Nanomicellar Lenalidomide-Fenretinide Combination Suppresses Tumor Growth in an MYCN Amplified Neuroblastoma Tumor.

PURPOSE: In a previous study, we demonstrated that the combination of fenretinide with lenalidomide, administered by a novel nanomicellar formulation (FLM), provided a strong antitumor effect in a neuroblastoma TrkB-expressing tumor. In this study, we tested the nanomicellar combination in an MYCN amplified neuroblastoma xenograft to assess its efficacy in different tumor genotypes and evaluate the interactions of the nanomicelles with the tumor cells. EXPERIMENTAL DESIGN: FLM was administered to mice bearing human NLF xenografts to evaluate its efficacy in comparison with the nanomicelles containing fenretinide alone (FM). Confocal laser-scanning fluorescence microscopy images of the NLF cells treated with FLM and FM allowed us to estimate the nanomicelle ability to transport the encapsulated drugs inside the tumor cells. Flow cytometric analysis of the cells from treated tumors was performed to assess the effect of treatment on GD2 expression and NK cell infiltration. RESULTS: FLM and FM decreased the growth of NLF xenografts at comparable extents during the treatment period. Afterwards, FLM induced a progressive tumor regression without regrowth, while FM treatment was followed by regrowth within 15-20 days after the end of treatment. Both FLM and FM were able to penetrate the tumor cells transporting the encapsulated drugs. FLM transported higher amount of fenretinide inside the cells. Also, FLM treatment strongly increased GD2 expression in treated tumors and slightly decreased the NK infiltration compared to FM. CONCLUSION: FLM treatment induced a superior antitumor response than FM in NLF xenografts, presumably due to the combined effects of fenretinide cytotoxicity and lenalidomide antiangiogenic activity. The ability of FLM to penetrate tumor cells, transporting the encapsulated drugs, substantially improved the therapeutic efficiency of this system. Moreover, the enhancement of GD2 expression in FLM treated tumors offers the possibility to further increase the antitumor effect by the use of anti-GD2 CAR-T cells and anti-GD2 antibodies in combination with FLM in multimodal therapies.

Microencapsulation of amorphous solid dispersions of fenretinide enhances drug solubility and release from PLGA in vitro and in vivo.

The purpose of this study was to develop solid dispersions of fenretinide(4HPR), incorporate them into poly(lactic-co-glycolic)(PLGA) millicylindrical implants, and evaluate the resulting implants in vitro and in vivo for future applications in oral cancer chemoprevention. Due to the extreme hydrophobicity of 4HPR, 4HPR-polyvinylpyrrolidone (PVP) amorphous solid dispersions(ASDs) were prepared for solubility enhancement. The optimal PVP-4HPR ratio of 9/1(w/w) provided a 50-fold solubility enhancement in aqueous media, which was sustained over 1 week. PVP-4HPR ASD particles were loaded into PLGA millicylinders and drug release was evaluated in vitro in PBST and in vivo by recovery from subcutaneous injection in rats. While initial formulations of PLGA PVP-4HPR millicylinders only released 10% 4HPR in vitro after 28 days, addition of the plasticizer triethyl-o-acetyl-citrate(TEAC) into PVP-4HPR ASDs resulted in a 5.6-fold total increase in drug release. Remarkably, the TEAC-PVP-4HPR PLGA implants demonstrated slow, continuous, and nearly complete release over 1 month in vivo compared to a 25% release for our previously reported formulation incorporating solubilizers and pore-forming agents. Hence, a combination of PLGA plasticizer and ASD formation provides an avenue for long-term controlled release in vivo for the exceptionally difficult drug to formulate, 4HPR, and a suitable formulation for future evaluation in rodent models of oral cancer.

Design, Synthesis, Radiosynthesis and Biological Evaluation of Fenretinide Analogues as Anticancer and Metabolic Syndrome-Preventive Agents.

