Comparison of Technetium-99m-Labeled Pentapeptides as Bone Imaging Agents: Influence of Different Types of Acidic Amino Acids

Abstract

Bone-seeking radiopharmaceuticals are essential for the early detection of bone metastases. In this study, we developed three Technetium-99m (^99mTc)-labeled oligopeptides composed of acidic amino acids and evaluated their bone-targeting ability. Hydrazinonicotinamide (HYNIC)-conjugated oligopeptides with five residues of aspartic acid (Asp), glutamic acid (Glu), or γ-carboxyglutamic acid (Gla) were synthesized and radiolabeled with ^99mTc using tricine and 3-acetylpyridine as co-ligands. Their hydroxyapatite binding, in vitro stability, biodistribution, and single-photon emission computed tomography (SPECT)/CT imaging in normal mice were evaluated. Among the three tracers, [^99mTc]Tc-HYNIC-(tricine)(AcP)-(dl-Gla)₅ ([^99mTc]3) showed the highest hydroxyapatite binding and bone uptake, with clear visualization in SPECT/CT. All compounds exhibited high radiochemical purity and in vivo stability. Oligopeptides containing Gla residues exhibited superior bone affinity and imaging performance, suggesting that Gla-based oligopeptides are promising scaffolds for the development of ^99mTc-labeled bone imaging agents.

Introduction

Bone is a frequent site of metastasis for various human malignancies—including breast cancer, lung cancer, and prostate cancer—due to its microenvironment that is rich in growth factors. Bone metastases significantly contribute to morbidity and mortality, making an early diagnosis and treatment essential for improving QOL.^1,2) Nuclear medicine imaging techniques, such as gamma scintigraphy, single-photon emission computed tomography (SPECT), and positron emission tomography (PET), after the injection of bone-seeking radiopharmaceuticals can detect bone disorders such as bone metastases with high sensitivity before structural changes become visible on radiography.^3,4) Bone-seeking radiopharmaceuticals with high affinity for hydroxyapatite highly accumulate at sites of increased bone metabolic activity, specifically osteoblastic activity, enabling early diagnosis of osteoblastic bone metastases. This high accumulation is known to be dependent on the fact that the surface area of hydroxyapatite is large due to its amorphous structure and increased blood flow at the site of osteoblastic bone metastases.^5,6)

Technetium-99m (^99mTc) is one of the most widely used radionuclides in clinical nuclear medicine owing to its favorable physical properties, including a suitable half-life (t₁/₂ = 6.01 h), appropriate gamma-ray energy (141 keV) for SPECT, and convenient availability from ⁹⁹Mo/^99mTc generators. These advantages have stimulated the development of various ^99mTc-labeled bone imaging agents. Clinically used agents, such as [^99mTc]Tc-methylenediphosphonate ([^99mTc]Tc-MDP)⁷⁾ and [^99mTc]Tc-hydroxy-methylenediphosphonate ([^99mTc]Tc-HMDP),⁸⁾ have long been used as standards for bone scintigraphy. Meanwhile, novel agents—^99mTc-EC-AMDP,^{9) 99m}Tc-MAG3-HBP, ^99mTc-HYNIC-HBP,^{10) 99m}Tc(CO)₃(PzNN-BP), ^99mTc(CO)₃(PzNN-ALN), and ^99mTc(CO)₃(PzNN-PAM)^11,12)—have been explored to improve stability and targeting efficiency with the concept of stable ^99mTc-complex conjugated bisphosphonate. In addition to ^99mTc, other radiometals, such as rhenium, gallium, and yttrium, have been used to develop radiometal complex conjugated bisphosphonate derivatives for radiotheranostics of bone metastases.^13–20)

