To Target or Not to Target Schistosoma mansoni Cyclic Nucleotide Phosphodiesterase 4A?

Schistosomiasis is a neglected tropical disease with high morbidity. Recently, the Schistosoma mansoni phosphodiesterase SmPDE4A was suggested as a putative new drug target. To support SmPDE4A targeted drug discovery, we cloned, isolated, and biochemically characterized the full-length and catalytic domains of SmPDE4A. The enzymatically active catalytic domain was crystallized in the apo-form (PDB code: 6FG5) and in the cAMP- and AMP-bound states (PDB code: 6EZU). The SmPDE4A catalytic domain resembles human PDE4 more than parasite PDEs because it lacks the parasite PDE-specific P-pocket. Purified SmPDE4A proteins (full-length and catalytic domain) were used to profile an in-house library of PDE inhibitors (PDE4NPD toolbox). This screening identified tetrahydrophthalazinones and benzamides as potential hits. The PDE inhibitor NPD-0001 was the most active tetrahydrophthalazinone, whereas the approved human PDE4 inhibitors roflumilast and piclamilast were the most potent benzamides. As a follow-up, 83 benzamide analogs were prepared, but the inhibitory potency of the initial hits was not improved. Finally, NPD-0001 and roflumilast were evaluated in an in vitro anti-S. mansoni assay. Unfortunately, both SmPDE4A inhibitors were not effective in worm killing and only weakly affected the egg-laying at high micromolar concentrations. Consequently, the results with these SmPDE4A inhibitors strongly suggest that SmPDE4A is not a suitable target for anti-schistosomiasis therapy.

Itraconazole, a cytochrome P450 inhibitor, enhanced the efficacy of praziquantel against Schistosoma mansoni infection and alleviated liver injury in mice.

Treatment of schistosomiasis is heavily reliant on the single antischistosomal drug praziquantel (PZQ). The use of synergistic drug-drug interactions is one possible solution, which could be used to mitigate PZQ's poor and variable bioavailability. Itraconazole (ITZ), a triazole antifungal agent, is a potent CYP3A inhibitor that can cause significant drug-drug interactions when used with CYP3A substrates. This study investigates the effect of ITZ as adjuvant therapy with PZQ on worm load, egg deposition and maturation, and the consequent histopathology and biochemical abnormalities in the liver during the immature and mature stages of Schistosoma mansoni (S. mansoni) infection. S. mansoni-infected mice were divided into five groups of eight-ten mice each: (I) infected untreated, (II) infected and treated with PZQ 3 weeks PI, (III) infected and treated with both ITZ and PZQ 3 weeks PI, (IV) infected and treated with PZQ 7 weeks PI, and (V) infected and treated with both ITZ and PZQ 7 weeks PI. All mice were killed by rapid decapitation 9 weeks PI. Data revealed that ITZ in combination with PZQ at both immature and mature stages improved the parasitological criteria of cure, and greatly reduced inflammation, granuloma and fibrotic tissue formation, and apoptosis versus PZQ alone. Furthermore, it showed the greatest impact on improving liver injury and oxidative stress markers. Notably, the effect was considerably stronger at the mature stage of S. mansoni infection. These findings support the notion that ITZ increased PZQ's antischistosomal activity by inhibiting CYP450 expression, potentially reducing PZQ metabolism and increasing systemic exposure.

Cloning and functional complementation of ten Schistosoma mansoni phosphodiesterases expressed in the mammalian host stages.

