In Vitro and In Vivo Activities of Synthetic Trioxolanes against Major Human Schistosome Species

Abstract

Schistosomiasis is a parasitic disease that remains of considerable public health significance in tropical and subtropical environments. Since the mainstay of schistosomiasis control is chemotherapy with a single drug, praziquantel, drug resistance is a concern. Here, we present new data on the antischistosomal properties of representative synthetic 1,2,4-trioxolanes (OZs). Exposure of adult Schistosoma mansoni for 24 h to a medium containing 20 μg/ml OZ209 reduced worm motor activity, induced tegumental alterations, and killed worms within 72 h. While exposure of S. mansoni to OZ78 had no apparent effect, addition of hemin reduced worm motor activity and caused tegumental damage. Administration of single 200-mg/kg of body weight oral doses of OZ78, OZ209, and OZ288 to mice harboring a juvenile S. mansoni infection resulted in worm burden reductions of 82.0 to 95.4%. In the adult infection model in mice, single 400-mg/kg doses of these compounds resulted in a maximum total worm burden reduction of 52.2%. High worm burden reductions (71.7 to 86.5%) were observed after administration of single 200-mg/kg doses of OZ78 and OZ288 to hamsters infected with either juvenile or adult S. mansoni. A single 200-mg/kg dose of OZ78 to hamsters infected with adult Schistosoma japonicum resulted in total and female worm burden reductions of 94.2 to 100%. Our results, along with the low toxicity, metabolic stability, and good pharmacokinetic properties of the OZs, indicate the potential for the development of novel broad-spectrum antischistosomal OZ drug candidates.

There is growing awareness of the huge public health significance of the so-called neglected tropical diseases (NTDs). The global burden due to NTDs is estimated to be similar to human immunodeficiency virus/AIDS, malaria, and tuberculosis (12, 17). Schistosomiasis is one of the most significant NTDs (12, 16). The epidemiology, pathogenesis, and progress made in the control of schistosomiasis have been reviewed recently (5, 6, 9, 26). The three most important species parasitizing humans are Schistosoma haematobium, Schistosoma japonicum, and Schistosoma mansoni. An estimated 779 million people are at risk of schistosomiasis, over 200 million are infected (19), and 20 million suffer from severe disease manifestations, i.e., chronic hepatic and intestinal fibrosis (S. mansoni and S. japonicum) and ureteric and bladder fibrosis and calcification of the genitourinary tract (S. haematobium) (9, 23).

There is no vaccine available for the control of schistosomiasis (2), but new research focusing on tetraspanin, a recombinant protein of S. mansoni, showed promising results (22). Since changing human water contact behavior and the control of intermediate host snails are particularly challenging, morbidity control remains the mainstay of schistosomiasis control (28). At present, morbidity control is based on a single drug, praziquantel (3, 23). In high-burden areas, the goal is to regularly administer praziquantel to school-age children and other high-risk groups (28). National control programs utilizing praziquantel have been implemented for several decades in China and Egypt, and several African countries have initiated praziquantel-based morbidity control programs (5, 6).

The semisynthetic artemisinin derivatives (e.g., artemether and artesunate) are currently the most potent antimalarial drugs available and are being used on a very large scale, particularly in combination with other antimalarials (1, 11). The artemisinins also exhibit trematocidal properties (26). With regard to schistosomiasis, the highest activities of the artemisinins are confined to the young developmental stages of the parasites and, hence, the artemisinins have been successfully developed as prophylactic agents against schistosomiasis (25, 29). When compared to the artemisinins, 1,2,4-trioxolanes (secondary ozonides, or OZs) are characterized by structural simplicity, ease of synthesis, and improved pharmacokinetic parameters. The OZs have been assessed primarily for their antimalarial activities, and OZ277 has been selected as an antimalarial drug development candidate (27). Interestingly, OZ78 shows strong activity against Echinostoma caproni (an intestinal fluke) and Fasciola hepatica and Clonorchis sinensis (liver flukes) (14, 15). Here, we present the first data from detailed in vitro and in vivo investigations of representative OZs against juvenile and adult stages of S. mansoni and S. japonicum.

