Monica Loi

Drug delivery systems : application of liposomal anti-tumor agents to neuroectodermal cancer treatment

Disseminated neuroectoderma-derived tumors, mainly neuroblastoma in childhood and melanoma in the adulthood, are refractory to most current therapeutic regimens and hence the prognosis remains very poor. Preclinical research studies have indicated several agents that show promising therapeutic potential for these neoplasms. However, there appears to be a limitation to their in vivo applicability, mainly due to unfavorable pharmacokinetic properties that lead to insufficient drug delivery to the tumor or metastatic sites or to high systemic or organ-specific toxicity. In this scenario, the focus is on targeted cancer therapy. Encapsulating anticancer drugs in liposomes enables targeted drug delivery to tumor tissue and prevents damage to the normal surrounding tissue. Indeed, sterically stabilized liposomes have been shown to enhance the selective localization of entrapped drugs to solid tumors, with improvements in therapeutic indices. The identification of tumor-associated antigens and/or genes and the relative ease of manipulating the physicochemical features of liposome hold promise for the development of novel therapeutic strategies that selectively target tumor cells. Combined targeting is still investigated, especially the availability to simultaneously target and kill both the cancer cells and the tumor vasculature. Animal models make it possible to link molecular genetics and biochemistry information to the physiological basis of disease and are important predictive tools that offer a frontline testing system for studying the involvement of specific genes and the efficacy of novel therapeutics approaches. Relevant experimental models of human neuroblastoma and melanoma, which better reflect the tumor behavior in patients, are required to evaluate the effectiveness of the various targeted liposomal formulations and their possible systemic and organ-specific toxicity. The most multifunctional targeted liposomes are herein described, with primary attention on testing their efficacy in clinically relevant animal models for the treatment of neuroblastoma and melanoma.

Anti-IL-10R antibody improves the therapeutic efficacy of targeted liposomal oligonucleotides

High-risk Neuroblastoma (NB) has still a poor prognosis. Liposomes targeted to NB cells and encapsulating antisense CpG-containing oligonucleotides (TL-asCpG) had increased anti-tumour efficacy in NB xenografts compared to free asCpG. Interleukin 10 (IL-10) suppresses antigen presenting cell activation contributing to tumour-mediated immune suppression. In principle, combination of TL-asCpG and antibodies against IL-10 receptor (aIL-10R) could prolong immune system activation, leading to better therapeutic results. Mice treated with TL-asCpG 4 h after human NB cell inoculation survived significantly longer than controls. An increased life span was achieved also in mice receiving TL-asCpG 24 and 72 h after NB cell challenge. The addition of aIL-10R to TL-asCpG in the 4-h protocol significantly increased the percentage of long term survivors compared to TL-asCpG only. Surviving mice treated with the combined strategy were completely cured. In contrast, long term surviving mice treated only with TL-asCpG presented lymph node infiltration with NB cells. TL-asCpG plus aIL-10R treatment was significantly superior to TL-asCpG alone also for the 24-h protocol. Ex vivo experiments demonstrated that the combined therapy evoked a stronger and more prolonged immune system activation compared to monotherapy. These results support the feasibility of a clinical trial with TL-asCpG and aIL-10R in advanced NB patients.

Abstract 3625: Novel phage-display derived peptides for tumor- and vasculature-targeted therapies in neuroblastoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Disseminated neuroblastoma (NB) is refractory to most current therapeutic regimens. The therapeutic index of anticancer drugs is increased by liposome encapsulation and further improvements is obtained by coupling tumor-targeting ligands to the surface of the lipidic envelop. Phage display technology is a powerful tool in discovering novel ligands specific to receptors on the surface of tumor epithelial and endothelial cells. Therapeutic targeting to tumor blood vessels combines blood vessel destruction with the expected anti-tumor activities of the drug, resulting in increased efficacy and reduced toxicity. To find NB-specific targeting moieties, we established a protocol for the isolation of heterogeneous cell populations by tissue fractionation of primary tumors and metastases from two models of human NB (with tumor cells injected either intravenously, to mimic minimal residual disease, or orthotopically in the adrenal gland of mice, to reflect the growth of advanced NB in children with large adrenal gland tumors and small metastatic lesions) and from stage IV, stroma poor, NB-derived specimens immediately after surgical removal. Cells extracted from corresponding healthy organs from mice and patients were used both in a negative pre-selection step and as a negative control for specific phage enrichment. The NB cell suspensions were subjected to multi-step screenings with the phage-displayed peptide library CX7C (where C = cysteine and X = any aminoacid). We globally isolated 135 NB-binding peptides. Of these, 31 were selected for binding to the primary tumor mass, 16 to the metastatic mass, 63 to tumor endothelial cells, and 25 to endothelial cells of metastases. Several proteins presenting sequence homology with the discovered peptides have been identified by BLAST analysis and were evaluated for their expression in NB tumors, derived from both mouse xenogratfs and patient specimens. Specifically, 5 novel phage display derived-peptides showed specific binding on NB specimens and homing to tumor cells and tumor vasculature, 10 minutes and 24 hours after injection through the tail vein of NB-bearing mice. We are testing the new molecular, tumor- and vasculature-specific peptides for generating novel tumor-specific liposomal therapies against NB. The availability of novel ligands binding to additional tumor-associated antigens and to targets on both endothelial and perivascular tumor cells will allow to design more sophisticated liposomal targeted anticancer strategies that exhibit high levels of selective toxicity for the cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3625. doi:10.1158/1538-7445.AM2011-3625

