U.S. patent application number 11/912964 was filed with the patent office on 2009-03-19 for transdermal absorption enhancer.
This patent application is currently assigned to JAPAN SCIENCE AND TECHNOLOGY AGENCY. Invention is credited to Rie Igarashi, Yoko Yamaguchi.
Application Number | 20090075860 11/912964 |
Document ID | / |
Family ID | 37308040 |
Filed Date | 2009-03-19 |
United States Patent
Application |
20090075860 |
Kind Code |
A1 |
Yamaguchi; Yoko ; et
al. |
March 19, 2009 |
TRANSDERMAL ABSORPTION ENHANCER
Abstract
An object of the present invention is to provide a transdermal
absorption enhancer by which various active ingredients are
transdermally absorbed. In accordance with a transdermal absorption
enhancer of the present invention which effective ingredient is
lyotropic liquid crystal which has been utilized as a basic
material for pharmaceutical preparations for external application
and for cosmetics, transdermal absorption of a macromolecular
substance and a water-soluble substance was able to be
improved.
Inventors: |
Yamaguchi; Yoko; (Kanagawa,
JP) ; Igarashi; Rie; (Kanagawa, JP) |
Correspondence
Address: |
WESTERMAN, HATTORI, DANIELS & ADRIAN, LLP
1250 CONNECTICUT AVENUE, NW, SUITE 700
WASHINGTON
DC
20036
US
|
Assignee: |
JAPAN SCIENCE AND TECHNOLOGY
AGENCY
Kawaguchi-shi, Saitama
JP
NANOEGG RESEARCH LABORATORIES, INC.
Kawasaki-shi, Kanagawa
JP
|
Family ID: |
37308040 |
Appl. No.: |
11/912964 |
Filed: |
April 28, 2006 |
PCT Filed: |
April 28, 2006 |
PCT NO: |
PCT/JP2006/308975 |
371 Date: |
October 29, 2007 |
Current U.S.
Class: |
514/1.1 ;
514/44R; 514/772; 514/772.3; 514/785 |
Current CPC
Class: |
A61P 3/10 20180101; A61P
3/02 20180101; A61K 9/0014 20130101; A61P 17/02 20180101; A61K
9/1274 20130101; A61K 47/12 20130101; A61K 9/06 20130101; A61K
31/23 20130101; A61K 47/44 20130101; A61K 31/203 20130101 |
Class at
Publication: |
514/2 ; 514/785;
514/772.3; 514/772; 514/44 |
International
Class: |
A61K 38/02 20060101
A61K038/02; A61K 47/44 20060101 A61K047/44; A61K 47/34 20060101
A61K047/34; A61K 47/06 20060101 A61K047/06; A61K 31/711 20060101
A61K031/711 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 28, 2005 |
JP |
2005-130962 |
Claims
1. A transdermal absorption enhancer, characterized in that,
lyotropic liquid crystal is an effective ingredient.
2. The transdermal absorption enhancer according to claim 1,
wherein said lyotropic liquid crystal contains 5% by weight to 80%
by weight of a surfactant and 5% by weight to 80% by weight of
water.
3. The transdermal absorption enhancer according to claim 2,
wherein said surfactant is a nonionic surfactant and/or
lecithin.
4. The transdermal absorption enhancer according to claim 3,
wherein said nonionic surfactant is at least one member selected
from the group consisting of polyoxyethylene alkyl ether,
polyoxyethylene sorbitan fatty acid ester and polyoxyethylene
hydrogenated castor oil.
5. The transdermal absorption enhancer according to claim 2,
wherein said lyotropic liquid crystal further contains 1% by weight
to 80% by weight of oil.
6. The transdermal absorption enhancer according to claim 5,
wherein said oil is squalane.
7. The transdermal absorption enhancer according to claim 2,
wherein said lyotropic liquid crystal further contains 1% by weight
to 55% by weight of a polyhydric alcohol.
8. The transdermal absorption enhancer according to claim 7,
wherein said polyhydric alcohol is glycerol.
9. The transdermal absorption enhancer according to claim 2,
wherein said lyotropic liquid crystal further contains 0.01% by
weight to 10% by weight of an auxiliary surfactant.
10. The transdermal absorption enhancer according to claim 9,
wherein said auxiliary surfactant is cholesterol.
11. A transdermal absorption composition, characterized in that,
lyotropic liquid crystal is compounded with an active
ingredient.
12. The transdermal absorption composition according to claim 11,
wherein said active ingredient is at least one member selected from
the group consisting of organic compound, peptide, protein,
oligonucleotide, DNA and RNA.
13. The transdermal absorption composition according to claim 11,
wherein said active ingredient is a macromolecular substance where
molecular weight is not less than 1,000 or a water-soluble
substance.
14. The transdermal absorption composition according to claim 11,
wherein said active ingredient is compounded in a form of being
included in the inside of fine particles of inorganic acid salt
with divalent metal.
15. The transdermal absorption composition according to claim 11,
wherein said active ingredient is compounded in an amount of 0.01%
by weight to 50% by weight to the lyotropic liquid crystal.
Description
TECHNICAL FIELD
[0001] The present invention relates to a transdermal absorption
enhancer by which various active ingredients are transdermally
absorbed.
BACKGROUND ART
[0002] Transdermal administration is easy and convenient as
compared with oral administration and administration by injection,
and may also be advantageous in terms of duration of the effect and
reduction of expression of the side effects, whereby that is an
excellent administration method. However, in order to permeate the
active ingredient into the body by transdermal administration, the
active ingredient is to be penetrated through the skin which
constitutes the primary barrier of the living body and, therefore,
its bioavailability (amount of the drug absorbed with a blood flow)
is inherently low. Accordingly, in order to achieve the improvement
of bioavailability of active ingredients, it has been conducted
that dipropylene glycol, hexylene glycol, isoparaffin, sodium
laurylsulfate, an ethylene oxide adduct of lauryl alcohol,
polyethylene glycol fatty acid ester, polyoxyethylene sorbitan
fatty acid ester, propyl carbonate, sodium pyrrolidonecarboxylate,
urea, lactic acid, sodium lactate, lecithin, dimethyl sulfoxide,
pyrrolidonecarboxylate, nicotinate, N-methylproline ester,
cholesteryl oleate, amine oxide or the like is compounded with
preparations for external application as a transdermal absorption
enhancer.
