In vivo models for endocrine-dependent breast carcinomas: special considerations of clinical relevance.

Tumours in hormone-regulated organs such as the breast, prostate or ovaries are among the most frequent malignancies. Because of their endocrine-dependent development and growth, they offer a unique opportunity for antihormonal treatment either single or long-term or in combination with radio- or chemotherapy. A prominent example is breast carcinoma, for which the anti-oestrogen tamoxifen has been used successfully for several years. Unfortunately, a substantial number of tumours are intrinsically tamoxifen-resistant, despite oestrogen-receptor positivity, and, eventually, almost all breast carcinomas acquire resistance towards tamoxifen. The recently developed pure anti-oestrogen Faslodex and the third-generation aromatase inhibitors (Letrozol, anastrozole (Arimidex) offer the possibility of alternative therapies. Preclinical models are needed, as most of the mechanisms of hormonal tumour dependence and the causes of the appearance of antihormone resistance are not yet fully understood. This review focuses on the development and characterisation of breast cancer xenografts derived directly from surgical resections. With their help, a deeper insight into the mechanisms of hormone regulation and anti-oestrogen resistance can be gained. The xenograft models have already been used in differential gene-expression analysis on DNA microarrays and for the evaluation of approaches to overcoming tamoxifen resistance.

The influence of cholesterol and charge on the membrane domains of alkylphospholipid liposomes as studied by EPR.

Alkylphospholipids are physiologically active derivatives of lipids effective in the treatment of breast cancer. Among them, octadecyl-(1,1-dimethyl-4-piperidino-4-yl)-phosphate (OPP) was demonstrated recently to have the strongest antitumor effect in micellar as well as in sterically stabilised liposome suspension with a low cholesterol content. In this work electron paramagnetic resonance (EPR) was used to study the influence of cholesterol, charge, and sterical stabilisation by PEG2000DSPE on the domain structure and fluidity characteristics of the membrane of OPP liposomes. As a spin probe 5-doxylpalmitoyl methyl ester was used. By computer simulation of the EPR spectra it was found that the experimental spectra are composed of three spectral components, which were attributed to three types of domains with different fluidity characteristics. The EPR parameters as well as the proportions of the individual domains were found to be mainly dependent on the amount of cholesterol, and only to a minor degree on charge and sterical stabilisation. There was a pronounced increase in the proportion of membrane domains with low order parameter, when the molar ratio of cholesterol to OPP was decreased below 1. At the same time the order parameters of all domains decreased, pointing to a transition from a less to a more fluid membrane organisation. These results coincide with an improved therapeutic activity of formulations with a low molar ratio of cholesterol to OPP and indicates that the fluidity characteristics of the membrane may be important for the effectiveness of liposomal alkylphospholipids against breast cancer cells.

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.

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.

Sialyl Lewis(x)-liposomes as vehicles for site-directed, E-selectin-mediated drug transfer into activated endothelial cells.

E-selectin, exclusively expressed on activated endothelial cells, is a potential target for site-directed delivery of agents. We and others have shown that sialyl LewisX-liposomes (sLe(x)-liposomes) are recognized by E-selectin. We now report an approach employing sLe(x)-liposomes to deliver antisense oligonucleotides (AS-ODNs) directed against the adhesion molecule ICAM-1 to activated vascular endothelial cells. ICAM-1 expression was analyzed at the protein level by immunofluorescence and a cell surface ELISA, and at the RNA level by RT-PCR. We have investigated two different AS-ODNs complementary to the 3' untranslated region and the AUG translation initiation codon of ICAM-1 mRNA. Both inhibited protein expression, but did not influence the mRNA level, pointing to a hybridization of AS-ODNs with the mRNA in the cytoplasm. Our results demonstrate the feasibility of a novel approach for the delivery of agents to activated endothelial cells by glycoliposomes targeted to E-selectin.

Liposomal bleomycin: increased therapeutic activity and decreased pulmonary toxicity in mice.

