| Background and ObjectiveIn the past decades, great efforts have been undertaken to establish radio labeled receptor-binding peptides in nuclear oncology for targeted tumor diagnosis and therapy, notably peptides based on somatostatin receptors (SSTRs). SSTRs are G-protein-coupled receptors expressed on cell membranes and five receptor subtypes (SSTR1-SSTR5) have been identified to date. They are overexpressed in many human tumor types, such as small cell lung cancers, neuroendocrine tumors(NETs), meningioma, glioma etc. Hence, there has been considerable interest in developing high-affinity somatostatin-derived ligands targeting to SSTRs. Octreotide [(D)-Phe1-c (Cys2-Phe3-(D)-Trp4-Lys5-Thr6-Cys7)Thr(ol)8], an 8-amino acid analogue of somatostatin, binds with high affinity to SSTR2 and to a lesser extent SSTR5. Substitution of Phe3 by Tyr3, as well as C-terminal Thr(ol)8 by Thr8, gives TATE [(D)-Phe1-c(Cys2-Tyr3-(D)-Trp4-Lys5-Thr6-Cys7)Thr8], leading to improved sensitivity and affinity for SSTR2, to a higher rate of internalization, and eventually to a higher uptake of the peptide in adrenals, pancreas, pituitary, and tumors.In the United States and Europe,111In-DTPA-otreotide (Octreoscan) is the current clinical gold standard in the diagnosis of NETs. However, its application is somewhat restricted by the high cost of the cyclotron-produced 111In and its suboptimal nuclear characteristics such as the medium-energy photons (171 keV, 245 keV)’51, which lead to lower spatial resolution in comparison to positron-emitting radionuclides. For positron emission tomography (PET) imaging, octreotide derivatives have been labeled with the positron emitters 68Ga,64Cu,18F, etc. Of these, certain 68Ga-labeled octreotide analogues such as 68Ga-DOTATATE, 68Ga-DOTATOC and 68Ga-DOTANOC have shown potential for NETs imaging and are now finding increasing clinical application. Nevertheless,68Ga-PET imaging is not generally available because of the limited daily production of the generator for each single elution (300-700MBq) and the lack of Food and Drug Administration-approved 68Ga-labeled pharmaceuticals and 68Ge/68Ga generators.Compared with the radiometals such as 68Ga and 64Cu,18F is a favored and the most widely used radionuclide and has ideal properties:its relatively long half-life (t1/2=110 min), low positron energy (0.64 MeV) and lack of side emissions. In addition,18F labeled compounds has the potential for delayed imaging, high starting activities enabling multidose preparation, and improved spatial resolution in PET. These emphasize the importance of developing the radiofluorinated somatostatin analogues.Numerous I8F-labeled octreotide analogues have been developed for NETs imaging, including 2-18F-FP-OC, Gluc-Lys([18F]FP)-TOCA, Gluc-S-Dpr([18F]FBOA)TOCA and Cel-S-Dpr([18F]FBOA)TOCA. However, all these compounds suffer from laborious multistep radiosynthetic procedures with moderate labeling yields, hindering their widespread use as routine tracers in the clinic. Moreover, it is quite challenging to make these multistep radiosynthetic processes fully automatic, which in turn sets a high technical barrier for putting these PET probes widely applied in clinical. Therefore, new labeling approaches on one-step 18F labeling methods have been developed, based on fluorine-silicon chemistry, fluorine-boron chemistry, and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-AIF chelation chemistry, especially over the last decade. Octreotide derivative TATE has been have been labeled with the former two methods and imaged with relative success. Herein, the 18F labeling of octreotide derivative TATE was described, using the new 1-step,1-pot Al18F chelation chemistry labeling method. This method uses the formation of aluminum fluoride and its complexation with NOTA, forming a stable Al18F-chelate peptide complex in an efficient 1-pot process. Besides, to increase the hydrophilcity, a PEG6 spacer is introduced, which improves the radioactivity accumulation in the target tissue (e.g., tumor). The in vitro affinity, the in vivo tumor targeting characteristics and Micro PET imaging of 18F-labeled NOTA-PEG6-TATE were also determined in mice bearing xenografts of human malignant glioblastoma cell line U87 MG, which overexpressed SSTR2.Methods1. RadiolabelingNOTA-PEG6-TATE was was purchased from Chinese Peptide Company (Hangzhou, China) and labeled with 18F using the Al18F chelation labeling method based on two completely different reaction solution, sodium acetate buffer (pH 4) and acetonitrile.1.1 Based on sodium acetate bufferAn aqueous 18F solution was obtained from an in-house PETtrace cyclotron and loaded onto QMA cartridge, and then 18F" was eluted from the cartridge with a 0.9% saline solution (0.2 mL) into a sealed vial. AICl3 (2 mM,3 μL) in 0.1 M sodium acetate buffer (pH 4) and 100 μL of NOTA-PEG6-TATE [1 mg/mL in 0.5 M sodium acetate buffer (pH 4)] were then added to the reaction solution sequentially. The reaction mixture was incubated at 100℃ for 20 min. The radiolabeled peptide was purified on C18 column. The radiochemical yield (RCY) and radiochemical purity was checked by analytical HPLC.1.2 Based on acetonitrile18F" was obtained from an in-house PETtrace cyclotron and eluted from the QMA cartridge with 0.5 mL K2CO3/K222 solution into a 5 mL reaction vial and then co-boiled together with acetonitrile at 105℃ for three times to eliminate the water completely. To this,36 μ.L of 1g/L AICl3 was added to the vial, followed by 200μL of NOTA-PEG6-TATE (0.5mg/mL in acetonitrile with 300μL metal-free glacial acetic acid). The reaction mixture was incubated at 105℃ for 20 min. The radiolabeled product was purified by a similar procedure.2. Octanol-water partition coefficientTo determine the lipophilicity of Al18F-NOTA-PEG6-TATE, 10μL (1.85MBq) of the radiolabeled peptide were diluted in 0.5 mL of phosphate-buffered saline, and then 0.5 mL octanol was added. After the tube was vigorously stirred by a vortex mixer for 2 min at room temperature, the two layers were separated by centrifugation (1000rpm × 5min). Samples of 10μL were taken from each layer, radioactivity was measured in a well-type y-counter. The LogP octanol-water value is reported as an average of three different measurements (mean±SD).3. In vitro stability studiesThe in vitro stability of Al18F-NOTA-PEG6-TATE in PBS and bovine serum was evaluated by incubating the radiolabeled probe (3.7MBq) in 0.5 mL of the corresponding solution/medium at 37 ℃. At different time points (0.5,1,1.5 and 2 h), an aliquot of PBS solution was taken and the radiochemical purity was determined by RP-HPLC under the same conditions. Whereas, for the bovine serum, an equal volume of acetonitrile was added and stirred by a vortex mixer for 2 min at room temperature, then followed by centrifugation (1000rpm × 5 min). The supernatant was analyzed by RP-HPLC as described above.4. Cell lines and animal modelsThe human glioblastoma cell line U87MG was grown in MEM medium supplemented with 10%(v/v) fetal bovine serum,100 IU/mL penicillin, and 100 μg/mL streptomycin at 37℃ in an atmosphere containing 5% CO2. The medium was changed every other day. A confluent monolayer was detached with 0.25% trypsin/0.02% trypsin-ethylenediaminetetraacetate (EDTA) and 0.01 M PBS (pH 7.4), and dissociated into a single-cell suspension for further cell culture. The U87MG tumor model was generated by subcutaneous injection of 5 × 106 cells into the right front flank of male nude mice. The mice were used for Micro PET studies when the tumors grow to a size of 0.8-1 cm in diameter (4-5 weeks after inoculation). The animal experiments were approved by the local animal welfare committee and performed according to national regulations.5. Cell uptake and efflux studiesU87MG cells were seeded into 24-well plates at a density of 1×105 cells per well and grown overnight. The culture medium was removed,0.5mL fresh MEM with 185 KBq of Al18F-NOTA-PEG6-TATE was added to each well, and cells were incubated at 37 ℃ for 10,30,60,90, or 180 min. After