Author/Editor	¹¹¹¹¹	Sibinovska, Nadica; Kristan, Katja; ªakelj, Simon
Title	¹¹¹¹¹	Suitability and reliability of in vitro epithelial cell models for nasal and pulmonary drug permeability determination
Translated title	¹¹¹¹¹	Ustreznost in zanesljivost in vitro epitelijskih celi§nih modelov za dolo§anje nazalne in pulmonalne permeabilnosti zdravilnih u§inkovin
Type	¹¹¹¹¹	monografija
Place	¹¹¹¹¹	Ljubljana
Publisher	¹¹¹¹¹	[N. Sibinovska]
Publication year	¹¹¹¹¹	2021
Volume	¹¹¹¹¹	str. 185 str.
Language	¹¹¹¹¹	eng
Abstract	¹¹¹¹¹	Uporaba ustreznih in zanesljivih in vitro modelov nosnih in plju§nih epitelijskih barier je klju§nega pomena pri vrednotenju permeabilnostnih lastnosti potencialnih zdravilnih u§inkovin in formulacij, ki so namenjene nazalni in pulmunarni aplikaciji. In vitro modeli morajo ustrezno posnemati barierne lastnosti nosne in dihalne sluznice, za«elena pa je tudi podobna ekspresija proteinov, ki tvorijo tesne stike in prena©alcev zdravilnih u§inkovin kot tudi jsko elektri§no upornost (TEER). Pred uporabo za visoko zmogljivo testiranje permeabilnosti zdravilnih u§ijsko elektri§no upornost (TEER). Pred uporabo za visoko zmogljivo testiranje permeabilnosti zdravilnih u§iezana tkiva, primarne celi§ne kulture in nesmrtne celi§ne linije, pri §emer ima vsak model svoje prednosti in slabrezana tkiva, primarne celi§ne kulture in nesmrtne celi§ne linije, pri §emer ima vsak model svoje prednosti in slabosti. Vendar pa so nesmrtne celi§ne linije v zgodnji fazi raziskav obi§ajno model prvega izbora, saj uporaba primarnih celic ali izrezane sluznice, kljub temu da bolje posnemata epitelijske bariere in vivo, ni primerna za visoko zmogljivo testiranje permeabilnosti zdravilnih u§inkovin, prav tako lahko pride do vpliva na variabilnost. RPMI 2650 in Calu-3 celi§ni liniji sta §love©kega izvora izvora) in predstavljata potenci izvora) in predstavljata potenciauvodu smo predstavili pregled an uvodu smo predstavili pregled ana modele epitelijskih barier. Prav tako smo podali podroben pregled «e objavljenih podatkov o karakterizaciji ceo modele epitelijskih barier. Prav tako smo podali podroben pregled «e objavljenih podatkov o karakterizaciji celic RPMI 2650 in Calu-3, saj sta bili ti dve celi§ni liniji v zadnjem desetletju predmet obse«ne raziskave glede optimalnih pogojev gojenja celic, morfologije in karakterizacije ultrastrukture, pa tudi glede genskega, proteinskega in funkcionalnega izra«anja prena©alcev zdravilnih u§inkovin. Ugotovitev, da rast na meji zrak-teko§ina (A-L) omogo§a ve§jo podobnost gojenih nesmrtnih celic z in vivo nosnim in dihalnim epitelijem, kave glede celi§nih linij RPMI 2650 in Calu-3. Vendar vrednotenje ustreznosti teh celi§nih linij kot modelov nosnegskave glede celi§nih linij RPMI 2650 in Calu-3. Vendar vrednotenje ustreznosti teh celi§nih linij kot modelov nosnegakega nabora vzorcev ©e ni bilo narejeno. Cilj na©e raziskave je bil raziskati ustreznost RPMI 2650 in Calikega nabora vzorcev ©e ni bilo narejeno. Cilj na©e raziskave je bil raziskati ustreznost RPMI 2650 in Calu-3 celi§nih linij, gojenih na meji A-L in L-L (teko§ina-teko§ina), kot modelov nosnega in dihalnega epitelija za dolo§anje permeabilnosti zdravilnih u§inkovin, pri §emer smo upo©tevali najnovej©e ICH (International Council for ements for Pharmaceuticals for Human Use (ICH) and US Food and Drug Administration (FDA) guidelines on showing suirements for Pharmaceuticalsrazvrstitev zdravilnih u§inkovin razvrstitev zdravilnih u§inkovin glede na njihovo permeabilnost. Ko smo dolo§ili, kateri pogoj rasti vodi do primernej©ega modela za testiranje permeabilnosti zdravilnih u§inkovin, smo raziskali uporabnost posameznih modelov za vrednotenje in vitro permeacije intranazalno apliciranih zdravilnih u§inkovin iz razli§nih formulacij, ki