In Vivo Bioluminescence Imaging Reveals Differences in Leishmania infantum Parasite Killing Kinetics by Antileishmanial Reference Drugs.

The bioluminescent Leishmania infantum BALB/c mouse model was used to evaluate the parasiticidal drug action kinetics of the reference drugs miltefosine, paromomycin, sodium stibogluconate, and liposomal amphotericin B. Infected mice were treated for 5 days starting from 7 days post-infection, and parasite burdens were monitored over time via bioluminescence imaging (BLI). Using nonlinear regression analyses of the BLI signal, the parasite elimination half-life (t1/2) in the liver, bone marrow, and whole body was determined and compared for the different treatment regimens. Significant differences in parasiticidal kinetics were recorded. A single intravenous dose of 0.5 mg/kg liposomal amphotericin B was the fastest acting with a t1/2 of less than 1 day. Intraperitoneal injection of paromomycin at 320 mg/kg for 5 days proved to be the slowest with a t1/2 of about 5 days in the liver and 16 days in the bone marrow. To conclude, evaluation of the cidal kinetics of the different antileishmanial reference drugs revealed striking differences in their parasite elimination half-lives. This BLI approach also enables an in-depth pharmacodynamic comparison between novel drug leads and may constitute an essential tool for the design of potential drug combinations.

Antileishmanial effects and drugability characteristics of a heterocyclic copper complex: An in silico, in vitro and molecular study.

Leishmaniasis caused by the protozoan Leishmania presents a severe illness, principally in tropical and subtropical areas. Antileishmanial metal complexes, like Glucantime®ï¸ with proven activity, are routinely studied to probe their potency. We investigated the effects of a Cu (II) homoleptic complex coordinated by two dimethyl-bipyridine ligands against Leishmania major stages in silico and in vitro. The affinity of this heterocyclic Cu (II) complex (CuDMBP) towards a parasitic metacaspase was studied by molecular docking. Key pharmacokinetic and pharmacodynamic properties of the complex were predicted using three web-based tools. CuDMBP was tested for in vitro antileishmanial activities using MTT assay, model murine macrophages, flow cytometry, and quantitative real-time polymerase chain reaction (qPCR). Molecular docking confirmed the tendency between the target macromolecule and the complex. ADMET evaluations highlighted CuDMBP's key pharmacological features, including P-glycoprotein-associated GI absorption and lack of trans-BBB permeability. MTT showed significant inhibitory effects against promastigotes. CuDMBP significantly increased the level of cellular IL-12 expression (p < 0.05), while the upregulation observed in the expression of iNOS was considered not significant (p > 0.05). It decreased the expression of IL-10 significantly (p < 0.05). Findings demonstrated that CuDMBP deserves to be introduced as a leishmanicidal candidate provided further studies are carried out.

4-Arylthiosemicarbazide derivatives - Pharmacokinetics, toxicity and anti-Toxoplasma gondii activity in vivo.

The increasing resistance of Toxoplasma gondii to drugs and side effects of therapy indicate that specific treatment for these parasites is still needed. The 4-arylthiosemicarbazide derivatives seem to be a solution to this challenge because they have low cytotoxicity against host cells and high anti-T. gondii activity. The molecular mechanism for these compounds is related to the inhibition of tyrosine amino acids involved in the proliferation and parasitophorous vacuole formation. The pharmacokinetic analysis shows that 1-(4-Methylimidazol-5-oyl)-4-(4-nitrophenyl)thiosemicarbazide and 4-(3-Iodophenyl)-1-(4-methylimidazol-5-oyl)thiosemicarbazide administered intragastrically pass into the bloodstream and cross the blood-brain barrier, and the absorption of both compounds is first-order absorption. Toxicity analysis shows that our derivatives possess lower toxicity than the routinely used drugs trimethoprim, sulfadiazine and pyrimethamine, as was observed in the level of liver enzymes and creatinine. Both derivatives are highly potent antiparasitic agents against T. gondii, prolonged survival and cure parasite-infected mice. Additionally, significant reductions in cyst formation in the brain and heart were observed, but the highest decreases were noted in muscle and the level of bradyzoites was similar to these observed in mice treated with commercially used drugs. Collectively, the obtained results support the conclusion that both compounds are highly efficacious in a mouse model of acute and chronic toxoplasmosis.

