Evaluation of the Near Infrared Compound Indocyanine Green as a

In vivo imaging indicated enhanced cerebral ICG distribution with valspodar (brain – foot area under the concentration–time curves of 3.0 × 1010,...
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Evaluation of the Near Infrared Compound Indocyanine Green as a Probe Substrate of P‑Glycoprotein Emma Portnoy,†,‡ Marina Gurina,† Shlomo Magdassi,‡ and Sara Eyal*,† †

Institute of Drug Research, School of Pharmacy, Faculty of Medicine, and ‡Casali Institute, Institute of Chemistry and Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem, Israel S Supporting Information *

ABSTRACT: The efflux transporter P-glycoprotein (P-gp) affects the pharmacokinetics of many drugs. Currently used methods for characterization of P-gp’s functional activity in vivo involve the use of radiolabeled substrates, are costly, and are technically demanding. Our objective was to evaluate whether the FDA-approved near-infrared compound indocyanine green (ICG) can be used as a probe substrate of P-gp. We also characterized the interaction of ICG with another efflux transporter, the breast cancer resistance protein (BCRP). We evaluated ICG accumulation and transport in MDCK cells overexpressing P-gp or BCRP (MDCKMDR1 and MDCK-BCRP, respectively) compared to control MDCK cells, in the presence or the absence of transporter inhibitors. In vivo imaging of ICG biodistribution in mice was conducted over 3.5 h using valspodar as the P-gp inhibitor. The EC50 values for ICG accumulation in control MDCK and MDCK-MDR1 cells were 9.0 × 10−6 ± 5.7 × 10−7 M and 1.5 × 10−5 ± 1.1 × 10−6 M, respectively. The efflux ratio for ICG in MDCK-MDR1 cells was 6.8-fold greater than in control cells. P-gp inhibition attenuated ICG efflux from MDR1-MDCK cells, and their effects in those cells were greater than in control MDCK cells. In contrast, BCRP level of expression or pharmacological inhibition did not significantly affect ICG cellular accumulation. In vivo imaging indicated enhanced cerebral ICG distribution with valspodar (brain − foot area under the concentration−time curves of 3.0 × 1010, 5.6 × 1010 and 3.7 × 1010 h·[p/s/sr]/μW in valspodar-treated mice vs 9.0 × 109 and 5.3 × 109 h·[p/s/sr]/μW in controls). The findings from this pilot study suggest that near-infrared imaging using ICG as the probe substrate should be further characterized as a methodology for in vivo evaluation of P-gp activity. KEYWORDS: indocyanine green, near-infrared optical imaging, P-glycoprotein, MDR1, breast cancer resistance protein, BCRP, multidrug resistance associated protein, MRP2, Mdr2

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enantiomer.5−8 However, PET scanning is associated with health risk since it involves ionizing radiation, is technically demanding, and is costly. More recently, On et al.9 reported on a method to evaluate P-gp activity in vivo by near-infrared (NIR) optical imaging, using rhodamine 800 as the P-gp substrate. Nevertheless, to date rhodamine 800 is not approved by the FDA for use in humans and thus cannot be applied for clinical studies. Here, we evaluated the P-gp-mediated transport of the only FDA-approved NIR molecule, indocyanine green (ICG). ICG is used for determination of cardiac output, liver function, and visualization of retinal and choroid vessels,10,11 and can be incorporated into nanoparticles for tracking drug delivery.12,13 Our findings suggest that ICG is a substrate for Pgp, but not BCRP, and may be combined with selective P-gp inhibitors for evaluation of P-gp activity by simple optical imaging.

