Therapeutic Effect of Diminazene Aceturate on Parasitic Blood Fluke Schistosoma mansoni Infection

Praziquantel is currently the only drug available to treat schistosomiasis, a disease of enormous public health significance caused by a blood fluke of the genus Schistosoma. Diminazene, a drug approved by the FDA, has been successfully used to treat diseases caused by blood protozoan parasites. In this study, we evaluated the antiparasitic properties of diminazene against Schistosoma mansoni ex vivo and in mice harboring either chronic or early S. mansoni infections.

ABSTRACT

Praziquantel is currently the only drug available to treat schistosomiasis, a disease of enormous public health significance caused by a blood fluke of the genus Schistosoma. Diminazene, a drug approved by the FDA, has been successfully used to treat diseases caused by blood protozoan parasites. In this study, we evaluated the antiparasitic properties of diminazene against Schistosoma mansoni ex vivo and in mice harboring either chronic or early S. mansoni infections. In vitro, we monitored phenotypic and tegumental changes as well as the effects of the drug on pairing and egg production. In mice infected with either adult (chronic infection) or immature (early infection) worms, diminazene was administered intraperitoneally (10 to 100 mg/kg of body weight) or by oral gavage (100 to 400 mg/kg), and we studied the influence of the drug on worm burden and egg production. Liver and spleen pathologies and serum aminotransferase levels were also analyzed. In vitro, 50% effective concentrations (EC₅₀) and EC₉₀ revealed that diminazene is able to kill both immature and adult parasites, and its effect was time and concentration dependent. In addition, confocal laser scanning microscopy showed morphological alterations in the teguments of schistosomes. In an animal model, the influence of the drug on worm burden, egg production, hepatomegaly, and splenomegaly depended on the dosing regimen applied and the route of administration. Diminazene also caused a significant reduction in aminotransferase levels. Comparatively, diminazene treatment was more effective in chronic infection than in early infection. In tandem, our study revealed that diminazene possesses anthelmintic properties and inhibits liver injury caused by Schistosoma eggs.

INTRODUCTION

Schistosomiasis is a disease of enormous human and animal health significance. It is caused by blood-dwelling flatworms of the genus Schistosoma and ranks among the most important parasitic diseases worldwide. Human schistosomiasis is endemic in 78 countries, with more than 230 million people infected and approximately 800 million people at risk of infection (1). Animal schistosomiasis also affects hundreds of millions of animals, and the disease is a problem of considerable economic significance, which often causes of animal morbidity and mortality (2). The life cycle of schistosomes involves a snail as the intermediate host and a definitive mammalian host. After cercariae penetrate the skin of the mammalian host, the larvae (schistosomula) reach the lung and next arrive in the hepatic portal system, where they mature and pair up. In the blood vessels of the mammalian host, these adult parasites can survive for several years, and they are therefore well known for their serious threat to human and animal health (3). Among Schistosoma species, Schistosoma mansoni represents one of the most important pathogenic species for humans. Morbidity due to schistosomiasis results from inflammation based on the immune response against eggs lodged in the host tissue, either as regulated chronic inflammation or as a condition resulting in fibrotic lesions. The liver carries the main burden of S. mansoni infection (1).

Despite considerable efforts over recent decades, no protective vaccine is yet in sight for schistosomiasis. Only one efficient and safe drug is currently available, namely, praziquantel. The development of resistance raises concerns about the urgent need for alternative drugs to control this disease in the long term (4). In addition, even though praziquantel has its advantages, which include tolerability, safety, efficacy, and low cost, it does not protect individuals from reinfection and is not active against the juvenile stages of the worm (5). Therefore, new and efficient antischistosomal compounds should be found (4, 6).

Although there are efforts to develop a new antischistosomal drug, this will take at least another decade from lead drug candidates (4, 7). In this context, drug repurposing (also called repositioning or reprofiling) may be considered a highly effective and promising strategy in drug discovery for parasitic diseases such as schistosomiasis (8). Diminazene aceturate, an aromatic diamidine (Fig. 1), has been successfully used as a veterinary drug to treat diseases caused by blood protozoan parasites, such as trypanosomiasis and babesiosis, since 1955 (9). It is also used to treat human African trypanosomiasis, also known as sleeping sickness (10). Diminazene has also been shown to have anti-inflammatory properties (10), and a recent study described the potential of diminazene to inhibit liver injury in experimental animals (11).

