Pharmacokinetics, biodistribution, and activity of Amphotericin B-loaded nanocochleates on the Leishmania donovani murine visceral leishmaniasis model.

Amphotericin B (AmB) is an effective drug to treat visceral leishmaniasis but its use is limited by its poor oral bioavailability. This article describes the in-vivo evaluation of AmB-loaded, lipid-based cochleate systems designed for the oral route. Two different cochleate formulations were studied: one based on the synthetic phospholipid dioleoylphosphatidylserine (DOPS) and another optimized formulation based on a naturally occurring phosphatidylserine (Lipoid PSP70) that would render the formulation more affordable in developing countries. Their antiparasitic activity was evaluated in a mouse model of visceral leishmaniasis. Limited efficacy was observed for the DOPS-based cochleates after three doses of AmB at 1 mg/kg. The Lipoid PSP70-based cochleates were administered either as a buffered suspension or in enteric-coated capsules. AmB-loaded cochleates administered as a suspension at a high dose (3 × 20 mg/kg) exhibited significant antiparasitic activity while AmB-loaded cochleates in enteric-coated capsules at a lower dose (3 × 5 mg/kg) presented a slightly higher significant activity. A pharmacokinetic and biodistribution study in rats was performed with the Lipoid PSP70-based cochleates, with a single oral dose of 7.5 mg AmB/kg. Cochleates in both administration forms led to lower concentrations of Amphotericin B in the plasma than intravenous AmBisome®. However, more accumulation in the organs of interest (liver, spleen) was observed for both presentations of cochleates than for AmBisome® by the oral route. Therefore, cochleate formulations of AmB that could be produced at a cost accessible for developing countries show promise for the treatment of visceral leishmaniasis.

The Potential of 2-Substituted Quinolines as Antileishmanial Drug Candidates.

There is a need for new, cost-effective drugs to treat leishmaniasis. A strategy based on traditional medicine practiced in Bolivia led to the discovery of the 2-substituted quinoline series as a source of molecules with antileishmanial activity and low toxicity. This review documents the development of the series from the first isolated natural compounds through several hundred synthetized molecules to an optimized compound exhibiting an in vitro IC50 value of 0.2 µM against Leishmania donovani, and a selectivity index value of 187, together with in vivo activity on the L. donovani/hamster model. Attempts to establish structure-activity relationships are described, as well as studies that have attempted to determine the mechanism of action. For the latter, it appears that molecules of this series act on multiple targets, possibly including the immune system, which could explain the observed lack of drug resistance after in vitro drug pressure. We also show how nanotechnology strategies could valorize these drugs through adapted formulations and how a mechanistic targeting approach could generate new compounds with increased activity.

Repurposing chlorpromazine for anti-leukaemic therapy by nanoparticle encapsulation.

Treatment of acute myeloid leukaemia (AML) relies on decades-old drugs, and while recent years have seen some breakthroughs, AML is still characterised by poor prognosis and survival rate. Drug repurposing can expedite the preclinical development of new therapies, and by nanocarrier encapsulation, the number of potentially viable drug candidates can be further expanded. The anti-psychotic drug chlorpromazine (CPZ) has been identified as a candidate for repurposing for AML therapy. Nanoencapsulation may improve the suitability of CPZ for the treatment of AML by reducing its effect on the central nervous system. Using the emulsion-evaporation technique, we have developed PEGylated PLGA nanoparticles loaded with CPZ for AML therapy. The nanoparticles were characterised to be between 150 and 300 nm by DLS, of spherical morphology by TEM, with a drug loading of at least 6.0% (w/w). After an initial burst release of adsorbed drug, the remaining 80% of the drug was retained in the PLGA nanoparticles for at least 24 h. The CPZ-loaded nanoparticles had equal cytotoxic potential towards AML cells to free CPZ, but acted more slowly, in line with the protracted drug release. Crucially, nanoparticles injected intravenously into zebrafish larvae did not accumulate in the brain, and nanoencapsulation also prevented CPZ from crossing an artificial membrane model. This demonstrates that the purpose for nanoencapsulation of CPZ is fulfilled, namely avoiding effects on the central nervous system while retaining the anti-AML activity of the drug.

