METHODS OF TREATMENT FOR MODIFYING HEMODYNAMICS
20220370543 · 2022-11-24
Assignee
Inventors
Cpc classification
A61P7/04
HUMAN NECESSITIES
A61K38/04
HUMAN NECESSITIES
International classification
Abstract
This disclosure provides method of treatment comprising administering an AQGV peptide, or a functional analog thereof, to a human subject, the human subject optionally having impaired kidney function, wherein the treatment of administering an 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).
Claims
1.-48. (canceled)
49. A method of treating a subject in need of maintaining or improving hemodynamic stability, a reduction in adverse vascular permeability, and/or a reduction in fluid retention, the method comprising: administering to the subject peptide(s) comprising at least 50% amino acids that are autophagy inhibiting amino acids, wherein the autophagy inhibiting amino acids are selected from the group consisting of alanine, glutamine, glycine, valine, leucine, isoleucine, proline, and arginine.
50. The method according to claim 49, wherein the peptide(s) comprise(s) at least 75% of autophagy inhibiting amino acids.
51. The method according to claim 49, wherein the peptide(s) consist(s) of said autophagy inhibiting amino acids.
52. The method according to claim 49, wherein the peptide(s) comprise(s) at least 50% alanine, glutamine, and leucine.
53. The method according to claim 49, wherein the peptide(s) comprise(s) at most 30% amino acids selected from the group consisting of glycine, valine, isoleucine, proline, and arginine.
54. The method according to claim 49, wherein at least two different peptides are administered to the subject, each said peptide comprising at least 50% amino acids selected from the group consisting of alanine, glutamine, glycine, valine, leucine, isoleucine, proline, and arginine.
55. The method according to claim 49, wherein at least two peptides vary in length from 4 to 30 amino acids.
56. The method according to claim 49, wherein the subject has been subjected to severe trauma.
57. The method according to claim 49, wherein the subject has been subjected to cancer treatment.
58. The method according to claim 49, wherein the subject is suffering from capillary leakage syndrome.
59. The method according to claim 56, wherein the severe trauma is surgery.
60. The method according to claim 57, wherein the cancer treatment comprises treatment with an antineoplastic or immunomodulating agent.
61. The method according to claim 58, wherein the subject is suffering capillary leakage syndrome from an adverse drug reaction.
62. The method according to claim 49, wherein the subject has impaired kidney function.
63. The method according to claim 49, further comprising: reducing use by the subject of a vasopressive agent.
64. The method according to claim 49, further comprising: reducing the subject's fluid intake.
65. The method according to claim 49, wherein the peptide(s) comprise(s) at least 50% amino acids selected from the group consisting of alanine, glutamine, glycine, and valine.
66. The method according to claim 49, wherein the peptide(s) consist(s) of amino acids selected from the group consisting of alanine, glutamine, glycine, and valine.
67. The method according to claim 49, wherein at least one peptide is/are in the form of a salt of peptide-organic acid.
68. A pharmaceutical formulation comprising at least one peptide comprising at least 50% of autophagy inhibiting amino acids amino acids, wherein an autophagy inhibiting amino acid is selected from the group consisting of alanine, glutamine, glycine, valine, leucine, isoleucine, proline, and arginine.
69. The pharmaceutical formulation of claim 68, wherein the at least one peptide comprises at least 75% of autophagy inhibiting amino acids.
70. The pharmaceutical formulation of claim 69, wherein the at least one peptide consists of 100% autophagy inhibiting amino acids.
71. The pharmaceutical formulation of claim 68, wherein the at least one peptide comprises at least 50% amino acids selected from the group consisting of alanine, glutamine, and leucine.
72. The pharmaceutical formulation of claim 68, wherein the at least one peptide comprises at most 30% amino acids selected from the group consisting of glycine, valine, isoleucine, proline, and arginine.
73. The pharmaceutical formulation of claim 68, comprising at least two different peptides each comprising at least 50% amino acids selected from the group consisting of alanine, glutamine, glycine, valine, leucine, isoleucine, proline, and arginine.
74. The pharmaceutical formulation of claim 68, wherein the peptides vary in length from 4 to 30 amino acids.
75. The pharmaceutical formulation of claim 68, comprising at least 0.85 mol/L of the peptide(s).
76. The pharmaceutical formulation of claim 68, together with at least one pharmaceutically acceptable excipient.
77. The pharmaceutical formulation of claim 68, wherein the peptide(s) comprise(s) at least 50% amino acids selected from the group consisting of alanine, glutamine, glycine, and valine.
78. The pharmaceutical formulation of claim 77, wherein the peptide(s) consist(s) of amino acids selected from the group consisting of autophagy inhibiting amino acids alanine, glutamine, glycine, and valine.
79. The pharmaceutical formulation of claim 78, wherein the peptide(s) is a salt selected from the group consisting of peptide-acetate, peptide-tartrate or peptide-citrate.
80. A method of treating a subject suffering from Clarkson's disease (CLS), the method comprising: administering the pharmaceutical formulation of claim 68 to the subject so as to treat the CLS.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0087] Peptide Synthesis
[0088] Hemodynamic peptides are for example synthesized using classical solid phase synthesis, or other methods known in the art. Purity of the peptides is confirmed by high performance liquid chromatography and/or by fast atom bombardment mass spectrometry. Traditionally, peptides are defined as molecules that consist of between 2 and 50 amino acids, whereas proteins are made up of 50 or more amino acids. In addition, peptides tend to be less well defined in structure than proteins, which can adopt complex conformations known as secondary, tertiary, and quaternary structures. Functional distinctions may also be made between peptides and proteins. In fact, most researchers, as well as this disclosure, use the term peptide to refer specifically to peptides, or otherwise relatively short amino acid chains of up to 50 amino acids, with the term polypeptide being used to describe proteins, or chains of >50 or many more amino acids.
[0089] Peptide Administration
[0090] As shown in clinical trial protocol (Groenendael et al., JMIR Res Protoc 2019 February; 8(2): e11441), study medication EA-230 formulation is packed and provided in sterile 5-mL glass vials, containing 1500 mg/vial, dissolved in water for injection at a final concentration of 300 mg/mL with an osmolality of 800 to 1000 mOsm/kg. The placebo formulation comprises sodium chloride diluted in water for injection in identical sterile 5-mL glass vials containing 29 mg/mL to reach a solution with an identical osmolality. EA-230 and placebo are prepared for continuous intravenous infusion with an osmolality of <400 mOsm/kg by adding the appropriate amount of EA-230 or placebo to 1000 mL normal saline under aseptic conditions.
