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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.

13 p.-7 fig.-5 tab.-1 graph. abst., 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., This study received funding from the EC 7th Framework Programme (FP7-HEALTH-2013-INNOVATION-1, PDE4NPD no.602666), RICET (RD16/0027/0010), FEDER funds and MECD [Grant FPU15/1465 to V. S.-P.].

9 p.-5 fig.-2 tab., 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 posttreatment.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., This work is part of the PDE4NPD consortium supported by Framework Program 7 of the European Commission No: 602666.Funding from RICET/FEDER funds (RD16/0027/0010), and the Ministry of Education, Culture and Sport (MECD) of Spain (Grant FPU15/1465 to V. S.-P.) is also acknowledged. Y. Z. was supported by a grant of the Chinese Science Council; A. R. B. and I. J. P. dE. were supported by the Netherlands Science Foundation.

This study investigates the therapeutic potential of Vilda in a NASH model with liver fibrosis and elucidates the underlying molecular mechanisms.To induce NASH, male Sprague-Dawley rats were fed a high-fat diet for 24 weeks with a single dose of STZ (40 mg/kg, IP). Vilda was orally administered at two doses (10 and 20 mg/kg) for 20 weeks.The induction of NASH was validated by abnormalities in hepatotoxicity indices, lipid profile, oxidative stress markers, and pathologically by marked fat deposition in hepatic tissues together with severe inflammatory cell infiltration. Moreover, NASH-affected rats demonstrated reduced insulin sensitivity manifested as elevated fasting blood glucose levels and disrupted homeostasis model assessment for insulin resistance. Vilda, at both doses, effectively abrogated all these pathological features of NASH. Mechanistically, these hepatoprotective properties of Vilda can be attributed to its antioxidant effects, anti-inflammatory effects (by inhibiting the TNF-α, NF-κB, JNK, and JAK/STAT pathways), and insulin-sensitizing effect (by upregulating the IRS-1/PI3K/Akt pathway). Besides, Vilda successfully counteracted NASH-associated liver fibrosis by downregulating the TGF-β1 pathway.The hepatoprotective and antifibrotic effects of Vilda were mostly dose-dependent. Collectively, this study offered a promising therapeutic avenue for Vilda as a novel strategy for counteracting the pathological progression of NASH and associated liver fibrosis.

Non-alcoholic fatty liver disease (NAFLD) constitutes a major health problem worldwide and intimately links with obesity and diabetes. This study aimed to explore the therapeutic impact of early treatment with metformin (MTF) alone or in combination with Lactobacillus reuteri DSM 17938 ( L. reuteri) + metronidazole (MTZ) in male Sprague Dawley rats with high-fat diet (HFD)-induced NAFLD. Hepatic steatosis was induced by feeding rats HFD for 6 weeks. MTF (150 mg/kg/day) or L. reuteri (2 × 109 colony forming unit/day) were given orally for 4 weeks; meanwhile, MTZ (15 mg/kg/day, p.o.) was administered for 1 week. Administration of L. reuteri + MTZ in combination with MTF produced a superior effect concerning insulin resistance (IR), lipid profile, liver function, oxidative stress, inflammatory and autophagic markers than using each treatment alone. Besides, this combination resulted in disappearance of steatosis, inflammation and vacuolation within hepatic architecture. Moreover, it normalized short chain fatty acids (SCFAs) as well as Firmicutes and Bacteroidetes faecal contents. In conclusion, early treatment with L. reuteri + MTZ in combination with MTF could prevent NAFLD progression and liver injury through targeting gut dysbiosis, inflammation and autophagic pathways.

Background Schistosomiasis is responsible for a considerable global disease burden. This work aimed to improve the therapeutic outcome of the only available antischistosomal drug worldwide, praziquantel (PZQ), by incorporating it into a novel carrier, “solid lipid nanoparticles (SLNs)”, to enhance its solubility, bioavailability and efficacy. A simple, cost-effective method was used to prepare SLN-PZQ. Results Compared to market PZQ (M-PZQ), SLN-PZQ was more bioavailable, as denoted by higher serum concentrations in both normal and infected mice where elevated Ka, AUC0–24, Cmax, and t1/2e with a decrease in kel were demonstrated. The AUC0–24 for SLN-PZQ in normal and Schistosoma mansoni-infected groups was almost nine- and eight-fold higher, respectively, than that for M-PZQ in corresponding groups. In normal and S. mansoni-infected mice, SLN-PZQ was detectable in serum at 24 h, while M-PZQ completely vanished 8 h post-treatment. Additionally, enhanced absorption with extended residence time was recorded for SLN-PZQ. Compared to M-PZQ, SLN-PZQ revealed superior antischistosomal activity coupled with enhanced bioavailability in all treated groups where higher percentages of worm reduction were recorded with all dosages tested. This effect was especially evident at the lower dose levels. The ED95 of SLN-PZQ was 5.29-fold lower than that of M-PZQ, with a significantly higher reduction in both the hepatic and intestinal tissue egg loads of all treated groups and almost complete disappearance of immature deposited eggs (clearly evident at the low dose levels). Conclusions SLN-PZQ demonstrated enhanced PZQ bioavailability and antischistosomal efficacy with a safe profile despite the prolonged residence in the systemic circulation.

Hibiscus sabdariffa L. (Malvaceae) is a common traditional tea that has many biological activities.To evaluate the hepatoprotective effect and study the metabolic profile of the anthocyanin-rich extract of H. sabdariffa calyces (HSARE).The hepatoprotective activity of HSARE was assessed (100 mg/kg/d for 4 weeks) by examining the hepatic, inflammatory, oxidative stress markers and performing a histopathological examination in rats with thioacetamide (TAA)-induced hepatotoxicity. HSARE was analyzed using ultra-performance liquid chromatography-quadrupole-time-of-flight-photodiode array-mass spectrometry (UPLC-qTOF-PDA-MS).The UPLC-qTOF-PDA-MS analysis of HSARE enabled the identification of 25 compounds represented by delphinidin and its derivatives, cyanidin, kaempferol, quercetin, myricetin aglycones and glycosides, together with hibiscus lactone, hibiscus acid and caffeoylquinic acids. Compared to the TAA-intoxicated group, HSARE significantly reduced the serum levels of alanine aminotransferase, aspartate aminotransferase and hepatic malondialdehyde by 37.96, 42.74 and 45.31%, respectively. It also decreased hepatic inflammatory markers, including tumour necrosis factor alpha, interleukin-6 and interferon gamma (INF-γ), by 85.39, 14.96 and 70.87%, respectively. Moreover, it decreased the immunopositivity of nuclear factor kappa-B and CYP2E1 in liver tissue, with an increase in the effector apoptotic marker (caspase-3 positive cells), restoration of the altered hepatic architecture and increases in the activities of superoxide dismutase (SOD) and glutathione by 150.08 and 89.23%, respectively.HSARE revealed pronounced antioxidant and anti-inflammatory potential where SOD and INF-γ were significantly improved. HSARE possesses the added value of being more water-soluble and of natural origin with fewer side effects expected compared to silymarin.