MATERIALS AND METHODS

Drugs.

1,2,4-Trioxolanes OZ01, OZ03, OZ78, OZ209, and OZ288, 1,2,4-trioxanes ST01 and ST03, and 1,3-dioxolanes NP17 and NP18 (Fig. 1) were synthesized as previously described (4, 20, 21, 27). The compounds were prepared as suspensions in 7% (vol/vol) Tween 80 and 3% (vol/vol) ethanol before oral administration.

FIG. 1.

Chemical structures of 1,2,4-trioxolanes OZ01, OZ03, OZ78, OZ209, OZ288, 1,2,4-trioxanes ST01 and ST03, and 1,3-dioxolanes NP17 and NP18.

Animals and parasites.

Experiments with S. mansoni (Liberian strain) were carried out at the Swiss Tropical Institute (Basel, Switzerland), in accordance with Swiss national animal welfare regulations. The experiments with S. japonicum (Anhui strain) were undertaken at the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Shanghai, China). For the studies carried out in Switzerland, female NMRI mice (n = 109; weight, ∼20 to 22 g) were purchased from RCC (Itingen, Switzerland) and male Syrian golden hamsters (n = 56; weight, ∼60 to 80 g) were obtained from Charles River (Sulzfeld, Germany). The Chinese studies were performed in male Syrian golden hamsters (n = 10; weight, ∼60 to 80 g) purchased from Shanghai Animal Center (Shanghai, China) and rabbits (New Zealand strain) of both sexes (n = 8; weight, ∼2.0 to 2.4 kg) obtained from the Shanghai Institute of Biological Products (Shanghai, China).

Cercariae of S. mansoni and S. japonicum were obtained following standard operating procedures at our laboratories from infected intermediate host snails, namely, Biomphalaria glabrata and Oncomelania hupensis, respectively.

In vitro studies with S. mansoni.

Adult S. mansoni specimens were incubated in 24-well Falcon plates (Costar), using two male and two female specimens per well. The experiments were carried out in duplicates. Culture medium in each well contained 2 ml Hanks' balanced salt solution (Gibco) supplemented with 20% calf serum, 300 IU/ml of penicillin, and 300 μg/ml of streptomycin (Gibco). In one set of experiments, 160 μl of hemin solution was added. The hemin solution was prepared as follows: 5 mg of hemin was dissolved in 1 ml of 0.1 M aqueous solution of NaOH, and 3.95 ml of PBS (pH 7.4) and 0.05 ml of 1 M HCl were added to adjust the pH to 7.2 to 7.4. The final concentration of the medium was 80 μg/ml. Stock solutions of OZ at 1 mg/ml were prepared in 60% (vol/vol) dimethyl sulfoxide.

S. mansoni cultures were incubated with 20 μg/ml drug for up to 72 or 96 h. The control well contained the highest concentration of solvent, i.e., 1.2% dimethyl sulfoxide. Cultures were kept at 37°C in an atmosphere of 5% CO₂ and were observed 24 h, 48 h, and 72 h, and worms were incubated with hemin also at 96 h after exposure under a dissecting microscope. The effects of the drugs on S. mansoni were assessed qualitatively with an emphasis on changes in worm motor activity, tegumental alterations, and/or occurrence of death.

In vivo studies with S. mansoni.

Mice and hamsters were infected subcutaneously with 80 and 120 S. mansoni cercariae, respectively. Twenty-one days (juvenile infection) or 49 days (adult infection) postinfection, groups of three to five animals were treated orally with OZs at single 50- to 400-mg/kg doses. Groups of untreated animals served as controls. Twenty-eight days posttreatment, animals were sacrificed by blood letting and dissected. The liver was removed and placed into a 20- by 20-cm transparent plastic folder and compressed between two glass plates, and the parenchyma was examined under a stereoscopic microscope. All S. mansoni worms were removed, sexed, and counted. The small and large intestines were removed and placed in a petri dish. The mesenteric veins were systematically examined under a stereoscopic microscope, and all S. mansoni were removed, sexed, and counted.

