JP5138299B2 - Dermal preparation - Google Patents

Description

  The present invention relates to an acne preventive agent or acne ameliorating agent, an agent for skin treatment for treatment, antibacterial property, which contains an L-carnosine zinc complex having a high skin safety and having no skin irritation composed of L-carnosine and zinc which are biological components. The present invention relates to a dermatological agent, an anti-Acne gonococcal skin agent, an anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) dermatological agent, and a skin care method using an L-carnosine zinc complex.

  The cause of acne (acne vulgaris) is said to involve a variety of physiological and bacteriological factors. Propionibacterium acnes, known as acne, and Staphylococcus aureus (Staphylococcus aureus) is considered the main cause of acne. Therefore, antibiotics such as clindamycin, erythromycin, gentamicin sulfate and chloramphenicol are generally widely used for the treatment of acne, but the emergence of resistant bacteria becomes a problem in the use of antibiotics. . Recently, the emergence of methicillin-resistant Staphylococcus aureus (MRSA), to which antibiotics do not work, has become a major social problem, but MRSA is also considered to be one of the causes of acne.

  Furthermore, regarding the use of antibiotics, the emergence of bacterial cross-resistance between compounds with similar chemical structures has become a problem that cannot be ignored. In addition, currently used antibiotics are problematic because they have side effects such as pruritus, redness, itching, irritation, dampness, liver damage, kidney damage, and stomach damage.

  Accordingly, there is a need for a dermatological agent that has no harmful side effects, has antibacterial action against acne bacilli, Staphylococcus aureus and MRSA, and can effectively prevent, ameliorate or treat acne. However, to date, no antibacterial compound that has solved these problems, particularly a safe compound that is effective against MRSA and does not cause skin irritation has not yet been found.

It has been reported that a wound healing promoter with a skin defect containing an L-carnosine zinc complex as an active ingredient exhibits antibacterial activity against Staphylococcus aureus (see, for example, Patent Document 1). However, nothing is known about the effect of L-carnosine zinc complex on koji mold and MRSA.
JP-A-10-29939

  In view of such circumstances, the present invention has no problems such as resistant bacteria, skin irritation, skin safety, harmful side effects and the like, and has high skin safety for prevention or improvement of acne, dermatological agent for treatment, antibacterial dermatological agent Another object is to provide an anti-MRSA dermatological agent and a skin care method.

  As a result of intensive studies to solve the above problems, the present inventors have found that an L-carnosine zinc complex, which is a complex of zinc and L-carnosine, which is a biological component, is not only S. aureus, It was found for the first time that it exhibits antibacterial activity against methicillin-resistant Staphylococcus aureus. Furthermore, since the L-carnosine zinc complex has no skin irritation and high skin safety, it has been found that the L-carnosine zinc complex is useful as a dermatological agent for preventing or improving acne and treating it. The present invention has been completed based on the new findings.

That is, the present invention
(1) An acne-preventing or acne-improving, therapeutic skin preparation characterized by containing an L-carnosine zinc complex as an active ingredient,
(2) The acne-preventing or acne-improving, therapeutic skin preparation according to (1), wherein the content of the L-carnosine zinc complex is 0.01 to 20.0% by mass,
(3) an antibacterial dermatological agent characterized by containing an L-carnosine zinc complex as an active ingredient,
(4) an anti-Acne gonorrhea dermatological agent characterized by containing an L-carnosine zinc complex as an active ingredient, and
(5) An anti-methicillin-resistant Staphylococcus aureus skin preparation characterized by containing an L-carnosine zinc complex as an active ingredient,
About.

The present invention also provides
(6) a cosmetic comprising an L-carnosine zinc complex, and
(7) A skin care method characterized by treating the skin with an L-carnosine zinc complex,
About.

  The agent for preventing acne or improving and treating acne according to the present invention (hereinafter also simply referred to as a dermatological agent) is an active ingredient L-carnosine zinc complex (CZ), acne gonococcus, which causes acne, yellow It exhibits superior antibacterial activity against staphylococci and methicillin-resistant Staphylococcus aureus (MRSA) compared to commercially available antibacterial agents, popidone iodine, and has no harmful side effects, allowing long-term administration with high skin safety. It is useful for preventing or improving acne and treating acne. In particular, since no highly safe compound exhibiting antibacterial action against MRSA has been found so far, it has been difficult to prevent and treat acne caused by MRSA. However, when the skin preparation of the present invention is used, Acne caused by MRSA can also be effectively prevented, ameliorated or treated.