This study investigates the involvement of liver dysfunction in the modulation of paracetamol pharmacokinetic profile in genotype-4 HCV patients treated with either paracetamol alone (Para) or in combination with caffeine (Para-Caf). Twenty healthy volunteers and 20 Child-Pugh B HCV patients, each divided into two equal subgroups, were examined, whose liver/kidney functions were correlated with their main clinical manifestation. After an overnight fasting, healthy and hepatic subjects received either a single dose of Para (1000 mg paracetamol) or Para-Caf (1000 mg paracetamol/130 mg caffeine). Two milliliters of saliva samples were collected prior to and at different time-intervals after drug administration and analyzed using HPLC. There was a noticeable increase in the mean concentration time profile of salivary paracetamol concentrations in hepatic patients, with concomitant decrease in paracetamol clearance (CLT), along with induction in the primary pharmacokinetic (PK) parameters, C max, AUC(0–8 h) and AUC(0–∞) (by about 95, 82, and 64 %, respectively, after treatment with Para, and 98, 96, and 101 %, respectively, after treatment with Para-Caf), when compared with the corresponding parameters in healthy subjects. Additionally, the healthy subjects treated with Para-Caf exhibited bioinequivalent increase in C max, K a, and t 1/2 with decrease in T max when compared with the healthy individuals treated with Para alone. A similar pattern was recorded in hepatic patients after addition of caffeine to paracetamol, with even augmented significant increase in K a and t 1/2 (by 100 and 32 %, respectively). Liver dysfunction modified the PK of paracetamol expressed as earlier effective paracetamol concentration, with obvious decrease in its clearance. Caffeine induced faster absorption (evidenced by shorter T max and higher K a) and prolonged t 1/2 of paracetamol, the effects that were more profound in hepatic patients. Further studies are needed to evaluate the influence of liver damage on paracetamol pharmacokinetics whenever repeated dosing is applied, to avoid possible drug accumulation.

Abstrct Gut microbiota is a crucial factor in pathogenesis of non-alcoholic steatohepatitis (NASH). Therefore, targeting the gut–liver axis might be a novel therapeutic approach to treat NASH. This study aimed to investigate the therapeutic effects of a probiotic (Lactobacillus reuteri) and metronidazole (MTZ) (an antibiotic against Bacteroidetes) either alone or in combination with metformin (MTF) in experimentally-induced NASH. NASH was induced by feeding rats high fat diet (HFD) for 12 weeks. MTF (150 mg/kg/day) or L. reuteri (2x109 colony forming unit/day) were given orally for 8 weeks; meanwhile, MTZ (15 mg/kg/day, p.o.) was administered for 1 week. Treatment with L. reuteri and MTZ in combination with MTF showed additional benefit compared to MTF alone concerning lipid profile, liver function, oxidative stress, inflammatory and autophagic markers. Furthermore, combined regimen succeeded to modulate acetate: propionate: butyrate ratios as well as Firmicutes and Bacteroidetes fecal contents with improvement of insulin resistance (IR). Yet, the administration of MTF alone failed to normalize Bacteriodetes and acetate contents which could be the reason for its moderate effect. In conclusion, gut microbiota modulation may be an attractive therapeutic avenue against NASH. More attention should be paid to deciphering the crosstalk mechanisms linking gut microbiota to non-alcoholic fatty liver disease (NAFLD) to identify new therapeutic targets for this disease.

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., Author summary Schistosomiasis is a serious and often fatal disease that is caused by infection with parasitic worms of the Schistosoma species. It affects several hundreds of millions of people world-wide, mostly in the tropics, through contact of skin with infected water, usually lakes and rivers. Only one drug, praziquantel, exists for its treatment, so resistance to it would leave the disease untreatable and new drugs are urgently needed. Here, we report on a strategy to develop new anti-schistosomal agents by inhibiting the regulatory systems of the worm. Cyclic nucleotides cAMP and cGMP are key regulators of cellular activity, and their activity is determined by enzymes called phosphodiesterases or PDEs, that can degrade them. We identified and cloned the genes coding for ten of these PDEs from Schistosoma mansoni, a species causing a very high disease burden. By expressing these genes in specialized cell lines of the yeast Saccharomyces cerevisiae and the protozoan parasite Trypanosoma brucei, under conditions where growth becomes conditional on the expression of the introduced Schistosoma PDE, we show that most of these genes indeed code for functional PDEs. For several PDEs we also determined, using a third expression system, the yeast Schizosaccharomyces pombe, whether they regulate cAMP or cGMP. We also identify a number of inhibitors for one of these PDEs and show that all are expressed more in male than in female worms. This work is essential to map and characterise the regulatory pathways of schistosomes and will facilitate the development of inhibitors of the key enzymes.

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.

Background and study aims THEBERA is a project funded by the European Union (EU), as an ERA-WIDE FP7 project, aiming to strengthen the Theodor Bilharz Research Institute (TBRI) capacities. Materials and methods A SWOT (strength/weakness/opportunities/threats) analysis of human, structural and organisational existing resources was performed in light of an extensive analysis of liver disease research and clinical management in Egypt, for a full understanding of TBRI needs. Results Strength and weakness features were identified and analysed, so were actions to be implemented and targets to be accomplished, to develop a business plan gathering the required critical mass (political, scientific, industrial, social) to select investment priorities, to sacrifice non-strategic areas of research, to promote national and international connections and industrial innovations, to update diagnostics and research device technologies and clinical management processes at European levels, to implement fundraising activities, to organise and properly assess training activities for young researchers, physicians, nurses, and technicians. Conclusions Research institute self assessment is a priority need for sustainable capacity building and for future build-up of a competent health care research institute. Sustainable capacity building strategies must be designed on needs assessment, involving salient requirements: clear strategy, leverage of administrative capacities, industrial support and connections, systematised training programmes and enhancement of mobility of health care staff implemented within ill-defined boundaries and continuously re-evaluated with multiple feedback loops in order to build a complex, adaptable and reliable system based on value.

