Repurposing of Anti-malarial SynriamTM and testing its efficacy against Egyptian strain of Schistosoma hematobium CURRENT

Background : Trials for discovering an anti-malarial drug, which can compete Schistosomal infection in co-endemic areas, are ongoing. Some preliminary studies were done on Synriam (SYN), antimalarial drug combination (arterolane maleate and piperaquine phosphate) released from Ranbaxy, to test its anti-schistosomal effect. However, in vitro incubation of SYN with different Schistosoma hematobium stages was not fully assessed. The aim of this study is to determine the antischistosomal in vitro effect of SYN on adult and juvenile stages of Schistosoma hematobium- Egyptian strain. Methods : The adult and juvenile worms were incubated with ascending concentrations of SYN and with praziquantel as positive control. Viability, survival, morphological and ultrastructural changes were assessed at different time points. Results : Higher concentrations of 60, 80 µg/ml showed rapid and lethal effects on adult and juvenile stages of both species, with prominent ultrastructural alterations. Concentrations of (10, 20, 40 µg/ml) showed mild to moderate effect on adult schistosomes . However, contrarily to praziquantel, larval immature stages responded significantly and rapidly to low concentration of SYN with 100% death rate. Conclusion : The present findings are consistent with the evaluation of anti-schistosomal therapeutic effect of SYN to be utilized in malaria co-endemic areas.


Introduction
Schistosomiasis is ranked as the second most prevalent devastating tropical disease in Africa after malaria. It affects over 250 million people worldwide [1]. Digenetic worms of the genus Schistosoma; Schistosoma mansoni (S. mansoni), Schistosoma hematobium (S. hematobium), and Schistosoma japonicum (S. japonicum) are incriminated in most of human schistosomiasis infections. Control programs were faced by clinical emergence of praziquantel-resistant strains and praziquantel therapeutic deficient activity against immature juvenile stages [2,3]. Accordingly, there is an urgent need to discover and develop new better acting drugs.
Within the same context, being the current treatment of choice for resistant strains of malaria [4], Artemisinin derivatives based on trioxane (ARTs), have shown reliable in vitro and in vivo antischistosomal effect, especially on immature juvenile stages [5][6][7]. Moreover, the trioxolanes, OZ78, OZ277 and OZ209 in particular, showed potent activity against juvenile S. mansoni and S. japonicum infections in the mouse model [8].
In 2011, after Ranbaxy Laboratories Limited (India) has licensed Synriam TM (SYN)-arterolane maleate (OZ277) and piperaquine-, to be used as a treatment for malaria patients, Mossallam and his team proved the powerful activity of this synthetic derivative in mice infected with S. mansoni especially against the parasite juvenile stages [9]. Working on the promising use of a single effective drug against both blood parasites in co-endemic areas is imperative. In the framework of Synriam testing, this study aimed to investigate the in vitro schistosomicidal properties of Synriam using multiple ascending concentrations against S. haematobium developmental stages using light and scanning electron microscopic (SEM) observations.

Animals and parasites:
Syrian golden hamsters (Mesocricetus auratus) of both sexes, aged 4-6 weeks were purchased form the Schistosome Biological Supply Program (SBSP), Theodor Bilharz Research Institute (TBRI), Giza, Egypt, and kept under favorable conditions of 25˚C temperature, 70% humidity, 12-hr light and 12-hr dark cycle and adapted for 1 week before infection. Animals were infected percutaneously with freshly shed Egyptian strain of S. hematobium cercariae from experimentally infected Bulinus trucatus snails according to [10].
Early stages Schistosomulae were obtained by mechanical transformation of S. hematobium cercariae [11,12] while that of hepatic stages were recovered by portal-mesenteric perfusion at the 42nd day post infection. Moreover, adult S. hematobium worms were recovered by perfusion of the vesical venous plexus at the 90th day post infection [13].

Preparation of the parasites and experimental design for in vitro culturing:
Recovered juvenile and adult (male and female) worms were washed 10 times in RPMI medium, taking great care for parasite tegument integrity. Worms were placed in wells of sterile, flat-bottom, 24-well plates (Corning, NY) containing 2 ml RPMI 1640 medium /well and incubated at 37°C and 5% CO2 atmosphere [9]. To mimic an in-vivo physiological environment, serum albumin (SA) and human alpha acidic glycoprotein (AGP) were added to the culture medium [15].
Before the beginning of the experiment, Worms were being assessed hourly, using light microscope (Olympus Inverted Microscope Model IX70; Olympus, Tokyo, Japan). Only viable, transparent, contractile worms showing total tegument integrity were included in the experiment while parasites that were contracted or had acquired an opaque appearance were considered dead and discarded.
Group c is further subdivided into subgroups by using different concentrations of the test drug (SYN was used to obtain final concentrations of 5 to 80 μg/ml [5,10,20,40, 60, and 80 μg/ml]).
In each experiment, the survival was monitored in (1 st , 3 rd , 24 th and 48 th hours) post-incubation with SYN, for body motility and occurrence of death using the dissecting microscope. Worms showed no body movement for at least 30 seconds observation was considered as dead [8]. All tests were repeated three times, viability data from one experiment was presented as range of scores (0-3) based on the motility of the worms; 0 (all worms are dead), 1 (minimal motor activity), 2 (slow motor 5 activity) and 3 (normal motor activity) [16,17]. The mean ±SD were determined for each independent experiment over the groups.

