U.S. patent application number 16/798085 was filed with the patent office on 2021-01-21 for methods for reducing oxidative damage.
This patent application is currently assigned to Cornell Research Foundation, Inc.. The applicant listed for this patent is Cornell Research Foundation, Inc.. Invention is credited to Hazel H. Szeto.
Application Number | 20210015887 16/798085 |
Document ID | / |
Family ID | 1000005120469 |
Filed Date | 2021-01-21 |
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United States Patent
Application |
20210015887 |
Kind Code |
A1 |
Szeto; Hazel H. |
January 21, 2021 |
METHODS FOR REDUCING OXIDATIVE DAMAGE
Abstract
The invention provides a method for reducing oxidative damage in
a mammal, a removed organ, or a cell in need thereof The method
comprises administering an effective amount of an aromatic cationic
peptide. The aromatic cationic peptide has (a) at least one net
positive charge; (b) a minimum of three 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 3 p.sub.m is the largest number
that is less than or equal to r+1; (e) a relationship between the
minimum number of aromatic groups (a) and the total number of net
positive charges (p.sub.t) wherein 3a or 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; and (f) at least one tyrosine or tryptophan
amino acid.
Inventors: |
Szeto; Hazel H.; (New York,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cornell Research Foundation, Inc. |
Ithaca |
NY |
US |
|
|
Assignee: |
Cornell Research Foundation,
Inc.
Ithaca
NY
|
Family ID: |
1000005120469 |
Appl. No.: |
16/798085 |
Filed: |
February 21, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15956941 |
Apr 19, 2018 |
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16798085 |
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14955412 |
Dec 1, 2015 |
9950026 |
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15956941 |
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14100626 |
Dec 9, 2013 |
9623069 |
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14955412 |
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12843333 |
Jul 26, 2010 |
8618061 |
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14100626 |
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11428188 |
Jun 30, 2006 |
7781405 |
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12843333 |
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11040242 |
Jan 21, 2005 |
7550439 |
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11428188 |
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60538841 |
Jan 23, 2004 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/08 20130101;
C07K 5/1019 20130101; A61K 38/10 20130101; A61K 38/07 20130101 |
International
Class: |
A61K 38/07 20060101
A61K038/07; A61K 38/08 20060101 A61K038/08; A61K 38/10 20060101
A61K038/10; C07K 5/11 20060101 C07K005/11 |
Goverment Interests
GOVERNMENT INTEREST
[0002] This invention was made with government support from the
National Institute on Drug Abuse under Grant No. P01 DA08924. The
U.S. government has certain rights in this invention.
Claims
1. A method for preventing loss of dopamine-producing neurons in a
mammal having or suspected of having Parkinson's disease, the
method comprising administering to the mammal an effective amount
of a peptide having the formula D-Arg-Dmt-Lys-Phe-NH.sub.2.
2. The method according to claim 1, wherein the mammal is a
human.
3. The method according to claim 1, wherein the peptide is
administered orally, topically, intranasally, systemically,
intravenously, subcutaneously, intramuscularly,
intracerebroventricularly, intrathecally, or transdermaliy.
4. The method of claim 1, wherein the peptide is mixed with a
pharmaceutically acceptable carrier.
5. A method for treating amyotrophic lateral sclerosis (ALS) in a
mammal, the method comprising administering to the mammal an
effective amount of a peptide having the formula
D-Arg-Dmt-Lys-Phe-NH.sub.2.
6. The method according to claim 5, wherein the mammal is a
human.
7. The method according to claim 5, wherein the peptide is
administered orally, topically, intranasally, systemically,
intravenously, subcutaneously, intramuscularly,
intracerebroventricularly, intrathecally, or transdermally.
8. The method of claim 5, wherein the peptide is mixed with a
pharmaceutically acceptable carrier.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/956,941, filed Apr. 19, 2018, which is a
continuation of U.S. patent application Ser. No. 14/955,412, filed
Dec. 1, 2015, now U.S. Pat. No. 9,950,026, which is a continuation
of U.S. patent application Ser. No. 14/100,626, filed Dec. 9, 2013,
now U.S. Pat. No. 9,623,069, which is a continuation of U.S. patent
application Ser. No. 12/843,333, filed Jul. 26, 2010, now U.S. Pat.
No. 8,618,061, which is a continuation of U.S. patent application
Ser. No. 11/428,188, filed Jun. 30, 2006, now U.S. Pat. No.
7,781,405, which is a continuation application of U.S. patent
application Ser. No. 11/040,242 filed on Jan. 21, 2005, now U.S.
Pat. No. 7,550,439, which claims priority to U.S. Provisional
Patent Application No. 60/538,841 filed on Jan. 23, 2004, the
contents of which are hereby incorporated by reference in their
entireties.
BACKGROUND OF THE INVENTION
[0003] Mitochondria are essential to cell survival as the main
producers of ATP via oxidative phosphorylation. However, the
mitochondria respirator chain is also a major source of oxidative
free radicals. For example, radical production can occur as a
result of the reaction of mitochondrial electron carriers, such as
ubiquinol, with oxygen to form a superoxide. Superoxides react by
dismutation to hydrogen peroxide, which can decompose to hydroxyl
radical. In addition, superoxides react with nitric oxide to form
peroxynitrite and other reactive oxidants.
[0004] Aging is associated not only with increased reactive oxygen
species (ROS) production, but also a decrease in the endogenous
antioxidant defense mechanisms. Mitochondria are particularly
vulnerable to oxidative stress because they are continuously
exposed to ROS. As a consequence, mitochondria decay is often
associated with aging.
[0005] Free radicals, including ROS, and reactive nitrogen species
(RNS) produce diverse non-specific damage to biological molecules,
including lipids, proteins, RNA and DNA. Such damage of these
molecules has been implicated in numerous clinical disorders, such
as atherosclerosis, preeclampsia, Alzheimer's disease, Parkinson's
disease and arthritis.
[0006] Antioxidant therapy can potentially delay the aging process,
and be beneficial in a host of human diseases and conditions such
as those described above. However, the development of specific
mitochondrial therapies has been hampered by the difficulty of
delivering antioxidant molecules to mitochondria in vivo. For
example, the molecule must first be taken up across the plasma
membrane into the cytoplasm, and then targeted selectively to
mitochondria.
[0007] None of the currently available antioxidant compounds
specifically target mitochondria. The endogenous antioxidants,
superoxide dismutase and catalase, are poorly absorbed orally, have
short half-lives, and do not cross the blood-brain barrier. The
natural antioxidants (e.g., Vitamin E, coenzyme Q, polyphenols) are
not water-soluble and tend to accumulate in cell membranes and only
cross the blood-brain barrier slowly.
[0008] Therefore, there is a need for improved methods of reducing
oxidative damage with antioxidative compounds that cross cell
membranes. In addition, it would also be beneficial for the
antioxidative compounds to specifically target mitochondria.
SUMMARY OF THE INVENTION
[0009] These and other objectives have been met by the present
invention which provide a method for reducing oxidative damage in a
mammal in need thereof. The method comprises administering to the
mammal an effective amount of an aromatic cationic peptide. The
aromatic cationic peptide have (a) at least one net positive
charge; (b) a minimum of three 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; (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; and (f) at least one tyrosine or tryptophan amino
acid.
[0010] In another embodiment, the invention also provides a method
of reducing oxidative damage in a removed organ of a mammal. The
method comprises administering to the removed organ an effective
amount of an aromatic-cationic peptide. The aromatic-cationic
peptide have (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;
(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; and (f) at least
one tyrosine or tryptophan amino acid.
[0011] In a further embodiment, the invention provides a method of
reducing oxidative damage in a mammal in need thereof. The method
comprises administering) to the mammal an effective amount of an
aromatic-cationic peptide. The aromatic-cationic peptide have (a)
at least one net positive charge; (b) a minimum of three 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;
(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, and (f) at least
one tyrosine or tryptophan amino acid.
[0012] In yet a further embodiment the invention provides a method
of reducing oxidative damage in a removed organ of a mammal. The
method comprises administering to the removed organ an effective
amount of an aromatic-cationic peptide. The aromatic cationic
peptide have (a) at least one net positive charge; (b) a minimum of
three 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;
(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, and (f) at least
one tyrosine or tryptophan amino acid.
[0013] In yet another embodiment, the invention provides a method
of reducing, oxidative damage in a cell in need thereof. The
aromatic cationic peptide have (a) at least one net positive
charge; (b) a minimum of three 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; (e) a relationship between the minimum
number of aromatic groups (a) and the total number of net positive
charges (p.sub.t) wherein 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, and (f) at least one tyrosine or tryptophan amino acid.
[0014] In an additional embodiment, the invention provides a method
of reducing oxidative damage in a cell in need thereof. The
aromatic cationic peptide have (a) at least one net positive
charge; (b) a minimum of three 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; (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, and (f) at least one tyrosine or tryptophan amino
acid.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIGS. 1A-1B. (FIG. 1A) SS-02 and (FIG. 1B) SS-05
dose-dependently scavenge H202.
[0016] FIGS. 2A-2B. (FIG. 2A) SS-02 dose-dependently inhibits
linoleic acid peroxidation induced by ABAP and (FIG. 2B) SS-02
SS-05, SS-29, SS-30, SS-31, SS-32 and Dmt reduced the rate of
linoleic acid peroxidation induced by ABAP.
[0017] FIGS. 3A-3B. (FIG. 3A) SS-02 dose-dependently inhibits LDL
oxidation induced by 10 mM CuSO.sub.4 and (FIG. 3B) SS-02, SS-05,
SS-29, SS-30, SS-31, SS-32 and Dmt reduced rate of LDL
oxidation.
[0018] FIGS. 4A-4B. (FIG. 4A) SS-02 inhibits mitochondrial
production of hydrogen peroxide as measured by luminol
chemiluminescence under basal conditions and upon stimulation by
antimycin. (FIG. 4B) SS-02, SS-29, SS-30 and SS-31 reduced
spontaneous generation of hydrogen peroxide generated by isolated
mitochondria.
[0019] FIGS. 5A-5B. (FIG. 5A) SS-31 inhibits spontaneous production
of hydrogen hydroperoxide by isolated mitochondria and (FIG. 5B)
SS-31 inhibits hydrogen peroxide production stimulated by
antimycin.
