Uptake and effect of praziquantel and the major human oxidative metabolite, 4-hydroxypraziquantel, by Schistosoma japonicum

After exposure to praziquantel in vitro at a concentration of 1 microgram/ml for 0.5-2 hr, amounts of praziquantel in Schistosoma japonicum varied from 2.1 +/- 1.2 to 3.7 +/- 1.6 ng/male worm and 1.3 +/- 1.2 to 2.2 +/- 1.5 ng/female worm during the time studied. At 30 micrograms/ml, praziquantel amounts were 11-33-fold higher. However, within 2 hr after removal from a medium containing 30 micrograms/ml praziquantel, 95% of the drug was released from the parasites. When S. japonicum worm pairs were incubated in vitro with 1, 10, and 30 micrograms/ml of 4-hydroxypraziquantel, the major human oxidative metabolite of praziquantel, 0.2 +/- 0.2, 3.8 +/- 1.3, and 7.4 +/- 1.3 ng/worm pair, respectively, were found after a 2-hr incubation. 15-30-fold lower than corresponding worm pair amounts of praziquantel. In vivo, when 4- or 5-wk S. japonicum-infected mice were treated orally with praziquantel (300 mg/kg), peak concentrations of praziquantel in plasma determined by high pressure liquid chromatography were 14.7 +/- 1.5 micrograms/ml (4-wk infection) and 16.7 +/- 2.8 micrograms/ml (5-wk infection) 15 min after treatment. Corresponding in vivo worm praziquantel amounts were 1.8 +/- 0.4 ng/male worm and 2.4 +/- 1.1 ng/female worm, respectively, in the 4-wk infection and 4.6 +/- 1.6 ng/male worm and 5.6 +/- 1.2 ng/female worm in the 5-wk infection. Peak plasma concentrations of 4-hydroxypraziquantel were similar but corresponding in vivo worm amounts were 1-20-fold lower, depending on the time after drug administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Praziquantel derivatives exhibit activity against both juvenile and adult Schistosoma japonicum

A praziquantel analog 10-hydroxy praziquantel and eight praziquantel/peroxide conjugates were synthesized. The biological activity of these compounds was evaluated against juvenile and adult stages of Schistosoma japonicum. Unlike praziquantel, 10-hydroxy praziquantel exhibits activity against both juvenile and adult Schistosoma japonicumin. All hybrid compounds displayed modest to significant worm killing activity. The present study has important significance for the development of hybrid antischistosomal drugs.     

Effect of praziquantel together with artemether on Schistosoma japonicum parasites of different ages in rabbits

The efficacy of combined treatment with praziquantel and artemether against infection with Schistosoma japonicum was tested on infected rabbits, in which 7-to 14-day-old schistosomules and 42-day-old adult schistosomes were simultaneously present. Rabbits were treated orally with praziquantel and artemether using various dosages and schedules. The therapeutic effects were evaluated by estimating the mean total worm burden (TWB) and female worm burden (FWB) and comparing them with the worm burdens in control animals treated with praziquantel or artemether alone. When the rabbits received praziquantel in a single dose (50 mg/kg), or daily for 2-6 days (30-60 mg/kg), the TWB was reduced by 28-66% and the FWB by 26-65%. In rabbits treated with artemether the reductions were 44-56% and 35-54%, respectively. Treatment with praziquantel in combination with artemether resulted in a significantly greater reduction of worm burden than was found for the groups treated with praziquantel or artemether alone, using the same dosages and schedules. TWB was reduced by 79-92%, and FWB by 80-93%. The results demonstrated that when rabbits infected simultaneously with schistosomules and adult schistosomes were treated with praziquantel in combination with artemether, the effects of the individual drugs could be increased significantly.     

Sex- and stage-related sensitivity of Schistosoma mansoni to in vivo and in vitro praziquantel treatment

The efficacy of praziquantel against a Puerto Rican strain of Schistosoma mansoni was assessed using both in vivo and in vitro approach. The drug effective dose (50%) in the infected mouse model was about 30 times higher when determined against 28-day-old infections than against 7-week-old parasites. Single-sex female infections were also largely refractory to treatment and single-sex male infections moderately refractory, in comparison with bisexual infections. The in vitro approach consisted of overnight exposure of parasite cultures to various drug concentrations, followed by several days of culture in drug-free medium. In vitro results confirmed in vivo data and allowed for the observation of schistosome morphological phenomena after praziquantel exposure. Early worm contraction was observed in all cases, even after exposure to sub-lethal concentrations of praziquantel or upon exposure of the largely refractory 28-day-old schistosomes. In these instances, however, worms resumed movements and normal shape upon drug removal and were able to survive. The inference of these observations on the clinical use of praziquantel and on its mechanism of action is discussed.     

