AU2019257440B2 - Methods for reducing CD36 expression - Google Patents

Abstract

The invention provides a method of reducing CD36 expression in a cell. The method comprises contacting the cell with an effective amount of an aromatic-cationic 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.

Description

METHODS FOR REDUCING CD36 EXPRESSION

This application is a divisional application from Australian application 2018200395. The full disclosure of AU 2018200395 is incorporated herein by reference.

[0001] The application asserts priority to U.S. Provisional Application Serial No. /718,170 filed on September 16, 2005. The specification of U.S. Provisional Application Serial No. 60/718,170 is hereby incorporated by reference.

[0002] The invention described in this application was funded by the National Institute of Drug Abuse, Grant No. PO DA08924, the National Institute of Neurological Diseases and Stroke, Grant No. R21 NS48295, and the National Heart, Lung and Blood Institute, Grant No. RO 1 HL0825 11. The United States Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] CD36 is a transmembrane protein of the class B scavenger receptor family. The protein is widely expressed on numerous cells, such as microvascular endothelium, macrophages, platelets, adipocytes, epithelial cells ( e.g., intestinal epithelial and renal tubular cells, etc.), pancreatic islet cells and cardiac muscle. The receptor may interact with multiple extracellular ligands, such as thrombospondin-1, long-chain fatty acids, and oxidized low-density lipoprotein.

[0004] Abnormal expression of CD36 has been implicated in a wide variety of diseases and conditions. For example, mice lacking CD36 have less atherosclerotic lesions when fed a Western diet compared to wild-type mice. Further, CD36 knock out mice were reported to be protected against acute cerebral ischemia.

[0005] Therefore, methods for reducing expression of CD36 expression are beneficial for treating a disease or condition characterized by abnormal expression of CD36.

[0005a] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

[0005b] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

SUMMARY OF THE INVENTION

[0005c] According to a first aspect, the present invention provides a use of an aromatic-cationic peptide having:

(a) at least one net positive charge;

(b) a minimum of four amino acids;

(c) a maximum of about twenty amino acids;

(d) 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

(e) 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 the manufacture of a medicament for treating one or more complications of diabetes in a mammal in need thereof, wherein the one or more complications of diabetes is associated with CD36 expression.

[0005d] According to a second aspect, the present invention provides a method of treating one or more complications of diabetes in a mammal in need thereof, which comprises administering to a subject an effective amount of an aromatic-cationic peptide having:

(a) at least one net positive charge;

(b) a minimum of four amino acids;

(c) a maximum of about twenty amino acids;

(d) 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

(e) 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,

la wherein the one or more complications of diabetes is associated with CD36 expression.

[0005e] Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

[0006] In one embodiment, the invention provides a method for reducing CD36 expression in a cell. The method comprises contacting the cell with an effective amount of an aromatic-cationic peptide.

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[0007] In another embodiment, the invention provides a method for reducing CD36 expression in a mammal in need thereof. The method comprises administering to the mammal an effective amount of an aromatic-cationic peptide.

[0008] In yet another embodiment, the invention provides a method for treating a disease or condition characterized by increased CD36 expression in a mammal in need thereof. The method comprises administering to the mammal an effective amount of an aromatic-cationic peptide.

[0009] In a further embodiment, the invention provides a method for treating ureteral obstruction in a mammal in need thereof. The method comprises administering to the mammal an effective amount of an aromatic-cationic peptide.

[0010] In yet a further embodiment, the invention provides a method for treating diabetic nephropathy in a mammal in need thereof. The method comprises administering to the mammal an effective amount of an aromatic-cationic peptide.

[001 1] In another embodiment, the invention provides a method for reducing CD36 expression in a removed organ or tissue. The method comprises administering to the mammal an effective amount of an aromatic-cationic peptide.

[0012] The aromatic-cationic peptides useful in the methods of the present invention have 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.

BRIEF DESCRIPTION OF THE FIGURES

[00 13] Figure 1. SS-3 1 reduced oxLDL-induced CD36 mRNA expression, CD36 protein expression, and foam cell formation in mouse peritoneal macrophages.

[0014] Figure 2. SS-3 1 treatment reduced infarct volume and hemispheric swelling in wild-type mice subjected to acute cerebral ischemia.

[0015] Figure 3. SS-3 1 treatment reduced the decrease in reduced glutathione (GSH) in post-ischemic brain in wild-type mice.

[0016] Figure 4. SS-3 1 had no effect in reducing infarct volume or hemispheric swelling in CD36 knock-out mice subjected to acute cerebral ischemia.

[0017] Figure 5. SS-31 did not reduce GSH depletion in post-ischemic brain from CD36 knock-out mice.

[001 8] Figure 6. SS-3 1 reduced CD36 mRNA expression in post-ischemic brain in wild-type mice.

[0019] Figure 7. SS-31 decreases CD36 expression on renal tubular cells after unilateral ureteral obstruction (UUO). Contralateral unobstructed kidney (fig. 7A); obstructed kidney in animals treated with saline (fig. 73B); and obstructed kidneys obtained from rats treated with SS-31 (fig. 7C).

[0020] Figure 8. SS-3 1 reduces lipid peroxidation in kidney after UUO. Tubular cells inthe obstructed kidney (fig. 8B), contralateral unobstructed control (fig. 8A); obstructed kidneys from rats treated with SS-31 (fig. 8C).

[0021] Figure 9. SS-3 1 reduced tubular cell apoptosis in obstructed kidney after UUO. Obstructed kidney from saline-treated animals (fig. 9B); contralateral unobstructed control (fig. 9A); obstructed kidney from SS-31 treated animals (fig. 9C).

[0022] Figure 10. SS-31 reduced macrophage infiltration in obstructed kidney induced by UUO. Obstructed kidney (fig. 10B); contralateral unobstructed control (fig. A); rats treated with SS-31 (fig. 10C).

[0023] Figure 11. SS-3 1 reduced interstitial fibrosis in obstructed kidney after UUO. Obstructed kidney (fig. lIB); contralateral unobstructed control (fig. lIA); rats treated with SS-31 (fig. 1IC).

[0024] Figure 12. Cold storage of isolated hearts with SS-3 1 or SS-20 prevented upregulation of CD36 expression. The "background" control (figs. 12A and 12B) represents two sections from a normal non-ischemic heart that were not treated with the primary anti CD36 antibody. "Normal heart" (figs. 12C and 12D) represents two sections obtained from a non-ischemic heart. The sections from arepresentative heart stored in St. Thomas solution (figs. 12E and 12F) for 18 hours at 4 0 C showed increased CD36 staining compared to "Normal heart." CD36 staining was significantly reduced in hearts stored with either 1 nM SS-31 (figs. 12G and 12H) or 100 nM SS-20 (figs. 121 and 12J) in St. Thomas solution.

[0025] Figure 13. SS-31 and SS-20 reduced lipid peroxidation in isolated guinea pig hearts subjected to warm reperfusion after prolonged cold ischemia. HNE staining in hearts subjected to 18 hours of cold storage in St. Thomas solution (fig. 13B) compared to non-ischemic hearts (fig. 13A). FNE staining was reduced in hearts stored in SS-31 (fig. 9C) or SS-20 (fig. 13D).

[0026] Figure 14. SS-31 and SS-20 abolished endothelial apoptosis in isolated guinea pig hearts subjected to warm reperfusion after prolonged cold ischemia. Hearts subjected to 18 hours of cold storage in St. Thomas solution (figs. 14C and 14D); non, ischemic normal hearts (figs. 14A and 14B). Apoptotic cells were not observed in hearts stored in SS-31 (figs. 14E and 14F) or SS-20 (figs. 14G and 14H).

[0027] Figure 15. SS-31 and SS-20 preserves coronary flow in isolated guinea pig hearts subjected to warm reperfusion after prolonged cold ischemia. Guinea pig hearts perfused with a cardioplegic solution (St. Thomas solution) alone or St. Thomas solution containing either 1 nM SS-3 1 (fig. 15A) or 100 nM SS-20 (fig. 15B) for 3 min. and then subjected to 18 hours of cold ischemia (4 0C).

