Note: Descriptions are shown in the official language in which they were submitted.
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"TREATING EPIDERI~YOSIS BTJI~I,OSA WITH THYMOSIN BETA 4"
BACKGROUND OF THE INVENTION
CROSS-REFERENCE TO RELATED APPLICATION
The present application claims the benefit of U.S. Provisional Application
Serial
No. 60/291,326, filed May 17, 2001.
1. FIELD OF THE INVENTION
The present invention relates to the field of healing or preventing
inflammatory
degenerative, immunological and other disorders of the skin and surrounding
tissue that
occur due to Epidermolysis Bullosa, and all of its subtypes.
2. DESCRIPTION OF THE BACKGROUND ART
The phenomenon called Epidermolysis Bullosa (EB) is a rare genetic disorder
that
afflicts all ethnic and racial groups. EB is a group of diseases characterized
by blister
formation after minor trauma to the skin. This may lead to open sores,
ulcerations and
scars. This family of disorders range in severity from mild to the severely
disabling,
mutilating and life threatening diseases of the skin. Unlike burns, these
afflictions
sometimes never go away. The most severe cases require great lifestyle
adjustments.
Afflicted children should never ride a bike, skate, or participate in sports,
because the
normal play of children causes chronic sores. Such sores may cover as much as
75
percent of the child's body. Blistering and scarring also occur in the mouth
and
esophagus. Therefore, frequently a diet of only liquids or soft foods is
possible. Scarring
also causes the fingers and toes to fuse, leaving deformities which severely
limit function.
Much of their life is tied to hospitals for treatment, blood transfusions,
biopsies and
surgeries. The eyes often blister preventing sight for days. Chronic anemia
reduces
energy, and growth is retarded. The life span for an individual afflicted with
EB is usually
not longer than 30 years.
There are three main types of EB: EB Simplex, Dystrophic EB (dominant or
recessive) and functional EB. The severity of symptoms varies between these
types. In
general terms, EB causes blisters which may be restricted to specific areas,
for example
hands or feet, or may affect large areas of the body. In the milder forms the
blisters heal
normally without leaving permanent damage to the skin. In the other forms, the
blisters
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heal with scarring which can result in permanent change to the skin, for
example angers
may fuse and hands contract, reducing movement. Some forms of functional EB
are life
threatening in infancy.
EB results from deleterious changes in the physiological, biochemical and
immunological properties of the skin. All forms of EB are genetic in origin
and the genes
responsible for several different subtypes of the condition are now known. The
genetic
defects result in the skin layers not adhering properly to each other, causing
areas of
structural weakness. This fragile skin is particularly vulnerable to damage
from mild
friction, causing the blisters which are the characteristic feature of the
condition. Skin is
an important barrier to infection, it is the first line of defense of the
immune system. The
fragile skin of those afflicted with EB loses this important defense
mechanism. Such
changes in vasculature decrease capacity to repair damage, increase propensity
for skin
cancers such as squamous cell carcinoma, and increase risk of infection. In
addition, the
open sores in the oral and digestive cavity can lead to increased dehydration
and
malnutrition.
There have been many attempts to treat EB, but none have had a substantial
impact on prevention or treatment of EB. Various growth factors, synthetic
skins,
antibiotics and other therapies have failed to adequately and effectively
treat EB. While
EB is a genetic disease, treatment that would more rapidly heal or better heal
the sores
would be extremely important. Further, preventative therapy would clearly
confer
substantial, perhaps life-saving benefit to the patient.
Numerous pharmaceutical, nutriceutical or cosmeceutical formulations have been
proposed to reduce or reverse EB or its affects.
There remains a need in the art for improved methods and compositions for
healing or preventing the blisters and sores associated with EB.
SUMMARY OF THE INVENTION
In accordance with the present invention, a method of treatment for promoting
healing or prevention of blisters, sores or skin degeneration associated with
EB involves
administration to a subject or patient in need of such treatment an effective
amount of a
composition comprising an EB-inhibiting polypeptide comprising amino acid
sequence
LKKTET or a conservative variant thereof having EB-inhibiting activity.
