U.S. patent application number 14/908053 was filed with the patent office on 2016-06-16 for methods and compositions for the prevention and treatment of friedreich's ataxia.
This patent application is currently assigned to Stealth Bio Therapeutics Corp. The applicant listed for this patent is STEALTH PEPTIDES INTERNATIONAL, INC.. Invention is credited to D. Travis Wilson.
Application Number | 20160166633 14/908053 |
Document ID | / |
Family ID | 52432489 |
Filed Date | 2016-06-16 |
United States Patent
Application |
20160166633 |
Kind Code |
A1 |
Wilson; D. Travis |
June 16, 2016 |
METHODS AND COMPOSITIONS FOR THE PREVENTION AND TREATMENT OF
FRIEDREICH'S ATAXIA
Abstract
The disclosure provides methods of preventing or treating
Friedreich's ataxia in a mammalian subject, reducing risk factors,
signs and/or symptoms associated with Friedreich's ataxia, and/or
reducing the likelihood or severity of Friedreich's ataxia. The
methods comprise administering to the subject an effective amount
of an aromatic-cationic peptide to e.g., reduce oxidative stress,
increase mitochondrial metabolism, or a combination thereof.
Inventors: |
Wilson; D. Travis; (Newton,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
STEALTH PEPTIDES INTERNATIONAL, INC. |
des Citronniers |
|
MC |
|
|
Assignee: |
Stealth Bio Therapeutics
Corp
Monaco
MC
|
Family ID: |
52432489 |
Appl. No.: |
14/908053 |
Filed: |
August 4, 2014 |
PCT Filed: |
August 4, 2014 |
PCT NO: |
PCT/US2014/049633 |
371 Date: |
January 27, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61861806 |
Aug 2, 2013 |
|
|
|
Current U.S.
Class: |
514/17.7 |
Current CPC
Class: |
A61K 38/07 20130101;
A61K 31/122 20130101; A61K 31/7048 20130101; A61K 31/401 20130101;
A61K 31/4412 20130101; A61K 31/7048 20130101; A61K 38/28 20130101;
A61K 45/06 20130101; A61K 31/4412 20130101; C07K 5/1019 20130101;
A61K 38/28 20130101; A61K 31/122 20130101; A61K 31/401 20130101;
A61K 38/07 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 38/07 20060101
A61K038/07; A61K 45/06 20060101 A61K045/06; C07K 5/11 20060101
C07K005/11 |
Claims
1. A method for treating or preventing Friedreich's ataxia or the
signs or symptoms of reduced frataxin levels or activity in a
subject in need thereof, comprising administering to the subject a
therapeutically effective amount of the peptide
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 or a pharmaceutically acceptable
salt thereof.
2. The method of claim 1, wherein the subject displays reduced
levels of frataxin expression compared to a normal control
subject.
3. The method of claim 1, wherein the peptide is administered daily
for 6 weeks or more.
4. The method of claim 1, wherein the peptide is administered daily
for 12 weeks or more.
5. The method of claim 1, wherein the subject has been diagnosed as
having Friedreich's ataxia.
6. The method of claim 5, wherein the Friedreich's ataxia comprises
one or more of muscle weakness, loss of coordination, vision
impairment, hearing impairment, slurred speech, curvature of the
spine, diabetes, and heart disorders.
7. The method of claim 1, wherein the subject is human.
8. The method of claim 1, wherein the peptide is administered
orally, topically, systemically, intravenously, subcutaneously,
intraperitoneally, or intramuscularly.
9. The method of claim 1, further comprising separately,
sequentially or simultaneously administering to the subject one or
more therapeutic agents selected from the group consisting of ACE
inhibitors, digoxin, enalapril, or lisinopril, diuretics, beta
blockers, idebenone, deferiprone, and insulin.
10. The method of claim 1, wherein the pharmaceutically acceptable
salt comprises acetate or trifluoroacetate salt.
11. A method for reducing mitochondrial iron in a mammalian subject
having or suspected of having Friedreich's ataxia, the method
comprising: administering to the subject a therapeutically
effective amount of the peptide D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 or
a pharmaceutically acceptable salt thereof.
12. The method of claim 11, wherein the mammalian subject has
decreased expression of frataxin compared to a normal control
subject.
13. The method of claim 11, wherein the peptide is administered
daily for 6 weeks or more.
14. The method of claim 11, wherein the peptide is administered
daily for 12 weeks or more.
15. The method of claim 11, wherein the Friedreich's ataxia
comprises one or more of muscle weakness, loss of coordination,
motor control impairment, vision impairment, hearing impairment,
slurred speech, curvature of the spine, diabetes, and heart
disorders.
16. The method of claim 11, wherein the subject is human.
17. The method of claim 11, wherein the peptide is administered
orally, topically, systemically, intravenously, subcutaneously,
intraperitoneally, or intramuscularly
18. The method of claim 11, further comprising separately,
sequentially or simultaneously administering to the subject one or
more therapeutic agents selected from the group consisting of ACE
inhibitors, digoxin, enalapril, or lisinopril, diuretics, beta
blockers, idebenone, deferiprone, and insulin.
19. (canceled)
20. The method of claim 9, wherein the combination of peptide and
an additional therapeutic agent has a synergistic effect in the
prevention or treatment of Friedreich's ataxia.
21. The method of claim 18, wherein the combination of peptide and
an additional therapeutic agent has a synergistic effect in the
prevention or treatment of Friedreich's ataxia.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to U.S.
Application No. 61/861,806, filed Aug. 2, 2013, the content of
which is incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The present technology relates generally to compositions and
methods for ameliorating or treating Friedreich's ataxia and/or
reducing the severity of Friedreich's ataxia. In particular, the
present technology relates to administering an effective amount of
an aromatic-cationic peptide to a subject suffering from
Friedreich's ataxia.
BACKGROUND
[0003] The following description is provided to assist the
understanding of the reader. None of the information provided or
references cited is admitted to be prior art to the compositions
and methods disclosed herein.
[0004] Friedreich's ataxia is an inherited autosomal recessive
disease that causes progressive damage to the nervous system. The
ataxia results from the degeneration of nerve tissue in the spinal
cord, in particular, sensory neurons essential for directing muscle
movement of the arms and legs. The spinal cord becomes thinner and
nerve cells lose some of their myelin sheath.
[0005] Friedreich's ataxia occurs when the FXN gene contains
amplified intronic GAA repeats. The mutant FXN gene contains
expanded GAA triplet repeats in the first intron; in a few
pedigrees, point mutations have also been detected. Since the
defect is located in an intron, which is removed from the mRNA
transcript between transcription and translation, the mutated FXN
gene does not result in the production of abnormal proteins.
Instead, the mutation causes gene silencing, i.e., the mutation
decreases the transcription of the gene, through induction of a
heterochromatin structure in a manner similar to position-effect
variegation.
[0006] The FXN gene encodes the protein frataxin. GAA repeat
expansion causes frataxin levels to be reduced. Frataxin is an iron
binding protein responsible for forming iron-sulphur clusters. One
result of frataxin deficiency is mitochondrial iron overload.
SUMMARY
[0007] In one aspect, the present disclosure provides methods for
treating or preventing Friedreich's ataxia, and/or treating or
preventing the signs or symptoms of reduced levels of frataxin or
frataxin activity in a subject in need thereof by administering to
the subject a therapeutically effective amount of an
aromatic-cationic peptide such as D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2
or a pharmaceutically acceptable salt thereof.
[0008] In some embodiments, the subject displays reduced levels of
frataxin compared to a normal control subject.
[0009] In some embodiments, the peptide is administered daily for 6
weeks or more. In some embodiments, the peptide is administered
daily for 12 weeks or more.
[0010] In some embodiments, the subject has been diagnosed as
having Friedreich's ataxia.
[0011] In some embodiments, the Friedreich's ataxia includes one or
more of muscle weakness, loss of coordination, vision impairment,
hearing impairment, slurred speech, curvature of the spine,
diabetes, and heart disorders.
[0012] In some embodiments, the subject is human.
[0013] In some embodiments, the peptide is administered orally,
topically, systemically, intravenously, subcutaneously,
intraperitoneally, or intramuscularly
[0014] In some embodiments, the method also includes separately,
sequentially or simultaneously administering to the subject one or
more agents selected from the group consisting of ACE inhibitors,
digoxin, enalapril, or lisinopril, diuretics, beta blockers,
idebenone, deferiprone, and insulin. In some embodiments, there is
a synergistic effect between the peptide and the additional agent
in this regard.
[0015] In some embodiments, the pharmaceutically acceptable salt
comprises acetate or trifluoroacetate salt.
[0016] In one aspect, the present technology provides a method for
reducing mitochondrial iron in a mammalian subject having or
suspected of having Friedreich's ataxia, the method comprising:
administering to the subject a therapeutically effective amount of
the peptide D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 or a pharmaceutically
acceptable salt thereof. In some embodiments, the mammalian subject
has decreased expression of frataxin compared to a normal control
subject. In some embodiments, the subject is human.
[0017] In some embodiments, the peptide is administered daily for 6
weeks or more. In some embodiments, the peptide is administered
daily for 12 weeks or more.
[0018] In some embodiments, the Friedreich's ataxia comprises one
or more of muscle weakness, loss of coordination, vision
impairment, hearing impairment, slurred speech, curvature of the
spine, diabetes, and heart disorders.
[0019] In some embodiments, the peptide is administered orally,
topically, systemically, intravenously, subcutaneously,
intraperitoneally, or intramuscularly
[0020] In some embodiments, the method includes administering
separately, sequentially or simultaneously to the subject one or
more therapeutic agents selected from the group consisting of ACE
inhibitors, digoxin, enalapril, or lisinopril, diuretics, beta
blockers, idebenone, deferiprone, and insulin.
[0021] In some embodiments, the pharmaceutically acceptable salt
comprises acetate or trifluoroacetate salt.
[0022] In some embodiments, the combination of peptide and an
additional therapeutic agent has a synergistic effect in the
reduction of mitochondrial iron and/or prevention or treatment of
Friedreich's ataxia.
[0023] In one aspect, the present technology provides for methods
for reducing the risk, signs or symptoms of Friedreich's ataxia in
a mammalian subject having decreased expression of frataxin
compared to a normal control subject. In some embodiments, the
method includes administering to the subject a therapeutically
effective amount of the peptide D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 or
a pharmaceutically acceptable salt thereof
[0024] In one aspect, the present technology provides for methods
of stabilizing mitochondrial metabolism in a mammalian subject
having or suspected of having Friedreich's ataxia and/or having
lower than control or normal levels of frataxin. In some
embodiments, the method includes administering to the subject a
therapeutically effective amount of the peptide
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 or a pharmaceutically acceptable
salt thereof.
