U.S. patent application number 08/896196 was filed with the patent office on 2002-05-30 for compositions and method for stimulating hair growth.
Invention is credited to NIELSEN, THOR B., SUN, LIYING.
Application Number | 20020065314 08/896196 |
Document ID | / |
Family ID | 23726553 |
Filed Date | 2002-05-30 |
United States Patent
Application |
20020065314 |
Kind Code |
A1 |
NIELSEN, THOR B. ; et
al. |
May 30, 2002 |
COMPOSITIONS AND METHOD FOR STIMULATING HAIR GROWTH
Abstract
A method for regulation of hair growth in an adult mammal, in
which a trichogenic composition is applied to the skin. Also
disclosed are methods for inducing skin differentiation and
stimulating hair growth, wherein a formulation of a trichogenic
composition is applied to the skin.
Inventors: |
NIELSEN, THOR B.;
(ROCKVILLE, MD) ; SUN, LIYING; (GAITHERSBURG,
MD) |
Correspondence
Address: |
FLESHNER & KIM
PO BOX 221200
CHANTILLY
VA
201531200
|
Family ID: |
23726553 |
Appl. No.: |
08/896196 |
Filed: |
July 17, 1997 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
08896196 |
Jul 17, 1997 |
|
|
|
08434994 |
May 4, 1995 |
|
|
|
Current U.S.
Class: |
514/526 |
Current CPC
Class: |
A61K 31/277 20130101;
A61K 31/275 20130101 |
Class at
Publication: |
514/526 |
International
Class: |
A61K 031/275; A01N
037/34 |
Claims
We claim:
1. A method for promoting hair growth in skin of a mammal, inducing
hair follicle differentiation in said skin, inducing a shift in
hair growth cycle from telogen to anagen in said skin, increasing
the rate of hair shaft elongation in said skin, or improving the
skin of a mammal, comprising the step of: applying to the skin of
said mammal a composition which comprises: i) from 0.0001 to 99% by
weight of a cyanocarboxylic acid derivative; and ii) from 1 to
99.999% by weight of a vehicle for component i).
2. The method according to claim 1, wherein said cyanocarboxylic
acid derivative has the formula: 6wherein R is an ester, an amide,
an alkyl amide, a dialkyl amide, an-alkoxyalkylamide, an anhydride,
a halide, a nitrile or an amino group; and R.sup.2 is
C.sub.1-C.sub.10 alkyl, cycloalkyl, alkenyl, alkoxyalkenyl,
alkynyl, aryl, alkaryl or aralkyl.
3. The method according to claim 1, wherein said cyanocarboxylic
acid derivative has the formula: 7wherein R is an ester of
C.sub.1-C.sub.20 alkyl, cycloalkyl, alkenyl, alkynyl, aryl,
alkaryl, aralkyl, hydroxyalkyl, or mono- or poly- alkoxyalkyl, an
alkyl amide of C.sub.1-C.sub.20, a dialkyl amide of
C.sub.1-C.sub.20 an alkoxyalkylamide, an anhydride of
C.sub.1-C.sub.20, a halide, a nitrile, or an amino group; and
R.sup.2 is C.sub.1-C.sub.10 alkyl, cycloalkyl, alkenyl,
alkoxyalkenyl, alkynyl, aryl, alkaryl, aralkyl, or H.
4. The method according to claim 1, wherein said cyanocarboxylic
acid derivative has the formula: 8wherein R is C.sub.1-C.sub.20
alkyl, cycloalkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl, or
mono- or poly- alkoxyalkyl; and R.sup.2 is C.sub.1-C.sub.10 alkyl,
cycloalkyl, alkenyl, alkoxyalkenyl, alkynyl, aryl, alkaryl,
aralkyl, or H.
5. The method according to claim 1, wherein said cyanocarboxylic
acid derivative has the formula: 9
6. The method according to claim 1, wherein said cyanocarboxylic
acid derivative has the formula: 10
7. The method according to claim 1, wherein said applying step
comprises applying a trichogenically effective amount of said
cyanocarboxylic acid derivative to said skin.
8. The method of claim 2, wherein said applying step comprises
applying a trichogenically effective amount of said cyanocarboxylic
acid derivative to said skin.
9. The method of claim 3, wherein said applying step comprises
applying a trichogenically effective amount of said cyanocarboxylic
acid derivative to said skin.
10. The method of claim 4, wherein said applying step comprises
applying a trichogenically effective amount of said cyanocarboxylic
acid derivative to said skin.
11. The method of claim 5, wherein said applying step comprises
applying a trichogenically effective amount of said cyanocarboxylic
acid derivative to said skin.
12. The method of claim 6, wherein said applying step comprises
applying a trichogenically effective amount of said cyanocarboxylic
acid derivative to said skin.
13. The method of claim 1, wherein said applying step comprises
applying a physiologically effective composition to said skin.
14. The method of claim 2, wherein said applying step comprises
applying a physiologically effective composition to said skin.
15. The method according to claim 1, wherein said cyanocarboxylic
acid derivative is selected from: ethoxyethyl 2-cyanoacrylate
butoxyethyl 2-cyanoacrylate n-butyl 2-cyanoacrylate isobutyl
2-cyanoacrylate n-propyl 2-cyanoacrylate isopropyl 2-cyanoacrylate
n-hexyl 2-cyanoacrylate isohexyl 2-cyanoacrylate cyclohexyl
2-cyanoacrylate benzyl 2-cyanoacrylate glycerol 2-cyanoacrylate
ethoxybutyl 2-cyanoacrylate n-pentyl 2-cyanoacrylate isopentyl
2-cyanoacrylate n-heptyl 2-cyanoacrylate isoheptyl 2-cyanoacrylate
n-octyl 2-cyanoacrylate isooctyl 2-cyanoacrylate n-nonyl
2-cyanoacrylate isononyl 2-cyanoacrylate n-decyl 2-cyanoacrylate
isodecyl 2-cyanoacrylate n-butyl 2-cyano-3-methoxyacrylate isobutyl
2-cyano-3-methoxyacrylate n-butyl 2-cyano-3-phenylacrylate isobutyl
2-cyano-3-phenylacrylate n-butyl-2-cyano-2-butenoate
isobutyl-2-cyano-2-butenoate n-butyl-2-cyano-2-pentenoate
isobutyl-2-cyano-2-pentenoate n-butyl-2-cyano-2-hexenoate
isobutyl-2-cyano-2-hexenoate n-butyl-2-cyano-2-heptenoate
isobutyl-2-cyano-2-heptenoate n-butyl-2-cyano-2-octenoate
isobutyl-2-cyano-2-octenoate n-butyl-2-cyano-2-nonenoate
isobutyl-2-cyano-2-nonenoate n-butyl-2-cyano-2-decenoate
isobutyl-2-cyano-2-decenoate N-propyl-2-cyanoacrylamide
N-butyl-2-cyanoacrylamide N-pentyl-2-cyanoacrylamide
N-hexyl-2-cyanoacrylamide N-heptyl-2-cyanoacrylamide
N-octyl-2-cyanoacrylamide N-nonyl-2-cyanoacrylamide
N-decyl-2-cyanoacrylamide N-benzyl-2-cyanoacrylamide
N-cyclohexyl-2-cyanoacrylamide N-ethoxyethyl-2-cyanoacrylamide
N-ethoxypropyl-2-cyanoacrylamide N-ethoxybutyl-2-cyanoacrylamide
N-ethoxypentyl-2-cyanoacrylamide N-ethoxyhexyl-2-cyanoacrylamide
N-ethoxyheptyl-2-cyanoacrylamide N-ethoxyoctyl-2-cyanoacrylamide
N-ethoxynonyl-2-cyanoacrylamide N-ethoxydecyl-2-cyanoacrylamide
N-propoxyethyl-2-cyanoacrylamide N-propoxypropyl-2-cyanoacrylamide
N-propoxybutyl-2-cyanoacrylamide N-propoxypentyl-2-cyanoacrylamide
N-propoxyhexyl-2-cyanoacrylamide N-propoxyheptyl-2-cyanoacrylamide
N-propoxyoctyl-2-cyanoacrylamide N-propoxynonyl-2-cyanoacrylamide
N-propoxydecyl-2-cyanoacrylamide N-butoxyethyl-2-cyanoacrylamide
N-butoxypropyl-2-cyanoacrylamide N-butoxybutyl-2-cyanoacrylamide
N-butoxypentyl-2-cyanoacrylamide N-butoxyhexyl-2-cyanoacrylamide
N-butoxyheptyl-2-cyanoacrylamide N-butoxyoctyl-2-cyanoacrylamide
N-butoxynonyl-2-cyanoacrylamide
N-butoxydecyl-2-cyanoacrylamide.
16. A composition for promoting hair growth in the skin of a
mammal, inducing hair follicle differentiation in said skin,
inducing a shift in hair growth cycle from telogen to anagen in
said skin, increasing the rate of hair shaft elongation in said
skin, or improving the skin of a mammal, consisting essentially of:
11
17. The method of claim 1, wherein said applying step comprises
applying one of n-butyl cyanoacrylate and isobutyl cyanoacrylate to
said skin.
18. The method of claim 1, wherein said applying step comprises one
of: topically applying said composition to the skin, applying said
composition via a dermal patch, applying said composition via
intradermal injection, and subcutaneously injecting said
composition.
19. The method of claim 17, wherein said applying step comprises
one of: topically applying said one of n-butyl cyanoacrylate and
isobutyl cyanoacrylate to the skin, applying said composition via a
dermal patch, applying said composition via intradermal injection,
and subcutaneously injecting said composition.
20. The method of claim 1, wherein said applying step comprises
applying said composition to the skin of a human.
21. The method of claim 1, wherein said applying step comprises
applying said composition to the skin of a mammal, wherein said
skin is no longer attached to said mammal.
22. The method of claim 1, wherein said applying step comprises
applying said composition to the skin of a sheep.
23. The method of claim 1, wherein said applying step comprises
applying said composition to the skin of a mouse.
24. The method according to claim 1, wherein said cyanocarboxylic
acid derivative is at least one of the following formulas: 12
25. The method of claim 24, wherein said applying step comprises
applying said composition to the skin of a human.
26. The method of claim 24, wherein said applying step comprises
applying said composition to the skin of a sheep.
27. The method of claim 1, wherein said applying step comprises
applying said composition containing at least 25% of said component
i) to said skin.
28. The method of claim 24, wherein said applying step comprises
applying said composition containing at least 25% of said component
i) to said skin.
29. The method of claim 24, wherein said applying step comprises
applying said composition containing at least 50% of said component
i) to said skin.
30. The method of claim 1, wherein said applying step comprises:
topically spraying said composition to the skin.
31. The method of claim 1, wherein said applying step comprises
applying said composition to the skin of an animal from which fur
is to be obtained.
32. The method of claim 24, wherein said applying step comprises
applying said composition to the skin of an animal from which fur
is to be obtained.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates generally to the stimulation of hair
growth in animals. In particular, this invention relates to the
stimulation of hair growth in mammals by the application of a
trichogenic formulation.
[0003] This invention also relates to a method for stimulating hair
growth in mammals, involving the application of a trichogenic
formulation to the skin, a method for increasing the rate of hair
shaft elongation, a method for inducing the de novo development of
hair follicles, a method for increasing the number of hair
follicles present in the treated skin, a method to induce hair
growth along a surgical incision, a method to improve hair regrowth
in a healed wound site, a method to keep hair in subjects who
receive chemotherapy or radiotherapy, a method for establishment of
animal models for research on hair follicle development and
formation, a method for establishment of animal model for research
on melanogenesis metabolism, and a method for establishment of
experimental models to study cytokine production and cell
proliferation.
[0004] 2. Background of the Related Art
[0005] Hair loss and baldness (alopecia) are common phenomena in
mammals, including humans. (see, for example, A. G. Messenger
(1993) J. Investig. Dermatol. 101:4S-9S; R. P. R. Dawber (1987)
Dermatologica 175:23-28; D. G. Brodland, S. A. Muller (1991) Cutis
47:173-176; J. R. Spindler, J. L. Data (1992) Dermatol.
