New 99mTc(CO)3 Mannosylated Dextran Bearing S-Derivatized

Article pubs.acs.org/molecularpharmaceutics

New 99mTc(CO)3 Mannosylated Dextran Bearing S-Derivatized Cysteine Chelator for Sentinel Lymph Node Detection I. Pirmettis,*,† Y. Arano,‡ T. Tsotakos,† K. Okada,‡ A. Yamaguchi,‡ T. Uehara,‡ M. Morais,§ J. D. G. Correia,§ I. Santos,§ M. Martins,∥ S. Pereira,∥ C. Triantis,† P. Kyprianidou,† M. Pelecanou,⊥ and M. Papadopoulos† †

Institute of Radioisotopes and Radiodiagnostic Products, NCSR “Demokritos”, 15310 Ag. Paraskevi, Athens, Greece Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan § Unidade de Ciências Químicas e Radiofarmacêuticas, ITN, Instituto Superior Técnico, Universidade Técnica de Lisboa, Estrada Nacional 10, 2686-953 Sacavém, Portugal ∥ CICECO, Universidade de Aveiro, Portugal ⊥ Institute of Biology, NCSR “Demokritos”, 15310 Ag. Paraskevi, Athens, Greece ‡

S Supporting Information *

ABSTRACT: The aim of the present study is to synthesize new mannosylated dextran derivative that can be labeled with Tc-99m for potential use in sentinel lymph node detection (SLND). The compound was designed to have a dextran with molecular weight of 10 kDa as a backbone, mannose for binding to mannose receptors of the lymph node and S-derivatized cysteine as a suitable chelator for labeling with [99mTc(H2O)3(CO)3]+ precursor. Reaction of allyl bromide with dextran (MW 11800) yielded the intermediate allyl-dextran (1) with about 40% coupling. Addition of cysteine to allyl-dextran resulted in the S-derivatized cysteine, compound DC15 (2). The final product DCM20 (3) was obtained in good yield after in situ hydrolysis and activation of cyanomethyl tetraacetyl-1-thio-D-mannopyranoside and coupling to DC15. All derivatives were purified by ultrafiltration and characterized by NMR. DC15 and DCM20 were quantitatively labeled with 99mTc (>95% radiochemical purity) using the fac-[99mTc(OH2)3(CO)3]+ precursor and ligand concentration of 1.5 × 10−6 M at neutral pH. Both 99mTc-labeled compounds 99mTc(CO)3−DC15 (6) and 99m Tc(CO)3−DCM20 (7) remained stable after 6 h incubation at 37 °C in the presence of excess histidine or cysteine, as well as even after 20-fold dilution and incubation for 24 h at room temperature. The characterization of the compounds 6 and 7 was performed by comparing their HPLC radiochromatograms with those of their rhenium surrogates Re(CO)3−DC15 (4) and Re(CO)3−DCM20 (5) respectively that were prepared using the precursor [NEt4]2 fac-[ReBr3(CO)3] and characterized by IR and NMR spectroscopy. When injected subcutaneously from the foot pad of mice, 99mTc-labeled mannosylated dextran (7) showed accumulation in the popliteal lymph node (SLN in this model) higher than that of non-mannosylated analogue (6) and the 99mTc-phytate serving as standard. Compound 7 also exhibited lower radioactivity levels at the injection site compared to 99mTc-phytate. The SPECT/CT studies in mice confirmed that 7 accumulated in the popliteal lymph node allowing its clear visualization. The present findings demonstrate that compound 7 (99mTc(CO)3-DCM20) is promising and merits further evaluation as a radiopharmaceutical for sentinel lymph node detection. KEYWORDS: Tc-99m, Re, dextran, mannose, cysteine, sentinel lymph node

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SLN to detect the presence of metastasis. If these first-draining SLNs show no evidence of metastasis, then the unnecessary

INTRODUCTION Sentinel lymph node biopsy (SLNB) is rapidly entering common practice in the management of patients with tumors. Sentinel lymph nodes are the first lymph nodes to receive lymphatic flow as well as metastatic cells from the primary tumor sites. In cases of breast cancer and melanoma, sentinel lymph node detection is followed by excision and biopsy of the © 2012 American Chemical Society

Received: Revised: Accepted: Published: 1681

January 9, 2012 April 11, 2012 April 20, 2012 April 20, 2012 dx.doi.org/10.1021/mp300015s | Mol. Pharmaceutics 2012, 9, 1681−1692

Molecular Pharmaceutics

Article

however, DTPA cannot be considered an ideal chelator to stabilize 99mTc.17,18 Other research attempts to develop SLND agents employ instead of DTPA the MAG-3 chelator15 and, recently, the pyrazolyl-diamine chelator with promising results.16 Aiming at the development of a novel 99mTc-radiopharmaceutical for SLND with improved characteristics, the design and synthesis of a new mannosylated dextran derivative DCM20, 3 (Chart 2), is reported in the present study. In DCM20 the

excision of the remaining lymph nodes is avoided, thus improving the patients’ quality of life.1−6 Sentinel lymph node detection (SLND) in nuclear medicine is performed by injecting small radiolabeled particles (20 to 500 nm) in the area where a tumor is located. 99mTc-labeled colloidal particles like 99mTc-sulfur colloid, 99mTc-antimony sulfur colloid, 99m Tc-rhenium sulfide colloid and 99mTc-HSA are currently being used as radiopharmaceuticals for SLND, while 99mTcphytate has been recently approved in Japan for this purpose.7,8 The radiolabeled particles migrate from the injection site into the lymphatic channel mainly by passive diffusion, and they are cleared from the lymph as foreign matter based on active saturable phagocytosis. Particle size and surface characteristics influence the rate of colloid drainage from the injection site to the dermal lymphatic capillaries and phagocytosis by lymph node macrophages. Particles smaller than 5 nm leak to the bloodstream, whereas particles larger than 100 nm are trapped in the interstitial space, resulting in masking of the SLNs that are usually situated only a few centimeters away from the injection site. Besides the 99mTclabeled colloidal particles, 99mTc-labeled human serum albumin (HSA, 6 nm) has been also applied as a radiopharmaceutical for SLND. Due to its small size 99mTc-labeled HSA rapidly clears from injection sites, however, it travels through the whole lymph node chain to the systemic circulation, a fact that makes the identification of the SLN difficult. Overall, the radiopharmaceuticals currently applied for SLN detection suffer either from slow clearance rate from the injection sites or low residence times in the SLN, or from both of the two, and they are not universally approved by authorities for this specific application. The introduction of mannose molecules to 99mTc-labeled macromolecules like albumin,9,10 polylysine11 and dextrans12−16 (Chart 1) showed that these agents, although of few nano-

Chart 2. Schematic Representation and Group Density of Mannosylated Dextran Having S-Functionalized Cysteine as Chelator for 99mTc, DCM20 and Its Technetium-99m Complex 99mTc(CO)3−DCM20

dextran carries S-derivatized cysteines that can serve as efficient SNO chelators for the fac-[99mTc(CO)3]+ core19−22 and at the same time as anchor points for the attachment of mannose moieties. The complexation of 3 with the fac-[M(CO)3]+ (M = Re, 99mTc) core, the characterization of the resulting complexes Re(CO)3−DCM20 (5) and 99mTc(CO)3−DCM20 (7), and the preliminary biological testing of 7 are also reported.

