Hexadecylphosphocholine Research Articles

Cytotoxicity Effects of Miltefosine and Niosomal form on Human Umbilical Vein Endothelial Cells: Colorimetric Assay, Apoptosis, and Gene Expression Profiling

Background: Miltefosine (MIL), hexadecyl phosphocholine, is the only oral medicine used to treat leishmaniasis. This drug has a major limitation and is expensive and potentially teratogenic. Objective: This study aimed to evaluate the toxic effect of MIL and its niosomal form on human umbilical vein endothelial cells (HUVECs), the expression genes, and the profile associated with apoptosis in the mitochondrial permeabilization regulated. Methods: Miltefosine niosome (MN) prepared by the thin-film hydration method and characterized. HUVECs were treated with MIL (100–1000μg/ml), and MN (10-50μg/ml) for 24, 48, and 72 h, and the persistence was assessed by colorimetric assay flow cytometry and real-time PCR. Results: Lesser toxicity was detected on cell proliferation for MN while both forms decreased Bcl-2 and elevated the expression of Bak/Bax and caspases-3, -8, and -9. The data demonstrated that MIL significantly exerted its cytotoxicity on HUVECs compared to MN. Conclusion: This drug should be considered embryotoxic during pregnancy, while in niosomal form, it released slowly and remained safe. The mechanism of action of MIL associated with programmed cell death.

The effects of miltefosine on the structure and dynamics of DPPC and DPPS liposomes mimicking normal and cancer cell membranes: FTIR and DSC studies

Miltefosine (MLT,hexadecylphosphocholine (HePC)) is an alkylphospholipid (APL) clinically used against leishmaniasis and the topical treatment of cancer. Although the MLT mechanism of action has not yet been clarified, it is thought to be related to its incorporation into the plasma membrane, affecting lipid homeostasis. An essential feature of APLs is that they possess a lethal effect that acts only on malignant cells without affecting normal cells; this property indicates the potential selective antitumor properties of these types of drugs. However, the exact mechanism of APLs action at the molecular level is not yet known in detail. This study aimed to elucidate the molecular mechanisms of the interaction of the antitumor alkylphospholipid drug miltefosine with molecular models of normal and cancer cell membranes. The interaction of MLT with dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposomes (MLVs) mimicking normal cell membrane and dipalmitoyl phosphatidylserine (DPPS) multilamellar liposomes (MLVs) mimicking cancer cell membrane was studied in the presence of (1 mol%,3 mol%, 6 mol%, 9 mol%, 15 mol%, 24 mol%, 30 mol% and 35 mol%) and in the absence of MLT by using two different non-invasive techniques; Fourier Transform Infrared (FTIR) spectroscopy and Differential Scanning Calorimetry (DSC). The comparative results show that miltefosine affects DPPS/MLT systems modeled cancer cells more than the DPPC/MLT system modeled healthy cells. Our results may be proof at the molecular level that miltefosine has selective antitumor properties.

Interfacial behaviour and quantitative analysis of hexadecyl phosphocholine drug at a polarized liquid/liquid interface.

The interfacial behaviour of the amphiphilic drug hexadecyl phosphocholine (HePC, also called miltefosine) was analysed by cyclic voltammetry applied at the water/1,2-dichloroethane interface. HePC is the only oral drug currently approved for the treatment of visceral, mucosal and cutaneous leishmaniasis. Because of its amphiphilic character, it can interact with biological membranes, solubilizing their compounds and leading to cell disruption. These interactions are responsible for its side effects and toxicity; therefore, HePC quantification in biological fluids and pharmaceutical preparations is extremely important. However, the lack of a chromophore in its structure prevents its spectroscopic determination. For this reason, the main challenge of this work was to propose an electroanalytical method for the quantification of this drug, which constitutes a simpler alternative than liquid chromatography-tandem mass spectrometry already reported. With this aim, in the first part of this work, the mechanism of the electrochemical process occurring after polarizing the interface was studied. By varying the experimental conditions, it was possible to determine that in a first step, at open circuit or at low potential values, HePC spontaneously adsorbed to the interface. Later, as the potential increased, the transfer of the anions present in the organic phase towards the aqueous side of the interface, where the HePC polar head groups were present, occurred thus forming adsorbed "ion pairs" and producing an increase in positive current. Subsequently, in the negative sweep, the "ion pairs" dissociated and desorbed giving rise to a negative peak. In this way, both negative and positive currents were considered useful for quantitative purposes. In the second part of this work, an appropriate experimental procedure was designed and proposed as a quantitative methodology for the HePC determination, which consisted of cleaning the interface and controlling the time at open circuit, followed by the voltammetric analysis. A linear response of both, positive or negative, peak currents with drug concentration was obtained within an acceptable range, providing a simple solution for the HePC quantification problem. Future studies will be carried out to evaluate the quantification and selectivity in real matrices containing polymer micelles working as HePC nanocarriers with the aim of avoiding the adverse effects of HePC when it is orally or intravenously administered.

