General
68Ga was obtained from a commercially available generator system (Eckert & Ziegler Isotope Products Inc.), eluted and purified according to manufacturer’s instructions. All starting compounds and reagents were used as obtained without further purification. Anhydrous solvents were obtained commercially and used only where indicated. Diluted HCl for elution of 68Ge/68Ga generator was prepared from HCl (trace metal) and high-purity water. High-purity water was also used for all radiochemical works, including the preparation of solutions of precursors and buffers. Hydrochloric acid (99.999%), apo-transferrin (≥ 98.0%), DTPA (≥ 99%), were obtained from Sigma-Aldrich (Saint-Louis, MO, USA). Iron chloride (FeCl3·6H2O, 99%), cobalt chloride (CoCl2 6H2O, 97%) and magnesium chloride (MgCl2, 98%) were obtained from Fisher Scientific (Ottawa, ON, CA). Copper chloride (CuCl2, 99%) and nickel chloride (NiCl2 6H2O, 98%) were obtained from Sigma-Aldrich (Saint-Louis, MO, USA). Acetonitrile (CH3CN) (HPLC grade, 99.9%) and High-purity water (Optima LC/MS, ultra-high-performance liquid chromatography ultraviolet grade, 0.03 mm filtered) were purchased from Fisher Scientific (Ottawa, ON, CA). 1H and 13C NMR spectra were recorded in deuterated solvents on a Brucker Ascend 400 NMR instrument. The residual solvent peaks have been used as internal references. The peak multiplicities are described as follows: s (singlet), d (doublet), t (triplet), q (quartet), quin (quintet), m (multiplet), and br (broad). High-resolution mass spectrum (HRMS) was recorded on a Triple TOF 5600, ABSciex mass spectrometer. Analytical HPLC was performed on an Agilent 1200 system (Agilent Technologies, Mississauga, Ontario, Canada) equipped with a Zorbax Eclipse XDB C18 reversed-phase column (4.6 × 250 mm, 5 μ) and Agilent 1200 series diode array UV–vis (Agilent Technologies) using a linear gradient of CH3CN (0.05% TFA) into H2O (0.025% TFA) (0–76%) over 23 min followed by CH3CN into H2O (76–100%) over 1 min, and CH3CN into H2O (100–0%) over 6 min with a flow rate of 1 mL/min. UPLC chromatograms were obtained from Waters UPLC H-Class equipped with an ELSD detector and a γ-counter from Eckert-Zeigler (Washington D.C. U.S.A.). The column used was a Waters BEH C18 column (2.1 × 50 mm) 1.7 µm and eluent was a gradient of CH3CN (0.05% AcOH)/H2O (0.05% AcOH). Alburex-25 (Human plasma, USP) was supplied by Grifols Canada Ltd. (formerly Talecris), CSL Behring. Instant thin-layer chromatography (ITLC-SG) was acquired from Agilent Technology (Santa Clara, CA). All glassware was cleaned with chromic sulfuric acid (Fisher Scientific). The radio-TLC were scanned using an Instant Imager scanner (Bioscan, DC, U.S.A.). Benzenesulfonic resin (CUBCX123) was bought from UCT, Inc (Bristol, PA, USA). Radioactivity measurements were performed in an ionization chamber (CRC-25PET; Capintec) on the 68Ga setting to control process efficiency. The labeling efficiency of [68Ga]Ga-4HMSA, [68Ga]Ga-NOTA and [68Ga]Ga-DOTA was assessed using ITLC-SG with 0.1 M sodium citrate (pH = 5) as eluant.
Preparation of [68Ga]Ga-citrate
The 68Ge/68Ga generator was eluted with 0.1 M HCl and the radioactivity was transferred to a cation exchange cartridge (CUBCX123). The cartridge was rinsed with 5 mL of physiological saline to remove potential 68Ge residues. 68Ga was eluted from the cartridge with 2 mL of sodium citrate solution (68 mM, pH 5.5) and then diluted with 3 mL of saline solution in the product vial. The radiochemical purity of [68Ga]Ga-citrate solution was evaluated in two solvent mixtures and two stationary phases (ITLC-SG and Whatman paper no2) and compared with the retention time of the [68Ga]GaCl3.
