stable infected hek293 oatp cells for transporter analysis

Stable transfected HEK293-OATP cells for transporter analysis

A model system for the assay of drug uptake.

PRIMACYT Cell Culture Technology GmbH, Hagenower Str. 73, D-19061 Schwerin, GermanyE-mail: [email protected], Phone.: +49 (0) 385-3993-600, Fax: +49 (0) 385-3993-602

www.primacyt.com

!

1PRIMACYT is a registered trade mark of PRIMACYT Cell Cure Technology GmbH - Copyright © 2013-2014 by PRIMACYT

!

2

The knowledge of drug 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 co-transporting 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.

!

HEK293 Substrates Inhibitors

HEK293-OATP1A2 Estrone 3-sulfateTaurocholic acid

FexofenadineNaringin



HEK293-OATP1B1 BromosulfophthaleinEstrone 3-sulfate

RifampicinCremophor


RifampicinCremophor

HEK293-OATP1B3 BromosulfophthaleinEstradiol 17ß-glucuronide

RifampicinCremophor


RifampicinCremophor


AtorvastatinRifampicin



3

Characterization of the transfected transporter proteins using specific substrates and inhibitors

Atorvastatin are trademarks of their respective owners.

! Cells for Science

4

Generation of 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.

!Cells for Science

5

Characterization of stable transfected HEK293-OATP1A2

Immunofluorescence and Western blot

Immunofluorescence analysis of HEK293-OATP1A2 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.

!!

! Cells for Science

6


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.

! Cells for Science

7


Functionality test

The uptake of Estrone 3-sulfate by HEK293-OATP1A2 was efficiently inhibited by Fexofenadine with an IC50 of 77.9 µmol/l.

The uptake of Estrone 3-sulfate by HEK293-OATP1A2 was efficiently inhibited by Naringin with an IC50 of 21.3 µmol/l.

!







RifampicinCremophor


RifampicinCremophor


RifampicinCremophor


RifampicinCremophor





8Atorvastatin are trademarks of their respective owners.


!Cells for Science

9

Characterization of stable transfected HEK293-OATP1B1


Immunofluorescence analysis of HEK293-OATP1B1 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.

! Cells for Science

10


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.

! Cells for Science

11


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%.

!







RifampicinCremophor


RifampicinCremophor

HEK293-OATP1B3 BromosulfophthaleinEstradiol-17ß-glucuronide

RifampicinCremophor


RifampicinCremophor







!Cells for Science

13





! Cells for Science

14


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.

! Cells for Science

15


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%.

!







RifampicinCremophor


RifampicinCremophor


RifampicinCremophor


RifampicinCremophor

HEK293 - OATP2B1 BromosulfophthaleinEstrone 3-sulfate


HEK293 - OATP2B1 BromosulfophthaleinEstrone 3-sulfate




!Cells for Science

17





! Cells for Science

18


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 HEK293-OATP2B1 was efficiently inhibited by Atorvastatin with IC50 of 0.4 µmol/l.

! Cells for Science

19


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.

Thank you!See also our Blog on primacyt.com

PRIMACYT Cell Culture Technology GmbH, Hagenower Str. 73, D-19061 Schwerin, GermanyE-mail: [email protected], Phone.: +49 (0) 385-3993-600, Fax: +49 (0) 385-3993-602

www.primacyt.com

!

20PRIMACYT is a registered trade mark of PRIMACYT Cell Culture Technology GmbH - Copyright © 2013-2014 by PRIMACYT

stable infected hek293 oatp cells for transporter analysis

Health & Medicine

hek293 cells

hek293oatp1a2 cells

hek293vc cells

hek293vc vector control

recombinant oatp1a2

drugdrug interaction

drug development

different transporter