| United States Patent |
5,958,384 |
| Holick |
September 28, 1999 |
Use of emu oil for stimulating skin
and hair growth
Abstract
The present invention is directed to the discovery that topical
or parenteral administration of emu oil to a mammal
stimulates the proliferation of skin. Emu oil
can be used to treat skin wrinkles and rejuvenate aged and photo-damaged
skin. It has also been discovered that emu oil
can be topically applied to stimulate melanogenesis in the skin
and to stimulate hair growth. Thus, emu
oil is useful to treat pigmentation disorders such as hypopigmentation,
stimulating melanogenesis to enhance skin tanning, and treating
disorders relating to disturbances in hair cycling
such as alopecia, male pattern baldness, female baldness, and
chemotherapy-induced alopecia.
| Inventors: |
Holick; Michael F. (31 Bishop
La., Sudbury, MA 01776) |
| Appl. No.: |
066598 |
| Filed: |
April 27, 1998 |
| Current U.S. Class: |
424/60; 424/522
|
| Intern'l Class: |
A61K 007/44; A01N 059/22 |
| Field of Search:
|
424/60,522 |
References Cited [Referenced
By]
U.S. Patent Documents
| 5431924 |
Jul., 1995 |
Ghosh et al. |
424/522. |
| 5472713 |
Dec., 1995 |
Fein et al. |
424/522. |
Primary Examiner: Nutter; Nathan M.
Attorney, Agent or Firm: Sterne, Kessler, Goldstein &
Fox P.L.L.C.
Parent Case Text
BACKGROUND OF THE INVENTION
This application is a divisional of U.S. application Ser. No.
08/433,789, filed May 3, 1995, now U.S. Pat. No. 5,744,128.
Claims
What is claimed is:
1. A method for treating skin conditions of skin slackness, wrinkles,
dry skin and insufficient sebum secretion, wherein said conditions
are a result of aging, comprising topically or parenterally applying
to the skin of a mammal emu oil, or a biologically
active fraction thereof, in an amount effective to increase the
proliferation of epidermal cells, with the proviso that said conditions
are not the result of photodamaged skin.
2. The method of claim 1, wherein non-fractionated emu
oil is applied to the skin.
3. The method of claim 1, wherein said emu oil or
biologically active fraction thereof, is applied as a mixture
with an active vitamin D compound.
4. The method of claim 1, wherein said emu oil is
applied as a mixture with a peptide at least 3 amino acids long,
having 10% or greater homology with a region of human parathyroid
hormone or human parathyroid hormone related peptide, and having
the ability to enhance proliferation of mammalian cells.
5. The method of claim 4, wherein said peptide has greater than
50% homology with said region.
6. The method of claim 5, wherein said peptide is PTH (7-34).
Description
FIELD OF THE INVENTION
The invention relates to methods of topically, orally or parenterally
applying emu oil or a fraction thereof to mammalian
skin or hair for the purposes of stimulating skin
and hair growth, or to enhance pigmentation of the
skin. The invention further relates to compositions for topical,
oral or parenteral application that are useful for treating skin
disorders, said compositions comprising emu oil
in combination with other substances known to have therapeutic
effects on mammalian skin.
For the purposes of this specification, the term "emu
oil" refers to oils and preparations of oils derived from the
emu (Dromais Novae--Hollandiae).
RELATED ART
International application WO92/08470, published May 29, 1992,
discloses pharmaceutical compositions including emu
oil and dermal transport enhancing compounds that are useful as
topical anti-inflammatory treatments. The published application
also discloses the isolation of a biologically active fraction
of emu oil that displays topical anti-inflammatory
activity. The inventors surmise that the potent anti-elastase
activity demonstrated to be present in the emu oil
could provide a local anti-inflammatory, as well as an anti-degenerative
effect, to dermal tissues that could be particularly relevant
during dermal inflammation where cell and tissue damage produced
by exposure to strong UV radiation, as in sunburn, occurs.
Oils extracted or rendered from emu body fats are
known to contain triglyceride esters of long chain fatty acids
including oleic acid and linoleic acid as well as the saturated
fatty acids, palmitic acid and stearic acid. See Hilditch, T.
P. and Williams, P. N., The Chemical Constitution of Natural Fats,
4th Edition, Chapman and Hall, London (1964). Emu
oil is predominantly composed of triglyceride esters of saturated
and unsaturated fatty acids. The overall fatty acid composition
of emu oil preparations is not too dissimilar to
that of chicken. However, while chicken oils are colorless, emu
oils are invariably yellow colored. Mammalian fats are known to
provide a depot for other naturally occurring lipophilic compounds.
These compounds would include the fat soluble vitamins such as
vitamin A, D and E as well as their precursors and metabolites.
The natural diet of the emu consists of seeds, berries,
grasses, leaves and plants present within the Australian bush
which would be expected to contain a large variety of carotenoids,
vitamins, terpenes, saponagens, flavones and other naturally occurring
bioactive occurring compounds.