Next, the present invention will be specifically described.
L-carnosine constituting the L-carnosine zinc complex used in the skin preparation of the present invention is a dipeptide biological component present in human and animal muscles, and zinc is an essential trace component present in the living body. Therefore, there is almost no concern about harmful side effects due to localized administration of the L-carnosine zinc complex, but the skin irritation test using rabbits and the skin sensitization test using guinea pigs are highly safe and harmful to the skin. Confirmed that there were no side effects.

  The L-carnosine zinc complex may be a complex of L-carnosine and zinc, and either a crystalline L-carnosine zinc complex or an amorphous type L-carnosine zinc complex can be used.

  The crystalline L-carnosine zinc complex is, for example, as described in Japanese Patent Publication No. 7-116160 (1995), Pharmaceutical Journal Vol. 125, pages 829-832 (2005), and the like. Can be manufactured from. Specifically, usually at 1 room temperature or under heating, in an anhydrous or hydrous polar organic solvent, or in the presence of a small amount of water, in the presence or absence of an alkali metal compound, with respect to 1 mol of L-carnosine. By carrying out the reaction using 1 mol of zinc salt, a crystalline L-carnosine zinc complex can be obtained.

  Amorphous type L-carnosine zinc complexes are described in, for example, Weitzel G. et al. Schneider F .; , Fretzdorff A .; M.M. , Hoppe-Seyler's Z. Physiol. Chem. , 307, 23-25 (1957), Pharmaceutical Journal Vol. 125, pages 829-832 (2005), and the like. Specifically, an L-carnosine zinc complex of amorphous type can be obtained by reacting 1 mol of L-carnosine and 1 mol of zinc salt in the presence or absence of an alkaline solution. The reaction is usually carried out at room temperature in the presence of water or a small amount of water, and the reaction is completed in several tens of minutes to several hours. After the reaction, the precipitated carnosine zinc complex is filtered or collected and dried to obtain an amorphous type L-carnosine zinc complex.

  As the zinc salt used in the production of the L-carnosine zinc complex, both a salt with an inorganic acid and a salt with an organic acid can be used. Examples of the salt of inorganic acid and zinc include zinc oxide, zinc halide, zinc sulfate, zinc nitrate, and zinc perchlorate. Examples of the salt of organic acid and zinc include zinc acetate and zinc lactate. Examples thereof include zinc salts of carboxylic acids and zinc acetylacetone, and any zinc salt can be used as long as the reaction proceeds for the production of an L-carnosine zinc complex.

  The concentration of the L-carnosine zinc complex in the dermatological agent is generally about 0.01 to 20.0% by mass in 100% by mass of the dermatological agent. Considering, it is preferable to set it as about 0.1-10.0 mass% in 100 mass% of dermatological agents.

  For the dermatological agent, in addition to the L-carnosine zinc complex, if necessary, other acne active ingredients such as resorcin, isopropylmethylphenol, ibuprofen piconol, tocopherol, ascorbic acid, betamethasone valerate, hydrocortisone, triamcinolone, Plant extract ingredients such as paraoxybenzoic acid alkyl ester, arbutin, placenta extract and herb extract can be used in any proportion.

  As other components added to the preparation of the dermatological agent, components generally used in dermatological agents, such as humectants, preservatives, antioxidants, surfactants, pH adjusters, oils, alcohols, Thickeners, thickeners, amino acids, sunscreen agents, fragrances, pigments and the like can be arbitrarily used in any ratio.

  The base used in the dermatological agent of the present invention is also a base generally used in dermatological agents, such as white petrolatum, yellow petrolatum, water-absorbing petrolatum, macrogol, paraffin, liquid paraffin, plastic base, silicone, beef tallow, wax. Lanolin, vegetable oil, hydroxypropyl cellulose, and a mixture thereof can be arbitrarily used.

  There are no particular limitations on the external dosage form of the dermatological agent, ointment, cream, emulsion, lotion, oil, emulsion, gel, powder, nanopowder, inclusion, pap, bandage As long as it is a pharmaceutical dosage form suitable for the skin, such as spray, liniment, etc., any dosage form may be used.

  The method for producing the dermatological agent is not particularly limited, and can be produced according to a known method in the pharmaceutical, quasi-drug, and cosmetic industry depending on the dosage form.

  Below, although the typical formulation example of the dermatological agent of this invention is shown, this invention is not limited to these. In the formulation examples, “ratio”, “part”, and “%” mean “mass ratio”, “part by mass”, and “mass%”, respectively, based on mass.

