U.S. patent application number 16/841183 was filed with the patent office on 2020-10-15 for methods for the prevention and treatment of burn injuries and secondary complications.
This patent application is currently assigned to The General Hospital Corporation d/b/a Massachusetts General Hospital, The General Hospital Corporation d/b/a Massachusetts General Hospital. The applicant listed for this patent is The General Hospital Corporation d/b/a Massachusetts General Hospital, The General Hospital Corporation d/b/a Massachusetts General Hospital. Invention is credited to Jeevendra A. Martyn, Laurence Rahme, Ronald G. Tompkins, A. Aria Tzika, Yong-Ming Yu.
Application Number | 20200323945 16/841183 |
Document ID | / |
Family ID | 1000004925910 |
Filed Date | 2020-10-15 |
![](/patent/app/20200323945/US20200323945A1-20201015-C00001.png)
![](/patent/app/20200323945/US20200323945A1-20201015-C00002.png)
![](/patent/app/20200323945/US20200323945A1-20201015-C00003.png)
![](/patent/app/20200323945/US20200323945A1-20201015-C00004.png)
![](/patent/app/20200323945/US20200323945A1-20201015-D00001.png)
![](/patent/app/20200323945/US20200323945A1-20201015-D00002.png)
![](/patent/app/20200323945/US20200323945A1-20201015-D00003.png)
![](/patent/app/20200323945/US20200323945A1-20201015-D00004.png)
![](/patent/app/20200323945/US20200323945A1-20201015-D00005.png)
![](/patent/app/20200323945/US20200323945A1-20201015-D00006.png)
![](/patent/app/20200323945/US20200323945A1-20201015-D00007.png)
View All Diagrams
United States Patent
Application |
20200323945 |
Kind Code |
A1 |
Tompkins; Ronald G. ; et
al. |
October 15, 2020 |
METHODS FOR THE PREVENTION AND TREATMENT OF BURN INJURIES AND
SECONDARY COMPLICATIONS
Abstract
The disclosure relates to methods for treating a subject
suffering from a burn injury or associated complications by
administering to the subject an effective amount of an
aromatic-cationic peptide. For example, a burn injury may be
associated with distant pathophysiological effects, such as
hypermetabolism, skeletal muscle dysfunction, and organ damage. The
disclosure also relates to methods for protecting a subject from a
burn injury by administering an effective amount of an
aromatic-cationic peptide to a subject at risk of a burn
injury.
Inventors: |
Tompkins; Ronald G.;
(Boston, MA) ; Tzika; A. Aria; (Jamaica Plain,
MA) ; Yu; Yong-Ming; (Cambridge, MA) ; Rahme;
Laurence; (Jamaica Plain, MA) ; Martyn; Jeevendra
A.; (Winchester, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The General Hospital Corporation d/b/a Massachusetts General
Hospital |
Boston |
MA |
US |
|
|
Assignee: |
The General Hospital Corporation
d/b/a Massachusetts General Hospital
Boston
MA
|
Family ID: |
1000004925910 |
Appl. No.: |
16/841183 |
Filed: |
April 6, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15266522 |
Sep 15, 2016 |
|
|
|
16841183 |
|
|
|
|
14061370 |
Oct 23, 2013 |
9457057 |
|
|
15266522 |
|
|
|
|
12727647 |
Mar 19, 2010 |
|
|
|
14061370 |
|
|
|
|
61162060 |
Mar 20, 2009 |
|
|
|
61249658 |
Oct 8, 2009 |
|
|
|
61258533 |
Nov 5, 2009 |
|
|
|
61259349 |
Nov 9, 2009 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/0014 20130101;
A61K 9/0019 20130101; A61K 38/07 20130101 |
International
Class: |
A61K 38/07 20060101
A61K038/07; A61K 9/00 20060101 A61K009/00 |
Claims
1.-20. (canceled)
21. A method for treating a subject suffering from a systemic
injury or condition secondary to a burn, the method comprising
administering to the subject in need thereof an effective amount of
a peptide having the formula
D-Arg-2'6'-dimethyltyrosine-Lys-Phe-NH.sub.2.
22. The method of claim 21, wherein the systemic injury is organ
damage, organ dysfunction or organ failure.
23. The method of claim 22, wherein the peptide is administered
following a burn injury but prior to the onset of symptoms of organ
dysfunction or organ failure.
24. The method of claim 22, wherein the peptide is administered
following the onset of symptoms of organ dysfunction or organ
failure.
25. The method of claim 22, wherein the organ dysfunction is
reduced myocardial contractility.
26. The method of claim 22, wherein the organ dysfunction, organ
damage or organ failure is liver dysfunction, liver damage or liver
failure.
27. The method of claim 22, wherein the organ dysfunction, organ
damage or organ failure is kidney dysfunction, kidney damage or
kidney failure.
28. The method of claim 22, wherein the organ dysfunction or organ
damage is bowel dysfunction or bowel damage.
29. The method of claim 22, wherein the organ dysfunction or organ
damage is lung dysfunction or lung damage.
30. The method of claim 21, wherein the systemic injury is
hypermetabolism.
31. The method of claim 30, wherein the peptide is administered
following a burn injury but prior to the onset of symptoms of
hypermetabolism.
32. The method of claim 30, wherein the peptide is administered
following the onset of symptoms hypermetabolism.
33. The method of claim 30, wherein the peptide in administered to
provide prophylaxis for hypermetabolism.
34. The method of claim 21, wherein the subject is a human.
35. The method of claim 21, wherein the peptide is administered
orally, subcutaneously, transdermally, intraperitoneally, or
topically.
36. The method of claim 21, wherein the peptide is administered
intravenously.
37. The method of claim 22, wherein the peptide is administered
intravenously.
38. The method of claim 30, wherein the peptide is administered
intravenously.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 61/259,349, filed Nov. 9, 2009, U.S. Provisional
Application No. 61/258,533, filed Nov. 5, 2009, U.S. Provisional
Application No. 61/249,658, filed Oct. 8, 2009, and U.S.
Provisional Application No. 61/162,060, filed Mar. 20, 2009, the
entire contents of which are hereby incorporated by reference in
their entirety.
TECHNICAL FIELD
[0002] The present technology relates generally to methods of
preventing or treating burn injuries and associated complications
by administration of an aromatic-cationic peptide.
BACKGROUND
[0003] The following description is provided to assist the
understanding of the reader. None of the information provided or
the references cited are admitted to be prior art to the present
invention.
[0004] Burn trauma causes approximately two million injuries,
100,000 hospital admissions, and 10,000 deaths every year in the
United States. In the past, many victims did not survive the
initial resuscitation period. Current survival rates and clinical
outcomes have progressively improved with the advent of aggressive
burn wound excision techniques, graft therapy, and superior
intensive care facilities, along with a better understanding of
post-burn physiological factors and fluid requirements.
[0005] Systemic injury, such as the dysfunction or failure of an
organ secondary to a severe burn injury and which is not
attributable to the burn injury, remains a continuing source of
morbidity and mortality. A severe burn is associated with release
of inflammatory mediators which ultimately cause local and distant
pathophysiological effects. Mediators including Reactive Oxygen
Species (ROS) and Reactive Nitrogen Species (RNS) are increased in
affected tissue, which are implicated in pathophysiological events
observed in burn patients. Free radicals have been found to have
beneficial effects on antimicrobial action and wound healing.
However, following a burn, there is an enormous production of ROS
which is harmful and implicated in inflammation, systemic
inflammatory response syndrome, immunosuppression, infection and
sepsis, tissue damage and multiple organ failure. Thus, clinical
response to burn is dependent on the balance between production of
free radicals and its detoxification.
SUMMARY
[0006] In one aspect, the present disclosure provides methods for
treating a subject suffering from a burn injury. The methods
include administering to the subject an effective amount of an
aromatic-cationic peptide. The aromatic-cationic peptic may 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 3
p.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 D-Arg-Dmt-Lys-Phe-NH.sub.2 (SS-31).
[0007] In another aspect, the present disclosure provides a method
of protecting a subject against the secondary effects of a burn
injury. In one embodiment, peptide is administered to a subject
following a burn injury to treat or ameliorate the incidence of
hypermetabolism. In one embodiment, peptide is administered to a
subject following burn injury to treat or ameliorate damage to the
liver, which occurs secondary to the burn injury.
[0008] In another aspect, the present disclosure provides a method
for protecting a subject against the primary effects of a burn by
administering an effective amount of an aromatic-cationic peptide
prior to exposure of the subject to an agent capable of causing a
burn, e.g., sunlight (UV), thermal radiation, or radiation
associated with radiotherapy. For example, the peptide may be
administered topically to a subject at risk for receiving a
burn.
[0009] In one embodiment, the systemic injury is organ dysfunction
or failure, such as organ dysfunction or failure that affects one
or more of the lung, liver, kidneys, or bowel. In one embodiment,
the peptide is administered following a burn injury but prior to
the onset of symptoms of organ dysfunction or failure. In another
embodiment, the peptide is administered following the onset of
symptoms of organ dysfunction or failure.
[0010] In one embodiment, the systemic injury is hypermetabolism.
In one embodiment, the peptide is administered following a burn
injury, but prior to the onset of symptoms of hypermetabolism. In
another embodiment, the peptide is administered following the onset
of symptoms hypermetabolism.
[0011] In one embodiment, the systemic injury is skeletal muscle
dysfunction, such as skeletal muscle wasting and cachexia. In one
embodiment, the peptide is administered following a burn injury,
but prior to the onset of symptoms of skeletal muscle dysfunction.
In another embodiment, the peptide is administered following the
onset of symptoms skeletal muscle dysfunction.
[0012] In one aspect, the present disclosure provides methods for
increasing ATP synthesis rate in a mammalian tissue, the method
comprising administering to a subject an effective amount of an
aromatic-cationic peptide. In one embodiment, the aromatic-cationic
peptide is a peptide having the formula
D-Arg-2'6'-dimethyltyrosine-Lys-Phe-NH.sub.2.
[0013] In one embodiment, following administration of the peptide,
the ATP synthesis rate in the mammalian tissue is increased
compared to a control tissue. In one embodiment, the control tissue
is tissue from a mammalian subject not administered the peptide. In
one embodiment, increasing the ATP synthesis rate is by recovery of
the mitochondrial redox status. In one embodiment, increasing the
ATP synthesis rate is by increasing the expression or activity of
the peroxisome proliferator activated receptor-gamma
coactivator-1.beta. (PGC-1.beta.) protein.
[0014] In one aspect, the present disclosure provides methods for
treating a disease or condition characterized by a reduced ATP
synthesis rate, the method comprising administering to a mammal in
need thereof an effective amount of an aromatic-cationic peptide.
In one embodiment, the aromatic-cationic peptide is a peptide
having the formula D-Arg-2'6'-dimethyltyrosine-Lys-Phe-NH.sub.2. In
one embodiment, the disease or condition is a burn injury.
