Reinhard Zeisig

Influence of hexadecylphosphocholine on the release of tumor necrosis factor and nitroxide from peritoneal macrophages in vitro

Hexadecylphosphocholine (HPC) has been investigated intensively for its cancerostatic properties. One explanation for the mechanism of action of HPC assumes that it plays a role in stimulation of the immune system. In particular, its potency to activate macrophages has already been recognised for different lyso- and ether lipids. Important steps in the cascade for developing cytotoxic effects of macrophages on tumor cells are the release of nitric oxide radicals (NO) and/or tumor necrosis factor (TNF). The aim of our study was to examine the role of HPC as primer and/or trigger for macrophage activation to cytotoxicity. In our experiments we used HPC in free (micellar) or liposomal form in different primer/trigger combinations with lipopolysaccharide (LPS). A weak change in morphology was revealed by electron microscopy, if macrophages were harvested from mice previously treated with HPC or HPC multilamellar vesicles. This observation was quantified by the measurement of NO, TNF and cytotoxic activity of the peritoneal macrophages. A specific release of NO was induced by the combination of in vivo treatment with liposomal HPC and subsequent stimulation by LPS in vitro. This process started only after 12 h of in vitro incubation of macrophages with the endotoxin. The release of TNF was dependent of the primer/trigger combination used. A moderate priming effect was obtained with HPC in liposomal form independently of the trigger. On the other hand, liposomes as priming agents were found to induce a dramatic increase in TNF release after in vitro coculture with the trigger LPS. The high release of NO and TNF is accompanied by only a weak increase in tumor cytostasis. The best results were once more found with macrophages primed with liposomal HPC and then triggered with LPS.

Physical properties and pharmacological activity in vitro and in vivo of optimised liposomes prepared from a new cancerostatic alkylphospholipid.

Liposomes from octadecyl-(1,1-dimethyl-4-piperidino-4-yl)-phosphate (OPP), a new alkylphospholipid derivative with an improved cancerostatic activity, were prepared for the first time and the activity in vitro and in vivo was characterised. The formation of liposomes (MLV, SUV and LUVET) differing in cholesterol content, charge, and sterical stabilisation is possible without serious problems, despite the lysolipid-like structure of the OPP. Liposomes with a low amount of cholesterol and with PEG2000DSPE-coating were the most stable OPP liposomes, both in buffer and in serum. The cytotoxicity of micellar or liposomal OPP against breast cancer cell lines in vitro was in the range of 20-60 microM. The cytotoxicity of the liposomal formulation was inversely related to the content of cholesterol, whereas the sterical stabilisation and/or the incorporation of a positive charge had only a very moderate modulating effect on the inhibition of cell proliferation. The strongest antitumour effect on the xenotransplanted breast cancer MT-3 in vivo was obtained with sterically stabilised OPP liposomes with low CH content. The beneficial therapeutic effect of these liposomes was accompanied by better tolerance and a significant inhibition of haemolysis compared to micellar OPP.

Antitumor effects of alkylphosphocholines in different murine tumor models: use of liposomal preparations

Hexadecylphosphocholine (HPC) and its analogs with a longer alkyl chain (C18 and C20) were examined for antineoplastic activity in the murine tumor models P388 leukemia, B 16 melanoma, the mammary carcinoma C3H and Ca 755, and the human MT-1 mammary tumor in nude mice. The maximum tolerated doses were determined and found to be higher in mice than in rats. The toxicity of the alkylphosphocholines increases with chain length. The murine mammary carcinoma C3H and the human MT-1 mammary carcinoma showed response to HPC whereas the classical screening models did not respond to the synthetic lipids. Furthermore, HPC showed activity in a mitoxantrone-resistant P388 leukemia. Treated/control values between 120 and 160% in survival time could be obtained following a daily application of the lipid. Examination of the activity of possible cleavage products of HPC gave no information about the mechanism of action of the used etherlipids. Liposomes with encapsulated mitoxantrone, formed from alkylphosphocholines, cholesterol and dicetylphosphate had the same activity against P388 mouse leukemia as the free drug. The hemolytic activity of the three lipids tested in vivo was assumed to be related to toxic deaths of single animals; hemolytic activity was observed to be sometimes independent of schedule and dose.