Chart 1. Schematic Representation and Group Density of Mannosylated Dextran Having DTPA,12,14 MAG,15 and Pyrazolyl-diamine16 as Chelator for 99mTc

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EXPERIMENTAL SECTION Materials and Methods. All laboratory chemicals were reagent grade and were used without further purification. Solvents for high-performance liquid chromatography (HPLC) were HPLC-grade. Dextran-8 (MW 11800 g/mol) was purchased from Serva Electrophoresis GmbH. The cyanomethyl 2,3,4,6-tetra-O-acetyl-1-thio-β-D-mannopyranoside and the starting material (NEt4)2[ReBr3(CO)3] were prepared according to the literature.23−25 Na99mTcO4 was obtained in physiological saline as a commercial 99Mo/99mTc generator eluate (Mallinckrodt Medical BV, The Netherlands, or Ultra-Techne Kow, Fujifilm RI Pharma Co., Ltd., Tokyo, Japan). The radioactive precursor fac-[99mTc(H2O)3(CO)3]+ was prepared using a commercially available IsoLink kit (Mallinckrodt Medical BV, The Netherlands) according to the manufacturer’s instructions or using a homemade kit containing 5.5 mg of NaBH4, 4 mg of Na2CO3 and 10 mg of Na−K tartarate that was purged with CO gas prior to addition of Na99mTcO4, as described elsewhere.26 99mTc-phytate (Fujifilm RI Pharma) was prepared according to the manufacturer’s instructions. IR spectra were recorded on a Nicolet 6700 ATR-IR spectrometer from Thermo Scientific in the region 4000−400 cm−1. NMR spectra were acquired in D2O at 25 °C on a 500 MHz Bruker DRX Avance spectrometer, using two-dimensional 1 H−1H (COSY, NOESY) and 1H−13C (HSQC, HMBC) correlation techniques. HPLC analysis was performed by two

meters in size, can be trapped by the sentinel node in a saturable mode due to their recognition by the mannose receptors of lymph node macrophages. Among them, Lymphoseek, the most studied and promising agent, currently in clinical trials, consists of a dextran backbone (MW 10 kDa), 23 amino, 55 mannose and 8 diethylene triaminepentaacetic acid (DTPA) units per dextran.12−14 The DTPA units serve as attachment sites for labeling the macromolecule with 99mTc, 1682

dx.doi.org/10.1021/mp300015s | Mol. Pharmaceutics 2012, 9, 1681−1692


Article

ammonium persulfate, and the resulting solution was stirred for 4 h at 50 °C under nitrogen atmosphere. The pH was adjusted to 4.0 using 0.1 N NaOH, and the solution was left at room temperature for 24 h. The volume was fixed to 50 mL with 0.02 M sodium acetate buffer pH 4.0, and after filtration (5 μm) the filtrate was transferred into an ultrafiltration cell (model 8400, Millipore Corp, Bedford, MA) fitted with an ultrafiltration membrane (YM03, MW cutoff 3000). The volume was fixed to 250 mL with 0.02 M sodium acetate buffer pH 4.0 and then concentrated to 10 mL by applying gas (dinitrogen) pressure directly to the ultrafiltration cell. Subsequently, the retentate was diluted with 250 mL of 0.1 M bicarbonate buffer, concentrated to 10 mL, as above; the retentate was diluted with 250 mL of deionized water, reconcentrated to 10 mL and finally lyophilized. Yield: 2.00 g (78%). MWcalculated = 16630 g/mol. 1H NMR (D2O, ppm): 5.16 (subst. dextran anomeric H-1), 4.97 (dextran anomeric H-1), 3.98, 3.75 (dextran H-6), 3.98−3.51 (subst. dextran H-4−H-6), 3.90 (dextran H-5), 3.82, 3.76 (OCH2CH2CH2S), 3.79 (cysteine SCH2CH), 3.73 (subst. dextran H-3), 3.71 (dextran H-3), 3.56 (dextran H-2), 3.52 (dextran H-4), 3.39 (subst. dextran H-2), 3.05, 2.97 (cysteine SCH2CH), 2.69 (OCH2CH2CH2S), 1.90 (OCH2CH2CH2S). 13C NMR (D2O, ppm): 177.87 (cysteine CO, broad), 100.41 (dextran anomeric C-1), 98.53 (subst. dextran anomeric C-1), 82.01 (subst. dextran C-2), 76.10 (dextran C-3), 74.11 (dextran C-2), 72.90 (dextran C-5), 72.24 (dextran C-4), 75.12 (subst. dextran C-3), 72.04 (OCH2CH2CH2S), 69.7−65.0 (subst. dextran C-4−C-6), 68.23 (dextran C-6), 56.75 (cysteine SCH2CH), 36.16 (cysteine SCH2CH), 31.52 (OCH2CH2CH2S), 30.64 (OCH2CH2CH2S). Synthesis of DCM20 (3). To a methanolic suspension of cyanomethyl 2,3,4,6-tetra-O-acetyl-1-thio-β-D-mannopyranoside (1.41 g in 33 mL of methanol) was added 2 mL of sodium methanoxide solution (21.6 mg), and the mixture was agitated periodically. After 24 h, 20 mL of the solution was transferred to a dried recovery flask and methanol was removed by rotary evaporation, affording 2-imino-2-methoxyethyl-1-thioβ-D-mannopyranoside as a thick golden syrup. Immediately after, a solution of DC15 (2) (0.18 g) in 7.5 mL of 0.2 M borate buffer (pH 9.0) was added to the flask and left to react for 20 h under periodical stirring. After filtration (5 μm) the filtrate was transferred into an ultrafiltration cell (model 8050, Millipore Corp, Bedford, MA) fitted with an ultrafiltration membrane (YM03). The volume was fixed to 50 mL with 0.1 M bicarbonate buffer and then concentrated to 5 mL by applying gas (dinitrogen) pressure directly to the ultrafiltration cell. Subsequently, the retentate was diluted with 50 mL of deionized water, concentrated to 5 mL, as above (twice), and finally, the retentate was lyophilized. Yield: 0.21 g (88%). MWcalculated = 22270 g/mol. 1H NMR (D2O, ppm): 5.44 (mannose anomeric H-1), 5.16 (subst. dextran anom. H-1), 4.97 (dextran anom. H-1), 4.32 (subst. cysteine SCH2CH), 4.10 (mannose H-2), 3.99, 3.75 (dextran H-6), 3.94−3.72 (mannose H-3−H-6), 3.90 (dextran H-5), 3.89−3.53 (subst. dextran H-3-H-6), 3.81, 3.89 (OCH2CH2CH2S), 3.80 (free cysteine SCH2CH), 3.74 (dextran H-3), 3.58 (dextran H-2), 3.52 (dextran H-4), 3.49, 3.42 (NHCCH2S), 3.40 (subst. dextran H-2), 3.20, 3.05 (subst. cysteine SCH2CH), 3.08, 2.98 (free cysteine SCH2CH), 2.72 (OCH2CH2CH2S), 1.90 (OCH2CH2CH2S). 13C NMR (D2O, ppm): 175.99 (mannose subst. cysteine CO), 174.05 (HNCCH2S), 100.45 (dextran anomeric C-1), 98.60 (subst. dextran anomeric C-1), 87.31 (mannose anomeric C-1), 82.04 (subst. dextran C-2), 76.47 (mannose C-3), 76.12 (dextran C-3), 75.17 (subst. dextran C-3),