Nurs.4:93-99; A. K. C. Leung, W. L. M. Robson (1993) J. Roy. Soc.
Health 113:252-256). Hair loss may be naturally occurring (primary
alopecia) or it may be induced by chemical or physical agents
(secondary alopecia). See, for example, M. B. Brodin (1987)
Dermatol. Clin. 5:571-579; A Tosi, et al. (1994) Drug Saf.
10:310-317; H. J. Carson, et al. (1994) J. Cutan. Pathol. 21:67-70.
Hair loss may also result from specific disease states, such as
mange, or formation of scar tissue from bites, and with increasing
age (D. A. Mehregan, et al. (1992) J. Am. Acad. Dermatol.
27:935-942; D. A. Slagle, T. A. Martin (1991) Am. Fam. Physician
43:2019-2024; L. V. Spencer, J. P. Callen (1987) Dermatol. Clin.
5:565-570. Hair loss is an extremely common condition in healthy
adult male humans, and occurs frequently in adult female humans. In
fact, some degree of alopecia on the vertex from puberty onwards is
thought to be a universal phenomenon in both men and women (R. P.
R. Dawber (1987) Dermatologica 175:23-28). Alopecia is also
frequently observed in both pre- and post-pubertal patients as a
side effect of anti-cancer chemotherapy (A. M. Hussein, et al.
(1990) Science 249:1564-1566; B. W. Cline, (1984) Cancer Nursing
7:221-228; A. F. Hood (1986) Med. Clin. North Am. 70:187-209).
[0006] The physical phenomenon of hair loss may lead to
psychological problems in the patient, decreased social activity,
and the development of psychological diseases. In the case of
cancer patients, the likelihood of chemotherapy-induced alopecia
may lead to a refusal to accept treatment. As a result of the
prevalence of alopecia, and its potentially devastating impact,
there is immense interest in the development of effective clinical
treatments, both to prevent hair loss and to stimulate regrowth of
lost hair.
[0007] Abnormal hair loss in animals is also commonly observed, and
is associated with certain disease conditions, including skin
wounds and mange. Hair growth in domestic animals is of economic
concern, both from a cosmetic standpoint in pets and show animals,
and in the production of fiber and pelts used in the textiles and
garment industries. Many domesticated animals (e.g. sheep) are used
as a source of fiber, including wool and fur. The coat is either
harvested (clipped) on a periodic basis throughout the life of the
animal, such as in the case of sheep; or the pelage together with
the skin is removed following sacrifice, e.g. mink. The skin of
many domesticated animals is used as a commercial source of leather
and suede. These materials are manufactured directly from the skin
of an animal by the process of tanning. Therefore, improvements in
the quality and thickness of skin prior to sacrifice can benefit
the commercial production of skin-derived products. Furthermore,
many animals, especially those with pedigrees, are shown publicly
in competitions for judgement of the best specimen in their class.
Such animals include, but are not limited to, the following
categories: horses, cattle, sheep, dogs, cats, and rabbits. In many
instances, an important criterion on which judgement is based is
the appearance of the coat or pelage. Thus there is a need for
physiologically effective treatment to improve the nature and
appearance of animal coats during the life of the animal.
[0008] Despite the widespread occurrence of alopecia, the need for
prevention and therapy, and extensive research efforts to find
suitable remedies, there remains an urgent need for effective
treatment. For example, lack of a proven and effective treatment
for alopecia has caused many afflicted individuals to adopt the
practice of wearing a wig or toupee. Another extreme measure used
to combat alopecia, hair transplant surgery, is not available as an
option in many cases, e.g. following chemotherapy, and offers, at
best, only a partial remedy. At the same, the latter treatment
suffers from a number of disadvantages, including the need for
surgery.
[0009] A common non-surgical treatment for stimulating hair growth
which is currently used clinically is minoxidil (The Upjohn
Company, Kalamazoo, Mich.). A solution of minoxidil as active
ingredient is known as Rogaine.sup.R. As stated in the
Rogaine.sup.R Patient Information Booklet (The Upjohn Company,
Kalamazoo, Mich., revised Jun., 1992) minoxidil is a vasodilatory
drug which has serious side effects when administered orally for
the treatment of hypertension. At the same time, topical
application of minoxidil for the treatment of alopecia is only
partially effective and suffers from a number of disadvantages. For
example, it is only recommended for treatment of male pattern
alopecia of the vertex (cf. frontal recession), has to be applied
twice daily for at least four months, and requires a normal scalp
with no local abrasions, dermatitis or sunburn--conditions that can
increase absorption into the blood stream and the concomitant risk
of side effects. Further, minoxidil is of limited effectiveness:
based on the investigator's evaluation, there is no significant
increase in terminal hair regrowth between minoxidil and placebo
treatment groups after four months of treatment (refer to the
Rogaine.sup.R Patient Information Booklet, The Upjohn Company,
Kalamazoo, Mich., revised Jun., 1992). In patients who do respond
to minoxidil treatment, the new hair is likely to be shed within a
few months after stopping treatment. Likewise, the effect of
minoxidil in stimulating hair growth in a macaque monkey model was
found to be transient: substantially all hair grown during
minoxidil treatment was lost within six months of treatment being
discontinued (P. A. Brigham, et al. (1988) Clinics in Dermatol.
6:177-187).
SUMMARY OF THE INVENTION
[0010] The methods and compositions of the present invention may be
used to promote hair growth/regrowth in adult mammals. The instant
methods, and compositions used therein, also induce major
physiological, developmental, and structural changes in the skin of
adult mammals including: skin differentiation, wound tissue
remodelling in healed incision/excision wound sites, follicle
development and regeneration, an increase in the number of hair
follicles, morphological and functional change of hair follicles in
different stages of the hair cycle, melanogenesis, hair shaft
elongation, and accelerated hair growth rate. The methods and
compositions of the present invention demonstrate the involvement
of various growth factors in follicular development and regulation
of the hair growth cycle. The methods and compositions of this
invention also demonstrate hair follicle differentiation and the
hair growth process in adult mammals in response to a single
application of an extraneous composition.
[0011] In accordance with one embodiment of the invention, the hair
growth stimulating method comprises topically treating the area of
skin affected by hair loss. The method may comprise topical
treatment as a single application, or it may comprise periodic
treatment over an extended treatment time period as needed.
Alternatively, the method may include a slow-release mechanism from
a suitable carrier, or via any of several drug delivery mechanisms
known in the art.
[0012] In accordance with a method of the invention, a trichogenic
composition may be applied at the point of an incision in the skin.
Such an incision may be made by a scalpel as a part of the
treatment protocol, in order to induce regeneration or the de novo
development of hair follicles within the dermis and subcutaneous
layer. Alternatively, an incision may pre-exist, for example, due
to cranial or facial injury, prior to treatment with the hair
growth stimulator.
[0013] In accordance with an embodiment of the invention, a
trichogenic composition may be applied at the site of an excision
in the skin. Such an excision may be due to various accident
injuries in cranial, facial, arm, leg, etc., in order to induce new
hair follicle formation and to promote tissue remodeling to normal
in the wounded site.
[0014] In accordance with a method of the invention, a trichogenic
composition may be applied during or after plastic surgery at the
sites of eyebrow, mustache, or beard to improve cosmetics.
[0015] In accordance with an embodiment of the invention, the
method may be applied to a subject who is receiving chemotherapy or
radiotherapy and suffering hair loss. Such a situation may occur as
a part of cancer treatment protocol, in order to induce hair
follicle formation. The embodiment of the invention also includes
the situation that the hair growth stimulator can be used for the
subject who is going to receive chemotherapy or radiotherapy, to
avoid hair loss, or who received chemotherapy or radiotherapy
before and is suffering a permanent hair loss. Alternatively, the
hair growth stimulator may be used on a subject who suffers hair
loss from exposure to a toxic chemical or radioactive source. Such
a situation may result from an industrial toxic chemical accident,
explosion of chemical or nuclear plant. or accident administration
of toxic chemicals or toxic drugs.
[0016] In accordance with still another embodiment of the
invention, the method may include administration by subcutaneous
injection to the treatment area.
[0017] Those skilled in the art of drug application know how to
determine the manner and frequency of application, the formulation
of the active ingredient, and the dose will be varied according to
the nature and severity of the condition being treated, the area of
skin affected, and the like.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1A is a photomicrograph of epidermis and dermis of a
C57BL/KsJ db/+ mouse 3 days after n-butyl cyanoacrylate
application, with the surface of the skin at the top. Epithelial
pegs project into the dermal layer, and antibody to TGF-.beta.1 is
localized in the epidermis and hair follicles in dermis (see brown
stain). The arrows indicate examples of the stain in epithelial
pegs and hair follicles. The bar represents 100 .mu.m.
[0019] FIG. 1B is a photomicrograph of a skin section of a
C57BL/KsJ db/+ mouse 3 days after n-butyl cyanoacrylate
application, showing the demarcation between the treated area (left
side) and the untreated area (right side). The treated area (big
arrows) has a multilayered epidermis, thickened dermis, and
epidermal peg elongation. The small arrows indicate examples of
epidermal peg elongation. The bar represents 100 .mu.m.
[0020] FIG. 2 is a photomicrograph of a skin section of a C57BL/KsJ
db/+ mouse 10 days after n-butyl cyanoacrylate application, showing
an increase in the number of mature hair follicles. The arrows
indicate examples of mature hair follicles. The bar represents 100
.mu.m.
[0021] FIG. 3A is a graph comparing the thickness of the epidermis
layer between the n-butyl cyanoacrylate-treated and adjacent
untreated areas. The maximum increase in thickness in the treated
areas (filled triangles) is about 2 fold the maximum level in the
untreated areas (open triangles). The epidermis returns to normal
thickness at about 40 days post treatment. Each time point
represents 2-6 mice of the strain C57BL/KsJ db/+.
[0022] FIG. 3B is a graph comparing the thickness of the dermis
layer between the n-butyl cyanoacrylate-treated and the untreated
areas. The thickness in the treated areas (filled triangles) is
about 1.3 fold that of untreated areas (open triangles) at the
first day after treatment, reaches 1.5 fold increase at days 16-20
post treatment, and returns to normal at about 70 days post
treatment. Each time point represents 2-6 mice of the strain
C57BL/KsJ db/+.
[0023] FIG. 3C is a photomicrograph of a skin section of a
C57BL/KsJ db/+, showing large mature hair follicles traversing the
entire thickness of the skin 16 days following application of
n-butyl cyanoacrylate. The arrows indicate examples of large mature
hair follicles. The bar represents 100 .mu.m.
[0024] FIG. 4A is a photomicrograph of a skin section of a
C57BL/KsJ db/+ mouse at 19 days after the left side was treated
topically with n-butyl cyanoacrylate (big arrows). A high
concentration of large mature hair follicles (cut in cross-section
and indicated by the small arrows) occupies much of the
subcutaneous space in the treated area. However the adjacent
untreated area has few hair follicles in the subcutaneous space.
There is a clear boundary between the treated and untreated areas
in terms of hair follicle number and the thickness of the skin. The
bar represents 100 .mu.m.
[0025] FIG. 4B is a graph comparing the hair follicle number
between the n-butyl cyanoacrylate-treated (filled triangles) and
adjacent untreated skin (open triangles). In the strain C57BL/KsJ
db/+, the maximum increase in the number of hair follicles in the
treated areas is about 2 fold that of untreated areas. Each time
point represents 2-6 mice.
[0026] FIG. 5A is a scanning electron photomicrograph of a specimen
of C57BL/KsJ db/+ mouse skin, showing a clear boundary between
treated and untreated areas. At 23 days after n-butyl cyanoacrylate
treatment, there is a higher density of hairs and deeper hair roots
in the treated area (arrows) compared to the adjacent untreated
area. The bar represents 200 .mu.m.
