Animal age-, dose- and cell line-dependent growth of human neuroblastoma in nude mice. A statistical analysis.

Cells lines from human neuroblastoma (NB) and T/lymphoma (T-L) were injected subcutaneously (sc) in female CD1 nu/nu athymic nude mice. Results obtained after the observation of tumour growth were statistically analyzed by SAS System. The following four parameters were considered: 1) dose of injected cells, 2) type of injected tumour (NB or T-L), 3) age of mice after individuation of three groups of animals (group A, 4-9 weeks old, group B, 9-20 weeks old, group C, >20 weeks old), 4) injected cell line within the same tumour type. Latency time (LT), corresponding to the interval between cell inoculum and the appearance of a 5 mm diameter subcutaneous mass, and survival time (ST), corresponding to the interval between cell inoculum and the appearance of a 20 mm diameter subcutaneous tumour mass, were considered to evaluate tumour growth. Results showed that mass progression is affected by the number of injected cells and both LT and ST are age- and dose- dependent; furthermore, significant differences were recorded by using different NB and T-L cell lines. Group C showed longer LT than other groups; group B animals showed a statistically significant longer ST than groups A and C (p<0.001). Our results indicate that growth of human NB in athymic mice is faster in young animals, which also show a significantly poorer prognosis, while better ST was observed in old and middle-aged animals. Results show statistically significant differences of both LT and ST in animals differing for age and in animals inoculated with different cell amounts. These results seem not to be related with biological properties of NB cells too, since neither the occurrence of MYCN amplification nor chromosome 1p deletion significantly modified such behaviour

Antitumor activity of Sodium ascorbate against neuroblastoma cell lines in vitro

none4BACKGROUND: Neuroblastoma (NB) is an extra-cranial solid tumour of childhood. In spite of the good clinical response to first-line therapy, complete eradication of NB cells is rarely achieved. Thus, new therapeutic strategies are needed to eradicate surviving NB cells and prevent relapse. Sodium ascorbate has been recently reported to induce apoptosis of B16 melanoma cells through down-regulation of the transferrin receptor, CD71. Since NB and melanoma share the same embryologic neuroectodermal origin, we used different human NB cell lines to assess whether the same findings occurred. RESULTS: We could observe dose- and time-dependent induction of apoptosis in all NB cell lines. Sodium ascorbate decreased the expression of CD71 and caused cell death within 24 h. An increase in the global and specific caspase activity took place, as well as an early loss of the mitochondrial transmembrane potential. Moreover, intracellular iron was significantly decreased after exposure to sodium ascorbate. Apoptotic markers were reverted when the cells were pretreated with the iron donor ferric ammonium citrate (FAC), further confirming that iron depletion is responsible for the ascorbate-induced cell death in NB cells. CONCLUSION: Sodium ascorbate is highly toxic to neuroblastoma cell lines and the specific mechanism of vitamin C-induced apoptosis is due to a perturbation of intracellular iron levels ensuing TfR-downregulation.noneCarosio R; Zuccari G; Orienti I; Montaldo PG.Carosio, R; Zuccari, G; Orienti, I; Montaldo, Pg

ENHANCEMENT OF ANTI-TUMOUR ACTIVITY OF OLEYL ALCOHOL BY COMPLEXATION IN MODIFIED POLYVINYLALCOHOL POLYMER MICELLES.

The anti-tumour activity of fatty acids has been extensively investigated upon a wide range of tumour cell lines. The fatty alcohols activity, on the contrary, has been scarcely studied. It has been reported that the alcohols are more active than the corresponding acids and that unsaturated fatty alcohols inhibits DNA synthesis and growth of Erlich ascites tumour cells more efficiently than their saturated analogs. The interest in oleyl alcohol arises from dietary effects of essential fatty acids and alcohols and their involvement in cardiovascular disorders and cancers. Moreover, oleyl alcohol (cis-9-octadecen-1-ol) was reported to be one of the most active among a series of saturated and unsaturated fatty alcohols and the presence of a single insaturation makes it more stable to oxidation than multiple unsaturations. Since activity is proportionally correlated to the fatty alcohol intracellular accumulation, which depends on the balance of their hydrophobic/ hydrophilic properties and, primarily, on their ability to be solubilized in aqueous media, in this work we evaluate the possibility to improve oleyl alcohol aqueous solubilization by complexation with non toxic amphiphilic polymers self-assembling in aqueous environment. The aim is to develop safe parenteral formulations holding oleyl alcohol solubilization levels suitable to elicit therapeutic responses. The amphiphilic polymers were prepared by partial substitution of polyvinyl alcohol with oleyl chains through a succinyl spacer, in the hypothesis, previously demonstrated in an almost similar situation concerning doxorubicin [1, 2], that structural similarities should improve mutual affinity between the solubilizer and the solubilizing agent

Micellar complexes of all-trans retinoic acid with polyvinylalcohol-nicotinoyl esters as new parenteral formulations in neuroblastoma.