Ultrastructural studies using scanning electron microscope:
Ultrastructural features of samples of juvenile and adult Shistosomes were examined and compared to those of the control groups for the effect of the tested drugs. Worms were washed out in PBS from the culture solution, fixed in Karnovsky's solution, for 10 hours (hrs), and then cleared by keeping them overnight at 4˚C in PBS. The samples were immediately processed according to [18]. The samples were fixed in equal volumes of glutaraldehyde 4% + cacodylate 0.2 % for 2 hrs. Then, washed in equal volumes of sucrose 0.4 % and cacodylate 0.2 % for 2 hrs followed by fixation in equal volumes of osmic acid 2% and cacodylate 0.3 % for 1 hr and then washed with distilled water.

Statistical analysis of data:
Data was entered and analyzed using SPSS Version 21 (IBM Corporation, NY, USA). Survival analysis was done using the Kaplan-Meier method. The log-rank test was used for pairwise comparison between different groups in each experiment. P value less than or equal to 0.05 was considered significant. Viability scoring was calculated using Kruskal-Wallis Test (non parametric test) and displayed as Viability score box-plot. Viability data from one experiment having average replicate was presented as range of scores. Mean values were determined for each independent experiment over the group. The mean ± SD of three viability mean values (of three independent experiments) for each group was calculated at different time points and used to examine if differences between the groups were statistically significant.

Anti-schistosomal effect of SYN on survival and viability of S. hematobium stages:
Assessment of survival and viability of adult and juvenile stages of S. hematobium during in vitro 6 incubation with different concentrations (conc.) of SYN are illustrated in Figure 1 (Fig 1).

Adult S. hematobium stage:
Similar to control group, incubation with 5 µg/ml revealed normal activity (motility score: 3) and outline till the second day of incubation. Diminished motility and abnormal body attitude were observed in a gradual progressive manner, starting form 1 hr and 3 hrs of incubation with 40 µg/ml, 20 µg/ml, respectively. Stretching and thinning of the whole worm up to complete deformed outline and loss of sucking ability, as evidenced by detachment of worms from sides of wells, were significant with higher concentrations (60 µg/ml, 80 µg/ml), since the 1 st hour of incubation (P <0.005). Motility and viability of worms was affected significantly according to dose of SYN and time of exposure (P <0.001). Mortality was reported initially using 40 µg/ml (16.7%) and reached 75% after 24 hours.
Meanwhile, death rate was 91.7% and 100% when the adult worms were incubated with 60 µg/ml and 80 µg/ml, respectively. All worms were dead by the end of the experiment upon incubation with all concentrations under study.

Juvenile S. hematobium Stages:
Immature stages were susceptible to lower concentrations of SYN. Normal body movement (motility score: 3) was noticed after 3 hours of incubation with 5 µg/ml. In both early and late stages, reduced motility and body stiffness was significant at conc. of 10 µg/ml within 1 st hour, which increased progressively with incubation duration. Unlike the adult, 100 % death rate was reached after 24 hrs of incubation starting from dose of 20 µg/ml. Interestingly, Kaplan-Meier survival analysis revealed significant effect on 3-hour and 42-days aged schistosomula stages using 40 µg/ml and 20 µg/ml, respectively, in comparison to PZQ-treated groups (P <0.001). (Fig 2 and 3):

Adult S. hematobium stage:
SEM micrographs revealed normal appearance of tegument and suckers (Fig 3A) using low concentrations (5,10 µg/ml) at the beginning of the experiment, followed by slight puffiness of the oral sucker and scattered vesicles by the end of the first day (Fig 3B). Upon early exposure to 20, 40 µg/ml, blunting of tubercles, slight irregularities in the inter-tubercular ridges ( Fig 3C) and edematous suckers were observed. This effect was progressively augmented using higher concentrations (60, 80 µg/ml) in the form of wrinkled surface, deeply fissured tegument with widespread loss of tubercles ( Fig 3D). Suckers were severely affected in the form of total disappearance of sucker vicinity (Fig 3E) and tegumental erosion of oral sucker (Fig 3F).

Juvenile S. hematobium Stages:
Tegument appeared corrugated with alternating bulging and depressions and invaginated acetabulum ( Fig 3G). The developed nodules and vesicles increased in size and depth, in proportion with dose and time, causing sloughing of the external tegument and accumulation of debris on and around the worm body ( Fig 3H).

