JP2002335995A - Method for evaluation of antimicrobial agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for exactly evaluating the physiological activity of an antifungal agent that can compare the growth inhibitory activity of the agent in the skin tissue as in the case of serpigo of guinea pig foot to combine the growth inhibitory activity with the MIC(minimum inhibitory concentration) to correlate them whereby the physiological activity of the antifungal agent can be exactly evaluated. SOLUTION: The model of serpigo of guinea pig foot as a preclinical test is useful but needs considerable amounts of time and labor. In this invention, horny cell layers are placed on a salt medium including an antifungal agent and the antifungal agent is inoculated into the horny cell layers to know the growth-inhibitory concentration of the antifungal agent and correlate the drug activity in the tissue with the MIC. In other words, skin indigenous microorganisms are placed on the corny cell layer on an agar medium, and the antifungal agent that is separated from the original position of the microorganism and the original position of the antifungal agent through the honey cell layer is allowed to include in the agar medium and cultured in the agar medium to know MIC and the MIC is correlated to the physiological activity of the antifungal agent cultured on the corny cell layer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for evaluating an antimicrobial agent, and more particularly to an evaluation method for accurately evaluating the efficacy of an antifungal agent using a salt medium and / or a stratum corneum.

[0002]

2. Description of the Related Art Fungi are widely distributed in nature, and it is known that there are tens of thousands of species. Of these, about 50 are said to be pathogenic to humans. There are two types of fungal infections, i.e., dermatomycosis, and are classified into superficial dermatomycosis and deep dermatomycosis depending on whether the site where the lesion occurs is shallow or deep. In superficial dermatomycosis, fungal infections remain on the surface of the skin, such as the keratin and epidermis, and are accustomed to ringworm such as athlete's foot. The part of the body is vulgaris (tinea in the crotch), the one in the body is ecchymotic tinea cruris (tinea cruris, tinea corporis), skin candidiasis, tinea versicolor,
Malassezia folliculitis, chronic mucocutaneous candidiasis, oral candidiasis, vulvar candidiasis, etc. are 90% of cutaneous mycosis.
Account for%. On the other hand, deep dermatomycosis is infection in which the infection extends from the dermis to the subcutaneous tissue and further deep, and includes spotricosis, chromomycosis, cutaneous cryptococcosis, and tinea pedis. Diagnosis of ringworm is not so easy, and it is important to confirm the fungal element by microscopic examination for follow-up.

[0003] In this method, fungi invade the stratum corneum. When the skin infected with Candida is observed with an electron microscope, an image in which the tough stratum corneum is dissolved is observed. This suggests that the bacteria enzymatically dissolved the keratinocytes and keratin fibers. Candida bacteria produce proteolytic proteases and invade the stratum corneum, and at the same time, proliferate using peptides and amino acids of the degradation products of these proteins as nutrient sources. The same applies to Trichophyton, but Trichophyton is known to produce serine proteases, decompose and use skin keratin, hair and nails for invasion. In addition, superficial fungi can penetrate only to the lowest layer of the stratum corneum because the serum component flows into the granular layer as tissue fluid. Inhibitors of bacterial invasion on the living body inhibit transferrin, which chelates iron required for bacterial growth, lysozyme and basic proteins that kill epidermal cells, and protease activity required for bacterial invasion and growth Known protease inhibitors, which are present in serum and epidermal cells, are said to act complementarily to prevent fungi from penetrating below the granular layer.

On the other hand, as to antifungal agents, recently developed terbinafine hydrochloride and butenafine hydrochloride have a stronger antifungal activity in vitro compared to the conventional mainstream imidazole antifungal agents such as bifonazole. Its efficacy is increasing in clinical practice.

[0005] In developing an antifungal agent, at the stage of drug synthesis, MIC value and MC value are determined by a simple in vitro test.
By determining the C value, a primary screening for normal drug efficacy is performed. However, the obtained results often do not reflect the in vivo drug efficacy. Therefore, the drug efficacy is further evaluated in an animal model test using guinea pigs. As a drug efficacy evaluation model, a tinea corporis model in which the back is infected with tricoptone mentagrophytes has been established as a test system.
It has been widely used to determine the therapeutic effect of antifungal agents. However, this tinea model has a lesion significantly different from that of human tinea, and cannot be a disease model. On the other hand, tricoptone
Mentagrophytes (Trichophyton mentagrophytes)
The tinea pedis model infected with) is similar to human tinea pedis in terms of gross, histological, and non-natural healing, and is used in antifungal treatment experiments. Is coming.

[0006] However, although the guinea pig tinea pedis model is extremely useful as a preclinical test, it requires considerable time and effort, so that the antifungal activity of the drug is as well as the in vivo guinea pig tinea pedis model. A new experimental system for evaluation was desired. Also, by placing the stratum corneum on a salt medium containing an antifungal agent and inoculating the antifungal agent thereon, bacteria cannot grow at a certain concentration of the antifungal agent. As in the guinea pig tinea pedis model, the growth activity of the drug in the tissue can be compared, and there is no report that it has a correlation with the MIC.