Cochleate drug delivery systems: An approach to their characterization.

Cochleate systems formed from phospholipids have very useful properties as drug delivery systems with sustained release capabilities, which are able to improve bioavailability and efficacy, reduce toxicity and increase the shelf-life of encapsulated molecules. These nanometric or micrometric structures are usually obtained after interaction of negatively charged liposomes with a positively charged bridging agent. Many different methods are now available to prepare cochleates and there are also numerous techniques that can be used to characterize them, some of which can be easily applied while others require more sophisticated equipment or analysis. The present review describes the important features of this drug delivery system; including their structural properties and potential applications, as well as a brief account of methods for their preparation and an extensive description of the techniques used for their characterization. This information could guide formulators in their choice of methods of characterization that would be best suited to their needs in terms of time, precision and technological difficulty.

Cochleate formulations of Amphotericin b designed for oral administration using a naturally occurring phospholipid.

The purpose of this work was to formulate the poor soluble antifungal and antiparasitic agent Amphotericin B (AmB) in cost-effective lipid-based formulations suitable for oral use in developing countries, overcoming the limitations of poor water solubility, nephrotoxicity and low oral bioavailability. The antifungal agent was formulated, at different molar proportions, in cochleate nanocarriers prepared using an accessible naturally occurring phospholipid rich in phosphatidylserine (Lipoid PSP70). These nanoassemblies were prepared by condensation of negatively charged phospholipid membrane vesicles with divalent cations (Ca2+). Small-angle X-ray scattering studies revealed the Ca2+-triggered condensation of loosely packed multilamellar vesicles into tightly packed bilayers of strongly dehydrated multilamellar organization characterized by narrow Bragg peaks. Transmission electron microscopy and quasi-elastic light scattering studies demonstrated the formation of nanosized particles. AmB drug loading was above 55% in all formulations. Circular dichroism demonstrated the prevalence of monomeric and complexed forms of AmB over toxic aggregates. The stability of AmB in gastric medium was improved by loading in cochleates and its release in gastrointestinal media was retarded. Confocal microscopy studies revealed the in-vitro interactions of Lipoid PSP70-based cochleates with Caco2 intestinal cell monolayers. The results suggest that the low-cost AmB-loaded cochleates may increase the therapeutic range of this drug.

Cyclodextrin complexation studies as the first step for repurposing of chlorpromazine.

The antipsychotic drug chlorpromazine (CPZ) has potential for the treatment of acute myeloid leukemia, if central nervous system side-effects resulting from its passage through the blood-brain barrier can be prevented. A robust drug delivery system for repurposed CPZ would be drug-in-cyclodextrin-in-liposome that would redirect the drug away from the brain while avoiding premature release in the circulation. As a first step, CPZ complexation with cyclodextrin (CD) has been studied. The stoichiometry, binding constant, enthalpy, and entropy of complex formation between CPZ and a panel of CDs was investigated by isothermal titration calorimetry (ITC). All the tested CDs were able to include CPZ, in the form of 1:1, 1:2 or a mixture of 1:1 and 1:2 complexes. In particular, a substituted γ-CD, sugammadex (the octasodium salt of octakis(6-deoxy-6-S-(2-carboxyethyl)-6-thio)cyclomaltooctaose), formed exclusively 1:2 complexes with an extremely high association constant of 6.37 × 109 M-2. Complexes were further characterized by heat capacity changes, one- and two-dimensional (ROESY) nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations. Finally, protection of CPZ against photodegradation by CDs was assessed. This was accelerated rather than reduced by complexation with CD. Altogether these results provide a molecular basis for the use of CD in delayed release formulations for CPZ.

Interaction of dequalinium chloride with phosphatidylcholine bilayers: A biophysical study with consequences on the development of lipid-based mitochondrial nanomedicines.

