Traditionally, the transporter function has been characterized using radiolabeled substrates. The use of radiolabeled substrates bears disadvantages and risks. It may cause potential health risks and in order to perform experiments a specific equipment like scintillation counter and an isotope laboratory are required. We have analysed the uptake function with fluorescent substrates, which can be performed in normal labs

1. Photo by Ole M -­ Creative Commons Attribution-­NonCommercial-­ShareAlike License https://www.flickr.com/photos/99095175@N00 Created with Haiku Deck

2. Validation of a cell platform for intestinal and
hepatic transporters using fluorescent
substances
A presentation of PRIMACYT
Internet App Stores
info@primacyt.com
www.primacyt.com
Check for Primacyt LIVE
PRIMACYT Cell Culture Technology GmbH
Hagenower Str. 73, D-­19061 Schwerin, Germany
Phone.: +49 (0) 385-­3993-­600, Fax: +49 (0) 385-­3993-­602
Copyright © 2015 by Primacyt Cell Culture Technology GmbH

3. Ø Traditionally, the transporter function has been characterized using
radiolabeled substrates.
Ø The use of radiolabeled substrates bears disadvantages and risks:
a) it may cause potential health risks
b) to perform experiments a specific equipment like scintillation
counter and an isotope laboratory are required.
Ø We have analysed the uptake function with fluorescent substrates, which
can be performed in normal labs.
Objective

4. Ø Membrane transporters are major variables for disposition, efficacy and
safety of many drugs.
Ø Organic anion transporting polypeptides (OATPs, gene family: SLCO) and
Na+-­taurocholate co-­transporting polypeptide (NTCP) belong to the uptake
transporters and mediate the uptake of a broad range of substrates
including several widely prescribed drugs into cells.
Ø We have developed a cell platform using stably transfected cells
expressing pharmacologic relevant uptake transporters.
Background

5. Characterization of transporter proteins using
fluorescent substances and inhibitors
HEK293 Substrate Inhibitor
HEK-­OATP1A2 Rhodamine 123 Rifampicin (Rifa)
HEK-­OATP1B1
Fluorescein methotrexate (FMTX)
Fluorescein
Rifampicin (Rifa)
HEK-­OATP1B3 Fluorescein methotrexate (FMTX) Rifampicin (Rifa)
HEK-­NTCP Cholyl-­Lysyl-­Fluorescein (CLF) Cholate

6. Characterization of stably transfected HEK-­OATP1A2
90 kDa
70 kDa
Localization and expression of transporter Protein OATP1A2
were analysed by immunofluorescence and Western blot
analysis.
Ø Immunofluorescence analysis of HEK-­OATP1A2 cells
showed that OATP1A2 was localized in the plasma
membrane.
Ø Western blot analysis of HEK-­OATP1A2 and HEK-­VC
(vector control) cells showed two bands at the molecular
mass of approximately 70 and 90 kDa in HEK-­OATP1A2
cells, which were not detectable in HEK-­VC cells.

7. Characterisation of stably transfected HEK-­OATP1A2
Functionality test
The uptake function of HEK-­OATP1A2 was
characterized with Rhodamine 123 as
substrate. Rifampicin served as inhibitor of
Rhodamine 123 uptake.
Ø HEK-­OATP1A2 showed an uptake of
Rhodamine 123 with a Km of 3.8 µmol/l
and Vmax of 283.2 pmol/mg x min.
Ø OATP1A2 mediated uptake of Rhodamine
123 was efficiently inhibited by Rifampicin
with an IC50 of 40.9 µmol/l.
0 2 4 6 8 10
0
100
200
300
Rhodamine (µM)
UptakeofRhodamine
(pmol/mg*min)
Km = 3.8 ± 1.3 µmol/l
Vmax = 283.2 ± 41.8 pmol/mg × min
0.1 1 10 100 1000
0
50
100
150
Rifa (µM)
UptakeofRhodamine123
(%)
IC50 = 40.9 (22.1;; 75.7) µmol/l

8. Characterization of stably transfected HEK-­OATP1B1
Localization and expression of transporter Protein OATP1B1
were analysed by immunofluorescence and Western blot
analysis.
Ø Immunofluorescence analysis of HEK-­OATP1B1 cells
showed that transporter proteins was localized in the
plasma membrane.
Ø Western blot analysis of HEK-­OATP1B1 and HEK-­VC
(vector control) cells showed a strong band at the
molecular mass of approximately 84 kDa in HEK-­
OATP1B1 cells, which was not detectable in HEK-­VC
cells.
84 kDa

