1,721,042 research outputs found
Bisoprolol pharmacokinetics and body composition in patients with chronic heart failure: a longitudinal study
No full textDevelopment of a rat blood plasma and brain tissue sample preparation procedure for the quantification of novel cholinestease inhibitors
Full text linkAlzheimerjeva 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.Alzheimer\u27s 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
Development of method for determination of simvastatin and simvastatin acid in rat serum by liquid chromatography coupled with tandem mass spectrometry
Full text linkSimvastatin je laktonsko predzdravilo, ki ima obsežen metabolizem prvega prehoda v jetrih. Njegov glavni presnovni produkt je simvastatinska kislina, ki je aktivna oblika simvastatina in deluje zaviralno na 3-hidroksi-3-metil glutaril koencim A reduktazo ter s tem zmanjša sintezo holesterola v jetrih. Simvastatin ima zaradi slabe vodotopnosti in obširnega metabolizma prvega prehoda zelo nizko biološko uporabnost.
V podporo predklinični študiji na podganah, katere namen je primerjava farmakokinetičnih lastnosti novega predzdravila in različnih na UL-FFA razvitih formulacij s simvastatinom, smo želeli razviti občutljivo, selektivno, točno in natančno metodo za merjenje koncentracij simvastatina in simvastatinske kisline v majhnih (100 µL) volumnih podganjega seruma. Poskusili smo tri različne načine priprave vzorcev, tako da smo izvedli obarjanje proteinov, ekstrakcijo na trdnem nosilcu z različnimi kartušami in tekočinsko ekstrakcijo z različnimi organskimi topili. Vzorce smo analizirali s tekočinsko kromatografijo sklopljeno s tandemsko masno detekcijo, pri čemer smo primerjali tudi uporabo različnih kolon in njihov vpliv na odziv spojin. Razvoj metode je potekal na vzorcih s človeško plazmo, končni preizkus izbrane optimalne metode s tekočinsko ekstrakcijo s terc-butil metil etrom, pa smo izvedli na vzorcih s podganjim serumom.
Selektivnost in natančnost metode smo ovrednotili na koncentracijskem območju med 0,5 µg/L in 50 µg/L. Ugotovili smo, da je simvastatin težavna spojina za analizo, ker ima problematično stabilnost in slabo ponovljivost priprave vzorcev. Boljšo ponovljivost je izkazovala simvastatinska kislina. Dokazali smo, da pri razviti metodi ni prisotnega učinka matrice, učinkovitost procesa priprave vzorca pa je okoli 15 % za simvastatin in 30 % za simvastatinsko kislino. Kljub nizkim vrednostim pa smo metodo preverili in zagotovili njeno ustreznost ter jo tudi potrdili v praksi na velikem številu vzorcev iz farmakokinetične študije.Simvastatin is a lactonic prodrug which has a large first pass metabolism in the liver. Its most important metabolite is simvastatin acid, an active form of simvastatin that is HMG-CoA reductase inhibitor and that causes decrease in cholesterol synthesis in liver. Because of the simvastatin’s low aqueous solubility and its large first pass metabolism simvastatin has extremely low bioavailability.
The aim of our work was to develop a sensitive, accurate and precise method for determination of simvastatin and simvastatin acid in small aliquots of rat plasma (100 µL). The developed method should serve as analytical support the pharmacokinetic preclinical study, where a novel simvastatin prodrug and various simvastatin formulations were compared in terms of their pharmacokinetic properties To select the optimal sample preparation technique, three methods were tested: protein precipitation, solid phase extraction using different cartridges and liquid extraction using different organic solvents. The samples were analysed by liquid chromatography coupled to tandem mass spectrometry. Additionally two different columns and their influence on analyte response were tested and compared. The method development was performed using human plasma as a surrogate, while the final method which utilized the liquid-liquid extraction by terc-buthyl methyl ether was accomplished on rat serum.
