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WO2016004441A1 - Méthodes de prévention ou de traitement d'une lésion d'infarctus aigu du myocarde - Google Patents

Méthodes de prévention ou de traitement d'une lésion d'infarctus aigu du myocarde Download PDF

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Publication number
WO2016004441A1
WO2016004441A1 PCT/US2015/039270 US2015039270W WO2016004441A1 WO 2016004441 A1 WO2016004441 A1 WO 2016004441A1 US 2015039270 W US2015039270 W US 2015039270W WO 2016004441 A1 WO2016004441 A1 WO 2016004441A1
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Prior art keywords
peptide
arg
lys
phe
subject
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D. Travis Wilson
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Stealth Peptides International Inc
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Stealth Peptides International Inc
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Priority claimed from US14/640,633 external-priority patent/US20160030501A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides

Definitions

  • the present technology relates generally to compositions and methods of preventing or treating cardiac ischemia-reperfusion injury.
  • the present technology relates to administering aromatic-cationic peptides in effective amounts to prevent or treat cardiac ischemia-reperfusion injury in mammalian subjects.
  • the present technology relates generally to the treatment of myocardial infarction in subjects diagnosed with hypertension through administration of therapeutically effective amounts of aromatic-cationic peptides to subjects in need thereof.
  • the peptide is administered at one or more of the following time points: prior to an episode of cardiac ischemia, prior to a heart procedure (e.g., bypass surgery, thrombolysis or angioplasty), after an episoe of cardiac ischemia, after a heart procedure (e.g., bypass surgery, thrombolysis or angioplasty).
  • the subject is human.
  • the peptide is administered to provide an effective amount at a concentration of peptide in the target tissue of about 10 "8 to 10 "6 molar. In some embodiments, the peptide is administered orally, topically, systemically, intravenously, subcutaneously, intraperitoneally, or intramuscularly. In some embodiments, the peptide is D-Arg-2',6'-Dmt-Lys-Phe-NH 2 .
  • the present technology is related to methods for reducing infarct size in a mammalian subject treated with a metal based stent, comprising administering to the mammalian subject a therapeutically effective amount of the peptide D-Arg-2'6'-Dmt-Lys- Phe- H2.
  • the peptide is administered at one or more of the following time points: prior to an episode of cardiac ischemia, prior to a heart procedure (e.g., bypass surgery, thrombolysis or angioplasty), after an episoe of cardiac ischemia, after a heart procedure (e.g., bypass surgery, thrombolysis or angioplasty).
  • the subject is human.
  • the peptide is administered to provide an effective amount at a concentration of peptide in the target tissue of about 10 ⁇ 8 to 10 ⁇ 6 molar. In some embodiments, the peptide is administered orally, topically, systemically, intravenously, subcutaneous ly, intraperitoneally, or intramuscularly. In some embodiments, the peptide is D- Arg-2',6'-Dmt-Lys-Phe-NH 2 .
  • the disclosure provides a method of treating or preventing cardiac ischemia-reperfusion injury, comprising administering to a mammalian subject a
  • the aromatic-cationic peptide is a peptide having:
  • the mammalian subject is a human.
  • 2p m is the largest number that is less than or equal to r+1, and a may be equal to pt.
  • the aromatic-cationic peptide may be a water-soluble peptide having a minimum of two or a minimum of three positive charges.
  • the peptide comprises one or more non-naturally occurring amino acids, for example, one or more D-amino acids.
  • the C-terminal carboxyl group of the amino acid at the C-terminus is amidated.
  • the peptide has a minimum of four amino acids. The peptide may have a maximum of about 6, a maximum of about 9, or a maximum of about 12 amino acids.
  • the peptide comprises a tyrosine or a 2',6'-dimethyltyrosine (Dmt) residue at the N-terminus.
  • the peptide may have the formula Tyr-D-Arg- Phe-Lys-NH2 or 2',6'-Dmt-D-Arg-Phe-Lys-NH2.
  • the peptide comprises a phenylalanine or a 2',6'-dimethylphenylalanine residue at the N-terminus.
  • the peptide may have the formula Phe-D-Arg-Phe-Lys-NH2 or 2',6'-Dmp-D-Arg- Phe-Lys-NH2.
  • the aromatic-cationic peptide has the formula D- Arg-2',6'-Dmt-Lys-Phe-NH 2 .
  • the peptide is defined by formula I:
  • R 1 and R 2 are each independently selected from
  • R J and R 4 are each independently selected from
  • halogen encompasses chloro, fluoro, bromo, and iodo
  • R 5 , R 6 , R 7 , R 8 , and R 9 are each independently selected from
  • halogen encompasses chloro, fluoro, bromo, and iodo; and n is an integer from 1 to 5.
  • R 1 and R 2 are hydrogen; R 3 and R 4 are methyl; R 5 , R 6 , R 7 , R 8 , and R 9 are all hydrogen; and n is 4.
  • the peptide is defined by formula II:
  • R and R are each independently selected from
  • R , R , R , R , R , R , R and R are each independently selected from
  • halogen encompasses chloro, fluoro, bromo, and iodo; and n is an integer from 1 to 5.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are all hydrogen; and n is 4.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , and R 11 are all hydrogen; R 8 and R 12 are methyl; R 10 is hydroxyl; and n is 4.
  • the aromatic-cationic peptides may be administered in a variety of ways.
  • the peptides may be administered orally, topically, intranasally,
  • intraperitoneally intravenously, subcutaneously, or transdermally (e.g., by iontophoresis).
  • the subject is administered a dosage of the aromatic-cationic peptide at least about 1 femtomole peptide; at least 10 femtomole peptide; at least 100 femtomole peptide; at least 1 picomole peptide; at least 10 picomole peptide; at least about 100 picomole peptide; at least 1 nanomole peptide; at least 10 nanomole peptide; at least 100 nanomole peptide; at least 1 micromole peptide; at least 10 micromole peptide; at least 100 micromole peptide; at least 1 millimole peptide; at least 10 millimole peptide; at least 100 millimole peptide; at least 1 mole peptide; at least 10 mole peptide; at least 100 mole peptide; at least 10 mole peptide; at least 100 mole peptide; at least 1000 mole peptide; at least 10,000 mole peptide; at least 100,000 mole
  • the subject is administered a dosage of the aromatic-cationic peptide at least about 1 femtogram peptide; at least 10 femtogram peptide; at least 100 femtogram peptide; at least 1 picogram peptide; at least 10 picogram peptide; at least about 100 picogram peptide; at least 1 nanogram peptide; at least 10 nanogram peptide; at least 100 nanogram peptide; at least 1 microgram peptide; at least 10 microgram peptide; at least 100 microgram peptide; at least 1 milligram peptide; at least 10 milligram peptide; at least 100 milligram peptide; at least 1 gram peptide; at least 10 gram peptide; or at least 100 gram peptide.
