Note: Descriptions are shown in the official language in which they were submitted.
BAKUCHIOL COMPOSITIONS FOR TREATMENT OF POST INFLAMMATORY
HYPERPIGMENTATION
BACKGROUND
Technical Field
The present invention generally relates to bakuchiol compositions and
their use for treatment of post inflammatory hyperpigmentation.
Description of the Related Art
Post inflammatory hyperpigmentation (PIH) is a unique skin
pigmentation condition that involves increased melanin synthesis and
deposition. PIH
is also characterized by apoptosis of melanocyte cells due to oxidative stress
and
assaults from mediators and cytokines of inflammatory and immune responses.
The
melanin deposition (i.e., hyperpigmentation) occurs beyond the epidermal
level, with
significant melanin being released into the papillary dermis and trapped by
large
immune cells. These unique histological characteristics of PIH present a
number of
difficulties for treatment of PIH with traditional agents.
Common treatments for PIH are focused on prevention of further
pigment development by controlling inflammation with corticosteroids and using
photoprotection agents. Chemical peeling compounds, such as salicylic acid and
glycolic acid, are also used to facilitate the skin renewal function and to
remove or
diminish the pigmentation. Topical retinoids have also been used to treat PIH,
but such
methods require up to 40 weeks before significant benefits are seen.
Tyrosinase inhibitors, or skin whiteners, such as hydroquinone, azelaic
acid, kojic acid and licorice extract, have also been employed for treatment
of PIH.
One significant disadvantage of using traditional skin whitening agents or
tyrosinase
inhibitors is the non-specific discolorization of the regular skin near the
PIH site. This
effect reduces the color of the background skin and makes the PIH sites more
prominent. Thus, these agents must be applied very carefully over the site of
the PIH.
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Date Recue/Date Received 2021-08-20
In addition, tyrosinase inhibitors are only effective for epidermal
hyperpigmentation
since this is the location of melanin synthesis by tyrosinase. Because post
inflammatory
pigmentation is in a deep layer of the skin (e.g., papillary dermis), it takes
more than 6
months of continued application of hydroquinone medication before visual
changes of
the dark marks are seen. Finally, hydroquinone type skin whiteners or
tyrosinase
inhibitors are associated with side effects including skin irritation,
dryness,
teratogenicity and induction of vitilago and skin cancers.
Post inflammatory hyperpigmentation can be derived from endogenous
inflammatory skin disorders such as acne, atopic dermatitis, allergic contact
dermatitis,
incontinent pigmenti, lichen planus, lupus erythematosus, morphea. Other
causes of
PIH include exogenous inflammatory stimuli such as mechanical trauma, ionizing
and
nonionizing radiation, burns, laser therapies and skin infections. Current
therapeutic
agents for the above skin disorders are ineffective for preventing,
alleviating, reducing
or treating PIH. For example, the above skin disorders are often treated with
anti-
inflammatory agents, such as retinoids, COX inhibitors (e.g., salicylic acid),
nonsteroidal anti-inflammatory drugs (NSAIDs), antimicrobial agents or
hormonal
drugs, but these treatments have been shown to be ineffective against PIH.
While significant advances have been made in the field, there continues
to be a need in the art for methods for preventing, alleviating, reducing or
treating
excess pigmentation. For example, methods for treatment of post inflammatory
hyperpigmentation are needed. The present invention fulfills these needs and
provides
further related advantages.
BRIEF SUMMARY
In general terms, the current invention is directed to methods for
preventing, alleviating, reducing or treating excess pigmentation. The
excess
pigmentation may be a result of a condition derived from an inflammatory skin
condition. For example, one embodiment of the present invention is a method
for
preventing, alleviating, reducing or treating post inflammatory
hyperpigmentation
(PIH). Such PIH may be derived from any number of skin disorders, including
acne.
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Date Recue/Date Received 2021-08-20
The method comprises administering an effective amount of a composition
comprising
bakuchiol and less than 500 ppm total furanocoumarin impurities to a mammal.
In contrast to other skin lightening agents, the presently disclosed
bakuchiol compositions are not tyrosinase inhibitors. Thus, the disclosed
compositions
specifically decolorize at the PIH site and are useful for treating
hyperpigmentation in
the deep layers of skin (e.g., papillary dermis). Accordingly, the presently
disclosed
methods comprise certain advantages over previous methods for treatment of
hyperpigmentation and/or PIH.
Accordingly, one embodiment of the present disclosure is directed to a
method for preventing, alleviating, reducing or treating excess pigmentation
resulting
from a condition derived from an inflammatory skin disorder, the method
comprising
administering to a mammal an effective amount of a composition comprising
bakuchiol,
or a pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier
and less than 500 ppm total furanocoumarin impurities.
In some embodiments, the condition is post inflammatory
hyperpigmentation. In other embodiments, the composition comprises less than
100
ppm total furanocoumarin impurities. In other embodiments, the furanocoumarin
impurities comprise psoralen, isopsoralen or combinations thereof. In
certain
embodiments, the composition shows no tyrosinase inhibition activity relative
to a kojic
acid control.
In yet other embodiments, the bakuchiol is chemically synthesized or
isolated from a plant. For example, in some embodiments the bakuchiol is
isolated
from a plant. In some further embodiments, the plant is from the Psoralea
genus of
plants, for example, Psoralea corylifolia L. (Luguminosae) or Psoralea
glandulosa L.
(Papilionaceae).
In other embodiments, the bakuchiol is isolated from seeds, stems, bark,
twigs, tubers, roots, root bark, young shoots, rhixomes, flowers or other
reproductive
organs, leaves or other aerial parts, or combinations thereof.
In some other embodiments, the post inflammatory hyperpigmentation
(PIH) is derived from acne, atopic dermatitis, allergic contact dermatitis,
incontinent
pigmenti, lichen planus, lupus erythematosus, morphea, mechanical trauma,
ionizing or
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Date Recue/Date Received 2021-08-20
nonionizing radiation, burns, laser or drug therapies, skin infection or
combinations
thereof. For examples, in certain aspects the post inflammatory
hyperpigmentation
(PIH) is derived from acne.
In other embodiments, the composition comprises 0.001 % to 99.9 % by
total weight of bakuchiol and a pharmaceutically, dermatologically or
cosmetically
acceptable carrier. For example, in some aspects the composition comprises
from 0.1
% to 2.0 % by total weight of bakuchiol, 1.0 % by total weight of bakuchiol or
0.5 % by
total weight of bakuchiol.
In other embodiments, the dermatologically acceptable carrier comprises
a nonsticking gauze, a bandage, a swab, a cloth wipe, a patch, a mask or a
protectant.
In some other embodiments, the cosmetically acceptable carrier comprises a
cleanser or
an antiseptic.
In some aspects, the composition is formulated for topical
administration. For example, in some aspects the composition further comprises
a
cream, a lotion, an ointment, a gel, an emulsion, a liquid, a paste, a soap, a
powder or
combinations thereof.
In other embodiments the composition further comprises an adjuvant,
skin penetration enhancer or liposomes. In yet other embodiments, the adjuvant
comprises a-hydroxyacids, salicylic acid, linoleic acid, retinoids, benzoyl
peroxide,
sodium sulfacetamide, clindamycin, erythromycin, dapsone, tetracycline,
doxycyclin,
minocyclin, zinc, estrogen or derivatives thereof, anti-androgens, sulfur,
corticosteroids,
cortisone, tazarotene, curcumin extract, acacia extract, scutellaria extract,
green tea
extract, grape seed extract or combinations thereof.
In certain embodiments, the composition is formulated in a capsule, for
example, controlled release capsule. In other embodiments, the composition is
administered topically, by aerosol, by suppository, intradermically,
intramuscularly or
intravenously.
In some aspects, the method prevents excess pigmentation. In other
aspects, the method alleviates excess pigmentation. In yet other aspects, the
method
reduces excess pigmentation. In
still other aspects, the method treats excess
pigmentation.
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Date Recue/Date Received 2021-08-20
In other embodiments, the excess pigmentation occurs in a deep layer of
skin, for example, in a papillary dermis layer of skin. In other embodiments,
the
method further comprises reducing super oxide anion. In some other
embodiments, the
method further comprises reducing melanogenesis. In yet other embodiments, the
method further comprises reducing melanocyte proliferation. In
still other
embodiments, the method further comprises preventing melanocyte apotosis.
In certain other embodiments, the mammal is a human. In some other
embodiments, the mammal is in need of preventing, alleviating, reducing or
treating
excess pigmentation resulting from a condition derived from an inflammatory
skin
disorder. For example, the mammal may be in need of treatment for PIH.
In another embodiment, the present disclosure is directed to a method for
reducing melanogenesis, reducing melanocyte proliferation or preventing
melanocyte
apotosis, the method comprising administering to a mammal an effective amount
of a
composition comprising bakuchiol, or a pharmaceutically acceptable salt
thereof, and a
pharmaceutically acceptable carrier and less than 500 ppm total furanocoumarin
impurities. In some further embodiments, the method further comprises reducing
super
oxide anion.
In other embodiments, the composition comprises less than 100 ppm
total furanocoumarin impurities. In other embodiments, the furanocoumarin
impurities
comprise psoralen, isopsoralen or combinations thereof. In certain
embodiments, the
composition shows no tyrosinase inhibition activity relative to a kojic acid
control.
In yet other embodiments, the bakuchiol is chemically synthesized or
isolated from a plant. For example, in some embodiments the bakuchiol is
isolated
from a plant. In some further embodiments, the plant is from the Psoralea
genus of
plants, for example, Psoralea corylifolia L. (Luguminosae) or Psoralea
glandulosa L.
(Papilionaceae).
In other embodiments, the bakuchiol is isolated from seeds, stems, bark,
twigs, tubers, roots, root bark, young shoots, rhixomes, flowers or other
reproductive
organs, leaves or other aerial parts, or combinations thereof.
In some embodiments, the melanogenesis, the melanocyte proliferation
or the melanocyte apotosis is a result of post inflammatory hyperpigmentation
(PIH).
Date Recue/Date Received 2021-08-20
In some other embodiments, the post inflammatory hyperpigmentation (PIH) is
derived
from acne, atopic dermatitis, allergic contact dermatitis, incontinent
pigmenti, lichen
planus, lupus erythematosus, morphea, mechanical trauma, ionizing or
nonionizing
radiation, burns, laser or drug therapies, skin infection or combinations
thereof. For
examples, in certain aspects the post inflammatory hyperpigmentation (PIH) is
derived
from acne.
In other embodiments, the composition comprises 0.001 % to 99.9 % by
total weight of bakuchiol and a pharmaceutically, dermatologically or
cosmetically
acceptable carrier. For example, in some aspects the composition comprises
from 0.1
% to 2.0 % by total weight of bakuchiol, 1.0 % by total weight of bakuchiol or
0.5 % by
total weight of bakuchiol.
