U.S. patent application number 16/569573 was filed with the patent office on 2020-02-06 for methods for preventing or treating mitochondrial permeability transition.
This patent application is currently assigned to Cornell Research Foundation, Inc.. The applicant listed for this patent is Cornell Research Foundation, Inc., Institut De Recherches Cliniques de Montreal. Invention is credited to Peter W. Schiller, Hazel H. Szeto, Kesheng Zhao.
Application Number | 20200038472 16/569573 |
Document ID | / |
Family ID | 32853391 |
Filed Date | 2020-02-06 |
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United States Patent
Application |
20200038472 |
Kind Code |
A1 |
Szeto; Hazel H. ; et
al. |
February 6, 2020 |
METHODS FOR PREVENTING OR TREATING MITOCHONDRIAL PERMEABILITY
TRANSITION
Abstract
The invention provides a method of reducing or preventing
mitochondrial permeability transitioning. The method comprises
administering an effective amount of an aromatic-cationic peptide
having at least one net positive charge; a minimum of four amino
acids; a maximum of about twenty amino acids; a relationship
between the minimum number of net positive charges (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 a
relationship between the minimum number of aromatic groups (a) and
the total number of net positive charges (p.sub.t) wherein 2 a 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.
Inventors: |
Szeto; Hazel H.; (New York,
NY) ; Schiller; Peter W.; (Montreal, CA) ;
Zhao; Kesheng; (Jackson Heights, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cornell Research Foundation, Inc.
Institut De Recherches Cliniques de Montreal |
Ithaca
Montreal |
NY |
US
CA |
|
|
Assignee: |
Cornell Research Foundation,
Inc.
Ithaca
NY
Institut De Recherches Cliniques de Montreal
Montreal
|
Family ID: |
32853391 |
Appl. No.: |
16/569573 |
Filed: |
September 12, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14595946 |
Jan 13, 2015 |
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16569573 |
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13778994 |
Feb 27, 2013 |
8957030 |
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14595946 |
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13247648 |
Sep 28, 2011 |
8404646 |
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13778994 |
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12753403 |
Apr 2, 2010 |
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13247648 |
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11427804 |
Jun 30, 2006 |
7718620 |
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12753403 |
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10771232 |
Feb 3, 2004 |
7576061 |
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11427804 |
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60444777 |
Feb 4, 2003 |
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60535690 |
Jan 8, 2004 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 1/04 20180101; C07K
5/1024 20130101; A61P 43/00 20180101; A61P 9/10 20180101; A61K
38/03 20130101; C07K 5/1019 20130101; C07K 5/1016 20130101; C07K
5/1008 20130101; A61P 19/08 20180101; A61P 39/00 20180101; A61P
25/00 20180101; A61K 38/07 20130101; A61P 9/00 20180101; A61P 1/00
20180101; A61P 9/08 20180101; A61P 21/02 20180101; A61P 25/28
20180101; A61P 3/00 20180101; A61P 3/08 20180101; A61P 21/04
20180101; A61P 25/16 20180101; A61P 21/00 20180101; A61P 25/14
20180101 |
International
Class: |
A61K 38/07 20060101
A61K038/07; A61K 38/03 20060101 A61K038/03; C07K 5/103 20060101
C07K005/103; C07K 5/107 20060101 C07K005/107; C07K 5/11 20060101
C07K005/11; C07K 5/117 20060101 C07K005/117 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support from the
National Institute on Drug Abuse under Grant No. POI DA08924-08.
The U.S. Government has certain rights in this invention.
Claims
1. 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 an aromatic-cationic peptide having: (a) at
least one net positive charge; (b) a minimum of four amino acids;
(c) a maximum of about twenty amino acids; (d) a relationship
between the minimum number of net positive charges (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 (e) 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, except
that when a is 1, p.sub.t may also be 1.
2. The method according to claim 1, wherein 2 p.sub.m is the
largest number that is less than or equal to r+1.
3. The method according to claim 1, wherein a is equal to
p.sub.t.
4. The method according to claim 1, wherein the peptide has a
minimum of two positive charges.
5. The method according to claim 1, wherein the peptide has a
minimum of three positive charges.
6. The method according to claim 1, wherein the peptide is water
soluble.
7. The method according to claim 1, wherein the peptide comprises
one or more D-amino acids.
8. The method according to claim 1, wherein the C-terminal carboxyl
group of the amino acid at the C-terminus is amidated.
9. The method according to claim 1, wherein the peptide comprises
one or more non-naturally occurring amino acids.
10. The method according to claim 1, wherein the peptide has a
minimum of four amino acids.
11. The method according to claim 1, wherein the peptide has a
maximum of about twelve amino acids.
12. The method according to claim 1, wherein the peptide has a
maximum of about nine amino acids.
13. The method according to claim 1, wherein the peptide has a
maximum of about six amino acids.
14. The method according to claim 1, wherein the peptide has
mu-opioid receptor agonist activity.
15. The method according to claim 1, wherein the peptide does not
have mu-opioid receptor agonist activity.
16. The method according to claim 1, wherein the mammal is a human.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/595,946, filed Jan. 13, 2015, which is a
continuation of U.S. patent application Ser. No. 13/778,994, filed
Feb. 27, 2013, now U.S. Pat. No. 8,957,030, which is a continuation
of U.S. patent application Ser. No. 13/247,648, filed Sep. 28,
2011, now U.S. Pat. No. 8,404,646, which is a continuation of U.S.
patent application Ser. No. 12/753,403, filed Apr. 2, 2010, now
abandoned, which is a continuation of U.S. patent application Ser.
No. 11/427,804, filed on Jun. 30, 2006, now U.S. Pat. No.
7,718,620, which is a continuation-in-part of U.S. patent
application Ser. No. 10/771,232, filed on Feb. 3, 2004, now U.S.
Pat. No. 7,576,061, which claims priority to U.S. Provisional
Patent Application No. 60/444,777, filed Feb. 4, 2003, and U.S.
Provisional Patent Application No. 60/535,690, filed Jan. 8, 2004.
The contents of these applications are incorporated herein by
reference in their entireties.
BACKGROUND OF THE INVENTION
[0003] Mitochondria exist in virtually all eukaryotic cells, and
are essential to cell survival by producing adenosine triphosphate
(ATP) via oxidative phosphorylation. Interruption of this vital
function can lead to cell death.
[0004] Mitochondria also play a major role in intracellular calcium
regulation by accumulating calcium (Ca.sup.2+). Accumulation of
calcium occurs in the mitochondria) matrix through a membrane
potential-driven uniporter.
[0005] The uptake of calcium activates mitochondrial
dehydrogenases, and may be important in sustaining energy
production and oxidative phosphorylation. In addition, the
mitochondria serve as a sink for excessive cytosolic Ca.sup.2+,
thus protecting the cell from Ca.sup.2+ overload and necrotic
death.
[0006] Ischemia or hypoglycemia can lead to mitochondrial
dysfunction, including ATP hydrolysis and Ca.sup.2+ overload. The
dysfunction causes mitochondrial permeability transition (MPT). MPT
is characterized by uncoupling of oxidative phosphorylation, loss
of mitochondrial membrane potential, increased permeability of the
inner membrane and swelling.
[0007] In addition, the mitochondria intermembrane space is a
reservoir of apoptogenic proteins. Therefore, the loss of
mitochondrial potential and MPT can lead to release of apoptogenic
proteins into the cytoplasm. Not surprisingly, there is
accumulating evidence that MPT is involved in necrotic and
apoptotic cell death (Crompton, Biochem J., 341:233-249 (1999)).
Milder forms of cellular insult may lead to apoptosis rather than
necrosis.
[0008] Cyclosporin can inhibit MPT. Blockade of MPT by cyclosporin
A can inhibit apoptosis in several cell types, including cells
undergoing ischemia, hypoxia, Ca.sup.2+ overload and oxidative
stress (Kroemer et al., Annu Rev Physiol., 60:619-642 (1998)).
[0009] Cyclosporin A, however, is less than optimal as a treatment
drug against necrotic and apoptotic cell death. For example,
cyclosporin A does not specifically target the mitochondria. In
addition, it is poorly delivered to the brain. Furthermore, the
utility of cyclosporin A is reduced by its immunosuppressant
activity.
[0010] The tetrapeptide [Dmt.sup.1]DALDA
(2',6'-dimethyltyrosine-D-Arg-Phe-Lys-NH.sub.2, SS-02) has a
molecular weight of 640 and carries a net three positive charge at
physiological pH. [Dmt.sup.1]DALDA readily penetrates the plasma
membrane of several mammalian cell types in an energy-independent
manner (Zhao et al., J Pharmacol Exp Ther., 304:425-432, 2003) and
penetrates the blood-brain barrier (Zhao et al., J Pharmacol Exp
Ther., 302:188-196, 2002). Although [Dmt.sup.1]DALDA has been shown
to be a potent mu-opioid receptor agonist, its utility has not been
expanded to include the inhibition of MPT.
[0011] Thus, there is a need to inhibit MPT in conditions such as
ischemia-reperfusion, hypoxia, hypoglycemia, and other diseases and
conditions which result in pathological changes as a result of the
permeability transitioning of the mitochondrial membranes. Such
diseases and conditions include many of the common
neurodegenerative diseases.
SUMMARY OF THE INVENTION
[0012] These and other objectives have been met by the present
invention which provides a method for reducing the number of
mitochondria undergoing a mitochondria permeability transition
(MPT), or preventing mitochondrial permeability transitioning in
any mammal that has need thereof. The method comprises
administering to the mammal an effective amount of an
aromatic-cationic peptide having:
[0013] a. at least one net positive charge;
[0014] b. a minimum of three amino acids;
[0015] c. a maximum of about twenty amino acids;
[0016] 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
[0017] e. 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, except that when a is 1, p.sub.t may also be 1.
[0018] In another embodiment, the invention provides a method for
reducing the number of mitochondria undergoing a mitochondrial
permeability transition (MPT), or preventing mitochondrial
permeability transitioning in a removed organ of a mammal. The
method comprises administering to the removed organ an effective
amount of an aromatic-cationic peptide having:
[0019] a. at least one net positive charge;
[0020] b. a minimum of three amino acids;
[0021] c. a maximum of about twenty amino acids;
[0022] 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
[0023] e. 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, except that when a is 1, p.sub.t may also be 1.
