U.S. patent application number 12/107666 was filed with the patent office on 2008-10-16 for methods and compositions for preventing and treating aging or photodamaged skin.
This patent application is currently assigned to PROCYTE CORPORATION. Invention is credited to Leonard M. Patt.
Application Number | 20080255032 12/107666 |
Document ID | / |
Family ID | 36757854 |
Filed Date | 2008-10-16 |
United States Patent
Application |
20080255032 |
Kind Code |
A1 |
Patt; Leonard M. |
October 16, 2008 |
METHODS AND COMPOSITIONS FOR PREVENTING AND TREATING AGING OR
PHOTODAMAGED SKIN
Abstract
Disclosed are methods for treating aging and photodamaged skin
employing topical application of compositions which comprise at
least one peptide manganese complex. Also disclosed are methods
wherein the composition further comprises retinol, at least one
retinol derivative, or a mixture thereof and methods wherein the
composition further comprises active agents selected from active
drug substances, emollients, sunscreen agents, skin lightening
agents, skin protectants, skin conditioning agents, humectants, and
mixtures thereof.
Inventors: |
Patt; Leonard M.; (Seattle,
WA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE, SUITE 5400
SEATTLE
WA
98104
US
|
Assignee: |
PROCYTE CORPORATION
Redmond
WA
|
Family ID: |
36757854 |
Appl. No.: |
12/107666 |
Filed: |
April 22, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11376728 |
Mar 15, 2006 |
7384916 |
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12107666 |
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60662577 |
Mar 16, 2005 |
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Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
A61K 2800/58 20130101;
A61P 17/02 20180101; A61K 8/64 20130101; A61K 38/05 20130101; A61Q
19/08 20130101; A61K 8/19 20130101 |
Class at
Publication: |
514/6 |
International
Class: |
A61K 38/06 20060101
A61K038/06 |
Claims
1. A method for treating photodamaged skin comprising contacting an
area of skin in need thereof with an effective amount of a
composition, wherein the composition consists of at least one
peptide manganese complex and optionally an inert and
physiologically-acceptable carrier or diluent, a skin lightening
agent, a sunscreen agent, a skin conditioning agent, a skin
protectant, an emollient, a humectant, a fatty alcohol, a fatty
acid, an organic base, an inorganic base, a preserving agent, a wax
ester, a steroid alcohol, a triglyceride ester, a phospholipid, a
polyhydric alcohol ester, a fatty alcohol ether, a hydrophilic
lanolin derivative, a hydrophilic beeswax derivative, a cocoa
butter wax, a silicon oil, a pH balancer, a cellulose derivative, a
hydrocarbon oil, an emulsifying agent, a surfactant, a thickening
agent, an excipient, retinol, a retinol derivative or a mixture
thereof, and wherein the at least one peptide manganese complex is
L-glycyl-L-histidyl-L-lysine:manganese(II).
2. The method of claim 1 wherein the molar ratio of peptide to
manganese in the at least one peptide manganese complex ranges from
about 1:1 to about 3:1.
3. The method of claim 2 wherein the molar ratio of peptide to
manganese in the at least one peptide manganese complex ranges from
about 1:1 to about 2:1.
4. The method of claim 1 wherein the at least one peptide manganese
complex is present at a concentration ranging from about 0.01% to
about 5% by weight of the composition.
5. The method of claim 4 wherein the at least one peptide manganese
complex is present at a concentration ranging from about 0.025% to
about 1% by weight of the composition.
6. The method of claim 5 wherein the at least one peptide manganese
complex is present at a concentration ranging from about 0.05% to
about 0.5% by weight of the composition.
7. The method of claim 1 wherein the at least one peptide manganese
complex is encapsulated in a liposome, microsponge or polymer
matrix adapted to aid in the delivery of the peptide manganese
complex to the area of skin or to enhance the stability of the
composition.
8. The method of claim 1 wherein the at least one peptide manganese
complex is formulated in an instrument adapted to deliver the
peptide manganese complex to the area of skin via
iontophoresis.
9. The method of claim 1 wherein the composition further comprises
an inert and physiologically-acceptable carrier or diluent.
10. The method of claim 9 wherein the inert and
physiologically-acceptable carrier or diluent is water,
physiological saline, bacteriostatic saline, a petrolatum based
cream, a pharmaceutically acceptable gel, a short chain alcohol, or
a short chain glycol.
11. The method of claim 1 wherein the composition further comprises
a skin lightening agent, a sunscreen agent, a skin conditioning
agent, a skin protectant, an emollient, a humectant, or a mixture
thereof.
12. The method of claim 1 wherein the composition further comprises
a fatty alcohol, a fatty acid, an organic base, an inorganic base,
a preserving agent, a wax ester, a steroid alcohol, a triglyceride
ester, a phospholipid, a polyhydric alcohol ester, a fatty alcohol
ether, a hydrophilic lanolin derivative, a hydrophilic beeswax
derivative, a cocoa butter wax, a silicon oil, a pH balancer, a
cellulose derivative, a hydrocarbon oil, or a mixture thereof.
13. The method of claim 1 wherein the composition further comprises
an emulsifying agent, a surfactant, a thickening agent, an
excipient, or a mixture thereof.
14. The method of claim 1 wherein the composition is in the form of
a solution, cream, gel, fluid cream, lotion, or oil.
15. The method of claim 1 wherein the composition further comprises
retinol, a retinol derivative, or a mixture thereof.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/376,728, filed Mar. 15, 2006 (now allowed),
which claims the benefit under 35 U.S.C. .sctn. 119(e) of U.S.
Provisional Patent Application No. 60/662,577 filed Mar. 16, 2005.
These applications are incorporated herein by reference in their
entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention generally relates to the treatment of
dermatological conditions and, more specifically, to the treatment
of conditions related to aging or photodamaged skin, by topical
application of a composition comprising a peptide manganese
complex.
[0004] 2. Description of the Related Art
[0005] Aging and photodamage result in a number of changes in the
structure and function of skin. Primary among these changes is a
thinning of the skin due to lower levels of collagen, elastin and
other components of the skin's connective tissue. Lower levels of
synthesis and activity (i.e., proliferation and viability) of
fibroblasts responsible for the synthesis of collagen and elastin
are also characteristic of aging skin.
