Synthesis,Quality Control,and Bench-to-Bed Translation of a New [ 68 Ga]Ga-Labeled NOTA-Conjugated Bisphosphonate for Imaging Skeletal Metastases by Positron Emission Tomography

Abstract

Background

: Early detection of skeletal metastasis is of great interest to determine the prognosis of cancer. Positron emission tomography-computed tomography (PET-CT) imaging provides a better temporal and spectral resolution than single photon emission computed tomography-computed tomography (SPECT-CT) imaging, and hence is more suitable to detect small metastatic lesions. Although [¹⁸F]NaF has been approved by U.S. FDA for a similar purpose, requirement of a medical cyclotron for its regular formulation restricts its extensive utilization. Efforts have been made to find suitable alternative molecules that can be labeled with ⁶⁸Ga and used in PET-CT imaging.

Objective:

The main objective of this study is to synthesize and evaluate a new [⁶⁸Ga]Ga-labeled NOTA-conjugated geminal bisphosphonate for its potential use in early detection of skeletal metastases using PET-CT.

Methods:

The authors performed a multistep synthesis of a new NOTA-conjugated bisphosphonic acid using thiourea linker and radiolabeled the molecule with ⁶⁸Ga. The radiolabeled formulation was evaluated for its in vitro stability, affinity for hydroxyapatite (HA) particles, preclinical biodistribution in animal models, and PET-CT imaging in patients.

Results:

The bifunctional chelator (NOTA)-conjugated bisphosphonate was synthesized with 97.8% purity and radiolabeled with ⁶⁸Ga in high yield (>98%). The radiolabeled formulation was found to retain its stability in vitro to the extent of >95% up to 4 h in physiological saline and human serum. The formulation also showed high affinity for HA particles in vitro with K_d = 907 ± 14 mL/g. Preclinical biodistribution studies in normal Wistar rats demonstrated rapid and almost exclusive skeletal accumulation of the complex. PET-CT imaging in a patient confirmed its ability to detect small metastatic skeletal lesions.

Conclusions:

The newly synthesized [⁶⁸Ga]Ga-labeled NOTA-conjugated bisphosphonate is a promising radiotracer for PET-CT imaging for skeletal metastases.

Introduction

Tumors, particularly of breast and prostate origin, metastasize into bone, leading to poor prognosis of cancer.^1,2 Bone metastasis patients experience severe loss in quality of life, mainly originating from severe pain, loss of mobility, and hypercalcemia.³ Early diagnosis of bone metastasis is important to determine the therapeutic modalities.⁴ Currently, bone-seeking radiopharmaceuticals having a bisphosphonate moiety are the agents of choice for diagnosis of bone metastasis.

In principle, bone metastasis may be visualized by either single photon emission computed tomography (SPECT) or positron emission tomography (PET). Among these, PET offers a better spectral and temporal resolution and, therefore, leads to the detection of very small metastases, which otherwise could not be imaged by SPECT.⁵ Although [^99mTc]Tc-labeled bisphosphonate namely [^99mTc]Tc-MDP (MDP = methylene diphosphonate) is extensively used in clinics for SPECT imaging, use of PET imaging agents for detecting bone metastases are limited.

Among the PET tracers, [¹⁸F]NaF is the only formulation approved by U.S. FDA and considered a gold standard in detection of skeletal metastases due to its excellent human pharmacokinetics.⁶ Although [¹⁸F]FDG PET is useful in detection of bone malignancies, it is often found ineffective in detection of prostate cancer-derived bone metastasis due to low glycolytic rates in metastatic lesions.⁷ In addition, despite the advantages of [¹⁸F]NaF, the requirement of a medical cyclotron for its regular formulation restricts extensive utilization. Consequently, development of an alternative PET radiotracer with suitable properties for early diagnosis of skeletal metastasis and that can be easily made available at the PET clinic is of great importance.

Introduction and availability of the ⁶⁸Ge/⁶⁸Ga generator system has opened up many alternative paths for designing new PET tracers for efficient detection of small metastatic skeletal lesions, which otherwise was not possible using conventional [^99mTc]Tc-MDP scintigraphy. Although ethylenediamine tetramethylene phosphonic acid (ETDMP) has emerged as a bone-targeting radiopharmaceutical ligand, particularly with particulate emitting radiolanthanides such as ¹⁵³Sm and ¹⁷⁷Lu,⁸ suboptimal accumulation of [⁶⁸Ga]Ga-EDTMP in skeleton has limited its diagnostic uses.⁹

In the past decade, geminal bisphosphonates have emerged as the most preferred choice in the design of bone-seeking metalloradiopharmaceuticals owing to their strong affinity for Ca(II) ions prevalent in hydroxyapatite (HA), the main inorganic component of bone surface.¹⁰ The bisphosphonates preferentially accumulate at the metastatic bone lesion sites, where the bone resorption and desorption occur much aggressively as compared with normal bones. However, a bifunctional chelator where the metal chelation sites have been separated from the HA binding sites is generally advantageous.