so na trgu. V prvem delu disertacije (Poglavje 1) smo v skladu z regulativnimi smernicami ovrednotili dva celi§na modela RPMI 2650 (A-L in L-L) za napovedovanje nazalneodelov tako, da smo izvedli teste permeabilnosti s ©tevilnimi ozna§evalci ni§elne permeabilnosti (dekstran, kmodelov tako, da smo izvedli teste permeabilnosti s ©tevilnimi ozna§evalci ni§elne permeabilnosti (dekstran, koednosti za model A-L RPMI 2650, sklepamo, da so bili celi§ni ve§plastni sloji manj prepustni, §e smo jih gojili narednosti za model A-L RPMI 2650, sklepamo, da so bili celi§ni ve§plastni sloji manj prepustni, §e smo jih gojili na ji. V obeh modelih smo s pomo§jo dvosmernih transportnih ©tudij z uporabo ustreznih prena©alnih substeji. V obeh modelih smo s pomo§jo dvosmernih transportnih ©tudij z uporabo ustreznih prena©alnih substratov in zaviralcev prikazali zanemarljivo funkcionalno ekspresijo prena©alcev, ki pospe©ujejo izlo§anje, kot sta npr. P-gp (P-glikoprotein) in BCRP (protein odpornosti za raka dojke, angl. Breast Cancer Resistance Protein). Z namenom, da bi ocenili permeabilnost 23 modelnih zdravilnih u§inkovin z nizko, srednjo in visoko permeabilnost, smo izvedli dvosmerne transportne ©tudije. Model A-L RPMI 2650 je omogo§al jasno razlikovanje med visoko permeabilnimi u§inkovinami (razreda 1 in 2 po biofarmacevtskem klasifikacijskem sistem (BCS)) in u§inkovinami z zmerno in nizko permeabilnostjo (BCS razreda 3 in 4), §esar pa model L-L ni razlikoval. Vrednosti za permeabilnost, ki smo jih dolo§ili dvanajstim modelnim zdravilnim u§inkovinam s celi§nim modelom A-L RPMI 2650, so dobro korelirale (Pearsonov koeficient korelacije (r = 0.96)) z vrednostmi pri popolnoma diferenciranem modelu rdili smo, da celi§ni model A-L RPMI 2650 predstavlja obetaven model nosnega epitelija za napovedovanje permeabilnostrdili smo, da celi§ni model A-L RPMI 2650 predstavlja obetaven model nosnega epitelija za napovedovanje permeabilnosti zdravilnih u§inkovin za nazalno aplikacijo od modela L-L. Ugotovili smo, da sta korelaciji med vrednostmi za permeabilnosti 22 modelnih zdravilnih u§inkovin v A-L RPMI 2650 modelu in tistimi, ki so bile dolo§ene vna§in raziskali ustreznost dveh na§in raziskali ustreznost dveh celi§nih modelov Calu-3 (A-L in L-L) za napovedovanje permeabilnosti zdravilnih u§inkovin skozi epitelij dihalnih poti. Ugotovili smo, da imata oba modela visoko barierno integriteto, kar je bilo razvidno iz zelo nizkih vrednosti za permeabilnost testiranih FD z visoko molekulsko maso. Z uporabo ABC (angl. ATP-binding cassette) transportnih substratov in zaviralcev smo izvedli dvosmerne transportne ©tudije, prav takonkovin z razli§no (nizko, srednjo in visoko) permeabilnostjo smo dolo§ili z dvosmernimi permeabilnostnimiinkovin z razli§no (nizko, srednjo in visoko) permeabilnostjo smo dolo§ili z dvosmernimi permeabilnostnimi (i.e. efluksno razmerje (ER) > (i.e. efluksno razmerje (ER) >elnih zdravilnih u§inkovin z obema celi§nima modeloma Calu-3. Ne glede na na§in rasti smo permeabilndelnih zdravilnih u§inkovin z obema celi§nima modeloma Calu-3. Ne glede na na§in rasti smo permeabilnost nizko permeabilnih zdravilnih u§inkovin zlahka lo§ili od permeabilnosti visoko permeabilnih zdravilnih u§inkovin, saj je bila o§itna razlika v Papp vrednostih, ki so bile znotraj obmo§ja dveh redov velikosti. Dodatno smo ugotovili, da so bile izra§unane Papp (navidezni permeabilnostni koeficient) vrednosti modelnih zdravilnih u§inkovin za oba celi§na modela v splo©nem znotraj istega velikostnega razreda, pri §emer so Papp vrednosti pri ve§ini zdravilnih u§inkovin ni«je za L-L Calu-3 model. Papp vrednosti, ki smo jih dolo§ili s Calu-3 celi§nima modeloma, so imele enak red velikosti kot Papp vrednosti, ki so bile dolo§ene s Caco-2 modelom. Ker je bila