Treatment of neuroblastoma and rhabdomyosarcoma using RGD-modified liposomal formulations of patupilone (EPO906).

BACKGROUND: Patupilone (EPO906) is a microtubule stabilizer with a potent antitumor effect. Integrin αVß3-binding (RGD) liposomes were loaded with EPO906, and their antitumor efficacy was evaluated in two pediatric tumor models, ie, neuroblastoma and rhabdomyosarcoma. METHODS: Integrin αVß3 gene expression, RGD-liposome cellular association, and the effect of EPO906 and liposomal formulations of EPO906 on cell viability were assessed in vitro in human umbilical vein endothelial cells (HUVEC), in the RH-30 rhabdomyosarcoma cell line, and in the Kelly neuroblastoma cell line. In vivo, mice bearing neuroblastoma or rhabdomyosarcoma tumors were treated with EPO906, EPO906-liposomes, or EPO906-RGD-liposomes. Tumor growth, cumulative survival, and toxicity were monitored. RESULTS: Integrin αVß3 was highly expressed in HUVEC and RH-30, but not in Kelly cells. Accordingly, RGD-liposomes were highly associated with HUVEC and RH-30 cells in vitro, but not with the Kelly cells. EPO906 and its liposomal formulations inhibited HUVEC, RH-30, and Kelly cell viability to the same extent. In vivo, EPO906 1.5 mg/kg and liposomal EPO906 potently inhibited tumor growth in both xenograft models without triggering major toxicity. At this dose, liposomal EPO906 did not enhance the antitumor effect of EPO906 in neuroblastoma, but tended to have an increased antitumor effect in rhabdomyosarcoma. Using a lower dose of EPO906-RGD-liposomes significantly enhanced cumulative survival in rhabdomyosarcoma compared with EPO906 alone. CONCLUSION: EPO906 shows a strong antitumor effect in neuroblastoma and rhabdomyosarcoma, without triggering major side effects. Its liposomal encapsulation does not alter its activity, and enhances cumulative survival when EPO906-RGD-liposomes are used at low dose in rhabdomyosarcoma.

Liposomal doxorubicin-based treatment in a preclinical model of adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a rare endocrine tumor entity with poor prognosis. Medical treatment is limited to common cytotoxic agents, which are associated with low treatment responses. Thus, lack of therapeutic efficacy demands innovative treatment options for patients with advanced ACC. Recently, we have developed and characterized anti-IGF1 receptor (IGF1-R) immunoliposomes (SSLD-1H7) for the treatment of neuroendocrine tumors of the gastroenteropancreatic system. As previous results indicated putative applicability also for other IGF1-R-overexpressing tumor entities, we initiated testing of liposomal preparations in in vitro and in vivo models of ACC. Adrenocortical NCIh295 cells were used for in vitro association studies with different liposomal formulations. Thereby, flow cytometry revealed high cellular association and internalization of anti-IGF1-R immunoliposomes (soy phosphatidylcholine (SPC)/cholesterol (Chol)-polyethyleneglycol (PEG)-1H7, 50.1±2.2%). Moreover, internalization of pegylated liposomes (SPC/Chol-PEG, 57.1±2.4%) and an even higher uptake of plain liposomes (84.6±0.8%; P<0.0001) were detectable in adrenocortical tumor cells. In vivo, liposomal treatments were investigated on NCIh295 tumor xenografts in pharmacokinetic and therapeutic experiments. A significant reduction in tumor size was detectable in NCIh295 tumor-bearing mice after a single treatment with SSLD-1H7 (0.89±0.15 cm; P=0.006) and a diminished efficacy for SSLD-PEG+ (1.01±0.19 cm; P=0.04) in comparison with untreated controls (1.5±0.0 cm). Thus, anti-IGF1-R immunoliposomes have been successfully tested in vitro and in vivo in a preclinical model for ACCs and could, therefore, represent a promising therapeutic approach for this tumor entity. Moreover, a combination of mitotane plus liposomally encapsulated cytostatic agents instead of free drugs could also be an interesting novel treatment option for ACC in the future.

The encapsulation of idarubicin within liposomes using the novel EDTA ion gradient method ensures improved drug retention in vitro and in vivo.

The purpose of this study was to design a new stable liposomal formulation for the anticancer drug idarubicin. Idarubicin is a relatively hydrophobic member of the anthracycline family. It exhibits pronounced bilayer interactions leading to rapid in vivo drug release from liposomes. This rapid drug leakage is due to the presence of cholesterol and charged lipids in the liposomal bilayer. Therefore, a novel method of remote drug loading was developed to prevent rapid drug release from PEGylated cholesterol-containing liposomes. This method uses EDTA disodium or diammonium salt as an agent to form low solubility complexes between the drug and EDTA molecules inside the liposomes, thus yielding improved drug retention. The efficiency of idarubicin encapsulation is close to 98% at a drug to lipid molar ratio of 1:5. An in vitro long-term storage experiment confirmed the high stability of the liposomes. The in vivo studies also showed the superiority of the new idarubicin formulation over the recently used remote loading methods. The plasma level of idarubicin was much higher when EDTA liposomes were used. The presented results fully demonstrate the superiority of the proposed method of idarubicin encapsulation over existing methods. The method offers the possibility of encapsulating not only all the anthracyclines, but also other weakly amphiphilic bases within the liposomes.

