A radiogallium–DOTA-based bivalent peptidic ligand targeting a chemokine receptor, CXCR4, for tumor imaging

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1386–1388

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A radiogallium–DOTA-based bivalent peptidic ligand targeting a chemokine receptor, CXCR4, for tumor imaging Kohei Sano a,b, Ryo Masuda c, Hayato Hisada b, Shinya Oishi c, Kenta Shimokawa a, Masahiro Ono b, Nobutaka Fujii c, Hideo Saji b, Takahiro Mukai a,d,⇑ a

Department of Biomolecular Recognition Chemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachimachi, Sakyo-ku, Kyoto 606-8501, Japan Department of Bioorganic Medical Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachimachi, Sakyo-ku, Kyoto 606-8501, Japan d Department of Biophysical Chemistry, Kobe Pharmaceutical University, 4-19-1 Motoyama Kitamachi, Higashinada-ku, Kobe 658-8558, Japan b c

a r t i c l e

i n f o

Article history: Received 26 November 2013 Revised 10 January 2014 Accepted 11 January 2014 Available online 21 January 2014 Keywords: Radiogallium DOTA CXCR4 Bivalent effect

a b s t r a c t We have developed a novel radiogallium (Ga)–DOTA-based bivalent peptidic ligand targeting a chemokine receptor, CXCR4, for tumor imaging. A CXCR4 imaging probe with two CXCR4 antagonists (Ac-TZ14011) on Ga–DOTA core, Ga–DOTA-TZ2, was synthesized, and the affinity and binding to CXCR4 was evaluated in CXCR4 expressing cells in vitro. The affinity of Ga–DOTA-TZ2 for CXCR4 was 20-fold greater than the corresponding monovalent probe, Ga–DOTA-TZ1. 67Ga–DOTA-TZ2 showed the significantly higher accumulation in CXCR4-expressing tumor cells compared with 67Ga–DOTA-TZ1, suggesting the bivalent effect enhances its binding to CXCR4. The incorporation of two CXCR4 antagonists to Ga–DOTA could be effective in detecting CXCR4-expressing tumors. Ó 2014 Elsevier Ltd. All rights reserved.

Gallium (Ga)-67 and Ga-68 are radionuclides that possess physical properties appropriate for clinical nuclear imaging. Ga-67 is a pure c-ray emitter [93 keV (40%), 184 keV (20%), 300 keV (17%), and 393 keV (5%); t1/2 = 3.3 d], suitable for single photon emission computed tomography (SPECT). Ga-68 is an excellent positron emitter (511-keV annihilation radiation; t1/2 = 68 min) suitable for positron emission tomography (PET)1 and is available from a 68 Ge/68Ga generator, which renders it independent of an on-site cyclotron. As a chelating agent with gallium, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) has been generally utilized because of its intense chelating potency and facile conjugation to ligand probes using various DOTA derivatives. Previous crystallographic studies demonstrated that the four nitrogen atoms of the cyclen ring and two oxygen atoms of the opposite carboxyl groups in the DOTA are coordinated to the gallium metal.2 Given this arrangement, the two remaining free carboxyl groups of the radiogallium–DOTA complex could be utilized for coupling to functional moieties, which could be available to recognize molecular targets for in vivo imaging without reducing the stability of radiogallium–DOTA complex. We have developed a Ga–DOTA complex conjugated with two nitroimidazole derivatives (Ga– DOTA-MN2) for use in hypoxic tumor nuclear imaging,3,4 which exhibited significant accumulation in the hypoxic regions of the tumor with high stability in vivo. ⇑ Corresponding author. Tel.: +81 78 441 7540; fax: +81 78 441 7541. E-mail address: [email protected] (T. Mukai). 0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2014.01.031