[0025] In one aspect, the disclosure provides a method of treating
or preventing Friedreich's ataxia in a mammalian subject,
comprising administering to said mammalian subject a
therapeutically effective amount of an aromatic-cationic peptide.
In some embodiments, the aromatic-cationic peptide is a peptide
having:
[0026] at least one net positive charge;
[0027] a minimum of four amino acids;
[0028] a maximum of about twenty amino acids;
[0029] a relationship between the minimum number of net positive
charges (p.sub.m) and the total number of amino acid residues (r)
wherein 3p.sub.m is the largest number that is less than or equal
to r+1; and a relationship between the minimum number of aromatic
groups (a) and the total number of net positive charges (p.sub.t)
wherein 2a is the largest number that is less than or equal to
p.sub.t+1, except that when a is 1, p.sub.t may also be 1. In
particular embodiments, the mammalian subject is a human.
[0030] In one embodiment, 2p.sub.m is the largest number that is
less than or equal to r+1, and may be equal to p.sub.t. The
aromatic-cationic peptide may be a water-soluble peptide having a
minimum of two or a minimum of three positive charges.
[0031] In one embodiment, the peptide comprises one or more
non-naturally occurring amino acids, for example, one or more
D-amino acids. In some embodiments, the C-terminal carboxyl group
of the amino acid at the C-terminus is amidated. In certain
embodiments, the peptide has a minimum of four amino acids. The
peptide may have a maximum of about 6, a maximum of about 9, or a
maximum of about 12 amino acids.
[0032] In one embodiment, the peptide may have the formula
Phe-D-Arg-Phe-Lys-NH.sub.2 or 2',6'-Dmp-D-Arg-Phe-Lys-NH.sub.2. In
a particular embodiment, the aromatic-cationic peptide has the
formula D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2.
[0033] In one embodiment, the peptide is defined by formula I:
##STR00001##
[0034] wherein R.sup.1 and R.sup.2 are each independently selected
from
[0035] (i) hydrogen;
[0036] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
##STR00002##
R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9,
R.sup.10, R.sup.11 and R.sup.12 are each independently selected
from
[0037] (i) hydrogen;
[0038] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
[0039] (iii) C.sub.1-C.sub.6 alkoxy;
[0040] (iv) amino;
[0041] (v) C.sub.1-C.sub.4 alkylamino;
[0042] (vi) C.sub.1-C.sub.4 dialkylamino;
[0043] (vii) nitro;
[0044] (viii) hydroxyl;
[0045] (ix) halogen, where "halogen" encompasses chloro, fluoro,
bromo, and iodo; and
n is an integer from 1 to 5.
[0046] 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.
[0047] In one embodiment, the peptide is defined by formula II:
##STR00003##
[0048] wherein R.sup.1 and R.sup.2 are each independently selected
from
[0049] (i) hydrogen;
[0050] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
##STR00004##
R.sup.3 and R.sup.4 are each independently selected from
[0051] (i) hydrogen;
[0052] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
[0053] (iii) C.sub.1-C.sub.6 alkoxy;
[0054] (iv) amino;
[0055] (v) C.sub.1-C.sub.4 alkylamino;
[0056] (vi) C.sub.1-C.sub.4 dialkylamino;
[0057] (vii) nitro;
[0058] (viii) hydroxyl;
[0059] (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
[0060] (i) hydrogen;
[0061] (ii) linear or branched C.sub.1-C.sub.6 alkyl;
[0062] (iii) C.sub.1-C.sub.6 alkoxy;
[0063] (iv) amino;
[0064] (v) C.sub.1-C.sub.4 alkylamino;
[0065] (vi) C.sub.1-C.sub.4 dialkylamino;
[0066] (vii) nitro;
[0067] (viii) hydroxyl;
[0068] (ix) halogen, where "halogen" encompasses chloro, fluoro,
bromo, and iodo; and
n is an integer from 1 to 5.
[0069] The aromatic-cationic peptides may be administered in a
variety of ways. In some embodiments, the peptides may be
administered orally, topically, intranasally, intravenously,
subcutaneously, or transdermally (e.g., by iontophoresis).
DETAILED DESCRIPTION
[0070] 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 technology. 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 technology belongs.
[0071] 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.
[0072] 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), or
topically. Administration includes self-administration and the
administration by another.
[0073] 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 those 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.
[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 that reduces, ameliorates or
delays the onset of the physiological symptoms of Friedreich's
ataxia. In the context of therapeutic or prophylactic applications,
in some embodiments, 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. In some embodiments,
it will also depend on the degree, severity and type of disease.
The skilled artisan will be able to determine appropriate dosages
depending on these and other factors. The compositions can also be
administered in combination with one or more additional therapeutic
compounds. In the methods described herein, aromatic-cationic
peptides, such as D-Arg-2'6'-Dmt-Lys-Phe-NH.sub.2, or a
pharmaceutically acceptable salt thereof, such as acetate or
trifluoroacetate salt, may be administered to a subject having one
or more signs, symptoms, or risk factors of Friedreich's ataxia,
such as, e.g., muscle weakness, especially in the arms and legs,
loss of coordination, motor control impairment, vision impairment,
hearing impairment, slurred speech, curvature of the spine,
diabetes, and heart disorders. For example, a "therapeutically
effective amount" of the aromatic-cationic peptides includes levels
at which the presence, frequency, or severity of one or more signs,
symptoms, or risk factors of Friedreich's ataxia are reduced or
eliminated. In some embodiments, a therapeutically effective amount
reduces or ameliorates the physiological effects of Friedreich's
ataxia, and/or the risk factors of Friedreich's ataxia, and/or
delays the progression or onset of Friedreich's ataxia.
[0075] As used herein, "isolated" or "purified" polypeptide or
peptide refers to a polypeptide or peptide that is substantially
free of cellular material or other contaminating polypeptides from
the cell or tissue source from which the agent is derived, or
substantially free from chemical precursors or other chemicals when
chemically synthesized. For example, an isolated aromatic-cationic
peptide would be free of materials that would interfere with
diagnostic or therapeutic uses of the agent. Such interfering
materials may include enzymes, hormones and other proteinaceous and
nonproteinaceous solutes.
[0076] 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.
[0077] As used herein, the term "simultaneous" therapeutic use
refers to the administration of at least two active ingredients by
the same route and at the same time or at substantially the same
time.
[0078] As used herein, the term "separate" therapeutic use refers
to an administration of at least two active ingredients at the same
time or at substantially the same time by different routes.
[0079] As used herein, the term "sequential" therapeutic use refers
to administration of at least two active ingredients at different
times, the administration route being identical or different. More
particularly, sequential use refers to the whole administration of
one of the active ingredients before administration of the other or
others commences. It is thus possible to administer one of the
active ingredients over several minutes, hours, or days before
administering the other active ingredient or ingredients. There is
no simultaneous treatment in this definition.
[0080] As used herein, the terms "treating" or "treatment" or
"alleviation" refers to therapeutic treatment, wherein the object
is to reduce, alleviate or slow down (lessen) the targeted
pathologic condition or disorder. By way of example, but not by way
of limitation, a subject is successfully "treated" for Friedreich's
ataxia if, after receiving a therapeutic amount of the
aromatic-cationic peptides, such as
D-Arg-2'6'-Dmt-Lys-Phe-NH.sub.2, or a pharmaceutically acceptable
salt thereof, such as acetate or trifluoroacetate salt, 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 Friedreich's ataxia, such as but not limited to, e.g.,
muscle weakness, especially in the arms and legs, loss of
coordination, motor control impairment, vision impairment, hearing
impairment, slurred speech, curvature of the spine, diabetes, and
heart disorders. It is also to be appreciated that the various
modes of treatment of medical conditions as described are intended
to mean "substantial," which includes total but also less than
total treatment, and wherein some biologically or medically
relevant result is achieved. Treating Friedreich's ataxia, as used
herein, also refers to treating the signs and symptoms related to
reduced frataxin activity or frataxin expression levels
characteristic of Friedreich's ataxia.
[0081] As used herein, "prevention" or "preventing" of a disease or
condition, e.g., Friedreich's ataxia refers to results that, in a
statistical sample, exhibit a reduction in the occurrence of the
disorder or condition in the treated sample relative to an
untreated control sample, or exhibit a delay in the onset of one or
more symptoms of the disorder or condition relative to the
untreated control sample. As used herein, preventing Friedreich's
ataxia includes preventing or delaying the initiation of,
preventing, delaying, or slowing the progression or advancement of
Friedreich's ataxia. As used herein, prevention of Friedreich's
ataxia also includes preventing a recurrence of one or more signs
or symptoms of Friedreich's ataxia.
Aromatic-Cationic Peptides
[0082] The present technology relates to methods and compositions
for preventing or treating Friedreich's ataxia in a subject in need
thereof. In some embodiments, the methods and compositions prevent
one or more signs or symptoms of Friedreich's ataxia in a subject.
In some embodiments, the methods and compositions increase the
level of frataxin expression in a subject. In some embodiments, the
methods and compositions reduce the likelihood that a subject with
risk factors for Friedreich's ataxia will develop one or more signs
or symptoms of Friedreich's ataxia, or will delay the onset of
Friedreich's ataxia. In some embodiments, the methods and
compositions include an aromatic-cationic peptide such as
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a pharmaceutically acceptable
salt thereof, such as acetate or trifluoroacetate salt.
[0083] It is known in the art that aromatic-cationic peptides of
the present technology, such as D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2,
possess anti-oxidant properties, including the capacity to reduce
the rate of lipid oxidation, peroxidation, mitochondrial
H.sub.2O.sub.2 production, and intracellular reactive oxygen
species (ROS) production. It is further known in the art that
aromatic-cationic peptides of the present technology, such as
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, localize to the mitochondria, and
have the capacity to inhibit caspase activation and apoptosis. It
has also been shown that aromatic-cationic peptides, such as
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, restore mitochondria membrane
potential. These and other properties of aromatic-cationic peptides
of the present technology, such as
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, are demonstrated in U.S.
application Ser. No. 11/040,242 (U.S. Pat. No. 7,550,439) and Ser.
No. 10/771,232 (U.S. Pat. No. 7,576,061). Accordingly,
aromatic-cationic peptides of the present technology, such as
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, are useful in the prevention and
treatment of diseases and conditions caused by, resulting from, or
otherwise associated with such cellular events, such as
Friedreich's ataxia.