Dequalinium (DQ) has been proposed as a mitochondrial targeting ligand for nanomedicines, including liposomes, given the implication of these organelles in many diseases. This original study focuses on the interactions of DQ with phosphatidylcholine bilayers during the formation of liposomes. Firstly, PEGylated liposomes suitable for drug delivery were studied and were found to be more stable when made in water than in phosphate-buffered saline, emphasizing the role of electrostatic interactions between positive charges on DQ and the polar head groups of the lipids. To gain more information, differential scanning calorimetry, small- and wide-angle X-ray scattering and diffraction, 31P and 2H NMR spectroscopy and freeze-fracture electron microscopy were performed on dimyristoylphosphatidylcholine (DMPC) model membranes in the presence of DQ. This molecule was shown to be located at the level of polar head groups and to induce electrostatic repulsions between adjacent lipid bilayers leading to membrane budding in water. These findings indicate that DQ is not completely inert towards lipid membranes and therefore is not an ideal candidate for encapsulation in liposomes. Overall, our work stresses the necessity for thorough physico-chemical characterization to better understand the mechanisms underlying the development of nanomedicines.

In-vitro and in-vivo antileishmanial activity of inexpensive Amphotericin B formulations: Heated Amphotericin B and Amphotericin B-loaded microemulsion.

Amphotericin B (AmB) is effective against visceral leishmaniasis (VL), but the renal toxicity of the conventional form, mixed micelles with deoxycholate (M-AmB), is often dose-limiting, while the less toxic lipid-based formulations such as AmBisome® are very expensive. Two different strategies to improve the therapeutic index of AmB with inexpensive ingredients were evaluated on this work: (i) the heat treatment of the commercial formulation (H-AmB) and (ii) the preparation of an AmB-loaded microemulsion (ME-AmB). M-AmB was heated to 70 °C for 20 min. The resulting product was characterized by UV spectrophotometry and circular dichroism, showing super-aggregates formation. ME-AmB was prepared from phosphate buffer pH 7.4, Tween 80®, Lipoid S100® and Mygliol 812® with AmB at 5 mg/mL. The droplet size, measured by dynamic light scattering, was about 40 nm and transmission electron microscopy confirmed a spherical shape. Rheological analysis showed low viscosity and Newtonian behavior. All the formulations were active in vitro and in vivo against Leishmania donovani (LV9). A selectivity index (CC50 on RAW/IC50 on LV9) higher than 10 was observed for ME-AmB, H-AmB and AmBisome®. Furthermore, no important in vivo toxicity was observed for all the samples. The in-vivo efficacy of the formulations after IV administration was evaluated in Balb/C mice infected with LV9 (three doses of 1 mg/kg AmB) and no significant difference was observed between H-AmB, M-AmB, ME-AmB and AmBisome®. In conclusion, these two inexpensive alternative formulations for AmB showing good efficacy and selectivity for Leishmania donovani merit further investigation.

Antitumor activity of nanoliposomes encapsulating the novobiocin analog 6BrCaQ in a triple-negative breast cancer model in mice.

In this study, we investigated the anticancer efficacy of pegylated liposomes containing 6BrCaQ, an hsp90 inhibitor derived from novobiocin. 6BrCaQ has been previously identified as the most potent compound in a series of quinoleic novobiocin analogs but is poorly water-soluble. We investigated, for the first time, the anti-proliferative effects of this drug in vivo in an orthotopic breast cancer model (MDA-MB-231 luc) using pegylated liposomes to allow its administration. Hsp90, hsp70 and hsp27 protein and mRNA expressions were not strongly affected after treatment meaning it did not induce a heat shock response often associated with resistance and poor prognosis. Liposomal delivery of 6BrCaQ retarded tumor growth at a low dose (1 mg/kg, injected once a week for 4 weeks). Histological analysis of tumors revealed necrosis and a lower proportion of proliferative cells in treated mice indicating that this drug has potential for breast cancer therapy when encapsulated in liposomes.

