Stability of calcium levofolinate, 5-fluorouracil and oxaliplatin (FOLFOX) mixture.

FOLFOX is the most common chemotherapy combination prescribed in colorectal cancer. It is composed of calcium levofolinate, 5-fluorouracil and oxaliplatin which demonstrated synergistic outcome. Nowadays, the lack of all-in-one formulation is due to the chemical composition of the pharmaceutical products and the highly pH-dependent stability of each drug. Herein, we aimed to investigate the stability of a ternary mixture of 5-fluorouracil, oxaliplatin and calcium levofolinate, knowing that coadministering these drugs would improve their efficacy. The effect of three pHs (5.0, 6.0 and 7.5) and two drug concentrations (8/3/6 and 1/1/1 mg/ml for 5-fluorouracil, oxaliplatin and calcium levofolinate, respectively) were examined. A high-performance liquid chromatography method was developed to separate and quantify the three drugs in one run. At higher concentrations, the ternary mixture was unstable regardless of pH. By reducing concentration, drug stability and compatibility in the mixture was improved at pH 5.0 for up to 3 days at +5°C ± 3 °C. In addition, binary mixtures provided stable properties at defined pHs. 5-fluorouracil/oxaliplatin mixture was stable at pH 5.0 over 48 hours while 5-fluorouracil/calcium levofolinate mixture was stable at pHs 6.0 and 7.5 up to 7 days.

Combination of tumor cell anti-adhesion and anti-tumor effect to prevent recurrence after cytoreductive surgery in a mice model.

One of the main problems of colorectal cancer is not the treatment of the primary tumor but the metastatic stage. Means of metastatic spread is the invasion of the peritoneal cavity which leads to peritoneal metastasis (PM). PM cannot be easily cured, and the current treatments is rather heavy, combining cytoreductive surgery with intravenous and intraperitoneal chemotherapy. This therapeutic procedure is associated with significant morbidity, altered patient quality of life and poor prognosis. We postulated that development of a prophylactic treatment could be of high interest in this context. In this study, we formulated an anti-adhesive thermogel which contains chemotherapeutics to play a role of a barrier against tumor cells implantation, avoiding their adhesion and treating the remaining tumor cells with chemotherapy intraperitoneally in a mice model of PM. The bioavailability of the thermogel was tested intraperitoneally in mice. No sign of toxicity was observed in terms of change in body weight, anatomopathology and blood biomarkers. In vitro experiments proved that the thermogel induced limited adhesion of the tumor cells. Loading of oxaliplatin (Ox) and 5-Fluorouracil (5-FU) into the thermogel were able to significantly decreased peritoneal carcinomatosis index (PCI) (-58%) and ascites (-70%) in a murine model of peritoneal metastases. These pre-clinical results confirmed that smart thermogel associated with standard chemotherapy 5-FU and Ox could be a good candidate to decrease the risk of tumor cell implantation during cytoreductive surgery and prevent future metastatic process.

Thermosensitive hydrogels for local delivery of 5-fluorouracil as neoadjuvant or adjuvant therapy in colorectal cancer.

Spurred by high risk for local tumor recurrence and non-specific toxicity of systemic chemotherapy, clinicians have recently granted a growing interest to locoregional therapeutic strategies. In this perspective, we recently developed a multipurpose thermosensitive hydrogel based on reversible thermogelling properties of poloxamers P407 and P188, a bioadhesive excipient and antineoplastic effect of 5-fluorouracil (5-FU) for the local treatment of colorectal cancer (CRC) in ectopic CT26 murine models. Antitumor efficacy was assessed in mice following intratumoral (IT) injection mimicking neoadjuvant therapy and subcutaneous (SC) application after tumor excision simulating adjuvant therapy. Rheological characterization disclosed that P407/P188/alginate 20/2/1% w/v thermosensitive hydrogel is an injectable free-flowing solution at ambient temperature that undergoes a SOL-GEL transition at 26.0 °C ± 0.6 °C and thereby forms in situ a non-flowing gel at physiological temperature. The generated gel presented an elastic behavior and responded according to a shear-thinning fluid upon shear rate. Although delayed by the addition of alginate 1% w/v, 5-FU is released mainly by diffusion mechanism. The local delivery of 5-FU from P407/P188/alginate/5-FU 20/2/1/0.5% w/v hydrogel in the preclinical tumor models led to a significant tumor growth delay. These results demonstrated that poloxamer-based thermosensitive hydrogels provide a simple and efficient means for local chemotherapeutics delivery.

Mucoadhesive thermosensitive hydrogel for the intra-tumoral delivery of immunomodulatory agents, in vivo evidence of adhesion by means of non-invasive imaging techniques.

Intratumoral injection of biocompatible gels is increasingly used for the sustained delivery of drugs and vaccines to enhance the anti-cancer immune response. Granulocyte-macrophage colony stimulating factor (GM-CSF) has become an attractive adjuvant thanks to its ability to boost the antitumor immune response by inducing proliferation, maturation and migration of the dendritic-cells (DCs) and the differentiation of lymphocytes. Killed Mycobacteria, such as Heat-killed Mycobacterium tuberculosis (HKMT) have been used in several studies as TLR-2 agonist to increase maturation of DCs. In this study, we designed a mucoadhesive thermosensitive formulation for the local delivery of GM-CSF and HKMT in order to enhance DCs activation and improve the local antitumor immune response. This formulation was selected based on its elastic and mucoadhesive properties obtained thanks to rheological studies. More importantly, intratumoral residence time of the labelled gel and protein were evidenced by means of MRI and non invasive in vivo optical imaging. Then, the efficacy of the combination of immunomodulators loaded thermogel was demonstated in vitro and in vivo. The selected thermogel exhibits rheological properties which confer a good elasticity and increased residence time of the immunostimulatory agents in the tumor, thus increasing the recruitment of DCs and T cytotoxic CD8+ lymphocytes.

