HK1090571B - Methods for preventing mitochondrial permeability transition - Google Patents

Description

Method for preventing mitochondrial permeability transition

This application claims priority from U.S. provisional application No. 60/444,777 filed on 4/2/2003 and U.S. provisional application No. 60/535,690 filed on 8/1/2004. The contents of U.S. provisional application nos. 60/444,777 and 60/535,690 are incorporated herein by reference.

The invention was made with government support approval number PO1DA08924-08, provided by the national institute of drug abuse. The united states government has certain rights in this invention.

Background

Mitochondria are present in almost all eukaryotic cells and produce Adenosine Triphosphate (ATP) by oxidative phosphorylation and are thus essential for cell survival. Blocking this important function can lead to cell death.

Mitochondria also accumulate calcium (Ca) by²⁺) And plays an important role in calcium regulation in cells. Calcium accumulation within the mitochondrial matrix occurs via membrane potential driven monodirectional transporters.

Calcium uptake activates mitochondrial dehydrogenases, which may be important in maintaining energy production and oxidative phosphorylation. In addition, mitochondria can act as excess cytosolic Ca²⁺Thereby protecting the cells from Ca²⁺Overload and necrosis.

Ischemia or hypoglycemia can lead to mitochondrial dysfunction, including ATP hydrolysis and Ca²⁺And (4) overload. This malfunction causes mitochondrial permeability alterations (mitochondrial permeability transition, MPT). MPT is characterized by: oxidative phosphorylation uncoupling, mitochondrial membrane potential loss, intimal permeability increase and swelling.

In addition, the mitochondrial membrane-space is the reservoir for apoptotic gene proteins (apoptotic proteins). Thus, loss of mitochondrial potential and MPT can result in release of apoptotic gene proteins into the cytoplasm. There is increasing evidence that MPT is associated with necrotic and apoptotic cell death (Crompton, Biochem J.341: 233-249, 1999), which is not surprising. Slight damage to the cells may result in apoptosis rather than necrosis.

Cyclosporin inhibits MPT. Blocking of MPT by cyclosporin A inhibits apoptosis in a variety of cell types, including those undergoing ischemia, hypoxia, Ca²⁺Overloaded and oxidatively stressed cells (Kroemer et al, Annu Rev physiol.60: 619 Across 642, 1998).

However, cyclosporin a is not the best as a therapeutic agent against necrotic and apoptotic cell death. For example, cyclosporin a cannot specifically target mitochondria. In addition, it is difficult to deliver to the brain. Furthermore, the range of application of cyclosporin A is reduced due to its immunosuppressive activity.

Tetrapeptide [ Dmt¹]DALDA (2 ', 6' -dimethyltyrosine-D-arginine-phenylalanine-lysine-NH)₂(ii) a SS-02) has a molecular weight of 640 and carries a net positive charge of 3 at physiological pH. [ Dmt¹]DALDA readily crosses the plasma membrane of many mammalian cell types in an energy-independent manner (Zhao et al, J Pharmacol exp. ther. 304: 425-432, 2003) and across the blood-brain barrier (Zhao et al, JPharmacol exp. ther. 302: 188-196, 2002). Although [ Dmt¹]DALDA has been shown to be a potential mu-opioid (opioid) receptor agonist, but its use has not yet been extended to include aspects that inhibit MPT.

Therefore, in the case of other diseases and conditions such as ischemia-reperfusion, hypoxia, hypoglycemia, and pathological changes due to mitochondrial membrane permeability changes (mitochondrial permeability transition), it is desirable to inhibit MPT. Such diseases and disorders include a variety of common neurodegenerative diseases or disorders.

Disclosure of Invention

These and other objects are achieved by the present invention which provides a method for reducing the number of mitochondria undergoing Mitochondrial Permeability Transition (MPT) or preventing mitochondrial permeability transition in any mammal in need thereof. The method comprises administering to the mammal an effective amount of an aromatic-cationic peptide having the following characteristics:

(a) at least one net positive charge;

(b) a minimum of 3 amino acids;

(c) up to about 20 amino acids;

(d) minimum number of net positive charges (p)_m) And the total number of amino acid residues (r) is: wherein 3p_mIs the maximum number less than or equal to r + 1; and

(e) minimum number of aromatic groups (a) and total number of net positive charges (p)_t) The relationship between them is: wherein 2a is less than or equal to p_tMaximum of +1, except when a is 1, p_tOr may be 1.

In another embodiment, the invention provides a method of reducing the number of mitochondria undergoing Mitochondrial Permeability Transition (MPT) or preventing mitochondrial permeability transition in an isolated organ of a mammal. The method comprises administering to the isolated organ an effective amount of an aromatic-cationic peptide having the following characteristics:

(a) at least one net positive charge;

(b) a minimum of 3 amino acids;

(c) up to about 20 amino acids;

(d) minimum number of net positive charges (p)_m) And the total number of amino acid residues (r) is: wherein 3p_mIs the maximum number less than or equal to r + 1; and

(e) minimum number of aromatic groups (a) and total number of net positive charges (p)_t) The relationship between them is: wherein 2a is less than or equal to p_tMaximum of +1, except when a is 1, p_tOr may be 1.

In yet another embodiment, the invention provides a method of reducing the number of mitochondria undergoing Mitochondrial Permeability Transition (MPT) or preventing mitochondrial permeability transition in a mammal in need thereof. The method comprises administering to the mammal an effective amount of an aromatic-cationic peptide having the following characteristics:

(a) at least one net positive charge;

(b) a minimum of 3 amino acids;

(c) up to about 20 amino acids;

(d) minimum number of net positive charges (p)_m) And the total number of amino acid residues (r) is: wherein 3p_mIs the maximum number less than or equal to r + 1; and

(e) minimum number of aromatic groups (a) and total number of net positive charges (p)_t) The relationship between them is: wherein 3a is less than or equal to p_tMaximum of +1, except when a is 1, p_tOr may be 1.

In a further embodiment, the present invention provides a method of reducing the number of mitochondria undergoing Mitochondrial Permeability Transition (MPT) or preventing mitochondrial permeability transition in an isolated organ of a mammal. The method comprises administering to the isolated organ an effective amount of an aromatic-cationic peptide having the following characteristics:

(a) at least one net positive charge;

(b) a minimum of 3 amino acids;

(c) up to about 20 amino acids;

(d) minimum number of net positive charges (p)_m) And the total number of amino acid residues (r) is: wherein 3p_mIs the maximum number less than or equal to r + 1; and

(e) minimum number of aromatic groups (a) and total number of net positive charges (p)_t) The relationship between them is: wherein 3a is less than or equal to p_tMaximum of +1, except when a is 1, p_tOr may be 1.

Drawings

FIG. 1: in mitochondria [ Dmt¹]Cellular internalization and aggregation of DALDA (SS-02). (A) Mitochondrial uptake of SS-19 (ex/em: 320/420) was measured using a fluorescence spectrophotometer. Addition of isolated mouse liver mitochondria (0.35mg/ml) resulted in rapid quenching of SS-19 fluorescence intensity (gray line). Mitochondrial reduction quenching with FCCP (1.5. mu.M) pretreatment<20% (black line). (B) The isolated mitochondria use³H]SS-02 was incubated at 37 ℃ for 2 minutes. The uptake was stopped by centrifugation (16000 Xg) at 4 ℃ for 5 min and the radioactivity in the pellet was determined. Mitochondrial inhibition pretreated with FCCP³H]SS-02 intake is 20%. Data are expressed as mean ± sem; n-3, P tested with Student's t<0.05. (C) Mitochondrial swelling induced by Procalotin lost TMRM taken up by isolated mitochondria, while SS-19 taken up was maintained at high concentrations. The black line is TMRM; the red line is SS-19. (D) Addition of SS-02 (200. mu.M) to isolated mitochondria did not alter mitochondrial potential as determined by TMRM fluorescence. Addition of FCCP (1.5. mu.M) causes rapid depolarization, while Ca²⁺(150 μ M) results in depolarization and a progressive onset of MPT.

Fig. 2 [ Dmt¹]DALDA (SS-02) protects mitochondria from Ca²⁺Overload and 3-nitropropionic acid (3NP) -induced Mitochondrial Permeability Transition (MPT). (A) Pretreatment of the isolated mitochondria with 10 μ M SS-02 (addition indicated by the lower arrow) prevented by Ca²⁺Occurrence of overload induced MPT (upper arrow). The black line is a buffer solution; the red line is SS-02. (B) Pretreatment of the separated mitochondria with SS-02 increases the doubling of Ca addition prior to MPT²⁺Mitochondrial tolerance of (a). Arrows indicate addition of buffer or SS-02. Line 1 is buffer; line 2 is 50 μ M SS-02; line 3 is 100 μ M SS-02. (C) SS-02 dose-dependently delayed the onset of MPT induced by 1mM3 NP. Arrows indicate addition of buffer or SS-02. Line 1 is buffer, line 2 is 0.5 μ M SS-02; line 3 is 5 μ M SS-02; line 4 was 50 μ M SS-02.

Fig. 3 [ Dmt¹]DALDA (SS-02) inhibits mitochondrial swelling and cell proliferationReleasing pigment C. (A) The isolated mitochondria were pretreated with SS-02, which dose-dependently inhibited Ca at 200. mu.M²⁺Mitochondrial swelling induced in a dose-dependent manner. Swelling was measured by absorbance at 540 nm. (B) SS-02 inhibition of Ca in isolated mitochondria²⁺Induced cytochrome C release. The amount of cytochrome C released is expressed as the percentage of total cytochrome C in the mitochondria. Data are presented as mean ± standard error, n ═ 3. (C) SS-02 can also inhibit MPP⁺(300. mu.M) induced mitochondrial swelling.