[0003] Up to now, the present inventors have energetically carried
out research and development for transdermal absorption of active
ingredients and, as a result, they have found that, when retinoic
acid having an action of enhancing regeneration of the skin by
enhancing differentiation and growth of keratinocytes is included
into capsules of a nanometer level (nano-particles) followed by
applying to the skin surface, retinoic acid is able to be
transdermally absorbed in efficient and sustained-releasing manner
without compounding of the transdermal absorption enhancers as
mentioned above (Non-Patent Document 1 and Non-Patent Document
2).
[0004] Non-Patent Document 1: Yoko Yamaguchi, "Novel
Nano-Technology for Transdermal Delivery", Bio Venture, vol. 4, no.
6, pages 62 to 64, 2004
[0005] Non-Patent Document 2: Y. Yamaguchi, T. Nagasawa, N.
Nakamura, M. Takenaga, M. Mizoguchi, S. Kawai, Y. Mizushima and R.
Igarashi, "Successful Treatment of Photo-Damaged Skin of Nano-Scale
atRA Particles Using a Novel Transdermal Delivery", 104, 29 to 40,
2005.
DISCLOSURE OF THE INVENTION
Problems that the Invention is to Solve
[0006] Although the above-mentioned nano-particle including
retinoic acid therein is expected for its clinical application as a
method for transdermal absorption of retinoic acid with little
irritation of retinoic acid to the skin, investigation of methods
for transdermal absorption of various active ingredients is still
meaningful.
[0007] Accordingly, an object of the present invention is to
provide a transdermal absorption enhancer by which various active
ingredients are transdermally absorbed.
Means for Solving the Problems
[0008] In view of the above, the present inventors have repeatedly
conducted intensive investigations and, as a result, they have
found that lyotropic liquid crystal (refer, for example, to
Japanese Patent Nos. 2,547,151 and 3,459,253) which has been
already known as a basic material for pharmaceutical preparations
for external application and for cosmetics has an action of
enhancing the transdermal absorption of various active
ingredients.
[0009] The transdermal absorption enhancer of the present invention
achieved on the basis of the above finding is characterized in that
lyotropic liquid crystal is an effective ingredient as mentioned in
claim 1.
[0010] The transdermal absorption enhancer mentioned in claim 2 is
characterized in that, in the transdermal absorption enhancer
according to claim 1, the lyotropic liquid crystal contains 5% by
weight to 80% by weight of a surfactant and 5% by weight to 80% by
weight of water.
[0011] The transdermal absorption enhancer mentioned in claim 3 is
characterized in that, in the transdermal absorption enhancer
according to claim 2, the surfactant is a nonionic surfactant
and/or lecithin.
[0012] The transdermal absorption enhancer mentioned in claim 4 is
characterized in that, in the transdermal absorption enhancer
according to claim 3, the nonionic surfactant is at least one
member selected from the group consisting of polyoxyethylene alkyl
ether, polyoxyethylene sorbitan fatty acid ester and
polyoxyethylene hydrogenated castor oil.
[0013] The transdermal absorption enhancer mentioned in claim 5 is
characterized in that, in the transdermal absorption enhancer
according to claim 2, the lyotropic liquid crystal further contains
1% by weight to 80% by weight of oil.
[0014] The transdermal absorption enhancer mentioned in claim 6 is
characterized in that, in the transdermal absorption enhancer
according to claim 5, the oil is squalane.
[0015] The transdermal absorption enhancer mentioned in claim 7 is
characterized in that, in the transdermal absorption enhancer
according to claim 2, the lyotropic liquid crystal further contains
1% by weight to 55% by weight of a polyhydric alcohol.
[0016] The transdermal absorption enhancer mentioned in claim 8 is
characterized in that, in the transdermal absorption enhancer
according to claim 7, the polyhydric alcohol is glycerol.
[0017] The transdermal absorption enhancer mentioned in claim 9 is
characterized in that, in the transdermal absorption enhancer
according to claim 2, the lyotropic liquid crystal further contains
0.01% by weight to 10% by weight of an auxiliary surfactant.
[0018] The transdermal absorption enhancer mentioned in claim 10 is
characterized in that, in the transdermal absorption enhancer
according to claim 9, the auxiliary surfactant is cholesterol.
[0019] In addition, a transdermal absorption composition of the
present invention is characterized in that, lyotropic liquid
crystal is compounded with an active ingredient as mentioned in
claim 11.
[0020] The transdermal absorption composition mentioned in claim 12
is characterized in that, in the transdermal absorption composition
according to claim 11, the active ingredient is at least one member
selected from the group consisting of organic compound, peptide,
protein, oligonucleotide, DNA and RNA.
[0021] The transdermal absorption composition mentioned in claim 13
is characterized in that, in the transdermal absorption composition
according to claim 11, the active ingredient is a macromolecular
substance where molecular weight is not less than 1,000 or a
water-soluble substance.
[0022] The transdermal absorption composition mentioned in claim 14
is characterized in that, in the transdermal absorption composition
according to claim 11, the active ingredient is compounded in a
form of being included in the inside of fine particles of inorganic
acid salt with divalent metal.
[0023] The transdermal absorption composition mentioned in claim 15
is characterized in that, in the transdermal absorption composition
according to claim 11, the active ingredient is compounded in an
amount of 0.01% by weight to 50% by weight to the lyotropic liquid
crystal.
ADVANTAGES OF THE INVENTION
[0024] In accordance with the present invention, there is provided
a transdermal absorption enhancer as a novel pharmaceutical use of
lyotropic liquid crystal which has been utilized as a basic
material for pharmaceutical preparations for external application
and for cosmetics and, in the transdermal absorption enhancer of
the present invention, various active ingredients are able to be
transdermally absorbed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 is a graph which shows the changes in concentration
of retinoic acid in blood in Example 1.