[0091] Need for Stock-Solution with Higher Concentration Active Substance.
[0092] A vial with EA-230 formulation (stock-solution) used in herein referenced clinical trial contained 1.5 gram EA-230, each vial containing 5 ml a 300 mg/ml [(300 g/L=0.8 mol/L) AQGV (SEQ ID NO:1) having a molecular weight of 373 g/mol]. In the trial, a best-treatment practice was established when infusion with active substance lasted at least 1.5 hours, preferably at least 2.5 hours, preferably at least, 3.5 hours, more preferably at least 4.5 hours, at 90 mg/kg per hour. As a consequence, and also depending on bodyweight, often more than 12-17 vials were needed for continuing effective treatment, an administration requirement that takes (too) much labor in the operating room or ICU for the required care. This disadvantage of treatment with too weak amounts of stock of EA-230 formulation brings forward a need to provide more and better concentrated stock-solutions than available.
[0093] Determination of Aggregation Points
[0094] It is recognized herein that many drug-like molecules can self-aggregate in aqueous media and aggregates may have physicochemical properties that skew experimental results and clinical decisions. The aggregation of peptide drugs is one of the most common and troubling processes encountered in almost all phases of biological drug development. Aggregation can take several different forms and the term is used to describe a number of different processes during which peptide molecules associate into larger species comprising multiple polypeptide chains. Aggregates can be amorphous or highly structured, e.g., amyloid fibrils, and can form in solution or on surfaces due to adsorption. They can arise as a result of the non-covalent association of polypeptide chains, or from covalent linkage of chains. In some cases, aggregation is reversible while in others it is effectively irreversible. In either case, it reduces the physical stability of the peptide in question, not only leading to a loss in activity but also other critical problems such as toxicity and immunogenicity.
[0095] Salts have complex effects on the physical stability of biomolecules affecting both conformational and colloidal stability. Their effects frequently vary according to the surface charge on the peptide and the overall effect of a salt on physical stability is a balance of different and multiple mechanisms by which salt interacts with water and biomolecules. Various salts can influence physical stability by altering the properties of the peptide-solvent system (Hofmeister effects) and by altering electrostatic interactions (Debye-Hückel effects).
[0096] One aim was to investigate the solubility of seven different salts on prototype autophagy inhibiting peptide AQGV (SEQ ID NO:1), using the modified shake flask method. At first, the AQGV-Ac salt will be converted to the free base, extracted with an organic solvent and concentrated in vacuo. Subsequently the citrate, maleate, sulfate (KHSO4), adenosine mono-phosphate, adenosine, acetate and tartaric acid salts will be prepared and screened for their solubility thereafter.
[0097] Results
[0098] Conversion to the Free Base.
[0099] Extraction of AQGV-Ac (SEQ ID NO:1-Ac) with organized solvents from neutralized solution (pH=6-7) turned out not to be possible. Therefore a solution of AQGV-Ac in water was transferred to an ion-exchange column (Amberlite, approximately 100 mL; IR120, H resin) The column was flushed using demi-water followed by 1N ammonia-solution. The first 3 basic fractions were concentrated to afford 4.7 g of the free base AQGV (SEQ ID NO:1; 1H-NMR).
[0100] Solubility Measurements.
[0101] At first attempts were made to mix a solution of the free base and an acid in order to achieve a concentrated DMSO solution of the salt and subsequently dilute this in water in order to determine solubility. However the salts attempted (adenosine and citric acid) did not dissolve in DMSO at all. In fact, the mixture became clear after the addition of a little water. Therefore the solubility determinations could not be conducted as planned originally. It was decided to determine the solubility of the salts required by dilution of known amount of salts (not soluble) till a clear solution is obtained.
[0102] For citric acid, 1 mmol AQGV (SEQ ID NO:1) and 1 mmol citric acid were mixed in 0.5 mL 0.9% NaCl. This afforded a clear solution. More material of both AQGV (SEQ ID NO:1) and citric acid were added (amounts of 0.5 and 0.25 mmol) until a total of 2.75 mmol was dissolved in the 0.5 mL 0.9% NaCl. The mixture remains clear but got very thick/viscous. The remaining experiments have been conducted differently: 1 or 0.5 mmol salt was weighed in a 4 ml vial and small amounts of 0.9% NaCl were added until a clear solution was obtained, which remained clear for more than a week. In case of adenosine and adenosine-monophosphate no clear solution could be obtained.
[0103] Based on the results depicted in Table 1 the concentration below which an aggregated peptide-salt tends to resolve of neutral and autophagy inhibiting peptide-salts screened were determined (aggregation points, see Table 2). It can be concluded that changing the anion significantly influences the solubility characteristics of AQGV (SEQ ID NO:1). Higher solubility (solubility in 0.9% NaCl) and therewith higher aggregation points were observed for the AQGV-citric acid (AQGV-citrate, >5.5 mol/L) and -tartaric acid (AQGV-tartrate) salt, whereas maleic acid and KHSO4 salts showed lower solubility, compared to AQGV-Ac (2 mol/L). Using adenosine-monophosphate or adenosine did not provide solubility. Citric acid seems to be a special case. Highly concentrated solution does not crystallize or aggregate but tend to form a highly viscous solution.