Conventional and sterically stabilized liposomes derived from phosphatidylcholine or the antitumor agents, hexadecylphosphocholine and octadecyl-(1,1-dimethyl-4-piperidino-4-yl)-phosphate, as bilayer forming constituents, containing bleomycin, were developed and tested. Liposomal encapsulation of bleomycin enhanced strongly the antitumor activity against P388 leukemia and the Lewis lung carcinoma. This effect was clearly dependent on the size and lipid composition of the bleomycin-containing liposomes. The therapeutic effects were nearly equal for liposomal and free bleomycin in the B16 melanoma. The partial replacement of phosphatidylcholine by alkylphospholipids and the inclusion of polyethylene glycol modified lipids for sterical stabilization did not further improve the therapeutic efficacy but increased, in some cases, the toxicity of liposomes. Bleomycin-induced lung injury was not observed if liposomal bleomycin was administered.

Cancerostatic octadecylpiperidinoylphosphate liposomes: effect of composition on uptake by and toxicity to J774 mouse macrophage cells and MT1 breast cancer cells in vitro.

Liposomes prepared from the cancerostatic octadecyl-(N,N-dimethylpiperidino-4-yl)-phosphate (OPP) were investigated in order to characterize the influence of composition on cytotoxicity and to minimize side effects on the immune system. Differently composed liposomes with respect to charge, cholesterol content and steric stabilization were used. Fluorescence measurements and the MTT assay were applied to investigate the effect of uptake and cytotoxicity, respectively, on J774 mouse macrophages and MT1 human breast cancer cells in vitro. Because of their endocytotic capability, uptake was generally higher for macrophages compared with tumour cells. OPP liposomes, which are negatively charged, cholesterol-poor and sterically stabilized, showed the lowest total and internal uptake by both cell lines. On the other hand, these liposomes were also the most cytotoxic ones for both cell lines investigated, with an inhibitory concentration of between 50 and 80 microM. Cytotoxicity does not correlate with cellular uptake and is most likely caused by other mechanisms. The results demonstrate that cancerostatic liposomes have composition-dependent toxic effects on macrophages which have to be seriously considered. For therapeutic experiments in vivo liposomes should be negatively charged and sterically stabilized and composed of OPP and cholesterol in a molar ratio of approximately 1.

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.

Phospholipid- and fatty acid-composition in the erythrocyte membrane of the one-humped camel [Camelus dromedarius] and its influence on vesicle properties prepared from these lipids.

The lipid composition of the membrane of erythrocytes from the one-humped camel (Camelus dromedarius) concerning the different lipid classes and the fatty acids was investigated for the first time. The most frequently occurring lipids are sphingomyelin (28.8% of total lipids), phosphatidylcholine and phosphatidylserine (each about 12%). The cholesterol/lipid ratio was calculated to be 0.30 (w/w). The fatty acids consist of shorter and/or more unsaturated chains, which resulted in a more fluid membrane compared to human RBC membranes. Liposomes prepared from erythrocyte lipid extracts were used to investigate the membrane properties resulting from this special lipid mixture. These vesicles were sufficient stable in buffer and significantly more stable than vesicles prepared from human erythrocyte lipid extracts in serum at 37 degrees C. Lipoplexes prepared from cationic erythrocyte lipid liposomes and a reporter gene showed in vitro an improved transfection on two colon carcinoma cells with an enhancement of up to 400% in beta-galactosidase activity in comparison to Lipofectin.

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.

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.

Antineoplastic activity of sterically stabilized alkylphosphocholine liposomes in human breast carcinomas.

New sterically stabilized liposomes derived from the antitumor agent hexadecylphosphocholine with reduced uptake by the mononuclear phagocyte system and improved antitumor activities were developed and tested. The bilayer of such sterically stabilized liposomes consists of hexadecylphosphocholine, cholesterol and polyethylene glycol-linked phosphoethanolamine. The measurement of carbon clearance in mice shows that these stabilized liposomes, in contrast to conventional alkylphosphocholine liposomes, are not largely engulfed by the mononuclear phagocyte system. Their therapeutic activity on experimental human breast carcinomas MaTu. MT-1 and MT-3 was tested in nude mice. Especially in the MaTu models the sterically stabilized hexadecylphosphocholine liposomes resulted in significantly reduced tumor growth in comparison to conventional hexadecylphosphocholine liposomes or free hexadecylphosphocholine. The enhanced therapeutic efficacy of sterically stabilized hexadecylphosphocholine liposomes is probably related to the extended circulation time of the formulation and its accumulation in tumors.

