U.S. patent application number 14/285226 was filed with the patent office on 2014-09-11 for methods for reducing cd36 expression.
This patent application is currently assigned to Cornell Research Foundation, Inc.. The applicant listed for this patent is Sunghee Cho, Shaoyl Liu, Hazel H. Szeto. Invention is credited to Sunghee Cho, Shaoyl Liu, Hazel H. Szeto.
Application Number | 20140256620 14/285226 |
Document ID | / |
Family ID | 37889419 |
Filed Date | 2014-09-11 |
United States Patent
Application |
20140256620 |
Kind Code |
A1 |
Szeto; Hazel H. ; et
al. |
September 11, 2014 |
METHODS FOR REDUCING CD36 EXPRESSION
Abstract
The invention provides a method for treating one or more
complications of diabetes in a mammal. The method comprises
administering to a mammal in need thereof an effective amount of an
aromatic-cationic peptide having at least one net positive charge;
a minimum of four amino acids; a maximum of about twenty amino
acids; a relationship between the minimum number of net positive
charges (p.sub.m) and the total number of amino acid residues (r)
wherein 3 p.sub.m is the largest number that is less than or equal
to r+1; and a relationship between the minimum number of aromatic
groups (a) and the total number of net positive charges (p.sub.t)
wherein 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.
Inventors: |
Szeto; Hazel H.; (New York,
NY) ; Liu; Shaoyl; (Palisades Park, NJ) ; Cho;
Sunghee; (Scarsdale, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Szeto; Hazel H.
Liu; Shaoyl
Cho; Sunghee |
New York
Palisades Park
Scarsdale |
NY
NJ
NY |
US
US
US |
|
|
Assignee: |
Cornell Research Foundation,
Inc.
Ithaca
NY
|
Family ID: |
37889419 |
Appl. No.: |
14/285226 |
Filed: |
May 22, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14075686 |
Nov 8, 2013 |
|
|
|
14285226 |
|
|
|
|
12850079 |
Aug 4, 2010 |
8603971 |
|
|
14075686 |
|
|
|
|
12434216 |
May 1, 2009 |
7811987 |
|
|
12850079 |
|
|
|
|
11532764 |
Sep 18, 2006 |
7541340 |
|
|
12434216 |
|
|
|
|
60718170 |
Sep 16, 2005 |
|
|
|
Current U.S.
Class: |
514/4.8 ;
514/6.9 |
Current CPC
Class: |
C07K 5/1019 20130101;
C07K 14/705 20130101; A61P 9/00 20180101; A61P 25/28 20180101; A61K
38/03 20130101; A61P 15/00 20180101; C07K 5/1016 20130101; A61P
3/06 20180101; C07K 5/101 20130101; A61P 3/10 20180101; A61P 9/10
20180101; A61P 13/02 20180101; A61P 29/00 20180101; A61P 25/00
20180101; A61K 38/06 20130101; A61K 38/08 20130101; A61P 27/02
20180101; A61K 38/00 20130101; A61P 3/04 20180101; A61P 43/00
20180101; C07K 5/1008 20130101; C07K 5/1024 20130101; A61P 13/12
20180101 |
Class at
Publication: |
514/4.8 ;
514/6.9 |
International
Class: |
C07K 5/103 20060101
C07K005/103; C07K 5/107 20060101 C07K005/107 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] The invention described in this application was funded by
the National Institute of Drug Abuse, Grant No. P01 DA08924, the
National Institute of Neurological Diseases and Stroke, Grant No.
R21 NS48295, and the National Heart, Lung and Blood Institute,
Grant No. RO1 HL082511. The United States Government has certain
rights in this invention.
Claims
1.-10. (canceled)
11. A method for treating tubular epithelial cell apoptosis in a
subject in need thereof comprising administering to the subject an
effective amount of an aromatic-cationic peptide having the formula
D-Arg-2'6'-Dmt-Lys-Phe-NH.sub.2 or Phe-D-Arg-Phe-Lys-NH.sub.2 or a
pharmaceutically acceptable salt thereof.
12. The method of claim 11, wherein the peptide has the formula
D-Arg-2'6'-Dmt-Lys-Phe-NH.sub.2.
13. The method of claim 11, wherein the peptide has the formula
Phe-D-Arg-Phe-Lys-NH.sub.2.
14. The method of claim 11, wherein the subject is a human.
15. The method of claim 11, wherein the peptide is administered
orally, topically, intranasally, systemically, intravenously,
subcutaneously, intramuscularly or transdermally.
16. The method of claim 11, wherein the tubular epithelial cell
apoptosis is associated with unilateral ureteral obstruction.
17. The method of claim 11, wherein the method further treats
interstitial fibrosis in the subject.
18. The method of claim 11, wherein the method further reduces
macrophage infiltration in the subject.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/434,216, filed May 1, 2009, which is a continuation of U.S.
application Ser. No. 11/532,764, filed Sep. 18, 2006, which claims
priority to U.S. Provisional Application No. 60/718,170, filed Sep.
16, 2005, the entire contents of all of which are incorporated
herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0003] CD36 is a transmembrane protein of the class B scavenger
receptor family. The protein is widely expressed on numerous cells,
such as microvascular endothelium, macrophages, platelets,
adipocytes, epithelial cells (e.g., intestinal epithelial and renal
tubular cells, etc.), pancreatic islet cells and cardiac muscle.
The receptor may interact with multiple extracellular ligands, such
as thrombospondin-1, long-chain fatty acids, and oxidized
low-density lipoprotein.
[0004] Abnormal expression of CD36 has been implicated in a wide
variety of diseases and conditions. For example, mice lacking CD36
have less atherosclerotic lesions when fed a Western diet compared
to wild-type mice. Further, CD36 knock out mice were reported to be
protected against acute cerebral ischcmia.
[0005] Therefore, methods for reducing expression of CD36
expression are beneficial for treating a disease or condition
characterized by abnormal expression of CD36.
SUMMARY OF THE INVENTION
[0006] In one embodiment, the invention provides a method for
reducing CD36 expression in a cell. The method comprises contacting
the cell with an effective amount of an aromatic-cationic
peptide.
[0007] In another embodiment, the invention provides a method for
reducing CD36 expression in a mammal in need thereof. The method
comprises administering to the mammal an effective amount of an
aromatic-cationic peptide.
[0008] In yet another embodiment, the invention provides a method
for treating a disease or condition characterized by increased CD36
expression in a mammal in need thereof. The method comprises
administering to the mammal an effective amount of an
aromatic-cationic peptide.
[0009] In a further embodiment, the invention provides a method for
treating ureteral obstruction in a mammal in need thereof. The
method comprises administering to the mammal an effective amount of
an aromatic-cationic peptide.
[0010] In yet a further embodiment, the invention provides a method
for treating diabetic nephropathy in a mammal in need thereof. The
method comprises administering to the mammal an effective amount of
an aromatic-cationic peptide.
[0011] In another embodiment, the invention provides a method for
reducing CD36 expression in a removed organ or tissue. The method
comprises administering to the mammal an effective amount of an
aromatic-cationic peptide.
[0012] The aromatic-cationic peptides useful in the methods of the
present invention have at least one net positive charge; a minimum
of four amino acids; a maximum of about twenty amino acids; a
relationship between the minimum number of net positive charges
(p.sub.m) and the total number of amino acid residues (r) wherein
3p.sub.m is the largest number that is less than or equal to r+1;
and a relationship between the minimum number of aromatic groups
(a) and the total number of net positive charges (p.sub.t) wherein
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.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1. SS-31 reduced oxLDL-induced CD36 mRNA expression,
CD36 protein expression, and foam cell formation in mouse
peritoneal macrophages.
[0014] FIG. 2. SS-31 treatment reduced infarct volume and
hemispheric swelling in wild-type mice subjected to acute cerebral
ischemia.
[0015] FIG. 3. SS-31 treatment reduced the decrease in reduced
glutathione (GSH) in post-ischemic brain in wild-type mice.
[0016] FIG. 4. SS-31 had no effect in reducing infarct volume or
hemispheric swelling in CD36 knock-out mice subjected to acute
cerebral ischemia.
[0017] FIG. 5. SS-31 did not reduce GSH depletion in post-ischemic
brain from CD36 knock-out mice.
[0018] FIG. 6. SS-31 reduced CD36 mRNA expression in post-ischemic
brain in wild-type mice.