[0028] Figure 16. SS-31 prevented damage to proximal tubules in diabetic mice. Diabetes was induced by streptozotocin (STZ) injection for 5 d. Kidney sections obtained after 3 weeks showed loss of brush border in STZ-treated animals (fig. 16A, panel B) that was not seen in mice not treated with STZ (panel A). The loss of brush border was not seen in STZ-treated animal that received daily SS-31 (3 mg/kg) (panel C).

[0029] Figure 17. SS-3 1 prevented renal tubular epithelial cell apoptosis in diabetic mice. Diabetes was induced by streptozotocin (STZ) injection for 5d. Kidney sections obtained after 3 weeks showed dramatic increase in apoptotic cells in proximal tubules in STZ-treated animals (fig. 17A, panel b) that was not seen in mice not treated with STZ (fig. 17A, panel a). The STZ-induced apoptosis was not seen in mice that received daily SS-31 (3 mg/kg) (fig. 17A, panel c). The percent of apoptotic cells caused by STZ was significantly reduced by SS-31 treatment (fig. 17B).

DETAILED DESCRIPTION OF THE INVENTION

Peptides

[0030] The invention is directed to the reduction of CD36 expression by certain aromatic-cationic peptides. The aromatic-cationic peptides are water-soluble and highly polar. Despite these properties, the peptides can readily penetrate cell membranes.

[003 1] The aromatic-cationic peptides useful in the present invention include a minimum of three amino acids, and preferably include a minimum of four amino acids, covalently joined by peptide bonds.

[0032] The maximum number of amino acids present in the aromatic-cationic peptides of the present invention is about twenty amino acids covalently joined by peptide bonds. Preferably, the maximum number of amino acids is about twelve, more preferably about nine, and most preferably about six. Optimally, the number of amino acids present in the peptides is four.

[0033] The amino acids of the aromatic-cationic peptides useful in the present invention 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. Preferably, at least one amino group is at the a position relative to a carboxyl group.

[0034] The amino acids may be naturally occurring. Naturally occurring amino acids include, for example, the twenty most common levorotatory (L) amino acids normally found in mammalian proteins, i.e., alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (GIn), glutamic acid (GIu), glycine (Gy), histidine

(His), isoleucine (lieu), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe),

proline (Pro), serine (Ser), threonine (Thr), tryptophan, (Trp), tyrosine (Tyr), and valine

(Val).

[0035] 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 include

hydroxyproline (Hyp).

[0036] The peptides useful in the present invention 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.

[0037] 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 useful in the present

invention preferably are also not recognized by common proteases.

[0038] 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.

[0039] The non-natural amino acids may, for example, comprise alkyl, aryl, or

alkylaryl groups not found in natural amino acids. Some examples of non-natural alkyl

amino acids include a-aminobutyric acid, P-aminobutyric acid, 7-aminobutyric acid, 6

aminovaleric acid, and C-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 7-phenyl- aminobutyric acid.

[0040] Non-naturally occurring amino acids include derivatives of naturally

occurring amino acids. The derivatives ofnaturally occurring amino acids may, for example,

include the addition of one or more chemical groups to the naturally occurring amino acid.

[0041] For example, one or more chemical groups can be added to one or more of the 2', 3', 4', 5', or 6' position of the aromatic ring of a phenylalanine or tyrosine residue, or the 4', 5', 6', or 7' position of the benzo ring of a tryptophan residue. The group can be any chemical group that can be added to an aromatic ring. Some examples of such groups include branched or unbranched C -C 4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C -C 4 alkyloxy (i.e., alkoxy), amino, Ci-C4 alkylamino and Ci-C 4 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 (NIe).

[0042] Another example of a modification of an amino acid in a peptide useful in the methods of the present invention 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.

[0043] 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 ofthe Ci-C 4 alkyl groups mentioned above, such as an acetyl or propionyl group.

[0044] The non-naturally occurring amino acids are preferably resistant, and more preferably insensitive, to common proteases. Examples ofnon-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.

[0045] In order to minimize protease sensitivity, the peptides useful in the methods of the invention 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.

[0046] 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.

[0047] It is important that the aromatic-cationic peptides have a minimum number ofnet positive charges at physiological pH in comparison to the total number of amino acid residues in the peptide. The minimum number ofnet positive charges at physiological pH will be referred to below as (pj. The total number of amino acid residues in the peptide will be referred to below as (r).

[0048] The minimum number ofnet 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.

[0049] "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.

[0050] 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.

[005 1] As an example of calculating net charge, the peptide Tyr-Arg-Phe-Lys Glu-His-Trp-Arg has one negatively charged amino acid (i.e., GIu) 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.

[0052] In one embodiment of the present invention, the aromatic-cationic peptides have a relationship between the minimum number ofnet positive charges at physiological pH

(p) 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:

(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

[0053] In another embodiment, the aromatic-cationic peptides have a relationship between the minimum number of net positive charges (p) 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 ofnet positive charges (pm) and the total number of amino acid residues (r) is as follows:

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

(pm) 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10

[0054] In one embodiment, the minimum number ofnet positive charges (pm) and the total number of amino acid residues (r) are equal. In another embodiment, the peptides have three or four amino acid residues and a minimum of one net positive charge, preferably, a minimum of two net positive charges and more preferably a minimum of three net positive charges.

[0055] It is also important that the aromatic-cationic peptides have a minimum number of aromatic groups in comparison to the total number of net positive charges (pt). The minimum number of aromatic groups will be referred to below as (a).

[0056] Naturally occurring amino acids that have an aromatic group include the amino acids histidine, tryptophan, tyrosine, and phenylalanine. For example, the hexapeptide Lys-Gln-Tyr-Arg-Phe-Trp has a net positive charge of two (contributed by the lysine and arginine residues) and three aromatic groups (contributed by tyrosine, phenylalanine and tryptophan residues).

[0057] In one embodiment of the present invention, the aromatic-cationic peptides useful in the methods of the present invention have a relationship between the minimum number of aromatic groups (a) and the total number of net positive charges at physiological pH (pt) wherein 3a is the largest number that is less than or equal to pt + 1, except that when

Pt is 1, a may also be 1. In this embodiment, the relationship between the minimum number of aromatic groups (a) and the total number ofnet positive charges (Pt) is as follows:

(pt) 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

[0058] In another embodiment, the aromatic-cationic peptides have a relationship between the minimum number of aromatic groups (a) and the total number ofnet positive charges (pt) wherein 2a is the largest number that is less than or equal to pt + 1. In this embodiment, the relationship between the minimum number of aromatic amino acid residues (a) and the total number of net positive charges (pt) is as follows:

(Pt) 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

[0059] In another embodiment, the number of aromatic groups (a) and the total number ofnet positive charges (p) are equal.

[0060] Carboxyl groups, especially the terminal carboxyl group of a C-terminal amino acid, are preferably amidated with, for example, ammonia to form the C-terminal amide. Alternatively, the terminal carboxyl group of the C-terminal amino acid may be amidated with any primary or secondary amine. The primary or secondary amine may, for example, be an alkyl, especially a branched or unbranched C -C 4 alkyl, or an aryl amine. Accordingly, the amino acid at the C-terminus of the peptide may be converted to an amido, N-methylamido, N-ethylamido, N,N-dimethylamido, N,N-diethylamido, N-methyl-N ethylamido, N-phenylamido or N-phenyl-N-ethylamido group.

[0061] The free carboxylate groups of the asparagine, glutamine, aspartic acid, and glutamic acid residues not occurring at the C-terminus of the aromatic-cationic peptides of the present invention may also be amidated wherever they occur within the peptide. The amidation at these internal positions may be with ammonia or any of the primary or secondary amines described above.