Remote loading of anthracyclines into liposomes.

The following chapter introduces a remote loading procedure for anthracyclines focussing on the well-established drug doxorubicin. The key advantage of remote loading is that it leads to higher drug to lipid ratios and encapsulation efficiencies compared to conventional passive trapping techniques like hydration of dried lipid films with aqueous drug solutions. The method presented is appropriate to produce sterile liposomal doxorubicin formulations with a final concentration of 2 mg/mL doxorubicin, which can be applied not only in vitro but also in vivo.

Anti insulin-like growth factor I receptor immunoliposomes: a single formulation combining two anticancer treatments with enhanced therapeutic efficiency.

CONTEXT: Through overexpression and aberrant activation in many human tumors, the IGF system plays a key role in tumor development and tumor cell proliferation. Different strategies targeting IGF-I receptor (IGFI-R) have been developed, and recent studies demonstrated that combined treatments with cytostatic drugs enhance the potency of anti-IGFI-R therapies. OBJECTIVE: The objective of the study was to examine the IGFI-R expression status in neuroendocrine tumors of the gastroenteropancreatic system (GEP-NETs) in comparison with healthy tissues and use potential overexpression as a target for novel anti-IGFI-R immunoliposomes. EXPERIMENTAL DESIGN: A human tumor tissue array and samples from different normal tissues were investigated by immunohistochemistry. An IGFI-R antagonistic antibody (1H7) was coupled to the surface of sterically stabilized liposomes loaded with doxorubicin. Cell lines from different tumor entities were investigated for liposomal association studies in vitro. For in vivo experiments, neuroendocrine tumor xenografts were used for evaluation of pharmacokinetic and therapeutic properties of the novel compound. RESULTS: Immunohistochemistry revealed significant IGFI-R overexpression in all investigated GEP-NETs (n = 59; staining index, 229.1 +/- 3.1%) in comparison with normal tissues (115.7 +/- 3.7%). Furthermore, anti-IGFI-R immunoliposomes displayed specific tumor cell association (44.2 +/- 1.6% vs. IgG liposomes, 0.8 +/- 0.3%; P < 0.0001) and internalization in human neuroendocrine tumor cells in vitro and superior antitumor efficacy in vivo (life span 31.5 +/- 2.2 d vs. untreated control, 19 +/- 0.6, P = 0.008). CONCLUSION: IGFI-R overexpression seems to be a common characteristic of otherwise heterogenous NETs. Novel anti-IGFI-R immunoliposomes have been developed and successfully tested in a preclinical model for human GEP-NETs. Moreover in vitro experiments indicate that usage of this agent could also present a promising approach for other tumor entities.

Novel 3-arylideneindolin-2-ones as inhibitors of NAD+ -dependent histone deacetylases (sirtuins).

Class III histone deacetylases (sirtuins) play pivotal roles in many cellular processes. They are linked to extended lifespan and to the pathogenesis of cancer and neuronal disorders. We present novel sirtuin inhibitors based on a 6,7-dichloro-2-oxindole scaffold with low micromolar activity. In vitro activity was rationalized by docking studies, and hyperacetylation of sirtuin targets could be demonstrated in cell culture.

Receptor-specific targeting with liposomes in vitro based on sterol-PEG(1300) anchors.

PURPOSE: The challenge in developing liposomes to be used in active drug targeting is to design a method that can be used for modifying liposomal membranes that is applicable for a number of different specific ligands. In this study, the post insertion technique was used with activated sterol-PEG(1300) anchors and was evaluated with regard to its effectiveness in active targeting in vitro. The key advantage of these anchors is that the insertion step into the liposomal membrane takes place at room temperature and is very fast. MATERIALS AND METHODS: For in vitro experiments, neuroblastoma cell lines overexpressing GD2 antigen on their surface as a target structure were chosen. This allowed the use of anti-GD2 antibodies coupled to the liposomal surface for testing of specific binding. These modified liposomes were labelled with rhodamine-PE and their cellular association was analyzed by flow cytometry. RESULTS: It was shown that the activated sterol-PEG(1300) anchors allow specific and significant interactions of the modified liposomes with GD2 positive cells. CONCLUSION: Coupling using sterol-PEG(1300) anchors is both simple and rapid. It is reproducible and applicable for all ligands bearing amino groups. This method demonstrates the advantage of a ready-to-use system for the modification of pre-formed liposomes with different ligands.