Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND ~ ~ODS FOR ENHANCING THE GROWTH OF HAIR AND
K~-l-O~ING HAIR COLOR
FIELD OF THE lNV~N-'l'lON
.
The present invention relates to methods for stimulat-
ing hair follicles and promoting the growth of hair in intact
skin, especially the scalp. More particularly, the invention
relates to a method for inducing angiogenesis and causing
increased vascularization and increased circulation (vasodila-
tion) of intact skin tissue and stimulating dormant, dying or
weak hair follicles to produce hair or promote the growth of
hair in such skin and reduce hair loss. The present invention
also relates to a method for stimulating nail growth and
producing stronger nail (ungual) tissue. In addition, the
present invention also relates to a method for promoting
melanogenesis and restoring the natural color to hair in which
the color is diminished or ~;~;n;~h;ng, such as in gray or
graying hair. The compositions of the present invention which
are used to enhance hair and nail growth and promote
melanogenesis and restore natural hair color are compositions
which are based on a minimum essential medium in combination
with preferably at least two non-steroidal anabolic hormones
one of which hormones is insulin and the other which is
selected from triiodothyronine, thyroxine and growth hormone.
Three anabolic hormones, including insulin, triiodothyronine
or thyroxine and growth hormone in combination with minimum
essential media may be preferably used. Preferably, an
enriched growth media such as MCDB 153 or related enriched
growth media is used in combination with insulin,
triiodothyronine or thyroxine and growth hormone. Composi-
tions for promoting hair and nail growth may also contain an
effective amount of a penetration enhancement agent for
~ promoting the penetration of the active constituents of the
compositions through the surface of the skin where
angiogenesis, melanogenesis and vascularization may be
promoted.
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BACKGROUND OF THE INVENTION
Hair follicles produce different types of hair at dif-
ferent stages in an animal's life. The hairs formed may
change quite dramatically in thickness, color, and length.
Thus, hair follicles have a transforming ability which allows
An; ~ls to adjust to seasonal changes and to life cycle
changes (i.e., hormonal or metabolic changes) over the course
of an ~n; ~1 's life. This allows the rapid distinction
between young animals and adults and between the adult sexes.
Thus, hair growth plays a major role in social and sexual com-
munication in mammals and explains why hair disorders such as
hirsutism, androgenetic alopecia and alopecia areata often
create psychological problems.
To enable hair follicle life cycle changes, hair fol-
licles pass through regular cycles of hair development and
growth (anagen) followed by periods of resting (telegen). The
hair produced after a resting period may be very similar to
the previous one produced, as seen in many hair follicle
cycles on the eyelids and the young human scalp; or, it may be
slightly or even markedly different. In certain instances,
for example during aging, stress conditions and the like in a
cycle known as catagen, the hair may actually regress in qual-
ity and cease growing. The precise mechanism of how altera-
tions occur in the type of hair produced by a hair follicle
are unknown. Methods which are shown to stimulate hair fol-
licles and enhance the growth of hair may be used to regulate
hair follicle disorders and increase hair production by
domesticated animals such as sheep.
The hair follicle is composed of epithelial components
(the matrix and outer root sheath) and dermal components (the
dermal papilla and connective tissue sheath). Hair growth is
effected by the division of the hair follicle matrix cells
under the control of the dermal papilla. Three distinct
stages of hair growth can be identified, an active phase
(anagen) during which hair growth occurs, an intermediate
regressive phase (catagen) during which hair actually ceases
growing or even regresses and a resting phase (telogen) during
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which no cell proliferation occurs. The factors that regulate
cell division are poorly understood, although growth factors,
steroid hormones, dermoepithelial interactions and the immune
system have been implicated. Philpott, et al., J. Cell Sci.,
97 (Pt3), 463 (1990), recently demonstrated that growth factors
such as epidermal growth factor (EGF) mimic the in vivo
depilatory action of EGF resulting in the formation of a club
hair-like structure; and that transforming growth factor
(TGF)-*1 may serve as a negative growth regulatory factor for
the hair follicle.
One area of investigation into hair growth involves
the use of androgens (steroidal hormones) where it has been
shown that androgens modulate the type of hair produced by
hair follicles in humans.
A number of recent publications evidence that human
scalp cells and hair may be grown in vitro. Several publica-
tions report the use of hormonally supplemented serum-free
cell culture medium of the MCDB series as an optimal medium
for growing human hair cells in culture. Kitano, et al., J.
Dermatol., l9(11), 793, (1992), used MCDB 153 supplemented
with amino acids, hydrocortisone, insulin, EGF and bovine
pituitary extract to grow isolated human scalp hair follicles
n vitro. Tanigaki, et al., Arch. Dermatol. Res., 282(6),
402, (1990), used MCDB supplemented with 7 growth factors to
induce hair cell differentiation of C3H mice hair cells in
culture. M.P. Philpott, et al., Ann.NY Acad. Sci., 642, 148
(1991), reported that serum inhibited hair follicle cell
growth n vitro while serum-free medium supported its growth.
Tobin, et al., Arch. Dermatol. Res., 285(3), 158 (1993), main-
tained human hair follicles in culture in serum-free medium.
The main source of energy for hair follicle cells in
the culture medium was reported by Williams, et al., J.
Invest. Dermatol., 100(6), 834 (1993). That group observed
that the glucose-glutamin cycle supports human hair follicle
growth rate. Philpott, et al., J. Cell Sci., 97 (Pt3), 463
(1990), demonstrated that growth factors such as TGF-beta in
vitro and TGF-alpha and EGF in vivo had negative effects on
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hair follicle growth. Thyroxine participation in in vivo
scalp hair growth in hyper- and hypothyroidism was reported by
Kiesewetter, et al., Z. Hautkr., 65(12), 1120 (1990). Li, et
al., Proc. Natl. Acad. Sci. USA, 89(18), 8764 (1991), reported
success in maintaining intact human scalp skin in culture.
The organ culture t~chn; que reported in this reference evi-
denced hair elongation, thymidine incorporation and a con-
tinued hair growth cycle. The presence of mast cells was
associated with the anagen phase while macrophages were seen
during the catagen phase of the cycle, suggesting the role of
the circulatory cells in the process of the hair growth cycle.
In vivo experiments investigating hair growth recently
have been reported by a number of investigators. Skoutelis,
et al., J. Invest. Dermatol., 95(2), 139 (1990) indicated that
neovascularization is a necessary component in the anagen
phase of hair growth, suggesting that adequate blood supply to
the skin is vital for hair growth. Randall, et al., J.
Invest. Dermatol., 101(1), 114s-120s (1993), described the ebb
and wax of the human hair growth annual cycle. These cycli-
cal changes must be taken into account when conducting in vivo
experiments in order to ensure accuracy.
Studies on the in vitro culturing of follicular cells
invariably refer to the usage of serum-free medium. In some
cases, MCDB 153 and Williams media are specifically mentioned.
Where serum supplement was added to the media, the result was
an inhibition of hair growth. In the in vitro studies in the
art, only intact hair follicles were capable of growing hair
and then only for a limited period of time whereupon senes-
cence of the culture cells ensued. In studies where the cells
were dispersed, either by trypsinization or other mechanical
methods, hair growth did not occur. This may suggest that
intact hair follicles may be capable of being revitalized.
In other studies, the effect of proteoglycans in the
hair growth cycle was determined. Chondroitan 6 sulphate,
unsulphated condroitin, dermatan sulphate and heparin sulphate
were studied for their effects of hair growth. A direct cor-
relation between the presence of chondroitan proteoglycans and
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hair growth was established. Still other studies have shown
that keratin growth factor may stimulate hair follicles as
well as sebaceous glands.
The importance of the vascularization of the dermis to
the process of hair growth is well known. Thus, any factor
which negatively affects the skin microvasculature will most
likely also undermine hair growth.
The presently available product Minoxidiltm has been
shown to be efficacious in enhancing hair growth. Its
mechAn;~m of action is based upon its vasodilatory effect
which causes shifting of the fluid volume from the vascular
compartment into the extracellular compartment, especially in
the dermal area. This, in turn, may cause an increase in
blood circulation in the dermis and increased supply of
nutrients to the dermis affecting the hair follicles.
In experiments on wound healing, MCDB 153 supplemented
with non-steroidal anabolic hormones was used as treatment on
surgical wounds. The skin on the dorsum of all the An; ~ls
was depilated prior to the extirpation of the skin patches.
During the change of bandages it was noted that the depilated
hair on the skin adjacent to the wound edges was growing at a
faster rate than the hair on areas further away from the
treated wound. The application of MCDB 153 including anabolic
hormones induced vascularized granulation tissue formatio~ as
well as epithelialization. The barrier for penetration
through the skin, the keratinized layer, was overcome by the
opening in the wound area. This condition allows for
nh;n~ered contact between the medium and the deeper layers of
the skin including the hair follicle cells and the capillary
endothelial cells.
Unpublished data by the present inventor on angiogenic
activity of supplemented MCDB 153 (with insulin, growth
hormone and triiodothyronine or thyroxine) performed on the
CAM (chorioallantoic membrane) indicated that the application
of the gelled medium did not elicit vascular response. The
gel did not appear to penetrate the external epithelial layers
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of the CAM. However, clinical experience has repeatedly shown
that in healing wounds, either burn wounds or surgical wounds,
where angiogenic activity is evident, faster rate of hair
growth was found adjacent to the wound area.