[0027] FIG. 5B is a photograph of the dorsal aspect of a C57BL/KsJ
db/+ mouse taken 17 days post application of n-butyl cyanoacrylate,
showing a triangle pattern of hair growth, which occurred exactly
along the pattern of the application. The untreated adjacent skin
remained hairless.
[0028] FIG. 6A is a photograph of the dorsal aspect of a diabetic
C57BL/KsJ db/db mouse taken 27 days post application of n-butyl
cyanoacrylate. The hair growth appears in a rectangle pattern with
three hair growth dots above the rectangle (arrows), exactly along
the pattern of the application. The untreated adjacent skin
remained hairless.
[0029] FIG. 6B. is a graph comparing the hair follicle number
between the n-butyl cyanoacrylate-treated and adjacent untreated
skin. In the diabetic strain C57BL/KsJ db/db, the maximum number of
hair follicles in treated areas (filled triangles) is about 4 fold
that of untreated areas (open triangles). Each time point
represents 2-6 mice.
[0030] FIG. 6C is a photograph of the dorsal aspect of a Balb/cBYj
nu/+ mouse taken 19 days post application of n-butyl cyanoacrylate.
The hair growth appears in a circle pattern (arrows), exactly along
the pattern of the application. The untreated adjacent area
remained hairless.
[0031] FIG. 7A is a photograph of the dorsal aspect of a Sprague
Dawley rat taken 23 days post application of n-butyl cyanoacrylate.
The hair growth appears as a rectangle pattern (arrows) exactly
along the pattern of the application. The untreated adjacent area
remained hairless.
[0032] FIG. 7B is a photomicrograph of a skin section of a Sprague
Dawley rat, showing a much higher density of hair follicles in
treated area (big arrows) compared to the untreated area. There is
a boundary between the treated and untreated areas in terms of hair
follicle number. The small arrows indicate examples of hair
follicles. The bar represents 400 .mu.m.
[0033] FIG. 8A is a photograph of dorsal aspect of C57BL/KsJ db/+
mouse with an incision wound treated with n-butyl cyanoacrylate,
showing profound hair growth along the linear wound margin (arrow)
at day 17 post treatment.
[0034] FIG. 8B is a photomicrograph of an incisional wound to which
n-butyl cyanoacrylate was applied, showing the development of many
new hair follicles and little scar tissue in the wound site
(between the big arrows) at day 17 post treatment. There are fewer
and smaller hair follicles farther from the wound site, indicating
that the process of remodelling and normalizing of the wound was
accelerated. The small arrows indicate examples of hair follicles.
The bar represents 200 .mu.m.
[0035] FIG. 8C is a photomicrograph of skin section of a C57BL/KsJ
db/+ mouse, showing few hair follicles but plenty of scar tissue in
the incisional wound site (between the big arrows) at day 17 post
treatment with PBS, a control for FIG. 8B. The bar represents 200
.mu.m.
[0036] FIG. 9 is a photomicrograph of skin section of a C57BL/KsJ
db/+ mouse, showing the development of new hair follicles in the
treated site (big arrows) and few hair follicles in the untreated
site 68 days after subcutaneous injection with n-butyl
cyanoacrylate. The small arrows indicate examples of hair follicles
in the dermis. The bar represents 100 .mu.m.
[0037] FIG. 10A is a photograph of the dorsal aspect of a C57BL/KsJ
db/+ mouse (pre-treated with cyclophosphamide) taken 31 days post
application of n-butyl cyanoacrylate. There is a profound growth of
new unpigmented gray hairs in the treated area (arrows). The
adjacent untreated skin is hairless and shows a lack of
pigmentation.
[0038] FIG. 10B is a photograph of the dorsal aspect of a C57BL/KsJ
db/+ mouse (pre-treated with doxorubicin) taken 31 days post
application of n-butyl cyanoacrylate. There is a profound growth of
hair with normal pigmentation in the treated area (arrows). The
untreated skin is hairless and has normal pigmentation.
[0039] FIG. 10C is a photograph of the dorsal aspect of a C57BL/KsJ
db/+ mouse (pre-treated with cyclophosphamide) taken 202 days post
application of n-butyl cyanoacrylate, showing the lack of
pigmentation in the new growth hairs in treated area (arrows).
[0040] FIG. 11 is a photograph of the organ culture of skin not
treated, and adjacent skin treated with n-butyl cyanoacrylate then
cultured in Dulbecco's Modified Eagle Medium supplemented with 10%
fetal bovine serum. At 14 days in organ culture after treatment
hair growth is apparent in the treated area (arrow), but the
adjacent untreated part of the skin is hairless.
[0041] FIG. 12A is a graph comparing the effect of extracts of skin
treated with n-butyl cyanoacrylate and of untreated skin in a cell
proliferation assay. The filled triangles indicate extracts of
treated tissue harvested at day 20 post-treatment. Open triangles
denote treated tissue harvested at day 10. Filled lozenges denote
untreated tissue harvested at day 20, and open lozenges are the
mouse albumin control.
[0042] FIG. 12B is a graph comparing the effect of extracts of skin
treated with n-butyl cyanoacrylate and of untreated skin, with and
without fractionation to exclude molecules larger than about 30
kDa, in a cell proliferation assay. The filled triangles indicate
extracts of treated tissue harvested at day 20 post-treatment. Open
triangles are the filtrate of the treated tissue harvested at day
20. Filled lozenges are the untreated tissue harvested at day 20.
Open lozenges denote the filtrate of the untreated tissue extract.
The filtration step removes stimulation, indicating that the
proliferative agent or agents are larger than 30 kDa in mass.
[0043] FIG. 13 is a photomicrograph of a skin section stained with
anti-TGF-.beta.1 antibody 6 days after treatment with n-butyl
cyanoacrylate, showing localization of TGF-.beta.1 in sebaceous
glands, epithelial cells and hair follicles in the treated area.
The small arrow indicates examples of the stain in sebaceous glands
and hair follicles. Bar represents 100 .mu.m.
[0044] FIG. 14 is a photograph of the dorsal aspect of a C57BL/KsJ
db/+ mouse taken 14 days post application of n-butyl cyanoacrylate,
showing that new hair growth was present at sites 2 (which received
topical n-butyl cyanoacrylate only) and 3 (which received topical
n-butyl cyanoacrylate and subcutaneous PBS/0.1% BSA). There was a
lack of hair growth-at site 1 which received topical n-butyl
cyanoacrylate and subcutaneous anti-TGF-.beta.1 neutralizing
antibody. This experiment suggests that TGF-.beta.1 was a necessary
part of the response to n-butyl cyanoacrylate.
[0045] FIG. 15 is a photograph of the dorsal aspect of a C57BL/KsJ
+/+ mouse taken 19 days post application of isobutyl cyanoacrylate,
showing a "T" pattern of hair growth (arrows) in the treated area
and the hairless skin in the shaved but untreated adjacent
area.
DETAILED DESCRIPTION OF THE INVENTION
A. Terminology
[0046] The term "hair" as used herein shall mean filamentous
appendages from the skin of vertebrates, including the pelage,
coat, fur or wool of mammals, and the feathers of birds.
[0047] The term "hair growth" as used herein shall mean any
increase in the total quantity of hair, an increase in the number
of active hair follicles, an increase in the number of terminal
hairs, an increase in the length of one or more hair shafts, an
increase in the rate of hair shaft elongation, or an increase in
the diameter of one or more hair shafts, on a given area of
skin.
[0048] The term "hair growth cycle" as used herein shall mean
progression through the phases known as anagen, the growth phase;
catagen, the regressing phase; and telogen, the resting phase. The
length of each phase varies with species, strains, individuals, and
body site; as well as environmental factors, intrinsic hormone
levels, and other factors.
[0049] The expression "terminal hair" as used herein shall mean
readily visible, relatively coarse hair that is typically
pigmented; such as that normally found on the scalp of young adult
humans. In animals, terminal hairs comprise the pelage and
whiskers. Terminal hair is contrasted with "vellus hair" which is
extremely fine, short, unpigmented and almost invisible.
[0050] The term "hair loss" as used herein shall mean a net
decrease in the amount of hair, in the number of terminal hairs, or
in the number of hair follicles, on a given area of skin.
[0051] The term "alopecia" as used herein means a condition in
which hair is being lost or has been lost, or a pre-existing
condition of congenital baldness.
[0052] The term "growth factor" as used herein means a biologically
active substance which influences proliferation and/or
differentiation of various cell types, and may effect
developmental, morphological and functional changes, either alone
or when modulated by other substances. A growth factor herein may
be a proteinaceous entity comprising one or more polypeptide
chains.
[0053] The term "TGF" as used herein means generally transforming
growth factor, and may refer to one or more members of the class of
transforming growth factors, or collectively to the entire class of
transforming growth factors.
[0054] The expression "de novo hair follicle differentiation" as
used herein means the formation of new hair follicles, as a result
of the proliferation of germinative cells and the further
differentiation of mesenchymal cells in the proximity of the
germinative cells.
[0055] The term "trichogenically effective amount" means that
amount which is effective in increasing: the total amount of hair,
the overall length or diameter of one or more hairs, the total
number of terminal hairs, the total number of hair follicles, or
the ratio of hair follicles in anagen:telogen. Such effects may be
due to prolongation of the anagen phase, delay in the transition
from anagen to telogen, or de-novo hair follicle development.
[0056] As used herein, a "physiologically effective formulation" is
a composition that stimulates an increase in hair growth of an
animal, or improves the overall appearance of the pelage of an
animal, or hair of a human.
B. General Methods
[0057] The skin or integumentary system is the largest organ of the
human body. It acts as an interface between the internal and
external environment, and fulfills thermoregulatory, barrier, and
sensory functions, among others. Histologically, three major tissue
layers are identified. The uppermost layer, the epidermis, is a
relatively thin stratified squamous epithelium which is itself
composed of five strata. Subjacent to the epidermis is the dermis,
a dense fibroelastic connective tissue stroma. The third layer,
lying beneath the dermis is the subcutaneous layer composed of
fatty connective tissue.
[0058] There are two types of skin: hair-bearing skin, which covers
the vast majority of the body surface; and hairless skin confined
to areas such as the palms of the hands, soles of the feet, and
mucous membranes. The two skin types are differentiated on the
basis of the presence or absence of the pilosebaceous apparatus:
the hair follicle and the accompanying sebaceous gland.
[0059] Hairs (or pili) are filamentous, keratinized structures
derived from the epidermis. Hairs have a number of roles, including
thermoregulation, sensory perception, and social communication. The
density of hairs per unit area of skin varies with species, strain,
and skin site. For example in humans, it ranges from about 600
cm.sup.-2 to about 60 cm.sup.-2, with the highest density being on
the face.
[0060] Hairs show enormous variation in the length and diameter of
the hair shaft: from <1 mm to >1,000 mm in length, and from
0.005 mm to 0.5 mm in diameter. There are also major differences,
within a given individual, in the degree of pigmentation. Two broad
categories of hairs are recognized: vellus hairs are short and
narrow, and are present over most of the body surface; while
terminal hairs are longer, thicker, and often heavily pigmented.
Terminal hairs include those of the scalp, eyebrows and eyelashes,
as well as the post-pubertal hair of the axillae and pubis, and the
facial and body hair in many males.
[0061] Each hair consists of a shaft and a root. The hair shaft is
composed of specialized cells (keratinocytes) containing a
particularly strong form of keratin, providing a filament of
material with high tensile strength. The root lies within the hair
follicle, which is an invagination of the epidermis. The hair
follicle may extend deeply into the hypodermis or may be more
superficial in the dermis. The proximal end of the root is expanded
to form the hair bulb. The bulb is deeply indented on its deep
surface by a conical vascular dermal papilla. (For a general
description of the components of the skin, its appendages, and the
pilosebaceous apparatus see, for example, R. F. Oliver (1980) in
The Skin of Vertebrates pp. 199-213, edited by R. I. C. Spearmen
& P. A. Riley, Academic Press; and P. L. Williams, et al.