Preparation of (N1, N4, N9-tri-tert-butoxycarbonyl)-1,12-diamino-4,9-diazadodecane 3
The tri-Boc-protected derivative was prepared according to the literature [14] with slight modifications. Spermine (4.0 g, 20 mmol) was dissolved in 30 mL of CH2Cl2 at 0 °C. Ethyl trifluoroacetate (2.4 mL, 20 mmol) in 5 mL of CH2Cl2 was added dropwise and the mixture was then left stirring for 1 h at room temperature. After evaporation of the solvent, the crude residue was taken up in 60 mL of THF and NEt3 (14.0 mL, 60 mmol) was added. A solution of (Boc)2O (17.5 g, 60 mmol) in 20 mL of THF was added and the mixture was stirred for 3 h. After evaporation of the solvent, the residue obtained was taken up with 100 mL of 8/2 MeOH/H2O mixture and Cs2CO3 (20.0 g, 62 mmol) was added and the whole mixture was heated to reflux for 3 h. The solvents were evaporated, and the residue treated with diisopropylether and aqueous solution of HCl (0.5 M). The oily insoluble organic material in both aqueous and organic solvents was decanted from a three-layer system. After drying, the compound was obtained as a pale-yellow oil. The product 3 was used without further purification. Yellow oil. (50%). 1H NMR (400 MHz, CDCl3): δ = 1.33 (s, 27H), 1.38–1.34 (m, 4H), 1.55–1.52 (m, 2H), 1.84–1.75 (m, 2H), 2.81 (t, J = 7.3 Hz, 2H), 3.00–2.96 (m, 2H), 3.18–3.07 (m, 6H), 3.28–3.25 (m, 2H). 13C NMR (100 MHz, CDCl3): δ = 156.0, 99.7, 28.6, 28.21, 25.5.
Preparation of benzyl (9,14-bis(tert-butoxycarbonyl)-2,2-dimethyl-4-oxo-3-oxa-5,9,14-triazaheptadecan-17-yl) glycinate 4 [15]
A solution of tri-Boc-spermine 3 (6.0 g, 10 mmol), benzyl bromoacetate (1.9 mL, 12 mmol) and TEA (2.6 mL, 16.7 mmol) in THF (120 mL) was stirred at room temperature overnight. The precipitate was filtered off and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel chromatography (100% diethylether, then 10% MeOH in CH2Cl2) to give 4 (5.3 g, 72%) as a pale yellow coloured viscous liquid. 1H NMR (400 MHz, CDCl3): δ 1.44–1.48 (m, 31H), 1.64–1.73 (m, 4H), 2.72 (t, J = 7.0, 2H), 3.10–3.26 (m, 10H), 3.44 (s, 2H), 5.16 (s, 2H), 7.32–7.37 (m, 5H). ESI MS (LC–ESI–MS) m/z: calcd, (650.84); found, 651.51 (M + 1).
Preparation of benzyl (3-((4-((3-aminopropyl)amino)butyl)amino)propyl) glycinate sodium salt. 5
A solution of 4 M HCl in dioxane (50 mL) was added to a stirring solution of 4 (3.0 g, 4.6 mmol) in CH2Cl2 (10 mL) at 25 °C under nitrogen. After 2 h, the solution was concentrated in vacuo and co-evaporated with toluene (3 × 10 mL) to yield quantitatively compound 5 as a poly-HCl white salt. 1H NMR (400 MHz, D2O): δ 1.62 (t, J = 7.2 Hz, 2H), 2.15–1.95 (m, 4H), 3.12–2.90 (m, 12H), 3.95 (s, 2H), 5.12 (s, 2H), 7.22–7.37 (m, 5H). 13C NMR (100 MHz, D2O): δ = 166.85, 134.51, 129.02, 128.89, 128.76, 128.62, 71.55, 70.71, 68.54, 60.27, 57.41, 47.42, 47.00, 44.52, 44.43, 43.21, 36.52, 23.72, 22.74, 22.49, 16.78. ESI MS (LC–ESI–MS) m/z: calcd, (350.51); found, 351.29 (M + 1).
