Radiosynthesis of [2-11C-carbonyl]dantrolene using [11C]phosgene for PET

ARTICLE IN PRESS Applied Radiation and Isotopes 68 (2010) 1715–1720

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Radiosynthesis of [2-11C-carbonyl]dantrolene using [11C]phosgene for PET Yuuki Takada a,b, Masanao Ogawa a,c, Hisashi Suzuki a, Toshimitsu Fukumura a,n a b c

Molecular Probe Group, Molecular Imaging Center, National Institute of Radiological Science, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan Department of Radiology, School of Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan SHI Accelerator Service Ltd., 1-17-6 Osaki, Shinagawa-ku, Tokyo 141-8686, Japan

a r t i c l e in fo

abstract

Article history: Received 2 November 2009 Received in revised form 6 March 2010 Accepted 17 March 2010

Automated radiosynthesis of [2-11C-carbonyl]dantrolene, the substrate of breast cancer resistance protein (BCRP/ABCG2), was performed for the first time through a multi-step/one-pot labeling sequence that started with ethyl 2-{2-[5-(4-nitrophenyl)furfurylidene]hydrazino}acetate and used [11C]phosgene as a labeling agent. After optimization of the automated synthesis conditions and parameters, [2-11Ccarbonyl]dantrolene was obtained at a radiochemical yield of 34.0 7 8.4% (decay-corrected). The radiochemical purity was greater than 98% and the specific activity was 46.8 7 15.2 GBq/mmol at the end of the synthesis. & 2010 Elsevier Ltd. All rights reserved.

Keywords: [2-11C-carbonyl]dantrolene [11C]phosgene Positron emission tomography (PET)

1. Introduction ATP-binding cassette (ABC) transporters, as typified by P-glycoprotein (P-gp/ABCB1) and the multidrug resistance-associated protein (MRP/ABCC) family, are assumed to play significant roles in the blood–brain barrier (BBB) to limit the movement of drugs and other xenobiotic compounds from the blood into the brain (Gottesman et al., 2002; Borst and Oude, 2002). Breast cancer resistance protein (BCRP/ABCG2), which was isolated from atypical multidrug resistant MCF-7 human breast cancer cells, is an ABC transporter and mediates the efflux transport of xenobiotics (Doyle et al., 1998; Ishikawa, 2009). BCRP is expressed in various normal tissues (Maliepaard et al., 2001) as well as in tissue barriers such as those in brain, testis, and placenta similarly to P-gp and MRPs (Bart et al., 2004; Zhang et al., 2003). In addition, the substrate specificity of BCRP shows considerable overlap with those of P-gp and MRPs (Enokizono et al., 2008). Recently, Enokizono et al. (2007, 2008) investigated the transport activity and substrate specificity of BCRP using phytoestrogens, dietary carcinogens, and drugs with Bcrp–/– mice. Among these substances, dantrolene was reported as a BCRPspecific substrate (Enokizono et al., 2008). P-gp and MRPs have been targets for cancer therapy, and many tracers for positron emission tomography (PET) have been developed to study the function of P-gp in vivo (Ishiwata et al., 2007). However, there are few studies of the PET tracers that might enable noninvasive imaging of BCRP-mediated transport

n

Corresponding author. Tel.: + 81 43 206 4041; fax: +81 43 206 3261. E-mail address: [email protected] (T. Fukumura).

0969-8043/$ - see front matter & 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2010.03.012

activity in vivo (Sharma et al., 2005; Kawamura et al., 2009; ¨ Dorner et al., 2009). Additionally, it is not necessarily an effective tracer (Sharma et al., 2005). In this paper, we describe the synthesis of [2-11C-carbonyl]dantrolene (1: Fig. 1) as a tool for the evaluation of brain penetration-mediated BCRP utilized [11C]phosgene, which was produced using an original automated synthesis apparatus (Ogawa et al., 2010).

