The hERG cavity can trap very bulky compounds, without perturbing normal gate closure.

Increased activity of efflux transporters, e.g., P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), at the blood-brain barrier is a pathological hallmark of many neurological diseases, and the resulting multiple drug resistance represents a major clinical challenge. Noninvasive imaging of transporter activity can help to clarify the underlying mechanisms of drug resistance and facilitate diagnosis, patient stratification, and treatment monitoring. We have developed a metabolically activated radiotracer for functional imaging of P-gp/BCRP activity with positron emission tomography (PET). In preclinical studies, the tracer showed excellent initial brain uptake and clean conversion to the desired metabolite, although at a sluggish rate. Blocking with P-gp/BCRP modulators led to increased levels of brain radioactivity; however, dynamic PET did not show differential clearance rates between treatment and control groups. Our results provide proof-of-concept for development of prodrug tracers for imaging of P-gp/BCRP function in vivo but also highlight some challenges associated with this strategy.

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)-[(11)C]verapamil and [(11)C]-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)-[(11)C]verapamil or [(11)C]-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 [(3)H]verapamil and [(3)H]-N-desmethyl-loperamide in ABCB1 and Abcb1a overexpressing cell lines. Moreover we conducted in vivo PET experiments and biodistribution studies with (R)-[(11)C]verapamil and [(11)C]-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 [(3)H]verapamil and [(3)H]-N-desmethyl-loperamide by ABCB1 and Abcb1a and its inhibition by tariquidar. [(3)H]-N-Desmethyl-loperamide was transported with a 5 to 9 times higher transport ratio than [(3)H]verapamil in ABCB1- and Abcb1a-transfected cells. In vivo, brain radioactivity concentrations were lower for [(11)C]-N-desmethyl-loperamide than for (R)-[(11)C]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)-[(11)C]verapamil and 1329 nM (95% CI: 980-1801) for [(11)C]-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)-[(11)C]verapamil and [(11)C]-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)-[(11)C]verapamil and [(11)C]-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 [(11)C]-N-desmethyl-loperamide but not (R)-[(11)C]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 [(11)C]-N-desmethyl-loperamide but not (R)-[(11)C]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.

Resveratrol shows beneficial effects in inflammation-based diseases like cancer, cardiovascular and chronic inflammatory diseases. Therefore, the molecular mechanisms of the anti-inflammatory resveratrol effects deserve more attention. In human epithelial DLD-1 and monocytic Mono Mac 6 cells resveratrol decreased the expression of iNOS, IL-8 and TNF-α by reducing mRNA stability without inhibition of the promoter activity. Shown by pharmacological and siRNA-mediated inhibition, the observed effects are SIRT1-independent. Target-fishing and drug responsive target stability experiments showed selective binding of resveratrol to the RNA-binding protein KSRP, a central post-transcriptional regulator of pro-inflammatory gene expression. Knockdown of KSRP expression prevented resveratrol-induced mRNA destabilization in human and murine cells. Resveratrol did not change KSRP expression, but immunoprecipitation experiments indicated that resveratrol reduces the p38 MAPK-related inhibitory KSRP threonine phosphorylation, without blocking p38 MAPK activation or activity. Mutation of the p38 MAPK target site in KSRP blocked the resveratrol effect on pro-inflammatory gene expression. In addition, resveratrol incubation enhanced KSRP-exosome interaction, which is important for mRNA degradation. Finally, resveratrol incubation enhanced its intra-cellular binding to the IL-8, iNOS and TNF-α mRNA. Therefore, modulation of KSRP mRNA binding activity and, thereby, enhancement of mRNA degradation seems to be the common denominator of many anti-inflammatory effects of resveratrol.