Preparation of benzyl 12,17,21-tris(4-((benzyloxy)(methyl)amino)-4-oxobutanoyl)-3-methyl-4,7-dioxo-1-phenyl-2-oxa-3,8,12,17,21-pentaazatricosan-23-oate 6
To a solution of 5 (0.5 g, 1.4 mmol) in DMF (5 mL) was added DIEA (1 mL, 5.7 mmol) and the mixture was left stirring for 1 h at room temperature. N-methyl-N (benzyloxy) succinimide (1.4 g, 5.7 mmol), DIEA (2 mL, 11.4 mmol) and HATU (2.2 g, 5.7 mmol) were stirred in anhydrous DMF (10 mL) for 1 h. This solution was added at 0 °C to the first suspension and the reaction mixture was left stirring to room temperature for 24 h. The reaction mixture was washed with 10% NaHCO3 solution followed by water. The organic phase was dried over anhydrous Na2SO4 and was then removed with a rotary evaporator. The crude product was purified by Biotage flash chromatography to yield 83% of protected 4HMSA 6 as a fluffy beige solid after lyophilisation. The product purity was confirmed to be 100% by analytical reversed phase HPLC: retention time of 24.1 min. 1H NMR (400 MHz, CDCl3): δ 1.52–1.85 (m, 8H), 2.40–2.85 (m, 16H), 3.20–3.45 (m, 24H), 4.12–4.25 (m, 4H), 4.85–4.95 (m, 8H), 5.2 (M, 2H), 7.45–7.55(m, 24H), 8.50–7.50 (s broad, 2H). 13C NMR (100 MHz, CDCl3): (mixture of rotamers), 174.12, 172.95, 172.93, 172.61, 172.57, 172.44, 172.39, 171.85, 169.44, 169.41, 169.32, 135.56, 135.52, 135.43, 135.11, 135.02, 134.61, 129.27, 129.08, 128.87, 128.68, 128.59, 128.56, 128.47, 128.45, 128.41, 128.38, 76.46, 67.45, 67.33, 66.94, 65.85, 50.83, 48.29, 47.52, 47.02, 46.70, 45.32, 45.19, 44.83, 43.51, 42.78, 42.60, 36.92, 36.21, 33.66, 30.58, 27.62, 27.50, 27.48, 27.35, 27.28, 27.24, 27.10, 27.04, 26.32, 26.17, 26.13, 26.09, 25.90, 25.73, 25.05, 25.00, 24.92. ESI MS (LC–ESI–MS) m/z: calcd, (1227.5); found, 1228.6 (M + 1), 615.4 (M/2 + 1).
Preparation of 2-hydroxy-11,16,20-tris(4-(hydroxy(methyl)amino)-4-oxobutanoyl)-3,6-dioxo-2,7,11,16,20-pentaazadocosan-22-oic acid 7
A solution of protected 4HMSA 6 and 10% Pd/C (20% w/w) was suspended in methanol. The reaction mixture was purged with hydrogen gas at room temperature overnight. The crude was filtered over celite and washed with methanol. The solvent was evaporated co-evaporated with diethyether to give quantitatively 4HMSA 7 as a colorless solid. The compound is very hygroscopic and must be kept under nitrogen at − 20 °C. The HPLC chromatogram revealed a mixture of three conformational isomers or conformers in variable compositions. The product purity was confirmed to be 100% with a retention time of 11.0 min. 1H NMR (400 MHz, DMSO): δ 1.42–1.75 (m, 8H), 2.35–2.75 (m, 18H), 2.85–3.38 (m, 24H), 3.85 (m, 1H), 4.17 (m, 1H) 7.71–7.92 (m, 1H), 9.80 (s broad, 3H). 13C NMR (100 MHz, DMSO): (mixture of rotamers), 172.85, 172.73, 172.67, 172.01, 171.90, 171.89, 171.85, 171.59, 171.48, 171.43, 171.38, 49.06, 47.86, 45.18, 45.16, 44.86, 44.85, 37.84, 36.81, 36.60, 36.26, 36.18, 30.21, 30.26, 29.03, 29.02, 28.09, 28.07, 27.78, 27.71, 27.52, 27.48, 27.44, 27.26, 27.22, 27.19, 27.10, 27.06, 26.28, 25.21, 25.19, 25.11. LRMS (LC–ESI–MS) m/z: calcd, (777.8); found, 778.4 (M + 1), 390.0 (M/2 + 1).
Radiolabeling of 4HMSA 7
68Ga was eluted from a 68Ge/68Ga generator using 0.1 M HCl. The 68Ga elution was pre-purified and concentrated on a CUBCX123 column. The purified 68Ga3+ was obtained by eluting the column with a mixture of 12.5 μL of 5.5 M HCl and 500 μL of 5 M NaCl. To this eluate was slowly added a solution of 100–150 μL ammonium acetate buffer 1 M, pH = 4.5 and mixed by brief shaking, resulting in a pH ranging from 3.5–3.8. Varying amounts (0.03–10 nmol) of 4HMSA dissolved in water were mixed with 37–40 MBq of neutralized [68Ga]GaCl3 as described above. The labeling mixtures were allowed to react for 5–10 min at room temperature. The radiolabeling yield for [68Ga]Ga-4HMSA was confirmed by radio-TLC using 0.1 M sodium citrate (pH = 5) as a mobile phase. In this system, free 68Ga eluted with the solvent front while [68Ga]Ga-4HMSA complex remained at the origin.
