JPH08103292A - Evaluation of antifungal agent - Google Patents

Abstract

PURPOSE: To quantitatively measure the viable distribution of fungi in each sole skin strip by inoculating a fungus to an animal sole, giving a specimen to be tested, then stripping the sole skin and separately culturing the skin strips, thus evaluate the efficacy of antifungal agents in each part CONSTITUTION: A superficial disease-causative fungus is inoculated on a mammalian sole (rat, guinea pig, rabbit, mouse or pig) and a specimen to be tested is percutaneously given to the animal. Then, the sole skin is cut off in strips, for example, in the width direction and separated into groups, preferably the skins are further finely stripped in each group. Then, each strip is separately cultured to determine the distribution of the cell number of the fungus in individual positions of the sole.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for evaluating an antifungal agent for foot skin, and more particularly to an method for evaluating an antifungal agent by accurately measuring the distribution of the number of fungi on the skin of the foot.

[0002]

2. Description of the Related Art There are 120 superficial mycosis patients in Japan.
It is estimated to be 0 to 18 million. The cause of the large number of patients is considered to be the intensity of fungal transmission, which is thought to be due to the living environment, lifestyle, and lifestyle, and the difficulty of curative treatment due to repeated infection. This is because the site of the disease of the mycosis is the skin, especially the skin on the soles of the feet, so the way the drug works is not uniform, and even if it seems to be cured, the pathogen is not completely exterminated, This is because it often causes a recurrence. Moreover, since the effect of the drug varies depending on the site of the foot, the antifungal effect obtained even when the drug is administered to the ineffective site is small. Therefore, it is necessary to use a drug adapted to the affected site depending on the affected site, but a method for evaluating the difference in the efficacy of the drug depending on the site has not been known.

Conventionally, antifungal agents have been evaluated by in vitro antifungal activity using colony formation as an index and in vivo, antiinfective agents were applied to infected animals and the skin was collected. A plurality of small fragments were cut and taken out, planted in a medium, the number of fungal fragments was counted from this, and the value divided by the total number of fragments was used as an index for evaluation. However, in vitro evaluation cannot consider the most important pharmacokinetics in antifungal evaluation, and in the conventional in vivo method, the value is only a multiple of the value obtained by dividing 1 by the intercept number, Therefore, it is discontinuous, and the fragments that grow only in small quantities and the fragments that grow in large quantities are treated in the same way. In addition, it is undeniable that the factor of contingency is too strong, and therefore it was obtained accidentally. Although it was possible to determine the presence or absence of fungi on the skin pieces, a correct quantification was not possible, and a correct evaluation in consideration of site differences was not possible.

[0004]

SUMMARY OF THE INVENTION Therefore, the object of the present invention is to provide a method for quantitatively measuring the survival distribution of fungi in the skin of the foot and evaluating antifungal agents according to actual conditions. .

[0005]

[Means for Solving the Problems] Based on the above situation, the inventors of the present invention have conducted earnest studies for a means for quantifying fungi, and as a result, after infecting the skin of the soles of experimental animals, the antifungal agent was used. It was found that the anti-fungal agent can accurately evaluate and evaluate the action of the antifungal agent on each part of the foot by treating the skin of the foot with each part and quantifying the number of fungi at each part cut out. The invention was completed.

Therefore, according to the present invention, a fungus is planted on the sole of an animal, a subject is administered to the animal, and then the skin on the sole of the foot is cut into small pieces, and the obtained slices are cultured to prepare a piece of the foot. It is intended to provide a method for evaluating an antifungal agent for foot skin, which is characterized by quantifying the distribution of the number of fungi on each site of the back skin.

In the present invention, the term antifungal agent refers to
1) a compound having an antifungal action, 2) a composition containing the compound of 1), which means both a dosage form which is an artificial composition and a non-artificial composition such as a natural extract. Is included.

Here, the fungus which is the subject of the method for evaluating the antifungal action of the present invention is a causative bacterium of superficial mycoses, and if it is specifically exemplified, it is of the genus Trichophyton.
yton), Microsporum, Epidermophyton and other imperfect filamentous fungi, Candida, Malassezia (Malassezi)
Incomplete yeast of a), and these mutant strains are mentioned.
Such mutant strains include resistant strains that naturally acquired resistance to drugs, nutrient-dependent mutant strains that have become nutritionally dependent, artificial mutant strains that have been artificially mutated by gene transfer, etc. Etc. can be illustrated.

