CN102617728B - Preparation method of 18F labeled product of Exendin-4 - Google Patents

Abstract

The invention discloses a [ ¹⁸ F]FB-Exendin-4 labeled product and its preparation method and application. The intermediate is prepared through a series of reactions of ^{18 F-} and ethyl 4-trimethylamine benzoate trifluorosulfonate. N-succinimide 4-[ ¹⁸ F]fluorobenzoate ([ ¹⁸ F]SFB) is then combined with the polypeptide Exendin-4 to obtain the [ ¹⁸ F]FB-Exendin-4 labeled polypeptide. The radiochemical purity of the labeled product disclosed by the invention is greater than 98%, the labeling rate is 35%, and the stability in vitro is good.

Description

A kind of preparation method of 18F labeled product of Exendin-4

technical field

The present invention relates to the field of radiopharmaceuticals. the

Background technique

The localization diagnosis methods of insulinoma mainly include functional test and imaging examination. Functional tests include transarterial calcium stimulated hepatic vein blood sampling for insulin measurement (ASVS), percutaneous transhepatic portal vein segmental blood sampling for insulin measurement (PTPC), segmental blood sampling through splenic portal vein puncture for rapid insulin determination (PVS ), and methods such as endoscopic ultrasonography and DSA are highly sensitive, but they are all invasive examinations and are difficult to be widely used clinically. Conventional imaging positioning methods include CT, MRI, etc., but the sensitivity is not high. the

Somatostatin receptor (SSTR) imaging was first applied to the localization of insulinomas, and ¹¹¹ In-labeled Octreotide was the most widely used, but the detection rate of insulinomas was less than 50%. This is because ¹¹¹ In-Octreotide mainly binds to two subtypes of SSTR, SSTR ₂ and SSTR ₅ , and the distribution of these two receptor subtypes in insulinoma β cells is not much, so the sensitivity is reduced. Since insulinomas highly express GLP-1 receptors, radionuclide-labeled GLP-1 and its analogs can be used for specific imaging and localization of insulinomas.

GLP-1 is a small molecular polypeptide composed of 30 amino acids, which can mediate the secretion of insulin through the specific GLP-1 receptor on the surface of pancreatic beta cells. However, GLP-1 can be rapidly degraded by dipeptidyl peptidase-IV (DPP-IV), and its half-life in vivo is only about 2 minutes. Therefore, its clinical application is greatly limited. GLP-1 analogs, such as Exendin-4 and Exendin-3, have similar biological activities to GLP-1, can resist the degradation of dipeptidyl peptidase-IV (dipeptidyl peptidase-IV), and have a long half-life in vivo. Therefore, the imaging of islet cell tumors and the monitoring of islet β-cell transplantation by radionuclide-labeled GLP-1 analogs have been developed rapidly. the

In the early days, Gotthardt et al. conducted in vivo imaging studies of rat insulinoma models by using ¹²³ I-labeled GLP-1 and its analogue Exendin-3. Studies have shown that insulinoma can specifically bind to ¹²³ I-labeled Exendin-3. However, due to the lack of tyrosine in the primary structure of Exendin-3, radioactive iodine labeling is difficult. Afterwards, Wild et al. labeled the GLP-1 analog Exendin-4 by ¹¹¹ In, namely [Lys ⁴⁰ (Ahx-DTPA- ¹¹¹ In)NH ₂ ]exendin-4, and carried out through pinhole (pinhole) SPECT/MRI and SPECT/CT Insulinoma imaging of Rip1Tag2 transgenic mice showed that tumor uptake was significantly higher than that in lung and pancreas tissues. Considering that Exendin-4 labeled with ¹¹¹ In may cause a relatively high radiation absorption dose to patients, Wild et al. labeled Exendin-4 with ⁶⁸ Ga and ^99m Tc respectively. SPECT and PET showed that the target/primary ratio of the tumor was high, which may be Shows smaller insulinomas. However, ⁶⁸ Ga is a positron nuclide prepared by the generator, and its spatial resolution is low, so its clinical application is limited. Although ^99m Tc is a commonly used radionuclide, the sensitivity of clinical SPECT imaging is lower than that of PET imaging. PET imaging has the characteristics of high spatial resolution and sensitivity, which is more suitable for the clinical diagnosis of insulinoma. ¹⁸ F is the most commonly used positron nuclide for PET/CT imaging, and the source is convenient. Therefore, we tried to study the feasibility of insulinoma imaging by labeling Exendin-4 with ¹⁸ F.

