, harmful extravasation of leukocytes, and hypotension, and often disease progression.

[0131] For example, it is well recognized that kinins, such as bradykinin, are involved in a series of physiological and sometimes pathological vascular responses that affect endothelial barrier function. Most of their actions are mediated by activation of two G protein-coupled receptors, designated B1 and B2. Activation of kinin receptors may play an important role in regulating atherosclerosis risk through promoting microangiogenesis, inhibiting vascular smooth muscle cell proliferation, coronary vasodilation, increasing local nitric oxide synthesis, or exerting antithrombotic effects. While the bradykinin B1 receptor (B1R) is typically absent under physiological conditions, it is highly inducible after tissue injury, stress, burns, and traumatic injury, as recently reported, for example, in COVID-19 disease.

[0132] Tissue injury-induced damage can result in a significant and time-dependent increase in des-Arg9 bradykinin (des-Arg9-BK) responsiveness, comparable to B1R mRNA expression, which induces activation of several members of the mitogen-activated protein kinase (MAPK) family, namely extracellular signal-regulated kinase (ERK) and p38 MAPK. Blockade of p38 MAPK, but not the ERK pathway, by selective inhibitors results in a significant reduction in the upregulated contractile response produced by the selective B1R agonist des-Arg9-BK and largely prevents the induction of B1R mRNA expression, enhancing the deleterious vascular permeability induced by tissue injury.

[0133] Among other stress stimuli, exposure to hypoxia as a result of impaired blood flow or impaired gas exchange between the alveoli and the surrounding capillaries also induces structural changes in the endothelial cell layer of blood vessels and alters their permeability and interaction with leukocytes and platelets. These structural changes again result in impaired endothelial cell barrier function, leading to deleterious effects on blood vessels, such as adverse vascular permeability with edema, vascular leakage, leukocyte extravasation, and hypotension (see also Figure 1), and may further impair gas exchange from the lungs to the blood and from the blood to the tissues, and vice versa.

[0134] Among the multiple well-characterized cytoskeletal changes in response to stress, one involves the reorganization of the actin cytoskeleton and the formation of stress fibers. Kayyali et al. (J Biol Chem (2002) 277(45):42596-602) described cytoskeletal changes in pulmonary microvascular endothelial cells in response to hypoxia and the potential mechanisms involved in this process. Hypoxia-induced actin redistribution appears to be mediated by downstream components of the MAPK p38, which is activated in pulmonary endothelial cells in response to hypoxia. Results indicate that the kinase MK2, a substrate of p38, becomes activated by hypoxia, resulting in the phosphorylation of one of its substrates, HSP27. As another example, F-actin reorganization is also an early event in burn-induced endothelial barrier dysfunction, and HSP27, a target of the p38 MAPK / MK2 pathway, plays an important role in actin dynamics. Because HSP27 phosphorylation is known to alter actin distribution and therefore cell contractility, Kayyali et al. provide that the p38-MK2-HSP27 pathway results in changes in vascular permeability due to actin redistribution, as observed, for example, in hypoxia.

[0135] Taken together, these results indicate that tissue injury stimulates the p38-MK2-HSP27 pathway, leading to significant changes in the actin cytoskeleton. It has also been previously shown that inhibition of the p38 MAPK pathway improves vascular dysfunction by significantly reducing endothelial cell contraction (Wang et al., APMIS (2014) 122(9):832).

[0136] Recently, another pathway, the PI3K / AKT / mTOR [phosphatidylinositol-3'-kinase (PI3K), protein kinase B (PKB or AKT), and mammalian target of rapamycin (mTOR)] pathway, has also been identified as essential for regulating endothelial cell contractility. Tsuji, Tamura, and Ogawa have indeed identified inhibitors of the PI3K-Akt pathway and mammalian target of rapamycin complex 1 (mTORC1) as potent inducers of endothelial cell elongation, which is necessary for the restoration of endothelial cell-controlled vascular permeability (Journal of Cell Science 2016 129: 1165-1178). Such elongation is necessary to bridge the gaps that form between endothelial cells when they contract following cytoskeletal reorganization by p38-MK2-HSP27 and / or PI3K / AKT / mTOR signaling. It is these gaps through which harmful leakage and extravasation occur that explain the resulting edema, vascular leakage, harmful extravasation of leukocytes, and loss of vascular fluid with the risk of hypotension (see again Figure 1).

[0137] Closure of these gaps is generally governed by the ratio of various angiogenic factors, such as angiopoietin-1 to angiopoietin-2, at sites of increased vascular permeability, whereby angiopoietin-2 generally induces endothelial cell apoptosis (promoting gap formation), and angiopoietin-1 counteracts gap formation by promoting endothelial cell elongation and gap closure. Inhibition of the p38 pathway, but not the ERK1 / 2 pathway, attenuates angiopoietin-2-mediated endothelial cell apoptosis (Li et al., Exp Ther Med. 2018 Dec;16(6):4729-4736. Published online 2018 Oct 1). In addition, the PI3K / AKT / mTOR pathway regulates the expression of other angiogenic factors, such as nitric oxide and angiopoietins (Karar and Mayti, Front. Mol. Neurosci., 02 December 2011, https: / / doi.org / 10.3389 / fnmol.2011.00051).

[0138] Thus, inhibiting signaling events in the p38 / p38-MK2-HSP27 and / or PI3K / AKT / mTOR pathways that signal cytoskeletal contraction reduces vascular permeability and thereby reduces harmful permeability and leakage, resulting in edema, vascular leakage, exacerbated leukocyte leakage, and vascular fluid loss with associated risk of hypotension. Methods and means for such inhibition are the object of the present invention.

[0139] Use of EA-230 in mitigating ventilation requirements and ventilation-associated lung injury in COVID-19 Infection with the SARS-CoV-2 virus, which causes COVID-19, is mostly mild or even uneventful in over 80% of infected people. The majority of patients experience an upper respiratory tract infection of the nose and throat accompanied by a dry cough, which usually resolves within 2 to 12 days, after which the virus will be eliminated from the body. These patients may or may not experience typical flu-like symptoms, such as fever, fatigue, headache, and muscle aches, during the period of viral infection. They may not require treatment with the AQGV-peptide of the present invention.

[0140] In the remaining cases, a distinct pathological state may develop (Figures 1, 2, and 3), often beginning in mild cases around the time when viral infection appears to be steadily declining due to the emergence of an immune response directed against the virus. Viral pneumonia appears first, increasing vascular permeability in the lungs (pulmonary phase), diffusely and massively affecting the deeper airways and alveoli of both lungs, resulting in reduced oxygen uptake and respiratory failure. This bilateral pneumonia can be rapidly followed by full-blown systemic disease with generalized fatigue, high fever, and ultimately organ failure (kidneys, liver, and heart). Patients with these symptoms and respiratory distress typically require hospitalization, admission to the intensive care unit (ICU), mechanical ventilation, and induced coma, even though death may occur in severe cases. These patients may be significantly helped by treatment with AQGV-peptide.

