COMPOSITION AND METHOD FOR ENHANCING ELASTICITY OF TISSUE

COMPOSITION ET PROCEDE DESTINE A AMELIORER L'ELASTICITE DES TISSUS

English Abstract

The present invention is directed to a composition and method used to enhance
the elasticity and/or appearance of tissue. Specifically, the present
invention is directed to a composition formulated from peptides having low
molecular weights and which substantially correspond to sequences found in
elastin.

French Abstract

Cette invention concerne une composition et un procédé utilisés en vue d'améliorer l'élasticité et/ou l'apparence des tissus. Plus particulièrement, l'invention concerne une composition préparée à partir de peptides possédant de faibles poids moléculaires et correspondant sensiblement à des séquences trouvées dans l'élastine.

Claims

WHAT IS CLAIMED IS:
1. A composition useful in treating a condition of mammalian tissue, wherein
said composition comprises a peptide or biological equivalent thereof,
selected from the
group consisting of SEQ ID 42, SEQ ID 43, SEQ ID 44, SEQ ID 45, SEQ ID 46, SEQ
ID 47,
SEQ ID 48, SEQ ID 49, SEQ ID 50, SEQ ID 51, SEQ ID 52, SEQ ID 53, SEQ ID 54,
SEQ ID 55, SEQ ID 56, SEQ ID 57, SEQ ID 58, SEQ ID 59, SEQ ID 60, SEQ ID 61,
SEQ ID 62, SEQ ID 63, SEQ ID 64, SEQ ID 65 SEQ ID 66, SEQ ID 67, SEQ ID 68,
SEQ ID 69, SEQ ID 70, SEQ ID 71, SEQ ID 72, SEQ ID 73, SEQ ID 74, SEQ ID 75.
2. The composition of claim 1, wherein said composition is a cosmetic
preparation.
3. The composition of claim 2, wherein said cosmetic preparation is formulated
as a topical preparation to be applied to a patient's skin.
4. The composition of claim 3, wherein said topical preparation is selected
from
the group consisting of an emulsion, lotion, spray, aerosol, powder, ointment,
cream and
foam.
5. The composition of claim 1, wherein the mammalian tissue being treated is a
blood vessel.
6. The composition of claim 1, wherein the composition is useful for treating
a
condition selected from the group consisting of hypertension, coronary heart
disease,
arteriosclerosis, angina, coronary thrombosis, chronic obstructive pulmonary
disease, and
restenosis post angioplasty.
7. The composition of claim 1, wherein said peptide is useful in improving
tissue
turgor.
42


8. The composition of claim 1, wherein said composition further comprises a
pharmaceutically acceptable carrier.
9. A method of enhancing tissue elasticity, said method comprises
administering
a therapeutically effective concentration of a peptide or biological
equivalent thereof, selected
from the group consisting of SEQ ID 42, SEQ ID 43, SEQ ID 44, SEQ ID 45, SEQ
ID 46,
SEQ ID 47, SEQ ID 48, SEQ ID 49, SEQ ID 50, SEQ ID 51, SEQ ID 52, SEQ ID 53,
SEQ ID 54, SEQ ID 55, SEQ ID 56, SEQ ID 57, SEQ ID 58, SEQ ID 59, SEQ ID 60,
SEQ ID 61, SEQ ID 62, SEQ ID 63, SEQ ID 64, and SEQ ID 65 SEQ ID 66, SEQ ID
67,
SEQ ID 68, SEQ ID 69, SEQ ID 70, SEQ ID 71, SEQ ID 72, SEQ ID 73, SEQ ID 74,
SEQ ID 75.
10. The method of claim 9, wherein the peptide is SEQ ID 55.
11. The method of claim 9, wherein the peptide is SEQ ID 60.
12. The method of claim 9, wherein the tissue is selected from the group
consisting of a blood vessel, lung tissue, and skin.
13. The method of claim 9, wherein administration of the peptide is repeated
over
a predetermined time period.
14. The method of claim 9, wherein the peptide is an active ingredient in a
cosmetic formulation.
15. A composition comprising:
a skin enhancing agent;
and a peptide,
said peptide being selected from the group consisting of: SEQ ID 1,
SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5, SEQ ID 6, SEQ ID 7, SEQ ID 8, SEQ ID
9,
SEQ ID 10, SEQ ID 11, SEQ ID 12, SEQ ID 13, SEQ ID 14, SEQ ID 15, SEQ ID 16,
43


SEQ ID 17, SEQ ID 18, SEQ ID 19, SEQ ID 20, SEQ ID 21, SEQ ID 22, SEQ ID 23,
SEQ ID 24, SEQ ID 25, SEQ ID 26, SEQ ID 27, SEQ ID 28, SEQ ID 29, SEQ ID 30,
SEQ ID 31, SEQ ID 32, SEQ ID 33, SEQ ID 34, SEQ ID 35, SEQ ID 36, SEQ ID 37,
SEQ ID 38, SEQ ID 39, SEQ ID 40, SEQ ID 41, SEQ ID 42, SEQ ID 43, SEQ ID 44,
SEQ ID 45, SEQ ID 46, SEQ ID 47, SEQ ID 48, SEQ ID 49, SEQ ID 50, SEQ ID 51,
SEQ ID 52, SEQ ID 53, SEQ ID 54, SEQ ID 55, SEQ ID 56, SEQ ID 57, SEQ ID 58,
SEQ ID 59, SEQ ID 60, SEQ ID 61, SEQ ID 62, SEQ ID 63, SEQ ID 64, SEQ ID 65,
SEQ ID 66, SEQ ID 67, SEQ ID 68, SEQ ID 69, SEQ ID 75, SEQ ID 71, SEQ ID 72,
SEQ ID 73, SEQ ID 74, SEQ ID 75, their biological equivalents and combinations
of any of
SEQ IDs 1-75.
16. The composition of claim 15, wherein said skin enhancing agent is a
retinoid.
17. The composition of claim 16, wherein said retinoid is all-trans retinoic
acid.
18. The composition of claim 16, wherein said retinoid is Retin A.
19. The composition of claim 16, wherein said retinoid is tretinoin.
20. The composition of claim 15, wherein said peptide is SEQ ID 17.
21. The composition of claim 15, wherein said peptide is SEQ ID 19.
22. The composition of claim 15, wherein said peptide is selected from the
group
consisting of SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5, SEQ ID 6, SEQ
ID 7,
SEQ ID 8, SEQ ID 9, SEQ ID 10, SEQ ID 11, SEQ ID 12, SEQ ID 13, SEQ ID 14,
SEQ ID 15, SEQ ID 16, SEQ ID 17, SEQ ID 18, SEQ ID 19, SEQ ID 20, SEQ ID 21,
SEQ ID 22, SEQ ID 23, SEQ ID 24, SEQ ID 25, SEQ ID 26, SEQ ID 27, SEQ ID 28,
SEQ ID 29, SEQ ID 30, SEQ ID 31, SEQ ID 32, SEQ ID 33, SEQ ID 34, SEQ ID 35,
SEQ ID 36, SEQ ID 37, SEQ ID 38, SEQ ID 39, SEQ ID 40, SEQ ID 41.
23. A peptide having a formula of R1-Valyl-Valyl-Prolyl-R2, wherein R1 is an
amino portion modified to include an amine, amide, or amino acid sequence
having 1-10
44


amino acids and R2 is a carboxy portion modified to include an amide, ester,
or carboxy
terminus sequence having 1-10 amino acids.
24. The peptide of claim 23, wherein the peptide is selected from the group
consisting of SEQ ID 15, SEQ ID 42, SEQ ID 43, SEQ ID 44, SEQ ID 45, SEQ ID
46,
SEQ ID 47, SEQ ID 48, SEQ ID 49, SEQ ID 50, SEQ ID 51, SEQ ID 52, SEQ ID 53,
SEQ ID 54, SEQ ID 55, SEQ ID 56, SEQ ID 57, SEQ ID 58, SEQ ID 59, SEQ ID 60,
SEQ ID 61, SEQ ID 62, SEQ ID 63, SEQ ID 64, and SEQ ID 65.
25. The peptide of claim 23, wherein the peptide is SEQ ID 17.
26. The peptide of claim 23, wherein R2 is selected from the group consisting
of a
glutamine residue and modified glutamine residue.
27. A peptide having a formula of R1-Valine-Valine-Proline-Asparagine-R2,
wherein R1 is an amino portion modified to include an amine, amide, or amino
acid sequence
having 1-10 amino acids and R2 is a carboxy portion modified to include an
amide, ester, or
carboxy terminus sequence having 1-10 amino acids.
28. The peptide of claim 27, wherein the peptide is SEQ ID 55.
29. The peptide of claim 27, wherein the peptide is SEQ ID 60.