Although bisphosphonates remain a major class of bone-targeting moieties, several noncollagenous bone proteins, such as osteopontin and bone sialoprotein, contain sequences rich in acidic amino acids (e.g., aspartic acid (Asp) and glutamic acid (Glu)), which are known to bind hydroxyapatite.^21,22) Inspired by this, acidic amino acid-composed oligopeptides have emerged as alternative bone-targeting carriers.²³⁾ Previously, we reported that radiogallium-labeled oligopeptides consisting of Asp, Glu, or γ-carboxyglutamic acid (Gla) exhibited high hydroxyapatite affinity and bone accumulation in mice.^6,24–26) Bisphosphonate scaffolds have long been employed in bone-targeting radiopharmaceuticals due to their high hydroxyapatite affinity. However, their limited functionalization sites and multi-step derivatization often necessitate protection/deprotection steps and harsh phosphorylation conditions, thereby restricting structural modifications and complicating the synthesis of multifunctional conjugates.^27–29) By contrast, acidic oligopeptide-based tracers (Asp/Glu/Gla) can be synthesized in a modular fashion via solid-phase peptide synthesis, enabling sequence tuning and orthogonal functionalization without compromising on hydroxyapatite binding. This chemical flexibility facilitates straightforward conjugation of imaging chelators or therapeutic payloads. Notably, (Asp)_n-based multitarget derivatives^30–32) and a boron-labeled (Asp)_n derivative for boron neutron capture therapy³³⁾ have already been developed.

Based on these findings, the current study aimed to explore ^99mTc-labeled oligopeptides as bone-seeking agents by conjugating ^99mTc complexes with pentapeptides composed of Asp, Glu, or Gla. Hydrazinonicotinamide (HYNIC) was selected as the bifunctional chelator, with tricine and 3-acetylpyridine (AcP) as co-ligands, due to their ability to form stable ^99mTc complexes with high radiochemical yield.^10,34) We selected five-residue peptides because they are synthetically accessible and sufficient to evaluate the influence of acidic amino acid type on bone-targeting. Accordingly, we synthesized and evaluated [^99mTc]Tc-HYNIC-(tricine)(AcP)-(l-Asp)₅ ([^99mTc]1), [^99mTc]Tc-HYNIC-(tricine)(AcP)-(l-Glu)₅ ([^99mTc]2), and [^99mTc]Tc-HYNIC-(tricine)(AcP)-(dl-Gla)₅ ([^99mTc]3) (Fig. 1), to assess the impact of the acidic amino acid type on hydroxyapatite binding, bone accumulation, and biodistribution in normal mice.

Fig. 1. Chemical Structures of [^99mTc]1, [^99mTc]2, and [^99mTc]3

Results and Discussion

In this study, we developed and evaluated three novel bone-seeking radiotracers—[^99mTc]1, [^99mTc]2, and [^99mTc]3—consisting of ^99mTc-HYNIC conjugated oligopeptides containing five residues of acidic amino acids—Asp, Glu, and Gla, respectively (Fig. 1). Our primary goal was to investigate how the type of acidic amino acid influences hydroxyapatite binding and bone accumulation in normal mice, aiming to identify a suitable scaffold for SPECT bone imaging agents.

Synthesis of Precursors

HYNIC-(l-Asp)₅, HYNIC-(l-Glu)₅, and HYNIC-(dl-Gla)₅ as precursors were synthesized by a standard 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase methodology using tert-butoxycarbonyl (Boc)-HYNIC-OH and Fmoc-l-Asp(OtBu)-OH, Fmoc-l-Glu(OtBu)-OH, or Fmoc-dl-Gla(OtBu)₂-OH as materials. The overall yields of HYNIC-(l-Asp)₅, HYNIC-(l-Glu)₅, and HYNIC-(dl-Gla)₅ were 49, 54, and 46%, respectively.