Spiral steroids as potential markers for pre-eclampsia: A pilot study

Pre-eclampsia is a condition that complicates 3–6% of pregnancies. At present, there is no FDA approved diagnostic method to evaluate risk or progression. We would like to report our observation of elevated levels of the spiral steroid phosphoester precursor in patients with pre-eclampsia.Samples from 20 normotensive pregnant women and from 20 women with pre-eclampsia were purchased from Global Alliance for the Prevention of Prematurity and Stillbirth (GAPPS). After addition of miltefosine (hexadecyl phosphocholine) as an internal control, each sample was extracted and evaluated by tandem MS. The method detected 2 spiral steroid phosphoesters and their common precursor. For each mass ion, the ion counts obtained were compared to the ion counts of miltefosine. The samples from the normotensive women were used to establish the mean and standard deviation. Then, Z-scores were determined for each of the serum components. Of the samples from the women with pre-eclampsia, 12 of 19 (63%) samples had Z-scores over 2.0 for at least one of the steroid phosphoesters. In contrast, current markers under development for risk of pre-eclampsia have prediction scores ranging from 8 to 33%.As the spiral steroids are lactones, similar to spironolactone and related compounds, they could function as endogenous potassium sparing hormones. However, as only about half of the affected patients had elevated levels of the spiral steroids, it may not be the only underlying pathology. Parturition would end transfer of placental spiral steroids and account for the termination of the symptoms of pre-eclampsia/eclampsia. Previous investigators have proposed inadequate placental function as the critical pathology of pre-eclampsia, but it is hard to imagine how inadequate placental function leads to the maternal pathology without invoking an endogenous potassium sparing hormone originating in the placenta.

Accessing lipophilicity of drugs with biomimetic models: A comparative study using liposomes and micelles

Lipophilicity is a physicochemical property of crucial importance in drug discovery and drug design. Biomimetic models, such as liposomes and micelles, constitute a valuable tool for the assessment of lipophilicity through the determination of partition coefficients (log Kp). However, the lack of standardization hampers the judgment about which model or method has the best and broadest passive drug permeation predictive capacity. This work provides a comparative analysis between the methodologies based on biomimetic models to determine the partition coefficient (log Kp). For that purpose, a set of reference substances preconized by the Organization for Economic Cooperation and Development (OECD) guidelines was used. The biomimetic models employed were liposomes and micelles composed by 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC) and hexadecylphosphocholine (HePC), respectively. Both lipids were used as representative phospholipids of natural membranes. The partition coefficients between biomimetic models and aqueous phases were determined by derivative spectroscopy at physiological conditions (37 °C and pH 7.4). The partition coefficients obtained using biomimetic models are quite different and more reliable than the ones obtained using an octanol/water system. Comparing the performance of the two biomimetic models, micelles revealed to be suitable only for substances with high molar absorption coefficient and log Kp > 3, but in general liposomes are the best model for accessing lipophilicity of drugs. Furthermore, a comparison between experimental data and the partition coefficients determined by the computational method COSMOmic is also provided and discussed. As a final summarizing result, a decision tree is provided in order to guide the selection of a tool for assessing the lipophilicity of drugs.

