Development and Validation of Asenapine and its Metabolite by Bioanalytical Methods Using Liquid Chromatography- Tandem Mass Spectroscopy (LC-MS/MS).

The objective of this study was to validate a simple and precise Ultra Performance liquid chromatographic method with Tandem Mass Spectrometry- (AB SCIEX) method for the determination of Asenapine and N-Desmethyl Asenapine (metabolite) in human plasma using Asenapine Maleate 13C D3 as Internal Standard (IS). The precision and accuracy data have to fulfill the requirements for quantification of the analytes in biological matrices to generate data for bioequivalence, bioavailability, pharmacokinetic or toxicology investigations. A Hypersil GOLD C18, 5μ column having 4.6 x 50 mm internal diameter in binary gradient mode with flow rate was 0.6 mL/min of mobile phase containing acetonitrile and ammonium formate (90:10) were used. The experiments were performed by loading in UPLC with a triple quadruple mass spectrometer, operating in the multiple reaction monitoring (MRM) modes. The method was validated over the concentration range of 0.1080 – 35.314 ng/mL(ANALYTE) and 0.1060 – 34.47 ng/mL (METABOLITE), by using 500 μL plasma samples.The mean recovery of Asenapine (81%) and N-Desmethyl Asenapine (80%) from spiked plasma samples was consistent and reproducible. The method was validated for linearity, accuracy, precision, specificity, and robustness. The intra- and inter-day precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. The developed assay method was applied to a clinical pharmacokinetic study in human volunteers

Influence of Nickel Coating on Flexural and Dynamic Behaviour of Aluminium

AbstractElectroless deposition is an autocatalytic chemical technique to deposit a layer of metal on a thin work piece in the presence of a reducing agent. In this work the changing structure of nickel deposits on aluminum and its alloys at the early stage of electroless nickel deposition using sodium hypophosphite ion as a reducing agent has been studied. The influences of nickel coating on flexural and dynamic behaviour of aluminium are investigated using experimental and numerical methods. Three-point bending tests are performed on coated & uncoated aluminium. The natural frequency of coated specimen and uncoated specimen has been studied. The nickel coating increases the natural frequency in aluminium. Experimental results are compared with finite element Analysis

Chronic Otitis Media (Squamous Disease): Clinical Predictors for Hearing Outcome

  Introduction Postoperative hearing outcome after tympanomastoid surgery may sometimes be disappointing. This study aims to identify the factors influencing hearing outcome in squamous type of chronic otitis media (COM). Materials and Methods Prospective descriptive study on consecutive patients undergoing tympano-mastoidectomy for squamous COM. Results Analysis of 40 ears revealed that 10% had discharge less than one year with majority (75%) having more than three years. Pars flaccida and postero-superior pars tensa retraction pockets were most common findings with frank cholesteatoma only in 10%. Though mean preoperative pure tone average was significantly better (p=0.004) in those with ear discharge less than one year, 50% required type IV tympanoplasty, irrespective of duration of discharge. Type IV tympanoplasty revealed significant worsening of hearing (p=0.05), unlike type I-III where intact stapes suprastructure showed a significant postoperative improvement (p = 0.036). Successful graft uptake with discharge free ear was noted in 97.5% by 12 weeks. Conclusion Majority of our patients availed otolaryngologist opinion after more than three years of onset of discharge. Significant ossicular erosion occurs early in the disease. Preoperative better audiogram is not a predictor of ossicular status. Lack of significant symptoms and subtle clinical finding in the tympanic membrane, overlooked by the patient as well as the primary health care provider are confounding factors for early referral and surgical intervention by otolaryngologist. There appears to be a considerable delay for the common person in reaching the services of otolaryngologist; a relevant issue which need to be addressed at a national level to reduce the burden of the disease

Transcriptional regulation of the urokinase receptor (u-PAR) - A central molecule of invasion and metastasis