[0104] Heeding aggregation risk, a vial with a stock solution of hemodynamic peptide for use in a clinical trial hitherto contained no more than (0.8 mol/L) active substrate in solution. Based on this disclosure, such a stock solution of an AQGV (SEQ ID NO:1)-salt of an organic acid, in particular of AQGV peptide-maleate, AQGV peptide-acetate AQGV peptide-tartrate or AQGV peptide-citrate (but neither adenosine nor adenosine monophosphate) now is provided with 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, most preferably at least 1.8 mol/L, of the hemodynamic peptide-acetate, hemodynamic peptide tartrate or hemodynamic peptide-citrate. In a more preferred embodiment, this disclosure provides a stock-solution of the hemodynamic peptide-tartrate or the hemodynamic peptide-citrate wherein the concentration of the hemodynamic peptide is in the range of 2 mol/L to 2.5 mol/L. In a more preferred embodiment, this disclosure provides a stock-solution of the hemodynamic-peptide-citrate wherein the concentration of the peptide-citrate is in the range of 2.5 mol/L to 3 mol/L. In a more preferred embodiment, this disclosure provides a stock-solution of the peptide-citrate wherein the concentration of the peptide-citrate is in the range of 3 mol/L to 3.5 mol/L. In a more preferred embodiment, this disclosure provides a stock-solution of the peptide-citrate wherein the concentration of the peptide-citrate is in the range of 3.5 mol/L to 4.5 mol/L. In a more preferred embodiment, this disclosure provides a stock-solution of the peptide-citrate wherein the concentration of the peptide-citrate is in the range of 4.5 mol/L to 5.5 mol/L. In a more preferred embodiment, this disclosure provides a stock-solution of the peptide-citrate wherein the concentration of the peptide-citrate is equal to or larger than 5.5 mol/L. It is preferred that the stock solution is an aqueous solution.
[0105] In describing protein or peptide composition, structure and function herein, reference is made to amino acids. In the present specification, amino acid residues are referred to using the following abbreviations. Also, unless explicitly otherwise indicated, the amino acid sequences of peptides and proteins are identified from N-terminal to C-terminal, left terminal to right terminal, the N-terminal being identified as a first residue. Ala: alanine residue; Asp: aspartate residue; Glu: glutamate 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: tryptophane residue; Tyr: tyrosine residue; Cys: cysteine residue. The amino acids may also be referred to by their conventional one-letter code abbreviations; 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.
[0106] Inhibition of Autophagy by Selected Amino Acids.
[0107] Autophagy is a degradation pathway that delivers extra cellular and cytoplasmic materials to lysosomes via double-membraned vesicles designated autophagosomes. Cytoplasmic constituents are sequestered into autophagosomes, which subsequently fuse with lysosomes, where the cargo is degraded. Extracellular materials are taken up by endocytosis or phagocytosis, which subsequently fuse with lysosomes, again where the cargo is degraded. Autophagy is a crucial mechanism involved in many aspects of cell function, including cellular metabolism and energy balance; and alterations in autophagy have been linked to various human pathological processes. Autophagy is a natural mechanism in which the cell removes and degrades cellular components with autolysosomes.
[0108] As recently reviewed (Cell. 2019 July; 8(7)), where the role of autophagy in the maintenance of tissue homeostasis is relatively well documented, its role during tissue repair and regeneration has only recently been appreciated. This disclosure provides that hemodynamic peptides, i.e., peptides enhanced with distinct amino acids or combinations thereof control the balance between on the one hand proteogenesis (mTOR kinase activities) and on the other hand proteolysis (autophagy) more than others, therewith identifying peptides enriched in autophagy inhibiting amino acids as better enhancers of proteogenesis underlying tissue repair than other peptides not being enriched in the amino acids. The mechanistic target of rapamycin complex I (mTORC1) is a central regulator of cellular and organismal growth and this pathway is 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, its site of activation. Recent and older data identify leucine (L), valine (V), isoleucine (I), alanine (A), glutamine (Q), arginine (R), glycine (G), proline (P), either alone or in combination, as more potent activators of mTOR or inhibitors of autophagy than other amino acids, such as glutamate (E), threonine (T), serine (S), lysine (K), threonine (T), phenylalanine (F), tyrosine (Y), and methionine (M) that have been reported to have no or opposite effects. Hence, as herein provided for inclusion in a hemodynamic peptide according to this disclosure, peptides enriched with leucine (L), valine (V), isoleucine (I), alanine (A), glutamine (Q), arginine (R), glycine (G), proline (P), either alone or (preferably) in combination, are most preferred activators of mTOR or inhibitors of autophagy for use in human cells, for packaging and targeting to cells. It is preferred that a hemodynamic peptide comprises at least 50%, more preferably at least 75% and most preferably 100% amino acids selected from the group A, Q, G, V, L, P, I and R. Preferably, a hemodynamic peptide as provided herein has a length in the range of 4-12 amino acids, more preferably 4-8 amino acids. Preferably, such a hemodynamic peptide is a linear peptide. A functional hemodynamic peptide analog according to this disclosure may be more preferably selected from the group of peptides comprising a dipeptide sequence selected from the group of AQ, LQ, PQ, VQ, GQ. A functional hemodynamic peptide according to this disclosure may be more preferably selected from the group of peptides comprising a tripeptide sequence selected from the group of AQL, LQL, PQL, VQL, GQL, PLQ, LQG, PQV, VGQ, LQP, LQV, AQG, QPL, PQV, VGQ, GQG. Amino acids leucine (L), alanine (A), glutamine (Q), and proline (P) are reported to have most prominent mTOR associated autophagic inhibitory effects on human cells (A J Meijer et al. Amino Acids 2015, 47, 2037-2063). Glycine (G; Zhong Z, Wheeler M D, Li X, Froh M, Schemmer P, Yin M, Bunzendaul H, Bradford B, Lemasters J J. 1-Glycine: a novel anti-inflammatory, immunomodulatory, and cytoprotective agent. Curr Opin Clin Nutr Metab Care 6: 229-240, 2003) improves amino-acid-stimulated mammalian target of rapamycin (mTOR) complex 1 activation. Hence, as herein provided for inclusion in a hemodynamic peptide according to this disclosure, leucine (L), alanine (A), glutamine (Q), glycine (G) and proline (P), either alone or (preferably) in combination, are preferred activators of mTOR or inhibitors of autophagy for use in human cells. It is more preferred that a hemodynamic peptide comprises at least 50%, more preferably at least 75% and most preferably 100% amino acids selected from the group A, Q, G, V, L, and P. In a more preferred embodiment, a hemodynamic peptide comprises at least 75% and most preferably 100% amino acids selected from the group A, Q, G, and V.
[0109] In a most preferred embodiment, a hemodynamic peptide according to this disclosure is a tetrapeptide that comprises 100% amino acids selected from the group A, Q, G, and V. Typical preferred examples of such a preferred tetrapeptide are AQGV (SEQ ID NO:1), LQGV (SEQ ID NO:4), VGQA (SEQ ID NO:3), VGQL (SEQ ID NO:6), AQVG (SEQ ID NO:2), and LQVG (SEQ ID NO:5). Most typically preferred is AQGV (SEQ ID NO:1) having been subject of a human clinical trial as provided below.
