Edited by: Matthias M. Herth, University of Copenhagen, Denmark
Reviewed by: Yoichi Shimizu, Kyoto University, Japan; Lucia Baratto, Stanford University, United States
This article was submitted to Radiopharmacy and Radiochemistry, a section of the journal Frontiers in Nuclear Medicine
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Bisphosponates are an interesting molecular class and in recent years their application has found its way into radiopharmaceutical research and thus into molecular imaging. In addition to great imaging of bone metastases, bisphospnate-based tracers for imaging also have some significant drawbacks. For example, their synthesis is often difficult. Additionally, this can lead to complex and almost impossible purification and quality control. This has limited the production and labeling of suitable molecular and their widespread use to a few facilities. Our squaric acid-based approach provides a way to overcome these problems and makes the synthesis as well as the purification of the compounds much easier. In addition, we were able to demonstrate that labeling with 68Ga is possible under the typical conditions.
Bone metastases are common in late stages of prostate, breast and lung cancer (
Bisphosphonates (BP) are a stable modification of pyrophosphates and all have a P-C-P motif (
In bisphosphonate-chelator conjugates the bisphosphonate serves as targeting vector and complexes different nuclides by means of a chelator and can thus be used for the diagnosis or therapy of bone metastases (
For example, NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) bisphosphonates are particularly suitable for diagnosis using PET, as they have better properties for complexing 68Ga than DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) derivatives (
Coupling by means of squaric acid esters offers a solution to these problems for several reasons: (i) Typically, for coupling with squaric acid esters, aqueous buffers can be used without any hydrolysis of the squaric acid ester. (ii) The squaric acid structure might play a similar role for binding to HAP as the imidazole in zoledronate (see
For the detection of bone metastases in clinics, bone scan is still the most used method. The radionuclide Tc-99m used for this purpose is bound to BPs, such as hydroxymethylene bisphosphonate (HMDP), hydroxyethylene bisphosphonate (HEDP), methylene diphosphonate (MDP) or diphosphono-1,2-propanedicarboxylic acid (DPD), and the patient is examined using a gamma camera. General advantages of this method are good availability, moderate cost, and high sensitivity. Nonetheless, there are a variety of advantages using PET/CT scans over bone scans to image bone metastases. Especially the increased accuracy in detecting bone metastases, an additional increased sensitivity, better differentiation between benign and malignant tumors, higher specificity, multidimensional information, and higher resolution makes the more expensive PET/CT-scan a better tool for some investigations. In addition, the time spent by the patient is significantly reduced, on the one hand due to the reduced general examination time and on the other hand due to the potential need for further scans. As a result, patient management can be significantly improved.
All chemicals were commercially available at Acros Organics (Nidderau, Germany), Merck (Darmstadt, Germany), Sigma Aldrich (Steinheim, Germany) or VWR (Darmstadt, Germany) and were used without further purification. Deuterated solvents for NMR spectroscopy were purchased from Deutero (Kastellaun, Germany). Silica gel (particle size: 0.040–0.063 mm) for column chromatography was purchased from VWR (Darmstadt, Germany). The measurements of 1H- and 13C-NMR spectra were performed on a Bruker Avance II 400 (400 MHz). Mass spectra were recorded on an Agilent Technologies 6130B Single Quadrupole LC/MS system. Semipreparative HPLC was performed on a Merck Hitachi LaChrom L-7100. Following column was used: Phenomenex Luna C18 (250 × 10 mm) 10 μm. Radioactivity was measured with a dose calibrator (ISOMED 2010; MED Nuklear-Medizintechnik Dresden GmbH, Dresden, Germany). Radio thin-layer chromatography (radio-TLC) was analyzed with a RITA* TLC imager (Elysia-Raytest, Straubenhardt, Germany) and an evaluation software (GinaStar TLC; Elysia-Raytest, Straubenhardt, Germany).
