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Stable infected HEK293 OATP Cells for Transporter Analysis
Stable transfected HEK293 cells expressing various drug transporters like OATP1A2, OATP1B1, OATP1B3, and OATP2B1 were generated. The transporter expression was confirmed and the cells were characterized by determining transport of specific substrates and inhibition by known inhibitors. Uptake assays found the cells actively transported representative substrates for each transporter and this uptake was inhibited by known inhibitors, demonstrating the functional expression of each transporter in the respective cell lines.
Stable infected HEK293 OATP Cells for Transporter Analysis
- 1. ! Stable transfected HEK293-OATPcells for transporter analysis A model system for the assay of drug uptake. PRIMACYT Cell Culture Technology GmbH, Hagenower Str. 73, D-19061 Schwerin, Germany E-mail: info@primacyt.com, Phone.: +49 (0) 385-3993-600, Fax: +49 (0) 385-3993-602 www.primacyt.com PRIMACYT is a registered trade mark of PRIMACYT Cell Cure Technology GmbH - Copyright © 2013-2014 by PRIMACYT 1
- 2. ! The knowledge ofdrug affinity and drug-drug interaction (DDI) and the role of organic anion transporting polypeptides (OATPs) and other transporter proteins like P-glycoprotein (MDR1, P-gp, ABCB1) is a basic requirement in drug development and is also recommended by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA). OATPs of the SLCO (former SLC21) superfamily are of fundamental importance in the transport of drugs across cell membranes, e.g. in intestine, liver, kidney, brain, skeleton muscle and placenta. Hepatic OATPs (OATP1B1, OATP1B3, OATP2B1) but also the sodium taurocholate cotransporting polypeptide (NTCP) are expressed at the sinusoidal membrane of human hepatocytes and transport several compounds into hepatocytes for biotransformation. In intestine, absorption of several compounds is mediated by e.g. OATP2B1 and the bile acid transporter ASBT (apical sodium-dependent bile salt transporter) which both are expressed at the brush border membrane. Primacyt utilizes well characterized stable transfected human embryonic kidney cells (HEK293) expressing different transporter proteins to study uptake of drugs and chemicals in vitro. 2
- 3. ! Characterization of thetransfected transporter proteins using specific substrates and inhibitors HEK293 Substrates Inhibitors HEK293-OATP1A2 Estrone 3-sulfate Taurocholic acid Fexofenadine Naringin HEK293-OATP1B1 Bromosulfophthalein Estrone 3-sulfate Rifampicin Cremophor HEK293-OATP1B3 Bromosulfophthalein Estradiol 17ß-glucuronide Rifampicin Cremophor HEK293-OATP2B1 Bromosulfophthalein Estrone 3-sulfate Atorvastatin Rifampicin Atorvastatin are trademarks of their respective owners. 3
- 4. ! Cells for Science Generationof stable transfected HEK293-OATP1A2 Coding sequence of SLCO1A2 was cloned into the retroviral expression vector pQCXIN. HEK293 cells were infected according to the instructions of the manufacturer. HEK293 cells expressing recombinant OATP1A2 were selected by neomycin. Equivalent Procedures were used for OATP1B1, OATP1B3 and OATP2B1with corresponding SLCO genes. 4
- 5. ! Cells for Science Characterizationof stable transfected HEK293-OATP1A2 Immunofluorescence and Western blot Immunofluorescence analysis of HEK293OATP1A2 cells showed that OATP1A2 was localized in the plasma membrane. ! Western blot analysis of HEK293-OATP1A2 and HEK293-VC (vector control) cells showed a strong band at the molecular mass of approximately 70 kDa in HEK293-OATP1A2 cells, which was not detectable in HEK293-VC cells. ! 5
- 6. ! Cells for Science Characterizationof stable transfected HEK293-OATP1A2 Functionality test The uptake function of HEK293-OATP1A2 was characterized with Taurocholic acid and Estrone 3-sulfate as substrates. Fexofenadine and Naringin served as inhibitors of Estrone 3-sulfate uptake. HEK293-OATP1A2 showed an uptake of Taurocholic acid with a Km of 80.5 µmol/l and Vmax of 20.5 pmol/mg × min. HEK293-OATP1A2 showed an uptake of Estron 3-sulfate with a Km value of 23.1 µmol/l and a Vmax value of 87.8 pmol/ mg×min. 6
- 7. ! Cells for Science Characterizationof stable transfected HEK293-OATP1A2 Functionality test The uptake of Estrone 3-sulfate by HEK293OATP1A2 was efficiently inhibited by Fexofenadine with an IC50 of 77.9 µmol/l. The uptake of Estrone 3-sulfate by HEK293OATP1A2 was efficiently inhibited by Naringin with an IC50 of 21.3 µmol/l. 7
- 8. ! Characterization of thetransfected transporter proteins using specific substrates and inhibitors HEK293 Substrates Inhibitors HEK293-OATP1A2 Estrone 3-sulfate Taurocholic acid Fexofenadine Naringin HEK293-OATP1B1 Bromosulfophthalein Estrone 3-sulfate Rifampicin Cremophor HEK293-OATP1B3 Bromosulfophthalein Estradiol 17ß-glucuronide Rifampicin Cremophor HEK293-OATP2B1 Bromosulfophthalein Estrone 3-sulfate Atorvastatin Rifampicin Atorvastatin are trademarks of their respective owners. 8
