N-trifluoroacetyladriamycin-14-valerate, an analog with greater experimental antitumor activity and less toxicity than adriamycin.

N-Trifluoroacetyladriamycin-14-valerate (AD 32), an analog of adriamycin, exhibits significantly greater antitumor activity than does adriamycin or daunorubicin in two experimental mouse tumor systems under similar assay conditions (C57BL X DBA/2 F1 male mice, agents administered i.p. each day for Days 1 to 4). Against the P388 leukemia at optimal dosages, AD 32 gave a +429% increase in median life-span with 3 of 5 60-day survivors compared to +132% for adriamycin (no 30-day survivors). In the L1210 leukemia system, AD 32 at several dosages consistently and reproducibly effected an increase in lifespan in excess of 445%, with a high percentage of 60+-day survivors compared to adriamycin (+42 to +54% ILS; no 30-day survivors). The reduced toxicity of AD 32 was evidenced by its optimal dose range, which is significantly greater than the lethal dose for 100% of mice of adriamycin, and by its lack of delayed toxicity. In vitro, AD 32 was somewhat less effective than was adriamycin in inhibiting the growth of CCRF-CEM cells; enzymatic conversion of AD 32 by cell-free culture medium was not observed. The unique growth-inhibitory properties of this analog indicate that the therapeutic effectiveness of the anthracycline antitumor antibiotics can be retained or enhanced by substitution on the glycosidic amino group.

Differences in cellular uptake and cytofluorescence of adriamycin and N-trifluoroacetyladriamycin-14-valerate.

Adriamycin-specific fluorescence appears slowly in living cells and is localized in nuclei and chromosomes. N-Trifluoroacetyladriamycin-14-valerate, a recently synthesized adriamycin analog, differs from the parent anthracycline in the rapid appearance of its fluorescence in the cytoplasm of living cells and the lack of any fluorescent binding to nuclei and chromosomes.

Membrane lipid modification and sensitivity of leukemic cells to the thioether lipid analogue BM 41.440.

Since the ether lipid anticancer drugs are membrane targeted, we examined the effect of membrane lipid structural alteration on their cytotoxicity. Enrichment with docosahexaenoic acid increased the sensitivity to the thioether lipid BM 41.440, compared to control cells enriched with oleic acid. The effect was dependent upon drug concentration, time, and the extent of cellular fatty acid enrichment. Other polyunsaturated fatty acids had a similar effect, which was proportional to the degree of unsaturation of the molecule inserted. Depletion of cellular glutathione with buthionine sulfoximine increased the sensitivity to ether lipid, but prooxidants such as Fe2+ and antioxidants such as vitamin E had little effect. The addition of serum to the incubation medium markedly diminished the cytotoxicity of ether lipids for cells modified with both docosahexaenoic acid and oleic acid, probably due to binding of the drug to serum components. The toxicity of another ether lipid, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, was not affected appreciably by membrane alteration. Drug uptake studies with a radiolabeled BM 41.440 analogue, 1-[3H]hexadecylthio-2-ethyl-rac- glycero-3-phosphocholine, demonstrated no difference in transport at early time points and no difference in accumulation up to 60 min. We conclude that increases in cellular and/or membrane fatty acid polyunsaturation heighten the cytotoxic effect of a membrane-active ether lipid. The effect is not due to a change in drug transport or accumulation. It may be related to a change in oxidative events. These observations provide further confirmation of the membrane being the target of ether lipid action, using biochemical rather than morphological techniques. Most importantly, this observation offers a potential innovative approach to therapy.

Cytotoxic interactions of heat and an ether lipid analogue in human ovarian carcinoma cells.