[0141] Increased vascular permeability leads to respiratory failure.

[0142] An immediate and serious complication of COVID-19 is the accumulation of fluid within the lungs, resulting in a significant decline in lung function (Figure 3). This pulmonary edema is caused by increased permeability of blood vessels within the lungs in response to the viral infection. Fluid from the vascular network surrounding the alveoli leaks into the lungs, destroying lung tissue and eliminating oxygen-transporting cells. The diffuse and massive increase in harmful fluid and necrotic (dead) cells in both lungs dramatically increases the distance oxygen must travel from the air to the blood through the lungs and vascular tissue, impeding the exchange of oxygen from the air to tissues throughout the body. Conversely, the diffusion of CO2 from the blood in the lungs to the air is also impeded. These patients typically develop acute respiratory failure in response to the intense dyspnea they experience, compensating for the oxygen deficit they experience.

[0143] COVID-19 in hospital cohorts

[0144] Approximately one-third of COVID-19 patients with the two comorbidities mentioned above (approximately 5–10% of patients currently infected with SARS-CoV-2) have severe illness requiring treatment in a hospital. In some countries, most of these patients are admitted to an intensive care unit (ICU). In other countries, a small group is selected for ICU treatment, while other patients are either expected to recover without intensive care or left to die (with only palliative care). Due to the exponential increase in infection rates seen in the pandemic, the number of patients admitted to a hospital or ICU at any given time can be enormous. As of the end of June 2020, COVID-19 had been confirmed in over 8 million cases worldwide, with a confirmed case fatality rate currently approaching 6%. There is an urgent need for effective treatment of this cohort of severely ill COVID-19 patients.

[0145] Ventilator-associated lung injury (VALI) and ventilator-induced lung injury (VILI)

[0146] Current management of severe COVID-19 is supportive, and respiratory failure is the leading cause of death (Ruan et al. Intensive Care Med. 2020; DOI: 10.1007 / s00134-020-05991-x). In the ICU, COVID-19 patients are typically connected to a mechanical ventilation system to facilitate breathing. However, mechanical ventilation itself can induce ventilator-assisted lung injury (VALI; www.ncbi.nlm.nih.gov / pubmed / 12559881) and ventilator-induced lung injury (VILI) with increased edema and worsening hypoxemia. VALI and VILI (https: / / www.ncbi.nlm.nih.gov / pubmed / 24283226) are recognized herein as playing distinct roles in accelerating the multiple organ failure associated with COVID-19. Patients requiring mechanical ventilation consume a disproportionate amount of medical resources, both in the ICU and after discharge. The short- and long-term mortality rates of these patients are high, and they suffer from a significant symptom burden for extended periods. Hospitalized survivors have significant functional and cognitive limitations and a high rate of rehospitalization. Some are at high risk of death even after discharge. Long-term hospitalization for PMV patients at high risk of mortality does not meet current cost-effectiveness criteria. Consequently, minimizing the need for mechanical ventilation, thereby minimizing the risk of VALI and VILI, may paradoxically be key to reducing mortality during COVID-19. Currently, no pharmacological methods to combat VALI of VILI, similar to the AQGV peptide provided herein, are available to address these issues.

[0147] The AQGV peptide EA-230 reduces harmful vascular fluid permeability.

[0148] Surprisingly, EA-230 has been found to effectively regulate vascular permeability. In particular, EA-230 significantly improves hemodynamic stability after open-heart surgery in humans, even in the absence of inflammatory activity in the patient. Permeability governs the amount of fluid that leaks from blood vessels. The administration of fluid therapy generally increases leakage. Based on patient observations in a Phase II study, a significant reduction in harmful fluid retention (fluid leakage due to fluid overload) was observed in patients treated with EA-230 (p=0.03). EA-230 was shown to be safe and well-tolerated throughout surgery. EA-230 administered intraoperatively showed significant improvements in postoperative patient recovery compared with placebo patients. Patients treated with EA-230 were discharged earlier from the intensive care unit (p=0.0232) and hospital (p=0.0015). EA-230 improved hemodynamic stability (p=0.006) and renal function (p=0.003). Patients' long-term recovery was significantly improved with EA-230. By improving vascular permeability, EA-230 can be used to reduce the development of harmful fluid in the lungs associated with COVID-19, thereby also reducing the need for mechanical ventilation, which has deleterious systemic effects.

[0149] The AQGV peptide EA-230 allows for point-of-care determination of its effect on the development of COVID-19.

[0150] Furthermore, EA-230 has a very short half-life, which facilitates intermittent administration of the drug at the bedside and determination of its actual effect in order to assess the patient's progress during treatment and make prompt decisions about continuing or discontinuing treatment. EA-230 showed a very short elimination half-life and a large distribution (LPS study: geometric mean and 95% confidence interval: 0.17 [0.12-0.24] hours and 2.2 [1.3-3.8] L / kg, respectively).

[0151] Respiratory failure is a common complication of COVID-19 and other respiratory diseases caused not only by influenza but also by coronaviruses such as SARS and MERS. This phenomenon became more widely known after the 2005 outbreak of the avian H5N1 influenza virus, also known as "bird flu," when high mortality rates were associated with uncontrollable systemic multiple organ failure. Now we have a SARS variant; what if we next have to face a MERS variant? Vaccines and antivirals may be different, but the fight against respiratory failure remains the same. The AQGV peptide can still be used.

[0152] Summary of the effects of AQGV peptide EA-230

[0153] Early administration led us to detect novel and truly beneficial effects of EA-230 on hemodynamics, renal function, ICU and hospital length of stay, including improved hemodynamic stability.

[0154] Treatment of patients with EA-230 during surgery significantly reduced the need for hemodynamic therapy (combined fluid therapy and blood pressure medications; p=0.006). In addition to these improved hemodynamics, EA-230 significantly improved renal function (as determined by its effect on glomerular filtration rate) and plasma levels of creatinine, a renal function biomarker (p=0.003). It also significantly reduced ICU recovery time and hospital length of stay. On average, patients treated with EA-230 required approximately 8 days of hospital care, compared with approximately 10 days for placebo-treated patients. Patients treated with EA-230 also had fewer readmissions than placebo-treated patients.