Fenretinide (4-HPR) is a synthetic derivative of all-trans-retinoic acid (ATRA) characterised by improved therapeutic properties and toxicological profile relative to ATRA. 4-HPR has been mostly investigated as an anti-cancer agent, but recent studies showed its promising therapeutic potential for preventing metabolic syndrome. Several biological targets are involved in 4-HPR's activity, leading to the potential use of this molecule for treating different pathologies. However, although 4-HPR displays quite well-understood multitarget promiscuity with regards to pharmacology, interpreting its precise physiological role remains challenging. In addition, despite promising results in vitro, the clinical efficacy of 4-HPR as a chemotherapeutic agent has not been satisfactory so far. Herein, we describe the preparation of a library of 4-HPR analogues, followed by the biological evaluation of their anti-cancer and anti-obesity/diabetic properties. The click-type analogue 3 b showed good capacity to reduce the amount of lipid accumulation in 3T3-L1 adipocytes during differentiation. Furthermore, it showed an IC50 of 0.53±0.8 µM in cell viability tests on breast cancer cell line MCF-7, together with a good selectivity (SI=121) over noncancerous HEK293 cells. Thus, 3 b was selected as a potential PET tracer to study retinoids in vivo, and the radiosynthesis of [18 F]3b was successfully developed. Unfortunately, the stability of [18 F]3b turned out to be insufficient to pursue imaging studies.

A Cationic Nanomicellar Complex of the Quaternary Amphiphilic Amine RC16+ with Fenretinide as a New Multitasking System for Antitumor Therapy.

OBJECTIVES: This study investigated the antitumor effect of a new nanomicellar complex obtained by combining the antitumor agent fenretinide with a quaternary amphiphilic amine RC16+ also endowed with antitumor activity. METHODS: The complex (Fen-RC16+) strongly improved the aqueous solubility of fenretinide (from 1,71 ± 0.08 µg/ml, pure fenretinide to 1500 ± 164 µg /ml, Fen-RC16+ complex) and provided a cytotoxic effect on SH-SY5Y neuroblastoma cell lines resulting from the intrinsic activity of both the complex components. Moreover, the mean size of the nanomicellar complex (ranging from 20 ± 1.97 nm to 40 ± 3.05 nm) was suitable for accumulation to the tumor site by the enhanced permeability and retention effect and the positive charge provided by the quaternary RC16+ induced adsorption of the complex on the tumor cell surface improving the intracellular concentration of fenretinide. RESULTS: All these characteristics made the Fen-RC16+ complex a multitasking system for antitumor therapy. CONCLUSION: Indeed its in vivo activity, evaluated on SH-SY5Y xenografts, was strong, and the tumor growth did not resume after the treatment withdrawal.

A novel oral micellar fenretinide formulation with enhanced bioavailability and antitumour activity against multiple tumours from cancer stem cells.

BACKGROUND: An increasing number of anticancer agents has been proposed in recent years with the attempt to overcome treatment-resistant cancer cells and particularly cancer stem cells (CSC), the major culprits for tumour resistance and recurrence. However, a huge obstacle to treatment success is the ineffective delivery of drugs within the tumour environment due to limited solubility, short circulation time or inconsistent stability of compounds that, together with concomitant dose-limiting systemic toxicity, contribute to hamper the achievement of therapeutic drug concentrations. The synthetic retinoid Fenretinide (4-hydroxy (phenyl)retinamide; 4-HPR) formerly emerged as a promising anticancer agent based on pre-clinical and clinical studies. However, a major limitation of fenretinide is traditionally represented by its poor aqueous solubility/bioavailability due to its hydrophobic nature, that undermined the clinical success of previous clinical trials. METHODS: Here, we developed a novel nano-micellar fenretinide formulation called bionanofenretinide (Bio-nFeR), based on drug encapsulation in an ion-pair stabilized lipid matrix, with the aim to raise fenretinide bioavailability and antitumour efficacy. RESULTS: Bio-nFeR displayed marked antitumour activity against lung, colon and melanoma CSC both in vitro and in tumour xenografts, in absence of mice toxicity. Bio-nFeR is suitable for oral administration, reaching therapeutic concentrations within tumours and an unprecedented therapeutic activity in vivo as single agent. CONCLUSION: Altogether, our results indicate Bio-nFeR as a novel anticancer agent with low toxicity and high activity against tumourigenic cells, potentially useful for the treatment of solid tumours of multiple origin.

Overcoming Biological Barriers in Neuroblastoma Therapy: The Vascular Targeting Approach with Liposomal Drug Nanocarriers.