(1989) Hairs in Gray's Anatomy, pp. 90-94, edited by P. L.
Williams, et al., Churchill Livingston).
[0062] The hair bulb comprises the germinative matrix, a zone of
great mitotic activity which generates the hair and its surrounding
inner root sheath, and the keratogenous zone, in which cells are
keratinized. The germinative matrix consists of a mass of
pluripotent cells capping the dermal papilla. Cells arising
mitotically from this group move apically, and may differentiate
along several different routes. The activity of the hair bulb, and
of the whole root complex involves various morphogenetic processes
in which different cell shapes, chemical forms of keratin, and
cellular migration patterns are produced.
[0063] The formation of hair follicles results from interactions
between the epidermis and mesenchyme during fetal development (R.
F. Oliver & C. A. B. Jahoda (1988) Clinics in Dermatology
6:74-82). The dermal components of the hair follicle, namely the
dermal papilla and dermal sheath, are derived from an aggregate of
mesenchymal cells. Follicle initiation and development begin with
the aggregation of dermal fibroblasts and epidermal keratinocytes.
The epidermal cells proliferate and penetrate the dermis as plugs.
Subsequently, the epidermally derived cells encircle a dermal
aggregation and incorporate it into a pocket of tissue, the dermal
papilla.
[0064] It is known that follicular development relies on a series
of messages between dermis and epidermis. The initial,
dermis-derived message is common, not only within mammalian
species, but to all classes of vertebrate. The next signal, from
the epidermis is class-specific, and instructs the dermis to form a
dermal papilla. Thereafter, a second dermal message instructs the
epidermal placode to form the class-specific appendage (e.g. hair
in mammals) (see, for example, A. G. Messenger (1993) J. Investig.
Dermatol. 101:4S-9S; D. L. du Cros (1993) J. Investig. Dermatol.
101:106S-113S).
[0065] Grafting studies have shown that the dermal papilla is
necessary for normal hair follicle function and production of the
shaft. The dogma regarding hair follicle development in an
individual is that the population of hair follicles and dermal
papillae is established during embryogenesis with no further
development subsequent to the first few days after birth (P. L.
Williams, et al. (1989) Hairs in Gray's Anatomy, pp. 90-94, edited
by P. L. Williams, et al., Churchill Livingston; D. H. Cormack
(1987) Hairs in Ham's Histology 9th Ed., ed. by D. H. Cormack, pub.
J. B. Lippincott Co.).
[0066] Hair growth is effected by proliferation of the hair
follicle matrix cells under control of the dermal papilla, and is
cyclical. Three distinct stages in the hair growth cycle are
recognized: anagen, an active phase when hair growth occurs;
catagen, the transition stage during which follicle activity
declines; and telogen, the resting phase when no cell proliferation
occurs. In simple terms, alopecia can be explained as degeneration
of the hair follicles and a shift in the population of follicles
from the anagen phase to the telogen phase.
[0067] The dynamics of the hair growth cycle vary from species to
species, between different body sites of the same species, and
between different follicle types in the same body site. Synchrony
of the hair growth cycle during the neonatal period occurs in many
animals, including humans. In many mammals, characteristic molt
waves continue into adult life. In many wild species, the molt is
regulated by environmental stimuli, particularly the photoperiod,
resulting in seasonal changes in the quality and quantity of the
pelage. In humans, follicular activity rapidly becomes
asynchronous, and local mechanisms of control of the hair cycle
predominate. However, systemic modulation of the human hair growth
cycle does occur during pregnancy and postpartum. It is also
reported that human hair growth does show vestiges of seasonal
variation (A. G. Messenger (1993) J. Investig. Dermatol.
101:4S-9S). In the mouse, the first follicles to appear during
embryogenic development are those of the vibrissa on the snout. Of
the pelage follicles, up to 30% are initiated prenatally, and the
remainder develop within the first few days following birth. Mature
murine pelage follicles undergo a hair growth cycle of
approximately four weeks duration. The various phases of the hair
growth cycle are accompanied by characteristic changes in the
thickness of the epidermis, dermis, and adipose layer (D. L. du
Cros (1993) J. Investig. Dermatol. 101:106S-113S).
[0068] Numerous factors may be involved in regulating the
proliferation of hair follicle matrix cells, and control the hair
growth cycle. For example, various growth factors, steroid
hormones, dermo-epithelial interaction, and the immune system have
been implicated. An increased vascularity in the dermis is known to
stimulate hair growth (J. R. Matias, et al. (1989) Arch. Dermatol.
Res. 281:247-253).
[0069] Growth factors are secretory molecules, generally
polypeptides, which mediate intercellular communication in
metazoans. Thus, various growth factors have been implicated in the
control of complex processes occurring during embryogenic
development and in tissue repair and regeneration (J. Massague
(1990) Annu. Rev. Cell Biol. 6:597-641). In addition, most of the
major growth factor families and their receptors have been
implicated in regulating skin cell function, including for example:
epidermal growth factor (EGF), keratinocyte growth factor (KGF),
transforming growth factor-.alpha. (TGF-.alpha.), transforming
growth factor-.beta. (TGF-.beta.), fibroblast growth factor (FGF),
bone morphogenetic protein-4 (BMP-4), and insulin-like growth-1
(IGF-1)(see A. G. Messenger (1993) J. Investig. Dermatol.
101:4S-9S; D. L. du Cros (1993) J. Investig. Dermatol.
101:106S-113S and references cited therein). Furthermore, several
growth factors have been implicated in hair follicle morphogenesis
and/or control of hair growth, including EGF (D. L. du Cros (1993)
J. Investig. Dermatol. 101:106S-113S; A. G. Messenger, J. Investig.
Dermatol. 101:4S-9S; M. P. Philpott, et al. (1990) J. Cell Science
97:463-471), FGF (D. L. du Cros (1993) ibid.), and KGF (G. F.
Pierce, et al. (1994) J. Exp. Med. 179:831-840), and TGF-.beta. (A.
G. Messenger (1993) ibid.; M. P. Philpott, et al. (1990)
ibid.).
[0070] Studies of the induction of hair follicle development and of
the hair growth cycle have been hampered, in part, by the lack of
suitable in vivo animal models, and by the paucity of appropriate
in vitro experimental systems. Numerous species and strains of
animals have been used to investigate the hair growth process in
vivo and/or to simulate human alopecia. Most studies have focused
on either new-born or weanling rats and mice, genetically impaired
or mutated mice, or stump-tailed macaque monkeys.
[0071] Members of a macaque species native to S. E. Asia, which
show a balding pattern similar to that associated with human
androgenetic alopecia, were used by Brigham et al. to study the
effect of topical minoxidil on the balding process analyzed by
folliculogram (P. A. Brigham, et al. (1988) Clinics in Dermatology
6:177-187). A. M. Hussein et al. (A. M. Hussein, et al. (1990)
Science 24:1564-1566) used young (6-8 day old) rats, treated with
cytosine arabinoside, doxorubicin or cyclophosphamide, as a model
for chemotherapy induced alopecia (A. M. Hussein, et al. (1990)
Science 24:1564-1566). A mutant strain of the mouse which expressed
androgen-dependent baldness was developed by Matias et al. as a
model of androgenetic alopecia (J. R. Matias, et al. (1989) Arch.
Dermatol. Res. 281:247-253). Kligman used a hairless mouse strain
as a model for evaluating hair growth promoters (L. H. Kligman
(1988) Clinics in Dermatology 6:163-168). Neonatal mouse skin was
used by du Cros (D. L. du Cros (1993) Developmental Biology
156:444-453) to investigate the influence of FGF in the development
and cycling of murine hair follicles (D. L. du Cros (1993)
Developmental Biology 156:444-453). The role of cyclosporin in hair
growth was investigated by A. Gilhar et al. using human
split-thickness skin grafts which were transplanted to nude rats
(A. Gilhar, et al. (1990) Dermatologica 181:117-121), or to nude
mice (A. Gilhar, et al. (1991) Acta Derm. Venereol. (Stockh)
71:327-330).
[0072] In vitro models include culture of excised, intact, human
anagen hair follicles (M. P. Philpott, et al. (1990) J. Cell
Science 97:463-471); organ culture of human hair follicles in
serum-free medium (R. Imai, et al. (1993) Arch. Dermatol. Res.
284:466-471); and the use of a collagen matrix system during
culture of a heterogeneous preparation of murine hair follicles, or
co-culture of murine hair follicle buds with immortalized rat
vibrissa dermal papilla cells (S. H. Yuspa, et al. (1993) J.
Investig. Dermatol. 101:27S-32S). All of the experimental models
described above have one or more significant disadvantages and/or
limitations to their use and effectiveness in studying mammalian
skin differentiation and hair growth.
[0073] The present invention demonstrates cyanoacrylates as strong
hair growth stimulators that can avoid the shortcomings of earlier
procedures. The adhesive properties of certain cyanoacrylate esters
was discovered by Coover in 1959 (H. W. Coover, et al. (1959) J.
Soc. Plast. Eng. 15:5). Over the past two decades cyanoacrylates,
in particular n-butyl cyancacrylate and iso-butyl cyanoacrylate,
have been widely used in surgery as tissue adhesives and as wound
coverings (M. L. Ronis, et al. (1984) Laryngoscope 94:210-213; S.
Sabanathan (1993) Eur. J Cardiothorac. Surg. 7:657-660; A. B.
Leahey, et al. (1993) Ophthalmology 100:173-180). N-butyl
cyanoacrylate has been used in more than one thousand eye surgeries
and larynx repairs (see, for example, A. B. Leahey, et al. (1993)
Ophthalmology 100:173-180, and references cited therein). Various
formulations of cyanoacrylate (as the Nexaband.sup.R family of
products, Tri-Point Medical, Raleigh, N.C.) are widely used in
veterinary medicine as wound dressings.
[0074] The present invention provides for skin differentiation,
hair follicle development, melanogenesis, and hair shaft elongation
in adult mammals following treatment with a trichogenic
composition. Several in vitro systems for investigating hair
follicle growth exist, focusing on cell proliferation or hair shaft
elongation, but not new follicle morphogenesis (S. Arase, et al.
(1990) J. Dermatol. 17:667-676; R. Imai, et al. (1993) Arch.
Dermatol. Res. 284:466-471; R. M. Philpott, et al. (1990) J. Cell
Science 97:463-471). The invention is unique in providing the de
novo differentiation and development of fully functional hair
follicles in adult mammals.
[0075] Any of several laboratory animals may be used in conjunction
with the present invention including, but not limited to, the
Sprague-Dawley strain of rat, and the following strains of mice:
C57BL/KsJ +/+, C57BL/KsJ db/+, C57BL/KsJ db/db, Balb/cBYj +/+,
Balb/cBYj nu/+, HRS/J Hr/+, and RHJ LeJ hr.sup.rh-j/+. Hair growth
was profoundly stimulated locally in response to a single topical
application of a formulation of a functional group derivative of a
carboxylic acid. For example, a trichogenic formulation comprising
an esterified derivative of acrylic acid may be used. In a
preferred embodiment a formulation of butyl cyanoacrylate is found
to be effective. Either a formulation of n-butyl cyanoacrylate or a
formulation of iso-butyl cyanoacrylate may be used. In each case,
the butyl cyanoacrylate is formulated with a suitable stabilizer to
prevent spontaneous polymerization.
[0076] Preparations of a trichogenic composition comprising a
functional group derivative of a carboxylic acid which is labile or
tends to polymerize may be formulated with a suitable stabilizer to
inhibit or delay chemical change to the active ingredient. In the
case of n-butyl cyanoacrylate and iso-butyl cyanoacrylate,
effective stabilizers are dibutyl sebacic acid and methyl
hydroquinone, respectively.