[0006] There are many treatments available to alter the appearance
of aging skin, including various creams and oils that primarily
serve to rehydrate the skin. Such re-hydration temporarily lessens
the appearance of fine line and wrinkles. There are also numerous
compounds that enhance the synthesis of certain components of the
skin, such as collagen and glycosaminoglycans, and the underlying
connective tissue. For example, all-trans retinoic acid has been
shown to stimulate collagen synthesis in UVB irradiated skin (see
Chen S. et al., Invest. Dermatol. 98(2):248-254 (1992)) and topical
growth factors have been shown to increase collagen synthesis and
produce a thickening of the epidermis (see Fitzpatrick R. E. at
al., Journal of Cosmetic and Laser Therapy 5(1):25-34 (2003)).
Ascorbic acid and its derivatives have also been shown to stimulate
increases in collagen synthesis (see Geesin J. C. et al., Skin
Pharmacology 6(1):65-71 (1993) and Murad S. et al., J. Invest.
Dermatol. 81(2):158-162 (1983)). Furthermore, it has been shown
that the biosynthesis of another component of the extracellular
matrix, elastin, is increased by topical application of L-fucose or
certain fucose-rich polysaccharides (see Robert L. et al.,
Biomedicine & Pharmacotherapy 58(2):123-128 (2004)).
Nonetheless, aging skin, and the corresponding fine lines, wrinkles
and other external appearances, remain a concern.
[0007] Manganese is an essential nutrient involved in the formation
of bone and in amino acid, cholesterol, and carbohydrate
metabolism. Enzymes, which utilize manganese for activity, include
arginase, glutamine synthetase, manganese superoxide dismutase,
prolidase, and some carbohydrate transferases. The Adequate Intake
levels for men and women have been set at 2.3 and 1.8 mg/day
respectively. The enzyme Superoxide Dismutase is one of the most
important defenses against oxidative damage in the body. There are
two types in humans, namely, the Cu--Zn Superoxide Dismutase
(SOD1), which is found mainly in the cytosol of the cell, and the
Mn-Superoxide Dismutase (SOD2), which is found in the mitochondria
(see Kobayashi et al., Acta Dermato-Venereologica 73(1):41-45
(1993)).
[0008] Small molecular weight complexes of manganese have been
shown to possess superoxide dismutase activity. For example, U.S.
Pat. Nos. 5,223,538 and 5,227,405 to Fridovich et al. describe
water-soluble manganese complexes useful to reduce and prevent
superoxide radical induced toxicity. In addition, U.S. Pat. No.
5,118,665 to Pickart discloses peptide manganese complexes with
superoxide dismutase activity useful for enhancing or restoring the
resistance of an animal to oxidative or inflammatory damage.
[0009] Manganese is also an important component of the enzyme
Prolidase. This is a manganese dependent exopeptidase (i.e., a
protease which cuts off amino acids from the end of the peptide
chain). Prolidase cleaves proline from peptides inside the cell and
provides a vital source of proline for new collagen synthesis. The
addition of manganese to increase intracellular manganese increases
the activity of Prolidase in deficient cells (see Hechtman et al.,
Pediatric Research 24(6):709-712 (1998)). Another manganese
requiring enzyme is Arginase. Arginase is an enzyme responsible for
the conversion of the amino acid arginine to urea in keratinocytes.
The addition of L-arginine and manganese to keratinocyte cultures
results in the increase of urea synthesis (see Wohlrab et al., Skin
Pharmacology and Applied Skin Physiology 15(1):44-54 (2002)).
[0010] Although there have been advances in the art, there remains
a need for more effective and otherwise improved methods for
treating dermatological conditions related to aging or photodamaged
skin, such as fine lines and wrinkles. In particular, there remains
a need for treatment methods that provide for increased
proliferation and viability of dermal fibroblasts and other
components of the dermal connective tissue, such as collagen. The
present invention addresses these needs and provides further
related advantages.
BRIEF SUMMARY OF THE INVENTION
[0011] In brief, the present invention is directed to the treatment
of dermatological conditions related to aging or photodamaged skin
by topical application of a composition comprising at least one
peptide manganese complex to an area of affected skin. It has been
surprisingly found that such compositions can be used to enhance
dermal fibroblast proliferation and viability and to stimulate
collagen synthesis, thereby substantially diminishing signs of
aging and photodamage in treated skin.
[0012] In one embodiment, the present invention is directed to a
method for treating aging or photodamaged skin, by contacting an
area of skin in need thereof with an effective amount of a
composition comprising at least one peptide manganese complex. In a
further embodiment, the composition further comprises retinol, a
retinol derivative, or a mixture thereof. Topical application of an
effective amount of such compositions to areas of skin in need of
such treatment results in significant reduction of the signs and
symptoms of aging and photodamage found on the areas contacted.
[0013] In other further embodiments, the present invention is
directed to methods for such treatment wherein the at least one
peptide manganese complex is encapsulated in a liposome,
microsponge, polymer matrix or other encapsulation technology
adapted to aid in the delivery of the peptide manganese complex to
the areas of skin need thereof, or to enhance the stability of the
composition. In yet other further embodiments, the at least one
peptide manganese complex is formulated in an instrument adapted to
deliver the peptide manganese complex via iontophoresis or
ultrasound to the areas of affected skin.
[0014] In yet other further embodiments, the composition further
comprises an inert and physiologically-acceptable carrier or
diluent, a skin lightening agent, a sunscreen agent, a skin
conditioning agent, a skin protectant, an emollient, a humectant,
or a mixture thereof in addition to the at least one peptide
manganese complex. In other related embodiments, the composition
further comprises an active drug substance or an active cosmetic
substance.
[0015] In still other further embodiments, the composition further
comprises an emulsifying agent, a surfactant, a thickening agent,
an excipient, or a mixture thereof, and/or the composition is in
the form of a liquid, cream, gel, fluid cream, lotion, oil,
emulsion or microemulsion.
[0016] These and other aspects of this invention will be evident
upon reference to the following detailed description of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0017] As noted above, in one embodiment, disclosed is a method for
treating aging or photodamaged skin by topically applying, to areas
of skin in need thereof, an effective amount of a composition
comprising at least one peptide manganese complex. As used herein
the word "treat," "treating" or "treatment" refers to using the
compositions of the present invention either prophylactically to
prevent signs of aging and photodamage, or therapeutically to
ameliorate an existing condition characterized by aging or
photodamage.
[0018] The compositions utilized may be in any form suitable for
topical application, including: a liquid, a cream, a lotion, a gel,
a fluid cream, an oil, an emulsion or a microemulsion. Some
examples of compositions formulated as cosmetic preparations,
useful for cleansing and protecting, in addition to treating, skin
are: creams for the face, hands, feet, or the entire body (i.e.,
day creams, night creams, make-up removal creams, and foundation
creams); make-up removal formulations; protective or skin care body
milks; skin care lotions, gels, or foams (such as cleansing or
disinfecting lotions); bath compositions; deodorant compositions;
and aftershave and pre-shave gels or lotions.
