U.S. patent application number 14/363035 was filed with the patent office on 2014-10-02 for aromatic-cationic peptides and uses of same.
The applicant listed for this patent is Stealth Peptides International, Inc., D. Travis WILSON. Invention is credited to Marc W. Andersen, Elizabeth Mead, D. Travis Wilson.
Application Number | 20140294796 14/363035 |
Document ID | / |
Family ID | 48574838 |
Filed Date | 2014-10-02 |
United States Patent
Application |
20140294796 |
Kind Code |
A1 |
Wilson; D. Travis ; et
al. |
October 2, 2014 |
AROMATIC-CATIONIC PEPTIDES AND USES OF SAME
Abstract
The disclosure provides compositions and methods relating to
aromatic-cationic peptides. The methods comprise administering to
the subject an effective amount of an aromatic-cationic peptide to
subjects in need thereof. For example, the peptides may be
administered to subjects in need of a mitochondrial-targeted
antioxidant.
Inventors: |
Wilson; D. Travis; (Newton,
MA) ; Andersen; Marc W.; (Raleigh, NC) ; Mead;
Elizabeth; (Hertfordshire, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
WILSON; D. Travis
Stealth Peptides International, Inc. |
Monaco |
|
US
MC |
|
|
Family ID: |
48574838 |
Appl. No.: |
14/363035 |
Filed: |
December 5, 2012 |
PCT Filed: |
December 5, 2012 |
PCT NO: |
PCT/US12/67984 |
371 Date: |
June 5, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61569120 |
Dec 9, 2011 |
|
|
|
Current U.S.
Class: |
424/94.1 ;
435/29; 514/21.9; 514/21.91; 514/415; 514/564; 530/330; 530/331;
548/504; 562/443 |
Current CPC
Class: |
C07K 5/06078 20130101;
G01N 33/68 20130101; C07K 5/0815 20130101; C07C 229/26 20130101;
C07K 5/10 20130101; C07K 5/06095 20130101; C07K 5/08 20130101; A61K
38/07 20130101; C07K 5/06 20130101; A61P 43/00 20180101; C07D
209/16 20130101; C07K 5/1019 20130101; A61P 39/06 20180101; C07K
5/1016 20130101; C07K 5/06086 20130101; A61K 38/06 20130101; C07K
5/0817 20130101; A61P 3/00 20180101; C07K 5/0812 20130101; A61P
25/28 20180101 |
Class at
Publication: |
424/94.1 ;
530/330; 514/21.9; 514/21.91; 530/331; 548/504; 514/415; 562/443;
514/564; 435/29 |
International
Class: |
C07K 5/10 20060101
C07K005/10; C07C 229/26 20060101 C07C229/26; C07D 209/16 20060101
C07D209/16; C07K 5/06 20060101 C07K005/06; C07K 5/08 20060101
C07K005/08 |
Claims
1. An aromatic-cationic peptide selected from the group consisting
of: TABLE-US-00010 6-Butyric acid CoQ0-Phe-D-Arg-Phe-Lys-NH.sub.2
6-Decanoic acid CoQ0-Phe-D-Arg-Phe-Lys-NH.sub.2 Arg-Arg-Dmt-Phe
Arg-Cha-Lys Arg-Dmt Arg-Dmt-Arg Arg-Dmt-Lys Arg-Dmt-Lys-Phe
Arg-Dmt-Lys-Phe-Cys Arg-Dmt-Phe Arg-Dmt-Phe-Lys Arg-Lys-Dmt-Phe
Arg-Lys-Phe-Dmt Arg-Phe-Dmt-Lys Arg-Phe-Lys Arg-Trp-Lys Arg-Tyr-Lys
Arg-Tyr-Lys-Phe D-Arg-D-Dmt-D-Lys-D-Phe-NH.sub.2
D-Arg-D-Dmt-D-Lys-L-Phe-NH.sub.2 D-Arg-D-Dmt-L-Lys-D-Phe-NH.sub.2
D-Arg-D-Dmt-L-Lys-L-Phe-NH.sub.2 D-Arg-Dmt-D-Lys-NH.sub.2
D-Arg-Dmt-D-Lys-Phe-NH.sub.2 D-Arg-Dmt-Lys-D-Phe-NH.sub.2
D-Arg-Dmt-Lys-NH.sub.2 D-Arg-Dmt-Lys-Phe-Cys D-Arg-Dmt-NH.sub.2
D-Arg-L-Dmt-D-Lys-D-Phe-NH.sub.2 D-Arg-L-Dmt-D-Lys-L-Phe-NH.sub.2
D-Arg-L-Dmt-L-Lys-D-Phe-NH.sub.2 D-Arg-Phe-Lys-NH.sub.2
D-Arg-Trp-Lys-NH.sub.2 D-Arg-Tyr-Lys-NH.sub.2 Dmt-Arg Dmt-Lys
Dmt-Lys-D-Phe-NH.sub.2 Dmt-Lys-NH.sub.2 Dmt-Lys-Phe Dmt-Lys-Phe
Dmt-Lys-Phe-NH.sub.2 Dmt-Phe-Arg-Lys H-Arg-D-Dmt-Arg-NH.sub.2
H-Arg-D-Dmt-Lys-NH.sub.2 H-Arg-D-Dmt-Lys-Phe-NH.sub.2
H-Arg-D-Dmt-NH.sub.2 H-Arg-Dmt-Lys-Phe-NH.sub.2
H-D-Arg-2,6-dichloro-L-tyrosine-L-Lys-L-Phe-NH.sub.2
H-D-Arg-2,6-dichlorotyrosine-Lys-Phe-NH.sub.2
H-D-Arg-2,6-difluoro-L-tyrosine-L-Lys-L-Phe-NH.sub.2
H-D-Arg-2,6-difluorotyrosine-Lys-Phe-NH.sub.2
H-D-Arg-2,6-dimethyl-L-phenylalanine-L-Lys-L-Phe-NH.sub.2
H-D-Arg-2,6-dimethylphenylalanine-Lys-Phe-NH.sub.2
H-D-Arg-4-methoxy-2,6-dimethyl-L-tyrosine-L-Lys-L-Phe-NH.sub.2
H-D-Arg-4-methoxy-2,6-dimethyltyrosine-Lys-Phe-NH.sub.2
H-D-Arg-Arg-Dmt-Phe-NH.sub.2 H-D-Arg-Cha-Lys-NH.sub.2
H-D-Arg-D-Dmt-D-Lys-D-Phe-NH.sub.2 H-D-Arg-D-Dmt-Lys-Phe-NH.sub.2
H-D-Arg-D-Dmt-NH.sub.2 H-D-Arg-Dmt-D-Lys-D-Phe-NH.sub.2
H-D-Arg-Dmt-Lys-2,6-dimethylphenylalanine-NH.sub.2
H-D-Arg-Dmt-Lys-3-hydroxyphenylalanine-NH.sub.2
H-D-Arg-Dmt-Lys-NH.sub.2 H-D-Arg-Dmt-Lys-OH H-D-Arg-Dmt-Lys-Phe-OH
H-D-Arg-Dmt-N6-acetyllysine-Phe-NH.sub.2 H-D-Arg-Dmt-OH
H-D-Arg-Dmt-Phe-Lys-NH.sub.2 H-D-Arg-Dmt-Phe-NH.sub.2
H-D-Arg-D-Phe-L-Lys-L-Phe-NH.sub.2
H-D-Arg-D-Trp-L-Lys-L-Phe-NH.sub.2
H-D-Arg-D-Tyr-L-Lys-L-Phe-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-2,6-dimethyl-L-phenylalanine-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-3-hydroxy-L-phenylalanine-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-D-Dmt-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-D-Trp-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-D-Tyr-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-L-Trp-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-L-Tyr-NH.sub.2
H-D-Arg-L-Dmt-L-Phe-L-Lys-NH.sub.2
H-D-Arg-L-Dmt-N6-acetyl-L-lysine-L-Phe-NH.sub.2
H-D-Arg-L-Lys-L-Dmt-L-Phe-NH.sub.2
H-D-Arg-L-Lys-L-Phe-L-Dmt-NH.sub.2
H-D-Arg-L-Phe-L-Dmt-L-Lys-NH.sub.2
H-D-Arg-L-Phe-L-Lys-L-Dmt-NH.sub.2
H-D-Arg-L-Phe-L-Lys-L-Phe-NH.sub.2
H-D-Arg-L-Trp-L-Lys-L-Phe-NH.sub.2
H-D-Arg-L-Tyr-L-Lys-L-Phe-NH.sub.2 H-D-Arg-Lys-Dmt-Phe-NH.sub.2
H-D-Arg-Lys-Phe-Dmt-NH.sub.2 H-D-Arg-Phe-Dmt-Lys-NH.sub.2
H-D-Arg-Phe-Lys-Dmt-NH.sub.2 H-D-Arg-Tyr-Lys-Phe-NH.sub.2
H-D-Dmt-Arg-NH.sub.2 H-D-His-L-Dmt-L-Lys-L-Phe-NH.sub.2
H-D-Lys-L-Dmt-L-Lys-L-Phe-NH.sub.2 H-Dmt-D-Arg-Lys-Phe-NH.sub.2
H-Dmt-D-Arg-NH.sub.2 H-Dmt-D-Arg-Phe-Lys-NH.sub.2
H-Dmt-D-Phe-Arg-Lys-NH.sub.2 H-Dmt-Lys-D-Arg-Phe-NH.sub.2
H-Dmt-Lys-Phe-D-Arg-NH.sub.2 H-Dmt-Phe-D-Arg-Lys-NH.sub.2
H-Dmt-Phe-Lys-D-Arg-NH.sub.2
H-D-N2-acetylarginine-Dmt-Lys-Phe-NH.sub.2
H-D-N8-acetylarginine-Dmt-Lys-Phe-NH.sub.2
H-D-Phe-D-Arg-D-Phe-D-Lys-NH.sub.2
H-L-Dmt-D-Arg-L-Lys-L-Phe-NH.sub.2
H-L-Dmt-D-Arg-L-Phe-L-Lys-NH.sub.2
H-L-Dmt-L-Lys-D-Arg-L-Phe-NH.sub.2
H-L-Dmt-L-Lys-L-Phe-D-Arg-NH.sub.2
H-L-Dmt-L-Phe-D-Arg-L-Lys-NH.sub.2
H-L-Dmt-L-Phe-L-Lys-D-Arg-NH.sub.2
H-L-His-L-Dmt-L-Lys-L-Phe-NH.sub.2
H-L-Lys-D-Arg-L-Dmt-L-Phe-NH.sub.2
H-L-Lys-D-Arg-L-Phe-L-Dmt-NH.sub.2
H-L-Lys-L-Dmt-D-Arg-L-Phe-NH.sub.2
H-L-Lys-L-Dmt-L-Lys-L-Phe-NH.sub.2
H-L-Lys-L-Dmt-L-Phe-D-Arg-NH.sub.2
H-L-Lys-L-Phe-D-Arg-L-Dmt-NH.sub.2
H-L-Lys-L-Phe-L-Dmt-D-Arg-NH.sub.2
H-L-Phe-D-Arg-L-Dmt-L-Lys-NH.sub.2
H-L-Phe-D-Arg-L-Lys-L-Dmt-NH.sub.2
H-L-Phe-L-Dmt-D-Arg-L-Lys-NH.sub.2
H-L-Phe-L-Dmt-L-Lys-D-Arg-NH.sub.2
H-L-Phe-L-Lys-D-Arg-L-Dmt-NH.sub.2
H-L-Phe-L-Lys-L-Dmt-D-Arg-NH.sub.2 H-Lys-D-Arg-Dmt-Phe-NH.sub.2
H-Lys-D-Arg-Phe-Dmt-NH.sub.2 H-Lys-Dmt-D-Arg-Phe-NH.sub.2
H-Lys-Dmt-Phe-D-Arg-NH.sub.2 H-Lys-D-Phe-Arg-Dmt-NH.sub.2
H-Lys-Phe-D-Arg-Dmt-NH.sub.2 H-Lys-Phe-Dmt-D-Arg-NH.sub.2
H-N2-acetyl-D-arginine-L-Dmt-L-Lys-L-Phe-NH.sub.2
H-N7-acetyl-D-arginine-Dmt-Lys-Phe-NH.sub.2
H-Phe(d5)-D-Arg-Phe(d5)-Lys-NH.sub.2 H-Phe-Arg-Phe-Lys-NH.sub.2
H-Phe-D-Arg-Dmt-Lys-NH.sub.2 H-Phe-D-Arg-Dmt-Lys-NH.sub.2
H-Phe-D-Arg-D-Phe-Lys-NH.sub.2 H-Phe-D-Arg-Lys-Dmt-NH.sub.2
H-Phe-D-Arg-Phe-D-Lys-NH.sub.2
H-Phe-D-Arg-Phe-Lys-Glu-Cys-Gly-NH.sub.2
H-Phe-D-Dmt-Arg-Lys-NH.sub.2 H-Phe-Dmt-D-Arg-Lys-NH.sub.2
H-Phe-Dmt-Lys-D-Arg-NH.sub.2 H-Phe-Lys-D-Arg-Dmt-NH.sub.2
H-Phe-Lys-Dmt-D-Arg-NH.sub.2 L-Arg-D-Dmt-D-Lys-D-Phe-NH.sub.2
L-Arg-D-Dmt-D-Lys-D-Phe-NH.sub.2 L-Arg-D-Dmt-D-Lys-L-Phe-NH.sub.2
L-Arg-D-Dmt-D-Lys-L-Phe-NH.sub.2 L-Arg-D-Dmt-L-Lys-D-Phe-NH.sub.2
L-Arg-D-Dmt-L-Lys-D-Phe-NH.sub.2 L-Arg-D-Dmt-L-Lys-L-Phe-NH.sub.2
L-Arg-D-Dmt-L-Lys-L-Phe-NH.sub.2 L-Arg-L-Dmt-D-Lys-D-Phe-NH.sub.2
L-Arg-L-Dmt-D-Lys-D-Phe-NH.sub.2 L-Arg-L-Dmt-D-Lys-L-Phe-NH.sub.2
L-Arg-L-Dmt-D-Lys-L-Phe-NH.sub.2 L-Arg-L-Dmt-L-Lys-D-Phe-NH.sub.2
L-Arg-L-Dmt-L-Lys-D-Phe-NH.sub.2 L-Arg-L-Dmt-L-Lys-L-Phe-NH.sub.2
L-Arg-L-Dmt-L-Lys-L-Phe-NH.sub.2 Lys-Dmt-Arf Lys-Dmt-D-Arg-NH.sub.2
Lys-Phe Lys-Phe-Arg-Dmt Lys-Phe-NH.sub.2 Lys-Trp-Arg
Lys-Trp-D-Arg-NH.sub.2 Phe-Arg-Dmt-Lys Phe-Arg-Phe-Lys
Phe-Arg-Phe-Lys Phe-Arg-Phe-Lys Phe-Arg-Phe-Lys Phe-Arg-Phe-Lys
Phe-Arg-Phe-Lys-Glu-Cys-Gly Phe-Dmt-Arg-Lys Phe-Lys-Dmt
Phe-Lys-Dmt-NH.sub.2 Succinic monoester
CoQ0-Phe-D-Arg-Phe-Lys-NH.sub.2
2. A pharmaceutical composition comprising one or more
aromatic-cationic peptides of claim 1 and pharmaceutically
acceptable salts thereof.
3. The pharmaceutical composition of claim 2 further comprising a
pharmaceutically acceptable carrier.
4. A method of reducing the number of mitochondria undergoing
mitochondrial permeability transition (MPT), or preventing
mitochondrial permeability transitioning in a mammal in need
thereof, the method comprising administering to the mammal an
effective amount of one or more aromatic-cationic peptides of claim
1.
5. A method for reducing oxidative damage in a mammal in need
thereof, the method comprising administering to the mammal an
effective amount of one or more aromatic-cationic peptides of claim
1.
6. A method for increasing the ATP synthesis rate in a mammal in
need thereof, the method comprising administering to the mammal an
effective amount of one or more aromatic-cationic peptides of claim
1.