Schistosomiasis, a high volume neglected tropical disease affecting more than 200 million people worldwide, can only be effectively treated by the tetrahydroisoquinoline drug praziquantel (PZQ). Herein, we describe an efficient approach to access PZQ derivatives by the Ugi 4-component reaction followed by the Pictet-Spengler reaction in a two-step, one-pot procedure. 30 novel PZQ derivatives are described based on the Ugi 4-component reaction and an X-ray structure of a novel derivative revealing different conformation compared with PZQ is discussed. Several analogues comparable in activity to the drug PZQ have been identified based on an in vitro Schistosoma mansoni worm viability assay.

The present work was undertaken to improve the efficacy of praziquantel (PZQ, CAS 55268-74-1, EMBAY 8440) by its combination with the anti-fecundity vaccine, recombinant glutathione S-transferase 26 (rSm GST26). Five groups of mice were used. One group received multiple doses of rSm GST26 (1 microgram x 4) 2 days apart starting 7 days before infection. The second group received PZQ (2 x 500 mg/kg) 8 weeks post infection. The third group received combined therapy of PZQ and multiple rSm GST26 in the same doses and at the same previously mentioned timings. Infected untreated and multiple bacterial lysate treated groups served as respective controls. All animals from all groups were sacrificed 10 and 12 weeks post infection. Hepatic histopathology granuloma diameter, liver functions, parasitological and immunological parameters were studied. Multiple injections of rSm GST26 alone decreased worm count, number of eggs per female worm, viability of eggs, granuloma diameter and accelerated the ova destruction. Treatment with PZQ alone induced incomplete eradication of worms, moderate reduction in granuloma diameter, decreased number of ova per g tissue and normalized the liver function. Combined therapy with rSmGST26 and PZQ showed dual effects as evident by the complete eradication of worms, pronounced reduction in the number of ova per g tissue, acceleration of egg destruction, marked reduction in granuloma diameter and amelioration of egg-induced pathology with increase in anti-GST immunoglobulin G.

Botros SS, Hammam O, Mahmoud M, Bergquist R. Praziquantel efficacy in mice infected with PZQ non-susceptible S. mansoni isolate treated with artemether: parasitological, biochemical and immunohistochemical assessment. APMIS 2010; 118: 692–702. Based on the fact that artemether (ART) affects immature schistosomes and that the effect of praziquantel (PZQ) mainly targets mature schistosomes, this work investigates the possible enhanced efficacy of PZQ in combination with ART in mice harboring a PZQ non-susceptible Schistosoma mansoni isolate. Associated schistosomal, inflammatory, hepatic histopathological changes have been investigated by examining the tissue markers expressing apoptosis using FAS (CD95), anti-apoptosis (Bcl2) and angiogenesis [vascular endothelial growth factor (VEGF)]. A batch of Swiss albino mice infected with a PZQ non-susceptible (EE10) S. mansoni isolate was divided into 12 groups. Animals of the first group were left without treatment as infected controls, while groups 2–6 received PZQ in increasing doses. The animals of group 7 received ART in double doses. Those comprising groups 8–12 received combined therapy of PZQ and ART in the same doses and at the same timings postinfection (PI) as those belonging to groups 2–6. Parasitological parameters, liver function, and histopathological and immunohistochemical studies of FAS, Bcl2 and VEGF antibodies were assessed. Combined administration of ART and PZQ reduced the ED50 (the dose at which the worm burden was decreased by 50%) of PZQ. Typical granulomas were not seen in animals treated with ART alone and combined with PZQ, with least expression of FAS and VEGF and increased expression of Bcl2. The minimal histopathological changes recorded in mice treated with both ART and PZQ could be related to a synergistic/additive effect of ART, markedly reducing the intensity of infection. Improved liver function tests support the less severe histopathological changes under the influence of this treatment protocol. This study encourages human trials especially in areas where malaria is not endemic, and differing combination doses should be investigated in view of the antagonistic effect noticed with some dose regimens.

9-( S )-[3-Hydroxy-2-(phosphonomethoxy)propyl]adenine [( S )-HPMPA] has been reported to have antischistosomal activity. Ether lipid esters of ( S )-HPMPA and cidofovir (CDV) have greatly increased activities in antiviral assays and in lethal animal models of poxvirus diseases. To see if ether lipid esters of CDV and ( S )-HPMPA enhance antischistosomal activity, we tested their alkoxyalkyl esters using Schistosoma mansoni worm killing in vitro. Hexadecyloxypropyl (HDP)-cyclic-( S )-HPMPA and HDP-cyclic-CDV exhibited significant in vitro antischistosomal activities and may offer promise alone or in combination with praziquantel.

In a recent study, the fasciolicidal activity of Mirazid (a myrrh-derived drug) and its effect on the function and histopathology of host liver were investigated in Egyptian sheep, with triclabendazole (TCBZ) used as the positive control. Sheep were infected with metacercariae (150/animal) and treated 3 months later, either with Mirazid (10 mg/kg/day for six consecutive days) or TCBZ (a single dose of 10 mg/kg), or left untreated, as controls. When the animals were killed 4 weeks after the end of treatment, no Fasciola flukes or eggs could be found in the animals given TCBZ but the number of flukes found in the Mirazid-treated animals was only 6% lower than that recorded in the untreated sheep (a statistically insignificant difference). In terms of their Fasciola egg loads, serum concentrations of hepatic enzymes and hepatic histopathological changes, the Mirazid-treated sheep appeared very similar to the untreated, infected animals. The TCBZ-treated animals, in contrast, showed remarkably little evidence of hepatic pathology. It therefore appears that, in the treatment of ovine fascioliasis, at least some batches of Mirazid have little, if any, value.

