In Vivo Activity of Aryl Ozonides against Schistosoma Species

Abstract

We evaluated the in vivo antischistosomal activities of 11 structurally diverse synthetic peroxides. Of all compounds tested, ozonide (1,2,4-trioxolane) OZ418 had the highest activity against adult Schistosoma mansoni, with total and female worm burden reductions of 80 and 90% (P < 0.05), respectively. Furthermore, treatment of S. haematobium-infected mice with OZ418 reduced the total worm burden by 86%. In conclusion, OZ418 is a promising antischistosomal lead compound.

TEXT

Schistosomiasis, caused by blood flukes of the genus Schistosoma, is an important cause of morbidity and mortality, mainly among the rural poor in sub-Saharan Africa (5, 16). The need for new antischistosomal drugs is quite evident, given that praziquantel is the only drug available and is in widespread use in population-based morbidity control programs (16).

Various classes of synthetic peroxides, including trioxaquines (1,2,4-trioxanes) (1, 12), the tetraoxaspirononadecane N-89 (15), and ozonides (1,2,4-trioxolanes) (19), have been studied for their antischistosomal properties. Our previous data demonstrate that, like the artemisinins, ozonides possess promising antischistosomal properties (10, 19). We investigated three representative ozonides, namely, carboxylic acid OZ78, amine OZ209, and phenol OZ288 (Fig. 1). Of these, 8′-aryl ozonide OZ288 had the highest antischistosomal activity; this ozonide reduced the total worm burden by 95% (200 mg/kg body weight) in mice harboring juvenile Schistosoma mansoni and by 52% (400 mg/kg) in mice harboring adult worms (19). Although 8′-alkyl ozonides OZ78 and OZ209 had activities greater than 80% against juvenile worms, lower activities (0 and 17%, respectively) were observed for these two ozonides against adult worms. We suggest that the increased iron(II) stabilities of 8′-aryl ozonides compared to those of 8′-alkyl ozonides (2) might contribute to the superior antischistosomal efficacy of OZ288.

Fig 1.

Structures of ozonides OZ78, OZ209, and OZ288.

The aim of the present study was to evaluate in a first step the in vivo antischistosomal activities of nine structurally diverse OZ288 analogs (Fig. 2), including antimalarial drug candidate OZ439, currently in phase II clinical testing (2). Since schistosomiasis and malaria are coendemic in many parts of the world, it is important to assess the potential auxiliary effects of antimalarial drug candidates such as OZ439 on Schistosoma species.

Fig 2.

Structures of OZ288 analogs, 1,2,4-trioxane ST28, and 1,2,4,5-tetraoxane MT23.

Our animal studies were carried out following national and cantonal regulations on animal welfare (permission no. 2070). All compounds were synthesized as described in previous publications and patents (3, 4, 17, 18). Compounds were freshly prepared in homogenous aqueous suspensions in 7% Tween 80 and 3% ethanol.

Female NMRI mice (n = 147; age, 4 weeks; weight, ∼20 g) were purchased from Charles River (Sulzfeld, Germany) and Harlan Laboratories (Blackthorn, United Kingdom). Mice were maintained in groups of 10 in Makrolon cages under environmentally controlled conditions (temperature, ∼25°C; humidity, ∼50%; 12-h light and 12-h dark cycle) with free access to water and food. Mice were acclimatized for 1 week prior to infections.

Each mouse was infected subcutaneously with ∼80 S. mansoni cercariae. Twenty-one days (pre-patent infection) or 49 days (patent infection) after the experimental infection, groups of 3 to 5 mice were treated orally with the test drugs at 400 mg/kg (adult infection) or 200 mg/kg (juvenile infection). In order to assess the effect of OZ418 against S. haematobium, 16 mice were similarly infected with ∼300 S. haematobium cercariae per mouse. Ninety days postinfection (patent infection), mice (n = 4) were treated orally with 400 mg/kg OZ418. Infected untreated mice served as controls in both experiments. By use of CO₂, S. mansoni- and S. haematobium-infected mice were killed 21 days posttreatment; they were dissected, and schistosomes were sexed and counted. For statistical analysis, the Kruskal-Wallis (KW) test was used (Statsdirect statistical software, version 2.4.5; Cheshire, United Kingdom).

