Oxamniquine derivatives overcome Praziquantel treatment limitations for Schistosomiasis.

Human schistosomiasis is a neglected tropical disease caused by Schistosoma mansoni, S. haematobium, and S. japonicum. Praziquantel (PZQ) is the method of choice for treatment. Due to constant selection pressure, there is an urgent need for new therapies for schistosomiasis. Previous treatment of S. mansoni included the use of oxamniquine (OXA), a drug that is activated by a schistosome sulfotransferase (SULT). Guided by data from X-ray crystallography and Schistosoma killing assays more than 350 OXA derivatives were designed, synthesized, and tested. We were able to identify CIDD-0150610 and CIDD-0150303 as potent derivatives in vitro that kill (100%) of all three Schistosoma species at a final concentration of 71.5 µM. We evaluated the efficacy of the best OXA derivates in an in vivo model after treatment with a single dose of 100 mg/kg by oral gavage. The highest rate of worm burden reduction was achieved by CIDD -150303 (81.8%) against S. mansoni, CIDD-0149830 (80.2%) against S. haematobium and CIDD-066790 (86.7%) against S. japonicum. We have also evaluated the ability of the derivatives to kill immature stages since PZQ does not kill immature schistosomes. CIDD-0150303 demonstrated (100%) killing for all life stages at a final concentration of 143 µM in vitro and effective reduction in worm burden in vivo against S. mansoni. To understand how OXA derivatives fit in the SULT binding pocket, X-ray crystal structures of CIDD-0150303 and CIDD-0150610 demonstrate that the SULT active site will accommodate further modifications to our most active compounds as we fine tune them to increase favorable pharmacokinetic properties. Treatment with a single dose of 100 mg/kg by oral gavage with co-dose of PZQ + CIDD-0150303 reduced the worm burden of PZQ resistant parasites in an animal model by 90.8%. Therefore, we conclude that CIDD-0150303, CIDD-0149830 and CIDD-066790 are novel drugs that overcome some of PZQ limitations, and CIDD-0150303 can be used with PZQ in combination therapy.

Addressing the oxamniquine in vitro-in vivo paradox to facilitate a new generation of anti-schistosome treatments.

The antischistosomal drug oxamniquine, OXA, requires activation by a sulfotransferase within the parasitic worm to enable killing. Examination of the pharmacokinetic/pharmacodynamic (PK/PD) relationship for OXA identified an in vitro-in vivo paradox with the maximal clinical plasma concentrations five-to ten-times lower than the efficacious concentration for in vitro schistosomal killing. The parasite resides in the vasculature between the intestine and the liver, and modeling the PK data to determine portal concentrations fits with in vitro studies and explains the required human dose. In silico models were used to predict murine dosing to recapitulate human conditions for OXA portal concentration and time course. Follow-up PK studies verified in mice that a 50-100 mg/kg oral gavage dose of OXA formulated in acetate buffer recapitulates the 20-40 mg/kg dose common in patients. OXA was rapidly cleared through a combination of metabolism and excretion into bile. OXA absorbance and tissue distribution were similar in wild-type and P-gp efflux transporter knockout mice. The incorporation of in vitro efficacy data and portal concentration was demonstrated for an improved OXA-inspired analog that has been shown to kill S. mansoni, S. haematobium, and S. japonicum, whereas OXA is only effective against S. mansoni. Second-generation OXA analogs should optimize both in vitro killing and physiochemical properties to achieve high portal concentration via rapid oral absorption, facilitated by favorable solubility, permeability, and minimal intestinal metabolism.

Disulfiram and dithiocarbamate analogues demonstrate promising antischistosomal effects.

