METHODS FOR THE PREVENTION OR TREATMENT

OF ACUTE MYOCARDIAL INFARCTION INJURY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.

62/020,673, filed July 3, 2014, and U.S. Application 14/640,633 filed March 6, 2015, which is a continuation of U.S. Patent Application No. 14/149,606 filed on January 7, 2014 (now abandoned), which is a continuation of U.S. Application No. 13/519,780 filed October 8, 2012 (now abandoned), which is the U.S. National Stage application of

PCT/US2010/062538, filed December 30, 2010, which claims the benefit of and priority to U.S Provisional Application 61/291,699 filed December 31, 2009, and U.S. Provisional Application No. 61/363, 138 filed July 9, 2010, the contents of which are hereby incorporated by reference their entireties.

TECHNICAL FIELD

[0002] The present technology relates generally to compositions and methods of preventing or treating cardiac ischemia-reperfusion injury. In particular, the present technology relates to administering aromatic-cationic peptides in effective amounts to prevent or treat cardiac ischemia-reperfusion injury in mammalian subjects.

SUMMARY

[0003] In one aspect, 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. In some embodiments, 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). In some embodiments, the subject is human. In some embodiments, 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 . [0004] In one aspect, 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. In some embodiments, 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). In some embodiments, the subject is human. In some embodiments, 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 .

[0005] In another aspect, the disclosure provides a method of treating or preventing cardiac ischemia-reperfusion injury, comprising administering to a mammalian subject a

therapeutically effective amount of an aromatic-cationic peptide. In some embodiments, the aromatic-cationic peptide is a peptide having:

at least one net positive charge;

a minimum of four amino acids;

a maximum of about twenty amino acids;

a relationship between the minimum number of net positive charges (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 ; and a relationship between the minimum number of aromatic groups (a) and the total number of net positive charges (p _t ) wherein 2a is the largest number that is less than or equal to p _t + 1, except that when a is 1, p _t may also be 1. In particular embodiments, the mammalian subject is a human.

[0006] In one embodiment, 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.

[0007] In one embodiment, the peptide comprises one or more non-naturally occurring amino acids, for example, one or more D-amino acids. In some embodiments, the C-terminal carboxyl group of the amino acid at the C-terminus is amidated. In certain embodiments, 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.

[0008] In one embodiment, the peptide comprises a tyrosine or a 2',6'-dimethyltyrosine (Dmt) residue at the N-terminus. For example, the peptide may have the formula Tyr-D-Arg- Phe-Lys-NH2 or 2',6'-Dmt-D-Arg-Phe-Lys-NH2. In another embodiment, the peptide comprises a phenylalanine or a 2',6'-dimethylphenylalanine residue at the N-terminus. For example, the peptide may have the formula Phe-D-Arg-Phe-Lys-NH2 or 2',6'-Dmp-D-Arg- Phe-Lys-NH2. In a particular embodiment, the aromatic-cationic peptide has the formula D- Arg-2',6'-Dmt-Lys-Phe-NH ₂ .

[0009] In one embodiment, the peptide is defined by formula I:

wherein R ¹ and R ² are each independently selected from

(i) hydrogen;

alkyl; where m = 1-3;

(v) [0011] R ^J and R ⁴ are each independently selected from

(i) hydrogen;

(ii) linear or branched Q-C6 alkyl;

(iii) C1-C6 alkoxy;

(iv) amino;

(v) C1-C4 alkylamino;

(vi) Ci-C4 dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where "halogen" encompasses chloro, fluoro, bromo, and iodo;

[0012] R ⁵ , R ⁶ , R ⁷ , R ⁸ , and R ⁹ are each independently selected from

(i) hydrogen;

(ii) linear or branched C1-C6 alkyl;

(iii) C 1-C6 alkoxy;

(iv) amino;

(v) C1-C4 alkylamino;

(vi) Ci-C4 dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where "halogen" encompasses chloro, fluoro, bromo, and iodo; and n is an integer from 1 to 5.

[0013] In a particular embodiment, 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.

[0014] In one embodiment, the peptide is defined by formula II:

[0015] wherein R and R are each independently selected from

(i) hydrogen;

(ii) linear or branched Ci-Ce alkyl; m = 1-3;

[0016] R , R , R , R , R , R , R , R , R and R are each independently selected from

(i) hydrogen;

(ii) linear or branched C1-C6 alkyl;

(iii) C1-C6 alkoxy;

(iv) amino;

(v) C1-C4 alkylamino;

(vi) Ci-C4 dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where "halogen" encompasses chloro, fluoro, bromo, and iodo; and n is an integer from 1 to 5. [0017] In a particular embodiment, 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. In another embodiment, 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.

