CN106084005A - Al18F-NOTA-PEG6-TATE targeting somatostatin receptor and its preparation method and application - Google Patents

Abstract

The invention discloses Al ¹⁸ F-NOTA-PEG ₆ -TATE targeting somatostatin receptors, a preparation method and application thereof. The preparation method of the marker is as follows: the somatostatin derivative TATE is connected with the bifunctional chelating agent NOTA by means of PEG to generate a radiolabeled precursor, and then the positron nuclide ¹⁸ F is labeled by a "one-pot method", namely The ¹⁸ F-labeled somatostatin derivative Al ¹⁸ F-NOTA-PEG ₆ -TATE can be obtained. The method of the invention can be carried out in an acetate buffer solution system or an acetonitrile reaction system, and the method has the advantages of simple operation, high labeling rate and high product purity. The somatostatin receptor-targeting positron marker prepared by the invention can be applied to the preparation of PET/CT molecular probes or tumor treatment drugs, and provides a new idea for the synthesis of clinical PET/CT molecular probes.

Description

Al18F-NOTA-PEG6-TATE targeting somatostatin receptor and its preparation method and application

technical field

The invention belongs to the field of radiolabeling, and in particular relates to Al ¹⁸ F-NOTA-PEG ₆ -TATE targeting somatostatin receptors and its preparation method and application.

Background technique

In recent years, radionuclide-labeled receptor-binding peptides have become one of the research hotspots in tumor-targeted diagnosis, and tumor imaging based on somatostatin receptor (SSTR) has attracted much attention. As a member of the G protein-coupled receptor family, SSTR has five subtypes, namely SSTR1-5, which can be used in small cell lung cancer, neuroendocrine tumor (neuroendocrine tumor, NET), meningioma and glioma and other tumors. Highly expressed on the cell surface. Based on this, more and more scholars believe that finding somatostatin-derived ligands with high affinity for SSTR may provide a reference for new targets for tumor diagnosis. Octreotide (octreotide, OC), is a cyclic octapeptide [D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr(ol)] synthesized after the natural somatostatin is artificially modified, it has an effect on SSTR2 Has high affinity, mild affinity for SSTR5. If the 3-position Phe is replaced by Tyr, and the carbon-terminal Thr (ol) is replaced by Thr, then octreotide can be derived into TATE, and its amino acid sequence is: D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr, The affinity of this derivative to SSTR2 is further increased, thereby increasing the internalization rate of tumor cells, and finally makes the peptide molecules have significantly high uptake in SSTR high-expression tissues (pancreas, adrenal gland, pituitary and SSTR high-expression tumors).

In European and American countries, ¹¹¹ In-DTPA-otreotide (Octreoscan) is currently the gold standard for clinical diagnosis of NETs, but its wide application is limited due to the high cost of cyclotron-generated ¹¹¹ In, and compared with positron nuclides, ¹¹¹ In The lower energy (171keV, 245keV) makes the spatial resolution of the final imaging of ¹¹¹ In-DTPA-otreotide lower. For positron electron PET imaging, ⁶⁸ Ga, ⁶⁴ Cu, ¹⁸ F, etc. have been used to label octreotide derivatives. Among them, ⁶⁸ Ga-labeled octreotide derivatives ( ⁶⁸ Ga-DOTATATE, ⁶⁸ Ga-DOTATOC and ⁶⁸ Ga-DOTANOC, etc.) have great potential in neuroendocrine tumor imaging, and their clinical use has increased significantly in recent years. However, due to the low daily output of ⁶⁸ Ge/ ⁶⁸ Ga generators, which can only elute 300-700MBq each time, coupled with the lack of FDA-approved ⁶⁸ Ga-labeled drugs and ⁶⁸ Ge/ ⁶⁸ Ga generators, ⁶⁸ Ga PET imaging is currently It is not widely used clinically.

