THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)
“α-LIPOIC ACID NANOPARTICLE AND METHOD FOR
PRODUCING THE SAME”
(1) EZAKI GLICO CO., LTD. of 6-5, Utajima 4-chome,
Nishiyodogawa-ku, Osaka-shi Osaka, 5558502, Japan
(2) NANOEGG RESEARCH LABORATORIES, INC. of 16-
1, Sugao 2-chome, Miyamae-ku, Kawasaki-shi, Kanagawa,
2168512, Japan
The following specification particularly describes the invention and the manner
in which it is to be performed.
- 1 - EG026
DESCRIPTION
α-LIPOIC ACID NANOPARTICLES AND METHODS FOR PREPARING THEREOF
5 TECHNICAL FIELD
[0001] The present invention relates to nanoparticles
comprising α-lipoic acid, and a method for producing thereof.
10 BACKGROUND ART
[0002] α-Lipoic acid is one kind of coenzyme which is
contained in the living body and acts on the glycolytic
metabolism and the cycling through the TCA cycle, and is
15 a substance in the form of yellow crystals having the
structural formula C8H14O2S2, the molecular weight of 206.3,
and the melting point of 60 to 62°C. α-Lipoic acid is also
present in the human body, and is contained in many foods
such as broccoli and red meat. Therefore, α-lipoic acid can
20 be said to be a highly safe substance. In terms of function,
α-lipoic acid is recognized as having strong antioxidant
capacity in the living body thereby reducing oxidative stress,
and as a chelating agent that is effective in the discharge
of heavy metals. α-Lipoic acid is currently formulated into
25 pharmaceutical products as “thioctic acid,” and thioctic
acid preparations are usually sold as injections. As the
efficacy and effect of the thioctic acid preparations,
supplementation upon an increase in the demand of thioctic
acid (at the time of vigorous physical labor), Leigh syndrome
30 (subacute necrotic encephalomyelitis), and toxic (due to
streptomycin or kanamycin) and noise-induced (occupational)
inner ear hearing impairment are described in Drugs in Japan,
Ethical Drugs (Non-Patent Document 1).
- 2 - EG026
[0003] α-Lipoic acid had been approved for use in foods
and cosmetics as a result of recent relaxation of regulations,
and therefore, further applications thereof in these fields
5 are expected.
[0004] α-Lipoic acid is in the form of a yellow powder,
but since it is hardly soluble in water, its uses are limited.
Furthermore, α-lipoic acid is very unstable to heat and light,
10 and is difficult to be present stably in the preparation.
Furthermore, it is a problem of α-lipoic acid that it has
a characteristic sulfurous odor, and the odor becomes
stronger when α-lipoic acid is degenerated and that it becomes
gummy by heat. Thus, when α-lipoic acid is used in foods,
15 cosmetics and pharmaceutical products, there is a serious
problem in terms of the product quality and after-use feel.
[0005] In order to solve such problems as described above,
Patent Document 1 suggests a water-soluble preparation
20 containing α-lipoic acid or a pharmacologically acceptable
salt thereof, and a sulfite or a hydrate thereof.
[0006] Patent Document 2 suggests a method for preparing
a water-soluble preparation by dissolving α-lipoic acid in
25 an organic solvent such as ethanol, subsequently adding an
emulsifying agent and a polyhydric alcohol thereto, and
bringing α-lipoic acid to an emulsified state by the physical
action of an emulsifying machine or the like.
30 [0007] Furthermore, Patent Document 3 and Patent Document
4 suggest methods for preparing a water-soluble α-lipoic
acid preparation, which methods enhance dispersibility in
water and emulsification stability by mixing α-lipoic acid
- 3 - EG026
with an organic solvent such as ethanol, an emulsifying agent
or a polyhydric alcohol.
[0008] However, the methods of bringing an emulsified state
5 as described in these patent documents require a special
apparatus called emulsifying machine, and when the particle
size of the emulsification liquid is large, and when the
distribution of the size of particle diameter is non-uniform,
the emulsified state becomes easily separable. Furthermore,
10 in other methods, it has been pointed out that since the
dispersion of α-lipoic acid by emulsification is incomplete,
the sulfurous odor characteristic to α-lipoic acid is
strongly generated during storage.
15 [0009] Meanwhile, there are documents related to
nanoparticle formation from retinoic acid. Patent
Documents 5 to 8 disclose polyvalent metal inorganic
salt-coated retinoic acid nanoparticles.
20 [0010] However, since retinoic acid is completely different
from α-lipoic acid in structure, it would not be easily
conceivable and had never been believed that α-lipoic acid
is used instead of the retinoic acid of Patent Documents
5 to 8.
25
[0011] More detailed description will be given in this regard.
First, as can be seen from the structures shown below, α-lipoic
acid and retinoic acid have completely different structures
except that they both contain one carboxyl group.
30 Furthermore, α-lipoic acid is also completely different from
retinoic acid in that α-lipoic acid contains sulfur atoms
in the molecule and does not have any double bond. From the
points as discussed above, it would not be easily conceivable
- 4 - EG026
to use α-lipoic acid instead of retinoic acid in the methods
described in Patent Documents 5 to 8.
[0012] [Chem. 1] α-Lipoic acid
5 Retinoic acid
Secondly, retinoic acid is said to be an important in
vivo hormone, which is, in the living body, involved with
the growth and differentiation of cells, maintenance of
10 homeostasis of the living body, morphogenesis, and the
expression control of various genes by means of the binding
to intranuclear retinoic acid receptors is proposed as a
mechanism of action. On the other hand, as a coenzyme of
the glycolytic system, α-lipoic acid catalyzes the oxidative
15 decarbonation reaction from pyruvic acid to acetyl CoA, and
thus is said to be an indispensable nutrient for cellular
respiration and energy production. Furthermore, as a
well-known function of α-lipoic acid, an antioxidant action
is known. From the points as discussed above, retinoic acid
20 and α-lipoic acid are completely different in terms of the
functions in the living body and the expected pharmacological
effects, and therefore, even from the viewpoint of the
effectiveness demanded when its industrial application is
believed, it would not be easily conceivable to use α-lipoic
25 acid as a substitute for retinoic acid in the methods described
in the Patent Documents 5 to 8.
[0013] Thirdly, it is reported in p. 410 of Non-Patent
Document 2 that the pKa value of retinoic acid is 6.4, and
30 it is described in the Discussion section on p. 411 that
the pKa increases to 7 to 8 as retinoic acid forms micelles.
On the other hand, Non-Patent Document 3 describes that the
pKa value of α-lipoic acid is 4.76. From the points as
- 5 - EG026
described above, α-lipoic acid and retinoic acid are
completely different in the properties related to
dissociation. Therefore, it would not be easily conceivable
to use α-lipoic acid as a substitute for retinoic acid in
5 the methods described in Patent Documents 5 to 8.
Patent Document 1: Japanese Laid-open Patent Publication
No. 2005-2096
Patent Document 2: Japanese Laid-open Patent Publication
No. 2006-129841
10 Patent Document 3: Japanese Laid-open Patent Publication
No. 2006-257010
Patent Document 4: Japanese Laid-open Patent Publication
No. 2007-16000
Patent Document 5: Japanese Laid-open Patent Publication
15 No. 2004-161739
Patent Document 6: International Publication No.
2005/037267 pamphlet
Patent Document 7: International Publication No.
2005/037268 pamphlet
20 Patent Document 8: International Publication No.
2005/070413 pamphlet
Non-Patent Document 1: Drugs in Japan, Ethical Drugs,
Edition of 2007, Jiho, Inc., p. 1327 (2006)
Non-Patent Document 2: Robbert Creton, et al., Int. J.
25 Dev. Biol., 39:409-414 (1995)
Non-Patent Document 3: Lester J. Reed, et al., JOURNAL
OF THE AMERICAN CHEMICAL SOCIETY, Vol. 75:1267 (1953)
DISCLOSURE OF THE INVENTION
30
PROBLEMS TO BE SOLVED BY THE INVENTION
[0014] The present invention is intended to solve the
- 6 - EG026
problems described above, and it is an object of the invention
to provide stable α-lipoic acid.
MEANS TO SOLVE THE PROBLEMS
5
[0015] The inventors of the present invention conducted
intensive studies in order to solve the problems as described
above, and as a result, they found that when a nonionic
surfactant, a divalent metal ion, and a carbonate ion or
10 a phosphate ion are used in a specific order, stable α-lipoic
acid nanoparticles can be obtained. Thus, the inventors
completed the present invention based on this finding.
[0016] The present invention utilizes the amphiphilicity
15 of α-lipoic acid. α-Lipoic acid is hardly soluble in water
under acidic conditions or neutral conditions, but if an
alkali is added, the mixture becomes a transparent liquid.
The α-lipoic acid in an alkali solution is believed to form
spherical micelles in water. It is believed that if a
20 nonionic surfactant is subsequently added to the α-lipoic
acid, mixed micelles of α-lipoic acid and the nonionic
surfactant are formed. Furthermore, it is believed that when
divalent metal cations are allowed to bind to the negative
charges of the lipoic acid ions by further adding a halide,
25 acetate or gluconate of the divalent metal to the mixed
micelles, and thereby preventing aggregation and
precipitation of α-lipoic acid, spherical- or oval-shaped
micelles in which the divalent metal ions are bound to the
surface of the lipoic acid, are formed. Furthermore,
30 divalent anions are added thereto, and the divalent anions
are allowed to adsorb (bind) to the metal ions at the micelle
surface, thereby neutralizing the charge at the micelle
surface. As a result, it is believed that a coating of a
- 7 - EG026
polyvalent metal inorganic salt is formed at the micelle
surface, and thus, α-lipoic acid nanoparticles coated with
the polyvalent metal inorganic salt are prepared. Since this
production method of nanoparticles uses α-lipoic acid
5 micelles as a template, the encapsulation ratio corresponds
to the concentration excluding monodisperse α-lipoic acid
molecules, and thus, is thought to be close to 100%. It is
thought that the hydrophilic group of the nonionic surfactant
is exposed at the surface of the subject nanoparticles. The
10 nanoparticles of the present invention are dispersed
transparently in water. Also, although the crystals of the
polyvalent metal inorganic salt such as CaCO3 do not dissolve
in water, the crystals are believed to adopt a vaterite or
amorphous structure at the nanoparticle surface, which
15 dissolves slowly in the living body, and thus a DDS effect
of sustained release of α-lipoic acid is expected.
[0017] The inventors of the present invention also found
that α-lipoic acid is solubilized in a certain type of nonionic
20 surfactant, and that by dispersing this solubilized product
in water, mixed micelles of α-lipoic acid and the nonionic
surfactant are formed. By adding a halide, acetate or
gluconate of a divalent metal to these mixed micelles of
the α-lipoic acid-dissolved nonionic surfactant, a divalent
25 metal cation is allowed to bind to the negative charge of
the α-lipoic acid ion. It is believed that, during this,
the presence of the surfactant prevents aggregation and
precipitation of α-lipoic acid, and that thereby, sphericalor
oval-shaped micelles having the divalent metal ion bound
30 to the surface of the lipoic acid are formed. A divalent
anion (an alkali metal carbonate or alkali metal phosphate)
is further added to these micelles, and the divalent anion
is allow to adsorb (bind) to the metal ion at the micelle
- 8 - EG026
surface to thereby neutralize the charge of the micelle
surface. Thus, it is thought that as a result, a coating
of a polyvalent metal inorganic salt is formed at the micelle
surface, and polyvalent metal inorganic salt-coated α-lipoic
5 acid nanoparticles are prepared.
[0018] In order to achieve the objects described above, the
present invention provides, for example, the following means:
10 (Item 1) A method for producing α-lipoic acid nanoparticles,
the method comprising the steps of:
preparing an aqueous dispersion liquid containing
α-lipoic acid and a nonionic surfactant;
adding a divalent metal salt into the aqueous dispersion
15 liquid, wherein the divalent metal salt is a divalent metal
halide, a divalent metal acetate or a divalent metal
gluconate; and
adding an alkali metal carbonate or an alkali metal
phosphate into the aqueous dispersion liquid wherein the
20 divalent metal salt has been added, thereby forming α-lipoic
acid nanoparticles.
[0019] (Item 2) The method according to Item 1, wherein the
step of preparing an aqueous dispersion liquid containing
25 α-lipoic acid and a nonionic surfactant, comprises:
dissolving α-lipoic acid in the nonionic surfactant which
is in a liquid form, to obtain a surfactant solution; and
adding water or a liquid containing water to the surfactant
solution to obtain the aqueous dispersion liquid.
30
[0020] (Item 3) The method according to Item 1, wherein the
step of preparing an aqueous dispersion liquid containing
α-lipoic acid and a nonionic surfactant, comprises: producing
- 9 - EG026
a mixture of α-lipoic acid, an alkaline substance and water
to prepare an α-lipoic acid-containing aqueous dispersion
liquid; and adding the nonionic surfactant into the α-lipoic
acid-containing aqueous dispersion liquid.
5
[0021] (Item 4) The method according to any one of Items
1 to 3, wherein the divalent metal salt is selected from
the group consisting of calcium chloride, calcium bromide,
calcium fluoride, calcium iodide, magnesium chloride,
10 magnesium bromide, magnesium fluoride, magnesium iodide,
zinc chloride, zinc bromide, zinc fluoride, zinc iodide,
calcium acetate, magnesium acetate, zinc acetate, calcium
gluconate, magnesium gluconate and zinc gluconate.
15 [0022] (Item 5) The method according to any one of Items
1 to 4, wherein the divalent metal salt is selected from
the group consisting of calcium chloride, magnesium chloride
and zinc gluconate.
20 [0023] (Item 6) The method according to any one of Items
1 to 5, wherein the alkali metal carbonate or alkali metal
phosphate is selected from the group consisting of sodium
carbonate, potassium carbonate, sodium hydrogen carbonate,
potassium hydrogen carbonate, sodium phosphate and potassium
25 phosphate.
[0024] (Item 7) The method according to any one of Items
1 to 6, wherein the alkali metal carbonate or alkali metal
phosphate is selected from the group consisting of sodium
30 carbonate and disodium hydrogen phosphate.
[0025] (Item 8) The method according to any one of Items
1 to 7, wherein the nonionic surfactant is selected from
- 10 - EG026
the group consisting of polyoxyethylene hydrogenated castor
oils, polyoxyethylene alkyl ethers, polyoxyethylene
sorbitan fatty acid esters, polyoxyethylene
polyoxypropylene alkyl ethers, sucrose fatty acid esters
5 and polyglycerin fatty acid esters.
[0026] (Item 9) The method according to Item 8, wherein the
HLB value of the nonionic surfactant is 10 or more.
10 [0027] (Item 10) The method according to any one of Items
1 to 9, wherein the nonionic surfactant is selected from
the group consisting of polyoxyethylene (degree of
polymerization 10 to 20) octyl dodecyl ether, polyoxyethylene
(degree of polymerization 10 to 20) stearyl ether,
15 polyoxyethylene (degree of polymerization 10 to 20)
polyoxypropylene (degree of polymerization 4 to 8) cetyl
ether, polyoxyethylene (degree of polymerization 20 to 100)
hydrogenated castor oil, and sucrose lauric acid ester.
20 [0028] (Item 11) The method according to any one of Items
2 and 4-10, wherein in the step of preparing an aqueous
dispersion liquid containing α-lipoic acid and a nonionic
surfactant,
polyethylene glycol is mixed into the nonionic surfactant,
25 prior to the dissolving of α-lipoic acid in the nonionic
surfactant; or
water containing polyethylene glycol is used as the
liquid containing water, in the step of adding a liquid
containing water to the surfactant solution.
30
[0029] (Item 12) α-Lipoic acid nanoparticles comprising
α-lipoic acid, a nonionic surfactant, a divalent metal ion,
and a carbonate ion or a phosphate ion.
- 11 - EG026
[0030] (Item 13) The α-lipoic acid nanoparticles according
to Item 12, wherein the divalent metal ion is a calcium ion,
a zinc ion or a magnesium ion.
5
[0031] (Item 14) The α-lipoic acid nanoparticles according
to Item 12 or 13, wherein the nonionic surfactant is selected
from the group consisting of polyoxyethylene hydrogenated
castor oils, polyoxyethylene alkyl ethers, polyoxyethylene
10 sorbitan fatty acid esters, polyoxyethylene
polyoxypropylene alkyl ethers, sucrose fatty acid esters
and polyglycerin fatty acid esters.
[0032] (Item 15) The α-lipoic acid nanoparticles according
15 to any one of Items 12 to 14, further comprising polyethylene
glycol.
[0033] (Item 16) An external preparation for skin,
comprising the α-lipoic acid nanoparticles according to any
20 one of Items 12 to 15.
[0034] (Item 17) A pharmaceutical product comprising the
α-lipoic acid nanoparticles according to any one of Items
12 to 15.
25
[0035] (Item 18) A composition for oral cavity, comprising
the α-lipoic acid nanoparticles according to any one of Items
12 to 15.
30 [0036] (Item 19) A food comprising the α-lipoic acid
nanoparticles according to any one of Items 12 to 15.
[0037] The present invention also provides, for example,
- 12 - EG026
the following means:
(Item A1) A method for producing α-lipoic acid nanoparticles,
the method comprising the steps of:
5 producing a mixture of α-lipoic acid, an alkaline
substance and water to prepare an α-lipoic acid-containing
aqueous dispersion liquid;
adding the nonionic surfactant into the aqueous
dispersion liquid;
10 adding a divalent metal salt into the aqueous dispersion
liquid wherein the nonionic surfactant has been added,
wherein the divalent metal salt is a divalent metal halide,
a divalent metal acetate or a divalent metal gluconate; and
adding an alkali metal carbonate or an alkali metal
15 phosphate into the aqueous dispersion liquid wherein the
divalent metal salt has been added, thereby forming α-lipoic
acid nanoparticles.
[0038] (Item A2) The method according to Item A1, wherein
20 the divalent metal salt is selected from the group consisting
of calcium chloride, calcium bromide, calcium fluoride,
calcium iodide, magnesium chloride, magnesium bromide,
magnesium fluoride, magnesium iodide, zinc chloride, zinc
bromide, zinc fluoride, zinc iodide, calcium acetate,
25 magnesium acetate, zinc acetate, calcium gluconate,
magnesium gluconate and zinc gluconate.
[0039] (Item A3) The method according to Item A1, wherein
the divalent metal salt is selected from the group consisting
30 of calcium chloride, magnesium chloride and zinc gluconate.
[0040] (Item A4) The method according to Item A1, wherein
the alkali metal carbonate or alkali metal phosphate is
- 13 - EG026
selected from the group consisting of sodium carbonate,
potassium carbonate, sodium hydrogen carbonate, potassium
hydrogen carbonate, sodium phosphate and potassium
phosphate.
5
[0041] (Item A5) The method according to Item A1, wherein
the alkali metal carbonate or alkali metal phosphate is
selected from the group consisting of sodium carbonate and
disodium hydrogen phosphate.
10
[0042] (Item A6) The method according to Item A1, wherein
the nonionic surfactant is selected from the group consisting
of polyoxyethylene hydrogenated castor oils,
polyoxyethylene alkyl ethers, polyoxyethylene sorbitan
15 fatty acid esters, polyoxyethylene polyoxypropylene alkyl
ethers, sucrose fatty acid esters and polyglycerin fatty
acid esters.
[0043] (Item A7) The method according to Item A6, wherein
20 the HLB value of the nonionic surfactant is 10 or more.
[0044] (Item A8) The method according to Item A1, wherein
the nonionic surfactant is selected from the group consisting
of polyoxyethylene (degree of polymerization 10 to 20) octyl
25 dodecyl ether, polyoxyethylene (degree of polymerization
10 to 20) stearyl ether, polyoxyethylene (degree of
polymerization 10 to 20) polyoxypropylene (degree of
polymerization 4 to 8) cetyl ether, polyoxyethylene (degree
of polymerization 20 to 100) hydrogenated castor oil, and
30 sucrose lauric acid ester.
[0045] (Item A9) α-Lipoic acid nanoparticles comprising
α-lipoic acid, a nonionic surfactant, a divalent metal ion,
- 14 - EG026
and a carbonate ion or a phosphate ion.
[0046] (Item A10) The α-lipoic acid nanoparticles according
to Item A9, wherein the divalent metal ion is a calcium ion,
5 a zinc ion or a magnesium ion.
[0047] (Item A11) The α-lipoic acid nanoparticles according
to Item A9, wherein the nonionic surfactant is selected from
the group consisting of polyoxyethylene hydrogenated castor
10 oils, polyoxyethylene alkyl ethers, polyoxyethylene
sorbitan fatty acid esters, polyoxyethylene
polyoxypropylene alkyl ethers, sucrose fatty acid esters
and polyglycerin fatty acid esters.
15 [0048] (Item A12) An external preparation for skin,
comprising the α-lipoic acid nanoparticles according to Item
A9.
[0049] (Item A13) A pharmaceutical product comprising the
20 α-lipoic acid nanoparticles according to Item A9.
[0050] (Item A14) A composition for oral cavity, comprising
the α-lipoic acid nanoparticles according to Item A9.
25 [0051] (Item A15) A food comprising the α-lipoic acid
nanoparticles according to Item A9.
EFFECT OF THE INVENTION
30 [0052] The method for preparing nanoparticles of the present
invention uses α-lipoic acid micelles as templates, and thus
the encapsulation ratio corresponds to the concentration
excluding monodisperse α-lipoic acid molecules, thereby
- 15 - EG026
being close to 100%. It is believed that at the surface of
the nanoparticles of the present invention, the hydrophilic
group of the nonionic surfactant is exposed, and thus the
nanoparticles are dispersed transparently in water.
5 Furthermore, at the surface of the nanoparticles, a
polyvalent metal inorganic salt is believed to adopt a
vaterite or amorphous structure, which dissolves slowly in
the living body, and thus a DDS effect of sustained release
of α-lipoic acid is expected. Furthermore, since the
10 nanoparticles of the present invention are coated with a
polyvalent metal inorganic salt at the surface, generation
of the sulfurous odor characteristic to α-lipoic acid can
be significantly suppressed.
15 BRIEF DESCRIPTION OF THE DRAWINGS
[0053] [Fig. 1] FIG. 1 shows the result of the particle size
distribution of the α-lipoic acid-MgCO3 nanoparticles
produced by using distilled water in Example 1, as measured
20 by using a light scattering photometer (Otsuka Electronics
Co., Ltd., ELS-710TY).
[Fig. 2] FIG. 2 shows the result of the particle size
distribution of the α-lipoic acid nanoparticles produced
by using ion-exchanged water in Comparative Example 1A,
25 measured by using a light scattering photometer (Otsuka
Electronics Co., Ltd., ELS-710TY).
[Fig. 3] FIG. 3 shows the results of the residual ratio
of α-lipoic acid. Symbol �� represents the results for the
reagent α-lipoic acid which is a control, symbol �� represents
30 the results for the α-lipoic acid nanoparticles of
Comparative Example 1, and symbol �� represents the results
for the α-lipoic acid-MgCO3 nanoparticles of Example 1.
[Fig. 4] FIG. 4 shows the results of Test Example 3.
- 16 - EG026
[Fig. 5] FIG. 5 shows the results of Test Example 4.
[Fig. 6] FIG. 6 shows a replica of the wrinkles in Test
Example 4.
[Fig. 7] FIG. 7 shows the result of the particle size
5 distribution of the α-lipoic acid-CaCO3 nanoparticles
produced by using distilled water in Example 22A, as measured
using a light scattering photometer (Otsuka Electronics Co.,
Ltd., FPAR1000).
[Fig. 8] FIG. 8 shows the result of the particle size
10 distribution of the α-lipoic acid-MgCO3 nanoparticles
produced by using distilled water in Example 29B, as measured
using a light scattering photometer (Otsuka Electronics Co.,
Ltd., FPAR1000).
[Fig. 9] FIG. 9 shows the results of Test Example 5.
15 [Fig. 10] FIG. 10 shows the results of Test Example 6.
[Fig. 11] FIG. 11 shows the results obtained in Test
Example 7 by adding α-lipoic acid-MgCO3 nanoparticles into
a 3T3-L1 cell culture medium, staining the lipids accumulated
in immature adipocytes with Oil Red O, and measuring with
20 a spectrophotometer (wavelength 520 nm).
[Fig. 12] FIG. 12 shows the results obtained in Test
Example 8 by adding α-lipoic acid-MgCO3 nanoparticles into
a 3T3-L1 cell culture medium, staining the lipids accumulated
in mature adipocytes with Oil Red O, and measuring with a
25 spectrophotometer (wavelength 520 nm).
[Fig. 13] FIG. 13 shows the results obtained in Test
Example 9 by measuring the α-lipoic acid concentration in
the cell disrupted fluid obtained by disrupting immature
adipocytes cultured in a 3T3-L1 cell culture medium with
30 α-lipoic acid-MgCO3 nanoparticles added therein, using a high
performance liquid chromatograph-mass spectrometer.
[Fig. 14] FIG. 14 shows the results obtained in Test
Example 9 by measuring the α-lipoic acid concentration in
- 17 - EG026
the supernatant of the immature adipocyte culture cultured
in a 3T3-L1 cell culture medium with α-lipoic acid-MgCO3
nanoparticles added therein, using a high performance liquid
chromatograph-mass spectrometer.
5 [Fig. 15] FIG. 15 shows the example criteria for
evaluation of the wrinkle model mouse produced in Test Example
10.
[Fig. 16] FIG. 16 shows a mouse wrinkle replica obtained
by applying α-lipoic acid-MgCO3 nanoparticles on a wrinkle
10 model mouse produced in Test Example 10 for 6 weeks, and
the scores for wrinkle evaluation.
[Fig. 17] FIG. 17 shows the results of hyaluronic acid
staining of the wrinkle model mouse skin section produced
in Test Example 10.
15 [Fig. 18] FIG. 18 shows the wrinkle replica of Test Example
11.
[Fig. 19] FIG. 19 shows the results of hyaluronic acid
ELISA for the cell disrupted fluid fraction of Test Example
12.
20
BEST MODE FOR CARRYING OUT THE INVENTION
[0054] Hereinafter, the present invention will be described
in detail.
25
[0055] (1. Material for α-lipoic acid nanoparticles)
The α-lipoic acid nanoparticles of the present invention
are produced using α-lipoic acid, a nonionic surfactant,
a divalent metal salt, and an alkali metal carbonate or alkali
30 metal phosphate. Those ordinarily skilled in the art can
use other materials as necessary, such as an alkaline aqueous
solution, in the production method of the present invention.
