Evaluation of Ga-DOTA-(D-Asp)n as bone imaging agents: D-aspartic acid peptides as carriers to bone

67Ga-DOTA-(L-Asp)11 and 67Ga-DOTA-(L-Asp)14, which have been developed as bone imaging agents, showed a high accumulation in bone and a rapid blood clearance in mice. However, peptides composed of D-amino acids are more stable in vivo than those composed of their L-equivalents. In this study, 67Ga-DOTA-(D-Asp)n (n = 2, 5, 8, 11, or 14) were synthesized using the Fmoc-based solid-phase methodology and evaluated. In hydroxyapatite binding assay, binding of 67Ga-DOTA-(D-Asp)n tended to increase with increasing length of the amino acid chain. 67Ga-DOTA-(D-Asp)11 and 67Ga-DOTA-(D-Asp)14 caused a high accumulation of radioactivity in the bones of the mice. However, the results for 67Ga-DOTA-(D-Asp)n and 67Ga-DOTA-(L-Asp)n were comparable. In urine analyses, the proportion of intact complex after injection of 67Ga-DOTA-(D-Asp)14 was significantly higher than that of 67Ga-DOTA-(L-Asp)14. Although 67Ga-DOTA-(D-Asp)14 was more stable than 67Ga-DOTA-(L-Asp)14, the properties of 67Ga-DOTA-(D-Asp)n and 67Ga-DOTA-(L-Asp)n as bone imaging agents may be comparable.

Recently, the performance of X-ray computed tomography (CT) and magnetic resonance imaging (MRI) method has been greatly improved, particularly in terms of their spatial resolution and technology for reconstructing the acquired images. Nuclear medicine imaging has been considered to be the most sensitive approach for diagnosing bone disorders such as bone metastases due to its ability to enable the early detection of abnormalities, namely, visualization of lesion sites before anatomical changes. For a long time, 99m Tc-methylenediphosphonate ( 99m Tc-MDP) and 99m Tc-hydroxymethylenediphosphonate ( 99m Tc-HMDP) have been widely used in bone imaging [1][2][3][4][5] . Because 99m Tc has the convenient physical characteristics [moderate half-life (6.01 h) for clinical use, a generator-produced radionuclide, and appropriate gamma ray energy for imaging] and imaging methods using conventional gamma cameras are simple. 99m Tc-MDP and 99m Tc-HMDP are complexes of 99m Tc with bisphosphonate analogs having high affinity for bone since the phosphate groups in the bisphosphonate can be coordinated with calcium in hydroxyapatite crystals in bone.
The use of [ 18 F]NaF for bone imaging was initially reported by Blau et al. in 1962 6 and approved by the US Food and Drug Administration in 1972. [ 18 F]NaF accumulates at a high level in bone because of chemisorption with the exchange of fluoride anions with the hydroxyl groups in hydroxyapatite [Ca 10 (PO 4 ) 6 (OH) 2 ]. However, [ 18 F]NaF had not been widely used due to its limited availability and high cost, but it has recently been reevaluated. The images obtained using clinical positron emission tomography (PET) generally have high spatial resolution and PET/CT scanners have become widely available commercially. Although Even-Sapir et al. reported that [ 18 F]NaF PET imaging is significantly more sensitive than 99m Tc-MDP planar and 99m Tc-MDP single photon emission computed tomography (SPECT) imaging 7 , the problems of limited availability and the high cost of cyclotrons have remained unresolved.
In recent years, 68 Ga (T 1/2 = 68 min) has drawn substantial attention as a positron emission radionuclide for clinical PET because of its attractive radiophysical properties, such as reasonable half-life for clinical use; it has particularly been used as a generator-produced radionuclide. 68 Ga-PET does not require an on-site cyclotron because 68 Ga can be eluted from the generator on demand. Moreover, as the parent nuclide, 68 Ge (T 1/2 = 271 days) has a long half-life, a generator could be used for a long period. Therefore, the demand for 68 Ga-labeled compounds for the diagnosis of bone disorders, such as bone metastases, has increased. Some new radiogallium-labeled complexes for bone imaging have been developed in recent years [8][9][10][11][12][13][14] . Bisphosphonate analogs are used as carriers in these radiogallium-labeled complexes. For example, Fellner et al. reported that 68 Ga-DOTA-conjugated bisphosphonate, 68 Ga-BPAMD, showed high uptake in osteoblastic metastatic lesions in a first human PET study 15 . In addition, Suzuki et al. reported that 68 Ga-NOTA-conjugated bisphosphonate, 68 Ga-NOTA-BP, showed high bone affinity and rapid blood clearance in animal experiments 10 .

