Claudia Kuntner

Pgp-Mediated Interaction Between (R)-[11C]Verapamil and Tariquidar at the Human Blood–Brain Barrier: A Comparison With Rat Data

Using positron emission tomography (PET) imaging we assessed, in vivo, the interaction between a microdose of (R)‐[11C]verapamil (a P‐glycoprotein (Pgp) substrate) and escalating doses of the Pgp inhibitor tariquidar (3, 4, 6, and 8 mg/kg) at the blood–brain barrier (BBB) in healthy human subjects. We compared the dose–response relationship of tariquidar in humans with data obtained in rats using a similar methodology. Tariquidar was equipotent in humans and rats in its effect of increasing (R)‐[11C]verapamil brain uptake (expressed as whole‐brain volume of distribution (VT)), with very similar half‐maximum‐effect concentrations. Both in humans and in rats, brain VT approached plateau levels at plasma tariquidar concentrations >1,000 ng/ml. However, Pgp inhibition in humans led to only a 2.7‐fold increase in brain VT relative to baseline scans (before administration of tariquidar) as compared with 11.0‐fold in rats. The results of this translational study add to the accumulating evidence that there are marked species‐dependent differences in Pgp expression and functionality at the BBB.

Tariquidar-Induced P-Glycoprotein Inhibition at the Rat Blood–Brain Barrier Studied with (R)-11C-Verapamil and PET

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)-11C-verapamil 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)-11C-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) or vehicle (n = 2). Results: After tariquidar administration, the distribution volume (DV) of (R)-11C-verapamil was 12-fold higher than baseline (3.68 ± 0.81 vs. 0.30 ± 0.08; P = 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 (K1) of (R)-11C-verapamil into the brain, which was about 8-fold higher after tariquidar. A dose–response assessment with tariquidar provided an estimated half-maximum effect dose of 8.4 ± 9.5 mg/kg. Conclusion: Our data demonstrate that (R)-11C-verapamil PET combined with tariquidar administration is a promising approach to measure P-gp function at the BBB.

Synthesis and small-animal positron emission tomography evaluation of [11C]-elacridar as a radiotracer to assess the distribution of P-glycoprotein at the blood-brain barrier

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.

Synthesis and in vivo evaluation of [11C]tariquidar, a positron emission tomography radiotracer based on a third-generation P-glycoprotein inhibitor

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. O-Desmethyl-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 (5mg/kg, n=2), as well as in wild-type, Mdr1a/b((-/-)), Bcrp1((-/-)) and Mdr1a/b((-/-))Bcrp1((-/-)) mice (n=3). In vitro autoradiography was performed with [(11)C]-1 using brain sections of all four mouse types, with and without co-incubation with unlabeled 1 or elacridar (1 microM). 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-to-blood 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.

A Novel Positron Emission Tomography Imaging Protocol Identifies Seizure-Induced Regional Overactivity of P-Glycoprotein at the Blood–Brain Barrier

Approximately one-third of epilepsy patients are pharmacoresistant. Overexpression of P-glycoprotein and other multidrug transporters at the blood–brain barrier is thought to play an important role in drug-refractory epilepsy. Thus, quantification of regionally different P-glycoprotein activity in the brain in vivo is essential to identify P-glycoprotein overactivity as the relevant mechanism for drug resistance in an individual patient. Using the radiolabeled P-glycoprotein substrate (R)-[11C]verapamil and different doses of coadministered tariquidar, which is an inhibitor of P-glycoprotein, we evaluated whether small-animal positron emission tomography can quantify regional changes in transporter function in the rat brain at baseline and 48 h after a pilocarpine-induced status epilepticus. P-glycoprotein expression was additionally quantified by immunohistochemistry. To reveal putative seizure-induced changes in blood–brain barrier integrity, we performed gadolinium-enhanced magnetic resonance scans on a 7.0 tesla small-animal scanner. Before P-glycoprotein modulation, brain uptake of (R)-[11C]verapamil was low in all regions investigated in control and post-status epilepticus rats. After administration of 3 mg/kg tariquidar, which inhibits P-glycoprotein only partially, we observed increased regional differentiation in brain activity uptake in post-status epilepticus versus control rats, which diminished after maximal P-glycoprotein inhibition. Regional increases in the efflux rate constant k2, but not in distribution volume VT or influx rate constant K1, correlated significantly with increases in P-glycoprotein expression measured by immunohistochemistry. This imaging protocol proves to be suitable to detect seizure-induced regional changes in P-glycoprotein activity and is readily applicable to humans, with the aim to detect relevant mechanisms of pharmacoresistance in epilepsy in vivo.