Description

CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
COMPOSITION AND METHOD FOR ENHANCING ELASTICITY OF TISSUE
BACI~.GROUND OF THE INVENTION
The present invention relates to compositions suitable as therapeutics,
pharmaceutics, and/or cosmetics. The compositions) of the present invention
preferably
include a compound which is analogous with a portion of mammalian elastin. The
present
invention also relates to methods of using the compounds and compositions
described and
contemplated herein to achieve a therapeutic, pharmaceutical, or cosmetic
effect. It is
preferable that the compounds) and compositions) of the present invention be
administered
at a therapeutically effective concentration and/or as an active ingredient of
a pharmaceutical,
therapeutic and/or cosmetic preparation.
Elastin is a highly cross-lined complex polypeptide and a major component
of elastic fibers present in the tissue of animals. Elastin is l~nown to play
a very important
role in maintaining tone, structure, and turgor of tissue. Elastin is found in
most connective
tissue in conjmlction with collagen and polysaccharides. Large amounts of
elastin can be
found in blood vessel walls, particularly in the arch of the aorta near the
heart. Elastin is also
present in slcin, tendons, ligaments, nerves, loose connective tissue, and in
the uterus. In
normal mammalian shin, specifically human shin, elastic tissue proteins
represent a relatively
small fraction of the total dermal proteins.
Elastin fibers are capable of stretching to several times their length and
then
returning to their original size upon release of tension. Accordingly, elastin
contributes to the
physiological elasticity of tissue. It has been fotmd, for instance, that a
loss of elasticity in
the skin is associated with decrease in the tone and turgor of the shin. It is
speculated that the
decrease in skin tone and turgor occurs through degradation of elastin and
collagen. The
quality of aging shin is affected by a loss in elasticity that occurs
gradually. This loss of
elasticity is most prominent in the last decades of life. Loss of elasticity
is due in part to a
change in the quality of connective tissue proteins such as elastin and
collagen. The most
common signs of aging shin are fine lines and wrinkles, which are partially
brought on by
exposure to ultra violet radiation, diseases and smoking.
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
Attempts have been made to use elastin itself as a cosmetic agent, however,
the dense cross-linlced structure of elastin makes it very difficult to
solubilize.
SUMMARY OF THE PRESENT INVENTION
The present invention is directed to compositions that are pharmaceutical,
therapeutic, and/or cosmetic in nature. Compositions of the present invention
preferably
modify or appear to modify the physical characteristics of the tissue to which
it is applied.
As described herein, the compounds) which best accomplish an increase or
apparent increase in tissue elasticity and turgor are ones wluch are analogous
to or
substantially homologous with portions of elastin. Compounds contemplated
within the
present invention are those that mimic the action or functionality of amino
acid containing
peptides or peptide-life compounds of the present invention.
More specifically, the compounds or compositions of the present invention
mimic the actions or functionality of a peptide selected from the group
consisting of
SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5 , SEQ ID 6, SEQ ID 7 , SEQ ID
8,
SEQ ID 9, SEQ ID 10 , SEQ ID 11, SEQ ID 12, SEQ ID 13, SEQ ZD 14, SEQ ID 15,
SEQ ID 16, SEQ ID 17, SEQ ID 18, SEQ ID 19, SEQ ID 20, SEQ ID 21, SEQ ID 22,
SEQ ID 23, SEQ ID 24, SEQ ID 25, SEQ ID 26, SEQ ID 27, SEQ ID 28, SEQ ID 29,
SEQ ID 30, SEQ ID 31, SEQ ID 32, SEQ ID 33, SEQ ID 34, SEQ ID 35, SEQ ID 36,
SEQ ID 37, SEQ ID 38, SEQ ID 39, SEQ ID 40, SEQ ID 41, SEQ ID 42, SEQ ID 43,
SEQ ID 44, SEQ ID 45, SEQ ID 46, SEQ ID 47, SEQ ID 48, SEQ ID 49, SEQ ID 50,
SEQ ID 51, SEQ ID 52, SEQ ID 53, SEQ ID 54, SEQ ID 55, SEQ ID 56, SEQ ID 57,
SEQ ID 58, SEQ ID 59, SEQ ID 60, SEQ ID 61, SEQ ID 62, SEQ ID 63, SEQ ID 64,
SEQ ID 65, SEQ ID 66, SEQ ID 67, SEQ ID 68, SEQ ID 69, SEQ ID 75, SEQ ID 71,
SEQ ID 72, SEQ ID 73, SEQ ID 74 and SEQ ID 75 and their biological
equivalents.
Another embodiment of the present invention is directed to a peptide or
peptide-life compound having a formula of Rl-Valine-Valine-Proline-R2, wherein
Rl is an
amino portion modified to include an amine, amide, or amino acid sequence
having 1-10
2
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
amino acids and R2 is a carboxy portion modified to include an amide, ester,
or carboxy
terminus sequence having 1-10 amino acids.
Another embodiment of the present invention is directed to a peptide or
peptide-lilce compound having a formula of Rl-Valine-Valine-Proline-Asparagine-
RZ,
wherein Rl is an amino portion modified to include an amine, amide, or amino
acid sequence
having 1-10 amino acids and R2 is a carboxy portion modified to include an
amide, ester, or
carboxy terminus sequence having 1-10 amino acids.
Another embodiment of the present invention is directed to a peptide or
peptide-like compound having a formula of Rl-Valine-Valine-Proline-Glutamine-
R2, wherein
Rl is an amino portion modified to include an amine, amide, or amino acid
sequence having
1-10 amino acids and R~ is a carboxy portion modified to include an amide,
ester, or carboxy
terminus sequence having 1-10 amino acids.
Another embodiment of the present invention is directed to a peptide or
peptide-like compound having a formula of Rl-Leucine-Glycine-R2, wherein Rl is
an
amino portion modified to include an amine, amide, or amino acid sequence
having 1-10
amino acids and R~ is a carboxy portion modified to include an amide, ester,
or carboxy
terminus sequence having 1-10 amino acids.
Another embodiment of the present invention is directed to a peptide having a
formula of Rl-Leucine-Glycine-Alanne-Glycine-Glycine-Alanine-Glycine-R2,
wherein Rl
is an amino portion modified to include an amine, amide, or amino acid
sequence having 1-
amino acids and R~ is a carboxy portion modified to include an amide, ester,
or carboxy
terminus sequence having 1-10 amino acids.
Another embodiment of the present invention is directed to a method of
enhancing the functionality, tone, turgor, and/or elasticity of the tissue to
which it is
administered by administrating effective amounts of a peptide to the tissue.
When treating
shin with the compositions of the present invention, the appearance of the
shin is eWanced.
It is believed that this enhancement is a consequence of improving the
elasticity and overall
3
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
appearance of the skin. It is preferable that the administration step be
comprised of a number
of separate steps which are repeated over a predetermined time (e.g., twice
daily for one
weelc). It is preferable that the predetermined time exceeds one week of daily
administration
of the compound, more preferably two weeks, and most preferably at least a
month of daily
topical application (with twice daily of the peptide administration over the
month being even
more preferable).
The compounds and agents described herein are preferably administered at an
effective concentration within a therapeutic, pharmaceutical or cosmetic
composition. The
therapeutically effective concentration of the compounds) (i.e. the peptide or
peptide-like
compounds) is preferably in a range of about .0002% to about 90% by weight,
more
preferably in a range of about .0S% to about 50% by weight, even more
preferably in a range
of about 0.5% to about 10%, even more preferably in the range of about 1.0% to
about 2.0%,
and even more preferably about 1.8% by weight.
The composition of the present invention can be formulated as a cosmetic
preparation to be applied topically to the shin, such as in an emulsion,
lotion, spray, powder,
ointment, cream, or foam or in other suitable pharmaceutical vehicles or
carriers commonly
lcnown in the art for other types of administration (e.g., oral or
subcutaneous). The delivery
system of the present invention is preferably a topical delivery system but
also may be a
subcutaneous, transcutaneous, oral, nasal, aerosol, or patch. The compositions
of the present
invention have many other applications. For example, they may also be used to
coat surgical
devices such as stems and the like.
The composition of the present invention may be suitable to treat a variety of
diseases or conditions selected from the group consisting of conditions or
diseases of the
skin, tendons, sheaths and/or bone, hair, lip, back or spine, brain or nervous
system,
autoimmune system, lungs, muscle, joints, nails, blood vessels/lymphatics,
breast, cartilage,
ear, eye, genito-urinary tract, gastrointestinal tract, immunologic systems,
ulcerative, blood
vessels/heart (e.g., hypertension), and other body systems.
4
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
BRIEF DESCRIPTION OF THE DRAWINGS
The features, aspects, and advantages of the present invention will become
better understood in light of the following description, appended claims, and
accompanying
drawings wherein:
Fig. 1 is a bar graph illustrating increased elastin production as a result of
application of select compounds of the present invention to mammalian skin.
Fig. 2 is a micrograph illustrating the microvascular response of the slcin
tissue
with peptides of the present invention: Fig. 2a is a representative sample
from unshaven
control tissue; Fig. 2b is a representative saanple from shaven control
tissue; Fig. 2c is a
representative sample of tissue which received DHEA topical treatment; Fig. 2d
is a
representative sample of tissue treated topically with peptides of the present
invention.
Fig. 3 is a bar graph illustrating tritiated Thyrnidine incorporation with
selected peptide or peptide-life compounds.
Fig. 4 illustrates an elastin rtPCR assay;
Fig. 5 illustrates quantification of newly synthesized tropoelastin visualized
with an anti-tropoelastin antibody and FITC-labeled conjugate. (A) Basal level
of tropoelastin
(green) produced by non-treated adult human dermal fibroblasts. (B) Increased
level of
tropoelastin (green) after incubation with HEP for 24 hours. (C) Tropoelastin
levels increase
with time above basal level;
Fig. 6 shows human shin treated with the composition of the present invention
(i.e. 2% HEP). Top panels are as indicated, before and after, H&E stained
tissue sections for
nuclei. The lower panels are before and after, Verhoff's stain for elastin;
and
Fig. 7 is a bar graph illustrating the measurement of elasticity of the slcin
by a
Cutometer with various formulations in accordance with the present invention.
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
DETAILED DESCRIPTION OF THE PRESENT INVENTION
So that the invention described herein may be more fully understood, the
following detailed description is set forth. The description is in no way
meant to limit the
breadth of the claims, but rather to specifically point out novel aspects of
the present
invention.
The present invention relates to compositions useful in increasing
functionality, elasticity, tone, turgor, and/or appearance of tissue. The
present invention is
also directed to administering therapeutically effective concentrations of the
compositions.
Defifaitions Useful ih Understanding the Invehtioy~: As used herein, the term
"subject" or "patient " means any mammal in which elastin is utilized for
proper tissue
function or appearance. The methods herein for use contemplate prophylactic,
cosmetic, and
curative use.
As used herein, the term "about" means plus or minus 10% of the numerical
value of the number with which it is being used. Therefore, about 50% means in
the range of
45%-55%. As used herein, the teen "Dalton" (or "Da") refers to the unit of
mass which is
equivalent to the mass of a hydrogen atom (I.66 x 10'24 gram).
Generally spearing, the term "tissue" refers to any aggregation of similarly
specialized cells which are united in the performance of a particular
function. The term
"tissue", as usually used herein, refers to tissue which includes elastin as
part of its structure
and/or function. For example, connective tissue which is made up of, among
other things,
collagen fibrils and elastin fibrils satisfies the definition of "tissue".
Additionally, since
elastin appears to be inherently involved in the visco-elasticity of blood
vessels, veins, and
arteries, these would be encompassed in the definition of "tissue". The term
"skin" is
encompassed by the term "tissue" but specifically means the outer integument
or covering of
the body, including the dermis and the epidermis which rests upon subcutaneous
tissue.
6
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
The term "elastin peptide fragment" in either singular or plural form herein
refers to the fact that the peptide or amino acid sequence being discussed
corresponds to, is
the biological equivalent of, is analogous with, or is substantially
homologous with, a portion
of elastin. The term "elastin peptide fragment " is not meant to convey any
meaning
regarding the source or starting material or method of arriving at the elastin
peptide fragment.
As used herein, the term "providing" when used in conjunction with a
therapeutic, pharmaceutical, or cosmetic, means to administer an agent
directly into or onto a
target tissue or to administer a therapeutic to a patient whereby the
therapeutic positively
impacts the tissue to which it is targeted (either in a prophylactic, curative
or cosmetic
manner). Thus, as used herein, the term "providing", when used in conjunction
with an
ehastin peptide fragment and/or a skin enhancing agent, can include, but is
not limited to,
providing the co mpound(s) into or onto the target tissue; providing an
elastin peptide
fragment systemically to a patient (e.g., by providing intravenous injection
whereby tlae
therapeutic agent reaches the target tissue); and providing the therapeutic
agent in the form of
the encoding sequence thereof to the target tissue (e.g., by so-called gene-
therapy tecluuques)
whereby the elastin peptide fragment and/or skin enhancing agent is expressed
within the
target tissue. Details on techniques for formulation and administration of
pharmaceuticals
may be found in the latest edition of Remingtof2's Pharmaceutical Sciefaces
(Mack Publishing
Co, Easton Pa.). Although local topical delivery is desirable, there are other
acceptable
means of delivery, for example: oral, parenteral, aerosol, intramuscular,
subcutaneous,
transcutaneous, intamedullary, intrathecal, intraventricular, intravenous,
intraperitoneal, or
intranasal administration.
As used herein, the term "therapeutic" means an agent utilized to treat,
combat, ameliorate, prevent or improve a condition or disease of a patient. A
particular
condition treated in the present invention is deficient elastin in a
particular tissue, that is, a
need in the tissue for more elastin. As it applies to skin, "therapy" is often
measured by
turgor, tone, appearance, degree of wrinlcles, and youthfuh~ess. As the term
applies to blood
vessels it may be measured by the degree of elasticity or proper vasomotor
response
(vasodilatation/vasoconstriction) of the vessel. Accordinghy, therapeutic
treatment of blood
vessels may have implications in diseases associated with visco-elasticity,
including
7
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
hypertension, arteriosclerosis, angina, angiogenesis, myocardial infarction,
coronary
tluombosis, restenosis post angioplasty, and chronic obstructive puhnonary
disease.
The compounds and compositions of the present invention may also be useful
as an agent for modifying tissue, especially slcin. The term "modify" is used
to convey that
the present invention changes either the appearance, form, characteristics
and/or the physical
attributes of the tissue to which it is being provided, applied or
administered. The change in
form can be reflected in any of the following, alone or in combination:
enhanced appearance
of the skin; increased softness of the shin; increased turgor of the skin;
increased texture of
the skin; increased elasticity of the skin; decreased wrinkle formation and
increased
endogenous elastin production in the shin.
Finally, the term "cosmetic," as used herein, refers to a beautifying
substance
or preparation which preserves, restores, bestows, simulates, or enhances the
appearance of
bodily beauty, specifically as it relates to the appearance of tissue or skin.
Initial Methods, Materials, acad Fo~mulatioczs: Elastin itself can be used as
starting material in the digestion or cleavage methods described herein to
arnve at the peptide
portion of the composition. This elastin can be derived from a number of
sources known in
the art. The sequences of the present invention can either be isolated from
the digestion pool
(and chemically modified if desired) or the peptides may be synthesized with a
peptide
synthesizer. A particularly useful source of elastin is ligamesatum hzcchae.
Ligameyatum
fauclaae contains large amounts of elastin (approximately 70% of the dry
weight of this
ligament is elastin), especially in proportion to the amount of collagen. Due
to the relatively
high elastin content and relatively low collagen content, liga~r2ehtum uuchae
is an ideal
starting material to use in deriving the elastin peptide fragments of the
present invention.
Ligarneratmn rauchae may be cleaned first using a procedure similar to that
disclosed in U.S.
Patent No. 5,028,695, the cleaning portion of which is incorporated herein by
reference
thereto. Although a preferred source of starting elastin is ligamehtum
huclaae, other
ligaments, tendons, comzective tissue, tissue, and synthetic sources may also
be used. For
example, the arteries and lungs, and other animal tissue, especially those
which have
significant amounts of elastin, can be used (e.g., rat, sheep, and porcine
aorta can be used as a
8
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
source of elastin as described in L.B. Sandberg, Connective Tissue Research,
1990, Vol. 25,
pp. 139-148, incorporated herein in its entirety by reference thereto). Also,
elastin from
different sources, or elastin and collagen from the same or different sources
could be mixed
together to produce a particular advantageous mix suitable for digestion or
hydrolytic
cleavage.
The ligament extraction process is comprised of taping dissected ligan2ehtum
yauclaae ligaments and removing as much fat and excess connective tissue as
possible. These
"clean" ligaments are then chopped into about one centimeter square (1 cm2)
pieces and
washed with doubly distilled water ("DDW"). The clean ligaments are then
placed on a
metal mortar and pre-chilled to -20°F. Liquid nitrogen is added to
freeze the tissue. The
ligaments are then minced or pulverized with the appropriate tool and re-
suspend ed in 1%
aqueous NaCI at a ratio of about 100 grams of tissue to about three liters of
1% aqueous NaCl
and homogenized in a blaring blender at high speed for 30-60 seconds. The
homogenized
ligament is transferred to a four-liter beaker and stirred overnight at
4°C on a magnetic stirrer,
after which it is centrifuged at 32,500 x G and a pellet is formed. The
supernatant is checked
for protein content using the Biuret method for protein determination. The
Biuret reaction is
done by mixing 2 milliliters of extract with 3 milliliters of reagent and
immediately reading
the protein concentration of the supernatant either by simple visual
inspection or at 540
nanometers on a spectrophotometer. The supernatant is then discarded. The
pellet (referred
to hereinafter as the elastin pellet) is resuspended in 1 % aqueous NaCI and
homogenized.
The process of homogenizing in a blaring blender, stirring overnight and
centrifuging are
repeated three to four times until the supernatant is Biuret negative. After
centrifugation, the
elastin pellet is resuspended in DDW and autoclaved at 30 psi for six hours.
The resuspended
elastin pellet is centrifuged again and the supernatant is tested for protein
content via the
Biuret method. The elastin is washed with boiling DDW and then with DDW at
room
temperature. The washes are tested for protein content via the Biuret method.
If the washes
are Biuret negative, the elastin pellet is dried with chloroformlmethanol
solution at a ratio of
2 paxts chloroform to 1 part methanol. If the Biuret test is positive, the six
hour autoclave
procedure with wash step is repeated until the Biuret test is negative.
Finally, the elastin
residue is washed with five volumes of pure methanol and air-dried at room
temperature.
The elastin residue is transferred to a desiccator and dried over P205 for 24
hours or until the
9
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
weight of the elastin residue is stable. The elastin residue is then milled in
a Willey mill
through a 40-mesh screen followed by a 60-mesh screen.
For the thermolysin digestion, three times re-crystallized thermolysin product
from CalBiochem (10394 Pacific Center Court, San Diego, CA 92121) was used.
The
thermolysin preparation contains sufficient calcium to ensure maximal activity
of the
enzyme. The thermolysin digestion is done as follows: a waterbath is brought
to a 55 °C
temperature with a rotary shaker and five grams of the finely milled largely
insoluble elastin
residue is hydrated with one liter of DDW for fifteen minutes at room
temperature. After
hydration, the one liter of DDW which contains the five grams of elastin is
placed in the 55 C
bath and the pH of the elastinlwater mixture is brought to between 7 and 8
with 10%
methylamine. Fifty milligrams of thermolysin (bacillus thermoproteolyticus) is
added
directly to the elastiWwater mixture. The thermolysin contains about 60%
protein (60.2%),
about 13% (13.2%) sodium acetate, and about 25% (25.3%) calcium acetate, with
a specific
activity of about 8,720 LU./mg dry weight. The pH of the elastin water mixture
is monitored
with a pH meter or pH stat and adjusted with 10% methylamine to keep the pH
between 6.8
and 7.5. The digestion is allowed to continue for 75 minutes. Concentrated
hydrochloric
acid is then added to adjust the pH to 3.0 to terminate the digestion.
After digestion is terminated, the digested product is preferably filtered
through a PM 10 Diaflow 10,000 molecular weight cut-off ultra-filtration
membrane to filter
out any protein or peptides exceeding about 10,000 Da molecular weight. The
resulting
supernatant is a derived composition comprised of peptides having a molecular
weight of less
than about 10,000 Da. This step is useful in removing any unwanted higher
molecular weight
material from the compositions of the present invention. This may be
particularly useful in
removing any potentially harmful higher molecular weight agents, such as
prions or other
high molecular weight pathogens.
The elastin peptide fragment/water mixture (inclusive of SEQ IDs 1-41 in
Table I shown below) which is obtained upon digestion with thermolysin
described above is
flash evaporated to dryness, redissolved in a small volume of DDW, and if
desired, diluted
sufficiently with DDW for lyophilization to dryness. In the alternative,
rather than
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
redissolving the elastin peptide(s), the filtered product is fieeze dried
twice, resulting in a
powder which contains 30 weight % chemically-bound water and very little salt
(NaCI).
Table I is a list of peptide sequences which in combination with other skin
enhancing agents
exhibit desirable characteristics. It should be noted that SEQ IDs 23 and 32
appear to be a
cormnon sequence because Proline hydroxylation is a post-translational event.
A typical cream incorporating SEQ IDs 1-41 of the present invention is
described as follows in order of relative concentration of each of the
ingredients: Purified
Water, Hydrolyzed elastin peptides ("HEP") (i.e., SEQ IDs 1-41),
Polyglycerylmethacrylate
(and) propylene glycol, Petrolatum, Dicaprylylether, Peg- 5, Glycerylstearate,
Glycerine,
Dimethicone (and) dimethiconal, Cetyl alcohol, Sweet almond oil, Acrylates/C10-
30,
Tocotheryl acetate, Phenoxyethanol, Benzyl alcohol, Disodium EDTA, Sodium
hydroxide,
Lactic acid, Sodium chloride. The HEP is often stated in weight percentage
(e.g., 2% HEP is
2 wt/wt% HEP)
TABLEI
SEQ PEPTIDE MOL NAME (N- to C-terminal)
#