Only a single drug against schistosomiasis is currently available and new drug development is urgently required but very few drug targets have been validated and characterised. However, regulatory systems including cyclic nucleotide metabolism are emerging as primary candidates for drug discovery. Here, we report the cloning of ten cyclic nucleotide phosphodiesterase (PDE) genes of S. mansoni, out of a total of 11 identified in its genome. We classify these PDEs by homology to human PDEs. Male worms displayed higher expression levels for all PDEs, in mature and juvenile worms, and schistosomula. Several functional complementation approaches were used to characterise these genes. We constructed a Trypanosoma brucei cell line in which expression of a cAMP-degrading PDE complements the deletion of TbrPDEB1/B2. Inhibitor screens of these cells expressing only either SmPDE4A, TbrPDEB1 or TbrPDEB2, identified highly potent inhibitors of the S. mansoni enzyme that elevated the cellular cAMP concentration. We further expressed most of the cloned SmPDEs in two pde1Δ/pde2Δ strains of Saccharomyces cerevisiae and some also in a specialised strain of Schizosacharomyces pombe. Five PDEs, SmPDE1, SmPDE4A, SmPDE8, SmPDE9A and SmPDE11 successfully complemented the S. cerevisiae strains, and SmPDE7var also complemented to a lesser degree, in liquid culture. SmPDE4A, SmPDE8 and SmPDE11 were further assessed in S. pombe for hydrolysis of cAMP and cGMP; SmPDE11 displayed considerable preferrence for cGMP over cAMP. These results and tools enable the pursuit of a rigorous drug discovery program based on inhibitors of S. mansoni PDEs.

Deciphering the enzymatic target of a new family of antischistosomal agents bearing a quinazoline scaffold using complementary computational tools.

A previous phenotypic screening campaign led to the identification of a quinazoline derivative with promising in vitro activity against Schistosoma mansoni. Follow-up studies of the antischistosomal potential of this candidate are presented here. The in vivo studies in a S. mansoni mouse model show a significant reduction of total worms and a complete disappearance of immature eggs when administered concomitantly with praziquantel in comparison with the administration of praziquantel alone. This fact is of utmost importance because eggs are responsible for the pathology and transmission of the disease. Subsequently, the chemical optimisation of the structure in order to improve the metabolic stability of the parent compound was carried out leading to derivatives with improved drug-like properties. Additionally, the putative target of this new class of antischistosomal compounds was envisaged by using computational tools and the binding mode to the target enzyme, aldose reductase, was proposed.

The phosphodiesterase-4 inhibitor roflumilast impacts Schistosoma mansoni ovipositing in vitro but displays only modest antischistosomal activity in vivo.

Praziquantel (PZQ) is the sole drug used to treat schistosomiasis, and the probability of developing resistance is growing the longer it is relied upon, justifying the search for alternatives. Phosphodiesterases (PDEs), particularly the PDE4 family, have attracted considerable attention as drug targets, including in Schistosoma mansoni, and especially SmPDE4A. This study investigates the potential antischistosomal activity of human PDE4 and potent SmPDE4A inhibitor roflumilast, either alone or combined with PZQ. In vitro, roflumilast resulted in a significant, concentration-dependent reduction in egg production but not of worm viability. In vitro exposure to roflumilast in combination with a low concentration of PZQ was less effective than PZQ alone, pointing to antagonism. S. mansoni-infected mice treated with roflumilast showed significant reductions in worm burden (27%) as well as hepatic and intestinal egg burdens (~28%) two weeks post treatment. Scanning EM of worms isolated from roflumilast-treated and untreated mice did not reveal noticeable changes to their tegument. S. mansoni-infected mice treated with a fixed dosage of roflumilast and a variable dosage of PZQ resulted in a higher reduction in worm burden, reduced hepatic egg counts, absence of immature eggs and a marked increase in dead eggs, compared to PZQ alone. However, the combination resulted in increased animal mortality, probably attributable to pharmacodynamic interactions between the two drugs. Although this study marks the first report of in vivo antischistosomal potential by a PDE inhibitor, an important proof of concept, we conclude that the antischistosomal effects of roflumilast are insufficient to warrant further development.

Screening of a PDE-focused library identifies imidazoles with in vitro and in vivo antischistosomal activity.

We report the evaluation of 265 compounds from a PDE-focused library for their antischistosomal activity, assessed in vitro using Schistosoma mansoni. Of the tested compounds, 171 (64%) displayed selective in vitro activity, with 16 causing worm hypermotility/spastic contractions and 41 inducing various degrees of worm killing at 100 µM, with the surviving worms displaying sluggish movement, worm unpairing and complete absence of eggs. The compounds that did not affect worm viability (n = 72) induced a complete cessation of ovipositing. 82% of the compounds had an impact on male worms whereas female worms were barely affected. In vivo evaluation in S. mansoni-infected mice with the in vitro 'hit' NPD-0274 at 20 mg/kg/day orally for 5 days resulted in worm burden reductions of 29% and intestinal tissue egg load reduction of 35% at 10 days post-treatment. Combination of praziquantel (PZQ) at 10 mg/kg/day for 5 days with NPD-0274 or NPD-0298 resulted in significantly higher worm killing than PZQ alone, as well as a reduction in intestinal tissue egg load, disappearance of immature eggs and an increase in the number of dead eggs.