[0077] Compositions comprising a functional group derivative of a
carboxylic acid, with or without a suitable stabilizer, may be
formulated with a suitable carrier material or diluent. Carriers
may be used as an aid in application of the active ingredient to
the treatment site or to dilute the active ingredient to provide an
appropriate dose. Examples of suitable carriers include various
oils, including various vegetable oils and mineral oils, waxes, and
various organic solvents such as dimethyl sulfoxide and acetone.
The list is not inclusive.
[0078] Suitable carriers may also comprise ingredients commonly
used in the cosmetics industry. Thus physiologically acceptable
carriers may be solids or liquids and may include solvents,
diluents, humectants, and emollients. Such carriers may be used
singly or in combination. Suitable carriers may include, but are
not limited to, the following examples:
[0079] Solvents and diluents, for example,
[0080] castor oil,
[0081] ethylene glycol monobutyl ether,
[0082] diethylene glycol monoethyl ether,
[0083] dimethyl formamide,
[0084] corn oil,
[0085] dimethyl sulfoxide,
[0086] mineral oil,
[0087] soybean oil,
[0088] tetrahydrofuran,
[0089] Emollients, for example,
[0090] cetyl palmitate,
[0091] dimethylpolysiloxane,
[0092] glyceryl monoricinoleate,
[0093] glyceryl monostearate,
[0094] isobutyl palmitate,
[0095] isocetyl stearate,
[0096] isopropyl palmitate,
[0097] isopropyl stearate,
[0098] butyl stearate,
[0099] isopropyl laurate,
[0100] hexyl laurate,
[0101] decyl oleate,
[0102] di-n-butyl sebacate,
[0103] isopropyl myristate,
[0104] lanolin,
[0105] lauryl lactate,
[0106] mink oil,
[0107] palmitic acid,
[0108] polyethylene glycol,
[0109] stearic acid,
[0110] sesame oil,
[0111] coconut oil,
[0112] arachis oil,
[0113] castor oil,
[0114] mineral oil,
[0115] isostearic acid,
[0116] palmitic acid,
[0117] isopropyl linoleate,
[0118] lauryl lactate,
[0119] myristyl lactate,
[0120] decyl oleate,
[0121] myristyl myristate,
[0122] Formulation of the active ingredient for application to skin
under the invention can also include ingredients to preserve the
components of the formulation of the active ingredient and to
prevent proliferation of microorganisms Preservation by the
inclusion of chemical preservatives and water activity depressants
are well known in the cosmetic, food and pharmaceutical industries.
Components of the formulation can be preserved by the inclusion of
a suitable concentration of a chemical preservative, such as
benzoic acid, sodium benzoate, potassium sorbate, propionic acid,
and C1 to C4 esters of p-hydroxybenzoic acid. The composition can
also be preserved by the inclusion of a water activity depressant
in an amount sufficient to depress the water activity (a.sub.w)
value to <0.9, more preferably to <0.85. Examples of water
activity depressants include sorbitol, propylene glycol, sugars,
and alkali metal salts, including carboxylates, halides, and
sulfates.
[0123] The active ingredient plus stabilizer may be soluble or
insoluble in a liquid carrier. If the active ingredient and
stabilizer compound are both soluble in the carrier, the carrier
acts as solvent for the active ingredient. If the active ingredient
and stabilizer are both insoluble in the carrier, they are
dispersed in the carrier by means of, for example, a suspension,
emulsion, gel, cream or paste, and the like. A preferred form of
carrier, solvent or diluent for the active ingredient is in the
form of an oil, including either light or heavy mineral oil.
Vegetable oils, such as oils obtained from any of corn, sunflower,
safflower, soybean, canola, and the like, may also be used.
[0124] Delivery of the formulation may also be via a slow-release
mechanism, such as a dermal patch, or other mechanism well known in
the art (see, for example, M. A. Longer & J. R. Robinson (1990)
in Remington's Pharmaceutical Sciences, ed. by A. R. Gennaro, Mack
Publishing Co.).
[0125] The trichogenic composition may also be formulated with an
anti-inflammatory agent, for example an antihistamine.
Alternatively, the subject may be treated with an anti-inflammatory
material following treatment with the trichogenic agent.
[0126] The above list of carrier materials and methods for drug
delivery is not meant to be exhaustive, but is presented merely for
illustrative purposes and should not be construed as limiting the
invention in any way. Those skilled in the art will realize that
conventional carrier materials and drug delivery mechanisms may be
used within the scope of the invention.
[0127] In the present invention, increased hair growth is readily
observed in mammals following treatment with the formulation known
as Nexaband.sup.R Liquid (Tri-Point Medical, Raleigh, N.C.). This
composition is comprised of n-butyl cyanoacrylate (>85%),
sebacic acid dibutyl ester (ca. 15%), as an inhibitor of
spontaneous polymerization or stabilizer, and a small amount of a
blue, FDA-approved dye. The mammals here are a rat and multiple
strains of mice. In subsequent tests, the application of
Nexaband.sup.R Liquid gave a universal, consistent and strong
response: manifest as greatly increased hair growth at the site of
application. The application here means, but is not limited to, the
following situations: topical application on the intact surface of
normal skin; topical application on the intact surface of the skin
of mammals that are systemically pre-treated with anticancer drugs
(and thereby hair regrowth and/or melanogenesis metabolism is
inhibited); application to a full-thickness incisional or
excisional wound; and application to the dermis layer by
subcutaneous injection.
[0128] The trichogenic effect of n-butyl cyanoacrylate is at least
twofold: 1) existing hair follicles are stimulated to grow hair at
an accelerated rate, and 2) development of hair follicles is
induced de novo. Induced hair follicles subsequently mature and
produce terminal hairs. These findings are unexpected and
surprising to us, because popular opinion dictates that hair
follicle development only occurs during pre- and neo-natal periods
and not in the adult. Nevertheless, all of the morphogenetic events
related to pre-natal hair follicle development are accomplished by
our invention.
[0129] Application of a formulation of iso-butyl cyanoacrylate,
containing trace amounts (about 0.01%) of monomethyl hydroquinone
as stabilizer, gave a positive response in the form of increased
hair growth in, for example, several strains of mice. The response
to iso-butyl cyanoacrylate in mice and rats was very similar to the
response of hair growth induced by n-butyl cyanoacrylate.
[0130] In contrast, the stabilizer, sebacic acid dibutyl ester, the
surgical adhesive Rezifilm.sup.R (which contains methyl acrylate),
and the adhesive Weldwood.sup.R were not effective in stimulating
hair growth. Similarly, 2% minoxidil (Rogaine.sup.R, The Upjohn
Company, Kalamazoo, Mich.), applied topically once daily for 20
days in an amount of 20 .mu.l cm.sup.-2, was not effective in
inducing hair growth.
[0131] These observations show that the chemical structure
responsible for the observed effects is that of a functional group
derivative of a cyanocarboxylic acid. Thus, active ingredients
useful in the practice of this invention are broadly delineated by
the formula: 1
[0132] wherein R is an ester, an amide, an alkyl amide, a dialkyl
amide, an alkoxyalkylamide, an anhydride, a halide, a nitrile, or
an amino group; and R.sup.2 is C.sub.1-C.sub.10 alkyl, cycloalkyl,
alkenyl, alkoxyalkenyl, alkynyl, aryl, alkaryl or aralkyl.
[0133] Preferably, R is an ester of C.sub.1-C.sub.20 alkyl,
cycloalkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl, hydroxyalkyl,
or mono- or poly- alkoxyalkyl, an alkyl amide of C.sub.1-C.sub.20,
a dialkyl amide of C.sub.1-C.sub.20, an alkoxyalkylamide, an
anhydride of C.sub.1-C.sub.20, a halide, a nitrile, or an amino
group; and R.sup.2 is C.sub.1-C.sub.10 alkyl, cycloalkyl, alkenyl,
alkoxyalkenyl, alkynyl, aryl, alkaryl or aralkyl.
[0134] In a preferred embodiment, the active ingredient is a
functional group derivative of an unsaturated cyanocarboxylic acid
with the general formula: 2
[0135] wherein R is an ester of C.sub.1-C.sub.20 alkyl, cycloalkyl,
alkenyl, alkynyl, aryl, alkaryl, aralkyl, hydroxyalkyl, or mono- or
poly-alkoxyalkyl, an alkyl amide of C.sub.1-C.sub.20, a dialkyl
amide of C.sub.1-C.sub.20, an alkoxyalkylamide, an anhydride of
C.sub.1-C.sub.20, a halide, a nitrile, or an amino group; and
R.sup.2 is C.sub.1-C.sub.10 alkyl, cycloalkyl, alkenyl,
alkoxyalkenyl, alkynyl, aryl, alkaryl, aralkyl, or H.
[0136] More preferably, the functional group derivative of a
cyano-carboxylic acid is an unsaturated cyanocarboxylic acid ester
of the formula: 3
[0137] wherein R is C.sub.1-C.sub.20 alkyl, cycloalkyl, alkenyl,
alkynyl, aryl, alkaryl, aralkyl, or mono- or poly- alkoxyalkyl; and
R.sup.2 is C.sub.1-C.sub.10 alkyl, cycloalkyl, alkenyl, alkynyl,
aryl, alkaryl, aralkyl, or H. Most preferably, R.sup.2 is H, and R
is a C4 alkyl radical. Thus the most preferred cyanoacrylate ester
is a butyl cyanoacrylate, either n-butyl cyanoacrylate: 4
[0138] or isobutyl cyanoacrylate: 5
[0139] Thus R groups (in the second formula on p 36) under the
invention may include, but are not limited to, the following
examples:
[0140] alkyl groups, for example, methyl, ethyl, propyl, butyl,
pentyl;
[0141] cycloalkyl groups such as cyclopropyl, cyclobutyl,
cyclohexyl;
[0142] alkenyl groups such as propenyl, butenyl, pentenyl;
[0143] alkynyl groups such as propynyl, butynyl, pentynyl;
[0144] aryl groups such as phenyl, biphenyl;
[0145] monoalkoxyalkyl groups such as ethoxyethyl, methoxyethyl,
ethoxymethyl;
[0146] polyalkoxyalkyl groups such as (ethoxyethyl)n;
[0147] alkyl amide groups such as N-propyl, N-butyl;
[0148] a dialkyl amide group such as N-dibutyl; and
[0149] an alkoxyalkylamide, such as N-ethoxyethyl.
[0150] According to one aspect of the invention, a trichogenic
composition applied to skin under the invention may comprise
mixtures of two or more cyanocarboxylic acid derivatives.
