Antitumor activity of the R- and S-enantiomers of RS-2-[[hydroxy[[2-[ (octadecyloxy)methyl]tetrahydrofuran-2-yl]methoxy]-phosphinyl]oxy]-N, N,N,-trimethylethylaminium hydroxide inner salt.

The R- and S-enantiomers of 2-[[hydroxyl[[2-[(octadecyloxy) methyl]tetrahydrofuran-2-yl]methoxy]-phosphinyl]oxy]-N,N,N,- trimethylethylaminium hydroxide salt (SRI 62-834) have been evaluated in several assays to determine potential antitumor activity. The S-enantiomer showed slightly greater cytotoxic activity than the R- or RS-forms against several murine tumor cell lines. In the mouse Meth A fibrosarcoma model, the S-enantiomer was ca. 4 times more effective than the R-isomer in controlling size of tumor growth and increasing the number of survivors.

Preclinical pharmacology and possible mechanism of action of the novel antitumor agent 5-(4'-piperidinomethylphenyl)-2,3-dihydroimidazo [2,1-a]isoquinoline.

SDZ 62-434 (CAS 115621-95-9, 5-(4'-piperidinomethylphenyl)-2,3-dihydroimidazo [2,1-a]isoquinoline dihydrochloride), a member of a novel class of antitumor agents, exhibited direct and macrophage-induced cytotoxicity against a variety of murine tumor cell lines. It is more effective than edelfosine in increasing survivors and reducing tumor volume in the oral mouse Meth A fibrosarcoma model. Preliminary studies suggest that an undefined cytotoxic effect, macrophage activation and possible effects on signal transduction may account for its antitumor mechanism of action. SDZ 62-434 is currently in Phase I clinical trials as a potential antitumor agent.

Antitumor activity of 5-aryl-2,3-dihydroimidazo[2,1-a]isoquinolines.

A series of 5-aryl-2,3-dihydroimidazo[2,1-a]isoquinolines previously reported to be platelet activating factor (PAF) receptor antagonists were evaluated for potential antitumor activity. Several compounds, such as the 5-(4'-tert-butylphenyl) (65), 5-[4'-(trimethylsilyl)phenyl] (69), and 5-(4'-cyclohexylphenyl) (71) analogs showed very good cytotoxicity against several tumor cell lines. 5-[4'-(Piperidinomethyl)phenyl]-2,3-dihydroimidazo[2,1- a]isoquinoline (SDZ 62-434, 53) was more effective on a milligram per kilogram basis than the clinical cytostatic agent edelfosine (1) in increasing survivors and decreasing tumor volume in the oral mouse Meth A fibrosarcoma assay. It was selected for further development and is currently in phase I clinical trials in cancer patients.

Antitumor activity of a piperidine phospholipid.

A piperidine phospholipid ((+/-)-2-[hydroxy] [1-octadecyloxycarbonylpiperidin-3-yl]methoxy-phosphinyl] oxy]-N,N,N, trimethylethaniminium hydroxide inner salt, SDZ 62-826) has been prepared that exhibited weak direct cytotoxicity and strong macrophage-induced cytotoxicity in vitro against a variety of murine and one human tumor cell lines. This compound was found to be as effective as ET-18-OCH3 and SRI 62-834, phospholipids with both strong direct and macrophage-induced cytotoxicity, in increasing survivors and reducing tumor volume when given either orally or intravenously in the mouse MethA fibrosarcoma model. These findings suggest that the macrophage-induced cytotoxicity exhibited by ET-18-OCH3 and other phospholipids may play an important role in this tumor model.

Macrophage mediated tumor cell destruction measured by an alkaline phosphatase assay.

A new assay to measure the cytotoxic or growth-inhibitory activity of macrophages against tumor cells is described. The method is based on the fact that, in contrast to macrophages, natural killer cells or cytotoxic lymphocytes, a variety of tumor cells have a very high content of alkaline phosphatase. The strong linearity between tumor cell number and alkaline phosphatase activity in the cultures permits evaluation of macrophage function with standard ELISA equipment.

Age-related changes in interleukin production in BALB/cNNia and SJL/J mice and their modification after administration of foreign macromolecules.