[0026] FIG. 2 is a graph which shows the changes in production
amount of HB-EGF when each of the four kinds of samples is applied
in Example 2.
[0027] FIG. 3 is a graph which shows the changes in production
amount of HB-EGF when each of the four kinds of samples is applied
and production amount of HB-EGF of the skin to which nothing is
applied in Example 3.
[0028] FIG. 4 is a cross-sectional picture of the skin when each of
the five kinds of samples is applied and a cross-sectional picture
of the skin to which nothing is applied in Example 5.
[0029] FIG. 5 is a graph which shows the changes in concentration
of insulin in blood in Example 5.
[0030] FIG. 6 is a graph which shows the changes in rate of
niacinamide in total blood to the administered dose with the
passage of time in Example 6.
[0031] FIG. 7 is a graph which shows the changes in residual rate
of retinoic acid in the five kinds of samples with the passage of
time in Example 7.
[0032] FIG. 8 is a graph which shows the changes in residual rate
of retinol palmitate in the four kinds of samples with the passage
of time in Example 8.
[0033] FIG. 9 is a cross-sectional picture of the skin to which
lyotropic liquid crystal compounded with SOD is applied in Example
9.
[0034] FIG. 10 is a cross-sectional picture of the skin to which a
dispersion solution where SOD is dispersed in water is applied in
the same.
[0035] FIG. 11 is a cross-sectional picture of the skin to which
only water is applied in the same.
[0036] FIG. 12 is a cross-sectional picture of the skin to which
each of the six kinds of samples is applied in Example 10.
[0037] FIG. 13 is a fluorescent cross-sectional picture of the skin
to which lyotropic liquid crystal compounded with oligo-DNA
fluorescently labeled with FITC is applied in Example 11.
[0038] FIG. 14 is a fluorescent cross-sectional picture of the skin
to which a dispersion solution where oligo-DNA fluorescently
labeled with FITC is dispersed in water is applied in the same.
[0039] FIG. 15 is a fluorescent cross-sectional picture of the skin
to which lyotropic liquid crystal compounded with fluorescently
labeled dextran (molecular weight: 4,000) with FITC is applied in
Example 12.
[0040] FIG. 16 is a fluorescent cross-sectional picture of the skin
to which lyotropic liquid crystal compounded with fluorescently
labeled dextran (molecular weight: 70,000) with FITC is applied and
a fluorescent cross-sectional picture of the skin to which a
dispersion solution where fluorescently labeled dextran (the same
as above) is dispersed in water is applied in Example 13.
BEST MODE FOR CARRYING OUT THE INVENTION
[0041] The transdermal absorption enhancer of the present invention
is characterized in that lyotropic liquid crystal is an effective
ingredient. The lyotropic liquid crystal in accordance with the
present invention means such a thing that, in a system where
surfactant (amphipathic molecule having a hydrophilic part and a
hydrophobic (lipophilic) part in a molecule) and water are
coexisting, a liquid crystal state (a state where a predetermined
regularity in molecular orientation is maintained as if in the case
of crystal while fluidity is still available as if in the case of
liquid) is formed depending upon the mixing ratio of both parts and
upon temperature. Principally, it is able to be understood that, in
lyotropic liquid crystal, when water is added, within a
predetermined temperature range, to a surfactant in a solid state
having a crystal structure where hydrophobic parts (hydrophobic
groups such as alkyl group) are faced each other, said parts lose
regularity due to thermal movement resulting in a liquid state and
then the hydrophilic parts act each other due to hydrogen bond to
maintain for a long period whereby an associated structure (such as
hexagonal structure and lamella structure) is resulted (refer, if
necessary, to Toshiyuki Suzuki, "Liquid Crystal", vol. 2, pages 194
to 201, 1998).
[0042] With regard to the surfactant which is a constituting
component of the lyotropic liquid crystal, there is no particular
limitation so far as it is able to form a liquid crystal state (a
periodical structure where the interplanar spacing is 10 nm to 800
nm is particularly preferred) in a system coexisting with water
depending upon the mixing ratio with water and upon temperature.
Thus, it may be a surfactant of any of the types of nonionic type,
anionic type, cationic type and amphoteric type and may also be a
surfactant derived from nature such as lecithin (for example, egg
yolk lecithin and soybean lecithin) and saponin. A single
surfactant may be used solely or plural kinds thereof may be mixed
and used.
[0043] Examples of the nonionic surfactant are polyoxyethylene
alkyl ether, polyoxyethylene alkyl phenol ether, alkyl glucoside,
polyoxyethylene fatty acid ester, sucrose fatty acid ester,
sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid
ester, fatty acid alkanolamide and polyoxyethylene hydrogenated
castor oil. Examples of the anionic surfactant are soap (sodium
salt, potassium salt, etc. of fatty acid), alkylbenzenesulfonate
(such as sodium salt), higher alcohol sulfate salt (such as sodium
salt), polyoxyethylene alkyl ether sulfate (such as sodium salt),
.alpha.-sulfofatty acid ester, .alpha.-olefin sulfonate (such as
sodium salt), monoalkylphosphate salt (such as sodium salt) and
alkanesulfonate (such as sodium salt). Examples of the cationic
surfactant are alkyl trimethylammonium salt (such as chloride),
dialkyl dimethylammonium salt (such as chloride), alkyl
dimethylbenzylammonium salt (such as chloride) and amine salt (such
as acetate salt and hydrochloride salt). Examples of the amphoteric
surfactant are alkylamino fatty acid salt (such as sodium salt),
alkylbetaine and alkylamine oxide. Rate of the surfactant in the
lyotropic liquid crystal is preferably 5% by weight to 80% by
weight, more preferably 7% by weight to 70% by weight and, still
more preferably, 10% by weight to 65% by weight. HLB value of the
surfactant is preferably not less than 8, more preferably not less
than 10 and, still more preferably, not less than 12.
[0044] With regard to water which is a constituting component of
the lyotropic liquid crystal, distilled water or the like may be
used. Water used therefor may contain organic solvent which is
miscible with water such as ethanol and isopropanol. Rate of water
in the lyotropic liquid crystal is preferably 5% by weight to 80%
by weight, more preferably 10% by weight to 60% by weight and,
still more preferably, 13% by weight to 50% by weight.