[0015] In one embodiment, the peptide is defined by formula I:
##STR00001##
wherein R.sup.1 and R.sup.2 are each independently selected
from
[0016] (i) hydrogen;
[0017] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
##STR00002##
R.sup.1, R.sup.2, 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
[0018] (i) hydrogen;
[0019] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
[0020] (iii) C.sub.1-C.sub.6 alkoxy;
[0021] (iv) amino;
[0022] (v) C.sub.1-C.sub.4 alkylamino;
[0023] (vi) C.sub.1-C.sub.4 dialkylamino;
[0024] (vii) nitro;
[0025] (viii) hydroxyl;
[0026] (ix) halogen, where "halogen" encompasses chloro, fluoro,
bromo, and iodo; and n is an integer from 1 to 5.
[0027] 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.
[0028] In one embodiment, the peptide is defined by formula II:
##STR00003##
wherein R.sup.1 and R.sup.2 are each independently selected
from
[0029] (i) hydrogen;
[0030] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
##STR00004##
R.sup.3 and R.sup.4 are each independently selected from
[0031] (i) hydrogen;
[0032] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
[0033] (iii) C.sub.1-C.sub.6 alkoxy;
[0034] (iv) amino;
[0035] (v) C.sub.1-C.sub.4 alkylamino;
[0036] (vi) C.sub.1-C.sub.4 dialkylamino;
[0037] (vii) nitro;
[0038] (viii) hydroxyl;
[0039] (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
[0040] (i) hydrogen;
[0041] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
[0042] (iii) C.sub.1-C.sub.6 alkoxy;
[0043] (iv) amino;
[0044] (v) C.sub.1-C.sub.4 alkylamino;
[0045] (vi) C.sub.1-C.sub.4 dialkylamino;
[0046] (vii) nitro;
[0047] (viii) hydroxyl;
[0048] (ix) halogen, where "halogen" encompasses chloro, fluoro,
bromo, and iodo; and n is an integer from 1 to 5.
[0049] 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.
BRIEF DESCRIPTION OF THE FIGURES
[0050] FIG. 1 is a flow chart showing the protocol and dosing
schedule for the study presented in Example 1.
[0051] FIG. 2 is a graph showing the VCO.sub.2 concentration over
time of an exemplary rat that has been burned and administered the
SS-31 peptide.
[0052] FIG. 3 is a flowchart showing the protocol and dosing
schedule for the study presented in Example 2.
[0053] FIG. 4 is a series of micrographs showing sections of liver
tissue from the various treatment groups of Example 2.
[0054] FIG. 5 is photograph of a western blot analysis of caspase-3
cleavage from the various treatment groups of Example 2.
[0055] FIG. 6 is a graph showing caspase-3 activity from the
various treatment groups of Example 2.
[0056] FIG. 7 is a photograph of a western blot analysis showing
protein oxidation from the various treatment groups of Example
2.
[0057] FIG. 8 is a chart showing the wound size comparison between
untreated and SS-31 peptide treated groups.
[0058] FIG. 9 is a graph showing the reduction of the nitroxide in
the gastrocnemius muscle before and after a burn injury.
[0059] FIG. 10 is a graph showing the reduction in nitroxide in the
gastrocnemius at 0 h after a burn injury in subjects administered
saline or SS-31 peptide.
[0060] FIG. 11 is a graph showing the reduction in nitroxide in the
gastrocnemius at 3 h after a burn injury in subjects administered
saline or SS-31 peptide.
[0061] FIG. 12 is a graph showing the reduction in nitroxide in the
gastrocnemius at 6 h after a burn injury in subjects administered
saline or SS-31 peptide.
[0062] FIG. 13 is a graph showing the reduction in nitroxide in the
gastrocnemius at 24 h after a burn injury in subjects administered
saline or SS-31 peptide.
[0063] FIG. 14 is a graph showing the reduction in nitroxide in the
gastrocnemius at 48 h after a burn injury in subjects administered
saline or SS-31 peptide.
[0064] FIG. 15 is a graph of ATP synthesis rate (.mu.mol/g/s) in
control (C), control+SS-31 peptide (C+P), burned (B), and
burned+peptide SS-31 (B+P) as measured by .sup.31P NMR at 6 hours
after burn.
[0065] FIG. 16 is a graph of mitochondrial aconitase activity in
control, burn, and peptide-treated subjects.
DETAILED DESCRIPTION
[0066] The present disclosure is based on the surprising discovery
by the inventors that certain aromatic-cationic peptides can treat
or ameliorate the local and distant pathophysiological effects of
burn injury, including, but not limited to, hypermetabolism and
organ damage. 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.
[0067] 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, Freshncy, Ed. (1986); Immobilized Cells and Enzymes (IRL
Press, 1986); Perbal, A Practical Guide to Molecular Cloning; the
series, Meth. Enymol., (Academic Press, Inc., 1984); Gene Transfer
Vectorsfor Mammalian Cells, Miller & Calos, Eds. (Cold Spring
Harbor Laboratory, N Y, 1987); and Meth. Enzymol., Vols. 154 and
155, Wu & Grossman, and Wu, Eds., respectively.
[0068] 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.
[0069] 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.
[0070] As used herein, "about" will be understood by persons of
ordinary skill in the art and will vary to some extent depending
upon the context in which it is used. If there are uses of the term
which are not clear to persons of ordinary skill in the art, given
the context in which it is used, "about" will mean up to plus or
minus 10% of the enumerated value.
[0071] 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.
[0072] 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.
[0073] As used herein, the terms "burn" or "burn conditions" are
intended to encompass the full range of such conditions, including
those resulting from: excessive exposure to radiation, e.g., solar
radiation resulting in sunburn, thermal radiation, welding flash,
fires, electrical discharge, contact with chemicals, friction,
contact with very hot objects such as cooking apparatus elements or
hot fluids such as scalding water, hot oil, etc.
[0074] As used herein, the term "effective amount" refers to 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, a burn injury or one or more
conditions associated with a burn injury. In the context of
therapeutic or prophylactic applications, the amount of a
composition administered to the subject will depend on the type and
severity of the disease and on the characteristics of the
individual, such as general health, age, sex, body weight and
tolerance to drugs. It will also depend on the degree, severity and
type of the injury. The skilled artisan will be able to determine
appropriate dosages depending on these and other factors. The
compositions can also be administered in combination with one or
more additional therapeutic compounds.
[0075] 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 therapeutic uses of the agent. Such interfering
materials may include enzymes, hormones and other proteinaceous and
nonproteinaceous solutes.
[0076] 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 a burn injury or any associated conditions or
complications.
[0077] The term "organ" as used herein refers to a part or
structure of the body which is adapted for a special function or
functions, and includes, but is not limited to, the lungs, the
liver, the kidneys, and the bowel, including the stomach and
intestines. In particular, it is contemplated that organs which are
particularly susceptible to dysfunction and failure arising from a
burn to another part of the body are encompassed by this term.
[0078] The term "organ dysfunction" as used herein refers to a
continuum of indications ranging from a minor perturbation in the
normal function(s) of an organ to "organ failure," i.e., the
cessation of sufficient organ output to sustain life. Various
diagnostic and clinical markers known in the art can be used to
assess the function of organs.
[0079] 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, as well as in a voluminous research
literature.
[0080] As used herein, "prevention" or "preventing" of a disorder
or condition refers to a compound that, in a statistical sample,
reduces the occurrence of the disorder or condition in the treated
sample relative to an untreated control sample, or delays the onset
or reduces the severity of one or more symptoms of the disorder or
condition relative to the untreated control sample.
[0081] As used herein, the words "protect" or "protecting" refer 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., a burn injury or associated conditions or
complications. 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 an injury. In particular
embodiments, the peptides protect a subject against distant
pathophysiological effects of burn injury when the peptides are
administered after a subject receives a burn injury, but before the
onset of symptoms of systemic injury. In one embodiment, the
peptides will protect a subject from the primary burn injury when
administered topically or systemically prior to the subject's
exposure to an agent capable of causing a burn, e.g., sunlight or
radiation.
[0082] 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.
[0083] As used herein, the terms "treating," "treatment," or
"alleviation" refer 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 described herein, 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 a burn injury, treatment or prevention may include
a reduction in the size or severity of the burn wound; a reduction
in hypermetabolism, liver damage or function; and improved effects
on other organ systems. 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
[0084] The aromatic-cationic peptides useful in the present methods
are water-soluble and highly polar. Despite these properties, the
peptides can readily penetrate cell membranes. The
aromatic-cationic peptides useful in the present methods include a
minimum of three amino acids, and preferably include a minimum of
four amino acids, covalently joined by peptide bonds. The maximum
number of amino acids present in the aromatic-cationic peptides of
the present methods 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.
[0085] The amino acids of the aromatic-cationic peptides can be any
amino acid. 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). 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.
[0086] The peptides can optionally 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.
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 are not
recognized by common proteases.
[0087] 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. 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. 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.
[0088] 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).
[0089] Another example of a modification of an amino acid is the
derivatization of a carboxyl group of an aspartic acid or a
glutamic acid residue of the peptide. One example of derivatization
is amidation with ammonia or with a primary or secondary amine,
e.g., methylamine, ethylamine, dimethylamine or diethylamine.
Another example of derivatization includes esterification with, for
example, methyl or ethyl alcohol. Another such modification
includes derivatization of an amino group of a lysine, arginine, or
histidine residue. For example, such amino groups can be acylated.
Some suitable acyl groups include, for example, a benzoyl group or
an alkanoyl group comprising any of the C.sub.1-C.sub.4 alkyl
groups mentioned above, such as an acetyl or propionyl group.
[0090] The non-naturally occurring amino acids are suitably
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.
[0091] In order to minimize protease sensitivity, the peptides
should have less than five, less than four, less than three, less
than two contiguous L-amino acids recognized by common proteases,
irrespective of whether the amino acids are naturally or
non-naturally occurring. If the peptide contains protease sensitive
sequences of amino acids, at least one of the amino acids may be 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.
[0092] 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). 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.
[0093] "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. 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.
[0094] In one embodiment, the aromatic-cationic peptides have a
relationship between the minimum number of net positive charges at
physiological pH (p.sub.m) and the total number of amino acid
residues (r) wherein 3 p.sub.m is the largest number that is less
than or equal to r+1. In this embodiment, the relationship between
the minimum number of net positive charges (p.sub.m) and the total
number of amino acid residues (r) is as follows:
TABLE-US-00001 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
[0095] In another embodiment, the aromatic-cationic peptides have a
relationship between the minimum number of net positive charges
(p.sub.m) and the total number of amino acid residues (r) wherein 2
p.sub.m is the largest number that is less than or equal to r+1. In
this embodiment, the relationship between the minimum number of net
positive charges (p.sub.m) and the total number of amino acid
residues (r) is as follows:
TABLE-US-00002 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
[0096] 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. 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).
[0097] 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).
[0098] In one embodiment, the aromatic-cationic peptides 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
[0099] 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 (2a
.ltoreq. p.sub.t + 1 or a = p.sub.t = 1) (p.sub.t) 1 2 3 4 5 6 7 8
9 10 11 12 13 14 15 16 17 18 19 20 (a) 1 1 2 2 3 3 4 4 5 5 6 6 7 7
8 8 9 9 10 10
[0100] In another embodiment, the number of aromatic groups (a) and
the total number of net positive charges (p.sub.t) are equal.
