[68Ga]/[188Re] Complexed [CDTMP] Trans-1,2-Cyclohexyldinitrilotetraphosphonic Acid As a Theranostic Agent for Skeletal Metastases

Objective Metastasis of the osseous tissue is one of the frequent and severe aggravations as a result of several neoplastic conditions, such as metabolic disorders, infections, and cancer. The objective of this study was to evaluate the pertinence of [68Ga]-trans-1,2-cyclohexyldinitrilo tetramethylene phosphonic acid (CDTMP) as a potential bone imaging agent for positron emission tomography (PET) applications as well as to assess [188Re]-CDTMP for bone pain palliation in metastatic skeletal disorders. Methods 68Ga complex of CDTMP was prepared at 80°C at pH 3.5, and 188Re complex of CDTMP was prepared at room temperature. [68Ga]-CDTMP complex was investigated as PET tracer while the therapeutic efficacy was assessed for [188Re]-CDTMP. Labeling efficiency, biodistribution, myelotoxicity, and imaging studies were carried out for the complexes synthesized. Both PET and MicroPET imaging studies were performed for [68Ga]-CDTMP whereas SPECT acquisitions were acquired for [188Re]-CDTMP. Data were analyzed semiquantitatively for all the scintigraphic scans obtained. Results The radiolabeling efficiency was observed to be >70% for [68Ga]-CDTMP. High bone uptake of [68Ga]-CDTMP as compared to contralateral tissue was found in PET imaging in Balb/C mice and New Zealand rabbit; the similar result for bone uptake was correlated in the biodistribution study of the compound in BALB/c mice at different time intervals. Biodistribution experiments carried out in mice showed maximum uptake of 6.12 ± 1.22%ID/g at 45 min postinjection. For [188Re]-CDTMP, total skeletal uptake was 8.12 ± 1.11%ID/g observed at 1 h postinjection from biodistribution data. High renal uptake confirms renal route of excretion. A good hydroxyapatite binding too was seen for both the complexes. No evidence of destruction or adverse functioning of vital organs was observed for the 188Re complex. Conclusion [68Ga]-CDTMP complex can be used as a promising PET bone imaging agent and [188Re]-CDTMP as a surrogate moiety for therapeutic application. Owing to the short half-life of 68Ga (68 min), cyclotron-independent radiopharmacy, fast clearance, and rapid renal excretion as evidenced in preclinical animal models. Very low myelotoxicity and highly selective bone uptake prove the potential of [188Re]-CDTMP for therapeutic application.