[0020] FIGS. 6A-6C. SS-31 dose-dependently decreased intracellular
ROS (reactive oxygen species) (FIG. 6A) and increased cell survival
(FIG. 6B) in N2A cells exposed to a high dose of the pro-oxidant
t-butyl hydroperoxide (t-BHP; 0.5 mM), (FIG. 6C) S S-02 also
dose-dependently increased cell survival when N2A cells were
exposed to 1 mM t-BHP.
[0021] FIGS. 7A-7B. SS-31 dose-dependently prevented loss of cell
viability caused by low doses of t-BHP (0.05-0.1 mM) in neuronal
(FIG. 7A) SH-SYSY and (FIG. 7B) N.sub.2A cells.
[0022] FIG. 8. SS-31 dose-dependently decreased the percent of
cells showing increased caspase activity after treatment with a low
dose of t-BHP for 12 h in N2A cells.
[0023] FIG. 9. SS-31 dose-dependently reduced the rate of ROS
accumulation in N.sub.2A cells with 0.1 mM t-BHP over a 4 h
period.
[0024] FIGS. 10A-10C. SS-31 inhibited lipid peroxidation caused by
exposure of N.sub.2A cells to 1 mM t-BHP for 1 h. (FIG. 10A)
untreated cells; (FIG. 10B) cells treated with 1 mM t-BHP for 3 h;
(FIG. 10C) cells treated with 1 mM t-BHP and 10 nM SS-31 for 3
h.
[0025] FIG. 11. SS-31 prevented mitochondrial depolarization and
ROS accumulation in N2A cells exposed to t-BHP.
[0026] FIGS. 12A-12D. SS-31 prevents apoptosis induced by a low
dose of t-BHP. Apoptosis was evaluated by confocal microscopy with
the fluorescent probe Hoechst 33342. (FIG. 12A) a representative
field of cells not treated with t-BHP. (FIG. 12AA) Fluorescent
image showing a few cells with dense, fragmented chromatin
indicative of apoptotic nuclei. (FIG. 12B) A representative field
of cells treated with 0.025 mM t-BHP for 24 h. (FIG. 12BB)
Fluorescent image showing an increased number of cells with
apoptotic nuclei. (FIG. 12C) A representative field of cells
treated with 0.025 mM t-BHP and 1 nM SS-31 for 24 h. (FIG. 12CC)
Fluorescent image showing a reduced number of cells with apoptotic
nuclei. (FIG. 12D) SS-31 dose-dependently reduced the percent of
apoptotic cells caused by 24 h treatment with a low dose of T-BHP
(0.05 mM).
[0027] FIG. 13A(a-e). SS-02 and SS-31 reduced lipid peroxidation in
isolated guinea pig hearts subjected to warm reperfusion after a
brief period of ischemia. Immunohistochemical analysis of
4-hydroxy-2-nonenol (HNE)-modified proteins in paraffin sections
from guinea pig hearts aerobically perfused 30 min with (a) buffer;
(b) 100 nM SS-02; (c) 100 nM SS-20 and (d) 1 nM SS-31, then
subjected to 30 min ischemia and reperfused for 90 min with
corresponding peptides. Tissue slices were incubated with anti-HNE
antibody. (e) Background control: staining without primary
antibody.
[0028] FIG. 13B(a-e). SS-02 and SS-31 reduced lipid peroxidation in
isolated guinea pig hearts subjected to warm reperfusion after a
brief period of ischemia. Immunohistochemical analysis of
4-hydroxynonenol (HNE)-modified proteins in paraffin sections from
guinea pig hearts aerobically perfused 30 min with buffer, then
subjected to 30 min ischemia and reperfused with (a) buffer; (b)
100 nM SS-02; (c) 100 nM SS-20 and (d) 1 nM SS-31 for 90 min.
Tissue slices were incubated with anti-FINE antibody. (e)
Background control: staining without primary antibody.
[0029] FIG. 14A. SS-31 significantly improved coronary flow in
isolated guinea pig hearts subjected to warm reperfusion after
prolonged (18 h) cold ischemia. The shaded area represents 18 h of
ischemia at 4.degree. C.
[0030] FIG. 14B(a-c). Guinea pig hearts perfused with a
cardioplegic solution (St. Thomas solution) without (a) or with (b)
1 nM SS-31 for 3 min and then subjected to 18 h of cold ischemia
(4.degree. C.), (c) background staining with primary antibody. The
hearts were then reperfused with buffer at 34.degree. C. for 90
min.
[0031] FIG. 14C. SS-31 prevents apoptosis in endothelial cells and
myocytes in isolated guinea pig hearts subjected to warm
reperfusion after prolonged (18 h) cold ischemia. Guinea pig hearts
perfused with a cardioplegic solution (St. Thomas solution) without
or with nM SS-31 for 3 min and then subjected to 18 h of cold
ischemia (4.degree. C.). The hearts were then reperused with buffer
at 34.degree. C. for 90 min. Apoptosis was assessed by the TUNEL
stain (green) and nuclei are visualized by DAPI (blue).
[0032] FIG. 15A. SS-31 improves survival of islet cells isolated
from mouse pancreas as measured by mitochondrial potential. SS-31
(nM) was added to all isolation buffers used throughout the
isolation procedure. Mitochondrial potential was measured using
TMRM (red) with confocal microscopy.
[0033] FIGS. 15B & 15C. SS-31 reduces apoptosis and increases
viability in islet cells isolated from mouse pancreas as measured
by flow cytometry. SS-31 (1 nM) was added to all isolation buffers
used throughout the isolation procedure. Apoptosis was ascertained
using annexin V and necrosis by propidium iodide (PI).
[0034] FIGS. 16A-16C. SS-31 reduces oxidative damage in pancreatic
islet cells caused by t-butylhydroperoxide (tBHP). Mouse pancreatic
islet cells were untreated (FIG. 16A), or treated with 25 .mu.M
tBHP without (FIG. 16B) or with 1 nM SS-31 (FIG. 16C).
Mitochondrial potential was measured by TMRM (red) and reactive
oxygen species were measured by DCF (green) using confocal
microscopy.
[0035] FIG. 17A. SS-31 protects dopamine cells against MPP*
toxicity. SN-4741 cells were treated with buffer, 50 .mu.M MPP* or
50 .mu.M MPP* and 1 nM SS-31, for 48 h, and the incidence of
apoptosis was determined by fluorescent microscopy with Hoechst
33342. The number of condensed fragmented nuclei was significantly
increased by MPP* treatment. Concurrent treatment with SS-31
reduced the number of apoptotic cells.
[0036] FIGS. 17B & FIG. 17BB. SS-31 dose-dependently prevented
loss of dopamine neurons in mice treated with MPTP. Three doses of
MPTP (10 mg/kg) was given to mice (n=12) 2 h apart. SS-31 was
administered 30 min before each MPTP injection, and at 1 h and 12 h
after the last MPTP injection. Animals were sacrificed one week
later and striatal brain reactions were immunostained for tyrosine
hydroxylase activity (shown in black).
[0037] FIGS. 17C-17E. SS-31 dose-dependently increased striatal
dopamine, DOPAC (3,4-dihydroxyphenylacetic acid) and HVA
(homovanillic acid) levels in mice treated with MPTP. Three doses
of MPTP (10 mg/kg) was given to mice (n=12) 2 h apart. SS-31 was
administered 30 min before each MPTP injection, and at 1 h and 12 h
after the last injection. Animals were sacrificed one week later
and dopamine, DOPAC and HVA levels were quantified by high pressure
liquid chromatography.
[0038] FIGS. 18A-18B. SS-31 reduced tBHP-induced LDH release in
SH-SYSY (FIG. 18A) and N.sub.2A (FIG. 18B) cells. Cells were
treated with 100 .mu.M tBHP alone, or with SS-31, for 24 h.
*P<0.05, **P<0.01, ***P<0.001, compared to tBHP alone.
[0039] FIGS. 19A-19C. SS-31 reduced tBHP-induced apoptosis as
demonstrated by phosphatidylserine translocation. N.sub.2A cells
were incubated with 50 .mu.M tBHP for 6 h and stained with Annexin
V and propidium iodide (PI). (19A) Untreated cells showed little
Annexin V stain and no PI stain. (FIG. 19B) Cells treated with tBHP
showed intense Annexin V staining (green) in most cells. Combined
staining with Annexin V and PI (red) indicate late apoptotic cells.
(FIG. 19C) Concurrent treatment with 1 nM SS-31 resulted in a
reduction in Annexin V-positive cells and no PI staining.
[0040] FIGS. 20A-20C. SS-31 reduced tBHP-induced apoptosis as
demonstrated by nuclear condensation. (FIG. 20A)(a-c; a'-c')
N.sub.2A cells were treated with 50 .mu.M tBHP alone or with SS-31
for 12 h. Cells were stained with Hoechst 33342 for 20 min., fixed,
and imaged by fluorescent microscopy. (a) Untreated cells show
uniformly stained nuclei (a'). (b) Cells treated with tBHP were
smaller and showed nuclear fragmentation and condensation (b'). (c)
Cells treated with tBHP and 1 nM SS-31 had less nuclear changes
(c'). (FIG. 20B)-(FIG. 20C) SS-31 dose-dependently reduced percent
of apoptotic cells in N.sub.2A cells. Apoptotic cells were counted
using MetaMorph software. *P<0.01 compared to untreated cells;
*P<0.01 compared to tBHP alone. (SS) SS-31 dose-dependently
reduced percent of apoptotic cells in SH-SYSY cells. SH-SYSY cells
were treated with 25 .mu.M tBHP for 24 h. *P<0.01 compared to
untreated cells; *P<0.01 compared to tBHP alone.
[0041] FIGS. 21A-21B. SS-31 prevented caspase activation in N2A
cells treated with tBHP. (FIG. 21A) incubation of N2A cells with
100 .mu.M tBHP for 24 h resulted in a significant increase in
pancaspase activity that was dose-dependently prevented by
co-incubation with SS-31 (*P<0.01 compared to tBHP alone). (FIG.
21B)(a-c) N2A cells were treated with 50 .mu.M tBHP for 12 h and
stained with caspase-9 FLICA.TM. kit containing red fluorescent
inhibitor SR-LEHD-FMK and Hoechst 33342. (Panel a) Untreated cells
showed no caspase-9 stain and uniformly stained nuclei. (Panel b)
cells treated with tBHP showed intense caspase-9 activity (red) in
cells that also show condensed nuclei. (Panel c) Cells treated with
tBHP and 1 nM SS-31 showed fewer caspase-9 positive cells and fewer
condensed nuclei.
