H. Eibl

Novel Temperature-Sensitive Liposomes with Prolonged Circulation Time

Hyperthermia increases the efficiency of various chemotherapeutic drugs and is administered as an adjunct to chemotherapy for the treatment of cancer patients. The temperature-dependent effect can be strongly increased by the use of temperature-sensitive liposomes in combination with regional hyperthermia, which specifically releases the entrapped drug in the heated tumor tissue. The novel lipid 1.2-dipalmitoyl-sn-glycero-3-phosphoglyceroglycerol (DPPGOG), which is closely related to the naturally occurring 1.2-dipalmitoyl-sn-glycero-3-phosphoglycerol, in combination with 1.2-dipalmitoyl-sn-glycero-3-phosphocholine and 1.2-distearoyl-sn-glycero-3-phosphocholine provides long-circulating temperature-sensitive liposomes with favorable properties under mildly hyperthermic conditions (41–42°C). DPPGOG facilitates temperature-triggered drug release from these liposomes (diameter, 175 nm) and leads to a substantially prolonged plasma half-life for the encapsulated drug with t1/2 = 9.6 h in hamsters and t1/2 = 5.0 h in rats. Quantitative fluorescence microscopy of amelanotic melanoma grown in the transparent dorsal skin fold chamber of hamsters demonstrated a favorable drug accumulation in heated tissue after i.v. application of these liposomes (42°C for 1 h). The mean area under the curve for tissue drug concentration was increased by more than sixfold by application of the new liposomes compared with nonliposomal drug delivery. In summary, we present a new DPPGOG-based liposomal formulation enabling long circulation time combined with fast and efficient drug release under mild hyperthermia. This adds positively to the results with lipid-grafted polyethylenglycol used thus far in temperaturesensitive liposomes and widens the possibilities for clinical applications.

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.

Intracellular mediators of erucylphosphocholine-induced apoptosis

Induction of apoptosis contributes to the cytotoxic action of the intravenously applicable alkylphosphocholine erucylphosphocholine (ErPC). To define molecular requirements for ErPC-induced apoptosis, activation of caspases-8, -9 and -3 and cleavage of the caspase-3 substrates PARP and ICAD were tested in normal Jurkat T cells, Jurkat cells resistant to death receptor (CD95 or TNFα-related apoptosis inducing ligand (TRAIL)-induced apoptosis, Jurkat cells lacking caspase-8 or Fas-associated death domain (FADD) Jurkat cells expressing a dominant-negative caspase-9 or overexpressing Bcl-2 as well as BJAB B-lymphoma cells expressing a dominant-negative FADD (FADD-DN). ErPC induced a time- and dose-dependent apoptotic cell death in Jurkat and BJAB cells, which was characterized by breakdown of the phosphatidylserine asymmetry, depolarization of the mitochondrial membrane potential, release of cytochrome c, activation of caspases-9, -8 and -3, cleavage of PARP and ICAD, as well as chromatin condensation. ErPC-induced apoptosis was independent from CD95-receptor signaling and FADD since CD95- and TRAIL-resistant, caspase-8- and FADD-negative Jurkat cells, as well as BJAB cells expressing FADD-DN were sensitive to ErPC-induced apoptosis. In contrast, inhibition of caspase-9 and overexpression of Bcl-2 significantly reduced ErPC-induced caspase activation and apoptosis. Thus, ErPC triggers apoptosis via a Bcl-2-dependent mitochondrial but death receptor-independent pathway.

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.

Erucylphosphohomocholine, the first intravenously applicable alkylphosphocholine, is cytotoxic to acute myelogenous leukemia cells through JNK- and PP2A-dependent mechanisms

Alkylphospholipids and alkylphosphocholines (APCs) are promising antitumor agents, which target the plasma membrane and affect multiple signal transduction networks. We investigated the therapeutic potential of erucylphosphohomocholine (ErPC3), the first intravenously applicable APC, in human acute myelogenous leukemia (AML) cells. ErPC3 was tested on AML cell lines, as well as AML primary cells. At short (6–12 h) incubation times, the drug blocked cells in G2/M phase of the cell cycle, whereas, at longer incubation times, it decreased survival and induced cell death by apoptosis. ErPC3 caused JNK 1/2 activation as well as ERK 1/2 dephosphorylation. Pharmacological inhibition of caspase-3 or a JNK 1/2 inhibitor peptide markedly reduced ErPC3 cytotoxicity. Protein phosphatase 2A downregulation by siRNA opposed ERK 1/2 dephosphorylation and blunted the cytotoxic effect of ErPC3. ErPC3 was cytotoxic to AML primary cells and reduced the clonogenic activity of CD34+ leukemic cells. ErPC3 induced a significant apoptosis in the compartment (CD34+ CD38Low/Neg CD123+) enriched in putative leukemia-initiating cells. This conclusion was supported by ErPC3 cytotoxicity on AML blasts showing high aldehyde dehydrogenase activity and on the side population of AML cell lines and blasts. These findings indicate that ErPC3 might be a promising therapeutic agent for the treatment of AML patients.