FIG 1.

Chemical structure of diminazene.

Specifically, we wondered whether diminazene might possess effective anthelmintic properties against the blood fluke S. mansoni. We therefore evaluated its in vitro and in vivo activities against immature and adult stages of S. mansoni. In vitro, we monitored phenotypic and tegumental changes, as well as the effects of the drug on pairing and egg production by adult worms. In vivo, we assessed the efficacy of diminazene treatment in mice infected with either adult (chronic infection) or immature (early infection) stages of S. mansoni by measuring worm and egg burdens. Liver and spleen pathologies and serum aminotransferase levels were also analyzed.

RESULTS

Diminazene exhibits antischistosomal properties in vitro.

The biological efficacy of diminazene was evaluated against both larval (schistosomula) and adult (male and female worm pairs) stages of S. mansoni. The gold-standard antiparasitic compound praziquantel was used as a positive control. Fifty percent and 90% effective concentrations (EC₅₀ and EC₉₀, respectively) of diminazene obtained against the different stages are summarized in Table 1. EC₅₀ and EC₉₀ values revealed that diminazene was more active against schistosomula than against adult worms. For example, estimated EC₅₀ values of 21.4 μM and 78.4 μM were calculated for diminazene at 72 h on larval and adult stages, respectively.

TABLE 1.

EC₅₀ and EC₉₀ values of diminazene against larval and adult S. mansoni worms

Stage	Diminazene EC (μM)
	50%			90%
	24 h	72 h	120 h	24 h	72 h	120 h
Larval	56.6 ± 7.4	21.4 ± 5.8	6.4 ± 1.4	69.1 ± 10.6	30.8 ± 6.8	11.4 ± 1.8
Adult	148.4 ± 12.4	78.4 ± 11.6	24.2 ± 3.6	183.6 ± 15.6	126.2 ± 9.4	29.4 ± 2.6

Further analysis revealed that diminazene induces mortality in immature and adult parasites in a time- and concentration-dependent manner (Fig. 2A and B). When schistosomes were exposed to diminazene at different concentrations, the life span of adult worms was longer than that in the larval stage. Diminazene was more active than praziquantel against immature worms. In contrast, the compound was less active against adult worms than praziquantel, which caused worm mortality in a short time at a very low concentration. The influence of diminazene on the ability of S. mansoni females to lay eggs also depended on the dosing regimen applied. To further investigate this effect, adult worm pairs were treated for 120 h with diminazene at 50, 25, 12.5, or 6.25 μM. Our results indicated a pronounced reduction (75 to 100%) in oviposition after treatment with diminazene at 50 to 12.5 μM (Fig. 2C).

FIG 2.

In vitro antischistosomal properties of diminazene against S. mansoni. (A) Immature worms (newly transformed schistosomula). (B) Adult worms (7 weeks old). Parasites were monitored for as long as 120 h using a microscope, and results are expressed as the percentage of mortality recorded by Kaplan-Meier survival curves. (C) Numbers of eggs released by couples of worms exposed to diminazene and controls. Mean values were derived from a minimum of three experiments (n = 3), and each experiment was performed with five replicates. The control consisted of RPMI 1640 plus 0.5% DMSO. PZQ, praziquantel at 2 μM.

To determine whether diminazene causes morphological alterations to the schistosome tegument, we used confocal laser scanning microscopy. In the case of adult parasites, male worms treated with diminazene displayed substantial tegumental disruption throughout the whole body, with the spinous tubercles losing their natural shape compared to those of controls. This loss of the surface integrity of parasites occurred in a concentration-dependent manner; it was more pronounced at concentrations above 25 μM (Fig. 3).

FIG 3.

Confocal laser scanning microscopy of ex vivo S. mansoni male worms. (A) Control worms showing intact surfaces. (B through D) Worms exposed to diminazene at 25 μM (B), 50 μM (C), or 100 μM (D). Parasites were monitored at different times up to 72 h, and photographs of the midbody region of a male worm were obtained. Bars, 5 μm.

Diminazene reduces worm burden and egg production in chronic S. mansoni infection.