systems. System I: Waters 600 chromatography system coupled to both a Waters 991 photodiode array detector and a Gabi gamma detector from Raytest. HPLC solvents consisted of water containing 0.1% trifluoroacetic acid (solvent A) and methanol containing 0.1% trifluoroacetic acid (solvent B). For the radiochemical analysis, a Nucleosil C18 reverse-phase column (10 μm, 250 × 4 mm) was used. The HPLC system started with 100% of A from 0 to 1 min followed by a linear gradient to 30% A (70% B) in 9 min; this composition was held for another 10 min. The flow rate was 1 mL/min. System II: Waters 600E chromatography system coupled to both a Waters 486 UV detector and a GABI gamma detector from Raytest (γ trace for 99mTc). Separations were achieved on a size exclusion column (Shodex SB-803HQ (8 × 300 mm) eluted with water at a flow rate of 1 mL/min. Determination of Glucose Units on Dextran Molecules. The number of sugar units per dextran molecule was determined by the sulfuric acid−phenol colorimetric assay using glucose as standard. Briefly, a 5% phenol solution in H2O (0.55 mL) and concentrated H2SO4 (2.5 mL) were added to glucose solutions (5−50 μg in 0.55 mL of H2O) and the mixture was vortexed. After standing for 30 min at room temperature, absorbance readings were taken at 490 nm and a calibration curve was obtained using the Origin 7.5 software. Based on the calibration curve, the sugar content was subsequently determined in triplicate samples of DC15 (2) and DCM20 (3) containing approximately 25 μg in 0.55 mL of water. The experiment was repeated two times. Synthesis of Allyl Dextran (1). Dextran-8 (10.0 g) was dissolved in 75 mL of distilled water together with 2.5 g of NaOH and 0.1 g of sodium borohydride. The solution was warmed to 50 °C, and allyl bromide (17.5 g, 0.15 mol) was added. The pH was maintained at 11 by addition of 2.5 N NaOH. After 3 h at 50 °C the solution was neutralized (pH 7.0) with acetic acid and dextran was purified by precipitation after addition of ethanol. Further purification was performed by ultrafiltration. The white solid was dissolved in 50 mL of deionized water and filtered through a 5 μm filter, and the filtrate was transferred into an ultrafiltration cell (model 8400, Millipore Corp, Bedford, MA) fitted with an ultrafiltration membrane (YM03, MW cutoff 3000). The volume was fixed to 250 mL with deionized water and then concentrated to 15 mL by applying gas (dinitrogen) pressure directly to the ultrafiltration cell. The retentate was diluted with 250 mL of deionized water, reconcentrated to 10 mL and finally lyophilized. Yield: 9.50 g (86%). MWcalculated = 13030 g/mol. 1 H NMR (D2O, ppm): 5.97 (m, −OCH2CHCH2), 5.37, 5.29 (OCH2CHCH2), 5.14 (subst. dextran anomeric H-1), 4.97 (dextran anomeric H-1), 4.20 (OCH2CHCH2), 3.99, 3.76 (dextran H-6), 3.99−3.53, (subst. dextran H-4−H-6), 3.90 (dextran H-5), 3.75 (subst. dextran H-3), 3.72 (dextran H-3), 3.58 (dextran H-2), 3.53 (dextran H-4), 3.45 (subst. dextran H-2). 13C NMR (D2O, ppm): 136.62 (OCH2CH CH2), 121.66 (OCH2CHCH2), 100.41 (dextran anomeric C-1), 98.55 (subst. dextran anomeric C-1), 81.06 (subst. dextran C-2), 76.12 (dextran C-3), 74.64 (OCH2CH CH2), 74.11 (dextran C-2), 72.89 (dextran C-5), 72.24 (dextran C-4), 75.18 (subst. dextran C-3), 72.3−68.2 (subst. dextran C-4−C-6), 68.23 (dextran C-6). Synthesis of DC15 (2). To a solution of 2.0 g of allyl dextran (1) in 10 mL of distilled water were added 1.43 g of cysteine or 1.86 g of cysteine hydrochloride and 0.12 g of 1683



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74.15 (dextran C-2), 73.75 (mannose C-2), 73.75 −72.25 (mannose C-4- C-5), 72.91 (dextran C-5), 72.9−68.2 (subst. dextran C-4−C-6), 72.25 (dextran C-4), 69.61 (mannose C-6), 68.24 (dextran C-6), 63.49 (OCH2CH2CH2S), 60.14 (mannose subst. cysteine SCH2CH), 56.47 (free cysteine SCH2CH), 36.34 (free cysteine SCH2CH), 36.16 (NHCCH2S), 35.76 (mannose subst. cysteine SCH2CH), 31.73 (OCH2CH2CH2S), 31.05 (OCH2CH2CH2S). Synthesis of Re(I) Complexes. Re(CO)3−DC15 (4). A solution of 2 (55 mg, 3.3 μmol) and [NEt4]2[ReBr3(CO)3] (77 mg, 100 μmol) in 5 mL of water was stirred at 50 °C for 4 h and left overnight at room temperature. Addition of 10 mL of methanol resulted in the precipitation of 4 as a white solid, which was collected and washed with methanol and diethyl ether. Yield: 52 mg (76%). MWcalculated = 20680 g/mol; tR = 11.5 min (RP-HPLC). IR (KBr, cm−1): 3336 (O−H, br), 2916 (C−H, m), 2026, 1892 (CO, s), 1627 (CO, br, s). 1H NMR (D2O, ppm): 5.16 (subst. dextran anomeric H-1), 4.97 (dextran anomeric H-1), 4.47 (complexed cysteine SCH2CH), 3.98, 3.74 (dextran H-6), 3.98−3.55 (subst. dextran H-4−H-6), 3.95, 3.85 (OCH2CH2CH2S attached to complexed cysteine), 3.94 (free cysteine SCH2CH), 3.90 (dextran H-5), 3.81, 3.74 (OCH2CH2CH2S), 3.76 (subst. dextran H-3), 3.71 (dextran H3), 3.56 (dextran H-2), 3.51 (dextran H-4), 3.42 (subst. dextran H-2), 3.14, 3.05 (free cysteine SCH2CH), 3.13 (OCH2CH2CH2S attached to complexed cysteine), 2.9, 3.1 (broad, complexed cysteine SCH2CH), 2.71 (OCH2CH2CH2S), 2.05 (OCH2CH2CH2S attached to complexed cysteine), 1.90 (OCH2CH2CH2S). 13C NMR (D2O, ppm): 197.97, 197.25, 194.90 (carbonyl CO), 183.96 (complexed cysteine CO), 175.35 (free cysteine CO), 100.42 (dextran anomeric C-1), 98.55 (subst. dextran anomeric C-1), 82.06 (subst. dextran C-2), 76.11 (dextran C-3), 75.14 (subst. dextran C-3), 74.14 (dextran C-2), 72.91 (dextran C-5), 72.25 (dextran C-4), 72.25 (OCH2CH2CH2S), 71.10 (OCH2CH2CH2S attached to complexed cysteine), 68.24 (dextran C-6), 72.3−68.2 (subst. dextran C-4−C-6), 60.38 (complexed cysteine SCH2CH), 56.27 (free cysteine SCH2CH), 51.00 (OCH2CH2CH2S attached to complexed cysteine), 35.24 (complexed cysteine SCH2CH), 34.71 (free cysteine SCH2CH), 30.62 (OCH2CH2CH2S), 30.92 (OCH2CH2CH2S attached to complexed cysteine), 31.50 (OCH2CH2CH2S). Re(CO)3−DCM20 (5). A solution of 3 (80 mg, 3.6 μmol) and [NEt4]2[ReBr3(CO)3] (17 mg, 22 μmol) in 5 mL of water was stirred at 50 °C for 4 h and left overnight at room temperature. Addition of 10 mL of methanol resulted in the precipitation of 5 as a white solid, which was collected and washed with a small amount of methanol and diethyl ether. Yield: 67 mg (81%). MWcalculated = 23083 g/mol; tR = 12.0 min (RPHPLC). IR (KBr) (cm−1): 3240 (O−H, br), 2918 (C−H, m), 2019, 1895 (CO, s), 1619 (CO, br, s). 1H NMR (ppm, D2O): 5.43 (mannose anomeric H-1), 5.15 (subst. dextran anom. H-1), 4.97 (nonsubst. dextran anom. H-1), 4.45 (complexed cysteine SCH2CH), 4.31 (mannose subst. cysteine SCH2CH), 4.09 (mannose H-2), 4.04−3.67 (mannose H-3− H-6), 3.98, 3.75 (dextran H-6), 3.98−3.52 (subst. dextran H-4−H-6), 3.91 (free cysteine SCH2CH), 3.89 (dextran H-5), 3.89, 3.76 (OCH2CH2CH2S attached to complexed cysteine), 3.85, 3.79 (OCH2CH2CH2S), 3.73 (subst. dextran H-3), 3.72 (dextran H-3), 3.57 (dextran H-2), 3.53 (dextran H-4), 3.48, 3.41 (NHCCH2S), 3.40 (subst. dextran H-2). 3.12, 3.03 (free cysteine SCH2CH), 3.18, 3.04 (mannose subst. cysteine SCH2CH), 3.10 (OCH2CH2CH2S attached to complexed

