Benoit Denizot

NGF release from poly(d,l-lactide-co-glycolide) microspheres. Effect of some formulation parameters on encapsulated NGF stability

Poly(d,l-lactide-co-glycolide) (PLGA 37.5/25 and 25/50) biodegradable microparticles, which allow the locally delivery of a precise amount of a drug by stereotactic injection in the brain, were prepared by a W/O/W emulsion solvent evaporation/extraction method which had been previously optimized. The aim of this work was to study the influence of two formulation parameters (the presence of NaCl in the dispersing phase and the type of PLGA) on the NGF release profiles and NGF stability during microencapsulation. A honey-comb-like structure characterized the internal morphology of the microspheres. The initial burst was attributed to the rapid penetration of the release medium inside the matrix through a network of pores and to the desorption of weakly adsorbed protein from the surface of the internal cavities. The non-release fraction of the encapsulated protein observed after twelve weeks of incubation was accounted for firstly by the adsorption of the released protein on the degrading microparticles and secondly by the entanglement of the encapsulated protein in the polymer chains. The use of sodium chloride in the dispersing phase of the double emulsion markedly reduced the burst effect by making the microparticle morphology more compact. Unfortunately, it induced in parallel a pronounced NGF denaturation. Finally, it appeared that microparticles made from a hydrophilic uncapped PLGA 37.5/25 in the absence of salt, allowed the release of intact NGF at least during the first 24 h as determined by both ELISA and a PC12 cell-based bioassay.

A new generation of anticancer, drug-loaded, colloidal vectors reverses multidrug resistance in glioma and reduces tumor progression in rats

By focusing on rat glioma, we elucidated whether new lipid nanocapsules (LNC) were able to improve anticancer hydrophobic drug bioavailability while also overcoming multidrug resistance. Blank LNCs and LNCs loaded with the antineoplastic agent paclitaxel were formulated by an emulsion inversion phase process. Expression of efflux pumps by rat glioma cells was assessed by reverse transcription-PCR, Western blot, and immunohistochemistry, and their activity was followed using the tracer 99Tcm-methoxyisobutylisonitrile. Modalities of LNC action were addressed by using confocal microscopy detection of fluorescently labeled LNCs, fluorescence-activated cell sorting, high-performance liquid chromatography measurement of paclitaxel release, and analysis of tumor cell growth. This revealed an interaction between LNCs and efflux pumps that resulted in an inhibition of multidrug resistance in glioma cells, both in culture and in cell implants in animals. LNCs were able to target the intracellular compartment of glioma cells, a mechanism that was abrogated by using intracellular cholesterol inhibitors but not by clathrin-coated pit or caveolae uptake inhibitors. This result can be correlated to the LNC inhibitory effects on efflux pump activity that is itself known to be stimulated by intracellular cholesterol. In parallel, we showed that paclitaxel-loaded LNCs were active reservoirs from which paclitaxel could be released. Finally, we established that paclitaxel-loaded LNCs were more efficient than the commercially available paclitaxel formulation (Taxol) for clinical use, thus reducing tumor expansion in vitro and in vivo. Considering the physiologically compatible nature of LNC excipients, these data may represent an important step towards the development of new clinical therapeutic strategies against cancers. [Mol Cancer Ther 2006;5(7):1710–22]

188Re-SSS lipiodol: radiolabelling and biodistribution following injection into the hepatic artery of rats bearing hepatoma.

