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Comparison of In Vitro Assays in Selecting Radiotracers for In Vivo P-Glycoprotein PET Imaging

Department of Radiology & Nuclear Medicine, VU University Medical Center, De Boelelaan 1085C, 1081 HV Amsterdam, The Netherlands
Department of Nuclear Medicine and Molecular Imaging, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands
Dipartimento di Farmacia-Scienze del Farmaco, Università Degli Studi di Bari, via Orabona 4, 70125 Bari, Italy
Biofordrug slr, via Orabona 4, 70125 Bari, Italy
Microbiology Systems and Biology Group, Netherlands Organisation for Applied Scientific Research (TNO), Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Pharmaceuticals 2017, 10(3), 76;
Received: 18 August 2017 / Revised: 13 September 2017 / Accepted: 15 September 2017 / Published: 20 September 2017
PDF [6052 KB, uploaded 20 September 2017]


Positron emission tomography (PET) imaging of P-glycoprotein (P-gp) in the blood-brain barrier can be important in neurological diseases where P-gp is affected, such as Alzheimer´s disease. Radiotracers used in the imaging studies are present at very small, nanomolar, concentration, whereas in vitro assays where these tracers are characterized, are usually performed at micromolar concentration, causing often discrepant in vivo and in vitro data. We had in vivo rodent PET data of [11C]verapamil, (R)-N-[18F]fluoroethylverapamil, (R)-O-[18F]fluoroethyl-norverapamil, [18F]MC225 and [18F]MC224 and we included also two new molecules [18F]MC198 and [18F]KE64 in this study. To improve the predictive value of in vitro assays, we labeled all the tracers with tritium and performed bidirectional substrate transport assay in MDCKII-MDR1 cells at three different concentrations (0.01, 1 and 50 µM) and also inhibition assay with P-gp inhibitors. As a comparison, we used non-radioactive molecules in transport assay in Caco-2 cells at a concentration of 10 µM and in calcein-AM inhibition assay in MDCKII-MDR1 cells. All the P-gp substrates were transported dose-dependently. At the highest concentration (50 µM), P-gp was saturated in a similar way as after treatment with P-gp inhibitors. Best in vivo correlation was obtained with the bidirectional transport assay at a concentration of 0.01 µM. One micromolar concentration in a transport assay or calcein-AM assay alone is not sufficient for correct in vivo prediction of substrate P-gp PET ligands. View Full-Text
Keywords: apical and basolateral membrane; bidirectional transport; blood-brain barrier; calcein-AM; tritium labeling apical and basolateral membrane; bidirectional transport; blood-brain barrier; calcein-AM; tritium labeling

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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Raaphorst, R.M.; Savolainen, H.; Cantore, M.; Van de Steeg, E.; Van Waarde, A.; Colabufo, N.A.; Elsinga, P.H.; Lammertsma, A.A.; Windhorst, A.D.; Luurtsema, G. Comparison of In Vitro Assays in Selecting Radiotracers for In Vivo P-Glycoprotein PET Imaging. Pharmaceuticals 2017, 10, 76.

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