Ether lipid (EL) analogues of platelet activating factor are known to have a cell membrane-mediated antitumor activity. Although previous studies demonstrated additive interactions with EL and conventional DNA-interacting chemotherapeutic agents, little is known about the interaction of EL with heat. In this study, the cytotoxic interaction of one EL analogue, ET-18-OMe, with heat was measured at two different temperatures, 42 and 44 degrees C, using BG-1 human ovarian carcinoma cells. When the number of colonies, greater than or equal to 40 microns in diameter, was counted as a function of incubation time, the rate of colony formation was suppressed by treatment with ET-18-OMe alone at doses greater than or equal to 2.0 microM or with heat alone. The combination of ET-18-OMe with heat inhibited the colony formation of the slowest growing fraction of the heated cells. The dose-response curve for BG-1 cells after continuous exposure to ET-18-OMe alone was exponential with a small shoulder (Dq = 0.25 microM). The T0 value (the time to reduce survival on the exponential portion of the curve by a factor of 1/e) of the 44 degrees C dose-response curve (30 min) was reduced to half (15 min) by the addition of 0.25 to 1.0 microM ET-18-OMe, but increased again to 24 min when heat was combined with ET-18-OMe concentrations greater than or equal to 2.0 microM. The thermotolerant tail seen in the dose-response curve after continuous heating at 42 degrees C was removed by adding as little as 0.25 microM ET-18-OMe. Isobologram analysis for the combined treatments with 44 degrees C heat and ET-18-OMe at surviving fractions of 0.5, 0.3, 0.1, and 0.01 showed that the treatments were supraadditive at low concentrations (less than 0.5 microM) of ET-18-OMe and additive at moderate concentrations (0.5 to 1.0 microM) of ET-18-OMe. Similarly, the interaction of ET-18-OMe with 42 degrees C heat at surviving fractions of 0.3 and 0.1 was supraadditive at low concentrations (less than 0.5 microM) of the ET-18-OMe and additive with moderate concentrations (0.5 to 1.5 microM) of ET-18-OMe. Because the greatest interaction of ET-18-OMe and heat occurred at clinically achievable doses of both agents, this combination of agents should be considered for use in clinical trials.

In vitro antiproliferative activity of combinations of ether lipid analogues and DNA-interactive agents against human tumor cells.

Ether lipid analogues of platelet-activating factor (1-octadecyl-2-acetyl-sn-glycero-3-phosphocholine) possess a wide range of biological activities, including inhibition of neoplastic cell growth in vitro and in vivo. This activity is believed to be membrane mediated. Three different ether lipid analogues, 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine, 1-thiohexadecyl-2-ethyl-rac-glycero-3-phosphocholine, and 4-amino-methyl-1-[2,3-(di-n-decyloxy)-n-propyl]-4-phenylpiperidine , were combined with three DNA-interactive drugs, Adriamycin, 4-hydroperoxycyclophosphamide, and cisplatin, in the expectation that combinations of drugs with different mechanisms of action might show enhanced antitumor activity. The in vitro antiproliferative activity of the combinations was measured with a semisoft agarose clonogenic assay of an ovarian adenocarcinoma cell line. Various permutations of drug combinations were studied. Isobologram analyses and different treatment schedules were performed. Enhanced antiproliferative activity was found with combinations of ether lipids with DNA-interactive drugs in comparison with single agents. Statistical evaluation of the data indicated that the increase in activity was due to an additivity phenomenon. Neither synergism nor antagonism was found.

Correlation of ether lipid content of human leukemia cell lines and their susceptibility to 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine.

A number of synthetic ether-linked phospholipids are selectively cytotoxic to neoplastic cells. However, the mechanisms underlying this selective cytotoxicity are not known. We have investigated the ether-lipid content of HL-60 and K562 human leukemia cells in relation to their sensitivity to 1-O-alkyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3). HL-60 cells are much more sensitive than K562 cells to the cytotoxic effects of ET-18-OCH3 and, at the same time, they contain nearly twice as much ether lipid as the more resistant K562 cells. These observations suggested a relation between the cellular ether-lipid content and sensitivity to ET-18-OCH3. Further evidence linking these properties was obtained when the ether-lipid content of K562 cells was increased by incubating them in medium containing 1-O-hexadecyl-sn-glycerol. This supplementation not only increased the ether-lipid content of the cells but also increased their sensitivity to ET-18-OCH3. The 50% inhibitory concentration for ET-18-OCH3 decreased from 18.4 microM in the control cells to 9.83 microM in the supplemented cells.

Inhibition of the signalling enzyme phosphatidylinositol-3-kinase by antitumor ether lipid analogues.

