Severin Mairinger

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.

PET and SPECT Radiotracers to Assess Function and Expression of ABC Transporters in Vivo

Adenosine triphosphate-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp, ABCB1), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance-associated proteins (MRPs) are expressed in high concentrations at various physiological barriers (e.g. blood-brain barrier, blood-testis barrier, blood-tumor barrier), where they impede the tissue accumulation of various drugs by active efflux transport. Changes in ABC transporter expression and function are thought to be implicated in various diseases, such as cancer, epilepsy, Alzheimers and Parkinsons disease. The availability of a non-invasive imaging method which allows for measuring ABC transporter function or expression in vivo would be of great clinical use in that it could facilitate the identification of those patients that would benefit from treatment with ABC transporter modulating drugs. To date three different kinds of imaging probes have been described to measure ABC transporters in vivo: i) radiolabelled transporter substrates ii) radiolabelled transporter inhibitors and iii) radiolabelled prodrugs which are enzymatically converted into transporter substrates in the organ of interest (e.g. brain). The design of new imaging probes to visualize efflux transporters is inter alia complicated by the overlapping substrate recognition pattern of different ABC transporter types. The present article will describe currently available ABC transporter radiotracers for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) and critically discuss strengths and limitations of individual probes and their potential clinical applications.

A novel PET protocol for visualization of breast cancer resistance protein function at the blood–brain barrier

Breast cancer resistance protein (BCRP) is the most abundant multidrug efflux transporter at the human blood–brain barrier (BBB), restricting brain distribution of various drugs. In this study, we developed a positron emission tomography (PET) protocol to visualize Bcrp function at the murine BBB, based on the dual P-glycoprotein (P-gp)/Bcrp substrate radiotracer [11C]tariquidar in combination with the Bcrp inhibitor Ko143. To eliminate the contribution of P-gp efflux to [11C]tariquidar brain distribution, we studied mice in which P-gp was genetically knocked out (Mdri1a/b(−/−) mice) or chemically knocked out by pretreatment with cold tariquidar. We found that [11C]tariquidar brain uptake increased dose dependency after administration of escalating doses of Ko143, both in Mdr1a/b(−/−) mice and in tariquidar pretreated wild-type mice. After 15 mg/kg Ko143, the maximum increase in [11C]tariquidar brain uptake relative to baseline scans was 6.3-fold in Mdr1a/bf(−/−) mice with a half-maximum effect dose of 4.98 mg/kg and 3.6-fold in tariquidar (8 mg/kg) pretreated wild-type mice, suggesting that the presented protocol is sensitive to visualize a range of different functional Bcrp activities at the murine BBB. We expect that this protocol can be translated to the clinic, because tariquidar can be safely administered to humans at doses that completely inhibit cerebral P-gp.

Radioligands targeting P-glycoprotein and other drug efflux proteins at the blood-brain barrier.

Brain penetration of radiopharmaceuticals or therapeutic drugs may be restricted by adenosine triphosphate-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp), breast cancer resistance protein (BCRP), or the multidrug resistance-associated proteins. These transporters are expressed in the luminal membrane of brain capillary endothelial cells forming the blood-brain barrier (BBB), where they actively efflux a wide range of chemically unrelated compounds from the brain back into the blood. Most efforts to visualize ABC transporters at the BBB with positron emission tomography have concentrated on Pgp. Pgp imaging probes can be classified as radiolabeled substrates or inhibitors. The radiolabeled substrates (R)-[(11) C]verapamil and [(11) C]-N-desmethyl-loperamide have been successfully used to assess Pgp function at the BBB of animals and humans. Radiolabeled Pgp inhibitors, such as [(11) C]tariquidar, [(11) C]elacridar, or [(11) C]laniquidar, were developed to measure Pgp expression levels at the BBB, which has so far remained unsuccessful as these probes were unexpectedly recognized at tracer concentrations by Pgp and BCRP as substrates resulting in low brain uptake. Studies on positron emission tomography tracers for other ABC transporters than Pgp (BCRP and multidrug resistance-associated proteins) are still in their infancy. It is hoped that the experience gained with the imaging of Pgp will be successfully translated to the development of radiotracers to visualize other ABC transporters.

