Antifungal activity of aminopyrrolnitrin against Trichophyton verrucosum in a guinea pig model of dermatophytosis

Dermatophytosis is a common and major public health concern worldwide. Despite the increasing availability of antifungal drugs, relapses and untreated cases of dermatophyte infections are reported. Therefore, novel antifungal agents are required. Aminopyrrolnitrin (APRN) shows promise for dermatophytosis treatment because of its antifungal activity.


| Fungal strain
A total of 27 T. verrucosum strains were used in the experiment.
Twenty-six isolates were obtained from infected bovine in Korea, and one strain that was used as a reference strain (KACC 44577) was acquired from the Korean Agricultural Culture Collection (KACC) at the Rural Development Administration, Korea.All strains were used for antifungal susceptibility tests.The reference strain was used for the guinea pig model test.T. verrucosum was cultured on Sabouraud dextrose agar (SDA; Merck, Darmstadt, Germany) plates at 37°C for 2 weeks.The conidia were harvested by gentle rubbing with a loop in a sterile saline solution.The suspension was then filtered through sterile gauze to eliminate hyphal fragments.The number of conidia was counted using a haemocytometer, and its concentration was adjusted to 1.0 × 10 5 conidia/mL and 1.0 × 10 8 conidia/mL for use in the antifungal susceptibility tests and animal models.
Its molecular weight and chemical structure were confirmed using liquid chromatography-mass spectrometry (LCMS: 6545XT, Agilent, California, USA) and nuclear magnetic resonance (NMR: JMTC-400, JASTEC, Kobe, Japan).The APRN was initially dissolved in a solution containing 10% DMSO at a concentration of 30 mg/mL.Enilconazole was purchased from Sigma (Aldrich, SA.USA).For further preparation, the components were diluted with normal saline.

| Antifungal susceptibility test
The minimum inhibitory concentration (MIC) was determined according to a slightly modified CLSI M38 method. 12The conidia were suspended in RPMI 1640 medium (pH 7.0) with morpholinerpropanesulfonic acid (MOPS) and 2% glucose (m/v).An inoculum of 1.0 × 10 5 conidia/mL was employed for testing.The concentration ranges of APRN and enilconazole was 0.06-32 μg/mL, and the incubation took place at 37°C for 7 days.The MIC was defined as the lowest concentration that led to complete inhibition of the observable growth of fungi and duplicate measurements were taken.
To test antifungal susceptibility all chemicals were purchased from Sigma (Aldrich, SA.USA).

| Animal model
The in vivo experiment was designed based on Song et al.'s study. 13xteen male Hartley guinea pigs (SLC, Shizuoka, Japan, mean body weight 300 g) were housed in a temperature-controlled room at 22°C under a 12:12 h cycle of light: dark.These were then randomly grouped into four sets, as listed in Table 1.To induce the infection, a 2.5 cm × 2.5 cm (6.25 cm 2 ) area on the middle of the back of each guinea pig was clipped, shaved, and abraded with sandpaper.
Following this, a 1 mL suspension containing 1.0 × 10 8 conidia of T. verrucosum was applied and rubbed thoroughly onto the designated area.In this study, topical formulations were administered to infected regions beginning on the 10 day post-infection (dpi).The infected skin was treated with 1 mL of APRN at varying concentrations (4 and 8 μg/mL).In comparison, 1 mL of enilconazole (2 μg/mL) and normal saline were administered to the positive control (PC) and negative control (NC) groups, respectively.These treatments were administered once daily for 10 days, as shown in Figure 2.

| Clinical evaluation
The clinical evaluation involved scoring each infected skin area following the method described by Mikaeili et al. with slight modifications (Table S1). 3 The afflicted 2.5 cm × 2.5 cm infection site of each guinea pig was segmented into four identical quadrants.Subsequently, each 1.25 cm × 1.25 cm (1.56 cm 2 ) quadrant within this region was evaluated using a rating system ranging from 0 to 5. The combined scores for both sites in each guinea pig were used to assess the therapeutic efficacy of each component.The formula used to calculate the percentage clinical efficacy for all treatment groups was as follows: T represents the total score of the treatment group including a PC, and N is the total score of the NC group.Each group's total score reflects the average clinical score for the animals within that group.

