Browsing by Subject "transporter"

Investigating the role of cell membrane proteins has increased over the last decade, as drugdrug interactions and genetic polymorphisms have been found to cause changes in drug pharmacokinetics and dynamics. In this study the characteristics of the OATP1B1 transporter were reviewed and new in vitro research method to study protein functions was developed. Human Embryonic Kidney cells (HEK) is a human derived mammalian cell-line that is widely used in the study of OATP1B1 transporter. The Sf9 cell line is isolated from Spodoptera frugiperda insect and is one of the standard in vitro tools in a genetic engineering study. In the experimental part of this thesis the goal was to express OATP1B1 transporter in Sf9 and HEK293 cell lines. The wild-type SLCO1B1-gene encoding the OATP1B1 was virulent with baculovirus into the cells by the Bac-to-Bac® Baculovirus Expression System. For expression in the Sf9 cells, the aim of the study was to clone the SLCO1B1-gene into the pFastBac vector. The cloning was not successful in this study although attempts were made for several approaches. The expression of OATP1B1 transporter in HEK293 cells was successful. HEK293 cells expressing OATP1B1 transporter are well suited for the study of the SLCO1B1-gene. The in vitro method developed in this study remains in the research team as a tool to investigate the polymorphisms of the SLCO1B1-gene, the inhibition of the transporter and possible drug interactions.

Elucidation of transporter- and/or metabolic enzyme-mediated drug interactions is important part of early drug development. However the knowledge about clinical consequences of transporter-mediated drug-drug interactions is still limited and more investigation is needed to improve our understanding. MDR1 transporter, widely distributed on the pharmacokinetic barriers in the body (e.g. intestine) and has been shown no limit the bioavailability of drugs. Substrates of MDR1 are exposed to limited intestinal drug absorption and intestinal drug-drug interactions due to inhibition of the transporter. In predicting the clinical significance of an interaction, the principal obstacle has been the limited ability to appropriately scale the preclinical data into in vivo situation. In vitro-in vivo correlations on the extent of MDR1's influence on absorption and standardized predicting methods for drug-drug interactions using the inhibitory constants (IC50 and Ki) would greatly increase the value of in vitro studies. Current in vitro and in silico methods for prediction of the influence of MDR1 on intestinal absorption and related drug-drug interactions are discussed in the literature review. In addition, the latest regulatory draft guidances (FDA, EMA) are reviewed. Aliskiren has been shown to be a sensitive MDR1 substrate in vivo and high affinity substrate for the transporter in vitro. The objective of the experimental work was to study the MDR1-mediated transport of aliskiren and the related drug-drug interactions in vitro and in silico. Vesicular transport assay was used to obtain kinetic parameters for aliskiren (Km and Vmax) and inhibitor potencies (IC50) for ketoconazole, verapamil, itraconazole and its metabolite hydroxyitraconazole. Ki was further calculated for itraconazole and hydroxyitraconazole. Aliskiren showed high affinity to MDR1 transporter with a Km value 5 µM, consistent to what was reported previously in different assay systems. The interactions between aliskiren and the inhibitors in vitro correlated to the observed interactions in vivo in humans. In addition, hydroxyitraconazole was shown to be a potent inhibitor of MDR1-mediated transport of aliskiren in vitro. This suggests that hydroxyitraconazole may contribute to the pronounced interaction observed between aliskiren and itraconazole in a clinical interaction study. A compartmental absorption and transit (CAT) model with added enterocyte compartments and MDR1 efflux was used to describe the influence of MDR1 on intestinal absorption of aliskiren in humans. The integration of kinetic parameters (Km) from in vitro studies requires further optimization on how to describe the intracellular drug concentrations in the model. Aliskiren is however suitable MDR1 probe substrate to be used in in vitro and in vivo trials in humans and therefore gives a good basis for developing vitro-in vivo predictive models.

P-glycoprotein is an ATP-dependent efflux protein expressed in many tissues which participate in absorption, distribution and elimination of drug molecules. It can mediate clinically significant drug-drug interactions. Characteristics of P-gp have been studied widely and crystal structure of mouse P-gp has been successfully determined. P-gp binds its substrates either directly from cell membrane or from cytosol and it has at least three separate binding sites. P-gp has wide selection of substrates from many therapeutical groups. According to the latest computational models, a typical P-gp substrate can be defined with the help of molecule structural factors rather than physicochemical properties. However function of P-gp is very complex which is why drug-drug interactions should be studied for each drug pair separately. In addition expression of P-gp is regulated by nuclear receptors PXR and CAR thus P-gp induction is separate, which also complicates P-gp mediated interactions. P-gp substrates celiprolol, talinolol, aliskiren and fexofenadine have in vivo interactions with P-gp inhibitors or inducers. The objective the experimental work was to study suitability of two in vitro methods, MDCKII-cell permeability assay and MDR1-vesicle transport assay, for predicting in vivo effect of drug-drug interaction. ATP-dependent transport of substrates was determined in membrane vesicles extracted from human P-gp expressing Sf9 cells. Cell assay was used to determine efflux ratio (ER) for all the substrates alone and efflux ratio with P-gp inhibitor itraconazole for the substrates which have reported in vivo interaction with itraconazole. All compounds showed ATP dependent transport in MDR1-vesicles and celiprolol, talinolol and fexofenadine showed ER over 1 in MDCKII-MDR1 cells thus according to vesicle assay and ER-value they are P-gp substrates. However ER of talinolol and fexofenadine was not affected by inhibitor itraconazole, thus the drugs did not fulfil the inhibition criteria of FDA for P-gp substrates. The performing of interaction test was possible failed due lack of pre-incubation of the cells with the inhibitor. Talinolol had the highest ER in thus according to cell experiments talinolol has P-gp dependent transport. Aliskiren ER was not obtained because of the low recovery of the drug but it had clear ATP-dependent transport in the vesicle assay as was expected according to in vivo results. According to in vitro results and in vivo studies celiprolol is a poor P-gp substrate whereas fexofenadine showed P-gp mediated transport both in vitro and in vivo. The results suggest that significance of drug interaction is difficult to predict with the vesicle and the cell assay but they can be used to recognize P-gp substrates.