[0019] FIG. 7. SS-31 decreases CD36 expression on renal tubular
cells after unilateral ureteral obstruction (UUO). Contralateral
unobstructed kidney (FIG. 7A); obstructed kidney in animals treated
with saline (FIG. 73B); and obstructed kidneys obtained from rats
treated with SS-31 (FIG. 7C).
[0020] FIG. 8. SS-31 reduces lipid peroxidation in kidney after
UUO. Tubular cells in the obstructed kidney (FIG. 8B),
contralateral unobstructed control (FIG. 8A); obstructed kidneys
from rats treated with SS-31 (FIG. 8C).
[0021] FIG. 9. SS-31 reduced tubular cell apoptosis in obstructed
kidney after UUO. Obstructed kidney from saline-treated animals
(FIG. 9B); contralateral unobstructed control (FIG. 9A); obstructed
kidney from SS-31 treated animals (FIG. 9C).
[0022] FIG. 10. SS-31 reduced macrophage infiltration in obstructed
kidney induced by UUO. Obstructed kidney (FIG. 10B); contralateral
unobstructed control (FIG. 10A); rats treated with SS-31 (FIG.
10C).
[0023] FIG. 11. SS-31 reduced interstitial fibrosis in obstructed
kidney after UUO. Obstructed kidney (FIG. 11B); contralateral
unobstructed control (FIG. 11A); rats treated with SS-31 (FIG.
11C).
[0024] FIG. 12. Cold storage of isolated hearts with SS-31 or SS-20
prevented upregulation of CD36 expression. The "background" control
(FIGS. 12A and 12B) represents two sections from a normal
non-ischemic heart that were not treated with the primary
anti-CD-36 antibody. "Normal heart" (FIGS. 12C and 12D) represents
two sections obtained from a non-ischemic heart. The sections from
a representative heart stored in St. Thomas solution (FIGS. 12E and
12F) for 18 hours at 4.degree. C. showed increased CD36 staining
compared to "Normal heart." CD36 staining was significantly reduced
in hearts stored with either 1 nM SS-31 (FIGS. 12G and 12H) or 100
nM SS-20 (FIGS. 121 and 12J) in St. Thomas solution.
[0025] FIG. 13. SS-31 and SS-20 reduced lipid peroxidation in
isolated guinea pig hearts subjected to warm reperfusion after
prolonged cold ischemia. FINE staining in hearts subjected to 18
hours of cold storage in St. Thomas solution (FIG. 13B) compared to
non-ischemic hearts (FIG. 13A). FINE staining was reduced in hearts
stored in SS-31 (FIG. 9C) or SS-20 (FIG. 13D).
[0026] FIG. 14. SS-31 and SS-20 abolished endothelial apoptosis in
isolated guinea pig hearts subjected to warm reperfusion after
prolonged cold ischemia. Hearts subjected to 18 hours of cold
storage in St. Thomas solution (FIGS. 14C and 14D); non-ischemic
normal hearts (FIGS. 14A and 14B). Apoptotic cells were not
observed in hearts stored in SS-31 (FIGS. 14E and 14F) or SS-20
(FIGS. 14G and 14H).
[0027] FIG. 15. SS-31 and SS-20 preserves coronary flow in isolated
guinea pig hearts subjected to warm reperfusion after prolonged
cold ischemia. Guinea pig hearts perfused with a cardioplegic
solution (St. Thomas solution) alone or St. Thomas solution
containing either 1 nM SS-31 (FIG. 15A) or 100 nM SS-20 (FIG. 15B)
for 3 min. and then subjected to 18 hours of cold ischemia
(4.degree. C.).
[0028] FIG. 16. SS-31 prevented damage to proximal tubules in
diabetic mice. Diabetes was induced by streptozotocin (STZ)
injection for 5 d. Kidney sections obtained after 3 weeks showed
loss of brush border in STZ-treated animals (FIG. 16A, panel B)
that was not seen in mice not treated with STZ (panel A). The loss
of brush border was not seen in STZ-treated animal that received
daily SS-31 (3 mg/kg) (panel C).
[0029] FIG. 17. SS-31 prevented renal tubular epithelial cell
apoptosis in diabetic mice. Diabetes was induced by streptozotocin
(STZ) injection for 5 d. Kidney sections obtained after 3 weeks
showed dramatic increase in apoptotic cells in proximal tubules in
STZ-treated animals (FIG. 17A, panel b) that was not seen in mice
not treated with STZ (FIG. 17A, panel a). The STZ-induced apoptosis
was not seen in mice that received daily SS-31 (3 mg/kg) (FIG. 17A,
panel c). The percent of apoptotic cells caused by STZ was
significantly reduced by SS-31 treatment (FIG. 17B).
DETAILED DESCRIPTION OF THE INVENTION
Peptides
[0030] The invention is directed to the reduction of CD36
expression by certain aromatic-cationic peptides. The
aromatic-cationic peptides are water-soluble and highly polar.
Despite these properties, the peptides can readily penetrate cell
membranes.
[0031] The aromatic-cationic peptides useful in the present
invention include a minimum of three amino acids, and preferably
include a minimum of four amino acids, covalently joined by peptide
bonds.
[0032] The maximum number of amino acids present in the
aromatic-cationic peptides of the present invention is about twenty
amino acids covalently joined by peptide bonds. Preferably, the
maximum number of amino acids is about twelve, more preferably
about nine, and most preferably about six. Optimally, the number of
amino acids present in the peptides is four.
[0033] The amino acids of the aromatic-cationic peptides useful in
the present invention can be any amino acid. As used herein, the
term "amino acid" is used to refer to any organic molecule that
contains at least one amino group and at least one carboxyl group.
Preferably, at least one amino group is at the position relative to
a carboxyl group.
[0034] The amino acids may be naturally occurring. Naturally
occurring amino acids include, for example, the twenty most common
levorotatory (L) amino acids normally found in mammalian proteins,
i.e., alanine (Ala), arginine (Arg), asparagine (Asn), aspartic
acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu),
glycine (Gly), histidine (His), isoleucine (lieu), leucine (Leu),
lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro),
serine (Ser), threonine (Thr), tryptophan, (Trp) tyrosine (Tyr),
and valine (Val).
[0035] Other naturally occurring amino acids include, for example,
amino acids that are synthesized in metabolic processes not
associated with protein synthesis. For example, the amino acids
ornithine and citrulline are synthesized in mammalian metabolism
during the production of urea. Another example of a naturally
occurring amino acid include hydroxyproline (Hyp).
[0036] The peptides useful in the present invention optionally
contain one or more non-naturally occurring amino acids. Optimally,
the peptide has no amino acids that are naturally occurring. The
non-naturally occurring amino acids may be levorotary (L-),
dextrorotatory (D-), or mixtures thereof.
[0037] Non-naturally occurring amino acids are those amino acids
that typically are not synthesized in normal metabolic processes in
living organisms, and do not naturally occur in proteins. In
addition, the non-naturally occurring amino acids useful in the
present invention preferably are also not recognized by common
proteases.
[0038] The non-naturally occurring amino acid can be present at any
position in the peptide. For example, the non-naturally occurring
amino acid can be at the N-terminus, the C-terminus, or at any
position between the N-terminus and the C-terminus.
[0039] The non-natural amino acids may, for example, comprise
alkyl, aryl, or alkylaryl groups not found in natural amino acids.
Some examples of non-natural alkyl amino acids include
.alpha.-aminobutyric acid, .beta.-aminobutyric acid,
.gamma.-aminobutyric acid, .delta.-aminovaleric acid, and
.epsilon.-aminocaproic acid. Some examples of non-natural aryl
amino acids include ortho-, meta, and para-aminobenzoic acid. Some
examples of non-natural alkylaryl amino acids include ortho-,
meta-, and para-aminophenylacetic acid, and
.gamma.-phenyl-.beta.-aminobutyric acid.
[0040] Non-naturally occurring amino acids include derivatives of
naturally occurring amino acids. The derivatives of naturally
occurring amino acids may, for example, include the addition of one
or more chemical groups to the naturally occurring amino acid.