Design, Synthesis, and Evaluation of a Low-Molecular-Weight 11C-Labeled Tetrazine for Pretargeted PET Imaging Applying Bioorthogonal in Vivo Click Chemistry

A low-molecular-weight tetrazine labeled with the short-lived positron emitter carbon-11 was developed as a bioorthogonal PET probe for pretargeted imaging. A method for efficient and fast synthesis of this imaging agent is presented using radiolabeling of a readily available precursor. High reactivity with trans-cyclooctenes was observed and in vivo investigations including PET/MR scanning showed homogeneous biodistribution, good metabolic stability, and rapid excretion in naive mice. These properties are key to the success of bioorthogonal (11)C-PET imaging, which has been shown in a simple pretargeting experiment using TCO-modified mesoporous silica nanoparticles. Overall, this (11)C-labeled tetrazine represents a highly versatile and advantageous chemical tool for bioorthogonal PET imaging and enables pretargeting approaches using carbon-11 for the first time.

Breast Cancer Resistance Protein and P-Glycoprotein Influence In Vivo Disposition of 11C-Erlotinib

11C-erlotinib is a PET tracer to distinguish responders from nonresponders to epidermal growth factor receptor–targeted tyrosine kinase inhibitors and may also be of interest to predict distribution of erlotinib to tissues targeted for treatment. The aim of this study was to investigate if the known interaction of erlotinib with the multidrug efflux transporters breast cancer resistance protein (humans, ABCG2; rodents, Abcg2) and P-glycoprotein (humans, ABCB1; rodents, Abcb1a/b) affects tissue distribution and excretion of 11C-erlotinib and has an influence on the ability of 11C-erlotinib PET to predict erlotinib tissue distribution at therapeutic doses. Methods: Wild-type and Abcb1a/b or Abcg2 knockout mice underwent 11C-erlotinib PET/MR scans, with or without the coinjection of a pharmacologic dose of erlotinib (10 mg/kg) or after pretreatment with the ABCB1/ABCG2 inhibitor elacridar (10 mg/kg). Integration plot analysis was used to determine organ uptake (CLuptake) and biliary excretion (CLbile) clearances of radioactivity. Results: 11C-erlotinib distribution to the brain was restricted by Abcb1a/b and Abcg2, and CLuptake into the brain was only significantly increased when both Abcb1a/b and Abcg2 were absent (wild-type mice, 0.017 ± 0.004 mL/min/g of tissue; Abcb1a/b(−/−)Abcg2(−/−) mice, 0.079 ± 0.013 mL/min/g of tissue; P < 0.001). The pretreatment of wild-type mice with elacridar increased CLuptake into the brain to levels comparable to Abcb1a/b(−/−)Abcg2(−/−) mice (0.090 ± 0.007 mL/min/g of tissue, P < 0.001). The absence of Abcb1a/b and Abcg2 led to a 2.6-fold decrease in CLbile (wild-type mice, 0.025 ± 0.005 mL/min/g of tissue; Abcb1a/b(−/−)Abcg2(−/−) mice, 0.0095 ± 0.001 mL/min/g of tissue; P < 0.001). There were pronounced differences in distribution of 11C-erlotinib to the brain, liver, kidney, and lung and hepatobiliary excretion into intestine between animals injected with a microdose and pharmacologic dose of erlotinib. Conclusion: ABCG2, ABCB1, and possibly other transporters influence in vivo disposition of 11C-erlotinib and thereby affect its distribution to normal and potentially also tumor tissue. Saturable transport of erlotinib leads to nonlinear pharmacokinetics, possibly compromising the prediction of erlotinib tissue distribution at therapeutic doses from PET with a microdose of 11C-erlotinib. The inhibition of ABCB1 and ABCG2 is a promising approach to enhance brain distribution of erlotinib to increase its efficacy in the treatment of brain tumors.

