Combination Anthelmintic/Antioxidant Activity Against Schistosoma Mansoni

Abstract

Schistosomiasis is a major neglected tropical disease. Treatment for schistosomiasis with praziquantel (PZQ), which is effective against the parasite, by itself is not capable to counteract infection-associated disease lesions including hepatic fibrosis. There is a pressing need for novel therapies. Due to their biological properties, antioxidant biomolecules might be useful in treating and reverting associated pathological sequelae. Here, we investigated a novel therapy approach based on a combination of anthelmintic drugs with antioxidant biomolecules. We used a host-parasite model involving Bioamphalaria glabrata and newly transformed schistosomula (NTS) of Schistosoma mansoni. For in vitro drug screening assays, was selected several antioxidants and evaluated not only antischistosomal activity but also ability to enhance activity of the anthelmintic drugs praziquantel (PZQ) and artesunate (AS). The morphological alterations induced by compounds alone/combined were assessed on daily basis using an inverted and automated microscope to quantify NTS viability by a fluorometric-based method. The findings indicated that not only do some antioxidants improve antischistosomal activity of the two anthelmintics, but they exhibit activity per se, leading to high mortality of NTS post-exposure. The combination index (CI) of PZQ + Mel (CI = 0.80), PZQ + Resv (CI = 0.74), AS + Resv (CI = 0.34), AS + NAC (CI = 0.89), VDT + Flav (CI = 1.03) and VDT + Resv (CI = 1.06) reveal that they display moderate to strong synergism. The combination of compounds with discrete mechanisms of action might provide a valuable adjunct to contribution for treatment of schistosomiasis-associated disease.

Keywords: Schistosoma mansoni; anthelmintic drug; anticancer drug; antioxidant biomolecules; combination therapy; drug repurposing; newly transformed schistosomula.

Figure 1

Representative micrographs of newly transformed schistosomula (NTS) at 72 h of exposure to the anthelmintics praziquantel (PZQ), artesunate (AS), and flubendazole (FBZ) and anticancer drugs vandetanib (VDT), and imatinib (IMT) at 100 µM. Compared to controls, NTS incubated with drugs showed severe morphological alterations, and the morphological changes differed among the drugs. Scale bar, 100 µm.

Figure 2

Representative micrographs of NTS at 72 h of incubation in antioxidants at 100 µM. Notable differences were evident among controls and NTS incubated with 4-phenyl-1,2,5-oxadiazole-3-carbonile-2-oxide (OXA), curcumin (Curc), and flavone (Flav), which induced severe granularity and reduced activity. In contrast to OXA and Curc, Flav did not kill all the NTS. Resveratrol (Resv) also induced moderate morphological alterations but did not kill all NTS. N-acetylcysteina (NAC), melatonin (Mel) and dipeptide (DiPept) did not induce significant morphological alterations. Scale bar, 100 µm.

Figure 3

Representative micrographs of NTS at 72 h after exposure to combinations of praziquantel (PZQ) and artesunate (AS) with several antioxidants at 100 μM, and 1:1 constant ratio; scale bar, 100 μm.

Figure 4

Representatives micrographs of NTS incubated with combinations of FBZ with Flav, Resv, and DiPept at 100 µM and constant ratio 1:1 at 72 h post-exposure. Notably, the morphologic alterations in combinations were more similar to drug alone rather antioxidant, specifically FBZ + Flav. In combinations FBZ + Resv and FBZ + DiPept NTS were more swollen and altered in shape (white arrows). Scale bar, 100 µm.

Figure 5

Representative micrographs of NTS at 72 h of incubation in VDT, Flav and Resv either alone or combined at 100 µM and 1:1 constant ratio; scale bar, 100 µm.

Figure 6

Representative micrographs of NTS incubated with combinations of VDT, Flav, and Resv alone and in combination at 25 µM and 1:1 constant ratio at 72 h post exposure. Scale bar: 100 µm.

Figure 7

Micrographs of NTS at 72 h of exposure to IMT, Flav, and Resv, Mel alone or combined at a concentration of 100 µM and 1:1 constant ratio; x20; bright field; scale bar, 100 µm.

Figure 8

Representative micrographs of NTS at 72 h of exposure to Curc and OXA and combined, at 25 µM and 1:1 constant ratio; ×20; bright field; scale bar, 100 µm.