Recently, the authors reported a bifunctional 1,4,7,10-tetraazacyclododecane-1′,4′,7′,10′-tetraacetic acid (DOTA)-bisphosphonate moiety, which could be radiolabeled with either ⁶⁸Ga or ¹⁵³Sm, for diagnosis and therapy of bone metastasis.¹¹ However, it has been well established that NOTA, a hexadentate ligand capable of chelating ⁶⁸Ga with high thermodynamic stability [log K = 30.98] by virtue of its matching cavity size with the hydrodynamic radius of Ga⁺³, has an advantage in in vitro stability over similar DOTA derivatives.¹² Several NOTA-based bisphosphonates namely NOTA-BP, NOTAM^BP, NO2AP^BP, and NO1A2P were prepared and evaluated earlier.^13,14

Among these, geminal bisphosphonates namely NOTAM^BP and NO2AP^BP were found to be more effective in preclinical studies.¹⁴ Although [⁶⁸Ga]Ga-NOTAM^BP was not suitable for clinical applications due to its hydrolytic instability,¹⁴ [⁶⁸Ga]Ga-NO2AP^BP was successfully tested in humans.^14,15 However, the synthetic difficulty of NO2AP^BP with a concomitant poor yield (∼17% overall yield)¹⁵ motivated us to find a suitable alternative to the ligand. In general, in most of these radiotracers, the chelating moiety is linked to the bisphosphonate through a two-third carbon length linker.

Moreover, synthesis of a tracer with a bisphosphonate having an α-hydroxy group needs a rigorous multistep synthesis, which would eventually increase the cost of the radiotracer. The authors envisaged that a bisphosphonate with a minimal length linker will keep the overall size of the molecule as small as possible, which may lead to favorable pharmacokinetics, namely faster clearance from blood resulting in more favorable bone–blood ratio.

Herein, a potent bisphosphonate bifunctional ligand 2,2′,2’″-(2-(4-(3-(diphosphonomethyl)thioureido)benzyl)-1,4,7-triazonane-1,4,7-triyl)triacetic acid (3; Fig. 1) derived from NOTA was reported, which could be radiolabeled with ⁶⁸Ga, and the complex was evaluated in vitro and in vivo. Finally, the tracer was utilized for the detection of skeletal metastases in patients, with an objective to establish it as a potential PET imaging agent for early diagnosis of bone metastasis, particularly in centers without an on-site cyclotron.

FIG. 1.

Scheme for synthesis of ligand 3.

Materials and Methods

2-(4-Isothiocyanatobenzyl)-N,N′,N″-triazacyclononane-1,4,7,-triacetic acid (p-SCN-Bn-NOTA) was purchased from Macrocyclics Inc., USA, and used as such. Dibenzylamine (1), diethyl phosphite, and triethyl orthoformate (all AR grade) were purchased from Sigma-Aldrich, USA. Ammonium acetate (spectroscopic grade) and hydrochloric acid (HCl, Suparapur^®) were purchased from Merck, Germany. All these chemicals were used without further purification. Solvents (dimethylformamide [DMF] and ethanol) of analytical grades were procured from Merck, Germany and were redistilled before use for synthesis.

High performance liquid chromatography (HPLC)-grade methanol and water were procured from Merck, Germany, for HPLC studies and were filtered and degassed before use. For all the radiolabeling studies, MilliQ water (resistivity higher than 18.2MΩ.cm) was used. HA microparticles used for adsorption studies of the radiotracers were synthesized and characterized following the procedure reported by the authors elsewhere.¹⁶

Gallium-68 in the form of [⁶⁸Ga]GaCl₃ in 0.1 M HCl solution was sourced from a 740 MBq (20 mCi) ⁶⁸Ge/⁶⁸Ga generator prepared in-house using CeO₂-PAN composite sorbent as the column matrix (GALGEN-1)¹⁷ for use in all radiochemical and preclinical studies. Doses for human clinical uses were formulated at the hospital radiopharmacy using [⁶⁸Ga]GaCl₃ eluted from a commercial 1110 MBq (30 mCi) ⁶⁸Ge/⁶⁸Ga generator (Isotope Technology Garching GmBH).¹⁸

Thin-layer chromatography (TLC) was performed on TLC aluminum sheets with silica gel 60 F254 (Merck KGaA, Germany) to monitor the progress of reactions during synthesis. Purification of reaction intermediates (Fig. 1) was carried out by column chromatography using silica gel (100–200 mesh; Sisco Research Laboratories Pvt. Ltd., India) column. For the purification of ligand 3 (Fig. 1), Younglin reverse phase high performance liquid chromatography (RP-HPLC) semipreparative system Model YL9100 consisting of a quaternary pump (YL9110S), an UV detector (YL9120 Dual Absorbance Detector), a manual sample injector, an automated gradient controller, and a semipreparative column (Venusil XBP C 18) was used.