Due to the relatively complex molecular structure of polypeptides, it is impossible to directly undergo nucleophilic fluorination or electrophilic fluorination, therefore, ¹⁸ F-labeled polypeptide drugs must go through intermediates. At present, ¹⁸ F-labeled intermediates have been rapidly developed and widely used, such as acylation, amidation, imidization, alkylation, photochemical linkage, click chemistry, siliconation, boronation, and solid-phase synthesis. ^18F labeling of intermediates. However, different labeling methods have their advantages and limitations. Among them, the synthesized N-succinimide 4-[ ¹⁸ F]fluorobenzoate ([ ¹⁸ F]SFB) is labeled by the acylation pathway. And purification method, radiochemical yield and in vivo stability of the labeled probe, it is considered to be the most suitable intermediate for ¹⁸ F labeling. Moreover, [ ¹⁸ F]SFB has been widely used in the connection of monoclonal antibodies, RGD (arginine-glycine-aspartic acid), Annexin-V and other biomolecules.

Kiesewetter et al. (Eur J Nucl Med Mol Imaging 2012; 39:463–473) synthesized the polypeptide Exendin-4 by labeling the intermediate [ ¹⁸ F]FBEM ([ ¹⁸ F]FBEM-[Cys ⁴⁰ ]-exendin-4) , and conducted small animal PET imaging of INS-1 insulinoma model mice. The study showed that the radiochemical yield of [ ¹⁸ F]FBEM-[Cys ⁴⁰ ]-exendin-4 was 6.4% (with ¹⁸ F ^- Calculation), radiochemical purity >96%; insulinoma clearly visualized. The Exendin-4 analogs reported in the literature are obtained by structural modification on the basis of Exendin-4, that is, Cys is added to the carboxyl segment of Exendin-4. The polypeptide containing Cys is easily oxidized, and its stability is worse than that without Cys. Secondly, the comprehensive radiochemical yield of the labeling method reported in the literature is relatively low, only 6.4%.

Contents of the invention

The technical problem to be solved by the present invention is to provide a ¹⁸ F labeled product of Exendin-4 with good stability in vitro and high radiochemical yield.

The second technical problem to be solved by the present invention is to provide a method for preparing the ¹⁸ F-labeled product of Exendin-4.

The third technical problem to be solved by the present invention is to provide the application of [ ¹⁸ F]FB-Exendin-4 in the preparation of drugs for imaging and diagnosis of insulinoma.

To achieve the first objective of the present invention, the present invention discloses a ¹⁸ F labeling product of Exendin-4, the labeling product is [ ¹⁸ F]FB-Exendin-4.

In order to achieve the second purpose of the present invention, the present invention discloses a preparation method of [ ¹⁸ F]FB-Exendin-4, comprising the following steps:

(1) ¹⁸ F ^- reaction with ethyl 4-trimethylaminobenzoate trifluorosulfonate to prepare intermediate N-succinimide 4-[ ¹⁸ F]fluorobenzoate [ ¹⁸ F]SFB;

(2) [ ¹⁸ F]SFB is coupled with Eexdin-4 to obtain [ ¹⁸ F]FB-Exendin-4.

As a preferred solution, step (1) includes: preparing [ ¹⁸ F]F ^- by cyclotron PET trace, adding 4-trimethylaminobenzoic acid ethyl trifluorosulfonate to the reaction bottle containing activated [ ¹⁸ F]F ^- acid salt, react with NaOH and HCl in turn, the reaction solution is rinsed with hydrochloric acid through an activated Sep-Pak C18 column, dried with N ₂ , and then eluted with acetonitrile to obtain 4-[ ¹⁸ F]fluorobenzoic acid; add tetrahydroxide Propylamine, dried azeotropically, added O-(N-succinimide)-1,1,3,3-tetramethyluronium tetrafluoroborate TSTU to react, and then acidified with acetic acid; the above solution was passed through The activated Sep-Pak C18 column was rinsed with acetonitrile/water of V : V = 1/7 in sequence, and the Sep-Pak C18 column was dried with N ₂ , and eluted with dichloromethane; the radiochemical purity was measured by TLC and HPLC , the activity meter measures the activity and calculates the radiochemical yield.

Step (2) includes: dissolving [ ¹⁸ F]SFB in acetonitrile, adding Exendin-4, dissolving Exendin-4 in 0.1M borax-boric acid buffer solution with a pH value of 8.5, and reacting at room temperature for 30 minutes; For HPLC separation, the chromatographic column used is ZORBAX SB C18, 5 μm, 9.4 mm × 150 mm, and the HPLC system uses the following gradient to elute at a speed of 3 mL/min: phase A is acetonitrile containing 0.1% trifluoroacetic acid, phase B is It is water containing 0.1% trifluoroacetic acid; 0-20 min, A rises from 16% to 90%; 20-30 min, A is 90%; collect the peak at t _R =18-19.5 min, the resulting product solution, spin Evaporate to dryness and re-dissolve with 0.9% NaCl injection to obtain the target product compound [ ¹⁸ F]FB-Exendin-4. After Radio-TLC, the developer is ethyl acetate and dichloromethane, and the volume ratio of the two is 3: 1. Radio-HPLC characterization, the Radio-HPLC analysis method is the same as the preparation method, but the flow rate is 1mL/min, and the chromatographic column is μBondapak ^TM C18, 5 μm, 300 mm × 3.9 mm.