[0101] 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.
[0102] 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.
[0103] In one embodiment, the aromatic-cationic peptide is a
tripeptide having two net positive charges and at least one
aromatic amino acid. In a particular embodiment, the
aromatic-cationic peptide is a tripeptide having two net positive
charges and two aromatic amino acids.
[0104] Aromatic-cationic peptides include, but are not limited to,
the following exemplary peptides:
TABLE-US-00005 Lys-D-Arg-Tyr-NH.sub.2 Phe-D-Arg-His
D-Tyr-Trp-Lys-NH.sub.2 Trp-D-Lys-Tyr-Arg-NH.sub.2 Tyr-His-D-Gly-Met
Phe-Arg-D-His-Asp Tyr-D-Arg-Phe-Lys-Glu-NH.sub.2
Met-Tyr-D-Lys-Phe-Arg D-His-Glu-Lys-Tyr-D-Phe-Arg
Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH.sub.2
Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His
Gly-D-Phe-Lys-Tyr-His-D-Arg-Tyr-NH.sub.2
Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH.sub.2
Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-Lys
Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH.sub.2
Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-Lys
Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH.sub.2
D-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-His-D-Lys-Arg-Trp- NH.sub.2
Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-Phe
Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His- Phe
Phe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe- NH.sub.2
Phe-Try-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D- Tyr-Thr
Tyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr- His-Lys
Glu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-
Tyr-Arg-D-Met-NH.sub.2
Arg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-
Phe-Tyr-D-Arg-Gly D-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-
Tyr-D-Tyr-Arg-His-Phe-NH.sub.2
Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-
Trp-D-His-Tyr-D-Phe-Lys-Phe
His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-
His-Phe-D-Lys-Tyr-His-Ser-NH.sub.2
Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-
Asp-Tyr-Trp-D-His-Trp-His-D-Lys-Asp
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
[0105] In some embodiments, peptides are those peptides which have
a tyrosine residue or a tyrosine derivative. Suitable 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).
[0106] 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).
[0107] 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). In
another embodiment, the amino acid at the N-terminus is
phenylalanine or its derivative. Derivatives of phenylalanine
include 2'-methylphenylalanine (Mmp), 2',6'-dimethylphenylalanine
(Dmp), N,2',6'-trimethylphcnylalaninc (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).
[0108] 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.
[0109] Suitable substitution variants of the peptides include
conservative amino acid substitutions. Amino acids may be grouped
according to their physicochemical characteristics as follows:
[0110] (a) Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P)
Gly(G) Cys (C);
[0111] (b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q);
[0112] (c) Basic amino acids: His(H) Arg(R) Lys(K);
[0113] (d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V);
and
[0114] (e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His (H).
[0115] 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 are
generally more likely to alter the characteristics of the original
peptide. Examples of peptides include, but are not limited to, the
aromatic-cationic peptides shown in Table 5.
TABLE-US-00006 TABLE 5 Examples of Aromatic-Cationic Peptide Amino
Amino Amino Amino C- Acid Acid Acid Acid Terminal Position 1
Position 2 Position 3 Position 4 Modification D-Arg Dmt Lys Phe
NH.sub.2 D-Arg Dmt Phe Lys NH.sub.2 D-Arg Phe Lys Dmt NH.sub.2
D-Arg Phe Dmt Lys NH.sub.2 D-Arg Lys Dmt Phe NH.sub.2 D-Arg Lys Phe
Dmt NH.sub.2 D-Arg Dmt Lys Phe NH.sub.2 D-Arg Dmt Lys Phe NH.sub.2
D-Arg Dmt Lys Phe NH.sub.2 D-Arg Dmt Lys Phe 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 NH.sub.2 Phe D-Arg Phe Lys NH.sub.2 Phe
D-Arg Phe Lys NH.sub.2 Phe D-Arg Phe Lys NH.sub.2 Phe D-Arg Dmt Lys
NH.sub.2 Phe D-Arg Dmt Lys NH.sub.2 Phe D-Arg Dmt Lys NH.sub.2 Phe
D-Arg Dmt Lys NH.sub.2 Phe D-Arg Dmt Lys NH.sub.2 Phe D-Arg Lys Dmt
NH.sub.2 Phe Dmt D-Arg Lys NH.sub.2 Phe Dmt Lys D-Arg NH.sub.2 Lys
Phe D-Arg Dmt NH.sub.2 Lys Phe Dmt D-Arg NH.sub.2 Lys Dmt D-Arg Phe
NH.sub.2 Lys Dmt Phe D-Arg NH.sub.2 Lys D-Arg Phe Dmt NH.sub.2 Lys
D-Arg Dmt Phe NH.sub.2 D-Arg Dmt D-Arg Phe NH.sub.2 D-Arg Dmt D-Arg
Dmt NH.sub.2 D-Arg Dmt D-Arg Tyr NH.sub.2 D-Arg Dmt D-Arg Trp
NH.sub.2 Trp D-Arg Phe Lys NH.sub.2 Trp D-Arg Tyr Lys NH.sub.2 Trp
D-Arg Trp Lys NH.sub.2 Trp D-Arg Dmt Lys NH.sub.2 D-Arg Trp Lys Phe
NH.sub.2 D-Arg Trp Phe Lys NH.sub.2 D-Arg Trp Lys Dmt NH.sub.2
D-Arg Trp Dmt Lys NH.sub.2 D-Arg Lys Trp Phe NH.sub.2 D-Arg Lys Trp
Dmt NH.sub.2 Cha D-Arg Phe Lys NH.sub.2 Ala D-Arg Phe Lys NH.sub.2
Cha = cyclohexylalanine
[0116] Under certain circumstances, it may be advantageous to use a
peptide that also has opioid receptor agonist activity. Examples of
mu-opioid analogs include, but are not limited to, the
aromatic-cationic peptides shown in Table 6.
TABLE-US-00007 TABLE 6 Aromatic-Cationic Peptides with Opioid
Receptor Agonist Activity Amino Amino Amino C-Terminal Acid Acid
Acid Amino Acid Modifica- Position 1 Position 2 Position 3 Position
4 tion 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
NH.sub.2 2'6'Dmt D-Arg Phe Lys NH.sub.2 2'6'Dmt D-Arg Phe Lys
NH.sub.2 2'6'Dmt D-Arg Phe Lys-NH(CH.sub.2).sub.2-- NH.sub.2 NH-dns
2'6'Dmt D-Arg Phe Lys-NH(CH.sub.2).sub.2-- NH.sub.2 NH-atn 2'6'Dmt
D-Arg Phe dnsLys NH.sub.2 2'6'Dmt D-Cit Phe Lys NH.sub.2 2'6'Dmt
D-Cit Phe Lys NH.sub.2 2'6'Dmt D-Cit Phe Ahp NH.sub.2 2'6'Dmt D-Arg
Phe Orn NH.sub.2 2'6'Dmt D-Arg Phe Dab NH.sub.2 2'6'Dmt D-Arg Phe
Dap NH.sub.2 2'6'Dmt D-Arg Phe Ahp(2- NH.sub.2 aminoheptanoic acid)
Bio- D-Arg Phe Lys NH.sub.2 2'6'Dmt 3'5'Dmt D-Arg Phe Lys NH.sub.2
3'5'Dmt D-Arg Phe Orn NH.sub.2 3'5'Dmt D-Arg Phe Dab NH.sub.2
3'5'Dmt D-Arg Phe Dap NH.sub.2 Tyr D-Arg Tyr Lys NH.sub.2 Tyr D-Arg
Tyr Orn NH.sub.2 Tyr D-Arg Tyr Dab NH.sub.2 Tyr D-Arg Tyr Dap
NH.sub.2 2'6'Dmt D-Arg Tyr Lys NH.sub.2 2'6'Dmt D-Arg Tyr Orn
NH.sub.2 2'6'Dmt D-Arg Tyr Dab NH.sub.2 2'6'Dmt D-Arg Tyr Dap
NH.sub.2 2'6'Dmt D-Arg 2'6'Dmt Lys NH.sub.2 2'6'Dmt D-Arg 2'6'Dmt
Orn NH.sub.2 2'6'Dmt D-Arg 2'6'Dmt Dab NH.sub.2 2'6'Dmt D-Arg
2'6'Dmt Dap NH.sub.2 3'5'Dmt D-Arg 3'5'Dmt Arg NH.sub.2 3'5'Dmt
D-Arg 3'5'Dmt Lys NH.sub.2 3'5'Dmt D-Arg 3'5'Dmt Orn NH.sub.2
3'5'Dmt D-Arg 3'5'Dmt Dab NH.sub.2 3'5'Dmt D-Arg 2'6'Dmt Lys
NH.sub.2 Tyr D-Lys Phe Dap NH.sub.2 Tyr D-Lys Phe Arg NH.sub.2 Tyr
D-Lys Phe Arg NH.sub.2 Tyr D-Lys Phe Lys NH.sub.2 Tyr D-Lys Phe Orn
NH.sub.2 2'6'Dmt D-Lys Phe Dab NH.sub.2 2'6'Dmt D-Lys Phe Dap
NH.sub.2 2'6'Dmt D-Lys Phe Arg NH.sub.2 2'6'Dmt D-Lys Phe Lys
NH.sub.2 3'5'Dmt D-Lys Phe Orn NH.sub.2 3'5'Dmt D-Lys Phe Dab
NH.sub.2 3'5'Dmt D-Lys Phe Dap NH.sub.2 3'5'Dmt D-Lys Phe Arg
NH.sub.2 3'5'Dmt D-Lys Phe Arg NH.sub.2 Tyr D-Lys Tyr Lys NH.sub.2
Tyr D-Lys Tyr Orn NH.sub.2 Tyr D-Lys Tyr Dab NH.sub.2 Tyr D-Lys Tyr
Dap NH.sub.2 2'6'Dmt D-Lys Tyr Lys NH.sub.2 2'6'Dmt D-Lys Tyr Orn
NH.sub.2 2'6'Dmt D-Lys Tyr Dab NH.sub.2 2'6'Dmt D-Lys Tyr Dap
NH.sub.2 2'6'Dmt D-Lys 2'6'Dmt Lys NH.sub.2 2'6'Dmt D-Lys 2'6'Dmt
Orn NH.sub.2 2'6'Dmt D-Lys 2'6'Dmt Dab NH.sub.2 2'6'Dmt D-Lys
2'6'Dmt Dap NH.sub.2 2'6'Dmt D-Arg Phe dnsDap NH.sub.2 2'6'Dmt
D-Arg Phe atnDap NH.sub.2 3'5'Dmt D-Lys 3'5'Dmt Lys NH.sub.2
3'5'Dmt D-Lys 3'5'Dmt Orn NH.sub.2 3'5'Dmt D-Lys 3'5'Dmt Dab
NH.sub.2 3'5'Dmt D-Lys 3'5'Dmt Dap NH.sub.2 Tyr D-Lys Phe Arg
NH.sub.2 Tyr D-Orn Phe Arg NH.sub.2 Tyr D-Dab Phe Arg NH.sub.2 Tyr
D-Dap Phe Arg NH.sub.2 2'6'Dmt D-Arg Phe Arg NH.sub.2 2'6'Dmt D-Lys
Phe Arg NH.sub.2 2'6'Dmt D-Orn Phe Arg NH.sub.2 2'6'Dmt D-Dab Phe
Arg NH.sub.2 3'5'Dmt D-Dap Phe Arg NH.sub.2 3'5'Dmt D-Arg Phe Arg
NH.sub.2 3'5'Dmt D-Lys Phe Arg NH.sub.2 3'5'Dmt D-Orn Phe Arg
NH.sub.2 Tyr D-Lys Tyr Arg NH.sub.2 Tyr D-Orn Tyr Arg NH.sub.2 Tyr
D-Dab Tyr Arg NH.sub.2 Tyr D-Dap Tyr Arg NH.sub.2 2'6'Dmt D-Arg
2'6'Dmt Arg NH.sub.2 2'6'Dmt D-Lys 2'6'Dmt Arg NH.sub.2 2'6'Dmt
D-Orn 2'6'Dmt Arg NH.sub.2 2'6'Dmt D-Dab 2'6'Dmt Arg NH.sub.2
3'5'Dmt D-Dap 3'5'Dmt Arg NH.sub.2 3'5'Dmt D-Arg 3'5'Dmt Arg
NH.sub.2 3'5'Dmt D-Lys 3'5'Dmt Arg NH.sub.2 3'5'Dmt D-Orn 3'5'Dmt
Arg NH.sub.2 Mmt D-Arg Phe Lys NH.sub.2 Mmt D-Arg Phe Orn NH.sub.2
Mmt D-Arg Phe Dab NH.sub.2 Mmt D-Arg Phe Dap NH.sub.2 Tmt D-Arg Phe
Lys NH.sub.2 Tmt D-Arg Phe Orn NH.sub.2 Tmt D-Arg Phe Dab NH.sub.2
Tmt D-Arg Phe Dap NH.sub.2 Hmt D-Arg Phe Lys NH.sub.2 Hmt D-Arg Phe
Orn NH.sub.2 Hmt D-Arg Phe Dab NH.sub.2 Hmt D-Arg Phe Dap NH.sub.2
Mmt D-Lys Phe Lys NH.sub.2 Mmt D-Lys Phe Orn NH.sub.2 Mmt D-Lys Phe
Dab NH.sub.2 Mmt D-Lys Phe Dap NH.sub.2 Mmt D-Lys Phe Arg NH.sub.2
Tmt D-Lys Phe Lys NH.sub.2 Tmt D-Lys Phe Orn NH.sub.2 Tmt D-Lys Phe
Dab NH.sub.2 Tmt D-Lys Phe Dap NH.sub.2 Tmt D-Lys Phe Arg NH.sub.2
Hmt D-Lys Phe Lys NH.sub.2 Hmt D-Lys Phe Orn NH.sub.2 Hmt D-Lys Phe
Dab NH.sub.2 Hmt D-Lys Phe Dap NH.sub.2 Hmt D-Lys Phe Arg NH.sub.2
Mmt D-Lys Phe Arg NH.sub.2 Mmt D-Orn Phe Arg NH.sub.2 Mmt D-Dab Phe
Arg NH.sub.2 Mmt D-Dap Phe Amp 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
[0117] 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
[0118] 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 Treatment and Prevention of Burn Injury and Secondary
Complications