Summary Objective To investigate immune mechanisms possibly involved in the amelioration of histopathological changes in livers of Schistosoma mansoni -infected mice treated with artemether (ART), including liver functions and apoptotic changes. Methods Male CD-1 Swiss albino mice were infected with Schistosoma mansoni and treated with praziquantel (PZQ) 6weeks post-infection (PI) (500mg/kg/day x2) and/or ART in double dose (each of 400mg/kg) 4 and 6weeks PI. Parasitological parameters, liver functions and histopathological changes including T-lymphocyte profile and apoptotic changes were assessed. Results Eight weeks PI, although the reduction in worm burden in mice treated with ART plus PZQ was comparable to that in PZQ-treated mice, yet there was complete absence of eggs and typical granulomas. The ratio of T-helper/cytotoxic cells was in favor of T-helper in infected control and in mice treated with both drugs. This ratio was 0.9:1 and 0.7:1 in PZQ and ART-treated groups, respectively, with moderate apoptotic changes in the latter. All biochemical parameters expressing liver function were improved with all treatment regimens. Conclusions Administration of ART in addition to PZQ resulted in absence of eggs and typical granulomas with less apoptotic changes than in ART-treated mice. Improved liver functions with higher apoptosis in ART-treated mice may suggest enhanced necrotic cell death/regenerative changes.

In this study, 15 healthy volunteers and 96 patients with hepatitis C virus, classified according to Child-Pugh into 36 Child-A, 31 Child-B and 29 Child-C, were examined. All subjects ingested 600 mg antipyrine in the form of hard gelatinous capsules after overnight fasting. One milliliter of saliva was collected at 4 and 24 h after ingestion of antipyrine and analyzed using high-performance liquid chromatography. Blood samples were collected from all subjects for examination, using conventional liver function tests. The pharmacokinetic variables for antipyrine were determined using the two concentration time points selected. A cut-off value of 0.34 ml/min/kg was used to distinguish between cirrhotic and non-cirrhotic patients. Alanine aminotransferase, aspartate aminotransferase and gamma-glutamyl transferase values were significantly higher with significantly lower antipyrine clearance in Child-A, B, and C patients than in normal volunteers. The total protein concentration was significantly lower in Child-B and C patients. Moreover, AST was significantly higher in Child-C patients and antipyrine clearance was lower in Child-B and C patients than in Child-A patients. Antipyrine clearance showed a significant negative correlation with Child-Pugh scores, total protein, the international normalization ratio of prothrombin time and globulin, and a positive correlation with albumin and albumin-to-globulin ratio. Unlike most of the conventional liver function tests, antipyrine clearance, which represents the intrinsic clearance capacity of the liver, measured using saliva, proved to be a sensitive marker of liver function. It was significantly impaired in the Child-Pugh group A patients with the least hepatic impairment. The international normalization ratio of prothrombin time was just as informative as antipyrine clearance in identifying minimal hepatic impairment.

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.

This work investigated the possible use of AdDP as adjuvant therapy to praziquantel (PZQ) in mice infected with PZQ-insusceptible Schistosoma mansoni isolate in a trial to increase the susceptibility of this isolate to the drug. Two batches of C57 BL/6 mice were infected with PZQ-susceptible and -insusceptible S. mansoni isolates, and each batch was divided into five groups. Seven weeks postinfection, the experimental group received AdDP (5 mg/kg) in addition to PZQ in reduced dose (3x100 mg/kg). Three of the remaining four groups were treated controls; they received AdDP, PZQ in reduced dose and in full dose (2x500 mg/kg), and the fourth group was infected untreated. In mice infected with PZQ-susceptible or -insusceptible S. mansoni isolate, praziquantel alone, and in addition to AdDP, reduced worm and egg loads and increased percentage dead eggs. Also, they improved the histopathological changes (reduction in granuloma diameter, percentage fibrotic area with increased percentage degenerated eggs). Inducible nitric oxide synthase (iNOS), nitric oxide (NO) in culture of peritoneal macrophages, and number of CD68-positive cells were decreased with improved alanine amino transaminase. In mice receiving combined therapy AdDP+PZQ, the antischistosomal efficacy and the reductions in the inflammatory granulomatous reactions, NO in cultured peritoneal macrophages, percentage fibrotic areas recorded, were comparable to that in mice receiving full dose of PZQ, with significantly higher reduction in CD68 cells denoting enhanced antischistosomal efficacy and healing of the inflammatory reactions in the liver.

Schistosoma mansoni is known to be refractory to praziquantel treatment in the pre-patent period of infection. Since Schistosoma haematobium has a much longer pre-patent period (10-12 weeks vs. 5-6 for the former species), we asked the question whether a correspondingly longer period of insusceptibility exists in urinary schistosomiasis. In hamsters treated at different times after infection, S. haematobium was partially refractory to praziquantel when treatment was given at week 5, but showed practically full sensitivity at 7-8 weeks and later times. Schistosoma haematobium worms obtained at different times after infection and exposed in vitro to praziquantel were refractory to low drug concentrations between 4 and 6 weeks, but were clearly affected at higher concentrations and at later time points. We conclude that S. haematobium does not have a praziquantel-insensitive window longer than in S. manson, in spite of its much longer maturation period. In addition, refractoriness of immature stages can be overcome at higher drug concentrations.

A decade ago, a study revealed that praziquantel (PZQ) failed to cure 1.6% of those with intestinal schistomiasis in five villages of the Nile delta region. The recommended dosage of PZQ is a single 40 mg/kg oral dose, and each of these patients continued to pass viable Schistosoma mansoni eggs despite three successive doses at or above this level. The eggs passed by these uncured villagers produced adult worms that were, in most cases, significantly less responsive to PZQ in vitro. This report investigates the current sensitivity of S. mansoni infections to PZQ after 10 years of therapeutic pressure in the same villages, testing the hypothesis that the number of drug failures would have increased as continued drug pressure selected for worms with diminished sensitivity to PZQ. The data show that these villages have experienced a significant decrease in the prevalence and intensity of S. mansoni infections, with present infection rate of 10.9%, compared with 25.1% in 1994. The first treatment resulted in normal range of cure rates, between 73.8 and 92.3% in each of the five villages in the study. After three successive doses (40, 40, and 60 mg/kg, the same treatment protocol applied a decade ago) there were no uncured patients remaining in the study. This shows that there has not been an increase of drug failure, despite 10 years of therapeutic pressure in these villages where there had been resistant infections and worms with decreased response to PZQ.

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 be100 mg/kg (mean=70+/-7 SD; median=68), while all five putatively praziquantel-resistant isolates had estimated ED50s100 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.