FIG. 4. D-arginine-Dmt-lysine-phenylalanine-NH₂(SS-31) inhibits mitochondrial swelling and cytochrome C release. (A) Pretreatment of the isolated mitochondria with SS-31 (10. mu.M) prevented the formation of Ca²⁺The occurrence of induced MPT. The gray line is a buffer solution; the red line is SS-31. (B) Pretreatment of mitochondria with SS-31 (50. mu.M) inhibited the inhibition by 200mM Ca²⁺Induced mitochondrial swelling. Swelling was measured by measuring light scattering at 570 nm. (C) SS-02 and SS-31 inhibit the reaction of cyclosporin (CsA) with Ca²⁺Comparison of induced mitochondrial swelling and cytochrome C release. Cytochrome C release is expressed as the percentage of total cytochrome C in the mitochondria. Data are presented as mean ± standard error, n ═ 3.

FIG. 5 [ Dmt ]¹]DALDA (SS-02) and D-arginine-Dmt-lysine-phenylalanine-NH₂(SS-31) protection of myocardial contractility during ischemia-reperfusion of perfused isolated guinea pig hearts. Hearts were perfused with buffer or buffer containing SS-02(100nM) or SS-31(1nM) for 30 minutes, and then subjected to global ischemia for 30 minutes. Reperfusion was performed with the same perfusion solution. Significant differences were found in the 3 treatment groups (two-way anova, P)<0.001)。

FIG. 6. adding Dmt to cardioplegia solution¹]DALDA significantly enhanced contractile function of perfused isolated guinea pig hearts following prolonged ischemia. After 30 min of stabilization, the heart was either treated with St.Thomas Cardioplegia (CPS) or with [ Dmt ] at 100nM¹]CPS perfusion of DALDA was 3 minutes. By total occlusion of coronary arteriesInfusion was performed for 90 minutes to initiate total heart loss. Reperfusion was then performed for 60 minutes with oxidized Krebs-Henseleit solution. Upon receiving [ Dmt¹]Post-ischemic contractility was significantly improved in the DALDA group (P)<0.001)。

Detailed Description

The present invention is based on the surprising discovery by the inventors: certain aromatic-cationic peptides significantly reduce the number of mitochondria undergoing Mitochondrial Permeability Transition (MPT), even completely blocking MPT. Reducing the number of mitochondria undergoing MPT and preventing MPT is important because MPT is associated with a variety of common diseases and conditions in mammals. In addition, isolated organs of mammals are prone to MPT. These diseases and conditions are of particular clinical interest, as they affect a large percentage of the population at some stage of life.

Peptides

The aromatic-cationic peptides useful in the present invention are water-soluble and highly polar. Despite these properties, these peptides are able to readily cross cell membranes.

Aromatic-cationic peptides useful in the present invention include a minimum of 3 amino acids covalently linked by peptide bonds, and preferably include a minimum of 4 amino acids covalently linked by peptide bonds.

The maximum number of amino acids in the aromatic-cationic peptides of the invention is about 20 amino acids covalently linked by peptide bonds. The preferred maximum number of amino acids is about 12, more preferably about 9, and most preferably about 6. Most preferably, the number of amino acids present in the peptide is 4.

The amino acid in the aromatic-cationic peptide useful in the present invention may be any amino acid. The term "amino acid" refers herein to any organic molecule containing at least one amino group and at least one carboxyl group. Preferably, at least one amino group is located in the alpha position relative to the carboxyl group.

These amino acids may be naturally occurring. Naturally occurring amino acids include, for example, the 20 most common left-handed (L) amino acids found naturally in mammalian proteins, namely alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ileu), 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 unrelated to protein synthesis. For example, ornithine and citrulline are amino acids that are metabolised by mammals during the production of urine.

Peptides useful in the invention may contain one or more non-naturally occurring amino acids. These non-naturally occurring amino acids can be levorotatory (L), dextrorotatory (D), or mixtures thereof. Most preferably, the peptide does not contain naturally occurring amino acids.

Non-naturally occurring amino acids are those amino acids that are not normally synthesized in the normal metabolic processes of an organism and do not naturally occur in proteins. In addition, preferred useful non-naturally occurring amino acids of the invention are also not recognized by common proteases.

Non-naturally occurring amino acids can occur anywhere in the peptide. For example, the non-naturally occurring amino acid can be located at the N-terminus, C-terminus, or anywhere between the N-terminus and C-terminus.

For example, the non-naturally occurring amino acid can include alkyl, aryl, or alkaryl groups. Some examples of alkyl amino acids include: alpha-aminobutyric acid, beta-aminobutyric acid, gamma-aminobutyric acid, delta-aminopentanoic acid and epsilon-aminocaproic acid. Some examples of aryl amino acids include ortho-, meta-, and para-aminobenzoic acid. Some examples of alkylaryl amino acids include o-, m-, and p-aminophenylacetic acid, and gamma-phenyl-beta-aminobutyric acid.

Non-naturally occurring amino acids also include derivatives of naturally occurring amino acids. Derivatives of naturally occurring amino acids can include, for example, the addition of one or more chemical groups to the naturally occurring amino acid.

For example, one or more chemical groups may be added to one or more of the 2 ', 3 ', 4 ', 5 ' or 6 ' positions of the aromatic ring of a phenylalanine or tyrosine residue, or one or more of the 4 ', 5 ', 6 ' or 7 ' positions of the benzo ring of a tryptophan residue. The group is any chemical group that can be added to an aromatic ring. Some examples of such groups include branched or straight chain C₁-C₄Alkyl, e.g. methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl or tert-butyl, C₁-C₄Hydrocarbyloxy (i.e. alkoxy), amino, C₁-C₄Alkylamine and C₁-C₄Dialkylamines (e.g., methylamine, dimethylamine), nitro, hydroxy, halo (i.e., fluoro, chloro, bromo, or iodo). Some specific examples of non-naturally occurring derivatives of naturally occurring amino acids include norvaline (Nva), norleucine (Nle), and hydroxyproline (Hyp).

Another example of an amino acid modification in a useful peptide in the methods of the invention is carboxyl derivatization of an aspartic acid or glutamic acid residue of the peptide. An example of derivatization is amidation with ammonia or with primary or secondary amines such as methylamine, ethylamine, dimethylamine or diethylamine. Another example of derivatization includes esterification with, for example, methanol or ethanol.

Another such modification includes amino derivatization of lysine, arginine, or histidine residues. For example, these amino groups may be acylated. Some suitable acyl groups include, for example, any of the above C₁-C₄Benzoyl or alkanoyl of alkyl groups, such as acetyl or propionyl.

Preferably the non-naturally occurring amino acid is stable to common proteases, more preferably it is not susceptible thereto. Examples of non-naturally occurring amino acids that are stable or insensitive to proteases include dextrorotatory (D-) forms of any of the naturally occurring L-amino acids described above, as well as L-and/or D-forms of non-naturally occurring amino acids. D-amino acids are not normally present in proteins, and although they are found in certain antimicrobial peptides, they are synthesized by tools other than the normal ribosomal protein synthesizer of cells. As used herein, these D-amino acids can be considered non-naturally occurring amino acids.

To minimize sensitivity to proteases, peptides useful in the methods of the invention should have less than 5, preferably less than 4, more preferably less than 3, and most preferably less than 2 contiguous L-amino acids recognized by common proteases, whether or not these amino acids are naturally occurring or non-naturally occurring. Most preferably, the peptide contains only D-amino acids and no L-amino acids.

If the peptide contains a protease-sensitive amino acid sequence, at least one of these amino acids is preferably a non-naturally occurring D-amino acid (D-arginine), thereby providing protease resistance. Examples of protease sensitive sequences include 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.

It is important that the aromatic-cationic peptide has a minimum number of net positive charges relative to the total number of amino acid residues in the peptide at physiological pH. The minimum number of net positive charges at physiological pH is hereinafter denoted as (p)_m). The total number of amino acid residues in the peptide is hereinafter denoted as (r).

The minimum number of net positive charges discussed below are all at physiological pH. The term "physiological pH" refers herein to the normal pH in the tissue and organ cells of the mammalian body. For example, the physiological pH of humans is typically about 7.4, but the normal physiological pH in mammals can be any pH from about 7.0 to about 7.8.

"Net charge" herein refers to the difference between the number of positive and negative charges carried by the amino acids present in the peptide. In this specification, it is to be understood that the net charge is measured at physiological pH. Naturally occurring amino acids that have a positive charge at physiological pH include L-lysine, L-arginine, and L-histidine. Naturally occurring amino acids having a negative charge at physiological pH include L-aspartic acid and L-glutamic acid.

Typically, peptides have a positively charged N-terminal amino group and a negatively charged C-terminal carboxyl group. At physiological pH the charges cancel each other out. As an example of calculating the net charge, peptide: tyrosine-arginine-phenylalanine-lysine-glutamic acid-histidine-tryptophan-arginine (Tyr-Arg-Phe-Lys-Glu-His-Trp-Arg) has one negatively charged amino acid (i.e., glutamic acid) and four positively charged amino acids (i.e., two arginine residues, one lysine and one histidine). The peptide thus contains 3 net positive charges.