[0045] The lyotropic liquid crystal may further contain oil besides
the surfactant and water. When oil is contained therein, the liquid
crystal structure becomes similar to a lamella structure formed by
the intercellular lipid in a horny layer and, upon application to
the skin surface, a phase transfer of the intercellular lipid
structure is apt to happen and, as a result, an excellent enhancing
action of transdermal absorption is achieved for the active
ingredient. Examples of the oil are vegetable oil such as wheat
germ oil, corn oil, sunflower oil and castor oil; silicone oil;
ester oil such as isopropyl myristate, glyceryl trioctanoate,
diethylene glycol monopropylene pentaerythritol ether and
pentaerythrityl tetraoctanoate; squalane; squalene; liquid
paraffin; and polybutene. A single oil may be used solely or plural
kinds thereof may be mixed and used. Rate of the oil in the
lyotropic liquid crystal is preferably 1% by weight to 80% by
weight, more preferably 5% by weight to 70% by weight and, still
more preferably, 10% by weight to 65% by weight.
[0046] The lyotropic liquid crystal may further contain a
polyhydric alcohol. When a polyhydric alcohol is contained therein,
it is possible to attempt for making the formation of liquid
crystal structure easy (expansion of phase region) and for making
it stable. Examples of the polyhydric alcohol are polyalkylene
glycol (such as polyethylene glycol and polyalkylene glycol),
glycerol, propylene glycol, 1,3-propanediol, 2-butene-1,4-diol,
pentane-1,5-diol, 2,2-dimethylpropane-1,3-diol,
3-methylpentane-1,5-diol, pentane-1,2-diol,
2,2,4-trimethylpentane-1,3-diol, 2-methylpropane-1,3-diol, hexylene
glycol, 1,3-butylene glycol, dipropylene glycol, diethylene glycol
and triethylene glycol. A single polyhydric alcohol may be used
solely or plural kinds thereof may be mixed and used. Rate of the
polyhydric alcohol in the lyotropic liquid crystal is preferably 1%
by weight to 55% by weight, more preferably 3% by weight to 52% by
weight and, still more preferably, 5% by weight to 50% by
weight.
[0047] The lyotropic liquid crystal may further contain an
auxiliary surfactant such as cholesterol. When an auxiliary
surfactant is contained therein, reduction of surface membrane
curvature is able to be achieved even when various kinds of
surfactants are used and, therefore, it is able to attempt for
making the formation of liquid crystal structure easy and for
making it stable. Rate of the auxiliary surfactant in the lyotropic
liquid crystal is preferably 0.01% by weight to 10% by weight.
[0048] The lyotropic liquid crystal is able to be prepared by
mixing of the surfactant and water which are constituting
components thereof in a predetermined ratio at predetermined
temperature. If necessary, an operation where the constituting
component is temporarily warmed before or after mixing may be
carried out.
[0049] In the transdermal absorption enhancer of the present
invention, various active ingredients are able to be transdermally
absorbed independently of physical and chemical properties thereof.
Here, "active ingredient" means that which is able to act as a drug
for attempting prevention and treatment of various diseases and
maintenance and improvement of health and beauty for mammals
including human being and, to be more specific, its examples are
vitamins, prostaglandins, anti-cancer drug, growth hormones,
various growth factors, vaccine antigen, gene encoding useful
protein and other organic compound, peptide, protein,
oligonucleotide, DNA and RNA. The matter being worthy of special
mention for the transdermal absorption enhancer of the present
invention is that transdermal absorption of substances which have
been impossible or difficult to be penetrated into the skin and
permeated into the body such as macromolecular substances where
molecular weight is 1,000 or more (although there is no particular
limitation for the upper limit of the molecular weight, it is, for
example, 500,000 to 1,000,000) and water-soluble substances such as
niacinamide (nicotinamide) (the water-soluble substance may be
defined, for example, as "a substance which shows a transmittance
(1/absorbance) of within a range of 70% to 100% at 450 nm
wavelength in a state of being dispersed in water and, when its
appearance is observed, no macroscopic separation is noted") is now
made possible.
[0050] Since lyotropic liquid crystal which has been utilized as a
basic material for pharmaceutical preparations for external
application and for cosmetics is an effective ingredient of the
transdermal absorption enhancer of the present invention, it is now
possible to prepare a transdermally absorption composition when the
lyotropic liquid crystal is compounded with an active ingredient.
Compounding amount of the active ingredient to the lyotropic liquid
crystal is, for example, from 0.01% by weight to 50% by weight.
When the lyotropic liquid crystal is compounded, for example, with
a substance having an enhancing action of differentiation and
growth of keratinocytes, a substance having a suppressive action to
melanin pigment production or a substance having an enhancing
action for the synthesis of intercellular lipid of horny layer, it
is now possible to prepare a dermal regeneration enhancing
composition where aging of the skin, generation of spots, etc. are
effectively able to be suppressed. Examples of the substances
having an enhancing action of differentiation and growth of
keratinocytes are retinal, 3-dehydroretinal, retinoic acid,
3-dehydroretinoic acid, substances similar to retinoic acid,
retinol, retinol fatty acid ester and 3-dehydroretinol fatty acid
ester. Examples of the substances having a suppressive action to
melanin pigment production are ascorbic acid glucoside, arbutin and
superoxide dismutase (SOD). Examples of the substances having an
enhancing action for the synthesis of intercellular lipid of horny
layer are niacinamide, etc. Such a substance itself may be
uniformly dispersed in the lyotropic liquid crystal followed by
being incorporated among the phases of the liquid crystal structure
so that it is compounded, or it may be included in the inside of
fine particles of inorganic acid salt with divalent metal such as
fine particles where diameter is 100 nm to 1,000 nm comprising
calcium carbonate, magnesium carbonate, zinc carbonate, calcium
phosphate, magnesium phosphate and zinc phosphate (with regard to a
method therefor, refer, if necessary, to WO 02/096396) and the fine
particles (nano-particles) into which such a substance is included
are uniformly dispersed in the lyotropic liquid crystal followed by
being incorporated among the phases of the liquid crystal structure
so that they are compounded. In addition, a divalent metal ion and
a counterion thereof are adsorbed on the surface (surface membrane)
of the lyotropic liquid crystal so as to enhance the
viscoelasticity of the membrane, whereby the physical and chemical
stability of the substance incorporated among the phases is able to
be improved. It is further possible to utilize in such a manner
that the transdermal absorption enhancer of the present invention
is previously applied on the skin surface and then the active
ingredient is added thereto whereby transdermal absorption is
conducted.
