U.S. patent application number 16/415248 was filed with the patent office on 2019-09-05 for composition for promoting hair growth containing novel pantetheine derivative.
The applicant listed for this patent is KOREA INSTITUTE OF OCEAN SCIENCE AND TECHNOLOGY. Invention is credited to Hyi-Seung LEE, Jong Seok LEE, Min Ah LEE, Yeon-Ju LEE, Mojid MONDOL, Hee Jae SHIN.
Application Number | 20190269593 16/415248 |
Document ID | / |
Family ID | 62708688 |
Filed Date | 2019-09-05 |
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United States Patent
Application |
20190269593 |
Kind Code |
A1 |
SHIN; Hee Jae ; et
al. |
September 5, 2019 |
COMPOSITION FOR PROMOTING HAIR GROWTH CONTAINING NOVEL PANTETHEINE
DERIVATIVE
Abstract
Provided is a composition for promoting hair growth, containing,
as an active ingredient, a new compound represented by the
following formula 1 or a salt thereof, which exhibits an excellent
effect of promoting the growth of dermal papilla cells to thereby
exhibit the effect of promoting hair growth: ##STR00001## wherein R
is any one selected from the group consisting of 4-pentenoyl,
10-undecenoyl, isobutyl formate, and 2,4-dihydroxybenzoyl.
Inventors: |
SHIN; Hee Jae; (Gyeonggi-do,
KR) ; LEE; Min Ah; (Gyeonggi-do, KR) ; MONDOL;
Mojid; (Gyeonggi-do, KR) ; LEE; Hyi-Seung;
(Seoul, KR) ; LEE; Jong Seok; (Gyeonggi-do,
KR) ; LEE; Yeon-Ju; (Gyeonggi-do, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KOREA INSTITUTE OF OCEAN SCIENCE AND TECHNOLOGY |
Gyeonggi-do |
|
KR |
|
|
Family ID: |
62708688 |
Appl. No.: |
16/415248 |
Filed: |
May 17, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15394153 |
Dec 29, 2016 |
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16415248 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61Q 7/00 20130101; A61K
8/46 20130101 |
International
Class: |
A61K 8/46 20060101
A61K008/46; A61Q 7/00 20060101 A61Q007/00 |
Claims
1. A composition for promoting hair growth, containing, as an
active ingredient, a compound represented by the following formula
1 or a salt thereof: ##STR00004## wherein R is any one selected
from the group consisting of 4-pentenoyl, 10-undecenoyl, isobutyl
formate, and 2,4-dihydroxybenzoyl.
2. The composition of claim 1, wherein the compound represented by
formula 1 is any one selected from the group consisting of
4-pentenoyl-D-pantetheine, 10-undecenoyl-D-pantetheine, isobutyl
formate-D-pantetheine, and 2,4-dihydroxybenzoyl-D-pantetheine.
3. The composition of claim 1, wherein the compound represented by
formula 1 exhibits an effect of promoting growth of dermal papilla
cells.
4. The composition of claim 1, wherein the compound represented by
formula 1 is prepared using D-pantethine as a starting
material.
5. The composition of claim 1, wherein the composition is a
cosmetic composition for preventing hair loss and promoting hair
growth.
6. The composition of claim 5, wherein the cosmetic composition has
any one formation selected from the group consisting of hair
tonics, hair conditioners, hair essence, hair lotion, hair
nourishing lotion, hair shampoo, hair rinse, hair treatments, hair
cream, hair nourishing cream, hair moisturizer cream, hair massage
cream, hair wax, hair aerosols, hair packs, hair nourishing pack,
hair soap, hair cleansing foam, hair oil, hair drying preparations,
hair preservation treatments, hair colorants, hair weaving
preparations, color-removing preparations for hair, hair gel, hair
glazes, hair dressingers, hair lacquers, hair moisturizers, hair
mousse, and hair sprays.
7. The composition of claim 1, wherein the composition is a
pharmaceutical composition for preventing hair loss and promoting
hair growth.
8. The composition of claim 7, wherein the pharmaceutical
composition has any one formation selected from the group
consisting of ointments, pastes, gels, jellies, serums, aerosol
sprays, non-aerosol sprays, foams, creams, lotions, solutions, and
suspensions.
Description
CROSS REFERENCE
[0001] This is a continuation of application Ser. No. 15/394,153
which is now pending, whose entire contents are incorporated herein
by reference.
BACKGROUND
1. Technical Field
[0002] The present invention relates to a composition for promoting
hair growth, and more particularly to a composition for promoting
hair growth, which contains a new pantetheine derivative that
exhibits an excellent effect of promoting the growth of dermal
papilla cells to thereby promote hair growth.
2. Description of the Related Art
[0003] The known causes of hair loss include excessive male hormone
production, excessive sebum secretion, scalp function deterioration
caused by peroxides, bacteria, etc., genetic factors, aging,
stress, and the like. In addition, the hair loss population is
gradually increasing due to increased social stress, environmental
pollution, westernized eating habits such as the consumption of
instant foods, frequent permanent waves and hair color changes,
etc.
[0004] The hair cycle can be divided into four separate stages:
anagen where hair grows; catagen where hair growth stops and the
hair bulb shrinks; telogen where the dermal papilla stops its
activity and hair remains on the scalp; and exogen where the dermal
papilla initiates its activity or produces new hair to shed old
hair.
[0005] The anagen stage (2-7 years) is a stage where hair grows. It
is subdivided into two stages: a hair production stage where hair
extends from the hair bulb to the hair follicle; and a stage where
hard keratin is produced in the hair follicle. Hair continues to
grow until the catagen stage.
[0006] The catagen stage (2-3 weeks) following the anagen stage is
a stage where the metabolism of hair becomes slower while the shape
of hair is maintained. In this step, keratin is not produced. The
hair in the catagen stage occupies 1% of the total number of hairs.
In this stage, the hair bulb shrinks to be divided into hair
papillae, which are surrounded by hair follicles and move upward,
and cell division is arrested.