[0042] FIGS. 22A-22C. SS-31 dose-dependently reduced intracellular
ROS production in N2A cells treated with tBHP. (FIG. 22A) N2A cells
were loaded with DCFDA, and then exposed to 100 .mu.M tBHP alone,
or with SS-31. Intracellular ROS was quantified by the formation of
fluorescent DCF. Results shown are mean values (n=3). (FIG.
22B)(a-c) N.sub.2A cells were plated in glass bottom dishes and
treated with 50 .mu.M tBHP, alone or with 1 nM SS-31, for 6 h.
Cells were loaded with DCFDA (10 .mu.M) and imaged by confocal
laser scanning microscopy using ex/em of 495/525 nm. (FIG. 22C)
Effect of 1 nM SS-31 in reducing intracellular ROS induced by 50
.mu.M tBHP (*P<0.001 compared to untreated cells; *P<0.05
compared to tBHP alone).
[0043] FIGS. 23A-23B. SS-31 protected against tBHP-induced
mitochondrial viability. (FIG. 23A) SS-31 protected mitochondrial
viability in N2A cells treated with tBHP for 24 h. Mitochondrial
viability was evaluated using the MTT assay (*P<0.01 compared to
untreated cells, *P<0.05, P<0.01 compared to tBHP alone).
(FIG. 23B) SS-31 protected mitochondrial viability in SH-SYSY cells
treated with tBHP for 25 h (*P<0.01 compared to untreated cells;
**P<0.01 compared to tBHP alone).
[0044] FIG. 24. Increased hydrogen peroxide (H.sub.2O.sub.2)
sensitivity of G93A-SOD transfected murine neuroblastoma (N2A)
cells as compared to wildtype SOD-transfected N.sub.2A cells after
addition of 0.5 or 1 mM H.sub.2O.sub.2 for 1 h. Cell death was
quantified by measurement of the percentage of
H.sub.2O.sub.2-induced LDH release of total cellular LDH-content.
H.sub.2O.sub.2-induced LDH release was significantly reduced by
treatment of the cells with 1 to 100 .mu.M SS-31 after incubation
with H.sub.2O.sub.2. Values are means +S.D., n=4-5, *p<0.1,
**p<0.05, Student's t-test.
Black columns wildtype-SOD1-transfected cells, grey columns:
G93A-SOD1 transfected N2A cells
[0045] FIGS. 25A-25B. (FIG. 25A) Cumulative probability of disease
on set and survival with SS-31 5 mg/kg/day treatment (n=14) started
at symptom onset as compared to vehicle treatment (n=14). Survival
was significantly improved by SS-31 (p<0.05, Mantel-Cox log-rank
test). (FIG. 25B) Mean survival (days) of G93A mice treated with
vehicle or SS-31 5 mg/kg/day. (Data are mean.+-.SD, p<0.05,
Student's t-test).
[0046] FIG. 26. Effect of SS-31 mg/kg/day on motor performance
(seconds) tested by rotarod (Values are mean.+-.standard error of
means of the mice still alive at the respective time point): it was
significantly improved between day (d) 110 and day 130 in
SS-31-treated animals as compared to the vehicle-treated group
(p<0.005, Repeated Measures ANOVA followed by Fisher's
PLSD).
[0047] FIGS. 27A & 27B. Attenuation of motor neuron loss by
SS-31 in the ventral horn of the lumbar spinal cord of G93A mice.
Photomicrographs show cresyl violet stained sections through the
ventral horn of the lumbar spinal cord from non-transgenic control
(A) and G93A mice treated with vehicle (PBS) (B) or SS-31 (C) at
110 days of age. Stereological analysis revealed significantly
reduced numbers of surviving neurons in G93A mice treated with
vehicle as compared to non-transgenic controls (***, p<0.001).
This cell loss was significantly ameliorated by treatment with
SS-31 (**, p<0.01). Values are mean.+-.standard error of means.
Differences among means were analyzed using ANOVA followed by
Newman-Keuls post hoc test.
[0048] FIGS. 28A-28C. 4-hydroxynonenol immunostaining.
Photomicrographs of representative sections through the ventral
horn of the lumbar spinal cord of wild-type control (FIG. 28A), and
G93A mice treated with vehicle (FIG. 28B) or SS-31 (FIG. 28C) show
generalized reduction of 4-hydroxynonenal staining in neurons and
neurophils in drug-treated mice.
[0049] FIGS. 29A-29C. Nitrotyrosine immunostaining.
Photomicrographs of representative sections through the ventral
horn of the lumbar spinal cord of wild-type control (FIG. 29A), and
G93A mice treated with vehicle (FIG. 29B) or SS-31 (FIG. 29C) show
generalized reduction of nitrotyrosine staining in neurons and
neurophils in drug-treated mice.
[0050] FIGS. 30A-30F. Temporal changes of cysteine (FIG. 30A, FIG.
30B), ascorbate (FIG. 30C, FIG. 30D) and GSH (FIG. 30E, FIG. 30F)
levels in post-ischemic brain. C57BL/6 mice were subjected to 30
min MCAO. Values are expressed as nmol/mg protein in cortex (FIG.
30A, FIG. 30C, FIG. 30E) and striatum (FIG. 30B, FIG. 30D, FIG.
30F). Error bars indicate SEM (n=4 animals per group). #<0.05 vs
0 h Contral, *p<0.05 vs corresponding Contral, one-way ANOVA
with post hoc Fisher's PLSD test. Contral, contralateral side;
Ipsil, ipsilateral side; 0 h, sham non-ischemic animal.
[0051] FIGS. 31A-31C. Effect of SS-31 peptide on ischemia-induced
changes in cysteine (FIG. 31A) ascorbate (FIG. 31B), and GSH (FIG.
31C) levels, C57BL/6 mice were subjected to 30 min MCAO and treated
with vehicle, SS-31 (2 mg/kg body weight) or SS-20 (2 mg/kg body
eight) peptide immediately after reperfusion. Mice were sacrificed
at 6 h postischemia. Values are expressed as percent increase
(cysteine) or percent depletion (ascorbate and GSH) in ipsilateral
side versus contralateral side. Error bars indicate SEM (n=4-6
animals per group). Note that a difference was observed in percent
GSH depletion SS-31-treated cortex. *p<0.05 vs vehicle treated
group (Veh), one-way ANOVA with post hoc Fisher's PLSD test.
[0052] FIGS. 32A-32F. Effect of SS-31 peptide on ischemia-induced
infarct size and swelling in C57BL/6 mice. Shown are representative
serial sections (1.2 mm apart) stained with Cresyl Violet from mice
subjected to 30 min (FIG. 32A) and 20 min (FIG. 32B) MCAO and
treated with vehicle (Veh) or SS-31 (2 mg/kg body weight)
immediately after reperfusion, 6 h, 24 h, and 48 h. Infarct volumes
((FIG. 32C) and swelling (FIG. 32D) were estimated at 72 h
postischemia from 12 serial sections (600 Bm apart) per animal.
Mean % cerebral blood flow (CBF) reduction during MCAO (FIG. 32E)
and % reperfusion at 10 min postischemia (FIG. 32F) shows no
difference between two groups. Error bars indicate SEM (n=11
animals per group). *p<0.05 from vehicle treated group (Veh),
one-way ANOVA with post hoc Fisher's PLSD test.
[0053] FIGS. 33A-33B. SS peptides penetrate islet cells,
co-localize with mitochondria and preserve islet mitochondrial
membrane potential. (FIG. 33A) Islet cell uptake of SS-31. DBA/2
islet cells were incubated with 1 nM of tritium labeled SS-31 and 1
.mu.M unlabeled SS-31 at 37.degree. C. for 1 h. Following
incubation, radioactivity was measured in the medium and in cell
lysates and the radioactive counts in the medium were subtracted
from radioactive counts in the cell lysate and normalized to
protein content. In four consecutive experiments, the mean.+-.(SE)
[.sup.3H]SS-31 uptake was 70.2+/31 10.3 pmol/mg, of proteins (FIG.
33B)(i-iv) SS-31 preserves islet mitochondrial potential. DBA/2
mice were treated with SS-31 (3 mg/kg s.c. BID) or vehicle control
24 hours prior to pancreas harvest for islet isolation. SS-31
treated groups had 1 nM SS-31 added to the islet isolation
reagents. Following isolation, TMRM uptake was evaluated using
confocal laser scanning microscopy. Fluorescent (i) and phase (ii)
images of TMRM uptake in control mice demonstrate reduced
fluorescent uptake indicating mitochondrial depolarization. In
sharp contrast, fluorescent (iii) and phase (iv) images of TMRM
uptake in SS-31 treated mice demonstrate increased uptake and
preserved mitochondrial potential indicative of SS-31 protective
effect.
[0054] FIGS. 34A-34D. SS-31 reduces islet cell apoptosis. DBA/2
mice were pre-treated (24 and 12-hours hours prior to pancreas
harvest and islet isolation) with SS-31 (3 mg/kg, s.c., BID) or
vehicle control. SS-31 (1 nM was added to reagents used for the
isolation of islets from SS-31-treated mice. The islets were
dissociated in to single cells with trypsin/EDTA and were stained
with Annexin V-FITC (AnV) and propidium iodide (PI) and analyzed
with the use of dual parameter low cytometry. (FIG. 34A) Percentage
of cell undergoing early apoptosis (AnV+cells); P=0.03. (FIG. 34B)
Percentage of cells undergoing late apoptosis/early necrosis
(AnV+/PI+cells); P=0.03. (FIG. 34C) Percentage of necrotic cells
(PI+cells); P=1.0 (FIG. 34D) Percentage of viable cells
(AnV-/PI-cells); P=0.03. Data from individual pancreatic islet
isolations and mean.+-.SE are shown, N=number of separate islet
isolations. Two-tailed P-values were calculated using Mann-Whitney
t-test.
[0055] FIGS. 35A-35C. Reversal of diabetes following
transplantation of a marginal mass of syngeneic islets. Diabetic
DBA/2 mice received 200 syngeneic islet cells under the right
kidney capsule. Reversal of diabetes was defined as random
nonfasting blood glucose levels below 200 mg/dl on 3-consecutive
days. (FIG. 35A) Blood glucose levels of each individual control
mouse following transplantation of 200 syngeneic islets. (FIG. 35B)
Blood glucose levels of each individual SS-31 treated mouse
following transplantation of 200 syngeneic islets. (FIG. 35C)
Reversal of diabetes following transplantation of a marginal islet
cell mass in SS-31 treatment vs. control. Number of normoglycemic
mice by day 1, 3, 5, 10 and 14 post transplantation and two-tailed
P-valued calculated using chi-squared bivariate analysis are
shown.
