Materials
68Ga was obtained by eluting a 68Ge/68Ga generator (1850 MBq 68Ge/68Ga generator, ITG, Germany) with 0.05 M HCl. DOTA-TATE was purchased from ABX advanced biochemical compounds GmbH. The bis-t-butyl NOTA ligand was provided by Immunomedics, Inc. Throl resin and the protected amino acids were purchased from CreoSalus Inc. (Louisville, KY). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). A liquid chromatography mass spectrometer (Shimadzu, Inc., Japan) was used to analyse NOTA-TATE. The radiochemical purity was documented using HPLC (LabAlliance, SSI, Inc., USA). A gamma counter (SN-695B; Hesuo Rihuan Photoelectric Instrument Co., Shanghai, China) and a calibrator (CRC-15R; Capintec, Inc., Florham Park, NJ, USA) were used to measure the radioactivity of the samples. Human blood serum and human plasma were provided by the in vitro analysis laboratory of the Department of Nuclear Medicine of the Affiliated Hospital of Southwest Medical University. Scintigraphy was performed by PET/CT (Gemini TF/16, Philips, Netherlands) or micro-PET/CT (Siemens, Germany, Inveon MM gantry, serial number 3125). The AR42J cell line was purchased from Nanjing Kehao Biotechnology Co., Ltd. BALB/c nude mice were purchased from Beijing HFK Bioscience Co., Ltd. Kunming (KM) mice were also purchased (Animal Experiment Center [animal licence SCXK 2013-17], Southwest Medical University, Luzhou, Sichuan, China). All studies were approved by the Ethics Committee of Southwest Medical University.
Preparation of NOTA-TATE complexes
The octreotide peptide analogue IMP466, NOTA-D-Phe-cyclo[Cys-Phe-D-Trp-Lys-Thr-Cys]-Throl (MH+1305), was synthesized using standard Fmoc-based solid phase peptide synthesis. Throl resin was mixed with 4 equivalents of each Fmoc protected amino acid and 3.9 equivalents of HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) as the coupling reagent in DMF. After incubation for 4 h, the resin was washed with DMF 3 times and added to a mixture of 96:2:2 DMF/piperidine/DBU (v/v/v) for Fmoc deprotection. After 5 min of incubation, the solvent was removed, and the coupling step was repeated sequentially according to the desired peptide sequence. The peptide was cleaved from the resin with a CH2Cl2/acetic acid/trifluoroethanol solution (3:1:1 v/v/v) under mild stirring for 2 h. After the peptide was cleaved from the resin, the peptide was cyclized by overnight incubation with DMSO at room temperature. All buffers used for radiolabelling were metal-free. We determined the purity and structural confirmation analysis of precursor NOTA-TATE on a Shimadzu LC-MS, and the results are shown in Supplementary Materials 1. The chemical structure of NOTA-TATE is shown in Fig. 1.
Preparation of 68Ga-labelled NOTA-TATE and DOTA-TATE
In this experiment, NOTA-TATE and DOTA-TATE solutions (20 μg/10 μL), 68Ga solution (10–15 mCi/mL) and sodium acetate buffer (pH = 5.5) were prepared. The effects of the corresponding conjugate concentration, 68Ga activity, pH, temperature and reaction time on the radiochemical yield and purity of 68Ga-labelled NOTA-TATE and DOTA-TATE were investigated by independent reaction conditions. For comparing the distribution and imaging experiments of 68Ga-NOTA-TATE and 68Ga-DOTA-TATE, both were obtained under the following conditions: The amount of NOTA-TATE or DOTA-TATE was fixed at 20 μg, and the activity of 68GaCl3 varied (370 MBq-555 MBq in 1 mL of 0.05 M HCl). The pH of respective mixtures was adjusted by adding sodium acetate buffer. The pH of the mixture was adjusted to 3.5–4, and 68Ga-NOTA-TATE was obtained by incubating at 90 °C for 10 min. 68Ga-DOTA-TATE was obtained by adjusting the pH of the mixture to 4–4.5 and incubating at 90 °C for 10 min. The radiolabelled conjugates were purified using preconditioned C-18 reversed-phase Sep-Pak cartridges.
Quality control techniques
Characterization of the radiolabelled complexes was carried out using reversed-phase HPLC. Water (A) and acetonitrile (B) each mixed with 0.1% trifluoroacetic acid was used as the mobile phase with gradient elution (0–3 min, 0–10% B; 3–15 min, 10–70% B; 15–20 min, 70–10% B) to separate the free 68Ga from the 68Ga complexes. The elution was monitored by detecting the radioactivity signal using an NaI (Tl) detector and the UV signal at 254 nm. The flow rate was maintained at 1 mL/min for elution.