Heteroaryl ether analogues of an antileishmanial 7-substituted 2-nitroimidazooxazine lead afford attenuated hERG risk: In vitro and in vivo appraisal.

Previous investigation of the potent antileishmanial properties of antitubercular 7-substituted 2-nitroimidazo[2,1-b][1,3]oxazines with biaryl side chains led to our development of a new clinical candidate for visceral leishmaniasis (DNDI-0690). Within a collaborative backup program, a racemic monoaryl lead (3) possessing comparable activity in mice but a greater hERG liability formed the starting point for our pursuit of efficacious second generation analogues having good solubility and safety. Asymmetric synthesis and appraisal of its enantiomers first established that chiral preferences for in vivo efficacy were species dependent and that neither form afforded a reduced hERG risk. However, in line with our findings in a structurally related series, less lipophilic heteroaryl ethers provided significant solubility enhancements (up to 16-fold) and concomitantly attenuated hERG inhibition. One promising pyridine derivative (49) displayed 100% oral bioavailability in mice and delivered a 96% parasite burden reduction when dosed at 50 mg/kg in a Leishmania donovani mouse model of visceral leishmaniasis.

Nanoparticles Loaded with a New Thiourea Derivative: Development and In vitro Evaluation Against Leishmania amazonensis.

BACKGROUND: Leishmaniasis is a neglected tropical disease caused by protozoa of the genus Leishmania. Current treatments are restricted to a small number of drugs that display both severe side effects and a potential for parasites to develop resistance. A new N-(3,4-methylenedioxyphenyl)-N'- (2-phenethyl) thiourea compound (thiourea 1) has shown promising in vitro activity against Leishmania amazonensis with an IC50 of 54.14 µM for promastigotes and an IC50 of 70 µM for amastigotes. OBJECTIVE: To develop a formulation of thiourea 1 as an oral treatment for leishmaniasis, it was incorporated into Nanoparticles (NPs), a proven approach to provide long-acting drug delivery systems. METHODS: Poly (D,L-Lactic-co-Glycolic Acid) (PLGA) polymeric NPs containing thiourea 1 were obtained through a nanoprecipitation methodology associated with solvent evaporation. The NPs containing thiourea 1 were characterized for Encapsulation Efficiency (EE%), reaction yield (% w/w), surface charge, particle size and morphology by Transmission Electron Microscopy (TEM). RESULTS: NPs with thiourea 1 showed an improved in vitro leishmanicidal activity with a reduction in its cytotoxicity against macrophages (CC50>100 µg/mL) while preserving its IC50 against intracellular amastigotes (1.46 ± 0.09 µg/mL). This represents a parasite Selectivity Index (SI) of 68.49, which is a marked advancement from the reference drug pentamidine (SI = 30.14). CONCLUSION: The results suggest that the incorporation into NPs potentiated the therapeutic effect of thiourea 1, most likely by improving the selective delivery of the drug to the phagocytic cells that are targeted for infection by L. amazonensis. This work reinforces the importance of nanotechnology in the acquisition of new therapeutic alternatives for oral treatments.

Effects of coadministration of corn oil and ponazuril on serum and cerebrospinal fluid concentrations of ponazuril in horses.

