Surface-active properties of the antitumour ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine)

The surface activity and interaction with lipid monolayers and bilayers of the antitumour ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine) have been studied. Edelfosine is a surface-active soluble amphiphile, with critical micellar concentrations at 3.5 microM and 19 microM in water. When the air-water interface is occupied by a phospholipid, edelfosine becomes inserted in the phospholipid monolayer, increasing surface pressure. This increase is dose-dependent, and reaches a plateau at ca. 2 microM edelfosine bulk concentration. The ether lipid can become inserted in phospholipid monolayers with initial surface pressures of up to 33 mN/m, which ensures its capacity to become inserted into cell membranes. Upon interaction with phospholipid vesicles, edelfosine exhibits a weak detergent activity, causing release of vesicle contents to a low extent (<5%), and a small proportion of lipid solubilization. The weak detergent properties of edelfosine can be related to its very low critical micellar concentrations. Its high affinity for lipid monolayers combined with low lytic properties support the use of edelfosine as a clinical drug. The surface-active properties of edelfosine are similar to those of other "single-chain" lipids, e.g. lysophosphatidylcholine, palmitoylcarnitine, or N-acetylsphingosine.     

Involvement of lipid rafts in the localization and dysfunction effect of the antitumor ether phospholipid edelfosine in mitochondria

Lipid rafts and mitochondria are promising targets in cancer therapy. The synthetic antitumor alkyl-lysophospholipid analog edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) has been reported to target lipid rafts. Here, we have found that edelfosine induced loss of mitochondrial membrane potential and apoptosis in human cervical carcinoma HeLa cells, both responses being abrogated by Bcl-x_L overexpression. We synthesized a number of new fluorescent edelfosine analogs, which preserved the proapoptotic activity of the parent drug, and colocalized with mitochondria in HeLa cells. Edelfosine induced swelling in isolated mitochondria, indicating an increase in mitochondrial membrane permeability. This mitochondrial swelling was independent of reactive oxygen species generation. A structurally related inactive analog was unable to promote mitochondrial swelling, highlighting the importance of edelfosine molecular structure in its effect on mitochondria. Raft disruption inhibited mitochondrial localization of the drug in cells and edelfosine-induced swelling in isolated mitochondria. Edelfosine promoted a redistribution of lipid rafts from the plasma membrane to mitochondria, suggesting a raft-mediated link between plasma membrane and mitochondria. Our data suggest that direct interaction of edelfosine with mitochondria eventually leads to mitochondrial dysfunction and apoptosis. These observations unveil a new framework in cancer chemotherapy that involves a link between lipid rafts and mitochondria in the mechanism of action of an antitumor drug, thus opening new avenues for cancer treatment.     

In vitro and in vivo efficacy of ether lipid edelfosine against Leishmania spp. and SbV-resistant parasites

Background

The leishmaniases are a complex of neglected tropical diseases caused by more than 20 Leishmania parasite species, for which available therapeutic arsenal is scarce and unsatisfactory. Pentavalent antimonials (SbV) are currently the first-line pharmacologic therapy for leishmaniasis worldwide, but resistance to these compounds is increasingly reported. Alkyl-lysophospoholipid analogs (ALPs) constitute a family of compounds with antileishmanial activity, and one of its members, miltefosine, has been approved as the first oral treatment for visceral and cutaneous leishmaniasis. However, its clinical use can be challenged by less impressive efficiency in patients infected with some Leishmania species, including L. braziliensis and L. mexicana, and by proneness to develop drug resistance in vitro.

Methodology/Principal Findings

We found that ALPs ranked edelfosine>perifosine>miltefosine>erucylphosphocholine for their antileishmanial activity and capacity to promote apoptosis-like parasitic cell death in promastigote and amastigote forms of distinct Leishmania spp., as assessed by proliferation and flow cytometry assays. Effective antileishmanial ALP concentrations were dependent on both the parasite species and their development stage. Edelfosine accumulated in and killed intracellular Leishmania parasites within macrophages. In vivo antileishmanial activity was demonstrated following oral treatment with edelfosine of mice and hamsters infected with L. major, L. panamensis or L. braziliensis, without any significant side-effect. Edelfosine also killed SbV-resistant Leishmania parasites in in vitro and in vivo assays, and required longer incubation times than miltefosine to generate drug resistance.