Additional unpublished data by the present inventor
using supplemented MCDB 153 gel (insulin, growth hormone and
triiodothyronine/thyroxine) on surgically induced corneal
wounds yielded pronounced and unexpected angiogenic response
in all experimental test animals. The angiogenic response was
expressed by coiled corneal neovascularization of branches of
the superficial and deep anterior ciliary arterial plexus and
by engorgement of all the surrounding vascular bed. When the
gel was applied to the intact eye, no irritation or angiogenic
activity was found.
BRIEF DESCRIPTION OF THE INVENTION
The present invention relates to formulations and
methods for promoting hair or nail (ungual tissue) growth in
intact skin of ~n; ~1 s, especially including the scalp of
humans. The formulations according to the present invention
are also useful for promoting angiogenesis in the dermis and
for stimulating dormant, dying or weak hair follicles to pro-
duce hair or promote the growth of hair in such skin. In con-
cert with such enhanced growth, the natural hair color of the
growing hair is restored in hair diminished in its natural
color. This is an unexpected result. Compositions according
to the present invention are also useful for enhancing the
growth and increasing the strength of ungual tissue as well as
correcting irregularities in such tissue. Methods for enhanc-
ing angiogenesis and increasing vascularization and blood flow
in intact skin, restoring the natural hair color to growing
hair, as well as methods for revitalizing intact skin are also
embraced by the present invention. The formulations according
to the present invention are useful for enhancing hair growth
by causing increased vascularization of intact skin tissue and
stimulating dormant, dying or weak hair follicles to produce
hair or promote the growth of hair in such skin. The formula-
tions according to the present invention are also useful for
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promoting melanogenesis in hair follicles and for restoring
the hair color in hair which exhibits diminished hair color
such as in gray, graying and damaged hair.
The present formulations comprise a hair or nail
growth enhancing effective amount of a non-steroidal anabolic
hormone selected from insulin, growth hormone,
triiodothyronine and thyroxine (T3 or T4), and mixtures
thereof, most preferably a mixture of all three hormones, in
combination with a hair or nail growth enhancing effective
amount of minimum essential medium, preferably a supplemented
medium such as MCDB 153. The formulations may be adjusted to
enhance penetration of the individual components through
intact skin in order to promote angiogenesis of the underlying
dermal layers and consequently, to stimulate hair follicles
and ungual tissue and promote hair or ungual tissue growth.
All of the components used in the compositions according to
the present invention are included in amounts effective for an
intended use generally- enhancing or stimulating hair and/or
nail growth or restoring hair color in hair exhibiting
~; ;n;~hed color.
In preferred embodiments according to the present
invention, the non-steroidal anabolic hormone is a mixture of
insulin and at least one or more anabolic hormones such as
triiodothyronine, thyroxine and growth hormone. It has been
unexpectedly discovered that a mixture of the anabolic
hormones insulin and triiodothyronine, thyroxine or growth
hormone produces a synergistic enhancement in hair growth when
combined with a m; n; lm essential medium. In more preferred
embodiments according to the present invention, the anabolic
hormone comprises a mixture of an amount of insulin, growth
hormone and triiodothyronine or thyroxine in amounts effective
to substantially enhance the growth of hair or ungual tissue,
also synergistically. Embodiments in which the anabolic
hormone is a mixture of effective amounts of triiodothyronine
or thyroxine and growth hormone or insulin and growth hormone
are also contemplated by the present invention. Generally in
concert with enhanced hair growth, hair color restoration
occurs in hair which is diminished in hair color. Composi-
-
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-8-
tions which restore hair color are generally made of the same
componentry and such components are included in the same gen-
eral amounts which are useful for promoting hair growth.
In general, insulin is included in compositions
according to the present invention at concentrations ranging
from about 5 ng/ml (nanograms/ml) to about 100 ug/ml (micro-
grams/ml) (preferably, at least about 50 ng/ml within this
range), more preferably about 500 ng/ml to about 20 ug/ml.
When a non-steroidal anabolic hormone other than insulin is
included in compositions according to the present invention,
for example, triiodothyronine, thyroxine or human growth
hormone, among others, each of these other hormones is
included in an amount effective to enhance the growth of hair
or ungual tissue, i.e., in an amount of at least about 0.05
ng/ml of the formulation, with a preferred range of about 0.5
ng/ml to about 100 ng/ml. In compositions which are delivered
in solid or concentrated form, i.e. as a gel, creme or the
like, the anabolic hormone is included in concentrations
similar to those contained in the solutions (based upon the
general assumption that 1 ml of solution is approximately
equal to about 1 gram in weight of the final composition).
Percent weights may fall outside of these ranges, depending
upon the ability to deliver the individual components of the
formulations through the skin, the level of stability of the
hormone and other factors, as well recognized by one of
ordinary skill in the art.
In addition to insulin, the compositions according to
the present invention preferably include at least one anabolic
hormone selected from triiodothyronine, thyroxine, and growth
hormone and most preferably both growth hormone and
triidothyronine or thyroxine, in combination with at least a
m;n; lm essential medium, preferably a supplemented minimum
essential medium such as MCDB 153. When growth hormone is
used, the preferred growth hormone is human growth hormone,
preferably in combination with triiodothyronine (T3),
thyroxine (T4) or insulin and more preferably in combination
with both insulin and triiodothyronine (T3) or thyroxine (T4).
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_g _
The preferred amount of anabolic hormone other than
insulin used will generally depend on the extent and rate of
hair or nail growth desired or damage to hair, nail or skin
tissue which is to be corrected, but in most of the cases the
amount of hormone will fall within a preferred range of about
0.5 ng/ml and about 100 ng/ml by weight or more of the com-
position. In the case of compositions which are delivered as
a gel, growth hormone, preferably human growth hormone, is
included in an amount ranging from about 0.5 ng/ml to about 50
ng/ml by weight or more, more preferably about 0.5 ng/ml to
about 10 ng/ml. Triiodothyronine (T3) or thyroxine (T4) is
preferably included in amounts ranging from about 0.5 ng/ml to
about 100 ng/ml or more. Triiodothyronine (T3) may be
preferred over thyroxine (T4) because it has greater potency
and the same general activity as thyroxine. Thyroxine (T4),
however, is more storage stable than is triiodothyronine (T3 )
and thyroxine's stability should be taken into account and is
preferred when formulating compositions which are to be stored
for at least several weeks or more. Triiodothyronine (T3 ) and
thyroxine (T4) may be readily substituted for each other,
however, with the general rule that at least about three to
five times the amount of thyroxine (T4) is substituted for
triiodothyronine (T3 ) .
BRIEF DESCRIPTION OF THE FIGURES
Figures 1-2 represent the results of the experiments
performed and described in Example 3. These graphs show the
relative degree of increase in blood flow measured by Laser-
Doppler Flow changes on burn wounds treated with a composition
of the present invention and conventional therapy (Silverol).
DETAILED DESCRIPTION OF THE INVENTION
In describing the present invention in the specifica-
tion, a number of terms will be used.
The term "angiogenesis" is used throughout the speci-
fication to describe processes which result in the development
of new blood vessels and the vasculature (neovasculature).
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The term "melanogenesis" is used throughout the speci-
fication to describe processes which result in the development
or restoration of pigment or color in the hair as it grows.
The term "revitalizing skin" is used throughout the
specification to describe a process in which skin becomes
rejuvenated, i.e., smoother, more moist, softer, oily/waxy,
and more cosmetically pleasing to the vision and touch.
The term "delivery polymer" or "gelling agent" is used
throughout the specification to describe a polymer or other
gelling agent which can be used in combination with minimum
essential medium and non-steroidal anabolic hormones selected
from insulin, triiodothyronine or thyroxine and growth hormone
and mixtures thereof, and optionally, a penetration enhance-
ment agent, the amount of delivery polymer included in an
amount effective to gel the composition and hold it in place
on the intact skin to be treated without running off. As used
herein, the term delivery polymer and gelling agent are
synonymous where the amount of delivery polymer or gelling
agent included is effective to gel the composition. These
delivery polymers include, for example, numerous hydrogels in
hydrated or unhydrated form, such as those derived from
hydroxyethylmethacrylate (HEMA), glycerolmethacrylate (GMA)
and polyvinylpyrrolidone (PVP), polyethylene glycol (PEG),
various carbohydrates, cellulose, cellulose ethers, including
methyl cellulose, hydroxyethyl cellulose and hydroxypropyl
cellulose, dextran, polythyleneoxide, dextran-polyethylene,
acrylamide, polyacrylamide, amylose, collagen, gelatin,
sepharose, agarose (for example, as an agarose saturated gel),
related polymers and mixtures thereof, among numerous others.
The cellulose ethers, especially including methyl cel-
lulose, hydroxymethyl cellulose, hydroxyethylcellulose and
hydroxypropylcellulose are preferred and are preferably
included at a weight ratio of about 0.1% to about 20% by
weight, more preferably about 0.5% to about 10% by weight of
the hair growth compositions. Methylcellulose and
hydroxyethyl cellulose are preferred cellulose ethers for use
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in the hair gel compositions according to the present inven-
tion, with hydroxyethylcelllulose being more preferred. One
of ordinary skill in the art will recognize to vary the type
and amount of delivery polymer in compositions according to
the present invention to provide enhanced delivery of the hair
growth composition appropriate for topical delivery and pene-
tration of the individual components through intact skin. The
term delivery polymer is also used to describe polymers which
instill slow-release or sustained release characteristics to
the hair and nail growth formulations of the invention.