cysteine), 3.09, 2.95 (complexed cysteine SCH2CH), 2.71 (OCH2CH2CH2S), 2.07 (OCH2CH2CH2S attached to complexed cysteine), 1.89 (OCH2CH2CH2S). 13C NMR (D2O, ppm): 198.17, 197.17, 194.77 (carbonyl CO), 178.47 (complexed cysteine CO), 176.19 (mannose substituted cysteine CO), 175.82 (free cysteine CO), 174.33 (NH CCH2S), 100.45 (dextran anomeric C-1), 97.25 (subst. dextran anomeric C-1), 87.30 (mannose anomeric C-1), 82.07 (subst. dextran C-2), 76.49−69.62 (mannose C-3−C-6), 76.14 (dextran C-3), 75.20 (subst. dextran C-3), 74.20 (dextran C-2), 73.76 (mannose C-2), 72.93 (dextran C-5), 72.9−68.2 (subst. dextran C-4−C-6), 72.75 (OCH2CH2CH2S), 72.26 (dextran C4), 71.76 (OCH2CH2CH2S attached to complexed cysteine), 68.23 (dextran C-6), 60.37 (complexed cysteine SCH2CH), 60.15 (mannose subst. cysteine SCH2CH), 56.36 (free cysteine SCH2CH), 51.59 (OCH2CH2CH2S attached to complexed cysteine), 36.13 (NHCCH2S), 35.76 (mannose subst. cysteine SCH2CH), 35.19 (free cysteine SCH2CH), 31.76 (OCH2CH2CH2S), 31.52 (complexed cysteine SCH2CH), 31.36 (OCH2CH2CH2S attached to complexed cysteine), 31.06 (OCH2CH2CH2S). Kit Formulation. Forty milligrams of each dextran derivative (2 or 3) was dissolved in 100 mL of water for injection, and amounts of this solution were further diluted with water for injection to achieve a final concentration of 50 to 400 μg per mL. The solutions were filtered using a 0.22 μm Millipore filter for terminal sterilization. Under aseptic conditions 1 mL of the solution was dispensed into 10 mL serum vials and lyophilized at −4 °C for 24 h. At the end of the lyophilization cycle the vials were stoppered under vacuum. Preparation of Technetium-99m Complexes, 99mTc(CO) 3 −DC15 (6) and 99m Tc(CO) 3 −DCM20 (7). The precursor fac-[99mTc(CO)3(H2O)3]+ was prepared using either the Isolink kit (Mallinckrodt Medical BV, The Netherlands) or the homemade kit, and its radiochemical purity was checked by RP-HPLC. Method A. A solution of fac-[99mTc(CO)3(H2O)3]+ (0.5− 1.0 mL, 1−20 mCi), pH 7−8 was added to a capped vial, containing 50−400 μg of each of the dextran compounds 2 or 3. The mixture was incubated at 75 °C for 25 min and then analyzed by HPLC using both HPLC analysis systems. Method B. A vial containing 50 μg of freeze-dried 3 was reconstituted with 1.5 mL of [99mTcO4]− (370−740 MBq), and the solution was added to the Isolink kit formulation (Mallinckrodt Medical BV). The mixture was incubated at 95 °C for 30 min, and after cooling the solution was neutralized with 1 N HCl/PBS and analyzed by HPLC. The stability of the 99mTc complexes at room temperature was studied by HPLC for a period up to 24 h. Stability of 6 and 7 in the Presence of Cysteine and Histidine. To estimate the stability of fac-[99mTc(CO)3] complexes, aliquots of 6 and 7 were 20-fold diluted in phosphate buffer (0.1 M, pH 7.4) containing an excess (100:1) of histidine or cysteine. The samples were incubated at 37 °C, and aliquots were removed periodically and analyzed by HPLC. Physical Characterization. The hydrodynamic diameter and the zeta potential of 3, 5, and the starting dextran were determined in phosphate buffer (0.1 M, pH = 7.4) by DLS using a ZetaSizer Nano ZS from Malvern. Particle size was measured at 25 °C with a 173° scattering angle. The surface charge was determined by electrophoretic mobility using laser doppler velocimetry (LDV) and zeta potential cells. 1684



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Scheme 1. Synthesis of DCM20a

a (i) BrC3H5, NaOH (2.5 M), H2O; (ii) L-cysteine, (NH4)2S2O8, H2O, nitrogen; (iii) 2-imino-2-methoxethyl-1-thio-β-D-mannoside, borate buffer (0.01 M, pH 9).

Biodistribution Studies. Animal studies were conducted in accordance with our institutional guidelines and were approved by Chiba University Animal Care Committee. The preparation mixture of the 99mTc-labeled compound 7 was diluted with saline prior to injection to adjust the concentration of 3 at 2 × 10−12 mol/20 μL. The biodistribution studies were performed by subcutaneous injection of 7 to 5 week old male ddY mice. Twenty microliters of 7 was administered in three groups of three mice each to the rear footpad. After injection, the pad was massaged for 0.5 min. Twenty-five minutes after the injection, a 20 μL aqueous solution of 2% Patent Blue was administered to the footpad to visualize the lymph nodes. Five minutes after the second injection, each animal was sacrificed by decapitation. The popliteal and inguinal lymph nodes, injection site (right paw) and tissues of interest were removed and weighed, and the radioactivity counts were determined with an auto well gamma-counter. Biodistribution studies were also conducted after subcutaneous injection in the mouse rear footpad of 99mTc-phytate or of the preparation mixture of 6 containing 2 × 10−12 mol of 2, as described above. SPECT/CT Imaging. Mice weighing 20 to 25 g were used for SPECT imaging studies. A 20 μL solution of 6 or 7 (10−50 μCi containing 2 × 10−11 mol of 2 or 3) or 99mTc-phytate (50 μCi) was injected subcutaneously in the rear footpad. Two mice were used for each imaging study. After anesthetizing with isofluran, SPECT images were taken with SPECT/CT (FX-3000, Gamma Medica Ideas Inc., Northridge, CA, USA) at 30 min postinjection for 6 and 7, or at 6 h postinjection for 99mTc-phytate, for 30 min.