Dual effect of Algerian propolis on lung cancer: antitumor and chemopreventive effects involving antioxidant activity

Abstract The purpose of our study was to divulge the antiproliferative effect of an ethanolic extract of Algerian propolis (EEP) in the human lung adenocarcinoma cell line (A549) and reveal the chemopreventive role against benzo(a)pyrene-induced lung carcinogenesis in albino Wistar rats. Cytotoxicity of EEP was evaluated using the MTT assay and cell adhesion in A549 cells. Moreover, rats were given 25 mg/kg of propolis for 5 days before induction of experimental lung cancer by a single intraperitoneal dose of 200 mg/kg benzo(a)pyrene. Body weight, lung weight, lipid peroxidation, marker enzymes, and enzymatic and non-enzymatic antioxidants were estimated. The EEP demonstrated an inhibitory effect on proliferation of A549 at 24 and 72 hours in a dose-dependent manner and blocked adhesion of the cells by fibrinogen. Moreover, EEP reduced the oxidative stress generated by benzo(a)pyrene. The pre-treatment showed that enzymatic and non-enzymatic antioxidants increased and lipid peroxidation decreased. A histological analysis further supported these findings and showed a decrease in the number of side effects. These results are particularly important for both clinical applications of propolis and the possibility for its use as a potential chemotherapeutic agent.

A cell impedance-based real-time in vitro assay to assess the toxicity of amphotericin B formulations.

Aerosolized liposomal amphotericin B (L-AmB) has been investigated as prophylaxis against invasive aspergillosis. However, the clinical results are controversial and some trials suggest that toxicity could be a limitation for wider use. Our aim was to assess the dynamics of cell toxicity induced in a human alveolar epithelial cell line (A549) after exposure to L-AmB (50 to 400µg/ml) or amphotericin B deoxycholate (D-AmB; 50 to 200µg/ml) by monitoring real-time A549 cell viability using an impedance-based technology. Results were expressed as cell index values integrating cell adhesion, proliferation, and survival. In parallel, the gene expression of proinflammatory cytokines was quantified at 6 and 24h after drug addition by real-time RT-PCR on cell lysates. No sustained reduction of cell indexes was observed with L-AmB or empty liposomes, even at 400µg/ml. Only the highest concentration tested of L-AmB (400µg/ml) yielded transient significant 6-fold and 4-fold induction of TNF-α and IL-8 mRNAs, respectively. In contrast, D-AmB induced a decrease in cell indexes and only the 50µg/ml concentration of D-AmB was followed by cell recovery, higher concentrations leading to cell death. Significant 4-fold, 7-fold and 3-fold inductions of TNF-α, IL-8 and IL-33 mRNAs were also observed at 6h with 50µg/ml of D-AmB. In conclusion, continuous cell impedance measurement showed no toxicity on overall cellular behavior although a slight proinflammatory cytokine expression is possible after L-AmB challenge. Real-time kinetics of cell impedance is an interesting tool for initial screening of cell toxicity.

New nanoparticles obtained by co-assembly of amphiphilic cyclodextrins and nonlamellar single-chain lipids: Preparation and characterization.

This work aimed at preparing new nanoscale assemblies based on an amphiphilic bio-esterified ß-cyclodextrin (ß-CD), substituted at the secondary face with n-decanoic fatty acid chains (ß-CD-C10), and monoolein (MO) as new carriers for parenteral drug delivery. Stable binary (ß-CD-C10/MO) and ternary (ß-CD-C10/MO/stabilizer) nanoscale assemblies close to 100nm in size were successfully prepared in water by the solvent displacement method. The generated nanoparticles were fully characterized by dynamic light scattering, transmission electron microscopy, small-angle X-ray scattering, residual solvent analysis, complement activation and the contribution of each formulation parameter was determined by principal component analysis. The ß-CD-C10 units were shown to self-organize into nanoparticles with a hexagonal supramolecular packing that was significantly modulated by the molar ratio of the constituents and the presence of a steric or electrostatic stabilizer (DOPE-PEG2000 or DOPA/POPA, respectively). Indeed, nanoparticles differing in morphology and in hexagonal lattice parameters were obtained while the co-existence of multiple mesophases was observed in some formulations, in particular for the ß-CD-C10/MO/DOPA and ß-CD-C10/MO/POPA systems. The mixed ß-CD-C10/MO/DOPE-PEG2000 nanoparticles (49:49:2 in mol%) appeared to be the most suitable for use as a drug delivery system since they contained a very low amount of residual solvent and showed a low level of complement C3 activation.

Heat shock proteins and cancer: How can nanomedicine be harnessed?