Phosphatidylinositol-3-kinase (PtdIns-3-kinase) is an enzyme found associated with many growth factor receptor protein tyrosine kinases and oncogene protein tyrosine kinases. PtdIns-3-kinase appears to be important for mitogenesis and the malignant transformation of cells. The antitumor ether lipid analogue, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3), was found to be an inhibitor of Swiss mouse 3T3 fibroblast and bovine brain PtdIns-3-kinases. The concentration of ET-18-OCH3 causing 50% inhibition (IC50) was 35 microM. The inhibition of PtdIns-3-kinase by ET-18-OCH3 was noncompetitive with ATP. Other antitumor ether lipid analogues also inhibited PtdIns-3-kinase. The cyclic ether lipid analogue (+/-)-2-(hydroxy[tetrahydro-2-(octadecyloxy)methylfuran-2- yl]methoxyl)phosphinyloxy,N,N,N-trimethyethaniminium hydroxide inhibited with an IC50 of 42 microM and hexadexylphosphocholine with an IC50 of 48 microM. 1-O-Octadecyl-2-O-methyl-rac-3-glycerophospho-myo- inositol was a weaker inhibitor of PtdIns-3-kinase, with an IC50 of 96 microM and was itself phosphorylated by the enzyme. Lipid extracted from cells grown with ET-18-OCH3 for 18 h showed inhibition of PtdIns-3-kinase with endogenous PtdIns as substrate, with an ET-18-OCH3 IC50 of 18 microM. ET-18-OCH3 inhibited platelet-derived growth factor-stimulated phosphatidylinositol-3-phosphate formation by v-sis NIH 3T3 cells with an IC50 of 12.5 microM. The results of the study suggest that inhibition of PtdIns-3-kinase might contribute to the antiproliferative activity of the antitumor ether lipid analogues.

Selective inhibition of phosphatidylinositol phospholipase C by cytotoxic ether lipid analogues.

The ether lipid analogue 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) has been shown to be a direct inhibitor of Swiss 3T3 fibroblast and BG1 ovarian adenocarcinoma cell cytosolic phosphoinositide selective phospholipase C (PIPLC) using [3H]-phosphatidylinositol-(4, 5)-bisphosphate ([3H]PIP2) as the substrate. The inhibition occurred when ET-18-OCH3 was incorporated into the [3H]PIP2 substrate micelles, with 50% inhibition (IC50) occurring at a ET-18-OCH3: [3H]PIP2 ratio of 0.04, or an assay concentration of 0.4 microM, and when ET-18-OCH3 was added directly to the incubation, with an IC50 of 9.6 microM. Lipid prepared from cells exposed to cytotoxic concentrations of ET-18-OCH3 for 18 h also inhibited PIPLC with an IC50 less than 1 microM. The noncytotoxic analogue 1-O-alkyl-2-hydroxy-sn-glycero-3-phosphocholine inhibited PIPLC when incorporated into the [3H]PIP2 substrate micelles, but lipid from cells grown with 5 microM 1-O-alkyl-2-hydroxy-sn-glycero-3-phosphocholine did not inhibit PIPLC. BG1 cells, which were more sensitive than Swiss 3T3 fibroblasts to growth inhibition by ET-18-OCH3, had a cytosolic PIPLC activity one-third that of Swiss 3T3 cells. NIH 3T3 cells exhibited the same sensitivity to growth inhibition by ET-18-OCH3 as Swiss 3T3 cells and had a similar level of PIPLC. v-sis NIH 3T3 cells were relatively resistant (greater than 3-fold) to growth inhibition by ET-18-OCH3 and had a cytosolic PIPLC activity more than twice that of the wild type cells. ET-18-OCH3 was a weak inhibitor, IC50 greater than 100 microM, of phospholipase D activity in NIH 3T3 cell membranes. In intact NIH 3T3 cells ET-18-OCH3 at cytotoxic concentrations did not inhibit phospholipase D or phosphatidylcholine-selective phospholipase C activity. The results show that the ether lipid analogues at cytotoxic concentrations are selective inhibitors of PIPLC and that the inhibition of PIPLC may be related to the growth inhibitory activity of the ether lipid analogues.

Inhibition of growth factor-dependent inositol phosphate Ca2+ signaling by antitumor ether lipid analogues.