[0119] The aromatic-cationic peptides described herein are useful
in treating or preventing burn injuries and systemic conditions
associated with a burn injury. In some embodiments, the
aromatic-cationic peptides may be administered to a subject
following a burn and after the onset of detectable symptoms of
systemic injury. Thus, the term "treatment" is used herein in its
broadest sense and refers to use of an aromatic-cationic peptide
for a partial or complete cure of the burn and/or secondary
complications, such as organ dysfunction and hypermetabolism.
[0120] In other embodiments, the aromatic-cationic peptides of the
invention may be administered to a subject following a burn, but
before the onset of detectable symptoms of systemic injury in order
to protect against or provide prophylaxis for the systemic injury,
such as organ damage or hypermetabolism. Thus the term "prevention"
is used herein in its broadest sense and refers to a prophylactic
use which completely or partially prevents local injury to the skin
or systemic injury, such as organ dysfunction or hypermetabolism
following burns. It is also contemplated that the compounds may be
administered to a subject at risk of receiving burns.
[0121] Burns are generally classified accordingly to their
seriousness and extent. First degree burns are the mildest and
normally only affect the epidermis. The burn site is red, painful,
dry, no blisters, very sensitive to touch and the damaged skin may
be slightly moist from the leakage of fluid in the deeper layers of
the skin. The sensory nerve ends are also exposed and create pain.
Mild sunburn is typical of a first degree burn. Second degree burns
is where both the epidermis and dermis are affected. The damage is
deeper and blisters usually appear on the skin. The skin is still
painful and sensitive, as the nerves have been affected as well as
the sebaceous glands in the area. Third degree burns are the most
serious, as the tissues in all layers of the skin are dead.
Normally the damaged area goes down into the subcutaneous tissue.
Usually there are no blisters, but the burnt surface can have
several types of appearance, from white to black (charred) or
bright red from blood in the bottom of the wound. In most cases, it
can penetrate down through the superficial fascia, and into the
muscle layers where various arteries and veins may be affected.
Because the skin nerves are damaged the burn can be quite painless
and on touching the skin sometimes it has no sensation whatsoever.
The lack of sensation or blanching of the skin blood vessels on
pressure indicates damaged skin.
[0122] It is contemplated that the invention is applicable to the
treatment of burns from any cause, including dry heat or cold
burns, scalds, sunburn, electrical burns, chemical agents such as
acids and alkalis, including hydrofluoric acid, formic acid,
anhydrous ammonia, cement, and phenol, or radiation burns. Burns
resulting from exposure to either high or low temperature are
within the scope of the invention. The severity and extent of the
burn may vary, but secondary organ damage or hypermetabolism will
usually arise when the burns are very extensive or very severe
(second or third degree burns). The development of secondary organ
dysfunction or failure is dependent on the extent of the burn, the
response of the patient's immune system and other factors, such as
infection and sepsis.
[0123] In some embodiments, the aromatic-cationic peptides are used
to treat or prevent organ dysfunction secondary to a burn. The
chain of physiological processes which lead to organ dysfunction
following burns is complex. In subjects with serious burns, release
of catecholamines, vasopressin, and angiotensin causes peripheral
and splanchnic bed vasoconstriction that can compromise perfusion
of organs remote to the injury. Myocardial contractility also may
be reduced by the release of TNF-.alpha.. Activated neutrophils are
sequestered in dermal and distant organs such as the lung within
hours following a burn injury, resulting in the release of toxic
reactive oxygen species and proteases and producing vascular
endothelial cell damage. When the integrity of pulmonary capillary
and alveolar epithelia is compromised, plasma and blood leak into
the interstitial and intra-alveolar spaces, resulting in pulmonary
edema. A decrease in pulmonary function can occur in severely
burned patients, as a result of bronchoconstriction caused by
humoral factors, such as histamine, serotonin, and thromboxane
A2.
[0124] Severe burn injury also causes a coagulation necrosis of
tissue. This initiates a physiological response in every organ
system, the severity of which is related to the extent of the burn.
Tissue destruction also results in increased capillary
permeability, with profound egress of fluid from the intravascular
space to the tissues adjacent to the burn wound. Inordinate amounts
of fluid are lost by evaporation from the damaged surface, which is
no longer able to retain water. This increase in capillary
permeability, coupled with evaporative water loss, causes a
hypovolemic shock, which may also in turn contribute to remote
organ dysfunction or failure.
[0125] Subjects suffering from severe burns are also at great risk
of sepsis. Bacterial invasion occurs in a burn patient because the
skin no longer acts as a barrier to the entrance of microorganisms.
Because of their reduced ability to mount an effective systemic
immune response, severely burned patients are susceptible to the
development of sepsis and life-threatening septic shock. Sepsis is,
however, a separate complication from the organ dysfunction or
failure which occurs secondary to burns. Organ dysfunction or
failure secondary to burns may occur in the absence of sepsis.
[0126] Subjects suffering from a burn injury are also at risk for
skeletal muscle dysfunction. While not wishing to be limited by
theory, a major cause of the mitochondrial skeletal muscle
dysfunction in burns may result from defects in oxidative
phosphorylation (OXPHOS) via stimulation of mitochondrial
production of reactive oxygen species (ROS) and the resulting
damage to the mitochondrial DNA (mtDNA). In some embodiments, the
aromatic-cationic peptides induce ATP synthesis via a recovery of
the mitochondrial redox status or via the peroxisome proliferator
activated receptor-gamma coactivator-10 which is downregulated as
early as 6 hours after burn. Thus, the mitochondrial dysfunction
caused by burn injury recovers with the administration of the
aromatic-cationic peptide.
[0127] In one aspect, the methods relate to treating a wound
resulting from a burn injury by administering to a subject an
effective amount of the aromatic-cationic peptides. The peptides
may be administered systemically or topically to the wound. Burn
wounds are typically uneven in depth and severity. There are
significant areas around the coagulated tissue where injury may be
reversible and damage mediated by the inflammatory and immune cells
to the microvasculature of the skin could be prevented. In one
embodiment, the administration of the peptides will slow or
ameliorate the effects of wound contraction. Wound contraction is
the process which diminishes the size of a full-thickness open
wound, especially a full-thickness burn. The tensions developed
during contracture and the formation of subcutaneous fibrous tissue
can result in deformity, and in particular to fixed flexure or
fixed extension of a joint where the wound involves an area over
the joint. Such complications are especially relevant in burn
healing. No wound contraction will occur when there is no injury to
the tissue; maximum contraction will occur when the burn is
full-thickness and no viable tissue remains in the wound. In
another embodiment, the administration of the peptides prevent
progression of a burn injury from a second degree burn to a third
degree burn.
[0128] The method for the treatment of burn injury may also be
effective for decreasing scarring or the formation of scar tissue
attendant the healing process at a burn site. Scarring is the
formation of fibrous tissue at sites where normal tissue has been
destroyed. The present disclosure thus also includes a method for
decreasing scarring specifically at skin tissue areas of second or
third degree burn. This method comprises treating an animal with a
second or third degree burn with an effective amount of an aromatic
cationic peptide.
[0129] In a particular embodiment, the aromatic-cationic peptides
are administered a subject suffering from a burn in order to treat
or prevent damage to distant organs or tissues. In particular,
dysfunction or failure of the lung, liver, kidneys, and/or bowel
following burns to the skin or other sites of the body has a
significant impact on morbidity and mortality. While not wishing to
be limited by theory, it is believed that systemic inflammatory
responses arise in subjects following burn injury, and that it is
this generalized inflammation which leads to remote tissue injury
which is expressed as the dysfunction and failure of organs remote
from the injury site. Systemic injury, including organ dysfunction
and hypermetabolism, is typically associated with second and third
degree burns. A characteristic of the systemic injury, i.e., organ
dysfunction or hypermetabolism, is that the burn which provokes the
subsequent injury or condition does not directly affect the organ
in question. i.e., the injury is secondary to the burn.
