U.S. patent application number 10/376991 was filed with the patent office on 2003-08-28 for method of treating alopecia.
Invention is credited to Oh, Hye-Sun, Smart, Robert C..
Application Number | 20030162757 10/376991 |
Document ID | / |
Family ID | 24419644 |
Filed Date | 2003-08-28 |
United States Patent
Application |
20030162757 |
Kind Code |
A1 |
Smart, Robert C. ; et
al. |
August 28, 2003 |
Method of treating alopecia
Abstract
A method of enhancing hair growth or treating alopecia in a
subject uses topically administered estrogen receptor antagonists.
Pharmaceutical formulations comprising estrogen receptor
antagonists are described.
Inventors: |
Smart, Robert C.; (Raleigh,
NC) ; Oh, Hye-Sun; (Raleigh, NC) |
Correspondence
Address: |
MYERS BIGEL SIBLEY & SAJOVEC
PO BOX 37428
RALEIGH
NC
27627
US
|
Family ID: |
24419644 |
Appl. No.: |
10/376991 |
Filed: |
February 28, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10376991 |
Feb 28, 2003 |
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09697214 |
Oct 26, 2000 |
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6555532 |
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09697214 |
Oct 26, 2000 |
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09257396 |
Feb 25, 1999 |
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6204258 |
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09257396 |
Feb 25, 1999 |
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08604448 |
Feb 21, 1996 |
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5965551 |
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Current U.S.
Class: |
514/169 ;
514/176; 514/182 |
Current CPC
Class: |
A61K 8/41 20130101; A61P
17/14 20180101; A61K 31/565 20130101; A61K 31/4535 20130101; A61K
31/00 20130101; A61K 31/138 20130101; A61Q 7/00 20130101; A61K 8/46
20130101; A61K 8/42 20130101; A61K 31/4025 20130101; A61Q 7/02
20130101; A61K 8/63 20130101; Y10S 514/88 20130101; A61P 17/00
20180101; A61K 31/192 20130101 |
Class at
Publication: |
514/169 ;
514/176; 514/182 |
International
Class: |
A61K 031/56; A61K
031/58 |
Goverment Interests
[0002] This invention was made with Government support under grant
CA46637 from the National Cancer Institute and grant ES07046 from
the National Institute for Environmental Health Sciences. The
Government has certain rights to this invention.
Claims
That which is claimed is:
1. A method of treating alopecia in a subject in need thereof
comprising topically applying to the skin of said subject an
estrogen receptor antagonist or a pharmaceutically acceptable salt
thereof in an amount sufficient to treat alopecia.
2. A method according to claim 1, wherein said estrogen receptor
antagonist is a steroid
3. A method according to claim 1, wherein said subject is a female
subject.
4. A method according to claim 1, wherein said subject is a male
subject.
5. A method according to claim 1, wherein said estrogen receptor
antagonist is applied to the scalp of a subject.
6. A method according to claim 1, wherein said skin contains
telogen-phase hair follicles.
7. A method according to claim 1, wherein said estrogen receptor
antagonist is selected from the group consisting of
7.alpha.-alkylamide analogs and 11.beta.-alkylamide analogs of
estradiol.
8. A method according to claim 1, wherein said estrogen receptor
antagonist is selected from the group consisting of
7.alpha.-[9-(4,4,5,5,5,-pentafluoropentylsulfinyl)nonyl]estra-1,3,5(10)-t-
riene-3,17.beta.-diol,
N-n-butyl-N-methyl-11-(3,17.beta.-dihydroxyestra-1,-
3,5(10)-triene-7.alpha.-yl)undecanamide,
11.beta.-[4-[2-(dimethylaminoetho- xy]phenyl]-estradiol,
N-methyl-N-isopropyl-(3,17.beta.-dihydroxy-estra-1,2-
,5(10)-trien-11-.beta.-yl)-undecamide,
[6-hydroxy-2(4-hydroxyphenyl)benzo[- b]thien-3-yl]
[4-[3-(1-pyrrolidin-yl)ethoxy]phenyl]methanone,
3-[4-(1,2-Diphenylbut-1-enyl)phenyl]acrylic acid, tamoxifen,
clomiphene, and keoxifene.
9. A method according to claim 1, wherein said estrogen receptor
antagonist is a compound of Formula 1: 3wherein m is from 1 to 15;
n is from 1 to 5; R.sub.1 is hydrogen, lower alkyl, or halogen;
R.sub.2 is oxygen or hydroxyl; R.sub.3 is lower alkyl or haloalkyl;
and X is 4wherein R.sub.4 is loweralkyl, or a pharmaceutically
acceptable salt thereof.
10. A method according to claim 9, wherein m is 9; n is 3; R.sub.1
is hydrogen; R.sub.2 is hydroxyl; R.sub.3 is haloalkyl; and X is
--SO--.
