Pharmaceutical preparation and stock solution

Abstract

To provide a method of treatment of a human subject considered in need of maintaining or improving hemodynamic stability.SOLUTION: The present invention provides a method of treatment comprising administering an AQGV peptide or a functional analogue thereof to a human subject, optionally having impaired kidney function, wherein the treatment of administering the AQGV peptide comprises maintaining or improving hemodynamic stability in the human subject, such as a human subject suffering or considered suffering from Clarkson's disease (CLS).SELECTED DRAWING: None

Description

[Background technology]

[0001] When a human subject suffers a severe trauma, particularly trauma caused by a medical intervention such as surgery, for example, open-heart surgery, the human subject or patient is admitted to an intensive care unit (ICU) where vital signs can be closely monitored. The patient receives medical treatment that allows for recovery, and if the vital signs are within acceptable boundaries, the patient can be released from the ICU and referred to standard in-hospital care. If the patient demonstrates stability with standard care, particularly if the patient demonstrates sufficient hemodynamic stability, the patient can be released from the hospital and returned home. The patient can then be readmitted to the hospital if the need arises, for example, because the patient's condition worsens. Any improvement in the aforementioned vital signs, i.e., any improvement in the patient's health and recovery that affects the patient's length of stay in the ICU, the length of stay with standard care in the hospital, and / or patient readmission, provides significant benefits to healthcare and patients in general. Therefore, any means and methods that improve the patient's health and recovery (recovery rate) are noteworthy.

[0002] In ICUs or hospitals, patients are often treated with infusion fluids, for example, with salt-containing aqueous solutions such as normal saline (e.g., 0.9% NaCl, also called saline) or any other solution suitable for infusion. Sometimes, medications may be added to these solutions if determined necessary for the patient. Typically, medication-containing aqueous solutions are given intravenously (iv) by continuous (intravenous) infusion or by intravenous bolus injection. Other routes of fluid therapy include intraperitoneal administration by infusion or bolus administration of the aforementioned fluids. Fluid balance (renal function) is one of the determining criteria for patient outcomes in ICUs. For example, hypervolemia is a medical condition characterized by excessive water retention or fluid retention, or generally referred to as water or fluid overload, in which the body has too much fluid. Infusion therapy can result in fluid overload. Fluid overload can occur in human subjects, and its symptoms include, for example, weight gain and edema.

[0003] Fluid overload and the resulting inadequate blood flow or hemodynamic instability, often accompanied by the recognized need for fluid and / or vasopressor therapy, occur relatively frequently in critically ill patients and are frequently the result of critical care interventions using intravenous fluid therapy. Despite the common perception that it is benign, fluid overload in critically ill patients is independently associated with increased morbidity and mortality. Fluid extravasation into the interstitial space due to capillary leakage can adversely affect multiple organ systems, with symptoms ranging from cognitive impairment, impaired cardiac contractility, and tissue edema in skin and muscle leading to delayed wound healing, pressure ulcers, and wound infections. In the lungs, fluid overload causes increased extravascular lung water volume, accompanied by increased work of breathing and impaired gas exchange, leading to hypoxemia. A particularly serious complication of fluid overload is renal damage. Fluid overload is known to prolong intensive care unit stays by 60% and hospital stays by 30%.

[0004] Fluid overload (FO) generally contributes to delayed ICU recovery and prolonged hospital stays, leading to increased healthcare resource utilization and costs. A recent US study (Child D et al., Clinicoecon Outcomes Res., 2015;7:1-8) estimated that total hospital costs per visit for the FO cohort were approximately $15,000 higher than for the non-FO cohort, averaging over $21,000. ICU costs for the FO cohort were over $5,000 higher than for the non-FO cohort. FO patients had a 16% higher mortality rate and a 31% longer hospital stay. This, more importantly, resulted in an almost 60% longer ICU stay, a significant increase in 30-day readmission, and increased mechanical ventilation compared to the non-FO cohort (all P<0.05). Diuretics are the most commonly used medications to treat clinically diagnosed fluid overload. However, there is no conclusive evidence that treatment with diuretics alters major outcomes such as survival to discharge or hospital stay.

[0005] The kidney is a highly vascular, encapsulated organ that is exquisitely sensitive to inadequate (insufficient or excessive) blood flow. The kidney is particularly sensitive to venous congestion, and studies have shown that reduced venous return causes greater renal damage than lack of arterial flow. Intravenous fluid infusion inevitably causes interstitial edema when it exceeds the lymphatic drainage capacity of the microcirculation. In the kidney, interstitial edema increases subcapsular and intracapsular pressure, leading to reduced forward renal arterial blood flow, reduced venous return, and reduced lymphatic drainage, ultimately causing tissue hypoxia and AKI. Inadequate urine volume in AKI can further exacerbate tissue edema, creating a vicious cycle.

[0006] Acute kidney injury is characterized by a rapid loss of kidney function. Furthermore, acute injury often progresses to a chronic state, ultimately leading to end-stage renal disease. These patients are considered critically ill and require dialysis or renal replacement therapy. AKI not only contributes to multiple organ failure in critically ill patients, but also directly impacts other organs and systems. More than three million patients suffer from AKI each year, with a mortality rate of up to 70%. AKI is directly associated with short- and long-term complications, and the condition is associated with a mortality rate of 40-70%. The mortality rate for AKI patients is approximately one in four. Currently, the only treatment options for AKI are dialysis and supportive care, which do not address the underlying cause, limit further damage, or prevent progression. There are currently no approved drugs to treat this condition. Summary of the Invention [Problem to be solved by the invention]

[0007] 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 when treated patients were compared to controls. Despite the lack of immunomodulatory effects, the inventors surprisingly discovered that analysis of the data from the clinical trials revealed new, previously unobserved, potentially beneficial properties that could be attributed to the AQGV peptide, properties that are clearly independent of the known, previously observed immunomodulatory effects.

[0008] Surprisingly, despite no observed immunomodulatory effects, patients treated with the AQGV peptide experienced a significant reduction in ICU (intensive care unit) stay and overall hospital stay. A thorough analysis of the parameters monitored in human subjects during this study revealed that the use of the AQGV peptide favorably modulated hemodynamics in treated patients. Despite the lack of any observed immunomodulatory effects of treatment with the AQGV peptide in these patients, it was shown that parameters related to renal function in human patients were either significantly improved or maintained at a functional level without deterioration. Parameters related to renal function and / or hemodynamics are typically monitored in patients to determine the length of stay in either the ICU or hospital. The use of the AQGV peptide or its functional equivalents favorably improved the monitored parameters in human patients, thereby enabling a reduction in the length of stay in either the ICU or hospital (see, e.g., Figures 5, 10, 15, and 16).

[0009] Thus, the present invention relates to the use of peptides, also referred to herein as AQGV peptides, and analogues (functional equivalents) thereof, for improving the clinical parameters of human patients admitted to hospital and / or intensive care, so that the time from admission to hospital and / or intensive care until discharge can be shortened. [Means for solving the problem]

[0010] In one embodiment, the present invention provides a method of treating a human subject believed to be in need of maintaining or improving hemodynamic stability, comprising administering to the subject an AQGV peptide, wherein the AQGV peptide is defined as a peptide comprising at least 50%, more preferably at least 75%, and most preferably 100% of amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R). In a preferred embodiment, the AQGV peptide comprises at least 50%, more preferably at least 75%, and most preferably 100% of amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), glycine (G), valine (V), leucine (L), and proline (P). In a more preferred embodiment, the present invention provides a method of treating a human subject believed to be in need of maintaining or improving hemodynamic stability, wherein the AQGV peptide comprises at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), leucine (L), and proline (P). In another embodiment, the present invention provides a method of treating a human subject believed to be in need of maintaining or improving hemodynamic stability, wherein the peptide comprises at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), glycine (G), and valine (V). The AQGV peptide preferably consists of at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A in single-letter code), glutamine (Q), glycine (G), and valine (V).

[0011] In another preferred embodiment, the present invention provides a method of treating a human subject believed to be in need of maintaining or improving hemodynamic stability, wherein the subject has undergone severe trauma, such as surgery. In another embodiment, the present invention provides a method of treating a human subject believed to be in need of maintaining or improving hemodynamic stability, wherein the subject is undergoing cancer treatment, such as treatment with an anti-neoplastic agent (e.g., chemotherapy and / or radiation therapy) or an immunomodulatory agent. In another embodiment, the present invention provides a method of treating a human subject believed to be in need of maintaining or improving hemodynamic stability, wherein the subject is believed to be suffering from capillary leak syndrome, such as that seen in adverse drug reactions. In another embodiment, the present invention provides a method of treating a human subject believed to be in need of maintaining or improving hemodynamic stability, wherein the human subject has organ dysfunction, particularly renal dysfunction. In another embodiment, the present invention provides a method of treating a human subject believed to be in need of maintaining or improving hemodynamic stability, wherein the method comprises reduced use of vasopressors. In another embodiment, the present invention provides a method of treating a human subject believed to be in need of maintaining or improving hemodynamic stability, wherein the method comprises reduced water intake. In another embodiment, the present invention provides a method of treating a human subject believed to be in need of a reduction in harmful vascular permeability, comprising administering to the subject a peptide comprising at least 50% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single-letter code A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R). In a preferred embodiment, the AQGV peptide comprises at least 50%, more preferably at least 75%, and most preferably 100% of the amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single-letter code A), glutamine (Q), glycine (G), valine (V), leucine (L), and proline (P).In a more preferred embodiment, the present invention provides a method of treating a human subject believed to be in need of a reduction in harmful vascular permeability, wherein the AQGV peptide comprises at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), leucine (L), and proline (P). In another embodiment, the present invention provides a method of treating a human subject believed to be in need of a reduction in harmful vascular permeability, wherein the peptide comprises at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), glycine (G), and valine (V). The AQGV peptide preferably consists of at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A in single-letter code), glutamine (Q), glycine (G), and valine (V).

[0012] In another embodiment, the present invention provides a method of treating a human subject believed to be in need of a reduction in harmful vascular permeability, the subject having undergone severe trauma, such as surgery, comprising administering an AQGV peptide to the subject. In another embodiment, the present invention provides a method of treating a human subject believed to be in need of a reduction in harmful vascular permeability, the subject having undergone cancer treatment, such as treatment with an anti-neoplastic agent or an immunomodulatory agent. In another embodiment, the present invention provides a method of treating a human subject believed to be in need of a reduction in harmful vascular permeability, the subject having a capillary leak syndrome, such as that seen in adverse drug reactions. In another embodiment, the present invention provides a method of treating a human subject believed to be in need of a reduction in harmful vascular permeability, the human subject having impaired renal function. In another embodiment, the present invention provides a method of treating a human subject believed to be in need of a reduction in harmful vascular permeability, the method comprising reduced use of vasopressors. In another embodiment, the present invention provides a method of treating a human subject believed to be in need of a reduction in harmful vascular permeability, the method comprising reduced water intake. In another embodiment, the present invention provides a method of treating a human subject believed to be in need of reducing harmful fluid retention, comprising administering to the subject an AQGV peptide comprising at least 50%, more preferably at least 75%, and most preferably 100% of amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R). In a preferred embodiment, the AQGV peptide comprises at least 50%, more preferably at least 75%, and most preferably 100% of amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), glycine (G), valine (V), leucine (L), and proline (P).In a more preferred embodiment, the present invention provides a method of treating a human subject believed to be in need of reducing harmful fluid retention, wherein the AQGV peptide comprises at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), leucine (L), and proline (P). In another embodiment, the present invention provides a method of treating a human subject believed to be in need of reducing harmful fluid retention, wherein the peptide comprises at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), glycine (G), and valine (V). The AQGV peptide preferably consists of at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A in single-letter code), glutamine (Q), glycine (G), and valine (V).

[0013] In another preferred embodiment, the present invention provides a method for treating a human subject believed to be in need of reducing harmful fluid retention, wherein the subject has undergone severe trauma, such as surgery. The AQGV peptide preferably consists of at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), glycine (G), and valine (V). The peptide is preferably AQGV.

[0014] In another embodiment, the present invention provides a method for treating a human subject believed to be in need of reducing harmful fluid retention, wherein the subject is undergoing cancer treatment, such as treatment with an anti-neoplastic or immunomodulatory agent. Preferably, the AQGV peptide consists of at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), glycine (G), and valine (V). Preferably, the peptide is AQGV.

[0015] In another embodiment, the present invention provides a method for treating a human subject believed to be in need of reducing harmful fluid retention, wherein the subject is believed to be suffering from capillary leak syndrome, such as that seen in adverse drug reactions. The AQGV peptide preferably consists of at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), glycine (G), and valine (V). The peptide is preferably AQGV. In another embodiment, the present invention provides a method for treating a human subject believed to be in need of reducing harmful fluid retention, wherein the human subject has impaired renal function. The AQGV peptide preferably consists of at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), glycine (G), and valine (V). The peptide is preferably AQGV. In another embodiment, the present invention provides a method for treating a human subject believed to be in need of reducing harmful fluid retention, the method comprising reduced use of vasopressors. The AQGV peptide preferably consists of at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), glycine (G), and valine (V). The peptide is preferably AQGV or, for example, a dimer, trimer, tetramer, or pentamer thereof. In another embodiment, the present invention provides a method for treating a human subject believed to be in need of reducing harmful fluid retention, the method comprising reduced water intake. The AQGV peptide preferably consists of at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), glycine (G), and valine (V).Preferably, the peptide is AQGV.

[0016] In another embodiment, the present invention provides a method for use in treating a human subject believed to be in need of maintaining or improving hemodynamic stability, wherein the AQGV peptide is a salt of an organic acid, preferably the organic acid is selected from the group consisting of maleic acid, acetic acid, tartaric acid, and citric acid. In another embodiment, the present invention provides a method for use in treating a human subject believed to be in need of maintaining or improving hemodynamic stability, wherein the AQGV peptide is a salt of an organic acid such as maleic acid, more preferably acetic acid, more preferably tartaric acid, and most preferably citric acid.

[0017] In another embodiment, the present invention provides a method for use in treating a human subject believed to be in need of a reduction in harmful vascular permeability, wherein the AQGV peptide is a salt of an organic acid, preferably the organic acid is selected from the group consisting of maleic acid, acetic acid, tartaric acid, and citric acid. In another embodiment, the present invention provides a method for use in treating a human subject believed to be in need of a reduction in harmful vascular permeability, wherein the AQGV peptide is a salt of an organic acid such as maleic acid, more preferably acetic acid, more preferably tartaric acid, and most preferably citric acid.

[0018] In another embodiment, the present invention provides a method for use in treating a human subject believed to be in need of reducing harmful fluid retention, wherein the AQGV peptide is a salt of an organic acid, preferably the organic acid is selected from the group consisting of maleic acid, acetic acid, tartaric acid, and citric acid. In another embodiment, the present invention provides a method for use in treating a human subject believed to be in need of reducing harmful fluid retention, wherein the AQGV peptide is a salt of an organic acid such as maleic acid, more preferably acetic acid, more preferably tartaric acid, and most preferably citric acid.