INTRODUCTION The efflux transporters P-glycoprotein (P-gp/MDR1/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) affect drug pharmacokinetics and pharmacodynamics. BCRP and Pgp are expressed in cancer cells and in sites critical for drug disposition such as the intestine, liver, kidneys, and endothelial cells of brain capillaries that form the blood−brain barrier (BBB).1−3 The substrate specificity of these transporters is broad and partially overlaps, and includes many drugs, such as antiretroviral compounds, immunosuppressants, and chemotherapeutic agents.4 Despite the recognition that the extent of P-gp and BCRP function has the potential to affect drug efficacy and toxicity, until recently their activity in sites such as the BBB has not been measured directly in humans because of the difficulty of sampling brain drug concentrations. Such studies have become feasible only over the past few years, with the development of noninvasive imaging techniques. Among these, the most common is positron emission tomography (PET), using radiolabeled transporter substrates such as 11C-loperamide, 11 C-N-desmethyl-loperamide, and 11C-verapamil or its R© 2012 American Chemical Society

Received: Revised: Accepted: Published: 3595

April 11, 2012 October 22, 2012 October 25, 2012 October 25, 2012 dx.doi.org/10.1021/mp300472y | Mol. Pharmaceutics 2012, 9, 3595−3601

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Brief Article

EXPERIMENTAL SECTION Materials. ICG was purchased from Acros Organics (Geel, Belgium). Calcein AM was obtained from Invitrogen (Carlsbad, CA, USA), and Bodipy-verapamil and Bodipy-prazosin were obtained from Molecular Probes (Grand Island, NY, USA). Valspodar (PSC-833) was from Tocris Bioscience (Bristol, U.K.). C-219 was from Covance (Hertzelia, Israel). BXP-21 and the primary antibody for β-actin were from Abcam (Cambridge, MA, USA). Secondary antibodies were from Jackson ImmunoResearch (West Grove, PA, USA). Skim milk was obtained from Difco (Franklin Lakes, NJ). Cell culture reagents were from Biological Industries (Beit Haemek, Israel). All other reagents were from Sigma-Aldrich (Rehovot, Israel). Cell Culture. The Madin−Darby canine kidney (MDCK) II cells transfected with cDNA coding for MDR1 (MDCK-MDR1 cells) and parent (MDCK-CT) cells were kindly provided by Dr. Alfred Schinkel (The Netherlands Cancer Institute). MDCK cells transfected with pcDNA empty vector (MDCKvector) and cDNA coding for wild-type BCRP (MDCK-BCRP cells) were a generous gift from Dr. Qingcheng Mao (University of Washington). MDCK-MDR1 cells and MDCK-CT cells were grown in Dulbecco’s modified Eagle’s phenol-free low-glucose medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a 5% CO2 incubator. MDCK-BCRP cells and their controls were grown under similar conditions, except that the medium was Eagle’s minimum essential medium (MEM) and cells were supplemented in addition with 0.05 mg/mL gentamicin. The cells were harvested by trypsin−EDTA after achieving 80−90% confluence. SDS−Polyacrylamide Gel Electrophoresis. Cells were washed twice with cold phosphate buffered saline (PBS). Whole cell lysates were prepared in ice-cold lysis buffer, containing 200 μL of 0.01 M Tris-HCl, pH 7.5, 0.1% sodium dodecyl sulfate (SDS), 0.01 M MgCl2, and protease inhibitor cocktail. The cells were shaken with the lysis buffer for 1 h at 5 °C. The lysate was centrifuged for 15 min at 14,300 rpm. Protein concentrations were determined by the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL, USA). Following SDS−PAGE analysis under reducing conditions gels were electrotransferred to nitrocellulose membranes. Membranes were blocked in Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% milk powder and probed overnight at 4 °C with the primary antibodies BXP-21 at 1:250, C219 at 1:1000, and anti β-actin antibody at 1:2500. The blots were then incubated with peroxidase-conjugated goat anti-rabbit secondary antibody or goat anti-mouse IgG at 1:10000 for 1 h and developed by enhanced chemiluminesence. Accumulation Studies. MDCK-CT or MDCK-MDR1 cells were seeded at 20 × 104 cells/well in 24 well plates. Experiments were performed two days after achieving confluent monolayers. Prior to the experiment, cells were incubated for 1 h with 1 mL of DMEM with 5 mM HEPES, pH 7.3, in the presence or the absence of a P-gp inhibitor, 200 μM verapamil or 10 μM CsA, or the MRP inhibitor MK-571 (50 μM). In the accumulation phase cells were coincubated with 0.25 μM calcein AM, 1 μM Bodipy-verapamil, or 1 μM ICG in the presence or the absence of the inhibitor. After 1 h, the cells were washed three times with ice-cold PBS. Intracellular fluorescence of calcein AM was measured within 1 h with excitation wavelength 485 nm and emission wavelength 528 nm