In one embodiment of the invention, the aromatic-cationic peptide has a minimum number of net positive charges (p) at physiological pH_m) And the total number of amino acid residues (r) is: wherein 3P_mIs the maximum number less than or equal to r + 1. In this embodiment, the minimum number of net positive charges (p)_m) And the total number of amino acid residues (r) are as follows:

(r)	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20
																			(p)	1	1	2	2	2	3	3	3	4	4	4	5	5	5	6	6	6	7

in another embodiment, the aromatic-cationic peptide exhibits minimal number of net positive charges (p)_m) And the total number of amino acid residues (r) is: wherein 2p is_mIs the maximum number less than or equal to r + 1. In this embodiment, the minimum number of net positive charges (p)_m) And the total number of amino acid residues (r) are as follows:

(r)	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20
																			(p)	2	2	3	3	4	4	5	5	6	6	7	7	8	8	9	9	10	10

in one embodiment, the minimum number of net positive charges (p)_m) And the total number of amino acid residues (r). In another embodiment, the peptide contains 3 or 4 amino acid residues and has at least one net positive charge, preferably at least 2 net positive charges and more preferably at least 3 net positive charges.

The aromatic-cationic peptide has a relative net positive charge sum (p)_t) The minimum amount of aromatic groups is also important. The minimum number of aromatic groups is hereinafter denoted as (a).

Naturally occurring amino acids having aromatic groups include those of histidine, tryptophan, tyrosine and phenylalanine. For example, hexapeptides: lysine-glutamine-tyrosine-arginine-phenylalanine-tryptophan contain 2 net positive charges (contributed by lysine and arginine residues) and 3 aromatic groups (contributed by tyrosine, phenylalanine and tryptophan residues).

In one embodiment of the invention, aromatic-cationic peptides useful in the methods of the invention have a net positive charge sum (p) at minimum number of aromatic groups (a) and physiological pH_t) The relationship between them is: wherein 3a is less than or equal to p_tMaximum of +1, except when p_tIs 1, and a may be 1. In this example, the minimum number of aromatic groups (a) and the total number of net positive charges (p)_t) The relationship between them is as follows:

(p)	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20
																					(a)	1	1	1	1	2	2	2	3	3	3	4	4	4	5	5	5	6	6	6	7

in another embodiment, the aromatic-cationic peptide has a minimum number of aromatic groups (a) and a total number of net positive charges (p)_t) The relationship between them is: wherein 2a is less than or equal to p_tMaximum of + 1. In this example, the minimum number of aromatic amino acid residues (a) and the total number of net positive charges (p)_t) The relationship between them is as follows:

(p)	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20
																					(a)	1	1	2	2	3	3	4	4	5	5	6	6	7	7	8	8	9	9	10	10

in another embodiment, the number of aromatic groups (a) and the total number of net positive charges (p)_t) Are equal.

Carboxyl groupIn particular the terminal carboxyl group of the C-terminal amino acid, is preferably amidated with, for example, ammonia to form a 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 be, for example, an alkyl group, especially a branched or straight chain C₁-C₄Alkyl, or aryl amines. Accordingly, the amino acid at the C-terminal end of the peptide may be converted into an amide group, an N-methylamide group, an N-ethylamide group, an N, N-dimethylamide group, an N, N-diethylamide group, an N-methyl-N-ethylamide group, an N-phenylamide group or an N-phenyl-N-ethylamide group.

Even if the free carboxyl groups of asparagine residue, glutamine residue, aspartic acid residue and glutamic acid residue are not located at the C-terminus of the aromatic cationic peptide of the present invention, they may be amidated regardless of their location in the peptide. Amidation of these internal positions can be performed with ammonia or any of the primary or secondary amines described above.

In one embodiment, an aromatic-cationic peptide useful in the methods of the invention is a tripeptide having two net positive charges and at least one aromatic amino acid. In a particular embodiment, an aromatic-cationic peptide useful in the methods of the invention is a tripeptide having two net positive charges and two aromatic amino acids.

Aromatic-cationic peptides useful in the methods of the invention include, but are not limited to, the following examples of peptides:

lysine-dextro arginine-tyrosine-NH₂(Lys-D-Arg-Tyr-NH₂)，

Phenylalanine-dextro-arginine-histidine (Phe-D-Arg-His),

D-tyrosine-tryptophan-lysine-NH₂(D-Tyr-Trp-Lys-NH₂)，

Tryptophan-D-lysine-tyrosine-arginine-NH₂(Trp-D-Lys-Tyr-Arg-NH₂)，

tyrosine-histidine-D-glycine-methionine (Tyr-His-D-Gly-Met),

phenylalanine-arginine-dextro-histidine-aspartic acid (Phe-Arg-D-His-Asp),

tyrosine-dextro arginine-phenylalanine-lysine-glutamic acid-NH₂(Tyr-D-Arg-Phe-Lys-Glu-NH₂)，

Methionine-tyrosine-dextrolysine-phenylalanine-arginine (Met-Tyr-D-Lys-Phe-Arg),

dextro histidine-glutamic acid-lysine-tyrosine-dextro phenylalanine-arginine (D-His-Glu-Lys-Tyr-D-Phe-Arg),

lysine-dextroglutamine-tyrosine-arginine-dextrophenylalanine-tryptophan-NH₂(Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH₂)，

Phenylalanine-dextroarginine-lysine-tryptophan-tyrosine-dextroarginine-histidine (Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His),

glycine-dextro phenylalanine-lysine-tyrosine-histidine-dextro arginine-tyrosine-NH₂(Gly-D-Phe-Lys-Tyr-His-D-Arg-Tyr-NH₂)，

valine-D-lysine-histidine-tyrosine-D-phenylalanine-serine-tyrosine-arginine-NH₂(Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH₂)，

Tryptophan-lysine-phenylalanine-dextro-aspartic acid-arginine-tyrosine-dextro-histidine-lysine (Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-Lys),

lysine-tryptophan-dextro tyrosine-arginine-asparagine-phenylalanine-tyrosine-dextro histidine-NH₂(Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH₂)，

Threonine-glycine-tyrosine-arginine-dextro-histidine-phenylalanine-tryptophan-dextro-histidine-lysine (Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-Lys),

aspartic acid-D-tryptophan-lysine-tyrosine-D-histidine-phenylalanine-arginine-D-glycine-lysine-NH₂(Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH₂)，

D-histidine-lysine-tyrosine-D-phenylalanine-glutamic acid-D-aspartic acid-D-histidine-D-lysine-arginine-tryptophan-NH₂(D-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-His-D-Lys-Arg-Trp-NH₂)，

Alanine-dextro-phenylalanine-dextro-arginine-tyrosine-lysine-dextro-tryptophan-histidine-dextro-tyrosine-glycine-phenylalanine (Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-Phe),

tyrosine-dextro histidine-phenylalanine-dextro arginine-aspartic acid-lysine-dextro arginine-histidine-tryptophan-dextro histidine-phenylalanine (Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-Phe),

phenylalanine-dextrotyrosine-arginine-glutamic acid-aspartic acid-dextrolysine-arginine-dextroarginine-histidine-phenylalanine-NH₂(Phe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH₂)，

Phenylalanine-tyrosine-lysine-dextro arginine-tryptophan-histidine-dextro lysine-glutamic acid-arginine-dextro tyrosine-threonine (Phe-Try-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-Thr),

tyrosine-aspartic acid-dextrolysine-tyrosine-phenylalanine-dextrolysine-dextroarginine-phenylalanine-proline-dextrotyrosine-histidine-lysine (Tyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-Lys),

glutamic acid-arginine-dextro lysine-tyrosine-dextro valine-phenylalanine-dextro histidine-tryptophan-arginine-dextro glycineacid-tyrosine-arginine-dextro-methionine-NH₂(Glu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met-NH₂)，

arginine-D-leucine-D-tyrosine-phenylalanine-lysine-glutamic acid-D-lysine-arginine-D-tryptophan-lysine-D-phenylalanine-tyrosine-D-arginine-glycine (Arg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-Phe-Tyr-D-Arg-Gly),

d-glutamic acid-arginine-lysine-D-arginine-D-histidine-phenylalanine-D-valine-tyrosine-arginine-tyrosine-D-tyrosine-arginine-histidine-phenylalanine-NH₂(D-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH₂)，

Aspartic acid-arginine-dextro phenylalanine-cysteine-phenylalanine-dextro arginine-dextro lysine-tyrosine-arginine-dextro tyrosine-tryptophan-dextro histidine-tyrosine-dextro phenylalanine-lysine-phenylalanine (Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-Phe),

histidine-tyrosine-dextro arginine-tryptophan-lysine-phenylalanine-dextro aspartic acid-alanine-arginine-cysteine-dextro tyrosine-histidine-phenylalanine-dextro lysine-tyrosine-histidine-serine-NH₂(His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH₂)，

glycine-alanine-lysine-phenylalanine-D-lysine-glutamic acid-arginine-tyrosine-histidine-D-arginine-aspartic acid-tyrosine-tryptophan-D-histidine-tryptophan-histidine-D-lysine-aspartic acid (Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-Asp),

and

threonine-tyrosine-arginine-D-lysineAmino acid-tryptophan-tyrosine-glutamic acid-aspartic acid-dextrolysine-dextroarginine-histidine-phenylalanine-dextrotyrosine-glycine-valine-isoleucine-dextrohistidine-arginine-tyrosine-NH₂(Thr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-NH₂)。

In one embodiment, peptides useful in the methods of the invention have mu-opioid receptor agonist activity (i.e., activate the mu-opioid receptor). Activation of the mu-opioid receptor generally has an analgesic effect.