Antineoplastic activity in vitro of free and liposomal alkylphosphocholines.

We investigated the liposome forming properties of three homologues of alkylphosphocholines: hexadecylphosphocholine (HPC), octadecylphosphocholine (OPC) and eicosanylphosphocholine (EPC). In the presence of cholesterol and dicetylphosphate, alkylphosphocholines form liposomes with slow permeability for entrapped carboxyfluorescein. We studied the direct cytotoxicity of alkylphosphocholine vesicles and their ability to attack MethA sarcoma cells, human skin and muscle fibroblasts (M22, GUS, Moscow), and human mouth epidermoid carcinoma cells (KB, ATCC, CCL 17). All alkylphosphocholines show cytotoxic activity against the investigated cells, the degree of which depends on the number of carbon atoms in the alkyl chain, concentration and incubation time. Whereas the etherlipid liposomes are less toxic to MethA cells than the free compounds, the liposomal alkylphosphocholines are more toxic toward KB and M22 cells than the corresponding free lipids.

Cell Adhesion Inhibition by Glycoliposomes: Effects of Vesicle Diameter and Ligand Density

The Thomsen-Friedenreich antigen (TF) is a pancarcinoma marker which is involved in the development of liver metastasis by binding tumour cells to the asialoglycoprotein receptor on hepatocytes. Blocking of this receptor prevents metastasis under certain circumstances. We report on conditions for an effective inhibition of the adhesion of KG-1 leukaemia cells expressing TF by lactosylated liposomes. In order to reach strong inhibition, carbohydrate blocking probes must be multivalent. Glycoliposomes are able to carry a large number of glycolipids accommodated in the lipid bilayer. They should be able to adapt their glycolipid pattern in order to achieve multiple binding. We found that, in addition to the number of carbohydrates on the liposome surface, their size, and probably the arrangement of neutral glycolipids in clustered domains, determine the inhibitory properties of glycoliposomes.

The composition-dependent presence of free (micellar) alkylphospholipid in liposomal formulations of octadecyl-1,1-dimethyl-piperidino-4-yl-phosphate affects its cytotoxic activity in vitro.

Abstract. This study was performed to investigate the effect of cholesterol content, surface charge and sterical stabilization on the physico-chemical properties of liposomes prepared from the cancerostatic alkylphospholipid, octadecyl-1,1-dimethyl-piperidino-4-yl-phosphate (D21266), and their relationship to in vitro cytotoxicity. Stable incorporation of OPP into liposomes was found to be highly dependent on the cholesterol content. 31P-NMR spectroscopy as well as analysis of the lipid composition of OPP-containing liposome formulations revealed an increase in the amount of non-liposome-associated, micellar OPP as the cholesterol content decreased. The fraction of non-liposome-associated OPP constituted about 10% of total OPP when cholesterol was present in equimolar amounts (45.5/45.5 mol %) and increased to approximately 30% at a twofold excess of OPP over cholesterol (58.8/29.4 mol %). In monolayer incorporation studies it was shown that the existence of an increasing micellar pool of lipids leads to increased lipid transfer into the target monolayer. Liposome formulations containing more OPP than cholesterol were also found to display greater cytotoxicity. However, all liposome formulations were less cytotxic than pure (micellar) OPP. Cytotoxicity was not affected by the incorporation of N-methoxy-polyethyleneglycol2000-phosphoethanolamine, a lipid that is known to reduce liposome uptake into phagocytic cells.The results demonstrate that the increase in cell toxicity correlates with the increase in non-liposome-associated, micellar OPP, which can readily exchange into cellular membranes.