[0155] Effects of EA-230 in human patients A prospective, randomized, double-blind, placebo-controlled study was conducted, enrolling 180 elective patients undergoing on-pump coronary artery bypass grafting. Patients were randomly assigned in a 1:1 ratio to receive either EA-230 at 90 mg / kg / h or placebo, infused from the start of the surgical procedure until the end of cardiopulmonary bypass. The primary focus of this first-in-patient study was the safety and tolerability of EA-230. The primary efficacy endpoint was modulation of the inflammatory response by EA-230. The key secondary endpoint was the effect of EA-230 on renal function.

[0156] Design and configuration This study was a single-center, prospective, double-blind, placebo-controlled, randomized, single-dose phase II study with an adaptive design to evaluate the safety and immunomodulatory effects of EA-230 in patients undergoing coronary artery bypass grafting (CABG). 180 eligible patients were included and randomized 1:1 to receive either active agent or placebo treatment. This was a first-in-patient safety and tolerability study, and its primary efficacy endpoint was to evaluate the immunomodulatory effects of EA-230. The primary secondary efficacy endpoint was the effect of EA-230 on renal function. This study complied with the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines and is registered with clinicaltrials.gov under number NCT03145220.

[0157] Randomization and stratification Patients were randomly assigned to active or placebo treatment by unblinded, independent investigators. Investigators used Good Clinical Practice-approved data management software (Castor EDC, Amsterdam, The Netherlands) for this process. The Castor system applied stratified randomization to ensure equal distribution between active and placebo treatments for patients with known risk factors for adverse outcomes. Three strata were included: 1) CABG procedure; 2) estimated GFR ≤30, 31–90, and 90 ml / min / 1.73 m; 2 3) preoperative renal function less than 4 or more than 4 (Nashef et al. Eur J Cardiothora Surg 2012 Apr;41(4):734-44).

[0158] connectivity Double-blind conditions were maintained for all patients, investigators, and medical research team personnel involved in all blinded study procedures, data collection, and / or data analysis. Unblinded study personnel not involved in other study procedures prepared the study medication. Infusion systems and solutions for active and placebo treatments were identical in appearance and texture. Unblinding was approved by the sponsor after completion of the study, conduct of a blinded data review, and database lock.

[0159] research intervention An intravenous infusion of EA-230, 90 mg / kg / hour, or placebo was initiated at the moment of the initial surgical incision using an automatic infusion pump. The infusion rate was set at 250 mL / hour, and the infusion continued until cessation of CPB or after 4 hours of continuous infusion, whichever occurred first.

[0160] The EA-230 formulation was packaged in sterile 5 mL glass vials containing 1500 mg / vial dissolved in water for injection at a final concentration of 300 mg / mL with an osmolality of 800–1000 mOsm / kg. The placebo formulation consisted of sodium chloride diluted with water for injection and placed in an identical sterile 5 mL glass vial containing 29 mg / mL to achieve a solution of the same osmolality. EA-230 and placebo were prepared for continuous intravenous infusion at an osmolality of less than 400 mOsm / kg by adding the appropriate amount of EA-230 or placebo to 1000 mL of normal saline under aseptic conditions. Placebo treatment vials and active agent treatment vials were manufactured by HALIX BV (Leiden, The Netherlands).

[0161] Adverse events (AEs) All AEs were judged by the investigator according to severity ("mild, moderate, or severe") and their perceived relationship to the study drug ("definitely, probably, possibly, or unrelated / unlikely to be related") according to the Common Terminology Criteria of the Adverse Events guidelines 4.030. SAEs or SUSARs include death, life-threatening illness, persistent and / or significant disability and / or incapacity, and hospitalization and / or prolonged inpatient hospitalization.

[0162] Ethical considerations, data quality assurance and patient and public involvement This study was conducted in accordance with the ethical principles of the Declaration of Helsinki (ICH E6(R1), the Medical Research Involving Human Subjects Act, guidelines of Good Clinical Practice and European Directive (2001 / 20 / CE)). Informed consent was obtained before any study-specific procedures were performed. Data were treated confidentially and anonymously, and Good Clinical Practice standards were applied. The handling of patient data in this study complied with the Dutch Personal Data Protection Act (Dutch: Wet Bescherming Persoonsgegevens, WBP).

[0163] Patients and the public were not involved in the design and / or implementation of the study protocol.

[0164] Study results were distributed individually to all study participants. The burden of the intervention was assessed by independent ethical committees, the CMO and CCMO, which included lay members.

[0165] result When evaluating the data obtained during the clinical trial, it was surprising that no immunomodulatory effect was evident, since no significant differences in plasma levels were observed between the EA-230 and placebo groups for IL-8, IL-10, IL-1RA, IL-17, MCP-1, and ICAM, as well as for the other cytokines tested. This also applied to IL-6 plasma levels, the primary endpoint of the study.

[0166] Surprisingly, significantly fewer patients in the EA-230 treatment group suffered from fluid retention (see Table 1 below). Various parameters were further analyzed, and it was found that hemodynamic parameters (e.g., vasopressor use and / or fluid balance) and / or renal parameters were favorably affected by the use of EA-230 compared to placebo. The inventors conclude that the timing of EA-230 administration was too early, or at least not long enough during the hyperinflammatory state in CABG patients.

[0167] However, surprisingly, it was found that the length of stay in the ICU (intensive care unit) and also the length of hospital stay of patients treated with the AQGV peptide were significantly shortened, even though no immunomodulatory effect was observed. Detailed analysis of the parameters monitored in human subjects during the study revealed / discovered that the use of the AQGV peptide favorably regulated the hemodynamics of treated patients. It was also found that parameters related to renal function in human patients were shown to be significantly improved or maintained and not worsened, even though no immunomodulatory effect was observed in these patients. Parameters related to renal function and / or hemodynamics are generally monitored in patients and determine the length of stay in either the ICU or hospital. Therefore, the use of AQGV advantageously improves the parameters monitored in human patients, thereby making it possible to shorten the length of stay in either the ICU or hospital.

[0168] [Table 1] Table 1. Adverse events (AEs) in the EASI study AEs, serious adverse events (SAEs), and suspected unexpected serious adverse reactions (SUSARs) with differences between treatment groups are listed in the table. Significantly fewer (chi-squared P<0.05) adverse events were found in the EA-230 treatment group (217) than in the placebo treatment group (283). Significantly fewer patients in the EA-230 treatment group (n=2) suffered from fluid retention than in the placebo treatment group (n=11) (chi-squared P<0.05), p<0.05.

