Edelfosine nanoemulsions inhibit tumor growth of triple negative breast cancer in zebrafish xenograft model.

Triple negative breast cancer (TNBC) is known for being very aggressive, heterogeneous and highly metastatic. The standard of care treatment is still chemotherapy, with adjacent toxicity and low efficacy, highlighting the need for alternative and more effective therapeutic strategies. Edelfosine, an alkyl-lysophospholipid, has proved to be a promising therapy for several cancer types, upon delivery in lipid nanoparticles. Therefore, the objective of this work was to explore the potential of edelfosine for the treatment of TNBC. Edelfosine nanoemulsions (ET-NEs) composed by edelfosine, Miglyol 812 and phosphatidylcholine as excipients, due to their good safety profile, presented an average size of about 120 nm and a neutral zeta potential, and were stable in biorelevant media. The ability of ET-NEs to interrupt tumor growth in TNBC was demonstrated both in vitro, using a highly aggressive and invasive TNBC cell line, and in vivo, using zebrafish embryos. Importantly, ET-NEs were able to penetrate through the skin barrier of MDA-MB 231 xenografted zebrafish embryos, into the yolk sac, leading to an effective decrease of highly aggressive and invasive tumoral cells' proliferation. Altogether the results demonstrate the potential of ET-NEs for the development of new therapeutic approaches for TNBC.

Squalenoyl-gemcitabine/edelfosine nanoassemblies: Anticancer activity in pediatric cancer cells and pharmacokinetic profile in mice.

Despite the great advances accomplished in the treatment of pediatric cancers, recurrences and metastases still exacerbate prognosis in some aggressive solid tumors such as neuroblastoma and osteosarcoma. In view of the poor efficacy and toxicity of current chemotherapeutic treatments, we propose a single multitherapeutic nanotechnology-based strategy by co-assembling in the same nanodevice two amphiphilic antitumor agents: squalenoyl-gemcitabine and edelfosine. Homogeneous batches of nanoassemblies were easily formulated by the nanoprecipitation method. Their anticancer activity was tested in pediatric cancer cell lines and pharmacokinetic studies were performed in mice. In vitro assays revealed a synergistic effect when gemcitabine was co-administered with edelfosine. Squalenoyl-gemcitabine/edelfosine nanoassemblies were found to be capable of intracellular translocation in patient-derived metastatic pediatric osteosarcoma cells and showed a better antitumor profile than squalenoyl-gemcitabine nanoassemblies alone. The intravenous administration of this combinatorial nanomedicine in mice exhibited a controlled release behavior of gemcitabine and diminished edelfosine plasma peak concentrations. These findings make it a suitable pre-clinical candidate for childhood cancer therapy.

Transferrin-conjugated polymeric nanomedicine to enhance the anticancer efficacy of edelfosine in acute myeloid leukemia.

In this study, transferrin (Tf)-conjugated polyethylene glycol (PEG)-poly-l-lysine (PLL)-poly(lactic-co-glycolic acid) (PLGA) (PEG-PLL-PLGA)-based micellar formulations were successfully prepared for the delivery of edelfosine (EDS) in leukemia treatment. The micelles were nanosized and presented spherical shaped particles. Our in vitro data suggest that the nanoformulations maintain the biological activity of drugs for longer periods and lead to a continuous release of active drug. The enhanced cellular uptake of EDS-TM resulted in significantly higher cytotoxic effect in K562 leukemia cells. Cell cycle analysis further demonstrated the significantly higher G2/M phase arrest of cancer cells. Immunoblot analysis clearly revealed the potential of EDS-TM in inducing apoptosis of cancer cells which could improve the anticancer efficacy in leukemia. Importantly, EDS-M and EDS-TM significantly prolonged the circulation profile of EDS throughout until 24h, indicating the potential of targeted nanoparticulate delivery system. The prolonged blood circulation potential of micellar formulations might improve the therapeutic potential of drug by increasing its bioavailability in the serum. It would be worthwhile evaluating the effects of the EDS-loaded micelles on cancer cells in vivo for clinical application.