[0130] In one embodiment, the aromatic-cationic peptides are
administered to treat or protect damage to liver tissues secondary
to a burn. Methods for assessing liver function are well known in
the art and include, but are not limited to, using blood tests for
serum alanine aminotransferase (ALT) levels, alkaline phosphatase
(AP), or bilirubin levels. Methods for assessing deterioration of
liver structure are also well known. Such methods include liver
imaging (e.g., MRI, ultrasound), or histological evaluation of
liver biopsy.
[0131] In one embodiment, the aromatic-cationic peptides are
administered to treat or protect damage to liver tissues secondary
to a burn. Methods for assessing liver function are well known in
the art and include, but are not limited to, using blood tests for
serum creatinine, or glomerular filtration rate. Methods for
assessing deterioration of kidney structure are also well known.
Such methods include kidney imaging (e.g., MRI, ultrasound), or
histological evaluation of kidney biopsy.
[0132] In one embodiment, the aromatic-cationic peptides are
administered to prevent or treat hypermetabolism associated with a
burn injury. A hypermetabolic state may be associated with
hyperglycemia, protein losses, and a significant reduction of lean
body mass. Reversal of the hypermetabolic response may be
accomplished by administering the aromatic-cationic peptides and by
manipulating the subject's physiologic and biochemical environment
through the administration of specific nutrients, growth factors,
or other agents. As demonstrated in the examples, the present
inventors discovered that the aromatic-cationic peptides of the
invention may be administered to a subject suffering from a burn in
order to treat or prevent hypermetabolism.
[0133] In one aspect, the disclosure provides a method for
preventing in a subject, a burn injury or a condition associated
with a burn injury, by administering to the subject an
aromatic-cationic peptide. It is contemplated that the
aromatic-cationic peptides may be administered to a subject at risk
of receiving burns. In prophylactic applications, pharmaceutical
compositions or medicaments of aromatic-cationic peptides are
administered to a subject susceptible to, or otherwise at risk of a
burn injury to eliminate or reduce the risk, lessen the severity,
or delay the outset of the burn injury and its complications.
[0134] Another aspect of the disclosure includes methods of
treating burn injuries and associated complications in a subject
for therapeutic purposes. In therapeutic applications, compositions
or medicaments are administered to a subject already suffering from
a burn injury in an amount sufficient to cure, or at least
partially arrest, the symptoms of the injury, including its
complications and intermediate pathological phenotypes in
development of the disease. It is contemplated that the
aromatic-cationic peptides may be administered to a subject
following a burn, but before the development of detectable symptoms
of a systemic injury, such as organ dysfunction or failure, and
thus the term "treatment" as used herein in its broadest sense and
refers to a prophylactic use which completely or partially prevents
systemic injury, such as organ dysfunction or failure or
hypermetabolism following burns. As such, the disclosure provides
methods of treating an individual afflicted with a burn injury.
Modes of Administration and Effective Dosages
[0135] Any method known to those in the art for contacting a cell,
organ or tissue with a peptide may be employed. Suitable methods
include in vitro, ex vivo, or in vivo methods. In vivo methods
typically include the administration of an aromatic-cationic
peptide, such as those described above, to a mammal, preferably a
human. When used in vivo for therapy, the aromatic-cationic
peptides of the present invention are administered to the subject
in effective amounts (i.e., amounts that have desired therapeutic
effect). They will normally be administered parenterally,
topically, or orally. The dose and dosage regimen will depend upon
the degree of burn injury or secondary complications, the
characteristics of the particular aromatic-cationic peptide used,
e.g., its therapeutic index, the subject, and the subject's
history.
[0136] The effective amount may be determined during pre-clinical
trials and clinical trials by methods familiar to physicians and
clinicians. An effective amount of a peptide, preferably in a
pharmaceutical composition, may be administered to a mammal in need
thereof by any of a number of well-known methods for administering
pharmaceutical compounds. The peptide may be administered
systemically or locally.
[0137] The peptide may be formulated as a pharmaceutically
acceptable salt. The term "pharmaceutically acceptable salt" means
a salt prepared from a base or an acid which is acceptable for
administration to a patient, such as a mammal (e.g., salts having
acceptable mammalian safety for a given dosage regime). However, it
is understood that the salts are not required to be
pharmaceutically acceptable salts, such as salts of intermediate
compounds that are not intended for administration to a patient.
Pharmaceutically acceptable salts can be derived from
pharmaceutically acceptable inorganic or organic bases and from
pharmaceutically acceptable inorganic or organic acids. In
addition, when a peptide contains both a basic moiety, such as an
amine, pyridine or imidazole, and an acidic moiety such as a
carboxylic acid or tetrazole, zwitterions may be formed and are
included within the term "salt" as used herein. Salts derived from
pharmaceutically acceptable inorganic bases include ammonium,
calcium, copper, ferric, ferrous, lithium, magnesium, manganic,
manganous, potassium, sodium, and zinc salts, and the like. Salts
derived from pharmaceutically acceptable organic bases include
salts of primary, secondary and tertiary amines, including
substituted amines, cyclic amines, naturally-occurring amines and
the like, such as arginine, betaine, caffeine, choline,
N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,
2-dimethylaminoethanol, ethanolamine, ethylenediamine,
N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine,
histidine, hydrabamine, isopropylamine, lysine, methylglucamine,
morpholine, piperazine, piperadine, polyamine resins, procaine,
purines, theobromine, triethylamine, trimethylamine,
tripropylamine, tromethamine and the like. Salts derived from
pharmaceutically acceptable inorganic acids include salts of boric,
carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric or
hydroiodic), nitric, phosphoric, sulfamic and sulfuric acids. Salts
derived from pharmaceutically acceptable organic acids include
salts of aliphatic hydroxyl acids (e.g., citric, gluconic,
glycolic, lactic, lactobionic, malic, and tartaric acids),
aliphatic monocarboxylic acids (e.g., acetic, butyric, formic,
propionic and trifluoroacetic acids), amino acids (e.g., aspartic
and glutamic acids), aromatic carboxylic acids (e.g., benzoic,
p-chlorobenzoic, diphenylacetic, gentisic, hippuric, and
triphenylacetic acids), aromatic hydroxyl acids (e.g.,
o-hydroxybenzoic, p-hydroxybenzoic,
1-hydroxynaphthalene-2-carboxylic and
3-hydroxynaphthalene-2-carboxylic acids), ascorbic, dicarboxylic
acids (e.g., fumaric, maleic, oxalic and succinic acids),
glucoronic, mandelic, mucic, nicotinic, orotic, pamoic,
pantothenic, sulfonic acids (e.g., benzenesulfonic, camphosulfonic,
edisylic, ethanesulfonic, isethionic, methanesulfonic,
naphthalenesulfonic, naphthalene-1,5-disulfonic,
naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic
acid, and the like.
[0138] The aromatic-cationic peptides described herein can be
incorporated into pharmaceutical compositions for administration,
singly or in combination, to a subject for the treatment or
prevention of a disorder described herein. Such compositions
typically include the active agent and a pharmaceutically
acceptable carrier. As used herein the term "pharmaceutically
acceptable carrier" includes saline, solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. Supplementary active compounds can
also be incorporated into the compositions.
[0139] Pharmaceutical compositions are typically formulated to be
compatible with its intended route of administration. Examples of
routes of administration include parenteral (e.g., intravenous,
intradermal, intraperitoneal or subcutaneous), oral, inhalation,
transdermal (topical), transmucosal, and rectal administration.
Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0140] Pharmaceutical compositions suitable for injectable use can
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, a composition for
parenteral administration must be sterile and should be fluid to
the extent that easy syringability exists. It should be stable
under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms such
as bacteria and fungi.
[0141] The aromatic-cationic peptide compositions can include a
carrier, which can be a solvent or dispersion medium containing,
for example, water, ethanol, polyol (for example, glycerol,
propylene glycol, and liquid polyethylene glycol, and the like),
and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. Prevention of the action
of microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thiomersal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate or
gelatin.
[0142] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle, which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, typical methods of preparation
include vacuum drying and freeze drying, which can yield a powder
of the active ingredient plus any additional desired ingredient
from a previously sterile-filtered solution thereof.
[0143] Oral compositions generally include an inert diluent or an
edible carrier. For the purpose of oral therapeutic administration,
the active compound can be incorporated with excipients and used in
the form of tablets, troches, or capsules, e.g., gelatin capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash. Pharmaceutically compatible binding agents,
and/or adjuvant materials can be included as part of the
composition. The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth
or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
[0144] Systemic administration of a therapeutic compound as
described herein can also be by transmucosal or transdermal means.
For transmucosal or transdermal administration, penetrants
appropriate to the barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art, and
include, for example, for transmucosal administration, detergents,
bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art. In one embodiment, transdermal
administration may be performed my iontophoresis.
[0145] Dosage, toxicity and therapeutic efficacy of the therapeutic
agents can be determined by standard pharmaceutical procedures in
cell cultures or experimental animals, e.g., for determining the
LD50 (the dose lethal to 50% of the population) and the ED50 (the
dose therapeutically effective in 50% of the population). The dose
ratio between toxic and therapeutic effects is the therapeutic
index and it can be expressed as the ratio LD50/ED50. Compounds
which exhibit high therapeutic indices are preferred. While
compounds that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to other cells and, thereby, reduce side effects.
[0146] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose can be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC50 (i.e., the concentration of the test compound which achieves a
half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography.
[0147] Typically, an effective amount of the aromatic-cationic
peptides, sufficient for achieving a therapeutic or prophylactic
effect, range from about 0.000001 mg per kilogram body weight per
day to about 10,000 mg per kilogram body weight per day.
Preferably, the dosage ranges are from about 0.0001 mg per kilogram
body weight per day to about 100 mg per kilogram body weight per
day. For example, dosages can be 1 mg/kg body weight or 10 mg/kg
body weight every day, every two days or every three days or within
the range of 1-10 mg/kg every week, every two weeks or every three
weeks. In one embodiment, a single dosage of peptide ranges from
0.1-10,000 micrograms per kg body weight. In one embodiment,
aromatic-cationic peptide concentrations in a carrier range from
0.2 to 2000 micrograms per delivered milliliter. An exemplary
treatment regime entails administration once per day or once a
week. Thereafter, the patient can be administered a prophylactic
regime.
[0148] In some embodiments, a therapeutically effective amount of
an aromatic-cationic peptide may be defined as a concentration of
peptide at the target tissue of 10.sup.-11 to 10.sup.-6 molar,
e.g., approximately 10.sup.-7 molar. This concentration may be
delivered by systemic doses of 0.01 to 100 mg/kg or equivalent dose
by body surface area. The schedule of doses would be optimized to
maintain the therapeutic concentration at the target tissue, most
preferably by single daily or weekly administration, but also
including continuous administration (e.g., parenteral infusion or
transdermal application).