BackgroundAlthough intra-arterial radiation therapy with 131I-lipiodol is a useful therapeutic approach to the treatment of hepatocellular carcinoma, various disadvantages limit its use. AimTo describe the development of a method for the labelling of lipiodol with 188Re-SSS (188Re (S2CPh)(S3CPh)2 complex) and to investigate its biodistribution after injection into the hepatic artery of rats with hepatoma. Methods188Re-SSS lipiodol was obtained after dissolving a chelating agent, previously labelled with 188Re, in cold lipiodol. The radiochemical purity (RCP) of labelling was checked immediately. The 188Re-SSS lipiodol was injected into the hepatic artery of nine rats with a Novikoff hepatoma. They were sacrificed 1, 24 and 48 h after injection, and used for ex vivo counting. ResultsLabelling of 188Re-SSS lipiodol was achieved with a yield of 97.3±2.1%. The immediate RCP was 94.1±1.7%. Ex vivo counting confirmed a predominantly hepatic uptake, with a good tumoral retention of 188Re-SSS lipiodol, a weak pulmonary uptake and a very faint digestive uptake. The ‘tumour/non-tumoral liver’ ratio was high at 1, 24 and 48 h after injection (2.9±1.5, 4.1±4.1 and 4.1±0.7, respectively). ConclusionsUsing the method described here, 188Re-SSS lipiodol can be obtained with a very high yield and a satisfactory RCP. The biodistribution in rats with hepatoma indicates a good tumoral retention of 188Re-SSS lipiodol associated with a predominant hepatic uptake, a weak pulmonary uptake and a very faint digestive uptake. This product should be considered for intra-arterial radiation therapy in human hepatoma.

Assessment of myocardial viability in rats: Evaluation of a new method using superparamagnetic iron oxide nanoparticles and Gd-DOTA at high magnetic field.

The aim of this study was to detect salvageable peri‐infarction myocardium by MRI in rats after infarction, using with a double contrast agent (CA) protocol at 7 Tesla. Intravascular superparamagnetic iron oxide (SPIO) nanoparticles and an extracellular paramagnetic CA (Gd‐DOTA) were used to characterize the peri‐infarction zone, which may recover function after reperfusion occurs. Infarcted areas measured from T1‐weighted (T1‐w) images post Gd‐DOTA administration were overestimated compared to histological TTC staining (52% ± 3% of LV surface area vs. 40% ± 3%, P = 0.03) or to T2‐w images post SPIO administration (41% ± 4%, P = 0.04), whereas areas measured from T2‐w images post SPIO administration were not significantly different from those measured histologically (P = 0.7). Viable and nonviable myocardium portions of ischemically injured myocardium were enhanced after diffusive Gd‐DOTA injection. The subsequent injection of vascular SPIO nanoparticles enables the discrimination of viable peri‐infarction regions by specifically altering the signal of the still‐vascularized myocardium. Magn Reson Med 52:932–936, 2004.

Effect of a 188Re-SSS lipiodol/131I-lipiodol mixture, 188Re-SSS lipiodol alone or 131I-lipiodol alone on the survival of rats with hepatocellular carcinoma

Background and aimIt has been shown that the use of a cocktail of isotopes of different ranges of action leads to an increase in the effectiveness of metabolic radiotherapy. The purpose of the present study was to compare with a control group the effectiveness of three different treatments in rats bearing hepatocellular carcinoma (HCC), using (1) a mixture of lipiodol labelled with both 131I and 188Re, (2) lipiodol labelled with 131I alone and (3) lipiodol labelled with 188Re alone. Material and methodsFour groups were made up, each containing 14 rats with the N1-S1 tumour cell line. Group 1 received a mixture composed of 22 MBq of 188Re-SSS lipiodol and 7 MBq 131I-lipiodol. Group 2 received 14 MBq 131I-lipiodol. Group 3 received 44 MBq of 188Re-SSS lipiodol and group 4 acted as the control. The survival of the various groups was compared by a non-parametric test of log-rank, after a follow-up of 60, 180 and 273 days. ResultsCompared with the controls, the rats treated with a mixture of 188Re-SSS lipiodol and 131I-lipiodol show an increase in survival, but only from day 60 onwards (P=0.05 at day 60 and 0.13 at days 180 and 273). For the rats treated with 131I-lipiodol, there was a highly significant increase in survival compared with the controls at day 60, day 180 and day 273 (P=0.03, 0.04 and 0.04, respectively). There is no significant increase in survival for the rats treated with 188Re-SSS lipiodol, irrespective of the follow-up duration (P=0.53 at day 60, 0.48 at day 180, and 0.59 at day 273). ConclusionsIn this study, 131I-lipiodol is the most effective treatment in HCC-bearing rats, because this is the only method that leads to a prolonged improvement of survival. These results cannot necessarily be extrapolated to humans because of the relatively small size and unifocal nature of the lesions in this study. It appears necessary to carry out a study in humans with larger tumours in order to compare these three treatments, particularly with a view to replacing 131I-labelled lipiodol by 188Re-labelled lipiodol. However, this study clearly demonstrated that, for small tumours, as in an adjuvant setting for exemple, 131I-labelled lipiodol should be a better option than 188Re-labelled lipiodol.