[0018] The aromatic-cationic peptides may be administered in a variety of ways. In some embodiments, the peptides may be administered orally, topically, intranasally,

intraperitoneally, intravenously, subcutaneously, or transdermally (e.g., by iontophoresis).

[0019] In some embodiments, 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 1000 mole peptide; at least 10,000 mole peptide; at least 100,000 mole peptide.

[0020] In some embodiments, 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.

[0021] In some embodiments, 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.

[0022] In one embodiment, 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 .

BRIEF DESCRIPTION OF THE FIGURES

[0023] FIG. 1 is a graph showing typical LV pressure (red) and volume -conducted ECG (blue) recordings from an isolated guinea pig heart.

[0024] 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.

[0025] 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); and 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).

[0026] 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.

[0027] 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.

[0028] FIG. 6 is a schematic diagram of illustrative embodiments of study designs employed.

[0029] FIG. 7 is a schematic diagram of the inclusion criteria for selection subjects for the STEMI study.

[0030] 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 .

[0031] 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.

DETAILED DESCRIPTION

[0032] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the invention are described below in various levels of detail in order to provide a substantial understanding of the present invention.

[0033] In practicing the present invention, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. These techniques are well-known and are explained in, e.g., Current Protocols in Molecular Biology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989); DNA Cloning: A Practical Approach, Vols. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis, Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds. (1985); Transcription and Translation, Hames & Higgins, Eds. (1984); Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; the series, Meth. Enzymol, (Academic Press, Inc., 1984); Gene Transfer Vectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring Harbor Laboratory, NY, 1987); and Meth. Enzymol., Vols. 154 and 155, Wu & Grossman, and Wu, Eds., respectively.

[0034] The definitions of certain terms as used in this specification are provided below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0035] As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like.

[0036] As used herein, 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

subcutaneously), or topically. Administration includes self-administration and the administration by another.

[0037] As used herein, the term "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, γ-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.

[0038] As used herein, 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. In the context of therapeutic or prophylactic applications, 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. In the methods described herein, 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. For example, 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.

[0039] As used herein the term "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.

[0040] 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. For example, 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.

[0041] 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.

[0042] As used herein, 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. It is also to be appreciated that 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.

[0043] As used herein, "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. As used herein, 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. Methods of Preventing or Treating Acute Cardiac Ischemia-Reperfusion Injury

[0044] 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.

[0045] In some embodiments, 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.

[0046] In some embodiments, the subject is administered the peptide prior to a

revascularization procedure. In another embodiment, 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

revascularization procedure. In another embodiment, the subject is administered the peptide regularly (i.e., chronically) following an AMI and/or a revascularization procedure. In one embodiment, 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

revascularization procedure.

[0047] 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. Suitably, the maximum number of amino acids is about twelve, more preferably about nine, and most preferably about six. [0048] The amino acids of the aromatic -cationic peptides can be any amino acid. As used herein, the term "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). Other naturally occurring amino acids include, for example, amino acids that are synthesized in metabolic processes not associated with protein synthesis. For example, the 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).

[0049] The peptides optionally contain one or more non-naturally occurring amino acids. Optimally, 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. In addition, 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. For example, 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.

[0050] 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, β-aminobutyric acid, γ-aminobutyric acid, δ-aminovaleric acid, and ε-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. [0051] For example, 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).

[0052] Another example of a modification of an amino acid in a peptide is the

derivatization of a carboxyl group of an aspartic acid or a glutamic acid residue of the peptide. One example of derivatization 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. For example, such 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.

[0053] The non-naturally occurring amino acids are preferably resistant, and more preferably insensitive, to common proteases. Examples of 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.

[0054] In order to minimize protease sensitivity, 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. Optimally, 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. An example of a protease sensitive sequence includes two or more contiguous basic amino acids that are readily cleaved by common proteases, such as endopeptidases and trypsin. Examples of basic amino acids include arginine, lysine and histidine.

[0055] 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. The term "physiological pH" as used herein 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.

[0056] "Net charge" as used herein 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.

[0057] Typically, 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. As an example of calculating net charge, 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.

[0058] In one embodiment, 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. In this embodiment, 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)

[0059] In another embodiment, 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. In this embodiment, 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 2. Amino acid number and net positive charges (2p _m < p+1)

[0060] In one embodiment, the minimum number of net positive charges (p _m ) and the total number of amino acid residues (r) are equal. In another embodiment, 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.