korelacija med celi§nimi modeli zelo visoka (r = 0.93 za A-L Calu-3 in Caco-2 ter r = 0.92 za L-L Calu-3 in Caco-2), smo zaklju§ili, da celi§ni liniji Calu-3 in Caco-2 lo§ujeta med zdravilnimi u§inkovinami z razli§no peremabilnostjo na podoben na§in. Kljub druga§nemu anatomskemu izvoru predstavljajo Calu-3 celice obetaven model nosne epitelne bariere, kar smo dokazali z odli§no korelacijo z MucilAir modelom za 11 modelnih zdravilnih u§inkovin (r = 0.97 di z dobro korelacijo s celi§no linijo RPMI 2650 (r = 0.95 za A-L Calu-3 in A-L RPMI 2650). ¦eprav nismo mogli zakljuudi z dobro korelacijo s celi§no linijo RPMI 2650 (r = 0.95 za A-L Calu-3 in A-L RPMI 2650). ¦eprav nismo mogli zaklju§iti, da eden izmed Calu-3 celi§nih modelov (A-L in L-L) omogo§a bolj©e razlikovanje med zdravilnimi u§inkovinami z razli§no permeabilnostjo, smo se odlo§ili, da bomo za nadaljnje permeabilnostne teste formulacij za intranazalno uporabo uporabili le modele A-L RPMI 2, vi©je genske ekspresije prena©alcev zdravilnih u§inkovin pri A-L Calu-3 modelu ter zaradi tega,a, vi©je genske ekspresije prena©alcev zdravilnih u§inkovin pri A-L Calu-3 modelu ter zaradi tega, ker se TEER vrednosti omenjenjega modela zelo dobro ujemajo z objavljenimi vrednostmi TEER-a dihalnega epitelija kuncev. V 3. poglaCalu-3, gojene na na§in A-L, pol Calu-3, gojene na na§in A-L, polikarbonatne membrane Transwell z razli§no velikostjo por in trislojno membrano lipid-olje-lipid v PAMPA sistemu (test permeabilnosti s sistemom vzporednih umetnih membran) (angl. parallel artificial membrane permeability assay). Oba celi§na modela smo uporabili za permeabilnostne teste dveh kortikosteroidov prve generacije, medtem ko sta bili drugi dve metodi dodatno uporabljeni za in vitro vrednotenje permeacije kortikosteroidov druge generacije. Pokazali smo, da in vitro rezultati permeacije zdravilnih u§inkovin sovpadajo z rezultati farmakokineti§nih ©tudij razli§nih formulacij preiskovanih intranazalnih kortikosteroidov in pravilno napovedali (ne) eja rezultatov bioekvivalen§nega testiranja nosnih sprejev. V poglavju 4 smo za vrednotenje in vitro permeacijenja rezultatov bioekvivalen§nega testiranja nosnih sprejev. V poglavju 4 smo za vrednotenje in vitro permeacije intranazalno apliciranih zdravilnih u§inkovin z lokalnim in sistemskim u§inkom v obliki raztopin in suspenzij prav tako uporabili A-L RPMI 2650 in A-L Calu-3 celi§na modela, z namenom, da pojasnimo vpliv razli§nih formulacij na permeabilnost zdravilnih u§inkovin. Celi§na modela sta se izkazala za dovolj diskriminatorna, prav tako smo z njima zaznali razlike v in vitro permeaciji zdravilnih u§inkovin glede na in vivo biolo©ko uporabnost, pri §emer smo pri samo enem primeru opazili ve§je razlike med formulacijami in vitro, kot pa dejansko obstajajo v in vivo. Za nekatere zdravilne u§inkovine, ki so bile v obliki raztopin, smo dobili dobro korelacijo z objavljenimi podatki o biolo©ki uporabnosti. Oba celi§na modela sta izkazala ustreznost za vrednotenje razli§nih nosnih formulacij in sta zaznala vpliv sestave formulacije na permeabilnost zdravilne u§inkovine. Ta raziskava je pokazala, da sta celi§ni liniji RPMI 2650 in Calu-3 primerni za napovedovanje permeabilnosti zdravilnih u§inkovin koinami z razli§no permeabilnostjo v primerjavi z L-L modelom, medtem ko celi§na modela A-L in L-L Calu-3 lo§ita med zdrvinami z razli§no permeabilnostjo v primerjavi z L-L modelom, medtem ko celi§na modela A-L in L-L Calu-3 lo§ita med zdra650 in A-L Calu-3 sta primerna za vrednotenje form2650 in A-L Calu-3 sta primerna za vrednotenje formue o vplivu formulacije na permeabilnost zdravilne u§inkovine. In vitro modeli predstavljajo obetaven sisteje o vplivu formulacije na permeabilnost zdravilne u§inkovine. In vitro modeli predstavljajo obetaven