[0051] The transdermal absorption enhancer of the present invention
may be directly applied to the skin surface as a preparation for
external application or may be applied to the skin surface after
dispersing in an ointment base, a cream base or a lotion base. It
goes without saying that, in making into the preparations, known
components such as antiseptic, moisturizer or antioxidant is
appropriately added thereto.
EXAMPLES
Example 1
Step 1
[0052] Nano-particles comprising the three kinds of formulations as
mentioned in Table 1 in which retinoic acid (an all-trans
substance; hereinafter, it has the same meaning) as an active
ingredient was included were prepared as follows.
[0053] Retinoic acid, ethanol and a 1N aqueous solution of sodium
hydroxide were placed in a beaker so that retinoic acid was
uniformly dissolved. Then, glycerol and Emulgen 2020G-HA
(polyoxyethylene octyl dodecyl ether) which is a trade name of a
nonionic surfactant manufactured by Kao were added thereto followed
by stirring for about 10 minutes. Then distilled water was added
thereto and the mixture was stirred for about 10 minutes to give a
mixed micelle of retinoic acid and the nonionic surfactant. After
that, a 5M aqueous solution of magnesium chloride or a 5M aqueous
solution of calcium chloride was added thereto followed by stirring
for about 1 hour. Finally, a 1M aqueous solution of sodium
carbonate was added thereto and the mixture was stirred for about 1
hour to give nano-particles in which retinoic acid was included in
a thin film of magnesium carbonate or in a thin film of calcium
carbonate where the diameter was 10 nm to 1,000 nm.
TABLE-US-00001 TABLE 1 Formulation 1 Formulation 2 Formulation 3
Retinoic Acid 140 mg 280 mg 560 mg Ethanol 400 .mu.L 800 .mu.L 1600
.mu.L 1N Aqueous Solution 560 .mu.L 1120 .mu.L 2240 .mu.L of Sodium
Hydroxide Glycerol 5 mL 5 mL 5 mL Distilled Water 17.72 mL 16.76 mL
14.28 mL Nonionic Surfactant 2 mL 2 mL 2 mL 5M Aqueous Solution
46.5 .mu.L 93 .mu.L 186 .mu.L of MgCl.sub.2 or CaCl.sub.2 1M
Aqueous Solution 46.5 .mu.L 93 .mu.L 186 .mu.L of Sodium
Carbonate
Step 2
[0054] 31 mL of glycerol was added to a beaker in which 17 mL of
distilled water was placed so that it was uniformly dissolved. Then
28 mL of Emulgen 2020G-HA was added thereto and uniformly dispersed
therein. Since viscosity of the solution increased at that time,
such a phenomenon was used as a yardstick for the uniform
dispersion of each of the materials. After that, 20 mL of squalane
was added to uniformly mix therewith, then 10 mL of squalene was
further added and the mixture was stirred for about 5 minutes. More
5 mL of squalane was added and the mixture was stirred, whereupon
viscosity of the solution gradually rose and it was instantly
gelled. This phenomenon was used as a yardstick for the formation
of the liquid crystal. After that, stirring was still continued for
several minutes to give lyotropic liquid crystal (comprising 28.0%
by weight of surfactant, 16.0% by weight of water, 25.0% by weight
of oil and 31.0% by weight of polyhydric alcohol). The
nano-particles prepared in the step 1 were compounded with the
lyotropic liquid crystal so as to make the compounding amount of
retinoic acid 0.1% by weight (formulation 1), 0.2% by weight
(formulation 2) or 0.4% by weight (formulation 3) to the lyotropic
liquid crystal to give the lyotropic liquid crystal where the
nano-particles in which retinoic acid was included were uniformly
dispersed without degradation. Incidentally, all of the above
operations were carried out under shielding the light and the
nonionic surfactant was used after being melted at about 60.degree.
C. (hereinafter, that is also the same).
[0055] Back of Wistar rats (seven weeks age; male) was shaved, the
shaved part was washed with lukewarm water, each 30 mg of the
lyotropic liquid crystals compounded with nano-particles in which
the retinoic acid was included according to the above formulation 1
(liquid crystal compounded with fine particles of magnesium
carbonate in which retinoic acid was included and liquid crystal
compounded with fine particles of calcium carbonate in which
retinoic acid was included) was applied to an area of 2 cm.times.5
cm thereof and concentration of retinoic acid in blood was
measured. As a comparative example, retinoic acid in the same dose
was hypodermically injected and concentration of retinoic acid in
blood was measured. Further, instead of the lyotropic liquid
crystal compounded with the nano-particles in which retinoic acid
was included, each of vaseline compounded with nano-particles in
which retinoic acid was included (fine particles of calcium
carbonate in which retinoic acid was included) so as to make the
compounding amount of retinoic acid same as that of the liquid
crystal of the formulation 1 and vaseline compounded with retinoic
acid itself so as to make the compounding amount retinoic acid same
as that of the liquid crystal of the formulation 1 was applied and
concentration of retinoic acid in blood was measured. The result is
shown in FIG. 1. Incidentally, (A) to (E) in FIG. 1 are as
follows.