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DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on a discovery that actin-sequestering peptides
such as thymosin ~i4 (T~i4) and other actin-sequestering peptides or peptide
fragments
containing amino acid sequence LKKTET or conservative variants thereof,
promote
healing or prevention of blisters, sores and skin degeneration associated with
Epidermolysis Bullosa. Included are N- or C-terminal variants such as KLKKTET
and
LKKTETQ. Without being bound to any particular theory, these peptides may have
the
capacity to promote repair, healing and prevention by having the ability to
induce terminal
deoxynucleotidyl transferase (a non-template directed DNA polymerase), to
decrease the
levels of one or more inflammatory cytokines or chemokines, and to act as a
chemotactic
and/or angiogenic factor for endothelial cells and thus heal and prevent
degenerative
changes in the skin of patients with afflicted EB, even though EB is the
result of an
inherited defect.
Thymosin (34 was initially identified as a protein that is up-regulated during
endothelial cell migration and differentiation in vitro. Thymosin ~i4 was
originally isolated
from the thymus and is a 43 amino acid, 4.9 kDa ubiquitous polypeptide
identified in a
variety of tissues. Several roles have been ascribed to this protein including
a role in a
endothelial cell differentiation and migration, T cell differentiation, actin
sequestration and
vascularization.
In accordance with one embodiment, the invention is a method of treatment for
promoting healing and prevention of blisters, sores and skin degradation
associated with
EB comprising administering to a subject in need of such treatment an
effective amount
of a composition comprising an EB-inhibiting peptide comprising amino acid
sequence
LKKTET, or a conservative variant thereof having EB-inhibiting activity,
preferably
Thymosin X34, an isoform of Thymosin X34, oxidized Thymosin X34, Thymosin ~i4
sulfoxide,
or an antagonist of Thymosin X34.
Compositions which may be used in accordance with the present invention
include
Thymosin (34 (T[34), T(34 isoforms, oxidized T[34, Thymosin (34 sulfoxide,
polypeptides or
any other actin sequestering or bundling proteins having actin binding
domains, or
peptide fragments comprising or consisting essentially of the amino acid
sequence
LKKTET or conservative variants thereof, having EB-inhibiting activity.
International
Application Serial No. PCT/US99/17282, incorporated herein by reference,
discloses
isoforms of T~4 which may be useful in accordance with the present invention
as well as
amino acid sequence LKKTET and conservative variants thereof having EB-
inhibiting
activity, which may be utilized with the present invention. International
Application Serial
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No. PCT/GB99/00833 (WO 99/49883), incorporated herein by reference, discloses
oxidized Thymosin (34 which may be utilized in accordance with the present
invention.
Although the present invention is described primarily hereinafter with respect
to T(34 and
T(34 isoforms, it is to be understood that the following description is
intended to be equally
applicable to amino acid sequence LKKTET, peptides and fragments comprising or
consisting essentially of LKKTET, conservative variants thereof having EB-
inhibiting
activity, as well as oxidized Thymosin ~4.
In one embodiment, the invention provides a method for healing and preventing
blisters and sores of skin in a subject by contacting the skin with an EB-
inhibiting effective
amount of a composition which contains T(34 or a T(34 isoform. The contacting
may be
topically or systemically. Examples of topical administration include, for
example,
contacting the skin with a lotion, salve, gel, cream, paste, spray,
suspension, dispersion,
hydrogel, ointment, or oil comprising T~34, alone or in combination with at
least one agent
that enhances T~34 penetration, or delays or slows release of T~34 peptides
into the area
to be treated. Systemic administration includes, for example, intravenous,
intraperitoneal,
intramuscular or subcutaneous injections, or inhalation, transdermal or oral
administration
of a composition containing T(34 or a T~34 isoform, etc. A subject may be a
mammal,
preferably human.
T~i4, or its analogues, isoforms or derivatives, may be administered in any
suitable
EB-inhibiting amount. For example, T~34 may be administered in dosages within
the
range of about 0.1-50 micrograms of T~i4, more preferably in amounts of about
1-25
micrograms.