sistem za pravilno napovedovanje izida bioekvivalen§nih testiranj za generi§ne nosne formulacije.The use of suitable and reliable in vitro models of the nasal and pulmonary epithelial barriers is essential for evaluation of permeability properties of drug candidates and formulations intended for nasal or pulmonary drug adminissa, and it is desirable to observe similar expression of tight junction proteins and drug transporters, as wellosa, and it is desirable to observe similar expression of tight junction proteins and drug transporters, as well as similar transepithelial electrical resistance (TEER) values. In addition, validation of the in vitro models is needed prior to utilizing them ford cell lines are among the available in vitro models for this purpose, with each model having its owned cell lines are among the available in vitro models for this purpose, with each model having its own er, the immortalized cell lines are usually regarded as a reasonable choice during the early research phase, since thever, the immortalized cell lines are usually regarded as a reasonable choice during the early research phase, since the use of primary cells or excised mucosa, although resembling the epithelial barriers in vivo more closely, are not suitable for high-throughput assessment and can introduce variability. The RPMI 2650 and the Calu-3 cell lines are of human origin (nasal and tracheo-bronchial origin, osed as possibly useful models of the nasal (both the RPMI 2650 and the Calu-3 cells) and the airway (the Calu-3posed as possibly useful models of the nasal (both the RPMI 2650 and the Calu-3 cells) and the airway (the Calu-3 cell line) epithelia. A review of the anatomy and physiology of the nasal cavity and the tracheo-bronchial epithelium, as well as the available in vivo and in vitro models of the respective epithelial barriers is presented in the introduction of the thesis. Moreover, detailed overview of the published data on characterization of the RPMI 2650 and the Calu-3 cells has been given, as these two cell lines have been subject of extensive investigation in the past decade, with regard to the optimal cell culturing conditions, morphology and ultrastructural characterization, as well as gene, protein and functional expression of uptake and efflux drug transporters. The finding that the air-liquid (A-L) culturing interfaceithelia has shaped all further research regarding the RPMI 2650 and Calu-3 cell lines. However, evaluation of thpithelia has shaped all further research regarding the RPMI 2650 and Calu-3 cell lines. However, evaluation of theility prediction using large enough set of model drugs has not been made yet. The aim of our reseability prediction using large enough set of model drugs has not been made yet. The aim of our research was to explore the suitability of the RPMI 2650 and the Calu-3 cell lines cultured at the A-L and liquid-liquid (L-L) interface as models of the arriers for drug permeability determination, considering the most recent International Council forbarriers for drug permeability determination, considering the most recent International Council for ements for Pharmaceuticals for Human Use (ICH) and US Food and Drug Administration (FDA) guidelines on showing suirements for Pharmaceuticals for Human Use (ICH) and US Food and Drug Administration (FDA) guidelines on showing suitability of in vitro permeability methods for drug permeability classification. Moreover, after determining which culturing interface resulted in obtaining a more suitable model for drug permeability assessment, we investigated the appfrom different marketed formulat from different marketed formulations. The first part of the thesis (Chapter 1) is focused on assessing two RPMI 2650 cell models (cultured at