[0169] [Table 1]

[0170] [Table 2] Table 2. Mean age of patients, mean on-pump length of patients divided by pump length quartiles Q1, Q2, Q3 and Q4, and all patients tested (Q1–Q4).

[0171] [Table 2]

[0172] Hemodynamic stability during the study In general, vasopressor use was reduced in the EA-230 treated group.

[0173] Patients were divided into quartiles based on treatment duration. In Table 3 below, descriptive frequencies of two variables are shown: days of vasopression, and net fluid balance days 0-2 (first 72 hours).

[0174] Groups were divided into patients with and without acute kidney injury (AKI), and into untreated (placebo) and treated with EA-230 (active agent). EA-230 reduced net fluid balance in both patients with and without AKI. EA-230 reduced the need for vasopressors in patients with AKI.

[0175] [Table 3]

[0176] Fluid balance and vasopressor use with EA-230 treatment The effect of EA-230 versus placebo was tested in univariate and multivariate models (see Table 4 below).

[0177] Input / independent variable: treatment group (EA-230 or placebo). Output / dependent variables were: days in the first 72-hour fluid balance endpoint, vasoconstriction score, or pressor score (area under the curve). The effect of EA-230 versus placebo was tested in two combined variables: Model A: days in fluid balance + vasoconstriction in the first 72 hours, and Model B: fluid balance + pressor score AUC in the first 72 hours. The results of both multivariate models showed significant improvements in hemodynamic parameters in patients receiving EA-230. This was observed in Model A (days in fluid balance + vasoconstriction in the first 72 hours) p=0.006 and Model B (fluid balance + pressor score AUC in the first 72 hours) p=0.008. In the group of patients who did not exhibit AKI, the hemodynamic effect of EA-230 was also significantly better, indicating that improvement in hemodynamics may occur independently of renal failure.

[0178] [Table 4] Table 4. Goal-directed hemodynamic therapy with EA-230 Analyses are shown for Model A for the total group and for subgroups of acute kidney injury matching the RIFLE criteria: no AKI (placebo n=42, EA-230 n=50), risk (placebo n=31, EA-230 n=34), and injury (placebo n=16, EA-230 n=6). Corresponding p-values ​​are listed.

[0179] [Table 4]

[0180] Taken together, these results demonstrate that the use of EA-230 can improve and / or maintain hemodynamics in human patients, as assessed by, among other things, the duration of vasopressor use, the amount of vasopressor administered, and / or its effect on fluid balance. In particular, EA-230 improves hemodynamic stability in humans. Permeability governs the amount of fluid that leaks from blood vessels. Implementing fluid therapy generally increases leakage. Based on patient observations in a phase II study, we found a significant reduction in adverse fluid retention (fluid leakage) in patients treated with EA-230 (p=0.03). Contractility also governs tone. While often regulated by the administration of blood pressure medications, however, it can have significant adverse side effects. Based on patient observations in a phase II study, we found a significant reduction in the required use of blood pressure medications in the half of patients treated with EA-230 for the longest period (>156 minutes; p=0.093). The present inventors also determined the mean maximum concentration (mean Cmax) determined in vivo in humans for EA-230 in a Phase II clinical trial. The mean arterial Cmax was found to be: 30,500 ng / mL (range 12,500-57,500 ng / mL). The mean venous Cmax was found to be: 68,400 ng / mL (range 19,600-113,000 ng / mL). EA-230 has beneficial effects on renal function.

[0181] The effect of EA-230 on regulating the incidence of various stages of acute kidney injury (AKI) was investigated using the RIFLE criteria (RIFLE: risk, injury, failure, loss of kidney function, and end-stage kidney disease classification, Clin Kidney J. 2013 Feb;6(1):8-14). In the EA-230 group, the number of patients without AKI increased, while the number of patients in the RIFLE injury category decreased. Furthermore, the use of EA-230 significantly improved glomerular filtration rate (GFR). Creatinine clearance, a biomarker of renal function, was significantly improved in patients treated with EA-230. When renal function was taken into account, creatinine clearance was significantly improved with EA-230 when renal function was less than 60 mL / min. No difference was observed when renal function was greater than 60 mL / min. No differences were observed between groups when pre-treatment renal function was greater than 60 mL / min / 1.73 m. These results indicate that the use of EA-230 can improve and / or maintain renal function in human patients.

[0182] Length of stay in ICU, hospital, and readmission

[0183] The study investigated the impact on patients' length of stay in the ICU and hospital (inpatient care). Treatment with EA-230 significantly reduced ICU and hospital length of stay (LOS). ICU LOS and hospital LOS were reduced in the EA-230 group. Patients treated with EA-230 also demonstrated a significant (p=0.09) reduction in the number of readmissions up to 90 days after surgery (see Table 5 below).

[0184] [Table 5] Table 5. Number of readmissions in the EASI study (CABG study). Number of patients who had to be readmitted due to clinical illness in the post-procedure period. Readmission rates were scored for the 28-day period after surgery, the 29-90-day period after surgery, and the total period of the 90-day period after surgery. Readmissions were reduced in patients who received EA-230 (treatment group).

[0185] [Table 5]

[0186] Furthermore, in the AQGV-treated patient group, the number of patients suffering from AKI injury was reduced, and when patients did suffer from AKI injury, these patients did not have prolonged hospital stays, as observed in the placebo group, and their hospital stays were similar to those of patients without AKI or at risk for AKI.

[0187] Treatment with EA-230 shows a strong beneficial effect on recovery. EA-230-treated patients required significantly less hemodynamic therapy, had significantly faster recovery of renal function after surgery, and had shorter intensive care unit (ICU) and hospital stays compared to placebo-treated patients.

[0188] These novel hemodynamic effects of EA-230 are independent of EA-230's anti-inflammatory effects. In summary, the significant improvements in hemodynamic stability, renal function, and recovery in EA-230-treated patients are related to EA-230's novel effects on vascular permeability and vasoconstriction. EA-230 demonstrates significant improvements in patient recovery compared with placebo-treated patients. EA-230-treated patients are discharged earlier from the intensive care unit (p=0.0232) and hospital (p=0.0015). EA-230 improves hemodynamic stability (p=0.006) and renal function (p=0.003).

[0189] Although the primary endpoint of short-term reduction in inflammatory cytokines (IL-6) was not achieved, EA-230 significantly improved long-term patient recovery.

[0190] There was a significant improvement in hemodynamic stability (reduction in fluid therapy and blood pressure medications; p=0.006), a significant improvement in renal function (improved glomerular filtration rate reduces plasma creatinine; p=0.003), a significant reduction in patients with adverse fluid retention during recovery (2 cases with EA-230 vs. 9 cases with placebo; p=0.03), and a significant reduction in readmissions within 90 days of treatment (4 cases with EA-230 vs. 10 cases with placebo; p=0.09).