The antischistosomal activities of the nine OZ288 analogs against adult S. mansoni are summarized in Table 1. For comparison, data obtained with the drug of choice, praziquantel, are presented (7).

Table 1.

Effect of selected ozonides administered at single oral doses of 400 mg/kg against adult S. mansoni worms harbored in NMRI mice

Compound	Total worm burden reduction (%)	Female worm burden reduction (%)
Praziquantela	96	98
OZ288b	52	65
OZ27	20	NDc
OZ47	12	8
OZ154	0	8
OZ165	74	78
OZ401	0	5
OZ418	80	90
OZ439	35	43
OZ508	0	0
OZ513	24	23

^aTotal worm burden reduction reported in reference 7.

^bBased on the study of Xiao et al. in 2007 (19).

^cND, not determined.

In contrast to OZ288, the ozonides with neutral and weak base functional groups either were completely inactive or showed very weak activities (5 to 43%). This, combined with the lack of activity of OZ508, demonstrate that the 8′-aryl substructure of OZ288 is a necessary but insufficient requirement for antischistosomal activity. Total and female worm burden reductions of 35 and 43%, respectively, were documented for the antimalarial drug candidate OZ439. The only ozonides more effective than OZ288 were carboxylic acids OZ165 and OZ418. We also note that in contrast to the neutral and weak base ozonides (Fig. 2) with 50% inhibitory concentrations (IC₅₀s) less than 5 ng/ml against cultured Plasmodium falciparum (2, 4, 17), these two acidic ozonides are much less potent against P. falciparum, with respective IC₅₀s of 37 and 97 ng/ml (4, 17).

Since the highest activity was observed with OZ165 and OZ418, these ozonides, along with the 1,2,4-trioxane (ST28) and 1,2,4,5-tetraoxane (MT23) analogs of OZ165 (Fig. 2), were assessed in more detail (Table 2). Each of the four compounds achieved high worm burden reductions (82 to 100%) in mice infected with juvenile S. mansoni, consistent with previous observations that synthetic peroxides have high activities against the juvenile stage of the parasite (1, 19). However, the four compounds had significant differences in efficacy against adult S. mansoni. For example, low total and female worm burden reductions of 33 and 40% were observed with ST28 (P > 0.05). Treatment of mice with MT23 resulted in moderate total and female worm burden reductions of 62 (P = 0.037) and 57% (P > 0.05), respectively. A minor shift of worms from the mesenteric veins to the liver was observed with these two compounds. Treatment with OZ165 achieved significant total and female worm burden reductions of 74 and 78%. Thus, for this set of peroxide heterocycles, OZ165 was more effective than either ST28 or MT23. In contrast, we have recently shown (18) that ST28 is more effective than either OZ165 or MT23 against Fasciola hepatica, another pathogenic liver fluke. Of all of the compounds tested, OZ418 had the highest antischistosomal efficacy, with total and female worm burden reductions of 80 and 90%, respectively (both P values = 0.01). Note that worm counts (mean worm counts of 13 to 28 worms) in the present work were slightly lower than in previous studies (mean worm counts of 39 to 41 worms) (19). In this respect, clinical studies recently demonstrated that significantly higher cure rates were observed with the artemisinins in patients with lower infection intensities of Fasciola spp. and Schistosoma spp. (8, 11).

Table 2.