[0007]

DISCLOSURE OF THE INVENTION The present invention has been made under such circumstances, and, like the guinea pig tinea pedis model, it is possible to compare the growth activity of a drug in a tissue. It is an object of the present invention to provide a method for accurately evaluating the physiological activity of an antifungal agent by taking a correlation in combination with a correlation.

[0008]

In view of such circumstances, the inventors of the present invention placed a stratum corneum section on a salt medium containing an antifungal agent and developed a stratum corneum section for the development of an antifungal agent. By inoculating the fungus on the top, the fungus can no longer grow where it encounters the antifungal agent that has leached into the stratum corneum from the agar medium (dilution concentration), so the antifungal agent concentration in the stratum corneum at this time By comparing the content with the growth inhibitory concentration (MIC) and examining the correlation, a method for accurately evaluating the efficacy of the antifungal agent was found, and the invention was completed. That is, the present invention relates to the following technology. (1) A method for evaluating an antimicrobial agent for microorganisms resident on the skin, wherein a stratum corneum is placed on a medium, and the stratum corneum separates the original location of the microorganism from the original location of the antimicrobial agent. A method for evaluating an antimicrobial agent, comprising: (2) The antimicrobial according to (1), wherein the antimicrobial agent is contained in an agar medium, a stratum corneum is placed on the medium, the microorganism is inoculated into the stratum corneum, and cultured on an agar medium. Agent evaluation method. (3) The method for evaluating an antimicrobial agent according to (1) or (2), wherein the microorganism is a fungus. (4) the antimicrobial agent is an antifungal agent,
(1) The method for evaluating an antimicrobial agent according to any one of (1) to (3). (5) The method for evaluating an antimicrobial agent according to any one of (1) to (4), wherein the stratum corneum is derived from a mammal. (6) (1) to (5), wherein the stratum corneum derived from a mammal is derived from a guinea pig and / or a miniature pig.
The method for evaluating an antimicrobial agent according to any one of the above. (7) The medium is a salt medium, (1) to (1).
The method for evaluating an antimicrobial agent according to any one of (6). (8) The method for evaluating an antimicrobial agent according to any one of (1) to (7), wherein the microorganism cannot grow on the salt medium alone. (9) The method for evaluating an antimicrobial agent according to any one of (1) to (8), wherein the efficacy of the antifungal agent is accurately evaluated by using a salt medium.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION (1) Method for Specially Producing and Breeding Experimental Animals of the Present Invention As an animal for collecting a stratum corneum, approximately 300 to 350 g of a Hartley female guinea pig was preliminarily reared for 6 days before use in the experiment. The animals were bred one cage at a time and had free access to solid feed and water.

(2) Method for Preparing Stratum Corneum Derived from Mammals Used in the Present Invention In the present invention, keratin slices derived from guinea pig sole skin and / or back or shoulder skin of miniature pigs are preferred. As the stratum corneum derived from miniature pigs, it is preferable to use a commercially available product. The method for preparing the stratum corneum derived from guinea pig plantar skin comprises the following steps. 1) Cut the skin on the sole of the guinea pig with a scalpel. 2) Remove the fat layer from the inside of the skin with a scalpel.
3) The skin is heat-treated on a hot plate at 60 ° C. for 1-2 minutes. 4) Make a cut in the stratum corneum with a 6 mm biopsy. 5) Remove the stratum corneum with tweezers. 6)
Float in saline. 7) Store at -20 ° C.

(3) Method for Preparing Bacterial Strains Used and Inoculated Bacterial Solution Used in the Present Invention In the evaluation method of the present invention, microorganisms resident on the skin are used. Fungi are preferred as such microorganisms resident on the skin. Fightes (Trichophyton menta)
grophytes) are particularly preferred. Specifically, Trichopton Mentagrophytes (Trichoph
yton mentagrophytes) SM110 strain was used. This strain was transformed into Sabouraud Dextr agar (Sabouraud Dextr).
(ose Agar (Difco)) was cultured at 28 ° C. for 3 weeks, and then the slope was covered with sterile physiological saline, and conidia were released by shaking. This was filtered through a sterile gauze to remove mycelia and medium clumps. The conidia in the obtained conidia release solution were calculated using a Thorma hemocytometer, adjusted to a concentration of 5 × 10 4 conidia / ml, and used as an inoculum solution.

(4) Culture Experiment Method of the Present Invention The culture experiment system comprises the following steps in an aseptic operation. 1) Use an agar salt medium. 2) D on 48-hole plate
The drug dissolved in MSO and 2-fold diluted (drug dilution series) was added to 1
Agar medium 0.49 added with 0 μL, sterilized and cooled to 45 ° C.
Add mL and pour. As the drug, an antimicrobial agent is used, and among them, it is particularly preferable to use an antifungal agent. 3)
The stratum corneum is placed on an agar salt medium. 4) Bacterial liquid (5 × 10 4 conidia /
ml) from above.