DETAILED DESCRIPTION OF THE INVENTION
[0056] The invention is based on the surprising discovery by the
inventors that certain aromatic-cationic peptides reduce oxidative
damage. Reducing oxidative damage is important since free radicals,
such as ROS and RNS, produce diverse non-specific damage to lipids,
proteins. RNA, and DNA oxidative damage caused by free radicals is
associated with several diseases and conditions in mammals.
Peptides
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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), trypotophan, (Trp), tyrosine (Tyr),
and valine (Val).
[0062] 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.
[0063] The peptides useful in the present invention can contain one
or more non-naturally 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.
[0064] 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.
[0065] 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.
[0066] The non-natural amino acids may, for example, comprise
alkyl, aryl, or alkylaryl groups. Some examples of alkyl amino
acids include a-aminobutyric acid, 3-aminobutyric acid,
.gamma.-aminobutyric acid, A-aminovaleric acid, and
.DELTA.-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-aminophenyleacetic acid, and
.gamma.-phenyl-.beta.-aminobutyric acid.
[0067] 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.
[0068] 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, bromio, or iodo). Some specific
examples of non-naturally occurring derivatives of naturally
occurring amino acids include norvaline (Nva), norleucine (Nle),
and hydroxyproline (Hyp).
[0069] Another example of a modification of an amino acid in a
peptide useful in the methods of the present invention is the
derivatization of a carboxyl group of an aspartic acid or a
glutamic acid residue of the peptide. One example of derivatization
is amidation with ammonia or with a primary or secondary amine,
e.g. methylamine, ethylamine, dimethylamine or diethylamine.
Another example of derivatization includes esterification with, for
example, methyl or ethyl alcohol.
[0070] 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.
[0071] 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-naturally occurring amino acids. The D-amino
acids do 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 1-amino acids are considered to be
non-naturally occurring amino acids.
[0072] 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.
[0073] 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.
[0074] It is important that at least one of the amino acids present
in the aromatic-cationic peptide is a tyrosine or tryptophan
residue, or a derivative thereof.
[0075] It is also 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).
[0076] 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.
[0077] "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, L-histidine. The naturally occurring amino acids that
are negatively charged at physiological pH include L-aspartic acid
and L-glutamic acid.
[0078] 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.
[0079] 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 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) ad 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
[0080] 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-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
[0081] 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.
[0082] 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).
[0083] 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).
[0084] 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+) 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
[0085] 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+) 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
[0086] In another embodiment, the number of aromatic groups (a) and
the total number of net positive charges (p.sub.t) are equal.
[0087] Carboxyl groups, especially the terminal carboxyl group of a
C-terminal amino acid, are preferably amidated with, for example,
ammonia to form the C-terminal amide, Alternatively, the terminal
carboxyl group of the C-terminal amino acid may be amidated with
any primary or secondary amine. The primary or secondary amine may,
for example, be an alkyl, especially a branched or unbranched
C.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-dimethylyamido, N,N-diethylamido,
N-methyl-N-ethylamido N-phenylamido or N-phenyl-N-ethylamido
group.
[0088] 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.
[0089] 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.
[0090] Aromatic-cationic peptides useful in the methods of the
present invention include, but are not limited to, the following
peptide examples:
TABLE-US-00005 Lys-D-Arg-Tyr-NH.sub.2, D-Tyr-Trp-Lys-NH.sub.2,
Trp-D-Lys-Tyr-Arg-NH.sub.2, Tyr-His-D-Gly-Met,
Tyr-D-Arg-Phe-Lys-Glu-NH.sub.2, Met-Tyr-D-Arg-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-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-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-Tyr-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.sub.2,
Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-
Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-Phe,
His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-
Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH.sub.2,
Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-
Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D- Lys-Asp, and
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.
[0091] 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.
[0092] 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. For example, the acute diseases
and conditions can be associated with moderate or severe pain. In
these instances, the analgesic effect of the aromatic-cationic
peptide may be beneficial in the treatment regiment 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.
[0093] 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 patients or other
mammal.
[0094] Potential adverse effects may include sedation,
constipation, nervous system depression and respiratory depression.
In such instances aromatic-cationic peptide that does not activate
the mu-opioid receptor may be an appropriate treatment.
[0095] Examples of acute conditions include heart attack, stroke
and traumatic injury. Traumatic injury may include traumatic brain
and spinal cord injury.
[0096] Examples of chronic diseases or conditions include coronary
artery disease and any neurodegenerative disorders, such as those
described below.
[0097] Peptides useful in the methods of the present invention
which have mu-opioid receptor activity are typically those peptides
which have a trysine 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'-dimethlyltyrosine (2',6'Dmt), 3',5'-dimethyltryosine
(3'S'Dmt); N,2',6'-trimethyltyrosine (Tmt); and
2'-hydroxy-6'-methyltryosine (Hmt).
[0098] In a particular preferred embodiment, a peptide that has
mu-opioid receptor activity has the formula Tyr-D-Arg-Phe-Lys-NH2
(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-Arg-Phe-Lys-NH.sub.2
(i.e., Dmt.sup.1-DALDA, which is referred to herein as SS-02).
[0099] 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
non-naturally occurring amino acid other than tyrosine.
[0100] In one embodiment, the amino acid at the N-terminus is
phenylalanine or its derivative. Preferred derivatives of
phenylalanine include 2'-methylphenylalanine (Mmp),
2',6'-dimethylphenylalanine (Dmp), N,2',6'-trimethylphenylalanine
(Tmp) and 2'-hydroxy-6'-methylphenylalanine (Hmp). In another
preferred embodiment, the amino acid residue at the N-terminus is
arginine. An example of such a peptide is
D-Arg-2'6'-Dmt-Lys-Phe-NH.sub.2 (referred to in this specification
as SS-31).
[0101] Another aromatic-cationic peptide that does not have
mu-opioid receptor activity has the formula Phe-D-Arg-Dmt-Lys-NH2.
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-Dmt-Lys-NH.sub.2.
[0102] In a preferred embodiment, the amino acid sequence of
Dmt.sup.1-DALDA (SS-02) is rearranged such that Dmt 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 (SS-31).
[0103] DALDA, SS-31, and their derivatives can further include
functional analogs. A peptide is considered a functional analog of
DALDA or SS-31 if the analog has the same function as DALDA or
SS-31. The analog may, for example, be a substitution variant of
DALDA or SS-31, wherein one or more amino acid is substituted by
another amino acid.
[0104] Suitable substitution variants of DALDA or SS-31 include
conservative amino acid substitutions. Amino acids may be grouped
according to their physicochemical characteristics as follows:
[0105] (a) Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P)
Gly(G);
[0106] (b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q);
[0107] (c) Basic amino acids: His(H) Arg(R) Lys(K);
[0108] (d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V);
and
[0109] (e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His(H).
[0110] Substitutions of an amino acid link 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.
[0111] 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-00006 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-dns NH.sub.2
2'6'Dmt D-Arg Phe Lys-NH(CH.sub.2).sub.2--NH-atn NH.sub.2 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-aminoheptanoic acid) NH.sub.2 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 3'5'Dmt 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 2'6'Dmt 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 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 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 acid Dap = diaminopropionic acid Dmt =
dimethyltyrosine Mmt = 2'-methyltyrosine Tmt =
N,2',6'-trimethyltyrosine Hmt = 2'-hydroxy,6'-methyltyrosine dnsDap
= 3-dansyl-L-.alpha.,.beta.-diaminopropionic acid atnDap =
3-anthraniloyl-L-.alpha.,.beta.-diaminopropionic acid Bio =
biotin
[0112] 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-00007 TABLE 2 Amino Amino Amino Amino Amino Acid
C-Terminal Acid Acid Acid Acid Position 5 Modifica- Position 1
Position 2 Position 3 Position 4 (if present) tion 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- D-Arg NH.sub.2 Lys Dmt 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
NH.sub.2
[0113] The amino acids of the peptides shown in table 1 and 2 may
be in either the L- or the D-configuration.
Methods of Reducing Oxidative Damage
[0114] The peptides described above are 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.-).
[0115] 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. Reperfusion
refers to the restoration of blood flow to any organ or tissue in
which the flow of blood is decreased or blocked. The restoration of
blood flow during reperfusion leads to respiratory burst and
formation of free radicals.
[0116] Decreased or blocked blood flow may be due to hypoxia or
ischemia. The loss or severe reduction in blood supply during
hypoxia or ischemia may, for example, be due to thromboembolic
stroke, coronary atherosclerosis, or peripheral vascular
disease.
[0117] Numerous organs and tissues are subject to ischemia or
hypoxia. Examples of such organs include brain, heart, kidney,
intestine and prostate. The tissue affected is typically muscle,
such as cardiac, skeletal, or smooth muscle. 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.
[0118] 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.
[0119] 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. Reducing
oxidative damage associated with ischemia/hypoxia and reperfusion
is important because the tissue damage associated with
ischemia/hypoxia and reperfusion is associated with, for example,
myocardial infarction, stroke and hemorrhagic shock.
[0120] In another embodiment, a mammal in need thereof can be a
mammal with a disease or condition associated with oxidative
damage. The oxidative damage can occur in any cell, tissue or organ
of the mammal. Examples of cells, tissues or organs include, but
are not limited to, endothelial cells, epithelial cells, nervous
system cells, skin, heart, lung, kidney and liver. For example,
lipid peroxidation and an inflammatory process are associated with
oxidative damage for a disease or condition.
[0121] 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 per-oxidation. 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.
[0122] Inflammatory process refers to the 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.
[0123] 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.
[0124] 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
bacterial include Escherichia coli, Klebsiella pneumoniae, Proteus
species, Pseudomonas aeruginosa, Serratia, and Bacteroides.
Examples of gram-positive bacteria include pneumococci and
streptococci.
[0125] 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.
[0126] 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.
[0127] The methods of the present invention 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.
[0128] 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).
[0129] Other conditions which can be treated in accordance with the
present invention include preeclampsia, diabetes, and symptoms of
and conditions associated with aging, such as macular degeneration,
wrinkles.
[0130] In another embodiment, the peptides useful in the present
invention may also be used in reducing oxidative damage in an organ
of a mammal prior to transplantation. For example, a removed organ,
when subjected to reperfusion after transplantation can be
susceptible to oxidative damage. Therefore, the peptides can be
used to reduce oxidative damage from reperfusion of the
transplanted organ.