Tariquidar and Elacridar Are Dose-Dependently Transported by P-Glycoprotein and Bcrp at the Blood-Brain Barrier: A Small-Animal Positron Emission Tomography and In Vitro Study

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 transporter-knockout mice as well as in in vitro transport assays in MDT-overexpressing cells. Brain entrance of [11C]ELC and [11C]TQD administered in nanomolar tracer doses was found to be limited by Pgp- and Bcrp1-mediated efflux at the mouse blood-brain barrier. At higher, MDT-inhibitory doses, i.e., 15 mg/kg for TQD and 5 mg/kg for ELC, brain activity uptake of [11C]ELC at 25 minutes after tracer injection was 5.8 ± 0.3, 2.1 ± 0.2, and 7.5 ± 1.0-fold higher in wild-type, Mdr1a/b(−/−,) and Bcrp1(−/−) mice, respectively, but remained unchanged in Mdr1a/b(−/−)Bcrp1(−/−) mice. Activity uptake of [11C]TQD was 2.8 ± 0.2 and 6.8 ± 0.4-fold higher in wild-type and Bcrp1(−/−) mice, but remained unchanged in Mdr1a/b(−/−) and Mdr1a/b(−/−)Bcrp1(−/−) mice. Consistent with the in vivo findings, in vitro uptake assays in Pgp- and Bcrp1-overexpressing cell lines confirmed low intracellular accumulation of ELC and TQD at nanomolar concentrations and increased uptake at micromolar concentrations. As this study shows that microdoses can behave pharmacokinetically differently from MDT-inhibitory doses if a compound interacts with MDTs, conclusions from microdose studies should be drawn carefully.

A new fast and fully automated software based algorithm for extracting respiratory signal from raw PET data and its comparison to other methods

PURPOSE Respiratory gating in PET is an approach used to minimize the negative effects of respiratory motion on spatial resolution. It is based on an initial determination of a patients respiratory movements during a scan, typically using hardware based systems. In recent years, several fully automated databased algorithms have been presented for extracting a respiratory signal directly from PET data, providing a very practical strategy for implementing gating in the clinic. In this work, a new method is presented for extracting a respiratory signal from raw PET sinogram data and compared to previously presented automated techniques. METHODS The acquisition of respiratory signal from PET data in the newly proposed method is based on rebinning the sinogram data into smaller data structures and then analyzing the time activity behavior in the elements of these structures. From this analysis, a 1D respiratory trace is produced, analogous to a hardware derived respiratory trace. To assess the accuracy of this fully automated method, respiratory signal was extracted from a collection of 22 clinical FDG-PET scans using this method, and compared to signal derived from several other software based methods as well as a signal derived from a hardware system. RESULTS The method presented required approximately 9 min of processing time for each 10 min scan (using a single 2.67 GHz processor), which in theory can be accomplished while the scan is being acquired and therefore allowing a real-time respiratory signal acquisition. Using the mean correlation between the software based and hardware based respiratory traces, the optimal parameters were determined for the presented algorithm. The mean/median/range of correlations for the set of scans when using the optimal parameters was found to be 0.58/0.68/0.07-0.86. The speed of this method was within the range of real-time while the accuracy surpassed the most accurate of the previously presented algorithms. CONCLUSIONS PET data inherently contains information about patient motion; information that is not currently being utilized. We have shown that a respiratory signal can be extracted from raw PET data in potentially real-time and in a fully automated manner. This signal correlates well with hardware based signal for a large percentage of scans, and avoids the efforts and complications associated with hardware. The proposed method to extract a respiratory signal can be implemented on existing scanners and, if properly integrated, can be applied without changes to routine clinical procedures.

Development of a 18F‐Labeled Tetrazine with Favorable Pharmacokinetics for Bioorthogonal PET Imaging

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.