[0041] For example, one or more chemical groups can be added to one
or more of the 2', 3', 4', 5', or 5' position of the aromatic ring
of a phenylalanine or tyrosine residue, or the 4', 5', 6', or 7'
position of the benzo ring of a tryptophan residue. The group can
be any chemical group that can be added to an aromatic ring. Some
examples of such groups include branched or unbranched
C.sub.1-C.sub.4 alkyl, such as methyl, ethyl, n-propyl, isopropyl,
butyl, isobutyl, or t-butyl, C.sub.1-C.sub.4 alkyloxy (i.e.,
alkoxy), amino, C.sub.1-C.sub.4 alkylamino and C.sub.1-C.sub.4
dialkylamino (e.g., methylamino, dimethylamino), nitro, hydroxyl,
halo (i.e., fluoro, chloro, bromo, or iodo). Some specific examples
of non-naturally occurring derivatives of naturally occurring amino
acids include norvaline (Nva) and norleucine (Nle).
[0042] Another example of a modification of an amino acid in a
peptide useful in the methods of the present invention is the
derivatization of a carboxyl group of an aspartic acid or a
glutamic acid residue of the peptide. One example of derivatization
is amidation with ammonia or with a primary or secondary amine,
e.g. methylamine, ethylamine, dimethylamine or diethylamine.
Another example of derivatization includes esterification with for
example, methyl or ethyl alcohol.
[0043] Another such modification includes derivatization of an
amino group of a lysine, arginine, or histidine residue. For
example, such amino groups can be acylated. Some suitable acyl
groups include, for example, a benzoyl group or an alkanoyl group
comprising any of the C.sub.1-C.sub.4 alkyl groups mentioned above,
such as an acetyl or propionyl group.
[0044] The non-naturally occurring amino acids are preferably
resistant, and more preferably insensitive, to common proteases.
Examples of non-naturally occurring amino acids that are resistant
or insensitive to proteases include the dextrorotatory (D-) form of
any of the above-mentioned naturally occurring L-amino acids, as
well as L- and/or D-non-naturally occurring amino acids. The
D-amino acids do not normally occur in proteins, although they are
found in certain peptide antibiotics that are synthesized by means
other than the normal ribosomal protein synthetic machinery of the
cell. As used herein, the D-amino acids are considered to be
non-naturally occurring amino acids.
[0045] In order to minimize protease sensitivity, the peptides
useful in the methods of the invention should have less than five,
preferably less than four, more preferably less than three, and
most preferably, less than two contiguous L-amino acids recognized
by common proteases, irrespective of whether the amino acids are
naturally or non-naturally occurring. Optimally, the peptide has
only D-amino acids, and no L-amino acids.
[0046] If the peptide contains protease sensitive sequences of
amino acids, at least one of the amino acids is preferably a
non-naturally occurring D-amino acid, thereby conferring protease
resistance. An example of a protease sensitive sequence includes
two or more contiguous basic amino acids that are readily cleaved
by common proteases, such as endopeptidases and trypsin. Examples
of basic amino acids include arginine, lysine and histidine.
[0047] It is important that the aromatic-cationic peptides have a
minimum number 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).
[0048] 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.
[0049] "Net charge" as used herein refers to the balance of the
number of positive charges and the number of negative charges
carried by the amino acids present in the peptide. In this
specification, it is understood that net charges are measured at
physiological pH. The naturally occurring amino acids that are
positively charged at physiological pH include L-lysine,
L-arginine, and L-histidine. The naturally occurring amino acids
that are negatively charged at physiological pH include L-aspartic
acid and L-glutamic acid.
[0050] Typically, a peptide has a positively charged N-terminal
amino group and a negatively charged C-terminal carboxyl group. The
charges cancel each other out at physiological pH.
[0051] 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) and the total number of amino acid
residues (r) is as follows:
TABLE-US-00001 (r) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
(p.sub.m) 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7
[0052] 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
[0053] 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.
[0054] 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).
[0055] 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).
[0056] In one embodiment of the present invention, the
aromatic-cationic peptides useful in the methods of the present
invention have a relationship between the minimum number of
aromatic groups (a) and the total number of net positive charges at
physiological pH (p.sub.t) wherein 3a is the largest number that is
less than or equal to p.sub.t+1, except that when p.sub.t is 1, a
may also be 1. In this embodiment, the relationship between the
minimum number of aromatic groups (a) and the total number of net
positive charges (p.sub.t) is as follows:
TABLE-US-00003 (p.sub.t) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
18 19 20 (a) 1 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7
[0057] In another embodiment, the aromatic-cationic peptides have a
relationship between the minimum number of aromatic groups (a) and
the total number of net positive charges (p.sub.t) wherein 2a is
the largest number that is less than or equal to p.sub.t+1. In this
embodiment, the relationship between the minimum number of aromatic
amino acid residues (a) and the total number of net positive
charges (p.sub.t) is as follows:
TABLE-US-00004 (p.sub.t) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
18 19 20 (a) 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10
[0058] In another embodiment, the number of aromatic groups (a) and
the total number of net positive charges (p.sub.t) are equal.
[0059] 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-dimethylamido, N,N-diethylamido,
N-methyl-N-ethylamido, N-phenylamido or N-phenyl-N-ethylamido
group.
[0060] 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.
[0061] 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.
[0062] Aromatic-cationic peptides useful in the methods of the
present invention include, but are not limited to, the following
peptide examples:
Lys-D-Arg-Tyr-NH.sub.2,
Phe-D-Arg-His,
D-Tyr-Trp-Lys-NH.sub.2,
Trp-D-Lys-Tyr-Arg-NH.sub.2,
Tyr-His-D-Gly-Met,
Phe-Arg-D-His-Asp,
Tyr-D-Arg-Phe-Lys-Glu-NH.sub.2,
Met-Tyr-D-Lys-Phe-Arg,
D-His-Glu-Lys-Tyr-D-Phe-Arg,
Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH.sub.2,
Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His,
Gly-D-Phe-Lys-Tyr-His-D-Arg-Tyr-NH.sub.2,
Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH.sub.2,
Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-Lys,
Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH.sub.2,
Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-Lys,
Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH.sub.2,
D-His-Lys-Tyr-D-Phc-Glu-D-Asp-D-His-D-Lys-Arg-Trp-NH.sub.2,
Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-Phe,
Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-Phe,
Phe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH.sub.2,
Phe-Try-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-Thr,
Tyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-Lys,
Glu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met-NH.sub.2,
Arg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-Phe-Tyr-D-Arg-Gly,
D-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH.s-
ub.2,
Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-Ph-
e,
His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Se-
r-NH.sub.2,
Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-Hi-
s-D-Lys-Asp, and
Thr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D--
His-Arg-Tyr-Lys-NH.sub.2.
[0063] In one embodiment, the peptides useful in the methods of the
present invention have mu-opioid receptor agonist activity (i.e.,
they activate the mu-opioid receptor). Activation of the mu-opioid
receptor typically elicits an analgesic effect.
[0064] In certain instances, an aromatic-cationic peptide having
mu-opioid receptor agonist activity is preferred. For example,
during short-term treatment, such as in an acute disease or
condition, it may be beneficial to use an aromatic-cationic peptide
that activates the mu-opioid receptor. Such acute diseases and
conditions are often associated with moderate or severe pain. In
these instances, the analgesic effect of the aromatic-cationic
peptide may be beneficial in the treatment regimen of the human
patient or other mammal. An aromatic-cationic peptide which does
not activate the mu-opioid receptor, however, may also be used with
or without an analgesic, according to clinical requirements.
[0065] Alternatively, in other instances, an aromatic-cationic
peptide that does not have mu-opioid receptor agonist activity is
preferred. For example, during long-term treatment, such as in a
chronic disease state or condition, the use of an aromatic-cationic
peptide that activates the mu-opioid receptor may be
contraindicated. In these instances the potentially adverse or
addictive effects of the aromatic-cationic peptide may preclude the
use of an aromatic-cationic peptide that activates the mu-opioid
receptor in the treatment regimen of a human patient or other
mammal. Potential adverse effects may include sedation,
constipation and respiratory depression. In such instances an
aromatic-cationic peptide that does not activate the mu-opioid
receptor may be an appropriate treatment.
[0066] Peptides useful in the methods of the present invention
which have mu-opioid receptor agonist activity are typically those
peptides which have a tyrosine residue or a tyrosine derivative at
the N-terminus (i.e., the first amino acid position). Preferred
derivatives of tyrosine include 2'-methyltyrosine (Mmt);
2',6'-dimethyltyrosine (2'6'Dmt); 3',5'-dimethyltyrosine (3'5'Dmt);
N,2',6'-trimethyltyrosine (Tmt); and 2'-hydroxy-6'-methyltryosine
(Hmt).