- 18 - EG026
[0056] (1a. α-Lipoic acid)
α-Lipoic acid that is used in the present invention may
be any α-lipoic acid that is known in the art. The α-lipoic
acid is also known as thioctic acid. The α-lipoic acid may
5 be any of R,S-(+/-)-α-lipoic acid, R-(+)-α-lipoic acid, and
S-(-)-α-lipoic acid. The α-lipoic acid may be in the form
of an acid or may be in the form of a salt. Any commercially
available α-lipoic acid may be used. The α-lipoic acid can
be in the form of a powder or crystals.
10
[0057] (1b. Nonionic surfactant)
The nonionic surfactant that is used in the present
invention may be any surfactant as long as it is nonionic.
Examples of the nonionic surfactant used in the present
15 invention include, but are not particularly limited to,
polyoxyethylene hydrogenated castor oils, polyoxyethylene
alkyl ethers, polyoxyethylene sorbitan fatty acid esters,
polyoxyethylene polyoxypropylene alkyl ethers,
polyglycerin fatty acid esters, sucrose fatty acid esters,
20 propylene glycol fatty acid esters, monoglycerin fatty acid
esters, diglycerin fatty acid esters, sorbitan fatty acid
esters, polyoxyethylene fatty acid esters, and the like.
As for the nonionic surfactant used in the present inventions,
those having an HLB value of about 10 or more are particularly
25 preferable. As for the nonionic surfactant used in the
present inventions, a nonionic surfactant which is selected
from the group consisting of polyoxyethylene hydrogenated
castor oil, polyoxyethylene alkyl ethers, polyoxyethylene
sorbitan fatty acid esters, polyoxyethylene
30 polyoxypropylene alkyl ethers, sucrose fatty acid esters
and polyglycerin fatty acid esters, and which has an HLB
value of about 10 or more, is particularly preferable.
According to the present invention, it is more particularly
- 19 - EG026
preferable that the nonionic surfactant be selected from
the group consisting of polyoxyethylene (degree of
polymerization 10 to 20) octyl dodecyl ether, polyoxyethylene
(degree of polymerization 10 to 20) stearyl ether,
5 polyoxyethylene (degree of polymerization 10 to 20)
polyoxypropylene (degree of polymerization 4 to 8) cetyl
ether, polyoxyethylene (degree of polymerization 20 to 100)
hydrogenated castor oil and sucrose lauric acid ester. In
the present inventions, one kind of nonionic surfactant may
10 be used, or two or more kinds of nonionic surfactant may
be used in combination. The HLB value of the polyoxyethylene
sorbitan fatty acid ester is preferably about 10 or more,
more preferably about 12 or more, and most preferably about
14 or more. The HLB value of the polyoxyethylene sorbitan
15 fatty acid ester is preferably about 20 or less, more
preferably about 18 or less, and most preferably about 16
or less.
[0058] The nonionic surfactant may be those which are solid
20 at room temperature (that is, a surfactant having a melting
point which is higher than room temperature), or may be those
which are liquid at room temperature (that is, a surfactant
having a melting point which is lower than room temperature).
The term “nonionic surfactant in a liquid form” is used in
25 the present specification in relation to both the embodiment
of using a nonionic surfactant which is a liquid at room
temperature and the embodiment of using a nonionic surfactant
which is a solid at room temperature, in a liquid form by
heating to melt.
30
[0059] As used in the present specification, the “HLB value”
refers to the Hydrophile Lipophile Balance value, and is
generally calculated by 20 × MH/M, wherein MH is the molecular
- 20 - EG026
weight of the hydrophilic group moiety, and M is the molecular
weight of the whole molecule. The HLB value is 0 when the
amount of hydrophilic groups in the molecule is 0%, and is
20 when the amount of hydrophilic groups is 100%. The HLB
5 value indicates, in connection with the surfactant, the size
and strength of the hydrophilic and hydrophobic groups that
form the surfactant molecule, so that a surfactant having
high hydrophobicity has a small HLB value, and a surfactant
having high hydrophilicity has a large HLB value.
10
[0060] Examples of the polyoxyethylene hydrogenated castor
oils that are preferably used in the present invention
includes polyoxyethylene hydrogenated castor oils having
any degree of polymerization of ethylene oxide. For example,
15 polyoxyethylene hydrogenated castor oils having the degree
of polymerization of ethylene oxide of about 10 or more are
preferred, and polyoxyethylene hydrogenated castor oils
having the degree of polymerization of ethylene oxide of
about 200 or less are preferred. Examples of even more
20 preferable polyoxyethylene hydrogenated castor oils include
polyoxyethylene hydrogenated castor oil 40, polyoxyethylene
hydrogenated castor oil 60, and polyoxyethylene hydrogenated
castor oil 80. Note that these numbers indicate the extent
of the degree of polymerization of ethylene oxide, and for
25 example, polyoxyethylene hydrogenated castor oil 40
indicates that the number of added moles of ethylene oxide
is 40.
[0061] Examples of the polyoxyethylene alkyl ethers that
30 are preferably used in the present invention include
polyoxyethylene alkyl ethers having any degree of
polymerization of ethylene oxide. Polyoxyethylene alkyl
ethers having the degree of polymerization of ethylene oxide
- 21 - EG026
of about 10 or more are preferred, and polyoxyethylene alkyl
ethers having the degree of polymerization of ethylene oxide
of about 20 or less are preferred. Examples of even more
preferable polyoxyethylene alkyl ethers include, for example,
5 polyoxyethylene (20) stearyl ether (also described as POE(20)
stearyl ether), polyoxyethylene (20) octyl dodecyl ether
(also described as POE(20) octyl dodecyl ether) and
polyoxyethylene (20) isostearyl ether (POE(20) isostearyl
ether). This number of (20) indicates that the degree of
10 polymerization of ethylene oxide is 20.
[0062] Examples of the polyoxyethylene sorbitan fatty acid
esters that are preferably used in the present invention
include polyoxyethylene sorbitan fatty acid esters having
15 any degree of polymerization of ethylene oxide.
Polyoxyethylene sorbitan fatty acid esters having the degree
of polymerization of ethylene oxide of about 10 or more are
preferred, and polyoxyethylene sorbitan fatty acid esters
having the degree of polymerization of ethylene oxide of
20 about 20 or less are preferred. Examples of even more
preferable polyoxyethylene sorbitan fatty acid esters
include, for example, polyoxyethylene (20) sorbitan
monooleate (also described as POE(20) sorbitan monooleate),
polyoxyethylene (20) sorbitan monolaurate (also described
25 as POE(20) sorbitan monolaurate), polyoxyethylene (20)
sorbitan monostearate (also described as POE(20) sorbitan
monostearate), polyoxyethylene (20) sorbitan monopalmitate
(also described as POE(20) sorbitan monopalmitate), and
polyoxyethylene (20) sorbitan trioleate (also described as
30 POE(20) sorbitan trioleate). This number of (20) indicates
that the degree of polymerization of ethylene oxide is 20.
[0063] Examples of the polyoxyethylene polyoxypropylene
- 22 - EG026
alkyl ethers that are preferably used in the present invention
include polyoxyethylene polyoxypropylene ethers having any
degree of polymerization of ethylene oxide.
Polyoxyethylene polyoxypropylene alkyl ethers having the
5 degree of polymerization of the polyoxyethylene moiety of
about 10 or more are preferred, and polyoxyethylene
polyoxypropylene alkyl ethers having the degree of
polymerization of the polyoxyethylene moiety of about 20
or less are preferred. Polyoxyethylene polyoxypropylene
10 alkyl ethers having the degree of polymerization of the
polyoxypropylene moiety of about 4 or more are preferred,
and polyoxyethylene polyoxypropylene alkyl ethers having
the degree of polymerization of the polyoxypropylene moiety
of about 8 or less are preferred. Examples of the even more
15 preferable polyoxyethylene polyoxypropylene alkyl ether
include, for example, polyoxyethylene (20) polyoxypropylene
(8) cetyl ether (also described as POE(20) POP(8) cetyl ether),
polyoxyethylene (20) polyoxypropylene (4) cetyl ether (also
described as POE(20) POP(4) cetyl ether), polyoxyethylene
20 (34) polyoxypropylene (23) cetyl ether (also described as
POE(34) POP(23) cetyl ether), polyoxyethylene
polyoxyethylene propylene decyl tetradecyl ether (also
described as POEPOE propylene decyl tetradecyl ether), and
polyoxyethylene (20) isostearyl ether (also described as
25 POE(20) isostearyl ether).
[0064] Examples of the polyglycerin fatty acid esters that
are preferably used in the present invention include, for
example, decaglycerin monolaurate, decaglycerin
30 monomyristate, decaglycerin monooleate and decaglycerin
monostearate. The HLB value of the polyglycerin fatty acid
ester used is not particularly limited, but the HLB value
is preferably about 8 or more, more preferably about 10 or
- 23 - EG026
more, and even more preferably about 12 or more. The HLB
value is preferably about 20 or less, more preferably about
19 or less, and even more preferably about 18 or less.
5 [0065] Examples of the sucrose fatty acid esters that are
preferably used in the present invention include, for example,
sucrose stearic acid ester, sucrose palmitic acid ester,
sucrose myristic acid ester and sucrose lauric acid ester.
Among them, sucrose lauric acid ester is more preferably
10 used.
[0066] In the present inventions, the content of the
surfactant in the α-lipoic acid nanoparticles varies with
the kinds of surfactant. The amount of the surfactant is
15 preferably about one-fold or more, more preferably about
2-fold or more, even more preferably about 3-fold or more,
particularly preferably about 4-fold or more, and most
preferably about 5-fold or more, of the weight of α-lipoic
acid. The amount of the surfactant is preferably about
20 40-fold or less, more preferably about 35-fold or less, even
more preferably about 30-fold or less, particularly
preferably about 25-fold or less, and most preferably about
20-fold or less, of the weight of α-lipoic acid. If the amount
of the surfactant relative to the amount of α-lipoic acid
25 is too small, the nanoparticles may become prone to aggregate,
and it may become difficult to obtain transparent and stable
particles. If the amount of surfactant relative to the amount
of α-lipoic acid is too large, even if the amount of addition
is increased, the effect obtainable thereby does not
30 significantly increase, and there may occur problems such
as that the α-lipoic acid content is relatively decreased,
the handling at the time of use becomes poor, and when the
subject nanoparticles are used in foods, the taste derived
- 24 - EG026
from the surfactant is manifested, thereby lowering the
product value.
[0067] (1c. Divalent metal salt)
5 In the present inventions, a divalent metal salt is used.
Examples of the divalent metal salt that can be used include
divalent metal halides, divalent metal acetates and divalent
metal gluconates.
10 [0068] The divalent metal acetate is a salt formed of acetic
acid and a divalent metal, and is also referred to as acetic
acid divalent metal salt. The divalent metal gluconate is
a salt formed of gluconic acid and a divalent metal, and
is also referred to as gluconic acid divalent metal salt.
15 The divalent metal salt is preferably selected from the group
consisting of calcium chloride, calcium bromide, calcium
fluoride, calcium iodide, magnesium chloride, magnesium
bromide, magnesium fluoride, magnesium iodide, zinc chloride,
zinc bromide, zinc fluoride, zinc iodide, calcium acetate,
20 magnesium acetate, zinc acetate, calcium gluconate,
magnesium gluconate and zinc gluconate, and is more
preferably selected from the group consisting of magnesium
chloride, calcium chloride and zinc gluconate. Commercially
available divalent metal salts can be used. One kind of
25 divalent metal salt may be used, or two or more kinds of
divalent metal salts may be used in mixture. It is preferable
to use one kind of divalent metal salt.
[0069] (1d. Alkali metal carbonate or alkali metal
30 phosphate)
In the present inventions, an alkali metal carbonate
or an alkali metal phosphate is used. Examples of the alkali
metal in the alkali metal carbonate or alkali metal phosphate
- 25 - EG026
include sodium, potassium, lithium, rubidium, cesium and
francium. The alkali metal is preferably sodium or potassium,
and is more preferably sodium. Examples of the alkali metal
carbonate that can be used in the present inventions include,
5 for example, sodium carbonate, potassium carbonate, sodium
hydrogen carbonate and potassium hydrogen carbonate, and
sodium carbonate is preferred. Examples of the alkali metal
phosphate that can be used in the present inventions include,
for example, sodium phosphate and potassium phosphate. The
10 sodium phosphate may be sodium metaphosphate, disodium
hydrogen phosphate, sodium dihydrogen phosphate, trisodium
phosphate, sodium pyrophosphate or sodium hydrogen
pyrophosphate, and is preferably disodium hydrogen phosphate.
The potassium phosphate may be potassium dihydrogen phosphate,
15 dipotassium hydrogen phosphate or tripotassium phosphate,
and is preferably dipotassium hydrogen phosphate.
[0070] Commercially available alkali metal carbonates and
alkali metal phosphates can be used. One kind of alkali metal
20 carbonate or alkali metal phosphate may be used, and two
or more kinds of alkali metal carbonate or alkali metal
phosphate may be used in mixture. It is preferable to use
one kind of alkali metal carbonate or alkali metal phosphate.
25 [0071] (1e. Additive)
In the present inventions, an additive can be used. The
additive is preferably a water-soluble polymer. Examples
of the additive include polyethylene glycol, Plant-derived
macromolecules, microorganism-derived macromolecules,
30 animal-derived macromolecules, starches and dextrins,
celluloses, vinylic-type macromolecules and acrylic-type
macromolecules.
- 26 - EG026
[0072] It is believed that by using the additive, a micelle
aggregation suppressive effect and dispersive effect by
adsorbing the water-soluble polymer to the micelle surface;
a micelle aggregation suppressive effect by steric hindrance
5 caused by the presence of polymer compounds in the water
(continuous phase) between micelles; and a micelle
aggregation suppressive effect by viscosity increase, and
the like may be obtained.
10 [0073] Polyethylene glycol is a substance represented by
HO(CH2CH2O)nH. Polyethylene glycol is a polyether having a
structure which is believed to be produced by dehydration
polycondensation of ethylene glycol, and having hydroxyl
groups at both ends. Various polyethylene glycols having
15 a molecular weight of about 200 to about 20,000 are known.
Polyethylene glycol is liquid when the molecular weight is
about 200 to about 600, and is solid when the molecular weight
exceeds about 1000. Since polyethylene glycol is a polymer,
it is usually marketed as mixtures of molecules having various
20 molecular weights. The number average molecular weight of
polyethylene glycol is preferably about 500 or more, more
preferably about 600 or more, even more preferably about
700 or more, still more preferably about 800 or more,
particularly preferably about 900 or more, and most
25 preferably about 1,000 or more. The number average molecular
weight of polyethylene glycol is preferably about 10,000
or less, more preferably about 9,000 or less, even more
preferably about 8,000 or less, still more preferably about
7,000 or less, particularly preferably about 6,500 or less,
30 and most preferably about 20,000 or less. Examples of
polyethylene glycol that is preferably used in the present
inventions include polyethylene glycol 1000, polyethylene
glycol 4000, and polyethylene glycol 6000.
- 27 - EG026
[0074] Plant-derived macromolecules refer to
macromolecules extracted or purified from plants. Examples
of the plant-derived macromolecules include gum arabic,
5 tragacanth gum, galactan, guar gum, locust bean gum,
carrageenan, pectin, quince seed (Cydonia oblonga seed)
extract, brown alga powder, and the like.
[0075] Microorganism-derived macromolecules refer to
10 macromolecules extracted or purified from microorganisms.
Examples of the microorganism-derived macromolecules
include xanthan gum, dextran, pullulan and the like.
[0076] Animal-derived macromolecules refer to
15 macromolecules extracted or purified from animals.
Examples of the animal-derived macromolecules include
collagen, casein, albumin, gelatin, hyaluronic acid, and
the like.
20 [0077] Starches and dextrins refer to starch and dextrin,
as well as chemical modification products, enzymatic
treatment products and physical treatment products thereof.
The starches are preferably chemically modified starches.
Examples of the starches include carboxymethyl starch,
25 methylhydroxy starch, and the like.
[0078] Celluloses refer to celluloses, and chemical
modification products, enzymatic treatment products and
physical treatment products thereof. Examples of the
30 celluloses include methylcellulose, nitrocellulose,
ethylcellulose, methylhydroxypropylcellulose,
hydroxyethylcellulose, cellulose sulfate,
hydroxypropylcellulose, carboxymethylcellulose,
- 28 - EG026
crystalline cellulose, cellulose powders, and the like.
[0079] Vinylic-type macromolecules refer to macromolecules
obtained by polymerizing vinyl monomers. Examples of the
5 vinylic-type macromolecules include polyvinyl alcohol,
polyvinyl methyl ether, polyvinylpyrrolidone, carboxyvinyl
polymers, and the like.
[0080] Acrylic-type macromolecules refer to macromolecules
10 obtained by polymerizing acrylic monomers. Examples of the
acrylic-type macromolecules include polyacrylic acid and
salts thereof, polyacrylimide, and the like.
[0081] (2. Method for producing α-lipoic acid
15 nanoparticles)
The method for producing α-lipoic acid nanoparticles
of the present invention comprises the steps of: preparing
an aqueous dispersion liquid containing α-lipoic acid and
a nonionic surfactant; adding a divalent metal salt into
20 the aqueous dispersion liquid, wherein the divalent metal
salt is a divalent metal halide, a divalent metal acetate
or a divalent metal gluconate; and adding an alkali metal
carbonate or an alkali metal phosphate into the aqueous
dispersion liquid wherein the divalent metal salt has been
25 added, thereby forming α-lipoic acid nanoparticles.
[0082] In a preferred embodiment, the step of preparing an
aqueous dispersion liquid containing α-lipoic acid and a
nonionic surfactant, comprises: dissolving α-lipoic acid
30 in the nonionic surfactant which is in a liquid form, to
obtain a surfactant solution; and adding water or a liquid
containing water to the surfactant solution to obtain the
aqueous dispersion liquid. In this embodiment, α-lipoic
- 29 - EG026
acid nanoparticles can be produced by carrying out steps
including “2a-1”, “2b-1”, “2c” and “2d” described below.
[0083] In another preferred embodiment, the step of
5 preparing an aqueous dispersion liquid containing α-lipoic
acid and a nonionic surfactant, comprises: producing a
mixture of α-lipoic acid, an alkaline substance and water
to prepare an α-lipoic acid-containing aqueous dispersion
liquid; and adding the nonionic surfactant into the α-lipoic
10 acid-containing aqueous dispersion liquid. In this
embodiment, α-lipoic acid nanoparticles can be produced by
carrying out steps including “2a-2”, “2b-2”, “2c” and “2d”
described below.
15 [0084] In a specific preferred embodiment, the method of
the present invention is a method for producing α-lipoic
acid nanoparticles and comprise the steps of: producing a
mixture of α-lipoic acid, an alkaline substance and water
to prepare an α-lipoic acid-containing aqueous dispersion
20 liquid (it is believed that, in the aqueous dispersion liquid,
the α-lipoic acid forms micelles); adding the nonionic
surfactant into the aqueous dispersion liquid (it is believed
that mixed micelles of the α-lipoic acid with the nonionic
surfactant form); adding a divalent metal salt into the
25 aqueous dispersion liquid, wherein the divalent metal salt
is a divalent metal halide, a divalent metal acetate or a
divalent metal gluconate; and adding an alkali metal
carbonate or an alkali metal phosphate into the aqueous
dispersion liquid wherein the divalent metal salt has been
30 added, thereby forming α-lipoic acid nanoparticles.
[0085] (2a-1. Step of dissolving α-lipoic acid in nonionic
surfactant in liquid form)
- 30 - EG026
An embodiment of initially dissolving α-lipoic acid in
a nonionic surfactant will be described. In this embodiment,
the nonionic surfactant is used as a solvent. That is, a
surfactant solution is prepared. In this embodiment, first,
5 α-lipoic acid is dissolved in a nonionic surfactant in a
liquid form, and thereby a surfactant solution is obtained.
This α-lipoic acid may be added directly to the nonionic
surfactant, or may be added indirectly. The phrase “added
indirectly” refers to adding after mixed with another
10 substance. For example, α-lipoic acid may be added to the
nonionic surfactant after being mixed with an additive.
α-Lipoic acid is usually marketed in the form of crystals
or powder. In this embodiment, α-lipoic acid dissolves
almost completely in the nonionic surfactant in a liquid
15 form. If the nonionic surfactant is liquid at room
temperature, this dissolution operation can be carried out
at room temperature, but if necessary, the nonionic
surfactant may be heated and then the dissolution operation
can be carried out. If the nonionic surfactant is solid at
20 room temperature, the nonionic surfactant is heated to a
liquid form, and then this dissolution operation is carried
out. Upon preparing this surfactant solution, the nonionic
surfactant may have the above-mentioned additive added
therein, as necessary.
25
[0086] When this surfactant solution is prepared,
preferably, water is not used. That is, the amount of water
incorporated upon preparing a surfactant solution is
preferably set at about 50 parts by weight or less, more
30 preferably about 20 parts by weight or less, even more
preferably about 10 parts by weight or less, still more
preferably about 5 parts by weight or less, and particularly
preferably about 1 part by weight or less, relative to 100
- 31 - EG026
parts by weight of α-lipoic acid. The lower limit of the
water amount is not particularly defined, but conditions
in which water of about 0.001 parts by weight or more, about
0.01 parts by weight or more, or about 0.1 parts by weight
5 or more, relative to 100 parts by weight of α-lipoic acid
is mixed may be employed.
[0087] α-Lipoic acid can be dissolved in alcohol, but in
the present inventions, it is preferable not to use alcohol
10 substantially. When alcohol is used, adverse effects may
be exerted on the efficiency of micelle formation by α-lipoic
acid. Therefore, for example, it is preferable to set the
amount of use of the alcohol at about 10 parts by weight
or less, more preferably at about 5 parts by weight or less,
15 even more preferably at about 1 part by weight or less,
particularly preferably at about 0.5 parts by weight or less,
and most preferably at about 0.1 parts by weight or less,
relative to 100 parts by weight of α-lipoic acid. Provided
that, in the case of using alcohol according to necessity,
20 the lower limit of the amount of use of the alcohol is not
particularly defined, but for example, it is possible to
set the amount of use of the alcohol at about 0.01 parts
by weight or more, relative to 100 parts by weight of α-lipoic
acid.
25
[0088] It is noted that in the embodiment that will be
described later, an alkaline compound is used when α-lipoic
acid is dissolved, but in the present embodiment, there is
no need to use an alkaline compound to dissolve α-lipoic
30 acid. In the present embodiment, when α-lipoic acid is
dissolved in the nonionic surfactant, preferably, the
dissolution operation is carried out without using any
material other than α-lipoic acid and the nonionic surfactant.
- 32 - EG026
For example, the dissolution operation can be carried out
without substantially using an alkaline compound.
Therefore, in regard to the amount of the alkaline compound
used upon carrying out the dissolution operation, for example,
5 the amount of use of the alkaline compound can be set at
5 parts by weight or less, can be set at about 1 part by
weight or less, can be set at about 0.5 parts by weight or
less, can be set at about 0.1 parts by weight or less, can
be set at about 0.05 parts by weight or less, and can also
10 be set at about 0.01 parts by weight or less, relative to
100 parts by weight of α-lipoic acid.
[0089] It is noted that when water is added after α-lipoic
acid is dissolved in the nonionic surfactant, alkali may
15 be added simultaneously with water, or alkaline water (for
example, an aqueous solution of a basic compound) may be
added, as necessary.
[0090] A nonionic surfactant which has a melting point above
20 room temperature, is preferably heated to melt. The heating
may be carried out such that the temperature of the nonionic
surfactant used reaches the temperature which is sufficient
for the nonionic surfactant to melt. Since it is feared that
heating may decompose α-lipoic acid, excessive heating, such
25 that the temperature of the nonionic surfactant reaches about
70°C or above, is not preferable. The temperature of the
nonionic surfactant at the time of adding α-lipoic acid,
is preferably higher than the melting temperature of this
nonionic surfactant, and is preferably (melting point + 20°C)
30 or lower, more preferably (melting point + 15°C) or lower,
and most preferably (melting point + 10°C) or lower.
[0091] During the production of a mixture of the nonionic
- 33 - EG026
surfactant and α-lipoic acid, another substance may be
additionally mixed, as long as the substance does not
substantially exert any adverse effects on the mixing
(micelle formation) of α-lipoic acid and the nonionic
5 surfactant. For example, the mixture may be produced by
mixing the nonionic surfactant and an additive (for example,
polyethylene glycol) and then adding α-lipoic acid.
Alternatively, an additive (for example, polyethylene
glycol) may be added to a mixture of nonionic surfactant
10 and α-lipoic acid.
[0092] It is preferable to stir the mixture
satisfactorily after α-lipoic acid has been added.
15 [0093] The amount of α-lipoic acid is selected such that
the concentration of α-lipoic acid in the α-lipoic
acid-containing aqueous dispersion liquid obtained in step
2b-1 is at or above the critical micelle concentration. The
concentration of α-lipoic acid in the α-lipoic
20 acid-containing aqueous dispersion liquid is preferably
about 0.1% by weight or more, more preferably about 0.5%
by weight or more, and more preferably about 1.0% by weight
or more. The concentration of α-lipoic acid in the α-lipoic
acid-containing aqueous dispersion liquid is preferably
25 about 20% by weight or less, more preferably about 16% by
weight or less, even more preferably about 14% by weight
or less, particularly preferably about 12% by weight or less,
and most preferably about 10% by weight or less.
30 [0094] The amount of the nonionic surfactant used to dissolve
α-lipoic acid can be arbitrarily selected, but when the amount
of α-lipoic acid is taken as 100, the amount of the nonionic
surfactant is, on the basis of weight, preferably about 100%
- 34 - EG026
or more, more preferably about 200% or more, even more
preferably about 300% or more, particularly preferably about
400% or more, and most preferably about 500% or more. The
amount of the nonionic surfactant that is added through this
5 step is, when the amount of α-lipoic acid is taken as 100%,
on a weight basis, preferably about 4000% or less, more
preferably about 3500% or less, even more preferably about
3000% or less, particularly preferably about 2500% or less,
and most preferably about 2000% or less.
10
[0095] (2b-1. Step of obtaining α-lipoic acid-containing
aqueous dispersion liquid by adding water to a mixture of
nonionic surfactant and α-lipoic acid)
Subsequently, an α-lipoic acid-containing aqueous
15 dispersion liquid is obtained by adding water to the mixture
of the nonionic surfactant and α-lipoic acid. During the
production of the α-lipoic acid-containing aqueous
dispersion liquid, another substance may be additionally
mixed, as long as the substance does not substantially exert
20 adverse effects on the mixing (micelle formation) of α-lipoic
acid and the nonionic surfactant.