Schistosomiasis is a neglected tropical disease with more than 200 million new infections per year. It is caused by parasites of the genus Schistosoma and can lead to death if left untreated. Currently, only two drugs are available to combat schistosomiasis: praziquantel and, to a limited extent, oxamniquine. However, the intensive use of these two drugs leads to an increased probability of the emergence of resistance. Thus, the search for new active substances and their targeted development are mandatory. In this study the substance class of "dithiocarbamates" and their potential as antischistosomal agents is highlighted. These compounds are derived from the basic structure of the human aldehyde dehydrogenase inhibitor disulfiram (tetraethylthiuram disulfide, DSF) and its metabolites. Our compounds revealed promising activity (in vitro) against adults of Schistosoma mansoni, such as the reduction of egg production, pairing stability, vitality, and motility. Moreover, tegument damage as well as gut dilatations or even the death of the parasite were observed. We performed detailed structure-activity relationship studies on both sides of the dithiocarbamate core leading to a library of approximately 300 derivatives (116 derivatives shown here). Starting with 100 µm we improved antischistosomal activity down to 25 µm by substitution of the single bonded sulfur atom for example with different benzyl moieties and integration of the two residues on the nitrogen atom into a cyclic structure like piperazine. Its derivatization at the 4-nitrogen with a sulfonyl group or an acyl group led to the most active derivatives of this study which were active at 10 µm. In light of this SAR study, we identified 17 derivatives that significantly reduced motility and induced several other phenotypes at 25 µm, and importantly five of them have antischistosomal activity also at 10 µm. These derivatives were found to be non-cytotoxic in two human cell lines at 100 µm. Therefore, dithiocarbamates seem to be interesting new candidates for further antischistosomal drug development.

Drug Discovery and Target Identification against Schistosomiasis: A Reality Check on Progress and Future Prospects.

Schistosomiasis ranks among the most important infectious diseases, with over 200 million people currently being infected and > 280,000 deaths reported annually. Chemotherapeutic treatment has relied on one drug, praziquantel, for four decades, while other drugs, such as oxamniquine and metrifonate, are no longer preferred for clinical use due to their narrow spectrum of activity - these are only active against S. mansoni and S. haematobium, respectively. Despite being cheap, safe, and effective against all schistosome species, praziquantel is ineffective against immature worms, which may lead to reinfections and treatment failure in endemic areas; a situation that necessitates repeated administration besides other limitations. Therefore, novel drugs are urgently needed to overcome this situation. In this paper, an up to date review of drug targets identified and validated against schistosomiasis while also encompassing promising clinical and preclinical candidate drugs is presented. While there are considerable efforts aimed at identifying and validating drug targets, the pipeline for new antischistosomals is dry. Moreover, the majority of compounds evaluated preclinically are not really advanced because most of them were evaluated in very small preclinical species such as mice alone. Overall, it appears that although a lot of research is going on at discovery phases, unfortunately, it does not translate to advanced preclinical and clinical evaluation.

An iterative process produces oxamniquine derivatives that kill the major species of schistosomes infecting humans.

Currently there is only one method of treatment for human schistosomiasis, the drug praziquantel. Strong selective pressure has caused a serious concern for a rise in resistance to praziquantel leading to the necessity for additional pharmaceuticals, with a distinctly different mechanism of action, to be used in combination therapy with praziquantel. Previous treatment of Schistosoma mansoni included the use of oxamniquine (OXA), a prodrug that is enzymatically activated in S. mansoni but is ineffective against S. haematobium and S. japonicum. The oxamniquine activating enzyme was identified as a S. mansoni sulfotransferase (SmSULT-OR). Structural data have allowed for directed drug development in reengineering oxamniquine to be effective against S. haematobium and S. japonicum. Guided by data from X-ray crystallographic studies and Schistosoma worm killing assays on oxamniquine, our structure-based drug design approach produced a robust SAR program that tested over 300 derivatives and identified several new lead compounds with effective worm killing in vitro. Previous studies resulted in the discovery of compound CIDD-0066790, which demonstrated broad-species activity in killing of schistosome species. As these compounds are racemic mixtures, we tested and demonstrate that the R enantiomer CIDD-007229 kills S. mansoni, S. haematobium and S. japonicum better than the parent drug (CIDD-0066790). The search for derivatives that kill better than CIDD-0066790 has resulted in a derivative (CIDD- 149830) that kills 100% of S. mansoni, S. haematobium and S. japonicum adult worms within 7 days. We hypothesize that the difference in activation and thus killing by the derivatives is due to the ability of the derivative to fit in the binding pocket of each sulfotransferase (SmSULT-OR, ShSULT-OR, SjSULT-OR) and to be efficiently sulfated. The purpose of this research is to develop a second drug to be used in conjunction with praziquantel to treat the major human species of Schistosoma. Collectively, our findings show that CIDD-00149830 and CIDD-0072229 are promising novel drugs for the treatment of human schistosomiasis and strongly support further development and in vivo testing.

Why does oxamniquine kill Schistosoma mansoni and not S. haematobium and S. japonicum?

