doktorska disertacija
Melita Hribar (Author), Jurij Trontelj (Mentor)

Abstract

V 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 gibanj; pri 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 dejavnikov; obenem 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.

Keywords

farmacevtske oblike;trdne farmacevtske oblike;peroralne farmacevtske oblike;zdravilne učinkovine;sproščanje;farmakokinetika;prebavila;želodec;črevesje;peristaltika;simulacija;aparature;modeli;vrednotenje;optimizacija;disertacije;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL FFA - Faculty of Pharmacy
Publisher: [M. Hribar]
UDC: 615.453.015:612.32/.33(043.3)
COBISS: 295486464 Link will open in a new window
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Secondary language: English
Secondary title: 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
Secondary abstract: 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's wall, therefore influencing the shape and movement of the container's 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' 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 capsules; and 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 repeatable experimental conditions, including the control of the hydrodynamic influences, as well as the direct physical contact of the silicone wall and the dosage form and the proper media selection, can lead to a design of a dissolution method showing discriminatory results, especially for mechanically susceptible dosage forms. The results of the current thesis additionally outline the prospects for future research, where the goal is to achieve an even better predictive power by closer replication of the true (measured) in vivo conditions inside the gastrointestinal tract.
Type (COBISS): Dissertation
Thesis comment: Univ. v Ljubljani, Fak. za Farmacijo
Pages: 186 str.
ID: 15645133