The synthetic synergistic cinnamon oil CIN-102 is active against Madurella mycetomatis, the most common causative agent of mycetoma

Abstract

Mycetoma is a devastating neglected tropical infection of the subcutaneous tissue and most commonly caused by the fungus Madurella mycetomatis. Treatment of mycetoma consists of a combination of a long term antifungal treatment with itraconazole and surgery. However, treatment is associated with low success rates. Therefore, there is a need to identify novel treatments for mycetoma. CIN-102 is a synthetic partial copy of cinnamon oils with activity against many pathogenic bacteria and fungi. In this study we determined the in vitro activity of CIN-102 against 21 M. mycetomatis isolates and its in vivo efficacy in a M. mycetomatis infected Galleria mellonella larval model. In vitro, CIN-102 was active against M. mycetomatis with MICs ranging from 32 μg/mL to 512 μg/mL. 128 μg/mL was needed to inhibit the growth in 50% of tested isolates. In vivo, concentrations below the MIC of 40 mg/kg and 80 mg/kg CIN-102 prolonged larval survival, but higher concentrations of CIN-102 did not.

Fig 1. Antifungal susceptibility of 21 M. mycetomatis isolates to CIN-102 and itraconazole expressed as MICs, determined by the XTT assay.

Fig 2. In vivo efficacy and toxicity of different dosages CIN-102 and the used treatment dosage itraconazole within G. mellonella larvae, compared to the solvent (PBS 10% tween80).

Dotted lines indicate uninfected larvae as toxicity controls, which were treated at the same time points as the infected groups. It is observed that none of the shown dosages effect the survival of uninfected larvae. The full lines indicate infected larvae treated with the corresponding dosages 4-, 28- and 52-hours post-infection. Larvae treated with solvent and itraconazole survive up until day 8, while larvae treated with 40 mg/kg and 80 mg/kg survive up until day 10 and significantly differ from the PBS control (*p = 0.0133**p = 0.0091).

Fig 3

The total number and size of grains in M. mycetomatis infected G. mellonella larvae treated with solvent (black dots), 80 mg/kg CIN-102 (grey dots), or 5.71mg/kg itraconazole (brown dots). Total number of grains counted in CIN-102 treated larvae do not differ at both one- and three-days post-infection (Man-Whitney, p = 0.39 and p = 0.57 respectively). The same is found for total grain size, for which no difference is found between CIN-102 treated larvae and the control, both one- and three-days post-infection (Man-Whitney, p = 0.31 and p = 0.69 respectively). Total number of grains counted in itraconazole treated larvae differed both one- and three-days post-infection (Man-Whitney, p = 0.01 and p = 0.05 respectively). Similarly, the total grain size differed between itraconazole and the control in both one- and three-days post-infection (Man-Whitney, p = 0.03 and p = 0.22 respectively).

Fig 4. M. mycetomatis grains in Galleria mellonella larvae, 200x magnification.

A: M. mycetomatis grain 24h after infection and treated with solvent. Cement material (CM) is present. No clear capsule is formed surrounding this grain. Some individual hemocytes (H) are present in the cement material. B: M. mycetomatis grain 24h after infection and treated with 80mg/kg CIN-102. Cement material is present. A capsule (C) is surrounding the grain, individual hemocytes are found in the grain. C: M. mycetomatis grain 24h after infection and treated with 5.71 mg/kg itraconazole (ITZ). Partial formation of cement material and encapsulation of the grain are observed. Individual hemocytes (H) are present in the cement material. D: M. mycetomatis grain 72h after infection and treated with solvent. Dense cement material seen, no individual hemocytes inside the grain. A capsule is surrounding the grain. E: M. mycetomatis grain 72h after infection and treated with 80mg/kg CIN-102. Cement material is less dense compared to the control (panel C). Capsule is surrounding the grain. Some large hemocytes (LH) are seen surrounding the grain and is not seen in solvent or itraconazole treated larvae. F: M. mycetomatis grain 72h after infection and treated with 5.71 mg/kg itraconazole (ITZ). Both cement material (CM) and encapsulation (C) of the grain are observed.