In other embodiments, the dermatologically acceptable carrier comprises
a nonsticking gauze, a bandage, a swab, a cloth wipe, a patch, a mask or a
protectant.
In some other embodiments, the cosmetically acceptable carrier comprises a
cleanser or
an antiseptic.
In some aspects, the composition is formulated for topical
administration. For example, in some aspects the composition further comprises
a
cream, a lotion, an ointment, a gel, an emulsion, a liquid, a paste, a soap, a
powder or
combinations thereof.
In other embodiments the composition further comprises an adjuvant,
skin penetration enhancer or liposomes. In yet other embodiments, the adjuvant
comprises a-hydroxyacids, salicylic acid, linoleic acid, retinoids, benzoyl
peroxide,
sodium sulfacetamide, clindamycin, erythromycin, dapsone, tetracycline,
doxycyclin,
minocyclin, zinc, estrogen or derivatives thereof, anti-androgens, sulfur,
corticosteroids,
cortisone, tazarotene, curcumin extract, acacia extract, scutellaria extract,
green tea
extract, grape seed extract or combinations thereof.
In certain embodiments, the composition is formulated in a capsule, for
example, controlled release capsule. In other embodiments, the composition is
administered topically, by aerosol, by suppository, intradermically,
intramuscularly or
intravenously.
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Date Recue/Date Received 2021-08-20
In some aspects, the method prevents excess pigmentation. In other
aspects, the method alleviates excess pigmentation. In yet other aspects, the
method
reduces excess pigmentation. In
still other aspects, the method treats excess
pigmentation. In some embodiments, the excess pigmentation occurs in a deep
layer of
skin, for example, in a papillary dermis layer of skin.
In some other embodiments, the method reduces melanogenesis. In yet
other embodiments, the method reduces melanocyte proliferation. In still other
embodiments, the method prevents melanocyte apotosis.
In certain other embodiments, the mammal is a human. In some other
embodiments, the mammal is in need of treatment to reduce melanogenesis,
reduce
melanocyte proliferation or prevent melanocyte apotosis.
In still other embodiments, the composition further comprises salicylic
acid or a pharmaceutically acceptable salt thereof.
Other embodiments of the present disclosure are directed the use of a
composition comprising bakuchiol or pharmaceutically acceptable salt thereof
and
salicylic acid or a pharmaceutically acceptable salt thereof and a
pharmaceutically
acceptable carrier for treating inflammatory or non-inflammatory lesions in a
mammal,
the use of a composition comprising bakuchiol or pharmaceutically acceptable
salt
thereof and salicylic acid or a pharmaceutically acceptable salt thereof and a
pharmaceutically acceptable carrier for the manufacture of a medicament for
treating
inflammatory or non-inflammatory lesions in a mammal as well as a composition
comprising bakuchiol or pharmaceutically acceptable salt thereof and salicylic
acid or a
pharmaceutically acceptable salt thereof and a pharmaceutically acceptable
carrier for
treating inflammatory or non-inflammatory lesions in a mammal. For example, in
some
embodiments the lesions comprise inflammatory acne lesions. In other
embodiments,
the method treats inflammatory and non-inflammatory lesions.
In certain other embodiments of the foregoing, the mammal is a human.
In some other embodiments, the mammal is in need of treatment of inflammatory
or
non-inflammatory lesions.
In other embodiments, the present invention includes a composition
comprising bakuchiol or pharmaceutically acceptable salt thereof and salicylic
acid or a
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Date Recue/Date Received 2021-08-20
pharmaceutically acceptable salt and a pharmaceutically acceptable carrier. In
certain
embodiments, the composition is formulated for topical administration.
These and other aspects of the invention will be apparent upon reference
to the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
In the figures, identical reference numbers identify similar elements.
The sizes and relative positions of elements in the figures are not
necessarily drawn to
scale and some of these elements are arbitrarily enlarged and positioned to
improve
figure legibility. Further, the particular shapes of the elements as drawn are
not
intended to convey any information regarding the actual shape of the
particular
elements, and have been solely selected for ease of recognition in the
figures.
Figure 1 depicts a chromatogram of bakuchiol, psoralen and isopsoralen
standards.
Figure 2 shows chromatograms of bakuchiol compositions before and
after hydrolysis.
Figure 3 presents data showing the strong antioxidant properties of
bakuchiol compositions.
Figure 4 is a graph of tyrosinase inhibition activity of bakuchiol
compositions and kojic acid.
Figure 5 shows changes in PIH severity of individual test subjects.
Figure 6 presents a graph of the percent change of PIH affected facial
area of individual test subjects.
Figure 7 demonstrates mean percentage change in PIH and PIH severity
of five test subjects.
Figure 8 depicts the mean grade level reduction of PIH and PIH severity
at each visit compared to baseline.
Figure 9 shows photos of two study participants at various time intervals.
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Date Recue/Date Received 2021-08-20
DETAILED DESCRIPTION
In the following description, certain specific details are set forth in order
to provide a thorough understanding of various embodiments. However, one
skilled in
the art will understand that the invention may be practiced without these
details. In
other instances, well-known structures have not been shown or described in
detail to
avoid unnecessarily obscuring descriptions of the embodiments. Unless the
context
requires otherwise, throughout the specification and claims which follow, the
word
"comprise" and variations thereof, such as, "comprises" and "comprising" are
to be
construed in an open, inclusive sense, that is, as "including, but not limited
to."
Further, headings provided herein are for convenience only and do not
interpret the
scope or meaning of the claimed invention.
Reference throughout this specification to "one embodiment" or "an
embodiment" means that a particular feature, structure or characteristic
described in
connection with the embodiment is included in at least one embodiment. Thus,
the
appearances of the phrases "in one embodiment" or "in an embodiment" in
various
places throughout this specification are not necessarily all referring to the
same
embodiment. Furthermore, the particular features, structures, or
characteristics may be
combined in any suitable manner in one or more embodiments. Also, as used in
this
specification and the appended claims, the singular forms "a," "an," and "the"
include
plural referents unless the content clearly dictates otherwise. It should also
be noted
that the term "or" is generally employed in its sense including "and/or"
unless the
content clearly dictates otherwise.
Definitions
As used herein, and unless the context dictates otherwise, the following
terms have the meanings as specified below.
"Bakuchiol" as used herein refers to the compound having the following
formula:
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Date Recue/Date Received 2021-08-20
ThY
OH
Bakuchiol
wherein the benzylic double-bond may be either cis or trans. As used herein,
bakuchiol
includes pharmaceutically acceptable salts and tautomers of bakuchiol.
Phenolic
compounds structurally related to bakuchiol are also included within this
definition.
"BakutrolTM" is a composition comprising bakuchiol and may also
further comprise fatty acids extracted from Psoralea plants.
"UP256" refers to a 0.5% (wt/wt) formulation of bakuchiol.
"Preventing", "prevention" and "prevent" in the context of the disclosed
methods all refer to prophylactic methods which hinder or stop the occurrence
of a
particular condition, for example PIH.
"Alleviating", "alleviation" and "alleviate" in the context of the
disclosed methods all refer to lessening or mitigating the effects or symptoms
of a
particular condition, for example PIH.
"Reducing", "reduction" and "reduce" in the context of the disclosed
methods all refer to decreasing the effects or symptoms of a particular
condition, for
example PIH.
"Treating", "treatment" and "treat" in the context of the disclosed
methods all refer to techniques or methods intended to improve the symptoms of
or
decrease or stop the occurrence of a particular condition, for example PIH.
"Impurity" includes any substance that is not wanted in the bakuchiol
composition, typically resulting from the isolation of bakuchiol from natural
sources.
The term impurity includes, but is not limited to furanocoumarin compounds
including,
but not limited to, psoralen, isopsoralen and other coumarin type impurities.
Impurities
also refer to impurities resulting from synthetic processes to obtain these
compositions.
Date Recue/Date Received 2021-08-20
"Therapeutic" includes treatment and/or prophylaxis. When used,
therapeutic refers to humans as well as other animals.
"Pharmaceutically, cosmetically or therapeutically effective dose or
amount" refers to a dosage level sufficient to induce a desired biological or
functional
result. That result may be the alleviation of the signs, symptoms or causes of
a disease,
a skin condition or any other alteration of a biological system that is
desired.
"Placebo" refers to the substitution of the pharmaceutically or
therapeutically effective dose or amount dose sufficient to induce a desired
biological
that may alleviate the signs, symptoms or causes of a disease with a non-
active
substance.
A "host" or "subject" or "patient" is a living subject, human or animal,
into which the compositions described herein are administered. Thus, the
compositions
described herein may be used for veterinary as well as human applications and
the
terms "patient" or "subject" or "host" should not be construed in a limiting
manner. In
the case of veterinary applications, the dosage ranges can be determined as
described
below, taking into account the body weight of the animal.
As noted above, one embodiment of the present disclosure relates to use
of a composition comprising bakuchiol essentially free of furanocoumarin
impurities
for prevention, alleviation, reduction or treatment of excess pigmentation
resulting from
a condition derived from an inflammatory skin disorder. For example, the
disclosed
methods are useful for treatment of post inflammatory hyperpigmentation (PIH).
In
certain embodiments, the PIH may be derived from acne. The disclosed method
has
demonstrated human clinical efficacy in prevention, alleviation, reduction,
and
treatment of post inflammatory hyperpigmentation derived from skin disorders
such as
acne, atopic dermatitis, allergic contact dermatitis, incontinent pigmenti,
lichen planus,
lupus erythematosus, morphea; and post inflammatory hyperpigmentation caused
by
mechanical trauma, ionizing and nonionizing radiation, burns, laser and drug
therapies,
and skin infections by using a synthetic bakuchiol or furanocoumarin free
psoralea
extract bakuchiol composition. These and other aspects and various embodiments
of
the present disclosure will become evident upon reference to the description
which
follows.
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Date Recue/Date Received 2021-08-20
A. Bakuchiol Compositions
In one embodiment, the present disclosure provides a composition
comprising bakuchiol, which is substantially free of impurities, particularly
furanocoumarin impurities. This composition is also referred to herein as
BakutrolTM.
In some embodiments, the composition is obtained by organic synthesis from
simple
compounds as demonstrated in the literature (Hongli Chen and Yuanchao Li,
Letters in
Organic Chemistry, 2008, 5, 467-469) or from a plant. In certain embodiments,
the
bakuchiol composition is isolated from a plant. Plant sources of bakuchiol
include the
family of plants including, but not limited to Luguminosae, Papilionaceae,
Lauraceae
and Magnoliaceae and the genus of plants including, but not limited to
Psorlea,
Sassafras, Magnolia and Astractylodes. For example, the bakuchiol compositions
may
be isolated from Psoralea corylifolia L. (Luguminosae) or Psoralea glandulosa
L.
