Characterization of major zinc containing myonecrotic and procoagulant metalloprotease 'malabarin' from non lethal trimeresurus malabaricus snake venom with thrombin like activity: its neutralization by chelating agents

A major myonecrotic zinc containing metalloprotease 'malabarin' with thrombin like activity was purified by the combination of gel permeation and anion exchange chromatography from T. malabaricus snake venom. MALDI-TOF analysis of malabarin indicated a molecular mass of 45.76 kDa and its N-terminal sequence was found to be Ile-Ile-Leu- Pro(Leu)-Ile-Gly-Val-Ile-Leu(Glu)-Thr-Thr. Atomic absorption spectral analysis of malabarin raveled the association of zinc metal ion. Malabarin is not lethal when injected i.p. or i.m. but causes extensive hemorrhage and degradation of muscle tissue within 24 hours. Sections of muscle tissue under light microscope revealed hemorrhage and congestion of blood vessel during initial stage followed by extensive muscle fiber necrosis with elevated levels of serum creatine kinase and lactate dehydrogenase activity. Malabarin also exhibited strong procoagulant action and its procoagulant action is due to thrombin like activity; it hydrolyzes fibrinogen to form fibrin clot. The enzyme preferentially hydrolyzes Aα followed by B subunits of fibrinogen from the N-terminal region and the released products were identified as fibrinopeptide A and fibrinopeptide B by MALDI. The myonecrotic, fibrinogenolytic and subsequent procoagulant activities of malabarin was neutralized by specific metalloprotease inhibitors such as EDTA, EGTA and 1, 10-phenanthroline but not by PMSF a specific serine protease inhibitor. Since there is no antivenom available to neutralize local toxicity caused by T. malabaricus snakebite, EDTA chelation therapy may have more clinical relevance over conventional treatment.     

eEF-2 kinase dictates cross-talk between autophagy and apoptosis induced by Akt Inhibition, thereby modulating cytotoxicity of novel Akt inhibitor MK-2206

Inhibition of the survival kinase Akt can trigger apoptosis, and also has been found to activate autophagy, which may confound tumor attack. In this study, we investigated regulatory mechanisms through which apoptosis and autophagy were modulated in tumor cells subjected to Akt inhibition by MK-2206, the first allosteric small molecule inhibitor of Akt to enter clinical development. In human glioma cells, Akt inhibition by MK-2206 or siRNA-mediated attenuation strongly activated autophagy, whereas silencing of eukaryotic elongation factor-2 (eEF-2) kinase, a protein synthesis regulator, blunted this autophagic response. Suppression of MK-2206-induced autophagy by eEF-2 silencing was accompanied by a promotion of apoptotic cell death. Similarly, siRNA-mediated inhibition of eEF-2 kinase potentiated the efficacy of MK-2206 against glioma cells. Together, these results showed that blunting autophagy and augmenting apoptosis by inhibition of eEF-2 kinase could modulate the sensitivity of glioma cells to Akt inhibition. Our findings suggest that targeting eEF-2 kinase may reinforce the antitumor efficacy of Akt inhibitors such as MK-2206.     

Decision rules and associated sample size planning for regional approval utilizing multiregional clinical trials

Multiregional clinical trials provide the potential to make safe and effective medical products simultaneously available to patients globally. As regulatory decisions are always made in a local context, this poses huge regulatory challenges. In this article we propose two conditional decision rules that can be used for medical product approval by local regulatory agencies based on the results of a multiregional clinical trial. We also illustrate sample size planning for such trials.     

Efficacy and Cost Comparisons of Bronchodilatator Administration Between Metered Dose Inhalers with Disposable Spacers and Nebulizers for Acute Asthma Treatment

Background: Despite demonstration of equivalent efficacy of beta agonist delivery using a metered dose inhaler (MDI) with spacer vs. nebulizer in asthma patients, use of a nebulizer remains standard practice. Objectives: We hypothesize that beta agonist delivery with a MDI/disposable spacer combination is an effective and low-cost alternative to nebulizer delivery for acute asthma in an inner-city population. Methods: This study was a prospective, randomized, double-blinded, placebo-controlled trial with 60 acute asthma adult patients in two inner-city emergency departments. Subjects (n = 60) received albuterol with either a MDI/spacer combination or nebulizer. The spacer group (n = 29) received albuterol by MDI/spacer followed by placebo nebulization. The nebulizer group (n = 29) received placebo by MDI/spacer followed by albuterol nebulization. Peak flows, symptom scores, and need for rescue bronchodilatator were monitored. Median values were compared with the Kolmogorov-Smirnov test. Results: Patients in the two randomized groups had similar baseline characteristics. The severity of asthma exacerbation, median peak flows, and symptom scores were not significantly different between the two groups. The median (interquartile range) improvement in peak flow was 120 (75–180) L/min vs. 120 (80–155) L/min in the spacer and nebulizer groups, respectively (p = 0.56). The median improvement in the symptom score was 7 (5–9) vs. 7 (4–9) in the spacer and nebulizer groups, respectively (p = 0.78). The median cost of treatment per patient was $10.11 ($10.03–$10.28) vs. $18.26 ($9.88–$22.45) in the spacer and nebulizer groups, respectively (p < 0.001). Conclusion: There is no evidence of superiority of nebulizer to MDI/spacer beta agonist delivery for emergency management of acute asthma in the inner-city adult population. MDI/spacer may be a more economical alternative to nebulizer delivery.