Radiolabeling with ^99mTc

The radiolabeled compounds [^99mTc]1, [^99mTc]2, and [^99mTc]3 were synthesized by the complexation of the precursors with ^99mTc using the co-ligands tricine and 3-acetylpyridine. The radiochemical yields of [^99mTc]1, [^99mTc]2, and [^99mTc]3 were 87, 88, and 87%, respectively. After reversed-phase HPLC (RP-HPLC) purification, all radiolabeled compounds had more than 98% radiochemical purities. Due to the difficulty in synthesizing the corresponding stable Re-HYNIC complex as a reference compound, ligand exchange experiments and comparative HPLC chromatograms (supporting information) were used to characterize [^99mTc]1, [^99mTc]2, and [^99mTc]3 complexes. Namely, for comparison, we also synthesized [^99mTc]Tc-HYNIC-(tricine)₂-(l-Asp)₅ ([^99mTc]4), [^99mTc]Tc-HYNIC-(tricine)₂-(l-Glu)₅ ([^99mTc]5), and [^99mTc]Tc-HYNIC-(tricine)₂-(dl-Gla)₅ ([^99mTc]6). The chromatograms of [^99mTc]4–6 with (tricine)₂ as coligands show that all peaks eluted before 5 min, whereas those of [^99mTc]1–3 with tricine and AcP as coligands eluted after 10 min. This shift in retention time clearly indicates the increased lipophilicity conferred by the AcP moiety.

In Vitro Stability

The radiochemical purities of [^99mTc]1, [^99mTc]2, and [^99mTc]3 after incubation for 3 h in phosphate-buffered saline (PBS) were 98.0 ± 0.1%, 98.2 ± 0.3%, and 96.3 ± 0.3%, (mean ± S.D. for three samples), respectively. The radiochemical purities of [^99mTc]1, [^99mTc]2, and [^99mTc]3 after incubation for 1 h in murine plasma were 97.3 ± 0.1%, 97.5 ± 0.2%, and 95.2 ± 0.2% (mean ± S.D. for three samples), respectively.

Hydroxyapatite-Binding Assays

Figure 2 shows the percentage of each [^99mTc]1, [^99mTc]2, and [^99mTc]3 that are bound to hydroxyapatite beads. Each binding to the beads increased with an increasing amount of hydroxyapatite. The binding ratios of [^99mTc]3 to hydroxyapatite at all concentrations were significantly superior to those of [^99mTc]1 and [^99mTc]2. In addition, the binding ratios of [^99mTc]1 were comparable to those of [^99mTc]2.

Fig. 2. Hydroxyapatite Binding Assay of [^99mTc]1, [^99mTc]2, and [^99mTc]3; Data Are Expressed as the Mean ± S.D. for the Four Samples

Significance was determined using a one-way ANOVA followed by Tukey’s post hoc. test (*p < 0.05, **p < 0.001).

Biodistribution Experiments

The biodistribution results of [^99mTc]1, [^99mTc]2, and [^99mTc]3 in normal mice are summarized in Tables 1–3, respectively. [^99mTc]3 was highly accumulated and retained in the bone compared with [^99mTc]1 and [^99mTc]2, which exhibited hardly any accumulation in bone. All the radiolabeled compounds show rapid clearance from nontargeted organs, and their radioactivity in almost all tissues—except bones and kidneys—was undetectable after 3 h postinjection. The renal clearance of radioactivity was slower than that from other tissues.

Table 1. Biodistribution of [^99mTc]1 in Mice

Tissues	Time after injection
	10 min	60 min	180 min
Blood	3.15 (0.42)	0.53 (0.05)	0.12 (0.01)
Liver	0.78 (0.19)	0.37 (0.04)	0.19 (0.02)
Kidney	11.31 (0.96)	3.66 (1.15)	1.33 (0.15)
Small intestine	0.97 (0.10)	0.42 (0.04)	0.18 (0.03)
Large intestine	0.78 (0.12)	0.17 (0.03)	0.47 (0.02)
Spleen	0.67 (0.11)	0.14 (0.03)	0.07 (0.01)
Pancreas	0.90 (0.08)	0.27 (0.08)	0.04 (0.01)
Lung	2.32 (0.20)	0.62 (0.24)	0.13 (0.00)
Heart	1.13 (0.24)	0.23 (0.03)	0.05 (0.00)
Stomach‡	0.44 (0.15)	0.30 (0.06)	0.15 (0.03)
Bone (Femur)	5.80 (1.10)	4.50 (0.80)	2.52 (0.24)
Muscle	0.96 (0.08)	0.15 (0.00)	0.03 (0.00)
Brain	0.13 (0.02)	0.03 (0.00)	0.01 (0.00)
Thyroid‡	0.05 (0.01)	0.01 (0.00)	0.01 (0.00)
Femur-to-blood ratio	1.84 (0.21)	8.49 (1.74)	20.13 (5.36)

Data are expressed as percent injected dose per gram of tissue. Each value represents the mean (S.D.) for four mice. ^‡Data are expressed as percent injected dose.