Tat peptide and hexadecylphosphocholine introduction into pegylated liposomal doxorubicin: An in vitro and in vivo study on drug cellular delivery, release, biodistribution and antitumor activity

We have investigated the co-addition of hexadecylphosphocholine (HePC) and a Tat derived peptide (Tat), coupled to Maleimide-PEG2000-DSPE pegylated liposomal doxorubicin (PLD) in many respects, including drug and liposome cellular delivery, drug release, biodistribution, in vivo cell delivery and antitumor activity. The liposomes were HePC-free and -containing liposomes, from which liposomes with 25, 50, 100 and 200 numbers of Tat/liposome were prepared. Similarly, DiI-C18 (3)-model liposomes (DiI-L and DiI-HePC-L) were prepared. HePC and Tat increased cellular delivery of Dox and cytotoxicity in B16F0 melanoma and C26 colon carcinoma cells. Tat enhanced liposome-cell interaction and caused Dox burst release. HePC and Tat reduced the serum retention time of liposomal Dox, slightly and dramatically, respectively. In comparison, Tat-liposomes enhanced Dox delivery to liver and spleen cells 3h post-injection. Likewise, Dox content of these tissues and tumor was lower at 24h. The naïve liposomes retarded tumor growth more effectively and their related median survival time of the treated C26 bearing BALB/c mice was longer than those of Tat-liposomes (MST>45days versus MST<38days). Overall liposomes exhibiting sustained drug release and negligible cell interaction were more suitable delivery systems in targeting cancerous tumors and suppressing their growth.

Platelet-activating factor receptor contributes to antileishmanial function of miltefosine.

Miltefosine [hexadecylphosphocholine (HPC)] is the only orally bioavailable drug for the disease visceral leishmaniasis, which is caused by the protozoan parasite Leishmania donovani. Although miltefosine has direct leishmanicidal effects, evidence is mounting for its immune system-dependent effects. The mechanism of such indirect antileishmanial effects of miltefosine remains to be discovered. As platelet-activating factor and HPC share structural semblances and both induce killing of intracellular Leishmania, we surmised that platelet-activating factor (PAF) receptor had a significant role in the antileishmanial function of miltefosine. The proposition was supported by molecular dynamic simulation of HPC docking into PAF receptor and by comparison of its leishmanicidal function on PAF receptor-deficient macrophages and mice under HPC treatment. We observed that compared with wild-type macrophages, the PAF receptor-deficient macrophages showed 1) reduced binding of a fluorescent analog of HPC, 2) decreased TNF-α production, and 3) lower miltefosine-induced killing of L. donovani. Miltefosine exhibited significantly compromised leishmanicidal function in PAF receptor-deficient mice. An anti-PAF receptor Ab led to a significant decrease in miltefosine-induced intracellular Leishmania killing and IFN-γ production in a macrophage-T cell coculture system. These results indicate significant roles for PAF receptor in the leishmanicidal activity of HPC. The findings open new avenues for a more rational understanding of the mechanism of action of this drug as well as for improved therapeutic strategies.