In vivo studies with S. japonicum.

Ten hamsters and eight rabbits were infected with 100 and 200 S. japonicum cercariae, respectively, via shaved abdominal skin. Thirty-five days postinfection, hamsters and rabbits were administered single 200-mg/kg and 15-mg/kg oral doses of OZ, respectively. Untreated animals served as controls. Animals were sacrificed by blood letting, and S. japonicum was recovered from the hepatic and portomesenteric veins using a perfusion technique (34).

Statistical analysis.

All statistical analyses were done with version 2.4.5 of the Statsdirect statistical software package (Cheshire, United Kingdom). The Kruskal-Wallis (KW) test was employed to compare the medians of the responses between the treatment and control groups. A difference in median was considered to be significant at a level of 5%.

RESULTS

In vitro studies with S. mansoni.

Results of the in vitro studies with 49-day-old adult S. mansoni exposed to OZ78 (with or without hemin) and OZ209 at concentrations of 20 μg/ml are summarized in Table 1. There was no effect on the motor activity, and the tegument remained unchanged when schistosomes were incubated in a medium containing OZ78 for as long as 72 h. However, addition of hemin to the culture medium resulted in slightly or significantly reduced worm motor activity in five out of eight S. mansoni at 24 h after exposure. After 96 h, seven worms were dead and the remaining worm had a significantly reduced motor activity. Meanwhile, extensive tegumental alterations were observed in all worms.

TABLE 1.

In vitro effects of OZ78 (with and without hemin) and OZ209 against 49-day-old adult S. mansoni

Groupa	No. of worms investigated	Incubation period (h)	No. of dead worms	Motor activity reduction		Tegumental alteration
				Slight	Significant	Partial	Extensive
Control	8	24	0	0	0	0	0
	8	48	0	0	0	0	0
	8	72	0	0	0	0	0
OZ78	8	24	0	0	0	0	0
	8	48	0	0	0	0	0
	8	72	0	0	0	0	0
OZ209	8	24	0	2	6	2	2
	8	48	2	0	6	3	5
	8	72	8	0	0	0	8
Control plus hemin	8	24	0	0	0	0	0
	8	48	0	0	0	0	0
	8	72	0	0	0	0	0
	8	96	0	5	0	0	0
OZ78 plus hemin	8	24	0	4	3	0	0
	8	48	0	2	4	2	0
	8	72	1	0	7	1	4
	8	96	7	0	1	0	8

^aAll compounds were used at a concentration of 20 μg/ml.

The results for OZ209 were considerably different. At 24 h postexposure, half of the worms showed partial or extensive tegumental alterations, and motor activities were significantly reduced in six out of eight worms. All worms were dead after exposure for 72 h.

In vivo studies with S. mansoni.

The effects of single 200-mg/kg oral doses of selected OZs (1,2,4-trioxolanes) and their 1,2,4-trioxane and nonperoxidic 1,3-dioxolane counterparts on worm burden reductions in 21-day-old juvenile S. mansoni harbored in mice, including changes in worm distributions, are summarized in Table 2. The 1,2,4-trioxanes ST01 and ST03 and the nonperoxidic 1,3-dioxolanes NP17 and NP18 had no apparent effect on juvenile S. mansoni. Administration of the 1,2,4-trioxolane OZ01 resulted in slight total (22.9%) and female (26.4%) worm burden reductions. These reductions, however, were not statistically significant.

TABLE 2.

