Injection Site Radioactivity of 99mTc-Labeled Mannosylated Dextran

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On the Injection Site Radioactivity of 99mTc-Labeled Mannosylated Dextran for Sentinel Lymph Node Mapping Aiko Yamaguchi, Hirofumi Hanaoka, Ioannis Pirmettis, Tomoya Uehara, Yoshito Tsushima, Minas Papadopoulos, and Yasushi Arano Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp500592e • Publication Date (Web): 25 Nov 2014 Downloaded from http://pubs.acs.org on November 29, 2014

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Molecular Pharmaceutics

On the Injection Site Radioactivity of 99mTc-Labeled Mannosylated Dextran for Sentinel Lymph Node Mapping Aiko Yamaguchi,†,‡ Hirofumi Hanaoka,†,‡ Ioannis Pirmettis,§ Tomoya Uehara,† Yoshito Tsushima,‡ Minas Papadopoulos,§ Yasushi Arano†,*

Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan ‡

Gradiate School of Medicine, Gunma University, 39-22 Showa-machi 3-chome, Maebashi 371-

8511, Japan §

Institute IPRETEA, NCSR “Demokritos”, 15310 Ag. Paraskevi Athens, Greece

Keywords: sentinel lymph node, injection site, mannosylated dextran, mannose receptor, macrophage, dendritic cell.

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ABSTRACT

The high and persistent radioactivity at the injection site hinders the accuracy and expansion of sentinel lymph node (SLN) mapping. We investigated the mechanism underlying the undesirable radioactivity after subcutaneous injection of

99m

Tc-labeled mannosylated dextran (99mTc(CO)3-

DCM20), a SLN mapping agent targeting mannose receptors on macrophages and dendritic cells, in a mouse model. Biodistribution studies were performed 1 h after subcutaneous injection of 99m

Tc(CO)3-DCM20 from the rear footpad of mice in the presence of varying molar amounts of

DCM20 or DC15, a modified dextran without mannose. Biodistribution studies were also conducted after subcutaneous injection of [125I]radioiodinated mannosyl-neoglycoalbumin (125INMA) from the rear footpad. The distribution of fluorescence-labeled DCM20 and DC15 at the injection site was also compared 1 h after subcutaneous injection by immunofluorescent histochemistry. The radioactivity levels of 99mTc(CO)3-DCM20 at the injection site and popliteal lymph node, a SLN in this model, decreased with an increase in molar amounts of DCM20, whereas no significant changes in biodistribution were observed after injection of DCM20 with varying molar amounts of DC15.

125

99m

Tc(CO)3-

I-NMA exhibited rapid elimination of

radioactivity from both the popliteal lymph node and the injection site. The fluorescence-labeled DCM20 co-localized well with CD68-positive cells such as macrophages and dendritic cells at the injection site. While partial co-localization was observed between DC15 and CD68-positive cells, the signal intensity was very weak. These findings suggest that specific binding of 99m

Tc(CO)3-DCM20 to the mannose receptor on macrophages and dendritic cells would be

responsible for the sustained radioactivity levels at the injection site. These results also imply that discriminated blockage of 99mTc(CO)3-DCM20 binding to mannose receptors at the injection sites would reduce the radioactivity at the injection site.


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Introduction Sentinel lymph node (SLN) mapping after peritumoral injection of technetium-99m (99mTc) labeled colloidal particles has become the standard of care for melanoma and breast cancer patients with non-clinically detectable metastases 1, 2. The application of this procedure to other types of tumors, however, remains limited, since high radioactivity levels at the injection site hinder identification of SLN in tumor types where lymphatic drainage is more complex and SLNs are within close proximity to other nodes or the primary tumor 3-6. Efforts have been made to overcome the drawbacks, and mannose-functionalized albumin 7, 8, polylysine 9 and dextran 10 have been developed and evaluated as alternatives to 99mTc labeled colloidal particles. These studies demonstrated that mannose-functionalized nano-carriers targeting mannose receptors on macrophages and dendritic cells hold potential as SLN mapping agents. Indeed, preclinical and clinical studies show that 99mTc-DTPA-mannosyl-dextran (Lymphoseek) exhibited faster injection site clearance and higher accumulation in SLN than did 99m

Tc-labeled colloidal particles 11. Based on the promising results of Lymphoseek, new

mannosylated dextran derivatives with well-defined radiochemistry such as dextran-pyrazoylmannose 12 and dextran-S-cysteine-mannose (DCM20) 13 (Figure 1a) have been developed. The chemical structures of the two compounds were defined by IR and NMR using non-radioactive rhenium, a congener of Tc. They are small-sized particles (8.4 ± 0.5 nm for dextran-pyrazolmannose and 6.5 ± 0.5 nm for dextran-S-cysteine-mannose) and both compounds exhibited higher SLN accumulation and faster injection site clearance than 99mTc-labeled colloidal particles in animal models 14, 15. However, both compounds still exhibited persistent radioactivity levels at the injection site. Although the sensitivity of SLN mapping would be improved with a hybrid single-photon


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emission computed tomography and low-dose CT scanner 16, a new radiopharmaceutical that reduces the radioactivity at the injection site is needed to increase accuracy and expand the procedure to a variety of tumors. However, the mechanisms underlying the undesirable radioactivity at the injection site still remain uncertain. The rational design of new radiopharmaceuticals that reduce the undesirable radioactivity can be developed after understanding the precise mechanism of radioactivity localization. In the present study, the mechanism responsible for the sustained radioactivity levels at the injection site after subcutaneous injection of 99mTc(CO)3-DCM20 was investigated in a mouse model. A rationale strategy to reduce the undesirable radioactivity will be discussed.