Objective: Metastasis of the osseous tissue is one of the frequent and severe aggravations as a result of several neoplastic conditions, such as metabolic disorders, infections, and cancer. The objective of this study was to evaluate the pertinence of [ 68 Ga]trans-1,2-cyclohexyldinitrilo tetramethylene phosphonic acid (CDTMP) as a potential bone imaging agent for positron emission tomography (PET) applications as well as to assess [ 188 Re]-CDTMP for bone pain palliation in metastatic skeletal disorders.
Methods: 68 Ga complex of CDTMP was prepared at 80°C at pH 3.5, and 188 Re complex of CDTMP was prepared at room temperature. [ 68 Ga]-CDTMP complex was investigated as PET tracer while the therapeutic efficacy was assessed for [ 188 Re]-CDTMP. Labeling efficiency, biodistribution, myelotoxicity, and imaging studies were carried out for the complexes synthesized. Both PET and MicroPET imaging studies were performed for [ 68 Ga]-CDTMP whereas SPECT acquisitions were acquired for [ 188 Re]-CDTMP. Data were analyzed semiquantitatively for all the scintigraphic scans obtained.
results: The radiolabeling efficiency was observed to be >70% for [ 68 Ga]-CDTMP. High bone uptake of [ 68 Ga]-CDTMP as compared to contralateral tissue was found in PET imaging in Balb/C mice and New Zealand rabbit; the similar result for bone uptake was correlated in the biodistribution study of the compound in BALB/c mice at different time intervals. Biodistribution experiments carried out in mice showed maximum uptake of 6.12 ± 1.22%ID/g at 45 min postinjection. For [ 188 Re]-CDTMP, total skeletal uptake was 8.12 ± 1.11%ID/g observed at 1 h postinjection from biodistribution data. High renal uptake confirms renal route of excretion. A good hydroxyapatite binding too was seen for both the complexes. No evidence of destruction or adverse functioning of vital organs was observed for the 188 Re complex.
conclusion: [ 68 Ga]-CDTMP complex can be used as a promising PET bone imaging agent and [ 188 Re]-CDTMP as a surrogate moiety for therapeutic application. Owing to the short half-life of 68 Ga (68 min), cyclotron-independent radiopharmacy, fast clearance, and rapid renal excretion as evidenced in preclinical animal models. Very low myelotoxicity and highly selective bone uptake prove the potential of [ 188 Re]-CDTMP for therapeutic application. [  inTrODUcTiOn Abnormal growth associated with neoplastic conditions, such as anomalous metabolic disorders, contagions, and malignancies result in metastasis in the skeletal tissue. Bone metastasis is prevalent among the majority of patients with metastatic conditions, which results in a high percentage of mortality and morbidity. These skeletal-related events that involve hypercalcemia caused by malignancy, pathological fractures, etc., and thus arises the requirement for surgical intervention or radiation treatment in bone. Published reports indicate toward the early occurrence of bone metastases; a tumor disease, however, the condition remains clinically asymptomatic and is recognized rather late (1,2). Bone imaging is one of the medical imaging techniques in nuclear medicine in which whole body metastasis assessment is performed using radiotracers such as [ 99m Tc]-phosphonates and [ 18 F]-fluoride. In the current scenario, a variety of [ 99m Tc] based tracers are being used for skeletal metastasis evaluation. Radiolabeled phosphonates are of utmost interest in nuclear medicine for metastasis evaluation as well as for therapy as many common tumors are associated with metastasis. Bone-targeting radiopharmaceuticals labeled with β emitters are acceptable adjuvant to external beam therapy for treatment of bone pain in osteoblastic bone metastatic condition. It has been reported that bone pain from metastatic disease occurs more profoundly in patients with malignancies in organs such as prostate, breast, and lung. Bone imaging and bone pain palliation are two distinct procedures followed in nuclear medicine. Phosphonates and phosphonic acids show high affinity for inorganic hydroxide and oxide materials and hence explored as the ideal targeting groups for hydroxyapatite (HAp) and bone. Bisphosphonate moieties radiolabeled with therapeutic isotopes are the category of pharmaceuticals that are widely accepted options for treatment of osteoporosis; a condition associated with an imbalance between osteoblastic and osteoclastic activities. Minimum exposure and maximum resolution, which signify a minimum radiation burden to the patient, as well as minimization of interfering signals in the scintigraphic image, require high specific skeletal uptake of the radiotracer (3). Ga-68-based radiopharmaceuticals for imaging bone thus found interest over 18 F-NaF because of its longer half-life of 110-min and high radiation burden imposed to the patient (4-6). A therapeutic agent employed in radionuclide therapy consists of two components; α particle, β particle or Auger electron-emitting radioisotope and the molecular targeting vector that ensures maximum accumulation at or in desired tumor cells (7). Polyphosphonates (PPs) such as MDP (methylenediphosphonate), DPD (3,3-diphosphono-1,2-propandicarboxylic acid), and EDTMP (ethylenediamino-N,N,N′,N′-tetrakismethylenephosphoric acid) are already established as suitable ligands for targeting therapeutic radionuclide into bone tissue (8). Various radiopharmaceuticals are in clinical use like [ 153 Sm]ethylene diamine tetramethylene phosphonate (Quadramet) and rhenium-186-hydroxyethylidenediphosphonic acid ([ 186 Re]-HEDP), all having unique characteristics (9,10).
As compared to [ 99m Tc]-PPs, [ 18 F]-fluoride has potential advantages of high sensitivity and increased spatial resolution due to advanced positron emission tomography (PET) technology (11,12), thus arises a need for ligand-based tracers with improved diagnostic accuracy for PET application. Apart from 18 F, 68 Ga is another potential radionuclide for PET imaging. Because of advances and commercial availability of 68 Ge/ 68 Ga generators, 68 Ga-radiolabeled PET tracers have found considerable interest.
[ 68 Ga]-phosphonates for PET bone imaging earns special mention, because of its easy availability from an in-house generator thus, overcoming the requirement of an on-site cyclotron facility. [ 68 Ga]-labeled phosphonates also prevail over the practical question of higher costs associated with the cyclotron-based production of [ 18 F]-fluoride as compared to a generator-based production and easy radiopharmacy, along with the limitation of on-site cyclotron access. 188 Re is known to possess an excellent therapeutic potential with a short half-life value of 16.9 h, maximum particle energy of 2.1 MeV, and 10 mm of maximum beta range in tissue (13). Availability of ( 188 W/ 188 Re) generator offers momentous advantages regarding easy access and convenience when compared to other therapeutic radionuclides in clinical use. We were thus motivated to evaluate already existed PP, CDTMP as an alternative for [ 68 Ga]-labeled known compounds with expected good uptake in bone as well as a therapeutic alternative for bone pain palliation in skeletal metastasis. The present study focuses on    negative ion mode) were performed on in-house Agilent 6310 system ion trap.

