Mervat Z. El-Azzouni

Miltefosine, a promising novel agent for schistosomiasis mansoni.

This research aims towards developing an alternative antischistosomal drug using miltefosine, which is primarily used in the treatment of leishmaniasis. The treatment and control of schistosomiasis, a notable neglected tropical disease (NTD), rely on a single drug, praziquantel (PZQ). The dependency on PZQ exclusively is quite alarming, given the spread of the disease (over 200 million people infected and close to 800 million people at risk in three continents) and the threat of drug resistance. This study shows that the oral administration of miltefosine in a daily dose of 20mg/kg for five successive days to mice infected with either invasive, juvenile or adult stages of Schistosoma mansoni resulted in significant reduction of worm burden, hepatic granulomata size and amelioration of hepatic pathology. Scanning Electron Microscopy revealed that miltefosine induced severe tegumental damage in adult schistosomes. In conclusion, we believe this is the first study highlighting miltefosine as a promising novel agent for schistosomiasis mansoni.

Miltefosine Lipid Nanocapsules for Single Dose Oral Treatment of Schistosomiasis Mansoni: A Preclinical Study.

Miltefosine (MFS) is an alkylphosphocholine used for the local treatment of cutaneous metastases of breast cancer and oral therapy of visceral leishmaniasis. Recently, the drug was reported in in vitro and preclinical studies to exert significant activity against different developmental stages of schistosomiasis mansoni, a widespread chronic neglected tropical disease (NTD). This justified MFS repurposing as a potential antischistosomal drug. However, five consecutive daily 20 mg/kg doses were needed for the treatment of schistosomiasis mansoni in mice. The present study aims at enhancing MFS efficacy to allow for a single 20mg/kg oral dose therapy using a nanotechnological approach based on lipid nanocapsules (LNCs) as oral nanovectors. MFS was incorporated in LNCs both as membrane-active structural alkylphospholipid component and active antischistosomal agent. MFS-LNC formulations showed high entrapment efficiency (EE%), good colloidal properties, sustained release pattern and physical stability. Further, LNCs generally decreased MFS-induced erythrocyte hemolytic activity used as surrogate indicator of membrane activity. While MFS-free LNCs exerted no antischistosomal effect, statistically significant enhancement was observed with all MFS-LNC formulations. A maximum effect was achieved with MFS-LNCs incorporating CTAB as positive charge imparting agent or oleic acid as membrane permeabilizer. Reduction of worm load, ameliorated liver pathology and extensive damage of the worm tegument provided evidence for formulation-related efficacy enhancement. Non-compartmental analysis of pharmacokinetic data obtained in rats indicated independence of antischistosomal activity on systemic drug exposure, suggesting possible gut uptake of the stable LNCs and targeting of the fluke tegument which was verified by SEM. The study findings put forward MFS-LNCs as unique oral nanovectors combining the bioactivity of MFS and biopharmaceutical advantages of LNCs, allowing targeting via the oral route. From a clinical point of view, data suggest MFS-LNCs as a potential single dose oral nanomedicine for enhanced therapy of schistosomiasis mansoni and possibly other diseases.

Initial characterization of an autoclaved Toxoplasma vaccine in mice

Toxoplasmosis is a zoonotic protozoal disease that has a major significance from the perspectives of public health and veterinary medicine. Therefore, an obvious long-term goal of many scientists would be the development of an effective vaccine. In this study, autoclaved vaccine was evaluated for its ability to protect mice against Toxoplasma gondii RH challenge as an acute infection model. Results showed that autoclaved Toxoplasma vaccine (ATV) when combined with BCG as an adjuvant was effective in triggering cell mediated immunity as shown by a significant increase in the percentage of splenic CD8+ T-lymphocytes. Following challenge, death of mice vaccinated with ATV was delayed for nine days. There was a significant decrease in parasite density in different organs, and a marked reduction of pathological changes in the liver suggesting that significant immune responses were mounted following vaccination. Future studies are warranted to test the vaccine against challenge with brain cysts as a chronic infection model and to evaluate it with other recent immunization strategies that can further enhance its immunogenicity.