Schistosome worm muscle tension and [45Ca2+]-uptake were tested as possible correlates of susceptibility to praziquantel (PZQ) assessed by estimating the drug ED50. Schistosoma mansoni cercariae of PZQ sensitive (S-CD, S-MOC and S-GP) and insensitive S. mansoni isolates (I-EE2, I-BANL and I-Senegal 47) were used to infect batches of CD-1 Swiss albino mice. Seven weeks after infection, animals of each batch were divided into six groups. Five of them received PZQ in doses of 12.5, 25, 50, 100 or 200 mg/kg PZQ, respectively, for five consecutive days, while the sixth was left as untreated controls. Two weeks after treatment mice were sacrificed, perfused and PZQ ED50's were estimated. Male worms recovered from infected untreated controls were examined for their muscle tension increase in response to PZQ using a physiological recorder coupled to a photooptic transducer. [45Ca2+]-uptake of male worms in the presence and absence of PZQ was determined using a liquid scintillation beta counter. Data revealed that PZQ insensitive isolates had significantly higher drug ED50 (>130 mg/kg) than PZQ sensitive isolates with ED50's

After placement in mice of PZQ-sensitive and -insensitive S. mansoni isolates obtained from villagers responding and not responding to PZQ, parasitological criteria reflecting their biological development and also the host anti-immature worm immunoglobulin isotypes were examined 8 and 10 weeks post infection. Hepatic granuloma diameter, hepatic histopathological changes and immunolocalization of IgG and IgM on the surface of PZQ-sensitive and -resistant worms were also examined 10 weeks post infection. Data showed that parasitological criteria were not significantly different between mice infected with the PZQ-sensitive and -insensitive S. mansoni isolates. As regards serum immunoglobulins, in mice infected with the PZQ-insensitive S. mansoni isolate, IgG and IgG1 were significantly (p0.05) lower 8 and 10 weeks post infection, respectively (1.41+/-0.07 and 1.08+/-0.10 and 1.35+/-0.06 and 1.09+/-0.07) than in mice infected with the PZQ-sensitive S. mansoni isolate (1.73+/-0.15 and 1.38+/-0.10 and 1.73+/-0.17 and 1.54+0.21) after the same observation periods. IgM level was nearly the same while IgE was lower than that recorded in mice infected with the PZQ-sensitive S. mansoni isolate. IgG immunofluorescence was also lower (60%+/-6.78) on the surface of resistant worms than that of sensitive worms (66.6%+/-5.27); meanwhile, hepatic granuloma diameter was significantly larger (296.5+/-3.0 vs 283.6+/-4.0) in mice infected with the PZQ-insensitive S. mansoni isolate with higher percentage of intact eggs. Differences in the immunogenic make up of PZQ-sensitive and -insensitive S. mansoni isolates qualitatively and/or quantitatively favoring a certain Th cell subpopulation response could be the underlying reason for such differences recorded in the host immunoglobulin isotype response and also the egg-induced hepatic histopathological changes.

Consideration of existing compounds always simplifies and shortens the long and difficult process of discovering new drugs specifically for diseases of developing countries, an approach that may add to the significant potential cost savings. This study focused on improving the biological characteristics of the already-existing antischistosomal praziquantel (PZQ) by incorporating it into montmorillonite (MMT) clay as a delivery carrier to overcome its known bioavailability drawbacks. The oral bioavailability of a PZQ-MMT clay nanoformulation and its in vivo efficacy against Schistosoma mansoni were investigated. The PZQ-MMT clay nanoformulation provided a preparation with a controlled release rate, a decrease in crystallinity, and an appreciable reduction in particle size. Uninfected and infected mice treated with PZQ-MMT clay showed 3.61- and 1.96-fold and 2.16- and 1.94-fold increases, respectively, in area under the concentration-time curve from 0 to 8 h (AUC 0–8 ) and maximum concentration of drug in serum ( C max ), with a decrease in elimination rate constant ( k el ) by 2.84- and 1.35-fold and increases in the absorption rate constant ( k a ) and half-life ( t 1/2 e ) by 2.11- and 1.51-fold and 2.86- and 1.34-fold, respectively, versus the corresponding conventional PZQ-treated groups. This improved bioavailability has been expressed in higher efficacy of the drug, where the dose necessary to kill 50% of the worms was reduced by >3-fold (PZQ 50% effective dose [ED 50 ] was 20.25 mg/kg of body weight for PZQ-MMT clay compared to 74.07 mg/kg for conventional PZQ), with significant reduction in total tissue egg load and increase in total immature, mature, and dead eggs in most of the drug-treated groups. This formulation showed better bioavailability, enhanced antischistosomal efficacy, and a safer profile despite the longer period of residence in the systemic circulation. Although the conventional drug's toxicity was not examined, animal mortality rates were not different between groups receiving the test PZQ-clay nanoformulation and conventional PZQ.

The aggressive use of praziquantel to combat schistosomiasis in Egpyt raises concern about the possible emergence of resistance. Eggs from Egyptian patients with praziquantel-resistant infections (not cured by 3 doses of praziquantel) have been used to establish infection-specific schistosome isolates in mice. The response of these worms to the drug was observed in vitro, in order to determine if the isolates obtained from these resistant infections were, in fact, less responsive to praziquantel. One of the hallmark effects of praziquantel on schistosomes in vitro is a disruption of the worm's outer surface, the tegument. Here, praziquantel-induced tegumental damage is observed in 3 distinct isolates, 2 derived from resistant infections and 1 from an infection cured by a single dose. The isolates from the resistant infections were less susceptible to praziquantel-induced tegumental damage in vitro, suggesting that the worms are in some way less responsive to the drug.

This study was undertaken to evaluate the effect of recombinant Schistosoma mansoni-26 Glutathione S-transferase (rSm 26 GST) or soluble egg antigen (SEA) alone and in addition to praziquantel (PZQ) on the state of resistance to S. mansoni reinfection. The associated changes in the immune responses were evaluated. The experimental group of mice were injected intravenously before S. mansoni infection (80 cercariae/mouse) either with rSm26 GST (1 microgx4) or SEA (10 microgx4) in addition to PZQ (2x500 mg/kg) administered 6 weeks post-infection. Seven control groups were used, three of them were the infected (80 cercariae/mouse), the challenged (240 cercariae/mouse) and the infected challenged controls (80+240 cercariae/mouse). The rest of the four groups were the treated controls receiving: the GST-Lyzate, rSmGST, SEA and PZQ in the same doses and at the same timings. Challenge infection was conducted for all the groups 8 weeks post-infection. Animals were sacrificed 3 weeks post-challenge. After sacrifice animals were perfused and percentage resistance to reinfection was calculated. Immune responses were assessed by the measurement of hepatic granuloma diameter, intralesional T-cell phenotypes and serum immunoglobulin isotypes. The highest percentage of resistance to reinfection was observed in rGST-treated group while the lowest percentage of resistance was detected in PZQ-treated group. Whereas in mice receiving combined rGST or SEA and PZQ, percentage resistance to reinfection was significantly higher than that in PZQ treated mice. The remarkable reduction in granuloma diameter in rGST-treated group with or without PZQ was associated with decrease in the intralesional L(3)T(4)(+) and increase in Lyt(2)(+) T-cell phenotypes. However, no special relationship was observed between the percentage of resistance and the changes in granuloma diameter or intralesional T-cell phenotypes. The increase in percentage resistance to reinfection was found accompanied by increased anti SWAP IgE. Combined rGST and PZQ provided the complementary goals of improved state of resistance to reinfection 'which was compromized after cure with PZQ' and the maximal reduction in granuloma diameter.