C-2 carbon is shifted downfield by 6.95 ppm and the H-2 proton is shifted upfield by 0.13 ppm compared to their shifts in unsubstituted dextran. The opposite trend is recorded for the adjacent anomeric site with C-1 being shifted upfield by 1.86 ppm and the anomeric proton H-1 shifted downfield by 0.17 ppm. Comparison in the 1H NMR spectra of the integral of each of the allyl peaks to the total integral of the anomeric protons of both the substituted and the unsubstituted glucose units of dextran (Figure 1A) indicates that approximately 41% of the glucose units are allylated. The ratio of the anomeric signal at 5.14 ppm assigned to the 2-O-allyl substituted glucoses relative to the rest of the anomeric protons all appearing under the 4.97 ppm anomeric peak indicates that approximately 61% of the total allylation takes place at the 2-OH while the rest takes place at other hydroxyls, most likely the 3-OH and 4-OH.29 Addition of L-cysteine quantitatively converted 1 to DC15 2 through thiol−ene “click” reaction31,32 resulting in the incorporation of S-derivatived cysteines in the dextran molecule. The reaction proceeded quantitatively as witnessed by the disappearance of the three resonances of the allyl groups and the appearance of characteristic peaks belonging to the propylene ether chain at 2.69 and 1.90 ppm (Figure 1B). The βCH2 protons of the introduced cysteine moiety at 3.05 and 2.97 ppm are clearly located in the 1H spectrum and correlate (Figure S1 in the Supporting Information) with a peak at 3.79 ppm hidden under the glucose peaks assigned to the αCH cysteine proton. The chemical shifts of S-derivatized cysteinyl protons are very close to those reported for the αCH and βCH2 protons of S-propyl cysteine21 in CD3OD and S-benzyl cysteine in D2O (our data). In the next step, reaction of 2 with freshly prepared 2-imino2-methoxyethyl-1-thio-β-D-mannoside solution yielded the mannosylated dextran 3 (DCM20) in which mannose groups are introduced in high yield through formation of an amidino linkage with the amino groups of S-derivatized cysteines. In the 1 H NMR spectrum of 3 characteristic peaks belonging to the anomeric (H-1) and the H-2 protons of mannose (Figure 1C) provide direct evidence for the presence of mannose in the sample with the rest of the protons overlapping with the glucose protons of dextran. In the 1H−13C long-range correlation spectra (HMBC) the NHC amidino carbon at 167.43 ppm correlates with the αCH of cysteine, proving the existence of the amidino linkage between cysteine and mannose. Upon mannosylation, the αCH of cysteine group is shifted downfield by 0.53 ppm relative to its position in 2 and becomes visible in the 1H spectrum (Figure 1C) while the βCH2 protons are also shifted downfield by an average of 0.1 ppm. Based on the intensity of mannose-related peaks relative

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RESULTS Synthesis and Characterization of Mannosylated S-Derivatized Dextran DCM20 (3). The mannosylated dextran 3 containing free S-derivatized cysteine moieties was prepared in three steps as depicted in Scheme 1. In all steps the products were purified through ultrafiltration and their identity was established by full 1H and 13C NMR assignments based on the combined analysis of a series of 1H−1H and 1H−13C correlation experiments. In the first step, reaction of dextran (MW 11800, 73 glucopyranose units) with allyl bromide in excess following the published procedure27 yielded the intermediate allyl dextran 1. The incorporation of the allyl group was evident in the 1H NMR spectrum by the presence of three multiplets at 5.97, 5.33, and 4.20 ppm (Figure 1A). Allyl substitution is mainly expected to occur at the 2-OH of the dextran glucose units being the most reactive site in the sugar moiety,28 and, indeed, characteristic 1H and 13C shifts associated with substitution at this site28−30 are present in the NMR spectra. Specifically, the 1685



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Synthesis and Characterization of the Rhenium Complexes 4 and 5. Both dextran derivatives 2 and 3, bearing S-derivatized cysteines as SNO chelators, reacted with equivalent amounts of the precursor [NEt4]2 fac-[ReBr3(CO)3] in aqueous solution leading to the corresponding rhenium derivatives 4 and 5 (Scheme 2). In the IR spectra of 4 and 5 the strong bands at 2026/1892 cm −1 and 2019/1895 cm−1 respectively, attributed to the ν(CO) stretching modes, denote the presence of the [Re(CO)3]+ core. Furthermore, in the 13C NMR spectra of 4 and 5 three peaks attributed to the carbonyl groups of the fac-[Re(CO)3]+ core can be clearly seen in the range 198−195 ppm in agreement with other SNO complexes in the literature.19,34,35 Analysis of the NMR spectra was first effected for the Re(CO)3+ complex 4 of the non-mannosylated derivative 2, which is expected to give simpler NMR spectra and facilitate the delineation of the mode of coordination of the S-derivatized cysteine moieties. Upon complexation, broadening of the peaks of the methylene protons adjacent to the S atom of cysteine (i.e., the βCH2 of cysteine and the OCH2CH2CH2S of the propylene chain) takes place, becoming barely visible; the peaks rise from the baseline and become evident as the temperature of the sample is raised (Figure 2). This broadening provides a solid sign for the coordination of the S atom of cysteine to the Re(CO)3+ core.19,21,36 The downfield shifts of the αCH proton of cysteine and of the methylene OCH2CH2CH2S group of the propylene chain by 0.68 and 0.44 ppm respectively, and of their corresponding carbons by 3.6 and 3.5 ppm, as well as the downfield shift of the cysteine carboxyl CO by 6 ppm are also in agreement with SNO coordination of the S-derivatized cysteines with the Re(CO)3+ core19,21 and our data on the shifts of S-benzyl-cysteine in methanol-d4. The slight upfield shift of the βCH2 protons of cysteine by approximately 0.05 ppm on average is also in agreement with literature data on Re(CO)3+ complexes of cysteine.19,21 Complex NMR spectra and broadened peaks are associated in the literature with S-derivatized L-cysteine complexes and attributed to the fact that the thioether S-atom represents a prochiral center generating diastereomers upon complexation, and furthermore, to the presence of rotamers that depending on the bulkiness of the S-substituent may have long enough life span to cause broadening of the NMR peaks. However, it should be noted than in our case the 1H chemical shifts of the coordinated cysteine are very close to those reported in CD3OD for the well-characterized through X-ray crystallography major isomer of the S-propyl-L-cysteine with the Re(CO)3+ core where the thioether group is anti to the coordinated amine group.21 Based on these data and the lack of any signals in the NMR spectra of 4 clearly attributable to a second isomer, it appears plausible that coordination of the S-derivatized cysteines takes place in a fashion similar to the above mode leading to one major isomer. In this case, restriction of rotation of the S-propyl chain attached to the very bulky dextran in combination with the overall polymeric nature of 4 may account for the broadness of the NMR peaks. The same structural features are present in the spectra of complex 5 indicating that complexation of the fac-[Re(CO)3]+ core takes place through the SNO atoms of the S-derivatized cysteines. No structural features are present in the spectra that could indicate the presence of alternative type of complexation, which even if present would account for a small percentage of the metalated product. Based on the intensity of the αCH peak of the Re-coordinated cysteine (Figure 3), it can be estimated

