Characterization of uptake and efflux transporters in freshlyisolated and cryopreserved primary hepatocytes of different

species by using radiolabeled substrates.

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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: info@primacyt.de

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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