A New Nitroimidazole-Based Drug Attenuates Skeletal Myositis and Overcomes Benznidazole Resistance in Experimental Chagas Disease

Figures

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  1. Chemical reactions used to synthesize the novel nitroimidazole-based drug 1-(2-(2-methoxy-6-nitro-4-propylphenoxy)­ethyl)-2-methyl-5-nitro-1H-imidazole [3] investigated alone and combined with benznidazole against T. cruzi in vitro and in vivo. [1] 2-Methyl-5-nitroimidazole-1-ethanol and [2] 4-propyl guaiacol.

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  2. Chemical characterization of the novel nitroimidazole-based drug 1-(2-(2-methoxy-6-nitro-4-propylphenoxy)­ethyl)-2-methyl-5-nitro-1H-imidazole (ME) by nuclear magnetic resonance (1H NMR).

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  3. Liquid chromatography–mass spectrometry (LCMS ESI/Q-TOF) profile of the novel nitroimidazole-based drug 1-(2-(2-methoxy-6-nitro-4-propylphenoxy)­ethyl)-2-methyl-5-nitro-1H-imidazole (ME).

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  4. Predictive physicochemical characteristics of the novel nitroimidazole-based drug 1-(2-(2-methoxy-6-nitro-4-propylphenoxy)­ethyl)-2-methyl-5-nitro-1H-imidazole (ME) compared to the reference trypanocidal drug benznidazole (BZ). The pink zone in the bioavailability radar is the suitable physicochemical space for oral bioavailability based on the optimal range for lipophilicity, size, polarity, insolubility, instauration, and flexibility.

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  5. Effect of the novel nitroimidazole-based drug 1-(2-(2-methoxy-6-nitro-4-propylphenoxy)­ethyl)-2-methyl-5-nitro-1H-imidazole (ME) and benznidazole (BZ) treatment on parasite viability (A,D), host cells infection rate (B,E), and cell parasitism (C,F) in trypomastigotes and C2C12 skeletal myocytes in vitro. Data are expressed as mean and standard deviation. Statistical difference among the groups (P < 0.05), compared to * infected untreated (IU), φ 2.4 μM, † 2.4 and 4.8 μM, # 2.4, 4.8, and 9.6 μM, ‡ 2.4, 4.8, 9.6, and 19.21 μM in BZ-treated parasites/cells; and compared to * IU, † 1.72, and 3.43 μM, # 1.72, 3.43, and 6.86 μM, and ‡ 1.72, 3.43, 6.86, and 13.73 μM in ME-treated parasites/cells.

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  6. Blood (A) and skeletal muscle (B) parasitism in T. cruzi-infected mice untreated and treated with the novel nitroimidazole-based drug 1-(2-(2-methoxy-6-nitro-4-propylphenoxy)­ethyl)-2-methyl-5-nitro-1H-imidazole (ME) alone and combined with benznidazole (BZ). Groups: UN = uninfected, INF = infected untreated, ME = infected treated with 100 mpk ME, BZ = infected treated with 100 mpk Bz, ME1 + BZ = infected treated with 50 mpk ME + 100 mpk Bz, and ME2 + BZ = infected treated with 100 mpk ME + 100 mpk Bz. Data are expressed as median (A) and mean and standard deviation (B, each point indicates individual values). Statistical difference among the groups (P < 0.05), compared to * INF, † ME, ‡ ME, BZ, and ME1 + BZ.

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  7. Microscopic images of the skeletal muscle from uninfected and T. cruzi-infected mice, untreated and treated with the novel nitroimidazole-based drug 1-(2-(2-methoxy-6-nitro-4-propylphenoxy)­ethyl)-2-methyl-5-nitro-1H-imidazole (ME) alone and combined with benznidazole (BZ) (hematoxylin and eosin staining, bright field microscopy, scale bars = 50 μm). Groups: UN = uninfected, INF = infected untreated, ME = infected treated with 100 mpk ME, BZ = infected treated with 100 mpk Bz, ME1 + BZ = infected treated with 50 mpk ME + 100 mpk Bz, and ME2 + BZ = infected treated with 100 mpk ME + 100 mpk Bz. Arrow = T. cruzi nest. Arrowhead = interstitial space. Sm= skeletal myocyte. II = inflammatory infiltrate.