Cytotoxic ether lipid analogues have been studied for their ability to inhibit growth factor-dependent [Ca2+]i signaling in Swiss 3T3 fibroblasts. 1-Octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) inhibited 45Ca2+ uptake and inositol(1,4,5)trisphosphate-induced 45Ca2+ release in saponin permeabilized cells with concentration producing 50% inhibition values of 55 and 360 microM, respectively. When cells were exposed to ET-18-OCH3 for 18 h before permeabilization there was selective inhibition of inositol(1,4,5)trisphosphate-induced 45Ca2+ release with a concentration producing 50% inhibition value of 20 microM, but no effect on 45Ca2+ uptake, or on 45Ca2+ release by arachidonic acid. The concentration of ET-18-OCH3 with continuous exposure to inhibit cell growth 50% was 19 microM. The ether lipid analogues 1-hexadecylthio-2-ethyl-rac-glycero-3- phosphocholine and 1-S-octadecyl-2-O-methylthiopropyl-3-N,N-dimethyl-gamma-hydroxy pro pyl ammonium iodide had effects similar to those of ET-18-OCH3 but the noncytotoxic analogue 1-alkyl-2-hydroxy-sn-glycero-3- phosphocholine was without effect. Exposure of cells to 10 microM ET-18-OCH3 produced 81% inhibition of platelet-derived growth factor-stimulated inositol phosphate formation and 66% inhibition of fluoroaluminate anion-stimulated inositol phosphate formation. Addition of ET-18-OCH3 to cells in medium with 10% fetal calf serum gave a transient increase in [Ca2+]i without causing an increase in resting [Ca2+]i, while the addition of ET-18-OCH3 to cells in medium without serum gave a sustained increase in resting [Ca2+]i. Cells exposed to 5 microM ET-18-OCH3 for 18 h showed no increase in resting [Ca2+]i but there was 95% inhibition of the [Ca2+]i response to platelet-derived growth factor, 63% inhibition of the response to bradykinin, and 55% inhibition of the response to vasopressin. The block by ether lipid analogues of inositol phosphate-mediated [Ca2+]i signaling suggests a mechanism for preventing the action of growth factors that could contribute to the inhibition of cell proliferation by the agents.

Effects of antineoplastic ether lipids on model and biological membranes.

Differential scanning calorimetry and electron spin resonance were utilized to measure the effects of di-ether glycerophospholipid analogs (EL) on the physical properties of model membranes and on the membrane fluidity of HL60 leukemic cells. 1-Octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OMe) and 1-thiohexadecyl-2-ethyl-rac-glycero-3-phosphocholine (ET-16S-OEt) lower the transition temperature of dimyristoylphosphatidylcholine vesicles in a range of concentrations between 0.5 and 15 mol %. Studies conducted on the interaction of EL with a wide spectrum of different phospholipids, namely dipalmitoylphosphatidylcholine, 1-hexadecyl-2-palmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, and dielaidoylphosphatidylethanolamine confirmed the ability of EL to effect the physical properties of model membranes. Changes in calorimetric enthalpy were observed only with phosphatidylethanolamine-containing phospholipids. ET-18-OMe and ET-16S-OEt increased the membrane fluidity of HL60 leukemic cells labeled with the fatty acid spin label probe 5-nitroxystearate. These data demonstrate the ability of EL to partition into phospholipidic domains and to change their physical properties. Furthermore, they affect the membrane fluidity of whole cells. These effects indicate an interaction between EL and the plasma membrane which may be of importance in determining the cytotoxic activity against tumor cells exerted by EL.

Pharmacokinetics of actinoymcin D in patients with malignant melanoma.

The distribution and excretion of tritiated actinomycin D have been determined in 3 adult patients with disseminated malignant melanoma. In the blood, the drug was preferentially taken up into nucleated cells. The urinary and fecal excretion was prolonged and only about 30 per cent of the dose of actinomycin was recovered in 9 days. There was evidence that the drug was concentrated in bone marrow and tumor cells, but did not readily cross the blood-brain barrier. The long tissue half-lige of actinomycin D suggeststhat an intermittenr schedule of administration would be the most effective.

Methotrexate analogs. 6. Replacement of glutamic acid by various amino acid esters and amines.

A series of methotrexate (MTX) analogs was prepared in which the glutamic acid moiety is replaced by various amino acid esters and amines. The synthetic method consisted of the reaction of 4-amino-4-deoxy-N10-methylpteroic acid with various reagents to form intermediate mixed anhydrides, which then reacted with amino acid esters or amines to give the MTX analogs. These compounds were tested for antibacterial activity against Streptococcus faecium and for antitumor activity against L1210 leukemia in mice. Several compounds showed significant antibacterial activity; the MTX homocysteinethiolactone and MTX aspartate analogs showed marginal in vivo antitumor activity.