Synthesis and evaluation of some pyrimidine analogs against toxoplasmosis

Toxoplasmosis is a worldwide infection caused by the obligate intracellular protozoan parasite Toxoplasma gondii. Some pyrimidine analogs were synthesized to be evaluated for their antitoxoplasma effects in animal experiments. Results were assessed by studying parasite density, estimation of serum Toxoplasma antigen, studying the ultrastructural changes of the parasite, and the histopathological changes of the affected organs and inhibitory activity for dihydrofolate reductase (DHFR) isolated from Cryptosporidium parvum. The result showed that 4 out of 12 synthesized compounds have promising antitoxoplasma potentials. The animals that received these four compounds showed statistically significant decrease in the mean number of the parasite count in the liver and the spleen when compared to the corresponding control group. Moreover, Toxoplasma antigen was very low or even absent in the serum of animals receiving these compounds. Light microscopic examination of the peritoneal exudates of animals receiving these compounds showed stoppage of movement and deformity in shape of the tachyzoites, whereas scanning electron microscopy revealed that the organisms had lost their crescent shape, with dimples and deep ridges on their surfaces. Very mild histopathological changes were noticed in liver, spleen, and lungs of the groups of animals receiving these compounds in comparison to the other groups. Thus, these compounds proved their effectiveness in eradication of the experimental Toxoplasma infection and inhibition of DHFR from C. parvum.

Miltefosine lipid nanocapsules: Intersection of drug repurposing and nanotechnology for single dose oral treatment of pre-patent schistosomiasis mansoni

A dual drug repurposing/nanotechnological approach was used to develop an alternative oral treatment for schistosomiasis mansoni using miltefosine (MFS), an anticancer alkylphosphocholine, and lipid nanocapsules (LNCs) as oral nanovectors. We demonstrated earlier that MFS possesses significant activity against different developmental stages of Schistosoma mansoni in the mouse model using 5 successive 20mg/kg/day oral doses. Moreover, an effective single dose (20mg/kg) oral treatment against the adult stage of S. mansoni in mice was developed using LNCs, particularly modified with CTAB, a positive charge imparting agent (MFS-LNC-CTAB(+)), or oleic acid as membrane permeabilizer (MFS-LNC-OA). Efficacy enhancement involved, at least in part, targeting of the worm tegument with MFS-LNCs as a new therapeutic entity. As the tegument surface charge and composition may differ in pre-patent stages of the parasite, it was of importance in the present study to assess the efficacy of a single oral dose of the two MFS-LNC formulations against invasive and immature stages for potential advantage relative to praziquantel. Results indicated potent schistosomicidal effects against both invasive and immature stages of S. mansoni in infected mice, efficacy being both formulation and developmental stage dependent. This was indicated by the significant reduction in the total worm burden of the invasive stage by 91.6% and 76.8% and the immature stage by 82.7% and 96.7% for MFS-LNC-CTAB+ and MFS-LNC-OA, respectively. Histopathological findings indicated amelioration of hepatic pathology with regression of the granulomatous inflammatory reaction and reduction in granulomas number and size, verifying marked improvement in architecture of hepatic lobules. From a clinical perspective, MFS-LNCs offer potential as an alternative single oral dose nanomedicine with a wide therapeutic profile for the mass chemotherapy of schistosomiasis mansoni.

Treatment of Schistosoma mansoni with miltefosine in vitro enhances serological recognition of defined worm surface antigens