2. Materials and methods 2.1. General All commercial reagents and solvents were used without further purification, unless otherwise specified. 1H and 13C nuclear magnetic resonance (NMR) spectra were measured using a JEOL JNM-AL300 NMR spectrometer in CDCl3 or DMSO-d6 solution. Chemical shift data for the proton resonances were reported in parts per million relative to internal standard TMS (d0.0). FAB-MS spectra were obtained with a JEOL MStation JMS700 spectrometer. Glycerol was used as a matrix for FAB-MS measurements. A dose calibrator (IGC-3R Curiemeter; Aloka, Tokyo, Japan) was used for all radioactivity measurements unless otherwise stated. High-performance liquid chromatography (HPLC) was performed using a JASCO HPLC system; effluent radioactivity was measured using a NaI (Tl) scintillation detector system. Column chromatography was performed using Wako gel C-200. Analytical thin-layer chromatography (TLC) was performed using TLC plates precoated with Merck Silica gel 60F254 (0.25 mm layer thickness).

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O C NH N N O

2.4. Multi-step manual synthesis of [2-11C-carbonyl]dantrolene

11

O2N

O

Fig. 1. Structure of [2-11C-carbonyl]dantrolene.

2.2. Precursor for the radiosynthesis of [2-11C-carbonyl]dantrolene 2.2.1. Ethyl 2-{2-[5-(4-nitrophenyl)furfurylidene]hydrazino} acetate (4) Ethyl hydrazinoacetate hydrochloride salt (3: 1.5 mmol) dissolved in a mixture of water 15 mL) and MeCN (15 mL) was treated with diisopropylethylamine (1.5 mmol) at 0 1C. This solution was slowly added to a solution of 5-(4-nitrophenyl)-2furaldehyde (2: 1.0 mmol) in MeCN (20 mL) at 0 1C. The mixture was stirred at room temperature overnight. The reaction mixture was diluted with water (50 mL) and cooled to 0 1C. The resulting precipitate was filtered and washed with water. The obtained pellet was dried under reduced pressure to afford 234.5 mg of compound 4 (0.74 mmol, 74.0%) as orange powder. Analytical TLC: Rf ¼0.42 (1:1 EtOAc/hexanes); 1H NMR (CDCl3) d 1.31 (t, 3H, J¼7.0 Hz), 4.06 (s, 2H), 4.24 (q, 2H, J¼7.0 Hz), 6.61 (d, 1H, J¼3.7 Hz), 6.90 (d, 1H, J¼ 3.7 Hz), 7.57 (s, 1H), 7.82 (d, 2H, J¼8.8 Hz), 8.24 (d, 2H, J¼8.8 Hz), NH signal was not observed; 13C NMR (CDCl3) dc 14.2 (q), 50.4 (t), 61.4 (t), 111.0 (d), 111.3 (d), 124.0 (d), 124.3 (d), 128.5 (s), 135.9 (s), 146.4 (s), 151.5 (s), 152.2 (s), 170.7 (s); HR-FABMS: [M+ H] + m/z 318.1109 (m/z 318.1090 calcd. for C15H16N3O5).

2.2.2. 2-{2-[5-(4-Nitrophenyl)furfurylidene]hydrazino} acetamide (5) Hydrazone 4 (827.5 mg, 2.61 mmol) dissolved in a mixture of CH2Cl2 and MeOH was treated with NH3OH at room temperature overnight. After the reaction mixture was evaporated, the resulting residue was purified by open column chromatography (silica gel, THF/CHCl3 ¼5/95) to afford 432.9 mg (1.50 mmol, 57.6%) of compound 5 as vermilion powder. Analytical TLC: Rf ¼0.13 (5: 95 MeOH/CHCl3); 1H NMR (DMSO-d6) d 3.75 (d, 2H, J¼4.8 Hz), 6.67 (d, 1H, J ¼3.7 Hz), 7.13 (brs, 2H), 7.35 (d, 1H, J¼3.7 Hz), 7.48 (s, 1H), 7.87 (brt, 1H, J¼4.8 Hz), 7.92 (d, 2H, J¼8.8 Hz), 8.26 (d, 2H, J¼8.8 Hz); 13C NMR (DMSO-d6) dc 50.4 (t), 109.9 (d), 112.5 (d), 123.6 (d), 124.0 (s), 124.4 (d), 135.7 (s), 145.4 (s), 149.8 (s), 153.4 (s), 171.3 (s); HR-FABMS: [M+ H] + m/z 289.0976 (m/z 289.0937 calcd. for C13H13N4O4).