11. A method according to claim 10, wherein R.sub.3 is
--CF.sub.2CF.sub.3.
12. A method according to claim 9, wherein m is 10; n is 3; R.sub.1
is hydrogen; R.sub.2 is hydroxyl; R.sub.3 is lower alkyl; X is
5R.sub.4 is methyl.
13. A method of promoting hair growth in a subject in need thereof
comprising topically applying to the skin of said subject a
compound selected from the group consisting of estrogen receptor
antagonists and pharmaceutically acceptable salts thereof in an
amount sufficient to promote hair growth.
14. A method according to claim 13, wherein said subject is
afflicted with hair loss, and said compound is administered in an
amount effective to treat hair loss.
15. A topical pharmaceutical formulation comprising an estrogen
receptor antagonist or a pharmaceutically acceptable salt thereof
in an amount effective to treat alopecia in a pharmaceutically
acceptable topical carrier.
16. A topical pharmaceutical formulation of claim 15, wherein said
estrogen receptor antagonist is a steroid.
17. A topical pharmaceutical formulation of claim 15, wherein said
estrogen receptor antagonist is
7.alpha.-[9-(4,4,5,5,5-pentafluoropentyls- ulfinyl)nonyl]estra-
1,3,5(10)-triene-3,17.beta.-diol.
18. A topical pharmaceutical formulation of claim 15, wherein said
formulation is a gel.
19. A topical pharmaceutical formulation of claim 15, wherein said
formulation is a spray.
Description
RELATED APPLICATION INFORMATION
[0001] This application is a continuation of prior co-pending U.S.
patent application Ser. No. 09/697,214, filed Oct. 26, 2000, which
in turn is a continuation of U.S. patent application Ser. No.
09/257,396, filed Feb. 25, 1999, now issued as U.S. Pat. No.
6,204,258, which in turn is a continuation of prior U.S. patent
application Ser. No. 08/604,448, filed Feb. 21, 1996, now issued as
U.S. Pat. No. 5,965,551, the disclosures of which are incorporated
by reference herein in their entirety.
FIELD OF THE INVENTION
[0003] This invention relates to methods of treating hair loss and
promoting hair growth, and more particularly to a method of using
estrogen receptor antagonists to promote hair growth in a subject
in need of such treatment.
BACKGROUND OF THE INVENTION
[0004] Alopecia (baldness) is a deficiency of hair, either normal
or abnormal, and is primarily a cosmetic problem in humans,
although the negative psychological impact of hair loss is well
known. See C. H. Mortimer et al., Clin. Exp. Dermatol. 9, 342-350
(1984). Dermatologists recognize many different types of alopecia,
with androgenic alopecia being the most common cause of hair loss
in both men and women. As this type of hair loss is more common and
more severe in males, it is typically referred to as "male pattern
baldness." However, it is thought that androgenic alopecia affects
more that one third of individuals of either sex who have a strong
family history of hair loss. See W. F. Bergfield, Clin. Dermatol.
6, 102-107 (1988).
[0005] One traditional treatment for alopecia is the method of hair
transplantation. Typically, this method involves transplanting
plugs of natural hair from areas of the scalp where hair is growing
to bald areas. This procedure is costly, time-consuming, painful,
and meets with only limited success.
[0006] Another common treatment for hair loss is the application of
a chemical or drug for the purpose of stimulating hair growth. For
example, U.S. Pat. No. 5,177,061 to Pickart proposes the topical
application of glycyl-L-histidyl-L-lycine:copper(II) (GHL-Cu) and
its derivatives to promote hair growth in warm-blooded animals
(applicant specifically intends the disclosure of this and all
other patent references cited herein to be incorporated herein by
reference in their entirety). U.S. Pat. No. 4,832,946 to Green
proposes a composition for topical application to mammalian hair or
skin, comprising an amount of the cell-free supernatant from a
culture of dermal papilla fibroblasts, which is said to increase
hair growth in the rat. U.S. Pat. No. 5,358,714 to Green proposes
the use of diacylglycerol activators of protein kinase C in order
to increase or maintain hair growth in mammals, while U.S. Pat. No.
5,068,315 to Buultjens et al. proposes the application of purified
hair growth regulating peptides (HGRP) to stimulate hair growth. It
has also been suggested that retinoids, substituted pyrimidines,
and immunosuppressants be used as possible treatments for hair
loss, although methods utilizing these compounds have not been
entirely successful in producing a reliable and safe method of
inducing hair growth. See G. Bazzano et al., J. Invest. Dermatol.
101 (1 Supplement), 138S-142S (1993); H. Jiang et al., J. Invest.
Dermatol. 104(4), 523-525 (1995).
