Analiza literaturnih podatkov fizioloških vrednosti aktivnosti prebavnih lipaz in optimizacija metode lipolize in vitro s postopkom menjave medijev

Herlec, Ajda (avtor), Bogataj, Marija (mentor), Rede, Katarina (komentor)

Ena izmed rešitev za povečanje biološke uporabnost slabo topnih učinkovin je njihova vgradnja v lipidne dostavne sisteme. Po peroralni aplikaciji so ti podvrženi kompleksnim procesom razgradnje, ki jih klasični testi za vrednotenje sproščanja učinkovin ne zmorejo ponazoriti. Zaradi tega se razvijajo novi testi na osnovi in vitro lipolize, ki v večji meri upoštevajo pogoje, ki vplivajo na razgradnjo lipidnih dostavnih sistemov. Eden izmed pomembnih vplivov so prebavne lipaze, ki hidrolizirajo estrske vezi lipidov in jih tako spremenijo v oblike, ki se lahko absorbirajo. Namen te naloge je bil analizirati fiziološke podatke o aktivnosti prebavnih lipaz iz literature in optimizirati metodo in vitro lipolize s postopkom menjave medija. V prvem delu naloge smo v podatkovni bazi Pubmed sistematično iskali članke s podatki o aktivnosti želodčne in pankreasne lipaze, določene pri zdravih prostovoljcih v stanjih na tešče in po obroku. Našli smo 17 ustreznih člankov, jih analizirali in pridobljene podatke grafično prikazali. Pri analizi člankov smo ugotovili, da so vrednosti aktivnosti želodčne in pankreasne lipaze zelo odvisne od parametrov določanja aktivnosti, kot sta struktura zaužitega obroka (trden ali tekoč obrok) in metoda določanja aktivnosti lipaz (izbira metode, uporabljen substrat). Aktivnosti pankreasne lipaze pa niso odvisne od mesta odvzema vzorcev v dvanajstniku. Ideja izvedbe metode in vitro lipolize s postopkom menjave medija je, da lipidne dostavne sisteme dispergiramo v 0,01 M HCl, ki ponazarja kisle želodčne pogoje, nato pa z dodatkom prebavnega medija ponazorimo prehod vsebine želodca v tanko črevo. Preizkusili smo več mešanic 0,01 M HCl in maleatnega pufra v različnih razmerjih z različnimi koncentracijami komponent ter pH vrednostmi maleatnega pufra. Izbrali smo mešanice, ki so imele pH 7,5 oz. 6,5. V nadaljevanju smo maleatnemu pufru dodali natrijev deoksiholat in fosfatidilholin in tako pripravili prebavni medij. Pri tem smo optimizirali postopek priprave prebavnega medija. Z razvitim postopkom menjave medija (0,01 M raztopina HCl, ki ji dodamo prebavni medij, pripravljen iz 100 mM maleatnega pufra s pH 7,8 z optimiziranim postopkom) smo uspešno nadgradili metodo in vitro lipolize. Z metodo in vitro lipolize s postopkom menjave medija smo vrednotili vpliv aktivnosti pankreatina na sproščanje dipiridamola iz samo-mikroemulgirajočega sistema (SMES), pri čemer smo primerjali tri aktivnosti (250, 600 in 1000 TBU/mL) pankreatina, izbrane na podlagi analize literature. Določili smo koncentracijo sproščenega dipiridamola v celokupnih vzorcih, v vzorcih vodne faze in ostanku. Iz neznanega razloga so bili rezultati v celokupnih vzorcih in celotni količini (seštevek dipiridamola v ostanku in mase dipiridamola v odvzetih vzorcih) nižji od 100 %. Iz vzorcev vodne faze pa smo opazili, da so bili deleži sproščenega dipiridamola najvišji pri najnižji aktivnosti pankreasne lipaze in najnižji pri najvišji aktivnosti, kar ni bilo v skladu z našimi pričakovanji.

• Jezik: Slovenski jezik
• Ključne besede: aktivnost želodčne lipaze, aktivnost pankreasne lipaze, prebavni medij, in vitro lipoliza
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2021
• PID: 20.500.12556/RUL-128198
• Datum objave v RUL: 06.07.2021

HERLEC, Ajda, 2021, Analiza literaturnih podatkov fizioloških vrednosti aktivnosti prebavnih lipaz in optimizacija metode lipolize in vitro s postopkom menjave medijev [na spletu]. Magistrsko delo. [Dostopano 5 maj 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=128198

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Analysis of literature data on digestive lipase activity physiological values and optimization of in vitro lipolysis method using medium exchange process

Izvleček

One of the solutions to increase the bioavailability of poorly water-soluble active pharmaceutical ingredients is their incorporation into lipid-based drug delivery system. After oral administration lipid-based drug delivery systems are digested in complex processes that cannot be simulated by conventional tests to evaluate the release of active ingredients. New tests based on in vitro lipolysis are being developed that take greater account of the conditions that affect digestion of lipid-based delivery systems. One of the important influences are digestive lipases, which hydrolyse lipid ester bonds and thus convert them into forms that are more easily absorbed. The aim of this thesis was to analyse physiological data on the activity of digestive lipases from literature and to optimize the in vitro lipolysis method using medium exchange process. In the first part, we systematically searched the Pubmed database for articles with data on fasted and fed gastric and pancreatic lipase activity determined in healthy volunteers. We found 17 relevant articles, analysed them and graphically presented the obtained data. In the analysis of the articles, we found the values of gastric and pancreatic lipase activity are highly dependent on activity determination parameters such as structure of solid or liquid meal and lipase activity determination method (choice of method, substrate used). However, pancreatic lipase activities do not depend on the sampling site in duodenum. The idea of the in vitro lipolysis method using medium exchange process is to disperse lipid-based delivery systems in 0.01 M HCl, which illustrates acidic gastric conditions, and then by adding digestive medium we demonstrate the passage of gastric contents into the small intestine. We tested several mixtures of 0,01 M HCl and maleate buffer in different ratios and with different concentrations of components and pH values of maleate buffer. We selected mixtures that had a pH of 7.5 or 6.5. Sodium deoxycholate and phosphatidylcholine were then added to the maleate buffer to prepare the digestive medium. We optimized the process of preparation of the digestive medium. The in vitro lipolysis method was successfully upgraded with a developed medium exchange process: 0.01 M HCl solution to which digestive medium prepared from 100 mM maleate buffer pH 7.8 is added with an optimized process of preparation. The effect of pancreatic activity on the release of dipyridamole from the self-microemulsifying system (SMES) was evaluated with the in vitro lipolysis method using medium exchange process, comparing three activities of pancreatin (250, 600 in 1000 TBU/mL) selected based on literature analysis. We determined the concentration of released dipyridamole in the whole samples, in the samples of aqueous phase and in the residue. For some unknown reason, the results in the whole samples and the total amount of dipyridamole (sum of dipyridamole in the residue and mass of dipyridamole in the samples taken) were lower than 100 %. From the aqueous phase samples, we observed that the proportions of dipyridamole released were highest at the lowest pancreatic lipase activity and lowest at the highest pancreatic lipase activity, which was not within our expectations.

• Ključne besede: gastric lipase activity, pancreatic lipase activity, digestive medium, in vitro lipolysis