SUMMARY OF THE INVENTION
It has now been found that effective treatment of various skin
and hair loss conditions can be achieved with compositions
that include an effective amount of emu oil or a
biologically active fraction thereof. It has been discovered that
application of emu oil can: stimulate the proliferation
of cells in mammalian skin tissue, stimulate melanogenesis in
mammalian skin tissue, and stimulate hair follicle
development and growth in mammalian skin tissue.
In one embodiment, the present invention relates to a method for
treating skin conditions of skin slackness, wrinkles, dry skin,
and insufficient sebum secretion comprising topically or parenterally
applying to the skin of a mammal a composition comprising emu
oil or a biologically active fraction thereof.
In a second embodiment, the present invention relates to a cosmetic
preparation for skin rejuvenation and hydration comprising emu
oil, or a biologically active fraction thereof, and one or more
active vitamin D compounds.
In a third embodiment, the present invention relates to a method
for treating hypopigmentation disorders by topically or parenterally
administering to the skin of a mammal a composition comprising
a pigmentation-increasing amount of emu oil, or
a biologically active fraction thereof.
In a fourth embodiment, the present invention relates to a method
for enhancing skin tanning of mammalian skin by applying a composition
comprising emu oil, or a biologically active fraction
thereof, to mammalian skin and/or hair in an amount
effective to enhance skin tanning.
In a fifth embodiment, the present invention relates to a method
for stimulating growth of mammalian hair comprising
applying a composition comprising emu oil, or a
biologically active fraction thereof, to the skin and/or hair
of a mammal in an amount effective to stimulate hair
growth.
In a sixth embodiment, the present invention relates to a method
of accelerating the healing of topical wounds by topically or
parenterally applying a composition comprising emu
oil, or biologically active fraction thereof, and one or more
parathyroid hormone peptides to a wound.
In a seventh embodiment, the present invention relates to a method
of treating burns by topically applying a composition comprising
emu oil, or a biologically active fraction thereof,
and one or more parathyroid hormone peptides to a burn.
In an eighth embodiment, the present invention relates to a method
for inhibiting cell proliferation and inducing cell differentiation
in a mammal suffering from psoriasis, or cancer or precancer of
the skin by topically or parenterally applying emu
oil, or a biologically active fraction thereof, in admixture with
a PTH/PTHrP peptide selected from the group consisting of PTH
(1-34), PTH (3-34) and PTHrP (1-34).
Other features and advantages of the invention will be apparent
from the following description of the preferred embodiments thereof,
and from the claims.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a bar graph that shows the effect of topical administration
of emu oil on tritiated thymidine (.sup.3 H-thymidine)
incorporation into the skin of C57 BL/6 mice, versus topical administration
of corn oil.
FIG. 2 is a photograph of C57 BL/6 mice after 19 days of topical
application of 0.1 ml of either corn oil (two mice on left side
of picture) or emu oil (three mice on right side
of picture) on the nape of the neck daily.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
One aspect of the present invention generally involves enhancing
proliferation of a mammalian cell by contacting the cell with
emu oil or biologically active fraction thereof.
This aspect of the present invention has particular application
in the promotion of skin growth in patients with skin ulcerations,
as well as in the stimulation of epidermal regrowth in people
who have decreased epidermal cell proliferation due to aging.
Additionally, this aspect of the invention has cosmetic applications
in rejuvenating aged and photodamaged skin.
A second aspect of the present invention generally involves enhancing
melanogenesis in mammalian skin tissue by contacting the skin
tissue with emu oil or a biologically active fraction
thereof. This aspect of the invention has particular utility in
treating pigmentation disorders such as hypopigmentation disorders,
and for enhancing skin tanning.
A third aspect of the present invention generally involves enhancing
hair follicle development and growth in mammalian
skin tissue by contacting the skin tissue with emu
oil or a biologically active fraction thereof. Alopecia, the disease
of hair loss, may arise from various causes. In
each instance, individual hairs cannot complete their normal hair
cycle to reach the telogen state. In order to decrease baldness
and accelerate hair generation, it is necessary
to bring the hair follicles from the telogen state
into the normal anagen state. It has now been found that hair
growth is stimulated by administering to a mammal emu
oil or a biologically active fraction thereof. This aspect of
the present invention has particular utility in the promotion
of new hair growth or stimulation of the rate of
hair growth, e.g., following chemotherapeutic treatment
or for treating a form of alopecia, e.g., male pattern baldness
and female hair loss.
For the purposes of this specification, the term "emu
oil" refers to oils and preparations of oils derived from the
emu (Dromais Novae--Hollandiae). The emu
oil, its partially purified active fractions or the active components
themselves may be administered to mammals and man topically or
parenterally.
The term "biologically active fraction" of emu oil
includes those fractions or active components of emu
oil that are useful as therapeutic agents in treating disorders
such as aging, photodamaged skin and skin ulcerations, where a
maintenance or stimulation of cell proliferation is desired. The
ability of a particular fraction or component to maintain or stimulate
cell proliferation can be determined by measuring the effect of
a particular function or component on .sup.3 H-thymidine incorporation
into the skin of mice. See, Holick et al., Proc. Natl. Acad. Sci.