Labeling of compounds was performed in sodium acetate buffer (0.2 M, pH 4.5) with 68Ga eluate from a 68Ge/68Ga generator (iThembaLab, South Africa) followed by anionic postprocessing (0.6 M HCl). Labeling of NODAGA.SA.PAM and DOTAGA.SA.PAM was performed with 5, 10, and 15 nmol molecule in 450 μL of sodium acetate buffer (0.2 M, pH 4.5) with 50–100 MBq 68Ga in 150 μL eluate for 15 min at 95 °C. The pH of the labeling solution was adjusted to approximately 7, and the solution was used for injection after dilution with isotonic saline to the correct volume activity. Labeling of DOTA-ZOL was performed as described in previous literature (
Stability studies were performed in HS and PBS solution (pH adjusted to 7 by PBS buffer) in triplicate. HS (human male AB plasma, USA origin) were bought from Sigma Aldrich, phosphate buffered saline (PBS) pH = 7.4 was purchased from Sigma Aldrich as well. The final procedure used 50–70 μl of the labeling solution (5–10 MBq) added to 1 ml of either HS or PBS. The pH was controlled to ensure no influence of the labeling buffer on the solution. Radio-TLC in a solution of acetone, acetylacetone and conc. hydrochloric acid (10:10:1) was used to determine the stability.
The Wistar rats were housed in the animal facility of the Helmholtz-Zentrum Dresden-Rossendorf and experiments were performed according to the guidelines of the European and German Regulations for Animal Welfare approved by the local Ethics Committee for Animal Experiments (Landesdirektion Dresden; file numbers 24-9165.40-4/2013, 24-9168.21-4/2004 1).
Male Wistar rats (RjHan:WI, Janvier Labs, France), 5–7 weeks old, were kept in a pathogen-free facility with ad libitum access to water and food. Biodistribution studies were performed with [68Ga]Ga-NODAGA.SA.PAM (
NH2-DOTAGA (30 mg; 58 μmol; 1 eq.) and squaric acid diethyl ester (26 μL; 30 mg 176 μmol; 3 eq.) were dissolved in phosphate buffer (0.5 M; pH 7; 0.5 mL) and stirred for 2 h at RT. The pH value of the reaction was controlled and, if necessary, adjusted to pH 7–7.5 with sodium hydroxide solution (1 M). The product DOTAGA.SA (28 mg; 43.5 μmol; 75 %) was isolated by semi-preparative HPLC (column: Phenomenex Luna C18 (250 x 10 mm) 10 μm; flow rate: 5 mL/min; solvent: H2O/MeCN +0.1 % TFA; gradient: 0–18 % MeCN in 15 min; Rt = 13 min) and obtained as a colorless solid after lyophilisation. 1H-NMR (600 MHz, D2O): δ (ppm) = 1.30 (dt: 3H, J3HH = 7.1 Hz; J = 12 Hz; CH3); 1.81 (br: 2H; CH2); 2.37 (br: 2H; CH2); 2.77–3.99 (m: 27H); 4.58 (dq: 2H; J3HH = 7.1 Hz; J = 21 Hz; CH2-CH3). 13C-NMR (151 MHz, D2O): δ (ppm) = 15.01 (CH3); 33.04 (CH2-CH2); 39.45 (CH2-CH2); 39.45 (CH2-CH2); 39.75 (CH2-CH2); 43.62 (CH2-CH2); 43.80 (CH2-CH2); 55.05 (CH2); (70.64 (CH2-CH3); 113.32 (COOH); 115.26 (COOH); 117.19 (COOH); 119.12 (COOH); 162.54 (CH2); 162.78 (CH2); 163.25 (CH2); 173.74 (CO-NH); 176.75 (C.SA); 177.22 (C.SA); 183.34 (C.SA); 188.72 (C.SA). MS (ESI positive): m/z (%): 322.2 [M+2H]2+; 643.3 [M+H]+; [M] calculated: 642.29; UPLC (Gradient: 0-100 % B in 15 min): [M] Rt = 3.01 min.
