Stereospecific synthesis of antitumor active thioether PAF analogs

A novel stereospecific synthesis of antitumor active thioether analogs of platelet-activating factor (PAF) is reported. The synthesis is based upon: i) the use ofD-serine to provide the chiral center for the construction of the optically active phospholipid molecule; ii) development of thesn-1-thioalkyl function via thioacetate displacement of methanesulfonate-activated primary hydroxyl group followed by alkylation of thesn-1-thiolate function; and iii) introduction of the phosphocholine moiety through the 2-chloro-2-oxo-1,3,2-dioxaphospholane/trimethylamine sequence. The entire scheme relies on the use of a single protecting group. The synthetic thioether phospholipid 1-S-hexadecyl-2-N-acetamidodeoxy-sn-glycero-3-phosphocholine has been shown to be a potent antitumor active phospholipid, exhibiting tumor cytotoxicity against a lymphoblastoid lymphoma (Li-A) cell line and a malignant histiocytic (DHL-4) cell line of human origin at the same level of potency as ET-18-OMe and 1-O-octadecyl-2-N-acetamidodeoxy-sn-glycero-3-phosphocholine. The synthetic method described has a great deal of flexibility, providing a convenient general route to a wide range of thioether PAF analogs. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Cytotoxic effects of ether lipids and derivatives in human nonneoplastic bone marrow cells and leukemic cells in vitro

The effects of 2-lysophosphatidylcholine (2-LPC), the alkyl lysophospholipid derivatives (ALP) 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃) and 1-O-hexadecyl-sn-glycero-3-phospho-trimethyl-ammonio-hexanol, the 2-acetamide analog of platelet-activating factor (PAF) 1-O-octadecyl-2-acetamide-sn-glycero-3-phosphocholine, the thioether lysophospholipid derivative (TLP) BM 41.440 and the ether-linked lipoidal amine CP-46,665 on tritiated thymidine uptake and trypan blue dye exclusion were tested in vitro in various freshly explanted cell samples from human nonneoplastic bone marrow and human leukemias. In both assay systems, a dose range of 1–20 μg/ml of the compounds was tested after 24, 48 and 72 hr of coincubation with the cells. The trypan blue dye exclusion revealed statistically significant preferential cytotoxicity in leukemic cells for three compounds with the order of quantitative selectiveness: ET-18-OCH₃>BM41.440>2-acetamide analog of PAF. CP-46,665 was the most toxic compound, but did not reveal significant differences between nonneoplastic bone marrow and leukemic cells when added in concentrations greater than 1 μg/ml. The trimethyl-ammoniohexanol compound showed only minor activity in the majority of tests, when added at concentrations <20 μg/ml. 2-LPC was rather ineffective. The tritiated thymidine uptake showed only preferential antiproliferative effects towards leukemic cells of ET-18-OCH₃ and, sometimes, within the dose time frame tested of BM 41.440. All compounds tested except 2-LPC and the trimethyl-ammonio-hexanol compound were active also in this assay (inhibition of uptake>50% of the controls). Based on these results, ET-18-OCH₃ and BM 41.440 are recommended for experimental bone marrow purging.     

The degradation of platelet-activating factor and related lipids: Susceptibility to phospholipases C and D