[0067] In a particular preferred embodiment, a peptide that has
mu-opioid receptor agonist activity has the formula
Tyr-D-Arg-Phe-Lys-NH.sub.2 (for convenience represented by the
acronym: DALDA, which is referred to herein as SS-01). DALDA has a
net positive charge of three, contributed by the amino acids
tyrosine, arginine, and lysine and has two aromatic groups
contributed by the amino acids phenylalanine and tyrosine. The
tyrosine of DALDA can be a modified derivative of tyrosine such as
in 2',6'-dimethyltyrosine to produce the compound having the
formula 2',6'-Dmt-D-Arg-Phe-Lys-NH.sub.2 (i.e., Dmt.sup.1-DALDA),
which is referred to herein as SS-02).
[0068] Peptides that do not have mu-opioid receptor agonist
activity generally do not have a tyrosine residue or a derivative
of tyrosine at the N-terminus (i.e., amino acid position 1). The
amino acid at the N-terminus can be any naturally occurring or
non-naturally occurring amino acid other than tyrosine.
[0069] 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).
[0070] Another aromatic-cationic peptide that does not have
mu-opioid receptor agonist activity has the formula
Phe-D-Arg-Phe-Lys-NH.sub.2 (i.e., Phe.sup.1-DALDA, which is
referred to herein as SS-20). Alternatively, the N-terminal
phenylalanine can be a derivative of phenylalanine such as
2',6'-dimethylphenylalanine (2'6'Dmp). DALDA containing
2',6'-dimethylphenylalanine at amino acid position 1 has the
formula 2',6'-Dmp-D-Arg-Phe-Lys-NH.sub.2, (i.e.
2'6'Dmp.sup.1-DALDA).
[0071] 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 agonist activity has the formula
D-Arg-2'6'Dmt-Lys-Phe-NH.sub.2 (referred to in this specification
as SS-31).
[0072] DALDA, Phe.sup.1-DALDA, SS-31, and their derivatives can
further include functional analogs. A peptide is considered a
functional analog of DALDA, Phe.sup.1-DALDA, or SS-31 if the analog
has the same function as DALDA, Phe.sup.1-DALDA, or SS-31. The
analog may, for example, be a substitution variant of DALDA,
Phe.sup.1-DALDA, or SS-31, wherein one or more amino acids are
substituted by another amino acid.
[0073] Suitable substitution variants of DALDA, Phe.sup.1-DALDA, or
SS-31 include conservative amino acid substitutions. Amino acids
may be grouped according to their physiochemical characteristics as
follows: [0074] (a) Non-polar amino acids: Ala(A) Ser(S) Thr(T)
Pro(P) Gly(G); [0075] (b) Acidic amino acids: Asn(N) Asp(D) Glu(E)
Gln(Q); [0076] (c) Basic amino acids: His(H4) Arg(R) Lys(K); [0077]
(d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V); and
[0078] (e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His (H).
[0079] Substitutions of an amino acid in a peptide by another amino
acid in the same group is referred to as a conservative
substitution and may preserve the physiological 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.
[0080] Examples of analogs useful in the practice of the present
invention that activate mu-opioid receptors include, but are not
limited, to the aromatic-cationic peptides shown in Table 1.
TABLE-US-00005 TABLE 1 Amino Amino Amino Amino C- Acid Acid Acid
Acid Amino Acid Terminal Position Position Position Position
Position 5 Modi- 1 2 3 4 (if present) fication 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.sub.2
NH(CH.sub.2).sub.2--NH- dns 2'6'Dmt D-Arg Phe Lys- NH.sub.2
NH(CH.sub.2).sub.2--NH- atn 2'6'Dmt D-Arg Phe dnsLys NH.sub.2
2'6'Dmt D-Cit Phe Lys NH.sub.2 2'6'Dmt D-Cit Phe Ahp NH.sub.2
2'6'Dmt D-Arg Phe Orn NH.sub.2 2'6'Dmt D-Arg Phe Dab NH.sub.2
2'6'Dmt D-Arg Phe Dap NH.sub.2 2'6'Dmt D-Arg Phe Ahp (2- NH.sub.2
aminoheptanoic acid) Bio- D-Arg Phe Lys NH.sub.2 2'6'Dmt 3'5'Dmt
D-Arg Phe Lys NH.sub.2 3'5'Dmt D-Arg Phe Orn NH.sub.2 3'5'Dmt D-Arg
Phe Dab NH.sub.2 3'5'Dmt D-Arg Phe Dap NH.sub.2 Tyr D-Arg Tyr Lys
NH.sub.2 Tyr D-Arg Tyr Orn NH.sub.2 Tyr D-Arg Tyr Dab NH.sub.2 Tyr
D-Arg Tyr Dap NH.sub.2 2'6'Dmt D-Arg Tyr Lys NH.sub.2 2'6'Dmt D-Arg
Tyr Orn NH.sub.2 2'6'Dmt D-Arg Tyr Dab NH.sub.2 2'6'Dmt D-Arg Tyr
Dap NH.sub.2 2'6'Dmt D-Arg 2'6'Dmt Lys NH.sub.2 2'6'Dmt D-Arg
2'6'Dmt Orn NH.sub.2 2'6'Dmt D-Arg 2'6'Dmt Dab NH.sub.2 2'6'Dmt
D-Arg 2'6'Dmt Dap NH.sub.2 3'5'Dmt D-Arg 3'5'Dmt Arg NH.sub.2
3'5'Dmt D-Arg 3'5'Dmt Lys NH.sub.2 3'5'Dmt D-Arg 3'5'Dmt Orn
NH.sub.2 3'5'Dmt D-Arg 3'5'Dmt Dab NH.sub.2 Tyr D-Lys Phe Dap
NH.sub.2 Tyr D-Lys Phe Arg NH.sub.2 Tyr D-Lys Phe Lys NH.sub.2 Tyr
D-Lys Phe Orn NH.sub.2 2'6'Dmt D-Lys Phe Dab NH.sub.2 2'6'Dmt D-Lys
Phe Dap NH.sub.2 2'6'Dmt D-Lys Phe Arg NH.sub.2 2'6'Dmt D-Lys Phe
Lys NH.sub.2 3'5'Dmt D-Lys Phe Orn NH.sub.2 3'5'Dmt D-Lys Phe Dab
NH.sub.2 3'5'Dmt D-Lys Phe Dap NH.sub.2 3'5'Dmt D-Lys Phe Arg
NH.sub.2 Tyr D-Lys Tyr Lys NH.sub.2 Tyr D-Lys Tyr Orn NH.sub.2 Tyr
D-Lys Tyr Dab NH.sub.2 Tyr D-Lys Tyr Dap NH.sub.2 2'6'Dmt D-Lys Tyr
Lys NH.sub.2 2'6'Dmt D-Lys Tyr Orn NH.sub.2 2'6'Dmt D-Lys Tyr Dab
NH.sub.2 2'6'Dmt D-Lys Tyr Dap NH.sub.2 2'6'Dmt D-Lys 2'6'Dmt Lys
NH.sub.2 2'6'Dmt D-Lys 2'6'Dmt Orn NH.sub.2 2'6'Dmt D-Lys 2'6'Dmt
Dab NH.sub.2 2'6'Dmt D-Lys 2'6'Dmt Dap NH.sub.2 2'6'Dmt D-Arg Phe
dnsDap NH.sub.2 2'6'Dmt D-Arg Phe atnDap NH.sub.2 3'5'Dmt D-Lys
3'5'Dmt Lys NH.sub.2 3'5'Dmt D-Lys 3'5'Dmt Orn NH.sub.2 3'5'Dmt
D-Lys 3'5'Dmt Dab NH.sub.2 3'5'Dmt D-Lys 3'5'Dmt Dap NH.sub.2 Tyr
D-Lys Phe Arg NH.sub.2 Tyr D-Orn Phe Arg NH.sub.2 Tyr D-Dab Phe Arg