[0110] This disclosure includes the use of a hemodynamic peptide, or a functional analog thereof, for use in the treatment of a human subject to improve the subject's length of stay at the ICU, further to shorten the subject's length of stay at the ICU. One way in which this may be attained is by modifying fluid retention in the human subject. The use of hemodynamic peptide, or a functional analog thereof, in accordance with this disclosure, includes the treatment of human patients that are believed to be at risk from treatment with a vasopressor or an inotropic medication and/or anticipated to require hemodynamic therapy with fluid therapy. Such human patients include patients that are or are to be admitted, or are expected to be admitted, into intensive care, and for which shortening length-of-stay at ICU is desired. Hence, the use of hemodynamic peptide, or a functional analog thereof, includes a use for the treatment of human patients that are believed to be at risk from treatment or expected to need treatment with vasopressor or inotropic medication and/or with fluid therapy, is provided as shown e.g., in the examples. Preferably, in use for shortening a subject's length of stay at the ICU, in human patients that are believed to be at risk, the hemodynamic peptide is administered at a rate that is at least 10 mg/kg patient weight per hour (mg/kg/hr). Preferably the administration rate is at least 20 mg, at least 30, at least 40 or, most preferably, at least 50 mg/kg/hr. Preferably, the hemodynamic peptide is administered for at least 1 hour, more preferably at least 1.5 hours, most preferably at least 2 hours. Preferably, the administration of the hemodynamic peptide is at a rate of at least 20 mg/kg/hr and administered 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.
[0111] Embodiment 1: A hemodynamic peptide, or a functional analog thereof, for use in the treatment of a human subject, the use comprising modifying hemodynamics in the human subject.
[0112] Embodiment 2: A hemodynamic peptide, or a functional analog thereof, for use in the treatment of a human subject considered at risk or suffering from fluid overload, the use comprising modifying hemodynamics in the human subject.
[0113] Embodiment 3: A hemodynamic peptide, or a functional analog thereof, for use in the treatment of a human subject considered at risk or suffering from excess vasopressor/inotropic use, the use comprising modifying hemodynamics in the human subject.
[0114] Embodiment 4: A hemodynamic peptide, or a functional analog thereof, for use in the treatment of a human subject, wherein the human subject is subjected to induced trauma and wherein the use comprises modifying hemodynamics in the human subject.
[0115] Embodiment 5: A hemodynamic peptide, or a functional analog thereof, for use in the treatment of a human subject having impaired kidney function, the use comprising modifying hemodynamics in the human subject.
[0116] Embodiment 6: A hemodynamic peptide, or a functional analog thereof, for use as in accordance with any one of embodiments 1-5, wherein the use reduces fluid retention in the human subject.
[0117] Embodiment 7: A hemodynamic peptide, or a functional analog thereof, for use in accordance with any one of embodiments 1-6 wherein the use comprises a reduced use of vasopressive agents.
[0118] Embodiment 8: A hemodynamic peptide, or a functional analog thereof, for use in accordance with any one of embodiments 1-7 wherein the use comprises a reduced fluid intake.
[0119] Embodiment 9: A hemodynamic peptide, or a functional analog thereof, for use in accordance with embodiment 7, wherein the reduced use of vasopressive agents comprises a reduced duration of vasopressive agent use.
[0120] Embodiment 10: A hemodynamic peptide, or a functional analog thereof, for use in accordance with any one of embodiments 6-9, wherein the subject is subjected to induced trauma.
[0121] Embodiment 11: A hemodynamic peptide, or a functional analog thereof, for use in accordance with any one of embodiments 6-10 wherein the use improves kidney function in the human subject.
[0122] Embodiment 12: A hemodynamic peptide, or a functional analog thereof, for use in accordance with embodiment 11, wherein the improved kidney function involves an improved GFR rate.
[0123] Embodiment 13: A hemodynamic peptide, or a functional analog thereof, for use in accordance with any one of embodiments 6-12, wherein the human subject has impaired kidney function the impaired kidney function being AKI.
[0124] Embodiment 14: A hemodynamic peptide, or a functional analog thereof, for use as in accordance with any one of embodiments 1-13, wherein the use reduces leakage of plasma from the blood to peripheral tissue and/or organs.
[0125] Embodiment 15: A hemodynamic peptide, or a functional analog thereof, for use in accordance with any one of embodiments 1-14, wherein the use is in a human subject suffering from or at risk of heart failure.
[0126] Embodiment 16: A hemodynamic peptide, or a functional analog thereof, for use in accordance with any one of embodiments 1-15, wherein the use is in a human subject at risk of having edema.
[0127] Embodiment 17: A hemodynamic peptide, or a functional analog thereof, for use in accordance with any one of embodiments 4-16, wherein the human subject has been subjected to induced trauma, the induced trauma being surgery.
[0128] Embodiment 18: A hemodynamic peptide, or a functional analog thereof, for use in accordance with embodiment 17, wherein the surgery requires a cardiopulmonary bypass.
[0129] Embodiment 19: A hemodynamic peptide, or a functional analog thereof, for use in accordance with any one of embodiments 1-18, wherein the peptide is administered into the bloodstream.
[0130] Embodiment 20: A hemodynamic peptide, or a functional analog thereof, for use in accordance with embodiment 19, wherein the peptide is administered at a rate of at least 70 mg/kg body weight/hour.
[0131] Embodiment 21: A hemodynamic peptide, a functional analog thereof, for use in accordance with embodiment 19 or embodiment 20, wherein the peptide is administered for at least 1 hour.
[0132] Embodiment 22: A hemodynamic peptide, a functional analog thereof, for use in accordance with any one of embodiments 17-21, wherein the administration is during surgery.
[0133] Embodiment 23: A hemodynamic peptide, or a functional analog thereof, for use in accordance with any one of embodiments 1-22, wherein the administration is during anti-cancer treatment.
[0134] Embodiment 24: A hemodynamic peptide, or a functional analog thereof, for use in accordance with any one of embodiments 1-23, wherein the administration is during an adverse drug reaction.
[0135] Embodiment 25: A hemodynamic peptide, or a functional analog thereof, for use in accordance with any one of embodiments 1-24, wherein the human subject is admitted into intensive care, and wherein the use improves parameters measured of the human subject, the parameters of the human subject determined to assess remaining in intensive care.