[0131] The removed organ can be any organ suitable for
transplantation. Examples of such organs include, the heart, liver,
kidney, lung, and pancreatic islets. The removed organ is placed in
a suitable medium, such as in a standard buffered solution commonly
used in the art.
[0132] 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 0.01 nM to about 10 .mu.M, preferably
about 0.1 nM to about 10 .mu.M, more preferably about 1 .mu.M to
about 5 .mu.M, and even more preferably about 1 nM to about 100
nM.
[0133] In yet another embodiment, the invention provides a method
for reducing oxidative damage in a cell in need thereof. Cells in
need of reducing oxidative damage are generally those cells in
which the cell membrane or DNA of the cell has been damaged by free
radicals, for example, ROS and/or RNS. Examples of cells capable of
being subjected to oxidative damage include the cells described
herein. Suitable examples of cells include pancreatic islet cells,
myocytes, endothelial cells, neuronal cells, stem cells, etc.
[0134] The cells can be tissue culture cells. Alternatively the
cells may be obtained from a mammal. In one instance, the cells can
be damaged by oxidative damage as a result of an insult. Such
insults include, for example, a disease or condition (e.g.,
diabetes, etc.) or ultraviolet radiation (e.g., sun, etc.). For
example pancreatic islet cells damaged by oxidative damage as a
result of diabetes can be obtained from a mammal.
[0135] The peptides described above can be administered to the
cells by any method known to those skilled in the art. For example,
the peptides can be incubated with the cells under suitable
conditions. Stick conditions can be readily determined by those
skilled in the art.
[0136] Due to reduction of oxidative damage, the treated cells may
be capable of regenerating. Such regenerated cells may be
administered back into the mammal as a therapeutic treatment for a
disease or condition. As mentioned above, one such condition is
diabetes.
[0137] Oxidative damage is considered to be "reduced" if the amount
of oxidative damage in a mammal, a removed organ, or a cell is
decreased after administration of an effective amount of the
aromatic cationic peptides described above. Typically, the
oxidative damage is considered to be reduced if the oxidative
damage is decreased by at least about 10%, preferably at least
about 25%, more preferably at least about 50%, even more preferably
at least about 75%, and most preferably at least about 90%.
Synthesis of the Peptides
[0138] 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
[0139] The peptide useful in the methods of the present invention
is administered to a mammal in an amount effective in reducing
oxidative damage. The effective amount is determined during
pre-clinical trials and clinical trials by methods familiar to
physicians and clinicians.
[0140] An effective amount of a peptide useful in the methods of
the present invention, preferably in a pharmaceutical composition,
may be administered to a mammal in need thereof by any of a number
of well-known methods for administering pharmaceutical
compounds.
[0141] The peptide may be administered systemically or locally. In
one embodiment, the peptide is administered intravenously. For
example, the aromatic-cationic peptides useful in the methods of
the present invention may be administered via rapid intravenous
bolus injection. Preferably, however, the peptide is administered
as a constant rate intravenous infusion.
[0142] The peptide can be injected directly into coronary artery
during for example, angioplasty of or coronary bypass surgery, or
applied onto coronary stents.
[0143] 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.
[0144] 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. This intracerebroventicular 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.
[0145] 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.
[0146] A description of methods for delivering a compound by
controlled release can be found in PCT Application No. WO
02/083106. The PCT application is incorporated herein by reference
in its entirety.
[0147] 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.
[0148] For systemic, intracerebroventricular, intrathecal, topical,
intranasal, subcutaneous, or transdermal administration,
formulations of the aromatic-cationic peptides useful in the
methods of the present invent oils 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
stabilizing 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.
[0149] 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.
[0150] The surfactant is preferably a nonionic surfactant, such as
a polysorbate. Some examples of suitable surfactants include
Tween20, Tween80; a polyethylene glycol or a polyoxyethylene
polyoxypropylene glycol, such as Pluronic F-68 at from about 0.001%
(w/v) to about 10% (w/v).
[0151] The salt or buttering agent may be any salt or buffering
agent such as for example, sodium chloride, or sodium/potassium
phosphate, respectively. Preferably, the buttering 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.
[0152] 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.
[0153] The mammal treated in accordance with the invention can be
any mammal, including, for example, farm animals, such as sheep,
pigs, cows, and horses pet animals such as dogs and cats,
laboratory animals, such as rats, mice and rabbits. In a preferred
embodiment, the mammal is a human.
EXAMPLES
Example 1
[Dmt.sup.1]DALDA Penetrates Cell Membrane
[0154] 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-warmed HBSS, and then incubated with 0.2 ml
of HBSS containing either 250 nM [.sup.3H][Dmt.sup.1]DALDA at
37.degree. C. for 4.degree. C. for various times up to 1 h.
[0155] [.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 1% 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-SYSY, HEK293
and CRFK cells. The intracellular concentration of [Dmt.sup.1]DALDA
was estimated to be approximately 50 times higher than
extracellular concentration.
[0156] 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 ml 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.
[0157] 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.
[0158] 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
[0159] 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-diamino-propionic acid) was prepared. The
analog contained .beta.-anthraniloyl-1-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.
[0160] 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 63.times.1.2W corr
objective.
[0161] 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 356 nm, and emission was
measured at 560 nm.
[0162] 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
Scavenging of Hydrogen Peroxide by SS-02 and SS-05 (FIG. 1)
[0163] Effect of SS-02 and SS-05 (Dmt-D-Arg-Phe Orn-NH.sub.2) on
H.sub.2O.sub.2 as measured by luminol-induced chemiluminescence, 25
.mu.M luminol and 0.7 IU horseradish peroxidase were added to the
solution of H.sub.2O.sub.2 (4.4 nmol) and peptide, and
chemiluminescence was monitored with a Chronolog Model 560
aggregometer (Havertown, Pa.) for 20 min at 37.degree. C.
[0164] Results show that SS-02 and SS-05 dose-dependently inhibited
the luminol response suggesting that these peptides can scavenge
H.sub.2O.sub.2.
Example 4
Inhibition of Lipid Peroxidation (FIG. 2)
[0165] Linoleic acid peroxidation was induced by a water-soluble
initiator. ABAP (2,2'-azobis(2-amidinopropane)), and lipid
peroxidation was detected by the formation of conjugated dienes,
monitored spectrophotometrically at 236 nm (B. Longoni, W. A.
Pryor, P. Marchiafava, Biochem. Biophys. Res. Commun. 233, 778-780
(1997)).
[0166] 5 ml of 0.5 M ABAP and varying concentrations of SS-02 were
incubated in 2.4 ml linoleic acid suspension until autoxidation
rate became constant. Results show that SS-02 dose-dependently
inhibited the peroxidation of linoleic acid.
[0167] Various peptides were added in concentration of 100 .mu.M.
The data are presented as the slope of diene formation. With the
exception of SS-20 (Phe-D-Arg-Phe-Lys-NH.sub.2), SS-21
(Cyclohexyl-D-Arg-Phe-Lys-NH.sub.2) and SS-22
(Ala-D-Arg-Phe-Lys-NH.sub.2), all other SS peptides reduced the
rate of linoleic acid peroxidation. Note that SS-20, SS-21 and
SS-22 do not contain either tyrosine or dimethyltyrosine residues.
SS-01, which contains Tyr rather than Dmt is not as effective in
preventing linoleic acid peroxidation. SS-29 is Dmt-D-Cit-Phe
Lys-NH.sub.2. SS-30 is Phe-D-Arg-Dmt-Lys-NH.sub.2, SS-32 is
Dmt-D-Arg-Phe-Ahp(2-aminoheptanoic acid)-NH.sub.2.
Example 5
Inhibition of LDL Oxidation (FIG. 3)
[0168] Human LDL (low density lipoprotein) was prepared fresh from
stored plasma. LDL oxidation was induced catalytically by the
addition of 10 mM CuSO.sub.4, and the formation of conjugated
dienes was monitored at 24 nm for 5 h at 37.degree. C. (B. Moosmann
and C. Behl, Mol Pharmacol., 61, 260-268 (2002)).
[0169] (A) Results show that SS-02 dose-dependently inhibited the
rate of LDL oxidation.
[0170] (B) Various peptides were added in concentration of 100
.mu.M. With the exception of SS-20 (Phe-D-Arg-Phe-Lys NH.sub.2)
SS-21. (Cyclohexyl-D-Arg-Phe-Lys-NH.sub.2) and SS-22
(Ala-D-Arg-Phe-Lys-NH.sub.2), all other SS peptides reduced the
rate of linoleic acid peroxidation (reduced rate of formation of
conjugated dienes). Note that SS-20, SS-21 and SS-22 do not contain
either tyrosine or dimethyltyrosine residues. SS-29 is
Dmt-D-Cit-Phe-Lys-NH.sub.2, SS-30 is Phe-D-Arg-Dmt-Lys-NH.sub.2,
SS-32 is Dmt-D-Arg-Phe-Ahp(2-aminoheptanoic acid)-NH.sub.2.
Example 6
Hydrogen Peroxide Production by Isolated Mouse Liver Mitochondria
(FIG. 4)
[0171] Because mitochondria are a major source of ROS production,
the effect of SS-02 on H.sub.2O.sub.2 formation in isolated
mitochondria under basal conditions as well as after treatment with
antimycin, a complex III inhibitor was examined. Livers were
harvested from mice and homogenized in ice-cold buffer and
centrifuged at 13800.times.g for 10 m. The pellet was washed once
and then re-suspended in 0.3 ml of wash butter and placed on ice
until use H.sub.2O.sub.2 was measured using luminol
chemiluminescence as described previously (Y. Li, H. Zhu, M. A.
Trush, Biochim. Biophys. Acta 1428, 1-12 (1999)). 0.1 mg
mitochondrial protein was added to 0.5 ml potassium phosphate
buffer (100 mM, pH 8.0) in the absence or presence of SS peptides
(100 .mu.M). 25 mM luminol and 0.7 IU horseradish peroxidase were
added, and chemilumunescence was 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 was quantified as the area under
the curve (AUC) over 20 min, and all data were normalized to AUC
produced by mitochondria alone.
[0172] (A) The amount of H.sub.2O.sub.2 production was
significantly reduced in the presence of 10 .mu.M SS-02. Addition
of antimycin (1 .mu.M) significantly increased H.sub.2O.sub.2
production by isolated mitochondria, and the increase was
completely blocked by 1.0 .mu.M Dmt.sup.1-DALDA (also referred to
as dDALDA in the specification).