The term "~;n;~um essential medium" is used throughout
the specification to describe a medium or mixture which con-
tains no serum, and in combination with anabolic hormone and
optionally, a penetration enhancement agent, comprises the
compositions according to the present invention. The term
minimum essential medium is readily understood by those in the
art to comprise a nutrient media which supports cellular
growth. The ; n; um essential medium according to the present
invention preferably comprises the following elements: (a)
essential amino acids; (b) non-essential amino acids; (c)
vitA ;~ selected from the group consisting of biotin, folate,
lipoate, niacinamide, pantothenate, pyridoxine, riboflavin,
thiamin and vitamin B12 and mixtures thereof, preferably a
vitamin mixture comprising folate, niacinamide, pantothenate,
pyridoxine, riboflavin and thiamin; (d) glucose; and (e) a
mixture of inorganic ions selected from the group consisting
of calcium, sodium, potassium, magnesium, chloride and mix-
tures thereof, preferably a mixture comprising calcium,
sodium, potassium, magnesium and chloride. Optionally,
vitamin C may be included as one of the preferred vit~;n~ in
the present formulations. It is noted that a minimum essen-
tial medium for use in the present invention may exclude non-
essential amino acids (b), but preferably, non-essential amino
acids are included in combination with essential amino acids.
Especially preferred amino acids include glutamine, serine and
cysteine.
All of the above-described elements (a), (b), (c), (d)
and (e) are included with the anabolic hormone mixture in con-
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centrations and/or amounts effective for enhancing or
stimulating the growth of hair and/or nails or revitalizing
skin tissue. It is noted that amounts of these components
which stimulate or enhance the growth of hair also quite unex-
pectedly restore the natural hair color to the growing hair in
many instances where the hair color is diminished and is char-
acterized as being gray or graying. The preferred concentra-
tion of essential and optional non-essential amino acids used
in the present invention ranges from about 5.0 um (10-6 mole)
to about 50 mmol. (10-3 mole). The preferred concentrations
of vitamins used in the present invention ranges from about 1
nanomole (10-9 mol.) to about 10 um. The preferred concentra-
tions of glucose used in the invention ranges from about 1
umol. to about 10 or more mmol. In the case of element (e),
these inorganic ions are preferably included in the present
compositions at a concentration range of about 1 umol to about
50 mmol. Optionally, a penetration enhancing agent is also
included in the formulations.
In addition to the elements (a), (c), (d) (e) and
optionally (b), the nutrient medium according to the present
invention may also contain any one or more of the following
elements: (f) purines and pyrimidines; (g) other organic com-
pounds; (h) other inorganic ions; (i) trace elements; and (j)
buffers and indicators. All of these optional elements (f),
(g), (h), (i) and (j), when they are included in the nutrient
medium according to the present invention, are included in
amounts effective, in combination with the anabolic
hormone(s), for promoting angiogenesis, enhancing vasculariza-
tion and/or stimulating dormant, dying or weak hair follicles
to promote hair growth and, in certain instances, to restore
the growing hair to its natural pigment or color. In the case
of nail growth compositions, these components are added to
stimulate or enhance the growth of ungual tissue, including
nail tissue of humans. In the case of skin revitalizing com-
positions, these components are added in amounts effective to
stimulate, enhance and revitalize the growth and appearance of
intact skin tissue of animals, including humans. Preferably,
components (f), (g), (j) and (k) range in concentration from
about 1 nmol. to about 10 mmol. In the case of components (h)
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and (j), the concentration preferably ranges from about 1
umol. to about 50 mmol. One of ordinary skill in the art will
be able to readily modify the type and amount of the com-
ponents of the m;n; 1~ essential medium consistent with the
teachings of the present invention.
In addition to serum free minimum essential medium,
the present invention may also make use of medium containing
serum, although the use of a serum containing cellular
nutrient medium is generally less preferred than is serum free
medium. Examples of such nutrient medium include, among
numerous others, DMEM, HAM F12 and HAM F10, all containing
serum. The term "minimum essential medium" is used throughout
the specification to describe all types of nutrient medium
contemplated for use in the present invention which contain at
least the basic elements as described hereinabove, as well as
supplemental components.
MCDB 153 is a preferred supplemented minimum essential
medium for use in the present invention. MCDB 153 is believed
to contain the components which are associated with an
enhanced rate of hair growth as well as other components which
enhance the rate of angiogenesis and vascularization of tissue
surrounding the hair follicle and consequently, the growth of
hair from the follicle and, in many instances, the restoration
of natural hair color in growing hair.
The minimum essential medium according to the present
invention may include one or more commercially available media
in solution or lyophilate (solid) form. The cellular nutrient
medium used may be in the form of a lyophilate which may be
reconstituted with water, preferably sterilized, distilled
water and then supplemented with an anabolic hormone such as
insulin, triiodothyronine, thyroxine, growth hormone or mix-
tures thereof, and optionally, certain penetration enhancing
agents or other additives. Lyophilized forms of insulin,
growth hormone and triiodothyronine or thyroxine may also be
used in the present compositions. Alternatively, the medium
may be used directly in formulations according to the present
invention in the form of a lyophilate, or related solid-type
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material, rather than a solution, especially when a gel is to
be used for delivery. It is clearly preferred when utilizing
solid-type materials for delivering the wound healing composi-
tions according to the present invention that the delivery
system in the form of a gel or other form contain moistening
quantities of water.
Many of the commercially available media (preferably,
serum free) which may be used in the instant invention are
available from suppliers such as Collaborative Research Incor-
porated, Bedford Massachusetts, USA, GIBCO (Grand Island
Biological Company), USA or Biological Industries, Beth
HaEmek, Israel. These media may be used as purchased or
modified within the scope and practice of the present inven-
tion.
The term "non-steroidal anabolic hormone" is used
throughout the specification to describe the primary hormones
which are included in the instant invention in combination
with ; n; um essential media to enhance or stimulate hair or
nail growth. These primary hormones include insulin,
triiodothyronine, thyroxine, and growth hormone, among others.
When growth hormone is used, it is preferred to use it in com-
bination with triiodothyronine or thyroxine or insulin or most
preferably with a mixture of triiodothyronine or thyroxine and
insulin. As used herein, the term non-steroidal anabolic
hormone includes naturally isolated (preferably, human) or
synthetically produced versions of these hormones which are
known to function substantially the same as the naturally
occuring hormones and includes, where relevant, compounds pro-
duced by genetic engineering processes and recombinant techni-
ques. While not being limited by way of theory, it is
believed that the inclusion of at least one non-steroidal
anabolic steroid selected from insulin and triiodothyronine or
thyroxine (preferably, insulin) serves to enhance the effect
of the minimum essential media in unexpectedly increasing the
rate and quality of hair (particularly, with respect to
restoration of hair color) and nail growth. A combination of
at least two anabolic hormones, e.g., insulin and growth
hormone, insulin and triiodothyronine or thyroxine, or growth
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hormone and triiodothyronine or thyroxine is preferably used
because a combination of non-steroidal anabolic hormones and
minimum essential medium creates an enhancement of tissue
growth (hair, ungual tissue, skin revitalization) which is
greater than the sum of the individual parts. Three anabolic
hormones (insulin, triiodothyronine or thyroxine and growth
hormone) in effective amounts in combination with ~;n;~ll~
essential medium acts synergistically to promote angiogenesis,
vascularization and consequently, hair growth and in many
instances, melanogenesis and hair color restoration. Thus, it
is believed that the non-steroidal anabolic hormone actually
enables the cells to utilize or process the nutrients in the
media, which action results in angiogenesis, and an enhance-
ment of vascularization and rate of hair and nail growth and
skin revitalization. Restoration of hair color is an
auxiliary beneficial result which occurs along with enhanced
hair growth in many instances.
The term "penetration enhancement agent" is used
throughout the specification to describe compounds which are
used to treat the skin before or during treatment with the
hair or nail growth promoting compositions according to the
present invention or which are added to the other components
in the present formulations in amounts effective to enhance
the penetration through the skin of the non-steroidal anabolic
hormones and the individual components of the minimum essen-
tial medium. It is noted that compositions according to the
present invention may be used with or without the inclusion of
a penetration enhancement agent. It is an unexpected result
that compositions which exclude a penetration enhancement
agent will be absorbed into intact skin and evidence a sur-
prising degree of activity (i.e., hair or nail growth enhance-
ment or skin revitalization). While not being limited by way
of theory, it is believed that the components used in the
present composition are absorbed through the skin by way of
hair roots and pores in the skin in a manner sufficient to
evidence significant activity.
Exemplary penetration enhancement agents which may be
used to treat the skin before applying the present formula-
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tions include, for example, soaps and detergents (solvents for
removing natural skin oils), depilatories, epilatories such as
trichloroacetic acid and phenol, enzymes which detach an
epithelial layer from the dermal substratum including trypsin,
collagenase, hyaluronidase, elastase and dispase and keratin
solvents such as urea and DMSO. In certain preferred
instances, water may be used as a penetration enhancement
agent, as hydration of the skin is associated with better pen-
etration. All of these agents are used in concentrations and
amounts which are effective for treating the skin in order to
enhance the penetration of the individual components in the
hair and nail growth compositions according to the present
invention through intact skin.
In the case of the enzymes trypsin, collagenase, dis-
pase, hyaluronidase and elastase, these enzymes are used to
treat intact skin in an amount ranging from about 0.1 to about
10 mg/ml., more preferably about l mg/ml. In the case of
trichloroacetic acid, this epilatory should be used in dilute
concentrations in water- generally about 1% by weight or less.
In the case of the use of urea, this is generally used as a
mixture of 20% by weight urea in lanolin (water may be sub-
stituted for the lanolin). DMSO may also be used, generally
as a dilute solution in water (0.25% to about 5% by weight,
preferably about 1% to about 2% by weight of the solution).
Water may also be used preferably as a penetration enhancer in
certain instances. Where water is used, the amount used is
that which will wet or hydrate the surface of the skin or
scalp to which the compositions of the present invention are
applied.