Effects of praziquantel and oxamniquine on a Saudi Arabian strain of Schistosoma mansoni in mice

The prophylactic and curative effects of praziquantel and oxamniquine on a Saudi Arabian strain of Schistosoma mansoni in MF-1 mice were assessed. The drugs were administered orally. At 240 mg/kg praziquantel, there was a reduction of 89.1% in adult worm recovery and a marked reduction in tissue deposited eggs. The reduction in adult worm recovery after dosing with 50 mg/kg oxamniquine was 89.2%. At low doses (40 mg/kg praziquantel and 30 mg/kg oxamniquine) administered at 11 days, 5 days and 3 h before and 5, 21 and 49 days after infection, the reduction in adult worm recovery was 0.0%, 65.1%, 58.8%, 33.6%, 0.0% and 76.0% for praziquantel and 0.0%, 66.0%, 60.0%, 41.3%, 10.8% and 79.0% for oxamniquine. Numbers of lung schistosomula and the size of hepatic granulomata were also reduced.     

Drug effects on the 5-HT response of Schistosoma mansoni

Several vertebrate 5-HT antagonists at concentrations around 0.1 mM reduced 5-HT-induced increases in the motor activity of the parasitic blood fluke Schistosoma mansoni. The order of potency for 5-HT response antagonism was haloperidol greater than cyproheptadine greater than mianserin greater than trazodone greater than spiperone greater than methysergide. Nisoxetine, a 5-HT uptake inhibitor in vertebrate preparations, was also a potent antagonist of the 5-HT response in schistosomes. The potent antischistosomal praziquantel reduced the 5-HT response similarly to the other antiserotonergic drugs, but at much lower concentrations, beginning around 0.1 microM. The 5-HT agonist quipazine stimulated worm activity at 1-0.1 mM when applied alone, but reduced the 1 mM 5-HT response when quipazine and 5-HT were administered concurrently. Dopamine (DA) alone had no effect on the overall activity of S. mansoni. Although no drug was found to have absolute species specificity, quantitative differences were observed between the relative activity of drugs in schistosomes and vertebrates.     

In vitro responses of praziquantel-resistant and -susceptible Schistosoma mansoni to praziquantel

The resistance status of five praziquantel-susceptible and five praziquantel-resistant isolates was confirmed by chemotherapy in CD(1) mice with 3 x 200mg/kg micronised praziquantel. Micronised praziquantel had higher efficacy than two other praziquantel formulations (prepared without milling). The five resistant isolates were less responsive to praziquantel than the five susceptible isolates (59-74% reduction in worm burden in resistant isolates compared with 92-100% in susceptible isolates). Observations were made on the in vitro responses of different stages of 10 isolates to praziquantel. There were different in vitro responses to praziquantel at the egg, miracidial, cercarial and adult stages of Schistosoma mansoni between praziquantel-resistant and praziquantel-susceptible isolates. There were differences in the response of resistant and susceptible isolates following exposure of freshly hatched miracidia to 10(-6)M praziquantel for 1 min and observing the percent change in shape. Using this test it should be possible to determine whether failed therapy in patients infected with S. mansoni is due to the presence of praziquantel-resistant worms. Similarly, by exposing freshly shed cercariae to 4 x 10(-7)M praziquantel and observing the percent of tail shedding over 80 min it should be possible to monitor for the presence of praziquantel-resistant worms in snails collected in the field.     

