JP3401476B2 - Cosmetics - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] TECHNICAL FIELD The present invention relates to a cytokine activity.
Treatment of diseases in which the action of potentiators and cytokines is reduced
Agent. The present invention particularly relates to the responsiveness of cytokines.
By increasing, cytokai decreased due to aging etc.
Activates the reactivity of cytokines or reduces cytokines
The present invention relates to cosmetics that can treat the abnormalities that occur. [0002] 2. Description of the Related Art Cytochrome is used for repairing aging and rough skin.
Is known to be closely related to
You. Cytokines are also significantly involved in disease
Attempts to use cytokines in therapy
Is being actively conducted. Specifically, as a therapeutic drug,
Tocaine itself orally or parenterally by injection, transdermal, etc.
And the like. But until complete healing
Require the use of large amounts of expensive cytokines
There is a great financial burden on patients as a therapeutic drug. On the other hand, the production of these cytokines is promoted.
When using a substance that does
Etc. are administered parenterally to humans. However, after all this
Cytokine
Similarly, the financial burden on patients is great. [0004] In addition, large dose administration of cytokines alone
May disturb the metabolic balance and may be used externally
May not be expected to have a local effect in terms of decomposition and absorption.
May cause more safe skin metabolism.
An active method was desired. [0005] SUMMARY OF THE INVENTION Accordingly, the present invention provides
Exists in the living body by administering itself to the living body
Providing cosmetics that can enhance cytokine response
The purpose is to provide. [0006] SUMMARY OF THE INVENTION The present invention is directed to the above-mentioned object.
To achieve1,3-diamino-2-propanol,
Selected from the group consisting of 1-amino-2-butanolD
Cosmetics containing a tanolamine derivative or a salt thereofTo
provide. [0007] BEST MODE FOR CARRYING OUT THE INVENTION Used in the present inventionDTanorua
Min derivatives1,3-diamino-2-propanol,
Also selected from the group consisting of 1-amino-2-butanol
ofIs good. [0008] Also,D used in the present inventionTanorua
The salt of the min derivative is not particularly limited, but is
Biologically acceptable hydrochloride, hydrobromide, sulfate, phosphorus
Inorganic acid salts such as acid salts and acetates, fumarate, maleate
Phosphate, tartrate, citrate, p-toluene sulfone
Organic acid salts such as acid salts can be mentioned. [0010] As the cytokine activated in the present invention,
Is a platelet-derived growth factor (Platelet-Deri)
ved Growth Factor, hereinafter PDGF
Abbreviated), fibroblast growth factor (Fibroblas)
t Growth Factor, hereinafter abbreviated as FGF
), Epidermal growth factor (Epidermal Growth)
h Factor, hereinafter abbreviated as EGF), transformation
Growth Factor (Transforming Growth)
Factor, hereinafter abbreviated as TGF), osteogenic factor
(Bone Morphogenetic Prote
in, hereinafter abbreviated as BMP), interferon (I
interferon (hereinafter abbreviated as IFN), granulocytes
Colony stimulating factor (Granulocyte Colo)
any-Stimulating Factor, hereinafter G
-CSF), macrophage colony stimulating factor
(Macrophage Colony-Stimu)
rating Factor, hereinafter abbreviated as M-CSF
), Insulin-like growth factor (Insulin-Li)
ke Growth Factor, hereinafter abbreviated as IGF
Do), hepatocyte growth factor (Hepatocyte Gr)
owth Factor (hereinafter abbreviated as HGF), bone
Medullary stem cell growth factor (Stem Cell Facto)
r, hereinafter abbreviated as SCF), nerve growth factor (Nerv
e Growth Factor, hereinafter abbreviated as NGF
Vascular endothelial growth factor (Vascular Endo)
therial Cell Growth Facto
r, hereinafter abbreviated as VEGF), interleukin (a
Interleukin Network, Kodansha, 1992)
And the like. Among them, especially, receptors
Tyrosine kinase, serine or
Cytokine having leonine phosphorylation or kinase activity
It is preferable because a high effect can be seen in the in. Tyrosine quina is a subunit of the receptor
, Serine or threonine phosphorylation or kinner
Cytokines having the activity
EGF, IGF, KGF, FGF, PDG
F, M-CSF, SCF, VEGF, NGF, HGF,
IL-2, TGF and the like. Basic FGF (basic FGF, hereinafter)
bFGF) and EGF (Hyundai Chemical Special Edition 16, East
Kyoto Chemical Dojinsha, p. 131), PDGF (Hyundai Chemical Special Edition)
4, Tokyo Chemical Dojinsha, 114 pages, 1985) or E
TGFalpha that binds to the GF receptor (TGFα,
Cytokines, Medical View, 10, p. 1991
Years), acidic FGF (acidic FGF, Molec)
ULTRA MEDICINE, Vol. 30, p. 986, 19
1993) is used as a treatment for stroke, pressure sores, wounds, and ulcers.
It is used as an anti-gastric ulcer and for the improvement of myocardial infarction.
There are also examples. On the other hand, BMP will enter an aging society in the future
As a treatment for osteoporosis
(Experimental Medicine Vol. 10, No. 15, 2010, 1992
Year). Further, TGF beta 1 (hereinafter referred to as TGF-β
1) is a fracture healing agent, progelatinase A or less
Suppression of matrix metalloprotease production outside
Tissue inhibitor of metalloproteiner
-Zes (Tissue Inhibitor of M
etalloproteinases, hereinafter TIMP
Abbreviated) (Experimental Medicine, 10, 15
No. 1860, 1992).
To further promote type I collagen synthesis,
It is expected as a wound healing agent. Hepatocyte growth factor (Hepatocyte)
(Growth Factor, hereinafter abbreviated as HGF)
Is an organ regenerating agent for recent organ transplantation
To suppress the growth of tumor cells (FEBS Let
ters, Vol. 2, p. 229, 1991)
It is also expected to be a therapeutic drug for cancer, which has the highest mortality rate of the person
I have. IFN gamma (IFNγ) and G-CS
F is used as a tumor treatment because it enhances the immune system.