Effects of single 200-mg/kg oral doses of selected synthetic trioxolanes administered to mice harboring a 21-day-old juvenile S. mansoni infection on worm burden, stratified by sex and worm distribution^a

Group	No. of mice investigated	No. of mice cured	Mean no. of worms (SD)					Total worm burden			Female worm burden
			Liver	Mesenteric veins	Total	Males	Females	% Reduction	KW	P value	% Reduction	KW	P value
Control	10		4.6 (3.4)	36.4 (11.0)	41.0 (9.5)	17.9 (4.1)	23.1 (6.7)
ST01	5	0	4.0 (2.0)	34.8 (8.5)	38.8 (9.3)	16.6 (5.1)	22.2 (6.3)	5.4	0	>0.999	3.9	0.18	0.666
ST03	5	0	4.0 (2.5)	38.6 (11.9)	42.6 (10.7)	19.0 (5.9)	23.6 (5.8)	0	NA	NA	0	NA	NA
NP17	5	0	7.4 (3.4)	41.0 (6.2)	48.4 (6.2)	22.8 (4.4)	25.6 (3.6)	0	NA	NA	0	NA	NA
NP18	5	0	3.0 (2.3)	37.2 (10.7)	40.2 (10.6)	18.2 (4.5)	22.0 (6.1)	2.0	0.03	0.853	4.8	0.03	0.853
OZ01	5	0	4.0 (1.9)	27.6 (11.7)	31.6 (11.6)	14.6 (6.0)	17.0 (5.8)	22.9	1.50	0.220	26.4	1.99	0.157
OZ03	5	0	5.6 (2.7)	1.8 (1.5)	7.4 (4.0)	2.6 (1.1)	4.8 (3.3)	82.0	9.42	0.002	79.2	9.44	0.002
Control	5		7.0 (1.9)	30.2 (15.4)	37.2 (15.7)	21.5 (8.9)	15.7 (7.8)
OZ78	3	0	4.7 (1.5)	2.0 (3.5)	6.7 (4.7)	4.7 (2.9)	2.0 (2.0)	82.0	5.40	0.020	87.3	5.40	0.020
Control	7		2.7 (2.4)	35.3 (7.0)	38.0 (7.2)	21.4 (4.4)	16.6 (3.7)
OZ209	3	0	1.0 (0)	6.0 (6.0)	5.7 (1.5)	3.0 (1.0)	2.7 (0.6)	85.0	5.72	0.017	83.7	5.79	0.016
Control	3		6.0 (2.0)	53.0 (9.6)	59.0 (9.5)	29.3 (5.0)	26.4 (3.8)
OZ288	5	0	2.0 (1.0)	0.7 (1.2)	2.7 (1.5)	1.7 (1.2)	1.0 (1.0)	95.4	3.86	0.049	96.2	3.85	0.049

^aSD, standard deviation; KW, Kruskal-Wallis; NA, not applicable.

Single 200-mg/kg oral doses of OZ03, OZ78, and OZ209 resulted in significant reductions in total and female worm burden reductions (79.2 to 87.3%). These reductions were accompanied with changes in the worm distribution. The highest activity in mice harboring a 21-day-old S. mansoni infection was observed following administration of OZ288, yielding total and female worm burden reductions of 95.4% and 96.2%, respectively.

Based on the promising results with OZ03, OZ78, OZ209, and OZ288 obtained with juvenile S. mansoni, these compounds progressed into the adult S. mansoni infection model. As summarized in Table 3, OZ288, administered orally at a single 400-mg/kg dose, was the only compound that exerted a significant reduction in total (52.2%) and female (64.9%) worm burdens. At this same dose, OZ209 was toxic; two mice died 48 to 96 h posttreatment.

TABLE 3.

Effects of single 400-mg/kg oral doses of selected OZ compounds administered to mice harboring a 49-day-old adult S. mansoni infection on worm burden, stratified by sex and worm distribution^a