Identification of oxadiazoles as new drug leads for the control of schistosomiasis

Treatment for schistosomiasis, which is responsible for more than 280,000 deaths annually, depends almost exclusively on praziquantel. Millions of people are treated annually with praziquantel, and drug-resistant parasites thus are likely to evolve. Phosphinic amides and oxadiazole 2-oxides, identified from a quantitative high-throughput screen, were shown to inhibit a parasite enzyme, thioredoxin glutathione reductase (TGR), with activities in the low micromolar to low nanomolar range. Incubation of parasites with these compounds led to rapid inhibition of TGR activity and parasite death. The activity of the oxadiazole 2-oxides was associated with a donation of nitric oxide. Treatment of schistosome-infected mice with 4-phenyl-1,2,5-oxadiazole-3-carbonitrile-2-oxide led to marked reductions in worm burdens from treatments against multiple parasite stages and egg-associated pathologies. The compound was active against the three major schistosome species infecting humans. These protective effects exceed benchmark activity criteria set by the World Health Organization for lead compound development for schistosomiasis.     

Effect of praziquantel treatment on pulmonary lesions of rats infected with Paragonimus iloktsuenensis

An experimental pathological study was performed to observe the effect of praziquantel treatment on the pulmonary lesions of the rat lung fluke, Paragonimus iloktsuenensis. The metacercariae were obtained from the freshwater crab, Sesarma dehaani, and 40 rats (wistar) were fed each with 10 metacercariae. On 20 rats praziquantel treatment (100mg/kg/day x 5 days) was done at 5 weeks after the infection while remaining 20 rats were kept untreated for use as controls. The drug-treated rats and the untreated ones were sacrificed 3, 7, 14, 21 or 28 days later for the observation of lung pathology. The rats infected with P. iloktsuenensis showed remarkable pulmonary changes; gross features of hemorrhagic and nodular worm capsules protruded on to the surface of the lung, and histologically local atelectasis, inflammatory cell infiltration, and egg granuloma around the worm capsules each containing one or two worms. Praziquantel treatment of the rats was shown to be highly effective in killing the worms and to lead them to degenerate, as early as in 3 days post-treatment. Almost all worms in the lung were dead and absorbed by the host cells in 21 days post-treatment, except a few living ones seen in a rat of 14-day post-treatment group. In most of the rats treated the pulmonary lesions showed the signs of resolution; regression of worm capsules with mummification of worms, decrease of inflammatory cell infiltration, improvement in the degree of atelectasis, and decreases in the frequency and size of the egg granuloma. From the results it is concluded that praziquantel is highly effective for the treatment of rat P. iloktsuenensis infection in the lung, not only by its direct killing effect of the worms but also due to the excellent resolution capacity of the pulmonary tissues.     

Egg Laying Capacity of Haplorchis taichui (Digenea: Heterophyidae) in Humans

Quantitative fecal egg counts represented as the number of eggs per gram of feces (EPG) are generally a reliable parameter to estimate the worm burden of intestinal and hepatic parasitoses. Although Haplorchis taichui (Digenea: Heterophyidae) is one of the most common minute human intestinal flukes, little is known about the relationship between EPG and the actual worm burden in patients or the severity of the disease. In the present study, fecal samples were collected from 25 villagers in northern Thailand before and after praziquantel treatment. The EPG values of each participant were determined by the modified cellophane thick smear method, and adult worms were collected from the whole stool after the treatment. Eggs per day per worm (EPDPW) of H. taichui were estimated 82 from egg counts and expelled worms. The EPG was not well correlated with the worm burden, and a reverse correlation was observed between the EPDPW and the worm burden.     

The Schistosoma mansoni Cytochrome P450 (CYP3050A1) Is Essential for Worm Survival and Egg Development

Schistosomiasis affects millions of people in developing countries and is responsible for more than 200,000 deaths annually. Because of toxicity and limited spectrum of activity of alternatives, there is effectively only one drug, praziquantel, available for its treatment. Recent data suggest that drug resistance could soon be a problem. There is therefore the need to identify new drug targets and develop drugs for the treatment of schistosomiasis. Analysis of the Schistosoma mansoni genome sequence for proteins involved in detoxification processes found that it encodes a single cytochrome P450 (CYP450) gene. Here we report that the 1452 bp open reading frame has a characteristic heme-binding region in its catalytic domain with a conserved heme ligating cysteine, a hydrophobic leader sequence present as the membrane interacting region, and overall structural conservation. The highest sequence identity to human CYP450s is 22%. Double stranded RNA (dsRNA) silencing of S. mansoni (Sm)CYP450 in schistosomula results in worm death. Treating larval or adult worms with antifungal azole CYP450 inhibitors results in worm death at low micromolar concentrations. In addition, combinations of SmCYP450-specific dsRNA and miconazole show additive schistosomicidal effects supporting the hypothesis that SmCYP450 is the target of miconazole. Treatment of developing S. mansoni eggs with miconazole results in a dose dependent arrest in embryonic development. Our results indicate that SmCYP450 is essential for worm survival and egg development and validates it as a novel drug target. Preliminary structure-activity relationship suggests that the 1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethan-1-ol moiety of miconazole is necessary for activity and that miconazole activity and selectivity could be improved by rational drug design.     

