U.S. patent application number 15/667087 was filed with the patent office on 2018-06-28 for methods for prevention and treatment of acute renal injury.
The applicant listed for this patent is Cornell University. Invention is credited to Diane Felsen, Hazel H. Szeto.
Application Number | 20180177840 15/667087 |
Document ID | / |
Family ID | 41013650 |
Filed Date | 2018-06-28 |
United States Patent
Application |
20180177840 |
Kind Code |
A1 |
Szeto; Hazel H. ; et
al. |
June 28, 2018 |
METHODS FOR PREVENTION AND TREATMENT OF ACUTE RENAL INJURY
Abstract
The disclosure relates to a method for protecting a kidney from
renal injury. For example, acute renal injury may be associated
with decreased or blocked blood flow in the subject's kidney or
exposure to a nephrotoxic agent, such as a radiocontrast dye. The
methods include administering to the subject an effective amount of
an aromatic-cationic peptide to a subject in need thereof.
Inventors: |
Szeto; Hazel H.; (New York,
NY) ; Felsen; Diane; (Brooklyn, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cornell University |
Ithaca |
NY |
US |
|
|
Family ID: |
41013650 |
Appl. No.: |
15/667087 |
Filed: |
August 2, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15172397 |
Jun 3, 2016 |
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15667087 |
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14598036 |
Jan 15, 2015 |
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15172397 |
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14063792 |
Oct 25, 2013 |
8940696 |
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14598036 |
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13402620 |
Feb 22, 2012 |
8592373 |
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14063792 |
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12392565 |
Feb 25, 2009 |
8143219 |
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13402620 |
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61031585 |
Feb 26, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 39/02 20180101; C07K 5/1016 20130101; A61P 13/02 20180101;
A61P 13/12 20180101; A61K 38/06 20130101; C07K 5/1019 20130101;
A61P 29/00 20180101; A61P 1/16 20180101; A61K 38/07 20130101 |
International
Class: |
A61K 38/07 20060101
A61K038/07 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The invention was made with United States government support
awarded by the following agencies: NIH DK58355; NIH DA08924; and
NIH DK73595. The United States has certain rights in this
invention.
Claims
1.-19. (canceled)
20. A method for protecting a kidney from renal fibrosis in a
subject in need thereof, the method comprising administering to the
subject an effective amount of a peptide selected from the group
consisting of Phe-D-Arg-Phe-Lys-NH.sub.2 and
D-Arg-2'6'Dmt-Lys-Phe-NH.sub.2.
21. The method of claim 20, wherein the peptide is
Phe-D-Arg-Phe-Lys-NH.sub.2.
22. The method of claim 20, wherein the peptide is
D-Arg-2'6'Dmt-Lys-Phe-NH.sub.2.
23. The method of claim 20, wherein the renal fibrosis is
associated with ureteral obstruction.
24. The method of claim 20, wherein the renal fibrosis comprises
increased renal interstitial volume compared to a healthy control
subject.
25. The method of claim 20, wherein the peptide is administered
orally, topically, intranasally, intramuscularly,
intraperitoneally, subcutaneously, or transdermally.
26. The method of claim 20, wherein the subject is a human.
27. A method for treating or preventing renal fibrosis in a subject
in need thereof, the method comprising administering to the subject
an effective amount of a peptide selected from the group consisting
of Phe-D-Arg-Phe-Lys-NH.sub.2 and
D-Arg-2'6'Dmt-Lys-Phe-NH.sub.2.
28. The method of claim 27, wherein the peptide is
Phe-D-Arg-Phe-Lys-NH.sub.2.
29. The method of claim 27, wherein the peptide is
D-Arg-2'6'Dmt-Lys-Phe-NH.sub.2.
30. The method of claim 27, wherein the renal fibrosis is
associated with ureteral obstruction.
31. The method of claim 27, wherein the renal fibrosis comprises
increased renal interstitial volume compared to a healthy control
subject.
32. The method of claim 27, wherein the peptide is administered
orally, topically, intranasally, intramuscularly,
intraperitoneally, subcutaneously, or transdermally.
33. The method of claim 27, wherein the subject is a human.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/172,397, filed Jun. 3, 2016, which is a continuation of U.S.
patent application Ser. No. 14/598,036, filed Jan. 15, 2015 which
is a continuation of U.S. patent application Ser. No. 14/063,792,
filed Oct. 25, 2013, to be issued as U.S. Pat. No. 8,940,696 on
Jan. 27, 2015, which is a continuation of Ser. No. 13/402,620,
filed Feb. 22, 2012, now U.S. Pat. No. 8,592,373, issued Nov. 26,
2013, which is a continuation of U.S. patent application Ser. No.
12/392,565, filed Feb. 25, 2009, now U.S. Pat. No. 8,143,219,
issued Mar. 27, 2012, which claims priority to U.S. Provisional
Application No. 61/031,585, filed Feb. 26, 2008. The entire
contents of the foregoing applications are hereby incorporated
herein by reference.
BACKGROUND
[0003] Acute Renal Injury (ARI), also known as acute renal failure
(ARF) or acute kidney injury (AKI), is a serious medical condition
of the kidneys. ARI is characterized by a decline of glomerular
filtration rate, urine output, or both. ARI is accompanied by an
inflammatory response that if unchecked can lead to renal fibrosis
and chronic renal failure. ARI usually occurs over a period of
hours or days and is potentially reversible. ARI is a common
complication in hospitalized patients, and its incidence has risen
significantly in the past 15 years. A particular type of ARI,
contrast-induced nephropathy, is caused by the toxic effects of
some radiocontrast agents.
SUMMARY
[0004] The present invention provides a method for protecting a
kidney from acute renal injury in a subject in need thereof. The
method includes administering to the subject 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;
and (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.
[0005] In one embodiment, peptide is administered to a subject
prior to or simultaneously with a radiocontrast agent, in order to
prevent or ameliorate the incidence of contrast-induced
nephropathy. In one embodiment, the peptide is selected from the
group consisting of Phe-D-Arg-Phe-Lys-NH.sub.2 (SS-20) or
D-Arg-2'6'Dmt-Lys-Phe-NH2 (SS-31).
[0006] In one embodiment, the acute renal injury is associated with
exposure of the subject to a nephrotoxic agent. In one embodiment,
the peptide is administered to the subject prior to or
simultaneously with the exposure to the nephrotoxic agent.
[0007] In one embodiment, the nephrotoxic agent is a radiocontrast
dye, e.g., acetrizoate; diatrizoate; iodamide; ioglicate;
iothalamate; ioxithalamate; metrizoate; metrizamide; iohexol;
iopamidol; iopentol; iopromide; and ioversol.
[0008] In one embodiment, the nephrotoxic agent is a drug or
chemical. In illustrative embodiments, the drug or chemical is one
or more of the compounds selected from the group consisting of:
cisplatin; gentamicin; cephaloridine; cyclosporine; amphotericin;
carbon tetrachloride; trichloroethylene; and dichloroacetylene.
[0009] In another aspect, the invention provides a method of
treating acute or chronic renal injury in a subject in need
thereof. The method includes administering to the subject an
effective amount of an aromatic-cationic peptide. The
aromatic-cationic peptides 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; and (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. In one embodiment, the peptide is administered after
exposure of the subject to a nephrotoxic agent.
[0010] In one embodiment, the peptide is selected from the group
consisting of Phe-D-Arg-Phe-Lys-NH.sub.2 (SS-20) and
D-Arg-2'6'Dmt-Lys-Phe-NH.sub.2 (SS-31) peptide.
[0011] In one embodiment, the peptide is defined by formula I:
##STR00001##
wherein R.sup.1 and R.sup.2 are each independently selected from
[0012] (i) hydrogen; [0013] (ii) linear or branched C.sub.1-C.sub.6
alkyl;
##STR00002##
[0013] R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.10, R.sup.11 and R.sup.12 are each independently
selected from
[0014] (i) hydrogen;
[0015] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
[0016] (iii) C.sub.1-C.sub.6 alkoxy;
[0017] (iv) amino;
[0018] (v) C.sub.1-C.sub.4 alkylamino;
[0019] (vi) C.sub.1-C.sub.4 dialkylamino;
[0020] (vii) nitro;
[0021] (viii) hydroxyl;
[0022] (ix) halogen, where "halogen" encompasses chloro, fluoro,
bromo, and iodo; and n is an integer from 1 to 5.
[0023] In a particular embodiment, R.sup.1, R.sup.2, R.sup.3,
R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10,
R.sup.11, and R.sup.12 are all hydrogen; and n is 4. In another
embodiment, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8, R.sup.9, and R.sup.11 are all hydrogen; R.sup.8
and R.sup.12 are methyl; R.sup.10 is hydroxyl; and n is 4.
[0024] In one embodiment, the peptide is defined by formula II:
##STR00003##
wherein R.sup.1 and R.sup.2 are each independently selected
from
[0025] (i) hydrogen;
[0026] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
##STR00004##
R.sup.3 and R.sup.4 are each independently selected from
[0027] (i) hydrogen;
[0028] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
[0029] (iii) C.sub.1-C.sub.6 alkoxy;
[0030] (iv) amino;
[0031] (v) C.sub.1-C.sub.4 alkylamino;
[0032] (vi) C.sub.1-C.sub.4 dialkylamino;
[0033] (vii) nitro;
[0034] (viii) hydroxyl;
[0035] (ix) halogen, where "halogen" encompasses chloro, fluoro,
bromo, and iodo; R.sup.5, R.sup.6, R.sup.7, R.sup.8, and R.sup.9
are each independently selected from
[0036] (i) hydrogen;
[0037] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
[0038] (iii) C.sub.1-C.sub.6 alkoxy;
[0039] (iv) amino;
[0040] (v) C.sub.1-C.sub.4 alkylamino;
[0041] (vi) C.sub.1-C.sub.4 dialkylamino;
[0042] (vii) nitro;
[0043] (viii) hydroxyl;
[0044] (ix) halogen, where "halogen" encompasses chloro, fluoro,
bromo, and iodo; and n is an integer from 1 to 5.
[0045] In a particular embodiment, R.sup.1 and R.sup.2 are
hydrogen; R.sup.3 and R.sup.4 are methyl; R.sup.5, R.sup.6,
R.sup.7, R.sup.8, and R.sup.9 are all hydrogen; and n is 4.
[0046] In one aspect, the disclosure provides a method for
protecting a kidney from chronic renal injury in a subject in need
thereof, the method comprising administering to the subject an
effective amount of a peptide selected from the group consisting of
Phe-D-Arg-Phe-Lys-NH2 (SS-20) and D-Arg-2'6'Dmt-Lys-Phe-NH2
(SS-31). In one embodiment, the chronic renal injury is associated
with administration of or exposure to a nephrotoxic agent, e.g., a
drug or chemical capable of causing chronic kidney injury. In a
particular embodiment, the drug or chemical is carbon
tetrachloride.
[0047] In some embodiments, the peptides are useful for the
prevention and/or treatment of acute hepatic injury caused by
ischemia, drugs (acetaminophen, alcohol), viruses, obesity
(non-alcoholic steatohepatitis), and obstruction (bile duct
obstruction, tumors). In some embodiments, the aromatic-cationic
peptides of the invention are administered to a subject to prevent
or treat acute liver failure (ALF).
BRIEF DESCRIPTION OF THE FIGURES
[0048] FIGS. 1A-1D shows micrographs of renal sections stained for
apoptotic cells using TUNEL.
[0049] FIGS. 2A-2H shows micrographs of renal sections stained with
H&E and PAS.
[0050] FIG. 3 is a chart showing that treatment with SS-20 or SS-31
significantly improved histopathological score resulting from 45
min ischemia and 24 h reperfusion.
[0051] FIGS. 4A and 4B show that treatment with SS-20 or SS-31
significantly increased rate of ATP production after reperfusion.
FIG. 4A shows the ATP content in renal tissue at the end of 15 min
ischemia. FIG. 4B shows the ATP content in renal tissue after 1 h
reperfusion following 45 min ischemia.
[0052] FIG. 5 is a chart showing pretreatment with SS-20 or SS-31
significantly improved renal mitochondrial respiration after 45 min
ischemia.
[0053] FIGS. 6A-6D show that SS-1 decreases medullary fibrosis in a
14-day unilateral ureteral obstruction (UUO) model. FIGS. 6A-6C
show inflammation or fibrosis in tubules, glomeruli, or
interstitium of contralateral unobstructed kidney (CK), obstructed
kidney (OK), or SS-31 treated kidneys, respectively. FIG. 6D is a
chart showing the interstitial in the CK, OK, and SS-31 treated
kidney.
[0054] FIG. 7 is a chart showing that SS-31 decreases fibroblast
expression in a 14-day UUO model.
[0055] FIG. 8 is a chart showing that SS-31 decreases tubular
apoptosis in a 14-day UUO model.
[0056] FIG. 9 is a chart showing that SS-31 decreases macrophage
expression in a 14-day UUO model.
[0057] FIG. 10 is a chart showing that SS-31 increases tubular
proliferation in a 14-day UUO model.
[0058] FIGS. 11A-11C show that SS-31 decreases oxidative damage in
a 14-day UUO model. FIG. 11A is a photograph of the results of
RT-PCR to analyze for HO-1 expression.
[0059] FIG. 11B and FIG. 11C are micrographs of the results of 8-OH
dG staining in both tubular and interstitial compartments of the
obstructed kidney.
[0060] FIG. 12 is a chart showing SS-31 reduced renal dysfunction
caused by radiocontrast dye.
[0061] FIGS. 13A-13C are a series of micrographs showing that SS-31
protected renal tubules from radiocontrast dye injury. PAS staining
revealed control rat kidneys with normal morphology, demonstrated
by intact brush borders on proximal tubules. Dye treatment resulted
in loss of the characteristic renal brush border in proximal tubule
cells, flattening of the epithelial cells, as well as some
vacuolization. These effects were attenuated with SS-31 treatment.
PAS staining in these samples revealed intact brush borders and
normal glomeruli.
[0062] FIGS. 14A-14C are a series of micrographs showing that SS-31
prevented renal tubular apoptosis induced by radiocontrast dye
injury. The TUNEL stain was used to visualize apoptotic renal
tubules. Control kidneys showed few apoptotic cells/hpf.
Vehicle-treated, dye-injected kidneys had numerous apoptotic cells
per high power field (hpf). This effect was greatly attenuated in
SS-31-treated, dye injected kidneys.
[0063] FIG. 15 is a flow chart showing the protocol and dosing
schedule for the study presented in Example 3, Experiment 2.
[0064] FIGS. 16A-16C are a series of PAS-stained micrographs
showing that dye treatment resulted in a loss of the characteristic
renal brush border in proximal tubule cells, as well as some
vacuolization (FIG. 16A). These effects were attenuated with SS-31
treatment, PAS staining in these samples revealed intact brush
borders and normal glomeruli (FIG. 16B).
[0065] FIG. 16C shows the control rat kidneys with normal
morphology, demonstrated by intact brush borders on proximal
tubules.
[0066] FIG. 17A is a chart showing the change in serum Cystatiin C
in control and SS-31 treated rats after radiocontrast dye
administration. FIG. 17B is a chart showing the change in GFR in
control and SS-31 treated rats following radiocontrast dye
administration.
[0067] FIG. 18 is a chart showing the creatinine clearance in
control and SS-31 treated rats before and after radiocontrast dye
administration.
[0068] FIG. 19 is a flow chart showing the protocol and dosing
schedule for the study presented in Example 4, Experiment 3.
[0069] FIG. 20 is a flow chart showing the protocol and dosing
schedule for the study presented in Example 5, Experiment 1.
[0070] FIG. 21A is a chart showing the GFR in control and SS-31
treated rats in a glycerin model of CIN before and after
radiocontrast dye administration. FIG. 21B is a chart showing the
urinary albumin in control and SS-31 treated rats in a glycerin
model of CIN before and after radiocontrast dye administration.
[0071] FIG. 22A shows a series of micrographs of PAS staining of
renal sections from rats treated with glycerin, Diatrizoate, and a
control (PBS). FIG. 22B shows a series of micrographs of PAS
staining of renal sections from rats treated with glycerin,
Diatrizoate, and SS-31.
[0072] FIG. 23 is a flow chart showing the protocol and dosing
schedule for the study presented in Example 5, Experiment 2.
[0073] FIG. 24 is a chart showing the GFR in control and SS-31
treated rats in a glycerin model of CIN before and after
radiocontrast dye administration.
[0074] FIG. 25 is a chart showing the urine protein in control and
SS-31 treated rats in a glycerin model of CIN before and after
radiocontrast dye administration.
[0075] FIG. 26A shows a series of micrographs of PAS staining of
renal sections from rats treated with glycerin, Diatrizoate, and a
control (PBS). FIG. 26B shows a series of micrographs of PAS
staining of renal sections from rats treated with glycerin,
Diatrizoate, and SS-31.
[0076] FIG. 27 is a flow chart showing the protocol and dosing
schedule for the study presented in Example 5, Experiment 3.
[0077] FIG. 28 is a chart showing the GFR in control and SS-31
treated rats in a glycerin model of CIN before and after
radiocontrast dye administration.
[0078] FIG. 29 is a chart showing the urine protein in control and
SS-31 treated rats in a glycerin model of CIN before and after
radiocontrast dye administration.