[0096] It is thought that when water is added and mixed with
the mixture of the nonionic surfactant and α-lipoic acid,
25 mixed micelles of α-lipoic acid and the nonionic surfactant
are spontaneously formed. In this embodiment, since it is
believed that mixed micelles are formed from a state in which
the α-lipoic acid and the nonionic surfactant are regularly
arranged, at one time through the addition of water, it is
30 thought that the mixed micelles can be formed very stably.
It is preferable to satisfactorily stir the solution after
water has been added. The stirring is preferably continued
for a certain length of time. The stirring time is preferably
- 35 - EG026
about 10 minutes or longer, more preferably about 20 minutes
or longer, even more preferably about 25 minutes or longer,
and most preferably about 30 minutes or longer. There is
no particular upper limit in the stirring time. For example,
5 the stirring time can be set at any value, such as about
48 hours or less, about 24 hours or less, about 18 hours
or less, about 12 hours or less, about 6 hours or less, about
4 hours or less, about 2 hours or less, about 1 hour or less,
about 50 minutes or less, about 40 minutes or less, or about
10 35 minutes or less.
[0097] In this manner, an aqueous dispersion liquid
containing α-lipoic acid and a nonionic surfactant is
obtained.
15
[0098] (2a-2. Step of mixing α-lipoic acid and alkali)
In the method of the present invention according to the
embodiment of initially mixing α-lipoic acid with an alkali,
first, a mixture of α-lipoic acid, the alkaline substance
20 and water is produced, and thus an α-lipoic acid-containing
aqueous dispersion liquid is prepared. α-Lipoic acid is
usually marketed in the form of crystals or powder. When
α-lipoic acid is added to water, α-lipoic acid undergoes
dispersion but is never completely dissolved. α-Lipoic acid
25 is dissolved in alcohol, but in the present inventions, it
is preferable not to use alcohol. If alcohol is used, adverse
effects may be exerted on the efficiency of micelle formation
by α-lipoic acid. The alkaline substance may be any alkaline
substance, but is preferably a strong base, and more
30 preferably sodium hydroxide.
[0099] The α-lipoic acid-containing aqueous dispersion
liquid can be produced by, for example, first adding α-lipoic
- 36 - EG026
acid into water to mix them, and adding an alkaline solution
to the mixture to mix therewith. The α-lipoic
acid-containing aqueous dispersion liquid can also be
produced by adding α-lipoic acid into water to mix them,
5 and adding an alkaline substance to the mixture to mix
therewith. The α-lipoic acid-containing aqueous dispersion
liquid can also be produced by adding α-lipoic acid into
an alkaline solution, and mixing them. The α-lipoic
acid-containing aqueous dispersion liquid can also be
10 produced by adding α-lipoic acid and an alkaline substance
into water, and mixing them.
[0100] During the production of the α-lipoic
acid-containing aqueous dispersion liquid, another
15 substance may be additionally mixed thereinto, as long as
the substance does not substantially exert adverse effects
on the mixing (micelle formation) of α-lipoic acid and the
alkali.
20 [0101] The amount of α-lipoic acid used for the production
of the α-lipoic acid-containing aqueous dispersion liquid,
is selected such that the concentration of α-lipoic acid
in the α-lipoic acid-containing aqueous dispersion liquid
is at or above the critical micelle concentration. The
25 concentration of α-lipoic acid in the α-lipoic
acid-containing aqueous dispersion liquid is preferably
about 0.1% by weight or more, more preferably about 0.5%
by weight or more, and even more preferably about 1.0% by
weight or more. The concentration of α-lipoic acid in the
30 α-lipoic acid-containing aqueous dispersion liquid is
preferably about 20% by weight or less, more preferably about
16% by weight or less, even more preferably about 14% by
weight or less, particularly preferably about 12% by weight
- 37 - EG026
or less, and most preferably about 10% by weight or less.
[0102] The amount of the alkaline substance used for the
production of the α-lipoic acid-containing aqueous
5 dispersion liquid can be any amount, as long as the amount
is capable of allowing α-lipoic acid to be dispersed in water.
The amount of the alkaline substance is preferably an amount
that brings the pH of the α-lipoic acid-containing aqueous
dispersion liquid to about 6.5 or higher. The amount of the
10 alkaline substance is preferably an amount that brings the
pH of the α-lipoic acid-containing aqueous dispersion liquid
to about 13.5 or lower, more preferably an amount that brings
the pH of the α-lipoic acid-containing aqueous dispersion
liquid to about 13.0 or lower, and particularly preferably
15 an amount that brings the pH of the α-lipoic acid-containing
aqueous dispersion liquid to about 12.5 or lower.
[0103] In this manner, an α-lipoic acid-containing aqueous
dispersion liquid is obtained.
20
[0104] (2b-2. Step of adding α-lipoic acid-containing
aqueous dispersion liquid and nonionic surfactant)
Subsequently, a nonionic surfactant is added to this
α-lipoic acid-containing aqueous dispersion liquid. Since
25 the surfaces of the micelles of α-lipoic acid are in the
state of being covered with negative charges, divalent metal
ions, for example, calcium ions (Ca2+), can easily be adsorbed
(bound) to cause an exchange reaction with sodium ions. In
this case, since divalent metal ions have higher adsorption
30 capacity (binding capacity) as compared with sodium ions,
the micelles having the divalent metal ions adsorbed thereto
become insoluble in water as the charges at the micelle surface,
become difficult to dissociate, and the micelles precipitate.
- 38 - EG026
When precipitation occurs, aggregation between particles
occurs, and very large particles are formed. In order to
prevent aggregation of particles at this stage, a nonionic
surfactant is added. The nonionic surfactant forms mixed
5 micelles together with α-lipoic acid, and protrudes
hydrophilic groups at the micelle surface. Thus, it is
thought that even if polyvalent metal ions are adsorbed
(bound) to the micelle surface, the presence of the
hydrophilic group protruded at the micelle surface prevents
10 the precipitation of micelles.
[0105] The amount of the nonionic surfactant that is added
in this step can be arbitrarily selected, but when the
concentration of α-lipoic acid is taken as 100, the amount
15 of the nonionic surfactant is, on a weight basis, preferably
about 100% or more, more preferably about 200% or more, even
more preferably about 300% or more, particularly preferably
about 400% or more, and most preferably about 500% or more.
The amount of the nonionic surfactant that is added in this
20 step, when the concentration of α-lipoic acid is taken as
100%, on a weight basis, is preferably about 4000% or less,
more preferably about 3500% or less, even more preferably
about 3000% or less, particularly preferably about 2500%
or less, and most preferably about 2000% or less.
25
[0106] It is speculated that mixed micelles of α-lipoic
acid and the nonionic surfactant are spontaneously formed
when the nonionic surfactant is added to the α-lipoic
acid-containing aqueous dispersion liquid to mix. It is
30 preferable to satisfactorily stir the solution after the
nonionic surfactant has been added. The stirring is
preferably continued for a certain length of time. The
stirring time is preferably about 10 minutes or longer, more
- 39 - EG026
preferably about 20 minutes or longer, even more preferably
about 25 minutes or longer, and most preferably about 30
minutes or longer. There is no particular upper limit in
the stirring time. For example, the stirring time can be
5 set at any value, such as about 48 hours or less, about 24
hours or less, about 18 hours or less, about 12 hours or
less, about 6 hours or less, about 4 hours or less, about
2 hours or less, about 1 hour or less, about 50 minutes or
less, about 40 minutes or less, or about 35 minutes or less.
10
[0107] In this manner, an aqueous dispersion liquid
containing α-lipoic acid and a nonionic surfactant is
obtained.
15 [0108] (2c. Step of adding divalent metal salt)
Subsequently, a divalent metal salt is added to the
aqueous dispersion liquid prepared in the above step 2b-1
or step 2b-2. The divalent metal salt may be directly added
to this aqueous dispersion liquid, or may be added in the
20 form of an aqueous solution, but preferably, the divalent
metal salt is added as an aqueous solution of the divalent
metal salt.
[0109] The aqueous dispersion liquid to which the divalent
25 metal salt is to be added, can be directly used as received
from the previous step, but preferably, the pH is adjusted
immediately before the metal salt is added, in accordance
with the metal salt used.
30 [0110] The inventors of the present invention found that
with regard to α-lipoic acid, the pH that is preferable for
the dispersion of α-lipoic acid is different from the pH
that is preferable for the addition of the divalent metal
- 40 - EG026
salt, and that when the divalent metal ion is added to the
aqueous dispersion liquid containing the mixed micelles
containing α-lipoic acid and the nonionic surfactant, there
exists a pH value which is preferable depending on the kind
5 of metal ion. This pH is desirably about 12.0 or lower when
the divalent metal ion is Mg2+, about 12.0 or lower in the
case of Ca2+, and about 9.5 or lower in the case of Zn2+, and
more desirably about 11.5 or lower in the case of Mg2+, about
11.5 or lower in the case of Ca2+, and about 8.8 or lower
10 in the case of Zn2+.
[0111] When the divalent metal salt is calcium chloride,
calcium bromide, calcium fluoride, calcium iodide, calcium
acetate or calcium gluconate, the pH of the aqueous dispersion
15 liquid immediately before the addition of divalent metal
salt is preferably about 3.4 or higher, more preferably about
3.6 or higher, particularly preferably about 3.8 or higher,
and most preferably about 4.0 or higher; and the pH of the
aqueous dispersion liquid immediately before the addition
20 of divalent metal salt is preferably about 12.0 or lower,
more preferably about 11.9 or lower, particularly preferably
about 11.7 or lower, and most preferably about 11.5 or lower.
[0112] When the divalent metal salt is magnesium chloride,
25 magnesium bromide, magnesium fluoride, magnesium iodide,
magnesium acetate or magnesium gluconate, the pH of the
aqueous dispersion liquid immediately before the addition
of divalent metal salt is preferably about 3.4 or higher,
more preferably about 3.6 or higher, particularly preferably
30 about 3.8 or higher, and most preferably about 4.0 or higher;
and the pH of the aqueous dispersion liquid immediately before
the addition of divalent metal salt is preferably about 12.0
or lower, more preferably about 11.9 or lower, particularly
- 41 - EG026
preferably about 11.7 or lower, and most preferably about
11.5 or lower.
[0113] When the divalent metal salt is zinc chloride, zinc
5 bromide, zinc fluoride, zinc iodide, zinc acetate or zinc
gluconate, the pH of the aqueous dispersion liquid
immediately before the addition of divalent metal salt is
preferably about 3.5 or higher, more preferably about 3.7
or higher, and most preferably about 3.9 or higher; and the
10 pH of the aqueous dispersion liquid immediately before the
addition of divalent metal salt is preferably about 9.5 or
lower, more preferably about 9.2 or lower, and most preferably
about 8.8 or lower.
15 [0114] The amount of the divalent metal salt added in this
step can be arbitrarily selected, but when the concentration
of α-lipoic acid is taken as 100, the amount is, on a molar
basis, preferably about 10% or more, more preferably about
20% or more, even more preferably about 30% or more,
20 particularly preferably about 40% or more, and most
preferably about 50% or more. The amount of the divalent
metal salt added in this step, when the concentration of
α-lipoic acid is taken as 100%, on a molar basis, is preferably
about 200% or less, more preferably about 160% or less, even
25 more preferably about 140% or less, particularly preferably
about 120% or less, and most preferably about 100% or less.
[0115] It is thought that by adding the divalent metal salt
into the aqueous dispersion liquid and mixing therewith,
30 divalent metal ions bind to the negative charges at the surface
of the mixed micelles, and thereby aggregation and
precipitation of the micelles of α-lipoic acid are prevented.
It is preferable to satisfactorily stir the solution after
- 42 - EG026
the divalent metal salt has been added. The stirring is
preferably continued for a certain length of time. The
stirring time is preferably about 10 minutes or longer, more
preferably about 20 minutes or longer, even more preferably
5 about 25 minutes or longer, and most preferably about 30
minutes or longer. There is no particular upper limit in
the stirring time. For example, the stirring time can be
set at any value, such as about 48 hours or less, about 24
hours or less, about 18 hours or less, about 12 hours or
10 less, about 6 hours or less, about 4 hours or less, about
2 hours or less, about 1 hour or less, about 50 minutes or
less, about 40 minutes or less, or about 35 minutes or less.
[0116] (2d. Step of adding alkali metal carbonate or alkali
15 metal phosphate)
Subsequently, an alkali metal carbonate or an alkali
metal phosphate is added to this aqueous dispersion liquid
to which the divalent metal salt has been added.
20 [0117] The amount of the alkali metal carbonate and alkali
metal phosphate (also referred to as “salt carrying a divalent
anion”) can be selected to be any amount, but when the amount
of the divalent metal salt added is taken as 1, the amount
of the salt carrying a divalent anion is, on a molar basis,
25 preferably about 0.01 or more, more preferably about 0.02
or more, and even more preferably about 0.1 or more. When
the amount of the divalent metal salt added is taken as 1,
the amount of the salt carrying a divalent anion is, on a
molar basis, preferably about 0.80 or less, more preferably
30 about 0.70 or less, and even more preferably about 0.60 or
less. In a particular embodiment, when the amount of the
divalent metal salt added is taken as 1, the amount of the
salt carrying a divalent anion may be, for example, on a
- 43 - EG026
molar basis, about 0.60 or less, about 0.50 or less, or about
0.40 or less. When the amount of the divalent metal salt
added is taken as 1, the amount of the salt carrying a divalent
anion is most preferably 0.2 on a molar basis. If the amount
5 of the salt carrying a divalent anion is too small relative
to the amount of the divalent metal salt, the positive charge
at the micelle surface is not neutralized, and the efficiency
of preventing the aggregation and precipitation of micelles
may be lowered. If the amount of the salt carrying a divalent
10 anion is too large relative to the amount of the divalent
metal salt, precipitation may become prone to occur.
[0118] For example, when the molar ratio of magnesium
chloride and sodium carbonate is set at 1:1, precipitation
15 occurs when the mixture is left to stand for a whole day,
but when the molar ratio is set at 1:0.01 to 0.8, and
particularly up to 1:0.4, the mixture remains transparent
and precipitation does not occur even when left to stand
for a long period time. When the mixture becomes turbid or
20 precipitation occurs, it is because the particle size of
the formed particles is too large. If the particle size is
too large, skin permeability becomes poor, and there would
be inconvenience even in the case of performing injection.
However, when the mixture is transparent and precipitation
25 does not occur, the particle size of the formed particles
is small, and the distribution is narrow. Therefore, skin
permeability is good, and inconvenience does not occur upon
performing injection.
30 [0119] In this manner, α-lipoic acid nanoparticles are
formed in an aqueous dispersion liquid.
[0120] The amount of the salt carrying a divalent anion that
- 44 - EG026
is added in this step can be selected to be any amount, but
when the concentration of α-lipoic acid is taken as 100,
the amount is, on a molar basis, preferably about 0.1% or
more, more preferably about 0.5% or more, even more preferably
5 about 1.0% or more, particularly preferably about 1.5% or
more, and most preferably about 2.0% or more. The amount
of the salt carrying a divalent anion that is added in this
step is, when the concentration of α-lipoic acid is taken
as 100%, on a molar basis, preferably about 80% or less,
10 more preferably about 74% or less, even more preferably about
68% or less, particularly preferably about 62% or less, and
most preferably about 60% or less. In a particular embodiment,
the amount of the salt carrying a divalent anion that is
added in this step is, when the concentration of α-lipoic
15 acid is taken as 100%, for example, on a molar basis, about
50% or less, about 46% or less, about 44% or less, about
42% or less, or about 40%.
[0121] It is thought that by adding the salt carrying a
20 divalent anion into the aqueous dispersion liquid which has
been added with a divalent metal salt and mixing therewith,
the divalent anions bind to the divalent metal ions that
are bound to the micelle surface. It is thought that by
binding the divalent anions to the divalent metal salt that
25 is bound to the micelle surface, the charge at the micelle
surface is substantially neutralized. It is thought that
at the micelle surface, the divalent metal ions and the
divalent anions bind to each other to form a polyvalent metal
inorganic salt. It is thought that as such, a coating of
30 a polyvalent metal inorganic salt is formed at the micelle
surface, and as a result, precipitation due to binding between
micelles is prevented.
- 45 - EG026
[0122] It is preferable to satisfactorily stir the solution
after the alkali metal carbonate or alkali metal phosphate
has been added. The stirring is preferably continued for
a certain length of time. The stirring time is preferably
5 about 10 minutes or longer, more preferably about 20 minutes
or longer, even more preferably about 25 minutes or longer,
and most preferably about 30 minutes or longer. There is
no particular upper limit in the stirring time. For example,
the stirring time can be set at any value, such as about
10 48 hours or less, about 24 hours or less, about 18 hours
or less, about 12 hours or less, about 6 hours or less, about
4 hours or less, about 2 hours or less, about 1 hour or less,
about 50 minutes or less, about 40 minutes or less, or about
35 minutes or less.
15
[0123] (2e. Other steps)
By carrying out the respective steps described above,
nanoparticles of α-lipoic acid are formed in an aqueous
dispersion liquid. This aqueous dispersion liquid can be
20 dried to obtain a powder, as necessary. Drying can be carried
out according to any method that is known in the art. The
drying is carried out by, for example, freeze-drying, spray
drying, drum drying or the like. Freeze-drying is preferred.
The powder containing the α-lipoic acid nanoparticles
25 produced according to the method of the present invention
is, if added to water, easily dispersed to form a transparent
liquid.
[0124] (3. α-Lipoic acid nanoparticles)
30 The α-lipoic acid nanoparticles of the present invention
contain α-lipoic acid, a nonionic surfactant, a divalent
metal ion, and a carbonate ion or a phosphate ion.
- 46 - EG026
[0125] The amount of the nonionic surfactant in the α-lipoic
acid nanoparticles of the present invention, when the
concentration of α-lipoic acid is taken as 100, on a weight
basis, is preferably about 100% or more, more preferably
5 about 200% or more, even more preferably about 300% or more,
particularly preferably about 400% or more, and most
preferably about 500% or more. The amount of the nonionic
surfactant in the α-lipoic acid nanoparticles of the present
invention, when the concentration of α-lipoic acid is taken
10 as 100%, on a weight basis, is preferably about 4000% or
less, more preferably about 3500% or less, even more
preferably about 3000% or less, particularly preferably about
2500% or less, and most preferably about 2000% or less.
15 [0126] The amount of the divalent metal ion in the α-lipoic
acid nanoparticles of the present invention, when the
concentration of α-lipoic acid is taken as 100, on a molar
basis, is preferably about 10% or more, more preferably about
20% or more, even more preferably about 30% or more,
20 particularly preferably about 40% or more, and most
preferably about 50% or more. The amount of the divalent
metal ion in the α-lipoic acid nanoparticles of the present
invention, when the concentration of α-lipoic acid is taken
as 100%, on a molar basis, is preferably about 200% or less,
25 more preferably about 160% or less, even more preferably
about 140% or less, particularly preferably about 120% or
less, and most preferably about 100% or less.
[0127] The amount of the carbonate ion or phosphate ion (also
30 referred to as divalent anion) in the α-lipoic acid
nanoparticles of the present invention, when the
concentration of α-lipoic acid is taken as 100, on a molar
basis, is preferably about 0.1% or more, more preferably
- 47 - EG026
about 0.5% or more, even more preferably about 1.0% or more,
particularly preferably about 1.5% or more, and most
preferably about 2.0% or more. The amount of the carbonate
ion or phosphate ion in the α-lipoic acid nanoparticles of
5 the present invention, when the concentration of α-lipoic
acid is taken as 100%, on a molar basis, is preferably about
80% or less, more preferably about 74% or less, even more
preferably about 68% or less, particularly preferably about
62% or less, and most preferably about 60% or less. In a
10 particular embodiment, the amount of the salt carrying a
divalent anion that is added in this step, when the
concentration of α-lipoic acid is taken as 100%, on a molar
basis, is for example, about 50% or less, about 46% or less,
about 44% or less, about 42% or less, or about 40% or less.
15
[0128] The divalent metal ion in the α-lipoic acid
nanoparticles of the present invention is preferably calcium
ion, zinc ion or magnesium ion.
20 [0129] When the amount of the divalent metal ion in the
α-lipoic acid nanoparticles of the present invention is taken
as 1, the amount of the divalent anion, on a molar basis,
is preferably about 0.01 or more, more preferably about 0.10
or more, and even more preferably about 0.20 or more. When
25 the amount of the divalent metal ion in the α-lipoic acid
nanoparticles of the present invention is taken as 1, the
amount of the divalent anion, on a molar basis, is preferably
about 0.80 or less, more preferably about 0.50 or less, and
even more preferably about 0.40 or less. When the amount
30 of the divalent metal ion in the α-lipoic acid nanoparticles
of the present invention is taken as 1, the amount of the
divalent anion is most preferably about 0.2 on a molar basis.
- 48 - EG026
[0130] (4. Uses of α-lipoic acid nanoparticles)
The α-lipoic acid nanoparticles of the present invention
can be used in various applications where α-lipoic acid has
been conventionally used. Examples of these applications
5 include an external preparation for skin, a pharmaceutical
product (including injection liquid), a composition for oral
cavity and a food.
[0131] (4a. External preparation for skin containing
10 α-lipoic acid nanoparticles)
The external preparation for skin of the present
invention contains the α-lipoic acid nanoparticles of the
present invention.
15 [0132] In the present specification, the term “external
preparation for skin” refers to a preparation to be used
for the skin, which achieves a desired effect when in contact
with the skin. The present invention is particularly
effective in the applications where the preparation is
20 continuously contacted with the skin for a long period of
time (for example, an application where the preparation is
continuously contacted with the skin for about one hour or
longer, or an application where the preparation is
continuously contacted with the skin for about 5 hours or
25 longer).
[0133] A preferred example of the external preparation for
skin is a cosmetic preparation.
30
[0134] Preferred examples of the cosmetic preparations
include skin care cosmetic preparations. Specific examples
of the cosmetic preparations include skin care cosmetic
- 49 - EG026
preparations such as skin lotion, emulsion and cream;
cosmetics such as foundation, eye shadow, lipstick and rouge
for cheek; hair cosmetic preparations, emollient cream,
emollient lotion, cream, cream rinse, cold cream, vanishing
5 cream, lotion, facial mask, gel, face pack, soap, body soap,
shampoo, conditioner, rinse, bath agent, bath medicine, face
wash, shaving cream, hair cream, hair lotion, hair treatment,
hair pack, gloss, lip cream, cake, and the like. The present
invention is particularly effective in applications in which
10 a moisturizing effect is desired. For example, the present
invention is effective as a skincare cosmetic preparation.
The invention is particularly effective in applications
contacting with the skin for a long period of time, but also
effective in applications such as face wash and shampoo where
15 it is washed away after use for a short period of time.
[0135] As described above, cosmetics are also included in
the cosmetic preparations. Cosmetics are classified into
cosmetics for cleaning, cosmetics for hair, basic cosmetics,
20 makeup cosmetics, fragrant cosmetics, cosmetics for sun-burn,
cosmetics for anti-sunburn, nail cosmetics, eye liner
cosmetics, eye shadow cosmetics, rouge for cheek, lip
cosmetics, oral cavity cosmetics, and the like. The present
invention is effective in any application of them.
25
[0136] Furthermore, the external preparation for skin may
be a pharmaceutical product or a quasi-drug. For example,
the α-lipoic acid nanoparticles may be blended to an ointment
containing a pharmaceutically effective component.
30
[0137] Blending of α-lipoic acid nanoparticles to an
external preparation for skin (for example, cosmetic or quasi
drug such as emulsion, skin lotion, cream, shampoo, or face
- 50 - EG026
wash) results in an external preparation for skin that is
effective in the prevention and treatment of wrinkles, spots,
freckles, pigmentation and the like. The external
preparation for skin of the present invention also increases
5 skin moisturization and is effective for alleviation of
symptoms such as dry skin, skin roughening, allergy and atopic
dermatitis. The external preparation for skin of the present
invention activates skin metabolism by exerting an
antioxidant capacity. Further, the external preparation
10 for skin of the present invention rapidly removes melanin
dye and active oxygen generated by ultraviolet irradiation,
thus exhibiting a whitening effect, and is effective for
preventing skin damage. Therefore, the external
preparation for skin containing the α-lipoic acid
15 nanoparticles of the present invention is effective to
alleviate adverse effect on the skin caused by drying and
ultraviolet ray, to improve pigmentation disorder such as
spots or freckles, and to delay aging phenomena such as
dullness, wrinkles, sag and alopecia.
20
[0138] Examples of the dosage forms of the external
preparation for skin of the present invention include
ointments, thickening gel systems, lotions, water in oil
emulsions, oil in water emulsions, solids, sheets, powders,
25 gels, mousse and sprays. The external preparation for skin
may be a product in the shape of a sheet impregnated with
the preparation such as a makeup removing facial mask.
[0139] When the dosage form of the external preparation for
30 skin is a lotion, emulsion, thickening gel system or the
like, in terms of improvement of its effect, it is preferable
to blend among the components above, in particular among
the thickeners, a water-soluble thickener consisting of,
- 51 - EG026
for example, a plant-derived macromolecule such as gum arabic,
tragacanth gum, galactan, guar gum, carrageenan, locust bean
gum, pectin, quince seed (Cydonia oblonga seed) extract,
or brown algae powder; a microbial-derived macromolecule
5 such as xanthan gum, dextran, or pullulan; an animal-derived
macromolecule such as collagen, casein, albumin, gelatin
or hyaluronic acid; starches such as carboxymethyl starch
or methylhydroxy starch; celluloses such as methylcellulose,
nitrocellulose, ethyl cellulose, methylhydroxypropyl
10 cellulose, hydroxyethyl cellulose, cellulose sulfate salt,
hydroxypropyl cellulose, carboxymethyl cellulose,
crystalline cellulose, or cellulose powder; a vinyl-type
macromolecule such as polyvinyl alcohol, polyvinyl methyl
ether, polyvinyl pyrrolidone or carboxy vinyl polymer; an
15 acrylic-type macromolecule such as polyacrylic acid or the
salt thereof or polyacrylimide; an organic thickener such
as glycyrrhizic acid or alginic acid; an inorganic thickener
such as bentonite, hectolite, labonite, magnesium aluminum
silicate, or silicic anhydride; in combination with a lower
20 alcohol such as ethanol or isopropanol among the alcohols.
[0140] The external preparation for skin of the present
invention can be produced by a known method.
25 [0141] In the present specification, the concept of the
external preparation for skin also encompasses clothes
containing the α-lipoic acid nanoparticles used in the
utilization methods in which, by binding the α-lipoic acid
nanoparticles to the fibers, mixing the α-lipoic acid
30 nanoparticles into the fiber material, impregnating the
α-lipoic acid nanoparticles into fibers, or applying the
α-lipoic acid nanoparticles on the surface of fabric, the
α-lipoic acid nanoparticles are transdermally absorbed when
- 52 - EG026
the clothes (for example, underwear or the like) produced
from the fibers or fabric are contacted with the skin.