Using a murine model of schistosomiasis, intraperitoneal (i.p.) (10 to 100 mg/kg of body weight) or oral gavage (p.o.) (100 to 400 mg/kg) treatment with diminazene was carried out 49 days postinfection (chronic infection). The antischistosomal efficacies of different doses of diminazene are summarized in Table 2. For comparison, data obtained with the drug of reference, praziquantel at 400 mg/kg p.o., are also presented. Administration of diminazene resulted in significant worm burden reductions (WBR) in comparison to burdens in infected control mice. The highest WBR were seen with diminazene at 100 mg/kg i.p. (WBR = 87.2%) and 400 mg/kg p.o. (WBR = 72.3%). Fifty percent effective doses (ED₅₀) of 44.6 mg/kg and 226.8 mg/kg were calculated for diminazene administered i.p. and p.o., respectively. Praziquantel resulted in a total WBR of 93.2%.

TABLE 2.

Effect of diminazene on worm burden in a schistosomiasis model of mice infected with adult Schistosoma mansoni worms^a

Drug, route, and dose (mg/kg)b	No. of mice	No. of wormsc	Worm burden reduction (%)d	ED50 (mg/kg)
Diminazene
Intraperitoneal
0e	10	36.6 ± 5.7		44.6 ± 4.2
10	5	22.1 ± 3.9	31.3 ± 3.9*
25	5	19.4 ± 4.4	47.8 ± 9.4**
50	5	14.8 ± 3.8	60.6 ± 8.7***
100	10	4.8 ± 1.5	87.2 ± 3.9****
Oral
0e	10	34.4 ± 5.7		226.8 ± 8.9
100	5	19.1 ± 1.4	36.7 ± 6.5*
200	5	20.2 ± 1.3	46.3 ± 3.9**
300	5	13.6 ± 1.8	61.1 ± 4.8***
400	10	9.8 ± 1.3	72.3 ± 3.9****
PZQ, oral, 400	5	2.6 ± 1.5	93.2 ± 4.2****

^aPatent infection. Except for the numbers of mice, values are means ± SD.

^bSingle dose on day 49 after infection. PZQ, praziquantel.

^cTotal worm burden (male plus female).

^dAsterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001) from the control group.

^eControl (vehicle-treated mice).

S. mansoni egg production is a key mechanism for both transmission and pathogenesis (1). The influence of diminazene on egg burden also depended on the dosing regimen applied and the route of administration (Table 3). With regard to the intestinal egg load, all egg stages (immature, mature, and dead) were observed in the intestines of infected control and treated mice. However, diminazene at 50 to 100 mg/kg i.p. and 200 to 400 mg/kg p.o. achieved high reductions in the number of immature eggs, demonstrating the efficacy of treatment at the highest doses, mainly intraperitoneally. Interestingly, the oogram showed enormous increases in the proportion of dead eggs. The numbers of eggs in the feces with both treatment regimens, i.p. and p.o, were also markedly lower than those for untreated control animals. The reference drug praziquantel produced highly significant egg reductions in the intestines (immature eggs) and feces.

TABLE 3.

Effect of diminazene on egg burden in a schistosomiasis model of mice infected with adult Schistosoma mansoni worms^a

Drug, route, and dose (mg/kg)b	No. of mice	Intestinal egg burden (%)c			Fecal egg burdend
		Immature eggs	Mature eggs	Dead eggs
Diminazene
Intraperitoneal
0e	10	60.3 ± 5.5	38.7 ± 4.1	0.8 ± 0.2	1,094.6 ± 117.3
10	5	44.8 ± 3.6	51.3 ± 5.3	5.6 ± 2.7	786.2 ± 101.3*
25	5	33.4 ± 4.2	59.1 ± 4.4	8.8 ± 1.5	638.9 ± 84.2**
50	5	15.7 ± 4.8	69.4 ± 3.8	29.6 ± 2.7	231.2 ± 67.9****
100	10	4.6 ± 1.1	46.2 ± 5.2	49.8 ± 4.5	107.8 ± 40.6 ****
Oral
0e	10	58.6 ± 4.7	40.4 ± 3.6	0.5 ± 0.5	1,216.8 ± 227.8
100	5	51.4 ± 3.6	48.7 ± 4.8	2.4 ± 2.2	647.8 ± 103.5**
200	5	40.5 ± 3.7	48.1 ± 2.6	12.9 ± 1.9	456.4 ± 64.9***
300	5	36.9 ± 3.9	43.2 ± 4.1	19.7 ± 1.8	203.2 ± 76.4****
400	10	24.8 ± 2.5	50.8 ± 5.6	23.4 ± 3.2	163.2 ± 52.1****
PZQ, oral, 400	5	2.9 ± 1.6	49.6 ± 6.6	44.8 ± 4.1	39.6 ± 22.7****

^aChronic infection. Except for the numbers of mice, values are means ± SD.