by a plate reader (Synergy HT, BioTek, Winooski, VT, USA). The emission of Bodipy-verapamil was detected by Typhoon FLA 9500 biomolecular imager (GE Healthcare Life Sciences, Piscataway Township, NJ, USA). Intracellular emission of ICG was detected by Odyssey NIR scanner (Li-Cor, Lincoln, NE, USA) with excitation wavelength 780 nm and emission wavelength 800 nm. Evaluation of kinetic parameters was conducted at 8 ICG concentrations, ranging from 1 × 10−7 M to 1 × 10−12 M. For MDCK-vector and MDCK-BCRP cells, experiments were conducted as described for MDCK-MDR1 cells, except that prior to the experiment cells were incubated with 1 mL of MEM with 5 mM HEPES, pH 7.3, in the presence or absence of 10 μM fumitremorgin C (FTC). In the accumulation phase the cells were coincubated with 500 nM Bodipy-prazosin or 1 μM of ICG in the presence or the absence of 10 μM FTC. After 1 h incubation, cells were washed three times with ice-cold PBS. Intracellular fluorescence of Bodipy-prazosin was measured within 1 h with excitation wavelength 485 nm and emission wavelength 528 nm. The emission of ICG, calcein AM, and Bodipy-prazosin was normalized to protein determined in cell lysates. The studies were performed in triplicate on two separate days in a humidified incubator. Directional Permeability Assays. The Transwell transport assay was the same as described previously14 with minor modifications. In brief, transport of ICG across cell monolayers was evaluated using micrporous polycarbonate membrane filters (3 μm pore size, 24 mm diameter, Costar Corning Life Sciences, Acton, MA). MDCK-CT cells or MDCK-MDR1 cells were seeded at 2 × 106 cells/well. The cells were grown with replacement of medium every day. The resistance was measured with Millicell-ERS (Millipore Corporation, Billerica, MA). Transport assays were conducted when the average transepithelial resistance was 200 Ω or greater. One hour before the experiment the medium was replaced with DMEM containing 5 mM HEPES buffer, pH 7.3. The volumes at the apical and the basolateral compartments were 1.5 and 2.5 mL, respectively. The experiments were started by replacing the medium with fresh DMEM containing 1 μM ICG in the presence or the absence of 200 μM verapamil in the apical and the basolateral compartments. Transport of ICG was tested in two directions (apical to basolateral, A to B, and basolateral to apical, B to A). Aliquots of 100 μL were taken from the receiver compartments every hour up to 4 h with replacement with fresh DMEM with or without 200 μM verapamil. ICG concentration was measured by Odyssey NIR scanner as described above. The studies were performed in triplicate in a humidified incubator on two different days. The efflux ratio (ER) was obtained by apparent permeability coefficients (Papp): ER = PappB/A /PappA/B

where PappB/A and PappA/B represent the apparent permeability of test compound from the basal to apical and apical to basal side of the cellular monolayer, respectively.15 Papp was calculated based on the following equation:15 Papp = (Vr/C0)(1/S)(dC /dt )

where Papp = apparent permeability, Vr is the volume of medium in the receiver chamber, C0 is the concentration of the test drug in the donor chamber, S is the surface area of monolayer, and dC/dt is the is the linear slope of the drug 3596

