Hypothetical diagram of lipid nanocapsules incorporating miltefosine.

<p>Hypothetical diagram of lipid nanocapsules incorporating miltefosine.</p

Miltefosine release profiles.

<p>Miltefosine release from selected lipid nanocapsule formulations in PBS pH 7.4 at 37°C over a 24 h-study period. Data represent mean ± SD (n = 3). The inset is an enlargement of the release profiles for the first 8h of the release study.</p

Effect of miltefosine lipid nanocapsule formulations (MFS-LNCs) on <i>S</i>. <i>mansoni</i> worm burden as compared with control groups(n = 6).

<p>* F test (ANOVA). % R₁: % reduction in each of the study groups relative to infected control. %R₂:% reduction in each of the study groups relative to miltefosine solution control</p><p>^a: significant with infected untreated control</p><p>^b: significant with MFS solution control</p><p>^c: significant with group I</p><p>^d: significant with group II</p><p>^e: significant with group III</p><p>^f: significant with group IV</p><p>^g: significant with group V</p><p>F* = 78.369 (p<0.001).</p

Scanning Electron Microscopy (SEM) of a male <i>Schistosoma mansoni</i> worm from a mouse treated with MFS-LNC-OA showing.

<p>(a) Marked tegumental irregularity and disfigurement (X 3,500); (b) Tegumental surface blebbing (X 7,500); (c) Edema, flattening and sloughing of the whole tubercles with partial to complete loss of the spines (X 5,000); (d) and (e) nano-objects of similar size to lipid nanocapsules in between spines and on damaged schistosomal surface, respectively (X35,000). SEM of a normal male worm showing: (f) and (g) normal dorsal tegumental surface and papilla (X5,000 and 35,000 respectively)</p

Effect of miltefosine lipid nanocapsule formulations (MFS-LNCs) on hepatic granulomas size in μm as compared with control groups.

<p>* F test (ANOVA), % R₁: % reduction in each of the study groups relative to infected control %R₂: % reduction in each of the study groups relative to miltefosine solution control</p><p>^a: significant with infected untreated control</p><p>^b: significant with MFS solution control</p><p>^c: significant with group I</p><p>^d: significant with group II</p><p>^e: significant with group III</p><p>^f: significant with group IV</p><p>^g: significant with group V</p><p>F* = 110.924 (p<0.001).</p

TEM images of miltefosine lipid nanocapsules (MFS-LNCs).

<p>.(a) Plain MFS-LNCs, (b) MFS-LNC-CTAB⁺, (c) MFS-LNC-OA and (d) MFS-LNC-OA-CTAB⁺ at X 7,500 magnification. The scale bar represents 100nm.</p

Mean plasma levels of miltefosine following oral administration of a 10 mg/kg dose of miltefosine as solution and lipid nanocapsule formulations to rats (n = 5).

<p>Error bars represent standard error of the mean.</p

Pharmacokinetic parameters of miltefosine after a single oral dose (10 mg/kg) of MFs solution or MFS-LNCSs formulations in rats.

<p>^ap<0.05</p><p>Pharmacokinetic parameters of miltefosine after a single oral dose (10 mg/kg) of MFs solution or MFS-LNCSs formulations in rats.</p

Treatment of <i>Schistosoma mansoni</i> with miltefosine <i>in vitro</i> enhances serological recognition of defined worm surface antigens

<div><p>Background</p><p>Miltefosine, an anti-cancer drug that has been successfully repositioned for treatment of <i>Leishmania</i> infections, has recently also shown promising effects against <i>Schistosoma</i> spp targeting all life cycle stages of the parasite. The current study examined the effect of treating <i>Schistosoma mansoni</i> adult worms with miltefosine on exposure of worm surface antigens <i>in vitro</i>.</p><p>Methodology/Principal findings</p><p>In an indirect immunofluorescence assay, rabbit anti-<i>S</i>.<i>mansoni</i> adult worm homogenate and anti-<i>S</i>. <i>mansoni</i> infection antisera gave strong immunofluorescence of the <i>S</i>. <i>mansoni</i> 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 <i>S</i>. <i>mansoni</i> 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.</p><p>Conclusion/Significance</p><p>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.</p></div

Characterization of worm surface antigens recognized by rabbit anti-SmI antibodies after miltefosine- and PZQ-treatment.

<p>(i) Immunofluorescent staining of PZQ- (6 μg/ml for 24 hours) (A and C) and miltefosine- (40 μg/ml for 48 hours) (B and D) treated <i>S</i>. <i>mansoni</i> adult worms detected with rabbit anti-SmI IgG antibodies (A and B) and IgG antibodies from a normal rabbit serum (C and D). Scale bar = 100 μm. (ii) Western immunoblots of a <i>S</i>. <i>mansoni</i> crude worm homogenate preparation (SmWH) were probed with rabbit anti-SmI antiserum (lane 1), rabbit anti-SmI antibodies eluted from PZQ- (6 μg/ml for 24 hours) (lane 2) and miltefosine- (40 μg/ml for 48 hours) (lane 3) treated worms. A blot of SmWH probed with a normal rabbit serum (lane 4) was used as a control. Lane M, protein molecular weight markers. Antigenic extract was loaded onto the gel with 10 μg protein/lane. Blots were detected using HRP-conjugated anti-rabbit IgG secondary antibodies. Detected antigens with the most intense reactivities are arrowed.</p