BACKGROUND: Ponazuril is used for the treatment of equine protozoal myeloencephalitis (EPM). Coadministration of ponazuril with oil could result in higher serum and cerebrospinal fluid (CSF) concentrations of ponazuril. HYPOTHESIS: Coadministration of corn oil will result in higher serum and CSF concentrations of ponazuril than when ponazuril is administered alone. ANIMALS: Ten resident university-owned adult horses of either sex and >2 years of age. METHODS: Cohort study. Ponazuril oral paste (5 mg/kg BW; ponazuril treatment group (PON); n = 5), or ponazuril oral paste (5 mg/kg BW; ponazuril and oil treatment group (PONOIL; n = 5) coadministered with 2 oz of corn oil q24h for 21 days. Horses were treated once daily, for 21 days. Blood was collected on days 0, 7, 14, and 21 before dosing. In addition, CSF was collected on days 1, 7, 14, and 21. The concentration of ponazuril was determined in serum and CSF and results compared using repeated measures ANOVA. RESULTS: Coadministration of ponazuril with 2 oz of corn oil resulted in higher concentrations of ponazuril in serum (at steady state) than that found in horses given ponazuril alone (6.2 ± 0.9 mg/L versus 4.5 ± 1.0 mg/L; P = .004) (mean ± 1 SD). Cerebrospinal fluid concentrations of ponazuril were also greater in horses that received ponazuril and oil (0.213 mg/L ± 0.04 versus 0.162 ± 0.04 mg/L) (P = .03). CONCLUSIONS AND CLINICAL IMPORTANCE: Results suggest that coadministration of corn oil with ponazuril might enhance the effectiveness of treatment with ponazuril.

Highly sensitive UPLC-MS/MS method for the quantification of paromomycin in human plasma.

A highly sensitive method was developed to quantitate the antileishmanial agent paromomycin in human plasma, with a lower limit of quantification of 5 ng/mL. Separation was achieved using an isocratic ion-pair ultra-high performance liquid chromatographic (UPLC) method with a minimal concentration of heptafluorobutyric acid, which was coupled through an electrospray ionization interface to a triple quadrupole - linear ion trap mass spectrometer for detection. The method was validated over a linear calibration range of 5 to 1000 ng/mL (r2≥0.997) with inter-assay accuracies and precisions within the internationally accepted criteria. Volumes of 50 µL of human K2EDTA plasma were processed by using a simple protein precipitation method with 40 µL 20 % trichloroacetic acid. A good performance was shown in terms of recovery (100 %), matrix effect (C.V. ≤ 12.0 %) and carry-over (≤17.5 % of the lower limit of quantitation). Paromomycin spiked to human plasma samples was stable for at least 24 h at room temperature, 6 h at 35 °C, and 104 days at -20 °C. Paromomycin adsorbs to glass containers at low concentrations, and therefore acidic conditions were used throughout the assay, in combination with polypropylene tubes and autosampler vials. The assay was successfully applied in a pharmacokinetic study in visceral leishmaniasis patients from Eastern Africa.

In Vitro Culture of Cryptosporidium parvum Using Hollow Fiber Bioreactor: Applications for Simultaneous Pharmacokinetic and Pharmacodynamic Evaluation of Test Compounds.

Hollow fiber technology is a powerful tool for the culture of difficult-to-grow cells. Cryptosporidium parvum has a multistage sexual and asexual life cycle that has proved difficult to culture by conventional in vitro culture methods. Here, we describe a method utilizing a hollow fiber bioreactor for the continuous in vitro growth of C. parvum that produces sexual and asexual stages. The method enables the evaluation of potential therapeutic compounds under conditions that mirror the dynamic conditions found in the gut facilitating preliminary pharmacokinetic and pharmacodynamic data to be obtained.

Model System Identifies Kinetic Driver of Hsp90 Inhibitor Activity against African Trypanosomes and Plasmodium falciparum.

Hsp90 inhibitors, well studied in the laboratory and clinic for antitumor indications, have promising activity against protozoan pathogens, including Trypanosoma brucei which causes African sleeping sickness, and the malaria parasite, Plasmodium falciparum To progress these experimental drugs toward clinical use, we adapted an in vitro dynamic hollow-fiber system and deployed artificial pharmacokinetics to discover the driver of their activity: either concentration or time. The activities of compounds from three major classes of Hsp90 inhibitors in development were evaluated against trypanosomes. In all circumstances, the activities of the tested Hsp90 inhibitors were concentration driven. By optimally deploying the drug to match its kinetic driver, the efficacy of a given dose was improved up to 5-fold, and maximal efficacy was achieved with a significantly lower drug exposure. The superiority of concentration-driven regimens was evident in vitro over several logs of drug exposure and was predictive of efficacy in a mouse model of African trypanosomiasis. In studies with P. falciparum, antimalarial activity was similarly concentration driven. This experimental strategy offers an expedient and versatile translational tool to assess the impact of pharmacokinetics on antiprotozoal activity. Knowing kinetic governance early in drug development provides an additional metric for judging lead compounds and allows the incisive design of animal efficacy studies.