USA 91:8014-8016 (1994). The term "biologically active fraction"
also includes fractions or active components that cause an increase
in skin pigmentation and/or hair growth as determined
by the method described in the Example herein.
Emu oil is commercially available from Emu
Products, Western Australia Pty. Ltd., Perth, Australia, or from
New World Technology, Inc., Greenwich, Conn., under the name "Kalaya
oil." Active emu oil fraction can be isolated according
to the following procedure disclosed in PCT published application
WO92/08470. Emu oil is diluted 1:1 with hexane and
fractionated on an activated florisil column (1 g of oil per 12
g of florisil). Additional hexane (100 ml per g of oil) is passed
through the column followed by dichloromethane (100 ml per g of
oil) and 10% methanol in dichloromethane (100 ml per g of oil).
The material eluting in the hexane and the dicholoromethane fraction
(0.89 g) is colorless and the material eluting in the 10% methanol
in dichloromethane fraction (0.11 g) was a yellow color.
The material eluting in the 10% methanol in dichloromethane fraction
is diluted 1:1 with hexane and applied to a silica column (1 g
of yellow material per 12 g of silica). Additional hexane (100
ml per g of oil) is passed through the column followed by dichloromethane
(100 ml per g of oil) and 10% methanol in dichloromethane (100
ml per g of oil).
The material eluting in the hexane and the dichloromethane fraction
is colorless (0.64 g) and the material eluting in the 10% methanol
in dichloromethane fraction (0.36 g) is a yellow color. Pure yellow
component is separated from the methanol/dichloromethane by evaporation.
If the 10% methanol in dichloromethane fraction from the silica
column is analyzed by gas chromatography using an on-column injection
technique, the material is shown to be free of triglycerides,
consisting principally of two closely eluting peaks. These two
peaks correspond to two peaks observed when the unpurified oil
is analyzed using the same technique.
Hydrolysis with sodium methoxide of the 10% methanol in dichloromethane
fraction from the silica column shows that this fraction is composed
of saturated and unsaturated fatty acids esterified with a series
of unidentified compounds. Indications are that the saturated
and unsaturated fatty acids are C.sub.16 -C.sub.18 with some shorter
and longer chain length acids present. The resulting biologically
active yellow-colored component(s) may be included in topical
and systemic compositions for practicing the methods of this invention.
Compositions comprising emu oil, or a biologically
active fraction thereof, and pharmaceutical preparations thereof,
are intended for topical, oral or parenteral, e.g., subcutaneous
injection, administration for prophylactic and/or therapeutic
or cosmetic treatment. Preferably, the pharmaceutical compositions
are administered topically, as an oil, paste, cream or salve.
For administration by parenteral injection the purified active
fractions of emu oil may be used directly. Alternatively,
the purified active fractions may be admixed with a neutral vehicle
such as a vegetable oil, or acacia gum and injected as a dispersed
suspension.
For oral administration, the emu oil, or a biologically
active fraction thereof, can be employed in dosage forms such
as gelatin capsules, liquid solutions, suspensions or elixirs.
Preferably, the compositions employed in each aspect of the present
invention consist of 0.01-100% by weight of emu
oil, or biologically active fraction thereof, by volume combined
with 99.99-0% by weight of suitable diluents, carriers, excipients
and other active agents. Repeated applications of the compositions
to obtain the desired results are envisioned.
In accordance with the first aspect of the present invention,
emu oil is employed in topical and parenteral formulations
thereof and methods of using for the treatment of such skin conditions
as dry skin (lack of dermal hydration), undue skin slackness (i.e.,
insufficient skin firmness) and insufficient sebum secretion.
The methods and compositions are also effective in general preservating,
conditioning, hydrating and protecting of skin, e.g., against
wrinkles.
One or more additional substances which have therapeutic effects
on the skin may also be incorporated in the compositions. Thus,
in one embodiment of this invention the composition also contains
one or more compounds capable of increasing cyclic-AMP levels
in the skin. Suitable compounds include adenosine or a nucleic
acid hydrolysate in an amount of about 0.1-1% and papaverine,
in an amount of about 0.5-5%, both by weight based on the weight
of the composition. Also suitable are .beta.-adrenergic agonists
such as isoproterenol, in an amount of about 0.1-2% or cyclic-AMP,
in an amount of about 0.1-1%, again both by weight based on the
weight of the composition. Other suitable types of additional
active ingredients which may be incorporated in the compositions
of this invention include any compounds known to have a beneficial
effect on skin. Such compounds include retinoids such as Vitamin
A, in an amount of about 0.003-0.3% by weight and chromanols such
as Vitamin E or a derivative thereof in an amount of about 0.1-10%
by weight, both based on the weight of the composition. Additionally,
anti-inflammatory agents and keratoplastic agents may be incorporated
in the cosmetic composition. A typical anti-inflammatory agent
is a corticosteroid such as hydrocortisone or its acetate in an
amount of about 0.25-5 % by weight, or a corticosteroid such as
dexamethasone in an amount of about 0.025-0.5 % by weight, both
based on the weight of the composition. A typical keratoplastic
agent is coal tar in an amount of about 0.1-20% or anthralin in
an amount of about 0.05-2% by weight, both based on the weight
of the composition. Especially preferred additional components
for purposes of the present invention are "active vitamin D compounds,"
parathyroid hormone (PTH) peptides or parathyroid hormone related
peptides (PTHrP), each described in detail, below.