  • the subject is administered a dosage of the aromatic-cationic peptide at least about 1 femtomoles peptide per liter/kilogram body weight/hour to the subject; at least 10 femtomoles peptide per liter/kilogram body weight/hour to the subject; at least 100 femtomole peptide per liter/kilogram body weight/hour to the subject; at least 1 picomole peptide per liter/kilogram body weight/hour to the subject; at least 10 picomole peptide per liter/kilogram body weight/hour to the subject; at least about 100 picomole peptide per liter/kilogram body weight/hour to the subject; at least 1 nanomole peptide per liter/kilogram body weight/hour to the subject; at least 10 nanomole peptide per
  • liter/kilogram body weight/hour to the subject at least 100 nanomole peptide per liter/kilogram body weight/hour to the subject; at least 1 micromole peptide per liter/kilogram body weight/hour to the subject; at least 10 micromole peptide per liter/kilogram body weight/hour to the subject; at least 100 micromole peptide per liter/kilogram body weight/hour to the subject; at least 1 millimole peptide; at least 10 millimole peptide per liter/kilogram body weight/hour to the subject; at least 100 millimole peptide per
  • liter/kilogram body weight/hour to the subject at least 1 mole peptide per liter/kilogram body weight/hour to the subject; at least 10 mole peptide per liter/kilogram body weight/hour to the subject; at least 100 mole peptide; at least 1000 mole peptide per liter/kilogram body weight/hour to the subject; at least 10,000 mole peptide per liter/kilogram body weight/hour to the subject; at least 100,000 mole peptide per liter/kilogram body weight/hour to the subject.
  • therapeutically effective amount provides a concentration of peptide in a target tissue of about 10 "15 to 10 "3 molar; about 10 "12 to 10 "6 ; about 10 “8 to 10 "6 ; about 10 "7 to 10 “6 ; or about ; about 10 "7 .
  • FIG. 1 is a graph showing typical LV pressure (red) and volume -conducted ECG (blue) recordings from an isolated guinea pig heart.
  • FIG. 2 is an illustration of the study design for animals used in the study. Hatched bars represent periods of global ischemia, accomplished by turning off the perfusion to the heart.
  • FIG. 3 is a series of charts showing the effects of the peptide.
  • Panel A left ventricular pressure LVDP
  • Panel B shows coronary flow rates
  • Panel C is a representative ECG trace from a guinea pig heart in the study
  • Panel D shows the QT interval (as assessed by the volume-conducted ECG)
  • Panel E shows the QTc interval (QT interval normalized to the RR interval using Bazzett's formula). Addition of the peptide to the buffer had no effect on any of the baseline experimental parameters examined. All error bars represent standard error of the mean (sem).
  • FIG. 4 presents data showing infarct size (expressed as a function of the zone at risk) for hearts exposed to 20 min of global ischemia:
  • FIG. 4A presents representative images from hearts in the 20 min ischemia group;
  • FIGs. 4B and 4C are graphs quantifying infarct sizes across groups for 20 min of ischemia. Numbers above graphs represent t-tests for comparisons with control, and error bars represent the standard error of the mean.
  • FIG. 5 is a series of graphs showing characterization of arrhythmias for the 2 hour reperfusion period from hearts exposed to 20 min of global ischemia:
  • Panel A is a representative picture showing a heart going into arrhythmia during reperfusion. ECG trace in blue, LVP trace in Red, with an arrow denoting the onset of VT and subsequent loss of developed pressure after the onset of fatal arrhythmia.
  • Panel B is a graph of arrhythmia severity quantified with a scoring system as described in text.
  • Panel C is a graph of time to sustained (>1 min) arrhythmia during reperfusion. There were no significant differences between groups in B or C.
  • FIG. 6 is a schematic diagram of illustrative embodiments of study designs employed.
  • FIG. 7 is a schematic diagram of the inclusion criteria for selection subjects for the STEMI study.
  • FIG. 8A-8C are charts comparing: 8A) area under the curve (AUC) for subjects with bare metal stents in ITT analysis either treated or untreated with D-Arg-2'6'-Dmt-Lys-Phe- H2; 8B) infarct size at day 4 for hypertensive subjects with bare metal stents in ITT analysis either treated or untreated with D-Arg-2'6'-Dmt-Lys-Phe-NH2; and 8C) infarct size at day 4 for hypertensive patients with bare metal stents in PAP either treated or untreated with D- Arg-2'6'-Dmt-Lys-Phe-NH 2 .
  • AUC area under the curve
  • FIG. 9 is a chart showing that subjects with metal based stents treated with D-Arg- 2'6'-Dmt-Lys-Phe-NH2 showed a decrease in serum creatine kinase MB as compared to the untreated subjects with metal based stents.
  • the "administration" of an agent, drug, or peptide to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or
  • Administration includes self-administration and the administration by another.
  • amino acid includes naturally-occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally-occurring amino acids.
  • Naturally-occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally-occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally-occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally- occurring amino acid.
  • Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • the term "effective amount" refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in, cardiac ischemia-reperfusion injury or one or more symptoms associated with cardiac ischemia-reperfusion injury.
  • the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • the compositions can also be administered in combination with one or more additional therapeutic compounds.
  • the aromatic-cationic peptides may be administered to a subject having one or more signs or symptoms of acute myocardial infarction, such as chest pain described as a pressure sensation, fullness, or squeezing in the midportion of the thorax; radiation of chest pain into the jaw or teeth, shoulder, arm, and/or back; dyspnea or shortness of breath; epigastric discomfort with or without nausea and vomiting; and diaphoresis or sweating.
  • a "therapeutically effective amount" of the aromatic-cationic peptides is meant levels in which the physiological effects of an ischemia-reperfusion injury are, at a minimum, ameliorated.
  • ischemia reperfusion injury refers to the damage caused first by restriction of the blood supply followed by a sudden resupply of blood and the attendant generation of free radicals. Ischemia is a decrease in the blood supply to the tissue and is followed by reperfusion, a sudden perfusion of oxygen into the deprived tissue.
  • An "isolated” or “purified” polypeptide or peptide is substantially free of cellular material or other contaminating polypeptides from the cell or tissue source from which the agent is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • an isolated aromatic-cationic peptide would be free of materials that would interfere with diagnostic or therapeutic uses of the agent.
  • Such interfering materials may include enzymes, hormones and other proteinaceous and nonproteinaceous solutes.
  • polypeptide As used herein, the terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
  • Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, generally referred to as proteins.
  • Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
  • Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art.
  • the terms “treating” or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder.
  • a subject is successfully "treated” for ischemia reperfusion injury if, after receiving a therapeutic amount of the aromatic-cationic peptides according to the methods described herein, the subject shows observable and/or measurable reduction in or absence of one or more signs and symptoms of ischemia reperfusion injury, such as, e.g., reduced infarct size.
  • the various modes of treatment or prevention of medical conditions as described are intended to mean “substantial”, which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved.
  • prevention or "preventing” of a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.
  • preventing ischemia-reperfusion injury includes preventing oxidative damage or preventing mitochondrial permeability transitioning, thereby preventing or ameliorating the harmful effects of the loss and subsequent restoration of blood flow to the heart.