[0007] The telogen stage (3 months) is a stage where the hair
papilla and the hair follicle gradually contracts and where the
hair root is pushed upward and falls out. This stage where hair
falls out continues for 3-4 months until the next anagen stage is
started.
[0008] Normal persons have a relatively large number of
anagen-stage hairs, whereas persons with alopecia have a relatively
large number of telogen-stage hairs, and thus have visible hair
loss. As hair loss progresses, the period of the anagen stage
becomes shorter, and for this reason, hair gradually becomes
thinner. Therefore, for the treatment of hair loss, it is important
to facilitate telogen-stage hair follicles to enter the anagen
stage and to extend the shortened anagen stage.
[0009] Male pattern alopecia (androgenetic alopecia) is caused by
the male hormone testosterone. If testosterone is converted to the
highly active hormone dihydrotestosterone (DHT) by the enzyme
5.alpha.-reductase, the hormone dihydrotestosterone will act on
hair follicles to induce anagen-stage hair follicles to enter the
catagen stage, thereby causing hair loss. For this reason, for the
treatment of androgenetic alopecia, methods for inhibiting DHT
production caused by 5.alpha.-reductase have been mainly used.
[0010] Female pattern alopecia is caused mainly by a decrease in
estrogen level after the menopause. As a therapeutic agent against
female pattern alopecia, minoxidil or estrogen has been mainly
used.
[0011] Alopecia areata is caused by autoimmune diseases, mental
stress, or genetic factors. The causes of alopecia areata
fundamentally differ from those of androgenetic alopecia, and a
method for treating alopecia areata also differs from a method for
treating androgenetic alopecia. Thus, for the treatment of alopecia
areata, a method of applying minoxidil to a hair loss area or
artificial stimulation in a hair loss area has been used.
[0012] However, agents such as minoxidil or trichosaccharides,
known to date to have effects on hair loss prevention and hair
growth promotion, have no distinct effect and cause side-effects
such as induction of skin irritation. Thus, there is an urgent need
to develop compositions having demonstrated safety and desired
effects.
SUMMARY OF THE INVENTION
[0013] An object of the present invention is to provide a hair
growth stimulant, which is capable of promoting the proliferation
of dermal papilla cells to thereby prevent, alleviate or treat hair
loss and exhibit excellent effects on hair growth or the like, and
a cosmetic or pharmaceutical composition for promoting hair growth,
which contains the hair growth stimulant.
[0014] To accomplish the above object, the present invention
provides a composition for promoting hair growth, containing, as an
active ingredient, a compound represented by the following formula
1 or a salt thereof:
##STR00002##
wherein R is any one selected from the group consisting of
4-pentenoyl, 10-undecenoyl, isobutyl formate, and
2,4-dihydroxybenzoyl.
[0015] In the composition for promoting hair growth according to
the present invention, the compound represented by formula 1 is
preferably any one selected from the group consisting of
4-pentenoyl-D-pantetheine, 10-undecenoyl-D-pantetheine, isobutyl
formate-D-pantetheine, and 2,4-dihydroxybenzoyl-D-pantetheine.
[0016] In the composition for promoting hair growth according to
the present invention, the compound represented by formula 1
exhibits the effect of promoting the growth of dermal papilla
cells.
[0017] In the composition for promoting hair growth according to
the present invention, the compound represented by formula 1 may be
prepared using D-pantethine as a starting material.
[0018] The composition for promoting hair growth according to the
present invention may be a cosmetic composition for preventing hair
loss and promoting hair growth.
[0019] In the composition for promoting hair growth according to
the present invention, the cosmetic composition preferably has any
one formation selected from the group consisting of hair tonics,
hair conditioners, hair essence, hair lotion, hair nourishing
lotion, hair shampoo, hair rinse, hair treatments, hair cream, hair
nourishing cream, hair moisturizer cream, hair massage cream, hair
wax, hair aerosols, hair packs, hair nourishing pack, hair soap,
hair cleansing foam, hair oil, hair drying preparations, hair
preservation treatments, hair colorants, hair weaving preparations,
color-removing preparations for hair, hair gel, hair glazes, hair
dressingers, hair lacquers, hair moisturizers, hair mousse, and
hair sprays.
[0020] The composition for promoting hair growth according to the
present invention may be a pharmaceutical composition for
preventing hair loss and promoting hair growth.
[0021] In the composition for promoting hair growth according to
the present invention, the pharmaceutical composition preferably
has any one formation selected from the group consisting of
ointments, pastes, gels, jellies, serums, aerosol sprays,
non-aerosol sprays, foams, creams, lotions, solutions, and
suspensions.
[0022] The hair growth stimulant according to the present invention
exhibits the effect of promoting the proliferation of human dermal
papilla cells to thereby prevent hair loss and promote hair growth.
Thus, the hair growth stimulant may be effectively used as an
active ingredient in cosmetic or pharmaceutical compositions for
preventing hair loss and promoting hair growth.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The above and other objects, features and advantages of the
present invention will be more clearly understood from the
following detailed description taken in conjunction with the
accompanying drawings, in which:
[0024] FIG. 1 shows a process of isolating new compounds (S1 and
S2) for hair loss prevention and hair growth promotion from a
culture broth of a 102CH635-3 strain;
[0025] FIG. 2 shows a method of synthesizing new D-pantetheine
derivatives according to the present invention; and
[0026] FIG. 3 shows the effects of new D-pantetheine derivatives
according to the present invention on the promotion of
proliferation of dermal papilla cells.
DETAILED DESCRIPTION OF THE INVENTION
[0027] Hereinafter, the present invention will be described in
detail.
[0028] The development and growth of hair and the hair cycle are
controlled by hair papilla which is a tissue derived from the
mesoderm of the hair follicle base. It is known that the hair
papilla interacts with hair matrix cells and stimulates these cells
to differentiate into several types of cells that form hair
follicles and hairs.