"Active vitamin D compounds" useful as additional substances in
the cosmetic and dermatological compositions of the first and
second aspects of the present invention are characterized structurally
as side chain unsaturated and side chain saturated homologs of
vitamin D, and preferably of 1,25-dihydroxyvitamin D.sub.3, in
which the side chain is elongated by insertion of one or more
methylene units into the chain at the carbon 24 position. See
U.S. Pat. No. 5,276,061, herein fully incorporated by reference.
They may be represented, therefore, by the following general structure:
##STR1## where R.sub.4 and R.sub.5 represent hydrogen or when
taken together R.sub.4 and R.sub.5 represent a carbon-carbon double
bond or a carbon-carbon triple bond, Z represents hydrogen, hydroxy
or protected-hydroxy, U represents hydrogen, fluoro, hydroxy,
protected-hydroxy or an alkyl group, X and Y which may be the
same or different are hydrogen or a hydroxy-protective group,
R.sub.1 represents the group --(CH.sub.2).sub.q --H or --CF.sub.3
and R.sub.2 represents the group --(CH.sub.2).sub.p --H or --CF.sub.3,
and where n, q and p are integers having independently the values
of 1 to 5, and R.sub.1 and R.sub.2 when taken together represent
the group --(CH.sub.2).sub.m where m is an integer having the
value of 2 to 5 (i.e., cycloalkyl).
The term "hydroxy-protective group" refers to any group commonly
used for the protection of hydroxy functions during subsequent
reactions, including, for example, acyl or alkylsilyl groups such
as triethylsilyl, triethylsilyl, t-butyldimethylsilyl and analogous
alkylated silyl radicals, or alkoxyalkyl groups such as methoxymethyl,
ethoxymethyl, methoxyethoxymethyl, tetrahydrofuranyl or tetrahydropyranyl.
A "protected-hydroxy" is a hydroxy function derivatized by one
of the above hydroxy-protecting groupings. "Alkyl" represents
a straight-chain or branched hydrocarbon radical of 1 to 10 carbons
in all its isomeric forms, such as methyl, ethyl, propyl, isopropyl,
butyl, isobutyl, pentyl, etc., and the terms "hydroxyalkyl" and
"fluoroalkyl" refer to such an alkyl radical substituted by one
or more hydroxy or fluoro groups respectively. An acyl group is
an alkanoyl group of 1 to 6 carbons in all its isomeric forms,
or an aroyl group, such as benzoyl, or halo-, nitro- or alkyl-
substituted benzoyl groups, or a dicarboxylic acyl group such
as oxalyl, malonyl, succinoyl, glutaroyl, or adipoyl. The term
"aryl" signifies a C.sub.6 to C.sub.14 aromatic group, e.g. a
phenyl or naphthyl group.
It should be noted in this description that the term "24-dihomo"
refers to the addition of two methylene groups at the carbon 24
position in the side chain. Likewise, the term "trihomo" refers
to the addition of three methylene groups. Also, the term "26,27-dimethyl"
refers to the addition of a methyl group at the carbon 26 and
27 positions so that for example R.sub.1 and R.sub.2 are ethyl
groups. Likewise, the term "26,27-diethyl" refers to the addition
of an ethyl group at the 26 and 27 positions so that R.sub.1 and
R.sub.2 are propyl groups.