[0019] In more specific embodiments, the at least one peptide
manganese complex comprises
glycyl-L-histidyl-L-lysine:manganese(II) ("GHK-Mn"),
L-alanyl-L-histidyl-L-lysine:manganese(II) ("AHK-Mn"),
L-valyl-L-histidyl-L-lysine:manganese(II) ("VHK-Mn"),
L-leucyl-L-histidyl-L-lysine:manganese(II) ("LHK-Mn"),
L-isoleucyl-L-histidyl-L-lysine:manganese(II) ("IHK-Mn"),
L-phenylalanyl-L-histidyl-L-lysine:manganese(II) ("FHK-Mn"),
L-prolyl-L-histidyl-L-lysine:manganese(II) ("PHK-Mn"),
L-seryl-L-histidyl-L-lysine:manganese(II) ("SHK-Mn"), or
L-threonyl-L-histidyl-L-lysine:manganese(II) ("THK-Mn").
[0020] As used herein, the abbreviations for the naturally
occurring amino acids are: [0021] Alanine Ala A [0022] Arginine Arg
R [0023] Asparagine Asn N [0024] Aspartic Acid Asx D [0025]
Cysteine Cys B [0026] Glycine Gly G [0027] Glutamine Gln Q [0028]
Glutamic Acid Glu E [0029] Histidine His H [0030] Isoleucine Ile I
[0031] Leucine Leu L [0032] Lysine Lys K [0033] Methionine Met M
[0034] Phenylalanine Phe F [0035] Proline Pro P [0036] Serine Ser S
[0037] Threonine Thr T [0038] Tryptophan Trp W [0039] Tyrosine Tyr
Y [0040] Valine Val V
[0041] As used herein, the expression "peptide manganese complex"
generally refers to a coordination compound comprising a peptide
molecule and a manganese(II) ion non-covalently complexed with the
peptide. As is well understood in the art, manganese (II)
designates a manganese ion having a valence of 2 (i.e., Mn.sup.+2).
The peptide molecule serves as the complexing agent by donating
electrons to the manganese ion to yield the non-covalent complex.
The peptide molecule is a chain of two or more amino acid units or
amino acid derivative units covalently bonded together via amide
linkages, the formation of such linkages being accompanied by the
elimination of water.
[0042] Generally, an amino acid consists of an amino group, a
carboxyl group, a hydrogen atom, and an amino acid side-chain
moiety--all bonded, in the case of an alpha-amino acid, to a single
carbon atom that is referred to as an alpha-carbon. The amino acid
units of the present invention may be provided by amino acids other
than alpha-amino acids. For example, the amino acids may be beta-
or gamma-amino acids, such as the following:
##STR00001##
where X is the amino acid side-chain moiety bonded, along with the
amino group and hydrogen, to an alpha-, beta-, or gamma-carbon
atom.
[0043] As another example, the amino acids of the present invention
include, but are not limited to, naturally occurring alpha-amino
acids. Naturally occurring amino acids are those from which the
amino acids units of naturally occurring proteins are derived. Some
of these amino acids, along with their respective amino acid side
chain moieties, are shown below in Table 1. The naturally occurring
amino acids shown are all in the L configuration, referring to the
optical orientation of the alpha carbon or other carbon atom
bearing the amino acid side chain. A peptide molecule of the
present invention may also comprise amino acids that are in the D
optical configuration, or a mixture of D and L amino acids.
TABLE-US-00001 TABLE 1 Naturally Occurring Amino Acid Side-Chain
Moieties Amino Acid Side Chain Moiety Amino Acid --H Glycine
--CH.sub.3 Alanine --CH(CH.sub.3).sub.2 Valine
--CH.sub.2CH(CH.sub.3).sub.2 Leucine --CH(CH.sub.3)CH.sub.2CH.sub.3
Isoleucine --(CH.sub.2).sub.4NH.sub.3.sup.+ Lysine
--(CH.sub.2).sub.3NHC(NH.sub.2)NH.sub.2.sup.+ Arginine ##STR00002##
Histidine --CH.sub.2COO-- Aspartic Acid --CH.sub.2CH.sub.2COO--
Glutamic Acid --CH.sub.2CONH.sub.2 Asparagine
--CH.sub.2CH.sub.2CONH.sub.2 Glutamine ##STR00003## Phenylalanine
##STR00004## Tyrosine ##STR00005## Tryptophan --CH.sub.2SH Cysteine
--CH.sub.2CH.sub.2SCH.sub.3 Methionine --CH.sub.2OH Serine
--CH(OH)CH.sub.3 Threonine ##STR00006## Proline
Other naturally occurring amino acids include hydroxyproline and
gamma-carboxyglutamate.
[0044] Representative amino acid derivatives include those set
forth in Table 2.
TABLE-US-00002 TABLE 2 Amino Acid Derivatives ##STR00007## where
X.sub.2 = H or the following moieties: --(CH.sub.2).sub.nCH.sub.3
where n = 1-20 --(CH.sub.2).sub.nCH(CH.sub.3)(CH.sub.2).sub.m
CH.sub.3 where n, m = 0-20 (when n = 0, m .noteq. 0 or 1 and when n
= 1, m .noteq. 0) --(CH.sub.2).sub.nNH.sub.2 where n = 1-20 (n
.noteq. 4) --(CH.sub.2).sub.nCONH.sub.2 where n = 3-20
--(CH.sub.2).sub.nCOOH where n = 3-20 ##STR00008## ##STR00009##
##STR00010## --(CH.sub.2).sub.nSH where n = 2-20
--(CH.sub.2).sub.nS(CH.sub.2).sub.mCH.sub.3 where n, m = 1-20 (when
n = 2, m .noteq. 0) --(CH.sub.2).sub.nCH.sub.2OH where n = 1-20
--(CH.sub.2).sub.nCH(CH.sub.3)OH where n = 1-20, and where X.sub.1
= H or the following moieties: --(CH.sub.2).sub.nCH.sub.3 where n =
0-20 --(CH.sub.2).sub.nCH(CH.sub.3)(CH.sub.2).sub.mCH.sub.3 where
n, m = 0-20.
[0045] In addition, histidine derivatives of this invention include
compounds having the structure:
##STR00011##
where n=1-20, and Y.sub.1 and Y.sub.2 are independently selected
from alkyl moieties containing from 1-12 carbon atoms or aryl
moieties containing from 6-12 carbon atoms. In certain embodiments,
n is 1, Y.sub.2 is methyl, and Y.sub.1 is H (i.e., 3-methyl
histidyl) or Y.sub.2 is H and Y.sub.1 is methyl (i.e., 5-methyl
histidine).