Antitumoral Alkylphospholipids Alter Cell Lipid Metabolism

Alkylphospholipid (APL) analogues are promising candidates in the search for treatments for cancer. In contrast to standard chemotherapeutic drugs, these lipophilic agents target the cell membrane without interacting directly with DNA. A variety of mechanisms have been suggested to explain the actions of these compounds, which can induce apoptosis and/or cell growth arrest. In this review, we focus on recent advances in our understanding of the actions of clinically-relevant APLs, such as hexadecylphosphocholine (HePC), edelfosine, erucylphosphocholine (ErPC) and perifosine on the human hepatoma HepG2 cell line, which is commonly used for lipid metabolism studies with a special emphasis on cholesterol metabolism. One consistent finding is that HePC and other APLs cause a reduction in the biosynthesis of phosphatidylcholine (PC) by inhibiting the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase (CT). Our research group has been at the forefront in demonstrating that exposure to APLs affects cholesterol homeostasis in mammalian cells. Treatment with HePC, for example, causes a marked enhancement in cholesterol synthesis, which has been related to an impairment in the arrival of cholesterol at the endoplasmic reticulum (ER). In a similar way to HePC, edelfosine, ErPC and perifosine increase the de novo synthesis and uptake of cholesterol and also inhibit the arrival of plasma-membrane cholesterol at the ER, which induces a significant cholesterogenic response in these cells, involving an increase in gene expression and higher levels of several proteins related to the biosynthetic pathway and receptor-mediated uptake of cholesterol. It is generally accepted nowadays that the maintenance of a tightly controlled free-cholesterol/PC ratio is crucial to optimum cell behaviour and that alterations to this ratio may lead to necrosis and/or apoptosis. Our results have considerable bearing on this idea because an increase in cholesterol biosynthesis associated with a decrease in the synthesis of choline-containing phospholipids and cholesterol esterification leads to a modification in the free-cholesterol/PC ratio in cells exposed to APLs. It is well accepted that cholesterol is critical for the formation of lipid rafts and therefore drugs that alter cell cholesterol content should modify the properties of these membrane domains and consequently the signal-transduction pathways, which depends upon lipid-raft integrity. Results on the whole show that APLs share a common active mechanism consisting of disrupting PC and sphingomyelin (SM) biosyntheses and cholesterol homeostasis, all of which leads to a disturbance in the native membrane structure, thus affecting signaling processes vital to cell survival and growth.

Manipulation of phospholipid composition alters protein binding to lipid droplets (606.3)

Lipid droplets are cytoplasmic organelles that store excess lipid. Each lipid droplet consists of a core of neutral lipid surrounded by a phospholipid monolayer. Proteins interact with the phospholipid surface and regulate lipid droplet synthesis and degradation. We have shown that the phospholipid composition of synthetic lipid droplets can influence protein binding in an in vitro assay. We aimed to determine if protein binding to lipid droplets is influenced by the lipid droplet phospholipid composition in cells. We altered the phospholipid composition of lipid droplets in NIH3T3 cells with hexadecylphosphocholine (HePC), an inhibitor of phosphatidylcholine (PC) synthesis. Isolation of lipid droplets from inhibitor treated and control cells and separation of phospholipids by thin layer chromatography, demonstrated a decrease in PC relative to phosphatidylethanolamine in HePC‐treated cells. Furthermore, HePC treatment prevented the lipid droplet localization of CTP:phosphocholine cytidylyltransferase, the enzyme that catalyzes the rate‐limiting step in PC synthesis. Other changes in protein localization to lipid droplets were observed by SDS‐PAGE and silver staining of lipid droplet proteins in HePC‐treated and control cells. Current and future experiments continue to examine how changes in phospholipid composition alter the protein composition of this important storage compartment.Grant Funding Source: Supported by an award from Research Corporation for Science Advancement

Dynamics of Micelle Formation from Mixed Lipid Droplets

Amphiphilic lipid molecules can form various micelles depending on not only their molecular composition but also their self-assembly pathway. In this work, coarse-grained molecular dynamics simulations have been applied to study the micellization behaviors of mixed dipalmitoylphosphatidylcholine (DPPC)/hexadecylphosphocholine (HPC) droplets. By varying DPPC/HPC composition and the size of lipid droplets, various micelles such as spherical and nonspherical (oblate or prolate) vesicles, disk-like micelles, double or single ring-like and worm-like micelles were observed. It is found that the lipid droplet as an initial state favors forming vesicles and ring-like micelles due to in situ micellization. Our simulation results demonstrate that using special initial conditions combined with various molecular compositions is an effective way to tune lipid micellar structure.