Increased gene delivery efficiency and specificity of a lipid-based nanosystem incorporating a glycolipid.

Hepatocellular carcinoma (HCC) is the third most common cause of death related to cancer diseases worldwide. The current treatment options have many limitations and reduced success rates. In this regard, advances in gene therapy have shown promising results in novel therapeutic strategies. However, the success of gene therapy depends on the efficient and specific delivery of genetic material into target cells. In this regard, the main goal of this work was to develop a new lipid-based nanosystem formulation containing the lipid lactosyl-PE for specific and efficient gene delivery into HCC cells. The obtained results showed that incorporation of 15% of lactosyl-PE into liposomes induces a strong potentiation of lipoplex biological activity in HepG2 cells, not only in terms of transgene expression levels but also in terms of percentage of transfected cells. In the presence of galactose, which competes with lactosyl-PE for the binding to the asialoglycoprotein receptor (ASGP-R), a significant reduction in biological activity was observed, showing that the potentiation of transfection induced by the presence of lactosyl-PE could be due to its specific interaction with ASGP-R, which is overexpressed in HCC. In addition, it was found that the incorporation of lactosyl-PE in the nanosystems promotes an increase in their cell binding and uptake. Regarding the physicochemical properties of lipoplexes, the presence of lactosyl-PE resulted in a significant increase in DNA protection and in a substantial decrease in their mean diameter and zeta potential, conferring them suitable characteristics for in vivo application. Overall, the results obtained in this study suggest that the potentiation of the biological activity induced by the presence of lactosyl-PE is due to its specific binding to the ASGP-R, showing that this novel formulation could constitute a new gene delivery nanosystem for application in therapeutic strategies in HCC.

In vitro intestinal co-culture cell model to evaluate intestinal absorption of edelfosine lipid nanoparticles.

Nanotechnology is providing a new therapeutic paradigm by enhancing drug efficacy and preventing side-effects. Edelfosine is a synthetic ether lipid analogue of platelet activating factor with high antitumor activity. The encapsulation of this potent antitumor drug in lipid nanoparticles increases its oral bioavailability; moreover, it prevents the hemolytic and gastrointestinal side-effects of the free drug. The literature points towards lymphatic absorption of lipid nanoparticles after oral administration, and previous in vitro and in vivo studies stress the protection against toxicity that these nanosystems provide. The present study is intended to assess the permeability of lipid nanoparticles across the intestinal barrier. Caco-2 monoculture and Caco-2/Raji co-culture were used as in vitro models of enterocytes and Microfold cells respectively. Results showed that free drug is internalized and possibly metabolized in enterocytes. These results do not correlate with those observed in vivo when edelfosine-lipid nanoparticles were administered orally in mice, which suggests that the microfold model is not a good model to study the absorption of edelfosine-lipid nanoparticles across the intestinal barrier in vitro.

Lactosylated liposomes for targeted delivery of doxorubicin to hepatocellular carcinoma.

BACKGROUND: N-lactosyl-dioleoylphosphatidylethanolamine (Lac-DOPE) was synthesized and evaluated as a liver-specific targeting ligand via asialoglycoprotein receptors for liposomal delivery of doxorubicin. METHODS: Lactosylated liposomes encapsulating calcein (Lac-L-calcein) or doxorubicin (Lac-L-DOX) composed of egg phosphatidylcholine, cholesterol, monomethoxy polyethylene glycol 2000-distearoyl phosphatidylethanolamine, and Lac-DOPE at 50:35:5:10 (mol/mol) were prepared by polycarbonate membrane extrusion and evaluated in human hepatocellular carcinoma HepG2 cells. Cellular uptake of Lac-L-calcein was monitored by confocal microscopy and by flow cytometry. The cytotoxicity of Lac-L-DOX was evaluated by MTT assay. The pharmacokinetic properties of Lac-L-DOX were studied in normal mice, and its biodistribution and antitumor activity were studied in nude mice with HepG2 xenografts. RESULTS: The size of Lac-L-DOX was less than 100 nm and the liposomes demonstrated excellent colloidal stability. In vitro uptake of Lac-L-calcein by HepG2 cells was four times greater than that of non-targeted L-calcein. In the presence of 20 mM lactose, the uptake of Lac-L-calcein was inhibited, suggesting that asialoglycoprotein receptors mediated the observed cellular uptake. Lac-L-DOX exhibited enhanced in vivo cytotoxicity compared with the nontargeted liposomal doxorubicin (L-DOX), and its pharmacokinetic parameters indicate that Lac-L-DOX has a long blood circulation time (t(1/2) 8.73 hours). Tissue distribution and therapeutic efficacy studies in nude mice bearing HepG2 xenografts show that Lac-L-DOX had significantly stronger tumor inhibitory activity compared with L-DOX and free doxorubicin, along with a higher accumulation of drug within the tumor site and greater cellular uptake by tumor cells. CONCLUSION: These data suggest that lactosylated liposomes are promising drug delivery vehicles for hepatocellular carcinoma.