[0079] FIG. 30A shows a series of micrographs of PAS staining of
renal sections from rats treated with glycerin, Diatrizoate, and a
control (PBS). FIG. 30B shows a series of micrographs of PAS
staining of renal sections from rats treated with glycerin,
Diatrizoate, and SS-31.
[0080] FIGS. 31A-31C are a series of micrographs showing H&E
staining of a CCl.sub.4 control group (FIG. 31A), an SS-31-treated
group (FIG. 31B), and a naive control group (FIG. 31C).
[0081] FIGS. 32A-32E are a series of charts showing the effects of
the aromatic-cationic peptides of the invention on total body
weight (FIG. 32A), kidney weight (FIG. 32B), serum creatinine (FIG.
32C), BUN (FIG. 32D), and creatinine clearance (FIG. 32E) in rats
administered the nephrotoxic agent cisplatin.
DETAILED DESCRIPTION
[0082] The invention is based on the surprising discovery by the
inventors that certain aromatic-cationic peptides can prevent
and/or treat acute organ failure, e.g., acute renal injury or acute
liver failure in a subject. In some aspects, aromatic-cationic
peptides can prevent and/or treat chronic organ damage, e.g.,
chronic renal injury. It is to be appreciated that certain aspects,
modes, embodiments, variations and features of the invention are
described below in various levels of detail in order to provide a
substantial understanding of the present invention.
[0083] In practicing the present invention, many conventional
techniques in molecular biology, protein biochemistry, cell
biology, immunology, microbiology and recombinant DNA are used.
These techniques are well-known and are explained in, e.g., Current
Protocols in Molecular Biology, Vols. I-III, Ausubel, Ed. (1997);
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Ed.
(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989); DNA Cloning: A Practical Approach, Vols. I and II, Glover,
Ed. (1985); Oligonucleotide Synthesis, Gait, Ed. (1984); Nucleic
Acid Hybridization, Hames & Higgins, Eds. (1985); Transcription
and Translation, Hames & Higgins, Eds. (1984); Animal Cell
Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes (IRL
Press, 1986); Perbal, A Practical Guide to Molecular Cloning, the
series, Meth. Enzymol., (Academic Press, Inc., 1984); Gene Transfer
Vectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring
Harbor Laboratory, NY, 1987); and Meth. Enzymol., Vols. 154 and
155, Wu & Grossman, and Wu, Eds., respectively. All references
cited herein are incorporated herein by reference in their
entireties and for all purposes to the same extent as if each
individual publication, patent, or patent application was
specifically and individually incorporated by reference in its
entirety for all purposes.
[0084] The definitions of certain terms as used in this
specification are provided below. Unless defined otherwise, all
technical and scientific terms used herein generally have the same
meaning as commonly understood by one of ordinary skill in the art
to which this invention belongs.
[0085] As used in this specification and the appended claims, the
singular forms "a", "an" and "the" include plural referents unless
the content clearly dictates otherwise. For example, reference to
"a cell" includes a combination of two or more cells, and the
like.
[0086] As used herein, phrases such as element A is "associated
with" element B mean both elements exist, but should not be
interpreted as meaning one element necessarily is causally linked
to the other.
[0087] As used herein, the "administration" of an agent, drug, or
peptide to a subject includes any route of introducing or
delivering to a subject a compound to perform its intended
function. Administration can be carried out by any suitable route,
including orally, intranasally, parenterally (intravenously,
intramuscularly, intraperitoneally, or subcutaneously), rectally,
or topically. Administration includes self-administration and the
administration by another.
[0088] As used herein, the term "amino acid" includes
naturally-occurring amino acids and synthetic amino acids, as well
as amino acid analogs and amino acid mimetics that function in a
manner similar to the naturally-occurring amino acids.
Naturally-occurring amino acids are those encoded by the genetic
code, as well as those amino acids that are later modified, e.g.,
hydroxyproline, .gamma.-carboxyglutamate, and O-phosphoserine.
Amino acid analogs refers to compounds that have the same basic
chemical structure as a naturally-occurring amino acid, i.e., an
.alpha.-carbon that is bound to a hydrogen, a carboxyl group, an
amino group, and an R group, e.g., homoserine, norleucine,
methionine sulfoxide, methionine methyl sulfonium. Such analogs
have modified R groups (e.g., norleucine) or modified peptide
backbones, but retain the same basic chemical structure as a
naturally-occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally-occurring amino acid. Amino acids
can be referred to herein by either their commonly known three
letter symbols or by the one-letter symbols recommended by the
IUPAC-IUB Biochemical Nomenclature Commission.
[0089] An "isolated" or "purified" polypeptide or peptide is
substantially free of cellular material or other contaminating
polypeptides from the cell or tissue source from which the agent is
derived, or substantially free from chemical precursors or other
chemicals when chemically synthesized. For example, an isolated
aromatic-cationic peptide would be free of materials that would
interfere with diagnostic or therapeutic uses of the agent. Such
interfering materials may include enzymes, hormones and other
proteinaceous and nonproteinaceous solutes.
[0090] As used herein, the term "medical condition" includes, but
is not limited to, any condition or disease manifested as one or
more physical and/or psychological symptoms for which treatment
and/or prevention is desirable, and includes previously and newly
identified diseases and other disorders. For example, a medical
condition may be acute renal injury or acute liver failure or any
associated symptoms or complications. In one embodiment, the
medical condition is acute renal injury associated with
radiocontrast dye administration.
[0091] As used herein, the terms "polypeptide", "peptide" and
"protein" are used interchangeably herein to mean a polymer
comprising two or more amino acids joined to each other by peptide
bonds or modified peptide bonds, i.e., peptide isosteres.
Polypeptide refers to both short chains, commonly referred to as
peptides, glycopeptides or oligomers, and to longer chains,
generally referred to as proteins. Polypeptides may contain amino
acids other than the 20 gene-encoded amino acids. Polypeptides
include amino acid sequences modified either by natural processes,
such as post-translational processing, or by chemical modification
techniques that are well known in the art. Such modifications are
well described in basic texts and in more detailed monographs, as
well as in a voluminous research literature.
[0092] As used herein, the word "protect" or "protecting" refers to
decreasing the likelihood and/or risk that the subject treated with
a peptide of the invention will develop a given disease or
disorder, e.g., acute renal injury or acute liver failure.
Typically, the likelihood of developing the disease or disorder is
considered to be reduced if the likelihood 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%, in comparison to the likelihood
and/or risk that the same subject untreated with a peptide of the
invention will develop tissue injury, e.g., acute renal injury or
acute liver failure. In particular embodiments, the peptides
protect a subject from acute renal injury caused by a contrast
agent (i.e., contrast-induced nephropathy) when the peptides are
administered prior to or simultaneously with the contrast
agent.
[0093] The term "subject" as used herein refers to a member of any
vertebrate species. The methods of the presently disclosed subject
matter are particularly useful for warm-blooded vertebrates.
Provided herein is the treatment of mammals such as humans, as well
as those mammals of importance due to being endangered, of economic
importance (animals raised on farms for consumption by humans)
and/or social importance (animals kept as pets or in zoos) to
humans. In particular embodiments, the subject is a human.
[0094] As used herein, the terms "treating" or "treatment" or
"alleviation" refers to both therapeutic treatment and prophylactic
or preventative measures, wherein the object is to prevent or slow
down (lessen) the targeted pathologic condition or disorder. A
subject is successfully "treated" for a disease or condition if,
after receiving a therapeutic amount of the aromatic-cationic
peptides according to the methods of the present invention, the
subject shows observable and/or measurable reduction in or absence
of one or more signs and symptoms of a particular disease or
condition. For example, for acute renal injury, treatment or
prevention may include a reduction in metabolic acidosis,
hyperkalaemia, oliguria or anuria, restoration in body fluid
balance, and improved effects on other organ systems. Kidney
function may also be assessed by measuring serum creatinine levels,
serum creatinine clearance, or blood urea nitrogen levels. It is
also to be appreciated that the various modes of treatment or
prevention of medical conditions as described are intended to mean
"substantial", which includes total but also less than total
treatment or prevention, and wherein some biologically or medically
relevant result is achieved.
Peptides
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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 c position relative
to the carboxyl group.
[0099] The amino acids may be naturally occurring. Naturally
occurring amino acids include, for example, the twenty most common
levorotatory (L) amino acids normally found in mammalian proteins,
i.e., alanine (Ala), arginine (Arg), asparagine (Asn), aspartic
acid (Asp), cysteine (Cys), glutamine (Glu), glutamic acid (Glu),
glycine (Gly), histidine (His), isoleucine (Ileu), leucine (Leu),
lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro),
serine (Ser), threonine (Thr), tryptophan, (Trp), tyrosine (Tyr),
and valine (Val).
[0100] 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.
[0101] 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. The peptide may have no amino acids that are naturally
occurring.
[0102] 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.
[0103] 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.
[0104] The non-natural amino acids may, for example, comprise
alkyl, aryl, or alkylaryl groups. Some examples of alkyl amino
acids include .alpha.-aminobutyric acid, .beta.-aminobutyric acid,
.gamma.-aminobutyric acid, .delta.-aminovaleric acid, and
.epsilon.-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-aminophenylacetic acid, and .gamma.-phenyl-.beta.-aminobutyric
acid.
[0105] 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.
[0106] For example, one or more chemical groups can be added to one
or more of the 2', 3', 4', 5', or 6' position of the aromatic ring
of a phenylalanine or tyrosine residue, or the 4', 5', 6', or 7'
position of the benzo ring of a tryptophan residue. The group can
be any chemical group that can be added to an aromatic ring. Some
examples of such groups include branched or unbranched
C.sub.1-C.sub.4 alkyl, such as methyl, ethyl, n-propyl, isopropyl,
butyl, isobutyl, or t-butyl, C.sub.1-C.sub.4 alkyloxy (i.e.,
alkoxy), amino, C.sub.1-C.sub.4 alkylamino and C.sub.1-C.sub.4
dialkylamino (e.g., methylamino, dimethylamino), nitro, hydroxyl,
halo (i.e., fluoro, chloro, bromo, or iodo). Some specific examples
of non-naturally occurring derivatives of naturally occurring amino
acids include norvaline (Nva), norleucine (Nle), and hydroxyproline
(Hyp).
[0107] 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.
[0108] 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.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] In suitable embodiments, 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).
[0113] 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.
[0114] "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.
[0115] 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.
[0116] 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 TABLE 1 Amino acid number and net positive charges
(3p.sub.m .ltoreq. p + 1) (r) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
18 19 20 (p.sub.m) 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7
[0117] 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 TABLE 2 Amino acid number and net positive charges
(2p.sub.m .ltoreq. p + 1) (r) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
18 19 20 (p.sub.m) 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10
[0118] 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.
[0119] In suitable embodiments, 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).
[0120] Naturally occurring amino acids that have an aromatic group
include the amino acids histidine, tryptophan, tyrosine, and
phenylalanine. For example, the hexapeptide
Lys-Gln-Tyr-D-Arg-Phe-Trp has a net positive charge of two
(contributed by the lysine and arginine residues) and three
aromatic groups (contributed by tyrosine, phenylalanine and
tryptophan residues).
[0121] 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 TABLE 3 Aromatic groups and net positive charges (3a
.ltoreq. p.sub.t + 1 or a = p.sub.t = 1) (p.sub.t) 1 2 3 4 5 6 7 8
9 10 11 12 13 14 15 16 17 18 19 20 (a) 1 1 1 1 2 2 2 3 3 3 4 4 4 5
5 5 6 6 6 7
[0122] 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 TABLE 4 Aromatic groups and net positive charges (3a
.ltoreq. p.sub.t + 1 or a = p.sub.t = 1) (p.sub.t) 1 2 3 4 5 6 7 8
9 10 11 12 13 14 15 16 17 18 19 20 (a) 1 1 2 2 3 3 4 4 5 5 6 6 7 7
8 8 9 9 10 10
[0123] In another embodiment, the number of aromatic groups (a) and
the total number of net positive charges (p.sub.t) are equal.
[0124] Carboxyl groups, especially the terminal carboxyl group of a
C-terminal amino acid, may be 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.
[0125] 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.
[0126] 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.
[0127] 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,
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-Lys-Phe-Arg,
D-His-Glu-Lys-Tyr-D-Phe-Arg,
Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH.sub.2,
Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His,
Gly-D-Phe-Lys-Tyr-His-D-Arg-Tyr-NH.sub.2,
Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH.sub.2,
Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-Lys,
Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH.sub.2,
Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-Lys,
Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH.sub.2,
D-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-His-D-Lys-Arg-Trp-NH.sub.2,
Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-Phe,
Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-Phe,
Phe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH.sub.2,
Phe-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.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.
[0128] In some embodiments, peptides useful in the methods of the
present invention are those peptides which have a tyrosine residue
or a tyrosine derivative. 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).
[0129] In one embodiment, the peptide has the formula
Tyr-D-Arg-Phe-Lys-NH.sub.2 (referred to herein as SS-01). SS-01 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 SS-01 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 (referred to herein as
SS-02).
[0130] In a suitable 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 herein as SS-31).
[0131] In another 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). An example of
such a peptide is Phe-D-Arg-Phe-Lys-NH.sub.2 (referred to herein as
SS-20). In one embodiment, the amino acid sequence of SS-02 is
rearranged such that Dmt is not at the N-terminus. An example of
such an aromatic-cationic peptide has the formula
D-Arg-2'6'Dmt-Lys-Phe-NH.sub.2 (SS-31).
[0132] In yet another embodiment, the aromatic-cationic peptide has
the formula Phe-D-Arg-Dmt-Lys-NH.sub.2 (referred to herein as
SS-30). Alternatively, the N-terminal phenylalanine can be a
derivative of phenylalanine such as 2',6'-dimethylphenylalanine
(2'6'Dmp). SS-01 containing 2',6'-dimethylphenylalanine at amino
acid position one has the formula
2',6'-Dmp-D-Arg-Dmt-Lys-NH.sub.2.
[0133] SS-20, SS-31, and their derivatives can further include
functional analogs. A peptide is considered a functional analog of
SS-20 or SS-31 if the analog has the same function as SS-20 or
SS-31. The analog may, for example, be a substitution variant of
SS-01 or SS-31, wherein one or more amino acid is substituted by
another amino acid.
[0134] Suitable substitution variants of SS-20 or SS-31 include
conservative amino acid substitutions. Amino acids may be grouped
according to their physicochemical characteristics as follows:
[0135] (a) Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P)
Gly(G) Cys (C);
[0136] (b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q);
[0137] (c) Basic amino acids: His(H) Arg(R) Lys(K);
[0138] (d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V);
and
[0139] (e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His (H).
[0140] Substitutions of an amino acid in a peptide by another amino
acid in the same group is referred to as a conservative
substitution and may preserve the physicochemical characteristics
of the original peptide. In contrast, substitutions of an amino
acid in a peptide by another amino acid in a different group is
generally more likely to alter the characteristics of the original
peptide. Examples of analogs useful in the practice of the present
invention include, but are not limited to, the aromatic-cationic
peptides shown in Table 5.
TABLE-US-00005 TABLE 5 Examples of Peptide Analogs Amino Amino
Amino Amino Amino Amino Amino Acid Acid Acid Acid Acid Acid Acid
C-Terminal Position 1 Position 2 Position 3 Position 4 Position 5
Position 6 Position 7 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
D-Arg Dmt Lys Phe Cys NH.sub.2 D-Arg Dmt Lys Phe Glu Cys Gly
NH.sub.2 D-Arg Dmt Lys Phe Ser Cys NH.sub.2 D-Arg Dmt Lys Phe Gly
Cys NH.sub.2 Phe Lys Dmt D-Arg NH.sub.2 Phe Lys D-Arg Dmt NH.sub.2
Phe D-Arg Phe Lys NH.sub.2 Phe D-Arg Phe Lys Cys NH.sub.2 Phe D-Arg
Phe Lys Glu Cys Gly NH.sub.2 Phe D-Arg Phe Lys Sar Cys NH.sub.2 Phe
D-Arg Phe Lys Gly Cys NH.sub.2 Phe D-Arg Dmt Lys NH.sub.2 Phe D-Arg
Dmt Lys Cys NH.sub.2 Phe D-Arg Dmt Lys Glu Cys Gly NH.sub.2 Phe
D-Arg Dmt Lys Ser Cys NH.sub.2 Phe D-Arg Dmt Lys Gly Cys NH.sub.2
Phe D-Arg Lys Dmt NH.sub.2 Phe Dmt D-Arg Lys NH.sub.2 Phe Dmt Lys
D-Arg NH.sub.2 Lys Phe D-Arg Dmt NH.sub.2 Lys Phe Dmt D-Arg
NH.sub.2 Lys Dmt D-Arg Phe NH.sub.2 Lys Dmt Phe D-Arg NH.sub.2 Lys
D-Arg Phe Dmt NH.sub.2 Lys D-Arg Dmt Phe NH.sub.2 D-Arg Dmt D-Arg
Phe NH.sub.2 D-Arg Dmt D-Arg Dmt NH.sub.2 D-Arg Dmt D-Arg Tyr
NH.sub.2 D-Arg Dmt D-Arg Trp NH.sub.2 Trp D-Arg Phe Lys NH.sub.2
Trp D-Arg Tyr Lys NH.sub.2 Trp D-Arg Trp Lys NH.sub.2 Trp D-Arg Dmt
Lys NH.sub.2 D-Arg Trp Lys Phe NH.sub.2 D-Arg Trp Phe Lys NH.sub.2
D-Arg Trp Lys Dmt NH.sub.2 D-Arg Trp Dmt Lys NH.sub.2 D-Arg Lys Trp
Phe NH.sub.2 D-Arg Lys Trp Dmt NH.sub.2 Cha D-Arg Phe Lys NH.sub.2
Ala D-Arg Phe Lys NH.sub.2 Cha = cyclohexyl
[0141] Under certain circumstances, it may be advantageous to use a
peptide that also has opioid receptor agonist activity. Examples of
analogs useful in the practice of the present invention include,
but are not limited to, the aromatic-cationic peptides shown in
Table 6.