- 9. ! Cells for Science Characterizationof stable transfected HEK293-OATP1B1 Immunofluorescence and Western blot Immunofluorescence analysis of HEK293OATP1B1 cells showed that OATP1B1 was localized in the plasma membrane. Western blot analysis of HEK293-OATP1B1 and HEK293-VC (vector control) cells showed a strong band at the molecular mass of approximately 84 kDa in HEK293-OATP1B1 cells, which was not detectable in HEK293-VC cells. 9
- 10. ! Cells for Science Characterizationof stable transfected HEK293-OATP1B1 Functionality test The uptake function of HEK293-OATP1B1 was characterized with Bromosulfophthalein and Estrone 3-sulfate as substrates. Rifampicin and Cremophor served as inhibitors. HEK293-OATP1B1 showed an uptake of Bromosulfophthalein (BSP) with a Km value of 4.2 µmol/l and a Vmax value of 52.9 pmol/ mg×min. The uptake of BSP was effectively inhibited by Rifampicin with an IC50 of 14.2 µmol/l. 10
- 11. ! Cells for Science Characterizationof stable transfected HEK293-OATP1B1 Functionality test HEK293-OATP1B1 showed an uptake of Estrone 3-sulfate (E3S) with a Km value of 1.0 µmol/l and and a Vmax of 2.2 pmol/mg×min). The uptake of E3S was effectively inhibited by Cremophor with IC50 of 0.2%. 11
- 12. ! Characterization of thetransfected transporter proteins using specific substrates and inhibitors HEK293 Substrates Inhibitors HEK293-OATP1A2 Estrone 3-sulfate Taurocholic acid Fexofenadine Naringin HEK293-OATP1B1 Bromosulfophthalein Estrone 3-sulfate Rifampicin Cremophor HEK293-OATP1B3 Bromosulfophthalein Estradiol-17ß-glucuronide Rifampicin Cremophor HEK293-OATP2B1 Bromosulfophthalein Estrone 3-sulfate Atorvastatin Rifampicin Atorvastatin are trademarks of their respective owners. 12
- 13. ! Cells for Science Characterizationof stable transfected HEK293-OATP1B3 Immunofluorescence and Western blot Immunofluorescence analysis of HEK293OATP1B3 cells showed that OATP1B3 was localized in the plasma membrane. Western blot analysis of HEK293-OATP1B3 and HEK293-VC (vector control) cells showed a strong band at the molecular mass of approximately 120 kDa in HEK293-OATP1B3 cells, which was not detectable in HEK293-VC cells. 13
- 14. ! Cells for Science Characterizationof stable transfected HEK293-OATP1B3 Functionality test The uptake function of HEK293-OATP1B3 was characterized with Bromosulfophthalein (BSP) as substrate. Rifampicin and Cremophor were used as inhibitors. HEK293-OATP1B3 showed an uptake of BSP with a Km value of 10.3 µmol/l and a Vmax value of 24.2 pmol/mg×min. 14
- 15. ! Cells for Science Characterizationof stable transfected HEK293-OATP1B3 Functionality test BSP uptake of HEK293-OATP1B3 cells could be efficiently inhibited by Rifampicin with an IC50 of 2.5 µmol/l. HEK293-OATP1B3 mediated BSP uptake could be efficiently inhibited by Cremophor with an IC50 of 0.005%. 15
- 16. ! Characterization of thetransfected transporter proteins using specific substrates and inhibitors HEK293 Substrates Inhibitors HEK293-OATP1A2 Estrone 3-sulfate Taurocholic acid Fexofenadine Naringin HEK293-OATP1B1 Bromosulfophthalein Estrone 3-sulfate Rifampicin Cremophor HEK293-OATP1B3 Bromosulfophthalein Estradiol-17ß-glucuronide Rifampicin Cremophor HEK293 - OATP2B1 Bromosulfophthalein Estrone 3-sulfate Atorvastatin Rifampicin Atorvastatin are trademarks of their respective owners. 16
- 17. ! Cells for Science Characterizationof stable transfected HEK293-OATP2B1 Immunofluorescence and Western blot Immunofluorescence analysis of HEK293OATP2B1 cells showed that OATP2B1 was localized in the plasma membrane. Western blot analysis of HEK293-OATP2B1 and HEK293-VC (vector control) cells showed a strong band at the molecular mass of approximately 85 kDa in HEK293-OATP2B1 cells, which was not detectable in HEK293-VC cells. 17
- 18. ! Cells for Science Characterizationof stable transfected HEK293-OATP2B1 Functionality test The uptake function of HEK293-OATP2B1 was characterized with Bromosulfophthalein and Estrone 3-sulfate as substrates. Atorvastatin and Rifampicin served as inhibitors. HEK293-OATP2B1 showed an uptake of Bromosulfophthalein with a Km value of 11.1 µmol/l and a Vmax value of 22.4 pmol/ mg×min. Bromosulfophthalein uptake by HEK293OATP2B1 was efficiently inhibited by Atorvastatin with IC50 of 0.4 µmol/l. 18
- 19. ! Cells for Science Characterizationof stable transfected HEK293-OATP2B1 Functionality test HEK293-OATP2B1 showed an uptake of Estrone 3-sulfate with a Km of 21.9 µmol/l and a Vmax of 55.7 pmol/mg×min. The uptake of Estrone 3-sulfate could be efficiently inhibited by Rifampicin with an IC50 of 3.8 µmol/l. 19
- 20. ! Thank you! See alsoour Blog on primacyt.com PRIMACYT Cell Culture Technology GmbH, Hagenower Str. 73, D-19061 Schwerin, Germany E-mail: info@primacyt.com, Phone.: +49 (0) 385-3993-600, Fax: +49 (0) 385-3993-602 www.primacyt.com PRIMACYT is a registered trade mark of PRIMACYT Cell Culture Technology GmbH - Copyright © 2013-2014 by PRIMACYT 20