WT


1. AVG 245 Alanine-Valine-Glycine


2. VGAG 302 Valine-Glycine-Alanine-Glycine


3. IGG 302 Isoleucine-Glycine-Glycine


4. LG 188 Leucine-Glycine


5. IGAG 316 Isoleucine-Glycine-Alanine-Glycine


6. LGG 245 Leucine-Glycine-Glycine


7. VAPG 342 Valine-Alanine-Proline-Glycine


8. LGPG 342 Leucine-Glycine-Proline-Glycine


9. LGAG 316 Leucine-Glycine-Alalune-Glycine


10. VGPG 328 Valine-Glycine-Proline-Glycine


11. FGPG 376 Phenylalanine-Glycine-Proline-Glycine


11
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
12. VGPQ 399 Valine-Glycine-Proline-Glutamine


13. LGA 259 Leucine-Glycine-Alanine


14. VGPA 342 Valine-Glycine-Proline-Alanine


15. VVPG 370 Valine-Valine-Proline-Glycine


16. AVPG 342 Alanine-Valine-Proline-Glycine


17. VVPQ 441 Valine-Valine-Proline-Glutamine


18. VAARPG 569 Valine-Alanine-Alanine-Arginine-Proline-


Glycine


19. LGAGGAG 501 Leucine-Glycine-Alanine-Glycine-Glycine-


Alaiune-Glycine


20. AIPG 356 Alanine-Isoleucine-Proline-Glycine


21. LGPGG 399 Leucine-Glycine-Proline-Glycine-Glycine


22. AAAQA 430 Alanine-Alanine-Alanine-Glutamine-Alanine


23. VGVHypG 444 Valine-Glycine-Valine-Hydroxyproline-Glycine


24. VYPGG 491 Valine-Tyrosine-Proline-Glycine-Glycine


25. IGGVGG 458 Isoleucine-Glycine-Glycine-Valine-Glycine-


Glycine


26. VAPGVG 498 Valine-Alanine-Proline-Glycine-Valine-Glycine


27. LGVGG 401 Leucine-Glycine-Valine-Glycine-Glycine


28. VLPG 384 Valine-Leucine-Proline-Glycine


29. FR.A.AA 534 Phenylalanine-Arginine-Alanine-Alanine-


Alanine


30. VGGVPG 484 Valine-Glycine-Glycine-Valine-Proline-Glycine


31. FGPGG 433 Phenylalanine-Glycine-Proline-Glycine-Glycine


32.x' VGVPG 427 Valine-Glycine-Valine-Proline-Glycine


33. VLPGAG 512 Valine-Leucine-Proline-Glycine-Alanine-


Glycine


12
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
34. VGLHypG 458 Valine-Glycine-Leucine-Hydroxyproline-


Glycine


35. LGVGA 415 Leucine-Glycine-Valine-Glycine-Alanine


36. AFPG 390 Alanine-Phenylalanine-Proline-Glycine


37. AFPGA 461 Alanine-Phenylalanine-Proline-Glycine-Alanine


38. VGIPA 455 Valine-Glycine-Isoleucine-Proline-Alanine


39. VGGIPT 542 Valine-Glycine-Glycine-Isoleucine-Proline-


Threonine


40. VGVGVPG 583 Valine-Glycine-Valine-Glycine-Valine-Proline-


Glycine


41. LGPGVG 498 Leucine-Glycine-Proline-Glycine-Valine-


Glycine


It would appear that certain groups of the peptides described in Table I (SEQ
IDs 1-41, inclusive) have preferred characteristics as they relate to
cosmetic, pharmaceutical
or therapeutic application to the slcin. The elastin peptide mixtures isolated
from thermolysin
digestion of elastin (i.e., SEQ IDs 1-41, inclusive) were collected as they
came off of a HPLC
column. Instead of isolating each of the thermolysin peptide fragments
individually, 5
fractions or clusters of peptides were collected in the S-50 minute range and
tested for
activity utilizing a bromodeoxyuridine Triphosphate (BrdUTP) incorporation
assay. The
assay measures production of mRNA involved in protein synthesis. Table II
measures the
green fluorescence intensity as a measure of increased mRNA in RFL-6 cells in
response to
the pooled elastin fragment.
13
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
TABLE II
Fraction # Approximate Elution time Approximate % Change Minus
Control
1 5 . 3 min. -11. 8 min 1
2 11.8 min - 23.0 min 4%
3 23 .0 min - 44.1 min 41
4 44.1 min - 45. 8 min 10%
45.8 min - 50.0 min 2%
6 Unfractionalized mixture (SEQ IDs 1-41) 52%
Each of the fractions show an increase in mRNA in RFL-6 cells over the
control group. From the test, however, it appears that Fraction #3 alone
and/or in
combination with other fractions (e.g., as seen with Fraction #6) has a
preferred composition
when increasing elasticity, turgor, and/or appearance of tissue, specifically
shin. Fraction 3
includes SEQ IDs 14-31. It should be noted that in light of the ease in
obtaining the
unfractionalized mixture (as described above) it may be more preferable to use
the
unfractionalized mixture than to isolate the most active ingredient.
Fraction or Cluster 3 was sub-fractionated into 10 fractions corresponding to
the ten major peals identified on the HPLC (at 215 mn). Table III below
illustrates the green
fluorescence intensity as a measure of increased mRNA ll1 RFL-6 cells in
response to sub-
fractionated portions of Fraction No. 3.
14
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
TABLE III
Expected Sed. No. % Change