[0007] In recent years, the topical application of minoxidil has
been a widely-used method for treating androgenic alopecia. See A.
R. Zapacosta, N. Eng. J. Med. 303, 1480-81 (1980). U.S. Pat. No.
4,139,619 to Chidsey, proposes atopical composition of minoxidil
and related iminopyrimidines to stimulate the conversion of vellus
hair to terminal hair and increase the rate of growth of terminal
hair. However, despite its popularity, minoxidil has not performed
in a completely satisfactory fashion in promoting hair growth in
all target populations.
SUMMARY OF THE INVENTION
[0008] A first aspect of the present invention is a method of
treating alopecia in a subject in need thereof. The method
comprises topically applying to the skin of the subject an estrogen
receptor antagonist or a pharmaceutically acceptable salt thereof
(an "active compound") in an amount sufficient to treat
alopecia.
[0009] A more general aspect of the present invention is a method
of promoting hair growth in a subject in need thereof. The method
comprises topically applying to the skin of the subject an active
compound as given above in an amount sufficient to promote hair
growth.
[0010] A second aspect of the present invention is a topical
pharmaceutical formulation comprising an active compound as given
above in an amount effective to treat alopecia or promote hair
growth, in a pharmaceutically acceptable topical carrier.
[0011] A third aspect of the present invention is the use of an
active compound as given above for the preparation of a medicament
useful for carrying out a method as given above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1A is a photomicrograph showing the immunohistochemical
localization of estrogen receptors in telogen hair follicle tissue
from mouse skin, where immunohistochemical staining was conducted
with hematoxylin counterstaining.
[0013] FIG. 1B is photomicrograph showing the immunohistochemical
localization of estrogen receptors in telogen hair follicle tissue
from mouse skin, where immunohistochemical staining was conducted
without hematoxylin counterstaining.
[0014] FIG. 1C is photomicrograph showing the immunohistochemical
localization of estrogen receptors in early-anagen hair follicle
tissue from mouse skin, where immunohistochemical staining was
conducted with hematoxylin counterstaining.
[0015] FIG. 1D is photomicrograph showing the immunohistochemical
localization of estrogen receptors in early-anagen hair follicle
tissue from mouse skin, where immunohistochemical staining was
conducted without counterstaining.
[0016] FIG. 2 is a graph illustrating that the estrogen receptor
antagonist ICI 182,780 causes hair growth during the second telogen
phase in mice. Closed triangles represent mice treated with 10 nmol
ICI 182,780 in 200 .mu.L acetone applied topically to an area of
clipped hair. Open circles represent control mice treated with 200
.mu.L acetone alone.
[0017] FIG. 3A is a photograph of a mouse treated with 200 .mu.L of
acetone applied topically to an area of clipped hair twice a
week.
[0018] FIG. 3B is a photograph of a mouse treated with 10 nmol ICI
182,780 in 200 .mu.L acetone applied topically to an area of
clipped hair twice a week.
[0019] FIG. 4A is a photomicrograph of skin tissue from a
six-week-old mouse treated with 200 .mu.L of acetone applied
topically to an area of clipped hair twice a week until seven weeks
of age. The tissue was stained with hematoxylin/eosin.
[0020] FIG. 4B is a photomicrograph of skin tissue from a
six-week-old mouse treated with 10 nmol ICI 182,780 in 200 .mu.L
acetone applied topically to an area of clipped hair twice a week
until seven weeks of age. The tissue was stained with
hematoxylin/cosin.
[0021] FIG. 4C is a photomicrograph of skin tissue from a
six-week-old mouse treated with 200 .mu.L of acetone applied
topically to an area of clipped hair twice a week until eight weeks
of age. The tissue was stained with hematoxylin/cosin.
[0022] FIG. 4D is a photomicrograph of skin tissue from a
six-week-old mouse treated with 10 nmol ICI 182,780 in 200 .mu.L
acetone applied topically to an area of clipped hair until eight
weeks of age. The tissue was stained with hematoxylin/eosin.
[0023] FIG. 5 is a graph illustrating that topical
17.beta.-estradiol blocks hair regrowth in mice. Six week old mice
(5 mice per group) were clipped and treated with topical
applications of either 10 nmol 17.beta.-estradiol in 200 .mu.L
acetone (closed circles) or acetone alone (open circles), twice a
week, up to sixteen weeks of age.
[0024] FIG. 6A is a photomicrograph of skin tissue from a mouse,
where 200 .mu.L of acetone was applied topically to shaved skin
twice a week for three weeks. Tissue is stained with
hematoxylin/eosin.
[0025] FIG. 6B is a photomicrograph of skin tissue from a mouse,
where 10 nmol 17.beta.-estradiol in 200 .mu.L acetone was applied
topically to shaved skin twice a week for three weeks. Tissue is
stained with hematoxylin/eosin.