Effect of sterical stabilization on macrophage uptake in vitro and on thickness of the fixed aqueous layer of liposomes made from alkylphosphocholines.

A serious problem using liposomes for therapeutic purposes is the fast removal from blood circulation by components of the reticuloendothelial system (RES) most likely after opsonization of the vesicles. This study was performed to quantify the reduction in macrophage uptake in vitro of sterically stabilized liposomes (PEG-liposomes) prepared from hexadecylphosphocholine, cholesterol and poly(ethylene glycol2000) distearoylphosphoethanolamine (PEG2000DSPE) for the first time. The uptake was determined using HPC-liposomes of different defined size (125, 250 and 1000 nm) without and with sterical stabilization by incorporating 5 mol% of PEG2000DSPE. HPTS was used as fluorescence marker allowing the discrimination between general uptake and the part of liposomes internalized into the low pH-compartment (Daleke, L.D., Hong, K. and Papahadjopoulos. D. (1990) Biochim. Biophys. Acta 1024, 352-366). Liposomal uptake by J774 mouse macrophage-like cells was time-dependent. Both the uptake and internalization were clearly reduced for PEG-liposomes compared to plain liposomes. Sterical stabilization reduced the general uptake of liposomes in vitro by more than 50% and the internalization by about 50-60%. PEG-liposomes additionally showed a delay in internalization into the macrophages during the first 6 h. Size of used liposomes had only a minor influence on liposomal uptake but highest concentration of lipid was found for large multilammelar vesicles (MLV). The fixed aqueous layer thickness (FALT) was determined by zeta potential measurements of plain and sterically stabilised HPC-liposomes (100 nm) in solutions of different ion concentrations. The calculation of the thickness was based on the linear correlation between ln zeta (zeta-potential) and kappa (Debye Hückel-Parameter). FALT was calculated and found to be for plain HPC-liposomes 0.83 +/- 0.17 nm and for PEG-HPC-liposomes 3.57 +/- 0.17 nm. Exchange of the HPC by an alkylphospholipid with different head group has no or only minor effect (PEG-OPP-liposomes 3.44 +/- 0.31 nm). Thus the reduced uptake of HPC-LUVET correlates with an increased thickness of the fixed aqueous layer around these liposomes and could support the hypothesis that the thickness is an important property responsible for preventing opsonization and resulting finally in a reduced macrophage uptake.

Preparation and properties of sterically stabilized hexadecylphosphocholine (miltefosine)-liposomes and influence of this modification on macrophage activation.

The aim of the study was to investigate for the first time the preparation, physical properties and macrophage activating effect of sterically stabilized liposomes made from hexadecylphosphocholine (HPC, Miltefosine) using different poly(ethylene glycol) lipids for coating. We could demonstrate that it is possible to prepare different liposomal vesicle types (MLV, SUV and LUVET) without any problem and with a high stability in buffer (release of hydrophilic marker was < 5% after half a year) and in plasma (t1/2 up to several days). The preparation method, including size of polycarbon membrane filter used for the preparation of LUVETs had the main influence on vesicle size and size distribution. The addition of a charged lipid like DCP and different amounts of PEG-lipid up to 10% had no effect on size and stability of PEG-LUVETs. A comparison of activating potency of PEG-HPC-vesicles with commonly used HPC-liposomes was performed with mouse peritoneal macrophages. HPC-liposomes induced a clear release of NO and TNF from mouse peritoneal macrophages especially in a synergistical action with LPS. On the contrary the effect of PEG-liposomes was similar to control cells after a combined activation in vivo/in vitro. The reduced interaction of these liposomes with the MPS was also demonstrated by an unchanged carbon ink uptake after treatment of mice (i.p.) with liposomes prepared with and without PEG-lipid. PEG-HPC-liposomes combine the advantages of HPC, liposomes and PEG-coating, resulting in a promising preparation for treatment of mammary cancers.

Alkylphosphocholine-induced production of nitric oxide and tumor necrosis factor alpha by U 937 cells.