[0029] Hair follicles undergo repeated hair cycles, each consisting
of anagen phase-catagen phase-telogen phase, and hair grows and
falls out during each cell cycle. This cell cycle process is
controlled by the interactions between mesodermal and ectodermal
cells, and dermal papilla cells play a key role in this control.
Mesodermal (dermal papilla) cells induce the production of hair in
the anagen phase, and then secrete a substance that stimulates the
growth of hair. The catagen phase begins in response to a change in
dermal papilla cells, and as hair follicles degenerate, hair
papilla moves upward and is placed immediately below the hair bulge
in which hair stem cells exist. Following the telogen phase, the
anagen phase begins in which hair stem cells divide by the signal
of dermal papilla cells to form new hair follicles.
[0030] According to the literature, the size and volume of dermal
papillae in alopecia patients are significantly smaller than those
in normal persons. The number of dermal papillae increases in the
anagen phase of the hair cycle, and the volume thereof is dependent
on the number of cells forming these dermal papillae. Furthermore,
the volume and division of the epithelial tissue of the hair bulb
are also dependent on the volume of dermal papillae.
[0031] When a new anagen phase begins following the telogen stage,
signals similar to those in the exogen stage act. It was reported
that, in the initial stage of the anagen stage, the signal of Wnt
protein in the epithelial cells of the hair bulge adjacent to
dermal papilla cells is very strong, and Wnt/.beta.-catenin signals
or the like play an important role.
[0032] It is reported that the Wnt/.beta.-catenin signaling system
promotes the formation of hair follicles ("WNT signals are required
for the initiation of hair follicle development." Andl T, et al.
(2002) Dev Cell. 2: 643-653), and plays an important role in
maintaining and activating genes that are expressed during the
anagen stage of the hair cycle ("Wnt signaling maintains the
hair-inducing activity of the dermal papilla." Kishimoto J, et al.
(2000) Genes Dev 14: 1181-1185), and promote differentiation from
stem cells to keratinocytes (".beta.-catenin controls hair follicle
morphogenesis and stem cell differentiation in the skin." Huelsken
J, et al. (2001) Cell 105: 533-545).
[0033] The present inventors have found that a composition
containing, as an active ingredient, a compound represented by the
following formula 1 or a salt thereof, promotes the proliferation
of dermal papilla cells to thereby increase the gene expression
level of dermal papilla cells related to hair loss prevention or
hair growth promotion, thereby completing the present
invention:
##STR00003##
wherein R is any one selected from the group consisting of
2-methylbutyryl, 3-methylbutyryl, cinnamoyl, 4-pentenoyl,
10-undecenoyl, isobutyl formate, 2,4-dihydroxybenzoyl, geranyl,
farnesyl, acryloyl, propanone, 2-pentanone,
1-(4-hydroxyphenyl)ethanone, 1-(2,4-dihydroxyphenyl)ethanone,
pentanoic acid, 2-hydroxypropanoic acid, 2-phenylacetic acid,
2-(4-(propanoyl)phenyl)acetic acid, 4-methylbenzoic acid,
4-(4-phenyl)-4-oxobutanoic acid, 2-oxoethyl acetyl,
2-phenoxyacetyl, 2-(benzyloxy)acetyl, 4-methoxybenzoyl,
3,5-dimethylphenol, 6-methoxybenzene-1,4-diol, propenylbenzene, and
4-hydroxycoumarin.
[0034] Preferably, the compound represented by formula 1 is any one
selected from the group consisting of
2-methylbutyryl-D-pantetheine, 3-methylbutyryl-D-pantetheine,
cinnamoyl-D-pantetheine, 4-pentenoyl-D-pantetheine,
10-undecenoyl-D-pantetheine, isobutyl formate-D-pantetheine,
2,4-dihydroxybenzoyl-D-pantetheine, and geranyl-D-pantetheine.
[0035] It is to be understood that the compound of formula 1, which
is used in the present invention, may be provided not only as a
free compound, but also as a pharmaceutically acceptable salt
thereof, a pharmaceutically acceptable solvate thereof, a
pharmaceutically acceptable polymorph thereof, or a
pharmaceutically acceptable prodrug thereof. In addition, the
active ingredient may be used alone or in combination with one or
more other pharmacologically active compounds.
[0036] A salt of the compound of formula 1, which is used as an
active ingredient in the composition for promoting hair growth is
not specifically limited, as long as it may be used in medical or
cosmetic preparations. The salt may include an inorganic salt or an
organic salt, and may be an acidic salt or an alkaline salt.
[0037] The composition according to the present invention may
further contain one or more other drugs or additives for the
prevention or treatment of hair loss or the promotion of hair
growth. The other drugs or additives include, but are not limited
to, retinoic acid, minoxidil, finasteride, zinc peptides, zinc
oxide, biotin, genistein, onion extracts, pumpkin seed oil, Emu
oil, green tea extracts, Willow bark extracts, and the like.
[0038] The composition for promoting hair growth according to the
present invention may contain, based on the total amount of the
composition, about 0.01-25 wt % of the compound of formula 1, and
the content of the compound of formula 1 in the composition may
vary depending on the kind of compound of formula 1.
[0039] Where the composition for promoting hair growth according to
the present invention is used as a pharmaceutical composition, may
be applied topically to a portion in need of the prevention or
treatment of hair loss or the promotion of hair growth, once or
twice a day. When the content of the active ingredient in the
composition is 1 wt %, the amount applied per day may be about
0.5-3 mg/cm.sup.2 (skin surface area), and may vary depending on
the area of a portion to which the active ingredient is to be
applied. The amount and frequency of this application may be
suitably determined depending on the patient's age and sex and the
severity of hair loss.
[0040] A pharmaceutical composition containing the compound of
formula 1 or a salt thereof may further contain a suitable carrier,
excipient and/or diluent, which is generally used in the
preparation of pharmaceutical compositions.