Neuroblastoma is a rare pediatric cancer characterized by a wide clinical behavior and adverse outcome despite aggressive therapies. New approaches based on targeted drug delivery may improve efficacy and decrease toxicity of cancer therapy. Furthermore, nanotechnology offers additional potential developments for cancer imaging, diagnosis, and treatment. Following these lines, in the past years, innovative therapies based on the use of liposomes loaded with anticancer agents and functionalized with peptides capable of recognizing neuroblastoma cells and/or tumor-associated endothelial cells have been developed. Studies performed in experimental orthotopic models of human neuroblastoma have shown that targeted nanocarriers can be exploited for not only decreasing the systemic toxicity of the encapsulated anticancer drugs, but also increasing their tumor homing properties, enhancing tumor vascular permeability and perfusion (and, consequently, drug penetration), inducing tumor apoptosis, inhibiting angiogenesis, and reducing tumor glucose consumption. Furthermore, peptide-tagged liposomal formulations are proved to be more efficacious in inhibiting tumor growth and metastatic spreading of neuroblastoma than nontargeted liposomes. These findings, herein reviewed, pave the way for the design of novel targeted liposomal nanocarriers useful for multitargeting treatment of neuroblastoma.

Cytotoxicity and molecular activity of fenretinide and metabolites in T-cell lymphoid malignancy, neuroblastoma, and ovarian cancer cell lines in physiological hypoxia.

OBJECTIVE: All-trans-N-(4-hydroxyphenyl)retinamide or fenretinide (4-HPR) acts by reactive oxygen species (ROS) and dihydroceramides (DHCers). In early-phase clinical trials 4-HPR has achieved complete responses in T-cell lymphomas (TCL) and neuroblastoma (NB) and signals of activity in ovarian cancer (OV). We defined the activity of 4-HPR metabolites in N-(4-methoxyphenyl)retinamide (MPR), 4-oxo-N-(4-hydroxyphenyl)retinamide (oxoHPR), and the 4-HPR isomer 13-cis-fenretinide (cis-HPR) in NB, OV, and TCL cell lines cultured in physiological hypoxia. METHODS: We compared the effect of 4-HPR, cis-HPR, oxoHPR, and MPR on cytotoxicity, ROS, and DHCers in a panel of TCL, NB, and OV cell lines cultured in bone marrow level physiological hypoxia (5% O2), utilizing a fluorescence-based cytotoxicity assay (DIMSCAN), flow cytometry, and quantitative mass spectrometry. RESULTS: 4-HPR (10 µmol/l) achieved more than three logs of cell kill in nine of 15 cell lines. Cytotoxicity of 4-HPR and oxoHPR was comparable; in some cell lines, cis-HPR cytotoxicity was lower than 4-HPR, but additive when combined with 4-HPR. MPR was not cytotoxic. ROS and DHCers were equivalently increased by 4-HPR and oxoHPR in all cell lines (P<0.01), to a lesser extent by cis-HPR (P<0.01), and not increased in response to MPR (P>0.05). Mitochondrial membrane depolarization, caspase-3 cleavage, and apoptosis (TUNEL) were all significantly increased by 4-HPR and oxoHPR (P<0.01). CONCLUSION: Cytotoxic and pharmacodynamic activity was comparable with 4-HPR and oxoHPR, lower with cis-HPR, and MPR was inactive. Neither MPR or cis-HPR antagonized 4-HPR activity. These data support focusing on achieving high 4-HPR exposures for maximizing antineoplastic activity.

Lipid nano-bubble combined with ultrasound for anti-keloids therapy.

Keloids were characterized by excessive growth of fibrous tissues, and shared several pathological characteristics with cancer. They did put physical and emotional stress on patients in that keloids could badly change appearance of patients. N-(4-hydroxyphenyl) retinamide (4HPR) showed cytotoxic activity on a wide variety of invasive-growth cells. Our work was aim to prepare N-(4-hydroxyphenyl) retinamide-loaded lipid microbubbles (4HPR-LM) combined with ultrasound for anti-keloid therapy. 4HPR-loaded liposomes (4HPR-L) were first prepared by film evaporation method, and then 4HPR-LM were manufactured by mixing 4HPR-L and perfluoropentane (PFP) with ultrasonic cavitation method. The mean particle size and entrapment efficiency 4HPR-LM were 113 nm and 95%, respectively. The anti-keloids activity of 4HPR-LM was assessed with BALB/c nude mice bearing subcutaneous xenograft keloids model. 4HPR-LM, combined with ultrasound, could significantly induce apoptosis of keloid fibroblasts in vitro and inhibited growth of keloids in vivo. Thus, 4HPR-LM could be considered as a promising agent for anti-keloids therapy.