[0061] It is also important that the 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. For example, 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).

[0062] 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. In this embodiment, the relationship between the minimum number of aromatic groups (a) and the total number of net positive charges (p _t ) is as follows: TABLE 3. Aromatic groups and net positive charges (3a < p _t +l or a= p _t =l)

[0063] In another embodiment, 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. In this embodiment, 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:

TABLE 4. Aromatic groups and net positive charges (2a < p _t +l or a= p _t =l)

[0064] In another embodiment, the number of aromatic groups (a) and the total number of net positive charges (p _t ) are equal.

[0065] 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.

Alternatively, 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. Accordingly, the amino acid at the C-terminus of the peptide may be converted to an amido, N- methylamido, N-ethylamido, N,N-dimethylamido, Ν,Ν-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.

[0066] In one embodiment, 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. [0067] Aromatic-cationic peptides include, but are not limited to, the following peptide examples:

Lys-D-Arg-Tyr-NH ₂

Phe-D-Arg-His

D-Tyr-Trp-Lys-NH ₂

Trp-D-Lys-Tyr-Arg-NH ₂

Tyr-His-D-Gly-Met

Phe-Arg-D-His-Asp

Tyr-D-Arg-Phe-Lys-Glu-NH ₂

Met-Tyr-D-Ly s -Phe-Arg

D-His -Glu-Lys -Tyr-D-Phe- Arg

Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH ₂

Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His

Gly-D-Phe-Lys-Tyr-His-D-Arg-Tyr-NH ₂

Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH ₂

Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-Lys

Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH ₂

Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-Lys

Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg- D-Gly-Lys-NH ₂

D-His-Lys-Tyr- D-Phe-Glu- D-Asp- D-His- D-Lys-Arg-Trp-NH ₂

Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-Phe

Tyr-D-His-Phe- D-Arg-Asp-Lys- D-Arg-His-Trp-D-His-Phe

Phe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH ₂

Phe-Try-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-Thr

Tyr-Asp-D-Lys-Tyr-Phe- D-Lys- D-Arg-Phe-Pro-D-Tyr-His-Lys

Glu-Arg-D-Lys-Tyr- D-Val-Phe- D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met-NH ₂

Arg-D-Leu-D-Tyr-Phe-Lys-Glu- D-Lys-Arg-D-Trp-Lys- D-Phe-Tyr-D-Arg-Gly

D-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr -Arg-His-Phe- NH ₂

Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Ty r-D-Phe-Lys- Phe His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D- Lys-Tyr-His- Ser-NH ₂

Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Tr p-D-His-Trp- His-D-Lys-Asp

Thr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr- Gly-Val-Ile-D- His-Arg-Tyr-Lys-NH ₂

[0068] In one embodiment, 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. In certain instances, an aromatic-cationic peptide having mu-opioid receptor agonist activity is preferred. 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.

[0069] Alternatively, in other instances, an aromatic-cationic peptide that does not have mu-opioid receptor agonist activity is preferred. For example, during long-term treatment, such as in a chronic disease state or condition, the use of an aromatic-cationic peptide that activates the mu-opioid receptor may be contraindicated. In these instances, 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. In such instances an aromatic-cationic peptide that does not activate the mu-opioid receptor may be an appropriate treatment.

[0070] 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).

[0071] In one embodiment, 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).

[0072] 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. In one embodiment, 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).

[0073] An example of an aromatic-cationic peptide that does not have mu-opioid receptor agonist activity has the formula Phe-D-Arg-Phe-Lys-NH2. Alternatively, 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 . In one embodiment, 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 .

[0074] 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:

(a) Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P) Gly(G) Cys (C);

(b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q);

(c) Basic amino acids: His(H) Arg(R) Lys(K);

(d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V); and

(e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His (H).

[0075] 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. In contrast, 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.

[0076] In some embodiments, one or more naturally occurring amino acids in the aromatic - cationic peptides are substituted with amino acid analogs. Examples of analogs that activate mu-opioid receptors include, but are not limited to, the aromatic -cationic peptides shown in Table 5.

TABLE 5. Peptide Analogs with Mu-Opioid Activity

Dab = diaminobutyric

Dap = diaminopropionic acid

Dmt = dimethyltyrosine

Mmt = 2'-methyltyrosine

Tmt = N, 2',6'-trimethyltyrosine

Hmt = 2'-hydroxy,6'-methyltyrosine

dnsDap = P-dansyl-L-a,P-diaminopropionic acid

atnDap = P-anthraniloyl-L-a,P-diaminopropionic acid

Bio = biotin

[0077] Examples of analogs that do not activate mu-opioid receptors include, but are not limited to, the aromatic-cationic peptides shown in Table 6.