[0026] FIG. 6C is a photomicrograph of skin tissue from a mouse,
where 200 .mu.L of acetone was applied topically to shaved skin
twice a week for five weeks. Tissue is stained with
hematoxylin/eosin.
[0027] FIG. 6D is a photomicrograph of skin tissue from a mouse,
where 10 nmol 17.beta.-estradiol in 200 .mu.L acetone was applied
topically to shaved skin twice a week for five weeks. Tissue is
stained with hematoxylin/eosin.
[0028] FIG. 6E is a photomicrograph of skin tissue from a mouse,
where 200 .mu.L of acetone was applied topically to shaved skin
twice a week for seven weeks. Tissue is stained with
hematoxylin/eosin.
[0029] FIG. 6F is a photomicrograph of skin tissue from a mouse,
where 10 nmol 17.beta.-estradiol in 200 .mu.L acetone was applied
topically to shaved skin twice a week for seven weeks. Tissue is
stained with hematoxylin/eosin.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The method of the present invention is useful in the
treatment of alopecia in mammals, and as such may be used to
promote, increase, or assist in the growth of hair. Subjects may be
male or female. As used herein, the term alopecia refers to the
complete absence of hair in skin which typically exhibits hair
growth, as well as a loss or diminution in the amount of hair.
Multiple types and causes of alopecia are recognized in humans,
including male pattern baldness, chemotherapy induced hair loss,
congenital alopecia, and alopecia areata. As used herein, the term
`treating alopecia` refers to both the treatment of skin with a
total absence of hair growth as well as treatment of skin having
reduced or patchy hair growth.
[0031] The present invention is concerned primarily with the
treatment of human subjects but may also be employed for the
treatment of other mammalian subjects, such as dogs, cats, and
sheep, for veterinary purposes (e.g., in the treatment of hair loss
due to mange or other causes, or for enhancing wool or pelt
production).
[0032] Hair, or pili, are fine threadlike appendages of the skin
which normally cover the entire body (with the exception of the
palms of the hands and soles of the feet, and the flexor surfaces
of joints). A hair comprises a root embedded in the hair follicle
and a free portion (the stem or shaft). As used herein, hair refers
to mature hair as well as the soft, downy hair known as vellus
hair.
[0033] The hair bulb, or follicle, is a compact structure located
in the dermis layer of the skin and is composed of three main
cellular groups. The first comprises a compact group of fibroblasts
known as the dermal papilla which includes a capillary system. The
second group comprises germinative epithelial cells of the hair
bulb which proliferate and differentiate to give rise to the mature
hair shaft. The third group of fibroblasts exists around the
outside of the bulb in the connective tissue sheath.
[0034] The present invention is, in general, carried out with
estrogen receptor antagonists. The term "antagonist," as used
herein, refers to compounds that act as complete or partial
inhibitors of the activity of a biological substance by
specifically binding to the receptor thereof. Numerous estrogen
receptor antagonists are known in the art. It is specifically
intended that both full and partial estrogen receptor antagonists
are embraced by the scope of the invention, although full estrogen
receptor antagonists are preferred.
[0035] Estrogen receptor antagonists may be steroids or non-steroid
compounds. In one preferred embodiment of the invention, the
estrogen receptor antagonist is a steroid. Particularly preferred
are 7.alpha.-alkylamide analogs of estradiol, 11.beta.-alkylamide
analogs of estradiol and 16.alpha.-halo-17.beta.-estradiols that
have pure antiestrogenic activity.
[0036] In a preferred embodiment of the invention, the estrogen
receptor antagonist is a compound of Formula I: 1
[0037] wherein m is from 1 to 15;
[0038] n is from 1 to 5;
[0039] R.sub.1 is hydrogen, lower alkyl, or halogen;
[0040] R.sub.2 is oxygen or hydroxyl;
[0041] R.sub.3 is lower alkyl or haloalkyl; and
[0042] X is 2
[0043] wherein R.sub.4 is loweralkyl.
[0044] Estrogen receptor antagonists suitable for use in the
present invention include, but are not limited to,
7.alpha.-[9-(4,4,5,5,5,-pentaf-
luoropentylsulfinyl)nonyl]estra-1,3,5(10)-triene-3,17.beta.-diol
(also known as ICI 182,780),
N-n-butyl-N-methyl-11-(3,17.beta.-dihydroxyestra-1-
,3,5(10)-triene-7.alpha.-yl)undecanamide (also known as ICI
164,384), 11.beta.-[4-[2-(dimethylaminoethoxy]phenyl]-estradiol
(also known as RU 39411),
N-methyl-N-isopropyl-(3,17.beta.-dihydroxy-estra-1,2,5(10)-trien--
11-.beta.-yl)-undecamide (RU 51625), its 17.alpha.-ethynyl
derivative (RU 53637),
[6-hydroxy-2(4-hydroxyphenyl)benzo[b]thien-3-yl]
[4-[3-(1-pyrrolidin-yl)ethoxy]phenyl]methanone (LY117018),
tamoxifen, clomiphene, keoxifene,
3-[4-(1,2-Diphenylbut-1-enyl)phenyl]acrylic acid, and the like,
including the active metabolites of these compounds. These
compounds may be made in accordance with known procedures which
will be apparent to those skilled in the art. See, e.g., A. E.
