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We investigated the potential role of scavenger receptor B-I (SR-BI) in the selective removal of liposomal markers from blood by hepatocytes. Liposomes were labeled with [(3)H]cholesteryloleyl-ether ([(3)H]COE), 1,2-di[1-(14)C]palmitoyl-phosphatidylcholine ([(14)C]PC), and N-(lissamine rhodamine-B sulfonyl)-phosphatidylethanolamine (N-Rh-PE). The radiolabels were eliminated at identical rates from plasma, while N-Rh-PE was cleared twice as fast. Involvement of SR-BI in the selective removal of N-Rh-PE from liposomes was studied in transfected Chinese hamster ovary cells over-expressing SR-BI. Uptake of N-Rh-PE from liposomes containing phosphatidylserine was higher than [(3)H]COE, and was further enhanced by apolipoprotein A-I, confirming involvement of SR-BI in the selective uptake of liposomal N-Rh-PE by cells.

Background: As a majority of ovarian cancer patients will ultimately develop recurrent disease, there is an urgent need for alternative or additional approaches in the treatment of this cancer., Materials and Methods: The antitumour effect of i.p. administered cisplatin, liposomal muramyltripeptide phosphatidylethanulamine (L-MTP-PE) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were investigated using an i.p. growing murine ovarian tumour. Tumour growth was followed by measuring weight and survival of the mice., Results: An i.p. injection of L-MTP-PE in non-tumour bearing mice resulted in an approximately 10-fold increase in the number of peritoneal cells, which were highly cytotoxic. Nonetheless, treatment of mice inoculated with MOT cells with cisplatin, L-MTP-PE and GM-CSF using different treatment schedules did not result in inhibited tumour growth when compared to treatment with cisplatin alone., Conclusion: Although L-MTP-PE showed an enormous increase in peritoneal cells with high tumour cytotoxic capacity, the immunotherapeutic treatment with GM-CSF and L-MTP-PE, aimed at the recruitment and activation of the peritoneal cell population, failed to result in a significant prolongation of survival.

The antitumor effects of muramyl tripeptide phosphatidylethanolamine, incorporated within the lipophilic phase of liposomes (lipMTP-PE) were studied using a model of liver metastasis of colon cancer in the rat. Intravenous immunotherapy with lipMTP-PE, when started 2 days before the inoculation of tumor cells and given twice a week, significantly reduced subsequent tumor growth in the liver. The main effect of treatment appeared to be a substantial local increase in the number of tumoricidal macrophages and lymphocytes. Tumor cell lysis by isolated macrophages in vitro, however, appeared not to be elevated above the level triggered by tumor growth alone. Therefore, the observed therapeutic effect of lipMTP-PE probably results from a combination of (1) an increase in the number of cytotoxic macrophages at the onset of metastatic growth in the liver, thus increasing the probability of lethal contacts between tumoricidal effectors and tumor cells and (2) indirect effects of lipMTP-PE, via the induction of cytokine production by liver macrophages, leading to increased numbers and/or activity of cytotoxic lymphocytes and natural killer cells.

In this study, we investigated different functional characteristics of the rat liver macrophage population after a single i.v. injection of liposome-encapsulated muramyl dipeptide (MDP) or its lipophilic derivative muramyl tripeptide-phosphatidylethanolamine (MTP-PE). The in situ induced tumoricidal activity of the liver macrophage population was determined in vitro against C26 colon adenocarcinoma cells. For investigating which cells are responsible for the observed cytotoxic effects, subfractions of the liver macrophage population, differing in cell size, were isolated at different intervals after injection of the liposomal muramyl peptides. From these subfractions, the number of cells, degree of cytotoxicity, and the response to an additional activation with free MDP in vitro were determined. Maximal induction of tumoricidal activity of the liver macrophage population was reached between 12 and 24 hours after injection of liposomal MDP, while no significant differences between the subfractions were observed. Heterogeneity of tumor cytolytic capacity was observed in subfractions of macrophages isolated at 2 and 48 hours after injection. At these time points, highest cytolytic activity was observed for the small to intermediate-size macrophages. No significant cytotoxicity was detectable in any subfraction 72 hours after injection of liposomal MDP. An identical pattern of macrophage tumoricidal activity was observed after injection of liposomal MTP-PE, although slightly lower cytotoxicity levels were found. When isolated during the first 12 hours after injection of liposomal MDP, the macrophage population was unable to respond to a subsequent in vitro exposure to MDP, with respect to tumor cytotoxicity. Twenty-four and 48 hours after injection, the smallest cells could be slightly reactivated, whereas the larger cells still remained unresponsive.(ABSTRACT TRUNCATED AT 250 WORDS)

We have investigated the processing of the non-exchangeable fluorescent phospholipid analogue phosphatidyl(N-sulphorhodamine B sulphonyl)ethanolamine (N-Rh-PE) by rat liver cells. In the hepatocyte couplet system, N-Rh-PE was incorporated into the plasma membrane at 2 degrees C and readily internalized upon warming to 37 degrees C. Fluorescence was initially found to be concentrated in vesicles clustered throughout the cell, but subsequently it started to accumulate in pericanalicular vesicles, tentatively identified as lysosomes, and in the bile canalicular lumen. Analysis of cells and media by t.l.c. revealed the slow formation of at least two metabolites. After intravenous injection into bile-fistula rats of [9,10-3H-oleoyl]N-Rh-PE incorporated in small unilamellar liposomes, the initial rates of elimination from plasma of 3H and rhodamine label were virtually identical. However, biliary secretion of the 3H label (5.5% of dose at 2 h) was much slower than that of the rhodamine label (49.3% at 2 h). The rhodamine label in bile was chloroform-soluble, but not identical to the native molecule, and was resistant to phospholipase A2 and alkaline hydrolysis. To gain insight in the mechanism of the rapid bile secretion of this metabolite, we compared the processing of N-Rh-PE, its deacylated form [glycerophospho(N-sulphorhodamine B sulphonyl)ethanolamine; Gly-N-Rh] and the rhodamine label itself (sulphorhodamine B sulphonyl chloride; SRho). Intravenous injection of chloroform-soluble N-Rh-PE and of methanol/water-soluble Gly-N-Rh complexed with albumin both resulted in rapid bile secretion of chloroform-soluble fluorescent compounds (60.2% and 86.3% respectively at 2 h), which showed behaviour identical to that of the metabolite of liposomal N-Rh-PE on t.l.c. Methanol/water-soluble SRho was also rapidly secreted into bile (89.5% at 2 h) without being metabolized. Bile secretion of the chloroform-soluble metabolite of N-Rh-PE and of SRho was markedly impaired (-31% and -52% respectively) in GY Wistar rats, which express a genetic defect in the hepatobiliary transport of organic anions. Our data show that the rat hepatocyte is capable of modifying the structure of N-Rh-PE, a process which proceeds considerably faster in vivo than in vitro. The chloroform-soluble metabolite is subsequently rapidly removed via the bile. The canalicular organic anion transporting system, which is deficient in GY rats, appears to be involved in the excretion of this apolar product of hepatic metabolism.