[0024] In yet another embodiment, the invention provides a method
of reducing the number of mitochondria undergoing mitochondrial
permeability transition (MPT), or preventing mitochondria)
permeability transitioning in a mammal in need thereof. The method
comprises administering to the mammal an effective amount of an
aromatic-cationic peptide having:
[0025] a. at least one net positive charge;
[0026] b. a minimum of three amino acids;
[0027] c. a maximum of about twenty amino acids;
[0028] d. a relationship between the minimum number of net positive
charges (p.sub.m) and the total number of amino acid residues (r)
wherein 3 p.sub.m is the largest number that is less than or equal
to r+1; and
[0029] 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.
[0030] In a further embodiment, the invention provides a method of
reducing the number of mitochondria undergoing mitochondrial
permeability transition (MPT), or preventing mitochondrial
permeability transitioning in a removed organ of a mammal. The
method comprises administering to the removed organ an effective
amount of an aromatic-cationic peptide having:
[0031] a. at least one net positive charge;
[0032] b. a minimum of three amino acids;
[0033] c. a maximum of about twenty amino acids;
[0034] 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
[0035] 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.
BRIEF DESCRIPTION OF THE FIGURES
[0036] FIGS. 1A-1D: Cellular internalization and accumulation of
[Dmt.sup.1] DALDA (SS-02) in mitochondria. (FIG. 1A) Mitochondrial
uptake of SS-19 was determined using fluorescence spectrophotometry
(ex/em=320/420 nm). Addition of isolated mouse liver mitochondria
(0.35 mg/ml) resulted in immediate quenching of SS-19 fluorescence
intensity (gray line). Pretreatment of mitochondria with FCCP (1.5
.mu.M) reduced quenching by <20% (black line). (FIG. 1B)
Isolated mitochondria were incubated with [.sup.3H]SS-02 at
37.degree. C. for 2 min. Uptake was stopped by centrifugation
(16000.times.g) for 5 min at 4.degree. C., and radioactivity
determined in the pellet. Pretreatment of mitochondria with FCCP
inhibited [.sup.3H]SS-02 uptake by .about.20%. Data are shown as
mean.+-.s.e.; n=3. *, P<0.05 by Student's t-test. (FIG. 1C)
Uptake of TMRM by isolated mitochondria is lost upon mitochondrial
swelling induced by alamethicin, while uptake of SS-19 is retained
to a large extent. Black line, TMRM; red line, SS-19. (FIG. 1D)
Addition of SS-02 (200 .mu.M) to isolated mitochondria did not
alter mitochondrial potential, as measured by TMRM fluorescence.
Addition of FCCP (1.5 .mu.M) caused immediate depolarization while
Ca.sup.2+ (150 .mu.M) resulted in depolarization and progressive
onset of MPT.
[0037] FIGS. 2A-2C. [Dmt.sup.1]DALDA (SS-02) protects against
mitochondrial permeability transition (MPT) induced by Ca.sup.2+
overload and 3-nitroproprionic acid (3NP). (FIG. 2A) Pretreatment
of isolated mitochondria with 10 .mu.M SS-02 (addition indicated by
down arrow) prevented onset of MPT caused by Ca.sup.2+ overload (up
arrow). Black line, buffer; red line, SS-02 (FIG. 2B) Pretreatment
of isolated mitochondria with SS-02 increased mitochondrial
tolerance of multiple Ca.sup.2+ additions prior to onset of MPT.
Arrow indicates addition of buffer or SS-02. Line 1, buffer; line
2, 50 .mu.M SS-02; line 3, 100 .mu.M SS-02. (FIG. 2C) SS-02
dose-dependently delayed the onset of MPT caused by 1 mM 3NP. Arrow
indicates addition of buffer or SS-02. Line 1, buffer; line 2, 0.5
.mu.M SS-02; line 3, 5 .mu.M SS-02; line 4, 50 .mu.M SS-02.
[0038] FIGS. 3A-3C. [Dmt.sup.1]DALDA (SS-02) inhibits mitochondrial
swelling and cytochrome c release. (FIG. 3A) Pretreatment of
isolated mitochondria with SS-02 dose dependently inhibited
mitochondrial swelling induced by 200 .mu.M Ca.sup.2+ in a
dose-dependent manner. Swelling was measured by absorbance at 540
nm. (FIG. 3B) SS-02 inhibited Ca.sup.2+-induced release of
cytochrome c from isolated mitochondria. The amount of cytochrome c
released was expressed as percent of total cytochrome c in
mitochondria. Data are presented as mean.+-.s.e., n=3. (FIG. 3C)
SS-02 also inhibited mitochondrial swelling induced by MPP.sup.+
(300 .mu.M).
[0039] FIGS. 4A-4C. D-Arg-Dmt-Lys-Phe-NH.sub.2 (SS-31) inhibits
mitochondrial swelling and cytochrome c release. (FIG. 4A)
Pretreatment of isolated mitochondria with SS-31 (10 .mu.M)
prevents onset of MPT induced by Ca.sup.2+. Gray line, buffer; red
line, SS-31. (FIG. 4B) Pretreatment of mitochondria with SS-31 (50
.mu.M) inhibited mitochondrial swelling induced by 200 mM
Ca.sup.2+. Swelling was measured by light scattering measured at
570 nm. (FIG. 4C). Comparison of SS-02 and SS-31 with cyclosporine
(CsA) in inhibiting mitochondrial swelling and cytochrome c release
induced by Ca.sup.2+, The amount of cytochrome c released was
expressed as percent of total cytochrome c in mitochondria. Data
are presented as mean.+-.s.e., n=3.
[0040] FIG. 5. [Dmt.sup.1]DALDA (SS-02) and
D-Arg-Dmt-Lys-Phe-NH.sub.2 (SS-31) protects myocardial contractile
force during ischemia-reperfusion in the isolated perfused guinea
pig heart. Hearts were perfused with buffer or buffer containing
SS-02 (100 nM) or SS-31 (1 nM) for 30 min and then subjected to
30-min global ischemia. Reperfusion was carried out using the same
perfusion solution. Significant differences were found among the
three treatment groups (2-way ANOVA, P<0.001).
[0041] FIG. 6. Addition of [Dmt.sup.1]DALDA to cardioplegic
solution significantly enhanced contractile function after
prolonged ischemia in the isolated perfused guinea pig heart. After
30 min stabilization, hearts were perfused with St. Thomas
cardioplegic solution (CPS) or CPS containing [Dmt.sup.1]DALDA at
100 .mu.m for 3 min. Global ischemia was then induced by complete
interruption of coronary perfusion for 90 min. Reperfusion was
subsequently carried out for 60 min with oxygenated Krebs-Henseleit
solution. Post-ischemic contractile force was significantly
improved in the group receiving [Dmt.sup.1]DALDA (P<0.001).
[0042] FIG. 7. SS-31 protected against tBHP-induced mitochondrial
depolarization and viability. N.sub.2A cells were plated in glass
bottom dishes and treated with (A) control, or with (B) 50 .mu.M
tBHP, alone or with (e) 1 nM SS-31, for 6 hours. Cells were loaded
with TMRM (20 nM) and imaged by confocal laser scanning microscopy
using ex/em of 552/570 nm.
[0043] FIG. 8. Representative rat heart slices stained; (a) is
myocardial ischemia area at risk, as determined by Evans blue dye
(unstained by blue dye), and (b) is infarct myocardium as
determined by TTC staining and formalin fixation (the pink and
white areas unstained by TTC).
[0044] FIG. 9. The area at risk relative to the left ventricle
after 1 hour ischemia followed by 1 hour of reperfusion in rats
treated with control, SS-31 or SS-20 administered 30 min before
ligation and 5 min before reperfusion. Bars represent group mean,
brackets indicated S.E.M.
[0045] FIG. 10. Infarct size relative to the left ventricle after 1
hour ischemia followed by 1 hour of reperfusion in rats treated
with control, SS-31 or SS-20 administered 30 min before ligation
and 5 min before reperfusion. Bars represent group mean, brackets
indicate S.E.M.
DETAILED DESCRIPTION OF THE INVENTION
[0046] The invention is based on the surprising discovery by the
inventors that certain aromatic-cationic peptides significantly
reduce the number of mitochondria undergoing, or even completely
preventing, 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. In addition, a removed organ of a mammal is
susceptible to MPT. These diseases and conditions are of particular
clinical importance as they afflict a large proportion of the human
population at some stage during their lifetime.
Peptides
[0047] The aromatic-cationic peptides useful in the present
invention are water-soluble and highly polar. Despite these
properties, the peptides can readily penetrate cell membranes.
[0048] The aromatic-cationic peptides useful in the present
invention include a minimum of three amino acids, and preferably
include a minimum of four amino acids, covalently joined by peptide
bonds.
[0049] The maximum number of amino acids present in the
aromatic-cationic peptides of the present invention is about twenty
amino acids covalently joined by peptide bonds. Preferably, the
maximum number of amino acids is about twelve, more preferably
about nine, and most preferably about six. Optimally, the number of
amino acids present in the peptides is four.
[0050] The amino acids of the aromatic-cationic peptides useful in
the present invention can be any amino acid. As used herein, the
term "amino acid" is used to refer to any organic molecule that
contains at least one amino group and at least one carboxyl group.
Preferably, at least one amino group is at the a position relative
to the carboxyl group.
[0051] 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 (Glu), glutamic acid (Glu),
glycine (Gly), histidine (His), isoleucine (Ileu), leucine (Leu),
lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro),
serine (Ser), threonine (Thr), tryptophan, (Trp), tyrosine (Tyr),
and valine (Val).
[0052] 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.
[0053] The peptides useful in the present invention can contain one
or more nonnaturally occurring amino acids. The non-naturally
occurring amino acids may be L-, dextrorotatory (D), or mixtures
thereof. Optimally, the peptide has no amino acids that are
naturally occurring.
[0054] Non-naturally occurring amino acids are those amino acids
that typically are not synthesized in normal metabolic processes in
living organisms, and do not naturally occur in proteins. In
addition, the non-naturally occurring amino acids useful in the
present invention preferably are also not recognized by common
proteases.
[0055] 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.
[0056] The non-natural amino acids may, for example, comprise
alkyl, aryl, or alkylaryl groups. Some examples of alkyl amino
acids include a-aminobutyric acid, (aminobutyric acid,
y-aminobutyric acid, 6-aminovaleric acid, and E-aminocaproic acid.
Some examples of aryl amino acids include ortho-, meta, and
para-aminobenzoic acid. Some examples of alkylaryl amino acids
include ortho-, meta-, and para aminophenyl acetic acid, and
y-phenyl-R-aminobutyric acid.