[0151] Compounds suitable for use in the present invention
include:
[0152] ethoxyethyl 2-cyanoacrylate
[0153] butoxyethyl 2-cyanoacrylate
[0154] n-butyl 2-cyanoacrylate
[0155] isobutyl 2-cyanoacrylate
[0156] n-propyl 2-cyanoacrylate
[0157] isopropyl 2-cyanoacrylate
[0158] n-hexyl 2-cyanoacrylate
[0159] isohexyl 2-cyanoacrylate
[0160] cyclohexyl 2-cyanoacrylate
[0161] benzyl 2-cyanoacrylate
[0162] glycerol 2-cyanoacrylate
[0163] ethoxybutyl 2-cyanoacrylate
[0164] n-pentyl 2-cyanoacrylate
[0165] isopentyl 2-cyanoacrylate
[0166] n-heptyl 2-cyanoacrylate
[0167] isoheptyl 2-cyanoacrylate
[0168] n-octyl 2-cyanoacrylate
[0169] isooctyl 2-cyanoacrylate
[0170] n-nonyl 2-cyanoacrylate
[0171] isononyl 2-cyanoacrylate
[0172] n-decyl 2-cyanoacrylate
[0173] isodecyl 2-cyanoacrylate
[0174] n-butyl 2-cyano-3-methoxyacrylate
[0175] isobutyl 2-cyano-3-methoxyacrylate
[0176] n-butyl 2-cyano-3-phenylacrylate
[0177] isobutyl 2-cyano-3-phenylacrylate
[0178] n-butyl-2-cyano-2-butenoate
[0179] isobutyl-2-cyano-2-butenoate
[0180] n-butyl-2-cyano-2-pentenoate
[0181] isobutyl-2-cyano-2-pentenoate
[0182] n-butyl-2-cyano-2-hexenoate
[0183] isobutyl-2-cyano-2-hexenoate
[0184] n-butyl-2-cyano-2-heptenoate
[0185] isobutyl-2-cyano-2-heptenoate
[0186] n-butyl-2-cyano-2-octenoate
[0187] isobutyl-2-cyano-2-octenoate
[0188] n-butyl-2-cyano-2-nonenoate
[0189] isobutyl-2-cyano-2-nonenoate
[0190] n-butyl-2-cyano-2-decenoate
[0191] isobutyl-2-cyano-2-decenoate
[0192] N-propyl-2-cyanoacrylamide
[0193] N-butyl-2-cyanoacrylamide
[0194] N-pentyl-2-cyanoacrylamide
[0195] N-hexyl-2-cyanoacrylamide
[0196] N-heptyl-2-cyanoacrylamide
[0197] N-octyl-2-cyanoacrylamide
[0198] N-nonyl-2-cyanoacrylamide
[0199] N-decyl-2-cyanoacrylamide
[0200] N-benzyl-2-cyanoacrylamide
[0201] N-cyclohexyl-2-cyanoacrylamide
[0202] N-ethoxyethyl-2-cyanoacrylamide
[0203] N-ethoxypropyl-2-cyanoacrylamide
[0204] N-ethoxybutyl-2-cyanoacrylamide
[0205] N-ethoxypentyl-2-cyanoacrylamide
[0206] N-ethoxyhexyl-2-cyanoacrylamide
[0207] N-ethoxyheptyl-2-cyanoacrylamide
[0208] N-ethoxyoctyl-2-cyanoacrylamide
[0209] N-ethoxynonyl-2-cyanoacrylamide
[0210] N-ethoxydecyl-2-cyanoacrylamide
[0211] N-propoxyethyl-2-cyanoacrylamide
[0212] N-propoxypropyl-2-cyanoacrylamide
[0213] N-propoxybutyl-2-cyanoacrylamide
[0214] N-propoxypentyl-2-cyanoacrylamide
[0215] N-propoxyhexyl-2-cyanoacrylamide
[0216] N-propoxyheptyl-2-cyanoacrylamide
[0217] N-propoxyoctyl-2-cyanoacrylamide
[0218] N-propoxynonyl-2-cyanoacrylamide
[0219] N-propoxydecyl-2-cyanoacrylamide
[0220] N-butoxyethyl-2-cyanoacrylamide
[0221] N-butoxypropyl-2-cyanoacrylamide
[0222] N-butoxybutyl-2-cyanoacrylamide
[0223] N-butoxypentyl-2-cyanoacrylamide
[0224] N-butoxyhexyl-2-cyanoacrylamide
[0225] N-butoxyheptyl-2-cyanoacrylamide
[0226] N-butoxyoctyl-2-cyanoacrylamide
[0227] N-butoxynonyl-2-cyanoacrylamide
[0228] N-butoxydecyl-2-cyanoacrylamide
[0229] Compounds which may prove useful in the practice of the
invention include:
[0230] n-butyl 2-cyano-3-aminoacrylate
[0231] isobutyl 2-cyano-3-aminoacrylate
[0232] thio-n-butyl-2-cyanoacrylic acid
[0233] thio-isobutyl-2-cyanoacrylic acid
[0234] thio-n-propyl-2-cyanoacrylic acid
[0235] thio-isopropyl-2-cyanoacrylic acid
[0236] thio-n-pentyl-2-cyanoacrylic acid
[0237] thio-isopentyl-2-cyanoacrylic acid
[0238] 1-cyano-2-propenyl butyl sulfoxide
[0239] 1-cyano-l-propenyl butyl sulfoxide
[0240] 1-cyano-ethyl butyl sulfoxide
[0241] 2-cyanoaniline
[0242] 2-amino-3-cyanotoluene
[0243] 2,4-diamino-3-cyanotoluene
[0244] 2-butyl-5-cyano-1,4-benzoquinone
[0245] 2-cyano-1,4-benzoquinone
[0246] 2-amino-3-cyano-1,4-benzoquinone
[0247] 2-butyl-6-cyano-2,5-cyclohexadiene-1-one
[0248] 5,6-dihydro-2-oxo-2-H-pyran-3-carbonitrile
[0249] 5-hydro-6-methyl-2-oxo-2-H-pyran-3-carbonitrile
[0250] 5,6,7-trihydro-2-oxo-3-oxepin-carbonitrile
[0251] The dosage of a trichogenic composition under the invention
required for stimulation of hair growth depends on the species of
the subject animal, as well as the age, gender, and overall
condition of the subject, and the degree and cause of the alopecia
or injury to hair-bearing skin. Dosage also depends on the potency
of the active ingredient, its formulation, and mode of application.
Consequently, a precise dosage for each type of treatment is not
given; instead appropriate dosage can be determined by the
experimentalist or caregiver by routine experimentation, for
example, using one or more animal systems as described herein.
Dosages and associated regimens are routine in the art. This
process can be performed for any mammal and, if necessary, for each
recipient prior to a full dose application. The composition can be
simply applied to the skin surface and need not be rubbed into the
skin. In certain situations it may be desirable to apply the
composition by spraying it over a larger skin surface. Such a
spraying might be a preferable approach to application if the
mammals are animals such as sheep, (improved fleece yield), cattle
(improved leather), or valuable fur animals such as minks, etc . .
. .
[0252] One approach involves applying a composition which includes
a cyanocarboxylic acid derivative and a vehicle for that
cyanocarboxylic acid derivative. The ratio of amounts of these can
begin with a composition of 0.0001% by weight of cyanocarboxylic
acid derivative and 99.999% of the vehicle for that derivative and
the results observed over a period of days. Then, the relative
percentage by weight of cyanocarboxylic acid derivative versus the
vehicle is increased until the desired result within the desired
time frame.
[0253] In general, an effective dose of topically applied
trichogenic composition per unit area of skin depends on the active
ingredient and its formulation. In the case of butyl cyanoacrylate,
the dose of active ingredient per unit area of skin surface which
is effective in stimulating hair growth ranges from about 1 .mu.g
cm.sup.-2 to about 20 mg cm.sup.-2. More preferably the dose of
butyl cyanoacrylate is in the range from about 10 .mu.g cm.sup.-2
to about 20 mg cm.sup.-2. Most preferably the dose of butyl
cyanoacrylate is in the range from about 5 mg cm.sup.-2 to about 20
mg cm.sup.-2.
[0254] In other situations it might be preferable to add the
composition to shampoo--for animals or even humans. The amount to
be added to the shampoo varies depending on the amount of hair
growth activity desired. For example, if a significant amount of
hair growth is desired the relative amount of the composition would
be greater than if the rate of hair growth is to be maintained. The
various shampoos would then indicate the level of strength.
C. Experimental
[0255] C.1 Cutaneous Changes Associated with the Application of a
Trichogenic Composition Comprising n-Butyl Cyanoacrylate
[0256] The dorsal aspect of C57BL/KsJ db/+ female mice was shaved
and a single topical dose of n-butyl cyanoacrylate (formulated as
Nexaband.sup.R Liquid) was applied. Within six hours of application
a slight thickening of the treated skin was observed. The response
correlates temporally with inflammation in the dermis and
subcutaneous layer; the keratin becomes irregular and the
epithelial layer shows signs of intermittent disruption;
inflammatory cell infiltrate can be seen in the dermis, and new
aggregates of cells are found in the subcutaneous layer. At this
early stage some of these new aggregates form laminae and appear to
constitute neo-angiogenesis. At day 1 post application, the gross
appearance of inflammation was still apparent, and there was
microscopic evidence for the formation of a lumen from an aggregate
of cells in the subcutaneous layer. At day 2 post application, an
aggregate of cells constituting a lumen in the subcutaneous layer
was shown to be positive for vimentin (indicating mesenchymally
derived cells). At day 3 post application, evidence of
trichogenesis was clearly seen at the treatment site: the epidermis
was multilayered and multiple complex epidermal projections
appeared to constitute hair follicle anlage (FIG. 1). These changes
were only observed in the treated area. At day 10 post application,
microscopic observation showed that the new epithelial pegs had
developed into mature hair follicles (in anagen (active)
phase)(FIG. 2). Between eight and 12 days post application, hair
follicles and new hair were present at the sites of application;
the remainder of the shaved area remained hairless. Epidermal and
dermal thickening and new hair follicle development in treated skin
were pronounced until days 10-20 post application (FIGS. 3A-3B), at
which time large mature hair follicles traversed the entire
thickness of the dermis and subcutaneous layer (FIG. 3C). Hair
follicle density reached a maximum level at about days 20-30 post
application, at which time the epithelium and connective tissue
elements began to return to their pre-treatment appearance (FIGS.
4A-4B).
[0257] At the gross level, the hair growth in the shaved treated
area occurs at 8-12 days and reaches full length at about 14-20
days post treatment, however, the remainder of the shaved untreated
area remains hairless and the hair follicles small (in telogen
(resting) phase)(5A-5B). The response of hair growth to the n-butyl
cyanoacrylate stimulus is very similar in 6 other strains of mice
(C57BL/KsJ +/+, C57BL/KsJ db/db, Balb/cBYj +/+, Balb/cBYj nu/+,
HRS/J hr/+, and RHJ/LeJ hr.sup.rh-j/+) (FIGS. 6A-6C) and Sprague
Dawley rats (FIGS. 7A-7B). The hair growth rate in the fastest
growth period (at about 10-15 days post treatment) reaches 1 mm per
day. This is entirely consistent with the microscopically observed
stimulation of hair follicles following treatment.
[0258] The evidence at both micro- and macro-levels demonstrates
that the net effect of the treatment of skin with n-butyl
cyanoacrylates is induction of de novo development of new hair
follicles and a shift in the hair growth cycle from telogen to
anagen.
[0259] C.2 Induction of Hair Follicles by Subdermal Application of
n-Butyl Cyanoacrylate in vivo.
[0260] The effect of n-butyl cyanoacrylate on the dermis is shown
as follows. An incisional, full-thickness wound, about 2 cm long
was made through the dorsal skin of five mice (strain C57BL/KsJ
db/+). A single dose of about 10 mg of n-butyl cyanoacrylate (in 10
.mu.l of Nexaband.sup.R Liquid) was applied to the bottom of the
wound, and the wound closed with two surgical clips. Control
animals which received 10 .mu.l of phosphate buffered saline (PBS:
0.144 g/l KH.sub.2PO.sub.4, 9.0 g/l NaCl, 0.795 g/l
Na.sub.2HPO.sub.4.7H.sub.2O; pH 7.2) were processed identically.
During the period of 1-90 days post application, the effect of
n-butyl cyanoacrylate treatment was examined by macroscopic
observation. Histological effects were observed by folliculogram:
skin samples containing treated and untreated areas were harvested,
fixed in 10% buffered formalin, embedded in paraffin, sectioned at
5 .mu.m, and stained with Hematoxylin & Eosin or Masson
trichrome in preparation for microscopic examination.
[0261] In a group of five C57BL/KsJ db/+ mice, a full-thickness
excisional wound was made with a biopunch (6 mm diameter) on the
dorsal skin. A single dose of 9 mg of n-butyl cyanoacrylate (in 10
.mu.l of Nexaband.sup.R Liquid) was applied to the bottom of the
wound, and the wound was left open. Control animals which received
10 .mu.l of PBS were treated identically.
[0262] In another example, 20 .mu.l of n-butyl cyanoacrylate was
applied subdermally by subcutaneous injection to a group of
C57BL/KsJ db/+ mice.