Other penetration enhancement agents which may be used
in the present compositions include, for example, ionophoresis
agents such as the mucopolysaccharides, for example
chondroitin, chondroitin-6-sulphate and dermatan sulphate,
among others. These compounds are generally included in com-
positions according to the present invention in effective
amounts to enhance penetration of the individual components
through intact skin.
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In addition to the above agents, a negatively charged
synthetic membrane (which generates a static charge) may also
be used for enhancing the individual components in composi-
tions according to the present invention.
The amount of each penetration enhancement agent which
is used in the formulations according to the present invention
will depend upon the requirement for hair or nail growth or
skin revitalization, but each component is included in an
amount effective for producing the intended results, for exam-
ple, substantially enhancing the rate of penetration of the
formulations through the skin. This amount will vary accord-
ing to the type of agent used. One of ordinary skill in the
art will know to vary the amount and type of agent within the
weight ranges defined above to enhance penetration through
intact skin and promote the efficacy of compositions according
to the present invention.
In general, in embodiments according to the present
invention, the formulations include an anabolic hormone other
than insulin at a concentration of at least about 0.05 ng/ml,
preferably about 0.5 ng/ml to about 100 ng/ml or more. In the
case of formulations containing insulin, the amount of insulin
generally falls outside of this range. Preferably, the
anabolic hormone comprises a mixture of insulin,
triiodothyronine or thyroxine and growth hormone because of
the known benefits these hormones have in promoting the growth
and elaboration of cells and their general absence of
toxicity. In addition, it is this combination of anabolic
hormones which evidences unexpected synergistic activity in
promoting angiogenesis, vascularization and hair or nail
growth in the instant invention.
The preferred insulin is human insulin (more
preferably human recombinant or genetically engineered
insulin), which is a well-known protein which is readily
available commercially from a number of sources (for example,
Novo Nordisk, Copenhagen, Denmark, among others). It is con-
stituted from a number of amino acids (approximately 51) with
a total molecular weight of about 5,500. Human insulin for
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use in the present invention is generally prepared using
genetic engineering techniques. Depending upon the manufac-
turer, the insulin may have slightly different activity based
upon weight, however the activity of insulin defined in units
is, of course, st~n~rd. While not being limited by way of
theory, in the present invention, it is believed that the
insulin promotes hair or nail growth at least in part by
enhancing and stimulating the transport and utilization of
glucose as an energy source by the hair or nail growing cells.
Growth hormone may also be used in the present inven-
tion, preferably in combination with insulin or
triiodothyronine or thyroxine and most preferably in combina-
tion with both triiodothyronine or thyroxine and insulin. The
preferred human growth hormone is a well-known defined protein
which is readily available and results from a pituitary secre-
tion into the blood system. It is constituted from a number
of amino acids with a total molecular weight of about 193,000.
The human growth hormone which may be used in the present
invention can be obtained from a variety of sources, including
genetic engineering processes and techniques. While not being
limited by way of theory, it is believed that the growth
hormone serves to stimulate and enhance angiogenesis, i.e.,
the elaboration and development of the vascular system which
is believed to also enhance the blood supply and the delivery
of nutrients to hair follices and nail tissue so as to produce
an enhancement or stimulation in hair or nail growth and, in
certain instances, restoration of natural color. The action
of growth hormone is believed to be synergistic with insulin
and/or triiodothyronine or thyroxine.
The present invention also contemplates the inclusion
of effective amounts of triiodothyronine or thyroxine either
alone, but preferably in combination with other non-steroidal
anabolic hormones. The preferred triiodothyronine is human
triiodothyronine, which is a well-known defined hormone and
readily available commercially. Triiodothyronine and
thyroxine are naturally occurring amino acids of the thyroid
gland which exert a stimulating effect on metabolism.
Although virtually identical in metabolic effects,
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triiodothyronine is more potent than is thyroxine but is less
stable. Where thyroxine is substituted for triiodothyronine,
to obtain the same effect as triiodothyronine, thyroxine is
added in an amount between about three and five times that of
triiodothyronine. Thyroxine, being more stable, is preferred
for use in the present invention in those compositions which
advantageously have storage stable characteristics. While not
being limited by way of theory, it is believed that the
triiodothyronine or thyroxine utilized in the present inven-
tion stimulates vascularization and facilitates the resupply
of blood borne components in cells responsible for the growth
of hair.
A particularly preferred composition according to the
present invention comprises a mixture of an effective amount
of human growth hormone in the presence of an effective amount
of insulin and triiodothyronine (T3) or thyroxine (T4),
preferably in a serum free cellular nutrient medium, most
preferably MCDB 153. In this preferred embodiment of the
instant invention, the anabolic hormones other than insulin,
i.e., growth hormone or triiodothyronine, are generally
included in the final composition in a concentration range of
about 0.05 ng/ml to about 100 ng/ml, preferably about 0.5
ng/ml to about 20 ng/ml or more and most preferably about 1
ng/ml to about 20 ng/ml. In the case of the inclusion of
thyroxine as a substitute for triiodothyronine, thyroxine is
generally included in an amount ranging from about 0.5 ng/ml
to about 100 ng/ml or more, generally at a concentration of at
least about three-five times that of triiodothyronine.
In the most preferred embodiment which includes three
anabolic hormones, insulin is also included in an effective
amount, generally an amount which is substantially greater
than the other anabolic hormones. The amount of insulin is
preferably included in amounts ranging from about 5ng/ml to
about 100 ug/ml more preferably about 50 ng/ml to about 20
ug/ml and even more preferably about 500 ng/ml to about 20
ug/ml. One of ordinary skill in the art will know to vary the
amount of anabolic hormones within effective ranges based upon
the type and potency of the preparation of the compound in
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order to enhance or stimulate the growth of hair or nail tis-
sue according to the present invention.
Compositions according to the present invention
preferably comprise effective amounts of ~; n; um essential
medium in combination with effective amounts of insulin,
triiodothyronine or thyroxine and growth hormone. It is a
combination of these three anabolic hormones in combination
with ; n;- essential medium which has exhibited the greatest
synergistic activity in promoting angiogenesis, vasculariza-
tion, increased blood flow and hair or nail growth. Suppl-
mented m;n;~llm essential medium is preferably used and MCDB
153 is the most preferred media to be used in the instant
invention. In addition to the other components, the inclusion
of effective concentrations of selenide are optional. In
addition, the inclusion of effective amounts of vitamin C
(ascorbic acid) is preferred. The compositions are preferably
transferrin-free, although transferrin may be added.
The m; n; - essential medium which is used in the
present invention is any medium having the effect of promoting
hair growth when used in effective amounts in combination with
the non-steroidal anabolic hormones. In preferred embodiments
according to the present invention, the nutrient media, com-
prised of the componentry set forth hereinabove, is mixed with
an effective amount of the non-steroidal anabolic hormone(s)
to form the compositions according to the present invention.
The term "~;n; ~llm essential medium" is readily recog-
nized by those of ordinary skill in the art. ~;n;~um essen-
tial medium is known to preferably comprise effective amounts
of the following constituents: (a) essential amino acids; (b)
non-essential amino acids; (c) vitamins as previously
described; (d) inorganic ions as previously described and (e)
glucose; and optionally, (f) purines and pyrimidines; (g)
other organic compounds; (h) other inorganic ions; (i) trace
elements; (j) buffers and indicators and (k) other supple-
ments. Preferably, the medium used herein also contains
effective amounts of elements (f) through (k). Serum free
nutrient medium is preferred. It is noted that non-essential
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amino acids (b) are preferably added, but are not required.
The preferred serum free nutrient medium is modified MCDB 153,
a well-known medium. Mixtures of standard commercial nutrient
media may also be used with favorable results in the instant
invention.
While not being limited by way of theory, it is
believed that one plausible explanation of the mechanism of
the accelerated growth of hair and nails, hair color restora-
tion and skin revitalization is that the presence of the
anabolic hormones, and in particular, the combination of
insulin, triiodothyronine or thyroxine and human growth
hormone in the formulations according to the present inven-
tion, synergistically promotes the utilization of the
nutrients from the nutrient medium and consequently, the
promotion of angiogenesis and melanogenesis and vasculariza-
tion of the tissue surrounding the hair follicle and nail
growth tissue. The result is the stimulation of hair fol-
licles and related nail tissue and consequently enhanced rate
of growth of hair and nails and hair color restoration. In
addition, not only is the rate of growth of hair enhanced, but
also the number, quality and in most instances, melanogenesis
of active hair-growing follicles growing hair also increases,
primarily due to the stimulation of dormant, dying or weak
follicles to produce hair. In effect, the quality of the
growth of hair, nail and skin tissue is enhanced using the
compositions of the present invention. Hair color restoration
is an auxiliary effect.
With regard to the enhancement in angiogenesis and the
promotion of vascularization, the ?ch~nism which might be
assumed is that new capillaries appear in the tissue surround-
ing the hair follicles and related nail tissue from the first
day on and reach their ~; um levels after one week or so.
The new vessels in granulation tissue originate as budlike
structures on nearby vessels, enhance vascularization, become
canalized and ramify throughout the dermis in proximity to the
hair follicles and nail growth tissue. The resulting increase
in vascularization provides more nutrition and stimulatory
factors for the hair follicle and nail and skin tissue, the
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consequence of which is the stimulation of the hair follicle
and related nail and skin tissue and the enhancement of pro-
duction of hair, nail and skin tissue.
