Methods for the prevention or treatment of heart failure   

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 61/248,681, filed Oct. 5, 2009, and U.S. Provisional Application No. 61/289,483, filed Dec. 23, 2009, the entire contents of all of which are hereby incorporated by reference in their entirety.

This invention was made with United States government support awarded by the following agency: NIH R01 HL101186, P30 AG013280, and P01 AG001751. The United States government has certain rights in this invention.

TECHNICAL FIELD

The present technology relates generally to compositions and methods of preventing or treating heart failure. In particular, the present technology relates to administering aromatic-cationic peptides in effective amounts to prevent or treat heart failure in mammalian subjects.

The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art to the present invention.

Heart failure is a leading cause of mortality and morbidity worldwide. In the United States, it affects nearly 5 million people and is the only major cardiovascular disorder on the rise. It is estimated that 400,000 to 700,000 new cases of heart failure are diagnosed each year in the U.S. and the number of deaths in the U.S. attributable to this condition has more than doubled since 1979, currently averaging 250,000 annually. Although heart failure affects people of all ages, the risk of heart failure increases with age and is most common among older people. Accordingly, the number of people living with heart failure is expected to increase significantly as the elderly population grows over the next few decades. The causes of heart failure have been linked to various disorders including coronary artery disease, past myocardial infarction, hypertension, abnormal heart valves, cardiomyopathy or myocarditis, congenital heart disease, severe lung disease, diabetes, severe anemia, hyperthyroidism, arrhythmia or dysrhythmia.

Heart failure (HF), also called congestive heart failure, is commonly characterized by decreased cardiac output, decreased cardiac contractility, abnormal diastolic compliance, reduced stroke volume, and pulmonary congestion. The clinical manifestations of heart failure reflect a decrease in the myocardial contractile state and a reduction in cardiac output. Apart from deficiencies in cardiac contractility, the HF disease state may arise from left ventricular failure, right ventricular failure, biventricular failure, systolic dysfunction, diastolic dysfunction, and pulmonary effects. A progressive decrease in the contractile function of cardiac muscle, associated with heart disease, often leads to hypoperfusion of critical organs.

SUMMARY

The present technology relates generally to the treatment or prevention of heart failure in mammals through administration of therapeutically effective amounts of aromatic-cationic peptides to subjects in need thereof. In particular embodiments, the aromatic-cationic peptides treat or prevent heart failure by enhancing mitochondrial function in cardiac tissues.

In one aspect, the disclosure provides a method of treating or preventing heart failure or hypertensive cardiomyopathy, comprising administering to said 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 (pm) and the total number of amino acid residues (r) wherein 3pm 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 (pt) wherein 2a is the largest number that is less than or equal to pt+1, except that when a is 1, pt may also be 1. In particular embodiments, the mammalian subject is a human.

In one embodiment, 2pm 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.

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.

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 (SS-01) or 2′,6′-Dmt-D-Arg-Phe-Lys-NH2 (SS-02). 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 (SS-20) 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-NH2 (referred to interchangeably as SS-31, MTP-131, or Bendavia™)

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

wherein R1 and R2 are each independently selected from

(i) hydrogen;

(ii) linear or branched C1-C6 alkyl;

R3 and R4 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) C1-C4 dialkylamino;

(vii) nitro;

(viii) hydroxyl;

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

R5, R6, R7, R8, and R9 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) C1-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.

In a particular embodiment, R1 and R2 are hydrogen; R3 and R4 are methyl; R5, R6, R7, R8, and R9 are all hydrogen; and n is 4.

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

wherein R1 and R2 are each independently selected from

(i) hydrogen;

(ii) linear or branched C1-C6 alkyl;

R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12 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) C1-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.

In a particular embodiment, R1, R2, R3, R4, R5, R6, R7, R8R9, R10, R11, and R12 are all hydrogen; and n is 4. In another embodiment, R1, R2, R3, R4, R6, R7, R8, R9, and R11 are all hydrogen; R8 and R12 are methyl; R10 is hydroxyl; and n is 4.

In one embodiment, the subject is suffering from heart failure. In one embodiment, the heart failure results from hypertension; ischemic heart disease; exposure to a cardiotoxic compound; myocarditis; thyroid disease; viral infection; gingivitis; drug abuse; alcohol abuse; pericarditis; atherosclerosis; vascular disease; hypertrophic cardiomyopathy; acute myocardial infarction; left ventricular systolic dysfunction; coronary bypass surgery; starvation; an eating disorder; or a genetic defect. In one embodiment, the subject is suffering hypertensive cardiomyopathy.

In one embodiment, myocardial contractility and cardiac output in the subject administered the peptide are increased compared to a control subject not administered the peptide. In one embodiment, the myocardial contractility and cardiac output in the subject are increased at least 10% compared to a control subject not administered the peptide.

