In Vitro Susceptibilities of the Microsporidia Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis to Albendazole and Its Sulfoxide and Sulfone Metabolites

Abstract

In vitro comparisons demonstrated that the efficacy of albendazole, albendazole-sulfoxide, and albendazole-sulfone against pathogenic Encephalitozoon species was proportional to the degree of oxidation at a concentration of >10⁻³ μg/ml. Furthermore, at a concentration of <10⁻² μg/ml, benzimidazoles were more effective against Encephalitozoon cuniculi and Encephalitozoon hellem than against Encephalitozoon intestinalis.

The microsporidia Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis are emerging obligate intracellular pathogens causing infections in human immunodeficiency virus-infected patients (24). Albendazole has been successfully used in encephalitozoonoses (2, 5, 10, 11, 19, 20). However, incomplete response (25) and relapses have also been documented (15, 23, 26). These clinical investigations have been accompanied by a handful of noncomparative laboratory studies; however, all but one of these (14) have not assessed the role of albendazole-sulfoxide and albendazole-sulfone active metabolites (8). We therefore compared the experimental activities of albendazole, albendazole-sulfoxide, and albendazole-sulfone on Encephalitozoon species in MRC5 cell culture.

The three benzimidazoles (SmithKline Beecham, Nanterre, France) were dissolved at 1 mg/ml in sterile dimethyl sulfoxide (DMSO), and 10-fold serial dilutions (10⁰ to 10⁻⁴ μg/ml) were prepared in minimum essential medium (MEM) (Eurobio, Paris, France). Spores of E. cuniculi, E. hellem, and E. intestinalis strains (kindly provided by T. van Gool, University of Amsterdam, Amsterdam, The Netherlands) were cocultivated with MRC5 embryonic lung fibroblasts (BioMérieux, Lyon, France) in a mixture of MEM, 1% glutamine, and 10% heat-inactivated fetal calf serum (Flow Laboratories, Paris, France) at 35°C in a 5% CO₂ atmosphere. The infection rate was monitored by rapid-heating Gram-chromotrope staining (21) and microscopic counting of spores in a Kova-slide (Hycor Biomedical, Inc., Irvine, Calif.). For antimicrosporidian activity assays, MRC5 cells subcultured at confluence on glass coverslips in a 24-well microplate were incubated with 100 μl of a suspension of 10⁶ spores/ml for 5 h at 37°C. One milliliter of culture medium, with or without benzimidazole, was then added, and the plate was incubated at 37°C in a 5% CO₂ incubator for 6 days. For each plate, a benzimidazole-free positive control and benzimidazole dilutions of 1 to 10⁻⁴ μg/ml were tested. After incubation, the mean and standard deviation of the number of microsporidia per field were determined by microscopic examination of rapidly heated Gram-chromotrope-stained coverslips (20 fields observed). The percentage of microsporidian growth inhibition was calculated as [1 − (mean number of infected cells in replicate cultures with benzimidazole/mean number of infected cells in control cultures)] × 100 (± standard error). The benzimidazole concentration inhibiting 90% of microsporidian growth in a control culture (IC₉₀) was estimated from plots of spore number versus log benzimidazole concentration. Each benzimidazole was tested in triplicate. The potential toxicities of benzimidazole and DMSO on MRC5-cultured cells were examined by using a microplaque colorimetric assay adaptated from that previously reported (22). Analysis of variance was used to compare the median and standard deviation values of optical density in the toxicity test and to compare the percentages of growth inhibition for each microsporidian species and for each concentration of each benzimidazole. The Kruskall-Wallis test was used when variances were not homogeneous according to Bartlett’s test.