NH.sub.2 Tyr D-Dap Phe Arg NH.sub.2 2'6'Dmt D-Arg Phe Arg NH.sub.2
2'6'Dmt D-Lys Phe Arg NH.sub.2 2'6'Dmt D-Orn Phe Arg NH.sub.2
2'6'Dmt D-Dab Phe Arg NH.sub.2 3'5'Dmt D-Dap Phe Arg NH.sub.2
3'5'Dmt D-Arg Phe Arg NH.sub.2 3'5'Dmt D-Lys Phe Arg NH.sub.2
3'5'Dmt D-Orn Phe Arg NH.sub.2 Tyr D-Lys Tyr Arg NH.sub.2 Tyr D-Orn
Tyr Arg NH.sub.2 Tyr D-Dab Tyr Arg NH.sub.2 Tyr D-Dap Tyr Arg
NH.sub.2 2'6'Dmt D-Arg 2'6'Dmt Arg NH.sub.2 2'6'Dmt D-Lys 2'6'Dmt
Arg NH.sub.2 2'6'Dmt D-Orn 2'6'Dmt Arg NH.sub.2 2'6'Dmt D-Dab
2'6'Dmt Arg NH.sub.2 3'5'Dmt D-Dap 3'5'Dmt Arg NH.sub.2 3'5'Dmt
D-Arg 3'5'Dmt Arg NH.sub.2 3'5'Dmt D-Lys 3'5'Dmt Arg NH.sub.2
3'5'Dmt D-Orn 3'5'Dmt Arg NH.sub.2 Mmt D-Arg Phe Lys NH.sub.2 Mmt
D-Arg Phe Orn NH.sub.2 Mmt D-Arg Phe Dab NH.sub.2 Mmt D-Arg Phe Dap
NH.sub.2 Tmt D-Arg Phe Lys NH.sub.2 Tmt D-Arg Phe Orn NH.sub.2 Tmt
D-Arg Phe Dab NH.sub.2 Tmt D-Arg Phe Dap NH.sub.2 Hmt D-Arg Phe Lys
NH.sub.2 Hmt D-Arg Phe Orn NH.sub.2 Hmt D-Arg Phe Dab NH.sub.2 Hmt
D-Arg Phe Dap NH.sub.2 Mmt D-Lys Phe Lys NH.sub.2 Mmt D-Lys Phe Orn
NH.sub.2 Mmt D-Lys Phe Dab NH.sub.2 Mmt D-Lys Phe Dap NH.sub.2 Mmt
D-Lys Phe Arg NH.sub.2 Tmt D-Lys Phe Lys NH.sub.2 Tmt D-Lys Phe Orn
NH.sub.2 Tmt D-Lys Phe Dab NH.sub.2 Tmt D-Lys Phe Dap NH.sub.2 Tmt
D-Lys Phe Arg NH.sub.2 Hmt D-Lys Phe Lys NH.sub.2 Hmt D-Lys Phe Orn
NH.sub.2 Hmt D-Lys Phe Dab NH.sub.2 Hmt D-Lys Phe Dap NH.sub.2 Hmt
D-Lys Phe Arg NH.sub.2 Mmt D-Lys Phe Arg NH.sub.2 Mmt D-Orn Phe Arg
NH.sub.2 Mmt D-Dab Phe Arg NH.sub.2 Mmt D-Dap Phe Arg NH.sub.2 Mmt
D-Arg Phe Arg NH.sub.2 Tmt D-Lys Phe Arg NH.sub.2 Tmt D-Orn Phe Arg
NH.sub.2 Tmt D-Dab Phe Arg NH.sub.2 Tmt D-Dap Phe Arg NH.sub.2 Tmt
D-Arg Phe Arg NH.sub.2 Hmt D-Lys Phe Arg NH.sub.2 Hmt D-Orn Phe Arg
NH.sub.2 Hmt D-Dab Phe Arg NH.sub.2 Hmt D-Dap Phe Arg NH.sub.2 Hmt
D-Arg Phe Arg NH.sub.2 Dab = diaminobutyric Dap = diaminopropionic
acid Dmt = dimethyltyrosine Mmt = 2'-methyltyrosine Tmt =
N,2',6'-trimethyltyrosine Hmt = 2'-hydroxy,6'-methyltyrosine dnsDap
= .beta.-dansyl-L-.alpha.,.beta.-diaminopropionic acid atnDap =
.beta.-anthraniloyl-L-.alpha.,.beta.-diaminopropionic acid Bio =
biotin
[0081] Examples of analogs useful in the practice of the present
invention that do not activate mu-opioid receptors include, but are
not limited to, the aromatic-cationic peptides shown in Table
2.
TABLE-US-00006 TABLE 2 Amino Acid Amino Acid Amino Acid Amino Acid
C-Terminal Position 1 Position 2 Position 3 Position 4 Modification
D-Arg Dmt Lys Phe NH.sub.2 D-Arg Dmt Phe Lys NH.sub.2 D-Arg Phe Lys
Dmt NH.sub.2 D-Arg Phe Dmt Lys NH.sub.2 D-Arg Lys Dmt Phe NH.sub.2
D-Arg Lys Phe Dmt NH.sub.2 Phe Lys Dmt D-Arg NH.sub.2 Phe Lys D-Arg
Dmt NH.sub.2 Phe D-Arg Dmt Lys NH.sub.2 Phe D-Arg Lys Dmt NH.sub.2
Phe Dmt D-Arg Lys NH.sub.2 Phe Dmt Lys D-Arg NH.sub.2 Lys Phe D-Arg
Dmt NH.sub.2 Lys Phe Dmt D-Arg NH.sub.2 Lys Dmt D-Arg Phe NH.sub.2
Lys Dmt Phe D-Arg NH.sub.2 Lys D-Arg Phe Dmt NH.sub.2 Lys D-Arg Dmt
Phe NH.sub.2 D-Arg Dmt D-Arg Phe NH.sub.2 D-Arg Dmt D-Arg Dmt
NH.sub.2 D-Arg Dmt D-Arg Tyr NH.sub.2 D-Arg Dmt D-Arg Trp NH.sub.2
Trp D-Arg Phe Lys NH.sub.2 Trp D-Arg Tyr Lys NH.sub.2 Trp D-Arg Trp
Lys NH.sub.2 Trp D-Arg Dmt Lys NH.sub.2 D-Arg Trp Lys Phe NH.sub.2
D-Arg Trp Phe Lys NH.sub.2 D-Arg Trp Lys Dmt NH.sub.2 D-Arg Trp Dmt
Lys NH.sub.2 D-Arg Lys Trp Phe NH.sub.2 D-Arg Lys Trp Dmt NH.sub.2
Cha = cyclohexyl
[0082] The amino acids of the peptides shown in table 1 and 2 may
be in either the L- or the D-configuration.
Methods
[0083] The aromatic-cationic peptides described above are useful in
reducing CD36 expression in a cell. For the purposes of this
specification CD36 expression in a cell is considered to be reduced
if the expression of CD36 is decreased by about 10%, preferably by
about 25%, more preferably by about 50%, even more preferably by
about 75%. Optimally, CD36 is reduced to about normal levels in a
cell.
[0084] CD36 is expressed on a wide variety of cells. Examples of
such cells include macrophages, platelets, adipocytes, endothelial
cells such as microvascular endothelial cells and umbilical vein
endothelial cells; epithelial cells such as intestinal epithelial
cells, gall bladder epithelial cells, bladder epithelial cells,
bronchial epithelial cells and alvelolar epithelial cells; renal
tubular cells; pancreatic islet cells; hepatocytes; skeletal muscle
cells; cardiac muscle cells; neuronal cells; glia cells; pancreas
cells; sperm cells; etc.
[0085] For the purposes of this specification, cells expressing
about 10%, typically about 25%, about typically about 50%, and even
more typically about 75% more CD36 than normal cells are considered
to express increased levels of CD36.
[0086] In one embodiment, the invention provides a method for
reducing CD36 expression in a cell. Any cell that expresses CD36
can be used in the method of the invention, and include those
mentioned above. The method for reducing CD36 expression in a cell
comprises contacting the cell with an effective amount of an
aromatic-cationic peptide described above.
[0087] In another embodiment, the invention provides a method for
reducing CD36 expression in a mammal in need thereof. The method
for reducing CD36 expression in the mammal comprises administering
to the mammal an effective amount of an aromatic-cationic peptide
described herein.
