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

Journal Pre-proof The phosphodiesterase-4 inhibitor roflumilast impacts Schistosoma mansoni ovipositing in vitro but displays only modest antischistosomal activity in vivo Sanaa S. Botros, Naglaa M. El-Lakkany, Sayed H. Seif el-Din, Samia William, AbdelNasser Sabra, Olfat A. Hammam, Harry P. de Koning PII:

S0014-4894(19)30377-7

DOI:

https://doi.org/10.1016/j.exppara.2019.107793

Reference:

YEXPR 107793

To appear in:

Experimental Parasitology

Received Date: 27 August 2019 Revised Date:

14 October 2019

Accepted Date: 7 November 2019

Please cite this article as: Botros, S.S., El-Lakkany, N.M., Seif el-Din, S.H., William, S., Sabra, A.-N., Hammam, O.A., de Koning, H.P., The phosphodiesterase-4 inhibitor roflumilast impacts Schistosoma mansoni ovipositing in vitro but displays only modest antischistosomal activity in vivo, Experimental Parasitology (2019), doi: https://doi.org/10.1016/j.exppara.2019.107793. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

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The phosphodiesterase-4 inhibitor Roflumilast impacts Schistosoma mansoni ovipositing in vitro but displays only modest antischistosomal activity in vivo Sanaa S. Botros a, Naglaa M. El-Lakkany a, Sayed H. Seif el-Din a, Samia William b, AbdelNasser Sabra a, Olfat A. Hammam c, Harry P. de Koning d,*

Departments of

a

Pharmacology,

b

Parasitology and

c

Pathology, Theodor Bilharz Research

Institute, Warrak El-Hadar, Imbaba, P.O. Box 30, Giza 12411, Egypt. d

Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, United

Kingdom.

Correspondence to: Harry P. de Koning: [email protected]

Keywords: Schistosoma mansoni; phosphodiesterase; PDE4; roflumilast; mouse model; drug discovery; hepatic granuloma.

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ABSTRACT 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 particularly 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.

1. Introduction Schistosomiasis, one of the most important neglected tropical diseases, is endemic in 75 countries worldwide in Africa, Asia, South America and the Middle East (Hotez and Kamath, 2009; Gray et al., 2011), where over 200 million individuals are infected, with approximately 280,000 fatalities every year; more than 200 million people require preventive annual treatment (WHO, 2018).There are no vaccines against schistosomiasis (Molehin et al., 2016) and the control of schistosomiasis relies on a single drug, praziquantel (PZQ), which has been used in clinical practice for almost four decades (WHO, 2012). Naturally, the long-term use of this drug in endemic areas, including mass-administration programs, brings concerns about reduced efficacy in field studies (Botros et al., 2005; Wang et al., 2012) and, as with all anthelmintics, the 2

inevitable risk of the development of resistant strains (Bergquist et al., 2017; De Koning, 2017). This problem is further emphasized by the well-known lack of PZQ efficacy against juvenile worms (Vale et al., 2017), which is a frequent cause of treatment failure in endemic areas. For these reasons, the development of new schistosomicidal drugs is urgently required. Phosphodiesterases (PDEs) are cyclic adenosine (cAMP)- or guanosine (cGMP)monophosphate specific enzymes that are present in most eukaryotic cells. Eleven human PDE families have been identified to date (Boswell-Smith et al., 2007) and their activities contribute to the control of the intracellular concentrations of these cyclic nucleotides that influence many signaling pathways in health and disease (Maurice et al., 2014; Kametani and Haga, 2015). A number of drugs based on inhibition of human PDEs are already on the market for a variety of clinical conditions. Of these, PDE4 has attracted considerable attention over the past decade as a drug target and selective inhibitors have shown success in a variety of diseases (Lipworth 2005; Maurice et al., 2014; Eskandari et al., 2015; Gurney et al., 2015; Klussmann, 2016). For instance, rolipram, roflumilast and cilomilast are used to treat chronic obstructive pulmonary disease (Fan Chung, 2006; Kumar et al., 2013). PDE4 homologues and their inhibitors have increasingly been investigated for therapeutic potential in parasitic protozoa as well (Shakur et al., 2011; Sebastián-Pérez et al., 2018; De Heuvel et al., 2019a,b; Siciliano de Araújo et al., 2019). Validation of trypanosomal PDE's as drug targets revealed a number of potent hits (De Koning et al., 2012) which have since been optimized for selectivity over hPDE4 (Blaazer et al., 2018; De Heuvel et al., 2019a,b). Indeed, a screening of several hundred potential PDE inhibitors by the PDE4NPD consortium found numerous new compounds with anti-schistosomal activities including gender-specific worm killing, and the complete cessation of ovipositing (Botros et al., 2019). For Schistosoma spp, SmPDE4A has received particular attention as it was reported that a series of oxaborole huPDE4A elicited effects like hypermotility and degeneration of S. mansoni in vitro. This effect correlated with inhibition of SmPDE4a, and expression of this enzyme in a Caenorhabditis elegans PDE4- mutant restored those worms to sensitivity to these inhibitors (Long et al., 2017). These authors identified four SmPDE4 orthologs and showed a correlation between inhibitory IC50 values of recombinant SmPDE4A and the antischistosomal effects of a series of benzoxaboroles. They also found that the hPDE4 inhibitor roflumilast, but not the related catechol rolipram, was a sub-nanomolar inhibitor of SmPDE4A. This sparked an effort 3