[0191] Further analysis of biomarkers associated with vasoconstriction and / or vasodilation

[0192] Given the observed effects on hemodynamics and pulmonary function, plasma samples will be further analyzed for selected biomarkers. Plasma samples from control patients and patients receiving EA-230 will be analyzed for the biomarkers endothelin-1, VEGF, angiotensin II, the ANG2 / ANG1 ratio, as well as cAMP and natriuretic peptides.

[0193] In vitro effects of EA-230 and AQGV analogs

[0194] In an in vitro transwell assay, the effects of the AQGV peptide (EA-230) and its analogs were tested on human endothelial cells. Briefly, endothelial cells were cultured in transwell culture dishes, and the medium was supplemented with the AQGV peptide and its analogs, or control compounds known to affect endothelial permeability, vasoconstriction, and / or vasodilation.

[0195] Suitable human endothelial cells are, for example, HUVEC (Park et al., Stem Cell Rev. 2 (2): 93-102, 2006; Jimenez et al., Cytotechnology 65, 1-14, 2012) and HMEC-1 (Ades EW, et al. J. Invest. Dermatol. 99(6): 683-690, 1992). The permeability of the endothelial layer is determined by measuring the permeation of macromolecules. Furthermore, biomarker levels are also determined in the culture medium. Experiments are performed, for example, as outlined in Cox et al., Shock, 43(4): 322-6; 2015. In the HUVEC permeability test, established human endothelial vascular cells (HUVECs), capable of lining blood vessels, were grown in sieve cell culture (i.e., n=5) in multiple test formats, allowing the determination of leak-through products in response to various test concentrations of EA-230 peptide or placebo control used, establishing the pharmacological parameters of EA-230 peptide effects on permeability in human cells with or without effectors such as thrombin, bradykinin, lipopolysaccharide (LPS), coronavirus spike protein, coronavirus nucleic acid, high mobility group box 1 (HMGB1) protein, and reversing these effects with AQGV-peptide.

[0196] Bravo et al. (J Pharmacol Toxicol Methods. 2018 Jan-Feb;89:47-53) also developed an impedance-based contraction assay using the xCELLigence RTCA MP system. This technique utilizes a specialized 96-well E-plate with gold microelectrode arrays printed within individual wells to monitor cell adhesion by recording electrical impedance in real time. Impedance change (percentage vs. control) can be used as a readout for cell contraction. Established human aortic smooth muscle cells (HaSMCs), capable of contracting blood vessels, were grown in cell culture (i.e., n=3) on gold electrodes in multiple test formats, allowing for electrical impedance measurements of endothelin-1-induced smooth muscle cell contraction in response to various test concentrations of EA-230 peptide or placebo control, establishing pharmacological parameters for EA-230 effects on contractility in human cells. Additionally, isolated aneurysm (n=3) / control (n=3) patient human aortic smooth muscle cells (APaSMCs) were grown in cell culture on gold electrodes in multiple test formats, allowing for electrical impedance measurements of ionomycin-induced smooth muscle cell contraction in patient versus control cells, depending on various test concentrations of EA-230 peptide or placebo control used, and the effects of EA-230 on patient cells were detected with or without effectors such as thrombin, bradykinin, lipopolysaccharide (LPS), coronavirus spike protein, coronavirus nucleic acid, and high mobility group box 1 (HMGB1) protein, and their reversal with AQGV-peptide.Similar studies were conducted using various test concentrations of EA-230 peptide or a placebo control to detect the effects of EA-230 in human lung organoid cultures with or without effectors such as thrombin, bradykinin, lipopolysaccharide (LPS), coronavirus spike protein, coronavirus nucleic acid, and high-mobility group box 1 (HMGB1) protein, and reversing these effects with AQGV-peptide.Similar studies were conducted using various test concentrations of EA-230 peptide or a placebo control to detect the effects of EA-230 in experimental mice equipped with the human ACE2 receptor with or without effectors such as thrombin, bradykinin, lipopolysaccharide (LPS), coronavirus spike protein, coronavirus nucleic acid, and high-mobility group box 1 (HMGB1) protein, and reversing these effects with AQGV-peptide.

[0197] An example of a pharmaceutical composition for use in a method for reducing the permeability of the endothelial layer of a blood vessel in a subject, the method comprising providing the endothelial layer with a substance that reduces the ratio of angiopoietin-2 to angiopoietin-1 at a site of increased permeability as a result of infection.

[0198] Example 1 AQGVLPGQ-Malate To prepare 1 L of composition, mix the following: AQGVLPGQ-Malate - 1.8 mol 0.9% NaCl - 1L

[0199] Example 2 LQGVLPGQ-Malate To prepare 1 L of composition, mix the following: LQGVLPGQ-Maleate - 1.8 mol 0.9% NaCl - 1L

[0200] Example 3 AQGLQPGQ-maleate To prepare 1 L of composition, mix the following: AQGLQPGQ-maleate - 1.8 mol 0.9% NaCl - 1L

[0201] Example 4 LQGLQPGQ-Malate To prepare 1 L of composition, mix the following: LQGLQPGQ-maleate - 1.8 mol 0.9% NaCl - 1L

[0202] Example 5 AQGV-Malate To prepare 1 L of composition, mix the following: AQGV-maleate - 1.8 mol 0.9% NaCl - 1L

[0203] Example 6 LQGVL-Malate To prepare 1 L of composition, mix the following: LQGVL-Malate - 1.8 mol 0.9% NaCl - 1L

[0204] Example 7 AQGLQ-Malate To prepare 1 L of composition, mix the following: AQGLQPGQ-maleate - 1.8 mol 0.9% NaCl - 1L

[0205] Example 8 LQGLQ-Malate To prepare 1 L of composition, mix the following: LQGLQ-Maleate - 1.8 mol 0.9% NaCl - 1L

[0206] Example 9 AQGVLPGQ-Acetate To prepare 1 L of composition, mix the following: AQGVLPGQ-acetate - 1.8 mol 0.9% NaCl - 1L

[0207] Example 10 LQGVLPGQ-Acetate To prepare 1 L of composition, mix the following: LQGVLPGQ-acetate - 1.8 mol 0.9% NaCl - 1L

[0208] Example 11 AQGLQPGQ-Acetate To prepare 1 L of composition, mix the following: AQGLQPGQ-acetate - 1.8 mol 0.9% NaCl - 1L

[0209] Example 12 LQGLQPGQ-Acetate To prepare 1 L of composition, mix the following: LQGLQPGQ-acetate - 1.8 mol 0.9% NaCl - 1L