The selectivity, accuracy and precision were tested in concentration range between 0.5 µg/L and 50 µg/L. We concluded that simvastatin is a problematic analyte due to high variability during the sample preparation and poor stability. Better precision was achieved with simvastatin acid. We proved that the chosen method does not suffer from matrix effect, and the process efficiency is around 15% for simvastatin and around 30% for simvastatin acid. Regardless of the low process efficiency, the method has been successfully applied to a large number of study samples
Evaluation of different media for implementation of dissolution testing of a matrix tablet on an apparatus simulating the movement of human intestine
Full text linkPreskus sproščanja peroralnih farmacevtskih oblik se običajno izvaja bodisi za namene kontrole kakovosti, kjer preverjamo skladnost izdelka, bodisi za namene raziskav in razvoja, kjer si želimo poustvariti čim bolj biorelevantno okolje, ki nam omogoča visoko stopnjo in vitro-in vivo korelacije. V ta namen se poslužujemo uporabe kompleksnih in vitro modelov in metod, s katerimi lahko dosegamo visoko in vivo napovedno moč. Glavni cilj magistrske naloge je bil opredeliti uporabnost novega in vitro modela, razvitega v podjetju Lek d.d. Ljubljana, ki posnema črevesno gibanje, za sproščanje trdnih peroralnih farmacevtskih oblik v črevesu. S tem namenom smo preskusili sproščanje učinkovine iz treh formulacij hidrofilnih ogrodnih tablet z različnimi kinetikami sproščanja (hitra, srednje hitra, počasna formulacija) in iz referenčnega zdravila. Pri ogrodni tableti je učinkovina porazdeljena v ogrodju, ki nam, v primerjavi z oblikami s takojšnjim učinkom, omogoča nadzorovano sproščanje skozi daljše časovno obdobje. Ovrednotili smo pet različnih metod za izvedbo preskusa sproščanja. V acetatnem pufru pH-4,5 (program P03), kjer smo preverjali vpliv pH na sproščanje učinkovine, je le-to potekalo hitreje kot pri drugi primerljivi metodi (fosfatni pufer pH-6,8, program P04) zaradi večje topnosti oziroma hitrosti raztapljanja modelne učinkovine. V umetnem črevesnem soku, ki posnema pogoje na tešče (FaSSIF), smo dosegli najmanjša razlikovanja med formulacijami z različnimi kinetikami sproščanja in najmanjše ujemanje z in vivo opaženo kinetiko (razmerja med deleži sproščene učinkovine iz testnih formulacij in reference). Dodatek NaCl fosfatnemu pufru oziroma povišanje ionske jakosti je zaradi izsoljevanja upočasnilo sproščanje učinkovine v primerjavi s pufrom brez dodatka NaCl. Pri vseh preskušenih medijih oziroma metodah smo z modelom uspeli doseči razlikovanja med formulacijami z različnimi kinetikami sproščanja. Kot najboljša za izbrano formulacijo se je izkazala metoda s kalijevim fosfatnim pufrom pH-6,8 in programom P12, saj smo na ta način dosegli največje ujemanje z in vivo opaženo kinetiko. Prav tako smo z metodo dosegli boljše ujemanje z in vivo razmerji kot s klasično napravo z vesli (USP II). S predstavljenim primerom želimo prikazati uporabnost in fleksibilnost naprave oziroma novega in vitro modela, za katerega pričakujemo, da bo lahko uspešno simuliral sproščanje tudi drugih podobnih formulacij.Dissolution testing of oral dosage forms is usually performed either for quality control purposes testing the compliance of batches or for research and development purposes simulating a biorelevant environment that allows high degree of in vitro-in vivo correlation. For this purpose we use complex in vitro models and methods that reach a high level of in vivo prediction. The main objective of this master’s thesis has been to assess the efficacy of a new in vitro model developed at Lek d.d. Ljubljana simulating the movement of human intestine for evaluation of dissolution of oral dosage forms in human intestine. Therefore, we performed dissolution testing of three different hydrophilic matrix tablets with different release kinetics (fast, medium and slow release) and a reference product. In a matrix tablet the active ingredient is distributed in the matrix allowing for a controlled release over a longer time period compared to an immediate release form. We evaluated five different methods for dissolution testing. Acetate buffer pH-4,5 (program P03) where we examined the influence of pH on the release of the active ingredient showed a faster release rate in comparison to the other equivalent method (phosphate buffer pH-6,8, program P04) due to its higher solubility in acidic environment. Fasted state simulated intestinal fluid showed the least discrimination between formulations with different release rates as well as the greatest discrepancy to in vivo observed kinetics (test formulations/reference ratios from in vivo study). Due to salting out, addition of NaCl to phosphate buffer (increase in ionic strength) reduced release rates as compared to a buffer without addition of NaCl. The model was able to show discrimination between formulations with different release kinetics in all tested media. Owing to the greatest correlation with in vivo observed kinetics the method with phosphate buffer pH-6,8 and program P12 was established as the best formulation. Compared to the conventional USP II paddle method, the aforementioned method showed better correlation to observed in vivo ratios. The presented case aims to show efficacy and flexibility of the method and the novel in vitro model which is expected to successfully simulate dissolution of other similar formulations
Optimization of conditions in the new stomach dissolution model and comparison of the results of selected formulation with in vivo data and a test with the device II according to the US Pharmacopoeia
No full textDevelopment and validation of a method for determination of empagliflozin in blood plasma and its adjustment for microsampling
No full textEmpagliflozin je peroralno zdravilo, ki se uporablja za zniževanje glukoze v krvi pri odraslih bolnikih z neustrezno urejeno sladkorno boleznijo tipa 2 in za zdravljenje bolnikov s srčnim popuščanjem. Načrtuje se farmakokinetična raziskava različnih odmerkov empagliflozina na bolnikih s srčnim popuščanjem. V okviru magistrske naloge smo zato nameravali razviti občutljivo, selektivno, točno in natančno metodo za merjenje koncentracij empagliflozina v krvni plazmi ter postaviti izhodišče za prilagoditev metode na mikrovzorčenje krvi. Preizkusili smo tekočinsko ekstrakcijo ter njeno izboljšano in novejšo različico, podprto tekočinsko ekstrakcijo. V okviru tekočinske ekstrakcije smo preizkusili različnie vrste in volumne organskih topil, pri podprti tekočinski ekstrakciji pa smo preizkusili različne kartuše. Končna metoda, ki smo jo uspešno validirali, temelji na tekočinski ekstrakciji z etil acetatom kot ekstrakcijskim topilom, ki ji sledi detekcija s tekočinsko kromatografijo, sklopljena s tandemsko masno spektrometrijo (LC-MS/MS). Razvito metodo smo ovrednotili v skladu s smernicami ICH M10. Delovno območje analizne metode smo potrdili na koncentracijskem območju med 1 ng/mL in 1000 ng/mL. Izkoristek priprave vzorca je pri nizkem in visokem kontrolnem vzorcu znašal nad 70 %. Večji vpliv ozadja in slabšo ponovljivost smo zaznali le pri najnižjem testiranem kalibracijskem vzorcu pri 0,5 ng/mL. Preverili smo relativni vpliv ozadja, pri čemer je vrednost RSD pri QCL znašala 6,98 % in pri QCH 4,17 %. Potrdili smo ustrezno točnost vseh treh nivojev kontrolnih vzorcev in LLOQ, ki je znašala med 89,2 % in 108,7 %. Prav tako smo pri vseh treh nivojih kontrolnih vzorcev in LLOQ potrdili ustrezno ponovljivost, saj so se vrednosti RSD gibale v območju med 0,45 % ter 12,6 %. Dokazali smo ustrezno dolgoročno stabilnost (vsebnost glede na t = 0 h je po 90 dneh na -20 °C na nivoju QCL znašala 88,3 %, na nivoju QCM 104,7 % in na nivoju QCH 104,6 %) in ustrezno stabilnost v času rokovanja z vzorcem za empagliflozin.