to identify new inhibitors of SmPDE4A, through in silico approaches with a virtual library and a carefully modeled structure of the enzyme, and subsequently the crystal structure of SmPDE4A was also elucidated (Sebastián-Pérez et al., 2019). Meanwhile, roflumilast, as a drug that is already in clinical use (for chronic obstructive pulmonary disease), is particularly attractive. In view of PDE inhibitors, anti-parasitic potential and the successful repurposing of some of the current anthelminthic drugs, specifically against neglected tropical diseases (Panic et al., 2014), PDE's inhibitors may serve as lead compounds for new drugs against schistosomiasis, with the major advantage of a well-understood pharmacology and toxicology in the human host. The possibility of repositioning clinically used PDE inhibitors for use against schistosomiasis is particularly attractive, as it would enormously reduce the developmental cost (and time) for such a treatment. This study was conducted to investigate the potential antischistosomal activity of the PDE4 inhibitor roflumilast, previously identified as a strong inhibitor of SmPDE4A (Long et al., 2017), using an in vitro schistosome worm killing assay and an in vivo animal model of Schistosoma mansoni infection. This study constitutes the first in vivo evaluation of a specific PDE4 inhibitor against murine schistosomiasis.

2. Materials and methods 2.1. Drugs Praziquantel (PZQ) (Distocide, Egyptian International Pharmaceutical Industries Company, EIPICO) and roflumilast (white powder) was provided by Professor R. Leurs of the Free University of Amsterdam (VUA), The Netherlands.

2.2. In vitro studies A. Preparation of drugs PZQ and roflumilast were prepared as stock solutions of 5 mM in pure DMSO. On the day of the experiment, different concentrations of the drugs (100 µM, 50 µM, 25 µM 10 µM and 5 µM) were freshly prepared in RPMI-1640 medium.

B. Compound potency testing on mature S. mansoni worm killing

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Worms were obtained from the Schistosome Biology Supply Center (SBSC), Theodor Bilharz Research Institute (TBRI), where 6−8 worms were placed/well in a 12-well tissue culture plate, and fresh RPMI-1640 medium (plus glutamine, 20% new born calf serum, and antibiotics [streptomycin, penicillin, and gentamicin]), containing the indicated concentration of the test compound, was added (Pica-Mattoccia and Cioli, 2004; Botros et al., 2005). Worms were incubated overnight in a CO2 incubator at 37 ºC. On the 2nd day, worms were examined by microscopy, washed three times with normal physiological saline, fresh medium was added and the incubation was continued. On the 3rd day, worm motility was observed and on the 4th day, medium was again changed. On day 5 (end of the observation period) worms were microscopically examined for their motility and appearance. Each concentration was tested in duplicate wells, and the final recording of percent worm mortality was determined as the number of dead worms [contracted and opaque] divided by the total number of worms ×100; the average of the duplicate wells was taken as the final %mortality for that experiment. Negative controls used medium without additions or medium with 2% DMSO; positive control media containing identical concentrations of PZQ were tested in parallel.

C. Ovipositing capacity Roflumilast was tested for its effect on worm ability to oviposit. 12-well tissue culture plates containing from 6−8 worms/well including at least one worm couple were used. Each concentration was tested in duplicate wells and on the 4th day, eggs were counted and discarded and the medium was changed. On day 5 (end of observation period) newly deposited eggs were counted. The final egg number is the total count of day four and five for each concentration tested, averaged for the duplicate wells. The data presented in Table 1 is the average of three independent experiments, each performed in duplicate. Statistical difference from untreated controls was determined using Student’s unpaired, two-tailed T-test.

2.3. In vivo study A. Infection of animals Male Swiss albino mice (CD-1) were obtained from SBSC of TBRI, Giza, Egypt, weighing 18–20 g each, and housed at an environmentally controlled room temperature of 20–22 °C, a 12 h light/dark cycle and 50−60% humidity with access to food and water ad libitum throughout the 5

acclimatization and experimental periods. Mice were infected with S. mansoni cercariae (provided by SBSC) using body immersion (Liang et al., 1987) and exposure to 80 ± 10 cercariae/mouse. All the animal experiments were conducted in accordance with the Guide for Care and Use of Laboratory Animals and were approved by the Institutional Review Board of TBRI.

B. Compounds tested in vivo Roflumilast was freshly prepared in 30% polyethylene glycol (PEG400) + 0.5% Tween-80 + 5% propylene glycol. Praziquantel (PZQ) was freshly suspended in 2% Cremphore-EL (SigmaAldrich, St Louis, MO, USA) before use.

C. Experimental design At the beginning of experiment, one hundred and thirty S. mansoni-infected mice were divided into two batches. Of the first batch (40 mice), 20 were treated 7 weeks post infection with roflumilast with a dose of 5 mg/kg/day for 5 days. These animals were sacrificed at 24 h and two weeks post end of treatment. Mice in a PZQ-treated group (10 mice) were administered the standard antischistosomal drug, PZQ, orally at a dose of 200 mg/kg/day for 5 consecutive days, and were sacrificed in parallel to untreated group (10 mice). The second batch (90 mice) was divided into nine groups, 10 mice each. Four of them were treated orally with a fixed dose of roflumilast (5 mg/kg/day/5 days) in addition to variable doses of PZQ, of 25, 50, 100 and 200 mg/kg/day, respectively, for five consecutive days starting from the 7th week post infection. Another four parallel groups of mice were administered PZQ alone in the scheduled doses for 5 consecutive days, while the ninth group was only administered the vehicle. All animals of this batch were sacrificed 10 days post end of treatment.

D. Assessment of parasitological criteria of cure Mice were sacrificed, perfused, and the worm burden was quantified as described (Duvall and De Witt, 1967) and sexed for estimation of percentage worm reduction and assessment of the PZQ ED50 (Cioli et al., 2004) in the presence and absence of roflumilast. The number of eggs per gram of liver or intestinal tissue was counted (Cheever, 1968). The percentage of egg developmental stages (oogram pattern) was studied (Pellegrino et al., 1962), identifying and

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counting eggs at different stages of maturity in three fragments of the intestine; the mean number of each stage was calculated.