[0210] Example 13 AQGV - Acetate To prepare 1 L of composition, mix the following: AQGV - Acetate - 1.8 mol 0.9% NaCl - 1L

[0211] Example 14 LQGVL-Acetate To prepare 1 L of composition, mix the following: LQGVL-acetate - 1.8 mol 0.9% NaCl - 1L

[0212] Example 15 AQGLQ-Acetate To prepare 1 L of composition, mix the following: AQGLQPGQ-acetate - 1.8 mol 0.9% NaCl - 1L

[0213] Example 16 LQGLQ-Acetate To prepare 1 L of composition, mix the following: LQGLQ-acetate - 1.8 mol 0.9% NaCl - 1L

[0214] Example 17 AQGVLPGQ-tartrate To prepare 1 L of composition, mix the following: AQGVLPGQ-tartrate - 1.8 mol 0.9% NaCl - 1L

[0215] Example 18 LQGVLPGQ-tartrate To prepare 1 L of composition, mix the following: LQGVLPGQ-tartrate - 1.8 mol 0.9% NaCl - 1L

[0216] Example 19 AQGLQPGQ-tartrate To prepare 1 L of composition, mix the following: AQGLQPGQ-tartrate - 1.8 mol 0.9% NaCl - 1L

[0217] Example 20 LQGLQPGQ-tartrate To prepare 1 L of composition, mix the following: LQGLQPGQ-tartrate - 1.8 mol 0.9% NaCl - 1L

[0218] Example 21 AQGV-tartrate To prepare 1 L of composition, mix the following: AQGV-tartrate - 1.8 mol 0.9% NaCl - 1L

[0219] Example 22 LQGVL-tartrate To prepare 1 L of composition, mix the following: LQGVL-tartrate - 1.8 mol 0.9% NaCl - 1L

[0220] Example 23 AQGLQ-tartrate To prepare 1 L of composition, mix the following: AQGLQPGQ-tartrate - 1.8 mol 0.9% NaCl - 1L

[0221] Example 24 LQGLQ-tartrate To prepare 1 L of composition, mix the following: LQGLQ-tartrate - 1.8 mol 0.9% NaCl - 1L

[0222] Example 25 AQGVLPGQ-citrate To prepare 1 L of composition, mix the following: AQGVLPGQ-citrate - 1.8 mol 0.9% NaCl - 1L

[0223] Example 26 LQGVLPGQ-citrate To prepare 1 L of composition, mix the following: LQGVLPGQ-citrate - 1.8 mol 0.9% NaCl - 1L

[0224] Example 27 AQGLQPGQ-citrate To prepare 1 L of composition, mix the following: AQGLQPGQ-citrate - 1.8 mol 0.9% NaCl - 1L

[0225] Example 28 LQGLQPGQ-citrate To prepare 1 L of composition, mix the following: LQGLQPGQ-citrate - 1.8 mol 0.9% NaCl - 1L

[0226] Example 29 AQGV-citrate To prepare 1 L of composition, mix the following: AQGV-citrate - 1.8 mol 0.9% NaCl - 1L

[0227] Example 30 LQGVL-citrate To prepare 1 L of composition, mix the following: LQGVL-citrate - 1.8 mol 0.9% NaCl - 1L

[0228] Example 31 AQGLQ-citrate To prepare 1 L of composition, mix the following: AQGLQPGQ-citrate - 1.8 mol 0.9% NaCl - 1L

[0229] Example 32 LQGLQ-citrate To prepare 1 L of composition, mix the following: LQGLQ-citrate - 1.8 mol 0.9% NaCl - 1L