Abstract 5144: Oligonucleotide-based inhibition of the pro-angiogenic activity of Hepatoma-derived growth factor related protein 3 in neuroblastoma

Hepatoma-derived growth factor related protein 3 (HDGFRP3) belongs to the HDGF polypeptide family. Expression of HDGF is a prognostic factor for NSCLC, pancreatic and gastrointestinal tumors, and silencing of HDGF suppressed cell proliferation, migration and invasion of squamous lung cancer cells. Here we investigated the expression of HDGFRP3 in various tumor cell lines by quantitative PCR and western blot analysis. HDGFRP3 is highly expressed and secreted from neuroblastoma (NB) cell lines. The presence of high levels of HDGFRP3 was confirmed in two animal models of human NB. Since HDGFRP3 shares a conserved Pro-Trp-Trp-Pro motif (PWWP) domain with its family members, we assessed whether it could promote proliferation, migration and angiogenesis in analogy to HDGF. Recombinant HDGFRP3 inhibited migration of endothelial cells in a modified Boyden chamber experiment, while cell survival was not affected. Nevertheless, the protein promoted angiogenesis in vitro, as assessed through vascular sprouting and tubulogenesis assays. Starting from the structural insight of the PWWP domain, an oligonucleotide-based inhibitor was designed and tested to block the pro-angiogenic effect of HDGFRP3. A single stranded DNA inhibited the pro-angiogenic properties of HDGFRP3 in vitro. After modifications to improve its serum stability, the cytotoxicity of the designed oligonucleotide-based molecule (BN210) was tested by an MTT cell viability assay on various NB cell lines. BN210 slightly reduced NB cells proliferation. To determine if BN210 could be used both as single anti-angiogenic agent and in combination with chemotherapy in vivo, orthotopic NB tumor-bearing mice were randomly divided in different groups of treatment: 1 mg/kg of vincristine (VCR), once a week for 4 weeks; 7-21 mg/kg of BN210, either i.p. (5 days/week) or i.v (every 2 days). Other mice were treated with a combination of VCR and BN210. NB-bearing mice treated with BN210 alone showed a partial increase in life span, compared to control mice. However, mice treated with the combination of VCR plus BN210 had statistically significant increased life span, compared to both the control and the single treatment groups, with long term survivors obtained with the combined schedule when BN210 was injected i.v. Thus, the oligonucleotide-based inhibition of the pro-angiogenic activity of HDGFRP3 can be considered as a novel strategy for combined anti-tumor therapy in NB. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5144. doi:10.1158/1538-7445.AM2011-5144

Abstract C215: EZN‐2208, a novel pegylated SN‐38 drug conjugate, markedly inhibits tumor growth and metastatic spreading in preclinical models of human neuroblastoma