(5) Culture Experiment System of the Present Invention In the present invention, a corneal slice of a plantar portion of a guinea pig or a corneal slice of a miniature pig is placed on a nutrient-free salt medium and cultured with fungi. In this culture system, the nutrients exist only in the stratum corneum, so the bacteria live in the stratum corneum. That is, Trichophyton mentagrophytes (Trichophyton mentagrophytes)
tes) When inoculated with a conidium solution of SM110 strain and cultured, the fungus grows as fast as on the agar medium. Addition of an antifungal agent at a two-fold dilution to agar also changes the drug concentration in the stratum corneum, that is, the higher the concentration of antifungal agent in agar, the higher the concentration of antifungal agent in corneal slice At a certain concentration, the fungus cannot grow. The growth inhibitory concentration is defined as the activity of the antifungal agent in the tissue, and the correlation with the MIC value is examined. Also, in the present invention, the essential component is that the original position of the test antimicrobial agent (the place where it first existed) and the original position of the test microorganism are separated from each other by the stratum corneum. Can be administered on the stratum corneum.However, in such a form, the concentration of the drug occurs, and the dose effect may be impaired. preferable. In the present invention, the antimicrobial agent in
Since the antimicrobial activity as a sum of the antimicrobial activity in vitro and the stratum corneum permeability is observed, the evaluation method of the present invention is more
It can be said that it is a simple test method close to vivo.

[0014]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

<Example 1> (Culture experiment system) 1) An agar salt medium is used. 2) DMS on 48-well plate
Add 10 μL of an antifungal agent (terbinafine, lanconazole, bifonazole) dissolved in O and 2-fold diluted, add sterile 0.49 mL of agar medium cooled to 45 ° C. after sterilization, and pour (concentration after dilution 10 μg / ml to 0.3 ng) / ml). 3) Place stratum corneum on agar salt medium. 4) 5 μL of a conidia solution (5 × 10 4 conidia / ml) of Trichophyton mentagrophytes SM110 strain is dispensed from above. 5) The stratum corneum subjected to the same operation from 1) to 3) is left while judging the growth of bacteria. 6) After standing, the keratin is taken out into a test tube, methanol is added, and the drug is extracted. 7) Analyze the concentration of the extracted drug by instrumental analysis (HPLC, LC / MS / MS, etc.). That is, when the concentration of the antifungal agent in the agar is high, the concentration of the antifungal agent in the stratum corneum also increases, and the fungus cannot grow at a certain concentration. Table 1 shows the concentration in the stratum corneum,
By comparing this concentration with the minimum growth inhibitory concentration (MIC), the activity of the antifungal agent in the tissue was compared, and a correlation with the MIC was obtained. The results showed a good correlation count value of r = 0.999. As shown in the above results, the antifungal agent content in the stratum corneum at this time was compared with the growth inhibitory concentration (MIC), and a correlation was obtained. It is possible to compare the physiological activities of the drugs, and it is clear that this is an evaluation method for accurately evaluating the efficacy of an antifungal agent.

[0016]

[Table 1]

[0017]

According to the present invention, as in the guinea pig tinea pedis model, the physiological activity of the drug in the tissue can be compared, and this can be combined with the minimum inhibitory concentration (MIC) and the correlation. Thus, a method for accurately evaluating the physiological activity of the antifungal agent can be provided.

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Claims (9)

[Claims] 1. A method for evaluating an antimicrobial agent against microorganisms resident on the skin, wherein a stratum corneum is placed on a medium, and the stratum corneum separates the original location of the microorganism from the original location of the antimicrobial agent. A method for evaluating an antimicrobial agent. 2. The method according to claim 1, wherein the antimicrobial agent is contained in an agar medium, a stratum corneum is placed on the medium, the microorganism is inoculated into the stratum corneum, and cultured on an agar medium. Evaluation method of antimicrobial agent. 3. The method for evaluating an antimicrobial agent according to claim 1, wherein the microorganism is a fungus. 4. The method for evaluating an antimicrobial agent according to claim 1, wherein the antimicrobial agent is an antifungal agent. 5. The method for evaluating an antimicrobial agent according to any one of claims 1 to 4, wherein the stratum corneum is derived from a mammal. 6. The stratum corneum derived from a mammal is derived from guinea pigs and / or miniature pigs.
The method for evaluating an antimicrobial agent according to any one of Items 1 to 5. 7. The method for evaluating an antimicrobial agent according to claim 1, wherein the medium is a salt medium. 8. The method for evaluating an antimicrobial agent according to claim 1, wherein the microorganism cannot grow on the salt medium alone. 9. The method for evaluating an antimicrobial agent according to any one of claims 1 to 8, wherein the efficacy of the antifungal agent is accurately evaluated by using a salt medium.