Compared with radioactive metal nuclides ⁶⁸ Ga and ⁶⁴ Cu, ¹⁸ F is more widely used because of its relatively long half-life (t _1/2 = 110min), low energy (0.64MeV) and lack of scattering. feature. In addition, ¹⁸ F-labeled compounds can be used for delayed imaging, and their high initial specific activity allows high-dose preparation, and ¹⁸ F-labeled compounds have high spatial resolution, so the use of radioactive ¹⁸ F-labeled somatostatin analogues has more obvious The advantages.

At present, there are many radioactive ¹⁸ F-labeled octreotide analogs (such as 2- ¹⁸ F-FP-OC, Gluc-Lys([ ¹⁸ F]FP)-TOCA, Gluc-S-Dpr([ ¹⁸ F]FBOA) TOCA And Cel-S-Dpr ([ ¹⁸ F]FBOA) TOCA, etc.) is used for imaging of neuroendocrine tumors. However, the radioactive preparation process of these compounds is cumbersome and the yield is low, and its clinical routine use is limited, and its preparation process is difficult to automate. For example: in 2003, Wester et al. synthesized glycosylated Gluc-Lys([ ¹⁸ F]FP)-TOCA through the prosthetic group method, first synthesized the intermediate product ¹⁸ F-NFP, and then through the acylation of ¹⁸ F, used ¹⁸ F -NFP reacts with N ^α -(1-deoxy-D-fructosyl)-Lys ⁰ -Tyr ³ -Lys ⁵ (Dde)-octreotate to synthesize glycosylated Gluc-Lys([ ¹⁸ F]FP)-TOCA, Among them, lysine (Lys) can be linked to ¹⁸ F-NFP, sugar groups and octreotide derivatives at the same time, the total synthesis time is about 3 hours, and the radiochemical yield is between 20% and 30%.

Contents of the invention

The object of the present invention is to provide a PET imaging molecular probe targeting somatostatin receptors and its preparation method and application.

The technical scheme that the present invention takes is:

A PET molecular probe targeting somatostatin receptor is Al ¹⁸ F-NOTA-PEG ₆ -TATE, and its structural formula is as follows:

Application of PET molecular probe Al ¹⁸ F-NOTA-PEG ₆ -TATE in somatostatin receptor-targeted tumor imaging. Including neuroendocrine tumors and non-neuroendocrine tumors (such as glioma, etc.).

Application of PET molecular probe Al ¹⁸ F-NOTA-PEG ₆ -TATE in imaging of neuroendocrine tumors targeting non-bone metastasis somatostatin receptors, or application in glioma.

Application of PET molecular probe Al ¹⁸ F-NOTA-PEG ₆ -TATE in molecular imaging diagnosis of neuroendocrine tumors.

A method for preparing a small molecule probe, comprising the following steps: adding AlCl ₃ and a small molecule compound linked by a bifunctional chelating agent NOTA to a reaction container filled with ¹⁸ F ions, reacting at 90-105° C. for 15-25 minutes, and then Separation and purification were carried out to prepare Al ¹⁸ F-NOTA-labeled small molecule probes.

Preferably, the small molecular compound is selected from small molecular polypeptides or vitamins.

Preferably, the vitamin is folic acid.

Preferably, the small molecule compound is PEG ₆ -TATE.

Preferably, the reaction buffer is acetate solution or anhydrous acetonitrile.

Preferably, the acetate buffer solution has a concentration of 0.1-0.5 mol/L and a pH of 3-6.5.

Preferably, the acetate buffer solution has a concentration of 0.1-0.5 mol/L and a pH of 3.8-4.2.

Preferably, in acetate buffer, 6 nmol of AlCl ₃ and 54.5-136 nmol of NOTA-PEG ₆ -TATE are added per 0.55-1.11 GBq of ¹⁸ F ions.

Preferably, in anhydrous acetonitrile, 52-260 nmol of AlCl ₃ , 26.4-264.5 nmol of NOTA-PEG ₆ -TATE, and 26.4-264.5 nmol of glacial acetic acid are added per 0.55-1.11 GBq of ¹⁸ F ions.

