Radiogallium Complex-Conjugated Bifunctional Peptides for

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Radiogallium complex-conjugated bifunctional peptides for detecting primary cancer and bone metastases simultaneously Kazuma Ogawa, Jing Yu, Atsushi Ishizaki, Masaru Yokokawa, Masanori Kitamura, Yoji Kitamura, K Shiba, and Akira Odani Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00186 • Publication Date (Web): 18 Jun 2015 Downloaded from http://pubs.acs.org on June 22, 2015

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Radiogallium complex-conjugated bifunctional peptides for detecting primary cancer and bone metastases simultaneously Kazuma Ogawa,*,†,‡ Jing Yu,† Atsushi Ishizaki,† Masaru Yokokawa,† Masanori Kitamura,† Yoji Kitamura,§ Kazuhiro Shiba,§ Akira Odani†

†

Graduate School of Medical Sciences, Kanazawa University, Kanazawa,

920-1192 Japan; ‡

Institute for Frontier Science Initiative, Kanazawa University, Kanazawa,

920-1192 Japan; §

Advanced Science Research Center, Kanazawa University, Kanazawa,

920-8640 Japan;

*Corresponding Author Division of Pharmaceutical Sciences; Graduate School of Medical Sciences; Kanazawa University; Kakuma-machi, Kanazawa 920-1192; Japan, Telephone: 81-76-234-4460; Fax: 81-76-234-4459 E-mail: [email protected]

ACS Paragon Plus Environment


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Abstract 68

Ga (T1/2 = 68 min, a generator-produced nuclide) is an interesting

radionuclide for clinical positron emission tomography (PET). Recently, it was reported that radiogallium-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated (Asp)n peptide [Ga-DOTA-(Asp)n] has great potential for bone metastases imaging. In the current study, a compound containing an aspartic acid peptide linker (D11) as a carrier to bone metastases, an RGD peptide [c(RGDfK) peptide] as a carrier to the primary cancer, and Ga-DOTA as a stable radiometal complex for imaging in one molecule, Ga-DOTA-D11-c(RGDfK), was designed, prepared, and evaluated to detect both the primary cancer and bone metastases simultaneously using 67Ga, which is easy to handle. After DOTA-D11-c(RGDfK) was synthesized using Fmoc-based solid-phase methodology, 67Ga-DOTA-D11-c(RGDfK) was prepared by complexing DOTA-D11-c(RGDfK) with 67Ga. Hydroxyapatite binding assays, integrin binding assays, biodistribution experiments, and single photon emission tomography (SPECT) imaging using tumor-bearing mice were performed using 67Ga-DOTA-D11-c(RGDfK).


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67

Ga-DOTA-D11-c(RGDfK) was prepared with a radiochemical purity of

>97 %. In vitro, 67Ga-DOTA-D11-c(RGDfK) had a high affinity for hydroxyapatite and αvβ3 integrin. In vivo, 67Ga-DOTA-D11-c(RGDfK) exhibited high uptake in bone and tumor. The accumulation of 67

Ga-DOTA-D11-c(RGDfK) in tumor decreased when it was co-injected

with c(RGDfK) peptide. 68Ga-DOTA-D11-c(RGDfK) has great potential as a PET tracer for the diagnosis of both the primary cancer and bone metastases simultaneously.



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Introduction The bone is a suitable environment for tumor to metastasize and grow because it contains abundant proliferation factors; therefore, many kinds of malignant tumors frequently metastasize to the bone.1, 2 With the advances made in treatments for bone metastases, the early detection of bone metastases has become increasingly important. Although progress has been made in anatomical imaging modalities, such as X-ray computed tomography (CT) and magnetic resonance imaging (MRI), over the past few decades, bone scintigraphy in nuclear medicine is the optimum method for detecting bone metastases because of its high sensitivity. Examples include bone-seeking diagnostic radiopharmaceuticals, such as technetium-bisphosphonate complexes [99mTc-methylenediphosphonate (99mTc-MDP) and 99mTc-hydorxymethylenediphosphonate (99mTc-HMDP)] for single photon emission computed tomography (SPECT) and [18F]NaF for positron emission tomography (PET), which usually localize in metastatic lesions before the appearance of symptoms and radiographic changes.3 These techniques allow the whole body to be evaluated easily. Recently, 68Ga has attracted much attention in nuclear medicine as a