Have been used. Interleukin-2 (IL-2) is an evil
In addition to reported cases of therapeutic drugs for vascular cell types, LAK
Used in therapy. LAK therapy can be used to
Cultivated by adding IL-2 to cytotoxic T cells
(CTL) and natural killer cells (NK cells)
Increase numbers back into the body and treat cancer by boosting immunity
(Iwanami Shinsho Publishing, “Cancer
Therapy ", Hiroshi Kobayashi, 292, pp. 151-154, 1
993). Has an effect similar to that of IL-2, and is stronger than IL-2.
Interleukin-12 (I
L-12) (Experimental Medicine, Vol. 10, No. 3, pp. 395)
399, 1992). About this cytokine
Are also being studied for application to anticancer drugs, LAK therapy, etc.
(Nikkei Biotech, 277, pp. 2-3, 1993
Year). Promotion of cytokine production used in the present invention
As a substance, for example, streptococcusStreptococ
us pyogenesWith penicillin (O)
K-432), glycyrrhizic acid, glycyrrhetinic acid
And so on. OK-432 is used as a tumor treatment
In the spleen cells of mice administered intraperitoneally.
To produce IFN. Also,
Tyrritic acid is used as a therapeutic agent for hepatitis,
Has an IFN-producing effect. In the present invention, the action of the cytokine is low.
A disease that has been reduced or has reduced cytokine levels
Or decreased reactivity of cytokine itself
Mean disease. Specifically, ulcers such as pressure sores, gastric ulcers, and lung fibers
Fibrosis, organ fibrosis such as cirrhosis, osteoporosis, and gas
Diseases such as immune system decline. The cosmetic of the present invention is used according to the purpose of use.
By adding known components that are commonly used,
To prepare compositions in various dosage forms such as solid preparations, semi-solid preparations, and liquid preparations.
It is possible to manufacture. Specifically, as a solid agent
For tablets, granules, fine granules, powders, powders, hard capsules
Ointments, gels, chestnuts and the like.
Liquids such as syrups and elixirs.
Sill, soft capsule, lotion, spray, paste
Agents and the like. Examples of commonly used known components include, for example,
For example, hydrocarbons such as petrolatum, squalane, liquid paraffin
High-grade alcohols such as hydrogen, stearyl alcohol, and cetanol
, Myristate isopropyl, palmitate isop
Lower alkyl esters of higher fatty acids such as ropir, lanoli
Animal fats and oils such as glycerin, propylene glycol
Such as polyhydric alcohol, Macrogol 400, Macrogo
Polyethylene glycol such as 4000
Glycerin fatty acid esters such as glycerin acid, lauri
Sodium sulfate, polyethylene monostearate
Coal, polyoxyethylene alkyl ether phosphoric acid
(Product name NIKKOL DDP-2, Nippon Surfactor
Surfactants, waxes, resins, water
And, if necessary, butyl paraoxybenzoate,
Mix with preservatives such as methyl benzoate and manufacture according to standard methods.
Can be These compositions may be in one-dose form.
Is an ethanolamine derivative represented by the general formula (I).
Or its salt, and cytokines or cytokine production
The excipient may be divided into two dosage forms. Two dose form
In such a case, it may be used together at the time of use. In that case, the two dosage form
The route of administration may be different. Ethanolamine represented by the general formula (I)
The content of the derivative depends on the dosage form, but the composition to be applied
0.0001 to 2 weight, preferably based on the total weight of the product
%, More preferably 0.001 to 1% by weight. Was
However, those that are diluted at the time of use, such as bath salts,
The content can be increased. Further, in the LAK therapy, the culture is performed outside the body.
When adding and culturing cytokines to lymphocytes to be cultured,
Ethanolamine derivative represented by general formula (I) of the present invention
The body-containing cytokine activity enhancer is added to the medium.
It can also be used as an auxiliary. In this case
The addition amount is determined based on the ethanolamine derivative represented by the general formula (I).
As a body volume, based on the total amount of the medium, preferably 0.0
001 to 2% by weight, more preferably 0.001 to 0.
5% by weight. The cytokine activity enhancer of the present invention
Therapeutic agents for diseases in which the activity of Itokine has been reduced are researched and tested.
It can be used as an experimental reagent when added to cultured cell lines.
As an active ingredient in ordinary pharmaceuticals and cosmetics
You can also be. [0028] The therapeutic agent for cytokine of the present invention comprises the aforementioned LA
In addition to using in addition to cultured lymphoid cells in K therapy,
Can be used for oral administration, injection, transdermal administration, etc.
You. The dosage form for oral administration includes tablets, condyles
Besides solid preparations such as granules, powders, fine granules and hard capsules,
Includes liquid preparations such as syrups, elixirs, and soft capsules.
I will. Tablets, granules, powders and fine granules are generally
A compound of formula (I) or a pharmaceutically acceptable salt thereof;
For example, lactose, starch, crystalline cellulose, stearin
Magnesium acid, hydroxypropyl cellulose, tall
It is produced by mixing with ordinary pharmaceutical additives such as For hard capsules, the above fine granules and powders are suitable.
It is manufactured by filling into capsules. Syrup is white
Sugar, D-sorbitol, carboxymethyl cellulose, etc.
Aqueous solution containing methyl parahydroxybenzoate,
Formula (I) together with a preservative such as propyl benzoate
Dissolve or suspend the compound or its pharmaceutically acceptable salt
Produced turbid. Elixirs include compounds of the general formula (I)
Or a solution of its pharmaceutically acceptable salt in ethanol.
Lyserine, orange oil, lemon oil, coriander oil, a
It is manufactured by mixing varnish oil, talc and the like. Soft capsules are made up of lipid excipients, such as
General formula for natural oils, oily emulsions, glycols, etc.
Dissolves the compound of (I) or a pharmaceutically acceptable salt thereof
Alternatively, it is produced by suspending and filling in a soft capsule. An injection is a compound of the formula (I) or a compound thereof.
A pharmaceutically acceptable salt of saline or, for example,
Lipid excipients such as vegetable oils, oily emulsions, and glycols
Dissolve or emulsify in an aseptic ampoule or vial
It is manufactured by encapsulating in a file. Using Bayer
If necessary, use a cytokine or site
After simultaneously adding a drug that promotes the production of
It is also possible to manufacture. The dosage form for transdermal administration includes ointments,
Semi-solid preparations such as gels and creams, lotions, cataplasms
Agents, sprays, and liquid preparations such as patches. The cytokine therapeutic agent of the present invention can be administered orally or
Is administered parenterally. For example, organ tumors, heart disease, etc.