[0046] Similarly, arginine derivatives of this invention include
compounds having the structure:
##STR00012##
where n=1-20 (excluding n=3)
[0047] As used herein, "alkyl" means a straight chain or branched,
cyclic or noncyclic, substituted or unsubstituted, saturated or
unsaturated aliphatic hydrocarbon containing from 1 to 18 carbon
atoms. Representative saturated straight chain alkyls include
methyl, ethyl, n-propyl and the like; while saturated branched
alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,
isopentyl, and the like. Representative, saturated cyclic alkyls
include cyclopropyl, cyclobutyl, cyclopentyl, --CH.sub.2cyclohexyl,
and the like; while unsaturated cyclic alkyls include
cyclopentenyl, cyclohexenyl, and the like. Unsaturated alkyls
contain at least one double or triple bond between adjacent carbon
atoms (referred to as an "alkenyl" or "alkynyl," respectively).
Representative alkenyls include ethylenyl, 1-butenyl, isobutylenyl,
2-methyl-2-butenyl, and the like; while representative alkynyls
include acetylenyl, 2-butynyl, 3-methyl-1-butynyl, and the
like.
[0048] Also, as used herein, "aryl" means an aromatic carbocyclic
moiety such as phenyl or naphthyl, and may be substituted or
unsubstituted. "Arylalkyl," as used herein, means an alkyl having
at least one alkyl hydrogen atom replaced with a substituted or
unsubstituted aryl moiety, such as benzyl (i.e., --CH.sub.2phenyl,
--(CH.sub.2).sub.2phenyl, --(CH.sub.2).sub.3phenyl,
--CH(phenyl).sub.2, and the like).
[0049] A peptide manganese complex of the present invention may
have the formula [R.sub.1--R.sub.2--R.sub.3]:manganese(II) where
R.sub.3 is at least one amino acid or amino acid derivative, as
defined above, bonded to R.sub.2 by a peptide bond. Where R.sub.3
is a single amino acid or amino acid derivative, then the peptide
of the peptide manganese complex is generally classified as a
tripeptide. As another example, a peptide manganese complex of the
present invention may have the formula
[R.sub.1--R.sub.2--R.sub.3]:manganese(II) where R.sub.3 is a
chemical moiety bonded to the R.sub.2 moiety by an amide bond. The
expression "chemical moiety," as used herein and with reference to
R.sub.3, includes any chemical moiety having an amino group capable
of forming an amide bond with the carboxyl terminus of R.sub.2
(i.e., the carboxyl terminus of histidine, arginine, or derivatives
thereof).
[0050] As a more particular example, where R.sub.3 is a chemical
moiety bonded to the R.sub.2 moiety by an amide bond, R.sub.3 is
--NH.sub.2, an alkylamino moiety having from 1-20 carbon atoms, or
an arylamino moiety having from 6-20 carbon atoms. As used herein,
an "alkylamino moiety" encompasses alkyl moieties containing an
amino moiety, wherein the alkyl moiety is as defined above, and
includes, but is not limited to, octyl amine and propyl amine.
Similarly, an "arylamino moiety" encompasses aryl moieties
containing an amino moiety, wherein the aryl moiety is as defined
above, and includes, but is not limited to, benzylamine and
benzyl-(CH.sub.2).sub.1-14-amine. Further examples of suitable
chemical moieties having amino groups capable of forming an amide
linkage with the carboxyl terminus of R.sub.2 include polyamines
such as spermine and sperimidine.
[0051] It should be understood that R.sub.3 may include more than
one chemical moiety. For example, additional amino acids or amino
acid derivatives may be bonded to the above-described peptide
manganese complexes comprising tripeptides to yield peptide
manganese complexes comprising peptides having four or more amino
acids and/or amino acid derivatives. For purposes of illustration,
Table 3, shown below, presents various representative examples of
peptide manganese complexes of the present invention.
TABLE-US-00003 TABLE 3 Representative Peptide-Manganese Complexes
Examples of [R.sub.1-R.sub.2]:manganese(II)
glycyl-histidine:manganese alanyl-histidine:manganese
glycyl-(3-methyl)histidine:manganese
alanyl-(3-methyl)histidine:manganese
glycyl-(5-methyl)histidine:manganese
alanyl-(5-methyl)histidine:manganese glycyl-arginine:manganese
alanyl-arginine:manganese (N-methyl)glycine-histidine:manganese
(N-methyl)glycine-arginine:manganese Examples of
[R.sub.1-R.sub.2-R.sub.3]:manganese(II) where R.sub.3 is Chemical
Moiety Linked by Amide Bond glycyl-histidyl-NH.sub.2:manganese
glycyl-arginyl-NH.sub.2:manganese
glycyl-(3-methyl)histidyl-NH.sub.2:manganese
alanyl-(3-methyl)histidyl-NH.sub.2:manganese
glycyl-arginyl-NH.sub.2:manganese alanyl-arginyl-NH.sub.2:manganese
(N-methyl)glycine-histidyl- (N-methyl)glycine-arginyl-
NH.sub.2:manganese NH.sub.2:manganese
glycyl-histidyl-NHoctyl:manganese glycyl-arginyl-NHoctyl:manganese
Examples of [R.sub.1-R.sub.2-R.sub.3]:manganese(II) where R.sub.3
is Amino Acid or Amino Acid Derivative Linked by Peptide Bond
glycyl-histidyl-lysine:manganese glycyl-arginyl-lysine:manganese
glycyl-(3-methyl)histidyl- glycyl-(5-methyl)histidyl-
lysine:manganese lysine:manganese alanyl-histidyl-lysine:manganese
alanyl-arginyl-lysine:manganese alanyl-(3-methyl)histidyl-
alanyl-(5-methyl)histidyl- lysine:manganese lysine:manganese
glycyl-histidyl-phenylalanine:manganese
glycyl-arginyl-phenylalanine:manganese glycyl-(3-methyl)histidyl-
glycyl-(5-methyl)histidyl- phenylalanine:manganese
phenylalanine:manganese alanyl-histidyl-phenylalanine:manganese
alanyl-arginyl-phenylalanine:manganese alanyl-(3-methyl)histidyl-
alanyl-(5-methyl)histidyl- phenylalanine:manganese
phenylalanine:manganese glycyl-histidyl-lysyl-phenylalanyl-
glycyl-arginyl-lysyl-phenylalanyl- phenylalanyl:manganese
phenylalanyl:manganese glycyl-(3-methyl)histidyl-lysyl-
glycyl-(5-methyl)histidyl-lysyl-
phenylalanyl-phenylalanyl:manganese
phenylalanyl-phenylalanyl:manganese
(N-methyl)glycyl-histidyl-lysine:manganese
(N-methyl)glycyl-arginyl-lysine:manganese
valyl-histidyl-lysine:manganese
glycyl-histidyl-lysyl-prolyl-phenylalanyl-
prolyl-histidyl-lysine:manganese proline:manganese
glycyl-D-histidyl-L-lysine:manganese
Leucyl-histidyl-lysine:manganese
seryl-histidyl-lysine:manganese
[0052] In addition, the expression "peptide manganese complex," as
used herein, encompasses peptide manganese complex derivatives. The
expression "peptide manganese complex derivative," as used herein,
refers to a peptide manganese complex where the peptide molecule
thereof has: 1) at least one amino acid side chain moiety that is a
modification and/or variation of a naturally occurring, amino acid
side-chain moiety; and/or 2) at least one of the hydrogens, bonded
to an amide linkage nitrogen atom, substituted with a different
moiety; and/or 3) the carboxyl group of the carboxyl terminal
residue esterified or otherwise modified; and/or 4) at least one
hydrogen, bonded to the nitrogen atom of the amino-terminal
residue, substituted with a different moiety. Accordingly, the
method of the present invention, in another embodiment, employs a
composition comprising at least one peptide manganese complex
derivative. For example, derivatives of GHK-Mn have the general
formula:
[glycyl-histidyl-lysine-R]:manganese(II)
wherein R may be, for example, an alkyl moiety containing from one
to eighteen carbon atoms, an aryl moiety containing from six to
twelve carbon atoms, an alkoxy moiety containing from one to twelve
carbon atoms, or an aryloxy moiety containing from six to twelve
carbon atoms.