Treatment of neuroblastoma (NB) is successful in less than half of patients with high‐risk disease. Camptothecin and its analog irinotecan (CPT‐11), hold great promise but several limitations suggest that chemical modifications may improve the therapeutic index. EZN‐2208 is a water soluble pegylated SN38 drug conjugate, composed of a four‐arm 40 KDa polyethylene glycol (PEG) linked via glycine residue to SN38. In various preclinical models of solid tumors EZN‐2208 was more efficacious than CPT‐11. In phase I trials in adult tumors, EZN‐2208 was well‐tolerated with neutropenia as the dose limiting toxicity. Here, the anti‐tumor activity of EZN‐2208 was first evaluated in preclinical, pseudometastatic and an orthotopic, models of human NB. Mice were treated every other day for 5 total doses with 10 mg/kg of CPT‐11 or with the SN38 equivalents of EZN‐2208. In the first model, mice treated with EZN‐2208 displayed significant increased life span compared to control mice or those treated with CPT‐11. After 150d post cell implantation, all EZN‐2208‐treated mice were still disease‐free, while control and CPT‐11‐treated animals died with metastatic disease. In the orthotopic model, mice treated with EZN‐2208 showed a dramatic arrest and regression in primary tumor growth compared to control mice. While CPT‐11‐treated mice died with widespread tumor masses within 80 days, long term survival was seen in 100% of EZN‐2208‐treated animals. 21 days after the end of treatment tumors had almost disappeared, as assessed by staining histological sections of the tumors with antibodies recognizing NB cells and the cell proliferation marker, Ki‐67. In a second set of in vivo experiments, MTD doses of both CPT‐11 and EZN‐2208 were compared in immunodeficient (GI‐LI‐N cells) and immunocompetent (NXS2 cells) orthotopic NB animal models. While CPT‐11 at MTD dose led to a partial increased in long term survival, EZN‐2208‐treated, GI‐LI‐N‐bearing mice were 100% cured after 180 days post cells implantation. In the very aggressive syngeneic NB animal model (NXS2 cells), while CPT‐11 did not exert any anti‐tumor effect, EZN‐2208 led to 100% and 40% of long term survivors, in mice challenged with 5x104 and 5x105 tumor cells, respectively. The differences in the anti‐angiogenic activity between CPT‐11 and EZN‐2208 was evaluated by chorioallantoic membrane (CAM) assay. Incubation of the CAMs with EZN‐2208 significantly reduced the number of radiating vessels that invaded the implant compared to either specimens alone or CAMs incubated CPT‐11. In the last set of in vivo experiments, the effects of EZN‐2208 and CPT‐11 were compared in a luciferase‐transfected human NB cells inoculated in the right flank of mice. EZN‐2208 led to a significant tumor regression compared to CPT‐11. Finally, mechanistic experiments showed enhanced TUNEL and Histone H2ax staining in tumors removed from mice treated with EZN‐2208, indicating its effect on tumor cell apoptosis. In conclusion, EZN‐2208 could be considered as a new, promising anti‐NB agent, to be administered alone and/or in combination with traditional chemotherapeutics. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C215.

Abstract A130: Effects of a novel liposomal formulation of fenretinide on human neuroblastoma cell growth, apoptosis and angiogenesis

Neuroblastoma (NB) is an embryonal tumor originating from the simpatico‐adrenal lineage of the neural crest and developing virtually at any site of the sympathetic nervous system. It approximately accounts for about 15% of all paediatric oncology deaths. NB is characterized by different clinical presentations and courses. Metastatic tumors at diagnosis remains a clinical challenge, despite advances in multimodal therapy. Retinoids are a class of compounds known to induce both terminal differentiation and apoptosis/necrosis of NB cells. Among them, fenretinide (HPR), thanks to its good safety profile in humans, has been considered one of the most promising synthetic retinoids tested as chemopreventive agent but it is only partially efficacious due to both a scanty oral delivery and a rapid methabolism. Here, we have developed a novel liposomal HPR by which the drug was encapsulated into sterically stabilized liposomes [SL‐(HPR)] according to the reverse‐phase evaporation method. This procedure led to a higher drug encapsulation and to better size and stability in organic fluids when compared to our previously HPR‐entrapped liposome preparation (Pagnan et al., Int.J.Cancer 1999;81:268). Moreover, liposomes were coupled with NGR peptides that target the tumor endothelial cell marker, the aminopeptidase N [NGR‐SL‐(HPR)] (Pastorino et al, Cancer Res 2006;66:10073). The in vivo sensitivity of NB to the HPR liposomal preparations was tested against an established orthotopic NB xenograft animal model. Tumor‐bearing mice treated with NGR‐SL‐(HPR) lived statistically longer than mice un‐treated or treated with free HPR [NGR‐SL‐(HPR) vs both control and HPR: P The use of this novel targeted delivery system for the apoptotic and anti‐angiogenic drug, fenretinide, could be considered as an adjuvant tool in the future treatment of NB patients. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A130.