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

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Abstract

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 [11C]methyl triflate to afford [11C]-1. Small-animal PET imaging of [11C]-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 wild-type, Mdr1a/b(−/−), Bcrp1(−/−) and Mdr1a/b(−/−)Bcrp1(−/−) mice (n = 3). In vitro autoradiography was performed with [11C]-1 using brain sections of all four mouse types, with and without co-incubation 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-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 [11C]-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 [11C]-1 interacts specifically with P-gp and BCRP in the BBB. However, further investigations are needed to assess if [11C]-1 behaves in vivo as a transported or a non-transported inhibitor.

Introduction

The ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp, ABCB1) was initially discovered in the 1970s as an active efflux transporter involved in the multidrug resistance of cancer cells, and was the first drug efflux transporter to be detected in endothelial cells at the human blood–brain barrier (BBB).1, 2 It has been suggested that P-gp serves as a general defense mechanism in the mammalian BBB, protecting the brain from intoxication by potentially harmful lipophilic compounds from natural sources and other lipophilic xenobiotics that otherwise could penetrate the BBB by simple diffusion without any limitation.3 Regional overactivity of P-gp and related transporters in the BBB contributes to the phenomenon of drug resistance in neurological disorders, such as epilepsy and depression, by impeding therapeutically effective concentrations of central nervous system (CNS) drugs at their sites of action.3

A promising strategy to enhance drug penetration across the BBB and thereby overcome drug resistance is the administration of third-generation P-gp inhibitors, which were originally developed as an adjunctive therapy for drug-resistant cancers and are characterized by a high potency in the nanomolar range, lack of toxicity and lack of cytochrome P450 interactions.4 The anthranilic acid derivative tariquidar (1, Scheme 1) is one of the most potent and selective third-generation P-gp inhibitors known to date.5 Unlike first- and second-generation P-gp inhibitors, such as verapamil, cyclosporine A or valspodar (PSC 833), 1 is a non-competitive inhibitor and not a substrate of P-gp.6 Compound 1 also inhibits breast cancer resistance protein (BCRP, ABCG2), another ABC-type transporter expressed in the BBB, but at higher concentrations than those at which it inhibits P-gp.7 However, unlike first- and second-generation P-gp inhibitors, 1 does not inhibit multidrug resistance-associated proteins (MRPs), such as MRP1.8 Compound 1 was initially tested in clinical trials in cancer patients in combination with anticancer drugs that are P-gp substrates (e.g., paclitaxel, docetaxel, doxorubicin, vinorelbine), which demonstrated treatment response in some patients.5 However, some of these trials had to be discontinued because of increased toxicity of treatment regimens, which has been attributed to enhanced systemic exposure to anticancer drugs due to peripheral P-gp inhibition.4, 5

In contrast to its well-studied role in blocking P-gp in chemoresistant cancer cells, considerably less is known about the capacity of 1 to inhibit P-gp in the BBB and thereby facilitate brain entry of CNS drugs. A number of animal studies have shown that brain levels of antiepileptics, cytostatics and other CNS drugs were increased following administration of 1.9, 10, 11, 12 Only few human studies assessing the effect of 1 on P-gp in the BBB have been conducted to date. Kurnik et al. studied brain penetration of the P-gp substrate loperamide by measuring reduction in pupil diameter as a surrogate marker for central opioid effect, with and without co-administration of 1 at a dose of 2 mg/kg body weight.13 This study failed to demonstrate an effect of 1 on loperamide’s central effects, whereas inhibition of P-gp in lymphocytes was >90%, suggesting that P-gp in the BBB is more resistant to inhibition as compared to peripheral P-gp.10 In a study recently conducted at our center, the effect of 1 (2 mg/kg) on brain penetration of the P-gp substrate (R)-[11C]verapamil was measured by PET imaging in healthy subjects.14 Our study showed a modest but significant (+24 ± 15%) increase in (R)-[11C]verapamil brain distribution volume, which suggested that higher doses of 1 might be needed in the clinic in order to improve the clinical efficacy of CNS drugs in therapy-refractory patients. On the other hand, in rats comparable plasma levels of 1 to those achieved in humans in our study caused an increase of approximately +300% in (R)-[11C]verapamil brain distribution volume, which points to pronounced species difference in tariquidar-induced P-gp inhibition.15 Another human study assessed the effect of different doses of 1 on brain penetration of the P-gp substrate radiotracer [11C]-N-desmethyl-loperamide and found a fourfold increase in brain radioactivity uptake after 6 mg/kg of 1.16