The multivalent effect, whereby the binding affinity of a probe containing multiple binding ligands to specific receptors expressed on target cells is enhanced relative to the corresponding monovalent probe, has generally become a well-accepted concept in the design of molecular imaging probes for specific signal amplification.5–7 Bivalent ligands targeting a chemokine receptor, CXCR4, with appropriate linker between two CXCR4 ligands have been reported to improve the interaction with CXCR4.8 CXCR4 is up-regulated in at least 23 different types of cancers,9 including malignant and metastatic cancer, as well as cancer stem cells,10,11 and has been correlated with poor survival,12 aggressiveness,13 metastasis,14 and recurrence.15 These findings motivated us to develop bivalent CXCR4 imaging probes based on a Ga–DOTA chelate for the in vivo imaging of malignant and metastatic tumors. To date, several probes for CXCR4 have been developed, which were derived from CXCR4 agonists and antagonists using fluorophores or radionuclides.16–23 Among them, we have developed a potent peptidic CXCR4 antagonist, Ac-TZ14011, and used indium111 labeling to image CXCR4 in the tumor.16 However, its tumor uptakes and tumor to normal tissue ratios were low (0.51% dose/ g at 1 h), which lead to their low applicability.16 This can be attributed primarily to the suboptimal affinity of the probe for CXCR4. To improve the affinity to CXCR4, we designed two AcTZ14011-conjugated DOTA (DOTA-TZ2; Fig. 1A) probe, employing an Ac-TZ14011 moiety as the recognition site of CXCR4 in the tumors. We also evaluated radiogallium–DOTA-TZ2 as a CXCR4 imaging probe in high and low CXCR4 expressing cells.

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B

CO2H O

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Figure 1. Chemical structures of bifunctional ligands for radiopharmaceuticals, (A) DOTA-TZ2 and (B) DOTA-TZ1.

1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,7bis(tert-butyl) ester [DOTA(tBu)2] was prepared according to our previous report.3 The Ac-TZ14011 peptide was synthesized as previously reported.16 Because the D-Lys8 residue is not required for the interaction with CXCR4,16 the e-amino side chain of D-Lys8 was used for the conjugation of Ac-TZ14011 to two carboxylic acid groups of DOTA(tBu)2, providing DOTA(tBu)2-TZ2 as a main product (see Supplementary Fig. 1A, 20 min). The purified DOTA(tBu)2TZ2 (see Supplementary Fig. 1B) was subjected to TFA treatment to afford the desired DOTA-TZ2 (see Supplementary Fig. 1C).

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For the comparison of CXCR4 binding activity, the corresponding monovalent ligand, DOTA-TZ1, was also designed and synthesized (Fig. 1B). DOTA-TZ1 was obtained by on-resin conjugation of tri-tert-butyl 1,4,7,10-tetraaza-cyclododecane-1,4,7-triacetate onto the bromoacetyl group of D-Lys and the subsequent deprotection.18 In brief, the peptide chain was constructed on TGR-resin by Fmoc solid phase peptide synthesis using Fmoc-D-Lys(Mtt)-OH. The Mtt protecting group was specifically removed by 1,1,1,3,3,3-hexafluoropropanol (HFIP). The free e-amino group of D-Lys was modified with bromoacetic acid/diisopropyl carbodiimide (DIC). The resulting peptide resin was treated with tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4-7-triacetate followed by cleavage from the resin and deprotection of the other protecting groups. The crude peptide was oxidized by air oxidation to form disulfide bonds between Cys residues, and then purified using HPLC (see Supplementary Fig. 2A). The subsequent treatment of each DOTA-conjugated CXCR4 antagonist with nonradioactive GaNO3 provided the Ga–DOTA-conjugated CXCR4 antagonists Ga–DOTA-TZ2 and Ga–DOTA-TZ1 (see Supplementary Figs. 1D and 2B). The binding activity of DOTA derivatives for CXCR4 was evaluated in a competitive binding assay against 125I-SDF-1 (Supplementary Fig. 3).18 Ga–DOTA-TZ1 showed equipotent CXCR4 binding activity to the parent compound Ac-TZ14011, whereas Ga–DOTA-TZ2 was approximately 20 times more inhibitory toward SDF-1 binding to CXCR4 compared with Ga–DOTA-TZ1 (IC50: Ac-TZ14011, 210 nM; Ga–DOTA-TZ1, 140 nM; Ga–DOTATZ2, 6.8 nM). The increased binding activity could be due to the presence of two molecules of Ac-TZ14011, which is consistent with a previous report that bivalent CXCR4 ligands with linker improve the interaction with CXCR4.8

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Figure 2. Analytical HPLC profiles of (A) 67Ga–DOTA-TZ2 and (B) 67Ga–DOTA-TZ1. The analyses were performed using a Cosmosil 5C18-AR-II column (4.6  250 mm) at a flow rate of 1.0 mL/min with a mixture of water and acetonitrile (80:20 and 83:17 for 67Ga–DOTA-TZ2 and 67Ga–DOTA-TZ1, respectively) containing 0.1% TFA. (C) The stability of probes in PBS. Probes were incubated in PBS for 24 h at 37 °C.

