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.     

Effect of lipid derivatives on invasion in vitro and on surface glycoproteins of three rodent cell types

The antiinvasive activity on MO₄ mouse cells of the following lipid derivatives was tested in vitro: an alkyllysophospholipid derivative (BM 41.440), an alkyl-linked lipoidal amine (CP-46,665) and a naturally occurring ester-linked phospholipid (2-LPC). In this test, BM 41.440 had the same antiinvasive potency as ET-18-OCH₃, whereas CP-46,665 and 2-LPC had no effect on invasion. Comparison of the antiinvasive effect of ET-18-OCH₃ on three types of cells showed the following ranking: 12R1C-RK rat kidney adenovirus type 12 transfected cells>MO₄ mouse cells>LLC-H61 Lewis lung carcinoma cells. This ranking was not reflected in ET-18-OCH₃-induced changes of cell surface exposed glycopeptides derived from the three types of cells metabolically labeled with radioactive fucose. The present and previous experiments suggested that changes in invasion caused by lipid derivatives depended upon relative cell surface fucosylglycopeptide alterations in both the invasive cells and the normal tissue.     

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.     

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.     

Increased membrane permeability for an antitumoral alkyl lysophospholipid in sensitive tumor cells

We have investigated cellular sensitivity to the antitumoral alkyl lysophospholipid (ALP) 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃) in vitro. The permeation of this lipid into the cell was not influenced by metabolic inhibitors of ATP biosynthesis. ET-18-OCH₃ uptake was not saturable within sublytic concentrations, but could be inhibited in part by cytochalasin B (CB) and dipyridamole. The activation energy of the CB-sensitive uptake process was increased up to threefold compared to CB-insensitive uptake. ET-18-OCH₃ influx and equilibrium binding of ET-18-OCH₃ were decreased in a fibrosarcoma cell variant (MethA) selected for ET-18-OCH₃ resistance. The resistant MethA cells were also less sensitive to cytolysis by lysophosphatidylcholine and other ALP. After 72 hr, the resistant MethA cells had metabolized only 11.8% more of the absorbed ET-18-OCH₃ than sensitive MethA cells. However, they tolerated at least a 30-fold concentration of this ALP. The uptake mechanism, which could be inhibited by CB, was less active in resistant MethA cells and several other ALP-resistant cell lines. The concentration of CB, required for maximal uptake inhibition, was increased more than four times in the ALP-sensitive tumor cell lines. CB-specific ET-18-OCH₃ uptake was also enhanced after virus transformation of 3T3 fibroblasts by SV 40. Dipyridamole retarded the ET-18-OCH₃-mediated cell destruction.     

Clinical phase I pilot study of the alkyl lysophospholipid derivative ET-18-OCH3 1

Sixteen patients suffering from widespread malignant disease, the majority pretreated and found in poor general health, were treated in a phase I pilot study with the alkyl lysophospholipid derivative 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (E-18-OCH₃). Eleven patients were treated intravenously, and five were given oral therapy. Prolonged IV administration of 15–20 mg/kg/day at a concentration of 5 mg ET-18-OCH₃ per 1 ml 20% human serum albumin could be continued safely. The maximum-tolerated dose was either 50 mg/kg as a single injection or 20 mg/kg during daily dispensions. Grade 2–4 toxicity, as pulmonary edema and impairment of hepatic function, then occurred during daily treatment. Toxicity was reversible. Mitogen stimulation and mixed lymphocyte culture studies revealed possible immunosuppressive effects of higher doses of ET-18-OCH₃. There were no chromosomal changes in cytogenetic studies. Frequent post-mortem examinations revealed no further toxicity. IV and oral treatment showed few encouraging response data since there were two partial remissions in non-small cell lung cancers and a reduction of leukemic blasts to less than 10% in an acute myelomonocytic leukemia.     

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.     

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.     

Antitumor activity of synthetic alkylphospholipids with or without PAF activity

1-O-Octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OMe) has been reported to possess definite antitumor activity in vivo. Twenty-two alkyl lysophospholipid analogs were chemically synthesized, and their antitumor activity against mouse experimental tumors (Sarcoma 180, MM46, P388) was examined. Among them, 1-O-octadecyl-2-O-acetoacetyl-rac-glycerol-3-phosphocholine was found to show antitumor activity similar to ET-18-OMe with less acute toxicity. Intravenous injection of the ET-18-OMe withsn-3 configuration retarded the subcutaneous growth of Sarcoma 180 cells effectively, while the growth inhibition by thesn-1 isomer was much less effective. This stereospecificity was similar to that observed in their activities as platelet-activating factor (PAF) agonists. The acetoacetyl compound, another PAF agonist, showed similar stereospecific antitumor action in vivo. These findings suggest that some alkyl lysophospholipids may activate host cells to a cytostatic stage against tumor cells in vivo through binding to a PAF receptor. Our preliminary results indicated that the responsible cells under these conditions might be primarily immature macrophages present in the bone marrow. No appreciable or even adverse stereospecificity was observed in the different sets of experiments where the activity of ET-18-OMe against MM46 tumor cells in vivo or the direct cytotoxicity against human promyelocytic leukemia HL-60 cells in vitro was examined. Under, some conditions, the antitumor activity of ET-18-OMe in vivo may be revealed through direct cytotoxicity and/or modulation of the host defense system by “nonspecific” mechanisms. Some alkylphospholipids without PAF activity may also show antitumor activity through similar, “nonspecific” mechanisms.     

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.     