Combined Approach of Backbone Amide Linking and On-Resin N-Methylation for the Synthesis of Bioactive and Metabolically Stable Peptides.

Rhabdopeptides are a large class of nonribosomal peptides from the bacteria Xenorhabdus and Photorhabdus with low micromolar activity against different protozoa, which are the causative agents of several tropical diseases. The development of a facile and flexible synthesis combining backbone amide linking with on-resin peralkylation for the synthesis of permethylated rhabdopeptides is described. This strategy allows the fast generation of permethylated naturally occurring and artificial rhabdopeptides for a structure-activity study. Furthermore, in vitro experiments revealed their superior properties regarding their stability and passive membrane diffusion.

Pharmacodynamics and cellular accumulation of amphotericin B and miltefosine in Leishmania donovani-infected primary macrophages.

Objectives: We examined the in vitro pharmacodynamics and cellular accumulation of the standard anti-leishmanial drugs amphotericin B and miltefosine in intracellular Leishmania donovani amastigote-macrophage drug assays. Methods: Primary mouse macrophages were infected with L. donovani amastigotes. In time-kill assays infected macrophages were exposed to at least six different concentrations of serially diluted drugs and the percentage of infected macrophages was determined after 6, 12, 24, 48, 72 and 120 h of exposure. Cellular drug accumulation was measured following exposure to highly effective drug concentrations for 1, 6, 24, 48 and 72 h. Data were analysed through a mathematical model, relating drug concentration to the percentage of infected cells over time. Host cell membrane damage was evaluated through measurement of lactate dehydrogenase release. The effect of varying the serum and albumin concentrations in medium on the cellular accumulation levels of miltefosine was measured. Results: Amphotericin B was more potent than miltefosine (EC50 values of 0.65 and 1.26 µM, respectively) and displayed a wider therapeutic window in vitro. The kinetics of the cellular accumulation of amphotericin B was concentration- and formulation-dependent. At an extracellular concentration of 10 µM miltefosine maximum cellular drug levels preceded maximum anti-leishmanial kill. Miltefosine induced membrane damage in a concentration-, time- and serum-dependent manner. Its cellular accumulation levels increased with decreasing amounts of protein in assay medium. Conclusions: We have developed a novel approach to investigate the cellular pharmacology of anti-leishmanial drugs that serves as a model for the characterization of new drug candidates.

Nigella sativa oil entrapped polycaprolactone nanoparticles for leishmaniasis treatment.

This study is the first to investigate the antileishmanial activities of Nigella sativa oil (NSO) entrapped poly-ɛ-caprolactone (PCL) nanoparticles on Leishmania infantum promastigotes and amastigotes in vitro. NSO molecules with variable initial doses of 50, 100, 150, and 200 mg were successfully encapsulated into PCL nanoparticles identified as formulations NSO1, NSO2, NSO3, and NSO4, respectively. This process was characterised by scanning electron microscope, dynamic light scattering, Fourier transform infrared, encapsulation efficiency measurements, and release profile evaluations. The resulting synthetised nanoparticles had sizes ranging between 200 and 390 nm. PCL nanoparticles encapsulated 98% to 80% of initial doses of NSO and after incubation released approximately 85% of entrapped oil molecules after 288 h. All investigated formulations demonstrated strong antileishmanial effects on L. infantum promastigotes by inhibiting up to 90% of parasites after 192 h. The tested formulations decreased infection indexes of macrophages in a range between 2.4- and 4.1-fold in contrast to control, thus indicating the strong anti-amastigote activities of NSO encapsulated PCL nanoparticles. Furthermore, NSO-loaded PCL nanoparticles showed immunomodulatory effects by increasing produced nitric oxide amounts within macrophages by 2-3.5-fold in contrast to use of free oil. The obtained data showed significant antileishmanial effects of NSO encapsulated PCL nanoparticles on L. infantum promastigotes and amastigotes.