Design of Controlled Release PLGA Microspheres for Hydrophobic Fenretinide.

Fenretinide, a chemotherapeutic agent for cancer, is water-insoluble and has a very low oral bioavailability. Hence, the objective was to deliver it as an injectable depot and improve the drug solubility and release behavior from poly(lactide-co-glycolide) (PLGA) microspheres by incorporating nonionic surfactants with fenretinide. Enhancement of drug solubilization was observed with Brij 35 or 98, Tween 20, and Pluronic F127, but not Pluronic F68. Co-incorporation of Brij 98 with fenretinide significantly changed the microsphere morphology and improved the fenretinide release profile. The most optimal microsphere formulation, with 20% Brij 98 as excipient, showed an initial in vitro burst around 20% and a sustained release over 28 days in a solubilizing release medium at 37 °C. The effect of addition of MgCO3, drug loading, and polymer blending on the release of fenretinide from PLGA microspheres was also investigated and observed to enhance the drug release. Two sustained release formulations, one incorporating 20% Brij 98 and the other incorporating 3% MgCO3 in the oil phase, were selected for dosing in Sprague-Dawley rats and compared to a single injection of an equivalent dose of fenretinide drug suspension. These two formulations were chosen due to their high encapsulation efficiency, high cumulative release, and desirable in vitro release profile. The drug suspension resulted in a higher initial release in rats compared to the polymeric formulations, however, sustained release was also observed beyond 2 weeks, which may be attributed to the physiological disposition of the drug in vivo. The two PLGA based test formulations provided the desired low initial burst of fenretinide followed by 4 weeks of in vivo sustained release.

Water-soluble derivatives of 4-oxo-N-(4-hydroxyphenyl) retinamide: synthesis and biological activity.

A novel series of 4-oxo-N-(4-hydroxyphenyl) retinamide (4-oxo-4-HPR) derivatives were synthesized with the aim of increasing the poor solubility of the parent compound in biological fluids, while maintaining the cytotoxic activity and the dual mechanism of action. The most promising compound 13a showed antiproliferative/apoptotic activity. The analysis of its mechanism of action revealed that it retained the particular characteristic of 4-oxo-4-HPR which is able to induce cell cycle arrest during the mitotic phase, coupled with the formation of aberrant mitotic spindles.

Cell Line-Dependent Variability of Coordinate Expression of p75NTR and CRABP1 and Modulation of Effects of Fenretinide on Neuroblastoma Cells.

Neuroblastoma is a childhood neural crest tumor. Fenretinide, a retinoic acid analogue, induces accumulation of mitochondrial reactive oxygen species and consequent apoptosis in neuroblastoma cells. The p75 neurotrophin receptor (p75NTR) enhances the antineuroblastoma cell efficacy of fenretinide in vitro. We examined the role of the retinoid binding protein, CRABP1, in p75NTR-mediated potentiation of the efficacy of fenretinide. Knockdown and overexpression, respectively, of either p75NTR or CRABP1 were effected in neuroblastoma cell lines using standard techniques. Expression was determined by qRT-PCR and confirmed at the protein level by Western blot. Metabolic viability was determined by Alamar blue assay. While protein content of CRABP1 correlated roughly with that of p75NTR in the three neuroblastoid or epithelioid human neuroblastoma cell lines studied, manipulation of p75NTR expression resulted in cell line-dependent, variable change in CRABP1 expression. Furthermore, in some cell lines, induced expression of CRABP1 in the absence of p75NTR did not alter cell sensitivity to fenretinide treatment. The effects of manipulation of p75NTR expression on CRABP1 expression and the effects of CRABP1 expression on fenretinide efficacy are therefore neuroblastoma cell line-dependent. Potentiation of the antineuroblastoma cell effects of fenretinide by p75NTR is not mediated solely through CRABP1.

Replication-Competent Influenza Virus and Respiratory Syncytial Virus Luciferase Reporter Strains Engineered for Co-Infections Identify Antiviral Compounds in Combination Screens.