[0088] Mammals in need of reducing CD36 expression include, for
example, mammals that have increased CD36 expression. The increased
expression of CD36 is associated with various diseases and
conditions. Examples of diseases and conditions characterized by
increased CD36 expression include, but are not limited to,
atherosclerosis, inflammation, abnormal angiogenesis, abnormal
lipid metabolism, abnormal removal of apoptotic cells, ischemia
such as cerebral ischemia and myocardial ischemia, ischemia
reperfusion, ureteral obstruction, stroke, Alzheimer's Disease,
diabetes, diabetic nephropathy and obesity. A discussion on the
involvement of CD36 in atherosclerosis may be found in "Targeted
disruption of the class B scavenger receptor CD36 protects against
atherosclerotic lesion development in mice," Febbraio M, Podrez E
A, Smith J D, Hajjar D P, Hazen S L et al., J Clinical
Investigation, 105:1049-1056, 2000, and "CD36: a class B scavenger
receptor involved in angiogenesis, atherosclerosis, inflammation,
and lipid metabolism," Febbraio M., Hajjar D P and Silverstein R L,
Journal of Clinical Investigation, 108:785-791, 2001.
[0089] Mammals in need of reducing CD36 expression also include
mammals suffering from complications of diabetes. Some
complications of diabetes include, in addition to nephropathy,
neuropathy, retinopathy, coronary artery disease, and peripheral
vascular disease associated with diabetes.
[0090] In another embodiment, the invention relates to a method for
reducing CD36 expression in removed organs and tissues. The method
comprises contacting the removed organ or tissue with an effective
amount of an aromatic-cationic peptide described above. An organ or
tissue may, for example, be removed from a donor for autologous or
heterologous transplantation. Some examples of organs and tissues
include heart, lungs, pancreas, kidney, liver, skin, etc.
Synthesis of the Peptides
[0091] The peptides useful in the methods of the present invention
may be synthesized by any of the methods well known in the art.
Suitable methods for chemically synthesizing the protein include,
for example those described by Stuart and Young in "Solid Phase
Peptide Synthesis," Second Edition, Pierce Chemical Company (1984),
and in "Solid Phase Peptide Synthesis," Methods Enzymol., 289,
Academic Press, Inc, New York (1997).
Modes of Administration
[0092] Any method known to those in the art for contacting a cell,
organ or tissue with a peptide may be employed. Suitable methods
include in vitro, ex vivo, or in vivo methods.
[0093] In vitro methods typically include cultured samples. For
example, a cell can be placed in a reservoir (e.g., tissue culture
plate), and incubated with an aromatic-cationic peptide under
appropriate conditions suitable for reducing CD36 expression.
Suitable incubation conditions can be readily determined by those
skilled in the art.
[0094] Ex vivo methods typically include cells, organs or tissues
removed from a mammal, such as a human. The cells, organs or
tissues can, for example, be incubated with the peptide under
appropriate conditions. The contacted cells, organs or tissues are
normally returned to the donor, placed in a recipient, or stored
for future use. Thus, the peptide is generally in a
pharmaceutically acceptable carrier.
[0095] In vivo methods are typically limited to the administration
of an aromatic-cationic peptide, such as those described above, to
a mammal, preferably a human. The peptides useful in the methods of
the present invention are administered to a mammal in an amount
effective in reducing expression CD36 or treating the mammal. The
effective amount is determined during pre-clinical trials and
clinical trials by methods familiar to physicians and
clinicians.
[0096] An effective amount of a peptide useful in the methods of
the present invention, preferably in a pharmaceutical composition,
may be administered to a mammal in need thereof by any of a number
of well-known methods for administering pharmaceutical compounds.
The peptide may be administered systemically or locally.
[0097] 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.
[0098] 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.
[0099] Other routes of administration include
intracerebroventricularly or intrathecally.
Intracerebroventiculatly refers to administration into the
ventricular system of the brain. Intrathecally refers to
administration into the space under the arachnoid membrane of the
spinal cord. Thus intracerebroventricular or intrathecal
administration may be preferred for those diseases and conditions
which affect the organs or tissues of the central nervous
system.
[0100] 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.
[0101] A description of methods for delivering a compound by
controlled release can be found in international PCT Application
No. WO 02/083106. The PCT application is incorporated herein by
reference in its entirety.
[0102] 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.
[0103] For systemic, intracerebroventricular, intrathecal, topical,
intranasal, subcutaneous, or transdermal administration,
formulations of the aromatic-cationic peptides useful in the
methods of the present inventions may utilize conventional
diluents, carriers, or excipients etc., such as those known in the
art to deliver the peptides. For example, the formulations may
comprise one or more of the following: a stabilizer, a surfactant,
preferably a nonionic surfactant, and optionally a salt and/or a
buffering agent. The peptide may be delivered in the form of an
aqueous solution, or in a lyophilized form.
[0104] 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.
[0105] 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).
[0106] The salt or buffering agent may be any salt or buffering
agent, such as for example, sodium chloride, or sodium/potassium
phosphate, respectively. Preferably, the buffering agent maintains
the pH of the pharmaceutical composition in the range of about 5.5
to about 7.5. The salt and/or buffering agent is also useful to
maintain the osmolality at a level suitable for administration to a
human or an animal. Preferably the salt or buffering agent is
present at a roughly isotonic concentration of about 150 mM to
about 300 mM.
[0107] 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.
[0108] The mammal treated in accordance with the invention can be
any mammal, including, for example, farm animals, such as sheep,
pigs, cows, and horses; pet animals such as dogs and cats;
laboratory animals, such as rats, mice and rabbits. In a preferred
embodiment, the mammal is a human.
EXAMPLES
Example 1
SS-31 Reduced Oxidized Low-Density Lipoprotein (oxLDL)-Induced CD36
Expression and Foam Cell Formation in Mouse Peritoneal
Macrophages
[0109] Atherosclerosis is thought to develop as a result of lipid
uptake by vascular-wall macrophages leading to the development of
foam cells and the elaboration of cytokines and chemokines
resulting in smooth muscle-cell proliferation. CD36 is a scavenger
receptor that mediates uptake of oxLDL into macrophages and
subsequent foam-cell development. CD36 knock out mice showed
reduced uptake of oxLDL and reduced atherosclerosis.
[0110] CD36 expression is regulated at the transcriptional level by
various cellular stimuli, including glucose and oxLDL. Macrophages
were harvested from mice peritoneal cavity and culture overnight in
the absence or presence of oxLDL (50 .mu.g/ml) for 48 h. Incubation
with oxLDL significantly increased CD36 mRNA (FIG. 1A). Inclusion
of SS-31 (10 nM or 1 .mu.M) to the culture medium abolished the
up-regulation of CD36 (FIG. 1A). SS-31 by itself had no effect on
CD36 expression.
[0111] Expression of CD36 protein, as determined by western blot,
was also significantly increased after 48 h incubation with 25
.mu.g/ml of oxLDL (oxL) when compared to vehicle control (V) (FIG.
1B). Other controls included CD36 expression from mouse heart (H)
and macrophages obtained from CD36 knockout mice (KO). The amount
of CD36 protein was normalized to .beta.-actin. Incubation with
SS-31 (1 .mu.M) (S) significantly reduced CD36 protein expression
compared to macrophages exposed to vehicle control (V) (P<0.01,
ANOVA with posthoc Neuman Keuls test). Concurrent incubation with
SS-31 (1 .mu.M) also significantly inhibited the upregulation of
CD36 protein expression in macrophages exposed to 25 .mu.g/ml oxLDL
for 48 h (oxL/S) (P<0.01, ANOVA with posthoc Neuman Keuls
test).
[0112] Incubation of macrophages with oxLDL for 48 h also increased
foam cell formation (FIG. 1C). Foam cell is indicated by oil red 0
which stains lipid droplets red. Inclusion of SS-31 (1 .mu.M)
prevented oxLDL-induced foam cell formation (FIG. 1C).
[0113] Incubation of macrophages with oxLDL increased apoptotic
cells from 6.7% to 32.8%. Concurrent treatment with SS-31 (1 nM)
significantly reduced the percentage of apoptotic cells induced by
oxLDL to 20.8%.
Example 2
SS-31 Protected Mice from Acute Cerebral Ischemia
[0114] Cerebral ischemia initiates a cascade of cellular and
molecular events that lead to brain damage. One such event is
postischemic inflammation. Using a mouse model of cerebral
ischemia-reperfusion (20 min. occlusion of the middle cerebral
artery), it was found that CD36 was upregulated in microglia and
macrophages in the post-ischemic brain, and there was increased
reactive oxygen species production. CD36 knock out mice had a
profound reduction in reactive oxygen species after ischemia and
improved neurological function compared to wild type mice.