Figure 1. 1H spectra (range δH 6.15−1.75) of (A) the allyl derivative 1, (B) the S-derivatized cysteinyl dextran 2 (DC15), and (C) the mannosylated dextran 3 (DCM20) in D2O at 25 °C.

to other peaks in the 1H spectra (e.g., the propylene chain peaks) the extent of cysteine mannosylation appears to be approximately 81%. The sugar content of 2 and 3 was estimated by the sulfuric acid−phenol assay.33 In the case of 2, 73 ± 2 glucose units were found, while in the mannosylated compound 3, 94 ± 3 sugar units were measured. The additional sugar content is attributed to the presence of mannose units, and is in agreement with the extent of mannosylation determined by NMR. 1686



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Scheme 2. Synthesis of Rhenium and Technetium-99m Complexesa

a

(i) [NEt4]2 fac-[ReBr3(CO)3] in H2O at 50 °C for 5 h; (ii) fac-[99mTc(CO)3(H2O)3]+ 25 min at 70 °C.

Figure 3. 1H spectrum (range δH 5.60 - 1.70) of the Re derivative of DCM20 5 (Re(CO)3−DCM20) in D2O at 25 °C. Note that the peak at 2.07 ppm, assigned to the middle OCH2CH2CH2S methylene group of the propylene chain having the S-atom coordinated to Re, coincides with one unidentified peak commonly present in the spectra of and tentatively assigned to S-oxidized compounds; this peak therefore cannot be used for estimation of the extent of complexation of cysteines.

Physical Characterization of Compounds 3 and 5. The hydrodynamic diameter and the zeta potential of 3, 5 and starting dextran were determined in phosphate buffer (0.1 M, pH 7.4) in order to mimic the physiological conditions. Table 1 summarizes the final average composition of the dextran derivatives, their physical parameters and the calculated molecular weight for each derivative. Figure 4 shows the size distribution histograms of starting dextran, as well as of compounds 3 and 5. The hydrodynamic diameter of the particle increases slightly with the modifications introduced on the dextran backbone. Addition of 30 propylene-S-cysteine groups, 24 of them being mannosylated, resulted in a nanocompound

Figure 2. 1H spectra (range δH 5.40−1.60) of the Re derivative of DC15 4 (DC15-Re) in D2O at (A) 25 °C and (B) 55 °C.

that on average 2−3 fac-[Re(CO)3]+ cores are coordinated per molecule of dextran. 1687



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Table 1. Group Density, Hydrodynamic Diameter, Zeta Potential and Calculated Molecular Weight of Dextran, 3, 4, and 5 group density (units/mol dextran) compound dextran allyl dextran (1) DC15 (2) DCM20 (3) Re(CO)3−DC (4) Re(CO)3−DCM (5) a

allyl

cysteine

mannose

diama (nm)

zeta potentiala (mV)

MW calcdb (g/mol)

24 0 24

4.7 ± 0.3 nd nd 6.5 ± 0.5 nd 8.3 ± 0.5

−9.9 ± 0.5 nd nd −6.3 ± 0.1 nd −6.3 ± 0.1

11800 13030 16630 22270 20680 23083

30 30 6 30 6

Mean ± SD. bBased on NMR estimation.

based on a comparative chromatographic study (RP- and SECHPLC) using as reference the analogous Re complexes 4 and 5 respectively. As an example, Figure 5 shows the RP- and SECHPLC chromatograms obtained for 7 (γ-detection) and 5 (UVdetection). Periodic HPLC analysis of 6 and 7 for up to 24 h did not show any significant changes in the chromatograms, even after 1:20 dilution in saline and PBS suggesting that the 99m Tc-complexes are stable. The in vitro stability of 6 and 7 toward transchelation was also studied by incubating with a large excess of the amino acids cysteine or histidine that display high affinity for the fac-[99mTc(CO)3]+ synthon. In both cases, the 99mTc-complexes presented high radiochemical purity (≥90%), even after long incubation times, as shown in Figure 6 for histidine. Biological Evaluation. The biological evaluation of the technetium-99m labeled mannosylated dextran 99mTc(CO)3− DCM20 (7) was performed by biodistribution studies and SPECT/CT imaging in mice after subcutaneous injection in murine footpad. For comparison purposes the non-mannosylated analogue 99mTc(CO)3−DC15 (6) and 99mTc-phytate, a radiopharmaceutical approved in Japan for SLND, were also tested under the same conditions. For the biodistribution studies 20 μL of the preparation solution of 6 and 7, containing 2 × 10−12 mol of 2 and 3 respectively, was administered in mice, and the results are shown in Figure 7. The uptake in popliteal lymph node (SLN in this case) for the mannosylated compound 7 is 9.2, 7.8 and 7.0% ID at 0.5, 1, and 6 h postinjection respectively, while for the 99mTc-phytate the uptake is 3% ID at all time points (Figure 7C). The radioactivity levels at the injection site after injection of 7 dropped to 67% at 30 min p.i. and then to 55% ID at 1 h remaining stable for up to 6 h (Figure 7D). For 99mTc-phytate, 80% of the injected dose was measured at the injection site at all times studied. The radioactivity levels in the blood and liver were very low for both 7 and 99mTc-phytate (Figure 7A,B). The non-mannosylated compound 6 showed fast clearance of radioactivity from the injection site (Figure 7D), however, only marginal uptake in the popliteal lymph node was observed (Figure 7C) accompanied with high radioactivity levels in the blood (Figure 7A). SPECT/CT images of mice after subcutaneous injection in the footpad of 7 or 6 (containing 2 × 10−11 mol of 3 or 2 respectively) or 99mTc-phytate are shown in Figure 8. The popliteal lymph node (SLN in this case) is delineated in the SPECT/CT image of the mouse 6 h after injection of 99mTcphytate (Figure 8C), while most of the radioactivity remains in the injection site, resulting in a ratio of injection site to SLN of 70.4. In the SPECT/CT image taken 30 min after injection of the mannosylated compound 7, the SLN is clearly delineated (Figure 8A). The high uptake in the SLN together with the faster injection site clearance resulted in a ratio of injection site

Figure 4. Hydrodynamic size of starting dextran (A), 3 (B) and 5 (C), determined by dynamic light scattering (detection angle of 173°).