Myxoviruses such as influenza A virus (IAV) and respiratory syncytial virus (RSV) are major human pathogens, mandating the development of novel therapeutics. To establish a high-throughput screening protocol for the simultaneous identification of pathogen- and host-targeted hit candidates against either pathogen or both, we have attempted co-infection of cells with IAV and RSV. However, viral replication kinetics were incompatible, RSV signal window was low, and an IAV-driven minireplicon reporter assay used in initial screens narrowed the host cell range and restricted the assay to single-cycle infections. To overcome these limitations, we developed an RSV strain carrying firefly luciferase fused to an innovative universal small-molecule assisted shut-off domain, which boosted assay signal window, and a hyperactive fusion protein that synchronized IAV and RSV reporter expression kinetics and suppressed the identification of RSV entry inhibitors sensitive to a recently reported RSV pan-resistance mechanism. Combined with a replication-competent recombinant IAV strain harboring nanoluciferase, the assay performed well on a human respiratory cell line and supports multicycle infections. Miniaturized to 384-well format, the protocol was validated through screening of a set of the National Institutes of Health Clinical Collection (NCC) in quadruplicate. These test screens demonstrated favorable assay parameters and reproducibility. Application to a LOPAC library of bioactive compounds in a proof-of-concept campaign detected licensed antimyxovirus therapeutics, ribavirin and the neuraminidase inhibitor zanamivir, and identified two unexpected RSV-specific hit candidates, Fenretinide and the opioid receptor antagonist BNTX-7. Hits were evaluated in direct and orthogonal dose-response counterscreens using a standard recRSV reporter strain expressing Renilla luciferase.

Formulation and evaluation of biodegradable nanoparticles for the oral delivery of fenretinide.

Fenretinide is an anticancer drug with low water solubility and poor bioavailability. The goal of this study was to develop biodegradable polymeric nanoparticles of fenretinide with the intent of increasing its apparent aqueous solubility and intestinal permeability. Three biodegradable polymers were investigated for this purpose: two different poly lactide-co-glycolide (PLGA) polymers, one acid terminated and one ester terminated, and one poly lactide-co-glycolide/polyethylene glycol (PLGA/PEG) diblock copolymer. Nanoparticles were obtained by using an emulsification solvent evaporation technique. The formulations were characterized by differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and particle size analysis. Dissolution studies and Caco-2 cell permeation studies were also carried out for all formulations. Ultra high performance liquid chromatography coupled with mass spectrometry (UPLC/MS) and ultraviolet detection was used for the quantitative determination of fenretinide. Drug loading and the type of polymer affected the nanoparticles' physical properties, drug release rate, and cell permeability. While the acid terminated PLGA nanoparticles performed the best in drug release, the ester terminated PLGA nanoparticles performed the best in the Caco-2 cell permeability assays. The PLGA/PEG copolymer nanoparticles performed better than the formulations with ester terminated PLGA in terms of drug release but had the poorest performance in terms of cell permeation. All three categories of formulations performed better than the drug alone in both drug release and cell permeation studies.

Colloidal silver nanoparticles improve anti-leukemic drug efficacy via amplification of oxidative stress.

Recently, increased reactive oxygen species (ROS) levels and altered redox status in cancer cells have become a novel therapeutic strategy to improve cancer selectivity over normal cells. It has been known that silver nanoparticles (AgNPs) display anti-leukemic activity via ROS overproduction. Hence, we hypothesized that AgNPs could improve therapeutic efficacy of ROS-generating agents against leukemia cells. In the current study, N-(4-hydroxyphenyl)retinamide (4-HPR), a synthetic retinoid, was used as a drug model of ROS induction to investigate its synergistic effect with AgNPs. The data exhibited that AgNPs with uniform size prepared by an electrochemical method could localize in the lysosomes, mitochondria and cytoplasm of SHI-1 cells. More importantly, AgNPs together with 4-HPR could exhibit more cytotoxicity and apoptosis via overproduction of ROS in comparison with that alone. Taken together, these results reveal that AgNPs combined with ROS-generating drugs could potentially enhance therapeutic efficacy against leukemia cells, thereby providing a novel strategy for AgNPs in leukemia therapy.

Preparation and in vitro evaluation of hydrophilic fenretinide nanoparticles.