2.4. Histological examination Liver specimens were fixed in 10% formalin, processed to paraffin blocks, sectioned to five sections/mouse, each of 4 µm thick; each section was 250 µm apart from the preceding one. Sections were stained with hematoxylin/eosin (H&E) and Masson trichrome for light microscopy examination of hepatic granulomas and associated histological changes. An ocular micrometer was used to measure noncontiguous granulomas, each with a single egg in its center. The overall mean granuloma diameter represents the measurements of 30 granulomas/5 sections/mouse. The cellular profile, the state of the S. mansoni eggs, and the associated histopathological changes were examined.

2.5. Scanning electron microscopy (SEM) Untreated worms and those recovered 24 h post treatment with either roflumilast or PZQ were incubated for 24 h at 37 °C in a 5% CO2 atmosphere, washed with sodium cacodylate buffer (0.1 M), fixed in 2.5% glutaraldehyde (pH 7.4) (Merck) over 24 h and then fixed in 1% osmium tetroxide for 1 h. Specimens were dehydrated in increasing concentrations of ethanol (50%, 70%, 80%, 90%, 95% and 100%) for 30 min each, dried in a critical point dryer, mounted on stubs, metalized with gold particles using a sputter coater and finally analyzed and photographed using a Jeol JSM-820 electron microscope.

2.6. Statistical analysis and ED50 calculation The percentage reduction of worm/egg burden in each treated group was calculated according to the following equation: % reduction = [(No. of worms/eggs in control group) − (No. of worms/eggs in treated group)] / (No. of worms/eggs in control group) × 100. ED50 (the effective dose of PZQ required to kill 50% of adult worms) values were calculated using Prism (GraphPad; Version 5.0) computer software using a variable slope for the sigmoidal curve. Results were expressed as mean ± SEM. A two-tailed Student’s t-test was used to detect the significance of difference between the means of different groups. Results were considered significant when the P value is <0.05. 7

3. Results 3.1. In vitro effects of roflumilast When roflumilast was tested in vitro, no worm killing was recorded, even at the highest concentration tested (100 µM), yet a significant and concentration-dependent reduction in egg production (35−52%; P<0.05, n=3) was recorded despite the continuous presence of intact live couples (Table 1). PZQ, in the range of 5 - 100 µM, resulted in 100% worm killing with complete absence of eggs; at 0.25 µM PZQ killed only the male worms. At this low PZQ concentration, the addition of 5 - 100 µM of roflumilast had the surprising effect of keeping both male and female worms alive yet uncoupling was recorded with 100% inhibiting of egg deposition (Table 2). The data in Table 2 does not allow a conclusion as to whether the roflumilast contributed to either the uncoupling or the reduced ovipositing, or whether these phenomena should be attributed to the low dose PZQ. The conclusion of the in vitro exposure experiments is that roflumilast has only a dose-dependent effect on ovipositing but does not possess any inherent schistosomicidal activity.

3.2. Activity of roflumilast in a mouse model of schistosomiasis In S. mansoni-infected mice, significant reductions in total worm burdens were recorded at 24 h and two weeks post treatment with roflumilast (23.9% and 26.7%, respectively, P<0.05), when compared to infected untreated mice (Table 3). Two weeks post treatment, the numbers of male and female worms (Table 3), and total worm burden were almost equally reduced (24.9%, 29.5% and 26.8%, respectively (P<0.05); Table 3), with an equivalent reduction in hepatic (28.5%) and intestinal (28.3%) tissue egg loads (P<0.05); however, eggs of all developmental stages were present and in very similar proportions as the untreated controls (Table 4). These results show that the 5×5 mg/kg dose of roflumilast resulted in a small but significant reduction in worm burden and similar reduction in egg counts, but did not affect the maturation of eggs. Thus, in contrast to the in vitro observations, roflumilast monotherapy did appear to affect ovipositing in vivo, as the reduction in egg count was virtually identical to the reduction in worm numbers. Combination of 5×5 mg/kg roflumilast with a low dose of PZQ significantly enhanced the antischistosomal potency versus PZQ alone (Table 5). This was evidenced by enhanced worm 8

reductions (86.7% vs 72.3% for PZQ alone at a dose of 5×25 mg/kg; P<0.05), a complete absence of immature eggs at any dosage, a reduction in mature eggs, and a marked increase in dead eggs (P<0.05 at 5×25 mg/kg PZQ; Fig. 1). At the higher PZQ doses, it became harder to distinguish the effects of the combination from the already very substantial effects of PZQ alone, and there was an increase in mortality in the drug combination groups, leading to a smaller sample size. However, a significantly higher reduction in the hepatic tissue egg load was observed at doses of 5×50 and 5×100 mg/kg in the PZQ-roflumilast-treated group compared to PZQ alone; similar reductions in intestinal egg loads were also observed but did not reach statistical significance (Table 5, Fig. 1). The co-administration with fixed-dose roflumilast thus substantially potentiated PZQ but the mortality among the treated mice point to a dangerous drug interaction, and this would need to be addressed through a rationally designed administrative protocol, if possible. Overall, it appears that the maximum tolerated roflumilast dose did cause a minor improvement in therapeutic outcomes for low doses of PZQ, but this is more than offset by the increased toxicity observed.