(3) with diameter 6.5 ± 0.5 nm. Its coordination with the rhenium tricarbonyl unit resulted in a slight increase of the hydrodynamic diameter (8.3 ± 0.5 nm) of the resulting nanocompound (5). Radiolabeling. The synthesized dextrans 2, 3 were labeled with 99mTc using the [99mTc(CO)3(OH2)3]+ precursor at ligand concentration of 10−5−10−6 M leading to radiolabeled dextrans 6 and 7 respectively. In particular DCM20 (3) could be quantitatively labeled by dissolving 50 μg in 1.5 mL of 99m TcO4−, transferring the solution into the Isolink kit and boiling the resulting solution for 30 min. In each case, HPLC analysis using C18 column showed the absence of any 99m TcO4− (tR = 3.5 min) and the quantitative (>95%) conversion of the precursor (tR = 4.5 min) to a single radiolabeled product, 6 (tR = 11.9) or 7 (tR = 12.5 min). Analogous results were obtained by using a size exclusion column with the 99mTclabeled dextrans 6 and 7 eluting at 6.0 and 5.9 min respectively. The characterization of the 99mTc-labeled dextrans 6 and 7 was 1688



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Figure 5. Chromatograms (γ-detection) of 99mTc(CO)3−DCM20, 7, obtained by RP-HPLC (A) and SEC-HPLC (C) together with the chromatograms of its Re congener 5 (UV-detection), obtained by RP-HPLC (B) and SEC-HPLC (D).

pyrazolyl-diamine for coordinating 99mTc. The synthesis of these nanocompounds is carried out in three major steps. In the first one, amino terminal leashes are attached to the hydroxyl units of dextran by the addition of cysteamine to allyl dextran. In the second step the chelator activated is coupled to the amino terminal leashes and, subsequently, in the third step mannosylation of most of the remaining amino groups takes place. In the case of MAG3,15 mannosylation preceded the coupling of the chelator, affording a compound with higher mannosylation degree. In the present study, the development of a novel mannosylated dextran derivative DCM20, 3, that can be labeled with 99mTc using the fac-[99mTc(OH2)3(CO)3]+ precursor, is reported. Instead of cysteamine, which is used in the synthesis of Lymphoseek and other mannosylated dextrans for attaching the mannose and the chelator, cysteine was used for the preparation of our compound. This modification provides the amino group of S-derivatized cysteine for the coupling of mannose while the remaining non-mannosylated cysteine groups serve as SNO chelators for the [99mTc(CO)3]+ core. Employment of cysteine instead of cysteamine reduces the overall synthetic steps since there is no need for the synthesis, activation and coupling of an additional bifunctional agent. Synthesis of 3 proceeded smoothly, with about 80% yield in each step. The products were purified by ultrafiltration, which proved especially efficient in the removal of cysteine and activated mannose reactants. Based on NMR spectroscopy and in agreement with the sugar content determination, the new dextran derivative DCM20 (3, MWcalculated 22270) contains on average 24 mannoses while 6 S-derivatized cysteines remain free for labeling with the fac-[99mTc(CO)3]+ core.

Figure 6. In vitro stability of 99mTc(CO)3−DC15, 6 (square), and 99m Tc(CO)3−DCM20, 7 (diamond), in the presence of excess histidine, at different time points. Histidine challenge [His] = 870 μM, 37 °C.

to SLN of 4.7. The second and third lymph nodes, accumulating lesser amount of radioactivity, could also be visualized with an injection site to SLN ratio of 15.2 and 49.3 respectively. The non-mannosylated compound 6 registered faint radioactivity at the SLN 30 min after injection (Figure 8B). About 50% of the radioactivity was cleared from the injection site and accumulated in the urinary bladder, resulting in a ratio of injection site to SLN of 47.3.

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DISCUSSION Mannosylatate dextrans labeled with 99mTc have been explored for sentinel lymph node detection. These nanocompounds (7− 10 nm) consist of a 10 kDa dextran backbone, several mannose units for recognition by the mannose receptors of the lymph node macrophages, and a chelating agent like MAG3, DTPA or 1689



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Figure 7. Biodistribution of radioactivity after subcutaneous injection of 99mTc(CO)3−DCM20, 7 (diamond), 99mTc(CO)3−DC15, 6 (square) and 99m Tc-phytate (triangle) in the blood (A), liver (B), popliteal lymph node (C), and injection site (D).

studies provided clear evidence for coordination taking place through the SNO atoms of the non-mannosylated cysteines. Dynamic light scattering (DLS) studies showed that the average particle size of 3 is 6.5 ± 0.5 nm while that of its Re(CO)3+ complex 5 is 8.3 ± 0.5 nm. Based on these values, it can be deduced that the analogous 99mTc radioactive nanocompound 7 may display a hydrodynamic diameter in the range 6.5−8.3 nm. The hydrodynamic diameter of 3 and 5 is similar to the DLS values found for DTPA-mannosyl-dextran12 (7.1 ± 0.9 nm), MAG3-mannosyl-dextran15 (5.5 ± 2.4 nm), and pyrazolyl-mannosyl-dextran16 (7.0 ± 0.7) and much smaller than the colloidal radiopharmaceuticals in clinical use (80−100 nm). Zeta potential measurements indicated a negative charge for 3 (−6.3 ± 0.1 mV). Further measurements have shown that complexation of 3 with the Re(CO)3+ core does not affect the charge of the final compound 5 (−6.3 ± 0.1 mV). The mannosylated compound 3, as well as its non-mannosylated 2 derivative, were successfully labeled with 99mTc affording compounds 99mTc(CO)3−DC15 (6) and 99mTc(CO)3−DCM20 (7) respectively, in high radiochemical yields (95%) even at low ligand concentration (1.5 × 10−6 M) using the fac-[99mTc(OH2)3(CO)3]+ precursor. To our knowledge this is the lowest concentration reported for the preparation of 99m Tc-labeled dextrans. The labeling was also performed in one pot by mixing and boiling for 30 min 50 μg of freeze-dried 3 with 1.5 mL of pertechnetate into the Isolink vial, demonstrating the feasibility of developing a single vial kit containing all the required ingredients for labeling. Both labeled compounds remained stable under diluted conditions and in the presence of excess histidine or cysteine. All the above parameters are very important because the compound aims to target mannose receptors on macrophages of lymph nodes. The preliminary biological testing of the mannosylated compound 99mTc(CO)3−DCM20, 7, in mice has shown rapid and high accumulation in the popliteal lymph node (9.2% ID at 30 min p.i.) that remains almost stable up to 6 h (7.0% ID). In addition, fast clearance from the injection site was observed (67% ID at 30 min p.i.). Because our data cannot be directly compared with the reported values for other radiotracers for SLND, such as filtered 99mTc-labeled sulfur colloid or [99mTc]DTPA-mannosyl-dextran (Lymphoseek) as these compounds were evaluated using different animal models, a comparative study with the 99mTc-phytate, a radiopharmaceutical

Figure 8. SPECT/CT images in mice after subcutaneous injection of 99m Tc(CO)3−DCM20, 7 (A), 99mTc(CO)3−DC15, 6 (B) and 99mTcPhytate (C). IS, injection site; SLN, sentinel (popliteal) lymph node; 2nd, secondary (inguinal) lymph node; 3rd, third (lumber) lymph node; B, bladder.

For the investigation of the coordination of 3 with the fac[99mTc(CO)3]+ core, macroscopic amounts of the rhenium complexes of both 2 (that contains no mannose) and 3 were prepared. Rhenium is the group VIIB congener of technetium and is often used as a nonradioactive alternative to technetium for large scale synthesis and structural characterization. Reaction of 2 or 3 with the [NEt4]2[ReBr3(CO)3] precursor led to the efficient formation of complexes 4 and 5 respectively (Scheme 2). Infrared spectroscopy of 4 and 5 showed the presence of the fac-[Re(CO)3]+ core, and NMR spectroscopic 1690



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Education, Culture, Sports, Science and Technology, Japan. M. Morais thanks the Portuguese Science Foundation (FCT) for a PhD grant (SFRH/BD/48066/2008).

which has been recently approved in Japan for SLND, was performed (Figure 7). The study showed that the mannosylated compound 99mTc(CO)3−DCM20 7 registered faster clearance of radioactivity from the injection site and about 3-fold higher uptake in the popliteal lymph node at all times studied. The SPECT images of mice (Figure 8) confirmed the ability of 7 to clearly visualize the SLN. Its rapid elimination rate from the injection site was also confirmed. The second and third lymph nodes, accumulating lesser amount of radioactivity, could also be visualized. The visualization of the second and the third lymph nodes might be probably attributed to the saturation of the mannose receptors by the injected amount of 3 (2 × 10−11 moles) in combination with the small size of the mouse popliteal lymph node (0.002 g). Thus, it can be reasonably assumed that this will not be observed in bigger animals or humans where the size of the lymph node is 0.2 to 2 g. The hypothesis of interaction of 7 with mannose receptors is supported by the low popliteal uptake of the non-mannosylated analogue 6.