Radiosynthesis and in vivo evaluation of 1-[18F]fluoroelacridar as a positron emission tomography tracer for P-glycoprotein and breast cancer resistance protein.

Aim of this study was to label the potent dual P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) inhibitor elacridar (1) with (18)F to provide a positron emission tomography (PET) radiotracer to visualize Pgp and BCRP. A series of new 1- and 2-halogen- and nitro-substituted derivatives of 1 (4a-e) was synthesized as precursor molecules and reference compounds for radiolabelling and shown to display comparable in vitro potency to 1 in increasing rhodamine 123 accumulation in a cell line overexpressing human Pgp (MDCKII-MDR1). 1-[(18)F]fluoroelacridar ([(18)F]4b) was synthesized in a decay-corrected radiochemical yield of 1.7±0.9% by a 1-step no-carrier added nucleophilic aromatic (18)F-substitution of 1-nitro precursor 4c. Small-animal PET imaging of [(18)F]4b was performed in naïve rats, before and after administration of unlabelled 1 (5 mg/kg, n=3), as well as in wild-type and Mdr1a/b((-/-))Bcrp1((-/-)) mice (n=3). In PET experiments in rats, administration of unlabelled 1 increased brain activity uptake by a factor of 9.5 (p=0.0002, 2-tailed Students t-test), whereas blood activity levels remained unchanged. In Mdr1a/b((-/-))Bcrp1((-/-)) mice, the mean brain-to-blood ratio of activity at 60 min after tracer injection was 7.6 times higher as compared to wild-type animals (p=0.0002). HPLC analysis of rat brain tissue extracts collected at 40 min after injection of [(18)F]4b revealed that 93±7% of total radioactivity in brain was in the form of unchanged [(18)F]4b. In conclusion, the in vivo behavior of [(18)F]4b was found to be similar to previously described [(11)C]1 suggesting transport of [(18)F]4b by Pgp and/or BCRP at the rodent BBB. However, low radiochemical yields and a significant degree of in vivo defluorination will limit the utility of [(18)F]4b as a PET tracer.

Factors Governing P-Glycoprotein-Mediated Drug–Drug Interactions at the Blood–Brain Barrier Measured with Positron Emission Tomography