[0053] Further examples of the peptide manganese complex and
peptide manganese complex derivatives encompassed by the present
invention include, but are not limited to, those disclosed and
described in the above- and below-cited U.S. Patents that are
directed to peptide manganese complexes, as well as those disclosed
and described in the published PCT application having the
International Publication Number WO 94/03482, which is incorporated
herein by reference in its entirety.
[0054] The synthesis of the above-described peptide manganese
complexes is described in detail in the above-referenced patents.
For example, the peptides of the peptide manganese complexes
disclosed herein may be synthesized by either solution or solid
phase techniques known to one skilled in the art of peptide
synthesis. The general procedure involves the stepwise addition of
protected amino acids to build up the desired peptide sequence. The
resulting peptide may then be complexed to manganese (at the
desired molar ratio of peptide to manganese) by dissolving the
peptide in water, followed by the addition of manganese chloride or
another suitable manganese salt and adjusting the pH to greater
than 4.0. The peptide manganese complex thus formed may be used as
a solution or as a dry powder after, for example, freeze-drying or
spray drying.
[0055] The compositions of the present invention may be prepared
from aqueous solutions of peptide manganese complexes. Such aqueous
solutions are prepared by methods that are well known to those
skilled in the art. For example, an amount of dried peptide
manganese complex, suitable for a desired concentration, is readily
dissolved in water with mixing and gentle heating. An alternative
method is to prepare a solution of the desired peptide, followed by
the addition of a manganese salt in the desired molar ratio to
yield the desired solution of the peptide manganese complex.
Examples of manganese salts that may be used are manganese chloride
and manganese acetate. When aqueous solutions of peptide manganese
complexes are prepared, the solutions are neutralized, typically
with NaOH or HCl.
[0056] In yet another embodiment of the present invention, the
peptide moiety of the at least one peptide manganese complex may be
of natural origin. In this embodiment, the peptide is formed by the
hydrolysis of naturally occurring proteins, polypeptides, or larger
peptides of either plant, microbial, or animal origin. Hydrolysis
may be by enzymatic treatment or by acid or base hydrolysis. The
manganese complex of this type of peptide manganese complex is
formed by addition of a suitable manganese salt to the aqueous
solution of the peptide. Alternatively, the peptide manganese
complex may be formed during the manufacturing of a formulation by
separate additions of the peptide and manganese salt in a suitable
solvent.
[0057] In more specific embodiments, the composition of the present
invention comprises at least one peptide manganese complex, where
the concentration of the peptide manganese complex, by weight of
the composition, ranges from about 0.01% to about 5%, from about
0.025% to about 1%, or from about 0.05% to about 0.5%,
respectively. In other more specific embodiments, the molar ratio
of peptide to manganese in the peptide manganese complex ranges
from about 1:1 to about 3:1 in some embodiments, and from about 1:1
to about 2:1 in other embodiments. In yet other more specific
embodiments, the pH of the composition is from about 4.0 to about
8.0.
[0058] In additional embodiments of the method disclosed herein,
the composition used comprises at least one peptide manganese
complex that is encapsulated in a liposome or microsponge adapted
to aid in the delivery of the peptide manganese complex to the area
of skin being treated; and, in other embodiments, is formulated in
an instrument adapted to deliver the peptide manganese complex via
iontophoresis to the area of skin in need of treatment. Exemplary
methods of encapsulating pharmaceutical or cosmetic actives are
disclosed in the U.S. Pat. Nos. 6,572,892, 6,572,870, 6,565,886,
6,565,873, 6,548,690, 6,534,549 and 6,455,088.
[0059] As noted above, in certain embodiments, the composition
utilized in the method of the present invention further comprises
retinol, a retinol derivative, or a mixture thereof, in addition to
a peptide manganese complex. Retinol is also known as vitamin A and
has the formula
3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6,8-nonatetraen-1--
ol. Other terms that are used for retinol are axerophthol and
vitamin A alcohol. In certain specific embodiments of the present
invention that use compositions comprising retinol, the isomeric
forms of the retinol used are: all-trans-retinol; 1,3-cis-retinol;
3,4-didehydro-retinol; and 9-cis-retinol, respectively. In other
specific embodiments of the present invention that use compositions
comprising a retinol derivative, the latter is an ester of retinol
selected from C.sub.1-C.sub.30 esters of retinol; C.sub.2-C.sub.20
esters of retinol; and C.sub.2, C.sub.3, and C.sub.16 esters of
retinol, respectively. More specifically, the ester of retinol may
be retinyl palmitate, retinyl acetate and retinyl propionate. Other
retinol derivatives that may be used are retinoic acid and retinyl
aldehyde. The concentration of the retinol, retinol derivative, or
mixture thereof, ranges from about 0.001% to about 10% in some
embodiments; from about 0.01% to about 1% in other embodiments; and
from about 0.01% to about 0.5% in yet other embodiments, by weight
of the composition.