[0019] In another embodiment, the present invention provides a method for use in treating a human subject believed to be in need of maintaining or improving hemodynamic stability, wherein the AQGV peptide is provided from a stock solution, preferably an aqueous solution, of AQGV peptide-acetate, AQGV peptide-tartrate, or AQGV peptide-citrate, preferably comprising or prepared to contain 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 method for use in treating a human subject believed to be in need of maintaining or improving hemodynamic stability, wherein the AQGV peptide is provided from a stock solution of the AQGV peptide-tartrate or the AQGV peptide-citrate, wherein the concentration of the AQGV peptide ranges from 2 mol / L to 2.5 mol / L. In a more preferred embodiment, the present invention provides a method for use in treating a human subject believed to be in need of maintaining or improving hemodynamic stability, wherein the AQGV peptide is provided from a stock solution of the AQGV peptide-citrate, wherein the concentration of the AQGV peptide-citrate ranges from 2.5 mol / L to 3 mol / L. In a more preferred embodiment, the present invention provides a method for use in treating a human subject believed to be in need of maintaining or improving hemodynamic stability, wherein the AQGV peptide is provided from a stock solution of the peptide-citrate, wherein the concentration of the peptide-citrate ranges from 3 mol / L to 3.5 mol / L.In a more preferred embodiment, the present invention provides a method for use in treating a human subject believed to be in need of maintaining or improving hemodynamic stability, wherein the AQGV peptide is provided from a peptide-citrate stock solution, wherein the peptide-citrate concentration is in the range of 3.5 mol / L to 4.5 mol / L. In a more preferred embodiment, the present invention provides a method for use in treating a human subject believed to be in need of maintaining or improving hemodynamic stability, wherein the AQGV peptide is provided from a peptide-citrate stock solution, wherein the peptide-citrate concentration is in the range of 4.5 mol / L to 5.5 mol / L. In a more preferred embodiment, the present invention provides a method for use in treating a human subject believed to be in need of maintaining or improving hemodynamic stability, wherein the AQGV peptide is provided from a peptide-citrate stock solution, wherein the peptide-citrate concentration is 5.5 mol / L or greater. Preferably, the stock solution is an aqueous solution.

[0020] In another embodiment, the present invention provides a method for use in treating a human subject believed to be in need of a reduction in deleterious vascular permeability, wherein the AQGV peptide is provided from a stock solution, preferably an aqueous solution, of AQGV peptide-acetate, AQGV peptide-tartrate, or AQGV peptide-citrate, preferably comprising or prepared to contain 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 method for use in treating a human subject believed to be in need of a reduction in harmful vascular permeability, wherein the AQGV peptide is provided from a stock solution of the AQGV peptide-tartrate or the AQGV peptide-citrate, wherein the concentration of the AQGV peptide ranges from 2 mol / L to 2.5 mol / L. In a more preferred embodiment, the present invention provides a method for use in treating a human subject believed to be in need of a reduction in harmful vascular permeability, wherein the AQGV peptide is provided from a stock solution of the AQGV peptide-citrate, wherein the concentration of the AQGV peptide-citrate ranges from 2.5 mol / L to 3 mol / L. In a more preferred embodiment, the present invention provides a method for use in treating a human subject believed to be in need of a reduction in harmful vascular permeability, wherein the AQGV peptide is provided from a stock solution of the peptide-citrate, wherein the concentration of the peptide-citrate ranges from 3 mol / L to 3.5 mol / L.In a more preferred embodiment, the present invention provides a method for use in treating a human subject believed to be in need of a reduction in harmful vascular permeability, wherein the AQGV peptide is provided from a peptide-citrate stock solution, wherein the peptide-citrate concentration ranges from 3.5 mol / L to 4.5 mol / L. In a more preferred embodiment, the present invention provides a method for use in treating a human subject believed to be in need of a reduction in harmful vascular permeability, wherein the AQGV peptide is provided from a peptide-citrate stock solution, wherein the peptide-citrate concentration ranges from 4.5 mol / L to 5.5 mol / L. In a more preferred embodiment, the present invention provides a method for use in treating a human subject believed to be in need of a reduction in harmful vascular permeability, wherein the AQGV peptide is provided from a peptide-citrate stock solution, wherein the peptide-citrate concentration is 5.5 mol / L or greater. Preferably, the stock solution is an aqueous solution.

[0021] In another embodiment, the present invention provides a method for use in treating a human subject believed to be in need of reducing harmful fluid retention, wherein the AQGV peptide is provided from a stock solution, preferably an aqueous solution, of AQGV peptide-acetate, AQGV peptide-tartrate, or AQGV peptide-citrate, preferably comprising or prepared to contain 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 method for use in treating a human subject believed to be in need of a reduction in harmful fluid retention, wherein the AQGV peptide is provided from a stock solution of the AQGV peptide-tartrate or the AQGV peptide-citrate, wherein the concentration of the AQGV peptide ranges from 2 mol / L to 2.5 mol / L. In a more preferred embodiment, the present invention provides a method for use in treating a human subject believed to be in need of a reduction in harmful fluid retention, wherein the AQGV peptide is provided from a stock solution of the AQGV peptide-citrate, wherein the concentration of the AQGV peptide-citrate ranges from 2.5 mol / L to 3 mol / L. In a more preferred embodiment, the present invention provides a method for use in treating a human subject believed to be in need of a reduction in harmful fluid retention, wherein the AQGV peptide is provided from a stock solution of the peptide-citrate, wherein the concentration of the peptide-citrate ranges from 3 mol / L to 3.5 mol / L.In a more preferred embodiment, the present invention provides a method for use in treating a human subject believed to be in need of reducing harmful fluid retention, wherein the AQGV peptide is provided from a peptide-citrate stock solution, wherein the peptide-citrate concentration ranges from 3.5 mol / L to 4.5 mol / L. In a more preferred embodiment, the present invention provides a method for use in treating a human subject believed to be in need of reducing harmful fluid retention, wherein the AQGV peptide is provided from a peptide-citrate stock solution, wherein the peptide-citrate concentration ranges from 4.5 mol / L to 5.5 mol / L. In a more preferred embodiment, the present invention provides a method for use in treating a human subject believed to be in need of reducing harmful fluid retention, wherein the AQGV peptide is provided from a peptide-citrate stock solution, wherein the peptide-citrate concentration is 5.5 mol / L or greater. Preferably, the stock solution is an aqueous solution.

[0022] In another embodiment, the present invention provides a method for use in treating a human subject suffering from or suspected of suffering from Clarkson's disease (CLS), wherein the AQGV peptide is provided from a stock solution, preferably an aqueous solution, of AQGV peptide-acetate, AQGV peptide-tartrate, or AQGV peptide-citrate, preferably comprising or prepared to contain 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 method for use in treating a human subject suffering from or suspected of suffering from Clarkson's disease (CLS), wherein the AQGV peptide is provided from a stock solution of the AQGV peptide-tartrate or the AQGV peptide-citrate, wherein the concentration of the AQGV peptide ranges from 2 mol / L to 2.5 mol / L. In a more preferred embodiment, the present invention provides a method for use in treating a human subject suffering from or suspected of suffering from Clarkson's disease (CLS), wherein the AQGV peptide is provided from a stock solution of the AQGV peptide-citrate, wherein the concentration of the AQGV peptide ranges from 2.5 mol / L to 3 mol / L. In a more preferred embodiment, the present invention provides a method for use in treating a human subject suffering from or suspected of suffering from Clarkson's disease (CLS), wherein the AQGV peptide is provided from a stock solution of the peptide-citrate, the concentration of the peptide-citrate being in the range of 3 mol / L to 3.5 mol / L.In a more preferred embodiment, the present invention provides a method for use in treating a human subject suffering from or suspected of suffering from Clarkson's disease (CLS), wherein the AQGV peptide is provided from a peptide-citrate stock solution, wherein the peptide-citrate has a concentration ranging from 3.5 mol / L to 4.5 mol / L. In a more preferred embodiment, the present invention provides a method for use in treating a human subject suffering from or suspected of suffering from Clarkson's disease (CLS), wherein the AQGV peptide is provided from a peptide-citrate stock solution, wherein the peptide-citrate has a concentration ranging from 4.5 mol / L to 5.5 mol / L. In a more preferred embodiment, the present invention provides a method for use in treating a human subject suffering from or suspected of suffering from Clarkson's disease (CLS), wherein the AQGV peptide is provided from a peptide-citrate stock solution, wherein the peptide-citrate has a concentration of 5.5 mol / L or greater. Preferably, the stock solution is an aqueous solution.

[0023] In another embodiment, the invention provides an AQGV peptide for use in treating a human subject believed to be in need of maintaining or improving hemodynamic stability, said peptide comprising at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R); and more preferably, said AQGV peptide is selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), valine (V), leucine (L), and proline (P). and wherein at least 50%, more preferably at least 75%, and most preferably 100% of the amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single-letter code A), glutamine (Q), leucine (L), and proline (P); or wherein at least 50%, more preferably at least 75%, and most preferably 100% of the amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single-letter code A), glutamine (Q), glycine (G), and valine (V). Most preferably, the peptide consists of at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), and valine (V). Preferably, the peptide for use in treating a human subject believed to be in need of maintaining or improving hemodynamic stability is AQGV.

[0024] In another embodiment, the invention relates to an AQGV peptide for use in treating a human subject believed to be in need of a reduction in deleterious vascular permeability, said peptide comprising at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R); and more preferably, said AQGV peptide is selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), valine (V), leucine (L), and proline (P). The present invention provides a peptide in which at least 50%, more preferably at least 75%, and most preferably 100% of the amino acids in the AQGV peptide are selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), leucine (L), and proline (P), and the AQGV peptide comprises at least 50%, more preferably at least 75%, and most preferably 100% of the amino acids in the AQGV peptide are selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), and valine (V). Most preferably, the peptide consists of at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), and valine (V). Preferably, the peptide for use in treating a human subject believed to be in need of maintaining or improving hemodynamic stability is AQGV.

[0025] In another embodiment, the present invention provides an AQGV peptide treatment of a human subject believed to be in need of reducing harmful fluid retention, wherein the peptide comprises at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R), and more preferably at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), valine (V), leucine (L), and proline (P). The present invention provides a method for treating a cancer cell line comprising administering to a patient a cancer treatment ... Most preferably, the peptide consists of at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), and valine (V). Preferably, the peptide for use in treating a human subject believed to be in need of reducing harmful fluid retention is AQGV.

[0026] In another embodiment, the present invention provides an AQGV peptide treatment of a human subject believed to be in need of reducing harmful fluid retention, wherein the peptide comprises at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R), and more preferably at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), valine (V), leucine (L), and proline (P). The present invention provides a method for treating a cancer cell line comprising administering to a patient a cancer treatment ... Most preferably, the peptide consists of at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), and valine (V). Preferably, the peptide for use in treating a human subject believed to be in need of reducing harmful fluid retention is AQGV.

[0027] In another embodiment, the present invention provides an AQGV peptide according to the present invention, wherein the subject has undergone severe trauma, such as surgery.

[0028] In another embodiment, the invention provides a peptide according to the invention, wherein the subject is undergoing cancer treatment, such as treatment with an anti-neoplastic agent or an immunomodulatory agent. In another embodiment, the invention provides an AQGV peptide according to the invention, wherein the subject is thought to be suffering from capillary leak syndrome, such as that seen in adverse drug reactions.

[0029] In another embodiment, the present invention provides an AQGV peptide according to the present invention, wherein the human subject has impaired renal function. In another embodiment, the present invention provides an AQGV peptide according to the present invention, wherein use thereof comprises (results in) reduced use of vasopressors. In another embodiment, the present invention provides an AQGV peptide according to the present invention, wherein use thereof comprises (results in) reduced fluid intake. In another embodiment, the present invention provides an AQGV peptide according to the present invention, wherein the peptide is a salt of an organic acid, preferably the organic acid is selected from the group consisting of maleic acid, acetic acid, tartaric acid, and citric acid. In another embodiment, the present invention provides an AQGV peptide according to the present invention, wherein the AQGV peptide is a salt of an organic acid such as maleic acid, more preferably acetic acid, more preferably tartaric acid, and most preferably citric acid. In another embodiment, the present invention provides a method wherein the AQGV peptide is provided from a stock solution, preferably an aqueous solution, of the AQGV peptide (preferably, the AQGV peptide is AQGV peptide-acetate, AQGV peptide-tartrate, or AQGV peptide-citrate), preferably wherein the stock solution comprises or is prepared to contain 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 stock solution of the AQGV peptide-tartrate or the AQGV peptide-citrate, wherein the concentration of the AQGV peptide ranges from 2 mol / L to 2.5 mol / L, or more preferably, the stock solution of the AQGV peptide-citrate, wherein the concentration of the AQGV peptide ranges from 2.5 mol / L to 3 mol / L.In a more preferred embodiment, the present invention provides a stock solution of peptide-citrate, wherein the concentration of the peptide-citrate ranges from 3 mol / L to 3.5 mol / L. In a more preferred embodiment, the present invention provides a stock solution of peptide-citrate, wherein the concentration of the peptide-citrate ranges from 3.5 mol / L to 4.5 mol / L. In a more preferred embodiment, the present invention provides a stock solution of peptide-citrate, wherein the concentration of the peptide-citrate ranges from 4.5 mol / L to 5.5 mol / L. In a more preferred embodiment, the present invention provides a stock solution of peptide-citrate, wherein the concentration of the peptide-citrate is 5.5 mol / L or more. Preferably, the stock solution is an aqueous solution.

[0030] In another embodiment, the present invention provides a pharmaceutical formulation comprising an AQGV peptide according to the present invention. In another embodiment, the present invention provides a pharmaceutical formulation according to the present invention and at least one pharmaceutically acceptable excipient. Preferably, the formulation is a stock solution of the AQGV peptide. Preferably, the stock solution is an AQGV peptide-acetate, AQGV peptide-tartrate, or AQGV peptide-citrate stock solution, preferably an aqueous solution, and preferably the stock solution comprises or is prepared to contain 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 pharmaceutical formulation comprising a stock solution of the AQGV peptide-tartrate or the AQGV peptide-citrate, wherein the concentration of the AQGV peptide ranges from 2 mol / L to 2.5 mol / L. In a more preferred embodiment, the present invention provides a pharmaceutical formulation comprising a stock solution of the AQGV peptide-citrate, wherein the concentration of the AQGV peptide-citrate ranges from 2.5 mol / L to 3 mol / L. In a more preferred embodiment, the present invention provides a method for treating a human subject believed to be in need of maintaining or improving hemodynamic stability, wherein the AQGV peptide is provided from a stock solution of the peptide-citrate, wherein the concentration of the peptide-citrate ranges from 3 mol / L to 3.5 mol / L. In a more preferred embodiment, the present invention provides a pharmaceutical formulation comprising a stock solution of the AQGV peptide-citrate, wherein the concentration of the AQGV peptide-citrate ranges from 3.5 mol / L to 4.5 mol / L. In a more preferred embodiment, the present invention provides a pharmaceutical formulation comprising a stock solution of said AQGV peptide-citrate, wherein the concentration of said AQGV peptide-citrate ranges from 4.5 mol / L to 5.5 mol / L.In a more preferred embodiment, the present invention provides a pharmaceutical formulation comprising a stock solution of the AQGV peptide-citrate, the concentration of which is 5.5 mol / L or more, and the stock solution is preferably an aqueous solution.

[0031] In one embodiment, the use of the AQGV peptide and analogs thereof is for use in medical therapy to alter hemodynamics in human subjects, particularly in subjects with consequent renal dysfunction. In a further embodiment, the use in human subjects to alter hemodynamics involves reduced unwanted fluid retention (i.e., unwanted fluid overload) and / or reduced use of pressor / inotropic agents in human subjects, particularly in subjects with consequent renal dysfunction. In another embodiment, the use of the AQGV peptide and analogs thereof is for use in human subjects with capillary leak, particularly in subjects with consequent renal dysfunction.

[0032] In one embodiment, the AQGV peptide or a functional analog thereof is provided for use in the treatment of a human subject, including therapy for altering hemodynamics in a human subject. Hemodynamics involves the dynamics of blood flow, i.e., the physical factors governing blood flow through the human body. Hemodynamics in a human patient can be monitored, for example, by measuring blood pressure and / or fluid balance. When a human patient has low blood pressure and / or disturbed fluid balance, vasopressors or inotropes can be used and / or administered intravenously, for example. Inotropes and vasopressors are biologically and clinically important vasoactive agents that come from different pharmacological groups and act at some of the most fundamental receptors and signaling systems in the body. More than 20 of these agents are in common clinical use, and few reviews of their pharmacology exist outside of physiology and pharmacology textbooks. Despite their widespread use in critically ill patients, the clinical effects of these drugs in pathology are poorly understood. The adverse effects of vasopressors and inotropes depend on the mechanism of action. With beta-agonist drugs, arrhythmias are one of the most common adverse effects that one hopes to reduce.