Na podlagi rezultatov validacije metode lahko potrdimo, da je metoda ustrezna za določitev koncentracij empagliflozina v plazemskih vzorcih bolnikov na terapiji z empagliflozinom za npr. namen terapevtskega spremljanja koncentracij ali izvedbe farmakokinetičnih študij. Metoda je dobro izhodišče za prilagoditev na mikrovzorčenje, kar smo tudi preverili na manjši seriji posušenih krvnih madežev v območju 5-250 ng/mL.Empagliflozin is an oral medicine used to lower blood glucose in adult patients with inadequately controlled type 2 diabetes. It is also used to treat patients with heart failure. A pharmacokinetic study of different empagliflozin doses in patients with heart failure is planned. As part of the master\u27s thesis, we therefore wanted to develop a sensitive, selective, accurate and precise method for measuring empagliflozin concentrations in blood plasma, and set a starting point for adapting the method to blood microsampling. We tested two different methods of plasma sample preparation, specifically liquid–liquid extraction and its improved and newer version, supported liquid extraction. As part of the liquid–liquid extraction, we focused on testing different types and volumes of organic solvents, and in supported liquid extraction, we tested different cartridges. The final method, which we successfully validated, is based on liquid–liquid extraction with ethyl acetate as the extraction solvent, followed by detection by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The developed method was evaluated according to ICH M10 guidelines. The operating range of the analytical method was confirmed in the concentration range between 1 ng/mL and 1000 ng/mL. The sample preparation recovery was above 70% for the low and high control samples. A greater matrix effect and lower reproducibility were detected only for the lowest calibration sample tested at 0.5 ng/mL. We checked the relative matrix effect, with the RSD values being 6.98% and 4.17% for QCL and QCH, respectively. We confirmed the appropriate accuracy of all three levels of control samples and the LLOQ, which ranged between 89.2% and 108.7%. We also confirmed adequate reproducibility at all three levels of control samples and LLOQ, as RSD values ranged between 0.45% and 12.6%. Furthermore, we demonstrated adequate long-term stability and bench-top stability during sample handling for empagliflozin.
Based on the method validation results, we can confirm that the method is suitable for determining empagliflozin concentrations in plasma samples of patients on empagliflozin therapy for, e.g., the purpose of therapeutic concentration monitoring or for the purpose of conducting pharmacokinetic studies. The method is a good starting point for adaptation to microsampling, which we also verified on a smaller series of dried blood spots in the range of 5–250 ng/mL
Optimization of a method for determination of bleomycin in biological samples
No full textBleomicin je zmes glikopeptidnih antibiotikov izoliranih iz Streptomyces verticillus, ki so učinkoviti pri zdravljenju številnih vrst raka. Ker ima tudi bleomicin neželene učinke, zlasti toksično delovanje na pljuča, je med terapijo pomembno spremljanje njegove koncentracije v krvi. Zaradi kompleksnosti njegove strukture, polarnosti in sposobnosti keliranja kovin pa je metodo za določanje bleomicina v bioloških vzorcih izjemno zahtevno razviti. V magistrski nalogi smo skušali optimizirati metodo, ki so jo razvili Kosjek s sodelavci, in sicer predvsem s ciljem prenosa ekstrakcije na trdnem nosilcu iz vakuumskega sistema s kolonami na nadtlačni sistem z mikrotitrskimi ploščami, ki je primernejši za ekstrakcijo večjega števila bioloških vzorcev. Na ta način smo želeli poenostaviti pripravo vzorca, skrajšati čas priprave in izboljšati izkoristek ekstrakcije bleomicina iz vzorcev. Izkazalo se je, da je izkoristek ekstrakcije BLM po prenosu metode na mikrotitrske plošče sicer malenkost nižji, ampak ta razlika ni pomembna, če upoštevamo poenostavitev metode. Na mikrotitrskih ploščah smo optimizirali tudi sestavo elucijskega topila. Nadalje smo poskušali postopek skrajšati tako, da smo plazemske oz. serumske vzorce injicirali direktno po denaturaciji in centrifugiranju, torej brez ekstrakcije na SPE ploščah. Rezultati so bili obetavni zgolj pri serumskih vzorcih, pri čemer je bil izkoristek boljši pri nižjem volumnu seruma kot pri višjem. Matriks vzorca namreč inhibira signal našega analita v masnem spektrometru, kar je intenzivneje pri večjem volumnu seruma. Inhibicija je pri plazmi še izrazitejša, zato smo odločili, da ta metoda ni primerna za direktno injiciranje.
