Drug/lactose co-micronization by jet milling to improve aerosolization properties of a powder for inhalation.

The aim of this work was to formulate a powder for inhalation with fusafungine, a drug substance initially highly cohesive. The classical approach based on micronization by jet milling to prepare respirable drug particles and then blending with a carrier was first applied. A fractional factorial experimental design was implemented to screen six formulation parameters. The effect of drug/lactose co-micronization on aerosolization was then evaluated. In vitro deposition studies were performed with the twin stage glass impinger and the inhaler Spinhaler. Micronization did not induce DSC-detectable amorphization and gave a highly cohesive, poor flowable powder with a theoretical aerodynamic diameter of 5 microm. The powder was then blended with coarse lactose and optionally fine lactose. Unfortunately, the respirable fraction could not be optimized and remained below 10%. On the other hand, a co-micronized powder drug/fine lactose 50:50 gave a respirable fraction of 16%. Following blending with a carrier, the respirable fraction and the emitted dose fraction reached 23% and 69%, respectively. The use of a fine lactose grade for co-micronization was essential. In conclusion, this study demonstrated that co-micronization with a fine lactose is an efficient and simple strategy to formulate a powder for inhalation with enhanced aerosolization properties, especially for highly cohesive drug substance.

Intraseptal implantation of NGF-releasing microspheres promote the survival of axotomized cholinergic neurons.

Neurotrophic factors therapy requires their precise delivery to the targeted neuronal population. For this purpose, a wide range of strategies have been developed, and among them the stereotaxic implantation of biodegradable microparticles. To assess the in vivo activity of NGF-releasing PLGA microspheres, unloaded and NGF-loaded microparticles were implanted in the rat brain, near the septal cholinergic neurons, axotomized by an unilateral transection of the fornix-fimbria. Histological analysis at two and six weeks after implantation revealed a non-specific astro- and micro-glial reaction around the microspheres, identical for both unloaded and NGF-loaded microspheres. No neuronal toxicity was noticed, and healthy looking neurons were observed in contact with the microspheres. In the non-treated animals, the percentage of axotomized surviving neurons, when compared to the contralateral intact side, was 31 +/- 2 and 27 +/- 1% at two and six weeks, respectively. Unloaded microspheres caused no protective nor neurotoxic effects (40 +/- 9 and 39 +/- 6% of surviving cholinergic neurons at two and six weeks, respectively). In contrast, NGF-loaded microspheres showed a significant effect on the survival of axotomized cholinergic neurons at two and six weeks after implantation (66 +/- 9 and 61 +/- 5% when compared to the contralateral intact side, respectively). These results show that PLGA microparticles present no neurotoxicity and release sufficient amounts of bioactive NGF to significantly limit the lesion-induced disappearance of cholinergic neurons in the septum during at least six weeks. PLGA microparticles can be used in the future to administer neurotrophic factors in central nervous system disorders.

Intracerebral implantation of NGF-releasing biodegradable microspheres protects striatum against excitotoxic damage.

Intrastriatal implantation of genetically modified cells synthesizing nerve growth factor (NGF) constitutes one way to obtain a long-term supply of this neurotrophic factor and a neuronal protection against an excitotoxic lesion. We have investigated if NGF-loaded poly(d,l-lactide-co-glycolide) microspheres could represent an alternative to cell transplantations. These microspheres can be implanted stereotaxically and locally release the protein in a controlled and sustained way. In order to test this paradigm, the NGF release kinetics were characterized in vitro using radiolabeled NGF, immunoenzymatic assay, and PC-12 cells bioassay and then in vivo after implantation in the intact rat striatum. These microspheres were thus implanted into the rat striatum 7 days prior to infusing quinolinic acid. Control animals were either not treated or implanted with blank microspheres. The extent of the lesion and the survival of ChAT-, NADPH-d-, and DARPP-32-containing neurons were analyzed. In vitro studies showed that microspheres allowed a sustained release of bioactive NGF for at least 1 month. Microspheres implanted in the intact striatum still contained NGF after 2.5 months and they were totally degraded after 3 months. After quinolinic acid infusion, the lesion size in the group treated with NGF-releasing microspheres was reduced by 40% when compared with the control groups. A marked neuronal sparing was noted, principally concerning the cholinergic interneurons, but also neuropeptide Y/somatostatin interneurons and GABAergic striatofuge neurons. These results indicate that implantation of biodegradable NGF-releasing microspheres can be used to protect neurons from a local excitotoxic lesion and that this strategy may ultimately prove to be relevant for the treatment of various neurological diseases.

Why does PEG 400 co-encapsulation improve NGF stability and release from PLGA biodegradable microspheres?

PURPOSE: The aim of this work was to understand the mechanism by which co-encapsulated PEG 400 improved the stability of NGF and allowed a continuous release from PLGA 37.5/25 microspheres. METHODS: Microparticles were prepared according to the double emulsion method. PEG 400 was added with NGF in the internal aqueous phase (PEG/PLGA ratio 1/1 and 1.8/1). Its effect was investigated through interfacial tension studies. Protein stability was assessed by ELISA. RESULTS: A novel application of PEG in protein stabilization during encapsulation was evidenced by adsorption kinetics studies. PEG 400 limited the penetration of NGF in the interfacial film of the primary emulsion. Consequently, it stabilized the NGF by reducing the contact with the organic phase. In addition, it avoided the NGF release profile to level off by limiting the irreversible NGF anchorage in the polymer layers. On the other hand, the amount of active NGF released in the early stages was increased. During microparticle preparation, NaCl could be added in the external aqueous phase to modify the structure of microparticles. This allowed to reduce the initial release rate without affecting the protein stability always encountered in the absence of PEG. CONCLUSIONS: PEG 400 appeared of major interest to achieve a continuous delivery of NGF over seven weeks from biodegradable microparticles prepared by the double emulsion technique.

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.