Conclusions/Significance

Our data reveal that edelfosine is the most potent ALP in killing different Leishmania spp., and it is less prone to lead to drug resistance development than miltefosine. Edelfosine is effective in killing Leishmania in culture and within macrophages, as well as in animal models infected with different Leishmania spp. and SbV-resistant parasites. Our results indicate that edelfosine is a promising orally administered antileishmanial drug for clinical evaluation.     

Synthesis of sterols with modified side chains.

Synthesis of sterols with side chain containing from four to nine carbons are described.     

Quantitative determination of the antitumor alkyl ether phospholipid edelfosine by reversed-phase liquid chromatography-electrospray mass spectrometry: application to cell uptake studies and characterization of drug delivery systems

Edelfosine is a synthetic alkyl ether phospholipid that represents a promising class of antitumor agents. However, analytical methods to measure these type compounds are scarce. The lack of a reliable methodology to quantify edelfosine is a major problem in ongoing and scheduled preclinical and clinical trials with this drug. We evaluated the applicability of high-performance liquid chromatography-mass spectrometry to determine edelfosine in biological samples and polymeric delivery systems. Sample pre-treatment involved polymer precipitation or cell lysis with methanol. HPLC separation was performed on an Alltima RPC(18) narrow-bore column and edelfosine quantification was done by electrospray ionization mass spectrometry (ESI-MS) using positive ion mode and selected ion monitoring. Assays were linear in the tested range of 0.3-10 microg/ml. The limit of quantification was 0.3 ng/sample in both matrices, namely biological samples and polymeric delivery systems. The interassay precision ranging from 0.79 to 1.49%, with relative errors of -6.7 and 12.8%. Mean extraction recovery was 95.6%. HPLC-ESI-MS is a reliable system for edelfosine analysis and quantification in samples from different sources, combining advantages of full automation (rapidity, ease of use, no need of extensive extraction procedures) with high analytical performance and throughput.     

Surface-active properties of the antitumour ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine)

The surface activity and interaction with lipid monolayers and bilayers of the antitumour ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine) have been studied. Edelfosine is a surface-active soluble amphiphile, with critical micellar concentrations at 3.5 μM and 19 μM in water. When the air-water interface is occupied by a phospholipid, edelfosine becomes inserted in the phospholipid monolayer, increasing surface pressure. This increase is dose-dependent, and reaches a plateau at ca. 2 μM edelfosine bulk concentration. The ether lipid can become inserted in phospholipid monolayers with initial surface pressures of up to 33 mN/m, which ensures its capacity to become inserted into cell membranes. Upon interaction with phospholipid vesicles, edelfosine exhibits a weak detergent activity, causing release of vesicle contents to a low extent (< 5%), and a small proportion of lipid solubilization. The weak detergent properties of edelfosine can be related to its very low critical micellar concentrations. Its high affinity for lipid monolayers combined with low lytic properties support the use of edelfosine as a clinical drug. The surface-active properties of edelfosine are similar to those of other “single-chain” lipids, e.g. lysophosphatidylcholine, palmitoylcarnitine, or N-acetylsphingosine.     

Biotransformation of sterols: selective cleavage of the side chain

This review elaborates on the recent development of microbial sterol biotransformation systems. Particular emphasis is laid on the new enzymatic approach investigating the cleavage of sterol side chain. New developments in the area of immobilized cell system and use of organic media along with recent reviews on side chain cleavage are discussed.     