TABLE-US-00006 TABLE 6 Peptide Analogs with Opioid Receptor Agonist
Activity 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
Tyr D-Arg Phe Lys Cys 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 Lys Cys
NH.sub.2 2',6'Dmt D-Cit Phe Ahp NH.sub.2 2',6'Dmt D-Arg Phe Orn
NH.sub.2 2',6'Dmt D-Arg Phe Dab NH.sub.2 2',6'Dmt D-Arg Phe Dap
NH.sub.2 2',6'Dmt D-Arg Phe Ahp(2- NH.sub.2 aminoheptanoic acid)
Bio-2',6'Dmt D-Arg Phe Lys NH.sub.2 3',5'Dmt D-Arg Phe Lys NH.sub.2
3',5'Dmt D-Arg Phe Orn NH.sub.2 3',5'Dmt D-Arg Phe Dab NH.sub.2
3',5'Dmt D-Arg Phe Dap NH.sub.2 Tyr D-Arg Tyr Lys NH.sub.2 Tyr
D-Arg Tyr Orn NH.sub.2 Tyr D-Arg Tyr Dab NH.sub.2 Tyr D-Arg Tyr Dap
NH.sub.2 2',6'Dmt D-Arg Tyr Lys NH.sub.2 2',6'Dmt D-Arg Tyr Orn
NH.sub.2 2',6'Dmt D-Arg Tyr Dab NH.sub.2 2',6'Dmt D-Arg Tyr Dap
NH.sub.2 2',6'Dmt D-Arg 2',6'Dmt Lys NH.sub.2 2',6'Dmt D-Arg
2',6'Dmt Orn NH.sub.2 2',6'Dmt D-Arg 2',6'Dmt Dab NH.sub.2 2',6'Dmt
D-Arg 2',6'Dmt Dap NH.sub.2 3',5'Dmt D-Arg 3',5'Dmt Arg NH.sub.2
3',5'Dmt D-Arg 3',5'Dmt Lys NH.sub.2 3',5'Dmt D-Arg 3',5'Dmt Orn
NH.sub.2 3',5'Dmt D-Arg 3',5'Dmt Dab NH.sub.2 2',6'Dmt D-Arg
2',6'Dmt Lys Cys NH.sub.2 Tyr D-Lys Phe Dap NH.sub.2 Tyr D-Lys Phe
Arg NH.sub.2 Tyr D-Lys Phe Arg Cys 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 3',5'Dmt D-Lys Phe Arg Cys 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
[0142] The amino acids of the peptides shown in Tables 5 and 6 may
be in either the L- or the D-configuration.
Synthesis of the Peptides
[0143] 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).
Methods of Protecting and Treating Renal Injury in a Subject
[0144] General.
[0145] The aromatic-cationic peptides described herein are useful
in protecting a subject's kidney from renal injury. Acute renal
injury (ARI) refers to a reduction of renal function to excrete
waste from a patient's blood. ARI is typically characterized as
including a decline of glomerular filtration rate (GFR) to a level
so low that little or no urine is formed. Therefore, substances
that the kidney usually eliminates remain in the body.
[0146] The causes of ARI are multifactorial, but they may be
classified into three categories: (1) prerenal ARI, in which the
kidneys fail to receive adequate blood supply, e.g., due to fall in
systemic blood pressure as in shock/cardiac arrest, or subsequent
to hemorrhage; (2) intrinsic ARI, in which the failure occurs
within the kidney, e.g., due to drug-induced toxicity; and (3)
postrenal ARI, caused by impairment of urine flow out of the
kidney, as in ureteral obstruction due to kidney stones or
bladder/prostate cancer. ARI may be associated with any one or a
combination of these categories.
[0147] Early diagnosis and treatment of acute renal injury can in
some cases prevent progression of acute renal injury to chronic
renal failure. ARI is accompanied by an inflammatory response that
if unchecked can lead to renal fibrosis and chronic renal failure.
Chronic renal failure (CRF) refers to a progressive loss of renal
function and irreversible kidney damage.
[0148] Methods for assessing renal function are well known in the
art and include, but are not limited to, measurements of blood
systemic and glomerular capillary pressure, proteinuria (e.g.,
albuminuria), microscopic and macroscopic hematuria, serum
creatinine level (e.g., one formula for estimating renal function
in humans equates a creatinine level of 2.0 mg/dl to 50 percent of
normal kidney function and 4.0 mg/dl to 25 percent), decline in the
glomerular filtration rate (GFR) (e.g., rate of creatinine
clearance), and degree of tubular damage. For example, such
assessment may include evaluating at least one kidney function
using biological and/or physiological parameters such as serum
creatinine level, creatinine clearance rate, 24-hour urinary
protein secretion, glomerular filtration rate (GFR), urinary
albumin creatinine ratio, albumin excretion rate, and renal
biopsy.
[0149] Methods for assessing deterioration of renal structure are
also well known. Illustrative methods are described in the
Examples. Such methods include renal imaging (e.g., MRI,
ultrasound), or histological evaluation of renal biopsy. In some
embodiments, the methods of the invention reduce deterioration of
renal structure as judged, for example, by the extent of
tubulointerstitial or glomerular damage and/or the degree of renal
fibrosis (e.g., deposition of collagen and fibronectin).
[0150] For a detailed review of renal function and disease states,
see The Kidney Physiology and Pathophysiology, eds. Seldin et al.,
3rd ed., Lippincott Williams & Wilkins Publishers, 2000.
Normally, less than 0.15 g of protein is excreted into the urine
per 24 hour period. Almost all types of kidney disease cause mild
(up to 500 mg per day) to moderate (up to 4 g per day) protein
leakage into the urine. The normal concentration of albumin in the
urine is less than 1.0 mg/dl. Generally, 30-300 mg/dl urinary
albumin is considered microalbuminuria, and 300 mg/dl and up is
considered macroalbuminuria. The normal values of serum creatinine
are 0.6-1.5 mg/dl for men and 0.6-1.1 mg/dl for women.
[0151] Methods of Preventing or Treating ARI Caused by
Ischemia.
[0152] An example of a condition in which kidneys fail to receive
adequate blood supply to the kidney is ischemia. Ischemia is a
major cause of ARI. Ischemia of one or both kidneys is a common
problem experienced during aortic surgery, renal transplantation,
or during cardiovascular anesthesia. Surgical procedures involving
clamping of the aorta and/or renal arteries, e.g., surgery for
supra- and juxtarenal abdominal aortic aneurysms and renal
transplantation, are also particularly liable to produce renal
ischemia, leading to significant postoperative complications and
early allograft rejection. In high-risk patients undergoing these
forms of surgery, the incidence of renal dysfunction has been
reported to be as high as 50%.
[0153] Ischemia may be caused by loss of blood, loss of fluid from
the body as a result of severe diarrhea or burns, shock, and
ischemia associated with storage of the donor kidney prior to
transplantation. In these situations, the blood flow to the kidney
may be reduced to a dangerously low level for a time period great
enough to cause ischemic injury to the tubular epithelial cells,
sloughing off of the epithelial cells into the tubular lumen,
obstruction of tubular flow that leads to loss of glomerular
filtration and acute renal injury.
[0154] Subjects may also become vulnerable to ARI after receiving
anesthesia, surgery, or .alpha.-adrenergic agonists because of
related systemic or renal vasoconstriction. Additionally, systemic
vasodilation caused by anaphylaxis, and anti-hypertensive drugs,
sepsis or drug overdose may also cause ARI because the body's
natural defense is to shut down, i.e., vasoconstriction of
non-essential organs such as the kidneys.
[0155] Accordingly, in one embodiment, a subject at risk for ARI
may be a subject undergoing an interruption or reduction of blood
supply or blood pressure to the kidney. These subjects may be
administered the aromatic-cationic peptides of the invention prior
to or simultaneously with such interruption or reduction of blood
supply. Likewise, aromatic-cationic peptides may be administered
after the therapeutic agent to treat ischemia.
[0156] Methods of Preventing or Treating ARI Caused by Drug-Induced
Toxicity.
[0157] Another cause of ARI includes drug-induced toxicity. For
example, nephrotoxins can cause direct toxicity on tubular
epithelial cells. Nephrotoxins include, but are not limited to,
therapeutic drugs, e.g., cisplatin, gentamicin, cephaloridine,
cyclosporin, amphotericin, radiocontrast dye (described in further
detail below), pesticides (e.g., paraquat), and environmental
contaminants (e.g., trichloriethylene and dichloroacetylene). Other
examples include puromycin aminonucleoside (PAN); aminoglycosides,
such as gentamicin; cephalosporins, such as cephaloridine;
calcineurin inhibitors, such as tacrolimus or sirolimus.
Drug-induced nephrotoxicity may also be caused by non-steroidal
anti-inflammatories, anti-retrovirals, anti-cytokines,
immunosuppressants, oncological drugs or ACE inhibitors. The
drug-induced nephrotoxicity may further be caused by nalgesic
abuse, ciprofloxacin, clopidogrel, cocaine, cox-2 inhibitors,
diuretics, foscamet, gold, ifosfamide, immunoglobin, Chinese herbs,
interferon, lithium, mannitol, mesalamine, mitomycin, nitrosoureas,
penicillamine, penicillins, pentamidine, quinine, rifampin,
streptozocin, sulfonamides, ticlopidine, triamterene, valproic
acid, doxorubicin, glycerol, cidofovir, tobramycin, neomycin
sulfate, colistimethate, vancomycin, amikacin, cefotaxime,
cisplatin, acyclovir, lithium, interleukin-2, cyclosporin or
indinavir.
[0158] In addition to direct toxicity on tubular epithelial cells,
some nephrotoxins also reduce renal perfusion, causing injury to
zones known to have limited oxygen availability (inner medullary
region). Such nephrotoxins include amphotericin and radiocontrast
dye. Renal failure can result even from clinically relevant doses
of these drugs when combined with ischemia, volume depletion,
obstruction, or infection. An example is the use of radiocontrast
dye in patients with impaired renal function. The incidence of
contrast-dye nephropathy (CIN) is 3-8% in the normal patient, but
increases to 25% for patients with diabetes mellitus. Most cases of
ARI occur in patients with predisposing comorbidities (McCombs, P.
R. & Roberts, B. Surg Gynecol Obstet, 148:175-178 (1979)).
[0159] Accordingly, in one embodiment, a subject at risk for ARI
may be receiving one or more therapeutic drugs that have a
nephrotoxic effect. These subjects may be administered the
aromatic-cationic peptides of the invention prior to or
simultaneously with such therapeutic agents. Likewise,
aromatic-cationic peptides may be administered after the
therapeutic agent to treat nephrotoxicity.
[0160] Methods of Preventing or Treating ARI Caused by
Radiocontrast Agents.
[0161] In one embodiment, the aromatic-cationic peptides of the
invention are administered to a subject at risk for
contrast-induced nephropathy (CIN), in order to protect the subject
from this condition. CIN is an important cause of acute renal
failure. CIN is defined as acute renal failure occurring within 48
h of exposure to intravascular radiographic contrast material. CIN
remains a common complication of radiographic procedures.
[0162] CIN arises when a subject is exposed to radiocontrast dye,
such as during coronary, cardiac, or neuro-angiography procedures.
Contrast dye is essential for many diagnostic and interventional
procedures because it enables doctors to visualize blocked body
tissues. Subjects at greatest risk for CIN are the elderly, and
those with diabetes, chronic kidney disease, or advanced heart
failure. The serum creatinine test can be used to monitor the onset
of CIN, monitor treatment, as well as monitor the effectiveness of
the peptides of the present invention in protecting subjects from
the onset of CIN.
[0163] In some embodiments, the aromatic-cationic peptides of the
invention are administered to a subject prior to or simultaneously
with the administration of a contrast agent in order to provide
protection against CIN. For example, the subject may receive the
peptides from about 1 to 2 hours, about 1 to 6 hours, about 1 to 12
hours, about 1 to 24 hours, or about 1 to 48 hours prior to
receiving the contrast agent. Likewise, the subject may be
administered the peptides at about the same time as the contrast
agent. Moreover, administration of the peptides to the subject may
continue following administration of the contrast agent. In some
embodiments, the subject may continue to receive the peptide at
intervals of about 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, and 48 hours
following administration of the contrast agent, in order to provide
a protective or prophylactic effect against CIN.
[0164] In some embodiments, the aromatic-cationic peptides of the
invention are administered to a subject after administration of a
contrast agent in order to treat CIN. For example, the subject may
receive the peptides from about 1 to 2 hours, about 1 to 6 hours,
about 1 to 12 hours, about 1 to 24 hours, about 1 to 48 hours, or
about 1 to 72 hours after receiving the contrast agent. For
instance, the subject may exhibit one or more signs or symptoms of
CIN prior to receiving the peptides of the invention, such as
increased serum creatinine levels and/or decreased urine volume.
Administration of the peptides of the invention improves one or
more of these indicators of kidney function in the subject compared
to a control subject not administered the peptides.
[0165] Types of Contrast Agents.
[0166] The incidence of CIN is influenced by the chemical structure
and particularly by the osmolarity and the ionic (or non-ionic)
structure of the contrast agent used (See Zirogiannis et al.,
"Contrast media induced nephropathy in patients undergoing cardiac
catheterization." Hellenic J Cardiol, 45:07-113 (2004)). A
"contrast agent" as used herein, refers to a compound employed to
improve the visibility of internal body structures in an image,
e.g., a CT or MRI scan. The term "contrast agent" is also referred
to herein as an "imaging agent." Imaging agents include those known
in the art, such as dyes, fluorescent dyes, gold particles, iron
oxide particles and other contrast agents including paramagnetic
molecules, x-ray attenuating compounds (for CT and x-ray), contrast
agents for ultrasound, .gamma.-ray emitting isotopes
(Scintigraphy), and positron-emitting isotopes (PET). Contrast
agents can be administered to the subject by, for example,
parenteral injection (e.g., intravenously, intra-arterially,
intra-thecally, intra-abdominally, subcutaneously,
intramuscularly), orally (e.g., as a tablet or a drink), rectally,
or via inhalation.
[0167] For example, an X-ray contrast agent can comprise barium
sulfate, or can comprise iodine in an organic (non-ionic) compound
or in an ionic compound. Examples of iodine contrast agents include
those shown in Table 7.
TABLE-US-00007 TABLE 7 Exemplary Iodinated Contrast Agents Iodine
Name Type Content Osmolality Level Diatrizoate (Hypaque 50) Ionic
Monomer 300 1550 High Osmolar Metrizoate (Isopaque Coronar 370)
Ionic 370 2100 High Osmolar Ioxaglate (Hexabrix) Ionic dimer 320
580 Low Osmolar Iopamidol (Isovue 370) Non-ionic monomer 370 796
Low Osmolar Iohexol (Omnipaque 350) Non-ionic 350 884 Low Osmolar
Ioxilan (Oxilan) Non-ionic Low Osmolar Iopromide Non-ionic Low
Osmolar Iodixanol (Visipaque 320) Non-ionic dimer 320 290 Iso
Osmolar
[0168] An MRI contrast agent can comprise a paramagnetic contrast
agent (such as a gadolinium compound), a super paramagnetic
contrast agent (such as iron oxide nanoparticles), a diamagnetic
agent (such as barium sulfate), and combinations thereof. Metal
ions preferred for MRI include those with atomic numbers 21-29,
39-47, or 57-83, and, more preferably, a paramagnetic form of a
metal ion with atomic numbers 21-29, 42, 44, or 57-83. Particularly
preferred paramagnetic metal ions are selected from the group
consisting of Gd(III), Fe(III), Mn(II and III), Cr(III), Cu(II),
Dy(III), Tb(III and IV), Ho(III), Er(III), Pr(III) and Eu(II and
III). Gd(III) is particularly useful. Note that as used herein, the
term "Gd" is meant to convey the ionic form of the metal
gadolinium; such an ionic form can be written as GD(III), GD3+,
etc. with no difference in ionic form contemplated. A CT contrast
agent can comprise iodine (ionic or non-ionic formulations),
barium, barium sulfate, Gastrografin (a diatrizoate meglumine and
diatrizoate sodium solution), and combinations thereof. In another
embodiment, a PET or SPECT contrast agent can comprise a metal
chelate.
[0169] As stated above, contrast agents include, but are not
limited to, molecules bearing one or more iodine moieties. Iodine
may be bound either in an organic compound or an ionic compound.
The earliest contrast agents were ionic, containing a sodium atom
that dissociated from the iodine in aqueous solution. Organic
compounds have fewer side effects as they do not dissociate into
component molecules.