Uptake and protein targeting of fluorescent oxidized phospholipids in cultured RAW 264.7 macrophages.

The truncated phospholipids 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) are oxidation products of 1-palmitoyl-2-arachidonoyl phosphatidylcholine. Depending on concentration and the extent of modification, these compounds induce growth and death, differentiation and inflammation of vascular cells thus playing a role in the development of atherosclerosis. Here we describe the import of fluorescent POVPC and PGPC analogs into cultured RAW 264.7 macrophages and the identification of their primary protein targets. We found that the fluorescent oxidized phospholipids were rapidly taken up by the cells. The cellular target sites depended on the chemical reactivity of these compounds but not on the donor (aqueous lipid suspension, albumin or LDL). The great differences in cellular uptake of PGPC and POVPC are a direct consequence of the subtle structural differences between both molecules. The former compound (carboxyl lipid) can only physically interact with the molecules in its immediate vicinity. In contrast, the aldehydo-lipid covalently reacts with free amino groups of proteins by forming covalent Schiff bases, and thus becomes trapped in the cell surface. Despite covalent binding, POVPC is exchangeable between (lipo)proteins and cells, since imines are subject to proton-catalyzed base exchange. Protein targeting by POVPC is a selective process since only a limited subfraction of the total proteome was labeled by the fluorescent aldehydo-phospholipid. Chemically stabilized lipid-protein conjugates were identified by MS/MS. The respective proteins are involved in apoptosis, stress response, lipid metabolism and transport. The identified target proteins may be considered primary signaling platforms of the oxidized phospholipid.

In vitro and in vivo efficacy of edelfosine-loaded lipid nanoparticles against glioma.

Edelfosine is the prototype molecule of a family of anticancer drugs collectively known as synthetic alkyl-lysophospholipids. This drug holds promise as a selective antitumor agent, and a number of preclinical assays are in progress. In this study, we observe the accumulation of edelfosine in brain tissue after its oral administration in Compritol® and Precirol® lipid nanoparticles (LN). The high accumulation of edelfosine in brain was due to the inhibition of P-glycoprotein by Tween® 80, as verified using a P-glycoprotein drug interaction assay. Moreover, these LN were tested in vitro against the C6 glioma cell line, which was later employed to establish an in vivo xenograft mouse model of glioma. In vitro studies revealed that edelfosine-loaded LN induced an antiproliferative effect in C6 glioma cell line. In addition, in vivo oral administration of drug-loaded LN in NMRI nude mice bearing a C6 glioma xenograft tumor induced a highly significant reduction in tumor growth (p<0.01) 14days after the beginning of the treatment. Our results showed that Tween® 80 coated Compritol® and Precirol® LN can effectively inhibit the growth of C6 glioma cells in vitro and suggest that edelfosine-loaded LN represent an attractive option for the enhancement of antitumor activity on brain tumors in vivo.

Anticancer alkylphospholipids: mechanisms of action, cellular sensitivity and resistance, and clinical prospects.