Specific and preferred examples of these compounds when the side
chain is unsaturated (i.e., R.sub.4 and R.sub.5 represent a double
bond) are: 24-dihomo-1,25-dihydroxy-22-dehydrovitamin D.sub.3,
i.e., the compound shown above, where X and Y are hydrogen, Z
is hydroxy, n equals 3, and R.sub.1 and R.sub.2 are each a methyl
group; 26,27-dimethyl-24-dihomo-1,25-dihydroxy-22-dehydrovitamin
D.sub.3, i.e., the compound shown above where X and Y are hydrogen,
Z is hydroxy, n equals 3, and R.sub.1 and R.sub.2 are each an
ethyl group; 24-trihomo-1,25-dihydroxy-22-dehydrovitamin D.sub.3,
i.e., the compound having the structure shown above, where X and
Y are hydrogen, Z is hydroxy, n equals 4, and R.sub.1 and R.sub.2
are each a methyl group; 26,27-dimethyl-24-trihomo-1,25-dihydroxy-22-dehydrovitamin
D.sub.3, i.e., the compound shown above where X and Y are hydrogen,
Z is hydroxy, n equals 4, and R.sub.1 and R.sub.2 are each an
ethyl group; 26,27-diethyl-24-dihomo-1,25-dihydroxy-22-dehydrovitamin
D.sub.3, i.e., the compound shown above where X and Y are hydrogen,
Z is hydroxy, n equals 3, and R.sub.1 and R.sub.2 are each a propyl
group; 26,27-diethyl-24-trihomo-1,25-dihydroxy-22-dehydrovitamin
D.sub.3, i.e., the compound shown above where X and Y are hydrogen,
Z is hydroxy, n equals 4, and R.sub.1 and R.sub.2 are each a propyl
group; 26,27-dipropyl-24-dihomo-1,25-dihydroxy-22-dehydrovitamin
D.sub.3, i.e., the compound shown above where X and Y are hydrogen,
Z is hydroxy, n equals 3, and R.sub.1 and R.sub.2 are each a butyl
group; and 26,27-dipropyl-24-trihomo-1,25-dihydroxy-22-dehydrovitamin
D.sub.3, i.e., the compound shown above where X and Y are hydrogen,
Z is hydroxy, n equals 4, and R.sub.1 and R.sub.2 are each a butyl
group.
Specific and preferred examples of these compounds when the side
chain is saturated (i.e., R.sub.4 and R.sub.5 each represent hydrogen)
are: 1,25-dihydroxyvitamin D.sub.3, 1,24-dihydroxyvitamin D.sub.3,
24-dihomo-1,25-dihydroxy-vitamin D.sub.3, i.e., the compound shown
above, where X and Y are hydrogen, Z is hydroxy, n equals 3, and
R.sub.1 and R.sub.2 are each a methyl group; 26,27-dimethyl-24-dihomo-1,25-dihydroxy-vitamin
D.sub.3, i.e., the compound shown above where X and Y are hydrogen,
Z is hydroxy, n equals 3, and R.sub.1 and R.sub.2 are each an
ethyl group; 24-trihomo-1,25-dihydroxy-vitamin D.sub.3, i.e.,
the compound having the structure shown above, where X and Y are
hydrogen, Z is hydroxy, n equals 4, and R.sub.1 and R.sub.2 are
each a methyl group; 26,27-dimethyl-24-trihomo-1,25-dihydroxy-vitamin
D.sub.3, i.e., the compound shown above where X and Y are hydrogen,
Z is hydroxy, n equals 4, and R.sub.1 and R.sub.2 are each an
ethyl group; 26,27-diethyl-24-dihomo-1,25-dihydroxy-vitamin D.sub.3,
i.e., the compound shown above where X and Y are hydrogen, Z is
hydroxy, n equals 3, and R.sub.1 and R.sub.2 are each a propyl
group; 26,27-diethyl-24-trihomo-1,25-dihydroxy-vitamin D.sub.3,
i.e., the compound shown above where X and Y are hydrogen, Z is
hydroxy, n equals 4, and R.sub.1 and R.sub.2 are each a propyl
group; 26,27-dipropyl-24-dihomo-1,25-dihydroxy-vitamin D.sub.3,
i.e., the compound shown above where X and Y are hydrogen, Z is
hydroxy, n equals 3, and R.sub.1 and R.sub.2 are each a butyl
group; and 26,27-dipropyl-24-trihomo-1,25-dihydroxy-vitamin D.sub.3,
i.e., the compound shown above where X and Y are hydrogen, Z is
hydroxy, n equals 4, and R.sub.1 and R.sub.2 are each a butyl
group.
Examples of the compounds of this invention wherein the side chain
is saturated can be prepared according to the general process
illustrated and described in U.S. Pat. No. 4,927,815 issued May
22, 1990, entitled "Compounds Effective in Inducing Cell Differentiation
And Process For Preparing Same," the description of which is specifically
incorporated herein by reference. Examples of the compounds of
this invention wherein the side chain is unsaturated can be prepared
according to the general process illustrated and described in
U.S. Pat. No. 4,947,012 issued Jul. 11, 1989, entitled "Vitamin
D Related Compounds And Process For Their Preparation," the description
of which is specifically incorporated herein by reference. Examples
of the compounds of this invention wherein R.sub.1 and R.sub.2
together represent a cyclopentano group can be prepared according
to the general process illustrated and described in U.S. Pat.
No. 4,851,401 issued Jul. 25, 1989, entitled "Novel Cyclopentano-Vitamin
D Analogs," the description of which is specifically incorporated
herein by reference.
Another synthetic strategy for the preparation of side-chain-modified
analogues of la-dihydroxy-cholecalciferol and 1.alpha.,25-dihydroxyetgocalciferol
is disclosed in Kutner et al., The Journal of Organic Chemistry
53:3450-3457 (1988). In addition, the preparation of 24-homo and
26-homo vitamin D analogs is disclosed in U.S. Pat. No. 4,717,721
issued Jan. 5, 1988 entitled "Sidechain Homo-Vitamin D Compounds
With Preferential Anti-Cancer Activity," the description of which
is specifically incorporated herein by reference.