Table 2. Biodistribution of [^99mTc]2 in Mice

Tissues	Time after injection
	10 min	60 min	180 min
Blood	2.65 (0.14)	0.27 (0.04)	0.10 (0.00)
Liver	0.55 (0.03)	0.15 (0.02)	0.08 (0.01)
Kidney	16.52 (1.48)	4.17 (1.16)	0.80 (0.13)
Small intestine	0.62 (0.10)	0.24 (0.03)	0.43 (0.13)
Large intestine	0.50 (0.05)	0.10 (0.01)	0.97 (0.27)
Spleen	0.63 (0.05)	0.13 (0.03)	0.05 (0.00)
Pancreas	0.81 (0.08)	0.15 (0.02)	0.04 (0.00)
Lung	2.10 (0.09)	0.29 (0.04)	0.08 (0.01)
Heart	0.94 (0.12)	0.10 (0.02)	0.04 (0.00)
Stomach‡	0.46 (0.06)	0.23 (0.03)	0.18 (0.05)
Bone (Femur)	2.20 (0.15)	0.45 (0.09)	0.32 (0.06)
Muscle	0.73 (0.18)	0.08 (0.00)	0.02 (0.00)
Brain	0.11 (0.02)	0.01 (0.00)	0.00 (0.00)
Thyroid‡	0.05 (0.00)	0.01 (0.00)	0.00 (0.00)
Femur-to-blood ratio	0.83 (0.05)	1.63 (0.11)	2.97 (0.36)

Data are expressed as percent injected dose per gram of tissue. Each value represents the mean (S.D.) for four mice. ^‡Data are expressed as percent injected dose.

Table 3. Biodistribution of [^99mTc]3 in Mice

Tissues	Time after injection
	10 min	60 min	180 min
Blood	2.07 (0.19)	0.42 (0.11)	0.30 (0.05)
Liver	0.54 (0.07)	0.25 (0.04)	0.23 (0.02)
Kidney	6.40 (1.60)	2.69 (0.51)	1.87 (0.22)
Small intestine	0.60 (0.02)	0.35 (0.26)	0.16 (0.03)
Large intestine	0.40 (0.02)	0.10 (0.01)	0.33 (0.06)
Spleen	0.46 (0.02)	0.13 (0.06)	0.10 (0.02)
Pancreas	0.57 (0.03)	0.08 (0.04)	0.04 (0.00)
Lung	1.51 (0.09)	0.26 (0.06)	0.16 (0.07)
Heart	0.75 (0.08)	0.18 (0.02)	0.11 (0.01)
Stomach‡	0.46 (0.07)	0.37 (0.02)	0.34 (0.12)
Bone (Femur)	15.85 (0.23)	25.03 (2.03)	20.48 (1.60)
Muscle	0.44 (0.05)	0.06 (0.01)	0.04 (0.00)
Brain	0.08 (0.02)	0.03 (0.05)	0.01 (0.00)
Thyroid‡	0.04 (0.00)	0.02 (0.01)	0.01 (0.00)
Femur-to-blood ratio	7.70 (0.74)	60.72 (23.02)	69.78 (19.80)

Data are expressed as percent injected dose per gram of tissue. Each value represents the mean (S.D.) for four mice. ^‡Data are expressed as percent injected dose.

Among the three candidates, [^99mTc]3—based on γ-carboxyglutamic acid (Gla)—showed superior hydroxyapatite binding and the highest bone uptake in biodistribution studies. These results are consistent with the known calcium-binding properties of Gla, which contains two carboxyl groups capable of forming stable chelates with hydroxyapatite components.⁶⁾ This supports the hypothesis that increasing the carboxyl group density within the peptide backbone enhances bone-targeting.