Co-administration of metformin and N-acetylcysteine with dietary control improves the biochemical and histological manifestations in rats with non-alcoholic fatty liver.

Non-alcoholic fatty liver disease (NAFLD) is a burgeoning health problem that affects 1/3 of the adult population and an increasing number of children in developed countries. Oxidative stress and insulin resistance are the mechanisms that seem to be mostly involved in its pathogenesis. This study was conceived in a NAFLD rat model to evaluate the efficacy of both metformin (MTF) and N-acetylcysteine (NAC) with dietary control on biochemical and histologic liver manifestations. Rats were classified into nine groups; normal (I), NAFLD-induced by feeding high-fat diet (HFD; II) for 12 weeks, NAFLD switched to regular diet (RD; III), NAFLD-HFD or -RD treated with MTF in a dose of 150 mg/kg (IV, V), NAC in a dose of 500 mg/kg (VI, VII) or MTF+NAC (VIII, IX) respectively for 8 weeks. After 20 weeks, the rats in group II showed notable steatosis, lobular inflammation, fibrosis accompanied with elevated (P < 0.05) serum alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transferase (γ-GT), cholesterol, triglycerides, LDL, VLDL, leptin, tumor necrosis factor (TNF-α), transforming growth factor (TGF-ß1) and hepatic malondialdehyde (MDA) compared with group I. Meanwhile, hepatic superoxide dismutase (SOD), glutathione GSH with serum HDL, adiponectin were significantly decreased (P < 0.05). These changes were to a less extent in group III. MTF or NAC individually resulted in improvement of most of these biochemical and histological parameters. These improvements were more pronounced in the combined groups VIII and IX versus each drug alone. NAC supplementation concomitant with MTF could be beneficial for the treatment of NAFLD and prevention of nonalcoholic steatohepatitis (NASH).

Hepatoprotective and antioxidant activity of Melaleuca styphelioides on carbon tetrachloride-induced hepatotoxicity in mice.

CONTEXT: Liver disease is a serious problem. Polyphenolic compounds have marked antioxidant effect and can prevent the liver damage caused by free radicals. In vitro studies have revealed the strong antioxidant activity of an ellagitannin-rich plant, namely, Melaleuca styphelioides Sm. (Myrtaceae). OBJECTIVE: In view of the limited therapeutic options available for the treatment of liver diseases, the hepatoprotective potential of the methanol extract of M. styphelioides leaves (MSE) was investigated against CCl4-induced liver injury in mice. MATERIALS AND METHODS: MSE was administered (500 and 1000 mg/kg/d p.o.) along with CCl4 for 6 weeks. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were determined in the serum. Glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione transferase (GST), and malondialdehyde (MDA) were estimated in the liver homogenate. The bioactive components of MSE were identified by NMR, UV and HRESI-MS/MS data. RESULTS: MSE treatment (500 and 1000 mg/kg/d) markedly inhibited the CCl4-induced increase in the levels of AST (31 and 38%), ALT (29 and 32%), ALP (13 and 19%), and MDA (22 and 37%) at the tested doses, respectively. MSE treatment markedly increased the levels of GSH (29 and 57%) and antioxidant enzymes compared with the CCl4-treated group. MSE was more effective than silymarin in restoring the liver architecture and reducing the fatty changes, central vein congestion, Kupffer cell hyperplasia, inflammatory infiltration, and necrosis induced by CCl4. The LD50 of MSE was more than 5000 mg/kg. CONCLUSION: MSE confers potent antioxidant and hepatoprotective effects against CCl4-induced toxicity.

