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.

Schistosomicidal and hepatoprotective activity of gamma-aminobutyric acid (GABA) alone or combined with praziquantel against Schistosoma mansoni infection in murine model.

OBJECTIVE: This study aimed to evaluate the efficacy of gamma-aminobutyric acid (GABA) alone or combined with praziquantel (PZQ) against Schistosoma (S) mansoni infection in a murine model. METHODS: Five groups, 8 mice each, were studied; GI served as normal controls; GII: S. mansoni-infected control group and the other three S. mansoni-infected groups received drug regimens for 5 consecutive days as follows GIII: Infected-PZQ treated group (200 mg/kg/day); GIV: Infected-GABA treated group (300 mg/kg/day) and GV: Infected-PZQ-GABA treated group (100 mg/kg/day for each drug). All animal groups were sacrificed two weeks later and different parasitological, histopathological and biochemical parameters were assessed. RESULTS: Combined GABA-PZQ treated group recorded the highest significant reduction in all parasitological, histopathological and biochemical parameters followed by PZQ and finally GABA groups. Combined GABA-PZQ treatment led to the complete disappearance of immature eggs and marked reduction of deposited eggs in liver tissues and improved liver pathology. Significant improvement in hepatic oxidative stress levels, serum albumin and total protein in response to GABA treatment alone or combined with PZQ. CONCLUSION: GABA had schistosomicidal, hepatoprotective and antioxidant activities against S. mansoni infection, GABA disrupted parasite pairing and activity, reduced the total number of worms recovered and the number of ova in the tissues. GABA may be considered an adjuvant therapy to potentiate PZQ antiparasitic activity and eradicate infection-induced liver damage and oxidative stress.

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.

A novel praziquantel solid lipid nanoparticle formulation shows enhanced bioavailability and antischistosomal efficacy against murine S. mansoni infection.

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.

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.

MCR synthesis of praziquantel derivatives.

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.

Further assessment of Mirazid as antischistosomal drug in experimental schistosomiasis hematobium.

Conflicting reports are found in the literature about the efficacy of Mirazid (MZ), which is a special formulation of myrrh obtained from the stem of Commiphora molmol (Nees), Engl. tree (Burseraceae), as an antischistosomal drug. This initiated the present study to further assess this drug in experimental schistosomiasis hematobium. The drug was administered orally to hamsters infected with Schistosoma hematobium ( Bilharz, 1852 ) using 500 mg/kg body weight for six successive days on an empty stomach. The drug effect was examined after three periods: 4, 8 and 12 weeks post-treatment. Emphasis was given to certain parameters such as change in worm load, number of ova/mg tissue, oogram pattern and number of ova/g stool, and tegumental changes in the worms by electron microscopy after prolonged observation periods. The results showed very slight 3.4% worm reduction by MZ after the longest evaluation period (12 weeks), versus very high reduction (100%) by the reference drug praziquantel (PZQ). In comparison with the untreated control no change was found in the number of ova/mg tissue in MZ-treated hamsters regardless of the date of observation (4-12 weeks), versus significantly high reduction (99.6%) observed in the case of PZQ treatment. However, a significant decrease (22%) in the ratio of immature and increase in dead ova in tissues of MZ-treated hamsters was obvious at 12 weeks post treatment. In MZ-treated animals, a slight reduction (18.3%) in the number of stool eggs versus absence of eggs in PZQ-treated animals 12 weeks after treatment. Scanning electron microscopic examination of S. hematobium worms revealed intact tubercles, spines and sensory bulbs and no effect of the ventral side after MZ treatment. Meanwhile, PZQ treatment revealed extensive disruption of the tegument worm. Therefore, this experimental study gives extra support to previously reported negative evaluation about the effectiveness of this drug in the treatment of schistosomiasis against many other published positive results. This controversy about the efficacy of MZ may be attributed to inconsistency of its material which is obtained from natural origin.

Antischistosomal activity of hexadecyloxypropyl cyclic 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine and other alkoxyalkyl esters of acyclic nucleoside phosphonates assessed by schistosome worm killing in vitro.

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.

Synthesis and in vitro evaluation of new benzothiazole derivatives as schistosomicidal agents.

