E. A. M. Fleer

Hexadecylphosphocholine, a new ether lipid analogue. Studies on the antineoplastic activity in vitro and in vivo.

Hexadecylphosphocholine (He-PC) is a new compound synthesized according to the minimal structural requirements deducted from studies with other ether lipids. In vitro studies on He-PC revealed remarkable antineoplastic activity on HL60, U937, Raji and K562 leukemia cell lines. In addition, He-PC, applied orally, showed a superior effect in the treatment of dimethylbenzanthracene-induced rat mammary carcinomas when compared to intravenously administered cyclophosphamide. After oral application He-PC was well absorbed from the intestine and metabolized in the liver by phospholipases C and D. During a 5-week treatment no hematotoxic effects were detected. In a clinical pilot study on breast cancer patients with widespread skin involvement, topically applied He-PC showed skin tumor regressions without local or systemic side effects.

Metabolism of ether phospholipids and analogs in neoplastic cells

The ether phospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (OM-GPC) is known to be a potent inhibitor of cell growth. Metabolic studies in both Raji and L1210 leukemic cells on OM-GPC,3H-labeled in the methyl groups of the choline moiety, showed a (diacyl)-phosphatidylcholine as the only labeled metabolite. Since the formation of radiolabeled (diacyl)-phosphatidylcholine showed a direct correlation with cell death, we tested other lipid analogs. One of these compounds, hexadecylphosphocholine (He-PC), which was3H-labeled in the methyl-choline groups, showed a formation of labaled (diacyl)-phosphatidylcholine similar to that found with OM-GPC. Again, there was a direct linear correlation between the formation of the labeled product and cell death. He-PC was found to be a potent cell toxin in in vitro experiments on cell cultures. However, analogs with an elongated phosphor to trimethylammonium distance showed no toxicity towards the cells in in vitro experiments. From the data, we conclude that the ether phospholipids are substrates for a phospholipase C or related enzyme. This substrate property may be responsible for the toxicity of the compounds in neoplastic cells.

Distribution and metabolism of hexadecylphosphocholine in mice

Distribution and metabolic fate of radiolabeled hexadecylphosphocholine (He-PC) has been studied in mice. It is demonstrated that He-PC is well-absorbed from the intestinal tract, intravenous (IV) and oral administration lead to similar distributions throughout the body, the highest accumulation of radioactivity occurs in liver, lung and kidney, and the metabolic products are radioactive choline, phosphocholine and 1,2-diacylphosphatidylcholine. The occurrence of these metabolites indicates that phospholipases C and D may be involved in He-PC breakdown.

Cytotoxic Activity of Lysophosphatidylcholine Analogues on Human Lymphoma Raji Cells

The cytotoxic activity of 21 lysophosphocholine analogues was tested on human lymphoma Raji cells. Structure-activity investigations revealed a more than 50-fold difference in the cytotoxicity between the different compounds. Whereas acyllysophosphocholines showed only borderline effects, the most pronounced toxic activity was observed with compounds which have an etherbond in the sn-1 position and a hydrogen, a methoxy or a methoxymethylgroup in position sn-2. Elongation of the phosphorous-nitrogen distance in the choline group markedly reduced the cytotoxicity of the compounds. From the results obtained it was concluded that a long chain fatty alcohol adjacent to a phosphocholine headgroup represents the minimal requirement for antineoplastic activity. Thus, a new group of antineoplastic compounds, the alkylphosphocholines, was developed in our laboratory, with in vitro cytotoxic activities just as effective as the most toxic alkyllysophosphocholines.

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.

Hexadecylphosphocholine, a new Antineoplastic Agent: Cytotoxic properties in leukaemic cells

Several gynecological tumors were tested for the i r suscept i b i l i t y to synthetic Alkyl-Lysophospholipids (ALP). The ALPs are new antimetabolic tumor agents that in ter fere with the phospholipid metabolism preferent ly of neoplastic ce l l s . In addit ion ALP proved to act ivate normal macrophages increasing the i r tumorcidal capacity. 2 ovarian, 2 cervix u te r i , and 2 endometrial carcinomas were plated in a methy lce lh lose monolayer colony assay. 2 x 105 ce l ls were plated in 30% feta l ca l f serum and Iscoves modified Dulbeccos medium containing methylcel lulose (0,9% w/v) as viscous support. ALP was added at concentrations ranging from 2-16 ~g/ml. The drug ef fect was expressed as percent reduction of colony formation as a function of the drug concentration. In a l l tumors a reduction of colony formation to 30-20% of control was observed. 3 tumors (I cerv ix , I endometrial, I ovarian carcinoma) were xenotransplanted into Balb-C nude mice. 10 animals/ group were treated p.o. with 2,5 mg/kg and 25 mg/kg every second day for 3 weeks. Growth inh ib i t ion was assessed by cont ro l l ing the tumor diameters. In the ovarian carcinoma a growth delay of 50% compared to the control animals was observed. In the group that was treated with 2,5 mg/kg, 6/I0 animals were free of tumor, in the group treated with 25 mg/kg 4/10 animals were free of tumors. No therapeutic e f fect was observed in the cervix uter i and the endometrial adenocarcinoma.

Ether lipids and analogues

Ether lipids, ether lysolipids and their analogues have been studied for their effects on cells and cell membranes for about two decades. The first observations on the effect of lysolipids on cells were made as early as 1964 by Munder et al., reporting the activation of peritoneal macrophages by ester lysolecithin. Because of the metabolic instability of ester lysolecithins, Westphal et al. used ether analogues in later studies. From then on, reports on the action of ether lipids on cells, including their cytotoxicity against tumor cells, slowly accumulated. Snyder and Wood reported higher levels of ether lipids in tumor cells as compared to normal cells. Tumor cells were found to be more sensitive for ether lysolipids than normal cells when tested under in vitro conditions. The Snyders group found that alkylglycero monooxigenase (AGMO), an alkyl cleavage enzyme that cleaves the ether bond of the long fatty alcohol chain of ether lipids, is absent or present at low levels in many tumor cells. Some years ago, Modolell et al. postulated a possible mechanism for the specific action of ether lipids on tumor cells, based on the lower levels of AGMO in many neoplastic cells. This postulate suggested a possible explanation for the accumulation of ether lipids in tumor tissues with concomitant disturbance of membrane metabolism. However, in later studies the hypothesis had to be discarded as more was discovered on the substrate specificity of AGMO. After this, new hypotheses on the mechanism of action of ether lipids have been proposed, based on (a) the interference of ether lipids with the metabolism of phospholipids, (b) the generation of possible toxic metabolites in tumor cells, or (c) effects of ether lipids on the activity of protein kinases. None of these new hy-