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promising positron-emitting radionuclide because of its radiophysical properties (T1/2 = 68 min).4 68Ga is a generator-produced nuclide that can be eluted at any time on demand from an in-house generator; it does not require a cyclotron on site. Moreover, unlike 99Mo/99mTc, which must be purchased frequently because of the short half-life of 99Mo (T1/2 = 66 h), the long half-life of the parent nuclide 68Ge (T1/2 = 271 d) gives it a long lifespan. Therefore, the generation of bone imaging agents using 68Ga is desirable. Some 68Ga-labeled bone-seeking compounds using bisphosphonate as a carrier to bone, which consist of Ga complex-conjugated bisphosphonate compounds, have been developed.5-7 In 2013, we reported that Ga-complex conjugated aspartic acid peptides exhibited high accumulation in bone and preferable biodistribution for bone imaging.8 Therefore, aspartic acid peptides could also function as carriers to bone. Some pathological processes, such as tumor development, are related to angiogenesis. Integrins are heterodimeric transmembrane glycoproteins that play roles in cell adhesion. Previous studies reported that αvβ3 integrin was a marker of angiogenic blood vessels and regulated



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angiogenesis.9-11 Moreover, αvβ3 integrin is expressed highly on endothelial cells during neovascularization in many kinds of cancers, such as melanoma, glioblastoma, ovarian cancer, and breast cancer, but it is expressed only at very low levels on quiescent endothelial cells.12 Therefore, αvβ3 integrin has garnered much interest as an important target for the imaging and treatment of tumors. Several compounds with a high affinity for αvβ3 integrin have been investigated and developed.13, 14 Cyclic pentapeptides containing an RGD (arginine-glycine-aspartic acid) sequence are typical ligands for the αvβ3 integrin, and have been investigated because of their high affinity. Radiolabeled RGD peptide-containing compounds have been used as tumor imaging agents for PET and SPECT.15, 16 In the current study, we assumed that the introduction of a carrier to the tumor lesion, a carrier to bone metastases, and a stable gallium complex into one molecule could be a promising PET radiotracer using 68Ga to detect both the primary lesion and bone metastases simultaneously. Based on this strategy, RGD peptide with high affinity for αvβ3 integrin, aspartic acid peptide for high affinity to hydroxyapatite in bone metastases, and Ga-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a


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stable gallium complex were selected. The novel complexes, Ga-DOTA-D8-c(RGDfK) and Ga-DOTA-D11-c(RGDfK) (compounds 17 and 18 in Scheme 1) were designed, synthesized, and evaluated in vitro and in vivo. In this drug design, D8 and D11 were selected because the sequence of more than eight aspartic acids in Ga-DOTA-Dn had demonstrated high affinity for bone in our previous study,8 and also because it is interesting to evaluate the effect of the introduction with different length of aspartic acid linkers in Ga-DOTA-Dn-c(RGDfK). The radiogallium complexes were prepared using the easy-to-handle radioisotope, 67Ga rather than 68Ga.



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Scheme 1. Syntheses scheme of Ga-DOTA-Dn-c(RGDfK) (n = 0, 8, or 11). (a) 30% HFIP; (b) diphenylphosphoryl azide, NaHCO3; (c) Pd(PPh3)4 : acetic acid : N-methylmorpholine (37:2:1); (d) 1,4,7,10-tetraazacyclododecane-1,4,7-tris(t-butyl acetate), HOBt, DIPCI; (e) 30% HFIP; (f) HOBt, DIPCI; (g) 95% TFA, 2.5% triisopropylsilane, 2.5% H2O; (h) Ga(NO3)3 or 67GaCl3.


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Results Preparation of the radiogallium complexes Precursors were synthesized by the standard Fmoc-based solid-phase methodology according to scheme 1 and the methods are described in experimental procedures section. The overall yields of the gallium complexes precursors, DOTA-D8-c(RGDfK), DOTA-D11-c(RGDfK), and DOTA-c(RGDfK) were 2.3%, and 3.3%, and 6.5%, respectively. The radiochemical yields of 67

Ga-DOTA-D8-c(RGDfK), 67Ga-DOTA-D11-c(RGDfK), and

67

Ga-DOTA-c(RGDfK) were 93.6%, 92.4%, and 90.0%, respectively. After

purification using HPLC, the radiochemical purities were all >97%. The retention times in HPLC chromatograms of 67Ga-DOTA-Dn-c(RGDfK) (radioactivity) and Ga-DOTA-Dn-c(RGDfK) (UV absorbance) were almost the same (n = 0, 8, and 11, respectively). The comparable retention times in the chromatograms indicate that the radiolabeled products were identical to their nonradioactive counterparts, determined using mass spectrometry.