Formulation Example 1 (1% ointment)
L-carnosine zinc complex 1 part white petrolatum / liquid paraffin (8: 2) mixture 99 parts (100 parts in total)

Formulation Example 2 (2% ointment)
L-carnosine zinc complex 2 parts white petrolatum / liquid paraffin (8: 2) mixture 98 parts (total 100 parts)

Formulation Example 3 (5% ointment)
L-carnosine zinc complex 5 parts white petrolatum / liquid paraffin (8: 2) mixture 95 parts (total 100 parts)

Formulation Example 4 (1% ointment)
L-carnosine zinc complex 1 part polyoxyethylene oleyl ether 5 parts white petrolatum / liquid paraffin (8: 2) mixture 94 parts (total 100 parts)

Formulation Example 5 (1% ointment)
L-carnosine zinc complex 1 part Water-soluble polymer 5 parts White petrolatum / liquid paraffin (8: 2) mixture 94 parts (100 parts in total)

Formulation Example 6 (1% ointment)
L-carnosine zinc complex 1 part Water-absorbing petrolatum 99 parts (total 100 parts)

Formulation Example 7 (Macrogol Base)
L-carnosine zinc complex 1 part Macrogol 400 70 parts Macrogol 4000 29 parts (100 parts in total)

Formulation Example 8 (sugar base)
L-carnosine zinc complex 1 part mantitol 30.5 parts mannitol 30.5 parts macrogol 400 10 parts macrogol 4000 3 parts 1,3-butylene glycol 7 parts medium chain fatty acid triglyceride 3.5 parts polysorbate 80 2.5 parts xanthan gum 0.01 parts purified water appropriate amount (total 100 parts)

Formulation Example 9
L-carnosine zinc complex (dissolved in HCl) 2 parts hydroxypropylcellulose aqueous base 98 parts (total 100 parts)

  The dermatological agent of the present invention is applied by applying, sticking, etc. to the affected part (acne) by a method of use according to the dosage form. The amount of the skin preparation of the present invention used is an appropriate amount several times a day, but is not particularly limited as long as the effects of the present invention are exhibited.

  The dermatological agent of the present invention has an antibacterial dermatological agent, anti-acne agent, because the L-carnosine zinc complex, which is an active ingredient, exhibits antibacterial action against acne bacilli, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA). It is also useful as a gonococcal skin agent, an anti-Staphylococcus aureus skin agent, or an anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) skin agent. Such antibacterial dermatological agents, anti-acne gonorrhoeae dermatological agents, and anti-methicillin-resistant Staphylococcus aureus dermatological agents are also one aspect of the present invention.

  Furthermore, since the L-carnosine zinc complex exerts the antibacterial action as described above, the use of the L-carnosine zinc complex on the skin such as the face will prevent the growth of acne bacilli, Staphylococcus aureus and methicillin resistant Staphylococcus aureus. Can be suppressed. Therefore, the L-carnosine zinc complex also exhibits a skin care effect, and a skin care method for treating the skin with the L-carnosine zinc complex is also one aspect of the present invention. Moreover, the cosmetics containing a L-carnosine zinc complex can be used suitably for beautiful skin care. In the skin care method of the present invention, as an embodiment using the L-carnosine zinc complex, for example, the above-mentioned dermatological agent or cosmetic containing the L-carnosine zinc complex is used, and water containing the L-carnosine zinc complex is added. Wash the face with water, wipe the skin by immersing water containing L-carnosine zinc complex in a towel or the like, or mix the powder of L-carnosine zinc complex with talc, titanium oxide, etc. in any proportion. Examples of the method include arbitrarily applying or spraying several times a day, but are not particularly limited. The amount of the L-carnosine zinc complex to be used is not particularly limited as long as it provides a skin care effect.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. In the examples, unless otherwise specified, “ratio”, “part” and “%” mean “mass ratio”, “part by mass” and “mass%”, respectively, based on mass.

(I) Skin irritation test The skin irritation test in rabbits of L-carnosine zinc complex (hereinafter sometimes abbreviated as CZ) was carried out by the following method.
Test substance:
White ointment containing 10% by mass or 20% by mass of L-carnosine zinc complex (hereinafter abbreviated as CZ-10 and CZ-20, respectively) The bases are Japanese Pharmacopoeia White Petrolatum and Japanese Pharmacopoeia Liquid Paraffin (Vaseline vs. Liquid Paraffin ) Was an 8 to 2 mixed base) and 3 test substances were used, which were only white ointment (base) containing no CZ (hereinafter abbreviated as CZ-0).