[0057] Non-naturally occurring amino acids also 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.
[0058] 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), norleucine (Nle), and hydroxyproline
(Hyp).
[0059] Another example of a modification of an amino acid in a
peptide useful in the methods of the present invention is the
derivatization of a carboxyl group of an aspartic acid or a
glutamic acid residue of the peptide. One example of derivatization
is amidation with ammonia or with a primary or secondary amine,
e.g. methylamine, ethylamine, dimethylamine or dethylamine. Another
example of derivatization includes esterification with, for
example, methyl or ethyl alcohol.
[0060] 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.
[0061] 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.
[0062] In order to minimize protease sensitivity, the peptides
useful in the methods of the invention should have less than five,
preferably less than four, more preferably less than three, and
most preferably, less than two contiguous L-amino acids recognized
by common proteases, irrespective of whether the amino acids are
naturally or non-naturally occurring. Optimally, the peptide has
only D-amino acids, and no L-amino acids.
[0063] If the peptide contains protease sensitive sequences of
amino acids, at least one of the amino acids is preferably a
non-naturally-occurring v-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.
[0064] It is important that the aromatic-cationic peptides 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).
[0065] 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.
[0066] "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.
[0067] 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-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.
[0068] In one embodiment of the present invention, 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 3 p.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-00001 (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
[0069] 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 2
p.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-00002 (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
[0070] 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,
preferably, a minimum of two net positive charges and more
preferably a minimum of three net positive charges.
[0071] 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).
[0072] Naturally occurring amino acids that have an aromatic group
include the amino acids histidine, tryptophan, tyrosine, and
phenylalanine. For example, the hexapeptide Lys-Gln-Tyr-Arg-Phe-Trp
has a net positive charge of two (contributed by the lysine and
arginine residues) and three aromatic groups (contributed by
tyrosine, phenylalanine and tryptophan residues).
[0073] In one embodiment of the present invention, the
aromatic-cationic peptides useful in the methods of the present
invention have a relationship between the minimum number of
aromatic groups (a) and the total number of net positive charges at
physiological pH (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-00003 (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
[0074] 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-00004 (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
[0075] In another embodiment, the number of aromatic groups (a) and
the total number of net positive charges (p.sub.t) are equal.
[0076] Carboxyl groups, especially the terminal carboxyl group of a
C-terminal amino acid, are preferably amidated with, for example,
ammonia to form the C-terminalamide. 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-dethyl amido,
Nmethyl-N-ethylamido, N-phenylamido or N-phenyl-N-ethylamido
group.
[0077] The free carboxylate groups of the asparagine, glutamine,
aspartic acid, and glutamic acid residues not occurring at the
C-terminus of the aromatic-cationic peptides of the present
invention may also be amidated wherever they occur within the
peptide. The amidation at these internal positions may be with
ammonia or any of the primary or secondary amines described
above.
[0078] In one embodiment, the aromatic-cationic peptide useful in
the methods of the present invention is a tripeptide having two net
positive charges and at least one aromatic amino acid. In a
particular embodiment, the aromatic-cationic peptide useful in the
methods of the present invention is a tripeptide having two net
positive charges and two aromatic amino acids.
[0079] Aromatic-cationic peptides useful in the methods of the
present invention include, but are not limited to, the following
peptide examples:
Lys-D-Arg-Tyr-NH.sub.2,
Phe-D-Arg-His,
D-Tyr-Trp-Lys-NH.sub.2,
Trp-D-Lys-Tyr-Arg-NH.sub.2,
Tyr-His-D-Gly-Met,
Phe-Arg-D-His-Asp,
Tyr-D-Arg-Phe-Lys-Glu-NH.sub.2,
Met-Tyr-D-Lys-Phe-Arg,
D-His-Glu-Lys-Tyr-D-Phe-Arg,
Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH.sub.2,
Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His,
Gly-D-Phe-Lys-Tyr-His-D-Arg-Tyr-NH.sub.2,
Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH.sub.2,
Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-Lys,
Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH.sub.2,
Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-Lys,
Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH.sub.2,
D-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-His-D-Lys-Arg-Trp-NH.sub.2,
Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-Phe,
Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-Phe,
Phe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH.sub.2,
Phe-Try-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-Thr,
Tyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-Lys,
Glu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met-NH.sub.2,
Arg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-Phe-Tyr-D-Arg-Gly,
D-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH.s-
ub.2,
Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-Ph-
e,
His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Se-
r-NH.sub.2,
Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-Hi-
s-D-Lys-Asp, and
Thr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D--
His-Arg-Tyr-Lys-NH.sub.2.
[0080] In one embodiment, the peptides useful in the methods of the
present invention have mu-opioid receptor agonist activity (i.e.,
activate the mu-opioid receptor). Activation of the mu-opioid
receptor typically elicits an analgesic effect.
[0081] In certain instances, an aromatic-cationic peptide having
mu-opioid receptor 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 patient
or other mammal, although an aromatic-cationic peptide which does
not activate the mu-opioid receptor may also be used with or
without an analgesic according to clinical requirements.
[0082] Alternatively, in other instances, an aromatic-cationic
peptide that does not have mu-opioid receptor 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.
[0083] 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.
[0084] Examples of acute conditions include heart attack, stroke
and traumatic injury. Traumatic injury may include traumatic brain
and spinal cord injury.
[0085] Examples of chronic diseases or conditions include coronary
artery disease and any neurodegenerative disorders, such as those
described below.
[0086] Peptides useful in the methods of the present invention
which have mu opioid receptor activity are typically those peptides
which have a tyrosine residue or a tyrosine derivative at the
N-terminus (i.e., the first amino acid position). Preferred
derivatives of tyrosine include 2'-methyltyrosine (Mmt);
2',6'-dimethyltyrosine (2'6'Dmt); 3',5'-dimethyltyrosine (3'5'Dmt);
N,2',6'-trimethyl tyrosine (Tmt); and 2'hydroxy-6'-methyltryosine
(Hmt).
[0087] In a particular preferred embodiment, a peptide that has
mu-opioid receptor activity has the formula
Tyr-D-Arg-Phe-Lys-NH.sub.2 (for convenience represented by the
acronym: DALDA, which is referred to herein as SS-01). DALDA has a
net positive charge of three, contributed by the amino acids
tyrosine, arginine, and lysine and has two aromatic groups
contributed by the amino acids phenylalanine and tyrosine. The
tyrosine of DALDA can be a modified derivative of tyrosine such as
in 2',6'dimethyltyrosine to produce the compound having the formula
2',6'-Dmt-D-Arg-PheLys-NH.sub.2 (i.e., Dmt.sup.1-DALDA, which is
referred to herein as SS-02).
[0088] Peptides that do not have mu-opioid receptor activity
generally do not have a tyrosine residue or a derivative of
tyrosine at the N-terminus (i.e., amino acid position one). The
amino acid at the N-terminus can be any naturally occurring or
nonnaturally occurring amino acids other than tyrosine.
[0089] In one embodiment, the amino acid at the N-terminus is
phenylalanine or its derivative. Preferred derivatives of
phenylalanine include 2'-methyl phenylalanine(Mmp),
2',6'-dimethylphenylalanine (Dmp), N,2',6'-trimethylphenylalanine
(Tmp), and 2'-hydroxy-6'-methylphenylalanine (Hmp).
[0090] Other aromatic-cationic peptide that does not have mu-opioid
receptor activity has the formula Phe-D-Arg-Phe-Lys-NH.sub.2 (i.e.,
Phe.sup.1-DALDA, which is referred to herein as SS-20).
Alternatively, the N-terminal phenylalanine can be a derivative of
phenylalanine such as 2',6'-dimethylphenylalanine (2'6'Dmp). DALDA
containing 2',6'-dimethylphenylalanine at amino acid position one
has the formula 2',6'-Dmp-D-Arg-Phe-Lys-NH.sub.2 (i.e.,
2'6'Dmp.sup.1-DALDA).
[0091] In a preferred embodiment, the amino acid sequence of
Dmt.sup.1-DALDA (SS-02) is rearranged such that Dint is not at the
N-terminus. An example of such an aromatic-cationic peptide that
does not have mu-opioid receptor activity has the formula
D-Arg-2'6'Dmt-Lys-Phe-NH.sub.2 (referred to in this specification
as SS-31).
[0092] DALDA, Phe.sup.1-DALDA, SS-31, and their derivatives can
further include functional analogs. A peptide is considered a
functional analog of DALDA, Phe.sup.1-DALDA, or SS-31 if the analog
has the same function as DALDA, Phe.sup.1-DALDA, or SS-31. The
analog may, for example, be a substitution variant of DALDA,
Phe.sup.1-DALDA, or SS-31, wherein one or more amino acid is
substituted by another amino acid.
[0093] Suitable substitution variants of DALDA, Phe.sup.1-DALDA, or
SS-31 include conservative amino acid substitutions. Amino acids
may be grouped according to their physicochemical characteristics
as follows:
[0094] a. Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P)
Gly(G);
[0095] b. Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q);
[0096] c. Basic amino acids: His(H) Arg(R) Lys(K);
[0097] d. Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V);
and
[0098] e. Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His (H).
[0099] 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.
[0100] Examples of analogs useful in the practice of the present
invention that activate mu-opioid receptors include, but are not
limited, to the aromatic-cationic peptides shown in Table 1.