The animals used in the method of the present invention include mammals such as rats, guinea pigs, rabbits, mice and pigs. Examples of the method for transplanting the fungus to the soles of these animals include a method of applying the fungus on the skin, a method of exposing the dermis and applying it on the dermis, a closed patch method, an intradermal injection method and the like. However, the closed patch method is preferable from the viewpoint of reproducibility. Here, the fungal transplant is preferably performed on the entire sole of the foot.

The administration of the subject varies depending on the type of the subject and includes transdermal administration, oral administration, intravenous administration and the like, but transdermal administration is preferred. In the case of transdermal administration, it is desirable to apply to all sites where the fungus is transplanted.

In the present invention, the method of cutting out the skin of the sole of the foot is not particularly limited as long as it cuts out the function of the skin, and it may be cut vertically into a line or horizontally. It may be linearly cut, or may be cut in a vertical and horizontal lattice pattern.
For example, it is preferable that after being cut out laterally in a linear shape, each site is divided into groups (see FIG. 1), and the skin in each group is further cut into small pieces and subjected to culture. Such a small section is
For example, 1 × 1 to 20 × 20 mm may be cut per piece. This small section is preferably washed with a bactericide solution such as benzalkonium chloride or hibitene gluconate for the purpose of removing bacteria other than fungi.

The medium used for culturing the obtained slices is not particularly limited as long as it is one that is usually used for culturing or bacterial isolation, and examples thereof include Sabouraud medium, modified Sabouraud medium, and Czapek agar medium. .

The culture is carried out at 10 to 40 ° C., preferably 20 to 40 ° C.
Sufficient time for colonies to grow at 40 ° C, eg 1
Static culture may be performed for up to 20 days.

Next, the distribution of the number of fungi in each site of the skin on the sole of the foot is quantified by measuring the number of colonies grown on the medium by culturing. For the colony measurement, the number of colonies may be measured or the staining intensity may be measured. Also, they may be secondarily measured by absorbance or the like. Alternatively, the growth rate of mycelia may be monitored using a video camera or the like. Furthermore, it is also possible to measure the size of a colony and use the numerical value. Among these,
Easy and most preferable is to measure the size of the colony, and the size of the colony is measured by measuring the major axis (1) and the minor axis (s) and determining the product (l × s). It is preferable to compare with a control because it is simple and convenient.

The number of fungi can be quantified by comparing the colony size and the like measured as described above with a calibration curve prepared separately. This calibration curve is a calibration curve showing the correlation between the size of colonies and the number of fungi, for example, culturing a predetermined number of fungi under the same conditions as above, the size of grown colonies, etc. It can be created by measuring. Here, the number of bacteria can be measured by staining the whole cells with a stain such as neutral red and measuring the number of cells according to the degree of coloring, or radiating the metabolism of nutrients marked with isotopes. The number of bacteria may be measured by quantifying the activity.
The most suitable method for counting the number of bacteria is to separate bacterial cells and conidia,
Conidia are counted with a blood cell counter or the like and used as a control for the number of bacteria. According to this method, it is possible to easily and reproducibly control the number of bacteria. The control fungus used here may be a standard strain or a clinical isolate.

From the number of fungi in each section, the distribution of fungi at each site on the sole of the foot, that is, the effect of the antifungal agent can be evaluated for each site.

When a composition is used as an analyte in the present invention, the composition can be evaluated (for example, evaluation of base material, evaluation of dosage form, etc.).

[0018]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but it goes without saying that the present invention is not limited to these examples.