Animals used:
Kitayama Labes Co., Ltd. Purchased five Japanese white female rabbits (kb1: JW, SPF) produced at Minowa Plant (Ina City, Nagano Prefecture), and after a 6-day quarantine / acclimation period, they grew smoothly. Animals 9 weeks old (weeks of dosing) were used for the study. About the administration animal, the back part was shaved and three animals with good skin condition (smooth skin) were used. The body weight of the three animals at this time was 1.75 to 1.97 kg.

The test substance was applied to the back skin of three animals (animal numbers 501, 502 and 503). Three sites each for healthy and damaged sites were provided on the back of each animal, for a total of 6 sites, and CZ-0, CZ-10 and CZ-20 were applied. FIG. 1 shows the back administration site. Draize et al. (JHDraize, G. Woodord and HO Calvery: Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes, J. Pharmacal. Exp. Ther., 82, 377-390 (1944) ), The application area was 6.25 cm ² (2.5 × 2.5 cm), the administration volume was 0.5 g / site, and the test substance was occluded once on the application sites of 3 rabbits for 24 hours.

  Prior to administration, the hair was shaved with an electric clipper on the day before administration (end of quarantine / acclimation period) so as not to injure the back. The next day (on the day of administration), the shaved portion is above the left and right three sections (2.5 x 2.5 cm) along the back midline (applied sites 1 and 2), inside (applied sites 3 and 4), and lower (applied) A total of 6 sections of sites 5 and 6) were provided. The sections of the application sites 1, 3 and 5 were scratched in a # shape using an 18G injection needle, taking care not to bleed, and were designated as damaged sites.

Application method:
After extending 0.5 g of the test substance on a lint cloth (2.5 × 2.5 cm), it was attached to a healthy site and a damaged site, respectively. The lint cloth was covered with dental moisture-proof rubber (3 × 3 cm), covered with an adhesive fixing elastic bandage, and then covered with a protective garment to prevent movement of the lint cloth. As shown in Table 1, each patch was rotated for each individual in order to avoid bias.

  24 hours after the occlusion, the test substance was gently wiped with cut cotton soaked in slightly warm water (38 ° C. water for injection: Lot No. 5J97N, Otsuka Pharmaceutical Factory, Japanese Pharmacopoeia).

Skin observation:
Each patch site was observed 0.5, 24, 48, and 72 hours after removal of the test substance, and the skin reaction score was determined according to the criteria in Table 2.

Calculation method of stimulation index (PI: Primary Irritation Index):
In accordance with Table 3, the scores after removal of the test substance 0.5 and 48 hours later (both erythema, crust and edema at the healthy site and the damaged site) were combined for each individual. I. I. Was calculated.

Safety category of the test substance I. I. From the mean P. of 3 of each test substance. I. I. And the irritation of the test substance was evaluated according to the safety category shown in Table 4.

Test Results For rabbits to which CZ-0, CZ-10 or CZ-20 were applied, in healthy sites, very mild erythema was observed after 0.5 hours of removal, and CZ-0 was applied to 2/3 cases of CZ-0. About 10 or CZ-20 was observed in 1/3 cases, but erythema disappeared 24 hours after removal.
At the injured site, very mild erythema 0.5 hours after removal was observed in 3/3 cases for CZ-0 application and in 2/3 cases for CZ-10 or CZ-20 application. Erythema disappeared by 24 hours after removal. No edema was observed at any site. The average P.D. calculated from the scores after 0.5 and 48 hours after removal (healthy site and damaged site). I. I. Was 0.4 for CZ-0 and 0.3 for CZ-10 or CZ-20, both of which were classified as weak irritants.

General state observation:
No abnormalities were observed in the general state of any animal before and after application on the application date of each application substance, or during the observation period after the next day.

Discussion From the test results, the average P.C. of CZ-0 (base only) was determined. I. I. Was 0.4. Moreover, the average P.A. of CZ-10 (white ointment containing 10% by mass of L-carnosine zinc complex). I. I. Of 0.3, CZ-20 (white ointment containing 20% by mass of L-carnosine zinc complex) I. I. Also. It was 0.3. From these results, all were evaluated as weak stimulants, but since the test result of the base (CZ-0) itself was 0.4, the average P.C. of CZ-10 and CZ-20. I. I. A value of 0.3 is considered to be derived from the base itself.
Therefore, it is considered that the L-carnosine zinc complex itself has no skin irritation, that is, a non-irritating substance.