Fractionin Fraction Peptide Seduence Green
#


Fluorescence


Intensity


1 SEQ ID 14 VGPA 39


2 SEQ IDs 15, 16 VVPG, AVPG 40


3 SEQ ID 17 VVPQ 85


4 SEQ IDs 18, 19 VAARPG, LGAGGAG 44


SEQ IDs 20, 21 AIl'G, LGPGG 42


6 SEQ ID 22 AAAQA 20


7 SEQ ID 23 VGVHypG 57


8 SEQ m 24 V~PGG 38


9 SEQ IDs 25, 26, 27, IGGVGG, VAPGVG, 10
28, 29


LGVGG, VLPG, FR.AAA


SEQ IDs. 30, 31 VGGVPG, FGPGG 23


Blanl~ 30
(B acl~ground)
As can be seen from Table III, it appears SEQ ID 17 (VVPQ) has the greatest
activity, followed by SEQ ID 23 (VGVHypG) and then SEQ ll~s 18 (VAARPG) and 19
(LGAGGAG). It would appear that SEQ IDs. 22 and 25-31 may actually have an
adverse
impact the overall therapeutic or cosmetic value of Fraction 3, at least to
the extent the
therapeutic value is attributed to elastin production. However, applicant does
not wish to be
bound by theory or speculation in that any fraction or combination of
fractions, while
lowering the green fluorescence intensity of the fractionated sample, may in
fact add a '
desirable characteristic to the intended use of the overall mixture when
combined with
another peptide (e.g., any of SEQ IDs 1-41, respectively). Other types of
testing may in fact
demonstrate the suitability of other peptides disclosed herein for
pharmaceutical andlor
cosmetic purposes.
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
The present invention can be formulated in a number of carrier vehicles, for
example, in a spray; an aerosol; a water and an oil-type emulsion; an oil and
water-type
emulsion; a face cream or body cream; a sun lotion or after-sun lotion; or
other topical
administration vehicle. U.S. Patent No. 4,327,078, which was referenced
earlier, is
illustrative of the different types of topical administrations which may be
employed to
administer a soluble elastin-based derivative, and is incorporated herein by
reference for this
purpose. The method of administering peptides and formulations of the present
invention
employs any of a number of lmown administrative routes such as oral, IV,
subcutaneous,
transcutaneous, and topical administration. A preferred method of the present
invention
employs a pharmaceutical or cosmetic composition which enhances the physical
appearance
of and/or the elasticity of tissue. Compositions of the present invention may
be in the form of
a peptide or peptides in combination with at least one other agent, such as
stabilizing
compound, which may be administered in any sterile, bio-compatible
pharmaceutical carrier,
including, but not limited to, saline, buffered saline, dextrose, and water.
The compositions
may be administered to a patient alone, or in combination with other agents,
drugs or
hormones. Pharmaceutically-acceptable carriers may also be comprised of
excipients and
auxiliaries which facilitate processing of the active compounds into
preparations which can
be used pharmaceutically. Further details on techniques for formulation and
administration
may be found in the latest edition of Rernifzgtora's Pharmaceutical Sciences.
The
pharmaceutical composition may be provided as a salt and can be formed with
many acids,
including but not limited to, hydrochloric, sulfuric, acetic, lactic,
tartaric, malic, succinic, etc.
Salts tend to be more soluble in aqueous or other protonic solvents than are
the coiTesponding
free base forms. After pharmaceutical compositions have been prepared, they
can be placed
in an appropriate container and labeled for treatment of an indicated
condition. Such labeling
would include amount, frequency, and method of administration.
With the aforementioned wide-spread applicability in mind, a number of
peptide or peptide-like compounds were isolated and/or synthesized and
analyzed as potential
therapeutic, pharmaceutical, or cosmetic agents.
As can be seen from Fig. 1, the topical treatment with a composition which
included peptide fragments (i.e., SEQ IDs 1-41) at a concentration of about
1.3% (wt/wt) of
16
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
the formulation when applied to the shin of a Sprague-Dawley male rat over a
one month
period illustrates a doubling of the elastin content of the shin, as compared
to both control
samples and similar applications and concentration of DHEA. In Figure 1, S
CONTR
represents the Shaven Control and US CONTR represents the Unshaven Control.
Fig. 1
illustrates that the compounds of the present invention have the advantageous
qualities of
enhancing the softness or elasticity of the skin. The peptides and
formulations of the present
invention also appear to improve the texture of slcin, specifically the
physical appearance of
the skin.
The Sprague-Dawley rats used to generate Fig. 1 were treated topically with a
1.3% concentration (wt/wt) of the preparation of the hydrophilic elastin
peptide for a period
of 30 days. Testing illustrated that the endogenous elastin (measured by
microgram (qg)
Elastin per milligram (mg) Slcin Fat Free Dry weight) of each of the rats to
which the
composition was applied doubled over that of a control sample and a sample
which was
treated with a 5% concentration of DHEA over a similar time period. Three
animals each
were used to generate the data for S CONTR, US CONT, and DHEA. Eleven animals
were
used for HEP. Three skin samples from the treated areas of each animal were
taken for
study, and the three results from each animal were averaged. The means of
these results are
as follows: S CONTR (.1.408); US CONTR (2.291); DHEA (1.753); HEP (3.175). The
elastin content of the skin was determined by a precise assay for rat elastin
developed by
Sandberg, et al. (Connective Tissue Research. 25: 139-48, 1990) the assay
portion of which
is hereby incorporated herein by reference thereto. An alpha level less than
0.001 for the data
of Fig. 1 as determined by analysis of variance is significant because there
is less than one
chance in a thousand that the findings occur by chance. The data of Fig. 1
further supports
the use of the cosmetic or pharmaceutical preparation over an extended period
preferably in
the range of one week to one month, more preferably in the range of seven days
to about
fourteen days and most preferably about fourteen days of daily administration
at about 1.5%
concentration (wt/wt) of elastin peptide or peptides.
Fig. 2 is a micrograph illustrating an increased appearance and beneficial
implication of the present invention. From Fig. 2, shin treated with an
elastin peptide
fragment appears to be healthier than untreated skin. This is evidenced under
a microscope
17
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
by an increase in vascular response. In Fig. 2, fixed tissue sections of rat
skin were labeled
with fluorescein conjugated antifibronectin antibodies. Fig. 2a is a
representative sample
from the unshaven control tissue; Fig. 2b is a representative sample from the
shaven control
sample; and Fig. 2c is a representative sample of the tissue which received
DHEA topical
treatment. Finally, Fig. 2d received treatment with the present invention in a
topical form in
accordance with the samples discussed above with regard to Fig. 1. The dermal
layer in the
control panels (Figs. 2a and 2b) is relatively uniform and thin compared to
the thickness of
both Figs. 2c and 2d. For convenience, in each of panels Figs. 2a - 2 d, the
dermal layer is
bracketed. Surprisingly, panel Fig. 2d illustrates an increased concentration
of capillary
venules in the subdermal region. The capillary venules are shown in this
figure as brightly
stained oval bodies that lie beneath the dermal layer. The increase in the
concentration of
endothelial cells in the subdermal region indicates an increase in capillary
density and
therefore illustrates the potential for the peptides and formulations of the
present invention to
be used for the formation of blood vessels or capillary venules. Thus,
compositions of the
present invention may be useful in neovasculaa-ization or angiogenesis.
Modifzcatioh ofActive Peptides: The bar graph of Fig. 3 illustrates the effect
of modifying sequences in a variety of ways. The results of modifying SEQ ID
17 (what
appears to be the most active peptide for many purposes) provide important
information on
the impact of such modifications. For instance, modification of SEQ JD NO 17
which result
in SEQ ID NOs 42 and 43 appear to adversely impact the suitability for these
purposes. SEQ
ID 4 (LG) resulted in about an 8% CPM above the control; SEQ ID 17 (WPQ)
resulted in
about a 28% CPM above the control; SEQ ID 19 (LGAGGAG) resulted in about an
18%
CPM above the control; SEQ ID 42 (VVPQ-NH2) resulted in about a 1% CPM above
the
control; SEQ ID 43 (Acetyl-VVPQ) resulted in about a 1% CPM above the control;
SEQ ID
48 (GAVVPQ--NH2) resulted in about a 25% CPM above the control; and SEQ ID 44
(Acetyl-GAVVPQ--NH2) resulted in about a 5% CPM above the controls. From Fig.
3 and
the genetic expression data presented herein, it appears that the synthetic
peptide SEQ ID 17
appears to have the same or nearly the same activity as SEQ ID 17 isolated
from the HPLC
fractionalization. Accordingly, focus should be placed upon this peptide. It
would also
appear that a GA residue attached to the N-terminus of the SEQ m 42 (resulting
in SEQ m
18
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
48) has a similar activity to the activity of SEQ ID 17. The ubiquity of the
GA residue in an
elastin's peptide sequence suggests that such a modification of other peptide
fragments may
augment their activity and/or otherwise may be desirable. Having an amide at
the carboxyl
terminus or an acetyl at the N-terminus may also beneficially affect activity
and/or solubility
of the subject peptide.
The information derived from Table III and Fig. 3 was utilized to
systematically synthesize peptides which would appear to be particularly
suitable as
pharmaceutical, cosmetic, and/or therapeutic compositions. A general method
for
synthesizing peptides is described in U.S. Patent No. 4,816,513, incorporated
herein by
reference thereto in its entirety, which describes a process for automatically
constructing a
polypeptide. Additionally, U.S. Patent No. 4,668,476, incorporated herein by
reference
thereto in its entirety, also describes an apparatus for automatically constl-
ucting a
polypeptide and a transfer system to transfer activated species from the
activator system to
the reaction vessel and to transfer amino acids, reagents, gases and solvents
from one part of
the apparatus to another. Generally, this synthesis process is conducted using
Fmoc
chemistry on automated solid phase synthesizers, (or in some cases by Boc
chemistry). In
most cases, the synthesized peptides would be purified by HPLC using reversed
phase C4
and C18 columns. Alternate purification methods include ion exchange and gel
filtration
chromatography.
Our studies indicate that sequences which include the critical residue VVP
have enhanced activity. SEQ ID 17 (VVPQ), for example, showed particularly
good activity.
Derivatives of SEQ ID 17 were synthesized. Table IV illustrates the three
types of
derivatives of SEQ ID 17 which were synthesized and detenniiied to be suitable
as
pharmaceutical, therapeutic, and or cosmetic compositions in accordance with
the present
invention. SEQ IDs 45-48 illustrate various modifications of VVPQ at either
the amino
terminus or carboxy terminus of the peptide. SEQ IDs 49-51 have been modified
to include a
cysteine residue at both the carboxy and amino terminus of the peptides. The
cysteine
residues provide a sulfhydryl group at each end of the chain which permits
convenient
formation of cyclic disulfide. Finally, SEQ IDs 52-54 are very similar to SEQ
IDs 49-51,
but they have copper as a chelating agent to form a cyclic structure.
19
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
TABLE IV (VVPQ derived peptides)
SEQ # PEPTIDE MOL NAME (N- to C-terminal)
WT
42 VVPQNH2 448 Alarsine-Valine-Proline-Glutamine-Amide
43 (CH3C0)VVPQ 475 Acetyl-Valine-Valine-Proline-Glutamine
44 (CH3C0) 610 Acetyl-Glycine-Alanine-Valine-Valine-Proline-
GAVVPQNHZ Glutamine-Amide
45 AVVPQ 512 Alarsine-Valine-Valine-Proline-Glutamine