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Figure 3. In vitro cellular uptake studies in cultured CXCR4+/CHO and CXCR4 /CHO cells. (A) Flow cytometry analyses of CXCR4 expression. (B) Time-dependent change of cellular uptake of 67Ga–DOTA-TZ1 and 67Ga–DOTA-TZ2 in CXCR4+/CHO and CXCR4 /CHO cells at 37 °C. ⁄⁄P <0.01, ⁄P <0.05 versus 67Ga–DOTA-TZ1 group. (C) The uptake of 67 Ga–DOTA-TZ1 and 67Ga–DOTA-TZ2 at 1 h in the absence or presence of varying concentrations of Ac-TZ14011.

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The desired 67Ga-labeled probes, 67Ga–DOTA-TZ1 and 67Ga– DOTA-TZ2, were prepared by reacting corresponding precursors (DOTA-TZ1 and DOTA-TZ2) with 67GaCl3 in 0.2 M ammonium acetate buffer (pH 4.8) at 95 °C for 30 min. The radiochemical yield and purity were >80% and >99% for both probes (67Ga–DOTA-TZ2 and 67Ga–DOTA-TZ1), respectively, which was confirmed using a reverse-phase HPLC (Fig. 2A and B). In the stability test, both probes, which were incubated in PBS for 24 h at 37 °C, were retained as intact forms (>99%) (Fig. 2C). Cellular uptake of 67Ga–DOTA-TZ1 and 67Ga–DOTA-TZ2 was evaluated in CXCR4+/CHO and CXCR4 /CHO cells. We first determined the level of CXCR4 expression in CXCR4+/CHO and CXCR4 /CHO cells using flow cytometry. CXCR4 was observed in only CXCR4+/CHO cells (Fig. 3A). 67Ga–DOTA-TZ1 and 67Ga– DOTA-TZ2 showed significantly higher uptake in CXCR4+/CHO cells than in CXCR4 /CHO cells over a period of 3 h (Fig. 3B). Furthermore, 67Ga–DOTA-TZ2 exhibited significantly higher accumulation in CXCR4+/CHO cells compared with 67Ga–DOTA-TZ1 over 2 h, except at the time point of 10 min. Ten minutes after incubation, the rapid binding of 67Ga–DOTA-TZ1 to CXCR4 on the surface of tumor cells was observed, whereas at later time points (after equilibrium binding), 67Ga–DOTA-TZ2 binds with a high affinity to CXCR4 compared with 67Ga–DOTA-TZ1, enabling the sensitive detection of tumor cells. The multivalent nature of the ligand arrangement increases its local concentration on the cell surface; thus, increasing the probability of the specific ligand–receptor interaction and enhancing target accumulation. The specificity of the binding of 67Ga–DOTA-TZ1 and 67Ga–DOTA-TZ2 to CXCR4 was also evaluated in CXCR4+/CHO cells by examining cellular uptake in the presence of varying concentrations of Ac-TZ14011 (Fig. 3C). Radioactivity accumulation in CXCR4+/CHO cells was decreased in an inhibitor dose-dependent manner. The radioactivity in CXCR4+/CHO cells when treated with high doses of the inhibitor was decreased to the same level as observed in CXCR4 /CHO cells. These results indicate the accumulation of 67Ga–DOTA-TZ1 and 67 Ga–DOTA-TZ2 in CXCR4+/CHO cells is CXCR4-specific. In summary, a novel CXCR4 imaging probe was designed and synthesized on the basis of the previous finding2 obtained by X-ray crystallography of Ga–DOTA chelates. This multivalent probe, Ga– DOTA-TZ2, contains two CXCR4 antagonists on a Ga–DOTA core. The affinity of Ga–DOTA-TZ2 for CXCR4 was 20-fold greater than the corresponding monovalent probe, Ga–DOTA-TZ1. 67Ga–DOTATZ2 exhibited significantly higher accumulation in CXCR4-expressing tumor cells compared with 67Ga–DOTA-TZ1, suggesting the bivalent nature of the probe enhances its binding to CXCR4. These results suggest that coupling of two CXCR4 antagonists to Ga–DOTA could be effective in detecting CXCR4-expressing tumors. Acknowledgments We would like to thank FUJIFILM RI Pharma Co., Ltd, Tokyo, Japan, for donating 67GaCl3. This work was supported in part by a

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