^bSingle dose on day 49 after infection. PZQ, praziquantel.

^cDetermined by oogram analysis.

^dNumber of eggs in the feces, determined by the Kato-Katz technique. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001) from the control group.

^eControl (vehicle-treated mice).

Diminazene reduces liver and splenic injury in chronic S. mansoni infection.

In mice harboring adult S. mansoni worms (chronic infection), there are typical clinical symptoms, including anemia, hepatosplenomegaly, liver granuloma and fibrosis, and ascites. To observe the influence of diminazene on organ disease caused by S. mansoni, the gross morphology of the liver and spleen, liver and spleen weights, and liver biochemistry function were evaluated and analyzed. As shown in Fig. 4, diminazene achieved significant reductions of hepatomegaly (Fig. 4A) and splenomegaly (Fig. 4B), as measured by weight, relative to symptoms in control infected mice, especially at higher doses, by both the enteral and parenteral routes (P < 0.01). Infection of mice with S. mansoni also resulted in elevation of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels over those in healthy (uninfected) controls. Administration of diminazene to infected animals caused significant reductions in AST (Fig. 4C) and ALT (Fig. 4D) levels from those in infected vehicle-treated animals, suggesting that diminazene reduces liver injury (P < 0.01).

FIG 4.

Effects of diminazene on organ pathology in a preclinical mouse model of schistosomiasis. (A) Liver weight; (B) spleen weight; (C) AST levels; (D) ALT levels. A single dose of diminazene, as indicated, was administered intraperitoneally or orally to each mouse harboring a chronic S. mansoni infection. On day 63 postinfection, all animals were humanely euthanized, and organ pathology was determined by liver and spleen weights as well as by serum transaminase (AST and ALT) levels. Data are presented as means ± SD (n, 5 per group). Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) from infected untreated control groups by Dunnett’s test. PZQ, praziquantel at a single oral dose of 400 mg/kg.

Diminazene has moderate, but significant, antischistosomal properties in early S. mansoni infection.

Having obtained encouraging results in chronic S. mansoni infection, we progressed with diminazene at the most effective doses, 100 mg/kg i.p. and 400 mg/kg p.o., to studies using mice harboring immature parasites (early infections), with praziquantel as a reference. As summarized in Table 4, diminazene significantly reduced worm and egg burdens. Although diminazene had moderate antischistosomal properties in a prepatent S. mansoni mouse model, this compound was more effective in early infection than praziquantel. Compared to those in control groups, liver and spleen weights were not significantly reduced with either diminazene or praziquantel.

TABLE 4.

Effect of diminazene in a schistosomiasis model of mice infected with immature Schistosoma mansoni worms^a

Drug, route, and dose (mg/kg)b	No. of mice	Reduction (%) in total burdens (male plus female)c			Wt (g) of:
		Worms	Intestinal eggs	Fecal eggs	Liver	Spleen
Diminazene
Intraperitoneal
0d	5				2.31 ± 0.2	0.71 ± 0.1
100	5	54.7** ± 5.3	46.4** ± 3.8	58.1** ± 7.1	2.23 ± 0.3	0.58 ± 0.1
Oral
0d	5				2.43 ± 0.3	0.66 ± 0.1
400	5	41.6* ± 4.9	34.5* ± 5.5	48.6* ± 6.3	2.18 ± 0.3	0.58 ± 0.1
PZQ, oral, 400	5	28.8* ± 5.7	22.3 ± 7.4	30.4* ± 3.9	2.11 ± 0.2	0.63 ± 0.1

^aEarly infection. Except for the numbers of mice, values are means ± SD.

^bSingle dose on day 21 after infection. PZQ, praziquantel.

^cAsterisks indicate significant differences (*, P < 0.05; **, P < 0.01) from the control group.

^dControl (vehicle-treated mice).

DISCUSSION

With no available alternative drug, praziquantel is the mainstay for controlling schistosomiasis and other flatworm infections in humans, domestic animals, and livestock. In this study, we demonstrate for the first time that the small-molecule drug diminazene aceturate exhibits anthelmintic properties against the blood fluke S. mansoni in vitro and also in a murine model of schistosomiasis for both early and chronic S. mansoni infections. This in vivo effect of the drug was associated with marked reductions in worm burdens and egg production, attenuation of hepatosplenomegaly, and a reduction of liver injury.