Investigations on the cellular uptake of hexadecylphosphocholine

The uptake of [(9,10)-3H]hexadecylphosphocholine (HePC) in six tumor cell lines was studied. All cell lines incorporated HePC in similar amounts, with the exception of the epidermoid cancer cell line KB, which took up higher amounts of HePC. The uptake of HePC at 37°C was shown to be time and concentration dependent. At 20°C, uptake was drastically reduced and at 4°C it was blocked completely. Binding of HePC, at 4°C, was not saturable at concentrations between 5 βg/mL (11.8 μM) and 100 μg/mL (235.3 μM), indicating that cell surface binding is not receptor-mediated. Furthermore, the effects of inhibitors of endocytosis were investigated. We observed a pronounced inhibitory effect by monensin and cytochalasin B. Colchicine was somewhat less effective whereas chloroquine was almost without effect. From these data we conclude that uptake of HePC is most probably mediatedvia a receptor-independent endocytotic mechanism.

Comparison of selective cytotoxicity of alkyl lysophospholipids.

Alkyl lysophospholipids have been shown to be cytooxic to a number of neoplastic tissues. One, ET-18-OCH3, has been used to selectively purge leukemic cells from mixtures with normal marrow progenitor cells,in vitro andin vivo. We have measured the 50% inhibitory (IC50) effect of a series of ether, lipids (EL) on leukemic cells (HL60, K562, Daudi, KG-1, KG-1a) and normal marrow progenitor cells. Cells were incubated with varying concentrations of EL for 4 hr and assayed for viability, [3H]thymidine incorporation and clonogenicity in semi-solid media. The effect on protein kinase C (PKC) activity was assayed for each compound. Compounds tested included three glycerophosphocholine analogs-ET-18-OCH3, ET-16-NHCOCH3, and BM 41.440. In addition, a lipoidal amine, CP 46665, an ethyleneglycolphospholipid, AEPL, and four single chain alkylphosphocholine analogs, HePC2, HePC3, HePC4 and HePC6 were also tested. During the period of incubation, the cells remained viable (>70%) as judged by trypan blue dye exclusion. The glycerophosphocholines were the most active and showed the highest therapeutic index. The lipoidal amine was active, but toxic to normal marrow progenitor cells. The ethyleneglycolphospholipid was active against HL60, but not against the other cell lines. The single chain alkylphosphocholine analogs were less active. All of the compounds inhibited PKC activity; however, the glycerophosphocholines were the most inhibitory.

Inhibition of calcium-dependent protein kinase C by hexadecylphosphocholine and 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine do not correlate with inhibition of proliferation of HL60 and K562 cell lines.

We investigated the hypothesis that the antiproliferative effect of hexadecylphosphocholine (HePC) and 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) is mediated through the inhibition of cellular protein kinase C (PKC). In the sensitive HL60 cell line, ID50 and LD50 values of 5.6 and 5.3 microM, respectively (HePC), and of 3.8 and 4.2 microM, respectively (ET-18-OCH3) were obtained. In the more resistant K562 cell line, these values were 69.1 and > 97 microM, respectively (HePC) and 7.8 and 76.8 microM, respectively (ET-18-OCH3). Treatment of both cell lines with HePC and ET-18-OCH3 (25 microM) for 2 h did not lead to PKC translocation. However, a 30% reduction of PKC activity, mainly due to a decrease in the cytosolic compartment, was found. Half maximal stimulation of PKC translocation by phorbolester (TPA) in HL60 and K562 cells, which were pretreated for 2 h with 25 microM of the lipids, resulted in a 20-30% decrease of membrane-bound PKC, whereas the cytosolic form was found to be unchanged. In the same experimental setting, dioctanoylglycerol (DIC8)-stimulated PKC translocation was not affected by HePC or ET-18-OCH3. However, a 10-20% reduction of PKC enzyme activity in the membrane and in the cytosolic fraction was obtained. These findings indicate that HePC and ET-18-OCH3 do not interfere with PKC translocation but rather mediate a general decrease of the enzyme activity in the membrane and cytosol of the cells. Since the extent of PKC inhibition was somewhat similar in the sensitive HL60 and the resistant K562 cell line, inhibition of PKC is probably not a prerequisite for the antiproliferative action of HePC and ET-18-OCH3.