animal studies
All the animal studies were done according to approved protocols of Institutional animal ethics committee (CPCSEA Regn No. 8/GO/RBi/S/99). New Zealand white Rabbits (2-3 kg), Wistar rats (200 ± 10 g), and Balb/C mice (25 ± 3 g) were used in experiments. Animals were housed under a controlled temperature condition of 22 ± 2°C and normal diet ad libitum.

PeT imaging experiments
Both PET and MicroPET acquisitions were performed. PET Imaging data were acquired on GE Discovery STE 16 system (PET/CT) system using New Zealand rabbits. PET data were reconstructed using vendor provided ordered-subsets expectation maximization. MicroPET imaging was performed on GE FLEX Triumph MicroPET/SPECT/CT scanner, which consists of a MicroPET module (LabPET 4) with 2 m × 2 m × 10 m, LYSO/ LGSO scintillators in an 8-pixel, quad-APD detector module arrangement. The rabbit was injected intravenously through ear vein whereas mice were injected through the tail vein, and imaging was done 45 min postinjection for [ 68 Ga]-CDTMP. The subject was anesthetized with ketamine (4.8 mg/kg), and midazolam (1.2 mg/kg) administered intramuscularly and was placed prone on PET scanner (37°C). Image reconstruction was done using Amira 4.1.1. Semiquantitative analysis was performed using Amide Software.
sPecT imaging experiment synthesis CDTMP (Figure 1) was synthesized by single step method previously reported by Panwar et al. (14). In brief, to a solution of trans-CDTA (0.5 g, 1.37 mmol) dissolved in 10 mL toluene, phosphorus acid (0.48 g, 6.85 mmol) was added. The mixture was then refluxed at 100°C while phosphorus trichloride (0.75 g, 0.755 mmol) in a volume of 300 mL was added drop wise to the refluxing mixture. After 3 h, the solvent was removed followed by addition of deionized water. The filtrate was concentrated under vacuum. The concentrated product was then precipitated by addition of ethanol to obtain the pure product. The formation of final product CDTMP was confirmed by Mass and NMR spectroscopy.

gallium complexation studies
To a solution of the CDTMP, 25 mg (0.05 mmol) in water was added Ga(NO3)3, 15 mg (0.06 mmol) at pH adjusted to 5.5 using sodium acetate. The reaction mixture was then heated at 90°C and stirred for 8 h. It was then allowed to cool and passed through the C-18 cartridge to remove unreacted species. The mixture was then filtered through a 0.22 μm syringe filter, freeze dried, to yield a white solid with a purity of >95% (15). Formation of the complex was confirmed by Mass spectroscopy.

radiochemical synthesis of [ 68 ga]-cDTMP
The 68 Ge/ 68 Ga generator was eluted according to manufacturer protocol (Eckert and Ziegler) with 5 mL of 0.1 N hydrochloric acid (330-520 MBq). 2 mL of above eluate was used for labeling. A stock solution of CDTMP (1 mg/mL water) was prepared, and a 200 µL aliquot was used in labeling studies. Labeling was carried out in Na-HEPES buffer with pH adjusted between 3 and 3.5. The reaction mixture was heated for 10 min at 80°C, passed through C-18 cartridge and eluted with ethanol. Radiolabeling yield was determined by ITLC/SG 1 cm × 8 cm strips (methanol/ammonium acetate 1 M; 1:1) for each step of the experiments.

HAp Binding Studies
To evaluate HAp binding, 20 mg of synthetic HAp was allowed to incubate in 1 mL of isotonic saline for 24 h. Labeled complex in a volume of 50 µL was then added, subjected to vortex for 10 s and incubated at ambient temperature for 10 min. After incubation, samples had been centrifuged, and the supernatant was removed. Hap fraction was washed twice with 0.5 mL saline, and all the washes were collected. Radioactivity was then checked in combined liquids and HAp fraction using gamma counter.