Binding of the α-lipoic acid nanoparticles to fibers can
be carried out by, for example, crosslinking or the like.
5 The method of binding a compound to fibers, the method of
mixing a compound into a fiber material, the method of
impregnating a compound into fibers, the method of applying
a compound on the surface of fabric, and the like are known
in the art.
10
[0142] Adding of the α-lipoic acid nanoparticles
synthesized by the method of the present invention, into
a external preparation for skin, does not require any special
process, and the α-lipoic acid nanoparticles are added
15 together with the raw materials in the early stage of the
production process of the external preparation for skin,
or added in the middle of the production process, or added
at the final stage of the production process. In regard to
the mode of addition, conventional methods such as mixing,
20 kneading, dissolution, immersion, spreading, spraying and
applying are selected in accordance with the kind and
properties of the external preparation for skin. The
external preparation for skin synthesized by the method of
the present invention can be prepared according to the methods
25 that are known to those skilled in the art.
[0143] When the content of the α-lipoic acid nanoparticles
contained in the external preparation for skin of the present
invention is converted to α-lipoic acid, the content is
30 preferably about 0.002% by weight or more, more preferably
about 0.01% by weight or more, even more preferably about
0.1% by weight or more, particularly preferably about 0.5%
by weight or more, and most preferably about 1.0% by weight
- 53 - EG026
or more. When the content of the α-lipoic acid nanoparticles
contained in the external preparation for skin of the present
invention is converted to α-lipoic acid, the content is
preferably about 10% by weight or less, more preferably about
5 8% by weight or less, even more preferably about 5% by weight
or less, particularly preferably about 4% by weight or less,
and most preferably about 3% by weight or less.
[0144] (4b. Sustained release external preparation for skin
10 containing α-lipoic acid nanoparticles)
The external preparation for skin of the present
invention may be a sustained release preparation. The
sustained release preparation may be a solid, a semi-solid
or a liquid, but is preferably a liquid.
15
[0145] Addition of the α-lipoic acid nanoparticles
synthesized by the method of the present invention, into
a sustained release preparation, does not require any special
process, and the α-lipoic acid nanoparticles are added
20 together with the raw materials in the early stage of the
production process of the sustained release preparation,
or added in the middle of the production process, or added
at the final stage of the production process. In regard to
the mode of addition, conventional methods such as mixing,
25 kneading, dissolution, immersion, spreading, spraying and
applying, are selected in accordance with the kind and
properties of the sustained release preparation. The
sustained release preparation of the present invention can
be prepared according to the methods that are known to those
30 skilled in the art.
[0146] When the content of the α-lipoic acid nanoparticles
contained in the sustained release preparation of the present
- 54 - EG026
invention is converted to α-lipoic acid, the content is
preferably about 0.002% by weight or more, more preferably
about 0.01% by weight or more, even more preferably about
0.1% by weight or more, particularly preferably about 0.5%
5 by weight or more, and most preferably about 1.0% by weight
or more. When the content of the α-lipoic acid nanoparticles
contained in the sustained release preparation of the present
invention is converted to α-lipoic acid, the content is
preferably about 10% by weight or less, more preferably about
10 8% by weight or less, even more preferably about 5% by weight
or less, particularly preferably about 4% by weight or less,
and most preferably about 3% by weight or less.
[0147] (4c. Composition for oral cavity containing α-lipoic
15 acid nanoparticles)
The composition for oral cavity of the present invention
contains the α-lipoic acid nanoparticles of the present
invention. The composition for oral cavity may be any
composition for oral cavity. The composition for oral cavity
20 may be a solid, a semisolid or a liquid, but is preferably
a liquid. Examples of the composition for oral cavity include
toothpaste (for example, cream toothpaste, powdered
toothpaste, and the like), dental cream, oral rinse
(including mouthwash), mouth spray, disintegrative film,
25 gel, and troche.
[0148] Adding of the α-lipoic acid nanoparticles
synthesized by the method of the present invention, into
a composition for oral cavity, does not require any special
30 process, and the α-lipoic acid nanoparticles are added
together with the raw materials in the early stage of the
production process of the composition for oral cavity, or
added in the middle of the production process, or added at
- 55 - EG026
the final stage of the production process. In regard to the
mode of addition, conventional methods such as mixing,
kneading, dissolution, immersion, spreading, spraying and
applying, are selected in accordance with the kind and
5 properties of the composition for oral cavity. The
composition for oral cavity of the present invention can
be prepared according to the methods that are known to those
skilled in the art.
10 [0149] When the content of the α-lipoic acid nanoparticles
contained in the composition for oral cavity of the present
invention is converted to α-lipoic acid, the content is
preferably about 0.002% by weight or more, more preferably
about 0.01% by weight or more, even more preferably about
15 0.1% by weight or more, particularly preferably about 0.5%
by weight or more, and most preferably about 1.0% by weight
or more. When the content of the α-lipoic acid nanoparticles
contained in the composition for oral cavity of the present
invention is converted to α-lipoic acid, the content is
20 preferably about 10% by weight or less, more preferably about
8% by weight or less, even more preferably about 5% by weight
or less, particularly preferably about 4% by weight or less,
and most preferably about 3% by weight or less.
25 [0150] (4e. Food containing α-lipoic acid nanoparticles)
The food of the present invention contains the α-lipoic
acid nanoparticles of the present invention. The food may
be any food. The food may be a solid, a semisolid or a liquid,
but is preferably a liquid. The food is preferably a health
30 food, and more preferably a health beverage, but the food
is not limited thereto. The health food may be used for the
same conventional applications as those of the α-lipoic acid
contained in the health food. Examples of the use and
- 56 - EG026
efficacy of the health food include wrinkles, spots, freckles,
pigmentation and the like.
[0151] The food may be, for example, frozen desserts (ice
5 cream, ice milk, iced dessert, and the like), favorite
beverages (for example, refreshing beverages, carbonated
drinks (cider, lemonade, and the like), flavoring drinks,
alcohol drinks, powdered juice and the like), dairy products
(milk, yogurt, ice cream, butter, margarine, cheese, whipping
10 cream, and the like), confectionery (Western confectionery,
Japanese confectionery, snacks, and the like, for example,
bean jam, bean jelly, buns with bean-jam filling, chocolate,
gum, jelly, agar, almond jelly, cake, castella, cookies,
rice crackers, tablet confectionery, and the like), bread,
15 rice cake, fishery processed products (kamaboko (boiled fish
paste), chikuwa (fish sausage), and the like), meat processed
products (sausage, ham, and the like), fruit processed
products (jam, marmalade, fruit sauce, and the like),
seasonings (dressing, mayonnaise, miso, and the like),
20 noodles (wheat noodles, buckwheat noodles, and the like),
pickles, and bottled products and canned products of meat,
fish and fruits, and the like.
[0152] Adding of the α-lipoic acid nanoparticles
25 synthesized by the method of the present invention, into
a food, does not require any special process, and the α-lipoic
acid nanoparticles are added together with the raw materials
in the early stage of the production process of the food,
or added in the middle of the production process, or added
30 at the final stage of the production process. In regard to
the mode of addition, conventional methods such as mixing,
kneading, dissolution, immersion, spreading, spraying and
applying, are selected in accordance with the kind and
- 57 - EG026
properties of the food. The food of the present invention
can be prepared according to the methods that are known to
those skilled in the art.
5 [0153] When the content of the α-lipoic acid nanoparticles
contained in the food of the present invention is converted
to α-lipoic acid, the content is preferably about 0.01% by
weight or more, more preferably about 0.05% by weight or
more, even more preferably about 0.1% by weight or more,
10 particularly preferably about 0.5% by weight or more, and
most preferably about 1.0% by weight or more. When the
content of the α-lipoic acid nanoparticles contained in the
food of the present invention is converted to α-lipoic acid,
the content is preferably about 10% by weight or less, more
15 preferably about 8% by weight or less, even more preferably
about 5% by weight or less, particularly preferably about
4% by weight or less, and most preferably about 3% by weight
or less.
20 [0154] (4f. Pharmaceutical product containing α-lipoic
acid nanoparticles)
The pharmaceutical product of the present invention
contains the α-lipoic acid nanoparticles of the present
invention. The pharmaceutical product may be any
25 pharmaceutical product. The form of the pharmaceutical
product may be any form. The pharmaceutical product of the
present invention may be a powder, a granule, a tablet, a
capsule, a pill, a liquid, a dispersion, an ointment, a cream
or the like. When the pharmaceutical product of the present
30 invention is used for the applications of oral administration,
the pharmaceutical product of the present invention is
preferably in the form of a tablet, powdered preparation,
liquid for internal use, capsule or the like. When the
- 58 - EG026
pharmaceutical product of the present invention is used for
the applications of parenteral administration, the
pharmaceutical product is preferably an injectable
preparation, an ointment or a cream, but is not limited thereto.
5 By using the pharmaceutical product of the present invention,
a sustained release effect can be obtained as the α-lipoic
acid nanoparticles slowly degrade in the body.
[0155] The pharmaceutical product of the present invention
10 may be used for the same conventional applications as those
of conventional pharmaceutical products containing α-lipoic
acid as a main ingredient. Examples of the use and efficacy
of the pharmaceutical product of the present invention
include supplementation upon an increase in the demand of
15 thioctic acid (at the time of vigorous physical labor), Leigh
syndrome (subacute necrotic encephalomyelitis), and toxic
(due to streptomycin or kanamycin) and noise-induced
(occupational) inner ear hearing impairment. Also, the
pharmaceutical product of the present invention may be an
20 infusion preparation or an injectable preparation for the
detoxification of heavy metals. The pharmaceutical product
of the present invention may also be a pharmaceutical product
for oral administration intended for the treatment of
diabetes.
25
[0156] Adding of the α-lipoic acid nanoparticles
synthesized by the method of the present invention, into
a pharmaceutical product, does not require any special
process, and the α-lipoic acid nanoparticles are added
30 together with the raw materials in the early stage of the
production process of the pharmaceutical product, or added
in the middle of the production process, or added at the
final stage of the production process. In regard to the mode
- 59 - EG026
of addition, conventional methods such as mixing, kneading,
dissolution, immersion, spreading, spraying and applying,
are selected in accordance with the kind and properties of
the pharmaceutical product. The pharmaceutical product of
5 the present invention can be prepared according to the methods
that are known to those skilled in the art.
[0157] When the content of the α-lipoic acid nanoparticles
contained in the pharmaceutical product of the present
10 invention is converted to α-lipoic acid, the content is
preferably about 0.01% by weight or more, more preferably
about 0.05% by weight or more, even more preferably about
0.1% by weight or more, particularly preferably about 0.5%
by weight or more, and most preferably about 1.0% by weight
15 or more. When the content of the α-lipoic acid nanoparticles
contained in the pharmaceutical product of the present
invention is converted to α-lipoic acid, the content is
preferably about 10% by weight or less, more preferably about
8% by weight or less, even more preferably about 5% by weight
20 or less, particularly preferably about 4% by weight or less,
and most preferably about 3% by weight or less.
EXAMPLES
25 [0158] In the following Examples and Comparative Examples,
the following substances were used as the reagents:
α-Lipoic acid: α-lipoic acid, special grade,
manufactured by Wako Pure Chemical Industries, Ltd. (purity
98% or higher, powder form);
30 Sucrose lauric acid ester: Ryoto Sugar Ester L-1695 (HLB
value about 15; linked fatty acid about 99%; monoester about
80%; di-, tri-, and poly-ester about 20%) manufactured by
Mitsubishi-Kagaku Foods Corporation;
- 60 - EG026
Polyoxyethylene (60) hydrogenated castor oil: NIKKOL
HCO-60 (HLB about 14; paste to solid, white to pale yellow
in color) manufactured by Nikko Chemicals Co., Ltd.;
Polyoxyethylene octyl dodecyl ether: EMULGEN 2020G-HA
5 (HLB value 13.0) manufactured by Kao Corporation;
POE(20)POP(8) cetyl ether: NIKKOL PBC44 (HLB about 12.5;
solid, white to pale yellow in color) manufactured by Nikko
Chemicals Co., Ltd.;
POE(20) stearyl ether: NIKKOL BS-20 (HLB 18.0; solid,
10 white to pale yellow in color) manufactured by Nikko Chemicals
Co., Ltd.;
Polyoxyethylene (20) sorbitan monooleic acid ester:
Polysorbate (80) (HLB about 15; colorless transparent liquid)
manufactured by NOF Corp.;
15 MgCl2: commercially available product, reagent grade;
CaCl2: commercially available product, reagent grade;
Zinc gluconate: commercially available product, reagent
grade;
Na2CO3: commercially available product, reagent grade;
20 and
Na2HPO4: commercially available product, reagent grade.
[0159] (Example 1: Production of α-lipoic acid-MgCO3
nanoparticles)
25 (Example 1A)
0.5 g of α-lipoic acid was added to 9 mL of ion-exchanged
water and mixed, and 5M NaOH was added to this mixed liquid
to adjust the pH of the mixed liquid to 7.2. When the pH
reached 7.2, the powder of α-lipoic acid disappeared, and
30 a transparent appearance such as in a solution was obtained.
Ion-exchanged water was added to this solution to result
in a volume of 10 mL. This solution was used as a stock solution,
and an aliquot of 100 μL was collected. This was added to
- 61 - EG026
0.9 mL of distilled water containing 0.1 g of Ryoto Sugar
Ester L-1695, and stirred satisfactorily. Stirring was
carried out for about 30 minutes, and then 20 μL of 0.5 M
MgCl2 was added to the resulting solution and stirred.
5 Stirring was carried out for 30 minutes, and then 20 μL of
0.1M Na2CO3 was added to this solution, and then further stirred.
Thereby, a transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained. This transparent
dispersion liquid was stirred for a whole day (24 hours),
10 and then the dispersion liquid was freeze-dried overnight
to obtain a paste. When these α-lipoic acid-MgCO3
nanoparticles were to be used in other tests, the paste after
freeze-drying was redispersed in distilled water to a
predetermined concentration before use. This paste after
15 freeze-drying was added to distilled water, satisfactorily
redispersed, and thereby, a transparent dispersion liquid
was obtained. This indicates that the α-lipoic acid-MgCO3
nanoparticles are stable after freeze-drying.
20 [0160] (Example 1B)
A paste containing α-lipoic acid-MgCO3 nanoparticles was
obtained by the same procedure as that in Example 1A, except
that the same amount of distilled water was used instead
of the ion-exchanged water for the preparation of the stock
25 solution. When these α-lipoic acid-MgCO3 nanoparticles were
to be used in other tests, the paste after freeze-drying
was redispersed in distilled water to a predetermined
concentration before use. This paste after freeze-drying
was added to distilled water. This paste was satisfactorily
30 redispersed, and thereby a transparent dispersion liquid
was obtained. This indicates that the α-lipoic acid-MgCO3
nanoparticles are stable after freeze-drying.
- 62 - EG026
[0161] (Example 2: Production of α-lipoic acid-MgCO3
nanoparticles)
(Example 2A)
0.25 g of α-lipoic acid was added to 9 mL of ion-exchanged
5 water and mixed, and 5M NaOH was added to this mixed liquid
to adjust the pH of the mixed liquid to 7.1. When the pH
reached 7.1, the powder of α-lipoic acid disappeared, and
a transparent appearance such as in a solution was obtained.
Ion-exchanged water was added to this solution to result
10 in a volume of 10 mL. This solution was used as a stock solution,
and an aliquot of 100 μL was collected. This was added to
0.9 mL of distilled water containing 0.1 g of Ryoto Sugar
Ester L-1695, and stirred satisfactorily. Stirring was
carried out for about 30 minutes, and then 20 μL of 0.5 M
15 MgCl2 was added to the resulting solution and stirred.
Stirring was carried out for 30 minutes, and then 20 μL of
0.1M Na2CO3 was added to this solution, and then further stirred.
Thereby, a transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained. This transparent
20 dispersion liquid was stirred for a whole day (24 hours),
and then the dispersion liquid was freeze-dried overnight
to obtain a paste. When these α-lipoic acid-MgCO3
nanoparticles were to be used in other tests, the paste after
freeze-drying was redispersed in distilled water to a
25 predetermined concentration before use. This paste after
freeze-drying was added to distilled water, satisfactorily
redispersed, and thereby, a transparent dispersion liquid
was obtained. This indicates that the α-lipoic acid-MgCO3
nanoparticles are stable after freeze-drying.
30
[0162] (Example 2B)
A paste containing α-lipoic acid-MgCO3 nanoparticles was
obtained by the same procedure as that in Example 2A, except
- 63 - EG026
that the same amount of distilled water was used instead
of the ion-exchanged water for the preparation of the stock
solution. When these α-lipoic acid-MgCO3 nanoparticles were
to be used in other tests, the paste after freeze-drying
5 was redispersed in distilled water to a predetermined
concentration before use. This paste after freeze-drying
was added to distilled water. This paste was satisfactorily
redispersed, and thereby, a transparent dispersion liquid
was obtained. This indicates that the α-lipoic acid-MgCO3
10 nanoparticles are stable after freeze-drying.
[0163] (Comparative Example 1: Production of α-lipoic acid
dispersion liquid)
(Comparative Example 1A)
15 0.5 g of α-lipoic acid was added to 9 mL of ion-exchanged
water and mixed, and 5M NaOH was added to this mixed liquid
to adjust the pH of the mixed liquid to 7.2. When the pH
reached about 7.2, the powder of α-lipoic acid disappeared,
and a transparent appearance such as in a solution was obtained.
20 Ion-exchanged water was added to this solution to result
in a volume of 10 mL. This solution was used as a stock solution,
and an aliquot of 100 μL was collected. This was added to
0.9 mL of distilled water containing 0.1 g of Ryoto Sugar
Ester L-1695, and stirred satisfactorily. This dispersion
25 liquid was stirred for a whole day (24 hours), and then the
dispersion liquid was freeze-dried overnight to obtain a
paste.
[0164] (Comparative Example 1B)
30 A paste was obtained by the same procedure as that in
Comparative Example 1A, except that the same amount of
distilled water was used instead of the ion-exchanged water
for the preparation of the stock solution.
- 64 - EG026
[0165] (Measurement Example 1: Measurement of particle
size)
0.3 g of each of the paste of α-lipoic acid-MgCO3
5 nanoparticles produced and used in Example 1A and the paste
of α-lipoic acid nanoparticles of Comparative Example 1A
produced without adding magnesium chloride and sodium
carbonate were respectively added to 3 mL of water, left
to stand at 4°C for about 3 hours, and then stirred for one
10 minute to disperse. The particle sizes were measured using
a light scattering photometer (Otsuka Electronics Co., Ltd.,
ELS-710 TY). As a result, it was confirmed that the particle
size of the α-lipoic acid-MgCO3 nanoparticles produced in
Example 1A was about 10 nm, and the particle size of the
15 α-lipoic acid nanoparticles of Comparative Example 1A
produced without adding magnesium chloride and sodium
carbonate was about 760 nm. The particle sizes when distilled
water was used and when ion-exchanged water was used were
almost the same. The results obtained by using Otsuka
20 Electronics Co., Ltd., ELS-710TY for measuring the particle
size distribution of the α-lipoic acid-MgCO3 nanoparticles
produced by using distilled water in Example 1A is shown
in FIG. 1, and the results obtained by using a light scattering
photometer (Otsuka Electronics Co., Ltd., ELS-710TY) for
25 measuring the particle size distribution of the α-lipoic
acid nanoparticles produced by using distilled water in
Comparative Example 1A is presented in FIG. 2.
[0166] (Example 3: Production of α-lipoic acid-MgCO3
30 nanoparticles)
(Example 3A)
0.5 g of α-lipoic acid was added to 9 mL of ion-exchanged
water and mixed, and 5M NaOH was added to this mixed liquid
- 65 - EG026
to adjust the pH of the mixed liquid to 7.0. When the pH
reached 7.0, the powder of α-lipoic acid disappeared, and
a transparent appearance such as in a solution was obtained.
Ion-exchanged water was added to this solution to result
5 in a volume of 10 mL. This solution was used as a stock solution,
and an aliquot of 100 μL was collected. This was added to
0.9 mL of distilled water containing 0.1 g of HCO-60, and
stirred satisfactorily. Stirring was carried out for about
30 minutes, and then 20 μL of 0.5 M MgCl2 was added to the
10 resulting solution and stirred. Stirring was carried out
for 30 minutes, and then 20 μL of 0.1M Na2CO3 was added to
this solution, and then further stirred. Thereby, a
transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained.
15
[0167] (Example 3B)
A transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained by the same procedure
as that in Example 3A, except that the same amount of distilled
20 water was used instead of the ion-exchanged water for the
preparation of the stock solution.
[0168] (Example 4: Production of α-lipoic acid-MgCO3
nanoparticles)
25 (Example 4A)
0.5 g of α-lipoic acid was added to 9 mL of ion-exchanged
water and mixed, and 5M NaOH was added to this mixed liquid
to adjust the pH of the mixed liquid to 7.3. When the pH
reached 7.3, the powder of α-lipoic acid disappeared, and
30 a transparent appearance such as in a solution was obtained.
Ion-exchanged water was added to this solution to result
in a volume of 10 mL. This solution was used as a stock solution,
and an aliquot of 50 μL was collected. This was added to
- 66 - EG026
0.95 mL of distilled water containing 0.02 g of EMULGEN
2020G-HA, and stirred satisfactorily. Stirring was carried
out for about 30 minutes, and then 10 μL of 0.5 M MgCl2 was
added to the solution and stirred. Stirring was carried out
5 for 30 minutes, and then 5 μL of 0.1M Na2CO3 was added to
this solution, and then further stirred. Thereby, a
transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained.
10 [0169] (Example 4B)
A transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained by the same procedure
as that in Example 4A, except that the same amount of distilled
water was used instead of the ion-exchanged water for the
15 preparation of the stock solution.
[0170] (Example 5: Production of α-lipoic acid-CaCO3
nanoparticles)
(Example 5A)
20 0.5 g of α-lipoic acid was added to 9 mL of ion-exchanged
water and mixed, and 5M NaOH was added to this mixed liquid
to adjust the pH of the mixed liquid to 7.1. When the pH
reached 7.1, the powder of α-lipoic acid disappeared, and
a transparent appearance such as in a solution was obtained.
25 Ion-exchanged water was added to this solution to result
in a volume of 10 mL. This solution was used as a stock solution,
and an aliquot of 50 μL was collected. This was added to
0.95 mL of distilled water containing 0.05 g of HCO-60, and
stirred satisfactorily. Stirring was carried out for about
30 30 minutes, then the pH of the solution was adjusted with
0.1M HCl or 0.1M NaOH to 6.6, and then 10 μL of 0.5M CaCl2
was added and stirred. Stirring was carried out for about
30 minutes, 10 μL of 0.1M Na2CO3 was added to this solution,
- 67 - EG026
and then further stirred. Thereby, a transparent dispersion
liquid containing α-lipoic acid-CaCO3 nanoparticles was
obtained. This transparent dispersion liquid was stirred
for a whole day (24 hours), and then the dispersion liquid
5 was freeze-dried overnight to obtain a paste.
[0171] (Example 5B)
A paste containing α-lipoic acid-CaCO3 nanoparticles was
obtained by the same procedure as that in Example 5A, except
10 that the same amount of distilled water was used instead
of the ion-exchanged water for the preparation of the stock
solution.
[0172] (Example 6: Production of α-lipoic acid-CaCO3
15 nanoparticles)
(Example 6A)
0.5 g of α-lipoic acid was added to 9 mL of ion-exchanged
water and mixed, and 5M NaOH was added to this mixed liquid
to adjust the pH of the mixed liquid to 7.0. When the pH
20 reached 7.0, the powder of α-lipoic acid disappeared, and
a transparent appearance such as in a solution was obtained.
Ion-exchanged water was added to this solution to result
in a volume of 10 mL. This solution was used as a stock solution,
and an aliquot of 20 μL was collected. This was added to
25 0.98 mL of distilled water containing 0.02 g of EMULGEN
2020G-HA, and stirred satisfactorily. Stirring was carried
out for about 30 minutes, then the pH of the solution was
adjusted with 0.1M HCl or 0.1M NaOH to 6.2, and then 5 μL
of 0.5M CaCl2 was added and stirred. Stirring was carried
30 out for 30 minutes, and then 5 μL of 0.1M Na2CO3 was added
to this solution, and then further stirred. Thereby, a
transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained.
- 68 - EG026
[0173] (Example 6B)
A transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained by the same procedure
5 as that in Example 6A, except that the same amount of distilled
water was used instead of the ion-exchanged water for the
preparation of the stock solution.
[0174] (Example 7: Production of α-lipoic acid-CaPO4
10 nanoparticles)
(Example 7A)
0.5 g of α-lipoic acid was added to 9 mL of ion-exchanged
water and mixed, and 5M NaOH was added to this mixed liquid
to adjust the pH of the mixed liquid to 6.9. When the pH
15 reached 6.9, the powder of α-lipoic acid disappeared, and
a transparent appearance such as in a solution was obtained.
Ion-exchanged water was added to this solution to result
in a volume of 10 mL. This solution was used as a stock solution,
and an aliquot of 20 μL was collected. This was added to
20 0.98 mL of distilled water containing 0.02 g of HCO-60, and
stirred satisfactorily. Stirring was carried out for about
30 minutes, then the pH of the solution was adjusted with
0.1M HCl or 0.1M NaOH to 6.4, and then 5 μL of 0.5M CaCl2
was added and stirred. Stirring was carried out for 30
25 minutes, 5 μL of 0.1M Na2HPO4 was added to this solution,
and then further stirred. Thereby, a transparent dispersion
liquid containing α-lipoic acid-CaPO4 nanoparticles was
obtained. This transparent dispersion liquid was stirred
for a whole day (24 hours), and then the dispersion liquid
30 was freeze-dried overnight to obtain a paste.
[0175] (Example 7B)
A paste containing α-lipoic acid-CaPO4 nanoparticles was
- 69 - EG026
obtained by the same procedure as that in Example 7A, except
that the same amount of distilled water was used instead
of the ion-exchanged water for the preparation of the stock
solution.
5
[0176] (Example 8: Production of α-lipoic acid-CaCO3
nanoparticles)
(Example 8A)
0.5 g of α-lipoic acid was added to 9 mL of ion-exchanged
10 water and mixed, and 1M NaOH was added to this mixed liquid
to adjust the pH of the mixed liquid to 11.7. When the pH
reached 11.7, the powder of α-lipoic acid disappeared, and
a transparent appearance such as in a solution was obtained.