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respectively. Based on results from the in vitro studies, we have also conducted a pilot in vivo imaging study in mice, using valspodar as the P-gp inhibitor. We first evaluated the contribution of P-gp to ICG accumulation in a concentration-dependent manner. The EC50 values in MDCK-CT and MDCK-MDR1 cells were 9.0 × 10−6 ± 5.7 × 10−7 M and 1.5 × 10−5 ± 1.1 × 10−6 M, respectively, indicating greater resistance to ICG accumulation in MDCK-MDR1 cells (data not shown). ICG accumulation was then evaluated at 1 μM, a concentration which represents those obtained in humans following intravenous injection of 10 mg of ICG19 and is lower than the EC50 values obtained in both MDCK-MDR1 and MDCK-CT cells. In MDCK-CT cells, cell protein concentration-normalized ICG fluorescence at 1 h was 1.8-fold greater, compared to MDCK-MDR1 cells (36388 ± 1539 au/μg protein/mL and 19939 ± 1845 au/μg protein/mL, respectively, P < 0.01; Figure 1A). Treatment with 200 μM verapamil increased the ICG emission 3.6-fold (P < 0.01) in MDCK-MDR1 cells, but only 1.8-fold in MDCK-CT cells (P < 0.01). The impact of verapamil on ICG accumulation in MDCK-CT cells presumably resulted from inhibition of endogenous canine MDR1, as previously reported20 (Figure 1C). Compared to verapamil, the effect of CsA on ICG accumulation in MDCK-MDR1 cells was lesser (1.5-fold increase; P < 0.01), with no effect in MDCK-CT cells. In comparison, calcein AM accumulation was 7.2-fold greater in MDCK-CT cells compared to MDCK-MDR1 cells (P < 0.01), and increased in the latter 14.3-fold (P < 0.01) and 11-fold (P < 0.01) with the coincubation with verapamil and CsA, respectively (Figure 1B). We further evaluated the contribution of MRPs vs P-gp to ICG efflux from MDCK-MDR1 cells. Despite MDR1 overexpression in these cells, MK-571 produced a greater effect than CsA on ICG cellular accumulation, in line with the established role of MRP2 in ICG cellular efflux. Combining CsA and MK-571 did not further enhance this effect, possibly due to inhibition of organic anion transporter polypeptides (OATPs) by CsA (Figure 1A in the Supporting Information). In comparison, accumulation of Bodipy-verapamil, an established P-gp substrate, was influenced predominantly by CsA and not by MK-571 (Figure 1B in the Supporting Information). Based on the results from the accumulation studies, the efflux ratio was evaluated with verapamil (200 μM) as the inhibitor (Figure 2; Figure 2 in the Supporting Information). In MDCKMDR1 cells, ICG efflux ratio was 6.8-fold greater than in MDCK-CT cells (6.7 vs 1.0, respectively) (Figure 2). The efflux ratio in MDCK-MDR cells was decreased by verapamil to 1.5 ± 0.6 (p < 0.05). In comparison, the previously reported rhodamine 800 efflux ratio in MDCK-MDR1 cells was 3, although this ratio was obtained over 1 h only permeability assay.9 The level of MDR1 expression affected mostly ICG basolateral-to-apical transfer (Figure 2A). Interestingly, ICG did not inhibit the MDR1 ATPase in an extracellular assay (Figure 3 in the Supporting Information). However, negative results were also obtained for ICG, an established MRP2 substrate,21 in an MRP2 ATPase assay (Figure 3 in the Supporting Information). False negative as well as false positive results have previously been reported with ATPase assays, and may be related to the species differences with regard to the source of the membrane preparation used in this assay.22 Overall, the Transwell assay is more reliable than ATPase assays for predicting P-gp efflux liability in vivo.22