[0056] (A): The result where the lyotropic liquid crystal
compounded with fine particles of magnesium carbonate in which
retinoic acid was included was applied
[0057] (B): The result where the lyotropic liquid crystal
compounded with fine particles of calcium carbonate in which
retinoic acid was included was applied
[0058] (C): The result where retinoic acid was hypodermically
injected
[0059] (D): The result where vaseline compounded with fine
particles of calcium carbonate in which retinoic acid was included
was applied
[0060] (E): The result where vaseline compounded with retinoic acid
itself was applied
[0061] As will be apparent from FIG. 1, concentrations of retinoic
acid in blood when the lyotropic liquid crystal compounded with
fine particles of magnesium carbonate in which retinoic acid was
included was applied and when the lyotropic liquid crystal
compounded with fine particles of calcium carbonate in which
retinoic acid was included was applied were nearly the same as the
concentration of retinoic acid in blood when retinoic acid was
hypodermically injected, whereupon it was found that the lyotropic
liquid crystal had an enhancing action of transdermal
absorption.
Example 2
[0062] Back of ddY mice (seven weeks age; male) was shaved, the
shaved part was washed with lukewarm water and each 30 mg of the
following four kinds of samples was applied to an area of 1.5
cm.times.1.5 cm thereof. Changes in the production amount of HB-EGF
(heparin-binding EGF-like growth factor) playing a role of dermal
regeneration function after 1 day, 2 days and 3 days from the
application date were measured (refer, if necessary, to Non-Patent
Document 2 for the details of the measuring means) whereby each
effects to transdermal absorption of retinoic acid was evaluated.
The result is shown in FIG. 2. As will be apparent from FIG. 2,
when fine particles of magnesium carbonate in which retinoic acid
was included was compounded with the lyotropic liquid crystal and
when fine particles of calcium carbonate in which retinoic acid was
included were compounded with the lyotropic liquid crystal, the
production amounts of HB-EGF after 2 days and 3 days from the
application date were much more as compared with the case where
they were compounded with vaseline, whereby it was found that
transdermal absorption of retinoic acid was improved by the
enhancing action of transdermal absorption of the lyotropic liquid
crystal.
[0063] (a) Lyotropic liquid crystal compounded with fine particles
of magnesium carbonate in which retinoic acid was included
according to the formulation 1 of Example 1 (Mg-Atra/Liquid
Crystal)
[0064] (b) Lyotropic liquid crystal compounded with fine particles
of calcium carbonate in which retinoic acid was included according
to the formulation 1 of Example 1 (Ca-atRA/liquid crystal)
[0065] (c) Vaseline Compounded with Fine Particles of Magnesium
carbonate in which retinoic acid was included so as to make the
compounding amount of retinoic acid same as that of the liquid
crystal of the formulation 1 of Example 1 (Mg-Atra/Vaseline)
[0066] (d) Vaseline compounded with fine particles of calcium
carbonate in which retinoic acid was included so as to make the
compounding amount of retinoic acid same as that of the liquid
crystal of the formulation 1 of Example 1 (Ca-atRA/vaseline)
Example 3
[0067] In accordance with the same manner as in Example 2, changes
in the production amounts of HB-EGF by application of the following
four kinds of samples were measured whereby each effects to
transdermal absorption of retinoic acid was evaluated. The result
is shown in FIG. 3 together with the production amount of HB-EGF of
the skin to which nothing was applied. As will be apparent from
FIG. 3, when retinoic acid was compounded with the lyotropic liquid
crystal, transdermal absorption of retinoic acid was improved and
production amount of HB-EGF increased independently of its
compounding form. The lyotropic liquid crystal itself also showed
an increasing action of HB-EGF production and the lyotropic liquid
crystal was found to be able to be an effective ingredient of a
dermal regeneration enhancer.
[0068] (a) Lyotropic liquid crystal compounded with fine particles
of magnesium carbonate in which retinoic acid was included
according to the formulation 1 of Example 1 (Mg-Atra/Liquid
Crystal)
[0069] (b) Lyotropic liquid crystal compounded with the mixed
micelle of retinoic acid and nonionic surfactant obtained in the
preparation of fine particles of magnesium carbonate in which
retinoic acid was included in the step 1 of Example 1 so as to make
the compounding amount of retinoic acid same as that of the liquid
crystal of the formulation 1 of Example 1 (atRA micelle/liquid
crystal)
[0070] (c) Lyotropic liquid crystal compounded with retinoic acid
itself so as to make the compounding amount of retinoic acid same
as that of the liquid crystal of the formulation 1 of Example 1
(atRA/liquid crystal)
[0071] (d) Lyotropic liquid crystal prepared in the step 2 of
Example 1 (liquid crystal only)
Example 4
[0072] Back of colored guinea pigs having melanin pigment-producing
cells (Weiser Maples; five weeks age; male) was shaved, the shaved
part was washed with lukewarm water and each 30 mg of the following
five kinds of samples was applied to an area of 2 cm.times.5 cm
thereof. After 3 days from the application date under irradiation
with any of UVA, UVB and UVA+UVB, skin of the part to which the
sample was applied was collected and the slice was fixed with
formalin, embedded in paraffin and stained by a Fontana-Masson
method where melanin pigment was stained out in black to evaluate
the dermal regeneration enhancing action. Cross-sectional pictures
of the skin to which each of the samples was applied are shown in
FIG. 4 together with the cross-sectional picture of the skin to
which nothing was applied. As will be apparent from FIG. 4,
thickening of the epidermis was noted in the case where the
lyotropic liquid crystal compounded with retinoic acid in various
forms was applied and in the case where the lyotropic liquid
crystal itself was applied.
[0073] (a) Lyotropic liquid crystal compounded with fine particles
of magnesium carbonate in which retinoic acid was included
according to the formulation 1 of Example 1 (Mg-Atra/Liquid
Crystal)
[0074] (b) Lyotropic liquid crystal compounded with the mixed
micelle of retinoic acid and nonionic surfactant obtained in the
preparation of fine particles of magnesium carbonate in which
retinoic acid was included in the step 1 of Example 1 so as to make
the compounding amount of retinoic acid same as that of the liquid
crystal of the formulation 1 of Example 1 (atRA micelle/liquid
crystal)
[0075] (c) Lyotropic liquid crystal compounded with retinoic acid
itself so as to make the compounding amount of retinoic acid same
as that of the liquid crystal of the formulation 1 of Example 1
(atRA/liquid crystal)
[0076] (d) Lyotropic liquid crystal prepared in the step 2 of
Example 1 (liquid crystal only)
[0077] (e) Vaseline compounded with fine particles of magnesium
carbonate in which retinoic acid was included so as to make the
compounding amount of retinoic acid same as that of the liquid
crystal of the formulation 1 of Example 1 (Mg-Atra/Vaseline)
Example 5
[0078] Lyotropic liquid crystal compounded with 0.5%, 1% and 3% (by
weight) of insulin (the lyotropic liquid crystal itself was a
product prepared by the step 2 of Example 1) was prepared and
concentration of insulin in blood was measured by the same manner
as in Example 1. As a comparative example, 0.2 mg/200 .mu.L of
insulin was hypodermically injected and concentration of insulin in
blood was measured. The result is shown in FIG. 5. Incidentally,
(A) to (D) in FIG. 5 are as follows.