A composition in accordance with the present invention can be administered
daily,
every other day, etc., with a single administration or multiple
administrations per day of
administration, such as applications 2, 3, 4 or more times per day of
administration.
T~34 isoforms have been identified and have about 70%, or about 75%, or about
80% or more homology to the known amino acid sequence of T(34. Such isoforms
include, for example, T(348'e, T(39, T(310, T(311, T(312, T~13, T~i14 and
T~i15. Similar to
T~i4, the T~i10 and T~315 isoforms have been shown to sequester actin. T~i4,
T~i10 and
T~315, as well as these other isoforms share an amino acid sequence, LKKTET,
that
appears to be involved in mediating actin sequestration or binding. Although
not wishing
to be bound to any particular theory, the activity of T(34 isoforms may be
due, in part, to
the ability to regulate the polymerization of actin. For example, T~i4 can
modulate actin
polymerization in skin (e.g. (3-thymosins appear to depolymerize F-actin by
sequestering
free G-actin). T(34's ability to modulate actin polymerization may therefore
be due to all,
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or in part, its ability to bind to or sequester actin via the LKKTET sequence.
Thus, as with
T(34, other proteins which bind or sequester actin, or modulate actin
polymerization,
including T(34 isoforms having the amino acid sequence LKKTET, are likely to
reduce EB,
alone or in a combination with T~4, as set forth herein.
Thus, it is specifically contemplated that known T(34 isoforms, such as
T~34a~a, T(39,
T(310, T~i11, T~i12, T(313, T~i14 and T(315, as well as T~i4 isoforms not yet
identified, will
be useful in the methods of the invention. As such T~34 isoforms are useful in
the
methods of the invention, including the methods practiced in a subject. The
invention
therefore further provides pharmaceutical compositions comprising T(34, as
well as T~i4
isoforms T~i4a~a, T(39, T~10, T~i11, T~12, T~13, T~314 and T~15, and a
pharmaceutically
acceptable carrier.
In addition, other proteins having actin sequestering or binding capability,
or that
can mobilize actin or modulate actin polymerization, as demonstrated in an
appropriate
sequestering, binding, mobilization or polymerization assay, or identified by
the presence
of an amino acid sequence that mediates actin binding, such as LKKTET, for
example,
can similarly be employed in the methods of the invention. Such proteins
include
gelsolin, vitamin D binding protein (DBP), profilin, cofilin, adsevertin,
propomyosin, fincilin,
depactin, DnaseI, vilin, fragmin, severin, capping protein, [3-actinin and
acumentin, for
example. As such methods include those practiced in a subject, the invention
further
provides pharmaceutical compositions comprising gelsolin, vitamin D binding
protein
(DBP), profilin, cofilin, depactin, DnaseI, vilin, fragmin, severin, capping
protein, ~-actinin
and acumentin as set forth herein. Thus, the invention includes the use of an
EB-
inhibiting polypeptide comprising the amino acid sequence LKKTET (which may be
within
its primary amino acid sequence) and conservative variants thereof.
As used herein, the term "conservative variant" or grammatical variations
thereof
denotes the replacement of an amino acid residue by another, biologically
similar residue.
Examples of conservative variations include the replacement of a hydrophobic
residue
such as isoleucine, valine, leucine or methionine for another, the replacement
of a polar
residue for another, such as the substitution of arginine for lysine, glutamic
for aspartic
acids, or glutamine for asparagine, and the like.
T~4 has been localized to a number of tissue and cell types and thus, agents
which stimulate the production of T(34 can be added to or comprise a
composition to
effect T~4 production from a tissue and/or a cell. Such agents include members
of the
family of growth factors, such as insulin-like growth factor (IGF-1 ),
platelet derived growth
factor (PDGF), epidermal growth factor (EGF), transforming growth factor beta
(TGF-(3),
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basic fibroblast growth factor (bFGF), thymosin a1 (Ta1) and vascular
endothelial growth
factor (VEGF). More preferably, the agent is transforming growth factor beta
(TGF-(3) or
other members of the TGF-(3 superfamily. T~34 compositions of the invention
may reduce
the affects of EB by effectuating growth of the connective tissue through
extracellular
matrix deposition, cellular migration and vascularization of the skin.