Role of sterols in the interaction of polyene antibiotics with lipid membranes

The problem whether the membrane sterols are indirect acceptors of polyenic antibiotics or they play the role of substances providing conditions (at the expense of putting in order the membrane phospholipids) for formation of conductive complexes (ionic canals) from the antibiotic molecules is discussed. The comparative study on the ability of sterols of various structure (ergosterol, 7-dehydrocholesterol, cholesterol, 5 alpha-cholestan-3 beta-ol) to interact with the membrane phospholipids and to increase the sensitivity of such membranes to amphotericin B showed no correlation between the levels of these properties. The value of the changes in the cross elasticity module (E) of artificial bilayer lipid membranes from egg lecithin on introduction of the above sterols into their composition was used as the criterion for the interaction level. The absence of correlation between the above properties of the sterols indicated that the role of the sterols in interaction of polyenic antibiotics with the membranes could not be considered as the only effect of the sterols on putting in order the phospholipids, which confirmed the hypothesis on the acceptor function of the sterols with respect to polyenic antibiotics. The study of the effect of amphotericin B on the elastic properties of the cholesterol-containing bilayer membranes isolated from egg lecithin showed tha the values of the longitudinal and cross elasticity modules of the membranes did not change during introduction into the membranes of the ionic canals.     

Systematic identification of proteins that elicit drug side effects

Side effect similarities of drugs have recently been employed to predict new drug targets, and networks of side effects and targets have been used to better understand the mechanism of action of drugs. Here, we report a large‐scale analysis to systematically predict and characterize proteins that cause drug side effects. We integrated phenotypic data obtained during clinical trials with known drug–target relations to identify overrepresented protein–side effect combinations. Using independent data, we confirm that most of these overrepresentations point to proteins which, when perturbed, cause side effects. Of 1428 side effects studied, 732 were predicted to be predominantly caused by individual proteins, at least 137 of them backed by existing pharmacological or phenotypic data. We prove this concept in vivo by confirming our prediction that activation of the serotonin 7 receptor (HTR7) is responsible for hyperesthesia in mice, which, in turn, can be prevented by a drug that selectively inhibits HTR7. Taken together, we show that a large fraction of complex drug side effects are mediated by individual proteins and create a reference for such relations.     

Molecular order and dynamics in planar lipid bilayers: effects of unsaturation and sterols

Angle-resolved fluorescence depolarization experiments were carried out on 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules embedded in macroscopically oriented multilayers of saturated [dimyristoylphosphatidylcholine (DMPC)] and unsaturated [palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dilineoylphosphatidylcholine (DLPC), plant digalactosyldiglyceride (DGDG)] lipids with and without cholesterol. In all the lipid systems studied the order parameter (P2) of TMA-DPH molecules was found to be higher than that for DPH. Considerations of the order parameter (P4), however, indicate that DPH molecules have a heterogeneous distribution in bilayers of unsaturated lipids, with a significant fraction of the molecules lying with their long axes parallel to the bilayer planes. Both the DPH and TMA-DPH molecules exhibit a decrease in the molecular order as well as a decrease in their rates of motion on increasing the unsaturation of the hydrocarbon chains. The addition of cholesterol tends to reverse this effect, with an increase in both the order and dynamics. Bilayers of DOPC, however, exhibit a somewhat different result. It is suggested that the discrepancies between these observations and findings with lipid vesicle systems simply reflect the effects of curvature on the behavior of the probe molecules. The results indicate that the concept of membrane fluidity must be used with great caution.     

Combination of carvacrol with simvastatin improves the lipid‐lowering efficacy and alleviates simvastatin side effects

The present investigation was designed to examine the possible additive hypolipidemic effect of carvacrol (CARV) in combination with simvastatin (SIM) on poloxamer 407 (P407)‐induced hyperlipidemia. Rats were injected with P407, (500 mg/ kg; i.p.), twice a week, for 30 days. Treatment was carried out by administration of SIM (20 mg/kg/day; p.o.) or CARV (50 mg/kg/day; p.o.) or combination of them. Treatment with CARV significantly decreased total cholesterol, triglycerides, low‐density lipoprotein, atherogenic index, leptin, and increased high‐density lipoprotein and adiponectin. Moreover, CARV potentiated the hypolipidemic effect of SIM. Both SIM and CARV alleviated the oxidative stress induced by P407. Interestingly, CARV, when combined with SIM, significantly ameliorated SIM‐induced liver and muscle injury by reducing the level of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, creatine kinase, and myoglobin and restoring the normal histological picture of both liver and muscle as well as apoptosis.     