concentration in the receptor chamber with time after correcting for dilution. ATPase Assay. The activation of P-gp, BCRP, and the multidrug resistance associated protein 2 (MRP2) ATPases was evaluated using PREDEASY kits (SOLVO Biotechnology, Szeged, Hungary), according to the manufacturer’s instructions. Animal Studies. The animal study protocol was approved by the Institutional Animal Care and Use Committee of the Hebrew University. Male FVB mice (8 weeks old) were purchased from Harlan Laboratories (Rehovot, Israel). The mice had free access to food (a standard diet) and water, and they were maintained on a 12/12-h automatically timed light/ dark cycle. Under isoflurane (1−2%, v/v) anesthesia, mice were shaved and underwent a baseline scan in an IVIS Kinetic in vivo imaging system (Caliper Life Sciences, Hopkinton, MA, USA). Valspodar (12.5 mg/kg or 25 mg/kg in polyethylene glycol 300:ethanol 80:20) or the vehicle, in a total volume of 100 μL, was administered into the tail vein. Forty five minutes later, ICG (8 mg/kg, in normal saline) was injected into the tail vein and the mice were repetitively scanned over a time period of 3.5 h. Blood samples were collected from the tails of naive mice (baseline) and at 0.25, 1, 2, 3, and 4 h after the injection. At the completion of the ICG scanning, mice were sacrificed under anesthesia by cervical dislocation and brains were harvested. Cardiac blood was collected in heparinized 96-well plates. Along the collection procedure, tissue and blood samples were protected from light and kept on ice. Immediately thereafter, blood and tissues were scanned by Typhoon FLA 9500 biomolecular imager. Data Analysis. Kinetic parameters (EC50, 50% effective concentration; Emax, maximal efficacy) of in vitro ICG accumulation were estimated by a sigmoidal dose−response nonlinear regression curve fit of the experimental data (n = 6 per ICG concentration; WinNonlin 6.2; Pharsight, Mountain View, CA). For analysis of in vivo ICG kinetics, regions of interest (ROIs) were drawn over the brains and hind feet and wells containing the plasma samples using the Living Image software, version 4.3.1 (Xenogen). The emission intensity of each sample was expressed in radiant efficiency units ([photons/second/steradian]/microwatt; [p/s/sr]/μW). Background (before ICG injection) emission intensity was subtracted from each ROI. Additional analysis included subtraction of the hind foot emission intensity from brain emission intensity, to correct for contamination from skin and blood vessels (suggested by the camera manufacturer for image analysis; http://www.caliperls.com/assets/023/8438.pdf). The hind foot was selected as the reference background region because it is remote from both the injection site and the brain, can be clearly identified, and does not contain tissues known to highly express P-gp. ICG area under the concentration−time curve (AUC) was calculated using WinNonlin. Unless otherwise stated, data are presented as mean ± SD. Analysis of variance was used to determine the statistical significance of difference (p < 0.05) between experimental groups (InStat; GraphPad, La Jolla, CA, USA).

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RESULTS AND DISCUSSION The current study evaluated ICG and NIR detection for assessing the activity of P-gp, BCRP, or both. We measured the emission of ICG from MDR1- and BCRP-overexpressing cells, compared to their controls, in the presence and the absence of transporter inhibitors. Calcein AM16 and Bodipy-prazosin17,18 were used as positive controls for P-gp and BCRP activity, 3597

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Figure 2. Bidirectional transport of ICG in MDCK-CT cells (CT), DMSO-treated MDCK-MDR1 cells (MDR1), and verapamil (200 μM)-treated MDCK-MDR1 cells. (A) Apical-to-basolateral (A to B) and basolateral-to-apical ICG transfer; a, significantly different from A to B in DMSO-treated MDCK-MDR1 cells and in MDCK-CT cells, P < 0.01; b, significantly different from A to B in verapamil-treated MDCK-MDR1 cells, P < 0.05; c, significantly different from B to A in MDCK-CT cells, P < 0.01. (B) ICG efflux ratios, based on A-to-B and B-to-A values. Results in A and B are presented as means ± SEM.

Figure 1. Cellular accumulation of ICG and calcein AM in MDCKMDR1 (MDR1) and MDCK-CT cells (CT). (A) Cell protein concentration-normalized NIR fluorescence of 1 μM ICG in MDCKMDR1 and MDCK-CT cells in the presence and the absence of the Pgp inhibitors verapamil (200 μM) and cyclosporine A (CsA; 10 μM) following 1 h incubation. (B) Calcein AM accumulation under the conditions described for ICG. (C) MDR1 protein concentrations in MDCK-MDR1 cells and in MDCK-CT cells. Also shown is the concentration of the reference protein β-actin. Results in A and B are presented as means ± SD; au, arbitrary units; a, significantly different from MDCK-CT cells, P < 0.01; b, significantly different from DMSOtreated MDCK-MDR1 cells, P < 0.01; c, significantly different from MDCK-CT cells, P < 0.05.

