The In Vitro Impact of Isoxazole Derivatives on Pathogenic Biofilm and Cytotoxicity of Fibroblast Cell Line

Abstract

The microbial, biofilm-based infections of chronic wounds are one of the major challenges of contemporary medicine. The use of topically administered antiseptic agents is essential to treat wound-infecting microorganisms. Due to observed microbial tolerance/resistance against specific clinically-used antiseptics, the search for new, efficient agents is of pivotal meaning. Therefore, in this work, 15 isoxazole derivatives were scrutinized against leading biofilm wound pathogens Staphylococcus aureus and Pseudomonas aeruginosa, and against Candida albicans fungus. For this purpose, the minimal inhibitory concentration, biofilm reduction in microtitrate plates, modified disk diffusion methods and antibiofilm dressing activity measurement methods were applied. Moreover, the cytotoxicity and cytocompatibility of derivatives was tested toward wound bed-forming cells, referred to as fibroblasts, using normative methods. Obtained results revealed that all isoxazole derivatives displayed antimicrobial activity and low cytotoxic effect, but antimicrobial activity of two derivatives, 2-(cyclohexylamino)-1-(5-nitrothiophen-2-yl)-2-oxoethyl 5-amino-3-methyl-1,2-oxazole-4-carboxylate (PUB9) and 2-(benzylamino)-1-(5-nitrothiophen-2-yl)-2-oxoethyl 5-amino-3-methyl-1,2-oxazole-4-carboxylate (PUB10), was noticeably higher compared to the other compounds analyzed, especially PUB9 with regard to Staphylococcus aureus, with a minimal inhibitory concentration more than x1000 lower compared to the remaining derivatives. The PUB9 and PUB10 derivatives were able to reduce more than 90% of biofilm-forming cells, regardless of the species, displaying at the same time none (PUB9) or moderate (PUB10) cytotoxicity against fibroblasts and high (PUB9) or moderate (PUB10) cytocompatibility against these wound cells. Therefore, taking into consideration the clinical demand for new antiseptic agents for non-healing wound treatment, PUB9 seems to be a promising candidate to be further tested in advanced animal models and later, if satisfactory results are obtained, in the clinical setting.

Keywords: Michael addition; Passerini reaction; anti-bacterial activity; antiseptics; cytotoxicity; isoxazole.

Figure 1

Designed isoxazole derivatives ((A) MAL1-5 series; (B) PUB1-10 series: R1-aldehyde/ketone residue; R2-isocyanide residue).

Figure 2

Synthesis of isoxazole linked maleimide conjugates (MAL1-5).

Figure 3

The [%] biofilm reduction of S. aureus 6538, P. aeruginosa 15,442 and C. albicans 103,231 displayed by PUB9 and PUB10 compounds at specific concentrations tested in microplate model. The red line shows level of 100% reduction while no reduction (0%, positive control of growth) was established for non-treated cells (maximal cellular growth).

Figure 4

The survivability [%] of L929 fibroblasts exposed to the spectrum of concentrations of PUB9 and PUB10 compounds. The asterisks show significant (p > 0.05, ANOVA test with Tukey’s multiple comparison test) difference in survivability between control setting (non-treated cells, considered 100% growth) and cells treated with 0.78–1.56 mg/mL of PUB9 or PUB10 as well as 0.20 mg/mL of PUB10.

Figure 5

The inhibition of staphylococcal growth zone resulting from release of PUB9 from BC carrier in modified disk-diffusion model. The diameter of BC carrier is 18 mm.

Figure 6

Comparison of cytocompatibility of isoxazole derivatives-fortified cellulosic carriers: PUB9-BC and PUB10-BC compared to control setting (cellulose carrier containing no isoxazole derivative) toward fibroblast cell line. Asterisks indicate significant differences (p < 0.05, ANOVA Test with Tukey’s multiple comparison test) between BC vs. PUB10-BC and PUB9-BC vs. PUB10-BC. The red line indicates the maximal potential cellular growth (of non-fortified cellulosic carrier).