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αvβ3 Integrin binding assay The αvβ3 integrin binding affinities of the Ga-DOTA complex-conjugated c(RGDfK) peptides were determined using purified human αvβ3 integrin in a competitive binding assay with 125I-c(RGDfK). Figure 1 shows typical 125I-c(RGDfK) displacement curves achieved using Ga-DOTA-D8-c(RGDfK), Ga-DOTA-D11-c(RGDfK), Ga-DOTA-c(RGDfK), and c(RGDfK). The IC50 values (nM) of Ga-DOTA-D8-c(RGDfK), Ga-DOTA-D11-c(RGDfK), Ga-DOTA-c(RGDfK), and c(RGDfK) were 13.5 ± 5.6, 10.7 ± 1.9, 7.7 ± 1.8, and 15.1 ± 8.1, respectively. Because these values were similar (statistically not significant), the Ga-DOTA complex and the aspartic acid peptide linker did not significantly impede the affinity of c(RGDfK) peptide for αvβ3 integrin.


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Figure 1. Typical displacement curves of competition binding assay to the αvβ3 integrin of 125I-c(RGDyK) with Ga-DOTA-D8-c(RGDfK), Ga-DOTA-D11-c(RGDfK), Ga-DOTA-c(RGDfK), and c(RGDfK).



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In vitro stability experiments 67

67

Ga-DOTA-D8-c(RGDfK), 67Ga-DOTA-D11-c(RGDfK), and

Ga-DOTA-c(RGDfK), which have the same DOTA ligand for chelating

with gallium, exhibited same degree of stability in PBS (radiochemical purities (%) of 94.2 ± 3.2, 96.1 ± 2.8, 94.6 ± 3.3 at 1 h, 91.7 ± 5.1, 92.4 ± 1.9, 91.1 ± 2.0 at 3 h, and 84.8 ± 2.4, 87.4 ± 3.0, 84.5 ± 2.3 at 24 h after incubation, respectively) and apo-transferrin solution (unbound ratios of radioactivity to apo-transferrin (%) of 97.5 ± 3.4, 96.1 ± 1.3, 98.9 ± 1.0 at 1 h, 98.1 ± 0.5, 95.2 ± 1.3, 96.7 ± 1.1 at 3 h, and 96.2 ± 3.7, 94.3 ± 1.2, 95.1 ± 0.6 at 24 h, respectively). After incubation at 37°C in PBS for 24 h, approximately 85% of the complexes remained intact. The results of incubation in apo-transferrin solution suggested that 67Ga in the complexes was hardly taken away by apo-transferrin.


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Hydroxyapatite binding assays The binding ratios of 67Ga-DOTA-D8-c(RGDfK), 67

Ga-DOTA-D11-c(RGDfK), 67Ga-DOTA-c(RGDfK), and 67Ga-DOTA to

hydroxyapatite beads are shown in Figure 2. 67Ga-DOTA and 67

Ga-DOTA-c(RGDfK), which do not contain an aspartic acid linker, did

not bind to the hydroxyapatite significantly. In contrast, the percentage of 67

Ga-DOTA-D8-c(RGDfK) and 67Ga-DOTA-D11-c(RGDfK) bound to the

hydroxyapatite increased with increasing amounts of hydroxyapatite. 67

Ga-DOTA-D11-c(RGDfK), which possesses a longer aspartic acid peptide

linker, exhibited a higher binding ratio to the hydroxyapatite than did 67

Ga-DOTA-D8-c(RGDfK).



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Figure 2. Binding ratios of 67Ga-DOTA-D8-c(RGDfK), 67

Ga-DOTA-D11-c(RGDfK), 67Ga-DOTA-c(RGDfK), and 67Ga-DOTA to

hydroxyapatite. Data are expressed as the mean ± SD for four samples.


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In vitro protein binding studies The percentages of 67Ga-DOTA-D8-c(RGDfK), 67

Ga-DOTA-D11-c(RGDfK), 67Ga-DOTA-c(RGDfK), and 67GaCl3 bound to

proteins in mouse serum were 19.4 ± 5.8%, 17.0 ± 1.6%, 14.3 ± 3.5%, and 95.7 ± 0.9%, respectively. 67GaCl3 showed significantly much higher ratio of the protein binding compared to 67Ga-DOTA-Dn-c(RGDfK) compounds. Among 67Ga-DOTA-Dn-c(RGDfK) compounds, the values of protein binding ratios were not significantly different.