this study further support a role for P-gp in ICG pharmacokinetics. Valspodar clearly increased systemic ICG concentrations, as indicated by the enhanced emission intensity in brain, feet, and blood (Figure 3A−C). The systemic effect on ICG accumulation appeared to be dose-dependent, although further studies are required to confirm this observation. Of note, the enhanced ICG accumulation was not necessarily mediated solely by P-gp, because valspodar is also an OATP inhibitor23 and ICG is an OATP substrate. In contrast, valspodar’s effect on cerebral ICG uptake was likely mediated by P-gp inhibition. In this preliminary study, we utilized two methods of data analysis: (1) subtraction of emission intensity from a reference background region (hind foot) from cerebral emission intensity, and (2) brain:blood concentration ratios. The subtraction method is consistent with conventional analyses of results from NIR optical imaging because it corrects for contamination from surrounding tissues as well as peripheral blood. Based on this method, valspodar increased cerebral ICG distribution, with AUC0−210min of 3.0 × 1010, 5.6 × 1010, and 3.7 × 1010 h·[p/s/sr]/μW in valspodar-treated mice vs 9.0 × 109 and 5.3 × 109 h·[p/s/sr]/μW in controls (Figure 3D). Compared to this analysis, brain:blood concentration ratios are less appropriate for this type of imaging, given the above-mentioned method limitations. Indeed, the ratios with

Unlike P-gp, BCRP did not appear to be involved in ICG transport, as indicated by the lack of effect of both BCRP overexpression and administration of the potent BCRP inhibitor FTC on ICG cellular accumulation (Figure 4A in the Supporting Information). In contrast, in MDCK-BCRP cells Bodipy-prazosin accumulation was 1.6-fold greater than in control cells (0.62 ± 0.10 vs 0.98 ± 0.07 respectively, P < 0.01; Figure 4B in the Supporting Information). Moreover, FTC significantly enhanced Bodipy-prazosin accumulation in MDCK-BCRP cells (1.4-fold increase, P < 0.01; Figure 4 in the Supporting Information). In extracellular assays, ICG did not activate BCRP ATPase activity (Figure 3 in the Supporting Information). To evaluate the feasibility of ICG as an in vivo probe for P-gp activity for experimental and clinical uses, we conducted a pilot study in mice using valpodar as the P-gp inhibitor. Results from 3598

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Figure 3. ICG fluorescence in mice in the presence and the absence of valspodar. Shown are individual ICG concentration−time profiles in brain (A), foot (B), and plasma (C). (D) Individual AUC values of ICG emission intensity in brain (foot emission intensity is subtracted) at 30, 60, 120, and 210 min after ICG injection. (E, F) Individual brain:plasma concentration ratios in vivo (E) and ex vivo (F). Also shown in F are ex vivo images of respective brains.

improving the signal-to-noise ratio compared to traditional fluorescent probes. In addition, NIR fluorescence can penetrate tissues up to several centimeters. Together, these advantages led to the growing use of ICG and NIR technologies in humans as well as for whole body imaging in small animals.12,13,26,27 Based on our pilot experiments, NIR optical imaging using ICG as the probe substrate should be further characterized as a methodology for in vivo evaluation of P-gp activity. If these studies are successful, ICG can be combined with selective P-gp inhibitors for in vivo imaging of the functional activity of P-gp before and during transporter inhibition. With the ongoing advance in NIR imaging in humans, this methodology can further be developed for simple, noninvasive evaluation of transporter function in health and disease. Furthermore, NIR with ICG can be used for rapid initial in vivo screening of new drugs for potential interference with hepatic nonenzymatic

subtraction of reference regions indicated greater increases in ICG emission intensity compared to estimates based on brain:blood concentration ratios (Figure 3E,F), possibly due to the high contamination from vascular ICG which obscures potential changes in emission from brain tissue. It has been reported that ICG is a substrate of Mdr2 (Pglycoprotein 2)24 and the multidrug resistance associated protein 2 (MRP2).21 However, another report proposed that the primary biliary transport system for ICG is not Mrp2.25 Our results suggest that the other transport system does not involve BCRP, and that ICG is a substrate of P-gp in addition to Mdr2. Whether P-gp is a primary biliary transport system for ICG will be evaluated in future studies. Although ICG has a narrower dynamic range as a P-gp probe compared to calcein AM (Figure 1), it has the advantage of emitting fluorescence in the NIR region (700−800 nm), thus 3599