3.3. Histopathological analysis after roflumilast treatment Liver sections of infected untreated mice and roflumilast-treated mice, sacrificed two weeks post treatment, revealed typical, large fibrocellular granuloma formed from central living ova including living miracidia surrounded with lymphocytes, epithelioid cells, eosinophils, polymorphonuclear cells and fibrous tissue. Treatment with roflumilast resulted in a reduced granuloma diameter (247 ± 34 µm vs 278 ± 19 µm) as well as a reduced percentage of degenerated ova (13.0 ± 4.0 vs 18.5 ± 9.0), but an increased number of fibrocellular granuloma (48.3 ± 23.2 vs 31.3 ± 14.7) two weeks post treatment; however, these changes were not statistically significant compared to the infected untreated control (Fig. 2).

3.4. SEM imaging of worms isolated after roflumilast treatment Worms recovered 24 h post treatment with roflumilast after a dose of 5 mg/kg/day for 5 days were examined for morphological changes using SEM and showed normal appearance of the tubercles with normal spines and intact ridges forming a pattern that was comparable to that in untreated worms (Fig. 3). On the other hand, PZQ caused a completely disrupted tegument, revealed as loss of spines with multiple blebs. 9

4. Discussion Currently, PZQ is the sole drug used to treat schistosomiasis and is widely used in masstreatment campaigns, all of which contributes to the ever growing probability of developing and spreading clinically significant levels of resistance to the drug. Meanwhile, the lack of drugs with antischistosomal potential in the discovery pipeline greatly encourages efforts to identify new drug candidates to replace PZQ or, alternatively, to be used in combinations that could prevent resistance from developing (Li et al., 2015). Drug repurposing is an efficient tool to find new drugs against helminthiases, reducing the time and costs of drug research and development (Panic et al., 2014). In the last decade, PDEs have attracted much attention as selective drug targets, including in parasitic diseases. Trypanosoma brucei express five PDEs (Berriman et al., 2005; Gould and De Koning, 2011) of which TbrPDEB1 and TbrPDEB2 are confirmed druggable targets (Bland et al., 2011; De Koning et al., 2012; Blaazer et al., 2015). In Leishmania, PDEs are also considered valuable therapeutic targets (Seebeck et al., 2011; Sebastián-Pérez et al., 2018). While some progress at last is being made in understanding protozoan cyclic nucleotide signaling pathways (Gould et al., 2013; Tagoe et al., 2015), for schistosomes the function(s) and potential of phosphodiesterases as a drug target are still almost unknown. Protasio et al. (2012) reported that G-protein-coupled receptors, adenylyl cyclases (AC) and protein kinase A (PKA) are present in the S. mansoni genome. A cAMP-dependent protein kinase was shown to control ciliary motion in the miracidial stage of S. mansoni (Matsuyama et al., 2004) and treatment of miracidia with AC modulators (Taft et al., 2010) inhibited transformation of miracidia to sporocysts. A screen of chemotypes associated with PDE inhibition identified many hits with various levels of antischistosomal activity, particularly impacting in vitro ovipositing and, in combination with PZQ, egg survival in vivo (Botros et al., 2019). Long et al. (2017) identified four SmPDE4 orthologs and showed a correlation between inhibitory IC50 values of recombinant SmPDE4A and the antischistosomal effects of a series of benzoxaboroles. These authors found that the hPDE4 inhibitor roflumilast, but not the related catechol rolipram, was a sub-nanomolar inhibitor of SmPDE4A. In this work, we further explore the effect of roflumilast on S. mansoni in vitro and in vivo, both as monotherapy and in combination with PZQ. The in vitro findings revealed no direct 10

killing effect of roflumilast on worms under the test conditions used, but a significant and concentration-dependent reduction in egg number (35−52%) was recorded despite the presence of intact live couples, possibly suggesting an effect of PDE4 inhibition on the male or female worm’s reproductive systems, although this clearly needs further investigation. This result was not dissimilar to those reported in the study by Botros et al. (2019), where the screening of 265 compounds identified as potential PDE inhibitors identified 171 compounds that caused antischistosomal insult, of which 28% displayed between 25% and 92% reduction in egg numbers despite the presence of live intact worm couples. All this suggests that, at least in vitro, PDE inhibitors might particularly affect ovipositing. As 82% of this series of PDE inhibitors impacted male worms, whereas females were barely affected, it could be speculated that ovipositing might be targeted through inhibition of PDEs in the male gonads. This is a distinct possibility as phosphodiesterases SmPDE4A and SmPDE4C (names as designated by Long et al. (2017)) are highly expressed in the S. mansoni testes (Lu et al., 2018). Long and coworkers (2017) also reported that, although roflumilast is an effective inhibitor of SmPDE4A, it only induced transient hypermotility in adult schistosomes (maximum 3.2-fold by worm assay), without major degenerative changes. This marginal phenotypic effect was attributed to a possible lack of penetration or rapid metabolism of the catechol by the parasite - yet the drug did induce hypermotility in their hands and we observed significant effects, with concentrations as low as 5 µM, on ovipositing, which was not investigated by the Caffrey team. It seems, then, that the logical conclusion might be that inhibition of SmPDE4A (and possibly other SmPDE4 homologues) has little effect on the survival of schistosomes in vitro. We also report here the first evaluation of activity of roflumilast, or any other known PDE inhibitor, against S. mansoni in an animal model. With the 5×5 mg/kg dosage regimen used, the drug was again observed to reduce egg production, by ~28% in both hepatic and intestinal tissues, but accompanied by a similar reduction in worm burden. As eggs of all developmental stages were observed, and in similar proportions as in the untreated control group, we conclude that it was the rate of egg production rather than their viability or maturation that was affected i.e. in vivo roflumilast acts on the worms rather than on the eggs. It is likely that the reduced egg load is the result of the decreased number of worms. However, that reduction was modest and the roflumilast dose could not be increased. Thus, we considered the possibility of combination with an otherwise non-curative concentration of PZQ. 11

