- 1 -
Lauroyl-gemcitabine loaded lipid nanocapsules for the treatment of metastases in mediastinal lymph nodes.
N. Wauthoz
1, E. Moysan
1, K. Kondo
2, M. Zandecki
3, V. Moal
3, M.-C. Rousselet
3, J.
Hureaux
1,3, G. Bastiat
1,, J.-P. Benoit
11
LUNAM Université – Micro et Nanomédecines Biomimétiques, INSERM – U1066 IBS-CHU, F-49933, Angers, France, email : guillaume.bastiat@univ-angers.fr;
2
Department of Oncological and Regenerative Surgery, University of Tokushima, Kuramoto-cho, Tokushima 770-8503, Japan, email : kondo@clin.med.tokushima-u.ac.jp;
3
Hematology, Biochemistry, Cell and Tissue Pathology and Pneumology Departments, Academic Hospital, Angers, F-49933, France, email : JoHureaux@chu-angers.fr;
INTRODUCTION
Lung cancers remain the leading cause of cancer-related mortality (both in males and females) around the world (Siegel et al., 2013). Once diagnosed, non-small cell lung cancers (NSCLC) 5-year survival rate reaches globally 15%
with the different therapeutic modalities such as surgery, radiotherapy and chemotherapy. For most patients, NSCLC had spread in mediastinal and supraclavicular lymph nodes (N2 disease, stage III) or in the contralateral lung, pleural cavity or beyond lung (M1 disease, stage IV) (Fung et al., 2012). These advanced stages are generally treated using sequential or concurrent chemotherapy and radiotherapy (stage III) or combined chemotherapies (stage IV). Because conventional chemotherapies are not specific and selective enough and are not able to reach the site of metastases, a therapeutic plateau with cytotoxic drugs has been reached for many years. In aim to protect the drug until the site of action, target the lymph nodes invaded by metastases and decrease the related systemic toxicities, nanomedicine could be a very great tool (Schroeder et al., 2012).
A new nanocarrier system, lipid nanocapsules (LNC) loaded with a lauroyl-modified gemcitabine (Gem-C12) have been developed and demonstrated the ability to form a hydrogel by nanoparticle assembly (Moysan et al., 2013).
This nanocarrier could be delivered as a gel by subcutaneous (sc) route or as a suspension (after dilution) for intravenous (iv) administration. The purpose of this study is to reach passively the lymph nodes in vivo, and to evaluate the antitumor efficacy of this new system in a human NSCLC metastatic model, which mimic the spreading of metastases in mediastinum from the primary tumor implanted in the lung of mice. The tolerance (myelosupression and hepatotoxicity) of this new treatment delivered either as a gel by sc route or as a suspension by iv route will be investigated.
EXPERIMENTAL METHODS
Gem-C12-loaded LNC formulation process
Gemcitabine modification and the LNC loading and characterization were already described (Moysan et al., 2013).
Orthotopic intrapulmonary implantation procedure The orthotopic intrapulmonary implantation procedure (Ma44-3 cell line derived from human NSCLC) was performed as previously reported (Ishikura et al., 2000), using male SCID-CB17 mice (4-5 weeks of age) (Charles River, l’Arbresle, France). Apparition of lung tumor and metastases was control using 5-aminolevulinic acid (ALA), metabolized in protophorphyrin IX (PtIX) in cancer cells.
Visualization of fluorescent LNC after iv or sc administrations
The visualization of LNC loaded with DiD (fluorescent probe) injected by iv or sc was performed on mice grafted with Ma44-3 cells. Fluorescence imaging of lung and mediastinum area were performed ex vivo using the CRI Maestro system (Woburn, USA).
In vivo antitumor efficacy
Treatments were started 5 days after the intrapulmonary implantation. At day 5, 7 and 9, NaCl 0.9% solution (saline iv), non-loaded LNC (non-loaded LNC iv), commercial gemcitabine hydrochloride (Gemcitabine iv), Gem-C12 (Gem-C12 iv) and liquid form of Gem-C12 loaded LNC (LNC Gem-C12 iv) were intravenously administered. At day 5 and 9, non-loaded LNC (non-loaded LNC sc) and gel form of Gem-C12 loaded LNC (LNC Gem-C12 sc) were subcutaneously administered. The total dose delivered in mice was 40 mg (molar equivalent gemcitabine hydrochloride) per kilo of body weight (n=10 per group).
In vivo hematologic and hepatic toxicity evaluation The hematological parameters were determined in the Haematology Ward of the Academic Hospital of Angers with an XE-2100 haematology analyser (Sysmex) and plasma biochemistry analyses were carried out at the Biochemistry Ward of the Academic Hospital of Angers on a Modular P® (Roche diagnostics). Mice (n=5 per group) blood samples were collected 13 days after intrapulmonary implantation.
- 2 - Statistical analysis
Survival analyses were carried out by means of Kaplan- Meier curves (log-rank test). Mean group comparisons were performed using Kruskal-Wallis test, with Dunn post-hoc tests for pairwise comparisons. Differences were considered statistically significant for p < 0.05.
RESULTS AND DISCUSSION
Figure 1. Visualization of lungs of mice at day 5 after tumor implantation with ALA oral administration and localization of DiD-loaded LNC delivered by sc (A/B) and iv (C/D) routes using PpIX (A/C) and DiD (B/D) visualization modes.
The nanocarrier loaded with a lipophilic derivative of gemcitabine (Gem-C12 loaded LNC) is able to passively target metastases in mediastinal lymph nodes (sc route).
Using iv route, both metastases and primary tumor in lungs were passively targeted (see Figure 1).
Concerning antitumor efficacy, significant difference was observed between controls (empty LNC and saline) and all gemcitabine treatments. Nevertheless, whatever the administration route of gemcitabine-loaded LNC, similar antitumor efficacies than the conventional systemic gemcitabine were observed in the lymphogenous metastatic preclinical model, with a schedule of twice a week for sc route in comparison to three times a week for the iv one (see Figure 2). The main advantage of sc route is the decrease of repeated administrations.
Moreover, using Gem-C12 loaded LNC (iv or sc routes), lower systemic side effects were observed in comparison to free gemcitabine after iv administration. No significant depletion in platelet count and in phosphatase alkaline were observed using Gem-C12 loaded LNC (data not shown), which means that a decrease in treatment toxicity.
ACKNOWLEDGMENTS
This work has been realized within the research program LYMPHOTARG financially supported by EuroNanoMed ERA-NET 09 and by the Région Pays de la Loire.
Figure 2. (A) The Kaplan-Meier survival curves and (B) the weight evolution of mice (n=10 per group) grafted with Ma44- 3 cells after various gemcitabine treatments (total dose of 40 mg/kg, molar equivalent gemcitabine hydrochloride).
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