The beneficial effects of the present invention are:

The present invention establishes a method for preparing PET molecular probes targeting somatostatin receptors, using the bifunctional chelating agent NOTA and the octreotide analog TATE modified by the hydrophilic group PEG to prepare the labeling precursor through the "one-pot method""Carry out ¹⁸ F labeling, that is to say, carry out the two-step reaction in the same container without intermediate purification steps, and finally perform purification to obtain the target product Al ¹⁸ F-NOTA-PEG ₆ -TATE. The labeling method of the invention is simple and time-saving, and the radiochemical yield is better than the traditional prosthetic group labeling method.

The NOTA-PEG ₆ -TATE of the present invention can be successfully labeled by the Al ¹⁸ F method. The preferred reaction buffer of the present invention is acetate solution or anhydrous acetonitrile. In the acetate buffer reaction system, 6nmol of AlCl ₃ and 54.5-136nmol of NOTA-PEG ₆ -TATE were added to every 0.55–1.11GBq of ¹⁸ F fluoride ion. Under this condition, the radiochemical yield of the target product is between 70% and 100%, so as to carry out large-dose labeling. In addition, the present invention uses anhydrous acetonitrile as the reaction solvent for the first time to carry out Al ¹⁸ F labeling of NOTA-PEG ₆ -TATE, and has achieved success. Under the anhydrous acetonitrile reaction system, 52-260nmol of AlCl ₃ and 26.4-264.5nmol of NOTA-PEG ₆ -TATE, and 26.4-264.5nmol of glacial acetic acid were added to each 0.55-1.11GBq of ¹⁸ F fluoride ion, and the obtained product released The chemical yields are between 64%-74%. After purification with C18 column, the radiochemical purity is above 95%, and the whole labeling and purification process can be completed within 1 hour.

The lipid-water partition coefficient experiment measured Log P=-2.45±0.38, indicating that Al ¹⁸ F-NOTA-PEG ₆ -TATE has strong water solubility, and this imaging agent can exist stably in PBS and calf serum for 2 hours; the cells take it up And excretion experiments proved that U87MG cells could rapidly uptake and excrete Al ¹⁸ F-NOTA-PEG ₆ -TATE; biodistribution experiments and Micro PET imaging indicated that the imaging agent was highly absorbed in tumors and excreted mainly through the kidneys.

In the present invention, human glioblastoma U87MG cells were selected for cell uptake and excretion experiments, and ^U87MG tumor _- bearing mice were used for biological distribution and Micro PET experiments. The higher uptake in highly expressed organs (adrenal gland and pancreas) indicates that U87MG cells do contain SSTR, which can specifically bind to Al ¹⁸ F-NOTA-PEG ₆ -TATE.

In vivo biodistribution experiments and Micro PET imaging results indicated that the uptake by the kidneys was relatively high, while the uptake by the liver and intestines was relatively low, indicating that the imaging agent was mainly excreted through the urinary system, and the hydrophilicity of the imaging agent was obtained from the lipid-water partition coefficient experiment. Strong conclusions are basically the same. In addition, the imaging agent has a low uptake in the brain, suggesting that the imaging agent does not pass through the blood-brain barrier, while ¹⁸ F-FDG, which is commonly used in clinical practice, can pass through the blood-brain barrier and have a high uptake in the brain, so Al ¹⁸ F-NOTA- PEG ₆ -TATE is superior to commonly used ¹⁸ F-FDG in imaging brain tumors.

In addition, the present invention has verified that the imaging agent Al ¹⁸ F-NOTA-PEG ₆ -TATE can specifically bind to the somatostatin receptor, so the imaging agent can also be used for imaging neuroendocrine tumors, especially the growth of non-bone metastases Imaging of neuroendocrine tumors targeted by statin receptors.