Wakeling et al., Canc. Res. 51, 3867-3873 (1991); D. Poirier et
al., J. Med. Chem. 37, 1115-1125 (1994); J. Bowler et al., Steroids
54, 71-79 (1989); C. Levesque et al., J. Med. Chem. 34, 1624-1630
(1991); A. Claussner et al., J. Steroid. Biochem. Mol. Biol. 41,
609-614 (1992).
[0045] The active compounds described herein can, as noted above,
be prepared in the form of their pharmaceutically acceptable salts.
Pharmaceutically acceptable salts are salts that retain the desired
biological activity of the parent compound and do not impart
undesired toxicological effects. Examples of such salts include (a)
acid addition salts formed with inorganic acids, for example
hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric
acid, nitric acid and the like; and salts formed with organic acids
such as, for example, acetic acid, oxalic acid, tartaric acid,
succinic acid, maleic acid, fumaric acid, gluconic acid, citric
acid, malic acid, ascorbic acid, benzoic acid, tannic acid,
palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic
acid, methanesulfonic acid, p-toluenesulfonic acid,
naphthalenedisulfonic acid, polygalacturonic acid, and the like;
(b) salts formed from elemental anions such as chlorine, bromine,
and iodine; and (c) salts derived from bases, such as ammonium
salts; alkali metal salts such as those of sodium and potassium;
alkaline earth metal salts such as those of calcium and magnesium;
and salts with organic bases such as dicyclohexylamine and
N-methyl-D-glucamine.
[0046] The dosage of the active compound is not particularly
critical so long as it achieves the desired effect. Actual dosage
will depend on factors such as the particular pharmaceutical
carrier, the particular skin region and condition being treated,
and the general health and condition of the subject. In one
embodiment of the invention, the active compound is topically
administered to the affected skin of a subject in an amount
sufficient to achieve a dose of at least 0.01 nanomoles (nmol), 0.1
nmol, or 1 nmol per 2 cm by 4.5 cm skin surface area, up to a dose
of 100 nmol, 1,000 nmol, or 10,000 nmol or more per 2 cm by 4.5 cm
skin surface area.
[0047] Depending on the solubility of the particular formulation of
active compound administered, the daily dose may be divided among
one or several unit dose administrations. The dose may be a single
unit dose, which may, for example, be administered several times a
week or from 1 to 3 times a day. Treatments may continue week to
week on a chronic basis as necessary (i.e., the active agent can be
administered chronically). Administration of the active compounds
may be carried out therapeutically or prophylactically, but
preferably the compounds are administered therapeutically, either
before substantial hair loss has occurred, or at a time when such
hair loss has just begun.
[0048] Pharmaceutical compositions for use in the present method
include those suitable for topical administration. The compositions
may conveniently be presented in unit dosage form and may be
prepared by any of the methods well known in the art. The most
suitable route of administration in any given case may depend upon
the anatomic location of the alopecia in the subject, the nature
and severity of the condition being treated, and the particular
active compound which is being used.
[0049] Pharmaceutical compositions useful in the present methods
are preferably applied topically at the site of the affected skin.
In alternative embodiments of the invention, pharmaceutical
compositions useful in the present invention may be administered to
a subject orally, parenterally (e.g., subcutaneously,
intravenously, or intramuscularly), or transdermally (e.g., at a
site other than the affected area to achieve systemic
administration).
[0050] In a further aspect of the present invention, estrogen
receptor antagonists may be used alone or in combination with one
or more anti-alopecics for the prophylaxis or treatment of
alopecia.
[0051] In the manufacture of a medicament according to the
invention (a "formulation"), active agents or the physiologically
acceptable salts thereof (the "active compound") are typically
admixed with, inter alia, an acceptable carrier. The carrier must,
of course, be acceptable in the sense of being compatible with any
other ingredients in the formulation and must not be deleterious to
the patient. The carrier may be a solid or a liquid, or both, and
is preferably formulated with the compound as a unit-dose
formulation, for example, an aliquot of ointment which may contain
from 0.001% to 99% by weight of the active compound. One or more
active compounds may be incorporated in the formulations of the
invention (e.g., the formulation may contain one or more additional
anti-alopecia agents as noted above), which formulations may be
prepared by any of the well known techniques of pharmacy consisting
essentially of admixing the components, optionally including one or
more accessory therapeutic ingredients.