A gradient elution technique using 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% TFA in 60% aqueous methanol (v/v) (B) as the solvents was used with the following gradient: 5% B to 95% B in 30 min. The flow rate was maintained at 2.0 mL/min. The absorbance was monitored at a wavelength of 254 nm. The analysis of the purified 3 was performed in a JASCO HPLC system (analytical column Kromasil^® C18) with a flow rate of 1.0 mL/min using the same solvents and gradient as mentioned above. The absorbance was monitored at a wavelength of 254 nm.

All the organic intermediates including ligand 3 were characterized by ¹H NMR using Bruker AC-200 instrument or 500 MHz Varian NMR spectrometer, and the spectra were processed using ACDLABS 12.0 1D NMR processor software. Melting points (m.p.) were determined using a Büchi B-540 apparatus. The elemental analyses were carried out with an Elementar Vario micro cube.

Assay of ⁶⁸Ga activity was carried out using Curiementor 3 (PTW Freiburg, Germany) isotope dose calibrator. For radionuclidic purity assay, γ ray spectra of ⁶⁸Ga sample aliquots were recorded using an HPGe detector (Canberra Eurisys, France) coupled to a 4K multichannel analyzer (MCA) system. An ¹⁵²Eu reference source, obtained from Amersham, Inc. (USA), was used for energy and efficiency calibration of the HPGe detector. All other radioactivity measurements were carried out by using a well-type NaI(Tl) scintillation counter (Mucha, Raytest, Germany).

Instant thin layer chromatography (ITLC) was performed using ITLC-SG paper strips obtained from Agilent Technologies Pvt. Ltd. (India) for analyses of the radiochemical purities of the ⁶⁸Ga-labeled radiotracer synthesized.

Millex-GP syringe filter (0.22 μm) units used for sterile filtration of ⁶⁸Ga-labeled radiotracer were procured from Merck-Millipore, Germany. The integrity of the filters before and after use was ascertained at 3.45 × 10⁵ N/m² pressure adopting bubble point method.¹⁸ Sterility of the solutions was tested in tryptic soya broth media and fluid thioglycollate media using Himedia Labs sterility test kits. Pyrogenicity of the solutions was checked by PTS (point-of-use test system; Charles River, USA). Clinical PET images were recorded by using a dedicated Siemens Biograph 6 LSO PET/CT scanner.

Synthesis, purification, and characterization of ligand 2,2′,2″-(2-(4-(3-(diphosphonomethyl)thioureido)benzyl)-1,4,7-triazonane-1,4,7-triyl)triacetic acid (3)

The scheme for synthesis of ligand 3 is shown in Figure 1. Aminomethylbisphosphonic acid (2) was synthesized using dibenzylamine (1) as the starting material following the procedure reported earlier.¹¹ Et₃N (75 μL) was added to a suspension of 2 (5.0 mg, 0.026 mmol) in 200 μL of distilled water and stirred at room temperature till a clear solution was obtained. To that mixture, a solution of p-SCN-Bn-NOTA (11.2 mg, 0.025 mmol) in DMF (200 μL) was added dropwise, and was stirred for 15 h at room temperature.

Thereafter, the solvent was evaporated under vacuum, and the solid residue was dissolved in 1 mL of 10% aqueous solution of K₂CO₃ (w/v). The product (K-salt of compound 3) was purified by RP-HPLC as described above, and was characterized by ¹H and ³¹P NMR spectroscopies. Yield: 12.1 mg, 76%, ligand purity 97.8%; white solid; RP-HPLC (Column: Kromasil^® C18; solvent A: 0.1% TFA–H₂O; solvent B: 0.1% TFA–60% aqueous ACN; gradient: 5% B–95% B in 30 min @ 1.0 mL/min, λ = 254 nm): R_t = 13.8 min (SupplementaryFig. S1).

¹H NMR (D₂O, 800 MHz): δ 2.49–2.58 (m, 1H), 2.60–2.70 (m, 2H), 2.87–3.17 (m, 9H), 3.17–3.30 (m, 4H), 3.31–3.40 (m, 1H), 3.42–3.53 (m, 3H), 3.69 (t, J = 15.0 Hz, 1H), 7.09–7.28 (m, 4H) (Supplementary Fig. S2). ³¹P NMR (D₂O, 600 MHz): δ 14.38 (s) (Supplementary Fig. S3). Anal. Calcd. for C₂₁H₃₃N₅O₁₂P₂S: C, 39.32; H, 5.19; N, 10.92; S, 5.00. Found: C, 39.59; H, 5.33; N, 10.81; S, 4.93% (Supplementary Fig. S4).

Synthesis of [⁶⁸Ga]Ga–3

Under the optimized reaction protocol, [⁶⁸Ga]Ga-2,2′,2″-(2-(4-(3-(diphosphonomethyl)thioureido)benzyl)-1,4,7-triazonane-1,4,7-triyl)triacetic acid complex ([⁶⁸Ga]Ga–3) was synthesized as per the following procedure. Stock solution of 3 was made in MilliQ water. [⁶⁸Ga]GaCl₃ eluate in 0.1 M HCl from ⁶⁸Ge/⁶⁸Ga generator (0.5 mL, 148–185 MBq ⁶⁸Ga) was added to a mixture of 0.1 mL aqueous solution of 3 (25 μg, 38.9 nmol) and 0.4 mL of 0.25 M sodium acetate. The reaction mixture was vortexed thoroughly and the pH was found to be ∼3.5.