Description of drawings

Figure 1 is the structural formula of Al ¹⁸ F-NOTA-PEG ₆ -TATE;

Figure 2 is the HPLC spectrum of Al ¹⁸ F-NOTA-PEG ₆ -TATE after labeling (a) and after purification (b);

Figure 3 shows the stability of Al ¹⁸ F-NOTA-PEG ₆ -TATE in PBS buffer and fetal bovine serum;

Fig. 4 is a graph of U87MG uptake (a) and elution (b) of Al ¹⁸ F-NOTA-PEG ₆ -TATE;

Fig. 5 is a graph showing the biological distribution of Al ¹⁸ F-NOTA-PEG ₆ -TATE in U87MG tumor-bearing mice 1 hour after injection;

Figure 6 shows the normal imaging of U87MG tumor-bearing mice, 1 hour and 2 hours after injection of Al ¹⁸ F-NOTA-PEG ₆ -TATE, and block imaging after co-injection of the precursor NOTA-PEG ₆ -TATE for 1 hour;

Figure 7 is an immunohistochemical diagram of the expression of SSTR in U87MG (A) and KB (B) tumor tissue sections.

detailed description

The present invention will be further described below in conjunction with specific examples, but is not limited thereto.

Materials used

All chemical reagents were purchased from professional chemical companies, were of analytical grade and used without further purification. NOTA-PEG ₆ -TATE[p-SCN-Bn-NOTA-PEG ₆ -(D)-Phe ¹ -c(Cys ² -Tyr ³ -(D)-Trp ⁴ -Lys ⁵ -Thr ⁶ -Cys ⁷ )- Thr ⁸ ] was designed by our laboratory and synthesized by a designated biological company. The chemical purity was greater than 92%, and it was confirmed by reverse-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry. The positron nuclide ¹⁸ F was produced by the center's cyclotron PET/CTtrace (Siemens, Germany), and after activation Light Accell plus QMA anion exchange column. The reverse-phase extraction column C ₁₈ Sep-Pak separation column was purchased from Waters Company (Milford, MA, USA), and was activated with ethanol and water sequentially before use. All radioactive compounds were analyzed by RP-HPLC (Shimadzu, China). The C18 column specification was 5 μm, 250×4.6 mm. The flow rate during analysis was 1 ml/min. The mobile phase conditions were as follows: Trifluoroacetic acid in water, B buffer is acetonitrile containing 0.1% trifluoroacetic acid, the analysis gradient is 0-18min, 30%B-60%B, 18-20min, 60%B-30%B.

The acetate buffer configuration is as follows:

1) The configuration of 0.5mol/L (ie 0.5M) acetate buffer solution: dissolve 20.5075g sodium acetate (molecular weight: 82.03) in water, then add 80.478mL glacial acetic acid (density 1.049g/mL) to constant volume 500mL, made into 0.5mol/L acetate buffer;

2) Configuration of 0.1mol/L (ie 0.1mM) acetate buffer: Take 60mL of 0.5mol/L acetate buffer and dilute to 300mL with water to prepare 0.1mol/L acetate buffer .

All the data obtained from the experiments in the following examples are represented by mean±standard deviation, and the results are statistically significant when p<0.05 by unpaired t-test.

Example 1 Radiolabeling

NOTA-PEG ₆ -TATE is labeled with ¹⁸ F by Al ¹⁸ F chemical method, based on two completely different reaction systems, acetate buffer (pH=4) and acetonitrile, both in the same reaction vessel, the reaction No intermediate products are produced in the process.

1.1 Based on acetate buffer

Take 1ml ¹⁸ F aqueous solution from the accelerator of our center, about 0.55–1.11GBq (15–30mCi), pass it through the activated QMA column, and then use 0.2ml 0.9% normal saline to elute ¹⁸ F ions into a 1.5ml EP tube , and then sequentially added 3 μL of 2 mM AlCl ₃ (6 nmol) acetate buffer (0.1 M, pH=4) and 54.5 nmol of NOTA-PEG ₆ -TATE precursor solution into the EP tube. After sealing, the reaction solution was reacted at 100°C for 20 minutes, cooled, and diluted with 0.5ml PBS buffer, then passed through the activated C18 column, and the column was first washed with 5ml deionized water to remove ¹⁸ F- and Al ¹⁸ F ²⁺ , and then eluted the target compound with 500 μl 7:3 ethanol/water mixture, and eluted 3 times in total. The radiochemical yield and radiochemical purity of the final product were measured by analytical HPLC, and diluted with PBS for in vivo and in vitro experiments.