The effect of two synthetic phospholipids on cell proliferation and phosphatidylcholine biosynthesis in Madin-Darby canine kidney cells

The concentration-dependent effects of two different synthetic phospholipids on cell proliferation and phosphatidylcholine biosynthesis were compared in Madin-Darby canine kidney (MDCK) cells. The alkyllysophospholipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine and the alkylphosphocholine, hexadecylphosphocholine, inhibited cell proliferation with half-inhibitory concentrations (IC₅₀) of 75 and 135 μmol/L, respectively. The agents also inhibited phosphatidylcholine biosynthesis in confluent and proliferating MDCK cells. The IC₅₀ of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine was 40 μmol/L in confluent cells and 20 μmol/L in proliferating cells, whereas the IC₅₀ of hexadecylphosphocholine was higher in both experimental systems (67 μmol/L in confluent cells and 40 μmol/L in proliferating cells). Further experiments revealed that the effect of both agents on phosphatidylcholine biosynthesis was reversible and that the inhibition was mediated by translocation of the rate-limiting enzyme of this pathway, CTP: phosphocholine cytidylyltransferase (EC 2.7.7.15), from membranes to the cytosol, where it is inactive. The present findings suggest that the inhibition of phosphatidylcholine biosynthesis by both synthetic phospholipids might be related, in part, to their antiproliferative effects.     

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.     

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.     

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.     

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.     

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.     

Characterization of an HL-60 cell variant resistant to the antineoplastic ether lipid 1-O-octadecyl-2-o-methyl-rac-glycero-3-phosphocholine

A resistant cell line (HL-60R) was selected by incubating HL-60 cells with increasing concentrations of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃) and used to examine the mechanism of resistance to the antineoplastic ether-linked lipid. The HL-60R cells exhibited a>10-fold increase in resistance when measured by [³H]-thymidine incorporation in comparison to the HL-60 cell line. ET-18-OCH₃ binding occurred at 4°C and was not saturable at the concentrations tested (1–100 μM), indicating that the binding was receptor-independent. At 4°C, association of ET-18-OCH₃ was low for each cell line. At 37°C, uptake in the HL-60 cells was approximately 5-fold greater in comparison to HL-60R cells at each concentration tested. However, when the cellular content of ET-18-OCH₃ was equal, both cell lines experienced similar declines in cell growth. Cellular incorporation of ether lipid was determined using serum-free media and in the presence of serum albumin or lipoproteins. Reduced uptake by the resistant cell line was observed only in the presence of albumin. A greater proportion of ether lipid could be removed from prelabeled HL-60R cells than from HL-60 cells, by an albumin wash procedure, indicating an increased rate of internalization and retention by the sensitive cell line. ET-18-OCH₃ uptake in the HL-60 cell line was also more sensitive to treatment with endocytic (chloroquine, monensin) or metabolic (NaF, KCN) inhibitors. These results suggest that uptake is the principal determinant influencing sensitivity of the resistant cell line and consists of receptor-independent binding followed by internalization. Differential uptake requires the presence of serum albumin and is dependent on the energy-dependent endocytosis of the ether lipid.     

Cytotoxic activity of the thioether phospholipid analogue BM 41.440 in primary human tumor cultures

The inhibitory effect of the new thioether alkyl lysophospholipid analogue 1-hexadecylmercapto-2-methoxymethyl-rac-glycero-3-phosphocholine (BM 41.440, Ilmofosine) on colony formation of different spontaneous human tumors was studied in vitro using a methyl cellulose monolayer assay. The most sensitive tumors were lung (small cell, squamous cell and adenocarcinomas), gastrointestinal and ovarian cancers and hyernephromas. On the basis of the current difinition of sensitivity by the National Cancer Institute, Bethesda, MD, i.e. more than 70% inhibition of colony formation at an arbitrary concentration of 10 μg/ml, 34 out of 64 malignancies tested were susceptible to BM 41.440.     

Lyso platelet activating factor (LysoPAF) and its enantiomer. Total synthesis and carbon-13 NMR spectroscopy

Described is a reaction sequence for the total synthesis of lyso platelet activating factor (lysoPAF; 1-O-alkyl-sn-glycero-3-phosphocholine) and its enantiomer. The procedure is versatile and yields optically pure isomers of defined chain length. The synthesis is equally suited for the preparation of lysoPAF analogues and its enantiomers with unsaturation in the long aliphatic chain. First,rac-1(3)-O-alkylglycerol is prepared by alkylation ofrac-isopropylideneglycerol with alkyl methanesulfonate followed by acid-catalyzed removal of the ketal group. The primary hydroxy group of alkylglycerol is then protected by tritylation, the secondary hydroxy group is acylated, and the protective trityl group is removed under mild acidic conditions with boric acid on silicic acid, essentially without acyl migration. Condensation of the diradylglycerol with bromoethyl dichlorophosphate in diethyl ether, hydrolysis of the resulting chloride, and nucleophilic displacement of the bromine with trimethylamine givesrac-1-O-alkyl-2-acylglycero-3-phosphocholine in good overall yield. The racemic alkylacylglycerophosphocholine is finally treated with snake venom phospholipase A₂ (Ophiophagus hannah) which affords 1-O-alkyl-sn-glycero-3-phosphocholine (lysoPAF) of natural configuration in optically pure form. The “unnatural” 3-O-alkyl-2-O-acyl-sn-glycerol-1-phosphocholine enantiomer, which is not susceptible to phospholipase A₂ cleavage, gives 3-O-alkyl-sn-glycero-1-phosphocholine upon deacylation with methanolic sodium hydroxide. Homogeneity and structure of the intermediates and final products were ascertained by carbon-13 nuclear magnetic resonance spectroscopy on monomeric solutions.     

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).