Prevention of Schistosoma japonicum infection in mice with long-acting praziquantel implants

This work reports the prevention outcomes of a praziquantel (PZQ) implant against the infection of Schistosoma japonicum in mice. The PZQ implant produced stable plasma PZQ concentrations in a range of 100-1300 ng/mL for a period of 70 days, by releasing PZQ in subcutaneous tissues in a sustained manner. To assess the prevention effects, the mice were infected at varying times after implantation. All the mice were sacrificed at 6 weeks after infection for worm and egg recovery and counting, worm morphological examination, determination of egg-hatching rates, and analysis of hepatic histology. The infection was successfully prevented for mice with early infection times (within 2-3 weeks), as nearly no worms, paired worms, eggs, or miracidia were recovered. However, in mice with late infection times (after 3 weeks), the prevention effects were diminished due to the decreased plasma PZQ concentrations at late times. Interestingly, the implants showed robust prevention effects on repeated infection at 1 and 3 weeks. In the infection-prevented mouse livers, no granuloma formation or granulomatous inflammation was observed. The results demonstrated that by blocking the development of infecting miracidia and by deactivating the eggs, the PZQ implants encouragingly prevented the S. japonicum infection and avoided liver damage.     

Adverse effects of praziquantel treatment of Schistosoma japonicum infection: involvement of host anaphylactic reactions induced by parasite antigen release

The present study using a murine model heavily infected with Schistosoma japonicum aimed to elucidate the pathogenesis of adverse effects of praziquantel treatment of schistosome-infected subjects. Inbred BALB/c mice were infected with S. japonicum (Yamanashi strain) before being treated with a single dose of praziquantel at 4 or 8 weeks p.i. All the mice treated at 8 weeks p.i. exhibited signs typical of systemic anaphylaxis until half of them died shortly after praziquantel administration. At autopsy, these mice exhibited remarkable intestinal alterations characterised by increased mucosal permeability, mucosal oedema and petechial haemorrhage, which are changes typical of immediate intestinal anaphylaxis. In these mice treated at 8 weeks p.i., degranulation of intestinal mast cells was frequently observed, which was particularly remarkable around S. japonicum eggs hatched as an effect of praziquantel. Furthermore, the plasma histamine concentration just after praziquantel treatment was much higher in mice at 8 weeks p.i. than that in uninfected mice or in S. japonicum-infected mice without drug treatment. In contrast, none of these intestinal changes was observed in untreated or uninfected control mice, or in mice administered praziquantel at 4 weeks p.i., in which worm pairs had just reached sexual maturation and begun egg-laying. The finding by ELISA that serum IgM and IgA levels specific to S. japonicum eggs decreased immediately after praziquantel treatment, together with the results of immunohistochemistry, revealed the sudden release of parasite antigens from the eggs hatched by praziquantel treatment. The results of this study demonstrate that adverse effects of praziquantel treatment of schistosomiasis characterised by abdominal signs depend on anaphylactic reactions due to parasite antigens, especially antigens from eggs hatched as an effect of praziquantel.     