Group	No. of mice investigated	No. of mice cured	Mean no. of worms (SD)					Total worm burden			Female worm burden
			Liver	Mesenteric veins	Total	Males	Females	% Reduction	KW	P value	% Reduction	KW	P value
Control	10	0	3.2 (2.0)	33.3 (8.0)	38.5 (9.5)	21.1 (4.9)	17.4 (5.3)
OZ03	3	0	3.3 (2.5)	24.0 (6.1)	27.3 (6.7)	15.0 (4.0)	12.3 (2.9)	29.1	2.88	0.089	29.3	1.41	0.235
Control	10	0	2.3 (2.5)	16.4 (12.3)	18.7 (13.7)	10.7 (8.0)	8.0 (5.9)
OZ78	5	0	1.6 (1.9)	20.6 (13.5)	22.2 (15.1)	13.4 (9.3)	8.8 (6.0)	0	NA	NA	0	NA	NA
OZ209	3	0b	4.3 (2.1)	11.3 (11.8)	15.6 (13.4)	9.7 (7.4)	6.0 (6.1)	16.6	0.06	0.799	25.0	0.47	0.493
Control	5	0	6.4 (3.8)	29.2 (7.2)	35.6 (5.4)	22.2 (4.0)	13.4 (2.7)
OZ288	3	0	4.0 (1.7)	13.0 (11.4)	17.0 (10.4)	12.3 (7.6)	4.7 (3.1)	52.2	4.46	0.034	64.9	5.0	0.025

^aSD, standard deviation; KW, Kruskal-Wallis; NA, not applicable.

^bTwo mice died 48 to 96 h after treatment.

The dose-response relationships of OZ78 and OZ288 in 21-day-old juvenile and 49-day-old adult S. mansoni harbored in hamsters are summarized in Tables 4 and 5. At the lowest dose of OZ288 investigated (50 mg/kg) in the juvenile infection model, statistically significant (P = 0.016) total and female worm burden reductions of 78.7 to 82.7% were observed. Slightly lower total and female worm burden reductions were observed after administration of OZ78 (67.0 to 73.4%). At 200 mg/kg, the total worm burden reductions were 80.2% and 86.5% for OZ78 and OZ288, respectively.

TABLE 4.

Dose-response relationship of OZ78 and OZ288 against 21-day-old juvenile S. mansoni harbored in hamsters^a

Group	Dose (mg/kg)	No. of hamsters investigated	No. of hamsters cured	Mean no. of worms (SD)					Total worm burden			Female worm burden
				Liver	Mesenteric veins	Total	Males	Females	% Reduction	KW	P value	% Reduction	KW	P value
Control		7	0	6.7 (4.2)	45.1 (9.0)	51.9 (10.4)	29.7 (6.5)	22.1 (4.9)
OZ78	50	4	0	2.3 (2.2)	11.5 (3.0)	13.8 (3.8)	6.5 (1.0)	7.3 (2.9)	73.4	7.00	0.008	67.0	7.06	0.008
	100	4	0	0.5 (1.0)	4.4 (2.2)	8.8 (4.3)	4.0 (2.2)	4.8 (2.2)	83.0	7.00	0.008	78.3	7.03	0.008
	200	4	0	1.3 (1.2)	8.8 (2.2)	10.3 (2.9)	4.8 (1.5)	5.5 (1.7)	80.2	7.03	0.008	75.1	7.06	0.008
OZ288	50	3	0	0.3 (0.6)	8.7 (3.1)	9.0 (2.6)	4.3 (1.5)	4.7 (1.2)	82.7	5.72	0.016	78.7	5.79	0.016
	100	3	0	0.7 (0.6)	7.7 (4.7)	8.3 (5.1)	4.0 (2.0)	4.3 (3.2)	84.0	5.72	0.016	80.5	5.76	0.016
	200	3	1	1.0 (1.7)	6.0 (7.2)	7.0 (8.9)	4.0 (5.3)	3.0 (3.6)	86.5	5.72	0.016	86.4	5.76	0.016

^aSD, standard deviation; KW, Kruskal-Wallis.

TABLE 5.

