Protein production and applications of NMR in pharmaceutical sciences
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The objective of Part I (Protein production for structural elucidation using NMR) is to design and develop strategies of producing amyloid beta protein for structural analysis using NMR. Amyloid beta oligomers are considered to be key elements causing Alzheimer’s disease and the elucidation of the structure of amyloid beta oligomers could be helpful in therapeutic development. Methods to express, purify and characterize amyloid beta protein are presented. Amyloid beta proteins are successfully produced from two constructs using E. coli expression system. The yield is 3 mg/L growth for unlabeled Aβ and 1 mg/L for uniformly labeled Aβ. The purity of Aβ was more than 95% based on MALDI-‐TOF analysis. Aβ fibrils and oligomers are prepared and analyzed by TEM. The results of 2D HSQC experiments indicate the C-terminus of Aβ might be the core region for aggregation. Moreover, CP2 has the ability to disaggregate Aβ oligomers but the action of disaggregation is immediate. Solid state NMR REDOR experiment will be performed in the future for identification of the binding site of CP2-‐ Aβ oligomers. The objective of Part II (Applications of NMR in pharmaceutical sciences) is to apply NMR techniques in pharmaceutical sciences. In chapter 7, a rapid, specific, and direct method for the real-‐time quantification of in vitro tenofovir (TNF) release from pH-‐sensitive microparticles using a Varian 400 MHZ 1H nuclear magnetic resonance (1H-‐NMR) spectrometer is introduced. Various analytical performance parameters such as linearity, precision, accuracy, limit of quantification (LOQ), limit of detection (LOD), and robustness were validated according to International Conference on Harmonization (ICH) guidelines. The results indicated this method had good linearity up to 5 mM and the method was specific, precise, and robust with all RSE% (relative standard error) less than 2%. The in vitro release of TNF from microparticles in both simulated vaginal fluid (VFS) and the mixture (VSFS) of VFS and simulated semen fluid (SFS) was monitored and quantified in real-‐time using 1H-‐NMR. The free TNF release using dialysis system was evaluated to compare with this new approach and the data demonstrated NMR was a more reliable and accurate method than other techniques which require dialysis systems for determination of drug release. The capability of real-‐ time quantification of in vitro drug release from microparticles not only provides a more accurate prediction of its biological behavior in vivo, but is also independent of potential interference from the dialysis membrane
Table of Contents
Introduction -- Protein expression, purification and characterization -- NMR basics and experiments -- Amyloid beta peptides expression, purification and oligomers preparation for structural elucidation using solid state NMR -- Introduction -- Quantitative NMR -- Real-time quantification of tenofovir in simulated biological fluids from ph-sensitive microparticles using 1H-NMR -- Other application of NMR in pharmaceutical science --
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Ph.D.