[0136] Embodiment 26: A hemodynamic peptide, or a functional analog thereof, for use in accordance with embodiment 25, wherein the improvement in parameters results in a reduced length of stay at intensive care.
[0137] Embodiment 27: A hemodynamic peptide, or a functional analog thereof, for use as in accordance with any one of embodiments 1-26, wherein the uses induces vasoconstriction.
[0138] Embodiment 28: A method of treatment comprising administering a hemodynamic peptide, or a functional analog thereof, to a human subject, the human subject being in need of maintaining hemodynamic stability.
[0139] Embodiment 29: A method of treatment comprising administering a hemodynamic peptide, or a functional analog thereof, to a human subject, the human subject being in need of improving hemodynamic stability.
[0140] Embodiment 30: A method of treatment comprising administering a hemodynamic peptide, or a functional analog thereof, to a human subject, the human subject having impaired kidney function, wherein the treatment of administering a hemodynamic peptide comprises maintaining or improving hemodynamic stability in the human subject.
[0141] Embodiment 31: A method of treatment comprising administering a hemodynamic peptide, or a functional analog thereof, to a human subject, the human subject being in need of improving an adverse drug reaction.
[0142] Embodiment 32: A method of treatment comprising administering a hemodynamic peptide, or a functional analog thereof, to a human subject, the human subject having or suspected of having Clarkson's disease (CLS).
[0143] Embodiment 33: A method of treatment comprising administering a hemodynamic peptide, or a functional analog thereof, to a human subject, the human subject having or suspected of having an adverse drug reaction affecting capillary leakage.
[0144] Embodiment 34: A method of treatment comprising administering a hemodynamic peptide, or a functional analog thereof, to a human subject, the human subject being in need of maintaining hemodynamic stability.
[0145] Embodiment 35: A method of treatment comprising administering a hemodynamic peptide, or a functional analog thereof, to a human subject, the human subject being in need of improving hemodynamic stability.
[0146] Embodiment 36: A method of treatment comprising administering a hemodynamic peptide, or a functional analog thereof, to a human subject, the human subject having impaired kidney function, wherein the treatment of administering a hemodynamic peptide comprises maintaining or improving hemodynamic stability in the human subject.
Examples
[0147] A Phase 2 clinical trial was designed to test EA-230 (tetrapeptide AQGV; SEQ ID NO:1) in cardiac surgery (CABG) patients who frequently develop hemodynamic imbalances leading to organ dysfunction. The study was finalized in 2019 and results demonstrate significantly improved renal function after treatment with EA-230, with a statistically highly significant reduction in LOS (length of stay in ICU and Hospital) and a reduction of hospital re-admission. These beneficial effects of EA 230 translate into a significant better recovery of open-heart-surgery patients treated with EA 230, providing a distinct economic benefit. This positive outcome likely emerged from the during this Phase 2 trial unexpectedly shown and greatly beneficial effects of EA-230 on hemodynamic stability of intensive care patients that leads to a reduction in fluid overload, reduction of vasopressor use, and improvement in kidney functionality.
[0148] During cardiac surgery, 180 patients received (double-blinded, placebo-controlled, randomized) 90 mg/kg/hour EA-230 or placebo. The primary endpoint was safety. Efficacy was assessed by immunomodulation (plasma interleukin (IL)-6 concentrations), renal function (glomerular filtration rate using iohexol and creatinine [GFRiohexol, eGFRMDRD] and the incidence of acute kidney injury [AKI, RIFLE criteria]), cardiovascular effects (fluid balance, vasoactive agents) and general outcome (length-of-stay).
[0149] Median [IQR] age was 68 [62-74] years, 158/180 males. No safety concerns emerged. EA-230 did not modulate IL-6 (area under the curve 2730 [1968-3760] vs. 2680 [2090-3570] pg/ml*hour for EA-230 and placebo group respectively, p=0.80). GFR increased following surgery (meanΔ±SEM GFRiohexol 19±2 vs. 16±2 ml/min/1.73 m2 respectively, p=0.13, eGFRMDRD 6±1 vs. 2±1 ml/min/1.73 m2, respectively, p=0.01). EA-230 tended to prevent AKI (stage Injury: 7% vs. 18%, respectively, p=0.07). Patients in the EA-230 group needed less fluids compared to placebo-treated patients (217±108 vs. 605±103 ml, respectively, p=0.01), while the use of vasoactive agents was similar in both groups (p=0.39). Hospital length-of-stay was shorter in EA-230 treated patients (8 [7-11] vs. 10 [8-12] days, respectively, p=0.001).
[0150] Safety of EA-230
[0151] Final analysis of the EASI-study showed an excellent safety profile of treatment with EA-230. A continuous infusion of 90 mg/kg/hour EA-230 for up to 4 hours was well tolerated by patients undergoing elective CABG surgery. Pharmacokinetic studies indicate that EA-230 is rapidly (within 5-10 minutes) cleared from the circulation when infusion is terminated. Patients that received EA-230 seemed to experience less (serious) adverse events and less mayor clinical adverse events. In conclusion, the safety profile of EA-230 in patients undergoing elective CABG surgery was comparable if not better to the safety profile of patients receiving a continuous placebo infusion.