Ion-exchanged water was added to this solution to result
15 in a volume of 10 mL. This solution was used as a stock solution,
and an aliquot of 100 μL was collected. This was added to
0.9 mL of distilled water containing 0.1g of POE(20)POP(8)
cetyl ether (PBC44), and stirred satisfactorily. Stirring
was carried out for about 30 minutes, then the pH of the
20 solution was adjusted with 0.1M HCl or 0.1M NaOH to 11.0,
and then 40 μL of 0.5M CaCl2 was added and stirred. Stirring
was carried out for 30 minutes, and then 4 μL of 0.1M Na2CO3
was added to this solution, and then further stirred. Thereby,
a transparent dispersion liquid containing α-lipoic
25 acid-CaCO3 nanoparticles was obtained.
[0177] (Example 8B)
A transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained by the same procedure
30 as that in Example 8A, except that the same amount of distilled
water was used instead of the ion-exchanged water for the
preparation of the stock solution.
- 70 - EG026
[0178] (Example 9: Production of α-lipoic acid-CaCO3
nanoparticles)
(Example 9A)
0.5 g of α-lipoic acid was added to 9 mL of ion-exchanged
5 water and mixed, and 1M NaOH was added to this mixed liquid
to adjust the pH of the mixed liquid to 11.5. When the pH
reached 11.5, the powder of α-lipoic acid disappeared, and
a transparent appearance such as in a solution was obtained.
Ion-exchanged water was added to this solution to result
10 in a volume of 10 mL. This solution was used as a stock solution,
and an aliquot of 100 μL was collected. This was added to
0.9 mL of distilled water containing 0.02g of POE(20) stearyl
ether, and stirred satisfactorily. Stirring was carried out
for about 30 minutes, then the pH of the solution was adjusted
15 with 0.1M HCl or 0.1M NaOH to 10.8, and then 40 μL of 0.5M
CaCl2 was added and stirred. Stirring was carried out for
30 minutes, and then 4 μL of 0.1M Na2CO3 was added to this
solution, and then further stirred. Thereby, a transparent
dispersion liquid containing α-lipoic acid-CaCO3
20 nanoparticles was obtained.
[0179] (Example 9B)
A transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained by the same procedure
25 as that in Example 9A, except that the same amount of distilled
water was used instead of the ion-exchanged water for the
preparation of the stock solution.
[0180] (Example 10: Production of α-lipoic acid-ZnCO3
30 nanoparticles)
(Example 10A)
0.5 g of α-lipoic acid was added to 9 mL of ion-exchanged
water and mixed, and 5M NaOH was added to this mixed liquid
- 71 - EG026
to adjust the pH of the mixed liquid to 6.8. When the pH
reached 6.8, the powder of α-lipoic acid disappeared, and
a transparent appearance such as in a solution was obtained.
Ion-exchanged water was added to this solution to result
5 in a volume of 10 mL. This solution was used as a stock solution,
and an aliquot of 100 μL was collected. This was added to
0.9 mL of distilled water containing 0.1 g of HCO-60, and
stirred satisfactorily. Stirring was carried out for about
30 minutes, and then the pH of the solution was adjusted
10 to 5.0 with 0.1M HCl, and then 20 μL of 5% zinc gluconate
solution was added and stirred. Stirring was carried out
for 30 minutes, and then 20 μL of 0.1M Na2CO3 was added to
this solution, and then further stirred. Thereby, a
transparent dispersion liquid containing α-lipoic
15 acid-ZnCO3 nanoparticles was obtained.
[0181] (Example 10B)
A transparent dispersion liquid containing α-lipoic
acid-ZnCO3 nanoparticles was obtained by the same procedure
20 as that in Example 10A, except that the same amount of distilled
water was used instead of the ion-exchanged water for the
preparation of the stock solution.
[0182] (Example 11: Production of α-lipoic acid-ZnCO3
25 nanoparticles)
(Example 11A)
0.5 g of α-lipoic acid was added to 9 mL of ion-exchanged
water and mixed, and 5M NaOH was added to this mixed liquid
to adjust the pH of the mixed liquid to 6.9. When the pH
30 reached 6.9, the powder of α-lipoic acid disappeared, and
a transparent appearance such as in a solution was obtained.
Ion-exchanged water was added to this solution to result
in a volume of 10 mL. This solution was used as a stock solution,
- 72 - EG026
and an aliquot of 100 μL was collected. This was added to
0.9 mL of distilled water containing 0.1 g of HCO-60, and
stirred satisfactorily. Stirring was carried out for about
30 minutes, and then the pH of the solution was adjusted
5 to 5.0 with 0.1M HCl, and then 20 μL of 0.5M zinc acetate
solution was added and stirred. Stirring was carried out
for 30 minutes, and then 20 μL of 0.1M Na2CO3 was added to
this solution, and then further stirred. Thereby, a
transparent dispersion liquid containing α-lipoic
10 acid-ZnCO3 nanoparticles was obtained.
[0183] (Example 11B)
A transparent dispersion liquid containing α-lipoic
acid-ZnCO3 nanoparticles was obtained by the same procedure
15 as that in Example 11A, except that the same amount of distilled
water was used instead of the ion-exchanged water for the
preparation of the stock solution.
[0184] (Example 12: Production of α-lipoic acid-MgCO3
20 nanoparticles)
(Example 12A)
0.5 g of α-lipoic acid was added to 9 mL of ion-exchanged
water and mixed, and 5M NaOH was added to this mixed liquid
to adjust the pH of the mixed liquid to 6.9. When the pH
25 reached 6.9, the powder of α-lipoic acid disappeared, and
a transparent appearance such as in a solution was obtained.
Ion-exchanged water was added to this solution to result
in a volume of 10 mL. This solution was used as a stock solution,
and an aliquot of 100 μL was collected. This was added to
30 0.9 mL of distilled water containing 0.1 g of Ryoto Sugar
Ester L-1695, and stirred satisfactorily. Stirring was
carried out for about 30 minutes, and then the pH of the
solution was adjusted to 6.8 with 0.1M HCl, and then 40 μL
- 73 - EG026
of 0.5M magnesium chloride solution was added and stirred.
Stirring was carried out for 30 minutes, and then 80 μL of
0.1M Na2CO3 was added to this solution, and then further stirred.
Thereby, a transparent dispersion liquid containing α-lipoic
5 acid-MgCO3 nanoparticles was obtained.
[0185] (Example 12B)
A transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained by the same procedure
10 as that in Example 12A, except that the same amount of distilled
water was used instead of the ion-exchanged water for the
preparation of the stock solution.
[0186] (Example 13: Production of α-lipoic acid-ZnCO3
15 nanoparticles)
(Example 13A)
0.5 g of α-lipoic acid was added to 9 mL of ion-exchanged
water and mixed, and 5M NaOH was added to this mixed liquid
to adjust the pH of the mixed liquid to 6.9. When the pH
20 reached 6.9, the powder of α-lipoic acid disappeared, and
a transparent appearance such as in a solution was obtained.
Ion-exchanged water was added to this solution to result
in a volume of 10 mL. This solution was used as a stock solution,
and an aliquot of 100 μL was collected. This was added to
25 0.9 mL of distilled water containing 0.1 g of HCO-60, and
stirred satisfactorily. Stirring was carried out for about
30 minutes, and then the pH of the solution was adjusted
to 3.9 with 0.1M HCl, and then 20 μL of 0.5M zinc acetate
solution was added and stirred. Stirring was carried out
30 for 30 minutes, and then 20 μL of 0.1M Na2CO3 was added to
this solution, and then further stirred. Thereby, a
transparent dispersion liquid containing α-lipoic
acid-ZnCO3 nanoparticles was obtained.
- 74 - EG026
[0187] (Example 13B)
A transparent dispersion liquid containing α-lipoic
acid-ZnCO3 nanoparticles was obtained by the same procedure
5 as that in Example 13A, except that the same amount of distilled
water was used instead of the ion-exchanged water for the
preparation of the stock solution.
[0188] (Example 14: Production of α-lipoic acid-CaCO3
10 nanoparticles)
(Example 14A)
0.5 g of α-lipoic acid was added to 9 mL of ion-exchanged
water and mixed, and 5M NaOH was added to this mixed liquid
to adjust the pH of the mixed liquid to 10.9. When the pH
15 reached 10.9, the powder of α-lipoic acid disappeared, and
a transparent appearance such as in a solution was obtained.
Ion-exchanged water was added to this solution to result
in a volume of 10 mL. This solution was used as a stock solution,
and an aliquot of 100 μL was collected. This was added to
20 0.9 mL of distilled water containing 0.1 g of HCO-60, and
stirred satisfactorily. Stirring was carried out for about
30 minutes, and then the pH of the solution was adjusted
to 6.4 with 0.1M HCl, and then 40 μL of 0.5M calcium chloride
solution was added and stirred. Stirring was carried out
25 for 30 minutes, and then 40 μL of 0.1M Na2CO3 was added to
this solution, and then further stirred. Thereby, a
transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained.
30 [0189] (Example 14B)
A transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained by the same procedure
as that in Example 14A, except that the same amount of distilled
- 75 - EG026
water was used instead of the ion-exchanged water for the
preparation of the stock solution.
[0190] (Example 15: Production of α-lipoic acid-CaCO3
5 nanoparticles)
(Example 15A)
0.5 g of α-lipoic acid was added to 9 mL of ion-exchanged
water and mixed, and 5M NaOH was added to this mixed liquid
to adjust the pH of the mixed liquid to 8.7. When the pH
10 reached 8.7, the powder of α-lipoic acid disappeared, and
a transparent appearance such as in a solution was obtained.
Ion-exchanged water was added to this solution to result
in a volume of 10 mL. This solution was used as a stock solution,
and an aliquot of 100 μL was collected. This was added to
15 0.9 mL of distilled water containing 0.1 g of HCO-60, and
stirred satisfactorily. Stirring was carried out for about
30 minutes, and then the pH of the solution was adjusted
to 6.3 with 0.1M HCl, and then 40 μL of 0.5M calcium chloride
solution was added and stirred. Stirring was carried out
20 for 30 minutes, and then 40 μL of 0.1M Na2CO3 was added to
this solution, and then further stirred. Thereby, a
transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained.
25 [0191] (Example 15B)
A transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained by the same procedure
as that in Example 15A, except that the same amount of distilled
water was used instead of the ion-exchanged water for the
30 preparation of the stock solution.
[0192] (Example 16: Production of α-lipoic acid-CaCO3
nanoparticles)
- 76 - EG026
(Example 16A)
0.5 g of α-lipoic acid was added to 9 mL of ion-exchanged
water and mixed, and 5M NaOH was added to this mixed liquid
to adjust the pH of the mixed liquid to 6.9. When the pH
5 reached 6.9, the powder of α-lipoic acid disappeared, and
a transparent appearance such as in a solution was obtained.
Ion-exchanged water was added to this solution to result
in a volume of 10 mL. This solution was used as a stock solution,
and an aliquot of 100 μL was collected. This was added to
10 0.9 mL of distilled water containing 0.1 g of HCO-60, and
stirred satisfactorily. Stirring was carried out for about
30 minutes, and then the pH of the solution was adjusted
to 6.4 with 0.1M HCl, and then 20 μL of 0.5M calcium chloride
solution was added and stirred. Stirring was carried out
15 for 30 minutes, and then 40 μL of 0.1M Na2CO3 was added to
this solution, and then further stirred. Thereby, a
transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained.
20 [0193] (Example 16B)
A transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained by the same procedure
as that in Example 16A, except that the same amount of distilled
water was used instead of the ion-exchanged water for the
25 preparation of the stock solution.
[0194] (Example 17: Production of α-lipoic acid-CaCO3
nanoparticles)
(Example 17A)
30 0.5 g of α-lipoic acid was added to 9 mL of ion-exchanged
water and mixed, and 5M NaOH was added to this mixed liquid
to adjust the pH of the mixed liquid to 6.9. When the pH
reached 6.9, the powder of α-lipoic acid disappeared, and
- 77 - EG026
a transparent appearance such as in a solution was obtained.
Ion-exchanged water was added to this solution to result
in a volume of 10 mL. This solution was used as a stock solution,
and an aliquot of 100 μL was collected. This was added to
5 0.9 mL of distilled water containing 0.1 g of EMULGEN 2020G-HA,
and stirred satisfactorily. Stirring was carried out for
about 30 minutes, and then the pH of the solution was adjusted
to 6.7 with 0.1M HCl, and then 20 μL of 0.5M calcium chloride
solution was added and stirred. Stirring was carried out
10 for 30 minutes, and then 40 μL of 0.1M Na2CO3 was added to
this solution, and then further stirred. Thereby, a
transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained.
15 [0195] (Example 17B)
A transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained by the same procedure
as that in Example 17A, except that the same amount of distilled
water was used instead of the ion-exchanged water for the
20 preparation of the stock solution.
[0196] (Example 18: Production of α-lipoic acid-CaCO3
nanoparticles)
(Example 18A)
25 0.5 g of α-lipoic acid was added to 9 mL of ion-exchanged
water and mixed, and 5M NaOH was added to this mixed liquid
to adjust the pH of the mixed liquid to 11.8. When the pH
reached 11.8, the powder of α-lipoic acid disappeared, and
a transparent appearance such as in a solution was obtained.
30 Ion-exchanged water was added to this solution to result
in a volume of 10 mL. This solution was used as a stock solution,
and an aliquot of 100 μL was collected. This was added to
0.9 mL of distilled water containing 0.1 g of HCO-60, and
- 78 - EG026
stirred satisfactorily. Stirring was carried out for about
30 minutes, and then the pH of the solution was adjusted
to 10.9 with 0.1M HCl, and then 20 μL of 0.5M CaCl2 was added
and stirred. Stirring was carried out for 30 minutes, and
5 then 20 μL of 0.1M Na2CO3 was added to this solution, and
then further stirred. Thereby, a transparent dispersion
liquid containing α-lipoic acid-CaCO3 nanoparticles was
obtained.
10 [0197] (Example 18B)
A transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained by the same procedure
as that in Example 18A, except that the same amount of distilled
water was used instead of the ion-exchanged water for the
15 preparation of the stock solution.
[0198] (Example 19: Production of α-lipoic acid-MgCO3
nanoparticles)
(Example 19A)
20 0.5 g of α-lipoic acid was added to 9 mL of ion-exchanged
water and mixed, and 5M NaOH was added to this mixed liquid
to adjust the pH of the mixed liquid to 9.1. When the pH
reached 9.1, the powder of α-lipoic acid disappeared, and
a transparent appearance such as in a solution was obtained.
25 Ion-exchanged water was added to this solution to result
in a volume of 10 mL. This solution was used as a stock solution,
and an aliquot of 100 μL was collected. This was added to
0.9 mL of distilled water containing 0.1 g of Ryoto Sugar
Ester L-1695, and stirred satisfactorily. Stirring was
30 carried out for about 30 minutes, and then the pH of the
solution was adjusted to 8.5 with 0.1M HCl, and then 20 μL
of 0.5M MgCl2 solution was added and stirred. Stirring was
carried out for 30 minutes, and then 20 μL of 0.1M Na2CO3
- 79 - EG026
was added to this solution, and then further stirred. Thereby,
a transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained.
5 [0199] (Example 19B)
A transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained by the same procedure
as that in Example 19A, except that the same amount of distilled
water was used instead of the ion-exchanged water for the
10 preparation of the stock solution.
[0200] (Example 20: Production of α-lipoic acid-MgCO3
nanoparticles)
(Example 20A)
15 0.28g of 1M NaOH was added to 0.05 g of α-lipoic acid,
mixed and stirred until it was completely dissolved. To this,
9.328ml of water for injection (Japan Parmacopeia water for
injection manufactured by Otsuka Pharmaceutical Co., Ltd.)
was added and mixed. To this mixed liquid, 0.3g of POE(20)
20 stearyl ether was added and stirred for 30 minutes or longer,
and then the pH of the solution was adjusted to 7.0 with
5N HCl. To this, 40 μL of 2.5M MgCl2 was added and stirred
satisfactorily, and then 2 μL of 1M Na2CO3 was added and further
stirred, and water for injection was added to result in a
25 volume of 10mL. Thereby, a transparent dispersion liquid
containing α-lipoic acid-MgCO3 nanoparticles was obtained.
[0201] (Example 20B)
A transparent dispersion liquid containing α-lipoic
30 acid-MgCO3 nanoparticles was obtained by the same procedure
as that in Example 20A, except that ion-exchanged water was
used instead of the water for injection.
- 80 - EG026
[0202] (Example 21: Production of α-lipoic acid-MgCO3
nanoparticles)
(Example 21A)
950μL of 0.26M NaOH was added to 0.05 g of α-lipoic acid,
5 mixed and stirred until it was completely dissolved. To this,
0.25g of POE(20) stearyl ether was added and stirred
satisfactorily, and then 3.626 mL of ion-exchanged water
was added to this and stirred for 30 minutes or longer. pH
of the solution was adjusted to 5.5 with 5N HCl. To this,
10 48 μL of 2.5M MgCl2 was added and stirred satisfactorily,
and then 48 μL of 1M Na2CO3 was added and further stirred,
and ion-exchanged water was added to result in 5mL. Thereby,
a transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained.
15
[0203] (Example 21B)
A transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained by the same procedure
as that in Example 21A, except that the same amount of distilled
20 water was used instead of the ion-exchanged water.
[0204] (Comparative Example 22-1: Production of α-lipoic
acid dispersion liquid)
(Comparative Example 22-1A)
25 To 4.0 g of polyoxyethylene hydrogenated castor oil
(HCO-60) which has been previously heated and melted, 0.5
g of an α-lipoic acid powder was added and mixed to dissolve
the α-lipoic acid. About 35 ml of distilled water was added
to this mixture and mixed for 30 minutes or longer. The pH
30 was adjusted to 6.8 with 5M NaOH, and then distilled water
was further added to result in a volume of 50 ml. Thereby,
an α-lipoic acid dispersion liquid was obtained.
- 81 - EG026
[0205] (Comparative Example 22-1B)
An α-lipoic acid dispersion liquid was obtained by the
same procedure as that in Comparative Example 22-1A, except
that the same amount of ion-exchanged water was used instead
5 of the distilled water.
[0206] (Comparative Example 22-2: Production of α-lipoic
acid dispersion liquid)
(Comparative Example 22-2A)
10 To 5.0 g of polyoxyethylene hydrogenated castor oil
(HCO-60) which has been previously heated and melted, 0.5
g of an α-lipoic acid powder was added and mixed to dissolve
the α-lipoic acid. About 35 ml of ion-exchanged water was
added to this mixture and mixed for 30 minutes or longer.
15 The pH was adjusted to 7.0 with 5M NaOH, and then ion-exchanged
water was further added to result in a volume of 50 ml. Thereby,
an α-lipoic acid dispersion liquid was obtained.
[0207] (Comparative Example 22-2B)
20 An α-lipoic acid dispersion liquid was obtained by the
same procedure as that in Comparative Example 22-2A, except
that the same amount of distilled water was used instead
of the ion-exchanged water.
25 [0208] (Example 22: Production of α-lipoic acid-CaCO3
nanoparticles)
(Example 22A)
To 4.0 g of polyoxyethylene hydrogenated castor oil
(HCO-60) which has been previously heated and melted, 0.5
30 g of an α-lipoic acid powder was added and mixed to dissolve
the α-lipoic acid. About 35 ml of distilled water was added
to this mixture and mixed for 30 minutes or longer. The pH
was adjusted to 4.6 with 5M NaOH. To this, 0.48 ml of 5 M
- 82 - EG026
CaCl2 aqueous solution was added and mixed, and then 0.96
ml of 1 M Na2CO3 aqueous solution was added and further mixed.
The pH of this solution was measured and adjusted to pH 6.7
with 1M NaOH or 1 M HCl. Distilled water was further added
5 to result in a volume of 50 ml. Thereby, a transparent
dispersion liquid containing α-lipoic acid-CaCO3
nanoparticles was obtained.
[0209] (Example 22B)
10 A transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained by the same procedure
as that in Example 22A, except that the same amount of
ion-exchanged water was used instead of the distilled water.
15 [0210] (Example 23: Production of α-lipoic acid-MgCO3
nanoparticles)
(Example 23A)
To 4.0 g of polyoxyethylene hydrogenated castor oil
(HCO-60) which has been previously heated and melted, 0.5
20 g of an α-lipoic acid powder was added and mixed to dissolve
the α-lipoic acid. About 35 ml of ion-exchanged water was
added to this mixture and mixed for 30 minutes or longer.
The pH was adjusted to 4.6 with 5M NaOH. To this, 0.96 ml
of 2.5 M MgCl2 aqueous solution was added and mixed, and then
25 0.96 ml of 1 M Na2CO3 aqueous solution was added and further
mixed. The pH of this solution was measured and adjusted
to pH 6.8 with 1M NaOH or 1 M HCl. Ion-exchanged water was
further added to result in a volume of 50 ml. Thereby, a
transparent dispersion liquid containing α-lipoic
30 acid-MgCO3 nanoparticles was obtained.
[0211] (Example 23B)
A transparent dispersion liquid containing α-lipoic
- 83 - EG026
acid-MgCO3 nanoparticles was obtained by the same procedure
as that in Example 23A, except that the same amount of distilled
water was used instead of the ion-exchanged water.
5 [0212] (Example 24: Production of α-lipoic acid-CaCO3
nanoparticles)
(Example 24A)
To 4.0 g of polyoxyethylene hydrogenated castor oil
(HCO-60) which has been previously heated and melted, 0.5
10 g of an α-lipoic acid powder was added and mixed to dissolve
the α-lipoic acid. About 35 ml of distilled water was added
to this mixture and mixed for 30 minutes or longer. The pH
was adjusted to 4.3 with 5M NaOH. To this, 0.24 ml of 5 M
CaCl2 aqueous solution was added and mixed, and then 0.24
15 ml of 1 M Na2CO3 aqueous solution was added and further mixed.
The pH of this solution was measured and adjusted to pH 6.7
with 1M NaOH or 1 M HCl. Distilled water was further added
to result in a volume of 50 ml. Thereby, a transparent
dispersion liquid containing α-lipoic acid-CaCO3
20 nanoparticles was obtained.
[0213] (Example 24B)
A transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained by the same procedure
25 as that in Example 24A, except that the same amount of
ion-exchanged water was used instead of the distilled water.
[0214] (Example 25: Production of α-lipoic acid-MgCO3
nanoparticles)
30 (Example 25A)
To 3.5 g of polyoxyethylene hydrogenated castor oil
(HCO-60) which has been previously heated and melted, 0.5
g of an α-lipoic acid powder was added and mixed to dissolve
- 84 - EG026
the α-lipoic acid. About 35 ml of distilled water was added
to this mixture and mixed for 30 minutes or longer. The pH
was adjusted to 4.5 with 5M NaOH. To this, 0.48 ml of 2.5M
MgCl2 aqueous solution was added and mixed, and then 0.24
5 ml of 1 M Na2CO3 aqueous solution was added and further mixed.
The pH of this solution was measured and adjusted to pH 6.3
with 1M NaOH or 1M HCl. Distilled water was further added
to result in a volume of 50 ml. Thereby, a transparent
dispersion liquid containing α-lipoic acid-MgCO3
10 nanoparticles was obtained.
[0215] (Example 25B)
A transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained by the same procedure
15 as that in Example 25A, except that the same amount of
ion-exchanged water was used instead of the distilled water.
[0216] (Example 26: Production of α-lipoic acid-CaCO3
nanoparticles)
20 (Example 26A)
To 3.5 g of polyoxyethylene hydrogenated castor oil
(HCO-60) which has been previously heated and melted, 0.5
g of an α-lipoic acid powder was added and mixed to dissolve
the α-lipoic acid. About 35 ml of distilled water was added
25 to this mixture and mixed for 30 minutes or longer. The pH
was adjusted to 4.2 with 5M NaOH. To this, 0.24 ml of 5 M
CaCl2 aqueous solution was added and mixed, and then 0.72
ml of 1 M Na2CO3 aqueous solution was added and further mixed.
The pH of this solution was measured and adjusted to pH 6.9
30 with 1M NaOH or 1 M HCl. Distilled water was further added
to result in a volume of 50 ml. Thereby, a transparent
dispersion liquid containing α-lipoic acid-CaCO3
nanoparticles was obtained.
- 85 - EG026
[0217] (Example 26B)
A transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained by the same procedure
5 as that in Example 26A, except that the same amount of
ion-exchanged water was used instead of the distilled water.
[0218] (Example 27: Production of α-lipoic acid-CaCO3
nanoparticles)
10 (Example 27A)
To 4.5 g of polyoxyethylene hydrogenated castor oil
(HCO-60) which has been previously heated and melted, 0.5
g of an α-lipoic acid powder was added and mixed to dissolve
the α-lipoic acid. About 35 ml of ion-exchanged water was
15 added to this mixture and mixed for 30 minutes or longer.
The pH was adjusted to 4.5 with 5M NaOH. To this, 0.24 ml
of 5 M CaCl2 aqueous solution was added and mixed, and then
0.24 ml of 1 M Na2CO3 aqueous solution was added and further
mixed. The pH of this solution was measured and adjusted
20 to pH 6.8 with 1M NaOH or 1 M HCl. Ion-exchanged water was
further added to result in a volume of 50 ml. Thereby, a
transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained.
25 [0219] (Example 27B)
A transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained by the same procedure
as that in Example 27A, except that the same amount of distilled
water was used instead of the ion-exchanged water.
30
[0220] (Example 28: Production of α-lipoic acid-MgCO3
nanoparticles)
(Example 28A)
- 86 - EG026
To 4.5 g of polyoxyethylene hydrogenated castor oil
(HCO-60) which has been previously heated and melted, 0.5
g of an α-lipoic acid powder was added and mixed to dissolve
the α-lipoic acid. About 35 ml of ion-exchanged water was
5 added to this mixture and mixed for 30 minutes or longer.
The pH was adjusted to 4.5 with 5M NaOH. To this, 0.48 ml
of 2.5 M MgCl2 aqueous solution was added and mixed, and then
0.48 ml of 1 M Na2CO3 aqueous solution was added and further
mixed. The pH of this solution was measured and adjusted
10 to pH 6.8 with 1M NaOH or 1 M HCl. Ion-exchanged water was
further added to result in a volume of 50 ml. Thereby, a
transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained.
15 [0221] (Example 28B)
A transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained by the same procedure
as that in Example 28A, except that the same amount of distilled
water was used instead of the ion-exchanged water.