Human ether-a-go-go related gene (hERG) channel inhibitors can be trapped in the channels at rest. The structural peculiarities of hERG blockers that enable trapping or alternatively resting state dissociation are currently unknown. Propafenone (small molecule, MW 341 g/mol) is efficiently trapped in the closed hERG channel pore (1). To investigate whether the size of the blocking molecule plays a role in trapping we synthesized bulky propafenone derivatives containing benzoyl and trimethylphenyl side chains, attached by piperazine linkers, with molecular weights of 500 (Fba212) and 650 g/mol (Fba213) respectively. hERG channels were expressed in Xenopus laevis oocyte and potassium current inhibition was studied using the two - microelectrode voltage clamp technique. It was found: first, both compounds are potent hERG blockers with IC50 3.7μM (Fba212) and 52μM (Fba213). Secondly, channel block by Fba212 and 213 was prevented by mutations Y652A and F656A as previously shown for propafenone. Third, both propafenone derivatives were trapped at rest. To obtain insights into the molecular mechanism of channel block docking experiments with Fba212 and Fba213 in closed and open conformation were performed. Both compounds interact with the propafenone binding site (Y652A and F656A). Fba213 was found to exceed the size of the closed channel cavity of our hERG homology model. We conclude that drug trapping in hERG channels does not necessarily require full closure of the activation gate. 1. Witchel HJ, Dempsey CE, Sessions RB, Perry M, Milnes JT, Hancox JC, Mitcheson JS. Mol Pharmacol. 2004 Nov; 66(5):1201-12.

HM30181, a potent and selective inhibitor of the adenosine triphosphate-binding cassette transporter P-glycoprotein (Pgp), was shown to enhance oral bioavailability and improve antitumour efficacy of paclitaxel in mouse tumour models. In search for a positron emission tomography (PET) radiotracer to visualise Pgp expression levels at the blood–brain barrier (BBB), we examined the ability of HM30181 to inhibit Pgp at the murine BBB. HM30181 was shown to be approximately equipotent with the reference Pgp inhibitor tariquidar in inhibiting rhodamine 123 efflux from CCRF-CEM T cells (IC50, tariquidar: 8.2±2.0nM, HM30181: 13.1±2.3nM). PET scans with the Pgp substrate (R)-[11C]verapamil in FVB wild-type mice pretreated i.v. with HM30181 (10 or 21mg/kg) failed to show significant increases in (R)-[11C]verapamil brain uptake compared with vehicle treated animals. PET scans with [11C]HM30181 showed low and not significantly different brain uptake of [11C]HM30181 in wild-type, Mdr1a/b(−/−) and Bcrp1(−/−) mice and significantly, i.e. 4.7-fold (P<0.01), higher brain uptake, relative to wild-type animals, in Mdr1a/b(−/−) Bcrp1(−/−) mice. This was consistent with HM30181 being at microdoses a dual substrate of Pgp and breast cancer resistance protein (Bcrp). In vitro autoradiography on low (EMT6) and high (EMT6Ar1.0) Pgp expressing murine breast tumour sections showed 1.9 times higher binding of [11C]HM30181 in EMT6Ar1.0 tumours (P<0.001) which was displaceable with unlabelled tariquidar, elacridar or HM30181 (1μM). Our data suggest that HM30181 is not able to inhibit Pgp at the murine BBB at clinically feasible doses and that [11C]HM30181 is not suitable as a PET tracer to visualise cerebral Pgp expression levels.

Aim of this study was to label the potent dual P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) inhibitor elacridar (1) with 18F 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-[18F]fluoroelacridar ([18F]4b) was synthesized in a decay-corrected radiochemical yield of 1.7±0.9% by a 1-step no-carrier added nucleophilic aromatic 18F-substitution of 1-nitro precursor 4c. Small-animal PET imaging of [18F]4b was performed in naïve rats, before and after administration of unlabelled 1 (5mg/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 Student’s t-test), whereas blood activity levels remained unchanged. In Mdr1a/b(−/−)Bcrp1(−/−) mice, the mean brain-to-blood ratio of activity at 60min 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 40min after injection of [18F]4b revealed that 93±7% of total radioactivity in brain was in the form of unchanged [18F]4b. In conclusion, the in vivo behavior of [18F]4b was found to be similar to previously described [11C]1 suggesting transport of [18F]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 [18F]4b as a PET tracer.