DMSO at a final concentration of 10⁻³ μg/ml had no toxic effect on MRC5 cells, whereas a statistically significant, moderate toxic effect was observed with albendazole at 10⁰ to 10⁻⁴ μg/ml, with albendazole-sulfoxide at 10⁰ μg/ml, and with albendazole-sulfone at 10⁰ to 10⁻¹ μg/ml (P < 0.05) (Table 1). Any benzimidazole concentration tested was significantly effective in inhibiting the growth of any of the three Encephalitozoon species (P < 0.0001) (Table 1). The percentage of E. hellem growth inhibition varied from 97.34 to 90.68% for albendazole, from 96.39 to 92.9% for albendazole-sulfoxide, and from 98.78 to 91.73% for albendazole-sulfone. The IC₉₀s were 2.7 × 10⁻⁴ μg/ml for albendazole and <10⁻⁴ μg/ml for albendazole-sulfoxide and -sulfone. The percentages of E. cuniculi growth inhibition varied from 97.29 to 53% for albendazole, from 96 to 75.98% for albendazole-sulfoxide, and from 100 to 71.63% for albendazole-sulfone. The IC₉₀s were 3.3 × 10⁻² μg/ml for albendazole, 6 × 10⁻³ μg/ml for sulfoxide, and 2 × 10⁻³ μg/ml for sulfone. The percentages of E. intestinalis growth inhibition varied from 100 to 29.16% for albendazole, from 100 to 53.43% for albendazole-sulfoxide, and from 98.94 to 50.39% for albendazole-sulfone. The IC₉₀s were 7.1 × 10⁻² μg/ml for albendazole, 3.8 × 10⁻² μg/ml for sulfoxide, and 10⁻¹ μg/ml for sulfone.

TABLE 1.

Intracellular efficacy of albendazole and its sulfoxide and sulfone metabolites on microsporidial growth and estimation of MRC5 host cell viability

Benzimidazole or metabolite	Drug concn (μg/ml)	% Microsporidian growth inhibitiona(Pb)	% Cell viabilityc(Pb)
E. hellem
Albendazole	100	97.34 ± 1.98 (<10−5)	85 ± 2 (<10−3)
	10−1	96.0 ± 1.14 (<10−5)	86 ± 1 (<10−3)
	10−2	94.77 ± 2.61 (<10−5)	92 ± 1.7 (0.045)
	10−3	94.6 ± 4.07 (<10−5)	88 ± 1 (<10−3)
	10−4	90.68 ± 6.98 (<10−5)	92 ± 0.7 (<10−3)
Albendazole-sulfoxide	100	96.39 ± 2.08 (<10−5)	92.5 ± 1.9 (0.024)
	10−1	96.39 ± 1.77 (<10−5)	94 ± 1.5 (NS)
	10−2	96.4 ± 1.71 (<10−5)	94 ± 1 (NS)
	10−3	97.43 ± 1.38 (<10−5)	95 ± 0.8 (NS)
	10−4	92.9 ± 2.54 (<10−5)	96 ± 0.8 (NS)
Albendazole-sulfone	100	98.78 ± 0.66 (<10−5)	90 ± 2 (0.013)
	10−1	98.79 ± 0.37 (<10−5)	92 ± 1.8 (0.042)
	10−2	97.65 ± 0.8 (<10−5)	93 ± 1 (NS)
	10−3	96.54 ± 1.23 (<10−5)	95 ± 0.7 (NS)
	10−4	91.73 ± 4.32 (<10−5)	95 ± 0.6 (NS)
E. cuniculi
Albendazole	100	97.29 ± 1.28 (<10−5)
	10−1	97.83 ± 0.85 (<10−5)
	10−2	97.83 ± 1.35 (<10−5)
	10−3	75.6 ± 2.1 (<10−5)
	10−4	53 ± 15.2 (<10−5)
Albendazole-sulfoxide	100	96.0 ± 2.0 (<10−5)
	10−1	96.0 ± 2.76 (<10−5)
	10−2	96.36 ± 1.7 (<10−5)
	10−3	90.4 ± 3.78 (<10−5)
	10−4	75.98 ± 16.62 (<10−5)
Albendazole-sulfone	100	100 ± 0 (<10−5)
	10−1	100 ± 0 (<10−5)
	10−2	100 ± 0 (<10−5)
	10−3	98.33 ± 0.87 (<10−5)
	10−4	71.63 ± 7.91 (<10−5)
E. intestinalis
Albendazole	100	100 ± 0 (<10−5)
	10−1	99.5 ± 0.4 (<10−5)
	10−2	98.97 ± 0.69 (<10−5)
	10−3	35.42 ± 33.81 (3 × 10−3)
	10−4	29.16 ± 32.51 (6 × 10−3)
Albendazole-sulfoxide	100	100 ± 0 (<10−5)
	10−1	100 ± 0 (<10−5)
	10−2	99.53 ± 0.39 (<10−5)
	10−3	57.99 ± 18.1 (<10−5)
	10−4	53.43 ± 16.91 (<10−5)
Albendazole-sulfone	100	98.94 ± 0.77 (<10−5)
	10−1	97.9 ± 1.2 (<10−5)
	10−2	71.35 ± 6.85 (<10−5)
	10−3	67.73 ± 12.35 (<10−5)
	10−4	50.39 ± 16.47 (<10−5)