[0263] Application of n-butyl cyanoacrylate to incisional and
excisional wounds stimulated hair growth along the wound margin
(FIGS. 8A-8C). Moreover, microscopic examination revealed that
subdermal treatment by application to a full thickness incisional
or excisional wound induced de novo hair follicle formation in the
subcutaneous adipose layer, in the dermis, as well as in the wound
site (FIG. 8B). In each case, new hair follicles subsequently grow
in the wound site through what would normally be scar devoid of
skin appendages as shown in FIG. 8C, indicating an additional
advantage is that the de novo hair follicle development in wound
site accelerates the process of wound remodelling (normalizing).
The direct delivery of n-butyl cyanoacrylate into subcutaneous
layer by subcutaneous injection also induces de novo hair follicle
formation and the thickening of full-thickness skin, compared to
the untreated site (FIG. 9). In the above three cases, the
phenomenon that the farther the distance from the treated site is,
the fewer and the smaller the hair follicles are, again
demonstrating the localized nature of the effect of n-butyl
cyanoacrylate on hair growth
[0264] C.3 The Stimulation of Hair Growth by n-Butyl Cyanoacrylate
in Mice Previously Treated with Cyclophosphamide and
Doxorubicin
[0265] The induction of hair loss following treatment with certain
anticancer drugs, such as cyclophosphamide or doxorubicin, is well
documented (A. Tierney & J. Taylor (1991) Nurs. Stand 5:29-31;
R. R. Love, et al. (1989) Cancer 63:604-612; B. W. Cline (1984)
Cancer Nurs. 7:221-228). The following experiment was performed to
determine the effect of n-butyl cyanoacrylate on hair regrowth in
mice pre-treated with cyclophosphamide or doxorubicin. Twenty mice
of strain C57BL/KsJ db/+ were peritoneally injected with either
cyclophosphamide in PBS (20 mg per kilogram of body weight) or
Doxorubicin in PBS (2 mg per kilogram of body weight) for 10
consecutive days. Then a single 10 .mu.l dose of n-butyl
cyanoacrylate was applied topically to an area of the shaved dorsum
of each animal. Control mice, which were injected with PBS alone,
were treated similarly. The effects of hair regrowth were
determined both by phototrichogram (macroscopic observation using
photography) and by folliculogram (microscopic observation of
histological changes in the treated and untreated skin).
[0266] All mice pre-treated with cyclophosphamide, doxorubicin and
PBS showed rapid hair regrowth in the areas where n-butyl
cyanoacrylate was applied: beginning at 8-11 days and growing to
full length at 15-18 days post treatment with n-butyl
cyanoacrylate. In contrast, in the adjacent skin not treated with
n-butyl cyanoacrylate in the groups pre-treated with
cyclophosphamide or doxorubicin, hair regrowth did not occur until
80 days after the beginning of the experiment (FIGS. 10A-10B). In
the animals pre-treated with cyclophosphamide, lack of pigmentation
was observed in the new hair growth as well as in the skin of the
untreated hairless area. The new hair occurred only in the areas
treated with n-butyl cyanoacrylate and remained until the mice were
sacrificed 335 days after treatment with n-butyl cyanoacrylate
(FIG. 10C). This is a good indicator that the hairs induced by
n-butyl cyanoacrylate are persistent and join into the last pelage.
Thus the dual treatment with cyclophosphamide and n-butyl
cyanoacrylate together can be used to study the mechanism of the
melanogenesis metabolism, the prevention and the treatment of
various diseases of abnormal pigmentation metabolism.
[0267]
[0268] C.4 The Effect of n-Butyl Cyanoacrylate Treatment on Hair
Growth in ex vivo
[0269] The studies on induction of hair follicle or elongation of
hair shaft with organ culture methods are well documented (R. F.
Oliver (1970) J. Embryol. Exp. Morphol. 23:219-236; C. A. B. Jahoda
(1992) Development 115:1103-1109; C. A. B. Jahoda & A. J.
Reynolds (1993) J. Investg. Dermatol. 101:33S-38S). In those
studies, the elongation of the hair shaft is too short and required
microscopic observation. The following experiment was performed to
determine how strong the effect of n-butyl cyanoacrylate on hair
growth was in the condition of organ culture and if the hair growth
was visible.
[0270] The shaved dorsal skin of mice (C57BL/KsJ db/+ and C57BL/KsJ
db/db) was treated with n-butyl cyanoacrylate by a single topical
application at a dose of 10 mg cm.sup.2. One hour later, the skin
(0.5.times.1.0 cm) containing both treated and untreated areas was
excised, rinsed in PBS, cultured in Dulbecco's Modified Eagle
Medium (Gibco BRL, Gaithersburg, Md.) supplemented with 10% fetal
bovine serum (HyClone Laboratories, Logan, Utah) at an atmosphere
of 95% O2 and 5% CO2, 37.degree. C. Hair growth was recorded by
macroscopy. Hair growth in the treated area was observed at days
7-12 post treatment and lasted to the end of the experiment (at day
25 post treatment); the untreated area remained hairless (FIG.
11).
[0271] C.5 The Effect of n-Butyl Cyanoacrylate Treatment on Growth
Factor Activity in Skin Extracts
[0272] A single topical application of 20 mg of n-butyl
cyanoacrylate was made to an area of shaved skin on the dorsum of
strain C57BL/KsJ db/+ mice. At days 10 and 20 post-treatment, skin
samples were excised from treated areas. Skin tissue was frozen in
dry ice, minced, homogenized in ice-cold PBS, and centrifuged at
15,000 g for 30 minutes at 4.degree. C. The protein concentration
of supernatants was adjusted to 1.0 mg/ml by the BioRad assay
procedure (BioRad Laboratories, Richmond, Calif.). Skin samples
from untreated areas of the same strain were harvested and
processed identically. Mouse serum albumin was prepared at a
concentration of 1.0 mg/ml in PBS as a control. An aliquot of each
supernatant extract was fractionated according to size with a
nominal 30 kilo Dalton (kDa) cutoff, and the low molecular weight
fraction (.ltoreq.30 kDa) was included in the fibroblast cell
proliferation assay, as follows.
[0273] The supernatant extracts from treated and untreated skin
samples, and mouse serum albumin control, were added to a quartet
of wells in a 96 well plate, and 2-fold serially diluted 11 times.
NIH/3T3 fibroblasts (ex American Type Culture Collection,
Rockville, Md.) were harvested at about 80% confluence, seeded into
each well at a density of 5,000 cells per well, and supplemented
with serum-free assay medium (QBSF 56, Quality Biological Inc.,
Gaithersburg, Md.). Cell proliferation was determined according to
the protocol of the CellTitre 96 Non-Radioactive Cell Proliferation
Assay (Promega, Madison, Wis.). The results (FIGS. 12A-12B)
indicated that protein extracts of treated skin were more active in
stimulating proliferation of NIH/3T3 cells, as compared with the
protein extract of untreated skin. The extract from skin harvested
at 20 days post-treatment was more active than the extract from
skin harvested at 10 days post-treatment. In contrast, the low
molecular weight fraction of both treated and untreated skin,
comprising proteins of 30 KDa or less, showed no discernible effect
on cell proliferation. This indicates that component(s) of treated
skin which are active in the cell proliferation assay have
molecular weights in excess of 30 KDa. The control protein
preparation of mouse serum albumin showed no effect on NIH/3T3 cell
proliferation.
[0274] C.6 The Relationship between the Dose of n-Butyl
Cyanoacrylate and the Response of Hair Growth
[0275] The effect of n-butyl cyanoacrylate concentration on
stimulation of hair growth was investigated as follows. Five mice
of each strains C57BL/KsJ db/+ and C57BL/KsJ db/db were shaved on
the dorsum, treated with a single topical application of either
undiluted Nexaband.sup.R Liquid (containing about 10 mg of n-butyl
cyanoacrylate per 10 .mu.l), or Nexaband.sup.R Liquid diluted to
50% or 25% with vegetable oil. Hair growth was macroscopically
scored at 49, 61 and 86 days after treatment. All animals treated
with undiluted Nexaband.sup.R Liquid were rated at the maximum
score at all three time points. Nexaband.sup.R Liquid diluted by
50% was less effective in stimulating hair growth than undiluted
Nexaband.sup.R, while a 25% dilution was even less effective (Table
1). However, hair growth in the area treated with a 25% dilution
was still much greater than in the adjacent untreated area, thereby
demonstrating that the effect of the invention on stimulating hair
growth is dose-dependent, i.e., adjustable in practical use.
1TABLE 1 Effect of Various Concentrations of n-Butyl Cyanoacrylate
on Hair growth Number of Animals Exhibiting Hair Growth* 49 61 86
Concen- Score: tration Strain 0 1+ 2+ 3+ 0 1+ 2+ 3+ 0 1+ 2+ 3+ 25%
db/+.sup.1 4 1 2 3 3 2 db/db.sup.2 2 2 1 2 1 1 1 3 2 50%
db/db.sup.2 2 3 1 4 2 3 100%.sup.3 db/+.sup.1 5 5 5 db/db.sup.2 5 5
5 *:The scoring scale used is : 0, no obvious hair growth; 1+, mild
hair regeneration in an area defined as less than 10% of the
treated area; 2+, moderate hair regeneration in an area larger than
10% but less than 50% of the treated area; 3+, high hair
regeneration with an area larger than 50% of treated area. .sup.1,
C57BL/KsJ db/+ mice; .sup.2, C57BL/KsJ db/db mice; .sup.3, 10 mg/10
.mu.l of n-butyl cyanoacrylate.
[0276] C.7 Influence of n-Butyl Cyanoacrylate on Concentration
& Localization of Growth Factors in Skin.
[0277] Forty stock female mice of strain C57BL/KsJ db/+ at eight
weeks of age were treated with a single dose of n-butyl
cyanoacrylate applied topically to the shaved dorsum. Animals were
sacrificed at days 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22
post-treatment, and skin samples were excised to yield an area of
n-butyl cyanoacrylate treated skin together with a contiguous,
adjacent sample of untreated skin. In preparation for
immunohistochemistry, samples were washed in PBS, fixed in 10%
formalin, and secondarily fixed in Bouin's solution prior to
paraffin embedding. Multiple 4-5 .mu.m sections were placed on
slides pre-coated with 3-aminopropylethoxysilane.
[0278] Antibody detection staining was performed using the
avidin/biotin peroxidase complex method (J. M. Elias, M. Margiotta,
& D. Gabore (1989) J. Am. Clin. Pathol. 92:62). The following
primary antibodies were used: anti-TGF-.beta.1 neutralizing
antibody; anti-TGF-.beta.2,3; and anti-EGF receptor.
[0279] Localization of the various growth factors was observed as
follows: The overall staining pattern for the presence of
TGF-.beta.1 in skin tissue treated topically with n-butyl
cyanoacrylate is shown in FIG. 13. Skin treated with n-butyl
cyanoacrylate showed a specific spatial and temporal distribution
of TGF-.beta.1. TGF-.beta.1 was detected in the sebaceous glands,
the epithelial cells of the epidermis, hair follicles, and
connective tissues (FIGS. 1A-1B). The intensity of staining for
TGF-.beta.1 reached a maximum at day 4 and declined to a relatively
low level by day 15 post-treatment. This distribution pattern for
TGF-p1 implicates TGF-.beta.1 in the differentiation and
development of new hair follicles, and indicates its involvement in
regulating the hair growth process. TGF-.beta.1 staining was more
intense in connective tissue subjacent to treated skin, as compared
with untreated skin. A difference in TGF-.beta.1 distribution was
observed between developing and mature hair follicles: staining was
fairly uniform throughout epithelial cells of developing follicles,
but appears to be confined to the outer root sheath in mature
follicles.
[0280] The distribution of TGF-.beta.2,3 and EGF in treated and
untreated skin tissues was similar to that for TGF-.beta.1.