An alternative approach to using radiolabeled P-gp substrates, such as (R)-[11C]verapamil,12, 14 [11C]-N-desmethyl-loperamide,17, 18 or 6,7-dimethoxy-2-[3-(5-[11C]methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)-propyl]-1,2,3,4-tetrahydro-isoquinoline ([11C]MC-266)19 for studying the interaction of 1 with P-gp at the BBB would be to perform PET experiments directly with radiolabeled 1. Such a radiotracer might, for example, help to better understand species differences in tariquidar-induced P-gp inhibition as well as the differential sensitivity of cerebral and peripheral P-gp to inhibition. As opposed to P-gp substrate radiotracers, radiolabeled 1 is expected to bind to P-gp at the BBB without being transported by it and thereby allow mapping of the distribution of cerebral P-gp.

The aim of this study was to develop carbon-11-labeled 1 ([11C]-1) as a PET tracer to directly study the interaction of 1 with P-gp and BCRP in vivo. Here, we report on the precursor synthesis, 11C-labeling and small-animal PET evaluation of [11C]-1.

Section snippets

Chemistry and radiolabeling

The radiolabeling precursor of [11C]-1, O-desmethyl-tariquidar (7), was synthesized as outlined in Scheme 1. Commercially available 4,5-dimethoxy-2-nitrobenzoic acid was first mono-demethylated using aq KOH to yield the corresponding 5-hydroxy derivative 2 (see Supplementary data).20 Derivative 2 was then pivaloyl-protected to give compound 3 (see Supplementary data), which was transformed into its acid chloride and coupled with 6,7-dimethoxy-2-(4-aminophenethyl)-1,2,3,4-tetrahydroisochinoline

Discussion

The aim of this study was to label 1 with a positron-emitting radionuclide and to assess the interaction of radiolabeled 1 with P-gp and BCRP in the BBB. As 1 contains four OCH3 groups, the most straightforward radiolabeling approach was O-11C-methylation of O-desmethyl-1, which offers the advantage that the radiotracer retains the chemical structure of native 1 (Scheme 1). During the preparation of this manuscript a study by Kawamura et al. has appeared in the literature, which also reported

General

All chemicals were purchased from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany), Merck (Darmstadt, Germany) or Apollo Scientific Ltd (Bredbury, UK) at analytical grade and used without further purification. The dimesylate of 1 was obtained from Xenova Ltd (Slough, UK). The dual P-gp/BCRP inhibitor elacridar hydrochloride was obtained from Glaxo SmithKline (Research Triangle Park, NC, USA). For administration, 1 dimesylate was freshly dissolved on each experimental day in 2.5% (w/v) aqueous

Conclusions

[11C]-1 was synthesized and a first in vivo evaluation performed using small-animal PET imaging. Our data suggest that [11C]-1 interacts specifically with P-gp and BCRP in the BBB. However, further experiments are needed to clarify if [11C]-1 behaves in vivo as a transported or as a non-transported P-gp/BCRP inhibitor.

Acknowledgments

The research leading to these results has received funding from the Austrian Science Fund (FWF) project ‘Transmembrane Transporters in Health and Disease’ (SFB F35) and from the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement number 201380 (‘Euripides’). The authors thank Gloria Stundner (AIT), Thomas Filip and Maria Zsebedics (Seibersdorf Laboratories GmbH) for their skillful help with laboratory animal handling. Elacridar hydrochloride was kindly

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    Florian Bauer and Claudia Kuntner contributed equally to this study.

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