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| ORIGINAL ARTICLE |
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| Year : 2022 | Volume
: 17
| Issue : 6 | Page : 707-722 |
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Development and efficacy of tryptophol-containing emulgel for reducing subcutaneous fungal nodules from Scedosporium apiospermum eumycetoma
Thitinan Kitisin1, Watcharamat Muangkaew1, Sumate Ampawong2, Nichapa Sansurin3, Natthawut Thitipramote4, Passanesh Sukphopetch3
1 Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, 10400, Bangkok, Thailand., Thailand
2 Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University, 10400, Bangkok, Thailand., Thailand
3 Northeast Laboratory Animal Center, Khon Kaen University, 40002, Khon Kaen, Thailand., Thailand
4 Center of Excellence in Natural Products Innovation, Mae Fah Luang University, 57100, Chiang Rai, Thailand
| Date of Submission | 08-Mar-2022 |
| Date of Decision | 26-Jun-2022 |
| Date of Acceptance | 20-Sep-2022 |
| Date of Web Publication | 29-Oct-2022 |
Correspondence Address:
Passanesh Sukphopetch
Northeast Laboratory Animal Center, Khon Kaen University, 40002, Khon Kaen, Thailand.
Thailand
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/1735-5362.359437
Background and purpose: Subcutaneous infections caused by Scedosporium apiospermum present as chronic eumycetomatous manifestations in both immunocompromised and immunocompetent individuals. Serious adverse effects/toxicities from the long-term use of antifungal drugs and antifungal resistance have been reported in patients with S. apiospermum infections. The present study aimed to determine the anti-S. apiospermum activities of fungal quorum sensing molecule known as tryptophol (TOH) and to develop a TOH-containing emulgel for treating S. apiospermum eumycetoma.
Experimental approach: Anti-S. apiospermum activities of TOH were determined and compared with voriconazole. Effects of TOH on S. apiospermum biofilm formation and human foreskin fibroblast (HFF)-1 cell cytotoxicity were determined. Moreover, TOH-containing emulgel was developed and physical properties, in vitro, and in vivo antifungal activities against S. apiospermum eumycetoma were evaluated.
Findings/Results: The minimal concentration of TOH at 100 µM exhibited anti-S. apiospermum activities by reducing growth rate, germination rate, and biofilm formation with less cytotoxicity to HFF-1 cells than voriconazole. Further study on the development of an emulgel revealed that TOH-containing emulgel exhibited excellent physical properties including homogeneity, consistency, and stability. Treatment by TOH-containing emulgel significantly reduced subcutaneous mass in a mouse model of S. apiospermum eumycetoma. The histopathological assessment showed marked improvement after 14 days of TOH-containing emulgel treatment.
Conclusion and implications: TOH could be used as an anti-fungal agent against S. apiospermum infections. A novel and stable TOH-containing emulgel was developed with excellent anti-S. apiospermum activities suggesting the utilization of TOH-containing emulgel as an innovative therapeutic approach in the treatment of S. apiospermum eumycetoma.
Keywords: Emulgel; Eumycetoma; Quorum sensing molecule, Scedosporium apiospermum; Tryptophol.
How to cite this article:
Kitisin T, Muangkaew W, Ampawong S, Sansurin N, Thitipramote N, Sukphopetch P. Development and efficacy of tryptophol-containing emulgel for reducing subcutaneous fungal nodules from Scedosporium apiospermum eumycetoma. Res Pharma Sci 2022;17:707-22 |
How to cite this URL:
Kitisin T, Muangkaew W, Ampawong S, Sansurin N, Thitipramote N, Sukphopetch P. Development and efficacy of tryptophol-containing emulgel for reducing subcutaneous fungal nodules from Scedosporium apiospermum eumycetoma. Res Pharma Sci [serial online] 2022 [cited 2023 Jan 28];17:707-22. Available from: https://www.rpsjournal.net/text.asp?2022/17/6/707/359437 |
| Introduction | |  |
Scedosporium fungi are increasingly recognized as the causes of opportunistic fungal infections but also play an important role as primary pathogens [1]. S. apiospermum is an emerging opportunistic filamentous fungus that can be isolated from impacted environments caused by human activities including urban areas, manure-enriched or polluted soils, and sewage water [2]. Traumatic localized infections of S. apiospermum can occur both in immunocompromised and immunocompetent individuals and are mostly developed from mycetomatous and chronic progressive subcutaneous infections to systemic or disseminated infections [3],[4].