The combination of roflumilast and PZQ in vitro yielded unexpected results. Whereas 0.25 µM PZQ alone killed all male (but not female) worms in vitro, in the presence of roflumilast (5−100 µM) all worms survived this PZQ concentration. This observation appears to point to an interaction between the two drugs that antagonized the action of PZQ, to the effect that only uncoupling of worms was observed. In contrast, roflumilast in vivo enhanced the antischistosomal effects of PZQ, reducing its estimated ED50 by approximately 10-fold. Thus, a dosage of roflumilast (5×5 mg/kg) enhanced the dose-dependent antischistosomal activity of PZQ, although the effect is probably additive rather than potentiating given the modest reduction in worm burden already observed with the same dosage of roflumilast in monotherapy. The coadministration with roflumilast also significantly reduced hepatic, intestinal and total egg counts relative to PZQ alone. At the lowest PZQ dosage, roflumilast brought the already low percentage of immature eggs down to zero, indicating a complete cessation of viable egg depositing. At the same time, the percentage of mature eggs also declined faster in the presence of roflumilast with a concomitant rise in the percentage of dead eggs. Thus, in vivo, a fixed dosage of roflumilast favorably shifts the PZQ dose-response curve with respect to parasite survival, egg production and egg viability. Whereas this would be a potentially important advantage where PZQ efficacy is diminishing through overuse, the increased mouse mortality in the drug combination groups constitutes a potential red flag. The observed difference in antischistosomal activity of the PZQ/roflumilast combination in vivo may be related to a pharmacological interaction at the metabolism level, resulting in higher efficacy and animal mortality. Hatzelmann and Schudt (2001) reported that roflumilast inhibits hPDE4 two to three times more potently than its metabolite, roflumilast N-oxide. However, the area under the curve concentration of roflumilast N-oxide was approximately 10 times greater than that of roflumilast in pharmacokinetic trials, suggesting that most of its pharmacological effects (possibly up to 90%) can be attributed to its N-oxide metabolite (Bethke and Lahu, 2011). This hypothesis potentially explains some of the observed differences between the in vivo and in vitro experiments, especially the leftward shift in the PZQ dose-response curve in the mouse model. Previous studies indicated that PZQ undergoes significant first-pass metabolism through the liver enzyme cytochrome P450 (CYP450) 3A4 (Li et al., 2003) and roflumilast similarly undergoes extensive hepatic metabolism by both phase 1 cytochrome P450s and phase 2 (conjugation) reactions (Daliresp, 2011). The higher antischistosomal 12

efficacy, coupled with higher mortality (~33-83%) recorded in mice receiving both PZQ and roflumilast, might thus be in part attributable to both drugs competing on the same active site of CYP450 enzymes during their metabolism, leading to an increased availability of PZQ and/or roflumilast and hence higher efficacy, but also the potential for toxic overdosing. In schistosomiasis, the morbidity is mainly attributed to the eggs because of the granulomatous inflammatory reaction, caused by the immune response to egg antigens (Pyrrho et al., 2002). Lenzi (1998) has described the granuloma as a dynamic and complex structure, which consists of eosinophils, neutrophils, lymphocytes, macrophages, giant cells, and fibroblasts surrounding schistosome eggs trapped in the liver. Preclinical studies investigating the anti-inflammatory mechanism of action of roflumilast in asthma, chronic obstructive pulmonary disease and colitis revealed suppression of cytokine synthesis (Sanz et al., 2005) and lymphocyte proliferation (Hatzelmann and Schudt, 2001). In the current study, roflumilast appeared to suppress the granulomatous inflammatory reaction, although this was not statistically significant, and the granulomas were fibrocellular compared to those in infected untreated controls. The apparent anti-inflammatory activity might have reached significant intensity with longer treatment, which was only 5 days in this work – considerably shorter than the 14-days roflumilast treatment used in bleomycin-induced lung injury (Cortijo et al., 2009) or the 11-days treatment in experimental colitis (Rieder et al., 2013). In conclusion, in vitro roflumilast displayed significant impact on worm ovipositing capacity but not worm viability, and the addition of a low, fixed concentration (0.25 µM) of PZQ with roflumilast actually antagonized the schistosomicidal power of PZQ. In an animal model of schistosomiasis, roflumilast displayed only a modest effect on worm burden and this compound does not appear sufficiently potent for monotherapy. We also observed harmful complications from PZQ combinations, probably attributable to pharmacodynamic interactions. Overall, we must conclude that roflumilast, despite its reported low nanomolar inhibition of SmPDE4A, is not a suitable candidate for a new schistosomiasis therapeutic, and SmPDE4A may not be a good drug target.

Acknowledgment This work was undertaken as part of the PDE4NPD consortium, supported by Framework Program 7 of the European Commission, grant number 602666. 13

Conflicts of interest The authors declare that they have no conflicts of interest.