Heat shock protein (hsp90) is an interesting target for cancer therapy because it is involved in the folding and stabilization of numerous proteins, including many that contribute to the development of cancer. It is part of the chaperone machinery that includes other heat shock proteins (hsp70, hsp27, hsp40) and is mainly localized in the cytosol, although many analogues or isoforms can be found in mitochondrion, endoplasmic reticulum and the cell membrane. Many potential inhibitors of hsp90 have been tested for cancer therapy but their usefulness is limited by their poor solubility in water and their ability to reach the target cells and the correct intracellular compartment. Nanomedicine, the incorporation of active molecules into an appropriate delivery system, could provide a solution to these drawbacks. In this review, we explain the rationale for using nanomedicine for this sort of cancer therapy, considering the properties of the chaperone machinery and of the different hsp90 analogues. We present some results that have already been obtained and put forward some strategies for delivery of hsp90 analogues to specific organelles.

Remote loading of doxorubicin into liposomes by transmembrane pH gradient to reduce toxicity toward H9c2 cells.

The use of doxorubicin (DOX) is limited by its dose-dependent cardiotoxicity. Entrapped DOX in liposome has been shown to reduce cardiotoxicity. Results showed that about 92% of the total drug was encapsulated in liposome. The release experiments showed a weak DOX leakage in both culture medium and in PBS, more than 98% and 90% of the encapsulated DOX respectively was still retained in liposomes after 24 h of incubation. When the release experiments were carried out in phosphate buffer pH5.3, the leakage of DOX from liposomes reached 37% after 24 h of incubation. Evaluation of cellular uptake of the liposomal DOX indicated the possible endocytosis of liposomes because the majority of visible fluorescence of DOX was mainly in the cytoplasm, whereas the nuclear compartment showed a weak intensity. When using unloaded fluorescent-liposomes, the fluorescence was absent in nuclei suggests that liposomes cannot cross the nuclear membrane. MTT assay and measurement of LDH release suggest that necrosis is the form of cellular death predominates in H9c2 cells exposed to high doses of DOX, while for weak doses apoptosis could be the predominate form. Entrapped DOX reduced significantly DOX toxicity after 3 and 6 h of incubation, but after 20 h entrapped DOX is more toxic than free one.

Freeze-drying of emulsified systems: A review.

Colloidal systems such as emulsions, microemulsions and nanoemulsions are able to transport active molecules, enhance their solubility and stability and minimize their side effects. However, since they are dispersions with an aqueous continuous phase they have some disadvantages such as the risk of microbiological contamination, degradation by hydrolysis, physico-chemical instability and loss of pharmacological activity of the drug. Freeze drying, in which the water is removed from the preparation by sublimation under vacuum, has been suggested as a means to resolve these problems. Lyophilized products are very stable and are easy to transport and store. However, there is very little information in the literature about the application of this technique to emulsified systems. The aim of this review is to evaluate the lyophilization process as a tool for increasing the shelf life of emulsified systems such as emulsions, microemulsions and nanoemulsions. In addition, the mechanism of cryoprotection and the techniques that can be used to characterize the freeze-dried systems are discussed.

Formulation and in vitro efficacy of liposomes containing the Hsp90 inhibitor 6BrCaQ in prostate cancer cells.

6BrCaQ is a promising anti-cancer agent derived from novobiocin, which has been shown to inhibit Hsp90. 6BrCaQ was loaded into nanometer-scaled phospholipid vesicles (liposomes) suitable for drug delivery to solid tumors. The effective incorporation of the drug within the phospholipid bilayer was investigated by differential scanning calorimetry. Liposomal 6BrCaQ showed good activity on PC-3 cell lines in vitro in terms of apoptosis induction and cell growth arrest in G2/M. Liposomes containing 6BrCaQ were also shown to slow down migration of PC-3 cells in presence of chemokine ligand 2 and to synergize with doxorubicin. Several Hsp90 targeting molecules like geldanamycin induce accumulation of Hsp70, leading to cytoprotection and often correlated with poor prognosis. In this study, we did not report any Hsp70 induction after treatment with liposomal 6BrCaQ but a decrease in Hsp90 and CDK-4 protein expression, indicating an effect on the chaperon machinery. Liposomal encapsulation of 6BrCaQ revealed promising anti-cancer effects and a better understanding of its mechanism of action.