1.2 Acetonitrile-based reaction system

Take 1ml of ¹⁸ F aqueous solution from the accelerator of this center, about 0.55–1.11GBq (15–30mCi), pass it through the activated QMA column, and then use 0.5ml of 0.5mM K ₂ CO ₃ solution to elute into a 5ml reaction bottle , add 0.5ml acetonitrile at 105°C to azeotropically remove water three times, then add 10μl 0.026mol/L (260nmol) AlCl ₃ solution and 200μl NOTA-PEG ₆ -TATE precursor solution (dissolve 0.5mg NOTA- TATE (52.9nmol) was added with 30ul glacial acetic acid (52nmol), and the reaction solution was reacted at 105°C for 20 minutes, cooled, and the purification method was the same as above.

1.3 The results of radioactive labeling are as follows:

The structural formula of Al ¹⁸ F-NOTA-PEG ₆ -TATE is shown in Figure 1. The entire radiolabeling process is completed within 60 minutes at 100-105°C, and the labeling rate based on the acetate reaction system after attenuation correction is between 70% and 100%. Between, the labeling rate based on the acetonitrile reaction system is between 64%-74% (see Figure 2. Figure 2 is based on the HPLC figure of the acetonitrile reaction system, wherein 2-a is after the reaction, and 2-b is after the reaction Purified HPLC figure). After purification by Sep-Pak C18 column separation column and detection by HPLC, no matter which reaction system it is based on, the purity is greater than 98%, and the specific activity is greater than 16.9GBq/umol.

The present invention proves that NOTA-PEG ₆ -TATE can be successfully labeled by the Al ¹⁸ F chelation chemical method, and the radiochemical yield of the novel Al ¹⁸ F chelation chemistry is higher than that of the traditional ¹⁸ F prosthetic group labeling method. Al ¹⁸ F-NOTA-PEG ₆ -TATE can be successfully labeled no matter it is based on acetate buffer or acetonitrile, but the radiochemical yield of the latter is slightly lower than that of the former. In addition, the labeling method based on the acetonitrile system requires azeotropic water removal, and the overall process takes longer than that of the acetate system. Any one of the above systems can be selected for Al ¹⁸ F chemical labeling according to the situation. The final labeled product can be separated and purified by C18 column without HPLC purification.

Embodiment 2 Fat-water partition coefficient is measured

Take 10μl (1.85MBq) of Al ¹⁸ F-NOTA-PEG ₆ -TATE reaction solution, add it to a 1.5ml EP tube containing 0.5ml n-octanol and 0.5ml phosphate buffer (PBS), seal it and vortex at room temperature 2min, centrifuged at 1000r/min for 5min. Sequentially take out 50 μl of the organic phase and the aqueous phase with a sample gun, place them in a gamma counter tube, and measure the count with a gamma counter. The average LogP value of the marker was calculated by the formula LogP=lg (count n-octanol/count PBS), and repeated three times.

The Log P of Al ¹⁸ F-NOTA-PEG ₆ -TATE was determined by experiments = -2.45±0.38, indicating that the molecular probe is obviously hydrophilic, so it can be speculated that the probe is excreted in the body through the urinary system.

Embodiment 3 in vitro stability test

Take 20 μl (about 3.7 MBq) of ¹⁸ F-NOTA-PEG ₆ -TATE solution, place them in 0.5 mL of PBS buffer (pH=7.24, 0.02 mol/L) or fetal bovine serum (FBS) respectively, place at 37 After incubation at ℃, the radiochemical purity was measured by HPLC after 0.5, 1, 1.5 and 2 hours to observe its in vitro stability. However, for fetal bovine serum, first dilute it with an equal volume of acetonitrile, seal it, vortex it at room temperature for 2 minutes, centrifuge it at 1000 r/min for 5 minutes, and take the supernatant for analysis by HPLC.