For diseases of the organs, psoriasis, oral
For epithelial diseases such as Lloyd, by percutaneous or local injection,
Preferably, the cytokine activity enhancer of the present invention is administered.
No. The dosage is determined by the patient's age, weight, symptoms, and administration.
Methods or co-administered cytokines or cytokines
It depends on the amount of the substance that promotes
In general, the amount of compound (I) is generally 0.5 to 1 per dose.
An amount of 000 mg is administered 1-3 times daily. And an example
For example, for oral diseases such as organ tumors and heart disease,
Or, when administered systemically by injection, 30 to 1
A dosage of 2,000 mg is appropriate, for organ diseases, psoriasis,
Topical transdermal administration for epithelial diseases such as keloids
In some cases, a dose of 1 to 50 mg is appropriate.
You. In these cases, the cytokine or support used together
The therapeutic amount of the substance that promotes production of itokines is higher than usual.
It can be used with a reduction of 20-90%. Next, prior to the examples, the effects of the present invention will be described.
The following describes test examples. However, the present invention is described in the test examples.
It is not limited to the specified raw materials and the mixing ratio. What
The definitions of the terms used in each test example are described below. (A) MEM medium Minimum Essential Medium
(Dainippon Pharmaceutical Co., Ltd., 10-101) 10.6g to 1.2
g Add sodium bicarbonate, add distilled water and add 1 liter.
After that, the pH was adjusted to about 7 by blowing carbon dioxide gas (hereinafter referred to as “pH”).
Below, abbreviated as MEM medium). (B) FBS Fetal bovine serum (Fetal Bovine Serum) (C) Buffer for measurement 0.2 M sodium chloride, 5 mM calcium chloride and
0.05% (W / V) Bridge-35 (trademark, Nakarai
Tesk Co., Ltd., polyoxyethylene (23E.0.) Lauryl
Ether) containing 50 mM Tris aqueous solution with hydrochloric acid
Buffer adjusted to pH 7.5 at room temperature. (D) Collagenase As a collagenase, a scaffold derived from human fibrosarcoma cells
Dependent cells were produced in serum-free and protein-free medium
Human procollagenase was purchased from CM-Sepharose (trademark,
Pharmacia) and zinc chelating Sepharose
(Trademark, manufactured by Pharmacia) and used for measurement.
Dissolve in the buffer solution and add trypsin (sig
Ma, Type 12) and added at 35 ° C. for 5 minutes
After incubation, soybean trypsin inhibitor
(Merck) to inactivate trypsin
(See Japanese Patent Application No. 1-238941). (E) Procollagenase production In this test, the amount of procollagenase produced was measured using trypsin.
It was quantified as collagenase activity obtained by activation. (F) TIMP production In this test, the amount of TIMP production was
It was quantified as sex. (G) bFGF responsiveness enhancement rate In this study, bFGF promotes procollagenase production
Are known to increase the bFGF activity of the compound.
The strong factor is that the amount of procollagenase production is compared with the addition of purified water.
Was calculated. (H) TGF-β1 responsiveness enhancement rate In this study, TGF-β1 promotes TIMP production
Is known to increase the TGF-β1 activity of the compound.
The power factor was calculated by comparing the amount of TIMP production with the addition of purified water.
Was. (I) PDGF responsiveness enhancement rate In this study, PDGF promotes procollagenase production
To increase the PDGF activity of the compound.
The strong factor is that the amount of procollagenase production is compared with the addition of purified water.
Was calculated. (J) Increase bFGF responsiveness in animal experiments
Power In this study, bFGF was found to promote angiogenesis.
The bFGF activity enhancement rate of the compound
The amount of hemoglobin in Matrigel injected subcutaneously in the abdomen
Produced in comparison with no additive. [0049] EXAMPLES Test Example-1 Normal human fibroblast cell line (collected from the skin of Caucasian women
CCD45SK (ATCC CRL 1506)]
Cell density was 10% (V / V) FBS and 1% (V / V)
In MEM medium containing non-essential amino acids (Dainippon Pharmaceutical)
1 × 10^FiveAnd adjust it to a 12-well plate.
Seed 0.8 ml each (8 × 10^Four5 pieces / hole)
The cells were cultured at 37 ° C. under% carbon dioxide and saturated steam. The cytokine activity described in Example 1 described below
0.6% (V / V-methyl-L-serine)
V) Diluted with MEM medium supplemented with FBS to 1 mM
To obtain an additive solution. After 24 hours, the culture solution was removed by suction, and the final concentration was
Cells in MEM medium supplemented with 0.6% (V / V) FBS
Was washed twice, and 0.8 ml of an addition solution was added, followed by 2 days
Cultured. After 2 days, the culture solution was removed by suction and the added solution was removed.
0.8 ml was added, and the cells were cultured for 2 days. Do this further
Repeat once, medium containing cytokine activity enhancer
For a total of 6 days. After the above operation, the culture solution is removed by suction.
3 ng / ml bFGF (Boehringer Mannheim)
Medium containing 0.6% (V / V) FBS
Was added and cultured for 3 days to obtain a culture supernatant. To 500 μl of the obtained culture supernatant, add 10 mM
Squirrel HCl buffer [adjusted to pH 7.8 at 4 ° C, 1 mM chloride
Calcium, including 0.05% (W / V) Bridge-35
3.5 ml), and equilibrated with the same buffer.
Pharos CL-6B (trademark, manufactured by Pharmacia, Inc.
(Volume of 0.5 ml). Next, the mixture containing 125 mM sodium chloride was added.
Inhibitor was removed with 0.5 ml of the same buffer as described above (total
4 times, 2 ml in total), containing 500 mM sodium chloride.