(Papilionaceae). The compositions may be obtained from the whole plant or from
one
or more individual parts of the plant including, but not limited to the seeds,
stems, bark,
twigs, tubers, roots, root bark, young shoots, rhixomes, flowers and other
reproductive
organs, leaves and other aerial parts or combinations thereof. Methods for
isolation of
bakuchiol from plants may include solvent extraction, supercritical fluid
extraction,
distillation, physical compressing or combinations thereof.
Bakuchiol, the structure of which is illustrated below, is a phenolic
compound having a single hydroxyl group on the aromatic ring and an
unsaturated
hydrocarbon chain. Although represented as trans in the structure below, the
benzylic
double bond of bakuchiol may also be cis.
/
/
OH
Bakuchiol
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Date Recue/Date Received 2021-08-20
The amount of Bakuchiol (i.e., weight percent (w/w%)) in the purified
plant extract depends on the method of extraction and the extent of
purification of the
crude extract. In one embodiment the amount of bakuchiol in the extract is in
the range
from 13.7% to 29.1% as shown in the Table 2. In other embodiments the amount
of
bakuchiol in the extract is at least 30%, at least 35%, at least 40%, at least
45%, at least
50%., at least 60%, at least 70%, at least 80% or at least 90%. In certain
embodiments,
the amount of Bakuchiol in the extract is 100%. In other certain embodiments,
the
amount of bakuchiol in the composition is not less than 60%. Examples 6-8
provide
examples of extracts comprising various amounts of bakuchiol.
Although bakuchiol is a biologically active natural product having a
great deal of potential for use in the prevention and treatment of various
diseases and
conditions, there are a number of limitations associated with the use of this
compound.
Some limitations include its low concentration in natural sources and the
presence of
co-existing toxic components in the bakuchiol source. The impurities present
in the
bakuchiol compositions will vary with the source of the bakuchiol. For
example,
psoralens, also known as furanocoumarins, are naturally occurring secondary
metabolites in Psoralea genus plants (a source of bakuchiol) and also exist in
many
fruits and vegetables. Examples of furanocoumarins often found co-existing
with
bakuchiol include psoralen and isopsoralen.
Z 0
0 0 0 0
/ 0
Psomlen Isopsoralen
A number of health risks have been associated with the handling, topical
application and ingestion of psoralen-containing plants and synthetic
psoralens.
Psoralens are well known to be phototoxic agents, which increase the
sensitivity of skin
to ultra violet radiation and promotes skin cancer (Epstein (1999) Med. Surg.
18(4):274-284). Psoralen has been shown to induce growth inhibition in rats
(Diawara
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Date Recue/Date Received 2021-08-20
et al. (1997) Cancer Lett. 114(1-2):159-160). Gonadal toxicity from crude
extracts of
Psoralea plants has been linked directly with the disruption of the
hypothalamus-
pituitary-gonadal axis (Takizawa et al. (2002) J. Toxicological Sciences
27(2):97-105).
Oral administration of the psoralens, bergapten (5-methoxypsoralen) and
xanthotoxin
(8-methoxypsoralen), in the diet of female rats reduced birthrates, the number
of
implantation sites, pups, corpora lutea, full and empty uterine weight and
circulating
estrogen levels in a dose-dependent manner (Diawara et al. (1999) J. Biochem.
Molecular Toxicology 13(3/4):195-203). Psoralens have also been shown to
induce the
mRNAs of the liver enzymes CYP1A1 and UGT1A6, suggesting that enhanced
metabolism of estrogens by psoralens may explain the reproductive toxicity and
the
observed reduction of ovarian follicular function and ovulation (Diawara et
al. (May-
June 2003) Pediatr Pathol Mol Med. 22(3):247-58.) Because of the toxicity of
furanocoumarins, it is important to remove psoralen and isopsoralen from
bakuchiol
compositions intended for treating post inflammatory hyperpigmentation or
other
conditions.
Psoralen and isopsoralen account for about 0.1-2% of the dry weight of
Psoralea seeds and about 1-20% of the weight in solvent or super-critical
fluid extracts.
Crude extracts from a Psoralea genus plant can be obtained by solvent
extraction, or
super-critical fluid extraction, distillation, physical compressing or a
combination of
above extraction methods. An enriched Bakuchiol composition can be obtained by
chromatographic separations, solvent partitions (India patent publication
#00570/KOL/2005), distillations, recrystallizations and other wet chemistry
and
physical processes. Published US Patent Application No. 2006/0251749 discloses
a
solvent extraction followed by hydroxylation to break down furanocoumarin
rings and
obtain an enriched bakuchiol composition essentially free of furanocoumarin
impurities
(e.g., less than 500 ppm, or less than 100 ppm furanocoumarin impurities). The
published method comprises the steps of extraction of the compound from a
plant
source, hydrolysis of the crude extract with a basic solution under heat, and
purification
by a method including but not limited to column chromatography, extraction
followed
by crystallization, solvent partition, recrystallization and combinations
thereof. The
present Applicants have discovered that such a composition of a bakuchiol
enriched
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Date Recue/Date Received 2021-08-20
psoralea extract essentially free of furanocoumarin impurities can be utilized
for
prevention, alleviation, reduction or treatment of excess pigmentation. For
example,
the disclosed bakuchiol compositions are effective for prevention,
alleviation,
reduction, or treatment of post inflammatory hyper pigmentation (PIH).
The present disclosure is also directed to methods for isolating and
purifying crude compositions of bakuchiol and related compounds obtained from
natural sources. The method for isolating and purifying these compositions
comprises
the steps of extraction of the compounds from a plant source, hydrolysis of
the crude
extract with a basic solution, and purification by a method including but not
limited to
column chromatography, extraction followed by crystallization, solvent
partition,
recrystallization and combinations thereof. Crude extracts purified in this
manner are
essentially free of furanocoumarin impurities such as psoralen and
isopsoralen. Thus,
the potential phototoxicity, topical irritation, carcenogenecity, and
reproductive toxicity
associated with these compounds are essentially eliminated.
In certain embodiments, the disclosed compositions comprise less than
500 ppm, less than 250 ppm, less than 100 ppm, or less than 50 ppm total
furanocoumarin impurities. The concentration of furanocoumarin impurities may
be
determined by any means known to one skilled in the art. For example, in one
embodiment the furanocoumarin content may be determined by HPLC.
The efficiency of bakuchiol extraction from plant sources was evaluated
using six different organic solvent systems under two sets of extraction
conditions as
described in Example 2. The results are set forth in Table 2. With reference
to Table 2,
it can be seen that bakuchiol can be extracted from Psoralea plants with any
number of
organic solvents and/or combinations thereof. The amount of bakuchiol in the
various
extracts ranged from 13.7% to 29.1% by weight. Other extraction methods
include, but
are not limited to, CO2 super-critical fluid extraction and water
distillation. Squeeze
exudates from fresh plant parts such as seeds, can also be utilized to obtain
Bakuchiol
compositions from natural sources.
The efficacy of purification of crude bakuchiol extracts by column
chromatography is demonstrated in Example 3 and Table 3. Eight different types
of
resins were evaluated specifically for their ability to separate bakuchiol
from
Date Recue/Date Received 2021-08-20
furanocoumarin impurities. Both
silica gel and CG-161 resins demonstrated
satisfactory separation. Column chromatographic separation of crude plant
extracts on
an industrial scale, however, is typically not economically feasible because
it requires
expensive equipment and reagents and experienced personnel. The extremely low
loading capacity of these samples due to the complexity of crude plant
extracts also
makes industrial scale column chromatography difficult.
Example 4 describes an economical method for separating bakuchiol
from furanocoumarin impurities. The method comprises treatment of compositions
containing furanocoumarin impurities with a base. As illustrated by the
following
Scheme 1, using NaOH for purposes of illustration, heating with a base opens
up the
lactone ring of the furanocoumarins, thereby converting them into the
corresponding
salts of carboxylic acids. These salts can then be easily separated from the
remainder of
the mixture by a variety of methods. The disclosed method allows preparation
of
bakuchiol compositions essentially free of furanocoumarin impurities (e.g.,
less than
500 ppm). Such highly pure bakuchiol compositions are not attainable using
standard
chromatographic techniques without the disclosed hydrolysis.
16
Date Recue/Date Received 2021-08-20
Reaction Scheme 1. Hydrolysis of Furanocoumarins
o o o HO 0
+¨
Na 00C
V 0 NaOH (55 InM) V 0
A
HO
0
0
_
_
+¨
Na 00C
The basic solution may comprise any base capable of opening lactone
rings, including, but not limited to sodium hydroxide, potassium hydroxide,
calcium
hydroxide, lithium hydroxide or combinations thereof. The solution can have
different
concentration and pH values to maximize the conversion to the acid salt. The
reaction
mixture can also be heated under different temperature and pressures to
maximize the
reaction rate, efficiency and yield.
The course of the reaction can be followed by HPLC to ensure complete
conversion of the furanocoumarins into their respective carboxylic acid salts.
HPLC
chromatograms of the crude composition before and after hydrolysis are
illustrated in
Figures 2. Upon completion of the reaction (as determined by HPLC), the
reaction
solution can be processed using various methods, including but are not limited
to
column chromatography, crystallization, solvent partition, precipitation,
solvent wash or
combinations thereof. Organic solvents that can be used for solvent
partitioning
include, but are not limited to petroleum ether, ethyl acetate, ethyl ether,
hexane,
chloroform, propanol, butanol, and methylene chloride, as well as other water
immiscible organic solvents.
Crude extracts purified in this manner are essentially free of
furanocoumarin impurities such as psoralen and isopsoralen. For example, the
purified
extract may comprise less than 500 ppm, less than 250 ppm, less than 100 ppm
or even
less than 50 ppm furanocoumarin impurities. Additionally, the color of these
highly
17
Date Recue/Date Received 2021-08-20
pure furanocoumarin free, bakuchiol compositions is light brown or red and
they are
very stable with respect to both color and composition of the active agent,
making them
particularly suitable for formulation, storage and cosmetic applications.
Also included in the present disclosure is a method for analyzing
compositions of bakuchiol, which enables detection and quantification of
impurities. In
this embodiment, the method for analyzing compositions of bakuchiol is
comprised of
the step of analyzing the compositions by high-pressure liquid chromatography
(HPLC). Analysis by HPLC enables quantification of the various components in
the
mixture and also provides a means to track bakuchiol, psoralen, isopsoralen
and other
natural components in Psoralea plants to guide the extraction, hydrolysis and
purification processes. A method for analyzing compositions of bakuchiol using
high
pressure liquid chromatography (HPLC) is described in Example 1 (Table 1).