[0170] Iodinated contrast media may be divided into water-soluble,
water-insoluble, and oily contrast media. Water-insoluble contrast
media include aqueous suspension of propyliodone (Dionosil.RTM.),
used in bronchography. Oily contrast media include Lipiodol, a
stable compound of 40% iodine in poppy seed oil, and Lipiodol
Ultra.RTM. Fluid and Ethiodol.RTM., ethyl esters of iodinated fatty
acids of poppy seed oil containing 48% and 37% iodine,
respectively. These oils are used for lymphography, and by some
also for hysterosalpingography. Iodophenylundecylic acid
(iophendylate) is a contrast medium for oil myelography (brand
names: Pantopaque.RTM., Myodil.RTM.).
[0171] The water-soluble contrast media represent by far the
largest group of iodinated contrast media. These contrast media may
be classified into high-osmolality, low-osmolality, and
iso-osmolality contrast media. The osmolality is related to some of
the adverse events of these contrast media. Monomeric ionic
contrast media constitute two main groups, oral cholegraphic
contrast media and uro/angiographic media. Examples of these media
include: acetrizoate (Diaginol.RTM., Urokon.RTM.); diatrizoate
(Angiografin.RTM., Hypaque.RTM., Renografin.RTM., Urografin.RTM.,
Urovison.RTM.); iodamide (Uromiro.RTM.); ioglicate (Rayvist.RTM.);
iothalamate (Conray.RTM.); ioxithalamate (Telebrix.RTM.); and
metrizoate (Isopaque.RTM., Triosil.RTM.).
[0172] The uro/angiographic media are all salts of derivatives of
tri-iodinated benzoic acid, and differ only in the side-chains at
position 3 and 5. The cations of the salts are mainly either sodium
or meglumine, or a mixture of both. Sodium salts are generally more
toxic to vascular endothelium and to blood-brain barrier and neural
tissue than meglumine. A mixture of sodium and meglumine has lower
cardiotoxicity than either salts alone. Some manufacturers have
partially replaced sodium with calcium and magnesium to reduce
toxicity.
[0173] The ionic monomeric contrast media for intravascular use are
so-called high-osmolar contrast media (HOCM), having an osmolality
seven to eight times that of plasma in ordinary clinical use. This
hyperosmolality is responsible for several subjective and objective
adverse effects such as pain, endothelial damage, thrombosis and
thrombophlebitis, disturbance of the blood-brain barrier,
bradycardia in cardioangiography and increased pressure in the
pulmonary circulation. The introduction of low-osmolar contrast
media (LOCM) has substantially reduced these side effects.
Osmolality is dependent upon the number of molecules per volume
unit solution. The ionic monomeric agents are salts that dissociate
into two molecules, one anion containing the radiopaque property
due to three iodine atoms, and one cation without radiopaque
properties. These agents are also called 3:2 or ratio 1.5 agents
(three iodine atoms per two molecules). There are three strategies
to obtain contrast media with lower osmolality without loss of
X-ray absorption: the production of 1) non-ionic monomers, 2) ionic
dimers or 3) non-ionic dimers. Non-ionic contrast media do not
dissociate and their water-solubility is generally achieved by
several hydrophilic hydroxyl groups. Tri-iodinated non-ionic
monomers have approximately half the osmolality of that of ionic
monomers (they are 3:1 or ratio 3 agents). Several agents are
available in convenient ready-to-use solutions of non-ionic
monomeric contrast media have appeared, and include: metrizamide
(Amipaque.RTM.); iohexol (Omnipaque.RTM.); iopamidol
(Iopamiro.RTM., Isovue.RTM., Niopam.RTM., Solutrast.RTM.); iopentol
(Imagopaque.RTM.); iopromide (Ultravist.RTM.); and ioversol
(Optiray.RTM.).
[0174] These contrast media have hydrophilic hydroxyl groups
attached to all three side chains, a feature that has also
contributed to reducing their toxicity. In addition to osmolality,
toxicity is also dependent upon chemical structure, an intrinsic
toxicity sometimes named chemotoxicity. Although the mechanism of
contrast medium toxicity is not completely understood, it is
believed that chemotoxicity is related to the protein-binding
capacity of the medium, which is caused by non-specific weak
interactions between the contrast medium and surrounding biological
molecules (e.g., enzymes). The interaction is mediated through
electrical forces, which are present only for ionic agents, and
through interaction between hydrophobic portions of the molecule,
which for contrast media is mainly the benzene ring. The many
hydrophilic side groups of the second generation non-ionic monomers
protect the inner hydrophobic benzene ring from interaction,
thereby reducing chemotoxicity. The relative
hydrophobicity/hydrophilicity of the molecule is called its
partition coefficient, a low partition coefficient being
advantageous because a high hydrophilicity contributes to low
protein binding.
[0175] Dimeric contrast media with two tri-iodinated benzene rings
constitute three main groups; ionic intravenous cholegraphic
contrast media, monoacidic ionic contrast media, and non-ionic
contrast media. The two latter groups both belong to the so-called
low-osmolar contrast media. There is only one monoacidic ionic
dimer; sodium meglumine ioxaglate (Hexabrix.RTM.). The dimeric
anion has one carboxyl group, the other has been replaced by a
non-ionizing side group. This gives six iodine atoms per two
particles; the contrast medium is a 6:2 (=3:1) or ratio 3 agent
(similar to the non-ionic monomers). Owing partly to its ionic
character, partly to its fewer hydroxyl groups, ioxaglate is more
toxic than the non-ionic monomers with similar osmolality (but less
toxic than the ionic monomers owing to lower osmolality).
[0176] The non-ionic dimers yield six iodine atoms per molecule
(ratio 6 agents). Examples of non-ionic dimer contrast media
include: iotrolan (Isovist.RTM.) and iodixanol (Visipaque.RTM.).
Iotrolan is nearly iso-osmolar to plasma (0.32 osm/kg H.sub.2O),
while iodixanol is actually hypo-osmolar to plasma. Iso-osmolarity
to plasma (0.29 osm/kg H.sub.2O) is achieved at any iodine
concentration by addition of an appropriate amount of saline. These
agents have a very low toxicity owing partly to their
iso-osmolarity, partly their non-ionic character, and partly to the
fact that they possess a very large number of hydroxyl groups (low
partition coefficient).
[0177] The various water-soluble contrast media for urography and
angiography (ionic and non-ionic monomers, non-ionic dimers) are
all extracellular contrast media, and are excreted unmetabolized by
glomerular filtration. Approximately 85-90% of the injected dose is
found in urine within the first 6 hours; 95-100% within the first
24 hours. Generally less than 2% may be found in feces.
[0178] Non-ionic contrast agents (containing 6 iodine anions in
their molecule) were believed to be less nephrotoxic than ionic
contrast agents (containing 3 iodine anions in their molecule).
However, in a recent study where 443 patients were randomly
assigned to a non-ionic (iopamidol) and an ionic (diatrizoate)
contrast agent, the incidence of CIN was not statistically
different (8.2 and 10.2% respectively) (Zirogiannis et al.,
"Contrast media induced nephropathy in patients undergoing cardiac
catheterization." Hellenic J Cardiol 45:107-113 (2004)).
Furthermore, recent evidence also points to an association between
exposure to a gadolinium-containing contrast agents during MRI
studies and incidence of nephrogenic fibrosing dermopathy in
patients with advanced renal disease (Agahtehrani and Moussa,
Vascular Disease Management, 4:49-59 (2007)). Thus, regardless of
the type of contrast agent, the mechanism for renal failure appears
to be the same, and the aromatic-cationic peptides of the invention
are useful in the prevention or treatment of ARI caused by these
agents.
[0179] Additional Applications for Aromatic-Cationic Peptides in
ARI.
[0180] In a further embodiment, a subject in need thereof may be a
subject having impairment of urine flow. Obstruction to the flow of
urine can occur anywhere in the urinary tract and has many
different possible causes. Such causes include kidney stones or
bladder/prostate cancer. Unilateral ureteral obstruction (UUO) is a
common clinical disorder associated with obstructed urine flow. It
is also 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. Thus, a
subject having or at risk for UUO may be administered the
aromatic-cationic peptides of the invention to protect or treat the
subject from ARI.
[0181] In yet another aspect of the invention, a method for
protecting a kidney from renal fibrosis in a mammal in need thereof
is provided. The method comprises administering to the mammal an
effective amount of an aromatic-cationic peptide as described
above. The peptides described above can be administered to a mammal
in need thereof, as described above, by any method known to those
skilled in the art.
[0182] The term "fibrosis" refers to abnormal processing of fibrous
tissue, or fibroid or fibrous degeneration. Fibrosis can result
from various injuries or diseases, and can often result from
chronic transplant rejection relating to the transplantation of
various organs. Fibrosis typically involves the abnormal
production, accumulation, or deposition of extracellular matrix
components, including overproduction and increased deposition of,
for example, collagen and fibronectin. As used herein, the term
"renal fibrosis" refers to fibrosis of the kidney. For example,
renal fibrosis is associated with overproduction or abnormal
deposition of extracellular matrix components, particularly
collagen, leading to the degradation or impairment of kidney
function.
[0183] In another aspect of the invention, a method for treating
acute renal injury in a mammal in need thereof is provided. The
method comprises administering to the mammal an effective amount of
an aromatic-cationic peptide as described above. The peptides
described above can be administered to a mammal in need thereof, as
described above, by any method known to those skilled in the
art.
[0184] The methods of the invention may be particularly useful in
patients with renal insufficiency, renal failure, or end-stage
renal disease attributable at least in part to a nephrotoxicity of
an drug or chemical. Other indications may include creatinine
clearance levels of lower than 97 (men) and 88 (women) ml/min,
blood urea of 20-25 mg/dl or higher. Furthermore, the treatment may
be useful in patients with microalbuminuria, macroalbuminuria,
and/or proteinuria levels of over 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
g or more per a 24 hour period, and/or serum creatinine levels of
about 1.0, 1.5, 2.0, 2.5, 3, 3.5, 4.0, 4.5, 5, 5.5, 6, 7, 8, 9, 10
mg/dl or higher.
[0185] The methods of the invention can be used to slow or reverse
the progression of renal disease in patients whose renal function
is below normal by 25%, 40%, 50%, 60%, 75%, 80%, 90% or more,
relative to control subjects. In some embodiments, the methods of
the invention slow the loss of renal function by at least 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, relative to
control subjects. In other embodiments, the methods of the
invention improve the patient's serum creatinine levels,
proteinuria, and/or urinary albumin excretion by at least 10%, 20%,
30%, 40%, 50%, 60%, 70%, or more, relative to control subjects.
Non-limiting illustrative methods for assessing renal function are
described herein and, for example, in WO 01/66140.
[0186] In another embodiment, the peptides useful in the present
invention may also be used in protecting a subject's kidney from
acute renal injury prior to transplantation. For example, a removed
kidney can be placed in a 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, about 0.1 nM to about 10 .mu.M, about 1 .mu.M to
about 5 .mu.M, or about 1 nM to about 100 nM.
[0187] Methods of Treating ARI.
[0188] In another aspect of the invention, the invention provides a
method for treating acute renal injury in a subject in need
thereof. The method comprises administering to the subject an
effective amount of an aromatic-cationic peptide as described
above. The peptides described above can be administered to a
subject in need thereof, as described above, by any method known to
those skilled in the art.
[0189] Subjects in need of protection from or treatment of acute
renal injury also include subjects 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.-).
[0190] Ischemic or toxic insults can lead to a secondary condition
of an acute, often reversible loss of renal function called acute
tubular necrosis (ATN), which is another common cause of ARI. The
course of ATN is triphasic. The initiating phase of ATN is
characterized by a precipitous drop in glomerular filtration rate
(GFR), triggered by ischemia and associated hemodynamic changes,
and accompanied by sublethal or lethal tubular epithelial injury.
The established or persistent phase of ATN is characterized by
persistent reduction in GFR. Sublethal tubular injury includes loss
of brush border, vacuolization and flattening of proximal tubules,
and dilation of tubules. Cellular necrosis may be seen in the
proximal tubule. The renal interstitium may be edematous, whereas
leukocytic infiltration may be observed in the medulla. The distal
tubular lumen may be occluded by casts, and distal, rather than
proximal, tubular epithelial cells are more likely involved by
apoptosis. Such structural and functional derangements resolve
during the recovery or diuretic phase wherein reparative and
regenerative responses restore renal architecture and normalize
GFR.
[0191] Common mechanisms of ARI caused by ischemia and
nephrotoxicants involve mitochondrial dysfunction and ATP deficits,
which result in renal proximal tubular cell (RPTC) injury and
necrosis. RPTCs are highly specialized both in terms of morphology
and function allowing for efficient transport of water, ions and
macromolecules via specific transport mechanisms. RPTCs contain
very high density of mitochondria, and oxygen is used to sustain
oxidative phosphorylation and synthesis of ATP, which is needed in
large quantities for renal tubular transport processes. During
ischemia, these transport processes are severely disrupted due to
loss of intracellular ATP. Restoration of blood flow will help to
limit cell death, but recovery of mitochondrial function depends on
the duration of ischemia. With prolonged ischemia, there is
evidence that the rate of oxidative phosphorylation continues to
deteriorate despite reperfusion. Progressive ischemia leads to
inhibition of several components of the mitochondrial electron
transport chain, including complex I, complex V, and the adenine
nucleotide translocator, and decrease in ATP production. In
addition, ischemia decreases cardiolipin and cytochrome c content
in cardiac mitochondria, both of which will inhibit cytochrome c
oxidase activity. Thus, mitochondria can suffer damage during
ischemia that would limit their capacity to generate ATP upon
restoration of oxygen and substrates with reperfusion.
[0192] Furthermore, damaged mitochondria generate enormous amounts
of ROS during reperfusion, and there is an excess of hydroxyl
radicals as a result of iron overload following ischemia.
Mitochondria are normally protected from oxidative damage by a
multi-layer network of mitochondrial antioxidant systems, but they
can undergo oxidative damage when ROS production exceeds the
antioxidant capacity of mitochondria. ROS can initiate damage to
nucleic acids, proteins and lipids in mitochondria. Protein
oxidation and nitration result in altered function of many enzymes
in the mitochondrial electron transport chain, while oxidation of
the adenine nucleotide translocator impairs the influx of ADP into
the matrix for ATP synthesis. ROS have been shown to damage renal
mitochondria by enhancing membrane permeability, decreasing
F.sub.0F.sub.1ATPase activity and reducing ATP production.
[0193] Mitochondrial dysfunction may therefore lead to necrosis or
apoptosis. Necrosis is characterized by cell swelling and
disruption of the cell membrane, leading to release of cellular
contents, especially proteolytic enzymes, which may result in
destruction of neighboring cells. Apoptosis, on the other hand, is
defined as programmed cell death wherein the organism eliminates
senescent, abnormal cells without affecting surrounding cells, and
is deemed to be preferable for the survival of the organism since
it eliminates dying cells by phagocytosis. The decision step
between death by apoptosis or necrosis appears to be dependent on
intracellular ATP content. Apoptosis involves energy-requiring
steps, especially in the formation of the apoptosome complex
between Apaf-1 and cytochrome c. Thus in the event of significant
cellular ATP depletion, death can only occur by necrosis.
[0194] There is accumulating evidence for a role of mitochondrial
ROS in the acute renal toxicity due to nephrotoxicants (Baliga et
al. Drug Metab Rev, 31:971-997 (1999)). Cisplatin induced
mitochondrial oxidative stress, decreased ATP and glutathione,
cardiolipin peroxidation and apoptosis, and cisplatin
nephrotoxicity can be ameliorated with hydroxyl radical scavengers
(Santos, N. A., et al., Cancer Chemother Pharmacol, 61:145-155
(2008)). Radiocontrast dyes increase the rate of renal tubular cell
apoptosis in vivo (Beeri, R., et al. Rapid DNA fragmentation from
hypoxia along the thick ascending limb of rat kidneys. Kidney Int,
47:1806-1810 (1995)) and in vitro (Hizoh et al., Nephrol Dial
Transplant, 13:911-918 (1998); Heinrich et al., Radiology,
235:843-849 (2005)). However, nephrotocixity of radiocontrast dye
media cannot solely be explained by oxidative stress, and
mitochondrial energetics and integrity appear to play an important
role as well (Zager et al., Radiographic contrast media-induced
tubular injury: evaluation of oxidant stress and plasma membrane
integrity. Kidney Int, 64:128-139 (2003); Humes et al., Am J
Physiol, 252:F246-255 (1987)). Oxidative damage has also been
implicated in postrenal obstructive ARI, and there is evidence that
the intrinsic mitochondria-mediated apoptotic pathway is involved
in stretch-induced tubular cell apoptosis (Zhang, G., et al., Exp
Nephrol, 9:71-80 (2001)).
[0195] When epithelial cells are damaged, they release inflammatory
mediators, including IL-1.beta. and TNF.alpha., that lead to an
intense inflammatory response initiated by the infiltration of
leukocytes and the release of proinflammatory cytokines, chemokines
and ROS. Leukocyte infiltration is observed with renal
ischemia-reperfusion, ureteral obstruction, and nephrotoxins, and
the inflammatory process contribute to ARI. Infiltrating
lymphocytes and macrophages become activated and begin secreting
profibrotic cytokines and growth factors, such as TGF.beta., IL-13
and PDGF, which further activate macrophages and fibroblasts.