[0062] In one embodiment, the aromatic-cationic peptide useful in the methods of the present invention is a tripeptide having two net positive charges and at least one aromatic amino acid. In a particular embodiment, the aromatic-cationic peptide useful in the methods of the present invention is a tripeptide having two net positive charges and two aromatic amino acids.

[0063] Aromatic-cationic peptides useful in the methods of the present invention include, but are not limited to, the following peptide examples:

Lys-D-Arg-Tyr-NH 2 ,

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

Trp-D-Lys-Tyr-Arg-NH 2 ,

Tyr-His-D-Gly-Met, Phe-Arg-D-His-Asp,

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

, Met-Tyr-D-Lys-Phe-Arg, D-His-Glu-Lys-Tyr-D-Phe-Arg, Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH 2

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

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

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

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

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

Phe-Try-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-Thr, Tyr-Asp-D-Lys-Tyr-Phe- D-Lys- D-Arg-Phe-Pro-D-Tyr-His-Lys, Glu-Arg-D-Lys-Tyr- D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met-NH 2 ,

Arg-D-Leu-D-Tyr-Phe-Lys-Glu- D-Lys-Arg-D-Trp-Lys- D-Phe-Tyr-D-Arg-Gly, D-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH 2 ,

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

Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-Asp, and Tlir-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-Lys NH 2

[0064] In one embodiment, the peptides useful in the methods of the present invention have mu-opioid receptor agonist activity (i.e., they activate the mu-opioid receptor). Activation of the mu-opioid receptor typically elicits an analgesic effect.

[0065] In certain instances, an aromatic-cationic peptide having mu-opioid receptor agonist activity is preferred. For example, during short-term treatment, such as in an acute disease or condition, it may be beneficial to use an aromatic-cationic peptide that activates the mu-opioid receptor. Such acute diseases and conditions are often associated with moderate or severe pain, In these instances, the analgesic effect of the aromatic-cationic peptide may be beneficial in the treatment regimen of the human patient or other mammal. An aromatic-cationic peptide which does not activate the mu-opioid receptor, however, may also be used with or without an analgesic, according to clinical requirements.

[0066] Alternatively, in other instances, an aromatic-cationic peptide that does not have mu-opioid receptor agonist activity is preferred. For example, during long-term treatment, such as in a chronic disease state or condition, the use of an aromatic-cationic peptide that activates the mu-opioid receptor may be contraindicated. In these instances the potentially adverse or addictive effects ofthe aromatic-cationic peptide may preclude the use of an aromatic-cationic peptide that activates the mu-opioid receptor in the treatment regimen of a human patient or other mammal. Potential adverse effects may include sedation, constipation and respiratory depression. In such instances an aromatic-cationic peptide that does not activate the mu-opioid receptor may be an appropriate treatment.

[0067] Peptides useful in the methods of the present invention which have mu opioid receptor agonist activity are typically those peptides which have a tyrosine residue or a tyrosine derivative at the N-terminus (i.e., the first amino acid position). Preferred derivatives of tyrosine include 2'-methyltyrosine (Mmt); 2',6'-dimethyltyrosine (2'6'Dmt); 3',5'-dimethyltyrosine (3'5'Dmt); N,2',6'-trimethyltyrosine (Tmt); and 2'-hydroxy-6' methyltryosine (Hmt).

[0068] In a particular preferred embodiment, a peptide that has mu-opioid receptor agonist activity has the formula Tyr-D-Arg-Phe-Lys-NH 2 (for convenience represented by the acronym: DALDA, which is referred to herein as SS-01). DALDA has a net positive charge of three, contributed by the amino acids tyrosine, arginine, and lysine and has two aromatic groups contributed by the amino acids phenylalanine and tyrosine. The tyrosine of DALDA can be a modified derivative of tyrosine such as in 2', 6' dimethyltyrosine to produce the compound having the formula 2',6'-Dmt-D-Arg-Phe-Lys NH 2 (i.e., Dint -DALDA, which is referred to herein as SS-02).

[0069] Peptides that do not have mu-opioid receptor agonist activity generally do not have a tyrosine residue or a derivative of tyrosine at the N-terminus (i.e., amino acid position 1). The amino acid at the N-terminus can be any naturally occurring or non naturally occurring amino acid other than tyrosine.

[0070] In one embodiment, the amino acid at the N-terminus is phenylalanine or its derivative. Preferred derivatives of phenylalanine include 2'-methylphenylalanine (Mmp), 2',6'-dimethylphenylalanine (Dmp), N,2',6'-trimethylphenylalanine (Tmp), and 2'-hydroxy 6'-methylphenylalanine (Hmp).

[0071] Another aromatic-cationic peptide that does not have mu-opioid receptor agonist activity has the formula Phe-D-Arg-Phe-Lys-NH2 (i.e., Phe^DALDA, which is referred to herein as SS-20). Alternatively, the N-terminal phenylalanine can be a derivative of phenylalanine such as 2',6'-dimethylphenylalanine (2'6'Dmp). DALDA containing 2',6' dimethylphenylalanine at amino acid position 1has the formula 2',6'-Dmp-D-Arg-Phe-Lys NH2 (i.e., 2'6'DiV-DALDA).

[0072] In a preferred embodiment, the amino acid sequence of Dmt^DALDA (SS-02) is rearranged such that Dint is not at the N-terminus. An example of such an aromatic-cationic peptide that does not have mu-opioid receptor agonist activity has the formula D-Arg-2'6'Dmt-Lys-Phe-NH2 (referred to in this specification as SS-3 1).

[0073] DALDA, Phe'-DALDA, SS-3 1, and their derivatives can further include functional analogs. A peptide is considered a functional analog of DALDA, Phel-DALDA, or SS-3 1 ifthe analog has the same function as DALDA, Phe^DALDA, or SS-3 1. The analog may, for example, be a substitution variant of DALDA, Phel-DALDA, or SS-3 1, wherein one or more amino acids are substituted by another amino acid.

[0074] Suitable substitution variants of DALDA, Phe^DALDA, or SS-3 1 include conservative amino acid substitutions. Amino acids may be grouped according to their physicochemical characteristics as follows:

(a) Non-polar amino acids: AIa(A) Ser(S) Thr(T) Pro(P) GIy(G); (b) Acidic amino acids: Asn(N) Asp(D) GIu(E) GIn(Q); (c) Basic amino acids: His(H) Arg(R) Lys(K); (d) Hydrophobic amino acids: Met(M) Leu(L) He(I) VaI(V); and (e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His (H).

[0075] Substitutions of an amino acid in a peptide by another amino acid in the same group is referred to as a conservative substitution and may preserve the physicochemical characteristics of the original peptide. In contrast, substitutions of an amino acid in a peptide by another amino acid in a different group is generally more likely to alter the characteristics of the original peptide.

[0076] Examples of analogs useful in the practice of the present invention that activate mu-opioid receptors include, but are not limited, to the aromatic-cationic peptides shown in Table 1.