7. A method for determining the presence or amount of an
administered aromatic-cationic peptide in a subject, the method
comprising: detecting the administered aromatic-cationic peptide in
a biological sample from the subject, wherein the aromatic-cationic
peptide is selected from the group consisting of: TABLE-US-00011
6-Butyric acid CoQ0-Phe-D-Arg-Phe-Lys-NH.sub.2 6-Decanoic acid
CoQ0-Phe-D-Arg-Phe-Lys-NH.sub.2 Arg-Arg-Dmt-Phe Arg-Cha-Lys Arg-Dmt
Arg-Dmt-Arg Arg-Dmt-Lys Arg-Dmt-Lys-Phe Arg-Dmt-Lys-Phe-Cys
Arg-Dmt-Phe Arg-Dmt-Phe-Lys Arg-Lys-Dmt-Phe Arg-Lys-Phe-Dmt
Arg-Phe-Dmt-Lys Arg-Phe-Lys Arg-Trp-Lys Arg-Tyr-Lys Arg-Tyr-Lys-Phe
D-Arg-D-Dmt-D-Lys-D-Phe-NH.sub.2 D-Arg-D-Dmt-D-Lys-L-Phe-NH.sub.2
D-Arg-D-Dmt-L-Lys-D-Phe-NH.sub.2 D-Arg-D-Dmt-L-Lys-L-Phe-NH.sub.2
D-Arg-Dmt-D-Lys-NH.sub.2 D-Arg-Dmt-D-Lys-Phe-NH.sub.2
D-Arg-Dmt-Lys-D-Phe-NH.sub.2 D-Arg-Dmt-Lys-NH.sub.2
D-Arg-Dmt-Lys-Phe-Cys D-Arg-Dmt-NH.sub.2
D-Arg-L-Dmt-D-Lys-D-Phe-NH.sub.2 D-Arg-L-Dmt-D-Lys-L-Phe-NH.sub.2
D-Arg-L-Dmt-L-Lys-D-Phe-NH.sub.2 D-Arg-Phe-Lys-NH.sub.2
D-Arg-Trp-Lys-NH.sub.2 D-Arg-Tyr-Lys-NH.sub.2 Dmt-Arg Dmt-Lys
Dmt-Lys-D-Phe-NH.sub.2 Dmt-Lys-NH.sub.2 Dmt-Lys-Phe Dmt-Lys-Phe
Dmt-Lys-Phe-NH.sub.2 Dmt-Phe-Arg-Lys H-Arg-D-Dmt-Arg-NH.sub.2
H-Arg-D-Dmt-Lys-NH.sub.2 H-Arg-D-Dmt-Lys-Phe-NH.sub.2
H-Arg-D-Dmt-NH.sub.2 H-Arg-Dmt-Lys-Phe-NH.sub.2
H-D-Arg-2,6-dichloro-L-tyrosine-L-Lys-L-Phe-NH.sub.2
H-D-Arg-2,6-dichlorotyrosine-Lys-Phe-NH.sub.2
H-D-Arg-2,6-difluoro-L-tyrosine-L-Lys-L-Phe-NH.sub.2
H-D-Arg-2,6-difluorotyrosine-Lys-Phe-NH.sub.2
H-D-Arg-2,6-dimethyl-L-phenylalanine-L-Lys-L-Phe-NH.sub.2
H-D-Arg-2,6-dimethylphenylalanine-Lys-Phe-NH.sub.2
H-D-Arg-4-methoxy-2,6-dimethyl-L-tyrosine-L-Lys-L-Phe-NH.sub.2
H-D-Arg-4-methoxy-2,6-dimethyltyrosine-Lys-Phe-NH.sub.2
H-D-Arg-Arg-Dmt-Phe-NH.sub.2 H-D-Arg-Cha-Lys-NH.sub.2
H-D-Arg-D-Dmt-D-Lys-D-Phe-NH.sub.2 H-D-Arg-D-Dmt-Lys-Phe-NH.sub.2
H-D-Arg-D-Dmt-NH.sub.2 H-D-Arg-Dmt-D-Lys-D-Phe-NH.sub.2
H-D-Arg-Dmt-Lys-2,6-dimethylphenylalanine-NH.sub.2
H-D-Arg-Dmt-Lys-3-hydroxyphenylalanine-NH.sub.2
H-D-Arg-Dmt-Lys-NH.sub.2 H-D-Arg-Dmt-Lys-OH H-D-Arg-Dmt-Lys-Phe-OH
H-D-Arg-Dmt-N6-acetyllysine-Phe-NH.sub.2 H-D-Arg-Dmt-OH
H-D-Arg-Dmt-Phe-Lys-NH.sub.2 H-D-Arg-Dmt-Phe-NH.sub.2
H-D-Arg-D-Phe-L-Lys-L-Phe-NH.sub.2
H-D-Arg-D-Trp-L-Lys-L-Phe-NH.sub.2
H-D-Arg-D-Tyr-L-Lys-L-Phe-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-2,6-dimethyl-L-phenylalanine-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-3-hydroxy-L-phenylalanine-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-D-Dmt-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-D-Trp-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-D-Tyr-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-L-Trp-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-L-Tyr-NH.sub.2
H-D-Arg-L-Dmt-L-Phe-L-Lys-NH.sub.2
H-D-Arg-L-Dmt-N6-acetyl-L-lysine-L-Phe-NH.sub.2
H-D-Arg-L-Lys-L-Dmt-L-Phe-NH.sub.2
H-D-Arg-L-Lys-L-Phe-L-Dmt-NH.sub.2
H-D-Arg-L-Phe-L-Dmt-L-Lys-NH.sub.2
H-D-Arg-L-Phe-L-Lys-L-Dmt-NH.sub.2
H-D-Arg-L-Phe-L-Lys-L-Phe-NH.sub.2
H-D-Arg-L-Trp-L-Lys-L-Phe-NH.sub.2
H-D-Arg-L-Tyr-L-Lys-L-Phe-NH.sub.2 H-D-Arg-Lys-Dmt-Phe-NH.sub.2
H-D-Arg-Lys-Phe-Dmt-NH.sub.2 H-D-Arg-Phe-Dmt-Lys-NH.sub.2
H-D-Arg-Phe-Lys-Dmt-NH.sub.2 H-D-Arg-Tyr-Lys-Phe-NH.sub.2
H-D-Dmt-Arg-NH.sub.2 H-D-His-L-Dmt-L-Lys-L-Phe-NH.sub.2
H-D-Lys-L-Dmt-L-Lys-L-Phe-NH.sub.2 H-Dmt-D-Arg-Lys-Phe-NH.sub.2
H-Dmt-D-Arg-NH.sub.2 H-Dmt-D-Arg-Phe-Lys-NH.sub.2
H-Dmt-D-Phe-Arg-Lys-NH.sub.2 H-Dmt-Lys-D-Arg-Phe-NH.sub.2
H-Dmt-Lys-Phe-D-Arg-NH.sub.2 H-Dmt-Phe-D-Arg-Lys-NH.sub.2
H-Dmt-Phe-Lys-D-Arg-NH.sub.2
H-D-N2-acetylarginine-Dmt-Lys-Phe-NH.sub.2
H-D-N8-acetylarginine-Dmt-Lys-Phe-NH.sub.2
H-D-Phe-D-Arg-D-Phe-D-Lys-NH.sub.2
H-L-Dmt-D-Arg-L-Lys-L-Phe-NH.sub.2
H-L-Dmt-D-Arg-L-Phe-L-Lys-NH.sub.2
H-L-Dmt-L-Lys-D-Arg-L-Phe-NH.sub.2
H-L-Dmt-L-Lys-L-Phe-D-Arg-NH.sub.2
H-L-Dmt-L-Phe-D-Arg-L-Lys-NH.sub.2
H-L-Dmt-L-Phe-L-Lys-D-Arg-NH.sub.2
H-L-His-L-Dmt-L-Lys-L-Phe-NH.sub.2
H-L-Lys-D-Arg-L-Dmt-L-Phe-NH.sub.2
H-L-Lys-D-Arg-L-Phe-L-Dmt-NH.sub.2
H-L-Lys-L-Dmt-D-Arg-L-Phe-NH.sub.2
H-L-Lys-L-Dmt-L-Lys-L-Phe-NH.sub.2
H-L-Lys-L-Dmt-L-Phe-D-Arg-NH.sub.2
H-L-Lys-L-Phe-D-Arg-L-Dmt-NH.sub.2
H-L-Lys-L-Phe-L-Dmt-D-Arg-NH.sub.2
H-L-Phe-D-Arg-L-Dmt-L-Lys-NH.sub.2
H-L-Phe-D-Arg-L-Lys-L-Dmt-NH.sub.2
H-L-Phe-L-Dmt-D-Arg-L-Lys-NH.sub.2
H-L-Phe-L-Dmt-L-Lys-D-Arg-NH.sub.2
H-L-Phe-L-Lys-D-Arg-L-Dmt-NH.sub.2
H-L-Phe-L-Lys-L-Dmt-D-Arg-NH.sub.2 H-Lys-D-Arg-Dmt-Phe-NH.sub.2
H-Lys-D-Arg-Phe-Dmt-NH.sub.2 H-Lys-Dmt-D-Arg-Phe-NH.sub.2
H-Lys-Dmt-Phe-D-Arg-NH.sub.2 H-Lys-D-Phe-Arg-Dmt-NH.sub.2
H-Lys-Phe-D-Arg-Dmt-NH.sub.2 H-Lys-Phe-Dmt-D-Arg-NH.sub.2
H-N2-acetyl-D-arginine-L-Dmt-L-Lys-L-Phe-NH.sub.2
H-N7-acetyl-D-arginine-Dmt-Lys-Phe-NH.sub.2
H-Phe(d5)-D-Arg-Phe(d5)-Lys-NH.sub.2 H-Phe-Arg-Phe-Lys-NH.sub.2
H-Phe-D-Arg-Dmt-Lys-NH.sub.2 H-Phe-D-Arg-Dmt-Lys-NH.sub.2
H-Phe-D-Arg-D-Phe-Lys-NH.sub.2 H-Phe-D-Arg-Lys-Dmt-NH.sub.2
H-Phe-D-Arg-Phe-D-Lys-NH.sub.2
H-Phe-D-Arg-Phe-Lys-Glu-Cys-Gly-NH.sub.2
H-Phe-D-Dmt-Arg-Lys-NH.sub.2 H-Phe-Dmt-D-Arg-Lys-NH.sub.2
H-Phe-Dmt-Lys-D-Arg-NH.sub.2 H-Phe-Lys-D-Arg-Dmt-NH.sub.2
H-Phe-Lys-Dmt-D-Arg-NH.sub.2 L-Arg-D-Dmt-D-Lys-D-Phe-NH.sub.2
L-Arg-D-Dmt-D-Lys-D-Phe-NH.sub.2 L-Arg-D-Dmt-D-Lys-L-Phe-NH.sub.2
L-Arg-D-Dmt-D-Lys-L-Phe-NH.sub.2 L-Arg-D-Dmt-L-Lys-D-Phe-NH.sub.2
L-Arg-D-Dmt-L-Lys-D-Phe-NH.sub.2 L-Arg-D-Dmt-L-Lys-L-Phe-NH.sub.2
L-Arg-D-Dmt-L-Lys-L-Phe-NH.sub.2 L-Arg-L-Dmt-D-Lys-D-Phe-NH.sub.2
L-Arg-L-Dmt-D-Lys-D-Phe-NH.sub.2 L-Arg-L-Dmt-D-Lys-L-Phe-NH.sub.2
L-Arg-L-Dmt-D-Lys-L-Phe-NH.sub.2 L-Arg-L-Dmt-L-Lys-D-Phe-NH.sub.2
L-Arg-L-Dmt-L-Lys-D-Phe-NH.sub.2 L-Arg-L-Dmt-L-Lys-L-Phe-NH.sub.2
L-Arg-L-Dmt-L-Lys-L-Phe-NH.sub.2 Lys-Dmt-Arf Lys-Dmt-D-Arg-NH.sub.2
Lys-Phe Lys-Phe-Arg-Dmt Lys-Phe-NH.sub.2 Lys-Trp-Arg
Lys-Trp-D-Arg-NH.sub.2 Phe-Arg-Dmt-Lys Phe-Arg-Phe-Lys
Phe-Arg-Phe-Lys Phe-Arg-Phe-Lys Phe-Arg-Phe-Lys Phe-Arg-Phe-Lys
Phe-Arg-Phe-Lys-Glu-Cys-Gly Phe-Dmt-Arg-Lys Phe-Lys-Dmt
Phe-Lys-Dmt-NH.sub.2 Succinic monoester
CoQ0-Phe-D-Arg-Phe-Lys-NH.sub.2
8. The method of claim 7, wherein detecting is performed during
administration of the peptide.
9. The method of claim 7, wherein detecting is performed after
administration of the peptide.
10. The method of any one of claim 7, wherein detecting comprises
HPLC.
11. The method of claim 10, wherein the HPLC comprises reverse
phase HPLC.
12. The method of claim 10, wherein the HPLC comprises ion exchange
HPLC.
13. The method of claim 7, wherein detecting comprises mass
spectrometry.
14. The method of claim 7, wherein the biological sample comprises
a fluid.
15. The method of claim 7, wherein the biological sample comprises
a cell.
16. The method of claim 7, wherein the biological sample comprises
a tissue.
17. The method of any one of claim 7, wherein the biological sample
comprises a biopsy.
18. An aromatic-cationic peptide comprising formula VII or a
stereoisomer thereof ##STR00022## wherein the chiral centers of
formula III are defined as
H--(R)-Arg-(S)-DMT-(S)-Lys-(S)-Phe-NH.sub.2, and wherein
stereoisomers are described by the formulas R--S--S--S, S--R--R--R,
S--S--S--S, R--R--R--R, R--R--S--S, S--S--R--R, S--R--S--S,
R--S--R--R, R--S--R--S, S--R--S--R, R--R--S--R, S--S--R--S,
R--R--R--S, S--S--S--R, R--S--S--R, and S--R--R--S.
19. An aromatic-cationic peptide comprising formula VII or a
constitutional thereof ##STR00023## selected from the group
consisting of Arg-Dmt-Lys-Phe-NH.sub.2, Phe-Dmt-Arg-Lys-NH.sub.2,
Phe-Lys-Dmt-Arg-NH.sub.2, Dmt-Arg-Lys-Phe-NH.sub.2,
Lys-Dmt-Arg-Phe-NH.sub.2, Phe-Dmt-Lys-Arg-NH.sub.2,
Arg-Lys-Dmt-Phe-NH.sub.2, or Arg-Dmt-Phe-Lys-NH.sub.2.
20. An aromatic-cationic peptide comprising formula VIII
##STR00024## wherein R is selected from (i) OMe, and (ii) H.
21. An aromatic-cationic peptide comprising formula IX ##STR00025##
wherein R is selected from (i) F, (ii) Cl, and (iii) H.
22. An aromatic-cationic peptide comprising formula X ##STR00026##
wherein R1-R4 are selected from (i) Ac, (ii) H, (iii) H, (iv) H,
(i) H, (ii) Ac, (iii) H, (iv) H, (i) H, (ii) H, (iii) Ac, (iv) H,
and (i) H, (ii) H, (iii) H, (iv) OH.
23. An aromatic-cationic peptide comprising formula XI ##STR00027##
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to U.S.
Provisional Application No. 61/569,120 filed Dec. 9, 2011, which is
incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The present technology relates generally to compositions and
methods of preventing or treating disease. In particular, the
methods relate to the administration of aromatic-cationic peptides
to a subject in need thereof.
BACKGROUND
[0003] The aromatic-cationic peptides disclosed herein are useful
in therapeutic applications relating to oxidative damage and cell
death. When administered to a mammal in need thereof, the peptides
localize to the mitochondria and improve the integrity and function
of the organelle. Administration of the peptides to a subject in
need thereof reduces the number of mitochondria undergoing
mitochondrial permeability transition, reduces the level of
oxidative damage to cells and tissues, and increases the rate of
mitochondrial ATP synthesis.
SUMMARY
[0004] In one aspect, the present invention provides an
aromatic-cationic peptide or a pharmaceutically acceptable salt
thereof. In some embodiments, the salt comprises trifluoroacetate
salt or acetate salt. In some embodiments, the peptide is selected
from the group consisting of:
TABLE-US-00001 6-Butyric acid CoQ0-Phe-D-Arg-Phe-Lys-NH.sub.2
6-Decanoic acid CoQ0-Phe-D-Arg-Phe-Lys-NH.sub.2 Arg-Arg-Dmt-Phe
Arg-Cha-Lys Arg-Dmt Arg-Dmt-Arg Arg-Dmt-Lys Arg-Dmt-Lys-Phe
Arg-Dmt-Lys-Phe-Cys Arg-Dmt-Phe Arg-Dmt-Phe-Lys Arg-Lys-Dmt-Phe
Arg-Lys-Phe-Dmt Arg-Phe-Dmt-Lys Arg-Phe-Lys Arg-Trp-Lys Arg-Tyr-Lys
Arg-Tyr-Lys-Phe D-Arg-D-Dmt-D-Lys-D-Phe-NH.sub.2
D-Arg-D-Dmt-D-Lys-L-Phe-NH.sub.2 D-Arg-D-Dmt-L-Lys-D-Phe-NH.sub.2
D-Arg-D-Dmt-L-Lys-L-Phe-NH.sub.2 D-Arg-Dmt-D-Lys- NH.sub.2
D-Arg-Dmt-D-Lys-Phe-NH.sub.2 D-Arg-Dmt-Lys-D-Phe-NH.sub.2
D-Arg-Dmt-Lys-NH.sub.2 D-Arg-Dmt-Lys-Phe-Cys D-Arg-Dmt-NH.sub.2
D-Arg-L-Dmt-D-Lys-D-Phe-NH.sub.2 D-Arg-L-Dmt-D-Lys-L-Phe-NH.sub.2
D-Arg-L-Dmt-L-Lys-D-Phe-NH.sub.2 D-Arg-Phe-Lys-NH.sub.2
D-Arg-Trp-Lys-NH.sub.2 D-Arg-Tyr-Lys-NH.sub.2 Dmt-Arg Dmt-Lys
Dmt-Lys-D-Phe-NH.sub.2 Dmt-Lys-NH.sub.2 Dmt-Lys-Phe Dmt-Lys-Phe
Dmt-Lys-Phe-NH.sub.2 Dmt-Phe-Arg-Lys H-Arg-D-Dmt-Arg-NH.sub.2
H-Arg-D-Dmt-Lys-NH.sub.2 H-Arg-D-Dmt-Lys-Phe-NH.sub.2
H-Arg-D-Dmt-NH.sub.2 H-Arg-Dmt-Lys-Phe-NH.sub.2
H-D-Arg-2,6-dichloro-L-tyrosine-L-Lys-L-Phe-NH.sub.2
H-D-Arg-2,6-dichlorotyrosine-Lys-Phe-NH.sub.2
H-D-Arg-2,6-difluoro-L-tyrosine-L-Lys-L-Phe-NH.sub.2
H-D-Arg-2,6-difluorotyrosine-Lys-Phe-NH.sub.2
H-D-Arg-2,6-dimethyl-L-phenylalanine-L-Lys-L-Phe-NH.sub.2
H-D-Arg-2,6-dimethylphenylalanine-Lys-Phe-NH.sub.2
H-D-Arg-4-methoxy-2,6-dimethyl-L-tyrosine-L-Lys-L-Phe-NH.sub.2
H-D-Arg-4-methoxy-2,6-dimethyltyrosine-Lys-Phe-NH.sub.2
H-D-Arg-Arg-Dmt-Phe-NH.sub.2 H-D-Arg-Cha-Lys-NH.sub.2
H-D-Arg-D-Dmt-D-Lys-D-Phe-NH.sub.2 H-D-Arg-D-Dmt-Lys-Phe-NH.sub.2
H-D-Arg-D-Dmt-NH.sub.2 H-D-Arg-Dmt-D-Lys-D-Phe-NH.sub.2
H-D-Arg-Dmt-Lys-2,6-dimethylphenylalanine-NH.sub.2