[0149] In some embodiments, the dosage of the aromatic-cationic
peptide is provided at a "low," "mid," or "high" dose level. In one
embodiment, the low dose is provided from about 0.001 to about 0.5
mg/kg/h, suitably from about 0.01 to about 0.1 mg/kg/h. In one
embodiment, the mid-dose is provided from about 0.1 to about 1.0
mg/kg/h, suitably from about 0.1 to about 0.5 mg/kg/h. In one
embodiment, the high dose is provided from about 0.5 to about 10
mg/kg/h, suitably from about 0.5 to about 2 mg/kg/h.
[0150] The skilled artisan will appreciate that certain factors may
influence the dosage and timing required to effectively treat a
subject, including but not limited to, the severity of the disease
or disorder, previous treatments, the general health and/or age of
the subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of the therapeutic
compositions described herein can include a single treatment or a
series of treatments.
[0151] The mammal treated in accordance with the invention can be
any mammal, including, for example, farm animals, such as sheep,
pigs, cows, and horses; pet animals, such as dogs and cats;
laboratory animals, such as rats, mice and rabbits. In a preferred
embodiment, the mammal is a human.
EXAMPLES
[0152] The present invention is further illustrated by the
following examples, which should not be construed as limiting in
any way.
Example 1--SS-31 Attenuates the Hypermetabolism after Burn Injury
in a Rat Model
[0153] Hypermetabolism (HYPM) is a hallmark feature of metabolic
disturbance after burn injury. The increased energy expenditure
(EE) is associated with accelerated substrate oxidations and shifts
of fuel utilization with increased contribution of lipid oxidation
to total energy production. Mitochondria are the organelle where
the substrate oxidations take place. Mitochondrial dysfunction
occurs after burn. It is closely related to the development of HYPM
and the altered substrate oxidations. SS-31
(D-Arg-2',6'-dimethyltyrosine-Lys-Phe-NH.sub.2) is a tetrapeptide
which penetrates into mitochondria, inhibits mitochondrial
swelling, and reduces oxidative cell death. This Example tested the
SS-31 peptide's potential function on total EE and subsequently,
the substrate oxidation after burn injury.
[0154] Sprague Dawley rats were randomized into three groups;
sham-burn(SB), burn with saline treatment (B) and burn with peptide
treatment (BP). Catheters were surgically placed into jugular vein
and carotid artery. B and BP animals received 30% total body
surface area full thickness burns by immersing the dorsal part into
100.degree. C. water for 12 seconds with immediate fluid
resuscitation. BP animals received IV injection of SS-31 (2 mg/kg
every 12 h) for three days (FIG. 1). The EE of the animals were
constantly monitored for 12 hours in a TSE Indirect Calorimetry
System (TSE Co. Germany).
[0155] The results of the experiment are shown in Table 7 and FIG.
2. Three days after burn injury, animals in the B group showed
significant increases in VO.sub.2, VCO.sub.2 and FE compared to
animals in the SB group. SS-31 treatment significantly reduced
VO.sub.2, VCO.sub.2 and EE (BP versus B P<0.05).
TABLE-US-00008 TABLE 7 Burn-SS-31 P (between Sham Burn-saline (2
mg/kg q12h) burn-saline (n = 12) (n = 3) (n = 3) and burn-SS-31)
VO.sub.2 615 .+-. 7 864 .+-. 22 765 .+-. 17 0.02 (ml/kg/h)
VCO.sub.2 408 .+-. 6 585 .+-. 15 515 .+-. 16 0.03 (ml/kg/h) Energy
3.457 .+-. 4.927 .+-. 0.216 4.165 .+-. 0.093 0.03 expenditure 0.062
(kcal/kg/h) RER 0.652 .+-. 0.672 .+-. 0.002 0.669 .+-. 0.005 0.65
0.006
[0156] The results indicate that treatment with SS-31 in rats with
a burn injury can attenuate burn-induced HYPM. As such, the
aromatic-cationic peptides of the invention are useful in methods
of treating burn injuries and secondary complications in subjects
in need thereof.
Example 2--SS-31 Protects the Liver from Burn-Induced Apoptosis in
the Mouse
[0157] Systemic inflammatory response syndrome (SIRS) and multiple
organ failure (MOF) are leading causes of morbidity and mortality
in severe burn patients. In this Example, the effects of the
aromatic-cationic peptides of the invention on liver damage in a
mouse model of burn injury were examined. Six to eight week old
male C57BL mice were subjected to 30% total body surface area
(TBSA) burn injury and subsequently injected daily with saline or
SS-31 peptide (5 mg/kg body weight). A weight- and time-matched
sham-burn group subjected to lukewarm (.about.37.degree. C.) water
but otherwise treated exactly the same served as controls (FIG. 3).
The liver tissues were collected 1, 3, and 7 days after burn injury
treatment and used to examine apoptosis by TUNEL method, activated
caspase protein levels by Western blots, and caspase activity by an
enzymatic reaction assay.
[0158] Burn injury increased the rate of apoptosis in the liver on
all days examined, but the most dramatic increase occurred on day 7
post-burn injury. However, treatment with SS-31 peptide minimized
the number of cells undergoing apoptosis with its effects most
obvious on day 7 of post-burn (FIG. 4). Western blot analysis
revealed a progressive increase in the protein levels of activated
form of caspase-3 with time following burn injury compared to sham
control group (FIG. 5). The SS-31 peptide reversed the increase in
the activated form of caspase-3 protein levels on day 3 and 7
post-burn to the levels similar to those of sham control animals.
Similar to the changes in the protein levels, the caspase activity
increased significantly on post-burn day 7, but the treatment with
SS-31 peptide reduced the caspase activity to a level that was
statistically not different from that of sham control group (FIG.
6). There was a trend for the increased caspase activity following
burn injury to reverse with the SS-31 peptide treatment on day 3
(FIG. 6). There was also decreased protein oxidation following burn
injury in mice treated with the SS-31 peptide (FIG. 7).
[0159] Thus, this study provides evidence that the SS-31 peptide
can reduce burn-induced activation of caspase signaling pathways
and subsequently attenuate apoptosis in the liver of mice. As such,
the aromatic-cationic peptides of the invention are useful in
methods to prevent or treat systemic organ damage, such as liver
damage, secondary to a burn.
Example 3--SS-31 Prevents Wound Contraction after Burn Injury
[0160] Burn wounds are typically uneven in depth and severity.
There are significant areas around the coagulated tissue where
injury may be reversible and damage mediated by the inflammatory
and immune cells to the microvasculature of the skin could be
prevented. Wound contraction is the process which diminishes the
size of a full-thickness open wound, especially a full-thickness
burn. The tensions developed during contracture and the formation
of subcutaneous fibrous tissue can result in deformity, and in
particular to fixed flexure or fixed extension of a joint where the
wound involves an area over the joint. Such complications are
especially relevant in burn healing. No wound contraction will
occur when there is no injury to the tissue; maximum contraction
will occur when the burn is full-thickness and no viable tissue
remains in the wound. This Example demonstrates the ability of the
aromatic-cationic peptides of the invention to reduce or prevent
wound contraction.
[0161] Sprague-Dawley rats (male, 300-350 g) were pre-treated with
1 mg SS-31 peptide administered IP (this corresponds to about 3
mg/kg) one hour prior to burn (65.degree. C. water for 25 sec on
the lower back), followed by topical treatment (1 mg on top of the
wound), and 1 mg SS-31 peptide administered IP every 12 h for 72 h.
Wounds were observed up to .about.3 weeks post-burn. In general,
the wounds took the appearance of a hard scab, and for the purpose
of this experiment, the area of the scab was quantified as a
measure of wound size. A slower rate of wound contraction was
observed in the peptide-treated group at time points .gtoreq.8 days
post-burn (p=0.06 by ANOVA) (FIG. 8). Treatment with SS-31 slowed
the wound contraction, which indicates the burn injury was less
severe compared to burns in animals without the peptide treatment.
As such, the aromatic-cationic peptides of the invention are useful
in methods of treating wounds associated with a burn injury.
Example 4--SS-31 Alleviates Skeletal Muscle Dysfunction after Burn
Injury
[0162] To demonstrate the treatment and prevention of post-burn
complications, the aromatic-cationic peptides of the invention were
tested on a murine model of mammalian burn injury. A major cause of
the mitochondrial skeletal muscle dysfunction in burns may result
from defects in oxidative phosphorylation (OXPHOS) via stimulation
of mitochondrial production of reactive oxygen species (ROS) and
the resulting damage to the mitochondrial DNA (mtDNA). This
hypothesis is supported by data indicating that the ATP synthesis
rate significantly decreases and ROS production increases in
skeletal muscle in response to burn injury. This progression
underlies the burn pathophysiology, which includes skeletal muscle
wasting and cachexia. Thus, this study investigated the potential
of aromatic-cationic peptides as a treatment modality to alleviate
skeletal muscle dysfunction after burns.
[0163] A clinically relevant murine nonlethal local burn injury
model was used to address whether aromatic-cationic peptides can
prevent/attenuate the adverse effects of burn injury on
mitochondrial dysfunction and ER stress. The redox state of the
gastrocnemius muscle immediately below a local cutaneous burn
(90.degree. C. for 3 sec) was evaluated by nitroxide EPR. It was
found that the redox state in the muscle of burned mice was
compromised, with the most dramatic effect at 6 h postburn
(P<0.05 compared to day 0 control, n=4), as evidenced by the low
rate of nitroxide reduction (FIG. 8).
[0164] Next, the effect of SS-31 peptide treatment administered IP
30 min before burn, and immediately after burn (3 mg/kg each dose)
was tested. At the 6 h timepoint, peptide treatment significantly
increased the rate of nitroxide reduction (lowering the curve of
intensity vs. time, FIG. 9). The effect was statistically
significant, suggesting that peptide treatment does decrease
oxidative stress in muscle underneath the burn. These data indicate
that the aromatic-cationic peptides of the invention are useful in
methods of preventing or treating secondary complications of a burn
injury, such as skeletal muscle dysfunction.
Example 5--SS-31 Treats Burn Wounds and Attenuates Progression of
Tissue Damage Following a Burn (Prophetic)
[0165] To demonstrate the treatment burn injuries, the
aromatic-cationic peptides of the invention are tested on a rat
model of mammalian burn injuries. The purpose of these experiments
is to determine whether the mitochondrial-directed anti-oxidant
peptide SS-31 improves wound healing (i.e., accelerates healing or
leads to less scarring) in a partial thickness burn wound. The
hypothesis is that SS-31 prevents apoptosis and other deleterious
processes, such as oxidative stress, microvascular damage, etc.
that lead to expansion of the burn wound (both in depth and area)
in the timeframe of 0 to 48 h after the initial injury. Thus, by
preventing expansion of the burn wound, it is expected that wound
healing will be faster, lead to less scarring, and better
appearance after healing. The results shown in Example 3 are
consistent with a protective effect of SS-31 in wound healing and
suggest that SS-31 can reduce wound contraction in a rat partial
thickness burn model. Additional experiments will test whether
SS-31 treatment leads to faster healing of burn wounds in a rat
model.