TABLE 6. Pe tide Analo s Lackin Mu-O ioid Activit

D-Arg Dmt D-Arg Phe NH ₂

D-Arg Dmt D-Arg Dmt NH ₂

D-Arg Dmt D-Arg Tyr NH ₂

D-Arg Dmt D-Arg Trp NH ₂

Trp D-Arg Phe Lys NH ₂

Trp D-Arg Tyr Lys NH ₂

Trp D-Arg Trp Lys NH ₂

Trp D-Arg Dmt Lys NH ₂

D-Arg Trp Lys Phe NH ₂

D-Arg Trp Phe Lys NH ₂

D-Arg Trp Lys Dmt NH ₂

D-Arg Trp Dmt Lys NH ₂

D-Arg Lys Trp Phe NH ₂

D-Arg Lys Trp Dmt NH ₂

Cha D-Arg Phe Lys NH ₂

Ala D-Arg Phe Lys NH ₂

Cha = cyclohexyl alanine

[0078] The amino acids of the peptides shown in Table 5 and 6 may be in either the L- or the D- configuration.

Synthesis of the Peptides

[0079] 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).

Prophylactic and Therapeutic Uses of Aromatic-Cationic Peptides.

[0080] General. The aromatic-cationic peptides described herein are useful to prevent or treat disease. Specifically, the disclosure provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) cardiac ischemia-reperfusion injury. Accordingly, 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.

[0081] Determination of the Biological Effect of the Aromatic-Cationic Peptide-Based Therapeutic. In various embodiments, 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 various embodiments, 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.

[0082] Prophylactic Methods. In one aspect, 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. In prophylactic applications, pharmaceutical 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.

[0083] Therapeutic Methods. Another aspect of the technology includes methods of treating cardiac ischemia-reperfusion injury in a subject for therapeutic purposes. In therapeutic applications, 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. As such, the invention provides methods of treating an individual afflicted with cardiac ischemia-reperfusion injury.

Treating Subjects with ST-Elevation Myocardial Infarction (STEMI) and Hypertension

[0084] In some embodiments, the aromatic-cationic peptides of the present technology are useful in the treatment of subjects suffering from ST-Elevation Myocardial Infarction (STEMI) and hypertension. In some embodiments, at least one aromatic-cationic peptide of the present technology is administered to subjects with ST-Elevation Myocardial Infarction (STEMI) and hypertension. In some embodiments, 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). In some embodiments, 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 .

Reduction of Infarct Size in Subjects Treatment with Metal Based Stents

[0085] In some embodiments, the aromatic-cationic peptides of the present technology are useful in the reducing infarct size in subjects treated with metal based stents. In some embodiments, 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. In some embodiments, treatment with aromatic-cationic peptides reduces serum creatine kinase in subjects treated with metal based stents. In some embodiments, 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 .

Modes of Administration and Effective Dosages

[0086] 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.

[0087] 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. [0088] The peptide may be formulated as a pharmaceutically acceptable salt. The term "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

pharmaceutically acceptable inorganic or organic acids. In addition, when a peptide contains both a basic moiety, such as an amine, pyridine or imidazole, and an acidic moiety such as a carboxylic acid or tetrazole, zwitterions may be formed and are included within the term "salt" as used herein. 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, Ν,Ν'-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

pharmaceutically acceptable organic acids include salts of aliphatic hydroxyl acids (e.g., citric, gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids), aliphatic

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, edisylic, ethanesulfonic, isethionic, methanesulfonic, naphthalenesulfonic, naphthalene- 1,5-disulfonic, naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic acid, and the like. [0089] The aromatic-cationic peptides described herein can be incorporated into pharmaceutical 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. As used herein the term

"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.

[0090] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of 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. The parenteral preparation can be enclosed in ampuoles, disposable syringes or multiple dose vials made of glass or plastic. For convenience of the patient or treating physician, 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).

[0091] Pharmaceutical 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. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, 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.

[0092] 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. 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. In many cases, it will be preferable to include 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.

[0093] 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. Generally, 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. In the case of sterile powders for the preparation of sterile injectable solutions, 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.

[0094] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, 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.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like 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.

[0095] For administration by inhalation, 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. Such methods include those described in U.S. Pat. No. 6,468,798.

[0096] Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal

administration can be accomplished through the use of nasal sprays. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. In one embodiment, transdermal administration may be performed my iontophoresis.