To achieve maximum yield of ⁶⁸Ga–3 complex, the mixture was left for 15 min at room temperature. Finally, the solution was filtered through Millex-GP syringe filter (0.22 μm) into a sealed sterile glass vial. Percentage yield of [⁶⁸Ga]Ga–3 complex and its radiochemical purity were determined by employing ascending ITLC-SG using 0.1 M citrate buffer (pH ∼4.5) as the eluting solvent. In this system, free [⁶⁸Ga]Ga³⁺ moves toward the solvent front (R_f = 0.9), while the complex remains at the point of spotting (R_f = 0), thereby enabling the determination of yield and purity. Typical ITLC radiochromatograms of [⁶⁸Ga]Ga–3 synthesized using optimized protocol and [⁶⁸Ga]GaCl₃ are shown in Figure 2.

FIG. 2.

Typical ITLC radiochromatograms of (a) [⁶⁸Ga]Ga–3 synthesized using optimized protocol and (b) uncomplexed [⁶⁸Ga]GaCl₃. ITLC, instant thin layer chromatography.

The optimized reaction protocol for synthesis of [⁶⁸Ga]Ga–3 was established by carrying out detailed studies on the influence of various experimental parameters such as amount of ligand, pH of the reaction mixture, reaction time, and temperature on the radiolabeling yield. The amount of ligand (3) was varied from 5 to 50 μg (7.8–78.0 nmol) in a reaction volume of 1 mL and the radiolabeling yields were determined in each case at various time intervals (5–20 min) while keeping the reaction mixture at both room temperature and 90°C. Also, radiolabeling was carried out for 15 min at room temperature by maintaining the pH of the reaction mixture at 2.0, 3.5, 4.5, and 6.0 using 25 μg (38.9 nmol) of ligand to ascertain the effect of pH on radiolabeling yield.

Determination of partition coefficient (log p) of [⁶⁸Ga]Ga–3

To ascertain the lipophilicity of [⁶⁸Ga]Ga–3 complex, partition coefficient was determined following the procedure reported by the authors earlier.¹⁹

In vitro stability studies

The in vitro stability of [⁶⁸Ga]Ga–3 was studied in physiological saline as well as in human serum. Aliquot of the formulation (0.1 mL) was mixed with 1 mL of physiological saline incubated at 37°C up to 4 h. The radiochemical purity of the formulation was determined at various time intervals postpreparation employing radio-TLC technique as described previously. To determine the in vitro stability of the formulation in human serum, 0.1 mL aliquot of the preparation was added to 1 mL of freshly separated human serum incubated at 37°C for 4 h.

Aliquots were withdrawn from the mixtures at different time intervals, ethanol was added to precipitate the serum protein, centrifuged, and the supernatant was analyzed by ITLC. Kinetic rigidity of the [⁶⁸Ga]Ga–3 complex toward transmetalation was ascertained by challenging the complex prepared using optimized protocol with the addition of excess amount of metal ions (Cu²⁺, Fe²⁺, Ca²⁺, and Zn²⁺), such that metal ion/ligand molar ratio is maintained at 1000.²⁰ The radiochemical purities (% RCP) of the complexes were determined as a function of time after incubating the [⁶⁸Ga]Ga–3 complex at room temperature in the presence of challenging metal ions.

Adsorption of radiotracers on HA particles

Affinity of [⁶⁸Ga]Ga–3 for HA particles in vitro was ascertained by adsorption study of the complex. For this, 0.1 mL aliquot of [⁶⁸Ga]Ga–3 formulation (∼10 MBq) was mixed with a suspension of HA particles containing 1, 2, 5, and 10 mg quantities in 1.9 mL of MillQ water. The mixture was vortexed thoroughly for 15 s and subsequently kept at room temperature under constant stirring until the adsorption of the complex formulation reached an equilibrium. For determination of percentage adsorption, the mixture was centrifuged at 280 g for 3 min, and ⁶⁸Ga radioactivity of an aliquot (typically, 20 μL) withdrawn from the supernatant solution was measured.

The same aliquot was withdrawn from “blank” and ⁶⁸Ga activity was measured. The “blank” consists of a 0.1 mL aliquot of the respective radiotracer diluted with 1.9 mL of MilliQ water, where no HA particle was added. The percentage adsorption of the complex on HA was calculated from Equation (1) using the data. $% A d s o r p t i o n = \frac{A_{i} - A_{e q}}{A_{i}} \times 100$ (1)

where “A_i ” is the measured ⁶⁸Ga activity per milliliter of the blank solution and “A _eq” is the measured ⁶⁸Ga activity per milliliter of supernatant solution of the test mixture under equilibrium condition. The distribution ratio (K_d ) of [⁶⁸Ga]Ga–3 for HA was further calculated using the standard expression [Eq. (2)] employing A_i and A _eq values for the concentration of HA particle for which % adsorption reaches the maxima. $K_{d} = \frac{V \times (A_{i} - A_{e q})}{A_{e q} \times m} m L ∕ g$ (2)

where “V” is the volume of the solution in milliliters and “m” is the mass of the adsorbent (HA) in grams.