20
[0222] (Example 29: Production of α-lipoic acid-MgCO3
nanoparticles)
(Example 29A)
To 5.0 g of polyoxyethylene hydrogenated castor oil
25 (HCO-60) which has been previously heated and melted, 0.5
g of an α-lipoic acid powder was added and mixed to dissolve
the α-lipoic acid. About 35 ml of ion-exchanged water was
added to this mixture and mixed for 30 minutes or longer.
The pH was adjusted to 4.6 with 5M NaOH. To this, 0.96 ml
30 of 2.5 M MgCl2 aqueous solution was added and mixed, and then
1.44 ml of 1 M Na2CO3 aqueous solution was added and further
mixed. The pH of this solution was measured and adjusted
to pH 6.8 with 1M NaOH or 1 M HCl. Ion-exchanged water was
- 87 - EG026
further added to result in a volume of 50 ml. Thereby, a
transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained.
5 [0223] (Example 29B)
A transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained by the same procedure
as that in Example 29A, except that the same amount of distilled
water was used instead of the ion-exchanged water.
10
[0224] (Example 30: Production of α-lipoic acid-CaCO3
nanoparticles)
(Example 30A)
To 5.0 g of polyoxyethylene hydrogenated castor oil
15 (HCO-60) which has been previously heated and melted, 0.5
g of an α-lipoic acid powder was added and mixed to dissolve
the α-lipoic acid. About 35 ml of ion-exchanged water was
added to this mixture and mixed for 30 minutes or longer.
The pH was adjusted to 4.6 with 5M NaOH. To this, 0.48 ml
20 of 5 M CaCl2 aqueous solution was added and mixed, and then
0.48 ml of 1 M Na2CO3 aqueous solution was added and further
mixed. The pH of this solution was measured and adjusted
to pH 6.6 with 1M NaOH or 1 M HCl. Ion-exchanged water was
further added to result in a volume of 50 ml. Thereby, a
25 transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained.
[0225] (Example 30B)
A transparent dispersion liquid containing α-lipoic
30 acid-CaCO3 nanoparticles was obtained by the same procedure
as that in Example 30A, except that the same amount of distilled
water was used instead of the ion-exchanged water.
- 88 - EG026
[0226] (Example 31: Production of α-lipoic acid-CaCO3
nanoparticles)
(Example 31A)
To 4.0 g of polyoxyethylene (20) stearyl ether which
5 has been previously heated and melted, 0.5 g of an α-lipoic
acid powder was added and mixed to dissolve the α-lipoic
acid. About 35 ml of distilled water was added to this mixture
and mixed for 30 minutes or longer. The pH was adjusted to
4.3 with 5M NaOH. To this, 0.24 ml of 5 M CaCl2 aqueous solution
10 was added and mixed, and then 0.48 ml of 1 M Na2CO3 aqueous
solution was added and further mixed. The pH of this solution
was measured and adjusted to pH 6.9 with 1M NaOH or 1 M HCl.
Distilled water was further added to result in a volume of
50 ml. Thereby, a transparent dispersion liquid containing
15 α-lipoic acid-CaCO3 nanoparticles was obtained.
[0227] (Example 31B)
A transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained by the same procedure
20 as that in Example 31A, except that the same amount of
ion-exchanged water was used instead of the distilled water.
[0228] (Example 32: Production of α-lipoic acid-CaCO3
nanoparticles)
25 (Example 32A)
To 7.0 g of EMULGEN 2020G-HA which has been previously
heated and melted, 1.0 g of an α-lipoic acid powder was added
and mixed to dissolve the α-lipoic acid. About 70 ml of
distilled water was added to this mixture and mixed for 30
30 minutes or longer. The pH was adjusted to 4.6 with 5M NaOH.
To this, 0.24 ml of 5 M CaCl2 aqueous solution was added and
mixed, and then 0.24 ml of 1 M Na2CO3 aqueous solution was
added and further mixed. The pH of this solution was measured
- 89 - EG026
and adjusted to pH 6.8 with 1M NaOH or 1 M HCl. Distilled
water was further added to result in a volume of 50 ml. Thereby,
a transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained.
5
[0229] (Example 32B)
A transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained by the same procedure
as that in Example 32A, except that the same amount of
10 ion-exchanged water was used instead of the distilled water.
[0230] (Example 33: Production of α-lipoic acid-CaCO3
nanoparticles)
(Example 33A)
15 To 7.0 g of Polysorbate (80) which has been previously
heated and melted, 0.25 g of an α-lipoic acid powder was
added and mixed to dissolve the α-lipoic acid. About 35 ml
of distilled water was added to this mixture and mixed for
30 minutes or longer. The pH was adjusted to 4.3 with 5M
20 NaOH. To this, 0.12 ml of 5 M CaCl2 aqueous solution was
added and mixed, and then 0.12 ml of 1 M Na2CO3 aqueous solution
was added and further mixed. The pH of this solution was
measured and adjusted to pH 6.5 with 1M NaOH or 1 M HCl.
Distilled water was further added to result in a volume of
25 50 ml. Thereby, a transparent dispersion liquid containing
α-lipoic acid-CaCO3 nanoparticles was obtained.
[0231] (Example 33B)
A transparent dispersion liquid containing α-lipoic
30 acid-CaCO3 nanoparticles was obtained by the same procedure
as that in Example 33A, except that the same amount of
ion-exchanged water was used instead of the distilled water.
- 90 - EG026
[0232] (Example 34: Production of α-lipoic acid-MgCO3
nanoparticles)
(Example 34A)
To 7.0 g of Polysorbate (80) which has been previously
5 heated and melted, 0.25 g of an α-lipoic acid powder was
added and mixed to dissolve the α-lipoic acid. About 35 ml
of distilled water was added to this mixture and mixed for
30 minutes or longer. The pH was adjusted to 4.5 with 5M
NaOH. To this, 0.24 ml of 2.5 M MgCl2 aqueous solution was
10 added and mixed, and then 0.12 ml of 1 M Na2CO3 aqueous solution
was added and further mixed. The pH of this solution was
measured and adjusted to pH 6.8 with 1M NaOH or 1 M HCl.
Distilled water was further added to result in a volume of
50 ml. Thereby, a transparent dispersion liquid containing
15 α-lipoic acid-MgCO3 nanoparticles was obtained.
[0233] (Example 34B)
A transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained by the same procedure
20 as that in Example 34A, except that the same amount of
ion-exchanged water was used instead of the distilled water.
[0234] (Example 35: Production of α-lipoic acid-CaCO3
nanoparticles)
25 (Example 35A)
To 7.0 g of Polysorbate (80) which has been previously
heated and melted, 0.25 g of an α-lipoic acid powder was
added and mixed to dissolve the α-lipoic acid. About 35 ml
of distilled water was added to this mixture and mixed for
30 30 minutes or longer. The pH was adjusted to 4.3 with 5M
NaOH. To this, 0.24 ml of 5 M CaCl2 aqueous solution was
added and mixed, and then 0.48 ml of 1 M Na2CO3 aqueous solution
was added and further mixed. The pH of this solution was
- 91 - EG026
measured and adjusted to pH 6.5 with 1M NaOH or 1 M HCl.
Distilled water was further added to result in a volume of
50 ml. Thereby, a transparent dispersion liquid containing
α-lipoic acid-CaCO3 nanoparticles was obtained.
5
[0235] (Example 35B)
A transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained by the same procedure
as that in Example 35A, except that the same amount of
10 ion-exchanged water was used instead of the distilled water.
[0236] (Example 36: Production of α-lipoic acid-MgCO3
nanoparticles)
(Example 36A)
15 To 7.0 g of Polysorbate (80) which has been previously
heated and melted, 0.25 g of an α-lipoic acid powder was
added and mixed to dissolve the α-lipoic acid. About 35 ml
of distilled water was added to this mixture and mixed for
30 minutes or longer. The pH was adjusted to 4.5 with 5M
20 NaOH. To this, 0.48 ml of 2.5 M MgCl2 aqueous solution was
added and mixed, and then 0.48 ml of 1 M Na2CO3 aqueous solution
was added and further mixed. The pH of this solution was
measured and adjusted to pH 6.9 with 1M NaOH or 1 M HCl.
Distilled water was further added to result in a volume of
25 50 ml. Thereby, a transparent dispersion liquid containing
α-lipoic acid-MgCO3 nanoparticles was obtained.
[0237] (Example 36B)
A transparent dispersion liquid containing α-lipoic
30 acid-MgCO3 nanoparticles was obtained by the same procedure
as that in Example 36A, except that the same amount of
ion-exchanged water was used instead of the distilled water.
- 92 - EG026
[0238] (Example 37: Production of α-lipoic acid-CaCO3
nanoparticles)
(Example 37A)
To 4.0 g of polyoxyethylene (20) stearyl ether and 1.0
5 g of polyethylene glycol (1000) which have been previously
heated and melted, 0.5 g of an α-lipoic acid powder was added
and mixed to dissolve the α-lipoic acid. About 35 ml of
distilled water was added to this mixture and mixed for 30
minutes or longer. The pH was adjusted to 4.5 with 5M NaOH.
10 To this, 0.24 ml of 5 M CaCl2 aqueous solution was added and
mixed, and then 0.24 ml of 1 M Na2CO3 aqueous solution was
added and further mixed. The pH of this solution was measured
and adjusted to pH 6.6 with 1M NaOH or 1 M HCl. Distilled
water was further added to result in a volume of 50 ml. Thereby,
15 a transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained.
[0239] (Example 37B)
A transparent dispersion liquid containing α-lipoic
20 acid-CaCO3 nanoparticles was obtained by the same procedure
as that in Example 37A, except that the same amount of
ion-exchanged water was used instead of the distilled water.
[0240] (Example 38: Production of α-lipoic acid-CaCO3
25 nanoparticles)
(Example 38A)
To 4.0 g of polyoxyethylene (20) stearyl ether and 1.5
g of polyethylene glycol (4000) which have been previously
heated and melted, 0.5 g of an α-lipoic acid powder was added
30 and mixed to dissolve the α-lipoic acid. About 35 ml of
distilled water was added to this mixture and mixed for 30
minutes or longer. The pH was adjusted to 4.3 with 5M NaOH.
To this, 0.24 ml of 5 M CaCl2 aqueous solution was added and
- 93 - EG026
mixed, and then 0.24 ml of 1 M Na2CO3 aqueous solution was
added and further mixed. The pH of this solution was measured
and adjusted to pH 6.8 with 1M NaOH or 1 M HCl. Distilled
water was further added to result in a volume of 50 ml. Thereby,
5 a transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained.
[0241] (Example 38B)
A transparent dispersion liquid containing α-lipoic
10 acid-CaCO3 nanoparticles was obtained by the same procedure
as that in Example 38A, except that the same amount of
ion-exchanged water was used instead of the distilled water.
[0242] (Example 39: Production of α-lipoic acid-CaCO3
15 nanoparticles)
(Example 39A)
To 4.0 g of polyoxyethylene (20) stearyl ether and 2.0
g of polyethylene glycol (4000) which have been previously
heated and melted, 0.5 g of an α-lipoic acid powder was added
20 and mixed to dissolve the α-lipoic acid. About 35 ml of
distilled water was added to this mixture and mixed for 30
minutes or longer. The pH was adjusted to 4.5 with 5M NaOH.
To this, 0.24 ml of 5 M CaCl2 aqueous solution was added and
mixed, and then 0.24 ml of 1 M Na2CO3 aqueous solution was
25 added and further mixed. The pH of this solution was measured
and adjusted to pH 6.5 with 1M NaOH or 1 M HCl. Distilled
water was further added to result in a volume of 50 ml. Thereby,
a transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained.
30
[0243] (Example 39B)
A transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained by the same procedure
- 94 - EG026
as that in Example 39A, except that the same amount of
ion-exchanged water was used instead of the distilled water.
[0244] (Example 40: Production of α-lipoic acid-CaCO3
5 nanoparticles)
(Example 40A)
To 4.0 g of polyoxyethylene (20) stearyl ether and 2.0
g of polyethylene glycol (1000) which have been previously
heated and melted, 0.5 g of an α-lipoic acid powder was added
10 and mixed to dissolve the α-lipoic acid. About 35 ml of
distilled water was added to this mixture and mixed for 30
minutes or longer. The pH was adjusted to 4.4 with 5M NaOH.
To this, 0.24 ml of 5 M CaCl2 aqueous solution was added and
mixed, and then 0.24 ml of 1 M Na2CO3 aqueous solution was
15 added and further mixed. The pH of this solution was measured
and adjusted to pH 6.9 with 1M NaOH or 1 M HCl. Distilled
water was further added to result in a volume of 50 ml. Thereby,
a transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained.
20
[0245] (Example 40B)
A transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained by the same procedure
as that in Example 40A, except that the same amount of
25 ion-exchanged water was used instead of the distilled water.
[0246] (Example 41: Production of α-lipoic acid-MgCO3
nanoparticles)
(Example 41A)
30 To 4.0 g of polyoxyethylene (20) stearyl ether and 1.5
g of polyethylene glycol (1000) which have been previously
heated and melted, 0.5 g of an α-lipoic acid powder was added
and mixed to dissolve the α-lipoic acid. About 35 ml of
- 95 - EG026
ion-exchanged water was added to this mixture and mixed for
30 minutes or longer. The pH was adjusted to 4.5 with 5M
NaOH. To this, 0.48 ml of 2.5 M MgCl2 aqueous solution was
added and mixed, and then 0.24 ml of 1 M Na2CO3 aqueous solution
5 was added and further mixed. The pH of this solution was
measured and adjusted to pH 6.4 with 1M NaOH or 1 M HCl.
Ion-exchanged water was further added to result in a volume
of 50 ml. Thereby, a transparent dispersion liquid
containing α-lipoic acid-MgCO3 nanoparticles was obtained.
10
[0247] (Example 41B)
A transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained by the same procedure
as that in Example 41A, except that the same amount of distilled
15 water was used instead of the ion-exchanged water.
[0248] (Example 42: Production of α-lipoic acid-MgCO3
nanoparticles)
(Example 42A)
20 To 4.0 g of polyoxyethylene (20) stearyl ether and 1.5
g of polyethylene glycol (4000) which have been previously
heated and melted, 0.5 g of an α-lipoic acid powder was added
and mixed to dissolve the α-lipoic acid. About 35 ml of
ion-exchanged water was added to this mixture and mixed for
25 30 minutes or longer. The pH was adjusted to 4.6 with 5M
NaOH. To this, 0.48 ml of 2.5 M MgCl2 aqueous solution was
added and mixed, and then 0.24 ml of 1 M Na2CO3 aqueous solution
was added and further mixed. The pH of this solution was
measured and adjusted to pH 6.8 with 1M NaOH or 1 M HCl.
30 Ion-exchanged water was further added to result in a volume
of 50 ml. Thereby, a transparent dispersion liquid
containing α-lipoic acid-MgCO3 nanoparticles was obtained.
- 96 - EG026
[0249] (Example 42B)
A transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained by the same procedure
as that in Example 42A, except that the same amount of distilled
5 water was used instead of the ion-exchanged water.
[0250] (Example 43: Production of α-lipoic acid-MgCO3
nanoparticles)
(Example 43A)
10 To 5.0 g of polyoxyethylene (20) stearyl ether and 1.5
g of polyethylene glycol (1000) which have been previously
heated and melted, 0.5 g of an α-lipoic acid powder was added
and mixed to dissolve the α-lipoic acid. About 35 ml of
ion-exchanged water was added to this mixture and mixed for
15 30 minutes or longer. The pH was adjusted to 4.5 with 5M
NaOH. To this, 0.48 ml of 2.5 M MgCl2 aqueous solution was
added and mixed, and then 0.48 ml of 1 M Na2CO3 aqueous solution
was added and further mixed. The pH of this solution was
measured and adjusted to pH 6.7 with 1M NaOH or 1 M HCl.
20 Ion-exchanged water was further added to result in a volume
of 50 ml. Thereby, a transparent dispersion liquid
containing α-lipoic acid-MgCO3 nanoparticles was obtained.
[0251] (Example 43B)
25 A transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained by the same procedure
as that in Example 43A, except that the same amount of distilled
water was used instead of the ion-exchanged water.
30 [0252] (Example 44: Production of α-lipoic acid-CaCO3
nanoparticles)
(Example 44A)
To 5.0 g of polyoxyethylene (20) stearyl ether and 1.5
- 97 - EG026
g of polyethylene glycol (1000) which have been previously
heated and melted, 0.5 g of an α-lipoic acid powder was added
and mixed to dissolve the α-lipoic acid. About 35 ml of
ion-exchanged water was added to this mixture and mixed for
5 30 minutes or longer. The pH was adjusted to 4.6 with 5M
NaOH. To this, 0.24 ml of 5 M CaCl2 aqueous solution was
added and mixed, and then 0.72 ml of 1 M Na2CO3 aqueous solution
was added and further mixed. The pH of this solution was
measured and adjusted to pH 6.8 with 1M NaOH or 1 M HCl.
10 Ion-exchanged water was further added to result in a volume
of 50 ml. Thereby, a transparent dispersion liquid
containing α-lipoic acid-CaCO3 nanoparticles was obtained.
[0253] (Example 44B)
15 A transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained by the same procedure
as that in Example 44A, except that the same amount of distilled
water was used instead of the ion-exchanged water.
20 [0254] (Example 45: Production of α-lipoic acid-MgCO3
nanoparticles)
(Example 45A)
To 5.0 g of polyoxyethylene (20) stearyl ether and 1.5
g of polyethylene glycol (1000) which have been previously
25 heated and melted, 0.5 g of an α-lipoic acid powder was added
and mixed to dissolve the α-lipoic acid. About 35 ml of
ion-exchanged water was added to this mixture and mixed for
30 minutes or longer. The pH was adjusted to 4.5 with 5M
NaOH. To this, 0.48 ml of 2.5 M MgCl2 aqueous solution was
30 added and mixed, and then 0.72 ml of 1 M Na2CO3 aqueous solution
was added and further mixed. The pH of this solution was
measured and adjusted to pH 6.6 with 1M NaOH or 1 M HCl.
Ion-exchanged water was further added to result in a volume
- 98 - EG026
of 50 ml. Thereby, a transparent dispersion liquid
containing α-lipoic acid-MgCO3 nanoparticles was obtained.
[0255] (Example 45B)
5 A transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained by the same procedure
as that in Example 45A, except that the same amount of distilled
water was used instead of the ion-exchanged water.
10 [0256] (Example 46: Production of α-lipoic acid-CaCO3
nanoparticles)
(Example 46A)
To 5.0 g of polyoxyethylene (20) stearyl ether which
has been previously heated and melted, 0.5 g of an α-lipoic
15 acid powder was added and mixed to dissolve the α-lipoic
acid. To this, 15ml of polyethylene glycol (1000) solution,
which was made by dissolving 10 g of polyethylene glycol
(1000) in ion-exchanged water to result in 100ml, was added
and mixed. About 20 mL of ion-exchanged water was further
20 added and mixed for 30 minutes or longer. The pH was adjusted
to 4.5 with 5M NaOH. To this, 0.24 ml of 5 M CaCl2 aqueous
solution was added and mixed, and then 0.24 ml of 1 M Na2CO3
aqueous solution was added and further mixed. The pH of this
solution was measured and adjusted to pH 6.7 with 1M NaOH
25 or 1 M HCl. Ion-exchanged water was further added to result
in a volume of 50 ml. Thereby, a transparent dispersion
liquid containing α-lipoic acid-CaCO3 nanoparticles was
obtained.
30 [0257] (Example 46B)
A transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained by the same procedure
as that in Example 46A, except that the same amount of distilled
- 99 - EG026
water was used instead of the ion-exchanged water.
[0258] (Example 47: Production of α-lipoic acid-CaCO3
nanoparticles)
5 (Example 47A)
To 5.0 g of polyoxyethylene (20) stearyl ether which
has been previously heated and melted, 0.5 g of an α-lipoic
acid powder was added and mixed to dissolve the α-lipoic
acid. To this, 15ml of polyethylene glycol (4000) solution,
10 which was made by dissolving 10 g of polyethylene glycol
(4000) in ion-exchanged water to result in a volume of 100ml,
was added and mixed. About 20 ml of ion-exchanged water was
further added and mixed for 30 minutes or longer. The pH
was adjusted to 4.3 with 5M NaOH. To this, 0.24 ml of 5 M
15 CaCl2 aqueous solution was added and mixed, and then 0.24
ml of 1 M Na2CO3 aqueous solution was added and further mixed.
The pH of this solution was measured and adjusted to pH 6.6
with 1M NaOH or 1 M HCl. Ion-exchanged water was further
added to result in a volume of 50 ml. Thereby, a transparent
20 dispersion liquid containing α-lipoic acid-CaCO3
nanoparticles was obtained.
[0259] (Example 47B)
A transparent dispersion liquid containing α-lipoic
25 acid-CaCO3 nanoparticles was obtained by the same procedure
as that in Example 47A, except that the same amount of distilled
water was used instead of the ion-exchanged water.
[0260] (Example 48: Production of α-lipoic acid-CaCO3
30 nanoparticles)
(Example 48A)
To 5.0 g of polyoxyethylene (20) stearyl ether which
has been previously heated and melted, 0.5 g of an α-lipoic
- 100 - EG026
acid powder was added and mixed to dissolve the α-lipoic
acid. To this, 15ml of polyethylene glycol (6000) solution,
which was made by dissolving 10 g of polyethylene glycol
(6000) in ion-exchanged water to result in a volume of 100ml,
5 was added and mixed. About 20 ml of ion-exchanged water was
further added and mixed for 30 minutes or longer. The pH
was adjusted to 4.4 with 5M NaOH. To this, 0.24 ml of 5 M
CaCl2 aqueous solution was added and mixed, and then 0.24
ml of 1 M Na2CO3 aqueous solution was added and further mixed.
10 The pH of this solution was measured and adjusted to pH 6.8
with 1M NaOH or 1 M HCl. Ion-exchanged water was further
added to result in a volume of 50 ml. Thereby, a transparent
dispersion liquid containing α-lipoic acid-CaCO3
nanoparticles was obtained.
15
[0261] (Example 48B)
A transparent dispersion liquid containing α-lipoic
acid-CaCO3 nanoparticles was obtained by the same procedure
as that in Example 48A, except that the same amount of distilled
20 water was used instead of the ion-exchanged water.
[0262] (Example 49: Production of highly concentrated
α-lipoic acid-MgCO3 nanoparticles)
(Example 49A)
25 2.85ml of 0.26M NaOH was added to 0.15 g of α-lipoic
acid, mixed and stirred until it was completely dissolved.
To this, 0.75g of POE(20) stearyl ether was added and stirred
satisfactorily, and then 0.5 ml of distilled water was added
to this and stirred for 30 minutes or longer. The pH of the
30 solution was adjusted to 5.5 with 5N HCl. To this, 144 μL
of 2.5M MgCl2 was added and stirred for 12 hours or longer,
and then 144 μL of 1M Na2CO3 was added and stirred for further
12 hours or longer, and to this, distilled water was added
- 101 - EG026
to result in a volume of 5.0ml. Thereby, a transparent
dispersion liquid containing α-lipoic acid-MgCO3
nanoparticles was obtained.
5 [0263] (Example 49B)
A transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained by the same procedure
as that in Example 49A, except that ion-exchanged water was
used instead of the distilled water.
10
[0264] (Example 50: Production of α-lipoic acid-MgCO3
nanoparticles)
(Example 50A)
0.28g of 1M NaOH was added to 0.05 g of α-lipoic acid,
15 mixed and stirred until it was completely dissolved. To this,
9.35ml of water for injection (Japan Parmacopeia water for
injection manufactured by Otsuka Pharmaceutical Co., Ltd.)
was added and mixed. To this mixed liquid, 0.3 g of
polyoxyethylene hydrogenated castor oil (HCO-60) was added
20 and stirred for 30 minutes or longer. Then, the pH of the
solution was adjusted to 7.0 with 5N HCl. To this, 40 μL
of 2.5M MgCl2 was added and stirred satisfactorily, and then
20 μL of 1M Na2CO3 was added and further stirred, and water
for injection was added to result in a volume of 10mL. Thereby,
25 a transparent dispersion liquid containing α-lipoic
acid-MgCO3 nanoparticles was obtained.
[0265] (Example 50B)
A transparent dispersion liquid containing α-lipoic
30 acid-MgCO3 nanoparticles was obtained by the same procedure
as that in Example 50A, except that ion-exchanged water was
used instead of the water for injection.
- 102 - EG026
[0266] The results of these Examples 1A to 50B and
Comparative Examples 1A, 1B, 22-1A to 22-2B are summarized
in the following Table 1-1 to Table 1-3. It is noted that
in Examples 1A to 21B and 49A to 50B, a procedure was used
5 in which an α-lipoic acid-containing aqueous dispersion
liquid is prepared and then a nonionic surfactant is added,
and in Examples 22A to 48B, a procedure was used in which
α-lipoic acid is dissolved in a nonionic surfactant and then
water is added.