Hepatoprotective and antioxidant effect of Bauhinia hookeri extract against carbon tetrachloride-induced hepatotoxicity in mice and characterization of its bioactive compounds by HPLC-PDA-ESI-MS/MS.

The hepatoprotective and antioxidant activity of Bauhinia hookeri ethanol extract (BHE) against CCl4-induced liver injury was investigated in mice. BHE was administered (500 and 1000 mg/kg/day) along with CCl4 for 6 weeks. The hepatic marker enzymes: alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were determined in the serum. The antioxidant parameters: glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione transferase (GST), and malondialdehyde (MDA) were estimated in the liver homogenate. BHE treatment significantly inhibited the CCl4-induced increase in ALT (44 and 64%), AST (36 and 46%), ALP (28 and 42%), and MDA (39 and 51%) levels at the tested doses, respectively. Moreover, BHE treatment markedly increased the activity of antioxidant parameters GSH, GPx, GR, GST, and SOD. Histological observations confirmed the strong hepatoprotective activity. These results suggest that a dietary supplement of BHE could exert a beneficial effect against oxidative stress and various liver diseases by enhancing the antioxidant defense status, reducing lipid peroxidation, and protecting against the pathological changes of the liver. The hepatoprotective activity of BHE is mediated, at least in part, by the antioxidant effect of its constituents. The active constituents of BHE were identified by HPLC-PDA-ESI/MS/MS.

Pharmacological and antioxidant actions of garlic and.or onion in non-alcoholic fatty liver disease (NAFLD) in rats.

Non-alcoholic fatty liver disease (NAFLD) includes a broad spectrum of fat-induced liver injury, ranging from mild steatosis to cirrhosis and liver failure. This study investigates the hepatoprotective properties of garlic and onion in NAFLD rat model. Ninety male Sprague-Dawley rats were randomly divided into 9 groups; normal (I), NAFLD induced with high fat diet (HFD; II), NAFLD switched to regular diet (RD; III), NAFLD-HFD or NAFLD-RD treated with garlic (IV, V), onion (VI, VII) or the combined garlic+onion (VIII, IX) respectively. A NAFLD rat model was established by feeding the animals with a high-fat diet for 12 wk. These animals were then treated with garlic or/and onion or vehicle for 8 wk (weeks 13-20) and then killed to obtain serum samples and liver tissues. Liver histology, lipids, parameters of oxidative stress, TNF-α and TGF-ß were measured. The liver in NAFLD-HFD showed typical steatosis, accompanied with mild to moderate lobular inflammatory cell infiltration. Serum levels of ALT, AST, ALP, leptin, cholesterol, triglycerides, TNF-α, TGF-ß and hepatic MDA' were significantly increased (P < 0.05) compared with normal group. This was accompanied with reduction of hepatic GSH, GR, GPx, GST, SOD and serum adiponectin. These changes were to a less degree in NAFLD-RD group. Combined administration of garlic+onion produced a better and significant decrease in liver steatosis, serum liver enzymes, oxidative markers and lipid peroxidation versus each one alone. In the same time, NAFLD-induced inflammation was also mitigated via reduction of TNF-α and TGF-ß. In addition, these results were better in the group IX versus group VIII.

Effect of ketoconazole, a cytochrome P450 inhibitor, on the efficacy of quinine and halofantrine against Schistosoma mansoni in mice.

The fear that schistosomes will become resistant to praziquantel (PZQ) motivates the search for alternatives to treat schistosomiasis. The antimalarials quinine (QN) and halofantrine (HF) possess moderate antischistosomal properties. The major metabolic pathway of QN and HF is through cytochrome P450 (CYP) 3A4. Accordingly, this study investigates the effects of CYP3A4 inhibitor, ketoconazole (KTZ), on the antischistosomal potential of these quinolines against Schistosoma mansoni infection by evaluating parasitological, histopathological, and biochemical parameters. Mice were classified into 7 groups: uninfected untreated (I), infected untreated (II), infected treated orally with PZQ (1,000 mg/kg) (III), QN (400 mg/kg) (IV), KTZ (10 mg/kg)+QN as group IV (V), HF (400 mg/kg) (VI), and KTZ (as group V)+HF (as group VI) (VII). KTZ plus QN or HF produced more inhibition (P<0.05) in hepatic CYP450 (85.7% and 83.8%) and CYT b5 (75.5% and 73.5%) activities, respectively, than in groups treated with QN or HF alone. This was accompanied with more reduction in female (89.0% and 79.3%), total worms (81.4% and 70.3%), and eggs burden (hepatic; 83.8%, 66.0% and intestinal; 68%, 64.5%), respectively, and encountering the granulomatous reaction to parasite eggs trapped in the liver. QN and HF significantly (P<0.05) elevated malondialdehyde levels when used alone or with KTZ. Meanwhile, KTZ plus QN or HF restored serum levels of ALT, albumin, and reduced hepatic glutathione (KTZ+HF) to their control values. KTZ enhanced the therapeutic antischistosomal potential of QN and HF over each drug alone. Moreover, the effect of KTZ+QN was more evident than KTZ+HF.