[0041] The active ingredient of the composition of the present
invention may be formulated with excipients, such as fillers,
extenders, binders, wetting agents, disintegrants, surfactants or
the like, or diluents, which are commonly used. The composition of
the present invention may further comprise an anti-coagulant, a
lubricant, fragrance, an emulsifier, a preservative or the like,
and may be formulated using a method well known in the art so as to
provide quick, sustained or delayed release of the active
ingredient after administration to mammals.
[0042] The pharmaceutical composition according to the present
invention may be prepared as a conventional pharmaceutical
formulation known in the field to which the present invention
pertains. Preferably, it may be prepared as a formulation for
transdermal administration or a skin external preparation for
topical application.
[0043] Specifically, the composition of the present invention may
be prepared as any formulations for skin application, for example,
ointments, pastes, gels, jellies, serums, aerosol sprays,
non-aerosol sprays, foams, creams, lotions, solutions, or
suspensions.
[0044] A functional cosmetic composition containing, as an active
ingredient, the hair growth stimulant of the present invention, may
be prepared as any formulation for skin application. More
specifically, the functional cosmetic composition may be prepared
as a formulation selected from among hair tonic, hair conditioner,
hair essence, hair lotion, hair nourishing lotion, hair shampoo,
hair rinse, hair treatment, hair cream, hair nourishing cream, hair
moisturizer cream, hair massage cream, hair wax, hair aerosol, hair
pack, hair nourishing pack, hair soap, hair cleansing foam, hair
oil, hair drying preparations, hair preservation treatments, hair
colorants, hair weaving preparations, color-removing preparations
for hair, hair gel, hair glazes, hair dressingers, hair lacquers,
hair moisturizers, hair mousse, and hair sprays. In addition, it
may be formulated in the form of skin contact materials such as
cosmetic products, detergents or fibers.
[0045] In a cosmetic composition having each formulation according
to the present invention, components other than the component of
formula 1 or a salt thereof may be suitably selected and added by a
person skilled in the art within a range that does not impair the
purpose and effect of the present invention. Examples of components
that may be added to the cosmetic composition of the present
invention include oil and fat components, skin moisturizers,
emollient agents, surfactants, organic or inorganic pigments,
organic powder, UV-absorbing agents, preservatives, sterilizers,
antioxidants, plant extracts, pH adjusting agents, alcohols,
pigments, fragrance, blood circulation promoters, antiperspirants
purified water, and the like.
[0046] Hereinafter, the present invention will be described in
further detail with reference to examples. However, it will be
obvious to those skilled in the art that these examples are for
illustrative purposes only and various changes and modifications
are possible departing from the scope and technical spirit of the
present invention as disclosed in the appended claims.
[0047] 1. Isolation and Identification of Bacillus sp. 102CH635-3
Strain
[0048] Microorganisms were isolated from marine sediment collected
from the vicinity of the Chuuk Lagoon, Micronesia, in February
2010. 1 g of the marine sediment was heat-treated at 60.degree. C.
for 20 minutes, and then streaked on a BN agar plate. The
microorganisms were cultured at 28.degree. C. for 7 days while
being observed, and an orange single colony of a 102CH635-3 strain
growing while spreading flatly was isolated. The 102CH635-3 strain
was preserved in 40% glycerol at -70.degree. C.
[0049] For 16S rRNA sequencing of 102CH635-3, genomic DNA was
amplified by PCR using 16S rRNA primers (27F 5'(AGA GTT TGA TCM TGG
CTC AG)3' and 1492R 5'(TAC GGY TAC CTT GTT ACG ACT T)3').
Specifically, a mixture of 20 ng of genomic DNA and 30 .mu.l of
EF-Taq (SolGent, Korea) reaction solution was subjected to PCR for
35 cycles. The PCR reaction was performed under the following
conditions: 35 cycles, each consisting of denaturation at
95.degree. C. for 1 min, primer annealing at 55.degree. C. for 1
min, and chain extension at 72.degree. C. for 1 min; followed by
final chain extension at 72.degree. C. for 10 minutes. A nucleotide
sequence having a length of 1,380 bp or more was obtained from the
amplified DNA, and analyzed using an ABI prism 3730XL DNA analyzer
(Applied Biosystems, Foster City, Calif.). The homology of the
nucleotide sequence of the obtained marine microorganisms to those
of strains deposited in the NCBI was analyzed by Blast search. As a
result, the 102CH635-3 strain showed a sequence homology of 99% to
Bacillus subtilis and a sequence homology of 99% to Bacillus
amyloliquefaciens, and thus it was identified to be Bacillus
sp.
[0050] 2. Establishment of Culture Conditions
[0051] A single colony of Bacillus sp. 102CH635-3 was inoculated
into a flask containing 50 ml of BN broth and was cultured at
28.degree. C. for 7 days. The culture broth was extracted with
ethyl acetate and concentrated under reduced pressure to obtain a
crude extract. The analysis of .sup.1H NMR data for the crude
extract indicated that the strain produced an interesting
substance. Thus, the strain was selected as a useful strain for
isolation of an active compound. Prior to mass culture of the
strain, an experiment for establishing optimal culture conditions
under which an active compound is produced was performed. As shown
in Table 1 below, the BN medium from which the strain was isolated,
and an SWNB medium prepared by adding sea salt to an NB medium that
is generally used for Bacillus sp., were used.
TABLE-US-00001 TABLE 1 Composition of the culture media BN SWNB
Components g/L D-glucose 10 -- Tryptone 2 -- Peptone -- 5 Yeast
extract 1 -- Beef extract 1 3 Glucose 5 -- Temperature (.degree.
C.) 28 28 Shaking speed 140 140 (rpm) Sea salt 32 32 pH 7.0 7.0
Culture duration 7 7 (days)
[0052] The 102CH635-3 strain was inoculated into 200 mL of each of
broths having different medium compositions, after which it was
cultured at 28.degree. C. for 7 days, and then extracted with ethyl
acetate and concentrated under reduced pressure, thereby obtaining
crude extracts. The .sup.1H NMR data of the culture crude extracts
obtained using the different medium compositions were analyzed. As
a result, it could be seen that the use of the SWNB medium was
effective for production of a useful compound. Thus, the SWNB
medium was used as an optimal culture medium for mass culture.