2.3. One-step manual synthesis of [2-11C-carbonyl]dantrolene [11C]Phosgene for radiosynthesis was prepared from cyclotron-produced [11C]CO2 as described previously (Ogawa et al., 2010). Briefly, [11C]CO2 was converted to [11C]CH4 using a methanizer, and then a mixture of [11C]CH4 and Cl2 gas was passed through a heated quartz tube to afford [11C]CCl4, which was oxidized to [11C]phosgene using a Kitagawa gas detection system. The generated [11C]phosgene was bubbled into a reaction vial containing a solution of compound 5 (2 mM, 500 mL). The aliquots of radioactive mixture were subjected to analytical HPLC (column: inertsil ODS-3, 4.6 mm ID  250 mm, UV at 380 nm; mobile phase: CH3CN/50 mM HCOONH4-HCOOH (40/60) at a flow rate of 1.0 mL/min).

The generated [11C]phosgene was bubbled into a reaction vial containing a solution of compound 4 in CH2Cl2 (2 mM, 500 mL) at  40 1C in a dry ice–acetonitrile bath. After radioactivity reached a plateau, the reaction vial was warmed to room temperature. After adding 7 M NH3 in MeOH solution (100 mL), a solution of 0.1 M NaOMe in MeOH (100 mL) was added to the reaction vial. The aliquots of radioactive mixture were subjected to analytical HPLC (column: inertsil ODS-3, 4.6 mm ID  250 mm, UV at 380 nm; mobile phase: CH3CN/50 mM HCOONH4-HCOOH (40/60) at a flow rate of 1.0 mL/min). 2.5. Multi-step automated radiochemical synthesis For automatic preparation of [2-11C-carbonyl]dantrolene, we used a versatile synthesis system for multiple PET radiopharmaceuticals, which was developed at the National Institute of Radiological Sciences, and created a sequence program in accordance with manual synthesis results (Ogawa et al., 2010; Fukumura et al., 2007). The generated [11C]phosgene was bubbled into a reaction vial containing a solution of compound 4 in CH2Cl2 (2 mM, 500 mL) at  20 to  30 1C. After radioactivity reached a plateau, the reaction vial was heated to 30 1C with an air heater and kept at that temperature for 5 min. After adding 8 M NH3 in MeOH solution (100 mL), the solvent was removed from the reaction mixture under N2 flow at 80 1C. The solution of NaOMe in MeOH/DME (0.1 M, 400 mL, 3:1) was added to the reaction vial, and then the radioactive mixture was subjected to semi-preparative HPLC. The purification was completed on an inertsil ODS-3 column (10 mm ID  250 mm) using a mobile phase of CH3OH/50 mM HCOONH4HCOOH (70/30) at the flow rate of 4.0 mL/min for monitoring radioactivity and UV absorption at 380 nm. The retention time for [2-11C-carbonyl]dantrolene was approximately 8 min. The collected radioactive fraction corresponding to [2-11C-carbonyl]dantrolene was evaporated to dryness under reduced pressure. The residue was dissolved in 3 mL of sterile normal saline containing polysorbate 80 (110 mL) and ethanol (90 mL). The product was collected in a sterile vial. 2.6. Radiochemical purity and specific activity determinations Radiochemical purity was assayed by analytical HPLC (column: inertsil ODS-3, 4.6 mm ID  250 mm, UV at 380 nm; mobile phase: CH3CN/50 mM HCOONH4-HCOOH (40/60)). The retention time for [2-11C-carbonyl]dantrolene was approximately 8.1 min at the flow rate of 1.0 mL/min. The identity was confirmed by co-injecting with the corresponding nonradioactive sample. Radiochemical purity was 498% for [2-11C-carbonyl]dantrolene (n¼6) and the average specific activity was determined to be 46.8 GBq/mmol.

3. Results and discussion 3.1. Synthesis study of [2-11C-carbonyl]dantrolene Firstly, we carried out one-step radiosynthesis of [2-11Ccarbonyl]dantrolene from precursor (5) and [11C]phosgene. Commercially available ethyl hydrazinoacetate hydrochloride salt (3) was treated with 5-(4-nitrophenyl)-2-furaldehyde (2) to afford ester 4 at a 74% yield (Palnitkar et al., 1999). Ester 4 was sufficiently pure ( 498%) to be used directly for the next step of the reaction. Ester 4 was treated with ammonia solution to provide precursor 5 at 57% yield (Scheme 1).