In one embodiment, the method further comprises separately, sequentially or simultaneously administering a cardiovascular agent to the subject. In one embodiment, the cardiovascular agent is selected from the group consisting of: an anti-arrhthymia agent, a vasodilator, an anti-anginal agent, a corticosteroid, a cardioglycoside, a diuretic, a sedative, an angiotensin converting enzyme (ACE) inhibitor, an angiotensin II antagonist, a thrombolytic agent, a calcium channel blocker, a throboxane receptor antagonist, a radical scavenger, an anti-platelet drug, a β-adrenaline receptor blocking drug, α-receptor blocking drug, a sympathetic nerve inhibitor, a digitalis formulation, an inotrope, and an antihyperlipidemic drug.

In another aspect, the disclosure provides a method for increasing myocardial contractility and cardiac output in a subject suffering from heart failure or hypertensive cardiomyopathy comprising administering to the subject a therapeutically effective amount of the peptide D-Arg-2′6′-Dmt-Lys-Phe-NH2 or Phe-D-Arg-Phe-Lys-NH2.

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).

FIG. 1 is a graph of flow cytometric analysis of neonatal cardiomyocytes stimulated with Ang II (1 μM) and loaded with Mitosox (5 μM), an indicator of mitochondrial ROS.

FIG. 2 is a series of charts showing the effects of SS-31 on blood pressure after a pressor dose of Ang II. FIG. 2A: Representative blood pressure tracings of mice at baseline and after Ang II (1.1 mg/kg/d) administered with a subcutaneous pump. FIG. 2B: Ang II significantly increased systolic blood pressure by 27.2 mm Hg and diastolic pressure by 24.8 mm Hg.

FIG. 3 is a series of charts showing that SS-31 ameliorates Ang-II-induced cardiac hypertrophy and diastolic dysfunction. FIG. 3A: Ang II (1.1 mg/kg/d) for 4 weeks substantially increased LVMI in WT control mice. Simultaneous administration of SS-31 (3 mg/kg/d) significantly attenuated the Ang II-induced increase in LVMI (left panel), to a similar extent as that observed in mice with inducible overexpression of mitochondrial catalase (i-mCAT, right panel). FIG. 3B and FIG. 3C: Left ventricular end-diastolic diameter (LVEDD) and fractional shortening (FS,%) were not significantly changed after 4 weeks of Ang II in the presence or absence of mitochondrial antioxidants. FIG. 3D: Diastolic function measured by tissue Doppler imaging of Ea/Aa significantly reduced after 4 weeks of Ang II, but this is significantly ameliorated by SS-31 or genetic overexpression of mCAT.

FIG. 4 is a series of charts showing SS-31 attenuates Ang-II induced cardiac hypertrophy and fibrosis. FIG. 4A: Ang II significantly increased heart weight (normalized to tibia length) and this was significantly attenuated by SS-31. FIG. 4B: Quantitative PCR showed a dramatic increase in atrial natriuretic peptide (ANP) gene expression, which was significantly prevented by SS-31. FIG. 4C: Representative histopathology shows substantial perivascular fibrosis (PVF) and interstitial fibrosis (IF) after Ang II, which was better protected in SS-31 treated hearts. FIG. 4D: Quantitative analysis of blue trichrome staining demonstrated a significant increase in ventricular fibrosis after Ang II, and this was substantially attenuated by SS-31. FIG. 4E: Quantitative PCR showed upregulation of pro-collagen 1a2 mRNA after Ang II, which was significantly reduced in SS-31 hearts.

FIG. 5 is a series of charts showing mitochondrial protein carbonyl and signaling for mitochondrial biogenesis increased after 4 weeks of Ang II treatment, which was prevented by SS-31. FIG. 5A: Ang II for 4 weeks significantly increased cardiac mitochondrial protein carbonyl content, an indicator of protein oxidative damage, and this was significantly ameliorated by SS-31. FIG. 5B: Quantitative PCR revealed significant upregulation of genes in mitochondrial biogenesis, all of which were attenuated by SS-31. *p<0.05 compared with saline group, #p<0.05 compared with Ang II treated group.

FIG. 6 is a series of charts showing SS-31 acts downstream of NADPH oxidase and reduces activation of p38 MAPK and apoptosis in response to Ang II. FIG. 6A: NADPH oxidase activity was significantly increased after Ang II. No significant effect of SS-31 was observed. FIG. 6B: Ang II for 4 weeks substantially induced apoptosis, as shown by increase in cleaved (activated) caspase 3 and this was significantly attenuated by SS-31. FIG. 6C: Phosphorylation of p38 MAP kinase significantly increased after Ang II, which was substantially lower in SS-31 treated hearts (upper panel). Protein levels of p38 MAP kinase also increased after Ang II.

FIG. 7 is a series of charts showing SS-31 ameliorated cardiac hypertrophy and failure in Gαq overexpressing mice. Echocardiography of Gαq mice with or without SS-31 treatment and WT littermates at 16 weeks of age. FIG. 7A: SS-31 (3 mg/kg/d) for 4 weeks (from age 12 to 16 weeks) significantly ameliorated the decline in systolic function, as indicated by FS, in Gαq overexpressing mice. FIG. 7B and FIG. 7C: Chamber enlargement and impairment of diastolic function in Gαq mice were slightly attenuated by SS-31 with borderline significance, p=0.08 and 0.06, respectively. FIG. 7D: Worsening of myocardial performance index (MPI) in Gαq mice was significantly ameliorated by SS-31. FIG. 7E: An increase in normalized heart weight in Gαq mice was substantially protected by SS-31, while increased normalized lung weight displayed a modest effect from SS-31 with borderline significance (p=0.09).