Myelotoxicity Studies
Myelotoxicity studies were performed on male Wistar rats. Myelotoxicity was evaluated by blood cell count (RBC, WBC, and platelets) post 75 MBq [ 188 Re]-CDTMP intravenous injection at selected time points up to 16 days. Blood samples were withdrawn using heparinized capillaries in EDTA coated vials, and blood cell count was recorded on an automatic analyzer (CellTac α MEK6450K, Nihon Kohden).

Decay Correction
All the data including blood clearance studies, biodistribution, and imaging were subjected to decay correction. resUlTs radiolabeling Before radiocomplexation of CDTMP, cold complexation of CDTMP was performed with cold gallium nitrate, which was characterized by mass spectroscopy (see Figure S1 in Supplementary Material). Radiochemical yield of [ 68 Ga]-CDTMP was optimized for various conditions of pH, temperature, and compound concentration chromatographically using ITLC-SG (Figure 2). The optimum pH for labeling was 3-3.5, and deterioration of radiolabeling yield was observed at higher pH. The radiolabeling efficiency was found to be 70 ± 1%. [ 188 Re-CDTMP] too was labeled with radiochemical purity higher than 97 ± 1.2%. It was found that addition of 100 µL of 0.1 M ascorbic acid increases the stability of complex. Radiochemical purity for [ 68 Ga]-CDTMP was assessed using EZ-TLC Scanner (see Figure  S2 in Supplementary Material) Radio-HPLC analysis after tc-18 cartridge purification was performed for [ 68 Ga]-CDTMP, which showed the presence of only one radiolabeled species at a retention time of 4.35 min (see Figure S3 in Supplementary Material  (Figure 3).

Blood Clearance
Blood kinetic studies in rabbits showed that the complex [ 68 Ga]-CDTMP followed a biphasic pattern of clearance. There was a rapid clearance of the [ 68 Ga]-CDTMP from the body. After 1 h, the clearance followed a slow pattern, and at 4 h approximately 18% of the activity remained in the blood (Figure 4). The biological half-life was obtained to be t1/2 (fast): 12 min; t1/2 (slow): 3 h and 10 min. A rapid washout of [ 68 Ga]-CDTMP favors the early imaging after administration as well as reduces the radiation burden to the subject.

Scintigraphy Studies
[ 68 Ga]-CDTMP PET and MicroPET imaging were performed on New Zealand rabbit and Balb/C mice, respectively. Imaging was done at 45 min postinjection for both mice and rabbit. High bone uptake as compared to contralateral tissue was observed, which attributes to its use as a bone imaging agent. Both PET and MicroPET images were validated by CT, and fused PET/CT   images were also obtained (Figures 5 and 6). Volume of interest analysis of the PET showed a bone to muscle ratio of 21.258 ± 1.3. Semiquantitative analysis of the MicroPET images depicts bone to muscle ratio of 24.146 ± 0.98, bone to kidney ratio of 7.976 ± 0.78. Imaging the [ 68 Ga]-CDTMP uptake in a healthy animals both mice and rabbits showed that the tracer accumulation in bones was prominent. Under physiological conditions, a high activity in the joints was observed. Thus, the [ 68 Ga]-CDTMP accumulation observed was greater in the shoulder and along the backbone as shown in [ 68 Ga] PET and MicroPET images suggestive of preferential accumulation of radiotracer in the metabolically active regions of bone. Scintigraphic images of [ 188 Re]-CDTMP for skeletal uptake in rabbits were taken at 1, 2, and 24 h postinjection. Images revealed high selective skeletal uptake with insignificant non-osseous tissue accumulation (Figure 7).

Biodistribution Studies
Biodistribution experiments for [ 68 Ga]-CDTMP carried out in mice showed maximum bone uptake of 6.12 ± 1.22 at 45 min postinjection (Figure 8). High kidney uptake observed for both the complexes reveals the renal route of excretion. For [ 188 Re]-CDTMP, total skeletal uptake was 8.12 ± 1.11%ID/g observed at 1 h from biodistribution studies (Figure 9). Biodistribution data for blood for both radiolabeled complexes depict a shorter     Figure 10A). Bone to blood ratio was also calculated for both the radiolabeled complexes, which clearly showed significant bone uptake (Figures 10B,C).