Imidazolium ionic liquids as solvents of pharmaceuticals: Influence on HSA binding and partition coefficient of nimesulide

In this work, the influence of imidazolium ionic liquids (ILs) on bio-chemical parameters that influence the in vivo behavior of nimesulide was evaluated. In this context, the binding of nimesulide to human serum albumin (HSA), in IL media, was studied. In parallel, the evaluation of the interaction of drug-IL systems, with micelles of hexadecylphosphocholine (HDPC), enabled the calculation of partition coefficients (K(p)). Both assays were performed in buffered media in the absence and in the presence of emim [BF(4)], emim [Ms] and emim [TfMs] 1%. Even though there was an increase of the dissociation constant (K(d)) in IL media, nimesulide still binds to HSA by means of strong interactions. The thermodynamic analysis indicates that the interaction is spontaneous for all the tested systems. Moreover, the studied systems exhibited properties that are favorable to the interaction of the drug with biological membranes, with K(p) values 2.5-3.5 higher than in aqueous environment. The studied nimesulide-IL systems presented promising characteristics regarding the absorption and distribution of the drug in vivo, so that the studied solvents seem to be good options for drug delivery.

Proapoptotic miltefosine nanovesicles show synergism with paclitaxel: Implications for glioblastoma multiforme therapy

Hexadecylphosphocholine (HePC) or miltefosine based proapoptotic lipid nanovesicles encapsulating paclitaxel for synergistic anticancer effect of paclitaxel and miltefosine in chemoresistant human glioblastoma multiforme (U-87 MG) overexpressing multidrug resistance 1 (MDR1) gene product P-glycoprotein (P-gp), were developed in this study. The nanovesicles had 100–200nm size and a negative zeta potential (∼−25mV) and optimized for miltefosine content based on their physiochemical properties. With a high encapsulation efficiency of 94%, the nanovesicles showed sustained release of paclitaxel in nasal fluid and triggered release in the cerebrospinal fluid. Synergistic action of paclitaxel and miltefosine was observed with a low IC50 of 162±5nM. The nanovesicle also overcame drug resistance and showed ATP dependent uptake via clathrin mediated pathway in glioblastoma cells. An improved therapeutic efficacy in comparison to Taxol®, the current clinical formulation of paclitaxel was observed. Efficient brain uptake of the nanovesicles upon intranasal administration was observed in vivo and the nanovesicles were also found to be able to cross blood brain barrier. These studies therefore suggest the therapeutic potential of proapoptotic lipid nanovesicles and their feasibility for intranasal administration in the treatment of chemoresistant glioblastoma.

Hemolytic properties of miltefosine in liposomes of various lipid compositions

The hemolytic activity of hexadecylphosphocholine (HePC) included in liposomes of phosphatidylcholine (PC), phosphatidylethanol (PET), and their mixture in various ratios has been studied. Spectroscopic data suggest that supramolecular particles containing less than 15% HePC occur as liposomes. Hemolysis at the same HePC concentration is less pronounced for liposomes (10% HePC) than for micelles (30% HePC). The inclusion of anionic phosphatidylethanol into the liposomal composition does not affect significantly the degree of hemolysis.

High-throughput microplate assay for the determination of drug partition coefficients

Partition coefficients (K(p)) of drugs between the phospholipid bilayer and the aqueous phase provide useful information in quantitative structure-activity relationship studies. Hexadecylphosphocholine (HePC) micelles, composed of a zwitterionic hydrophilic surface and a hydrophobic core, mimic the biomembranes and have several advantages over other lipid structures to assess K(p) values. Their preparation is easy, fast and avoids the use of toxic organic solvents, and the output has fewer spectroscopic interferences. Here, we describe a high-throughput microplate protocol for assessing the K(p) of drugs using HePC micelles as membrane models and derivative spectrophotometry as the detection technique. Moreover, the time-consuming data treatment to assess K(p) values is easily performed by a dedicated Excel routine developed here and described in detail. The K(p) values of nonsteroidal anti-inflammatory drugs (acemetacin, clonixin, diclofenac and indomethacin) were determined to show the simplicity of the method and to validate this protocol, which provides K(p) values (n = 3) of two drugs in ∼ 2 h.

Disruption of cellular cholesterol transport and homeostasis as a novel mechanism of action of membrane-targeted alkylphospholipid analogues.