Dose-response relationship of OZ78 and OZ288 against 49-day-old adult S. mansoni harbored in hamsters^a

Group	Dose (mg/kg)	No. of hamsters investigated	No. of hamsters cured	Mean no. of worms (SD)					Total worm burden			Female worm burden
				Liver	Mesenteric veins	Total	Males	Females	% Reduction	KW	P value	% Reduction	KW	P value
Control		7	0	6.7 (4.2)	45.1 (9.0)	51.9 (10.4)	29.7 (6.5)	22.1 (4.9)
OZ78	50	4b	0	3.5 (2.1)	24.5 (16.3)	28.0 (18.4)	14.5 (10.6)	13.5 (7.8)	46.1	3.08	0.079	38.9	2.16	0.141
	100	4	0	4.5 (4.5)	11.5 (6.1)	16.0 (9.7)	11.5 (6.4)	4.5 (3.7)	69.2	7.00	0.008	79.6	7.03	0.008
	200	4	0	2.0 (0.8)	5.8 (3.5)	7.8 (3.8)	6.3 (2.4)	1.5 (1.7)	85.0	7.00	0.008	93.2	7.06	0.008
OZ288	50	3	0	4.0 (1.7)	23.7 (16.8)	27.7 (18.1)	13.3 (10.1)	14.0 (8.2)	46.6	3.32	0.067	36.7	1.31	0.251
	100	3	0	2.7 (2.3)	21.0 (7.0)	24.7 (5.9)	11.7 (2.5)	13.0 (3.5)	52.4	5.72	0.016	41.2	5.79	0.016
	200	3	0	2.0 (2.6)	11.7 (14.2)	14.7 (15.9)	8.0 (8.7)	6.7 (7.2)	71.7	5.72	0.016	69.7	5.76	0.016

^aSD, standard deviation; KW, Kruskal-Wallis.

^bTwo hamsters were not infected and, hence, they were excluded from the analysis.

In hamsters harboring an adult S. mansoni infection, significant total and female worm burden reductions of 85.0 to 93.2% were achieved with a single-200 mg/kg dose of OZ78. At half this dose, total and female worm burden reductions were slightly lower (69.2 to 79.6%), but these reductions were still statistically significant. Administration of OZ288 at doses of 100 mg/kg also resulted in significant total and female worm burden reductions (41.2 to 71.7%). At the lowest doses investigated (50 mg/kg), moderate, but statistically insignificant, total and female worm reductions were observed for both OZ78 (38.9 to 46.1%) and OZ288 (36.7 to 46.6%).

S. japonicum in vivo studies.

Table 6 shows the results obtained after administration of a single 200-mg/kg dose of OZ78 to hamsters infected with 49-day-old adult S. japonicum and a single dose of 15 mg/kg of the same compound given to rabbits with an adult S. japonicum infection. Highly significant total and female worm burden reductions (94.2 to 100%) were achieved in the hamster model. The statistically insignificant respective total and female worm burden reductions in the rabbit model were 40.7 to 42.3%.

TABLE 6.

Effect of a single oral dose of OZ78 administered to hamsters and rabbits harboring a 49-day-old adult S. japonicum infection^a

Animal host	Group	Dose (mg/kg)	No. of animals investigated	No. of animals cured	Mean no. of worms (SD)			Total worm burden			Female worm burden
					Total	Males	Females	% Reduction	KW	P value	% Reduction	KW	P value
Hamster	Control		6		69.0 (13.1)	35.0 (6.2)	33.7 (6.8)
	OZ78	200	4	2	4.0 (4.9)	4.0 (4.9)	0	94.2	6.63	0.010	100	6.96	0.008
Rabbit	Control		4		97.0 (16.4)	50.3 (7.8)	46.8 (8.8)
	OZ78	15	4	0	57.5 (32.0)	30.5 (16.5)	27.0 (15.6)	40.7	4.08	0.043	42.3	3.00	0.083

^aSD, standard deviation; KW, Kruskal-Wallis.

DISCUSSION

The impetus for this investigation arose from the present situation in which praziquantel is virtually the only drug available for the individual treatment and community-based morbidity control of schistosomiasis, one of the most prevalent parasitic diseases in the tropics and subtropics (3, 7, 23). In view of renewed efforts to control schistosomiasis in high-burden areas of sub-Saharan Africa by means of large-scale deployment of praziquantel, there is concern about parasite resistance development (8, 10).

