The Antifungal and Anti-Pneumocystis Activities of the Novel Compound A3IS (Mycosinate)

ABSTRACT

A3IS (Mycosinate) is a synthetic product which only contains ingredients found naturally within honey. A3IS is a broad-spectrum antimicrobial product which produces a sustained release of hydrogen peroxide at low but therapeutic levels. The product elicits this release through an enzymatic reaction between glucose oxidase and the substrate glucose once the product is hydrated. As medical uses for different honeys are being re-evaluated, the purpose of this study was to evaluate the in vitro effects of A3IS against a comprehensive panel of human pathogens, including Pneumocystis species, providing a unique assessment against a panel of eukaryotic pathogens. Without exception, A3IS exhibited significant efficacy at 50% and 100% inhibitory concentrations against a broad spectrum of human pathogens including yeasts, molds (both hyaline and dematiaceous), and dimorphic fungi. Notably, A3IS was effective against fungal strains with a high level of resistance to fluconazole or voriconazole. The 50% inhibitory concentrations for Pneumocystis carinii and P. murina (surrogates for P. jirovecii) were considered “Marked” and “Moderate” on an established rank scale, and would be considered for in vivo studies, based on an established in vitro-in vivo pipeline. These results indicate that A3IS is a novel anti-fungal agent against an extensive range of human fungal pathogens.

INTRODUCTION

A3IS (Mycosinate) is a synthetic product which only contains ingredients found naturally within honey and was developed from research based on the antimicrobial properties of honey. From the recently completed review of medical grade and off the shelf honey, A3IS shows similar or better levels of antimicrobial activity compared to those reviewed in Nolan et al. (1). The medical effects of honeys are being reconsidered due to the rise of antimicrobial resistance in bacteria and fungi but also because of the increased interest in natural health therapies (2, 3). A survey of several commercial honeys found antifungal activities against dermatophytes and Candida albicans (3) and significant anti-inflammatory and wound healing properties (4).

A3IS produces a sustained release of hydrogen peroxide at low but therapeutic levels. The product elicits this release through an enzymatic reaction between glucose oxidase and the substrate glucose once the product is hydrated, see Equation 1, below.

$$\mathit{Glucose}\hspace{0.17em}+\hspace{0.17em}\mathit{Water}\hspace{0.17em}+\hspace{0.17em}\mathit{Oxygen}\stackrel{\mathit{Glucose}\hspace{0.17em}\mathit{Oxidase}}{\to }\mathit{Gluconic}\mathit{\hspace{0.17em}Acid}\hspace{0.17em}+\hspace{0.17em}\mathit{Hydrogen}\mathit{\hspace{0.17em}Peroxide}$$ (1)

The sustained release of hydrogen peroxide is the main antimicrobial characteristic of A3IS, but it also contains a high level of sugars, which can disrupt bacteria through osmotic pressure, essentially removing water, without which the bacteria cannot grow or divide (5). Osmotic pressure induced by honey has been shown to decrease survival of the pathogenic fungi, Cryptococcus neoformans and Candida species (6, 7).

The purpose of the present study was to evaluate the in vitro effects of the synthetic A3IS against a panel of pathogenic fungi, including Pneumocystis species, providing a unique assessment against a diverse group of eukaryotic pathogens. Identification of novel and potentially efficacious compounds against fungi is an important endeavor as resistance to commercially available antifungal agents frequently renders them ineffective or decreases their ability to prevent or eradicate these infections (8). Additionally, products like A3IS affecting multiple targets within the microbe, may offer a better chance at averting resistance than those targeting a specific enzyme (9).

Cytotoxicity.

Cytotoxicity of BALB/c 3T3 mouse fibroblast cells was observed at concentrations of 100 μg/mL and higher for the wells with 10% and 5% of the glucose oxidase; for 1% at 10,000 μg/mL; and no cytotoxicity at 0.1% or 0.01% (10 μg/mL and 1 μg/mL). The standard formulation for A3IS is 0.5% of the active ingredient.