Alkylphospholipid (APL) analogues constitute a new class of synthetic anti-tumour agents that act directly on cell membranes. We have previously demonstrated that hexadecylphosphocholine (HePC) alters intracellular cholesterol traffic and metabolism in HepG2 cells. We now extended our studies to analyse the effects of other clinically relevant APLs, such as edelfosine, erucylphosphocholine and perifosine on intracellular cholesterol homeostasis. Using radiolabelled substrates we determined the effect of APLs on cholesterol metabolism and cholesterol traffic from the plasma membrane to the endoplasmic reticulum (ER). Protein levels and gene expression of the main proteins involved in cholesterol homeostasis were analysed by Western blot and RT-PCR respectively. Membrane raft and non-raft fractions were isolated from HepG2 cells by a detergent-free method. All APLs inhibited the transport of cholesterol from the plasma membrane to the ER, which induced a significant cholesterogenic response in HepG2 cells. This response involved an increased gene expression and higher levels of several proteins related to the biosynthesis and the receptor-mediated uptake of cholesterol. Cell exposure to the APL-representative HePC enhanced the content of cholesterol mainly in the membrane raft fractions, compared with the untreated cells. Membrane-targeted APLs exhibited a novel and common mechanism of action, through disruption of cholesterol homeostasis, which in turn affected specific lipid microdomains of cellular membranes.

Surface-active properties of nitrogen heterocyclic and dialkylamino derivates of hexadecylphosphocholine and cetyltrimethylammonium bromide

The physico-chemical properties of dialkylamino and nitrogen heterocyclic analogues of hexadecylphosphocholine (HPC) and cetyltrimethylammonium bromide (CTAB) were investigated. The surface properties, such as the critical micelle concentration (cmc), the surface tension value at the cmc (gamma(cmc)), and the surface area at the surface saturation per head group (A(cmc)) were determined by means of surface tension measurements. Micelle size was determined using the dynamic light scattering method. The influence of dialkylamino groups and heterocyclic ring size on surface-active properties was investigated. Surface activity and micellar size of prepared analogues of HPC and CTAB were mutually compared.

117 POSTER ANG1005, Paclitaxel conjugated to the angiopep brain transport vector for the treatment of brain cancer: preclinical studies

We have investigated the co-addition of hexadecylphosphocholine (HePC) and a Tat derived peptide (Tat), coupled to Maleimide-PEG2000-DSPE pegylated liposomal doxorubicin (PLD) in many respects, including drug and liposome cellular delivery, drug release, biodistribution, in vivo cell delivery and antitumor activity. The liposomes were HePC-free and -containing liposomes, from which liposomes with 25, 50, 100 and 200 numbers of Tat/liposome were prepared. Similarly, DiI-C18 (3)-model liposomes (DiI-L and DiI-HePC-L) were prepared. HePC and Tat increased cellular delivery of Dox and cytotoxicity in B16F0 melanoma and C26 colon carcinoma cells. Tat enhanced liposome-cell interaction and caused Dox burst release. HePC and Tat reduced the serum retention time of liposomal Dox, slightly and dramatically, respectively. In comparison, Tat-liposomes enhanced Dox delivery to liver and spleen cells 3 h post-injection. Likewise, Dox content of these tissues and tumor was lower at 24 h. The naïve liposomes retarded tumor growth more effectively and their related median survival time of the treated C26 bearing BALB/c mice was longer than those of Tat-liposomes (MST > 45 days versus MST < 38 days). Overall liposomes exhibiting sustained drug release and negligible cell interaction were more suitable delivery systems in targeting cancerous tumors and suppressing their growth.

