Methanol (CH3OH) fluxes were quantified above a managed temperate mountain grassland in the Stubai Valley (Tyrol, Austria) during the growing seasons 2008 and 2009. Half-hourly methanol fluxes were calculated by means of the virtual disjunct eddy covariance (vDEC) method using 3-dimensional wind data from a sonic anemometer and methanol volume mixing ratios measured with a proton-transfer-reaction mass spectrometer (PTR-MS). During (undisturbed) mature and growing phases methanol fluxes exhibited a clear diurnal cycle with close-to-zero fluxes during nighttime and emissions, up to 10 nmol m−2 s−1, which followed the diurnal course of radiation and air temperature. Management events were found to represent the largest perturbations of methanol exchange at the studied grassland ecosystem: Peak emissions of 144.5 nmol m−2 s−1 were found during/after cutting of the meadow reflecting the wounding of the plant material and subsequent depletion of the leaf internal aqueous methanol pools. After the application of organic fertilizer, elevated methanol emissions of up to 26.7 nmol m−2 s−1 were observed, likely reflecting enhanced microbial activity associated with the applied manure. Simple and multiple linear regression analyses revealed air temperature and radiation as the dominant abiotic controls, jointly explaining 47 % and 70 % of the variability in half-hourly and daily methanol fluxes. In contrast to published leaf-level laboratory studies, the surface conductance and the daily change in the amount of green plant area, used as ecosystem-scale proxies for stomatal conductance and growth, respectively, were found to exert only minor biotic controls on methanol exchange.
disjunct eddy covariance; flux; methanol; volatile organic compounds; management; grassland; PTR-MS
Increased lipid availability reduces insulin-stimulated glucose disposal in skeletal muscle, which is generally explained by fatty acid–mediated inhibition of insulin signaling. It remains unclear whether lipids also impair transcapillary transport of insulin and glucose, which could become rate controlling for glucose disposal. We hypothesized that lipid-induced insulin resistance is induced by inhibiting myocellular glucose uptake and not by interfering with the delivery of insulin or glucose. We measured changes in interstitial glucose and insulin in skeletal muscle of healthy volunteers during intravenous administration of triglycerides plus heparin or glycerol during physiologic and supraphysiologic hyperinsulinemia, by combining microdialysis with oral glucose tolerance tests and euglycemic-hyperinsulinemic clamps. Lipid infusion reduced insulin-stimulated glucose disposal by ∼70% (P < 0.05) during clamps and dynamic insulin sensitivity by ∼12% (P < 0.05) during oral glucose loading. Dialysate insulin and glucose levels were unchanged or even transiently higher (P < 0.05) during lipid than during glycerol infusion, whereas regional blood flow remained unchanged. These results demonstrate that short-term elevation of free fatty acids (FFAs) induces insulin resistance, which in skeletal muscle occurs primarily at the cellular level, without impairment of local perfusion or transcapillary transport of insulin and glucose. Thus, vascular effects of FFAs are not rate controlling for muscle insulin-stimulated glucose disposal.
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 (Mdr1a/b(−/−) mice) or chemically knocked out by pretreatment with cold tariquidar. We found that [11C]tariquidar brain uptake increased dose dependently 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/b(−/−) 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.
blood–brain barrier; breast cancer resistance protein; Ko143; P-glycoprotein; positron emission tomography; [11C]tariquidar
We investigate the behaviour of the recently proposed Quantum PageRank algorithm, in large complex networks. We find that the algorithm is able to univocally reveal the underlying topology of the network and to identify and order the most relevant nodes. Furthermore, it is capable to clearly highlight the structure of secondary hubs and to resolve the degeneracy in importance of the low lying part of the list of rankings. The quantum algorithm displays an increased stability with respect to a variation of the damping parameter, present in the Google algorithm, and a more clearly pronounced power-law behaviour in the distribution of importance, as compared to the classical algorithm. We test the performance and confirm the listed features by applying it to real world examples from the WWW. Finally, we raise and partially address whether the increased sensitivity of the quantum algorithm persists under coordinated attacks in scale-free and random networks.