Determination of apparent molar activity (AMA)
AMA (GBq/μmol) of [68Ga]Ga-4HMSA was calculated in triplicate by titration of the chelator with purified [68Ga]GaCl3. Solutions of 4HMSA (150 μL in 4 mL polypropylene tube) at different concentrations (1–12 × 10−2 nmol) were prepared via serial dilutions. [68Ga]GaCl3 solution was adjusted to a final pH of 3.5–3.8 with NH4OAc buffer (1 M pH = 4.5) and added (22 μL, ~ 15 MBq) to each tube to give a total volume of 250 μL. In addition, a blank tube with ~ 15 MBq of [68Ga]GaCl3 in 250 μL of water was prepared. After mixing and incubation at RT for 10 min, the AMA was determined by measuring [68Ga]Ga-4HMSA labeling efficiency in each tube by TLC using 0.1 M sodium citrate (pH = 5) as mobile phase. TLC plates were analyzed using a radio-TLC scanner. The percentages of complexation were plotted as a function of amount of chelator (nmol) and the AMA was determined when > 95% complexation was obtained.
Comparative 68Ga labeling of 4HMSA, DOTA, and NOTA
Concentration effect
A series of reactions were performed in which a solution of [68Ga]GaCl3 (approx. 25 MBq) was added to a solution of each chelator at a concentration in the range of 1 mM to 100 nM. The final pH of the reaction solution was 3.5–3.8 (using NH4OAc buffer 1 M pH = 4.5). A side-by-side comparison was made of the 68Ga-chelating efficiency of 4HMSA with NOTA and DOTA using instant thin layer chromatography (ITLC) at progressively lower chelator concentration (Fig. 4).
pH effect
A series of reactions were undertaken in which a solution of [68Ga]GaCl3 (approx. 25 MBq) was added to a solution of each chelator at 10 μM concentration. The final pH of the reaction solution was adjusted at 1–2, 2–3 and 3.5–3.8 using NH4OAc buffer 1 M pH = 4.5. Labeling of 4HMSA and NOTA were performed at room temperature and DOTA was labeled at 80 °C as a function of time (Fig. 5).
Apo-transferrin challenge studies
Apo-transferrin (77 kDa) was diluted with 10 mM of sodium carbonate (pH 6.5–7) to reach a final concentration of 10 mg/mL. Apo-transferrin (10 equiv) (820 μL, ∼1.3 × 105 nM) was added to diluted [68Ga]Ga-4HMSA (820 μL, ∼40 MBq). The solution was divided into four aliquots that were incubated at 37 °C for 30, 60 and 120 min. An aliquot was transferred to Amicon Ultra 0.5 mL 50 kDa filter (Merck KGaA, Darmstadt, Germany) and centrifuged at 6600 rpm for 12 min. In this system, [68Ga]Ga-4HMSA passed through the filter, while apo-transferrin and [68Ga]Ga-apo-transferrin remained on the filter. The radioactivity in each fraction was measured using a dose calibrator. The percentage of transchelation was then calculated. The study was performed in triplicate.
Transchelation studies using DTPA
4HMSA (100 μL, 100 μM) was labeled [68Ga]GaCl3 (15 MBq) at room temperature for 10 min and then incubated with 50 and 1000-fold excess of DTPA (200 μL of 5 and 50 mM), respectively, at 37 °C and pH = 7.2 for a period of 80 min. To maintain the pH, 100 μL of sodium acetate buffer (0.5 M, pH ≈ 5.5) was added to the solutions. Each solution was prepared in triplicate, and the quality control was performed, as described above for different time points.
Transmetallation challenge studies
4HMSA (1 mL, 200 μM) was labeled with 100 MBq of neutralized [68Ga]GaCl3 diluted in 1 mL of water at room temperature for 10 min for a total volume of 2 mL. The chelator was then incubated with tenfold excess of various metal cations [iron(III) chloride, cobalt(II) chloride, copper(II) chloride, magnesium(II) chloride and nickel (II) chloride] at pH = 2.0 and 7.4 (Table 3). Each metal solution (200 μL) was added to 200 μL of [68Ga]Ga-4HMSA, and the solutions were incubated at 37 °C. To maintain the pH, sodium acetate buffer (0.1 M, pH ≈ 5.5) was added to the solutions. The samples were monitored for 60 min by radio-TLC to determine the percentage of the intact [68Ga]Ga-4HMSA. All the studies were done in triplicate.