  • the present technology relates to the treatment or prevention of acute cardiac ischemia-reperfusion injury by administration of certain aromatic-cationic peptides. Also provided is a method of treating a myocardial infarction in a subject to prevent injury to the heart upon reperfusion.
  • the subject is administered the peptide during and after the ischemia. In yet another embodiment, the subject is administered the peptide continuously before, during, and after ischemia. In another embodiment, the subject is administered the peptide during and after the reperfusion. In yet another embodiment, the subject is administered the peptide continuously before, during, and after reperfusion. In one embodiment, the subject is administered the peptide as a continuous IV infusion from immediately prior to reperfusion for about 1 to 3 hours after reperfusion. Thereafter, the subject may be administered the peptide chronically by any route of administration.
  • the subject is administered the peptide prior to a
  • the subject is administered the peptide after the revascularization procedure. In another embodiment, the subject is administered the peptide during and after the revascularization procedure. In yet another embodiment, the subject is administered the peptide continuously before, during, and after the
  • the subject is administered the peptide regularly (i.e., chronically) following an AMI and/or a revascularization procedure.
  • the subject is administered an aromatic-cationic peptide of the present technology, such as D-Arg-2'6'-Dmt-Lys-Phe-NH2 or a pharmaceutically acceptable salt thereof, for at least one week, at least one month or at least one year after the
  • the aromatic-cationic peptides are water-soluble and highly polar. Despite these properties, the peptides can readily penetrate cell membranes.
  • the aromatic-cationic peptides typically include a minimum of three amino acids or a minimum of four amino acids, covalently joined by peptide bonds.
  • the maximum number of amino acids present in the aromatic-cationic peptides is about twenty amino acids covalently joined by peptide bonds.
  • the maximum number of amino acids is about twelve, more preferably about nine, and most preferably about six.
  • the amino acids of the aromatic -cationic peptides can be any amino acid.
  • amino acid is used to refer to any organic molecule that contains at least one amino group and at least one carboxyl group. Typically, at least one amino group is at the a position relative to a carboxyl group.
  • the amino acids may be naturally occurring.
  • Naturally occurring amino acids include, for example, the twenty most common levorotatory (L) amino acids normally found in mammalian proteins, i.e., alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (He), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan, (Trp), tyrosine (Tyr), and valine (Val).
  • L levorotatory
  • Naturally occurring amino acids include, for example, amino acids that are synthesized in metabolic processes not associated with protein synthesis.
  • amino acids ornithine and citrulline are synthesized in mammalian metabolism during the production of urea.
  • Another example of a naturally occurring amino acid includes hydroxyproline (Hyp).
  • the peptides optionally contain one or more non-naturally occurring amino acids.
  • the peptide has no amino acids that are naturally occurring.
  • the non-naturally occurring amino acids may be levorotary (L-), dextrorotatory (D-), or mixtures thereof.
  • Non- naturally occurring amino acids are those amino acids that typically are not synthesized in normal metabolic processes in living organisms, and do not naturally occur in proteins.
  • the non-naturally occurring amino acids suitably are also not recognized by common proteases.
  • the non-naturally occurring amino acid can be present at any position in the peptide.
  • the non-naturally occurring amino acid can be at the N-terminus, the C-terminus, or at any position between the N-terminus and the C-terminus.
  • the non-natural amino acids may, for example, comprise alkyl, aryl, or alkylaryl groups not found in natural amino acids.
  • Some examples of non-natural alkyl amino acids include a-aminobutyric acid, ⁇ -aminobutyric acid, ⁇ -aminobutyric acid, ⁇ -aminovaleric acid, and ⁇ -aminocaproic acid.
  • Some examples of non-natural aryl amino acids include ortho-, meta, and para-aminobenzoic acid.
  • Some examples of non-natural alkylaryl amino acids include ortho-, meta-, and para-aminophenylacetic acid, and y-phenyl-p-aminobutyric acid.
  • Non-naturally occurring amino acids include derivatives of naturally occurring amino acids.
  • the derivatives of naturally occurring amino acids may, for example, include the addition of one or more chemical groups to the naturally occurring amino acid.
  • one or more chemical groups can be added to one or more of the 2', 3', 4', 5', or 6' position of the aromatic ring of a phenylalanine or tyrosine residue, or the 4', 5', 6', or 7' position of the benzo ring of a tryptophan residue.
  • the group can be any chemical group that can be added to an aromatic ring.
  • Some examples of such groups include branched or unbranched C 1 -C 4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C 1 -C 4 alkyloxy (i.e., alkoxy), amino, C 1 -C 4 alkylamino and C 1 -C4 dialkylamino (e.g., methylamino, dimethylamino), nitro, hydroxyl, halo (i.e., fluoro, chloro, bromo, or iodo).
  • Some specific examples of non-naturally occurring derivatives of naturally occurring amino acids include norvaline (Nva) and norleucine (Me).
  • derivatization of a carboxyl group of an aspartic acid or a glutamic acid residue of the peptide is amidation with ammonia or with a primary or secondary amine, e.g. methylamine, ethylamine, dimethylamine or diethylamine.
  • Another example of derivatization includes esterification with, for example, methyl or ethyl alcohol.
  • Another such modification includes derivatization of an amino group of a lysine, arginine, or histidine residue.
  • amino groups can be acylated.
  • Some suitable acyl groups include, for example, a benzoyl group or an alkanoyl group comprising any of the Ci- C 4 alkyl groups mentioned above, such as an acetyl or propionyl group.
  • the non-naturally occurring amino acids are preferably resistant, and more preferably insensitive, to common proteases.
  • non-naturally occurring amino acids that are resistant or insensitive to proteases include the dextrorotatory (D-) form of any of the above-mentioned naturally occurring L-amino acids, as well as L- and/or D- non- naturally occurring amino acids.
  • the D-amino acids do not normally occur in proteins, although they are found in certain peptide antibiotics that are synthesized by means other than the normal ribosomal protein synthetic machinery of the cell. As used herein, the D-amino acids are considered to be non-naturally occurring amino acids.
  • the peptides should have less than five, preferably less than four, more preferably less than three, and most preferably, less than two contiguous L-amino acids recognized by common proteases, irrespective of whether the amino acids are naturally or non-naturally occurring.
  • the peptide has only D- amino acids, and no L-amino acids. If the peptide contains protease sensitive sequences of amino acids, at least one of the amino acids is preferably a non-naturally-occurring D-amino acid, thereby conferring protease resistance.
  • protease sensitive sequence includes two or more contiguous basic amino acids that are readily cleaved by common proteases, such as endopeptidases and trypsin.
  • basic amino acids include arginine, lysine and histidine.
  • the aromatic-cationic peptides should have a minimum number of net positive charges at physiological pH in comparison to the total number of amino acid residues in the peptide.
  • the minimum number of net positive charges at physiological pH will be referred to below as (p m ).
  • the total number of amino acid residues in the peptide will be referred to below as (r).
  • the minimum number of net positive charges discussed below are all at physiological pH.
  • physiological pH refers to the normal pH in the cells of the tissues and organs of the mammalian body. For instance, the physiological pH of a human is normally approximately 7.4, but normal physiological pH in mammals may be any pH from about 7.0 to about 7.8.