Discussion
It has been shown that the bisphosphonate structure is very useful as a carrier of physiologically active molecules or compounds with medicinal properties. This is particularly true for bone lesions because of the high affinity of bisphosphonate for hydroxyapatite, which is plentiful in bone but not in soft tissues 21,22 . Stable radiometal complex-conjugated bisphosphonate compounds have been designed as bone-seeking radiopharmaceuticals; they have been synthesized and evaluated for the diagnosis and therapy of bone metastases 11,[23][24][25][26][27][28][29][30] . The available data show that bisphosphonate is an excellent carrier of radioisotopes to bone lesions. Our recent study has shown that L-aspartic acid peptides could also work as carriers of radioisotopes to bone lesions; L-aspartic acid peptides have high affinity for hydroxyapatite 19,31 . Thus, we assumed that D-aspartic acid peptides might be even better carriers. They should have a similar degree of affinity for hydroxyapatite but higher stability in vivo than the L-aspartic acid compounds.
SCIEntIFIC REPORTs | 7: 13971 | DOI:10.1038/s41598-017-14149-7 In the hydroxyapatite-binding assay, 67 Ga-DOTA-(D-Asp) n with a longer amino acid chain showed higher affinity for hydroxyapatite than the short-chain compounds. The binding patterns of 67 Ga-DOTA-(D-Asp) n were almost the same as those of 67 Ga-DOTA-(L-Asp) n 19 . A previous study reported that the dissociation constants and the maximal binding rates of Fmoc-peptide compounds for hydroxyapatite show no significant differences among Fmoc-(L-Asp) n , Fmoc-(D-Asp) n , and Fmoc-(L-Glu) n (n = 2, 4, 6, 8, 10) 32 . This is consistent with the results of hydroxyapatite binding assay in our study. We found that aspartic acid peptides had the same degree of affinity for hydroxyapatite regardless of their optical isomeric form. Moreover, there were no differences between the affinities of aspartic acid peptides and glutamic acid peptides for hydroxyapatite.
In in vivo studies, it is known that the peptides that composed of D-amino acids are more stable than the L-amino acid peptides 20 . A study examining the Fmoc compounds reported that, after a single i.v. administration, the plasma concentration of Fmoc-(L-Asp) 6 decreased more rapidly than the concentration of Fmoc-(D-Asp) 6 . Degradation products did not appear in the plasma after the injection of Fmoc-(D-Asp) 6 , but Fmoc-(L-Asp) 4 and Fmoc-(L-Asp) 2 were detected in plasma after the injection of Fmoc-(L-Asp) 6 32 . Therefore, we had expected to observe increased accumulation in bone after the injection of 67 Ga-DOTA-(D-Asp) n , caused by their superior in vivo stability. In urine analyses, 67 Ga-DOTA-(L-Asp) 14 metabolized to more hydrophilic complexes, which should be 67 Ga-DOTA conjugated with shorter aspartic acid peptides, because of the cleavage of an amide bond in the peptide. These compounds were diluted before the full-length compound during the RP-HPLC using an ODS column. This indicates that 67 Ga-DOTA-(D-Asp) 14 is more stable than 67 Ga-DOTA-(L-Asp) 14 . Since 67 Ga-DOTA conjugated with shorter aspartic acid peptides should show lower accumulation in bone than 67 Ga-DOTA conjugated with long aspartic acid peptides, we expected that 67 Ga-DOTA-(D-Asp) n , which has higher stability, would show higher accumulation in bone than 67 Ga-DOTA-(L-Asp) n . However, against our expectations, the accumulation of radioactivity in bone was comparable for 67 14 , and the 67 Ga-DOTA-conjugated D-glutamic acid peptide, 67 Ga-DOTA-(D-Glu) 14 , also showed rapid clearance from the blood and high accumulation in bone, similarly to 67 Ga-DOTA-(L-Asp) 14 and 67 Ga-DOTA-(D-Asp) 14 . Generally, radiometal-labeled peptides tend to show a high accumulation of radioactivity in the kidneys. It has been reported that the accumulation of radioactivity in the kidneys after the injection of 111 In-labeled peptides is affected by their molecular charges 33,34 . As the renal brush border membrane is negatively