the A-L and L-L interface) forthod suitability. Firstly, the cell layer integrity of the two cell models was investigated through cethod suitability. Firstly, the cell layer integrity of the two cell models was investigated through coith high molecular weight), and lower permeability values were obtained for the A-L RPMI 2650 model, indicating that thwith high molecular weight), and lower permeability values were obtained for the A-L RPMI 2650 model, indicating that theNegligible functional expression Negligible functional expression of the P-glycoprotein (P-gp) and Breast Cancer Resistance Protein h models was shown by bidirectional transport studies including appropriate substrates and inhibitors of drug transpth models was shown by bidirectional transport studies including appropriate substrates and inhibitors of drug transpof 23 model drugs form the low, moderate and high permeability category. The A-L RPMI 2650 model was able to cleof 23 model drugs form the low, moderate and high permeability category. The A-L RPMI 2650 model was able to clea 1 and 2) and drugs with moderate and low) 1 and 2) and drugs with moderate and low nd 4). On the other hand, no such clear difference in the permeability could be made with the L-Land 4). On the other hand, no such clear difference in the permeability could be made with the L-L model. Moreover, the model drug permeability for 12 model drugs determined in the A-L RPMI 2650 cell model correlated well (Pearson correlation coefficient (r) = 0.96) with the fully differentiated nasal epithelial model (MucilAir). We confirmed that the A-L RPMI 2650 cell model is a promising model of the nasal epithelial barrier, used for nasal drug permeability prediction. Good, but slightly lower correlations between the drug permeability of 22 model drugs in the investigated nasal cell models and those determined in the intestinal models (Caco-2 cells and isolated rat jejunum) were also established. Utilizing the same concept, we investigated the suitability of two Calu-3 cell models (A-L and L-L) for prediction of drug permeability across the airway epithelia in Chapter 2. It was shown that the two models have high barrier integrity, reflected through the very low permeability of the tested FDs with high molecular weight. Bidirectional transport studies using ATP-binding cassette (ABC) transporter substrates and inhibitors were carried out, and functional activity of P-gp, but not of BCRP was revealed. Moreover, the permeability of 22 model drugs belonging to different (low, moderate or high) permeability categories was assessed by bidirectional permeability assaysmodel drugs with both the A-L an model drugs with both the A-L andble model drugs could be readily distinguished from the permeability of highly permeable compoundable model drugs could be readily distinguished from the permeability of highly permeable compoundsders of magnitude. Another observation from our study was that the obtained Papp values for the model drugs with drders of magnitude. Another observation from our study was that the obtained Papp values for the model drugs with dier of magnitude, with the Papp values of most of the drugs being lower for the L-L Calu-3 model. Morder of magnitude, with the Papp values of most of the drugs being lower for the L-L Calu-3 model. Moreover, since the Papp values determined with the two Calu-3 cell models had the same order of magnitude as the Papp values determined with the Caco-2 model, and the established correlation between the cell were able to conclude that the Calu-3 and Caco-2 cell lines distinguish between drugs with differente were able to conclude that the Calu-3 and Caco-2 cell lines distinguish between drugs with different lar way. The potential of the Calu-3 cells