In some cases, aromatic-cationic peptides with mu-opioid receptor activity are preferred. For example, in short-term treatments such as acute diseases and conditions, it is beneficial to use aromatic-cationic peptides that activate μ -opioid receptors. These acute diseases and conditions are often associated with moderate or severe pain. In these cases, the analgesic effect of the aromatic-cationic peptide is beneficial in a therapeutic regimen for a patient or other mammal, although it does not activate the aromatic-cationic peptide of the mu-opioid receptor, but may be used in combination with or without an analgesic, depending on clinical needs.

In other cases, another alternative is preferably an aromatic-cationic peptide that does not have mu-opioid receptor activity. For example, the use of aromatic-cationic peptides that activate the μ -opioid receptor may be contraindicated during long-term treatment such as chronic diseases and conditions. In these cases, the potential side effects or addictive effects of the aromatic-cationic peptides may prevent the use of aromatic-cationic peptides having mu-opioid receptor activation in therapeutic regimens for patients or other mammals.

Potential side effects may include sedation, constipation, and respiratory depression. In these cases, aromatic-cationic peptides that do not activate the μ -opioid receptor may be an appropriate therapeutic agent.

Examples of acute disorders include: heart attack, stroke (stroke) and trauma. Trauma may include brain trauma and spinal cord trauma.

Examples of chronic diseases and conditions include: coronary artery disease and any neurodegenerative disease as described below.

Peptides having mu-opioid receptor activity useful in the methods of the invention are typically those that contain a tyrosine residue or N-terminal (i.e., first amino acid position) derivative of tyrosine. Preferred tyrosine derivatives include: 2' -methyl tyrosine (Mmt); 2 ', 6' -dimethyltyrosine (2 '6' Dmt); 3 ', 5' -dimethyltyrosine (3 '5' Dmt); n, 2 ', 6' -trimethyltyrosine (Tmt); and 2 '-hydroxy-6' -methyl tyrosine (Hmt).

In a particularly preferred embodiment, the peptide having mu-opioid receptor activity has the formula: tyrosine-dextro arginine-phenylalanine-lysine-NH₂(Tyr-D-Arg-Phe-Lys-NH₂) (for convenience, the acronym is: DALDA, referred to herein as SS-01). DALDA has 3 net positive charges provided by the amino acids tyrosine, arginine and lysine, and 2 aromatic groups provided by phenylalanine and tyrosine. The tyrosine of DALDA may be a modified tyrosine derivative such as 2 ', 6' -dimethyltyrosine, to produce a tyrosine derivative having the formula 2 ', 6' -dimethyltyrosine-d-arginine-phenylalanine-lysine-NH₂(2’，6’-Dmt-D-Arg-Phe-Lys-NH₂) (i.e., Dmt)¹-DALDA, herein referred to as SS-02).

Peptides that do not have mu-opioid receptor activity typically do not contain a tyrosine residue or tyrosine derivative at the N-terminus (i.e., position 1 of the amino acid). 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 N-terminal amino acid is phenylalanine or a derivative thereof. Preferred phenylalanine derivatives include: 2 ' -Methylphenylalanine (Mmp), 2 ', 6 ' -dimethylphenylalanine (Dmp), N, 2 ', 6 ' -trimethylphenylalanine (Tmp) and 2 ' -hydroxy-6 ' -methylphenylalanine (Hmp).

Another aromatic-cationic peptide that does not have mu-opioid receptor activity has the formula: phenylalanine-dextro-arginine-phenylalanine-lysine-NH₂(Phe-D-Arg-Phe-Lys-NH₂) (i.e., Phe)¹DALDA, referred to herein as SS-20). Alternatively, the N-terminal phenylalanine may be a derivative of phenylalanine such as 2 ', 6' -dimethylphenylalanine (2 '6' Dmp). The molecular formula of DALDA containing 2 ', 6' -dimethylphenylalanine at amino acid position 1 is 2 ', 6' -Dmp-dextro-arginine-phenylalanine-lysine-NH₂(2’，6’-Dmp-D-Arg-Phe-Lys-NH₂) (i.e., 2 '6' Dmp)¹-DALDA)。

In a preferred embodiment, Dmt¹The amino acid sequence of DALDA (SS-02) is rearranged so that Dmt is not located at the N-terminus. Examples of such aromatic-cationic peptides that do not have mu-opioid receptor activity have the formula: d-arginine-2 '6' Dmt-lysine-phenylalanine-NH₂(D-Arg-2’6’Dmt-Lys-Phe-NH₂) (referred to as SS-31 in this specification).

DALDA、Phe¹the-DALDA, SS-31, and derivatives thereof may further include functional analogs. If the analogue has the same chemical formula as DALDA, Phe¹A similar function of DALDA or SS-31, the peptide is then referred to as DALDA, Phe¹-functional analogs of DALDA or SS-31. For example, the analogue may be DALDA, Phe¹-substitution variants of DALDA or SS-31 in which one or more amino acids are substituted by other amino acids.

DALDA、Phe¹Suitable substitution variants of DALDA or SS-31 include conservative amino acid substitutions. Amino acids can be grouped according to their physicochemical properties as follows:

(a) non-polar amino acids: alanine (a) serine (S) threonine (T) proline (P) glycine (G) (ala (a) ser (S) thr (T) pro (P) gly (G));

(b) acidic amino acids: asparagine (N) aspartic acid (D) glutamic acid (E) glutamine (Q) (asn (N) asp (D) glu (E) gln (Q));

(c) basic amino acids: histidine (H) arginine (R) lysine (K) (his (H) arg (R) lys (K));

(d) hydrophobic amino acids: methionine (M) leucine (L) isoleucine (I) valine (V) (met (M) leu (L) ile (I) val (V)); and

(e) aromatic amino acids: phenylalanine (F) tyrosine (T) tryptophan (W) histidine (H) (phe (F) tyr (y) trp (W) his (H)).

Substitution of one amino acid in a peptide with another amino acid of the same group is called conservative substitution, and this can preserve the physicochemical properties of the original peptide. Conversely, substitution of one amino acid in a peptide with another amino acid from a different set is generally more likely to alter the properties of the original peptide.

Examples of useful analogs that activate the mu-opioid receptor in the practice of the present invention include, but are not limited to, the aromatic-cationic peptides shown in Table 1.

TABLE 1

Amino acid position 5C-terminal

Position 1 position 2 position 3 position 4 (if present) modification

Tyrosine dextral arginine phenylalanine lysine NH₂

Tyrosine dextro arginine phenylalanine ornithineAlkali (Orn) NH₂

Tyrosine dextral arginine phenylalanine Dab NH₂

Tyrosine dextral arginine phenylalanine Dap NH₂

2 '6' Dmt D-arginine phenylalanine lysine NH₂

2 '6' Dmt D-arginine phenylalanine lysine cysteine NH₂

2 '6' Dmt D-arginine phenylalanine lysine-NH (CH)₂)₂-NH-dns NH₂

2 '6' Dmt D-arginine phenylalanine lysine-NH (CH)₂)₂-NH-atn NH₂

2 '6' Dmt D-arginine phenylalanine dns lysine NH₂

2 '6' Dmt D-citicoline phenylalanine lysine NH₂

(D-Cit)

2 '6' Dmt D-citicoline phenylalanine Ahp NH₂

(D-Cit)