1-O-Octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃) is an ether-linked lipid that exhibits selective cytotoxicity toward several types of tumor cells and is relatively inactive toward normal cells under the same conditions of treatment. The mechanism of this selective cytotoxicity is unknown. We conducted studies to determine whether this compound is metabolized by phospholipases C and D and, if so, whether sensitive and resistant cells differ in their ability to degrade ET-18-OCH₃ by these enzymes. We have examined the metabolism of the L-isomer of ET-18-OCH₃, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (L-ET-18-OCH₃), by lysophospholipase D of rat liver microsomes and by a phospholipase D from the marine bacteriumVibrio damsela. The metabolism of L-ET-18-OCH₃ was also examined in cell culture using Madin-Darby canine kidney cells, human promyelocytic leukemia cells and human myelocytic leukemia cells. In these studies, L-ET-18-OCH₃ and related 1-O-alkyl-linked phosphocholine analogs radiolabeled with³H in the 1-O-alkyl chain were used. L-ET-18-OCH₃ was not hydrolyzed by lysophospholipase D from rat liver microsomes under conditions where cleavage of 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine was observed. However, phospholipase D from the marine bacteriumV. damsela readily hydrolyzed L-ET-18-OCH₃ to 1-O-[³H]octadecyl-2-O-methyl-sn-glycero-3-phosphate, demonstrating that L-ET-18-OCH₃ can be degraded by a phospholipase D. Platelet-activating factor (PAF) and lyso-PAF were also substrates for the bacterial phospholipase D. When intact cells were incubated with radiolabeled L-ET-18-OCH₃ a product was formed that was identified as 1-O-[³H]octadecyl-2-O-methyl-sn-glycerol. There are two mechanisms that could account for the appearance of this product. The first involves cleavage of the compound by a phospholipase C, resulting in direct release of the diglyceride. The second possible mechanism involves cleavage by a phospholipase D to form the phosphatidic acid analog with subsequent hydrolysis to the diglyceride by a phosphohydrolase. Preliminary data support the phospholipase C-type mechanism. Regardless of which mechanism operates in intact cells, the metabolic degradation of L-ET-18-OCH₃ does not appear to be a significant factor in the selective cytotoxicity of this antitumor agent.     

Effect of synthetic phospholipids on platelet aggregation and serotonin release

1-O-Hexadecyl-2-O-acetyl-sn-glycerol-3-phosphocholine (platelet-activating factor, PAF) is known to stimulate platelet aggregation and serotonin release in concentrations ranging from 10^?10–10^?5 M. Since a variety of synthetic PAF analogues are potent antineoplastic agents in vitro and in vivo, it was the aim of this study to examine the PAF-like activity of 15 analogues, including 1-O-actadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃) and a thioether analogue. In platelet-rich plasma from human blood, platelet aggregation and serotonin release were studied to compare the effects on PAF and the analogues. Platelet function was controlled by testing their response to adenosine diphosphate, arachidonic acid, collagen and epinephrine. Our results show that only PAF was able to induce platelet aggregation and serotonin release in concentrations from 10^?9 to 10^?5 M, whereas all the tested analogues up to a concentration of 10^?3 M failed to induce these effects.     

Structure-cytotoxicity studies on alkyl lysophospholipids and some analogs in leukemic blasts of human origin in vitro

Eleven lipids have been tested for cytotoxic (trypan blue dye exclusion) activity in cells from eight freshly explanted human leukemias in vitro. 4-Aminomethyl-1-[2,3-(di-N-decyloxy)N-propyl]-4-phenylpiperidine (CP-46,665), 1-mercapto-hexadecyl-2-methoxymethyl-rac-glycero-3-phosphocholine (BM 41.440), the 2-acetamide analog of platelet-activating factor (PAF) and 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃) were found among the most active compounds. 2-Lysophosphatidylcholine (2-LPC) showed the lowest activity. However, in addition there was variation among the results regarding the activity of the 1-octadecyl-rac-glycero-3-phosphocholine (ET-18-OH) and its D- and L-forms, but a significantly higher cytotoxic activity of D-ET-18-OH compared with L-ET-18-OH on the basis of 2-LPC as control after an incubation time of 48 hr. We conclude that with the limited number of structures available, this type of study is not sufficient to yield further information about the mode of the accumulation and toxicity of this type of lipids.     