to be used as a Doctoral thesis Abstract 3 model of the nasal ilar way. The potential of the Calu-3 cells to be used as a Doctoral thesis Abstract 3erent anatomical source, was demonstrated by the obtained excellent correlation with MucilAir for 11 mferent anatomical source, was demonstrated by the obtained excellent correlation with MucilAir for 11 mo2650 cell line (r = 0.95 for the 2650 cell line (r = 0.95 for the A-L Calu-3 vs. A-L RPMI 2650). Although we could not claim that when it comes to the A-L and L-L Calu-3 cells, one cell model enables better differentiationthe A-L RPMI 2650 and the A-L Ca the A-L Rered formulations, due to the observed presence of mucus and higher gene expression of drug transporters in the A-L tered formulations, due to the observed presence of mucus and higher gene expression of drug transporters in the A-L Calu-3 cells, and TEER values of thisvitro methods for nasal spray ev vitro methods for nasal spray evabranes with different pore size and lipid-oil-lipid tri-layer membrane in the parallel artificial membrane pembranes with different pore size and lipid-oil-lipid tri-layer membrane in the parallel artificial membrane permeability assay (PAMPA) system. The cell lines were implemented for permeability assays of two first-generation corticosteroids, while the other two methods were additionally utilized for in vitro permeation assessment of two-secoesults of pharmacokinetic studies of different formulations of the investigated intranasal corticosteroids and corresults of pharmacokinetic studies of different formulations of the investigated intranasal corticosteroids and correctly predicted (non)equivalence of the nasal sprays. Thus, the three in vitro methods have potential to predict the results of bioequivalence testing of nasal spray products. In Chapter 4, we employed the A-L RPMI 2650 and A-L Calu-3 cell models for evaluation of in vitro permeation of intranasally administered drugs having local and systemic effect from solution- and suspension-based fown to be sufficiently discriminative and revealed differences in the in vitro drug permeation comparable to theown to bee in vivo bioavailability, while in only one case they showed much higher observed differences between fhe in vivo bioavailability, while in only one case they showed much higher observed differences between fobtained for a limited number of drugs incorporated in solution-based formulations. The two cell models were shoobtained for a limited number of drugs incorporated in solution-based formulations. The two cell models were showosition on the permeability of the active drug. This research has shown that the RPMI 2650 and the Calu-3 cell lposition on the permeability of the active drug. This research has shown that the RPMI 2650 and the Calu-3 cell lihe A-L RPMI 2650 model allows better differentiation between drugs with different peThe A-L RPMI 2650 model allows better differentiation between drugs with different permeability characteristics in comparison with the RPMI 2650 cells grown at L-L interface, while the A-L and L-L Calu-3 cell models differentiate between drugs with low, moderate and high permeability designation in a similar manner. Tuspension-based formulations for intranasal administration and can provide valuable information about the influencesuspension-based formulations for intranasal administration and can provide valuable information about the influence of the formulation on the permeability of the active drug. The in vitro models have the potential to correctly predict the outcome of bioequivalence testing for generic nasal formulations.
Descriptors	¹¹¹¹¹	Zdravilne u§inkovine Permeabilnost
Keywords	¹¹¹¹¹	permeabilne lastnosti celi§na linija Calu-3 celi§ni modeli celi§ni model RPMI 2650