H-D-Arg-Dmt-Lys-3-hydroxyphenylalanine-NH.sub.2
H-D-Arg-Dmt-Lys-NH.sub.2 H-D-Arg-Dmt-Lys-OH H-D-Arg-Dmt-Lys-Phe-OH
H-D-Arg-Dmt-N6-acetyllysine-Phe-NH.sub.2 H-D-Arg-Dmt-OH
H-D-Arg-Dmt-Phe-Lys-NH.sub.2 H-D-Arg-Dmt-Phe-NH.sub.2
H-D-Arg-D-Phe-L-Lys-L-Phe-NH.sub.2
H-D-Arg-D-Trp-L-Lys-L-Phe-NH.sub.2
H-D-Arg-D-Tyr-L-Lys-L-Phe-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-2,6-dimethyl-L-phenylalanine-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-3-hydroxy-L-phenylalanine-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-D-Dmt-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-D-Trp-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-D-Tyr-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-L-Trp-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-L-Tyr-NH.sub.2
H-D-Arg-L-Dmt-L-Phe-L-Lys-NH.sub.2
H-D-Arg-L-Dmt-N6-acetyl-L-lysine-L-Phe-NH.sub.2
H-D-Arg-L-Lys-L-Dmt-L-Phe-NH.sub.2
H-D-Arg-L-Lys-L-Phe-L-Dmt-NH.sub.2
H-D-Arg-L-Phe-L-Dmt-L-Lys-NH.sub.2
H-D-Arg-L-Phe-L-Lys-L-Dmt-NH.sub.2
H-D-Arg-L-Phe-L-Lys-L-Phe-NH.sub.2
H-D-Arg-L-Trp-L-Lys-L-Phe-NH.sub.2
H-D-Arg-L-Tyr-L-Lys-L-Phe-NH.sub.2 H-D-Arg-Lys-Dmt-Phe-NH.sub.2
H-D-Arg-Lys-Phe-Dmt-NH.sub.2 H-D-Arg-Phe-Dmt-Lys-NH.sub.2
H-D-Arg-Phe-Lys-Dmt-NH.sub.2 H-D-Arg-Tyr-Lys-Phe-NH.sub.2
H-D-Dmt-Arg-NH.sub.2 H-D-His-L-Dmt-L-Lys-L-Phe-NH.sub.2
H-D-Lys-L-Dmt-L-Lys-L-Phe-NH.sub.2 H-Dmt-D-Arg-Lys-Phe-NH.sub.2
H-Dmt-D-Arg-NH.sub.2 H-Dmt-D-Arg-Phe-Lys-NH.sub.2
H-Dmt-D-Phe-Arg-Lys-NH.sub.2 H-Dmt-Lys-D-Arg-Phe-NH.sub.2
H-Dmt-Lys-Phe-D-Arg-NH.sub.2 H-Dmt-Phe-D-Arg-Lys-NH.sub.2
H-Dmt-Phe-Lys-D-Arg-NH.sub.2
H-D-N2-acetylarginine-Dmt-Lys-Phe-NH.sub.2
H-D-N8-acetylarginine-Dmt-Lys-Phe-NH.sub.2
H-D-Phe-D-Arg-D-Phe-D-Lys-NH.sub.2
H-L-Dmt-D-Arg-L-Lys-L-Phe-NH.sub.2
H-L-Dmt-D-Arg-L-Phe-L-Lys-NH.sub.2
H-L-Dmt-L-Lys-D-Arg-L-Phe-NH.sub.2
H-L-Dmt-L-Lys-L-Phe-D-Arg-NH.sub.2
H-L-Dmt-L-Phe-D-Arg-L-Lys-NH.sub.2
H-L-Dmt-L-Phe-L-Lys-D-Arg-NH.sub.2
H-L-His-L-Dmt-L-Lys-L-Phe-NH.sub.2
H-L-Lys-D-Arg-L-Dmt-L-Phe-NH.sub.2
H-L-Lys-D-Arg-L-Phe-L-Dmt-NH.sub.2
H-L-Lys-L-Dmt-D-Arg-L-Phe-NH.sub.2
H-L-Lys-L-Dmt-L-Lys-L-Phe-NH.sub.2
H-L-Lys-L-Dmt-L-Phe-D-Arg-NH.sub.2
H-L-Lys-L-Phe-D-Arg-L-Dmt-NH.sub.2
H-L-Lys-L-Phe-L-Dmt-D-Arg-NH.sub.2
H-L-Phe-D-Arg-L-Dmt-L-Lys-NH.sub.2
H-L-Phe-D-Arg-L-Lys-L-Dmt-NH.sub.2
H-L-Phe-L-Dmt-D-Arg-L-Lys-NH.sub.2
H-L-Phe-L-Dmt-L-Lys-D-Arg-NH.sub.2
H-L-Phe-L-Lys-D-Arg-L-Dmt-NH.sub.2
H-L-Phe-L-Lys-L-Dmt-D-Arg-NH.sub.2 H-Lys-D-Arg-Dmt-Phe-NH.sub.2
H-Lys-D-Arg-Phe-Dmt-NH.sub.2 H-Lys-Dmt-D-Arg-Phe-NH.sub.2
H-Lys-Dmt-Phe-D-Arg-NH.sub.2 H-Lys-D-Phe-Arg-Dmt-NH.sub.2
H-Lys-Phe-D-Arg-Dmt-NH.sub.2 H-Lys-Phe-Dmt-D-Arg-NH.sub.2
H-N2-acetyl-D-arginine-L-Dmt-L-Lys-L-Phe-NH.sub.2
H-N7-acetyl-D-arginine-Dmt-Lys-Phe-NH.sub.2
H-Phe(d5)-D-Arg-Phe(d5)-Lys-NH.sub.2 H-Phe-Arg-Phe-Lys-NH.sub.2
H-Phe-D-Arg-Dmt-Lys-NH.sub.2 H-Phe-D-Arg-Dmt-Lys-NH.sub.2
H-Phe-D-Arg-D-Phe-Lys-NH.sub.2 H-Phe-D-Arg-Lys-Dmt-NH.sub.2
H-Phe-D-Arg-Phe-D-Lys-NH.sub.2
H-Phe-D-Arg-Phe-Lys-Glu-Cys-Gly-NH.sub.2
H-Phe-D-Dmt-Arg-Lys-NH.sub.2 H-Phe-Dmt-D-Arg-Lys-NH.sub.2
H-Phe-Dmt-Lys-D-Arg-NH.sub.2 H-Phe-Lys-D-Arg-Dmt-NH.sub.2
H-Phe-Lys-Dmt-D-Arg-NH.sub.2 L-Arg-D-Dmt-D-Lys-D-Phe-NH.sub.2
L-Arg-D-Dmt-D-Lys-D-Phe-NH.sub.2 L-Arg-D-Dmt-D-Lys-L-Phe-NH.sub.2
L-Arg-D-Dmt-D-Lys-L-Phe-NH.sub.2 L-Arg-D-Dmt-L-Lys-D-Phe-NH.sub.2
L-Arg-D-Dmt-L-Lys-D-Phe-NH.sub.2 L-Arg-D-Dmt-L-Lys-L-Phe-NH.sub.2
L-Arg-D-Dmt-L-Lys-L-Phe-NH.sub.2 L-Arg-L-Dmt-D-Lys-D-Phe-NH.sub.2
L-Arg-L-Dmt-D-Lys-D-Phe-NH.sub.2 L-Arg-L-Dmt-D-Lys-L-Phe-NH.sub.2
L-Arg-L-Dmt-D-Lys-L-Phe-NH.sub.2 L-Arg-L-Dmt-L-Lys-D-Phe-NH.sub.2
L-Arg-L-Dmt-L-Lys-D-Phe-NH.sub.2 L-Arg-L-Dmt-L-Lys-L-Phe-NH.sub.2
L-Arg-L-Dmt-L-Lys-L-Phe-NH.sub.2 Lys-Dmt-Arf Lys-Dmt-D-Arg-NH.sub.2
Lys-Phe Lys-Phe-Arg-Dmt Lys-Phe-NH.sub.2 Lys-Trp-Arg
Lys-Trp-D-Arg-NH.sub.2 Phe-Arg-Dmt-Lys Phe-Arg-Phe-Lys
Phe-Arg-Phe-Lys Phe-Arg-Phe-Lys Phe-Arg-Phe-Lys Phe-Arg-Phe-Lys
Phe-Arg-Phe-Lys-Glu-Cys-Gly Phe-Dmt-Arg-Lys Phe-Lys-Dmt
Phe-Lys-Dmt-NH.sub.2 Succinic monoester
CoQ0-Phe-D-Arg-Phe-Lys-NH.sub.2
wherein Cha is cyclohexylalanine
[0005] In one embodiment, the peptide is defined by formula I:
##STR00001##
wherein R.sup.1 and R.sup.2 are each independently selected
from
[0006] (i) hydrogen;
[0007] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
[0008] (iii)
##STR00002##
where m=1-3;
[0009] (iv)
##STR00003##
[0010] (v)
##STR00004##
R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9,
R.sup.10, R.sup.11 and R.sup.12 are each independently selected
from
[0011] (i) hydrogen;
[0012] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
[0013] (iii) C.sub.1-C.sub.6 alkoxy;
[0014] (iv) amino;
[0015] (v) C.sub.1-C.sub.4 alkylamino;
[0016] (vi) C.sub.1-C.sub.4 dialkylamino;
[0017] (vii) nitro;
[0018] (viii) hydroxyl;
[0019] (ix) halogen, where "halogen" encompasses chloro, fluoro,
bromo, and iodo; and n is an integer from 1 to 5.
[0020] Ina particular embodiment, R.sup.1, R.sup.2, R.sup.3,
R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10,
R.sup.11, and R.sup.12 are all hydrogen; and n is 4. In another
embodiment, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8, R.sup.9, and R.sup.11 are all hydrogen; R.sup.8
and R.sup.12 are methyl; R.sup.10 is hydroxyl; and n is 4.
[0021] In one embodiment, the peptide is defined by formula II:
##STR00005##
wherein R.sup.1 and R.sup.2 are each independently selected
from
[0022] (i) hydrogen;
[0023] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
[0024] (iii)
##STR00006##
where m=1-3;
[0025] (iv)
##STR00007##
[0026] (v)
##STR00008##
R.sup.3 and R.sup.4 are each independently selected from
[0027] (i) hydrogen;
[0028] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
[0029] (iii) C.sub.1-C.sub.6 alkoxy;
[0030] (iv) amino;
[0031] (v) C.sub.1-C.sub.4 alkylamino;
[0032] (vi) C.sub.1-C.sub.4 dialkylamino;
[0033] (vii) nitro;
[0034] (viii) hydroxyl;
[0035] (ix) halogen, where "halogen" encompasses chloro, fluoro,
bromo, and iodo;
R.sup.5, R.sup.6, R.sup.7, R.sup.8, and R.sup.9 are each
independently selected from
[0036] (i) hydrogen;
[0037] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
[0038] (iii) C.sub.1-C.sub.6 alkoxy;
[0039] (iv) amino;
[0040] (v) C.sub.1-C.sub.4 alkylamino;
[0041] (vi) C.sub.1-C.sub.4 dialkylamino;
[0042] (vii) nitro;
[0043] (viii) hydroxyl;
[0044] (ix) halogen, where "halogen" encompasses chloro, fluoro,
bromo, and iodo; and
n is an integer from 1 to 5.
[0045] In a particular embodiment, R.sup.1 and R.sup.2 are
hydrogen; R.sup.3 and R.sup.4 are methyl; R.sup.5, R.sup.6,
R.sup.7, R.sup.8, and R.sup.9 are all hydrogen; and n is 4.
[0046] In one embodiment, the aromatic-cationic peptides have a
core structural motif of alternating aromatic and cationic amino
acids. For example, the peptide may be a tetrapeptide defined by
any of formulas III to VI set forth below:
Aromatic-Cationic-Aromatic-Cationic (Formula III)
Cationic-Aromatic-Cationic-Aromatic (Formula IV)
Aromatic-Aromatic-Cationic-Cationic (Formula V)
Cationic-Cationic-Aromatic-Aromatic (Formula VI)
wherein, Aromatic is a residue selected from the group consisting
of: Phe (F), Tyr (Y), Trp (W), and Cyclohexylalanine (Cha); and
Cationic is a residue selected from the group consisting of: Arg
(R), Lys (K), Norleucine (Nle), and 2-amino-heptanoic acid
(Ahe).
[0047] In some embodiments, the peptide is defined by formula
VII:
##STR00009##
[0048] In some embodiments, the peptide is an isomer of formula
VII, wherein the chiral centers of formula III are defined as
H--(R)-Arg-(S)-DMT-(S)-Lys-(S)-Phe-NH.sub.2, and wherein
stereoisomers are described by the formulas
R--S--S--S
S--R--R--R
S--S--S--S
R--R--R--R
R--R--S--S
S--S--R--R
S--R--S--S
R--S--R--R
R--S--R--S
S--R--S--R
R--R--S--R
S--S--R--S
R--R--R--S
S--S--S--R
R--S--S--R
S--R--R--S
[0049] In some embodiments, the peptide is a constitutional isomer
of formula VII selected from the group consisting of:
Arg-Dmt-Lys-Phe-NH.sub.2
Phe-Dmt-Arg-Lys-NH.sub.2
Phe-Lys-Dmt-Arg-NH.sub.2
Dmt-Arg-Lys-Phe-NH.sub.2
Lys-Dmt-Arg-Phe-NH.sub.2
Phe-Dmt-Lys-Arg-NH.sub.2
Arg-Lys-Dmt-Phe-NH.sub.2
Arg-Dmt-Phe-Lys-NH.sub.2
[0050] In some embodiments, the peptide is defined by formula
VIII:
##STR00010##
wherein R is selected from [0051] (i) OMe, and [0052] (ii) H.
[0053] In some embodiments, the peptide is defined by formula
IX:
##STR00011##
wherein R is selected from [0054] (i) F, [0055] (ii) Cl, and [0056]
(iii) H.
[0057] In some embodiments, the peptide is defined by formula
X:
##STR00012##
wherein R1-R4 are selected from [0058] (i) Ac, (ii) H, (iii) H,
(iv) H, [0059] (i) H, (ii) Ac, (iii) H, (iv) H, [0060] (i) H, (ii)
H, (iii) Ac, (iv) H, and [0061] (i) H, (ii) H, (iii) H, (iv)
OH.
[0062] In one embodiment, the peptide is defined by formula XI:
##STR00013##
[0063] In some aspects, pharmaceutical compositions are provided
herein. In some embodiments, the pharmaceutical compositions
include one or more aromatic-cationic peptides or a
pharmaceutically acceptable salt thereof, such as acetate salt or
trifluoroacetate salt. In some embodiments, the pharmaceutical
composition includes one or more pharmaceutically acceptable
carriers.
[0064] In one aspect, the disclosure provides a method of reducing
the number of mitochondria undergoing mitochondrial permeability
transition (MPT), or preventing mitochondrial permeability
transitioning in a mammal in need thereof, the method comprising
administering to the mammal an effective amount of one or more
aromatic-cationic peptides described herein, or a pharmaceutically
salt thereof such as acetate salt or trifluoroacetate salt. In
another aspect, the disclosure provides a method for increasing the
ATP synthesis rate in a mammal in need thereof, the method
comprising administering to the mammal an effective amount of one
or more aromatic-cationic peptides described herein or a
pharmaceutically salt thereof such as acetate salt or
trifluoroacetate salt. In yet another aspect, the disclosure
provides a method for reducing oxidative damage in a mammal in need
thereof, the method comprising administering to the mammal an
effective amount of one or more aromatic-cationic peptides
described herein or a pharmaceutically salt thereof such as acetate
salt or trifluoroacetate salt.
[0065] In some aspects, a method for determining the presence or
amount of an aromatic-cationic peptide present in a subject is
provided. Typically, the methods include detecting the peptide in a
biological sample from the subject. In some embodiments, the
peptide is detected during administration of the peptide to a
subject; in some embodiments, the peptide is detected after
administration of the peptide to a subject. In some embodiments,
detecting includes HPLC, for example, reverse phase HPLC or ion
exchange HPLC. In some embodiments, detection includes mass
spectrometry.
[0066] In some embodiments, the biological sample comprises a
fluid; in some embodiments, the biological sample comprises a cell.
In some embodiments, the biological sample comprises a tissue. In
other embodiments, the biological sample comprises a biopsy.