[0033] The inventors have discovered that the use of the AQGV peptide or a functional analog thereof significantly improves hemodynamics in a human patient after trauma, 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 (as defined herein) improves hemodynamic stability in a human patient, as described herein. Altering or optimizing hemodynamics in a human subject is important after surgery or injury, for example, when the human subject suffers from trauma and / or blood loss. Thus, the AQGV peptide or an analog thereof can be advantageously used in hemodynamic therapy. Hemodynamic therapy, i.e., optimizing hemodynamics in a patient, includes perioperative hemodynamic therapy and / or goal-directed hemodynamic therapy. Such therapy may include fluid management and / or the use of vasopressors in the patient.

[0034] Functional AQGV analogs are defined herein as peptides that perform similar effects or functions as the AQGV peptides described herein, although not necessarily in terms of quantity. They may be used in accordance with the present invention as single peptides or in combination with other analogs and / or AQGV peptides in any desired ratio to adjust the half-life of the resulting mixture. Functional AQGV analogs may have sequence identity, i.e., may comprise at least a portion or the entire AQGV peptide. Preferably, functional AQGV analogs are structural analogs of the AQGV peptide. A preferred structural analog may be the LQGV peptide. A structural analog of the AQGV peptide may be selected from peptides containing amino acids selected from the group consisting of alanine (single-letter A), glutamine (Q), glycine (G), valine (V), leucine (L), and proline (P). A, Q, G, V, and L are preferred. A, Q, G, and V are most preferably 4-30 amino acids in length, preferably 4-12 amino acids, in any order and ratio relative to the others. In a preferred embodiment, the present invention provides structural analogs of AQGV that contain at least 50%, more preferably at least 75%, and most preferably 100% of amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single-letter code A), glutamine (Q), glycine (G), valine (V), leucine (L), proline (P), and arginine (R). While the ratio between amino acids can vary, the peptide must contain at least three different amino acids, and Q must be present. Preferably, structural analogs of the AQGV peptide have a length ranging from 4-30 amino acids, more preferably 4-12 amino acids. Preferably, such structural analogs are linear peptides. Suitable structural analogs of AQGV may have a length of less than four amino acids, e.g., three amino acids, but such lengths may require higher doses of such peptides because the half-life of such peptides is shorter and therefore more preferred. Longer structural analogs, e.g., those longer than 30 residues, are less preferred due to the potential immunogenicity of such longer 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, VGQL, LQPL, LQVG, LQGA, AQGA, QPLA, PQVP, VGQA, QVGQ, VGQG.

[0035] Vasopressors are a class of drugs that can increase hypotension. Some vasopressors act as vasoconstrictors, others sensitize adrenergic receptors to catecholamine-glucocorticoids, and others can increase cardiac output. Regardless of the vasopressor used, the present invention allows for a reduction in vasopressor use. A reduction in vasopressor use involves a reduction in the amount of vasopressor used, i.e., a reduction in the duration of vasopressor use and / or a reduction in the dose of vasopressor. Examples of vasopressors include epinephrine, noradrenaline, phenylephrine, dobutamine, dopamine, and vasopressin. Fluid management in a patient involves, for example, monitoring oral, enteral, and / or intravenous fluid intake and fluid output (e.g., urinary), followed by managing fluid intake in case, for example, fluid retention is observed (i.e., fluid intake exceeds fluid output, indicating a condition of fluid overload). Notably, the use of the AQGV peptide or its analogs can reduce fluid retention (also referred to herein as fluid overload). Thus, the AQGV peptide or its functional analogs can be used in addition to known interventions for improving hemodynamics in human patients, thereby resulting in a more rapid improvement in hemodynamics than would occur without the AQGV peptide or its analogs.

[0036] In another embodiment, the AQGV peptide or a functional analog thereof is provided for use in treating a human subject with impaired renal function. In a further embodiment, the impaired renal function is acute kidney injury (AKI). In one embodiment, the AQGV peptide or a functional analog thereof is provided for use in treating a human subject to improve renal function. Renal function can be assessed by determining the glomerular filtration rate (GFR), for example, by assessing the clearance of iohexol from plasma. Renal function can also be assessed by measuring the plasma level of creatinine and calculating the estimated GFR (eGFR) function, also known as the Modification of Diet in Renal Disease (MDRD) formula or equation, from the plasma level, taking into account patient characteristics such as gender, age, and race. Renal function can be assessed based on the GFR measurement (or its estimate based on MDRD) by applying the RIFLE criteria (see Figure 3). Having a RIFLE score, which is a stage of risk, injury, failure, loss, or ESKD, can indicate renal damage and / or impaired renal function. It is standard clinical practice to assess renal function in humans (e.g., by determining GFR, creatinine clearance, and / or eGFR / MDRD). Improvement in renal function compared to not receiving the AQGV peptide can include progression of renal function stage assessed under the RIFLE criteria to a less severe stage (e.g., progression of the patient from injury to at risk of injury or without AKI). Improvement in renal function also includes having an improvement in GFR or eGFR score. Regardless of which assessment is made, use of the AQGV peptide or analog thereof can improve renal dysfunction in subjects lacking immunomodulatory effects and / or renal function in humans with renal impairment.

[0037] The use of AQGV peptide can improve renal function and may also prevent the decline and / or impairment of renal function (see, e.g., Figures 6, 7, and 8). As a result, AKI may be prevented. Preferably, for prophylaxis of human subjects with impaired renal function, the AQGV peptide is administered at a rate of at least 50 mg / kg of patient body weight per hour (mg / kg / hr). Preferably, this administration rate is at least 60 mg, at least 70, at least 80, 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 AQGV peptide is administered at a rate of at least 70 mg / kg / hr for at least 1 hour, more preferably at least 1.5 hours, and most preferably at least 2 hours, although administration may be longer, such as more than 3 hours. The actual administration time may be determined by a physician. Thus, in one embodiment, the use of the AQGV peptide or a functional analog thereof allows for the maintenance of renal function in a human patient. Thus, the use of the AQGV peptide or an analog thereof allows for the protection of renal function in a human patient. In another embodiment, the use of the AQGV peptide or an analog thereof prevents the decline and / or impairment of renal function in a human patient. For example, a human patient who may be classified as having no AKI or at risk for renal impairment (such as AKI) may maintain that state instead of progressing to a more severe stage of renal dysfunction if such patient is treated with an AQGV peptide. Thus, a human patient at risk for developing renal impairment, e.g., due to (induced) trauma, may maintain their renal functional state as a result of treatment with an AQGV peptide or an analog thereof.

[0038] In another embodiment, an AQGV peptide or a functional analog thereof is provided for use in a human subject with impaired renal function, the use comprising altering hemodynamics in the human subject. Treatment of renal function and treatment of hemodynamic stability can be combined herein, and the use of an AQGV peptide or a functional analog thereof according to the present invention can be advantageously used to protect and / or improve renal function and alter hemodynamics. Such combined use can result, for example, in improved and / or maintained renal function, reduced use of vasopressors, and / or improved fluid management in the human subject (see, e.g., Tables 1, 3, 4, and Figures 14, 18, 19, and 20). Preferably, in treating a human subject with impaired renal function, the AQGV peptide is administered at a rate of at least 50 mg / kg of patient body weight per hour (mg / kg / hr). Preferably, this administration rate is at least 60 mg, at least 70, at least 80, or most preferably at least 90 mg / kg / hr. Preferably, the AQGV peptide is administered over a period of at least 1 hour, more preferably at least 1.5 hours, and most preferably at least 2 hours. Preferably, the AQGV peptide is administered at a rate of at least 70 mg / kg / hour over a period of at least 1 hour, more preferably at least 1.5 hours, most preferably at least 2 hours (such as at least 2.5 hours), more preferably at least 3.5 hours, and more preferably at least 4.5 hours.

[0039] In a further embodiment, the present invention provides for reduced use of vasopressors. Reduced use of vasopressors is understood to include reducing the amount of vasopressor used. Vasopressor use can be reduced by reducing the duration of vasopressor use. Vasopressor use can be reduced by reducing the amount of vasopressor (e.g., reducing the amount per dose and / or increasing the time interval between doses). Vasopressor use can be reduced by reducing the amount of vasopressor and the duration of vasopressor use. By reducing vasopressor use, the human subject advantageously recovers more quickly than a human subject not receiving the AQGV peptide or analog thereof. Preferably, for use in reducing the amount and / or duration of vasopressor use, the AQGV peptide is administered at a rate of at least 50 mg / kg patient body weight per hour (mg / kg / hr). Preferably, this administration rate is at least 60 mg, at least 70, at least 80, 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 AQGV peptide is administered at a rate of at least 70 mg / kg / hour for at least 1 hour, more preferably at least 1.5 hours, and most preferably at least 2 hours (e.g., 2.5 hours or more). In another embodiment, the use of the AQGV peptide or a functional analog thereof reduces harmful fluid retention in a human subject. Leaky capillaries, fluid retention, or fluid overload can occur in a human subject, symptoms of which include, for example, weight gain and edema. Fluid retention (otherwise known as swelling or edema) or capillary leak is the accumulation of fluid in the body. As fluid leaks from the bloodstream, blood volume and blood pressure can decrease. This can deprive tissues in the kidneys, brain, and liver of oxygen and nutrients necessary for the organs to function normally. Such swelling most often affects the appendages (such as the feet, ankles, and hands), but swelling can also affect other parts of the body, such as organ or abdominal cavities or the brain. The cause of swelling may be related to medications, heart disease, liver disease, or kidney failure.Cancer treatments, such as radiation therapy or some chemotherapy drugs, can cause fluid retention in the body. This form of cancer swelling is most noticeable in the feet, ankles, hands, and face. It is a vascular reaction that causes an increased ability of fluid in capillaries to "leak" through the skin layers, resulting in swelling. This occurs much less frequently than hives alone. Fluid retention commonly causes swelling in the tongue, lips, or eyelids. Airway swelling can lead to breathing difficulties, airway obstruction, and, in severe cases, death. Brain swelling is often associated with or follows blood-brain barrier dysfunction and edema formation after neurotrauma. Traumatic brain injury (TBI) is a leading cause of death and long-term disability in developing countries, particularly among young and elderly people. One of the major clinical problems associated with TBI, as well as other types of brain injuries such as subarachnoid hemorrhage or intracerebral hemorrhage, is the formation of cerebral edema (rapid swelling of neural tissue that may be fatal if uncontrolled).

[0040] Another concern is capillary leak syndrome (CLS), also known as systemic capillary leak syndrome (SCLS) or Clarkson's disease (CLS), a disorder characterized by frequent flare-ups of massive leakage of plasma from blood vessels into adjacent body cavities and muscles. This can lead to a sudden drop in blood pressure, which, if untreated, can lead to organ failure and death. Capillary leak syndrome (CLS) is a rare disease involving deep-seated vascular leakage, which is associated with a high mortality rate. It can also occur in cancer patients, and effective treatment strategies have yet to be established. CLS can occur spontaneously or secondary to autoimmune diseases, hematologic malignancies, snakebites, and medical treatments (e.g., chemotherapy and therapeutic growth factors). It has become increasingly clear in recent years that drugs harmfully associated with CLS are actually commonly used. There have been multiple reports of CLS as an adverse effect of anticancer drugs and therapies, and the incidence of CLS according to the type of anticancer drug has been systematically evaluated (PMID: 30691103). The majority of studies have reported on the incidence of CLS during interleukin-2 (IL-2) treatment, followed by anti-CD (anti-CD) agents. Furthermore, the use of IL-2 plus antineoplastic or immunomodulatory agents, including anticancer drugs and anticancer immunotherapies such as monoclonal antibodies (mAbs) such as imatinib mesylate and rituximab, has shown a dose-dependent increase in the incidence of CLS as an adverse event of anticancer treatment. Similarly, Clarkson's disease (CLS) is a common adverse response to experimental or investigational drugs and is commonly reported as a suspected adverse drug reaction (ADR) in human clinical trials.

[0041] Preferably, in treating a human subject with fluid retention, the AQGV peptide, as defined herein, is administered at a rate of at least 50 mg / kg of patient body weight per hour (mg / kg / hr). Preferably, this administration rate is at least 60 mg, at least 70, at least 80, 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 AQGV peptide is administered at a rate of at least 70 mg / kg / hr for at least 1 hour, more preferably at least 1.5 hours, most preferably at least 2 hours (such as at least 2.5 hours), more preferably at least 3.5 hours, and more preferably at least 4.5 hours. Thus, in one embodiment, the use of the AQGV peptide or an analog thereof allows for the treatment of fluid retention in a human subject. Thus, the use of the AQGV peptide or an analog thereof prevents fluid retention in a human subject. In a preferred embodiment, the use of the AQGV peptide or an analog thereof prevents fluid retention, such as Clarkson's disease (CLS), in human patients undergoing anti-cancer treatment, such as treatment with an anti-neoplastic or immunomodulatory agent.

[0042] In another embodiment, the AQGV peptide or its analog is used to prevent fluid retention in a human patient. For preventing fluid retention in a human subject, the AQGV peptide is administered at a rate of at least 50 mg / kg of patient body weight per hour (mg / kg / hr). Preferably, this administration rate is at least 60 mg, at least 70, at least 80, 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 AQGV peptide is administered at a rate of at least 70 mg / kg / hr for at least 1 hour, more preferably at least 1.5 hours, and most preferably at least 2 hours or at least 2.5 hours or more. In a preferred embodiment, the AQGV peptide or its analog is used to prevent fluid retention, such as Clarkson's disease (CLS), in a human patient undergoing anti-cancer treatment, such as treatment with an anti-neoplastic or immunomodulatory agent that affects capillary leakage. In another preferred embodiment, the use of the AQGV peptide or an analog thereof prevents fluid retention, such as in Clarkson's disease (CLS), in human patients with adverse drug reactions, such as after (experimental) treatment with drugs that affect capillary leakage, which are often antineoplastic or immunomodulatory drugs.

[0043] Fluid retention can be the result of reduced renal function and / or impaired hemodynamics. Therefore, because the use of the AQGV peptide can affect renal function and / or hemodynamics in a human subject, the use of the AQGV peptide can similarly affect fluid retention. Fluid retention can be the result of leaky capillaries. Therefore, the use of the AQGV peptide and / or its analogs can have an effect on capillary leakiness and potentially reduce plasma leakage from blood vessels into surrounding tissues and / or organs. Most preferably, edema can be reduced and / or avoided by the use of the AQGV peptide. Edema may also be referred to as harmful fluid retention because of its adverse effects on patients. Regardless of the cause of fluid retention, the use of the AQGV peptide and / or its functional analogs can improve fluid retention in a human subject, thereby alleviating symptoms associated with fluid retention, such as weight gain and edema, which may result in reduced use of diuretics. Preferably, for use in human subjects with fluid retention, the AQGV peptide, as defined herein, is administered at a rate of at least 50 mg / kg of patient body weight per hour (mg / kg / hr). Preferably, this administration rate is at least 60 mg, at least 70, at least 80, 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 AQGV peptide is administered at a rate of at least 70 mg / kg / hr for at least 1 hour, more preferably at least 1.5 hours, most preferably at least 2 hours (such as at least 2.5 hours), more preferably at least 3.5 hours, and more preferably at least 4.5 hours.