  • Net charge refers to the balance of the number of positive charges and the number of negative charges carried by the amino acids present in the peptide. In this specification, it is understood that net charges are measured at physiological pH.
  • the naturally occurring amino acids that are positively charged at physiological pH include L- lysine, L-arginine, and L-histidine.
  • the naturally occurring amino acids that are negatively charged at physiological pH include L-aspartic acid and L-glutamic acid.
  • a peptide has a positively charged N-terminal amino group and a negatively charged C-terminal carboxyl group. The charges cancel each other out at physiological pH.
  • the peptide Tyr-Arg-Phe-Lys- Glu-His-Trp-D-Arg has one negatively charged amino acid (i.e., Glu) and four positively charged amino acids (i.e., two Arg residues, one Lys, and one His). Therefore, the above peptide has a net positive charge of three.
  • the aromatic-cationic peptides have a relationship between the minimum number of net positive charges at physiological pH (p m ) and the total number of amino acid residues (r) wherein 3p m is the largest number that is less than or equal to r + 1.
  • the relationship between the minimum number of net positive charges (p m ) and the total number of amino acid residues (r) is as follows: TABLE 1. Amino acid number and net positive charges (3p m ⁇ p+1)
  • the aromatic -cationic peptides have a relationship between the minimum number of net positive charges (p m ) and the total number of amino acid residues (r) wherein 2p m is the largest number that is less than or equal to r + 1.
  • the relationship between the minimum number of net positive charges (p m ) and the total number of amino acid residues (r) is as follows:
  • the minimum number of net positive charges (p m ) and the total number of amino acid residues (r) are equal.
  • the peptides have three or four amino acid residues and a minimum of one net positive charge, suitably, a minimum of two net positive charges and more preferably a minimum of three net positive charges.
  • aromatic -cationic peptides have a minimum number of aromatic groups in comparison to the total number of net positive charges (p t ).
  • the minimum number of aromatic groups will be referred to below as (a).
  • Naturally occurring amino acids that have an aromatic group include the amino acids histidine, tryptophan, tyrosine, and phenylalanine.
  • the hexapeptide Lys-Gln-Tyr-D-Arg-Phe-Trp has a net positive charge of two (contributed by the lysine and arginine residues) and three aromatic groups (contributed by tyrosine, phenylalanine and tryptophan residues).
  • the aromatic-cationic peptides should also have a relationship between the minimum number of aromatic groups (a) and the total number of net positive charges at physiological pH (p t ) wherein 3a is the largest number that is less than or equal to p t + 1, except that when p t is 1, a may also be 1.
  • the aromatic-cationic peptides have a relationship between the minimum number of aromatic groups (a) and the total number of net positive charges (pt) wherein 2a is the largest number that is less than or equal to p t + 1.
  • the relationship between the minimum number of aromatic amino acid residues (a) and the total number of net positive charges (p t ) is as follows:
  • the number of aromatic groups (a) and the total number of net positive charges (p t ) are equal.
  • Carboxyl groups especially the terminal carboxyl group of a C-terminal amino acid, are suitably amidated with, for example, ammonia to form the C-terminal amide.
  • the terminal carboxyl group of the C-terminal amino acid may be amidated with any primary or secondary amine.
  • the primary or secondary amine may, for example, be an alkyl, especially a branched or unbranched C1-C4 alkyl, or an aryl amine.
  • the amino acid at the C-terminus of the peptide may be converted to an amido, N- methylamido, N-ethylamido, N,N-dimethylamido, ⁇ , ⁇ -diethylamido, N-methyl-N- ethylamido, N-phenylamido or N-phenyl-N-ethylamido group.
  • the free carboxylate groups of the asparagine, glutamine, aspartic acid, and glutamic acid residues not occurring at the C- terminus of the aromatic-cationic peptides may also be amidated wherever they occur within the peptide.
  • the amidation at these internal positions may be with ammonia or any of the primary or secondary amines described above.
  • the aromatic-cationic peptide is a tripeptide having two net positive charges and at least one aromatic amino acid. In a particular embodiment, the aromatic-cationic peptide is a tripeptide having two net positive charges and two aromatic amino acids.
  • Aromatic-cationic peptides include, but are not limited to, the following peptide examples:
  • the peptides have mu-opioid receptor agonist activity (i.e., they activate the mu-opioid receptor).
  • Mu-opioid activity can be assessed by radioligand binding to cloned mu-opioid receptors or by bioassays using the guinea pig ileum (Schiller et al, Eur J Med Chem, 35:895-901, 2000; Zhao et al, J Pharmacol Exp Ther, 307:947-954, 2003).
  • Activation of the mu-opioid receptor typically elicits an analgesic effect.
  • an aromatic-cationic peptide having mu-opioid receptor agonist activity is preferred.
  • an aromatic-cationic peptide that activates the mu-opioid receptor For example, during short-term treatment, such as in an acute disease or condition, it may be beneficial to use an aromatic-cationic peptide that activates the mu-opioid receptor. Such acute diseases and conditions are often associated with moderate or severe pain. In these instances, the analgesic effect of the aromatic-cationic peptide may be beneficial in the treatment regimen of the human patient or other mammal. An aromatic-cationic peptide which does not activate the mu-opioid receptor, however, may also be used with or without an analgesic, according to clinical requirements.
  • an aromatic-cationic peptide that does not have mu-opioid receptor agonist activity is preferred.
  • the use of an aromatic-cationic peptide that activates the mu-opioid receptor may be contraindicated.
  • the potentially adverse or addictive effects of the aromatic-cationic peptide may preclude the use of an aromatic-cationic peptide that activates the mu-opioid receptor in the treatment regimen of a human patient or other mammal. Potential adverse effects may include sedation, constipation and respiratory depression.
  • an aromatic-cationic peptide that does not activate the mu-opioid receptor may be an appropriate treatment.
  • Peptides which have mu-opioid receptor agonist activity are typically those peptides which have a tyrosine residue or a tyrosine derivative at the N-terminus (i.e., the first amino acid position).
  • Suitable derivatives of tyrosine include 2'-methyltyrosine (Mmt); 2',6'- dimethyltyrosine (2'6'-Dmt); 3',5'-dimethyltyrosine (3'5'Dmt); N,2',6'-trimethyltyrosine (Tmt); and 2'-hydroxy-6'-methyltryosine (Hmt).
  • a peptide that has mu-opioid receptor agonist activity has the formula Tyr-D-Arg-Phe-Lys-NH 2 .
  • This peptide has a net positive charge of three, contributed by the amino acids tyrosine, arginine, and lysine and has two aromatic groups contributed by the amino acids phenylalanine and tyrosine.
  • the tyrosine can be a modified derivative of tyrosine such as in 2 ',6 '-dimethyltyrosine to produce the compound having the formula 2',6'- Dmt-D-Arg-Phe-Lys-NH 2 .
  • This peptide has a molecular weight of 640 and carries a net three positive charge at physiological pH. This peptide readily penetrates the plasma membrane of several mammalian cell types in an energy-independent manner (Zhao et al. , J. Pharmacol Exp Ther., 304:425-432, 2003).
