Targeted delivery and triggered release of liposomal doxorubicin enhances cytotoxicity against human B lymphoma cells.

Dioleoylphosphatidylethanolamine (DOPE)-containing liposomes that demonstrated pH-dependent release of their contents were stabilized in the bilayer form through the addition of a cleavable lipid derivative of polyethylene glycol (PEG) in which the PEG was attached to a lipid anchor via a disulfide linkage (mPEG-S-S-DSPE). Liposomes stabilized with either a non-cleavable PEG (mPEG-DSPE) or mPEG-S-S-DSPE retained an encapsulated dye at pH 5.5, but treatment at pH 5.5 of liposomes stabilized with mPEG-S-S-DSPE with either dithiothreitol or cell-free extracts caused contents release due to cleavage of the PEG chains and concomitant destabilization of the DOPE liposomes. While formulations loaded with doxorubicin (DXR) were stable in culture media, DXR was rapidly released in human plasma. pH-Sensitive liposomes, targeted to the CD19 epitope on B-lymphoma cells, showed enhanced DXR delivery into the nuclei of the target cells and increased cytotoxicity compared to non-pH-sensitive liposomes. Pharmacokinetic studies suggested that mPEG-S-S-DSPE was rapidly cleaved in circulation. In a murine model of B-cell lymphoma, the therapeutic efficacy of an anti-CD19-targeted pH-sensitive formulation was superior to that of a stable long-circulating formulation of targeted liposomes despite the more rapid drug release and clearance of the pH-sensitive formulation. These results suggest that targeted pH-sensitive formulations of drugs may be able to increase the therapeutic efficacy of entrapped drugs.

Intrathecal application with liposome-entrapped Fasudil for cerebral vasospasm following subarachnoid hemorrhage in rats.

To date, the pharmacological approach to cerebral vasospasm following subarachnoid hemorrhage has been hampered in part by an inability to attain sufficiently high concentrations of vasodilator drugs in the cerebrospinal fluid (CSF). To overcome this limitation of current drug therapy, we have developed a sustained-release preparation of protein kinase inhibitor Fasudil. Cerebral vasospasm in rats was induced by double-injection method. Treated rats received 0.417 mg liposome-entrapped Fasudil via the cisterna magna and control rats received drug-free liposomes in the same manner. The diameter of the basilar artery was assessed at 7 days after the initial blood injection. Vasoconstriction of the rat basilar artery was significantly reduced in group treated with liposomal Fasudil compared to the control group (treated group: 87.7 +/- 6.18%, n= 10; control group: 66.3 +/- 9.82%, n = 10; ***P< 0.001). This new approach for cerebral vasospasm may have significant potential for use in the clinical setting.

Neuroprotection by intrathecal application of liposome-entrapped fasudil in a rat model of ischemia.

Pharmacological treatment for cerebral ischemia cannot attain sufficiently high concentrations of the drugs in the cerebrospinal fluid (CSF) without precipitating systemic side effects. The objective of this study is the development of a liposomal drug delivery system that maintains effective concentrations of protein kinase inhibitors fasudil in the CSF, resulting in neuroprotection against cerebral ischemia. Focal cerebral ischemia in rats was induced by middle cerebral artery occlusion using an intraluminal suture technique. Treated rats received 0.25 mg liposome-entrapped fasudil via the cisterna magna 2 hours after ischemic insult. Control rats received drug-free liposomes. Neurological condition and the infarct size were assessed at 24 and 72 hours after ischemia. The concentration of liposome-entrapped fasudil in the CSF was measured before sacrifice. Treated animals showed significantly improved neurological outcomes after the 24-hour observation period compared to the control group (p < 0.001). Treatment with 0.25 mg liposomal fasudil resulted in a reduction in the infarct area (24 hours: 29.0 +/- 4.4%, 72 hours: 28.1 +/- 3.9% of total brain slices) compared to controls (49.6 +/- 4.6%, p < 0.001), but there was no statistical difference between 24 and 72 hours. At 24 hours post-administration, CSF concentrations of liposome-entrapped fasudil were 45.4 +/- 31.5 micrograms/ml (20% of the injected dose). A single intrathecal injection of liposomal fasudil can maintain a therapeutic drug concentration in the CSF over a period of time, significantly decreasing infarct size in a rat model of acute ischemia.

Correlations between the rate of intracellular release of endocytosed liposomal Doxorubicin and cytotoxicity as determined by a new assay.