[0053] 3. Mass Culture, and Isolation and Structural Determination
of New Compounds (S1 and S2) for Hair Loss Prevention/Hair Growth
Promotion
[0054] A single colony of the Bacillus sp. 102CH635-3 strain was
inoculated and seed-cultured at 28.degree. C. and 120 rpm for 7
days. For mass culture, the seed strain was inoculated into a
large-scale fermenter containing 30 liters of SWNB medium and was
cultured at 28.degree. C. for 13 days. 30 liters of the resulting
culture broth was centrifuged by a continuous centrifuge, and the
culture filtrate was extracted twice with the same amount of ethyl
acetate, and the mycelium was extracted twice with methanol. The
extract was concentrated by a vacuum evaporator to obtain a crude
extract which was then stored at -20.degree. C. until use as a
sample for isolation.
[0055] The ethyl acetate extract of the culture filtrate was
fractionated by reverse-phase chromatography. As an elution
solvent, a water/methanol mixture was used, and the ethyl acetate
extract was fractionated into 5 fractions (from a 20% methanol
fraction to a 100% methanol fraction). The 60% methanol fraction
was concentrated under reduced pressure, dried, and then recovered.
This fraction was purified by C18 reversed-phase HPLC (column:
YMC-ODS-A, 5 .mu.m, 10.times.250 mm; solvent: 45% MeOH; elution
rate: 1.5 ml/min; RI detector) to isolate two single compounds. The
finally isolated new compounds were 2-methylbutyryl-D-pantetheine
(S1; 0.5 mg) and 3-methylbutyryl-D-pantetheine (S2; 1.9 mg) (see
FIG. 1).
[0056] Structural elucidation by analysis of spectrometric data
such as 1D NMR, 2D NMR and HR-ESIMS indicated that S1 and S2 are
new pantetheine derivatives. S1 showed a peak at m/z 385.1778
[M+Na].sup.+ in HR-ESIMS and was determined to have a molecular
formula of C.sub.16H.sub.30N.sub.2O.sub.5S. The .sup.1H NMR
spectrum of S1 showed that S1 has 4 methyl groups and 6 methylene
groups. The COSY spectrum of S1 showed the couplings of four
methylene groups, H-5/H-6 and H-8/H-9, and the HMBC spectrum
revealed a partial structure of S1 by showing the correlations
between the amide carbons, C-4 (.delta. 176.2) and C-7 (.delta.
174.0), and the neighboring hydrogen atoms. In addition, comparison
with that reported in the literature indicated that S1 has a
partial structure of pantetheine. Furthermore, the HMBC spectrum of
S1 showed HMBC correlations between the carbonyl carbon at C-10
(.delta. 205.0) and H-9 (.delta. 3.00), H-12 (.delta. 1.48, 1.71)
and H-14 (.delta. 1.15), indicating that S1 is a new compound
having a structure in which a 2-methylbutyl group is attached to
pantetheine. Moreover, in order to determine the stereochemistry of
C-3, the [.alpha.].sub.D value of S1 was compared with that of
D-pantetheine reported in the literature, and as a result, it was
determined that S1 and D-pantetheine have similar [.alpha.].sub.D
values of +12.2 (c 3.45, H.sub.2O) and +23.7 (c 0.5, H.sub.2O),
respectively. Thus, S1 was determined to be
2-methylbutyryl-D-pantetheine. The HR-ESIMS of S2 showed that S2
has a molecular formula of C.sub.16H.sub.30N.sub.2O.sub.5S,
suggesting that S2 has the same molecular weight and molecular
formula as those of S1. Analysis of various 1D and 2D NMR data,
including 1H NMR, .sup.13C NMR and HMBC data, indicated that S2 has
a structure very similar to that of S1. However, the COSY and HMBC
spectra indicated that S2 has a partial structure of a
3-methylbutyl group in which the methyl group at .delta. 0.95 (d,
J=5.0) is attached to a position different from that in S1. Thus,
the structure of S2 was determined to be
3-methylbutyryl-D-pantetheine. In addition, the results of database
search indicated that S2 is a new compound that has not yet been
reported.
[0057] S1: [.alpha.].sub.D +23.7 (c 0.5, H.sub.2O), .sup.1H NMR
(CD.sub.3OD, 500 MHz) 0.92 (s, CH.sub.3), 0.92 (s, CH.sub.3), 0.92
(t, CH.sub.3), 1.15 (d, J=5.0, CH.sub.3), 1.48-1.71 (m, CH.sub.2),
2.41 (t, CH.sub.2), 2.59 (m, CH), 3.00 (t, CH.sub.2), 3.32 (t,
CH.sub.2), 3.38-3.46 (d, J=10.0, CH.sub.2), 3.47 (m, CH.sub.2),
3.89 (s, CH), .sup.13C NMR (CD.sub.3OD, 125 MHz) 12.1, 17.8, 21.1,
21.5, 28.4, 29.0, 36.5, 36.6, 40.3, 40.5, 51.5, 70.5, 77.4, 174.0,
176.2, 205.0, HR-ESIMS m/z 385.1778 [M+Na].sup.+ (calculated for
C.sub.16H.sub.30N.sub.2O.sub.5NaS, 385.1773).
[0058] S2: [.alpha.].sub.D +7.7 (c 0.5, H.sub.2O), .sup.1H NMR
(CD.sub.3OD, 500 MHz) 0.92 (s, CH.sub.3), 0.92 (s, CH.sub.3), 0.95
(d, J=5.0, CH.sub.3), 0.95 (d, J=5.0, CH.sub.3), 2.13 (m, CH), 2.41
(t, CH.sub.2), 2.46 (d, J=5.0, CH.sub.2), 3.00 (t, CH.sub.2), 3.32
(t, CH.sub.2), 3.38-3.46 (d, J=10.0, CH.sub.2), 3.47 (m, CH.sub.2),
3.89 (s, CH), .sup.13C NMR (CD.sub.3OD, 125 MHz) 21.1, 21.5, 22.7,
22.7, 27.8, 29.3, 36.5, 36.6, 40.3, 40.5, 53.8, 70.5, 77.4, 174.0,
176.2, 200.2, HR-ESIMS m/z 385.1779 [M+Na].sup.+ (calculated for
C.sub.16H.sub.30N.sub.2O.sub.5NaS, 385.1773).