Synthetic anticancer alkylphospholipids (APLs), such as edelfosine, miltefosine and perifosine, are a group of structurally related lipids that act on cellular membranes rather than the DNA. APLs have essentially one long hydrocarbon chain that allows easy partitioning into membrane lipid bilayers, but they resist catabolic degradation. APLs therefore accumulate in cell membranes and can interfere with normal lipid metabolism and lipid-dependent signal transduction. This action, often leading to apoptosis, is most effective in metabolically active, proliferating cells, such as cancer cells, but not in quiescent normal cells. This review describes the general mechanisms of APL cellular uptake and action. Most important for their biological effect are the inhibition of phosphatidylcholine synthesis, the inhibition of the MAP-kinase/ERK proliferative and phosphatidylinositol 3-kinase/ Akt survival pathways and the stimulation of the Stress-activated protein kinase/JNK pathway, which may lead to apoptosis in cancer cells. APLs are most promising in combination with conventional cancer therapies. For example, ALPs increase the cancer cell sensitivity to radiotherapy in vitro and in vivo. We highlight the clinical potential of perifosine, an orally available APL.

Synthesis and structure-activity relationship effects on the tumor avidity of radioiodinated phospholipid ether analogues.

Radioiodinated phospholipid ether analogues have shown a remarkable ability to selectively accumulate in a variety of human and animal tumors in xenograft and spontaneous tumor rodent models. It is believed that this tumor avidity arises as a consequence of metabolic differences between tumor and corresponding normal tissues. The results of this study indicate that one factor in the tumor retention of these compounds in tumors is the length of the alkyl chain that determines their hydrophobic properties. Decreasing the chain length from C12 to C7 resulted in little or no tumor accumulation and rapid clearance of the compound in tumor-bearing rats within 24 h of administration. Increasing the chain length had the opposite effect, with the C15 and C18 analogues displaying delayed plasma clearance and enhanced tumor uptake and retention in tumor-bearing rats. Tumor uptake displayed by propanediol analogues NM-412 and NM-413 was accompanied by high levels of liver and abdominal radioactivity 24 h postinjection to tumor-bearing rats. Addition of a 2-O-methyl moiety to the propanediol backbone also retarded tumor uptake significantly. A direct comparison between NM-404 and its predecessor, NM-324, in human PC-3 tumor bearing immune-compromised mice revealed a dramatic enhancement in both tumor uptake and total body elimination of NM-404 relative to NM-324. On the basis of imaging and tissue distribution studies in several rodent tumor models, the C18 analogue, NM-404, was chosen for follow-up evaluation in human lung cancer patients. Preliminary results have been extremely promising in that selective uptake and retention of the agent in tumors is accompanied by rapid clearance of background radioactivity from normal tissues, especially those in the abdomen. These results strongly suggest that extension of the human trials to include other cancers is warranted, especially when NM-404 is radiolabeled with iodine-124, a new commercially available positron-emitting isotope. The relatively long physical half-life of 4 days afforded by this isotope appears well-suited to the pharmacodynamic profile of NM-404.

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.

Phase I and pharmacokinetic study of the cytotoxic ether lipid ilmofosine administered by weekly two-hour infusion in patients with advanced solid tumors.