[0060] In further embodiments of the methods of the present
invention, the compositions used may comprise at least one active
agent in addition to the peptide manganese complex. In one such
embodiment, the composition is formulated as a pharmaceutical
preparation and comprises at least one active drug substance, such
as a sunscreen active. In another such embodiment, the composition
further comprises at least one active agent for rendering the
composition suitable as a cosmetic preparation. Active agents, as
defined herein, are compounds that provide benefits to the skin
and/or provide desirable properties to a composition formulated as
a cosmetic preparation. Some examples of active agents, other than
drug substances, are skin lightening agents, tanning agents, skin
conditioning agents, skin protectants, emollients and
humectants.
[0061] Representative sunscreen drugs are active ingredients that
absorb, reflect, or scatter radiation in the UV range at
wavelengths from 290 to 400 nanometers. Specific examples include
benzophenone-3 (oxybenzone), benzophenone-4 (sulisobenzone),
benzophenone-8 (dioxybenzone), butyl methoxydibenzoylmethane
(Avobenzone), DEA-methoxycinnamate (diethanolamine
methoxycinnamate), ethyl dihydroxypropyl PABA (ethyl
4-[bis(hydroxypropyl)] aminobenzoate), ethylhexyl dimethyl PABA
(Padimate O), ethylhexyl methoxycinnamate (octyl methoxycinnamate),
ethylhexyl salicylate (octyl salicylate), homosalate, menthyl
anthranilate (Meradimate), octocrylene, PABA (aminobenzoic acid),
phenylbenzimidazole sulfonic acid (Ensulizole), TEA-salicylate
(trolamine salicylate), titanium dioxide, and zinc oxide. One
skilled in the art will appreciate that other sunscreen agents may
be used in the compositions and preparations of the present
invention.
[0062] Representative skin lightening agents include, but are not
limited to, ascorbic acid and derivatives thereof, kojic acid and
derivatives thereof, hydroquinone and derivatives thereof, azelaic
acid, various plant extracts such as those from licorice, grape
seed and bear berry, and mixtures of any one or more of the
foregoing. Those skilled in the art will appreciate that other skin
lightening agents may be included in the compositions used for some
of the methods of the present invention.
[0063] Hydroquinone (p-dihydroxybenzene or p-hydroxyphenol) is a
phenolic compound having the following structure:
##STR00013##
Derivatives of hydroquinone include other substituted phenolic
compounds such as N-acetyl-4-S-cysteaminylphenol (4-S-CAP), Arbutin
(hydroquinone-beta-D-glucopyranoside), t-butyl hydroquinone:
##STR00014##
and other alkyl substitutions. Esters of hydroquinone are also
possible, such as Hydroquinone mono-methyl ether
(p-Hydroxyanisole).
[0064] Kojic acid (5-hydoxy-4-pyran-4-one-2-methyl) is a fungal
metabolic product having the following structure:
##STR00015##
Derivatives of kojic acid consist of fatty acid esters such as
kojic acid dipalmitate (Hexadecanoic Acid,
4-Oxo-6-[[(1-Oxohexadecyl)Oxy]Methyl]-4H-Pyran-3-yl Ester):
##STR00016##
monopalmitate, iso-palmitate, and the like.
[0065] As noted above, the compositions may further comprise skin
conditioning agents. Such agents comprise substances that enhance
the appearance of dry or damaged skin, as well as materials that
adhere to the skin to reduce flaking, restore suppleness, and
generally improve the appearance of skin. Representative examples
of skin conditioning agents include: acetyl cysteine, N-acetyl
dihydrosphingosine, acrylates/behenyl acrylate/dimethicone acrylate
copolymer, adenosine, adenosine cyclic phosphate, adensosine
phosphate, adenosine triphosphate, alanine, albumen, algae extract,
allantoin and derivatives, aloe barbadensis extracts, aluminum PCA,
amyloglucosidase, arbutin, arginine, azulene, bromelain, buttermilk
powder, butylene glycol, caffeine, calcium gluconate, capsaicin,
carbocysteine, carnosine, beta-carotene, casein, catalase,
cephalins, ceramides, chamomilla recutita (matricaria) flower
extract, cholecalciferol, cholesteryl esters, coco-betaine,
coenzyme A, corn starch modified, crystallins,
cycloethoxymethicone, cysteine DNA, cytochrome C, darutoside,
dextran sulfate, dimethicone copolyols, dimethylsilanol
hyaluronate, DNA, elastin, elastin amino acids, epidermal growth
factor, ergocalciferol, ergosterol, ethylhexyl PCA, fibronectin,
folic acid, gelatin, gliadin, beta-glucan, glucose, glycine,
glycogen, glycolipids, glycoproteins, glycosaminoglycans,
glycosphingolipids, horseradish peroxidase, hydrogenated proteins,
hydrolyzed proteins, jojoba oil, keratin, keratin amino acids, and
kinetin.
[0066] Other examples of skin conditioning agents are: lactoferrin,
lanosterol, lauryl PCA, lecithin, linoleic acid, linolenic acid,
lipase, lysine, lysozyme, malt extract, maltodextrin, melanin,
methionine, mineral salts, niacin, niacinamide, oat amino acids,
oryzanol, palmitoyl hydrolyzed proteins, pancreatin, papain, PEG,
pepsin, phospholipids, phytosterols, placental enzymes, placental
lipids, pyridoxal 5-phosphate, quercetin, resorcinol acetate,
riboflavin, RNA, saccharomyces lysate extract, silk amino acids,
sphingolipids, stearamidopropyl betaine, stearyl palmitate,
tocopherol, tocopheryl acetate, tocopheryl linoleate, ubiquinone,
vitis vinifera (grape) seed oil, wheat amino acids, xanthan gum,
and zinc gluconate. Skin conditioning agents, other than those
listed above, may also be used, as is readily appreciated by those
skilled in the art.
[0067] In other embodiments, the compositions may include a skin
protectant, defined herein as a compound that protects injured or
exposed skin or mucous membrane surfaces from harmful or irritating
external compounds. Representative examples thereof include: algae
extract, allantoin, aluminum hydroxide, aluminum sulfate, betaine,
camellia sinensis leaf extract, cerebrosides, dimethicone,
glucuronolactone, glycerin, kaolin, lanolin, malt extract, mineral
oil, petrolatum, potassium gluconate, and talc. Those skilled in
the art will readily appreciate that skin protectants, other than
those listed above, may be included in the compositions used for
the methods of the present invention.