[0084] The aromatic-cationic peptides are water-soluble and highly
polar. Despite these properties, the peptides can readily penetrate
cell membranes. The aromatic-cationic peptides typically include a
minimum of three amino acids or a minimum of four amino acids,
covalently joined by peptide bonds. The maximum number of amino
acids present in the aromatic-cationic peptides is about twenty
amino acids covalently joined by peptide bonds. Suitably, the
maximum number of amino acids is about twelve, about nine, or about
six.
[0085] The amino acids of the aromatic-cationic peptides can be any
amino acid. As used herein, the term "amino acid" is used to refer
to any organic molecule that contains at least one amino group and
at least one carboxyl group. Typically, at least one amino group is
at the a position relative to a carboxyl group. The amino acids may
be naturally occurring. Naturally occurring amino acids include,
for example, the twenty most common levorotatory (L) amino acids
normally found in mammalian proteins, i.e., alanine (Ala), arginine
(Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys),
glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine
(His), isoleucine (Ile), 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. Another example of a naturally occurring amino
acid includes hydroxyproline (Hyp).
[0086] The peptides optionally contain one or more non-naturally
occurring amino acids. Optimally, the peptide has no amino acids
that are naturally occurring. The non-naturally occurring amino
acids may be levorotary (L-), dextrorotatory (D-), or mixtures
thereof. 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 suitably are also
not recognized by common proteases. 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.
[0087] The non-natural amino acids may, for example, comprise
alkyl, aryl, or alkylaryl groups not found in natural amino acids.
Some examples of non-natural alkyl amino acids include
.alpha.-aminobutyric acid, .beta.-aminobutyric acid,
.gamma.-aminobutyric acid, .delta.-aminovaleric acid, and
.epsilon.-aminocaproic acid. Some examples of non-natural aryl
amino acids include ortho-, meta-, and para-aminobenzoic acid. Some
examples of non-natural alkylaryl amino acids include ortho-,
meta-, and para-aminophenylacetic acid, and
.gamma.-phenyl-.beta.-aminobutyric acid. Non-naturally occurring
amino acids include derivatives of naturally occurring amino acids.
The derivatives of naturally occurring amino acids may, for
example, include the addition of one or more chemical groups to the
naturally occurring amino acid.
[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) and norleucine (Nle).
[0089] Another example of a modification of an amino acid in a
peptide 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 or 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, or
less than two contiguous L-amino acids recognized by common
proteases, irrespective of whether the amino acids are naturally or
non-naturally occurring. Optimally, the peptide has only D-amino
acids, and no L-amino acids. If the peptide contains protease
sensitive sequences of amino acids, at least one of the amino acids
is, in some embodiments, 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] The aromatic-cationic peptides should 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 is 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.
[0094] Typically, a peptide has a positively charged N-terminal
amino group and a negatively charged C-terminal carboxyl group. The
charges cancel each other out at physiological pH. As an example of
calculating net charge, the peptide
Tyr-Arg-Phe-Lys-Glu-His-Trp-D-Arg has one negatively charged amino
acid (i.e., Glu) and four positively charged amino acids (i.e., two
Arg residues, one Lys, and one His). Therefore, the above peptide
has a net positive charge of three.
[0095] 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 3p.sub.m is the largest number that is less
than or equal to r+1. In this embodiment, the relationship between
the minimum number of net positive charges (p.sub.m) and the total
number of amino acid residues (r) is as follows:
TABLE-US-00001 TABLE 2 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
[0096] In another embodiment, the aromatic-cationic peptides have a
relationship between the minimum number of net positive charges
(p.sub.m) and the total number of amino acid residues (r) wherein
2p.sub.m is the largest number that is less than or equal to r+1.
In this embodiment, the relationship between the minimum number of
net positive charges (p.sub.m) and the total number of amino acid
residues (r) is as follows:
TABLE-US-00002 TABLE 3 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
[0097] 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,
suitably, a minimum of two net positive charges or a minimum of
three net positive charges.
[0098] It is also important that the aromatic-cationic peptides
have a minimum number of aromatic groups in comparison to the total
number of net positive charges (p.sub.t). The minimum number of
aromatic groups will be referred to below as (a). 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).
[0099] The aromatic-cationic peptides should also 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 4 Aromatic groups and net positive charges (3a
.ltoreq. p.sub.t + 1 or a = p.sub.t = 1) (p.sub.t) 1 2 3 4 5 6 7 8
9 10 11 12 13 14 15 16 17 18 19 20 (a) 1 1 1 1 2 2 2 3 3 3 4 4 4 5
5 5 6 6 6 7
[0100] 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 5 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
[0101] In another embodiment, the number of aromatic groups (a) and
the total number of net positive charges (p.sub.t) are equal.
[0102] Carboxyl groups, especially the terminal carboxyl group of a
C-terminal amino acid, are suitably 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. 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 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 peptide examples:
TABLE-US-00005 TABLE 6 EXEMPLARY PEPTIDES
2',6'-Dmp-D-Arg-2',6'-Dmt-Lys-NH.sub.2
2',6'-Dmp-D-Arg-Phe-Lys-NH.sub.2 2',6'-Dmt-D-Arg-PheOrn-NH.sub.2
2',6'-Dmt-D-Arg-Phe-Ahp(2-aminoheptanoicacid)-NH.sub.2
2',6'-Dmt-D-Arg-Phe-Lys-NH.sub.2 2',6'-Dmt-D-Cit-PheLys-NH.sub.2
Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-Phe
Arg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-Phe-Tyr-D-
Arg-Gly
Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-
D-Phe-Lys-Phe Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH.sub.2
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2
D-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-
His-Phe-NH.sub.2 D-His-Glu-Lys-Tyr-D-Phe-Arg
D-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-Asp-D-His-D-Lys-Arg-Trp-NH.sub.2
D-Tyr-Trp-Lys-NH.sub.2
Glu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met-
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. Gly-D-Phe-Lys-His-D-Arg-Tyr-NH.sub.2
His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-
Tyr-His-Ser-NH.sub.2 Lys-D-Arg-Tyr-NH.sub.2
Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH.sub.2
Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH.sub.2
Met-Tyr-D-Arg-Phe-Arg-NH.sub.2 Met-Tyr-D-Lys-Phe-Arg
Phe-Arg-D-His-Asp Phe-D-Arg-2',6'-Dmt-Lys-NH.sub.2 Phe-D-Arg-His
Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His Phe-D-Arg-Phe-Lys-NH.sub.2
Phe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH.sub.2
Phe-Tyr-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-Thr
Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-Lys
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 Trp-D-Lys-Tyr-Arg-NH.sub.2
Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-Lys
Tyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-Lys
Tyr-D-Arg-Phe-Lys-Glu-NH.sub.2 Tyr-D-Arg-Phe-Lys-NH.sub.2
Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-Phe
Tyr-His-D-Gly-Met Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH.sub.2
[0105] In one embodiment, the peptides have mu-opioid receptor
agonist activity (i.e., they activate the mu-opioid receptor).
Peptides, which have mu-opioid receptor agonist activity, are
typically those peptides that have a tyrosine residue or a tyrosine
derivative at the N-terminus (i.e., the first amino acid position).
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, a peptide that has mu-opioid receptor
agonist activity has the formula Tyr-D-Arg-Phe-Lys-NH.sub.2.
Tyr-D-Arg-Phe-Lys-NH.sub.2 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
Tyr-D-Arg-Phe-Lys-NH.sub.2 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.
2',6'-Dmt-D-Arg-Phe-Lys-NH.sub.2 has a molecular weight of 640 and
carries a net three positive charge at physiological pH.
2',6'-Dmt-D-Arg-Phe-Lys-NH.sub.2 readily penetrates the plasma
membrane of several mammalian cell types in an energy-independent
manner (Zhao, et al., J. Pharmacol Exp Ther., 304:425-432,
2003).
[0107] Alternatively, in other instances, the aromatic-cationic
peptide does not have mu-opioid receptor agonist activity. For
example, during long-term treatment, such as in a chronic disease
state or condition, the use of an aromatic-cationic peptide that
activates the mu-opioid receptor may be contraindicated. In these
instances, the potentially adverse or addictive effects of the
aromatic-cationic peptide may preclude the use of an
aromatic-cationic peptide that activates the mu-opioid receptor in
the treatment regimen of a human patient or other mammal. Potential
adverse effects may include sedation, constipation and respiratory
depression. In such instances an aromatic-cationic peptide that
does not activate the mu-opioid receptor may be an appropriate
treatment. Peptides that do not have mu-opioid receptor agonist
activity generally do not have a tyrosine residue or a derivative
of tyrosine at the N-terminus (i.e., amino acid position 1). The
amino acid at the N-terminus can be any naturally occurring or
non-naturally occurring amino acid other than tyrosine. In one
embodiment, the amino acid at the N-terminus is phenylalanine or
its derivative. Exemplary derivatives of phenylalanine include
2'-methylphenylalanine (Mmp), 2',6'-dimethylphenylalanine
(2',6'-Dmp), N, 2',6'-trimethylphenylalanine (Tmp), and
2'-hydroxy-6'-methylphenylalanine (Hmp).
[0108] An example of an aromatic-cationic peptide that does not
have mu-opioid receptor agonist activity has the formula
Phe-D-Arg-Phe-Lys-NH.sub.2. Alternatively, the N-terminal
phenylalanine can be a derivative of phenylalanine such as
2',6'-dimethylphenylalanine (2',6'-Dmp). Tyr-D-Arg-Phe-Lys-NH.sub.2
containing 2',6'-dimethylphenylalanine at amino acid position 1 has
the formula 2',6'-Dmp-D-Arg-Phe-Lys-NH.sub.2. In one embodiment,
the amino acid sequence of 2',6'-Dmt-D-Arg-Phe-Lys-NH.sub.2 is
rearranged such that Dmt is not at the N-terminus. An example of
such an aromatic-cationic peptide that does not have mu-opioid
receptor agonist activity has the formula
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2.
[0109] Suitable substitution variants of the peptides listed herein
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 are 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.
[0116] Examples of peptides that activate mu-opioid receptors
include, but are not limited to, the aromatic-cationic peptides
shown in Table 7.