TABLE 1 Amino Acid C-Terminal Amino Acid Amino Acid Amino Acid Amino Acid Position 5 (if Modification Position 1 Position 2 Position 3 Position 4 present) Tyr D-Arg Phe Lys N H2 Tyr D-Arg Phe Orn NH 2 Tyr D-Arg Phe Dab NH 2 Tyr D-Arg Phe Dap NH 2 2'6'Dmt D-Arg Phe Lys NH 2 2'6'Dmt D-Arg Phe Lys Cys NH 2 2'61 DiTit D-Arg Phe Lys-NH(CH 2)2-NH-dns NH 2 2'6'Dmt D-Arg Phe Lys-NH(CH 2)2-NH-atn NH 2 2'6'Dmt D-Arg Phe dnsLys NH 2 2'6'Dmt D-Cit Phe Lys NH 2 2'6'Dmt D-Cit Phe Ahp NH 2 2'6'Dmt D-Arg Phe Orn NH 2 2'6'Dmt D-Arg Phe Dab NH 2 2'6'Dmt D-Arg Phe Dap NH 2 2'6'Dmt D-Arg Phe Ahp(2-aminoheptanoic acid) NH 2 Bio-2'6'Dmt D-Arg Phe Lys NH 2 3'5'Dmt D-Arg Phe Lys NH 2 3'5'Dmt D-Arg Phe Om NH 2 3'5'Dmt D-Arg Phe Dab NH 2 3'5'Dmt D-Arg Phe Dap NH 2 Tyr D-Arg Tyr Lys NH 2 Tyr D-Arg Tyr Orn NH 2 Tyr D-Arg Tyr Dab NH 2 Tyr D-Arg Tyr Dap NH 2 2'6'Dmt D-Arg Tyr Lys NH 2 2'6'Dmt D-Arg Tyr Om NH 2 2'6'Dmt D-Arg Tyr Dab NH 2 2'6'Dmt D-Arg Tyr Dap NH 2 2'6'Dmt D-Arg 2'6'Dmt Lys NH 2 2'6'Dmt D-Arg 2'6'Dmt Orn NH 2 2'6'Dmt D-Arg 2'6'Dmt Dab NH 2 2'6'Dmt D-Arg 2'6'Dmt Dap NH 2 3'5'Dmt D-Arg 3'5'Dmt Arg NH 2

3'5'Dmt D-Arg 3'5'Dmt Lys NH 2 3'5'Dmt D-Arg 3'5'Dmt Orn NH 2 3'5'Dmt D-Arg 3'5'Dmt Dab NH 2 ryr D-Lys Phe Dap NH 2 ryr D-Lys Phe Arg NH 2 ryr D-Lys Phe Lys NH 2 ryr D-Lys Phe Orn NH 2 2'6'Dmt D-Lys Phe Dab NH 2 ?'6'Dmt D-Lys Phe Dap NH 2 ?'6'Dmt D-Lys Phe Arg NH 2 ?'6'Dmt D-Lys Phe Lys NH 2 3'5'Dmt D-Lys Phe Orn NH2 3'5'Dmt D-Lys Phe Dab NH 2 3'5'Dmt D-Lys Phe Dap NH 2 3'5'Dmt D-Lys Phe Arg NH 2 ryr D-Lys Tyr Lys NH 2 Fyr D-Lys Tyr Om NH 2 Fyr D-Lys Tyr Dab NH 2 Fyr D-Lys Tyr Dap NH 2 ?'6'Dmt D-Lys Tyr Lys NH 2 ?'6'Dmt D-Lys Tyr Orn NH 2 ?'6'Dmt D-Lys Tyr Dab NH 2 ?'6'Dmt D-Lys Tyr Dap NH 2 ?'6'Dmt D-Lys 2'6'Dmt Lys NH 2 ?'6'Dmt D-Lys 2'6'Dmt Om NH 2 ?'6'Dmt D-Lys 2'6'Dmt Dab NH 2 ?'6'Dmt D-Lys 2'6'Dmt Dap NH 2 '6'Dmt D-Arg Phe dnsDap NH 2 ?'6'Dmt D-Arg Phe atnDap NH 2 '5'Dmt D-Lys 3'5'Dmt Lys NH 2 '5'Dmt D-Lys 3'5'Dmt Orn NH 2 '5'Dmt D-Lys 3'5'Dmt Dab NH 2 V'5'Dmt D-Lys 3'5'Dmt Dap NH2 Tyr D-Lys Phe Arg NH2 Tyr D-Orn Phe Arg NH2 Tyr D-Dab Phe Arg NH2 Tyr D-Dap Phe Arg NH2 2'6'Dmt D-Arg Phe Arg NH 2 2'6'Dmt D-Lys Phe Arg NH 2 2'6'Dmt D-Om Phe Arg NH 2 2'6'Dmt D-Dab Phe Arg NH 2 3'5'Dmt D-Dap Phe Arg NH 2 3'5'Dmt D-Arg Phe Arg NH 2 3'5'Dmt D-Lys Phe Arg NH 2 3'5'Dmt D-Orn Phe Arg NH 2 Tyr D-Lys Tyr Arg NH 2 Tyr D-Orn Tyr Arg NH 2 Tyr D-Dab Tyr Arg NH 2 Tyr D-Dap Tyr Arg NH 2 2'6'Dmt D-Arg 2'6'Dmt Arg NH 2 2'6'Dmt D-Lys 2'6'Dmt Arg NH 2 2'6'Dmt D-Orn 2'6'Dmt Arg NH 2

?'6'Dmt D-Dab 2'6'Dmt Arg NH 2 3'5'Dmt D-Dap 3'5'Dmt Arg NH 2 3'5'Dmt D-Arg 3'5'Dmt Arg NH 2 3'5'Dmt D-Lys 3'5'Dmt Arg NH 2 3'5'Dmt D-Orn 3'5'Dmt Arg NH 2 Mint D-Arg Phe Lys NH 2 Mint D-Arg Phe Orn NH 2 Mint D-Arg Phe Dab NH 2 Mint D-Arg Phe Dap NH 2 rmt D-Arg Phe Lys NH 2 rmt D-Arg Phe Om NH 2 rmt D-Arg Phe Dab NH 2 rmt D-Arg Phe Dap NH 2 Hint D-Arg Phe Lys NH 2 Hint D-Arg Phe Orn NH 2 Hint D-Arg Phe Dab NH 2 Hint D-Arg Phe Dap NH 2 Mint D-Lys Phe Lys NH 2 Mint D-Lys Phe Orn NH 2 Mint D-Lys Phe Dab NH 2 Mint D-Lys Phe Dap NH 2 Mint D-Lys Phe Arg NH 2 rmt D-Lys Phe Lys NH 2 Fint D-Lys Phe Orn NH 2 Fint D-Lys Phe Dab NH 2 Fint D-Lys Phe Dap NH 2 Fint D-Lys Phe Arg NH 2 Hint D-Lys Phe Lys NH 2 Hint D-Lys Phe Orn NH 2 Hint D-Lys Phe Dab NH 2 Hint D-Lys Phe Dap NH 2 Hint D-Lys Phe Arg NH 2 Mint D-Lys Phe Arg NH 2 Mint D-Orn Phe Arg NH 2 Mint D-Dab Phe Arg NH 2 Mint D-Dap Phe Arg NH 2 Mint D-Arg Phe Arg NH 2 Tint D-Lys Phe Arg NH 2 Tint D-Orn Phe Arg NH 2 Tint D-Dab Phe Arg NH 2 Tint D-Dap Phe Arg NH 2 Tint D-Arg Phe Arg NH 2 Hint D-Lys Phe Arg NH 2 Hint D-Orn Phe Arg NH 2 Hint D-Dab Phe Arg NH 2 Hint D-Dap Phe Arg NH 2 Hint D-Arg Phe Arg NH 2

Dab = diaminobutyric Dap = diaminopropionic acid

Dmt= dimethyltyrosine Mmt= 2'-methyltyrosine Tmt =N, 2',6'-trimethyltyrosine Hmt =2'-hydroxy,6'-methyltyrosine dnsDap= j-dansyl-L-a, -diaminopropionic acid

atnDap= j-anthraniloyl-L-ca, -diaminopropionic acid Bio = biotin

[0077] Examples of analogs useful in the practice of the present invention that do not activate mu-opioid receptors include, but are not limited to, the aromatic-cationic peptides shown in Table 2.