PURPOSE: A Phase I trial was performed to determine the dose-limiting toxicity and maximum tolerated dose, and to describe the pharmacokinetics of the alkyl-lysophospholipid, ilmofosine, when administered as a weekly 2-h infusion in patients with solid tumors. EXPERIMENTAL DESIGN: Thirty-nine patients were entered into a trial of ilmofosine administered weekly for 4 weeks followed by a 2-week rest period. Dose escalation occurred in 10 levels from 12 to 650 mg/m(2). RESULTS: Thirty-six patients were evaluable for toxicity. The median number of cycles per patient was 1 (range, 1-4). Dose-limiting gastrointestinal toxicity occurred at 650 mg/m(2) with grade 3 nausea in two patients and grade 3 vomiting and diarrhea in one patient. Grade 2 diarrhea was observed in four of six patients treated at 550 mg/m(2). In addition, two patients treated at 550 mg/m(2) and two patients treated at 650 mg/m(2) experienced a decline in performance status of two or more levels that was determined to be due to treatment. There were no tumor responses. Stabilization of disease for at least 8 weeks occurred in six patients. Plasma concentrations of ilmofosine and its sulfoxide metabolite were evaluated by high-pressure liquid chromatography. The elimination of both compounds was biexponential with terminal half-lives of approximately 40 h for ilmofosine and 48 h for the sulfoxide. The area under the concentration-time curve was dose-proportional for each compound, and there was no evidence of saturable kinetics. CONCLUSIONS: The dose-limiting toxicity of ilmofosine is gastrointestinal and the recommended dose for Phase II trials is 450 mg/m(2) as a 2-h weekly infusion. The relatively long half-life of ilmofosine and its active metabolite support the use of this intermittent schedule.

Activity, pharmacokinetics and tissue distribution of TLC ELL-12 (liposomal antitumor ether lipid) in rats with transplantable, s.c. methylnitrosourea-induced tumors.

TLC ELL-12 is a liposomal formulation of the novel antineoplastic compound 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (L-ET-18-OCH(3)). The purpose of these studies was to evaluate the activity and tissue distribution of L-ET-18-OCH(3) when administered i.v. as TLC ELL-12 to rats bearing solid tumors. Growth-inhibitory activity of L-ET-18-OCH(3) and TLC ELL-12 against methylnitrosourea (MNU)-induced tumors grown in vitro was evaluated. Female Buffalo rats were injected s.c. with transplantable MNU-induced tumor cells. Four days later, animals were treated i.v. with L-ET-18-OCH(3) administered as TLC ELL-12 once daily for 5 consecutive days. Another group of MNU-tumor bearing rats was given a single 12.5 mg/kg dose of TLC ELL-12 containing [14C]L-ET-18-OCH(3) by i.v. injection into a tail vein. The 50% growth inhibitory concentration for TLC ELL-12 against MNU tumor cells in vitro was 63 microM (about 30 microg/ml). Tumor growth was significantly inhibited in ELL-12-treated rats versus controls. After a single dose, whole blood L-ET-18-OCH(3) concentrations declined in a multiphasic fashion with C(max) and terminal half-life values of approximately 91.1 microg L-ET-18-OCH(3)/ml and 13.1 h, respectively. Tumor L-ET-18-OCH(3) levels increased through the first 16-24 h post-dosing to about 23 microg/g and remained elevated at the terminal time point with little evidence of metabolism. Concentration-time profiles for selected tissues indicate rapid distribution of L-ET-18-OCH(3) from the circulation into tissues with highest concentrations in spleen, liver, lungs, kidneys and gastrointestinal tract. L-ET-18-OCH(3) as TLC ELL-12 shows both in vitro and in vivo activity against the MNU tumor line. When i.v. administered, L-ET-18-OCH(3) from ELL-12 is well distributed and slowly eliminated by metabolism in tissues.

Lem3p is essential for the uptake and potency of alkylphosphocholine drugs, edelfosine and miltefosine.

The alkylphosphocholine class of drugs, including edelfosine and miltefosine, has recently shown promise in the treatment of protozoal and fungal diseases, most notably, leishmaniasis. One of the major barriers to successful treatment of these infections is the development of drug resistance. To understand better the mechanisms underlying the development of drug resistance, we performed a combined mutant selection and screen in Saccharomyces cerevisiae, designed to identify genes that confer resistance to the alkylphosphocholine drugs by inhibiting their transport across the plasma membrane. Mutagenized cells were first selected for resistance to edelfosine, and the initial collection of mutants was screened a second time for defects in internalization of a short chain, fluorescent (7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD))-labeled phosphatidylcholine reporter. This approach identified mutations in a single gene, YNL323W/LEM3, that conferred resistance to alkylphosphocholine drugs and inhibited internalization of NBD-labeled phosphatidylcholine. Loss of YNL323W/LEM3 does not confer resistance to N-nitroquinilone N-oxide or ketoconazole and actually increases sensitivity to cycloheximide. The defect in internalization is specific to NBD-labeled phosphatidylcholine and phosphatidylethanolamine. Labeled phosphatidylserine is internalized at normal levels in lem3 strains. LEM3 is a member of an evolutionarily conserved family and has two homologues in S. cerevisiae. Single point mutations that produce resistance to alkylphosphocholine drugs and inhibition of NBD-labeled phosphatidylcholine internalization were identified in several highly conserved domains. These data demonstrate a requirement for Lem3p expression for normal phosphatidylcholine and alkylphosphocholine drug transport across the plasma membrane of yeast.