[0044] In another embodiment, the use of the AQGV peptide or its functional analog according to the present invention is not limited to patients with renal impairment and / or in need of hemodynamic therapy. The use of the AQGV peptide and / or its functional analog according to the present invention includes the treatment of human patients who are considered to be at risk of renal impairment and / or who are expected to require hemodynamic therapy. Such human patients include patients who are scheduled or expected to be sent to intensive care. Therefore, the use of the AQGV peptide or its functional analog includes use for induced trauma, such as surgery, as shown in the Examples. Induced trauma includes any physical injury to the human body and typically includes blood loss and / or damage to the tissues of a human subject. Induced trauma includes, for example, surgery. Thus, in a preferred embodiment, the induced trauma is surgery. The use of the AQGV peptide for induced trauma, such as surgery, can be before, during, and / or after surgery. It may be preferable to use the AQGV peptide or its analog during surgery. In particular, surgery may more preferably require cardiopulmonary bypass. Advantageously, the use of the AQGV peptide as shown in the examples improved GFR, particularly in patients undergoing prolonged cardiopulmonary bypass and thus receiving prolonged infusions of the AQGV peptide, i.e., for more than 2.5 hours. Thus, in a further embodiment, the use of the AQGV peptide or its analog is during cardiopulmonary bypass for more than 2.5 hours, wherein the AQGV peptide or its (functional) analog is administered during cardiopulmonary bypass. Preferably, the AQGV peptide is administered at a rate of at least 50 mg / kg of patient body weight per hour (mg / kg / hour). Preferably, this administration rate is at least 60 mg, at least 70, at least 80, or most preferably at least 90 mg / kg / hour. 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 AQGV peptide is administered at a rate of at least 70 mg / kg / hour for at least 1 hour, more preferably at least 1.5 hours, most preferably at least 2 hours or at least 2.5 hours, more preferably at least 3.5 hours, and more preferably at least 4.5 hours. Typically, administration of the AQGV peptide (or combination thereof) of the present invention may continue throughout the interventional procedure, and sometimes even thereafter. However, it is possible to determine during the intervention whether adjusting fluid balance in the subject being treated requires administration of a composition or formulation of the present invention, and to begin such administration during the intervention. In another or further embodiment, the AQGV peptide or functional analog thereof for use in accordance with the present invention is for use in a human subject with heart failure. Preferably, in a human subject with heart failure, the AQGV peptide is administered at a rate of at least 50 mg / kg of patient body weight per hour (mg / kg / hour). Preferably, this administration rate is at least 60 mg, at least 70, at least 80, or most preferably at least 90 mg / kg / hour. Preferably, the AQGV peptide is administered over a period of at least 1 hour, more preferably at least 1.5 hours, and most preferably at least 2 hours. Preferably, the AQGV peptide is administered at a rate of at least 70 mg / kg / hour over a period of at least 1 hour, more preferably at least 1.5 hours, most preferably at least 2 hours (such as at least 2.5 hours), more preferably at least 3.5 hours, and more preferably at least 4.5 hours.

[0045] In another embodiment, the use of the AQGV peptide or a functional analog thereof according to the present invention is not limited to patients with renal impairment and / or in need of hemodynamic therapy. The present invention includes the use of the AQGV peptide or a functional analog thereof for use in treating a human subject suffering from or suspected of being at risk for fluid overload, including altering hemodynamics in the human subject. The use of the AQGV peptide or a functional analog thereof according to the present invention includes the treatment of human patients suspected of being at risk for fluid overload and / or expected to require hemodynamic therapy. Such human patients include those who are scheduled or expected to be referred to intensive care. Thus, the use of the AQGV peptide or a functional analog thereof includes use for the prevention of induced fluid overload, such as that caused by fluid therapy, as demonstrated in the Examples. Preferably, for use in preventing induced fluid overload, the AQGV peptide is administered at a rate of at least 70 mg / kg / hour for at least 1 hour, more preferably at least 1.5 hours, most preferably at least 2 hours or at least 2.5 hours, more preferably at least 3.5 hours, more preferably at least 4.5 hours.

[0046] In another embodiment, the use of the AQGV peptide or its functional analog according to the present invention is not limited to patients with renal impairment and / or in need of hemodynamic therapy. The present invention includes the use of the AQGV peptide or its functional analog for use in treating a human subject believed to be in need of vasopressor / inotropic therapy, which use involves modifying hemodynamics in a human subject. The use of the AQGV peptide or its functional analog according to the present invention includes the treatment of human patients believed to be at risk from treatment with vasopressor or inotropic agents and / or expected to require hemodynamic therapy. Such human patients include those who are scheduled or expected to be admitted to intensive care. Thus, the use of the AQGV peptide or its functional analog includes the treatment of human patients believed to be at risk from treatment with vasopressor or inotropic agents, such as treatment with an agent selected from the group consisting of dopamine, dobutamine, adrenaline, noradrenaline, phenylephrine, vasopressin, and milrinone, as shown in the examples. Preferably, for use in treating a human patient believed to be at risk from treatment with a vasopressor or inotropic agent, the AQGV peptide is administered at a rate of at least 50 mg / kg of patient body weight per hour (mg / kg / hr). Preferably, this administration rate is at least 60 mg, at least 70, at least 80, 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 AQGV peptide is administered at a rate of at least 70 mg / kg / hr for at least 1 hour, more preferably at least 1.5 hours, most preferably at least 2 hours (such as at least 2.5 hours), more preferably at least 3.5 hours, and more preferably at least 4.5 hours.

[0047] In another embodiment, the use of the AQGV peptide or its functional analog according to the present invention is not limited to patients with renal impairment and / or requiring hemodynamic therapy. The present invention includes the use of the AQGV peptide or its functional analog for use in treating a human subject to improve, or even shorten, the subject's length of stay in an ICU, which use includes modifying hemodynamics in a human subject. The use of the AQGV peptide or its functional analog according to the present invention includes the treatment of human patients believed to be at risk from treatment with vasopressors or inotropic agents and / or expected to require hemodynamic therapy with fluid therapy. Such human patients include those who are, will be, or are expected to be admitted to intensive care, thereby shortening their ICU stay. Thus, the use of the AQGV peptide or its functional analog includes use for treating human patients believed to be at risk from treatment with vasopressors or inotropic agents and / or with fluid therapy, for example, as shown in the Examples. Preferably, for use in reducing a subject's length of stay in an ICU, particularly in a human patient considered at risk upon ICU admission, the AQGV peptide is administered at a rate of at least 50 mg / kg of patient body weight per hour (mg / kg / hr). Preferably, this administration rate is at least 60 mg, at least 70, at least 80, or most preferably at least 90 mg / kg / hr. Preferably, the AQGV peptide is administered over a period of at least 1 hour, more preferably at least 1.5 hours, and most preferably at least 2 hours. Preferably, the AQGV peptide is administered at a rate of at least 70 mg / kg / hr over a period of at least 1 hour, more preferably at least 1.5 hours, and most preferably at least 2 hours (such as at least 2.5 hours), more preferably at least 3.5 hours, and more preferably at least 4.5 hours.

[0048] In another embodiment, the use of the AQGV peptide or its functional analog according to the present invention is not limited to patients with renal impairment and / or requiring hemodynamic therapy. The present invention includes the use of the AQGV peptide or its functional analog for use in treating a human subject to improve (i.e., reduce) the subject's length of stay in a hospital, which use involves modifying hemodynamics in a human subject. The use of the AQGV peptide or its functional analog according to the present invention includes the treatment of human patients believed to be at risk from treatment with vasopressors or inotropic agents and / or expected to require vasopressors or inotropic agents and / or expected to require hemodynamic therapy with fluid therapy. Such human patients include those who are, will be, or are expected to be referred to intensive care or a hospital, thereby shortening the length of hospital stay. Thus, the use of the AQGV peptide or its functional analog includes use for the treatment of human patients believed to be at risk from treatment with vasopressors or inotropic agents and / or with fluid therapy, for example, as shown in the Examples. Preferably, for use in reducing a subject's length of stay in an ICU, particularly in a human patient considered at risk upon ICU admission, the AQGV peptide is administered at a rate of at least 50 mg / kg of patient body weight per hour (mg / kg / hr). Preferably, this administration rate is at least 60 mg, at least 70, at least 80, or most preferably at least 90 mg / kg / hr. Preferably, the AQGV peptide is administered over a period of at least 1 hour, more preferably at least 1.5 hours, and most preferably at least 2 hours. Preferably, the AQGV peptide is administered at a rate of at least 70 mg / kg / hr over a period of at least 1 hour, more preferably at least 1.5 hours, and most preferably at least 2 hours (such as at least 2.5 hours), more preferably at least 3.5 hours, and more preferably at least 4.5 hours.

[0049] The present invention also provides an AQGV peptide for use in treating a human subject believed to be in need of maintaining or improving hemodynamic stability, the AQGV peptide comprising at least 50% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R). In a preferred embodiment, the AQGV peptide comprises at least 50%, more preferably at least 60%, and most preferably at least 70% of amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), and leucine (L), which have been shown to have the best autophagy-inhibiting properties. In a more preferred embodiment, the AQGV peptide contains at most 30%, more preferably at most 20%, and most preferably at most 10% of amino acids selected from the group of autophagy-inhibiting amino acids consisting of glycine (G), valine (V), isoleucine (I), proline (P), and arginine (R). The inclusion of these amino acids is desirable to confer proteolytic susceptibility to the AQGV peptide, as needed. In one embodiment, the AQGV peptide preferably contains amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single-letter code A), glutamine (Q), glycine (G), and valine (V). To improve solubility, the AQGV peptide is preferably in the form of a salt selected from the group of AQGV peptide-acetate, more preferably AQGV peptide-tartrate, and most preferably AQGV peptide-citrate. AQGV peptides vary in length from 4 to 30 amino acids.

[0050] The present invention also provides an AQGV peptide for use in treating a human subject believed to be in need of reduced harmful vascular permeability, the AQGV peptide comprising at least 50% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R). In a preferred embodiment, the AQGV peptide comprises at least 50%, more preferably at least 60%, and most preferably at least 70% of amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), and leucine (L), which have been shown to have the best autophagy-inhibiting properties. In a more preferred embodiment, the AQGV peptide contains at most 30%, more preferably at most 20%, and most preferably at most 10% of amino acids selected from the group of autophagy-inhibiting amino acids consisting of glycine (G), valine (V), isoleucine (I), proline (P), and arginine (R). The inclusion of these amino acids is desirable to confer proteolytic susceptibility to the AQGV peptide, as needed. In one embodiment, the AQGV peptide preferably contains amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single-letter code A), glutamine (Q), glycine (G), and valine (V). To improve solubility, the AQGV peptide is preferably in the form of a salt selected from the group of AQGV peptide-acetate, more preferably AQGV peptide-tartrate, and most preferably AQGV peptide-citrate. AQGV peptides vary in length from 4 to 30 amino acids.

[0051] The present invention also provides an AQGV peptide for use in treating a human subject believed to be in need of reducing harmful body fluids, the AQGV peptide comprising at least 50% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R). In a preferred embodiment, the AQGV peptide comprises at least 50%, more preferably at least 60%, and most preferably at least 70% of amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), and leucine (L), which have been shown to have the best autophagy-inhibiting properties. In a more preferred embodiment, the AQGV peptide contains at most 30%, more preferably at most 20%, and most preferably at most 10% of amino acids selected from the group of autophagy-inhibiting amino acids consisting of glycine (G), valine (V), isoleucine (I), proline (P), and arginine (R). The inclusion of these amino acids is desirable to confer proteolytic susceptibility to the AQGV peptide, as needed. In one embodiment, the AQGV peptide preferably contains amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single-letter code A), glutamine (Q), glycine (G), and valine (V). To improve solubility, the AQGV peptide is preferably in the form of a salt selected from the group of AQGV peptide-acetate, more preferably AQGV peptide-tartrate, and most preferably AQGV peptide-citrate. AQGV peptides vary in length from 4 to 30 amino acids.

[0052] The present invention also provides an AQGV peptide for use in treating a human subject suffering from or suspected of suffering from Clarkson's disease (CLS), the AQGV peptide comprising at least 50% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R). In a preferred embodiment, the AQGV peptide comprises at least 50%, more preferably at least 60%, and most preferably at least 70% of amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), and leucine (L), which have been shown to have the best autophagy-inhibiting properties. In a more preferred embodiment, the AQGV peptide contains at most 30%, more preferably at most 20%, and most preferably at most 10% of amino acids selected from the group of autophagy-inhibiting amino acids consisting of glycine (G), valine (V), isoleucine (I), proline (P), and arginine (R). The inclusion of these amino acids is desirable to confer proteolytic susceptibility to the AQGV peptide, as needed. In one embodiment, the AQGV peptide preferably contains amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single-letter code A), glutamine (Q), glycine (G), and valine (V). To improve solubility, the AQGV peptide is preferably in the form of a salt selected from the group of AQGV peptide-acetate, more preferably AQGV peptide-tartrate, and most preferably AQGV peptide-citrate. AQGV peptides vary in length from 4 to 30 amino acids.

[0053] The present invention also provides a pharmaceutical formulation according to the present invention, comprising at least two different AQGV peptides, each of which contains at least 50% of amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R). In a preferred embodiment, the AQGV peptide contains at least 50%, more preferably at least 60%, and most preferably at least 70% of amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), and leucine (L), which have been found to have the best autophagy-inhibiting properties. In a more preferred embodiment, the AQGV peptide contains at most 30%, more preferably at most 20%, and most preferably at most 10% of amino acids selected from the group of autophagy-inhibiting amino acids consisting of glycine (G), valine (V), isoleucine (I), proline (P), and arginine (R). The inclusion of these amino acids is desirable to confer proteolytic susceptibility to the AQGV peptide, as needed. In one embodiment, the AQGV peptide preferably contains amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single-letter code A), glutamine (Q), glycine (G), and valine (V). To improve solubility, the AQGV peptide is preferably in the form of a salt selected from the group of AQGV peptide-acetate, more preferably AQGV peptide-tartrate, and most preferably AQGV peptide-citrate. AQGV peptides vary in length from 4 to 30 amino acids. Furthermore, the pharmaceutical formulation preferably contains one or more of the AQGV peptides at a concentration of at least 0.85 mol / L. In another embodiment, the pharmaceutical formulation contains at least one pharmaceutically acceptable excipient. An example of such a formulation is a stock solution of the AQGV peptides as provided herein.

[0054] The invention also provides the use of a formulation or solution according to the invention for use in a method of treating a human subject having or suspected of having Clarkson's disease (CLS), in a method of treating a human subject suspected of needing to maintain or improve hemodynamic stability, in a method of treating a human subject suspected of needing to reduce harmful vascular permeability, or in a method of treating a human subject suspected of needing to reduce harmful fluid retention.

[0055] Preferably, the use of the AQGV peptide or its functional analogue according to the present invention as described above involves administration of said 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 AQGV 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 provide additional benefits to the human subject, and may also contain other medicinal compounds (e.g., vasopressors, typically in reduced proportions).

[0056] Preferably, the AQGV peptide is administered at a rate of at least 50 mg / kg of patient body weight per hour (mg / kg / hr). Preferably, this administration rate is at least 60 mg, at least 70, at least 80, 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 AQGV peptide is administered at a rate of at least 70 mg / kg / hr for at least 1 hour, more preferably at least 1.5 hours, most preferably at least 2 hours (such as at least 2.5 hours), more preferably at least 3.5 hours, and more preferably at least 4.5 hours. Preferably, administration occurs during surgery. More preferably, administration occurs during the entire duration of surgery.

[0057] As shown in the Examples, the mean arterial maximum concentration (mean Cmax) determined 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 was 68,400 ng / ml, with a range of 19,600 to 113,000 ng / ml. Therefore, any means and methods may be used to administer EA-230 (or AQGV), but preferably means and methods that enable obtaining 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 are contemplated. Therefore, the route of administration need not necessarily be limited to intravenous administration, but may include other routes of administration that result in similar venous and / or arterial Cmax concentrations.