  • Peptides that do not have mu-opioid receptor agonist activity generally do not have a tyrosine residue or a derivative of tyrosine at the N-terminus (i.e., amino acid position 1).
  • the amino acid at the N-terminus can be any naturally occurring or non-naturally occurring amino acid other than tyrosine.
  • the amino acid at the N-terminus is phenylalanine or its derivative.
  • Exemplary derivatives of phenylalanine include 2'- methylphenylalanine (Mmp), 2',6'-dimethylphenylalanine (2',6'-Dmp), N,2',6'- trimethylphenylalanine (Tmp), and 2'-hydroxy-6'-methylphenylalanine (Hmp).
  • an aromatic-cationic peptide that does not have mu-opioid receptor agonist activity has the formula Phe-D-Arg-Phe-Lys-NH2.
  • the N-terminal phenylalanine can be a derivative of phenylalanine such as 2',6'-dimethylphenylalanine (2'6'- Dmp).
  • the peptide containing 2',6'-dimethylphenylalanine at amino acid position 1 has the formula 2',6'-Dmp-D-Arg-Phe-Lys-NH 2 .
  • the amino acid sequence of the peptide is rearranged such that Dmt is not at the N-terminus.
  • An example of such an aromatic-cationic peptide that does not have mu-opioid receptor agonist activity has the formula D-Arg-2'6'-Dmt-Lys-Phe-NH 2 .
  • the peptides mentioned herein and their derivatives can further include functional analogs.
  • a peptide is considered a functional analog if the analog has the same function as the stated peptide.
  • the analog may, for example, be a substitution variant of a peptide, wherein one or more amino acids are substituted by another amino acid. Suitable substitution variants of the peptides include conservative amino acid substitutions.
  • Amino acids may be grouped according to their physicochemical characteristics as follows:
  • Non-polar amino acids Ala(A) Ser(S) Thr(T) Pro(P) Gly(G) Cys (C);
  • Aromatic amino acids Phe(F) Tyr(Y) Trp(W) His (H).
  • substitutions of an amino acid in a peptide by another amino acid in the same group is referred to as a conservative substitution and may preserve the physicochemical characteristics of the original peptide.
  • substitutions of an amino acid in a peptide by another amino acid in a different group is generally more likely to alter the characteristics of the original peptide.
  • one or more naturally occurring amino acids in the aromatic - cationic peptides are substituted with amino acid analogs.
  • analogs that activate mu-opioid receptors include, but are not limited to, the aromatic -cationic peptides shown in Table 5.
  • Tmt N, 2',6'-trimethyltyrosine
  • dnsDap P-dansyl-L-a,P-diaminopropionic acid
  • Examples of analogs that do not activate mu-opioid receptors include, but are not limited to, the aromatic-cationic peptides shown in Table 6.
  • amino acids of the peptides shown in Table 5 and 6 may be in either the L- or the D- configuration.
  • the peptides may be synthesized by any of the methods well known in the art. Suitable methods for chemically synthesizing the protein include, for example, those described by Stuart and Young in Solid Phase Peptide Synthesis, Second Edition, Pierce Chemical Company (1984), and in Methods Enzymol, 289, Academic Press, Inc, New York (1997).
  • the aromatic-cationic peptides described herein are useful to prevent or treat disease.
  • the disclosure provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) cardiac ischemia-reperfusion injury.
  • the present methods provide for the prevention and/or treatment of cardiac ischemia-reperfusion injury in a subject by administering an effective amount of an aromatic- cationic peptide to a subject in need thereof.
  • suitable in vitro or in vivo assays are performed to determine the effect of a specific aromatic-cationic peptide-based therapeutic and whether its administration is indicated for treatment.
  • in vitro assays can be performed with representative animal models, to determine if a given aromatic-cationic peptide-based therapeutic exerts the desired effect in preventing or treating ischemia- reperfusion injury.
  • Compounds for use in therapy can be tested in suitable animal model systems including, but not limited to rats, mice, chicken, pigs, cows, monkeys, rabbits, and the like, prior to testing in human subjects. Similarly, for in vivo testing, any of the animal model systems known in the art can be used prior to administration to human subjects.
  • the invention provides a method for preventing, in a subject, cardiac ischemia-reperfusion injury by administering to the subject an aromatic-cationic peptide that prevents the initiation or progression of the condition.
  • Subjects at risk for cardiac ischemia-reperfusion injury can be identified by, e.g., any or a combination of diagnostic or prognostic assays as described herein.
  • compositions or medicaments of aromatic-cationic peptides are administered to a subject susceptible to, or otherwise at risk of a disease or condition in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.
  • Administration of a prophylactic aromatic-cationic can occur prior to the manifestation of symptoms characteristic of the aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • the appropriate compound can be determined based on screening assays described above.
  • compositions or medicaments are administered to a subject suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease, including its complications and intermediate pathological phenotypes in development of the disease.
  • the invention provides methods of treating an individual afflicted with cardiac ischemia-reperfusion injury.
  • the aromatic-cationic peptides of the present technology are useful in the treatment of subjects suffering from ST-Elevation Myocardial Infarction (STEMI) and hypertension.
  • at least one aromatic-cationic peptide of the present technology is administered to subjects with ST-Elevation Myocardial Infarction (STEMI) and hypertension.
  • treatment of subjects with STEMI and hypertension with aromatic-cationic peptides will have one or more of the following effects: reduce infarct volume, reduce myocardial edema, improve ST-segment resolution, reduce infarct size, increase left ventricle ejection fraction, reduce the occurrence of a new congestive heart failure (CHF) event post percutaneous coronary intervention (PCI).
  • CHF congestive heart failure
  • the aromatic-cationic peptide is one or more of 2',6'-dimethyl-Tyr-D-Arg-Phe- Lys-NH 2 , Phe-D-Arg-Phe-Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 .
  • the aromatic-cationic peptides of the present technology are useful in the reducing infarct size in subjects treated with metal based stents.
  • at least one aromatic-cationic peptide of the present technology is administered to subjects treated with metal based stents for acute myocardial infarction or vessel occlusion.
  • treatment with aromatic-cationic peptides reduces serum creatine kinase in subjects treated with metal based stents.
  • the aromatic- cationic peptide is one or more of 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 .
  • any method known to those in the art for contacting a cell, organ or tissue with a peptide may be employed. Suitable methods include in vitro, ex vivo, or in vivo methods. In vivo methods typically include the administration of an aromatic-cationic peptide, such as those described above, to a mammal, suitably a human. When used in vivo for therapy, the aromatic-cationic peptides are administered to the subject in effective amounts (i.e., amounts that have desired therapeutic effect). The dose and dosage regimen will depend upon the degree of the injury in the subject, the characteristics of the particular aromatic-cationic peptide used, e.g., its therapeutic index, the subject, and the subject's history.
  • the effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians.
  • An effective amount of a peptide useful in the methods may be administered to a mammal in need thereof by any of a number of well- known methods for administering pharmaceutical compounds.
  • the peptide may be administered systemically or locally.