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.

[11C]Loperamide and [11C]N-desmethyl-loperamide ([11C]dLop) have been proposed as radiotracers for imaging brain P-glycoprotein (P-gp) function. A major route of [11C]loperamide metabolism is N-demethylation to [11C]dLop. We aimed to test whether inhibition of CYP3A4 with ketoconazole might reduce the metabolism of [11C]loperamide and [11C]dLop in mice, and thereby improve the quality of these radiotracers.Studies were performed in wild-type and P-gp knockout (mdr−1a/b −/−) mice. During each of seven study sessions, one pair of mice, comprising one wild-type and one knockout mouse, was pretreated with ketoconazole (50 mg/kg, ip), while another such pair was left untreated. Mice were sacrificed at 30 min after injection of [11C]loperamide or [11C]dLop. Whole brain and plasma samples were measured for radioactivity and analyzed with radio-high-performance liquid chromatography.Ketoconazole increased the plasma concentrations of [11C]loperamide and its main radiometabolite, [11C]dLop, by about twofold in both wild-type and knockout mice, whereas the most polar radiometabolite was decreased threefold. Furthermore, ketoconazole increased the brain concentrations of [11C]loperamide and the radiometabolite [11C]dLop by about twofold in knockout mice, and decreased the brain concentrations of the major and most polar radiometabolite in wild-type and knockout mice by 82% and 49%, respectively. In contrast, ketoconazole had no effect on plasma and brain distribution of administered [11C]dLop and its radiometabolites in either wild-type or knockout mice, except to increase the low plasma [11C]dLop concentration. The least polar radiometabolite of [11C]dLop was identified with LC-MSn as the N-hydroxymethyl analog of [11C]dLop and this also behaved as a P-gp substrate.In this study, ketoconazole (50 mg/kg, ip) proved partially effective for inhibiting the N-demethylation of [11C]loperamide in mouse in vivo but had relatively smaller or no effect on [11C]dLop.

Introduction: The multidrug efflux transporter breast cancer resistance protein (BCRP) is highly expressed in the blood-brain barrier (BBB), where it limits brain entry of a broad range of endogenous and exogenous substrates. Methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl)phenyl)amino-carbonyl)-2-(quinoline-2-carbonylamino)benzoate (1) is a recently discovered BCRP-selective inhibitor, which is structurally derived from the potent P-glycoprotein (P-gp) inhibitor tariquidar. The aim of this study was to develop a new PET tracer based on 1 to map BCRP expression levels in vivo.Methods: Compound 1 was labelled with C-11 in its methyl ester function by reaction of the corresponding carboxylic acid 2 with [C-11] methyl triflate. Positron emission tomography (PET) imaging of [C-11]-1 was performed in wild-type, Mdr1a/b((-/-)), Bcrp1((-/-)) and Mdr1a/b((-/-)) Bcrp1((-/-)) mice (n=3 per mouse type) and radiotracer metabolism was assessed in plasma and brain.Results: Brain-to-plasma ratios of unchanged [C-11]-1 were 4.8- and 10.3-fold higher in Mdr1a/b((-/-)) and in Mdr1a/b((-/-))Bcrpl((-/-)) mice, respectively, as compared to wild-type animals, but only modestly increased in Bcrpl((-/-)) mice. [C-11]-1 was rapidly metabolized in vivo giving rise to a polar radiometabolite which was taken up into brain tissue.Conclusion: Our data suggest that [C-11]-1 preferably interacts with P-gp rather than BCRP at the murine BBB which questions its reported in vitro BCRP selectivity. Consequently, [C-11]-1 appears to be unsuitable as a PET tracer to map cerebral BCRP expression. (C) 2010 Elsevier Inc. All rights reserved.