(II) Skin sensitization test In order to confirm the skin safety when the L-carnosine zinc complex was repeatedly used, a skin sensitization test in guinea pigs was performed by the following method.

Test substance:
Two test substances, CZ-0 (base only) and CZ-10 (white ointment containing 10% by mass of L-carnosine zinc complex) were used.

Target substances and reagents:
As a positive target substance, 2,4-dinitrochlorobenzene (DNCB) is used, and details thereof are shown below. Moreover, the following were used also about the other solvent and reagent.
DNCB (Manufacturer: Wako Pure Chemical Industries, Standard: Special reagent grade)
Water for injection (Manufacturer: Otsuka Pharmaceutical Factory, Standard: Japanese Pharmacopoeia)
Freund's Complete Adjuvant (FCA) (Manufacturer: Difco, Standard: Research)
Sodium Lauryl Sulfate (SLS) (Manufacturer: Wako Pure Chemical Industries, Standard: Biochemical)
San White ^R P-1 (highly purified vaseline) (manufacturer: Cosmetics Science, standard: cosmetic oils)
Ethanol (Manufacturer: Wako Pure Chemical Industries, Standard: Special reagent grade)
DNCB was stored at room temperature and protected from light. Other reagents were stored at room temperature.

Test system:
After purchasing 21 4-week-old male guinea pigs (Slc: Hartley, Japan SLC Co., Ltd.) and quarantine and acclimatization period for 7 days after arrival, 21 animals that grew smoothly were used. The assignment to each group was randomly made to each group by an appropriate stratification method using random numbers generated by a computer. The weight range at the time of grouping was 296 to 344 g, and 20 animals aged 5 weeks (weeks at the start of sensitization) were used in the test.

Preparation and storage of test substance / substance mixture with medium / water for injection / FCA emulsion: Add 7 ml of water for injection to FCA of equal volume in a glass bottle and repeat suction and discharge with a glass syringe. Mixed emulsification. The finished index was a state of floating (spherical) without spreading when dropped on the water surface.
Test substance and test substance base: A prescribed amount was weighed with an electronic balance and then used as it was.
-SLS mixture: 2.7 g of highly purified petrolatum was weighed in a PP sample tube with an electronic balance, 0.3 g of SLS was weighed and added to the highly purified petrolatum, and then subjected to hot water roasting (40-50 ° C) with a hot stirrer. Highly purified petrolatum was mixed to prepare 10 mass / mass%. Thereafter, two 2.5 mL disposable syringes were filled so as not to have bubbles.
DNCB solution: 0.01 g of DNCB was weighed with an electronic balance, transferred to a 10 mL graduated cylinder, dissolved in ethanol (applied with ultrasonic waves: using an ultrasonic cleaner), and then adjusted to a final 10 mL with ethanol (0. 1 mass / v%).
-Ethanol: 5 mL of ethanol was dispensed and used as it was.

  All of these doses were prepared at the time of use. The water for injection / FCA emulsion was placed in a brown sealed glass container, and the DNCB solution and ethanol were placed in a brown sealed PP container and stored at room temperature and protected from light, respectively. The SLS mixture was filled in 2.5 mL disposable syringes (2), then stored at room temperature and used.

Test method:
As shown in Table 5, a test substance group (S1), a test substance base group (S2), an untreated group (S3), and a positive control group (S4) were provided using five animals per group.

FIG. 2 shows the sensitization site and the induction site of guinea pigs. The sensitization site and the induction site in FIG. 2 are as follows, respectively.
Four corners in sensitized site broken line: intradermal injection site (each 0.1 g (mL)) and primary contact sensitization (occlusion patch, 1.5 × 1.5 cm, 0.1 g (mL))
Broken line display site: transdermal administration (occlusion sticking) (2 × 4 cm, 0.1 and 0.2 g (mL))
Induction site solid line display site (1-4 sites / animal): transdermal administration (open application (2 × 2 cm, 0.01 g (mL))