B. Treatment of Excess Pigmentation with Bakuchiol Compositions
One embodiment of the present disclosure relates to use of a
composition comprising bakuchiol essentially free of furanocoumarin impurities
for
prevention, alleviation, reduction or treatment of excess pigmentation
resulting from a
condition derived from an inflammatory skin disorder. For example, the
disclosed
methods include prevention, alleviation, reduction or treatment of post
inflammatory
hyperpigmentation (PIH). In certain embodiments, the PIH may be derived from
acne.
The disclosure includes the formulation of a bakuchiol composition in a
typical
cosmetic vehicle and also in skin care cream, gel lotion and other
formulations as
discussed in more detail below. As shown in the Examples, the present
Applicants have
demonstrated the un-expected human clinical efficacy of bakuchiol compositions
in
prevention, alleviation, reduction or treatment of post inflammatory
hyperpigmentation
(PIH) derived from skin disorders such as acne, atopic dermatitis, allergic
contact
dermatitis, incontinent pigmenti, lichen planus, lupus erythematosus, morphea;
and post
inflammatory hyperpigmentation caused by mechanical trauma, ionizing and
nonionizing radiation, burns, laser and drug therapies, and skin infections.
The disclosed methods comprise administering to a mammal (e.g., a
human patient) an effective amount of a composition comprising bakuchiol,
which is
18
Date Recue/Date Received 2021-08-20
substantially free of furanocoumarin impurities. For example, the compositions
may
comprise less than 500 ppm furanocoumarin impurities. The composition may
comprise from about 0.0001% to about 100% bakuchiol. For example, in certain
embodiments the composition comprises from about 0.1% to about 2% bakuchiol or
from about 0.5% to about 1% bakuchiol. In other examples the composition
comprises
about 0.5% or about 1.0% bakuchiol. In certain embodiments, the mammal is a
human,
and in other embodiments the mammal is in need of prevention, alleviation,
reduction
or treatment of excess pigmentation resulting from a condition derived from an
inflammatory skin disorder, for example the mammal may be in need of treatment
for
PIH.
The present disclosure demonstrates unexpectedly unique biological
properties of synthetic or natural bakuchiol compositions. As shown in Example
5 and
Table 4, a Bakutrol composition comprising about 57.35% bakuchiol has
unexpectedly
high anti-oxidation capacity, especially against super oxide anion (>69,000
pmole
TE/g) with a total ORAC value against five predominant reactive species at
>92,000
pmole TE/g.
Superoxide is an anion with the chemical formula 02-. A chronic
inflammatory condition, such as acne vulgaris, can have dramatically increased
superoxide anion production from keratinocytes, which are stimulated by a gram-
positive anaerobic bacterium such as P. acnes (Grange PA., et al. Plos
Pathogens 2009,
5(7) 1-14.). Superoxide is biologically quite toxic and is deployed by the
immune
system to kill invading microorganisms. In phagocytes, superoxide is produced
in large
quantities by the enzyme NADPH oxidase for use in oxygen-dependent killing
mechanisms of invading pathogens. Superoxide anion and other reactive oxygen
species in the inflamed skin can also induce melanogenesis, melanocyte
proliferation
and melanocyte apotosis, which is a major causative factor of post
inflammatory
hyperpigmentation.
Accordingly, one embodiment of the present disclosure is a
method for alleviating, reducing or treating excess pigmentation resulting
from a
condition derived from an inflammatory skin disorder by reducing superoxide
with a
composition comprising bakuchiol essentially free of furanocoumarin
impurities. In
one embodiment, the condition is PIH. In another embodiment, the present
disclosure
19
Date Recue/Date Received 2021-08-20
provides a method of reducing melanogenesis or melanocyte proliferation or
inhibiting
melanocyte apotosis, for example, by reducing superoxide anion. The method
comprises administering an effective amount of a composition comprising
bakuchiol
essentially free of furanocoumarin impurities to a mammal. In certain
embodiments,
the mammal is a human, and in other embodiments the mammal is in need of
reducing
melanogenesis or melanocyte proliferation or inhibiting melanocyte apotosis.
As demonstrated in Example 6 and Figure 3, a composition comprising
77.02% bakuchiol, which is substantially free of impurities, particularly
furanocoumarin impurities showed protective effect on oxidative stress induced
by 4-
teriaybutylphenol (4-TBP). The cytotoxicity to melanocytes from the reactive
oxygen
species generated by 4-TBP was protected by the bakuchiol compositions at the
two
concentrations tested. While not wishing to be bound by the theory, the
present
Applicants believe the unexpected clinical benefits from a synthetic or
natural
bakuchiol composition of reducing, alleviating, preventing or treating post
inflammatory hyperpigmentation (PIH) is derived from its unique and unexpected
capacity to neutralize reactive oxygen species, especially superoxide anion,
and protect
melanocytes from oxidative stress under inflammation conditions that lead to
reduced
epidermal melanosis and/or dermal melanosis.
In addition to its unexpectedly high anti-oxidation capacity, the present
Applicants have discovered that the disclosed bakuchiol compositions are not
tyrosinase
inhibitors. This is in contrast to other reports which disclose bakuchiol as a
skin
whitening agent via tyrosinase inhibition (Japanese Patent No. P1107123). This
unexpected discovery led the present Applicants to the currently disclosed
methods for
treating PIH where the pigmentation occurs in a deep skin layer and tyrosinase
inhibitors are ineffective. The lack of tyrosinase inhibition of the disclosed
bakuchiol
compositions is shown in Example 7 and Figure 4. Both pure Bakuchiol (100%)
and
enriched Bakuchiol (77.02%) with not more than 100 ppm furanocoumarins from
natural sources have no tyrosinase inhibition function at eight different
dosages.
Compositions comprising bakuchiol at concentrations of 86.54% and
77.02% bakuchiol, were evaluated for their safety profiles. As shown in
Example 9 and
Table 6, based on in vitro and human clinical tests, the Bakutrol (UP256)
compositions
Date Recue/Date Received 2021-08-20
showed no eye irritation, no skin irritation on the normal or scarified skin,
no skin
contact sensitization, no phototoxicity and no mutagenic toxicity. The topical
creams of
the Bakuchiol composition were well tolerated in all human and in vitro tests.
As demonstrated in Example 10, a natural bakuchiol composition
(BakutrolTM) comprising 77.02% bakuchiol and less than 100 ppm furanocoumarin
extracted and enriched from the seeds of Psoralea corylifolia was tested in a
human
clinical trial on subjects with post inflammatory hyperpigmentation (PIH)
derived from
mild or moderate acne vulgaris. The bakuchiol composition was formulated at
0.5%
bakuchiol for topical application. After daily topical application of the 0.5%
Bakutrol
cream, a dramatic reduction of post inflammatory hyperpigmentation (PIH) was
observed in all five subjects. As shown in Figure 5, all five subjects had at
least one
grade level reduction of PIH severity. More than 50% improvement of PIH
affected
facial area was achieved after 8 weeks of continued topical application of the
0.5%
Bakutrol cream (Figure 6). The
mean percentage and absolute grade level
improvements of both PIH and its severity are summarized in Figures 7 & 8.
Improvements of more than 40%, or more than one grade level reduction of both
PIH
and severity, were achieved as early as 4 weeks after using the bakuchiol
composition.
Substantial reduction of PIH on the affected facial skin sites is clearly
evident in the
photos of two subjects as shown in Figure 9. Both subjects showed progressive
improvement of skin Post-Inflammatory Hyperpigmentation (PIH) associated with
mild
and moderate acnes after topical application of a Bakutrol cream.
Table 7 (Example 10) summarizes the clinical out puts for using a
furanocoumarin free Bakuchiol composition (i.e., Bakutrol) in comparison to
popular
acne treatment products which contain either an anti-microbial or an anti-
inflammatory
or combinations thereof. The
data in Table 7 clearly demonstrates that the
furanocoumarin free Bakuchiol composition not only improved inflammatory and
non-
inflammatory lesion counts, but also significantly improved skin Post-
Inflammatory
Hyperpigmentation. The PIH benefit from the Bakuchiol composition is not
expected
based on its lacks of tyrosinase inhibition activity.
Table 8 (Example 11) presents data showing reduction in PIH grade (i.e.,
extent of pigmentation). The data clearly shows that bakuchiol is more
effective than
21
Date Recue/Date Received 2021-08-20
both placebo and salicylic acid for treatment of PIH. Furthermore, the present
applicants have also discovered that bakuchiol (or compositions comprising the
same)
are effective for treatment of inflammatory lesions, such as acne lesions.
Table 9
(Example 11) demonstrates the effectiveness of bakuchiol for treatment of
inflammatory lesions compared to treatment with placebo or salicylic acid.
In addition to methods comprising treatment with compositions
comprising bakuchiol, the present invention includes embodiments wherein a
mammal
is treated with a composition comprising bakuchiol and salicylic acid. For
example, the
present applicants have discovered that salicylic acid is effective for
treatment of non-
inflammatory lesions, while bakuchiol is effective for treatment of
inflammatory
lesions. Accordingly, one embodiment of the present invention is directed to a
method
of treating inflammatory lesions (e.g., acne lesions), the method comprising
administering an effective amount of a composition comprising bakuchiol or a
pharmaceutically acceptable salt thereof to a mammal. Another embodiment is
directed
to a method of treating inflammatory and/or non-inflammatory lesions (e.g.,
acne
lesions), the method comprising administering an effective amount of a
composition
comprising bakuchiol and salicylic acid (or pharmaceutically acceptable salts
thereof)
to a mammal. Other embodiments include treatment of non-inflammatory lesions
by
administering an effective amount of a composition comprising salicylic acid
or a
pharmaceutically acceptable salt thereof to a mammal. In certain embodiments
of the
foregoing, the mammal is a human. In other embodiments, the mammal is in need
of
treatment for inflammatory and/or non-inflammatory lesions, such as acne.
In addition to treatment of lesions, the combination of bakuchiol and
salicylic acid is effective for treating any of the foregoing conditions
(e.g., PIH,
reducing melanogenesis, reducing melanocyte proliferation or preventing
melanocyte
apotosis, etc.). Accordingly, some embodiments are directed to treatment with
a
composition comprising bakuchiol and salicylic acid. Other embodiments include
a
composition comprising bakuchiol or a pharmaceutically acceptable salt
thereof,
salicylic acid or a pharmaceutically acceptable salt thereof and a
pharmaceutically
acceptable carrier.
22
Date Recue/Date Received 2021-08-20
The foregoing methods are effective to substantially eliminate
inflammatory and/or non-inflammatory lesions. For example in some embodiments
the
methods reduce lesions from about 1% to about 99% or from about 10% to about
90%.