It is further believed that the function of the medium
is to provide nutrients to normal, distressed and injured fol-
licles of the dermis in order to stimulate the follicle and
enhance the growth of hair. In this way, the medium functions
with the non-steroidal anabolic hormone to promote the normal
processes of stimulation of hair follicles and the enhanced
growth of hair. In addition, in many instances, as in the
case of gray or graying hair, the color of the hair returns to
its natural color.
A number of nutrient media, preferably serum free,
alone or in combination, may be used in the present invention,
including commercially available media or other media well
known in the art. Examples of such media (all without serum
or having had the serum removed) include ADC-l, LPM (Bovine
Serum Albumin-free), F10 (HAM), F12 (HAM), DCCMl, DCCM2, RPMI
1640, MCDB 105, MCDB 110, MCDB 202, MCDB 402, MCDB 153, BGJ
Medium (with or without Fitton-Jackson Modification), Minimum
Essential Medium Eagle, Basal Medium Eagle (BME-with the addi-
tion of Earle's salt base), Dulbecco's Modified Eagle Medium
(DMEM-without serum), Glasgow Modification Eagle Medium
(GMEM), Leibovitz L-15 Medium, McCoy's 5A Medium, Medium M199
(M199E- with Earle's salt base), Medium M199 (M199H- with
Hank's salt base), Minimum Essential Medium Eagle (MEM-E- with
Earle's salt base), Minimum Essential Medium Eagle (MEM-H-
with Hank's salt base) and M;n; I Essential medium Eagle
(MEM-NAA- with non-essential amino acids), among numerous
others. These and other useful serum-free media are available
from Biological Industries, Bet HaEmek, Israel, among others.
In addition, serum-containing nutrient media may also
be used in compositions according to the present invention,
but the use of serum-containing media is less preferred
because of the possibility that the serum may be cont~;n~ted
with microbial agents and because the patient may develop
immunological reactions to certain antigenic components con-
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tained in the serum.
While a large number of serum free nutrient media may
be used in the present invention, a preferred nutrient media
for use in the present invention is modified MCDB 153.
Hereafter are enumerated the particular constituents
and concentrations of the above groups for the preferred
medium, MCDB 153:
Group (a): Concentration (M)
Arginine 1.0 x 10-3
Cysteine or Cystine 2.4 x 10-4
Glutamine 6.0 x 10-3
Histidine 8.0 x 10-5
Isoleucine 1.5 x 10-5
Leucine 5.0 x 10-4
Lysine 1.0 x 10-4
Methionine 3.0 x 10-5
Phenylalanine 3.0 x 10-5
Threonine 1.0 x 10-4
Tryptophan 1.5 x 10-5
Tyrosine 1.5 x 10-5
Valine 3.0 x 10-4
Group (b):
Alanine 1.0 x 10-4
Asparagine L.0 x 10-4
Aspartate 3.0 x 10-4
Glutamate 1.0 x 10-4
Glycine 1.0 x 10-4
Proline 3.0 x 10-4
Serine 6.0 x 10-4
Group (c):
Biotin 6.0 x 10-8
Folate 1.8 x 10-6
Lipoate 1.0 x 10-6
Niacinamide 3.0 x 10-7
Pantothenate 1.0 x 10-6
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Pyridoxine 3.0 x 10-7
Riboflavin 1.0 x 10-7
Thiamin 1.0 x 10-6
Vitamin B12 3.0 x 10-7
Group (d)
Glucose 6.0 x 10-3
Group (e):
Magnesium 6.0 x 10-4
Postassium 1.5 x 10-3
Sodium 1.5 x 10-1
Chloride 1.3 x 10-1
Calcium 0.1 mmol.
Group (f):
Adenine 1.8 x 10-4
Thymidine 3.0 x 10-6
Group (g):
Acetate 3.7 x 10-3
Choline 1.0 x 10-4
i-Inositol 1.0 x 10-4
Putrescine 1.0 x 10-6
Pyruvate 5.0 x 10-4
Group (h)
Phosphate 2.0 x 10-3
Sulfate 4.5 x 10-6
Group (i):
Copper 1.0 x 10-8
Iron 1.5 x 10-6
Zinc 3.0 x 10-6
Group (j):
Bicarbonate 1.4 x 10-2
HEPES 2.8 x 10-2
Weights of each of the above components in the medium
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may be varied within the concentrations described hereinabove
(in hair or nail growth enhancing effective amounts) to pro-
vide formulations workable within the description of the pres-
en~ lnven~lon.
Preferably, the non-steroidal anabolic hormone to be
incorporated into the modified MCDB 153 composition, according
to the present invention, is a mixture of at least two
hormones selected from insulin, triiodothyronine/thyronine and
growth hormone at hair growth enhancing effective concentra-
tions. Most preferably, the anabolic hormone includes a mix-
ture of human growth hormone, insulin (containing transferrin
or transferrin-free) and triiodothyronine (T3) or thyroxin
(T4), each hormone included in a hair growth enhancing effec-
tive amount. The three hormone combination exhibits an unex-
pected synergistic effect in promoting hair growth. Hormones
other than insulin are included in an amount ranging from at
least about 0.05ng/ml, preferably at least about 0.5ng/ml to
about 100 ng/ml, and more preferably about 1 ng/ml to about
100 ng/ml. In the case of thyroxine, it is generally sub-
stituted for triiodothyronine at a concentration of at least
about three to five times the concentration of
triiodothyronine used. In the case of insulin, the effective
amount of insulin generally ranges from about 5ng/ml to about
lOOug/ml and more preferably about 50 ng/ml to about 20ug/ml,
even more preferably about 500 ng/ml to about 20 ug/ml within
this range.
Insulin is a desirable constituent anabolic hormone,
found to impart a maturing stimulus of the growing culture.
Insulin may be commercially obtained and is generally provided
in mU quantities (about 41 ng of insulin). The International
Unit of Insulin (SI= System International) is the activity
contained in 0.04167 mg (41.67 ug) of the 4th International
St~n~d Preparation (1958). The Standard Preparation is a
quantity of purified Zinc Insulin crystals extracted 52% from
Bovine and 48% from Porcine pancreas (See, Martindale
Pharmacopoeia, 26th Ed.).
In addition to effective amounts of non-steroidal
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anabolic hormones and media as described above, formulations
according to the present invention may also contain transfer-
rin (which is believed to improve iron transport) in amounts
which preferably range from about 500 ng/ml to about 50 ug/ml,
more preferably about 5-10 ug/ml and selenite (preferably, in
the form of sodium selenite) in amounts preferably ranging
from about 0.5 to about 50 ng/ml, more preferably about 5-10
ng/ml. The inclusion of transferrin is not critical to the
use of the instant invention and in certain cases may be less
preferred.
The formulations according to the present invention
may also include an effective amount of an anti-dandruff agent
or other ingredients which are commonly applied to the scalp
or hair, including antimicrobial agents, where desirable,
generally in amounts found useful in topical applications. In
the case of nail growth formulations, the inclusion of an
anti-fungal agent for use in preventing the outgrowth or fur-
ther growth of nail fungus infections is also optional. One
of ordinary skill in the art can easily determine the type and
amount of anti-dandruff or antimicrobial agent chosen for use
in formulations according to the present invention.
In certain embodiments according to the present inven-
tion, the formulations as described herein are further for-
mulated with gelation agents or related delivery polymers for
gelling the formulations according to the present invention
for delivery to the area of intact skin to be treated. In
these embodiments, the formulations comprising effective
amounts of anabolic hormone(s) and nutrient media, either
alone or in addition to other optional components, especially
including an effective amount of a penetration enhancement
agent, are admixed with amounts of a delivery polymer effec-
tive for producing a gel, for example a hydrogel polymer
derived from HEMA (hydroxyethylmethacrylate) or NVP (N-
vinylpyrrolidone), polyethylene glycol (PEG), polyethylene,
gelatin, various carbohydrates, sepharose, agarose, methylcel-
lulose and related hydrophilic cellulose ethers including cel-
lulose, hydroxymethyl cellulose, hydroxyethyl cellulose and
hydroxypropylcellulose, dextran, polyethyleneoxide, dextran-
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polyethylene, acrylamide, polyacrylamide, amylose or collagen
to promote the delivery of the components to the surface of
the skin. The inclusion of a cellulose ether gellation agent
and in particular, the use of methyl cellulose or hydroxyethyl
cellulose, is clearly preferred. In general, the amount of
delivery polymer which is added to the formulations to produce
a gel is that amount which solidifies the composition to a
point where the composition does not easily flow off of the
intact skin to be treated and generally ranges from about 0.1%
by weight to about 20% by weight, preferably about 0.5% to
about 10% by weight, depending upon the type of delivery
polymer used. The gel compositions according to the present
invention preferably contain sufficient water or moisture to
maintain the area to be treated in a relatively moist state- a
condition shown to be beneficial for penetration enhancement
of the individual components of the formulations through the
skin.
In addition to solution, gel or hydrogel forms, com-
positions according to the present invention also may be for-
mulated as creams, elixirs, powders and the like for delivery
to the scalp or other area of intact skin to be treated for
the hair growth enhancing effects of the present compositions.
The various components of the compositions according to the
present invention may have to be varied in order to maintain
effective concentrations. When compositions according to the
present invention are formulated, these compositions may also
contain an amount of a pharmaceutically acceptable excipient
and, in addition, other additives such as diluents, compound-
ing agents, bulking agents, surfactants and the like. One of
ordinary skill in the art will recognize to vary the con-
centrations of the individual components as a function of the
type of delivery vehicle used for the hair growth composi-
tions.