Procollagenase was recovered in 0.5 ml of the same buffer (total
Four times, a total volume of 2 ml) was used as a test solution. After appropriately diluting the test solution with the measuring buffer,
μl with 25 μl of measurement buffer and trypsin solution
(Sigma, Type 12 in buffer for measurement, concentration 1m
g / ml), add 20 μl, and at 35 ° C. for 5 minutes
After incubation, soybean trypsin inhibitor
Solution (adjusted to a concentration of 3 mg / ml with measurement buffer) 30
Add tryl to inactivate the trypsin and remove collagenase solution.
A liquid was obtained. Fluorescein isothiocyanate (hereinafter referred to as fluorescein isothiocyanate)
Below, abbreviated as FITC)
(FITC-collagen acetate solution at a concentration of 1 mg / ml,
Cosmo Bio Co., Ltd.) as the substrate solution
Method (see Inflammation, Vol. 2, No. 2, p. 123, 1984)
Measure the collagenase activity (unit / ml) according to the
Was. And, by the above-mentioned trypsin treatment, procollagena
Collagenase generated from lysate per minute at 35 ° C
1 μg of type I collagen (FITC-collagen)
The amount to be solved is defined as one unit of procollagenase,
The amount of enzyme production (unit / ml of culture solution) was determined (this value was
X₁And). On the other hand, as Comparative Example 1, N-methyl-L-
Purified water is added instead of serine, and the same operation as above is performed.
Cytokine activity enhancer (N-methyl-L-serine)
Procollagenase production without adding (unit / culture)
(Ml of nutrient solution)₁And). Next, based on these values, the site
The Cain (bFGF) activity enhancement rate was calculated. The results below
It is shown in Table 1. bFGF activity enhancement rate (%) = [X₁/ Y₁] × 100 [0060] [Table 1] The cytokine activity enhancer of Example 1
Increases collagenase production,
(BFGF) activity. Test Example-2 (concentration dependence) The cytokine activity enhancer (N
-Methyl-L-serine) with 0.6% (V / V) FBS
Diluted with added MEM medium to 0.01-10 mM
To obtain an additive solution. After culturing in the same manner as in Test Example 1, the culture supernatant was
I got Procollagenase production in the obtained culture supernatant
Was quantified. The results are shown in Table 2 below. [0064] [Table 2]N-methyl-L-serine reduces the amount of procollagenase produced.
Increased cytokine (bFGF) activity
Had increased in a dependent manner. Test Example-3 A normal human fibroblast cell line was inoculated in the same manner as in Test Example-1.
Was. The cytokine activity enhancer described in Example 2 described below.
0.6% (V / V) FBS (diethanolamine)
Dilute to 1 mM with the added MEM medium to make an addition solution.
Was. After culturing in the same manner as in Test Example 1, the culture supernatant was
I got However, here, the amount of bFGF added was 10 n
g / ml. The results of the measurement are shown in Table 3 below. [0067] [Table 3] The cytokine activity enhancer of Example 2 was procollagen
Increases the amount of lysate, and cytokines (bFGF)
Activity was enhanced. Test Example-4 Normal human fibroblast cell line (collected from the skin of Caucasian women
Detroit-551 (ATCC CCL 11
0)] with 10% (V / V) FBS, final concentration
0.1% (W / V) lactalbumin enzyme hydrolyzate (SIG
1% (V / V) non-essential amino acids and 1 mM
Sodium pyruvate (both from Dainippon Pharmaceuticals)
1 × 10 in MEM medium containing^FiveAnd adjust to 1 / ml
Inoculate 0.8 ml each into a 2-well plate (8 × 10
^FourPer piece), 5% carbon dioxide gas, saturated steam, 37 ° C
And cultured. The cytokine activity described in Example 3 to be described later
0.6% (V / V) F
BS, final concentration 0.1% (W / V) lactalbumin enzyme
Hydrolyzate, 1% (V / V) non-essential amino acids and 1 mM
Diluted with MEM medium supplemented with sodium
mM, and used as an addition solution. After 24 hours, the culture solution was removed by suction and the final concentration was
Cells in MEM medium supplemented with 0.6% (V / V) FBS
Was washed twice, and 0.8 ml of an addition solution was added, followed by 4 days
Cultured. After 4 days, the culture solution was removed by suction, and 3 ng / m
lbFGF, 0.6% (V / V) FBS, final concentration 0.1
% (W / V) lactalbumin enzyme hydrolyzate, 1% (V / V)
V) Non-essential amino acids and 1 mM sodium pyruvate
0.8 ml of MEM medium containing
A culture supernatant was obtained. [0072] To 250 µl of the obtained culture supernatant, 10 mM
Squirrel HCl buffer [adjusted to pH 7.8 at 4 ° C, 1 mM chloride
Calcium, including 0.05% (W / V) Bridge-35
1.5 ml) and the CM-cell equilibrated with the same buffer.
Pharos CL-6B (trademark, bed capacity 0.5 ml)
Was served. CM-Sepharose CL-6B (trademark)
Of Procollagenase and Procollagenase Content
Was measured in the same manner as in Test Example-1. The results below
It is shown in Table 4. [0074] [Table 4] The cytokine activity enhancer of Example 3 was a procollagenizer.
Increases the amount of lysate, and cytokines (bFGF)
Activity was enhanced. Test Example-5 The cytokine activity enhancer (N
-Methylethanolamine) as in Test Example-4.
The enhancement of bFGF activity was examined as described above. Below are the measurement results
It is shown in Table 5. [Table 5] The cytokine activity enhancer of Example 4 was procollagen
Increases the amount of lysate, and cytokines (bFGF)
Activity was enhanced. Test Example-6 Normal human fibroblast cell line (collected from the skin of Caucasian women
Detroit-551 (ATCC CCL 110)
Cell density of 10% (V / V) FBS, final concentration 1% (V
/ V) Non-essential amino acids and 1 mM sodium pyruvate
MEM medium containing media (all manufactured by Dainippon Pharmaceutical Co., Ltd.)
(Hereinafter referred to as MEM2 medium) 1 × 10^FivePieces / ml
And inoculate 0.8 ml each into a 12-well plate
Seed (8 × 10^Four5% carbon dioxide, saturated water vapor
The cells were cultured at 37 ° C. under air. The cytokine activity described in Example 5 described later
(N-isopropanoyl-2-methyl-ethano)
MEM containing 0.6% (V / V) FBS
It was diluted with 2 medium to 3 mM to give an added solution. After 24 hours, the culture solution was removed by suction, and the final concentration was
In MEM2 medium supplemented with 0.6% (V / V) FBS,
After washing the cells twice, add 0.8 ml of the addition solution, and
The culture was continued for a while. Two days later, the culture solution was removed by suction, and added again.
