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

https://doi.org/10.1016/j.exppara.2019.107793Get rights and content

Highlights

  • 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.

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 especially SmPDE4A. This study investigates the potential antischistosomal activity of human PDE4 and potent SmPDE4A inhibitor roflumilast, either alone or combined with PZQ. In vitro, roflumilast resulted in a significant, concentration-dependent reduction in egg production but not of worm viability. In vitro exposure to roflumilast in combination with a low concentration of PZQ was less effective than PZQ alone, pointing to antagonism. S. mansoni-infected mice treated with roflumilast showed significant reductions in worm burden (27%) as well as hepatic and intestinal egg burdens (~28%) two weeks post treatment. Scanning EM of worms isolated from roflumilast-treated and untreated mice did not reveal noticeable changes to their tegument. S. mansoni-infected mice treated with a fixed dosage of roflumilast and a variable dosage of PZQ resulted in a higher reduction in worm burden, reduced hepatic egg counts, absence of immature eggs and a marked increase in dead eggs, compared to PZQ alone. However, the combination resulted in increased animal mortality, probably attributable to pharmacodynamic interactions between the two drugs. Although this study marks the first report of in vivo antischistosomal potential by a PDE inhibitor, an important proof of concept, we conclude that the antischistosomal effects of roflumilast are insufficient to warrant further development.

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 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 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.

Section snippets

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.

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

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

Discussion

Currently, PZQ is the sole drug used to treat schistosomiasis and is widely used in mass-treatment 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

Declaration of competing interest

The authors declare that they have no conflicts of interest.

Acknowledgment

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

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