Example 1 Trichophyton TIMM2789 strain was modified with 1/10 Saburo-agar medium (peptone 0.2% glucose 0.1%,
Potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.
1%, agar 1.5%), and cultured on a slope at 27 ° C.
After 2 weeks, the slope was covered with a sterilized physiological saline containing 0.1% (w / v) Tween 80, and the conidia were released by shaking. After removing the mycelium with sterilized gauze, the number of conidia in the conidia suspension was calculated using a hemocytometer, and the concentration was adjusted to 1 × 10 ⁸ conidia / ml to give an inoculum. Guinea pigs were infected by the method of Uchida et al., Which is a modification of the Fujita method. That is, first wipe the left and right hind limbs of the guinea pig thoroughly with alcoholic cotton, and soak the lint cotton part of the bandage with gauze (made by Band-Aid Johnson & Johnson) with 100 μl of the inoculum, and apply the same bandage vertically to the hind leg planter part of the guinea pig, Adhesion was made at two places of the nail and Ken Achilles, gum tape was prepared, the outside of the hind limb equipped with band-aid was covered, the surgical tape was vertically divided into two, and the guinea pig hind limbs were tied and fixed with this tape. ) 3 days later
The tape was cut with scissors and the bandage was removed to expose the foot. Animals were divided into groups after confirming the formation of lesions 14 days after the start of infection. 28 days after the start of infection
1% butenafine-containing hydrophilic ointment (Drug-A) daily,
Treated with 1% bifonazole-containing petrolatum (Drug-B) and vehicle, and sacrificed animals 44 days after infection,
After wiping the whole foot 3 times with cotton swab, cut the skin of the foot from the heel and cut it to the base of the finger, and then ultrasonically wash each tissue in 1% disinfectant and then wash 3 times with sterilized water, As indicated by the solid line in FIG. 1, ten small pieces were cut horizontally from the base of the finger to the heel at substantially equal intervals. The small section of the base is No. 1. Numbering is performed in the order of the sections below, and the heel portion is No. It was set to 10. Next, add each of the small pieces to cycloheximide 500 μg / ml and chloramphenicol 100 μ.
The cells were placed on a Sabouraud medium plate containing g / ml and 50 μg / ml of sisomycin and cultured at 27 ° C. Cultured tissue sections are
Ten days later, the one in which the tinea bacterium community had developed was regarded as positive, and the medicinal effect was determined and evaluated. At the same time, the size of the colony is the major axis ×
Measured with the shortest diameter. Separately from these, the same strain used in the infection experiment was diluted to prepare a 10-fold dilution series, and then the bacterial solution was placed on the agar plate and cultured. On the 10th day from the start of culture, the major axis (1) and the minor axis (s) were measured, and the multiplied value was also measured to draw a calibration curve. From this calibration curve, the size of the colony obtained from the above-mentioned animal experiment was used to measure The number of surviving bacteria was quantified. The site distribution of the viable cell count is shown in FIG. From this figure, the site distribution of viable cell count varies depending on the antifungal agent,
And it is clearly understandable how the antifungal agent works on the skin of the foot. Therefore, according to the evaluation method of the present invention, it is possible to accurately evaluate the survival method after treatment of a fungus, the characteristics when an antifungal agent acts, and the like. Further, by applying this method, it is possible to change the antifungal agent to be used depending on the location of the lesion, and to search for a dosage form of a substance having an antifungal action that is suitable for the application site. Further, the effect of the combination of two or more kinds of substances having antifungal action can be clarified.

[0020]

According to the method of the present invention, the efficacy of the antifungal agent can be evaluated accurately and in detail for each part of the sole of the foot.

[Brief description of drawings]

FIG. 1 is a view showing an example in which the skin on the sole of the foot is cut into small pieces.

FIG. 2 is a diagram showing the distribution of the number of fungi for each site where the sole of the foot is finely cut.

Claims (5)

[Claims] 1. A plant of fungi is planted on the sole of an animal, a subject is administered thereto, and then the skin of the sole of the foot is cut into small pieces. A method for evaluating an antifungal agent for foot skin, which comprises quantifying the distribution of the number of fungi in a region. 2. The quantification of the distribution of the number of fungi is carried out by culturing each section and then measuring the size of the colonies grown on the medium to quantify the distribution of the number of fungi on each part of the skin on the sole of the foot. The evaluation method according to claim 1, wherein 3. The quantification of the distribution of the number of fungi is carried out by culturing each section, measuring the size of colonies grown on the medium, and measuring the size of the obtained colonies with a calibration curve prepared separately. The evaluation method according to claim 1, wherein the distribution of the number of fungi on each part of the skin on the sole of the foot is quantified in comparison. 4. The evaluation method according to claim 1, wherein a part of the fungus is spores or aerial hyphae. 5. The evaluation method according to claim 1, wherein the fungus is one kind or two or more kinds selected from the genus Trichophyton or the genus Epidermophyton.