Animal experiments The biodistribution of 67Ga-DOTA-D8-c(RGDfK), 67

Ga-DOTA-D11-c(RGDfK), 67Ga-DOTA-c(RGDfK), and 67GaCl3 are

shown in Tables 1 and 2. The accumulation of 67Ga-DOTA-D11-c(RGDfK) in bone was significantly higher than that of 67Ga-DOTA-D8-c(RGDfK). 67

Ga-DOTA-c(RGDfK) did not accumulate in bone appreciably. On the

other hand, 67Ga-DOTA-D8-c(RGDfK), 67Ga-DOTA-D11-c(RGDfK), and 67

Ga-DOTA-c(RGDfK) exhibited high uptake in tumor. Although the



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reason is unclear, the radioactivity in blood was slightly higher after the injection of 67Ga-DOTA-D8-c(RGDfK) than that after the injection of 67

Ga-DOTA-D11-c(RGDfK) and 67Ga-DOTA-c(RGDfK). The three

radiogallium complexes exhibited similar biodistribution patterns in other organs, such as slight high uptake in kidney, liver, intestine, and spleen, and low uptake in pancreas, lung, heart, stomach, muscle, and brain. 67

GaCl3 accumulated at high levels in bone and tumor. However, the

radioactivity was also extremely high in blood and other organs. Thus, it would be difficult to use 68GaCl3 as a PET tracer for tumor or bone imaging because of the short half-life of 68Ga (68 min) and its delayed clearance from the blood and other non-targeted organs. To evaluate the specificity of the novel gallium complex to αvβ3 integrin in vivo, the biodistribution of 67Ga-DOTA-D11-c(RGDfK) at 1 h after injection with an excess amount of RGD peptide is shown in Table 1 (blocking). A significant reduced accumulation of 67

Ga-DOTA-D11-c(RGDfK) in tumor was observed by co-injection with

c(RGDfK) peptide. The accumulation of radioactivity in almost tissues (except bone) was decreased in the blocking experiment.


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TABLE 1. Biodistribution of radioactivity at 1 h after intravenous administration of 67Ga-DOTA-D8-c(RGDfK), 67Ga-DOTA-D11-c(RGDfK), 67 Ga-DOTA-c(RGDfK), or 67GaCl3 in U87MG tumor bearing mice. Blocking means biodistribution of radioactivity at 1 h after co-injection of 67 Ga-DOTA-D11-c(RGDfK) with c(RGDfK) (0.2 mg/mouse) in U87MG tumor bearing mice. 67

Tissue

Ga-DOTAD8-

Liver Kidney Small-intestine Large-intestine

Spleen Pancreas Lung Heart Stomach* Bone Muscle Brain

Ga-DOTAD11-

c(RGDfK)

Blood

67

1.16 (0.12) † 1.56 (0.21) 3.47 (0.61) 2.26 (0.72) 0.92 (0.14) 1.28 (0.09) 0.64 (0.07) 1.06 (0.14) † 0.66 (0.07) † 0.76 (0.24) † 1.21 (0.24) † 0.40 (0.05) 0.07 (0.01)

c(RGDfK)

0.64 (0.08) †,‡ 1.57 (0.13) 2.89 (0.59) 2.48 (0.14) 1.18 (0.20) 1.22 (0.08) 0.79 (0.07) †,‡ 0.80 (0.05) †,‡ 0.66 (0.04) † 0.56 (0.06) †,‡ 3.11 (0.32) †,‡ 0.46 (0.05) † 0.08 (0.01)

67

Ga-DOTA-

c(RGDfK)

0.24 (0.06) 1.31 (0.18) 2.77 (0.57) 1.76 (0.45) 1.17 (0.17) 1.17 (0.21) 0.50 (0.08) 0.56 (0.13) 0.44 (0.12) 0.29 (0.06) 0.72 (0.10) 0.36 (0.03) 0.08 (0.03)


67

GaCl3

17.87 (1.56) 4.76 (0.24) 5.59 (0.60) 5.19 (0.65) 3.53 (0.55) 3.90 (0.74) 5.33 (0.45) 10.77 (0.69) 4.86 (0.29) 0.68 (0.05) 11.96 (1.41) 2.02 (0.28) 0.55 (0.02)

Blocking 0.69 (0.48) 0.29 (0.16) § 2.90 (0.39) 1.47 (0.59) § 0.38 (0.12) § 0.24 (0.10) § 0.31 (0.20) § 0.53 (0.30) 0.27 (0.10) § 0.66 (0.09) § 6.47 (0.61) § 0.14 (0.07) § 0.03 (0.01) §