TABLE 2

Amino Acid Amino Acid Amino Acid Amino Acid C-Terminal Position 1 Position 2 Position 3 Position 4 Modification D-Arg Dmt Lys Phe NH 2 D-Arg Dmt Phe Lys NH 2 D-Arg Phe Lys Dmt NH 2 D-Arg Phe Dmt Lys NH 2 D-Arg Lys Dmt Phe NH 2 D-Arg Lys Phe Dmt NH 2 Phe Lys Dmt D-Arg NH 2 Phe Lys D-Arg Dmt NH 2 Phe D-Arg Dmt Lys NH 2 Phe D-Arg Lys Dmt NH 2 Phe Dmt D-Arg Lys NH 2 Phe Dmt Lys D-Arg NH 2 Lys Phe D-Arg Dmt NH 2 Lys Phe Dmt D-Arg NH 2 Lys Dmt D-Arg Phe NH 2 Lys Dmt Phe D-Arg NH 2 Lys D-Arg Phe Dmt NH 2 Lys D-Arg Dmt Phe NH 2 D-Arg Dmt D-Arg Phe NH 2 D-Arg Dmt D-Arg Dmt NH 2 D-Arg Dmt D-Arg Tyr NH 2 D-Arg Dmt D-Arg Trp NH 2 Trp D-Arg Phe Lys NH 2 Trp D-Arg Tyr Lys NH 2 Trp D-Arg Trp Lys NH 2 Trp D-Arg Dmt Lys NH 2 D-Arg Trp Lys Phe NH 2 D-Arg Trp Phe Lys NH 2

D-Arg Trp Lys Dmt NH 2 D-Arg Trp Dmt Lys NH 2 D-Arg Lys Trp Phe NH 2 D-Arg Lys Trp Dmt NH 2 Cha D-Arg Phe Lys NH 2 Ala D-Arg Phe Lys NH 2

Clm = cyclohexyl

[0078] The amino acids of the peptides shown in table 1 and 2 may be in either the L- or the D- configuration.

Methods

[0079] The aromatic-cationic peptides described above are useful in reducing CD36 expression in a cell. For the purposes of this specification, CD36 expression in a cell is considered to be reduced ifthe expression of CD36 is decreased by about 10%, preferably by about 25%, more preferably by about 50%, even more preferably by about 75%.

Optimally, CD36 is reduced to about normal levels in a cell.

[0080] CD36 is expressed on a wide variety of cells. Examples of such cells include macrophages, platelets, adipocytes, endothelial cells such as microvascular endothelial cells and umbilical vein endothelial cells; epithelial cells such as intestinal epithelial cells, gall bladder epithelial cells, bladder epithelial cells, bronchial epithelial cells and alverolar epithelial cells; renal tubular cells; pancreatic islet cells; hepatocytes; skeletal muscle cells; cardiac muscle cells; neuronal cells; glia cells; pancreas cells; sperm cells; etc.

[008 1] For the purposes of this specification, cells expressing about 10%, typically about 25%, about typically about 50%, and even more typically about 75% more

CD36 than normal cells are considered to express increased levels of CD36.

[0082] In one embodiment, the invention provides a method for reducing CD36 expression in a cell. Any cell that expresses CD36 can be used in the method of the invention, and include those mentioned above. The method for reducing CD36 expression in a cell comprises contacting the cell with an effective amount of an aromatic-cationic peptide described above.

[0083] In another embodiment, the invention provides a method for reducing CD36 expression in a mammal in need thereof. The method for reducing CD36 expression in the mammal comprises administering to the mammal an effective amount of an aromatic cationic peptide described herein.

[0084] Mammals in need of reducing CD36 expression include, for example, mammals that have increased CD36 expression. The increased expression of CD36 is associated with various diseases and conditions. Examples of diseases and conditions characterized by increased CD36 expression include, but are not limited to atherosclerosis, inflammation, abnormal angiogenesis, abnormal lipid metabolism, abnormal removal of apoptotic cells, ischemia such as cerebral ischemia and myocardial ischemia, ischemia reperfusion, ureteral obstruction, stroke, Alzheimer's Disease, diabetes, diabetic nephropathy and obesity. A discussion on the involvement of CD36 in atherosclerosis may be found in "Targeted disruption ofthe class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice". Febbraio M, Podrez EA, Smith JD, Hajjar DP, Hazen SL et al. J Clinical Investigation 105:1049-1056, 2000 and "CD36: a class B scavenger receptor involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism". Febbraio M., Hajjar DP and Silverstein RL. Journal of Clinical Investigation 108:785-791, 2001.

[0085] Mammals in need of reducing CD36 expression also include mammals suffering from complications of diabetes. Some complications of diabetes include, in addition to nephropathy, neuropathy, retinopathy, coronary artery disease, and peripheral vascular disease associated with diabetes.

[0086] In another embodiment, the invention relates to a method for reducing CD36 expression in removed organs and tissues. The method comprises contacting the removed organ or tissue with an effective amount of an aromatic-cationic peptide described above. An organ or tissue may, for example, be removed from a donor for autologous or heterologous transplantation. Some examples of organs and tissues include heart, lungs, pancreas, kidney, liver, skin, etc.

Synthesis of the Peptides

[0087] The peptides useful in the methods of the present invention may be synthesized by any of the methods well known in the art. Suitable methods for chemically synthesizing the protein include, for example those described by Stuart and Young in "Solid Phase Peptide Synthesis," Second Edition, Pierce Chemical Company (1984), and in "Solid Phase Peptide Synthesis," Methods Enzymol. 289, Academic Press, Inc, New York (1997).

Modes of Administration

[0088] Any method known to those in the art for contacting a cell, organ or tissue with a peptide may be employed. Suitable methods include in vitro, e vivo, or in vivo methods.

[0089] In vitro methods typically include cultured samples. For example, a cell can be placed in a reservoir (e.g., tissue culture plate), and incubated with an aromatic cationic peptide under appropriate conditions suitable for reducing CD36 expression. Suitable incubation conditions can be readily determined by those skilled in the art.

[0090] Ex vivo methods typically include cells, organs or tissues removed from a mammal, such as a human. The cells, organs or tissues can, for example, be incubated with the peptide under appropriate conditions. The contacted cells, organs or tissues are normally returned to the donor, placed in a recipient, or stored for future use. Thus, the peptide is generally in a pharmaceutically acceptable carrier.

[0091] In vivo methods are typically limited to the administration of an aromatic cationic peptide, such as those described above, to a mammal, preferably a human. The peptides useful in the methods of the present invention are administered to a mammal in an amount effective in reducing expression of CD36 or treating the mammal. The effective amount is determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians.

[0092] An effective amount of a peptide useful in the methods of the present invention, preferably in a pharmaceutical composition, may be administered to a mammal in need thereof by any of a number of well-known methods for administering pharmaceutical compounds. The peptide may be administered systemically or locally.

[0093] In one embodiment, the peptide is administered intravenously. For example, the aromatic-cationic peptides useful in the methods of the present invention may be administered via rapid intravenous bolus injection. Preferably, however, the peptide is administered as a constant rate intravenous infusion.

[0094] The peptide may also be administered orally, topically, intranasally, intramuscularly, subcutaneously, or transdermally. In a preferred embodiment, transdermal administration of the aromatic-cationic peptides by methods of the present invention is by iontophoresis, in which the charged peptide is delivered across the skin by an electric current.

[0095] Other routes of administration include intracerebroventricularly or intrathecally. Intracerebroventiculatly refers to administration into the ventricular system of the brain. Intrathecally refers to administration into the space under the arachnoid membrane of the spinal cord. Thus intracerebroventricular or intrathecal administration may be preferred for those diseases and conditions which affect the organs or tissues of the central nervous system.

[0096] The peptides useful in the methods ofthe invention may also be administered to mammals by sustained release, as is known in the art. Sustained release administration is a method of drug delivery to achieve a certain level of the drug over a particular period of time. The level typically is measured by serum or plasma concentration.

[0097] A description of methods for delivering a compound by controlled release can be found in international PCT Application No. WO 02/083 106. The PCT application is incorporated herein by reference in its entirety.

[0098] Any formulation known in the art of pharmacy is suitable for administration of the aromatic-cationic peptides useful in the methods of the present invention. For oral administration, liquid or solid formulations may be used. Some examples of formulations include tablets, gelatin capsules, pills, troches, elixirs, suspensions, syrups, wafers, chewing gum and the like. The peptides can be mixed with a suitable pharmaceutical carrier (vehicle) or excipient as understood by practitioners in the art. Examples of carriers and excipients include starch, milk, sugar, certain types of clay, gelatin, lactic acid, stearic acid or salts thereof, including magnesium or calcium stearate, talc, vegetable fats or oils, gums and glycols.