A class of peptides, termed PTH/PTHrP agonist and antagonist peptides,
that have been shown to be capable of regulating cell proliferation
and differentiation in mammals are useful as additional substances
in the compositions and methods of the present invention. See
PCT Published Application WO 92/04039, published Mar. 19, 1992.
These peptides provide two important therapeutic properties, one
involving inhibition of cell proliferation and enhancement of
cell differentiation (the agonist activity), which is useful in
the treatment of hyperproliferative disorders, such as psoriasis
and cancer, and one involving enhancement of cell proliferation
(the antagonist activity), which is useful for stimulating skin
and hair growth, and in wound healing. In addition,
some of the peptides possess the ability to enhance wound healing
and stimulate hair growth based on in vivo wound
healing activity or in vitro hair growth activity
rather than strict agonist or antagonist activity in vitro. Thus,
these peptides are useful as co-ingredients in the present invention,
especially in the first, second, fifth, sixth and seventh embodiments
of the invention.
Generally, peptides useful in the present invention include peptides
that are at least 3, and more preferably at least 8, amino acids
long, that have 10% or greater (more preferably, 50% or greater,
and most preferably 75% or greater) homology with a region (preferably
within the amino-terminal 34 amino acid region) of human parathyroid
hormone or human parathyroid hormone related peptide (PTHrP).
Examples of useful peptides include a 34-residue amino terminal
fragment of human PTH (1-34) (herein, PTH (1-34)), synthetic fragment
of PTHrP (›Tyr! PTHrP fragment (1-34) amide (herein, PTHrP
(1-34)), ›Nle.sup.8, Nle.sup.18, Tyr.sup.34 ! bovine (3-34)
PTH amide (herein, PTH (3-34)), ›Tyr.sup.34 ! bovine PTH
(7-34) amide (herein, PTH (7-34)), human PTH (7-34), human, chicken,
bovine, porcine or rat parathyroid hormone (herein, PTH (1-84)
and human parathyroid-related protein (herein, hPTHrP (1-141)).
The peptides having agonist activity (PTH/PTHrP agonists) have
particular application in the treatment of hyperproliferative
skin disorders such as psoriasis and cancer. Agonist peptides
are at least 3, and more preferably at least 8, amino acids long,
have 10% or greater (more preferably, 50% or greater, and most
preferably 75% or greater) homology with a region (preferably
within the amino-terminal 34 amino acid region) of human parathyroid
hormone or human PTHrP, and are capable of inhibiting proliferation
or enhancing the differentiation in vitro of cultured human keratinocytes.
These peptides may also be useful in the treatment of certain
cancers, by the inhibition of cancer cell proliferation and by
the induction of differentiation. Preferred agonist peptides include
PTH (1-34), PTH (3-34) or PTHrP (1-34) and their amide derivatives.
The peptides having antagonist activity (PTH/PTHrP antagonists)
are generally useful for enhancing proliferation of mammalian
cells. Antagonist peptides are at least 3, and more preferably
at least 8, amino acids long, have 10% or greater (more preferably,
50% or greater, and most preferably 75% or greater) homology with
a region (preferably within the amino-terminal 34 amino acid region)
of human parathyroid hormone or human parathyroid hormone related
peptide, and are capable of blocking the differentiation or the
inhibition of proliferation in vitro of cultured human keratinocytes
by PTH (1-34) or 1,25(OH).sub.2 D.sub.3 or PTHrP (1-34). A preferred
PTH/PTHrP antagonist peptide is PTH (7-34) and its amide derivative.
Other PTH/PTHrP peptides are useful for enhancing proliferation
of a mammalian cell. These peptides are at least 3, and more preferably
at least 8, amino acids long, have 10% or greater (more preferably,
50% or greater, and most preferably 75% or greater) homology with
a region (preferably within the amino-terminal 34 amino acid region)
of human parathyroid hormone or human parathyroid hormone related
peptide, and are capable of enhancing wound healing in an in vivo
skin punch assay. Preferred peptides for this aspect of the invention
include PTH (1-34), PTH (7-34), PTH (1-84), hPTHrP (1-141), PTHrP
(1-34), or PTHrP (7-34). These peptides have particular application
as co-ingredients in the methods and compositions for enhancing
wound healing and may also have applications in promoting skin
growth in patients with burns or skin ulcerations as well as stimulating
epidermal regrowth in people who have decreased epidermal cell
proliferation due to aging.
Hair growth is stimulated in mammals by PTH/PTHrP
peptides that are at least 3, and more preferably at least 8,
amino acids long, have 10% or greater (more preferably, 50% or
greater, and most preferably 75% or greater) homology with a region
(preferably within the amino-terminal 34 amino acid region) of
human parathyroid hormone or human parathyroid hormone related
peptide, and are capable of stimulating hair growth
in vitro. A preferred peptide in this aspect of the invention
is PTH (7-34). These peptides have application as a co-ingredient
in the methods of promoting new hair growth or stimulating
the rate of hair growth, and can be applied in an
amount of about 0.01 .mu.g to about 100 .mu.g per gm of composition.