Alkyl-lysophospholipid resistance in multidrug-resistant Leishmania tropica and chemosensitization by a novel P-glycoprotein-like transporter modulator.

Drug resistance has emerged as a major impediment in the treatment of leishmaniasis. Alkyl-lysophospholipids (ALP), originally developed as anticancer drugs, are considered to be the most promising antileishmanial agents. In order to anticipate probable clinical failure in the near future, we have investigated possible mechanisms of resistance to these drugs in Leishmania spp. The results presented here support the involvement of a member of the ATP-binding cassette (ABC) superfamily, the Leishmania P-glycoprotein-like transporter, in the resistance to ALP. (i) First, a multidrug resistance (MDR) Leishmania tropica line overexpressing a P-glycoprotein-like transporter displays significant cross-resistance to the ALP miltefosine and edelfosine, with resistant indices of 9.2- and 7.1-fold, respectively. (ii) Reduced expression of P-glycoprotein in the MDR line correlates with a significant decrease in ALP resistance. (iii) The ALP were able to modulate the P-glycoprotein-mediated resistance to daunomycin in the MDR line. (iv) We have found a new inhibitor of this transporter, the sesquiterpene C-3, that completely sensitizes MDR parasites to ALP. (v) Finally, the MDR line exhibits a lower accumulation than the wild-type line of bodipy-C(5)-PC, a fluorescent analogue of phosphatidylcholine that has a structure resembling that of edelfosine. Also, C-3 significantly increases the accumulation of the fluorescent analogue to levels similar to those of wild-type parasites. The involvement of the Leishmania P-glycoprotein-like transporter in resistance to drugs used in the treatment of leishmaniasis also supports the importance of developing new specific inhibitors of this ABC transporter.

Intracellular triggering of Fas, independently of FasL, as a new mechanism of antitumor ether lipid-induced apoptosis.

Antitumor ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH(3); edelfosine) induces apoptosis in cancer cells, sparing normal cells. We have found that the apoptotic action of ET-18-OCH(3) required drug uptake and Fas in the target cell. Failure to accomplish one of these requirements prevents cell killing by the ether lipid. In human lymphoid leukemic cells, ET-18-OCH(3) does not promote Fas or FasL expression and ET-18-OCH(3)-induced apoptosis is not inhibited by pre-incubation with an anti-Fas blocking antibody that abrogates cell killing mediated by Fas/FasL interactions. ET-18-OCH(3)-resistant normal human Fas-positive fibroblasts do not incorporate ET-18-OCH(3), but undergo apoptosis upon ET-18-OCH(3) microinjection. Murine fibroblasts L929 and L929-Fas, stably transfected with human Fas cDNA, do not incorporate ET-18-OCH(3) and are resistant to its action when added exogenously. Microinjection of ET-18-OCH(3) induces apoptosis in L929-Fas cells, but not in wild-type L929 cells. Confocal laser scanning microscopy shows that ET-18-OCH(3) induces Fas clustering and capping during triggering of ET-18-OCH(3)-induced apoptosis. Microinjection-induced apoptosis and Fas clustering are specific for the molecular structure of ET-18-OCH(3). Our data indicate that ET-18-OCH(3) induces apoptosis via Fas after the ether lipid is inside the cell, and this Fas activation is independent of the interaction of Fas with its natural ligand FasL. This explains the selective action of ET-18-OCH(3) on tumors since only cancer cells incorporate sufficient amounts of the drug.