2 '6' Dmt D-arginine phenylalanine ornithine ammonia base (Orn) NH₂

2 '6' Dmt D-arginine phenylalanine Dab NH₂

2 '6' Dmt D-arginine phenylalanine Dap NH₂

2 '6' Dmt D-arginine phenylalanine Ahp (2-aminoheptanoic acid) NH₂

Bio-2 '6' Dmt D-arginine phenylalanine lysine NH₂

3 '5' Dmt D-arginine phenylalanine lysine NH₂

3 '5' Dmt D-arginine phenylalanine ornithine ammonia base (Orn) NH₂

3 '5' Dmt D-arginine phenylalanine Dab NH₂

3 '5' Dmt D-arginine phenylalanine Dap NH₂

Tyrosine dextro arginineTyrosine lysine NH₂

Tyrosine dextro arginine tyrosine ornithine (Orn) NH₂

Tyrosine dextral arginine tyrosine Dab NH₂

Tyrosine dextro-arginine tyrosine Dap NH₂

2 '6' Dmt D-arginine tyrosine lysine NH₂

2 '6' Dmt D-arginine tyrosine ornithine ammonia base (Orn) NH₂

2 '6' Dmt D-arginine tyrosine Dab NH₂

2 '6' Dmt D-arginine tyrosine Dap NH₂

2 '6' Dmt D-arginine 2 '6' Dmt lysine NH₂

2 '6' Dmt D-arginine 2 '6' Dmt ornithine (Orn) NH₂

2 '6' Dmt D-arginine 2 '6' Dmt Dab NH₂

2 '6' Dmt D-arginine 2 '6' Dmt Dap NH₂

3 '5' Dmt D-arginine 3 '5' Dmt arginine NH₂

3 '5' Dmt D arginine 3 '5' Dmt lysine NH₂

3 '5' Dmt D-arginine 3 '5' Dmt ornithine (Orn) NH₂

3 '5' Dmt D-arginine 3 '5' Dmt Dab NH₂

Tyrosine D-lysine phenylalanine Dap NH₂

Tyrosine dextral lysine phenylalanine arginine NH₂

Tyrosine D-lysine phenylalanine lysine NH₂

Tyrosine dextro-lysine phenylalanine ornithine (Orn) NH₂

2 '6' Dmt D-lysine phenylalanine Dab NH₂

2 '6' Dmt D-lysine phenylalanine Dap NH₂

2 '6' Dmt D-lysine phenylalanine arginine NH₂

2 '6' Dmt D-lysine phenylalanine lysine NH₂

3 '5' Dmt D-lysine phenylalanine ornithine (Orn) NH₂

3 '5' Dmt D-lysine phenylalanine Dab NH₂

3 '5' Dmt D-lysine phenylalanine Dap NH₂

3 '5' Dmt D-lysine phenylalanine arginine NH₂

Tyrosine dextro-lysine tyrosine lysine NH₂

Tyrosine dextro lysine tyrosine ornithine (Orn) NH₂

Tyrosine dextro-lysine tyrosine Dab NH₂

Tyrosine dextro-lysine tyrosine Dap NH₂

2 '6' Dmt D-lysine tyrosine lysine NH₂

2 '6' Dmt D-lysine tyrosine ornithine (Orn) NH₂

2’6' Dmt D-lysine tyrosine Dab NH₂

2 '6' Dmt D-lysine tyrosine Dap NH₂

2 '6' Dmt D lysine 2 '6' Dmt lysine NH₂

2 '6' Dmt D-lysine 2 '6' Dmt ornithine (Orn) NH₂

2 '6' Dmt D-lysine 2 '6' Dmt Dab NH₂

2 '6' Dmt D-lysine 2 '6' Dmt Dap NH₂

2 '6' Dmt D-arginine phenylalanine dnsDap NH₂

2 '6' Dmt D-arginine phenylalanine atnDap NH₂

3 '5' Dmt D lysine 3 '5' Dmt lysine NH₂

3 '5' Dmt D-lysine 3 '5' Dmt ornithine (Orn) NH₂

3 '5' Dmt D-lysine 3 '5' Dmt Dab NH₂

3 '5' Dmt D-lysine 3 '5' Dmt Dap NH₂

Tyrosine dextral lysine phenylalanine arginine NH₂

Tyrosine dextro guanamine phenylalanine arginine NH₂

(Orn)

Tyrosine dextro Dab phenylalanine arginine NH₂

Tyrosine dextro Dap phenylalanine arginine NH₂

2 '6' Dmt D-arginine phenylalanine arginine NH₂

2 '6' Dmt D-lysine phenylalanine arginine NH₂

2 '6' Dmt D-ornithine phenylalanine arginine NH₂

(D-Orn)

2 '6' Dmt D-Dab Phe arginine NH₂

3 '5' Dmt D-Dap phenylalanine arginine NH₂

3 '5' Dmt D-arginine phenylalanine arginine NH₂

3 '5' Dmt D-lysinePhenylalanine arginine NH₂

3 '5' Dmt D-ornithine phenylalanine arginine NH₂

(D-Orn)

Tyrosine dextro lysine tyrosine arginine NH₂

Tyrosine dextro guanamine tyrosine arginine NH₂

(D-Orn)

Tyrosine dextro Dab tyrosine arginine NH₂

Tyrosine dextro Dap tyrosine arginine NH₂

2 '6' Dmt D-arginine 2 '6' Dmt arginine NH₂

2 '6' Dmt D lysine 2 '6' Dmt arginine NH₂

2 '6' Dmt D-ornithine 2 '6' Dmt arginine NH₂

(D-Orn)

2 '6' Dmt dextro Dab 2 '6' Dmt arginine NH₂

3’5’Dmt Dextro Dap 3 '5' Dmt arginine NH₂

3 '5' Dmt D-arginine 3 '5' Dmt arginine NH₂

3 '5' Dmt D lysine 3 '5' Dmt arginine NH₂

3 '5' Dmt D-ornithine 3 '5' Dmt arginine NH₂

(D-Orn)

Mmt D-arginine phenylalanine lysine NH₂

Mmt D-arginine phenylalanine ornithine (Orn) NH₂

Mmt D-arginine phenylalanine Dab NH₂

Mmt D-arginine phenylalanine Dap NH₂

Tmt D-arginine phenylalanine lysine NH₂

Tmt D-arginine phenylalanine ornithine (Orn) NH₂

Tmt D-arginine phenylalanine Dab NH₂

Tmt dextro argininePhenylalanine Dap NH acid₂

Hmt D-arginine phenylalanine lysine NH₂

Hmt D-arginine phenylalanine ornithine (Orn) NH₂

Hmt D-arginine phenylalanine Dab NH₂

Hmt D-arginine phenylalanine Dap NH₂

Mmt D-lysine phenylalanine lysine NH₂

Mmt D-lysine phenylalanine ornithine (Orn) NH₂

Mmt D-lysine phenylalanine Dab NH₂

Mmt D-lysine phenylalanine Dap NH₂

Mmt D-lysine phenylalanine arginine NH₂

Tmt D-lysine phenylalanine lysine NH₂

Tmt D-lysine phenylalanine ornithine (Orn) NH₂

Tmt D-lysine phenylalanine Dab NH₂

Tmt D-lysine phenylalanine Dap NH₂

Tmt D-lysine phenylalanine arginine NH₂

Hmt D-lysine phenylalanine lysine NH₂

Hmt D-lysine phenylalanine ornithine (Orn) NH₂

Hmt D-lysine phenylalanine Dab NH₂

Hmt D-lysine phenylalanine Dap NH₂

Hmt D-lysine phenylalanine arginine NH₂

Mmt D-lysine phenylalanine arginine NH₂

Mmt D-ornithine phenylalanine arginine NH₂

(D-Orn)

Mmt D-Dab phenylalanine arginine NH₂

Mmt D-Dap phenylalanine arginine NH₂

Mmt D-arginine phenylalanine arginine NH₂

Tmt D-lysine phenylalanine arginine NH₂

Tmt D-ornithine phenylalanine arginine NH₂

(D-Orn)

Tmt D-Dab phenylalanine arginine NH₂

Tmt D-Dap phenylalanine arginine NH₂

Tmt D-arginine phenylalanine arginine NH₂

Hmt D-lysine phenylalanine arginine NH₂

Hmt dextro ornithine phenylalanine arginine NH₂

(D-Orn)

Hmt dextro Dab phenylalanine arginine NH₂

Hmt dextro Dap phenylalanine arginine NH₂

Hmt D-arginine phenylalanine arginine NH₂

Dab ═ diaminobutyric acid

Dap ═ diaminopropionic acid

Dmt ═ dimethyl tyrosine

Mmt ═ 2' -methyl tyrosine

Tmt ═ N, 2 ', 6' -trimethyltyrosine

Hmt-2 '-hydroxy-6' -methyl tyrosine

dnsDap ═ beta-dansyl-L-alpha, beta-diaminopropionic acid

atnDap ═ beta-anthranoyll-alpha, beta-diaminopropionic acid

Bio ═ biotin.

Examples of useful analogs that do not activate the mu-opioid receptor in the practice of the present invention include, but are not limited to, the aromatic-cationic peptides shown in table 2.

TABLE 2

Amino acid C-terminus

Position 1 position 2 position 3 position 4 modification

D-arginine Dmt lysine phenylalanine NH₂

D-arginine Dmt phenylalanine lysine NH₂

D-arginine phenylalanine lysine Dmt NH₂

D-arginine phenylalanine Dmt lysine NH₂

D-arginine lysine Dmt phenylalanine NH₂

D-arginine lysine phenylalanine Dmt NH₂

Phenylalanine lysine Dmt dextro arginine NH₂

Phenylalanine lysine dextro arginine Dmt NH₂

Phenylalanine dextro-arginine Dmt lysine NH₂

Phenylalanine dextro-arginine lysine Dmt NH₂

Phenylalanine Dmt dextro-arginine lysine NH₂

Phenylalanine Dmt lysine dextro arginine NH₂

Lysine phenylalanine dextro-arginine Dmt NH₂

Lysine phenylalanine Dmt dextro arginine NH₂

Lysine Dmt D arginine phenylalanine NH₂

Lysine Dmt phenylalanine dextro arginine NH₂

Lysine dextro arginine phenylalanine Dmt NH₂

Lysine dextro arginine Dmt phenylalanine NH₂

D-arginine Dmt-D-arginine phenylalanine NH₂

D-arginine Dmt-D-arginine Dmt NH₂

D-arginine Dmt-D-arginine tyrosine NH₂

D-arginine Dmt-D-arginine tryptophan NH₂

Tryptophan-D-arginine-phenylalanine-lysine NH₂

Tryptophan-D-arginine-tyrosine-lysine NH₂

Tryptophan D-arginine Tryptophan lysine NH₂

Tryptophan-D-arginine-Dmt-lysine-NH₂

Color of dextro arginineL-alanine lysine phenylalanine NH₂

D-arginine tryptophan phenylalanine lysine NH₂

D-arginine tryptophan lysine Dmt NH₂

D-arginine tryptophan Dmt lysine NH₂

D-arginine lysine tryptophan phenylalanine NH₂

D-arginine lysine tryptophan Dmt NH₂

Cha D-arginine phenylalanine lysine NH₂

Alanine D-arginine phenylalanine lysine NH₂

Cha ═ cyclohexyl

The amino acids in the peptides shown in tables 1 and 2 may be in either the L-or D-conformation.

Method of treatment

The peptides described above may be used to treat any disease or disorder associated with MPT. Such diseases and conditions include, but are not limited to: ischemia and/or reperfusion of a tissue or organ, hypoxia, and any of a number of neurodegenerative diseases. Mammals in need of treatment or prevention of MPT are those mammals suffering from these diseases or disorders.