[0067] In some embodiments, the aromatic-cationic peptide that is
detected is selected from the group consisting one or more of:
TABLE-US-00002 D-Arg-Dmt-Lys-Phe-NH.sub.2
Dmt-D-Arg-Phe-Lys-NH.sub.2 Phe-D-Arg-Dmt-Lys-NH.sub.2 6-Butyric
acid CoQ0-Phe-D-Arg-Phe-Lys-NH.sub.2 6-Decanoic acid
CoQ0-Phe-D-Arg-Phe-Lys-NH.sub.2 Arg-Arg-Dmt-Phe Arg-Cha-Lys Arg-Dmt
Arg-Dmt-Arg Arg-Dmt-Lys Arg-Dmt-Lys-Phe Arg-Dmt-Lys-Phe-Cys
Arg-Dmt-Phe Arg-Dmt-Phe-Lys Arg-Lys-Dmt-Phe Arg-Lys-Phe-Dmt
Arg-Phe-Dmt-Lys Arg-Phe-Lys Arg-Trp-Lys Arg-Tyr-Lys Arg-Tyr-Lys-Phe
D-Arg-D-Dmt-D-Lys-D-Phe-NH.sub.2 D-Arg-D-Dmt-D-Lys-L-Phe-NH.sub.2
D-Arg-D-Dmt-L-Lys-D-Phe-NH.sub.2 D-Arg-D-Dmt-L-Lys-L-Phe-NH.sub.2
D-Arg-Dmt-D-Lys-NH.sub.2 D-Arg-Dmt-D-Lys-Phe-NH.sub.2
D-Arg-Dmt-Lys-D-Phe-NH.sub.2 D-Arg-Dmt-Lys-NH.sub.2
D-Arg-Dmt-Lys-Phe-Cys D-Arg-Dmt-NH.sub.2
D-Arg-L-Dmt-D-Lys-D-Phe-NH.sub.2 D-Arg-L-Dmt-D-Lys-L-Phe-NH.sub.2
D-Arg-L-Dmt-L-Lys-D-Phe-NH.sub.2 D-Arg-Phe-Lys-NH.sub.2
D-Arg-Trp-Lys-NH.sub.2 D-Arg-Tyr-Lys-NH.sub.2 Dmt-Arg Dmt-Lys
Dmt-Lys-D-Phe-NH.sub.2 Dmt-Lys-NH.sub.2 Dmt-Lys-Phe Dmt-Lys-Phe
Dmt-Lys-Phe-NH.sub.2 Dmt-Phe-Arg-Lys H-Arg-D-Dmt-Arg-NH.sub.2
H-Arg-D-Dmt-Lys-NH.sub.2 H-Arg-D-Dmt-Lys-Phe-NH.sub.2
H-Arg-D-Dmt-NH.sub.2 H-Arg-Dmt-Lys-Phe-NH.sub.2
H-D-Arg-2,6-dichloro-L-tyrosine-L-Lys-L-Phe-NH.sub.2
H-D-Arg-2,6-dichlorotyrosine-Lys-Phe-NH.sub.2
H-D-Arg-2,6-difluoro-L-tyrosine-L-Lys-L-Phe-NH.sub.2
H-D-Arg-2,6-difluorotyrosine-Lys-Phe-NH.sub.2
H-D-Arg-2,6-dimethyl-L-phenylalanine-L-Lys-L-Phe-NH.sub.2
H-D-Arg-2,6-dimethylphenylalanine-Lys-Phe-NH.sub.2
H-D-Arg-4-methoxy-2,6-dimethyl-L-tyrosine-L-Lys-L-Phe-NH.sub.2
H-D-Arg-4-methoxy-2,6-dimethyltyrosine-Lys-Phe-NH.sub.2
H-D-Arg-Arg-Dmt-Phe-NH.sub.2 H-D-Arg-Cha-Lys-NH.sub.2
H-D-Arg-D-Dmt-D-Lys-D-Phe-NH.sub.2 H-D-Arg-D-Dmt-Lys-Phe-NH.sub.2
H-D-Arg-D-Dmt-NH.sub.2 H-D-Arg-Dmt-D-Lys-D-Phe-NH.sub.2
H-D-Arg-Dmt-Lys-2,6-dimethylphenylalanine-NH.sub.2
H-D-Arg-Dmt-Lys-3-hydroxyphenylalanine-NH.sub.2
H-D-Arg-Dmt-Lys-NH.sub.2 H-D-Arg-Dmt-Lys-OH H-D-Arg-Dmt-Lys-Phe-OH
H-D-Arg-Dmt-N6-acetyllysine-Phe-NH.sub.2 H-D-Arg-Dmt-OH
H-D-Arg-Dmt-Phe-Lys-NH.sub.2 H-D-Arg-Dmt-Phe-NH.sub.2
H-D-Arg-D-Phe-L-Lys-L-Phe-NH.sub.2
H-D-Arg-D-Trp-L-Lys-L-Phe-NH.sub.2
H-D-Arg-D-Tyr-L-Lys-L-Phe-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-2,6-dimethyl-L-phenylalanine-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-3-hydroxy-L-phenylalanine-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-D-Dmt-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-D-Trp-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-D-Tyr-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-L-Trp-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-L-Tyr-NH.sub.2
H-D-Arg-L-Dmt-L-Phe-L-Lys-NH.sub.2
H-D-Arg-L-Dmt-N6-acetyl-L-lysine-L-Phe-NH.sub.2
H-D-Arg-L-Lys-L-Dmt-L-Phe-NH.sub.2
H-D-Arg-L-Lys-L-Phe-L-Dmt-NH.sub.2
H-D-Arg-L-Phe-L-Dmt-L-Lys-NH.sub.2
H-D-Arg-L-Phe-L-Lys-L-Dmt-NH.sub.2
H-D-Arg-L-Phe-L-Lys-L-Phe-NH.sub.2
H-D-Arg-L-Trp-L-Lys-L-Phe-NH.sub.2
H-D-Arg-L-Tyr-L-Lys-L-Phe-NH.sub.2 H-D-Arg-Lys-Dmt-Phe-NH.sub.2
H-D-Arg-Lys-Phe-Dmt-NH.sub.2 H-D-Arg-Phe-Dmt-Lys-NH.sub.2
H-D-Arg-Phe-Lys-Dmt-NH.sub.2 H-D-Arg-Tyr-Lys-Phe-NH.sub.2
H-D-Dmt-Arg-NH.sub.2 H-D-His-L-Dmt-L-Lys-L-Phe-NH.sub.2
H-D-Lys-L-Dmt-L-Lys-L-Phe-NH.sub.2 H-Dmt-D-Arg-Lys-Phe-NH.sub.2
H-Dmt-D-Arg-NH.sub.2 H-Dmt-D-Arg-Phe-Lys-NH.sub.2
H-Dmt-D-Phe-Arg-Lys-NH.sub.2 H-Dmt-Lys-D-Arg-Phe-NH.sub.2
H-Dmt-Lys-Phe-D-Arg-NH.sub.2 H-Dmt-Phe-D-Arg-Lys-NH.sub.2
H-Dmt-Phe-Lys-D-Arg-NH.sub.2
H-D-N2-acetylarginine-Dmt-Lys-Phe-NH.sub.2
H-D-N8-acetylarginine-Dmt-Lys-Phe-NH.sub.2
H-D-Phe-D-Arg-D-Phe-D-Lys-NH.sub.2
H-L-Dmt-D-Arg-L-Lys-L-Phe-NH.sub.2
H-L-Dmt-D-Arg-L-Phe-L-Lys-NH.sub.2
H-L-Dmt-L-Lys-D-Arg-L-Phe-NH.sub.2
H-L-Dmt-L-Lys-L-Phe-D-Arg-NH.sub.2
H-L-Dmt-L-Phe-D-Arg-L-Lys-NH.sub.2
H-L-Dmt-L-Phe-L-Lys-D-Arg-NH.sub.2
H-L-His-L-Dmt-L-Lys-L-Phe-NH.sub.2
H-L-Lys-D-Arg-L-Dmt-L-Phe-NH.sub.2
H-L-Lys-D-Arg-L-Phe-L-Dmt-NH.sub.2
H-L-Lys-L-Dmt-D-Arg-L-Phe-NH.sub.2
H-L-Lys-L-Dmt-L-Lys-L-Phe-NH.sub.2
H-L-Lys-L-Dmt-L-Phe-D-Arg-NH.sub.2
H-L-Lys-L-Phe-D-Arg-L-Dmt-NH.sub.2
H-L-Lys-L-Phe-L-Dmt-D-Arg-NH.sub.2
H-L-Phe-D-Arg-L-Dmt-L-Lys-NH.sub.2
H-L-Phe-D-Arg-L-Lys-L-Dmt-NH.sub.2
H-L-Phe-L-Dmt-D-Arg-L-Lys-NH.sub.2
H-L-Phe-L-Dmt-L-Lys-D-Arg-NH.sub.2
H-L-Phe-L-Lys-D-Arg-L-Dmt-NH.sub.2
H-L-Phe-L-Lys-L-Dmt-D-Arg-NH.sub.2 H-Lys-D-Arg-Dmt-Phe-NH.sub.2
H-Lys-D-Arg-Phe-Dmt-NH.sub.2 H-Lys-Dmt-D-Arg-Phe-NH.sub.2
H-Lys-Dmt-Phe-D-Arg-NH.sub.2 H-Lys-D-Phe-Arg-Dmt-NH.sub.2
H-Lys-Phe-D-Arg-Dmt-NH.sub.2 H-Lys-Phe-Dmt-D-Arg-NH.sub.2
H-N2-acetyl-D-arginine-L-Dmt-L-Lys-L-Phe-NH.sub.2
H-N7-acetyl-D-arginine-Dmt-Lys-Phe-NH.sub.2
H-Phe(d5)-D-Arg-Phe(d5)-Lys-NH.sub.2 H-Phe-Arg-Phe-Lys-NH.sub.2
H-Phe-D-Arg-Dmt-Lys-NH.sub.2 H-Phe-D-Arg-Dmt-Lys-NH.sub.2
H-Phe-D-Arg-D-Phe-Lys-NH.sub.2 H-Phe-D-Arg-Lys-Dmt-NH.sub.2
H-Phe-D-Arg-Phe-D-Lys-NH.sub.2
H-Phe-D-Arg-Phe-Lys-Glu-Cys-Gly-NH.sub.2
H-Phe-D-Dmt-Arg-Lys-NH.sub.2 H-Phe-Dmt-D-Arg-Lys-NH.sub.2
H-Phe-Dmt-Lys-D-Arg-NH.sub.2 H-Phe-Lys-D-Arg-Dmt-NH.sub.2
H-Phe-Lys-Dmt-D-Arg-NH.sub.2 L-Arg-D-Dmt-D-Lys-D-Phe-NH.sub.2
L-Arg-D-Dmt-D-Lys-D-Phe-NH.sub.2 L-Arg-D-Dmt-D-Lys-L-Phe-NH.sub.2
L-Arg-D-Dmt-D-Lys-L-Phe-NH.sub.2 L-Arg-D-Dmt-L-Lys-D-Phe-NH.sub.2
L-Arg-D-Dmt-L-Lys-D-Phe-NH.sub.2 L-Arg-D-Dmt-L-Lys-L-Phe-NH.sub.2
L-Arg-D-Dmt-L-Lys-L-Phe-NH.sub.2 L-Arg-L-Dmt-D-Lys-D-Phe-NH.sub.2
L-Arg-L-Dmt-D-Lys-D-Phe-NH.sub.2 L-Arg-L-Dmt-D-Lys-L-Phe-NH.sub.2
L-Arg-L-Dmt-D-Lys-L-Phe-NH.sub.2 L-Arg-L-Dmt-L-Lys-D-Phe-NH.sub.2
L-Arg-L-Dmt-L-Lys-D-Phe-NH.sub.2 L-Arg-L-Dmt-L-Lys-L-Phe-NH.sub.2
L-Arg-L-Dmt-L-Lys-L-Phe-NH.sub.2 Lys-Dmt-Arf Lys-Dmt-D-Arg-NH.sub.2
Lys-Phe Lys-Phe-Arg-Dmt Lys-Phe-NH.sub.2 Lys-Trp-Arg
Lys-Trp-D-Arg-NH.sub.2 Phe-Arg-Dmt-Lys Phe-Arg-Phe-Lys
Phe-Arg-Phe-Lys Phe-Arg-Phe-Lys Phe-Arg-Phe-Lys Phe-Arg-Phe-Lys
Phe-Arg-Phe-Lys-Glu-Cys-Gly Phe-Dmt-Arg-Lys Phe-Lys-Dmt
Phe-Lys-Dmt-NH.sub.2 Succinic monoester
CoQ0-Phe-D-Arg-Phe-Lys-NH.sub.2
wherein Cha is cyclohexylalanine
[0068] In some aspects, a kit for the detection of
aromatic-cationic peptides is provided. In some embodiments, the
kits include a biological sample collector to collect a sample from
the subject, and a sample storage device for preservation of the
biological sample. In some embodiments, the biological sample
comprises a fluid. In some embodiments, the biological sample
comprises a cell. In some embodiments, the biological sample
comprises a tissue sample. In some embodiments, the biological
sample comprises a biopsy.
DETAILED DESCRIPTION
[0069] It is to be appreciated that certain aspects, modes,
embodiments, variations and features of the invention are described
below in various levels of detail in order to provide a substantial
understanding of the present invention.
[0070] In practicing the present invention, many conventional
techniques in molecular biology, protein biochemistry, cell
biology, immunology, microbiology and recombinant DNA are used.
These techniques are well-known and are explained in, e.g., Current
Protocols in Molecular Biology, Vols. I-III, Ausubel, Ed. (1997);
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Ed.
(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989); DNA Cloning: A Practical Approach, Vols. I and II, Glover,
Ed. (1985); Oligonucleotide Synthesis, Gait, Ed. (1984); Nucleic
Acid Hybridization, Hames & Higgins, Eds. (1985); Transcription
and Translation, Hames & Higgins, Eds. (1984); Animal Cell
Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes (IRL
Press, 1986); Perbal, A Practical Guide to Molecular Cloning; the
series, Meth. Enzymol., (Academic Press, Inc., 1984); Gene Transfer
Vectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring
Harbor Laboratory, NY, 1987); and Meth. Enzymol., Vols. 154 and
155, Wu & Grossman, and Wu, Eds., respectively.
[0071] The definitions of certain terms as used in this
specification are provided below. Unless defined otherwise, all
technical and scientific terms used herein generally have the same
meaning as commonly understood by one of ordinary skill in the art
to which this invention belongs.
[0072] As used in this specification and the appended claims, the
singular forms "a", "an" and "the" include plural referents unless
the content clearly dictates otherwise. For example, reference to
"a cell" includes a combination of two or more cells, and the
like.
[0073] As used herein, the "administration" of an agent, drug, or
peptide to a subject includes any route of introducing or
delivering to a subject a compound to perform its intended
function. Administration can be carried out by any suitable route,
including orally, intranasally, parenterally (intravenously,
intramuscularly, intraperitoneally, or subcutaneously), or
topically. Administration includes self-administration and the
administration by another.
[0074] As used herein, the term "amino acid" includes
naturally-occurring amino acids and synthetic amino acids, as well
as amino acid analogs and amino acid mimetics that function in a
manner similar to the naturally-occurring amino acids.
Naturally-occurring amino acids are those encoded by the genetic
code, as well as those amino acids that are later modified, e.g.,
hydroxyproline, .gamma.-carboxyglutamate, and O-phosphoserine.
Amino acid analogs refers to compounds that have the same basic
chemical structure as a naturally-occurring amino acid, i.e., an
.alpha.-carbon that is bound to a hydrogen, a carboxyl group, an
amino group, and an R group, e.g., homoserine, norleucine,
methionine sulfoxide, methionine methyl sulfonium. Such analogs
have modified R groups (e.g., norleucine) or modified peptide
backbones, but retain the same basic chemical structure as a
naturally-occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally-occurring amino acid. Amino acids
can be referred to herein by either their commonly known three
letter symbols or by the one-letter symbols recommended by the
IUPAC-IUB Biochemical Nomenclature Commission.
[0075] As used herein, the term "biological sample" refers to
material derived from or contacted by living cells. The term
encompasses tissues, cells and biological fluids isolated from a
subject, as well as tissues, cells and fluids present within a
subject. Biological samples include but are not limited to, whole
blood, fractionated blood, semen, saliva, tears, urine, fecal
material, sweat, buccal, skin, cerebrospinal fluid, and hair.
Biological samples also includes biopsies of internal organs and
cancers. Biological samples can be obtained from subjects for
diagnosis or research or can be obtained from undiseased
individuals, as controls or for basic research.
[0076] As used herein, the term "effective amount" refers to a
quantity sufficient to achieve a desired therapeutic and/or
prophylactic effect. In the context of therapeutic or prophylactic
applications, the amount of a composition administered to the
subject will depend on the type and severity of the disease and on
the characteristics of the individual, such as general health, age,
sex, body weight and tolerance to drugs. It will also depend on the
degree, severity and type of disease. The skilled artisan will be
able to determine appropriate dosages depending on these and other
factors. The compositions can also be administered in combination
with one or more additional therapeutic compounds.
[0077] An "isolated" or "purified" polypeptide or peptide is
substantially free of cellular material or other contaminating
polypeptides from the cell or tissue source from which the agent is
derived, or substantially free from chemical precursors or other
chemicals when chemically synthesized. For example, an isolated
aromatic-cationic peptide would be free of materials that would
interfere with diagnostic or therapeutic uses of the agent. Such
interfering materials may include enzymes, hormones and other
proteinaceous and nonproteinaceous solutes.
[0078] As used herein, the terms "polypeptide", "peptide", and
"protein" are used interchangeably herein to mean a polymer
comprising two or more amino acids joined to each other by peptide
bonds or modified peptide bonds, i.e., peptide isosteres.
Polypeptide refers to both short chains, commonly referred to as
peptides, glycopeptides or oligomers, and to longer chains,
generally referred to as proteins. Polypeptides may contain amino
acids other than the 20 gene-encoded amino acids. Polypeptides
include amino acid sequences modified either by natural processes,
such as post-translational processing, or by chemical modification
techniques that are well known in the art.
[0079] As used herein, the terms "treating" or "treatment" or
"alleviation" refers to both therapeutic treatment and prophylactic
or preventative measures, wherein the object is to prevent or slow
down (lessen) the targeted pathologic condition or disorder. It is
also to be appreciated that the various modes of treatment or
prevention of medical conditions as described are intended to mean
"substantial", which includes total but also less than total
treatment or prevention, and wherein some biologically or medically
relevant result is achieved.
[0080] As used herein, "prevention" or "preventing" of a disorder
or condition refers to a compound that, in a statistical sample,
reduces the occurrence of the disorder or condition in the treated
sample relative to an untreated control sample, or delays the onset
or reduces the severity of one or more symptoms of the disorder or
condition relative to the untreated control sample.
Methods of Prevention or Treatment
[0081] The present technology relates to the treatment or
prevention of disease by administration of certain
aromatic-cationic peptides.
[0082] The aromatic-cationic peptides are water-soluble and highly
polar. Despite these properties, the peptides can readily penetrate
cell membranes. The aromatic-cationic peptides typically include a
minimum of two or three amino acids or a minimum of four amino
acids, covalently joined by peptide bonds. The maximum number of
amino acids present in the aromatic-cationic peptides is about
twenty amino acids covalently joined by peptide bonds. Suitably,
the maximum number of amino acids is about twelve, more preferably
about nine, and most preferably about six.
[0083] The amino acids of the aromatic-cationic peptides can be any
amino acid. As used herein, the term "amino acid" is used to refer
to any organic molecule that contains at least one amino group and
at least one carboxyl group. Typically, at least one amino group is
at the a position relative to a carboxyl group. The amino acids may
be naturally occurring. Naturally occurring amino acids include,
for example, the twenty most common levorotatory (L) amino acids
normally found in mammalian proteins, i.e., alanine (Ala), arginine
(Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys),
glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine
(His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine
(Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine
(Thr), tryptophan, (Trp), tyrosine (Tyr), and valine (Val). Other
naturally occurring amino acids include, for example, amino acids
that are synthesized in metabolic processes not associated with
protein synthesis. For example, the amino acids ornithine and
citrulline are synthesized in mammalian metabolism during the
production of urea. Another example of a naturally occurring amino
acid includes hydroxyproline (Hyp).
[0084] The peptides optionally contain one or more non-naturally
occurring amino acids. Optimally, the peptide has no amino acids
that are naturally occurring. The non-naturally occurring amino
acids may be levorotary (L-), dextrorotatory (D-), or mixtures
thereof. Non-naturally occurring amino acids are those amino acids
that typically are not synthesized in normal metabolic processes in
living organisms, and do not naturally occur in proteins. In
addition, the non-naturally occurring amino acids suitably are also
not recognized by common proteases. The non-naturally occurring
amino acid can be present at any position in the peptide. For
example, the non-naturally occurring amino acid can be at the
N-terminus, the C-terminus, or at any position between the
N-terminus and the C-terminus.
[0085] 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
.alpha.-aminobutyric acid, .beta.-aminobutyric acid,
.gamma.-aminobutyric acid, .delta.-aminovaleric acid, and
.epsilon.-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
.gamma.-phenyl-.beta.-aminobutyric acid. Non-naturally occurring
amino acids include derivatives of naturally occurring amino acids.
The derivatives of naturally occurring amino acids may, for
example, include the addition of one or more chemical groups to the
naturally occurring amino acid.
[0086] 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.sub.1-C.sub.4 alkyl, such as methyl, ethyl, n-propyl, isopropyl,
butyl, isobutyl, or t-butyl, C.sub.1-C.sub.4 alkyloxy (i.e.,
alkoxy), amino, C.sub.1-C.sub.4 alkylamino and C.sub.1-C.sub.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 (Nle).
[0087] Another example of a modification of an amino acid in a
peptide is the derivatization of a carboxyl group of an aspartic
acid or a glutamic acid residue of the peptide. One example of
derivatization is amidation with ammonia or with a primary or
secondary amine, e.g. methylamine, ethylamine, dimethylamine or
diethylamine. Another example of derivatization includes
esterification with, for example, methyl or ethyl alcohol. Another
such modification includes derivatization of an amino group of a
lysine, arginine, or histidine residue. For example, such amino
groups can be acylated. Some suitable acyl groups include, for
example, a benzoyl group or an alkanoyl group comprising any of the
C.sub.1-C.sub.4 alkyl groups mentioned above, such as an acetyl or
propionyl group.
[0088] The non-naturally occurring amino acids are preferably
resistant, and more preferably insensitive, to common proteases.