NH2-NODAGA (5 mg; 12 μmol; 1 eq.) and squaric acid diethyl ester (9 μL; 10 mg 60 μmol; 5 eq.) were dissolved in phosphate buffer (0.5 M; pH 7; 0.5 mL) and stirred for 2 h at RT. The pH value of the reaction was controlled and, if necessary, adjusted to pH 7–7.5 with sodium hydroxide solution (1 M). The product NODAGA.SA (6 mg; 11 μmol; 93 %) was isolated by semi-preparative HPLC (column: Phenomenex Luna C18 (250 × 10 mm) 10 μm; flow rate: 5 mL/min; solvent: H2O/MeCN + 0.1 % TFA; gradient: 0–30 % MeCN in 20 min; Rt = 13.1 min) and obtained as a colorless solid after lyophilisation.1H-NMR (300 MHz, D2O): δ (ppm) = 1.32 (dt: 3H, J3HH = 7 Hz; J = 7 Hz; CH3), 1.77–2.02 (m: 2H; CH2); 2.29 (dt; 2H; J3HH = 7.5 Hz; J3HH = 6.6 Hz; CH-CH2-CH2); 2.85-3.01 (m: 4H; cyclic-CH2); 3.02–3.22 (m: 8H; cyclic-CH2); 3.25–3.35 (m: 2H; CH2); 3.40–3.53 (m: 2H; CH2); 3.57–3.63 (m: 1H; CH); 3.78 (br: 4H; CH2-COOH); 4.61 (dq: 2H; J3HH = 7.1 Hz; J = 13 Hz; CH2-CH3). MS (ESI positive): m/z (%): 542.2 [M+H]+; 1084.3 [2M+H]+; [M] calculated: 541,24; UPLC (Gradient: 0–100 % B in 15 min): [M] Rt = 3.38 min.
β-Alanine (2.2 g, 25 mmol, 1 eq.) and phosphonic acid (4.1 g; 50 mmol; 2 eq.) were dissolved in sulfolane and then phosphorus trichloride (4.4 mL; 6.9 g; 50 mmol; 2 eq.) was added dropwise within 15 min. The reaction mixture was then stirred for 3 h at 75°C. The mixture was cooled to 0°C and diluted with water (25 mL). The mixture was then stirred for another 12 h at 105°C. Via crystallization by addition of ethanol (20 mL) and cooling of the reaction mixture to 0°C, pamidronate (2.1 g; 8.9 mmol; 36%) was obtained.1H NMR (300 MHz, D2O): δ (ppm) = 2.20 (tt: J3HH = 6.2 Hz; J3PH = 13 Hz; 2H; NH2-CH2-CH2); 3.27 (t: J3HH = 6.2 Hz; 2H; NH2-CH2). 31P NMR (121.5 MHz, D2O): δ (ppm) = 17.5 (s, 2P). MS (ESI positive): m/z (%): 236.0 (100) [M+H]+; 471.0 (25) [2M+H]+; (ESI negative): 233.9 (100) [M-H]−; 469.0 (90) [2M-H]−; 703.9 (80) [3M-H]−; 939.0 (70) [4M-H]−; 1174.0 (30) [5M-H]−; 1409.0 (5) [6M-H]−; [M] calculated: 235.0. UPLC (gradient: 0-30% B in 4 min): [M] Rt = 0.32 min.
NODAGA.SA (19.6 mg; 36.2 μmol; 1 eq.) was dissolved with pamidronate (10 mg; 42.55 μmol; 1.2 eq.) in phosphate buffer (0.5 M; pH 9; 1 mL) and stirred for 24 h at RT. The pH of the reaction was monitored and adjusted to pH 9–10 with sodium hydroxide solution (1 M) if necessary. By semipreparative HPLC (column: Phenomenex Luna C18 (250 × 10 mm) 10 μm; flow rate: 5 mL/min; running medium: H2O/MeCN + 0.1% TFA; gradient: 5–6.5% MeCN in 9 min; Rt = 7.5 min), the product NODAGA.SA.PAM (8 mg; 10.96 μmol; 30%) was isolated and obtained as a colorless solid after lyophilization.1H NMR (300 MHz, D2O): δ (ppm) = 1.87–2.03 (m: 2H; CH2); 2.09-2.38 (m: 2H; CH2); 2.85-3.04 (m: 4H; ring-CH2); 3.05–3.25 (m: 8H; ring-CH2); 3.27-3.39 (m: 2H; CH2); 3.49 (t: 1H; J3HH = 7 Hz; CH); 3.57–3.70 (br: 2H; CH2); 3.78 (br: 4H; CH2-COOH); 4.61 (dq: 2H; J3HH = 7.1 Hz; J = 13 Hz; CH2-CH3). 31P NMR (121.5 MHz, D2O): δ (ppm) = 18.20 (s, 2P). MS (ESI positive): m/z (%): 366.2 (35) [M+2H]2+; 731.1 (100) [M+H]+; [M] calculated: 730.2; [M(natGa)]: 399.2 (80)/400.1 (75) [M(Ga)+2H]2+; 797.0 (100)/799.0 (60) [M(Ga)+H]+; calculated: 796.2 (100)/798.2 (65). UPLC (gradient: 0–100% B in 15 min): [M] Rt = 0.81 min; [M(Ga)] Rt = 0.89 min.