  • the peptide may be formulated as a pharmaceutically acceptable salt.
  • pharmaceutically acceptable salt means a salt prepared from a base or an acid which is acceptable for administration to a patient, such as a mammal (e.g., salts having acceptable mammalian safety for a given dosage regime). However, it is understood that the salts are not required to be pharmaceutically acceptable salts, such as salts of intermediate compounds that are not intended for administration to a patient.
  • Pharmaceutically acceptable salts can be derived from pharmaceutically acceptable inorganic or organic bases and from
  • salts derived from pharmaceutically acceptable inorganic bases include ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, and zinc salts, and the like.
  • Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary and tertiary amines, including substituted amines, cyclic amines, naturally-occurring amines and the like, such as arginine, betaine, caffeine, choline, ⁇ , ⁇ '-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N- ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperadine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.
  • Salts derived from pharmaceutically acceptable inorganic acids include salts of boric, carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric or hydroiodic), nitric, phosphoric, sulfamic and sulfuric acids. Salts derived from
  • organic acids include salts of aliphatic hydroxyl acids (e.g., citric, gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids), aliphatic hydroxyl acids (e.g., citric, gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids), aliphatic hydroxyl acids (e.g., citric, gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids), aliphatic hydroxyl acids (e.g., citric, gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids), aliphatic hydroxyl acids (e.g., citric, gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids), aliphatic hydroxyl acids (e.g., citric, gluconic, glycolic, lactic, lactobionic, malic, and tart
  • monocarboxylic acids e.g., acetic, butyric, formic, propionic and trifluoroacetic acids
  • amino acids e.g., aspartic and glutamic acids
  • aromatic carboxylic acids e.g., benzoic, p- chlorobenzoic, diphenylacetic, gentisic, hippuric, and triphenylacetic acids
  • aromatic hydroxyl acids e.g., o-hydroxybenzoic, p-hydroxybenzoic, l-hydroxynaphthalene-2- carboxylic and 3-hydroxynaphthalene-2-carboxylic acids
  • ascorbic dicarboxylic acids (e.g., fumaric, maleic, oxalic and succinic acids), glucoronic, mandelic, mucic, nicotinic, orotic, pamoic, pantothenic, sulfonic acids (e.g., benzenesulfonic, camphosulfonic, ed
  • compositions for administration, singly or in combination, to a subject for the treatment or prevention of a disorder described herein.
  • Such compositions typically include the active agent and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.
  • compositions are typically formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral (e.g., intravenous, intradermal, intraperitoneal or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoretic, and transmucosal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants
  • the parenteral preparation can be enclosed in ampuoles, disposable syringes or multiple dose vials made of glass or plastic.
  • the dosing formulation can be provided in a kit containing all necessary equipment (e.g., vials of drug, vials of diluent, syringes and needles) for a treatment course (e.g., 7 days of treatment).
  • compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the aromatic-cationic peptide compositions can include a carrier, which can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • a carrier which can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thiomerasol, and the like.
  • Glutathione and other antioxidants can be included to prevent oxidation.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • typical methods of preparation include vacuum drying and freeze drying, which can yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
  • compositions can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such as sucrose or saccharin
  • the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • transdermal administration can be accomplished through the use of nasal sprays.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • transdermal administration may be performed my iontophoresis.
  • a therapeutic protein or peptide can be formulated in a carrier system.
  • the carrier can be a colloidal system.
  • the colloidal system can be a liposome, a phospholipid bilayer vehicle.
  • the therapeutic peptide is encapsulated in a liposome while maintaining peptide integrity.
  • there are a variety of methods to prepare liposomes See Lichtenberg et al, Methods Biochem. Anal., 33 :337-462 (1988); Anselem et al, Liposome Technology, CRC Press (1993)). Liposomal formulations can delay clearance and increase cellular uptake (See Reddy, Ann. Pharmacother., 34(7- 8):915-923 (2000)).
  • An active agent can also be loaded into a particle prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable or gastroretentive polymers or liposomes.
  • Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles and viral vector systems.
  • the carrier can also be a polymer, e.g., a biodegradable, biocompatible polymer matrix.
  • the therapeutic peptide can be embedded in the polymer matrix, while maintaining protein integrity.
  • the polymer may be natural, such as polypeptides, proteins or polysaccharides, or synthetic, such as poly a-hydroxy acids. Examples include carriers made of, e.g., collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin, and combinations thereof.
  • the polymer is poly-lactic acid (PLA) or copoly lactic/glycolic acid (PGLA).
  • PHA poly-lactic acid
  • PGLA copoly lactic/glycolic acid
  • the polymeric matrices can be prepared and isolated in a variety of forms and sizes, including microspheres and
  • the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylacetic acid.
  • Such formulations can be prepared using known techniques.
  • the materials can also be obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to specific cells with monoclonal antibodies to cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,81 1.
  • the therapeutic compounds can also be formulated to enhance intracellular delivery.
  • liposomal delivery systems are known in the art, see, e.g., Chonn and Cullis, "Recent Advances in Liposome Drug Delivery Systems," Current Opinion in Biotechnology 6:698-708 (1995); Weiner, “Liposomes for Protein Delivery: Selecting Manufacture and Development Processes,” Immunomethods, 4(3):201-9 (1994); and Gregoriadis, “Engineering Liposomes for Drug Delivery: Progress and Problems," Trends Biotechnol., 13(12):527-37 (1995).
  • Mizguchi et al, Cancer Lett., 100:63-69 (1996) describes the use of fusogenic liposomes to deliver a protein to cells both in vivo and in vitro.
  • Dosage, toxicity and therapeutic efficacy of the therapeutic agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • an effective amount of the aromatic-cationic peptides range from about 0.000000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day.
  • the dosage ranges are from about 0.0000001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day.
  • dosages can be 1 mg/kg body weight or 10 mg/kg body weight every day, every two days or every three days or within the range of 1-10 mg/kg every week, every two weeks or every three weeks.
  • a single dosage of peptide ranges from 0.1-10,000 micrograms per kg body weight.
  • aromatic-cationic peptide concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter.
  • An exemplary treatment regime entails administration once per day or once a week.
  • a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
  • a therapeutically effective amount of an aromatic-cationic peptide may be defined as a concentration of peptide at the target tissue of 10 "11 to 10 "6 molar, e.g., approximately 10 "7 molar. This concentration may be delivered by systemic doses of 0.01 to 100 mg/kg or equivalent dose by body surface area. The schedule of doses would be optimized to maintain the therapeutic concentration at the target tissue, most preferably by single daily or weekly administration, but also including continuous administration (e.g., parenteral infusion or transdermal application).
  • therapeutically effective amount provides a concentration of the aromatic-cationic peptide in a target tissue of about 10 "15 to 10 "3 molar; about 10 "12 to 10 "6 ; about 10 “8 to 10 “6 ; about 10 "7 to 10 “6 ; or about; about 10 "7 .
  • the dosage of the aromatic-cationic peptide is provided at an "ultralow", “low,” “mid,” or “high” dose level.