The phenomenon of tumor-associated proteolysis has been acknowledged as a decisive step in the progression of cancer. This short review focuses on the urokinase receptor (u-PAR), a central molecule involved in tumor-associated invasion and metastasis, and summarizes the transcriptional regulation of u-PAR. The urokinase receptor (u-PAR) is a heavily glycosylated cell surface protein and binds the serine protease urokinase specifically and with high affinity. It consists of three similar cysteine-rich repeats and is anchored to the cell membrane via a GPI-anchor. The u-PAR gene comprises 7 exons and is located on chromosome 19q13. Transcriptional activation of the u-PAR promoter region can be induced by binding of transcription factors (Sp1, AP-1, AP-2, NF-kappaB). One current study gives an example for transcriptional downregulation of u-PAR through a PEA3/ets transcriptional silencing element. Knowledge of the molecular regulation of this molecule in tumor cells could be very important for diagnosis and therapy in the near future

Suppression of uPA and uPAR Attenuates Angiogenin Mediated Angiogenesis in Endothelial and Glioblastoma Cell Lines

In our earlier reports, we showed that downregulation of uPA and uPAR inhibited glioma tumor angiogenesis in SNB19 cells, and intraperitoneal injection of a hairpin shRNA expressing plasmid targeting uPA and uPAR inhibited angiogenesis in nude mice. The exact mechanism by which inhibition of angiogenesis takes place is not clearly understood.In the present study, we have attempted to investigate the mechanism by which uPA/uPAR downregulation by shRNA inhibits angiogenesis in endothelial and glioblastoma cell lines. uPA/uPAR downregulation by shRNA in U87 MG and U87 SPARC co-cultures with endothelial cells inhibited angiogenesis as assessed by in vitro angiogenesis assay and in vivo dorsal skin-fold chamber model in nude mice. Protein antibody array analysis of co-cultures of U87 and U87 SPARC cells with endothelial cells treated with pU2 (shRNA against uPA and uPAR) showed decreased angiogenin secretion and angiopoietin-1 as well as several other pro-angiogenic molecules. Therefore, we investigated the role of angiogenin and found that nuclear translocation, ribonucleolytic and 45S rRNA synthesis, which are all critical for angiogenic function of angiogenin, were significantly inhibited in endothelial cells transfected with uPA, uPAR and uPA/uPAR when compared with controls. Moreover, uPA and uPAR downregulation significantly inhibited the phosphorylation of Tie-2 receptor and also down regulated FKHR activation in the nucleus of endothelial cells via the GRB2/AKT/BAD pathway. Treatment of endothelial cells with ruPA increased angiogenin secretion and angiogenin expression as determined by ELISA and western blotting in a dose-dependent manner. The amino terminal fragment of uPA down regulated ruPA-induced angiogenin in endothelial cells, thereby suggesting that uPA plays a critical role in positively regulating angiogenin in glioblastoma cells.Taken together, our results suggest that uPA/uPAR downregulation suppresses angiogenesis in endothelial cells induced by glioblastoma cell lines partially by downregulation of angiogenin and by inhibition of the angiopoietin-1/AKT/FKHR pathway