Ether lipids inhibit the effects of phorbol diester tumor promoters

Recent studies have shown that the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulates protein kinase C (PKC), whereas the ether-linked phospholipid 1-O-octadecyl-2-O-methyl-rac-glycerol-3-phosphocholine (ET-18-OCH₃) inhibits PKC activity in vitro. Therefore, the antitumor effects of ET-18-OCH₃ could be due to its inhibition of PKC activity and the effects of tumor promotion. TPA stimulates arachidonic acid release, prostaglandin synthesis, phosphatidylcholine synthesis and the degradation of phosphatidylcholine by phospholipase C in Madin Darby canine kidney (MDCK) cells. Therefore, we have determined the effects of ET-18-OCH₃ on these consequences of TPA stimulation. Preliminary experiments determined that ET-18-OCH₃ inhibited PKC partially purified from MDCK cells by ion-exchange chromatography on DEAE-cellulose. In addition, ET-18-OCH₃, inhibited the TPA-stimulated phosphorylation of a 40,000-dalton protein in intact MDCK cells. These data indicate that ET-18-OCH₃ is an effective inhibitor of PKC activity in MDCK cells. In addition, ET-18-OCH₃ was found to inhibit arachidonic acid release and prostaglandin synthesis. The inhibition of prostaglandin synthesis appears to be secondary to inhibition of arachidonic acid release, since ET-18-OCH₃ does not inhibit TPA-stimulated synthesis of prostaglandin H synthase or the activity of the enzyme directly (Parker, J., Daniel, L. W., and Waite, M. [1987]J. Biol. Chem. 262, 5385–5393). ET-18-OCH₃ also inhibits TPA-stimulated phosphatidylcholine synthesis and phosphatidylcholine degradation by phospholipase C. These data provide evidence that the antineoplastic ether lipids inhibit the biochemical effects of the tumor promoter TPA in intact cells and indicate that this inhibition may have a role in their biological activities.     

Effect of platelet-activating factor on cortisol and corticosterone secretion by perfused dog adrenal

Administration of platelet-activating factor (PAF) to perfused adrenal increased cortisol and corticosterone secretion. With hexadecyl PAF (C₁₆PAF; 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine), the increase was significant at 1 nM and maximal at 10 nM. The responses to 10 nM octadecyl PAF (C₁₈PAF; 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine) were one fourth of those to 10 nM C₁₆PAF. The addition of C₁₆PAF to dispersed adrenal cells significantly increased cortisol and corticosterone production at 0.1 nM and 10 nM, respectively. C₁₆PAF was about 1000 times more potent than histamine on a molar basis in respect to cortisol response in both perfused adrenal and dispersed adrenal cells. The results suggest that PAF induces cortisol release from dog adrenal. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989. The present data were also reported at the VIIth International Congress on Hormonal Steroids, Madrid, Spain, September, 1986 (J. Steroid Biochem. 25, 76S, 1986, Abstract).     

Platelet-activating factor acetylhydrolase activity in human tissues and blood cells

Human tissues, blood cells, and plasma have enzymes that catalyze the hydrolysis of PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). The activities are not due to phospholipases A₂ that hydrolyze long chain acyl groups at thesn-2 position of glycerophospholipids, since they are calcium-independent and are specific for hydrolysis of short chain acyl groups. We examined the biochemical properties of these PAF acetylhydrolase activities (EC 3.1.1.47) in homogenates of human liver and spleen, in white blood cells (neutrophils and monocytes), and in erythrocytes. The data suggest that the plasma and intracellular PAF acetylhydrolase activities are likely due to different proteins. Second, the intracellular PAF acetylhydrolase activities in liver and spleen share several biochemical features that differentiate them from the activities in blood cells. Third, the activities in monocytes and neutrophils have properties that differentiate them from the activity present in human erythrocytes. Finally, the erythrocyte activity has unique properties that place it in a separate category of short chain acylhydrolases. In conclusion, there is a family of distinct enzymes that can be identified as PAF acetylhydrolases based on their calcium-independence and specificity for a short residue at thesn02 position of phospholipids. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Immunomodulatory and therapeutic properties of alkyl lysophospholipids in mice

This paper describes the immunomodulatory and therapeutic properties of the alkyl lysophospholipids [ALP; 1-O-octadecyl-2-O-rac-glycero-3-phosphocholine (ET-18-OCH₃)]. ALP was able to activate macrophages both in vitro and in vivo as well as to act as an immunoadjuvant for syngeneic tumor vaccines. However, ALP appeared to be transferred, at least in part, to the macrophage membrane, and some of the tumoricidal macrophage-activating properties seem to be associated with the direct cytotoxic effect of membrane-released ALP. ALP also had some therapeutic activity for experimental and spontaneous metastases, requiring administration three but not two times weekly at near-toxic doses; this suggests that at least some of its therapeutic activity is due to direct cytotoxicity.     