[0068] An emollient, as the term is used herein, is a cosmetic
ingredient that can help the skin maintain a soft, smooth, and
pliable appearance. Emollients are able to provide these benefits,
largely owing to their ability to remain on the skin surface or in
the stratum corneum to act as a lubricant and reduce flaking. Some
examples of emollients, suitable for embodiments of this invention,
are: acetyl arginine, acetylated lanolin, algae extract, apricot
kernel oil PEG-6 esters, avocado oil PEG-11 esters, bis-PEG-4
dimethicone, butoxyethyl stearate, C.sub.18-C.sub.36 acid glycol
ester, C.sub.12-C.sub.13 alkyl lactate, caprylyl glycol, cetyl
esters, cetyl laurate, coconut oil PEG-10 esters,
di-C.sub.12-C.sub.13 alkyl tartrate, diethyl sebacate,
dihydrocholesteryl butyrate, dimethiconol, dimyristyl tartrate,
disteareth-5 lauroyl glutamate, ethyl avocadate, ethylhexyl
myristate, glyceryl isostearates, glyceryl oleate, hexyldecyl
stearate, hexyl isostearate, hydrogenated palm glycerides,
hydrogenated soy glycerides, hydrogenated tallow glycerides,
hydroxypropyl bisisostearamide MEA, isostearyl neopentanoate,
isostearyl palmitate, isotridecyl isononanoate, laureth-2 acetate,
lauryl polyglyceryl-6 cetearyl glycol ether, methyl gluceth-20
benzoate, mineral oil, myreth-3 palmitate, octyidecanol,
octyldodecanol, odontella aurita oil, 2-oleamido-1,3
octadecanediol, palm glycerides, PEG avocado glycerides, PEG castor
oil, PEG-22/dodecyl glycol copolymer, PEG shorea butter glycerides,
phytol, raffinose, stearyl citrate, sunflower seed oil glycerides,
and tocopheryl glucoside. Those skilled in the art will readily
appreciate that emollients, other than those listed above, may also
be used.
[0069] Humectants are cosmetic ingredients that help maintain
moisture levels in skin. Some examples of suitable humectants are:
acetyl arginine, algae extract, aloe barbadensis leaf extract,
betaine, 2,3-butanediol, chitosan lauroyl glycinate, diglycereth-7
malate, diglycerin, diglycol guanidine succinate, erythritol,
fructose, glucose, glycerin, honey, hydrolyzed wheat protein/PEG-20
acetate copolymer, hydroxypropyltrimonium hyaluronate, inositol,
lactitol, maltitol, maltose, mannitol, mannose, methoxy PEG,
myristamidobutyl guanidine acetate, polyglyceryl sorbitol,
potassium PCA, propylene glycol, sodium PCA, sorbitol, sucrose, and
urea. Other humectants may be used for yet additional embodiments
of this invention, as will be appreciated by those skilled in the
art.
[0070] In addition to the foregoing active agents, the compositions
employed in the methods of the present invention may also comprise
inert and physiologically acceptable carriers or diluents. Suitable
carriers or diluents include, but are not limited to: water,
physiological saline, bacteriostatic saline (e.g., saline
containing 0.9 mg/ml benzyl alcohol), petrolatum based creams
(e.g., USP hydrophilic ointments and similar creams), various types
of pharmaceutically acceptable gels, and short chain alcohols and
glycols (e.g., ethyl alcohol and propylene glycol).
[0071] In other further embodiments, the compositions employed may
comprise additional ingredients such as fatty alcohols, fatty
acids, organic or inorganic bases, preserving agents (such as
phenoxyethanol and mixtures of various parabens), wax esters,
steroid alcohols, triglyceride esters, phospholipids such as
lecithin and cephalin, polyhydric alcohol esters, fatty alcohol
ethers, hydrophilic lanolin derivatives, hydrophilic beeswax
derivatives, cocoa butter waxes, silicon oils, pH balancers,
cellulose derivatives, hydrocarbon oils such as palm oil, coconut
oil, and mineral oil, and mixtures thereof.
[0072] Additional ingredients may be included in the above
compositions to vary the texture, viscosity, color and/or
appearance thereof, as is appreciated by one of ordinary skill in
the art. Accordingly, in a further embodiment, the compositions, in
addition to at least one peptide manganese complex, comprise an
emulsifying agent, a surfactant, a thickening agent, an excipient
or a mixture thereof.
[0073] More specifically, emulsifiers and surfactants may be
included in those compositions used for the present invention that
are formulated as emulsions. Either water-in-oil or oil-in-water
emulsions may be formulated. Examples of suitable surfactants and
emulsifying agents include: nonionic ethoxylated and nonethoxylated
surfactants, abietic acid, almond oil PEG, beeswax, butylglucoside
caprate, C.sub.18-C.sub.36 acid glycol ester, C.sub.9-C.sub.15
alkyl phosphate, caprylic/capric triglyceride PEG-4 esters,
ceteareth-7, cetyl alcohol, cetyl phosphate, corn oil PEG esters,
DEA-cetyl phosphate, dextrin laurate, dilaureth-7 citrate,
dimyristyl phosphate, glycereth-17 cocoate, glyceryl erucate,
glyceryl laurate, hydrogenated castor oil PEG esters,
isosteareth-11 carboxylic acid, lecithin, lysolecithin,
nonoxynol-9, octyldodeceth-20, palm glyceride, PEG diisostearate,
PEG stearamine, poloxamines, polyglyceryls, potassium linoleate,
PPG's, raffinose myristate, sodium caproyl lactylate, sodium
caprylate, sodium cocoate, sodium isostearate, sodium tocopheryl
phosphate, steareths, TEA-C.sub.12-C.sub.13 pareth-3 sulfate,
tri-C.sub.12-C.sub.15 pareth-6 phosphate, and trideceths. Other
surfactants and emulsifiers may be used, as will be appreciated by
those skilled in the art.
[0074] Examples of thickening (i.e., viscosity increasing) agents
include, but are not limited to, those agents commonly used in skin
care preparations, such as: acrylamides copolymer, agarose,
amylopectin, bentonite, calcium alginate, calcium carboxymethyl
cellulose, carbomer, carboxymethyl chitin, cellulose gum, dextrin,
gelatin, hydrogenated tallow, hydroxytheylcellulose,
hydroxypropylcellulose, hydroxpropyl starch, magnesium alginate,
methylcellulose, microcrystalline cellulose, pectin, various PEG's,
polyacrylic acid, polymethacrylic acid, polyvinyl alcohol, various
PPG's, sodium acrylates copolymer, sodium carrageenan, xanthan gum,
and yeast beta-glucan. Thickening agents other than those listed
above may also be used in related embodiments of the present
invention.