TABLE-US-00006 TABLE 7 Peptide Analogs with Mu-Opioid Activity
Amino Acid Amino Acid Amino Acid Amino Acid C-Terminal Position 1
Position 2 Position 3 Position 4 Modification Tyr D-Arg Phe Lys
NH.sub.2 Tyr D-Arg Phe Orn NH.sub.2 Tyr D-Arg Phe Dab NH.sub.2 Tyr
D-Arg Phe Dap NH.sub.2 2'6'Dmt D-Arg Phe Lys NH.sub.2 2'6'Dmt D-Arg
Phe Lys-NH(CH.sub.2).sub.2--NH-dns NH.sub.2 2'6'Dmt D-Arg Phe
Lys-NH(CH.sub.2).sub.2--NH-atn NH.sub.2 2'6'Dmt D-Arg Phe dnsLys
NH.sub.2 2'6'Dmt D-Cit Phe Lys NH.sub.2 2'6'Dmt D-Cit Phe Ahp
NH.sub.2 2'6'Dmt D-Arg Phe Orn NH.sub.2 2'6'Dmt D-Arg Phe Dab
NH.sub.2 2'6'Dmt D-Arg Phe Dap NH.sub.2 2'6'Dmt D-Arg Phe
Ahp(2-aminoheptanoic acid) NH.sub.2 Bio-2'6'Dmt D-Arg Phe Lys
NH.sub.2 3'5'Dmt D-Arg Phe Lys NH.sub.2 3'5'Dmt D-Arg Phe Orn
NH.sub.2 3'5'Dmt D-Arg Phe Dab NH.sub.2 3'5'Dmt D-Arg Phe Dap
NH.sub.2 Tyr D-Arg Tyr Lys NH.sub.2 Tyr D-Arg Tyr Orn NH.sub.2 Tyr
D-Arg Tyr Dab NH.sub.2 Tyr D-Arg Tyr Dap NH.sub.2 2'6'Dmt D-Arg Tyr
Lys NH.sub.2 2'6'Dmt D-Arg Tyr Orn NH.sub.2 2'6'Dmt D-Arg Tyr Dab
NH.sub.2 2'6'Dmt D-Arg Tyr Dap NH.sub.2 2'6'Dmt D-Arg 2'6'Dmt Lys
NH.sub.2 2'6'Dmt D-Arg 2'6'Dmt Orn NH.sub.2 2'6'Dmt D-Arg 2'6'Dmt
Dab NH.sub.2 2'6'Dmt D-Arg 2'6'Dmt Dap NH.sub.2 3'5'Dmt D-Arg
3'5'Dmt Arg NH.sub.2 3'5'Dmt D-Arg 3'5'Dmt Lys NH.sub.2 3'5'Dmt
D-Arg 3'5'Dmt Orn NH.sub.2 3'5'Dmt D-Arg 3'5'Dmt Dab NH.sub.2 Tyr
D-Lys Phe Dap NH.sub.2 Tyr D-Lys Phe Arg NH.sub.2 Tyr D-Lys Phe Lys
NH.sub.2 Tyr D-Lys Phe Orn NH.sub.2 2'6'Dmt D-Lys Phe Dab NH.sub.2
2'6'Dmt D-Lys Phe Dap NH.sub.2 2'6'Dmt D-Lys Phe Arg NH.sub.2
2'6'Dmt D-Lys Phe Lys NH.sub.2 3'5'Dmt D-Lys Phe Orn NH.sub.2
3'5'Dmt D-Lys Phe Dab NH.sub.2 3'5'Dmt D-Lys Phe Dap NH.sub.2
3'5'Dmt D-Lys Phe Arg NH.sub.2 Tyr D-Lys Tyr Lys NH.sub.2 Tyr D-Lys
Tyr Orn NH.sub.2 Tyr D-Lys Tyr Dab NH.sub.2 Tyr D-Lys Tyr Dap
NH.sub.2 2'6'Dmt D-Lys Tyr Lys NH.sub.2 2'6'Dmt D-Lys Tyr Orn
NH.sub.2 2'6'Dmt D-Lys Tyr Dab NH.sub.2 2'6'Dmt D-Lys Tyr Dap
NH.sub.2 2'6'Dmt D-Lys 2'6'Dmt Lys NH.sub.2 2'6'Dmt D-Lys 2'6'Dmt
Orn NH.sub.2 2'6'Dmt D-Lys 2'6'Dmt Dab NH.sub.2 2'6'Dmt D-Lys
2'6'Dmt Dap NH.sub.2 2'6'Dmt D-Arg Phe dnsDap NH.sub.2 2'6'Dmt
D-Arg Phe atnDap NH.sub.2 3'5'Dmt D-Lys 3'5'Dmt Lys NH.sub.2
3'5'Dmt D-Lys 3'5'Dmt Orn NH.sub.2 3'5'Dmt D-Lys 3'5'Dmt Dab
NH.sub.2 3'5'Dmt D-Lys 3'5'Dmt Dap NH.sub.2 Tyr D-Lys Phe Arg
NH.sub.2 Tyr D-Orn Phe Arg NH.sub.2 Tyr D-Dab Phe Arg NH.sub.2 Tyr
D-Dap Phe Arg NH.sub.2 2'6'Dmt D-Arg Phe Arg NH.sub.2 2'6'Dmt D-Lys
Phe Arg NH.sub.2 2'6'Dmt D-Orn Phe Arg NH.sub.2 2'6'Dmt D-Dab Phe
Arg NH.sub.2 3'5'Dmt D-Dap Phe Arg NH.sub.2 3'5'Dmt D-Arg Phe Arg
NH.sub.2 3'5'Dmt D-Lys Phe Arg NH.sub.2 3'5'Dmt D-Orn Phe Arg
NH.sub.2 Tyr D-Lys Tyr Arg NH.sub.2 Tyr D-Orn Tyr Arg NH.sub.2 Tyr
D-Dab Tyr Arg NH.sub.2 Tyr D-Dap Tyr Arg NH.sub.2 2'6'Dmt D-Arg
2'6'Dmt Arg NH.sub.2 2'6'Dmt D-Lys 2'6'Dmt Arg NH.sub.2 2'6'Dmt
D-Orn 2'6'Dmt Arg NH.sub.2 2'6'Dmt D-Dab 2'6'Dmt Arg NH.sub.2
3'5'Dmt D-Dap 3'5'Dmt Arg NH.sub.2 3'5'Dmt D-Arg 3'5'Dmt Arg
NH.sub.2 3'5'Dmt D-Lys 3'5'Dmt Arg NH.sub.2 3'5'Dmt D-Orn 3'5'Dmt
Arg NH.sub.2 Mmt D-Arg Phe Lys NH.sub.2 Mmt D-Arg Phe Orn NH.sub.2
Mmt D-Arg Phe Dab NH.sub.2 Mmt D-Arg Phe Dap NH.sub.2 Tmt D-Arg Phe
Lys NH.sub.2 Tmt D-Arg Phe Orn NH.sub.2 Tmt D-Arg Phe Dab NH.sub.2
Tmt D-Arg Phe Dap NH.sub.2 Hmt D-Arg Phe Lys NH.sub.2 Hmt D-Arg Phe
Orn NH.sub.2 Hmt D-Arg Phe Dab NH.sub.2 Hmt D-Arg Phe Dap NH.sub.2
Mmt D-Lys Phe Lys NH.sub.2 Mmt D-Lys Phe Orn NH.sub.2 Mmt D-Lys Phe
Dab NH.sub.2 Mmt D-Lys Phe Dap NH.sub.2 Mmt D-Lys Phe Arg NH.sub.2
Tmt D-Lys Phe Lys NH.sub.2 Tmt D-Lys Phe Orn NH.sub.2 Tmt D-Lys Phe
Dab NH.sub.2 Tmt D-Lys Phe Dap NH.sub.2 Tmt D-Lys Phe Arg NH.sub.2
Hmt D-Lys Phe Lys NH.sub.2 Hmt D-Lys Phe Orn NH.sub.2 Hmt D-Lys Phe
Dab NH.sub.2 Hmt D-Lys Phe Dap NH.sub.2 Hmt D-Lys Phe Arg NH.sub.2
Mmt D-Lys Phe Arg NH.sub.2 Mmt D-Orn Phe Arg NH.sub.2 Mmt D-Dab Phe
Arg NH.sub.2 Mmt D-Dap Phe Arg NH.sub.2 Mmt D-Arg Phe Arg NH.sub.2
Tmt D-Lys Phe Arg NH.sub.2 Tmt D-Orn Phe Arg NH.sub.2 Tmt D-Dab Phe
Arg NH.sub.2 Tmt D-Dap Phe Arg NH.sub.2 Tmt D-Arg Phe Arg NH.sub.2
Hmt D-Lys Phe Arg NH.sub.2 Hmt D-Orn Phe Arg NH.sub.2 Hmt D-Dab Phe
Arg NH.sub.2 Hmt D-Dap Phe Arg NH.sub.2 Hmt D-Arg Phe Arg NH.sub.2
Dab = diaminobutyric Dap = diaminopropionic acid Dmt =
dimethyltyrosine Mmt = 2'-methyltyrosine Tmt =
N,2',6'-trimethyltyrosine Hmt = 2'-hydroxy,6'-methyltyrosine dnsDap
= .beta.-dansyl-L-.alpha.,.beta.-diaminopropionic acid atnDap =
.beta.-anthraniloyl-L-.alpha.,.beta.-diaminopropionic acid Bio =
biotin
[0117] Examples of peptides that do not activate mu-opioid
receptors include, but are not limited to, the aromatic-cationic
peptides shown in Table 8.
TABLE-US-00007 TABLE 8 Peptide Analogs Lacking Mu-Opioid Activity
Amino Acid Amino Acid Amino Acid Amino Acid C-Terminal Position 1
Position 2 Position 3 Position 4 Modification D-Arg Dmt Lys Phe
NH.sub.2 D-Arg Dmt Phe Lys NH.sub.2 D-Arg Phe Lys Dmt NH.sub.2
D-Arg Phe Dmt Lys NH.sub.2 D-Arg Lys Dmt Phe NH.sub.2 D-Arg Lys Phe
Dmt NH.sub.2 Phe Lys Dmt D-Arg NH.sub.2 Phe Lys D-Arg Dmt NH.sub.2
Phe D-Arg Phe 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 = cyclohexyl alanine
[0118] The amino acids of the peptides shown in Table 5 and 6 may
be in either the L- or the D-configuration.
[0119] The peptides may be synthesized by any of the methods well
known in the art. Suitable methods for chemically synthesizing the
protein include, for example, those described by Stuart and Young
in Solid Phase Peptide Synthesis, Second Edition, Pierce Chemical
Company (1984), and in Methods Enzymol., 289, Academic Press, Inc.,
New York (1997).
Friedreich's Ataxia
[0120] Friedreich's ataxia is an inherited autosomal recessive
disease that causes progressive damage to the nervous system. The
ataxia results from the degeneration of nerve tissue in the spinal
cord, in particular, sensory neurons essential for directing muscle
movement of the arms and legs. The spinal cord becomes thinner and
nerve cells lose some of their myelin sheath.