Superoxide production by macrophages stimulatedin vivo with synthetic ether lipids

The anticancer activity of synthetic ether lipids may depend in part upon their ability to activate cells of the monocyte/macrophage lineage. In the present study, we have sought to determine whether 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OMe) and related ether lipids enhance superoxide production by mouse peritoneal macrophages. Ether lipids were administered intraperitoneally to C57BL/6 mice 4 d after injection with thioglycollate broth. Elicited peritoneal macrophages were harvested and purified one day later, and superoxide production was detected by measuring the superoxide dismutase inhibitable reduction of cytochrome c. Low levels of superoxide were secreted by macrophages in the absence of phorbol 12-myristate 13-acetate (PMA). When PMA was addedin vitro to macrophages from ET-18-OMe-treated mice, these cells secreted 194.2 nmol superoxide/mg protein in comparison to 53.5 nmol superoxide/mg protein for PMA-treated control cells. Thein vitro treatment of the macrophages with ET-18-OMe was not effective in stimulating superoxide secretion. Macrophages harvested from mice treated with a series of ether lipids (with and without phosphorus) were examined, and superoxide secretion was found to vary with structure. AM-18-OEt and CP-7 were the most effective compounds, secreting 8.6 and 11.9 times more superoxide, respectively, than PMA-stimulated control cells. Moreover, direct cytotoxicity of the compounds for HL60 human promyelocytic leukemic cells did not necessarily correlate with the ability of each drug to increase macrophage superoxide production.     

Antineoplastic activity of conjugates of lipids and 1-β-d-arabinofuranosylcytosine

Five different lipid conjugates of 1-β-D-arabinofuranosylcytosine (ARA-C) were tested in comparison with ARA-C, the ether lipid ET-18-OCH₃ (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) and their equimolar mixtures. The compounds were tested in vitro for cytotoxicity in the trypan blue dye exclusion test with cells from six different leukemias, one glioblastoma and two bronchogenic carcinomas of human origin. The compounds were given in vivo to assess their therapeutic activity against 3-Lewis lung carcinoma (3-LL) of syngeneic C₅₇Bl₆ mice. Although some of the conjugates have shown cytotoxic activity in vitro against the cell samples tested, they have not revealed higher cytotoxicity than ET-18-OCH₃, ARA-C or their equimolar mixtures. In these experiments, ARA-CDP-D,L-MBA was the conjugate with the highest cytotoxicity. Some of the conjugates significantly inhibited tumor growth and also increased survival of C₅₇Bl₆ mice with intraperitoneally (ip) implanted 3-LL. In these experiments, ARA-CDP-D,L-PTBA, ARA-CDP-D,L-PBA, ARA-CDP-L-dipalmitin and ARA-CDP-D,L-PCA were more active than either the parent compounds ARA-C and ET-18-OCH₃ alone or their equimolar mixtures. Furthermore, when the conjugates were injected as adjuvant chemotherapy shortly after the surgical removal of the primary 3-LL, they inhibited the metastasis of 3-LL to the lungs of the animals, demonstrated by an increase of the survival time and the number of surviving animals. The mode of action of these new antineoplastic compounds still is speculative.     

Acylation of lyso platelet-activating factor by splenocytes of the rainbow trout,Oncorhyncus mykiss

In mammalian systems, platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, (PAF) is rapidly inactivated by a deacetylation/reacylation system that produces 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine which is highly enriched in arachidonic acid. There is some evidence that n−3 fatty acids may have an impact on this system in humans but the nature of this impact is unclear. In rainbow trout, n−3 fatty acids are known to be essential dietary components which are derived through the food chain. Substantial quantities of n−3 fatty acids are found in trout membrane phospholipids. We show here that in sharp contrast to mammalian cells, trout cells acylate lyso platelet-activating factor, alkyl-GPC, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine, (lyso-PAF) with a high degree of specificity for n−3 fatty acids. When [³H]lysoPAF was incubated with these cells, only three molecular species of alkylacylglycerophosphocholine were produced, and 92% contained n−3 fatty acids. Since isolated membranes yielded similar results, it appears that the acylation proceedsvia a coenzyme A-independent transacylase as found in mammalian systems.     