In vitro production of Interleukin-2, -3, -4 and -10 by activated lymphocytes of BALB/cNNia and SJL/J was studied. While IL-2 production in BALB/c mice remains constant throughout the life span of the animals (8-113 weeks), an increase in production from stimulated SJL cells was seen. Age-related increases in IL-3 and IL-4 production occur between young and middle age (8-60 weeks) in both strains. Some organ differences in quantity of lymphokine produced were seen; the direction of age-related changes was the same for lymphocytes of spleen and MLN. The exceptional feature of BALB/cNNia was the relative stabilization of the levels of interleukin production, as animals approach old age. BALB/cNNia and SJL, which are at the two opposite extremes of lifespan, differ also in their response to molecular interventions: in BALB/cNNia fetal sheep liver extract and hemocyanin increase the output of interleukins. This is in striking contrast to the effects observed in older SJL mice in which the extract reduced the output of IL-3 and IL-4 by old animals, whereas hemocyanin increased the output of IL-2 and IL-3 at all ages tested.

Differential modulation of the effects of lipopolysaccharide on macrophages by a major outer membrane protein of Proteus mirabilis.

We previously showed that a major protein isolated from purified cell walls of Proteus mirabilis (39-kDa protein) is a strong modulator of the specific immune responses to LPS from this bacterium in mice. When mixed with LPS before immunization, this protein enhances T cell-dependent, IgG antibody-producing cell responses specific for LPS. Furthermore, complexes of the 39-kDa protein with LPS drastically inhibit the production of oxygen radicals by murine macrophages activated with LPS, as measured in a chemiluminescence assay. In the present report, we have further investigated possible modulating effects of the protein at the level of LPS-macrophage interaction. When mixed with LPS, the 39-kDa protein inhibited IL-1 production by murine macrophages derived from bone marrow in a dose-dependent manner, as determined in an IL-2-dependent IL-1 assay. On the other hand, the protein had little effect on LPS-mediated suppression of MHC class II expression on the surface of macrophages induced with IFN-gamma. Some abrogation of suppression was observed, but the amounts of protein needed for this effect were quite large, in comparison with the amounts rendering inhibition of IL-1 production. In contrast, the 39-kDa protein enhanced the LPS-induced cytotoxicity of macrophages against L929 target cells, primarily as the result of production of TNF. These results show that the 39-kDa protein is a potent modulator of the interaction of LPS with macrophages, exerting its effects in a differential manner with respect to various parameters of LPS-induced activation of macrophages.

Alkyllysophospholipid prevents induction of experimental allergic encephalomyelitis.

Alkyllysophospholipids are synthetic analogues of natural phospholipids possessing a high immunomodulating and antitumoral capacity. Experimental autoimmune encephalomyelitis is a model disease for multiple sclerosis which can be induced by injecting rats with myelin basic protein, MBP. The effect of one alkyllysophospholipid, ET-18-OCH3, on the course of experimental autoimmune encephalomyelitis was investigated. It was found that animals treated with ET-18-OCH3 showed only weak signs of disease. MBP specific T-cell lines were co-cultivated with ET-18-OCH3. The compound suppressed T-cell proliferation markedly, suggesting that this might be its mode of action in vivo. Since ET-18-OCH3 has only low toxicity in man, it could be of interest to perform further studies on its effects on autoimmune, demyelinating disease.

Antitumor activity of Ilmofosine (BM 41.440) in the 3Lewis-lung carcinoma model.

Ilmofosine (1-hexadecylthio-2-methoxymethyl-1,3-propanediol-phosphocholine, BM 41.440) is a thioether phospholipid with cytostatic/cytotoxic properties. The antineoplastic activity of this compound was investigated in vivo in the 3Lewis-lung carcinoma system. 3Lewis lung tumor-bearing C57Bl/6 mice were treated with 0.625 to 40 mg Ilmofosine/kg per day p.o. either from days 1 to 9 or from days 11 to 28 after intrafoot-pad tumor cell inoculation. Ilmofosine caused a significant dose-related response on tumor growth and metastases, expressed in terms of tumor diameter, tumor weight, survival time and number of metastases-free animals as compared to sham-treated and positive (cyclophosphamide) controls. The results suggest that direct cytostatic/cytotoxic effects, rather than immune-modulatory mechanisms, preferentially contribute to the antitumor activity of Ilmofosine in vivo.

Cyclic oxygen analogues of alkyl-lysophospholipids. Synthesis and neoplastic cell growth inhibitory properties.