The human histiocytic cell line U 937, which expresses a number of monocyte markers and properties, was investigated with regard to its ability to be activated for NO and tumor necrosis factor (TNF) release after treatment with alkylphosphocholines (APC) and APC liposomes. Using APC multilamellar vesicles (MLV) a clear dose-dependent increase of NO production could be demonstrated for U 937 cells, whereas the corresponding soluble substances had no effect. The time course of NO release was characterised by a peak between 2 h and 12 h and a strong decrease after 24 h. LPS caused no NO release nor the production of TNF in U 937 cells. The simultaneous incubation of the cells with lipopolysaccharide and APC or APC-MLV, led to a strong increase in TNF production. Closer investigation of the time sequence of this synergistic effect demonstrated that cells, that had first been treated with hexadecylphosphocholine (HPC)-MLV and 4 h later with lipopolysaccharide secreted significantly more TNF into the supernatants than in the experiment where both substances were added simultaneously. From these results it was concluded that APC-MLV are possibly able to act as a primer in the process of lipopolysaccharide mediated TNF induction. Furthermore, a positive influence of phorbol 12-myristate 13-acetate (PMA) on the ability of U 937 cells to produce TNF following a treatment with HPC or HPC-MLV could be observed. PMA-pretreated cells were shown to release much more TNF compared to control cells, which led to the supposition that the immunomodifying activity of APC becomes effective only in more highly differentiated cell types.

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.

Cytotoxic effects of alkylphosphocholines or alkylphosphocholine-liposomes and macrophages on tumor cells.

The influence of the alkylphosphocholines (APC) on macrophage activation to tumor cytotoxicity was investigated in vitro with both mouse peritoneal and rat liver macrophages. For this purpose the compounds were used either in micellar or in liposomal form. The cytotoxic effect of micellar or liposomal APC was increased with prolongation of the aliphatic chain and was reduced for the liposomal form. Peritoneal macrophages incubated with APC-liposomes gave a comparable cytotoxic effect on MethA cells to that of the free, highly toxic APC alone. These liposomes can activate rat liver macrophages (Kupffer cells) in vitro to a moderate tumor cytotoxicity on C26 colon carcinoma cells, while the micellar APC were toxic to macrophages. A significant release of NO-radicals from peritoneal macrophages was obtained with Liposomes but not with micellar lipid. The release of tumor necrosis factor (TNF) was stimulated by incubation with micellar or liposomal HPC. Whereas the micellar HPC was comparable to lipopolysaccharide (LPS) in TNF release stimulation, the HPC-liposomes caused a much higher release.

Phospholipid analogues: side chain- and polar head group-dependent effects on phosphatidylcholine biosynthesis.

In recent studies we showed that the phospholipid analogue hexadecylphosphocholine inhibits phosphatidylcholine biosynthesis by affecting the translocation of the rate-limiting enzyme of phosphatidylcholine biosynthesis, CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15), to membranes, where it is active (Geilen et al. 1992. J. Biol. Chem. 267: 6719-6724). The present study was performed to investigate the structure-dependency of this effect. It is shown that the inhibitory properties of phospholipid analogues are dependent on their alkyl side chain length (dodecylphosphocholine < tetradecylphosphocholine < hexadecylphosphocholine < heptadecylphosphocholine < octadecylphosphocholine > eicosadecylphosphocholine). Furthermore, it is demonstrated that this inhibition of phosphatidylcholine biosynthesis by phospholipid analogues is also dependent on the polar head group (hexadecylphosphocholine >> hexadecylphosphoethanolamine = hexadecylphosphoserine). These effects result from an inhibition of the CTP:phosphocholine cytidylyltransferase and are not due to an inhibition of choline uptake or differences in the cellular uptake of the phospholipid analogues investigated.

Antineoplastic activity of alkylphosphocholines (APC) in human breast carcinomas in vivo and in vitro; use of liposomes.

This study examines the in vitro and in vivo activity of alkylphosphocholines (APC) in experimental human breast carcinomas. Three analogs, hexadecylphosphocholine (HPC), octadecylphosphocholine (OPC) and eicosanylphosphocholine (EPC) were investigated. Three hormone receptor negative cell lines were sensitive to all three APCs in vitro whereas the receptor positive MCF-7 line was more resistant. Sensitivity was seen in 4/6 hormone receptor negative tumors in vivo, with HPC being the most active analog. There were no antitumor effects in the four receptor positive models. The reasons for these differences in response between hormone receptor negative and -positive lines are not yet understood and require further study. Gastrointestinal toxicity and hemolysis, the major side effects of the APCs, were reduced by the use of liposomal preparations.

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.