Biotransformation of alkylglycerols in plant cell cultures: Production of platelet activating factor and other biologically active ether lipids

Plant cells in culture are capable of incorporating exogenous 1-O-alkyl-sn-glycerols into various neutral and ionic ether lipids. 1-O-Alkyl-2-acyl-sn-glycerol-3-phosphocholines, the major class of compounds thus formed, are used for the preparation of platelet activating factor (PAF) in high yields. Similarly, the prochiral 2-O-alkyl-sn-glycerols are transformed to chiral 2-O-alkyl glycerophospholipids from which compounds can be obtained that exhibit antiviral activity in plant and animal cells. Reaction of 1-O-alkyl-2-acyl-sn-glycerol-3-phosphocholines with phospholipase D in the presence of ethanolamine leads to 1-O-alkyl-2-acyl-sn-glycerol-3-phosphoethanolamines, which serve as starting material, for the preparation of 1-O-alkyl-2-acyl-sn-glycero-3-phospho-(N-acyl)ethanolamines, compounds known to have antitumor activity. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989. Dedicated to Professor Morris Kates, Ottawa, on the occasion of his retirement.     

Therapeutic activity of ET-18-OCH3 and hexadecylphosphocholine against mammary tumors in BD-VI rats

The present therapy experiments with two different transplantable mammary tumors were performed to compare the therapeutic efficacy in BD-VI rats of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃) and hexadecylphosphocholine (HPC). Both compounds were administered orally, subcutaneously or intracutaneously at equimolar doses ranging from 4.8 to 88 μmol/kg/day five times per week for two weeks. Under the experimental conditions, both transplanted mammary carcinomas were moderately sensitive to the therapy with either HPC or ET-18-OCH₃. Comparing both tumors, TMA2 was more sensitive than TMA1. The activity and toxicity of both compounds were dose-related in both tumor lines. Females seemed to be less sensitive with respect to antineoplastic activity and toxicity. Like ET-18-OCH₃, HPC was active also at low, probably noncytotoxic doses associated with no detectable toxicity according to body weight development. This suggests that there are at least two different mechanisms of action that lead to tumor growth inhibition.     

Compounds biologically similar to platelet activating factor are present in stored blood components

Agents which prime the neutrophil NADPH oxidase develop during routine storae of whole blood and packed red blood cells. This plasma priming activity can be inhibited by bepafant (WEB 2170), a specific platelet activating factor (PAF) receptor antagonist. Quantitation of the priming agent(s), by a commercially available radioimmunoassay for PAF, reproducibly demonstrated high levels of PAF activity. However, analysis of these plasma samples from stored blood components by gas chromatography/mass spectroscopy did not reveal any 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine. We conclude that the polyclonal antibody to PAF used in these studies may have recognized different epitopes of a family of heterogeneous, biologically active lipids that manifest their effects through the PFA receptor.     

Transient activation of 1-O-alkyl-sn-glycero-3-phosphocholine: Acetyl-CoA acetyltransferase during the incubation of macrophages

The activity of the platelet-activating factor (PAF)-synthesizing enzyme, 1-O-alkyl-sn-glycero-3-phosphocholine (lysoPAF):acetyl-CoA acetyltransferase (EC 2.3.1.67) in alveolar macrophage lysate was found to be elevated after warming the cells to 37°C. Such an increase in enzyme activity was detectable only when intact cells were warmed. The stimulation was transient, reaching a peak at 2 min, and then gradually decreased to the control level. We could not find increased PAF formation in warmed cells which had increased acetyltransferase activity, even though substantial amounts of lysoPAF were shown to be present within cells. In contrast, considerable amounts of PAF were formed after treatments of the cells with exogenous lysoPAF. These results suggest that the activation of acetyltransferase is not sufficient to induce PAF formation and that the increased availability of substrates, especially lysoPAF, in the cells is indispensable for triggering PAF biosynthesis in this type of cells.     

Modulation of chemical carcinogenesis in rats by alkyl lysophospholipids

The influence of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃) and 1-hexadecylmercapto-2-methoxymethyl-rac-propyl-3-phosphocholine (TLP, BM41.440) on methylnitrosourea (MNU)-induced rat mammary carcinomas and of ET-18-OCH₃ on 7,12-dimethylbenzanthracene (DMBA)-induced leukemias was investigated. Both agents effectively delayed MNU-induced mammary tumor formation at high, cytotoxic dosages but TLP had no influence at low “immunomodulatory” doses. ET-18-OCH₃ also significantly protected against leukemia development in DMBA-treated Long-Evans rats.     