[0058] In another embodiment, the AQGV peptide or a functional analog thereof is provided for any use according to the present invention described above, wherein a human subject is admitted to intensive care and the use improves a measured parameter of the human subject, which parameter is determined to assess whether the human subject should remain in intensive care. As described above, parameters evaluated when a human patient is in intensive care include parameters related to renal function and hemodynamics. In any case, the use of the AQGV peptide or an analog thereof is intended to improve such parameters, thereby reducing the length of stay in the intensive care unit. Not only does the use of the AQGV peptide or an analog thereof reduce the length of stay in intensive care, but the effect of the use of the AQGV peptide or an analog thereof also reduces the length of stay in hospital and reduces hospital readmissions.

[0059] In any case, the use of the AQGV peptide or a functional analog thereof has a profound effect on renal function and / or hemodynamics in a human subject, thereby advantageously helping the human subject, for example, when undergoing an induced trauma, such as when undergoing cardiac surgery and being on a cardiopulmonary bypass pump. Thus, in one embodiment, the use of the AQGV peptide or a functional analog thereof is for use in cardiac surgery. In another embodiment, the use of the AQGV peptide or a functional analog thereof is for use in a human patient on a cardiopulmonary bypass pump.

[0060] Without being bound by theory, the effect of the AQGV peptide or its functional analogues may have an effect on vasoconstriction. Vasoconstriction involves the narrowing of blood vessels resulting from the contraction of the muscular layer of the blood vessel. Thus, in one embodiment, the use of the AQGV peptide or its functional analogues according to the present invention involves inducing vasoconstriction. In particular, the use of the AQGV peptide or its analogues may induce peripheral vasoconstriction and / or vasoconstriction in efferent arterioles in the kidney. Peripheral vasoconstriction may improve hemodynamics, while vasoconstriction in efferent arterioles may improve renal function. [Brief explanation of the drawings]

[0061] [Figure 1]Summary of the procedural timeline for the EASI trial (JMIR Res. Protol. 2019 Feb. 6; 8(2):e11441; doi:10.2196 / 11441) from enrollment to end of follow-up. The EASI trial was a prospective, randomized, double-blind, placebo-controlled trial that enrolled 180 elective patients undergoing on-pump coronary artery bypass grafting (CABG) with or without concomitant valve surgery. Patients were randomly assigned in a 1:1 ratio to receive EA-230 (90 mg / kg / h) or placebo, infused from the start of surgery until the end of cardiopulmonary bypass (CBP). Eighty-nine patients received placebo and 91 patients received EA-230, administered intravenously via continuous intravenous infusion over 2–4 hours. [Figure 2] Vasopressor requirements during the first 24 hours in the intensive care unit (ICU) after surgery are shown. In all groups (top panel), they also correlated with the duration of treatment, which was variable because the study drug infusion continued as long as the patient was on cardiopulmonary bypass (bottom panel). Adjustments to vasopressor use during the first 24 hours of ICU admission were determined and expressed as a composite inotropic score: (dopamine dose x 1 μg / kg / min) + (doptamine dose x 1 μg / kg / min) + (epinephrine dose x 100 μg / kg / min) + (norepinephrine dose x 100 μg / kg / min) + (phenylephrine x 100 μg / kg / min) + (vasopressin (mUnits / kg / min) * 10,000) + (milrinone x 10 mcg / kg / min) (Pediatric critical care medicine: a journal of the Society of Critical Care Medicine and the World Federation of Pediatric Intensive and Critical Care Medicine Societies. 2010 Mar;11(2):234-8. PMID:19794327. doi:10.1097 / PCC.0b013e3181b806fc. Data were log-transformed for repeated measures two-way analysis of variance. [Figure 3A] The effect of EA-230 on the incidence of different stages of acute kidney injury (AKI) was determined based on the RIFLE criteria (RIFLE: classification into risk, injury, failure, loss of kidney function, and end-stage kidney disease, Clin. Kidney J., 2013 Feb;6(1):8-14). In the EA-230 group, the proportion of patients without AKI increased, while the proportion of patients in the injury category of the RIFLE criteria decreased. In Figure 3A, the results were obtained based on the complete RIFLE score. In Figure 3B, the results were obtained by grouping based only on creatinine and GFR data. [Figure 3B] See legend to Figure 3A. [Figure 4] The effect of EA-230 on glomerular filtration rate (GFR) was determined by MDRD (Am. J. Kidney Dis. 2002 Feb;39(2 Suppl 1):S1-266). Treatment with EA-230, but not placebo, significantly improved GFR after surgery (day +1) compared with preoperative (day -1) GFR (left panel). The right panel shows that the MDRD effect converges after day 1 when EA-230 treatment is stopped. [Figure 5] Effect of EA-230 on plasma creatinine concentrations as a biomarker of renal function. Treatment with EA-230, but not placebo, showed significant improvement in creatinine levels after surgery (day +1) compared to day -1 (pre-surgery). [Figure 6] The effect of EA-230 on plasma creatinine was related to preoperative renal function. EA-230 significantly improved creatinine levels (p=0.012) when baseline renal function was less than 60 ml / min / 1.73 m2, but no statistically significant differences were observed between groups when renal function was greater than 60 ml / min / 1.73 m2 (Repeated measures two-way ANOVA). [Figure 7]The effect of EA-230 on GFR (MDRD) was related to preoperative renal function. When baseline renal function was less than 60 ml / min / 1.73 m2, treatment with EA-230, but not placebo, significantly (p=0.021) improved estimated GFR after surgery (day +1) compared with estimated GFR before surgery (day -1). When renal function was greater than 60 ml / min / 1.73 m2, no statistically significant differences were found between groups. Repeated measures two-way ANOVA. [Figure 8] The effect of EA-230 on MDRD was related to the duration of cardiopulmonary bypass (CPB) and, therefore, the duration of study drug infusion. Treatment with EA-230, but not placebo, significantly improved GFR after surgery (day +1) compared with preoperative (day -1) GFR in patients with a longer duration of cardiopulmonary bypass (>median length) (and, therefore, longer study drug infusion). No differences were observed between groups when the duration of CPB (and, therefore, the duration of study drug infusion) was short. Repeated measures two-way analysis of variance. [Figure 9] There is no immunomodulatory effect of EA-230. EA-230 was well tolerated and showed a favorable safety profile. However, treatment with EA-230 did not result in significant changes in plasma IL-6, the primary endpoint. In this figure, the results of the IL-6 study are shown for the full set of patients with short or long cardiopulmonary bypass durations. [Figure 10] Effect of EA-230 on Length of Stay. The EASI trial investigated patients' length of stay in the ICU and hospital (inpatient care). Treatment with EA-230 resulted in a significant reduction in ICU and hospital length of stay (LOS). ICU and hospital length of stay was reduced in the EA-230 group; 24 hours after ICU admission, 12% of patients in the EA-230 group were still in the ICU compared with 22% in the control group (p=0.02), and hospital length of stay was 195 [171-265] hours and 234 [192-295] hours in the EA-230 and placebo groups, respectively (p=0.002). Patients treated with EA-230 also showed a significant (p=0.09) reduction in readmissions up to 90 days after surgery. [Figure 11]Correlation between renal impairment stage, as assessed by RIFLE score, and hospital length of stay by treatment group (placebo or AQGV). Generally, literature suggests that more severe renal impairment is associated with longer hospital length of stay, as observed in the placebo group in this figure. This association was observed in the placebo group but not in the AQGV group, suggesting that AKI was resolved more quickly in the AQGV group. In conclusion, fewer patients developed AKI in the AQGV-treated group, but when patients did develop AKI, these patients did not require the prolonged hospital length of stay observed in the placebo group, and their length of stay was similar to that of patients without or at risk for AKI. [Figure 12] Treatment with EA-230 did not result in significant changes in plasma IL-6 overall, the primary endpoint. In this figure, the results of the IL-6 study are shown for the full set of patients with short or long cardiopulmonary bypass duration. [Figure 13] Overall, treatment with EA-230 did not result in significant changes in plasma IL-8, IL-10, IL-1RA, IL-17, MIP-1a, MIP-1b, MCP-1, ICAM, VCAM, or any of the other cytokines tested, and no immunomodulatory effects were observed. Treatment with EA-230 had no effect on iohexol clearance. [Figure 14] The need for treatment of hemodynamic instability by postoperative use of vasopressors (left) and by use of fluid therapy to adjust net fluid balance was significantly improved during the first 24 hours in the intensive care unit (ICU) in patients given EA-230 peptide. Concomitantly, EA-230 given intraoperatively significantly improved postoperative hemodynamic recovery, resulting in a significant improvement in hemodynamic stability (reduced need for complex treatment consisting of fluid therapy and blood pressure medications; repeated measures two-way ANOVA, p=0.006). [Figure 15]Treatment of CABG patients with EA-230 during surgery resulted in a highly significant, nearly 40% shorter stay in the intensive care unit (ICU). On average, placebo-treated CABG patients spent 40 hours in the ICU, while those treated with EA-230 were allowed to leave after 25 hours, freeing up valuable ICU space. [Figure 16] Patients treated with EA-230 demonstrated a highly statistically significant reduction in hospital length of stay of over 20%. On average, placebo-treated patients required approximately 12 days of ongoing care in the hospital recovering from CABG surgery, while those treated with EA-230 recovered and left the hospital 2.5 days earlier. In conclusion, treatment with EA-230 resulted in a highly significant reduction in postoperative length of stay for patients undergoing elective CABG surgery. Furthermore, this beneficial reduction in length of stay did not increase the risk of readmission; instead, EA-230 resulted in a significantly lower risk of 90-day readmission (4 for EA-230 vs. 10 for placebo, p=0.09). [Figure 17] The beneficial effect of long-term (longer than the median) treatment with EA-230 on renal function, measured as glomerular filtration rate (GFR), compared with patients treated for shorter than the median treatment period. Findings show a highly significant statistical improvement in post-operative renal function with long-term treatment of patients with EA-230 during elective surgery. [Figure 18] Beneficial effect of prolonged (longer than median) treatment with EA-230 on postoperative hemodynamic stability (measured as need for vasopressors / inotropes or fluid therapy) compared to patients treated for shorter than median treatment. Hemodynamic stability significantly benefits from prolonged use of EA-230 during surgery (POD = post-operative day). [Figure 19]Summary of inflammatory, renal, cardiovascular, and overall efficacy endpoint results. (a) Inflammation. Left panel: Plasma interleukin (IL)-6 concentrations over time from preoperative (baseline) to the morning after the following surgery (postoperative morning, POM) (p=0.99). The blue box indicates the period during which the study drug was administered. Right panel: Area under the plasma concentration-time effect curve (AUEC) of IL-6. Data are presented as median and interquartile range. P values ​​were calculated using a repeated measures two-way analysis of variance (ANOVA, interaction term, left panel) or the Mann-Whitney U test (right panel). (b) Renal. Left panel: Renal function, expressed as GFR iohexol and eGFRMDRD, from the day before surgery (baseline) to the following POM. Data are presented as mean and standard error of the mean. P values ​​were calculated using a repeated measures two-way analysis of variance (ANOVA, interaction term). Right panel: Classification of acute kidney injury (AKI) according to the RIFLE criteria. Patients were classified as "No AKI" (n=50 in the EA-230 group, n=42 in the placebo group), "Risk" (n=34 in the EA-230 group, n=31 in the placebo group), or "Injury" (n=6 in the EA-230 group, n=16 in the placebo group), and no patients were staged as "Failure," "Loss of function," or "End stage of renal disease." Data are presented as percentages of patients. P values ​​were calculated using Pearson's chi-square test. (c) Cardiovascular. Left panel: Net cumulative fluid balance during the first 24 hours after intensive care unit (ICU) admission (p=0.97). Right panel: Postoperative net fluid balance on postoperative day 1 (POD1) (n = 90 in the EA-230 group, n = 89 in the placebo group), POD2 (n = 90 in the EA-230 group, n = 89 in the placebo group), and POD3 (n = 86 in the EA-230 group, n = 85 in the placebo group). PODs 4-7 are not shown due to limited data availability. Data are presented as mean and standard error of the mean. P values ​​were calculated using a repeated measures two-way analysis of variance (ANOVA, interaction term, left panel) or Student's t-test (right panel). (d) General. Left panel: Length of stay in the ICU (p = 0.02).Right panel: Length of stay in hospital (p=0.001). P-values ​​were calculated using the log-rank test. CPB: cardiopulmonary bypass. (e) GFR: (estimated) glomerular filtration rate. MDRD: modification of diet in renal disease. pg: picograms. ml: milliliters. h: hours. min: minutes. m: meters. [Figure 20] Post-hoc analysis using subgroups (split using medians) for short (n = 90) and long (n = 89) duration of surgery. (a) Area under the plasma concentration-time effect curve (AUEC) of interleukin (IL)-6 with a test between treatment groups (short: EA-230 vs. placebo: p = 0.88, and long: EA-230 vs. placebo: p = 0.41). Data presented as median and interquartile range (IQR). (b) Daily net fluid balance (Short: p=0.54, p=0.33, p=0.75, and p=0.84 for first intensive care unit (ICU) day, postoperative day 1 (POD1), POD2, and POD3, respectively. Long: p=0.09, p=0.008, p=0.09, and p=0.89 for first ICU day, POD1, POD2, and POD3, respectively). Data are presented as mean and standard error of the mean (SEM). (c) Vasopressors and inotropes administered during the first 24 hours of ICU admission, shown as inotropic score (Short: p=0.28, Long: 0.048). Data are presented as median and IQR. (d and e) Renal function is shown as GFR iohexol (d) and eGFRmdrd (e) (short: GFR iohexol: p = 0.47, and eGFRmdrd: p = 0.27. long: GFR iohexol: p = 0.02, and eGFRmdrd: p < 0.0001). Data are presented as mean and SEM. P values ​​were calculated using the Mann-Whitney U test or Student's T test. *: p < 0.05. ♯: p < 0.1. h: time. [Figure 21]Renal function parameters. Blue boxes indicate the study drug administration period. (a) Plasma creatinine concentrations (p = 0.022) and corresponding eGFRMDRD (p = 0.663) from baseline (the day before surgery) to postoperative day 7 (POD). (b) Baseline and POD 1 were samples collected for the study; POD 2–7 measurements were additional samples extracted from the electronic patient record and were not available daily for all patients. n = 60, 48, 68, 21, 21, and 17 for POD 2, 3, 4, 5, 6, and 7, respectively. (c) Renal function is shown as endogenous creatinine clearance (GFRECC) (p = 0.74). Sample collection for this parameter occurred in the morning from the start of surgery through postoperative day 1. Urinary (d) creatinine (p = 0.029) and (e) urea (p = 0.004) concentrations. (f) Proenkephalin plasma concentration (p=0.53). Data for (a), (b), and (c) are presented as means ± standard error of the mean. Data for (d), (e), and (f) are presented as medians with interquartile ranges. P values ​​for (a), (b), (d), (e), and (f) were calculated using a repeated measures two-way analysis of variance (ANOVA, interaction term). P value for (c) was calculated using Student's t-test. CPB: cardiopulmonary bypass. POD: days after surgery. h: hours. (e) GFR: (estimated) glomerular filtration rate derived using the modification of diet in renal disease formula. [Figure 22]Urinary kidney injury markers per millimolar (mmol) creatinine (Cr) over time from preoperative (baseline) to the morning after surgery (POM): (a) interleukin (IL)-18 (p=0.78), (b) kidney injury molecule (KIM) 1 (p=0.21), (c) neutrophil gelatinase-associated lipocalin (NGAL) (p=0.92), (d) liver-type fatty acid-binding protein (L-FABP) (p=0.23), and (e) N-acetyl-β-D-glucosaminidase (NAG) (p=0.14). Blue boxes indicate the duration of study drug administration. Data are presented as medians with interquartile ranges. P values ​​were calculated using a two-way repeated measures analysis of variance (ANOVA, interaction term). ng: nanograms. CPB: cardiopulmonary bypass. μg: micrograms. h: hours. [Figure 23] Summary and results of dissolution experiments. [Figure 24] Based on the results shown in Figure 22, the concentration (aggregation point) of the screened neutral peptide salts below which the aggregated peptide salts become soluble was determined. It can be concluded that changing the anion significantly affects the solubility of AQGV. Compared to AQGV-acetate, higher solubility (solubility in 0.9% NaCl) and, therefore, higher aggregation points were observed for the AQGV-citric acid (AQGV-citrate) and AQGV-tartaric acid (AQGV-tartrate) salts, while the maleate and KHSO4 salts showed lower solubility. Using adenosine monophosphate or adenosine did not improve solubility. Citric acid appears to be a special case. Highly concentrated solutions did not crystallize or aggregate, but tended to form highly viscous solutions. DETAILED DESCRIPTION OF THE INVENTION

[0062] (Peptide synthesis) AQGV peptides are synthesized using, for example, classical solid-phase synthesis or other methods known in the art. Peptide purity is confirmed by high-performance liquid chromatography and / or fast atom bombardment mass spectrometry. Typically, peptides are defined as molecules consisting of between 2 and 50 amino acids, while proteins are composed of more than 50 amino acids. Peptides also tend to have less well-defined structures than proteins, which can adopt complex three-dimensional structures known as secondary, tertiary, and quaternary structures. A functional distinction may also be made between peptides and proteins. Indeed, most researchers, as well as the present application, use the term peptide to specifically refer to peptides or otherwise relatively short amino acids up to 50 amino acids, while the term polypeptide is used to describe proteins or chains of more than 50 or many more amino acids.

