The Activity of 2,3-Dimethylquinoxaline against Madurella mycetomatis

Objective: Madurella mycetomatis is the most prevalent causative species of the eumycetoma, a chronic progressive neglected tropical disabling infectious disease of the subcutaneous tissue. It carries a significant social and economic burden to the endemic areas. Unfortunately, the available treatment is challenging, requires prolonged therapy, resulting in low cure rates and a high incidence of recurrence. Screening new compounds for antifungal activity against Madurella mycetomatis is needed. Design: In vitro : the susceptibility of a Madurella mycetomatis isolate toward the 2,3-dimethylquinoxaline was determined. In vivo: the efficacy of 2,3-dimethylquinoxaline 1% topical gel against the same strain was evaluated in the BALB/c mouse eumycetoma model (n=6; 6-8 weeks old). Results: 2,3-Dimethylquinoxaline inhibited the growth of Madurella mycetomatis at MIC 312 µg/ml (1.9 mM). The infected mouse neck’s granuloma disappeared after 14 days of the treatment with 2,3-dimethylquinoxaline 1 % gel. Conclusion: 2,3-Dimethylquinoxaline showed good antifungal activity and worth further optimization towards developing a new eumycetoma treatment.


Introduction
Mycetoma is a chronic progressive inflammatory disease [1]. It is classified as either eumycetoma caused by fungi or actinomycetoma caused by bacteria [1]. Mycetoma was first reported in Madura's Indian town and was initially called Madura foot [2]. It commonly affects young adults in developing countries after a traumatic inoculation of the causative microorganism into subcutaneous tissue. Mycetoma is endemic in tropical and subtropical areas at the "mycetoma belt," which includes ten countries distributed over three continents [3]. It commonly involves the extremities, gluteal, and back region. It is characterized by a painless mass with multiple discharge grains. It often spreads to involve deep structures and bone, resulting in deformity and loss of function [1].
Madurella mycetomatis is the most common causative species of eumycetoma and accounts for 70% of infections [4]. Morphologic features of grains associated with Madurella mycetomatis are black, hard, and relatively large in diameter (1-2 mm) [5]. Clinical improvement is judged by mass size reduction, while the cure is considered when the mass disappears and confirmed clinically, cytologically, and radiologically [6].
Treatment of eumycetoma should be started as early as possible to avoid amputation and disability [7]. Unfortunately, there is a minimal option for eumycetoma management [7].
The current antifungal therapy mainly helps to localize the disease for easily surgical excision. Itraconazole replaced ketoconazole as the standard gold therapy after concerns of adrenal insufficiency and liver injury [9,10]. Posaconazole was found to be a clinically successful antifungal therapy for refractory cases (Table 1) [11]. Eumycetoma with medium to large lesions should be treated with Itraconazole for six months pre-surgical excision, followed by a minimum of further six months until there is clear evidence of cure.

Ketoconazole
Not recommended since 2013 The FDA limits ketoconazole usage due to potentially fatal liver injury, risk of drug interaction, and adrenal gland problems.
Voriconazole; Posaconazole Second line Few cases report shown a good response with mixed results.

Fosravuconazole
Promising new line It is currently undergoing clinical trials (NCT03086226).

Fluconazole
Poor activity Not effective and has no place in therapy.
Anidulafungin, Caspofungin, Micafungin Poor activity Not effective and has no place in therapy.

Amphotericin B Poor outcome
It is associated with significant adverse effects and a high relapse rate.

Add to Itraconazole in resistant cases
Terbinafine alone showed lower cure rates than Itraconazole.
The current treatment is unsatisfactory and involves long-term antimicrobial therapy and aggressive surgical excisions [12]. Drug discovery and development often create additional challenges for being a neglected tropical disease. Efforts are needed to discover a new treatment for Madurella mycetomatis. There is a need for an affordable and more effective therapy for endemic developing countries.

DMQ 1% topical gel formulation
All chemicals were of analytical grade. DMQ was obtained from

In vitro susceptibility testing
Microdilution assay was used as described previously to determine the susceptibility of Madurella mycetomatis towards 2,3-dimethylquinoxaline [20]. The isolate was cultured in RPMI1640 medium (Gibco) with MOPS (Sigma) at 37˚C for 7 days, then harvested by centrifugation, homogenized by sonication, and adjusted to obtain transmission of 70% at 660nm.
2,3-Dimethylquinoxaline was tested at concentrations range from 4.8 µg/ml to 2.5 mg/ml (0.03 to 15.8 mM). The assay were perfomed in duplicate and repeated in triplicate.
All infected mice (n=6) with Madurella mycetomatis developed a well-defined granuloma on their necks ( Figure 1A). For all treated mice (n=3) with DMQ 1 % gel for 14 days, the granuloma disappeared, leaving a mild scar ( Figure 1B). A) The granuloma developed in infected mouse model.
B) The improvement after treatment with DMQ 1% gel.
Madurella mycetomatis known to produce translationally controlled tumor protein (TCTP) [23]. This protein has a vital role in controlling the cell cycle and stress response [24]. TCTP gene expression is considered a virulent factor with an anti-apoptotic effect that stabilizes cells from programmed cell death. Quinoxalines have been shown to induce programmed cell death in treated cells [25]. Further studies are essential to explicate how precisely fungal cell death happened and how other antifungal drugs can interact for possible enhancement of the treatment outcome.
Minimal reports addressed DMQ pharmacology and pharmacokinetic. The current application of DMQ is in the laboratory to determine the amount of diacetyl in foods and beverages [26]. Few reports indicate that DMQ is a competitive hepatic P-450 enzyme inducer [27,28].
Given the properties and uses of approved quinoxalines, one of their most prominent characteristics is their ability to reach target tissues at an appropriate concentration (Table 2) [29]. In contrast, amphotericin B has minimal or no value in Madurella mycetomatis, although it has excellent in vitro activity, explained by the limited drug distribution into the infected tissues [30]. The results obtained from this study revealed a promising activity of DMQ against Madurella mycetomatis and merited further optimization. The antifungal effects probably rely on a new mechanism of action.

Disclosure Conflict of interest
No conflict of interest associated with this work.