Dual role of hexadecylphosphocholine (miltefosine) in thermosensitive liposomes: Active ingredient and mediator of drug release

Lysolipid-based thermosensitive liposomes have been successfully introduced as efficient drug delivery system with fast drug release upon heat treatment. Hexadecylphosphocholine (HePC) is structurally related to 1-palmitoyl-2-lyso- sn-glycero-3-phosphocholine (P-lyso-PC) but chemically and metabolically more stable, thereby offering strong antineoplastic, antiprotozoal and antifungal activity. We investigated the properties of HePC in low temperature sensitive (LTSL) and high temperature sensitive liposomes (HTSL) based on 1,2-dipalmitoyl- sn-glycero-3-phosphoglyceroglycerol (DPPGOG). Therefore liposomes composed of DPPC/DSPC = 8:2 (molar ratio), DPPC/DSPC/DPPGOG = 5:2:3, HePC/DPPC/DSPC/DPPGOG = 1:4:2:3, HePC/DSPC/DPPGOG = 1:6:3 and P-lyso-PC/DPPC/DSPC/DPPGOG = 1:4:2:3 were prepared by the lipid film hydration and extrusion method and compared with regard to stability at 37 °C and the release kinetics under heating conditions (38 °C–45 °C) in the presence of fetal calf serum. Each formulation was characterized for size distribution, ζ-potential, encapsulation efficiency and phase transition temperature ( T m). The influence of heat on the cytotoxic activity of HePC in TSL was investigated using the WST-1 assay in BFS-1 fibrosarcoma and C6 glioma cells for the low (HePC-LTSL, T m = 42.9 °C) and the high (HePC-HTSL, T m = 48.5 °C) temperature sensitive formulations and compared to micellar HePC or plain TSL. The cellular HePC uptake after 15 min incubation at 37 °C or 42 °C was determined by liquid chromatography tandem–mass spectrometry (LC–MS/MS). As expected, HePC increases the release rate of TSL similar to lysolipid. The cytotoxicity of HePC in TSL was heat inducible and stronger than the one induced by micellar HePC which did not respond to heat. A possible explanation for this raise in cytotoxicity of HePC in TSL is the increased cellular transfer of HePC under heating conditions demonstrated by LC–MS/MS.

Lysosomal and mitochondrial pathways in miltefosine-induced apoptosis in U937 cells

Hexadecylphosphocholine (HePC) is an anticancer agent whose effect has been shown to involve apoptosis induction but the signaling pathways leading to apoptosis remain to be elucidated. We show here that HePC induces activation of caspase-9, -3, and -8 via the intrinsic pathway, release of cytochrome c, activation and relocation of Bax to the mitochondria as well as the cleavage of Bid. Moreover, a lysosomal pathway characterized by partial lysosomal rupture, cathepsin B activation and relocation from lysosomes to the cytosol, is involved in HePC-induced apoptosis. A cathepsin B/L inhibitor partially suppresses caspase activation and apoptosis induction, indicating signaling between lysosomes and mitochondria. Conversely, the pancaspase inhibitor Q-VD-OPH inhibits lysosomal rupture, but only at early time points, suggesting that immediate lysosomal rupture involves caspases. Overexpression of Bcl-2, an anti-apoptotic protein known to prevent mitochondrial dysfunction, totally abrogates lysosomal destabilization and cell death.

Physicochemical Characterization of Molecular Assemblies of Miltefosine and Amphotericin B

This study describes the interactions between two amphiphilic molecules with antileishmanial activity, amphotericin B (AmB) and miltefosine [hexadecylphosphocholine (HePC)], the latter being effective by the oral route. The effect of HePC on the aggregation state of AmB in aqueous solution and the interactions between the two agents were monitored using absorption spectroscopy and circular dichroism. Structural characterization of the mixed aggregates formed in water by dynamic light scattering (DLS) and cryofracture electron microscopy was performed. At concentrations above its critical micelle concentration, HePC was shown to interact with AmB, leading to an increase in the proportion of AmB in its monomeric form as a result of a micellar solubilization mechanism with a capacity of 26 +/- 3 mmol of AmB solubilized/mol of HePC, that is, nearly 40 molecules of HePC per molecule of AmB in the mixed micelles. These were revealed as individual and spherical aggregates close to 10 nm in diameter by both electron microscopy and DLS. Such a micellar formulation provides a new AmB-based system which might be useful in delivering AmB orally for visceral leishmaniasis bitherapy.