[0063] (Peptide administration) As outlined in the clinical trial protocol (Groenendael et al., JMIR Res Protoc, 2019 Feb;8(2):e11441), the test drug, EA-230, was dissolved in water for injection at a final concentration of 300 mg / mL with an osmolality of 800 to 1000 mOsm / kg and provided in sterile 5 mL glass vials at 1500 mg / vial. The placebo formulation consisted of sodium chloride diluted with water for injection at 29 mg / mL in the same sterile 5 mL glass vial to provide a solution with the same osmolality. EA-230 and placebo were prepared for 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.

[0064] (Necessity of stock solutions containing highly concentrated active substances) The vials containing the EA-230 formulation (stock solution) used in the clinical trials referenced herein contained 1.5 grams of EA-230, with each vial containing 5 ml of 300 mg / ml [(300 g / L = 0.8 mol / L) AQGV has a molecular weight of 373 g / mol]. In these trials, best treatment practices were established when the infusion of active substance was continued for at least 1.5 hours, preferably at least 2.5 hours, preferably at least 3.5 hours, and more preferably at least 4.5 hours at 90 mg / kg per hour. As a result, and depending on body weight, often more than 12-17 vials were required to maintain effective treatment, a requirement for administration that was too laborious in the operating room or ICU for the required care. This shortcoming of treatment with too weak a stock amount of EA-230 formulation highlighted the need to provide more and better concentrated stock solutions than those available.

[0065] (Determination of the coagulation point) It is recognized herein that many drug-like molecules can self-aggregate in aqueous media, and that aggregates have physicochemical properties that distort experimental results and clinical decisions. Peptide drug aggregation is one of the most common and troublesome processes encountered in almost all phases of biologic drug development. While aggregation can take several different forms, the term is used to describe a number of different processes during which peptide molecules associate to form larger species consisting of multiple polypeptide chains. Aggregates can be amorphous or highly structured, e.g., amyloid fibrils, and can form on surfaces or in solution upon absorption. Aggregates can arise as a result of noncovalent association of polypeptide chains or from covalent linkages between the chains. In some cases, aggregation is reversible; in others, it is virtually irreversible. In either case, aggregation reduces the physical stability of the peptide in question, leading not only to loss of activity but also to other potentially devastating problems, such as toxicity and immunogenicity.

[0066] Salts have complex effects on the physical stability of biomolecules, affecting both conformational and colloidal stability. These effects frequently vary according to the surface charge on the peptide, and the overall effect of a given salt on physical stability is a balance of the different mechanisms by which the salt interacts with water and biomolecules. Various salts can affect physical stability by altering the properties of the peptide-solvent system (Hofmeister effect) or by altering electrostatic interactions (Debye-Hückel effect).

[0067] We aimed to investigate the solubility of seven different salts of the prototype autophagy inhibitor peptide AQGV using a modified shake-flask method. First, AQGV-acetate was converted to the free base, extracted with an organic solvent, and concentrated in vacuo. Subsequently, citrate, maleate, sulfate (KHSO), adenosine monophosphate, adenosine salt, acetate, and tartrate salts were prepared and subsequently screened for their solubility.

[0068] (result) (Conversion to free base) Extraction of AQGV-acetic acid from the neutralized solution (pH = 6-7) with organic solvents proved impossible. Therefore, the aqueous solution of AQGV-acetic acid was transferred to an ion-exchange column (Amberlite, approximately 100 mL; IR120, H resin). The column was flushed with demineralized water followed by a 1N ammonia solution. The first three basic fractions were concentrated to give 4.7 g of free base AQGV (H-NMR).

[0069] (Solubility measurement) In a first attempt, the free base solution was mixed with the acid to obtain a concentrated DMSO solution of the salt, which was then diluted in water to determine solubility. However, the salts tested (adenosine and citric acid) were not soluble in DMSO at all. In fact, the mixture became clear after adding a small amount of water. Therefore, the solubility determination could not be performed as originally planned. Therefore, it was decided to determine the solubility of the required salt by diluting a known amount of the (insoluble) salt until a clear solution was obtained.

[0070] For citric acid, 1 mmol of AQGV and 1 mmol of citric acid were mixed in 0.5 mL of 0.9% NaCl. This resulted in a clear solution. More of both AQGV and citric acid materials were added (0.5 and 0.25 mmol amounts), resulting in a total of 2.75 mmol dissolved in 0.5 mL of 0.9% NaCl. The mixture remained clear but was very thick / viscous. The remaining experiments were performed differently, with 1 or 0.5 mmol of salt being weighed into a 4 mL vial and small amounts of 0.9% NaCl added until a clear solution was obtained, which remained clear for over a week. In the case of adenosine and adenosine monophosphate, no clear solutions were obtained.

[0071] Based on the results shown in Table 1, the neutral pH concentration (aggregation point, see Table 2) of the screened autophagy-inhibitory peptide salts was determined, below which the aggregated peptide salts became soluble. It can be concluded that changing the anion significantly affected the solubility of AQGV. Compared with AQGV-acetate (2 mol / L), higher solubility (solubility in 0.9% NaCl) and, therefore, higher aggregation points were observed for AQGV-citric acid (AQGV-citrate, >5.5 mol / L) and AQGV-tartrate (AQGV-tartrate) salts, whereas maleate and KHSO4 salts showed lower solubility. Using adenosine monophosphate or adenosine did not improve solubility. Citric acid appears to be a special case. Highly concentrated solutions did not crystallize or aggregate, but tended to form highly viscous solutions.

[0072] Due to the risk of aggregation, vials containing stock solutions of AQGV peptides for use in clinical trials have previously contained at most 0.8 mol / L of active substance in solution. According to the present invention, such stock solutions of AQGV salts of organic acids, in particular AQGV peptide-maleate, AQGV peptide-acetate, AQGV peptide-tartrate, or AQGV peptide-citrate stock solutions (but excluding adenosine or adenosine monophosphate), can now be prepared to comprise or contain 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 stock solution of the AQGV peptide-tartrate or the AQGV peptide-citrate, wherein the concentration of the AQGV peptide ranges from 2 mol / L to 2.5 mol / L. In a more preferred embodiment, the present invention provides a stock solution of the AQGV peptide-citrate, wherein the concentration of the AQGV peptide-citrate ranges from 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 ranges from 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 ranges from 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 ranges from 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.

[0073] The composition, structure, and function of proteins or peptides are described herein by reference to amino acids. Amino acid residues are referred to herein using the abbreviations described below. Unless otherwise specified, the amino acid sequences of peptides and proteins are identified from N-terminus to C-terminus, left to right, with the N-terminus being 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, and Cys: cysteine ​​residue. Amino acids are sometimes referred to by their conventional single-letter designations: 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.

[0074] (Inhibition of autophagy by selected amino acids) Autophagy is a degradation pathway that delivers extracellular and cytoplasmic materials to lysosomes via double-membrane vesicles called autophagosomes. Cytoplasmic components are trapped within autophagosomes, which then fuse with lysosomes, where the cargo is degraded. Extracellular materials are taken up by endocytosis or phagocytosis, which then fuse with lysosomes, where the cargo is also degraded. Autophagy is an important mechanism involved in many aspects of cellular function, including cellular metabolism and energy balance, and alterations in autophagy have been linked to various human pathological processes. Autophagy is a natural mechanism by which cells remove and degrade cellular components via autolysosomes.

[0075] While the role of autophagy in maintaining tissue homeostasis is relatively well described in a recent review (Cell., July 2019;8(7)), its role during tissue repair and regeneration has only recently begun to be appreciated. The present invention demonstrates that AQGV peptides, i.e., peptides enriched with distinctive amino acids or their combinations, better control the balance between proteogenesis (mTOR kinase activity) on the one hand and proteolysis (autophagy) on the other. It also identifies peptides enriched with autophagy-inhibiting amino acids as better enhancers of proteogenesis, underlying tissue repair, than other peptides without such amino acids. The target of rapamycin complex 1 (mTORC1) is a central regulator of cell and organismal growth, and this pathway has been implicated in the pathogenesis of many human diseases. mTORC1 promotes cell and tissue growth in response to the availability of nutrients, such as amino acids, which drive mTORC1 to the lysosomal surface, where it is activated. Recent and older data identify leucine (L), valine (V), isoleucine (I), alanine (A), glutamine (Q), arginine (R), glycine (G), and proline (P), alone or in combination, as more potent activators of mTOR or inhibitors of autophagy than other amino acids, such as glutamic acid (E), threonine (T), serine (S), lysine (K), threonine (T), phenylalanine (F), tyrosine (Y), and methionine (M), which have been reported to have no or opposite effects. Thus, as provided herein for inclusion in the AQGV peptides of the present invention, peptides augmented with leucine (L), valine (V), isoleucine (I), alanine (A), glutamine (Q), arginine (R), glycine (G), proline (P), alone or (preferably) in combination, are the most preferred activators of mTOR or inhibitors of autophagy for use in human cells, with respect to packaging and targeting into cells.Preferably, the AQGV peptide comprises at least 50%, more preferably at least 75%, and most preferably 100% of amino acids selected from the group consisting of A, Q, G, V, L, P, I, and R. Preferably, the AQGV peptides provided herein have a length ranging from 4-12 amino acids, more preferably 4-8 amino acids. Preferably, such AQGV peptides are linear peptides. More preferably, functional AQGV peptide analogs of the present invention may be selected from the group consisting of peptides comprising a dipeptide sequence selected from the group consisting of AQ, LQ, PQ, VQ, and GQ. More preferably, functional AQGV peptides of the present invention may be selected from the group consisting of peptides comprising a tripeptide sequence selected from the group consisting of AQL, LQL, PQL, VQL, GQL, PLQ, LQG, PQV, VGQ, LQP, LQV, AQG, QPL, PQV, VGQ, and GQG. Amino acids such as leucine (L), alanine (A), glutamine (Q), and proline (P) have been reported to have the most potent inhibitory effects on mTOR-associated autophagy in human cells (AJ Meijer et al., Amino Acids, 2015, 47, 2037-2063). Glycine (G; Zhong Z, Wheeler MD, Li X, Froh M, Schemmer P, Yin M, Bunzendaul H, Bradford B, Lemasters JJ. l-Glycine: a novel antiinflammatory, immunomodulatory, and cytoprotective agent. Curr Opin Clin Nutr Metab Care 6:229) ameliorates amino acid stimulation of mammalian target of rapamycin (mTOR) complex 1 activation. Thus, as provided herein for inclusion in the AQGV peptides of the present invention, leucine (L), alanine (A), glutamine (Q), glycine (G), and proline (P), alone or (preferably) in combination, are preferred activators of mTOR or inhibitors of autophagy for use in human cells.More preferably, the AQGV peptide comprises at least 50%, more preferably at least 75%, and most preferably 100% of the amino acids selected from the group A, Q, G, V, L, and P. In a more preferred embodiment, the AQGV peptide comprises at least 75%, and most preferably 100% of the amino acids selected from the group A, Q, G, and V.

[0076] In a most preferred embodiment, the AQGV peptide of the present invention is a tetrapeptide comprising 100% amino acids selected from the group A, Q, G, and V. Typical preferred examples of such preferred tetrapeptides are AQGV, LQGV, VGQA, VGQL, AQVG, LQVG. Most typical and preferred is AQGV, which is the subject of human clinical trials as provided below.

[0077] The present invention includes the use of the AQGV peptide or a functional analog thereof for use in treating a human subject to improve or even shorten the subject's length of stay in an ICU. One way this may be achieved is by altering fluid retention in the human subject. Uses of the AQGV peptide or a functional analog thereof according to the present invention include the treatment of human patients believed to be at risk from treatment with vasopressors or inotropic agents and / or expected to require hemodynamic therapy with fluid therapy. Such human patients include those who are, will be, or are expected to be referred to intensive care, thereby shortening the length of stay in the ICU. Accordingly, the use of the AQGV peptide or a functional analog thereof includes use for the treatment of human patients believed to be at risk from or expected to require treatment with vasopressors or inotropic agents and / or fluid therapy, for example, as shown in the Examples. Preferably, for use in reducing the length of stay of a subject in an ICU in a human patient considered at risk, the AQGV peptide is administered at a rate of at least 10 mg / kg of patient body weight per hour (mg / kg / hr). Preferably, this administration rate is at least 20 mg, at least 30, at least 40, or most preferably at least 50 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 AQGV peptide is administered at a rate of at least 20 mg / kg / hr for at least 1 hour, more preferably at least 1.5 hours, most preferably at least 2 hours, such as at least 2 hours or at least 2.5 hours, more preferably at least 3.5 hours, more preferably at least 4.5 hours.