Examples of non-naturally occurring amino acids that are resistant
or insensitive to proteases include the dextrorotatory (D-) form of
any of the above-mentioned naturally occurring L-amino acids, as
well as L- and/or D-non-naturally occurring amino acids. The
D-amino acids do not normally occur in proteins, although they are
found in certain peptide antibiotics that are synthesized by means
other than the normal ribosomal protein synthetic machinery of the
cell. As used herein, the D-amino acids are considered to be
non-naturally occurring amino acids.
[0089] In order to minimize protease sensitivity, the peptides
should have less than five, preferably less than four, more
preferably less than three, and most preferably, less than two
contiguous L-amino acids recognized by common proteases,
irrespective of whether the amino acids are naturally or
non-naturally occurring. Optimally, the peptide has only D-amino
acids, and no L-amino acids. If the peptide contains protease
sensitive sequences of amino acids, at least one of the amino acids
is preferably a non-naturally-occurring D-amino acid, thereby
conferring protease resistance. An example of a protease sensitive
sequence includes two or more contiguous basic amino acids that are
readily cleaved by common proteases, such as endopeptidases and
trypsin. Examples of basic amino acids include arginine, lysine and
histidine.
[0090] The aromatic-cationic peptides should have a minimum number
of net positive charges at physiological pH in comparison to the
total number of amino acid residues in the peptide. The minimum
number of net positive charges at physiological pH will be referred
to below as (p.sub.m). The total number of amino acid residues in
the peptide will be referred to below as (r). The minimum number of
net positive charges discussed below are all at physiological pH.
The term "physiological pH" as used herein refers to the normal pH
in the cells of the tissues and organs of the mammalian body. For
instance, the physiological pH of a human is normally approximately
7.4, but normal physiological pH in mammals may be any pH from
about 7.0 to about 7.8.
[0091] "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.
[0092] Typically, a peptide has a positively charged N-terminal
amino group and a negatively charged C-terminal carboxyl group. The
charges cancel each other out at physiological pH. As an example of
calculating net charge, the peptide
Tyr-Arg-Phe-Lys-Glu-His-Trp-D-Arg has one negatively charged amino
acid (i.e., Glu) and four positively charged amino acids (i.e., two
Arg residues, one Lys, and one His). Therefore, the above peptide
has a net positive charge of three.
[0093] In one embodiment, the peptide is defined by formula I:
##STR00014##
wherein R.sup.1 and R.sup.2 are each independently selected
from
[0094] (i) hydrogen;
[0095] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
[0096] (iii)
##STR00015##
where m=1-3;
[0097] (iv)
##STR00016##
[0098] (v)
##STR00017##
R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9,
R.sup.10, R.sup.11 and R.sup.12 are each independently selected
from
[0099] (i) hydrogen;
[0100] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
[0101] (iii) C.sub.1-C.sub.6 alkoxy;
[0102] (iv) amino;
[0103] (v) C.sub.1-C.sub.4 alkylamino;
[0104] (vi) C.sub.1-C.sub.4 dialkylamino;
[0105] (vii) nitro;
[0106] (viii) hydroxyl;
[0107] (ix) halogen, where "halogen" encompasses chloro, fluoro,
bromo, and iodo; and
n is an integer from 1 to 5.
[0108] Ina particular embodiment, R.sup.1, R.sup.2, R.sup.3,
R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10,
R.sup.11, and R.sup.12 are all hydrogen; and n is 4. In another
embodiment, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8, R.sup.9, and R.sup.11 are all hydrogen; R.sup.8
and R.sup.12 are methyl; R.sup.10 is hydroxyl; and n is 4.
[0109] In one embodiment, the peptide is defined by formula II:
##STR00018##
wherein R.sup.1 and R.sup.2 are each independently selected
from
[0110] (i) hydrogen;
[0111] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
[0112] (iii)
##STR00019##
where m=1-3;
[0113] (iv)
##STR00020##
[0114] (v)
##STR00021##
R.sup.3 and R.sup.4 are each independently selected from
[0115] (i) hydrogen;
[0116] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
[0117] (iii) C.sub.1-C.sub.6 alkoxy;
[0118] (iv) amino;
[0119] (v) C.sub.1-C.sub.4 alkylamino;
[0120] (vi) C.sub.1-C.sub.4 dialkylamino;
[0121] (vii) nitro;
[0122] (viii) hydroxyl;
[0123] (ix) halogen, where "halogen" encompasses chloro, fluoro,
bromo, and iodo;
R.sup.5, R.sup.6, R.sup.7, R.sup.8, and R.sup.9 are each
independently selected from
[0124] (i) hydrogen;
[0125] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
[0126] (iii) C.sub.1-C.sub.6 alkoxy;
[0127] (iv) amino;
[0128] (v) C.sub.1-C.sub.4 alkylamino;
[0129] (vi) C.sub.1-C.sub.4 dialkylamino;
[0130] (vii) nitro;
[0131] (viii) hydroxyl;
[0132] (ix) halogen, where "halogen" encompasses chloro, fluoro,
bromo, and iodo; and
n is an integer from 1 to 5.
[0133] In a particular embodiment, R.sup.1 and R.sup.2 are
hydrogen; R.sup.3 and R.sup.4 are methyl; R.sup.5, R.sup.6,
R.sup.7, R.sup.8, and R.sup.9 are all hydrogen; and n is 4.
[0134] In one embodiment, the aromatic-cationic peptides have a
core structural motif of alternating aromatic and cationic amino
acids. Fr example, the peptide may be a tetrapeptide defined by any
of formulas III to VI set forth below:
Aromatic-Cationic-Aromatic-Cationic (Formula III)
Cationic-Aromatic-Cationic-Aromatic (Formula IV)
Aromatic-Aromatic-Cationic-Cationic (Formula V)
Cationic-Cationic-Aromatic-Aromatic (Formula VI)
wherein, Aromatic is a residue selected from the group consisting
of: Phe (F), Tyr (Y), Trp (W), and Cyclohexylalanine (Cha); and
Cationic is a residue selected from the group consisting of: Arg
(R), Lys (K), Norleucine (Nle), and 2-amino-heptanoic acid
(Ahe).
[0135] In one aspect, the disclosure provides a method of reducing
the number of mitochondria undergoing mitochondrial permeability
transition (MPT), or preventing mitochondrial permeability
transitioning in a mammal in need thereof, the method comprising
administering to the mammal an effective amount of one or more
aromatic-cationic peptides described herein. In another aspect, the
disclosure provides a method for increasing the ATP synthesis rate
in a mammal in need thereof, the method comprising administering to
the mammal an effective amount of one or more aromatic-cationic
peptides described herein. In yet another aspect, the disclosure
provides a method for reducing oxidative damage in a mammal in need
thereof, the method comprising administering to the mammal an
effective amount of one or more aromatic-cationic peptides
described herein.
[0136] In one embodiment, the aromatic-cationic peptides have a
relationship between the minimum number of net positive charges at
physiological pH (p.sub.m) and the total number of amino acid
residues (r) wherein 3p.sub.m is the largest number that is less
than or equal to r+1. In this embodiment, the relationship between
the minimum number of net positive charges (p.sub.m) and the total
number of amino acid residues (r) is as follows:
TABLE-US-00003 TABLE 1 Amino acid number and net positive charges
(3p.sub.m .ltoreq. p + 1) (r) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
18 19 20 (p.sub.m) 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7
[0137] In another embodiment, the aromatic-cationic peptides have a
relationship between the minimum number of net positive charges
(p.sub.m) and the total number of amino acid residues (r) wherein
2p.sub.m is the largest number that is less than or equal to r+1.
In this embodiment, the relationship between the minimum number of
net positive charges (p.sub.m) and the total number of amino acid
residues (r) is as follows:
TABLE-US-00004 TABLE 2 Amino acid number and net positive charges
(2p.sub.m .ltoreq. p + 1) (r) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
18 19 20 (p.sub.m) 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10
[0138] In one embodiment, the minimum number of net positive
charges (p.sub.m) and the total number of amino acid residues (r)
are equal. In another embodiment, the peptides have three or four
amino acid residues and a minimum of one net positive charge,
suitably, a minimum of two net positive charges and more preferably
a minimum of three net positive charges.
[0139] It is also important that the aromatic-cationic peptides
have a minimum number of aromatic groups in comparison to the total
number of net positive charges (p.sub.t). The minimum number of
aromatic groups will be referred to below as (a). Naturally
occurring amino acids that have an aromatic group include the amino
acids histidine, tryptophan, tyrosine, and phenylalanine. For
example, the hexapeptide Lys-Gln-Tyr-D-Arg-Phe-Trp has a net
positive charge of two (contributed by the lysine and arginine
residues) and three aromatic groups (contributed by tyrosine,
phenylalanine and tryptophan residues).
[0140] The aromatic-cationic peptides should also have a
relationship between the minimum number of aromatic groups (a) and
the total number of net positive charges at physiological pH
(p.sub.t) wherein 3a is the largest number that is less than or
equal to p.sub.t+1, except that when p.sub.t is 1, a may also be 1.
In this embodiment, the relationship between the minimum number of
aromatic groups (a) and the total number of net positive charges
(p.sub.t) is as follows:
TABLE-US-00005 TABLE 3 Aromatic groups and net positive charges (3a
.ltoreq. p.sub.t + 1 or a = p.sub.t = 1) (p.sub.t) 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
[0141] In another embodiment, the aromatic-cationic peptides have a
relationship between the minimum number of aromatic groups (a) and
the total number of net positive charges (p.sub.t) wherein 2a is
the largest number that is less than or equal to p.sub.t+1. In this
embodiment, the relationship between the minimum number of aromatic
amino acid residues (a) and the total number of net positive
charges (p.sub.t) is as follows:
TABLE-US-00006 TABLE 4 Aromatic groups and net positive charges (2a
.ltoreq. p.sub.t + 1 or a = p.sub.t = 1) (p.sub.t) 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
[0142] In another embodiment, the number of aromatic groups (a) and
the total number of net positive charges (p.sub.t) are equal. In
one embodiment, the aromatic-cationic peptide may have
[0143] (a) at least one net positive charge;
[0144] (b) a minimum of three amino acids;
[0145] (c) a maximum of about twenty amino acids;
[0146] (d) a relationship between the minimum number of net
positive charges (p.sub.m) and the total number of amino acid
residues (r) wherein 3p.sub.m is the largest number that is less
than or equal to r+1; and
[0147] (e) a relationship between the minimum number of aromatic
groups (a) and the total number of net positive charges (p.sub.t)
wherein 3a is the largest number that is less than or equal to
p.sub.t+1, except that when a is 1, p.sub.t may also be 1.
[0148] Carboxyl groups, especially the terminal carboxyl group of a
C-terminal amino acid, are suitably amidated with, for example,
ammonia to form the C-terminal amide. Alternatively, the terminal
carboxyl group of the C-terminal amino acid may be amidated with
any primary or secondary amine. The primary or secondary amine may,
for example, be an alkyl, especially a branched or unbranched
C.sub.1-C.sub.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. The free carboxylate groups of the asparagine, glutamine,
aspartic acid, and glutamic acid residues not occurring at the
C-terminus of the aromatic-cationic peptides may also be amidated
wherever they occur within the peptide. The amidation at these
internal positions may be with ammonia or any of the primary or
secondary amines described above.
[0149] Aromatic-cationic peptides include, but are not limited to,
the following exemplary peptides:
TABLE-US-00007 6-Butyric acid CoQ0-Phe-D-Arg-Phe-Lys-NH.sub.2
6-Decanoic acid CoQ0-Phe-D-Arg-Phe-Lys-NH.sub.2 Arg-Arg-Dmt-Phe
Arg-Cha-Lys Arg-Dmt Arg-Dmt-Arg Arg-Dmt-Lys Arg-Dmt-Lys-Phe
Arg-Dmt-Lys-Phe-Cys Arg-Dmt-Phe Arg-Dmt-Phe-Lys Arg-Lys-Dmt-Phe
Arg-Lys-Phe-Dmt Arg-Phe-Dmt-Lys Arg-Phe-Lys Arg-Trp-Lys Arg-Tyr-Lys
Arg-Tyr-Lys-Phe D-Arg-D-Dmt-D-Lys-D-Phe-NH.sub.2
D-Arg-D-Dmt-D-Lys-L-Phe-NH.sub.2 D-Arg-D-Dmt-L-Lys-D-Phe-NH.sub.2
D-Arg-D-Dmt-L-Lys-L-Phe-NH.sub.2 D-Arg-Dmt-D-Lys-NH.sub.2
D-Arg-Dmt-D-Lys-Phe-NH.sub.2 D-Arg-Dmt-Lys-D-Phe-NH.sub.2
D-Arg-Dmt-Lys-NH.sub.2 D-Arg-Dmt-Lys-Phe-Cys D-Arg-Dmt-NH.sub.2
D-Arg-L-Dmt-D-Lys-D-Phe-NH.sub.2 D-Arg-L-Dmt-D-Lys-L-Phe-NH.sub.2
D-Arg-L-Dmt-L-Lys-D-Phe-NH.sub.2 D-Arg-Phe-Lys-NH.sub.2
D-Arg-Trp-Lys-NH.sub.2 D-Arg-Tyr-Lys-NH.sub.2 Dmt-Arg Dmt-Lys
Dmt-Lys-D-Phe-NH.sub.2 Dmt-Lys-NH.sub.2 Dmt-Lys-Phe Dmt-Lys-Phe
Dmt-Lys-Phe-NH.sub.2 Dmt-Phe-Arg-Lys H-Arg-D-Dmt-Arg-NH.sub.2
H-Arg-D-Dmt-Lys-NH.sub.2 H-Arg-D-Dmt-Lys-Phe-NH.sub.2
H-Arg-D-Dmt-NH.sub.2 H-Arg-Dmt-Lys-Phe-NH.sub.2
H-D-Arg-2,6-dichloro-L-tyrosine-L-Lys-L-Phe-NH.sub.2
H-D-Arg-2,6-dichlorotyrosine-Lys-Phe-NH.sub.2
H-D-Arg-2,6-difluoro-L-tyrosine-L-Lys-L-Phe-NH.sub.2
H-D-Arg-2,6-difluorotyrosine-Lys-Phe-NH.sub.2
H-D-Arg-2,6-dimethyl-L-phenylalanine-L-Lys-L-Phe-NH.sub.2
H-D-Arg-2,6-dimethylphenylalanine-Lys-Phe-NH.sub.2
H-D-Arg-4-methoxy-2,6-dimethyl-L-tyrosine-L-Lys-L-Phe-NH.sub.2
H-D-Arg-4-methoxy-2,6-dimethyltyrosine-Lys-Phe-NH.sub.2
H-D-Arg-Arg-Dmt-Phe-NH.sub.2 H-D-Arg-Cha-Lys-NH.sub.2
H-D-Arg-D-Dmt-D-Lys-D-Phe-NH.sub.2 H-D-Arg-D-Dmt-Lys-Phe-NH.sub.2
H-D-Arg-D-Dmt-NH.sub.2 H-D-Arg-Dmt-D-Lys-D-Phe-NH.sub.2
H-D-Arg-Dmt-Lys-2,6-dimethylphenylalanine-NH.sub.2
H-D-Arg-Dmt-Lys-3-hydroxyphenylalanine-NH.sub.2
H-D-Arg-Dmt-Lys-NH.sub.2 H-D-Arg-Dmt-Lys-OH H-D-Arg-Dmt-Lys-Phe-OH
H-D-Arg-Dmt-N6-acetyllysine-Phe-NH.sub.2 H-D-Arg-Dmt-OH
H-D-Arg-Dmt-Phe-Lys-NH.sub.2 H-D-Arg-Dmt-Phe-NH.sub.2
H-D-Arg-D-Phe-L-Lys-L-Phe-NH.sub.2
H-D-Arg-D-Trp-L-Lys-L-Phe-NH.sub.2
H-D-Arg-D-Tyr-L-Lys-L-Phe-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-2,6-dimethyl-L-phenylalanine-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-3-hydroxy-L-phenylalanine-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-D-Dmt-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-D-Trp-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-D-Tyr-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-L-Trp-NH.sub.2
H-D-Arg-L-Dmt-L-Lys-L-Tyr-NH.sub.2
H-D-Arg-L-Dmt-L-Phe-L-Lys-NH.sub.2
H-D-Arg-L-Dmt-N6-acetyl-L-lysine-L-Phe-NH.sub.2
H-D-Arg-L-Lys-L-Dmt-L-Phe-NH.sub.2
H-D-Arg-L-Lys-L-Phe-L-Dmt-NH.sub.2
H-D-Arg-L-Phe-L-Dmt-L-Lys-NH.sub.2
H-D-Arg-L-Phe-L-Lys-L-Dmt-NH.sub.2
H-D-Arg-L-Phe-L-Lys-L-Phe-NH.sub.2
H-D-Arg-L-Trp-L-Lys-L-Phe-NH.sub.2
H-D-Arg-L-Tyr-L-Lys-L-Phe-NH.sub.2 H-D-Arg-Lys-Dmt-Phe-NH.sub.2
H-D-Arg-Lys-Phe-Dmt-NH.sub.2 H-D-Arg-Phe-Dmt-Lys-NH.sub.2
H-D-Arg-Phe-Lys-Dmt-NH.sub.2 H-D-Arg-Tyr-Lys-Phe-NH.sub.2
H-D-Dmt-Arg-NH.sub.2 H-D-His-L-Dmt-L-Lys-L-Phe-NH.sub.2
H-D-Lys-L-Dmt-L-Lys-L-Phe-NH.sub.2 H-Dmt-D-Arg-Lys-Phe-NH.sub.2
H-Dmt-D-Arg-NH.sub.2 H-Dmt-D-Arg-Phe-Lys-NH.sub.2
H-Dmt-D-Phe-Arg-Lys-NH.sub.2 H-Dmt-Lys-D-Arg-Phe-NH.sub.2
H-Dmt-Lys-Phe-D-Arg-NH.sub.2 H-Dmt-Phe-D-Arg-Lys-NH.sub.2
H-Dmt-Phe-Lys-D-Arg-NH.sub.2
H-D-N2-acetylarginine-Dmt-Lys-Phe-NH.sub.2
H-D-N8-acetylarginine-Dmt-Lys-Phe-NH.sub.2
H-D-Phe-D-Arg-D-Phe-D-Lys-NH.sub.2
H-L-Dmt-D-Arg-L-Lys-L-Phe-NH.sub.2
H-L-Dmt-D-Arg-L-Phe-L-Lys-NH.sub.2
H-L-Dmt-L-Lys-D-Arg-L-Phe-NH.sub.2
H-L-Dmt-L-Lys-L-Phe-D-Arg-NH.sub.2
H-L-Dmt-L-Phe-D-Arg-L-Lys-NH.sub.2
H-L-Dmt-L-Phe-L-Lys-D-Arg-NH.sub.2
H-L-His-L-Dmt-L-Lys-L-Phe-NH.sub.2
H-L-Lys-D-Arg-L-Dmt-L-Phe-NH.sub.2
H-L-Lys-D-Arg-L-Phe-L-Dmt-NH.sub.2
H-L-Lys-L-Dmt-D-Arg-L-Phe-NH.sub.2
H-L-Lys-L-Dmt-L-Lys-L-Phe-NH.sub.2
H-L-Lys-L-Dmt-L-Phe-D-Arg-NH.sub.2
H-L-Lys-L-Phe-D-Arg-L-Dmt-NH.sub.2
H-L-Lys-L-Phe-L-Dmt-D-Arg-NH.sub.2
H-L-Phe-D-Arg-L-Dmt-L-Lys-NH.sub.2
H-L-Phe-D-Arg-L-Lys-L-Dmt-NH.sub.2
H-L-Phe-L-Dmt-D-Arg-L-Lys-NH.sub.2
H-L-Phe-L-Dmt-L-Lys-D-Arg-NH.sub.2
H-L-Phe-L-Lys-D-Arg-L-Dmt-NH.sub.2
H-L-Phe-L-Lys-L-Dmt-D-Arg-NH.sub.2 H-Lys-D-Arg-Dmt-Phe-NH.sub.2
H-Lys-D-Arg-Phe-Dmt-NH.sub.2 H-Lys-Dmt-D-Arg-Phe-NH.sub.2
H-Lys-Dmt-Phe-D-Arg-NH.sub.2 H-Lys-D-Phe-Arg-Dmt-NH.sub.2
H-Lys-Phe-D-Arg-Dmt-NH.sub.2 H-Lys-Phe-Dmt-D-Arg-NH.sub.2
H-N2-acetyl-D-arginine-L-Dmt-L-Lys-L-Phe-NH.sub.2
H-N7-acetyl-D-arginine-Dmt-Lys-Phe-NH.sub.2
H-Phe(d5)-D-Arg-Phe(d5)-Lys-NH.sub.2 H-Phe-Arg-Phe-Lys-NH.sub.2
H-Phe-D-Arg-Dmt-Lys-NH.sub.2 H-Phe-D-Arg-Dmt-Lys-NH.sub.2
H-Phe-D-Arg-D-Phe-Lys-NH.sub.2 H-Phe-D-Arg-Lys-Dmt-NH.sub.2
H-Phe-D-Arg-Phe-D-Lys-NH.sub.2
H-Phe-D-Arg-Phe-Lys-Glu-Cys-Gly-NH.sub.2
H-Phe-D-Dmt-Arg-Lys-NH.sub.2 H-Phe-Dmt-D-Arg-Lys-NH.sub.2
H-Phe-Dmt-Lys-D-Arg-NH.sub.2 H-Phe-Lys-D-Arg-Dmt-NH.sub.2
H-Phe-Lys-Dmt-D-Arg-NH.sub.2 L-Arg-D-Dmt-D-Lys-D-Phe-NH.sub.2
L-Arg-D-Dmt-D-Lys-D-Phe-NH.sub.2 L-Arg-D-Dmt-D-Lys-L-Phe-NH.sub.2
L-Arg-D-Dmt-D-Lys-L-Phe-NH.sub.2 L-Arg-D-Dmt-L-Lys-D-Phe-NH.sub.2
L-Arg-D-Dmt-L-Lys-D-Phe-NH.sub.2 L-Arg-D-Dmt-L-Lys-L-Phe-NH.sub.2
L-Arg-D-Dmt-L-Lys-L-Phe-NH.sub.2 L-Arg-L-Dmt-D-Lys-D-Phe-NH.sub.2
L-Arg-L-Dmt-D-Lys-D-Phe-NH.sub.2 L-Arg-L-Dmt-D-Lys-L-Phe-NH.sub.2
L-Arg-L-Dmt-D-Lys-L-Phe-NH.sub.2 L-Arg-L-Dmt-L-Lys-D-Phe-NH.sub.2
L-Arg-L-Dmt-L-Lys-D-Phe-NH.sub.2 L-Arg-L-Dmt-L-Lys-L-Phe-NH.sub.2
L-Arg-L-Dmt-L-Lys-L-Phe-NH.sub.2 Lys-Dmt-Arf Lys-Dmt-D-Arg-NH.sub.2
Lys-Phe Lys-Phe-Arg-Dmt Lys-Phe-NH.sub.2 Lys-Trp-Arg
Lys-Trp-D-Arg-NH.sub.2 Phe-Arg-Dmt-Lys Phe-Arg-Phe-Lys
Phe-Arg-Phe-Lys Phe-Arg-Phe-Lys Phe-Arg-Phe-Lys Phe-Arg-Phe-Lys
Phe-Arg-Phe-Lys-Glu-Cys-Gly Phe-Dmt-Arg-Lys Phe-Lys-Dmt
Phe-Lys-Dmt-NH.sub.2 Succinic monoester
CoQ0-Phe-D-Arg-Phe-Lys-NH.sub.2
wherein Cha is cyclohexylalanine
[0150] In one embodiment, the peptides have mu-opioid receptor
agonist activity (i.e., they activate the mu-opioid receptor).