Discussion
Artemisinins and trioxolanes are among the firstly discovered anti-malarial drugs with promising antischistosomal effect [19]. Subsequently, more researchers tried to shed light on the anti-schistosomal powers of artemisinins and their synthetic derivatives [3,20,21]. Up on characterization of artemisinin chemically, a new class of anti-schistosomal bioactive peroxides named Ozonides (1,2,4trioxolanes) started to exist. Their potency against Schistosoma species was discovered especially against the developing than the adult [8].
The newly released anti-malarial combination of arterolane maleate (Ozonide OZ277) and piperaquine phosphate, SYN, exhibited encouraging anti-schistosomal properties against S. mansoni [9], which provides a good opportunity for curing Schistosoma and Malaria in co-endemic areas. In the same context, a field trial had been done by Barda and her team to test the effect of multiple effective antimalarial drugs including Synriam on S. mansoni and S. haematobium infected adolescents in Cote d' voir [22]. Surprisingly, results of this trial have shown weak performance against S. haematobium, which were not coinciding with ours. It could be hypothesized that, Barda et al exploratory study was restricted to single age group "adolescents" and done on relatively small sample size of patients, which may play a role in such discrepancy in statistical significances. Therefore, as a proof-ofconcept, more laboratory and clinical trials would be necessary to confirm these findings.
To extend the available knowledge about this antimalarial drug, in vitro effect of ascending SYN concentrations on S. haematobium developmental stages were tested at selected time points. Since motility scoring, number of dead worms and tegument structural alterations are common parasitological parameters, which were often evaluated to indicate the biological activity of any antischistosomal drug [6,[23][24][25][26][27], we assessed these parameters to determine the effect of SYN on adult, early and late juvenile schistosomes in respect to the drug of choice, praziquantel.
As shown in results, stages under study responded to SYN preparations according to "dose and time of exposure" pattern. This observation indicates that SYN can target all the intra-mammalian developmental stages of the parasite. This complies with similar studies on artemesinins tested for anti-schistosomal properties [28][29][30][31].
Up on comparing to the significant effect of ascending concentrations of SYN, increasing the concentration of PZQ from 1 g /ml up to 30 g /ml didn't produce any discrepant anti-schistosomal effect on S. japonicam in vitro [32].
It is believed that ART-mediated lethal effect is due to alkylation of internal and external cellular proteins (enzymes), such as sarcoplasmic endoplasmic reticulum ATPase PfATP6, which is crucial to parasite survival [33]. The carbon-centred free radicals incriminated in such process were produced by an irreversible redox reaction between the peroxides in artemisinins and hemoglobin degradation product, Haem [34,35].
In this study, SYN exhibited significant effect on juvenile stages of S. haematobium. The killing ability was detected at concentration of 10 g /ml after 1 hour of exposure, reaching the highest efficacy at 20 g /ml within 2 hours of incubation (100% mortality). However, PZQ had minimal effect on larval and immature stages. It was hypothesized that the juveniles' high potential gene activity in transcriptional up regulation of multidrug resistance-associated protein 1 (SmMRP1) can be responsible in such resistance against PZQ [36].
Our observation is supported by other studies testing the in vitro anti-schistosomal properties of natural or synthetic ARTs on S. mansoni, S. haematobium and S. japonicum [8,37,38]. Eventually, early (skin stage) and late (hepatic) schistosomula were susceptible to SYN as evident by the process 9 of pathological alterations in vivo. Although Mossallam and his colleagues had proved that adding haem in the culture media is more efficient than using SYN alone [9], our results exhibited, beyond doubt, a slower onset of action of SYN on adult schistosomes using the lowest tested concentrations, while the effect was significantly augmented and hastened after the exposure to the higher doses (60 g/ml, 80 g/ml) without the addition of haem. This can be explained that haem is needed to intensify the effect of lower concentrations of SYN, while higher concentrations can produce enough killing effect by its one. However, their results using 20 g/ml SYN were apparently discrepant than ours using the same concentration within the same time point. A finding which need more trials to explain the pharmacokinetics and metabolism of the drug on the cellular level and its impact inside different strains of the parasite.
To document the effect of any anti-schistosomal drug, observations should be done on the ultrastructural level. Tegument, being the target for most of the anti-schistosomal drugs, is usually assessed using electron micrograph studies. We observed prominent tegumental affection of juvenile and adult stages, which was escalated as the time of incubation and concentration of SYN increased.
Bleb formation, vesiculation, focal erosions and disintegration were typical features observed in this study. Likewise, ARTs derivates' effect on the schistosomal tegument was reported clearly in [8,21,38,39]. Additionally, the tegumental damage reported with juvenile schistosomes was correlated with the high killing efficacy of SYN on larval stages. To conclude, SYN is considered a promising antischistosomal drug covering the inherited defects of PZQ in terms of effectiveness against different developmental stages of schistosomes, which provide both prophylaxis and treatment advantages.
However, further studies are important to clarify the pharmacokinetics and bioavailability of the drug and consequently, to allocate suitable dose schedule in treating human infection.

Ethics approval and consent to participate
Scientific research ethical committee of TBRI, Giza, Egypt, approved the protocol of this study. All the animal experiments were performed in accordance with the Egyptian National Animal Welfare Standards and under recommendations of the TBRI Ethical Committee for laboratory animals research guidelines.

Availability of data and materials
Not applicable

Competing interests
The authors declare that they have no competing interests

Authors' contributions
All authors contributed to every activity of the manuscript; idea of the research, study design, collection of materials, methodology, writing the paper and revising it. All authors read and approved the final manuscript.