[0173] (B) The amount of H.sub.2O.sub.2 generated was significantly
reduced by peptides SS-02, SS-29, SS-30 and SS-31. SS-21 and SS-22
had no effect on H.sub.2O.sub.2 production. Note that SS-21 and
SS-22 do not contain a tyrosine or dimethyltryosine residue. The
amino acid Dmt dimethyltyrosine) alone also inhibited
H.sub.2O.sub.2 generated.
Example 7
SS-31 inhibits H.sub.2O.sub.2 Generation by Isolated Mitochondria
(FIG. 5)
[0174] H.sub.2O.sub.2 was measured using luminol chemiluminescence
as described previously (Y, Li, H. Zhu, M. A. Trush, Biochim.
Biophys. Acta 1428, 1-12 (1999)). 0.1 mg mitochondrial protein was
added to 0.5 ml potassium phosphate buffer (100 mM, pH 8.0) in the
absence or presence of SS-31. 25 mM luminol and 0.7 IU horseradish
peroxidase were added, and chemilumunescence was 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 was quantified
as the area under the curve (AUC) over 20 min, and all data were
normalized to AUC produced by mitochondria alone.
[0175] (A) SS-31 dose-dependently reduced the spontaneous
production of H.sub.2O.sub.2 by isolated mitochondria.
[0176] (B) SS-31 dose-dependently reduced the production of
H.sub.2O.sub.2 induced by antimycin in isolated mitochondria.
Example 8
SS-02 and SS-31 Reduced Intracellular ROS and Increased Cell
Survival (FIG.
[0177] To show that the claimed peptides are effective when applied
to whole cells, neuronal N.sub.2A cells were 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 were washed twice and replaced with medium alone or medium
containing varying concentrations of SS-02 or SS-31 for 4 hr.
Intracellular ROS was measured by carboxy-H2DCFDA (Molecular
Probes, Portland, Oreg.). Cell death was assessed by a cell
proliferation assay (MTS assay, Promega, Madison, Wis.).
[0178] Incubation with tBHP resulted in dose-dependent increase in
intracellular ROS (A) and decrease in cell viability, (B and C).
Incubation of these cells with either SS-31 or SS-02
dose-dependently reduced intracellular ROS (A) and increased cell
survival (B and C), with EC50 in the nM range.
Example 9
SS-31 Prevented Loss of Cell Viability (FIG. 7)
[0179] Neuronal N2A and SH-SYSY cells were plated in 96-well plate
at a density of 1.times.10.sup.4/well and allowed to grow for 2
days before treatment with t-butyl hydroperoxide (tBHP) (0.05-0.1
mM) with or without SS-31 (10.sup.-12 M to 10.sup.-9 M for 24 h.
Cell death was assessed by a cell proliferation assay (MTS assay,
Promega, Madison, Wis.).
[0180] Treatment of N.sub.2A and SH-SYSY cells with low doses of
t-BHP (0.05-0.1 mM) for 24 h resulted in a decrease in cell
viability. (A) 0.05 mM t-BHP induced 50% loss of cell viability in
N.sub.2A cells and 30% in SH-SYSY cells. (B) 0.1 mM t-BHP resulted
in a greater reduction in cell viability in SH-SYSY cells.
Concurrent treatment of cells with SS-31 resulted in a
dose-dependent reduction of t-BHP-induced cytotoxicity. Complete
protection against t-BHP was achieved by 1 nM SS-31.
Example 10
SS-31 Decreased Capase Activity (FIG. 8)
[0181] N.sub.2A cells were grown on 96-well plates, treated with
t-BHP (0.05 mM) in the absence or presence of SS-31 (10.sup.-11
M-10.sup.-8 M) at 37.degree. C. for 12-24 h. All treatments were
carried out in quadriplicates. N.sub.2A cells were incubated with
t-BHP (50 mM) with or without SS-31 at 37.degree. C. for 12 h.
Cells were gently lifted from the plates with a cell detachment
solution (Accutase, Innovative Cell Technologies, Inc., San Diego,
Calif) and washed twice in PBS. Caspase activity was assayed using
the FLICA kit (Immunochemistry Technologies LLC, Bloomington,
Minn.) According to the manufacturer's recommendation, cells were
resuspended (approx. 5.times.10.sup.6 cells/ml) in PBS and labeled
with pan-caspase inhibitor FAM-VAD-FMK for 1 h at 37.degree. C.
under 5% CO.sub.2 and protected from the light. Cells were then
rinsed to remove the unbound reagent and fixed. Fluorescence
intensity in the cells was measured by a laser scanning cytometer
(Beckman-Coulter XL, Beckman Coulter, Inc., Fullerton, Calif.)
using the standard emission filters for green (FL1). For each run,
10,000 individual events were collected and stored in list-mode
files for off-line analysis.
[0182] Caspase activation is the initiating trigger of the
apoptotic cascade, and our results showed a significant increase in
caspase activity after incubation of SH-SYSY cells with 50 mM t-BHP
for 12 h which was dose-dependently inhibited by increasing
concentrations of SS-31.
Example 11
SS-31 Reduced Rate of ROS Accumulation (FIG. 9)
[0183] Intracellular ROS was evaluated using the fluorescent probe
DCFH-DA (5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein
diacetate). DCFH-DA enters cells passively and is then deacetylated
to nonfluorescent DCFH. DCFH reacts with ROS to form DCF, the
fluorescent product. N.sub.2A cells in 96 sell plates were washed
with HBSS and loaded with 10 .mu.M of DCFDA for 30 min. for 30 min.
at 37.degree. C. Cells were washed 3 times with BSS and exposed to
0.1 mM of t-BHP, alone or with SS-31. The oxidation of DCF was
monitored in real time by a fluorescence microplate reader
(Molecular Devices) using 485 nm for excitation and 530 nm for
emission.
[0184] The rate of ROS accumulation in N.sub.2A cells treated with
0.1 mM t-BHP was dose-dependently inhibited by the addition of
SS-31.
Example 12
SS-31 Inhibited Lipid Peroxidation in Cells Exposed to Oxidative
Damage (FIG. 10)
[0185] SS-31 inhibited lipid peroxidation in N2A cells treated with
t-BHP. Lipid peroxidation was evaluated by measuring HNE Michael
adducts. 4-HNE is one of the major aldehydic products of the
peroxidation of membrane polyunsaturated fatty acids. N.sub.2A
cells were seeded on glass bottom dish 1 day before t-BHP treatment
(1 mM, 3 h, 37.degree. C., 5% CO.sub.2) in the presence of absence
of SS-31 (10.sup.-8 to 10.sup.-10 M). Cells were then washed twice
with PBS and fixed 30 min with 4% paraformaldehyde in PBS at RT and
then washed 3 times with PBS. Cells were then permeabilized,
treated with rabbit-anti-HNE antibody followed by the secondary
antibody (goat anti-rabbit IgG conjugated to biotin). Cells were
mounted in Vectashield and imaged using a Zeiss fluorescence
microscope using an excitation wavelength of 460.+-.20 nm and a
longpass filter of 505 nm for emission.
[0186] (A) Untreated cells (B) cells treated with 1 mM t-BHP for 3
h; (C) cells treated with 1 mM t-BHP and 10 nM SS-31 for 3 h.
Example 13
SS-02 Inhibits Loss of Mitochondrial Potential in Cells Exposed to
Hydrogen Peroxide
[0187] Caco-2 cells were treated with tBHP (1 mM) in the absence or
presence of SS-02 (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 tBHP, the TMRM fluorescence is much reduced,
suggesting generalized depolarization. In contrast, concurrent
treatment with SS-02 protected against mitochondrial depolarization
caused by tBHP.
Example 14
SS-31 Prevents Loss of Mitochondrial Potential and Increased ROS
Accumulation in N.sub.2A Cells Caused by Exposure to t-BHP (FIG.
11)
[0188] N.sub.2A cells in glass bottom dish were treated with 0.1 mM
t-BHP, alone or with 1 nM SS-31, for 6 h. Cells were then loaded
with 10 .mu.m of dichlorofluorescin (ex/em=485/530) for 30 min at
37.degree. C., 5% CO.sub.2. Then cells were subjected 3 times wash
with HBSS and stained with 20 nM of Mitotracker TMRM (ex/em=550/575
nm) for 15 min at 37.degree. C. and examined by confocal laser
scanning microscopy.
[0189] Treatment of N.sub.2A cells with t-BHP resulted in loss of
TMRM fluorescence indicating mitochondrial depolarization. There
was also a concomitant increase in DCF fluorescence indicating
increase in intracellular ROS. Concurrent treatment with 1 nM SS-31
prevented mitochondrial depolarization and reduced ROS
accumulation.
Example 15
SS-31 Prevents Apoptosis Caused by Oxidative Stress (FIG. 12)
[0190] SH-SYSY cells were grown on 96-well plates, treated with
t-BHP (0.025 mM) in the absence or presence of SS-31
(10.sup.-12M-10.sup.-9M) at 37.degree. C. for 24 h. All treatments
were carried out in quadriplicates. Cells were then stained with 2
mg/ml Hoechst 33342 for 20 min, fixed with 4% paraformaldehyde, and
imaged using a Zeiss fluorescent microscope (Axiovert 200M)
equipped with the Zeiss Acroplan .times.20 objective. Nuclear
morphology was evaluated using an excitation wavelength of
350.+-.10 nm and a longpass filter of 400 nm for emission. All
images were processed and analyzed using the MetaMorph software
(Universal Imaging Corp., West Chester, Pa.). Uniformly stained
nuclei were scored as healthy, viable neurons, while condensed or
fragmented nuclei were scored as apoptotic.
[0191] SS-31 prevents apoptosis induced by a low dose of t-BHP.
Apoptosis was evaluated by confocal microscopy with the fluorescent
probe Hoechst 33342 (A1) a representative field of cells not
treated with t-BHP. (A2) Fluorescent image showing a few cells with
dense, fragmented chromatin indicative of apoptotic nuclei. (A3) A
representative field of cells treated with 0.025 mM t-BHP for 24 h.
(A4) Fluorescent image showing an increased number of cells with
apoptotic nuclei. (A5) A representative field of cells treated with
0.025 mM t-BHP and 1 nM SS-31 for 24 h. (A6) Fluorescent image
showing a reduced number of cells with apoptotic nuclei.