Human schistosomiasis is a disease which globally affects over 229 million people. Three major species affecting humans are Schistosoma mansoni, S. haematobium and S. japonicum. Previous treatment of S. mansoni includes the use of oxamniquine (OXA), a prodrug that is enzymatically activated in S. mansoni but is ineffective against S. haematobium and S. japonicum. The OXA activating enzyme was identified and crystallized, as being a S. mansoni sulfotransferase (SmSULT). S. haematobium and S. japonicum possess homologs of SmSULT (ShSULT and SjSULT) begging the question; why does oxamniquine fail to kill S. haematobium and S. japonicum adult worms? Investigation of the molecular structures of the sulfotransferases indicates that structural differences, specifically in OXA contact residues, do not abrogate OXA binding in the active sites as previously hypothesized. Data presented argue that the ability of SULTs to sulfate and thus activate OXA and its derivatives is linked to the ability of OXA to fit in the binding pocket to allow the transfer of a sulfur group.

Schistosomal Sulfotransferase Interaction with Oxamniquine Involves Hybrid Mechanism of Induced-fit and Conformational Selection.

BACKGROUND: Sulfotransferase family comprises key enzymes involved in drug metabolism. Oxamniquine is a pro-drug converted into its active form by schistosomal sulfotransferase. The conformational dynamics of side-chain amino acid residues at the binding site of schistosomal sulfotransferase towards activation of oxamniquine has not received attention. OBJECTIVE: The study investigated the conformational dynamics of binding site residues in free and oxamniquine bound schistosomal sulfotransferase systems and their contribution to the mechanism of oxamniquine activation by schistosomal sulfotransferase using molecular dynamics simulations and binding energy calculations. METHODS: Schistosomal sulfotransferase was obtained from Protein Data Bank and both the free and oxamniquine bound forms were subjected to molecular dynamics simulations using GROMACS-4.5.5 after modeling it's missing amino acid residues with SWISS-MODEL. Amino acid residues at its binding site for oxamniquine was determined and used for Principal Component Analysis and calculations of side-chain dihedrals. In addition, binding energy of the oxamniquine bound system was calculated using g_MMPBSA. RESULTS: The results showed that binding site amino acid residues in free and oxamniquine bound sulfotransferase sampled different conformational space involving several rotameric states. Importantly, Phe45, Ile145 and Leu241 generated newly induced conformations, whereas Phe41 exhibited shift in equilibrium of its conformational distribution. In addition, the result showed binding energy of -130.091 ± 8.800 KJ/mol and Phe45 contributed -9.8576 KJ/mol. CONCLUSION: The results showed that schistosomal sulfotransferase binds oxamniquine by relying on hybrid mechanism of induced fit and conformational selection models. The findings offer new insight into sulfotransferase engineering and design of new drugs that target sulfotransferase.

A novel cell-free method to culture Schistosoma mansoni from cercariae to juvenile worm stages for in vitro drug testing.

BACKGROUND: The arsenal in anthelminthic treatment against schistosomiasis is limited and relies almost exclusively on a single drug, praziquantel (PZQ). Thus, resistance to PZQ could constitute a major threat. Even though PZQ is potent in killing adult worms, its activity against earlier stages is limited. Current in vitro drug screening strategies depend on newly transformed schistosomula (NTS) for initial hit identification, thereby limiting sensitivity to new compounds predominantly active in later developmental stages. Therefore, the aim of this study was to establish a highly standardized, straightforward and reliable culture method to generate and maintain advanced larval stages in vitro. We present here how this method can be a valuable tool to test drug efficacy at each intermediate larval stage, reducing the reliance on animal use (3Rs). METHODOLOGY/PRINCIPAL FINDINGS: Cercariae were mechanically transformed into skin-stage (SkS) schistosomula and successfully cultured for up to four weeks with no loss in viability in a commercially available medium. Under these serum- and cell-free conditions, development halted at the lung-stage (LuS). However, the addition of human serum (HSe) propelled further development into liver stage (LiS) worms within eight weeks. Skin and lung stages, as well as LiS, were submitted to 96-well drug screening assays using known anti-schistosomal compounds such as PZQ, oxamniquine (OXM), mefloquine (MFQ) and artemether (ART). Our findings showed stage-dependent differences in larval susceptibility to these compounds. CONCLUSION: With this robust and highly standardized in vitro assay, important developmental stages of S. mansoni up to LiS worms can be generated and maintained over prolonged periods of time. The phenotype of LiS worms, when exposed to reference drugs, was comparable to most previously published works for ex vivo harvested adult worms. Therefore, this in vitro assay can help reduce reliance on animal experiments in search for new anti-schistosomal drugs.