DOTAGA.SA (27 mg; 42 μmol; 1 eq.) was dissolved with pamidronate (24.7 mg; 105 μmol; 2.5 eq.) in phosphate buffer (0.5 M; pH 9; 1 mL) and stirred for 24 h at RT. The pH of the reaction was monitored and adjusted to pH 9 to 10 with sodium hydroxide solution (1 M) if necessary. By semipreparative HPLC (column: Phenomenex Luna C18 (250 × 10 mm) 10 μm; flow rate: 5 mL/min; running medium: H2O/MeCN + 0.2% TFA; gradient: 0–8% MeCN in 12 min; Rt = 11 min), the product DOTAGA.SA.PAM (17 mg; 20.5 μmol; 49%) was isolated and obtained as a colorless solid after lyophilization. 1H NMR (300 MHz, D2O): δ (ppm) = 1.88 (br: 2H; CH2); 2.12–2.36 (m: 2H; CH2); 2.47 (br: 2H; CH2); 2.90–4.00 (m: 29H). 31P NMR (121.5 MHz, D2O): δ (ppm) = 18.51 (s, 2P). MS (ESI positive): m/z (%): 416.7 (50) [M+2H]2+; 832.2 (100) [M+H]+; [M] calculated: 831.25; [M(natGa)]: 449.7 (100)/ 450.5 (65) [M(Ga)+2H]2+; 898.2/899.2 (5) [M(Ga)+H]+; calculated: 897.25 (100)/899.25 (65); [M(natLu)]: 335.5 (20)/ 450.5 (65) [M(Lu)+3H]3+; 502.6 (100) [M(Lu)+2H]2+; calculated: 1003.25; UPLC (gradient: 0-10% B in 4 min): [M] Rt = 0.48 min; [M(Ga)] Rt = 0.55 min; [M(Lu)] Rt = 0.56 min.
The synthesis of DOTAGA.SA.PAM (4) and NODAGA.SA.PAM (5) are straight forward and comparable to the procedures already published (
For NODAGA.SA.PAM, further
PET/CT imaging: Orthogonal sections
The PET images (midframe time 90 min) show that both compounds have increased retention in the epiphyses, again confirming specific uptake into areas of active bone remodeling. Apart from the skeleton only the bladder is otherwise highlighted. Thus, there is no retention of the bisphosphonates in the soft tissue and both compounds have a preferential renal excretion. [68Ga]Ga-NODAGA.SA.PAM (40 ± 4 %IDrenal, 60 min p.i.) has a slightly higher renal excretion compared to [68Ga]Ga-DOTA-ZOL (33 ± 17%IDrenal, 60 min p.i.) (see
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
The animal study was reviewed and approved by Landesdirektion Dresden; file numbers 24-9165.40-4/2013, 24-9168.21-4/2004 1.
LG, NE, and RB: conceptualization. LG, NE, DM, and RB: methodology and formal analysis. LG, NE, TG, DM, and RB: software. NE and LG: investigation. LG, RB, and DM: data curation. LG and TG: writing—original draft preparation. LG, NE, DM, TG, FR, and RB: supervision. FR and RB: writing—review and editing. All authors contributed to the article and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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We would like to thank the International Center of Precision Oncology (ICPO) for finical support for open access publication. We also thank the Max Planck Graduate Center Mainz (MPGC) for supporting Lukas Greifenstein and Tilmann Grus.