Background Miltefosine, an anti-cancer drug that has been successfully repositioned for treatment of Leishmania infections, has recently also shown promising effects against Schistosoma spp targeting all life cycle stages of the parasite. The current study examined the effect of treating Schistosoma mansoni adult worms with miltefosine on exposure of worm surface antigens in vitro. Methodology/Principal findings In an indirect immunofluorescence assay, rabbit anti-S.mansoni adult worm homogenate and anti-S. mansoni infection antisera gave strong immunofluorescence of the S. mansoni adult worm surface after treatment with miltefosine, the latter antiserum having previously been shown to synergistically enhance the schistosomicidal activity of praziquantel. Rabbit antibodies that recognised surface antigens exposed on miltefosine-treated worms were recovered by elution off the worm surface in low pH buffer and were used in a western immunoblotting assay to identify antigenic targets in a homogenate extract of adult worms (SmWH). Four proteins reacting with the antibodies in immunoblots were purified and proteomic analysis (MS/MS) combined with specific immunoblotting indicated they were the S. mansoni proteins: fructose-1,6 bisphosphate aldolase (SmFBPA), Sm22.6, alkaline phosphatase and malate dehydrogenase. These antibodies were also found to bind to the surface of 3-hour schistosomula and induce immune agglutination of the parasites, suggesting they may have a role in immune protection. Conclusion/Significance This study reveals a novel mode of action of miltefosine as an anti-schistosome agent. The immune-dependent hypothesis we investigated has previously been lent credence with praziquantel (PZQ), whereby treatment unmasks parasite surface antigens not normally exposed to the host during infection. Antigens involved in this molecular mechanism could have potential as intervention targets and antibodies against these antigens may act to increase the drug’s anti-parasite efficacy and be involved in the development of resistance to re-infection.

Immuno-therapeutic potential of Schistosoma mansoni and Trichinella spiralis antigens in a murine model of colon cancer

SummaryConsiderable evidence indicates a negative correlation between the prevalence of some parasitic infections and cancer and their interference with tumor growth. Therefore, parasitic antigens seem to be promising candidates for cancer immunotherapy. In this study, the therapeutic efficacy of autoclaved Schistosoma mansoni and Trichinella spiralis antigens against a colon cancer murine model was investigated. Both antigens showed immunomodulatory potential, as evidenced by a significant decrease in serum IL-17, a significant increase in serum IL-10, and the percentage of splenic CD4+T-cells and intestinal FoxP3+ Treg cells. However, treatment with S. mansoni antigen yielded protection against the deleterious effect of DMH-induced colon carcinogenesis only, with a significant decrease in the average lesion size and number of neoplasias per mouse. For the first time, we report an inhibitory effect of S. mansoni antigen on the progression of chemically induced colon carcinogenesis, but the exact mechanism has yet to be clarified. This anti-tumor strategy could introduce a new era of medicine in which a generation of anticancer vaccines of parasitic origin would boost the therapy for incurable cancers.

Praziquantel–lipid nanocapsules: an oral nanotherapeutic with potential Schistosoma mansoni tegumental targeting

Purpose Lipid nanocapsules (LNCs) have shown potential to increase the bioavailability and efficacy of orally administered drugs. However, their intestinal translocation to distal target sites and their implication in pharmacokinetic (PK)–pharmacodynamic (PD) relationships are yet to be elucidated. In this study, the effect of LNCs on the PD activity and pharmacokinetics of praziquantel (PZQ), the mainstay of schistosomiasis chemotherapy, was investigated. Materials and methods The composition of LNCs was modified to increase PZQ payload and to enhance membrane permeability. PZQ–LNCs were characterized in vitro for colloidal properties, entrapment efficiency (EE%), and drug release. PD activity of the test formulations was assessed in Schistosoma mansoni-infected mice 7 days post-oral administration of a single 250 mg/kg oral dose. Pharmacokinetics of the test formulations and their stability in simulated gastrointestinal (GI) fluids were investigated to substantiate in vivo data. Results PZQ–LNCs exhibited good pharmaceutical attributes in terms of size (46–62 nm), polydispersity index (0.01–0.08), EE% (>95%), and sustained release profiles. Results indicated significant efficacy enhancement by reduction in worm burden, amelioration of liver pathology, and extensive damage to the fluke suckers and tegument. This was partly explained by PK data determined in rats. In addition, oral targeting of the worms was supported by the stability of PZQ–LNCs in simulated GI fluids and scanning electron microscopy (SEM) visualization of nanostructures on the tegument of worms recovered from mesenteric/hepatic veins. Cytotoxicity data indicated tolerability of PZQ–LNCs. Conclusion Data obtained provide evidence for the ability of oral LNCs to target distal post-absorption sites, leading to enhanced drug efficacy. From a practical standpoint, PZQ–LNCs could be suggested as a potential tolerable single lower dose oral nanomedicine for more effective PZQ mass chemotherapy.