[0078] Embodiment 1: An AQGV peptide or a functional analogue thereof for use in the treatment of a human subject, said use comprising modifying hemodynamics in said human subject. Embodiment 2: An AQGV peptide or a functional analogue thereof for use in treating a human subject suffering from or believed to be at risk from fluid overload, said use comprising modifying hemodynamics in said human subject. Embodiment 3: An AQGV peptide or a functional analogue thereof for use in treating a human subject suffering from or believed to be at risk from excessive vasopressor / inotropic agent use, said use comprising modifying hemodynamics in said human subject. Embodiment 4: An AQGV peptide or a functional analogue thereof for use in the treatment of a human subject, wherein the human subject has undergone an induced trauma, the use comprising modifying hemodynamics in the human subject. Embodiment 5: An AQGV peptide or a functional analogue thereof for use in treating a human subject having impaired renal function, said use comprising modifying hemodynamics in said human subject. Embodiment 6: The AQGV peptide or a functional analogue thereof for use according to any one of embodiments 1-5, wherein said use reduces fluid retention in said human subject. Embodiment 7: The AQGV peptide or a functional analogue thereof for use according to any one of embodiments 1-6, wherein said use comprises a reduced use of vasopressors. Embodiment 8: The AQGV peptide or a functional analogue thereof for use according to any one of embodiments 1-7, wherein said use comprises reduced water intake. Embodiment 9: The AQGV peptide or a functional analogue thereof for use according to embodiment 7, wherein said reduced use of vasopressors comprises a reduced duration of use of vasopressors. Embodiment 10: The AQGV peptide or a functional analogue thereof for use according to any one of embodiments 6-9, wherein the subject has undergone an induced trauma. Embodiment 11: The AQGV peptide or a functional analogue thereof for use according to any one of embodiments 6-10, wherein said use improves renal function in said human subject. Embodiment 12: The AQGV peptide or a functional analogue thereof for use according to embodiment 11, wherein the improved renal function is accompanied by an improved GFR rate. Embodiment 13: The AQGV peptide or a functional analog thereof for use according to any one of embodiments 6-12, wherein the human subject has renal dysfunction that is AKI. Embodiment 14: The AQGV peptide or a functional analogue thereof for use according to any one of embodiments 1 to 13, wherein said use reduces plasma leakage from the blood into surrounding tissues and / or organs. Embodiment 15: The AQGV peptide or a functional analogue thereof for use according to any one of embodiments 1-14, wherein said use is in a human subject suffering from or at risk of heart failure. Embodiment 16: The AQGV peptide or a functional analogue thereof for use according to any one of embodiments 1-15, wherein said use is in a human subject at risk of having edema. Embodiment 17: The AQGV peptide or a functional analogue thereof for use according to any one of embodiments 4-16, wherein the human subject has undergone an induced trauma that is surgery. Embodiment 18: The AQGV peptide or a functional analogue thereof for use according to embodiment 17, wherein the surgery requires cardiopulmonary bypass. Embodiment 19: The AQGV peptide or a functional analogue thereof for use according to any one of embodiments 1 to 18, wherein the peptide is administered into the bloodstream. Embodiment 20: The AQGV peptide or a functional analogue thereof for use according to embodiment 19, wherein the peptide is administered at a rate of at least 70 mg / kg body weight / hour. Embodiment 21: The AQGV peptide or a functional analogue thereof for use according to embodiment 19 or 20, wherein the peptide is administered over a period of at least 1 hour. Embodiment 22: The AQGV peptide or a functional analogue thereof for use according to any one of embodiments 17-21, wherein said administration is during surgery. Embodiment 23: The AQGV peptide or a functional analogue thereof for use according to any one of embodiments 1 to 22, wherein said administration is during anti-cancer treatment. Embodiment 24: The AQGV peptide or a functional analogue thereof for use according to any one of embodiments 1-23, wherein said administration is during an adverse drug reaction. Embodiment 25: The AQGV peptide or a functional analogue thereof for use according to any one of embodiments 1-24, wherein the human subject is sent to intensive care and the use improves a measured parameter of the human subject, and the parameter of the human subject is determined to assess whether the subject should remain in intensive care. Embodiment 26: The AQGV peptide or a functional analogue thereof for use according to embodiment 25, wherein the improvement of the parameter results in a reduced length of stay in intensive care. Embodiment 27: The AQGV peptide or a functional analogue thereof for use according to any one of embodiments 1 to 26, wherein said use induces vasoconstriction. Embodiment 28: A method of treatment comprising administering an AQGV peptide or a functional analog thereof to a human subject in need of maintaining hemodynamic stability. Embodiment 29: A method of treatment comprising administering an AQGV peptide or a functional analog thereof to a human subject in need of improved hemodynamic stability. Embodiment 30: A method of treatment comprising administering an AQGV peptide or a functional analog thereof to a human subject having impaired renal function, wherein said treatment of administering an AQGV peptide comprises maintaining or improving hemodynamic stability in said human subject. Embodiment 31: A method of treatment comprising administering an AQGV peptide or a functional analog thereof to a human subject in need of ameliorating an adverse drug reaction. Embodiment 32: A method of treatment comprising administering an AQGV peptide or a functional analog thereof to a human subject having or suspected of having Clarkson's disease (CLS). Embodiment 33: A method of treatment comprising administering an AQGV peptide or a functional analog thereof to a human subject having or suspected of having an adverse drug reaction that affects capillary leakage. Embodiment 34: A method of treatment comprising administering an AQGV peptide or a functional analog thereof to a human subject in need of maintaining hemodynamic stability. Embodiment 35: A method of treatment comprising administering an AQGV peptide or a functional analog thereof to a human subject in need of improved hemodynamic stability. Embodiment 36: A method of treatment comprising administering an AQGV peptide or a functional analog thereof to a human subject having impaired renal function, wherein said treatment of administering an AQGV peptide comprises maintaining or improving hemodynamic stability in said human subject.

[0079] (Example) A Phase 2 clinical trial was designed to test EA-230 (tetrapeptide AQGV) in cardiac surgery (CABG) patients, who often develop hemodynamic imbalances that lead to organ failure. The trial was completed in 2019, and the results showed significantly improved renal function after treatment with EA-230, accompanied by a highly statistically significant reduction in LOS (length of stay in the ICU and hospital) and reduced readmissions. These beneficial effects of EA-230 translated into significantly better recoveries for open-heart surgery patients treated with EA-230, resulting in clear economic benefits. The positive outcomes likely resulted from the highly beneficial effects of EA-230 on hemodynamic stability in intensive care patients, which were unexpectedly demonstrated during this Phase 2 trial, leading to reduced fluid overload, reduced vasopressor use, and improved renal function.

[0080] During cardiac surgery, 180 patients (double-blind, placebo-controlled, randomized) received EA-230 at 90 mg / kg / h or placebo. The primary endpoint was safety. Efficacy was assessed by immunomodulation (plasma interleukin (IL)-6 concentration), renal function (glomerular filtration rate using iohexol and creatinine (GFR iohexol, eGFRMDRD), and incidence of acute kidney injury (AKI, RIFLE criteria)), cardiovascular effects (fluid balance, vasoactive agents), and general outcome (length of stay).

[0081] The median [IQR] age was 68 [62-74] years, and 158 / 180 were male. No safety concerns were raised. EA-230 did not modulate IL-6 (area under the curve: 2730 [1968-3760] vs. 2680 [2090-3570] pg / ml*hour for the EA-230 and placebo groups, respectively, p=0.80). GFR increased after surgery (mean delta ± SEM GFR iohexol: 19 ± 2 vs. 16 ± 2 ml / min / 1.73 m², p=0.13; eGFRMDRD: 6 ± 1 vs. 2 ± 1 ml / min / 1.73 m², p=0.01). EA-230 showed a trend toward preventing AKI (stage injury: 7% vs. 18%, respectively, p=0.07). Patients in the EA-230 group required less fluid administration than placebo-treated patients (217 ± 108 vs. 605 ± 103 ml, respectively, p = 0.01), but vasoactive medication use was similar in both groups (p = 0.39). Length of hospital stay was shorter in the EA-230-treated group (8 [7-11] vs. 10 [8-12] days, respectively, p = 0.001).

[0082] (EA-230 Safety) The final analysis of the EASI trial demonstrated a favorable safety profile of treatment with EA-230. Continuous infusion of EA-230 at 90 mg / kg / hour for up to 4 hours was well tolerated by patients undergoing elective CABG surgery. Pharmacokinetic studies indicate that EA-230 is rapidly cleared from the circulation (within 5-10 minutes) when the infusion is terminated. Patients receiving EA-230 appeared to experience fewer (serious) adverse events and fewer major clinical adverse events. In conclusion, the safety profile of EA-230 in patients undergoing elective CABG surgery was comparable to, if not superior to, the safety profile of patients receiving a continuous infusion of placebo.

[0083] (Effectiveness of EA-230) Continuous infusion of EA-230 at 90 mg / kg / hour for up to 4 hours in patients undergoing elective CABG surgery demonstrated an unexpected but clear clinical benefit of EA-230 treatment. Most notably, treatment with EA-230 (n=90) resulted in a highly significant reduction in total postoperative length of stay by approximately 3 days. While no effect on CABG-induced cytokine responses (based on IL-6, the primary endpoint), treatment with EA-230 clearly resulted in a significant overall improvement in hemodynamic stability, based on reduced need for blood pressure (vasopressor / inotropic) medications and fluid therapy, thereby preventing postoperative fluid overload. There was also a significant overall improvement in renal function. As detailed herein, prolonged treatment with EA-230 during surgery provides the clinical benefit of increased patient recovery rates after surgery. The present results also demonstrate a beneficial effect of prolonged (longer than the median) treatment with EA-230 on renal function, as measured by glomerular filtration rate (GFR), compared with patients treated for shorter than the median treatment period. Our findings (Figure 17) demonstrate a highly significant statistical improvement in postoperative renal function with prolonged treatment of patients with EA-230 during elective CABG surgery. Similarly, postoperative hemodynamic stability after surgery (Figure 18, measured as the need for vasopressors / inotropes and / or fluid therapy) significantly benefits from the prolonged use of EA-230 during surgery.

[0084] (Summary of EA-230 effects) Early administration led us to note novel and highly beneficial effects of EA-230 on hemodynamics, renal function, and length of stay in the ICU and hospital, all of which were associated with improved hemodynamic stability. Treatment of patients with EA-230 during surgery significantly reduced the need for postoperative 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). EA-230 also significantly shortened postoperative recovery time in the ICU and significantly reduced length of stay in the hospital (Figure 1). On average, EA-230-treated patients required approximately 8 days of in-hospital care, compared with approximately 10 days for placebo-treated patients. Additionally, fewer EA-230-treated patients required re-hospitalization compared with placebo-treated patients.

[0085] Effects of EA-230 in human patients A prospective, randomized, double-blind, placebo-controlled trial enrolled 180 elective patients undergoing on-pump coronary artery bypass surgery with or without concomitant valve surgery. Patients were randomly assigned in a 1:1 ratio to receive EA-230 (90 mg / kg / hour) or placebo, which was infused from the start of surgery until the end of cardiopulmonary bypass. The primary focus of this first-in-patient trial was the safety and tolerability of EA-230. The primary efficacy endpoint was modulation of the inflammatory response by EA-230, quantified as the change in interleukin-6 plasma concentrations after surgery. The key secondary endpoint was the effect of EA-230 on renal function.

[0086] (Design and configuration) This study was a single-center, prospective, randomized, double-blind, placebo-controlled, single-dose phase II trial with an adaptive design to evaluate the safety and immunomodulatory effects of EA-230 in patients undergoing on-pump cardiac surgery for on-pump coronary artery bypass grafting (CABG) with or without concomitant valve surgery. 180 eligible patients were enrolled and randomly assigned in a 1:1 ratio to receive either active or placebo treatment. This was a first-in-patient safety and tolerability study; the primary efficacy objective was to evaluate the immunomodulatory effects of EA-230. The primary secondary efficacy endpoint was the effect of EA-230 on renal function. This trial was written in accordance with the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) and registered at clinicaltrials.gov under NCT03145220.

[0087] (Randomization and Stratification) On the morning of surgery, patients were randomized to active or placebo treatment by an unblinded, independent investigator. During this process, investigators used data management software (Castor EDC, Amsterdam, The Netherlands) approved for Good Clinical Practice (GCP). The Castor system applied stratified randomization to ensure equal allocation of patients with known risk factors for adverse outcomes between active and placebo treatment. Three strata were included: 1) CABG surgery with or without concomitant valve surgery; 2) preoperative renal function with estimated GFR of ≤30, 31–90, and >90 ml / min / 1.73 m2; and 3) EuroSCORE II of <4 or ≥4 (Nashef et al., Eur. J. Cardiothora. Surg., 2012 Apr;41(4):734–44).

[0088] (blinded) Double-blind conditions were maintained for all patients, all blinded medical study team personnel involved in study procedures, data collection, and / or data analysis, and the principal investigator. 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 permitted by the sponsor after completion of the study, performance of a blinded data review, and database lock.

[0089] (Test Intervention) An intravenous infusion of EA-230 (90 mg / kg / hour) or placebo was initiated at the time of the initial surgical incision using an automatic infusion pump. The infusion rate was set at 250 ml / hour, and the infusion was continued until discontinuation of CPB or 4 hours of continuous infusion, whichever occurred first.

[0090] The EA-230 formulation was dissolved in water for injection at a final concentration of 300 mg / ml with an osmolality of 800 to 1000 mOsm / kg and filled into sterile 5 ml glass vials at a content of 1500 mg / vial. The placebo formulation consisted of sodium chloride diluted with water for injection in the same sterile 5 ml glass vial at a content of 29 mg / ml to yield a solution with the same osmolality. EA-230 and placebo were prepared for intravenous continuous 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 and active treatment vials were manufactured by HALIX BV (Leiden, The Netherlands).

[0091] (adverse event, AE) All AEs were judged by the investigator for severity ('mild, moderate, or severe') according to Common Terminology Criteria for Adverse Events guidelines 4.030 and their perceived relationship to study drug ('definitely, probably, possibly, or not / probably not related'). SAEs and SUSARs included death, life-threatening illness, long-term and / or significant disability and / or incapacity, and hospitalization and / or prolonged inpatient treatment.

[0092] (Ethical considerations, data quality assurance, and patient / public participation) 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, GCP guidelines, and European Directive 2001 / 20 / CE. Informed consent was obtained before any study-specific procedures were performed. Data were handled confidentially and anonymously, and GCP standards were applied. The handling of patient data in this study complied with the Dutch Personal Information Protection Act (Dutch: Wet Bescherming Persoonsgegevens, WBP). Patients and the public were not involved in the design and / or execution of the study protocol. Study outcomes were sent individually to all study participants. The burden of the intervention was assessed by independent institutional ethics committees, the CMO and CCMO, which include lay members.

[0093] (result) When the data obtained during the clinical trial were evaluated, notably, no significant differences were observed in the plasma levels of IL-8, IL-10, IL-1RA, IL-17, MCP-1, and ICAM, as well as the other cytokines tested, between the EA-230 and placebo groups, demonstrating no clear immunomodulatory or anti-inflammatory effects. This also applied to the primary endpoint of the study, IL-6 plasma levels (see Figure 12), and the inflammatory renal injury markers IL-18, KIM1, NGAL, L-FABP, and NAG (see Figure 22). Notably, significantly fewer patients in the EA-230-treated group suffered from fluid retention (see Table 1). Further analysis of various parameters revealed that hemodynamic parameters (vasopressor use and / or fluid balance) and / or renal parameters were favorably affected by the use of EA-230 compared to placebo.

[0094] 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 here. Significantly fewer (chi-squared P<0.05) AEs occurred in the EA-230 treatment group (n=2) compared with the placebo treatment group (n=283). Significantly fewer (chi-squared P<0.05) patients suffered from fluid retention in the EA-230 treatment group (n=2) compared with the placebo treatment group (n=11). [Table 1]

[0095] Table 2: Mean on-pump duration of patients divided into quartiles Q1, Q2, Q3, and Q4 of pump duration, and therefore treatment duration, along with patient mean age, and for all patients tested (Q1-Q4). [Table 2]

[0096] (Hemodynamic stability in the EASI study) Figure 2 shows the use of vasopressors. In general, vasopressor use was reduced in the group treated with EA-230. Patients were divided into quartiles based on duration of treatment. Table 3 shows descriptive frequencies for two variables: number of days receiving vasopressin and net fluid balance on days 0-2 (first 72 hours). Groups were divided into patients without treatment (placebo) and patients treated with EA-230 (active), as well as patients with and without acute kidney injury (AKI). EA-230 reduced net fluid balance in both patients with and without AKI. EA-230 reduced the need for vasopressors in patients with AKI. [Table 3]

[0097] (Adjustment of fluid balance and vasopressor use by treatment with EA-230) The effect of EA-230 versus placebo was tested in univariate and multivariate models (see Table 4). The input / independent variable was treatment group (EA-230 or placebo). The output / dependent variable was outcome measure: fluid balance in the first 72 hours, number of days on vasopressin, or vasopressor score (area under the curve). The effect of EA-230 versus placebo was tested for two combined variables in Model A (fluid balance in the first 72 hours + number of days on vasopressin) and Model B (fluid balance in the first 72 hours + vasopressor score AUC). Results from both multivariate models showed significant improvements in hemodynamic parameters in patients receiving EA-230. This was observed in Model A (fluid balance in the first 72 hours + number of days on vasopressin) (p = 0.006) and Model B (fluid balance in the first 72 hours + vasopressor score AUC) (p = 0.008). The hemodynamic effects of EA-230 were also significantly better in patients who did not develop AKI, demonstrating that hemodynamic improvement can occur independently of renal failure.