TABLE-US-00002 TABLE 2 NMR data for S1 and S2 in CD.sub.3OD S1 S2
No .delta..sub.C .delta..sub.H, mult. (J in Hz) HMBC .delta..sub.C
.delta..sub.H, mult. (J in Hz) HMBC 1 70.5 3.38, 3.46 d C2, C3 70.5
3.38, 3.46 d C3 (J = 10.0) (J = 10.0) 2 40.5 40.5 3 77.4 3.89, s
C2, C4, C15 77.4 3.89, s C4 4 176.2 176.2 5 36.5 3.47, m C4, C6, C7
36.5 3.47, m C4, C6, C7 6 36.6 2.41, t C5, C7 36.6 2.41, t C5, C7 7
174.0 174.0 8 40.3 3.32, t C7, C8 40.3 3.32, t C7, C9 9 29.0 3.00,
t C8, C10 29.3 3.00, t C8, C10 10 205.0 200.2 11 51.5 2.59, m C12
53.8 2.46, d (J = 5.0) C10, C12, C14 12 28.4 1.48, 1.71, m C10, C11
27.8 2.13, m 13 12.1 0.92, t C11, C12 22.7 0.95, d (J = 5.0) C11,
C12, C14 14 17.8 1.15, C10, C11, 22.7 0.95, d (J = 5.0) C11, C12,
C13 d (J = 5.0) C12 15 21.1 0.92, s C2, C3, 21.1 0.92, s C2, C3,
C16 C16 16 21.5 0.92, s C1, C15 21.5 0.92, s C1, C2, C15
[0059] 4. Synthesis of the New D-Pantetheine Compounds (S1 and S2)
and their Derivatives
[0060] It is known that pantethine and pantetheine are the
components of CoA (coenzyme A) and ACP (acyl carrier protein) and
are involved in fatty acid and carbohydrate metabolisms after
hydrolysis. It was reported that these compounds play a role in
metabolic reactions, including fatty acid oxidation, amino acid
degradation and cholesterol synthesis, and have effects against
hyperlipidemia, hematological diseases, renal diseases,
arteriolosclerosis, diabetes, and the like.
Calcium-D-pantetheine-S-sulfonate is known to have whitening
activity, moisturizing activity, melanin production inhibitory
activity and anti-aging activity such as skin inflammation
inhibitory activity, and thus has been used as an additive in
famous cosmetic products in Korea and other countries. Thus, in
order to synthesize the new D-pantetheine compounds isolated from
the 102CH635-3 strain and their derivatives, studies on the
synthesis of D-pantetheine compounds were conducted using
D-pantethine, thereby synthesizing not only the new natural
compounds (S1 and S2), but also their derivatives.
[0061] Using commercially available D-pantethine (P2125, SIGMA),
various new derivatives of D-pantetheine were synthesized according
to the method shown in FIG. 2. In a detailed synthetic method,
dithiothreitol (DTT) and H.sub.2O:MeOH (1:1, v/v) were added to
D-pantethine, and the mixture was allowed to react at 4.degree. C.
for 16 hours under N.sub.2 atmosphere. After it was confirmed by
TLC that D-pantethine disappeared, the reaction was terminated, and
the solvent was removed. Dry tetrahydrofuran (THF) and
triethylamine were added to the reaction product (pantetheine) from
which the solvent has been removed, and each of reagents having
various functional groups (R) was added thereto, after each of the
mixtures was allowed to react at 0.degree. C. for 2 hours and at
room temperature for 3 hours.
[0062] Herein, R is one of 2-methylbutyryl, 3-methylbutyryl,
cinnamoyl, 4-pentenoyl, 10-undecenoyl, isobutyl formate,
2,4-dihydroxybenzoyl, geranyl, farnesyl, acryloyl, propanone,
2-pentanone, 1-(4-hydroxyphenyl)ethanone,
1-(2,4-dihydroxyphenyl)ethanone, 1-(2,4-dihydroxyphenyl)ethanone,
pentanoic acid, 2-hydroxypropanoic acid, 2-phenylacetic acid,
2-(4-(propanoyl)phenyl)acetic acid, 4-methylbenzoic acid,
4-(4-phenyl)-4-oxobutanoic acid, 2-oxoethyl acetyl,
2-phenoxyacetyl, 2-(benzyloxy)acetyl, 4-methoxybenzoyl,
3,5-dimethylphenol, 6-methoxybenzene-1,4-diol, propenylbenzene, and
4-hydroxycoumarin.
[0063] After the reaction solvent was completely removed, the
remaining material was solvent-fractionated with H.sub.2O:ethyl
acetate (EtOAC) (1:1, v/v), and the ethyl acetate layer was
recovered, treated with MgSO.sub.4, and then concentrated to obtain
reaction products. It was confirmed by LR-APCIMS that the desired
compounds were synthesized, and the products were separated using
HPLC. Purification was performed using C18 reversed-phase HPLC
(column: YMC-ODS-A, 5 .mu.m, 10.times.250 mm; solvent: 60% MeOH;
elution rate: 1.5 mL/min; RI detector) to separate various new
derivatives.
[0064] Among the synthesized new derivatives,
2-methylbutyryl-D-pantetheine, 3-methylbutyryl-D-pantetheine,
cinnamoyl-D-pantetheine, 4-pentenoyl-D-pantetheine,
10-undecenoyl-D-pantetheine, isobutyl formate-D-pantetheine,
2,4-dihydroxybenzoyl-D-pantetheine and geranyl-D-pantetheine were
analyzed by .sup.1H NMR, .sup.13C NMR and MS spectrometry to
determine their structures. It is to be understood that new
derivatives not described in this example can also be synthesized
according to the proposed synthetic method.