[0115] Cerebral ischemia was induced by occlusion of the right
middle cerebral artery for 30 min. Wild-type (WT) mice were given
either saline vehicle (Veh) (ip, n=19) or SS-31 (2 mg/kg or 5
mg/kg, ip, n=6) at 0, 6, 24 and 48 h after ischemia. Mice were
killed 3 days after ischemia. Brains were removed, frozen, and
sectioned. Brain sections were stained by the Nissl stain. Infarct
volume and hemispheric swelling was determined using an image
analyzer. Data were analyzed by one-way ANOVA with posthoc
analysis.
[0116] Treatment of wild type mice with SS-31(2 mg/kg or 5 mg/kg,
ip, n=6) at 0, 6, 24 and 48 hours after 30 min. occlusion of the
middle cerebral artery resulted in a significant reduction in
infarct volume (FIG. 2A) and hemispheric swelling (FIG. 2B)
compared to saline controls. (*P<0.05 compared to Veh).
[0117] Thirty min. cerebral ischemia in WT mice resulted in
significant depletion in reduced glutathione (GSH) in the
ipsilateral cortex and striatum compared to the contralateral side
in vehicle-treated animals (FIG. 3). The depletion of GSH in the
ipsilateral cortex was significantly reduced in mice treated with
SS-31 (2 mg/kg ip at 0, 6, 24 and 48 h) (FIG. 3). The depletion of
GSH in the striatum was also reduced by SS-31 treatment but did not
reach statistical significance.
Example 3
SS-31 Mediated Protection Against Acute Cerebral Ischemia Mimics
Protection Observed in CD36 Knockout Mice
[0118] CD36 knockout (CD36 KO) mice were subjected to acute
cerebral ischemia as described under Example 2. CD36 KO mice were
given either saline vehicle (Veh) (ip, n=5) or SS-31 (2 mg/kg, i.p.
n=5) at 0, 6, 24 and 48 h after 30 min ischemia. Infarct volume
(FIG. 4A) and hemispheric swelling (FIG. 4B) in CD36 KO mice were
similar whether they received saline or SS-31.
[0119] Treatment of CD36 KO mice with SS-31 (2 mg/kg, i.p., n=5)
also failed to further prevent GSH depletion in the ipsilateral
cortex caused by 30 min ischemia (FIG. 5).
[0120] These data suggest that the protective action of SS-31
against acute cerebral ischemia may be mediated by inhibiting the
upregulation of CD36.
Example 4
SS-31 Reduced CD36 mRNA Expression in Post-Ischemic Brain
[0121] Transient occlusion of the middle cerebral artery has been
shown to significantly increase the expression of CD36 mRNA in
microglia and macrophages in the post-ischemic brain. Wild-type
mice were given saline vehicle (Veh, i.p., n=6) or SS-31 (5 mg/kg,
i.p., n=6) at 0 and 6 h after 30 min ischemia, and CD36 mRNA levels
were determined using real time PCR. CD36 expression was
upregulated almost 6-fold in the ipsilateral brain compared to the
contralateral brain in mice that received saline (FIG. 6). CD36
mRNA was significantly reduced in the ipsilateral brain in mice
that received SS-31 treatment (FIG. 6).
Example 5
SS-31 Suppressed Upregulation of CD-36 in Renal Tubular Cells
Following Unilateral Ureteral Obstruction
[0122] Unilateral ureteral obstruction (UUO) is a common clinical
disorder associated with tubular cell apoptosis, macrophage
infiltration, and interstitial fibrosis. Interstitial fibrosis
leads to a hypoxic environment and contributes to progressive
decline in renal function despite surgical correction. CD36 has
been shown to be expressed on renal tubular cells.
[0123] CD36 was found to have been upregulated in tubular cells
after UUO. UUO was performed in Sprague-Dawley rats. The rats were
treated with saline (ip, n=6) or SS-31 (1 mg/kg ip, n=6) one day
prior to induction of UUO, and once a day for 14 days after UUO.
Rats were killed, kidneys removed, embedded in paraffin and
sectioned. The slides were treated with the anti-CD36 polyclonal
IgG (Santa Cruz #sc-9154; 1:100 with blocking serum) at room
temperature for 1.5 hours. The slides were then incubated with the
second antibody conjugated with biotin (anti-rabbit IgG-G1; ABC
kit, PK-6101) at room temperature for 30 min. The slides were then
treated with avidin, developed with DAB and counterstained with 10%
hematoxylin. The contralateral unobstructed kidney served as the
control for each animal.
[0124] UUO resulted in tubular dilation and significant increase in
expression of CD36 on the tubular cells (FIG. 7). Tubular dilation
was also observed in rats treated with SS-31, but there was a
significant reduction in CD36 expression (FIG. 3). CD36 expression
(brown stain) is primarily found on tubular cells in the
contralateral unobstructed kidney (FIG. 7A). CD36 expression was
increased in the obstructed kidney in animals treated with saline
(FIG. 7B), but was much reduced in obstructed kidneys obtained from
rats treated with SS-31 (FIG. 7C).
[0125] To determine whether SS-31 reduces lipid peroxidation in
kidney after UUO, rats were treated with either saline (n=6) or
SS-31 (1 mg/kg ip, n=6) one day prior to induction of UUO, and once
a day for 14 days UUO. Rats were then killed, kidneys removed,
embedded in paraffin and sectioned. Slides were incubated with
anti-HNE rabbit IgG and a biotin-linked anti-rabbit IgG was used as
secondary antibody. The slides were developed with DAB. Lipid
peroxidation, which was increased by UUO, was reduced by SS-31
treatment (FIG. 8). HNE stain (brown) was significantly increased
in tubular cells in the obstructed kidney (FIG. 8B) compared to the
contralateral control (FIG. 8A). Obstructed kidneys from rats
treated with SS-31 showed significantly less HNE stain (FIG. 8C)
compared to saline-treated rats (FIG. 8B).
[0126] To determine whether SS-31 reduced tubular cell apoptosis in
obstructed kidney after UUO, rats were treated with either saline
(n=6) or SS-31 (1 mg/kg ip, n=6) one day prior to induction of UUO,
and once a day for 14 days after UUO. Rats were then killed,
kidneys removed, embedded in paraffin and sectioned. To quantitate
nuclei with fragmented DNA, the TUNEL assay were performed with in
situ TUNEL kit (Intergen, Purchase, N.Y.). Slides were developed
with DAB and counterstained with 10% hematoxylin. The upregulation
of CD36 in saline-treated controls associated with tubular cell
apoptosis was significantly inhibited by SS-31 treatment (FIG. 9).
Compared to the contralateral unobstructed control (FIG. 9A), a
significant increase in apoptotic cells was observed in the
obstructed kidney from saline-treated animals (FIG. 9B). The number
of apoptotic cells was significantly reduced in obstructed kidney
from SS-31 treated animals (FIG. 9C) (P<0.001; n=6).
[0127] Macrophage infiltration (FIG. 10) and interstitial fibrosis
(FIG. 11) were also prevented by SS-31 treatment. Rats were treated
with either saline (n=6) or SS-31 (1 mg/kg ip, n=6) one day prior
to induction of UUO, and once a day for 14 days after UUO. Rats
were then killed, kidneys removed, embedded in paraffin and
sectioned. Slides were treated with monoclonal antibody for ED1
macrophage (1:75; Serotec). Horseradish-peroxidase-linked rabbit
anti-mouse secondary antibody (Dako) was used for macrophage
detection. Sections were then counterstained with 10% hematoxylin.
The number of macrophages in the obstructed kidney in
saline-treated rats (FIG. 10B) was significantly increased compared
to the contralateral unobstructed control (FIG. 10A). Macrophage
infiltration was significantly reduced in rats treated with SS-31
(FIG. 10C) (P<0.05; t-test).
[0128] Rats were treated with either saline (n=6) or SS-31 (1 mg/kg
ip, n=6) one day prior to induction of UUO, and once a day for 14
days after UUO. Rats were then killed, kidneys removed, embedded in
paraffin and sectioned. Slides were stained with hematoxylin and
eosin and Masson's trichome for interstitial fibrosis (blue stain).
Obstructed kidneys from saline-treated rats showed increase
fibrosis (FIG. 11B) compared to the contralateral unobstructed
control (FIG. 11A); while obstructed kidneys from SS-31 treated
rats showed significantly less fibrosis (P<0.05; t-test).