[0097] 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. In one embodiment, the therapeutic peptide is encapsulated in a liposome while maintaining peptide integrity. As one skilled in the art would appreciate, 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. [0098] The carrier can also be a polymer, e.g., a biodegradable, biocompatible polymer matrix. In one embodiment, 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. In one embodiment, the polymer is poly-lactic acid (PLA) or copoly lactic/glycolic acid (PGLA). The polymeric matrices can be prepared and isolated in a variety of forms and sizes, including microspheres and

nanospheres. Polymer formulations can lead to prolonged duration of therapeutic effect. (See Reddy, Ann. Pharmacother., 34(7-8):915-923 (2000)). A polymer formulation for human growth hormone (hGH) has been used in clinical trials. (See Kozarich and Rich, Chemical Biology, 2:548-552 (1998)).

[0099] Examples of polymer microsphere sustained release formulations are described in PCT publication WO 99/15154 (Tracy et al), U.S. Pat. Nos. 5,674,534 and 5,716,644 (both to Zale et al), PCT publication WO 96/40073 (Zale et al), and PCT publication WO

00/38651 (Shah et al). U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073 describe a polymeric matrix containing particles of erythropoietin that are stabilized against aggregation with a salt.

[0100] In some embodiments, 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. 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.

[0101] The therapeutic compounds can also be formulated to enhance intracellular delivery. For example, 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.

[0102] 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.

[0103] 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. For any compound used in the methods, 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. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0104] Typically, an effective amount of the aromatic-cationic peptides, sufficient for achieving a therapeutic or prophylactic effect, range from about 0.000000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day.

Preferably, 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. For example 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. In one embodiment, a single dosage of peptide ranges from 0.1-10,000 micrograms per kg body weight. In one embodiment, aromatic-cationic peptide concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter.

[0105] An exemplary treatment regime entails administration once per day or once a week. In therapeutic applications, 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.

[0106] In some embodiments, 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). In some embodiments, 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 .

[0107] In some embodiments, the dosage of the aromatic-cationic peptide is provided at an "ultralow", "low," "mid," or "high" dose level. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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.

[0108] In some embodiments, 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 1000 mole peptide; at least 10,000 mole peptide; at least 100,000 mole peptide.

[0109] In some embodiments, 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.

[0110] In some embodiments, 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. [0111] The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, 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.

[0112] 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. In a preferred embodiment, the mammal is a human.

EXAMPLE

[0113] The present invention is further illustrated by the following example, which should not be construed as limiting in any way.

[0114] Example 1. Effects of aromatic-cationic peptides in protecting against cardiac ischemia-reperfusion injury in a guinea pig model

[0115] The effects of aromatic-cationic peptides in protecting against cardiac ischemia- reperfusion injury in a guinea pig model were investigated. The myocardial protective effect of the peptide D-Arg-2'6'-Dmt-Lys-Phe-NH2 were demonstrated by this Example.

Experimental Methods.

[0116] Procedures for the use of guinea pigs are in accordance with the guidelines established by the American Physiological Society and have been previously approved by the Institutional Animal Care and Use Committee at East Carolina University (Internal AUP # Q269). Adult male guinea pigs (200-3008) were anesthetized with a ketamine/xylazine cocktail (85/15 mg mL, respectively; ip delivery). Upon the absence of reflexes to ensure a deep plane of anesthesia, hearts were excised via midline thoracotomy and immersed in ice- cold saline. 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.

[0118] After a 10-min equilibration period, hearts were divided into the following different treatment groups: 1) Control followed by LR; 2) Administration of 1 nM peptide in the perfusate both before and after index ischemia; and 3) Post-ischemic administration of 1 nM peptide using both a bolus dose (also 1 nM, administered immediately prior to reperfusion) and peptide in the reperfusion solution (FIG. 2).

[0119] 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. At the end of the 2 h reperfusion protocol, 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).

Results

[0120] Effects of Peptide on Baseline Myocardial Function. The effects of peptide on baseline myocardial mechanical and electrical function were investigated. There were no discernable effects of peptide on left ventricular developed pressure, coronary flow, heart rate, or repolarization (as assessed by the QT interval, see FIG. 3D). Baseline hemodynamic values are presented in Table 7 below.

Table 7; Baseline hemod namic values for uinea i hearts used in the stud

[0121] Infarct Size. Infarct size from hearts exposed to 20 min of global ischemia are presented in FIGs. 4A, 4B and 4C. Peptide treatment reduced ventricular infarct size compared to placebo in an animal model that utilized 20 min of global no-flow ischemia followed by 120 min of restoration of blood flow. A trend towards myocardial protection was evident whether peptide was administered: (1) only in conjunction with reperfusion (n=4; 35% decrease in infarct size compared to placebo, or (2) pre-ischemia and in conjunction with reperfusion (n=3; 25% decrease in infarct size compared to placebo.