Fenretinide is an effective anti-cancer drug with high in vitro cytotoxicity and low in vivo systemic toxicity. In clinical trials, fenretinide has shown poor therapeutic efficacy following oral administration - attributed to its low bioavailability and solubility. The long term goal of this project is to develop a formulation for the oral delivery of fenretinide. The purpose of this part of the study was to prepare and characterize hydrophilic nanoparticle formulations of fenretinide. Three different ratios of polyvinyl pyrrolidone (PVP) to fenretinide were used, namely, 3:1, 4:1, and 5:1. Both drug and polymer were dissolved in a mixture of methanol and dichloromethane (2:23 v/v). Rotary evaporation was used to remove the solvents, and, following reconstitution with water, a high pressure homogenizer was used to form nanoparticles. The particle size and polydispersity index were measured before and after lyophilization. The formulations were studied by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and X-ray powder diffraction (XRPD). The effectiveness of the formulations was assessed by release studies and Caco-2 cell permeability assays. As the PVP content increased, the recovered particle size following lyophilization became more consistent with the pre-lyophilization particle size, especially for those formulations with less lactose. The DSC scans of the formulations did not show any fenretinide melting endotherms, indicating that the drug was either present in an amorphous form in the formulation or that a solid solution of the drug in PVP had formed. For the release studies, the highest drug release among the formulations was 249.2±35.5ng/mL for the formulation with 4:1 polymer-to-drug. When the permeability of the formulations was evaluated in a Caco-2 cell model, the mean normalized flux for each treatment group was significantly higher (p<0.05) from the fenretinide control. The formulation containing 4:1 polymer-to-drug ratio and 6:5 lactose-to-formulation ratio emerged as the optimal choice for further evaluation as a potential oral delivery formulation for fenretinide.

Anti-tumor activity of fenretinide complexed with human serum albumin in lung cancer xenograft mouse model.

Sufficient knowledge regarding cellular and molecular basis of lung cancer progression and metastasis would help in the development of novel and effective strategies for the treatment of lung cancer. 4HPR is a synthetic retinoid with potential anti-tumor activity but is still limited because of its poor bioavailability. The use of albumin as a complexing agent for a hydrophobic drug is expected to improve the water solubility and consequently their bioavailability.This study investigated the antitumor activity of a novel complex between albumin and 4-HPR in a mouse model of human lung cancer and focuses on role and mechanism of Cav-1 mainly involved in regulating cancer and ACSVL3 mainly connected with tumor growth. Their expressions were assayed by immunohistochemistry and qRT-PCR, to demonstrate the reduction of the tumor growth following the drug treatment. Our results showed a high antitumor activity of 4HPR-HSA by reduction of the volume of tumor mass and the presence of a high level of apoptotic cell by TUNEL assay. The downregulation of Cav-1 and ACSVL3 suggested a reduction of tumor growth. In conclusion, we demonstrated the great potential of 4HPR-HSA in the treatment of lung cancer. More data about the mechanism of drug delivery the 4HPR-HSA are necessary.

Novel PLA microspheres with hydrophilic and bioadhesive surfaces for the controlled delivery of fenretinide.

Novel polylactide (PLA) microspheres endowed with hydrophilic and bioadhesive surfaces as injectable formulations for the controlled release of fenretinide were prepared, using a novel technique based on the co-precipitation of PLA with gelatin, at the interface of a liquid dispersion formed by the addition of N-methylpyrrolidone containing PLA and dextrin (DX), towards an aqueous solution of gelatin (G). The resulting PLA-G-DX microspheres were compared with others prepared by the same technique using polylactide-co-glycolide (PLGA), with or without DX, and with or without phosphatidylcholine. Of the different systems, the PLA-G-DX microspheres had the best morphological, dimensional and functional characteristics. They had the highest drug loading, and their drug release was the most efficient over time without any burst effect. Their in vitro anti-tumoural activity was strongly enhanced with respect to the pure fenretinide. This paralleled the increased drug concentration inside the cells due to their marked bioadhesion to the tumour cell membranes as indicated by scanning electron microscope images.

Enhanced anti-tumor and anti-angiogenic efficacy of a novel liposomal fenretinide on human neuroblastoma.