The plasma and tumor dispositions of a commonly used antimelanoma agent, carboplatin, were evaluated in patients with cutaneous melanoma and compared with four different murine melanoma models (one genetically engineered mouse model, one human cell line xenograft, and two orthotopic syngeneic transplant models). The tumor pharmacokinetics of carboplatin in the a genetically engineered mouse model most closely resembled the tumor disposition in patients with melanoma.
Rodent studies are a vital step in the development of novel anticancer therapeutics and are used in pharmacokinetic (PK), toxicology, and efficacy studies. Traditionally, anticancer drug development has relied on xenograft implantation of human cancer cell lines in immunocompromised mice for efficacy screening of a candidate compound. The usefulness of xenograft models for efficacy testing, however, has been questioned, whereas genetically engineered mouse models (GEMMs) and orthotopic syngeneic transplants (OSTs) may offer some advantages for efficacy assessment. A critical factor influencing the predictability of rodent tumor models is drug PKs, but a comprehensive comparison of plasma and tumor PK parameters among xenograft models, OSTs, GEMMs, and human patients has not been performed.
In this work, we evaluated the plasma and tumor dispositions of an antimelanoma agent, carboplatin, in patients with cutaneous melanoma compared with four different murine melanoma models (one GEMM, one human cell line xenograft, and two OSTs).
Using microdialysis to sample carboplatin tumor disposition, we found that OSTs and xenografts were poor predictors of drug exposure in human tumors, whereas the GEMM model exhibited PK parameters similar to those seen in human tumors.
The tumor PKs of carboplatin in a GEMM of melanoma more closely resembles the tumor disposition in patients with melanoma than transplanted tumor models. GEMMs show promise in becoming an improved prediction model for intratumoral PKs and response in patients with solid tumors.
GEMM; Microdialysis; Carboplatin pharmacokinetics; Mouse tumor models; Melanoma models; Genetically engineered mouse models
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.
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.
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(−/−)).
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.
Positron emission tomography; (R)-[11C]verapamil; Blood–brain barrier; P-glycoprotein; Multidrug resistance protein 1; Breast cancer resistance protein
Aim of this study was to determine whether the carbon-11-labelled antiepileptic drug [11C]mephobarbital is a substrate of P-glycoprotein (Pgp) and can be used to assess Pgp function at the blood-brain barrier (BBB) with positron emission tomography (PET). We performed paired PET scans in rats, wild-type (FVB) and Mdr1a/b(−/−) mice, before and after intravenous administration of the Pgp inhibitor tariquidar (15 mg/kg). Brain-to-blood AUC0-60 ratios in rats and brain AUC0-60 values of [11C]mephobarbital in wild-type and Mdr1a/b(−/−) mice were similar in scan 1 and scan 2, respectively, suggesting that in vivo brain distribution of [11C]mephobarbital is not influenced by Pgp efflux. Absence of Pgp transport was confirmed in vitro by performing concentration equilibrium transport assay in cell lines transfected with MDR1 or Mdr1a. PET experiments in wild-type mice, with and without pretreatment with the multidrug resistance protein (MRP) inhibitor MK571 (20 mg/kg), and in Mrp1(−/−) mice suggested that [11C]mephobarbital is also not transported by MRPs at the murine BBB, which was also supported by in vitro transport experiments using human MRP1-transfected cells. Our results are surprising as phenobarbital, the N-desmethyl derivative of mephobarbital, has been shown to be a substrate of Pgp, which suggests that N-methylation abolishes Pgp affinity of barbiturates.
drug resistance; epilepsy; blood-brain barrier; P-glycoprotein; PET; [11C]mephobarbital
Background and Objective
In microdose studies, the pharmacokinetic (PK) profile of a drug in blood after administration of a dose up to 100 μg is measured with sensitive analytical techniques, such as accelerator mass spectrometry (AMS). As most drugs exert their effect in tissue rather than blood, methodology is needed for extending PK analysis to different tissue compartments. In the present study, we combined, for the first time, AMS analysis with positron emission tomography (PET) in order to determine the PK profile of the model drug verapamil in plasma and brain of humans. In order to assess PK dose-linearity of verapamil, data were acquired and compared after administration of an intravenous (iv) microdose and an iv microdose dosed concomitantly with an oral therapeutic dose.