  • the ultralow dose is provided from about 0.001 ng/kg/h to about 0.01 mg/kg/h, suitably from about 0.001 ng/kg/h to about 10 ng/kg/h.
  • the low dose is provided from about 0.01 to about 0.5 mg/kg/h, suitably from about 0.01 to about 0.1 mg/kg/h.
  • the mid- dose is provided from about 0.1 to about 1.0 mg/kg/h, suitably from about 0.1 to about 0.5 mg/kg/h.
  • the high dose is provided from about 0.5 to about 10 mg/kg/h, suitably from about 0.5 to about 2 mg/kg/h.
  • the subject is administered a dosage of the aromatic-cationic peptide at least about 1 femtomole peptide; at least 10 femtomole peptide; at least 100 femtomole peptide; at least 1 picomole peptide; at least 10 picomole peptide; at least about 100 picomole peptide; at least 1 nanomole peptide; at least 10 nanomole peptide; at least 100 nanomole peptide; at least 1 micromole peptide; at least 10 micromole peptide; at least 100 micromole peptide; at least 1 millimole peptide; at least 10 millimole peptide; at least 100 millimole peptide; at least 1 mole peptide; at least 10 mole peptide; at least 100 mole peptide; at least 10 mole peptide; at least 100 mole peptide; at least 1000 mole peptide; at least 10,000 mole peptide; at least 100,000 mole
  • the subject is administered a dosage of the aromatic-cationic peptide at least about 1 femtogram peptide; at least 10 femtogram peptide; at least 100 femtogram peptide; at least 1 picogram peptide; at least 10 picogram peptide; at least about 100 picogram peptide; at least 1 nanogram peptide; at least 10 nanogram peptide; at least 100 nanogram peptide; at least 1 microgram peptide; at least 10 microgram peptide; at least 100 microgram peptide; at least 1 milligram peptide; at least 10 milligram peptide; at least 100 milligram peptide; at least 1 gram peptide; at least 10 gram peptide; or at least 100 gram peptide.
  • the subject is administered a dosage of the aromatic-cationic peptide at least about 1 femtomoles peptide per liter/kilogram body weight/hour to the subject; at least 10 femtomoles peptide per liter/kilogram body weight/hour to the subject; at least 100 femtomole peptide per liter/kilogram body weight/hour to the subject; at least 1 picomole peptide per liter/kilogram body weight/hour to the subject; at least 10 picomole peptide per liter/kilogram body weight/hour to the subject; at least about 100 picomole peptide per liter/kilogram body weight/hour to the subject; at least 1 nanomole peptide per liter/kilogram body weight/hour to the subject; at least 10 nanomole peptide per
  • liter/kilogram body weight/hour to the subject at least 1 micromole peptide per liter/kilogram body weight/hour to the subject; at least 10 micromole peptide per liter/kilogram body weight/hour to the subject; at least 100 micromole peptide per liter/kilogram body weight/hour to the subject; at least 1 millimole peptide; at least 10 millimole peptide per liter/kilogram body weight/hour to the subject; at least 100 millimole peptide per
  • liter/kilogram body weight/hour to the subject at least 1 mole peptide per liter/kilogram body weight/hour to the subject; at least 10 mole peptide per liter/kilogram body weight/hour to the subject; at least 100 mole peptide; at least 1000 mole peptide per liter/kilogram body weight/hour to the subject; at least 10,000 mole peptide per liter/kilogram body weight/hour to the subject; at least 100,000 mole peptide per liter/kilogram body weight/hour to the subject.
  • treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.
  • the mammal treated in accordance present methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice and rabbits.
  • the mammal is a human.
  • Example 1 Effects of aromatic-cationic peptides in protecting against cardiac ischemia-reperfusion injury in a guinea pig model
  • Hearts were cannulated by the aorta and perfused with a modified Krebs- Henseleit buffer containing: 118 mM NaCl, 24 mM NaHC0 3 , 4.75 mM KC1, 1.2 mM KH 2 ,P0 4 , 1.2 mM MgS0 4 , 2.0 mM CaCl 2 , and 10 mM glucose (gassed with 95/5% 0 2 /C0 2 ). Hearts were placed in a buffer-filled perfusion chamber and maintained at 37°C for the duration of the experiments. [0117] Following the initiation of perfusion, hearts were instrumented for the simultaneous observation of mechanical and electrical function.
  • a buffer-filled latex balloon was inserted into the left ventricle (via the mitral valve) for the measurement of left ventricular developed pressure, with balloon volume adjusted to establish an end diastolic pressure of 5-8 mm Hg.
  • Three electrodes were placed into the buffer-filled perfusion chamber for the measurement of volume-conducted ECG.
  • a pre-established protocol of electrode placement was utilized to obtain a signal analogous to Lead II of a typical 12-lead ECG. All physiological parameters were continuously monitored and stored on a personal computer using commercially available software (Chart, AD Instruments). Typical baseline values for the guinea pig heart can be seen in FIG. 1.
  • Ischemia/Reperfusion Hearts were exposed to global no-flow ischemia by stopping perfusion. The duration of ischemia was 20 min. At the end of the index ischemia, static buffer from the perfusion lines was washed out (via an accessory port proximal to the aortic cannula) and reperfusion ensued for 120 min. Peptide administration in the perfusate was accomplished via dissolving the compound in solution prior to administration. The reperfusion bolus dose was delivered to the heart via syringe through a drug-delivery port just above the aortic cannula.
  • the LV was dissected, sliced into 5 mm-thick slices, incubated in triphenyltetrazolium chloride (TTC) for 10 min (37°C), and digitally photographed for subsequent infarct size analysis. Infarct sizes are expressed as the infarcted area as a percentage of the LV (in the global ischemia model, the entire LV constitutes the zone-at-risk).
  • TTC triphenyltetrazolium chloride
  • Coronary Flow Rates were monitored continuously and are expressed as mL/min*g of LV. At the onset of reperfusion, hearts that were perfused with peptide beginning at the baseline period had higher flow rates throughout most of reperfusion. The control flow rates with the peptide whole time were different.
  • Hearts were cannulated by the aorta and perfused with a modified Krebs-Henseleit buffer containing (in mM): 1 18 NaCl, 24 NaHC03, 4.75 KC1, 1.2 KH2P04, 1.2 MgS04, 2.0 CaC12, and 10 glucose (gassed with 95/5% 02/C02). Hearts were placed in a buffer-filled perfusion chamber and maintained at 37°C for the duration of the experiments.
  • a modified Krebs-Henseleit buffer containing 1 18 NaCl, 24 NaHC03, 4.75 KC1, 1.2 KH2P04, 1.2 MgS04, 2.0 CaC12, and 10 glucose (gassed with 95/5% 02/C02).
  • the LV was dissected, sliced into 5mm-thick slices, incubated in triphenyltetrazolium chloride (TTC) for 10 minutes (37 °C), and digitally photographed for subsequent infarct size analysis. Infarct sizes are expressed as the infarcted area as a percentage of the LV (in the global ischemia model, the entire LV constitutes the zone-at-risk).
