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

Abstract

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.

Influence of breast cancer resistance protein and P-glycoprotein on tissue distribution and excretion of Ko143 assessed with PET imaging in mice

“…On the other hand, the same authors found no in vitro transport of [ 3 H]Ko143 in human ABCB1- and mouse Abcb1a-overexpressing cells, which contradicts our in vivo PET results (Weidner et al, 2015). This makes the selection of candidate compounds for the development of PET tracers for ABC transporters very challenging, as in vitro data, even when acquired with low concentrations of 3 H-labeled compounds (Weidner et al, 2015; Raaphorst et al, 2017), do not necessarily predict the in vivo behavior of PET tracers.…”

The Role of P-Glycoprotein at the Blood–Brain Barrier in Neurological and Psychiatric Disease

“…Since [ 94m Tc]sestamibi is a substrate of both P-gp and ABCC1 and produces a relatively weak PET signal, this agent is no longer in use, and other tracers were developed to measure the function of P-gp (Kannan et al 2009c). In vitro (Raaphorst et al 2017) In vivo (mice, rats, NHP) (Hendrikse et al 1998b;Toyohara et al 2017;Syvänen et al 2006Syvänen et al , 2011 In humans (Bartels et al 2010;…”

Head-to-head comparison of (R)-[11C]verapamil and [18F]MC225 in non-human primates, tracers for measuring P-glycoprotein function

“…Nowadays, ( R )-[ 11 C]verapamil and [ 11 C]- N -desmethyl-loperamide are considered “gold standard” tracers for imaging the P-gp function, being the most extensively used in preclinical and clinical research [ 24 , 25 , 27 ]. However, these tracers have been identified as strong P-gp substrates [ 22 , 28 , 29 ]; i.e., the tracers are quickly transported from the brain to the blood. This results in a low tracer concentration inside the brain [ 25 ], precluding their use in the assessment of P-gp upregulation, which might occur in treatment-resistant depression [ 30 ] and patients with intractable epilepsy [ 13 ].…”