The effect of praziquantel on the pseudophyllidean cestode Bothriocephalus acheilognathi in vitro

Adult Bothriocephalus acheilognathi were incubated in solutions containing 0 (control), 0.1, 1.0, 10.0 and 100 micrograms praziquantel per ml (0, 10(2), 10(3), 10(4) and 10(5) micrograms l-1) of 0.9% saline for 5, 15 and 60 min at a temperature of 18 degrees C. The worms contracted immediately upon being placed in the drug. Scanning and transmission electron microscopy revealed considerable tegumental damage particularly in the neck region. Vacuolization and 'bubbling' of the tegument occurred in all of the drug solutions tested. Exposure to drug concentrations of more than 1.0 micrograms ml-1 (10(3) micrograms l-1) praziquantel for 15 min or greater resulted in many of the 'bubbles' bursting and releasing their contents to the exterior. Mature proglottides were distorted and had occasional large swellings resulting in the mass expulsion of eggs. Praziquantel had no ovicidal activity. Exposure to drug concentrations of 100 micrograms (10(5) micrograms l-1) praziquantel per ml saline for 24 h was not lethal to the worms.     

Artemether as adjuvant therapy to praziquantel in murine Egyptian schistosomiasis mansoni

We investigated the activity of artemether (ART) against different developmental stages of schistosomes alone and in addition to praziquantel (PZQ). ART was administered orally (400 mg/kg) 4 and 6 wk postinfection (PI), 4 and 5 wk PI, or 4 or 6 wk PI alone and in addition to oral PZQ (500 x 2 mg/kg) 6 wk PI. Mice were killed in parallel to infected untreated controls 8 wk PI. Parasitological parameters and histological changes in the liver were studied. ART given 4 and 6 wk PI reduced worm burdens by 59 and 55% and tissue egg load by 96 and 90%, respectively. Moreover, eggs in different developmental stages were not found. The reduction in worm and egg burden (63 and 58%, and 96 and 99%, respectively) in mice treated with ART 4 and 5 wk or 4 and 6 wk PI was comparable with that in ART-treated mice at 4 or 6 wk PI. Compared with PZQ alone, combined treatment of PZQ and ART (4 and 5 wk or 4 and 6 wk PI) did not enhance worm eradication, but there was a complete absence of parasite eggs. Livers revealed no granulomata when ART was given 4 and 5 wk or 4 and 6 wk PI, with minimal central necrosis in those treated 4 and 6 wk PI. In conclusion, combined treatment of ART (4 and 6 wk PI) and PZQ resulted in >90% worm eradication and amelioration of Schistosoma mansoni eggs from the tissues, with minor histological changes in the liver.     

Depletion of praziquantel in plasma and muscle tissue of cultured rockfish Sebastes schlegeli after oral and bath treatment

Depletion of praziquantel in plasma and muscle tissue after oral and bath treatments was studied in cultured rockfish Sebastes schlegeli. In the oral treatment, a single dose of 400 mg praziquantel kg(-1) body weight was administered by intubation of the stomach. A bath treatment at 100 ppm of praziquantel for 4 min was also carried out. Plasma and muscle tissue samples were collected at 3, 6, 12, 24, 48, 72, 96, 120, 144 and 168 h post-treatment, and analyzed for praziquantel by reversed-phase HPLC using diazepam as the internal standard. Following oral treatment, praziquantel was detected in plasma and muscle tissue until 96 h after treatment. In plasma the praziquantel concentration was highest at the 9 h sampling time and declined sharply at the 48 h sampling point. The concentrations of praziquantel in the muscle tissue were lower than those in the plasma, and the highest value was found at the 9 h sampling time. Following bath treatment, praziquantel was found in plasma and muscle tissue until 72 and 24 h after treatment, respectively. In plasma the praziquantel concentration was highest at the 12 h sampling time and declined sharply thereafter. The concentrations of praziquantel in the muscle tissue were significantly lower than those in the plasma, and the concentrations declined consistently with time.     

Stability and reproductive fitness of Schistosoma mansoni isolates with decreased sensitivity to praziquantel.