In other embodiments, the methods reduce lesions by greater than 50%.
The ratio of bakuchiol to salicylic acid is not particularly limited and can
be determined by one of ordinary skill in the art based on the desired result.
For
example, in some embodiments the weight ratio of bakuchiol to salicylic acid
ranges
from about 1:100 to about 100:1. In other embodiments, the weight ration
ranges from
about 10:90, about 20:80, about 30:70, about 40:60, about 50:50, about 60:40,
about
70:30, about 80:20 to about 10:90. The compositions may be formulated
according to
any of the formulations described herein.
C. Formulation of Bakuchiol Compositions
The bakuchiol compositions of the present disclosure can be formulated
by any means known to those of skill in the art. As shown in Example 8 and
Table 5,
the compositions of the present disclosure can be formulated as
pharmaceutical,
cosmetic or dermatological compositions, and can include other components such
as a
pharmaceutically and/or cosmetically acceptable actives, excipient, adjuvant,
carrier or
combinations thereof. An excipient is an inert substance used as a diluent or
vehicle for
dermatological and cosmetically accepted products and drugs. Examples of such
excipients include, but are not limited to water, buffers, saline, glycerin,
hydrated silica,
propylene glycol, aluminum oxide, carrageenan, cellulose gum, titanium
dioxide,
Ringer's solution, dextrose solution, mannitol, Hank's solution, preservatives
and other
aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as
fixed
oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other
useful
formulations include suspensions containing viscosity enhancing agents, such
as
sodium carboxymethylcellulose, sorbitol, or dextran.
In Example 8, compositions of the present disclosure were formulated in
transcutol, or caprylic triglyceride, or polysorbate-20, or purified water or
combinations
of two or more of the above vehicle. Excipients can also contain minor amounts
of
additives, such as EDTA, disodium DDTA, BHA, BHT, diammonium citrate,
23
Date Recue/Date Received 2021-08-20
nordihydroguaiaretic acid, propyl gallate, sodium gluconate, sodium
metabisulfite, t-
butyl hydroquinone, SnC12, H202, and 2,4,5-trihydroxybutyrophenone, vitamin C,
vitamin E, vitamin E acetate, phenonip, and other substances that enhance
isotonicity
and chemical stability.
Examples of substances for adjusting the pH of the formulation include
sodium hydroxide, sodium carbonate, sodium bicarbonate, pentasodium
triphosphate,
tetrasodium pyrophosphate, sodium lauryl sulfate, calcium peroxide, phosphate
buffer,
bicarbonate buffer, tris buffer, histidine, citrate, and glycine, or mixtures
thereof.
Examples of flavors include, but are not limited to thimerosal, m- or o-
cresol, formalin,
fruit extracts and benzyl alcohol. Standard formulations can either be liquid
or solids,
which can be taken up in a suitable liquid as a suspension or solution for
administration.
Thus, in a non-liquid formulation, the excipient can comprise dextrose, human
serum
albumin, preservatives, etc., to which sterile water or saline can be added
prior to
administration.
In one embodiment, the bakuchiol composition is formulated with other
active compounds that target a different mechanism of action for reduction of
skin
pigmentation. Such
actives include but are not limited to, hydroquinone,
monobenzylether, arbuting, deoxy arbutin, mequinol, N-acetyl-4- S-cy steaminy
1phenol,
kojic acid, azelaic acid, glycolic acid, gentisic acid, favonoids, aloesin,
stilbene and
stilbene derivatives, licorice extract, bearberry extract, mulberry extract,
aloe vera gel,
glabridin, vitamin C derivatives, magnesium ascorbyl phosphate,
tetrahexyldecyl
ascorbate, vitamin e derivatives, tranexamic acid and its derivatives,
biomimetric of
TGF-B proteins, centaureidin, niacinamide, PAR-2 inhibitors, lectins,
neoglycoproteins,
resorcinol and its derivatives, and NivitolTM.
In another embodiment, the composition comprises anti-inflammatory
and anti-microbial agents that can synergistically work with the bakuchiol
composition
to reduce the infection, infection related inflammation, and acceleration of
epidermal
turnover. Such actives include but are not limited to a-hydroxyacids,
salicylic acid,
linoleic acid, retinoids, benzoyl peroxide, sodium sulfacetamide, clindamycin,
erythromycin, dapsone, tetracycline, doxycyclin, minocyclin, zinc, estrogen
and its
24
Date Recue/Date Received 2021-08-20
derivatives, anti-androgens, sulfur, corticosteroids, cortisone, tazarotene,
curcumin
extract, acacia extract, scutellaria extract, green tea extract, and grape
seed extract.
In certain embodiments, the composition comprises an adjuvant or a
carrier. Adjuvants are typically substances that generally enhance the
biological
response of a mammal to a specific bioactive agent. Suitable adjuvants
include, but are
not limited to, Freund's adjuvant; other bacterial cell wall components;
aluminum,
calcium, copper, iron, zinc, magnesium, stannous based salts; silica;
microdermabrasion
agents, polynucleotides; toxoids; serum proteins; viral coat proteins; other
bacterial-
derived preparations; gamma interferon; block copolymer adjuvants, such as
Hunter's
Titermax adjuvant (Vaxcel.TM., Inc. Norcross, Ga.); Ribi adjuvants (available
from
Ribi ImmunoChem Research, Inc., Hamilton, Mont.); and saponins and their
derivatives, such as Quil A (available from Superfos Biosector A/S, Denmark).
Carriers are typically compounds that increase the half-life of a therapeutic
composition
in the treated host. Suitable carriers include, but are not limited to,
polymeric
controlled release formulations, biodegradable implants, liposomes, nano-
capsulation,
nano-particles, bacteria, viruses, oils, esters, and glycols.
In other examples, the composition is prepared as a controlled release
formulation, which slowly releases the composition into the host. As used
herein, a
controlled release formulation comprises a composition of bakuchiol in a
controlled
release vehicle. Suitable controlled release vehicles will be known to those
skilled in
the art.
Examples of controlled release formulations are biodegradable (i.e.,
bioerodible) and include capsules.
In one embodiment, a suitable ointment is comprised of the desired
concentration of UP256 (bakuchiol) that is an efficacious, nontoxic quantity
generally
selected from the range of 0.001% to 100% based on total weight of the topical
formulation, from 65% to 100% (for example, 75% to 96%) of white soft
paraffin, from
0% to 15% of liquid paraffin, and from 0% to 7% (for example 3 to 7%) of
lanolin or a
derivative or synthetic equivalent thereof. In another embodiment the ointment
may
comprise a polyethylene - liquid paraffin matrix.
In one embodiment, a suitable cream is comprised of an emulsifying
system together with the desired concentration of UP256 (bakuchiol)
synthesized and/or
Date Recue/Date Received 2021-08-20
isolated from a single plant or multiple plants as provided above. The
emulsifying
system is preferably comprised of from 2 to 10% of polyoxyethylene alcohols
(e.g., the
mixture available under the trademark CetomacrogolTM1000), from 10 to 25% of
stearyl alcohol, from 20 to 60% of liquid paraffin, and from 10 to 65% of
water;
together with one or more preservatives, for example from 0.1 to 1% of N,N"-
methylenebis[N'43-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyllureal (available
under
the name Imidurea USNF), from 0.1 to 1% of alkyl 4-hydroxybenzoates (for
example
the mixture available from Nipa Laboratories under the trade mark Nipastat),
from 0.01
to 0.1% of sodium butyl 4-hydroxybenzoate (available from Nipa Laboratories
under
the trade mark Nipabutyl sodium), and from 0.1 to 2% of phenoxyethanol.
In one embodiment, a suitable gel is comprised of a semi-solid system in
which a liquid phase is constrained within a three dimensional polymeric
matrix with a
high degree of cross-linking. The liquid phase may be comprised of water,
together
with the desired amount of UP256 (bakuchiol), from 0.01 to 20% of water-
miscible
additives, for example glycerol, polyethylene glycol, or propylene glycol, and
from 0.01
to 10%, preferably from 0.5 to 2%, of a thickening agent, which may be a
natural
product, for example tragacanth, pectin, carrageen, agar and alginic acid, or
a synthetic
or semi-synthetic compound, for example methylcellulose and
carboxypolymethylene
(carbopol); together with one or more preservatives, for example from 0.1 to
2% of
methyl 4-hydroxybenzoate (methyl paraben) or phenoxyethanol-differential.
Another
suitable formulation, is comprised of the desired amount of UP256 (bakuchiol),
together with from 70 to 90% of polyethylene glycol (for example, polyethylene
glycol
ointment containing 40% of polyethylene glycol 3350 and 60% of polyethylene
glycol
400, prepared in accordance with the U.S. National Formulary (USNF)), from 5
to 20%
of water, from 0.02 to 0.25% of an anti-oxidant (for example butylated
hydroxytoluene), and from 0.005 to 0.1% of a chelating agent (for example
ethylenediamine tetraacetic acid (EDTA)).
The term soft paraffin as used above encompasses the cream or ointment
bases white soft paraffin and yellow soft paraffin. The term lanolin
encompasses native
wool fat and purified wool fat. Derivatives of lanolin include in particular
lanolins
which have been chemically modified in order to alter their physical or
chemical
26
Date Recue/Date Received 2021-08-20
properties and synthetic equivalents of lanolin include in particular
synthetic or
semisynthetic compounds and mixtures which are known and used in the
pharmaceutical and cosmetic arts as alternatives to lanolin and may, for
example, be
referred to as lanolin substitutes.
One suitable synthetic equivalent of lanolin that may be used is the
material available under the trademark SoftisanTM known as Softisan 649.
Softisan
649, available from Dynamit Nobel Aktiengesellschaft, is a glycerine ester of
natural
vegetable fatty acids, of isostearic acid and of adipic acid; its properties
are discussed
by H. Hermsdorf in Fette, Seifen, Anstrichmittel, Issue No. 84, No.3 (1982),
pp. 3-6.
The other substances mentioned hereinabove as constituents of suitable
ointment or cream bases and their properties are discussed in standard
reference works,
for example US Pharmacopoeia.
Cetomacrogol 1000 has the formula
CH3(CH2).(OCH2CH2)n0H, wherein m may be 15 or 17 and n may be 20 to 24.
Butylated hydroxytoluene is 2,6-di-tert-butyl-p-cresol. Nipastat is a mixture
of methyl,
ethyl, propyl and butyl 4-hydroxybenzoates.
The compositions disclosed herein may be produced by conventional
pharmaceutical techniques. Thus the aforementioned compositions, for example,
may
conveniently be prepared by mixing together at an elevated temperature, for
example
60-70 C, the soft paraffin, liquid paraffin if present, and lanolin or
derivative or
synthetic equivalent thereof. The mixture may then be cooled to room
temperature,
and, after addition of the hydrated crystalline calcium salt of mupirocin,
together with
the corticosteroid and any other ingredients, stirred to ensure adequate
dispersion.