Assessment of tegumental damage to Schistosoma mansoni and S. haematobium after in vitro exposure to ferrocenyl, ruthenocenyl and benzyl derivatives of oxamniquine using scanning electron microscopy.

BACKGROUND: Schistosomiasis is one of the most harmful parasitic diseases worldwide, praziquantel being the only drug in widespread use to treat it. We recently demonstrated that ferrocenyl, ruthenocenyl and benzyl derivatives of oxamniquine (Fc-OXA, Rc-OXA and Bn-OXA) are promising antischistosomal drug candidates. METHODS: In this study we assessed the tegumental damage of these three derivatives of oxamniquine using scanning electron microscopy. Adult Schistosoma mansoni and S. haematobium were exposed to a concentration of 100 µM of each drug and incubated for 4-120 h, according to their onset of action and activity. RESULTS: While on S. mansoni the fastest acting compound was Fc-OXA, which revealed high activity after 4 h of incubation, on S. haematobium, Rc-OXA revealed the quickest onset, being lethal on all males within 24 h. In both species studied, the three derivatives showed the same patterns of tegumental damage consisting of blebs, sloughing and tegument rupturing all over the body. Additionally, on S. mansoni distinct patterns of tegumental damage were observed for each of the compounds: tissue ruptures in the gynaecophoric canal for Fc-OXA, loss of spines for Rc-OXA and oral sucker rupture for Bn-OXA. CONCLUSIONS: Our study confirmed that Fc-OXA, Rc-OXA and Bn-OXA are promising broad spectrum antischistosomal drug candidates. All derivatives show fast in vitro activity against S. mansoni and S. haematobium while validating the previous finding that the parent drug oxamniquine is less active in vitro under the conditions described. This work sets the base for further studies on the identification of a lead oxamniquine derivative, with the aim of identifying a molecule with the potential to become a new drug for human use.

Immunohistochemical Investigations of Treatment with Ro 13-3978, Praziquantel, Oxamniquine, and Mefloquine in Schistosoma mansoni-Infected Mice.

To date, there is only one drug in use, praziquantel, to treat more than 250 million people afflicted with schistosomiasis, a debilitating parasitic disease. The aryl hydantoin Ro 13-3978 is a promising drug candidate with in vivo activity superior to that of praziquantel against both adult and juvenile Schistosoma mansoni organisms. Given the drug's contrasting low activity in vitro and the timing of its onset of action in vivo, it was postulated that immune-assisted parasite clearance could contribute to the drug's in vivo activity. We undertook histopathological studies to investigate this hypothesis. Infected mice were treated with an effective dose of Ro 13-3978 (100 mg/kg of body weight) and were dissected before and after the drug's in vivo onset of action. The veins and livers were excised, paraffin-embedded, and sectioned, and macrophages (IBA-1), neutrophils (Neutro), B cells (CD45R), and T cells (CD3) were stained by immunohistochemistry. For comparison, samples from infected untreated mice and mice treated with effective doses of praziquantel (400 mg/kg), oxamniquine (200 mg/kg), and mefloquine (200 mg/kg) were examined. At 24 h after treatment with Ro 13-3978, significant macrophage recruitment to the veins was observed, along with a modest increase in circulating B cells, and at 48 h, neutrophils and T cells are also present. Treatment with praziquantel and oxamniquine showed similar patterns of recruitment but with comparatively higher cellular levels, whereas mefloquine treatment resulted in minimal cell recruitment until 3 days posttreatment. Our study sheds light on the immediate immune responses to antischistosomal treatment in mice and provides further insight into immune effector mechanisms of schistosome clearance.

Medicinal chemistry of antischistosomal drugs: Praziquantel and oxamniquine.