Unlike many previously developed bisphosphonate-based bone imaging agents, the current study focuses on oligopeptide-based tracers that may offer advantages in terms of synthesis simplicity and chemical flexibility. Although a direct comparison with earlier Tc-99m-labeled compounds, such as [^99mTc]Tc-HYNIC-HBP, is limited by species differences in previous studies, our results demonstrate that [^99mTc]3 achieves high bone uptake with rapid clearance from nontarget tissues in mice, which is comparable to the pharmacokinetic profiles reported for other bone-seeking agents.³⁵⁾

To further evaluate the bone-targeting properties of [^99mTc]3, SPECT/CT imaging was performed; the results are presented in the following section.

SPECT/CT Imaging

Figure 3 shows the SPECT/CT images of [^99mTc]3 in normal mice as coronal views (A and B) at 1 and 3 h postinjection. High radioactivity accumulation in the bone (especially bone joints) and no accumulation in other organs was observed after [^99mTc]3 injection. These results are consistent with the biodistribution of [^99mTc]3. At 3 h postinjection, SPECT/CT images demonstrated clearer bone accumulation of [^99mTc]3 compared with 1 h, with higher contrast relative to blood, indicating sustained retention in bone.

Fig. 3. Typical Coronal Maximum Intensity Projection (MIP) SPECT/CT Images of [^99mTc]3 in ddY Mice at (A) 1 h and (B) 3 h after Intravenous Injection

Nevertheless, this study has limitations that should be acknowledged. One limitation is that the evaluations were performed in healthy mice rather than disease models. As with other hydroxyapatite-targeting bone-seeking radiopharmaceuticals, [^99mTc]3 is expected to accumulate preferentially at sites of increased osteoblastic activity.³⁵⁾ The knee joint in mice is known to exhibit such increased osteoblastic activity, likely accounting for the prominent accumulation of [^99mTc]3 observed in this region in SPECT images. Thus, [^99mTc]3 may be a promising candidate for targeting osteoblastic bone metastases. Although the observed accumulation of [^99mTc]3 in bone reflects strong hydroxyapatite affinity, further validation using osteoblastic or mixed-type bone metastasis models is warranted to assess its clinical relevance.

In summary, Gla-based oligopeptides represent a promising bisphosphonate-free scaffold for ^99mTc-labeled bone-targeting radiotracers. The favorable pharmacokinetics, high in vivo stability, and excellent bone selectivity of [^99mTc]3 support its potential as a SPECT imaging agent for skeletal disorders.

Conclusion

[^99mTc]3 was synthesized with >98% radiochemical purity. It exhibited higher affinity for hydroxyapatite and superior bone accumulation in normal mice, along with faster clearance from nonspecific organs compared with [^99mTc]1 and [^99mTc]2. In SPECT/CT imaging studies, [^99mTc]3 enabled clear visualization of the entire skeleton, especially the joint regions, with minimal uptake in nontarget tissues. These results highlight the potential of [^99mTc]3 as a promising bone-seeking agent for SPECT/CT imaging.

Experimental

General Experimental Procedures

Pertechnetate ([^99mTc]TcO₄⁻) was eluted in a saline solution from a ⁹⁹Mo/^99mTc generator (Nihon Mediphysics, Tokyo, Japan). 2-Chlorotrityl chloride resin, Fmoc-l-Asp(OtBu)-OH, and Fmoc-l-Glu(OtBu)-OH were purchased from AmBeed (Chicago, IL, U.S.A.). Fmoc-dl-Gla(OtBu)₂-OH was synthesized according to a previous report.⁶⁾ 1,3-Diisopropylcarbodiimide (DIPCI) and 1-hydroxybenzotriazole hydrate (HOBt) were purchased from Watanabe Chemical Industries (Hiroshima, Japan). N,N-Diisopropylethylamine (DIPEA) was purchased from Nacalai Tesque (Kyoto, Japan). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Other chemicals and solvents were reagent grade and used as received. Electrospray ionization mass spectra (ESI-MS) were obtained with JEOL JMS-T100TD (JEOL, Tokyo, Japan). Purification was conducted using a RP-HPLC system (Prominence system, Shimadzu, Kyoto, Japan). The radioactivity was measured by an Auto Gamma System ARC-7010B (Aloka, Tokyo, Japan).