TABLE-US-00005 TABLE 1 Amino Acid Amino Acid Amino Acid Amino Acid
Amino Acid Position 5 C-Terminal Position 1 Position 2 Position 3
Position 4 (if present) Modification Tyr D-Arg Phe Lys NH.sub.2 Tyr
D-Arg Phe Orn NH.sub.2 Tyr D-Arg Phe Dab NH.sub.2 Tyr D-Arg Phe Dap
NH.sub.2 2'6'Dmt D-Arg Phe Lys NH.sub.2 2'6'Dmt D-Arg Phe Lys Cys
NH.sub.2 2'6'Dmt D-Arg Phe Lys-NH(CH.sub.2).sub.2- NH.sub.2 NH-dns
2'6'Dmt D-Arg Phe Lys-NH(CH.sub.2).sub.2- NH.sub.2 NH-atn 2'6'Dmt
D-Arg Phe dnsLys NH.sub.2 2'6'Dmt D-Cit Phe Lys NH.sub.2 2'6'Dmt
D-Cit Phe Ahp NH.sub.2 2'6'Dmt D-Arg Phe Orn NH.sub.2 2'6'Dmt D-Arg
Phe Dab NH.sub.2 2'6'Dmt D-Arg Phe Dap NH.sub.2 2'6'Dmt D-Arg Phe
Ahp (2- NH.sub.2 aminoheptanoic acid) Bio-2'6'Dmt D-Arg Phe Lys
NH.sub.2 3'5'Dmt D-Arg Phe Lys NH.sub.2 3'5'Dmt D-Arg Phe Orn
NH.sub.2 3'5'Dmt D-Arg Phe Dab NH.sub.2 3'5'Dmt D-Arg Phe Dap
NH.sub.2 Tyr D-Arg Tyr Lys NH.sub.2 Tyr D-Arg Tyr Orn NH.sub.2 Tyr
D-Arg Tyr Dab NH.sub.2 Tyr D-Arg Tyr Dap NH.sub.2 2'6'Dmt D-Arg Tyr
Lys NH.sub.2 2'6'Dmt D-Arg Tyr Orn NH.sub.2 2'6'Dmt D-Arg Tyr Dab
NH.sub.2 2'6'Dmt D-Arg Tyr Dap NH.sub.2 2'6'Dmt D-Arg 2'6'Dmt Lys
NH.sub.2 2'6'Dmt D-Arg 2'6'Dmt Orn NH.sub.2 2'6'Dmt D-Arg 2'6'Dmt
Dab NH.sub.2 2'6'Dmt D-Arg 2'6'Dmt Dap NH.sub.2 3'5'Dmt D-Arg
3'5'Dmt Arg NH.sub.2 3'5'Dmt D-Arg 3'5'Dmt Lys NH.sub.2 3'5'Dmt
D-Arg 3'5'Dmt Orn NH.sub.2 3'5'Dmt D-Arg 3'5'Dmt Dab NH.sub.2 Tyr
D-Lys Phe Dap NH.sub.2 Tyr D-Lys Phe Arg NH.sub.2 Tyr D-Lys Phe Lys
NH.sub.2 Tyr D-Lys Phe Orn NH.sub.2 2'6'Dmt D-Lys Phe Dab NH.sub.2
2'6'Dmt D-Lys Phe Dap NH.sub.2 2'6'Dmt D-Lys Phe Arg NH.sub.2
2'6'Dmt D-Lys Phe Lys NH.sub.2 3'5'Dmt D-Lys Phe Orn NH.sub.2
3'5'Dmt D-Lys Phe Dab NH.sub.2 3'5'Dmt D-Lys Phe Dap NH.sub.2
3'5'Dmt D-Lys Phe Arg NH.sub.2 Tyr D-Lys Tyr Lys NH.sub.2 Tyr D-Lys
Tyr Orn NH.sub.2 Tyr D-Lys Tyr Dab NH.sub.2 Tyr D-Lys Tyr Dap
NH.sub.2 2'6'Dmt D-Lys Tyr Lys NH.sub.2 2'6'Dmt D-Lys Tyr Orn
NH.sub.2 2'6'Dmt D-Lys Tyr Dab NH.sub.2 2'6'Dmt D-Lys Tyr Dap
NH.sub.2 2'6'Dmt D-Lys 2'6'Dmt Lys NH.sub.2 2'6'Dmt D-Lys 2'6'Dmt
Orn NH.sub.2 2'6'Dmt D-Lys 2'6'Dmt Dab NH.sub.2 2'6'Dmt D-Lys
2'6'Dmt Dap NH.sub.2 2'6'Dmt D-Arg Phe dnsDap NH.sub.2 2'6'Dmt
D-Arg Phe atnDap NH.sub.2 3'5'Dmt D-Lys 3'5'Dmt Lys NH.sub.2
3'5'Dmt D-Lys 3'5'Dmt Orn NH.sub.2 3'5'Dmt D-Lys 3'5'Dmt Dab
NH.sub.2 3'5'Dmt D-Lys 3'5'Dmt Dap NH.sub.2 Tyr D-Lys Phe Arg
NH.sub.2 Tyr D-Orn Phe Arg NH.sub.2 Tyr D-Dab Phe Arg NH.sub.2 Tyr
D-Dap Phe Arg NH.sub.2 2'6'Dmt D-Arg Phe Arg NH.sub.2 2'6'Dmt D-Lys
Phe Arg NH.sub.2 2'6'Dmt D-Orn Phe Arg NH.sub.2 2'6'Dmt D-Dab Phe
Arg NH.sub.2 3'5'Dmt D-Dap Phe Arg NH.sub.2 3'5'Dmt D-Arg Phe Arg
NH.sub.2 3'5'Dmt D-Lys Phe Arg NH.sub.2 3'5'Dmt D-Orn Phe Arg
NH.sub.2 Tyr D-Lys Tyr Arg NH.sub.2 Tyr D-Orn Tyr Arg NH.sub.2 Tyr
D-Dab Tyr Arg NH.sub.2 Tyr D-Dap Tyr Arg NH.sub.2 2'6'Dmt D-Arg
2'6'Dmt Arg NH.sub.2 2'6'Dmt D-Lys 2'6'Dmt Arg NH.sub.2 2'6'Dmt
D-Orn 2'6'Dmt Arg NH.sub.2 2'6'Dmt D-Dab 2'6'Dmt Arg NH.sub.2
3'5'Dmt D-Dap 3'5'Dmt Arg NH.sub.2 3'5'Dmt D-Arg 3'5'Dmt Arg
NH.sub.2 3'5'Dmt D-Lys 3'5'Dmt Arg NH.sub.2 3'5'Dmt D-Orn 3'5'Dmt
Arg NH.sub.2 Mmt D-Arg Phe Lys NH.sub.2 Mmt D-Arg Phe Orn NH.sub.2
Mmt D-Arg Phe Dab NH.sub.2 Mmt D-Arg Phe Dap NH.sub.2 Tmt D-Arg Phe
Lys NH.sub.2 Tmt D-Arg Phe Orn NH.sub.2 Tmt D-Arg Phe Dab NH.sub.2
Tmt D-Arg Phe Dap NH.sub.2 Hmt D-Arg Phe Lys NH.sub.2 Hmt D-Arg Phe
Orn NH.sub.2 Hmt D-Arg Phe Dab NH.sub.2 Hmt D-Arg Phe Dap NH.sub.2
Mmt D-Lys Phe Lys NH.sub.2 Mmt D-Lys Phe Orn NH.sub.2 Mmt D-Lys Phe
Dab NH.sub.2 Mmt D-Lys Phe Dap NH.sub.2 Mmt D-Lys Phe Arg NH.sub.2
Tmt D-Lys Phe Lys NH.sub.2 Tmt D-Lys Phe Orn NH.sub.2 Tmt D-Lys Phe
Dab NH.sub.2 Tmt D-Lys Phe Dap NH.sub.2 Tmt D-Lys Phe Arg NH.sub.2
Hmt D-Lys Phe Lys NH.sub.2 Hmt D-Lys Phe Orn NH.sub.2 Hmt D-Lys Phe
Dab NH.sub.2 Hmt D-Lys Phe Dap NH.sub.2 Hmt D-Lys Phe Arg NH.sub.2
Mmt D-Lys Phe Arg NH.sub.2 Mmt D-Orn Phe Arg NH.sub.2 Mmt D-Dab Phe
Arg NH.sub.2 Mmt D-Dap Phe Arg NH.sub.2 Mmt D-Arg Phe Arg NH.sub.2
Tmt D-Lys Phe Arg NH.sub.2 Tmt D-Orn Phe Arg NH.sub.2 Tmt D-Dab Phe
Arg NH.sub.2 Tmt D-Dap Phe Arg NH.sub.2 Tmt D-Arg Phe Arg NH.sub.2
Hmt D-Lys Phe Arg NH.sub.2 Hmt D-Orn Phe Arg NH.sub.2 Hmt D-Dab Phe
Arg NH.sub.2 Hmt D-Dap Phe Arg NH.sub.2 Hmt D-Arg Phe Arg NH.sub.2
Dab = diaminobutyric Dap = diaminopropionic acid Dmt =
dimethyltyrosine Mmt = 2'-methyltyrosine Tmt =
N,2'6'-trimethyltyrosine Hmt = 2'hydroxy,6'-methyltyrosine dnsDap =
.beta.-dansyl-L-.alpha.,.beta.-diaminopropionic acid antDap =
.beta.-anthraniloyl-L-.alpha.,.beta.-diaminopropionic acid Bio =
biotin
[0101] Examples of analogs useful in the practice of the present
invention that do not activate mu-opioid receptors include, but are
not limited to, the aromatic-cationic peptides shown in Table
2.
TABLE-US-00006 TABLE 2 Amino Acid Amino Acid Amino Acid Amino Acid
C-Terminal Position 1 Position 2 Position 3 Position 5 Modification
D-Arg Dmt Lys Phe NH.sub.2 D-Arg Dmt Phe Lys NH.sub.2 D-Arg Phe Lys
Dmt NH.sub.2 D-Arg Phe Dmt Lys NH.sub.2 D-Arg Lys Dmt Phe NH.sub.2
D-Arg Lys Phe Dmt NH.sub.2 Phe Lys Dmt D-Arg NH.sub.2 Phe Lys D-Arg
Dmt NH.sub.2 Phe D-Arg Dmt Lys NH.sub.2 Phe D-Arg Lys Dmt NH.sub.2
Phe Dmt D-Arg Lys NH.sub.2 Phe Dmt Lys D-Arg NH.sub.2 Lys Phe D-Arg
Dmt NH.sub.2 Lys Phe Dmt D-Arg NH.sub.2 Lys Dmt D-Arg Phe NH.sub.2
Lys Dmt Phe D-Arg NH.sub.2 Lys D-Arg Phe Dmt NH.sub.2 Lys D-Arg Dmt
Phe NH.sub.2 D-Arg Dmt D-Arg Phe NH.sub.2 D-Arg Dmt D-Arg Dmt
NH.sub.2 D-Arg Dmt D-Arg Tyr NH.sub.2 D-Arg Dmt D-Arg Trp NH.sub.2
Trp D-Arg Phe Lys NH.sub.2 Trp D-Arg Tyr Lys NH.sub.2 Trp D-Arg Trp
Lys NH.sub.2 Trp D-Arg Dmt Lys NH.sub.2 D-Arg Trp Lys Phe NH.sub.2
D-Arg Trp Phe Lys NH.sub.2 D-Arg Trp Lys Dmt NH.sub.2 D-Arg Trp Dmt
Lys NH.sub.2 D-Arg Lys Trp Phe NH.sub.2 D-Arg Lys Trp Dmt NH.sub.2
Cha D-Arg Phe Lys NH.sub.2 Ala D-Arg Phe Lys NH.sub.2 Cha =
cyclohexyl
[0102] The amino acids of the peptides shown in table 1 and 2 may
be in either the L- or the D-configuration.