All-trans-retinoic acid (ATRA) is now included in many antitumor therapeutic schemes for the treatment of acute promyelocytic leukemia, Kaposi's sarcoma, head and neck squamous cell carcinoma, ovarian carcinoma, bladder cancer, and neuroblastoma. Unfortunately, its poor aqueous solubility hampers its parenteral formulation, whereas oral administration of ATRA is associated with progressively diminishing drug levels in plasma, which is related to induction of retinoic acid-binding proteins and increased drug catabolism by cytochrome P450-mediated reactions. An ATRA formulation, obtained by complexation of the drug into polymeric micelles, might be suitable for parenteral administration overcoming these unwanted effects. To this purpose, amphiphilic polymers were prepared by polyvinylalcohol (PVA) partial esterification with nicotinoyl moieties and their functional properties evaluated with regard to ATRA complexation. The physicochemical characteristics of the polymers and the complexes were analyzed by 1H-NMR, Dynamic Light Scattering (DLS), Capillary Electophoresis (CE), and were correlated with the complex ability to improve the drug solubilization and release the free drug in an aqueous environment. Subsequently, the best complex, providing the highest ATRA solubilization and release, was evaluated in vitro to test its cytotoxicity towards neuroblastoma cell lines. The PVA substitution degree calculated from 1H-NMR was found to be 5.0%, 8.2%, 15.3% (nicotinoyl moiety:PVA monomer molar ratio), while capillary electrophoresis analysis on the complexes revealed that the drug loadings were 0.95%, 1.20%, 4.76% (ATRA:polymer w:w) for PVA substitution degrees of 5.0%, 8.2%, and 15.3%, respectively. Complexation strongly increased ATRA aqueous solubility, which reached 1.20 +/- 0.25 mg/mL. The DLS measurements of the polymers and the complexes in aqueous solutions revealed mean sizes always below 400 nm, low polydispersity (min 0.202 +/- 0.013, max 0.450 +/- 0.032), and size almost unaffected by concentration. Drug fractional release did not exceed 8% after 48 h. The cytotoxicity studies against neuroblastoma cell lines outlined a significant growth inhibition effect of complexed ATRA with respect to free ATRA. These data suggest that the systems analyzed may be suitable carriers for parenteral administration of ATRA and other hydrophobic antitumor drugs, where the carriers are required to improve drug aqueous solubility and delay drug release almost after their accumulation in solid tumors

Fenretinide-polyvinylalcohol Conjugates: New Systems Allowing Fenretinide Intravenous Administration

N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) has been shown to be active toward many tumors without appreciable side effects. However its in vitro activity does not match a correspondent efficacy in vivo. The main reason is that the drug's hydrophobicity hinders its bioavailability in the body fluids. Even if the drug is previously dissolved in organic solvents, such as ethanol or DMSO, the subsequent dilution in body fluids trigger its precipitation in fine aggregates characterized by very low dissolution efficiency, never reaching amounts suitable for therapeutic response. To date no intravenous formulation of 4-HPR exists on the market. The 4-HPR linkage to a hydrophilic polymer by a covalent bond easily hydrolyzable in aqueous environment is expected to increase the drug's aqueous solubility, providing the free drug after hydrolysis of the covalent bond. This may be a useful tool for the preparation of aqueous intravenous formulations of 4-HPR. For this purpose, we linked 4-HPR to polyvinylalcohol (PVA) by a carbonate bond at different drug/hydroxy vinyl monomer molar ratios. We demonstrated that conjugation increased 4-HPR aqueous solubility and strongly inhibited neuroblastoma cell proliferation. In addition, in an in vivo neuroblastoma metastatic model, we obtained a significant antitumor effect as a consequence of the improved drug bioavailability

Modified doxorubicin for improved encapsulation in PVA polymeric micelles

Polyvinylalcohol, partially substituted with lipophilic acyl chains, generates polymeric micelles in aqueous phase, containing a hydrophobic core able to encapsulate lipophilic drugs. Two types of polymers were obtained by conjugation of polyvinylalcohol with oleoyl or linoleoyl chains as pendant groups. The polymers, at a substitution degree of approximately 1%, are soluble in water and form polymeric micelles whose size increases with polymer concentration. Doxorubicin was hydrophobized, by linking an oleoyl chain via amide bond, to make the drug more similar to the substituted polymers and promote its encapsulation into the inner core of the micelles. The properties of the drug-polymer systems were evaluated in solution by dynamic light scattering technique and correlated to the physicochemical characteristics of the drug and the substituted polymers. Solubilization tests revealed that the similarity of the chain, in both the polymer and the drug, promotes better drug encapsulation in the oleoyl than linoleoyl derivative. The drug-polymer systems are stable in phosphate buffer saline (pH 7.4) at 37 degrees C, and the release of the drug is activated by the presence of the proteolytic enzyme pronase-E. The enzyme activated drug release and the size of the polymeric micelles, compatible with the pore dimensions of the tumor vessels, make these systems interesting for targeting lipophilic drugs to solid tumors, where the proteolytic enzyme concentration strongly raises with respect to the other body compartments

Enhanced anti-neuroblastoma activity of a fenretinide complexed form after intravenous administration

OBJECTIVES: The major limitation to successful chemotherapy of neuroblastoma (NB) is the toxicity and the poor bioavailability of traditional drugs. METHODS: We synthesised an amphiphilic dextrin derivative (DX-OL) able to host fenretinide (4-HPR) by complexation. In this study, we have investigated the effects of 4-HPR-loaded amphipilic dextrin (DX-OL/4-HPR) in comparison with 4-HPR alone both in vitro on human NB cells and in vivo in pseudometastatic NB models. The haemolysis assay was used as a measure of the potential damage caused by the pharmaceutical formulation in vivo. Pharmacokinetic experiments were performed to assess drug plasma levels in mice treated with free or complexed 4-HPR. KEY FINDINGS: DX-OL/4-HPR exerted a more potent cytotoxic activity on NB cells. Complexed 4-HPR significantly increased the proportion of sub-G1 cells with respect to free 4-HPR. Dextrin derivatives showed no haemolytic activity, indicating their suitability for parenteral administration. DX-OL/4-HPR increased the lifespan and the long-term survival of treated mice over controls. The analysis of drug plasma levels indicates that the complexed drug has a higher AUC due to a reduced clearance from the blood. CONCLUSIONS: Our data suggest that DX-OL/4-HPR is an injectable formulation that is able to improve drug aqueous solubility and bioavailability