Traumatic localized infections of S. apiospermum are mostly manifested as mycetomatous with 70-80% occurring in the skin of the hands or feet known as S. apiospermum eumycetoma [5]. The pathology of the disease is developed as granulomatous inflammation penetrating the deep dermis and the subcutaneous tissue [6]. Moreover, S. apiospermum eumycetoma is endemic among tropical and subtropical regions as well as in Thailand [4],[7],[8]. However, this pathogenic fungus is notorious for its nature of therapy-refractory and is highly resistant to several antifungal agents [9]. Thus, there is an urgent need to develop new treatment strategies for treating the therapeutic challenge of Scedosporium infections [10].
Several potential antifungal drugs have been extensively investigated over the past 30 years [11]. Currently, treatments are limited to three classes of antifungal drugs i.e., polyenes, azoles, and echinocandins [12]. Although the drugs effectiveness against drug-resistant-pathogenic fungi have been proven, complications of treatment and adverse outcomes due to the extensive treatment periods and the potentially toxic drugs used have been noted [13]. Thus, alternative natural compounds exhibiting antifungal activity have been introduced. Among candidates, naturally occurring indole-based compounds are of great interest due to their properties responsible for cell-cell communication as well as auto-antibiotic actions known as fungal quorum sensing molecules [14],[15],[16]. Lesser-known but just interesting is tryptophol (TOH), an indole-3-ethanol metabolite that can be found in terrestrial fungi [17]. TOH has been found to affect fungal morphogenesis and induced fungal cell apoptosis providing antifungal potential against different fungal species [18]. We previously reported that TOH exhibited antifungal activities against pathogenic Candida albicans in vitro [19]. However, the utilization of TOH for treating S. apiospermum eumycetoma is poorly determined.
TOH is soluble in organic solvents such as ethanol [17]. However, repeated topical application of ethanol-based-TOH as an antifungal treatment may adversely affect the skin and cause skin irritation and contact dermatitis [20]. To improve topical TOH delivery, an alternative drug delivery system is required. Gellified emulsions also known as emulgels are mainly used to formulate hydrophobic drugs topically, which can be formed by the addition of a gelling agent into the emulsion phase [21]. The formulated emulgel can entrap active drugs and slowly release-slowly absorb through the skin. Moreover, an emulgel provides good stability, is easily removable, greaseless, soothing, and non-staining than other available topical preparation such as creams or ointments [22]. Thus, the current study aimed to determine the antifungal effects of TOH against S. apiospermum infection and to design and develop a TOH-containing emulgel. The physical properties, in vitro, and in vivo antifungal activity against S. apiospermum eumycetoma were also evaluated.
| Materials and methods | | |
Fungal strain and growth condition
S. apiospermum CBS 117410 used in this study was kindly provided by Dr. Ana Alastruey-Izquierdo (Servicio de Micologίa, Instituto de Salud. Carlos III, Madrid, Spain) [23]. The conidial suspension of S. apiospermum CBS 117410 was prepared by suspending 5-day-old culture on Sabouraud dextrose agar (SDA; Oxoid Ltd., Hampshire, UK) in sterile 0.9% saline to 1 × 105 conidia/mL and incubated at 37 °C.
Antifungal susceptibility
TOH (C10H11NO) was purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). The working solution of TOH was prepared by diluting 1 M stock solution to 1000 mM (v/v) with absolute ethanol (EtOH). Antifungal activity of TOH against S. apiospermum CBS 117410 was evaluated using the agar spot assay according to Kitisin et al. [7]. Ten µL of S. apiospermum CBS 117410 conidia (concentration about 1 × 105 conidia/mL) were spotted on the surface of SDA plates containing either 10, 100, or 1000 µM of TOH or voriconazole (VOZ; Sigma-Aldrich Inc., St. Louis, MO, USA). The plates were incubated aerobically at 25 °C. Absolute EtOH and RPMI 1640 culture medium (Sigma-Aldrich Inc., St. Louis, MO, USA) at 1% (v/v) were used as the diluent controls for TOH and VOZ, respectively. The colony diameter (mm) was measured every day for 7 days. Un-inoculated SDA plate was used as a negative control.
Conidial germination
The inhibitory effect of TOH on S. apiospermum CBS 117410 germination was determined according to Rollin-Pinheiro et al. [24]. The fungal conidia (1 × 105/mL) were freshly added into a microplate containing RPMI 1640 medium and incubated at 37 °C for 12 h. Then, 500 µL of TOH or VOZ at 10, 100, or 1000 µM was added to each well and incubated for 24 h. A total of 500 cells were counted under an optical microscope, and the length of conidial germination was quantified by ImageJ (National Institutes of Health, Bethesda, MD, USA).