Methods of Treating
[0103] The peptides described above are useful in treating any
disease or condition that is associated with MPT. Such diseases and
conditions include, but are not limited to, ischemia and/or
reperfusion of a tissue or organ, hypoxia and any of a number of
neurodegenerative diseases. Mammals in need of treatment or
prevention of MPT are those mammals suffering from these diseases
or conditions.
[0104] 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.
[0105] 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.
[0106] 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.
[0107] 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.
[0108] 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.
[0109] The methods of the present invention 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 Gehrig's disease). The methods of the present
invention can be used to delay the onset or slow the progression of
these and other neurodegenerative diseases associated with MPT. The
methods of the present invention 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.
[0110] The peptides useful in the present invention may also be
used in preserving an organ of a mammal prior to transplantation.
For example, a removed organ can be susceptible to MPT due to lack
of blood flow. Therefore, the peptides can be used to prevent MPT
in the removed organ.
[0111] The removed organ can be placed in a standard buffered
solution, such as those commonly used in the art. For example, a
removed heart can be placed in a cardioplegic solution containing
the peptides described above. The concentration of peptides in the
standard buffered solution can be easily determined by those
skilled in the art. Such concentrations may be, for example,
between about 0.1 nM to about 10 .mu.M, preferably about 1 .mu.M to
about 10 .mu.M.
[0112] The peptides may also be administered to a mammal taking a
drug to treat a condition or disease. If a side effect of the drug
includes MPT, mammals taking such drugs would greatly benefit from
the peptides of the invention.
[0113] An example of a drug which induces cell toxicity by
effecting MPT is the chemotherapy drug Adriamycin.
Synthesis of the Peptides
[0114] The peptides useful in the methods of the present invention
may be chemically synthesized by any of the methods well known in
the art. Suitable methods for synthesizing the protein include, for
example those described by Stuart and Young in "Solid Phase Peptide
Synthesis," Second Edition, Pierce Chemical Company (1984), and in
"Solid Phase Peptide Synthesis," Methods Enzymol. 289, Academic
Press, Inc, New York (1997).
Modes of Administration
[0115] The peptide useful in the methods of the present invention
is administered to a mammal in an amount effective in reducing the
number of mitochondria undergoing, or preventing, MPT. The
effective amount is determined during pre-clinical trials and
clinical trials by methods familiar to physicians and
clinicians.
[0116] An effective amount of a peptide useful in the methods of
the present invention, preferably in a pharmaceutical composition,
may be administered to a mammalian need thereof by any of a number
of well-known methods for administering pharmaceutical
compounds.
[0117] The peptide may be administered systemically or locally. In
one embodiment, the peptide is administered intravenously. For
example, the aromaticcationic peptides useful in the methods of the
present invention may be administered via rapid intravenous bolus
injection. Preferably, however, the peptide is administered as a
constant rate intravenous infusion.
[0118] The peptide can be injected directly into coronary artery
during, for example, angioplasty or coronary bypass surgery, or
applied onto coronary stents.
[0119] The peptide may also be administered orally, topically,
intranasally, intramuscularly, subcutaneously, or transdermally. In
a preferred embodiment, transdermal administration of the
aromatic-cationic peptides by methods of the present invention is
by iontophoresis, in which the charged peptide is delivered across
the skin by an electric current.
[0120] Other routes of administration include
intracerebroventricularly or intrathecally.
Intracerebroventiculatly refers to administration into the
ventricular system of the brain. Intrathecally refers to
administration into the space under the arachnoid membrane of the
spinal cord. Thus intracerebroventricular or intrathecal
administration may be preferred for those diseases and conditions
which affect the organs or tissues of the central nervous system.
In a preferred embodiment, intrathecal administration is used for
traumatic spinal cord injury.
[0121] The peptides useful in the methods of the invention may also
be administered to mammals by sustained release, as is known in the
art. Sustained release administration is a method of drug delivery
to achieve a certain level of the drug over a particular period of
time. The level typically is measured by serum or plasma
concentration.
[0122] Any formulation known in the art of pharmacy is suitable for
administration of the aromatic-cationic peptides useful in the
methods of the present invention. For oral administration, liquid
or solid formulations may be used. Some examples of formulations
include tablets, gelatin capsules, pills, troches, elixirs,
suspensions, syrups, wafers, chewing gum and the like. The peptides
can be mixed with a suitable pharmaceutical carrier (vehicle) or
excipient as understood by practitioners in the art. Examples of
carriers and excipients include starch, milk, sugar, certain types
of clay, gelatin, lactic acid, stearic acid or salts thereof,
including magnesium or calcium stearate, talc, vegetable fats or
oils, gums and glycols.
[0123] For systemic, intracerebroventricular, intrathecal, topical,
intranasal, subcutaneous, or transdermal administration,
formulations of the aromatic-cationic peptides useful in the
methods of the present inventions may utilize conventional
diluents, carriers, or excipients etc., such as are known in the
art can be employed to deliver the peptides. For example, the
formulations may comprise one or more of the following: a
stabilizer, a surfactant, preferably a nonionic surfactant, and
optionally a salt and/or a buffering agent. The peptide may be
delivered in the form of an aqueous solution, or in a lyophilized
form.
[0124] The stabilizer may, for example, be an amino acid, such as
for instance, glycine; or an oligosaccharide, such as for example,
sucrose, tetralose, lactose or a dextran. Alternatively, the
stabilizer may be a sugar alcohol, such as for instance, mannitol;
or a combination thereof. Preferably the stabilizer or combination
of stabilizers constitutes from about 0.1% to about 10% weight for
weight of the peptide.
[0125] The surfactant is preferably a nonionic surfactant, such as
a polysorbate. Some examples of suitable surfactants include
Tween20, Tween80; a polyethylene glycolor a polyoxyethylene
polyoxypropylene glycol, such as Pluronic F-68 at from about 0.001%
(w/v) to about 10% (w/v).
[0126] The salt or buffering agent may be any salt or buffering
agent, such as for example, sodium chloride, or sodium/potassium
phosphate, respectively. Preferably, the buffering agent maintains
the pH of the pharmaceutical composition in the range of about 5.5
to about 7.5. The salt and/or buffering agent is also useful to
maintain the osmolality at a level suitable for administration to a
human or an animal. Preferably the salt or buffering agent is
present at a roughly isotonic concentration of about 150 mM to
about 300 mM.
[0127] The formulations of the peptides useful in the methods of
the present invention may additionally contain one or more
conventional additive. Some examples of such additives include a
solubilizer such as, for example, glycerol; an antioxidant such as
for example, benzalkonium chloride (a mixture of quaternary
ammonium compounds, known as "quats"), benzyl alcohol, chloretone
or chlorobutanol; anaesthetic agent such as for example a morphine
derivative; or an isotonic agent etc., such as described above. As
a further precaution against oxidation or other spoilage, the
pharmaceutical compositions may be stored under nitrogen gas in
vials sealed with impermeable stoppers.
[0128] The mammal can be any mammal, including, for example, farm
animals, such as sheep, pigs, cows, and horses; pet animals, such
as dogs and cats; laboratory animals, such as rats, mice and
rabbits In a preferred embodiment, the mammal is a human.
EXAMPLES
Example 1: [Dmt.sup.1]DALDA Penetrates Cell Membrane
[0129] The cellular uptake of [.sup.3H] [Dmt.sup.1]DALDA was
studied using a human intestinal epithelial cell line (Caco-2), and
confirmed with SH-SY5Y (human neuroblastoma cell), HEK293 (human
embryonic kidney cell) and CRFK cells (kidney epithelial cell).
Monolayers of cells were grown on 12-well plates (5.times.10.sup.5
cells/well) coated with collagen for 3 days. On day 4, cells were
washed twice with pre-wanned HBSS, and then incubated with 0.2 ml
of HBSS containing either 250 nM [.sup.3H][Dmt.sup.1]DALDA at
37.degree. C. or 4.degree. C. for various times up to 1 h.
[0130] [.sup.3H][Dmt.sup.1]DALDA was observed in cell lysate as
early as 5 min, and steady state levels were achieved by 30 min.
The total amount of [.sup.3H][Dmt.sup.1]DALDA recovered in the cell
lysate after 1 h incubation represented about I % of the total
drug. The uptake of [.sup.3H][Dmt.sup.1]DALDA was slower at
4.degree. C. compared to 37.degree. C., but reached 76.5% by 45 min
and 86.3% by 1 h. The internalization of [.sup.3H][Dmt.sup.1]DALDA
was not limited to Caco-2 cells, but was also observed in SH-SY5Y,
HEK293 and CRFK cells. The intracellular concentration of
[Dmt.sup.1]DALDA was estimated to be approximately 50 times higher
than extracellular concentration.
[0131] In a separate experiment, cells were incubated with a range
of [Dmt.sup.1]DALDA concentrations (1 .mu.M-3 mM) for 1 h at
37.degree. C. At the end of the incubation period, cells were
washed 4 times with HBSS, and 0.2 mI of 0.1N NaOH with 1% SDS was
added to each well. The cell contents were then transferred to
scintillation vials and radioactivity counted. To distinguish
between internalized radioactivity from surface-associated
radioactivity, an acid-wash step was included. Prior to cell lysis,
cells were incubated with 0.2 ml of 0.2 M acetic acid/0.05 M NaCl
for 5 min on ice.
[0132] The uptake of [Dmt.sup.1]DALDA into Caco-2 cells was
confirmed by confocal laser scanning microscopy (CLSM) using a
fluorescent analog of [Dmt.sup.1]DALDA
(Dmt-D-Arg-Phe-dnsDap-NH.sub.2; where
dnsDap=.beta.-dansyl-1-.alpha.,.beta.-diaminopropionic acid). Cells
were grown as described above and were plated on (35 mm) glass
bottom dishes (MatTek Corp., Ashland, Mass.) for 2 days. The medium
was then removed and cells were incubated with 1 ml of HBSS
containing 0.1 .mu.M to 1.0 .mu.M of the fluorescent peptide analog
at 37.degree. C. for 1 h. Cells were then washed three times with
ice-cold HBSS and covered with 200 .mu.l of PBS, and microscopy was
performed within 10 min at room temperature using a Nikon confocal
laser scanning microscope with a C-Apochromat 63x/1.2 W corr
objective. Excitation was performed at 340 nm by means of a UV
laser, and emission was measured at 520 nm. For optical sectioning
in z-direction, 5-10 frames with 2.0 .mu.m were made.