However, the intensity of TGF-.beta.2,3 stain in the epidermis was
greater than that of TGF-.beta.1 throughout the study period. In
connective tissue from treatment areas, much less staining activity
of EGF-receptor was found as compared with TGF-.beta.1.
[0281] C.8 The Inhibition of n-Butyl Cyanoacrylate-Stimulated Hair
Growth by Treatment with Anti-TGF-.beta.1-Neutralizing Antibody
[0282] Forty mice of strain C57BL/KsJ db/+ eight weeks old were
divided into four equal groups (A-D). The dorsal aspect of the mice
were shaved, and five different sites on the dorsum were designated
(sites 1-5). Each site was 5-6 mm.sup.2. Animals of group A were
treated topically at sites 1, 2 & 3 with n-butyl cyanoacrylate
only (10 .mu.l per site) formulated as Nexaband.sup.R Liquid.
Animals of group B were treated topically at sites 1, 2 & 3
with the same dose of n-butyl cyanoacrylate, but sites 1 and 3
subsequently received subcutaneous injections of either
anti-TGF-.beta.1 neutralizing antibody (1 .mu.g per g of body
weight) in PBS/0.1% BSA (at site 1), or PBS/0.1% BSA as a matched
control (at site 3). Group C animals were treated as for Group B
except that anti-EGF neutralizing antibody was injected at site 1
instead of anti-TGF-pi. In all groups, site 4 was a shaved area of
skin that did not receive any treatment, while site 5 was an area
of normal skin that remained unshaved and received no treatment.
Group D animals were shaved only and received no treatment.
[0283] Hair regeneration was documented by macroscopic observations
supported by serial photographs of treatment sites. Histological
observations were made on skin biopsies taken at frequent intervals
ranging from 6 hours to 21 days post-treatment.
[0284] Stimulation of hair growth at sites treated with n-butyl
cyanoacrylate alone was clearly evident by day 14 at site 2 and 3
in Group B. In contrast, hair growth was much less at site 1
injected with anti-TGF-.beta.1-neutralizing antibody (FIG. 14).
After about 15-20 days post-treatment (i.e. at about 10-15 days
after antibody injections were discontinued) hair at these sites
attained the thickness of that at sites treated with n-butyl
cyanoacrylate alone. Little effect was observed following injection
of anti-EGF neutralizing antibody. These results implicate
TGF-.beta.1 in stimulation of hair growth following treatment with
n-butyl cyanoacrylate.
[0285] C.9 Effect of Isobutyl Cyanoacrylate on Hair Growth
[0286] The following experiment was performed to identify the
effect of isobutyl cyanoacrylate, a structural analog of n-butyl
cyanoacrylate, on hair growth. The shaved backs of C57BL/KsJ db/+
and C57BL/KsJ db/db mice were treated topically at a dose of 10
.mu.l cm.sup.-2 with either isobutyl cyanoacrylate (Sigma Chemical
Co., St Louis, Mo.) or n-butyl cyanoacrylate. The hair growth
response stimulated by isobutyl cyanoacrylate in both strains was
nearly identical to that inducted by n-butyl cyanoacrylate: In
treated area many new hair follicles occurred 2-3 days post
treatment, hair growth was visible 8-12 days post treatment and
reached full length at 15-18 days post treatment; the untreated
areas were still hairless (FIG. 15 & Table 2), clearly
demonstrating that the cyanoacrylate group in either n-butyl
cyanoacrylate or isobutyl cyanoacrylate is the component
responsible for hair growth.
2TABLE 2 Effect of Cyanoacrylates on Hair Growth Agents
Species/strain No. of animals Hair growth* IBC C57BL/KsJ db/+ 10 3+
C57BL/KsJ db/db 10 3+ NBC C57BL/KsJ +/+ 10 3+ C57BL/KsJ db/db 10 3+
*The scoring scale used is: 0, no obvious hair growth; 1+, mild
hair regeneration in an area defined as less than 10% of the
treated area; 2+, moderate hair regeneration in an area larger than
10% but less than 50% of the treated area; 3+, high hair
regeneration with an area larger than 50% of the treated area. NBC,
n-butyl cyanoacrylate; IBC, isobutyl cyanoacrylate.
[0287] C.10 Characteristics of Hair Growth Stimulated by
Cyanoacrylate
[0288] Under this invention, two components in the family of
cyanoacrylate, n-butyl cyanoacrylate and isobutyl cyanoacrylate,
were tested in two species (rat and mouse) and seven strains of
mouse(see section C.1) and found to stimulate hair growth in normal
intact skin, in intact skin of the animals pre-treated with either
of two anticancer drugs, and at the edges of excisional and
incisional wounds.
[0289] The hair growth induced by n-butyl cyanoacrylate has the
following characteristics:
[0290] 1. A single application of the hair growth stimulator can
induce hair follicle shift from telogen to anagen and de novo
development of hair follicles, resulting in profound hair
growth.
[0291] 2. The reproducibility of hair growth induced by the
stimulator at the dose of 10 mg/10 .mu.l cm.sup.-2 is 100% in more
than 700 normal adult mice and rats, and 80 genetically
healing-impaired diabetic (db/db) animals (FIGS. 5-7).
[0292] 3. The ability of the stimulator to stimulate hair growth is
very strong and specific. New hair follicle formation in the
treated area occurs as early as 2-3 days after treatment (FIGS.
1A-1B); the skin thickened and the new hair growth can been seen as
early as 8-12 days in treated area (FIGS. 2-3); the hair growth
rate in the fastest growth period can reach 1 mm per day; and the
size of new hair follicles is larger; and the new hairs are more
pigmented, more coarse in diameter, and 1-2 mm longer than those in
adjacent untreated area (FIGS. 5-8). Hair growth also can be
induced ex vivo (FIG. 11), an useful model for studies on
mechanisms of hair follicle formation and for tests of the effects
of drugs on hair growth.
[0293] 4. The hair growth can be induced even when the
melanogenesis metabolism of hairs is severely suppressed by
administration of an anticancer drug, cyclophosphamide (FIG. 10).
Hair follicle regeneration and active pigment metabolism are
closely related to each other and up to now there were no models in
vitro or in vivo that could separate the two processes. Therefore,
one advantage of this invention is to establish an experimental
animal model to study the mechanism of melanogenesis.
[0294] 5. The induced hair is the last pelage of the animals, and
no shedding of hair has been found over 210 days in rats and 335
days in mice post treatment. Even in the genetically
healing-impaired diabetic mice, hair regrowth in treated areas
remains unchanged, but the shaved untreated areas are hairless more
than 216 days post treatment. In contrast to this, after a single
application of n-butyl cyanoacrylate, the hair keeps regrowing in
the treated areas even if the area. is subsequently shaved three
times at 20-day intervals.
[0295] 6. The induced hair growth is dose-dependent, i.e., the
extent of hair growth varies with the extent of dilution and is
adjustable.(Table 1).
[0296] 7. The hair growth response is completely localized to the
area where the stimulator is applied. A clear demarcation between
treated and untreated skin signals the localized nature of the
response. Scanning electron micrography revealed that hair
follicles in treated skin were located deeper within the dermis
than those in untreated skin (FIG. 5A).
[0297] 8. No any side effect or toxic effect or tumor-like tissue
formation after the treatment has been observed except for a mild
inflammation shortly after application.
EXAMPLES
Example 1
Induction of Hair Follicle Differentiation and Development in Adult
Mammals
[0298] The dorsal aspect of C57BL/KsJ db/+ female mice was shaved
and a single topical dose of n-butyl cyanoacrylate (formulated as
Nexaband.sup.R Liquid) was applied. Beginning at day 2 post
treatment, aggregates of cells formed in the adipose tissue of the
subcutaneous layer. During subsequent days the number of such
cellular aggregations increased. only the outer cells stains
positive for vimentin and are presumed to be of mesenchymal origin,
consistent with the induction of hair follicles in treated skin by
a mechanism similar to that occurring during the neo-natal
period.
[0299] The observed development of hair follicles outside the
dermis was confirmed above where n-butyl cyanoacrylate was applied
to incisional and excisional wounds or injected subdermally.
Example 2
Stimulation of Hair Growth in a Domesticated Animal by Treatment
with n-Butyl Cyanoacrylate
[0300] A domesticated animal, for example, having undergone hair
loss due to disease, cancer chemotherapy, aging, skin parasites, or
other causes, is treated with a trichogenically effective
formulation of a cyanocarboxylic acid derivative. Following
treatment, new, normal terminal hair grows from the treated area.
The treatment is repeated as necessary to provide hair growth at
the desired level.
Example 3
Localized Induction of Hair Regrowth in Animals Following
Injury
[0301] A domesticated animal, having undergone localized trauma to
hair-bearing skin is mildly anesthetized, the wound dried, and 10
mg/cm.sup.2 of n-butyl cyanoacrylate "painted" over the area of the
wound. After a few seconds the n-butyl cyanoacrylate polymerizes
and no other dressing is required. Within 10 days new hair grows
from the healing or healed area.
Example 4
Cyanoacrylate Esters as Cosmetic Products for Show Animals
[0302] Esters of cyanoacrylate are effective as cosmetic products
by providing show animals with increased quantity and improved
quality of hair growth. Under the invention a physiologically
effective formulation of a cyanoacrylate ester is applied topically
to the skin of the subject animal, by brushing it to the underlying
skin. Alternatively, the physiologically effective formulation may
be applied to the coat and underlying skin by means of a spraying
device or any other means known in the art. The dose and frequency
of the application are varied depending on the nature of the animal
and the type of results desired, as will be apparent to the skilled
artisan. Following treatment, the coat grows thicker, is more
pigmented and appears more healthy; as compared with the coat of a
similar but untreated animal.
Example 5
Stimulation of Growth of the Coat of a Domesticated Animal Used as
a Commercial Source of Fiber
[0303] A physiologically effective amount of a suitable formulation
of a cyanoacrylate ester is applied to an animal used as a
commercial source of fiber. Application is effected as described in
Example 4. Preferably treatment with cyanoacrylate ester occurs a
suitable period of time prior to clipping the coat (e.g. in the
case of sheep) or before sacrifice of the animal (e.g. mink).
Suitable treatment of animals with an ester of cyanoacrylate
improves both the quantity and quality of the fiber which they
produce.
Example 6
Treatment of Skin of Domesticated Animals for Improved Leather
& Suede Production
[0304] Domesticated animals are treated with a physiologically
effective amount of a formulation of an ester of cyanoacrylate, as
described in Example 5. Due to the nature of the response of
mammalian skin following treatment, only a single application may
be required. Preferably the application is made a few days before
slaughter. The optimum time for application depends on those
considerations well within the grasp of those skilled in the art.
This treatment provides superior leather products.
Example 7
Treatment of Alopecia in Humans with an Ester of Cyanoacrylate
[0305] A human patient with a hair loss problem is treated with a
topical formulation comprising a trichogenically effective dose of
an ester of cyanoacrylate which is applied to the affected area.
The formulation is allowed to remain in situ for a period of about
24 hours. Such treatment results in stimulated hair growth within a
period of a few days to a few weeks. The application may be
repeated as necessary.
Example 8
Treatment of a Incisional or Excisional Wound on a Special Site in
Humans
[0306] A human patient with a fresh incisional or excisional wound,
or a pre-existed incisional or excisional wound on scalp, the
site(s) of mustache, eyebrow, beard, etc., is treated with a
topical formulation comprising a trichogenically effective dose of
an ester of cyanoacrylate which is applied to the affected area.
Such treatment results in stimulated hair growth within a period of
a few days to a few weeks, with an additional advantage that the
normalizing process of the tissue (remodelling process) of the
incisional or excisional wound is accelerated and scar formation is
greatly decreased.
[0307] The present invention has been described in various
embodiments, it will be apparent to one of ordinary skill that many
modifications can be made thereto which nevertheless utilize the
methods and compositions of the invention as disclosed. The scope
of the invention is defined by the appended claims rather than by
the embodiments presented above.
* * * * *