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(1) Keshtgar, M. R. S.; Ell, P. J. Sentinel lymph node detection and imaging. Eur. J. Nucl. Med. 1999, 26 (1), 57−67. (2) Keshtgar, M.; Zaknun, J. J.; Sabih, D.; Lago, G.; Cox, C. E.; Leong, S. P. L.; Mariani, G. Implementing sentinel lymph node biopsy programs in developing countries: Challenges and opportunities. World J. Surg. 2011, 35 (6), 1159−1168. (3) Morton, D. L.; Chan, A. D. The concept of sentinel lymph node localization: how it started. Semin. Nucl. Med. 2000, 30 (1), 4−10. (4) Shayan, R.; Achen, M. G.; Stacker, S. A. Lymphatic vessels in cancer metastasis: bridging the gaps. Carcinogenesis 2006, 27 (9), 1729−1738. (5) Veronesi, U.; Galimberti, V.; Zurrida, S.; Pigatto, F.; Veronesi, P.; Robertson, C.; Paganelli, G.; Viale, G. Sentinel lymph node biopsy as an indicator for axillary dissection in early breast cancer. Eur. J. Cancer 2001, 37 (4), 454−458. (6) Leong, S. P. L. The role of sentinel lymph nodes in malignant melanoma. Surg. Clin. North Am. 2000, 80 (6), 1741−1757. (7) Wilhelm, A. J.; Mijnhout, G. S.; Franssen, E. J. F. Radiopharmaceuticals in sentinel lymph node detection - an overview. Eur. J. Nucl. Med. 1999, 26 (Suppl.), S36−S42. (8) Eshima, D.; Fauconnier, T.; Eshima, L.; Thornback, J. R. Radiopharmaceuticals for lymphoscintigraphy: Including dosimetry and radiation considerations. Semin. Nucl. Med. 2000, 30 (1), 25−32. (9) Takagi, K.; Uehara, T.; Kaneko, E.; Nakayama, M.; Koizumi, M.; Endo, K.; Arano, Y. 99mTc-labeled mannosyl-neoglycoalbumin for sentinel lymph node identification. Nucl. Med. Biol. 2004, 31 (7), 893− 900. (10) Jeong, J. M.; Hong, M. K.; Kim, Y. J.; Lee, J.; Kang, J. H.; Lee, D. S.; Chung, J.-K.; Lee, M. C. Development of 99mTc-neomannosyl human serum albumin (99mTc-MSA) as a novel receptor binding agent for sentinel lymph node imaging. Nucl. Med. Commun. 2004, 25 (12), 1211−1217. (11) Vera, D. R.; Wisner, E. R.; Stadalnik, R. C. Sentinel node imaging via a nonparticulate receptor-binding radiotracer. J. Nucl. Med. 1997, 38 (4), 530−535. (12) Vera, D. R.; Wallace, A. M.; Hoh, C. K.; Mattrey, R. F. A synthetic macromolecule for sentinel node detection: 99mTc-DTPAMannosyl-Dextran. J. Nucl. Med. 2001, 42 (6), 951−959. (13) Hoh, C. K.; Wallace, A. M.; Vera, D. K. Preclinical studies of 99m Tc-DTPA-mannosyl-dextran. Nucl. Med. Biol. 2003, 30 (5), 457− 464. (14) Wallace, A. M.; Hoh, C. K.; Darrah, D. D.; Schulteis, G.; Vera, D. R. Sentinel lymph node mapping of breast cancer via intradermal administration of Lymphoseek. Nucl. Med. Biol. 2007, 34 (7), 849− 853. (15) Vera, D. R.; Wallace, A. M.; Hoh, C. K. [99mTc]-MAG3Mannosyl-Dextran: a receptor-binding radiopharmaceutical for sentinel node detection. Nucl. Med. Biol. 2001, 28 (5), 493−498. (16) Morais, M.; Subramanian, S.; Pandey, U.; Samuel, G.; Venkatesh, M.; Martins, M.; Pereira, S.; Correia, J. D.G.; Santos, I. Mannosylated Dextran Derivatives Labeled with fac-[M(CO)3]+ (M = 99m Tc, Re) for Specific Targeting of Sentinel Lymph Node. Mol Pharmaceutics 2011, 8 (2), 609−620. (17) Eckelman, W. C. Radiolabeling with technetium-99m to study high-capacity and low-capacity biochemical systems. Eur. J. Nucl. Med. 1995, 22 (3), 249−263. (18) Liu, G.; Hnatowich, D. J. Labeling Biomolecules with Radiorhenium - a Review of the Bifunctional Chelators. Anticancer Agents Med. Chem. 2007, 7, 367−377. (19) Van Staveren, D. R.; Benny, P. D.; Waibel, R.; Kurz, P.; Pak, J.K.; Alberto, R. S-Functionalized Cysteine: Powerful Ligands for the Labelling of Bioactive Molecules with Triaquatricarbonyltechnetium99m(1+) ([99mTc(OH2)3(CO)3]+). Helv. Chim. Acta 2005, 88 (3), 447−460.

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CONCLUSIONS The new mannosylated nanocompound DCM20 (3) has a dextran backbone, S-derivatized cysteine as a suitable chelator for labeling with the [99mTc(CO)3]+ core, and mannose for binding to the lymph node mannose receptors. The design of 3 reduces the synthetic steps and facilitates a large-scale synthesis. The new compound provides stable 99mTc-labeled mannosylated dextran with high radiochemical yield (>95%) and high specific activity. The biodistribution and SPECT/CT studies showed that 99mTc(CO)3−DCM20 (7) exhibits fast injection site clearance, high uptake and retention in the sentinel lymph node. These attractive chemical and biological features, as well as the “one pot” labeling kit, justify further evaluation of 99mTc(CO)3−DCM20 as a new radiopharmaceutical for sentinel lymph node detection.

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ASSOCIATED CONTENT

S Supporting Information *

1

H−1H correlation spectrum of 3 and 1H−13C correlation spectra of 3 and 6. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Institute of Radioisotopes and Radiodiagnostic Products, NCSR “Demokritos”, 15310 Ag. Paraskevi, Athens, Greece. E-mail: [email protected]. Phone: +30 2106503921. Fax: +30 2106503829. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is part of the CRP “Development of 99mTc radiopharmaceuticals for sentinel node detection and cancer diagnosis” of the IAEA, which is acknowledged. The authors also wish to thank both John J. Zaknun and Ambi M. R. Pillai from the NA department of the IAEA, Vienna, for their role in initiating this cooperative research activity (CRP) on sentinel node imaging. This work was supported in part by a Special Fund for Education and Research (Development of SPECT Probes for Pharmaceutical Innovation) from the Ministry of 1691



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New 99mTc(CO)3 Mannosylated Dextran Bearing S-Derivatized

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