Rapid formulation assessment of filgrastim therapeutics by a thermal stress test.

The biosimilar versions of recombinant methionyl human granulocyte colony-stimulating factor (rh-Met-G-CSF, filgrastim) are now widely available. Because changes to the formulation often lead to subtle differences, there is a critical need to define techniques to test and insure the quality of these products. The present study was designed to compare formulation and thermal stress stability of filgrastim products. The formulation ingredients including acetate, polysorbate 80, and sorbitol were determined using state-of-the-art validated analytical methods. The formulation pH and osmolality were also measured. Moreover, the stability profiles of 8 filgrastim products using thermal stress at 57 °C for 4 h were assessed by size-exclusion high-performance liquid chromatography (SE-HPLC) and in vitro biological assay. The products had different stability profiles. More stable products were within the specification for formulation and less stable products were beyond the specification limits. Altogether, the results suggest that a short-time stress study at 57 °C and analysis of filgrastim by SE-HPLC could unveil formulation problems and is potentially useful for comparability studies.

Neuroprotective effects of three different sizes nanochelating based nano complexes in MPP(+) induced neurotoxicity.

Parkinson's disease (PD) is the world's second most common dementia, which the drugs available for its treatment have not had effects beyond slowing the disease process. Recently nanotechnology has induced the chance for designing and manufacturing new medicines for neurodegenerative disease. It is demonstrated that by tuning the size of a nanoparticle, the physiological effect of the nanoparticle can be controlled. Using novel nanochelating technology, three nano complexes: Pas (150 nm), Paf (100 nm) and Pac (40 nm) were designed and in the present study their neuroprotective effects were evaluated in PC12 cells treated with 1-methyl-4-phenyl-pyridine ion (MPP (+)). PC12 cells were pre-treated with the Pas, Paf or Pac nano complexes, then they were subjected to 10 µM MPP (+). Subsequently, cell viability, intracellular free Calcium and reactive oxygen species (ROS) levels, mitochondrial membrane potential, catalase (CAT) and superoxide dismutase (SOD) activity, Glutathione (GSH) and malondialdehyde (MDA) levels and Caspase 3 expression were evaluated. All three nano complexes, especially Pac, were able to increase cell viability, SOD and CAT activity, decreased Caspase 3 expression and prevented the generation of ROS and the loss of mitochondrial membrane potential caused by MPP(+). Pre-treatment with Pac and Paf nano complexes lead to a decrease of intracellular free Calcium, but Pas nano complex could not decrease it. Only Pac nano complex decreased MDA levels and other nano complexes could not change this parameter compared to MPP(+) treated cells. Hence according to the results, all nanochelating based nano complexes induced neuroprotective effects in an experimental model of PD, but the smallest nano complex, Pac, showed the best results.

Effect of Chronic Restraint Stress on HPA Axis Activity and Expression of BDNF and Trkb in the Hippocampus of Pregnant Rats: Possible Contribution in Depression during Pregnancy and Postpartum Period.

INTRODUCTION: Brain-Derived Neurotrophic Factor (BDNF) and its receptor, TrkB, in the hippocampus are targets for adverse effects of stress paradigms; in addition, BDNF and its receptor play key role in the pathology of brain diseases like depression. In the present study, we evaluated the possible role of hippocampal BDNF in depression during pregnancy. METHODS: To achieve the purpose, repeated restrain stress (1 or 3 hours daily for 7 days) during the last week of pregnancy was used and alteration in the gene expression of hippocampal BDNF and TrkB evaluated by semi-quantitative PCR. RESULTS: The results showed that in stress group the level of ACTH and Corticosterone is increased showing that our model was efficient in inducing psychological stress; we also found that BDNF and TrkB expression are decreased in 3 hours stress group but not in 1 hour stress compared to control group. DISCUSSION: Our results imply that decrease in BDNF and its receptor could contribute in some adverse effects of stress during pregnancy such as elevation of depressive like behavior.

Down Regulation of ackA-pta Pathway in Escherichia coli BL21 (DE3): A Step Toward Optimized Recombinant Protein Expression System.

BACKGROUND: One of the most important problems in production of recombinant protein is to attain over-expression of the target gene and high cell density. In such conditions, the secondary metabolites of bacteria become toxic for the medium and cause cells to die. One of these aforementioned metabolites is acetate, which enormously accumulated in the medium, so that both cell and protein yields are affected. OBJECTIVES: To overcome this problem, several strategies applied. In this research we used antisense RNA strategy, where the transcription of phosphotransacetylase (PTA) and acetate kinase (ACK), two acetate pathway key enzymes, could be controlled, which led to reduced acetate production. MATERIALS AND METHODS: In order to achieve this, recombinant plasmid harboring antisense sequences targeting both of pta and ackA was assembled, after transfecting to the cells, its effects on the cell growth and acetate accumulation in the minimal media was assessed and compared with the control, the plasmid without antisense cassette, in presence and absence of IPTG in Escherichia coli BL21 (DE3). RESULTS: It was observed that the mentioned strategy partially affect the growth and amount of excreted acetate in comparison with the control. In addition it was found that high down-regulation of the acetate production pathway reduces the growth rate of E. coli BL21 (DE3). CONCLUSIONS: The study principally proved the importance of this strategy in acetate excretion control.