Antifungal activity.

The results of in vitro testing against the panel of fungi are shown in Table 1. Overall, A3IS demonstrated broad spectrum in vitro activity against yeast, molds, and the dimorphic fungi Blastomyces dermatitidis and Coccidioides species. Against yeasts, the activity of A3IS ranged from ≤0.02% to 0.15% wt/vol with both the 50% and 100% inhibition of growth endpoints, and its activity was maintained against fluconazole resistant strains. Similar activity was observed against all mold isolates tested and ranged from ≤0.02% to 0.3% wt/vol with both the 50% and 100% inhibition of growth endpoints. As against the fluconazole resistant yeasts, the activity of A3IS was maintained against Aspergillus, Fusarium, Scedosporium, and Lomentospora isolates that demonstrated high-level in vitro resistance to voriconazole (i.e., voriconazole MIC ≥8 mg/mL). Finally, the growth of each B. dermatitidis and Coccidioides isolate tested was inhibited at the lowest concentration of A3IS tested (0.02% wt/vol), including several Coccidioides strains with reduced susceptibility or in vitro resistance to fluconazole.

TABLE 1.

In vitro activity of A3IS against different fungi as measured by broth dilution according to the CLSI M27 and M38 standards^a

Fungal organism	Strain no.	A3IS 50% inhibition	A3IS 100% inhibition	Fluconazole 50% inhibitionb	Voriconazole 100% inhibitionb	Posaconazole 100% inhibition
Candida parapsilosis	ATCC 22019	≤0.02%	0.04%	1
Candida krusei	ATCC 6258	≤0.02%	≤0.02%	16
Candida albicans	SC5314	0.15%	0.3%	0.25
	CA90028	0.08%	0.15%	0.25
	CA1	0.04%	0.15%	>64*
	CA2	0.04%	0.15%	>64*
Candida auris	DI17-47	0.08%	0.3%	>64*
	DI17-48	0.08%	0.3%	2
	DI17-46	0.08%	0.3%	>64*
	CAU1	≤0.02%	0.04%	1
Candida glabrata	CG1	≤0.02%	0.04%	64*
	CG2	≤0.02%	0.04%	8
	CG3	≤0.02%	0.15%	32
	CG4	≤0.02%	0.15%	2
Cryptococcus neoformans	USC1597	≤0.02%	≤0.02%	16
	H99	≤0.02%	≤0.02%	4
	CN1	≤0.02%	≤0.02%	4
	CN2	≤0.02%	≤0.02%	64*
Aspergillus flavus	ATCC 204304	0.3%	0.6%		1
	Aflav1	0.3%	0.6%		1
	Aflav2	0.3%	0.6%		1
	Aflav3	0.04%	0.04%		0.5
Aspergillus fumigatus	AF293	≤0.02%	≤0.02%		0.5
	DI15-106	0.08%	0.08%		>16*
	DI15-116	0.08%	0.15%		8*
	AF4	0.04%	0.04%		0.25
Fusarium species	F1 (F. oxysporum)	≤0.02%	≤0.02%		8*
	F2 (F. oxysporum)	≤0.02%	≤0.02%		8*
	F (F. oxysporum)	≤0.02%	≤0.02%		4
	F4 (F. solani)	0.04%	0.08%		>16*
Scedosporium species	S1 (S. boydii)	≤0.02%	≤0.02%		>16*
	S2 (L. prolificans)	≤0.02%	≤0.02%		>16*
	S3 (S. apiospermum)	≤0.02%	≤0.02%		1
	S4 (S. apiospermum)	≤0.02%	≤0.02%		1
Altenaria	Alt1	0.08%	0.15%		4
Curvularia	Curv1	0.15%	0.3%		0.25
Exophialia	Exoph1	≤0.02%	0.04%		0.25
Exserohilum	Exser1	≤0.02%	≤0.02%		0.06
Rhizopus arrhizus	99-880	≤0.02%	≤0.02%			0.5
	99-892	≤0.02%	≤0.02%			0.125
	RA3	≤0.02%	≤0.02%			0.25
	RA4	≤0.02%	≤0.02%			0.5
Blastomyces dermatitidis	BD1	≤0.02%	≤0.02%	16
	BD2	≤0.02%	≤0.02%	64*
	BD3	≤0.02%	≤0.02%	32*
	BD4	≤0.02%	≤0.02%	8
Coccidioides species	Cocci1	≤0.02%	≤0.02%	16
	Cocci2	≤0.02%	≤0.02%	64*
	Cocci3	≤0.02%	≤0.02%	32*
	DI17-143	≤0.02%	≤0.02%	8

