Anti-Pythium insidiosum activity of three novel triazole compounds: synthesis, pharmacokinetic and toxicological parameters

Abstract

Pythiosis, caused by Pythium insidiosum, is an infectious and non-transmissible disease affecting horses, dogs, and humans, with no effective drug treatment available. Triazoles are compounds of interest for their potential pharmacological properties against fungi and bacteria. In this study, we synthesized three new triazole compounds (C1, C2, and C3) to assess their in vitro activities against P. insidiosum and their safety on human leukocytes. Susceptibility testing was performed against P. insidiosum isolates (n = 15) to determine the minimum inhibitory concentration (MIC) and minimum oomicidal concentration (MOC). The leukocyte toxicity of triazoles was evaluated by measuring cell viability, morphological aspects, and oxidative stress endpoints. In silico prediction of the compounds absorption, distribution, metabolism, excretion and toxicity (ADMET) was determined using the pkCSM platform. Both triazoles C1 and C2 exhibited anti-Pythium insidiosum activity at concentrations from 2 to 64 µg/mL to MIC and MOC, while C3 MIC was 4-64 µg/mL and MOC 8-64 µg/mL. The three compounds did not induce viability loss and/or morphologic changes to human leukocytes, and showed absence of a pro-oxidant profile. ADMET properties prediction of the compounds was similar to the reference drug fluconazole. This study introduces novel triazole compounds exhibiting anti-P. insidiosum activity at concentrations non-toxic to human leukocytes.

Keywords: Azoles; Human leukocytes; Pythiosis; Susceptibility tests; Toxicity.

Fig. 1

Synthesis reaction of 1,2,3-triazoles-1,4-disubstituted (C1-3)

Fig. 2

Chemical structure of the three new triazole compounds

Fig. 3

Cell viability of human leukocytes exposed to the triazole compounds. Cells were treated with compounds C1 (A), C2 (B) and C3 (C) at concentrations ranging from 0.25 to 100 µM for 3 h at 37 °C. Results are expressed as mean ± SEM of 3 independent experiments (n = 3), and were evaluated by one-way ANOVA, followed by Tukey’s post-test

Fig. 4

Levels of reactive species (RS) and total thiols in human leukocytes exposed to the triazole compounds. RS was determined by flow cytrometry using the marker DHR-123 and total thiols by spectrophotometry using DTNB. Cells were exposed to compounds C1 (A and D), C2 (B and E) and C3 (C and F) at concentrations from 1 to 50 µM for 3 h at 37 ºC. RS levels were expressed as arbitrary fluorescence units and total thiols as % control. Data were analysed by one-way ANOVA, followed by Tukey’s post-test (n = 3 to 4)

Fig. 5

Morphological parameters of human leukocytes exposed to the triazole compounds. Cells were exposed to compounds C1, C2 and C3 in concentrations ranging from 1 to 50 µM for 3 h at 37 ºC. Size (A, B, C) and granularity (D, E, F) by flow cytometry. (G) Representative histograms indicating SSC-and FSC-A of control and treated cells. Results were expressed as arbitrary fluorescence units and analysed by one-way ANOVA followed by Tukey’s post-test (n = 4). 100.000 events were acquired for sample

Fig. 6

Lipid peroxidation. The lipid peroxidation levels were measured by TBARS method, using phosphatidylcholine samples. The samples were incubated in the presence or absence of compounds C1 (A), C2 (B) and C3 (C) at concentrations of 5 to 100 µM, using FeSO₄ as a pro-oxidant agent. Results are expressed as mean ± SEM of 4 independent experiments (n = 4), and the asterisk signifies p < 0.05 when compared with Control by one-way ANOVA, followed by Tukey’s post-test