FIG. 8 is a diagrammatic illustration of the proposed effect of mitochondrial antioxidant SS-31 on Ang II and Gαq-induced cardiomyopathy. Mitochondrial antioxidant SS-31 acts downstream to Angiotensin II receptor, Gαq, NADPH oxidase and upstream of p38 MAPK and apoptosis.

FIG. 9 is a series of charts showing both cardiac fibrosis and cardiac expression of Coll 1a2 gene were not significantly altered by 4 weeks of SS-31 treatment (FIG. 9A and FIG. 9B). FIG. 9C shows cardiac mitochondrial protein carbonyl content significantly increased after Ang II, which was reduced by SS-31.

DETAILED DESCRIPTION

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. 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.

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.

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.

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 α-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.

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, heart failure or one or more symptoms associated with heart failure. 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 heart failure, such as cardiomegaly, tachypnea, and hepatomegaly. For example, a “therapeutically effective amount” of the aromatic-cationic peptides is meant levels in which the physiological effects of a heart failure are, at a minimum, ameliorated.

As used herein, the terms “congestive heart failure” (CHF), “chronic heart failure”, “acute heart failure”, and “heart failure” are used interchangeably, and refer to any condition characterized by abnormally low cardiac output in which the heart is unable to pump blood at an adequate rate or in adequate volume. When the heart is unable to adequately pump blood to the rest of the body, or when one or more of the heart valves becomes stenotic or otherwise incompetent, blood can back up into the lungs, causing the lungs to become congested with fluid. If this backward flow occurs over an extended period of time, heart failure can result. Typical symptoms of heart failure include shortness of breath (dyspnea), fatigue, weakness, difficulty breathing when lying flat, and swelling of the legs, ankles or abdomen (edema). Causes of heart failure are related to various disorders including coronary artery disease, systemic hypertension, cardiomyopathy or myocarditis, congenital heart disease, abnormal heart valves or valvular heart disease, severe lung disease, diabetes, severe anemia hyperthyroidism, arrhythmia or dysrhythmia and myocardial infarction. The primary signs of congestive heart failure are: cardiomegaly (enlarged heart), tachypnea (rapid breathing; occurs in the case of left side failure) and hepatomegaly (enlarged liver; occurs in the case of right side failure).

As used herein, the term “hypertensive cardiomyopathy” refers to a weakened heart caused by the effects of hypertension (high blood pressure). Over time, uncontrolled hypertension causes weakness of the heart muscle. As hypertensive cardiomyopathy worsens, it can lead to congestive heart failure. Early symptoms of hypertensive cardiomyopathy include cough, weakness, and fatigue. Additional symptoms of hypertensive cardiomyopathy include leg swelling, weight gain, difficulty breathing when lying flat, increasing shortness of breath with activity, and waking in the middle of the night short of breath.

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.

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.

As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.

As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.

As used herein, the terms “treating” or “treatment” or “alleviation” refers to therapeutic treatment, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. A subject is successfully “treated” for heart failure 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 heart failure, such as, e.g., cardiac output, myocardial contractile force, cardiomegaly, tachonea, and/or hepahemogaly. 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. Treating heart failure, as used herein, also refers to treating any one or more of the conditions underlying heart failure, including, without limitation, decreased cardiac contractility, abnormal diastolic compliance, reduced stroke volume, pulmonary congestion, and decreased cardiac output.

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 heart failure includes preventing the initiation of heart failure, delaying the initiation of heart failure, preventing the progression or advancement of heart failure, slowing the progression or advancement of heart failure, delaying the progression or advancement of heart failure, and reversing the progression of heart failure from an advanced to a less advanced stage.

The present technology relates to the treatment or prevention of heart failure and related conditions by administration of certain aromatic-cationic peptides. 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.

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 (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), 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).

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.

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 α-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 γ-phenyl-β-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.

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 C1-C4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C1-C4 alkyloxy (i.e., alkoxy), amino, C1-C4 alkylamino and C1-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 (Nle).

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 C1-C4 alkyl groups mentioned above, such as an acetyl or propionyl group.

The non-naturally occurring amino acids are suitably resistant or 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.

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.

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 (pm). 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.

“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.

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.

In one embodiment, the aromatic-cationic peptides have a relationship between the minimum number of net positive charges at physiological pH (pm) and the total number of amino acid residues (r) wherein 3pm 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 (pm) and the total number of amino acid residues (r) is as follows:

TABLE 1 Amino acid number and net positive charges (3pm ≦ p + 1) (r) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (pm) 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have a relationship between the minimum number of net positive charges (pm) and the total number of amino acid residues (r) wherein 2pm 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 (pm) and the total number of amino acid residues (r) is as follows:

TABLE 2 Amino acid number and net positive charges (2pm ≦ p + 1)

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