Neglected tropical diseases (NTDs) are a group of diseases that, besides prevailing in poverty conditions, contribute to the maintenance of social inequality, being a strong barrier to a country development. Schistosomiasis, a NTD, is a tropical and subtropical disease caused by the trematode Schistosoma mansoni (Africa, Middle East, Caribbean, Brazil, Venezuela, Suriname), japonicum (China, Indonesia, the Philippines), mekongi (several districts of Cambodia and the Lao People's Democratic Republic), intercalatum and guianensis (areas of tropical rainforests in Central Africa) and hematobium (Middle East Africa, Corsica, France) whose adult forms inhabit the mesenteric vessels of the host, while the intermediate forms are found in the aquatic gastropod snails of the genus Biomphalaria. Currently, praziquantel (PZQ) is the first line drug chosen for the treatment of schistosomiasis according to the World Health Organization (WHO) Model List of Essential Medicines, 2015. PZQ chemotherapy is considered to be the most important development for decades in the treatment of schistosomiasis. Beside the PZQ, oxamniquine (OXA) has been first described in 1969 and launched in Brazil by Pfizer under the name of Mansil® for oral administration. It has a lower cost when compared to PZQ, being active in the intestinal and hepatosplenic infections caused exclusively by S. mansoni, single species in Brazil. Both PZQ and OXA have limitations, as low efficacy in the treatment of acute schistosomiasis, low activity against S. mansoni in immature stages and resistance or tolerance, which is the reason why further research are still necessary for the development of a second generation of antischistosomal drugs. For the development of new PZQ analogs, three main strategies can be adopted: (a) synthesis and evaluation of PZQ analogues; (b) rational design of new pharmacophores; (c) discovery of new active compounds from screening programs on a large scale. Such (b) approach is difficult as the target of PZQ still unknown, the synthesis of new active analogues is possible from delineation of structure-activity relationships for PZQ. Thus, we proposed for a review article an accurate analysis of PZQ and OXA medicinal properties and uses, focusing on the pharmacochemical aspects of both drugs through 178 bibliographic references. The mechanisms of action will be discussed, with the latest information available in the literature (for the first time in the case of the oxamniquine). Cases of resistance are also discussed. As both drugs are available as a racemic mixture the biological impact of their stereochemistry to activity and side effects are reviewed. The results obtained for the combination of PZQ and artemisinin derivatives against immature worms are also introduced in the discussion. Using the information about more than 200 PZQ new derivatives synthetized during almost 35years since its discovery, a deep structure-activity relationship (SAR) is also proposed in this study.

Independent origins of loss-of-function mutations conferring oxamniquine resistance in a Brazilian schistosome population.

Molecular surveillance provides a powerful approach to monitoring the resistance status of parasite populations in the field and for understanding resistance evolution. Oxamniquine was used to treat Brazilian schistosomiasis patients (mid-1970s to mid-2000s) and several cases of parasite infections resistant to treatment were recorded. The gene underlying resistance (SmSULT-OR) encodes a sulfotransferase required for intracellular drug activation. Resistance has a recessive basis and occurs when both SmSULT-OR alleles encode for defective proteins. Here we examine SmSULT-OR sequence variation in a natural schistosome population in Brazil ∼40years after the first use of this drug. We sequenced SmSULT-OR from 189 individual miracidia (1-11 per patient) recovered from 49 patients, and tested proteins expressed from putative resistance alleles for their ability to activate oxamniquine. We found nine mutations (four non-synonymous single nucleotide polymorphisms, three non-coding single nucleotide polymorphisms and two indels). Both mutations (p.E142del and p.C35R) identified previously were recovered in this field population. We also found two additional mutations (a splice site variant and 1bp coding insertion) predicted to encode non-functional truncated proteins. Two additional substitutions (p.G206V, p.N215Y) tested had no impact on oxamniquine activation. Three results are of particular interest: (i) we recovered the p.E142del mutation from the field: this same deletion is responsible for resistance in an oxamniquine selected laboratory parasite population; (ii) frequencies of resistance alleles are extremely low (0.27-0.8%), perhaps due to fitness costs associated with carriage of these alleles; (iii) that four independent resistant alleles were found is consistent with the idea that multiple mutations can generate loss-of-function alleles.

Structural and Functional Characterization of the Enantiomers of the Antischistosomal Drug Oxamniquine.