When selecting a candidate PTH/PTHrP agonist or antagonist peptide
for the present invention, a preferred first step is to choose
a peptide which includes a fragment which has at least 10%, and
more preferably 50% or greater, homology with an 8 or greater
amino acid long fragment within the amino terminal 34 amino acid
region of human PTH or PTHrP. By "homology" is meant amino acid
sequence identity. Because of the high degree of homology among
human PTH and PTH of other species, non-human as well as human
fragments or analogs can be used. For purposes of the present
invention, percent homology is determined by lining up a sequence
of interest (SOI) with a selected region (REGION) of human parathyroid
hormone (hPTH) or parathyroid hormone related peptide (PTHrP)
directly comparing amino acids of the two sequences beginning
at the amino terminus of each sequence; and calculating the ratio
of: ##EQU1## Homologous peptides must also be at least 3, and
more preferably at least 8, amino acids long. Further, the fragment
can be modified in any of a variety of standard chemical ways,
e.g., the carboxy-terminal amino acid residue can be made into
a terminal amide group; the amino-terminal residue can be modified
with groups to, e.g., enhance lipophilicity; the peptide can be
chemically glycosylated to increase solubility or in vivo half-life;
and D-amino acids can be substituted for L-isomers in the peptide.
Candidate peptides are tested for suitability as inhibitors of
cell proliferation and enhancers of differentiation using cultured
human keratinocytes, as described in U.S. Pat. No. 5,037,816.
Those peptides which inhibit proliferation and induce differentiation
in cultured keratinocytes are those potentially useful as therapeutic
agents in treating disorders, e.g., psoriasis, precancer, such
as actinic keratoses, and cancer, where suppression of cell proliferation
is desired.
Candidate peptides may be tested for suitability as enhancers
of cell proliferation using cultured human keratinocytes. Those
peptides which block the effect of agonist peptides or 1,25(OH).sub.2
D.sub.3 on cultured keratinocyte proliferation are those potentially
useful as therapeutic agents in treating disorders, e.g., wounds,
burns, or skin ulcerations, where maintenance or stimulation of
cell proliferation is desired.
Candidate peptides may be tested for their ability to enhance
wound healing by carrying out a skin punch biopsy test, described
in PCT Published Application WO92/04039, published Mar. 19, 1992.
Candidate peptides may be tested for suitability as stimulators
of hair growth using an in vitro hair
growth assay, such as is described in PCT Published Application
WO92/04039. Those peptides which stimulate hair
growth in vitro are those potentially useful for co-administering
with emu oil for stimulating hair
growth in vivo.
Compositions for use in the treatment of such skin conditions
as dry skin (lack of dermal hydration), undue skin slackness (i.e.,
insufficient skin firmness) and insufficient sebum secretion,
as well as compositions effective in general preservating, conditioning,
hydrating and protecting of skin, e.g., against wrinkles treatment
of skin, preferably comprise emu oil, or a biologically
active fraction thereof, and optionally one or more side chain
unsaturated or active vitamin D compounds, and/or one or more
PTH/PTHrP antagonists, and a suitable carrier. A preferred amount
of emu oil is about 0.01 to 99.99% by weight. Lesser
amounts of the biologically active fractions can be used. A cosmetically
effective amount of active vitamin D compounds for use in accordance
with this invention is from about 0.01 .mu.g to about 100 .mu.g
per gm of composition. A concentration of about 10 .mu.g active
vitamin D compound per gm of the composition is preferred. A cosmetically
or dermatologically effective amount of a PTH/PTHrP agonist or
PTH/PTHrP antagonist peptide for use in accordance with this invention
is about 0.01 .mu.g to about 100 .mu.g per gm of composition.
A concentration of about 10 .mu.g PTH/PTHrP agonist or antagonist
peptide per gm of composition is preferred.
The topical compositions of this invention are formulated preferably
as oils, creams, lotions, ointments and the like by choice of
appropriate carriers. Suitable carriers include vegetable or mineral
oils, white petrolatum (white soft paraffin), branched chain fats
or oils, animal fats and high molecular weight alcohol (greater
than C.sub.12). The preferred carriers are those in which the
active ingredient is soluble. Emulsifiers, stabilizers, humectants
and antioxidants may also be included as well as agents imparting
color or fragrance, if desired.
Creams are preferably formulated from a mixture of mineral oil,
self-emulsifying beeswax and water in which mixture the active
ingredient, dissolved in a small amount of an oil such as almond
oil, is admixed. A typical example of such a cream is one which
includes about 40 parts water, about 20 parts beeswax, about 40
parts mineral oil and about 1 part almond oil.
Ointments may be formulated by mixing a solution of the active
ingredient in a vegetable oil such as almond oil with warm soft
paraffin and allowing the mixture to cool. A typical example of
such an ointment is one which includes about 30% almond oil and
about 70% white soft paraffin by weight.