[0152] Efficacy of EA-230
[0153] Continuous infusion of 90 mg/kg/hour EA-230 for up to 4 hours in patients undergoing elective CABG surgery demonstrated unexpected but distinct clinical benefits of treatment with EA-230. Foremost, treatment with EA-230 (n=90) resulted in a highly significant reduction of about 3 days of total post-operative length-of-stay. While no effects on CABG-induced cytokine responses (with primary endpoint IL-6) were found, treatment with EA-230 distinctly caused an overall significant improvement of hemodynamic stability, based on reduced need of blood pressure (vasopressor/inotropic) medication and reduced need for fluid therapy, therewith preventing against post-operative fluid overload. Also, a significant overall improvement of kidney function was found. As detailed herein, prolonged treatment with EA-230 during surgery provides increased clinical benefits of patient recovery rates after surgery. The results also show beneficial effects of prolonged treatment (long=longer than median) with EA-230 on kidney function, measured as glomerular filtration rate (GFR), as compared with patients that were treated shorter than median treatment. The findings (
[0154] Summary of EA-230 Effects
[0155] Early administration led us to the detection of novel and highly beneficial effects of EA-230 on hemodynamics, kidney function, length of stay in ICU and hospital, which relate to improved hemodynamic stability. Treatment of patients with EA-230 during surgery significantly reduced the need for hemodynamic therapy (combined fluid therapy and blood pressure medication) after surgery (p=0.006). Besides these improved hemodynamics, EA-230 significantly improved kidney function (as determined by its effects on the glomerular filtration rate) and plasma levels of kidney function biomarker creatinine (p=0.003). It also significantly shortened post-surgery recovery stay at the ICU and significantly reduced length of stay in the hospital (
[0156] Effects of EA-230 in Human Patients
[0157] A prospective, randomized, double-blind, placebo-controlled study was performed in which 180 elective patients, undergoing on-pump coronary artery bypass grafting, with or without concomitant valve surgery, were enrolled. Patients were randomized in a 1:1 ratio and received either EA-230, 90 mg/kg/hour, or a placebo. These were infused at the start of the surgical procedure until the end of the use of the cardiopulmonary bypass. The main focus in this first-in-patient study was on safety and tolerability of EA-230. The primary efficacy endpoint was the modulation of the inflammatory response by EA-230 quantified as the change in interleukin-6 plasma concentrations after surgery. A key secondary endpoint was the effect of EA-230 on renal function.
[0158] Design and Setting
[0159] The present study was a single-center, prospective, double-blind, placebo-controlled, randomized, single-dose phase II study. It has an adaptive design to evaluate the safety and immunomodulatory effects of EA-230 in patients undergoing on-pump cardiac surgery for coronary artery bypass grafting (CABG) with or without concomitant valve surgery. 180 eligible patients were included and were randomized to receive either active or placebo treatment in a 1:1 ratio. This was a first-in-patient safety and tolerability study, of which the primary efficacy objective was to assess the immunomodulatory effects of EA-230. The key-secondary efficacy endpoint was the effect of EA-230 on renal function. This study was described in accordance with the Standard Protocol Items: Recommendations for Interventional Trial (SPIRIT) guidelines, and registered at clinicaltrials.gov under number NCT03145220.
[0160] Randomization and Stratification
[0161] On the morning of surgery, patients were randomized by non-blinded independent study personnel for active or placebo treatment. Study personnel used Good-Clinical-Practice-approved data management software (Castor EDC, Amsterdam, The Netherlands) in this process. The Castor system applies a stratified randomization to ensure equal distribution between active and placebo treatment of patients with known risk factors for adverse outcomes. Three strata were included: 1) a CABG procedure with or without concomitant valve surgery; 2) pre-operative renal function with an estimated GFR of ≤30, 31-90 and >90 ml/min/1.73 m2; and 3) a EuroSCORE II of <4 or ≥4 (Nashef et al., Eur. J. Cardiothora. Surg. 2012 April; 41(4):734-44).
[0162] Blinding
[0163] Double-blind conditions were maintained for all patients, the attending physicians and the medical study team personnel involved in all blinded study procedures, data collection and/or data analyses. Non-blinded study personnel not involved in any other study procedures prepared the study medication. Infusion systems and solutions for active and placebo treatment were identical in appearance and texture. Unblinding was authorized by the sponsor after completion of the study, performance of a blinded data review and locking of the database.
[0164] Study Intervention
[0165] Intravenous infusion of EA-230, 90 mg/kg/hour, or placebo, was initiated at the moment of first surgical incision using an automated infusion pump. Infusion rate was set at 250 ml/hour, and infusion was continued until cessation of the CPB, or after 4 hours of continuous infusion, whichever comes first.
[0166] The EA-230 formulation was packed in sterile 5 ml glass vials, containing 1500 mg/vial, dissolved in water for injection at a final concentration of 300 mg/ml with an osmolality of 800 to 1000 mOsm/kg. The placebo formulation consisted of sodium chloride diluted in water for injection in identical sterile 5 ml glass vials containing 29 mg/ml to reach a solution with an identical osmolality. EA-230 and placebo were prepared for continuous intravenous infusion with an osmolality of <400 mOsm/Kg by adding the appropriate amount of EA-230 or placebo to 1000 ml normal saline under aseptic conditions. Placebo and active treatment vials, were manufactured by HALIX BV (Leiden, the Netherlands).
[0167] Adverse Events (AEs)
[0168] All AEs were judged by the investigators with regard to severity (“mild, moderate, or severe”) according to Common Terminology Criteria for Adverse Events guidelines 4.030 and their perceived relation to the study drug (“definitely, probably, possibly, or unrelated/unlikely to be related”). SAEs or SUSARs include death, life-threatening disease, persistent and/or significant disability and/or incapacity, and hospitalization and/or prolongation of inpatient hospitalization.
[0169] Ethical Considerations, Data Quality Assurance & Patient and Public Involvement.
[0170] The study was conducted in accordance with the ethical principles of the Declaration of Helsinki (ICH E6(R1)), the Medical Research Involving Human Subjects Act, guidelines of Good Clinical Practice and European Directive (2001/20/CE). Informed consent was obtained before any study-specific procedures were performed. Data was handled confidentially and anonymously and Good-Clinical-Practice standards were applied. The handling of patient data in this study complies with the Dutch Personal Data Protection Act (in Dutch: Wet Bescherming Persoonsgegevens, WBP). Patients and the public were not involved in the design and/or the conduct of the study protocol. Study outcome was disseminated to all study participants individually. The burden of the intervention was assessed by the independent ethics committees CMO and CCMO, which include lay members.