Material and Methods Materials [99mTc] Pertechnetate (99mTcO4-) was eluted in a saline solution on a daily basis from 99m

Mo/99mTc generator (FUJIFILM RI Pharma Co., Ltd., Tokyo, Japan). Dextran-S-cysteine

(DC15), DCM20 and 99mTc(CO)3-DCM20 were prepared as previously reported 13. [125I]Radioiodinated mannosyl-neoglycoalbumin containing 44 mannose units per molecule of human serum albumin was prepared with a radiochemical purity of over 97%, according to the procedure as previously described 17. A succinimidyl ester of fluorescence dye, Hylite Fluor 555 (HF555), was obtained from Dojindo Molecular Technology Inc. (Kumamoto, Japan). Reversedphase HPLC (RP-HPLC) was performed with a COSMOSIL 5C18-AR-300 column (4.6 x 50 mm, Nacalai Tesque, Kyoto, Japan) at a flow rate of 1 mL/min with mobile phase starting from 100% A (0.1% aqueous TFA) to 10 min, 30% A and 70% B (acetonitrile with 0.1% TFA) at 20


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min, and the final composition was kept for an additional 10 min. In HPLC analyses, the eluent was monitored with a NaI (Tl) radiodetector (Gabi star, Raytest, Straubenhardt, Germany). Preparation of fluorescence labeled DC15 and DCM20 The HF555-labeled DCM20 and DC15 (Figures 1b and c) were prepared by conjugating DC15 and DCM20 with HF555, according to the manufacturer’s protocol. In brief, a 100 µL solution of DC15 or DCM20 (0.5 mg) in 0.3 M bicarbonate buffer (pH 8.3) was mixed with a 10 µL solution of HF555 succinimidyl ester (20 nmol) in DMSO at room temperature. After 60 min incubation, the mixture was transferred into an ultrafiltration cell (Vivaspin, 3,000 molecular weight cutoff, Sartorius, Goettingen, Germany), and was dialyzed with five exchange volumes of 0.3 M bicarbonate buffer pH 8.3, followed by five exchange volumes of 0.1 M phosphatebuffered saline (PBS; pH 7.4). A spectrophotometer was used to analyze the concentration of the dye from absorbance values at 555 nm. The concentrations of DC15 or DCM20 were determined by the phenol-sulfuric acid method 18. The average number of HF555 conjugated per molecule of DC15 and DCM20 was found to be 0.65 and 0.72, respectively. Biodistribution studies Animal studies were conducted in accordance to our institutional guidelines and were approved by both Chiba University Animal Care Committee and Gunma University Animal Care Committee. The concentrations of DCM20 were adjusted to 2 x 10-11, 2 x 10-10, 2 x 10-9 mol / 20 µL with 0.1 M phosphate buffer (pH 7.4) prior to injection. Groups of five male ddY mice (5 to 7-week old) each were subcutaneously administrated 20 µL of 99mTc(CO)3-DCM20 containing varying molar amounts of DCM20 from the rear footpad. After injection, the footpad was massaged for 0.5


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min. Each animal was sacrificed by decapitation 1 h after injection. To identify lymph nodes, mice were subcutaneously administrated 20 µL of 2% Patent Blue in water from the footpad 10 min before decapitation. The popliteal lymph node, injection site (left paw) and tissue of interest were removed, weighed and the radioactivity counts were determined with an auto well-type gamma counter (ARC-380M, Aloka Co. Ltd., Tokyo, Japan). Similar studies were performed by injecting 99mTc(CO)3-DCM20 (containing 2 x10-11 mol of DCM20) in the presence of 2 x 10-10 or 2 x 10-9 mol of DC15. Biodistribution studies were also conducted after subcutaneous injection of 20 µL (0.08 µg; 1 x 10-12 mol) of 125I-NMA from rear footpad. At 5, 30 min, 1 and 6 h postinjection, mice were sacrificed by decapitation, and the tissues of interest were removed, weighed and the radioactivity counts were determined by a well-type counter. Immunofluorescence staining Mice were subcutaneously administered 20 µL of HF555-labeled DC15 or DCM20 containing 2 x 10-11 mol of DC15 or DCM20 from the rear footpad. After injection, the footpad was massaged for 0.5 min. Each animal was sacrificed by decapitation 1 h after injection. The injection site was removed and frozen sections of murine footpad were constructed to determine the localization of DC15 or DCM20. The sections were fixed with cold acetone, air dried for 20 min, blocked with 10% normal goat serum and 0.1% Tween 20 in PBS (pH 7.4) for 30 min at room temperature, then the sections were incubated with 1 : 200 dilution of anti CD68 antibody (AbD serotec, Raleigh, NC, USA), a commonly used marker for macrophages and dendritic cells 19, for 1 h. Plates were washed (4 times, 5 min each) with PBS (pH 7.4) and incubated with 1 : 400 secondary antibody (Alexa Fluor 350 goat anti-rat IgG; Invitrogen). After washing 4 times,


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coverslips were mounted using a mounting medium (fluoromount/plus, Cosmobio, Tokyo, Japan). Samples were viewed using BZ9000 (Keyence, Osaka, Japan). Statistical analysis Data are expressed as mean ± SD where appropriate. Results were statistically analyzed using one-way ANOVA followed by the Tukey’s test. Differences were considered statistically significant when p was

Injection Site Radioactivity of 99mTc-Labeled Mannosylated Dextran

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