Add 0.8 ml of the solution, culture for 2 days, and culture for a total of 4 days.
Was. Two days later, the culture was aspirated off and 3 ng / ml bFG was removed.
F, MEM2 medium containing 0.6% (V / V) FBS
0.8 ml was added and cultured for 3 days to obtain a culture supernatant. C
Procollage from M-Sepharose CL-6B ™
Test for recovery of protease and measurement of procollagenase
-1. Procollagenase production in the obtained culture supernatant
The production was quantified. The results are shown in Table 6 below. [0081] [Table 6] N-isopropanoyl-2-methyl-ethanolamine
Has increased procollagenase production,
Cain (bFGF) activity was enhanced. Test Example-7 Normal human fibroblast cell line was inoculated in the same manner as in Test Example-6.
Was. The cytokine activity enhancer described in Example 6 described below.
0.6% of (D, L-2-amino-1-propanol)
(V / V) 3 mM after dilution with MEM2 medium containing FBS
And used as an addition solution. After culturing in the same manner as in Test Example 6, the culture supernatant was
I got Procollagenase production in the obtained culture supernatant
Was quantified. The results are shown in Table 7 below. [0084] [Table 7] D, L-2-amino-1-propanol is procollagen
It increases the production of the enzyme and increases the production of cytokines (bFG
F) The activity was enhanced. Test Example-8 Normal human fibroblast cell line was inoculated in the same manner as in Test Example-6.
Was. The cytokine activity enhancer described in Example 7 described below.
0.6% (V / V) of (2-amino-1-butanol)
Dilute to 3 mM with MEM2 medium containing FBS and add
The solution was used. After culturing in the same manner as in Test Example-6, culture supernatant
I got Procollagenase production in the obtained culture supernatant
Was quantified. The results are shown in Table 8 below. [0086] [Table 8] 2-amino-1-butanol produces procollagenase
Increased the amount of cytokine (bFGF) activity
Had been strengthened. Test Example-9 Normal human fibroblast cell line was inoculated in the same manner as in Test Example-6.
Was. The cytokine activity enhancer described in Example 8 described below.
0.6% of (1,3-diamino-2-propanol)
(V / V) 3 mM after dilution with MEM2 medium containing FBS
And used as an addition solution. Culture in the same manner as in Test Example-6
Thereafter, a culture supernatant was obtained. Procollage in the obtained culture supernatant
The amount of the enzyme produced was quantified. The results are shown in Table 9 below. [0088] [Table 9] 1,3-diamino-2-propanol is procollagena
Increased the production of cytokines (bFG
F) The activity was enhanced. Test Example-10 Normal human fibroblast cell line was inoculated in the same manner as in Test Example-6.
Was. The cytokine activity enhancer described in Example 9 described later.
0.6% (V / V) of (1-amino-2-butanol)
Dilute to 3 mM with MEM2 medium containing FBS and add
The solution was used. After culturing in the same manner as in Test Example-6, culture supernatant
I got Procollagenase production in the obtained culture supernatant
Was quantified. The results are shown in Table 10 below. [0090] [Table 10] 1-amino-2-butanol is
Genease production is increased, and cytokines (bF
GF) activity. Test Example-11 The cytokine activity enhancer (N
-Methyl-L-serine) with 0.6% (V / V) FBS
Dilute to 1 mM with the added MEM medium,
did. Six days with the added solution in the same manner as in Test Example 1.
Cultured. Six days later, 30 ng / ml PDGF-BB (E
-0.6%
(V / V) Add 0.8 ml of MEM medium containing FBS
And cultured for 3 days to obtain a culture supernatant. The cytokine (PDGF) activity enhancement rate was
As in Test Example 1, a cytokine activity enhancer was added.
The production amount of procollagenase in the culture supernatant_TwoAnd the ratio
Comparative Example 1 Procollage in culture supernatant to which purified water was added
Yase production_TwoWas calculated by the following equation. Cytokine (PDGF) activity enhancement rate
(%) = [X_Two/ Y_Two] × 100 Procollagenase production in the obtained culture supernatant
The production was quantified. The results are shown in Table 11 below. [0097] [Table 11]The cytokine activity enhancer of Example 1 was procollagen
Increases the amount of lysate, and cytokines (PDGF)
Activity was enhanced. Test Example-12 The cytokine activity enhancer (N
-Methyl-L-serine) with 0.6% (V / V) FBS
Dilute to 1 mM with the added MEM medium,
did. As in Test Example 1, the additive solution was added and
Cultured for days. After the culture, the medium was removed and 3 ng / ml T
GF-β1 (Austral Biologicals)
And MEM medium containing 0.6% (V / V) FBS at 0.
8 ml was added and cultured for 3 days to obtain a culture supernatant. Measurement of the amount of TIMP produced in the obtained culture supernatant
Was determined as follows. First, measure the culture supernatant
10- to 1000-fold dilution with a buffer solution. With each diluent
Mix an equal volume with a known amount (0.24 units) of collagenase solution
Test Example-1 using FITC-collagen as a substrate
The collagenase activity is measured in the same manner as described above. This measurement result
The inhibition curve was determined from this curve, and the medium that inhibited 50% from this inhibition curve
The dilution ratio of the culture supernatant was determined, and this dilution ratio was used as a unit. On the other hand, Comparative Example 1 was the same as Test Example-1.
Was performed. In addition, cytokine (TGF-β1)
The activity enhancement rate depends on the culture with the cytokine activity enhancer added.
The amount of TIMP produced in the supernatant was X_ThreeAnd as Comparative Example 1
The amount of TIMP produced in the culture supernatant to which water was added was determined as Y_Threeage
It was calculated by the following equation. Cytokine (TGF-β1) activity enhancement rate
(%) = [X_Three/ Y_Three] × 100 The amount of TIMP produced in the obtained culture supernatant was determined.