[0099] For systemic, intracerebroventricular, intrathecal, topical, intranasal, subcutaneous, or transdermal administration, formulations of the aromatic-cationic peptides useful in the methods of the present inventions may utilize conventional diluents, carriers, or excipients etc., such as those known in the art to deliver the peptides. For example, the formulations may comprise one or more of the following: a stabilizer, a surfactant, preferably a nonionic surfactant, and optionally a salt and/or a buffering agent. The peptide may be delivered in the form of an aqueous solution, or in alyophilized form.

[00100] The stabilizer may, for example, be an amino acid, such as for instance, glycine; or an oligosaccharide, such as for example, sucrose, tetralose, lactose or a dextran. Alternatively, the stabilizer may be a sugar alcohol, such as for instance, mannitol; or a combination thereof. Preferably the stabilizer or combination of stabilizers constitutes from about 0.1% to about 10% weight for weight of the peptide.

[0100] The surfactant is preferably a nonionic surfactant, such as a polysorbate. Some examples of suitable surfactants include Tween20, Tween80; a polyethylene glycol or a polyoxy ethylene polyoxypropylene glycol, such as Pluronic F-68 at from about 0.001% (w/v) to about 10% (w/v).

[0101] The salt or buffering agent may be any salt or buffering agent, such as for example, sodium chloride, or sodium/potassium phosphate, respectively. Preferably, the buffering agent maintains the pH of the pharmaceutical composition in the range of about 5.5 to about 7.5. The salt and/or buffering agent is also useful to maintain the osmolality at a level suitable for administration to a human or an animal. Preferably the salt or buffering agent is present at a roughly isotonic concentration of about 150mM to about 300mM.

[0102] The formulations of the peptides useful in the methods of the present invention may additionally contain one or more conventional additive. Some examples of such additives include a solubilizer such as, for example, glycerol; an antioxidant such as for example, benzalkonium chloride (a mixture of quaternary ammonium compounds, known as "quats"), benzyl alcohol, chloretone or chlorobutanol; anaesthetic agent such as for example a morphine derivative; or an isotonic agent etc., such as described above. As a further precaution against oxidation or other spoilage, the pharmaceutical compositions may be stored under nitrogen gas in vials sealed with impermeable stoppers.

[0103] The mammal treated in accordance with the invention can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice and rabbits. In a preferred embodiment, the mammal is a human.

EXAMPLES

Example 1: SS-31 reduced oxidized low-density lipoprotein (oxLDL)-induced CD36 expression and foam cell formation in mouse peritoneal macrophages.

[0104] Atherosclerosis is thought to develop as a result of lipid uptake by vascular-wall macrophages leading to the development of foam cells and the elaboration of cytokines and chemokines resulting in smooth muscle-cell proliferation. CD36 is a scavenger receptor that mediates uptake of oxLDL into macrophages and subsequent foam-cell development. CD36 knock out mice showed reduced uptake of oxLDL and reduced atherosclerosis.

[0105] CD36 expression is regulated at the transcriptional level by various cellular stimuli, including glucose and oxLDL. Macrophages were harvested from mice peritoneal cavity and culture overnight in the absence or presence of oxLDL (50 tg/ml) for 48 h. Incubation with oxLDL significantly increased CD36 mRNA (figure IA). Inclusion of SS-31 (10 nM or 1 M) to the culture medium abolished the up-regulation of CD36 (figure IA). SS-31 by itself had no effect on CD36 expression.

[0106] Expression of CD36 protein, as determined by western blot, was also significantly increased after 48 h incubation with 25 tg/ml of oxLDL (oxL) when compared to vehicle control (V) (figure IB). Other controls included CD36 expression from mouse heart (H) and macrophages obtained from CD36 knockout mice (KO). The amount of CD36 protein was normalized to P-actin. Incubation with SS-31 (1 M) (S) significantly reduced CD36 protein expression compared to macrophages exposed to vehicle control (V) (P<0.01,

ANOVA withposthoc Neuman Keuls test). Concurrent incubation with SS-31 (1 M) also significantly inhibited the upregulation of CD36 protein expression in macrophages exposed to25 tg/mloxLDLfor48h(oxL/S)(PO.01, ANOVA withposthoc Neuman Keuls test).

[0107] Incubation of macrophages with oxLDL for 48 h also increased foam cell formation (figure IC). Foam cell is indicated by oil red 0 which stains lipid droplets red. Inclusion of SS-3 1 (1 M) prevented oxLDL-induced foam cell formation (figure 1C).

[0108] Incubation of macrophages with oxLDL increased apoptotic cells from 6.7% to 32.8%. Concurrent treatment with SS-31 (1 nM) significantly reduced the percentage of apoptotic cells induced by oxLDL to 20.8%.

Example 2: SS-3 1Protected Mice from Acute Cerebral Ischemia

[0 109] Cerebral ischemia initiates a cascade of cellular and molecular events that lead to brain damage. One such event is postischemic inflammation. Using a mouse model of cerebral ischemia-reperfusion (20 min. occlusion of the middle cerebral artery), it was found that CD36 was upregulated in microglia and macrophages in the post-ischemic brain, and there was increased reactive oxygen species production. CD36 knock out mice had a profound reduction in reactive oxygen species after ischemia and improved neurological function compared to wild type mice.

[01 10] Cerebral ischemia was induced by occlusion of the right middle cerebral artery for 30 min. Wild-type (WT) mice were given either saline vehicle (Veh) (ip, n=9) or SS-3 1 (2 mg/kg or 5 mg/kg, ip, n=6) at 0, 6, 24 and 48 h after ischemia. Mice were killed 3 days after ischemia. Brains were removed, frozen, and sectioned. Brain sections were stained by the Nissl stain. Infarct volume and hemispheric swelling was determined using an image analyzer. Data were analyzed by one-way ANOVA with posthoc analysis.

[01 11] Treatment of wild type mice with SS-3 1 (2 mg/kg or 5 mg/kg, ip, n=6) at , 6, 24 and 48 hours after 30 min. occlusion of the middle cerebral artery resulted in a significant reduction in infarct volume (figure 2A) and hemispheric swelling (figure 2B) compared to saline controls.(*P<0.05 compared to Veh).

[01 12] Thirty min. cerebral ischemia in WT mice resulted in significant depletion in reduced glutathione (GSH) in the ipsilateral cortex and striatum compared to the contralateral side in vehicle-treated animals (figure 3). The depletion of GSH in the ipsilateral cortex was significantly reduced in mice treated with SS-31 (2 mg/kg ip at 0, 6, 24 and 48 h) (figure 3). The depletion of GSH in the striatum was also reduced by SS-31 treatment but did not reach statistical significance.

Example 3. SS-3 1 mediated protection against acute cerebral ischemia mimics protection observed in CD36 knockout mice.

[01 13] CD36 knockout (CD36 KO) mice were subjected to acute cerebral ischemia as described under Example 2. CD36 KO mice were given either saline vehicle (Veh) (ip, n=5) or SS-31 (2 mg/kg, i.p. n=5) at 0, 6, 24 and 48 h after 30 min ischemia.

Infarct volume (figure 4A) and hemispheric swelling (figure 4B) in CD36 KO mice were similar whether they received saline or SS-3 1.

[01 14] Treatment of CD36 KO mice with SS31 (2 mg/kg, i.p., n=5) also failed to further prevent GSH depletion in the ipsilateral cortex caused by 30 min ischemia (figure 5).

[01 15] These data suggest that the protective action of SS-3 1 against acute cerebral ischemia may be mediated by inhibiting the upregulation of CD36.

Example 4. SS-3 reduced CD36 mRNA expression in post-ischemic brain.