charged, a repulsive force could arise between this membrane and negatively charged compounds. Such repulsive force could inhibit the reabsorption of these compounds into renal proximal tubular cells. The introduction of negative charges into radiometal-labeled peptides has also been studied to develop a method of decreasing the accumulation of radioactivity in the kidneys 35 The extremely low accumulation of radioactivity in the kidneys after the injection of 67 Ga-DOTA-(L-Asp) 14 and  Table 3. Biodistribution of radioactivity after i.v. injection of 67 Ga-DOTA-(D-Asp) 8 in mice a . a Expressed as % injected dose. Each value represents the mean (SD) for five animals. b Expressed as % injected dose. c Femur:blood ratio. 67 Ga-DOTA-(D-Asp) 14 may have been caused by their negative charges. We had expected that 67 Ga-DOTA-(L-Glu) 14 and 67 Ga-DOTA-(D-Glu) 14 , being negatively charged like 67 Ga-DOTA-(L-Asp) 14 and 67 Ga-DOTA-(D-Asp) 14 , would also cause low accumulation of radioactivity in the kidneys. However, contrary to our expectations, high accumulation and retention or slower clearance of radioactivity in the kidneys were observed after the injection of 67 Ga-DOTA-(L-Glu) 14 or 67 Ga-DOTA-(D-Glu) 14 . The mechanism behind these phenomena are unclear, but we must conclude that the glutamic acid peptides are not appropriate as carriers to the bone in the nuclear medicine imaging because of their association with high radioactivity in the kidneys. In this study, no differences in the biodistributions between L-aspartic acid [ 67 Ga-DOTA-(L-Asp) n ] and D-aspartic acid [ 67 Ga-DOTA-(D-Asp) n ] compounds were observed, presumably because of their extremely rapid blood clearance. Recently, we have proposed a new concept of using a bifunctional peptide containing an aspartic acid peptide linker as a carrier to bone metastases and an RGD peptide, which has high affinity for α v β 3 integrin, as a carrier to primary cancer 31 . In this compound, L-aspartic acid is used as a composite component of the aspartic acid peptide linker. A D-aspartic acid peptide linker may be effective in the new approach. Higher stability of the D-aspartic acid peptide linker should be effective for higher accumulation in target tissues because the blood clearance of bifunctional peptide does not occur as rapidly as that of 67 2, 5, 8, 11, or 14). The protected peptidyl resin was manually constructed by an Fmoc-based solid-phase methodology using a method described previously 19 . After   the construction of the peptide chain on the resin, the Fmoc protecting group was removed using 20% piperidine in dimethylformamide (DMF), and a mixture containing two equivalents of DOTA-tris, 1,3-diisopropylcarbodiimide (DIPCDI), and 1-hydroxybenzotriazole hydrate (HOBt) in dimethylformamide (DMF) was added and allowed to react for 2 h. For the cleavage of peptides from the resin and deprotection, 0.5 mL of thioanisole and 5 mL of trifluoroacetic acid (TFA) were added to the completely protected peptide resin at 0 °C. After stirring at room temperature for 2 h, the resin was removed by filtration, and ether was added to the filtrate at 0 °C to precipitate crude peptide. The crude products were purified by reversed-phase (RP)-HPLC using a Hydrosphere 5C18 column (10 × 150 mm; YMC, Kyoto, Japan) at a flow rate of 4 mL/min with an isocratic mobile phase of water containing 0.1% TFA [in the case of DOTA-(D-Asp) 2 ] or using a Cosmosil 5C 18     Urine Analyses. 67 Ga-DOTA-(L-Asp) 14 was prepared according to a method described previously 19 . 67 Ga-DOTA-(L-Asp) 14 or 67 Ga-DOTA-(D-Asp) 14 solution (370 kBq / 200 μL) was intravenously injected to 6-week-old male ddY mice. At 1 h post-injection, mice were sacrificed and their urea samples were taken from the bladders. After ultrafiltration (Microcon-30, Merck KGaA), the filtrate samples were analyzed by RP-HPLC at a flow rate of 1 mL/min with a gradient mobile phase from water containing 0.1% TFA to 20% methanol in water containing 0.1% TFA for 20 min.