incubation, cells were washed three times with 0.5mL PBS and harvested by trypsinization with 0.25% trypsin/0.02% EDTA. The cell suspensions were collected and measured in a γ-counter. Cell uptake data was presented as percentage of total input radioactivity after decay correction. Experiments were performed twice with triplicate wells.For efflux studies, U87MG cells were first incubated with Al18F-NOTA-PEG6-TATE (about 185 kBq/well) in 24-well plates for 2 h at 37℃ to allow internalization. Then cells were washed three times with PBS, and incubated with cell culture medium for 0,15,30,60,90 and 120 min. After washing three times with PBS, cells were harvested by trypsinization with 0.25% trypsin/0.02%EDTA. The cell suspensions were collected and measured in a y counter. Efflux data was presented as percentage of total input radioactivity after decay correction. Experiments were conducted twice with triplicate wells.6. Animal biodistribution studiesThe male nude mice bearing U87MG xenografts were injected with about 3.7 MBq (100μCi) of Al18F-NOTA-PEG6-TATE through the tail vein anesthetized with 2% isoflurane. The blocking study of Al18F-NOTA-PEG6-TATE was performed by co-injection of the probe with NOTA-PEG6-TATE (10 mg/kg body weight) through the tail vein. The mice was sacrificed at 1.0 h post-injection (p.i.). The tumor and normal organs of interest were removed and weighed, and their radioactivity was measured in a gamma counter. The radioactivity uptakes in the tumor and normal organs were expressed as a percentage of the injected radioactive dose per gram of tissue (%ID/g).7. Micro PET imagingU87MG tumor-bearing mice under isoflurane anesthesia were injected intravenously with approximately 100 μL 3.7 MBq (100 μCi) of Al18F-NOTA-PEG6-TATE via the tail vein and representative coronal images were obtained at 60 min and 120min after injection. The blocking experiment was performed by co-injection with unlabeled peptide NOTA-PEG6-TATE at 10 mg/kg body weight and 3.7 MBq (100 uCi) of Al18F-NOTA-PEG6-TATE and representative coronal images were obtained at 60 min after co-injection. For each micro PET scan, regions of interest (ROIs) were drawn over the tumor and major organs on decay-corrected whole-body coronal images. The accumulation of radioactivity in tumor and other tissues was obtained from the maximum values measured by the ROIs, and then converted to %ID/g. The mice were sacrificed at the end of the study for PET imaging. The animal experiments were approved by the local animal welfare committee.8. Immunohistochemical stainingAfter imaging, mice were killed and tumors were removed, and then fixed prior to paraffin processing and embedding. Thin paraffin sections were immune stained with antibodies against SSTR2. We used paraffin sections of human epidermal carcinoma of the mouth (KB tumor) for negative control staining.9. Statistical analysisStatistics treatment was made with the software of SPSS 20.0. Data are expressed as mean±SD where appropriate. Differences were considered statistically significant when p values were less than 0.05.Results1. RadiolabelingThe whole radiosynthesis was complete within 60min, with a decay-corrected yield of 70%-100% based on sodium acetate buffer and 64%-74% based on acetonitrile. After purification, the both radiochemical purity were more than 98%. The specific activity was at least 16.9 GBq/μmol.2. Octanol-water partition coefficientTo establish lipophilicity of Al18F-NOTA-PEG6-TATE, the octanol-water partition coefficient was determined. The LogP value of Al18F-NOTA-PEG6-TATE was 2.45±0.38, suggesting that Al18F-NOTA-PEG6-TATE is quite hydrophilic.3. In vitro stability studiesFor the tracer, more than 95% of the radioactivity kept its initial form after 2 h incubation in PBS. In bovine serum (at 37 ℃) more than 80% of Al18F-NOTA-PEG6-TATE detected was still remained intact after 2 h, indicating that most Al18F-NOTA-PEG6-TATE is stable in vitro.4. Cell uptake and efflux studiesThe results demonstrated that