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Synthesis of cold dantrolene was attempted with precursor 5 using triphosgene. The reaction between 5 and triphosgene proceeded at room temperature, and the yield of cold dantrolene was ca. 20%. Then, we attempted to prepare [2-11C-carbonyl]dantrolene by a one-step ring closure reaction of 5 with [11C]phosgene in several solutions, for example, dimethoxyethane (DME), tetrahydrofuran (THF), and CH2Cl2; however, these were unsuccessful. Phosgene is often used as a dehydration reagent to convert amide to the corresponding nitrile (Cotarca and Eckert, 2004). Therefore, a trace amount of [11C]phosgene may have reacted preferentially with amide in hot syntheses. This unexpected result may be explained by deficient nucleophilicity of hydrazone nitrogen atoms due to a low pKa value of NH proton of hydrazone nitrogen atoms compared with amide protons in precursor 5. In other aprotic polar solvents such as N, Ndimethylformamide (DMF) and dimethylsulfoxide (DMSO), even cold dantrolene was not obtained. It is predicted that phosgene had been consumed for formation of Vilsmeier reagent from DMF as well as for formation of dimethylchlorosulfonium chloride as swern’s oxidation reagent from DMSO. By using an ester that would hardly react with phosgene instead of an amide such as compound 5, it was expected that phosgene could react with hydrazone nitrogen atoms. In addition, a hydantoin moiety was often formed by treatment of urea

O

O2 N

O H

+

(2)

through the utilization of acids or bases in solution and on solid phase synthesis (Dewitt et al., 1993; Hanessian et al., 1996; Wilson et al., 1998). Thus, we planned an alternative synthesis route by a three-step reaction utilizing compound 4 as a precursor, as shown in Scheme 2. Additionally, we planned a three-step/one-pot synthesis, because one-pot synthesis is an ideal technique to minimize the reagent, time, and effort used in comparison with the conventional multi-step synthesis, and removes the need for the separation and purification of the intermediate product. This enables the desired product to be obtained by a simple operation. It was necessary to proceed through the following steps to synthesize [2-11C-carbonyl]dantrolene. The first step was the reaction of 4 with [11C]phosgene to synthesize acid chloride (6), the second step was conversion to urea 7, and the third step was cyclization with cleavage of an ethoxy group using a base (Scheme 2). The reaction at each step proceeded smoothly in DME, even though the amount of trapped [11C]phosgene and the yield of [2-11C-carbonyl]dantrolene were unstable (Table 1). However, the presence of an unidentified radioactive by-product that lowered the radiochemical yield of [2-11C-carbonyl]dantrolene was revealed by analytical HPLC. In order to determine the stage at which the by-product was produced, analytical HPLC was carried out at each step. We

O

H HCl H2 N N

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OEt O

(3)

(4) ii

H N N

O

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NH 2 O

(5) O 11C

iii

X

N N

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O2 N

NH O

(1) Scheme 1. Synthesis of precursors and one-step synthesis of (1). Reagents. (i) iPr2NEt, MeCN, H2O; (ii) NH3OH, MeOH, CH2Cl2; (iii)

O

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N NH

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O C NH2 N N OEt 11

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OEt

(6) ii

O

C Cl

O2 N O

O

O

(7)

(8)

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O2 N

O

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NH O

(1) Scheme 2. Multi-step synthesis of (1). Reagents. (i)

11

COCl2, DME or CH2Cl2; (ii) NH3OH, MeOH; (iii) base solution.

11

COCl2, DME.