[0192] (B) SS-31 dose-dependently reduced the percent of apoptotic
cells caused by 24 h treatment with a low dose of t-BHP (0.05
mM).
Example 16
SS-31 Prevents Lipid Peroxidation in Hearts Subjected to Brief
Intervals of Ischemia-Reperfusion. (FIG. 13)
[0193] Isolated guinea pig hearts were perfused in a retrograde
manner in a Langendorff apparatus and subjected to various
intervals of ischemia-reperfusion. Hearts were then fixed
immediately and embedded in paraffin. Immunohistochemical analysis
of 4-hydroxy-2-nonenol (HNE)-modified proteins in the paraffin
sections was carried out using an anti-HNE antibody.
[0194] (A) Immunohistochemical analysis of 4-hydroxy-2-nonenol
(HNE)-modified proteins in paraffin sections from guinea pig hearts
aerobically perfused 30 min with (a) buffer; (b) 100 nM SS-02; (c)
100 nM SS-20 and (d) 1 nM SS-31, then subjected to 30 min ischemia
and reperfused for 90 min with same peptides. Tissue slices were
incubated with anti-FINE antibody. (e) Background control: staining
without primary antibody.
[0195] (B) Immunohistochemical analysis of HNE-modified proteins in
paraffin sections from guinea pig hearts aerobically perfused 30
min with buffer; then subjected to 30 min ischemia and reperfused
with (a) buffer; (b) 100 nM SS-02; (c) 100 nM SS-20 and (d) 1 nM
SS-31 for 90 min with same peptides. Tissue slices were incubated
with anti-HNE antibody. (e) Background control staining without
primary antibody.
Example 17
SS-31 Increases Coronary Flow and Reduces Lipid Peroxidation and
Apoptosis in Hearts Subjected to Prolonged Cold Ischemia Followed
by Warm Reperfusion (FIG. 14)
[0196] Isolated guinea pig hearts were perfused in a retrograde
manner in a Langendorff apparatus with a cardioplegic solution (St.
Thomas solution) without or with SS-31 (1 nM) for 3 min. and then
clamped and stored at 4.degree. C. for 18 h. Subsequently, the
hearts were remounted in the Langendorff apparatus and reperfused
with Krebs-Henseleit solution at 34.degree. C. for 90 min. Hearts
were then rapidly fixed and paraffin-embedded.
[0197] (A) SS-31 significantly improved coronary flow in hearts
after 18 h cold ischemic storage. The shaded area represents 18 h
of cold ischemia.
[0198] (B) Immunohistochemical analysis of HNE-modified proteins in
paraffin sections from guinea pig hearts stored without (a) or with
(b) SS-31 (1 nM). (c) Background staining without primary
antibody.
[0199] (C) SS-31 prevents apoptosis in endothelial cells and
myocytes in isolated guinea pig hearts subjected to warm
reperfusion after prolonged (18 h) cold ischemia. Apoptosis was
assessed by the TUNEL stain (green) and nuclei are visualized by
DAPI (blue).
Example 18
SS-31 Improves Survival of Islet Cells Isolated from Mouse Pancreas
(FIG. 15)
[0200] (A) SS-31 improves mitochondrial potential in islet cells
isolated from mouse pancreas. Pancreas was harvested from mice and
islet cells were prepared according standard procedures. In some
studies, SS-31 (1 nM) was added to all isolation buffers used
throughout the isolation procedure. Mitochondrial potential was
measured using TMRM (red) and visualized by confocal
microscopy.
[0201] (B) SS-31 reduces apoptosis and increases viability in islet
cells isolated from mouse pancreas. Pancreas was harvested from
mice and islet cells were prepared according standard procedures.
In some studies, SS-31 (1 nM) was added to all isolation buffers
used throughout the isolation procedure. Apoptosis was ascertained
by flow cytometry using annexin V and necrosis by propidium
iodide.
Example 19
SS-31 Protects Against Oxidative Damage in Pancreatic Islet Cells
(FIG. 16)
[0202] Mouse pancreatic islet cells were untreated (a), or treated
with 25 .mu.M tBHP without or with 1 nM SS-31 (c). Mitochondrial
potential was measured by TMRM (red) and reactive oxygen species
were measured by DCF (green) using confocal microscopy.
Example 20
SS-31 Protects Against Parkinson's Disease (FIG. 17)
[0203] MPTP is a neurotoxin that selectively destroys striatal
dopamine neurons and can be used as an animal model of Parkinson's
Disease. MPP.sup.+, a metabolite of MPTP, targets mitochondria,
inhibits complex I of the electron transport chain and increases
ROS production. MPP.sup.+ is used in cell culture studied because
cells are unable to metabolize MPTP to the active metabolite. MPTP
is used for animal studies.
[0204] (A) SS-31 protects dopamine cells against MPP.sup.+
toxicity. SN-4741 cells were treated with buffer, 50 .mu.M
MPP.sup.+ or 50 .mu.M MPP.sup.+ and 1 nM SS-31, for 48 h, and the
incidence of apoptosis was determined by fluorescent microscopy
with Hoechst 33342. The number of condensed fragmented nuclei was
significantly increased by MPP+ treatment. Concurrent treatment
with SS-31 reduced the number of apoptotic cells.
[0205] (B) SS-31 dose-dependently prevented loss of dopamine
neurons in mice treated with MPTP. Three doses of MPTP (10 mg/kg)
was given to mice (n=12) 2 h apart. SS-31 was administered 30 min
before each MPTP injection, and at 1 h and 12 h after the last MPTP
injection. Animals were sacrificed one week later and striatal
brain regions were immunostained for tyrosine hydroxylase
activity.
[0206] (C) SS-31 dose-dependently increased striatal dopamine DOPAC
(3,4-dihydroxyphenylacetic acid) and HVA (homovanillic acid) levels
in mice treated with MPTP. Three doses of MPTP (10mg/kg) was given
to mice (n=12) 2 h apart. SS-31 was administered 30 min before each
MPTP injection, and at 1 h and 12 h after the last MPTP
injection.
Animals were sacrificed one week later and dopamine, DOPAC and HVA
levels were quantified by high pressure liquid chromatography.
Example 21
SS-31 Protected SH-SYSY and N.sub.2A Cells Against tBHP Induced
Cytotoxicity (FIG. 18)
[0207] The loss of cell viability induced by 100 .mu.M tBHP was
accompanied by a significant increase in LDH release in SH-SYSY
(FIG. 18A) and N.sub.2A cell (FIG. 18B). (Concurrent treatment of
cells with SS-31 resulted in dose-dependent decrease in LDH release
in both SH-SYSY (P<0.01) and N.sub.2A cells (P<0.0001). LDH
release was reduced significantly by 0.1 and 1 nM of SS-31 in both
cell lines (P<0.05). SS-20, the control non-scavenging peptide,
did not protect against tBHP-induced cytotoxicity in N.sub.2A cells
(FIG. 18B).
Example 22
SS-31 Protected Against tBHP-Induced Apoptosis. (FIGS. 19 and
20)
[0208] The translocation of phosphatidylserine from the inner
leaflet of the plasma membrane to the outer leaflet is observed
early in the initiation of apoptosis. This can be observed with
Annexin V, a phospholipid binding protein with high affinity for
phosphatidylserine. FIG. 19A shows that untreated N2A cells showed
little to no Annexin V staining (green). Incubation of N2A cells
with 50 mM tBHP for 6 h resulted in Annexin V staining on the
membranes of most cells (FIG. 19B). Combined staining with Annexin
V and propidium iodide (red) showed many late apoptotic cells (FIG.
19B). Concurrent treatment of N2A cells with 1 nM SS-31 and 50
.mu.M tBHP resulted in a reduction in Annexin V-positive cells and
no propidium iodide staining (FIG. 19C), suggesting that SS-31
protected against tBHP-induced apoptosis.
[0209] The morphological appearance of cells treated with tBHP was
also consistent with apoptosis. N2A cells incubated with 50 .mu.M
tBHP for 12 h became rounded and shrunken (FIG. 20A, panel b).
Staining with Hoechst 33324 showed increased number of cells with
nuclear fragmentation and condensation (FIG. 20A, panel b). These
nuclear changes were abolished by concurrent treatment with 1 nM
SS-31 (FIG. 20A, panel c'). The number of apoptotic cells was
dose-dependently reduced by concurrent treatment with SS-31
(P<0.0001) (FIG. 20B).
[0210] An increased number of cells with condensed nuclei was also
observed when SH-SYSY cells were treated with 25 .mu.M tBHP for 24
h, and the number of apoptotic cells was dose-dependently reduced
by concurrent treatment with SS-31 (P<0.0001) (FIG. 20C).
Example 23
SS-31 Protected Against tBHP-Induced Caspase Activation (FIG.
21)
[0211] Incubation of N.sub.2A cells with 100 .mu.M tBHP for 24 h
resulted in a significant increase in pan-caspase activity that was
dose-dependently prevented by co-incubation with SS-31 (P<0.0001
(FIG. 21A). A N.sub.2A cells treated with 50 .mu.M tBHP for 12 h
showed intense staining (red) for caspase-9 activity (FIG. 21B,
panel b). Note that cells that show nuclear condensation all showed
caspase-9 staining. Concurrent incubation with 1 nM SS-31 reduced
the number of cells showing caspase-9 staining (FIG. 21B, panel
c).
Example 24
SS-31 Inhibited tBHP-Induced Increase in Intracellular ROS (FIG.
22)
[0212] Intracellular ROS production is an early and critical event
in oxidant-induced cytotoxicity. Treatment of N.sub.2A cells with
100 .mu.M tBHP resulted in rapid increase in intracellular ROS, as
measured by DCF fluorescence, over 4 h at 37.degree. C. (FIG. 22A).
Concurrent treatment with SS-31 dose-dependently reduced the rate
of ROS production, with 1 nM SS-31 effectively reducing ROS
production by >50%. The reduction in intracellular ROS was
confirmed by fluorescent microscopy with DCF (FIG. 22B). Treatment
with N.sub.2A cells with 50 .mu.M tBHP caused significant increase
in DCF fluorescence (green), and this was significantly reduced by
co-incubation with 1 nM SS-31 (FIG. 22C).
Example 25
SS-31 Prevented Loss of Mitochondrial Function Caused by tBHP (FIG.