Leishmanicidal candidate LASSBio-1736, a cysteine protease inhibitor with favorable pharmacokinetics: low clearance and good distribution.

1. LASSBio-1736 ((E)-1-4(trifluoromethyl) benzylidene)-5-(2-4-dichlorozoyl) carbonylhydrazine) is proposed to be an oral cysteine protease leishmanicidal inhibitor. 2. This work aimed to investigate plasma pharmacokinetics, protein binding and tissue distribution of LASSBio-1736 in male Wistar rats. 3. LASSBio-1736 was administered to male Wistar rats at doses of 3.2 mg/kg intravenously and 12.6 mg/kg oral and intraperitoneal. The individual plasma-concentration profiles were determined by HPLC-UV and evaluated by non-compartmental and population pharmacokinetic analysis (Monolix 2016R1, Lixoft). Tissue distribution was evaluated after iv injection of 3.2 mg/kg drug by non-compartmental approach. 4. After intravenous administration, Vdss (1.79 L/kg), t ½ (23.1 h) and CLtot (56.1 mL/h/kg) were determined, and they were statistically similar (α =0.05) to oral and intraperitoneal pharmacokinetic parameters. The plasma profiles obtained after intravenous, oral and intraperitoneal administration of the compound were best fitted to a three-compartment and one-compartment open model with first-order absorption. 5. The intraperitoneal and oral bioavailability were around 40 and 15%, respectively. 6. Liver, spleen and skin tissues showed penetration of 340, 130 and 40%, respectively, with t ½ like plasma values. 7. LASSBio-1736 protein binding was 95 ± 2%. 8. The t ½, CLtot and tissue distribution of the compound agreed with the desired drug characteristics for leishmanicidal activity.

Discovery of novel, orally bioavailable, antileishmanial compounds using phenotypic screening.

Leishmaniasis is a parasitic infection that afflicts approximately 12 million people worldwide. There are several limitations to the approved drug therapies for leishmaniasis, including moderate to severe toxicity, growing drug resistance, and the need for extended dosing. Moreover, miltefosine is currently the only orally available drug therapy for this infection. We addressed the pressing need for new therapies by pursuing a two-step phenotypic screen to discover novel, potent, and orally bioavailable antileishmanials. First, we conducted a high-throughput screen (HTS) of roughly 600,000 small molecules for growth inhibition against the promastigote form of the parasite life cycle using the nucleic acid binding dye SYBR Green I. This screen identified approximately 2,700 compounds that inhibited growth by over 65% at a single point concentration of 10 µM. We next used this 2700 compound focused library to identify compounds that were highly potent against the disease-causing intra-macrophage amastigote form and exhibited limited toxicity toward the host macrophages. This two-step screening strategy uncovered nine unique chemical scaffolds within our collection, including two previously described antileishmanials. We further profiled two of the novel compounds for in vitro absorption, distribution, metabolism, excretion, and in vivo pharmacokinetics. Both compounds proved orally bioavailable, affording plasma exposures above the half-maximal effective concentration (EC50) concentration for at least 12 hours. Both compounds were efficacious when administered orally in a murine model of cutaneous leishmaniasis. One of the two compounds exerted potent activity against trypanosomes, which are kinetoplastid parasites related to Leishmania species. Therefore, this compound could help control multiple parasitic diseases. The promising pharmacokinetic profile and significant in vivo efficacy observed from our HTS hits highlight the utility of our two-step phenotypic screening strategy and strongly suggest that medicinal chemistry optimization of these newly identified scaffolds will lead to promising candidates for an orally available anti-parasitic drug.

Chitosan coated PluronicF127 micelles for effective delivery of Amphotericin B in experimental visceral leishmaniasis.