Pharmacodynamics of mefloquine and praziquantel combination therapy in mice harbouring juvenile and adult Schistosoma mansoni.

Praziquantel (PZQ) is currently the only drug widely used for the treatment of schistosomiasis, but the antimalarial drug mefloquine (Mef) possesses interesting antischistosomal properties. Combination therapy with these two drugs has been suggested as a strategy for transmission control, as PZQ is active against adult worms and Mef is active against schistosomula. To examine the efficacy of combination therapy, Schistosoma mansoni-reinfected mice were separated into seven groups: untreated (I), treated with PZQ in doses of 200 mg/kg (II) or 1,000 mg/kg (III), treated with Mef in doses of 200 mg/kg (IV) or 400 mg/kg (V); each dose was divided equally and given on two consecutive days. Group VI was treated with doses of PZQ + Mef as in groups II and IV, respectively, while group VII was treated with PZQ + Mef as in groups III and V, respectively. PZQ + Mef at the reduced doses of 200 mg/kg each enhanced the therapeutic efficacy over the reduced PZQ dose alone as shown by a very high reduction in the total numbers of mature worms (95% vs. 49%), immature worms (96% vs. 29%) and the complete eradication of immature females, mature females and immature eggs. The reduction in worm burden was associated with the healing of hepatic granulomatous lesions and the normalisation of all liver enzymes. Therefore, the use of Mef with PZQ is more effective than PZQ alone and should be considered for clinical trials in humans as a potential treatment regimen to prevent treatment failures in areas with high rates of schistosomiasis.

Pharmacodynamics of mefloquine and praziquantel combination therapy in mice harbouring juvenile and adult Schistosoma mansoni

Praziquantel (PZQ) is currently the only drug widely used for the treatment of schistosomiasis, but the antimalarial drug mefloquine (Mef) possesses interesting antischistosomal properties. Combination therapy with these two drugs has been suggested as a strategy for transmission control, as PZQ is active against adult worms and Mef is active against schistosomula. To examine the efficacy of combination therapy, Schistosoma mansoni-reinfected mice were separated into seven groups: untreated (I), treated with PZQ in doses of 200 mg/kg (II) or 1,000 mg/kg (III), treated with Mef in doses of 200 mg/kg (IV) or 400 mg/kg (V); each dose was divided equally and given on two consecutive days. Group VI was treated with doses of PZQ + Mef as in groups II and IV, respectively, while group VII was treated with PZQ + Mef as in groups III and V, respectively. PZQ + Mef at the reduced doses of 200 mg/kg each enhanced the therapeutic efficacy over the reduced PZQ dose alone as shown by a very high reduction in the total numbers of mature worms (95 percent vs. 49 percent), immature worms (96 percent vs. 29 percent) and the complete eradication of immature females, mature females and immature eggs. The reduction in worm burden was associated with the healing of hepatic granulomatous lesions and the normalisation of all liver enzymes. Therefore, the use of Mef with PZQ is more effective than PZQ alone and should be considered for clinical trials in humans as a potential treatment regimen to prevent treatment failures in areas with high rates of schistosomiasis.

Effect of Ficus sycomorus and Azadirachta indica extracts on liver state of mice infected with Schistosoma mansoni.