[0065] S1 (2-methylbutyryl-D-pantetheine): [.alpha.].sub.D +9.9 (c
1.0, H.sub.2O), .sup.1H NMR (CD.sub.3OD, 500 MHz) 0.92 (s,
CH.sub.3), 0.92 (s, CH.sub.3), 0.92 (t, CH.sub.3), 1.15 (d, J=5.0,
CH.sub.3), 1.48-1.71 (m, CH.sub.2), 2.41 (t, CH.sub.2), 2.59 (m,
CH), 3.00 (t, CH.sub.2), 3.32 (t, CH.sub.2), 3.38-3.46 (d, J=10.0,
CH.sub.2), 3.47 (m, CH.sub.2), 3.89 (s, CH), .sup.13C NMR
(CD.sub.3OD, 125 MHz) 12.1, 17.8, 21.1, 21.5, 28.4, 29.0, 36.5,
36.6, 40.3, 40.5, 51.5, 70.5, 77.4, 174.0, 176.2, 205.0, LR-APCIMS
m/z 360.99 [M-H].sup.-, 362.87 [M+H].sup.+,
C.sub.16H.sub.30N.sub.2O.sub.5S.
[0066] S2 (3-methylbutyryl-D-pantetheine): [.alpha.].sub.D +5.9 (c
1.0, H.sub.2O), .sup.1H NMR (CD.sub.3OD, 500 MHz) 0.92 (s,
CH.sub.3), 0.92 (s, CH.sub.3), 0.95 (d, J=5.0, CH.sub.3), 0.95 (d,
J=5.0, CH.sub.3), 2.13 (m, CH), 2.41 (t, CH.sub.2), 2.46 (d, J=5.0,
CH.sub.2), 3.00 (t, CH.sub.2), 3.32 (t, CH.sub.2), 3.38-3.46 (d,
J=10.0, CH.sub.2), 3.47 (m, CH.sub.2), 3.89 (s, CH), .sup.13C NMR
(CD.sub.3OD, 125 MHz) 21.1, 21.5, 22.7, 22.7, 27.8, 29.3, 36.5,
36.6, 40.3, 40.5, 53.8, 70.5, 77.4, 174.0, 176.2, 200.2, LR-APCIMS
m/z 363.14 [M+H].sup.+, C.sub.16H.sub.30N.sub.2O.sub.5S.
[0067] S3 (cinnamoyl-D-pantetheine): .sup.1H NMR (CD.sub.3OD, 500
MHz) 0.92 (s), 2.42 (t), 3.15 (t), 3.35-3.51 (m), 3.89 (s), 6.86
(d, J=15.0), 7.41-7.42 (m), 7.62-7.66 (m), .sup.13C NMR
(CD.sub.3OD, 125 MHz) 21.1, 21.5, 29.4, 36.5, 36.6, 40.3, 40.5,
50.0, 70.5, 77.4, 125.9, 129.8, 130.3, 132.0, 135.6, 142.3, 176.2,
191.2, LR-APCIMS m/z 408.15 [M-H].sup.-, 409.02 [M+H].sup.+,
C.sub.20H.sub.28N.sub.2O.sub.5S.
[0068] S4 (4-pentenoyl-D-pantetheine): .sup.1H NMR (CD.sub.3OD, 500
MHz) 0.92 (s), 2.36-2.42 (m), 2.68 (t), 3.01 (t), 3.32 (t),
3.33-3.53 (m), 3.89 (s), 4.98 (d, J=10.0), 5.05 (d, J=15.0),
5.78-5.84 (m), .sup.13C NMR (CD.sub.3OD, 125 MHz) 21.1, 21.5, 29.3,
30.6, 36.4, 36.5, 40.2, 40.5, 44.1, 70.5, 77.4, 116.3, 137.6,
174.0, 176.1, 199.9, LR-APCIMS m/z 360.90 [M+H].sup.+, 721.25
[2M+H].sup.+, C.sub.16H.sub.28N.sub.2O.sub.5S.
[0069] S5 (10-undecenoyl-D-pantetheine): .sup.1H NMR (CD.sub.3OD,
500 MHz) 0.92 (s), 1.38 (t), 1.64 (m), 2.04 (q), 2.41 (t), 2.58
(t), 3.00 (t), 3.32-3.50 (m), 3.90 (s), 4.90-4.92 (dd), 4.96-5.00
(m), 5.77-5.81 (m) .sup.13C NMR (CD.sub.3OD, 125 MHz) 21.1, 21.4,
26.8, 29.2, 30.1, 30.2, 30.2, 30.4, 30.5, 35.0, 36.4, 36.5, 40.2,
40.5, 44.9, 70.5, 77.3, 114.9, 140.2, 173.9, 176.1, 200.7,
LR-APCIMS m/z 443.10 [M-H].sup.-, 415.10 [M+H].sup.+,
C.sub.22H.sub.40N.sub.2O.sub.5S.
[0070] S6 (isobutyl formate-D-pantetheine): 1H NMR (CD.sub.3OD, 500
MHz) 0.92 (s), 0.94 (d, J=10.0), 1.95 (m), 2.42 (t), 2.98 (t),
3.38-3.50 (m), 3.89 (s), 4.01 (d, J=5.0), .sup.13C NMR (CD.sub.3OD,
125 MHz) 19.3, 21.0, 21.5, 29.2, 31.3, 36.4, 36.5, 40.4, 40.5,
70.5, 74.6, 77.3, 172.1, 174.0, 176.1, LR-APCIMS m/z 376.95
[M-H].sup.-, 378.90 [M+H].sup.+,
C.sub.16H.sub.30N.sub.2O.sub.6S.