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determined that the by-product was produced at the first step, where [11C]phosgene was bubbled into a solution of 4. In general, amino acids are converted into N-carboxy-a-amino acid anhydrides (NCAs) via chloroformamides, and DME behaves as a phosgene-equivalent reagent (Cotarca and Eckert, 2004). The treatment of precursor 4 using [11C]phosgene in DME may have produced NCA 8 (Scheme 2). Optimization of the radiolabeling conditions was carried out using CH2Cl2 as a solvent. When compound 4 was treated with [11C]phosgene in CH2Cl2 at room temperature, the ratio of trapped [11C]phosgene and the yield of [2-11C-carbonyl]dantrolene were stable and increased. Further examination revealed that the trapping ratio and yield increased with decreasing temperature of the reaction vial. Better results were obtained by bubbling of [11C]phosgene at  40 1C in CH2Cl2. Under these conditions, the ratio of trapped [11C]phosgene and the yield of [2-11C-carbonyl]dantrolene reached satisfactory levels (Table 1). Urea 7 was

Table 1 Trapping ratio of [11C]phosgene and yield of [2-11C-carbonyl]dantrolene. Solvent

Temp. (oC)

Trapping ratio (%)a

Yield (%)b

DME CH2Cl2 CH2Cl2 CH2Cl2

r.t. r.t. 0  40

47.5 728.7 (n¼22) 54.4 710.8 (n ¼7) 72.3 710.0 (n¼ 4) 82.4 79.7 (n ¼9)

25.4 7 26.4 (n¼ 9) 38.6 7 3.4 (n¼ 2) 69.6 7 5.2 (n¼ 4) 74.6 7 8.4 (n¼ 7)

a b

Determined from ratio of radio activity of reaction vial and waste gas. Determined by radiochromatogram of analytical HPLC after decay correction.

produced rapidly by the addition of ammonia solution at room temperature. The final step, cyclization with cleavage of an ethoxy group, was achieved promptly using NaOMe solution, which was selected as a base due to its ease of preparation. In addition, the [2-11C-carbonyl]dantrolene was obtained in similar yield though KOtBu or NaOtBu solution was used as a substitute of NaOMe solution. On the basis of these results, we attempted to use an automatic synthesizer, because we achieved the synthesis of [2-11Ccarbonyl]dantrolene by a three-step/one-pot synthesis with no difficulty in the shortest possible time.

3.2. Automated synthesis of [2-11C-carbonyl]dantrolene We attempted automated synthesis of [2-11C-carbonyl]dantrolene utilizing an automatic production system for [11C]phosgene as previously reported (Ogawa et al., 2010). However, several problems occurred during the automated radiosynthesis procedure. The generated [11C]phosgene was bubbled into a solution of precursor 4 in CH2Cl2 from  25 to  40 1C. However, the addition of ammonia solution to the reaction vial at these low temperatures did not produce the corresponding intermediate acid chloride (6). Following various examinations, an appropriate temperature (30 1C) and time (5 min) were determined for the formation of 6. The complete elimination of CH2Cl2 in the reaction mixture before the addition of a base solution was necessary because the presence of CH2Cl2 in the reaction mixture disturbed the purification of [2-11C-carbonyl]dantrolene by reverse-phase

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Fig. 2. Preparative chromatograms of the radiosynthesis of [2-11C-carbonyl]dantrolene. The ratios of MeOH/DME are 1:1 (A) and 3:1 (B). The black line shows the UV peak at 380 nm, and the pink line shows radioactive peaks.

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minutes Fig. 3. Analytical HPLC chromatograms of purified [2-11C-carbonyl]dantrolene. The peak at a retention time of 8.58 min represents [2-11C-carbonyl]dantrolene. The upper panel shows the UV chromatogram at 380 nm and the lower panel shows the radioactive chromatogram.

semi-preparative HPLC by causing a shift in retention time, peak tailing, and overlapping. The resulting concentrates were dissolved in NaOMe solution for cyclization reaction; however, the concentrates did not completely dissolve. The presence of DME in NaOMe solution increased not only the solubility of the concentrates but also the formation of unidentified non-radioactive by-products. Fig. 2A and B shows HPLC chromatograms of the purification of [2-11Ccarbonyl]dantrolene, when the ratios of MeOH/DME were 1:1 and 3:1, respectively. When the ratio of MeOH/DME was adjusted to 3:1, the formation of impurities was reduced. The formation of urea and the cyclization with cleavage of an ethoxy group occurred very rapidly, which contributed to shortening of the total synthesis time. Purification and formulation of [2-11C-carbonyl]dantrolene from the radioactive mixture were conducted by reverse-phase semi-preparative HPLC followed by evaporation, dissolution, and filtration. An analytical chromatogram of the final product of [2-11C-carbonyl]dantrolene showed a single product with a retention time of 8.1 min (Fig. 3), although a partially overlapping impurity and a radioactive fraction were observed in the semi-preparative HPLC chromatogram (Fig. 2B). Radiochemical yield at EOB was 34.078.4% with a radiochemical purity of 498%, and the total synthesis time was about 44–49 min. The specific activity was 46.8715.2 GBq/mmol (EOS) (n¼6).