The potential role of hepatoprotective and antipathological effect of Ficus sycomorus and Azadirachta indica extracts was evaluated for scavenging the reactive oxygen species (ROS) and reduced the oxidative damage and pathological changes in the liver of S. mansoni infected mice. The levels of alanine aminotransferase (ALT), asparate aminotransferase (AST) and gamma glutamyl transferase (GGT) were evaluated in the infected mice and treated orally with each plant extract 12 weeks post infection (P.I.) in a dose of 500 mg/kg of each plant extract for five consecutive days and sacrificed two weeks P.I. The infection of mice showed an elevation of ALT, AST & GGT. Treatment of mice with 70% methanol extract of each plant extract reduced significantly ALT, AST & GGT elevation. The highest reduction was with the methanolic extract of F. sycomorus (42%, 35% &44% for ALT, AST & GGT respectively). Fractionation of the methanolic extract of each plant was carried out. The effect of ethyl acetate and butanolic fractions of each plant was also evaluated. The result showed that the two fractions lowered the levels of the tested enzymes and decreased the number and size of granuloma diameters with an increased in the percentage of degenerated ova.

Comparative efficacy and bioavailability of different praziquantel brands.

This study investigates the efficacy, bioavailability and drug metabolizing enzymes mainly involved in the metabolism of the commercial brands of praziquantel (PZQ) in Egypt in comparison with the original pure powder. Mice infected with PZQ-susceptible (CD) or PZQ-insusceptible (EE2) Schistosoma mansoni isolates were divided each into seven groups, six of them received PZQ brands (Distocide, Epiquantel, Biltricide, Bilharzid, Praziquantel, and pure PZQ), while the seventh one was left as infected untreated. Seven weeks post-infection, worms were quantified and hepatic CYP450 and CYT b5 were examined. For PZQ pharmacokinetics, groups of normal mice were given the different PZQ brands and divided into subgroups, killed at 2, 5, 15, 30, 60, 90, 120,150, 180, 240 and 360 min post-dosing. Physicochemical examination revealed better dissolution rates for Biltricide, Distocide and PZQ T3A rather than Epiquantel and Bilharzid. Significant decrease in worm burden was recorded in all groups of mice regardless of the brand of PZQ used, but with better results obtained with CD isolate rather than the EE2 isolate. Biltricide and Distocide showed higher C(max) and AUC(0-6h) in normal mice, in addition to higher worm reduction with least inhibition of CYP450 and CYT b5 in EE2-infected mice. PZQ T3A, Bilharzid and Epiquantel showed, in addition to lower efficacy, higher K(el), lower t(1/2e), C(max) and AUC(0-6h). The 32-46% reduction of their bioavailability reflected on their antischistosomal efficacy and recovery of drug metabolizing enzymes. Quality of generic PZQ should include, in addition to examining the physicochemical characteristics of the brands, biological testing including efficacy and bioavailability studies.

Response of Schistosoma mansoni isolates having different drug sensitivity to praziquantel over several life cycle passages with and without therapeutic pressure.

The stability of praziquantel (PZQ)-insusceptible S. mansoni isolates and the possible selection of PZQ-insusceptible parasites upon applying therapeutic pressure were examined over several life cycle passages (snails to mice). To test isolate stability, 3 PZQ-susceptible and 7 PZQ-insusceptible isolates were used to establish infection in mice, and they were passaged each for 2-5 life cycles. After each passage, 6 groups of mice were used to assess the PZQ dose at which the worm burden was decreased by 50% (ED50). Five of them were treated with doses of PZQ (12.5, 25, 50, 100, and 200 mg/kg for 5 days) 7 wk after infection; the last group represented infected, but untreated, controls. Possible selection of PZQ-insusceptible parasites under therapeutic pressure was examined by subjecting 1 PZQ-susceptible and 1 PZQ-insusceptible S. mansoni isolate to therapeutic pressure by PZQ for 8 passages. After the final passage, PZQ ED50 was estimated. All PZQ-susceptible S. mansoni isolates showed stable susceptibility to PZQ (mean PZQ ED50 = 85 mg/kg) over all passages. Two of the 7 PZQ-insusceptible S. mansoni isolates (847 and ER5) showed normal sensitivity to PZQ in 1-2 passages (although not the last passage, and without a declining ED50 profile), whereas the remaining passages kept a sustained insusceptibility to the drug (mean PZQ ED50 = 217 mg/kg). Worm maturity and sex were irrelevant to variability in drug ED50 within an individual isolate over different passages, revealing the heterogeneous nature of the parasite. Therapeutic pressure for limited life cycle passages did not result in a significant increase in drug ED50. The fact that reversion of some of the PZQ-insusceptible S. mansoni isolates to normal drug-sensitive state is not long lasting and that the therapeutic pressure by PZQ in the field is not comparable with that in the laboratory (unlimited), make monitoring the response of patients to the drug in the field an integral part of schistosomiasis control measures.