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  8. Interstitial cellularity (A), parenchyma and stroma distribution (B), myocytes thickness (C), and cross-sectional area (CSAD) in the skeletal muscle from T. cruzi-infected mice untreated and treated with the novel nitroimidazole-based drug 1-(2-(2-methoxy-6-nitro-4-propylphenoxy)­ethyl)-2-methyl-5-nitro-1H-imidazole (ME) alone and combined with benznidazole (BZ). Groups: UN = uninfected, INF = infected untreated, ME = infected treated with 100 mpk ME, BZ = infected treated with 100 mpk Bz, ME1 + BZ = infected treated with 50 mpk ME + 100 mpk Bz, and ME2 + BZ = infected treated with 100 mpk ME + 100 mpk Bz. Data are expressed as mean and standard deviation (each point indicates individual values). Statistical difference among the groups (P < 0.05), compared to * UN, # INF, † ME, ‡ ME, BZ, and ME1 + BZ.

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  9. Reactive oxygen species (ROSA) and nitric oxide (B) levels in the skeletal muscle from T. cruzi-infected mice untreated and treated with the novel nitroimidazole-based drug 1-(2-(2-methoxy-6-nitro-4-propylphenoxy)­ethyl)-2-methyl-5-nitro-1H-imidazole (ME) alone and combined with benznidazole (BZ). Groups: UN = uninfected, INF = infected untreated, ME = infected treated with 100 mpk ME, BZ = infected treated with 100 mpk Bz, ME1 + BZ = infected treated with 50 mpk ME + 100 mpk Bz, and ME2 + BZ = infected treated with 100 mpk ME + 100 mpk Bz. RFU = relative fluorescence units. Data are expressed as mean and standard deviation (each point indicates individual values). Statistical difference among the groups (P < 0.05), compared to * UN, # INF, † ME, ‡ ME, BZ, and ME1 + BZ.

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  10. Advanced oxidation protein products (chloramine T equivalentsChlor T, A), protein carbonyl (PCN, B), and lipid hydroperoxides in the skeletal muscle from T. cruzi-infected mice untreated and treated with the novel nitroimidazole-based drug 1-(2-(2-methoxy-6-nitro-4-propylphenoxy)­ethyl)-2-methyl-5-nitro-1H-imidazole (ME) alone and combined with benznidazole (BZ). Groups: UN = uninfected, INF = infected untreated, ME = infected treated with 100 mpk ME, BZ = infected treated with 100 mpk Bz, ME1 + BZ = infected treated with 50 mpk ME + 100 mpk Bz, and ME2 + BZ = infected treated with 100 mpk ME + 100 mpk Bz. Data are expressed as mean and standard deviation (each point indicates individual values). Statistical difference among the groups (P < 0.05), compared to * UN, # INF, † ME, ‡ ME, BZ, and ME1 + BZ.

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  11. Myeloperoxidase (MPOA) and N-acetyl-β-d-glucosaminidase (NAG, B) activity in the skeletal muscle from T. cruzi-infected mice untreated and treated with the novel nitroimidazole-based drug 1-(2-(2-methoxy-6-nitro-4-propylphenoxy)­ethyl)-2-methyl-5-nitro-1H-imidazole (ME) alone and combined with benznidazole (BZ). Groups: UN = uninfected, INF = infected untreated, ME = infected treated with 100 mpk ME, BZ = infected treated with 100 mpk Bz, ME1 + BZ = infected treated with 50 mpk ME + 100 mpk Bz, and ME2 + BZ = infected treated with 100 mpk ME + 100 mpk Bz. Data are expressed as mean and standard deviation (each point indicates individual values). Statistical difference among the groups (P < 0.05), compared to * UN, # INF, † ME, ‡ ME, BZ, and ME1 + BZ.

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  12. Interferon gamma (IFN-γA), tumor necrosis factor (TNFB), interleukin 6 (IL-6C), and interleukin 10 (IL-10D) levels in the skeletal muscle from T. cruzi-infected mice, untreated, and treated with the novel nitroimidazole-based drug 1-(2-(2-methoxy-6-nitro-4-propylphenoxy)­ethyl)-2-methyl-5-nitro-1H-imidazole (ME) alone and combined with benznidazole (BZ). Groups: UN = uninfected, INF = infected untreated, ME = infected treated with 100 mpk ME, BZ = infected treated with 100 mpk Bz, ME1 + BZ = infected treated with 50 mpk ME + 100 mpk Bz, and ME2 + BZ = infected treated with 100 mpk ME + 100 mpk Bz. Data are expressed as mean and standard deviation (each point indicates individual values). Statistical difference among the groups (P < 0.05), compared to * UN, # INF, † ME, ‡ ME, BZ, and ME1 + BZ.