[0133] CLSM confirmed the uptake of fluorescent
Dmt-D-Arg-Phe-dnsDap-NH.sub.2 into Caco-2 cells after incubation
with 0.1 .mu.M [Dmt.sup.1,DnsDap.sup.4]DALDA for 1 h at 37.degree.
C. The uptake of the fluorescent peptide was similar at 37.degree.
C. and 4.degree. C. The fluorescence appeared diffuse throughout
the cytoplasm but was completely excluded from the nucleus.
Example 2: Targeting of [Dmt.sup.1]DALDA to Mitochondria
[0134] To examine the subcellular distribution of [Dmt.sup.1]DALDA,
the fluorescent analog, [Dmt.sup.1, AtnDap.sup.4]DALDA
(Dmt-D-Arg-Phe-atnDap-NH.sub.2; where
atn=.beta.-anthraniloyl-1-.alpha.,.beta.,-diamino-propionic acid),
was prepared. The analog contained
.beta.-anthraniloyl-1-.alpha.,.beta.-diaminopropionic acid in place
of the lysine reside at position 4. The cells were grown as
described in Example 1 and were plated on (35 mm) glass bottom
dishes (MatTek Corp., Ashland, Mass.) for 2 days. The medium was
then removed and cells were incubated with 1 ml of HBSS containing
0.1 .mu.M of [Dmt.sup.1, AtnDap.sup.4]DALDA at 37.degree. C. for 15
min to 1 h.
[0135] Cells were also incubated with tetramethylrhodamine methyl
ester (TMRM, 25 nM), a dye for staining mitochondria, for 15 min at
37.degree. C. Cells were then washed three times with ice-cold HBSS
and covered with 200 .mu.l of PBS, and microscopy was performed
within 10 min at room temperature using a Nikon confocal laser
scanning microscope with a C-Apochromat 63x/1.2 W corr
objective.
[0136] For [Dmt.sup.1, AtnDap.sup.4]DALDA, excitation was performed
at 350 nm by means of a UV laser, and emission was measured at 520
nm. For TMRM, excitation was performed at 536 nm, and emission was
measured at 560 nm.
[0137] CLSM showed the uptake of fluorescent [Dmt.sup.1,
AtnDap.sup.4]DALDA into Caco-2 cells after incubation for as little
as 15 min at 37.degree. C. The uptake of dye was completely
excluded from the nucleus, but the blue dye showed a streaky
distribution within the cytoplasm. Mitochondria were labeled red
with TMRM. The distribution of [Dmt.sup.1, AtnDap.sup.4]DALDA to
mitochondria was demonstrated by the overlap of the [Dmt.sup.1,
AtnDap.sup.4]DALDA distribution and the TMRM distribution.
Example 3: Uptake of [Dmt.sup.1]DALDA into Mitochondria
[0138] To isolate mitochondria from mouse liver, mice were
sacrificed by decapitation. The liver was removed and rapidly
placed into chilled liver homogenization medium. The liver was
finely minced using scissors and then homogenized by hand using a
glass homogenizer.
[0139] The homogenate was centrifuged for 10 min at 1000.times.g at
4.degree. C. The supernatant was aspirated and transferred to
polycarbonate tubes and centrifuged again for 10 min. at
3000.times.g, 4.degree. C. The resulting supernatant was removed,
and the fatty lipids on the side-wall of the tube were carefully
wiped off.
[0140] The pellet was resuspended in liver homogenate medium and
the homogenization repeated twice. The final purified mitochondrial
pellet was resuspended in medium. Protein concentration in the
mitochondrial preparation was determined by the Bradford
procedure.
[0141] Approximately 1.5 mg mitochondria in 400 .mu.l buffer was
incubated with [.sup.3H][Dmt.sup.1]DALDA for 5-30 min at 37.degree.
C. The mitochondria were then centrifuged down and the amount of
radioactivity determined 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 [.sup.3H][Dmt.sup.1]DALDA in mitochondria was
found to be 200 times higher than in the buffer. Thus
[Dmt.sup.1]DALDA is concentrated in mitochondria.
[0142] Based on these data, the concentration of [Dmt.sup.1]DALDA
in mitochondria when the isolated guinea pig hearts were perfused
with [Dmt.sup.1]DALDA can be estimated:
TABLE-US-00007 Concentration of [Dmt.sup.1]DALDA in coronary
perfusate 0.1 .mu.M Concentration of [Dmt.sup.1]DALDA in myocyte 5
.mu.M Concentration of [Dmt.sup.1]DALDA in mitochondria 1.0 mM
Example 4: Accumulation of [Dmt.sup.1]DALDA by Isolated
Mitochondria (FIGS. 1A-1D)
[0143] To further demonstrate that [Dmt.sup.1]DALDA is selectively
distributed to mitochondria, we examined the uptake of [Dmt.sup.1,
AtnDap.sup.4]DALDA and [.sup.3H][Dmt.sup.1]DALDA into isolated
mouse liver mitochondria. The rapid uptake of [Dmt.sup.1,
AtnDap.sup.4]DALDA was observed as immediate quenching of its
fluorescence upon addition of mitochondria (FIG. 1 A). Pretreatment
of mitochondria with FCCP (carbonyl cyanide
p-(trifluoromethoxy)-phenylhydrazone), an uncoupler that results in
immediate depolarization of mitochondria, only reduced [Dmt.sup.1,
AtnDap.sup.4]DALDA uptake by <20%. Thus uptake of [Dmt.sup.1,
AtnDap.sup.4]DALDA was not potential-dependent.
[0144] To confirm that the mitochondrial targeting was not an
artifact of the fluorophore, we also examined mitochondrial uptake
of [.sup.3H][Dmt.sup.1]DALDA. Isolated mitochondria were incubated
with [.sup.3H][Dmt.sup.1]DALDA and radioactivity determined in the
mitochondrial pellet and supernatant. The amount of radioactivity
in the pellet did not change from 2 min to 8 min. Treatment of
mitochondria with FCCP only decreased the amount of
[.sup.3H][Dmt.sup.1]DALDA associated with the mitochondrial pellet
by .about.20% (FIG. 1B).
[0145] The minimal effect of FCCP on [Dmt.sup.1]DALDA uptake
suggested that [Dmt.sup.1]DALDA was likely to be associated with
mitochondrial membranes or in the intermembrane space rather than
in the matrix. We next examined the effect of mitochondrial
swelling on the accumulation of [Dmt.sup.1, AtnDap.sup.4]DALDA in
mitochondria by using alamethicin to induce swelling and rupture of
the outer membrane. Unlike TMRM, the uptake of [Dmt.sup.1,
AtnDap.sup.4]DALDA was only partially reversed by mitochondrial
swelling (FIG. 1C). Thus, [Dmt.sup.1]DALDA is associated with
mitochondrial membranes.
Example 5: [Dmt.sup.1]DALDA does not Alter Mitochondrial
Respiration or Potential (FIG. 1D)
[0146] The accumulation of [Dmt.sup.1]DALDA in mitochondria did not
alter mitochondrial function. Incubating isolated mouse liver
mitochondria with 100 .mu.M [Dmt.sup.1]DALDA did not alter oxygen
consumption during state 3 or state 4, or the respiratory ratio
(state 3/state 4) (6.2 versus 6.0). Mitochondrial membrane
potential was measured using TMRM (FIG. 1 D) Addition of
mitochondria resulted in immediate quenching of the TMRM signal
which was readily reversed by the addition of FCCP, indicating
mitochondrial depolarization. The addition of Ca.sup.2+ (150 .mu.M)
resulted in immediate depolarization followed by progressive loss
of quenching indicative of MPT. Addition of [Dmt.sup.1]DALDA alone,
even at 200 .mu.M, did not cause mitochondrial depolarization or
MPT.
Example 6: [Dmt.sup.1]DALDA Protects Against MPT Induced by
Ca.sup.2+ and 3-Nitropropionic Acid. (FIGS. 2A-2C)
[0147] In addition to having no direct effect on mitochondrial
potential, [Dmt.sup.1]DALDA was able to protect against MPT induced
by Ca.sup.2+ overload. Pretreatment of isolated mitochondria with
[Dmt.sup.1]DALDA (10 .mu.M) for 2 min prior to addition of
Ca.sup.2+ resulted only in transient depolarization and prevented
onset of MPT (FIG. 2A), [Dmt.sup.1]DALDA dose-dependently increased
the tolerance of mitochondria to cumulative Ca.sup.2+ challenges.
FIG. 2B shows that [Dmt.sup.1]DALDA increased the number of
Ca.sup.2+ additions that isolated mitochondria could tolerate prior
to MPT.
[0148] 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 caused
dissipation of mitochondrial potential and onset of MPT (FIG. 2C).
Pretreatment of mitochondria with [Dmt.sup.1]DALDA dose-dependently
delayed the onset of MPT induced by 3NP (FIG. 2C).
[0149] To demonstrate that [Dmt.sup.1]DALDA can penetrate cell
membranes and protect against mitochondrial depolarization elicited
by 3NP, Caco-2 cells were treated with 3NP (10 mM) in the absence
or presence of [Dmt.sup.1]DALDA (0.1 .mu.M) 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 was
much reduced, suggesting generalized depolarization. In contrast,
concurrent treatment with [Dmt.sup.1]DALDA protected against
mitochondrial depolarization caused by 3NP.
Example 7: [Dmt.sup.1]DALDA Protects Against Mitochondrial Swelling
and Cytochrome c Release
[0150] MPT pore opening results in mitochondrial swelling. We
examined the effects of [Dmt.sup.1]DALDA on mitochondrial swelling
by measuring reduction in absorbance at 540 nm (A.sub.540). The
mitochondrial suspension was then centrifuged and cytochrome c in
the mitochondrial pellet and supernatant determined by a
commercially-available ELISA kit. Pretreatment of isolated
mitochondria with SS-02 inhibited swelling (FIG. 3A) and cytochrome
c release (FIG. 3B) induced by Ca.sup.2+ overload. Besides
preventing MPT induced by Ca.sup.2+ overload, SS-02 also prevented
mitochondrial swelling induced by MPP.sup.+
(1-methyl-4-phenylpyridium ion), an inhibitor of complex I of the
mitochondrial electron transport chain (FIG. 3C).