46 GAVVPQ 569 Glycine-Alarsine-Valine-Valine-Proline-Glutamine


47 AVVPQNHZ 519 Alarsine-Valine-Valine-Proline-Glutamine-amide


48 GAVVPQNHZ 576 Glycine-Alarsine-Valine-Valine-Proline-Glutamine-


amide


49 CVVPQC 647 Cysteine-Valine-Valine-Proline-Glutamine-Cysteine
50 CAVVPQC 718 Cysteine-Alarsine-Valine-Valine-Proline-Glutamine-
Cysteine
51 CGAVVPQC 775 Cysteine-Glycine-Alanine-Valine-Valine-Proline-
Glutamine-Cysteine
52 ~C~ 64 Copper
C/V V P Q C 647 Cysteine-Valine-Valine-Proline-Glutamine-Cysteine
53 ~ Ci~ 64 Copper
C A V V P Q C 718 Cysteine-Alarsine-Valine-Valine-Proline-Glutamine-
Cysteine
54 /Cu\ 64 Copper
C/G A V V P Q\C 775 Cysteine-Glycine-Alarsine-Valine-Valine-Proline-Glutamine-
Cysteine
Based on the information gleaned through testing of derivatives and genetic
expression data, "VVP" appears to be aai important residue.. SEQ ID 55 was
synthesized to
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
replace the glutamine of SEQ m 17 with an asparagine (Asp - "N") residue, the
glutamine
residue and asparagine residue having similar charge properties.
Modifications were made to SEQ m 52 that were very similar to those made
to SEQ m 17. These modified or synthetic peptides are illustrated in Table V.
TABLE V (VVPN derived peptides)
SEQ # PEPTmE MOL NAME (N- to C-terminal)
WT
55 VVPN 427 Valine-Valine-Proline-Asparagine
56 AVVPN 498 Alanine-Valine-Valine-Proline-Asparagine
57 GAVVPN 555 Glycine-Alanine-Valine-Valine-Proline-Asparagine
58 AVVPNNH2 505 Alanine-Valine-Valine-Proline-Asparagine-Amide
59 GAVVPNNH2 562 Glycine-Alanine-Valine-Valine-Proline-Asparagine-
Amide
60 CVVPNC 633 Cysteine-Valine-Valine-Proline-Asparagine-Cysteine
61 CAVVPNC 704 Cysteine-Alanine-Valine-Valine-Proline-Asparagine-
Cysteine
62 CGAVVPNC 761 Cysteine-Glycine-Alanine-Valine-Valine-Proline-
Asparagine-Cysteine
63 /Cu\ 64 Copper
C/V V P N\C 633 Cysteine-Valine-Valine-Proline-Asparagine-Cysteine
64 a 64 Copper
704 C steine-Alanine-Valine-Valine-Proline-As
ara ine-
y p g


C A V V P N C Cysteine


65 Cu 64 Copper


C G A V V P N 761 Cysteine-Glycine-Alanine-Valine-Valine-Proline-
C


Asparagine-Cysteine


21
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
Since SEQ ID 19 (Leucine-Glycine-Alanine-Glycine-Glycine-Alanine-
Glycine) also indicated enhanced activity, it was used as a base model for the
synthesis of the
peptides shown in Table VI below.
TABLE VI (LGAGGAG derived peptides)
SEQ PEPTIDE MOL NAME (N- to C-terminal)
#