Previously, we showed that liposomes with surface-attached anti-CD19 were internalized into human B lymphoma cells through receptor-mediated endocytosis, resulting in improved anti-tumor efficacy 1-2 . In order to further increase the efficacy of antineoplastic drug-containing liposomes, we have taken advantage of this internalization process by producing triggered release liposomes that rapidly release drug from the enzyme-rich, acidic environment of lysosomes. To analyze the effectiveness of these triggered-release formulations, we developed a nuclear accumulation assay for doxorubicin (DXR) that allows us to determine the rate of cytoplasmic drug delivery subsequent to drug release from the endosomal/lysosomal compartments by examining the rate of accumulation of drug in cellular nuclei. We demonstrate the usefulness of this assay by comparing the kinetics of cytoplasmic drug delivery for DXR-containing, pH-sensitive, triggered release liposomes versus DXR-containing, non-sensitive, liposomal formulations. We see a significant correlation between the rate of nuclear accumulation of DXR and its in vitrocytotoxicity. This indicates that pH-sensitive formulations traffic drug to the cytoplasm and the nucleus significantly more rapidly than do non-sensitive formulations. We conclude that the development of triggered release liposomes is a promising strategy for further improving the therapeutic efficacy of liposomal antineoplastic drugs targeted selectively to cancer cells by surface-attached ligands that bind to internalizing epitopes.

N-(4-hydroxyphenyl) retinamide is cytotoxic to melanoma cells in vitro through induction of programmed cell death.

Melanoma is a highly malignant and increasingly common tumour. Since metastatic melanoma remains incurable, new treatment approaches are needed. Previously, we reported that the synthetic retinoid N-(4-hydroxyphenyl)retinamide (fenretinide, HPR) induces apoptosis in neuroblastoma cells, sharing a neuroectodermal origin with melanoma cells. Since no data exist thus far on the effects of HPR on human melanoma tumours, our purpose was to investigate the in vitro modulation of cell growth and apoptosis by HPR in melanoma cells. Ten human melanoma cell lines were exposed in vitro to increasing concentrations of HPR. Dose-dependent growth inhibition and cytotoxicity were observed. According to cytofluorimetric analysis, propidium iodide staining and TUNEL assay, HPR-treated melanoma cells were shown to undergo apoptosis. However, IC50 values ranged from 5 to 28 microM, while IC90 values were between 10 and 45 microM. These last concentrations are approximately 10-fold higher than those achievable in patients given oral HPR. To explore the potential of new delivery strategies, HPR was loaded at high concentrations into immunoliposomes directed to disialoganglioside GD2, a tumour-specific antigen extensively expressed by neuroectoderma-derived tumours. Treatment of melanoma cells for a short time (2 hr) with HPR-containing immunoliposomes followed by culture in drug-free medium gave rise to apoptosis of target cells, whereas cells treated for 2 hr with equivalent concentrations of the free drug survived. The efficacy of immunoliposomal HPR was strongly dependent on the density of GD2 expression in the different cell lines.

GD2-mediated melanoma cell targeting and cytotoxicity of liposome-entrapped fenretinide.

Melanoma is a highly malignant and increasingly common neoplasm. Because metastatic melanoma remains incurable, new treatment approaches are needed. Immunoliposomes have been previously shown to enhance the selective localization of immunoliposome-entrapped drugs to solid tumors with improvements in the therapeutic index of the drugs. Previously, we reported that the synthetic retinoid fenretinide (HPR) is an inducer of apoptosis in neuroblastoma (NB) cells, sharing the neuroectodermal origin with melanoma cells. HPR is a strong inducer of apoptosis also in melanoma cells, although at doses 10-fold higher than those achievable clinically. Thus, our purpose was to investigate the in vitro potentiation of its cytotoxic effect on melanoma cells in combination with long-circulating GD2-targeted immunoliposomes. GD2 is a disialoganglioside extensively expressed on tumors of neuroectodermal origin, including melanoma. Murine anti-GD2 antibody (Ab) 14.G2a and its human/mouse chimeric variant ch14.18 have been ligated to sterically stabilized liposomes by covalent coupling of Ab to the polyethylene glycol (PEG) terminus. Ab-bearing liposomes showed specific, competitive binding to and uptake by various melanoma cell lines compared with liposomes bearing non-specific isotype-matched Abs or Ab-free liposomes. Cytotoxicity was evaluated after 2 hr treatment, followed by extensive washing and 72 hr incubation. This treatment protocol was designed to minimize non-specific adsorption of liposomes to the cells, while allowing for maximum Ab-mediated binding. When melanoma cells were incubated with 30 microM HPR entrapped in anti-GD2 liposomes, a significant reduction in cellular growth was observed compared to free HPR, entrapped HPR in Ab-free liposomes or empty liposomes. Cytotoxicity was not evident in tumor cell lines of other origins that did not express GD2. Growth of NB cells was also inhibited by immunoliposomes with entrapped HPR.