Ischemia in mammalian tissues or organs is a multifaceted pathological condition caused by oxygen deficiency (hypoxia) and/or glucose (i.e., substrate) deficiency. Oxygen and/or glucose deficiency in the tissue or organ cells results in a reduction or complete loss of energy production capacity and subsequent loss of the function of active ion transport across the membrane. Oxygen and/or glucose deficiency may also lead to pathological changes in other cell membranes, including permeability changes within the mitochondrial membrane. Other molecules, such as apoptotic proteins that are normally blocked in mitochondria, can leak into the cytoplasm and cause apoptosis (apoptosis cell death). Severe ischemia can lead to cell necrosis.

Ischemia or hypoxia in a particular tissue or organ can be caused by a loss or severe reduction in blood supply to that tissue or organ. Loss or severe reduction of blood supply can be caused by, for example, thromboembolic stroke, coronary arteriosclerosis, or peripheral vascular disease. The tissue affected by ischemia or hypoxia is typically a muscle, such as cardiac, skeletal, or smooth muscle.

The organ affected by ischemia or hypoxia may be any organ that suffers from ischemia or hypoxia. Examples of organs affected by ischemia or hypoxia include brain, heart, kidney and prostate. For example, myocardial ischemia or hypoxia is often caused by arteriosclerosis or thrombotic blockage, which results in a reduction or loss of oxygen delivery to myocardial tissue through the blood supply of cardiac arteries and capillaries. Such myocardial ischemia or hypoxia can lead to pain and necrosis of the affected myocardium, and ultimately can lead to heart failure.

Ischemia or hypoxia in skeletal or smooth muscle can be caused by similar causes. For example, ischemia or hypoxia in small intestine smooth muscle or limb skeletal muscle can also be caused by arteriosclerosis or thrombotic occlusion.

Reperfusion is the restoration of blood flow to any tissue or organ that reduces or blocks blood flow. For example, blood flow may be restored to any tissue or organ affected by ischemia or hypoxia. Restoration of blood flow (reperfusion) may be achieved by any method known to those skilled in the art. Reperfusion of ischemic heart tissue can be achieved, for example, by angioplasty (revascularization), coronary artery bypass grafting or by the use of thrombolytic drugs.

The methods of the invention may also be used to treat or prevent neurodegenerative diseases associated with MPT. Neurodegenerative diseases associated with MPT include: such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS, also known as Logoherberg's disease). The methods of the invention may be used to delay the onset or slow the progression of these or other neurodegenerative diseases associated with MPT. The methods of the invention are particularly useful in treating patients and susceptible persons in early stages of a neurodegenerative disease associated with MPT.

The peptides useful in the present invention may also be used to preserve mammalian organs prior to transplantation. For example, ex vivo organs may be susceptible to MPT due to lack of blood flow. Thus, the peptides can be used to prevent MPT in ex vivo organs.

The body organ may be placed in standard buffers such as those commonly used in the art. For example, ex vivo hearts may be placed in cardioplegic solution containing the peptides described above. The concentration of the peptide in a standard buffer can be readily determined by one skilled in the art. For example, the concentration may be between about 0.1nM and about 10. mu.M, preferably about 1. mu.M to about 10. mu.M.

The peptides may also be administered to a mammal undergoing drug treatment for diseases and conditions. If the side effects of the drug include MPT, the mammal that is using the drug will receive great benefit from the peptides of the invention.

An example of a drug that induces cytotoxicity by affecting MPT is the chemotherapeutic drug doxorubicin.

Synthesis of skin

Peptides useful in the methods of the invention can be chemically synthesized by any method known in the art. Suitable methods for synthesizing the protein include: for example by Stuart and Young in "Solid Phase Peptide Systhesis," second edition, Pierce Chemical Company (1984), and in "Solid Phase Peptide Systhesis," Methods Enzymol.289The method described in Academic Press, Inc, New York (1997).

Method of administration

In the methods of the invention, a useful peptide is administered to a mammal in an amount effective to reduce the amount of mitochondrial MPT encountered or to prevent MPT. The effective amount is determined in preclinical testing and clinical testing using methods familiar to physicians and clinicians.

In the methods of the invention, an effective amount of a useful peptide, preferably in a pharmaceutical composition, can be administered to a mammal in need thereof by any of a number of well-known methods of administering pharmaceutical compounds.

The peptide may be administered systemically or locally. In one embodiment, the peptide is administered intravenously. For example, aromatic-cationic peptides useful in the methods of the invention can be administered by rapid bolus intravenous injection. However, it is preferred that the peptide is administered by constant rate intravenous infusion.

The peptides can be injected directly into the coronary arteries, for example, in angioplasty or coronary artery bypass surgery, or coated onto coronary artery stents.

The peptide may also be administered orally, topically, intranasally, intramuscularly, subcutaneously, or transdermally. In a preferred embodiment, the transdermal administration of the aromatic-cationic peptide in the method of the invention is effected by iontophoresis, wherein the charged peptide is transported through the skin by an electric current.

Other routes of administration include intracerebroventricular or intrathecal administration. Intraventricular administration refers to administration into the luminal system of the brain. Intrathecal administration refers to administration into the subarachnoid space of the spinal cord. Thus, intraventricular or intrathecal administration may be preferred for diseases and conditions affecting organs or tissues of the central nervous system. In a preferred embodiment, intrathecal administration is used for spinal cord trauma.

Peptides useful in the methods of the invention can also be administered to mammals by sustained release means, as is well known in the art. Sustained release drug delivery is a method of drug delivery that achieves a certain level of drug over a specified period of time. This level is usually determined from serum or plasma concentrations.

Any dosage form known in the pharmaceutical art is suitable for administration of the aromatic-cationic peptides useful in the methods of the invention. For oral administration, liquid or solid dosage forms may be used. Some examples of dosage forms include: tablets, gel capsules, pills, lozenges, elixirs, suspensions, syrups, wafers, chews (chewing gum), and the like. The peptide may be mixed with a suitable pharmaceutical carrier (vehicle) or excipient known to those skilled in the art. Examples of carriers or excipients include: starch, milk, sucrose, certain types of clays, gelatin, lactic acid, stearic acid or its salts (including magnesium or calcium stearate), talc, vegetable fats or oils, gums and glycols.

For systemic, intracerebroventricular, intrathecal, topical, intranasal, subcutaneous, or transdermal administration, the dosage forms of the aromatic-cationic peptides useful in the methods of the invention may utilize conventional diluents, carriers, or excipients, and the like, as may be used in the art, to deliver the peptides. For example, the dosage form may contain one or more of the following: a stabilizer, a surfactant, preferably a non-ionic surfactant, and optionally a salt and/or a buffer. The peptide may be delivered as an aqueous solution or as a lyophilisate.

The stabilizer may be, for example, an amino acid such as glycine; or oligosaccharides such as sucrose, tetrasaccharide, lactose or dextran. Alternatively, the stabilizer may be a sugar alcohol such as mannitol; or a combination thereof. Preferably, the stabilizer or stabilizer composition constitutes from about 0.1% to about 10% by weight of the peptide.

The surfactant is preferably a nonionic surfactant, such as a polysorbate. Examples of suitable surfactants include: tween 20, tween 80; polyethylene glycol or polyoxyethylene polyoxypropylene glycol (ether), such as Pluronic F-68 from about 0.001% (w/v) to about 10% (w/v).

The salt or buffer may be any salt or buffer such as sodium chloride or sodium/potassium phosphate, respectively. Preferably, the buffer maintains the pH of the pharmaceutical composition in the range of about 5.5 to about 7.5. Salts and/or buffers are also used to maintain osmolarity at a level suitable for administration to a human or animal. It is preferred that the salt or buffer exhibits a roughly isotonic concentration of about 150. mu.M to about 300. mu.M.

In the process of the invention, the dosage forms of the useful peptides may additionally contain one or more customary additives. Some examples of such additives include: a solubilizing agent such as glycerol; antioxidants such as benzalkonium chloride (a mixture of quaternary ammonium compounds, called "quaternary ammonium compounds"), benzyl alcohol, chlorotetrone, or chlorobutanol; anesthetics such as morphine derivatives; or the isotonic agents mentioned above and the like. To further prevent oxidation or other spoilage, the pharmaceutical composition may be stored under nitrogen in vials sealed with impermeable stoppers.

The mammal can be any mammal, including, for example, farm animals such as sheep, pigs, cattle and horses; pets such as dogs and cats; laboratory animals such as rats, mice and rabbits. In a preferred embodiment, the mammal is a human.

Examples

Example 1:[Dmt¹]DALDA penetrates cell membranes

Study Using human intestinal epithelial cell line (Caco-2)³H][Dmt¹]Cellular uptake of DALDA and was performed with SH-SY5Y cells (human neuroblastoma cells), HEK293 cells (human embryonic kidney cells) and CRFK cells (renal epithelial cells)And (5) confirming. On collagen-coated 12-well plates (5X 10)⁵Cells/well) single layer cells were cultured for three days. On the fourth day, the cells were washed twice with preheated HBSS, and then with 0.2ml of a solution containing 250nM [ 2 ]³H][Dmt¹]HBSS from DALDA was incubated at 37 ℃ or 4 ℃ for various periods up to 1 hour.

