A Quadrupole Dalton-based multi-attribute method for product characterization,process development,and quality control of therapeutic proteins

During manufacturing and storage process, therapeutic proteins are subject to various post-translational modifications (PTMs), such as isomerization, deamidation, oxidation, disulfide bond modifications and glycosylation. Certain PTMs may affect bioactivity, stability or pharmacokinetics and pharmacodynamics profile and are therefore classified as potential critical quality attributes (pCQAs). Identifying, monitoring and controlling these PTMs are usually key elements of the Quality by Design (QbD) approach. Traditionally, multiple analytical methods are utilized for these purposes, which is time consuming and costly. In recent years, multi-attribute monitoring methods have been developed in the biopharmaceutical industry. However, these methods combine high-end mass spectrometry with complicated data analysis software, which could pose difficulty when implementing in a quality control (QC) environment. Here we report a multi-attribute method (MAM) using a Quadrupole Dalton (QDa) mass detector to selectively monitor and quantitate PTMs in a therapeutic monoclonal antibody. The result output from the QDa-based MAM is straightforward and automatic. Evaluation results indicate this method provides comparable results to the traditional assays. To ensure future application in the QC environment, this method was qualified according to the International Conference on Harmonization (ICH) guideline and applied in the characterization of drug substance and stability samples. The QDa-based MAM is shown to be an extremely useful tool for product and process characterization studies that facilitates facile understanding of process impact on multiple quality attributes, while being QC friendly and cost-effective.     

Piperine analogs as potent Staphylococcus aureus NorA efflux pump inhibitors

Based on our recent findings that piperine is a potent Staphylococcus aureus NorA efflux pump inhibitor (EPI), 38 piperine analogs were synthesized and bioevaluated for their EPI activity. Twenty-five of them were found active with potentiating activity equivalent or more than known EPIs like reserpine, carsonic acid and verapamil. The inhibitory mechanism of the compounds was confirmed by efflux inhibition assay using ethidium bromide as NorA substrate. The present communication describes the synthesis, bioevaluation and structure related activity of these efflux pump inhibitors.     

Synthesis and antibacterial activity evaluation of metronidazole–triazole conjugates

Synthesis and antibacterial activity of metronidazole–triazole conjugates are reported. Total 21 hybrid compounds have been synthesized with different substitution pattern on the triazole ring in order to study their influence on the antibacterial activity. These compounds demonstrated potent to weak antibacterial activity against Gram-positive, and Gram-negative bacteria. Six compounds have shown equal or better antibacterial activity against Gram-negative strains than the reference compound.     

The L-4F mimetic peptide prevents insulin resistance through increased levels of HO-1, pAMPK, and pAKT in obese mice

We examined mechanisms by which L-4F reduces obesity and diabetes in obese (ob) diabetic mice. We hypothesized that L-4F reduces adiposity via increased pAMPK, pAKT, HO-1, and increased insulin receptor phosphorylation in ob mice. Obese and lean mice were divided into five groups: lean, lean-L-4F-treated, ob, ob-L-4F-treated, and ob-L-4F-{"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Food intake, insulin, glucose adipocyte stem cells, pAMPK, pAKT, CB1, and insulin receptor phosphorylation were determined. Subcutaneous (SAT) and visceral adipose tissue (VAT) were determined by MRI and hepatic lipid content by magnetic resonance spectroscopy. SAT and VAT volumes decreased in ob-L-4F-treated animals compared with control. L-4F treatment decreased hepatic lipid content and increased the numbers of small adipocytes (P < 0.05) and phosphorylation of insulin receptors. L-4F decreased CB1 in SAT and VAT and increased pAKT and pAMPK in endothelium. L-4F-mediated improvement in endothelium was prevented by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Inhibition of pAKT and pAMPK by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 was associated with an increase in glucose levels. Upregulation of HO-1 by L-4F produced adipose remodeling and increased the number of small differentiated adipocytes. The anti-obesity effects of L-4F are manifested by a decrease in visceral fat content with reciprocal increases in adiponectin, pAMPK, pAKT, and phosphorylation of insulin receptors with improved insulin sensitivity.     

Development and Evaluation of Sustained Release Gastroretentive Minimatrices for Effective Treatment of H. pylori Infection

In the present work, sustained release gastroretentive minimatrices of amoxicillin have been designed and optimized using central composite design. Effect of amount of xanthan gum, rate controlling polymers (HPMC K100M CR/PEO coagulant (1:1)), carbopol 974P, and gas generating couple (sodium bicarbonate/citric acid (3:1)) was studied on dependent (response) variables, i.e., buoyancy lag time, drug release at 1 h, time required for 95% drug release, swelling index, and bioadhesive strength. Minimatrices were prepared by non aqueous granulation method using solution of PVP K30 in isopropyl alcohol. All the formulations were found to contain 99.2% to 100.9% of amoxicillin per minimatrix. Optimum formulation (Formulation number AGT09) containing high level of the independent variables was having buoyancy lag time of 7 min and drug release at 1 h was 32.5%. It required 9.39 h for 95% drug release while swelling index and bioadhesive strength were 341 and 17.9 dyn/cm², respectively. This formulation was said to be optimum because it has minimum buoyancy lag time, requires maximum time for 95% drug release, and has higher bioadhesive capabilities. In vitro results of an optimized formulation indicate its sustained drug release and gastric retention capability, which may be very useful for effective treatment of H. pylori infection.     