The goal of study was to develop micellar formulation of Amphotericin B (AmB) to improve its antileishmanial efficacy. AmB loaded pluronic F127 (PF 127) micelles were developed and coated with chitosan (Cs-PF-AmB-M) to accord immunoadjuvant and macrophage targeting properties. Hemolysis and cytotoxicity studies demonstrated that Cs-PF-AmB-M was 7.93 fold (at 20µg/ml AmB concentration) and 9.35 fold less hemolytic and cytotoxic, respectively in comparison to AmB suspension. Flow cytometry studies indicated that Cs-PF-FITC-M was 21.97 fold higher internalized byJ774A.1 macrophage in comparison to PF-FITC-M.Cs-PF-AmB-M showed excellent in-vitro (1.82 fold in compared to AmB suspension) and in-vivo (75.84±7.91% parasitic inhibition) antileishmanial activity against macrophage resident intracellular promastigotes and Leishmania donovani infected Syrian hamsters, respectively. Chitosan coating stimulated a Th1 immune response mediating auxiliary immunotherapeutic action as judged by in-vitro and in-vivo cytokine and mRNA expression. Toxicity studies demonstrated normal blood urea nitrogen (BUN) and plasma creatinine (PC) level and no sign of abnormal histopathology upon intravenous administration of micellar formulations. Pharmacokinetic profiling and tissue distribution studies indicated that AmB was preferentially localized in macrophage harboring tissue instead of kidney, thereby circumventing the characteristic nephrotoxicity. Conclusively, Cs-PF-AmB-M could be a viable alternative for the current immuno and chemotherapy of visceral leishmaniasis (VL).

Hexadecylphosphocholine (Miltefosine) stabilized chitosan modified Ampholipospheres as prototype co-delivery vehicle for enhanced killing of L. donovani.

Lipid nanoparticles are stable, biodegradable and biocompatible carriers offering excellent therapeutic efficacy. Here, a novel effort has been made to develop Miltefosine (HePC- hexadecylphosphocholine) stabilized chitosan anchored nanostructured lipid carriers (NLC) of Amphotericin B (AmB) as co-delivery vehicle to enhance killing of L. donovani. The entrapment efficiency of AmB was achieved upto 85.3% for HePC-AmB-CNLCs with mean particle size of 150.8±8.4nm, and zeta potential value of +28.2±1.1mV, respectively. The cumulative amount of AmB released at even after the 24h was less than 65% from HePC-AmB-CNLCs and Tween-80-AmB-CNLCs. Intravenous administration of HePC-AmB-CNLCs revealed the significantly increased localization of AmB in both liver and spleen when estimated. FACS study represented enhanced uptake of FITC-HePC-CNLCs over FITC-HePC-NLCs in J774A.1 cell lines. Highly significant in vitro and in vivo anti-leishmanial activity (p<0.05 compared with Tween 80-AmB-CNLCs) was observed with HePC-AmB-CNLCs when tested against VL in Leishmania donovani-infected hamsters. The haemolysis and cytotoxicity studies showed the safety of HePC-AmB-CNLCs and Tween 80-AmB-CNLCs. The findings suggested that it would be preferable to deliver AmB through HePC stabilized chitosan anchored nanostructured lipid carriers for rapid and effective treatment with decreased adverse effects.

Bioinspired Calcium Phosphate Nanoparticles Featuring as Efficient Carrier and Prompter for Macrophage Intervention in Experimental Leishmaniasis.

PURPOSE: To develop a biocompatible and bioresorbable calcium phosphate (CaP) nanoparticles (NPs) bearing Amphotericin B (AmB) with an aim to provide macrophage specific targeting in visceral leishmaniasis (VL). MATERIALS & METHODS: CaP-AmB-NPs were architectured through emulsion precipitation method. The developed formulation was extensively characterized for various parameters including in-vitro and in-vivo antileishmanial activity. Moreover, plasma pharmacokinetics, tissue biodistribution and toxicity profile were also assessed. RESULTS: Optimized CaP-AmB-NPs exhibited higher entrapment (71.1 ± 6.68%) of AmB. No trend related to higher hemolysis was apparent in the developed formulation as evidenced in commercially available colloidal and liposomal formulations. Cellular uptake of the developed CaP-AmB-NPs was quantified through flow cytometry in J774A.1 cell line, and a 23.90 fold rise in uptake was observed. Fluorescent microscopy also confirmed the time dependent rise in uptake. In-vivo multiple dose toxicity study demonstrated no toxicity upto 5 mg/kg dose of AmB. Plasma kinetics and tissue distribution studies established significantly higher concentration of AmB in group treated with CaP-AmB-NPs in liver and spleen as compared to CAmB, LAmB and AmB suspension group. In-vivo animal experimental results revealed that the CaP-AmB-NPs showed higher splenic parasite inhibition compared to CAmB and LAmB in leishmania parasite infected hamsters. CONCLUSIONS: The investigated CaP-AmB-NPs are effective in provoking macrophage mediated uptake and collectively features lower toxicity and offers a suitable replacement for available AmB-formulations for the obliteration of intra-macrophage VL parasite.