WT


66 LGAGGAGV 600 Leucine-Glycine-Alanine-Glycine-Glycine-Alanine-


Glycine-Valine


67 LGAGGAGVL 713 Leucine-Glycine-Alanine-Glycine-Glycine-Alanine-


Glycine-Valine-Leucine


68 LGAGGAGVNHZ 607 Leucine-Glycine-Alanine-Glycine-Glycine-Alanine-


Glycine-Valine-Amide


69 LGAGGAGVLNHZ 720 Leucine-Glycine-Alanine-Glycine-Glycine-Alanne-


Glycine-Valine-Leucine-Amide


70 CLGAGGAGC 707 Cysteine-Leucine-Glycine-Alanine-Glycine-Glycine-


Alanine-Glycine-Cysteine


71 CLGAGGAGVC 806 Cysteine-Leucine-Glycine-Alanine-Glycine-Glycine-


Alanine-Glycine-V aline-Cysteine


72 CLGAGGAGVLC 919 Cysteine-Leucine-Glycine-Alanine-Glycine-Glycine-


Alanine-Glycine-V aline-Leucine-Cysteine


73 ~C~ 64 Copper
/
\


C L 707 Cysteine-Leucine-Glycine-Alanine-Glycine-Glycine-
G A G G A
G C


Alanine-Glycine-Cysteine


74 Cu 64 Copper


~
~


C L 806 Cysteine- Leucine-Glycine-Alanine-Glycine-Glycine-
G A


Alanine-Glycine-Valine-Cysteine


75 Cu 64 Copper


/


C L 919 Cysteine-Leucine-Glycine-Alanine-Glycine-Glycine-
G A G C


Alanine-Glycine-Valine-Leucine-Cysteine


22
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
In Tlity°o and In hivo Analysis of Select Active Peptides:
Example 1: The peptides VVPQ (SEQ ID 17) and LGAGGAG (SEQ ID 19)
were further analyzed using human shin fibroblasts. These peptides have
striping and distinct
influences on gene expression patterns of these cultured shin fibroblasts. h1
one study,
human skin fibroblasts were grown to confluency in T75 cell-culture flasl~s (2
passages)
using Dulbecco's modified essential media (DMEM) complemented with 10% fetal
calf
senun (FCS), penicillin, streptomycin and neomycin. After removing the media,
the cells
were washed with 10% phosphate buffered saline (PBS) and media supplemented
with either
~,g/ml WPQ or 10 ~,g/ml LGAGGAG, respectively. The control culture media
contained
no added peptides. The fibroblasts were treated in this manner for 4 hours
after seeding.
The media was subsequently removed and total RNA was extracted using RNA STAT-
60
(Tel-Test, Friendswood, TX).
Fifty micrograms of total RNA fiom each sample (either VVPQ, LGAGGAG,
or control) was treated with DNase I at 37°C for 1 hour in order to
remove possible genomic
DNA contamination. This treated total RNA was then size-separated by
electrophoresis
through a 1.2% denaturing gel to confirm that no significant degradation of
RNA had tal~en
place. Next, 5 g of treated total RNA from each cell sample was reverse
transcribed to
single-stranded cDNA using [-32P] dATP and reverse transcriptase, according to
the
manufacturer's protocol for the Clontech Atlas array (Palo Alto, CA). Column
chromatography of the labeled cDNA probes as well as hybridization to the
array membranes
was also performed according to the manufacturer's protocol. A Molecular
Dynamics
Phosphorlmager SI (Sunnyvale, CA) was used to expose and screen the membranes
following hybridization and post-hybridization washing.
The array experiments were performed in two sets: 1. untreated fibroblasts and
VVPQ - treated fibroblasts, 2. untreated fibroblasts and LGAGGAG - treated
fibroblasts.
Each set of data was quantitated, compared and analyzed using either
Clontech's Atlashnage
(Palo Alto, CA) or Molecular Dynamic's IrnageQuant (Sumiyvale, CA) software. A
Clontech atlas array (Palo Alto, CA) of 588 cDNAs was utilized to analyze the
changes in
steady state mRNA levels within all 6 functional cDNA groups using fibroblasts
treated and
untreated with the elastin peptides VVPQ and LGAGGAG. Autoradiograms of
duplicate
23
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
arrays probed with 32P-labeled cDNA obtained from total RNA isolated from VVPQ-
treated
and untreated skin fibroblasts were analyzed using ImageQuaslt (Sunnyvale, CA)
software.
In the DNA array analysis, the autoradiographic signal obtained from each
duplicate
cDNA set on each filter is compared to the control cDNAs on the same filter.
This intensity
ratio is then compared for the same duplicate cDNAs between filters. Increased
recovery of
signal using a cDNA probe from RNA isolated from treated fibroblasts is
interpreted as
increased mRNA levels. Decreased mRNA levels are observed as a decrease in
signal.
There were instances where there was no significant hybridization of labeled
cDNA to
membrane bound duplicate cDNA spots. All array signals that indicated up or
down
regulation of steady state mRNA levels of at least two-fold in response to
VVPQ treatment
were identified.
It appears that VVPQ treatment results in an alteration in steady state levels
of
relatively few niRNAs, any many of those upregulated in steady state levels
were involved in
either apoptosis or DNA repair. These include caspase 10 and RFC4. No
particular
functional class of mRNAs appear to predominate among the mRNAs which appear
to be
downregulated in response to VVPQ treatment. The hypothesis is that VVPQ has a
significant influence on apoptosis in these human fibroblast cultures, but the
mechanism of
this influence is only now being elucidated.
The peptide LGAGGAG appears to result in an increased steady state level of
many different mRNAs. Of these, many of the encoded proteins again appear to
be involved
in either apoptosis or DNA repair. Although the number of mRNAs altered by
treatment with
LGAGGAG was greater in our initial studies than with VVPQ, an influence by
both peptides
on apoptosis and or DNA repair is similar. The larger and distinctly different
influence of
LGAGGAG, however, appears to be on mRNAs encoding proteins involved in cell to
cell
communication. A large percentageof mRNAs experinced increased steady state
levels in
response to treatment of fibroblasts with LGAGGAG. These include mRNAs
encoding
proteins such as endothelin 2, oncostatin, TGF- and BMP2A. Interestingly, the
levels of only
a few mRNAs were noted to be significantly down-regulated in skin fibroblasts
following
treatment with LGAGGAG.
24
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
The influence of these two peptides (VVPQ and LGAGGAG) on the gene
expression patterns of cultured skin fibroblasts appear to be different. While
still a
preliminary study, it appears that the heptapeptide LGAGGAG has a marked
influence on the
steady state levels of a larger number and a greater variety of mRNAs in
treated fibroblasts
than the tetrapeptide VVPQ. While both peptides do influence the levels of
mRNAs
encoding proteins involved in DNA repair and apoptosis, LGAGGAG appears to
have
particular influence on niRNAs encoding several different cytokines l~nown to
influence the
expression of genes responsible for the synthesis of elastin and several
different collagen
types. It is entirely possible therefore, that the mRNA level changes
reflected in this array
analysis represent at least part of the signaling cascade influenced by
LGAGGAG. The
potential signaling cascade influences induced by exposure to the heptapeptide
may lead to
the connective tissue alterations responsible for phenotypic changes in the
shin reported
herein. In contrast, the tetrapeptide VVPQ appeared to have a relatively
quiescent effect on
cultured skin fibroblasts. It must be emphasized that these results are
limited by the limited
cDNA present in the array used to generate this data. With VVPQ, The mRNAs
that are
altered in levels are almost entirely those encoding proteins involved in
apoptosis and DNA
repair. While apoptotic changes can have a profound influence on connective
tissue
remodeling, tlus pathway may be different from the one illustrated by the
treatment with
LGAGGAG which appears to effect levels of mRNAs encoding proteins involved in
cell to
cell communication. The prediction from these initial results therefore, would
be that these
two elastin peptides have different influences on the biosynthetic profiles of
shin fibroblasts.
Even more information could be gained about the effects of these and other
elastin peptides
on fibroblasts by utilizing a larger array of cDNAs. The differences in gene
expression
detected by these array analyses may be manifested by significant phenotypic
differences on
the functional integrity of the dermis and will merit further study.
Example 2 - Elastiya mRNA levels afZd T~opeoElastifz:
When the hydrophilic elastin peptides (HEP) of the present invenion were
applied topically they appeared to return elasticity and resilience to shin
while helping to
lessen wrinkles and fine lines. The in vitro and in vivo effects of the
peptides of the present
invention follow.
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
MATERIALS, METHODS, & RESULTS
Elastin naRNA levels. To test the ability of HEP to stimulate the expression
of
the elastin gene, an rtPCR assay was developed to amplify newly synthesized
elastin mRNA.
Adult human dermal fibroblasts were grown to approximately 80% confluence
prior to being
incubated with 10 p,g/mL HEP for 2, 4, and 6 hours. A control sample having
received no
HEP was also run in parallel. After the incubation period, dermal fibroblasts
were lysed and
total RNA was purified from each sample. Total RNA was quantified and
subjected to rtPCR
using forward and reverse primers complementary to human elastin mRNA.
Amplified
cDNA products were then subjected to electrophoresis at 150 volts for 30 min.
on a 2% TBE
agarose gel containing ethidium bromide. The gel was then photographed and
bands were
quantified using the I~odalc 1D Gel Electrophoresis software. Fig. 4
illustrates this elastin
rtPCR assay. Lane (1) shows the net intensity of ampliFed elastin mRNA from
non-treated
cells. Lane (2) shows the net intensity of amplified elastin inRNA from
treated cells after 2
hours incubation with HEP. Lane (3) shows the net intensity of amplified
elastin mRNA from
treated cells after 4 hours incubation with HEP. Lane (4) shows the net
intensity of amplified
elastin mRNA from treated cells after 6 hours incubation with HEP. The net
intensities of the
PCR products indicate that HEP stimulates nearly a two-fold increase in
elastin mRNA
expression just after 2 hours of incubating adult human dermal fibroblasts
with HEP. Results
from this assay indicate that HEP causes an upregulation of the elastin gene
in adult human
dermal fibroblasts within hours. Specifically, HEP induces nearly a two-fold
increase in
elastin mRNA after only two hours incubation with adult human dermal
fibroblasts as
compared to control samples. The stimulus, however, appears to be somewhat
transient and
elastin mRNA levels begin to decrease in dermal fibroblasts after 4 and 6
hours incubation
with HEP.
T~opoelastin levels. Newly synthesized tropoelastin protein levels were
measured by quantitative immunocytochemistry in HEP-treated adult human dermal
fibroblasts using an LSC (laser scanning cytometer) and a fluorescein-labeled
anti-
tropoelastin antibody. Adult human dermal fibroblasts were grown to
approximately 80%
confluence on glass coverslips before being incubated with l Og/mL HEP for 0,
2, 4, 8 and 24
hours. After incubation, 100 ~,L of 1:100 diluted whole rabbit serum
containing anti-human
tropoelastin antibodies was added to the dermal fibroblasts after having been
fixed in 70%
26
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
ethanol for 24 hours. After incubating with whole rabbit serum at 4°C
for 24 hours, the cells
were washed and fluorescein-labeled anti-rabbit secondary antibody (5 ~,ghnL
final
concentration) was added for an additional 24 how-s at 4°C. Cells were
then washed and
counter stained with propidium iodide (5 ~,g/mL final concentration) for 20
min. before being
washed and mounted on slides. Fluorescence was then quantified using a laser
scanning
cytometer. Results show that tropoelastin increases steadily with time above
basal level (See
Fig. 5C). Figure 5 shows more green-fluorescent tropoelastin in treated adult
human dermal
fibroblasts (FigureSB) than in non-treated (Figure 5A). At 24 hours, there is
15-20% more
tropoelastin synthesized by HEP-treated adult human dermal fibroblasts as
compared to non-
treated (Figure 5C)
Cutometen measuf°ements. Cutometer measurements were performed on
patients who were using Elastica Skin Cream (2% HEP) twice daily. Facial
Cutometer
measurements were performed on the right outer canthus of patients using 2%
HEP cream
twice daily using a model SEM 575 Cutometer (Courage+I~hazaka, Koln, Germany).
Measurements were taken every 4 weeks for a period of 12 months. Cutometer
measurements
were performed using mode one consisting of one skin pull through a 2 mm
aperature using
400 mbar of constant negative pressure for 5 seconds. Mode one gave data
relevant to skin
elasticity. Results showed a mean increase in skin elasticity of 31% after
just 4 weelcs and
35% after 12 weeks treatment with Elastica Skin Cream. Cutometer measurements
taken 30
days post-treatment reveal a continued mean elevation in skin elasticity of
23% from basal
Cutometer measurements.
Human spin histologies. Tissue sections from 2mm punch biopsies (before and
after 6 weeks treatment With 2% HEP) of the right outer canthus were stained
with H&E for
morphology and Verhoff's stain for elastin. Images were taken on an Olympus BX-
60-based
microscope. The microscope was equipped with a 100-watt mercury lamp and epi-
fluorescent
filters with emission wavelengths of 470nm, 535riri1 and 630nm. The images
were viewed
through either a Cohu (black and white, range of magnification 16X-800X) or an
OLY-750
analog color camera (range of magnification 4X-200X) and captured with an
image
processing system (Image Pro Plus, Media Cybernetics, Silver Springs MD).
Quantitative
morphometry of cell nuclei was also performed using the H&E stained sections.
Sections of
2 mm punch biopsies from treated human shin show increased dermal thickness
and
27
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
increased cell density after six weeps of twice daily use of Elastica Shin
Cream. Initial
quantitative morphometric determinations estimate an enhanced cellularity of
approximately
15%. The differences can be seen in H&E and Verhoff stained tissue sections of
before and
after treatment punch biopsies shown in Fig. 6.
DISCUSSION. Both rtPCR and immunocytochemistry confirm that HEP
stimulates production of tropoelastin in adult human dermal fibroblasts in
vitro. Quantitative
morphometric data obtained from histologies of patient punch biopsies confirm
that the HEP
cream stimulates an increase in dermal cellularity. It is quite possible that
increased
tropoelastin levels and dermal cellularity combine to enhance slcin
viscoelasticity thereby
causing a faster recovery rate in treated versus non-treated stretched shin.
Together these
results offer a plausible explanation for observed increases in shin
elasticity through
Cutometer measurements. While not wishing to be bound by theory, it appears
that the
peptides of the present invention increase slcin elasticity when applied
topically to the shin.
Moreover, this increase in elasticity translates to a lessening in the
appearance of fine lines
and wrinkles as seen in before and after treatment photographs. Furthermore,
there were no
substantial side effects such as redness, peeling, irritation or sun
sensitivity in conjunction
with use of the compositions) of the present invention.
Applications of the Presezzt Izzvezztio>z: The peptide or peptide-like
compounds
of the present invention, as well as their corresponding therapeutic
compositions, are
expected to have a variety of important applications. The following
descriptions provide a
brief summary of the conditions these peptides) are likely to benefit.
Skif2 cohditiozzs: There are many shin conditions and diseases which would
benefit from elastin treatment. Beyond the obvious cosmetic applications
(i.e., increased
tone, turgor, and appearance), enhanced elastin production will produce long-
term beneficial
results. For example, the inherited disease Scleraderma is characterized by a
thickening and
stiffening of the slcin, and cutaneous ulcers due to the overproduction of
collagen (there are a
number of diseases which involve overproduction of collagen and which seem to
have an
adverse effect on elastin production/content and compromise the tissue). This
disease can
also have systemic effects on organs and blood vessels. The stiffiless and
difficulty in motion
along with the cutaneous ulcers would benefit greatly from incorporation of
elastin into the
28
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
skin. A similarly positive outcome would be expected with the treatment of
lupus and
rheumatoid related skin changes which are generally collagen-vascular diseases
involving a
decrease in elastin. Herpes Simplex and Shingles are also diseases which would
appear to
benefit from increased elastin activity.
Other shin conditions would appear to benefit from the present invention.
Conditions and problems such as acne rosacea, acne vulgaris, aging skin with
vascular
fragility, burn treatment, scar contractures from burns, radiation burns,
pruritis (or chronic
itching), psoriasis, urticaria (commonly referred to as hives), xerosis
(abnormal dryness of the
skin, eyes or mouth), vesicular dennatoses, cracked fingers and feet, drug
eruptions (from an
allergic reaction), epidermolysis ( a skin condition where the epidermis is in
a loosened state,
often with the formation of blebs and bullae either spontaneously or after
trauma), and
erythema multiforme would benefit from treatment with the elastin peptides) of
the present
invention.
There are also inheritable shin disorders such as cutis laxa and EDS or Ehlers
Danlos Syndrome (a group of connective tissue disorders in which the skin
hangs in loose
pendulous folds believed to be caused by decreased elastic tissue formation as
well as an
abnormality in elastin formation or an excess of collagen), elastoderma,
progeria, and
pseudoxanthoma elasticum (an inherited disorder in which elastic fibers found
in many
tissues slowly become calcified) which would benefit from an increase in
elastin in the
affected tissues.
It is believed that the application of the peptides of the present invention
alone
or in combination with the tissue enhancing agents of the present invention
would result in an
increase in tissue elastin and may provide effective treatment for serious
diseases such as
pemphigus.
Tendons, .Sheatlas czrad Bone: Tendons, sheaths and bone all are comprised in
part of elastin. Chronic, painful conditions affecting some of these tissues
include carpal
tmmel disease, fasciitis, flat feet, and tendonitis. These conditions and
similar ones will be
improved with increased levels of elastin in the affected tissue. Bone spurs,
fascial tears,
29
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
ligament tears and tendon tears will heal faster with supplemental elastin
provided by the
elastin peptides of the present invention. These tissues may even become
stronger as a result
of the expected stimulation of elastin production accompanying this treatment.
Additionally,
cartilage growth abnormalities may be corrected by application of elastin
peptides of the
present invention.
Treatment with the compound of the present invention will also be useful in
veterinary medicine for skin ulcerations in livestock such as horses and
cattle. Hoof
problems can be very painful and problematic for horses and other hoofed
animals. Hoof
conditions may benefit from increased keratinocyte production seen with
certain
compositions of the present invention.
Hair: Hair growth, color, and removal can all be improved by treatment with
elastin peptides which will make the hair stronger, more shiny, and improve
the condition and
healing of irritated skin upon removal of unwanted hair. Premature graying of
hair may also
be due to decreased elastin.
Lips: Chapped lips and chronic dermatitis or inflammation of the lips can be
greatly improved upon treatment with elastin peptides of the present
invention. Long-term
relief would be a potential benefit from the stimulation of endogenous elastin
in these tissues.
Back: The breakdown of elastin in the spine can contribute to herniated dislcs
and lead to acute and/or chronic pain. Replacing elastin with peptides of the
present
invention along with the stimulation of endogenous elastin could result in
improved healing
of the disk and reduce or eliminate the pain associated with this condition,
especially when
combined with other treatments, such as steroids.
Br~cisz and hey vous system: In nerve compression syndromes, treatment with
elastin peptides of the present invention will likely stimulate endogenous
elastin production
in certain neurological conditions and promote revascularization after stroke
and neural tissue
transplants. This revascularization could greatly improve the clinical outcome
of these
treatments.
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
Autoinunune diseases: Lupus and other rheumatoid related diseases are
characterized by localized destruction or degeneration of elastin in tissues
throughout the
body. These and similar diseases could greatly benefit from treatment with