7-substituted actinomycin D analogs. Chemical and growth-inhibitory studies.

The synthesis and biological activity of three 7-substituted actinomycin D derivatives are reported. Three such derivatives, 7-nitro-, 7-amino-, and 7-hydroxyactinomycin D, were synthesized via new methods which were first tested successfully with a chromophore model system. Of these, 7-nitro- and 7-aminoactinomycin D were assayed for growth inhibitory activity against mammalian cells (CCRF-CEM human lymphoblastic leukemia) in vitro and against the Ridgway osteogenic sarcoma and the L1210, P1534, and P388 murine leukemias in vivo. In these systems, the inhibitory activity of the 7-substituted analogs was comparable to actinomycin D. In two bacterial systems ( (L. casei and L. arabinosus) in vitro, on the other hand, these compounds showed inhibitory profiles which are distinctly different from actinomycin D. These studies demonstrate that substitution at the 7 position, which does not interfere with DNA binding, is capable of yielding experimental antitumor agents with significant activity against a variety of tumors.

Synthesis and evaluation of neoplastic cell growth inhibition of 1-N-alkylamide analogues of glycero-3-phosphocholine.

Previously unreported analogues of the synthetic antitumor phospholipid ET-18-OMe (1-octadecyl-2-methoxy-rac-glycero-3-phosphocholine), in which the 1-ether oxygen has been replaced by an amido group, have been prepared and evaluated for in vitro cytotoxic effects and for inhibition of protein kinase C. The title compounds exhibit cytotoxic effects against several tumor cell lines and are approximately equipotent to ET-18-OMe. The compounds were also found to inhibit protein kinase C in an in vitro assay. This work is a continuation of our previous structure-activity studies on thio-substituted derivatives of ET-18-OMe.

Synthesis of phosphocholine and quaternary amine ether lipids and evaluation of in vitro antineoplastic activity.

The in vitro antineoplastic activity of many phosphorus-containing (e.g., phosphocholines) and non-phosphorus-containing (e.g., quaternary ammonium salts) ether lipids has been evaluated in the HL-60 promyelocytic cell line. These compounds are analogues of ET-18-OMe (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine). Structural modification of 1-(alkylamido)-, -(alkylthio)-, and -(alkyloxy)propyl backbones has provided further insight into the structure-activity relationships of these lipids. In this study, a long saturated C-1 chain and a three-carbon backbone with a single short C-2 substituent were preferred. At the positively charged nitrogen of phosphocholines, fewer than three substituents caused a significant loss of activity, and substituents larger than methyl decreased activity slightly. In the nonphosphorus compounds, many nitrogen heterocycles and also a sulfonium moiety were incorporated without changing the degree of activity; however, a thiazolium group decreased activity. The most active compound, 29 [N-[3-(hexadecyloxy)-2-methoxypropyl]-3-(hydroxymethyl)pyridinium bromide], was approximately twice as active as the reference standard, ET-18-OMe, in a trypan blue dye exclusion assay.

Synthesis of sulfur analogues of alkyl lysophospholipid and neoplastic cell growth inhibitory properties.

Five sulfur-containing phospholipid analogues (compounds 1-5) of alkyl lysophospholipid (1-O-alkyl-2-O-methyl-rac-glycero-3-phosphocholine, ALP) were synthesized and tested for inhibition of neoplastic cell proliferation with two human ovarian carcinoma cell lines in a clonogenic assay and with the HL-60 promyelocytic leukemia cell line. Compared with 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OMe), the most active reference analogue, these thio analogues are at least as active against HL-60 cells, and the 1-S-hexadecyl-2-O-ethyl analogue (2) is twice as active in the clonogenic assays.

Synthesis of quaternary amine ether lipids and evaluation of neoplastic cell growth inhibitory properties.

Novel quaternary amine ether lipids have been synthesized and tested for inhibition of neoplastic cell proliferation with the HL-60 promyelocytic leukemia cell line. These compounds contain a positively charged quaternary amine functional group attached either directly to the glycerol backbone or at the end of an alkoxy chain. The biological testing has identified several analogues with activity equivalent to or greater than that exhibited by the reference compound in this assay, ET-18-OMe (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine). Among the most active analogues are compounds 11, [N,N,N-triethyl-3-(hexadecyloxy)-2-ethoxy-1-propylammonium bromide] and 22 [N-[4-[3-(hexadecyloxy)-2-ethoxypropoxy]-1-butyl]pyridinium bromide], which are approximately 3 times as active as the reference standard.