Biodistribution studies

Biodistribution studies of the [⁶⁸Ga]Ga–3 complex were carried out in normal Wistar rats. The radiotracer formulation was freshly prepared before the study following optimized protocol. Male Wistar rats, each weighing 200–225 g, were randomly divided into different groups each comprising 4 animals. Each animal was administered with ∼0.1 mL solution of [⁶⁸Ga]Ga–3 (∼3.7 MBq), appropriately diluted using physiological saline, through one of the lateral tail veins. One group of 4 animals were sacrificed at the end of 10, 30, 60, and 120 min postadministration by CO₂ asphyxiation.

Blood samples were withdrawn from the heart of the animal immediately after sacrifice into a previously weighed syringe. Various other organs and tissues were excised after sacrifice, washed with normal saline, dried using absorbent paper, weighed, and the radioactivity associated with each organ and tissue was determined using a flat-type NaI(Tl) counter (Electronic Enterprises, Mumbai, India). The weight of the organs and samples of various tissues was also determined by using an analytical balance.

The percentage injected activity (dose) in various organs/tissue (%ID/organ) was calculated from the above data. Appropriate decay correction on the activity injected into each animal initially was employed at the time of counting the radioactivity at various time points. This gives the biodistribution pattern of the formulation at various time points postadministration specified above. The total uptake in blood, bone, and muscles was calculated by assuming that 7%, 10%, and 40% of the body weight are constituted by these organs/tissue, respectively.^21,22

Clinical studies

Doses of [⁶⁸Ga]Ga–3 (∼148 MBq each) were prepared at the hospital radiopharmacy using [⁶⁸Ga]GaCl₃ eluted from a commercial ⁶⁸Ge/⁶⁸Ga generator (Isotope Technology Garching, GmBH) following the optimized protocol of formulation as detailed above. The prepared formulations are subjected to the quality control tests as described earlier.

Required ethical clearance for clinical studies with [⁶⁸Ga]Ga–3 was obtained from the local institutional ethics committee (IEC) of the nuclear medicine center. Written informed consents were obtained from the patients before enrollment in this clinical study. Six male patients suffering from prostate cancer in their advanced stages that likely give rise to skeletal metastases were enrolled for clinical PET imaging using ⁶⁸Ga–3. Whole body PET images were acquired in 3D mode using Siemens Biograph 6 LSO PET/CT scanner, 45 min after intravenous administration of ∼111 MBq of [⁶⁸Ga]Ga–3.

Results and Discussions

Synthesis of ligand 3

Commercially available p-SCN-Bn-NOTA and the in-house prepared aminomethylbisphosphonic acid (2, Fig. 1) were coupled together to get the desired ligand 3, in 76% yield and with 97.8% purity (Supplementary Fig. S1). The ligand was characterized using ¹H NMR spectroscopy (Supplementary Fig. S2), ³¹P NMR spectroscopy (Supplementary Fig. S3), and elemental analysis (Supplementary Fig. S4). Unlike the case of the structurally similar DOTA derivative reported by us earlier,¹¹ the purification yield of ligand 3 was much higher (76% compared with 46% in case of the DOTA derivative), and no decomposition products were observed during the purification process. This indicated the superior stability of ligand 3 during the purification conditions, and improved the overall yield to 29% in four steps.

To assess whether compound 3 can be successfully chelated to Ga, the Fourier Transform Infrared (FT-IR) and ¹H NMR spectra of the cold Ga–3 complex were recorded, and compared them with those recorded for compound 3 as its tripotassium salt. As demonstrated in the FT-IR spectra (Supplementary Fig. S5), for compound 3, the peak at 1573/cm is ascribed to the stretching vibration of –COO–.

In Ga–3, the stretching band of 1573/cm disappeared and a new vibration peak at 1617/cm was observed, validating the coordination of COO− with Ga. To further reveal the formation of Ga–3, ¹H NMR spectra of the tripotassium salt of 3 were recorded before and after complexation with Ga. As shown in Supplementary Figure S6, ¹H NMR results show significant changes when compound 3 is coordinated with Ga, compared with that of compound 3.

Synthesis of [⁶⁸Ga]Ga–3

Variation of radiolabeling yield of [⁶⁸Ga]Ga–3 as a function of amount of ligand and time of incubation of reaction mixture at room temperature is shown in Figure 3. It was observed that when the reaction mixture was incubated for 15 min at room temperature using minimum of 25 μg (38.9 nmol) of the bisphosphonate derivative 3, the yield of the ⁶⁸Ga-labeled formulation was >98%. Consequently, use of 25 μg (38.9 nmol) of 3 and 15 min incubation at room temperature was considered as the optimum condition for formulation of [⁶⁸Ga]Ga–3.