[0075] As heretofore noted, the compositions used for the methods
of the present invention, being products for topical application to
human skin, are, accordingly, formulated as a liquid, cream, gel,
fluid cream or milk, lotion, oil, emulsion or microemulsion. Also,
the above compositions may be further combined with suitable
excipients adapted for application to the face and neck. Suitable
excipients should have a high affinity for the skin, be well
tolerated, stable, and yield a consistency that allows for easy and
pleasant utilization.
[0076] Typically, for a method of the present invention, aside from
the content of the composition used, a small amount of the
composition (from about 1 ml to about 5 ml) is applied to exposed
areas of skin from a suitable container or applicator, and, if
necessary, the composition is then spread over and/or rubbed into
the skin using the hand, finger, or other suitable device. Each
composition disclosed herein is typically packaged in a container
that is appropriate in view of the composition's viscosity and
intended use by the consumer. For example, a lotion or fluid cream
may be packaged in a bottle, roll-ball applicator, capsule,
propellant-driven aerosol device, or a container fitted with a
manually operated pump. A cream may simply be stored in a
non-deformable bottle, or in a squeeze container, such as a tube or
a lidded jar.
[0077] The following examples are provided for the purpose of
illustration, not limitation.
EXAMPLES
[0078] The examples, which follow, illustrate the preparation,
characterization and utility of certain compositions used for
exemplary embodiments directed to the methods of the present
invention; and illustrate the effectiveness of such methods in
treating conditions related to aging and photodamaged skin.
Example 1
[0079] The composition of a representative moisturizing lotion used
for a method of the present invention is shown below.
TABLE-US-00004 REPRESENTATIVE MOISTURIZING LOTION Ingredients % w/w
Water 73.80% Glycerin 1.00% Hydroxyethylcellulose 0.50% diisopropyl
adipate 4.00% isocetyl palmitate 6.00% octyl stearate 10.00%
glyceryl monooleate 1.00% cetyl alcohol 1.00% stearyl alcohol 0.80%
behenyl alcohol 0.50% palmitic acid 0.25% stearic acid 0.25%
L-alanyl-L-histidyl-L-lysine manganese 0.30% complex propylene
glycol 0.55% Phenoxyethanol 0.30% iodopropynyl butylcarbonate 0.02%
total 100.00%
Example 2
[0080] The composition of a representative moisturizing cream used
for a method of the present invention is shown below.
TABLE-US-00005 REPRESENTATIVE MOISTURIZING CREAM Ingredients % w/w
purified water 77.35% ethylhexyl palmitate 8.00% Octyldodecanol
2.50% butyloctyl calicylate 2.00% Squalane 1.50% jojoba oil 0.50%
tocopheryl acetate 0.20% Bisabolol 0.20% Polyacrylamide 1.50%
laureth-7 0.50% Glycerin 3.00% Panthenol 0.60% Allantion 0.10%
Cyclomethicone 0.50% Hydroxyethylcellulose 0.10% polysorbate 20
0.20% glycyl-L-histidyl-L-lysine manganese 0.25% complex propylene
glycol 0.56% diazolidinyl urea 0.30% Methylparaben 0.11%
Propylparaben 0.03% total 100.00%
Example 3
Stimulation of Collagen Formation in Fibroblasts by a Peptide
Manganese Complex
[0081] The effect of peptide manganese complexes on collagen
formation was determined by determining the amount of collagen type
I produced by human fibroblasts in cell culture. Normal human
dermal fibroblasts were seeded into a 96-well cell culture plate
and grown to confluence in high glucose media supplemented with 10%
fetal calf serum for 3 days. The fibroblasts were then placed in
low glucose media with 2% serum and various amounts of a
representative peptide manganese complex. After 3 days, the culture
media was collected and analyzed for collagen type I content by a
sandwich ELISA assay using purified antibody to collagen type
I.
[0082] The results, shown in Table 4, show that the addition of
GHK-Mn complex (glycyl-L-histidyl-L-lysine manganese) stimulated
the formation of collagen type I.
TABLE-US-00006 TABLE 4 Stimulation of Collagen Type I Synthesis in
Dermal Fibroblasts By Glycyl-L-Histidyl-L-Lysine Manganese Complex
ELISA Collagen Type I Standard Concentration of Complex (%
Increase) Deviation 0 ug/ml control 48 .+-.4.2 10 ug/ml 61.5 (+28%)
.+-.3.5 100 ug/ml 102.5 (+129%) .+-.0.7
Example 4
Stimulation of the Growth and Viability of Fibroblasts by a Peptide
Manganese Complex
[0083] The effect of peptide manganese complexes on the growth and
viability of fibroblasts was determined. Normal human dermal
fibroblasts were seeded into a 96-well cell culture plate and grown
to confluence in high glucose media supplemented with 10% fetal
calf serum for 3 days. The fibroblasts were then placed in low
glucose media with 2% serum and various amounts of a representative
peptide manganese complex. After 3 days, the cells were incubated
with neutral red to assess their viability and stained with
sulforhodamin B and counted to determine the number of cells.
[0084] The results, shown in Tables 5 and 6, show that the addition
of GHK-Mn complex (glycyl-L-histidyl-L-lysine manganese) stimulated
the proliferation of normal human dermal fibroblasts and increased
their viability.
TABLE-US-00007 TABLE 5 Increase in Proliferation of Dermal
Fibroblasts By Glycyl-L-Histidyl-L-Lysine Manganese Complex
Sulforhodamin B Stain - Cell Number Standard Concentration of
Complex (% Increase) Deviation 0 ug/ml control 313 .+-.6 10 ug/ml
383 (+22%) .+-.15 100 ug/ml 535 (+71%) .+-.5
TABLE-US-00008 TABLE 6 Increase in Viability of Dermal Fibroblasts
By Glycyl-L-Histidyl-L-Lysine Manganese Complex Neutral Red Stain
Stain - Cell Viability Standard Concentration of Complex (%
Increase) Deviation 0 ug/ml control 212 .+-.12 10 ug/ml 231 (+16%)
.+-.5 100 ug/ml 253 (+19%) .+-.19
[0085] All of the above U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications and non-patent publications referred to in this
specification and/or listed in the Application Data Sheet, are
incorporated herein by reference, in their entirety.
[0086] From the foregoing, it will be appreciated that, although
specific embodiments of the present invention have been described
herein for purposes of illustration, various modifications may be
made without deviating from the spirit and scope of the invention.
Accordingly, the present invention is not limited except as by the
appended claims.
* * * * *