As early as 5 minutes, it was observed in the cell lysate³H][Dmt¹]DALDA, and reached steady state levels at 30 minutes. The term "recovered in cell lysate" after 1 hour of incubation³H][Dmt¹]The total amount of DALDA represents approximately 1% of the total drug. Although³H][Dmt¹]The uptake of DALDA was slower at 4 ℃ than at 37 ℃, but reached 76.5% at 45 minutes and 86.3% at 1 hour. [³H][Dmt¹]Internalization of DALDA is not limited to Caco-2 cells, but can also be observed in SH-SY5Y cells, HEK293 cells, and CRFK cells. [³H][Dmt¹]The intracellular concentration of DALDA is estimated to be approximately 50-fold higher than the extracellular concentration.

In a separate experiment, [ Dmt ] was used in a range of concentrations (1. mu.M-3 mM)¹]DALDA incubated the cells at 37 ℃ for 1 hour. After the incubation period was complete, the cells were washed 4 times with HBSS and 0.2ml of 0.1N NaOH containing 1% SDS was added to each well. The cell contents were then transferred to scintillation vials and counted for radioactivity. To distinguish between internalized radioactivity and surface-bound radioactivity, an acid wash step was introduced. Prior to cell lysis, cells were incubated with 0.2ml of 0.2M acetic acid/0.05M NaCl on ice for 5 minutes.

[Dmt¹]The uptake of DALDA into Caco-2 cells was performed by Confocal Laser Scanning Microscopy (CLSM) using a [ Dmt ]¹]Fluorescent analogs of DALDA (Dmt-D-Arg-Phe-dnsDap-NH)₂) To confirm; wherein the dnsDap is beta-dansyl-1-alpha, beta-diaminopropionic acid). The cells were cultured as described above and plated on (35mm) glass-bottom dishes (MatTek corp., Ashland, MA) for two days. The culture medium (medium) was discarded, and the cells were incubated with 1ml of HBSS containing 0.1. mu.M to 1.0. mu.M of the fluorescent peptide analog at 37 ℃For 1 hour. Cells were then washed 3 times with ice-cold HBSS and covered with 200 μ l of PBS, and then microimaging was completed within 10 minutes at room temperature with a Nikon confocal laser scanning microscope with a C-apochromatic 63 x/1.2W correction objective. Excitation by a UV laser was performed at 340nm and the emitted light was measured at 520 nm. For optical tomographic microscopy imaging in the Z-direction, 5-10 frames are cut every 2.0 μm.

CLSM confirmed the use of [ Dmt ] 0.1. mu.M¹，DnsDap⁴]After DALDA incubation at 37 ℃ for 1 hour, fluorescence Dmt-D-arginine-phenylalanine-dnsDap-NH₂Uptake into Caco-2 cells. Uptake of fluorescent peptide was similar at 37 ℃ and 4 ℃. Fluorescence is diffused throughout the cytoplasm, but is completely excluded from the nucleus.

Example 2:[Dmt¹]targeted localization of DALDA to mitochondria

To detect [ Dmt¹]Subcellular distribution of DALDA, preparation of fluorescent analogs [ Dmt¹，AtnDap⁴]DALDA (Dmt-D-arginine-phenylalanine-atnDap-NH₂(ii) a Wherein atn is beta-anthraniloyl-1-alpha, beta-diaminopropionic acid). The analog contains beta-anthranilic acid-1-alpha, beta-diaminopropionic acid instead of a lysine residue at position 4. The cell cultures were as described in example 1 and plated on (35mm) glass-bottom plates (MatTek corp., Ashland, MA) for 2 days. The culture medium was discarded, and 1ml of a medium containing 0.1. mu.M [ Dmt ]¹，AtnDap⁴]The cells were incubated with HBSS from DALDA at 37 ℃ for 15 minutes to 1 hour.

Cells were also incubated with tetramethylrhodamine methyl ester (TMRM, 25nM), a mitochondrial staining dye at 37 ℃ for 15 minutes. Cells were then washed 3 times with ice-cold HBSS and covered with 200 μ l of PBS and microscopic imaging was completed within 10 minutes at room temperature with a Nikon confocal laser scanning microscope with a C-apochromatic 63 x/1.2W correction objective.

For [ Dmt¹，AtnDap⁴]DALDA, excited by UV laser at 350nmThe emitted light was measured at 520 nm. For TMRM, excitation was performed at 536nm and emission was measured at 560 nm.

After incubation at 37 ℃ for at least 15 min, CLSM showed fluorescence [ Dmt¹，AtnDap⁴]DALDA was taken up into Caco-2 cells. Although uptake of the dye was completely excluded from the nucleus, the blue dye showed a striated distribution in the cytoplasm. Mitochondria were labeled red with TMRM. By superimposing [ Dmt¹，AtnDap⁴]Distribution of DALDA and distribution of TMRM to show [ Dmt [ ]¹，AtnDap⁴]Mitochondrial distribution of DALDA.

Example 3:[Dmt¹]uptake of DALDA into mitochondria

To separate mitochondria from mouse liver, mice were sacrificed by decapitation. The liver was removed and quickly placed in a cooled liver homogenate. Mouse livers were cut into small pieces with scissors and then homogenized manually with a glass homogenizer.

The homogenate was centrifuged at 1000 Xg for 10 minutes at 4 ℃. The supernatant was aspirated and transferred to a polycarbonate tube and centrifuged again at 3000 Xg for 10 min at 4 ℃. The supernatant was discarded and the grease on the tube side walls carefully removed.

The pellet was resuspended in liver homogenate and the homogenate repeated twice. The final purified mitochondrial pellet was resuspended in a homogenate. Protein concentration during mitochondrial preparation was measured by the Bradford method.

The mitochondria used in about 1.5mg in 400. mu.l of a buffer solution³H][Dmt¹]DALDA was incubated at 37 ℃ for 5-30 minutes. The mitochondria were then centrifuged off and the amount of radioactivity measured in the mitochondrial fraction and the buffer fraction. Assuming a mitochondrial matrix volume of 0.7. mu.l/mg protein (Lim et al, J.Physiol 545: 961-³H][Dmt¹]The concentration of DALDA in mitochondria was 200 times higher than in buffer. Thus, [ Dmt¹]DALDA was concentrated in the mitochondria.

Based on these data, [ Dmt ] is used¹]DALDA perfused isolated guinea pig hearts, [ Dmt [ Dmt ] ]¹]The concentration of DALDA in mitochondria can be estimated as:

[Dmt¹]the concentration of DALDA in the coronary perfusate was 0.1. mu.M

[Dmt¹]The concentration of DALDA in myocytes was 5. mu.M

[Dmt¹]The concentration of DALDA in mitochondria was 1.0mM

Example 4:isolated mitochondrial Pair [ Dmt¹]Accumulation of DALDA (FIG. 1)

To further prove [ Dmt¹]DALDA is selectively distributed in mitochondria, and we detected [ Dmt¹，AtnDap⁴]DALDA and [ 2 ]³H][Dmt¹]The uptake of DALDA into isolated liver mitochondria in mice. Rapid quenching of fluorescence was observed after mitochondrial addition, indicating that [ Dmt¹，AtnDap⁴]Rapid uptake of DALDA (fig. 1A). Mitochondria are pretreated with an uncoupler FCCP (p- (trifluoromethoxy) -phenylhydrazone carbonyl cyanide) which causes rapid depolarization of mitochondria, [ Dmt¹，AtnDap⁴]Only reduced uptake of DALDA<20 percent. Thus, [ Dmt¹，AtnDap⁴]Uptake of DALDA is not potential dependent.

To confirm that mitochondrial targeting is not an artifact of a fluorophore (artifact), we also examined [ 2 ]³H][Dmt¹]Mitochondrial uptake of DALDA. The isolated mitochondria use³H][Dmt¹]DALDA incubation and detection of radioactivity in mitochondrial pellet and supernatant. From 2 minutes to 8 minutes, the amount of radioactivity in the pellet did not change. The mitochondria treated with FCCP is only reduced in association with the mitochondrial precipitate³H][Dmt¹]Amount of DALDA-20% (fig. 1B).

FCCP Pair [ Dmt¹]The weak effect of DALDA uptake indicates: [ Dmt¹]DALDA may be attached to the mitochondrial membrane or located in the inter-membrane space rather than in the matrix. We subsequently examined the mitochondrial swelling pair [ Dmt ] by using procalcitonin to induce swelling and outer membrane disruption¹，AtnDap⁴]Effect of DALDA accumulation in mitochondria. Unlike TMRM, [ Dmt¹]The uptake of DALDA was only partially reversed by mitochondrial swelling (fig. 1C). Thus, [ Dmt¹]DALDA is attached to the mitochondrial membrane.

Example 5:[Dmt¹]DALDA did not alter mitochondrial respiration or potential (FIG. 1D)

[Dmt¹]Accumulation of DALDA in mitochondria does not alter mitochondrial function. With a [ Dmt ] of 100. mu.M¹]DALDA incubation of isolated mouse liver mitochondria did not alter oxygen consumption during stage 3 and stage 4, or alter respiration rate (stage 3/stage 4) (6.2/6.0). Mitochondrial membrane potential was measured with TMRM (fig. 1D). Addition of mitochondria resulted in a rapid quenching of the TMRM signal, which was rapidly restored by addition of FCCP, indicating mitochondrial depolarization. Adding Ca²⁺(150 μ M) resulted in rapid depolarization with a gradual loss of quench indication of MPT. Addition of [ Dmt ] alone¹]DALDA, even at 200 μ M, does not cause mitochondrial depolarization or MPT.