[0122] When the seven animals who were administered peptide with reperfusion (with or without treatment prior to onset of ischemia) were analyzed as a group, there was a 30% decrease in infarct size compared with placebo.

[0123] Incidence of Arrhythmia. The effect of peptide treatment against protection from arrhythmia was investigated. Using a scoring system previously established [19], the extent of arrhythmia in each group was quantified, with a higher score indicating a higher incidence of arrhythmia. Arrhythmias were scored for the entire 2 h reperfusion period as follows: 0 = 0 - 49 ventricular premature beats; 1 = 50-499 ventricular premature beats; 2 = >499 ventricular premature beats and/or 1 episode of spontaneously reverting VT or VF less than 60 s in total duration; 3 = > 1 episode of VT or VF that is <60 s total duration; 4 = reverting VT or VF or both that is < 120 s in total duration; 5 = VT or VT or both that is > 120 s in combined duration; 6 = non-reverting (fatal) VT or VF that began > 15 min after reperfusion; 7 = fatal VT/VF that began between 5 min and 15 min after reperfusion; 8 = fatal VT/VF that began less than 5 min after reperfusion; 9 = fatal VT/VF that began less than 1 min after reperfusion.

[0124] No differences in the severity of arrhythmia (as assessed by the arrhythmia score), or the time to sustained arrhythmia were found (FIG. 5B and 5C). For the latter, this reflects the minute in reperfusion when the heart went into sustained (>1 min) VT/VF, which is common in this model. On average, control hearts went into sustained VT/VF at about 20 min of reperfusion. There were no tendencies for this time point to be delayed in the peptide treatment group (indicates lack of protection).

[0125] Coronary Flow Rates. 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.

[0126] These results show that in a standardized guinea pig model of acute myocardial ischemia and reperfusion, peptide treatment was able to reduce myocardial infarct size compared to the control group. These results indicate that peptide treatment prevents pathologic and morphologic findings of acute cardiac ischemia-reperfusion injury. As such, aromatic-cationic peptides are useful in methods at preventing and treating a cardiac ischemia-reperfusion injury in mammalian subjects.

[01271 Example 2. Effects of aromatic-cationic peptides in protecting against cardiac ischemia-reperfusion injury in a guinea pig model

[0128] The effects of aromatic-cationic peptides in protecting against cardiac ischemia- reperfusion injury in a guinea pig model were investigated. The myocardial protective effect of the peptide D-Arg-2'6'-Dmt-Lys-Phe-NH2 were further demonstrated by this Example. The study designs employed in the studies is shown in Figure 6. Dotted bars represent periods of global ischemia accomplished by turning off the perfusion to the heart. [01291 Experimental Methods.

[0130] Procedures for the use of guinea pigs are in accordance with the guidelines established by the American Physiological Society. Adult male guinea pigs (200-3 OOg) were anesthetized with a ketamine/xylazine cocktail (85/15 mg mL, respectively; ip delivery). Upon the absence of reflexes to ensure a deep plane of anesthesia, hearts were excised via midline thoracotomy and immersed in ice-cold saline. 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.

[0131] 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 5-8mmHg. 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).

[0132] After a 10-minute equilibration period, hearts were divided into the following different treatment groups: 1. Control followed by I/R; 2. Administration of InM D-Arg-2'6'- Dmt-Lys-Phe-NH ₂ in the perfusate both before and after index ischemia; 3. Post-ischemic administration of InM D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ using both a bolus dose (also InM, administered immediately prior to reperfusion) and D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ in the reperfusion solution; 4. Positive control using ischemic preconditioning; 3 cycles of 5 min VR before index ischemia).

[01331 Methods: Ischemia/Reperfusion

[0134] Hearts were exposed to global no-flow ischemia by stopping perfusion for 20 minutes. 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 minutes. Administration of all compounds in the perfusate was accomplished via dissolving the compound(s) 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. At the end of the 2h reperfusion protocol, 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).

[01351 Results

[0136] 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.