In a method for enhancing the growth of hair and/or
restoring the natural color of the hair in intact skin
(scalp), especially including hair of the human scalp, the
formulations as described hereinabove are topically applied to
the skin tissue to be treated as a liquid or gel at least once
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a day and preferably at least twice a day and up to six or
more times a day. In many instances it may be advisable to
apply the compositions to the scalp or other area of the skin
to be treated at least once a day after showering or bathing.
In the case of formulations containing a delivery polymer,
preferably a moistened delivery polymer, the formulations may
be administered less frequently than when the formulations are
applied as a liquid. One of ordinary skill in the art will
readily be able to determine the amount and frequency of
administering the formulations according to the present inven-
tion.
one possible regimen for utilizing the compositions
according to the instant invention is to first treat the skin
with a penetration enhancement agent in an amount (i.e., at a
concentration) and for a time effective for enhancing pene-
tration of the components of the hair or nail growth composi-
tion. In this method, the penetration enhancement agent is
generally applied to the intact skin to be treated with the
hair growth composition for 30 seconds up to one half hour or
more (preferably from about 1 to about 5 minutes) to condition
the skin for penetration. It is noted that the inclusion of a
penetration enhancement agent is unexpectedly not required in
order to deliver effective amounts of the present compositions
to promote hair growth, nail growth and skin revitalization.
In cases where water is the penetration enhancer, the skin to
which the composition is to be applied is first wet with
water, after which time, the composition is applied. A cover-
ing, such as a polyethylene shower cap, may be used to keep
the composition in contact with the area to be treated.
In other methods according to the present invention
the scalp is simply washed with soap or detergents to remove
all natural oils from the scalp before application of the hair
growth enhancing composition. After a period of time suffi-
cient for treating the skin, the treated skin is then exposed
to the hair growth composition (preferably, after wetting with
water to promote penetration) and massaged into the scalp. In
certain preferred embodiments, the hair growth composition is
a gel which is rubbed onto the area of the skin to be treated
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and is left on the skin for a convenient period, generally at
least 1-3 hours and preferably for an eight hour period such
as overnight. A covering is preferably placed over the gel in
order to keep the gel in contact with the treated skin area.
To remove the composition, it can be washed off with clear
water.
The amount of material which is to be spread on the
skin or massaged into the scalp to be treated will be readily
apparent. The formulations should be applied in a manner
which is cosmetically appealing. In general, in solution or
gel form, about 0.5-2.0 cc of formulation is applied per 5-10
cm2 to the area of the scalp. Depending upon the requirement
of hair growth and the extent of baldness, an amount greater
or less than 0.5-1 cc of formulation per 5-10 cm2 of the wound
surface may be utilized. In many instances, the depth of the
formulation on the skin should be at least about 0.05-0.1 mm.
Greater depths may also be used, but generally at the expense
of the cosmetic appeal of the product. Obviously, the greater
the amount of composition and the greater the depth of gel on
the skin surface, the greater will be the delivery of composi-
tion to the surface of the skin. In certain cases where the
hair is already quite thick, for example, when treating the
hair to restore the natural color of the hair, more material
may have to be added to the hair in order to assure the indi-
vidual that a sufficient amount of the composition will come
into contact with the underlying skin or scalp area.
In the case of treating hair to restore natural color,
the treatment regimen and compositions used are virtually
identical to the method used for enhancing the growth of hair.
Restoration of hair color generally will occur as an auxiliary
result of enhanced growth of hair. Obviously, where the hair
color is diminished in color, for example, in gray, graying or
even white hair, the methods of use will be the same as if the
desired result is enhanced hair growth, but in such a case,
the more clearly desired result will be restoration of hair
color. Enhanced hair growth most likely will also occur.
In the case of treatment of nails or ungual tissue or
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skin tissue of ~n; ~ls, including humans, to strengthen and
enhance the growth of the nail tissue or revitalize the skin,
treatment may be at night or during the day. In the case of a
preferred night treatment, the tissue of the patient is
treated by applying the compositions according to the present
invention directly on the tissue after first wetting the tis-
sue and, in the case of ungual tissue, to the cuticle area of
the nail or tissue where the nail or ungual tissue is attached
to the nail or tissue bed. During this night treatment, about
1 ml per cm2 is placed on the tissue as described above and
the composition and tissue is covered with gauze and adhesive.
A cover may be advantageously applied to keep the composition
in contact with the treated tissue.
In the case of treatment during the day (following a
night treatment as described hereinabove), a preferred treat-
ment includes washing off the remainder of the gel under run-
ning water and then drying the tissue. After the drying step,
the compositions according to the invention may be applied as
for the night treatment, as above, or alternatively, the com-
positions are applied to the tissue liberally and is allowed
to be absorbed into the nail and surrounding skin.
Preferably, the skin is first moistened with water before
applying the composition. A protective covering may be used
in order to keep the composition in contact with the treated
scalp.
Preliminary bioassays to determine the acceleration of
hair growth which were carried out on rats, guinea pigs and on
selected clinical cases indicated that the formulations
according to the present invention exhibited a significant
beneficial result relative to traditional therapies, including
the use of Minoxidil. It is noteworthy that unlike Minoxidil,
which evidences hepatotoxicity, tachycardia and contact
dermatitis in vivo, the present invention exhibits little, if
any, toxicity. In the case of nail growth, clinical use of
the preferred formulation evidenced enhanced growth of both
finger and toe nails.
The invention will be described hereinafter by a num-
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ber of Examples which illustrate some actual tests carried out
on skin to enhance the growth of hair utilizing the composi-
tions according to the present invention. It should be under-
stood that the Examples are not exhaustive nor limiting and
are presented only for a better understanding of the inven-
tion.
EXAMPLES
Materials and Methods
1. Preparation of Hair Growth Gel
The whole procedure was performed under sterile condi-
tions.
a. Delivery System
Four grams of Methyl cellulose (Methocel MC 4000 cp,
Fluka AG) in 90 ml of double distilled water was autoclaved.
b. Media
The preferred media contained essential and non-
essential amino acids, VitA~; n~, other organic constituents,
major inorganic salts, trace ~lements and buffers and was sup-
plemented with CaCl and L-glutamine and with the non-steroidal
anabolic hormones, insulin, thyroxin, growth hormone and
insulin-like growth factor (IGF) at the concentrations as
indicated below.
Component Concentration in M
Amino Acids (L-enantiomers)
Alanine 1.0 X 10-4
Arginine HCl 1.0 x 10-3
Asparagine 1.0 X 10-4
Aspartic Acid 3.0 X 10-5
Cysteine HCl or Cystine 2.4 X 10-4
Glutamic Acid 1.0 X 10-4
Glutamine 6.0 X 10-3
Glycine 1.0 X 10-4
Histidine HCl 6.0 X 10-5
Isoleucine 1.5 X 10-5
Leucine 5.0 X 10-4
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Lysine HCl 1.0 X 10-4
Methionine 3.0 X 10-5
Phenylalanine 3.0 X 10-5
Proline 3.0 X 10-4
Serine 6.0 X 10-4
Threonine 1.0 X 10-4
Tryptophan 1.5 X 10-5
Tyrosine 1.5 X 10-5
Valine 3.0 X 10-4
Vit; inc
d-Biotin 6.0 X 10-8
Folic Acid 1.8 X 10-6
DL-a-lipoic acid 1.0 X 10-6
Niacinamide 3.0 X 10-7
D-pantothenate 1/20a 1.0 X 10-6
Pyridoxine HCl 3.0 X 10-7
Riboflavin 1.0 X 10-7
Thiamin HCl 1.0 X 10-6
Vitamin B12 3.0 X 10-7
L-Ascorbic Acid 9.9 X 10-5
Other Organic Constituents
Acetate 3.7 X 10-3
Adenine 1.8 X 10-4
Choline chloride 1.0 X 10-4
D-glucose 6.0 X 10-3
i-Inositol ~ 1.0 X 10-4
Putrescine 2HCl 1.0 X 10-6
Na Pyruvate 5.0 X 10-4
Thymidine 3.0 X 10-6
Major Inorganic Salts
CaCl2 3.0 X 10-5
KCl 1.5 X 10-3
MgCl2 6.0 X 10-4
NaCl l. 2 X lO--
Na2HPo4 2.0 X 10-3
Trace Elements
CuS04 1.1 X 10-8
FeS04 5.0 X 10-6
H2Se03 3.0 X 10-8
MnS04 1. 0 X 10--9
Na2Si03 5.0 X 10-7
(NH4)6M~7~24 1.0 X 10-9
NH4V03 5.0 X 10-9
NiCl2 5.0 X 10-l~
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SnC12 5.0 X lo-10
ZnS04 5.0 X 10-7
Buf~ers
Hepes 2.8 X 10-2
NaHC03 1.4 X 10-2
Phenol Red 3.3 X 10-6
Supplements
CaC12 14.7 ug/ml (Merck)
L-Glutamin 0.877 mg/ml
NaHC03 1.176 mg/ml
Human Growth Hormone 5 ng/ml (Biotechnology
General)
Insulin 5-10 ug/ml (Novo Nordisk)
(about 14 3 mU/ml)
Thyroxine(T4) 1.0 X 10-7M (Sigma)
(77.69 ng/ml)
Transferrin 5 ug/ml (Sigma)
Sodium Selenite 5 ng/ml (Sigma)
Vehicle
Methylcellulose (Methocel 4000 cp, Fluka AG) 4~ or
Hydroxyethylcellulose 6%
c. Preparation of Hair Growth Formulation
1. 4 grams of the methyl cellulose is autoclaved in
90 ml of double distilled water. The water/methyl cellulose
mixture is cooled to 4~C and stirred until the gel dissolves
and the solution clears (overnight with a magnetic stirrer in
the cold room). To the water/methyl cellulose mixture is
added 10 ml of the concentrated MCDB 153 solution (X 10 con-
centrated media available from Biological Industries, Beth
Haemek, Israel) containing the supplements as described above
(adjusted to a final volume of 100 ml of formulation). The
solution is then mixed well and then decanted into 50 ml
sterile test tubes. The formulation is stored at 4~C.
