Zeitschrift für Bioanalyse und Biomedizin

High performance liquid chromatographic tandom mass spectrometric method for the estimation of S-amlodipine in human plasma has been developed and validated using Tizanidine as internal standard. Sample process was accomplished by Liquid-Liquid Extraction technique. The processed sample was chromatographed and analysed on Phenomenex, Lux 3u Cellulose-2 (150 × 4.6) mm column using mobile phase [0.1% formic acid in water and Acetonitrile (50:50% v/v)]. S-amlodipine was chromatographed and analysed by MS detector. Amlodipine has two basic moiety (one primary and one secondary amino) so it shows pKa=9.45 with two ester linkages and one ether linkage, so it is highly lipid soluble (logP=2.22) whereas Tizanidine has two secondary amino and three tertiary amino groups, so it shows pKa=7.49. Primary amine –NH2 is aliphatic in nature in amlodipine and secondary amine –NH is in ring so it has less action of basicity. Tizanidine has secondary amine –NH is in chain which predominates the basicity (pKa=7.49) and other secondary and tertiary amines are in ring so they show less action with formic acid/ACN mobile phase. Formic acid ionizes (primary amino) –NH2 part, then (secondary amino) –NH part and then (tertiary amino) =N part. So amlodipine elutes 1st then tizanidine comes out. LogP of amlodipine is 2.22 and for tizanidine it is 1.4, so due to Formic acid-water/CAN mobile phase Amlodipine has higher retention time than tizanidine and comparison Rt. The analytical method described is valid the determination of S-amlodipine (over a range of 0.77 ng/ml to 50.98 ng/ml) using Tizanidine as internal standard in human plasma. Signals from the detector were captured in a computer and processed using Analyst software. This validation report provides results of various validation parameters.

Breast cancer has been a significant health problem in terms of both morbidity and mortality and has been the second leading cause of cancer deaths worldwide. Exploring and understanding the cellular and molecular mechanisms of BC tumorigenesis and progression as well as resistance to chemotherapeutic drugs involved will be critically important for advancing the overall efficacy of BC therapy. The increasing data demonstrates high levels of constitutively active AhR in mammary tumors, suggesting there is a putative role for the AhR in mammary gland tumorigenesis, while target the AhR for new drug discovery and development has been payed more attention in recent years. The present review aims to provide a relative detailed understanding and progression on the integrated role of AhR in BC tumorigenesis and in BC resistance to chemotherapeutic drugs.

Plasma lithography is a technique to space-selectively create hydrophilic surfaces on silicone materials with oxygen plasma treatment. Cells can thus be micropatterned within the modified surfaces, allowing for artificially controlling the geometry of individual cell colonies. Conventional plasma lithography employs a photolithographically microfabricated mask with which the pattern geometry is determined. However, fast on-demand design change to the micropattern may be limited due to the time and cost associated with the sophisticated photolithographic fabrication. Here we attempted to microfabricate a mask for plasma lithography in a novel, quick, low-cost manner. An infrared absorption film was processed using a low-power Nd:YAG laser on an optical microscope to produce a mask of arbitrary pattern geometries. Our experiments indicate that plasma-shielding masks with various geometries are promptly obtained at a spatial resolution of several tens of microns with a laser power of below 200 mW. We demonstrate that cells are indeed micropatterned on functionalized silicone substrates so as to conform to the geometry of the laser-processed mask, thus suggesting the potential of this technique as a low-cost, fast on-demand means for cell micropatterning.

Objective: To determine the role of norepinephrine (NE) on DNA damage and reactive oxygen species (ROS) generation in ovarian surface epithelial cells.

Method: Non-tumorigenic, immortalized ovarian surface epithelial cells were treated with NE, bleomycin, and bleomycin followed by NE. The comet assay was performed on each treatment group to determine the amount of single and double-strand breaks induced by treatments. ROS levels for each treatment group were measured using the H2DCF-DA fluorescence assay. Finally, RNA transcripts were measured for each treatment group with regards to the expression of DNA repair and oxidative stress genes.

Results: The mean tail moment of untreated cells was significantly greater than that of cells treated with NE (p=0.02). The mean tail moment of cells treated with bleomycin was significantly greater than that of cells treated with bleomycin followed by NE (p<0.01). Treatment with NE resulted in significantly less ROS generation than in untreated cells (p<0.01). NE treatment after hydrogen peroxide treatment resulted in a noticeable decrease in ROS generation. Genes associated with oxidative stress were upregulated in cells treated with bleomycin, however this upregulation was blunted when bleomycin-treated cells were treated subsequently with NE.

Conclusion: NE is associated with decreased DNA damage and ROS production in ovarian surface epithelial cells. This effect is protective in the presence of the oxidative-damaging agent bleomycin. These results suggest an additional physiologic role for the stress hormone NE, in protecting ovarian surface epithelial cells from oxidative stress.

Biopharmaceuticals like monoclonal antibodies are widely used in clinical medicine for various therapies e.g. cancer, inflammatory and autoimmune diseases. Immunogenicity is one of the issues for safety. Such undesired immunogenicity can also limit the use of biopharmaceuticals, particularly for the treatment of chronic diseases that necessitate repeated treatments over long period. Assessment of immunogenicity is an important component of drug safety evaluation, which is presently performed by estimating risk factors. Risk based approach considers both probability of induction of immune response and expected clinical consequences. A combination of the two may result in high, medium or low risk levels and will depend on the product, patient and treatment related characteristics. Well engineered cells, well designed formulation coupled with good manufacturing scheme may sometimes reduce some of the extrinsic and intrinsic factors and increase the stability of drug product. One of the proposal for remedies is to purify the drug product to homogeneity or near homogeneity retaining its stability and functional activity. On the other hand, quite a few biosimilar drugs, which are supposed to be economical version of branded biotherapeutics, is expected to be in the market in next several years. Unfortunately, biosimilar manufacturing is often different from the brand name drug due to variation in manufacturing processes. As a result, such variations may trigger unwanted immunogenicity. Risk based approach, like branded drug, is more likely be required for drug development of therapeutic proteins for evaluation of immunogenicity followed by a development plan for risk mitigation.