[0137] Incidence of Arrhythmia. The effect of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ against protection from arrhythmia was studied. The isolated guinea pig heart exposed to global ischemia exhibits reproducible ventricular arrhythmia at the onset of reperfusion. Almost all hearts in the study exhibited some degree of ventricular tachycardia and/or fibrillation (VT/VF) during the protocol. Hearts that received 0.01nM/0.1nM(30min R) showed protection against the incidence of VT/VF. A higher concentration of D-Arg-2'6'-Dmt-Lys- Phe- H ₂ (InM) was not effective in reducing the amount of time that hearts spent in VT/VF. Cyclosporin (0.2 μΜ) showed efficacy in reducing the time hearts spent in ventricular arrhythmia, but this slight decrease did not reach statistical significance.

[0138] Coronary Flow Rates. 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. lnM whole time; O.OlnM D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ to O. lnM at reperfusion; 0.2uM cyclosporin-A whole time; and 0.2uM cyclosporin-A at reperfusion. There were no differences observed in coronary flow rates. In groups receiving 0.0 InM D-Arg-2'6'-Dmt- Lys-Phe-NH ₂ , baseline flow rates were slightly lower than other groups.

[0139] Left Ventricular Developed Pressure. The effects of D-Arg-2'6'-Dmt-Lys-Phe- H ₂ on baseline myocardial mechanical function was examined. There were no discernable effects of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ (at any concentration (0.01 nM, 0.1 nM) or cyclosporin-A (0.2 μΜ) on baseline left ventricular developed pressure, heart rate, or rates of contraction/relaxation.

Example 3: Treating Subjects with ST-Elevation Myocardial Infarction (STEMI) and Hypertension

[0140] This example shows that aromatic-cationic peptides of the present technology are useful in the treatment of subjects diagnosed with STEMI and hypertension.

Methods and Materials

[0141] 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.

[0142] 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.

[0143] To assess efficacy in large Mis that had not sustained reperfusion injury, only subjects who met angiographic and post-PCI inclusion criteria were included in the primary analysis population (PAP) (Figure 7): (1) the presence of a proximal/mid left anterior descending (LAD) occlusion with pre-PCI TIMI flow 0 or 1 and no visible evidence of significant coronary collateral flow; (2) dilation of only the LAD; (3) successful PCI of the LAD with post-PCI TIMI flow > 2; and (4) no evidence of a second MI within 72 hours of the initial PCI which would confound the infarct size determination.

[0144] Subjects were randomized in a double blind 1 : 1 fashion to receive either D-Arg- 2',6'-Dmt-Lys-Phe-NH2 of at 0.05 mg/kg/hr or a control placebo by IV injection (Treatment group: n = 58; Control group: n = 60). 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. Follow-up visits were conducted at discharge, at 30 ± 7 days, 90 ± 14 days, and 6 ± 1.5 months post-PCI.

[0145] Baseline characteristics of the subjects are listed in Table 8.

Pre-PCI thrombus aspiration 71.7% (43) 65.5% (38) 0.47

Abbreviations: IQR=Interquartile range; LAD=Left anterior descending; SD=Standard deviation

[0146] The following end points were measured:

[0147] 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).19-21 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 (Δ) 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 (Δ) in these measures from day 4 ± 1 to day 30 ± 7, TIMI myocardial perfusion grade (TMPG), TIMI flow grade (TFG), corrected TIMI frame count post-PCI and complete ST-segment elevation resolution (>70%) by the Schroder criteria. Laboratory measures of congestive heart failure (CHF) (N -terminal pro-B type natriuretic protein), and renal function (serum creatinine, estimated glomerular filtration rate, cystatin C, and blood urea nitrogen) were also evaluated.

[0148] The incidence of the clinical composite endpoint of all-cause death, new-onset CHF beginning > 24 hours post-PCI and within the duration of the index hospitalization, and CHF re-hospitalization was also determined through 30 ± 7 days and 6 ± 1.5 months post-PCI.

Safety endpoints included treatment emergent adverse events (TEAE). The pharmacokinetic profile of D-Arg-2'6'-Dmt-Lys-Phe-NH2 was determined based on the drug serum

concentration.

[0149] Based on the significant number of hypertensive subjects in the study, the data points from subjects (both control and treated) having the baseline characteristic of

hypertension were compared.

Statistical Analysis [0150] Continuous variables were analyzed using analysis of covariance (ANCOVA) or a one-way analysis of variance (ANOVA). Categorical data was analyzed using the Fisher's Exact test. The primary efficacy analysis was based on the PAP, excluding subjects with insufficient CK-MB results (subjects with sufficient CK-MB results must have at least the 6 or 12 hour nominal time point and at least one time point at the 24 hour sampling time point or later). Unless otherwise noted, all statistical tests of hypotheses were two-sided and required a 5% level of significance. Statistical analyses were performed using the SAS System®, Version 9.3.