[0129] These results show that SS-31 suppresses the upregulation of
CD36 on renal tubular cells induced by UUO.
Example 6
SS-31 and SS-20 Reduced CD36 Expression in Isolated Hearts Upon
Reperfusion after Prolonged Cold Ischemic Storage
[0130] Organ transplantation requires hypothermic storage of the
isolated organ for transport to the recipient. Currently, cardiac
transplantation is limited by the short time of cold ischemic
storage that can be tolerated before coronary blood flow is
severely compromised (<4 hours). The expression of CD36 in
coronary endothelium and cardiac muscles is up-regulated in
isolated hearts subjected to prolonged cold ischemic storage and
warm reperfusion.
[0131] Isolated guinea pig hearts were perfused with St. Thomas
solution alone, or St. Thomas solution containing 1 nM SS-31 or 100
nM SS-20, for 3 min. and then stored in the same solution at
4.degree. C. for 18 hours. After ischemic storage, hearts were
reperfused with 34.degree. C. Kreb-Henseleit solution for 90 min.
Hearts freshly isolated from guinea pigs were used as controls.
[0132] The hearts were fixed in paraffin and sliced for
immunostaining with an anti-CD36 rabbit polyclonal antibody. The
results are shown in FIG. 12. Two sections are shown for each
treatment group. Antibody staining showed that CD36 is expressed in
endothelium and cardiac muscles in normal hearts. The "background"
(FIGS. 12A and 12B) represents two sections from a normal
non-ischemic heart that was not treated with the primary antibody.
"Normal heart" (FIGS. 12C and 12D) represents two sections obtained
from a non-ischemic heart. The sections from a representative heart
stored in St. Thomas solution (FIGS. 12E and 12F) for 18 hours at
4.degree. C. showed increased CD36 staining compared to "Normal
heart." CD36 staining was significantly reduced in hearts stored
with either 1 nM SS-31 (FIGS. 12G and 12H) or 100 nM SS-20 (FIGS.
121 and 12J) in St. Thomas solution for 18 h.
[0133] CD36 staining is increased in hearts that have undergone 18
hours of cold ischemic storage and warm reperfusion. However,
hearts that were stored with either 1 nM SS-31 or 100 nM SS-20 did
not show the upregulation of CD36 expression.
[0134] Lipid peroxidation in the hearts was also decreased by the
aromatic-cationic peptides. Guinea pig hearts were perfused with a
cardioplegic solution (St. Thomas solution) alone or St. Thomas
solution containing either 1 nM SS-31 or 100 nM SS-20 for 3 min.
and then subjected to 18 hours of cold ischemia (4.degree. C.). The
hearts were then reperfused with Krebs Henseleit buffer at
34.degree. C. for 90 min. Immunohistochemical analysis of
4-hydroxynonenol (HNE)-modified proteins in paraffin sections from
tissue slices were performed by incubation with an anti-FINE
antibody (Santa Cruz) and a fluorescent secondary antibody. FINE
staining was significantly increased in hearts subjected to 18
hours of cold storage in St. Thomas solution (FIG. 13B) compared to
non-ischemic hearts (FIG. 13A). FINE staining was reduced in hearts
stored in SS-31 (FIG. 13C) or SS-20 (FIG. 13D).
[0135] Further, the peptides dramatically reduced endothelial
apoptosis (FIG. 14). Guinea pig heats were perfused with a
cardioplegic solution (St. Thomas solution) alone or St. Thomas
solution containing either 1 nM SS-31 or 100 nM SS-20 for 3 min.
and then subjected to 18 hours of cold ischemia (4.degree. C.). The
hearts were then reperfused with Krebs Henseleit buffer at
34.degree. C. for 90 min. After deparaffinization, sections were
incubated with deoxynucleotidyl transferase (Tdt) with
digoxigenin-dNTP for 1 hour. The reaction was stopped with
terminating buffer. A fluorescent anti-digoxigenin antibody was
then applied. Hearts subjected to 18 hours of cold storage in St.
Thomas solution (FIGS. 14C and 14D) showed prominent endothelial
apoptosis whereas no endothelial apoptosis was observed in
non-ischemic normal hearts (FIGS. 14A and 14B). Apoptotic cells
were not observed in hearts stored in SS-31 (FIGS. 14E and 14F) or
SS-20 (FIGS. 14G and 14H).
[0136] A significant improvement of coronary blood flow after
prolonged cold ischemic storage and warm reperfusion occurred (FIG.
15). Guinea pigs hearts were perfused with a cardioplegic solution
(St. Thomas solution) alone or St. Thomas solution containing
either 1 nM SS-31 (FIG. 15A) or 100 nM SS-20) (FIG. 15B) for 3 min.
and then subjected to 18 hours of cold ischemia (4.degree. C.). The
hearts were then reperfused with Krebs Henseleit buffer at
34.degree. C. for 90 min. Coronary flow was significantly reduced
after prolonged ischemia compared to pre-ischemic control
(expressed as 100%). Preservation in either SS-31 or SS-20
significantly restored coronary flow to approximately 80% of
pre-ischemic flow.
Example 7
SS-31 Prevented Renal Damage in Diabetic Mice
[0137] CD36 expression is upregulated in a variety of tissues of
diabetic patients, including monocytes, heart, kidneys, and plasma.
High glucose is known to upregulate the expression of CD36 by
improving the translational efficiency of CD36 mRNA. Diabetic
nephropathy is a common complication of type 1 and type 2 diabetes,
and is associated with tubular epithelial degeneration and
interstitial fibrosis. CD36 has been identified as a mediator of
tubular epithelial apoptosis in diabetic nephropathy. High glucose
stimulates CD36 expression and apoptosis in proximal tubular
epithelial cells.
[0138] Streptozotocin (STZ) was used to induce diabetes in mice.
Three groups of CD-1 mice were studied. Group I--no STZ treatment;
Group II--STZ (50 mg/kg, ip) was given once a day for 5 d; Group
III--STZ (50 mg/kg, ip) was given once a day for 5 d, and SS-31 (3
mg/kg, ip) was given once a day for 16 d. STZ treatment resulted in
progressive increase in blood glucose. By week 3, blood glucose
values were: Group I (10.6.noteq.0.27 mmol/L); Group II
(24.5.noteq.1.15 mmol/L); Group III (21.3 1.48 mmol/L). Animals
were sacrificed after 3 weeks and kidney tissues preserved for
histopathology. Kidney sections were examined by Periodic Schiff
(PAS) staining for renal tubular brush border.
[0139] STZ treatment caused dramatic loss of brush border in
proximal tubules in the renal cortex (FIG. 16). In mice not treated
with STZ, the renal brush border in the cortex was stained red with
PAS (FIG. 16A, see white arrows). In mice treated with STZ, the
brush border was obliterated, and the tubular epithelial cells
showed small condensed nuclei (FIG. 16B). Daily treatment with
SS-31 (3 mg/kg, ip) presented the loss of brush border in the
STZ-treated mice (FIG. 16C), and the nuclei appeared normal (FIG.
16, top and bottom panels). In general, the architecture of the
proximal renal tubules was preserved in diabetic mice treated with
SS-31.
[0140] STZ treatment induced significant apoptosis in tubular
epithelial cells (FIG. 17). Kidney sections were examined for
apoptosis using the TUNEL assay. After deparaffinization, sections
were incubated with deoxynucleotidyl transferase (Tdt) with
digoxigenin-dNTP for 1 hour. The reaction was stopped with
terminating buffer. A fluorescent anti-digoxigenin antibody was
then applied. Kidney sections from mice treated with STZ showed
large number of apoptotic nuclei in the proximal tubules (PT) (FIG.
17A, panel b), compared to no apoptotic cells in mice not treated
STZ (FIG. 17A, panel a). Treatment with daily SS-31 dramatically
reduced apoptotic cells in the proximal tubule (FIG. 17A, panel c).
FIG. 17B shows the significant decrease in tubular cell apoptosis
provided by SS-31.
[0141] CD36 expression in proximal tubular epithelial cells is
known to be increased by high glucose and is upregulated in
diabetic models. SS-31. by reducing CD36 expression, was able to
inhibit tubular cell apoptosis and loss of brush border in mice
treated with STZ without affecting blood glucose.
* * * * *