  • TTC triphenyltetrazolium chloride
  • Infarct Size Hearts were exposed to 20 minutes of global ischemia. Hearts that were treated with either InM or 0.01nM/0.1nM(30min R) D-Arg-2'6'-Dmt-Lys-Phe-NH 2 had lower incidence of infarction when compared to control group. For these D-Arg-2'6'-Dmt- Lys-Phe-NH2 treatment groups, the time of drug administration (i.e., pre- versus postishemic) did not influence the magnitude of efficacy. Cyclosporin also attenuated LR injury, but only when administered prior to ischemia. There was a strong trend for cyclosporin to reduce infarct size when administered at reperfusion. The low dose (O.OlnM) of D-Arg-2'6'-Dmt-Lys-Phe-NH2 was the most effective.
  • Coronary Flow Rates were monitored continuously and expressed as mL/min*g of whole heart wet weight. Treatment groups were control, InM D- Arg-2'6'-Dmt-Lys-Phe-NH 2 whole time; InM D-Arg-2'6'-Dmt-Lys-Phe-NH 2 at reperfusion; O. lnM D-Arg-2'6'-Dmt-Lys-Phe-NH 2 at reperfusion; O.OlnM D-Arg-2'6'-Dmt-Lys-Phe-NH 2 to O.
  • Example 3 Treating Subjects with ST-Elevation Myocardial Infarction (STEMI) and Hypertension
  • This example shows that aromatic-cationic peptides of the present technology are useful in the treatment of subjects diagnosed with STEMI and hypertension.
  • the EMBRACE STEMI study is known in the art (see e.g., Chakrabarti et ah, Am Heart J, 165(4):509-514 (2013), the content of which is incorporate by reference in its entirety). Briefly, the EMBRACE STEMI trial is a multicenter, randomized, double-blind, placebo-controlled study. The study design was approved by institutional and national regulatory bodies and ethical committees. Multiple safety reviews were performed by an independent Data and Safety Monitoring Board throughout the course of the trial. All subjects provided written informed consent prior to randomization.
  • Subjects aged 18 to 85 years with anterior STEMI undergoing first-time percutaneous coronary intervention (PCI) plus stenting with an anticipated time from ischemic symptoms to time of balloon inflation ⁇ 4 hours, with > 0.1 mV ST-segment elevation in at least 2 contiguous precordial leads were enrolled.
  • Major exclusion criteria included a history of prior myocardial infarction (MI), previous heart failure (e.g., known left ventricle ejection fraction (LVEF) ⁇ 30% prior to the qualifying infarct), and cardiogenic shock.
  • MI myocardial infarction
  • previous heart failure e.g., known left ventricle ejection fraction (LVEF) ⁇ 30% prior to the qualifying infarct
  • cardiogenic shock included a history of prior myocardial infarction (MI), previous heart failure (e.g., known left ventricle ejection fraction (LVEF) ⁇ 30% prior to the qualifying infarct), and cardiogenic shock.
  • MI myocardial infarction
  • D-Arg-2',6'-Dmt-Lys-Phe-NH 2 was administered > 15 minutes, but ⁇ 60 minutes prior to PCI and for 1 hour following reperfusion.
  • Subjects underwent cardiac magnetic resonance imaging (MRI) at 4 ⁇ 1 days and again at 30 ⁇ 7 days post-PCI.
  • MRI cardiac magnetic resonance imaging
  • IQR Interquartile range
  • LAD Left anterior descending
  • SD Standard deviation
  • the primary end point of the study was infarct size, or the area under the curve (AUC) of creatine kinase-MB (CK-MB) enzyme over 72 hours following PCI with the values log transformed and adjusted for the percent of artery distal to the occlusion and duration of symptoms (two variables known to confound the estimation of infarct size).
  • Secondary end points included the AUCO-72 of troponin I, peak cardiac enzymes, the ratio of the volume of infarcted myocardium (late contrast gadolinium enhancement) to the left ventricular (LV) mass on cardiac MRI at day 4 ⁇ 1, day 30 ⁇ 7, and the change ( ⁇ ) in ratios from day 4 ⁇ 1 to day 30 ⁇ 7, measures of myocardial structure and function (left ventricular ejection fraction (LVEF), left ventricular end systolic volume (LVESV), and left ventricular end diastolic volume (LVEDV)) at day 4 ⁇ 1, day 30 ⁇ 7, and the change ( ⁇ ) in these
  • CHF congestive heart failure
  • renal function serum creatinine, estimated glomerular filtration rate, cystatin C, and blood urea nitrogen
  • Safety endpoints included treatment emergent adverse events (TEAE).
  • TEAE treatment emergent adverse events
  • the treated grouped also had reduced frequency of new onset CHF as compared to the control group during the first 8 hours post- PCI (11 incidents in the control group vs. 5 incidents in the treated group).
  • the primary end point of the study was infarct size, or the area under the curve (AUC) of creatine kinase-MB (CK-MB) enzyme over 72 hours following PCI with the values log transformed and adjusted for the percent of artery distal to the occlusion and duration of symptoms (two variables known to confound the estimation of infarct size).
  • AUC area under the curve
  • h-SOD human superoxide dismutase
  • Curtis MJ Walker MJ. Quantification of arrhythmias using scoring systems: an examination of seven scores in an in vivo model of regional myocardial ischaemia.
  • a range includes each individual member.
  • a group having 1-3 cells refers to groups having 1, 2, or 3 cells.
  • a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

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Abstract

L'invention concerne des méthodes de prévention ou de traitement d'une lésion de reperfusion d'ischémie cardiaque chez un sujet mammifère. Les méthodes comprennent l'administration d'une quantité efficace d'un peptide aromatique-cationique à des sujets le nécessitant.
PCT/US2015/039270 2014-07-03 2015-07-06 Méthodes de prévention ou de traitement d'une lésion d'infarctus aigu du myocarde Ceased WO2016004441A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10870678B2 (en) 2016-04-11 2020-12-22 Arcuate Therapeutics, Inc. Chiral peptides

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120130480A1 (en) * 2010-11-18 2012-05-24 Robert Falotico Local vascular delivery of adenosine a2a receptor agonists to reduce myocardial injury
US20130195837A1 (en) * 2009-12-31 2013-08-01 Stealth Peptides International, Inc. Methods for the prevention or treatment of vessel occlusion injury
US20140100166A1 (en) * 2009-10-05 2014-04-10 University Of Washington Methods for the prevention or treatment of heart failure

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Publication number Priority date Publication date Assignee Title
US20140100166A1 (en) * 2009-10-05 2014-04-10 University Of Washington Methods for the prevention or treatment of heart failure
US20130195837A1 (en) * 2009-12-31 2013-08-01 Stealth Peptides International, Inc. Methods for the prevention or treatment of vessel occlusion injury
US20120130480A1 (en) * 2010-11-18 2012-05-24 Robert Falotico Local vascular delivery of adenosine a2a receptor agonists to reduce myocardial injury

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10870678B2 (en) 2016-04-11 2020-12-22 Arcuate Therapeutics, Inc. Chiral peptides

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