Results

[0151] Control subjects with hypertension had a greater mean left ventricle (LV) mass as compared to subject with hypertension and treated with D-Arg-2'6'-Dmt-Lys-Phe-NH2 (162.2 ± 52.4 grams (control) vs. 141.5 ± 53.2 grams (treated); p=0.08).

[0152] Subjects treated with D-Arg-2'6'-Dmt-Lys-Phe-NH2 had a reduction in the mean infarct volume at day 4 ± 1 on cardiac MRI as compared to control subjects (52.6 ± 30.2 mL (control) vs. 35.8 ± 22.8 mL (treated); p=0.03)

[0153] Myocardial edema on cardiac MRI was also reduced among hypertensive subjects treated with D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ (60.9 ± 20.5 (control) vs. 48.9 ± 22.0 (treated) (p=0.053).

[0154] ST-segment resolution at 24 hours post-PCI among treated hypertensive subjects was greater than the untreated control group (proportion of complete or partial ST segment resolution was 88.2% in control vs. 100% in treated; p=0.05).

[0155] A total of 23 new onset CHF occurred < 24 hours post-PCI, in all hypertensive subjects (both control and treated). Hypertensive subjects treated with D-Arg-2'6'-Dmt-Lys- Phe-NFL had fewer incidents of new onset CHF <24 hours post-PCI (15 incidents in the control group vs. 8 incidents in the treated group). 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).

[0156] It was not expected that D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ would reduce infarct volume, reduce mean LV mass, reduce myocardial edema, improve ST-segment resolution, and reduce incidents of new onset CHF < 24 hours post-PCI of subjects with STEMI and hypertension.

[0157] These results show that D-Arg-2'6'-Dmt-Lys-Phe-NH2 is useful in the treatment of subjects suffering from acute myocardial infarction and hypertension. Accordingly, the methods and compositions of the present technology are useful in the treatment of subjects suffering from acute myocardial infarction and hypertension.

Example 4: Reduction of Infarct Size in Hypertensive Patients with Bare Metal Stents in ITT

[0158] As discussed in Example 3, subjects between the ages of 18 to 85 years with anterior STEMI undergoing first-time PCI plus stenting with an anticipated time from ischemic symptoms to time of balloon inflation of < 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 (known LVEF < 30% prior to the qualifying infarct), and cardiogenic shock.

[0159] Randomization and end points. Subjects that received bare metal stents or drug eluting stents were randomized in a double blind fashion to receive either IV D-Arg-2',6' Dmt-Lys-Phe- NH ₂ at 0.05 mg/kg/hr or placebo. Study drug 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. Follow-up visits were conducted at discharge, at 30 ± 7 days, 90 ± 14 days, and 6 ± 1.5 months post-PCI. The randomized trial was analyzed by the intention-to-treat approach (ITT).

[0160] 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).

[0161] Results: Fewer subjects randomized to the D-Arg-2'6'-Dmt-Lys-Phe- NH ₂ group were identified to have hypertension (Table 8). Given the higher proportion of hypertensive subjects in the placebo group (60.0% vs. 37.9%; p=0.02), the endpoints among hypertensive subjects were further evaluated in a non-prespecified analysis. [0162] There was no difference in the AUC of CK-MB in patients with bare metal stents under ITT analysis. Figure 8(A). However, Figure 8(B) shows that hypertensive patients that received bare metal stents exhibited a significant reduction in mean infarct size at day 4 when treated with D-Arg-2'6' Dmt-Lys-Phe-NH ₂ (41.14 in placebo (n=66) vs. 32.34 in D- Arg-2'6'-Dmt-Lys-Phe-NH ₂ (n=67); p=0.057) under ITT analysis, but not in the primary analysis population (PAP) (Figure 8(C).

[0163] Patients with metal based stents treated with D-Arg-2'6'-Dmt-Lys-Phe- H ₂ also showed a decrease in serum creatine kinase MB as compared to the untreated patients with metal based stents. FIG. 9.

[0164] These results demonstrate that hypertensive patients with bare metal stents exhibit a significant reduction in mean infarct size at day 4 when treated with D-Arg-2'6'-Dmt-Lys- Phe- H ₂ relative to that observed in the placebo group. Accordingly, the methods of the present technology are useful in reducing infarct size in hypertensive patients.

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[0165] The present invention is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the invention. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art.

Functionally equivalent methods and apparatuses within the scope of the invention, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this invention is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0166] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0167] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as "up to," "at least," "greater than," "less than," and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0168] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification. [0169] Other embodiments are set forth within the following claims.