Decreased sensitivity to 1-O-octadecyl-2-O-methyl-glycerophosphocholine in MCF-7 cells adapted for serum-free growth correlates with constitutive association of Raf-1 with cellular membranes.

We have previously shown that inhibition of MCF-7 cell proliferation by 1-O-octadecyl-2-O-methyl-glycerophosphocholine (ET-18-OCH3) is linked to a drug-induced decrease in membrane Raf-1 levels and the subsequent inhibition of mitogen-activated protein (MAP) kinase activation in response to growth factor stimulation. We now report that adaptation of MCF-7 cells for growth in a serum-free formulation results in decreased sensitivity to growth inhibition by ET-18-OCH3. The decrease in ET-18-OCH3 sensitivity occurred progressively during the adaptation process and correlated with the presence of increasing amounts of inactive Raf-1 that stably associated with MCF-7 cell membranes. ET-18-OCH3 sensitivity could be restored by growing the adapted cells in serum-containing medium, which resulted in the loss of membrane-associated Raf-1. In human normal mammary epithelial cells, which are insensitive to ET-18-OCH3, Raf-1 was also associated with membranes in quiescent cells. In both cell types, incubation with ET-18-OCH3 had no effect on the membrane-Raf-1 levels, suggesting that ET-18-OCH3-induced reduction of Raf-1 levels in growth factor-stimulated MCF-7 cells is due to inhibition of Raf translocation. The activation and termination of the MAP kinase pathway in response to growth factors in the adapted MCF-7 cells and HNME cells occurred without changes to membrane Raf-1 levels. Because membrane translocation is not required to activate Raf in these cells, inhibition of Raf translocation by ET-18-OCH3 subsequent to cell stimulation has no effect on the activation of the membrane-bound Raf and, consequently, the activation of the MAP kinase pathway. The ability of the cells to activate the MAP kinase pathway in the presence of the drugs enables them to resist the growth-inhibitory effects of the drug, leading to the observed ET-18-OCH3 insensitivity of the cells.

Intravitreal pharmacokinetics in rabbits of the foscarnet lipid prodrug: 1-O-octadecyl-sn-glycerol-3-phosphonoformate (ODG-PFA).

PURPOSE: To evaluate the intraocular distribution and metabolism of the lipid prodrug of foscarnet, 1-O-octadecyl-sn-glycerol-3- phosphonoformate (ODG-PFA), following intravitreal administration. METHODS: Twenty rabbits received ODG-[14C]PFA intravitreal injection, yielding 0.632 mM resultant intravitreal concentration. Two animals per group were sacrificed at different intervals post-injection. The drug levels in ocular tissues were determined with counting the radioactivity by Tracor Mark III Liquid Scintillation Counter. Four rabbits were used for analysis of the drug metabolism in vitreous by lipid extraction technique. RESULTS: The drug level in vitreous was 526 microM at day one and 227 microM at the fifth week. The vitreous half life was approximately four to five weeks. The retinal level of the drug was 292 microM at day one, 75 microM at the fifth week and 32 microM at the tenth week, which was still more than ten times higher than the IC90 against HCMV. Lipid extraction analysis showed that, in vivo, both ODG-PFA and PFA were present in vitreous, but in in vitro incubations with vitreous, ODG-PFA conversion to PFA was negligible. CONCLUSION: ODG-PFA possesses a long vitreous half life and sustained high drug level in retina. The vitreous did not metabolize drug but acted as a drug reservoir. Intravitreal liposomal ODG-PFA may be expected to be a long acting potent local therapy for CMV retinitis.

Undifferentiated HL-60 cells internalize an antitumor alkyl ether phospholipid more rapidly than resistant K562 cells.