FIG. 3.

Variation of radiolabeling yield [% radiolabeling yield ± SD (n = 3)] of [⁶⁸Ga]Ga–3 as a function of amount of ligand and time of incubation of reaction mixture at room temperature. SD, standard deviation.

When the reactions were carried out at different pH, it was found that the yield of [⁶⁸Ga]Ga–3 was maximum at pH 3.5 as shown in Supplementary Figure S7. Furthermore, attempt was made to prepare the product by heating the reaction mixture containing 10 μg (15.6 nmol) of 3 at 90° C using a dry bath. However, it was found that the yield was 88.5% ± 2.2% (n = 4) after heating for 20 min. Thus, the yield of [⁶⁸Ga]Ga–3 under optimized radiolabeling condition was 98.4% ± 0.3% (n = 4).

Determination of partition coefficient (log p) of [⁶⁸Ga]Ga–3

The log p value of [⁶⁸Ga]Ga–3 complex was found to be −2.13 ± 0.15 (n = 3), which indicated that the complex was sufficiently hydrophilic in nature as desirable for its fast clearance from blood.

In vitro stability studies

Figure 4a shows the radiochemical purities of [⁶⁸Ga]Ga–3 determined at various time intervals when the formulation was stored in physiological saline and human serum in vitro at 37°C. It was observed that radiochemical purity of [⁶⁸Ga]Ga–3 was high (>95%), even at 4 h of incubation in saline as well as human serum. This indicated that the complex is robust and does not undergo any significant degradation up to ∼4 half-lives of ⁶⁸Ga, even when it is exposed to competing metal ions and chelators (applicable when human serum is the storage medium).

FIG. 4.

(a) Radiochemical purities [% radiochemical purity ± SD (n = 3)] of [⁶⁸Ga]Ga–3 at various time intervals when the formulation was stored in physiological saline and human serum in vitro at 37°C, showing in vitro stability of the formulation, (b) radiochemical purities [% radiochemical purity ± SD (n = 3)] of [⁶⁸Ga]Ga–3 at various time intervals when challenged by addition of 1000-fold molar excess of Cu²⁺, Fe²⁺, Ca²⁺, and Zn²⁺.

When [⁶⁸Ga]Ga–3 complex was challenged by addition of 1000-fold molar excess of Cu²⁺, Fe²⁺, Ca²⁺, and Zn²⁺, it was observed that there was no significant reduction of its radiochemical purity studied up to 1 h postaddition of the metal ions (Fig. 4b), which indicates kinetic rigidity of the [⁶⁸Ga]Ga–3 complex toward transmetalation.

Adsorption of [⁶⁸Ga]Ga–3 on HA particles

The percentage adsorption of [⁶⁸Ga]Ga–3 on HA particles was found to increase with the amount of particles used in the adsorption studies and reached an optimum value of 84.5% ± 2.3% (n = 4) under equilibrium condition when 5 mg of the particles 10 MBq activity of ⁶⁸Ga–3 was used (Supplementary Fig. S8). The “K_d ” value of the formulation for HA particles determined from Equation (2) was found to be 907 ± 14 mL/g. These data exhibit high affinity of ⁶⁸Ga–3 for HA particles.

Biodistribution studies

The results of biodistribution studies carried out in normal Wistar rats after intravenous administration of [⁶⁸Ga]Ga–3 are illustrated in Figure 5. The synthesized complex demonstrated rapid accumulation in skeleton (40.38% ± 1.64% ID at 10 min postadministration, n = 3, p < 0.05). Skeletal accumulation was found to increase to 50.24% ± 2.61% ID at 60 min (n = 3, p < 0.05). The formulation undergoes rapid clearance from blood and other organ/tissue through the renal route.

FIG. 5.

Biodistribution pattern of [⁶⁸Ga]Ga–3 [% ID in different organs ± SD (n = 3)] in normal Wistar rats at different time points postadministration. Around ∼3.7 MBq activity of the formulation prepared using optimized protocol was administered intravenously into each rat. ID, injected dose.

Clearance of the formulation through the renal route was demonstrated by initial uptake of the radiotracer in the kidneys (4.76% ± 0.62% ID at 10 min postadministration, n = 3, p < 0.05) that gradually decreased with time (3.77% ± 0.33% ID at 10 min, 2.53% ± 0.28% ID at 60 min and 0.79% ± 0.12% ID at 120 min post injection, respectively, n = 3, p < 0.05). Comparative uptake (% ID/g) of [⁶⁸Ga]Ga–3 in femur and blood along with femur–blood ratio at various times postinjection in heathy Wistar rats is given in Table 1. Overall, the biodistribution pattern of [⁶⁸Ga]Ga–3 was found to be suitable for its use as a radiotracer for bone imaging.

Table 1.

