Purpose-Overactivity of the multidrug efflux transporter P-glycoprotein (P-gp) at the bloodbrain barrier (BBB) is believed to play an important role in resistance to central nervous system drug treatment. (R)-[ 11 C]verapamil (VPM) PET can be used to measure the function of P-gp at the BBB, but low brain uptake of VPM hampers the mapping of regional differences in cerebral P-gp function and expression. The aim of this study was to evaluate the dose-response relationship of two potent P-gp inhibitors and to investigate if increased brain uptake of VPM mediated by P-gp inhibition can be used to assess regional differences in P-gp activity.Methods-Two groups of Sprague-Dawley rats (n=12) underwent single VPM PET scans at 120 min after administration of different doses of the P-gp inhibitors tariquidar and elacridar. In an additional 6 rats, paired VPM PET scans were performed before and after administration of 3 mg/ kg tariquidar.Results-Inhibitor administration resulted in an up to 11-fold increase in VPM brain distribution volumes (DV) with ED 50 values of 3.0±0.2 and 1.2±0.1 mg/kg for tariquidar and elacridar, respectively. In paired PET scans, 3 mg/kg tariquidar resulted in regionally different enhancement of brain activity distribution, with lowest DV in cerebellum and highest DV in thalamus.Conclusion-Our data show that tariquidar and elacridar are able to increase VPM brain distribution in rat brain up to 11-fold over baseline at maximum effective doses, with elacridar being about 3 times more potent than tariquidar. Regional differences in tariquidar-induced modulation of VPM brain uptake point to regional differences in cerebral P-gp function and expression in rat brain.

A low-molecular-weight (18) F-labeled tetrazine derivative was developed as a highly versatile tool for bioorthogonal PET imaging. Prosthetic groups and undesired carrying of (18) F through additional steps were evaded by direct (18) F-fluorination of an appropriate tetrazine precursor. Reaction kinetics of the cycloaddition with trans-cyclooctenes were investigated by applying quantum chemical calculations and stopped-flow measurements in human plasma; the results indicated that the labeled tetrazine is suitable as a bioorthogonal probe for the imaging of dienophile-tagged (bio)molecules. In vitro and in vivo investigations revealed high stability and PET/MRI in mice showed fast homogeneous biodistribution of the (18) F-labeled tetrazine that also passes the blood-brain barrier. An in vivo click experiment confirmed the bioorthogonal behavior of this novel tetrazine probe. Due to favorable chemical and pharmacokinetic properties this bioorthogonal agent should find application in bioimaging and biomedical research.

The aim of this study was to develop a positron emission tomography (PET) tracer based on the dual P-glycoprotein (P-gp) breast cancer resistance protein (BCRP) inhibitor tariquidar (1) to study the interaction of 1 with P-gp and BCRP in the blood-brain barrier (BBB) in vivo. Odesmethyl-1 was synthesized and reacted with [ 11 C]methyl triflate to afford [ 11 C]-1. Small-animal PET imaging of [ 11 C]-1 was performed in naïve rats, before and after administration of unlabeled 1 (15 mg/kg, n=3) or the dual P-gp/BCRP inhibitor elacridar (5 mg/kg, n=2), as well as in wildtype, Mdr1a/b (−/−) , Bcrp1 (−/−) and Mdr1a/b (−/−) Bcrp1 (−/−) mice (n=3). In vitro autoradiography was performed with [ 11 C]-1 using brain sections of all 4 mouse types, with and without coincubation with unlabeled 1 or elacridar (1 μM). In PET experiments in rats, administration of unlabeled 1 or elacridar increased brain activity uptake by a factor of 3-4, whereas blood activity levels remained unchanged. In Mdr1a/b (−/−) , Bcrp1 (−/−) and Mdr1a/b (−/−) Bcrp1 (−/−) mice, brain-toblood ratios of activity at 25 min after tracer injection were 3.4, 1.8 and 14.5 times higher, respectively, as compared to wild-type animals. Autoradiography showed approximately 50% less [ 11 C]-1 binding in transporter knockout mice compared to wild-type mice and significant displacement by unlabeled elacridar in wild-type and Mdr1a/b (−/−) mouse brains. Our data suggest that [ 11 C]-1 interacts specifically with P-gp and BCRP in the BBB. However, further investigations are needed to assess if [ 11 C]-1 behaves in vivo as a transported or a non-transported inhibitor.

Elacridar (ELC) and tariquidar (TQD) are generally thought to be nontransported inhibitors of P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP), but recent data indicate that they may also be substrates of these multidrug transporters (MDTs). The present study was designed to investigate potential transport of ELC and TQD by MDTs at the blood-brain barrier at tracer doses as used in positron emission tomography (PET) studies. We performed PET scans with carbon-11-labeled ELC and TQD before and after MDT inhibition in wild-type and transporterknockout mice as well as in in vitro transport assays in MDToverexpressing cells.

