Pharmacological properties of radiotracers that measure p-glycoprotein function and density

Energy-dependent transporters of the ATP-binding cassette (ABC) family regulate the movement of molecules across cellular membranes. Several of these transporters are expressed in the endothelial cells of brain microvessels (blood-brain barrier) to protect brain tissue from exposure to toxins in the blood. Three of the most common ABC transporters at the blood-brain barrier are P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance protein 1 (MRP1). Changes in P-gp function and density are hypothesized to play a role in neurological disorders, mediating drug-resistant epilepsy, drug effectiveness against HIV infection of the brain, and Alzheimer disease. Therefore, to measure P-gp function and density in vivo, substrates (which are transported by P-gp) and inhibitors (which bind to P-gp) have been radiolabeled for use in the nuclear imaging technique positron emission tomography (PET). For accurate quantification, radiotracers must be selective for P-gp and have high signal strength. The purpose of this thesis was to evaluate whether two radiotracers that are used to image P-gp function and density fulfill these properties.

The selectivity and signal strength of the P-gp substrate N-desmethyl-loperamide (dLop) and the P-gp inhibitor tariquidar were assessed using pharmacology assays in human cell lines and post-mortem mouse brains, and using PET imaging in transgenic mice and healthy humans. We found that the radiotracer [11C]dLop is selective as a substrate for P-gp among the three major ABC transporters of the blood-brain barrier because accumulation of [3H]dLop was lowest in cells expressing P-gp, and the uptake of [11C]dLop was highest in brains of mice lacking P-gp. In addition to being selective, dLop is ionically trapped in acidic lysosomes; [3H]dLop accumulation decreased by 50% in human cells pretreated with compounds that raise lysosomal pH. This irreversible trapping mechanism of [11C]dLop amplifies the measured PET signal because radioactivity accumulates over time. However, the P-gp inhibitor tariquidar competes with dLop for lysosomal accumulation because it decreased the accumulation of [3H]dLop by 50% in human cells and that of [11C]dLop by 35-40% in lysosome-rich organs of P-gp knockout mice and healthy humans; competition was not observed in the brain. The lysosomal competition in the peripheral organs is problematic because tariquidar is used in combination with [11C]dLop to measure P-gp function in vivo and suggests that these two compounds cannot be used together to measure P-gp function in the periphery.

We also found that tariquidar is not a specific inhibitor of P-gp; it is also a substrate and inhibitor of BCRP. At low concentrations, [3H]tariquidar had highest accumulation in cells expressing P-gp and lowest accumulation in cells expressing BCRP, while at higher concentrations (100 nM), tariquidar inhibited the function of both P-gp and BCRP. In addition to not being selective, [11C]tariquidar has a low signal strength as a radiotracer because specific binding of [3H]tariquidar to P-gp in post-mortem mouse brains was only 20-30% of the total signal.

In conclusion, the selectivity and high signal strength of the radiotracer [11C]dLop allow it to selectively measure P-gp function at the blood-brain barrier and this radiotracer can be used to determine P-gp’s role in neurological disorders. In contrast, the lack of selectivity and low signal strength of [11C]tariquidar indicate that this inhibitor cannot measure P-gp density and that better inhibitor radiotracers are required.

List of scientific papers

I. Kannan P, Brimacombe KR, Zoghbi SS, Liow JS, Morse C, Taku A, Pike VW, Halldin C, Innis RB, Gottesman MM, Hall MD. N-desmethyl-loperamide is selective for P-glycoprotein among three ATP-binding cassette transporters at the blood-brain barrier. Drug Metab Disp. 38, 917-22 (2010).
https://doi.org/10.1124/dmd.109.031161

II. Kannan P, Brimacombe KR, Kreisl WC, Liow JS, Zoghbi SS, Telu S, Zhang Y, Pike VW, Halldin C, Gottesman MM, Innis RB, Hall MD. Lysosomal trapping of a radiolabeled substrate of P-glycoprotein as a mechanism for signal amplification in PET. Proc Natl Acad Sci U S A. 108, 2593-2598 (2011).
https://doi.org/10.1073/pnas.1014641108

III. Kannan P, Telu S, Shukla S, Ambudkar SV, Pike VW, Halldin C, Gottesman MM, Innis RB, Hall MD. The “specific” P-glycoprotein inhibitor tariquidar is also a substrate and an inhibitor for breast cancer resistance protein (BCRP/ABCG2). ACS Chem Neurosci. 2, 82-89 (2011).
https://doi.org/10.1021/cn100078a