Weighed. The results are shown in Table 12 below. [0104] [Table 12] The cytokine activity enhancer of Example 1
IMP production is increased, and cytokines (TGF
-Β1) The activity was enhanced. Test Example-13 The cytokine activity enhancer described in Example 3 described below (D
(Tanolamine) in MEM medium containing 0.6% FBS
Diluted to 1 mM to make an addition solution, as in Test Example-12.
A culture supernatant was obtained. However, addition of cytokine activity enhancer
Culture period for 4 days, and TGF-β1 added 6 days
The amount of TIMP produced in the subsequent culture supernatant was measured. Quantitative determination of TIMP production in the obtained culture supernatant
did. The results are shown in Table 13 below. [0108] [Table 13] The cytokine activity enhancer of Example 3
IMP production is increased, and cytokines (TGF
-Β1) The activity was enhanced. Test Example-14 Example 10 [84 mM (1% by weight) N-methyl-L-
Phosphorus] or Example 11 [75 mM (1% by weight)
Sopropanoyl-2-methyl-ethanolamine]
The ream was abdominal shaved from SD male rats (7 weeks old, body
Weight 210-230 g, 18-24 animals per group, CLEA Japan
1 ng / m after daily application for 21 days
lbFGF (Dainippon Pharmaceutical Co., Ltd.) and heparin (Gib
Co., Ltd. BRL Co., Ltd.) [MATRIG
EL ™, basement membrane matrix phenol red
Free, catalog number 40234C] under the abdomen
Each 1 ml was injected, and collected from the abdomen 8 days later. The collected Matrigel was added to a phosphate buffer (neat)
Hiscotron (trademark)
After homogenizing at Seisakusho Co., Ltd., centrifugation
The amount of hemoglobin is determined by hemoglobin-Test Wako (Wako Jun
(This value was calculated using X_FourAnd ). On the other hand, the cream of Comparative Example 2 described later was used.
After performing the same operation as above, increase cytokine activity.
F when no strong agent (N-methyl-L-serine) is added
The amount of moglobin was measured (this value was_FourAnd ). [0113] [Table 14] Next, based on these values, the site
The Cain (bFGF) activity enhancement rate was calculated. [0115] bFGF activity enhancement rate (%) = [X_Four/ Y_Four] × 100 The amount of hemoglobin in the obtained Matrigel
Was quantified. The results are shown in Table 15 below. [0117] [Table 15] N-methyl-L-serine and N-isoprop
Lopanoyl-2-methyl-ethanolamine is
Increases the amount of hemoglobin and increases cytokine (b
FGF) activity. Test Example-15 (Skin permeability test) Wistar male rats were shaved the day before the test.
After slaughter, the abdominal skin was immediately peeled off. The creams of Application Examples -28 to 30 to be described later
As a sample, a vertical diffusion cell device (Kelcoso
Engineering company, effective area 8cm^Two) Was used. Figure
In 1, 1 is a tetrafluoroethylene lid, 2
Is a sampling port, 3 is a drug sample, 4 is skin, 5 is O-
A ring, 6 is a receptor phase, and 7 is a stirrer. Using the above vertical diffusion cell device,
Place in a 32 ° C air bath and place an isotonic resin on the receptor side.
Acid buffer (1.44% sodium bicarbonate and 2.33%)
%) Prepared with 45% potassium dihydrogen phosphate
1 g of a sample was applied to the abdominal exfoliated skin of the rat side (n =
3) Buffering of the reservoir after 1, 2, 4 and 6 hours
Collect 0.2 ml of the solution and store immediately at -20 ° C
did. After thawing the frozen sample, add 10 mM
Diluted to a concentration of asparatylglycine (final concentration 40μ).
M) was added as an internal standard. Amino acid content in external standard
The same concentration of asparatylglycine was added to the analysis standard solution (AN type).
And N-methyl-L-serine, ethanolamine and
And N-methylethanolamine (final concentration 10 μM)
Used in addition. The quantification of these samples was performed using o-phthalic
Post-column amino acid analysis using the dehid method
(Analytical Biochemis)
try, 96, 298, 1979).
Was. The results for N-methyl-L-serine are shown in FIG.
It is shown in FIG. N-methyl-L-serine (Application Example 28)
Penetrates the skin, and penetrates for 2 to 6 hours after applying the cream
The degree is about 0.12μmol / cm^Two/ Hour (Figure
2). In addition, ethanolamine (Application Example 29) and N
-Methylethanolamine (Application 30) also penetrates the skin
(Table 16). [0124] [Table 16]Test Example-16 (Acute toxicity test) Water and an aqueous solution of N-methyl-L-serine
To 5 g / kg body weight) at 0.2 ml / k
g ICR male mouse (5 weeks old, weight 24
-28 g, 5 animals per group), and then administered mouse for 7 days.
Was observed. In the N-methyl-L-serine administration group,
As in the control group (water only), no deaths were observed.
Did not. Test Example-17 (Skin cumulative irritation test) N-methyl-L-serine, ethanolamine and N-
Each of methylethanolamine was treated with hydrochloric acid to pH 7
The adjusted aqueous solution was used as a test sample. Japanese native male
Using a rabbit (approximately 3 kg in weight), the Dratez method (Apprai
sal of the Safety of Chem
icals in Foods, Drags and
Cosmetics, 46, 1959, edite
d and published by the De
itorial Committee, Associate
Tion of Food and DrugOffi
vials of U.S.A. S. A. ). That is, the back of the rabbit (3 ×
4 cm) in a 0.1% (W / V) aqueous solution of the test compound
Apply 0.1ml once a day by open application for 4 days
Was. 24 hours later, erythema, edema, crusts and fissures on application
With core. [0127] [Table 17] The erythema score and edema score listed in Table 17
Cumulative irritation score by summing crust score and crack score
And Next, based on the cumulative stimulus score, the following Table 1 was used.
N-methyl-L-serine, ethanol
Lumin and N-methylethanolamine
Judged. The results are shown in Table 19 below. [0130] [Table 18] [0131] [Table 19] N-methyl-L-serine, ethanolami
And N-methylethanolamine accumulation on the skin
It was found that the productive irritation was low. Test Example-18 (Primary skin irritation test,
No. 04-1130) N-methyl-L-serine, ethanolamine and N-
Each of methylethanolamine was treated with hydrochloric acid to pH 7
The adjusted aqueous solution was used as a test sample. Using Japanese native male rabbits (weight: about 3 kg)
The test was performed according to the Drates method described above. That is, the back of a rabbit whose hair was cut was abraded.
