Summary This study reports on the development of a liposomal formulation of vincristine with significantly enhanced stability and biological properties. The in vitro and in vivo pharmacokinetic, tumour delivery and efficacy properties of liposomal vincristine formulations based on sphingomyelin (SM) and cholesterol were compared with liposomes composed of distearoylphosphatidylcholine (DSPC)

BackgroundChemotherapy for glioblastoma (GBM) patients is compromised in part by poor perfusion in the tumor. The present study evaluates how treatment with liposomal formulation of irinotecan (Irinophore C™), and other liposomal anticancer drugs, influence the tumor vasculature of GBM models grown either orthotopically or subcutaneously.MethodsLiposomal vincristine (2 mg/kg), doxorubicin (Caelyx®; 15 mg/kg) and irinotecan (Irinophore C™; 25 mg/kg) were injected intravenously (i.v.; once weekly for 3 weeks) in Rag2M mice bearing U251MG tumors. Tumor blood vessel function was assessed using the marker Hoechst 33342 and by magnetic resonance imaging-measured changes in vascular permeability/flow (Ktrans). Changes in CD31 staining density, basement membrane integrity, pericyte coverage, blood vessel diameter were also assessed.ResultsThe three liposomal drugs inhibited tumor growth significantly compared to untreated control (p < 0.05-0.001). The effects on the tumor vasculature were determined 7 days following the last drug dose. There was a 2-3 fold increase in the delivery of Hoechst 33342 observed in subcutaneous tumors (p < 0.001). In contrast there was a 5-10 fold lower level of Hoechst 33342 delivery in the orthotopic model (p < 0.01), with the greatest effect observed following treatment with Irinophore C. Following treatment with Irinophore C, there was a significant reduction in Ktrans in the orthotopic tumors (p < 0.05).ConclusionThe results are consistent with a partial restoration of the blood-brain barrier following treatment. Further, treatment with the selected liposomal drugs gave rise to blood vessels that were morphologically more mature and a vascular network that was more evenly distributed. Taken together the results suggest that treatment can lead to normalization of GBM blood vessel the structure and function. An in vitro assay designed to assess the effects of extended drug exposure on endothelial cells showed that selective cytotoxic activity against proliferating endothelial cells could explain the effects of liposomal formulations on the angiogenic tumor vasculature.

Serum proteins, acting as opsonins, are believed to contribute significantly to liposome-macrophage cell association and thus regulate liposome uptake by cells of the mononuclear phagocytic system (MPS). We studied the effect of serum protein on binding and uptake of phosphatidylglycerol-, phosphatidylserine-, cardiolipin-, and N,N-dioleyl-N,N-dimethylammonium chloride- (DODAC) containing as well as poly(ethylene glycol)- (PEG) containing liposomes by mouse bone marrow macrophages in vitro. Consistent with the postulated surface-shielding properties of PEG, protein-free uptake of liposomes containing 5 mol% PEG and either 20 mol% anionic phosphatidylserine or 20 mol% cationic DODAC was equivalent to uptake of neutral liposomes. In contrast to previous reports indicating that protein adsorption to liposomes increases uptake by macrophages, the presence of bound serum protein did not increase the uptake of these liposomes by cultured macrophages. Rather, we found that pre-incubating liposomes with serum reduced the uptake of liposomes containing phosphatidylserine. Surprisingly, serum treatment of PEG-containing liposomes also significantly reduced liposome uptake by macrophages. It is postulated that, in the case of phosphatidylserine liposomes, the bound serum protein can provide a non-specific surface-shielding property that reduces the charge-mediated interactions between liposomes and bone marrow macrophage cells. In addition, incubation of PEG-bearing liposomes with serum can result in a change in the properties of the PEG, resulting in a surface that is better protected against interactions with cells.

Conclusions: Although in vivo efficacy studies in two HER-2/neu overexpressing breast xenograft models showed that the combination of trastuzumab and gefitinib was effective, analyses of various cellular parameters failed to reveal beneficial effects and argue that this drug combination may not be favorable.

No abstract