7-Amino-actinomycin D as a cytochemical probe. I. Spectral properties.

The optical absorption and fluorescence characteristics of 7-animo-actinomycin D were determined to evaluate its potential as a fluorescent cytochemical probe. At pH 7.0, the absorption maximum and fluorescence excitation maximum are both at 503 nm; the fluorescence emission is at 675 nm. When this compound forms complexes with DNA in solution, the absorption and fluorescence excitation maxima shift to 543 nm and the fluorescence emission shifts to 655 nm. The fluorescence quantum yield is 0.016 for 7-amino-actinomycin D free in solution and 0.01-0.02 for complexes with native DNA. The 7-amino-actinomycin D also exhibits fluorescence shifts characteristic of binding when put into solution with poly(dG-dC) poly(dG-dC), but not with poly(dI-dC) poly(dI-dC). The spectral characteristics are the same at pH 7.0 whether the solvent is 0.01 M PO4 with 0.0001 M EDTA or Earle's salts with 0.025 M N-2-hydroxyethylpiperazine-N1-2-ethanesulfonic acid.

Novel membrane-interactive ether lipid analogs that inhibit infectious HIV-1 production and induce defective virus formation.

A new class of membrane-active ether lipid (EL) analogs of platelet-activating factor were studied for in vitro anti-HIV-1 activity. Human T-cell (CEM-ss) monolayers or suspension cultures were used to determine effects of structural modifications of Type A phosphorus-containing and Type B nonphosphorus EL analogs on (a) the inhibitory concentration50 (IC50) for HIV-1 syncytial plaque formation and cell growth, and, (b) virus budding at the cell plasma membrane. Results indicate that representative Type A and Type B EL inhibit HIV-1 but not herpes simplex virus type 2 plaque formation when added before or up to 2 days after viral infection. Anti-HIV-1 activity does not involve direct inactivation of virus infectivity. Type A EL (IC50 range = 0.2-1.4 microM) with alkyoxy, alkylthio, or alkyamido substitution at glycerol position 1 and ethoxy or methoxy substitution at position 2, and Type B compounds (IC50 range = 0.33-0.63 microM) with an inverse choline or nitrogen heterocyclic substitution at position 3 have selective activity against HIV-1-infected T-cells. EL treatment of HIV-1-infected cells is associated with subsequent release of reverse transcriptase activity, but infectious virus production is inhibited with time after infection. Electron microscopic examination of HIV-1-infected and EL-treated cells revealed absence of detectable budding virus at the plasma membrane but presence of intracytoplasmic vacuolar virus particles. In summary, these data suggest that EL analogs are a novel class of agents that induce defective intracytoplasmic vacuolar HIV-1 formation in T-cells. Being membrane interactive, EL are ideally suited for combination chemotherapy with DNA-interactive anti-HIV nucleoside analogs.

Unidirectional membrane uptake of the ether lipid antineoplastic agent edelfosine by L1210 cells.

We have studied the cellular uptake of edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; ET-18-OCH3), a membrane active anticancer drug of the ether lipid family, by L1210 murine leukemia cells. Initial unidirectional linear uptake velocity was 1.1 nmol/min per 2 x 10(6) cells; at about 30 min it reached a steady-state phase of accumulation of approximately 5 nmol/2 x 10(6) cells. Concentration studies indicated no saturation kinetics from 0 to 40 microM. Studies with metabolic inhibitors displayed no energy dependence. There was no effect of chloroquine, monensin or cytochalasin B, which are known inhibitors of endocytosis. The inhibitory effect of lower temperature on uptake was moderate in extent and compatible with passive diffusion. There was no efflux of drug from preloaded cells which indicates intense binding of incorporated drug to cells. In human serum, edelfosine bound to several protein components, primarily high density lipoprotein and albumin, and this may explain why cellular uptake was slowed considerably by the presence of serum or albumin in the incubation medium. We conclude that the lipophilic ether lipid derivative edelfosine is taken up by passive diffusion by the L1210 cell. It is tightly bound to cellular structures, probably by insertion into the membrane lipid bilayer.