Serum binding and in vitro stability studies
In vitro serum binding studies for both 68Ga-labelled NOTA-TATE and DOTA-TATE were carried out by following a generalized procedure summarized below. One millilitre of fresh anticoagulated (heparinized) human plasma was prepared. Freshly labelled compound (3.7 MBq, 0.1 mCi) was added to three centrifuge tubes, each containing 0.1 mL of human plasma. After incubation at 37 °C for 2 h, 1 mL of ice-cold methanol was added to each tube. The mixtures were separated by centrifugation (5 min, 3000 rpm) to collect the supernatant, which was repeated three times. A gamma counter was then used to measure the radioactivity of the supernatant (A) and the precipitate (B) in counts per minute (CPM); the plasma-protein binding rate was calculated as PPB = B/(A + B)100%, and the average of three tubes was recorded.
To evaluate the in vitro stability, the radiolabelled preparation was incubated with human blood serum in a water bath at a constant temperature of 37 °C. Aliquots (100 μL) were taken from the reaction mixture at 15 min, 30 min, 1 h, 2 h, 3 h and 4 h, followed by precipitation of serum proteins using acetonitrile. The solution mixture was centrifuged, and the supernatant was analysed by HPLC using the protocol mentioned above. In addition, we added the labelled formulations to physiological saline and incubated them at 37 °C. All the labelled compounds were tested by HPLC for radiochemical purity at each of the abovementioned time points.
Determination of the lipid-water partition (log Po/w)
The lipid-water partition (logP) of 68Ga-labelled complexes was determined in the octanol water system by following the procedure mentioned below. In brief, 490 μL of ultrapure water was added to each of three centrifuge tubes containing 0.5 mL of saturated n-octanol and then combined with 10 μL (1.85 MBq/0.05 mCi) of freshly prepared radiolabelled complexes by ultrasonication (3 min). The centrifuge tube was allowed to stand for approximately 1 min for the liquid to become stratified. The upper and lower liquids were defined as groups A (A1, A2, A3) and B (B1, B2, B3), accounting for the organic and aqueous phases, respectively. Then, 0.1 mL was retrieved from each of the 6 sections. The radioactivity (CPM) of both phases was determined using a gamma counter, and the lipid-water partition coefficient was calculated as follows: logP = log (A-background)/(B-background). The average of three tubes was recorded as the logP.
Biodistribution and imaging studies in AR42J pancreatic tumour xenografts
Tumour cells were subcutaneously injected into the left forelimb of each nude mouse. Biodistribution and imaging studies were conducted when the tumour volume reached at least 1.0 × 1.0 × 1.0 cm3.
The biodistribution of 68Ga-NOTA-TATE in AR42J tumour-bearing mice (weight range, 15–20 g) was studied. Each mouse was injected with 0.1 mL of 68Ga-NOTA-TATE (0.1 mCi, 8.8 GBq/mg) in the tail vein. At 15 min, 30 min, 1 h and 2 h after injection, groups of four tumour-bearing mice (2 females and 2 males) were sacrificed by cervical dislocation to determine the percent injected dose per gramme (%ID/g) in various samples of mouse tissues (i.e. the blood, liver, spleen, lung, kidney, gallbladder, stomach, intestine, femur, muscle and tumour) with a gamma counter. For comparison, biodistribution studies of 68Ga-DOTA-TATE were performed the same way.
Six (3 female and 3 male) mice with AR42J tumour xenografts (15–20 g) were used in the imaging study. All tumour-bearing mice were anaesthetized by intranasal inhalation of isoflurane. Each mouse was immediately injected with 0.1 mL of 68Ga-NOTA-TATE or 68Ga-DOTA-TATE at a dose of 2.96 MBq (0.08 mCi). Images were acquired and reconstructed using micro-PET/CT at 30 min and 2 h after intravenous injection.
Acute animal toxicity test of 68Ga-NOTA-TATE
Thirty KM mice were randomly divided into a control group, a 68Ga- NOTA-TATE low-dose group (100 μCi) and a high-dose group (1 mCi). Each group contained 10 mice, half female and half male. The dose of the appropriate compound was administered through the tail vein at an administration volume of 0.2 mL. The animal blood routine, body weight and animal feed consumption were performed before administration and on days 4, 7, 14, 21 and 28 after administration. At the end of the observation period, the surviving mice were dissected, and histopathological examination was performed on the myocardium, liver, spleen, lung, kidney, stomach, intestine, gonad, bone, muscle and brain of each mouse.
Volunteer imaging study
Twenty-two healthy volunteers gave informed consent and agreed to participate in the study. Twelve volunteers (6 males and 6 females, age 47.3 ± 12.1 years) underwent 68Ga-NOTA-TATE PET/CT scans, and ten volunteers (5 males and 5 females, age 42.9 ± 14.7 years) underwent 68Ga-DOTA-TATE PET/CT scans. All volunteers did not have a history of treatment with cold octreotide and did not fast before imaging. Each patient was injected with an average activity of 1.9 MBq/kg. The timing of image acquisition ranged from 30 to 45 min after injection (3 min/bed position). The PET image was attenuated and then reconstructed using the iterative reconstruction algorithm implemented.