Results from infected patients, not cured by multiple doses of praziquantel (PZQ), have been reported from different geographic locations, suggesting that resistance to the drug may be present. This has been coupled with several in vivo (e.g., studies on mice infected with 'resistant isolates') and in vitro tests (e.g., direct application and measurement of the effects of the drug on schistosomes maintained in culture) demonstrating a significant reduction in the drug's efficacy. Despite little field evidence that schistosomes are becoming less sensitive to the drug, 100% cure after PZQ is rarely achieved; meanwhile, the percentage of cure rates in endemic areas could be an overestimate if one accounts for the sensitivity of most egg counting methods coupled with the limited faecal sampling. To be proactive, the efficacy of PZQ has to be monitored on a systematic basis not only for cure, but also for the reduction of egg excretion complemented with periodical assessment for the susceptibility to the drug on local strains. Investigation of field isolates with confirmed diminished sensitivity to the drug will help in determining the frequency, epidemiology, genetic and physiologic basis for the observed resistance. Monitoring for changes in drug responsiveness in high transmission areas, where treatment failure as a result of immature or resistant parasites can not be differentiated, should be initiated. New chemotherapeutic alternatives and strategies in addition to a simple rapid, inexpensive test to detect resistance should be encouraged.

This study was undertaken to study the efficacy of praziquantel (PZQ) in potentially tolerized Schistosoma mansoni infected, egg-injected C57BL/6 mice, receiving multiple administrations of soluble egg antigen (SEA) intravenously (i.v.). Four animal groups were studied. Experimental group I received four injections of SEA (10 μg) intravenously on days −7, −5, −3 and −2 before infection and PZQ orally (500 mg/kg over two consecutive days) 7 weeks post-infection. Three control groups received the following treatment: group II received the same tolerizing dose of SEA without PZQ, group III received PZQ in the same dose and at the same timing. Group IV received S. mansoni infection and egg injection 8 weeks post-infection and served as an infected, egg-injected control. Egg injection was conducted 8 weeks post-infection using viable S. mansoni eggs via the tail vein. Animals were killed 16 days post-egg injection, i.e. 10 weeks post-infection. After sacrifice, lungs and livers were removed for histopathological study and measurement of granuloma diameters. Spleens and serum were collected for the assay of lymphoproliferative response to SEA and antischistosomal immunoglobulins. The worm and egg burdens were also studied. Compared to infected, egg-injected untreated controls, repeated i.v. administrations of SEA down-regulated egg-injected (pulmonary) and egg-deposited (hepatic) granulomas and the lymphoproliferative response to SEA. Antischistosomal IgG level was increased. Susceptibility to S. mansoni infection was not found to be different from that in the infected, egg-injected controls. PZQ in the dose used caused complete eradication of worms, disappearance of immature egg stages, decrease in the number of mature eggs and an increase in the number of dead eggs. Hepatic granuloma diameter, lymphoproliferative response to SEA and IgG level were reduced. In mice receiving a combined regimen of multiple SEA administrations and PZQ with down-regulated granuloma and reduced lymphoproliferative response to SEA, the efficacy of PZQ was the same as in mice receiving PZQ alone. This was shown by comparable grades of worm and egg reduction. The histopathological examination of liver and lung sections in the different treated groups revealed moderate to small-sized hypocellular granulomas. Although no statistically significant difference was recorded between the mean granuloma diameters of the experimental group receiving both the tolerizing dose of SEA and PZQ compared to the group receiving the tolerizing dose of SEA without chemotherapy, this experimental group showed the least associated histopathological parenchymal changes. It appears from this work that combined SEA and PZQ provided many complementary goals; a reduction of egg-induced pathology, minimal parenchymal changes and the eradication of worms.

This study was undertaken to assess the optimum conditions required to reduce the vigorous host granulomatous reaction around Schistosoma mansoni eggs. Soluble schistosomal egg antigen (SEA) at a concentration of 10 or 100 micrograms protein was administered i.p. or i.v. into unprimed C57BL/6 mice. SEA was injected either alone or in combination with cyclophosphamide (CY) 100 or 50 mg/kg via i.p. route. Seven or 14 days later viable eggs of S. mansoni were injected via the tail vein into treated groups and untreated normal controls. Mice were sacrificed 8, 16 and 24 days after the injection of eggs. The lungs were removed for histopathological study, measurement of granuloma diameter and phenotypic analysis of granuloma intralesional T-cell subsets. Compared to untreated controls, the lower concentration of SEA (10 micrograms) administered by the i.v. route 7 days before egg injection, induced a significant reduction in granuloma diameter 16 days after egg injection than that by the i.p. route or at a higher SEA concentration (100 micrograms). Compared to untreated controls, the higher dose of CY (100 mg/kg), given i.p. alone or in combination with 10 micrograms SEA by the i.v. or i.p. route, induced a significant reduction in granuloma diameter, while 50 mg/kg CY did not cause any reduction. The reduction in granuloma diameter by i.v. administration of low SEA concentration alone or in combination with CY IP, was associated with a decrease in the granuloma intralesional L3T4+/Lyt2+ ratio. The decrease in the ratio was due to an increase in Lyt2+ cells. The results suggest that the use of low dose SEA by the i.v. route alone or combined with an immunosuppressive drug ameliorates pathological changes concurrent with S. mansoni infection.

