Aminoguanidines: New leads for treatment of Giardia duodenalis infection

Abstract

Giardia duodenalis is an ubiquitous parasitic pathogen that causes significant morbidity and mortality worldwide. Failures in drug therapy are commonly due to poor patient compliance as a result of the need for repeated administration, off target drug effects and increasing parasite drug resistance. In this study the in vitro efficacy and selectivity of the aminoguanidine compound robenidine and 2 structural analogues against Giardia were determined. After 5 h exposure to each compound the IC₅₀ was as low as 0.2 μM with corresponding MLCs as low as 2.8 μM. This is in contrast to metronidazole which required 24 h to exhibit inhibitory activity. A modified adherence assay, developed for this study, demonstrated that three of the compounds inhibited in vitro adherence of the parasite. The lead compound exhibited rapid giardicidal activity (<5hr). In addition, microscopy studies demonstrated damage to the plasma membrane of trophozoites. In conclusion, a class of aminoguanidines, represented by robenidine, has shown antigiardial activity warranting further investigation.

Keywords: Adherence; Antigiardial; Drug development; Giardia duodenalis; Giardiasis; Giardicidal.

Graphical abstract

Fig. 1

Chemical structures of the four compounds investigated in this study. A. robenidine; B. NCL 062; C. NCL 099; D. Metronidazole; and E. Albendazole.

Fig. 2

Metabolic inhibitory activity of robenidine, NCL 062 and NCL 099 against Giardia duoenalis BAH2c2 strain, assemblage A. Trophozoites were exposed to the compounds for 5 h before removal of the compounds and addition of resazurin. Absorbance was measured (630 and 570 nm), percent reduction of resazurin calculated and data presented as a percentage of the growth control. Treatment groups are: square - metronidazole; triangle –NCL 099; diamond - robenidine; and circle - NCL 062. Error ± SEM, n = 3. Data represents a typical experiment. Note the break in the x-axis indicates a change in the scale of drug concentration.

Fig. 3

Inhibition of adherence of Giardia duodenalis by metronidazole (Mz), robenidine, NCL 062 and NCL 099. Cells were exposed to the compounds for 5 h before staining. Stained cells were imaged at 10x magnification and counted using DotCount™ software. Each assay was completed in triplicate and 5 images taken per sample. Top. Number of adherent trophozoites after exposure to 4 μM of each compound for 5 h; Bottom. An example of the Dotcount software identifying trophozoites at higher magnification. Left: stained trophozoites, Right: highlighted trophozoites identified by the Dotcount software. Each highlighted cell is counted individually. Error ± SEM. * p-value <0.05, ** p-value <0.0001.

Fig. 4

Morphological effect of Robenidine and NCL 099 on Giardia trophozoites. Giardia trophozoites were exposed to each compound for 24 h before fixation with glutaraldehyde and staining following the Diff quik protocol. A-control cells, B – cells treated with metronidazole (IC₅₀, 2 μM), C- robendine treated cells (IC₅₀, 2 μM), D – NCL 099 treated cells (IC₅₀, 3 μM), E − NCL 099 treated cells (2 x IC₅₀).

Fig. 5

Transmission Electron Microscopy of Giardia duodenalis trophozoites: A control, and after exposure to B metronidazole for 4 h (3 x IC₅₀) or C-E robenidine for 1 h (3 x IC₅₀). Membrane blebbing at the adhesive disc can be seen in robenidine treated cells (arrows) as well as membranous structures within the cytoplasm (arrow head), disintegration of the cytoplasmic space and rupturing of the cell membrane (star). Images taken with a FEI tecnai G2 Spirit Transmission Electron microscope.

Fig. 6

Scanning electron microscopy of Giardia duodenalis trophozoites: A control ventral, B control dorsal, C exposed to robenidine 2 h (3 x IC₅₀) ventral, D exposed to robenidine 4 h (3 x IC₅₀) dorsal. Multiple ruptures as well as blebbing at the adhesive disk can be seen on the ventral surface (C) while tiny blebbing and rupturing of the dorsal surface can be seen in D. E metronidazole exposed cells (3 x IC₅₀) 4 h, dorsal.

Fig. 7

Recovery assay of Giardia duodenalis exposed to robenidine and metronidazole. Giardia trophozoites were exposed to robenidine or metronidazole for 5 h at 5x the IC₅₀. Key: closed circle – growth control, triangle – metronidazole, square – robenidine, open circle – NCL 062, diamond – NCL 099. Cell numbers were determined at 24 and 48 h post exposure. Error ± SD.