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Tumor

3.07 2.83 3.45 10.57 0.62 (0.91) (1.38) (0.33) (2.69) (0.31) § Expressed as % injected dose per gram. Each value represents the mean (SD) for four animals. * Expressed as % injected dose. † p < 0.05 vs. 67Ga-DOTA-c(RGDfK) ‡ p < 0.05 vs. 67Ga-DOTA-D8-c(RGDfK) § p < 0.05 vs. 67Ga-DOTA-D11-c(RGDfK)


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TABLE 2. Biodistribution of radioactivity at 3 h after intravenous administration of 67Ga-DOTA-D8-c(RGDfK), 67Ga-DOTA-D11-c(RGDfK), 67 Ga-DOTA-c(RGDfK), or 67GaCl3 in U87MG tumor bearing mice. 67 Ga-DOTA- 67Ga-DOTA- 67 Ga-DOTA67 D8D11GaCl3 Tissue c(RGDfK) c(RGDfK) c(RGDfK) Blood 0.86 0.37 0.14 13.80 † †,‡ (0.09) (0.03) (0.03) (2.78) Liver 1.53 2.67 1.66 5.83 †,‡ (0.11) (0.22) (0.17) (0.76) Kidney 2.80 2.95 2.21 6.84 † † (0.31) (0.25) (0.10) (0.89) Small-intestine 1.24 2.53 2.06 5.33 † ‡ (0.38) (0.35) (0.47) (0.54) Large-intestine 4.53 2.90 5.84 2.25 † †,‡ (0.24) (0.36) (0.88) (0.29) Spleen 1.25 2.34 1.58 4.93 †,‡ (0.11) (0.45) (0.11) (1.43) 0.47 0.25 4.85 Pancreas 0.44 † † (0.04) (0.06) (0.04) (0.43) 0.84 0.60 0.42 9.11 Lung (0.09) † (0.05) ‡ (0.12) (0.80) 0.57 0.33 4.56 0.49 Heart † † (0.05) (0.04) (0.05) (0.97) * 0.27 0.33 0.27 0.77 Stomach (0.09) (0.06) (0.03) (0.09) 2.28 4.55 0.79 17.82 Bone † †,‡ (0.22) (0.53) (0.09) (2.96) 0.25 0.29 0.16 1.50 Muscle † † (0.05) (0.01) (0.04) (0.11) 0.04 0.04 0.04 0.38 Brain (0.01) (0.00) (0.01) (0.05) 3.66 6.92 5.10 9.81 Tumor † †,‡ (0.22) (0.56) (0.15) (1.55) Expressed as % injected dose per gram. Each value represents the mean (SD) for four animals.



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*

Expressed as % injected dose. p < 0.05 vs. 67Ga-DOTA-c(RGDfK) ‡ p < 0.05 vs. 67Ga-DOTA-D8-c(RGDfK) †


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SPECT images Figure 3 shows axial (A, D, and G), sagittal (B, E, and H), and coronal (C, F, and I) views of SPECT images captured at 2 h after the injection of 67Ga-DOTA-D11, 67Ga-DOTA-c(RGDfK), or 67

Ga-DOTA-D11-c(RGDfK). The time point of SPECT imaging was set at 2

h post-injection of tracers. Considering the results of biodistribution experiments, imaging at later time point should be better to obtain images of higher signal / noise ratios. However, as the application for the novel tracers to 68Ga-PET is expected, the imaging at earlier time point should be better due to the short half-life of 68Ga. 67Ga-DOTA-D11 exhibited a marked accumulation of radioactivity in bone (Figure 3A-3C). Very little accumulated radioactivity was observed in tissues other than bone, confirming the results of the biodistribution experiments in our previous report.8 67Ga-DOTA-c(RGDfK) showed a high accumulation of radioactivity surrounding the site of tumor cell inoculation. Although kidney, which is the route of excretion, also exhibited the accumulation of relatively high amounts of radioactivity, the accumulation of radioactivity was lower in the other tissues, including bone. In contrast, 67

Ga-DOTA-D11-c(RGDfK) exhibited high accumulation of radioactivity in



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both tumor and bone, which is consistent with the biodistribution experiments.