The efficacy of two brands (brand 1=Billricide; Bayer AG, Leverkusen, Germany; brand 2=Distocide; EPICO pharmaceuticals, Cairo, Egypt) of praziquantel (PZQ) in full and half doses (40 and 20 mg kg−1 monitored as percentage egg reduction and cure rate was investigated in S. mansoni infected school-children. A total of 506 school-children (8–16 years of age) were classified into three groups according to their intensity of infection, heavy [>500 eggs per grams (epg)], moderate (100–500 epg) and light (

A pool of 38 pan-African Centres of Excellence (CoEs) in health innovation has been selected and recognized by the African Network for Drugs and Diagnostics Innovation (ANDI), through a competitive criteria based process. The process identified a number of opportunities and challenges for health R&D and innovation in the continent: i) it provides a direct evidence for the existence of innovation capability that can be leveraged to fill specific gaps in the continent; ii) it revealed a research and financing pattern that is largely fragmented and uncoordinated, and iii) it highlights the most frequent funders of health research in the continent. The CoEs are envisioned as an innovative network of public and private institutions with a critical mass of expertise and resources to support projects and a variety of activities for capacity building and scientific exchange, including hosting fellows, trainees, scientists on sabbaticals and exchange with other African and non-African institutions.

Based on the fact that artemether (ART) affects immature schistosomes and that the effect of praziquantel (PZQ) mainly targets mature schistosomes, this work investigates the possible enhanced efficacy of PZQ in combination with ART in mice harboring a PZQ non-susceptible Schistosoma mansoni isolate. Associated schistosomal, inflammatory, hepatic histopathological changes have been investigated by examining the tissue markers expressing apoptosis using FAS (CD95), anti-apoptosis (Bcl2) and angiogenesis [vascular endothelial growth factor (VEGF)]. A batch of Swiss albino mice infected with a PZQ non-susceptible (EE10) S. mansoni isolate was divided into 12 groups. Animals of the first group were left without treatment as infected controls, while groups 2-6 received PZQ in increasing doses. The animals of group 7 received ART in double doses. Those comprising groups 8-12 received combined therapy of PZQ and ART in the same doses and at the same timings postinfection (PI) as those belonging to groups 2-6. Parasitological parameters, liver function, and histopathological and immunohistochemical studies of FAS, Bcl2 and VEGF antibodies were assessed. Combined administration of ART and PZQ reduced the ED(50) (the dose at which the worm burden was decreased by 50%) of PZQ. Typical granulomas were not seen in animals treated with ART alone and combined with PZQ, with least expression of FAS and VEGF and increased expression of Bcl2. The minimal histopathological changes recorded in mice treated with both ART and PZQ could be related to a synergistic/additive effect of ART, markedly reducing the intensity of infection. Improved liver function tests support the less severe histopathological changes under the influence of this treatment protocol. This study encourages human trials especially in areas where malaria is not endemic, and differing combination doses should be investigated in view of the antagonistic effect noticed with some dose regimens.

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.

SUMMARYTreatment with praziquantel (PZQ) has become virtually the sole basis of schistosomiasis control in sub-Saharan Africa and elsewhere, and the drug is reviewed here in the context of the increasing rate that it is being used for this purpose. Attention is drawn to our relative lack of knowledge about the mechanisms of action of PZQ at the molecular level, the need for more work to be done on schistosome isolates that have been collected recently from endemic areas rather than those maintained in laboratory conditions for long periods, and our reliance for experimental work mainly onSchistosoma mansoni, little work having been done onS. haematobium. There is no evidence that resistance to PZQ has been induced in African schistosomes as a result of its large-scale use on that continent to date, but there is also no assurance that PZQ and/or schistosomes are in any way unique and that resistant organisms will not be selected as a result of widespread drug usage. The failure of PZQ to produce complete cures in populations given a routine treatment should therefore solicit considerable concern. With few alternatives to PZQ currently available and/or on the horizon, methods to monitor drug-susceptibility in African schistosomes need to be devised and used to help ensure that this drug remains effective for as long a time as possible.

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.

This study investigates the development of the Egyptian strain of Schistosoma haematobium and the resultant immunohistopathology and biochemical changes in organs affected. In addition, the response of different developmental stages of S. haematobium worms to praziquantel (PZQ) was examined. Schistosoma haematobium-infected hamsters were classified into 4 groups and were treated at day 35, 55, 75, and 95 postinfection (PI), respectively. Each group was subdivided into 3 subgroups. Two of them were treated orally with PZQ (300 mg/kg or 500 mg/kg divided equally on 2 consecutive days), and the third group was left without treatment. Treated groups were killed 20 days posttreatment. Infection with S. haematobium became patent 73 days PI; tissue egg load and worm fecundity were higher at 95 days and maximal 115 days PI, with an oogram pattern comparable to that in Schistosoma mansoni infection. In the liver, small cellular granulomas were observed 75 days PI, with preponderance of CD4+ T-cell phenotypes. In the urinary bladder, only submucosal focal Brunn's-nest formation and angiogenesis without typical granulomas were observed. Ninety-five and 115 days PI, confluent granulomata with multiple eggs in the center were observed in the liver and urinary bladder, with a preponderance of CD8+ positive T cells in the liver and hyperplasia of the urinary bladder epithelium with cystitis cystica and papillae formation. One hundred percent worm eradication was recorded with the higher dose of PZQ in animals treated 75 and 95 days PI. In conclusion, in spite of the long prepatent period of the Egyptian strain of S. haematobium, sensitivity to PZQ was recorded soon after infection. Granulomata were similar to those of S. mansoni in the livers and urinary bladders, but they were confluent with multiple eggs in the centers, hyperplasia of the urinary bladder urothelium with cystitis cystica, papillae, and Brunn's-nest formation predictive of malignant changes with no hepatocyte dysplasia.

Resveratrol is a naturally occurring polyphenol, possesses several pharmacological activities including anticancer, antioxidant, antidiabetic, antinociceptive, and antiasthmatic activity. Little is known about its hepatoprotective action mechanisms. This study was conceived to explore the possible protective mechanisms of resveratrol compared with the hepatoprotective silymarin in thioacetamide (TAA)-induced hepatic injury in rats. Thirty-two rats were equally divided into four groups; normal control (i), TAA (100 mg/kg) (ii), TAA + silymarin (50 mg/kg) (iii), and TAA + resveratrol (10 mg/kg) (iv). Liver function and histopathology, pro-inflammatory cytokines, oxidative stress, and apoptotic markers were examined. Data were analyzed using ANOVA test followed by Tukey post hoc test. Compared to TAA-intoxicated group, resveratrol mitigated liver damage, and inflammation as noted by less inflammatory infiltration, hydropic degeneration with decreased levels of tumor necrosis factor-alpha, interleukin-6, and interferon-gamma by 78.83, 18.12, and 64.49%, respectively. Furthermore, it reduced (P < 0.05) alanine and aspartate aminotransferases by 36.64 and 48.09%, respectively, restored hepatic glutathione content and normalized superoxide dismutase and malondialdehyde levels. While it inhibited nuclear factor-kappa B, cytochrome 2E1, and enhanced apoptosis of necrotic hepatocytes via increasing caspase-3 activity. Our findings indicated that the potential hepatoprotective mechanisms of resveratrol are associated with inhibition of inflammation, enhancing the apoptosis of necrotic hepatocytes, and suppression of oxidative stress.