Diminazene has been explored previously for its antiparasitic capabilities against blood protozoa such as Trypanosoma and Babesia species (12). Of interest to the current study, both larval and adult stages of S. mansoni are susceptible to diminazene in vitro. Compared to the positive control praziquantel, diminazene exhibited significantly higher efficacy in the larval stage. However, praziquantel had a very fast onset of action on adult schistosomes and is much more effective than diminazene. As shown in the microscopic images, high concentrations of diminazene are required to cause tegumental damage in schistosomes. This variation in susceptibility with respect to parasite age has been described for several compounds (13, 14). Praziquantel, for example, primarily targets adult schistosomes. Significant morphological, physiological, and biochemical changes occur in the developing parasite, and disparities in drug susceptibility between immature and adult schistosomes may be related to existing differences in membrane structure and organization (15, 16). Interestingly, although diminazene was poor as a biocidal agent against adult worms, in vitro assays showed that diminazene acted on S. mansoni egg laying at sublethal concentrations. This reduction in oviposition suggests that diminazene meets an important criterion for the selection of anthelmintic compounds (17). In addition, it is known that some drugs that are slightly active in vitro can be highly effective in vivo (e.g., oxamniquine) (18).

Diminazene can be administered through either the enteral or the parenteral route. However, this compound is usually administered parenterally in a single dose or in repeated doses (for days or even a few weeks) in order to obtain a high concentration of diminazene in the circulating blood (9, 19). In this study, we chose both the intraperitoneal and oral routes, using different doses administered only once. These doses were selected according to previous studies of diminazene or other antischistosomal agents (4). Of note, a single dose is recommended for schistosomiasis treatment (4, 5). Additionally, since schistosomiasis is characterized as a chronic disease, we initially evaluated the effect of diminazene in a chronic S. mansoni infection. Interestingly, diminazene markedly reduced worm burdens. For example, an ED₅₀ of 226.8 mg/kg was calculated for diminazene given orally, which is close to the value for praziquantel (20). Intraperitoneally, diminazene had an ED₅₀ value as low as 44.6 mg/kg. However, no mice were cured even at the highest doses of diminazene (100 mg/kg i.p. or 400 mg/kg p.o.) or the standard dose of praziquantel (400 mg/kg p.o.). It is noteworthy that only moderate worm burden reductions were achieved when diminazene was administered to mice harboring early S. mansoni infections. Therefore, in contrast to the findings of in vitro studies, the best results were achieved in chronic infection. These differences between the therapeutic effects of diminazene against immature and adult stages of S. mansoni may be due to the host’s immune response (16).

Schistosoma eggs play a fundamental role in the transmission of schistosomiasis (1). Treatment of S. mansoni-infected mice with diminazene greatly reduced the number of eggs in feces. Quantitative and qualitative oogram analyses also revealed a significant reduction in the number of immature eggs and an increase in the frequency of dead eggs. This result could be attributed to a decrease in the number of adult schistosomes because of treatment with diminazene or a reduction of egg laying by female worms after treatment. Similar findings were also obtained with other antischistosomal compounds (21, 22).

Egg production is also a crucial factor in pathogenesis and can result in systemic pathological effects or organ-specific effects, leading to severe pathology, such as hepatosplenomegaly, periportal hepatic fibrosis, and even death (23). In the current study, we observed hepatomegaly and splenomegaly in untreated mice infected with S. mansoni. These results are consistent with the observation of elevated levels of aminotransferases (ALT and AST) in the sera of Schistosoma-infected animals over those for healthy (noninfected) mice. Similar findings for liver injury and elevations in liver enzyme levels were also obtained both in animal experimental studies (24) and in clinical medical studies of patients with schistosomiasis (25). In the present study, we found that diminazene achieved significant reductions of liver and spleen weights and lowered ALT and AST levels. This benefit of diminazene in reducing liver injury in Schistosoma-infected animals may be associated with a reduction in the number of worms and/or in egg burden. Recent studies have demonstrated the beneficial effect of diminazene in liver diseases (11, 26), and this drug could provide therapeutic benefits for the treatment and control of several chronic inflammatory diseases (21). Therefore, the reduction in liver injury observed in this study may also be due to the direct effect of diminazene.