[0230] SEQUENCE LISTING <110> Biotempt BV <120> METHODS AND MEANS FOR MODIFYING HEMODYNAMICS IN INFECTIONS <130> GAI22-64pc <140> PCT / NL2021 / 050223 <141> 2021-04-06 <150> US 63 / 005,999 <151> 2020-04-06 <150> US 63 / 045,737 <151> 2020-06-29 <150> US 63 / 085,771 <151> 2020-09-30 <160> 97 <170> PatentIn version 3.5 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> MPER sequence <400> 1 Lys Trp Pro Trp Ile Trp Leu 1 5 <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AQGV-peptide <400> 2 Ala Gln Gly Val 1 <210> 3 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> LQGV-peptide <400> 3 Leu Gln Gly Val 1 <210> 4 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AQGV analogue <400> 4 Ala Gln Leu Pro 1 <210> 5 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AQGV analogue <400> 5 Pro Leu Gln Ala 1 <210> 6 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AQGV analogue <400> 6 Leu Gln Gly Val 1 <210> 7 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AQGV analogue <400> 7 Leu Ala Gly Val 1 <210> 8 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AQGV analogue <400> 8 Pro Gln Val Gly 1 <210> 9 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AQGV analogue <400> 9 Pro Gln Val Ala 1 <210> 10 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AQGV analogue <400> 10 Pro Gln Val Arg 1 <210> 11 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AQGV analogue <400> 11 Val Gly Gln Leu 1 <210> 12 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AQGV analogue <400> 12 Leu Gln Pro Leu 1 <210> 13 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AQGV analogue <400> 13 Arg Gln Gly Val 1 <210> 14 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AQGV analogue <400> 14 Leu Gln Val Gly 1 <210> 15 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AQGV analogue <400> 15 Leu Gln Gly Ala 1 <210> 16 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AQGV analogue <400> 16 Leu Gln Gly Arg 1 <210> 17 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AQGV analogue <400> 17 Ala Gln Gly Ala 1 <210> 18 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AQGV analogue <400> 18 Gln Pro Leu Ala 1 <210> 19 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AQGV analogue <400> 19 Pro Gln Val Pro 1 <210> 20 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AQGV analogue <400> 20 Val Gly Gln Ala 1 <210> 21 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AQGV analogue <400> 21 Gln Val Gly Gln 1 <210> 22 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AQGV analogue <400> 22 Val Gly Gln Gly 1 <210> 23 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 23 Ala Gly Gln Val 1 <210> 24 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 24 Gln Gln Gly Val 1 <210> 25 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 25 Leu Gln Gly Val 1 <210> 26 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 26 Gly Gln Gly Val 1 <210> 27 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 27 Pro Gln Gly Val 1 <210> 28 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 28 Val Gln Gly Val 1 <210> 29 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 29 Ala Leu Gly Val 1 <210> 30 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 30 Leu Leu Gly Val 1 <210> 31 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 31 Gln Leu Gly Val 1 <210> 32 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 32 Gly Leu Gly Val 1 <210> 33 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 33 Pro Leu Gly Val 1 <210> 34 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 34 Val Leu Gly Val 1 <210> 35 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 35 Gln Leu Gly Val 1 <210> 36 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 36 Gln Ala Gly Val 1 <210> 37 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 37 Gln Gly Gly Val 1 <210> 38 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 38 Gln Pro Gly Val 1 <210> 39 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 39 Gln Val Gly Val 1 <210> 40 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 40 Leu Ala Gly Val 1 <210> 41 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 41 Leu Gly Gly Val 1 <210> 42 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 42 Leu Pro Gly Val 1 <210> 43 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 43 Leu Val Gly Val 1 <210> 44 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> SARS-COV-2 spike protein peptide-motif sequence <400> 44 Lys Trp Pro Trp Tyr Ile Trp Leu 1 5 <210> 45 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> SARS-COV-2 spike protein peptide-motif sequence variant <400> 45 Lys Trp Pro Trp Tyr Val Trp Leu 1 5 <210> 46 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> MPER peptide <400> 46 Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu 1 5 10 15 Gly Phe <210> 47 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> MPER peptide <400> 47 Lys Trp Pro Trp Tyr Val Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala 1 5 10 15 Ile Val <210> 48 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (8)..(8) <223> linked to maleate <400> 48 Ala Gln Gly Val Leu Pro Gly Gln 1 5 <210> 49 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (8)..(8) <223> Linked to maleate <400> 49 Leu Gln Gly Val Leu Pro Gly Gln 1 5 <210> 50 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (8)..(8) <223> linked to maleate <400> 50 Ala Gln Gly Leu Gln Pro Gly Gln 1 5 <210> 51 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (8)..(8) <223> linked to maleate <400> 51 Leu Gln Gly Leu Gln Pro Gly Gln 1 5 <210> 52 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (4)..(4) <223> linked to maleate <400> 52 Ala Gln Gly Val 1 <210> 53 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> linked to maleate <400> 53 Leu Gln Gly Val Leu 1 5 <210> 54 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> linked to maleate <400> 54 Ala Gln Gly Leu Gln 1 5 <210> 55 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> linked to maleate <400> 55 Leu Gln Gly Leu Gln 1 5 <210> 56 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (8)..(8) <223> linked to acetate <400> 56 Ala Gln Gly Val Leu Pro Gly Gln 1 5 <210> 57 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (8)..(8) <223> linked to acetate <400> 57 Leu Gln Gly Val Leu Pro Gly Gln 1 5 <210> 58 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (8)..(8) <223> linked to acetate <400> 58 Ala Gln Gly Leu Gln Pro Gly Gln 1 5 <210> 59 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (8)..(8) <223> linked to acetate <400> 59 Leu Gln Gly Leu Gln Pro Gly Gln 1 5 <210> 60 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (4)..(4) <223> linked to acetate <400> 60 Ala Gln Gly Val 1 <210> 61 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> linked to acetate <400> 61 Leu Gln Gly Val Leu 1 5 <210> 62 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> linked to acetate <400> 62 Ala Gln Gly Leu Gln 1 5 <210> 63 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> linked to acetate <400> 63 Leu Gln Gly Leu Gln 1 5 <210> 64 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (8)..(8) <223> linked to tartrate <400> 64 Ala Gln Gly Val Leu Pro Gly Gln 1 5 <210> 65 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (8)..(8) <223> linked to tartrate <400> 65 Leu Gln Gly Val Leu Pro Gly Gln 1 5 <210> 66 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (8)..(8) <223> linked to tartrate <400> 66 Ala Gln Gly Leu Gln Pro Gly Gln 1 5 <210> 67 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (8)..(8) <223> linked to tartrate <400> 67 Leu Gln Gly Leu Gln Pro Gly Gln 1 5 <210> 68 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (4)..(4) <223> linked to tartrate <400> 68 Ala Gln Gly Val 1 <210> 69 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> linked to tartrate <400> 69 Leu Gln Gly Val Leu 1 5 <210> 70 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> linked to tartrate <400> 70 Ala Gln Gly Leu Gln 1 5 <210> 71 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> linked to tartrate <400> 71 Leu Gln Gly Leu Gln 1 5 <210> 72 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (8)..(8) <223> linked to citrate <400> 72 Ala Gln Gly Val Leu Pro Gly Gln 1 5 <210> 73 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (8)..(8) <223> linked to citrate <400> 73 Leu Gln Gly Val Leu Pro Gly Gln 1 5 <210> 74 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (8)..(8) <223> linked to citrate <400> 74 Ala Gln Gly Leu Gln Pro Gly Gln 1 5 <210> 75 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (8)..(8) <223> linked to citrate <400> 75 Leu Gln Gly Leu Gln Pro Gly Gln 1 5 <210> 76 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (4)..(4) <223> linked to citrate <400> 76 Ala Gln Gly Val 1 <210> 77 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> linked to citrate <400> 77 Leu Gln Gly Val Leu 1 5 <210> 78 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> linked to citrate <400> 78 Ala Gln Gly Leu Gln 1 5 <210> 79 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> linked to citrate <400> 79 Leu Gln Gly Leu Gln 1 5 <210> 80 <211> 49 <212> PRT <213> Artificial Sequence <220> <223> CoV-229E spike protein <400> 80 Leu Gln Thr Leu Ile Asp Asn Ile Asn Ser Thr Leu Val Asp Leu Lys 1 5 10 15 Trp Leu Asn Arg Val Glu Thr Tyr Ile Lys Trp Pro Trp Trp Val Trp 20 25 30 Leu Cys Ile Ser Val Val Leu Ile Phe Val Val Ser Met Leu Leu Leu 35 40 45 Cys <210> 81 <211> 49 <212> PRT <213> Artificial Sequence <220> <223> CoV-NL63 spike protein <400> 81 Leu Gln Gly Leu Ile Asp Gln Ile Asn Ser Thr Tyr Val Asp Leu Lys 1 5 10 15 Leu Leu Asn Arg Phe Glu Asn Tyr Ile Lys Trp Pro Trp Trp Val Trp 20 25 30 Leu Ile Ile Ser Val Val Phe Val Val Leu Leu Ser Leu Leu Val Phe 35 40 45 Cys <210> 82 <211> 49 <212> PRT <213> Artificial Sequence <220> <223> CoV-HKU1 spike protein <400> 82 Ile Gln Glu Ser Ile Lys Ser Leu Asn Ser Ser Phe Ile Asn Leu Lys 1 5 10 15 Glu Ile Gly Thr Tyr Glu Met Tyr Val Lys Trp Pro Trp Tyr Ile Trp 20 25 30 Leu Leu Ile Val Ile Leu Phe Ile Ile Phe Leu Met Ile Leu Phe Phe 35 40 45 In the <210> 83 <211> 49 <212> PRT <213> Artificial Sequence <220> <223> CoV-OC43 spike protein <400> 83 Leu Gln Glu Pathway To Lys Val Leu Asn Gln Ser Tyr To Asn Leu Lys 1 5 10 15 Asp Ile Gly Thr Tyr Glu Tyr Tyr Val Lys Trp Pro Trp Tyr Val Trp 20 25 30 Leu Leu Ile Cys Leu Ala Gly Val Ala Met Leu Val Leu Leu Phe Phe 35 40 45 In the <210> 84 <211> 49 <212> PRT <213> Artificial Sequence <220> <223> MERS spike protein <400> 84 Leu Gln Gln Val Val Lys Ala Leu Asn Glu Ser Tyr Ile Asp Leu Lys 1 5 10 15 Glu Leu Gly Asn Tyr Thr Tyr Tyr Asn Lys Trp Pro Trp Tyr Ile Trp 20 25 30 Leu Gly Phe Ila Gly Leu Val Ala Leu Ala Leu Cys Val Phe Phe 35 40 45 In the <210> 85 <211> 49 <212> PRT <213> Artificial Sequence <220> <223> SARS-CoV spike protein <400> 85 Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln 1 5 10 15 Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp 20 25 30 Leu Gly Phe Ile Gly Leu Ile Ile Val Met Val Thr Ile Leu 35 40 45 Leu <210> 86 <211> 49 <212> PRT <213> Artificial Sequence <220> <223> SARS CoV-2 spike protein <400> 86 Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln 1 5 10 15 Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp 20 25 30 Leu Gly Phe Ile Gly Leu Ile Ile Val Met Val Thr Ile Met 35 40 45 Leu <210> 87 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> SARS-CoV-1 MPER peptide <400> 87 Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu 1 5 10 15 Gly Phe <210> 88 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> SARS-CoV-1 MPER peptide <400> 88 Lys Trp Pro Trp Tyr Val Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala 1 5 10 15 Ile Val <210> 89 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> SARS-CoV-1 Fusogenic sequence <400> 89 Lys Trp Pro Trp Tyr Val Trp Leu Gly Phe 1 5 10 <210> 90 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> SARS-CoV-2 Fusogenic sequence <400> 90 Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe 1 5 10 <210> 91 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Fusogenic sequence <400> 91 Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe 1 5 10 <210> 92 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> part of CoV-OC43 spike protein <400> 92 Met Asn Arg Leu Gln Glu Ala Ile Lys Val Leu Asn Gln Ser Tyr Ile 1 5 10 15 Asn Leu Lys Asp Ile Gly Thr Tyr Glu Tyr Tyr Val Lys Trp Pro Trp 20 25 30 Tyr Val Trp Leu 35 <210> 93 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> part of CoV-HKU-1 spike protein <400> 93 Met Asn Leu Ile Gln Glu Ser Ile Lys Ser Leu Asn Asn Ser Tyr Ile 1 5 10 15 Asn Leu Lys Asp Ile Gly Thr Tyr Glu Met Tyr Val Lys Trp Pro Trp 20 25 30 Tyr Val Trp Leu 35 <210> 94 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> part of CoV-229E spike protein <400> 94 Val Gln Lys Leu Gln Thr Leu Ile Asp Asn Ile Asn Ser Thr Leu Val 1 5 10 15 Asp Leu Lys Trp Leu Asn Arg Val Glu Thr Tyr Ile Lys Trp Pro Trp 20 25 30 Trp Val Trp Leu 35 <210> 95 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> part of CoV-NL63 spike protein <400> 95 Thr Val Glu Leu Gln Gly Leu Ile Asp Gln Ile Asn Ser Thr Tyr Val 1 5 10 15 Asp Leu Lys Leu Leu Asn Arg Phe Glu Asn Tyr Ile Lys Trp Pro Trp 20 25 30 Trp Val Trp Leu 35 <210> 96 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> part of SARS-CoV spike protein <400> 96 Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile 1 5 10 15 Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp 20 25 30 Tyr Val Trp Leu 35 <210> 97 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> part of SARS-CoV-2 spike protein <400> 97 Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile 1 5 10 15 Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp 20 25 30 Tyr Ile Trp Leo 35