The in vitro stability test results of Al ¹⁸ F-NOTA-PEG ₆ -TATE in PBS buffer and fetal bovine serum are shown in Figure 3. It can be seen from Figure 3 that the molecular probe is The radiochemical purity was still greater than 95% after incubation for 2 hours, and the radiochemical purity was greater than 80% after incubation in fetal bovine serum for 2 hours, indicating that most of Al ¹⁸ F-NOTA-PEG ₆ -TATE exists stably in vitro.

Example 4 Cell Culture and Model Establishment

The human glioblastoma U87MG tumor cell line was cultured in MEM medium supplemented with 10% fetal bovine serum, 100 IU/mL penicillin and 100 IU/ml streptomycin, and the culture medium was replaced every other day. Cells were cultured in humid air containing 5% CO ₂ at a temperature of 37°C. When the cells covered the culture dish, they were separated with 0.05% EDTA and 0.01mol/L phosphate buffer (pH7.4) for further cell culture. 5×10 ⁶ U87MG cells were inoculated subcutaneously on the right shoulder of each male nude mouse. When the tumor diameter reached 0.8–1 cm, it was used for in vivo Micro PET experiments (approximately 4–5 weeks after implantation). All animal experiments followed national standards and Principles of animal welfare.

Example 5 Cell uptake and elution experiments

Add 1×10 ⁵ U87MG tumor cells per well in a 24-well plate, then overnight to ensure their adherence, remove the medium the next day, and add serum-free culture containing 185KBq Al ¹⁸ F-NOTA-PEG ₆ -TATE to each well 0.5ml solution, incubate at 37°C for 10, 30, 60, 90, 180min, wash 3 times with 0.5ml/well frozen PBS, then digest the cells with 0.25% trypsin/0.02% EDTA, collect the cell suspension and use a γ-counter Measure count. Cell uptake data were expressed by cell binding force after decay correction, that is, percentage of added dose. Three parallel samples were set up in the experiment and repeated twice.

For the cell elution experiment, 1×10 ⁵ U87MG tumor cells per well were added to a 24-well plate, and co-incubated with 185 KBq/well Al ¹⁸ F-NOTA-PEG ₆ -TATE at 37°C for 2 hours to fully internalize. Then remove the medium, wash with frozen PBS 3 times, then add serum-free medium and incubate at 37°C for 0, 15, 30, 60, 90, 120min. Afterwards, rinse with frozen PBS solution for 3 times, and finally digest the cells with 0.25% trypsin/0.02% EDTA, collect the cell suspension and count it with a γ-counter. The cell excretion data were expressed by the cell retention rate, that is, the percentage of the added dose after decay correction. The experiment was set up with 3 parallel samples and repeated twice. The results are shown in Figure 4.

From the results in Figure 4, it can be seen that U87MG cells can quickly and efficiently bind Al ¹⁸ F-NOTA-PEG ₆ -TATE, as shown in Figure 4a, and with the extension of incubation time, the binding force of the imaging agent to U87MG cells is further enhanced , reached the peak when incubated for 3 hours, and the highest value of cell binding was 1.77%±0.07%. In the cell elution experiment, within the first 10 minutes, Al ¹⁸ F-NOTA-PEG ₆ -TATE was excreted fastest in U87MG cells, and the cell retention rate decreased from 1.35%±0.10% to 0.41%±0.01%. The imaging agent was excreted slowly in U87MG cells, and the cell retention was 0.29%±0.07% at the end of 2 hours, as shown in Figure 4b.

Embodiment 6 biodistribution experiment in vivo

The in vivo biological experiment was performed by injecting 3.7MBq (100μCi) of Al ¹⁸ F-NOTA-PEG ₆ -TATE through the tail vein of each U87MG tumor mouse under 2% isoflurane anesthesia, and the blocking experiment was co-injected through the tail vein The above tracer and precursor solution NOTA-PEG ₆ -TATE (10 mg/kg). The animals were sacrificed 1 h after the injection of the imaging agent, the tumor and major organs were separated and weighed, and then the radioactive count was measured with a γ-counter. ID/g) to represent.