4. Conclusion We have accomplished three-step/one-pot automated synthesis of [2-11C-carbonyl] dantrolene for the first time using [11C]phosgene, which was produced by our improved automated synthetic apparatus with high specific activity and reproducibility. [11C]Phosgene was shown to be a beneficial labeling agent for synthesis of carbonyl compounds such as urea, carbamate, and NCAs. We are hopeful that PET study using [2-11C-carbonyl]dantrolene will be a useful tool for the evaluation of BCRP-mediated transport activity in vivo.

Acknowledgments The authors thank the staff of the Cyclotron Operation Section and the Department of Molecular Probes of the National Institute of Radiological Sciences (NIRS) for their support with the operation of the cyclotron and the production of radioisotopes. This study was partially supported by a consignment grant for the Molecular Imaging Program on Research Base for PET Diagnosis from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT). The authors also wish to thank Mr. Kenji Furutuka for measurement of HR-FABMS. References Bart, J., Hollema, H., Groen, H.J., de Vries, E.G., Hendrikse, N.H., Sleijfer, D.T., Wegman, T.D., Vaalburg, W., van der Graaf, W.T., 2004. The distribution of drug-efflux pumps, P-gp, BCRP, MRP1 and MRP2, in the normal blood–testis barrier and in primary testicular tumors. Eur. J. Cancer 40, 2064–2070. Borst, P., Oude, E.R., 2002. Mammalian ABC transporters in health and disease. Annu. Rev. Biochem. 71, 537–592. Cotarca, L., Eckert, H., 2004. Phosgenations—A Handbook. Wiley-VCH. Dewitt, S.H., Kiely, J.S., Stankovic, C.J., Schroeder, M.C., Reynolds Cody, D.M., Pavia, M.R., 1993. ’’Diversomers’’: an approach to nonpeptide, nonoligomeric chemical diversity. Proc. Natl. Acad. Sci. USA 90, 6909–6913. ¨ Dorner, B., Kuntner, C., Bankstahl, J.P., Bankstahl, M., Stanek, J., Wanek, T., ¨ ¨ Stundner, G., Mairinger, S., Loscher, W., Muller, M., Langer, O., Erker, T., 2009. Synthesis and small-animal positron emission tomography evaluation of 11 [ C]-elacridar as a radiotracer to assess the distribution of P-glycoprotein at the blood–brain barrier. J. Med. Chem. 52, 6073–6082. Doyle, L.A., Yang, W., Abruzzo, L.V., Krogmann, T., Gao, Y., Rishi, A.K., Ross, D.D., 1998. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc. Natl. Acad. Sci. USA 95, 15665–15670. Enokizono, J., Kusuhara, H., Sugiyama, Y., 2007. Effect of breast cancer resistance protein (Bcrp/Abcg2) on the disposition of phytoestrogens. Mol. Pharmacol. 72, 967–975. Enokizono, J., Kusuhara, H., Ose, A., Schinkel, A.H., Sugiyama, Y., 2008. Quantitative investigation of the role of breast cancer resistance protein (Bcrp/Abcg2) in limiting brain and testis penetration of xenobiotic compounds. Drug Metab. Dispos. 36, 995–1002. Fukumura, T., Suzuki, H., Mukai, K., Zhang, M.R., Yoshida, Y., Nemoto, K., Suzuki, K., 2007. Development of versatile synthesis equipment for multiple production of PET radiopharmaceutical. J. Labelled Compd. Radiopharm. 50, s202. Gottesman, M.M., Fojo, T., Bates, S.E., 2002. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat. Rev. Cancer 2, 48–58.

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