Myelotoxicity Studies
In myelotoxicity studies after 4 h, 1, 3, and 5 days of [ 188 Re]-CDTMP injection in Wistar rats, there was a slight decrease in white blood cell counts of the animals postinjection ( Table 1).
After 7 days, there was no significant difference between RBC and Hb from baseline. In the hematological evaluation, platelets were found to be the most sensitive cells. With every therapeutic dose of [ 188 Re]-CDTMP, a decrease in the number of platelets was observed. Insignificant decrease of platelet counts was noted in the first period (7 days), which was found to recover in a period

DiscUssiOn
Development of [ 68 Ga]-labeled bone-seeking agents could lead to further improvement in bone imaging using PET independent of cyclotron-based radiopharmacy. After an easy and short radiolabeling procedure, [ 68 Ga]-CDTMP forms a stable complex assessed in vivo and thus has a good potential for use as radiotracer. An easy, fast and reliable kit-based preparation protocol could enable PET facilities without on-site cyclotron to perform PET bone scans. In clinical scenario, [ 99m Tc]-MDP and [ 99m Tc]-HMDP are widely accepted bone scintigraphy agents, but their optimized chemical and pharmaceutical perspective have not yet been optimized as these complexes are not pure single chemical species but are mixtures of short-chain and long-chain oligomers (17). Synthetic and radiosynthetic ([ 68 Ga]-CDTMP and [ 188 Re]-CDTMP) approach reported that CDTMP eliminates the formation of undesired intermediate products thereby obviating the need for its further purification.
One of the major factor considered for selective localization of a radiotracer in bone is active bone mineralization of HAp [Ca10(PO4)6(OH)2], which is the main mineral constituent of bones. HAp-binding assay was carried out to evaluate phosphonates interaction with skeletal tissue. The uncontrolled change in mineralization of HAp is the basis for phosphonates bone imaging. HAp-binding studies were performed to assess the adsorption behavior of radiolabeled complex. Gallium-based radiopharmacy of CDTMP overcomes the need of in-house cyclotron facility as well as enhanced spatial resolution, and quantification properties of PET technique are much superior to conventional SPECT technique. Much advanced imaging results of PET/CT, as compared to SPECT/CT, are clearly demonstrated by using [ 18 F]fluoride (18). 68 Ga possess a half-life of 68 min; positron emission fraction of β + 89%; and E + β max 1.9 MeV is a positron-emitting nuclide reported with a remarkable clinical imaging potential, as the radionuclide can be easily obtained on-site from 68 Ge/ 68 Ga generators (19)(20)(21). A review of published reports of bone-seeking agents such as [68Ga]-labeled citrate (22)   Rhenium nuclides have γ emissions, which allow for easy detection of distribution pattern and clearance by gamma scintigraphy. The success of the bone pain palliation therapeutic treatment by a particular radiopharmaceutical depends upon the activity administered and percentage uptake of bone. The radioactivity administered and the extent of radiopharmaceutical uptake by bone are the two most important properties considered as the determinants of the success of the treatment with therapeutic radioisotope. To evaluate the skeletal uptake of the radiolabeled phosphonates used in this study, we employed the method that is based on conventional ROI technique and allows compartmental calculation for bone and soft tissue. The preclinical evaluation shows its therapeutic efficacy as [ 188 Re]-CDTMP. [ 188 Re-CDTMP] was labeled with radiochemical purity higher than 97% and showed a bone uptake of ~70%. The evaluated compound CDTMP showed fast and high bone absorption and moderate soft tissue accumulation in soft tissue. The renal clearance time proved to be longer for [ 188 Re-CDTMP], and a high activity was also observed in the skeleton was constant for 96 h, which clearly reveals the long biological half-life of [ 188 Re-CDTMP] in the target tissue. Similar results were also observed for [ 177 Lu]-DOTAZOL and [ 68 Ga]-DOTAZOL (28). The rapid accumulation of agent in bone and high target-to-non-target uptake ratio showed that CDTMP has both therapeutic potential as when coupled to β-emitting radionuclides.
The present study suggests that [ 68 Ga]-labeled CDTMP may serve as a promising vector in nuclear medicine for PET imaging and bone pain palliation with a good bone accumulation property. In vivo PET evaluation revealed predominant uptake in bone with low non-target accumulation and rapid non-osseous clearance. Apart from present assessment, future clinical prospects of [ 68 Ga]-CDTMP require further evaluations and preclinical trials. Moreover, the present study also describes the therapeutic potential of [ 188 Re]-CDTMP as a good therapeutic probe in preclinical studies. Being generator-based radioisotopes 68 Ga and 188 Re have easy availability; single step synthetic strategy of CDTMP both facilitates an on-site simple radiopharmacy with low costs.

eThics sTaTeMenT
This study was carried out in accordance with the recommendations of (CPCSEA Regn No.8/GO/RBi/S/99). The protocol was