[0052] Formulations suitable for topical application to the skin
preferably take the form of an ointment, cream, lotion, paste, gel,
spray, aerosol, or oil. Carriers which may be used include
vaseline, lanoline, polyethylene glycols, alcohols, transdermal
enhancers, and combinations of two or more thereof.
[0053] Additionally, the compounds of the present invention may be
administered by a liposome delivery system, as detailed in U.S.
Pat. No. 4,828,837 to Uster et al.
[0054] The compounds useful in the method of the present invention
may be applied the surface of the skin at any anatomical location
in need of treatment on the subject, including the scalp, pubis,
face, chest, and legs. Administration of the compounds of the
present invention onto the scalp of the subject is a preferred
embodiment of the invention.
[0055] Formulations suitable for oral administration may be
presented in discrete units, such as capsules, cachets, lozenges,
or tablets, each containing a predetermined amount of the active
compound; as a powder or granules; as a solution or a suspension in
an aqueous or non-aqueous liquid; or as an oil-in-water or
water-in-oil emulsion. Such formulations may be prepared by any
suitable method of pharmacy which includes the step of bringing
into association the active compound and a suitable carrier (which
may contain one or more accessory ingredients as noted above). In
general, the formulations of the invention are prepared by
uniformly and intimately admixing the active compound with a liquid
or finely divided solid carrier, or both, and then, if necessary,
shaping the resulting mixture. For example, a tablet may be
prepared by compressing or molding a powder or granules containing
the active compound, optionally with one or more accessory
ingredients. Compressed tablets may be prepared by compressing, in
a suitable machine, the compound in a free-flowing form, such as a
powder or granules optionally mixed with a binder, lubricant, inert
diluent, and/or surface active/dispersing agent(s). Molded tablets
may be made by molding, in a suitable machine, the powdered
compound moistened with an inert liquid binder. Formulations for
oral administration may optionally include enteric coatings known
in the art to prevent degradation of the formulation in the stomach
and provide release of the drug in the small intestine.
[0056] Formulations of the present invention suitable for
parenteral administration comprise sterile aqueous and non-aqueous
injection solutions of the active compound, which preparations are
preferably isotonic with the blood of the intended recipient. These
preparations may contain anti-oxidants, buffers, bacteriostats and
solutes which render the formulation isotonic with the blood of the
intended recipient. Aqueous and non-aqueous sterile suspensions may
include suspending agents and thickening agents. The formulations
may be presented in unit dose or multi-dose containers, for example
sealed ampoules and vials, and may be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid carrier, for example, saline or water-for-injection
immediately prior to use. Extemporaneous injection solutions and
suspensions may be prepared from sterile powders, granules and
tablets of the kind previously described.
[0057] Formulations suitable for transdermal administration (either
at the site in need of treatment or at another site to achieve
systemic administration) may be presented as discrete patches
adapted to remain in intimate contact with the epidermis of the
recipient for a prolonged period of time. Formulations suitable for
transdermal administration may also be delivered by electrophoresis
or iontophoresis (see, e.g., Pharmaceutical Research 3, 318 (1986))
and typically take the form of an optionally buffered aqueous
solution of the active compound.
[0058] Although applicants do not wish to be bound to any
particular theory of the instant invention, it appears that
estrogen receptor antagonists cause telogen-phase hair follicles
(resting follicles) to enter the anagen phase (active follicle)
cycle, which results in the production of hair fiber and hair
growth. Normally, approximately 90% of scalp hairs are in the
anagen phase, with less than 1% in an intermediate catogen phase
and the rest in telogen. With the onset of patterned baldness,
however, a successively greater proportion of hairs are in telogen,
while correspondingly fewer are found in the anagen phase.
[0059] The following examples are provided to more hilly illustrate
the present invention and should not be construed as limiting
thereof. As used in the following examples, 17-.beta. Estradiol was
purchased from Sigma Chemical Company (St. Louis, Mo., USA); ICI
182,780 was a gift from Zeneca Pharmaceuticals (Cheshire, England);
link antibody, label antibody and 3,3'-diaminobenzidine
tetrahydrochloride/Super Sensitive Multi-link Immunostaining Kit
were purchased from Biogenex Laboratorie (San Ramon, Calif., USA);
10X Automation Buffer was purchased from Biomeda Corporation,
(Foster City, Calif., USA).