Create a site (damaged skin), that of damaged skin and normal skin
In each case, 0.1 ml of water or 1% (W /
V) 0.1 ml of the aqueous solution was applied to a bandage for patch test (1.
2x1.6cm, Ribbon Aid registered trademark, River tape
(Manufactured by Pharmaceutical Co., Ltd.). 24 hours later,
Peel off, observe skin for erythema and edema,
The same observation was made 48 hours after the plaster peeling. And the table
Erythema and edema scores were assigned according to 20 evaluation criteria.
I did. [0136] [Table 20] Each score listed in Table 21 was obtained, and the following formula was used.
More primary stimulus scores were calculated.                          A + BC + DE + FG + H       Primary stimulus score = + + +                          2 2 2 2 [0138] [Table 21] Next, based on the primary stimulus score,
Based on the criteria, the degree of irritation of N-methyl-L-serine was determined.
Was. The results are shown in Table 23 below. [0140] [Table 22] [0141] [Table 23] N-methyl-L-serine, ethanolami
And N-methylethanolamine have low skin irritation
(See JP-A-4-1130)
See). Test Example-19 (Skin irritation test) N-methyl-L-serine, ethanolamine and N-
Each of methylethanolamine was treated with hydrochloric acid to pH 7
The adjusted aqueous solution was used as a test sample. In addition, healthy person 19
The subject was the subject. The closed patch test method (Journa
l of the Society of Cosmet
ic Chemist, vol. 31, p. 97, 1980)
As a result, using the KI chamber on the subject's forearm,
0.05 ml of 1% (W / V) aqueous solution of test compound for 24 hours
Between the patch removal 1 hour and 24 hours after patch removal
A skin reaction was observed. N-methyl-L-serine, ethanolami
And N-methylethanolamine patches
In testing, one hour after patch removal and 24 hours
One subject had slight erythema at both later points in time.
It was judged that there was almost no skin irritation. Less than
Examples will be further described below with reference to examples of the present invention. Example 1 (N-methyl-L-serine) A 1 M aqueous solution of N-methyl-L-serine has a pore size
0.2 μm nitrocellulose membrane (Advantech East
Filtration and sterilization with DISMIC-25 manufactured by Western Corporation,
An in activity enhancer was obtained. Examples 2 to 4 (diethanolamine, ethanol
Nolamine and N-methylethanolamine) Diethanolamine adjusted to pH 7.5 with hydrochloric acid, ethanol
Nolamine or N-methylethanolamine 1M solution
The same procedure as in Example 1 was carried out except that the solution was used and cooled under ice.
Thus, a cytokine activity enhancer was obtained. Examples 5 to 9 (N-isopropanoyl-2
-Methyl-ethanolamine, D, L-2-amino-1
-Propanol, 2-amino-1-butanol, 1,3
-Diamino-2-propanol and 1-amino-2-
Butanol) Under ice cooling, N-isopropanoyl-2-methyl-ethanol
Amine, D, L-2-amino-1-propanol,
2-amino-1-butanol, 1,3-diamino-2-
Propanol and 1-amino-2-butanol
Each was adjusted to pH 7.5 with hydrochloric acid to make a 1M solution,
Nitrocellulose membrane with an arc size of 0.2 μm (Adobe
Sterilized by filtration with DISMIC-25) manufactured by Antec Toyo.
Then, a cytokine activity enhancer was obtained. Examples 10 to 11 (cream) N-methyl-L-serine as an active ingredient in 100 g
(Example 1) or N-isopropanoyl-2-methyl
-Contains 1000 mg of ethanolamine (Example 5)
Cream was prepared as shown in Table 24. [0150] [Table 24] N-methyl-L-serine or N-isoprop
Lopanoyl-2-methyl-ethanolamine, Palau
Methyl xyloxybenzoate, potassium hydroxide, concentrated glycerin,
1,3-butylene glycol, sodium cetyl sulfate and
And purified water by heating to 80 ° C in a hot water bath and mixing.
Stearic acid, cetanol heated to 80 ° C.,
Lipophilic glycerin monostearate, lanolin, liquid
Raffin, methylpolysiloxane and paraoxybenzoate
It was slowly added with stirring to the mixture of butyl perfumes. Next
In addition, a homogenizer (TOKUSHUKIKAIKOG)
Vigorous stirring for 2.5 minutes (2500 rpm)
m), after each component is sufficiently emulsified and dispersed, slowly stirred
Each was cooled to obtain a cream. Comparative Example 2 N-methyl-L-serine or N-isopropanoyl-
Purified water was added without adding 2-methyl-ethanolamine.
Except for 6.38 g, the same as in Examples 10 to 11
Thus, a cream of Comparative Example 2 was obtained. Examples 12 to 14 (tablets) To a mixture of the components (A) in Table 25, 30 g of the component (B) was added.
It was dissolved in water and added, and kneaded well. 20 parts of this kneaded material
And then granulated into granules through a sieve, and dried.
The component (C) is mixed with the granules thus obtained, and the mixture is compressed into tablets of 200 mg.
By tableting, one tablet contains 100 mg of the active ingredient.
Tablets were prepared. [0154] [Table 25] Examples 15 to 20 (capsules) Thoroughly mix each of the components in Table 26, 300 mg of the mixture
Into 2 capsules, and the active ingredient in 1 capsule
100 mg (Examples 15-17) or 200 mg (real
Examples 18 to 20) The contained capsules were prepared. [0156] [Table 26] Examples 21 to 26 (granules) In a mixture of the component (A) in Table 27, dissolve in 1000 g of water.
The added component (B) was added and kneaded sufficiently. This kneaded material is
Granulating through a mesh sieve, drying and sizing
30 g of the active ingredient per 1 g (Examples 21 to 21)
23) or 300 mg (Examples 24-26)
Granules were prepared. [0158] [Table 27] Examples 27 to 29 (syrups) Add component (B) in Table 28 to 400 ml of purified water at 90 ° C.