Mu-opioid activity can be assessed by radioligand binding to cloned
mu-opioid receptors or by bioassays using the guinea pig ileum
(Schiller et al., Eur J Med Chem, 35:895-901, 2000; Zhao et al., J
Pharmacol Exp Ther, 307:947-954, 2003). Activation of the mu-opioid
receptor typically elicits an analgesic effect. In certain
instances, an aromatic-cationic peptide having mu-opioid receptor
agonist activity is preferred. For example, during short-term
treatment, such as in an acute disease or condition, it may be
beneficial to use an aromatic-cationic peptide that activates the
mu-opioid receptor. Such acute diseases and conditions are often
associated with moderate or severe pain. In these instances, the
analgesic effect of the aromatic-cationic peptide may be beneficial
in the treatment regimen of the human patient or other mammal. An
aromatic-cationic peptide which does not activate the mu-opioid
receptor, however, may also be used with or without an analgesic,
according to clinical requirements. Peptides which have mu-opioid
receptor agonist activity are typically those peptides which have a
tyrosine residue or a tyrosine derivative at the N-terminus (i.e.,
the first amino acid position).
[0151] Alternatively, in other instances, an aromatic-cationic
peptide that does not have mu-opioid receptor agonist activity is
preferred. For example, during long-term treatment, such as in a
chronic disease state or condition, the use of an aromatic-cationic
peptide that activates the mu-opioid receptor may be
contraindicated. In these instances, the potentially adverse or
addictive effects of the aromatic-cationic peptide may preclude the
use of an aromatic-cationic peptide that activates the mu-opioid
receptor in the treatment regimen of a human patient or other
mammal. Potential adverse effects may include sedation,
constipation and respiratory depression. In such instances an
aromatic-cationic peptide that does not activate the mu-opioid
receptor may be an appropriate treatment. Peptides that do not have
mu-opioid receptor agonist activity generally do not have a
tyrosine residue or a derivative of tyrosine at the N-terminus
(i.e., amino acid position 1). The amino acid at the N-terminus can
be any naturally occurring or non-naturally occurring amino acid
other than tyrosine. In one embodiment, the amino acid at the
N-terminus is phenylalanine or its derivative. Exemplary
derivatives of phenylalanine include 2'-methylphenylalanine (Mmp),
2',6'-dimethylphenylalanine (2',6'-Dmp),
N,2',6'-trimethylphenylalanine (Tmp), and
2'-hydroxy-6'-methylphenylalanine (Hmp).
[0152] The peptides mentioned herein and their derivatives can
further include functional variants. A peptide is considered a
functional variant if the variant has the same function as the
stated peptide. The analog may, for example, be a substitution
variant of a peptide, wherein one or more amino acids are
substituted by another amino acid. Suitable substitution variants
of the peptides include conservative amino acid substitutions.
Amino acids may be grouped according to their physicochemical
characteristics as follows:
[0153] (a) Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P)
Gly(G) Cys (C);
[0154] (b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q);
[0155] (c) Basic amino acids: His(H) Arg(R) Lys(K);
[0156] (d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V);
and
[0157] (e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His (H).
[0158] 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.
[0159] In some embodiments, the peptides disclosed herein are
derived from precursors, such as peptide precursors. For example,
in some embodiments, the precursor comprises an aromatic-cationic
which is also a therapeutic agent or drug.
Synthesis of Aromatic-Cationic Peptides
[0160] The aromatic-cationic peptides disclosed herein may be
synthesized by any of the methods well known in the art. Suitable
methods for chemically synthesizing the protein include, for
example, liquid phase and solid phase synthesis, and those methods
described by Stuart and Young in Solid Phase Peptide Synthesis,
Second Edition, Pierce Chemical Company (1984), and in Methods
Enzymol., 289, Academic Press, Inc, New York (1997). Recombinant
peptides may be generated using conventional techniques in
molecular biology, protein biochemistry, cell biology, and
microbiology, such as those described in Current Protocols in
Molecular Biology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et
al., Molecular Cloning: A Laboratory Manual, Second Ed. (Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989);
DNA Cloning: A Practical Approach, Vols. I and II, Glover, Ed.
(1985); Oligonucleotide Synthesis, Gait, Ed. (1984); Nucleic Acid
Hybridization, Hames & Higgins, Eds. (1985); Transcription and
Translation, Hames & Higgins, Eds. (1984); Animal Cell Culture,
Freshney, Ed. (1986); Immobilized Cells and Enzymes (IRL Press,
1986); Perbal, A Practical Guide to Molecular Cloning; the series,
Meth. Enzymol., (Academic Press, Inc., 1984); Gene Transfer Vectors
for Mammalian Cells, Miller & Calos, Eds. (Cold Spring Harbor
Laboratory, NY, 1987); and Meth. Enzymol., Vols. 154 and 155, Wu
& Grossman, and Wu, Eds., respectively.
Detection and Characterization of Aromatic-Cationic Peptides
[0161] The aromatic-cationic peptides described herein may be
detected and/or characterized using methods known in the art.
Peptides in a sample may be detected, for example, using methods of
high performance liquid chromatography (HPLC) such as those
described in Aguilar, HPLC of Peptides and Proteins: Methods and
Protocols, Humana Press, New Jersey (2004). Peptides may be
detected, for example, using reverse-phase HPLC (RP-HPLC) or ion
exchange HPLC. High-performance liquid chromatography (or
high-pressure liquid chromatography, HPLC) is a chromatographic
technique that can separate a mixture of compounds and is used in
biochemistry and analytical chemistry to identify, quantify and
purify the individual components of the mixture. HPLC typically
utilizes different types of stationary phases, a pump that moves
the mobile phase(s) and analyte through the column, and a detector
to provide a characteristic retention time for the analyte. The
detector may also provide additional information related to the
analyte, (e.g., UV/Vis spectroscopic data for analyte if so
equipped). Analyte retention time varies depending on the strength
of its interactions with the stationary phase, the
ratio/composition of solvent(s) used, and the flow rate of the
mobile phase. Typically, with HPLC, a pump (rather than gravity)
provides the higher pressure required to move the mobile phase and
analyte through a relatively densely packed column. The increased
density arises from smaller particle sizes. This allows for a
better separation on columns of shorter length when compared to
ordinary column chromatography.
[0162] In some embodiments, peptides are detected and/or
characterized using reverse phase HPLC (RP-HPLC). Reversed phase
HPLC (RP-HPLC or RPC) typically includes a non-polar stationary
phase and an aqueous, moderately polar mobile phase. One common
stationary phase is a silica which has been treated with RMe2SiCl,
where R is a straight chain alkyl group such as C.sub.18H.sub.37 or
C.sub.8H.sub.17. With these stationary phases, retention time is
longer for molecules which are less polar, while polar molecules
elute more readily.
[0163] In some embodiments, peptides are detected and/or
characterized using ion exchange HPLC. Typically, in ion-exchange
chromatography, retention is based on the attraction between solute
ions and charged sites bound to the stationary phase. Ions of the
same charge are excluded. Typical types of ion exchangers include
but are not limited to the following. Polystyrene resins. These
resins allow cross linkage which increases the stability of the
chain. In general, higher cross linkage reduces swerving, which
increases the equilibration time and ultimately improves
selectivity. Cellulose and dextran ion exchangers (gels): These
possess larger pore sizes and low charge densities making them
suitable for protein separation. Controlled-pore glass or porous
silica.
[0164] In general, ion exchangers favor the binding of ions of
higher charge and smaller radius. Typically, an increase in counter
ion (with respect to the functional groups in resins) concentration
reduces the retention time. Typically, an increase in pH reduces
the retention time in cation exchange while a decrease in pH
reduces the retention time in anion exchange.
[0165] In addition, peptides in a sample may be characterized, for
example, using methods of mass spectrometry (MS). A general
reference related to methods of mass spectrometry is Sparkman, Mass
Spectrometry Desk Reference, Pittsburgh: Global View Pub
(2000).
[0166] One of skill in the art will understand that the
aromatic-cationic peptides described herein may be detected and/or
characterized using any number of conventional biochemical methods
known in the art. The HPLC and MS methods described herein are
illustrative and are not to be construed as limiting in any
way.
Prophylactic and Therapeutic Uses of Aromatic-Cationic Peptides
[0167] The aromatic-cationic peptides described herein are useful
to prevent or treat disease. Specifically, the disclosure provides
for both prophylactic and therapeutic methods of treating a subject
at risk of (or susceptible to) disease by administering the
aromatic-cationic peptides described herein. Accordingly, the
present methods provide for the prevention and/or treatment of
disease in a subject by administering an effective amount of an
aromatic-cationic peptide to a subject in need thereof.
[0168] In one embodiment, the peptides described above are useful
in treating any disease or condition that is associated with
mitochondrial permeability transition (MPT). Reducing the number of
mitochondria undergoing, and preventing, MPT is important, since
MPT is associated with several common diseases and conditions in
mammals. Such diseases and conditions include, but are not limited
to, ischemia and/or reperfusion of a tissue or organ, hypoxia,
neurodegenerative diseases, etc. Mammals in need of treatment or
prevention of MPT are those mammals suffering from these diseases
or conditions.
[0169] Ischemia in a tissue or organ of a mammal is a multifaceted
pathological condition which is caused by oxygen deprivation
(hypoxia) and/or glucose (e.g., substrate) deprivation. Oxygen
and/or glucose deprivation in cells of a tissue or organ leads to a
reduction or total loss of energy generating capacity and
consequent loss of function of active ion transport across the cell
membranes. Oxygen and/or glucose deprivation also leads to
pathological changes in other cell membranes, including
permeability transition in the mitochondrial membranes. In addition
other molecules, such as apoptotic proteins normally
compartmentalized within the mitochondria, may leak out into the
cytoplasm and cause apoptotic cell death. Profound ischemia can
lead to necrotic cell death. Ischemia or hypoxia in a particular
tissue or organ may be caused by a loss or severe reduction in
blood supply to the tissue or organ. The loss or severe reduction
in blood supply may, for example, be due to thromboembolic stroke,
coronary atherosclerosis, or peripheral vascular disease. The
tissue affected by ischemia or hypoxia is typically muscle, such as
cardiac, skeletal, or smooth muscle. The organ affected by ischemia
or hypoxia may be any organ that is subject to ischemia or hypoxia.
Examples of organs affected by ischemia or hypoxia include brain,
heart, kidney, and prostate. For instance, cardiac muscle ischemia
or hypoxia is commonly caused by atherosclerotic or thrombotic
blockages which lead to the reduction or loss of oxygen delivery to
the cardiac tissues by the cardiac arterial and capillary blood
supply. Such cardiac ischemia or hypoxia may cause pain and
necrosis of the affected cardiac muscle, and ultimately may lead to
cardiac failure. Ischemia or hypoxia in skeletal muscle or smooth
muscle may arise from similar causes. For example, ischemia or
hypoxia in intestinal smooth muscle or skeletal muscle of the limbs
may also be caused by atherosclerotic or thrombotic blockages.
[0170] Reperfusion is the restoration of blood flow to any organ or
tissue in which the flow of blood is decreased or blocked. For
example, blood flow can be restored to any organ or tissue affected
by ischemia or hypoxia. The restoration of blood flow (reperfusion)
can occur by any method known to those in the art. For instance,
reperfusion of ischemic cardiac tissues may arise from angioplasty,
coronary artery bypass graft, or the use of thrombolytic drugs.
[0171] The methods described herein can also be used in the
treatment or prophylaxis of neurodegenerative diseases associated
with MPT. Neurodegenerative diseases associated with MPT include,
for instance, Parkinson's disease, Alzheimer's disease,
Huntington's disease and Amyotrophic Lateral Sclerosis (ALS, also
known as Lou Gherig's disease). The methods disclosed herein can be
used to delay the onset or slow the progression of these and other
neurodegenerative diseases associated with MPT. The methods
disclosed herein are particularly useful in the treatment of humans
suffering from the early stages of neurodegenerative diseases
associated with MPT and in humans predisposed to these
diseases.
[0172] The aromatic-cationic peptides described above are also
useful in preventing or treating insulin resistance, metabolic
syndrome, burn injuries and secondary complications, heart failure,
diabetic complications (such as diabetic retinopathy), ophthalmic
conditions (such as choroidal neovascularization, retinal
degeneration, and oxygen-induced retinopathy).
[0173] The aromatic-cationic peptides described above are also
useful in reducing oxidative damage in a mammal in need thereof.
Mammals in need of reducing oxidative damage are those mammals
suffering from a disease, condition or treatment associated with
oxidative damage. Typically, the oxidative damage is caused by free
radicals, such as reactive oxygen species (ROS) and/or reactive
nitrogen species (RNS). Examples of ROS and RNS include hydroxyl
radical (HO.), superoxide anion radical (O.sub.2.sup.-), nitric
oxide (NO.), hydrogen peroxide (H.sub.2O.sub.2), hypochlorous acid
(HOCl) and peroxynitrite anion (ONOO.sup.-). In one embodiment, a
mammal in need thereof may be a mammal undergoing a treatment
associated with oxidative damage. For example, the mammal may be
undergoing reperfusion, ischemia, or hypoxia.
[0174] In another embodiment, the aromatic-cationic peptides can be
used to prevent lipid peroxidation and/or inflammatory processes
that are associated with oxidative damage for a disease or
condition. Lipid peroxidation refers to oxidative modification of
lipids. The lipids can be present in the membrane of a cell. This
modification of membrane lipids typically results in change and/or
damage to the membrane function of a cell. In addition, lipid
peroxidation can also occur in lipids or lipoproteins exogenous of
a cell. For example, low-density lipoproteins are susceptible to
lipid peroxidation. An example of a condition associated with lipid
peroxidation is atherosclerosis. Reducing oxidative damage
associated with atherosclerosis is important since atherosclerosis
is implicated in, for example, heart attacks and coronary artery
disease.