Lotions may be conveniently prepared by dissolving the active
ingredient, in a suitable high molecular weight alcohol such as
propylene glycol or polyethylene glycol.
The tissue healing compositions comprise emu oil
or a biologically active fraction thereof in admixture with an
active vitamin D compound or a PTH/PTHrP antagonist peptide, and
may include a conventional pharmaceutical carrier or excipient.
In addition, these compositions may include other medicinal agents,
growth factors, wound sealants, carriers, etc., that are known
or apparent to those skilled in the art. The tissue healing compositions
of the invention are administered to a warm-blooded animal, such
as human, already suffering from a wound, oxidative skin damage,
skin lesions or burns, in an amount sufficient to allow the healing
process to proceed more quickly than if the host were not treated.
Amounts effective for this use will depend on the severity of
the wound, sore or burn, and the general state of health of the
patient being treated. Maintenance dosages over a prolonged period
of time may be adjusted as necessary. For veterinary uses, higher
levels may be administered as necessary.
In the case of an animal suffering from decreased hair
growth, the compositions of the invention are administered in
an amount sufficient to increase the rate of hair
growth. Amounts effective for this use will depend on the extent
of decreased hair growth, and the general state
of health of the patient being treated. Maintenance dosages over
a prolonged period of time may be adjusted as necessary. For veterinary
uses, higher levels may be administered as necessary.
In the case of an animal suffering from hypopigmentation disorder(s),
the compositions of the invention are administered in an amount
sufficient to increase the pigmentation of affected skin tissue.
Amounts effective for this use will depend on the extent of hypopigmentation,
and the general state of health of the patient being treated.
Maintenance dosages over a prolonged period of time may be adjusted
as necessary.
For application as skin tanning enhancers, the compositions of
the invention are administered in an amount sufficient to enhance
the tanning of a subject's skin. The compositions can be applied
in conjunction with exposure to the sun, or artificial ultraviolet
radiation such as a suntanning bed, or the compositions can be
applied without subsequent exposure to the sun or tanning lights.
In either instance, the benefit of an enhanced skin tan will be
achieved.
Animals which may be treated according to the present invention
include all animals which may benefit therefrom. Such animals
include, but are not limited to, mammals such as humans.
The efficacy of emu oil in accordance with this
invention was determined by the following procedure. The following
example is illustrative, but not limiting, of the method and compositions
of the present invention. Other suitable modifications and adaptations
of the variety of conditions and parameters normally encountered
in clinical therapy and obvious to those skilled in the art are
in the spirit and scope of the invention.
EXAMPLE
Adolescent C57 BL/6 mice that were six to eight weeks old with
all of their hair follicles arrested in telogen
for several weeks were selected. The hair from the
back skin was removed by a wax/rosin mixture as previously described
by R. Paus et al., J. Invest. Dermatol. 103:143-147 (1994). After
depilation, three mice receive on the nape of the neck topically,
0.1 ml of emu oil and 2 mice received topically
0.1 ml of corn oil in a double-blinded fashion. For the next 19
days, the animals received a single topical application of either
emu oil or corn oil. On day 18, the animals received
.sup.3 H-thymidine intraperitoneally as previously described by
M. F. Holick et al., Proc. Natl. Acad. Sci. USA 91:8014-8016 (1994).
Twenty-four hours later the animals backs were photographed and
the animals were then sacrificed and the skin removed for analysis.
An evaluation of the incorporation of .sup.3 H-thymidine in the
epidermis revealed that the animals that were treated with emu
oil had a significant 29% increase in .sup.3 H-thymidine incorporation
when compared to the control animals that received a topical application
of corn oil (FIG. 1). The photograph of the animals just before
sacrifice demonstrated increased pigmentation and hair
over the upper back region of the three mice that received emu
oil compared to the two mice that received corn oil (FIG. 2).
A histologic evaluation confirmed the visual observation. There
was a more marked increase in the size and length of the hair
follicles and thickness of the skin in the mouse skin that was
treated with emu oil when compared to mouse skin
treated with corn oil.
It can be concluded from these studies that the topical application
of emu oil increased the synthesis of DNA in the
epidermis which is a measure of increase in the proliferative
activity of the epidermis. The increase in pigmentation and hair
in the photograph of animals receiving emu oil demonstrates
that the topical application of emu oil can stimulate
melanogenesis and hair follicle development and
growth. The histological analysis demonstrating an increase in
the thickness of the epidermis and size and length of the hair
follicle provides strong evidence that the topical application
of emu oil stimulates skin growth, hair
growth and induces the proliferation of the cells around the hair
follicle.
Having now fully described this invention it will be understood
to those of ordinary skill in the art that the same can be performed
within a wide and equivalent range of conditions, formulations,
and other parameters without affecting the scope of the invention
or any embodiment thereof. All patents and publications cited
herein are fully incorporated by reference herein in their entirety.
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