The adenosine triphosphate-binding cassette transporter P-glycoprotein (ABCB1/Abcb1a) restricts at the blood–brain barrier (BBB) brain distribution of many drugs. ABCB1 may be involved in drug–drug interactions (DDIs) at the BBB, which may lead to changes in brain distribution and central nervous system side effects of drugs. Positron emission tomography (PET) with the ABCB1 substrates (R)-[11C]verapamil and [11C]-N-desmethyl-loperamide and the ABCB1 inhibitor tariquidar has allowed direct comparison of ABCB1-mediated DDIs at the rodent and human BBB. In this work we evaluated different factors which could influence the magnitude of the interaction between tariquidar and (R)-[11C]verapamil or [11C]-N-desmethyl-loperamide at the BBB and thereby contribute to previously observed species differences between rodents and humans. We performed in vitro transport experiments with [3H]verapamil and [3H]-N-desmethyl-loperamide in ABCB1 and Abcb1a overexpressing cell lines. Moreover we conducted in vivo PET experiments and biodistribution studies with (R)-[11C]verapamil and [11C]-N-desmethyl-loperamide in wild-type mice without and with tariquidar pretreatment and in homozygous Abcb1a/1b(−/−) and heterozygous Abcb1a/1b(+/−) mice. We found no differences for in vitro transport of [3H]verapamil and [3H]-N-desmethyl-loperamide by ABCB1 and Abcb1a and its inhibition by tariquidar. [3H]-N-Desmethyl-loperamide was transported with a 5 to 9 times higher transport ratio than [3H]verapamil in ABCB1- and Abcb1a-transfected cells. In vivo, brain radioactivity concentrations were lower for [11C]-N-desmethyl-loperamide than for (R)-[11C]verapamil. Both radiotracers showed tariquidar dose dependent increases in brain distribution with tariquidar half-maximum inhibitory concentrations (IC50) of 1052 nM (95% confidence interval CI: 930–1189) for (R)-[11C]verapamil and 1329 nM (95% CI: 980–1801) for [11C]-N-desmethyl-loperamide. In homozygous Abcb1a/1b(−/−) mice brain radioactivity distribution was increased by 3.9- and 2.8-fold and in heterozygous Abcb1a/1b(+/−) mice by 1.5- and 1.1-fold, for (R)-[11C]verapamil and [11C]-N-desmethyl-loperamide, respectively, as compared with wild-type mice. For both radiotracers radiolabeled metabolites were detected in plasma and brain. When brain and plasma radioactivity concentrations were corrected for radiolabeled metabolites, brain distribution of (R)-[11C]verapamil and [11C]-N-desmethyl-loperamide was increased in tariquidar (15 mg/kg) treated animals by 14.1- and 18.3-fold, respectively, as compared with vehicle group. Isoflurane anesthesia altered [11C]-N-desmethyl-loperamide but not (R)-[11C]verapamil metabolism, and this had a direct effect on the magnitude of the increase in brain distribution following ABCB1 inhibition. Our data furthermore suggest that in the absence of ABCB1 function brain distribution of [11C]-N-desmethyl-loperamide but not (R)-[11C]verapamil may depend on cerebral blood flow. In conclusion, we have identified a number of important factors, i.e., substrate affinity to ABCB1, brain uptake of radiolabeled metabolites, anesthesia, and cerebral blood flow, which can directly influence the magnitude of ABCB1-mediated DDIs at the BBB and should therefore be taken into consideration when interpreting PET results.

Synthesis and preclinical evaluation of the radiolabeled P-glycoprotein inhibitor [11C]MC113

OBJECTIVES With the aim to develop a PET tracer to visualize P-glycoprotein (Pgp) expression levels in different organs, the Pgp inhibitor MC113 was labeled with (11)C and evaluated using small-animal PET. METHODS [(11)C]MC113 was synthesized by reaction of O-desmethyl MC113 with [(11)C]methyl triflate. Small-animal PET was performed with [(11)C]MC113 in FVB wild-type and Mdr1a/b((-/-)) mice (n=3 per group) and in a mouse model of high (EMT6Ar1.0) and low (EMT6) Pgp expressing tumor grafts (n=5). In the tumor model, PET scans were performed before and after administration of the reference Pgp inhibitor tariquidar (15mg/kg). RESULTS Brain uptake of [(11)C]MC113, expressed as area under the time-activity curve from time 0 to 60min (AUC(0-60)), was moderately but not significantly increased in Mdr1a/b((-/-)) compared with wild-type mice (mean±SD AUC(0-60), Mdr1a/b((-/-)): 88±7min, wild-type: 62±6min, P=0.100, Mann Whitney test). In the tumor model, AUC(0-60) values were not significantly different between EMT6Ar1.0 and EMT6 tumors. Neither in brain nor in tumors was activity concentration significantly changed in response to tariquidar administration. Half-maximum effect concentrations (IC(50)) for inhibition of Pgp-mediated rhodamine 123 efflux from CCRFvcr1000 cells were 375±60nM for MC113 versus 8.5±2.5nM for tariquidar. CONCLUSION [(11)C]MC113 showed higher brain uptake in mice than previously described Pgp PET tracers, suggesting that [(11)C]MC113 was only to a low extent effluxed by Pgp. However, [(11)C]MC113 was found unsuitable to visualize Pgp expression levels presumably due to insufficiently high Pgp binding affinity of MC113 in relation to Pgp densities in brain and tumors.