[0175] Inflammatory processes include and activation of the immune
system. Typically, the immune system is activated by an antigenic
substance. The antigenic substance can be any substance recognized
by the immune system, and include self-derived particles and
foreign-derived particles. Examples of diseases or conditions
occurring from an inflammatory process to self-derived particles
include arthritis and multiple sclerosis. Examples of foreign
particles include viruses and bacteria. The virus can be any virus
which activates an inflammatory process, and associated with
oxidative damage. Examples of viruses include, hepatitis A, B or C
virus, human immunodeficiency virus, influenza virus, and bovine
diarrhea virus. For example, hepatitis virus can elicit an
inflammatory process and formation of free radicals, thereby
damaging the liver. The bacteria can be any bacteria, and include
gram-negative or gram-positive bacteria. Gram-negative bacteria
contain lipopolysaccharide in the bacteria wall. Examples of
gram-negative bacteria include Escherichia coli, Klebsiella
pneumoniae, Proteus species, Pseudomonas aeruginosa, Serratia, and
Bacteroides. Examples of gram-positive bacteria include pneumococci
and streptococci. An example of an inflammatory process associated
with oxidative stress caused by a bacteria is sepsis. Typically,
sepsis occurs when gram-negative bacteria enter the
bloodstream.
[0176] Liver damage caused by a toxic agent is another condition
associated with an inflammatory process and oxidative stress. The
toxic agent can be any agent which causes damage to the liver. For
example, the toxic agent can cause apoptosis and/or necrosis of
liver cells. Examples of such agents include alcohol, and
medication, such as prescription and non-prescription drugs taken
to treat a disease or condition.
[0177] The methods disclosed herein can also be used in reducing
oxidative damage associated with any neurodegenerative disease or
condition. The neurodegenerative disease can affect any cell,
tissue or organ of the central and peripheral nervous system.
Examples of such cells, tissues and organs include, the brain,
spinal cord, neurons, ganglia, Schwann cells, astrocytes,
oligodendrocytes and microglia. The neurodegenerative condition can
be an acute condition, such as a stroke or a traumatic brain or
spinal cord injury. In another embodiment, the neurodegenerative
disease or condition can be a chronic neurodegenerative condition.
In a chronic neurodegenerative condition, the free radicals can,
for example, cause damage to a protein. An example of such a
protein is amyloid .beta.-protein. Examples of chronic
neurodegenerative diseases associated with damage by free radicals
include Parkinson's disease, Alzheimer's disease, Huntington's
disease and Amyotrophic Lateral Sclerosis (also known as Lou
Gherig's disease).
[0178] Determination of the Biological Effect of the
Aromatic-Cationic Peptide-Based Therapeutic.
[0179] In various embodiments, suitable in vitro or in vivo assays
are performed to determine the effect of a specific
aromatic-cationic peptide-based therapeutic and whether its
administration is indicated for treatment. In various embodiments,
in vitro assays can be performed with representative animal models,
to determine if a given aromatic-cationic peptide-based therapeutic
exerts the desired effect in preventing or treating a disease or
medical condition. Compounds for use in therapy can be tested in
suitable animal model systems including, but not limited to rats,
mice, chicken, pigs, cows, monkeys, rabbits, and the like, prior to
testing in human subjects. Similarly, for in vivo testing, any of
the animal model systems known in the art can be used prior to
administration to human subjects.
[0180] Prophylactic Methods.
[0181] In one aspect, the invention provides a method for
preventing, in a subject, disease by administering to the subject
an aromatic-cationic peptide that prevents the initiation or
progression of the condition. In prophylactic applications,
pharmaceutical compositions or medicaments of aromatic-cationic
peptides are administered to a subject susceptible to, or otherwise
at risk of a disease or condition in an amount sufficient to
eliminate or reduce the risk, lessen the severity, or delay the
outset of the disease, including biochemical, histologic and/or
behavioral symptoms of the disease, its complications and
intermediate pathological phenotypes presenting during development
of the disease. Administration of a prophylactic aromatic-cationic
can occur prior to the manifestation of symptoms characteristic of
the aberrancy, such that a disease or disorder is prevented or,
alternatively, delayed in its progression. The appropriate compound
can be determined based on screening assays described above.
[0182] Therapeutic Methods.
[0183] Another aspect of the technology includes methods of
treating disease in a subject for therapeutic purposes. In
therapeutic applications, compositions or medicaments are
administered to a subject suspected of, or already suffering from
such a disease in an amount sufficient to cure, or at least
partially arrest, the symptoms of the disease, including its
complications and intermediate pathological phenotypes in
development of the disease. As such, the invention provides methods
of treating an individual afflicted with a disease or medical
condition.
Modes of Administration and Effective Dosages
[0184] Any method known to those in the art for contacting a cell,
organ or tissue with a peptide may be employed. Suitable methods
include in vitro, ex vivo, or in vivo methods. In vivo methods
typically include the administration of an aromatic-cationic
peptide, such as those described above, to a mammal, suitably a
human. When used in vivo for therapy, the aromatic-cationic
peptides are administered to the subject in effective amounts
(i.e., amounts that have desired therapeutic effect). The dose and
dosage regimen will depend upon the degree of the injury in the
subject, the characteristics of the particular aromatic-cationic
peptide used, e.g., its therapeutic index, the subject, and the
subject's history.
[0185] The effective amount may be determined during pre-clinical
trials and clinical trials by methods familiar to physicians and
clinicians. An effective amount of a peptide useful in the methods
may be administered to a mammal in need thereof by any of a number
of well-known methods for administering pharmaceutical compounds.
The peptide may be administered systemically or locally.
[0186] The peptide may be formulated as a pharmaceutically
acceptable salt. The term "pharmaceutically acceptable salt" means
a salt prepared from a base or an acid which is acceptable for
administration to a patient, such as a mammal (e.g., salts having
acceptable mammalian safety for a given dosage regime). However, it
is understood that the salts are not required to be
pharmaceutically acceptable salts, such as salts of intermediate
compounds that are not intended for administration to a patient.
Pharmaceutically acceptable salts can be derived from
pharmaceutically acceptable inorganic or organic bases and from
pharmaceutically acceptable inorganic or organic acids. In
addition, when a peptide contains both a basic moiety, such as an
amine, pyridine or imidazole, and an acidic moiety such as a
carboxylic acid or tetrazole, zwitterions may be formed and are
included within the term "salt" as used herein. Salts derived from
pharmaceutically acceptable inorganic bases include ammonium,
calcium, copper, ferric, ferrous, lithium, magnesium, manganic,
manganous, potassium, sodium, and zinc salts, and the like. Salts
derived from pharmaceutically acceptable organic bases include
salts of primary, secondary and tertiary amines, including
substituted amines, cyclic amines, naturally-occurring amines and
the like, such as arginine, betaine, caffeine, choline,
N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,
2-dimethylaminoethanol, ethanolamine, ethylenediamine,
N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine,
histidine, hydrabamine, isopropylamine, lysine, methylglucamine,
morpholine, piperazine, piperadine, polyamine resins, procaine,
purines, theobromine, triethylamine, trimethylamine,
tripropylamine, tromethamine and the like. Salts derived from
pharmaceutically acceptable inorganic acids include salts of boric,
carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric or
hydroiodic), nitric, phosphoric, sulfamic and sulfuric acids. Salts
derived from pharmaceutically acceptable organic acids include
salts of aliphatic hydroxyl acids (e.g., citric, gluconic,
glycolic, lactic, lactobionic, malic, and tartaric acids),
aliphatic monocarboxylic acids (e.g., acetic, butyric, formic,
propionic and trifluoroacetic acids), amino acids (e.g., aspartic
and glutamic acids), aromatic carboxylic acids (e.g., benzoic,
p-chlorobenzoic, diphenylacetic, gentisic, hippuric, and
triphenylacetic acids), aromatic hydroxyl acids (e.g.,
o-hydroxybenzoic, p-hydroxybenzoic,
1-hydroxynaphthalene-2-carboxylic and
3-hydroxynaphthalene-2-carboxylic acids), ascorbic, dicarboxylic
acids (e.g., fumaric, maleic, oxalic and succinic acids),
glucoronic, mandelic, mucic, nicotinic, orotic, pamoic,
pantothenic, sulfonic acids (e.g., benzenesulfonic, camphosulfonic,
edisylic, ethanesulfonic, isethionic, methanesulfonic,
naphthalenesulfonic, naphthalene-1,5-disulfonic,
naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic
acid, and the like. In some embodiments, the salt comprises
trifluoroacetate salt or acetate salt.
[0187] The aromatic-cationic peptides described herein or a
pharmaceutically salt thereof such as acetate salt or
trifluoroacetate salt, can be incorporated into pharmaceutical
compositions for administration, singly or in combination, to a
subject for the treatment or prevention of a disease or medical
condition described herein. Such compositions typically include the
active agent and a pharmaceutically acceptable carrier. As used
herein the term "pharmaceutically acceptable carrier" includes
saline, solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. Supplementary
active compounds can also be incorporated into the
compositions.
[0188] Pharmaceutical compositions are typically formulated to be
compatible with its intended route of administration. Examples of
routes of administration include parenteral (e.g., intravenous,
intradermal, intraperitoneal or subcutaneous), oral, inhalation,
transdermal (topical), intraocular, iontophoretic, and transmucosal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. pH can be adjusted with acids or
bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic. For
convenience of the patient or treating physician, the dosing
formulation can be provided in a kit containing all necessary
equipment (e.g., vials of drug, vials of diluent, syringes and
needles) for a treatment course (e.g., 7 days of treatment).
[0189] Pharmaceutical compositions suitable for injectable use can
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, a composition for
parenteral administration must be sterile and should be fluid to
the extent that easy syringability exists. It should be stable
under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms such
as bacteria and fungi.
[0190] The aromatic-cationic peptide compositions or a
pharmaceutically salt thereof such as acetate salt or
trifluoroacetate salt, can include a carrier, which can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol, and
liquid polyethylene glycol, and the like), and suitable mixtures
thereof. The proper fluidity can be maintained, for example, by the
use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. Prevention of the action of microorganisms can be
achieved by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, ascorbic acid,
thiomerasol, and the like. Glutathione and other antioxidants can
be included to prevent oxidation. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate or
gelatin.
[0191] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle, which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, typical methods of preparation
include vacuum drying and freeze drying, which can yield a powder
of the active ingredient plus any additional desired ingredient
from a previously sterile-filtered solution thereof.
[0192] Oral compositions generally include an inert diluent or an
edible carrier. For the purpose of oral therapeutic administration,
the active compound can be incorporated with excipients and used in
the form of tablets, troches, or capsules, e.g., gelatin capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash. Pharmaceutically compatible binding agents,
and/or adjuvant materials can be included as part of the
composition. The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth
or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
[0193] For administration by inhalation, the compounds can be
delivered in the form of an aerosol spray from a pressurized
container or dispenser which contains a suitable propellant, e.g.,
a gas such as carbon dioxide, or a nebulizer. Such methods include
those described in U.S. Pat. No. 6,468,798.
[0194] Systemic administration of a therapeutic compound as
described herein can also be by transmucosal or transdermal means.
For transmucosal or transdermal administration, penetrants
appropriate to the barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art, and
include, for example, for transmucosal administration, detergents,
bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays.
For transdermal administration, the active compounds are formulated
into ointments, salves, gels, or creams as generally known in the
art. In one embodiment, transdermal administration may be performed
my iontophoresis.
[0195] A therapeutic peptide or a pharmaceutically salt thereof
such as acetate salt or trifluoroacetate salt, can be formulated in
a carrier system. The carrier can be a colloidal system. The
colloidal system can be a liposome, a phospholipid bilayer vehicle.
In one embodiment, the therapeutic peptide is encapsulated in a
liposome while maintaining peptide integrity. As one skilled in the
art would appreciate, there are a variety of methods to prepare
liposomes. (See Lichtenberg et al., Methods Biochem. Anal.,
33:337-462 (1988); Anselem et al., Liposome Technology, CRC Press
(1993)). Liposomal formulations can delay clearance and increase
cellular uptake (See Reddy, Ann. Pharmacother., 34(7-8):915-923
(2000)). An active agent can also be loaded into a particle
prepared from pharmaceutically acceptable ingredients including,
but not limited to, soluble, insoluble, permeable, impermeable,
biodegradable or gastroretentive polymers or liposomes. Such
particles include, but are not limited to, nanoparticles,
biodegradable nanoparticles, microparticles, biodegradable
microparticles, nanospheres, biodegradable nanospheres,
microspheres, biodegradable microspheres, capsules, emulsions,
liposomes, micelles and viral vector systems.
[0196] The carrier can also be a polymer, e.g., a biodegradable,
biocompatible polymer matrix. In one embodiment, the therapeutic
peptide can be embedded in the polymer matrix, while maintaining
protein integrity. The polymer may be natural, such as
polypeptides, proteins or polysaccharides, or synthetic, such as
poly .alpha.-hydroxy acids. Examples include carriers made of,
e.g., collagen, fibronectin, elastin, cellulose acetate, cellulose
nitrate, polysaccharide, fibrin, gelatin, and combinations thereof.
In one embodiment, the polymer is poly-lactic acid (PLA) or copoly
lactic/glycolic acid (PGLA). The polymeric matrices can be prepared
and isolated in a variety of forms and sizes, including
microspheres and nanospheres. Polymer formulations can lead to
prolonged duration of therapeutic effect. (See Reddy, Ann.
Pharmacother., 34(7-8):915-923 (2000)). A polymer formulation for
human growth hormone (hGH) has been used in clinical trials. (See
Kozarich and Rich, Chemical Biology, 2:548-552 (1998)).
[0197] Examples of polymer microsphere sustained release
formulations are described in PCT publication WO 99/15154 (Tracy et
al.), U.S. Pat. Nos. 5,674,534 and 5,716,644 (both to Zale et al.),
PCT publication WO 96/40073 (Zale et al.), and PCT publication WO
00/38651 (Shah et al.). U.S. Pat. Nos. 5,674,534 and 5,716,644 and
PCT publication WO 96/40073 describe a polymeric matrix containing
particles of erythropoietin that are stabilized against aggregation
with a salt.
[0198] In some embodiments, the therapeutic compounds are prepared
with carriers that will protect the therapeutic compounds against
rapid elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylacetic acid. Such formulations
can be prepared using known techniques. The materials can also be
obtained commercially, e.g., from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to specific cells with monoclonal antibodies to
cell-specific antigens) can also be used as pharmaceutically
acceptable carriers. These can be prepared according to methods
known to those skilled in the art, for example, as described in
U.S. Pat. No. 4,522,811.
[0199] The therapeutic compounds can also be formulated to enhance
intracellular delivery. For example, liposomal delivery systems are
known in the art, see, e.g., Chonn and Cullis, "Recent Advances in
Liposome Drug Delivery Systems," Current Opinion in Biotechnology
6:698-708 (1995); Weiner, "Liposomes for Protein Delivery:
Selecting Manufacture and Development Processes," Immunomethods,
4(3):201-9 (1994); and Gregoriadis, "Engineering Liposomes for Drug
Delivery: Progress and Problems," Trends Biotechnol., 13(12):527-37
(1995). Mizguchi et al., Cancer Lett., 100:63-69 (1996), describes
the use of fusogenic liposomes to deliver a protein to cells both
in vivo and in vitro.
[0200] Dosage, toxicity and therapeutic efficacy of the therapeutic
agents can be determined by standard pharmaceutical procedures in
cell cultures or experimental animals, e.g., for determining the
LD50 (the dose lethal to 50% of the population) and the ED50 (the
dose therapeutically effective in 50% of the population). The dose
ratio between toxic and therapeutic effects is the therapeutic
index and it can be expressed as the ratio LD50/ED50. Compounds
which exhibit high therapeutic indices are preferred. While
compounds that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0201] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the methods, the therapeutically effective
dose can be estimated initially from cell culture assays. A dose
can be formulated in animal models to achieve a circulating plasma
concentration range that includes the IC50 (i.e., the concentration
of the test compound which achieves a half-maximal inhibition of
symptoms) as determined in cell culture. Such information can be
used to more accurately determine useful doses in humans. Levels in
plasma may be measured, for example, by high performance liquid
chromatography.
[0202] Dosage may also be determined empirically by detecting
aromatic-cationic peptides in a biological sample from a subject.
Biological samples obtained from a subject who has been
administered aromatic-cationic peptides may be subjected to HPLC
and/or MS to detect and characterize the aromatic-cationic peptides
present in the subject's bodily fluids and tissues. Biological
samples include any material derived from or contacted by living
cells. Examples of biological samples include but are not limited
to whole blood, fractionated blood, semen, saliva, tears, urine,
fecal material, sweat, buccal, skin, cerebrospinal fluid, and hair.
Biological samples also include biopsies of internal organs, organs
removed for transplant or cancers. The presence of
aromatic-cationic peptides in the biological sample is established
by comparison to data obtained for reference samples such as those
provided in Example 6. The levels of aromatic-cationic peptides
present in the sample may serve as a basis to increase or decrease
the dosage of an aromatic-cationic peptide or a precursor thereof,
administered to a given subject, wherein the precursor may be an
aromatic-cationic which is also a therapeutic agent or drug.
[0203] Typically, an effective amount of the aromatic-cationic
peptides, sufficient for achieving a therapeutic or prophylactic
effect, range from about 0.000001 mg per kilogram body weight per
day to about 10,000 mg per kilogram body weight per day.
Preferably, the dosage ranges are from about 0.0001 mg per kilogram
body weight per day to about 100 mg per kilogram body weight per
day. For example dosages can be 1 mg/kg body weight or 10 mg/kg
body weight every day, every two days or every three days or within
the range of 1-10 mg/kg every week, every two weeks or every three
weeks. In one embodiment, a single dosage of peptide ranges from
0.1-10,000 micrograms per kg body weight. In one embodiment,
aromatic-cationic peptide concentrations in a carrier range from
0.2 to 2000 micrograms per delivered milliliter. An exemplary
treatment regime entails administration once per day or once a
week. In therapeutic applications, a relatively high dosage at
relatively short intervals is sometimes required until progression
of the disease is reduced or terminated, and preferably until the
subject shows partial or complete amelioration of symptoms of
disease. Thereafter, the patient can be administered a prophylactic
regime.
[0204] In some embodiments, a therapeutically effective amount of
an aromatic-cationic peptide may be defined as a concentration of
peptide at the target tissue of 10.sup.-11 to 10.sup.-6 molar,
e.g., approximately 10.sup.-7 molar. This concentration may be
delivered by systemic doses of 0.01 to 100 mg/kg or equivalent dose
by body surface area. The schedule of doses would be optimized to
maintain the therapeutic concentration at the target tissue, most
preferably by single daily or weekly administration, but also
including continuous administration (e.g., parenteral infusion or
transdermal application).