Six healthy male volunteers received an iv microdose (0.05 mg) (period 1) and an iv microdose dosed concomitantly with an oral therapeutic dose (80 mg) of verapamil (period 2) in a randomized, cross-over, two-period study design. The iv dose was a mixture of (R/S)-[14C]verapamil and (R)-[11C]verapamil and the oral dose was unlabelled racemic verapamil. Brain distribution of radioactivity was measured with PET whereas plasma PK of (R)- and (S)-verapamil was determined with AMS. PET data were analyzed by kinetic modeling to estimate the rate constants for transfer of radioactivity across the blood-brain barrier.
Most PK parameters of (R)- and (S)-verapamil as well as parameters describing exchange of radioactivity between plasma and brain (K1=0.030±0.003 and 0.031±0.005 mL·mL−1·min−1 and k2=0.099±0.006 and 0.095±0.008 min−1 for period 1 and 2, respectively) were not statistically different between the two periods although there was a trend for non-linear kinetics for the (R)-enantiomer. On the other hand, all PK parameters (except for t1/2) differed significantly between the (R)- and (S)-enantiomers for both periods. Cmax, AUC(0-24) and AUC(0-inf) were higher and CL, V and VSS were lower for the (R)- than for the (S)-enantiomer.
Combining AMS and PET microdosing allows long term PK data along with information on drug tissue distribution to be acquired in the same subjects thus making it a promising approach to maximize data output from a single clinical study.
Measurement of microvascular perfusion with Intravoxel Incoherent Motion (IVIM) MRI is gaining interest. Yet, the physiological influences on the IVIM perfusion parameters (“pseudo-diffusion” coefficient D*, perfusion fraction f, and flow related parameter fD*) remain insufficiently characterized. In this article, we hypothesize that D* and fD*, which depend on blood speed, should vary during the cardiac cycle. We extended the IVIM model to include time dependence of D* = D*(t), and demonstrate in the healthy human brain that both parameters D* and fD* are significantly larger during systole than diastole, while the diffusion coefficient D and f do not vary significantly. The results non-invasively demonstrate the pulsatility of the brain’s microvasculature.
About 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 co-administered tariquidar, which is an inhibitor of P-glycoprotein, we evaluated whether small-animal positron emission tomography (PET) 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.
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, Alzheimer’s and Parkinson’s 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.
ABC transporter; blood-brain barrier; breast cancer resistance protein; multidrug resistance-associated protein; P-glycoprotein; positron emission tomography; single-photon emission computed tomography
Tariquidar, a potent, nontoxic, third-generation P-glycoprotein (P-gp) inhibitor, is a possible reversal agent for central nervous system drug resistance. In animal studies, tariquidar has been shown to increase delivery of P-gp substrates into brain by several-fold. The aim of this study was to measure P-gp function at the human blood-brain barrier (BBB) after tariquidar administration using PET and the model P-gp substrate (R)–11C-verapamil. Methods: 5 healthy volunteers underwent paired (R)–11C-verapamil PET scans and arterial blood sampling, before and at 2 h 50 min after i.v. administration of tariquidar (2 mg/kg body weight). Inhibition of P-gp on CD56+ peripheral lymphocytes of each volunteer was determined by means of the rhodamine-123 efflux assay. Tariquidar concentrations in venous plasma were quantified using liquid chromatography/mass spectrometry. Results: Tariquidar administration resulted in significant increases (Wilcoxon test for paired samples) in the distribution volume (DV, +24±15%) and influx rate constant (K1, +49±36%) of (R)–11C-verapamil across the BBB (DV=0.65±0.13 and 0.80±0.07, p=0.043, K1=0.034±0.009 and 0.049±0.009, p=0.043, before and after tariquidar, respectively). A strong correlation was observed between change in brain DV after administration of tariquidar and tariquidar exposure in plasma (r=0.90, p=0.037). The mean plasma concentration of tariquidar achieved during the second PET scan (490±166 ng/mL) corresponded to 100% inhibition of P-gp function in peripheral lymphocytes. Conclusion: Tariquidar significantly increased brain penetration of (R)–11C-verapamil-derived activity, due to increased influx. As opposed to peripheral P-gp function, central P-gp inhibition appeared to be far from complete after the administered tariquidar dose.