BACKGROUND: For over two decades, a racemic mixture of oxamniquine (OXA) was administered to patients infected by Schistosoma mansoni, but whether one or both enantiomers exert antischistosomal activity was unknown. Recently, a ~30 kDa S. mansoni sulfotransferase (SmSULT) was identified as the target of OXA action. METHODOLOGY/PRINCIPAL FINDINGS: Here, we separate the OXA enantiomers using chromatographic methods and assign their optical activities as dextrorotary [(+)-OXA] or levorotary [(-)-OXA]. Crystal structures of the parasite enzyme in complex with optically pure (+)-OXA and (-)-OXA) reveal their absolute configurations as S- and R-, respectively. When tested in vitro, S-OXA demonstrated the bulk of schistosomicidal activity, while R-OXA had antischistosomal effects when present at relatively high concentrations. Crystal structures R-OXA•SmSULT and S-OXA•SmSULT complexes reveal similarities in the modes of OXA binding, but only the S-OXA enantiomer is observed in the structure of the enzyme exposed to racemic OXA. CONCLUSIONS/SIGNIFICANCE: Together the data suggest the higher schistosomicidal activity of S-OXA is correlated with its ability to outcompete R-OXA binding the sulfotransferase active site. These findings have important implications for the design, syntheses, and dosing of new OXA-based antischistosomal compounds.

Genetic and molecular basis of drug resistance and species-specific drug action in schistosome parasites.

Oxamniquine resistance evolved in the human blood fluke (Schistosoma mansoni) in Brazil in the 1970s. We crossed parental parasites differing ~500-fold in drug response, determined drug sensitivity and marker segregation in clonally derived second-generation progeny, and identified a single quantitative trait locus (logarithm of odds = 31) on chromosome 6. A sulfotransferase was identified as the causative gene by using RNA interference knockdown and biochemical complementation assays, and we subsequently demonstrated independent origins of loss-of-function mutations in field-derived and laboratory-selected resistant parasites. These results demonstrate the utility of linkage mapping in a human helminth parasite, while crystallographic analyses of protein-drug interactions illuminate the mode of drug action and provide a framework for rational design of oxamniquine derivatives that kill both S. mansoni and S. haematobium, the two species responsible for >99% of schistosomiasis cases worldwide.

Disease-drug pairs revealed by computational genomic connectivity mapping on GBA1 deficient, Gaucher disease mice.

We have reported that, in addition to recapitulating the classical human Gaucher disease (GD1) phenotype, deletion of the glucocerebrosidase (GBA1) gene in mice results in the dysfunction of a diverse population of immune cells. Most of immune-related, non-classical features of GD1, including gammopathies and autoimmune diathesis, are resistant to macrophage-directed therapies. This has prompted a search for newer agents for human GD1. Here, we used high-density microarray on splenic and liver cells from affected GBA1(-/-) mice to establish a gene "signature", which was then utilized to interrogate the Broad Institute database, CMAP. Computational connectivity mapping of disease and drug pairs through CMAP revealed several highly enriched, non-null, mimic and anti-mimic hits. Most notably, two compounds with anti-helminthic properties, namely albendazole and oxamniquine, were identified; these are particularly relevant for future testing as the expression of chitinases is enhanced in GD1.

Isothermal microcalorimetry to study drugs against Schistosoma mansoni.

Alternative antischistosomal drugs are required since praziquantel is virtually the only drug available for treatment and morbidity control of schistosomiasis. Manual microscopic reading is the current "gold standard" to assess the in vitro antischistosomal properties of test drugs; however, it is labor-intensive, subjective, and difficult to standardize. Hence, there is a need to develop novel tools for antischistosomal drug discovery. The in vitro effects of praziquantel, oxamniquine, artesunate, and mefloquine on metabolic activity and parasite motility of Schistosoma mansoni (newly transformed schistosomula [NTS] and 49-day-old adult worms) were studied using isothermal microcalorimetry (IMC). Results were compared to morphological readouts of viability. Results obtained for the four drugs tested with phenotypic evaluation by microscopy and IMC showed a good correlation, but IMC also identified drug effects that were not visible by microscopic evaluation, and IMC precisely determined the onset of action of the test drugs. Similar sensitivities on NTS and adult schistosomes were observed for praziquantel and mefloquine, while slight differences in the drug susceptibilities of the two developmental stages were noted with oxamniquine and artesunate. IMC is a useful tool for antischistosomal drug discovery that should be further validated. In addition, our data support the use of NTS in in vitro antischistosomal drug assays.

Similar cellular responses after treatment with either praziquantel or oxamniquine in Schistosoma mansoni infection.