Biofilm formation
S. apiospermum is able to form biofilms on the surface of sterile polystyrene microplates (96-well) or glass-bottom Petri dishes [25]. Biofilm formation of S. apiospermum CBS 117410 was performed by seeding 100 μL of conidial suspension (1 × 105/mL) to each well of the microplate and incubating at 37 °C for 3 h (adherence phase). As conidia adhered, the supernatant containing non-adherent cells was discarded and fresh RPMI 1640 supplemented with 2% glucose and 20% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) was added to each well and incubated for 24 h. A well containing only the growth medium was used as a negative control.
The biofilms were washed three times with sterile phosphate-buffered saline (PBS, pH 7.2) to remove non-adherent cells. Then, 200 µL of TOH or VOZ at 10, 100, or 1000 µM was added to each well and incubated for 24 h. Absolute EtOH and RPMI 1640 at 1% (v/v) were used as diluent controls. The inhibitory activity of TOH on S. apiospermum CBS 117410 biofilm formation was determined using two different approaches including crystal violet and XTT-reduction assays.
Crystal violet assay
Crystal violet assay was used to determine overall biomass including cells and the extracellular matrix of the biofilms by binding to negatively charged cell surface molecules and polysaccharides matrix of the fungi [25]. After washing the biofilms with sterile PBS, the remaining biofilm in each well was fixed with methanol for 20 min and stained with crystal violet (0.02%) for 30 min. The solution was removed, and the biofilms were washed twice with sterile PBS. The impregnated crystal violet was dissolved using a 30% acetic acid solution for 10 min. One hundred µL of dissolved solution in each well was collected and transferred into a new 96-well flat-bottom microplate and measured the absorbance at 590 nm using a spectrophotometer (Sunrise Tecan, Grödig, Austria).
XTT assay
XTT assay was used to determine the metabolic activity of the cells inside the biofilms indicating cell viability and density [26]. An orange formazan was converted from a yellow XTT dye by mitochondrial enzymes of the healthy cells. After washing the biofilms with sterile PBS, the remaining biofilm in each well was stained with 100 μL of an XTT:menadione solution (0.5 mg/mL:1 μM) as described previously [26]. The microplate was incubated in the dark at 37 °C for 2 h. The dissolved solution was transferred into a new microplate and measured the absorbance at 490 nm using a spectrophotometer (Sunrise Tecan, Grödig, Austria).
In vitro human foreskin fibroblast cell cytotoxicity
The human foreskin fibroblasts (HFF-1) cell line was obtained from American Type Culture Collection (ATCC®, Manassas, VA; catalog #SCRC-1041™). HFF-1 cells were routinely maintained in Dulbecco's modified eagle medium (DMEM; Gibco® by Life Invitrogen™, Carlsbad, USA) supplemented with 10% FBS and 100 units/mL of penicillin-streptomycin (Gibco® by Life Invitrogen™, Carlsbad, USA) and incubated under humidified conditions with 5% CO2 at 37 °C. Measurement of the cytotoxicity of TOH was carried out using two different assays, which are 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) activities.
MTT assay
HFF-1 cells were seeded at a concentration of 1 × 105 cells/mL onto a 96-well microplate and allowed to incubate overnight. Once reached 80-90% confluence, HFF-1 cells were washed with sterile PSB and treated with 100 µL of TOH or VOZ at 10, 100, or 1000 µM. The plate was incubated for 24 h. Absolute EtOH and RPMI 1640 at 1% (v/v) were used as diluent controls. HFF-1 cells incubated with culture medium alone were used as negative controls (100% viable). After 24 h incubation, the supernatant was aspirated from each well and HFF-1 cells were washed three times with sterile PBS. Then, 10 μL of MTT solution (Sigma-Aldrich Inc., St. Louis, MO, USA) was added and incubated at 37 °C for 2 h [27]. After carefully removing the supernatant, formazan was dissolved with 100 μL of 2% NP-40 (Sigma-Aldrich Inc., St. Louis, MO, USA). The absorbance was detected at 540 nm with a reference filter of 620 nm using a spectrophotometer (Sunrise Tecan, Grödig, Austria). The percentage of cell viability was calculated using the following equation:
LDH assay
LDH values were determined using the CytoTox 96® non-radioactive cytotoxicity assay (Promega Corporation, Madison, WI, USA), according to the manufacturer’s instructions [28]. Briefly, TOH and VOZ treatments on HFF-1 cells were prepared similar to the MTT assay. After 24 h incubation, 50 μL aliquots of cell supernatants were mixed with 50 μL of CytoTox 96® reagent in fresh 96-well flat clear bottom plates and incubated at room temperature in the dark for 30 min. Fifty µL of stop solution was then added and the absorbance was read at 490 nm using a spectrophotometer (Sunrise Tecan, Grödig, Austria). Untreated HFF-1 cells were used as negative controls, and the positive controls were provided in the assay kit. The percentage of cell cytotoxicity was calculated using the following equation:
Formulation of TOH-containing emulgel
Emulgel formula was developed according to Chen et al. [29] and Shahin et al. [30] as shown in [Table 1]. Briefly, emulgel base was prepared by a constant mechanical stirring of the gel-based SEPIGEL 305. The concentration of TOH that exhibited the highest antifungal activities against S. apiospermum CBS 117410 infection in vitro with the lowest cell cytotoxicity was chosen as an active ingredient. Then, TOH was slowly added under agitation to the emulgel base. The emulgel-base formulation was prepared with absolute EtOH and was used as a control emulgel.