| Mycological evaluation
Skin scrape samples from each quadrant of each guinea pig were collected at 21 dpi, after disinfection with a solution containing 70% isopropyl alcohol (Sigma, SA.USA) and 0.5% cycloheximide (Kisanbio, Seoul, Korea).To assess mycological activity, the specimens were cultured on SDA supplemented with chloramphenicol (Kisanbio, Seoul, Korea) and cycloheximide, incubated at 37°C for 7 days, and the absence of growth was used to define the successful elimination of fungi following the treatment.The efficacy percentages for various treatment and control groups were determined using the identical formula as applied in the assessment of mycological efficacy (refer to the above section).

| Histopathologic evaluation
Two randomly selected areas of each quadrant region were chosen as skin sample sites, and a biopsy punch (4 mm in diameter) at 21 dpi was performed at each site.The specimens were then rinsed three times with phosphate-buffered saline.They were then fixed in 10% neutral-buffered formalin and embedded in paraffin wax.The 4 μm-thick sectioned tissues were stained with haematoxylin and eosin using a standard laboratory protocol and periodic acid-Schiff (PAS) section staining.The histopathological lesions included hyperkeratosis (mild, moderate, severe), parakeratosis (focal, diffuse), neutrophil infiltration, spongiosis, and identification of fungi by PAS staining, as described by Wang et al. 14 with modifications.

| Statistical analysis
Clinical evaluation data are expressed as mean ± standard deviation.
Mycological clinical efficiency and clinical evaluation scores were statistically analysed using one-way analysis of variance (ANOVA) and Tukey's HSD test using SPSS 26.0 (IBM Corp., Armonk, NY, USA).Statistical significance was set at p < .05.

| In vitro antifungal activity of APRN
The results of the MIC of the 27 T. verrucosum strains are shown in Table 1.APRN showed antifungal activity against T. verrucosum with a MIC 90 and MIC range of 4.0 and 2-4 μg/mL, respectively.Therefore, APRN is a potential lead compound for further investigations as an antifungal drug.The MIC 90 of enilconazole was 2.0 μg/mL.

| Clinical evaluation
All guinea pigs infected with T. verrucosum exhibited clinical signs at 10 dpi.No significant differences were observed among the groups at 10 dpi when treatment was initiated.The clinical appearance of the guinea pig skin was photographed at 21 dpi (Figure 3).The control group exhibited patches of hair loss and readily visible ulcerated or scaly skin.Guinea pigs treated with enilconazole exhibited patches of hair loss and scaly skin.However, the daily application of different APRN concentrations resulted in scaly skin with minor hair loss.
The clinical scores of the groups were compared (Table 2).

| Histological evaluation
To determine the histological efficacy of APRN, histopathological parameters (H&E staining) and identification of fungal infection (PAS staining) in guinea pig skin infected with T. verrucosum were evaluated (Figure 4 and Table 3).The skin of all guinea pigs showed mild-to-severe hyperkeratosis.and PC (50%) groups.In the PAS stain, the guinea pig skin samples showed fungi in the epidermis and hair follicle in the NC (100%) and PC (50%) groups.No infections were observed in the treated groups.