Al18F-NOTA-PEG6-TATE had rapid and high cell uptake. Moreover, uptake of the tracer by U87MG cells increased with increasing incubation time, with a maximum uptake of 1.77%±0.07% of the total input radioactivity after 3 h of incubation. Note that the cell uptake protocol used in this study did not distinguish between cell surface-bound and internalized activity. For cell efflux study, Al18F-NOTA-PEG6-TATE had rather rapid elution within the first 10 minute, from 1.35%±0.10% to 0.41%±0.01%. Afterwards, the cell retention of Al18F-NOTA-PEG6-TATE decreased slowly with time, and to 0.29%± 0.07% after 2 h of incubation5. Animal biodistribution studiesThe tumor uptake of the radiotracer was 2.43± 0.40 %ID/g at 1h after injection, and it was significantly higher than that in the most normal organs, such as the blood, muscle, brain, heart, liver, lung and stomach, especially the muscle and brain. The tracer also showed high uptakes in the kidney (4.13±0.92%ID/g) owing to rapid renal clearance of the radiolabeled peptides. Physiological uptake can be seen obviously in the SSTR-overexpressed organs, such as pancreas (1.06± 0.19 %ID/g),stomach (1.62±0.25%ID/g) and adrenal glands (3.88±0.88%ID/g), indicating the high affinity of Al18F-NOTA-PEG6-TATE for somatostatin receptor. Unfortunately, there was obviously high uptake in the bone (67.18±10.78 %ID/g) and it was not reduced in an excess of unlabeled precursor, suggesting de-fluorination occurred for the probe in vivo. In the blocking study, a decrease of radioactivity can be seen in the tumors, pancreas, stomach and adrenal glands for Al18F-NOTA-PEG6-TATE in the presence of an excess of non-radiolabeled peptide, further suggesting the high affinity of Al18F-NOTA-PEG6-TATE for somatostatin receptor.6. Micro PET imagingThe in vivo pharmacokinetic profile and tumor targeting property of Al18F-NOTA-PEG6-TATE in U87MG tumor-bearing mice were evaluated by micro PET scans. The probe was rapidly cleared from the urinary system. The U87MG tumors were clearly visualized and the tumor uptake was 3.8±0.3 and 4.4±0.4% %ID/g at 1 and 2 h p.i. respectively. The tumor uptake was reduced to the background level in an excess of unbound precursor and the uptake values were 0.33±0.06 ID/g, confirming the SSTR-specific targeting of Al18F-NOTA-PEG6-TATE in the SSTR-positive U87MG tumor. Low levels of liver and muscle uptakes were also observed. It was similar to the biodistribution studies, de-fluorination was occurred for the Al18F-NOTA-PEG6-TATE in vivo. The uptake of femur was 14.4±2.7%ID/g and was not inhibited in an excess of unbound NOTA-TATE (14.4±1.8%ID/g), suggesting the high uptake is not the overexpression of SSTR in the bone further but the de-fluorination.7. Immunohistochemical stainingThe IHC analysis showed SSTR was overexpressed on the membranes of the tumor cells in U87MG models, whereas minimum SSTR expression was seen in KB models. These results were closely matched with the Al18F-NOTA-PEG6-TATE Micro PET findings, which indicated that focal uptake of Al18F-NOTA-PEG6-TATE in tumor sites was because of expression of SSTR not caused by nonspecific uptake.ConclusionsNOTA-TATE was successfully prepared and radiolabeled with 18F-fluoride via Al18F intermediate with good yield. This fluorination method for the somatostatin analogs was very simple and straightforward. The labeled product Al18F-NOTA-PEG6-TATE exhibits excellent in vitro serum stability and in vivo tumor imaging quality pharmacokinetics with specific tumor targeting, rapid blood clearance and predominantly renal excretion. It suggests that Al18F-NOTA-PEG6-TATE may be a potential PET tracer candidate for monitoring neuroendocrine neoplasm. The favorable in vivo performance, the easy production method of Al18F-NOTA-PEG6-TATE and unexpected de-fluorination warrant further optimization of this probe as well as the radiofluorination strategy so that the clinical translation of 18F-labeled somatostatin analogs can be accelerated. |
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