PET; (R)-11C-verapamil; tariquidar; P-glycoprotein; blood-brain barrier
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 11C and evaluated using small-animal PET.
[11C]MC113 was synthesized by reaction of O-desmethyl MC113 with [11C]methyl triflate. Small-animal PET was performed with [11C]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 (15 mg/kg).
Brain uptake of [11C]MC113, expressed as area under the time-activity curve from time 0 to 60 min (AUC0-60), was moderately but not significantly increased in Mdr1a/b(−/−) compared with wild-type mice (mean±SD AUC0-60, Mdr1a/b(−/−): 88±7 min, wild-type: 62±6 min, P=0.100, Mann Whitney test). In the tumor model, AUC0-60 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 (IC50) for inhibition of Pgp-mediated rhodamine 123 efflux from CCRFvcr1000 cells were 375±60 nM for MC113 versus 8.5±2.5 nM for tariquidar.
[11C]MC113 showed higher brain uptake in mice than previously described Pgp PET tracers, suggesting that [11C]MC113 was only to a low extent effluxed by Pgp. However, [11C]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.
P-glycoprotein; blood-brain barrier; tumor; tariquidar; MC113; MC18; positron emission tomography
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 4 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.
PET; [11C]tariquidar; P-glycoprotein; breast cancer resistance protein; 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 11C by reaction of O-desmethyl 1 with [11C]-methyl triflate. In vitro autoradiography and small-animal PET imaging of [11C]-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 [11C]-1 binding was displaceable by an excess of unlabeled 1. As the signal obtained with [11C]-1 appeared to be specific for P-gp at the BBB, its utility for the visualization of cerebral P-gp merits further investigation.
P-gp; elacridar; 11C; PET; blood-brain barrier
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±2.0 nM, HM30181: 13.1±2.3 nM). PET scans with the Pgp substrate (R)-[11C]verapamil in FVB wild-type mice pretreated i.v. with HM30181 (10 or 21 mg/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 (1 μM). 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.
P-glycoprotein; blood-brain barrier; HM30181; tariquidar; positron emission tomography; microdosing
Overactivity of the multidrug efflux transporter P-glycoprotein (P-gp) at the blood-brain barrier (BBB) is believed to play an important role in resistance to central nervous system drug treatment. (R)-[11C]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.
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.
Inhibitor administration resulted in an up to 11-fold increase in VPM brain distribution volumes (DV) with ED50 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.
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.
small animal PET; (R)–[11C]verapamil; tariquidar; elacridar; P-glycoprotein, blood-brain barrier, regional
The multidrug efflux transporter P-glycoprotein (P-gp) is expressed in high concentrations at the blood-brain barrier (BBB) and believed to be implicated in resistance to central nervous system drugs. We used small-animal positron emission tomography (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.
A group of 7 Wistar Unilever rats underwent paired (R)-11C-verapamil PET scans at an interval of 3 h, one baseline scan and one scan after i.v. injection of tariquidar (15 mg/kg, n=5) or vehicle (n=2).
Following tariquidar administration, the distribution volume DV of (R)-11C-verapamil was 12-fold higher as compared to baseline (3.68±0.81 versus 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 mainly be attributed 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 (ED50) of 8.4±9.5 mg/kg.
Our data demonstrate that (R)-11C-verapamil PET combined with tariquidar administration is a promising approach to measure P-gp function at the BBB.
Small-animal PET; (R)-11C-verapamil; tariquidar; P-glycoprotein; blood-brain barrier; drug resistance
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 (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 Student’s 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 [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 BCRP and/or Pgp 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.
PET; 1-[18F]fluoroelacridar; P-glycoprotein; breast cancer resistance protein; blood-brain barrier
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.
1 was labelled with 11C in its methyl ester function by reaction of the corresponding carboxylic acid 2 with [11C]methyl triflate. PET imaging of [11C]-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.