Thermodynamics of interaction of a fluorescent DNA oligomer with the anti-tumour drug netropsin.

Fluorescence spectroscopy was used to study the interaction between the minor-groove-binding drug netropsin and the self-complementary oligonucleotide d(CTGAnPTTCAG)2 containing the fluorescent base analogue 2-aminopurine (nP). The binding of netropsin to this oligonucleotide causes strong quenching of the 2-aminopurine fluorescence, observed by steady-state as well as time-resolved spectroscopy. From fluorescence titrations, binding isotherms were recorded and evaluated. The parameters showed one netropsin binding site/oligonucleotide duplex and an association constant of about 10(5) M-1 at 25 degrees C, 3-4 orders of magnitude weaker than for an exclusive adenine/thymine host sequence. From the temperature dependence of the association constant the thermodynamic parameters were obtained as delta G = -29 kJ/mol, delta H = -12 kJ/mol and delta S = +55 J.mol-1.K-1 at 25 degrees C. These parameters resemble those of the interaction of poly[(dG-dC).(dG-dC)] with netropsin, indicating a mainly entropy-driven reaction. The amino group of 2-aminopurine, like that of guanine, resides in the minor groove of DNA. Therefore the relatively weak binding of netropsin to d(CTGAnPTTCAG)2 is probably related to partial blockage of the tight fit of netropsin into the preferred minor groove of an exclusive adenine/thymine host sequence.     

Synthesis of sterols oxygenated in the terminal fragment of their side chains

Methods of stereoselective synthesis of oxysterols are considered by the examples of (25R)-26-hydroxycholesterol, (24S)-24,25-epoxycholesterol, and (24S)-24-hydroxycholesterol containing functional groups in the terminal fragments of their side chain. Special attention is paid to the problems of construction of chiral centers C24 and C25.     

Inhibition of cholesterol absorption by the combination of dietary plant sterols and ezetimibe: effects on plasma lipid levels

Consumption of plant sterols and treatment with ezetimibe both reduce cholesterol absorption in the intestine. However, the mechanism of action differs between the two treatments, and the consequences of combination treatment are unknown. Therefore, we performed a double-blind, placebo-controlled, crossover study for the plant sterol component with open-label ezetimibe treatment. Forty mildly hypercholesterolemic subjects were randomized to the following treatments for 4 weeks each: 10 mg/day ezetimibe combined with 25 g/day control spread; 10 mg/day ezetimibe combined with 25 g/day spread containing 2.0 g of plant sterols; 25 g/day spread containing 2.0 g of plant sterols; and placebo treatment consisting of 25 g/day control spread. Combination treatment of plant sterols and ezetimibe reduced low density lipoprotein cholesterol (LDL-C) by 1.06 mmol/l (25.2%; P < 0.001) compared with 0.23 mmol/l (4.7%; P = 0.006) with plant sterols and 0.94 mmol/l (22.2%; P < 0.001) with ezetimibe monotherapy. LDL-C reduction conferred by the combination treatment did not differ significantly from ezetimibe monotherapy (-0.12 mmol/l or -3.5%; P = 0.13). Additionally, the plasma lathosterol-to-cholesterol ratio increased with all treatments. Sitosterol and campesterol ratios increased after plant sterol treatment and decreased upon ezetimibe and combination therapy. Our results indicate that the combination of plant sterols and ezetimibe has no therapeutic benefit over ezetimibe monotherapy in subjects with mild hypercholesterolemia.     