Pharmacokinetics and pharmacodynamics of oleylphosphocholine in a hamster model of visceral leishmaniasis.

OBJECTIVES: This study evaluated the pharmacokinetic properties of oleylphosphocholine (OlPC) in hamsters following a single oral dose. Its prophylactic activity was tested to establish exposure-activity relationships, while a 5 + 5 day oral regimen at 20 mg/kg with long post-treatment follow-up was performed to assess its curative potential. METHODS: Single oral doses of 20, 50 and 100 mg/kg were administered for pharmacokinetic analysis while a 100 mg/kg single oral dose was given on day 7, 4 or 1, or 4 h prior to infection in the prophylactic efficacy study. The animals were infected on day 0 with Leishmania infantum and the resulting parasite burdens were measured in target organs on day 21. In the curative model, treatment started on day 21 post-infection at 20 mg/kg for 5 + 5 days and amastigote burdens were determined in target organs either on day 42 [10 days after the end of treatment (dpt)] or day 72 (40 dpt). RESULTS: OlPC showed elimination t1/2 of ∼50 h and dose-proportional exposure. The prophylactic action of OlPC was in agreement with model-simulated drug exposures, showing dose-dependent residual activity. Interestingly, the 100 mg/kg single dose administered 4 days before infection (day -4) still reduced the overall parasite burden by ∼50%. In the curative model, >99% clearance of infection was observed at 10 dpt in all OlPC-treated animals and remained so at 40 dpt. CONCLUSIONS: This study reveals that total plasma exposure (AUCt-∞) correlates well with the prophylactic and curative efficacy of OlPC in the L. infantum hamster model.

Topical amphotericin B in ultradeformable liposomes: Formulation, skin penetration study, antifungal and antileishmanial activity in vitro.

Aiming to improve the topical delivery of AmB to treat cutaneous fungal infections and leishmaniasis, ultradeformable liposomes containing amphotericin B (AmB-UDL) were prepared, and structural and functional characterized. The effect of different edge activators, phospholipid and AmB concentration, and phospholipid to edge activator ratio on liposomal deformability, as well as on AmB liposomal content, was tested. Liposomes having Tween 80 as edge activator resulted of maximal deformability and AmB/phospholipid ratio. These consisted of AmB-UDL of 107±8nm diameter, 0.078-polydispersity index and -3±0.2mV Z potential, exhibiting monomeric AmB encapsulated in the bilayer at a 75% encapsulation efficiency. After its cytotoxicity on keratinocytes (HaCaT cells) and macrophages (J774 cells) was determined, the in vitro antifungal activity of AmB-UDL was assayed. It was found that fungal strains (albicans and non-albicans Candida ATCC strains and clinical isolates of C. albicans) were more sensitive to AmB-UDL than mammal cells. Minimum inhibitory concentration values for AmB-UDL were 5-24 and 24-50 times lower than IC50 for J774 and HaCaT cells, respectively. AmB-UDL at 1.25µg/ml also displayed 100 and 75% anti- Leishmania braziliensis promastigote and amastigote activity, respectively. Finally, upon 1h of non-occlusive incubation, the total accumulation of AmB in human skin was 40 times higher when applied as AmB-UDL than as AmBisome. AmB-UDL provided a profound AmB penetration toward deep epithelial layers, achieved without classical permeation enhancers. Because of that, topical treatments of cutaneous fungal infection and leishmaniasis with AmB-UDL may be regarded of potential of clinical significance.

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.