elastin peptides
of the present invention which would promote restoration of damaged tissue and
even
provide long-teen benefit from the stimulation of endogenous elastin.
Lungs: Many lung diseases including chronic obstructive pulmonary disease,
laryngeal stenosis, pulmonary fibrosis, pulmonary sarcoid and tracheal
stenosis are associated
with a decrease in elastin, an important component in maintaining the
elasticity and proper
functioning of the lung. Often, these lung conditions are due to a decrease in
particular
proteases which normally balance the activity of elastin-degrading proteases,
refereed to
generally as elastases. An example of this type of deficiency is alpha 1
protease inhibitor
deficiency. A decrease in elastin due to this type of deficiency causes a
breakdown of the
lung matrix which is vital for proper lung function. Other factors, such as
smol~ing can also
lead to breakdown of the elastin component of the lung matrix.
Muscle: Muscles are often covered with a thin layer of connective tissue
which is comprised of elastin and other components such as collagen. Thus,
applying
peptides of the present invention to muscle tissue would increase muscle tone
and the healing
of muscle tears and generally strengthen muscles by increasing their elastin
content.
.Joints: Similarly, joints are comprised of comlective tissue, including
elastin.
hz many cases individuals suffer from joint pain and joint abnormalities as a
result of
inflammatory disease or from wear and tear which all generally result in
decreased amounts
of elastin present in the connective tissue of joints. Thus, many joint
diseases or problems
such as athletic joint injuries, torn cartilage and/or ligaments,
osteoarthritis, joint pain,
rheumatoid arthritis, and stiff j oints could benefit from treatment with
elastin peptides of the
present invention. These elastin peptides will have the capability to
stimulate endogenous
elastin in these tissues and may provide substantial and long-teen rebuilding
and maintenance
of the elastin in these tissues.
31
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
Nails: Elastin is useful in treating and preventing nail bi-ittleness, split
nails,
and to enhance the hardness of nails in general. Nails are comprised of
flattened epidermal
cells and have a high concentration of elastin in the nail bed. Thus,
increasing the elastin
content of these cells will result in a stronger and more flexible nail.
Blood vessels/ lynaplz.atics: Elastin is an important constituent of vessels.
Therefore, application of the peptides to the affected tissues in vascular
diseases would
appear to result in beneficial therapy. The vascular diseases contemplated
include those
which involve abnormalities of arteries or veins including atherosclerotic
occlusive disease,
chronic venous insufficiency, diabetic vasculitis (inflammation of a vessel
caused by
diabetes), fibrotic mediastinitis associated with superior vena cave syndrome
(an exuberant
inflammatory sclerogenic process of infectious, rheumatic, hemorrhagic, or
undetermined
origin, often accompanied by obstruction of mediastinal structure, especially
the vena cave),
varicose veins, temporal arteritis, stasis dermatitis, and lyrnphedema
(including elephantiasis,
which is a chronic unilateral or bilateral edema of the extremities due to
accumulation of
interstitial fluid as a result of the stasis of lymph, which is caused by an
obstruction of the
lymph vessels).
Breast: Capsule contractures secondary to breast implants are disorders of
fibers and are conditions of fixed high resistance (rigidity) to passive
stretch of a muscle.
Fibrocystic disease, selected cases of breast cancer where there is tissue
loss may also benefit
from treatment with elastin peptides.
Cartilage growth: Transformation of hyaline cartilage to elastin cartilage in
remaking of structures such as an ear, nose, larynx or any structure in which
elastic cartilage
would be beneficial, could be aided by treatment with elastin peptides.
Ear°: Chronic serous otitis media and hearing loss secondary to otitis
media as
well as other diseases causing scarring of the ear drum cam benefit from
replacement of
elastin which can serve to repair scarred ear drum tissue caused by these
chronic infections.
32
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
Eye: Eye disorders such as diabetic retinitis, retinal hemorrhages associated
with pseudoxanthoma elasticum (PXE), macular degeneration, and retinitis
pigmentosa all
involve abnormalities of the retina which is comprised in part of elastic
fibers. PXE is an
inherited disorder in which elastic fibers become slowly calcified, producing
characteristic
changes in the shin, the retina of the eye, and the cardiovascular system.
Incorporation of
healthy, normal elastin peptides to the retinas of individuals affected by
these disorders could
improve vision and lead to healing of the retina and prevention of further
damage caused by
the lack of or presence of malformed elastin is this tissue.
Genito-of°ihary tact: There are various genito-urinary conditions
which are
associated with either chronic inflammation or other conditions leading to a
decrease in
elasticity of connective tissue, or with the narrowing of canals or ducts
(strictures). The
replenishing of elastin or the reversal of the strictures by treatment with
elastin and the
stimulation of endogenous elastin would benefit a number of genito-urinary
conditions
including benign prostatic hyperplasia, chronic sclerosing vaginitis,
glornerular sclerosis,
ureteral stricture, uterine benign fibroids, and vaginal stenosis. Another
potential benefit of
the present invention in the Genito-urinary tract is the application of the
compostions of the
present inventions to treat Peyronies disease. Peyronies disease is an
induration of the
corpora cavernosa of the penis which produces a fibrous chordee. The
manifestation of this
disease is also known as fibrous cavernitis, penis plastica, and penile
induration.
Gast~oihtestif2al t~act.~ A number of gastrointestinal (GI) conditions are the
result of chronic inflammation, or abnormal thickening or calcification of GI
tissues
including anal fissures, chronic pancreatitis, esophageal stenosis, esophageal
varicies,
hemorrhoids, intestinal adhesions, and pyloric stenosis. Crohn's disease as
well as other
localized inflammatory/fibrotic bowel diseases are characterized by a chronic
granulomatous
inflammatory condition of unlmown etiology. Scarring and tluckening of the
bowel wall
frequently leads to intestinal obstruction and the formation of f stula and
abscesses. It is
likely that supplying elastin to these tissues may improve gastrointestinal
function in these
patients and restore the normal balance of connective tissue components in the
gastrointestinal tract. Similarly, in biliary cirrhosis and fibrotic liver
diseases such as liver
33
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
cirrhosis, diffuse and interlacing bands of fibrous tissue form and replace
the normal liver
lobules.
InammZOlogy: Enhancement of the immune response through cytokine
activation as well as suppression of immunity for prevention of transplant
rejection and for
treatment of autoimlnune disorders may be mediated by altering elastin levels.
It has been
shown that human activated lymphocytes express the elastin-laminin receptor.
The
expression of the elastin-laminin receptor is a general property of most
activated hwnan
lymphocytes, but is dependent upon lymphocyte subsets. Elastin peptides
activate these
receptors and trigger the stimulation of biosynthesis and release of an
elastase.
Ulce~atiofzs: Ulcers are defects or excavations of the surface of an organ or
tissue, produced by the sloughing of inflammatory tissue. Common ulcerative
disorders
include esophageal, duodenal, and gastric ulcers. It is believed that
providing ulcerative
tissues with elastin will speed the healing of the affected tissue and
possibly even strengthen
the tissue by stimulating endogenous elastin production.
Blood TlesselslHeart: Since large amounts of elastin are found in the walls of
blood vessels, particularly in the arch of the aorta near the heart, it is
important to maintain
the normal healthy balance of elastin in blood vessels and other vessels (such
as lymph
vessels). Additionally, in pulmonary tissues, the subendothelimn is comprised
of the internal
elastic lamina, a layer which normally separates the endothelium from the
underlying smooth
muscle cells. In many cardiac diseases such as congestive heart failure,
coronary artery
disease, homocystinuria, restrictive pericarditis, sclerosing endocarditis,
supra ventricular
aortic stenosis, this internal elastic lamina is compromised due to the
breakdown of elastin
resulting in a remodeling of this matrix layer. This breakdown is often the
result of an
imbalance in enzylne(s) (such as elastase) which degrade elastin. In some
cases, such as in
Marfan's syndrome, the elastin malformations are due to an autosomal dominant,
congenital
disorder affecting connective tissue. Thus, providing affected tissue with
normal elastin
peptides may be a useful treatment for strengthening the connective tissue in
individuals with
Marfan's syndrome.
34
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
A bacterial infection caused by the group A beta hemolytic Streptococci
resulting in rheumatic fever can sometimes lead to rheumatic heart disease, a
serious
condition characterized by inflammation and degeneration of connective tissue
structures of
the body, especially of the heart valves. Treatment of tissues affected by
rheumatic heart
disease with elastin peptides may allow these tissues to heal and be rebuilt.
Additional
clinical uses of supplemental elastin peptides include use as arterial stems
to enhance internal
elastic membrane regeneration in angioplasty procedures.
Hype~tensioyz: High arterial blood pressure (generally hypertension) can be
the result of multiple and diverse etiologies including congeal tal heart
defects, chronic lung
disease, hepatic disorders, and autoirmnune disease (particularly
scleroderma). Hypertension
is often marled by endothelial perturbations as well as abnormalities in the
subendothelium.
These subendothelial problems are manifested in the breakdown of the internal
elastic
lamina, often by an enzyme which degrades elastin. This breakdown results in
the
remodeling or rearrangement of the laminar matrix which may result in chronic
hypertension.
Correcting the elastin composition of the internal elastic lamina with
supplemental elastin
peptides would improve this condition and would likely augment the standard
treatment
which includes elastase inhibiting drugs.
With blood vessels and hypertension, a particularly suitable use of the
peptides
of the present invention would be along with a stmt. Depending on the nature
of the stent,
the stmt may have the therapeutic mixture (e.g., peptide(s) alone or in
combination with
other therapeutic uses) incorporated in the body of the stmt or coated
thereon. For
incorporation, a biodegradable plastic stmt will normally be used which will
release the
therapeutic agents while supporting the vessel and protecting against
restenosis. In the
fabrication of the stent, the biodegradable matrix may be formed by any
convenient means
known in the art. Alternatively, the stmt may be coated with the therapeutic
mixture, using
an adhesive or coating which will allow for controlled release of the
therapeutic mixture. The
stmt may be dipped, sprayed or otherwise coated with a composition containing
the NO
precursor agent or the therapeutic mixture and a matrix, such as biodegradable
polymers, a
physiologically acceptable adhesive, proteins, polysaccharides, or the like.
By appropriate
choice of the material for the stmt andlor the coating comprising the
therapeutic mixture, a
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
physiologically active amount of the therapeutic mixture may be maintained at
the site of the
vascular injury, usually for at least one day and up to a week or more.
I~ Combiraatioh with oth.efr Shin En7~aracihg Agents:
In light of the favorable results observed with the elastin peptide
composition
and the potential applications, the peptide or peptide-like compositions were
considered in
conjunction (e.g., stepwise administration) or in combination (e.g., a mixture
with other skin
enhancing agents). Vitamin C (ascorbic acid) and its derivatives are other
compounds which
have been topically applied as the active ingredient for the treatment of
various skin
conditions and which would appear useful in combination with the peptides in
SEQ IDs 1-75.
U.S. Pat. No. 4,983,382 describes the preparation of stabilized ascorbic acid
compositions for
topical application. It is well l~nown that ascorbic acid (or Vitamin C as it
is synonomously
referred to herein) is essential to the maintenance of a healthy and
attractive skin appearance
in humans. Vitamin C helps to stimulate and regulate the production of
collagen in human
shin tissue thus retarding the formation of wriu~les and otherwise helping to
avoid a
prematurely aged look to shin. This, in turn, helps to maintain a healthier
and younger
looking appearance longer. Vitamin C also acts to help prevent or minimize
lipid oxidation
and other forms of cellular damage resulting from prolonged exposure to the
sun's ultraviolet
rays, further counteracting premature aging of the skin. It is believed
further still that ascorbic
acid helps to inhibit (i) the formation of melanin which leads to skin
discoloration during the
aging process, and (ii) the release of histamine from cellular membranes
believed to be
responsible for many allergenic reactions, particularly among individuals
having so-called
sensitive skin. See also, U.S. Pat. Nos. 5,140,043 and 5,122,536, which are
incorporated
herein by reference.
Another skin enhancing agent that would appear to be suitable in combination
or conjunction with peptide SEQ IDs 1-75 would be salicylic acid. It is known
to use
salicyclic acid for the treatment of acne, see for example, U.S. Pat. Nos.
4,891,227 and
4,891,228, to Thaman et al., the disclosures of which are incorporated herein.
Further,
salicylic acid has been used for the removal of wart, corns and calluses; for
the treatment of
psoriasis, seborrheic dermatitis and dandruff; and for the topical treatment
of ringworm
infection. A listing of commercially available products containing salicylic
acid can be found
in the Physician's Desk Reference, 45th Edition, 1991, page 323.
36
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
However, none of these skin enhancing agents have yet demonstrated the
positive results that have been seen with retinoids. Retinoids ( e.g., Vitamin
A and its
derivatives) are substances which are known to have a broad spectrum of
biological activity.
These substances affect cell growth, differentiation and proliferation.
Retinoids affect the
differentiation, maintenance, and proliferation of many types of cells whether
they are of
ectodermal, endodermal or mesodermal origin; whether epithelial, fibroblastic
or
mesenchymal; or whether they are neoplastic, preneoplastic or non-neoplastic.
At present,
retinoids have found clinical utility in the treatment of severe cystic acne,
psoriasis, and other
disorders of keratinization. Possible uses of retinoids are being explored in
the prophylaxis
and treatment of cancer. For a review of developments in retinoid therapy, see
Pawson, B. A.
et al, "Retinoids at the Threshold: Their Biological Significance and
Therapeutic Potential",
Journal of Medicinal Chemistry 25:1269-1277 (1982). One aspect of the present
invention is
the combination of retinoids of the peptide or peptides in the group of SEQ ms
I-75. See,
e.g., A.S. Zelickson, J. Cutaneous Aging Cosmet. Dermatol., 1:41-47 (1988); J.
S. Weiss,
JAMA, 259:527-532 (1988); J. Bhawan, Arch. Dermatol., 127:666-672 (1991); and
L.H.
I~ligman, Connect. Tissue Res., 12:139-150 (1984). U.S. Pat. No. 4,603,146
also describes
the use of Vitamin A acid for retarding shin aging, and U.S. Pat. No.
4,877,805 describes the
use of retinoids generally for the same purpose (the disclosures of which are
both
incorporated herein by reference).
When considering the use of retinoids in shin care products, it is believed
that
certain retinoids such as, for example, retinol (Vitamin A alcohol), would be
preferred over
retinoic acid. This is because retinol is an endogenous compound naturally
occurnng in the
human body and essential for good growth, differentiation of epithelial
tissues and
reproduction. Retinol is also preferred because it has a much larger safety
margin than other
retinoids such as retinoic acid. Accordingly, attention has turned toward
formulating shin
care compositions which contain retinol.
Particularly useful in the present invention is Tretinoin. Tretinoin is a
yellow
to light orange crystalline powder having a characteristic floral odor. The
chemical name for
tretinoin is (all E)-3,7 dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-
2,4,6,8-nonatetraenoic
acid. Tretinoin is also referred to as all- traps-retinoic acid and has a
molecular weight of
37
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
300.44. Retin A and RENOVA~ are brands of tretinoin (a short for traps-
retinoic acid), a
substance related to but distinct from vitamin A. RENOVA~ differs from Retin A
in that it
contains a moisturizing cream. Retin A (RENOVA~) produces multiple effects in
the skin.
In particular, it increases the responsiveness of skin cells to epidermal
growth factor (EGF),
the natural hormone that stimulates skin growth.
Typical strength of topical tretinoin (Retin A, RENOVA~) creams is 0.025 -
0.1 percent. One study has found that 0.025 percent Retin A may be as
effective as 0.05 or
0.1 percent, but with lower incidence of skin irritation. For people with
sensitive skin,
0.025% Retin A (RENOVA ~) may be the optimal strength. According to the
studies,
improvement on tretinoin (Retin A, RENOVA~) may continue for up to a year of
continued
use.
Early studies by others indicate that RENOVA~ or Retin A actually
decreases the elasticity of the shin. (See Photodama~e by Barbara Gilchrest,
published by
Blaclcwell Science (1995)). Contrary to this we have found that in combination
with or in
conjunction with the peptides or peptide-like compounds of the present
invention, an
improvement in elasticity is detected when using a tretinoin. A Cutometer
(Courage &
I~hazaka, Germany) is used to quantify slcin elasticity. The Cutometer's
vacuum probe is
placed perpendicular to the skin surface to contact the skin and measure its
elasticity. This
device then generates data which includes several readings: immediate skin
deformation,
delayed distention, final deformation, and immediate retraction. The Cutometer
SEM 57S~
(available from Courage & Khazaka, Germany) allows relatively objective
determination of
elasticity of the skin (See Skin ~ Allergy News 30(2): 18, 1999). For details
on the
Cutometer and the testing methods cited herein one is directed to the text
Bioengineering of
the Skin Methods and Instrumentation (1995 Catalog Number 8374, ISBN:
0849383749, as
well as the 1998 version which are both hereby incorporated by reference
thereto in their
entirety) .
A number of volunteers were treated with the hydrolyzed elastin peptides (as
described herein, SEQ IDs 1-41) at a 2% weight concentration versus a group of
volunteers
who used Retin A (actually RENOVA~) first and then applied the 2% hydrolyzed
elastin
38
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
product. The RENOVA~ used in the present invention is a tretinoin emollient
cream at a
0.05% weight concentration. It contains the active tretinoin (a retinoid) in
an emollient cream
base. Tretinoin includes RENOVA~ at a concentration of 0.05% wt/wt in a water
in oil
emulsion formulation consisting of light mineral oil, NF, sorbitol solution
USP;
hydroxyoctacosanyl hydroxystearate; methoxy PEG-22ldodecyl glycol copolymer;
PEG-
45/dodecyl glycol copolymer; stearoxytrimethylane and stearyl alcohol;
dimethicone 50 cs;
methylparaben, NF; acetate disodium, USP; quaternium-15; butylated
hydroxytoluene, NF;
citric acid monohydrate, USP, fragrance; and purified water, USP.
As shown in Fig. 7, the hydrolyzed elastin peptides (SEQ IDs 1-41) in a 2%
by weight concentration was applied to various test subj ects. Over a two-
month period, the
R2 was between 24% and 23% increase and the R7 was 31%. Even more dramatic
were
those results obtained by using a 0.05% by weight RENOVA~ formulation followed
by
application of the 2% hydrolyzed elastin peptide cream of the present
invention. In these
patients the elasciticity as measured by R2 and R7 doubled on average.
Table VII illustrates samplings over 3 separate intervals (separated by
approximately one month) with 5 separate patients. But for the subject
identified as 45 (bg)
there was a consistent and dramatic increase in elasticity. Table VII is shown
below:
TABLE VII
1 Month 2 Month 3 Month