And then add component (C) at the same temperature and mix well.
And then cooled to 30 ° C. Ingredient (A) is added to this mixture.
Dissolved in 100 ml of purified water and added at 30 ° C. for 30 minutes
Stirred. Next, the component (D) is added and stirred for 20 minutes.
Add purified water to make the total volume 1000 ml, and perform aseptic filtration.
100 ml of the active ingredient in 1 ml
A syrup preparation was prepared. [0160] [Table 28] Examples 30 to 53 (injections) The amount of cytokine activity enhancer shown in Table 29 below was
Purified water for injection (10% human serum albumin and 20%
(Including mannitol) to make a 200 ml solution,
The bacteria were removed by aseptic filtration. N-methyl-L-seri
, Ethanolamine and diethanolamine
Put 2 ml in a vial with a capacity of 10 ml
Was dispensed. Aseptically purge with nitrogen, stopper and obtain injection.
Was. N-methylethanolamine, N-isopropanoy
Ru-2-methyl-ethanolamine, D, L-2-amido
No-1-propanol, 2-amino-1-butanol,
1,3-diamino-2-propanol and 1-amino
In the case of -2-butanol, the sterilized solution is used in a volume of 3 ml.
Dispense 2ml into brown amber ampoules and seal the ampules
To obtain an injection. The amount of the active ingredient in the table is 1 vial.
N-methyl-L-serine in medium or in one ampoule, eta
Nolamine, diethanolamine, N-methylethanol
Amine, N-isopropanoyl-2-methyl-eta
Nolamine, D, L-2-amino-1-propano
2-amino-1-butanol, 1,3-diamino-
With 2-propanol or 1-amino-2-butanol
The amount is expressed. [0162] [Table 29] Examples 54 to 71 (Ointments) Component (A) in Tables 30 and 31 was heated to 80 ° C in a hot water bath
And the mixture is heated to 80 ° C.
(B) was added gradually with stirring. Next, homogenous
1. With Iser (manufactured by TOKUSHUKIKAKOGYO)
Stir vigorously for 5 minutes (2500 rpm)
After emulsifying and dispersing, gradually cool while stirring, and finally
Ethanolamine derivative and bFGF or aFGF
By adding, 500m of the active ingredient in 100g
g and 2 mg of bFGF (Examples 54 to 62), or
1000 mg of active ingredient and 0.2 mg of aFGF
Examples 63-71) Ointments containing were prepared. [0164] [Table 30][0165] [Table 31]The following is an application example. The values in the table are
Unless indicated otherwise, the percentages are by weight. Application Examples 1 to 27 N-methyl-L-serine as an active ingredient in 100 g
(Application Examples 1, 10 and 19), diethanolamine
(Application Examples 2, 11 and 20), ethanolamine
Examples 3, 12 and 21), N-methylethanolamid
(Application Examples 4, 13 and 22), N-isopropanoy
2-methyl-ethanolamine (Application Examples 5, 14 and
And 23) or D, L-2-amino-1-propano
(Application Examples 6, 15 and 24), 2-amino-1-but
Tanol (Application Examples 7, 16 and 25), 1,3-dia
Mino-2-propanol (Application Examples 8, 17 and 26)
Or 1-amino-2-butanol (Application Examples 9, 18 and
And 100), 200 or 400 mg
Lotions were prepared as shown in Tables 32 and 33. [0168] [Table 32][0169] [Table 33]The active ingredients in the table and the component (A) were mixed in a hot water bath for 8 hours.
The mixture was heated to 0 ° C and mixed. On the other hand, component (B) also
The mixture was heated to 0 ° C and mixed, and the mixture was stirred.
Add slowly while stirring, and homogenizer (TOKUSH
Stir vigorously for 2.5 minutes with UKIKAIKOGYO)
(2500 rpm). Cool slowly to room temperature with stirring
I got a lotion. Application Examples 28 to 30 A cream having the composition shown in Table 34 was prepared. [0172] [Table 34] After uniformly mixing and dissolving component (A) at 80 ° C,
The mixture (B) was mixed and dissolved (mixture I). Aside from this,
After uniformly mixing and dissolving component (D) at 80 ° C.,
(C) was mixed and dissolved (mixture liquid II). Next, the mixed solution I
Slowly add the mixed solution II to a temperature of 30 ° C while stirring thoroughly.
To obtain a cream. Application Example 31 (Heart Tonic) [0174] [Table 35]Application Example 32 (Heart Tonic) [Table 36] Application Example 33 (Aerosol type coating agent) [0177] [Table 37] Application Example 34 (bath additive) 3 g of N-methyl-L-serine is mixed with the following components, and
100 g of a bath preparation was obtained. [0179] [Table 38] In addition, this bath agent is diluted about 3000 times at the time of use. [0180] EFFECT OF THE INVENTION The cytokine activity enhancer of the present invention is
Acts on skin cells to increase responsiveness to cytokines
Therefore, skin metabolism can be activated. Also, the general formula
Ethanolamine derivative represented by (I) or a salt thereof
Is able to penetrate the skin, so that
Can be enhanced. In addition, ethanolamine
Derivatives or their salts have low toxicity,
Can also enhance the action of cytokines. Follow
And an ethanolamine derivative of the general formula (I)
Salts are useful as cytokine activity enhancers.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing a vertical diffusion cell device used in Test Example-15. FIG. 2 is a diagram showing the results of a skin permeability test of N-methyl-L-serine in Test Example-15.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI A61K 31/133 A61K 31/133 A61P 43/00 107 A61P 43/00 107 121 121 (56) References JP-A-57-131711 ( JP, A) JP-A-60-246306 (JP, A) JP-A-4-74106 (JP, A) JP-A-4-74111 (JP, A) JP-A-4-95008 (JP, A) JP-A-4-95018 (JP, A) JP-A-5-194186 (JP, A) JP-B-47-46331 (JP, B1) Patent 3073862 (JP, B2) (58) Fields investigated (Int. Cl. ⁷⁾ A61K 7/00-7/50

Claims (1)

(57) [Claim 1] 1,3-diamino-2-propanol,
A cosmetic containing an ethanolamine derivative or a salt thereof selected from the group consisting of 1-amino-2-butanol .