In accordance with one embodiment, subjects are treated with an agent that
stimulates production in the subject of an EB-inhibiting peptide as defined
above.
Additionally, agents that assist or stimulate EB reduction may be added to a
composition along with T~34 or a T~4 isoform. Such agents include angiogenic
agents,
growth factors, agents that direct differentiation of cells, agents that
promote migration of
cells and agents that stimulate the provision of extracellular matrix material
in the skin.
For example, and not by way of limitation, T~34 or a T~i4 isoform alone or in
combination
can be added in combination with any one or more of the following agents:
VEGF, KGF,
FGF, PDGF, TGF~3, IGF-1, IGF-2, IL-1, prothymosin a and thymosin a1 in an
effective
amount.
The invention also includes a pharmaceutical composition comprising a
therapeutically effective amount of T~34 or a T~i4 isoform in a
pharmaceutically acceptable
carrier. Such carriers include those listed above with reference to parenteral
administration.
The actual dosage or reagent, formulation or composition that heals or
prevents
blisters, sores and skin degeneration associated with EB may depend on many
factors,
including the size and health of a subject. However, persons of ordinary skill
in the art
can use teachings describing the methods and techniques for determining
clinical
dosages as disclosed in PCT/US99/17282, supra, and the references cited
therein, to
determine the appropriate dosage to use.
Suitable topical formulations include T~4 or a T(34 isoform at a concentration
within the range of about 0.001 - 10% by weight, more preferably within the
range of
about 0.01 - 0.1 % by weight, most preferably about 0.05% by weight.
The therapeutic approaches described herein involve various routes of
. administration or delivery of reagents or compositions comprising the T~i4
or other
compounds of the invention, including any conventional administration
techniques (for
example, but not limited to, topical administration, local injection,
inhalation, or systemic
administration), to a subject. The methods and compositions using or
containing T~34 or
other compounds of the invention may be formulated into pharmaceutical
compositions
by admixture with pharmaceutically acceptable non-toxic excipients or
carriers.
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The invention includes use of antibodies which interact with T~4 peptide or
functional fragments thereof. Antibodies which consists essentially of pooled
monoclonal
antibodies with different epitopic specificities, as well as distinct
monoclonal antibody
preparations are provided. Monoclonal antibodies are made from antigen
containing
fragments of the protein by methods well known to those skilled in the art as
disclosed in
PCT/US99/17282, supra. The term antibody as used in this invention is meant to
include
monoclonal and polyclonal antibodies.
In yet another embodiment, the invention provides a method of treating a
subject
by administering an effective amount of an agent which modulates T~34 gene
expression.
The term "modulate" refers to inhibition or suppression of T(34 expression
when T(34 is
over expressed, and induction of expression when T(34 is under expressed. The
term
"effective amount" means that amount of T(34 agent which is effective in
modulating T~i4
gene expression resulting in reducing the symptoms of the T(34 associated EB.
An agent
which modulates T~4 or T~i4 isoform gene expression may be a polynucleotide
for
example. The polynucleotide may be an antisense, a triplex agent, or a
ribozyme. For
example, an antisense directed to the structural gene region or to the
promoter region of
T(34 may be utilized.
In another embodiment, the invention provides a method for utilizing compounds
that modulate T~4 activity. Compounds that affect T(34 activity (e.g.,
antagonists and
agonists) include peptides, peptidomimetics, polypeptides, chemical compounds,
minerals such as zincs, and biological agents.
While not be bound to any particular theory, the present invention may promote
healing or prevention of blisters, sores and skin degeneration associated with
Epidermolysis Bullosa by inducing terminal deoxynucleotidyl transferase (a non-
template
directed DNA polymerase), to decrease the levels of one or more inflammatory
cytokines,
or chemokines, and to act as a chemotactic factor for endothelial cells, and
thereby
promoting healing or preventing degenerative changes in skin brought about by
Epidermolysis Bullosa or other degenerative or environmental factors.
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