The effect of treatment with either oxamniquine or praziquantel on S.mansoni specific IFN-gamma, IL-4, IL-5 and IL-10 was compared on PBMC which were collected pretreatment, 6 and 18 weeks post treatment. Using sandwich ELISA on the supernatants harvested from the PBMC stimulation by crude S. mansoni SEA and SWAP antigens after 5 days the levels of PBMC proliferation and cytokine production were similar according to treatment with either praziquantel or oxamniquine. Before treatment, infected groups showed low ratios, of IL-4:IFN-gamma, IL-5:IFNgamma and IL-10:IFN-gamma, indicating that IFN-gamma was high in the infected individuals. The general increase in immuno-modulation was observed post-treatment with elevated immune reactivity and cytokine production in both treatment groups. Treatment induced significant increases in levels of IL-4 (p < 0.05), IL-5 (p < 0.0001) and IL-10 (p < 0.05) cytokines 6 and 18 weeks after treatment. There were no significant differences in the increase in IL-4, IL-5 and IL-10 between children treated with praziquantel or oxamniquine. Pre-treatment IFN-gamma and IL-5 levels were positively correlated with infection (p < 0.001), while post treatment IL-4 cytokine levels were negatively correlated with baseline infection status (p < 0.001). The results suggest that treatment-induced immune responses are similar for both common anti-schistosome drugs praziquantel or oxamniquine having similar and immunizing effect.

Síntese e atividade biológica do derivado 6-formil-oxamniquina / Synthesis and biological activity of 6-formyl-oxamniquine derivative

A esquistossomose, uma importante doença no Brasil, é causada por trematódeo pertencente ao gênero Schistosoma, atingindo milhões de pessoas, numa das maiores regiões endêmicas dessa doença em todo globo. O principal objetivo desse trabalho foi sintetizar o derivado 6-formil-oxamniquina e avaliar sua atividade biológica. O derivado 6-formil- oxamniquina foi obtido por oxidação da oxamniquina com dióxido de manganês empregando diclorometano como solvente, à temperatura ambiente, por 24 horas. Sua obtenção foi confirmada por espectrometria na região de infravermelho e espectroscopia de RMN 13C e ÕH, apresentando atividade similar quando comparada à oxamniquina comercial (Mansil®).

Schistosomiasis, an important disease in Brazil, is caused by a trematode of the genus Schistosoma, reaching millions of person in one of the most endemic region of this disease in the whole globe. The main goal of this work was to syntetize the 6-formyl-oxamniquine derivative and evaluate its biological activity. The 6-formyl-oxamniquine derivative was obtained by the oxidation of oxamniquine with MnO2, applying CH2Cl2 as solvent at room temperature for 24 hours. The obtaintion of 6-formyl-oxamniquine derivative compound was confirmed by IR spectroscopy and 13C NMR and ÕH NMR, presenting similar activity when compared to the commercial oxamniquine (Mansil®).

Association of oxamniquine praziquantel and clonazepam in experimental Schistosomiasis mansoni

The antischistosomal activity of clonazepam, when administered alone or in association with oxamniquine and praziquantel, was experimentally evaluated in mice infected with Schistosoma mansoni. The animals were treated 45 days post-infection with a single dose, by oral route, according to three treatment schedules: clonazepam 25 mg/kg and sacrificed 15 min, 1h or 4 h after treatment; clonazepam 1.0, 2.5 or 10.0 mg/kg and sacrificed 15 days post-treatment or with the dose of 10 mg/kg in association with oxamniquine 50 mg/kg or praziquantel 200 mg/kg, single dose, orally, every schedule with a control group. The efficacy of the drugs in vivo was assessed by means of worm counts and their distribution in mesentery and liver, mortality and oogram changes. In the chemotherapeutic schedules used, clonazepam did not present antischistosomal activity and the result of the association of this drug with oxamniquine or praziquantel was not significantly different from the one obtained when these two last drugs were administered alone. In the in vitro experiments, the worms exposed to 0.6 mg/mL clonazepam remained motionless throughout the 8-day-period of observation, without egg-laying, whereas the worms of the control group showed normal movements, egg-laying and hatching of miracidia on the last day of observation. The results obtained in the present study confirm the action of clonazepam on S. mansoni adult worm, in vitro, causing total paralysis of males and females. However, no additive or synergistic effects were observed when clonazepam were used in association with oxamniquine or praziquantel.