These studies are focused on schistosomes derived from human infections not cured by three successive doses of praziquantel that also produced infections in mice that were significantly more difficult to cure than infections with control worms. Half (three of six) of these isolates retained their decreased response to praziquantel after multiple passages through the life-cycle in the absence of therapeutic pressure. Two of the isolates, including the one initially least sensitive to praziquantel; reverted, to a sensitivity not significantly different from controls. For example, the EE6 isolate initially required 680 mg/kg praziquantel to affect a 50% reduction in worm load in murine infections, but after only six passages through the life cycle over 5 years this was reduced to 113 mg/kg, not different from control infections. The stability of some of the isolates and the reversion of others indicates that the biological or genetic factors conferring decreased praziquantel response varies among the isolates. The three isolates that retained decreased sensitivity to praziquantel all showed compromises in reproductive fitness in the laboratory, expressed most frequently as a decreased cercarial production from snails infected with those isolates compared to controls. For example, the total cercarial production of snails infected with the EE10 isolate was only 57% that of controls. The reversion of some of the isolates to a praziquantel sensitive state and the decreased reproductive fitness of those that did not revert suggest that there is some biological cost associated with the relative praziquantel insensitivity of these worms, which could help limit the impact of such isolates in the field. Infections with the less sensitive isolates also produced significantly less circulating schistosomal antigen in mice, suggesting that a decrease in the host immune response elicited by these worms could be one of the factors contributing to the diminished praziquantel efficacy.     

Schistosoma japonicum: treatment of different developmental stages in mice with long-acting praziquantel implants

This paper reports the effective treatment of Schistosoma japonicum in a mouse model with long-acting praziquantel (PZQ)-loaded poly(ε-caprolactone) implants. The implants yielded stable, high plasma PZQ concentrations ranging 100–1600 ng/mL during the 40-day investigation period. For assessment of efficacy, the implants were implanted into mice immediately after infection and at 1, 2, 3 and 4 weeks after infection to treat the schistosomes at different developmental stages. All the mice were sacrificed at 6 weeks after infection for worm and egg recovery, worm morphology examination, and histopathological analysis of implantation site tissues. The worm burdens, egg burdens, and numbers of miracidia hatched from the retrieved eggs for all the implant-treated groups (except groups T2-A, T4 and T5) were reduced by 100% when compared with the control group. From groups T2-A, T4 and T5, some schistosome debris was recovered. Eggs were found in only group T5 for which the time between infection and implantation was 4 weeks, which enabled the maturation of juvenile female schistosomes into adult ones that lay eggs. Histopathological observations of implantation tissue showed no evidence of granulomatous foreign-body or lymphoid cell aggregation, demonstrating good biocompatibility of the PZQ implants. These results demonstrate that the long-acting PZQ implants can kill schistosomes at any developmental stages and attenuate/avoid the associated liver damage.     

Ameliorative effects of Schisandrin B on Schistosoma mansoni-induced hepatic fibrosis in vivo

Schistosomiasis is second only to malaria as the most devastating parasitic disease in the world. It is caused by the helminths Schistosoma mansoni (S. mansoni), S. haematobium, or S. japonicum. Typically, patients with schistosomiasis suffer from symptoms of liver fibrosis and hepatosplenomegaly. Currently, patients were treated with praziquantel. Although praziquantel effectively kills the worm, it cannot prevent re-infection or resolve liver fibrosis. Also, current treatment options are not ample to completely cure liver fibrosis and splenic damages. Moreover, resistance of praziquantel has been reported in vivo and in vitro studies. Therefore, finding new effective treatment agents is urgently needed. Schisandrin B (Sch B) of Schisandra chinensis has been shown to protect against different liver injuries including fatty liver disease, hepatotoxicity, fibrosis, and hepatoma. We herein investigate the potential of using Sch B to treat S. mansoni-induced liver fibrosis. Results from the present study demonstrate that Sch B is beneficial in treating S. mansoni-induced liver fibrosis and splenic damages, through inhibition of inflammasome activation and apoptosis; and aside from that regulates host immune responses. Besides, Sch B treatment damages male adult worm in the mice, consequently helps to reduce egg production and lessen the parasite burden.     

Development of chiral praziquantel analogues as potential drug candidates with activity to juvenile Schistosoma japonicum

A series of chiral praziquantel analogues were synthesized and evaluated against Schistosoma japonicum both in vitro and in vivo. All compounds exhibited low to considerable good activity in vivo. Remarkably, worm reduction rate of R-3 was 60.0% at a single oral dose of 200 mg/kg against juvenile stage of Schistosoma japonicum. The target compounds displayed in vivo antischistosomal activity against both Schistosoma japonicum and Schistosoma mansoni. Furthermore, all R-isomers displayed stronger antischistosomal activity than S-isomers in vivo, indicating R-isomers were the active enantiomers, while S-isomers were less active ones. This structure activity relationship (SAR) could have important implications in further drug development for schistosomiasis.