9. 0 10 20 30 40 50
0
20
40
60
80
FMTX (µM)
UptakeofFMTX
(µmol/mg*min)
Characterization of stably transfected HEK-­OATP1B1
Functionality test
The uptake function of HEK-­OATP1B1 was
characterized with FMTX and Fluorescein as
substrates. Rifampicin served as inhibitor.
Ø HEK-­OATP1B1 showed an uptake of FMTX
with a Km of 4.7 µmol/l and Vmax of 59.1
pmol/mg x min.
Ø HEK-­OATP1B1 showed an uptake of
Fluorescein with a Km of 18.2 µmol/l and
Vmax of 147.9 pmol/mg x min.
Km = 4.7 ± 1.3 µmol/l
Vmax = 59.1 ± 4.7 pmol/mg × min
0 10 20 30 40 50
0
50
100
150
Fluorescein (µM)
UptakeofFluorescein
(pmol/mg*min)
Km = 18.2 ± 6.7 µmol/l
Vmax = 147.9 ± 23.8 pmol/mg × min

10. Characterization of stably transfected HEK-­OATP1B1
Functionality test
Ø OATP1B1 mediated uptake of FMTX was
efficiently inhibited by Rifampicin with an
IC50 of 0.70 µmol/l.
Ø The uptake of Fluorescein was efficiently
inhibited by Rifampicin with an IC50 of
1.06 µmol/l.
0.01 1 100
0
50
100
150
Rifa (µM)
UptakeofFMTX
(%)
0.01 1 100
0
50
100
150
Rifa (µM)
UptakeofFluorescein
(%)
IC50 = 0.70 (0.46;; 1.05) µmol/l
IC50 = 1.06 (0.77;; 1.48) µmol/l

11. Characterization of stably transfected HEK-­OATP1B3
Localization and expression of transporter Protein OATP1B3
were analysed by immunofluorescence and Western blot
analysis
Ø Immunofluorescence analysis of HEK-­OATP1B3 cells
showed that OATP1B3 was localized in the plasma
membrane.
Ø Western blot analysis of HEK-­OATP1B3 and HEK-­VC
(vector control) cells showed a strong band at the
molecular mass of approximately 120 kDa in HEK-­
OATP1B3 cells, which was not detectable in HEK-­VC cells.
120 kDa

12. Characterization of stably transfected HEK-­OATP1B3
Functionality test
The uptake function of HEK-­OATP1B3 was
characterized with FMTX as substrate.
Rifampicin served as inhibitor of FMTX
uptake.
Ø HEK-­OATP1B3 showed an uptake of FMTX
with a Km of 1.5 µmol/l and Vmax of 22.8
pmol/mg x min.
Ø OATP1B3 mediated uptake of FMTX was
efficiently inhibited by Rifampicin with an
IC50 of 0.38 µmol/l.
0 5 10 15 20
0
5
10
15
20
25
FMTX (µM)
UptakeofFMTX
(pmol/mg*min)
Km = 1.5 ± 0.4 µmol/l
Vmax = 22.8 ± 1.7 pmol/mg × min
0.01 0.1 1 10 100
0
50
100
150
Rifa (µM)
UptakeofFMTX
(%)
IC50 = 0.38 (0.31;; 0.47) µmol/l

13. Characterization of stably transfected HEK-­NTCP
Localization and expression of transporter Protein NTCP were
analysed by immunofluorescence and Western blot analysis.
Ø Immunofluorescence analysis of HEK-­NTCP cells showed
that NTCP was localized in the plasma membrane.
Ø Western blot analysis of HEK-­NTCP and HEK-­VC (vector
control) cells showed a strong band at the molecular
mass of approximately 120 kDa in HEK-­NTCP cells, which
was not detectable in HEK-­VC cells.
45 kDa

14. Characterization of stably transfected HEK-­NTCP
Functionality test
The uptake function of HEK-­NTCP was
characterized with CLF as substrate. Cholate
served as inhibitor of CLF uptake.
Ø HEK-­NTCP showed an uptake of CLF with
a Km of 1.7 µmol/l and Vmax of 2.9
pmol/mg x sec.
Ø NTCP mediated uptake of CLF was
efficiently inhibited by Cholate with an IC50
of 7.28 µmol/l.
0 2 4 6 8 10
0
1
2
3
4
CLF (µM)
UptakeofCLF
(pmol/mgxsec)
Km = 1.7 ± 0.2 µmol/l
Vmax = 2.9 ± 0.1 pmol/mg × sec
0.01 0.1 1 10 100 1000
0
50
100
150
Cholate (µM)
UptakeofCLF
(%)
IC50 = 7.28 (4.46;; 11.9) µmol/l

15. Conclusions
Ø Transport function of stably transfected HEK-­293 cells expressing
OATPs and NTCP can be characterized with fluorescent substances
Ø Fluorescent compounds are well suited as an unhazardous alternative
to radiolabeled chemicals.
Ø These methods can be used to characterize the transporter activities in
primary hepatocyte cultures.