Physical and biological properties of the developed TOH-containing emulgel including visual appearance, viscosity, spreadability, stability, pH, TOH content, in vitro and in vivo antifungal activity, and fungal identification of skin samples were determined. All experiments were performed in triplicate.
Visual inspection
For visual appearance, the prepared emulgel formulae were examined for their physical appearances including color, homogeneity, and phase separation [30].
Viscosity
The viscosity of prepared emulgel formulae was determined using a Brookfield viscometer (Brookfield, Model programmable DV2, USA) [30]. Briefly, about 0.5 g of each formula was placed into the beaker and left for equilibrium (30 min) at 25 °C. Measurements with the spindle were made at a moderate speed of 100 rpm for 10 min. The viscosity reading was recorded.
Spreadability
The spreadability of each emulgel formula was measured using two glass slides and a wooden block, which provided the basis for the slip and drag characteristics of the emulgel [30]. About 1 g of each prepared emulgel was placed onto the ground fixed glass plate and covered with another glass plate. Weight (20 g) was then placed on the top of this slide. The time (s) required to separate the two plates was noted. The spreadability was calculated using the following equation:
where S stands for spreadability, M, for weight tied to the upper slide; L is the length of glass slides; T, the time taken to separate the slides completely from each other.
Stability
The prepared emulgel formulae were tested for stability using centrifugation and temperature cycle tests [30]. For the centrifugation test, 10 g of each prepared emulgel formula was subjected to centrifugation at 9000 rpm for 30 min. The stability of emulgel after cooling-heating and freeze-thaw was determined using a temperature cycle test. Briefly, 2 g of each prepared emulgel formula was subjected to 10 temperature cycles, each cycle was 24 h, starting at -4 °C (8 h) and 40 °C (16 h). Satisfactory physical stability was evaluated at the end of these tests.
pH
The pH of all the formulated emulgels was measured by a digital pH meter [31].
TOH content of emulgel
To determine the TOH content in the emulgel, 1 g of the formulated emulgel was diluted with 100 mL EtOH. The diluted solution was filtered through a 0.45 μm Millipore™ filter and scanned at 278 nm spectrophotometrically. Standard TOH solution in each concentration of a calibration curve was prepared by dilution of the TOH stock solution with an absolute EtOH. The amount of TOH in the emulgel was determined from the standard plot of TOH [31].
In vitro antifungal activity of TOH-containing emulgel
Agar well diffusion method was adopted for evaluating the antifungal activity of the TOH-containing emulgel as described previously [32]. Briefly, S. apiospermum CBS 117410 conidia (1 × 105/mL) was poured onto SDA plates. Sterilize cork borer was used to make holes of 6 mm and formulated emulgels were aseptically applied. Commercial VOZ cream (100 µM) was used as a positive control. Inhibition zones were measured.
In vivo antifungal activity of TOH-containing emulgel
Female BALB/c mice (aged 8-week-old, body weight: 20-25 g) were purchased from Nomura Siam International Co, Ltd., Thailand, and further divided into 2 groups, 5 each; including uninfected mice and S. apiospermum-infected mice. The disseminated scedosporiosis mouse model was produced and validated as described previously [33]. Briefly, all mice were infected intraperitoneally with a single dose of 200 mg/kg cyclophosphamide to induce a neutropenic condition. On the following day, the dorsal hairs of the mice were shaved and the skin was sterilized with 70% EtOH. Mice were subcutaneously inoculated with 1 × 107 S. apiospermum CBS 117410 conidia per mouse. Eumycetoma was characterized as subcutaneous nodules with ulcer formation.