^aMICs were read at both 50% inhibition and 100% inhibition of growth for A3IS, 50% inhibition of growth for fluconazole, and 100% inhibition of growth for voriconazole and posaconazole. A3IS tested over concentration range of 0.02% to 10% wt/vol, fluconazole tested at 0.125–64 μg/mL, and voriconazole and posaconazole tested at 0.03–16 μg/mL.

^bStrains with high level resistance to fluconazole or voriconazole are indicated with an asterisk (*).

Anti-Pneumocystis activity.

The anti-Pneumocystis activities of A3IS were evaluated in 2 different species of rodent-derived fungi: Pneumocystis murina, from mice, and Pneumocystis carinii, from rats. These 2 species are considered suitable surrogate models for the species which infects humans, Pneumocystis jirovecii. No species of this genus of fungi can be grown outside the mammalian lung, and short-term cultures of rodent-derived Pneumocystis spp. have been used to predict activity for preclinical drug development (10). The IC₅₀ (Inhibitory Concentration) of A3IS for P. carinii was 0.6415 μg/mL and 4.155 μg/mL for P. murina, which are ranked as having “Marked” and “Moderate” activity (Table 2). Pentamidine isethionate, an agent that has been used to treat Pneumocystis jirovecii pneumonia (PjP) in humans, has an IC₅₀ of 0.300 μg/mL in this in vitro assay, which is ranked as having “Marked activity” (11).

TABLE 2.

Activity scale of 72-h IC₅₀ values

Activity rank	Concentration
Highly active	<0.010 μg/mL
Very marked	0.011 to 0.099 μg/mL
Marked	0.10 to 0.99 μg/mL
Moderate	1.0 to 9.99 μg/mL
Slight	10.0 to 49.9 μg/mL
None	≥50 μg/mL

In this manuscript, we presented in vitro MIC results against a range of different fungi, including yeasts, hyaline and dematiaceous molds, and endemic fungi for the investigational agent, A3IS. A3IS effects were similar against all fungi, including isolates that had reduced susceptibility/in vitro resistance to clinically available antifungals. These types of data are what are generally presented in early, proof-of-concept studies with novel investigational agents.

In keeping with the notable efficacy of A3IS across the pathogenic yeasts, molds, and fungi, the activity against the 2 rodent species of Pneumocystis was also notable and fell into the “Marked” and “Moderate” activity rankings. Candidate agents with this ranking would be recommended for evaluation in the mouse model of Pneumocystis pneumonia in the preclinical drug development pipeline our laboratory has established (10). Infections with P. jirovecii, (PjP) are increasingly reported in patients severely immunocompromised following allogeneic hematopoietic stem cell transplantation or solid organ transplantation, those with hematological malignancies receiving immunosuppressive therapies, patients with rheumatologic disorders, and those infected with HIV with CD4 counts < 200 cells/μL (12). In fact, patients with hematologic malignancies have superseded HIV-infected patients as the population with the highest incidence of PjP (12). Together with the increasing incidence of PjP in immunosuppressed patients and that treatment of this oftentimes fatal pneumonia is complicated by life-threatening allergies, toxicity, and only moderate efficacy (13), there is a real need for development of novel therapies and prevention strategies. The use of natural compounds and their synthetic counterparts have been increasingly reported in peer-reviewed literature and have gained favor because of their reduced side effects. Besides wound healing, natural compounds have been used in supportive treatment of cancer (14), to treat damaged nerves (15), and as anti-fibrotic agents (16). Thus, A3IS may hold promise as a novel treatment for Pneumocystis pneumonia.