[0079] (A): The result when the lyotropic liquid crystal compounded
with 0.5% (by weight) of insulin was applied
[0080] (B): The result when the lyotropic liquid crystal compounded
with 1% (by weight) of insulin was applied
[0081] (C): The result when the lyotropic liquid crystal compounded
with 3% (by weight) of insulin was applied
[0082] (D): The result when 0.2 mg/200 .mu.L of insulin was
hypodermically injected
[0083] As will be apparent from FIG. 5, although the initial
concentration of insulin in blood when the lyotropic liquid crystal
compounded with insulin was applied was less than the initial
concentration of insulin in blood when insulin was hypodermically
injected, the concentration of insulin in blood when insulin was
hypodermically injected thereafter quickly decreased while, in case
the lyotropic liquid crystal compounded with insulin was applied,
the concentration of insulin in blood was maintained in a
relatively high level. From the result as such, it was found that,
when insulin was compounded with the lyotropic liquid crystal,
insulin was now able to be transdermically absorbed in a
sustained-release manner.
Example 6
[0084] Back of Wistar rats (seven weeks age; male) was shaved, the
shaved part was washed with lukewarm water and each 30 mg of the
two kinds of samples which were (A) a lyotropic liquid crystal
compounded with 2% (by weight) of .sup.14C niacinamide (the
lyotropic liquid crystal itself was a product prepared by the step
2 of Example 1) and (B) an aqueous gel (carboxyvinyl polymer; trade
name: "Carbopol", Nikko Chemicals) compounded with 2% (by weight)
of .sup.14C niacinamide was applied to an area of 2 cm.times.2 cm
thereof. During the period of after 15 minutes to 6 hours from the
application, blood was collected and radiation activity of .sup.14C
contained in 200 .mu.L of blood was measured whereby the rate (%)
of niacinamide in total blood to the administered dose was
calculated. The result is shown in FIG. 6. As will be apparent from
FIG. 6, it was found that, when niacinamide was compounded with the
lyotropic liquid crystal, niacinamide was apt to be included into
blood and its concentration in blood was maintained in a relatively
highly level for a long period. The above result is believed to be
due to the fact that, although niacinamide is a substance which is
usually very difficult to be absorbed transdermically since it is a
water-soluble substance, it is permeated from the skin surface due
to a enhancing action of the lyotropic liquid crystal for
transdermal absorption whereby it is included into blood from
capillary blood vessels in dermis.
Example 7
[0085] Changes in the residual rate of retinoic acid with the
passage of time when the following five kinds of samples were
stored at 40.degree. C. were checked. The result is shown in FIG.
7. As will be apparent from FIG. 7, when fine particles of
inorganic acid salt with divalent metal in which retinoic acid was
included were compounded with the lyotropic liquid crystal, it was
found that retinoic acid remained stably even at the stage when 60
days elapsed from the preparation.
[0086] (A) Lyotropic liquid crystal compounded with fine particles
of zinc carbonate in which retinoic acid was included so as to make
the compounding amount of retinoic acid to the lyotropic liquid
crystal 0.1% by weight
[0087] (B) Lyotropic liquid crystal compounded with fine particles
of calcium carbonate in which retinoic acid was included in the
same manner
[0088] (C) Lyotropic liquid crystal compounded with fine particles
of calcium phosphate in which retinoic acid was included in the
same manner
[0089] (D) Lyotropic liquid crystal compounded with fine particles
of magnesium carbonate in which retinoic acid was included in the
same manner
[0090] (E) Vaseline where retinoic acid itself was compounded
therein so as to make the compounding amount of retinoic acid to
the lyotropic liquid crystal 0.1% by weight
[0091] Note: The sample (B) corresponds to the lyotropic liquid
crystal compounded with fine particles of calcium carbonate in
which retinoic acid was included according to the formulation 1 of
Example 1 while the sample (D) corresponds to the lyotropic liquid
crystal compounded with fine particles of magnesium carbonate in
which retinoic acid was included according to the formulation 1 of
Example 1. The samples (A) and (C) were prepared in accordance with
the method for the preparation of the samples (B) and (D).
Example 8
[0092] Changes in the residual rate of retinol palmitate with the
passage of time when the following four kinds of samples were
stored at 40.degree. C. were checked. The result is shown in FIG.
8. As will be apparent from FIG. 8, when retinol palmitate was
compounded with the lyotropic liquid crystal, it was found that
retinol palmitate remained stably even at the stage when 30 days
elapsed from the preparation.
[0093] (A) Lyotropic liquid crystal compounded with 0.1% (by
weight) of retinol palmitate (the lyotropic liquid crystal itself
was a product prepared by the step 2 of Example 1)
[0094] (B) 0.1% (w/w) ethanol solution of retinol palmitate
[0095] (C) Ethanol solution where an antioxidant (BHT) was added to
the ethanol solution (B)
[0096] (D) Nano-particles in which retinol palmitate was included,
prepared in accordance with the method for the preparation of the
nano-particles in which retinoic acid was included in the step 1 of
Example 1 (containing 0.1% by weight of retinol palmitate)
Example 9
[0097] Back of colored guinea pigs having melanin pigment-producing
cells (Weiser Maples; five weeks age; male) was shaved, the shaved
part was washed with lukewarm water, 30 mg of the lyotropic liquid
crystal compounded with 0.1% (by weight) of SOD having a
suppressive action to melanin pigment production (molecular weight:
32,000) was applied to an area of 1.5 cm.times.1.5 cm thereof and
the influence on the epidermis was checked by the method mentioned
in Example 4. A cross-sectional picture of the skin (stained by a
Fontana-Masson method) is shown in FIG. 9. Further, a
cross-sectional picture of the skin (stained by a Fontana-Masson
method) when 30 .mu.L of a dispersion solution where 0.1% (by
weight) of SOD was dispersed in water was applied is shown in FIG.