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Molehin, A.J., Rojo, J.U., Siddiqui, S.Z., Gray, S.A., Carter, D., Siddiqui, A.A., 2016. Development of a schistosomiasis vaccine. Expert Review of Vaccines 15, 619–627. Panic, G., Duthaler, U., Speich, B., Keiser, J., 2014. Repurposing drugs for the treatment and control of helminth infections. International Journal for Parasitology: Drugs and Drug Resistance 4, 185–200. Pellegrino, J., Oliveira, C.A., Faria, J., Cunha, A.S., 1962. New approach to the screening of drugs in experimental schistosomiasis mansoni in mice. The American Journal of Tropical Medicine and Hygiene 11, 201–215. Pica-Mattoccia, L, Cioli, D., 2004. Sex- and stage-related sensitivity of Schistosoma mansoni to in vivo and in vitro praziquantel treatment. International Journal of Parasitology 34, 527–533. Protasio, A.V., Tsai, I.J., Babbage, A., Nichol, S., Hunt, M., Aslett, M.A., De Silva, N., Velarde, G.S., Anderson, T.J., Clark, R.C., Davidson, C., Dillon, G.P., Holroyd, N.E., LoVerde, P.T., Lloyd, C., McQuillan, J., Oliveira, G., Otto, T.D., Parker-Manuel, S.J., Quail, M.A., Wilson, R.A., Zerlotini, A., Dunne, D.W., Berriman, M., 2012. A systematically improved high quality genome and transcriptome of the human blood fluke Schistosoma mansoni. PLOS Neglected Tropical Diseases 6, e1455. Pyrrho Ados, S., Ramos, J.A., Neto, R.M., Silva, C.S., Lenzi, H.L., Takiya, C.M., Gattass, C.R., 2002. Dexamethasone, a drug for attenuation of Schistosoma mansoni infection morbidity. Antimicrobial Agents and Chemotherapy 46, 3490–3498. Rieder, F., Siegmund, B., Bundschuh, D.S., Lehr, H.A., Endres, S., Eigler, A., 2013. The selective phosphodiesterase 4 inhibitor roflumilast and phosphodiesterase 3/4 inhibitor pumafentrine reduce clinical score and TNF expression in experimental colitis in mice. PLoS One 8, e56867. Sanz, M.J., Cortijo, J., Morcillo, E.J., 2005. PDE4 inhibitors as new anti-inflammatory drugs: effects on cell trafficking and cell adhesion molecules expression. Pharmacology and Therapeutics 106, 269–297. Sebastián-Pérez, V., Hendrickx, S., Munday, J.C., Kalejaiye, T., Martínez, A., Campillo, N.E., De Koning, H., Caljon, G., Maes, L., Gil, C., 2018. Cyclic nucleotide specific phosphodiesterases as potential drug targets for anti-Leishmania therapy. Antimicrobial Agents and Chemotherapy 62, e00603-18 19

Sebastián-Pérez V, Schroeder S, Munday JC, Van der Meer T, Zaldívar-Díez J, Siderius M, De Koning HP, Brown DG, Martínez A, Campillo NE, Leurs R, Gil C (2019) Discovery of novel Schistosoma mansoni PDE4A inhibitors as potential agents against schistosomiasis. Future Medicinal Chemistry 11, 1703–1720. Seebeck, T., Sterk, G.J., Ke, H., 2011. Phosphodiesterase inhibitors as a new generation of antiprotozoan drugs: exploiting the benefit of enzymes that are highly conserved between host and parasite. Future Medicinal Chemistry 3, 1289–1306. Shakur, Y., de Koning, H.P., Ke, H., Kambayashi, J., Seebeck, T., 2011. Therapeutic potential of phosphodiesterase inhibitors in parasitic diseases. Handbook of Experimental Pharmacology 204, 487–510. Siciliano de Araújo, J., Garcia-Rubia, A., Sebastian-Perez, V., Kalejaiye, T.D., Bernardino da Silva, P., Fonseca-Berzal, C.R., Maes, L., De Koning, H.P., Soeiro, M.N.C., Gil, C., 2019. Imidazole derivatives as promising agents for the treatment of Chagas disease. Antimicrobial Agents and Chemotherapy 63, e02156-18 Taft, A.S., Norante, F.A., Yoshino, T.P., 2010. The identification of inhibitors of Schistosoma mansoni miracidial transformation by incorporating a medium-throughput small-molecule screen. Experimental Parasitology 125, 84–94. Tagoe, D.N., Kalejaiye, T.D., De Koning, H.P., 2015. The ever unfolding story of cAMP signaling in trypanosomatids: vive la difference! Frontiers in Pharmacology 6, 185. Vale, N., Gouveia, M.J., Rinaldi, G., Brindley, P.J., Gartner, F., Correia da Costa, J.M., 2017. Praziquantel for schistosomiasis: single-drug metabolism revisited, mode of action, and resistance. Antimicrobial Agents and Chemotherapy 61, e02582-16. Wang, C., Ashton, T.D., Gustafson, A., Bland, N.D., Ochiana, S.O., Campbell, R.K., Pollastri, M.P., 2012. Synthesis and evaluation of human phosphodiesterases (PDE) 5 inhibitor analogs as trypanosomal PDE inhibitors. Part 1.Sildenafil analogs. Bioorganic and Medicinal Chemistry Letters 22, 2579–2581. Wang, W., Wang, L., Liang, Y.S., 2012. Susceptibility or resistance of praziquantel in human schistosomiasis: a review. Parasitology Research 111, 1871–1877. WHO, 2012. Progress Report 2001–2011 and Strategic Plan 2012–2020. Geneva, Switzerland: 2012.

20

WHO,

2018.

What

Is

Schistosomiasis?

Available

http://www.who.int/schistosomiasis/disease/en/ (accessed on 15 January 2018).

21

online:

Figure Legends Fig. 1. Effects of roflumilast co-administration on the in vivo antischistosomal activities of PZQ. A) Total worms/group. B) Hepatic egg count. C) Intestinal egg count. D) Percentage mature eggs. E) Percentage dead eggs. *, P<0.05 versus PZQ-treated group. Data displayed was taken from Table 5.

Fig. 2. Histopathological changes of liver sections stained with H&E or Masson trichrome, of S. mansoni-infected, untreated (A & B) and treated with roflumilast (C & D) at a dose of 5 mg/kg daily for 5 days.