[0098] Table 4: Goal-directed hemodynamic therapy with EA-230 Analyses for Model A are shown for the total group and for subgroups of renal dysfunction divided according to 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. [Table 4]

[0099] Combined, these results demonstrate that the use of EA-230 can improve and / or maintain hemodynamics in human patients, as assessed by affecting the duration of vasopressor use, the amount of vasopressor administered, and / or fluid balance. Specifically, EA-230 improves hemodynamic stability after open-heart surgery in humans. Permeability governs the amount of fluid that leaks from blood vessels. The administration of fluid therapy generally increases leakage. Based on the results of the Phase II patient observations, we found a significant reduction in adverse fluid retention (fluid leakage due to fluid overload) in patients treated with EA-230 (p=0.03). Additionally, contractility governs tone, which is often regulated by the administration of blood pressure medications, but these medications can have major adverse side effects. Based on the results of the Phase II patient observations, 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). We also determined the mean maximum concentrations (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 to 57,500 ng / ml). The mean venous Cmax was found to be 68,400 ng / ml (range 19,600 to 113,000 ng / ml).

[0100] (EA-230 has beneficial effects on renal function) The effect of EA-230 on modulating the incidence of different stages of acute kidney injury (AKI) was determined based on the RIFLE criteria (RIFLE: classification into risk, injury, failure, loss of kidney function, and end-stage kidney disease, 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 injury category of the RIFLE criteria decreased (see Figure 3). Furthermore, the use of EA-230 significantly improved GFR after surgery (Figure 4). Creatinine clearance, as a biomarker of renal function, was significantly improved after surgery in patients treated with EA-230 (Figure 5). When preoperative renal function was taken into account, creatinine clearance was significantly improved when EA-230 was used and preoperative renal function was less than 60 ml / min (Figure 6, left). No difference was observed when preoperative renal function was greater than 60 ml / min (Figure 6, right). Similar observations were made based on GFR parameters (Figure 7). Treatment with EA-230 significantly improved postoperative estimated GFR compared with preoperative estimated GFR, whereas treatment with placebo did not. No differences were observed between groups when renal function was greater than 60 ml / min / 1.73 m. Additionally, when patients had long-term cardiopulmonary bypass, treatment with EA-230 significantly improved postoperative GFR compared with preoperative GFR (Figure 8, treatment length >156 min, p = 0.001). Combined, these results indicate that the use of EA-230 can improve and / or maintain renal function in human patients.

[0101] (ICU, hospital length of stay, and readmission) The study investigated patient length of stay in the ICU and length of stay in the hospital (inpatient care) (see Figure 10). Treatment with EA-230 resulted in a significant reduction in ICU and hospital length of stay (LOS). ICU and hospital length of stay decreased in the EA-230 group. Patients treated with EA-230 also showed a significant (p=0.09) reduction in readmissions up to 90 days after surgery (see Table 5).

[0102] Table 5: Number of readmissions in the EASI study (CABG study). Number of patients who had to be readmitted to the hospital due to clinical illness in the post-operative period. Readmissions were scored for a total period of 90 days after surgery, at the 28-day post-operative period, and in the range of 29-90 days after surgery. Readmissions were reduced in the treatment group of patients receiving EA-230. [Table 5]

[0103] Furthermore, in the AQGV-treated patient group, the number of patients with AKI was reduced, but when patients developed AKI, these patients did not require the prolonged length of stay observed in the placebo group, and the length of stay was similar to that of patients without AKI or at risk of AKI (see Figure 11).

[0104] Together, treatment with EA-230 shows a potent beneficial effect on recovery: EA-230-treated patients required significantly less hemodynamic therapy, recovered renal function significantly more quickly after surgery, and stayed in the intensive care unit (ICU) and hospital for a shorter period of time compared to the placebo-treated group.

[0105] These novel hemodynamic effects of EA-230 appeared independent of EA-230's anti-inflammatory effects. In summary, the significant improvements in hemodynamic stability, renal function, and postoperative recovery in EA-230-treated patients are related to EA-230's novel effects on vascular permeability and vasoconstriction. EA-230 given intraoperatively demonstrates significant improvements in postoperative patient recovery over placebo patients. EA-230-treated patients are released more quickly from intensive care (p=0.0232) and hospital (p=0.0015). EA-230 improves hemodynamic stability (p=0.006) and renal function (p=0.003). Although the primary endpoint of short-term reduction in inflammatory cytokines (IL-6) was lost, long-term patient recovery was significantly improved with EA-230. EA-230 was shown to be safe and well tolerated throughout surgery. In conclusion, EA-230 given intraoperatively significantly improves postoperative recovery.

[0106] A significant improvement in hemodynamic stability was found (reduced fluid therapy and blood pressure medications, p=0.006), which was accompanied by a significant improvement in renal function (improved glomerular filtration rate reduced plasma creatinine, p=0.003), a significant reduction in patients suffering from adverse fluid retention during recovery (2 for EA-230 vs. 9 for placebo, p=0.03), and a substantial reduction in hospital readmissions at 90 days after surgery (4 for EA-230 vs. 10 for placebo, p=0.09).

[0107] (Further analyzed biomarkers related to vasoconstriction and / or vasodilation) Given the observed effects on hemodynamics and renal function, plasma samples were further analyzed for select biomarkers. Plasma samples from control patients and patients receiving EA-230 were analyzed for biomarkers such as endothelin-1, VEGF, angiotensin II, and cAMP, as well as natriuretic peptides. The assays described below were used to determine biomarker levels.

[0108] In vitro effects of EA-230 and AQGV analogs The effects of the AQGV peptide (EA-230) and its analogs were tested on human vascular endothelial cells in an in vitro transwell assay. Briefly, vascular 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. Suitable human vascular endothelial cells include HUVECs (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). Endothelial permeability was determined by measuring the passage of macromolecules. Additionally, biomarker levels were 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 assay, established human umbilical vein endothelial cells (HUVECs), which have the ability to line blood vessels, are grown in cell culture on sieves in multiple assay formats (i.e., n=5), allowing for determination of leakage in response to various test concentrations of the EA-230 peptide or placebo control used, and establishing pharmacological parameters for the effect of the EA-230 peptide on permeability in human cells.

[0109] Bravo et al. (J. Pharmacol. Toxicol. Methods, January-February 2018; 89:47-53) also developed an impedance-based contraction assay using the xCELLigence RTCA MP system. This technique uses a specialized 96-well E-plate with an array of gold microelectrodes printed in individual wells to monitor cell adhesion by recording electrical impedance in real time. The change in impedance (percentage of control) can be used as a readout for cell contraction. Established human aortic smooth muscle cells (HaSMCs), which have the ability to contract blood vessels, were grown in cell culture on gold electrodes in multiple test formats (i.e., n=3), allowing for the determination of the electrical impedance of endothelin-1-induced smooth muscle cell contraction in response to various test concentrations of the EA-230 peptide or placebo control. This established pharmacological parameter for the EA-230 peptide's effect on contractility in human cells. Additionally, isolated human aortic smooth muscle cells (APaSMCs) from aneurysm (n=3) / control (n=3) patients, which have the ability to differentially constrict blood vessels, were grown in cell culture on gold electrodes in multiple test formats, allowing electrical impedance determination of ionomycin-induced smooth muscle contraction of patient versus control cells in response to various test concentrations of EA-230 peptide or placebo control used, detecting the effects of EA-230 on patient cells.

[0110] [Note] [Appendix 1] A method for treating a human subject believed to be in need of maintaining or improving hemodynamic stability, comprising administering to the subject a peptide comprising at least 50% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R).

[0111] [Appendix 2] 1. A method of treating a human subject believed to be in need of a reduction in harmful vascular permeability, comprising administering to the subject a peptide comprising at least 50% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R).

[0112] [Appendix 3] 1. A method of treating a human subject believed to be in need of reducing harmful fluid retention, comprising administering to the subject a peptide comprising at least 50% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R).

[0113] [Appendix 4] 4. The method of any one of claims 1 to 3, wherein the peptide comprises at least 75% amino acids selected from the group of autophagy-inhibiting amino acids.

[0114] [Appendix 5] 5. The method of any one of claims 1 to 4, wherein the peptide consists of amino acids selected from the group of autophagy-inhibiting amino acids.

[0115] [Appendix 6] 6. The method of any one of claims 1 to 5, wherein the peptide comprises at least 50% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), and leucine (L).

[0116] [Appendix 7] 7. The method of any one of claims 1 to 6, wherein the peptide comprises at most 30% amino acids selected from the group of autophagy-inhibiting amino acids consisting of glycine (G), valine (V), isoleucine (I), proline (P), and arginine (R).

[0117] [Appendix 8] 8. The method of any one of claims 1 to 7, comprising administering to the subject at least two different peptides, each peptide comprising at least 50% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R).

[0118] [Appendix 9] 9. The method of any one of claims 1 to 8, wherein the one or more peptides are in the range of 4-30 amino acids in length.

[0119] [Appendix 10] 10. The method of any one of claims 1 to 9, wherein the subject has undergone severe trauma, such as surgery.

[0120] [Appendix 11] 10. The method of any one of claims 1 to 9, wherein the subject is undergoing cancer treatment, such as treatment with an anti-neoplastic agent or an immunomodulatory agent.

[0121] [Appendix 12] 10. The method of any one of claims 1 to 9, wherein the subject is believed to be suffering from capillary leak syndrome, an adverse drug reaction.

[0122] [Appendix 13] 13. The method of any one of claims 1 to 12, wherein the human subject has impaired renal function.

[0123] [Appendix 14] 14. The method of any one of claims 1 to 13, wherein the method comprises reduced use of vasopressors.

[0124] [Appendix 15] 15. The method of any one of clauses 1 to 14, wherein the method comprises reduced fluid intake.

[0125] [Appendix 16] 16. The method of any one of claims 1 to 15, wherein the peptide comprises at least 50% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), and valine (V).

[0126] [Appendix 17] 16. The method of any one of appendixes 1 to 15, wherein the peptide consists of amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), and valine (V).

[0127] [Appendix 18] 18. The method of any one of claims 1 to 17, wherein the peptide is a salt selected from the group of peptide-organic acid.

[0128] [Appendix 19] 1. A peptide for use in treating a human subject believed to be in need of maintaining or improving hemodynamic stability, said peptide comprising at least 50% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R).

[0129] [Appendix 20] 1. A peptide for use in treating a human subject believed to be in need of a reduction in deleterious vascular permeability, said peptide comprising at least 50% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R).

[0130] [Appendix 21] 1. A peptide for use in treating a human subject believed to be in need of reducing harmful fluids, said peptide comprising at least 50% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R).

[0131] [Appendix 22] 22. The peptide of any one of claims 19 to 21, wherein the subject has undergone a severe trauma, such as surgery.

[0132] [Appendix 23] 22. The peptide of any one of claims 19 to 21, wherein the subject is undergoing cancer treatment, such as treatment with an anti-neoplastic agent or an immunomodulatory agent.

[0133] [Appendix 24] 22. The peptide of any one of claims 19 to 21, wherein the subject is thought to be suffering from capillary leak syndrome, an adverse drug reaction.

[0134] [Appendix 25] 25. The peptide of any one of claims 19 to 24, wherein the human subject has impaired renal function.

[0135] [Appendix 26] 26. The peptide of any one of clauses 19 to 25, wherein the use comprises reduced use of vasopressors.

[0136] [Appendix 27] 27. The peptide of any one of clauses 19 to 26, wherein the use comprises reduced fluid intake.

[0137] [Appendix 28] 28. The peptide of any one of claims 19 to 27, wherein the peptide comprises at least 50% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), and valine (V).

[0138] [Appendix 29] 29. The peptide of claim 28, wherein the peptide consists of amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), and valine (V).

[0139] [Appendix 30] 30. The peptide of claim 29, wherein the peptide is a salt selected from the group consisting of peptide-acetate, peptide-tartrate, and peptide-citrate.

[0140] [Appendix 31] 31. A pharmaceutical formulation comprising a peptide according to any one of claims 19 to 30.

[0141] [Appendix 32] 32. The pharmaceutical formulation of claim 31, wherein the peptide comprises at least 75% amino acids selected from the group of autophagy-inhibiting amino acids.

[0142] [Appendix 33] 33. The pharmaceutical formulation of claim 31 or 32, wherein the peptide consists of 100% amino acids selected from the group of autophagy-inhibiting amino acids.

[0143] [Appendix 34] 34. The pharmaceutical formulation of any one of claims 31 to 33, wherein the peptide comprises at least 50% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (A), glutamine (Q), and leucine (L).

[0144] [Appendix 35] 35. The pharmaceutical formulation of any one of claims 31 to 34, wherein the peptide comprises at most 30% amino acids selected from the group of autophagy-inhibiting amino acids consisting of glycine (G), valine (V), isoleucine (I), proline (P), and arginine (R).

[0145] [Appendix 36] 36. The pharmaceutical formulation of any one of Appendices 31 to 35, comprising at least two different peptides, each comprising at least 50% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R).

[0146] [Appendix 37] 37. The pharmaceutical formulation of any one of claims 31 to 36, wherein one or more of the peptides vary in length by 4-30 amino acids.

[0147] [Appendix 38] 38. The pharmaceutical formulation of any one of claims 31 to 37, comprising at least 0.85 mol / L of one or more of the peptides.

[0148] [Appendix 39] 39. The pharmaceutical formulation of any one of claims 31 to 38, comprising at least one pharmaceutically acceptable excipient.

[0149] [Appendix 40] 40. A stock solution comprising the pharmaceutical formulation of any one of appendices 31 to 39.

[0150] [Appendix 41] 41. The formulation or solution of any one of claims 31 to 40 for use in a method of treatment of a human subject deemed to be in need of maintaining or improving hemodynamic stability.

[0151] [Appendix 42] 41. The formulation or solution of any one of claims 31 to 40 for use in a method of treating a human subject deemed in need of reducing deleterious vascular permeability.

[0152] [Appendix 43] 41. The formulation or solution of any one of Appendices 31 to 40 for use in a method of treating a human subject deemed in need of reducing harmful fluid retention.

[0153] [Appendix 44] 41. The formulation or solution of any one of Appendices 31 to 40 for use in a method of treating a human subject having or suspected of having Clarkson's disease (CLS).

[0154] [Appendix 45] 41. A method of treating a human subject believed to be in need of maintaining or improving hemodynamic stability, comprising administering to said subject a formulation or solution of any one of claims 31 to 40.

[0155] [Appendix 46] 41. A method of treating a human subject believed to be in need of a reduction in harmful vascular permeability, comprising administering to said subject a formulation or solution of any one of claims 31 to 40.

[0156] [Appendix 47] 41. A method of treating a human subject believed to be in need of reducing harmful fluid retention, comprising administering to said subject a formulation or solution of any one of claims 31 to 40.

[0157] [Appendix 48] 41. A method of treating a human subject suffering from or suspected of suffering from Clarkson's disease (CLS), comprising administering to the subject a formulation or solution according to any one of claims 31 to 40.

Claims

[Claim 1] 1. A method of treating a human subject believed to be in need of maintaining or improving hemodynamic stability, comprising administering to the subject a peptide comprising at least 50% amino acids selected from the group of autophagy-inhibiting amino acids consisting of alanine (single letter A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R).

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