Metodo za ekstrakcijo BLM iz seruma in tumorskih tkiv smo optimizirali ter ovrednotili vpliv mase tkiva oziroma volumna biološkega vzorca na odziv masnega detektorja. Pri validaciji metode smo sledili smernicam Ameriškega urada za hrano in zdravila (FDA), kjer je bilo to smiselno in mogoče. Zaradi odkrite slabše robustnosti kromatografije internega standarda epirubicina smo se odločili, da ni primeren za to nalogo. Uporabno območje razvite metode za serum je od 100 do 5000 ng/mL, za tumorsko tkivo pa od 50 do 2500 ng/g.Bleomycin is a mixture of cytotoxic glycopeptide antibiotics isolated from a strain of Streptomyces verticillus. It is efficient in treatment of many neoplastic tissues. Nevertheless, bleomycin has side effects, mostly on pulmonary tissue. Consequently, bleomycin blood level should be monitored. Because of structure complexity, polarity and ability to chelate metals it is very difficult to set sensitive and selective analytical method for its determination in biological tissues.
In our work, we tried to optimize the method which was developed in following research Kosjek et al. Mostly with goal to transfer solid phase extraction from cartridge where we used low pressure to microplates under high pressure, which are more appropriate for extraction of multiple biological samples.
The goal was to simplify sample preparation, reduce time and increase efficiency of extraction of bleomycin from samples. The method was successfully transferred although efficiency of bleomycin extraction on microplate was lower. Within microplate we optimized composition of the elution solvent. Additionally, we tried to shorten the process with direct injection of blood samples after denaturation and centrifugation without the extraction using SPE microplates step. Results were relevant only for serum samples. Better results were with lower volume samples than higher volume samples. In mass spectrometer bleomycin detection is suppressed by matrix, especially in higher volume serum samples. Suppression is even greater in plasma samples, which is why we decided that it is not appropriate for direct injection.
We optimised the method for extraction of bleomycin from serum and tumor tissue and evaluated how mass of tissue and volume of biological sample influence to the mass detector response. In validation process we followed U.S Food and drug administration guidelines where this was reasonable and possible. Due to the poor robustness of internal standard in chromatography we decided that it is not suitable for this task. The working range of analytical method for serum is from 100 ng/mL to 5000 ng/mL and for tumor tissue from 50 to 2500 ng/g
Development and evaluation of new models for drug dissolution from solid oral pharmaceutical formulations using the simulation of the peristalsis in human stomach and intestine
No full textV sklopu raziskovalnega dela za doktorsko disertacijo smo vrednotili dve aparaturi, ki sta bili razviti v sodelovanju podjetja Lek d.d., člana skupine Sandoz, in Fakultete za strojništvo v Ljubljani. Vsebina raziskovalnega dela je bila razdeljena na dva tematska sklopa in sicer na vrednotenje in optimizacijo delovanja aparature za posnemanje želodčnega gibanja ter aparature za posnemanje črevesnega gibanja. Obe temeljita na podobnem principu delovanja. Sestavljeni sta iz fleksibilne silikonske vreče, ki predstavlja lumen organa ter zaslonk, ki izvajajo zunanji pritisk in s tem vplivajo na obliko ter premik vsebine. Aparatura za posnemanje črevesnega gibanja dodatno sestoji še iz platforme, ki se nagiba v smeri dveh osi in s tem omogoča gibanje tekočine. Premikanje platforme in zaslonk, ki delujejo v sosledju, ustvari specifične hidrodinamske pogoje in nastanek gibanja, ki skuša posnemati vzorec peristaltičnega gibanja v želodcu in tankem črevesu. Nastavitve parametrov, kot so amplituda zaprtja zaslonk, hitrost in zakasnitev med njimi, je možno nastavljati preko računalniške programske opreme. Glede na veliko fizično podobnost omenjenih aparatur anatomiji človeškega želodca in črevesa smo želeli v doktorski nalogi raziskati in oceniti dejansko uporabnost novih modelov za napovedovanje kinetike sproščanja zdravilnih učinkovin (ZU) iz farmacevtskih oblik (FO).