[0171] Results
[0172] When assessing the data obtained during the clinical trial, strikingly, no immunomodulatory or anti-inflammatory effects were apparently observed as no significant difference of plasma levels between the EA-230 and placebo group were observed for IL-8, IL-10, IL-1RA, IL-17, MCP-1 and ICAM and other cytokines tested. This was also the case for IL-6 plasma levels, the primary endpoint of the study (see
TABLE-US-00001 TABLE 1 Adverse events (AEs) in the EASI-study. AEs, serious adverse events (SAE), and suspected unexpected serious adverse reaction (SUSAR) with differences between treatment groups are listed here. Significantly less (Chi Square P < 0.05) AEs were found in the EA-230 treatment group (217) than in the placebo treated group (283). Significantly less patients (Chi Square P < 0.05) suffering from fluid retention were found the EA-230 treatment group (n = 2) than in the placebo treated group (n = 11), p < 0.05. EA-230 (N = 91) Placebo (N = 89) Overall (N = 180) e n (%) e n (%) e n (%) Any AE 217 78 (85.7) 283 81 (91) 500 159 (88.3) Any SAE 23 12 (13.2) 19 17 (19.1) 32 29 (16.1) Any SUSAR 0 0 (0) 1 1 (1.1) 1 1 (0.6) AE of mild intensity 188 76 (83.5) 231 78 (87.6) 419 154 (85.6) AE of moderate intensity 23 22 (24.2) 45 27 (30.3) 68 49 (27.2) AE of severe intensity 6 5 (5.5) 7 4 (4.5) 13 9 (5) Blood and lymphatic system disorders Overall 6 5 (5.5) 8 8 (9) 14 13 (7.2) Anemia 5 5 (5.5) 8 8 (9) 13 13 (7.2) Hemorrhagic diathesis 1 1 (1.1) 0 0 (0) 1 1 (0.6) Gastrointestinal disorders Overall 27 21 (23.1) 36 23 (25.8) 63 44 (24.4) Nausea 15 15 (16.5) 12 12 (13.5) 27 27 (15) Infections and infestations Overall 15 13 (14.3) 22 17 (19.1) 37 30 (16.7) Metabolism and nutrition disorders Overall 10 10 (11) 22 14 (15.7) 32 24 (13.3) Fluid retention 2 2 (2.2) 9 9 (10.1) 11 11 (6.1) Psychiatric disorders Overall 9 9 (9.9) 14 13 (14.6) 23 22 (12.2) Delirium 5 5 (5.5) 9 9 (10.1) 14 14 (7.8) Renal and urinary disorders Overall 4 4 (4.4) 12 11 (12.4) 16 15 (8.3) N = Number of patients involved n = Number of patients experiencing the event e = Number of events
TABLE-US-00002 TABLE 2 Average on-pump length of patients with average age of patients, split in quartiles Q1, Q2, Q3 and Q4 of pump length, and thus of treatment duration, and of all patients tested (Q1-Q4). Treatment Average on-pump-length Age in years Quartiles group in minutes (+/−SD) (+/−SD) Q1 EA-230 112 +/− 12 68.5 +/− 7.3 Q1 Placebo 113 +/− 7 70.3 +/− 7.9 Q2 EA-230 137 +/− 5 66.5 +/− 9.5 Q2 Placebo 136 +/− 6 68.1 +/− 6.9 Q3 EA-230 164 +/− 8 66.3 +/− 8.7 Q3 Placebo 159 +/− 8 68.3 +/− 11.0 Q4 EA-230 211 +/− 24 65.0 +/− 7.4 Q4 Placebo 207 +/− 22 64.0 +/− 10.6 Q1-Q4 EA-230 156 +/− 37 66.5 +/− 8.3 Q1-Q4 Placebo 153 +/− 39 67.7 +/− 9.3
[0173] Hemodynamic Stability in the EASI-Study
[0174] In
TABLE-US-00003 TABLE 3 Frequencies Statistics RIFLE Netto score fluid (incl urine days on balance output) Treatment group vasopressin day 0-2 no AKI Placebo N Valid 42 42 Missing 0 0 Mean 1.38 1951.97 Median 1.00 1957.15 Std. Deviation .795 1027.255 Active N Valid 51 51 Missing 0 0 Mean 1.37 1348.47 Median 1.00 1213.70 Std. Deviation 1.131 1304.211 AKI Placebo N Valid 47 47 Missing 0 0 Mean 2.23 3342.04 Median 2.00 2965.20 Std. Deviation 2.108 2275.286 Active N Valid 39 39 Missing 0 0 Mean 1.97 2549.16 Median 2.00 2328.80 Std. Deviation 1.423 1517.623
Modulation of Fluid Balance and Vasopressor Use by Treatment with EA-230
[0175] The effects of EA-230 versus placebo were tested in uni- and multivariate models (see Table 4). Input/independent variable: treatment group (EA-230 or placebo). Output/dependent variables were: endpoint of fluid balance first 72 hours, days on vasopressin or vasopressor score (area under the curve). Effects of EA-230 versus placebo were tested on two combined variables in model A: fluid balance first 72 hours+days on vasopressin and model B: Fluid balance first 72 hours+vasopressor score AUC. The results of testing in both multivariate models showed significant improvement of hemodynamic parameters in patients receiving EA-230. This was observed in model A (fluid balance first 72 hours+days on vasopressin) p=0.006 and in model B (fluid balance first 72 hours+vasopressor score AUC) p=0.008. In the group of patients that showed no AKI, hemodynamic effects of EA-230 were significantly better as well, illustrating that improvement in hemodynamics can occur independent of kidney failure.