Drug-metabolizing enzymes and praziquantel bioavailability in mice harboring Schistosoma mansoni isolates of different drug susceptibilities.

The level of drug-metabolizing enzymes (cytochrome P450 [CYP450] and cytochrome b5 [cyt b5]) and the bioavailability of praziquantel (PZQ) were investigated in batches of mice infected with Schistosoma mansoni displaying either a decreased susceptibility to PZQ ("EE2" and "BANL"-isolates), or a normal susceptibility to the drug ("CD" isolate). Each batch was divided into 2 groups. The first group was further subdivided into 5 subgroups. Subgroups 1 to 4 were treated 7 wk postinfection (PI) with oral PZQ at 25, 50, 100, and 200 mg/kg for 5 consecutive days, whereas the fifth subgroup was administered the vehicle only as control. Animals were perfused 9 wk PI, and worms were counted to estimate PZQ ED50. CYP450 and cyt b5 were examined in hepatic microsomes of infected untreated mice and of infected mice treated with 25 and 200 mg/ kg PZQ. The second group was given PZQ 7 wk PI and was further subdivided into 11 subgroups, killed at 2, 5, 15, 30, 60, 90, 120, 150, 180, 240, and 360 min postdosing to study pharmacokinetic parameters of PZQ. Mice harboring S. mansoni isolates having higher PZQ ED50 (170.3 mg/kg for EE2 and 249.9 mg/kg for BANL vs. 82.96 mg/kg for CD) had higher levels of CYP450 and cyt b5, a PZQ Cmax decreased by 19-30% and area under the serum concentration-time curve0-6 hr decreased by 57-74%. Data suggest that S. mansoni isolates that are less sensitive to PZQ induce a lower inhibition of hepatic drug-metabolizing enzymes, with a consequently higher metabolic transformation of PZQ.

Determination of ED50 values for praziquantel in praziquantel-resistant and -susceptible Schistosoma mansoni isolates.

The dose of praziquantel required to kill 50% of adult worms in vivo (i.e. the ED50) was estimated for nine different isolates of Schistosoma mansoni in infected mice. Four of the isolates were selected because they had not knowingly been in contact with the drug (i.e. they were putatively praziquantel-susceptible). Five putatively praziquantel-resistant isolates were chosen because they had been selectively bred for drug-resistance in the laboratory and/or had previously been shown to be relatively resistant to praziquantel in the field. The work was performed in three laboratories in different countries using pre-agreed and comparable experimental protocols. All four praziquantel-susceptible isolates had ED50s estimated to be <100 mg/kg (mean=70+/-7 SD; median=68), while all five putatively praziquantel-resistant isolates had estimated ED50s >100 mg/kg (mean=209+/-48 SD; median=192). Thus, the five praziquantel-resistant isolates, including two that had been subjected to drug pressure during more than 20 passages in mice, had drug ED50s that were approximately three times as great as those of the praziquantel-susceptible isolates. Two of the five isolates in the putatively resistant group had previously been passaged 15 or more times in mice without administration of drug-pressure, but had ED50s consistent with the other three isolates in the group, indicating that the trait of praziquantel-resistance did not necessarily impair biological fitness during laboratory passage. The protocols used here to estimate the praziquantel ED50s of S. mansoni isolates should be useful for establishing and monitoring the drug susceptibility/resistance profiles of parasite isolates freshly obtained from endemic areas, particularly those in which increased usage of the drug is likely to occur.