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Publication
Towards a new approach to phenylketonuria treatment : stabilization of pheylalanine hydroxylase using nanobiomaterials
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Abstract(s)
Phenylketonuria (PKU; MIM# 261600), the most frequent inherited disorder of amino acid metabolism, is caused by a deficient activity of human phenylalanine hydroxylase (hPAH; EC 1.14.16.1). To prevent the development of a severe neurophysiological retardation a strict dietetic restriction must be implemented as soon as possible, after birth. However being the only universal treatment available to date, it presents a poor long term compliance with a late onset of neurological symptoms. Aiming at an enzymatic replacement therapy, the use of recombinant proteins is still limited due to its physicochemical instability resulting in a high tendency to form soluble aggregates and consequent loss of biological function throughout pharmaceutical production and storage. In previous studies we have shown that low-molecular-weight polyol compounds stabilize the recombinant hPAH protein, preserving its structure and activity. In the present work our goal was to further stabilize hPAH by combining the use of polyols with the stabilization effect conferred by lyophilisation. Three excipients (glucose, trehalose and melibiose) were found to be able to preserve hPAH structure and function upon lyophilisation. The reconstituted samples maintained 100 % enzyme activity and substrate activation even after one year storage at 4 °C. In addition, protein engineering and the use of polymeric nanobiomaterials were also investigated. Site directed mutagenesis was used in order to change the protein’s surface charge or introduce residues less susceptible to oxidation. The C29S variant showed improved functional and structural properties. Considering polymeric nanobiomaterials, chitosan, hyaluronic acid and cyclodextrins were used to prepare nanoparticulate systems. Bridging the knowledge obtained by in silico and experimental data, we were able to establish the critical parameters that impacted the mild formation of chitosan nanoparticles and optimize a suitable hPAH nanoformulation in terms of particle size, encapsulation efficiency and biocompatibility This project contributed to elucidate the stabilization mechanisms of hPAH, a model protein for the study of inborn errors of metabolism and protein misfolding disorders. It may also result in a novel and effective approach to PKU treatment.
Description
Tese de doutoramento, Farmácia (Tecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2015
Keywords
Teses de doutoramento - 2015