2. Autoclave 6 grams of hydroxyethyl cellulose, mid-
dle viscosity 1 (Fluka Cat. #54290) in 83 ml double distilled
water. Separately, prepare 10 ml of 10 times concentrated
medium containing the supplements as described above (adjusted
to a final volume of 100 ml of formulation). Cool the
hydroxyethyl cellulose solution to room temperature and stir
-
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until the gel dissolves and the solution clears (overnight
with magnetic stirrer, in refrigerator). Add the medium solu-
tion with all the supplements to the gel solution, mix well
and decant into 20 ml. sterile test tubes and store at 4~C.
d. Instructions for Use in Clinical Settings
The scalp is treated once a day, preferably before
going to sleep. If the scalp is oily or if other hair groom-
ing products are in use, the hair is hydrated (in certain
cases, also shampooed) to remove the oil or other deposits and
then towel dried. Massage the gel into the scalp and leave on
overnight. Preferably, the scalp is first moistened before
applying the composition. In the morning, wash out the
remains of the gel with clear water.
Examplç 1
Clinical Use of Hair Growth Formulations
The following represent results of the use of the
above-described hair growth formulation (formulation lc.2. in
materials and methods section, above) in the clinical setting.
Essentially, 15 individuals used the hair growth gel according
to the instructions provided in the materials and methods sec-
tion (ld.), as described above. The following set of ques-
tions was answered by each of the individuals who participated
in the clinical experiment. The results obtained from
patients answering the questionnaire appear in Table 1,
below.
1. Did you suffer any loss of hair during combining
before you started using the formulation?
2. If so, did the hair loss stop after you started
the treatment?
3. If so, how long after the start of the treatment
did it stop?
4. Is you hair growing now, after the start of the
treatment, at a faster rate?
5. If so, how long after you started the treatment
did you notice that?
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6. Do you or anybody else see new growth of hair?
7. if so, how long after you started the treatment
did you notice that?
pR~T.TMTNARY CLINICAL 'I'RTAT~ OF HAIR ~KO~ l~. FORMUI~TION
Patient Sex Age Alopecia Alopecia Etiology Pattern Dynamics
Type Duration of Defect P~y~ess
M.M. M 25 Diffuse 2 yrs. Heredity Combination Slow
B.A. M 20 Diffuse 2 yrs. Male Pattern Combin. Rapid
D.M. M 47 Areata 26 yrs. Male Patt. Bald/RHL* Slow
S.L. M 40 Areata 15 yrs. Heredity Bald/RHL* Slow
D.T. M 10 Areata 3 mos. Infection Bald pate Static
E.Z. M 16 Diffuse 1 yr. Heredity Combin. Rapid
A.W. M 46 Areata 16 yrs. Heredity Bald/RHL* Slow
M.L. M 44 Areata 6 yrs. Male Patt. Bald pate Slow
S.P. M 28 A-eata 3 yrs. Herdity Bald/RHL* Rapid
L.L. F 38 D ffuse 5 yrs. Unknown Comb n. Slow
F.B. F 46 D ffuse 20 yrs. Heredity Comb_n. Slow
M.G. F 67 D ffuse 5 yrs. Heredity Comb n. Slow
M.L. F 43 D'ffuse 10 yrs. Unknown Combin. Slow
R.G. F 53 D ffuse 20 yrs. Unknown Comb-n. Slow
T.B. F 14 Areata 3 mos. Trauma Bald pate Static
* ~C~ i ng Hair Line
Table 1
RESULTS OF 'rR~A~
Patient Cessation of Hair Growth De Novo
Loss Increase Hair Growth
M.M. l week 2 weeks 1 month
B.A. 2 weeks 1 month 1 month
D.M. 2 weeks 1 month 2 months
S.L. 2 weeks 2 months 2 months
D.T. 1 week 1 week 1 week
E.Z. 2 weeks 3 weeks 1 month
A.W. 2 weeks 1 month 2 months
M.L. 2 weeks 1 month 1 month
S.P. 2-3 weeks 3-4 weeks 3-4 weeks
L.L. 2 weeks 1 month 1 month
F.B. 2 weeks 1 month 1 month
M.G. 2 weeks 6 weeks 6 weeks
M.L. 3 weeks 6 weeks 6 weeks
R.G. 3 weeks 1 month 1 month
T.B. 1 week 1 week 1 week
In all cases, hair loss stopped within a period of 2-
3 weeks. In addition, all of the patients showed an increase
in hair growth and all patients experienced de novo hair
growth.
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Example 2
Further Clinical R~C~llts Using Hair Growth Formulation
The individuals treated in example 1 were studied for
a further period to determine the effects of the instant
invention on hair growth and cessation of hair loss. All of
the individuals, prior to the study suffered from alopecia
androgenetica or traumatic alopecia of variable severity. The
individuals were instructed to use the hair growth formulation
of the instant invention as described in example 1 (except
that transferrin was excluded from the formulation) and this
use continued for more than six months. Essentially, the
volunteers were instructed to apply the hair growth formula-
tion to the scalp once a day, before retiring, by massaging it
into the scalp area. The gel was left on during the night,
and washing of the remains of the gel was performed the fol-
lowing morning. The results of the study are presented in
Tables 2 and 3, below. As shown in Table 1 of Example 1, the
reaction to the treatment was seen 1-3 weeks after the start
of the treatment, when cessation of hair loss was reported by
all participants. In addition, generally within 4-6 weeks,
the increase in the rate of existing hair growth was reported
as well as the start of new hair growth. In the further
results, after four months of treatment, all of the
participants in the study evidenced moderate or dense hair
growth, with most show moderate hair growth. After more than
6 months of treatment several individuals within the treatment
group evidenced dense hair growth and two individuals
exhibited a complete cover of the deficit area.
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Table 2
~Uk...~ RESULTS OF ~r
Pat. Sex Age HAIR GROWTH
None Slight Moderate n~nc~
4m 6m+ 4m 6m+ 4m 6m+ 4m6m+
M.M. M 25 X X
B.A. M 20 X ?
D.M. M 47 X X
S.L. M 40 X X
D.T. M 10 X Comp.
E.Z. M 16 X X
A.W. M 46 X X
M.L. M 44 X X
S.P. M 28 X X
L.L. F 38 X X
F.B. F 46 X X
M.G. F 67 X X
M.L. F 43 X *
R.G. F 53 X *
T.B. F 14 X Comp.
4m - After 4 months of treatment.
6m+- After more than 6 months of treatment.
Comp- Complete cover of the deficit area.
*- Cessation of loss (main complaint).
Example 3
E~fects of Hair Growth Formulation on Hair Growth in Mice
To test the efficacy of the instant invention in
inducing hair growth in the animal mode, the model of Paus, et
al. (1991) was used.
In this experiment, ninety-six (96) C57BL mice aged 5-
6 weeks were divided into five groups. Two experimental
groups were treated with the instant invention (formulation
lc.2 with transferrin excluded) three times daily by applica-
tion of the gel to the dorsal depilated area of the mouse.
Three control groups were used, two of which were treated with
vehicle placebo in the same manner as the two experimental
groups. The third group was untreated.
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Group No. 1: Treatment with Hair Growth C~ __ ition starting
on Day O (three times daily) and continl~ for 30 days.
This group consisted of 20 mice, which were depilated
manually on Day 0 of the experiment. On Day 7, anagen ensued
(as judged by gray coloration produced by melanogenesis). On
Day 10, an injection of the cytoxic agent cytophosphan (0.5 ml
per gram) was made ip. On day 15, catagen resulted form the
administration of the cytophosphan. on Day 16-17, anagen was
seen and on that day blood samples were taken for CBC analy-
sis, as well as punch biopsies. On Day 18, the second
cytophosphan injection was given. On Day 26-27, catagen
occurred and on Day 28, anagen was present. Second blood
samples were taken on Day 30, at which time punch biopsies
were also taken.
Group No. 2: Treatment with Hair Growth G~ ition starting
on Day 10 (three times daily) after cytophosphan injection and
continl~ for 30 days.
This group consisted of 20 mice, which were depilated
manually on Day 0 of the exp~ri ?nt. On Days 7-9, anagen
occurred (Day 7 - 30%, Day 8 - 60~, Day 9 - 70%). On Day 10,
cytophosphan (0.5 ml per gram) was administered, all animals
were in anagen (100~) at that time. On Day 14, catagen
occurred and on Days 16-17, anagen was seen and on Day 17
blood samples were taken for analysis and biopsies were also
taken. On Day 18, the second cytophosphan injection was
given. On Day 26-27, catagen occurred and on Day 28-29,
anagen occurred. Second blood samples were taken on Day 30,
at which time punch biopsies were also taken.
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Group No. 3: Treatment with vehicle placebo starting on Day O
(three times daily) and con~; nlle-l for 28 days.
This group consisted of 20 mice, which were depilated
manually on Day 0 of the experiment. On Days 7-8, anagen
occurred. On Day 9, cytophosphan (0.5 ml per gram) was
administered and on Day 15, catagen occurred. On Days 16-17,
anagen was seen and on Day 20 vellus hair was seen. On Day
24, terminal hair was observed and on Day 28, the number of
~n; ~ls that had terminal hair was 4 (mild), 10 (moderate) and
6 (extensive terminal growth).
Group No. 4: Treatment with vehicle placebo starting on Day 9
following cytophocrh;~n injections and continued for 20 days.