Primary contact sensitization (1) The day before intradermal administration (Day-1), the site of intradermal administration on the dorsal skin of the neck (shown by a broken line) and the surrounding area with an electric clipper and electric razor I shaved.
(2) First day of sensitization (Day 0) The sensitized sites (2 × 4 cm, Fig. 2: Marking the four corners of the broken line section with oil-based ink) are provided on the shaved / shaved part, and water emulsion for injection is placed in the four corners Intradermal injections of 1 mL each.
(3) After intradermal injection, four scratches were made in a # shape using an injection needle (21G) in the compartment of the intradermal injection site.
(4) Test substance, 0.1 g of test substance base or 0.1 mL of 0.1% DNCB solution at 1.5 × 1.5 cm each in 4 intradermal injection sites (FIG. 2: four corners in broken line) A lint cloth was applied after being stretched or impregnated, covered with a dental moisture-proof rubber, and covered with an adhesive fixing elastic bandage.
(5) Twenty-four hours after the occlusion, the test substance, test substance base and DNCB on the application site were gently removed with cut cotton soaked in lukewarm water (38 ° C water for injection: Lot No. 5J97N, Otsuka Pharmaceutical Factory, Japanese Pharmacopoeia). Removed.
(6) The operations of (3) and (5) were performed once a day for a total of 3 days.
(7) For the untreated group, the treatments (1) and (2) were performed, and the treatments (3) to (6) were not performed.

Secondary contact sensitization (1) Day 6 (6th day from the first day of sensitization), inside and around the compartment of the intradermal injection site were shaved with an electric razor.
(2) Day 7, 0.1 mL of SLS mixture (10% in highly purified petroleum jelly) was applied in the same compartment (within the broken line shown in FIG. 2).
(3) Day 8 (about 24 hours after application of the SLS mixture), the SLS mixture was gently wiped with cut cotton soaked with diethyl ether, and then the test substance, test substance base 0.2 g and DNCB solution 0 2 mL of each 2 × 4 cm lint cloth was spread or impregnated and then applied, covered with 3 × 5 cm of dental moisture-proof rubber, and covered with an adhesive fixing elastic bandage.
(4) Day 10 (approximately 48 hours after occlusion), and the test substance, test substance base and DNCB at the applied site were removed in the same manner as the primary sensitization.
(5) For the untreated group, the treatments (1) to (4) were not performed.

Induction (1) The animal's back was shaved and shaved using Day 21, an electric clipper and an electric razor.
(2) Day 22, triggering site (each 2 × 2 cm) (solid line display site in FIG. 2; each 2 × 2 cm, four corners marked with black oil-based ink) 2 locations (alternate to 1 and 2 in FIG. 2 for each animal) The test substance, 0.1 g of the test substance base or DNCB solution, and 0.1 mL of the ethanol solution were spread or impregnated on a 2 × 2 cm lint cloth, and applied from there. Covered with × 2.5 cm of dental moisture-proof rubber and covered with an adhesive fixing elastic bandage. In addition, in order to avoid the bias | inclination by a sticking site | part, the origin part was rotated for every individual | organism | solid as shown in Table 6.
(3) Each inducing substance was gently wiped with cut cotton soaked in Day 23, (after 48 hours after occlusion), in warm water (38 ° C. water for injection: Lot No. 5J97N, Otsuka Pharmaceutical Factory, Japanese Pharmacopoeia).

Skin observation:
The skin condition of the inducing site 24 and 48 hours after removal of each inducing substance was observed according to the criteria shown in Table 2 to obtain a score. In addition, since it was thought that shaving was required during the skin observation period, shaving was performed as lightly as possible (minimizing physical irritation of the skin by shaving) at least 30 minutes before each observation.

Rating:
The degree and frequency of erythema and edema observed after the induction of each test substance group were compared with the test substance base group, the untreated group and the positive control group, and were evaluated comprehensively. In addition, an average evaluation point for each observation time in the reaction after the induction in each group (total of reaction evaluation points / total number of animals in the group) was calculated. In addition, when reaction was recognized in the inducing site of the test substance base group and the untreated group, the reaction was subtracted and evaluated. That is, a reaction in which the reaction observed at the time of starting the test substance group exceeded the reaction at the time of starting the same substance in the test substance base group or the untreated group was defined as a positive reaction.

Test results:
Skin reaction after induction (1) CZ-10 sensitization group In CZ-10 and CZ-0 induction, both 2 and 5 cases (No. 1, 4) had very mild erythema after 24 and 48 hours of removal, respectively. Admitted. Edema was not observed at any of the sites. The average evaluation points of CZ-10 and CZ-0 after 24 and 48 hours after removal were both 0.4.

(2) CZ-0 sensitization group In CZ-10 induction, 3/5 cases (No. 11, 13, 14) and 1/5 cases (No. 13). Edema was not observed at any of the sites. The average evaluation points at 24 and 48 hours after removal were 0.6 and 0.2, respectively.
In CZ-0 induction, very mild erythema was observed in 3/5 cases (No. 12 to 14) and 1/5 cases (No. 13) after 24 and 48 hours of removal, respectively. Edema was not observed at any of the sites. The average evaluation points at 24 and 48 hours after removal were 0.6 and 0.2, respectively.