Example 8: D-Arg-Dmt-Lys-Phe-NH.sub.2 (SS-31) can Protect Against
MPT, Mitochondrial Swelling and Cytochrome c Release
[0151] The non-opioid peptide SS-31 has the same ability to protect
against MPT (FIG. 4A), mitochondrial swelling (FIG. 4B), and
cytochrome c release (FIG. 4C), induced by Ca.sup.2+. The methods
for study are as described above for SS-02. In this example,
mitochondrial swelling was measured using light scattering
monitored at 570 nm.
Example 9: [Dmt.sup.1]DALDA (SS-02) and D-Arg-Dmt-Lys-Phc-NH.sub.2
(SS-31) Protects Against Ischemia-Reperfusion-Induced Myocardial
Stunning
[0152] Guinea pig hearts were rapidly isolated, and the aorta was
cannulated in situ and perfused in a retrograde fashion with an
oxygenated Krebs-Henseleit solution (pH 7.4) at 34.degree. C. The
heart was then excised, mounted on a modified Langendorff perfusion
apparatus, and perfused at constant pressure (40 cm H.sub.2O).
Contractile force was measured with a small hook inserted into the
apex of the left ventricle and the silk ligature tightly connected
to a force-displacement transducer. Coronary flow was measured by
timed collection of pulmonary artery effluent.
[0153] Hearts were perfused with buffer, [Dmt.sup.1]DALDA (SS-02)
(100 nM) or D-Arg-Dmt-Lys-Phe-NH.sub.2 (SS-31) (1 nM) for 30 min
and then subjected to 30 min of global ischemia. Reperfusion was
carried out with the same solution used prior to ischemia.
[0154] Two-way ANOVA revealed significant differences in
contractile force (P<0.001), heart rate (P=0.003), and coronary
flow (P<0.001) among the three treatment groups. In the buffer
group, contractile force was significantly lower during reperfusion
compared with before ischemia (FIG. 5). Both SS-02 and SS-31
treated hearts tolerated ischemia much better than buffer-treated
hearts (FIG. 5). In particular, SS-31 provided complete inhibition
of cardiac stunning. In addition, coronary flow is well-sustained
throughout reperfusion and there was no decrease in heart rate.
Example 10: [Dmt.sup.1]DALDA (SS-02) Enhances Organ
Preservation
[0155] For heart transplantation, the donor heart is preserved in a
cardioplegic solution during transport. The preservation solution
contains high potassium which effectively stops the heart from
beating and conserve energy. However, the survival time of the
isolated heart is still quite limited.
[0156] We examined whether [Dmt.sup.1]DALDA prolongs survival of
organs. In this study, [Dmt.sup.1]DALDA was added to a commonly
used cardioplegic solution (St. Thomas) to determine whether
[[Dmt.sup.1]DALDA enhances survival of the heart after prolonged
ischemia (model of ex vivo organ survival).
[0157] Isolated guinea pig hearts were perfused in a retrograde
fashion with an oxygenated Krebs-Henseleit solution at 34.degree.
C. After 30 min. of stabilization, the hearts were perfused with a
cardioplegic solution CPS (St. Tohomas) with or without
[Dmt.sup.1]DALDA at 100 nM for 3 min. Global ischemia was then
induced by complete interruption of coronary perfusion for 90 min.
Reperfusion was subsequently carried out for 60 min. with
oxygenated Krebs-Henseleit solution. Contractile force, heart rate
and coronary flow were monitored continuously throughout the
experiment.
[0158] The addition of [Dmt.sup.1]DALDA to cardioplegic solution
significantly enhanced contractile function (FIG. 6) after
prolonged ischemia.
Example 11: D-Arg-Dmt-Lys-Phe-NH.sub.2 (SS-31) Prevented
tBHP-Induced Mitochondrial Depolarization
[0159] To investigate whether SS-31 prevents mitochondria)
depolarization caused by tBHP, N2A cells were treated with 50 .mu.M
tBHP for 6 h. Treatment with tBHP resulted in a dramatic loss of
mitochondrial potential. Fluorescence intensity of TMRM (red), a
cationic indicator that is taken up into mitochondria in a
potential dependent manner, was significantly lower in cells
treated with 50 .mu.M tBHP (FIG. 7), and this was completely
blocked by concurrent treatment with 1 nM SS-31 (FIG. 7).
Example 12: D-Arg-Dmt-Lys-Phe-NH.sub.2 (SS-31) and
Phe-D-Arg-Phe-Lys-NH.sub.2 (SS-20) Prevents Myocardial Infarction
in Rats
Methods
Infarction/Reperfusion
[0160] Adult male Sprague-Dawley rats (F344 strain, National
Institute of Aging, maintained by Harian Sprague-Dawley Inc.),
weighting between 285 and 425 gm, were used. Twenty-four rats
(n=8/group) were randomly assigned to one of three groups: (1)
Control group was given 0.4 ml of saline as an intra-peritoneal
(IP) injection 30 minutes before the ligation of left anterior
descending coronary artery, followed by same IP dose injection 5
minutes before reperfusion; (2) SS-31 group was treated with SS-31
(3 mg/kg dissolved in 0.4 ml of saline) as an IP injection 30
minutes before the ligation followed by same IP dose injection as
maintenance 5 minutes before reperfusion; and (3) SS-20 group was
treated with SS-20 (3 mg/kg dissolved in 0.4 ml of saline) as an IP
injection 30 minutes before the ligation followed by same dose IP
injection as maintenance just 5 minutes before reperfusion. Also,
Sham-operated rats (n=3) were used to account for possible effects
related to the surgical protocol. All procedures were performed in
a blinded manner, with the groups assigned letters and their
identities unknown to the operators. Likewise, the two independent
investigators analyzing the data were blind to the treatment
assignments.
[0161] After anesthesia with ketamine (90 mg/kg IP) and xylazine (4
mg/kg IP), a tracheotomy was performed, and the rat was intubated
with polyethylene tube and ventilated (Harvard Rodent Ventilator
model 683) with room air and a tidal volume of 0.65 ml/100 gm of
body weight at 90 breaths per minute. Body temperature was
maintained at 37.degree. C. by using a heated operating table. The
internal jugular vein was surgically exposed and a polyethylene
tube was inserted for Evans blue dye solution injection. Peripheral
limb electrodes were inserted subcutaneously and electrocardiogram
was monitored throughout the procedure.
[0162] The chest was opened by left thoracotomy at the fourth
intercostal space to expose the heart. The pericardium was removed,
and the left atrial appendage was gently moved to reveal the
location of the left coronary artery. The vein descending along the
septum of the heart was used as the marker for the left coronary
artery. A suture ligature (7.0 Prolene) along with a snare occluder
was placed around the vein and left coronary artery close to the
place of origin. The left anterior descending coronary artery was
occluded by applying tension to the sling through a polyethylene 10
tubing and clamping. Successful occlusion was confirmed by
elevation of the ST segment or the presence of deep S wave on the
ECG and by cyanosis of the anterior wall of the left ventricle.
Sixty minutes after occlusion, the snare occluder was released and
reperfusion of the myocardium was visually confirmed. The heart was
then reperfused for sixty minutes. The heart was arrested in
diastole with an overdose of KCl and rapidly excised at the end of
the experiment. The sham-operated control group was subjected to
thoracotomy and passage of a silk ligature around the left coronary
artery without ligation.
Determination of Area at Risk and Area of Infarction
[0163] At the end of reperfusion, the left coronary artery was
briefly re-occluded and Evans blue dye solution (2 ml of 2%, Sigma)
was slowly injected into the jugular vein to distinguish the
perfused area (blue staining) from the area at risk (no staining).
The excised hearts were cut parallel to the atrioventricular groove
into 5 slices (.about.1 mm thick) from base to apex. After removing
all atrial and right ventricle tissues, all slices were scanned.
The slices were incubated in a 2% solution of triphenyl-tetrazolium
chloride (TTC, Sigma) in phosphate buffer for 20 min at 37.degree.
C. and pH of 7.4, and then immersed in 10% buffered formaldehyde
for 14 days to distinguish the infarct area (unstained) from the
viable myocardium (brick red staining). A prolonged formalin
fixation was used to make infarct border zones easier to visualize.
After a 14-day formalin immersion, the slices were scanned again
and all scanned areas were quantified with NIH Imagesoftware. Area
at risk (AAR) was expressed as a percentage of the left ventricle
(LV). Area of infarction (AI) was calculated as a percentage of the
AAR.
Assessment of Arrhythmias
[0164] An ECG was recorded continuously from a standard lead II (AC
AMP 700) inserted into the limbs of all rats. The ECG was printed
at 25 mm/second. The cardiac arrhythmias were monitored and
assessed in accordance with the Lambeth Convention. The assessment
was performed in a blinded manner using the original paper
recordings. A validated score was used to quantify the severity of
cardiac arrhythmias. The score consisted of six types: 0, no
ventricular extrasystoles (VES), ventricular tachycardia (VT) or
ventricular fibrillation (VF); 1=VES; 2=one to five episodes of VT
(more than four coupled VES); 3=more than five episodes of VT
and/or one VF; 4=two to five episodes of VF; 5=more than five
episodes of VF.
Statistical Analysis
[0165] All values are expressed as mean.+-.standard error of mean
(S.E.M.). Statistical analyses were performed using SPSS version
10. Differences among groups in body weight, heart rate,
arrhythinia, area at risk as a percentage of the left ventricle,
and area of infarction as a percentage of the area at risk were
analyzed using ANOVA. If significant differences were detected,
comparisons between the control group and the 2 treatment groups
were conducted using the Mann-Whitney U-test. A two-tailed
p<0.05 was regarded as significant.
Results
Risk Areas and Infarct Sizes
[0166] Representative slices of left ventricle form control, SS-31
and SS-20 groups are shown in FIG. 8. The AAR/LV ratio was similar
among the three groups (52.1.+-.2.5% in the control group,
55.9.+-.1.4%, p=0.38 in the SS-31 group, and 52.+-.2.1%, p=0.2 in
the SS-20 group; FIG. 9). However, the AI/AAR ratio (FIG. 10) was
significantly smaller in the SS-31 group (53.9.+-.1.1%, p<0.01),
in the SS-20 group (47.1.+-.1.4%, p<0.01) than in the control
group (59.9.+-.1). Meanwhile, AAR/LV ratio of sham group was
50.3.+-.4.6% (p=0.78 vs control) and AI/AAR ratio of sham group was
3.7.+-.3.7% (p<0.05 vs control).