Brain-to-plasma ratios of unchanged [11C]-1 were 4.8- and 10.3-fold higher in Mdr1a/b(−/−) and in Mdr1a/b(−/−)Bcrp1(−/−) mice, respectively, as compared to wild-type animals, but only modestly increased in Bcrp1(−/−) mice. [11C]-1 was rapidly metabolized in vivo giving rise to a polar radiometabolite which was taken up into brain tissue.
Our data suggest that [11C]-1 preferably interacts with P-gp rather than BCRP at the murine BBB which questions its reported in vitro BCRP selectivity. Consequently, [11C]-1 appears to be unsuitable as a PET tracer to map cerebral BCRP expression.
breast cancer resistance protein; P-glycoprotein; blood-brain barrier; PET; inhibitor; tariquidar
The aim of this study was to assess the influence of age on the functional activity of the multidrug efflux transporter P-glycoprotein (P-gp) at the human blood-brain barrier.
7 young (mean age: 27±4 years) and 6 elderly (mean age: 69±9 years) healthy volunteers underwent dynamic (R)-[11C]verapamil (VPM) positron emission tomography (PET) scans and arterial blood sampling. Parametric distribution volume (DV) images were generated using Logan linearisation and age groups were compared with statistical parametric mapping (SPM). Brain regions that SPM analysis had shown to be most affected by age were analysed by a region of interest (ROI)-based approach using a maximum probability brain atlas, before and after partial volume correction (PVC).
SPM analysis revealed significant clusters of DV increases in cerebellum, temporal and frontal lobe of elderly compared to younger subjects. In the ROI-based analysis, elderly subjects showed significant DV increases in amygdala (+30%), insula (+26%) and cerebellum (+25%), before PVC, and in insula (+33%), after PVC.
Increased VPM DV values in the brains of elderly subjects suggest a decrease in cerebral P-gp function with increasing age.
P-glycoprotein; blood-brain barrier; age; (R)-[11C]verapamil; positron emission tomography
One important mechanism for chemoresistance of tumours is overexpression of the adenosine triphosphate-binding cassette transporter P-glycoprotein (Pgp). Pgp reduces intracellular concentrations of chemotherapeutic drugs. Aim of this study was to compare the suitability of the radiolabelled Pgp inhibitors [11C]tariquidar and [11C]elacridar with the Pgp substrate radiotracer (R)-[11C]verapamil to discriminate tumours expressing low and high levels of Pgp using small-animal PET imaging in a murine breast cancer model.
Murine mammary carcinoma cells (EMT6) were continuously exposed to doxorubicin to generate a Pgp overexpressing, doxorubicin-resistant cell line (EMT6AR1.0 cells). Both cell lines were subcutaneously injected in female athymic nude mice. One week after implantation, animals underwent PET scans with [11C]tariquidar (n=7), [11C]elacridar (n=6) and (R)-[11C]verapamil (n=7), before and after administration of unlabelled tariquidar (15 mg/kg). Pgp expression in tumour grafts was studied by Western blotting.
[11C]Tariquidar showed significantly higher retention in Pgp overexpressing EMT6AR1.0 compared with EMT6 tumours (mean area under the time-activity curve in scan 1 from time 0 to 60 min, AUC0-60±SD: 38.8±2.2 min vs. 25.0±5.3 min, p=0.016, Wilcoxon matched pairs test). [11C]Elacridar and (R)-[11C]verapamil were not able to discriminate Pgp expression in tumour models. Following administration of unlabelled tariquidar, both EMT6Ar1.0 and EMT6 tumours showed increases in tumoural uptake of [11C]tariquidar, [11C]elacridar and (R)-[11C]verapamil.
Among the tested radiotracers, [11C]tariquidar performed best in discriminating high from low Pgp expressing tumours. Therefore [11C]tariquidar merits further investigation as a PET tracer to assess Pgp expression levels of solid tumours.