Synthesis and Radiolabeling:

Precursors and radiolabeled compounds of [^99mTc]Tc-HYNIC-(tricine)(AcP)-(l-Asp)₅ ([^99mTc]1), [^99mTc]Tc-HYNIC-(tricine)(AcP)-(l-Glu)₅ ([^99mTc]2), and [^99mTc]Tc-HYNIC-(tricine)(AcP)-(dl-Gla)₅ ([^99mTc]3) were prepared according to the procedure outlined in Chart 1.

Chart 1. Synthesis Scheme of [^99mTc]Tc-HYNIC-(tricine)(AcP)-(l-Asp)₅ ([^99mTc]1), [^99mTc]Tc-HYNIC-(tricine)(AcP)-(l-Glu)₅ ([^99mTc]2), and [^99mTc]Tc-HYNIC-(tricine)(AcP)-(dl-Gla)₅ ([^99mTc]3)

Reagents: (a) 2-Chlorotrityl chloride resin, DIPEA, DCM, (b) (1) 20% piperidine/DMF (2) Fmoc-l-Asp(OtBu)-OH or Fmoc-l-Glu(OtBu)-OH or Fmoc-dl-Gla(OtBu)₂-OH, HOBt, DIPCI, DMF, (c) Boc-HYNIC-OH, HOBt, DIPCI, DMF, and (d) 95% TFA 5% TIS, (e) [^99mTc]TcO₄^-, tricine, 3-acetylpyridine, SnCl₂.

Synthesis of Precursors:

Precursors, HYNIC-(l-Asp)₅, HYNIC-(l-Glu)₅, and HYNIC-(dl-Gla)₅ were synthesized manually using a standard Fmoc-based solid-phase methodology according to a previous report with a slight modification.³⁶⁾ In brief, Fmoc-l-Asp(OtBu)-OH (4.0 equiv.), Fmoc-l-Glu(OtBu)-OH (4.0 equiv.) or Fmoc-dl-Gla(OtBu)₂-OH (4.0 equiv.) and DIPEA (2.5 equiv.) were added to 2-chlorotrityl chloride resin (1.0 equiv.) suspended in dichloromethane (DCM). After shaking for 2 h, 1 mL of methanol was added to the mixture, and the mixing continued for another 30 min at room temperature. The peptide chain was constructed according to the cycle consisting of (I) 10 min of Fmoc deprotection with 20% piperidine in dimethylformamide (DMF) and (II) 1.5 h coupling of the Fmoc-l-Asp(OtBu)-OH (2.5 equiv.), Fmoc-l-Glu(OtBu)-OH (2.5 equiv.), or Fmoc-DL-Gla(OtBu)₂-OH (2.5 equiv.) with DIPCI (2.5 equiv.) and HOBt (2.5 equiv.) in DMF. The coupling reaction was repeated when the resin showed positive results in the Kaiser test to yield Resin-[l-Asp(OtBu)]₅, Resin-[l-Glu(OtBu)]₅, or Resin-[dl-Gla(OtBu)₂]₅. Furthermore, Boc-HYNIC-OH, which was synthesized according to the reported study,³⁷⁾ was coupled to the peptides in the same method to obtain Resin-[l-Asp(OtBu)]₅-HYNIC(Boc), Resin-[l-Glu(OtBu)]₅-HYNIC(Boc), or Resin-[dl-Gla(OtBu)₂]₅-HYNIC(Boc). Upon completion of the reaction, the reaction mixture was treated with triisopropylsilane : trifluoroacetic acid (TIS : TFA = 5 : 95). After 2 h of stirring at room temperature, the crude unprotected peptides, HYNIC-(l-Asp)₅, HYNIC-(l-Glu)₅, or HYNIC-(dl-Gla)₅ were obtained after crystallization of the mixture with ice-cooled diethyl ether. The crude HYNIC-(l-Asp)₅ and HYNIC-(l-Glu)₅ were purified by RP-HPLC on Cosmosil 5C₁₈-AR-II column (10 ID × 250 mm; Nacalai Tesque) at a flow rate of 4.0 mL/min with a gradient mobile phase of 10–25% methanol in water containing 0.1% TFA for 20 min. The crude HYNIC-(dl-Gla)₅ was purified by RP-HPLC on Cosmosil 5C₁₈-AR-II column (10 ID × 250 mm) at a flow rate of 4.0 mL/min with an isocratic mobile phase of 5% methanol in water containing 0.1% TFA for 20 min. Chromatograms were recorded by monitoring the UV absorption at 220 nm wavelength. After lyophilization, white solids of HYNIC-(l-Asp)₅, HYNIC-(l-Glu)₅, and HYNIC-(dl-Gla)₅ were obtained, and their molecular weights were determined by ESI-MS. LRMS (ESI+) analysis of HYNIC-(l-Asp)₅ calcd for C₂₆H₃₃N₈O₁₇ [M + H]⁺: m/z = 729.2 found 729.2. LRMS (ESI+) analysis of HYNIC-(l-Glu)₅ calcd for C₃₆H₄₃N₈O₁₇ [M + H]⁺: m/z = 799.3 found 799.4. LRMS (ESI+) analysis of HYNIC-(dl-Gla)₅ calcd for C₃₆H₄₃N₈O₂₇ [M + H]⁺: m/z = 1019.2 found 1019.2.