[01 16] Transient occlusion of the middle cerebral artery has been shown to significantly increase the expression of CD36 mRNA in microglia and macrophages in the post-ischemic brain. Wild-type mice were given saline vehicle (Veh, i.p., n=6) or SS-31 (5 mg/kg, i.p., n=6) at 0 and 6h after 30 min ischemia, and CD36 mRNA levels were determined using real time PCR. CD36 expression was upregulated almost 6-fold in the ipsilateral brain compared to the contralateral brain in mice that received saline (figure 6). CD36 mRNA was significantly reduced in the ipsilateral brain in mice that received SS-31 treatment (figure 6).

Example 5: SS-31 Suppressed Upregulation of CD36 in Renal Tubular Cells Following Unilateral Ureteral Obstruction

[01 17] Unilateral ureteral obstruction (UUO) is a common clinical disorder associated with tubular cell apoptosis, macrophage infiltration, and interstitial fibrosis. Interstitial fibrosis leads to a hypoxic environment and contributes to progressive decline in renal function despite surgical correction. CD36 has been shown to be expressed on renal tubular cells.

[0118] CD36 was found to have been upregulated in tubular cells after UUO. UUO was performed in Sprague-Dawley rats. The rats were treated with saline (ip, n=6) or SS-3 1 (1 mg/kg ip, n=6) one day prior to induction of UUO, and once a day for 14 days after UUO. Rats were killed, kidneys removed, embedded in paraffin and sectioned. The slides were treated with the anti-CD36 polyclonal IgG (Santa Cruz #sc-9154; 1:100 with blocking serum) at room temperature for 1.5 hours. The slides were then incubated with the second antibody conjugated with biotin (anti-rabbit IgG-Bl; ABC kit, PK-6101) at room temperature for 30 min. The slides were then treated with avidin, developed with DAB and counterstained with 10% hematoxylin. The contralateral unobstructed kidney served as the control for each animal.

[0119] UUO resulted in tubular dilation and significant increase in expression of CD36 on the tubular cells (fig. 7). Tubular dilation was also observed in rats treated with SS 31, but there was a significant reduction in CD36 expression (fig. 3). CD36 expression (brown stain) is primarily found on tubular cells in the contralateral unobstructed kidney (fig. 7A). CD36 expression was increased in the obstructed kidney in animals treated with saline (fig. 7B), but was much reduced in obstructed kidneys obtained from rats treated with SS-31 (fig. 7C).

[0120] To determine whether SS-31 reduces lipid peroxidation in kidney after UUO, rats were treated with either saline (n=6) or SS-3 1 (1 mg/kg ip, n=6) one day prior to induction of UUO, and once a day for 14 days UUO. Rats were then killed, kidneys removed, embedded in paraffin and sectioned. Slides were incubated with anti-HNE rabbit IgG and a biotin -linked anti-rabbit IgG was used as secondary antibody. The slides were developed with DAB. Lipid peroxidation, which was increased by UUO, was reduced by SS 31treatment (fig. 8). HNE stain (brown) was significantly increased in tubular cells in the obstructed kidney (fig. 8B) compared to the contralateral control (fig. 8A). Obstructed kidneys from rats treated with SS-31 showed significantly less HNE stain (Fig. 8C) compared to saline-treated rats (fig. 8B).

[0121] To determine whether SS-31 reduced tubular cell apoptosis in obstructed kidney after UUO, rats were treated with either saline (n=6) or SS-3 1 (1 mg/kg ip, n=6) one day prior to induction of UUO, and once a day for 14 days after UUO. Rats were then killed, kidneys removed, embedded in paraffin and sectioned. To quantitate nuclei with fragmented DNA, the TUNEL assay were performed with in situ TUNEL kit (Intergen, Purchase, NY). Slides were developed with DAB and counterstained with 10% hematoxylin. The upregulation of CD36 in saline-treated controls associated with tubular cell apoptosis was significantly inhibited by SS-31 treatment (fig. 9). Compared to the contralateral unobstructed control (fig. 9A), a significant increase in apoptotic cells was observed in the obstructed kidney from saline-treated animals (fig. 9B). The number of apoptotic cells was significantly reduced in obstructed kidney from SS-31 treated animals (fig. 9C) (PO.001; n=6).

[0122] Macrophage infiltration (fig. 10) and interstitial fibrosis (fig. 11) were also prevented by SS-31 treatment. Rats were treated with either saline (n=6) or SS-31 (1 mg/kg ip, n=6) one day prior to induction of UUO, and once a day for 14 days after UUO. Rats were then killed, kidneys removed, embedded in paraffin and sectioned. Slides were treated with monoclonal antibody for EDl macrophage (1:75; Serotec). Horseradish-peroxidase linked rabbit anti-mouse secondary antibody (Dako) was used for macrophage detection. Sections were then counterstained with 10% hematoxylin. The number of macrophages in the obstructed kidney in saline-treated rats (fig. 10B) was significantly increased compared to the contralateral unobstructed control (fig. 10A). Macrophage infiltration was significantly reduced in rats treated with SS-3 1 (fig. 10C) (PO.05; t-test).

[0123] Rats were treated with either saline (n=6) or SS-3 1 (1 mg/kg ip, n=6) one day prior to induction of UUO, and once a day for 14 days after UUO. Rats were then killed, kidneys removed, embedded in paraffin and sectioned. Slides were stained with hematoxylin and eosin and Masson's trichome for interstitial fibrosis (blue stain). Obstructed kidneys from saline-treated rats showed increase fibrosis (fig. 1IB) compared to the contralateral unobstructed control (fig. IJA); while obstructed kidneys from SS-3 treated rats showed significantly less fibrosis (P<0.05; t-test).

[0124] These results show that SS-31 suppresses the upregulation of CD36 on renal tubular cells induced by UUO.

Example 6: SS-31 and SS-20 Reduced CD36 Expression in Isolated Hearts Upon Reperfusion After Prolonged Cold Ischemic Storage

[0125] Organ transplantation requires hypothermic storage of the isolated organ for transport to the recipient. Currently, cardiac transplantation is limited by the short time of cold ischemic storage that can be tolerated before coronary blood flow is severely compromised (<4 hours). The expression of CD36 in coronary endothelium and cardiac muscles is up-regulated in isolated hearts subjected to prolonged cold ischemic storage and warm reperfusion.

[0126] Isolated guinea pig hearts were perfused with St. Thomas solution alone, or St. Thomas solution containing 1 nM SS-31, or 100 iiM SS-20, for 3 min. and then stored in the same solution at 4 0 C for 18 hours. After ischemic storage, hearts were reperfused with 34 0C Kreb-Henseleit solution for 90 min. Hearts freshly isolated from guinea pigs were used as controls.

[0127] The hearts were fixed in paraffin and sliced for immunostaining with an anti-CD36 rabbit polyclonal antibody. The results are shown in fig. 12. Two sections are shown for each treatment group. Antibody staining showed that CD36 is expressed in endothelium and cardiac muscles in normal hearts. The "background" control (figs. 12A and 12B) represents two sections from a normal non-ischemic heart that was not treated with the primary antibody. "Normal heart" (figs. 12C and 12D) represents two sections obtained from a non-ischemic heart. The sections from a representative heart stored in St. Thomas solution (figs. 12E and 12F) for 18 hours at 4 0C showed increased CD36 staining compared to "Normal heart." CD36 staining was significantly reduced in hearts stored with either 1 nM SS-31 (figs. 12G and 12H) or 100 nM SS-20 (figs. 121 and 12J) in St. Thomas solution for 18 h.

[0128] CD36 staining is increased in hearts that have undergone 18 hours of cold ischemic storage and warm reperfusion. However, hearts that were stored with either 1 nM SS-31 or 100 nM SS-20 did not show the upregulation of CD36 expression.