Example 6:[Dmt¹]DALDA protects mitochondria from Ca²⁺And 3-Nitropropionic acid induced MPT (FIG. 2)

In addition to having no direct effect on mitochondrial membrane potential, [ Dmt¹]DALDA is also able to protect mitochondria from Ca²⁺MPT due to overload. After adding Ca²⁺Before, with [ Dmt ]¹]DALDA (10 μ M) pre-treated isolated mitochondria for 2 min, resulting in only transient depolarization while preventing onset of MPT (fig. 2A). [ Dmt¹]DALDA dose-dependent increase in mitochondrial Pair of accumulated Ca²⁺Tolerance to irritation. FIG. 2B shows [ Dmt [ ]¹]DALDA increases the tolerance of isolated mitochondria prior to MPTAdded Ca of²⁺The amount of (c).

3-Nitropropionic acid (3NP) is an irreversible inhibitor of succinate dehydrogenase in the electron transport chain complex II. Addition of 3NP (1mM) to isolated mitochondria caused a drop in mitochondrial potential and initiated the onset of MPT (fig. 2C). By [ Dmt¹]DALDA pretreatment of mitochondria delayed the onset of MPT induced by 3NP dose-dependently (fig. 3C).

To prove [ Dmt¹]DALDA is able to cross the cell membrane and protect mitochondria from mitochondrial depolarization by 3NP in the absence or presence [ Dmt¹]Caco-2 cells were treated with 3NP (10mM) for 4 hours in the case of DALDA (0.1. mu.M), then incubated with TMRM and detected under LSCM. In the control cells, it can be clearly seen that mitochondria appear as fine streaks throughout the cytoplasm. In cells treated with 3NP, TMRM fluorescence was greatly reduced, indicating generalized depolarization. In contrast, [ Dmt ] is used simultaneously¹]DALDA treatment protects mitochondria from mitochondrial depolarization by 3 NP.

Example 7:[Dmt¹]DALDA protects mitochondria from mitochondrial swelling and cytochrome C release

MPT pore opening leads to mitochondrial swelling. We measured the absorbance at 540nm (A)₅₄₀) Is detected to detect [ Dmt¹]Effect of DALDA on mitochondrial swelling. The mitochondrial suspension was then centrifuged and cytochrome C in the mitochondrial pellet and supernatant was detected using a commercially available ELISA kit. Pretreatment of isolated mitochondria with SS-02 inhibits expression of Ca²⁺Swelling due to overload (fig. 3A) and release of cytochrome C (fig. 3B). Except for inhibiting the formation of Ca²⁺MPP, SS-02 caused by overload can also suppress MPP⁺(1-methyl-4-phenylpyridinium (phenylpyridium) ion) induced mitochondrial swelling, MPP⁺Is an inhibitor of complex I of the mitochondrial electron transport chain (fig. 3C).

Example 8:dextro arginine-Dmt-lysine-phenylalanine-NH₂(D-Arg-Dmt-Lys-Phe-NH₂) (SS-31) is able to protect mitochondria from MPT, mitochondrial swelling and cytochrome C release.

Non-opioid peptide SS-21 similarly protects mitochondria from Ca²⁺Induced MPT (fig. 4A), mitochondrial swelling (fig. 4B) and cytochrome C release (fig. 4C). Methods of investigation were as described above for SS-02. In this example, mitochondrial swelling was detected by monitoring light scattering at 570 nm.

Example 9:[Dmt¹]DALDA (SS-02) and D-arginine-Dmt-lysine-phenylalanine-NH₂(D-Arg-Dmt-Lys-Phe-NH₂) (SS-31) protection of the myocardium from myocardial stunning caused by ischemia-reperfusion.

The guinea pig heart was rapidly isolated, then cannulated in situ into the aorta and perfused retrograde with oxidized Krebs-Henseleit solution (pH7.4) at 34 ℃. The heart was then excised, placed on a modified Langendorff perfusion apparatus, and perfused at constant pressure (40cm water column). Contractile force was measured with a small hook inserted into the apex of the left ventricle and a ribbon firmly attached to the force-displacement transducer (transducer). Coronary flow is detected by timing the collection of the outflow from the pulmonary artery.

Mixing heart buffer solution, [ Dmt¹]DALDA (SS-02) (100nM) or D-arginine-Dmt-lysine-phenylalanine-NH₂(D-Arg-Dmt-Lys-Phe-NH₂) (SS-31) (1nM) was perfused for 30 min, followed by 30 min of systemic ischemia. Reperfusion was performed with the same solution used before ischemia.

Two-way anova showed significant differences in contractility (P <0.001), heart rate (P0.003), and coronary flow velocity (P <0.001) for the three treatment groups. In the buffer group, the contractility during reperfusion was greatly reduced compared to that before ischemia (fig. 5). Hearts treated with SS-02 and SS-31 had much better ischemic tolerance than hearts treated with buffer (FIG. 5). In particular SS-31 is able to completely suppress cardiac arrest. In addition, the coronary flow rate remained well stabilized throughout the reperfusion process without heart rate reduction.

Example 10:[Dmt¹]DALDA (SS-02) improves organ preservation

For heart transplantation, the donor heart is kept in cardioplegic solution during transport. The preservation solution contains high potassium, and can effectively stop heart beat and preserve energy. However, the survival time of ex vivo hearts is still very limited.

We are right to [ Dmt¹]DALDA was tested for prolonged organ survival. In these studies, [ Dmt ] will be¹]DALDA was added to commonly used cardioplegic solution (st. thomas) to detect after prolonged ischemia, [ Dmt¹]Whether DALDA can improve the survival of the heart (a model of organ survival in vitro in vivo).

Isolated guinea pig hearts were perfused retrograde with oxidized Krebs-Henseleit solution at 34 ℃. After 30 minutes of stabilization, the heart was treated with or without 100nM [ Dmt ]¹]DALDA cardioplegic CPS (st. tohomas) perfusion for 3 minutes. Systemic ischemia was then initiated by completely blocking coronary perfusion for 90 minutes. Reperfusion was then performed for 60 minutes with oxidized Krebs-Henseleit solution. Contractility, heart rate and coronary flow rate were continuously monitored throughout the experiment.

Adding Dmt to cardioplegia solution¹]DALDA significantly improved contractile function after prolonged ischemia (fig. 6).

Claims (30)

1. Use of an aromatic-cationic peptide in the manufacture of a medicament for reducing the number of mitochondria undergoing Mitochondrial Permeability Transition (MPT) or preventing mitochondrial permeability transition in a mammal in need thereof, wherein an effective amount of the aromatic-cationic peptide is administered to the mammal, the peptide being represented by any one of the following formulae: d-arginine-2 ', 6' -dimethyltyrosine-lysine-phenylalanine-NH₂2 ', 6' -dimethyltyrosine-D-arginine-phenylalanine-lysine-NH₂(Dmt¹-DALDA)。

2. The use according to claim 1, wherein the peptide is administered orally.

3. The use according to claim 1, wherein the peptide is administered topically.

4. The use of claim 1, wherein the peptide is administered intranasally.

5. The use of claim 1, wherein the peptide is administered systemically.

6. The use according to claim 3, wherein the peptide is administered intravenously.

7. The use of claim 1, wherein the peptide is administered subcutaneously.

8. The use according to claim 1, wherein the peptide is administered intramuscularly.

9. The use according to claim 1, wherein the peptide is administered intracerebroventricularly.

10. The use of claim 1, wherein the peptide is administered intrathecally.

11. The use according to claim 1, wherein the peptide is administered transdermally.

12. The use according to claim 11, wherein the transdermal administration is by iontophoresis.

13. The use of claim 1, wherein the mammal is suffering from ischemia.

14. The use of claim 1, wherein the mammal is undergoing reperfusion.

15. The use of claim 1, wherein the mammal is experiencing hypoxia.

16. The use of claim 13, wherein the ischemia is caused by stroke.

17. Use according to claim 13, wherein the ischemia is intestinal ischemia.

18. The use of claim 13, wherein the ischemia is present in muscle tissue.

19. Use according to claim 18, wherein the muscle tissue is myocardial tissue.

20. The use of claim 18, wherein the muscle tissue is skeletal muscle tissue.

21. Use according to claim 18, wherein the muscle tissue is smooth muscle tissue.

22. The use of claim 1, wherein the mammal is experiencing hypoxia.

23. The use of claim 1, wherein the mammal is suffering from a neurodegenerative disease or disorder.

24. The use of claim 23, wherein the neurodegenerative disease or disorder is parkinson's disease.

25. The use of claim 23, wherein the neurodegenerative disease or disorder is alzheimer's disease.

26. The use of claim 23, wherein the neurodegenerative disease or disorder is huntington's disease.

27. The use of claim 23, wherein the neurodegenerative disease or disorder is Amyotrophic Lateral Sclerosis (ALS).

28. The use of claim 1, wherein the mammal is undergoing drug induced MPT.

29. The use according to claim 1, wherein the mammal is a human.

30. A method of reducing the number of mitochondria undergoing Mitochondrial Permeability Transition (MPT) or preventing mitochondrial permeability transition in an isolated organ of a mammal, the method comprising administering to the isolated organ an effective amount of an aromatic-cationic peptide represented by any one of the following formulae: d-arginine-2 ', 6' -dimethyltyrosine-lysine-phenylalanine-NH₂2 ', 6' -dimethyltyrosine-D-arginine-phenylalanine-lysine-NH₂(Dmt¹-DALDA)。