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Study of PEGylation 12PA with Activity PEG Derivative

Author: WangPeng
Tutor: TangJun
School: Jilin University
Course: Polymer Chemistry and Physics
Keywords: exenatide 12PA PEGylation molecular dynamic simulation
CLC: TQ464.7
Type: Master's thesis
Year: 2010
Downloads: 208
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Abstract

The biotechnology revolution has produced novel peptides and proteins that have become important new drugs. Around a third of drug candidates in clinical trials are polypeptides. In 2000, sales of polypeptide drugs were estimated at US $15 billion worldwide. Despite these tremendous advances, polypeptide drugs possess several shortcomings that limit their usefulness for application, which include their short circulating Half-Life, propensity to generate neutralizing antibodies, susceptibility to destruction by proteolysis enzymes, low solubility, and rapid kidney clearance due to their structural stability. Scientists have searched for new techniques to over these problems during these years; more and more attentions have been focused on the research of PEGylation of peptides. Commonly used PEG derivative is Methoxy-PEG(M-PEG) with one end terminated by methoxy whose structure is CH3-(OCH2CH2)nOH. It’s structure determines a neutral、hydrophilic polymer when grafts onto the surface, a biocompatible interface and protection layer can be gained, which can reduce the immunogenicity of exogenous material, lower the kidney clearance speed of Pegylated drug because of its large volume and absorbability of proteins、cells and bacteria, thus raise the effective time of curing; Meanwhile, the PEG molecules clear quickly in body without changing its structure. It has been proved that molecular weight of PEG is the only factor of clearance speed. The condition of Pegylation reaction could be critical in practice to destroy the protein/peptides losing their activity. Modified PEG are needed to active PEG from its low reaction activity.In this research, we chose a commonly used activate PEG derivative m-SC-PEG as modifier to realize chemical modification of 12PA. The contents contained the characterization of chemical structure, three-dimensional dynamics simulation and measurement of bioactivity.NMR was used to determine the structure of 12PA, molecular weight was measured by MS to determine its chemical structure. There are two modified position available, one is the amino residual in Lysine and the other is the amino residual in Histidine. Molecular dynamic simulation was carried out to determine which position has higher modified activity.Molecular structure was built and minimum amount of processing was taken to reduce computations. Construct three-dimensional configuration of 12PA by using the theory of extrapolate field effect. After a more stable conformation was obtained by 10-step temperature programmed, the simulation results was tested by projects such as temperature、kinetic energy、potential energy and root mean square deviation,thus the accuracy and stability were confirmed. It is concluded that the chemical environment of these two different amino residues were of great difference by simulation. The Lysine residues on the chain were protrudable beyond 12PA, while the ends of Histidine molecules were embedded in the 12PA because of the adjacent three hydrogen bonds. PEG modification could be predicted by competition of chemical reaction and steric angles to be connected on amino residuals on Lysine, the following experimental part was designed by the results of simulation.According to typical reaction route, when a certain temperature and reaction time were given, PEGylation of 12PA could be regulated by changing pH and polarity of reaction solution. White powder fluffy product could be obtained by lyophilization with good solubility. The product was tested by MALDI-MS and RP-HPLC to analysis modified products with different modifying position, the result was accord with those results calculated by theoretical analysis. Mono substitutive on Lysine product was obtained,with little end- and bi-modified byproducts. Materials with good selectivity and stability were obtained.To search the potential value of Polyethylene glycol-modified products on biology, we carried out cell activity experiment using pancreatic tumor cells by the typical MTT method. The results showed that pure 12PA or PEG has little pharmacodynamics on cancer cells, while PEG-12A gained significant inhibition upon the cancer cells. Enhanced potency generated due to a certain synergy of the two, Which may provides theoretical support on the discovery of new drugs and expands potential biomedical applications.

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CLC: > Industrial Technology > Chemical Industry > Pharmaceutical chemical industry > Drug production of biological products > Amino acids,peptides,proteins
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