The results are shown in Figure 5. The results in Figure 5 show that the U87MG tumor uptake value (the third column in Figure 5) 1 hour after the injection of the imaging agent is 2.43±0.40%ID/g, which is higher than other normal organs, such as blood (1.22±0.09%ID/g) , muscle (0.86±0.12%ID/g), brain (0.89±0.09%ID/g), heart (1.68±0.25%ID/g), liver (1.71±0.14%ID/g) and lung (2.05±0.15% ID/g), especially muscle and brain. The uptake of the imaging agent in both kidneys was high, up to 4.13±0.02%ID/g, while the gastrointestinal uptake was low, indicating that Al ¹⁸ F-NOTA-PEG ₆ -TATE was excreted through the urinary system, not the liver and gallbladder. In addition, obvious physiological uptake can be seen in the pancreas and adrenal gland expressing SSTR, the uptake values were 1.06±0.18%ID/g and 3.88±0.88%ID/g, suggesting that Al ¹⁸ F-NOTA-PEG ₆ -TATE and somatostatin The receptor has high affinity. The uptake in bone was significantly increased, up to 67.18±0.78%ID/g, indicating that Al ¹⁸ F-NOTA-PEG ₆ -TATE is prone to defluorination in vivo.

Blocking experiments showed that the specific uptake of Al ¹⁸ F-NOTA-PEG ₆ -TATE by tumors, pancreas and adrenal glands could be significantly reduced in the presence of a large amount of non-radioactive precursors, further proving that Al ¹⁸ F-NOTA-PEG ₆ -TATE and The somatostatin receptor has high affinity. In addition, the imaging agent has a low uptake in the brain, suggesting that the imaging agent does not pass through the blood-brain barrier, while ¹⁸ F-FDG, which is commonly used in clinical practice, can pass through the blood-brain barrier and have a high uptake in the brain, so Al ¹⁸ F-NOTA- PEG ₆ -TATE is superior to commonly used ¹⁸ F-FDG in imaging brain tumors.

However, the uptake in bone did not decrease significantly in the presence of excess precursors, indicating that the high uptake in bone was due to the defluorination of Al ¹⁸ F-NOTA-PEG ₆ -TATE in vivo, rather than the high expression of SSTR in bone. At present, defluorination is still inevitable, but because the fluorine removed is very small, and the human body itself contains fluorine, there is no safety problem. The Al ¹⁸ F-NOTA-PEG ₆ -TATE of the present invention can be used for somatostatin receptor-targeted tumor imaging; in particular, non-bone metastases-targeted somatostatin receptor-targeted neuroendocrine tumor imaging.

Embodiment 7Micro PET imaging

Micro PET acquisition and image analysis were performed with Siemens Inevon small animal PET/CT. U87MG nude mice bearing glioma in the experimental group were injected with 100 μl of about 3.7 MBq (100 μCi) of Al ¹⁸ F-NOTA-PEG ₆ -TATE through the tail vein under 2% isoflurane anesthesia, and the samples were collected 60 minutes and 120 minutes after the injection of the imaging agent. static image. In the blocking group, the imaging agent 3.7MBq (100μCi) and 10mg/kg precursor solution NOTA-PEG ₆ -TATE were co-injected through the tail vein, and static images were collected 60min after the injection of the imaging agent. The experimental animals were fixed in the prone position. During the imaging process, the nasal mask with a connecting tube was used to continuously inhale isoflurane to maintain the animals under anesthesia. The body temperature of the experimental animals was kept stable by circulating the heating plate with water. Images were attenuated by CT and reconstructed using 2D ordered subset expectation maximization. After each PET scan, use InevonResearch Workplace 4.1 software to delineate the interest area of the tumor and major organs on the attenuation-corrected coronal image of the whole body, measure the radioactive uptake value, and express it in %ID/g. Animals were sacrificed according to animal welfare principles.

