The mechanisms that govern the activity and the factors that control the anticancer activity of synthetic ether lipids have not been fully elucidated. In this study, three factors were studied in relationship to cell survival after treatment with 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3): (1) exposure dose, (2) drug uptake, and (3) cell density and cell-cycle distribution. In BG-1 human ovarian carcinoma cells, cell survival was an exponential function of exposure dose and was dependent on drug concentration. Drug uptake was dependent on the concentration of ET-18-OCH3, whereas the reduction in cell survival was directly related to the uptake of drug only in the first decade of cell kill. When the quantity of cells per flask was tripled from 4 to 12×106 cells, ET-18-OCH3 failed to induce a G2 block. Furthermore, the cell kill induced by a 72-h exposure to 2 μM ET-18-OCH3 was decreased by a factor of 2 when the cell density increased. Therefore, exposure dose and cell density are important parameters in determining the cell kill induced by ET-18-OCH3.

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 added in 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. The in 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.

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.

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.

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.

The synthetic ether lipids ET-18-OCH3 and BM41.440 and a derivative, hexadecylphosphocholine, were tested for inhibition of [3H]-thymidine uptake into a Chinese hamster ovarian cell line (AUXB1) and its multidrug-resistant subline selected for colchicine resistance (CHRC5). The activity of all three compounds against the multidrug-resistant subline was equal to or higher than that against the parent line. The same result was found for their activity against a human leukemic lymphoblastic cell line (CEM/O) and its methotrexate-resistant subline (CEM/MTX). In contrast, two multidrug-resistant cell lines selected for resistance to Adriamycin, the mouse leukemia cell line P388/ADR and the murine sarcoma cell line S180/ADR, expressed modest cross-resistance to the lipids as measured by thymidine uptake. Experiments performed using the trypan-blue dye-exclusion assay yielded comparable results, although this system revealed a slightly different sensitivity in showing the cytotoxicity of the drugs. By this assay, modest cross-resistance for ET-18-OCH3 and BM41.440 to Adriamycin was found only after 24 h incubation and decreased after 48 h incubation, with almost equal sensitivity to both drugs being shown by the parental (P388/W) and resistant lines (P388/ADR). Furthermore, findings from a human tumor-cloning assay were in accordance with these data, although they did not indicate cross-resistance for the P388/ADR cell line. These results suggest that certain ether lipids and derivatives might represent valuable anticancer drugs warranting further study in the setting of resistant disease.

The synthesis of two novel glycero-3-phosphoinositol ether lipid analogues, rac-l-O-octadecyl-2-O methylglycero-3-phospho-myo-inositol 6 (an ether lipid analogue of rac-l-O-octadecyl-2-O-methylglycero-3-phosphocholine; ET-18-OMe) and rac-l-O-octadecyl-2-O-acetylglycero-3-phospho-myo-inositol 11 (an ether lipid analogue of platelet-activating factor), is described. The two target compounds and the synthetic intermediates were evaluated for inhibition of HL60, BG1, and BG3 human malignant cells in vitro and inhibition of protein kinase C. Tumor inhibitory activity was found for compounds 6 and 11 in all systems but not for their synthetic intermediates. However, compounds 6 and 11 as well as the synthetic intermediates 5 and 13, but not 9, exhibited protein kinase C inhibitory activity.

A rapid, reproducible HPLC method based on dansyl chloride derivatization has been developed for the determination of l-asparagine, l-aspartate, l-glutamine, and l-glutamate in mouse and human serum samples. This improved procedure has been designed for automation with an autoinjector system. Studies with mice bearing the sensitive and the asparaginase-resistant L5178Y leukemia show that this analytical method can be employed to monitor the effect of l-asparaginase on serum levels of these four amino acids. The method can be used to monitor serum amino acid levels in patients undergoing therapy with l-asparaginase.

Combinations of recombinant interferon alfa2 (IFN-α2) with doxorubicin, 4′-epidoxorubicin, 4′-deoxydoxorubicin, or 4-demethoxydaunorubicin were tested for antiproliferative activity against a panel of human tumor cell lines in a human tumor clonogenic assay. The histologies of the cell lines were ovary, cervix, breast, and melanoma. Each of the cytotoxic compounds showed dose-dependent antiproliferative effects against each of the cell lines, and the results indicated that doxorubicin derivatives were consistently more potent than the parent drug. In all instances, 4-demethoxydaunorubicin was the most potent derivative, requiring 2–20 times less drug to inhibit 70% of tumor colony formation. Combinations of IFN-α2, with doxorubicin or its derivatives may show additive or synergistic antiproliferative activity against certain tumor cell lines. The ovarian carcinoma cell line, BG-1, responded synergistically to each of the four compounds in combination with IFN-α2. The cervical carcinoma cell line, CaSki, and the breast carcinoma line, MCF-7, responded to the combinations in a manner best described as additive. In the melanoma line, SK-Mel-28, the drugs were found to be subadditive or even antagonistic. While the potency of the anthracyline derivatives ranked consistently across the different cell lines, the synergistic interaction with IFN-α2 is a cell line-specific phenomenon unrelated to sensitivity to either anthracyclines or interferon.

Bioactive phospholipid analogs of platelet-activating factor (PAF) represent a new approach to cancer chemotherapy. Various modifications of the basic structure of PAF lead to different ether lipid (EL) analogs. Data from the evaluation of thioalkyl and amidoalkyl glycerophosphocholine and of glycerophosphoinositol EL analogs against different experimental tumors in vitro (HL60 and K562 human leukemia cells, BG1 and BG3 ovarian adenocarcinomas) are presented. Exclusion of trypan blue after short exposure to the drugs determined cytotoxicity, and a soft agarose clonogenic assay measured the ability of the analogs to inhibit tumor cell proliferation. The thioalkyl EL are very active against the cell lines using both end points, and the amidoalkyl EL showed efficacy against the leukemic cell lines, whereas the phosphoinositol EL are active only at high concentrations. Combined use of EL analogs, which are membrane-interactive, with classical DNA-interactive chemotherapeutic drugs revealed that the combinations have additive antiproliferative effects. These results are promising leads in the development of the anticancer potential of ether lipid analogs.