We found that, unlike praziquantel, which has only minimal activity against juvenile schistosomes (18, 33), single oral doses of OZ78, OZ209, and OZ288 were highly effective against juvenile S. mansoni in mice. In addition, these OZs had high activities against both juvenile and adult stages of S. mansoni and adult S. japonicum in hamsters. For example, worm burden reductions above 80% were observed in hamsters infected with either juvenile or adult S. mansoni or adult S. japonicum following the administration of OZ78. Data available thus far suggest that the OZ compounds compare favorably to artemether, which is known for its high efficacy against juvenile S. mansoni and juvenile S. japonicum in the mouse model (24, 25, 31). It should be noted, however, that a new laboratory investigation with artemether revealed high worm burden reductions in adult S. mansoni harbored in hamsters (26). However, due to enhanced pharmacokinetic properties of the OZ compounds versus artemether, we would have expected greater antischistosomal properties of the former compared to the latter compound. For example, the oral bioavailability of OZ78 in rats is more than an order of magnitude higher than that of artemether (74% versus 1.4%) (27). This might at least partially explain that a fourfold higher dose of artesunate was necessary to cure F. hepatica-infected rats compared to OZ78 (13, 14).

The complete lack of activity of the nonperoxidic OZ03 isosteres NP17 and NP18 against both juvenile and adult stages of S. mansoni indicates that the OZ peroxide bond is essential for the antischistosomal activity. NP17 and NP18 both are also completely devoid of antimalarial activity (4). That the 1,2,4-trioxanes ST01 and ST03 had no antischistosomal activity indicates a certain structural specificity for the antischistosomal properties of the OZ class of synthetic peroxides. However, as illustrated with OZ78, the OZ structural requirements mandatory for significant antischistosomal, fasciocidal, and clonorchicidal activities (13-15) are different from those associated with good antimalarial activity (27). Hence, it should be possible to optimize trematocidal activity independent of antimalarial activity, which will reduce the probability of drug resistance development in the field. The differences in the activity of OZs against adult schistosomes between the hamster and the mouse model are noteworthy. We found that OZ78 was completely inactive against adult S. mansoni in mice, but as mentioned above, OZ78 was highly active against adult S. mansoni harbored in hamsters. Differences in the immunological responses or pharmacokinetics between the different animal species might explain these findings and remain to be elucidated. However, it is also evident that juvenile S. mansoni in the hamster model was also more susceptible to the OZs than the adult forms. While there was no dose-response effect against juvenile S. mansoni following the administration of 50 to 200 mg/kg OZ78 (Table 4) because the lowest dose was already at the top of the dose-response curve, there was a consistent dose-response effect against adult S. mansoni (Table 5).

Our in vitro experiments on adult schistosomes confirmed the promising in vivo results. An interesting observation was that OZ78 required interaction with hemin to significantly reduce motor activity and to alter the tegument, whereas OZ209 did not. In contrast to artemether, for which the current working hypothesis of its mechanism of action depends on the iron-dependent reduction of the endoperoxide bridge to sequentially generate carbon-centered free radicals that alkylate parasite proteins (30, 32), cleavage of the peroxide bridge may not be mandatory for OZ209. That OZ209 is killing the parasites by one or more iron-independent mechanisms is a hypothesis supported by its unusually high antimalarial activity and its relatively greater toxicity (27) compared to all of the other OZ compounds tested thus far. From this observation, we speculate that a nonperoxide antischistosomal compound or a compound with broad-spectrum activity against non-hemoglobin-digesting trematodes can be identified.

In conclusion, we have developed a promising starting point for identification of a new broad-spectrum antischistosomal drug candidate. Several OZ compounds contain a functional group which provides convenient chemical handles for focused chemical exploration. In addition, the low toxicity, metabolic stability, and improved pharmacokinetic properties of the OZs provide us with the confidence to move forward with this class of compounds.