The lesion was seen on day 5 post-inoculation and considered day 0 of treatment. Then, the TOH-containing emulgel and the commercial VOZ cream (100 µM) were topically applied once a day from day 1 to 21. Dissected skin of S. apiospermum induced eumycetoma in mice after treatment with TOH-containing emulgel and their controls were collected on day 14 and day 21 of treatments and fixed with 10% neutral buffer formalin for 48 h. The wounded tissues were dehydrated and embedded in paraffin wax. Sections of skin samples were further stained with hematoxylin and eosin (H&E) or Gomori methenamine silver (GMS) and examined under a light microscope. Changes in histologic disease activity of the tissues were semi-quantified by H-score (multiplication of the hyphae distribution of the positive area 0-100% as scored with 0 = absent, 1, 2, and 3 = mild, moderate, and severe, respectively) [33].
All animal experiments were conducted in accordance with the Animals for Scientific Purposes Act, B.E. 2558 (A.D. 2015), Thailand. Experiments on mice were approved by Institutional Animal Care and Use Committee (IACUC) of Khon Kaen University, Thailand (approval number: IACUC-KKU-44/63, reference number: 660201.2.11/271 [29]).
Statistical analysis
Experimental data were analyzed using GraphPad Prism, version 6.05, (GraphPad Software, San Diego, CA, USA). All experiments were performed in triplicate. Data were expressed by mean ± SD. Significant differences at P < 0.05 were calculated using an independent t-test or analyses of variance (ANOVA) followed by Tukey’s multiple comparison test.
| Results | | |
Antifungal susceptibility
[Table 2] showed the colonies diameters of S. apiospermum CBS 117410 after 3 days of incubation with TOH and standard antifungal drug VOZ by agar spot assay. SDA media containing either 1% EtOH (v/v) or 1% RPMI 1640 (v/v) were utilized as diluent controls for TOH or VOZ, respectively. The colony diameters of untreated S. apiospermum CBS 117410 were increased from day 3 to day 7 in a time-dependent manner similar to diluent control groups [Table 2]. VOZ treatments at the concentration ranging from 10 μM to 1000 μM remarkably lessened the growth rate of S. apiospermum CBS 117410, especially at moderate (100 μM) and high concentrations (1000 μM). Thus, the minimal effective concentration of VOZ at 100 μM was used for further subsequent analyses. | Table 2: Colony diameters of TOH-treated Scedosporium apiospermum CBS 117410 at 25 °C.
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An increase in S. apiospermum CBS 117410 colony diameter was observed from day 3 to day 5 in the similar manner to the untreated or diluent control groups [Table 2]. Treatments of 1% EtOH did not reduce the growth rate of S. apiospermum CBS 117410 from day 3 to day 7 when compared to the untreated controls (P > 0.05). However, treatments of TOH at concentration ranging from 10 μM to 1000 μM remarkably lessened the growth rate of S. apiospermum CBS 117410 in a concentration-dependent manner (P < 0.05). Interestingly, there was no growth of S. apiospermum CBS 117410 at any time point in the 100 μM and 1000 μM of TOH treated groups (P < 0.05). Therefore, the minimal effective concentration of TOH at 100 μM was used for further subsequent analyses.
Conidial germination
Germination of fresh S. apiospermum CBS 117410 conidia was lessened after treating with TOH or VOZ at 24 h of incubation in a concentration-dependent manner [Table 3]. EtOH at 1% (v/v) did no effect on germ tube formation. TOH or VOZ at 10 μM didn’t affect germ tube formation. Whereas the greatest effects on germ tube inhibition were observed in the highest concentrations (1000 μM) of TOH and VOZ (P < 0.05). However, treatments with TOH at moderate to high concentrations of 100 to 1000 μM significantly reduced the germ tube length of S. apiospermum CBS 117410 than VOZ. Thus, TOH at 100 μM was the minimal effective concentration for controlling S. apiospermum germination in vitro. | Table 3: Germ tube length of TOH-treated Scedosporium apiospermum CBS 117410 after 24 h incubation at 37 °C.
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Biofilm formation
Treatments with TOH remarkably reduced the fungal total biofilm biomass and metabolic activity rates of S. apiospermum CBS 117410 when compared to the VOZ-treated groups in a concentration-dependent manner as determined using crystal violet and XTT reduction assays, respectively [Table 4]. EtOH at 1% (v/v) had no effect. Moreover, treatments of TOH at 100 to 1000 μM significantly reduced the amount of biomass produced and metabolic activity rates than VOZ treatments (P < 0.05). Thus, TOH at 100 μM was the minimal effective concentration for controlling S. apiospermum biofilm formation in vitro. | Table 4: Biofilm formation of TOH-treated Scedosporium apiospermum CBS 117410 after 24 h incubation at 37 °C.