[0166] First, it is expected that SS-31 will accelerate wound
healing in a rat model of burn injury. Sprague Dawley rats will be
randomized into three groups; sham-burn (SB), burn with saline
treatment (B) and burn with peptide treatment (BP). B and BP
animals will receive a 30% total body surface area full thickness
burns by immersing the dorsal part into 100.degree. C. water for 12
seconds with immediate fluid resuscitation. BP animals will receive
IV injection of SS-31 (2 mg/kg every 12 h) for three days. Wound
reepithelialization, contraction, and depth are judged via gross
morphology and histologically over a period of 21 days. For this
purpose, immediately after wounding, dark marks are applied onto
the skin of the animals at the wound edges as well as 1 cm away
from the edges. Wounds are digitally photographed over 21 days, and
image analysis software used to measure the area of the wound
(defined as the scab). In addition, the distances of the marks away
from the wound site are used to assess contraction.
[0167] At selected time points, wounds will be harvested from the
animals. Since the conversion from a second to a third degree wound
is expected to occur primarily in the first 48 hours, samples are
harvested at 12, 24, and 48 hours. In addition, to monitor the
long-term impact on the wound healing process, samples are
harvested at 2, 7, 14, and 21 d. The tissues will be fixed and
embedded, and sections across the center of the wounds generated
for histology and staining with H&E as well as trichrome
staining. Slides will be visualized microscopically.
[0168] Second, analyses will determine whether or not SS-31
prevents conversion of a partial thickness burn to a full-thickness
burn. For this purpose, TUNEL staining and caspase-3 assays are
carried out to sec if apoptosis occurs in the hair follicles of the
skin. Skin samples obtained from time points between 0 and 48 h are
used for this purpose. Normal skin is used as a"blank" sample.
TUNEL assays are performed using commercial kits according to the
manufacturer's procedures. Active caspase-3 is detected on the
slides by immunofluorescence using a rabbit anti-active caspase-3
antibody. Quantification of TUNEL and caspase-3 positivity is done
on digitally acquired images at high power. The number of positive
cells per high power field is determined, and compared among the
groups.
[0169] Third, luminescence mapping is performed using Doppler
imaging to assess wound blood flow. Two hours after burn, the
dorsum of the animal is imaged on a scanning laser Doppler
apparatus to quantify the superficial blood flow distribution in
the skin within and outside of the burn area. For luminescence
mapping, 100 male Sprague-Dawley rats are used. Eighty animals
receive a large (covering 30% of the total body surface area)
full-thickness burn injury on the dorsum. This is a
well-established model. They are divided into 2 groups, one treated
with SS-31 and the other with placebo (saline) treatment. Each
group is further divided into 4 subgroups consisting of 4 time
points where animals will be sacrificed for further analysis. Prior
to sacrifice, luminescence imaging is carried out, followed by
euthanasia and skin tissue sampling for subsequent histology. The
remaining 20 animals will receive a "sham burn" and will be treated
with SS-31 or saline. Euthanasia is performed on two animals in
each of the corresponding 4 time points. On average, each animal
will be housed for 10 days (including the pre-burn days in the
animal farm) in separate cages.
[0170] It is predicted that SS-31 administration will accelerate
wound healing and attenuate the progression of burn injuries that
normally develops in the rat model. Measured outcomes include wound
contraction, reepithelialization distance, as well as any other
features that may be of interest in the dermis, such as cellularity
and collagen organization. Ki67 proliferation antigen will be
assessed, as well as TUNEL and caspase-3 positivity. Blood flow (as
measured by luminescence mapping) will also be measured. A
comparison is made between control rats and burned rats
administered SS-31. Successful treatment of burn injuries by the
aromatic-cationic peptides of the invention is indicated by a
reduction in one or more of the markers associated with burn injury
progression enumerated above.
Example 6--SS-31 Protects Against Protects Against Sunburn and
Attenuates Progression of Tissue Damage Following a Sunburn
(Prophetic)
[0171] In this Example, the effects of aromatic-cationic peptides
to protect against sunburn injury in a mammalian model are
examined. Hairless mice, with skin characteristics similar to
humans, are exposed to excessive UV radiation over the course of a
week. Subjects are randomly divided into three groups: (i)
burn-saline, (ii) burn-SS-31 (4 mgkg.sup.-1day.sup.-1; low dose
group), (ii) burn-SS-31 (40 mgkg.sup.-1day.sup.-1; high dose
group). Peptide dissolved in 1 ml/kg saline will be administered
intravenously twice a day for seven days.
[0172] It is predicted that SS-31 administration will accelerate
wound healing and attenuate the progression of sunburn injuries
that normally develops in the model. Measured outcomes include
wound contraction, reepithelialization distance, as well as any
other features that may be of interest in the dermis, such as
cellularity and collagen organization. Ki67 proliferation antigen
will be assessed, as well as TUNEL and caspase-3 positivity. Blood
flow (as measured by luminescence mapping) will also be measured. A
comparison is made between control rats and burned rats
administered SS-31. Controls include sham-burn (SB) and burn with
saline treatment (B). Successful prevention or amelioration of
sunburn injuries by the aromatic-cationic peptides of the invention
is indicated by a reduction in one or more of the markers
associated with burn injury progression enumerated above.
Example 7--SS-31 Attenuates the Burn Induced Hypermetabolism by the
Down Regulation of UCP-1 Expression in Brown Adipose Tissue
[0173] Hypermetabolism is the hallmark feature of metabolic
disturbance after burn injury. Mitochondria dysfunction occurs
after burns, and is closely related to the development of
hypermetabolism (and altered substrate oxidation). It was shown
that mitochondria targeted small peptide, SS-31, which penetrates
into mitochondria, inhibits mitochondrial swelling, and reduces
oxidative cell death, attenuates the hypermetabolism after burn
injury. Uncoupling protein 1 (UCP-1) is specifically expressed in
the brown adipose tissue, and plays a key role in producing heat.
The purpose of this example is to elucidate that the
down-regulation of UCP-1 is the key mechanism to attenuate the
hypermetabolism in burns treated with SS-31.
[0174] Methods.
[0175] Sprague Dawley rats were randomly divided into 5 groups;
sham (S), sham with saline treatment (SSal), sham with SS-31
treatment (SPep), burn with saline treatment (BSal) and burn with
SS-31 treatment (BPep). In burn group, the back of animal was
immersed into 100.degree. C. water for 12 seconds to produce III
degree 30% TBSA burns under general anesthesia. Sham burn was
produced by immersing the lukewarm water in the same manner. Both
groups of animals received 40 m/kg intraperitoneal saline injection
for the resuscitation following the injury. A venous catheter was
placed surgically into the right jugular vein subsequent to sham or
burn injury. SS-31 (2 mg/kg) or saline was injected as priming, and
infused for 7 days (4 mg/kg/day) using osmotic pump (Durect,
Calif.). S group meant the control group, which did not receive any
general anesthesia, sham or burn injury and catheter placement. The
indirect calorimetry was performed for 24 hours at 6 days after
burn injury in a TSE Indirect Calorimetry System (TSE Co. Germany),
and VO.sub.2, VCO.sub.2 and energy expenditure were recorded every
six minutes. Interscapullar brown adipose tissue was collected
after the indirect calorimetry, and UCP-1 expression in the brown
adipose tissue was evaluated by Western blot.
[0176] Results.
[0177] VO.sub.2, VCO.sub.2 and energy expenditure were
significantly increased in BSal group compared to SSal group
(p=0.000, p=0.000 and p=0.000, respectively). Those in BPep group
were significantly attenuated compared to BSal group (p<0.01,
p<0.05 and p<0.05, respectively). UCP-1 expression in BSal
group was 1.5 times higher than in SSal group (p<0.05).
Meanwhile, that in BPep group was 32% decreased compared to BSal
group (p=0.057).
[0178] These results show that SS-31 attenuates the burn induced
hypermetabolism by the down regulation of UCP-1 expression in brown
adipose tissue. As such, the aromatic cationic peptides described
herein are useful in methods for treating a subject suffering from
a burn injury.
Example 8--SS-31 Induces ATP Synthesis Rate Following a Burn in a
Mouse Burn Model
[0179] To demonstrate the treatment and prevention of post-burn
complications, the aromatic-cationic peptides of the invention were
tested on a murine model of mammalian burn injury. A major cause of
the mitochondrial skeletal muscle dysfunction in burns may result
from defects in oxidative phosphorylation (OXPHOS) via stimulation
of mitochondrial production of reactive oxygen species (ROS) and
the resulting damage to the mitochondrial DNA (mtDNA). This
hypothesis is supported by data indicating that the ATP synthesis
rate significantly decreases and ROS production increases in
skeletal muscle in response to burn injury. This progression
underlies the burn pathophysiology, which includes skeletal muscle
wasting and cachexia. Thus, this study investigated the potential
of aromatic-cationic peptides as a treatment modality to alleviate
skeletal muscle dysfunction after burns.
[0180] This example evaluated the effects of an aromatic cationic
peptide SS-31 in a clinically relevant burn trauma model using
.sup.31P NMR and electron paramagnetic resonance (EPR) in vivo. The
results showed that SS-31 peptide induces ATP synthesis rate by
causing recovery of the mitochondrial redox status at 6 hours after
burn.
[0181] Materials and Methods.
[0182] Male 6-week-old CD1 mice weighing 20-25 g were anesthetized
by intraperitoneal (i.p.) injection of 40 mg/kg pentobarbital
sodium. The left hind limb of all mice in all groups was shaved.
Each burned mouse was subjected to a nonlethal scald injury of 3-5%
total body surface area (TBSA) by immersing its left hind limb in
90.degree. C. water for 3 sec.
[0183] NMR spectroscopy is described in detail in Padfield, et al.,
Proc Natl Acad Sci USA 102: 5368-5373 (2005). Briefly, mice were
randomized into burn, burn+SS-31 peptide, control, and
control+peptide groups. The SS-31 peptide (3 mg/kg) was injected
intraperitoneally at 30 min prior to burn and a second injection
immediately after the burn. NMR experiments were performed in a
horizontal bore magnet (proton frequency 400 MHz, 21 cm diameter,
Magnex Scientific) using a Bruker Avance console. A 90.degree.
pulse was optimized for detection of phosphorus spectra (repetition
time 2 s, 400 averages, 4K data points). Saturation
90.degree.-selective pulse trains (duration 36.534 ms, bandwidth 75
Hz) followed by crushing gradients were used to saturate the
.gamma.-ATP peak. The same saturation pulse train was also applied
downfield of the inorganic phosphate (Pi) resonance, symmetrically
to the .gamma.-ATP resonance. T.sub.1 relaxation times of Pi and
phosphocreatine (PCr) were measured using an inversion recovery
pulse sequence in the presence of .gamma.-ATP saturation. An
adiabatic pulse (400 scans, sweep with 10 KHz, 4K data) was used to
invert Pi and PCr, with an inversion time between 152 ms and 7651
ms.