Neuroblastoma is an embryonal tumor originating from the simpatico-adrenal lineage of the neural crest. It approximately accounts for about 15% of all pediatric oncology deaths. Despite advances in multimodal therapy, metastatic neuroblastoma tumors at diagnosis remain a clinical challenge. Retinoids are a class of compounds known to induce both terminal differentiation and apoptosis/necrosis of neuroblastoma cells. Among them, fenretinide (HPR) has been considered one of the most promising anti-tumor agent but it is partially efficacious due to both poor aqueous solubility and rapid metabolism. Here, we have developed a novel HPR formulation, by which the drug was encapsulated into sterically stabilized nanoliposomes (NL[HPR]) according to the Reverse Phase Evaporation method. This procedure led to a higher structural integrity of liposomes in organic fluids for a longer period of time, in comparison with our previous liposomal formulation developed by the film method. Moreover, NL[HPR] were further coupled with NGR peptides for targeting the tumor endothelial cell marker, aminopeptidase N (NGR-NL[HPR]). Orthotopically xenografted neuroblastoma-bearing mice treated with NGR-NL[HPR] lived statistically longer than mice untreated or treated with free HPR (NGR-NL[HPR] vs both control and HPR: P<0.0001). Also, NL[HPR] resulted in a statistically improved survival (NL[HPR] vs both control and HPR: P<0.001) but to a less extent if compared with that obtained with NGR-NL[HPR] (NGR-NL[HPR] vs NL[HPR]: P<0.01). Staining of tumor sections with antibodies specific for neuroblastoma and for either pericytes or endothelial cells evidenced that HPR reduced neuroblastoma growth through both anti-tumor and anti-angiogenic effects, mainly when delivered by NGR-NL[HPR]. Indeed, in this group of mice a marked reduction of tumor progression, of intra-tumoral vessel counts and VEGF expression, together with a marked down-modulation of matrix metalloproteinases MMP2 and MMP9, was observed. In conclusion, the use of this novel targeted delivery system for the apoptotic and antiangiogenic drug, fenretinide, could be considered as an adjuvant tool in the future treatment of neuroblastoma patients.

Fenretinide causes emphysema, which is prevented by sphingosine 1-phoshate.

Sphingolipids play a role in the development of emphysema and ceramide levels are increased in experimental models of emphysema; however, the mechanisms of ceramide-related pulmonary emphysema are not fully understood. Here we examine mechanisms of ceramide-induced pulmonary emphysema. Male Sprague-Dawley rats were treated with fenretinide (20 mg/kg BW), a synthetic derivative of retinoic acid that causes the formation of ceramide, and we postulated that the effects of fenretinide could be offset by administering sphingosine 1-phosphate (S1P) (100 µg/kg BW). Lung tissues were analyzed and mean alveolar airspace area, total length of the alveolar perimeter and the number of caspase-3 positive cells were measured. Hypoxia-inducible factor alpha (HIF-1α), vascular endothelial growth factor (VEGF) and other related proteins were analyzed by Western blot analysis. Immunohistochemical analysis of HIF-1α was also performed. Ceramide, dihydroceramide, S1P, and dihydro-S1P were measured by mass spectrometer. Chronic intraperitoneal injection of fenretinide increased the alveolar airspace surface area and increased the number of caspase-3 positive cells in rat lungs. Fenretinide also suppressed HIF-1α and VEGF protein expression in rat lungs. Concomitant injection of S1P prevented the decrease in the expression of HIF-1α, VEGF, histone deacetylase 2 (HDAC2), and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein expression in the lungs. S1P injection also increased phosphorylated sphingosine kinase 1. Dihydroceramide was significantly increased by fenretinide injection and S1P treatment prevented the increase in dihydroceramide levels in rat lungs. These data support the concept that increased de novo ceramide production causes alveolar septal cell apoptosis and causes emphysema via suppressing HIF-1α. Concomitant treatment with S1P normalizes the ceramide-S1P balance in the rat lungs and increases HIF-1α protein expression via activation of sphingosine kinase 1; as a consequence, S1P salvages fenretinide induced emphysema in rat lungs.

Design and characterization of fenretinide containing organogels.

The stable, transparent, organogels, which are prepared by adding a minute amount of water to a solution of lecithin in biocompatible oil, are here studied as matrices for solubilization and percutaneous delivery of fenretinide (4 hydroxypropyl phenyl retinamide, 4HPR), a retinoic acid derivative. The influence of different types of oil, content of water and presence of hyaluronic acid was studied on gel properties. Rheology studies were carried out in order to detect the effect of these variables on gel viscosity. 4HPR diffusion from the different organogels was determined by in vitro Franz cell. It was found that diffusion coefficients (Jn) of 4HPR incorporated in organogels are about five fold lower than Jn of 4HPR in organic solution. Stability and shelf life stability studies demonstrate that 4HPR incorporated in organogels does not degrade and that organogels maintain 90% of 4HPR stability for periods up to 4 months.