TABLE-US-00004 TABLE 4 Goal-directed hemodynamic therapy by EA-230. An analysis is shown for model A for the total group and for subgroups of acute kidney injury split conform 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). The corresponding p-values are listed. TREATMENT EFFECTS ON: Univariate 1. Univariate Multivariate Significant improvement of Fluid balance 2. Days on 1 and 2 active over placebo first 72 hours vasopressin combined RIFLE stage Total group 0.441 0.807 0.006 No AKI 0.017 0.996 0.048 Risk 0.807 0.564 0.753 Injury 0.051 0.055 0.114 Uni- and multivariate general linear model analysis
[0176] Combined, these results indicate that the use of EA-230 can improve and/or maintain hemodynamics in human patients, as assessed by, among other things, affecting the duration of vasopressor use, amount of vasopressor administered and/or fluid balance. In particular, EA-230 improves hemodynamic stability after open heart surgery in humans. Permeability governs the amount of fluid leaking from blood vessels. Administration of fluid therapy generally increases leakage. Based on Phase II trial patient observations, a significant reduction of adverse fluid retention (fluid leakage with fluid overload) was found in patients treated with EA-230 (p=0.03). Also, contractility governs tone. It is often adjusted by administration of blood-pressure medications, which, however, may show major detrimental side effects. Based on Phase II trial patient observations, a considerable reduction of required blood pressure medication use was found in the half of patients treated longest with EA-230 (>156 min; p=0.093). Mean maximum concentrations (mean Cmax) were also found as determined in vivo in humans for EA-230 in the Phase II clinical trial. Mean arterial Cmax found: 30500 ng/ml (range 12500 to 57500 ng/ml). Mean venous Cmax found: 68400 ng/ml (range 19600 to 113000 ng/ml)
[0177] EA-230 has an Advantageous Effect on Kidney Function
[0178] Effects of EA-230 on modulation in incidence of different stages of acute kidney injury (AKI) were determined according to the RIFLE criteria (RIFLE: risk, injury, failure, loss of kidney function, and end-stage kidney disease classification, Clin. Kidney J. 2013 February; 6(1): 8-14). In the EA-230 group, the number of patients with no AKI increased, whereas the number of patients in the Injury category of the RIFLE criteria decreased (see
[0179] Length of Stay in ICU, Hospital and Readmissions
[0180] In the study, effects on length of stay at the ICU of patients and length of stay in the hospital (inpatient care) were investigated (see
TABLE-US-00005 TABLE 5 Number of readmissions in the EASI-study (CABG-study). The number of patients that had to be re-admitted to the hospital due to clinical disease in the period post-surgery. Readmittance was scored in the period of 28 days after operation, and in the period ranging from 29-90 days after operation, and for the total period of 90 days after operation. Readmittance was reduced in patients receiving EA-230 treated group. CABG-study CABG-study Total CABG- Table of Re- Re-admission Re-admission study Total admissions day 28 day 90 Re-admission patients Placebo 5 5 10 89 EA-230 2 2 4 91 Total 7 7 14 180
[0181] Furthermore, in the patient group treated with AQGV (SEQ ID NO:1), the number of patients suffering from AKI Injury was reduced, and when patients suffered AKI injury, these patients did not have a prolonged length-of-stay, as observed in the placebo group and length of stay was similar to patients having no AKI or patients being at risk of AKI (see
[0182] Treatment with EA-230 herewith shows strong beneficial effects on recovery. EA 230-treated patients required significantly less hemodynamic therapy, regained post-surgical kidney function significantly faster and remained for a shorter period of time in the Intensive Care Unit (ICU) and in the hospital, as compared to placebo-treated patients.
[0183] These novel hemodynamic effects of EA-230 appear to be independent of anti-inflammatory effects of EA-230. In short, significant improvements of hemodynamic stability, kidney function and post-operative recovery of EA-230 treated patients relate to novel effects of EA-230 on blood vessel-permeability and blood vessel-contractility. EA-230 given during surgery shows significant improvements in patient recovery after surgery, over placebo patient. EA-230 treated patients are released faster from intensive care (p=0.0232) and hospital (p=0.0015). EA-230 improves hemodynamic stability (p=0.006) and kidney function (p=0.003). Whilst the primary endpoint—short term inflammatory cytokine (IL-6) reduction—was missed, long-term patient recovery was significantly improved by EA-230. Throughout surgery, EA-230 was shown to be safe and well tolerated. In conclusion, EA-230 given during surgery significantly improves recovery after surgery.
[0184] Significant improvement was found of hemodynamic stability (reducing fluid therapy and blood pressure medication; p=0.006), with: significant improvement of kidney function (improved glomerular filtration rate reduces plasma creatinine; p=0.003), significant reduction of patients suffering from adverse fluid retention during recovery (2 for EA-230, 9 for placebo; p=0.03), and considerable reduction of re-admissions to the hospital in the 90 days after surgery (4 for EA 230, 10 for placebo; p=0.09).
[0185] Further Analysis Biomarkers Related to Vasostriction and/or Vasodilation
[0186] In view of the effects observed on hemodynamics and kidney function, plasma samples are further analyzed with regard to selected biomarkers. Plasma samples of control patients and patients receiving the EA-230 are analyzed with regard to biomarkers Endothelin-1, VEGF, Angiotensine II and cAMP and natriuretic peptides. The following assays are used to determine the levels of the biomarkers
[0187] In Vitro Effects of EA-230 and AQGV Analogs
[0188] In an in vitro transwell assay the effects of the AQGV (SEQ ID NO:1) peptide (EA-230), and analogs thereof, is tested on human endothelial cells. Briefly, endothelial cells are cultured in transwell culture dishes and culture medium is supplemented with hemodynamic peptides, and analogs thereof, or control compounds known to affect endothelial layer permeability, vasoconstriction and/or vasodilation. Suitable human endothelial cells are e.g., HUVECs (Park et al., Stem Cell Rev. 2 (2): 93-102, 2006; Jiménez et al., Cytotechnology 65, 1-14, 2012) and HMEC-1 (Ades E W, et al. J. Invest. Dermatol. 99(6): 683-690, 1992). The permeability of the endothelial layer is determined by measuring the penetration of a macromolecule. Furthermore, levels of biomarkers are also determined in culture medium. Experiments are carried as outlined e.g., in Cox et al., Shock, 43(4):322-6; 2015. In HUVEC permeability tests, established human endothelial vascular cells (HUVEC), capable of lining blood vessels, are grown in cell-culture (i.e., n=5) on sieves, in multiple test formats, allowing determination of leak-through products depending on various test-concentrations of EA-230 peptide or placebo controls used, establishing pharmacological parameters of EA-230-peptide-effects on permeability in human cells.
[0189] Also, Bravo et al (J. Pharmacol. Toxicol. Methods 2018 January-February; 89:47-53) developed an impedance-based contraction assay using the xCELLigence RTCA MP system. This technology utilizes special 96-well E-plates with gold microelectrode arrays printed in individual wells to monitor cellular adhesion by recording the electrical impedance in real time. The impedance change (percentage vs. control) can be used as the readout for cellular contraction. Established human aortic smooth muscle cells (HaSMC), capable of contracting blood vessels, are grown in cell-culture (i.e., n=3) on gold-electrodes, in multiple test formats, allowing electrical-impedance-determination of endothelin-1 induced smooth muscle cell contractions, depending on various test-concentrations of EA-230-peptide or placebo controls used, establishing pharmacological parameters of EA-230-effects on contractility in human cells. In addition, isolated aneurysmatic (n=3)/control (n=3) patient human aortic smooth muscle cells (APaSMC), differentially capable of contracting blood vessels, are grown in cell-cultures on gold-electrodes in multiple test formats, allowing electrical-impedance-determination of ionomycin-induced smooth muscle cell contractions of patient-versus-control cells, depending on various test-concentrations of EA-230-peptide or placebo controls used, detecting effects of EA-230 in patient cells.