Comparative Uptake (%ID/g) of [⁶⁸Ga]Ga–3 in Femur and Blood Along with Femur–Blood Ratio at Various Times Postinjection in Heathy Wistar Rats

Parameter	Time postinjection (min)
Parameter	10	30	60	120
%ID/g in femur	1.98 ± 0.11	2.32 ± 0.12	2.48 ± 0.18	2.37 ± 0.15
%ID/g in blood	0.19 ± 0.03	0.07 ± 0.02	0.02 ± 0.01	0.00 ± 0.00
Femur–blood	10.3 ± 0.8	32.5 ± 6.5	121.7 ± 18.6	Very high

Mean ± standard deviation data are presented. Four animals were used in each time point.

ID, injected dose.

Clinical studies

Doses of [⁶⁸Ga]Ga–3 (∼148 MBq each) were formulated at the hospital radiopharmacy with >98% radiochemical purity. The formulation was found to be sterile and the level of endotoxins in all the decayed formulations tested was found to be in the range of 2.5–3.5 EU/mL, which is within the acceptable limits. A representative whole body PET scan of a patient (65 years, male) suffering from primary carcinoma prostate recoded 30 min postadministration of 111 MBq [⁶⁸Ga]Ga–3 is shown in Figure 6a.

FIG. 6.

Whole body PET images of (a) 65 years man with primary carcinoma prostate and suffering from bone pain recorded 30 min postadministration of [⁶⁸Ga]Ga–3 and (b) 53 years man with primary carcinoma prostate and suffering from bone pain recorded 45 min postadministration of [⁶⁸Ga]Ga–BPAMD. Arrows indicate site-specific localization of the PET tracer in the small metastatic lesion sites. PET, positron emission tomography; BPAMD, (4-{[(bis(phosphonomethyl))carbamoyl]methyl}-7,10-bis(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl)acetic acid.

The PET scan shows that the synthesized radiotracer ([⁶⁸Ga]Ga–3) could effectively detect small metastatic skeletal lesions of the patient, as indicated by arrows, due to its site-specific localization in the micrometastatic sites in the skeleton. The skeletal lesion sites found to exhibit standardized uptake value (SUV) 13–16 times higher than that of normal bones. Similar results were obtained in other patients undergoing [⁶⁸Ga]Ga–3 PET scans. For comparison with [⁶⁸Ga]Ga–3 PET, a representative whole body PET scan of another patient (63 years male) of primary carcinoma prostate recorded 30 min postadministration of 111 MBq [⁶⁸Ga]Ga–(4-{[(bis(phosphonomethyl))carbamoyl]methyl}-7,10-bis(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl)acetic acid (BPAMD) formulation is shown in Figure 6b.

The radiotracer was prepared at the hospital radiopharmacy setup following the procedure reported earlier.²² The skeletal lesion sites in the PET scan using [⁶⁸Ga]Ga–BPAMD were found to exhibit SUV eight to nine times higher than that of normal bones. In comparison with [⁶⁸Ga]Ga–BPAMD, the developed radiotracer [⁶⁸Ga]Ga–3 would be advantageous in clinical applications in terms of ease of radiolabeling and potential improvements in the sensitivity of detection of metastatic lesions. Unlike BPAMD, radiolabeling of 3 with ⁶⁸Ga is easier and does not require heating, leading to a decrease in the time required for radiosynthesis (Table 2).

Table 2.

Comparison of Radiosynthesis, Biodistribution, In Vivo Pharmacokinetics, and Positron Emission Tomography Imaging Results of Other Promising [⁶⁸Ga]Ga–Bisphosphonate Radiotracers

Radiotracer	Radiosynthesis	Biodistribution, pharmacokinetics and PET imaging	References
[⁶⁸Ga]Ga–BPAMD	Requires heating at 95°C for 20 min	Accumulation of ∼50% of injected activity in skeleton at 60 min postinjection in healthy Wistar rat, bone-to-blood ratio >40.	^23,24
[⁶⁸Ga]Ga–BPAMD	Requires heating at 95°C for 20 min	In clinical studies, skeletal lesions were found to exhibit SUV 8–9 times compared with normal bones	^23,24
[⁶⁸Ga]Ga–DOTA^ZOL	Requires heating at 98°C for 15 min	Accumulation of 42.7% ± 5.2% of injected activity in skeleton at 60 min postinjection in healthy Wistar rats, bone-to-blood ratio 11.5.	^25,26
[⁶⁸Ga]Ga–DOTA^ZOL	Requires heating at 98°C for 15 min	Clinical studies showed high uptake in skeletal lesion compared with normal bone	^25,26
[⁶⁸Ga]Ga–DOTA-Bn-SCN-BP	Requires heating at 90°C for 15 min	Accumulation of 46.2% ± 2.7% of injected activity in skeleton at 60 min postinjection in healthy Wistar rats, bone-to-blood ratio ∼38	¹¹
[⁶⁸Ga]Ga–NODAGA^ZOL	Requires heating at 98°C for 10 min	Showed high femur accumulation at 60 min postinjection in healthy Wistar rats (SUV = 4.5 ± 0.3), femur-to-blood ratio 57 ± 5.	^27,28
[⁶⁸Ga]Ga–NODAGA^ZOL	Requires heating at 98°C for 10 min	Clinical PET scans showed very high uptake in skeletal lesions (SUV = 26.9)	^27,28
[⁶⁸Ga]Ga–NO2AP^BP	Requires milder condition (heating at 60°C for 10 min)	Showed high femur accumulation at 60 min postinjection in healthy Wistar rats (SUV = 6.5 ± 1.5).	^14,29
[⁶⁸Ga]Ga–NO2AP^BP	Requires milder condition (heating at 60°C for 10 min)	Clinical PET scans showed very high uptake in skeletal lesions (SUV = 17–30)	^14,29
[⁶⁸Ga]Ga–3	Can be achieved at room temperature in 10 min	Accumulation of 50.2% ± 2.6% of injected activity in skeleton at 60 min postinjection in healthy Wistar rats, bone-to-blood ratio ∼122.	This work
[⁶⁸Ga]Ga–3	Can be achieved at room temperature in 10 min	In clinical studies, skeletal lesions were found to exhibit SUV 13–16 times compared with normal bones	This work