(3) Untreated group In CZ-10-induced, very mild erythema was observed in 3/5 cases (No. 23-25) and 2/5 cases (No. 23, 24) after 24 and 48 hours of removal, respectively. Admitted. Edema was not observed at any of the sites. The average evaluation points at 24 and 48 hours after removal were 0.6 and 0.4, respectively.
In CZ-0 induction, very mild erythema was observed in 2/5 cases (No. 22, 24) and 1/5 cases (No. 22), respectively, 24 and 48 hours after removal. Edema was not observed at any of the sites. The average evaluation points at 24 and 48 hours after removal were 0.4 and 0.2, respectively.

(4) 0.1% DNCB group In 0.1% DNCB induction, 3/5 cases (No. 31, 33, 35) of moderate to strong erythema and mild crust formation after 24 hours of removal. Mild edema was observed in 2/5 cases (No. 32, 34), and mild edema was observed in scab formation 48 hours after removal. The average evaluation points at 24 and 48 hours after removal were 5.4 and 6.0, respectively, and the positive rate was 100%.
In ethanol induction, erythema and edema were not observed in any of the induction sites.

  During the observation period, no abnormalities were observed in any of the animals. During the observation period, each group showed a steady increase in body weight.

Discussion:
From the above results, in the test substance group and the test substance base group, CZ-10 and CZ-0 induction showed very mild erythema in 2/5 cases, similar to the same induction in the untreated group The average evaluation score was also an approximate value. From this, it is considered that the skin irritation derived from CZ-0, which is a test substance base, is involved in any of the reactions observed when CZ-10 is induced in the CZ-10 group.
In the DNCB induction of the positive target substance group, mild edema in scab formation was observed in 5/5 cases at any induction site. During the observation period, no abnormality was observed in the general state in any of the test substance group, the test substance base group, the no-treatment group, and the positive control substance group, and each group showed a smooth increase in body weight.
Therefore, it is judged that CZ-10 and its base CZ-0 under the test conditions have no skin sensitization.

(III) Antibacterial activity against Staphylococcus aureus, Acne bacilli and methicillin-resistant Staphylococcus aureus (MRSA) Next, the antibacterial activity against Staphylococcus aureus, acne bacilli and MRSA is tested by measuring the minimum inhibitory concentration (MIC). did.

Test substance:
L-carnosine zinc complex (CZ), L-carnosine, zinc oxide, one-to-one mixture of L-carnosine and zinc oxide, popidone iodine (commercially available antibacterial agent, iodine formulation, positive control agent).

Test bacteria:
Staphylococcus aureus IFO12732, Acne bacilli (Propionibacterium acnes JCM6425), methicillin-resistant Staphylococcus aureus NBRC12732

Medium used:
Bouillon for sensitivity measurement (Nissui Pharmaceutical), Sensitivity medium-N (Nissui Pharmaceutical), GAM agar medium (Nissui Pharmaceutical), GAM bouillon medium (Nissui Pharmaceutical), Tryptic Soy Agar (Difco).

(1) Test method for Staphylococcus aureus:
As the test substance, a dilution series was prepared using dimethyl sulfoxide. Use 0.5 mL of this diluted solution so that the test substance concentration in the medium is 2.0, 1.6, 1.2, 0.8, 0.4, 0.2, 0.1, 0.05%. The mixture was added to 9.5 mL of sensitivity medium-N, and the mixture was poured into a petri dish to obtain a solidified plate.
The inoculated bacteria were cultured in a sensitivity measuring broth at 37 ° C. for 20 hours to prepare a bacterial suspension of about 10 ⁶ CFU / mL. This suspension of test bacteria was streaked on a test substance mixed agar plate using a platinum loop and cultured at 37 ± 1 ° C. for 18 to 20 hours, and the MIC (Minimum Growth Inhibitory Concentration) was observed with a minimum concentration at which no growth was observed. ).