TGF.beta. plays a pivotal role in epithelial-mesenchymal transition
(EMT) by down-regulating E-cadherin and up-regulating .alpha.-SMA
expression, and increasing collagen production. Fibrosis occurs
when the synthesis of new collagen by myofibroblasts exceeds the
rate at which it is degraded, such that the total amount of
collagen increases over time. The chemokines, such as MCP-1, also
play a role in fibrosis by recruiting macrophages and
myofibroblasts to the site of injury. Collagen turnover and ECM
remodeling is regulated by various matrix metalloproteases
(MMPs).
Methods of Protecting or Treating a Subject from Tissue Injury or
Organ Failure
[0196] The discovery that the aromatic-cationic peptides of the
present invention are useful in preventing or treating ARI is also
applicable to tissue injury and organ failure in other systems
besides the kidney. For instance, the aromatic-cationic peptides of
the invention are predicted to minimize mitochondrial dysfunction,
cell death, inflammation, and fibrosis. In some embodiments, the
present invention provides a method of treating a subject having a
tissue injury, e.g., noninfectious pathological conditions such as
pancreatitis, ischemia, multiple trauma, hemorrhagic shock, and
immune-mediated organ injury.
[0197] The tissue injury can be associated with, for example,
aortic aneurysm repair, multiple trauma, peripheral vascular
disease, renal vascular disease, myocardial infarction, stroke,
sepsis, and multi-organ failure. In one aspect, the invention
relates to a method of treating a subject having a tissue such as
from heart, brain, vasculature, gut, liver, kidney and eye that is
subject to an injury and/or ischemic event. The method includes
administering to the subject a therapeutically effective amount of
an aromatic-cationic peptide to provide a therapeutic or
prophylactic effect. Another embodiment of the present invention
provides the administration of the peptides of the present
invention to improve a function of one or more organs selected from
the group consisting of: renal, lung, heart, liver, brain,
pancreas, and the like. In a particular embodiment, the improvement
in lung function is selected from the group consisting of lowered
levels of edema, improved histological injury score, and lowered
levels of inflammation.
[0198] In some embodiments, the peptides are useful for the
prevention and/or treatment of acute hepatic injury caused by
ischemia, drugs (acetaminophen, alcohol), viruses, obesity
(non-alcoholic steatohepatitis), and obstruction (bile duct
obstruction, tumors). In some embodiments, the aromatic-cationic
peptides of the invention are administered to a subject to prevent
or treat acute liver failure (ALF). ALF is a clinical condition
that results from severe and extensive damage of liver cells
leading to failure of the liver to function normally. ALF results
from massive necrosis of liver cells leading to hepatic
encephalopathy and severe impairment of hepatic function. It is
caused by various kinds of diseases, such as viral hepatitis (A, B,
C), drug toxicity, frequent alcohol intoxication, and autoimmune
hepatitis. ALF is a very severe clinical condition with high
mortality rate. Drug-related hepatotoxicity is the leading cause of
ALF in the United States.
[0199] In some embodiments, the aromatic-cationic peptides of the
invention are administered to a subject prior to or simultaneously
with the administration of an drug or agent known or suspected to
induced hepatotoxicity, e.g., acetaminophen, in order to provide
protection against ALF. For example, the subject may receive the
peptides from about 1 to 2 hours, about 1 to 6 hours, about 1 to 12
hours, about 1 to 24 hours, or about 1 to 48 hours prior to
receiving the drug or agent. Likewise, the subject may be
administered the peptides at about the same time as the drug or
agent to provide a prophylactic effect against ALF caused by the
drug or agent. Moreover, administration of the peptides to the
subject may continue following administration of the drug or agent.
In some embodiments, the subject may continue to receive the
peptide at intervals of about 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, and
48 hours following administration of the drug or agent, in order to
provide a protective or prophylactic effect.
[0200] In some embodiments, the aromatic-cationic peptides of the
invention are administered to a subject exhibiting one or more
signs or symptoms of ALF, including, but not limited to, elevated
levels of hepatic enzymes (transaminases, alkaline phosphatase),
elevated serum bilirubin, elevated serum ammonia, elevated serum
glucose, elevated serum lactate, or elevated serum creatinine.
Administration of the peptides of the invention improves one or
more of these indicators of liver function in the subject compared
to a control subject not administered the peptides. The subject may
receive the peptides from about 1 to 2 hours, about 1 to 6 hours,
about 1 to 12 hours, about 1 to 24 hours, about 1 to 48 hours, or
about 1 to 72 hours after the first signs or symptoms of ALF.
Modes of Administration
[0201] The peptides useful in the methods of the present invention
are administered to a subject in an amount effective in protecting
a subject's from acute renal injury or acute liver failure in a
subject in need thereof. Also, the peptides useful in the methods
of the present invention are administered to a subject in an amount
effective in treating acute renal injury or acute liver failure in
a subject in need thereof.
[0202] As used herein, the term "effective amount" or
"pharmaceutically effective amount" or "therapeutically effective
amount" of a composition, is a quantity sufficient to achieve a
desired therapeutic and/or prophylactic effect, e.g., an amount
which results in the prevention of, or a decrease in, the symptoms
associated with ARI or ALF. The amount of a composition of the
invention administered to the subject will depend on the type and
severity of the disease and on the characteristics of the
individual, such as general health, age, sex, body weight and
tolerance to drugs. It will also depend on the degree, severity and
type of disease. The skilled artisan will be able to determine
appropriate dosages depending on these and other factors. The
compositions of the present invention can also be administered in
combination with one or more additional therapeutic compounds. In
the methods of the present invention, the aromatic-cationic
peptides may be administered to a subject having one or more signs
of ARI caused by a disease or condition. Administration of an
effective amount of the aromatic-cationic peptides may improve at
least one sign or symptom of ARI in the subject, e.g., metabolic
acidosis (acidification of the blood), hyperkalaemia (elevated
potassium levels), oliguria or anuria (decrease or cessation of
urine production), changes in body fluid balance, and effects on
other organ systems. For example, a "therapeutically effective
amount" of the aromatic-cationic peptides is meant levels in which
the physiological effects of acute renal failure are, at a minimum,
ameliorated. Typically, the efficacy of the biological effect is
measured in comparison to a subject or class of subjects not
administered the peptides.
[0203] The effective amount is determined during pre-clinical
trials and clinical trials by methods familiar to physicians and
clinicians. An effective amount of a peptide useful in the methods
of the present invention, preferably in a pharmaceutical
composition, may be administered to a subject in need thereof by
any of a number of well-known methods for administering
pharmaceutical compounds.
[0204] In a preferred embodiment, 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.
[0205] The peptide may also be administered orally, topically,
intranasally, intramuscularly, intraperitoneally, subcutaneously,
or transdermally.
[0206] The peptides useful in the methods of the invention may also
be administered to subjects 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. A description of methods for delivering a compound
by controlled release can be found in PCT Application No. WO
02/083106.
[0207] 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.
[0208] For systemic, oral, interperitoneal, topical, intranasal,
subcutaneous, or transdermal administration, formulations of the
aromatic-cationic peptides useful in the methods of the present
inventions may utilize conventional diluents, carriers, or
excipients etc., such as are known in the art can be employed to
deliver the peptides. For example, the formulations may comprise
one or more of the following: a stabilizer, a surfactant,
preferably a nonionic surfactant, and optionally a salt and/or a
buffering agent. The peptide may be delivered in the form of an
aqueous solution, or in a lyophilized form.
[0209] 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.
[0210] 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).
[0211] 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. The salt or buffering agent may be present at a
roughly isotonic concentration of about 150 mM to about 300 mM.
[0212] 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.
EXAMPLES
Example 1. Protection Against Prerenal ARI Caused by
Ischemia-Reperfusion (I/R)
[0213] The effects of the aromatic-cationic peptides of the
invention in protecting a subject from ARI caused by
ischemia-reperfusion were investigated in an animal model of ARI
caused by I/R.
[0214] Sprague Dawley rats (250.about.300 g) were assigned to four
groups: (1) sham surgery group without I/R; (2) I/R+saline vehicle
treatment; (3) I/R+SS-20 treatment; (4) I/R+SS-31 treatment. Each
experimental group consisted of 8 rats. SS-20 and SS-31 (3 mg/kg,
dissolved in saline) were administered to rats 30 min before
ischemia and immediately before onset of reperfusion. The control
rats were given saline alone on the same schedule.
[0215] Rats were anesthetized with a mixture of ketamine (90 mg/kg,
i.p.) and xylazine (4 mg/kg, i.p.). The left renal vascular pedicle
was occluded temporarily using a micro-clamp for 30 or 45 min. At
the end of the ischemic period, reperfusion was established by
removing of the clamp. At that time, the contralateral right kidney
was removed. After 24 h reperfusion, animals were sacrificed and
blood samples were obtained by cardiac puncture. Renal function was
determined by blood urea nitrogen (BUN) and serum creatinine
(BioAssay Systems DIUR-500 and DICT-500).
[0216] Renal Morphologic Examination:
[0217] Kidneys were fixed in 10% neutral-buffered formalin and
embedded in paraffin wax. Three micron sections were stained with
hematoxylin-eosin (H&E) and periodic acid-Schiff (PAS) and
analyzed by light microscopy. Lesions were scored using the
following criteria: [1] mitosis and necrosis of individual cells;
[2] necrosis of all cells in adjacent proximal convoluted tubules
with survival of surrounding tubules; [3] necrosis confined to the
distal third of the proximal convoluted tubule with a band of
necrosis extending across the inner cortex; and [4] necrosis
affecting all three segments of the proximal convoluted tubule.
[0218] TUNEL Assay for Apoptosis:
[0219] Renal tissue sections were deparaffinized and rehydrated by
xylenes, graded alcohol series and deionized H.sub.2O, and then
incubated in 20 g/ml proteinase K for 20 min at RT. In situ cell
death detection POD kit (Roche, Ind., USA) was used according to
the manufacturer's instructions. Briefly, endogenous peroxidase
activity in the kidney sections was blocked by incubation for 10
min with 0.3% H.sub.2O.sub.2 in methanol. The sections were then
incubated in a humidified chamber in the dark for 30 min at
37.degree. C. with TUNEL reaction mixture. After washing, the
slides were incubated with 50-100 .mu.l Converter-POD in a
humidified chamber for 30 min at RT. The slides were incubated in
DAB solution (1-3 min), and counterstained with hemotoxylin,
dehydrated through a graded series of alcohol, and mounted in
Permount for microscopy.
[0220] Immunohistochemistry:
[0221] Renal sections were cut from paraffin blocks and mounted on
slides. After removal of paraffin with xylene, the slides were
rehydrated using graded alcohol series and deionized H.sub.2O.
Antigen Retrieval was heated in citrate buffer (10 mM Citric Acid,
0.05% Tween 20, pH 6.0). Endogenous peroxidase was blocked with
hydrogen peroxide 0.3% in methanol. Immunohistochemistry was then
performed using primary antibody against heme oxygenase-1 (HO-1)
(rat anti-HO-1/HMOX1/HSP32 monoclonal antibody (R&D Systems,
MN, USA) at 1:200 dilution)) and secondary antibody (HRP-conjugated
goat anti-rat IgG, VECTASTAIN ABC (VECTOR Lab Inc. MI, USA)).
Substrate reagent 3-amino-9-ethylcarbazole (AEC, Sigma, MO, USA)
was used to develop red color. Hematoxylin was used for
counterstaining.
[0222] Western Blotting:
[0223] Kidney tissue was homogenized in 2 ml of RIPA lysis buffer
(Santa Cruz, Calif., USA) on ice and centrifuged at 500.times.g for
30 min to remove cell debris. Aliquots of the supernatants were
stored at -80.degree. C. Thirty (30) g of protein of each sample
was suspended in loading buffer and boiled for 5 min then each
sample was subjected to a 10% SDS-PAGE gel electrophoresis. The
resolved proteins were transferred to a PVDF membrane. After
blocking the membrane in 5% non-fat dry milk and 1% bovine serum
albumin for 1 h, the nitrocellulose blot was incubated with a
1:2000 diluted anti-HO-1/HMOX1/HSP32 or a 1:1000 diluted
anti-AMPK.alpha.-1, monoclonal antibodies (R&D Systems, MN,
USA) then incubated with horseradish peroxidase-conjugated
secondary antibodies. The protein bands in the blot were detected
with the use of an Enhanced Chemi Luminescence detection system
(Cell Signaling, MA, USA) and X-ray film. Relative density
measurements provide quantification.
[0224] ATP Content Assay:
[0225] The kidney tissue was immediately put into 10 ml 5%
trichloroacetic acid with 10 mM DTT, 2 mM EDTA and cut into small
pieces. The tissue was homogenized on ice, incubated for another 10
min, centrifuged for 10 min at 2000.times.g, and neutralized with
10N KOH to pH 7.6. After centrifugation for 10 min at 2000.times.g,
aliquots of the resulting supernatant were stored at -80.degree. C.
ATP was measured by bioluminescence (ATP bioluminescent kit, Sigma,
MO, USA).
[0226] Results.
[0227] The effects of the aromatic-cationic peptides of the
invention on a model of ARI caused by ischemia-reperfusion were
investigated. Renal mitochondria were isolated in accordance with
the procedures described above and oxygen consumption was measured.
The results are as follows.
TABLE-US-00008 TABLE 8 BUN and Serum Creatinine Values Sham I/R +
Saline I/R + SS-20 I/R + SS-31 BUN (mg/dl) 40.8 .+-. 4.6 170 .+-.
1.7* 130.7 .+-. 5.5# 113.8 .+-. 11.4# Serum Creatinine 0.56 .+-.
0.04 1.73 .+-. 0.12* 1.06 .+-. 0.11# 1.05 .+-. 0.16# (mg/dl)
[0228] The above table shows treatment with either SS-20 or SS-31
improved BUN and serum creatinine values in rats after 45 min
ischemia and 24 h reperfusion. BUN and serum creatinine were
assayed using kits from BioAssay Systems (DIUR-500 and DICT-500).
The * indicates that p<0.05 from the sham group. The # indicates
p<0.05 from the IR=saline group.
[0229] FIG. 1 shows that treatment with either SS-20 or SS-31
prevented tubular cell apoptosis after 30 min ischemia and 24 h
reperfusion. Renal sections were stained for apoptotic cells using
TUNEL. Very few TUNEL-positive cells were observed in the medulla
region of animals not subjected to I/R (FIG. 1A). A large number of
TUNEL-positive epithelial cells were observed in the medulla of
animals after 30 min ischemia and 24 h reperfusion (FIG. 1B).
Treatment with SS-20 (3 mg/kg) or SS-31 (3 mg/kg) 30 min before
onset of 30 min ischemia and just prior to reperfusion
significantly prevented TUNEL-positive cells in the medulla (FIG.
1C and FIG. 1D).
[0230] FIG. 2 shows that treatment with either SS-20 or SS-31
prevented tubular cell injury after 45 min ischemia and 24 h
reperfusion. Renal sections were stained with H&E (FIG. 2A-2D)
and PAS (FIG. 2E-2H). Ischemia-reperfusion resulted in loss of
brush border, vacuolization and necrosis in the proximal tubules in
the inner medulla, and the distal tubules were occluded by casts
(FIG. 2B). Pretreatment with either SS-20 or SS-31 protected brush
border and prevented necrosis in the proximal tubules (FIG. 2C and
FIG. 2D).
[0231] FIG. 3 shows that treatment with SS-20 or SS-31
significantly improved histopathological score resulting from 45
min ischemia and 24 h reperfusion. Data are presented as the
mean.+-.SD. A one-way ANOVA and Scheffe's tests were used for
multiple pairwise comparisons.
[0232] FIG. 4 shows that treatment with SS-20 or SS-31
significantly increased rate of ATP production after reperfusion.
ATP content in renal tissue was determined at the end of 15 min
ischemia (FIG. 4A), and also after 1 h reperfusion following 45 min
ischemia (FIG. 4B). ATP was already significantly reduced after 15
min ischemia, and this was not altered by pretreatment with either
SS-20 or SS-31. ATP content was still very low after 1 h
reperfusion in the vehicle control group, but was significantly
higher in the animals treated with SS-20 or SS-31.
[0233] FIG. 5 shows the results of pretreatment with SS-20 or SS-31
significantly improved renal mitochondrial respiration after 45 min
ischemia. State 3 respiration was initiated with the addition of
ADP. Oxygen consumption was significantly reduced in mitochondria
isolated from rats after 45 min ischemia. Pretreatment with SS-20
or SS-31 significantly improved oxygen consumption such that
ischemic mitochondria were no different from non-ischemic
controls.
[0234] In summary, these results indicate that the peptide SS-31
and SS-20 are effective in reducing the incidence of ARI caused by
ischemia-reperfusion. As such, the aromatic-cationic peptides of
the invention are useful in methods of protecting a subject from
ARI caused by ischemia.
Example 2. Protection Against Postrenal ARI Caused by Ureteral
Obstruction
[0235] The effects of the aromatic-cationic peptides of the
invention in protecting a subject from ARI caused by ureteral
obstruction were investigated in an animal model of ureteral
obstruction. This Example describes the results of such
experiments.
[0236] Sprague-Dawley rats underwent unilateral ureteral ligation
(UUO) with 4-0 silk suture through a midline abdominal incision
under sterile conditions. Ureteral obstruction was carried out by
ligating the left ureter at the end of lower ureter, just above the
ureterovesical junction.
[0237] SS-31 (1 mg/kg or 3 mg/kg; n=8) was administered
intraperitoneally, one day prior to UUO and continuing for 14 days.