Ligand migration and hexacoordination in type 1 non-symbiotic rice hemoglobin

Type 1 non-symbiotic rice hemoglobin (rHb1) shows bis-histidyl heme hexacoordination and is capable of binding diatomic ligands reversibly. The biological function is as yet unclear, but the high oxygen affinity makes it unlikely to be involved in oxygen transport. In order to gain insight into possible physiological roles, we have studied CO rebinding kinetics after laser flash photolysis of rHb1 in solution and encapsulated in silica gel. CO rebinding to wt rHb1 in solution occurs through a fast geminate phase with no sign of rebinding from internal docking sites. Encapsulation in silica gel enhances migration to internal cavities. Site-directed mutagenesis of FB10, a residue known to have a key role in the regulation of hexacoordination and ligand affinity, resulted in substantial effects on the rebinding kinetics, partly inhibiting ligand exit to the solvent, enhancing geminate rebinding and enabling ligand migration within the internal cavities. The mutation of HE7, one of the histidyl residues involved in the hexacoordination, prevents hexacoordination, as expected, but also exposes ligand migration through a complex system of cavities. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.     

Desiccation induced oxidative stress and its biochemical responses in intertidal red alga Gracilaria corticata (Gracilariales, Rhodophyta)

Intertidal alga Gracilaria corticata growing in natural environment experiences various abiotic stresses during the low tides. The aim of this study was to determine whether desiccation exposure would lead to oxidative stress and its effect varies with exposure periods. This study gives an account of various biochemical changes in G. corticata following the exposure to desiccation for a period of 0 (control), 1, 2, 3 and 4 h under controlled conditions. During desiccation, G. corticata thalli showed dramatic loss of water by almost 47% when desiccated for 4 h. The enhanced production of reactive oxygen species (ROS) and increased lipid peroxidation observed during the exposure of 3-4 h were chiefly contributed by higher lipoxygenase (LOX) activity with the induction of two new LOX isoforms (LOX-2, ∼85 kDa; LOX-3, ∼65 kDa). The chlorophyll, carotenoids and phycobiliproteins (phycoerythrin and phycocyanin) were increased during initial 2 h exposure compared to control and thereafter declined in the succeeding exposure. The antioxidative enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), glutathione peroxidase (GPX) and the regeneration rate of reduced ascorbate (AsA) and glutathione (GSH) increased during desiccation up to 2-3 h. Further, the isoforms of antioxidant enzymes Mn-SOD (∼150 kDa), APX-4 (∼110 kDa), APX-5 (∼45 kDa), GPX-1 (∼80 kDa) and GPX-2 (∼65 kDa) responded specifically to the desiccation exposure. Compared to control, a relative higher content of both free and bound insoluble putrescine and spermine together with enhanced n-6 PUFAs namely C20:4(n-6) and C20:3(n-6) fatty acids found during 2 h exposure reveals their involvement in defence reactions against the desiccation induced oxidative stress.     

Outbreak of Abortions and Infertility in Thoroughbred Mares Associated with Waterborne <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

At a thoroughbred equine breeding farm near Hissar (Haryana), three mares aborted in their seventh month of pregnancy. The vaginal swabs of all aborted mares, and stomach contents, heart blood, liver, spleen and placenta of aborted fetuses yielded pure culture of Aeromonas hydrophila. In addition, A. hydrophila was also isolated from the vaginal swabs of three repeat breeding mares and faecal sample of a diarrheic foal. The source of infection was possibly water supply as all the water samples collected from taps, mother tank and storage tank were found to be positive for A. hydrophila. The antibiogram of all the isolates was similar showing resistance to ampicillin, carbenicillin, gentamicin, kanamycin and amikacin but sensitive to chloramphenicol, ciprofloxacin, cefuroxime, ceftriaxone, cotrimoxazole, cotrimazine, nitrofurantoin, streptomycin and tetracycline. All the 20 sera samples collected from three aborted and three repeat breeding, and eight in-contact mares, a diarrheic foal, three cows and two male buffaloes maintained at the same farm contained antibodies against A. hydrophila with titres ranging from 80 to 640. The water supply was instantly chlorinated using 0.05% sodium hypochlorite for three consecutive days and all the culturally positive mares were treated with intravaginal administration of 1 g ciprofloxacin, while the foal was given nitrofurantoin for three days. After one month, A. hydrophila could not be isolated either from mares or from their environment and antibody titre in all the seropositive animals showed a declining trend. Later, all the aborted and repeat breeding mares were confirmed to be pregnant. Thus, the present study indicated that water-borne A. hydrophila might be associated with equine abortions and infertility, and diarrhea in newborn foals.