Testirali smo več izbranih vrst FO z različnimi ZU in rezultate primerjali s klasičnimi in vitro preskusi sproščanja (na primer s farmakopejsko metodo z vesli). Na aparaturi za posnemanje želodčnega gibanja smo testirali običajne tablete, mini tablete in kapsule. Na aparaturi za posnemanje črevesnega gibanja smo testirali ogrodne tablete z dvema različnima polimeroma - polietilen oksidom (PEO) in hidroksipropilmetil celulozo (HPMC). Primerjava je pokazala, da aparaturi za posnemanje želodčnega oz. črevesnega gibanja uspešno in nazorno ločita med izbranimi (različnimi) vzorci formulacij ter jih smiselno razporedita glede na njihovo sestavo ali izbrane pogoje testa. Pomemben del našega dela je bila optimizacija delovanja aparatur, s čimer smo dosegli večjo ponovljivost (RSD vrednosti smo zmanjšali z začetnih 15-20% v povprečju na vrednosti pod 10%). S pripravo natančnega protokola izvedbe testov smo z eksperimentalno določenimi parametri (določitev najprimernejšega mesta aplikacije FO v aparaturo in načina nadomeščanja medija) bistveno zmanjšali variabilnost med meritvami in zagotovili ponovljivo izvedbo eksperimentov, kar je pogoj tako za razvojno kot tudi za rutinsko delo. Eksperimenti za zmanjšanje variabilnosti so poleg optimizacije izvedbe obsegali tudi mehansko optimizacijo. Pri aparaturi za posnemanje črevesnega gibanja smo določili končno obliko in dimenzije vreče, ustrezen izbor materialov ter v sodelovanju s specializiranim podjetjem izdelali model za industrijsko izdelavo. Na ta način smo zagotovili odsotnost večjih razlik med posameznimi vrečami, ki so bile prisotne pri ročni izdelavi, kar je priporočljivo tudi pri vreči v aparaturi za posnemanje želodčnega gibanja, ki je še v fazi razvoja. Med raziskovalnim delom smo ovrednotili vpliv pogojev med testiranjem. Pri aparaturi za posnemanje želodčnega gibanja smo z namenom primerjave testirali tako zaprt način delovanja kot tudi odprt način (vzpostavljen pretok skozi aparaturo) ter testirali vpliv različnih pretokov in programskih nastavitev na sproščanje ZU iz FO. Pri obeh aparaturah smo testirali programe z različno hitrostjo in intenziteto ter ovrednotili njihov vpliv na kinetiko sproščanja učinkovine. Spreminjali smo amplitudo in hitrost krčenja zaslonk (ter nagib in hitrost gibanja platforme) ter posledično vplivali na premikanje tekočine v notranjosti silikonskih vsebnikov. Na podlagi dobljenih rezultatov smo dokazali, da s spreminjanjem amplitude in periode gibanja spreminjamo hidrodinamske pogoje v notranjosti silikonske vreče in s tem vplivamo na kinetiko sproščanja ZU iz različnih FO. Programska oprema nam nudi, da se s frekvenco kontrakcij približamo in vivo vrednostim peristaltičnih kontrakcij. Aparaturi omogočata, da se tudi z nekaterimi drugimi parametri približamo določenim in vivo pogojem. Teste na aparaturah za posnemanje želodčnega in črevesnega gibanja izvajamo z volumni, ki so bliže dejanskim in vivo pogojem (≤ 250 mL) v primerjavi s farmakopejsko metodo z vesli, kjer se običajno uporabi volumen medija 500-900 mL. Pri aparaturi za posnemanje želodčnega gibanja smo namestili ventil, ki omogoča, da vzorce zbiramo na izstopni strani silikonske vreče in določamo že raztopljeno učinkovino v frakcijah, ki pritečejo skozi vrečo želodca. Na ta način je omogočeno, da raziskujemo ne samo, kako hitro se učinkovina raztopi, ampak tudi, kako hitro raztopljena učinkovina priteče iz želodca, kar je z vidika in vivo procesa absorpcije v tankem črevesu zelo pomemben dejavnik. Izberemo lahko poljubno vrednost pretoka v in iz silikonske vreče, med našimi eksperimenti pa smo običajno uporabili in vivo relevantne pretoke. Z mehansko optimizacijo smo ventil nadgradili tako, da omogoča spreminjanje pretoka v časovni odvisnosti med potekom eksperimenta, tako da aparatura za posnemanje želodčnega gibanja lahko ponazori tudi in vivo vzorec želodčnega praznjenja tekočin. Metoda ponuja možnost in vitro ponazoritve želodčnega praznjenja v stanju na tešče po zaužitju kozarca vode (240 mL), kar je po smernicah ameriške agencije za hrano in zdravila (angl. Food and Drug Administration) predpisan protokol za in vivo farmakokinetične študije. Trditev, da z aparaturama lahko posnemamo nekatere in vivo parametre, smo podprli z dodatnimi meritvami z endoskopsko kapsulo SmartPill®, s katero smo opravili eksperimente merjenja tlakov v notranjosti obeh omenjenih aparatur in rezultate primerjali z dostopnimi literaturnimi in vivo podatki. Za razliko od klasičnih aparatur aparaturi za posnemanje želodčnega in črevesnega gibanja omogočata oblikovanje takih programov gibanja, ki kapsulo v notranjosti silikonske vreče mehansko obremenijo, saj zaradi stiskanja zaslonk omogočijo neposreden kontakt stene vreče in kapsule ter dosežejo in vivo relevantne vrednosti tlaka. Prav tako smo pokazali, da obstaja odvisnost med amplitudo kontrakcije in izmerjenim tlakom. Večja kot je bila nastavitev zožanja zaslonke, večji je bil izmerjeni tlak kontrakcije. Pomembnost vzpostavitve odvisnosti med izbiro programa in intenziteto izmerjenega tlaka se kaže v možnosti oblikovanja vnaprej pripravljenih programov, ki bi ponazorili različne faze želodčnega gibanja.
Na podlagi do sedaj opravljenih eksperimentov in uspešnih in vitro-in vivo korelacij lahko zaključimo, da sta aparaturi za simulacijo želodčnega in črevesnega gibanja uporabni kot in vitro preskus sproščanja za različne FO. Aparaturi dobro ločita med izbranimi vzorci v več uporabljenih medijih z ustrezno ponovljivostjo. Obenem smo pokazali, da omogočata nadzorovano izvedbo eksperimenta, saj je možen nadzor nad različnimi parametri, ki vplivajo na kinetiko sproščanja ZU. Pri obeh aparaturah lahko zagotovimo izbiro ustreznega volumna medija v vreči, volumna vzorčenja, aplikacije FO, načina nadomeščanja medija ter uporabe različnih gibanjpri aparaturi za simulacijo želodčnega gibanja je možno nadzorovati in spreminjati tudi pretok skozi želodčno vrečo. Prednost aparatur v primerjavi s klasičnimi in vitro testi je v možnosti nadzora vseh omenjenih parametrov, tako posamezno, kadar želimo preučevati vpliv določenega dejavnika na sproščanje ZU iz izbrane FO neodvisno od drugih dejavnikovobenem pa aparaturi ponujata možnost izvedbe eksperimenta z nastavitvijo vseh parametrov hkrati, kar je pomembno predvsem pri približevanju in vivo pogojem, ki naj bi nam kar najbolje omogočili napoved in vivo obnašanje izbrane FO.