A separate group of animals was given saline, as a vehicle control
(n=16).
[0238] Renal Histology:
[0239] Trichrome sections of paraffin embedded specimens were
examined by a board-certified pathologist (SVS, renal pathology
specialist), and fibrosis scored on a scale of 0-+++
[0240] Immunohistochemical Analysis:
[0241] Immunohistochemical staining for macrophages was carried out
using a monoclonal antibody to ED-1 (Serotec) as previously
described. Macrophages were counted in 10 high-power fields
(.times.400) by two different independent investigators in a
blinded fashion. The TUNEL assay was performed as described in
Example 1. The presence of fibroblasts was examined using
immunohistochemistry, as described in Example 1. The antibody
utilized was DAKO # S100-A4 (1:100 dilution). The S100-A4 antigen
is also known as FSP-1 (fibroblast specific protein). Antigen was
retrieved by incubating cells with Proteinase K for 20 min in an
oven. The remaining immunoperoxidase protocol was carried out
according to routine procedures. Staining for S100-A4 was found in
spindle-shaped interstitial cells, and also in cells which were
round, and were identified as inflammatory cells by the
pathologist. Only spindle-shaped cells were included in the counts.
Samples incubated without primary antibody exhibited no staining.
8-OH dG staining was carried using Proteinase K for antigen
retrieval. The antibody used was from the Japan Institute Control
of Aging, and was used at a dilution of 1:200-1:500.
[0242] Polymerase Chain Reaction Analysis:
[0243] PCR for heme oxygenase-1 (HO-1) isolated from kidneys was
performed as follows. Rat kidneys were harvested and were kept at
-80.degree. C. until use. Total RNA was extracted using the
Trizol.RTM.-Chloroform extraction procedure. mRNA was purified
using the Oligotex mRNA extraction kit (Qiagen, Valencia, Calif.)
according to manufacturer's instructions. mRNA concentration and
purity were determined by measuring absorbance at 260 nm. RT-PCR
was preformed using Qiagen One-step PCR kit (Qiagen, Valencia,
Calif.). PCR was performed in an automated thermal cycler
ThermoHybrid, PX2 with an initial activation step for 15 min at
95.degree. C. followed by 35 cycles of denaturation for 45 s at
94.degree. C., annealing for 30 s at 60.degree. C., extension for
60 s at 72.degree. C. PCR products were separated by a 2% agarose
gel electrophoresis. Bands on gels were visualized by ethidium
bromide staining and analyzed using Image J densitometric analysis
software. Primers for HO-1 were: CTG AAG AAG ATT GCG CAG AA (SEQ ID
NO: 1) and ATG GCA TAA ATT CCC ACT GC (SEQ ID NO: 2) [427 bp].
GAPDH was used as a control.
[0244] Results.
[0245] In accordance with the procedures just described, the
effects of the aromatic-cationic peptides of the invention in
protecting a subject from ARI caused by ureteral obstruction were
investigated in an animal model of ureteral obstruction. UUO (14
days) produces a characteristic set of changes in the kidney
including increased interstitial fibrosis, tubular apoptosis,
macrophage infiltration and tubular proliferation. Animals were
pre-treated with SS-31 one day prior to UUO and daily through
14-days. Control animals received saline only throughout the period
of UUO.
[0246] Contralateral unobstructed kidney (FIG. 6A, CK) showed very
little, if any, inflammation or fibrosis in tubules, glomeruli or
interstitium (data not shown). The obstructed kidney (FIG. 6B, OK)
of the control vehicle treatment group showed moderate (1-2+)
medullary trichrome staining, along with areas of focal peripelvic
1+ staining. The cortex showed less fibrosis than the medulla. OK
also showed moderate inflammation, generally scored as 1+ in the
cortex and 2+ in the medulla. SS-31 treated kidneys (FIG. 6C)
showed significantly less trichrome staining, being 0-trace in the
cortex and tr-1+ in the medulla). When interstitial volume was
measured, medullary interstitial volume in the control OK was
69.2.+-.2.1% as compared to 0.5.+-.0.1% in the CK (FIG. 6D).
Treatment with 1 mg/kg SS-31 modulated the increase in interstitial
volume to 54.9.+-.2.3%; a higher dose of SS-31 was also effective.
Thus, SS-31 decreases medullary fibrosis in a 14-day UUO model.
[0247] Fibroblasts were visualized by immunoperoxidase for
fibroblast-specific protein (FSP-1). Increased expression of FSP-1
was found in the OK group (16.9.+-.2.3 cells/HPF), with only a
small number of interstitial fibroblasts present in the control CK
group (1.1.+-.0.3 FSP+ cells/HPF) (FIG. 7). SS-31 (1 mg/kg)
significantly decreased the amount of fibroblast infiltration in
the OK to 43.4% of control; SS-31 (3 mg/kg) treatment further
decreased the amount of fibroblast infiltration in the OK to 28.0%
of the untreated OK. Thus, SS-31 decreases fibroblast expression in
a 14-day UUO model.
[0248] In the untreated OK, 2 weeks of UUO resulted in a
significant increase in apoptotic tubular cells as compared to the
CK. Apoptotic cells were visualized by use of the TUNEL assay.
SS-31 at 1 mg/kg significantly decreased tubular apoptosis from
15.1.+-.3.1 apoptotic cells per HPF to 5.1.+-.0.5 cells per HPF
(p<0.05); SS-31 at 3 mg/kg caused a further significant decrease
in renal tubular apoptosis (3.0.+-.0.3 apoptotic cells/HPF) (FIG.
8). Thus, SS-31 decreases tubular apoptosis in a 14-day UUO
model.
[0249] There was a significant increase in macrophage infiltration
into the OK as compared to the CK after 2 weeks of UUO (33.8.+-.6.3
cells/HPF vs. 0.04.+-.0.03 cells/HPF). Macrophages were visualized
by immunoperoxidase for ED-1. Both 1 and 3 mg/kg SS-31
significantly decreased macrophage infiltration into the OK (FIG.
9). Thus, SS-31 decreases macrophage expression in a 14-day UUO
model.
[0250] The obstructed kidney was associated with increased
proliferation of renal tubular cells, as visualized by
immunoperoxidase for PCNA. SS-31 caused a significant increase in
renal tubular proliferation in the OK. Tubular proliferation was
increased 2-fold at the 1 mg/kg dose and 3.5-fold at 3 mg/kg at the
3 mg/kg dose (FIG. 10). Thus, SS-31 increases tubular proliferation
in a 14-day UUO model.
[0251] The obstructed kidney (OK) showed elevated oxidative damage,
as assessed by increased expression of heme oxygenase-1 (HO-1) and
8-OH dG. RT-PCR was used for HO-1 expression UUO was associated
with an increase in HO-1 expression (HO-1/GADPH 1.09 versus 0.07 in
the unobstructed control) (FIG. 11A). Treatment with SS-31
decreased HO-1 expression in the obstructed kidney. 8-OH dG
staining was detected in both tubular and interstitial compartments
of the obstructed kidney (FIG. 11B). The number of 8-OH dG positive
cells/HPF was significantly increased in the OK compared to the CK
(5.0.+-.1.4 cells/HPF vs. 1.4.+-.0.1 cells/HPF), and this was
significantly reduced with SS-31 treatment (FIG. 11C). Thus, SS-31
decreases oxidative damage in a 14-day UUO model.
[0252] In summary, these results indicate that the peptide SS-31 is
effective in reducing interstitial fibrosis, tubular apoptosis,
macrophage infiltration and tubular proliferation in a animal model
of API caused by ureteral obstruction. As such, the
aromatic-cationic peptides of the invention are useful in methods
of protecting a subject from acute renal injury caused by ureteral
obstruction.
Example 3. Treatment and Prevention of CIN in Indo/L-NAME Animal
Models
[0253] The effects of the aromatic-cationic peptides of the
invention in protecting a subject from CIN were investigated in an
animal model of ARI caused by radiocontrast dye administration.
This Example describes the results of such experiments.
Experiment 1
[0254] Animal Model:
[0255] A model of radiocontrast dye-induced renal failure as
described by Agmon et al. J Clin Invest 94:1069-1075 (1994) in this
Example. As in humans, radiocontrast dye is generally non-toxic
when administered to animals with normal renal function. However,
radiocontrast dye can induce ARI in animals with impaired renal
function. In this model, impaired renal function was induced by the
administration of indomethacin (10 mg/kg) and L-NAME (10 mg/kg).
The animals were divided into 3 groups: [0256] 1. Group (1) control
(n=8) [0257] 2. Group (2) Indomethcin/L-NAME (given 15 min apart)
followed by iothalamate (AngioConray 6 ml/kg) (n=7) [0258] 3. Group
(3) SS-31 (3 mg/kg, ip) was given 15 min prior to administration of
indomethacin/L-NAME/iothalamate, and a second dose of SS-31 (3
mg/kg) was repeated immediately after drug exposure (n=9)
[0259] Experimental Protocol:
[0260] Rats were kept in metabolic cages (Nalge Co., Rochester,
N.Y.) with free access to tap water and standard rat chow. After a
baseline 24-h urinary collection, the rats were anesthetized with a
mixture of ketamine (90 mg/kg, i.p.) and xylazine (4 mg/kg, i.p.).
The left femoral vein and artery were cannulated and a baseline
blood sample drawn (1 ml) for measurement of creatinine level. The
animals then received the various drug treatments as indicated
above. Iothalamate Meglumine 60% (contrast dye; AngioConray 6
ml/kg) was then injected through the arterial cannula. The left
femoral artery was then ligated and the cannula removed. Rats were
then returned to the metabolic cages for another 24-h urine
collection. At the end of this period, a blood sample was drawn
from the tail vein (1 ml). After this, the animals were
anesthetized, kidneys were removed, and the animals euthanized.
[0261] Renal Function:
[0262] Renal function was assessed by determining GFR at baseline
and 24 h following dye administration. GFR was determined by
creatinine clearance which was estimated over a 24 h interval
before and after dye administration. Creatinine clearance was
analyzed by measuring plasma (Pcr), urinary creatinine (Ucr) levels
(Bioassay Systems; DICT-500) and urine volume.
[0263] Renal Histology:
[0264] Kidneys were fixed in 10% neutral-buffered formalin and
embedded in paraffin wax. Three micron sections were stained with
hematoxylin-eosin (H&E) and periodic acid-Schiff (PAS) and
analyzed by light microscopy by a board certified pathologist.
[0265] Results:
[0266] In accordance with the procedures just described, the
effects of the aromatic-cationic peptides of the invention in
protecting a subject from CIN were investigated in an animal model
of ARI caused by radiocontrast dye administration. The results are
as follows.
[0267] In the control rats, there was no significant difference in
GFR between the first 24 h period (235.0.+-.30.5 .mu.l/min/g) and
the second 24 h period (223.7.+-.44.0 .mu.l/min/g) (FIG. 12). When
contrast dye was administered to animals pre-treated with
indomethacin and L-NAME, GFR declined from 230.8.+-.21.0
.mu.l/min/g to 87.6.+-.10.6 .mu.l/min/g in 24 h. Treatment with
SS-31 before and after dye administration reduced the decline in
renal function, and GFR declined only from 247.9.+-.20.4
.mu.l/min/g to 157.9.+-.26.6 .mu.l/min/g. The effect of SS-31 on
GFR following dye injection was significant (P<0.05 when
compared to the vehicle treated group). Six of nine SS-31 treated
rats were almost completely protected from the effects of the dye.
Thus, SS-31 reduced renal dysfunction caused by radiocontrast
dye.
[0268] PAS staining revealed control rat kidneys with normal
morphology, demonstrated by intact brush borders on proximal
tubules (FIG. 13A). Dye treatment resulted in a loss of the
characteristic renal brush border in proximal tubule cells, as well
as some vacuolization (FIG. 13B). These effects were attenuated
with SS-31 treatment, PAS staining in these samples revealed intact
brush borders and normal glomeruli (FIG. 13C). Thus, SS-31
protected renal tubules from radiocontrast dye injury.
[0269] The TUNEL stain was used to visualize apoptotic renal
tubules. Control kidneys showed few apoptotic cells/hpf (FIG. 14A).
Vehicle-treated, dye-injected kidneys had numerous apoptotic
cells/hpf (FIG. 14B). This effect was greatly attenuated in
SS-31-treated, dye injected kidneys (FIG. 14C). Thus, SS-31
prevented renal tubular apoptosis induced by radiocontrast dye
injury.
[0270] In summary, these results indicate that the peptide SS-31 is
effective in reducing renal dysfunction protecting renal tubules
from radiocontrast dye injury. As such, the aromatic-cationic
peptides of the invention are useful in methods of protecting a
subject from acute renal injury caused by contrast agents.
Experiment 2
[0271] Animal Model:
[0272] The same indomethacin and L-NAME model described above was
used in this experiment. In this model, impaired renal function was
induced by the administration of indomethacin (8 mg/kg) and L-NAME
(10 mg/kg). The animals were divided into 2 groups with the dosing
schedules and study protocol presented in FIG. 15.
[0273] Renal Function:
[0274] Renal function was assessed by determining serum and urinary
creatinine using DICT-kit from Bioassays Systems (Hayward, Calif.)
at baseline and 26 h following dye administration. Urinary protein
concentration was also determined at baseline and 26 h following
dye administration by BCA Protein Assay kit (Thermo Scientific,
Rockford, Ill.). Samples were analyzed by students t-test and
differences were considered significant at p<0.05.
[0275] Renal Histology:
[0276] Kidneys were fixed in 10% neutral-buffered formalin and
embedded in paraffin wax. Three micron sections were stained with
hematoxylin-eosin (H&E) and periodic acid-Schiff (PAS) and
analyzed by light microscopy by a board certified pathologist.
[0277] Results:
[0278] In accordance with the procedures just described, the
effects of the aromatic-cationic peptides of the invention in
protecting a subject from CIN were investigated in an animal model
of ARI caused by radiocontrast dye administration. The results are
as follows.
TABLE-US-00009 TABLE 9 Results of Renal Function Analysis Baseline
1 day A: Indo/L-Name/Dye/PBS No. of animals 9 7 Body Weight (g) 278
.+-. 2.5 263 .+-. 2.2 SCr (mg/dL) 0.61 .+-. 0.02 0.85 .+-. 0.07 Ucr
(mg/dL) 11.88 .+-. 3.11 58.97 .+-. 11.62 U vol (ml) N.A. N.A. U
protein (mg/ml) Bradford N.A. N.A. U protein (mg/ml) BCA 4.32 .+-.
1.65 21.72 .+-. 4.63 B: Indo/L-Name/Dye/SS-31 No. of animals 9 7
Body Weight (g) 278 .+-. 5 258 .+-. 5 SCr (mg/dL) 0.67 .+-. 0.09
0.73 .+-. 0.04 Ucr (mg/dL) 15.54 .+-. 3.87 38.12 .+-. 8.19 U vol
(ml) N.A. N.A. U protein (mg/ml) Bradford N.A. N.A. U protein
(mg/ml) BCA 4.28 .+-. 1.15 11.76 .+-. 2.71 Data presented were Mean
.+-. SEM
[0279] For the rats pre-treated with indomethacin and L-NAME, the
administration of contrast dye induced a serum creatinine increase
from the baseline level 0.61.+-.0.02 (mg/dL) to 0.85.+-.0.07
(mg/dL); and the urinary protein concentration increased from
4.32.+-.1.65 (mg/ml) to 21.72.+-.4.63 (mg/ml) at 26 h. This
represented a 40% Scr increase, and four-fold protein concentration
increase at 26 h post contrast dye administration. In contrast,
treatment with SS-31 before and after dye administration reduced
the decline in renal function, and the serum creatinine increased
only from the baseline level 0.67.+-.0.09 (mg/dL) to 0.73.+-.0.04
(mg/dL); and the urinary protein concentration increased from
4.28.+-.1.15 (mg/ml) to 11.76.+-.2.71 (mg/ml). This represented
only a 9% Scr increase, and 1.7-fold protein concentration increase
at 26 h post contrast dye administration. Five out of seven SS-31
treated rats were protected from the contrast dye induced
nephropathy (i.e., a Scr increase less than 25% at 26 h post
dye).
[0280] PAS staining revealed that dye treatment resulted in a loss
of the characteristic renal brush border in proximal tubule cells,
as well as some vacuolization (FIG. 16A). These effects were
attenuated with SS-31 treatment, PAS staining in these samples
revealed intact brush borders and normal glomeruli (FIG. 16B). FIG.
16C shows the control rat kidneys with normal morphology,
demonstrated by intact brush borders on proximal tubules. Thus,
SS-31 prevented renal tubular apoptosis induced by radiocontrast
dye injury.
[0281] In summary, these results indicate that the peptide SS-31 is
effective in reducing renal dysfunction protecting renal tubules
from radiocontrast dye injury. As such, the aromatic-cationic
peptides of the invention are useful in methods of protecting a
subject from acute renal injury caused by contrast agents.
Example 4. Treatment and Prevention of CIN in Diabetic Animal
Models
Experiment 1
[0282] Animal Model:
[0283] Impaired renal function caused by diabetes is one of the
major predisposing factors for contrast induced nephropathy
(McCullough, P et al., J. Am. College of Cardiology, 2008, 51,
1419-1428). In this experiment, a total of 57 SD rats were fed with
high fat diet for 6 weeks, followed by the injection with low dose
streptozotocin (30 mg/kg). After 9 more weeks, the blood glucose,
serum creatinine and Cystatin C was measured. Based on the
following selection criteria: Scr>250 M, Cystatin C>750 ng/ml
and blood glucose >=16.7 uM, 20 rats were selected to proceed
with the contrast induced nephropathy study.