23)
[0213] Treatment with low doses of tBHP (50-100 .mu.M) for 24 h
resulted in a significant decrease in mitochondrial function as
measured by the MTT assay in both cell lines. Only viable
mitochondria containing NADPH dehydrogenase activity are capable of
cleaving MTT to the formazan. A 50 .mu.M tBHP induced 50% loss of
mitochondrial function in N.sub.2A cells (FIG. 23A, P<-0.01) and
30% loss of mitochondrial function in SH-SYSY cells (FIG. 23B,
P<0.01). Concurrent treatment with SS-31 dose-dependently
reduced tBHP-induced mitochondrial toxicity in both N.sub.2A (FIG.
23A; P<0.0001) and SHSYSY cells (FIG. 23B; P<0.0001). The
non-scavenging peptide, SS-20, did not protect against tBHP-induced
mitochondrial dysfunction in N.sub.2A cells (FIG. 23A). Treatment
of N.sub.2A cells with SS-31 alone had no effect on mitochondrial
function.
Example 26
Increased Hydrogen Peroxide (H.sub.2O.sub.2) Sensitivity of
G93A-SOD Transfected Murine Neuroblastoma (N2a) Cells (FIG. 24)
[0214] In a cell culture model of N.sub.2A cells overexpressing
either wild type or mutant (69A-SOD1, the mutant cells were
significantly more sensitive to H.sub.2O.sub.2-induced cell death
both at 0.04 mM and 1 mM concentrations. This cell-death was
significantly reduced by addition of SS-31 in concentration between
1 and 100 .mu.M to the medium 1 h after exposure to H.sub.2O.sub.2
(FIG. 24).
Example 27
SS-31 Increased Survival of G93A Transgenic Familial ALS Mice (FIG.
25)
[0215] Treatment with 5 mg-kg SS-31 i.p. started at 30 days of age
of G93A transgenic familial amyotrophic lateral sclerosis (ALS)
mice (high copy number, B6SJL-Tg(SOD1-G93A)1Gur/J) led to a
significant delay of disease onset as defined by the appearance of
tremor and hind limb clasping as well as deterioration in the
rotarod performance, the average age of onset in the control group
was 88+7 days, in the SS-31 treated group 95+6 days (p<0.05,
Logrank (Mantel-Cox)). Survival was significantly increased by
SS-31 treatment from 130.+-.12 to 142+12 day (i.e., 9%) (p<0.05,
Logrank (Mantel-Cox)) (FIG. 25). Treatment was well tolerated and
no side effects were observed. There was a gender effect on
survival which has been observed in previous studies with this
model as well in G93A mice in this background with males having a
shorter life span than females (average of 5 days). This gender
difference was seen in both groups and not modified by the
treatment.
Example 28
Effect of SS-31 on Motor Performance of G93A Transgenic Familial
ALS Mice (FIG. 26)
[0216] Motor performance was significantly improved in the SS-31
treated mice between day 100 and 130 (p<0005, Repeated measures
ANOVA followed by Fisher's PLSD) (FIG. 26).
Example 29
Attenuation of Motor Neuron Loss by SS-31 in the Ventral Horn of
the Lumbar Spinal Cord of G93A Mice. (FIGS. 27, 28 and 29)
[0217] Stereological cell counts in the lumbar spinal cord revealed
a significant cell loss in the vehicle treated G93A mice as
compared to non-transgenic littermate control animals. The cell
loss was significantly ameliorated by administration of SS-31 (FIG.
27). Immunostaining for markers of oxidative and nitrosative stress
(4-hydroxynonenenal, 3-nitrotyrosine) showed increased levels of
lipid peroxidation and protein nitration in the spinal cord of G93A
as compared to control mice. This was markedly reduced in the SS-31
treated mice (FIG. 28, 4-hydroxynonenenal; FIG. 29,
3-nitrotyrosine).
Example 30
Reduced Ascorbate and GSH Levels in Post-Ischemic Brain (FIG.
30)
[0218] Ascorbate and GSH, in addition to cysteine were determined
in the postischemic cortex and striatum. While cysteine levels were
generally decreased in both hemispheres after ischemia, they were
significantly higher in the ipsilateral side compared to the
contralateral side (FIGS. 30A and B). By contrast, the levels of
ascorbate and GSH, the major water-soluble intracellular
antioxidants in brain, were progressively decreased in the
ipsilateral side within a few hours of reperfusion (FIGS. 30C-F).
Antioxidant reduction was significant in both cortex and striatum
at 6 h and was further reduced at 24 h, at a time when the infarct
is visible.
Example 31
Treatment with SS-31, But Not SS-20, Attenuates GSH Depletion in
Post-Ischemic Cortex (FIG. 31)
[0219] To test the efficacy of SS-3 lon redox status, cysteine,
ascorbate and GSH levels were determined 6 h after 30 min transient
middle cerebral artery occlusion (MCAO) in mice treated
intraperitoneally (i.p.) with saline (vehicle), SS-31 (2 mg/kg) or
SS-20 (2 mg/kg) upon reperfusion. Values in FIG. 31 are expressed
as percent increase (cysteine) and percent depletion (ascorbate and
GSH) in the ipsilateral side compared to the contralateral side.
Absolute values were originally expressed as nmoles/mg protein as
shown in FIG. 30 and converted to percent difference. The percent
ipsilateral cysteine increase was similar among vehicle-, SS-31-,
and SS-20-treated groups (FIG. 31A). Percent ipsilateral depletion
of ascorbate was marginally but not significantly affected in both
SS-31 and SS-20-treated animals (FIG. 31B). In contrast,
ischemia-induced GSH depletion in the cortex was significantly
attenuated in SS-31-treated animals compared to the vehicle-treated
group (FIG. 31C).
[0220] The degree of ipsilateral GSH depletion in SS-20-treated
mice was not significantly different from that of vehicle-treated
mice (FIG. 31C). The data show that SS-31 assists in maintaining
antioxidant status and protects against ischemia-induced depletion
of GSH in the cortex.
Example 32
Treatment with SS-31 Peptide Reduces Infarct Size and Swelling
(FIG. 32)
[0221] To address whether SS-31-induced attenuation in GSH
depletion is associated with neuroprotection, mice were subjected
to 30 or 20 min MCAO and then treated with vehicle or SS-31 (2
mg/kg, i.p.) upon reperfusion) and at 6 h, 24 h, 48 h after MCAO.
Infarct volumes and hemispheric swelling were determined at 3 days
after ischemia. SS-31 treatment resulted in moderate but
significant reduction in infarct volume following both 30 min (24%
reduction) and 20 min (30% reduction) ischemia, (FIGS. 32A-C).
Treatment with SS-31 also significantly attenuated hemispheric
swelling in both 30 and 20 min ischemic paradigms (FIG. 32D). There
was no difference in cerebral blood flow (CBF) reduction during
MCAO (FIG. 32E) and reperfusion at 10 min (FIG. 32F) between
vehicle- and SS-31 -treated groups, indicating that the
neuroprotective effect by SS-31 occurs via mechanisms other than
altered CBF during and after the ischemic insult.
Example 33
Islet Cell Uptake of SS-31 (FIG. 33)
[0222] Islets are tightly adherent cell clusters and entry of
peptides/proteins may be impaired given their architecture. FIG.
33A shows that SS-31 readily penetrates intact mouse islets; in
four consecutive experiments, the mean (.+-.SE) of [.sup.3H]SS-31
uptake was 70.2.+-.10.3 pmol/mg of protein.
Example 34
SS-31 Prevents Mitochondrial Depolarization (FIG. 33)
[0223] Mitochondrial depolarization and the release of cytochrome c
into the cytoplasm are critical antecedent events to cell death.
TMRM, a fluorescent cationic indicator is taken up into
mitochondria in a potential dependent manner. FIG. 33B confocal
laser scanning microscopic imaging of TMRM treated islets shows
that the islets from mice treated with SS-31 exhibit greater uptake
of TMRM compared to islets from mice not treated with SS-31.
Example 35
Optimization of Islet Isolation with SS-31
[0224] To investigate whether the SS-31 optimizes islet isolation
and results in increased islet yield, islet donor mice were
pre-treated with SS-31. SS-31 resulted in a significantly higher
islet cell yield compared to untreated mice the mean.+-.(SE) islet
yield from the pancreata harvested from SS-31 pre-treated mice was
291.+-.60 islets per pancreas (N=6 separate islet isolations from
28 pancreata) compared to 242.+-.53 islets per pancreas retrieved
from the control mice (N=6 separate islet isolations from 30
pancreata) (P=0.03, Two-tailed Pvalue calculated with Mann-Whitney
test).
Example 36
SS-31 Reduces Islet Cell Apoptosis (FIG. 34)
[0225] SS-31 treatment, in addition to enhancing islet yield
resulted in a significant decrease in early as well as late, islet
cell apoptosis. Dual parameter flow cytometry analyses of islet
cells stained with both Annexin V and propidium iodide demonstrated
that the treatment of islet donor mice with SS-31 reduced the
percentage of early apoptotic cells (Annexin V alone positive
cells) from 11.4.+-.2.4% to 5.5.+-.1.0% (FIG. 34A, P=0.03). SS-31
treatment reduced late apoptosis/early necrosis (Annexin
V+/PI+cells) from 22.7.+-.4.7% to 12.6.+-.1.8 (FIG. 34B, P=0.03)
and increased islet cell viability (Annexin V-/PI-cells) from
47.+-.5.1% to 62.+-.3.5% (FIG. 34D, P=0.03)). SS-31 treatment of
islet donor mice, however, did not reduce the percentage of
necrotic cells (PI+cells: 20.+-.3.2% vs. 19.+-.4.7%, P=1.0).
Example 37
SS-31 Improves Post-Transplant Islet Graft Function (FIG. 35)
[0226] The SS-31 treatment associated decrease in islet cell
apoptosis and enhanced viability had a beneficial functional
consequence. In a marginal islet cell mass transplantation model, 0
of 8 recipients of islets isolated from pancreas harvested from the
control mice had successful reversal of hyperglycemia (defined as
three consecutive blood glucose levels<200 mg/dl), whereas
sustained normoglycemia occurred in 5 of ten recipients of islets
isolated from pancreas from the SS-31 treated donor mice (FIG. 35).
It is worth noting that reversal of hyperglycemia was prompt (by
day 1 post-transplant) and discontinuation SS-31 treatment of the
islet graft recipient on day 10 did not result in return of
hyperglycemia demonstrating a sustained effect of SS-31 treatment
on islet cell function.
* * * * *