This group consisted of 20 mice which were depilated
on Day 0 and angagen was observed on Day 9, when the mice were
injected with cytophosphan. Catagen was observed on Day 15,
and anagen followed on Day 17--18. Vellus hairs were seen on
Day 20 and Day 24. On Day 28, terminal hair growth was mild
in five naimals, moderate in three animals and extensive in
two animals.
Group No. 5: No Treatment given to this ~ol.L-ol y~u~ used
as a h;~:~l in~ or the other studies.
- This group consisted of 16 mice, which were depilated
on Day 0 and anagen ensued on Days 9-10. On Day 10,
cytophosphan was injected and catagen occurred on Days 15--17.
on Days 18--21,anagen occurred and on Day 21, punch biopsies
were taken.
The various experimental procedures performed on the 5
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groups of mice as described above are detailed in the flow
chart set forth in Table 3, below.
Results: In the experimental mice, the use of the present
invention produced significant enhancement of the growth of
hair in experimental animals under conditions in which the
hair growth composition was used to treat the animals prior to
injection with cytophosphan and after cytophosphan. (Table 4,
below). Although the standard deviation of the results of the
test animals was rather large, nonetheless, the overall
results evidenced significant enhancement of hair growth in
the test animals which had been treated with the present
invention compared to untreated control animals. In addition,
the use of the present invention did not produce mortality in
any of the test animals (total of 234 animals).
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Table 3
FLOW CHART OF EXPFR~ AL PRO~K~
Treated Pre-Inj. Post-Inj. Pre-Inj. Post-Inj. None
Group 1 2 3 4
Day No.
0 Depil. Depil. Depil. Depil. Depil.
1-6
7 1st Anagen 1st Anagen 1st Anagen
8 Anagen 60% Anagen 95%
g Anagen 70% 1st Inject. 1st Anag. 1st Anag.
100%
1st Inject.
101st Inject. 1st Inject. Anagen 100~
1st Inject.
11-13
141st Catagen
1st Catagen 2nd Anagen 1st Catag. 1st Catag. 1st Catag.
30% 38%
16 2nd Anagen Anagen 2nd Anagen 2nd Anagen Catagen
50% 100~ 20% 30% 50%
17 Anagen 100% + 1st CBC + Anagen Anagen Catagen
1st CBC + Biopsy Biopsy 100% 100% 100%
18 2nd Inject. 2nd Inject. 2nd Anagen
100%
19
Vellus Hair Vell. Hair
21 Biopsy
22-23
24 Term. Hair Term. Hair
26 2nd Catagen 2nd Catagen
50% 50%
27 Catagen 100% Catagen 100%
28 3rd Catagen 100% 3rd Categen 50%
29 Anagen 100%
2nd CBC + 2nd CBC +
Biopsy Biopsy
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Table 4
HAIR LENGTH IN MICE
CuNlK~L PRE-INJECTIûN TR~A~M~NT POST-INJECTION ~RRATMRNT
No Treatment With Invention With Invention
1st 1st
Dav 7 Day 7
0.121 + SD 0.206 0.084 + 0.12
(29.4%) (66.6%)
DaY 11
0.097 + SD 0.13
(41%)
2nd 2nd
Dav 12 Day 12
0.79 + 0.25 0.65 + 0.31
(100%) (100%)
% Number of An; ~1 s with Hair
Example 4
Restoration of Hair Color
The following four cases evidence that the present inven-
tion may be used to restore the natural hair color in hair
treated using the present invention. Auxiliary to enhanced hair
growth, the same hair evidenced an unexpected hair color restora-
tion consistent with the enhancement or restoration of
melanogenesis by the present composition.
Case #l
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A man in his 40's, who had a diffuse vertex hair loss of
5 years duration has been using the present composition (composi-
tion lc.2) for 5 and 1/2 months. His natural hair coloring is
light-r~ h brown, but in the vertex area, where hair loss
appeared, the color had faded to yellowish-white. After using
the present composition, the natural hair coloring of hair in the
vertex area was restored. The hair has the same re~ h-brown
coloring as the res of the hair on the head.
Case #2
A woman in her late 40'S suffered diffuse, rapid progres-
sive hair loss throughout the frontal and vertex areas. Applica-
tion of the present composition for 4.5 months has resulted in
cessation of hair loss, new hair growth, increased rate of hair
growth and resoration of her original dark-brown pigmentation.
Case #3
A 69 year old female suffered from rapid progressive hair
loss in the frontal and vertex areas. She has been using the
present composition as recommended for the last 6 months. The
treatment has induced cessation of hair loss, increased the rate
of hair growth and the growth of new hair. Restoration of her
original red pigmentation was observed in hair that has been
white for years (notwithstanding the artificial hair rinses she
r uses).
Case #4
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A 54 year old caucasian male who has a beard in which the
original dark coloration became streaked with white used the
present composition as r~cl ~nded for 1 month (nighttime appli-
cation sufficient to allow contact of the formulation with the
underlying skin, washing off the composition in the morning).
The treatment has induced pigment change in which the original
coloration has returned in the newly growth hair, and in the
white hair, the roots have become black, evidencing restoration
of hair color in the newly grown portion of the hair.
Example 5
Experiments Det~ ; n; ng Angiogenesis in Ani 1~
The material as defined in the materials and methods sec-
tion, above, in liquid or gel form was used (with the exception
that triiodothyronine at 13.02 ng/ml was substituted for the
thyroxine, the amount of CaCl2 used was 4.0 X 10-5 and no Phenol
Red was included in the formulation- in liquid form, no thickener
was used- in gel form, 4 g of methyl cellulose was used) to treat
corneal ulcers in Hartley derived female guinea pigs.
Hartley derived female guinea pigs weighing 350-400g were
housed in individual cages and fed guinea pig chaw and vitamin C
enriched water ad libitum.
General anasthetics of the animals was obtained by injec-
tion of Imalgene (Katamin, Rhone Merieux, France) 20 mg/kg, and
local anasthetics with 1 drop of Localin (Benoxinate HCl 0.4~,
Fischer Pharmaceuticals, Ltd.).
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Under Zeiss dissecting Microscope, using a 3mm diameter
biopsy punch to delineate the ulcer margins, the corneal
epithelium was carefully scraped off the corneas of both eyes.
~ .
Experiment 1
Following the injury, the right eyes of 7 ~ni ~ls were
treated with the formulation as described above in liguid form
using two drops every two hours and in liguid form in the eve-
ning. After 24 hours and 48 hours, the eyes were .o~ ;ned under
the microscope and photographed.
Experiment 2
Six animals were used in this experiment. Following the
injury, in three animals both right and left eyes were treated
with the formulation as described above in liquid form using one
drop every three hours (until 11 PM and again starting at 6:30
AM). In the other three ~n;~l s~ both eyes were not treated.
After 24 hours and 48 hours the corneas of both eyes of all of
the ~n; ~ls were t~m; ned under the microscope and photographed.
Experiment 3
six ~n;m~l s were used in this experiment. Following
injury, in three ~n;~ls both eyes were treated with the formula-
tion as described above in liguid form, 2 drops every 2 hours and
in gel form, in the evening. In the other three animals, both
eyes were left untreated.
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Results
A total number of 19 guinea pig eyes were treated with
Cariel while a total number of 16 eyes were used as a control
(untreated).
The result of the treatment yielded the following:
1. A strong angiogenic reaction which was seen in and around
all treated corneas:
a. The limbal capillary bed was dilated.
b. Very fine radially oriented capillaries were found
adjacent to the corneal ulcer in every case where treatment
occurred.
2. Engorgement of the surrounding deep and superficial blood
vessels.
3. Edema of the cornea and swelling of the stroma surround-
ing the ulcer.
4. The results of the untreated controls yielded no
angiogenic response in and around the corneal ulcers.
Conclusions
1. Treatment with the above-described formulations, whether
in liquid or gel form induced an angiogenesis response in the
corneal ulcers.
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2. The ?ch~ni- of action of the formulation in enhancing
hair growth is partially due to its strong angiogenic activity
and increased blood flow.
Example 6
Experiments Performed on Guinea Pigs
Thermal Burn ~.d~
Hartley-derived albino female guinea-pigs weighing 250 g
were used in this study. The animals were housed in individual
cages and fed regular guinea-pig chow and water enriched with
Vitamin C ad libitum. All surgical procedures to impart burn
wounds to the animals were performed under general anaesthesia of
Katamin HCl 150 mg/kg i.m./d,1-2-(chlorophenyl)-2-
(methylamino)cyclohexanone hydrochloride, Parke Davis).
The ~ ls were divided into two groups. In the first
group, eight guinea pigs were treated with Silverol, which is
currently used as a preparation for burn wounds in all military
trauma units in Israel. The second group of eight animals was
treated essentially with composition lcl. of the materials and
methods section (5 ug/ml insulin, methyl cellulose as gelling
agent). The experiment was repeated on three different occa-
sions.
Laser Doppler Flowmeter measurements on burn wounds were
performed on days 2 and 8 after injury. Figures 1 and 2 present
the data obtained from the Laser Doppler Flowmeter measurements
in graph and hologram form. The results evidence that the
preferred embodiment evidences effective increase in blood flow
compared to conventional therapy. This increased blood flow is
believed to be at least partially responsible for the enhanced
growth to hair and nail tissue and the unexpected skin
revitalization activity these compositions evidence.
While the invention has been described in its preferred
embodiment, it is to be understood that the words which have been
used are words of description rather than limitation and that
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changes may be made within the purview of the appended claims
without departing from the true scope and spirit of the invention
in its broader aspects.