Finally, bakuchiol has a partition coefficient of log P = 6.13. The
partition coefficient of a chemical compound provides a thermodynamic measure
of its
hydrophilicity/lipophilicity balance and thus its potential bioavailability.
Having a
partition coefficient of 6.13 means this compound has high cell membrane
penetration
and bioavailability when formulated in a delivery system. The skin penetration
of the
active compound ¨ bakuchiol in a skin care cream was quantified in an ex-vivo
test on
isolated human skin. The results showed a good skin penetration and
bioavailability.
In certain embodiments, the disclosed compositions comprise a skin penetration
enhancer.
27
Date Recue/Date Received 2021-08-20
D. Administration of Bakuchiol Compositions
The compositions of the present disclosure can be administered by any
method known to one of ordinary skill in the art. For example, the disclosed
compositions can be administered internally or topically. The modes of
administration
include, but are not limited to, enteral (oral) administration, parenteral
(intravenous,
subcutaneous, and intramuscular) administration and topical application. In
certain
embodiments, the compositions are administered topically.
The content of a bakuchiol composition in the finished skin care
products for PIH can range from 0.001% to 99.9% by weight. In some
embodiments,
the composition comprises from 0.1% to 2% bakuchiol. In other examples the
composition comprises 0.5% or 1.0% bakuchiol. In certain embodiments the
amount of
bakuchiol composition in a PIH skin care cream ranges from 0.5-1%. The methods
according to this disclosure comprise administering internally or topically to
a mammal
a therapeutically effective amount of a composition comprising bakuchiol,
which is
totally synthesized or isolated from natural sources (or a combination
thereof) and is
substantially free of impurities, particularly furanocoumarin impurities
(e.g., less than
500 ppm).
The therapeutic agents of the instant disclosure can be administered
topically by any suitable means known to those of skill in the art for
topically
administering therapeutic compositions. Such modes of administration include,
but are
not limited to, as an ointment, gel, lotion, or cream base or as an emulsion,
as a patch,
dressing or mask, a nonsticking gauze, a bandage, a swab or a cloth wipe. Such
topical
application can be locally administered to any affected area, using any
standard means
known for topical administration. A therapeutic composition can be
administered in a
variety of unit dosage forms depending upon the method of administration. For
particular modes of delivery, the therapeutic compositions can be formulated
in an
excipient as discussed above. A therapeutic composition of the present
disclosure can
be administered to any host, preferably to mammals, and more preferably to
humans.
The particular mode of administration will depend on the condition to be
treated.
28
Date Recue/Date Received 2021-08-20
Regardless of the manner of administration, the specific dose is
calculated according to the approximate body weight of the host. Further
refinement of
the calculations necessary to determine the appropriate dosage for treatment
involving
each of the above mentioned formulations is routinely made by those of
ordinary skill
in the art and is within the scope of tasks routinely performed by them
without undue
experimentation, especially in light of the dosage information and assays
disclosed
herein. These dosages may be ascertained through use of the established assays
for
determining dosages utilized in conjunction with appropriate dose-response
data. In
certain embodiments, the dose of the composition comprising bakuchiol ranges
from
0.001 to 200 mg per kilogram of body weight.
The following examples are provided for purposes of illustration, not
limitation.
29
Date Recue/Date Received 2021-08-20
EXAMPLES
EXAMPLE 1
QUANTIFICATION OF BAKUCHIOL, PSORALEN AND ISOPSORALEN BY HPLC
The amount of bakuchiol, psoralen and isopsoralen in the extracts,
fractions, process materials, ingredients and final formulated products were
quantified
by high pressure liquid chromatography (HPLC) using a PhotoDiode Array
detector
(HPLC/PDA). The targeted compounds were eluted from a Luna Phenyl-hexyl column
(250 mm x 4.6 mm) using an acetonitrile (ACN) or methanol & water gradient
from
36% to 100% ACN over a period of 12 minutes, followed by 100% ACN for three
minutes. The detailed HPLC conditions used are set forth in Table 1. A
chromatogram
of the HPLC separation is shown in Figure 1. The targeted compounds were
identified
and quantified based on retention time and UV peak area using commercially
available
pure bakuchiol, psoralen and isopsoralen as quantification standards. The
retention
times for the bakuchiol, psoralen and isopsoralen were 18.19 minutes, 7.33
minutes and
7.95 minutes, respectively.
Table 1. HPLC Conditions for quantification of Bakuchiol, Psoralen and
Isopsoralen
Column Luna Phenyl-hexyl, 150 x 4.6mm
0-8 min 36% ACN/water
8-20 min 36% ACN/water to 100% ACN
Gradient
20-23 min 100% ACN
23-28 min 36% ACN/water
Flow rate 1 mL/min
0-11 min 246 nm (for psoralen and angelicin, 7-8
Detection min)
11-28 min 260 nm (for bakuchiol, 18-19min)
Temperature 35 C
Date Recue/Date Received 2021-08-20
0.1 mg/mL in Me0H for bakuchiol
Standard concentration
0.025 mg/mL for psoralen and angelicin
Extract preparation 0.2 mg/mL in Me0H
Linear range 0.0 1 mg/mL to 0.15mg/mL
EXAMPLE 2
GENERAL METHODS FOR EXTRACTION OF BAKUCHIOL FROM PSORALEA PLANTS
Method A - To a flask was added solvent (100 mL) and Psoralea
corylifolia seed powder (10 g), and the mixture was shaken on a wrist shaker
at room
temperature for one hour. The mixture was then passed through a filter and the
filtrate
collected. The extraction process was repeated one more time with fresh
solvent, the
filtrates were combined, the solvent removed on a rotoevaporator and the
residue dried
under high vacuum.
Method B - To a flask was added solvent (50 mL) and Psoralea
corylifolia seed powder (10 g), and the mixture was refluxed for 40 min. The
solution
was then filtered and the extraction process was repeated two more times with
fresh
solvent. The filtrates were combined, and the solvent was evaporated to obtain
a dried
extract.
Following the above extraction methods, sample plant material was
extracted with the following solvents: dichloromethane (DCM), ethyl acetate
(Et0Ac),
acetone, methanol (Me0H), petroleum ether (BP 35-60 C) and petroleum ether
(BP
60-90 C). The extracts and plant materials were then analyzed by HPLC
analysis as
described in Example 1. The results are set forth in Table 2.
Table 2. Quantification of Various Psoralea Corylifolia Extracts
Petroleum Petroleum Petroleum
Ether DCM Et0Ac Acetone Me0H Ether Ether
(35-60 C) (35-60 C) (60-90 C)
Extract 0.5833 1.7535 1.6710 1.8932 1.8795 0.6457 0.9203
wt. (g)
31
Date Recue/Date Received 2021-08-20
Petroleum Petroleum Petroleum
Ether DCM Et0Ac Acetone Me0H Ether Ether
(35-60 C) (35-60 C) (60-90 C)
%
Bakuchiol 29.1% 14.2% 13.7% 13.7% 13.9% 25.6% 27.2%
in Extract
%
Bakuchiol 1.7% 2.5% 2.3% 2.6% 2.6% 2.6% 2.7%
in Plant
Method Wrist shaker Reflux
(100 m1/10 g solid) (50 m1/10 g solid)
EXAMPLE 3
CHROMATOGRAPHIC METHODS FOR PURIFYING BAKUCHIOL EXTRACTS
Various chromatographic methods were utilized for purifying bakuchiol
from the crude solvent extract isolated from the seeds of Psoralea corylifolia
using the
method described in Example 2. The efficiency of a specific column enrichment
method was demonstrated as a means of obtaining high purity bakuchiol free of
contamination by furanocoumarins, particularly psoralen/isopsoralen
contamination.
Briefly, each empty column cathidge (1.3 cm internal diameter (ID) and 20 mL
capacity, from Bio-Rad) was packed with a different media and eluted with
different
solvents in an attempt to separate the furanocoumarin impurities from
bakuchiol. The
fractions (10 mL per fraction) were collected in test tubes and analyzed with
silica gel
TLC plates developed with 20% Et0Ac/petroleum ether. The targeted compounds,
bakuchiol, psoralen and isopsoralen, were identified based on their retention
times,
which were determined using solutions of standard compounds. The results are
set
forth in Table 3. Many of the methods described in Table 3 were useful for the
separation of furanocoumarins and bakuchiol from both synthetic and natural
sources,
32
Date Recue/Date Received 2021-08-20
however the cost of this methodology may not be economically feasible for
large scale
production.
Table 3. Summary of Column Chromatographic Separation of Bakuchiol from
Furanocoumarins in Crude Extracts of Psoralea Corylifolia
Column size/
Media Elution Solvent Results
Extract Loading
1. Petroleum ether
Al2O3 (neutral) Little
2 mL/25 mg 2. Et0Ac
separation
(IT. Baker)
3. Me0H
XAD-4 Me0H/water gradient
(amerlite in 20% increments
mL/19 mg No separation
polystyrene from 100% water to
resin) 100% Me0H
Pet. ether/Et0Ac
gradient in 20%
Some
increments from 100%
XAD-7 petroleum ether to separation
(amerlite 100% Me0H
8 mL/16 mg
polyacry late
resin) Me0H/water gradient
in 20% increments Little
from 100% water to separation
100% Me0H
1. Petroleum ether
2. 5% acetone/pet.
Polyamide 5 mL/50 mg No separation
ether
3. Acetone
LH-20 8 mL/50 mg Petroleum ether No separation
1. Petroleum ether
Good
Silica gel 5 mL/50 mg 2. 15% Et0Ac/pet. separation
ether
1. Petroleum ether
CG-71md 5 mL/50 mg No separation
2. Acetone
CG-161cd Petroleum ether No separation
5 mL/50 mg
33
Date Recue/Date Received 2021-08-20
Column size/
Media Elution Solvent Results
Extract Loading
Good
separation
6 mL/50 mg Me0H/water step
gradient
Low yield
EXAMPLE 4
HYDROLYSIS OF AN EXTRACT ISOLATED FROM THE SEEDS OF PSORALEA CORYLIFOLIA
A hexane extract or a CO2 super-critical fluid extract of seeds of
Psoralea corylifolia, which contained about 25% bakuchiol, was mixed with a 1M
NaOH solution. The solution was heated in a reaction vessel to a temperature
at or
above 80 C for at least one hour. A small portion of the solution was taken
from the
flask periodically and analyzed by HPLC as described in Example 1. The
reaction was
stopped after HPLC analysis showed that the peaks for psoralen and isopsoralen
had
completely disappeared. The reaction mixture was then cooled to room
temperature
and the aqueous phase was removed. After the solution was washed multiple
times
with saturated a NaCl solution, the organic layer was extracted with ethyl
acetate or
other organic solvents. The organic solution was filtered, washed, dried and
evaporated
to yield a brownish red syrup having a bakuchiol content not less than 50% and
a
combined total of no more than 100 ppm of psoralen and angelicin
(isopsoralen).