Ether phospholipids have demonstrated both in vitro and in vivo activity against a wide variety of tumor cell lines. The known cyclic ether phospholipid, SRI 62-834, was used as the model to prepare eight novel phospholipids containing a cyclic ether. All of the compounds were as effective as ET-18-OCH3 in their ability to activate macrophage-induced cytotoxicity against the Abelson-8.1 tumor cell line but varied in their direct cytotoxic effects. One of the new compounds, SDZ 62-406, was selected for in vivo studies and showed oral and i.v. activity in the mouse MethA fibrosarcoma model in the same range as ET-18-OCH3. No correlation was found between the direct or macrophage-activated cytotoxicity and the ability of the compounds to inhibit or promote platelet-activating factor (PAF)-induced aggregation of human platelets.

Influence of platelet activating factor and a nonmetabolizable analogue on superoxide production by bone marrow derived macrophages.

Serum-free cultured macrophages could be stimulated for lucigenin-dependent chemiluminescence by platelet activating factor (PAF) and phorbol myristate acetate (PMA). Stimulation with PMA resulted in a desensitization against PAF, whereas prestimulation with PAF had no influence on a following response caused by PMA. The PAF analogue, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (Et-18-OCH3), did not induce chemiluminescence by itself and desensitized the cells against PAF, like substimulating concentrations of PAF. PAF and PMA responsiveness was rapidly modulated in a similar manner during adherence of the cells to polystyrene tubes. At higher concentrations, Et-18-OCH3 as well as lysophosphatidylcholines potentiated PMA-induced chemiluminescence. The PAF analogue was most effective. Although PMA-induced chemiluminescence was stimulated at least 5-fold by Et-18-OCH3, this compound increased the PMA-induced activation of protein kinase C only 1.39-fold. The priming effect of Et-18-OCH3 was not reduced in the absence of extracellular Ca2+ and after cell membrane depolarisation.

Antitumor activity of SRI 62-834, a cyclic ether analog of ET-18-OCH3.

SRI 62-834, an analog of the antitumor agent ET-18-OCH3 in which the oxygen atom at carbon atom 2 has been incorporated into a five-membered heterocycle, has been prepared and evaluated as an antitumor agent. The compound exhibited good cytotoxicity in vitro against a variety of tumor cell lines and was as effective as ET-18-OCH3 given orally in the mouse Meth A sarcoma model. SRI 62-834 was shown to be an inhibitor of platelet-derived growth factor (PDGF), possibly at the receptor level, and platelet-activating factor (PAF) at the receptor level.

[Determination of the phagocytosis of granulocytes by the measurement of chemiluminescence]. / Bestimmung der Phagozytose von Granulozyten durch Messung der Chemilumineszenz.

During two successive years the phagocytic capacity of the granulocytes of 800 children from areas with environmental pollution and with clean air was examined and compared. To this end, the luminol chemiluminescence during phagocytosis of opsonized zymosan was measured in whole blood. In all areas the phagocytic activity of the granulocytes of boys was higher than that of girls. In area with environmental pollution it was higher than in clean-air areas. The cells disposition towards phagocytosis was lower in clean-air areas than in areas with an environmental burden. Boys showed a more rapid increase in phagocytosis than girls. Between both years of examination distinct differences were observed in the level of chemiluminescence. The phagocytic defense systems of children who grew up in areas rated as having an environmental burden, showed a distinct change in activity as opposed to that of children from so-called clean-air areas. It could be proved that the phagocytic defense system of clinically healthy children reacts to environmental irritants with a change of its activity state.

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.

Ether phospholipids as inhibitors of the arachidonoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine acyltransferase in macrophages.

1-Alkylglycerophosphatide analogs which are known to activate macrophages to enhanced tumor cytotoxicity are structurally closely related to 1-acyl-sn-glycero-3-phosphocholine. In this study we have examined the influence of some of these compounds and of platelet-activating factor (PAF-acether, 1-0-alkyl-2-0-acetyl-sn-glycero-3-phosphocholine) on the arachidonoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine acyltransferase (EC 2.3.1.23) in homogenate of bone-marrow-derived murine macrophages. This enzyme is suggested to be involved in the control of the availability of the icosanoid precursor, arachidonic acid. Kinetic experiments revealed apparent Km and V values for 1-palmitoyl-sn-glycero-3-phosphocholine of 6.0 microM and 16.10 nmol/mg protein per min, respectively. When the 1-palmitoyl-sn-glycero-3-phosphocholine concentration was equal to Km, the enzyme was dose-dependently inhibited by 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine with a 50% inhibition at 30 microM. The kinetic parameters in the presence of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (K'm = 10.0 microM, V' = 11.40 nmol X mg-1 X min-1) suggest that this alkyl phospholipid is a mixed-type inhibitor. All other alkyl analogs tested (1-O-methyl-2-O-octadecyl-rac-glycerol-3-phosphocholine, racemic PAF-acether, L-PAF-acether, D-1-O-hexadecyl-sn-glycero-3-phosphocholine, 1-O-octadecyl-rac-glycero-3-phosphocholine) inhibited the enzyme to various degrees. Arachidonic acid transfer to the 1-alkylglycerophosphatide analogs themselves could be ruled out under the assay conditions used. Therefore, we conclude that the arachidonoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine acyltransferase can be inhibited by synthetic and naturally occurring ether phospholipids in homogenate of bone-marrow-derived murine macrophages.

