| 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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