[0129] Lipid peroxidation in the hearts was also decreased by the aromatic cationic peptides. Guinea pig hearts were perfused with a cardioplegic solution (St. Thomas solution) alone or St. Thomas solution containing either 1 nM SS-3 1 or 100 nM SS-20 for 3 min. and then subjected to 18 hours of cold ischemia (4 0C). The hearts were then reperfused with Krebs Henseleit buffer at 34 0 C for 90 min. Immunohistochemical analysis of 4 hydroxynonenol (HNE)- modified proteins in paraffin sections from tissue slices were performed by incubation with an anti-HNE antibody (Santa Cruz) and a fluorescent secondary antibody. HNE staining was significantly increased in hearts subjected to 18 hours of cold storage in St. Thomas solution (fig. 13B) compared to non-ischemic hearts (fig. 13A). HNE staining was reduced in hearts stored in SS-31 (fig. 13C) or SS-20 (fig. 13D).

[0130] Further, the peptides dramatically reduced endothelial apoptosis (fig. 14). Guinea pig hearts were perfused with a cardioplegic solution (St. Thomas solution) alone or St. Thomas solution containing either 1 nM SS-31 or 100 nM SS-20 for 3 min. and then subjected to 18 hours of cold ischemia (4 0C). The hearts were then reperfused with Krebs Henseleit buffer at 34 0 C for 90 min. After deparaffinization, sections were incubated with deoxynucleotidyl transferase (Tdt) with digoxigenin-dNTP for 1 hour. The reaction was stopped with terminating buffer. A fluorescent anti-digoxigenin antibody was then applied. Hearts subjected to 18 hours of cold storage in St. Thomas solution (figs. 14C and 14D) showed prominent endothelial apoptosis whereas no endothelial apoptosis was observed in non-ischemic normal hearts (figs. 14A and 140B). Apoptotic cells were not observed in hearts stored in SS-31 (figs. 14E and 14F) or SS-20 (figs. 14G and 14H).

[0131] A significant improvement of coronary blood flow after prolonged cold ischemic storage and warm reperfusion occurred (fig. 15). Guinea pig hearts were perfused with a cardioplegic solution (St. Thomas solution) alone or St. Thomas solution containing either 1 nM SS-31 (fig. 15A) or 100 nM SS-20 (fig. 15B) for 3 min. and then subjected to 18 hours of cold ischemia (4 0 C). The hearts were then reperfused with Krebs Henseleit buffer at 34 0 C for 90 min. Coronary flow was significantly reduced after prolonged ischemia compared to pre-ischemic control (expressed as 100%). Preservation in either SS-3 1 or SS significantly restored coronary flow to approximately 80% of pre-ischemic flow.

Example 7: SS-31 prevented renal damage in diabetic mice

[0132] CD36 expression is upregulated in a variety of tissues of diabetic patients, including monocytes, heart, kidneys, and plasma. High glucose is known to upregulate the expression of CD36 by improving the translational efficiency of CD36 mRNA. Diabetic nephropathy is a common complication of type 1 and type 2 diabetes, and is associated with tubular epithelial degeneration and interstitial fibrosis. CD36 has been identified as a mediator of tubular epithelial apoptosis in diabetic nephropathy. High glucose stimulates CD36 expression and apoptosis in proximal tubular epithelial cells.

[0133] Streptozotocin (STZ) was used to induce diabetes in mice. Three groups of CD-I mice were studied: Group I - no STZ treatment; Group II - STZ (50 mg/kg, ip) was given once a day for 5d; Group III - STZ (50 mg/kg, ip) was given once a day for 5 d, and

SS-3 1 (3 mg/kg, ip) was given once a day for 16 d. STZ treatment resulted in progressive increase in blood glucose. By week 3, blood glucose values were: Group I (10.6 ±0.27 mmol/L); Group 11 (24.5± 1.15 mmol/L); Group III (21.3± 1.48 mmol/L). Animals were sacrificed after 3 weeks and kidney tissues preserved for histopathology. Kidney sections were examined by Periodic Schiff (PAS) staining for renal tubular brush border.

[0134] STZ treatment caused dramatic loss of brush border in proximal tubules in the renal cortex (figure 16). In mice not treated with STZ, the renal brush border in the cortex was stained red with PAS (figure 16A, see white arrows). Inmice treated with STZ, the brush border was obliterated, and the tubular epithelial cells showed small condensed nuclei (figure 16B). Daily treatment with SS-31 (3 mg/kg, ip) prevented the loss of brush border in the STZ-treated mice (figure 16C), and the nuclei appeared normal (figure 16C, top and bottom panels). In general, the architecture of the proximal renal tubules was preserved in diabetic mice treated with SS-31.

[0135] STZ treatment induced significant apoptosis in tubular epithelial cells (figure 17). Kidney sections were examined for apoptosis using the TUNEL assay. After deparaffinization, sections were incubated with deoxynucleotidyl transferase (Tdt) with digoxigenin-dNTP for 1 hour. The reaction was stopped with terminating buffer. A fluorescent anti-digoxigenin antibody was then applied. Kidney sections from mice treated with STZ showed large number of apoptotic nuclei in the proximal tubules (PT)(figure 17A, panel b), compared to no apoptotic cells in mice not treated with STZ (figure 17A, panel a). Treatment with daily SS-31 dramatically reduced apoptotic cells in the proximal tubule (figure 17A, panel c). Figure 17B shows the significant decrease in tubular cell apoptosis provided by SS-31.

[0136] CD36 expression in proximal tubular epithelial cells is known to be increased by high glucose and is upregulated in diabetic models. SS-31. by reducing CD36 expression, was able to inhibit tubular cell apoptosis and loss of brush border in mice treated with STZ without affecting blood glucose.

Claims (13)

What is claimed is:

1. Use of an aromatic-cationic peptide having:

(a) at least one net positive charge;

(b) a minimum of four amino acids;

(c) a maximum of about twenty amino acids;

(d) 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

(e) 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 the manufacture of a medicament for treating one or more complications of diabetes in

a mammal in need thereof, wherein the one or more complications of diabetes is

associated with CD36 expression.

2. The use of Claim 1, wherein the aromatic-cationic peptide comprises Phe-D-Arg-Phe

Lys-NH 2 (SS-20) and/or D-Arg-2'6'Dmt-Lys-Phe-NH 2 (SS-31).

3. The use of Claim 1 or Claim 2, wherein the one or more complications of diabetes are

selected from the group consisting of diabetic nephropathy, diabetic neuropathy,

increased body weight in the mammal compared to a mammal not suffering from

diabetes, increased blood glucose in the mammal compared to a mammal not suffering

from diabetes, diabetic apoptosis, diabetic retinopathy and any combination thereof.

4. The use of Claim 3, wherein the diabetic apoptosis is associated with diabetic

nephropathy.

5. The use of Claim 3 or Claim 4, wherein the diabetic apoptosis comprises tubular

epithelial cell apoptosis.

6. The use of any one of Claims 3 to 5, wherein the diabetic apoptosis comprises the loss of

proximal tubule brush border.

7. The use of any one of Claims 3 to 6, wherein the diabetic apoptosis is associated with

interstitial fibrosis.

8. The use of any one of Claims 3 to 7, wherein the diabetic apoptosis is associated with

elevated CD36 expression.

9. The use of any one of the preceding claims, wherein the mammal is a human.

10. The use of any one of the preceding claims, wherein the mammal is suffering from type I

diabetes.

11. The use of any one of the preceding claims, wherein the mammal is suffering from type II

diabetes.

12. The use of any one of the preceding claims, wherein the peptide is to be administered

orally, topically, systemically, intravenously, subcutaneously, or intramuscularly.

13. A method of treating one or more complications of diabetes in a mammal in need thereof,

which comprises administering to a subject an effective amount of an aromatic-cationic

peptide having:

(a) at least one net positive charge;

(b) a minimum of four amino acids;

(c) a maximum of about twenty amino acids;

(d) 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

(e) 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,

wherein the one or more complications of diabetes is associated with CD36 expression.