(R)-[11C]verapamil is selectively transported by murine and human P-glycoprotein at the blood–brain barrier, and not by MRP1 and BCRP

Introduction Positron emission tomography (PET) with [11C]verapamil, either in racemic form or in form of the (R)-enantiomer, has been used to measure the functional activity of the adenosine triphosphate-binding cassette (ABC) transporter P-glycoprotein (Pgp) at the blood–brain barrier (BBB). There is some evidence in literature that verapamil inhibits two other ABC transporters expressed at the BBB, i.e. multidrug resistance protein 1 (MRP1) and breast cancer resistance protein (BCRP). However, previous data were obtained with micromolar concentrations of verapamil and do not necessarily reflect the transporter selectivity of verapamil at nanomolar concentrations, which are relevant for PET experiments. The aim of this study was to assess the selectivity of verapamil, in nanomolar concentrations, for Pgp over MRP1 and BCRP. Methods Concentration equilibrium transport assays were performed with [3H]verapamil (5 nM) in cell lines expressing murine or human Pgp, human MRP1, and murine Bcrp1 or human BCRP. Paired PET scans were performed with (R)-[11C]verapamil in female FVB/N (wild-type), Mrp1(−/−), Mdr1a/b(−/−), Bcrp1(−/−) and Mdr1a/b(−/−)Bcrp1(−/−) mice, before and after Pgp inhibition with 15 mg/kg tariquidar. Results In vitro transport experiments exclusively showed directed transport of [3H]verapamil in Mdr1a- and MDR1-overexpressing cells which could be inhibited by tariquidar (0.5 μM). In PET scans acquired before tariquidar administration, brain-to-blood ratio (Kb,brain) of (R)-[11C]verapamil was low in wild-type (1.3 ± 0.1), Mrp1(−/−) (1.4 ± 0.1) and Bcrp1(−/−) mice (1.8 ± 0.1) and high in Mdr1a/b(−/−) (6.9 ± 0.8) and Mdr1a/b(−/−)Bcrp1(−/−) mice (7.9 ± 0.5). In PET scans after tariquidar administration, Kb,brain was significantly increased in Pgp-expressing mice (wild-type: 5.0 ± 0.3-fold, Mrp1(−/−): 3.2 ± 0.6-fold, Bcrp1(−/−): 4.3 ± 0.1-fold) but not in Pgp knockout mice (Mdr1a/b(−/−) and Mdr1a/b(−/−)Bcrp1(−/−)). Conclusion Our combined in vitro and in vivo data demonstrate that verapamil, in nanomolar concentrations, is selectively transported by Pgp and not by MRP1 and BCRP at the BBB, which supports the use of (R)-[11C]verapamil or racemic [11C]verapamil as PET tracers of cerebral Pgp function.

FDG uptake is a surrogate marker for defining the optimal biological dose of the mTOR inhibitor everolimus in vivo

This study aimed to test whether [18F]fluoro-D-glucose (FDG) uptake of tumours measured by positron emission tomography (PET) can be used as surrogate marker to define the optimal biological dose (OBD) of mTOR inhibitors in vivo. Everolimus at 0.05, 0.5, 5 and 15 mg kg−1 per day was administered to gastric cancer xenograft-bearing mice for 23 days and FDG uptake of tumours was measured using PET from day 1 to day 8. To provide standard comparators for FDG uptake, tumour volume, S6 protein phosphorylation, Ki-67 staining and everolimus blood levels were evaluated. Everolimus blood levels increased in a dose-dependent manner but antitumour activity of everolimus reached a plateau at doses ⩾5 mg kg−1 per day (tumour volume treated vs control (T/C): 51% for 5 mg kg−1 per day and 57% for 15 mg kg−1 per day). Correspondingly, doses ⩾5 mg kg−1 per day led to a significant reduction in FDG uptake of tumours. Dose escalation above 5 mg kg−1 per day did not reduce FDG uptake any further (FDG uptake T/C: 49% for 5 mg kg−1 per day and 52% for 15 mg kg−1 per day). Differences in S6 protein phosphorylation and Ki-67 index reflected tumour volume and changes in FDG uptake but did not reach statistical significance. In conclusion, FDG uptake might serve as a surrogate marker for dose finding studies for mTOR inhibitors in (pre)clinical trials.