Pharmacokinetics of sterically stabilized hexadecylphosphocholine liposomes versus conventional liposomes and free hexadecylphosphocholine in tumor-free and human breast carcinoma bearing mice

The pharmacokinetics of free and different liposomal formulations of hexadecylphosphocholine (HPC) was investigated in tumor-bearing (human mammary tumor MaTu) and tumor-free mice after intravenous and intraperitoneal administration. The levels of HPC were evaluated at different times in serum, normal tissues, and tumor. The purpose was to test the hypothesis that the enhanced therapeutic efficacy of sterically stabilized HPC liposomes in comparison to conventional vesicles and free HPC is due to its pharmacokinetics. Conventional non-compartmental pharmacokinetic analysis and an elaborate three- and four-compartmental model were used for explaining the experimental data. The serum levels of HPC obtained with sterically stabilized liposomes were only consistently higher in comparison to conventional vesicles and free HPC in the first 4 h. In the xenografted MaTu carcinoma, the differences of the HPC content between the different groups are unexpectedly low and do not reflect the high therapeutic activity [5] of sterically stabilized HPC liposomes.Detailed analysis shows that the liposomally encapsulated drug displays a modified pharmacokinetic behavior, which may also involve lymphatic absorption of the liposomal drug.

Lipoplexes with alkylphospholipid as new helper lipid for efficient in vitro and in vivo gene transfer in tumor therapy

To improve liposomal gene transfer we investigated the influence of membrane-interacting alkylphospholipids (APLs) on gene transfer efficiency in vitro and in vivo using the LacZ reporter gene and the cytosine deaminase (CD) suicide gene. Liposomes were first optimized concerning the kind and amount of APL and the additional liposome components. Thus, an up to 270% increase in the transfer efficiency of the LacZ gene into HCT15 and HCT116 human colon carcinoma cells could be obtained in vitro compared to lipofectin-mediated transfection by using a lipoplex consisting of tetradecylphosphocholine/dimethyldioctadecylamine/cholesterol/dioleylphosphoethanolamine-liposomes and the pSV40-βGal-plasmid. The in vivo experiments revealed that alkylphospholipid-lipoplexes (APL–LPs) were similarly effective in the transfer of the LacZ gene into colon carcinoma as formulations consisting of lipofectin. Using the CD-gene in combination with APL-LPs resulted in a significantly stronger inhibition of C26 colon carcinoma growth compared to lipofectin-mediated gene transfer following treatment of mice with the prodrug 5-fluorocytosine. The results of this study demonstrate for the first time that the utilization of membrane-active APLs as component of the liposomal part of lipoplexes enhances the efficacy of gene therapy in vitro and in vivo.

Abstract A14: Patient-derived tumor xenografts supporting development of targeted drugs.

Almost in parallel with the new millennium processes in drug development for cancer therapy have changed substantially. Insight in the cancer biology have revealed this disease as a complex genetic disorder leading to a high variety of phenotypes among the different individuals. Many tumors present a very specific pattern of molecular changes and this leads to a heterogenous individual response to drugs. The correct identification of predictive biomarkers selecting the most appropriate therapies and avoiding unnecessary treatments for an individual patient is still a challenge. Patient-derived tumor xenografts allow preclinical investigations in a clinically relevant way. We performed investigations to improve the understanding of cancer complexity and to draw rational conclusions for therapy decisions. Tumor models were established by direct transplantation of surgical specimens to immunodeficient mice and were maintained in early passages. We have shown a high correlation between original patient sample and xenograft both at gene and protein level. We established several large panels of tumor models: i.e. 10 breast, 30 colorectal, 25 lung, 6 ovarian, 10 sarcomas, and 30 leukemias (25 ALL and 5 AML). During the characterization of the xenografts for response towards clinically used anticancer drugs, the analysis for mutations and of gene expression revealed interesting correlations. In NSCLC models upregulation of hypoxia marker genes correlated with resistance to microtubule targeted drugs. In the colon cancer xenografts KRAS mutations predicted resistance to cetuximab. We further identified a potential set of 20 genes which may be predictive for oxaliplatin response. These results demonstrate, that the ability to assess anti-tumor activity in well-characterized xenografts in correlation with particular genetic or molecular characteristics may support the development of new therapeutic regimens. Conclusions from what we discussed are: drug discovery, systems biology, and translational research are moving together to address all the new hallmarks of cancer - increasing the success rate of drug discovery panels of patient derived xenograft models represent an important tool for translational research to address clinically relevant questions in a standardized and strictly controlled fashion predictive value of the preclinical models is increasing steadily - they show a high concordance with the clinical specimens concerning marker expression and response to therapy Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A14.