A series of benzothiazol-2-yl-dithiocarbamates 3a-d along with their copper complexes 4a-c were synthesized via the reaction of suitable alkyl, aralkyl or heteroaryl halides with the sodium salt of benzothiazol-2-yl-dithiocarbamic acid, followed by complexation with copper sulphate. N-(4-Acetyl-5-aryl-4,5-dihydro-1,3,4-thiadiazol-2-yl)-N-benzothiazol-2-yl-acetamides 7a-c were synthesized by cyclization of the appropriate thiosemicarbazones 6a-c in acetic anhydride. Selected compounds were screened for in vitro schistosomicidal activity against Schistosoma mansoni at three different dosage levels (10, 50 and 100 microg/mL). Three of these products, 4a-c, showed schistosomicidal activity similar to praziquantel, with 100% worm mortality at 10 microg/mL. These compounds would constitute a new class of potent schistosomicidal agents.

Effect of praziquantel on the immature stages of Schistosoma haematobium.

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.

Lack of evidence for an antischistosomal activity of myrrh in experimental animals.

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.

Validation of sensitivity to praziquantel using Schistosoma mansoni worm muscle tension and Ca2+-uptake as possible in vitro correlates to in vivo ED50 determination.

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 <100 mg/kg. Moreover, in response to PZQ they were found to possess significant reductions in their worm muscle tension and their [45Ca2+]-uptake were <100%. Both parameters showed a significant negative correlation to PZQ ED50 in vivo and a significant positive correlation to each other.

Activity of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine against Schistosomiasis mansoni in mice.

The activity of the acyclic nucleotide analogue 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine [(S)-HPMPA] against Schistosoma mansoni was investigated in mice. The compound was injected intraperitoneally, usually on two or five consecutive days, at 10 to 20 mg/kg of body weight/day. The treatment started before, at the time of, and after the onset of egg laying (oviposition) by S. mansoni. The animals were killed from 7 to 40 days after the cessation of treatment. Significant reductions in the total numbers of female and coupled worms were found. Female fecundity and both hepatic and intestinal egg loads were suppressed. These effects were more pronounced with dosing regimens launched before the time of oviposition. The complete disappearance of immature eggs and a significant reduction to the complete absence of mature eggs, with 99 to 100% of the eggs being dead, were produced. No hepatic egg-induced granulomas were present in mice treated at the time of oviposition, and the granulomas were smaller in mice treated before S. mansoni oviposition. These preliminary findings extend the knowledge of the antiparasitic properties of (S)-HPMPA.

Effect of simultaneous and/or consecutive administration of the broad spectrum anthelmintic flubendazole together with praziquantel in experimental Schistosoma mansoni infection.

This study is a trial to demonstrate the effect of the broad spectrum anthelmintic drug flubendazole (methyl 5-(p-fluoro-benzoyl)-2-benzimidazolecarbamate, CAS 31430-15-6), a mebendazole derivative, together with praziquantel (CAS 55268-74-1, EMBAY 8440, Biltricide) in murine schistosomiasis mansoni. Moreover, the relationship between the posttreatment worm burden, oogram pattern, tissue egg load and hepatic granuloma volume was also investigated. Three main groups of Swiss albino mice infected with Schistosoma mansoni cercariae were used in the experiment. Group I included infected untreated control mice. Group II: Subgroup II (a): Animals received 1/3 the dose of praziquantel 25 days post infection. Subgroup II (b): Mice were given 1/3 dose of flubendazole 25 days post infection. Subgroup II (c): Animals received the combination (1/3 dose of flubendazole + 1/3 the dose of praziquantel 25 days post infection. Group III: Subgroup III (a): Mice were given 1/3 the dose of praziquantel 7 weeks post infection. Subgroup III (b): Mice received 1/3 dose of flubendazole 25 days post infection. 24 days later, 1/3 the dose of praziquantel was given. Mice given the consecutive drug regimen (flubendazole 1/3 single oral dose 25 days post infection, then praziquantel 1/3 oral dose for two successive days 24 days later, revealed a significant reduction in the recovery of adult schistosomes after portal perfusion (95.9%), absence of immature stages of ova development, a higher level of dead ova in the oogram and the smallest granuloma mean diameter. These data were less conspicuous in mice given the simultaneous drug regimen.