[0121] Symptoms typically begin between the ages of 5 and 15 years,
although they sometimes appear in adulthood. The first symptom to
appear is usually gait ataxia, or difficulty walking. The ataxia
gradually worsens and slowly spreads to the arms and the trunk.
There is often loss of sensation in the extremities, which may
spread to other parts of the body. Other features include loss of
tendon reflexes, especially in the knees and ankles Most people
with Friedreich's ataxia develop scoliosis, which often requires
surgical intervention for treatment. Dysarthria (slowness and
slurring of speech) develops and can get progressively worse. Many
individuals with later stages of Friedreich's ataxia develop
hearing and vision loss.
[0122] Heart disease often accompanies Friedreich's ataxia, such as
hypertrophic cardiomyopathy (enlargement of the heart), myocardial
fibrosis (formation of fiber-like material in the muscles of the
heart), and cardiac failure. Heart rhythm abnormalities such as
tachycardia (fast heart rate) and heart block (impaired conduction
of cardiac impulses within the heart) are also common. Other
symptoms that may occur include chest pain, shortness of breath,
and heart palpitations.
[0123] About 20 percent of people with Friedreich's ataxia develop
carbohydrate intolerance and 10 percent develop diabetes. Most
individuals with Friedreich's ataxia tire very easily and find that
they require more rest and take a longer time to recover from
common illnesses such as colds and flu.
[0124] The rate of progression varies from person to person.
Generally, within 10 to 20 years after the appearance of the first
symptoms, the person is confined to a wheelchair, and in later
stages of the disease individuals may become completely
incapacitated. Friedreich's ataxia can shorten life expectancy, and
heart disease is the most common cause of death.
[0125] Friedreich's ataxia occurs when a mutated FXN gene contains
amplified intronic GAA repeats. The mutant FXN gene contains
expanded GAA triplet repeats in the first intron; in a few
pedigrees, point mutations have been detected. Since the defect is
located in an intron, which is removed from the mRNA transcript
between transcription and translation, the mutated FXN gene does
not result in the production of abnormal proteins. Instead, the
mutation causes gene silencing, i.e., the mutation decreases the
transcription of the gene, through induction of a heterochromatin
structure in a manner similar to position-effect variegation. The
GAA repeat expansion in FXN and subsequent gene silencing results
in the reduction of frataxin levels.
[0126] The FXN gene encodes the protein frataxin. Frataxin is a
highly conserved iron binding protein. Human frataxin is
synthesized as a 210 amino acid precursor that is imported to the
mitochondria via the mitochondrial targeting signal contained in
the N-terminus. The frataxin precursor is subsequently cleaved to a
mature 14 kDa protein (residues 81-210).
[0127] Frataxin binds both Fe.sup.2+ and Fe.sup.3+ ions in an
electrostatic manner and functions as an iron chaperone during
Fe--S cluster assembly. Frataxin directly binds to the central
Fe--S cluster assembly complex, which is composed of Nfs1 enzyme
and Isu scaffold protein. Nfs1 is a cysteine desulfurase used in
the synthesis of sulfur bioorganic derivatives and Isu is the
transient scaffold protein on which the Fe--S cluster assembles.
Frataxin increases the efficiency of Fe--S cluster formation, which
is required to activate the mitochondrial Kreb cycle enzyme
aconitase. Frataxin also plays a role in mitochondrial iron storage
and heme biosynthesis by incorporating mitochondrial iron into
protoporphyrin (PIX).
[0128] Loss of frataxin function results in the disruption of
iron-sulfur cluster biosynthesis, mitochondrial iron overload,
oxidative stress, impaired aerobic electron transport chain
respiration and cell death in the brain, spinal cord and heart.
Studies have also shown that frataxin protects dopaminergic
neuronal cells against MPTP-induced toxicity in a mouse model of
Parkinson's disease.
[0129] Mitochondrial iron overload leads to impaired
intra-mitochondrial metabolism and a defective mitochondrial
respiratory chain. A defective mitochondrial respiratory chain
leads to increased free radical generation and oxidative damage,
which may be considered as mechanisms that compromise cell
viability. Recent evidence suggests that frataxin might detoxify
reactive oxygen species (ROS) via activation of glutathione
peroxidase and elevation of thiols. (See e.g., Calabrese et al.,
Journal of the Neurological Sciences, 233(1): 145-162 (June
2005)).
[0130] In some embodiments, treatment with an aromatic-cationic
peptide, such as D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a
pharmaceutically acceptable salt thereof, such as acetate or
trifluoroacetate salt, stabilizes the mitochondrial metabolism in a
tissue or an organ in mammalian subjects that have suffered or are
at risk of suffering Friedreich's ataxia. By way of example, but
not by way of limitation, in some embodiments, mitochondrial
metabolism is increased in the spinal cord of a treated
subject.
[0131] In some embodiments, treatment with an aromatic-cationic
peptide, such as D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a
pharmaceutically acceptable salt thereof, such as acetate or
trifluoroacetate salt, reduces free radical generation, oxidative
stress, or both in a tissue or an organ in mammalian subjects that
have suffered or are at risk of suffering Friedreich's ataxia. By
way of example, but not by way of limitation, in some embodiments,
free radical generation, oxidative damage, or both are increased in
the spinal cord of a treated subject.
[0132] In some embodiments, treatment with an aromatic-cationic
peptide, such as D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a
pharmaceutically acceptable salt thereof, such as acetate or
trifluoroacetate salt, reduces build-up of iron in the mitochondria
in a tissue or an organ in mammalian subjects that have suffered or
are at risk of suffering Friedreich's ataxia. By way of example,
but not by way of limitation, in some embodiments, iron in the
mitochondria decreases in the spinal cord of a treated subject.
Therapeutic Methods
[0133] The following discussion is presented by way of example
only, and is not intended to be limiting.
[0134] One aspect of the present technology includes methods of
treating reduced frataxin expression in a subject diagnosed as
having, suspected as having, or at risk of having reduced frataxin
expression levels. One aspect of the present technology includes
methods of treating Friedreich's ataxia in a subject diagnosed as
having, suspected as having, or at risk of having Friedreich's
ataxia. In therapeutic applications, compositions or medicaments
comprising an aromatic-cationic peptide such as
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a pharmaceutically acceptable
salt thereof, such as acetate or trifluoroacetate salt, are
administered to a subject suspected of, or already suffering from
such a disease, such as, e.g., decreased frataxin expression levels
or Friedreich's ataxia, in an amount sufficient to reduce the
severity at least partially arrest or delay the onset of one or
more of the symptoms of the disease, including its complications
and intermediate pathological phenotypes in development of the
disease.
[0135] Subjects suffering from decreased frataxin expression levels
or Friedreich's ataxia can be identified by any or a combination of
diagnostic or prognostic assays known in the art. For example,
typical symptoms of Friedreich's ataxia include symptoms such as,
e.g., muscle weakness, especially in the arms and legs, loss of
coordination, motor control impairment, vision impairment, hearing
impairment, slurred speech, curvature of the spine, diabetes, and
heart disorders. In some embodiments, the subject may exhibit
reduced levels of frataxin expression compared to a normal subject,
which are measureable using techniques known in the art. In some
embodiments, the subject may exhibit one or more mutations in the
FXN gene associated with Friedreich's ataxia, which are detectable
using techniques known in the art.
Prophylactic Methods
[0136] In one aspect, the present technology provides a method for
preventing or delaying the onset of Friedreich's ataxia or symptoms
of Friedreich's ataxia in a subject at risk of having reduced
levels of frataxin expression compared to a normal subject. In some
embodiments, the subject may exhibit one or more mutations in the
FXN gene associated with Friedreich's ataxia, which are detectable
using techniques known in the art. Subjects at risk for reduced
frataxin expression levels or Friedreich's ataxia can be identified
by, e.g., any or a combination of diagnostic or prognostic assays
known in the art. In prophylactic applications, pharmaceutical
compositions or medicaments of aromatic-cationic peptides, such as
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a pharmaceutically acceptable
salt thereof, such as acetate or trifluoroacetate salt, are
administered to a subject susceptible to, or otherwise at risk of a
disease or condition such as e.g., Friedreich's ataxia, in an
amount sufficient to eliminate or reduce the risk, lessen the
severity, or delay the outset of the disease, including
biochemical, histologic and/or behavioral symptoms of the disease,
its complications and intermediate pathological phenotypes
presenting during development of the disease. Administration of a
prophylactic aromatic-cationic can occur prior to the manifestation
of symptoms characteristic of the disease or disorder, such that
the disease or disorder is prevented or, alternatively, delayed in
its progression.
[0137] Subjects or at risk for reduced frataxin expression levels
or Friedreich's ataxia may exhibit one or more of the following
non-limiting risk factors: cardiomyopathy, skeletal muscle
abnormalities, neutropenia, slow development, weak muscle tone,
increased levels of organic acids in the urine and blood, and/or
frequent bacterial infections, such as pneumonia.
[0138] For therapeutic and/or prophylactic applications, a
composition comprising an aromatic-cationic peptide, such as
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a pharmaceutically acceptable
salt thereof, such as acetate or trifluoroacetate salt, is
administered to the subject. In some embodiments, the peptide
composition is administered one, two, three, four, or five times
per day. In some embodiments, the peptide composition is
administered more than five times per day. Additionally or
alternatively, in some embodiments, the peptide composition is
administered every day, every other day, every third day, every
fourth day, every fifth day, or every sixth day. In some
embodiments, the peptide composition is administered weekly,
bi-weekly, tri-weekly, or monthly. In some embodiments, the peptide
composition is administered for a period of one, two, three, four,
or five weeks. In some embodiments, the peptide is administered for
six weeks or more. In some embodiments, the peptide is administered
for twelve weeks or more. In some embodiments, the peptide is
administered for a period of less than one year. In some
embodiments, the peptide is administered for a period of more than
one year.
[0139] For therapeutic and/or prophylactic applications, a
composition comprising an aromatic-cationic peptide, such as
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a pharmaceutically acceptable
salt thereof, such as acetate or trifluoroacetate salt, may be
administered in combination with one or more additional agents. In
some embodiments, there is a synergistic effect between the peptide
and the one or more additional agents.