Ectosomes from neutrophil-like cells down-regulate nickel-induced dendritic cell maturation and promote Th2 polarization.

DCs are the first immune cells to be exposed to allergens, including chemical sensitizers, such as nickel, a human TLR4 agonist that induces DC maturation. In ACD, DCs can interact with PMNs that are recruited and activated, leading, in particular, to ectosome release. The objective of this work was to characterize the effects of PMN-Ect on DC functions in an ACD context. We first developed a standardized protocol to produce, characterize, and quantify ectosomes by use of human PLB-985 cells, differentiated into mature PMN (PLB-Ect). We then studied the in vitro effects of these purified ectosomes on human moDC functions in response to NiSO4 and to LPS, another TLR4 agonist. Confocal fluorescence microscopy showed that PLB-Ect was internalized by moDCs and localized in the lysosomal compartment. We then showed that PLB-Ect down-regulated NiSO4-induced moDC maturation, as witnessed by decreased expression of CD40, CD80, CD83, CD86, PDL-1, and HLA-DR and by decreased levels of IL-1ß, IL-6, TNF-α, and IL-12p40 mRNAs. These effects were related to p38MAPK and NF-κB down-regulation. However, no increase in pan-regulatory DC marker genes (GILZ, CATC, C1QA) was observed; rather, levels of effector DC markers (Mx1, NMES1) were increased. Finally, when these PLB-Ect + NiSO4-treated moDCs were cocultured with CD4(+) T cells, a Th2 cytokine profile seemed to be induced, as shown, in particular, by enhanced IL-13 production. Together, these results suggest that the PMN-Ect can modulate DC maturation in response to nickel, a common chemical sensitizer responsible for ADC.

Focused ultrasound influence on calcein-loaded thermosensitive stealth liposomes.

Focused ultrasound (FUS) is a versatile technology for non-invasive thermal therapies in oncology. Indeed, this technology has great potential for local heat-mediated drug delivery from thermosensitive liposomes (TSLs), thus improving therapeutic efficacy and reducing toxicity profiles. In the present study we evaluated the influence of FUS parameters on the release of calcein from TSLs used to model a hydrophilic drug. Quantitative calcein release from TSLs (DPPC/CHOL/DSPE-PEG2000: 90/5/5) and non-thermosensitive liposomes (NTSLs) (DPPC/CHOL/DSPE-PEG2000: 65/30/5) was measured by spectrofluorimetry after both water bath and FUS-induced in vitro heating. The heating of TSLs at 42 °C in a water bath resulted in a maximum calcein release of 45%. No additional calcein release was observed at temperatures above 42 °C. A similar percentage of calcein release was achieved when TSLs were exposed to 1 MHz sinusoidal waves at peak negative pressure of 1.5 MPa, 40% duty cycle, for 10 min (i.e. above 42 °C). No release was detected when NTSLs were heated in a water bath. For both TSLs and NTSLs, the calcein release was increased by more than 10% for acoustic pressures ranging from 1.5 MPa to 2 MPa. This additional release was attributed to the mechanical stress generated by FUS, which was sufficient to disrupt the liposomal membrane. Furthermore, analysis of cryo-TEM images showed a significant decrease in liposome size (14%) induced by the thermal effect, whereas the liposome diameter remained unaffected by the FUS-triggered non-thermal effects.

Formulation and pharmacokinetics of thermosensitive stealth® liposomes encapsulating 5-Fluorouracil.

PURPOSE: We optimize the encapsulation and investigate the pharmacokinetics of 5-Fluorouracil (5-FU) delivered by thermosensitive stealth(®) liposomes (TSLs) designed to trigger drug release upon hyperthermia using focused ultrasound (FUS). METHODS: 5-FU was encapsulated into liposomes made of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol/1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000 either as a free molecule or complexed with copper-polyethylenimine. Heat-triggered drug release was evaluated using either a water bath or FUS. Formulation cytotoxicity was assessed on HT-29 cell line by MTS assay. Pharmacokinetics and biodistribution of 5-FU were evaluated in HT-29-tumor bearing mice. RESULTS: 5-FU was easily encapsulated using the lipid hydration method (encapsulation efficacy of 13%) but poorly retained upon dilution. 5-FU complexation with copper-polyethylenimine improved 5-FU retention into liposomes and allowed to obtain an encapsulation efficacy of 37%. At 42°C, heat-triggered 5-FU release from TSLs was 63% using a water bath and 68% using FUS, within 10 min, whereas it remained below 20% for the non-thermosensitive formulation. The MTS assay revealed that formulation toxicity arose from 5-FU and not from the excipients. In addition, 5-FU complex encapsulation into TSLs induces a reduction of the IC50 from 115 down to 49 µM. Pharmacokinetics reveals a longer circulation of encapsulated 5-FU and a more important body exposure, although tumor passive targeting is not significantly higher than free 5-FU. CONCLUSIONS: Complexation of 5-FU with copper-polyethylenimine appears an interesting strategy to improve 5-FU retention into TSLs in vitro and in vivo. TSLs allow heat-triggered release of the drug within 10 min at 42°C, a reasonable time for future in vivo experiments.