Network-based method to infer the contributions of proteins to the etiology of drug side effects

Studying the molecular mechanisms that underlie the relationship between drugs and the side effects they produce is critical for drug discovery and drug development. Currently, however, computational methods are still unavailable to assess drug-protein interactions with the aim of globally inferring the contributions of various classes of proteins toward the etiology of side effects. In this work, we integrated data reflecting drug-side effect relationships, drug-target relationships, and protein-protein interactions to develop a novel network-based probabilistic model, SidePro, to evaluate the contributions of proteins toward the etiology of side effects. For a given side effect, the method applies an expectation---maximization algorithm and a diffusion kernel-based approach to estimate each protein’s contribution. We applied this method to a wide range of side effects and validated the results using cross-validation and records from the Side Effect Resource database. We also studied a specific side effect, nephrotoxicity, which is known to be associated with the irrational use of the Chinese herbal compound triptolide, a diterpenoid epoxide in the Thunder of God Vine, Tripterygium wilfordii Lei-Gong-Teng. Using triptolide as an example, we scored the target proteins of triptolide using our model and investigated the high-scoring proteins and their related biological processes. The results demonstrated that our model could differentiate between the potential side effect targets and therapeutic targets of triptolide. Overall, the proposed model could accurately pinpoint the molecular mechanisms of drug side effects, thus making contribution to safe and effective drug development.     

Rapid analysis of drug effects on the cell cycle

Using a flow cytometric technique to analyse DNA content and chromatin structure simultaneously, the following parameters of cell cycle progression were estimated in control and drug-treated L1210 cell cultures: (a) the kinetics of cell exit from the G1 phase; (b) the probability of cell exit from the indeterminate portion of the G1 phase, measured as the half-time of cell residence in that state; (c) the duration of the deterministic portion of G1 phase; (d) the rates of cell transit through selected "windows" in S phase; (e) the rate of cell entrance into mitosis; (f) the mean duration of the cell cycle (Tc). These parameters are obtained in a single stathmokinetic experiment from measurements of individual samples withdrawn at 30 min-1 hr intervals from Vinblasatine-treated cultures. In the same experiment mitotic indices are obtained with high statistical accuracy, and may be used to determine the terminal point of drug action. In addition to cell cycle analysis the method makes it possible to detect drug-induced changes in nuclear chromatin that are manifested by varying sensitivity of DNA in situ to denaturation by acid. Such changes were found to be associated with defective chromatin condensation, altered histone modifications or intercalation of the drugs into DNA. Using this technique the effects of sodium n-butyrate and two new antitumor drugs on L1210 cells were investigated.     

The genome-wide DNA sequence specificity of the anti-tumour drug bleomycin in human cells

The cancer chemotherapeutic agent, bleomycin, cleaves DNA at specific sites. For the first time, the genome-wide DNA sequence specificity of bleomycin breakage was determined in human cells. Utilising Illumina next-generation DNA sequencing techniques, over 200 million bleomycin cleavage sites were examined to elucidate the bleomycin genome-wide DNA selectivity. The genome-wide bleomycin cleavage data were analysed by four different methods to determine the cellular DNA sequence specificity of bleomycin strand breakage. For the most highly cleaved DNA sequences, the preferred site of bleomycin breakage was at 5′-GT* dinucleotide sequences (where the asterisk indicates the bleomycin cleavage site), with lesser cleavage at 5′-GC* dinucleotides. This investigation also determined longer bleomycin cleavage sequences, with preferred cleavage at 5′-GT*A and 5′- TGT* trinucleotide sequences, and 5′-TGT*A tetranucleotides. For cellular DNA, the hexanucleotide DNA sequence 5′-RTGT*AY (where R is a purine and Y is a pyrimidine) was the most highly cleaved DNA sequence. It was striking that alternating purine–pyrimidine sequences were highly cleaved by bleomycin. The highest intensity cleavage sites in cellular and purified DNA were very similar although there were some minor differences. Statistical nucleotide frequency analysis indicated a G nucleotide was present at the ?3 position (relative to the cleavage site) in cellular DNA but was absent in purified DNA.