The development of highly selective and fast biocompatible reactions for ligation and cleavage has paved the way for new diagnostic and therapeutic applications of pretargeted in vivo chemistry. The concept of bioorthogonal pretargeting has attracted considerable interest, in particular for the targeted delivery of radionuclides and drugs. In nuclear medicine, pretargeting can provide increased target-to-background ratios at early time-points compared to traditional approaches. This reduces the radiation burden to healthy tissue and, depending on the selected radionuclide, enables better imaging contrast or higher therapeutic efficiency. Moreover, bioorthogonally triggered cleavage of pretargeted antibody–drug conjugates represents an emerging strategy to achieve controlled release and locally increased drug concentrations. The toolbox of bioorthogonal reactions has significantly expanded in the past decade, with the tetrazine ligation being the fastest and one of the most versatile in vivo chemistries. Progress in the field, however, relies heavily on the development and evaluation of (radio)labeled compounds, preventing the use of compound libraries for systematic studies. The rational design of tetrazine probes and triggers has thus been impeded by the limited understanding of the impact of structural parameters on the in vivo ligation performance. In this work, we describe the development of a pretargeted blocking assay that allows for the investigation of the in vivo fate of a structurally diverse library of 45 unlabeled tetrazines and their capability to reach and react with pretargeted trans -cyclooctene (TCO)-modified antibodies in tumor-bearing mice. This study enabled us to assess the correlation of click reactivity and lipophilicity of tetrazines with their in vivo performance. In particular, high rate constants (>50 000 M –1 s –1 ) for the reaction with TCO and low calculated log D 7.4 values (below −3) of the tetrazine were identified as strong indicators for successful pretargeting. Radiolabeling gave access to a set of selected 18 F-labeled tetrazines, including highly reactive scaffolds, which were used in pretargeted PET imaging studies to confirm the results from the blocking study. These insights thus enable the rational design of tetrazine probes for in vivo application and will thereby assist the clinical translation of bioorthogonal pretargeting.

The multidrug efflux transporter P-glycoprotein (P-gp) is expressed in high concentrations at the blood-brain barrier (BBB) and is believed to be implicated in resistance to central nervous system drugs. We used small-animal PET and (R)-11 Cverapamil together with tariquidar, a new-generation P-gp modulator, to study the functional activity of P-gp at the BBB of rats. To enable a comparison with human PET data, we performed kinetic modeling to estimate the rate constants of radiotracer transport across the rat BBB. Methods: A group of 7 Wistar Unilever rats underwent paired (R)-11 C-verapamil PET scans at an interval of 3 h: 1 baseline scan and 1 scan after intravenous injection of tariquidar (15 mg/kg, n 5 5) or vehicle (n 5 2). Results: After tariquidar administration, the distribution volume (DV) of (R)-11 C-verapamil was 12-fold higher than baseline (3.68 6 0.81 vs. 0.30 6 0.08; P 5 0.0007, paired t test), whereas the DVs were essentially the same when only vehicle was administered. The increase in DV could be attributed mainly to an increased influx rate constant (K 1 ) of (R)-11 C-verapamil into the brain, which was about 8-fold higher after tariquidar. A doseresponse assessment with tariquidar provided an estimated half-maximum effect dose of 8.4 6 9.5 mg/kg. Conclusion: Our data demonstrate that (R)-11 C-verapamil PET combined with tariquidar administration is a promising approach to measure P-gp function at the BBB.

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With the aim to develop a positron emission tomography (PET) tracer to assess the distribution of P-glycoprotein (P-gp) at the blood-brain barrier (BBB) in vivo, the potent third-generation P-gp inhibitor elacridar (1) was labeled with 11 C by reaction of O-desmethyl 1 with [ 11 C]-methyl triflate. In vitro autoradiography and small-animal PET imaging of [ 11 C]-1 was performed in rats (n=3), before and after administration of unlabeled 1, as well as in wild-type, Mdr1a/b (−/−) and Bcrp1 (−/−) mice (n=3). In PET experiments in rats, administration of unlabeled 1 increased brain activity uptake 5.4-fold, whereas blood activity levels remained unchanged. In Mdr1a/b (−/−) mice, brain activity uptake was 2.5-fold higher compared to wild-type animals, whereas in Bcrp1 (−/−) mice brain activity uptake was only 1.3-fold higher. In vitro autoradiography showed that 63% of [ 11 C]-1 binding was displaceable by an excess of unlabeled 1. As the signal obtained with [ 11 C]-1 appeared to be specific for P-gp at the BBB, its utility for the visualization of cerebral P-gp merits further investigation.