Toxicity data for diminazene have been reported in the medical and veterinary fields (for reviews, see references 9 and 27). With regard to the in vitro cytotoxic profile, it was shown that diminazene at 100 μM (a concentration below the 50% lethal concentrations [LC₅₀] described here for larval and adult stages of S. mansoni) does not alter the viability of several human and animal cell lines (see, e.g., references 28, to ,30). Animal toxicity is apparently species dependent. For example, diminazene has been shown to be toxic to camels, dogs, and horses, but not to cattle or the usual laboratory species, such as rats or mice (26, 27). Interestingly, studies conducted by Baldissera et al. (31) reported that diminazene is cytotoxic to liver and kidney cells. Conversely, diminazene has been associated with attenuation of liver injury (11). In humans, despite the use of this drug for patients suffering from trypanosomiasis, little toxicity has been documented (9, 27). For these reasons, although no sign of toxicity was noted in our study, the use of diminazene as an antischistosomal agent may be limited.

In conclusion, we have demonstrated that diminazene has in vitro anthelmintic properties against different stages of intravascular parasitic S. mansoni in a time- and concentration-dependent manner. Morphological studies using confocal laser scanning microscopy also revealed substantial tegumental alterations in adult parasites. We have also demonstrated that diminazene markedly reduces worm and egg burdens in mice harboring chronic S. mansoni infections. Since the liver carries the main burden of S. mansoni infection, we found that diminazene achieved significant reductions in hepatosplenomegaly and lowered ALT and AST levels, suggesting that the drug reduces liver injury. Diminazene, which is commonly used in veterinary practice, is FDA approved and has been successfully used in clinical practice to treat human and animal trypanosomiasis in tropical and subtropical countries. The present evidence suggests that diminazene has the potential to treat schistosomiasis. However, future studies should look at extending this work to other Schistosoma species and host-parasite models, as well as investigating animals infected with other trematodes and nematodes or protozoan parasites that affect the liver.

MATERIALS AND METHODS

Drug and reagents.

Diminazene aceturate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Praziquantel was kindly provided by Ecovet (São Paulo, SP, Brazil). Unless otherwise noted, all materials/reagents were obtained from commercial suppliers and used without further purification. RPMI 1640 culture medium, Basch medium 169, heat-inactivated fetal calf serum (FBS), and penicillin G-streptomycin solutions (10,000 U/ml penicillin G sodium salt, 10 mg/ml streptomycin sulfate) were obtained from Vitrocell (Campinas, SP, Brazil). HEPES buffer and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO). In all experiments, diminazene was solubilized in saline solution, whereas praziquantel was dissolved in DMSO.

Parasites and hosts.

The life cycle of Schistosoma mansoni (strain BH) was maintained using Biomphalaria glabrata snails and mice by following the standard procedures of our laboratory (32). Three-week-old Swiss mice (purchased from Anilab, São Paulo, Brazil) were infected by subcutaneous injection with approximately 150 S. mansoni cercariae each. Animals were maintained with free access to a rodent diet and water under environmentally controlled conditions (25°C; humidity, 50%). Animal experiments were carried out at the animal facility at Universidade Guarulhos (UNG) and were approved by the UNG Animal Ethics Committee (no. 031/17).

In vitro studies.

The in vitro antischistosomal properties were evaluated against immature (newly transformed schistosomula) worms and adult (49-day-old) worms ex vivo.

(i) Assay using immature schistosomes.

Drug testing was performed on immature worms as reported previously (13). Briefly, newly transformed schistosomula were obtained by mechanical transformation using a vortex mixer. Parasites were then cultivated for 3 h prior to the bioassay in medium 169 containing antibiotics and supplemented with 10% FBS and 1% (vol/vol) penicillin-streptomycin solution at 37°C under a 5% CO₂ atmosphere. For in vitro bioassays, schistosomula were transferred to 24-well culture microplates (Corning, New York, NY, USA) containing approximately 50 parasites/well and were cultured in medium 169. Parasites were maintained continuously in medium (with or without drugs) for 120 h at 37°C under a 5% CO₂ atmosphere. The following drug concentrations were evaluated: 200, 100, 50, 25, 12.5, 6.25, and 3.12 μM (equivalent to 100, 50, 25, 12.5, 6.25, 3.12, and 1.56 μg/ml, respectively). Each drug concentration was tested at least in triplicate, and the experiments were repeated three times. Drug-free medium served as a control. The viability of schistosomula was recorded visually at 2, 8, 24, 48, 72, 96, and 120 h using an inverted microscope (33).