Za vrednotenje in vivo relevantnosti novo razvitih aparatur smo profile sproščanja primerjali z in vivo podatki, kjer smo ugotovili, da se pri izbranih primerih lahko z novo razvitimi in vitro metodami bolje kot s klasičnimi farmakopejskimi metodami za preskušanje sproščanja približamo posnemanju sproščanja ZU in vivo. Na voljo so nam bili rezultati farmakokinetičnih študij za mini tablete s takojšnjim sproščanjem in za obe vrsti ogrodnih tablet. Pri primerjavi dveh vzorcev mini tablet smo testirali različne vrednosti pretokov ter različno intenzivne programe gibanja. Napovedno moč modela smo ovrednotili z izračunanimi determinacijskimi koeficienti (R2). Izkazalo se je, da aparatura za posnemanje želodčnega gibanja izkazuje boljšo korelacijo z in vivo podatki (R2=0,99) kot farmakopejska metoda z vesli (R2=0,87). Za PEO ogrodne tablete smo na aparaturi za posnemanje črevesnega gibanja razvili metodo, s katero smo dosegli boljše ujemanje z in vivo razmerji v primerjavi s farmakopejsko metodo z vesli. Pri tem smo testirali različne medije, njihove volumne in različne programe gibanja. Izračunali smo faktor podobnosti (f2) za farmakopejsko metodo z vesli in aparaturo za posnemanje črevesnega gibanja. Po smernicah FDA profila nista enaka, v kolikor je f2<50, kar smo dosegli le z aparaturo za posnemanje črevesnega gibanja. Raziskovalno delo smo nadaljevali s testiranjem HPMC ogrodnih tablet, kjer smo s testiranjem dveh različnih vzorcev dosegli in vitro-in vivo korelacijo najvišje stopnje (level A) in tako razširili dokaz uporabnosti modela za testiranje ogrodnih tablet. Upešno smo lahko napovedali profil koncentracije učinkovine v plazmi iz obeh vzorcev. Omenjene rezultate smo dosegli z uporabo enakih programskih nastavitev kot v primeru PEO ogrodnih tablet, kar govori v prid tudi izbranemu programu, ki tako ponuja dodatne možnosti za raziskovanje vplivov na kinetiko sproščanja iz drugih mehansko občutljivih FO.
V doktorski disertaciji smo predstavili inovativno metodologijo testiranja sproščanja z dvema novima modeloma, katerih prednost je predvsem čim večje približevanje pogojem in vivo. Z njima lahko raziskujemo vpliv mehanskih in hidrodinamskih obremenitev ter nekaterih drugih in vivo relevantnih pogojev na kinetiko sproščanja ZU iz FO. Na podlagi dobljenih rezultatov lahko predvidevamo, da se aparaturi za posnemanje želodčnega in črevesnega gibanja s svojo obliko in specifičnim vzorcem gibanja dobro približata nekaterim pogojem, ki in vivo vplivajo na kinetiko sproščanja ZU. Novi aparaturi smo uspešno uvedli v razvojno raziskovalno delo ter ovrednotili vpliv pogojev med testiranjem. Z dobro nadzorovanimi in ponovljivimi eksperimentalnimi pogoji, ki vključujejo tako dober nadzor hidrodinamskih vplivov, kot tudi izvajanja direktnih pritiskov stene na FO, ter s primernim izborom medijev za sproščanje, lahko vzpostavimo take pogoje, pri katerih se bodo lahko nazorno pokazale razlike, predvsem med različno mehansko občutljivimi FO. Izsledki dosedanjega dela hkrati ponujajo iztočnice za nadaljnje delo, saj bo imela napoved na podlagi in vitro rezultatov z nadaljnjim, še boljšim približevanjem fizikalnih pogojev znotraj lumna k dejansko izmerjenim in vivo vrednostim, še večjo težo.The focus of the research work for the doctoral thesis was to evaluate two apparatuses which were developed in collaboration with the company Lek Pharmaceuticals d.d., a Sandoz company, and the Faculty of Mechanical Engineering Ljubljana. The experimental work was divided into two sections to evaluate and optimize both, the Advanced Gastric Simulator (AGS) and the Intestine Model for Simulating the Peristaltic Action (IMSPA). The principle of action is similar for both apparatuses. They consist of a flexible silicone container, which represents the lumen of the organ, and the constriction mechanisms, which generate physical pressure on the container\u27s wall, therefore influencing the shape and movement of the container\u27s interior. The IMSPA additionally consists of a platform, with a two-axis actuator, to enable the movement of the fluid inside the container. The specific hydrodynamic conditions, closely resembling the peristaltic movement in the human stomach and the small intestine, are created by the action of the platform and the sequentially-acting constriction mechanisms. The settings of the parameters, such as the amplitude of the mechanisms\u27 aperture, the speed and the delay between the adjacent mechanisms, can be selected with the computer software. Considering the close physical similarity of the apparatuses with the anatomy of the human stomach and intestine, the purpose of this doctoral thesis was to research and evaluate the actual applicability of the new models for the prediction of drug release kinetics from the pharmaceutical dosage forms.