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elimination processes, whether they involve hepatic blood flow, OATPs, or P-gp/MDR3/MRP2 mediated transport. To this point, ICG’s interaction with a variety of uptake and efflux transporters should be taken into account when this compound is utilized for estimation of parameters such as blood flow in various tissues and overall hepatic function. Finally, despite its above-mentioned limitations, NIR imaging is overall a promising new technology for whole-body imaging of transporter function. For example, it can be used experimentally and clinically to assess the effects of medications and disease on transporter activity and for evaluation of new drugs as transporter modulators. Our future studies will evaluate additional NIR fluorescent molecules as transporter probes.

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(3) Maliepaard, M.; Scheffer, G. L.; Faneyte, I. F.; van Gastelen, M. A.; Pijnenborg, A. C.; Schinkel, A. H.; van De Vijver, M. J.; Scheper, R. J.; Schellens, J. H. Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues. Cancer Res. 2001, 61, 3458−64. (4) Eyal, S.; Hsiao, P.; Unadkat, J. D. Drug interactions at the bloodbrain barrier: fact or fantasy? Pharmacol. Ther. 2009, 123, 80−104. (5) Kannan, P.; John, C.; Zoghbi, S. S.; Halldin, C.; Gottesman, M. M.; Innis, R. B.; Hall, M. D. Imaging the function of P-glycoprotein with radiotracers: pharmacokinetics and in vivo applications. Clin. Pharmacol. Ther. 2009, 86, 368−77. (6) Sasongko, L.; Link, J. M.; Muzi, M.; Mankoff, D. A.; Yang, X.; Collier, A. C.; Shoner, S. C.; Unadkat, J. D. Imaging P-glycoprotein transport activity at the human blood-brain barrier with positron emission tomography. Clin. Pharmacol. Ther. 2005, 77, 503−14. (7) Seneca, N.; Zoghbi, S. S.; Liow, J. S.; Kreisl, W.; Herscovitch, P.; Jenko, K.; Gladding, R. L.; Taku, A.; Pike, V. W.; Innis, R. B. Human brain imaging and radiation dosimetry of 11C-N-desmethyl-loperamide, a PET radiotracer to measure the function of P-glycoprotein. J. Nucl. Med. 2009, 50, 807−13. (8) Bauer, M.; Zeitlinger, M.; Karch, R.; Matzneller, P.; Stanek, J.; Jäger, W.; Böhmdorfer, M.; Wadsak, W.; Mitterhauser, M.; Bankstahl, J. P.; Löscher, W.; Koepp, M.; Kuntner, C.; Müller, M.; Langer, O. Pgp-mediated interaction between (R)-[11C]verapamil and tariquidar at the human blood-brain barrier: a comparison with rat data. Clin. Pharmacol. Ther. 2012, 91, 227−33. (9) On, N. H.; Chen, F.; Hinton, M.; Miller, D. W. Assessment of Pglycoprotein activity in the Blood-Brain Barrier (BBB) using Near Infrared Fluorescence (NIRF) imaging techniques. Pharm. Res. 2011, 28, 2505−15. (10) Dzurinko, V. L.; Gurwood, A. S.; Price, J. R. Intravenous and indocyanine green angiography. Optometry 2004, 75, 743−55. (11) Sakka, S. G. Assessing liver function. Curr. Opin. Crit. Care 2007, 13, 207−14. (12) Larush, L.; Magdassi, S. Formation of near-infrared fluorescent nanoparticles for medical imaging. Nanomedicine (London) 2011, 6, 233−40. (13) Portnoy, E.; Lecht, S.; Lazarovici, P.; Danino, D.; Magdassi, S. Cetuximab-labeled liposomes containing near-infrared probe for in vivo imaging. Nanomedicine (London) 2011, 7, 480−8. (14) Zhang, Y.; Gupta, A.; Wang, H.; Zhou, L.; Vethanayagam, R. R.; Unadkat, J. D.; Mao, Q. BCRP transports dipyridamole and is inhibited by calcium channel blockers. Pharm. Res. 2005, 22, 2023−34. (15) FDA Draft Guidance for Industry: Drug Interaction StudiesStudy Design, 2006. (16) Rautio, J.; Humphreys, J. E.; Webster, L. O.; Balakrishnan, A.; Keogh, J. P.; Kunta, J. R.; Serabjit-Singh, C. J.; Polli, J. W. In vitro pglycoprotein inhibition assays for assessment of clinical drug interaction potential of new drug candidates: a recommendation for probe substrates. Drug Metab. Dispos. 2006, 34, 786−92. (17) Ni, Z.; Bikadi, Z.; Cai, X.; Rosenberg, M. F.; Mao, Q. Transmembrane helices 1 and 6 of the human breast cancer resistance protein (BCRP/ABCG2): identification of polar residues important for drug transport. Am. J. Physiol. 2010, 299, C1100−9. (18) Robey, R. W.; Honjo, Y.; van de Laar, A.; Miyake, K.; Regis, J. T.; Litman, T.; Bates, S. E. A functional assay for detection of the mitoxantrone resistance protein, MXR (ABCG2). Biochim. Biophys. Acta 2001, 1512, 171−82. (19) Niemann, C.; Henthorn, T.; Krejcie, T.; Shanks, C.; EndersKlein, C.; Avram, M. Indocyanine green kinetics characterize blood volume and flow distribution and their alteration by propranolol. Clin. Pharmacol. Ther. 2000, 67, 342−50. (20) Poller, B.; Wagenaar, E.; Tang, S. C.; Schinkel, A. H. Doubletransduced MDCKII cells to study human P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) interplay in drug transport across the blood−brain barrier. Mol. Pharmaceutics 2011, 8, 571−82. (21) Jansen, P. L.; van Klinken, J. W.; van Gelder, M.; Ottenhoff, R.; Oude Elferink, R. P. J. Preserved organic anion transport in mutant

