Differential proteomic analysis of STAT6 knockout mice reveals new regulatory function in liver lipid homeostasis.

Increased inflammatory signaling is a key feature of metabolic disorders. In this context, the role of increased pro-inflammatory signals has been extensively studied. By contrast, no efforts have been dedicated to study the contrasting scenario: the attenuation of anti-inflammatory signals and their role in metabolic homeostasis. IL-4 and IL-13 are anti-inflammatory cytokines signaling through the Signal Transducer and Activator of Transcription 6 (STAT6). Our study was aimed at evaluating the lack of STAT6 signaling on liver homeostasis. To this end we analyzed the liver proteome of wild type and STAT6 knock-out mice using 2D nanoscale LC-MS/MS with iTRAQ labeling technique. The coordinated changes in proteins identified by this quantitative proteome analysis indicated disturbed lipid homeostasis and a state of hepatocellular stress. Most significantly, the expression of the liver fatty acid binding protein (FABP1) was increased in the knock-out mice. In line with the elevated FABP1 expression we found latent liver lipid accumulation in the STAT6-deficient mice which was further aggravated when mice were challenged by a high fat diet. In conclusion, our study revealed a so far uncharacterized role for STAT6 in regulating liver lipid homeostasis and demonstrates the importance of anti-inflammatory signaling in the defense against the development of liver steatosis.

Biodegradable PLGA microparticles for sustained release of a new GnRH antagonist: part II. In vivo performance.

Poly (DL-lactide-co-glycolide) microparticles (MP) containing a highly potent peptidic gonadotropin releasing hormone antagonist (degarelix) of interest in the prostate cancer indication were screened for biological performance. Efficacy was tested in a castrated male rat model at 3 doses (0.4, 1.0 and 1.5 mg/kg) and assessed as inhibition of luteinizing hormone (LH) secretion. When increasing the dose, onset of inhibition was faster, inhibition was more intense, and duration of action was prolonged. The MP type was also highly influent. If spray-dried and microextrusion particles exhibited comparable potencies, double emulsion microspheres were significantly less potent, both for onset and duration of inhibition. Interestingly, for the latter type it was found that the degarelix fraction released upon reconstitution in the solution for injection was significantly lower (max 0.3%), in comparison to spray-dried MP (max 2%) or microextrusion (max 4%). With the three types of particles, increasing peptide content was detrimental for duration of action, but only little difference was noticed between particles based on different polymers. At 1.5 mg/kg, LH inhibition was achieved over 36 days with spray-dried MP based on 75/25 lactate/glycolate copolymer. This was superior by 1 week to the performance of unformulated degarelix given at the same dose.

Biodegradable microparticles for sustained release of a new GnRH antagonist--part I: Screening commercial PLGA and formulation technologies.

The formulation of a new GnRH antagonist (degarelix) in biodegradable poly(DL-lactide-co-glycolide) (PLGA) microparticles was investigated for the development of a 3-month sustained release formulation to treat prostate cancer. The aim was to screen formulation technologies and distinct copolymers to produce microparticles (MP) of different types with good entrapment efficiency (>85%) and peptide purity (>95%) after gamma sterilization. Basically, three types of degarelix-loaded MP (4, 8 and 16% w/w nominal content) were produced with solvent and non-solvent technologies, namely double-emulsion solvent evaporation, spray-drying and two extrusion methods. Besides composition, commercial copolymers differing in residual monomer content and functional group at the carboxylic terminus (acid or ester) were characterized and employed. Peptide loading capacity and purity, as well as shape, size characteristics, and porosity of the produced microparticles were discussed in relation to technology and copolymer choice. Spray-drying and micro-extrusion were the two preferred formulation technologies because of higher entrapment efficiency and better preservation of peptide purity during production and gamma-sterilization. The impact of formulation technologies on the MP characteristics overwhelmed the impact of copolymer selection. Nevertheless, one particular polymer was discarded since it was more susceptible towards radiolytic degradation. The resulting degarelix-MP will be tested in a biological assay for selection of the formulation based on performance.