EXAMPLE 1
Animals and Estrogen or Estrogen Receptor Antagonist Treatment
[0060] Female CD-1 mice, 4, 5, or 9 weeks of age, were purchased
from Charles River Laboratories, Raleigh, N.C. Mice were kept in an
animal facility at least 1 week prior to use and were fed rodent
chow (Agway Food, Granville Milling and Co., Creedmoor, N.C.) and
water ad libitum. The mice were kept on corn cob bedding and placed
on a 12 hour light/dark cycle. Female CD-I mice were clipped on the
dorsal region (approximately 4.times.2.5 cm area) with electric
clippers. The mice were then treated twice weekly on the clipped
dorsal surface with either (a) 10 nmol 17-.beta.-estradiol in 200
.mu.l acetone or (b) acetone alone. For the studies with the
estrogen receptor antagonist, ICI 182,780 was dissolved in acetone
and 200 .mu.l was topically applied twice weekly.
EXAMPLE 2
Localization of Estrogen Receptor in Mouse Skin
[0061] Seven-week-old female CD-1 mice were killed by cervical
dislocation and the dorsal skin area was excised. The skins were
fixed for 24 hours in a cold 10% neutral buffered formalin, then
changed to cold 70% ethanol and processed and embedded in paraffin.
Tissue sections were cut at 5 .mu.m and placed on SuperFrost Plus
slides for immunohistochemistry. In order to localize the estrogen
reception in skin, the paraffin sections were deparaffinized by two
changes of xylene and rehydrated in a graded series of ethanol
(100, 95 and 70%) followed by 1.times. automation buffer. The skin
sections were placed in 3% H.sub.2O.sub.2 for 10 min to quench the
endogenous peroxidase activity and then washed with 1.times.
automation buffer. The sections were treated with fresh trypsin of
0.15 mg/ml in 1.times. automation buffer for 4 min at room
temperature followed by 1.times. automation buffer twice and
incubated with fresh DNAase of 0.25 mg/ml in 1.times. automation
buffer for 3 min at room temperature followed by 1.times.
automation buffer twice. The sections were blocked with 10% normal
goat serum in 1.times. automation buffer for 15 min at room
temperature. After blocking, the excessive solution was drained,
and a prediluted primary antibody (Abbott ER-ICA monoclonal
antibody, Abbott Laboratories, North Chicago, Ill.) was applied and
incubated overnight at 4.quadrature.C. Slides were washed in
1.times. automation buffer for 5 min twice and a secondary antibody
(biotinylated goat anti-rat IgG, Boehringer Mannheim Corporation,
Indianapolis, Ind.) at a dilution of 1:50 in 1.times. automation
buffer was applied for 60 min at room temperature. After washing
with 1.times. automation buffer for 5 min twice, the sections were
incubated with peroxidase (HRP)-conjugated streptavidin (1:20
dilution, BioGenex, San Ramon, Fla.) for 30 min at room temperature
and washed with 1.times. automation buffer for 5 min and 0.05M
Tris-HCl (pH 7.5) for 5 min. The sections were then incubated with
DAB (prepared according to the manufacturer's instruction) for 10
min in a dark box. Slides were rinsed with 0.05M Tris-HCl buffer
twice and counterstained in hematoxylin for 5 seconds followed by
rinsing in distilled water five times. Finally, the sections were
dehydrated in a graded series of ethanol and xylene, and then
slides were permanently mounted with Permount.
[0062] Immunohistochemical staining for the estrogen receptor in
mouse skin revealed intense and specific staining of the nuclei of
cells with the dermal papilla of a telogen follicle, as shown in
the counterstained sample FIG. 1A. Non-counterstained samples (FIG.
1B) are provided to better demonstrate the areas and levels of
estrogen receptor expression. Estrogen receptor expression within
most telogen dermal papilla demonstrated a polarity, as nuclei of
cells within the lower half of the dermal papilli stained
intensely, while very little staining was observed in the upper
half of dermal papilla. In addition, the expression of the estrogen
receptor was hair cycle dependent as there was a weaker staining of
the dermal papilla of mid to late catagen or anagen follicles
(FIGS. 1C and 1D), and no detectable staining in dermal papilla of
mid to late anagen follicles (data not shown). Very light estrogen
receptor staining was observed in cells of the outer root sheath in
the isthmus of the telogen follicle as well as in some nuclei of
dermal fibroblasts.
EXAMPLE 3
Estrogen Receptor Antagonist ICI 182,780 Induces Hair Growth
[0063] Mice were treated with the estrogen receptor antagonist ICI
182,780 as provided in Example 1. Twice weekly treatment of clipped
dorsal skin was begun when the mice were 6 weeks of age, with
either (a) 10 nmol ICI 182,780 in acetone or (b) acetone alone. As
shown in FIG. 2 and FIG. 3, by 9 weeks of age, 60% of the ICI
182,780 treated mice demonstrated full hair regrowth while no hair
growth was observed in the acetone treated mice. By 10 weeks of age
all of the ICI 182,780 treated mice developed a full coat of hair
while only 40% of the acetone-treated mice demonstrated full hair
regrowth.