^aPercent inhibition = [1 − (mean number of infected cells in replicate cultures with the benzimidazole/mean number of infected cells in control cultures)] × 100 (± standard error). 

^bValues for test and control wells were compared by analysis of variance and the Kruskal-Wallis test. NS, not significant. 

^cThe toxicity of each benzimidazole concentration to uninfected cells was estimated by a colorimetric assay (see Materials and Methods) performed in three replicate wells. Optical density values were compared with those for untreated control cells. 

Statistical analysis indicated that albendazole-sulfoxide and albendazole-sulfone at concentrations >10⁻⁴ μg/ml were significantly (5 to 15 times) more effective than albendazole against E. intestinalis, and albendazole-sulfone was significantly (5 times) more effective than albendazole-sulfoxide (P < 0.05) against any of the three species. E. intestinalis was significantly less susceptible than the two other microsporidian species, regardless of the benzimidazole concentration (P < 0.0001). For benzimidazole concentrations <10⁻² μg/ml, E. cuniculi was significantly less susceptible than E. hellem (P < 0.0001).

The previously unreported toxicity for albendazole and, to a lesser extent, its metabolites, we observed could be due to the fact that the microplaque colorimetric assay we used is highly sensitive as a result of the large number of measurements collected for individual benzimidazole concentrations. This moderate toxic effect had a minimal effect on the IC₉₀ determinations. Previous IC determinations of albendazole activity against Encephalitozoon spp. were 2.5 to 0.008 μg/ml for E. cuniculi (1, 4, 9, 16, 27), 0.005 to 0.008 μg/ml for E. intestinalis (3, 14), and 0.008 μg/ml for E. hellem (4). The higher ICs we observed may have resulted from the experimental model we used, which included simultaneous inoculation and treatment; previously, albendazole had been added to well-established cultures. We found E. intestinalis to be less susceptible to albendazole and its two major derivatives than E. hellem and E. cuniculi. The new data were obtained as the three Encephalitozoon species were tested in parallel. Previous studies have included only one species, thus preventing the accurate comparison of interspecies susceptibilities. Albendazole acts by disrupting microsporidian microtubules through β-tubulin binding (17). Even if six β-tubulin residues identified as being predictive for benzimidazole susceptibility in parasites (13) exhibited a sequence predictive of susceptibility in E. cuniculi, E. hellem (7), and E. intestinalis (6), divergences in primary sequences may support differences in susceptibility. Alternative hypotheses include E. intestinalis-related altered intracellular penetration or metabolism of albendazole. We also confirmed that albendazole-sulfone is significantly more effective against the Encephalitozoon species than albendazole-sulfoxide, which in turn is more effective than albendazole. E. intestinalis has previously been found to be 1.7 times more susceptible to albendazole-sulfoxide than to albendazole (14), but no data have been presented for albendazole-sulfone. Indeed, in patients treated with oral albendazole, albendazole and albendazole-sulfone remained undetectable (i.e., <0.02 μg/ml) (18), whereas albendazole-sulfoxide concentrations varied between 0.1 and 0.5 μg/ml (12, 18).