The advantage of the present invention is that the present invention successfully synthesizes [ ¹⁸ F]SFB and the polypeptide Exendin-4 ([ ¹⁸ F]FB-Exendin-4), the radiochemical purity of the labeled product is greater than 98%, and the labeling rate is 35% (calculated with ^{18 F-} as the starting material), good stability in vitro.

Description of drawings

Figure 1 is the synthesis route of [ ¹⁸ F]SFB.

Fig. 2 is the radioactive TLC pattern of the labeled polypeptide [ ¹⁸ F]FB-Exendin-4.

Fig. 3 is the radioactive HPLC analysis spectrum of the labeled polypeptide [ ¹⁸ F]FB-Exendin-4.

Figure 4 is the in vitro competition binding curve. The IC50 value of exendin-4 analyzed by Graphpad Prism5 is 0.99nmol/L. the

Figure 5 shows the saturation curve, the equilibrium dissociation constant K _d value of ¹²⁵ I-exendin-4 and GLP-1 receptor is 56.64, and the maximum binding constant B _max is 4.651×10 ⁷ .

Fig. 6 is PET/CT imaging of ¹⁸ F-FB-Exendin-4 insulinoma nude mouse model at 30 minutes and 120 minutes in small animals.

Figure 7 shows the SUV values of each organ at 30 minutes and 120 minutes. the

Figure 8 shows the SUV values of each organ without blocking and blocking for 60 minutes. the

Figure 9 shows the %ID/g values of various organs of nude mice after 120 min. the

Detailed ways

The specific embodiments of the preparation method and application of [ ¹⁸ F]FB-Exendin-4 provided by the present invention will be described in detail below in conjunction with the accompanying drawings.

Example 1. Preparation of [ ¹⁸ F]FB-Exendin-4

Material

Exendin-4 was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. (Shanghai, China) following standard FMOC chemistry. 4-fluorobenzoic acid (SB) was purchased from Fluka, Switzerland. ¹⁸ F was provided by the Accelerator Center of Sinovac Pharmaceutical Co., Ltd., and obtained by proton irradiation of oxygen-enriched ¹⁸ OH ₂ 0 . High-performance liquid phase system Dionex P680 summit HPLC analysis system (Dionex, USA), equipped with Bioscan flow-count detector (Bioscan, USA); produced by cyclotron (GE, Waukesha, Wisconsin, USA).

(1) Synthesis of N-succinimide 4-[ ¹⁸ F]fluorobenzoate ([ ¹⁸ F]SFB)

[ ¹⁸ F]fluoride was produced by cyclotron PET trace (GE, Waukesha, Wisconsin, USA). The 18O(p, n)18F nuclear reaction is used to irradiate [ ¹⁸ O]H ₂ O contained in a 1.5ml silver target (silver target) by protons, the incident proton energy is 18MeV, and the electron beam current is 36μA. After [ ¹⁸ F]F ^- was prepared by a cyclotron, it was adsorbed onto a Sep-Pak light QMA column (Sigma) to be eluted. [ ¹⁸ F]F ^- was eluted into a V-shaped reaction bottle (ALTECH, USA) through 1.5ml of a mixed solution containing K ₂ CO ₃ and Kryptofix 222 (K222; Sigma), and anhydrous ¹⁸ F was obtained by azeotropic evaporation. ^- .

Synthetic reference method for labeled precursor [ ¹⁸ F]SFB (Kirchhoff TD, Merkesdal S, Frericks B, et al. Intraarterial calcium stimulation (ASVS) for pancreatic insulinoma: comparison of preoperative localization procedures. Radiologe, 2003(43):301 -305.; Suzuki K, Takahashi S, Aiura K, et al. Evaluation of the usefulness of percutaneous transhepatic portal catheterization for preoperatively diagnosing the localization of insulinoma. Pancreas, 2002,24:96-102.; Wang GJ, Dou JT, Wang ZQ, et al. Comparison of endoscopic ultrasonography and digital subtraction angiography in localization of insulinomas. Chinese Journal of practical International Medicine, 2006, 26(22):1829-1830.), see Figure 1 for the synthesis process.