Synthesis of trideuteratedO-alkyl platelet activating factor and lyso derivatives

Racemic heavy isotope analogs of 1-O-alkyl-sn-glycero-3-phosphocholine (lysoPAF) and 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (PAF) were prepared for use as internal standards to facilitate quantitative studies based on mass spectrometry. Starting from pentadencane-1,15-diol andrac-glycerol-1,2-acetonide, a convergent synthesis of 1-O-[16′-²H₃]hexadecyl and 1-O-[18′-²H₃]octadecylrac-glycero-3-phosphocholine and their acetyl derivatives is described. Three deuterium atoms were introduced at the terminal position of the 1-O-alkyl group by displacement of thep-toluensulfonyl group from 1-O-alkyl-15′-p-toluensulfonate and 1-O-alkyl-17′-p-toluensulfonate with [²H₃]-methylmagnesium iodide. The 1-O-alkyl-17′-p-toluensulfonate was obtained by reaction of the 1-O-alkyl-15′-p-toluensulfonate with allylmagnesium bromide, followed by reductive ozonolysis and treatment withp-toluenesulfonyl chloride. The hydroxyl group at C-2 was protected by a benzyl group and removed at a late stage in the synthesis. This provided the corresponding lysoderivatives or allowed preparation of racemic PAF by subsequent acetylation of the free hydroxy group. The phosphocholine moiety was introduced at glycerol C-3 by reaction with bromoethyldichlorophosphate and trimethylamine. The synthetic compounds were analyzed by FAB/MS and GC/NICIMS. They were shown to contain less than 0.6% protium impurity.     

Enzymatic acylation of ether and ester lysophospholipids in rat liver microsomes

The acylation of lysophospholipids by rat liver acyltransferases was studied. A comparison between ester and ether lysophospholipids as substrates revealed large differences in substrate properties. For instance, oleic acid from oleoyl-CoA and arachidonic acid from arachidonoyl-CoA were not incorporated into 1-O-octadecyl-sn-glycero-3-phosphocholine under experimental conditions that allowed an optimal transfer of oleic acid and arachidonic acid to 1-O-palmitoyl-sn-glycero-3-phosphocholine. However, we observed an acyl-CoA-independent transfer of arachidonic acid from 1-O-stearoyl-2-O-arachidonoyl-sn-glycero-3-phosphoinositol to 1-O-octadecyl-sn-glycero-3-phosphocholine.     

Growth inhibitory effects of liposome-associated 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine

The growth inhibitory effects of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH₃) and various liposome compositions of ET-18-OCH₃ were compared in a standardized growth inhibition assay utilizing a diverse tumor cell line panel including cell lines expressing multidrug resistance. ET-18-OCH₃ and ELL-12 (4∶3∶1∶2, dioleoylphosphatidylcholine/cholesterol/dioleoylphosphatidylethanolamine-glutaric acid/ET-18-OCH₃), an optimal liposomal ET-18-OCH₃ formulation, inhibited growth in the micromolar range in drug-sensitive and-resistant cells. In general, ET-18-OCH₃-liposomes were about twofold less growth inhibitory than ET-18-OCH₃. However, the known hemolytic effects of ET-18-OCH₃ were greatly reduced, up to 20 or more times, by liposome association. The effects of ET-18-OCH₃ and ELL-12 were compared in intracellular [Ca²⁺] modulation and DNA fragmentation assays. ET-18-OCH₃ elicited both concentration- and serum-dependent transient and permanent increases in intracellular [Ca²⁺]. In contrast, ELL-12 did not modulate intracellular [Ca²⁺]. ET-18-OCH₃ and ELL-12 similarly affected DNA fragmentation, which may be indicative of apoptosis. The results suggest that, although the specific growth inhibitory effects of ET-18-OCH₃ and ELL-12 are similar, associating ET-18-OCH₃ with stable well-characterized liposomes eliminates nonspecific cell membrane-associated lytic effects.