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In vitro human foreskin fibroblast cell cytotoxicity
After 24 h incubation, the HFF-1 cell viability did not show a significant change after treating with 10 and 100 μM of TOH as determined by MTT assay [Figure 1A]. However, HFF-1 cell viability (85 ± 3.30%) was significantly decreased at 1000 μM TOH concentration when compared to controls. Treatment with VOZ at 100 and 1000 μM significantly decreased HFF-1 cell viability compared to untreated controls [Figure 1B]. In addition, HFF-1 cell viability was decreased to lower than 80% when treated with VOZ at 1000 μM. The results from the LDH assay were correlated to the MTT data after 24 h generation times. The percentages of cell cytotoxicity in TOH and VOZ-treated groups were increased in a concentration-dependent manner when compared to controls in all treatments [Figure 1C] and[Figure D]. However, TOH treatments significantly exhibited less cytotoxic effect on HFF-1 cells than VOZ treatments. Collectively, the results suggested that TOH at 100 μM was the minimal effective concentration for controlling S. apiospermum CBS 117410 infections without compromising risks to human skin in vitro and thus were selected for further emulgel development.{Figure 1A}{Figure 1B}{Figure C}{Figure D}
Formulation of TOH-containing emulgel
An emulgel that has 100 μM of TOH as an active ingredient was successfully developed. The parameters of TOH-containing emulgel and control emulgel were presented in [Table 5].
Upon investigation, the prepared TOH-containing emulgel appeared as white, translucent, homogenous of semisolid consistency, and non-sticky skin feel. Moreover, there was no phase separation reported both in TOH-containing and control emulgels [Figure 2A]. The viscosity of the TOH-containing emulgel was 2401 cps, whereas the control emulgel was 2350 cps. The spreadability of the TOH-containing emulgel was 26.00 ± 0.50 g.cm/s., whereas the control emulgel was 25.00 ± 0.10 g.cm/s. The effects of accelerated stabilities including centrifugation and temperature cycle test were determined. The results showed no color change or signs of instability involving phase separation or inconsistency increases were observed [Figure 2B] and [Figure C] and [Table 5]. The pH of TOH-containing and control emulgels were 5.53 ± 0.01 and 5.40 ± 0.10, respectively. Moreover, the TOH content of the formulated TOH-containing emulgel was 99.98 %.{Figure 2A}{Figure 2A}{Figure 2C}
In vitro antifungal activity of TOH-containing emulgel
The antifungal activity of TOH-containing emulgel against S. apiospermum CBS 117410 using the agar diffusion method was investigated. From the results, the control emulgel base did not show any antifungal activity (no zone of inhibition) against S. apiospermum CBS 117410. However, TOH-containing emulgel exhibited antifungal activity against S. apiospermum CBS 117410 with a mean zone of inhibition (23.80 ± 1.09 mm), which was similar to the mean zone of inhibition exhibited by the commercial antifungal drug VOZ (19.50 ± 3.16 mm, [Table 6]). Moreover, the antifungal activity of TOH-containing emulgel was similar to TOH solution (100 µM) suggesting that the emulgel base formation does not negatively interfere with the antifungal medication of TOH. | Table 6: Diameter of the inhibition zone of TOH-containing emulgel-treated Scedosporium apiospermum CBS 117410 at 72 h post-incubation, 25 °C.
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In vivo antifungal activity of TOH-containing emulgel
After day one post-inoculation of S. apiospermum CBS 117410 into the subcutaneous layer of the skin, experimental mice showed the erythematous patch in the inoculated areas. Subcutaneous mass with few sinuses and few white grains were observed in the infected mice on day 3 post-inoculation. On day 5 post-inoculation, the eumycetoma was developed as hard consistency subcutaneous mass with multiple sinuses, white grains, and central necrosis [Figure 3A], which indicated as day 0 for applying topical tested emulgels. After 7 days and 14 days of experimental treatments, the photographs of eumycetoma at the back of mice were taken at a distance of 10 cm from the lesion site using a digital camera as shown in [Figure 3B]. There was an insignificant difference in the mass size between experimental groups at the first day of the treatments [Figure 3C]. Treatments with TOH-containing emulgel and commercial VOZ cream significantly reduced the mass size of S. apiospermum eumycetoma than the emulgel base treated groups after 7 days of treatments. Interestingly, treatment of TOH-containing emulgel significantly reduced the mass size than commercial VOZ cream after 14 days and 21 days of treatments [Figure 3B] and [Figure C].{Figure 3B}{Figure 3A}{Figure 3C}
Histopathological evaluation