- 103 - EG026
[0267] [Table 1-1]
Table of Examples and Comparative Examples (1)
Example
No.
pH of
α-LP
solution
Nonionic surfactant Kind of surfactant Adjust
ed pH
Divalent
metal salt
(A)
Salt
carrying
divalent
anion (B)
A:B
(molar
ratio)
1A,1B 7.2 Ryoto Sugar Ester L-1695 Sucrose fatty acid ester － MgCl2 Na2CO3 5:1
Comparative
Examples
1A, 1B
7.2 Ryoto Sugar Ester L-1695 Sucrose fatty acid ester － － － －
2A,2B 7.1 Ryoto Sugar Ester L-1695 Sucrose fatty acid ester － MgCl2 Na2CO3 5:1
3A,3B 7.0 HCO-60 Polyoxyethylene hydrogenated castor oil － MgCl2 Na2CO3 5:1
4A,4B 7.3 EMULGEN 2020G-HA Polyoxyethylene octyl dodecyl ether － MgCl2 Na2CO3 5:1
5A,5B 7.1 HCO-60 Polyoxyethylene hydrogenated castor oil 6.6 CaCl2 Na2CO3 5:1
6A,6B 7.0 EMULGEN 2020G-HA Polyoxyethylene octyl dodecyl ether 6.2 CaCl2 Na2CO3 5:1
7A,7B 6.9 HCO-60 Polyoxyethylene (60) hydrogenated
castor oil
6.4 CaCl2 Na2HPO４ 5:1
8A,8B 11.7 PBC44 POE(20)POP(8)cetyl ether 11.0 CaCl2 Na2CO3 50:1
9A,9B 11.5 POE(20)stearyl ether Polyoxyethylene alkyl ether 10.8 CaCl2 Na2CO3 50:1
10A,10B 6.8 HCO-60 Polyoxyethylene hydrogenated castor oil 5.0 Zinc
gluconate
Na2CO3 -
11A,11B 6.9 HCO-60 Polyoxyethylene hydrogenated castor oil 5.0 Zn(COOH)2 Na2CO3 5:1
12A,12B 6.9 Ryoto Sugar Ester L-1695 Sucrose fatty acid ester 6.8 MgCl2 Na2CO3 5:2
13A,13B 6.9 HCO-60 Polyoxyethylene hydrogenated castor oil 3.9 Zn(COOH)2 Na2CO3 5:1
14A,14B 10.9 HCO-60 Polyoxyethylene hydrogenated castor oil 6.4 CaCl2 Na2CO3 5:1
15A,15B 8.7 HCO-60 Polyoxyethylene hydrogenated castor oil 6.3 CaCl2 Na2CO3 5:1
16A,16B 6.9 HCO-60 Polyoxyethylene hydrogenated castor oil 6.4 CaCl2 Na2CO3 5:2
17A,17B 6.9 EMULGEN 2020G-HA Polyoxyethylene octyl dodecyl ether 6.7 CaCl2 Na2CO3 5:2
18A,18B 11.8 HCO-60 Polyoxyethylene hydrogenated castor oil 10.9 CaCl2 Na2CO3 5:1
19A,19B 9.1 Ryoto Sugar Ester L-1695 Sucrose fatty acid ester 8.5 MgCl2 Na2CO3 5:1
20A,20B - POE(20)stearyl ether Polyoxyethylene alkyl ether 7.0 MgCl2 Na2CO3 50:1
21A,21B - POE(20)stearyl ether Polyoxyethylene alkyl ether 5.5 MgCl2 Na2CO3 5:2
49A,49B - POE(20)stearyl ether Polyoxyethylene alkyl ether 5.5 MgCl2 Na2CO3 5:2
50A,50B - HCO-60 Polyoxyethylene hydrogenated castor oil 7.0 MgCl2 Na2CO3 5:1
- 104 - EG026
[0268] [Table 1-2]
Table of Examples and Comparative Examples (2)
Example
No.
pH before
addition
of metal
ion
Nonionic surfactant Kind of surfactant Final
pH
Divalent
metal
salt (A)
Salt
carrying
divalent
anion (B)
A:B
(molar
ratio)
22A,22B 4.6 HCO-60 Polyoxyethylene hydrogenated castor oil 6.7 CaCl2 Na2CO3 5:2
Comparative
Examples
22-1A,B
- HCO-60 Polyoxyethylene hydrogenated castor oil 6.8 － － －
Comparative
Examples
22-2A,B
- HCO-60 Polyoxyethylene hydrogenated castor oil 7.0 － － －
23A,23B 4.6 HCO-60 Polyoxyethylene hydrogenated castor oil 6.8 MgCl2 Na2CO3 5:2
24A,24B 4.3 HCO-60 Polyoxyethylene hydrogenated castor oil 6.7 CaCl2 Na2CO3 5:1
25A,25B 4.5 HCO-60 Polyoxyethylene hydrogenated castor oil 6.3 MgCl2 Na2CO3 5:1
26A,26B 4.2 HCO-60 Polyoxyethylene hydrogenated castor oil 6.9 CaCl2 Na2CO3 5:3
27A,27B 4.5 HCO-60 Polyoxyethylene hydrogenated castor oil 6.8 CaCl2 Na2CO3 5:1
28A,28B 4.5 HCO-60 Polyoxyethylene hydrogenated castor oil 6.8 MgCl2 Na2CO3 5:2
29A,29B 4.6 HCO-60 Polyoxyethylene hydrogenated castor oil 6.8 MgCl2 Na2CO3 5:3
30A,30B 4.6 HCO-60 Polyoxyethylene hydrogenated castor oil 6.6 CaCl2 Na2CO3 5:1
31A,31B 4.3 POE(20)stearyl ether Polyoxyethylene alkyl ether 6.9 CaCl2 Na2CO3 5:2
32A,32B 4.6 EMULGEN 2020G-HA Polyoxyethylene octyl dodecyl ether 6.8 CaCl2 Na2CO3 5:1
33A,33B 4.3 Polysorbate 80 Polyoxyethylene sorbitan fatty acid
ester 6.5 CaCl2 Na2CO3 5:1
34A,34B 4.5 Polysorbate 80 Polyoxyethylene sorbitan fatty acid
ester 6.8 MgCl2 Na2CO3 5:1
35A,35B 4.3 Polysorbate 80 Polyoxyethylene sorbitan fatty acid
ester 6.5 CaCl2 Na2CO3 5:2
36A,36B 4.5 Polysorbate 80 Polyoxyethylene sorbitan fatty acid
ester 6.9 MgCl2 Na2CO3 5:2
- 105 - EG026
[0269] [Table 1-3]
Table of Examples and Comparative Examples (3)
Example
No.
pH before
addition
of metal
ion
Nonionic surfactant Kind of surfactant Final pH
Divalent
metal salt
(A)
Salt
carrying
divalent
anion (B)
A:B
(molar
ratio)
37A,37B 4.5 POE(20)stearyl ether Polyoxyethylene alkyl ether 6.6 CaCl2 Na2CO3 5:1
38A,38B 4.3 POE(20)stearyl ether Polyoxyethylene alkyl ether 6.8 CaCl2 Na2CO3 5:1
39A,39B 4.5 POE(20)stearyl ether Polyoxyethylene alkyl ether 6.5 CaCl2 Na2CO3 5:1
40A,40B 4.4 POE(20)stearyl ether Polyoxyethylene alkyl ether 6.9 CaCl2 Na2CO3 5:1
41A,41B 4.5 POE(20)stearyl ether Polyoxyethylene alkyl ether 6.4 MgCl2 Na2CO3 5:1
42A.42B 4.6 POE(20)stearyl ether Polyoxyethylene alkyl ether 6.8 MgCl2 Na2CO3 5:1
43A,43B 4.5 POE(20)stearyl ether Polyoxyethylene alkyl ether 6.7 MgCl2 Na2CO3 5:2
44A,44B 4.6 POE(20)stearyl ether Polyoxyethylene alkyl ether 6.8 CaCl2 Na2CO3 5:3
45A,45B 4.5 POE(20)stearyl ether Polyoxyethylene alkyl ether 6.6 MgCl2 Na2CO3 5:3
46A,46B 4.5 POE(20)stearyl ether Polyoxyethylene alkyl ether 6.7 CaCl2 Na2CO3 5:1
47A,47B 4.3 POE(20)stearyl ether Polyoxyethylene alkyl ether 6.6 CaCl2 Na2CO3 5:1
48A,48B 4.4 POE(20)stearyl ether Polyoxyethylene alkyl ether 6.8 CaCl2 Na2CO3 5:1
43A,43B 4.5 POE(20)stearyl ether Polyoxyethylene alkyl ether 6.7 MgCl2 Na2CO3 5:2
44A,44B 4.6 POE(20)stearyl ether Polyoxyethylene alkyl ether 6.8 CaCl2 Na2CO3 5:3
45A,45B 4.5 POE(20)stearyl ether Polyoxyethylene alkyl ether 6.6 MgCl2 Na2CO3 5:3
46A,46B 4.5 POE(20)stearyl ether Polyoxyethylene alkyl ether 6.7 CaCl2 Na2CO3 5:1
47A,47B 4.3 POE(20)stearyl ether Polyoxyethylene alkyl ether 6.6 CaCl2 Na2CO3 5:1
48A,48B 4.4 POE(20)stearyl ether Polyoxyethylene alkyl ether 6.8 CaCl2 Na2CO3 5:1
POE(20)stearyl ether = Polyoxyethylene (20) stearyl ether
Polysorbate 80 = Polyoxyethylene (20) sorbitan monooleate, also referred to as oleic acid polyoxyethylene
sorbitan.
Examples 37A, 37B, 40A, 40B, 41A, 41B, 43A, 43B, 44A, 44B, 45A, 45B, 46A and 46B contain polyethylene glycol
(1000) as an additive.
Examples 38A, 38B, 39A, 39B, 42A, 42B, 47A and 47B contain polyethylene glycol (4000) as an additive.
Examples 48A and 48B contain polyethylene glycol (6000) as an additive.
- 106 - EG026
[0270] (Test Example 1: Thermostability test of
preparation)
The α-lipoic acid-MgCO3 nanoparticles produced in
Example 1A, and the α-lipoic acid nanoparticles produced
5 in Comparative Example 1A without adding magnesium chloride
and sodium carbonate were respectively heated at 60°C, and
the amounts of α-lipoic acid in the sample after one hour
of heating and after 3 hours of heating were analyzed by
HPLC. As a control, a reagent α-lipoic acid was used. The
10 amount of α-lipoic acid after heating was divided by the
amount of α-lipoic acid before heating, and the result was
multiplied by 100 such that the residual ratio of α-lipoic
acid was calculated. The results of the residual ratio of
α-lipoic acid are presented in Table 2 below and in FIG.
15 3. Symbol �� represents the results of control reagent of
α-lipoic acid, symbol �� represents the results of the α-lipoic
acid dispersion liquid of Comparative Example 1, and symbol
�� represents the results of the α-lipoic acid-MgCO3
nanoparticles of Example 1A.
20
[0271] [Table 2]
Heating time (hrs) 0 1 3
Control (reagent α-lipoic acid) 100 50.51 44.20
α-Lipoic acid nanoparticles of
Residual Comparative Example 1A 100 102.30 97.68
ratio (%) α-Lipoic acid-MgCO3
nanoparticles of Example 1A 100 104.22 93.59
As a result, while the amount of the α-lipoic acid of
a reagent was reduced by about 55% after 3 hours of heating,
25 in the α-lipoic acid-MgCO3 nanoparticles of Example 1A and
the α-lipoic acid nanoparticles produced without adding
magnesium chloride and sodium carbonate, no substantial
reduction in the amount of the α-lipoic acid was observed.
- 107 - EG026
As it is clear from a comparison with the control, it is
understood that the preparation of the present invention
is very excellent in the stability of α-lipoic acid.
5 [0272] (Test example 2: Improvement in sulfurous odor)
The paste after freeze-drying of the α-lipoic acid-MgCO3
nanoparticles produced in Example 1A, and the paste after
freeze-drying of the α-lipoic acid nanoparticles produced
in Comparative Example 1A without adding magnesium chloride
10 and sodium carbonate, were respectively dispersed in
distilled water such that the final concentration of α-lipoic
acid reached 0.1%. The dispersions were placed in
transparent test tubes made of resin, and the test tubes
were left to stand indoors under sunlight. As a control,
15 an aqueous dispersion liquid having α-lipoic acid dissolved
in water (final concentration of α-lipoic acid 0.1%), which
was prepared by adding an alkali (5M NaOH) to reagent α-lipoic
acid, and thereby adjusting the pH to 7 to 7.5, was also
left to stand in the same manner.
20
[0273] As a result, after a lapse of two weeks, the
characteristic sulfurous odor strongly emanated from the
control solution and the dispersion liquid of α-lipoic acid
nanoparticles produced in Comparative Example 1A without
25 adding magnesium chloride and sodium carbonate. The level
was the same as the level of the aqueous dispersion liquid
having α-lipoic acid dissolved in water, which was used as
the control. On the contrary, no odor emanated from the
α-lipoic acid-MgCO3 nanoparticle dispersion liquid.
30
[0274] (Test Example 3: Test for suppression effect of
α-lipoic acid-MgCO3 nanoparticles on ultraviolet-induced
pigmentation in colored guinea pig)
- 108 - EG026
The dorsal part of a colored guinea pig (Weiser Maples,
5 weeks old, male) having melanogenic cells, was shaved in
an area of 2 cm × 2 cm, and the α-lipoic acid-MgCO3 nanoparticle
dispersion liquid (containing 350 μg of α-lipoic acid)
5 obtained in Example 3A was applied in an amount of 80 mg
per day, once a day, 5 days per week (from Monday to Friday).
After the application, on each of the application initiation
days (Monday) and after 2, 4 and 7 days (Wednesday, Friday
and next Monday), irradiation with UV-A at 8 J/cm2 and UV-B
10 at 12 mJ/cm2 were carried out. As an index of melanogenesis
in the guinea pig skin, the brightness (L* value) of the
skin was measured using a color-difference meter, and the
amount of reduction of brightness was used as an index of
the degree of blackening. In regard to the brightness, a
15 larger L* value represents a whiter color. The absolute
values of the amount of change in the brightness (ΔL* value)
due to melanogenesis from the test initiation day were
compared. Guinea pigs that were applied with only water which
did not contain α-lipoic acid, were taken as a control group,
20 and a comparison was made.
[0275] As a result, the group applied with the α-lipoic
acid-MgCO3 nanoparticles exhibited less reduction in the
brightness as compared with the control group, that is,
25 blackening of the skin was suppressed, throughout the whole
test period. The absolute value of the ΔL* value at the time
of completion of the test was 8.3 for the control group,
while the absolute value was 6.6 for the group applied with
the α-lipoic acid-MgCO3 nanoparticles.
30
[0276] In Table 3 below and in FIG. 4, the absolute values
of the ΔL* values measured after zero days (Monday), after
4 days (Friday), after 7 days (Monday) and after 9 days
- 109 - EG026
(Wednesday) are presented. It was confirmed by the results
obtained as shown above, that the α-lipoic acid nanoparticles
are absorbed into the skin and can suppress pigmentation
caused by ultraviolet rays.
5
[0277] [Table 3]
Time (days)
Sample 0 4 7 9
Control (water)
0 3.0 7.8
8.3
α-Lipoic acid-MgCO3
nanoparticle dispersion
liquid
0 2.0 6.3 6.6
[0278] (Test Example 4: Test for verifying effects of
α-lipoic acid-MgCO3 nanoparticles on skin moisture, barrier
10 function and recovery from wrinkles in photoaging model
mouse)
The dorsal part of a hairless mouse (Hos:HR-1, 7 weeks
old, male) was irradiated with UV-B at 55 mJ/cm2 per day for
2 months, 5 days per week (that is, irradiated only from
15 Monday to Friday, not irradiated on Saturdays and Sundays),
and thereby a photoaging model mouse was produced. The
α-lipoic acid-MgCO3 nanoparticle dispersion liquid
(containing 350 μg of α-lipoic acid) obtained by Example
3A was applied on this mouse in an amount of 80 mg per day,
20 once a day, 5 days per week (that is, applied only from Monday
to Friday, not applied on Saturdays and Sundays) for 1 month.
The mouse skin was subjected, at the time of the initiation
of application and at the time of the completion of application,
to an observation of the skin condition by visual inspection,
25 and measurement of the amount of moisture in the stratum
corneum and the amount of transepidermal water loss (TEWL),
to evaluate the conditions of wrinkles and stratum corneum
moisture, and the skin barrier functions.
- 110 - EG026
[0279] As a result, as shown in Table 4 below and in FIG.
5, the group applied with the α-lipoic acid-MgCO3
nanoparticles was recognized to have a recovery of the amount
5 of moisture in the stratum corneum as compared with those
at the time of application initiation. On the other hand,
the control group (similarly applied with only water which
did not contain α-lipoic acid) was not recognized to have
a recovery in the amount of moisture in the stratum corneum.
10 The amount of moisture in the stratum corneum at the time
of completion of the test was 18.2 (μs) for the group applied
with the α-lipoic acid-MgCO3 nanoparticles, while the amount
was 5.6 (μs) for the control group. Furthermore, the TEWL
value on the last day of the test was 15.7 (g/h•m2) for the
15 group applied with the α-lipoic acid-MgCO3 nanoparticles,
while the TEWL value was 32.6 (g/h•m2) for the control group.
Thus, it was confirmed that application with the α-lipoic
acid-MgCO3 nanoparticles caused the recovery of the barrier
function of the skin. A photograph of the replica of wrinkles
20 is presented in FIG. 6. Any changes in the wrinkle state
as compared with the time of initiation of test was not
recognized in the control group, but in the group applied
with the α-lipoic acid-MgCO3 nanoparticles, an obvious
decrease in wrinkles was recognized. From the results shown
25 above, the following effects were confirmed. The α-lipoic
acid nanoparticles are absorbed into the skin and these
nanoparticles return a photoaged skin state into a healthy
state.
30 [0280] [Table 4]
Amount of moisture in stratum corneum
Time (days)
1 30
- 111 - EG026
α-Lipoic acid-MgCO3
nanoparticle dispersion
liquid
8.9 18.2
Control (water) 7.2 5.6
[0281] (Measurement Example 2: Measurement of particle
size)
The solution of α-lipoic acid-CaCO3 nanoparticles
5 produced in Example 22A was subjected to the measurement
of particle size with a light scattering photometer (Otsuka
Electronics Co., Ltd., FPAR1000). As a result, it was
confirmed that the particle size of the α-lipoic acid-CaCO3
nanoparticles produced in Example 22A was about 20 nm. The
10 particle sizes were almost the same both when distilled water
was used and when ion-exchanged water was used. The results
of the particle size distribution of the α-lipoic acid-CaCO3
nanoparticles produced in Example 22A using distilled water,
as measured using Otsuka Electronics Co., Ltd., FPAR1000
15 are presented in FIG. 7.
[0282] (Measurement Example 3: Measurement of particle
size)
The particle size of solution of α-lipoic acid-MgCO3
20 nanoparticles produced in Example 29A was measured by a light
scattering photometer (Otsuka Electronics Co., Ltd.,
FPAR1000). From the fact that the solution was perfectly
transparent and the results of the measurement of particle
size, it was confirmed that the α-lipoic acid-MgCO3
25 nanoparticles produced in Example 29A form weak clusters
having average particle sizes of 200 nm and 1700 nm in which
primary particles have an average particle size of about
12 nm. The particle sizes were almost the same both when
distilled water was used and when ion-exchanged water was
- 112 - EG026
used. The result of the particle size distribution of the
α-lipoic acid-MgCO3 nanoparticles produced by using
ion-exchanged water in Example 29A, as measured with Otsuka
Electronics Co., Ltd., FPAR1000, is presented in FIG. 8.
5
[0283] (Measurement Example 4: Measurement of particle
size)
For each of the solutions of α-lipoic acid-MgCO3
nanoparticles produced in Examples 24A, 24B, 25A, 25B, 33A,
10 33B, 36A and 36B, the particle size was measured with a light
scattering photometer (Otsuka Electronics Co., Ltd.,
FPAR1000).
[0284] The average particle sizes (nm) of the respective
15 α-lipoic acid nanoparticles measured in Measurement Examples
1 to 4 are summarized in Table 5 below.
[0285] [Table 5]
- 113 - EG026
Average particle
size (nm)
Examples 1A, 1B α-Lipoic acid-MgCO3
nanoparticles 10
Comparative
Example 1A
α-Lipoic acid
dispersion liquid 760
Examples 22A, B α-Lipoic acid-CaCO3
nanoparticles 19.3
Examples 24A, B α-Lipoic acid-CaCO3
nanoparticles 17.8
Examples 25A, B α-Lipoic acid-MgCO3
nanoparticles 55.9
Examples 29A, B α-Lipoic acid-MgCO3
nanoparticles 109.2
Examples 33A, B α-Lipoic acid-CaCO3
nanoparticles 82.7
Examples 36A, B α-Lipoic acid-MgCO3
nanoparticles 11
[0286] (Test Example 5: Thermostability test of
preparation)
The α-lipoic acid-CaCO3 nanoparticles produced in
5 Example 22A and the α-lipoic acid dispersion liquid produced
in Comparative Example 22-1A (neither calcium chloride nor
sodium carbonate were added) were respectively stored at
60°C (storage under heating), and the amount of α-lipoic acid
in the solution was analyzed by HPLC once every week up to
10 3 weeks. The amount of α-lipoic acid after storage under
heating was divided by the amount of α-lipoic acid before
heating, and the result was multiplied by 100 such that the
residual ratio of α-lipoic acid was calculated. The results
of the residual ratio of α-lipoic acid are presented in Table
15 6 below and in FIG. 9. Symbol �� indicates the results for
the α-lipoic acid dispersion liquid of Comparative Example
22-1A, and symbol �� indicates the results for the α-lipoic
acid-CaCO3 nanoparticles of Example 22A.
- 114 - EG026
[0287] [Table 6]
Time elapsed (days) 0 7 14 21
α-Lipoic acid dispersion
liquid of Comparative
Example 22-1A
100.0 97.5 93.9 82.1
Residual
ratio (%) α-Lipoic acid-CaCO3
nanoparticles of Example
22A
100.0 98.4 97.2 88.7
As a result, after a storage for 3 weeks at 60°C, the
5 α-lipoic acid nanoparticles produced in Comparative Example
22-1A without adding magnesium chloride and sodium carbonate
showed a reduction of about 18%, but in the α-lipoic acid-CaCO3
nanoparticles of Example 22A, the reduction of α-lipoic acid
was suppressed to about 11%. Accordingly, it is understood
10 that the preparation of the present invention is quite
excellent in the stability of α-lipoic acid.
[0288] (Test Example 6: Thermostability test of
preparation)
15 The α-lipoic acid-MgCO3 nanoparticles produced in
Example 29A and the α-lipoic acid nanoparticles produced
in Comparative Example 22-2A without adding magnesium
chloride and sodium carbonate were respectively stored at
60°C, and the amount of α-lipoic acid in the solution was
20 analyzed by HPLC once every week up to 3 weeks. The amount
of α-lipoic acid after storage under heating was divided
by the amount of α-lipoic acid before heating, and the result
was multiplied by 100 such that the residual ratio of α-lipoic
acid was calculated. The results of the residual ratio of
25 α-lipoic acid are presented in Table 7 below and in FIG.
10. Symbol �� indicates the results for the α-lipoic acid
nanoparticles of Comparative Example 22-2A, and symbol ��
indicates the results for the α-lipoic acid-MgCO3
- 115 - EG026
nanoparticles of Example 29A.
[0289] [Table 7]
Time elapsed (days) 0 7 14 21
α-Lipoic acid dispersion
liquid of Comparative
Example 22-2A
100.0 99.6 95.3 86.6
Residual
ratio (%) α-Lipoic acid-MgCO3
nanoparticles of Example
29A
100.0 98.1 96.1 91.5
5 As a result, after a storage for 3 weeks at 60°C, the
α-lipoic acid nanoparticles produced in Comparative Example
22-2A without adding magnesium chloride and sodium carbonate
showed a reduction of about 13%, but in the α-lipoic acid-MgCO3
nanoparticles of Example 29A, the reduction of α-lipoic acid
10 was suppressed to about 8%. Accordingly, it is understood
that the preparation of the present invention is quite
excellent in the stability of α-lipoic acid.
[0290] (Test Example 7: Test on function of α-lipoic
15 acid-MgCO3 nanoparticles in connection with differentiation
of preadipocytes)
1.5 ml of D-MEM medium (D-MEM medium supplemented with
10% FCS, 100 units/ml of penicillin, and 100 μg/ml of
streptomycin, all at final concentrations) was added to a
20 plastic petri dish having a diameter of 3.5 cm. To this,
5.0 × 104 cells of 3T3-L1 cells, which are preadipocytes,
were suspended, and precultured for 3 days to result in a
confluent state. Thereafter, the medium was replaced with
3 ml of an adipocyte differentiation induction medium (D-MEM
25 supplemented with 10% FCS, 100 units/ml of penicillin, 100
μg/ml of streptomycin, 5 μg/ml of insulin, 0.25 μM of
dexamethasone, and 0.5 mM of isobutyl-methylxanthine (IBMX),
all at final concentrations). After another 2 days, the
- 116 - EG026
medium was replaced with 3 ml of the adipocyte differentiation
induction medium of the same composition, and then the
culturing was carried out for 2 days. Thus, the culturing
in the adipocyte differentiation induction medium was carried
5 out for 4 days in total. During this culturing, an α-lipoic
acid solution or the α-lipoic acid-MgCO3 nanoparticle
solution of Example 20A was added to the adipocyte
differentiation induction medium to result in 0, 100, 250
or 500 μM of α-lipoic acid concentration. The culturing were
10 all carried out under the conditions of 5% CO2 and 37°C.
[0291] The amount of accumulated lipids in the cultured cells
thus obtained was measured. The cells were washed with 1
ml of a PBS buffer solution, and then the cells were fixed
15 for 5 minutes with neutral buffered formalin. The cells were
further washed with a 70% ethanol solution and distilled
water. Subsequently, 1 ml of an Oil Red O solution (a staining
solution prepared by mixing a saturated Oil Red O/isopropanol
solution and distilled water at a ratio of 6:4 and filtering
20 the mixture) was added, and left to stand for 15 minutes.
The staining solution was removed, and the cells were washed
with a 70% ethanol solution until the dye no longer diffused.
Then, 0.75 ml of a 4% Nonidet P-40/isopropanol solution was
added, stirred for 30 minutes, and the dye was allowed to
25 elute out. The whole amount of this solution was recovered,
and the absorbance at a wavelength of 520 nm was measured
with a spectrophotometer.
[0292] As a result, while the addition of α-lipoic acid
30 resulted the reduction of the accumulation of lipids, it
was recognized that the addition of α-lipoic acid-MgCO3
nanoparticles has an action of accumulating lipids in the
cells (FIG. 11). That is, it was suggested that the α-lipoic
- 117 - EG026
acid-MgCO3 nanoparticles has a function of allowing efficient
incorporation of sugar into immature adipocytes. Since the
α-lipoic acid nanoparticles accelerated the incorporation
of sugar in the test described above, an effect of improving
5 the blood glucose level, which has not been recognized with
α-lipoic acid alone, can be expected for the α-lipoic acid
nanoparticles, and this suggested the usefulness of the
nanoparticles as a therapeutic drug for diabetes.
10 [0293] (Test Example 8: Test on function of α-lipoic
acid-MgCO3 nanoparticles in connection with
dedifferentiation of mature adipocytes)
1.5 ml of D-MEM medium (D-MEM medium supplemented with
10% FCS, 100 units/ml of penicillin, and 100 μg/ml of
15 streptomycin, all at final concentrations) was added to a
plastic petri dish having a diameter of 3.5 cm. To this,
5.0 × 104 cells of 3T3-L1 cells, which are preadipocytes,
were suspended, and precultured for 3 days to result in a
confluent state. Thereafter, the medium was replaced with
20 3 ml of an adipocyte differentiation induction medium (D-MEM
medium supplemented with 10% FCS, 100 units/ml of penicillin,
100 μg/ml of streptomycin, 5 μg/ml of insulin, 0.25 μM of
dexamethasone, and 0.5 mM of isobutyl-methylxanthine, all
at final concentrations), and the culturing was carried out
25 for 4 days to induce the differentiation into adipocytes.