Fig. 3. Scanning electron micrographs of the tegument of dorsal surface of untreated (A) and roflumilast-treated (B) adult male schistosomes 24 h post end of treatment; showing normal appearance of the tubercles with normal spines and intact ridges in both A and B. (C) PZQ revealed completely disrupted tegument with multiple blebs 24 h post treatment.

22

Table 1: Schistosoma mansoni mature (6 weeks) worm killing and ovipositing after in vitro exposure to different concentrations of roflumilast, compared to untreated controls. End of observation (5 days) Test compounds

Concentration

Untreated control

% Worm killing

Worm ovipositing (n=3)

Couples

Total worms

♂

♀

No. of eggs/couple

% reduction

P value

0/17 (0%)

0/9 (0%)

0/8 (0%)

112 ± 10

-

-

0/7 0/6 (0%) 53.3 ± 4.2 52.2% 0.0024 (0%) 0/6 50 µM 0/12 (0%) 0/6 (0%) 56.7 ± 1.7 49.3% 0.0018 (0%) 0/8 Roflumilast 25 µM 0/14 (0%) 0/6 (0%) 65.0 ± 2.9 41.8% 0.0040 (0%) 0/8 10 µM 0/14 (0%) 0/6 (0%) 68.3 ± 6.0 38.8% 0.0101 (0%) 0/8 5 µM 0/13 (0%) 0/5 (0%) 72.5 ± 6.3 35.1% 0.0152 (0%) Roflumilast was prepared as a stock solution of 5 mM in pure DMSO on the day of the experiment. The represented data is the average results of duplicate wells/concentration. The ovipositing data are the average of three independent experiments, each performed in duplicate; the average and standard error of the mean is shown as well the percent reduction relatively to the average control value, and the level of statistical difference relatively to control, using Student’s unpaired, two-tailed t-test. 100 µM

23

0/13 (0%)

Intact

Intact Intact Intact Intact Intact

Table 2: In vitro Schistosoma mansoni mature (6 weeks old) worm killing and ovipositing under different concentrations of roflumilast in combination with PZQ at its EC50 concentration, compared to negative control. End of observation (5 days) Test Compounds

Untreated control 100 µM 50 µM 25 µM

PZQ* 10 µM 5 µM

Roflumilast + PZQEC50 (0.25 µM)

% Worm killing

Concentration

Ovipositing

Total worms

♂

♀

No. of eggs/couple

% Reduction

0/15 (0%)

0/8 (0%)

0/7 (0%)

100

-

Intact

0

100%

-

0

100%

-

0

100%

-

0

100%

-

0

100%

-

13/13 (100%) 13/13 (100%) 14/14 (100%) 13/13 (100%) 13/13 (100%)

7/7 (100%) 7/7 (100%) 8/8 (100%) 7/7 (100%) 7/7 (100%)

6/6 (100%) 6/6 (100%) 6/6 (100%) 6/6 (100%) 6/6 (100%)

0.25 µM

9/16 (56%)

9/9 (100%)

0/7 (0%)

0

100%

-

100 µM + 0.25 µM

0/16 (0%)

0/9 (0%)

0/7 (0%)

0

100%

Uncoupling

50 µM + 0.25 µM

0/14 (0%)

0/8 (0%)

0/6 (0%)

0

100%

Uncoupling

25 µM + 0.25 µM

0/14 (0%)

0/8 (0%)

0/6 (0%)

0

100%

Uncoupling

10 µM + 0.25 µM

0/15 (0%)

0/9 (0%)

0/6 (0%)

0

100%

Uncoupling

5 µM + 0.25 µM

0/14 (0%)

0/8 (0%)

0/6 (0%)

0

100%

Uncoupling

The represented data is the average results of duplicate wells/concentration. EC50 is the effective concentration of a drug that kills 50% of worms in vitro. *PZQ EC50 = 0.25 µM.

24

Couples

Table 3: Effect of roflumilast (5 mg/kg/day for 5 days) on worm load and sex in S. mansoniinfected mice sacrificed 24 h and two weeks post treatment.

Groups Infected control (n=8) Roflu (n=8)

Date of sacrifice Two weeks

Worm load and sex Total males

Total females

Total couples

Total Worms

12.9 ± 0.6

9.1 ± 0.5

5.6 ± 0.4

22.0 ± 0.9

10.0 ± 0.9 6.8 ± 1.4 4.0 ± 0.6 16.8 ± 2.0* (-22.4%) (-26.1%) (-29.0%) (-23.9%) * * Roflu (n=9) Two weeks 9.7 ± 1.3 6.4 ± 1.0 5.0 ± 0.5 16.1 ± 1.5* (-24.9%) (-29.5%) (-11.2%) (-26.8%) * * * PZQ (n=8) Two weeks 0.6 ± 0.3 0.1 ± 0.1 0.0 ± 0.0 0.8 ± 0.3* (-95.1%) (-98.6%) (-100%) (-96.6%) Roflumilast and PZQ were administered orally 7 weeks post S. mansoni infection in doses of 5 mg/kg/day and 200 mg/kg/day respectively each for 5 days. Results are presented as mean ± SEM. n= number of mice/group. Numbers in parentheses represent % change from infected control. * Significant difference from infected control, P<0.05.