Mice that inoculated with S. apiospermum CBS 117410 into the subcutaneous layer of the skin and received topical emulgel base treatment for 14 days exhibited histological pathology of eumycetoma. The H&E stained wound sections revealed a large mass area of fungal infection surrounded by mononuclear infiltrates [Figure 4A]. Moreover, GMS stained wound sections revealed a high density of fungal hyphae within the lesion area. S. apiospermum CBS 117410-infected mice that received topical TOH-containing emulgel treatment for 14 days exhibited significantly lower damage of fungal infection than commercial VOZ cream and emulgel-base treatments [Figure 4A] and [Figure C].{Figure 4A}{Figure C}
Reduction in eumycetoma pathology with a lessened amount of fungal hyphae was significantly observed on day 21 of topical TOH-containing emulgel treatment when compared to commercial VOZ cream and emulgel-base treatments [Figure 4B]. The lesion area of TOH-containing emulgel-treated group showed skin scab and skin reepithelization but not in other experimental conditions [Figure 4B]. Moreover, tissue inflammation and density of fungal hyphae within the lesion area was significantly decreased in topical TOH-containing emulgel treated group than other groups [Figure 4B] and [Figure C].{Figure 4B}
| Discussion | | |
Scedosporium species especially S. apiospermum have been found in clinical settings as well as in human-impacted areas including urban parks, agricultural soils, and polluted water [34]. Opportunistic infections of S. apiospermum are increasingly reported worldwide both in immunocompromised and immunocompetent individuals [3]. Disseminated disease with involvement of the lungs, brain, and skin infections is commonly reported and related to trauma or near-drowning events [35]. Moreover, traumatic localized infections in both immunocompromised and immunocompetent individuals are mostly manifested as eumycetoma, a chronic progressive subcutaneous infection that penetrates muscles and bones [36]. Successful treatments of Scedosporium spp. are largely dependent on accurate and prompt diagnosis and effective medications [37]. However, current surgical intervention or commercially available antifungal treatments may not be adequate enough to treat Scedosporium infections due to their intrinsic antifungal resistance and poor penetration of antifungal agents from debridement [38]. Thus, this study was designed to develop and evaluate the effectiveness of alternative topical applications to treat S. apiospermum eumycetoma using a TOH-containing emulgel.
In the present study, we found that treatment of TOH lessened the growth of S. apiospermum CBS 117410 in a concentration-dependent manner. In consistent with our previous reports, we found that a moderate concentration (100 μM) of TOH remarkably lessened the growth of S. apiospermum [7]. Although the drug of choice for S. apiospermum infections is VOZ [39], we found that TOH appeared to be as effective as VOZ. In the current study, an inhibitory effect of TOH on S. apiospermum CBS 117410 germination was investigated as a reduction of germination rate might be important for controlling its biofilm formation. As expected, TOH at 100 μM was able to decrease the germ tube length and effectively controlled S. apiospermum biofilm formation more than VOZ. Therefore, our study showed that treatment of TOH is able could be utilized as an alternative therapeutic potential for controlling the growth of S. apiospermum CBS 117410. Moreover, TOH treatments exhibited lesser cytotoxic effects on the human foreskin fibroblast (HFF-1) cells than VOZ treatments. The TOH belongs to a class of 3-substituted indole compounds featuring a 2-hydroxyalkyl side chain [14]. The previous study has suggested that TOH can disrupt fungal morphogenesis and induce fungal cell apoptosis, which can be used as an antifungal agent against different fungal species [17]. Therefore, our present study demonstrated that TOH can be utilized as a promising antifungal agent against S. apiospermum CBS 117410 infections without compromising risks to human skin and was selected for further emulgel development. Further study is required to investigate the molecular mechanisms of TOH on antifungal activity against cutaneous pathogenic fungi.
TOH at 100 μM was successfully incorporated into emulgel base containing SEPIGEL 305 as a gelling agent. SEPIGEL 305 is composed of polyacrylamide, polyoxyethylene 7 lauryl ether, and isoparaffin, which acted as a gelling agent, a non-ionic emulsifier, and fatty oil, respectively [40]. Thus, the addition of SEPIGEL 305 increases viscosity, a degree of cooling power, and optimal dermo-cosmetic qualities. Triethanolamine was added to neutralize the formulae to the pH range of 5.5-6.5 by uncoiling the polymer chains and forming the gel structure [30]. This pH range caused the TOH-containing emulgel to be compromised with the pH of the skin (pH 5) [30]. Propylene glycol acts as a humectant to the emulgel, which helps to increase the spreadability as well as the aesthetic feeling of the product [41]. Our study showed that TOH-containing emulgel exhibited an excellent level in terms of homogeneity, consistency, stability, and a high percentage of TOH content. We further demonstrated that TOH-containing emulgel exhibited antifungal activity against S. apiospermum CBS 117410 in vitro. Thus, our study suggested that the developed TOH-containing emulgel formulation was optimal for use as topical drug delivery in vivo.