Determination of the Biological Effect of the Aromatic-Cationic
Peptide-Based Therapeutic
[0140] In various embodiments, suitable in vitro or in vivo assays
are performed to determine the effect of a specific
aromatic-cationic peptide-based therapeutic and whether its
administration is indicated for treatment. In various embodiments,
in vitro assays can be performed with representative animal models,
to determine if a given aromatic-cationic peptide-based therapeutic
exerts the desired effect increasing frataxin expression, and
preventing or treating Friedreich's ataxia. Compounds for use in
therapy can be tested in suitable animal model systems including,
but not limited to rats, mice, chicken, cows, monkeys, rabbits, and
the like, prior to testing in human subjects. Similarly, for in
vivo testing, any of the animal model system known in the art can
be used prior to administration to human subjects. In some
embodiments, in vitro or in vivo testing is directed to the
biological function of D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a
pharmaceutically acceptable salt thereof, such as acetate or
trifluoroacetate salt.
Modes of Administration and Effective Dosages
[0141] Any method known to those in the art for contacting a cell,
organ or tissue with an aromatic-cationic peptide of the present
technology, such as D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a
pharmaceutically acceptable salt thereof, such as acetate or
trifluoroacetate salt, 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, suitably a human. When used in
vivo for therapy, the aromatic-cationic peptides, such as
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a pharmaceutically acceptable
salt thereof, such as acetate or trifluoroacetate salt, are
administered to the subject in effective amounts (i.e., amounts
that have desired therapeutic effect). The dose and dosage regimen
will depend upon the degree of the disease in the subject, the
characteristics of the particular aromatic-cationic peptide used,
e.g., its therapeutic index, the subject, and the subject's
history.
[0142] 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 useful in the methods
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.
[0143] 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),
glucuronic, 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. In some embodiments, the salt is an acetate or
trifluoroacetate salt.
[0144] The aromatic-cationic peptides described herein, such as
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a pharmaceutically acceptable
salt thereof, such as acetate or trifluoroacetate salt, 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.
[0145] 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), intraocular, iontophoretic, and transmucosal
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. For
convenience of the patient or treating physician, the dosing
formulation can be provided in a kit containing all necessary
equipment (e.g., vials of drug, vials of diluent, syringes and
needles) for a treatment course (e.g., 7 days of treatment).
[0146] 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.
[0147] 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, thiomerasol, and the like. Glutathione and other
antioxidants can be included to prevent oxidation. 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 that delays absorption, for example, aluminum monostearate
or gelatin.
[0148] 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
[0149] 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.
[0150] For administration by inhalation, the compounds can be
delivered in the form of an aerosol spray from a pressurized
container or dispenser, which contains a suitable propellant, e.g.,
a gas such as carbon dioxide, or a nebulizer. Such methods include
those described in U.S. Pat. No. 6,468,798.
[0151] 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.
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.
[0152] A therapeutic protein or peptide can be formulated in a
carrier system. The carrier can be a colloidal system. The
colloidal system can be a liposome, a phospholipid bilayer vehicle.
In one embodiment, the therapeutic peptide is encapsulated in a
liposome while maintaining peptide integrity. s one skilled in the
art would appreciate, there are a variety of methods to prepare
liposomes. (See Lichtenberg, et al., Methods Biochem. Anal.,
33:337-462 (1988); Anselem, et al., Liposome Technology, CRC Press
(1993)). Liposomal formulations can delay clearance and increase
cellular uptake (See Reddy, Ann. Pharmacother., 34(7-8):915-923
(2000)). An active agent can also be loaded into a particle
prepared from pharmaceutically acceptable ingredients including,
but not limited to, soluble, insoluble, permeable, impermeable,
biodegradable or gastroretentive polymers or liposomes. Such
particles include, but are not limited to, nanoparticles,
biodegradable nanoparticles, microparticles, biodegradable
microparticles, nanospheres, biodegradable nanospheres,
microspheres, biodegradable microspheres, capsules, emulsions,
liposomes, micelles and viral vector systems.
[0153] The carrier can also be a polymer, e.g., a biodegradable,
biocompatible polymer matrix. In one embodiment, the therapeutic
peptide can be embedded in the polymer matrix, while maintaining
protein integrity. The polymer may be natural, such as
polypeptides, proteins or polysaccharides, or synthetic, such as
poly .alpha.-hydroxy acids. Examples include carriers made of,
e.g., collagen, fibronectin, elastin, cellulose acetate, cellulose
nitrate, polysaccharide, fibrin, gelatin, and combinations thereof.
In one embodiment, the polymer is poly-lactic acid (PLA) or copoly
lactic/glycolic acid (PGLA). The polymeric matrices can be prepared
and isolated in a variety of forms and sizes, including
microspheres and nanospheres. Polymer formulations can lead to
prolonged duration of therapeutic effect. (See Reddy, Ann.
Pharmacother., 34(7-8):915-923 (2000)). A polymer formulation for
human growth hormone (hGH) has been used in clinical trials. (See
Kozarich and Rich, Chemical Biology, 2:548-552 (1998)).
[0154] Examples of polymer microsphere sustained release
formulations are described in PCT publication WO 99/15154 (Tracy,
et al.), U.S. Pat. Nos. 5,674,534 and 5,716,644 (both to Zale, et
al.), PCT publication WO 96/40073 (Zale, et al.), and PCT
publication WO 00/38651 (Shah, et al.). U.S. Pat. Nos. 5,674,534
and 5,716,644 and PCT publication WO 96/40073 describe a polymeric
matrix containing particles of erythropoietin that are stabilized
against aggregation with a salt.
[0155] In some embodiments, the therapeutic compounds are prepared
with carriers that will protect the therapeutic compounds against
rapid elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Such formulations
can be prepared using known techniques. The materials can also be
obtained commercially, e.g., from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to specific cells with monoclonal antibodies to
cell-specific antigens) can also be used as pharmaceutically
acceptable carriers. These can be prepared according to methods
known to those skilled in the art, for example, as described in
U.S. Pat. No. 4,522,811.
[0156] The therapeutic compounds can also be formulated to enhance
intracellular delivery. For example, liposomal delivery systems are
known in the art, see, e.g., Chonn and Cullis, "Recent Advances in
Liposome Drug Delivery Systems," Current Opinion in Biotechnology
6:698-708 (1995); Weiner, "Liposomes for Protein Delivery:
Selecting Manufacture and Development Processes," Immunomethods,
4(3):201-9 (1994); and Gregoriadis, "Engineering Liposomes for Drug
Delivery: Progress and Problems," Trends Biotechnol., 13(12):527-37
(1995). Mizguchi, et al., Cancer Lett., 100:63-69 (1996), describes
the use of fusogenic liposomes to deliver a protein to cells both
in vivo and in vitro.
[0157] 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
that 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 uninfected cells and, thereby, reduce side effects.
[0158] 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, in some embodiments,
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 methods, 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 determine useful doses in humans
accurately. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
[0159] 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. Suitably,
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 0.001-10,000
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. In therapeutic
applications, a relatively high dosage at relatively short
intervals is sometimes required until progression of the disease is
reduced or terminated, and in some embodiments, until the subject
shows partial or complete amelioration of symptoms of disease.
Thereafter, the patient can be administered a prophylactic
regime.
[0160] 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.-12 to 10.sup.-6 molar,
e.g., approximately 10.sup.-7 molar. This concentration may be
delivered by systemic doses of 0.001 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, and
in some embodiments, by single daily or weekly administration, but
also including continuous administration (e.g., parenteral infusion
or transdermal application).
[0161] 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.
[0162] The mammal treated in accordance present methods 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.
Combination Therapy with an Aromatic-Cationic Peptide and Other
Therapeutic Agents
[0163] In some embodiments, one or more additional therapeutic
agents are administered to a subject in combination with an
aromatic-cationic peptide, e.g., D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2,
or a pharmaceutically acceptable salt thereof, such as acetate or
trifluoroacetate salt, such that a synergistic therapeutic effect
is produced. A "synergistic therapeutic effect" refers to a
greater-than-additive therapeutic effect, which is produced by a
combination of at least two therapeutic agents, and which exceeds
that which would otherwise result from administration of any
individual therapeutic agent alone. Therefore, lower doses of one
or more of any individual therapeutic agent may be used in treating
a medical disease or condition, e.g., disruptions in mitochondrial
oxidative phosphorylation, resulting in increased therapeutic
efficacy and decreased side-effects. By way of example, but not by
way of limitation, exemplary additional therapeutic agents that can
be combined with aromatic-cationic peptides for the treatment or
prevention of Friedreich's ataxia include, but are not limited to,
ACE inhibitors, e.g., digoxin, enalapril, or lisinopril, diuretics,
beta-blockers, idebenone, deferiprone, and insulin.
[0164] The multiple therapeutic agents may be administered in any
order or even simultaneously. If simultaneously, the multiple
therapeutic agents may be provided in a single, unified form, or in
multiple forms (by way of example only, either as a single pill or
as two separate pills). One of the therapeutic agents may be given
in multiple doses, or both may be given as multiple doses. If not
simultaneous, the timing between the multiple doses may vary from
more than zero weeks to less than four weeks. In addition, the
combination methods, compositions and formulations are not to be
limited to the use of only two agents.
EXAMPLES
[0165] The present compositions and methods are further illustrated
by the following examples, which should not be construed as
limiting in any way.
Example 1
Aromatic-Cationic Peptides Rescue Friedreich's Ataxia Fibroblasts
from Iron-Oxidant Stress
[0166] This example demonstrates the effect of the
aromatic-cationic peptide D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 on
fibroblasts from Friedreich's ataxia (FRDA) patients that have
induced iron-oxidant stress.
[0167] Methods:
[0168] In the absence of FXN, it is widely accepted that deficient
cells will have an increased sensitivity to oxidative stress, which
most likely contributes to the cascade of events leading to
cytotoxicity. Iron with hydroquinone (HQ) induces oxidative stress
in cells because HQ forms a lipophilic chelate with iron and
rapidly transfers the metal across the normally impermeable plasma
membrane. HQ or Fe alone in culture media is not toxic to FRDA
fibroblasts even after an extended exposure of 24 hours.
[0169] FRDA fibroblasts are treated with 1-10 .mu.M
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 in culture media for 24 hours.
After 24 hours, the media is changed and the cells are treated with
5 .mu.m Fe/HQ for 5 hours. Controls include FRDA fibroblasts
without D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 and then treated with 5
.mu.m Fe/HQ for 5 hours and FRDA fibroblasts treated with
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 for 24 hours and no addition of
Fe/HQ.
[0170] Results:
[0171] It is anticipated that cells that are treated only with
Fe/HQ will show changes in the morphology and have loss of
adherence, which indicates that Fe/HQ is cytotoxic. It is
anticipated that cells that were treated with
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 prior to the addition of Fe/HQ
will be able to survive and show reduced evidence of cytotoxicity
as demonstrated by their morphologic appearance being substantially
identical to, or less deformed than cells treated only with
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2.