(2) Test method for acne bacilli:
Test substance CZ, zinc oxide, and a one-to-one mixture of L-carnosine and zinc oxide were prepared as test substance stock solutions with dimethyl sulfoxide. For L-carnosine and popidone iodine, test substance stock solutions were prepared with sterile water. The stock solution was prepared as a dilution series with dimethyl sulfoxide or sterilized water. 1 mL of this diluted solution was placed in a petri dish, and 19 mL of GAM agar medium was mixed so that the test substance concentration in the medium was 2.0, 1.0, 0.5, 0.25, and 0.125%.
The inoculum was anaerobically cultured in a GAM bouillon medium at 37 ° C. for 40 to 48 hours to prepare a bacterial suspension of about 10 ⁶ CFU / mL. 10 μL of the bacterial solution was inoculated on a test substance mixed medium plate, anaerobically cultured at 37 ° C. for 48 to 72 hours, and the minimum concentration at which no growth was observed was defined as MIC.

(3) Test method for methicillin-resistant Staphylococcus aureus (MRSA):
After sterilizing and dissolving 0.8 g of the test substance CZ, it was added to 20 mL of Tryptic Soy Agar kept at 50 ° C. and mixed well to obtain a concentration of 4%. Using this as a stock solution, a serial dilution series was prepared using Tryptic Soy Agar kept at 50 ° C. (2.0, 1.0, 0.5, 0.25, 0.125%). 10 mL of these agar media were poured into a petri dish and solidified.
The inoculum was pre-cultured at 35 ° C. for 24 hours in Tryptic Soy Agar, and the grown colony was suspended in sterilized purified water to prepare about 10 ⁶ CFU / mL, which was used as an inoculum solution. About 0.01 mL of this inoculum was applied to the test substance mixed medium with a platinum loop and cultured at 35 ° C. ± 1 ° C. for 2 days, and the minimum concentration at which no growth was observed was defined as MIC.

  The results of the antibacterial test are collectively shown in Table 7.

Considerations for test results:
The L-carnosine zinc complex (CZ) is a bacterial species that causes acne from L-carnosine, zinc oxide, a one-to-one mixture thereof, and popidone iodine, a commercially available antibacterial agent for external use. It has been found that it exhibits an excellent antibacterial action against Acne bacilli, Staphylococcus aureus, MRSA). That is, the present invention is the first to discover that CZ, which is a complex of L-carnosine and zinc, which is a biological component, has antibacterial activity against acne bacilli (acne) and MRSA.

(VI) Improving effect on acne and presence or absence of irritation (evaluation method) 5% CZ ointment (prescription example shown below) on the right side of 7 normal 20-30 year old boys with acne symptoms on the face Appropriate amounts were applied once a day, three times a week, and the presence or absence of acne improvement and irritation (rash, redness, pruritus) was determined according to the following criteria.
(Prescription example of 5% ointment)
L-carnosine zinc complex 5 parts white petrolatum / liquid paraffin (8: 2) mixture 95 parts (total 100 parts)

Evaluation points and criteria 5 points: Acne disappeared, clearly improved 4 points: Clearly improved 3 points: Improved 2 points: Slightly improved 1 point: No change

(Evaluation results and irritation)
As a result of comparing the determination of the acne improvement effect with the left side of the non-coated face, the average value of 7 people was 4.00, and the acne symptom could be effectively improved. In addition, none of the worsening cases, rashes, redness and pruritus occurred. The results are shown in Table 8.

  The dermatological agent for preventing or improving acne and treating acne according to the present invention is commercially available against Lactococcus acne, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus, whose active ingredients are L-carnosine zinc complexes. Since it exhibits an antibacterial action superior to that of popidone iodine, an external antibacterial agent, and has no harmful side effects, it is useful as a preventive or ameliorating or treating agent for acne that can be administered for a long period of time with high skin safety. In particular, since no highly safe compound exhibiting antibacterial action against MRSA has been found so far, it has been difficult to prevent and treat acne caused by MRSA. However, when the skin preparation of the present invention is used, Acne caused by MRSA is also useful because it can be effectively prevented, ameliorated or treated.

It is a schematic diagram which shows the back administration site | part (applying site | part) of a rabbit in the skin irritation test of an L-carnosine zinc complex. The numbers in the figure are the numbers of the application sites. It is a schematic diagram which shows the sensitization site | part and induction site | part of a guinea pig in the skin sensitization test of a L-carnosine zinc complex. The numbers in the figure are the numbers of the origin (sticking) sites.

Claims (3)

  A skin preparation for preventing or improving acne or treating acne, comprising an L-carnosine zinc complex as an active ingredient.   The dermatological agent for preventing or improving acne and treating acne according to claim 1, wherein the content of the L-carnosine zinc complex is 0.01 to 20.0% by mass. A skin preparation for anti-Acne gonorrhoeae comprising an L-carnosine zinc complex as an active ingredient.

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