The pharmacokinetic properties and tumor targeting characteristics of Al ¹⁸ F-NOTA-PEG ₆ -TATE in U87MG tumor-bearing mice can be observed by Micro PET/CT imaging. It can be seen from Figure 6 that U87MG tumor nodules are clearly displayed on the Micro PET image, and the uptake values of the tumor tissue were (3.8±0.3) and (4.4±0.4)%ID/g 1 hour and 2 hours after the injection of the imaging agent, respectively . In the presence of excess non-radioactive precursors, tumor uptake decreased significantly, close to background, to (0.33±0.06)%ID/g, confirming the effect of Al ¹⁸ F-NOTA-PEG ₆ -TATE on SSTR-positive U87MG tumor tissue There is obvious SSTR targeting. In addition, similar to the in vivo distribution experiment, the uptake in the kidneys is higher, and the uptake in the liver and muscle is lower. In vivo biodistribution experiments and Micro PET imaging results indicated that the uptake by the kidneys was relatively high, while the uptake by the liver and intestines was relatively low, indicating that the imaging agent was mainly excreted through the urinary system, and the hydrophilicity of the imaging agent was obtained from the lipid-water partition coefficient experiment. Strong conclusions are basically the same.

The results of in vivo distribution experiments were consistent, and Micro PET/CT imaging further confirmed that Al ¹⁸ F-NOTA-PEG ₆ -TATE had defluorinated in vivo. The femur uptake value in the normal group was higher (14.4±2.7)%ID/g, but there was no significant decrease in the blocking group (14.4±1.8)%ID/g, indicating that the high uptake in the bone was not due to SSTR was highly expressed, but defluorination occurred.

Example 8 Immunohistochemical experiment

After imaging, the U87MG tumor-bearing mice were killed by neck dissection, the tumor nodules were dissected out, fixed with 10% formalin, embedded in paraffin and sectioned to 4 μm, dewaxed and hydrated, antigen restored, and developed with DAB Methods Immunostaining with SSTR2 antibody (H-50, SANTA) was performed. Meanwhile, non-neuroendocrine tumor-human oral epithelial cancer cell line KB tumor nodules were used as negative control.

The results of immunohistochemistry showed that SSTR2 was highly expressed on the surface of U87MG tumor cell membrane, while the expression of KB tumor was low, as shown in Figure 7. These results were consistent with the results of Micro PET imaging, indicating that the tumor uptakes the imaging agent Al ¹⁸ F-NOTA-PEG ₆ -TATE is not due to non-specific uptake, but due to the high expression of SSTR2 on the surface of tumor cells.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (10)

1. a PET molecular probe for targeting somatostatin receptor, for Al¹⁸F-NOTA-PEG₆-TATE, its structural formula is as follows:

。

2.PET molecular probe Al¹⁸F-NOTA-PEG₆-TATE application in the tumor imaging of somatostatin receptor targeting.

3.PET molecular probe Al¹⁸F-NOTA-PEG₆-TATE application in neuroendocrine tumor molecular image diagnoses.

4. the preparation method of a Small-molecule probe, it is characterised in that comprise the following steps: filling¹⁸The reaction vessel of F ion Middle addition AlCl₃And bifunctional chelating agent NOTA connect micromolecular compound, 90-105 DEG C react 15-25 minute, then Isolated and purified, prepare Al¹⁸The Small-molecule probe of F-NOTA labelling.

Preparation method the most according to claim 4, it is characterised in that: micromolecular compound is raw selected from micromolecule polypeptide or dimension Element.

Preparation method the most according to claim 5, it is characterised in that: micromolecular compound is PEG₆-TATE。

Preparation method the most according to claim 4, it is characterised in that: reaction buffer is acetate solution or anhydrous second Nitrile.

Preparation method the most according to claim 7, it is characterised in that: the concentration of acetate buffer is 0.1-0.5mol/ L, pH are 3-6.5.

Preparation method the most according to claim 6, it is characterised in that: in acetate buffer, every 0.55 1.11 GBq 's¹⁸F ion adds the AlCl of 6nmol₃And the NOTA-PEG of 54.5-136nmol₆-TATE 。

Preparation method the most according to claim 6, it is characterised in that: in anhydrous acetonitrile, every 0.55 1.11 GBq 's¹⁸F ion adds the AlCl of 52-260nmol₃And 26.4-264.5nmol NOTA-PEG₆-TATE, and 26.4- 264.5nmol glacial acetic acid.