Effect of 1,2,4-trioxolanes (ozonides) OZ165 and OZ418, 1,2,4-trioxane ST28, and 1,2,4,5-tetraoxane MT23 against juvenile and adult S. mansoni worms harbored in NMRI mice

Infection (dose)	Compound tested	No. of mice investigated	No. of mice cured	Mean no. of worms (SD)e				Total worm burden reduction		Female worm burden reduction
				Liver	Mesenteric veins	Males	Females	%	P value	%	P value
None	Control 1	10	0	1.9 (1.7)	26.0 (13.1)	15.4 (8.1)	12.5 (7.0)
	Control 2	9	0	0.9 (1.1)	16.6 (4.9)	8.5 (3.3)	8.9 (3.4)
	Control 3	18	0	1.8 (2.3)	21.6 (13.3)	12.8 (7.4)	10.5 (6.6)
	Control 4	18	0	1.2 (2.3)	17.4 (13.7)	6.9 (5.0)	6.1 (5.0)
Adult infection (400 mg/kg)	OZ165c	6	0	1.2 (1.2)	4.8 (6.6)	3.6 (3.7)	2.3 (3.8)	74.1	0.0029	78.1	0.003
	ST28a	4	0	3.3 (2.5)	15.0 (6.7)	11.3 (3.2)	7.5 (3.8)	32.8	0.228	40.0	0.117
	MT23a	5	1	2.6 (2.6)	8.0 (10.3)	5.2 (5.0)	5.4 (5.8)	62.1	0.037	56.8	0.097
	OZ418d	5	0	0.5 (0.6)	2.0 (0.8)	2.0 (0.7)	0.6 (0.6)	80.0	0.01	90.2	0.01
Juvenile infection (200 mg/kg)	OZ165b	4	0	0.8 (0.5)	2.0 (2.3)	1.75 (1.0)	1.0 (1.2)	84.2	0.005	88.8	0.005
	ST28a	4	0	1.0 (1.4)	2.5 (1.0)	1.25 (0.5)	2.3 (1.9)	87.5	0.005	81.6	0.006
	MT23a	4	0	1.5 (1.3)	0.5 (1.0)	0.5 (0.6)	1.5 (1.3)	92.8	0.005	88.0	0.005
	OZ418d	4	4	0	0	0	0	100	0.005	100	0.005

^aData for this compound are relative to control 1.

^bData for this compound are relative to control 2.

^cData for this compound are relative to control 3.

^dData for this compound are relative to control 4.

^eSD, standard deviation.

Given the superior efficacy of OZ418, we tested this ozonide against S. haematobium, another major human-pathogenic schistosome species (14). Treatment of S. haematobium-infected mice with OZ418 resulted in significant (P = 0.01) total worm burden reduction of 86% (Table 3), demonstrating that OZ418 has high efficacy against two schistosome species.

Table 3.

Effects of ozonide OZ418 and praziquantel against adult S. haematobium worms harbored in NMRI mice

Compound tested	No. of mice investigated	No. of mice cured	Mean no. of worms (SD)			Total worm burden reduction
			Liver	Mesenteric veins	Total	%	P value
Control	12	0	2.3 (4)	3 (4)	5.3 (5)
OZ418 (400 mg/kg)	4	2	0 (0)	0.8 (1)	0.8 (1)	85.9	0.01
Control	14	0	2.1 (3.7)	2.7 (2.6)	4.9 (3.9)
Praziquantel (200 mg/kg)	4	3	0 (0)	0.25 (0.5)	0.25 (0.5)	94.9	0.003

In conclusion, OZ418 is a promising lead compound effective against several developmental stages of S. mansoni. The activity of OZ418 against adult S. mansoni compares favorably with that of praziquantel (50% effective dose [ED₅₀] of 172 mg/kg; worm burden reduction of 96% at 400 mg/kg) (Table 1) (7). In contrast to praziquantel, which lacks activity against juvenile worms (6), OZ418 also has high activity against the juvenile stage of S. mansoni. Importantly, this compound is also quite effective against S. haematobium, the causative agent of urinary schistosomiasis. Accordingly, OZ418 possesses many properties of an ideal antischistosomal compound (9, 13). Given the threat of praziquantel resistance and lack of orally active antischistosomal candidates in the drug development pipeline that show high activities against adult and juvenile schistosomes and are active against multiple schistosome species, we continue to investigate OZ418 and its analogs as potential anthelmintic drug development candidates.