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Razvoj postopka za pripravo vzorcev krvne plazme in možganov podgane za kvantifikacijo novih zaviralcev holinesteraze
ID Turk, Maja (Author), ID Trontelj, Jurij (Mentor) More about this mentor... This link opens in a new window, ID Žakelj, Simon (Co-mentor)

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Abstract
Alzheimerjeva bolezen in depresija sta pogosti nevrološki motnji. Zdravljenje prve poteka z zaviralci holin esteraz in antagonisti glutamatnih receptorjev, zdravljenje druge pa med drugim tudi z inhibitorji monoaminske oksidaze. Te spojine morajo biti sposobne prehajati krvno-možgansko bariero, da lahko dosežejo mesto delovanja v možganih. V okviru magistrskega dela smo razvili in optimizirali metodo priprave vzorca krvne plazme in možganov za merjenje koncentracij atenolola in zaviralcev holin esteraze GUK-901 in donepezila ter zaviralcev monoamin oksidaz GDK-494, GDK-490C, GDK487, SAD-18. Pri preučevanju prehoda krvno-možganske bariere atenolol in donepezil dva lahko uporabimo kot modelni referenčni spojini. Atenolol ima nizko permeabilnost, donepezil pa visoko. Človeško krvno plazmo in možgane poskusnih živali smo obogatili s standardnimi raztopinami preiskovanih spojin ter jih nato očistili biološkega matriksa z ekstrakcijo na trdni fazi. Biološki vzorci namreč vsebujejo proteine, fosfolipide in druge nečistote, ki lahko vplivajo na ionizacijo analitov. Zato morajo biti ustrezno očiščeni pred merjenjem s tekočinskim kromatografom ultra visoke ločljivosti, sklopljenim z masnim detektorjem. Med razvojem metode priprave vzorcev smo preverjali ponovljivost ekstrakcije na trdni fazi, vpliv biološkega matriksa, linearnost odzivov in določili spodnjo mejo kvantifikacije. Ponovljivost ekstrakcije smo ovrednotili z relativnim standardnim odklonom, vsi rezultati so bili znotraj želenih 15 %, razen spojin GDK-494 in SAD-18 v vzorcih plazme, ki sta imeli slabšo ponovljivost. Z umeritvenimi krivuljami smo preverjali linearnost odzivov. Pri obogatenih vzorcih plazme in eni umeritveni krivulji z obogatenim možganskim tkivom so bile vrednosti Pearsonovega korelacijskega koeficienta nad 0,999, ostale vrednosti so bile nad 0,99. Rezultate na tej stopnji razvoja ocenjujemo kot ustrezne, za raziskave in vivo na živalskem modelu pa bi bilo treba izvesti še validacijo metode. Na realnih bioloških vzorcih smo določali porazdeljevanje GUK-901 med krvno plazmo in možgani. Rezultati kažejo, da GUK-901 uspešno prehaja krvno-možgansko bariero.

Language:Slovenian
Keywords:Alzheimerjeva bolezen, ekstrakcija na trdni fazi, krvno-možganska bariera, zaviralci holin esteraz
Work type:Master's thesis/paper
Organization:FFA - Faculty of Pharmacy
Year:2020
PID:20.500.12556/RUL-113785 This link opens in a new window
Publication date in RUL:04.02.2020
Views:1194
Downloads:228
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Secondary language

Language:English
Title:Development of a rat blood plasma and brain tissue sample preparation procedure for the quantification of novel cholinestease inhibitors
Abstract:
Alzheimer's disease and depression are common neurological disorders. So far, the first one has been treated with cholinesterase inhibitors and glutamate receptor antagonists. For the second one monoamine oxidase inhibitors can be used among other things. In order to improve treatment, this moleculs must be able to cross the blood-brain barrier to reach the site of action in the brain. We have developed and optimized a method for preparing blood plasma and brain samples to measure the concentrations of atenolol and cholinesterase inhibitors GUK-901, donepezil and monoamine oxidase inhibitor GDK-494, GDK-490C, GDK487, SAD-18. In examining the passage of the blood-brain barrier, atenolol and donepezil can be used as model reference compounds. Atenolol has low permeability and donepezil has high permeability. Human blood plasma and the brains of experimental animals have been enriched with standard solutions of the test compounds, and these were, in turn, purified by solid-phase extraction. Biological samples contain proteins, phospholipids and other impurities that can affect the ionization of analytes. Therefore, impurties must be removed before the drug concentrations are measured with an ultra-high resolution liquid chromatograph connected to a mass detector. During the sample preparation method development, we have examined the repeatability of the solid phase extraction, the influence of the biological matrix, the linearity of the responses, and determined the lower limit of quantification. The repeatability of extraction has been evaluated by the relative standard deviation. All results were within the desired 15%, except for compounds GDK-494 and SAD-18 in the plasma samples, which had worse repeatability. The linearity of the responses has been checked with the calibration curves. For the enriched plasma samples and one calibration curve with enriched brain tissue, the Pearson correlation coefficient values were above 0.999, the other values were above 0.99. The results at this stage of development are considered relevant for the in vivo research. For further animal model studies, a validation of the method should be performed as well. The distribution of GUK-901 between blood plasma and brain has been determined on real biological samples. The results have shown that GUK-901 successfully crosses the blood-brain barrier.

Keywords:Alzheimer disease, solid phase extraction, blood-brain bariere, cholinesterase inhibitors

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