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 in90% worm eradication and amelioration of Schistosoma mansoni eggs from the tissues, with minor histological changes in the liver.

In a multicenter investigation of the potential antischistosomal activity of myrrh, a resin obtained from an African plant, different derivatives of the resin, including the commercial preparation Mirazid, were tested at different doses in mice and hamsters infected with Schistosoma mansoni. In mice infected with the Egyptian (CD) strain of S. mansoni, four of six groups treated with Mirazid did not show significant worm reduction, while the remaining groups showed significant but trivial reductions. In mice infected with the Puerto Rican (Mill Hill) strain of S. mansoni, a Mirazid solution was toxic for mice at high doses and produced modest or no worm reduction at lower doses. In hamsters and mice infected with Puerto Rican (NMRI) and Brazilian (LE) strains of S. mansoni and treated with the crude extract of myrrh in doses ranging from 180 to 10,000 mg/kg, no signs of antibilharzial activity were observed. Total tissue egg load and egg developmental stages were not affected by any of the treatment regimens. These results were in contrast to those obtained in praziquantel-treated animals in which 94% worm reduction and 100% egg reduction was observed. Based on the findings of this work, we cannot recommend the use of Mirazid in human cases of schistosomiasis.

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.

The effect of praziquantel in different concentrations on isolated rat hepatocytes as a cellular target was studied to detect any possible toxicity. Leakage of cytosolic enzymes, aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase (LDH) was monitored after one hour of incubation of all the cells with the drug. Levels of reduced glutathione (GSH) and cytochrome P450 were also assayed. The drug, in concentrations of 5, 25, 50 and 100 μg/ml, had no effect on any of these parameters. In contrast, the hepatotoxic compound trichloroethylene showed dose-dependent toxicity, as measured by trypan blue (TB) exclusion, LDH leakage, and reduction in GSH content in the present cellular model. These results suggest that praziquantel is a relatively safe drug with respect to liver function.

In two groups of mice infected with 60 (group I) and 120 (group II) Schistosoma mansoni cercariae, respectively, the effects of intensity and duration of infection, and of praziquantel therapy (curative vs subcurative dose) on the levels of circulating anodic antigen (CAA), were studied. CAA was measured in trichloracetic acid-treated serum samples with an avidin - biotin enzyme-linked immunosorbent assay (AB - ELISA) using the monoclonal anti-CAA antibody. Total worm burdens, oogram patterns and ova counts/g liver and intestine were followed up. The lowest detectable level of CAA was about 1.0 ng/ml, and was positive with a worm load of 3–5/mouse. CAA levels became already detectable as early as 1–2 weeks post-infection (pi) before any parasitological parameter and showed a significant drop from the 11th–12th week pi onwards. A positive correlation was demonstrated between the CAA level and worm load. Following successful praziquantel therapy, CAA disappeared earlier than any of the other parameters studied.

Murine schistosomiasis is usually associated with hepatic granulomatous lesions together with high serum and granuloma angiotensin converting enzyme (ACE) activity. Praziquantel (PRZ) which is known to reduce granuloma size was studied to show whether this effect is related to changes in ACE activity. Furthermore, captopril was studied to show whether by inhibiting ACE activity, the drug could also affect granuloma size. PRZ, captopril, and their combination led to significant reduction in liver granuloma. However, in normal mice, captopril was shown to increase rather than decrease serum ACE. The decrease in ACE activity by PRZ was correlated with its curative effect in infected mice. However, in experimentally induced pulmonary granulomata, the drug reduced granuloma size without affecting ACE activity of either serum or granuloma. It may be concluded that reduction in ACE activity may be beneficial as far as diminution of granuloma size is concerned and irrespective of whether there is an active infection or not. The possible use of Captopril as an antihypertensive in bilharzial infections associated with hypertension would probably not adversely affect the granulomatous lesions.

The schistosomicidal efficacy of praziquantel (2 × 500 mg/kg), oxamniquine (1 × 100 mg/kg) and combined one-third the curative dose of each of them (333 mg/kg praziquantel+33 mg/kg oxamniquine), were correlated to gonadal steroid hormonal changes and state of disease immunopathology, in mice infected with 100 Schistosoma mansoni (S. mansoni) cercariae. Maximum efficacy recorded with combination regimen particularly 4 weeks after treatment, was accompanied by maximum reduction in granuloma volume (65%), least fall in testosterone level (−56·2 and −60·2% in male and female mice respectively) and increased progesterone level (+ 33 · 9 and + 81 · 5% in male and female mice respectively). These findings revealed a potentiated effect of combination therapy on mature infection and the possible involvement of gonadal steroid hormones in affecting the efficacy of schistosomicidal drugs and state of disease immunopathology.

Cytophotometric measurement of the effect of praziquantel (500 mg/kg for 2 days) versus hycanthone (60 mg/kg for 3 days) on hepatocyte nuclear deoxyribonucleic acid (DNA) content was evaluated in Schistosoma mansoni infected mice. Drugs were given 8 weeks post-infection and repeated weekly for 4 weeks. DNA values of infected untreated control and infected drug treated groups were related to the median and upper diploid DNA values of normal control. Schistosoma mansoni infection per se did not change the hepatocyte DNA content, yet aneuploidy was 16.7%. Praziquantel did not result in significant change of DNA content or ploidy, while hycanthone resulted in marked significant increase of DNA content (328.9%) and aneuploidy (100%), compared to infected untreated control. Histopathological examination revealed hyperchromatic nuclei with mitosis in the hepatocytes of hycanthone treated mice, but not in praziquantel treated animals. These DNA changes were found to correlate with the reported safety of praziquantel and the carcinogenicity of hycanthone.