[0205] In some embodiments, the dosage of the aromatic-cationic
peptide is provided at a "low," "mid," or "high" dose level. In one
embodiment, the low dose is provided from about 0.01 to about 0.5
mg/kg/h, suitably from about 0.01 to about 0.1 mg/kg/h. In one
embodiment, the mid-dose is provided from about 0.1 to about 1.0
mg/kg/h, suitably from about 0.1 to about 0.5 mg/kg/h. In one
embodiment, the high dose is provided from about 0.5 to about 10
mg/kg/h, suitably from about 0.5 to about 2 mg/kg/h.
[0206] The skilled artisan will appreciate that certain factors may
influence the dosage and timing required to effectively treat a
subject, including but not limited to, the severity of the disease
or disorder, previous treatments, the general health and/or age of
the subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of the therapeutic
compositions described herein can include a single treatment or a
series of treatments.
[0207] In some embodiments, multiple doses, or varying amounts of
an aromatic-cationic peptide are administered to a subject. In some
embodiments, the multiple doses or varying amounts of the peptide
are administered throughout the course of a procedure (e.g., a
surgery) or are administered throughout the course of a disease or
conditions. For example, in some embodiments, the peptide is
administered intravenously, for example during a surgery, and the
amount of peptide provided to the subject is adjusted during the
procedure. In other embodiments, the subject is administered a dose
of peptide (e.g., orally or by injection, e.g., intradermal,
subcutaneous, intramuscular, intravenous, intraosseous, and
intraperitoneal) about once every 10 minutes, 15 minutes, 30
minutes, hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12
hours, once per day, once every other day, or once per week. In
some embodiments, the amount of peptide present in the subject (the
subject's peptide level) is monitored to determine the appropriate
dose and schedule needed to maintain a desired peptide level in the
subject. In some embodiments, peptide levels are determined
periodically during administration, and/or are determined at one or
more time points after administration. For example, in some
embodiments, peptide levels are determined within a few minutes of
administration, about 10 minutes, 15 minutes, 30 minutes, 1 hour, 2
hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, two
days, 3 days 5 days, 7 days or 14 days after administration. In
other embodiments, the subjects peptide levels are determined every
10 minutes, 15 minutes, 20 minutes, 30 minutes, hourly, every two
hours, every 4 hours, every 6 hours or every 12 hours, for a
predetermined time, such as during a surgical procedure. Depending
on the determined peptide level, one or more additional doses may
be provided to achieve a desired peptide level to maintain a
therapeutic effect. In some embodiments, peptide levels may be
found sufficient to delay an additional dose or doses.
[0208] In some embodiments, the detected levels of
aromatic-cationic peptide are compared to aromatic-cationic peptide
levels in a healthy control subject (e.g., a subject who has been
administered substantially the same dose of the peptide via
substantially the same route of administration). Additionally or
alternatively, in some embodiments, the level of the peptide is
determined in different organs, systems and/or fluids of a subject.
In some embodiments, the level of the peptide is determined in
different organs, systems and/or fluids of a subject and compared
to peptide levels in comparable systems, organs, and fluids of a
control subject. In some embodiments, such an analysis provides
information regarding the availability of the peptide, and the
transport of the peptide throughout the body of the subject as
compared to the control. For example, the route of administration
may be changed for a particular subject to optimize peptide
delivery to a particular tissue or organ (e.g., to achieve a more
localized distribution of the peptide). Additionally or
alternatively, the route of administration could be changed for a
particular subject to for a more systemic peptide distribution.
[0209] Method for identifying and determining the presence or
amount (level) of an aromatic-cationic peptide in a subject sample
are known in the art and include, but are not limited to HPLC and
mass spectrometry.
[0210] As noted previously, in some embodiments, peptide levels are
determined in a biological sample from the subject, and include,
without limitation, blood samples, tissues samples, (e.g., organ or
tumor biopsy samples), urine and saliva.
[0211] Also disclosed herein are kits for the detection of
aromatic-cationic peptides. In some embodiments, the kits include a
sample collection device to collect a sample from the subject, and
a sample storage device for preservation of the biological sample.
Depending on the intended method of detection, the sample is stored
in an appropriate buffer or preservative, also provided in the kit.
In some embodiments, a sample collector includes a container for
liquid, such as a vial or tube (e.g., for blood, blood products,
urine). In other embodiments, the sample collector is an absorbent
material, such as a sterile cotton swab (e.g., to collect a buccal
sample, saliva, nasal swabs, etc), a slide, a sterile paper, a
card, a syringe, etc. The sample storage device may be any device
that will encase and protect the sample during transport, shipment
and/or storage. For example, in some embodiments, the sample
storage device is a sealable tube.
[0212] In some embodiments, the kits also include instructions for
obtaining a sample and properly storing the sample until analysis.
In some embodiments, the sample includes a bodily fluid, one or
more cells, a tissue or portion of an organ, a biopsy sample,
and/and a portion of a tumor.
[0213] The mammal treated in accordance with the present methods
can be any mammal, including, for example, farm animals, such as
sheep, pigs, cows, and horses; pet animals, such as dogs and cats;
laboratory animals, such as rats, mice and rabbits. In a suitable
embodiment, the mammal is a human.
EXAMPLES
[0214] The present invention is further illustrated by the
following examples, which should not be construed as limiting in
any way.
Example 1
Aromatic-Cationic Peptides of the Present Technology Inhibit
Inhibits H.sub.2O.sub.2 Generation by Isolated Mitochondria
[0215] In this Example, the effects of the aromatic-cationic
peptides of the invention on H.sub.2O.sub.2 generation by isolated
mitochondria are investigated. H.sub.2O.sub.2 is measured using
luminol chemiluminescence as described in Y. Li, H. Zhu, M. A.
Trush, Biochim. Biophys. Acta 1428, 1-12 (1999)). Briefly, 0.1 mg
mitochondrial protein is added to 0.5 ml potassium phosphate buffer
(100 mM, pH 8.0) in the absence or presence of an aromatic-cationic
peptide. Luminol (25 mM) and 0.7 IU horseradish peroxidase are
added, and chemilumunescence is monitored with a Chronolog Model
560 aggregometer (Havertown, Pa.) for 20 min at 37.degree. C. The
amount of H.sub.2O.sub.2 produced is quantified as the area under
the curve (AUC) over 20 min, and all data are normalized to AUC
produced by mitochondria alone.
[0216] It is predicted that the aromatic-cationic peptide will
reduce the spontaneous production of H.sub.2O.sub.2 by isolated
mitochondria. As such, the aromatic-cationic peptides are useful
for reducing oxidative damage and are useful in the treatment or
prevention of diseases or conditions that relate to oxidative
damage.
Example 2
Aromatic-Cationic Peptides of the Present Technology Reduce
Intracellular ROS and Increase Cell Survival
[0217] To show that the claimed peptides are effective antioxidants
when applied to whole cells, neuronal N.sub.2A cells are plated in
96-well plates at a density of 1.times.10.sup.4/well and allowed to
grow for 2 days before treatment with tBHP (0.5 or 1 mM) for 40
min. Cells are washed twice and replaced with medium alone or
medium containing varying concentrations of aromatic-cationic
peptides (10.sup.-12 M to 10.sup.-9 M) for 4 h. Intracellular ROS
is measured by carboxy-H.sub.2DCFDA (Molecular Probes, Portland,
Oreg.). Cell death is assessed by a cell proliferation assay (MTS
assay, Promega, Madison, Wis.).
[0218] Incubation with tBHP will result in an increase in
intracellular ROS and decrease in cell viability. However, it is
predicted that incubation of these cells with an aromatic-cationic
peptide will reduce intracellular ROS and increase cell survival.
As such, the aromatic-cationic peptides are useful for reducing
oxidative damage and are useful in the treatment or prevention of
diseases or conditions that relate to oxidative damage.
Example 3
Aromatic-Cationic Peptides of the Present Technology Protect
Against MPT Induced by Ca.sup.2+ and 3-Nitropropionic Acid
[0219] To isolate mitochondria from mouse liver, mice are
sacrificed by decapitation. The liver is removed and rapidly placed
into chilled liver homogenization medium. The liver is finely
minced using scissors and then homogenized by hand using a glass
homogenizer. The homogenate is centrifuged for 10 min at 1000 g at
4.degree. C. The supernatant is aspirated and transferred to
polycarbonate tubes and centrifuged again for 10 min at 3000 g,
4.degree. C. The resulting supernatant is removed, and the fatty
lipids on the side-wall of the tube are carefully wiped off. The
pellet is resuspended in liver homogenate medium and the
homogenization repeated twice. The final purified mitochondrial
pellet is resuspended in medium. Protein concentration in the
mitochondrial preparation is determined by the Bradford
procedure.
[0220] To investigate the localization of the aromatic-cationic
peptides of the invention, approximately 1.5 mg mitochondria in 400
.mu.l buffer is incubated with labeled aromatic-cationic peptide
for 5-30 min at 37.degree. C. The mitochondria are then centrifuged
down and the amount of label is measured in the mitochondrial
fraction and buffer fraction. Assuming a mitochondrial matrix
volume of 0.7 .mu.l/mg protein (Lim et al., J Physiol 545:961-974,
2002), the concentration of peptide in mitochondria can be
determined. It is predicted that the claimed aromatic-cationic
peptides will be more concentrated in mitochondria compared to the
buffer fraction.
[0221] To investigate the effects of the aromatic-cationic peptides
of the invention on mitochondrial membrane potential, isolated
mouse liver mitochondria are incubated with 100-200 .mu.M
aromatic-cationic peptide. Mitochondrial membrane potential is
measured using tetramethyl rhodamine methyl ester (TMRM). Addition
of mitochondria results in immediate quenching of the TMRM signal
which is readily reversed by the addition of FCCP, indicating
mitochondrial depolarization. The addition of Ca.sup.2+ (150 .mu.M)
results in immediate depolarization followed by progressive loss of
quenching, indicative of MPT. Addition of aromatic-cationic peptide
alone, even at 200 .mu.M, is not predicted to cause mitochondrial
depolarization or MPT. It is also predicted that the
aromatic-cationic peptides will not alter mitochondrial function,
including oxygen consumption during state 3 or state 4, or the
respiratory ratio (state 3/state 4).
[0222] To show that the claimed peptides are effective at
protecting against MPT induced by Ca.sup.2+ overload, isolated
mitochondria are pre-treated with aromatic-cationic peptide (10
.mu.M) for 2 min prior to addition of Ca.sup.2+. It is predicted
that the aromatic-cationic peptides of the invention will increase
the tolerance of mitochondria to cumulative Ca.sup.2+
challenges.
[0223] 3-Nitropropionic acid (3NP) is an irreversible inhibitor of
succinate dehydrogenase in complex II of the electron transport
chain. Addition of 3NP (1 mM) to isolated mitochondria causes
dissipation of mitochondrial potential and onset of MPT.
Pretreatment of mitochondria with the aromatic-cationic peptides of
the invention is predicted to delay the onset of MPT induced by
3NP.
[0224] To demonstrate that the aromatic-cationic peptides of the
invention can penetrate cell membranes and protect against
mitochondrial depolarization elicited by 3NP, Caco-2 cells are
treated with 3NP (10 mM) in the absence or presence of the
aromatic-cationic peptides for 4 h, and then incubated with TMRM
and examined under LSCM. In control cells, the mitochondria are
clearly visualized as fine streaks throughout the cytoplasm. In
cells treated with 3NP, the TMRM fluorescence is much reduced,
indicating generalized depolarization. In contrast, it is predicted
that concurrent treatment with the aromatic-cationic peptides of
the invention will protect against mitochondrial depolarization
caused by 3NP.
[0225] As such, the aromatic-cationic peptides are useful for
preventing MPT and are useful in the treatment or prevention of
diseases or conditions that relate to MPT.
Example 4
Aromatic-Cationic Peptides of the Present Technology Protect
Against Mitochondrial Swelling and Cytochrome c Release
[0226] MPT pore opening results in mitochondrial swelling. This
Example examines the effects of the aromatic-cationic peptides of
the invention on mitochondrial swelling by measuring reduction in
absorbance at 540 nm (A.sub.540). Once the absorbance is measured,
the mitochondrial suspension is then centrifuged and cytochrome c
in the mitochondrial pellet and supernatant is determined by a
commercially-available ELISA kit. It is predicted that pretreatment
of isolated mitochondria with the aromatic-cationic peptides of the
invention will inhibit swelling and cytochrome c release induced by
Ca.sup.2+ overload. Besides preventing MPT induced by Ca.sup.2+
overload, it is predicted that the aromatic-cationic peptides of
the invention will also prevent mitochondrial swelling induced by
MPP.sup.+ (1-methyl-4-phenylpyridium ion), an inhibitor of complex
I of the mitochondrial electron transport chain.
[0227] As such, the aromatic-cationic peptides are useful for
preventing MPT and are useful in the treatment or prevention of
diseases or conditions that relate to MPT.
Example 5
The Peptides of the Present Technology Increase the Rate ATP
Synthesis in Isolated Mitochondria
[0228] This example will demonstrate the impact of peptides of the
present technology on the rate of mitochondrial ATP synthesis.
[0229] The rate of mitochondrial ATP synthesis will be determined
by measuring ATP in respiration buffer collected from isolated
mitochondria 1 min after addition of 400 mM ADP. ATP will be
assayed by HPLC. All experiments will be carried out in triplicate,
with n=3. It is predicted that addition of peptides of the present
technology to isolated mitochondria will increase the rate of ATP
synthesis in a dose-dependent manner.
[0230] This result will demonstrate the peptides of the present
technology are useful in methods and compositions for increasing
the rate of mitochondrial ATP synthesis.
Example 6
Characterization of Aromatic-Cationic Peptides
[0231] Aromatic-cationic peptides of the present technology can be
synthesized using solid phase synthesis and characterized using
HPLC and MS. Exemplary HPLC and MS methods are provided in Examples
7 and 8 below.
Example 7
Detection of Aromatic-Cationic Peptides in a Biological Sample
[0232] This example demonstrates the detection of aromatic-cationic
peptides in a biological sample by HPLC. Biological samples are
collected from subjects in a suitable manner depending on the
nature of the sample. Biological samples include any material
derived from or contacted by living cells. Examples of biological
samples include but are not limited to whole blood, fractionated
blood, semen, saliva, tears, urine, fecal material, sweat, buccal,
skin, cerebrospinal fluid, and hair. Biological samples also
include biopsies of internal organs or cancers. Once obtained, the
biological samples are stored in a manner compatible with the
methods of detection until the methods are performed to ensure the
preservation of aromatic-cationic peptides present in the
sample.
[0233] Samples are loaded onto a 250.times.4.6 (i.d.) mm C18 5
.mu.m column and subjected to a gradient of 0.1% trifluoroacetic
acid in acetonitrile (Solution A) and 0.1% trifluoroacetic acid in
HPLC-grade water (Solution B) according to the following
scheme:
TABLE-US-00008 TABLE 6 HLPC Methods A B 0.01 min 7% 93% 25 min 32%
68% 25.1 min 100% 0% Flow rate 1.0 ml/min Wave Length 220 nm Load
Volume 10 .mu.l
[0234] The presence of aromatic-cationic peptides in the biological
sample is established by comparison to data obtained for reference
samples such as those provided in Example 6.
[0235] The foregoing method is illustrative only, and should not be
construed as limiting in any way. One of skill in the art will
understand that the aromatic-cationic peptides described herein may
be analyzed by a number of HPLC methods, such as those describe in
Aguilar, HPLC of Peptides and Proteins: Methods and Protocols,
Humana Press, New Jersey (2004).
Example 8
Detection of Aromatic-Cationic Peptides in a Biological Sample by
MS
[0236] This example demonstrates the detection of aromatic-cationic
peptides in a biological sample by MS. Biological samples are
collected from subjects in a suitable manner depending on the
nature of the sample. Biological samples include any material
derived from or contacted by living cells. Examples of biological
samples include but are not limited to whole blood, fractionated
blood, semen, saliva, tears, urine, fecal material, sweat, buccal,
skin, cerebrospinal fluid, and hair. Biological samples also
include biopsies of internal organs or cancers. Once obtained, the
biological samples are stored in a manner compatible with the
methods of detection until the methods are performed to ensure the
preservation of aromatic-cationic peptides present in the
sample.
[0237] Samples are loaded in a 20 .mu.l volume and analyzed under
the following exemplary conditions.
TABLE-US-00009 TABLE 7 MS Methods Probe ESI Nebulizer Gas Flow 1.5
L/min Curved Desolvation -20.0 v Line (CDL) CDL Temp 250.degree. C.
Block Temp 200.degree. C. Probe Bias +4.5 kv Detector 1.5 kv T.
Flow 0.2 ml/min Buffer 50% H.sub.2O-50% Acetonitrile
[0238] One of skill in the art will understand, that the
aromatic-cationic peptides described herein may be analyzed by a
number of MS methods, such as those describe in Sparkman, Mass
Spectrometry Desk Reference, Pittsburgh: Global View Pub
(2000).
[0239] The present invention is not to be limited in terms of the
particular embodiments described in this application, which are
intended as single illustrations of individual aspects of the
invention. Many modifications and variations of this invention can
be made without departing from its spirit and scope, as will be
apparent to those skilled in the art. Functionally equivalent
methods and apparatuses within the scope of the invention, in
addition to those enumerated herein, will be apparent to those
skilled in the art from the foregoing descriptions. Such
modifications and variations are intended to fall within the scope
of the appended claims. The present invention is to be limited only
by the terms of the appended claims, along with the full scope of
equivalents to which such claims are entitled. It is to be
understood that this invention is not limited to particular
methods, reagents, compounds compositions or biological systems,
which can, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to be limiting.
[0240] In addition, where features or aspects of the disclosure are
described in terms of Markush groups, those skilled in the art will
recognize that the disclosure is also thereby described in terms of
any individual member or subgroup of members of the Markush
group.
[0241] As will be understood by one skilled in the art, for any and
all purposes, particularly in terms of providing a written
description, all ranges disclosed herein also encompass any and all
possible subranges and combinations of subranges thereof. Any
listed range can be easily recognized as sufficiently describing
and enabling the same range being broken down into at least equal
halves, thirds, quarters, fifths, tenths, etc. As a non-limiting
example, each range discussed herein can be readily broken down
into a lower third, middle third and upper third, etc. As will also
be understood by one skilled in the art all language such as "up
to," "at least," "greater than," "less than," and the like, include
the number recited and refer to ranges which can be subsequently
broken down into subranges as discussed above. Finally, as will be
understood by one skilled in the art, a range includes each
individual member. Thus, for example, a group having 1-3 cells
refers to groups having 1, 2, or 3 cells. Similarly, a group having
1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so
forth.
[0242] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification.
[0243] Other embodiments are set forth within the following claims.
Sequence CWU 1
1
515PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Arg Xaa Lys Phe Cys 1 5 24PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Arg
Tyr Lys Phe 1 34PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 3Phe Arg Phe Lys 1 44PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 4Phe
Arg Phe Lys 1 57PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 5Phe Arg Phe Lys Glu Cys Gly 1 5
* * * * *