[0284] The animals were divided into 2 groups. Group 1 rats were
fed administered iohexol and SS-31; Group 2 rats were administered
iohexol and a control vehicle. On day 1, serum samples were
collected from the rats in each group and total protein in urine
was measured using a Bradford assay. On days 2 and 3, 3 mg/kg SS-31
(Group 1) or a vehicle (Group 2) was administered s.c. 30 min prior
to the dye injection (6 mL/kg i.v. tail vein). SS-31 (Group 1) or
PBS (Group 2) administration was repeated at both 2 h and 24 h
post-dye administration. Serum and urine samples were collected at
days 4 and 5. The rats were euthanized on day 5 to harvest kidneys
and vital organs. Samples were analyzed by students t-test and
differences were considered significant at p<0.05.
[0285] Renal Function:
[0286] Renal function was assessed by determining serum and urinary
creatinine using Jiancheng Cr kit (Nanjing, P.R.C) at baseline, 48
h and 72 h following dye administration. The creatinine clearance
was calculated based on the Scr, Ucr and Urinary volume. Urinary
protein concentration was determined by Bradford Protein Assay kit
(Sigma, St. Louis, Mo.). The Cystatin C was measured Westang Rat
Cystatin C kit (Shanghai, P.R.C.)
[0287] Results:
[0288] In accordance with the procedures just described, the
effects of the aromatic-cationic peptides of the invention in
attenuating the CIN in a diabetic animal model were investigated.
The results are summarized in the Table 10 and FIG. 17A and FIG.
17B.
TABLE-US-00010 TABLE 10 Results of Renal Function Analysis Baseline
T1 T2 Group 1: HFD/STZ/ Iohexol/SS-31 No. of animals 10 Body
Weight(g) 478.5 .+-. 9.30 479.0 .+-. 8.5 474.9 .+-. 8.4 Scr(umol/L)
305.7 .+-. 15.1 285.4 .+-. 12.0 295.7 .+-. 20.6 Cystatin C(ng/ml)
708.6 .+-. 51.3 1008.1 .+-. 46.4 1303.6 .+-. 147.1 Ucr(.mu.mol/L)
3187.0 .+-. 196.9 2816 .+-. 163.6 2675.4 .+-. 100.8 U vol(ml) 15.4
.+-. 2.6 17.4 .+-. 1.6 20.5 .+-. 2.3 U protein(mg/4 h) 4.83 .+-.
1.03 4.71 .+-. 0.60 3.59 .+-. 1.0 GFR(.mu.l/min/100 g) 140.0 .+-.
11.1 149.3 .+-. 15.6 162.7 .+-. 18.7 U albumin(.mu.g/ml) 15.9 .+-.
2 17.6 .+-. 2.8 9.6 .+-. 3.3 U albumin(.mu.g/4 h) 241.5 .+-. 44.6
291.9 .+-. 46 187.2 .+-. 64.1 Group 2: HFD/STZ/ Iohexol/PBS No. of
animals 10 Body Weight(g) 489.3 .+-. 11.5 487.6 .+-. 11.5 482.2
.+-. 11.5 Scr(.mu.mol/L) 260.0 .+-. 12.0 257.2 .+-. 21.9 249.5 .+-.
18.8 Cystatin C(ng/ml) 655.9 .+-. 49.1 938.4 .+-. 57.9 1576.7 .+-.
149.9 Ucr(.mu.mol/L) 3367.5 .+-. 376.0 3498.0 .+-. 691.0 3079.4
.+-. 381.3 U vol(ml) 14.37 .+-. 2.0 13.2 .+-. 2.3 16.6 .+-. 2.8 U
protein(mg/4 h) 3.44 .+-. 0.75 3.04 .+-. 0.32 4.52 .+-. 1.0
GFR(.mu.l/min/100 g) 158.5 .+-. 18.6 153.6 .+-. 17.3 177.3 .+-.
23.0 U albumin(.mu.g/ml) 10.4 .+-. 1.4 22.4 .+-. 7.3 9 .+-. 2.2 U
albumin(.mu.g/4 h) 130.2 .+-. 15.6 184.7 .+-. 46.8 115.3 .+-.
17.7
[0289] When contrast dye was administered in diabetic rats, for the
vehicle treated group, the serum Cystatin C (an AKI biomarker)
level raised from 655.9.+-.49.1 (baseline) to 938.4.+-.57.9 (48 h
post dye) and 1576.7.+-.149.9 (ng/ml) at 72 h post dye
administration; whereas for the SS-31 treated group, the Cystatin C
only raised from 708.6.+-.51.3 to 1008.1.+-.46.4 and
1303.6.+-.147.1 (ng/ml) in the same period of time (FIG. 17A). At
72 hr, the effect of SS-31 in attenuating the increase of Cystatin
C was statistically significant (P<0.05).
[0290] When contrast dye was administered in diabetic rats, for the
vehicle treated group, the creatinine clearance decreased more than
20% at 48 h post the dye injection (FIG. 17B). In contrast, for the
SS-31 treated group, there was no significant change in creatinine
clearance before and after the dye injection in the same period of
time. The effect of SS-31 in attenuating the creatinine clearance
induced by dye administration was statistically significant
(P<0.05). Thus, SS-31 reduced renal dysfunction caused by
radiocontrast dye in a diabetic animal model. As such, the
aromatic-cationic peptides of the invention are useful in methods
of protecting a subject from acute renal injury caused by contrast
agents.
Experiment 2
[0291] In this experiment, 30 SD rats were fed with high fat diet
for 6 weeks, followed by the injection with low dose streptozotocin
(30 mg/kg). After 9 more weeks, the rats were used in the contrast
induced nephropathy study. The animals were divided into 2 groups
with the doses, dosing schedules and study protocol as follows. On
day 0, the weight of the animals was determined and glucose and
serum samples were collected. On day 1, Scr was assayed and spot
urine collection performed. On days 2 and 3, 3 mg/kg SS-31 (Group
1) or a vehicle (Group 2) was administered s.c. 30 min prior to the
dye injection (6 mL/kg i.v. tail vein). SS-31 (Group 1) or PBS
(Group 2) administration was repeated at 2 h, 1 day, 2, days, 3
days, 4 days, and 5 day post-dye administration. On day 8, serum
and urine samples were collected and the rats were euthanized to
harvest kidneys and vital organs.
[0292] Renal Function:
[0293] Renal function was assessed by determining serum and urinary
creatinine using Jiancheng Cr kit (Nanjing, P.R.C) at baseline, 24
h, 3 day and 6 days following contrast dye (Iohexol)
administration. The creatinine clearance was calculated based on
the Scr, Ucr and Urinary volume. Samples were analyzed by students
t-test and differences were considered significant at
p<0.05.
[0294] Results:
[0295] In accordance with the procedures just described, the
effects of the aromatic-cationic peptides of the invention in
attenuating the CIN in a diabetic animal model were investigated.
The results are presented in Table 11 and in FIG. 18.
TABLE-US-00011 TABLE 11 Results of Renal Function Analysis Baseline
T1 T2 T3 Group A: HFD/STZ/ Iohexol/SS-31 No. of animals 15 Body
weight (g) 541.9 .+-. 23.4 566.5 .+-. 20.3 565.6 .+-. 21 558.3 .+-.
20.1 Scr (.mu.mol/L) 214.6 .+-. 15.4 168.6 .+-. 21.9 236.2 .+-.
26.6 175.3 .+-. 25.9 Blood Glucose(mmol/L) 12.2 .+-. 2.4 19.9 .+-.
3.4 15.6 .+-. 2.4 Ucr (.mu.mol/L) 4182.5 .+-. 699.8 4488.9 .+-.
861.6 4744.8 .+-. 1350.6 4030.7 .+-. 620 U vol(ml) 18.1 .+-. 4.4
15.2 .+-. 4.3 12.8 .+-. 3.6 9.6 .+-. 2.3 U protein (mg/4 h) 5.4
.+-. 1.1 3.6 .+-. 0.8 4.3 .+-. 0.9 2.4 .+-. 0.4 GFR (.mu.l/min/100
g) 159.2 .+-. 25.1 159.9 .+-. 9 100.9 .+-. 16.7 121.5 .+-. 9.1
Group B: HFD/STZ/ Iohexol/PBS No. of animals 15 Body weight (g)
545.5 .+-. 21.8 561.8 .+-. 18.4 561.1 .+-. 19.3 554.6 .+-. 18.5 Scr
(.mu.mol/L) 217.4 .+-. 12.1 214.7 .+-. 33.6 253.7 .+-. 33 183.4
.+-. 28.8 Blood Glucose(mmol/L) 12.1 .+-. 2.2 19.1 .+-. 3.1 16.7
.+-. 2.3 Ucr (.mu.mol/L) 4584.4 .+-. 1270.8 4596.2 .+-. 699.2
4619.8 .+-. 693 3468.7 .+-. 521.2 U vol(ml) 20.5 .+-. 4.2 11.7 .+-.
3 8.8 .+-. 2 9.8 .+-. 2 U protein (mg/4 h) 6.5 .+-. 1.7 2.7 .+-.
0.4 2.8 .+-. 0.4 2.8 .+-. 0.6 GFR (.mu.l/min/100 g) 172 .+-. 16.1
125.2 .+-. 7.3 83.8 .+-. 7.5 125.4 .+-. 15.4
[0296] When contrast dye was administered in diabetic rats, for the
vehicle treated group, the creatinine clearance decreased more than
27% at 24 h post the dye injection from 172.+-.16.1 to 125.2.+-.7.3
(.mu.l/min/100 g) (FIG. 18). In contrast, for the SS-31 treated
group, there was no significant change in creatinine clearance
before and after the dye injection in the same period of time
159.2.+-.25.1 to 159.9.+-.9 (l/min/100 g). The effect of SS-31 in
attenuating the creatinine clearance induced by dye administration
was statistically significant (p<0.05). Thus, SS-31 reduced
renal dysfunction caused by radiocontrast dye in a diabetic animal
model. As such, the aromatic-cationic peptides of the invention are
useful in methods of protecting a subject from acute renal injury
caused by contrast agents.
Experiment 3
[0297] In this experiment, 20 SD rats were fed with high fat diet
for 6 weeks, followed by the injection with low dose streptozotocin
(35 mg/kg). After 15 more weeks, the rats were subjected to
contrast induced nephropathy study. The HFD/STZ animals were
divided into 2 groups with the doses, dosing schedules and study
protocol presented in FIG. 19. In addition, we included a normal
control group was included in which 9 rats from the same batch on
regular chow diet were subjected to the same protocol.
[0298] Renal Function:
[0299] Renal function was assessed by determining serum and urinary
creatinine using Jiancheng Cr kit (Nanjing, P.R.C) at baseline, 24
h, 3 day and 6 days following contrast dye (Iohexol)
administration. The creatinine clearance was calculated based on
the Scr, Ucr and urinary volume. Samples were analyzed by students
t-test and differences were considered significant at
p<0.05.
[0300] Results:
[0301] In accordance with the procedures just described, the
effects of the aromatic-cationic peptides of the invention in
attenuating the CIN in a diabetic animal model were investigated.
The results are presented in Table 12
TABLE-US-00012 TABLE 12 Serum Creatinine Results for Study Groups
Scr (.mu.M) Group No. Tb T22h % change T22 Group A: 1 30 30 0%
NPD/PBS/Dye/PBS 3 37 34 -8% 4 38 34 -11% 5 41 34 -17% 6 35 35 0% 7
37 33 -11% 8 35 38 9% 9 37 33 -11% Group B: 13 23 32 39%
HFD/STZ/PBS/Dye/PBS 20 21 27 29% 28 31 35 13% 30 21 27 29% 33 26 24
-8% 36 28 32 14% 41 27 25 -7% 46 24 29 21% 55 26 25 -4% 63 22 27
23% Group C: 34 25 25 0% HFD/STZ/SS- 38 25 26 4% 31/Dye/SS-31 45 27
24 -11% 60 23 25 9% 64 29 27 -7% 65 24 28 17% 67 25 30 20% 69 25 35
40% 72 29 19 -34%
[0302] From the above table, one can see that in the control
animals (group A), the Scr did not increase at 24 h post contrast
dye insult in any of the animals. For HFD/STZ rats, in the vehicle
treated group, 5 out 10 animals had elevated Scr more than 20% post
24 h contrast dye administration. In contrast, with the treatment
of SS-31, only 2 out 9 rats had an increase in Scr more than 20% in
the same period of time. Thus, SS-31 reduced renal dysfunction
caused by radiocontrast dye in a diabetic animal model. As such,
the aromatic-cationic peptides of the invention are useful in
methods of protecting a subject from acute renal injury caused by
contrast agents.
Example 5. Treatment and Prevention of CIN in a Glycerol-Induced
Rhabdomyolysis Animal Model
[0303] The effects of the aromatic-cationic peptides of the
invention in protecting and/or treating a subject from CIN were
investigated in a glycerol-induced rhabdomyolysis animal model of
ARI caused by radiocontrast dye administration. This Example
describes the results of such experiments.
Experiment 1
[0304] Animal Model:
[0305] Previous studies indicated that the administration of
contrast dye in animals with kidneys that were damaged by
glycerol-induced rhabdomyolysis can cause CIN (Parvez, Z et al.
Investigative Radiology, 1989, 24, 698-702; Duan et al. Acta
Radiologica, 2000, 41, 503-507). In this model, SD rats with body
weight of 300-400 g were dehydrated for 24 h followed by i.m.
injection of 25% glycerol solution (v/v) at the dose of 10 ml/kg.
Twenty-four hours (24 h) later, the rats were grouped and subjected
to a contrast induced nephropathy study protocol as detailed in
FIG. 20. The effects of SS-31 on ARI can be examined by comparing
the renal functions in animals from group C with those from group
B. Samples were analyzed by students t-test and differences were
considered significant at p<0.05.
[0306] Renal Function:
[0307] Renal function was assessed by determining serum and urinary
creatinine using Jiancheng Cr kit (Nanjing, P.R.C) at baseline, 24
h after dehydration and 48 h post the contrast dye administration.
The creatinine clearance was calculated based on the Scr, Ucr and
urinary volume. Urinary microalbumin concentration was determined
by a competition ELISA assay.
[0308] Results:
[0309] In accordance with the procedures just described, the
effects of aromatic-cationic peptides in attenuating the CIN in a
glycerol-induced rhabdomyolysis animal model were investigated. The
results are presented in FIG. 21 and FIG. 22.
[0310] When contrast dye was administered in glycerol induced
rhabdomyolysis rats, the creatinine clearance decreased 13% at 48 h
post the dye injection for the vehicle treated group (FIG. 21). In
contrast, there was no significant change in creatinine clearance
before and after the dye injection in the same period of time for
the SS-31 treated group (FIG. 21A).
[0311] Albuminuria is an indicator of increased permeability of the
glomerular membrane, and it can be induced by contrast dye. After
24 h dehydration (TO), the urinary microalbumin level was
385.1.+-.59.8 (g/24 h) for Gly/Dye/PBS group and 757.7.+-.462.9
(g/24 h) for Gly/Dye/SS-31 group (no peptide treatment at this time
point) (FIG. 21B). With the i.m. administration of glycerol, 24 h
post injection (T1), the urinary microalbumin level increased
substantially in both groups to due to the glycerol-induced
rhabdomyolysis. However, at T2 time point, the total albuminuria in
the SS-31 treated group decreased significantly compared to the
vehicle treated group, which suggests a protective effect for SS-31
on the permeability of the glomerular basement membrane caused by
the contrast dye in this glycerol-induced rhabdomyolysis model.
[0312] PAS staining revealed the dye treatment resulted in a loss
of the characteristic renal brush border in proximal tubule cells,
swelling glomeruli and marked protein cast deposition in the renal
tubule cells (FIG. 22A). These effects were attenuated with SS-31
treatment because PAS staining in these samples revealed intact
brush borders, normal glomeruli and minimum protein cast in the
tubular cells (FIG. 22B). Thus, SS-31 protected renal tubules from
radiocontrast dye injury.
Experiment 2
[0313] SD rats with body weight of 300-400 grams were dehydrated
for 24 h followed by i.m. injection with 25% glycerol solution
(v/v) at the dose of 10 ml/kg. Twenty-four hours later, the rats
were grouped and subjected to contrast induced nephropathy study
protocol. Sixteen SD rats with body weight of 300-350 g were
randomized into three groups with the doses, dosing schedules and
study protocol presented in FIG. 23. The effects of SS-31 on ARI
can be examined by comparing the renal functions in animals from
group A with that from group B. Samples were analyzed by Student's
t-test and differences were considered significant at
p<0.05.
[0314] Renal Function:
[0315] Renal function was assessed by determining serum and urinary
creatinine using Jiancheng Cr kit (Nanjing, P.R.C) at baseline, 24
h after dehydration and 48 h post the contrast dye administration.
The creatinine clearance was calculated based on the Scr, Ucr and
urinary volume. Urinary protein concentration was determined by
Bradford Protein Assay kit (Sigma, St. Louis, Mo.).