Plasma stability studies
Plasma stability studies were realized as previously described by our group [12]. Briefly, the study was carried out by incubating [68Ga]Ga-4HMSA (60 MBq in 200 µL PBS buffer) in 200 μL of human plasma and the solution was incubated at 37 °C for up to 2 h. The plasma was then mixed twice with (1:1) acetonitrile to precipitate all the proteins. The samples were subjected to vortex mixing for 1 min and then centrifugation for 15 min at 6600 rpm. Plasma proteins were separated from the soluble component by ultracentifugation and the radioactivity was counted. The supernatant fraction was assayed by radio-iTLC on C18 plates; free [68Ga]GaCl3 and [68Ga]Ga-4HMSA were used as standards. The radio-TLCs were eluted with 0.1 M sodium citrate buffer at pH 5.5 using an Instant Imager system (Bioscan, DC, U.S.A.) for the radiodetection.
Protein binding in rodent plasma
[68Ga]Ga-4HMSA (5 nmol / 250 MBq) was incubated in fresh rat plasma (250 μL) and in PBS (250 μL) (control) at 37 °C for various time points (15, 30, 45, 60, and 120 min). The plasma was then mixed twice with (1:1) acetonitrile to precipitate all the proteins. The samples were subjected to vortex mixing for 1 min and then centrifugation for 15 min at 6600 rpm. Plasma proteins were separated from the soluble component by ultracentifugation and the radioactivity was counted.
Animal studies
Animal experiments were performed in adult female BALB/c mice (Charles River Laboratories, Saint-Constant, QC, Canada). The animals were maintained in animal facility, under specific pathogen-free conditions. Housing and all procedures involving animals were performed in accordance with the guidelines of the Canadian Council on Animal Care (CCAC) and were approved by the Ethics Committee of the Université de Sherbrooke.
Induction of inflammation
Mice were injected subcutaneously in the left footpad with 10 μL of complete Freud adjuvant (CFA) solution (0.5 mg/mL), which contains components of Mycobacterium tuberculosis (M. tuberculosis). The solution was made with a water-in-oil emulsion using the heat-killed mycobacteria in mineral oil with saline in a ratio of 1:1. The inflammation was studied at 1, 2, 3- and 7-days post-CFA administration.
PET Imaging studies
The animals were anaesthetized by inhalation of isoflurane (induction 2–2.5%, maintained 1–1.5% oxygen flow 1–1.5 L/min) during i.v. injection and PET imaging procedures. A catheter was installed in the caudal vein for the administration of the radiotracer. The mouse was positioned in the field of view of the PET/CT scanner (Triumph/LabPET8™ platform (Gamma Medica, Northridge, CA), and a 45 min dynamic acquisition was acquired followed immediately by the administration of either [68Ga]Ga-4HMSA or [68Ga]Ga-citrate (∼ 9 MBq, 0.2 mL). The dynamic acquisition was followed by 2 min CT scan. Static PET/CT images were acquired between 45 and 60 min after injection of [68Ga]Ga-citrate for these mice groups: 2, 3- and 7-days post-inflammation. The images were reconstructed using the three-dimensional maximum likelihood estimation method algorithm, and analysis was performed using AMIDE software [16]. To quantify the radiotracer uptake, regions of interest (ROIs) were drawn around organs. The ROI activity was expressed as percent of injected dose per gram of tissue (% ID/g).
Biodistribution studies
Under isoflurane anesthesia (induction 2–2.5%, maintained 1–1.5% oxygen flow 1–1.5 L/min) the mice were injected via the caudal vein with either [68Ga]Ga-4HMSA or [68Ga]Ga-citrate (∼9 MBq, 0.2 mL). At 60 min post injection, the mice were euthanized by CO2 inhalation under isoflurane anesthesia and the organs of interest were collected, washed with PBS, blotted dry, weighted, and counted in a γ-counter (HIDEX AMG, Gamble Technologies Limited, Mississauga, Canada). The results were expressed in terms of percentage of injected dose per gram (% ID/g).
Statistical analysis
All statistical analyses were performed using Prism 7.03 for Window (GraphPad software, Inc., La Jolla, CA, USA). All results are reported as mean ± SD. The number of animals ranged from 2 to 8. Differences were considered statistically significant at p ≤ 0.05 (paired t-test).