Primary Laryngeal Aspergillosis in the Immunocompetent Patient

Introduction Fungal infections of the larynx are truly rare in immunocompetent people with only a handful of cases reported in the past five decades. Here we present a case series of 3 people (from 2019 to 2021), with no comorbid conditions, who presented with primary laryngeal aspergillosis. Materials and Methods Three patients with primary aspergillosis of the larynx were seen in the ENT outpatient department from 2019 to 2021, aged between 40-50 years of which two were females and one patient was a male. We describe the clinical presentation of each patient. Discussion Aspergillosis of the larynx usually occurs secondary to bronchopulmonary infections in the immunocompromised. The usual etiological factors include prolonged use of inhaled steroids, cytotoxic drugs, radiotherapy, smoking, mucosal injury, and antibiotic abuse. Persistent hoarseness of voice is the most common presenting symptom. Video laryngoscopy reveals erythema, oedema, hyperkeratosis, adherent white plaques, shallow ulcerations, and grey or white pseudo membrane formation over the vocal cords. Definitive diagnosis is done by the demonstration of hyphae either by KOH staining, culture in Sabouraud Dextrose Agar at 28 degrees Celsius, or tissue biopsy. Conclusion Primary aspergillosis of the larynx has occurred more frequently in recent times. Aspergillosis of the larynx can often mimic malignant or premalignant lesions. Persistent hoarseness of voice not responding to treatment should also raise a suspicion of fungal laryngitis, in the clinician and relatively simple treatment be done before significant morbidity occurs

Targeted Toxins in Brain Tumor Therapy

Targeted toxins, also known as immunotoxins or cytotoxins, are recombinant molecules that specifically bind to cell surface receptors that are overexpressed in cancer and the toxin component kills the cell. These recombinant proteins consist of a specific antibody or ligand coupled to a protein toxin. The targeted toxins bind to a surface antigen or receptor overexpressed in tumors, such as the epidermal growth factor receptor or interleukin-13 receptor. The toxin part of the molecule in all clinically used toxins is modified from bacterial or plant toxins, fused to an antibody or carrier ligand. Targeted toxins are very effective against cancer cells resistant to radiation and chemotherapy. They are far more potent than any known chemotherapy drug. Targeted toxins have shown an acceptable profile of toxicity and safety in early clinical studies and have demonstrated evidence of a tumor response. Currently, clinical trials with some targeted toxins are complete and the final results are pending. This review summarizes the characteristics of targeted toxins and the key findings of the important clinical studies with targeted toxins in malignant brain tumor patients. Obstacles to successful treatment of malignant brain tumors include poor penetration into tumor masses, the immune response to the toxin component and cancer heterogeneity. Strategies to overcome these limitations are being pursued in the current generation of targeted toxins

Downregulation of uPAR and Cathepsin B Induces Apoptosis via Regulation of Bcl-2 and Bax and Inhibition of the PI3K/Akt Pathway in Gliomas

Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes.In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU)-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results.In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas

Cell surface antigens in renal tumour cells: detection by immunoluminescence and enzymatic analysis

Two renal cell carcinoma cell lines (49RC 43STR and 75RC 2STR) were characterized by detection of the cell surface proteins: CD44(var), intercellular adhesion molecule-1 (ICAM-1), urokinase-type plasminogen activator (uPA) and its receptor and aminopeptidase N (APN). To detect their localization the immunoluminescent technique was used. In addition, the enzyme activity of uPA and APN was investigated in cell suspensions as well as in monolayers. The latter procedure was more advantageous since the additional use of HPLC permits a single registration of the fluorescent hydrolysis-product AMC (7-amino-4-methylcoumarin) without interference by cellular autofluorescence or non-reacted fluorescent substrate. Unlike 75RC 2STR, the cell line 49RC 43STR expressed high levels of uPA and APN. Contrary to that the cell line 75RC 2STR expressed high levels of ICAM-1 and CD44(v6), whereas 49RC 43STR showed a low level of ICAM-1 and no distinct light signal with anti-CD44(v6). The uPA activity was measured directly as well as indirectly (via plasmin) with the substrate Z-Gly-Gly-Arg-AMC. Both activator and plasmin activity were inhibited by D-Val-Phe-Lys-CMK and phenylmethylsulfonyl fluoride. The anti-catalytic antibody to uPA and that to uPA receptor were found to be inhibiting the uPA activity in a concentration-dependent manner. APN activity was assayed using alanine-p-nitroanilide. Peptidase activity was effectively inhibited by 1,10-phenanthroline and partly inhibited by ethylenediamine-tetraacetic acid. © 2001 Cancer Research Campaignhttp://www.bjcancer.co