characterization of uptake and efflux transporters in freshly isolated and cryopreserved primary...
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Characterization of uptake and efflux transporters in freshlyisolated and cryopreserved primary hepatocytes of different
species by using radiolabeled substrates.
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by P
RIM
ACYT
Cell
Cult
ure T
echn
olog
y G
mbH
, Sch
wer
in, G
erm
any
The 18th International Congress on In Vitro Toxicology ESTIV2014Egmond aan Zee, The Netherlands
Contact Data:PRIMACYT Cell Culture Technology GmbH, Hagenower Straße 73, 19061 Schwerin, Germany
Phone: +49 (0)385 - 3993 600, Email: [email protected]
BackgroundPrimary mammalian hepatocytes are the system of choice for multiple in-vitro applications such as drug metabolism, toxicology, and transporter assays.
Membrane transporters can be major determinants for the uptake and efflux of drugs.
Fig. 1: Human hepatic membrane transport proteins(Chandra and Brouwer, Pharmaceutical Research, 2004)
• Analysis of concentration dependent uptake of estrone-3 sulfate E1S in fresh isolated and cryopreserved plated hepatocytes.
• Characterization of efflux transporter P-gp with [3H]-talinolol (Tal) in presence or absence of its specific inhibitor PSC833.
• Examination of efflux transporter MRP2 with [3H]-estradiol-17β-glucoronide (E17β-Gln), with and without the inhibitor MK571.
Objectives
MethodsPrimary human, monkey (Cynomolgus) and dog (Beagle) hepatocytes have been cultured on 24well plates in HHMM (Human Hepatocyte Maintenance Medium). Assays with radiolabeled substrates +/- inhibitors were done on day 2 in culture after isolation or after thawing.
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Phase contrast images of freshly isolated monkey (A), dog (C), human (E) and cryopreserved monkey (B), dog (D) hepatocytes on day 1 in culture.
A - freshly isolated monkey
E - freshly isolated Human
D - cryopreserved dog
C - freshly isolated dog
B - cryopreserved monkey
Figure 2
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Concentration dependent uptake of E1S in primary human, dog and monkey hepatocytes. Blue curves: cryopreserved cells; Red and green curves: fresh hepatocytes in absence (red) or presence (green) of BSP (Bromosulfophthaleine), a competitive inhibitor for E1S uptake.
Figure 3
Notes: *p<0.05 vs. -BSP
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Time dependent efflux of Talinolol and E17β-Gln mediated by P-gp and MRP2, resp., in fresh human, dog and monkey hepatocytes. Figure 4
Notes: *p<0.05 vs. –PSC833, †p<0.05 vs. –MK571, $p<0.05 vs. human, §p<0.05 vs. dog
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E1S is a suitable model substrate to verify the functionality of uptake transporters in primary hepatocytes of different species.
Results generated in animal hepatocytes can not be transferred to humans.
P-gp mediated transport of Tal can be influenced by PSC833 in hepatocytes of all species.
In dog hepatocytes MRP2 efflux can be verified with E17β-Gln and inhibited by MK571.
Results and Conclusions
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Contributors
PRIMACYT Cell Culture Technology GmbH, Schwerin, GermanyA. Ullrich, J. Jia and D. Runge
Department of Clinical Pharmacology, University Medicine of Greifswald, GermanyM. Keiser, J.Jia and W. Siegmund
Department of Surgery, University Medicine of GreifswaldM. Patrzyk, A. Busemann and C. D. Heidecke
Major keywords are: uptake transporters, ef.lux transporters, radiolabeled substrates, membrane transporters, inhibitor PSC833, inhibitor MK571, time dependent ef.lux of Talinolol, freshly isolated hepatocytes,
cryopreserved hepatocytes, human hepatocytes, Cynomolgus monkey, hepatocytes, Beagle hepatocytes, 18th International Congress on In Vitro Toxicology ESTIV2014.
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