10 and a cross-sectional picture of the skin (stained by a
Fontana-Masson method) when 30 .mu.L of water only was applied is
shown in FIG. 11. As will be apparent from FIG. 9 to FIG. 11, when
the lyotropic liquid crystal compounded with SOD was applied,
amount of melanin pigment in the epidermis significantly decreased
(being judged from the fact that black spots and areas were little)
as compared with the case where a dispersion solution where SOD was
dispersed in water was applied or the case where only water was
applied. That was believed to be due to the fact that SOD
penetrated through horny layer and reached into the epidermis.
Incidentally, thickening of the epidermis noted upon application of
the lyotropic liquid crystal compounded with SOD was believed to be
due to the dermal regeneration enhancing action of the lyotropic
liquid crystal itself.
Example 10
[0098] According to the same manner as in Example 9, the influence
of the following six kinds of samples on the epidermis was checked.
Cross-sectional pictures of the skin (stained by a Fontana-Masson
method) are shown in FIG. 12. As will be apparent from FIG. 12,
amount of melanin pigment in the epidermis significantly decreased
when arbutin having a suppressive action to melanin pigment
production was compounded with the lyotropic liquid crystal and
applied. Since thickening of the epidermis was also noted even when
the lyotropic liquid crystal only was applied, it was confirmed
that the lyotropic liquid crystal itself had a dermal regeneration
enhancing action.
[0099] (a) Lyotropic liquid crystal compounded with 2% (by weight)
of .alpha.-arbutin (molecular weight: 272; Ezaki Clico Co.,
Ltd.)
[0100] (b) Lyotropic liquid crystal compounded with 2% (by weight)
of .beta.-arbutin (molecular weight: 272; Hayashibara Co.,
Ltd.)
[0101] (c) Lyotropic liquid crystal only
[0102] (d) Dispersion solution where 2% (by weight) of
.alpha.-arbutin was dispersed in water
[0103] (e) Dispersion solution where 2% (by weight) of
.beta.-arbutin was dispersed in water
[0104] (f) Water only
Example 11
[0105] Back of ddY mice (seven weeks age; male) was shaved, the
shaved part was washed with lukewarm water and 30 mg of the
lyotropic liquid crystal compounded with 2% (by weight) of
oligo-DNA (molecular weight: 8899) fluorescently labeled with
fluorescein isothiocyanate (FITC) was applied to an area of 1.5
cm.times.1.5 cm thereof. After two hours from the application, skin
of the part to which the sample was applied was collected, made
into a frozen slice and subjected to a fluorescent observation. A
fluorescent cross-sectional picture of the skin is shown in FIG.
13. Another fluorescent cross-sectional picture of the skin to
which 30 .mu.L of a dispersion solution where 2% (by weight) of
oligo-DNA fluorescently labeled with FITC was dispersed in water
was applied is shown in FIG. 14. As will be apparent from FIG. 13
and FIG. 14, the oligo-DNA was permeated into the epidermis after
two hours from the application when it was compounded with the
lyotropic liquid crystal and applied.
Example 12
[0106] According to the same manner as in Example 11,
penetratability into the skin was checked when the lyotropic liquid
crystal compounded with 5% (by weight) of dextran (molecular weight
4,000) fluorescently labeled with FITC was applied. A fluorescent
cross-sectional picture of the skin is shown in FIG. 15. As will be
apparent from FIG. 15, dextran was permeated into the epidermis
within 15 minutes and, with the passage of time, it was further
permeated thereinto when it was compounded with the lyotropic
liquid crystal and applied.
Example 13
[0107] According to the same manner as in Example 11,
penetratability into the skin was checked when the lyotropic liquid
crystal compounded with 5% (by weight) of dextran (molecular
weight: 70,000) fluorescently labeled with FITC was applied. Also,
penetratability into the skin was checked when 30 .mu.L of a
dispersion solution where 5% (by weight) of dextran (the same one
as above) fluorescently labeled with FITC was dispersed in water
was applied. Fluorescent cross-sectional picture of the skin for
each of the above cases is shown in FIG. 16. As will be apparent
from FIG. 16, dextran was permeated into the epidermis within 15
minutes and, with the passage of time, it was further permeated
thereinto when it was compounded with the lyotropic liquid crystal
and applied.
Example 14
[0108] A lyotropic liquid crystal comprising 16.819% by weight of
squalane, 8.931% by weight of soybean lecithin, 4.466% by weight of
cholesterol, 15.026% by weight of POE (60) hydrogenated castor oil,
38.897% by weight of glycerol and 15.860% by weight of distilled
water was prepared. When the lyotropic liquid crystal was
previously applied on the skin surface and then retinoic acid was
added thereto later, transdermal absorption of retinoic acid was
able to be improved.
Preparation Example 1
[0109] A commercially available antiseptic was added to the
lyotropic liquid crystal of Example 14 to prepare a product.
Preparation Example 2
[0110] The lyotropic liquid crystal of Example 14 was compounded
with a home-made lotion base (milky liquid) and then a commercially
available antiseptic was added thereto to prepare a lotion. The
lotion base was prepared by mixing of soybean lecithin,
cholesterol, PEG 4000, cyclic silicone, Carbopol (macromolecular
gelling agent), Keltrol (macromolecular gelling agent) and
distilled water followed by emulsifying.
INDUSTRIAL APPLICABILITY
[0111] The present invention has an industrial applicability in
such a respect that there is provided a transdermal absorption
enhancer as a novel pharmaceutical use of lyotropic liquid crystal
which has been utilized as a basic material for pharmaceutical
preparations for external application and for cosmetics.
* * * * *