PET, positron emission tomography; SUV, standardized uptake value.

Moreover, preclinical investigations indicated that a higher bone-to-blood ratio was obtained for [⁶⁸Ga]Ga–3 than for [⁶⁸Ga]Ga–BPAMD (Table 2). In clinical investigations also, [⁶⁸Ga]Ga–3 showed higher lesion-to-bone ratio than obtained using [⁶⁸Ga]Ga–BPAMD. This pointed toward slightly greater sensitivity and potential improvements in the patient images using [⁶⁸Ga]Ga–3 compared with using [⁶⁸Ga]Ga–BPAMD.

Conclusions

In vitro, in vivo, and clinical evaluations revealed that the ⁶⁸Ga complex of NOTA-conjugated bisphosphonic acid ([⁶⁸Ga]Ga–3) synthesized could be used as a promising new PET radiopharmaceutical for detection of micrometastatic skeletal lesions in cancer patients as it exhibited 13–16 times higher SUV in metastatic lesion sites than normal bones along with fast clearance from all nontarget organs. Ready availability of ⁶⁸Ge/⁶⁸Ga generator and the ligand will help in carrying out bone imaging at any time unlike [¹⁸F]NaF, the availability of which is not always assured.

Footnotes

Acknowledgments

The authors from Bhabha Atomic Research Centre are grateful to the Department of Atomic Energy, Government of India for financial support. The valuable service of the staff members of the animal house facility of Bhabha Atomic Research Centre in carrying out animal biodistribution studies is also acknowledged.

Authors' Contributions

S.C. contributed to conceptualization, data curation, formal analysis, writing—original draft, review and editing, and supervision. S.C., H.D.S., and R.N. were involved in data curation and formal analysis. R.C. carried out data curation, formal analysis, and writing—review and editing.

D.G. was in charge of conceptualization, data curation, formal analysis, and writing—review and editing. A.J. carried out formal analysis. M.R.A.P. took charge of formal analysis, writing—review and editing, and supervision.

Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

Supplementary Material

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Figure S4

Supplementary Figure S5

Supplementary Figure S6

Supplementary Figure S7

Supplementary Figure S8

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Synthesis,Quality Control,and Bench-to-Bed Translation of a New [ 68 Ga]Ga-Labeled NOTA-Conjugated Bisphosphonate for Imaging Skeletal Metastases by Positron Emission Tomography

Abstract

Background

Objective:

Methods:

Results:

Conclusions:

Introduction

Materials and Methods

Synthesis, purification, and characterization of ligand 2,2′,2″-(2-(4-(3-(diphosphonomethyl)thioureido)benzyl)-1,4,7-triazonane-1,4,7-triyl)triacetic acid (3)

Synthesis of [68Ga]Ga–3

Determination of partition coefficient (log p) of [68Ga]Ga–3

In vitro stability studies

Adsorption of radiotracers on HA particles

Biodistribution studies

Clinical studies

Results and Discussions

Synthesis of ligand 3

Synthesis of [68Ga]Ga–3

Determination of partition coefficient (log p) of [68Ga]Ga–3

In vitro stability studies

Adsorption of [68Ga]Ga–3 on HA particles

Biodistribution studies

Clinical studies

Conclusions

Footnotes

Acknowledgments

Authors' Contributions

Disclosure Statement

Funding Information

Supplementary Material

References

Supplementary Material

Synthesis of [⁶⁸Ga]Ga–3

Determination of partition coefficient (log p) of [⁶⁸Ga]Ga–3

Synthesis of [⁶⁸Ga]Ga–3

Determination of partition coefficient (log p) of [⁶⁸Ga]Ga–3

Adsorption of [⁶⁸Ga]Ga–3 on HA particles