[0172] These results will show that aromatic-cationic peptides of
the present disclosure, such as D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2,
or a pharmaceutically acceptable salt thereof, such as acetate or
trifluoroacetate salt, are useful in the prevention and treatment
of diseases and conditions associated with reduced frataxin
expression levels. It is further expected that administration of
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 in combination with one or more
additional therapeutic agents will have synergistic effects in this
regard. It is further anticipated that these results will show that
aromatic-cationic peptides of the present disclosure, such as
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a pharmaceutically acceptable
salt thereof, such as acetate or trifluoroacetate salt, are useful
in methods comprising administration of the peptide to subjects
having or susceptible to Friedreich's ataxia.
Example 2
Aromatic-Cationic Peptides Prolong Survival of FXN-Knockout
Mice
[0173] This example demonstrates the effect of the
aromatic-cationic peptide D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 on
survival of FXN-knockout (KO) mice.
[0174] Methods:
[0175] FXN-KO mice are treated with 1-10 .mu.M
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 or PBS beginning at Day 3 of life
for 60 days. The mice receive the aromatic peptide and PBS by
intraperitoneal (IP) injections three times per week. All mice will
need to reach an age of 10 days to be included in the study, and
all mice will be weaned at 28 days of age. Control animals include
of littermates heterozygous for the conditional allele and had no
clinical or biochemical phenotype. The control heterozygous
littermates receive equivalent volume injections of PBS.
[0176] Results:
[0177] It is anticipated that FXN-KO mice treated with
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 will have increased survival as
compared to FXN-KO mice treated with PBS. It is further expected
that administration of D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 in
combination with one or more additional therapeutic agents will
have synergistic effects in this regard.
[0178] These results will show that aromatic-cationic peptides of
the present disclosure, such as D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2,
or a pharmaceutically acceptable salt thereof, such as acetate or
trifluoroacetate salt, are useful in the prevention and treatment
of Friedreich's ataxia.
Example 3
Use of Aromatic-Cationic Peptides in the Treatment of Friedreich's
Ataxia
[0179] This example will demonstrate the use of aromatic-cationic
peptides, such as D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a
pharmaceutically acceptable salt thereof, such as acetate or
trifluoroacetate salt, in the treatment of Friedreich's ataxia.
[0180] Methods:
[0181] Friedreich's ataxia patients receive daily administrations
of a therapeutically effective (e.g., 1-10 mg/kg body weight)
amount of aromatic-cationic peptide, such as
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a pharmaceutically acceptable
salt thereof, such as acetate or trifluoroacetate salt. Peptides
may be administered orally, topically, systemically, intravenously,
subcutaneously, intraperitoneally, or intramuscularly according to
methods known in the art. Subjects are evaluated weekly for the
presence and/or severity of signs and symptoms associated with
Friedreich's ataxia, including, but not limited to, e.g., muscle
weakness, especially in the arms and legs, loss of coordination,
motor control impairment, vision impairment, hearing impairment,
slurred speech, curvature of the spine, diabetes, and heart
disorders. Treatments are maintained until such a time as symptoms
of Friedreich's ataxia are ameliorated or eliminated.
[0182] Results:
[0183] It is predicted that Friedreich's ataxia subjects receiving
therapeutically effective amounts of aromatic-cationic peptide,
such as D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a pharmaceutically
acceptable salt thereof, such as acetate or trifluoroacetate salt
will display reduced severity of symptoms associated with
Friedreich's ataxia. It is further expected that administration of
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 in combination with one or more
additional therapeutic agents will have synergistic effects in this
regard.
[0184] These results will show that aromatic-cationic peptides,
such as D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a pharmaceutically
acceptable salt thereof, such as acetate or trifluoroacetate salt
are useful in the treatment of Friedreich's ataxia. Accordingly,
the peptides are useful in methods comprising administering
aromatic-cationic peptides to a subject in need thereof for the
treatment of Friedreich's ataxia.
Example 4
Use of Aromatic-Cationic Peptides in Combination with Other Agents
to Reduce Symptoms of Friedreich's Ataxia
[0185] This example will demonstrate the synergetic effect from the
use of aromatic-cationic peptides, such as
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a pharmaceutically acceptable
salt thereof, such as acetate or trifluoroacetate salt, and another
agent, e.g., idebenone, in the treatment of Friedreich's
ataxia.
[0186] Methods:
[0187] Friedreich's ataxia patients are split into four groups.
Group 1 receives daily administrations of a therapeutically
effective amount of aromatic-cationic peptide (e.g., 1-10 mg/kg
body weight), such as D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a
pharmaceutically acceptable salt thereof, such as acetate or
trifluoroacetate salt. Peptides may be administered orally,
topically, systemically, intravenously, subcutaneously,
intraperitoneally, or intramuscularly according to methods known in
the art.
[0188] Group 2 receives daily administrations of a therapeutically
effective amount of a known agent used in the treatment of
Friedreich's ataxia, e.g., 100 mg idebenone. The known agent may be
administered orally, topically, systemically, intravenously,
subcutaneously, intraperitoneally, or intramuscularly according to
methods known in the art.
[0189] Group 3 receives daily administrations of
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 and the same agent as Group 2,
wherein the dosage of D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 and the
agent is the same amount used in Groups 1 and 2, respectively.
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 and the known agent may be
administered orally, topically, systemically, intravenously,
subcutaneously, intraperitoneally, or intramuscularly according to
methods known in the art. D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 and the
agent may be administered simultaneously, either as a single pill
or as two separate pills, in any order or not simultaneously, e.g.,
idebenone is given an hour after treatment with
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2.
[0190] The fourth groups receives a similar treatment as Group 3,
except at half the dosage of both D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2
and the known agent.
[0191] Subjects are evaluated weekly for the presence and/or
severity of signs and symptoms associated with Friedreich's ataxia,
including, but not limited to, e.g., muscle weakness, especially in
the arms and legs, loss of coordination, motor control impairment,
vision impairment, hearing impairment, slurred speech, curvature of
the spine, diabetes, and heart disorders. Treatments are maintained
until such a time as symptoms of Friedreich's ataxia are
ameliorated or eliminated.
[0192] Results:
[0193] It is predicted that Groups 1 and 2 will display reduced
severity of symptoms associated with Friedreich's ataxia. It is
predicted that Group 3 will show a greater reduction in the
severity of symptoms or elimination of symptoms associated with
Friedreich's ataxia. It is predicted that Group 4 will displayed
reduced severity of symptoms associated with Friedreich's ataxia
equal to or great than the reduction of symptoms in Groups 1 and
2.
[0194] These results will show that the combination of
aromatic-cationic peptides, such as
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a pharmaceutically acceptable
salt thereof, such as acetate or trifluoroacetate salt, and known
agents used to treat Friedreich's ataxia are useful in the
treatment of Friedreich's ataxia. The synergistic effect of the
combination of the two treatments can lead to a reduced dosage of
both compounds, thereby reducing possible side effects of the
compounds. Accordingly, the peptides are useful in methods
comprising administering aromatic-cationic peptides to a subject in
need thereof for the treatment of Friedreich's ataxia.
Example 5
Treatment of Friedreich's Ataxia Using
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2
[0195] This example will demonstrate the use of aromatic-cationic
peptides, such as D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a
pharmaceutically acceptable salt thereof, such as acetate or
trifluoroacetate salt, in the treatment of Friedreich's ataxia.
[0196] Methods:
[0197] 24 subjects diagnosed with Friedreich's ataxia are randomly
split into four groups (3 test groups and 1 control group) with six
subjects per group. Group 1 receives daily intravenous
administrations of D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 at 0.1 mg/kg of
body weight. Group 2 receives daily intravenous administrations of
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 at 0.5 mg/kg of body weight. Group
3 receives daily intravenous administrations of
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 at 1.0 mg/kg of body weight. Group
4 receives daily intravenous administrations of a control peptide
at 1.0 mg/kg of body weight.
[0198] Subjects are selected based on: 1) having a molecular
genetic diagnosis of Friedreich's ataxia (FRDA) consisting of a
GAA-repeat expansion on both alleles of the FXN gene; 2) FRDA
patients over the age of 18 years; 3) subjects must be well enough
and willing to provide written informed consent; and 4) a female
subject is eligible to participate if she is of: a)
non-childbearing potential defined as pre-menopausal females with a
documented tubal ligation or hysterectomy; or postmenopausal
defined as 12 months of spontaneous amenorrhea (in questionable
cases a blood sample with simultaneous follicle stimulating hormone
(FSH)>40 MlU/ml and estradiol <40 pg/ml (<140 pmol/L) is
confirmatory); b) child-bearing potential and agrees to use one of
the following contraception methods: abstinence, contraceptive
methods with a failure rate of <1%, oral contraceptive (either
combined or progestogen alone), injectable progestogen, implants of
levonorgestrel, estrogenic vaginal ring, percutaneous contraceptive
patches, intrauterine device (IUD) or intrauterine system (IUS)
that meets the <1% failure rate as stated in the product label,
male partner(s) sterilization (vasectomy with documentation of
azoospermia) prior to the female subject's entry into the study,
double barrier method, e.g., condom and occlusive cap (diaphragm or
cervical/vault caps) plus vaginal spermicidal agent
(foam/gel/film/cream/suppository).
[0199] FRDA subjects are excluded based on: 1) subjects with
significant clinical dysphagia; 2) subjects taking sodium valproate
or any other known histone deacetylase inhibitor; 3) subject's
participating in another clinical trial or who have done so within
30 days before screening; 4) subjects known to be positive for
human immunodeficiency virus (HIV); 5) subjects with any additional
medical condition or illness that, in the opinion of the
investigator would interfere with study compliance and/or impair
the patient's ability to participate or complete the study; 6)
concurrent diseases or conditions that may interfere with study
participation or safety include liver disease, bleeding disorders,
arrhythmias, organ transplant, organ failure, current neoplasm,
poorly controlled diabetes mellitus, poorly controlled
hypertension, clinically significant haematological or biochemical
abnormality; 7) subjects with a history of substance abuse (e.g.,
alcohol or drug abuse) within the previous 6 months before
enrollment; 8) subjects with a history of severe allergies; 9)
inability to provide informed consent; 10) female subjects who are
lactating or pregnant (positive pre-randomisation serum pregnancy
test) or plan to become pregnant during the study; and 11) subjects
unable or unwilling to provide written informed consent
[0200] Subjects are evaluated every two weeks for the presence
and/or severity of signs and symptoms associated with Friedreich's
ataxia, which including, but are not limited to, e.g., muscle
weakness, loss of coordination, motor control impairment, vision
impairment, hearing impairment, slurred speech, curvature of the
spine, diabetes, and heart disorders. Treatments and evaluations
are maintained for 12 months.