[0071] S7 (2,4-dihydroxybenzoyl-D-pantetheine): 1H NMR (CD.sub.3OD,
500 MHz) 0.91 (s), 2.43 (t), 2.72 (t), 3.35-3.51 (m), 3.85 (s),
3.89 (s), 6.27 (d, J=2.5), 6.36 (dd, J=2.0, 10.0), 7.72 (d,
J=10.0), .sup.13C NMR (CD.sub.3OD, 125 MHz) 21.0, 21.5, 32.8, 36.5,
36.6, 37.3, 39.6, 40.5, 70.5, 77.4, 103.9, 109.5, 112.7, 134.5,
166.9, 167.1, 174.0, 176.2, 201.0, LR-APCIMS m/z 427.13
[M-H].sup.-, 429.13 [M+H].sup.+,
C.sub.19H.sub.28N.sub.2O.sub.7S.
[0072] S8 (geranyl-D-pantetheine): .sup.1H NMR (CD.sub.3OD, 500
MHz) 0.92 (s), 1.61 (s), 1.67 (s), 1.68 (s), 2.05 (t), 2.12 (q),
2.42 (t), 2.57 (t), 3.18 (d, J=8.0), 3.34-3.53 (m), 3.89 (s), 5.10
(t), 5.23 (t) .sup.13C NMR (CD.sub.3OD, 125 MHz) 16.3, 17.9, 21.1,
21.5, 26.1, 27.7, 29.9, 31.3, 36.5, 36.6, 40.2, 40.5, 40.8, 70.5,
77.4, 122.1, 125.3, 132.6, 140.1, 173.9, 176.2, LR-APCIMS m/z
413.09 [M-H].sup.-, 414.84 [M+H].sup.+,
C.sub.22H.sub.40N.sub.2O.sub.5S.
[0073] 5. Effects of New D-Pantetheine Compounds on Hair Loss
Prevention/Hair Growth Promotion
[0074] Dermal papilla cells play a key role in the production and
growth of hair, and compounds that promote the growth of dermal
papilla cells are attracting attention as agents for preventing
hair loss and promoting hair growth. In the present invention, in
order to develop novel compounds for hair loss prevention and hair
growth promotion, the effects of the new natural compounds and
derivatives thereof on the promotion of growth of dermal papilla
cells were measured.
[0075] (1) Measurement of Viability of Dermal Papilla Cells
[0076] For cell culture, Dulbecco's Modified Eagle Medium (DMEM),
fetal bovine serum (FBS), 0.25% trypsin-1 mM EDTA, and
penicillin-streptomycin were used, and dermal papilla cells were
cultured at 37.degree. C. in a humidified 5% CO.sub.2. The medium
was replaced twice a week, and the cultured dermal papilla cells
were washed with PBS within 6-7 days. Next, the attached cells were
separated by 0.05% trypsin-0.02% EDTA, centrifuged, and then
subcultured for experiments. Specifically, 200 .mu.l of dermal
papilla cells were dispensed into each well of a 96-well plate at a
density of 1.times.10.sup.4 cells/well, and cultured under the
conditions of 37.degree. C. and 5% CO.sub.2 for 18 hours so as to
be attached to each well. After 18 hours, the medium was removed,
and 200 .mu.l of samples was added to each well having the cells
attached thereto, followed by culture for 24 hours. After the
medium was removed, 100 .mu.l of a solution of
3-(4,5-dimethylthiazol)-2,5-diphenyltetrazoliumbromide (MTT),
prepared by dissolving MTT in PBS at a concentration of 500
.mu.g/ml, was added to the cultured cells, followed by culture for
4 hours under the same conditions as described above. The formed
formazan crystal was dissolved in DMSO, and the absorbance at 570
nm was measured using an ELISA reader. The measured OD values
ranging from 0.00 to 3.50 were used for the experimental results.
The sample for treatment of the cells was used at concentrations of
0.5, 5 and 50 .mu.M.
[0077] (2) Effects on Promotion of Growth of Dermal Papilla
Cells
[0078] The results of the experiment for the new pantetheine
compounds obtained in the present invention (see FIG. 3 and Table
3) indicated that the pantetheine compounds showed excellent
effects on the promotion of growth of dermal papilla cells, and
particularly, in the case of S1 and S6 among these compounds, the
dermal papilla cell proliferation rate increased up to 140%.
[0079] Although the effects of all the compounds represented by
formula 1 according to the present invention on the promotion of
growth of dermal papilla cells were not examined, it will be
obvious that the compounds that were not examined show effects
equal to those of compounds S1-S7, because these compounds have the
same backbone structure as that of compounds S1-S7 and differ from
compounds S1-S7 only in terms of the substituent groups.
TABLE-US-00003 TABLE 3 Effect of compounds S1-S7 on the growth of
dermal papilla cells Concentration (.mu.M) Control 0.5 5 50 S1
(2-methylbutyryl-pantetheine) 100 113.31 129.65 139.19 S2
(3-methylbutyryl-pantetheine) 100 121.04 114.65 121.28 S3
(cinnamoyl-pantetheine) 100 119.77 122.38 133.33 S4
(4-pentenoyl-pantetheine) 100 116.88 122.89 111.97 S5
(10-undecenoyl-pantetheine) 100 111.24 93.01 101.62 S6 (isobutyl
formate-pantetheine) 100 130.57 112.94 135.95 S7
(2,4-dihydroxybenzoyl- 100 95.73 97.32 93.12 pantetheine) minoxidil
100 146.66 146.52 140.29
[0080] As described above, the compounds according to the present
invention showed activities of promoting the growth of dermal
papilla cells, at levels almost equal to that of minoxidil, and are
new compounds that have not yet been reported. These compounds of
the present invention have a low molecular weight and are also
easily synthesized. Thus, the possibility for these compounds to be
developed into cosmetic or medical products for hair loss
prevention and hair growth promotion is very high.
[0081] Although the specific embodiments of the present invention
have been disclosed for illustrative purposes, those skilled in the
art will appreciate that various modifications, additions and
substitutions are possible without departing from the scope and
spirit of the invention as disclosed in the accompanying
claims.
* * * * *