Multidrug resistance; P-glycoprotein; positron emission tomography; [11C]tariquidar; [11C]elacridar; (R)-[11C]verapamil
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) of healthy human subjects. We compared the dose-response relationship of tariquidar in humans with data obtained in rats using similar methodology. Tariquidar was equipotent in humans and rats to increase (R)-[11C]verapamil brain uptake, expressed as whole brain volume of distribution (VT), with very similar half-maximum effect concentrations. Both in humans and rats, brain VTs approached plateau levels at tariquidar plasma concentrations >1,000 ng/mL. However, Pgp inhibition in humans only led to 2.7-fold increase in brain VT relative to baseline scans without tariquidar compared to 11.0-fold in rats. The results of this translational study add to the accumulating evidence of marked species-dependent differences in Pgp expression and functionality at the BBB.
P-glycoprotein; blood-brain barrier; drug-drug interaction; positron emission tomography; (R)-[11C]verapamil; tariquidar; human; rat
Medications are frequently prescribed outside their regulatory approval (off-label) by physicians particularly where appropriate therapies are not available. However, the risk/benefit ratio of drugs in off-label use needs to be critically appraised because it may differ from approved on-label usage. Therefore, an extensive exploration of current evidence on clinical data is well-advised. The objective of this study was to develop a search strategy that facilitates detection of the off-label drug use documents in EMBASE via OvidSP.
We constructed two sets of gold standards from relevant records to off-label drug use by a sensitive search of MEDLINE and EMBASE. Search queries, including search words and strings, were conceived based on definition of off-label use of medications as well as text analysis of 500 randomly selected relevant documents. The selected terms were searched in EMBASE (from 1988 to 2011) and their retrieval performance was compared with the gold standards. We developed a sensitivity-maximizing, and a sensitivity- and precision-maximizing search strategy.
From 4067 records relevant to off-label drug use in our full gold standard set, 3846 records were retrievable from EMBASE. “off label*.af.” was the most sensitive single term (overall sensitivity 77.5%, sensitivity within EMBASE 81.9%, precision 88.1%). The highest sensitive search strategy was achieved by combining 36 search queries with overall sensitivity of 94.0% and precision of 69.5%. An optimal sensitive and precise search strategy was yielded precision 87.4% at the expense of decreasing overall sensitivity to 89.4%.
We developed highly sensitive search strategies to enhance the retrieval of studies on off-label drug use in OvidSP EMBASE.
Off-label use; Information retrieval; EMBASE; MEDLINE; Sensitivity
This study investigated the influence of P-glycoprotein (P-gp) inhibitor tariquidar on the pharmacokinetics of P-gp substrate radiotracer (R)-[11C]verapamil in plasma and brain of rats and humans by means of positron emission tomography (PET).
Data obtained from a preclinical and clinical study, in which paired (R)-[11C]verapamil PET scans were performed before, during, and after tariquidar administration, were analyzed using nonlinear mixed effects (NLME) modeling. Administration of tariquidar was included as a covariate on the influx and efflux parameters (Qin and Qout) in order to investigate if tariquidar increased influx or decreased outflux of radiotracer across the blood–brain barrier (BBB). Additionally, the influence of pilocarpine-induced status epilepticus (SE) was tested on all model parameters, and the brain-to-plasma partition coefficient (VT-NLME) was calculated.
Our model indicated that tariquidar enhances brain uptake of (R)-[11C]verapamil by decreasing Qout. The reduction in Qout in rats during and immediately after tariquidar administration (sevenfold) was more pronounced than in the second PET scan acquired 2 h after tariquidar administration (fivefold). The effect of tariquidar on Qout in humans was apparent during and immediately after tariquidar administration (twofold reduction in Qout) but was negligible in the second PET scan. SE was found to influence the pharmacological volume of distribution of the central brain compartment Vbr1. Tariquidar treatment lead to an increase in VT-NLME, and pilocarpine-induced SE lead to increased (R)-[11C]verapamil distribution to the peripheral brain compartment.
Using NLME modeling, we were able to provide mechanistic insight into the effects of tariquidar and SE on (R)-[11C]verapamil transport across the BBB in control and 48 h post SE rats as well as in humans.
Nonlinear mixed effects modeling; Positron emission tomography; (R)-[11C]verapamil; P-glycoprotein; Tariquidar; Pilocarpine-induced epilepsy; Species differences