Based on the in vitro activity shown in the present study, transition to pre-clinical evaluations should be feasible as A3IS can be administered as a gel (external settings such as skin or nails), a powder, or a spray, the latter two being more appropriate for systemic fungal infections with the advantage of a spray to target lung infections. The cytotoxicity assays indicate very low cytotoxic effects, supporting the feasibility of the transition.

A3IS.

The test product, A3IS batch number N675, was supplied in 25g squeezable aluminum tubes from Nektr Technologies (County Sligo, Ireland). The concentration of glucose oxidase was 5,000 μg/mL.

In vitro antifungal activity.

Testing for antifungal activity was performed by broth dilution methods as described in the Clinical and Laboratory Standards Institute (CLSI) M27 (17) and M38 (18) reference standards. A broad range of different fungal species known to cause invasive disease in humans were used. These included yeasts, molds (both hyaline and dematiaceous), and dimorphic fungi. Several were ATCC isolates used by CLSI as quality controls for antifungal susceptibility testing. Others were strains routinely used by various laboratories in the study of fungal pathogenesis and antifungal resistance, while many were clinical strains received by our reference laboratory for clinical diagnostic testing with a broad spectrum of susceptibility profiles, including many that are resistant to clinically available antifungals. This was done to test the range of antifungal activity of A3IS and activity against clinical isolates refractory to antifungal therapy. Subcultures were prepared on either Sabouraud’s dextrose agar (for yeasts) or potato flake agar (for molds) prior to in vitro testing. Suspensions of each isolate were prepared in sterile saline and further dilutions were prepared in RPMI 1640 growth medium (without sodium bicarbonate, with phenol red and 0.2% wt/vol glucose) buffered with 0.165M 3-(N-morpholino) propanesulfonic acid (MOPS) to pH 7.0. Stock solutions of A3IS were prepared by dissolving the compound in sterile distilled water at 10× concentrations of the final concentrations to be tested, with further 1:5 dilutions prepared in RPMI 1640. Stock solutions of the clinically available antifungals fluconazole, voriconazole, and posaconazole were prepared in DMSO at 100× concentrations of those to be tested, and further 1:50 dilutions were then prepared in RPMI 1640. RPMI 1640 media and all chemicals for susceptibility testing were purchased from Sigma-Aldrich (St. Louis, MO). The final concentration of DMSO in the assay was 1% vol/vol. The agents were then dispensed into either 96-well cell culture trays or 12 × 75 mm test tubes at 2× concentrations (Thermo Fisher Scientific, Waltham, MA), which were then inoculated 1:2 with the prepared fungal suspensions. Final concentrations of the fungal isolates ranged from 0.5 to 2 × 10³ cells/mL for yeasts and 0.5 to 2 × 10⁴ cells/mL for molds. The final concentrations of the agents tested were 0.02% to 10% wt/vol for A3IS, 0.125 to 64 μg/mL for fluconazole, and 0.03 to 16 μg/mL for voriconazole and posaconazole. MICs for A3IS were read visually at 50% and 100% growth inhibition after 24 to 72 h of incubation, depending upon the fungal species, at 50% inhibition of growth for fluconazole, and at 100% inhibition of growth for voriconazole and posaconazole, as recommended by CLSI.

In vitro anti-Pneumocystis activity.

