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In this study we determined the in vivo localization of recombinant proteins expressed by intraperitoneally (i.p.) injected recombinant Semliki Forest virus (SFV) particles. Subsequently, we investigated the influence of i.p. administered SFV particles encoding recombinant murine granulocytemacrophage colony-stimulating factor (rmGM-CSF) on intraperitoneal recruitment and activation of cells. Finally, the therapeutic effect of SFV-GM-CSF treatment on an i.p. growing ovarian tumor was determined. Intraperitoneal injections of recombinant SFV particles encoding the reporter protein luciferase resulted in a high level of luciferase activity in cells of the peritoneal lining and tumor cells in the peritoneal cavity. Low levels of luciferase activity were found in liver, spleen and lungs. Injection of SFV-GM-CSF particles resulted in a slight increase in the number of peritoneal macrophages and in a significant increase in the number of neutrophils. In contrast to multiple i.p. injections with commercially available recombinant GM-CSF, i.p. injected SFVGM-CSF particles activated the macrophages to tumor cytotoxicity. Although treatment of tumor-bearing mice with SFVGM-CSF particles did not result in prolonged survival, tumor growth was inhibited for 2 weeks. Our findings indicate that macrophage-activating cytokines expressed by the efficient and safe recombinant SFV system when administered i.p. may provide an immunotherapeutic treatment modality additional to current chemotherapeutic treatment of intraperitoneally growing cancers. [ABSTRACT FROM AUTHOR]

We have investigated the antiproliferative action towards CC531 colon adenocarcinoma cells of target cell-specific immunoliposomes containing the amphiphilic dipalmitoyl derivative of 5-fluorodeoxyuridine (FUdR-dP). FUdR-dP incorporated in immunoliposomes caused a 13-fold stronger inhibition of CC531 cell growth in vitro, during a 72-h treatment, than FUdR-dP in liposomes without antibody, demonstrating that the prodrug is efficiently hydrolysed to yield the active drug, FUdR, intracellularly. The intracellular release of active FUdR was confirmed by determining the fate of [SUP3]H-labelled immunoliposomal FUdR-dP. Treatments shorter than 72 h with FUdR-dP in immunoliposomes resulted in anti-tumour activities comparable to, or even higher than, that of free FUdR. The shorter treatments reflect more closely the in vivo situation and illustrate the potential advantage of the use of immunoliposomes over non-targeted liposomal FUdR-dP or free FUdR. Association of tumour cell-specific immunoliposomes with CC531 cells was up to tenfold higher than that of liposomes without antibody or with irrelevant IgG coupled, demonstrating a specific interaction between liposomes and target cells which causes an efficient intracellular delivery of the drug. Since biochemical evidence indicates a lack of internalization or degradation of the liposomes as such, we postulate that entry of the drug most likely involves the direct transfer of the prodrug from the immunoliposome to the cell membrane during its antigen-specific interaction with the cells, followed by hydrolysis of FUdR-dP leading to relatively high intracellular FUdR-levels. In conclusion, we describe a targeted liposomal formulation for the anticancer drug FUdR, which is able to deliver the active drug to colon carcinoma cells with high efficiency, without the need for the cells to internalize the liposomes as such. [ABSTRACT FROM AUTHOR]

We studied the effect of recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) on the cytotoxic potential of murine peritoneal cells. Mice received rmGM-CSF intraperitoneally using different dosages and injection schemes. At different time points after the last injection, mice were sacrificed, peritoneal cells isolated and their tumour cytotoxicity was determined by a cytotoxicity assay using syngeneic [methyl-[SUP3]H]thymidine-labelled colon carcinoma cells. Also, the cytotoxic response to a subsequent in vitro stimulation with lipopolysaccharide was determined. Upon daily injection of 6000-54 000 U rmGM-CSF over a 6-day period, the number of peritoneal cells increased over ten fold with the highest rmGM-CSF dose. Increases in cell numbers was mainly due to increases in macrophage numbers. Upon injection of three doses of 3000 U rmGM-CSF per day for 3 consecutive days, the number of macrophages remained elevated for minimally 6 days. Although the peritoneal cells from rmGM-CSF-treated mice were not activated to a tumoricidal state, they could be activated to high levels of cytotoxicity with an additional in vitro stimulation of lipopolysaccharide. Resident cells isolated from control mice could be activated only to low levels of tumour cytotoxicity with lipopolysaccharide. Tumour cytotoxicity strongly correlated with nitric oxide secretion. When inhibiting nitric oxide synthase, tumour cell lysis decreased. Thus, the expanded peritoneal cell population induced by multiple injections of rmGM-CSF has a strong tumour cytotoxic potential and might provide a favourable condition for immunotherapeutic treatment of peritoneal neoplasms. [ABSTRACT FROM AUTHOR]

A previous study has shown that lipophilic prodrugs can be delivered efficiently to normal lung endothelium by incorporation into liposomes covalently conjugated to monoclonal antibody (mAb) 34A against the lung endothelial anticoagulant protein thrombomodulin. In the present study, the potential use of these lung-targeted immunoliposomes (34A-liposomes) for delivery of a lipophilic prodrug, 3',5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine (dpFUdR), to the tumor-bearing lung was examined using BALB/c mice bearing experimental lung metastasis induced by i.v. injection of EMT-6 mouse mammary tumor cells. Immunohistochemical examination of the tumor-bearing lung showed specificity of mAb 34A to lung endothelium. Tumor cells appeared to localize just outside of the normal blood vessels and were within a small diffusion distance from the mAb 34A-binding sites. 111In-labeled 34A-liposomes containing monosialoganglioside (GM1) were prepared that included [3H]-dpFUdR at 3.0 mol% in the lipid mixture. In vitro cell binding studies further demonstrated that 34A-liposomes bound specifically to normal mouse lung cells that expressed thrombomodulin but not to EMT-6 cells. Biodistribution study showed efficient and immunospecific accumulation of [3H]-dpFUdR incorporated into 34A-liposomes in the lung at a level parallel with that of 111In-labeled 34A-liposomes, indicating that the drug is delivered to the target organ in intact liposomes. Liposomal dpFUdR appeared to be metabolized in the lung to the parent drug FUdR at a rate slower than in the liver and spleen. Furthermore, treatment of lung-metastasis-bearing mice with dpFUdR incorporated into 34A-liposomes on days 1 and 3 after tumor cell injection resulted in a significant increase in the median survival time of treated mice as compared with control mice (%T/C value, 165%). dpFUdR either dispersed in emulsion or incorporated into antibody-free liposomes was ineffective in prolonging the survival of mice. These results indicate the potential effectiveness of organ-specific immunoliposomes containing a lipophilic prodrug for the targeted therapy of metastatic tumors. [ABSTRACT FROM AUTHOR]