Infections of Scedosporium spp. into the subcutaneous layer of the skin cause a chronic progressive granulomatous infection known as eumycetoma [6]. In the present study, we successfully developed a eumycetoma mice model from S. apiospermum CBS 117410 subcutaneous infections. The pathological findings in the established mouse model in this study were similar to evidence in infected patients as characterized by the formation of grains containing aggregates of fungal hyphae [42]. VOZ is a new triazole antifungal agent that exhibits a broad spectrum of antifungal activity against Aspergillus spp., Cryptococcus spp., and Candida spp., as well as fluconazole-resistant groups [43]. VOZ has been reported to be effective in most patients, including children against Scedosporium spp. [44]. Although there were reports of successful treatment with VOZ for S. apiospermum eumycetoma, however, uptake of VOZ caused several side effects [36],[37],[39]. The most common side effect of VOZ has altered color discrimination, blurred vision, and photophobia have been noted [45]. Several adverse effects including hepatitis, photohypersensitivity, and pseudoporphyria have also been reported [39],[46]. Therefore, new antifungal agents are needed for the treatment of S. apiospermum infections.
Treatment of TOH-containing emulgel on eumycetoma mice model from S. apiospermum CBS 117410 infections showed positive antifungal activity in the control of eumycetoma mass and improved mouse skin lesions than commercial VOZ cream. Thus, our study suggested that TOH may help to promote tissue remodeling and significant inhibition of S. apiospermum CBS 117410 infection. Further studies are required to reveal the molecular mechanisms of immunological reaction during TOH-containing emulgel treatment. We previously reported that treatments of TOH reduced the C. albicans pathogenicity by increased cellular apoptosis [19]. In this report, we demonstrated that treatments of TOH remarkably elevate the apoptosis pathway in C. albicans by transcriptional upregulation of caspase recruitment domain-containing protein (CARD)-9 and Noxa and downregulation of Bcl-2. In general, the activation of apoptosis is positively regulated by the activation of CARD-9 and Bcl-10 through the nuclear factor-kappa B stress pathway [47]. Moreover, oxidative stress-induced apoptosis is coordinately regulated by anti-apoptotic Bcl-2 protein and Noxa (a pro-apoptotic protein of Bcl-2) [48]. Taken together, it is possible that TOH-containing emulgel can promote cellular apoptosis and lessen the virulence of S. apiospermum eumycetoma similar to the pathogenic C. albicans. Therefore, the collective results from the present study purpose that TOH-containing emulgel can be used for treating S. apiospermum eumycetoma, which provides an alternative way to utilize fungal quorum sensing molecules with potential applications for combating fungal infections. We plan to measure user satisfaction with TOH-containing emulgel for future registration as a topical antifungal drug. Nevertheless, we plan to deepen our study into the molecular mechanisms of TOH-containing emulgel-mediated pathogenicity attenuation of S. apiospermum and other cutaneous pathogenic fungi in the future.
| Conclusion | | |
A promising in vitro anti-S. apiospermum assay proved TOH to be highly effective. Treatment of TOH at 100 µM exhibited anti-S. apiospermum activities by reducing growth rate, germination rate, biofilm formation, and less cytotoxicity to HFF-1 cells than VOZ. TOH-containing emulgel was developed. Formulation of TOH-containing emulgel revealed an excellent physical property including homogeneity, consistency, and stability. Treatment of TOH-containing emulgel significantly ameliorated subcutaneous mass in S. apiospermum eumycetoma mice and improved skin histological architecture after 14 days of treatment. Therefore, our study demonstrated an innovative approach for efficient treatment against S. apiospermum eumycetoma using TOH-containing emulgel topical application. Further experimental and clinical trials are required to confirm the findings of the present study.
Acknowledgments
This research work was financially supported by the Health Systems Research Institute (Grant number: HSRI 64-051), the National Research Council of Thailand (Grant number: NRCT 230/2564), and Mahidol University (NDFR). The funding agencies had no role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript.
Conflicts of interest statement
The authors declared no conflicts of interest in this study.
Authors’ contribution
P. Sukphopetch designed and supervised the project; T. Kitisin, W. Muangkaew, S. Ampawong, and N. Thitipramote performed the experiments, analyzed, and interpreted the data; N. Sansurin provisioned the experimented animals. T. Kitisin contributed to the writing of the manuscript and revision; S. Ampawong and N. Thitipramote were involved in planning and supervising the work. The final version of the manuscript was approved by all authors.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]
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