[0064] These data indicate that ICI 182,780 caused the telogen
follicle to enter anagen during what should have been the second
synchronized telogen phase. The confirm this result, skin was
collected and prepared for histological analysis at 7, 8, 9, 10 and
11 weeks. At 7 weeks of age the ICI 182,780 treated mice
demonstrated follicles that were already in early to mid anagen,
while the follicles of the acetone-treated mice were synchronized
in second telogen (FIGS. 4A and 4B). By eight weeks of age the
follicles of the ICI 182,780 treated mice demonstrated follicles
that were in mid to late anagen while the hair follicles in
acetone-treated mice were in telogen (FIGS. 4C and 4D). These
results indicate that the estrogen receptor antagonist ICI 182,780
initiates the transition of a telogen follicle into anagen. At
eleven weeks of age, the follicles of mice treated with ICI 182,780
entered telogen, while control mice were in mid to late anagen
(data not shown), indicating that ICI 182,780 does not prolong
anagen.
EXAMPLE 4
17-.beta. Estradiol Inhibits Hair Growth
[0065] In order to determine if 17-.beta.-estradiol could influence
hair growth, six week old CD-1 female mice were clipped on the
dorsal Surface with electric clippers and then treated twice weekly
from the 6th week of age to the 16th week of age with topical
applications of either (a) 10 nmol 17-.beta.-estradiol or (b)
acetone vehicle alone. Treatment with 17-.beta.-estradiol had a
potent inhibitory effect on dorsal hair regrowth. As shown in FIG.
5, 100% of the acetone-treated mice demonstrated full hair regrowth
by 13 weeks of age. In contrast, mice treated with
17-.beta.-estradiol did not demonstrate any full hair regrowth by
16 weeks of age. Some of the 17-.beta.-estradiol-treated mice did
demonstrate a partial patchy hair regrowth that involved less than
10% of the total clipped area. At 12 weeks of age, 20% of the
17-.beta.-estradiol-treated mice demonstrated partial patchy hair
regrowth and by 15 weeks 40% of the 17-.beta.-estradiol-treated
mice demonstrated such partial patchy hair regrowth (data not
shown).
[0066] The above results demonstrate that 17-.beta.-estradiol
treatment just before and during the 2nd telogen phase potently
blocked hair growth. In order to determine if 17-.beta.-estradiol
could block hair growth in pre-existing anagen follicles, 10 week
old mice in the anagen phase of the hair cycle were treated twice
weekly with 17-.beta.-estradiol applied topically to clipped dorsal
skin. Within two weeks it was apparent that the
17-.beta.-estradiol-treated mice were no longer growing hair while
all the acetone mice developed a full coat of hair (data not
shown). Treatment with 17-.beta.-estradiol was continued for 10
weeks and during this period no hair growth was apparent. The
results of this experiment demonstrate that 17-.beta.-estradiol can
also block hair growth in mouse skin treated during the 3rd
synchronous anagen phase.
EXAMPLE 6
Effect of 17-.beta.-estradiol on the Hair Follicle Cycle
[0067] To determine the effect of 17-.beta.-estradiol on the hair
follicle cycle, mice were treated with 17-.beta.-estradiol or
acetone alone twice weekly from the 4th week of age to the 15th
week of age. Every week, histological analysis of the skin from 3
mice from each group was conducted to determine the phase of the
hair cycle. Representative skin histology sections from acetone-
and 17-.beta.-estradiol-treated mice at 7, 9, and 11 weeks of age
are shown in FIGS. 6A-6F. As expected, at 7 weeks of age all
follicles were in telogen in both the 17-.beta.-estradiol and the
acetone-treated mice. By 9 weeks of age, the hair follicles of the
acetone control mice entered anagen while the hair follicles of the
17-.beta.-estradiol mice remained in telogen. By 11 weeks of age,
the 20 hair follicles of the acetone treated mice were all in mid
to late anagen while the hair follicles of the
17-.beta.-estradiol-treated mice remained in telogen. At the
termination of the experiment, when the mice were 15 weeks of age,
the hair follicles of the 17-.beta.-estradiol-treated mice were
still arrested in telogen (data not shown). Collectively these data
demonstrate that the topical application of 17-.beta.-estradiol
prevents hair regrowth by arresting the hair follicle in the
telogen phase of the hair cycle.
[0068] The foregoing examples are illustrative of the present
invention, and are not to be construed as limiting thereof. The
invention is defined by the following claims, with equivalents of
the claims to be included therein.
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