In this study, we confirmed a previous finding that 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (methyl-PAF) expresses higher antineoplastic activity against the promyelocytic leukemia cell line HL-60, than against the erythroleukemic cell line K562, and intended to clarify the reason for this. Using an albumin back-exchange method, we measured the rates of binding and internalization of [3H]methyl-PAF by HL-60 and K562 cells. We found that methyl-PAF associated very rapidly and to similar extents with the two types of cells at low concentrations of extracellular bovine serum albumin, but that when bound to the cell surface, it was internalized into HL-60 cells faster than into K562 cells. The internalization of methyl-PAF by HL-60 cells was concentration-independent, intracellular ATP-independent and susceptible to thiol group-modifying reagents and cytochalasin B. Thus the inward transbilayer movement of methyl-PAF seems to occur by cytochalasin B-sensitive protein-mediated mechanism based on passive diffusion not requiring energy, in which SH-groups of protein play a critical role. We also found that the internalization of 1-hexadecanoyl-2-(4,4-difluoro-5,7- dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphocholine (Bodipy-C5-PC), whose structure resembles that of methyl-PAF, into HL-60 cells was faster than that into K562 cells. Using a combination of an albumin back-exchange method and observation by confocal laser scanning microscopy, we next examined the intracellular distribution of this fluorescent phospholipid probe after its internalization. Intracellular membranes, especially those peripheral to nuclei, were fluorescence-labeled in both HL-60 and K562 cells, but fluorescence of the nuclear membranes was weak, suggesting that this probe seems mainly to accumulate in intracellular granules, and may interact directly with several key enzymes for phospholipid metabolism, leading to cell injury. Because the difference between the internalization rates of methyl-PAF in HL-60 and K562 cells was correlated with their different susceptibilities to the cytotoxic effect of methyl-PAF, we suggest that the capacities for uptake of methyl-PAF and its accumulation in intracellular membranes are critical factor for its induction of apoptosis. (c) 1998 Elsevier Science B.V.

Functional interactions between synthetic alkyl phospholipids and the ABC transporters P-glycoprotein, Ste-6, MRP, and Pgh 1.

The ABC superfamily of transporters includes the mammalian P-glycoprotein family (Class I and Class II P-gps), the multidrug resistance-associated protein (MRP), the Pgh-1 product of Plasmodium falciparum gene pfmdr1, all of which are associated with cellular pleiotropic drug resistance phenomena. STE6, the yeast transporter for the farnesylated peptide pheromone a, is also a member of this family. Structural similarities in this family translate into functional homology as expression of mouse Mdr3S (P-gp), P. falciparum Pgh-1, and human MRP partially restore mating in a sterile yeast mutant lacking a functional STE6 gene. The demonstration that Class II P-gps function as phosphatidylcholine (PC) translocators raise the possibility that other ABC transporters may also interact with physiological lipids. We report the identification of the synthetic lipid and PC analog ET-18-OCH3 (edelfosine) as a substrate for not only Class II P-gp but also for Class I P-gps and surprisingly for the other ABC transporters MRP, Pgh-1, and STE6. Expression of these proteins in the yeast Saccharomyces cerevisiae JPY201 was found to confer cellular resistance to cytotoxic concentrations of this lipid by a factor of 4-20-fold in a growth inhibition assay. The noted activity of ABC transporters toward this synthetic lipid was specific as a mutant variant of Mdr3 (Mdr3F) with reduced activity could not convey cellular resistance to ET-18-OCH3. ET-18-OCH3 was also found capable of blocking a-peptide pheromone transport and STE6 complementation by these ABC proteins. The inhibitory effect of ET-18-OCH3 on cell growth and a-factor transport could be abrogated by incubation with the lipid acceptor protein BSA or by enzymatic cleavage by microsomal alkylglycerol mono-oxygenase (MAMO). MAMO and BSA reversal of the ether lipid effect was only seen in the presence of a functional transporter. These results suggest that the group of cytotoxic synthetic PC analogs studied reveal possible structural and functional aspects common to the ABC transporters tested. Furthermore, the studies with BSA and MAMO suggest that the mechanism of transport of ET-18-OCH3 by these ABC transporters may be related to the flippase mechanism of PC transport by Mdr2.