Browsing by Author "Tikkanen, Alli"

Organic Anion Transporting Polypeptide 2B1 (OATP2B1) is an influx transporter expressed widely throughout the body in tissues such as intestine, liver, brain, placenta and skeletal muscle. Since many clinically used drugs are transported by OATP2B1, changes in the function of the transporter due to genetic polymorphism could lead to altered pharmacokinetics or -dynamics of OATP2B1 substrate drugs. The aim of this Master’s thesis was to create and optimize a cellular uptake assay to study the function of OATP2B1. Furthermore, the aim was to study the effects of six naturally occurring nonsynonymous single nucleotide variants on OATP2B1 transport function in vitro. With site-directed mutagenesis, single nucleotide changes were introduced into the gene coding for OATP2B1. OATP2B1 variants were expressed in human derived HEK293 cell line using baculovirus expression system. A cellular uptake assay with estrone-3-sulfate and a fluorescent probe 4’, 5’-dibromofluorescein (DBF) as substrates was set up and optimized. With the assay, OATP2B1-mediated uptake of variants was compared to the transport activity of OATP2B1 wild type. Amino acid changes Ser486Phe and Cys520Ser impaired OATP2B1 transport function severely. In addition, variant Thr318Ile transported DBF and estrone-3-sulfate less efficiently compared to OATP2B1 wild type, but Arg312Gln, Thr392Ile and Ser532Arg transport function was not affected. A method to study OATP2B1 function was created successfully. According to the results, single amino acid changes in OATP2B1 can impair OATP2B1 function. The results and method can be utilized to understand findings from pharmacogenetic studies in vivo, and to predict consequences of especially rare variants, which can be difficult to detect in small sample populations in clinical studies. However, further studies on the expression level and cellular localization of OATP2B1 variants are needed to fully characterize the impact of the variants studied.