Radiolabeling with ^99mTc

An aliquot of 25 μg of HYNIC-(l-Asp)₅, HYNIC-(l-Glu)₅, or HYNIC-(dl-Gla)₅ was dissolved in 40 μL of 0.1 M borate buffer (pH 9.5). After adding 200 μL of tricine solution (30 mg/mL in 10 mmol/L citrate buffer, pH 5.2), 200 μL of 3-acetylpyridine solution (10 μL/mL in 10 mmol/L citrate buffer, pH 5.2), 100 μL of [^99mTc]TcO₄⁻ solution (7.4 MBq in saline), and 25 μL of SnCl₂ solution (1.0 mg/mL in 0.1 M HCl), respectively, the solution was heated at 90°C for 35 min. Tc-99m labeled compounds were purified by RP-HPLC on Cosmosil 5C₁₈-AR-II column (4.6 ID × 150 mm; Nacalai Tesque) at a flow rate of 1.0 mL/min with a gradient mobile phase of 15 to 40% methanol in water containing 0.1% TFA for 20 min for [^99mTc]1, 20 to 40% methanol in water containing 0.1% TFA for 20 min for [^99mTc]2, and 10 to 40% methanol in water containing 0.1% TFA for 20 min for [^99mTc]3. The column temperature was maintained at 40°C. Chromatograms were obtained by monitoring the UV absorption at a wavelength of 220 nm. An auto well gamma counter determined radiochemical yield and purity.

In Vitro Stability

The stabilities of radiotracers, [^99mTc]1, [^99mTc]2, or [^99mTc]3 in PBS and murine plasma were analyzed as described previously with a slight modification.³⁶⁾ Briefly, radiotracer solutions (37 kBq, 50 μL) in a sealed tube were diluted with 0.1 M PBS pH 7.4 (450 μL) and incubated at 37°C. After incubation for 3 h, the purities of radiotracers were analyzed by RP-HPLC. Meanwhile, for stability assay in murine plasma, radiotracers were mixed in murine plasma at a ratio of 1 : 10. After incubation at 37°C for 1 h, an equivalent amount of ice-cold acetonitrile was added. After centrifugation at 1000 × g at 4°C for 10 min, the supernatant was filtered through a 0.45-μm filter followed by RP-HPLC analysis as described above. Then, the radiochemical purities were determined.