The Use of Nanocarriers in Acute Myeloid Leukaemia Therapy: Challenges and Current Status.

Chemotherapy for AML is hampered by severe side-effects and failure to eliminate all the blasts that eventually leads to relapse. The use of nanosized particulate drug carriers such as liposomes and polymeric nanoparticles has the potential to improve AML therapy by delivering more of the drug to the disease site, thereby reducing toxicity. For example, encapsulation in liposomes reduces the cardiotoxicity of anthracyclines, giving an improved therapeutic index. Moreover, when the surface properties are engineered appropriately, nanocarriers remain in the circulation and extravasate in tissues with sinusoidal capillaries, one of which is bone marrow, leading to a more favourable distribution of the associated drug. Drug carrier technology contributes to the development of newer drugs, such as nucleic acids that can be protected from degradation and delivered into cells, thus opening the way for gene-silencing strategies. Furthermore, carrier systems provide a means of dispersing poorly water-soluble molecule for in vivo administration and thus increase the "druggability" of new lead compounds, such as heat-shock protein inhibitors. Particulate carriers can transport more than one active agent, allowing synergistic action and theranostic strategies. Notably, phase I and II clinical trials are being performed with CPX-351, a liposomal formulation containing cytarabine and daunorubicin at an optimal ratio. Finally, by attaching suitable ligands to the nanocarrier surface, specific targeting to AML cells can be achieved. In this review, we give examples of successful targeting to folate and transferrin receptors against AML.

Efficacy of multi-functional liposomes containing daunorubicin and emetine for treatment of acute myeloid leukaemia.

Despite recent advances in chemotherapy against acute myeloid leukaemia (AML), the disease still has high mortality, particularly for patients who tolerate extensive chemotherapy poorly. Nano-formulations have potential to minimise the adverse effects of chemotherapy. We present here a liposomal formulation encapsulating both the anthracycline daunorubicin (DNR) and emetine (Eme) for enhanced cytotoxic effect against AML cells. Eme could be loaded into the PEGylated liposomes together with DNR by the acid precipitation principle, with a loading efficiency of Eme at about 50% of that of DNR. The liposome surface was modified with folate to enhance drug loading into cells, giving higher cytotoxic activity. Both intracellular drug loading and cytotoxic activity could be further increased by anti-folate treatment of AML cells with methotrexate (MTX). The combination of DNR and Eme also increased drug loading in MTX-treated cells compared to DNR alone. Liposomes with both DNR and Eme were particularly efficient against AMLs with deficient p53. In conclusion, we have produced a multi-functional liposomal anti-leukaemic drug formulation designed to overcome some of the problems in anthracycline chemotherapy: (1) Combination of DNR and Eme to diminish drug resistance. (2) Using PEGylated stealth liposomes to minimise adverse side-effects. (3) Molecules on the liposomal surface target proteins on AML-cells ensure selectivity, which was enhanced by priming the leukaemia cells with MTX.

Development of oil-in-water microemulsions for the oral delivery of amphotericin B.

Amphotericin B (AmB) is a very efficient drug against serious diseases such as leishmaniasis and systemic fungal infections. However, its oral bioavailability is limited due to its poor solubility in water. Nevertheless, it is marketed as high-cost lipid parenteral formulations that may induce serious infusion-related side effects. In this study, oil-in-water (O/W) microemulsions (MEs) were developed and characterized with a view to their use as solubility enhancers and oral delivery systems for AmB. Therefore, different nonionic surfactants from the Tween(®) and Span(®) series were tested for their solubilization capacity in combination with several oils. Based on pseudoternary phase diagrams, AmB-loaded MEs with mean droplet sizes about 120 nm were successfully produced. They were able to improve the drug solubility up to 1000-fold. Rheological studies showed the MEs to be low-viscosity formulations with Newtonian behavior. Circular dichroism and absorption spectra revealed that part of the AmB in the MEs was aggregated as an AmB reservoir carrier. Cytotoxicity studies revealed limited toxicity to macrophage-like cells that may allow the formulations to be considered as suitable carriers for AmB.