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Figure 3. SPECT/CT images (A, D, G: axial images), (B, E, H: sagittal images), (C, F, I: coronal images) of tumor bearing mice at 2 h after the intravenous injection of (A-C) 67Ga-DOTA-D11, (D-F) 67

Ga-DOTA-c(RGDfK), or (G-I) 67Ga-DOTA-D11-c(RGDfK). Arrows

indicate the site where tumor cells were injected.



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DISCUSSION The purpose of this study was to develop a PET tracer that could detect osteoblastic bone metastases, primary cancer, and osteolytic metastases simultaneously. To achieve this, a stable gallium complex site for a PET radionuclide, an aspartic acid peptide linker as a carrier to osteoblastic bone metastases, and an RGD peptide sequence as a carrier to the primary cancer and osteolytic metastases were introduced into one molecule. 68

Ga is one of the most practical and interesting radionuclides for

clinical PET because 68Ga is an in-house generator-produced radionuclide that can be eluted at any time on-demand. The easy labeling of short half-life radionuclides is very important for clinical use. Although any differences between 68Ga and 67Ga for labeling DOTA derivatives are unclear, the radiochemical yields were very high (> 90%) in the current study. The next step for their clinical use is to optimize the labeling conditions using 68Ga to obtain higher radiochemical yields at low ligand concentration. Then, preparation of a kit for radiolabeling that does not require purification should be tested.


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In stability experiments in vitro, the results indicated that the radiogallium complexes were slowly decomposed in a time-dependently manner in PBS and were hardly taken away by apo-transferrin. The slight decomposition should not be critical problem. 67Ga-DOTA-Dn-c(RGDfK) complexes showed fast blood clearance and free gallium (67GaCl3) showed very slow blood clearance (Table 1 and Table 2). The protein binding ratios of 67Ga-DOTA-Dn-c(RGDfK) complexes were low and the protein binding ratio of 67GaCl3 was extremely high from the results of in vitro protein binding experiments. Then, if 67Ga was released from 67

Ga-DOTA-Dn-c(RGDfK), the blood clearance of radioactivity after

injection of 67Ga-DOTA-Dn-c(RGDfK) could be delayed. Moreover, if any peptide bonds in 67Ga-DOTA-Dn-c(RGDfK) were cleaved, tumor accumulation of radioactivity could be lower. Therefore, these results indicated that large proportions of 67Ga-DOTA-Dn-c(RGDfK) complexes were distributed to whole body without decomposition. Meanwhile, the blood clearance of radioactivity after injection of 67Ga-DOTA-Dn-c(RGDfK) was correlated with the protein binding ratios of 67Ga-DOTA-Dn-c(RGDfK) although the difference of the protein binding ratios among



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67

Ga-DOTA-Dn-c(RGDfK) complexes was not significant. The protein

binding does not always decide the blood clearance, but there is no conflict between the blood clearance in the biodistribution experiments and the results of the protein binding experiments in vitro. Recent reports suggested that aspartic acid-containing peptides could be useful carriers for bone imaging radiotracers.8, 17 The affinity of aspartic acid peptides for bone is derived from the high affinity of the acidic amino acid peptides for hydroxyapatite in bone,18, 19 which is similar to that of the bisphosphonate compounds that have been used as carriers for bone-seeking radiopharmaceuticals. In the current study, 67Ga-DOTA and 67

Ga-DOTA-c(RGDfK), which do not contain an aspartic acid peptide

linker, did not bind to hydroxyapatite significantly, whereas, 67

Ga-DOTA-D8-c(RGDfK) and 67Ga-DOTA-D11-c(RGDfK), which possess

an aspartic acid peptide linker, exhibited a high affinity for hydroxyapatite, as expected (Figure 2). Thus, in the current drug design, the aspartic acid peptide linker between the Ga-DOTA complex and the RGD peptide could function as a carrier to bone. In SPECT imaging, 67

Ga-DOTA-D11-c(RGDfK) exhibited a high accumulation of radioactivity


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in bone (Figure 3G-3I), whereas 67Ga-DOTA-c(RGDfK) did not accumulate in bone (Figure 3D-3F). Moreover, our recent report demonstrated that the affinity for hydroxyapatite increased as the number of aspartic acid residues increased.8 Therefore, the results of the hydroxyapatite binding assays and biodistribution experiments demonstrated that the affinity for bone was increased as the number of aspartic acid residues increased [Figure 2, Tables 1 and 2; 67

Ga-DOTA-c(RGDfK) < 67Ga-DOTA-D8-c(RGDfK)

Radiogallium Complex-Conjugated Bifunctional Peptides for

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