Several dosage forms with different active pharmaceutical ingredients were tested. The results were compared with the classical in vitro dissolution tests (USP II). The AGS was used for testing ordinary tablets, mini-tablets and capsulesand the IMSPA was used for evaluating the matrix tablets of two different polymer types – polyethylene oxide (PEO) and hydroxypropylmethyl cellulose (HPMC). The comparison showed, that the new models for the drug dissolution from solid oral pharmaceutical formulations using the simulation of the peristalsis in the human stomach and intestine, successfully and explicitly separated the tested (different) samples based on their composition or the selected test conditions.
An important part of our research work was the optimization of the apparatuses that led to larger repeatability (RSD values decreased from initial average values of 15-20% to values under 10%). Preparation of the precise protocol of the test execution with experimentally set parameters (defining the most appropriate position for the application of the dosage form into the apparatus and the manner of replacing the medium) significantly decreased the variability between the measurements and assured the repeatable test execution, which is a necessary factor for research and routine work. The experiments for decreasing the variability comprised not only the test procedure optimization, but also the mechanical optimization. The final shape and dimensions of the silicone tube and the suitable material selection were defined in the IMSPA. The model for industrial manufacturing of the silicone tube was designed in collaboration with a company, specializing in polymer materials, assuring the absence of major differences between the silicone tubes that were present when made by hand. This is recommended also for the silicone container in the AGS, which is still in the development phase.
During the research work we evaluated the influence of the test conditions. In the AGS, we compared the closed and open system (the flow of fluid through the apparatus) and tested the influence of various flow rates and programs on the drug release. Furthermore, we tested the programs with different speeds and intensities in both apparatuses to evaluate their influence on the drug release kinetics. The amplitude and the speed of contractions (and the amplitude and the speed of the platform movement) affecting the fluid movement inside the silicone containers were continually modified. Results showed that by modification of the amplitude and the period of movement, we can change the hydrodynamic conditions in the silicone interior and consequently influence the drug release from different dosage forms. The custom software enables us to reach in vivo values of the peristaltic contraction frequency.
The apparatuses enable the selection of certain other parameters to approach in vivo conditions. The volume of the medium used for the dissolution test can be selected to approach more realistic in vivo conditions (≤ 250 mL) compared to the USP II, where medium volume is normally 500-900 mL. A special valve was mounted in the AGS for simulating the pylorus and enabling the collection of the samples and the detection of the dissolved drug at the outlet of the container. In this manner, not only can the drug dissolution rate be determined but also, how fast the dissolved drug is emptied from the stomach, which is very likely the most important parameter for the estimation of the drug levels available for absorption further along the small intestine. Optional values of the flow rate in and out of the container can be selected, although in vivo relevant flows were chosen most frequently during our experiments. The pyloric valve was upgraded with a mechanical optimization in a way to enable time-dependent continuous flow rate adjustment during the test in order to simulate the in vivo pattern of the gastric emptying of fluids. This method offers the possibility of in vitro simulation of gastric emptying in the fasted state after drinking one glass of water (240 mL), which is a prescribed protocol for in vivo pharmacokinetic studies according to the U.S. agency FDA (Food and Drug Administration). The statement that with the apparatuses some of the in vivo parameters can be simulated, was supported by additional measurements with the endoscopic capsule SmartPill®, which enabled experiments with pressure measurements inside both apparatuses and direct comparison with the available literature in vivo data. In contrast with the conventional apparatuses, the AGS and the IMSPA enabled the design of programs to create direct mechanical influence in the silicone containers due to the physical contact of the silicone wall and the capsule created by the contractions, reaching in vivo relevant pressure values. We also showed that there is a dependency between the contraction amplitude and the measured pressure. The higher the constriction mechanism was set towards narrowing, the higher the measured pressure of the contraction became. The importance of establishing the relationship between the intensity of the different programs and the generated pressure lies in the possibility to develop various motility patterns for simulating the different phases of the specific gastric movement.
Based on the experiments and successfully determined in vitro-in vivo correlations, it can be concluded that the AGS and the IMSPA can be used for in vitro dissolution testing for several dosage forms. The apparatuses distinguish well between the tested samples in different dissolution media with the adequate repeatability. In addition, the models enable controlled test execution due to the possibility of controlling the parameters that influence the drug release kinetics. In both models, appropriate media volume in the container, sampling volume, insertion position of the dosage form, the manner of replacing the medium and various motion patterns can be selected. In the AGS, a controlled flow rate through the silicone container can be monitored and altered. The advantage of the novel apparatuses compared to the classical in vitro tests is in the possibility of controlling all the mentioned parameters, either isolated or combined. Solely the influence of one selected parameter on the drug release from the tested dosage form independently of other conditions can be studied as to whether it may play a role in the drug release process. Furthermore, the experiment combining all the parameters simultaneously is especially important when trying to simulate in vivo conditions with the goal to enable a good prediction of in vivo behavior of the dosage form.
To evaluate the in vivo relevance of the new models, the dissolution profiles were compared to in vivo data. The newly developed in vitro methods established a close simulation of the in vivo drug release in several examples. The results from the pharmacokinetic studies for immediate-release mini-tablets and both types of matrix tablets were available.