[0184] EPR spectroscopy is described in detail in Khan et al.,
"Burn Trauma in skeletal muscle results in oxidative stress as
assessed by in vivo electron paramagnetic resonance." Mol Med
Reports 1: 813-819 (2008). Briefly, mice were randomized into burn,
burn+SS-31 peptide and control groups. The SS-31 peptide (3 mg/kg)
was injected intraperitoneally at 0, 3, 6, 24, and 48 hours
post-burn. EPR measurements were carried out with a 1.2-GHz EPR
spectrometer equipped with a microwave bridge and external loop
resonator specially designed for in vivo experiments. The optimal
spectrometer parameters were: incident microwave power, 10 mW;
magnetic field center, 400 gauss; modulation frequency, 27 kHz. The
decay kinetics of intravenously-injected nitroxide (150 mg/kg) were
measured at the various time points, which indicated the
mitochondrial redox status of the muscle.
[0185] Results.
[0186] EPR was used to measure the redox status of burn and
burn+peptide groups at various times after the burn. FIG. 9 is a
graph showing the reduction of the nitroxide in the gastrocnemius
muscle before and after a burn injury. These results show that
subjects experience a significantly elevated redox status at 6 h
after a burn injury. FIGS. 10-14 show the reduction in nitroxide in
the gastrocnemius muscle before and after a burn injury in control,
burn and burn+peptide groups at 0, 3, 6, 24, and 48 h after a burn
injury, respectively. According to EPR, a significant decrease in
redox status of burn and burn+peptide groups as compared to control
was detected (p<0.05) at 6 h after burn; also, significant
increase (recovery) in redox status of burn+peptide group as
compared to burn was detected (p<0.05) (FIG. 15).
[0187] Burn injury caused significant reduction of ATP synthesis
rate at 6 hours (FIG. 11, Table 1). The peptide SS-31 induced a
significant increase in the ATP synthesis rate in burned mice and a
non-statistically significant increase in controls.
TABLE-US-00009 TABLE 8 Results of in vivo .sup.31P-NMR saturation
transfer experiments. Healthy Controls + Burn + Controls Peptide
Burn Peptide (n = 5) (n = 5) (n = 8) (n = 8) .DELTA.M/M.sub.0 0.24
.+-. 0.05 0.185 .+-. 0.02 0.23 .+-. 0.05 0.31 .+-. 0.06 (P = 0.097)
(P = 0.902) (P = 0.488) T.sub.1obs (s) 1.16 .+-. 0.14 1.16 .+-.
0.14 1.33 .+-. 0.27 1.33 .+-. 0.27 P.sub.i (.mu.mol/g) 1.01 .+-.
0.28 5.49 .+-. 0.28 0.34 .+-. 0.25 2.93 .+-. 0.56 (P = 0.0008) (P =
0.006) (P = 0.035) ATP synthesis 0.25 .+-. 0.09 0.74 .+-. 0.09 0.06
.+-. 0.02 0.63 .+-. 0.11 rate (.mu.mol/g/s) (P = 0.008) (P = 0.026)
(P = 0.046)
[0188] As shown in Table 8 and illustrated in FIG. 15: ATP
synthesis rate (Pi.fwdarw..gamma.ATP) at 6 hours after burn was
significantly reduced in burned (B) mice; and SS-31 treatment
resulted in significantly increased ATP synthesis rate in both
control (C+P) and burned (B+P) mice. Importantly, ATP synthesis
rate was significantly increased in burned mice injected with the
SS-31 (B+P), as compared to burned alone mice (B) (P=0.0001).
Moreover, when the ATP synthesis rate (reaction
PCr.fwdarw..gamma.ATP) was compared in burned mice and mice
injected with SS-31 the increase was statistically significant
(P=0.006)(Table 9). According to EPR, a significant decrease in the
redox status of burn and burn+SS-31 groups as compared to control
was detected (p<0.05); also, a significant increase (recovery)
in the redox status of burn+peptide group as compared to burn alone
was observed (p<0.05), (FIG. 12).
TABLE-US-00010 TABLE 9 Results of in vivo.sup.31P-NMR saturation
transfer experiments performed on the hindlimb skeletal muscle of
mice ATP synthesis rate (reaction PCr .fwdarw. .gamma.ATP) Healthy
Controls + Burn + Controls Peptide Burn Peptide (n = 5) (n = 5) (n
= 8) (n = 8) .DELTA.M/M.sub.0 0.24 .+-. 0.05 0.15 .+-. 0.02 0.23
.+-. 0.05 0.31 .+-. 0.06 (P = 0.097) (P = 0.902) (P = 0.488)
T.sub.1obs (s) 1.16 .+-. 0.14 1.16 .+-. 0.14 1.33 .+-. 0.27 1.33
.+-. 0.27 K.sub.f (s.sup.-1) 0.21 .+-. 0.04 0.14 .+-. 0.02 0.17
.+-. 0.04 0.24 .+-. 0.05 (P = 0.096) (P = 0.605) (P = 0.771) PCr
(.mu.mol/g) 2.28 .+-. 0.23 3.76 .+-. 0.51 1.16 .+-. 0.23 3.25 .+-.
0.29 (P = 0.047) (P = 0.01) (P = 0.054) ATP synthesis 0.50 .+-.
0.12 0.50 .+-. 0.06 0.24 .+-. 0.01 0.72 .+-. 0.11 rate
(.mu.mol/g/s) (P = 0.96) (P = 0.007) (P = 0.119)
[0189] In summary, the results show that SS-31 induces ATP
synthesis rate possibly via a recovery of the mitochondrial redox
status or via the peroxisome proliferator activated receptor-gamma
coactivator-1.beta. (PGC-1.beta.) which is downregulated as early
as 6 hours after burn. See Tzika et al., Int J Mol Med 21: 201-208,
2008. Thus, the mitochondrial dysfunction caused by burn injury
recovers with the administration of the SS-31 peptide.
Administration of the SS-31 peptide increased ATP synthesis rate
substantially even in control healthy mice. These data indicate
that the aromatic-cationic peptides of the invention are useful in
methods of preventing or treating secondary complications of a burn
injury, such as skeletal muscle dysfunction.
Example 9--SS-31 Effects on Mitochondrial Aconitase Activity
[0190] Mitochondrial aconitase is part of the TCA cycle and its
activity has been directly correlated with the TCA flux. Moreover,
its activity is inhibited by ROS and thus it is considered as an
index of oxidative stress. Here we present the local and systematic
effects of burn on the mitochondrial aconitase activity using a 5%
TBSA mice burn model. The effects of a mitochondrial peptide
(SS-31) had been also examined.
[0191] Although in burn, we would expect decreased levels of
mitochondrial aconitase activity due to the increased ROS
production, in our study we observed increased mitochondrial
aconitase activity both in burned (local burn effect) and
contralateral to burned leg (systemic burn effect), most probably
due to the hypermetabolism that burn injury induces (FIG. 16).
Thus, the increased ROS production known to occur in burn injury
and could inhibit this mitochondrial aconitase activity cannot
overcome the also existing hypermetabolism in burn, in terms of
mitochondrial aconitase activity and thus TCA flux. A similar
observation has been also shown in the case of exercise/repeated
contractions in intact human and isolated mouse skeletal muscle
although an increase in ROS is also observed in this situation.
Given the decreased ATP synthesis rate, we also observed in
skeletal muscle from the same burn model, this result could account
as an indirect evident that in this particular hypermetabolic
syndrome TCA is futile.
[0192] These results show that SS-31 administration in burned
animals decreased mitochondrial aconitase activity up to the
control levels, indicating thus that SS-31 recovers the TCA flux,
may be in response to a more effective aerobic respiration as this
is suggested by the increased ATP synthesis rate compared with
untreated burned animals.
Example 10--A Single Dose of SS-31 Induces ATP Synthesis Rate
Following a Burn in a Mouse Burn Model
[0193] This example evaluated the effects of an aromatic cationic
peptide SS-31 in a clinically relevant burn trauma model using
.sup.31P NMR in vivo. The results showed that SS-31 peptide induces
ATP synthesis rate by causing recovery of the mitochondrial redox
status after burn.
[0194] Male 6-week-old CD1 mice (20-25 g) were anesthetized by
intraperitoneal injection of 40 mg/kg pentobarbital sodium and the
left hind limb of all mice was shaved. Burn injury was inflicted by
a nonlethal scald injury of 3-5% total body surface area by
immersing the left hind limb in 90.degree. C. water for 3 sec. Mice
were randomized into burn (B), burn+SS-31 (B+P), control (C) and
control+SS-31 (C+P) groups. SS-31 (3 mg/kg) was injected
intraperitoneally at 30 min before burn and immediately after burn.
A separate group of burned animals received only one dose of the
SS-31 peptide immediately after burn.
TABLE-US-00011 TABLE 10 Healthy Controls + Burn + Burn + Controls
Peptide Burn Peptide Peptide* (n = 5) (n = 5) (n = 8) (n = 8) (n =
8) .DELTA.M/M.sub.0 0.24 .+-. 0.15 .+-. 0.02 0.23 .+-. 0.05 0.31
.+-. 0.06 0.39 .+-. 0.07 0.05 (P = 0.097) (P = 0.902) (P = 0.488)
(P = 0.072) Tl.sub.obs (s) 1.16 .+-. 1.16 .+-. 0.14 1.33 .+-. 0.27
1.33 .+-. 0.27 1.33 .+-. 0.27 0.14 Pi 1.01 .+-. 5.49 .+-. 0.28 0.34
.+-. 0.25 2.93 .+-. 0.56 1.41 .+-. 0.26 (.mu.mol/g) 0.28 (P =
0.0008) (P = 0.006) (P = 0.035) (P = 0.166) ATP 0.25 .+-. 0.74 .+-.
0.09 0.06 .+-. 0.02 0.63 .+-. 0.11 0.36 .+-. 0.08 synthesis 0.09 (P
= 0.008) ( P = 0.026) (P = 0.046) (P = 0.211) rate
(.mu.mol/g/s)
[0195] The results are shown in Table 10. Values are means.+-.SE;
.DELTA.M/M0 is the fractional change in Pi magnetization as a
result of saturation transfer; T1.sub.obs is the observed spin
lattice relaxation time of Pi during .gamma.ATP saturation in
seconds; ATP synthesis is calculated as [Pi].times.k; [Pi] is the
concentration of Pi extrapolated from the baseline NMR spectrum,
comparing Pi and .gamma.ATP peaks and ATP concentration measured
with bioluminescence assay; k is calculated as
(1/T1.sub.obs).times.(.DELTA.M/M0); P-values (unpaired Student's
t-test) are for comparisons between experimental and control
groups; * indicates only one dose of SS-31 peptide (3 mg/kg) was
injected to animals immediately after burn. Thus, the last column
of the table shows that SS-31 normalizes ATP synthesis rate even at
a single dose after burn. These data indicate that the
aromatic-cationic peptides of the invention are useful in methods
of preventing or treating secondary complications of a burn
injury.
[0196] 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.
[0197] 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.
[0198] 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.
[0199] 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.
[0200] Other embodiments are set forth within the following
claims.
* * * * *