[0316] Results:
[0317] In accordance with the procedures just described, the
effects of the aromatic-cationic peptides of the invention in
attenuating the CIN in a glycerol-induced rhabdomyolysis animal
model were investigated. The results are presented in FIGS. 24, 25,
and 26.
[0318] When contrast dye was administered in glycerol-induced
rhabdomyolysis rats, the creatinine clearance decreased 25% at 48 h
post the dye injection for the vehicle treated group (FIG. 24). In
contrast, the reduction of creatinine clearance was 13% in the same
period of time for the SS-31 treated group. Thus, SS-31 reduced
renal dysfunction caused by radiocontrast dye in a glycerol induced
rhabdomyolysis animal model.
[0319] Proteinuria is a sign of renal damage, and the presence of
excess protein in urine indicates either an insufficiency of
absorption or impaired filtration. It was previously reported that
glycerol and contrast media can increase the permeability of the
glomerular membrane and cause excessive amount of urinary protein
leaking (Thomsen H. S. et al., Acta Radiologica, 1989, 30,
217-222). In the vehicle treated group, the 24 h urinary protein
level at each different time point was: Tb (baseline)=15.8.+-.1.6
mg; TO (24 h post glycerin)=52.9.+-.9.1 mg; T1 (48 h post glycerin
and 24 h post dye)=54.1.+-.5.8 mg, T2 (72 h post glycerin and 48 h
post dye)=29.7.+-.3.6 mg; and T3 (96 h post glycerin and 72 h post
dye)=30.2.+-.5.1 mg (FIG. 25).
[0320] In the SS-31 treatment group, the 24 h urinary protein level
at Tb (15.5.+-.1.5 mg) and TO (50.2.+-.8.6 mg) was comparable to
the vehicle control group, when there was no peptide administration
at these time points. However, at the time point of T1 (48 h post
glycerin and 24 hrs post dye), the 24 h urinary protein in the
SS-31 treated group was reduced to 39.5.+-.3.5 mg (FIG. 25).
Compared to the vehicle treated group at the same time point, the
difference was statistically significant (p<=0.05). This
demonstrated that SS-31 peptide accelerated the recovery of the
glomerular basement membrane permeability dysfunction in this CIN
animal model. Thus, the aromatic-cationic peptides of the invention
are useful in methods for treating ARI caused by radiocontrast
dye.
[0321] PAS staining revealed the dye treatment resulted in a loss
of the characteristic renal brush border in proximal tubule cells,
swelling glomeruli and marked protein cast deposition in the renal
tubule cells (FIG. 26A). These effects were attenuated with SS-31
treatment because PAS staining in these samples revealed intact
brush borders, normal glomeruli and minimum protein cast in the
tubular cells (FIG. 26B). Thus, SS-31 protected renal tubules from
radiocontrast dye injury.
Experiment 3
[0322] SD rats with body weight of 300-400 grams were dehydrated
for 24 h followed by i.m. injection with 25% glycerol solution
(v/v) at the dose of 10 ml/kg. Twenty-four hours (24 h) later, the
rats were grouped and subjected to contrast induced nephropathy
study protocol. Eighteen SD rats with body weight of 350-400 g were
randomized into three groups with the doses, dosing schedules and
study protocol presented in FIG. 27. The effects of SS-31 on ARI
can be examined by comparing the renal functions in animals from
group A with that from group B.
[0323] Renal Function:
[0324] Renal function was assessed by determining serum and urinary
creatinine using Jiancheng Cr kit (Nanjing, P.R.C) at baseline, 24
h after dehydration and 48 h post the contrast dye administration.
The creatinine clearance was calculated based on the Scr, Ucr and
Urinary volume. Urinary protein concentration was determined by
Bradford Protein Assay kit (Sigma, U.S.A.). Samples were analyzed
by students t-test and differences were considered significant at
p<0.05.
[0325] Results:
[0326] In accordance with the procedures just described, the
effects of the aromatic-cationic peptides of the invention in
attenuating the CIN in a glycerol-induced rhabdomyolysis animal
model were investigated. The results are presented in FIG. 28 and
FIG. 29.
[0327] When contrast dye was administered in glycerol-induced
rhabdomyolysis rats, for the vehicle treated group, the creatinine
clearance decreased 24% at 48 h post the dye injection (FIG. 28).
In contrast, for the SS-31 treated group, there was no significant
change in the creatinine clearance in the same period of time (FIG.
28). Thus, SS-31 reduced renal dysfunction caused by radiocontrast
dye in a glycerol-induced rhabdomyolysis animal model.
[0328] As discussed above, proteinuria is a sign of renal damage,
and the presence of excess protein in urine indicates either an
insufficiency of absorption or impaired filtration. Comparing the
protein level at different time point in the vehicle treated group
with the SS-31 treated group, the peptide treated group recovered
faster than the vehicle group (FIG. 29). This demonstrated that
SS-31 peptide accelerated the recovery of the glomerular basement
membrane permeability dysfunction in this CIN animal model. Thus,
the aromatic-cationic peptides of the invention are useful in
methods for the treatment of ARI caused by radiocontrast dye.
[0329] PAS staining revealed the dye treatment resulted in a loss
of the characteristic renal brush border in proximal tubule cells,
swelling glomeruli and marked protein cast deposition in the renal
tubule cells (FIG. 30A). These effects were attenuated with SS-31
treatment because PAS staining in these samples revealed intact
brush borders, normal glomeruli and minimum protein cast in the
tubular cells (FIG. 30B). Thus, SS-31 protected renal tubules from
radiocontrast dye injury.
[0330] In summary, the results from the above examples clearly
demonstrated the usefulness of the aromatic-cationic peptides of
the invention in protecting a subject from acute kidney injury
caused by contrast agents in several animal models. In addition, it
accelerated the recovery of the injured kidneys evidenced in
Experiment 7 and 8.
Example 6: Treatment and Prevention of Nephrotoxicity in the
CCl.sub.4 Chronic Kidney Injury Model
[0331] Animal Model:
[0332] Generation of reactive radicals has been implicated in
carbon tetrachloride-induced nephrotoxicity, which are involved in
lipid peroxidation, accumulation of dysfunctional proteins, leading
to injuries in kidneys (Ozturk, F. et al. Urology, 2003, 62,
353-356). This Example describes the effect of administration of
aromatic-cationic peptides for the prevention of carbon
tetrachloride (CCl.sub.4)-induced chronic nephrotoxicity.
[0333] Study Design and Experimental Protocol:
[0334] In this model, SD rats with body weight of 250 g were fed
with 0.35 g/L phenobarbital solution (Luminal water) for two weeks,
then divided into three groups and treated as follows.
TABLE-US-00013 TABLE 13 Treatment Groups # of Inducing Agent Dose
Vol. Dose Group rats For 7 weeks Compound (ml/kg) (mg/kg) Schedule
Vehicle 1 9 Luminal water + PBS only 2 N/A 5 days a PBS Naive Olive
oil i.g., s.c. week, q.d., 2 ml/kg control 1 ml/kg, twice for 7 wks
per week 2 12 Luminal water + PBS only 2 N/A 5 days a PBS CCl4 50%
CCl.sub.4 i.g., s.c. week, q.d., control 2 ml/kg, twice for 7 wks
per wk 3 12 Luminal water + SS-31 2 10 mg/kg 5 days a PBS SS-31 50%
CCl.sub.4 i.g., s.c. week, q.d., 2 ml/kg, twice for 7 wks per
wk
[0335] At the end of fifth week, four rats from each group were
sacrificed for liver histopathological sectioning and fibrosis
examination. In the end of 7th week, all remaining rats in each
group were sacrificed, and kidney and liver tissues were harvest
for histopathological sectioning and examination.
[0336] Renal Histology:
[0337] Kidneys were fixed in 10% neutral-buffered formalin and
embedded in paraffin wax. Three micron sections were stained with
hematoxylin-eosin (H&E) and analyzed by light microscopy by a
certified pathologist.
[0338] Results:
[0339] In accordance with the procedures described above, the
effects of the aromatic-cationic peptides of the invention in
protecting against CCl.sub.4 induced chronic nephrotoxcity were
investigated. The results are presented in Table 14 below:
TABLE-US-00014 TABLE 14 Summary of Renal Histology Results Tubular
epithelial cell Tubular epithelial Glomeruli degeneration cell
necrosis Naive group 5/5 - 5/5 - 5/5 - CCl.sub.4 group 6/6 - 4/6 ++
2/6 ++ 2/6 + 1/6 + 3/6 - CCl.sub.4 group + 5/5 - 5/5 - 5/5 - SS-31
Note: "-" = No abnormal findings; ".+-." = Minimal; "+" = Slight;
"++" = Moderate; "+++" = severe
[0340] SS-31 Protected Renal Tubules from CCl.sub.4
Nephrotoxicity.
[0341] H&E staining revealed the CCl.sub.4 treatment resulted
in tubular epithelial cell degeneration and necrosis (FIG. 31A); in
contrast, the renal histology of SS-31 treated animals (FIG. 31B)
showed no observable histopathological change, and it was almost
identical to the naive control animal (FIG. 31C).
[0342] Thus, the results from the above example demonstrated the
usefulness of the aromatic-cationic peptides of the invention in
protecting a subject from chronic kidney injury caused by CCl.sub.4
nephrotoxicity.
Example 7. Prevention of ARI Caused by Cisplatin
[0343] The effects of the aromatic-cationic peptides of the
invention in protecting a subject from cisplatin-induced ARI were
investigated in an animal model of ARI caused by cisplatin.
[0344] Sprague-Dawley rats (350-400 g) were given a single dose of
cisplatin (7 mg/kg) intraperitoneally on Day 1. One group of rats
(n=8) received one dose of SS-31 (3 mg/kg) subcutaneously just
prior to cisplatin administration, and repeated once daily for 3
more days. A second group of rats (n=8) A second group of rats
(n=8) received an equal volume of saline on the day of cisplatin
administration and for 3 more days after.
[0345] Experimental Protocol:
[0346] Rats had free access to tap water and standard rat chow
prior to cisplatin administration. Control blood samples were
obtained from the tail vein before cisplatin administration. Saline
or SS-31 (dissolved in saline) was administered to the rats
subcutaneously prior to cisplatin injection. Cisplatin (dissolved
in saline) was administered at a dose of 7 mg/kg intraperitoneally.
All rats then received either saline or SS-31 (3 mg/kg) daily for 3
days. Rat were placed in metabolic cages for the last 24 hours for
urine collection. At the end of this period, a blood sample was
withdrawn from the tail vein. After this, the animals were
anesthetized, kidneys were removed, and the animals euthanized.
[0347] Renal Function:
[0348] Renal function was assessed by blood urea nitrogen (BUN),
serum creatinine, urine creatinine, and urine protein (Beckman ALX
Chemical Analyzer). GFR was estimated from creatinine clearance
which was determined from urinary creatinine, urine flow rate, and
serum creatinine.
[0349] Renal Histology:
[0350] Kidneys were fixed in 10% neutral-buffered formalin and
embedded in paraffin wax. Three micron sections were stained with
periodic acid-Schiff (PAS) and analyzed by light microscopy.
[0351] Results:
[0352] In accordance with the procedures just described, the
effects of the aromatic-cationic peptides of the invention in
protecting a subject from cisplatin nephrotoxicity were
investigated in an animal model of ARI caused by cisplatin
administration. Between group comparisons were determined using the
Student's t-test.
[0353] In the saline treated rats, there was a significant
reduction in body weight 3 days after cisplatin administration
(333.+-.12.6 g) when compared to body weight before cisplatin
administration (378.4.+-.8.2 g) (p<0.0001) (FIG. 32A). Body
weight in the SS-31 treatment group was similar to saline group
before cisplatin treatment (376.9.+-.5.2 g; p=0.673), and body
weight did not change 3 days after cisplatin treatment (384.+-.28
g; p=0.46). There was a significant difference in the effect of
cisplatin treatment on body weight in the SS-31 treatment group
compared to saline (p=0.004). Kidney weight was also higher in the
SS-31 treated group (1.6.+-.0.2 g) compared to the saline treated
group (1.4.+-.0.2 g) (p=0.036) (FIG. 32B).
[0354] In the saline treated rats, serum creatinine increased from
0.4.+-.0.09 mg/dl to 2.5.+-.2.8 mg/dl) 3 days after cisplatin
treatment (p=0.065) (FIG. 32C). In contrast, serum creatinine only
increased from 0.4.+-.0.09 mg/dl to 0.8.+-.0.73 mg/dl in the SS-31
treatment group. The variation among rats was too large to reach
statistical significance. However, 5 out of 8 rats that received
SS-31 had no change in serum creatinine (<0.4 mg/dl), whereas
only one out of 8 saline rats had serum creatinine less than 1.0
mg/dl. Creatinine clearance was significantly higher in the SS-31
treated rats 3 days after cisplatin treatment (2.2.+-.1.3 ml/min)
compared to the saline group (0.5.+-.0.6 ml/min) (p=0.024) (FIG.
32E).
[0355] In the saline treated rats, BUN increased from 17.3.+-.1.5
mg/dl to 114.4.+-.105 mg/dl) 3 days after cisplatin treatment
(p=0.035) (FIG. 32D). In contrast, BUN only increased from
15.5.+-.1.7 mg/dl to 29.3.+-.25 mg/dl in the SS-31 treatment group
(p=0.167). BUN was significantly lower in the SS-31 treated group 3
days after cisplatin treatment (p=0.042).
[0356] In summary, SS-31 protected kidneys from damage caused by
cisplatin. As such, the aromatic-cationic peptides of the invention
are useful in methods of protecting a subject from acute renal
injury caused by nephrotoxic agents.
Example 8. Protection and Treatment of ALF by Aromatic-Cationic
Peptides
[0357] To demonstrate the effects on prevention and/or treatment of
ALF, the aromatic-cationic peptides of the invention are tested on
animal models of ALF. Suitable animal models induce ALF in
experimental animals using surgical procedures, toxic liver injury,
or a combination of both (See Belanger and Butterworth, "Acute
Liver Failure: A Critical Appraisal of Available Animal Models."
Metabolic Brain Disease, 20:409-423 (2005)).
[0358] To test the effect of the aromatic-cationic peptides of the
invention on the prevention of ALF, the peptides are administered
prior to or simultaneously with the drug or surgical insult. A
comparison of hepatic function following the insult is made between
subjects who received the peptides and those subjects who did not
receive the peptides. Hepatic function is assessed using one or
more indicators such as levels of serum hepatic enzymes
(transaminases, alkaline phosphatase), serum bilirubin, serum
ammonia, serum glucose, serum lactate, or serum creatinine.
Efficacy of the aromatic-cationic peptides of the invention in
preventing ALF is indicated by a reduction in the occurrence or
severity of the ALF (indicated by the markers above) as compared to
control subjects.
[0359] To test the effect of the aromatic-cationic peptides of the
invention on the treatment of ALF, the peptides are administered
following the drug or surgical insult used to induce the ALF in the
animal model. Following a course of treatment (ranging from several
hours to several days), a comparison of hepatic function is made
between subjects who received the peptides and those subjects which
did not receive the peptides. Hepatic function is assessed using
one or more indicators such as levels of serum hepatic enzymes
(transaminases, alkaline phosphatase), serum bilirubin, serum
ammonia, serum glucose, serum lactate, or serum creatinine.
Efficacy of the aromatic-cationic peptides of the invention in
treating ALF is indicated by a reduction in one or more signs or
symptoms of ALF (indicated by the markers above) as compared to
control subjects.
EQUIVALENTS
[0360] The present invention is not to be limited in terms of the
particular embodiments described in this application, which are
intended as single illustrations of individual aspects of the
invention. Many modifications and variations of this invention can
be made without departing from its spirit and scope, as will be
apparent to those skilled in the art. Functionally equivalent
methods and compositions within the scope of the invention, in
addition to those enumerated herein, will be apparent to those
skilled in the art from the foregoing descriptions. Such
modifications and variations are intended to fall within the scope
of the appended claims. The present invention is to be limited only
by the terms of the appended claims, along with the full scope of
equivalents to which such claims are entitled. It is to be
understood that this invention is not limited to particular
methods, reagents, compounds compositions or biological systems,
which can, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to be limiting.
[0361] In addition, where features or aspects of the disclosure are
described in terms of Markush groups, those skilled in the art will
recognize that the disclosure is also thereby described in terms of
any individual member or subgroup of members of the Markush
group.
[0362] As will be understood by one skilled in the art, for any and
all purposes, particularly in terms of providing a written
description, all ranges disclosed herein also encompass any and all
possible subranges and combinations of subranges thereof. Any
listed range can be easily recognized as sufficiently describing
and enabling the same range being broken down into at least equal
halves, thirds, quarters, fifths, tenths, etc. As a non-limiting
example, each range discussed herein can be readily broken down
into a lower third, middle third and upper third, etc. As will also
be understood by one skilled in the art all language such as "up
to," "at least," "greater than," "less than," and the like, include
the number recited and refer to ranges which can be subsequently
broken down into subranges as discussed above. Finally, as will be
understood by one skilled in the art, a range includes each
individual member. Thus, for example, a group having 1-3 cells
refers to groups having 1, 2, or 3 cells. Similarly, a group having
1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so
forth.
[0363] Other embodiments are set forth within the following claims.
Sequence CWU 1
1
2120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1ctgaagaaga ttgcgcagaa 20220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2atggcataaa ttcccactgc 20
* * * * *