Claims

1. An agent used to improve hemodynamic stability in a human subject receiving intrapulmonary fluid therapy in order to reduce infection-associated occurrences of harmful fluids in the lungs of the subject, wherein the agent comprises a peptide of 4 to 12 amino acids, and the peptide comprises a peptide consisting of the amino acid sequence of AQGV or a functional analog thereof.

2. The agent according to claim 1, wherein the functional analogue is selected from the group consisting of AQLP, PLQA, LQGV, LAGV, PQVG, PQVA, PQVR, VGQL, LQPL, RQGV, LQVG, LQGA, LQGR, AQGA, QPLA, PQVP, VGQA, QVGQ, VGQG, or other permutations of peptides consisting of 4 to 12 amino acids, particularly other permutations of peptides consisting of 4 to 12 amino acids composed of the amino acids of the above-mentioned tetrapeptides.

3. The agent according to claim 1 or 2, wherein the occurrence of the harmful fluid associated with infection is caused by a viral respiratory infection.

4. The agent according to any one of claims 1 to 3, wherein the outbreak of the harmful fluid associated with infection is caused by influenza or coronavirus.

5. The agent according to any one of claims 1 to 3, wherein the outbreak of the harmful fluid associated with infection is caused by a coronavirus.

6. The agent according to any one of claims 1 to 5, wherein the peptide is administered intravenously to the subject.

7. The agent according to claim 6, wherein the peptide is administered intravenously at a rate of at least 50 mg / kg / hour.

8. The agent according to claim 6 or 7, wherein the peptide is administered intravenously at a rate of at least 60 mg / kg / hour.

9. The agent according to any one of claims 6 to 8, wherein the peptide is administered intravenously for at least one hour.

10. The agent according to any one of claims 1 to 9, wherein the subject is a patient receiving mechanical ventilation.

11. The agent according to any one of claims 1 to 10, wherein the subject has experienced or is thought to have experienced COVID-19 or a similar infectious disease.

12. A pharmaceutical formulation comprising the agent according to any one of claims 1 to 11, for use in improving hemodynamic stability in a human subject in order to reduce infection-associated occurrences of harmful fluids in the lungs of the human subject.

13. The formulation according to claim 12, further comprising an additive suitable for parenteral administration.

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