Thereafter, the medium was replaced with an adipocyte
maturation medium (D-MEM medium supplemented with 10% FCS,
100 units/ml of penicillin, 100 μg/ml of streptomycin, and
5 μg/ml of insulin, all at final concentrations), and the
30 culturing was carried out for 7 days. Thereafter, the medium
was replaced with a test medium, and culturing was carried
out for another 4 days. The test medium was prepared by adding
an α-lipoic acid solution or the α-lipoic acid-MgCO3
- 118 - EG026
nanoparticle solution of Example 20A to the adipocyte
maturation medium to result in 0, 100, 250 or 500 μM of α-lipoic
acid concentration. Each of the media was replaced with the
same medium every other day during the culture. The culture
5 was all carried out under the conditions of 5% CO2 and 37°C.
[0294] The amount of accumulated lipids in the cultured cells
thus obtained was measured. The cells were washed with 1
ml of a PBS buffer solution, and then the cells were fixed
10 for 5 minutes with neutral buffered formalin. The cells were
further washed with a 70% ethanol solution and distilled
water. To this, 1 ml of an Oil Red O solution was added,
and left to stand for 15 minutes. After the staining, the
cells were washed with a 70% ethanol solution until the dye
15 no longer diffused. To this, 0.75 ml of a 4% Nonidet
P-40/isopropanol solution was added, stirred for 30 minutes,
and the dye was allowed to elute out. The total amount of
this solution was recovered, and the absorbance at a
wavelength of 520 nm was measured with a spectrophotometer.
20
[0295] As a result, in the α-lipoic acid-added group, the
amount of lipid accumulation was almost indifferent from
that of the non-added group; however, in the group added
with the α-lipoic acid-MgCO3 nanoparticles, an action of
25 accumulating lipids in the cells was recognized (FIG. 12).
That is, it is suggested that, similarly to Test Example
7, the α-lipoic acid-MgCO3 nanoparticles have an action of
allowing efficient incorporation of sugar into mature
adipocytes. Particularly with regard to adipocytes, it was
30 confirmed that they have the action of allowing accumulation
of lipids at all stages of differentiation. From the results
obtained above, a high effect of improving blood glucose
levels, which has not been recognized with α-lipoic acid
- 119 - EG026
alone, can be expected for the α-lipoic acid nanoparticles,
and this suggested the usefulness of the nanoparticles as
a therapeutic drug for diabetes.
5 [0296] (Test Example 9: Analysis of stability of α-lipoic
acid-MgCO3 nanoparticles in culture medium and cellular
localization of the nanoparticles)
Culturing was carried out in the same manner as those
in Test Example 7. α-Lipoic acid and the α-lipoic acid-MgCO3
10 nanoparticles of Example 20A were respectively added at a
final concentration of 250 μM. The total amount of the
supernatant of these culture media of cells was recovered
and was designated as a culture supernatant fraction. The
cells were further washed with a PBS buffer solution, and
15 then recovery and washing of the cells was carried out by
conventional methods. The cells were precipitated by
centrifugation, and the cells were suspended in 500 μL of
purified water and disrupted by ultrasonication. This
disrupted cell fluid was centrifuged for 15 minutes at 4°C
20 at 15000 rotations per minute, and the supernatant was
recovered and designated as a cell disrupted fluid fraction.
The residual α-lipoic acid concentration of each fraction
was quantified using a high performance liquid
chromatography-mass spectrometer.
25
[0297] As a result, no difference in concentration was
recognized in the cell disrupted fluid fraction (FIG. 13).
However, in the culture supernatant fraction, a high α-lipoic
acid residual was confirmed for the experimental group added
30 with α-lipoic acid-MgCO3 nanoparticles (FIG. 14). From the
results obtained above, it was confirmed that the α-lipoic
acid-MgCO3 nanoparticles were quite stable in the culture
medium. Furthermore, it was suggested that the difference
- 120 - EG026
in the action between the α-lipoic acid and the α-lipoic
acid-MgCO3 nanoparticles as observed in Test Example 7 is
not due to the difference in the α-lipoic acid concentration
in the cells, but is due to the difference in the
5 physicochemical properties possessed by the α-lipoic
acid-MgCO3 nanoparticles.
[0298] (Test Example 10: Test on anti-wrinkle effect of
α-lipoic acid-MgCO3 nanoparticle application in wrinkle
10 model mouse)
A hairless mouse (Hr/kud, 9 weeks old, male) was
irradiated with ultraviolet rays, and thus a wrinkle model
mouse was produced. In the production of this wrinkle model,
the mouse was irradiated with ultraviolet rays over 13 weeks
15 (5 days/week, from Monday to Friday), such that the total
exposure doses of UVA and UVB were 148.99 J/cm2 and 3.49 J/cm2,
respectively. After the production of the wrinkle model,
a commercially available cosmetic product containing 0.01%
α-lipoic acid and the 0.01% α-lipoic acid-MgCO3
20 nanoparticle-containing aqueous dispersion liquid of
Example 21A were applied on the dorsal part of the mouse
in an amount of 30 mg/cm2/day each, 5 times per week (from
Monday to Friday), and this was carried out for 6 weeks.
As control groups, an unapplied group in which the mice were
25 bred for 6 weeks without applying any preparation and an
untreated group in which the mice were bred concurrently
without being subjected to wrinkle formation by UV
irradiation, were used. The produced wrinkle model was
evaluated by a replica method. Based on the originally
30 established scoring criteria shown in FIG. 15, the degrees
of wrinkles were compared by visual inspection, and scoring
of the wrinkle model mouse was performed. Furthermore,
paraffin-embedded sections of the mouse dorsal skin were
- 121 - EG026
produced, were stained for hyaluronic acid and thereby their
total amounts were compared. The staining for hyaluronic
acid was carried out using a method in which biotin-labeled
hyaluronic acid-bound protein (biotin-labeled HABP,
5 Seikagaku Corp.) was used as a probe and was detected by
a streptavidin-labeled fluorescent dye (Cy3 streptavidin,
Jackson ImmunoResearch LABORATORIES).
[0299] As a result, from the analysis of the replica of the
10 mouse dorsal part where 6 weeks application was carried out,
a higher wrinkle improving effect was observed in the group
applied with a 0.01% α-lipoic acid-MgCO3
nanoparticle-containing aqueous dispersion liquid as
compared with the 0.01% α-lipoic acid-containing commercial
15 product (FIG. 16). Also, in the scoring, a wrinkle improving
effect was confirmed (Table 8).
[0300] Staining for hyaluronic acid of the same mouse skin
sections was carried out, and as a result, reduction of
20 hyaluronic acid was observed in the group of 0.01% α-lipoic
acid-containing commercial product or unapplied group, but
accumulation of hyaluronic acid to the same extent as that
of the untreated group was observed in the group applied
with 0.01% α-lipoic acid-MgCO3 nanoparticles (FIG. 17). It
25 is known that reduction of hyaluronic acid in the dermis
is related to the formation of wrinkles. From the results
shown above, it was confirmed that the α-lipoic acid-MgCO3
nanoparticles have an action of increasing the amount of
hyaluronic acid, which is an extracellular matrix, in the
30 dermal layer which has been damaged by ultraviolet rays,
and wrinkles were improved by such an effect.
[0301] [Table 8]
- 122 - EG026
Wrinkle improving effect by application of α-lipoic
acid-MgCO3 nanoparticles
Test group Before test After 6 weeks
α-Lipoic acid
nanoparticles (n=5) 2.94 ± 0.75 2.40 ± 0.59
Commercial product (n=6) 3.00 ± 0.71 2.63 ± 0.77
Wrinkle
model
Unapplied (n=5) 3.00 ± 0.70 3.04 ± 0.61
Untreated (n=5) 1.00 ± 0.00 1.03 ± 0.07
Average ± S.D.
5 [0302] (Test Example 11: Test on human wrinkle improving
effect by application of α-lipoic acid-MgCO3 nanoparticles)
A male subject, whose age was in the thirties (Subject
1) was made to evenly apply a 0.01% α-lipoic acid-containing
aqueous dispersion liquid on one half of his face and the
10 0.01% α-lipoic acid-MgCO3 nanoparticle-containing aqueous
dispersion liquid of Example 21A on the other half of his
face, two times a day everyday. Also, a female subject, whose
age was in the fifties (Subject 2) was made to evenly apply
the 0.01% α-lipoic acid-MgCO3 nanoparticle-containing
15 aqueous dispersion liquid of Example 21A on one half of her
face everyday, and the other half of her face was left unapplied.
The test period was 16 weeks respectively, and the evaluation
of wrinkles was carried out by producing replicas of the
crow's feet area before the test and after 16 weeks.
20
[0303] As a result, in both of the subjects, an improvement
in wrinkles was observed on the side applied with the 0.01%
α-lipoic acid-MgCO3 nanoparticle-containing aqueous
dispersion liquid, as compared with the opposite side, which
25 was applied with the 0.01% α-lipoic acid-containing aqueous
dispersion liquid or unapplied (FIG. 18). From the results
shown above, it was confirmed that α-lipoic acid-MgCO3
nanoparticles have an effect of improving human wrinkles.
- 123 - EG026
[0304] (Test Example 12: Test for hyaluronic acid
accumulation by α-lipoic acid-MgCO3 nanoparticles)
3 ml of D-MEM medium (D-MEM medium supplemented with
5 10% FCS, 100 units/ml of penicillin, and 100 μg/ml of
streptomycin, all at final concentrations) was added to a
plastic petri dish having a diameter of 6.0 cm. To this,
1.5 × 105 cells of 3T3-L1 cells, which are preadipocytes,
were suspended, and precultured for 3 days to result in a
10 confluent state. Thereafter, the medium was replaced with
3 ml of an adipocyte differentiation induction medium (D-MEM
supplemented with 10% FCS, 100 units/ml of penicillin, 100
μg/ml of streptomycin, 5 μg/ml of insulin, 0.25 μM of
dexamethasone, and 0.5 mM of isobutyl-methylxanthine, all
15 at final concentrations). After another 2 days, the medium
was replaced with 3 ml of the adipocyte differentiation
induction medium of the same composition, and then the
culturing was carried out for 2 days. Thus, the culturing
in the adipocyte differentiation induction medium was carried
20 out for 4 days in total. Either an α-lipoic acid solution
or the α-lipoic acid-MgCO3 nanoparticle solution of Example
50A was added to the adipocyte differentiation induction
medium to result in 0, 100, 250 or 500 μM of α-lipoic acid
concentration. After the culturing, 1 ml of a PBS buffer
25 solution was added to the petri dish, from which the culture
supernatant was removed, and the cells were harvested with
a cell scraper. The cells thus harvested were disrupted by
ultrasonication to result in a cell disrupted fluid. The
amount of hyaluronic acid contained therein was quantified
30 by an enzyme-linked immunosorbent assay (ELISA). The
experimental method of hyaluronic acid ELISA was carried
out according to the method described in Annica Jacobson,
et al., Int. J. Cancer, 102:212-219 (2002). The culture was
- 124 - EG026
all carried out under the conditions of 5% CO2 and 37°C.
[0305] As a result, the α-lipoic acid-MgCO3
nanoparticle-added group showed a higher hyaluronic acid
5 amount as compared with the α-lipoic acid-added group (FIG.
19). From the results shown above, it was confirmed that
the α-lipoic acid-MgCO3 nanoparticles have the action of
causing hyaluronic acid to be accumulated on the cell surface.
Accordingly, it was suggested that the α-lipoic acid-MgCO3
10 nanoparticles improve wrinkles by enhancing the water
retentivity of the dermal layer of the skin. Furthermore,
an effect of reducing damages between joint cartilage tissues
by accumulating and concentrating hyaluronic acid at the
cartilage cell surfaces in joints, is expected, and thus
15 the usefulness of the nanoparticles as a therapeutic drug
for osteoarthritis was also suggested.
[0306] (Example 51: Production of an ointment for external
use)
20 An ointment for external use is produced by mixing the
materials of the formulation shown in Table 9 below according
to a method conventionally carried out in the art.
[0307] [Table 9]
25 Formulation of ointment for external use
α-Lipoic acid-phosphate Ca nanoparticle
paste of Example 7A
White petrolatum
Carboxymethylcellulose
Methylparaben
5.0
93.5
1.2
0.3
Total 100.00
parts by weight
- 125 - EG026
[0308] (Example 52: Production of a cosmetic emulsion)
A cosmetic emulsion is produced by mixing the materials
of the formulation shown in Table 10 below according to a
5 method conventionally carried out in the art.
[0309] [Table 10]
Formulation of a cosmetic emulsion
α-Lipoic acid-CaCO3 nanoparticle dispersion
liquid of Example 14A
Cetyl alcohol
Petrolatum
Liquid paraffin
Polyoxyethylene (10) sorbitan monostearate
Polyethylene glycol (1500)
Triethanolamine
Tocopherol acetate
Sodium hydrogen sulfite
Carboxyvinyl polymer
Fragrance
Methylparaben
Water
0.10
1.5
12.00
8.00
10.00
3.00
1.00
0.30
0.01
0.05
Appropriate
amount
Appropriate
amount
Balance
Total 100.00
parts
by weight
10 [0310] (Example 53: Production of a toothpaste)
A toothpaste is produced by mixing the materials of the
formulation shown in Table 11 below according to a method
conventionally carried out in the art.
15 [0311] [Table 11]
Formulation of a toothpaste
- 126 - EG026
α-Lipoic acid-ZnCO3 nanoparticle dispersion
liquid of Example 10B
Calcium hydrogen phosphate
Glycerin
Sorbitol
Carboxymethylcellulose sodium
Sodium lauryl sulfate
Saccharin sodium
Flavor
Sodium benzoate
Water
2.00
45.00
8.00
20.00
1.00
1.50
0.10
1.00
0.30
Balance
Total 100.00
parts by
weight
[0312] (Example 54: Production of a tablet)
A tablet is produced by mixing the materials of the
formulation shown in Table 12 below according to a method
5 conventionally carried out in the art.
[0313] [Table 12]
Formulation of a tablet
Polydextrose
Sugar ester
Flavor
Sorbitol
Palatinose
α-Lipoic acid-CaCO3 nanoparticle paste of
Example 1A
9.7
2.0
0.3
27.0
60.0
1.0
Total 100.0 parts
by weight
10 [0314] (Example 55: Production of an injection liquid)
An injection liquid is produced by mixing the materials
of the formulation shown in Table 13 below according to a
method conventionally carried out in the art.
15 [0315] [Table 13]
- 127 - EG026
Formulation of an injection liquid
Physiological saline of Japanese Pharmacopoeia
α-Lipoic acid-CaCO3 nanoparticle paste of
Example 5B
95.0
5.0
Total 100.0
parts by
weight
[0316] (Example 56: Production of a skin toner)
A skin toner is produced by mixing the materials of the
5 formulation shown in Table 14 below according to a method
conventionally carried out in the art.
[0317] [Table 14]
Formulation of a skin toner
α-Lipoic acid-Ca nanoparticle dispersion
liquid of Example 22A
1,3-Butylene glycol
Polyethylene glycol 1000
Glycerin
1% Hyaluronic acid
Methylparaben
Water
1.0
1.0
1.0
1.0
1.0
0.1
94.9
Total 100.0
parts by
weight
10
[0318] (Example 57: Production of an external lotion for
skin)
A external lotion for skin is produced by mixing the
materials of the formulation shown in Table 15 below according
15 to a method conventionally carried out in the art.
[0319] [Table 15]
Formulation of a lotion
- 128 - EG026
α-Lipoic acid-Ca nanoparticle dispersion
liquid of Example 22B
Dipropylene glycol
Hydroxyethylcellulose
Xanthan gum
Glycerin
1% Hyaluronic acid Na
Dextrin
Methylparaben
Water
5.0
3.0
0.2
0.1
2.0
2.0
0.8
0.2
86.7
Total 100.00 parts
by weight
[0320] (Test Example 13: Test for verifying the effect of
α-lipoic acid nanoparticle-containing external lotion for
skin, on the decrease of barrier function of hairless mouse
5 skin due to UV-B irradiation)
The dorsal part of a hairless mouse (Hos:HR-1, 25 weeks
old, male) was irradiated one time with UV-B at 70 mJ/cm2.
After the ultraviolet irradiation, the α-lipoic acid-CaCO3
nanoparticle-containing external lotion for skin obtained
10 in Example 57 was applied on this mouse using 100 μl per
day, once a day, for consecutive 4 days. Transepidermal water
loss (TEWL) was measured immediately before ultraviolet
irradiation and on the 4th day and 5th day from the day of
ultraviolet irradiation, and thereby the state of skin
15 barrier function was checked. The amount of increase of the
TEWL value from TEWL immediately before the ultraviolet
irradiation to TEWL on each measurement day, was defined
as ΔTEWL, and this was used as a criterion for a decrease
in the skin barrier function.
20
[0321] As shown in Table 16, it was confirmed that for all
of the measurement days, the group applied with the α-lipoic
acid-CaCO3 nanoparticle-containing external lotion for skin
(n=3) was suppressed an increase in the TEWL value, that
- 129 - EG026
is, a decrease in the skin barrier function was suppressed,
as compared with those for the control groups (applied
similarly with only water that did not contain α-lipoic acid,
n=3). From the results shown above, it was confirmed that
5 the α-lipoic acid-CaCO3 nanoparticle-containing external
lotion for skin acts on the skin after ultraviolet irradiation,
and exhibits an effect of reducing the functional disorder
of the skin due to ultraviolet stimulation.
10 [0322] [Table 16]
Time elapsed (days) 0 3 4
Group applied with distilled
water 0 63.2 165.4
ΔTEWL
(g•m2/h)
average
value
Group applied with α-lipoic
acid-CaCO3
nanoparticle-containing
external lotion for skin of
Example 57
0 31.9 137.0
[0323] (Example 58: Production of a drink preparation)
A drink preparation was produced by mixing the materials
of the formulation shown in Table 17 below according to a
15 method conventionally carried out in the art.
[0324] [Table 17]
Formulation of a drink preparation
α-Lipoic acid-Ca nanoparticle dispersion
liquid of Example 22B
Sucrose
Water
10.0
27.0
Balance
Total 100.00 parts
by weight
20 A comparative article was produced by replacing the
α-lipoic acid-Ca nanoparticle solution in the Table 17
described above, with a 1% aqueous solution obtained by
- 130 - EG026
neutralizing and dissolving α-lipoic acid using a minimal
amount of 0.25 M aqueous sodium hydroxide solution. 5 expert
panelists performed an organoleptic evaluation of the drink
preparation of Example 58 and the comparative article. As
5 a result, all of the experts evaluated that the drink
preparation of the present Example has a reduced sulfurous
odor and a reduced tingling sensation of the tongue which
are caused by α-lipoic acid, as compared with the comparative
article, thus having excellent palatability.
10
[0325] (Example 59: Production of a refreshing beverage)
A refreshing beverage was produced by mixing the
materials of the formulation shown in Table 18 below according
to a method conventionally carried out in the art.
15
[0326] [Table 18]
Formulation of a refreshing beverage
α-Lipoic acid-Mg nanoparticle dispersion
liquid of Example 29B
Sucrose
Citric acid
Malic acid
Water
10.0
9.0
1.4
0.5
Balance
Total 100.00 parts
by weight
A comparative article was produced by replacing the
20 α-lipoic acid-Mg nanoparticle solution in Table 18 described
above, with a 1% aqueous solution obtained by neutralizing
and dissolving α-lipoic acid using a minimal amount of 0.25
M aqueous solution of sodium hydroxide. 5 expert panelists
performed an organoleptic evaluation of the refreshing
25 beverage (drink preparation) of Example 59 and the
comparative article. As a result, all of the panelists
evaluated that the refreshing beverage (drink preparation)
- 131 - EG026
of the present Example has a reduced sulfurous odor and a
reduced tingling sensation at the tongue which are caused
by α-lipoic acid, as compared with the comparative article,
thus having excellent palatability.
5
[0327] As such, the present invention has been exemplified
using preferred embodiments of the present invention, but
the present invention should not be construed to be limited
to these embodiments. It is understood that the scope of
10 the present invention should be interpreted only by the claims.
It is understood that a person skilled in the art will
understand that from the descriptions of the specific
preferred embodiments of the present invention, equivalent
scope can be carried out based on the description of the
15 present invention and common technical knowledge. It is
understood that all patents, patent applications, and
documents cited in this specification should be herein
incorporated by reference for the content thereof to the
same extent as if the contents themselves were specifically
20 described in the present specification.
INDUSTRIAL APPLICABILITY
[0328] The subject nanoparticles maintain the form of a
25 transparent solution when dissolved in water, and are less
irritant since α-lipoic acid is coated with a coating of
a polyvalent metal inorganic salt. Therefore, it is possible
to administer such in the form of subcutaneously and
intravenously injectable preparations.
30
[0329] When the subject nanoparticles are administered by
applying in the form of an external preparation, or through
the oral mucosa such as the gingiva in the form of a composition
- 132 - EG026
for oral cavity, the nanoparticles are satisfactorily
transdermally absorbed, and do not cause inflammation since
the nanoparticles are not irritant. α-Lipoic acid is
released from the nanoparticles in a sustained release manner,
5 and thus effects such as activation of the skin, suppression
of photoaging, recovery from photoaging, and suppression
of melanogenesis due to ultraviolet stimulation can be
manifested.
10 [0330] When the subject nanoparticles are utilized in foods,
since the sulfurous odor characteristic of α-lipoic acid
is reduced, the value as an article of preference is enhanced,
as well as the amount of formulation of α-lipoic acid can
also be increased. Thus, a composition which may more easily
15 exhibit the effectiveness of α-lipoic acid can be obtained.
Furthermore, since the subject nanoparticles have a very
large specific surface, they are very satisfactorily absorbed
into the body. Moreover, since the subject nanoparticles
are water-soluble, use in a wide variety of forms of foods
20 such as beverages is made possible.
- 133 - EG026
CLAIMS
1. A method for producing α-lipoic acid nanoparticles, the
method comprising the steps of:
5 preparing an aqueous dispersion liquid containing
α-lipoic acid and a nonionic surfactant;
adding a divalent metal salt into the aqueous dispersion
liquid, wherein the divalent metal salt is a divalent metal
halide, a divalent metal acetate or a divalent metal
10 gluconate; and
adding an alkali metal carbonate or an alkali metal
phosphate into the aqueous dispersion liquid wherein the
divalent metal salt has been added, thereby forming α-lipoic
acid nanoparticles.
15
2. The method according to claim 1, wherein the step of
preparing an aqueous dispersion liquid containing α-lipoic
acid and a nonionic surfactant, comprises:
dissolving α-lipoic acid in the nonionic surfactant which
20 is in a liquid form, to obtain a surfactant solution; and
adding water or a liquid containing water to the
surfactant solution to obtain the aqueous dispersion liquid.
3. The method according to claim 1, wherein the step of
25 preparing an aqueous dispersion liquid containing α-lipoic
acid and a nonionic surfactant, comprises:
producing a mixture of α-lipoic acid, an alkaline
substance and water to prepare an α-lipoic acid-containing
aqueous dispersion liquid; and
30 adding the nonionic surfactant into the α-lipoic
acid-containing aqueous dispersion liquid.
4. The method according to claim 1, wherein the divalent
- 134 - EG026
metal salt is selected from the group consisting of calcium
chloride, calcium bromide, calcium fluoride, calcium iodide,
magnesium chloride, magnesium bromide, magnesium fluoride,
magnesium iodide, zinc chloride, zinc bromide, zinc fluoride,
5 zinc iodide, calcium acetate, magnesium acetate, zinc acetate,
calcium gluconate, magnesium gluconate and zinc gluconate.
5. The method according to claim 1, wherein the divalent
metal salt is selected from the group consisting of calcium
10 chloride, magnesium chloride and zinc gluconate.
6. The method according to claim 1, wherein the alkali metal
carbonate or alkali metal phosphate is selected from the
group consisting of sodium carbonate, potassium carbonate,
15 sodium hydrogen carbonate, potassium hydrogen carbonate,
sodium phosphate and potassium phosphate.
7. The method according to claim 1, wherein the alkali metal
carbonate or alkali metal phosphate is selected from the
20 group consisting of sodium carbonate and disodium hydrogen
phosphate.
8. The method according to claim 1, wherein the nonionic
surfactant is selected from the group consisting of
25 polyoxyethylene hydrogenated castor oils, polyoxyethylene
alkyl ethers, polyoxyethylene sorbitan fatty acid esters,
polyoxyethylene polyoxypropylene alkyl ethers, sucrose
fatty acid esters and polyglycerin fatty acid esters.
30 9. The method according to claim 8, wherein the HLB value
of the nonionic surfactant is 10 or more.
10. The method according to claim 1, wherein the nonionic
- 135 - EG026
surfactant is selected from the group consisting of
polyoxyethylene (degree of polymerization 10 to 20) octyl
dodecyl ether, polyoxyethylene (degree of polymerization
10 to 20) stearyl ether, polyoxyethylene (degree of
5 polymerization 10 to 20) polyoxypropylene (degree of
polymerization 4 to 8) cetyl ether, polyoxyethylene (degree
of polymerization 20 to 100) hydrogenated castor oil, and
sucrose lauric acid ester.
10 11. The method according to claim 2, wherein in the step
of preparing an aqueous dispersion liquid containing α-lipoic
acid and a nonionic surfactant,
polyethylene glycol is mixed into the nonionic surfactant,
prior to the dissolving of α-lipoic acid in the nonionic
15 surfactant; or
water containing polyethylene glycol is used as the
liquid containing water, in the step of adding a liquid
containing water to the surfactant solution.
20 12. α-Lipoic acid nanoparticles comprising α-lipoic acid,
a nonionic surfactant, a divalent metal ion, and a carbonate
ion or a phosphate ion.
13. The α-lipoic acid nanoparticles according to claim 12,
25 wherein the divalent metal ion is a calcium ion, a zinc ion
or a magnesium ion.
14. The α-lipoic acid nanoparticles according to claim 12,
wherein the nonionic surfactant is selected from the group
30 consisting of polyoxyethylene hydrogenated castor oils,
polyoxyethylene alkyl ethers, polyoxyethylene sorbitan
fatty acid esters, polyoxyethylene polyoxypropylene alkyl
ethers, sucrose fatty acid esters and polyglycerin fatty
- 136 - EG026
acid esters.
15. The α-lipoic acid nanoparticles according to claim 12,
further comprising polyethylene glycol.
5
16. An external preparation for skin, comprising the α-lipoic
acid nanoparticles according to claim 12.
17. A pharmaceutical product comprising the α-lipoic acid
10 nanoparticles according to claim 12.
18. A composition for oral cavity, comprising the α-lipoic
acid nanoparticles according to claim 12.
15 19. A food comprising the α-lipoic acid nanoparticles
according to claim 12.