Production of new thermoluminescent mini-dosimeters

A method of producing CaSO4:Dy thermoluminescent mini-dosimeters was reported in 1986 by B W Wessels for determination of the in vivo absorbed dose in radioimmunotherapy, a field in which absorbed dose gradients are important. These dosimeters, which undergo dissolution when used in a liquid environment, showed a sensitivity loss of up to 30% after 4 days of immersion in our tests. Moreover, several studies have shown that biocompatibility problems can occur during in vivo studies in animals. This paper describes the production and testing of a new type of thermoluminescent mini-dosimeter obtained by microextrusion of a mixture of LiF:Mg,Cu,P polypropylene and plastic adjuvants. These dosimeters, in the form of long 400 microm diameter filaments, can be cut to the desired length. The production process allows an LiF:Mg,Cu,P load of up to 50%. Results obtained in external irradiation indicate that these new miniature LiF:Mg,Cu,P dosimeters have good sensitivity (about 1.6 times that of CaSO4:Dy mini-TLDs), homogeneous response within a production batch (mean +/-4%), response stability in water (0.7% of variation in sensitivity after 2 weeks of immersion) and stability in aqueous solutions at different pH. LiF:Mg,Cu,P mini-dosimeters appear to be highly promising for internal dosimetry, and evaluation is in progress in animals.

Effect of stabilized iodized oil emulsion on experimentally induced hepatocellular carcinoma in rats.

PURPOSE Previous studies have shown that the use of Lipiodol UltraFluid (LUF) emulsified with water leads to an increase in the tumoral uptake of iodine I 131-labeled LUF and reduced pulmonary uptake. Although emulsions containing LUF are currently used for chemoembolization of hepatocellular carcinomas (HCCs), this approach is impossible with intraarterial radiation therapy (RT) because of the problems of radiation protection linked to instability of the emulsions. The aims of this study were to develop stabilized emulsions of radiolabeled LUF of different particle sizes and viscosities and to study its biodistribution in rats with HCC. MATERIALS AND METHODS An emulsifier made of polyethylene glycol and hydrogenated castor oil was used to stabilize emulsions containing water and technetium Tc 99m-labeled Super Six Sulfur LUF. The various emulsions were injected in the hepatic arteries of rats with HCC. Twenty-four hours after injection, the rats were killed and the liver, tumor, and lungs were removed to perform ex-vivo gamma-counting to quantify tumoral, hepatic, and pulmonary uptake. RESULTS Emulsions of oil in water and water in oil of different viscosities (0.68-1.06 Pa.S) and particle size distributions (21-45 mum) were prepared and kept stable for more than 24 hours. Whatever the type of emulsion, the observed effect on tumoral uptake was the opposite of that expected. Indeed, a decrease in tumoral activity was observed (P < .05 in three of five cases) and a tendency toward increased pulmonary activity was observed (P < .05 in two of five cases) rather than any significant decrease. CONCLUSIONS This study made it possible to develop emulsions of radiolabeled iodized oil that remain stable for more than 24 hours. However, studies of biodistribution in rats with HCC failed to demonstrate any improvement in tumoral targeting, but rather showed a decrease in tumoral uptake that renders this approach impractical for intraarterial radiolabeled iodized oil RT as well as for intraarterial iodized oil chemoembolization. These results may possibly be explained by the use of an emulsifier containing lipophilic and hydrophilic components that modify the properties of LUF.