Heart Rate and Cardiac Arrhythmias
[0167] Compared with controls, there were no significant
differences in body weight and heart rate in any of the two groups
during the study period (Table 3). Almost all arrhythmias occurred
between 2 and 15 min after coronary occlusion. These rats with
arrhythmias showed isolated ventricular extrasystole or
nonsustained ventricular tachycardia (VT), but not ventricular
fibrillation. They were transient and recovered without therapy.
Severity and occurrence rate of cardiac arrhythmias in SS-31 group
(5 points, p<0.05) and SS-20 group (3 points, p<0.005) showed
a significant reduction compared with control (13 points) during
the entire study period.
TABLE-US-00008 TABLE 3 Body weight of all rats and heart rate
during the course of the experiment. Heart Rate Body Open weight
Baseline chest Ischemia Ischemia Reperfusion Reperfusion Control
351 .+-. 18 246 .+-. 4 212 .+-. 9 186 .+-. 10 166 .+-. 8 176 .+-.
16 161 .+-. 4 (n = 8) SS-31 340 .+-. 12 257 .+-. 10 236 .+-. 8 184
.+-. 14 169 .+-. 15 161 .+-. 12 164 .+-. 13 (n = 8) SS-20 349 .+-.
18 255 .+-. 5 211 .+-. 10 191 .+-. 17 184 .+-. 15 182 .+-. 16 183
.+-. 12 (n + 8) All values are expressed as means .+-. S.E.M. There
was no difference among study groups (P < 0.05).
Sequence CWU 1
1
2718PRTartificial sequencearomatic-cationic peptide 1Tyr Arg Phe
Lys Glu His Trp Arg1 526PRTartificial sequencearomatic-cationic
peptide 2Lys Gln Tyr Arg Phe Trp1 535PRTartificial
sequencearomatic-cationic peptideMISC_FEATURE(2)..(2)D-amino
acidMISC_FEATURE(5)..(5)amidation 3Tyr Arg Phe Lys Glu1
545PRTartificial sequencearomatic-cationic
peptideMISC_FEATURE(3)..(3)D-amino acid 4Met Tyr Lys Phe Arg1
556PRTartificial sequencearomatic-cationic
peptideMISC_FEATURE(1)..(1)D-amino acidMISC_FEATURE(5)..(5)D-amino
acid 5His Glu Lys Tyr Phe Arg1 566PRTartificial
sequencearomatic-cationic peptideMISC_FEATURE(2)..(2)D-amino
acidMISC_FEATURE(5)..(5)D-amino acidMISC_FEATURE(6)..(6)amidation
6Lys Gln Tyr Arg Phe Trp1 577PRTartificial
sequencearomatic-cationic peptideMISC_FEATURE(2)..(2)D-amino
acidMISC_FEATURE(6)..(6)D-amino acid 7Phe Arg Lys Trp Tyr Arg His1
587PRTartificial sequencearomatic-cationic
peptideMISC_FEATURE(2)..(2)D-amino acidMISC_FEATURE(6)..(6)D-amino
acidMISC_FEATURE(7)..(7)amidation 8Gly Phe Lys Tyr His Arg Tyr1
598PRTartificial sequencearomatic-cationic
peptideMISC_FEATURE(2)..(2)D-amino acidMISC_FEATURE(5)..(5)D-amino
acidMISC_FEATURE(8)..(8)amidation 9Val Lys His Tyr Phe Ser Tyr Arg1
5108PRTartificial sequencearomatic-cationic
peptideMISC_FEATURE(4)..(4)D-amino acidMISC_FEATURE(7)..(7)D-amino
acid 10Trp Lys Phe Asp Arg Tyr His Lys1 5118PRTartificial
sequencearomatic-cationic peptideMISC_FEATURE(3)..(3)D-amino
acidMISC_FEATURE(8)..(8)D-amino acid and amidation 11Lys Trp Tyr
Arg Asn Phe Tyr His1 5129PRTartificial sequencearomatic-cationic
peptideMISC_FEATURE(5)..(5)D-amino acidMISC_FEATURE(8)..(8)D-amino
acid 12Thr Gly Tyr Arg His Phe Trp His Lys1 5139PRTartificial
sequencearomatic-cationic peptideMISC_FEATURE(2)..(2)D-amino
acidMISC_FEATURE(5)..(5)D-amino acidMISC_FEATURE(8)..(8)D-amino
acidMISC_FEATURE(9)..(9)amidation 13Asp Trp Lys Tyr His Phe Arg Gly
Lys1 51410PRTartificial sequencearomatic-cationic
peptideMISC_FEATURE(1)..(1)D-amino acidMISC_FEATURE(4)..(4)D-amino
acidMISC_FEATURE(6)..(8)D-amino acidMISC_FEATURE(10)..(10)amidation
14His Lys Tyr Phe Glu Asp His Lys Arg Trp1 5 101510PRTartificial
sequencearomatic-cationic peptideMISC_FEATURE(2)..(3)D-amino
acidMISC_FEATURE(6)..(6)D-amino acidMISC_FEATURE(8)..(8)D-amino
acid 15Ala Phe Arg Tyr Lys Trp His Tyr Gly Phe1 5
101611PRTartificial sequencearomatic-cationic
peptideMISC_FEATURE(2)..(2)D-amino acidMISC_FEATURE(4)..(4)D-amino
acidMISC_FEATURE(7)..(7)D-amino acidMISC_FEATURE(10)..(10)D-amino
acid 16Tyr His Phe Arg Asp Lys Arg His Trp His Phe1 5
101711PRTartificial sequencearomatic-cationic
peptideMISC_FEATURE(3)..(3)D-amino acidMISC_FEATURE(7)..(7)D-amino
acidMISC_FEATURE(9)..(9)D-amino acidMISC_FEATURE(11)..(11)amidation
17Phe Phe Tyr Arg Glu Asp Lys Arg Arg His Phe1 5
101812PRTartificial sequencearomatic-cationic
peptideMISC_FEATURE(4)..(4)D-amino acidMISC_FEATURE(7)..(8)D-amino
acidMISC_FEATURE(11)..(11)D-amino acid 18Phe Tyr Lys Arg Trp His
Lys Lys Glu Arg Tyr Thr1 5 101912PRTartificial
sequencearomatic-cationic peptideMISC_FEATURE(3)..(3)D-amino
acidMISC_FEATURE(6)..(7)D-amino acidMISC_FEATURE(10)..(10)D-amino
acid 19Tyr Asp Lys Tyr Phe Lys Arg Phe Pro Tyr His Lys1 5
102013PRTartificial sequencearomatic-cationic
peptideMISC_FEATURE(3)..(3)D-amino acidMISC_FEATURE(5)..(5)D-amino
acidMISC_FEATURE(7)..(7)D-amino acidMISC_FEATURE(10)..(10)D-amino
acidMISC_FEATURE(13)..(13)D-amino acid and amidation 20Glu Arg Lys
Tyr Val Phe His Trp Arg Gly Tyr Arg Met1 5 102114PRTartificial
sequencearomatic-cationic peptideMISC_FEATURE(2)..(3)D-amino
acidMISC_FEATURE(7)..(7)D-amino acidMISC_FEATURE(9)..(9)D-amino
acidMISC_FEATURE(11)..(11)D-amino
acid13MISC_FEATURE(13)..(13)D-amino acid 21Arg Leu Tyr Phe Lys Glu
Lys Arg Trp Lys Phe Tyr Arg Gly1 5 102215PRTartificial
sequencearomatic-cationic peptideMISC_FEATURE(1)..(1)D-amino
acidMISC_FEATURE(4)..(5)D-amino acidMISC_FEATURE(8)..(8)D-amino
acidMISC_FEATURE(12)..(12)D-amino
acidMISC_FEATURE(15)..(15)amidation 22Glu Asp Lys Arg His Phe Phe
Val Tyr Arg Tyr Tyr Arg His Phe1 5 10 152316PRTartificial
sequencearomatic-cationic peptideMISC_FEATURE(3)..(3)D-amino
acidMISC_FEATURE(6)..(7)D-amino acidMISC_FEATURE(10)..(10)D-amino
acidMISC_FEATURE(12)..(12)D-amino acidMISC_FEATURE(14)..(14)D-amino
acid 23Asp Arg Phe Cys Phe Arg Lys Tyr Arg Tyr Trp His Tyr Phe Lys
Phe1 5 10 152417PRTartificial sequencearomatic-cationic
peptideMISC_FEATURE(3)..(3)D-amino acidMISC_FEATURE(7)..(7)D-amino
acidMISC_FEATURE(11)..(11)D-amino acidMISC_FEATURE(14)..(14)D-amino
acidMISC_FEATURE(17)..(17)amidation 24His Tyr Arg Trp Lys Phe Asp
Ala Arg Cys Tyr His Phe Lys Tyr His1 5 10 15Ser2519PRTartificial
sequencearomatic-cationic peptideMISC_FEATURE(5)..(5)D-amino
acidMISC_FEATURE(10)..(11)D-amino acidMISC_FEATURE(15)..(15)D-amino
acidMISC_FEATURE(18)..(18)D-amino acid 25Gly Ala Lys Phe Lys Glu
Arg Tyr His Arg Arg Asp Tyr Trp His Trp1 5 10 15His Lys
Asp2620PRTartificial sequencearomatic-cationic
peptideMISC_FEATURE(4)..(4)D-amino acidMISC_FEATURE(9)..(10)D-amino
acidMISC_FEATURE(13)..(13)D-amino acidMISC_FEATURE(17)..(17)D-amino
acidMISC_FEATURE(20)..(20)amidation 26Thr Tyr Arg Lys Trp Tyr Glu
Asp Lys Arg His Phe Tyr Gly Val Ile1 5 10 15His Arg Tyr Lys
20275PRTArtificial sequencearomatic-cationic
peptideMISC_FEATURE(1)..(1)2'6'
dimethyltyrosineMISC_FEATURE(2)..(2)D-amimo
acidMISC_FEATURE(5)..(5)amidation 27Tyr Arg Phe Lys Cys1 5
* * * * *