| Efficacy of APRN in the guinea pig model
Clinical, mycological and histological efficacy was measured in the APRN-treated groups and compared to that in the untreated group (Table 2).Clinical efficacy was calculated by clinical evaluation of APRN treatment compared to the NC group (no treatment) and PC group (enilconazole treatment).The clinical efficacy of the treatments was as follows: PC (39.58%);APRN4 (70.83%); and APRN8 (70.83%).Skin scrapings inoculated into the SDA with chloramphenicol and cycloheximide are considered reliable for the diagnosis of dermatophytosis.Sixteen samples were collected from each group.
The results of the 7-day culture used for mycological evaluation showed PC (37.5%),APRN4 (87.5%), and APRN8 (75%).Histological efficacy was calculated by the identification of fungi and compared with the NC, showing PC (50%), APRN4 (100%), and APRN8 (100%).Enilconazole is one of the most widely used treatments for T. verrucosum. 15Pyrrolnitrin is biosynthesised from L-Tryptophan, which is affected by the prnABCD gene cluster. 16Our previous study showed S. grimesii MRS-1 produces APRN due to the lack of a gene cluster. 7Nevertheless, the concertation of APRN produced by S. grimesii MRS-1 was insufficient to test for antifungal activity (data not shown).Consequenly, manufactured APRN and enilconazole were used in this study.
In vitro, the antifungal activity of APRN against T. verrucosum demonstrated a MIC 90 value of 4 μg/mL, including the reference strain.This result indicates that APRN has the potential to block the growth of T. verrucosum and provides information to determine the capacity of APRN in vivo for further experiments.
Guinea pigs have been used as models to investigate the impact of different zoophilic dermatophytes on skin physiology and to assess the effectiveness of antifungal therapeutic agents. 17,18In this study, T. verrucosum caused redness, scaling, and hair loss in guinea pigs at 10 dpi, and these clinical signs remained until 21 dpi in the NC group.This result is similar to that of Song et al 13 but differs from that of Mikaeili et al, 3 where clinical signs began at 3 dpi.This might be due to the use of clinically isolated fungi with different characteristics at the sources of isolation. 19,20In this study, fungi from the KACC, isolated from humans, were used to obtain more reliable results, and before treatment, all fungi were detected by microscopic evaluation (data not shown).
The therapeutic effect of daily topical application of APRN was assessed in a guinea pig model using 4 and 8 μg/mL of chemical.In gross observations, the application of different concentrations of APRN significantly improved the clinical signs of the fungal infection.Compared with the PC group, the application of APRN showed higher anti-dermatophyte activity.In this study, mycological culture was used to reflect the elimination of fungi.The results showed that APRN had the highest eliminating efficacy.The clearance rates of fungi by APRN4 (87.5%) and APRN8 (75%) were higher than that of enilconazole (37.5%).The mycological efficacy was slightly higher than the clinical efficacy.It is possible that, although the fungi were eliminated, the skin did not completely recover during the treatment.Ahn et al. 21described that even through laser treatment histopathological lesions did not recover completely.
Histopathological evaluation was not routinely used during treatment of dermatophyte infections.Invasion of dermatophytes causes an inflammatory response and histological clue which demonstrates infection. 22Histopathological examination of the skin reveals focal hyperkeratosis and guinea pigs exhibit epidermal hyperkeratosis as a result of surgical manipulation or stretching, according to previous reports. 23,24However, spongiosis and neutrophil infiltration, parameters of the inflammatory response due to fungal infection, were detected only in the control group.In addition, basket-weave patterns, which are a histological indicator of fungal infections, were only detected in the untreated group, providing a histological clue suggesting fungal infection. 22S staining clearly demonstrates infection, by highlighting fungal cell walls.In this study, no fungi were detected in APRN4 and 8 but some hyphae were detected in the enilconazole-treated group.However, unlike in the control group, hyphae were observed in the stratum corneum (sandwich sign).Histopathological evaluation showed that APRN eliminated epidermal fungal infection.The histological efficacy was higher than the mycological efficacy.No hyphae were present in the tissue collected for PAS staining, likely because of resolution caused by the heightened self-renewal of the skin. 25is study had a limitation in that there was no information on the mechanism of action of APRN's antifungal activity.APRN is the precursor of pyrrolnitrin and its antifungal effect might be related to pyrrolnitrin.The structure-activity analysis indicates that pyrrolnitrin's main effect is on the cell membrane, where it obstructs the synthesis of proteins, RNA, and DNA, while also disrupting the typical flow of electrons in the respiratory electron transport chain. 26wever, future studies will need to confirm the exact mechanism behind the antifungal activity of APRN.
To our knowledge, this is the first study to evaluate the efficacy of APRN in a guinea pig model.Further large-scale studies are needed to examine the pharmacokinetics, biodistribution, and side effects, such as liver toxicity, of APRN topical skin therapy.
In conclusion, we found that APRN has antifungal activity against writing -review and editing; conceptualization; supervision; project administration.

F I G U R E 2 F I G U R E 3
Abbreviations: APRN, aminopyrrolnitrin; NC, negative control; PC, positive control.

F I G U R E 4
Abbreviations: APRN, aminopyrrolnitrin; NC, negative control; PC, positive control.

T
. verrucosum in vitro and exerts significant clinical, mycological, and histopathological effects against dermatophytosis in a guinea pig model when applied topically.Thus, APRN is a potential candidate for the treatment of dermatophytosis.AUTH O R CO NTR I B UTI O N SHanGyu Lee: Investigation; writing -original draft; visualization; conceptualization.Eun-Yeong Bok: Investigation; formal analysis; writing -original draft.Young-Hun Jung: Writing -review and editing; methodology.Tai-Young Hur: Writing -review and editing; funding acquisition; supervision.Young-Ok Kim: Supervision; writing -review and editing.Hee Jeong Kong: Writing -review and editing; resources.Dong-Gyun Kim: Visualization; writingreview and editing.Young-Sam Kim: Conceptualization; writingoriginal draft.Jae Ku Oem: Investigation; writing -original draft;