Interval 1 Pull 3 Pulls1 Pull3 Pulls1 Pull3 Pulls


Subject: 41 (Gross) +55% +62% +54% +64% +57% +62%
(th) Fitz:
III Gender F-48


5 (Net) +70% +72% +66% +77% +55% +68%


7 (Portion)+60% +64% +56% +61% +49% +61%


Subject: 42 R2 (Gross)+75% +63% +76% +76% +85% +68%
(vb) Fitz: *L Cheel~N/A +78% +40%* +82% +65%
IV Gender: F-54


5 (Net) +86% +81 +77% +82% +$6% +81
*L Cheel~% +88% +10%* +88% +81%
N/A


39
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
7 (Portion)- +76% +67% +68% +76% +82% +73%


*L Cheek N/A +81% +29% +83% +70%


Subject 41 (Gross) +68% +67% +73% +67% +60% +69%
(ce) Fitz:


III Gender: *L CheelcN/A +26% +17%* +9% +4%
F-41


5 (Net) +71% +77% +86% +89% +74% +68%


*L CheelcNlA +39%* +50%* -18% -14%


7 (Portion)+64% +64% +74% +72% +67% +58%


*L Cheek N/A +26%* +39%* +2% +2%


Subject: 44 (Gross) +55% +65% +65% +65% +42% +59%


(ph) Fitz: *L CheelcN/A
II Gender:


-50


5 (Net) +77% +79% +81% +79% +76% +84%


*L Cheek N/A


7 (Portion)+65% +66% +53% +66% +60% +71%


*L Cheek N/A


Subject: 45 (Gross) +15% -23% +2% 8% +1% +3%


(bg) Fitz: **+16%
III Gender:


-52 * *+22



**+5%


5 (Net) +6% -28% +25% +26% +38% +4%


**+42% **+25



**-20%


7 (Portion)+6% -28% -9% -16% +1% -22%


**+17% ***+8



*-10%


*2nd Biopsy taken L. Cheek
* *Monthly interval variances showed a wide range of + and - readings
Please note, tlus groups' Cutometer readings represent one and three pulls.
One pull = total
elasticity; three pulls=fatigue and recovery
Figure 7 and Table VII illustrate the desirable results obtained when using
various embodiments of the present invention. All readings on the Cutometer
have been taken
in Mode 1, which is constant negative pressure: 5 seconds on, 5 seconds off.
The pull of
SUBSTITUTE SHEET (RULE 26)


CA 02415225 2003-O1-06
WO 01/91700 PCT/USO1/17384
three curves indicates the fatigue and recovery of the shin, while one pull
indicates the
elasticity of the curve. Group 4 studies include both one and three pulls. R2
= gross
elasticity. It is represented by Ua/Uf which indicates the total pull and
relaxation of the skin
under negative pressure. RS = net elasticity. It is represented by Ur/LJe
which indicates the
perpendicular pull and relaxation of the shin under negative pressure. R7 =
portion elasticity.
It is represented by Ur/LTf and is indicative of the portion of elasticity
relative to the entire
curve. Based upon the Cutometer readings and the in vivo analysis, it appears
that the
peptides of the present invention stimulate or simulate elasticity of the
tissue.
While the foregoing has been set forth in considerable detail, the sequences
are presented for elucidation, and not limitation. Modifications and
improvements, including
equivalents, of the technology disclosed above which are within the purview
and abilities of
those in the art are included within the scope of the claims appended hereto.
It will be readily
apparent to those skilled in the art that numerous modifications, alterations
and changes can
be made with respect to the specifics of the above description without
departing from the
inventive concept described herein. For example, the compounds can be
administered via
many alternative drug delivery vehicles known in the art and the peptides can
be derived
from digestion of elastin or by amino acid sequencing (either solid state or
liquid), as well as
from over-expression in a bacterial system. Modification (either chemical or
enzymatic) of
the basic sequences described herein are also witlun the purview of the
present invention.
Therefore the disclosed sequences may be modified to include this residue at
either the amino
or carboxyl ends of the peptides. The sequences may also be chemically
modified to increase
their activity (e.g., amidation of the carboxyl terminus portion of a
sequence). Computational
chemistry can be used to predict structure-function relationship, and
compounds thus
predicted and synthesized may mimic the structure and function of a particular
peptide or
peptide-like compound disclosed herein and may be utilized. The peptides may
be
chemically modified to increase their activity. Accordingly, all such
variances should be
viewed as being within the scope of the present invention as set forth in the
claims below.
41
SUBSTITUTE SHEET (RULE 26)