EXAMPLE 5
ANTI-OXIDATION PROPERTY OF FURANOCOUMARIN FREE BAKUCHIOL COMPOSITION
A natural bakuchiol composition (Lot# UP256-0906MP) comprising
57.35% bakuchiol and a combined total of less than 100 ppm psoralen and
angelicin
(isopsoralen) was evaluated for its antioxidant capacity against peroxy
radicals,
hydroxyl radicals, peroxynitrite, super oxide anion and singlet oxygen at
Brunswick
laboratories, Norton, MA USA. The total oxygen radical absorbance capacity of
the
bakuchiol composition was measured according to the published methodology (Ou,
B.
et al., J Agric and Food Chem, 2001, 49 (10): 4619-4626; Prior, RL. et al., J
Agric and
Food Chem, 2005, 53: 4290-4302). Results are tabulated in Table 4.
34
Date Recue/Date Received 2021-08-20
Table 4: Anti-oxidation Profile of BakutrolTM (UP256)
Test Resultt Units
Antioxidant power against peroxyl radicals 12,848 pmole TE/gram
Antioxidant power against hydroxyl radicals 6,262 pmole TE/gram
Antioxidant power against peroRnitrite 289 pmole TE/gram
Antioxidant power against super oxide anion 69,929 pmole TE/gram
Antioxidant power against singlet oxygen 3,067 pole TE/gram
Total ORACFN (sum of above) 92,395 pole TE/gram
* The acceptable precision of the ORAC assay is < 15% relative standard
deviation.
t Liquid samples weighed and extracted due to viscosity.
There are five cstdorninant reactive species found in the body.. peroxyl
rndicals. hydroxyl rfidicals,
peroxynitrite. tiper oxide anion. and singlet oxygen. Total ORACFN provides a
ineauut of the total
autiolilutt pima of a foothillaitionpLoduci vinq die five pretkuninaut
ireaoive
EXAMPLE 6
EVALUATION OF A BAKUCHIOL COMPOSITION FOR ANTIOXIDATION PROTECTIVE EFFECT
ON 4-TBP CYTOTOXICITY
A natural bakuchiol composition comprising 77.02% bakuchiol and a
combined total of less than 100 ppm psoralen and angelicin (isopsoralen) was
tested for
its antioxidant property by assessing its ability to prevent 4-
tertiarybutylphenol (4-TBP)
induction of oxidative stress during a 5-day treatment period using compound
concentrations at the 95% viability dose. Oxidative stress was determined by
assaying
the generation of Reactive Oxygen Species (ROS) using the Image-iT Live Green
Reactive Oxygen Species Detection Kit (InVitrogen). In this assay, carboxy-
2',7'-
dichlorodihydrofluorescein diacetate is added to cultured cells for 30 minutes
where it
diffuses into melanocytes and is hydrolyzed by intracellular esters to 2',7'-
dichlorofluorescein (DCF) which reacts with ROS to generate fluorescent DCF.
After 5
days of treatment with 200 pM or 400 p,M of 4-TBP, the generation of ROS in
melanocytes demonstrated a dose response (i.e., moderate to robust,
respectively),
Date Recue/Date Received 2021-08-20
whereas the untreated and DMSO treated melanocytes exhibited no ROS
generation.
When the treatment protocol included the test compounds, UP256 (bakuchiol)
demonstrated strong antioxidant property as shown in Figure 3.
EXAMPLE 7
TYROSINASE INHIBITION ACTIVITY OF A BAKUCHIOL COMPOSITION
Two natural bakuchiol compositions comprising 77.02% bakuchiol
(100% purity) were tested for tyrosinase inhibition activity. Both materials
contained a
combined total of less than 100 ppm total psoralen and angelicin
(isopsoralen).
A tyrosinase inhibition assay was carried out using the method reported
by Jones et al. (2002) Pigment. Cell Res. 15:335. Using this method, the
conversion of
L-Dopa, a substrate of tyrosinase, into dopachrome was followed by monitoring
absorption at 450 nm. Tyrosinase was prepared in 50 mM potassium phosphate
buffer,
pH 6.8 (assay buffer) at 2000 U/ml and stored at -20 C in 1 ml aliquots prior
to use.
For use in assays, stock enzyme solutions were thawed and diluted to 200 U/ml
with
assay buffer. A 2 mM working solution of substrate, L-DOPA, was prepared in
assay
buffer for each assay. Samples were dissolved in 10% DMSO (0.5 ml) and diluted
to 5
ml with assay buffer. The reaction mixture comprised 0.050 ml 2 mM L-DOPA,
0.050
ml 200 U/ml mushroom tyrosinase and 0.050 ml inhibitor. Reaction volume was
adjusted to 200 [11 with assay buffer. Assays were performed in 96 well Falcon
3097
flat-bottom microtiter plates (Beckton Dickinson, NJ). Appearance of
dopachrome was
measured with a WALLAC 1420 Multilable Counter (Turku, Finland). Average
velocity was determined from linear enzyme rate as measured by change in
absorbance
(AA450) at 450 nm per minute. Percent inhibition of tyrosinase by test samples
was
determined by comparison of absorbance of samples versus control using formula
(1):
(Negative control absorption - sample absorption)/Negative control absorption
x 100
(1)
36
Date Recue/Date Received 2021-08-20
As shown in Figure 4, both bakuchiol compositions showed no
tyrosinase inhibition activity, while the positive control (kojic acid) showed
dose
responsive tyrosinase inhibition with and IC50 value of 63.9 uM.
EXAMPLE 8
FORMULATION OF A BAKUCHIOL COMPOSITION IN COSMETIC CREAM, GEL, AND LOTION
Two natural bakuchiol compositions comprising 86.54% bakuchiol and
77.02% bakuchiol were formulated in a cosmetic vehicle or complicated skin
care
cream, gel or lotions as demonstrated below.
Formulation A
Bakuchiol 1.0%
Vitamin E acetate 0.1%
Phenonip 0.5%
Transcutol 98.4%
Formulation B
Bakuchiol 1.0%
Vitamin E acetate 0.1%
Caprylic triglyceride 98.4%
Phenonip 0.5%
Formulation C
Bakuchiol 1.0%
Polysorbate-20 15.0%
Transcutol 5.0%
Vitamin E acetate 0.1%
Purified water 78.2%
Phenonip 0.5%
Table 5. Formulation D
37
Date Recue/Date Received 2021-08-20
No. Material INCI Name
1 D.I.WATER Water
2 Gemseal 40 C15-19 Alkane
3 GLYCERIN Glycerin
4 1.3-B.G Butylene Glycol
Carbopol#940 Carbomer
6 ARLATON 2121 Sorbitan Stearate/Sucrose Cocoate
7 Salacos 816T C15-19 Alkane
8 Sunflower Oil Helianthus Annuus(Sunflower) Seed Oil
9 TREHALOSE Trehalose
ERITHRITOL Erythritol
11 Dow Corning #345 Cyclomethicone
12 CALCOL 68 (CETANOL) Cetanol
STEARIC ACID
13 Stearic Acid
(EMERSOL#132)
CITHROL GMS
14 Glyceryl Stearate/PEG-100 Stearate
A/S(AR#165)
GMS #205 Glyceryl Stearate SE
16 BEES WAX Beeswax
SODIUM
17 Sodium Hyaluronate
HYALURONATE(Hyasol)
18 Bakuchiol at 0.5% Hydrolyzed Psoralea corylifolia extract
38
Date Recue/Date Received 2021-08-20
EXAMPLE 9
EVALUATION OF THE SAFETY PROFILE OF A BAKUCHIOL COMPOSITION
Two natural bakuchiol compositions comprising 86.54% bakuchiol and
77.02% bakuchiol and a combined total of less than 100 ppm psoralen and
angelicin
(isopsoralen) were formulated in a cosmetic vehicle or complicated skin care
cream and
tested on in vitro models or in human clinical trials for their safety
profile. As
demonstrated in Table 6, the bakuchiol compositions showed no eye irritation,
no skin
irritation, no skin allergic contact sensitization and no phototoxicity. The
compositions
had a solid safety profile at a broad range of concentration levels (20% to
100% by
weight of bakuchiol) with good skin penetration properties.
Table 6: Results of BakutrolTM Safety Testing
BRIEF DESCRIPTION OF THE TEST
TEST NAME RESULT
PROCEDURE
This is a biological assay to evaluate Test Product at the
EPIOCULAR
ocular toxicity or irritating potential of 1%
concentration
MTT
a test article by determining the ET50 was classified in
the
VIABILITY
for MTT viability of EpiOcular minimal to non-
ASSAY
samples. irritating category.
This is a clinical test to assess the
The effects of 0.5%
irritating potential of chemical
of the test product
CHAMBER compounds. The test is performed on
on scarified skin
SCARIFICATIO human subjects whose skin was
N sensitized by scratching. The variable were
comparable to
the saline control at
being tested is the compound's ability
72 hours.
to cause irritation of compromised skin.
This is a clinical test to assess both
Under study
irritating and allergenic potentials of
conditions the test
chemical compounds. The test is
product did not
REPEATED .. performed by repetitive application of
indicate a potential
INSULT PATCH the compounds to the skin of healthy
for dermal irritation
volunteers. The variables being tested
or allergic contact
are the compound's ability to cause
sensitization.
erythema or edema.
39
Date Recue/Date Received 2021-08-20
BRIEF DESCRIPTION OF THE TEST
TEST NAME RESULT
PROCEDURE
This is a clinical test to assess
phototoxic potential of test compounds. The test product
The test is performed on human was
considered
PHOTO-
subjects by application of the non-phototoxic
TOXICITY
compounds to the skin, followed by according to
UV-irradiation, and up to 1 week post- reference at the
irradiation period. The variables being 0.5% concentration
tested are the compound's ability to tested.
cause adverse or unexplained reactions.
This is a biological assay to assess the
mutagenic potential of chemical Test product was
compounds. The test uses strains of the not associated with
AMES bacterium that carry specific mutations. any
mutagenic
The variable being tested is the changes at doses up
mutagen's ability to cause a reversion to 3 mg/plate
of these mutations.
At 0.5%
This is an ex-vivo test to assess skin
formulation
penetration of chemical compounds.
concentration, the
PERCUTA- The test is performed by single
data indicates a
NEOUS application to cadaver skin. The
ABSORBTION variable being tested is the percentage good
penetration
profile and
of test compound that is absorbed into
absorption into
skin over a certain period of time.
skin over 48 hours.