(ii) Assay using adult schistosomes.

Drug testing on adult worms was performed as reported previously (34, 35). Briefly, adult schistosomes were collected from the hepatic portal and mesenteric veins of an infected rodent. Parasites (one pair per well) were then incubated in a 24-well culture plate containing RPMI 1640 culture medium supplemented with 10% FBS and 1% (vol/vol) penicillin-streptomycin solution at 37°C under 5% CO₂. Diminazene was dissolved in saline solution to obtain final test concentrations as mentioned above (3.12 to 200 μM) in culture plates with a final volume of 2 ml. Each drug concentration was tested at least in triplicate, and the experiments were repeated three times. Drug-free medium served as a control. Cultures were incubated at 37°C under 5% CO₂, and the parasites were kept for 120 h and were monitored at 2, 8, 24, 48, 72, 96, and 120 h using both a light microscope and a stereomicroscope. The effect of the drug was assessed, with emphasis on changes in worm motor activity, oviposition, and alteration in the tegument (36, 37).

(iii) Microscopy analysis.

During the in vitro experiments, immature and adult worms were monitored using an inverted light microscope (INV100; BEL Engineering, Monza [MB], Italy) and a stereomicroscope (EZ4E; Leica Microsystems, Wetzlar, Germany) (38). In addition, morphological analysis on the teguments of schistosomes was performed using a confocal laser scanning microscope (LSM 510 Meta; Zeiss, Göttingen, Germany) whose experimental protocols have been published previously (39, 40). Briefly, the parasites were fixed in a formalin-acetic acid-alcohol (FAA) solution, and autofluorescence was excited with an argon-ion laser at 488 nm.

In vivo chemotherapeutic studies.

For in vivo efficacy studies, 3-week-old mice were infected subcutaneously with 80 S. mansoni cercariae each. First, animals were randomly divided into experimental groups (5 mice per group), and diminazene (10 to 100 mg/kg) was administered intraperitoneally (i.p.) or by oral gavage (p.o.) 49 days postinfection (adult stage; chronic infection). In another experiment, diminazene was administered at 100 mg/kg i.p. and 400 mg/kg p.o. to groups of five S. mansoni-infected mice 21 days postinfection (immature stage; early infection). For each experiment, infected but only vehicle-treated mice (5 mice per group) served as controls.

On day 63 postinfection, animals in all groups were weighed and euthanized, and then adult worms were perfused in RPMI 1640 medium (21). For the measurement of worm burdens, schistosomes were collected from the hepatic portal system and mesenteric veins, sexed, and counted. Therapeutic efficacy was also determined on the basis of the technique of qualitative and quantitative oograms in intestines, as well as the Kato-Katz method for quantitative fecal examination (41, 42). Finally, to examine the effect of diminazene on hepatosplenomegaly induced by S. mansoni infection, the blood was collected for biochemical analysis, and both the liver and the spleen were excised and weighed. Groups of five healthy (uninfected) animals also served as controls.

Assessment of biochemical parameters.

Serum was obtained by centrifugation of animal blood at 3,500 rpm for 15 min to examine liver function. Alanine aminotransferase (ALT) and aspartate transaminase (AST) were measured using commercial kits (Labtest, Lagoa Santa, MG, Brazil) according to the manufacturer’s instructions.

Statistical analysis.

Statistical analyses were performed using GraphPad Prism software, and they are in accordance with recommendations in the pharmacology field (41). All data from the in vitro anthelmintic experiments are presented as means ± standard deviations (SD) for at least three independent assays. Fifty percent effective concentrations (EC₅₀) and EC₉₀ in vitro, and 50% effective doses (ED₅₀) in vivo, were calculated using sigmoid dose-response curves and 95% confidence intervals (41, 43). Kaplan-Meier survival analyses were also used to compare in vitro survival data. For in vivo studies, a parametric Dunnett test was applied to compare the control group with the treated group. The level of statistical significance was set to a P value of <0.05.

Randomization and blinding.

Animal studies are reported in compliance with the ARRIVE guidelines of the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) (40, 44). Mice were randomly assigned to the experimental groups, and pharmacological treatments were performed randomly as well. The mice were also euthanized in a random manner inside the groups. All parameters were measured by different people, and experiments were carried out by at least two different investigators. In order to eliminate bias in interpretation, the researchers who conducted the experiments were not the same as those who analyzed the data.