When comparing the two samples of the mini-tablets, various flow rates and motion programs with different intensities were tested. The predicting power of the model was evaluated with calculated determination coefficients (R2). It was shown that the AGS provided a better correlation with the in vivo data (R2=0.99) compared to the USP II (R2=0.87).
In the IMSPA the method for testing the PEO matrix tablets was developed, which showed better correlation with observed in vivo ratios compared to the USP II. Various media, media volume and motion patterns were tested. The similarity factor (f2) was calculated for the USP II method as well as for the IMSPA method. According to the FDA guidelines, the dissolution profiles are not considered to be equal, if f2<50, which was achieved when using the IMSPA method only. The research work was continued with the testing of the two HPMC matrix tablets, where the highest level of in vitro - in vivo correlation (Level A) was achieved, which confirmed the further applicability of the model for testing the matrix tablets. Successful prediction of the plasma concentration profiles for HPMC matrix tablets was established. The results were achieved by using the same software program as in the case of the PEO matrix tablets, which additionally presents the benefit of this particular program to further research possibilities for determining influences on the drug release kinetics for other mechanically susceptible dosage forms.
In this doctoral thesis, we presented the innovative methodology for drug dissolution testing using the two new models with the advantage of the close resemblance to the in vivo conditions. The influence of mechanical and hydrodynamic stress, as well as some other in vivo relevant conditions on the drug release kinetics, can be researched. Based on the obtained results, it can be assumed that the apparatuses simulate well, some of the parameters influencing the drug release kinetics due to its shape and the specific movement. The new apparatuses were successfully implemented in the regular research work and the main parameters influencing the test conditions were evaluated. Fully controlled and
Development and validation of a stability-indicating analytical method for determination of aggregates in a medicinal product with a peptide drug substance
No full textTerapevtski peptidi so poseben razred zdravilnih učinkovin, ki združujejo nekatere prednosti majhnih molekul in terapevtskih proteinov. Glavna terapevtska področja njihove uporabe so metabolne bolezni, onkologija in kardiovaskularne bolezni. Za zagotavljanje varnosti, kakovosti in učinkovitosti peptidnih zdravil regulativne agencije zahtevajo vrednotenje oligomernih in agregacijskih stanj. V ta namen se najpogosteje uporablja velikostno izključitvena kromatografija.
Cilj magistrske naloge je bil razviti analizno metodo na osnovi velikostno izključitvene kromatografije za določanje agregatov v zdravilu s peptidno zdravilno učinkovino. Ključni želeni lastnosti metode sta bili zadostna občutljivost ter ločljivost med agregati in zdravilno učinkovino. Preizkusili smo različne kromatografske kolone ter proučili vpliv osnovnih kromatografskih pogojev: temperature kolone, pretoka in sestave mobilne faze. Ugotovili smo, da je bila za kakovostno ločbo ključna izbira kolone s čim manjšim premerom delcev stacionarne faze in ustrezno velikostjo por, poleg tega pa sta nanjo v veliki meri vplivala delež trifluoroocetne kisline in acetonitrila v mobilni fazi. Odločili smo se za dokončni razvoj metode na koloni, namenjeni UHPLC analizam. Optimizacija je vključevala povečanje temperature kolone, pretoka in deleža organskega modifikatorja propan-1-ola v mobilni fazi.
Končno analizno metodo smo validirali v skladu z internim načrtom in kriteriji podjetja Lek d. d. Določili smo ponovljivost, rigidnost, mejo zaznave, mejo določitve, linearnost, točnost, robustnost in selektivnost.
Validirano analizno metodo smo uporabili za določanje agregatov v stabilnostnih vzorcih proučevanega zdravila. Ugotovili smo, da vsebnost agregatov s temperaturo in časom narašča, s čimer smo potrdili uporabnost in primernost metode za spremljanje stabilnosti peptidne učinkovine v končnem izdelku.Therapeutic peptides are a special class of medicinal agents that combine certain advantages of small molecules and therapeutic proteins. Their main therapeutic areas of application include metabolic diseases, oncology, and cardiovascular diseases. To ensure the safety, quality, and efficacy of peptide drugs, regulatory agencies require the evaluation of oligomeric and aggregation states. For this purpose, size-exclusion chromatography is most commonly used.
The aim of this master\u27s thesis was to develop a stability-indicating analytical method based on size-exclusion chromatography for determination of aggregates in a medicinal product with a peptide drug substance. The key desired properties of the method were sufficient sensitivity and resolution between the aggregates and the drug substance. We tested different chromatographic columns and studied the influence of basic chromatographic conditions: column temperature, flow rate, and mobile phase composition. We found that the key to quality separation was to choose a column with the smallest possible particle diameter of the stationary phase and appropriate pore size. Additionally, the content of trifluoroacetic acid and acetonitrile in the mobile phase had a significant impact. We decided to finalize the method development on a column intended for UHPLC analyses. Optimization involved increasing the column temperature, the flow rate and the content of the organic modifier propan-1-ol in the mobile phase.
The final analytical method was validated according to the internal plan and criteria of Lek d. d. We determined repeatability, rigidity, limit of detection, limit of quantification, linearity, accuracy, robustness and selectivity.
The validated analytical method was then used to determine the content of aggregates in stability samples of the studied drug product. The content of aggregates was found to increase with temperature and time, thus confirming the usefulness and suitability of the method for monitoring the stability of the peptide drug substance in the studied drug product
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