EXAMPLE 10
CLINICAL EVALUATION OF A FURANOCOUMARIN-FREE BAKUCHIOL COMPOSITION
A natural bakuchiol composition (BakutrolTM) extracted and enriched
from the seeds of Psoralea corylifolia and comprising 77.02% bakuchiol and
less than
100 ppm furanocoumarin was formulated in a cosmetic skin care cream
(formulation D,
Example 8) and tested in a human clinical trial. The study was a pilot, open
label
human study to evaluate the clinical benefits of BakutrolTM at 0.5%
concentration after
topical application. The study included 5 subjects meeting the
exclusion/inclusion
criteria for evaluation of the benefits of the natural bakuchiol composition
for
improvement of Post-Inflammatory Hyperpigmentation (PIH). The duration of the
study was 12 weeks. The subjects were instructed to apply the BakutrolTM 0.5%
cream
twice a day, morning and night, and return to the site for a total of 9
visits, including the
screening visit. The evaluation included Investigator Global Assessment of
skin
Date Recue/Date Received 2021-08-20
conditions (IGA), and Evaluation of overall grade and severity of skin Post-
Inflammatory Hyperpigmentation (PIH) and other associated skin conditions,
including
Erythema, Dryness, Peeling, Oiliness, Safety, and Tolerability. The
subject
questionnaires included safety and compliance questions in relation to
irritation, skin
comfort, use of other products and sunscreens. Photographs were taken at
baseline,
Week 4, Week 8 and Week 12. Change from baseline in PIH severity, change from
baseline in the PIH Grade, and the proportion of success according to the IGA
scale
were recorded and analyzed. The following 6 levels of PIH grade of severity
(0=
absent, 1= slight, 2=mild, 3=moderate, 4=moderately severe, 5= severe) and
total area
of PIH affected facial surface were utilized for the clinical output analyses.
As shown in Figure 5, all five subjects treated with a topical cream
comprising BakutrolTM at 0.5% had at least one grade reduction of PIH
severity. The
percentage improvement of PIH affected facial area was more than 50% after 8
weeks
of continued application (see Figure 6). The mean percentage and absolute
grade level
improvements of both PIH and its severity are summarized in Figures 7 & 8.
Improvements of more than 40%, or more than one grade level reduction of both
PIH
and severity, were achieved as early as 4 weeks after using a natural
bakuchiol
composition extracted and enriched from the seeds of Psoralea corylifolia and
comprising 77.02% bakuchiol and less than 100 ppm furanocoumarin. Substantial
reduction of PIH on the affected facial skin sites is clearly evident in the
photos of two
subjects as shown in Figure 9. Both subjects showed progressive improvement of
skin
Post-Inflammatory Hyperpigmentation (PIH) associated with mild and moderate
acnes
after topical application of a BakutrolTM cream.
Table 7 summarizes the clinical outputs for using a furanocoumarin-free
Bakuchiol composition compared to popular acne treatment products that contain
either
an anti-microbial or an anti-inflammatory or a combination thereof. The data
clearly
demonstrate that the furanocoumarin-free Bakuchiol composition not only
improved
inflammatory and non-inflammatory lesion counts but also significantly
improved skin
Post-Inflammatory Hyperpigmentation. The
PIH benefit from the Bakuchiol
composition is unexpected based on its lack of tyrosinase inhibition as
demonstrated in
Example7.
41
Date Recue/Date Received 2021-08-20
Table 7. Clinical Report Summary of Bakutrol and OTC Drugs
Study 1 Study 2 Study 3 Study 4
week study * 12 week study * 12 week 12 week
Study * Study *
Benzaclin (5% Benzoyl Clindamycin Vehicle ClindaGel Bakutrol
Benzoyl Peroxide peroxide
& 1% (1%) N=120 (1% (UP256)
Clindamycin) (5%) Clindamycin) 0.5%
N=120 N=120 N=120
N=162 N=13
Mean percent reduction in inflammatory lesion counts
46% 32% 16% +3% 51% 33%
Mean percent reduction in non-inflammatory lesion counts
22% 22% 9% +1% 25% 16%
Mean percent reduction in total lesion counts
36% 28% 15% 0.2% 38% 26%
Improvement of Post Inflammatory Hyperpigmentation
0 0 0 0 0 62%
EXAMPLE 11
EVALUATION OF A FURANOCOUMARIN-FREE BAKUCHIOL COMPOSITION ON DIMINISHING
THE EFFECTS OF POST-INFLAMMATORY HYPERPIGMENTATION
The safety and efficacy of a 0.5% (wt/wt) cream of bakuchiol (UP256)
was evaluated in a double blind placebo and positive controlled study for the
treatment
of PIH related to acne. The study evaluated a bakuchiol cream at 0.5%
concentration, a
2% Salicylic acid cream and a placebo cream (Vehicle) in an Asian population.
The
participants were instructed to apply the study creams to the face twice a day
(AM/PM).
Study participants were given instructions for application and provided with
sunscreen.
The study population consisted of male and female subjects older than
18 and younger than 40 years and in generally good health as determined by a
medical
42
Date Recue/Date Received 2021-08-20
history. The bakuchiol study arm enrolled 18 subjects, the Salicylic acid
(SAL) study
arm enrolled 20 subjects, and the placebo study arm enrolled 19 subjects.
The Investigators and study staff discussed and agreed upon a clear
definition of PIH as related to acne or other tissue injury (which did not
include freckles
(Ephilides), solar lentigo (lentigines), or melisma). The PIH grade is a
measure of the
severity of the hyperpigmentation (higher number = more severe pigmentation).
The
patient population had a PIH grade >3 and acne was mild to moderate Grade 2-
3, the
key factor was the PIH as related to current or past inflammatory acne.
Primary Study Objectives:
1. PIH IGA Time Frame: Baseline and weeks 2, 4 and 8
2. PIH % Distribution Time Frame: Baseline and weeks 2, 4 and 8
PIH efficacy was evaluated based on changes from baseline (p<0.05) and over
other
treatment groups (p<0.05) using t-test or /and ANOVA.
Secondary Objectives: Safety Assessments
Subject's Assessment questionnaires and Tolerability Assessments were
collected at Baseline and weeks 2, 4, and 8. Urine pregnancy tests for females
of
childbearing potential were collected at Baseline and week 8.
Data Analysis
The following data was collected:
1. Change from baseline in PIH Severity;
2. Change from baseline in the PIH Grade; and
3. Change from baseline in the Lesion counts
Results:
The data was analyzed from Investigator evaluations at each visit and
from photographs taken at the specified time points. The photographs from
baseline,
week 4, and week 8 were evaluated by a second group of investigators and two
independent dermatologists before the study was unblended to further confirm
that the
43
Date Recue/Date Received 2021-08-20
population met criteria. The data analyzed is based on confirmed evaluations,
there was
no photo for week two therefore week two data is not included in the analysis.
Table 8
summarizes the data.
Table 8. PIH Grade Changes for Study Groups
Bakutrol Baseline Week Week ANOVA
4 8
(UP256) ( p value)
Mean 3.44 2.83 2.5 0.0083
SD 0.78 0.86 0.99
SEM 0.18 0.2 0.23
N 18 18 18
(t -test) 0.0005 0.0003
p value
PLACEBO Baseline Week Week 0.9712
4 8
Mean 3 3.05 3
SD 0.82 0.71 0.67
SEM 0.19 0.16 0.15
N 19 19 .. 19
p value 0.5778 1
2% SAL Baseline Week Week 0.9798
4 8
Mean 3.15 3.2 3.2
SD 0.93 0.89 0.89
SEM 0.21 0.2 0.2
N 20 20 20
p value 0.3299 0.3299
44
Date Recue/Date Received 2021-08-20
The Bakutrol (UP256) group showed significant change in PIH grade
(i.e., lower PIH grade) from baseline at week 8 (p<0.05). Additionally, the
Bakutrol
group showed significant change over placebo at week 8 (p< 0.05). The data
also show
that the bakutrol treated group is the only group that has a time and
treatment
significant p value (p=0.0083).
Table 9. Inflammatory Acne Lesions for All Groups
Bakutrol Baseline Week Week ANOVA OVER
4 8 TREATMENT TIME
(UP256)
Mean 13.11 9 5.67 0.016
SD 9 6.93 4.67
SEM 2.12 1.63 1.1
N 18 18 18
p value 0.0014 0.0001
Salicylic Baseline Week 4 Week 8 0.513
Acid
Mean 11.2 9.25 8.65
SD 6.83 8.33 6.47
SEM 1.53 1.86 1.45
N 20 20 20
p value 0.0242 0.0159
PLACEBO Baseline Week 4 Week 8 0.0985
Mean 8.42 7 5.68
SD 4.38 3.83 3.22
SEM 1 0.88 0.74
Date Recue/Date Received 2021-08-20
N 19 19 19
p value 0.1367 0.0021
The Bakutrol (UP256) group showed significant change from baseline at
weeks 4 and 8 (p<0.001). The Bakutrol group also showed significant change
over
placebo at week 8 (p< 0.05). In addition UP256 is the only group that reached
a greater
than 50% decrease of inflammatory lesions at 8 weeks (57%).
There were no significant changes for the Bakutrol (UP256) group in
the non-inflammatory lesion category (data not shown); however, the salicylic
acid
treated groups in the non-inflammatory lesion category reached a p value <0.05
at 4
weeks of treatment.
Table 10: Summary of Questionnaire and Investigator Safety Evaluation
0.5% Bakutrol PLACEBO 2.0% Salicylic acid Subject Assessment
Category
UP256
1 2 4 Itching/Burning
0 0 2 Skin Discomfort
Conclusion:
The results show that the Bakutrol (UP256) cream significantly reduced
Post Inflammatory Hyperpigmentation (PIH) related to acne after only 4 weeks
of
topical application. UP256 also significantly reduced inflammatory acne
lesions (<
0.05) by 57% after 8 weeks.
Bakutrol (UP256) cream had a good safety profile and was well tolerated
by study participants. The cosmetic acceptability of the cream was rated as
better than,
or as good as, that of their previous topical over-the-counter (OTC) acne
therapy.
EXAMPLE 12
TREATMENT OF INFLAMMATORY AND NON-INFLAMMATORY ACNE LESIONS
46
Date Recue/Date Received 2021-08-20
A composition comprising both bakuchiol and salicylic acid is prepared.
Patients having both inflammatory, non-inflammatory, or both types of acne of
lesions
are treated with the composition. The composition is effective to treat both
inflammatory and non-inflammatory lesions with a p value <0.05 at 4 weeks of
treatment. The reduction in lesions ranges from about 10% to about 90%.
47
Date Recue/Date Received 2021-08-20