25

24 h

Table 4: Effect of roflumilast (5 mg/kg/day for 5 days) on tissue egg load and percentage egg developmental stages in S. mansoni-infected mice sacrificed 24 h and two weeks post treatment. Groups Date of sacrifice Infected untreated control (n=8) Roflu (n=8)

Tissue egg load

% of egg developmental stages

Hepatic count ×10³

Intestinal count ×10³

% immature

% mature

% dead

12.4 ± 1.3

18.4 ± 1.8

52.8 ± 2.5

39.4 ± 1.8

7.9 ± 1.1

Two weeks

24 h

9.2 ± 0.8 14.3 ± 0.9 50.9 ± 2.5 40.6 ± 2.1 8.5 ± 1.4 (-26.5%) (-22.2%) Roflu (n=9) Two weeks 8.9 ± 0.7* 13.2 ± 1.1* 51.7 ± 0.9 40.3 ± 0.8 8.0 ± 0.5 (-28.5%) (-28.3%) PZQ (n=8) Two weeks 1.3 ± 0.4* 0.6 ± 0.1* 0.0 ± 0.0* 0.0 ± 0.0* 100 ± 0.0 * (-89.8%) (-96.9%) Roflumilast and PZQ were administered orally 7 weeks post S. mansoni infection in doses of 5 mg/kg/day and 200 mg/kg/day respectively each for 5 days. Results are presented as mean ± SEM. n= number of mice/group. Numbers in parentheses represent % change from infected control. * Significant difference from infected control at P<0.05.

26

Table 5: Effect of praziquantel alone and in combination with roflumilast on total worm load, tissue egg load and egg development in S. mansoni-infected mice sacrificed 10 days post treatment. Total worms Tissue egg load % of egg developmental stages (%reduction) Hepatic count Intestinal % immature % mature % dead Groups ×10³ count ×10³ (% reduction) (% reduction) Infected untreated control 30.1 ± 1.3 16.4 ± 1.0 28.8 ± 1.6 52.1 ± 1.1 39.1 ± 1.2 8.8 ± 0.5 (n=10) PZQ (5×25 mg/kg) (n=6)

8.3 ± 0.8* (72.3%)

8.8 ± 0.6* (46.3%)

7.3 ± 0.8* (74.8%)

6.7 ± 3.3*

33.3 ± 4.9

60.0 ± 3.7*

Roflu (5×5 mg/kg) + PZQ (5×25 mg/kg) (n=2)

4.0 ± 0.0*# (86.7%)

6.6 ± 1.3* (60.0%)

5.2 ± 0.5* (81.8%)

0 ± 0*#

21.0 ± 4.0*

79.0 ± 4.0*#

PZQ (5×50 mg/kg) (n=7)

5.7 ± 0.4* (81.0%)

8.4 ± 0.5* (48.8%)

6.5 ± 0.7* (77.4%)

0 ± 0*

12.1 ± 2.6*

87.9 ± 2.6*

Roflu (5×5 mg/kg) + PZQ (5×50 mg/kg) (n=4)

5.0 ± 1.7* (83.4%)

5.1 ± 0.9*# (68.8%)

3.7 ± 1.1* (87.3%)

0 ±0*

6.8 ± 1.4*

93.3 ± 1.4*

PZQ (5×100 mg/kg) (n=10)

2.7 ± 0.5* (91%)

7.5 ± 0.8* (54.2%)

6.4 ± 0.6* (77.9%)

0 ± 0*

6.2 ± 2.3*

93.8 ± 2.3*

Roflu (5×5 mg/kg) + PZQ (5×100 mg/kg) (n=4)

2.0 ± 0.7* (93.4%)

5.0 ± 0.6*# (69.7%)

5.0± 0.5* (82.5%)

4.0 ± 0.7*

96.0 ± 0.7*

PZQ (5×200 mg/kg) (n=8)

1.0 ± 0.3* (96.7%)

5.7 ± 1.0* (65.3%)

3.7 ± 0.8* (87.1%)

2.5 ± 1.6*

97.5 ± 1.6*

Roflu (5×5 mg/kg) + PZQ (5×200 mg/kg) (n=2)

1.5 ± 0.5* (95.0%)

3.7 ± 0.4* (77.4%)

3.2 ± 0.8* (89.0%)

0.5 ± 0.5*

99.5 ± 0.5*

0 ± 0* 0 ± 0* 0 ± 0*

PZQ was given orally 7 weeks post S. mansoni infection in doses of 25, 50, 100 and 200 mg/kg/day for 5 days alone and in addition to oral roflumilast (5 mg/kg/day for 5 days). All groups contained 10 mice at the start of the experiment. A higher mortality (~ 60-80%) was recorded in the PZQ+roflumilast groups than with PZQ alone. Results are presented as mean ± SEM.

27

n= number of mice/group. Numbers in parentheses represent % change from infected control. *, # Significant difference from infected untreated and PZQ treated groups, respectively; P<0.05.

28

Total worms / group

% mature eggs

% dead eggs

Intestinal egg count

Figure 1

H&E

Figure 2

Untreated

Masson Trichrome

B

C

D

Roflumilast

A

Fig. 2: Histopathological changes of liver sections stained with H&E and Masson trichrome, of S. mansoni-infected, untreated (A & B) and treated with roflumilast (C & D) at a dose of 5 mg/kg daily for 5 days. Scale bars are 100 µm.

A

B

C

Fig. 3: Scanning electron micrographs of the tegument of dorsal surface of untreated (A) and roflumilast treated (B) adult male schistosomes 24 h post end of treatment; showing normal appearance of the tubercles with normal spines and intact ridges in both A and B. (C) PZQ revealed completely disrupted tegument with multiple blebs 24 h post treatment.

• • • • •

Cyclic nucleotide phosphodiesterases (PDEs) have been proposed as potential drug targets in Schistosoma. The PDE4 inhibitor roflumilast modestly reduced worm burden and hepatic/intestinal egg burdens in S. mansoni-infected mice. Combinations with praziquantel were more effective in vivo, but also more toxic. This study marks the first report of in vivo antischistosomal potential by a PDE inhibitor. Roflumilast is insufficiently active for further development.