A major drawback of infusions of the vinca alkaloids is the lengthy period of hospitalization which is often required for this novel technique of cancer therapy. A potentially useful system to deliver outpatient therapy has been investigated in a preclinical study. A self-contained infusion pump powered by a self-charging fluorocarbon system has been implanted SC in three dogs. The performance of two pumps which had been factory-calibrated to deliver 2.5 and 4.5 ml/day, respectively, was evaluated during 22 infusions of the vinca alkaloids (vincristine, 7; vinblastine, 7; and vindesine, 8). Infusions were given over a 5- to 7-day period and were repeated at 3-week intervals. No malfunctioning of the pumps occurred in over 500 cumulative days of use. The flow rates of the pumps were quite stable except in one animal whose increased flow rate was probably a consequence of fever due to self-induced inflammation about the pump pocket. No local or distant tissue reactions to the pump were observed. Decompostion of vincristine and vinblastine in the infusate at the end of 5- or 7-day infusions was minimal as determined by high-pressure liquid chromatography. The amount of decomposition of vindesine in the infusate was variable. Steady-state concentrations of vincristine during infusion were always > 10-9 M, and were similar to those previously determined in our clinical infusion trials using a dosage of 0.5 mg/m2/day. Clinical evaluation of this system for prolonged infusions of vincristine and other vinca alkaloids appears to be warranted.

The pharmacokinetics and metabolism of dibutyl methotrexate (DBMTX) and gamma-monobutyl methotrexate (gamma-MBMTX) were studied in Rhesus monkeys. When a bolus IV dose of either [3H]DBMTX or [3H] gamma-MBMTX was given, the principal species in serum for up to 1 h was the monoester, with MTX accounting for less than 10% of the total radioactivity. Products other than gamma-MBMTX and MTX were formed in substantial amounts with DBMTX, but not with gamma-MBMTX. Total radioactivity recovered in the bile 5 h after [3H]DBMTX injection accounted for 32% of the administered dose, indicating high hepatic extraction for this lipophilic compound. Serum and CSF levels of unchanged gamma-MBMTX, as well as of MTX arising via esterase cleavage, were measured by HPLC after IV infusion of gamma-MBMTX (10 g/m2). Efflux of monoester from CSF was slower than disappearance from serum. However, gamma-MBMTX levels in CSF were no higher than could be attained by infusing MTX itself at the same dose rate. While CSF/serum ratios were ca. 10-fold higher for gamma-MBMTX than for MTX, this difference could be explained on the basis of the very different affinities of the two compounds for serum proteins. HPLC analysis of serum processed by methanol precipitation as opposed to ultrafiltration of the proteins showed gamma-MBMTX to be greater than 99% bound, whereas for MTX this value was 50% or less. When gamma-MBMTX and MTX levels measured after ultrafiltration were corrected for this difference in serum protein binding the total amount of the two drugs in serum became almost equivalent.

A stable mutant of human leukemia CCRF/CEM cells has recently been isolated which is transport resistant for methotrexate (MTX). Encapsulation of MTX in cationic unilamellar liposomes increased the association of the drug 5-fold with the sensitive, and 50-fold with the resistant, cells as compared to the uptake of free drug. The liposome-mediated associations of MTX with sensitive and transport deficient cell lines were similar. Cytostatic studies demonstrated that liposome encapsulation increased MTX activity 4-fold towards the transport resistant cell line. The addition of cholesterol to the vesicles decreased their effectiveness. A 4-fold increase in drug sensitivity due to encapsulation may allow such transport resistant tumor cells to become responsive to chemotherapeutic doses of MTX which are currently feasible in human clinical protocols.

The structure-activity relationship of human thymidylate synthetase (EC 2.1.1.45) was studied with two groups of folate analogs: (1) methotrexate (MTX) analogs modified at the glutamate residue and N10; and (2) tetrahydrofolate (H4PteGlu) analogs modified at N5 and N10. With respect to MTX analogs, it was found that: (1) substitution of the glutamate side chain by α-aminoadipic acid. α-aminopimelic acid or β-aminoglutaric acid slightly affects its Ki; (2) a free α-carboxyl group on the amino acid side chain of MTX, or any free carboxyl group in that vicinity plays an important role in the inhibitory potency of MTX analogs to the enzyme; (3)esterification or amidation of the α-carboxyl group of MTX decreases the inhibitory potency; and (4) free aspartyl or glutamyl conjugation through a peptide linkage to the γ-carboxyl group of the glutamate side chain decreases its Ki to the enzyme by 5- and 8-fold respectively. Tetrahydrofolate analogs formed by inserting an ethylene, iminyl or a carbonyl bridge between the nitrogen at N5 and N10 or by substitution at the N5 position were found to be poor inhibitors under our assay conditions.

We describe a new method for the mapping of mammalian genes, utilizing in situ hybridization of mRNA to DNA of chromosomes. It involves the hydrogen bonding of the polyadenylic acid at the 3' end of hybridized mRNA to the polyuridylic acid tail of a highly fluorescent latex microsphere. The resultant double hybrid can be visualized by fluorescence microscopy. The chromosomal localization of human alpha + beta globin genes has been explored by this method. Our data point ot the long arms of chromosomes 4 and 5 as the loci for the human globin genes.