ASSOCIATED CONTENT

* Supporting Information S

Additional figures as discussed in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Institute of Drug Research, Room 304, School of Pharmacy, Faculty of Medicine, The Hebrew University, Ein Kerem, Jerusalem, 91120, Israel. Phone: 972-2-675-8667. Fax: 972-2675-7246. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Miriam Shmuel at the Institute of Drug Research, The Hebrew University, for her assistance with assay establishment and to Natalie Corchia at the Genetic Therapy Laboratories, Hadassah Medical Center, for her help with the in vivo studies. We thank Dr. Qingcheng Mao (University of Washington) for providing the MDCK-vector and MDCKBCRP cell lines. We thank Dr. Alfred Schinkel (The Netherlands Cancer Institute) for providing the MDCK and MDCK-MDR1 cell lines. The study was supported by a research grant from the David R. Bloom Center for Pharmacy. This work is abstracted from the Ph.D. thesis of Ms. Emma Portnoy in partial fulfillment of the Ph.D. requirements of the Hebrew University of Jerusalem.

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ABBREVIATIONS USED au, arbitrary units; BCRP, breast cancer resistance protein; BBB, blood−brain barrier; CsA, cyclosporine A; CT, control cells; EC50, 50% effective concentration; Emax, maximal efficacy; ER, efflux ratio; FTC, fumitremorgin C; ICG, indocyanine green; MDR, multidrug resistance protein; MDCK, Madin− Darby canine kidney; MRP, multidrug resistance associated protein; NIR, near-infrared; P-gp, P-glycoprotein; Papp, apparent permeability coefficient

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