[0201] Methods for measuring loss of coordination include, but are
not limited to, Functional Reach Test, Pediatric Clinical Test of
Sensory Interaction for Balance, the Pediatric Balance Scale, the
Timed "Up & Go" Test, the Timed "Up and Down Stairs" Test, and
the measurement of static standing.
[0202] Methods for measuring loss of coordination include, but are
not limited to, force control measurements of various muscle groups
using dynamometer in the isometric testing mode.
[0203] Results:
[0204] It is anticipated that Groups 1, 2, and 3 will display
reduced severity of symptoms associated with Friedreich's ataxia as
compared to Group 4. It is also anticipated that Groups 1, 2, and 3
will show a dose dependent reduction in the severity of symptoms
associated with Friedreich's ataxia.
[0205] These results will show that the combination of
aromatic-cationic peptides, such as
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a pharmaceutically acceptable
salt thereof, such as acetate or trifluoroacetate salt, and known
agents used to treat Friedreich's ataxia are useful in the
treatment of Friedreich's ataxia. The synergistic effect of the
combination of the two treatments can lead to a reduced dosage of
both compounds, thereby reducing possible side effects of the
compounds. Accordingly, the peptides are useful in methods
comprising administering aromatic-cationic peptides to a subject in
need thereof for the treatment of Friedreich's ataxia.
Example 6
Aromatic-Cationic Peptides Restore Mitochondrial Membrane Potential
and Translocation of Frataxin into Mitochondria
[0206] This example will demonstrate that aromatic-cationic
peptides, such as D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a
pharmaceutically acceptable salt thereof, such as acetate or
trifluoroacetate salt, restore mitochondrial membrane potential and
increase translocation of frataxin into the mitochondria.
Methods and Materials
[0207] Cell Line:
[0208] P131 is a lymphoblast cell line with deficient frataxin
expression. P131 is transfected with a pcDFRDAHA1 vector that
contains the 210 amino acid human frataxin tagged with a HA1
epitope. The transcriptional unit is under the control of the CMV
immediate-early promoter. The plasmid also encodes the geneticin
resistance gene for selection of transfectants. The inserted
sequence is confirmed by DNA sequencing. Plasmid DNA is prepared
using a DNA miniprep commercial kit (Promega, Madison, Wis.). DNA
quality is determined by restriction endonuclease digestion and
quantified by UV spectrophotometry.
[0209] Transfected lymphoblast line P131 is prepared by growing
P131 in fresh medium for 16 hours, and then transiently
transfecting P131 with 2 .mu.g/ml pcDFRDAHA1 expression vector or
pcDFRDAHA1 empty vector, or 1 .mu.g/ml of the reporter gene plasmid
pCMV.sport-.beta. gal using DMRIE-C (Life-Tech, CA) according to
the manufacturer's protocol for suspension cells. Each transfection
is performed in triplicate in 6-well plates with 2 .mu.g of plasmid
DNA, 6 .mu.l of DMRIE-C and 2.times.10.sup.6 cells mixed in 1.2
ml/well of OPTI-MEM low-serum medium. Five hours after
transfection, fresh culture medium is added.
[0210] 24 hours after transfection, cells are stained with X-gal to
determine transfection efficiency and selected with 400 .mu.g/ml
geneticin for 12 days. Frataxin gene expression is examined by
semiquantitative and quantitative RT-PCR and anchored-RT-PCR,
western blot and dot blot as described below. Cell lines expressing
low (i.e., having similar frataxin expression levels as cells from
a subject diagnosed with Friedreich's ataxia), and high frataxin
levels are selected for assays, and aliquots of cells are frozen
for experiments. Frataxin mRNA expression levels are periodically
examined by quantitative RT-PCR on the lightcycler.
[0211] Measuring Mitochondrial Potential:
[0212] Transfected P131 cells are plated on a dish and treated with
0.1 mM t-butyl hydroperoxide (t-BHP), alone or with 1 nM
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, for 6 hours. Cell untreated with
t-BHP and D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 are used as a control.
Cells are then treated with 10 .mu.m of dichlorofluorescin
(ex/em=485/530) for 30 minutes at 37.degree. C., 5% CO.sub.2. The
cells are subjected to a wash with HBSS three time and stained with
20 nM of Mitotracker TMRM (ex/em=550/575 nm) for 15 minutes at
37.degree. C. The cells are then examined by confocal laser
scanning microscopy.
[0213] Measuring Translocation of Frataxin: Transfected P131 cells
that exhibit low or high expression of frataxin are plated onto six
dishes, wherein three dishes are treated with 1 nM
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 and three dishes are not treated,
i.e., control cells. After 6 hours, each dish is washed with wash
buffer and fix for staining Frataxin is fluorescently tagged by
treating the cells with FITC anti-HA1 antibodies for about one hour
at room temperature. Each plate is then examined by fluorescence
microscope (Axiovert.TM.). Transfected P131 cells that exhibit low
expression of frataxin mimic the disease state of Friedreich's
ataxia. A parallel assay using transfected P131 cells that exhibit
high expression of frataxin is performed.
Results
[0214] It is anticipated that t-BHP treated transfected P131 cells
without D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 treatment will show a loss
of TMRM fluorescence, which indicates mitochondrial depolarization.
It is anticipated that t-BHP treated transfected P131 cells with
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 treatment will show TMRM
fluorescence, which indicates prevention of mitochondrial
depolarization and restoration of the membrane potential. It is
anticipated that cell not treated with either t-BHP or
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 will also show a loss of TMRM
fluorescence, however the loss will be less than the t-BHP only
treated cells.
[0215] It is anticipated that transfected P131 cells that exhibit
low and high frataxin expression level when treated with
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 will show an increase in frataxin
localized to the inner membrane of the mitochondria as compared to
transfected P131 cells not treated with
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2.
[0216] These results will show that
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 is useful for restoring
mitochondrial membrane potential. The results will also show that
maintaining the mitochondrial membrane potential results in the
translocation of frataxin to the inner mitochondrial membrane.
Example 7
Use of Aromatic-Cationic Peptides in Treating Mitochondrial Iron
Loading in Friedreich's Ataxia Mouse Model
[0217] This example will demonstrate the use of aromatic-cationic
peptides, such as D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2, or a
pharmaceutically acceptable salt thereof, such as acetate or
trifluoroacetate salt, in treating mitochondrial iron loading in
Friedreich's ataxia.
[0218] Mouse Model.
[0219] This example uses the muscle creatine kinase (MCK)
conditional frataxin knockout mice described by Puccio et al., Nat.
Genet. 27:181-186 (2001). In this model, the tissue-specific Cre
transgene under the control of MCK promoter results in the
conditional deletion of frataxin in only the heart and skeletal
muscle.
[0220] Eight-week-old mutant mice are administered a daily dose of
0.25 mg/kg/day of D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 or saline
vehicle only (control) subcutaneously for two weeks. Total RNA is
isolated from hearts of two 10-week-old wild-type mice, two
10-week-old untreated mutant mice and two 10-week-old treated
mutant mice. Total RNA is isolated using TRIzol (Invitrogen).
First-strand cDNA synthesis and biotin-labeled cRNA are performed
and hybridized to the mouse Affymetrix GeneChip 430 2.0. A 2-phase
strategy is used to identify differentially expressed genes. First,
genome-wide screening is performed using Affymetrix GeneChips.
Then, low-level analysis is performed with Affymetrix GeneChip
Operating Software 1.3.0, followed by the GC robust multiarray
average (GCRMA) method for background correction and
quantile-quantile normalization of expression. Tukey's method for
multiple pairwise comparisons is applied to acquire fold-change
estimations. Tests for significance are calculated and adjusted for
multiple comparisons by controlling the false discovery rate at
5%.
[0221] Definitive evidence of differential expression is obtained
from RT-PCR assessment of samples used for the microarray analysis
and at least 3 other independent samples. Principal component
analysis is performed by standard methods. Western blot analysis is
performed using antibodies against frataxin (US Biological); Tfr1
(Invitrogen); Fpn1 (D. Haile, University of Texas Health Science
Center); Hmox1 (AssayDesigns); Sdha, Gapdh, and Iscu1/2 (Santa Cruz
Biotechnology); Fech (H. Dailey, University of Georgia, Biomedical
and Health Sciences Institute); Hfe2 (S. Parkkila, University of
Tampere, Institute of Medical Technology); Nfs1, Uros, and Alad
(Abnova); Sec15l1 (N. C. Andrews, Duke University); Ftl1, Fth1,
Ftmt (S. Levi, San Raffaele Institute); and Hif1.alpha. (BD
Biosciences).
[0222] For heme assays, hearts are exhaustively perfused and washed
with PBS (0.2% heparin at 37.degree. C.) to remove blood. After
homogenization, heme is quantified using the QuantiChrom Heme Assay
(BioAssay Systems). Tissue iron is measured via inductively coupled
plasma atomic emission spectrometry
[0223] For iron loading measurement assays, hearts are exhaustively
perfused and washed with PBS (0.2% heparin at 37.degree. C.) to
remove blood. Mitochondria from the hearts are isolated using a
mitochondrial isolation kit (Thermo Scientific, Rockford, Ill.).
The iron concentration of the mitochondria is determined by the
Ferene S-based Iron Assay Kit (BioVision, Milpitas, Calif.)
according to the manufacturer's protocol.
[0224] It is anticipated that untreated mutant mice will exhibit
decreased expression of genes involved in heme synthesis,
iron-sulfur cluster assembly, and iron storage (FRDA Control) as
compared to wild-type mice (Normal). However, it is anticipated
that mutant mice treated with D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 will
show expression levels that are similar to normal subjects with
respect to genes involved in these three mitochondrial iron
utilization pathways. It is further expected that administration of
the present technology will have synergistic effects in this
regard. It is also anticipated that mice treated with
D-Arg-2',6'-Dmt-Lys-Phe-NH.sub.2 will show an decrease in iron
within the isolated mitochondria as compared to untreated mice.
[0225] These results will show that aromatic-cationic peptides of
the present technology are useful in treating mitochondrial iron
loading in Friedreich's ataxia or in subjects with lower frataxin
expression or activity.
EQUIVALENTS
[0226] 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 apparatuses 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.
[0227] 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.
[0228] 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 sub-ranges 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.
[0229] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification.
[0230] Other embodiments are set forth within the following
claims.
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