A31S was diluted directly into culture media at dilutions of 20, 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039, 0.02, 0.01, and 0.005% (wt/vol). Setup was performed in a BSLII Cabinet and the compound was added within 30 minutes of dilutions. Culture media consists of RPMI 1640 (Gibco, Grand Island, NY) containing 20% fetal bovine serum (HyClone, South Logan, UT), 1% MEM vitamin solution (Gibco, Grand Island, NY), 1% MEM NEAA (Gibco, Grand Island, NY), 200 units/mL penicillin, and 0.2 mg/mL streptomycin (Gibco, Grand Island, NY). Negative controls were media alone and with 10 μg/mL ampicillin. Positive control was 1 μg/mL pentamidine isethionate (Sigma-Aldrich, St. Louis, MO).

(i) Pneumocystis ATP assay. Cryopreserved P. carinii (Pc) isolated from rat lung tissue and P. murina (Pm) isolated from mouse lung tissue were distributed into triplicate wells of 48-well plates with a final volume of 500 μL and a final concentration of 2.5 × 10⁷ nuclei/mL Pc and 2.5 × 10⁶ nuclei/mL Pm. Controls and compound dilutions were added and incubated at 36°C, 5% CO₂. At 24, 48, and 72 h, 10% of the well volume was removed and the organisms were lysed with a proprietary lysis solution that inactivates the endogenous ATPases (by raising the pH via a 0.1M alkaline solution), then suspended in a substrate buffer solution containing recombinant firefly luciferase and its substrate d-luciferin (Perkin Elmer ATPlite kit, Waltham, MA). ATP drives light emission in a linear fashion. This assay is extremely sensitive; most luminometers can detect as little as 0.1 picomole of ATP. The luminescence generated by the ATP content of the samples was measured at 560 nm by a BioTek Synergy HTX (Winooski, VT) spectrophotometer. A sample of each group was examined microscopically on the final assay day to rule out the presence of bacteria.

(ii) Calculations. A3IS was run in triplicate at each concentration for both P. carinii and P. murina. Background luminescence was subtracted, triplicate well readings were averaged, and standard deviation was determined. Using EXCEL software, % reduction in ATP for all groups was calculated (experimental/vehicle control ×100 – vehicle control). 50% Inhibitory Concentrations (IC₅₀) were calculated using the GraphPad Prism software nonlinear fit of log-dose vs response.

Cytotoxicity assays.

Cytotoxicity assays were performed by an accredited external laboratory (No. 1206.3, Expertise No. 081143), under the National Institute of Public Health, Prague. BALB/c 3T3 mouse fibroblasts were seeded into individual wells of 96-well microtiter plates at a density of 1 × 10⁴ cells in Dulbecco’s Minimum Essential Medium (DMEM) (Sevapharma, Prague) with 10% inactivated calf serum. After 24 h preincubation at 37°C, 7.5% CO₂, the medium was removed and replaced with the test agent (A3IS) diluted in DMEM without serum at different concentrations of glucose oxidase from 10% to 0.01% (1 μg/mL to 10,000 μg/mL). Dodecylsulphate sodium salt (SDS) (Sigma-Aldrich, Prague) at concentrations of 1, 10, and 20 μg/mL was used as a positive control, and culture medium without serum served as the cell control. All test samples and controls were run in quadruplicates. At the end of 24 h, the medium was removed, and the cells were stained with Neutral Red dye according to INVITTOX Protocol No. 46 (https://norecopa.no/norina/invittox) (0.2 mL Neutral Red solution per well, 3 h incubation, desorb solution-ethanol/acetic acid). The Neutral Red uptake was measured fluorometrically (530 nm excitation, 590 nm emission). The Fluorescent Units, FSU, were compared to the untreated control wells and converted to a percentage. Viability was ranked as follows: viability of 80% or higher, no cytotoxicity; viability higher or equal to 60% and lower than 80%, mild cytotoxicity; viability higher or equal to 40% and lower than 60%, moderate cytotoxicity; viability below 40%, severe cytotoxicity.