Hydroxyapatite-Binding Assays

Hydroxyapatite-binding assays were conducted according to previously described procedures.³⁰⁾ In brief, hydroxyapatite beads (Bio-Gel; Bio-Rad, Hercules, CA, U.S.A.) were suspended in Tris–HCl-buffered saline (50 mM, pH 7.4) at 1.0, 2.5, 10, and 25 mg/mL. For the solutions of [^99mTc]1, [^99mTc]2, or [^99mTc]3, the ligand concentrations were adjusted to 19.5 μM by adding HYNIC-(l-Asp)₅, HYNIC-(l-Glu)₅, or HYNIC-(dl-Gla)₅, respectively. Two hundred microliters of each of [^99mTc]1, [^99mTc]2, or [^99mTc]3 solution was added to 200 μL of the hydroxyapatite suspension, and the samples were gently shaken (500 rpm) for 1 h at room temperature. After centrifugation at 10000 × g for 5 min, the radioactivity of the supernatants was measured using an auto well gamma counter. Control experiments were performed using the same procedure without hydroxyapatite beads. The binding ratios of hydroxyapatite were determined as follows:

  

$$\begin{array}{l}Hydroxyapatitebinding\left(\text{\%}\right)\\ =\left(1-\frac{samplesupernatantradioactivity}{controlsupernatantradioactivity}\right)\times 100\text{\%}\end{array}$$

Animal

Animal experiments were conducted under the Guidelines for the Care and Use of Laboratory Animals of Kanazawa University. The Committee on Animal Experimentation of Kanazawa University approved the animal experimental procedure of Kanazawa University. The animals were housed with free access to food and water at 23°C with a 12 h alternating light/dark schedule.

Biodistribution Experiments

Biodistribution experiments were performed after an intravenous administrating of each diluted tracer solution (92–130 kBq/100 mL) to 6-week-old male ddY mice (27–32 g, Japan SLC, Hamamatsu, Japan). Four mice at each time point of each compound were sacrificed at 10, 60, and 180 min postinjection. Tissues of interest were removed and weighed. Complete left femurs were isolated as representative bone samples, radioactivity counts were determined by an auto well gamma counter, and counts were corrected for background radiation and physical decay during counting.

SPECT/CT Imaging and Data Reconstruction

SPECT-CT imaging experiments of [^99mTc]3 in normal mice were performed using a small animal SPECT system (VECTor/CT, MIlabs, Houten, The Netherlands). SPECT scanning was performed for 1 and 3 h postinjection of [^99mTc]3 (approximately 37 MBq). Data were acquired in list mode, and photopeak windows were set after the acquisition. The energy windows employed were 126–154 keV. Data were reconstructed using pixel-based order-subsets expectation maximization, with correction for attenuation on CT, in 16 subsets and 6 iterations. The voxel size was 0.8 × 0.8 × 0.8 mm. The obtained SPECT/CT images were analyzed using an image-processing application (AMIDE Imaging software, version 1.0.4).

Statistical Analysis

The statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, U.S.A.). Significance in hydroxyapatite binding assay was determined using ANOVA followed by Tukey’s post hoc test. Results were considered statistically significant at p < 0.05, **p < 0.001.

Acknowledgments

This work was supported in part by SGH Foundation, Terumo life science foundation, the Directorate of Research, Technology & High Education, Ministry of Education, Culture, Research, and Technology of The Republic of Indonesia through Regular Fundamental Grant 2024 (111/E5/PG.02.00.PL/2024), The Management Talenta, National Research and Innovation Agency (BRIN), Indonesia through Visiting Researcher Program (3/II/HK/2023), and also The Indonesia Endowment Funds for Education (LPDP) through the Research and Innovation for Advanced Indonesia Batch 3 (12/II.7/HK/2023).

Author Contributions

Conceptualization, N.E. and K.O.; methodology, N.E., M.M., and K.O.; validation, N.E., M.M., A.M., A.R.P., B.B.P., R.R., X.H., T.F., K.M., R.J., and K.O.; formal analysis, N.E., M.M., and K.O.; investigation, N.E.; resources, H.W. and S.K.; writing—original draft preparation, N.E. and K.O.; writing—review and editing, N.E. and K.O.; visualization, N.E and B.B.P.,; supervision, K.O.; project administration, K.O.; funding acquisition, N.E., A.M., A.R.P., and K.O. All authors have read and agreed to the published version of the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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