Effect of a lysolecithin analogue on nonspecific resistance to infection of mice.

The effect of racemic 1-octadecyl-2-methoxy-sn-glycero-3 phosphorylcholine (ET-18-OCH3) on the nonspecific resistance of mice to infection with Salmonella typhimurium was investigated. Two S. typhimurium strains with different virulence were studied and no effect was observed in either case at concentrations of ET-18-OCH3 up to 100 micrograms/mouse. However, a concentration of 500 micrograms/mouse caused decreased resistance to S. typhimurium, correlating with a depression of carbon clearance. Treatment of macrophages with ET-18-OCH3 in vitro inhibited phagosome-lysosome fusion, but had no effect on zymosan-induced luminol-dependent chemiluminescence. The relationship between the adjuvant and nonspecific anti-infectious activity of ET-18-OCH3 and other compounds is discussed.

Tumor cytotoxicity of human macrophages after incubation with synthetic analogues of 2-lysophosphatidylcholine.

Human alveolar macrophages as well as macrophages derived from Teflon culture of blood-borne monocytes were incubated with synthetic analogues of 2-lysophosphatidylcholine and then tested for their cytotoxic capacity against an allogeneic lymphoma cell line. Metabolic, rather stable analogues enhanced macrophage cytotoxicity significantly. This phenomenon was shown both in a growth-inhibition assay as well as in the 51Cr release assay. Macrophage activation was dose- and time-dependent and was potentiated at temperatures above 37 degrees C. Incubation of the macrophages with the active compounds induced characteristic changes in cell morphology as revealed by scanning electron microscopy.

Influence of the alkyllysophospholipid ET-18-OCH3 on methylnitrosourea-induced rat mammary carcinomas.

This study describes the efficacy of the alkyllysophospholipid 1-octadecyl-2-methoxy-Sn-racglycero-3-phosphocholine (ET-18-OCH3) in inhibiting the growth of methylnitrosourea-induced mammary carcinomas in Sprague-Dawley rats. In experiment A 2 X 10 mg/kg Et-18-OCH3 were administered daily for 10 weeks prior to manifestation of mammary carcinomas which resulted in a significant inhibition of median tumor number and median tumor volume per rat. Treatment of established tumors (experiment B) with 6 and 60 mg/kg ET-18-OCH3 daily for 3 weeks effected a stagnation in tumor growth for the higher dosage only with 90% tumor inhibition in comparison to untreated controls; at the same time, however, clear toxic effects were seen, thus indicating a narrow therapeutic index of ET-18-OCH3 in single-drug therapy. Combination of ET-18-OCH3 with compounds possessing a different toxicity spectrum is suggested.

Temperature dependence of leukemic cell destruction by alkyl-lysophospholipids (NSC 324368).

Alkyl-analogs (ALP) of 2-lysophosphatidylcholine induce a progressive destruction of neoplastic cells by interfering with the continuous turnover of membrane phospholipids. Using leukemic blast cells from patients with acute forms of leukemia the effect of temperature was evaluated. It was found that temperature strongly influences the cytotoxic activity of ALP. High temperatures potentiate whereas a slight decrease in temperature reduces leukemic cell destruction by ALP. At temperatures below 30 degrees C even high doses of ALP will not destroy these tumor cells. Furthermore, cell destruction initiated at 37 degrees C can be abolished by lowering the incubation temperature to 25 degrees C. These biological data have been confirmed by biochemical studies, showing a temperature dependence of ALP adsorption not accompanied by a corresponding increase of alkyl-cleavage enzyme activity. The rate of membrane phospholipid turnover seems to be essential for temperature dependent ALP induced cell destruction.