Briefly described as adding 4-trimethylaminobenzoic acid ethyl trifluorosulfonate (5 mg, 16 μmol, dissolved in 0.3 mL of anhydrous acetonitrile) to a reaction flask containing activated anhydrous ^{18 F-} , heating at 90 °C 10 min. After the reaction liquid is cooled, add 0.7 mL of 1 mol/L NaOH, react at 100°C for 5 min, add 0.9 mL of 1 mol/L HCl after cooling, and pass the reaction liquid through an activated Sep-Pak C18 column, first with 2 mL Wash with 0.01 mol/L hydrochloric acid, dry the Sep-Pak C18 column with N ₂ , and then elute with 3 mL of acetonitrile to obtain 4-[ ¹⁸ F]fluorobenzoic acid. Add 20 μL of tetrapropylamine hydroxide aqueous solution, azeotropically dry at 100°C, add 12 mg of O-(N-succinimide)-1,1,3,3-tetramethylurea tetrafluoroboron acid ester (TSTU) (dissolved in 0.25 mL of acetonitrile), react at 90°C for 5 min. It was then acidified with 3 mL of 5% acetic acid and diluted with 6 mL of water. Pass the above solution through an activated Sep-Pak C18 column, rinse with 10 mL of acetonitrile/water ( V : V =1/7) sequentially, dry the Sep-Pak C18 column with _N2 , and wash with 2 mL of dichloromethane take off. The radiochemical purity was measured by TLC and HPLC, the activity was measured by an activity meter, and the radiochemical yield was calculated.

(2) Coupling of [ ¹⁸ F]SFB to Eexdin-4

Dissolve [ ¹⁸ F]SFB in 50 μL of acetonitrile, add 100 μg of Exendin-4 (dissolved in 200 μL of 0.1M borax-boric acid buffer with a pH value of 8.5), and react at room temperature for 30 min. After the reaction solution was filtered, it was separated by HPLC. The chromatographic column used was ZORBAX SB C18, 5 μm, 9.4 mm × 150 mm. The HPLC system used the following gradient to elute at a speed of 3 mL/min: Phase A contained 0.1% trifluoroacetic acid Acetonitrile and phase B are water containing 0.1% trifluoroacetic acid; 0-20 min, A rises from 16% to 90%; 20-30 min, A is 90%. Collect the peak at t _R =18-19.5 min, and the obtained product solution is evaporated to dryness by rotary evaporation, and re-dissolved with 0.9% NaCl injection solution to obtain the target product compound [ ¹⁸ F]FB-Exendin-4. After Radio-TLC, the developer is selected It is ethyl acetate and dichloromethane, the volume ratio of the two is 3:1, Radio-HPLC characterization, Radio-HPLC analysis method is the same as the preparation method, but the flow rate is 1mL/min, the chromatographic column is μBondapak ^TM C18, 5 μm, 300 mm × 3.9 mm.

Example 2. Determination of in vitro stability of ¹⁸ F-FB-Exendin-4

The two markers were placed in normal saline and fetal bovine serum, respectively, and placed in a constant temperature water bath at 37°C, and the radiochemical purity of the markers was detected by HPLC after 1, 4, 8, and 12 h, respectively.

Firstly, the labeling precursor ethyl-4-trimethylamine benzoate-trifluorosulfonate was successfully synthesized, which was synthesized by a three-step one-pot method, and then separated by a Sep-Pak C18 column to obtain the intermediate N-succinimide -4-[ ¹⁸ F]fluorobenzoate ([ ¹⁸ F]SFB). The radiochemical purity of [ ¹⁸ F]SFB is greater than 95%. Exendin-4 reacts with [ ¹⁸ F]SFB to obtain ¹⁸ F-FB-Exendin-4. Radioactive TLC showed an R _f value of 0.0 ^for18F -FB-Exendin-4 (Figure 2). Purification of the crude product by HPLC revealed a major radioactive peak, ¹⁸ F-FB-Exendin-4 peak, with retention time t _R =17.2 min (Fig. 3). The labeling rate of ¹⁸ F-FB-Exendin-4 was (35.6±2.3)% (n=4), and the radiochemical purity was greater than 98%. ¹⁸ F-FB-Exendin-4 has good stability in fetal bovine serum and normal saline, and its radiochemical purity can reach more than 90% when placed in a constant temperature water bath at 37°C for more than 12 hours.

Embodiment 3, in vitro cell competition binding experiment

Culture of RIN-m5f cells

Insulinoma cell line RIN-m5f cells (gifted by Cancer Hospital, Chinese Academy of Medical Sciences) were cultured in a 10 cm diameter culture medium with RPMI1640 medium (Sigma Company) containing 15% FBS (Hyclone), 1% penicillin and streptomycin (GBICO Company) Dish (Corning Company) and cultured in a constant temperature incubator (Heraeus Company) at 37°C and 5% CO ₂ .

Saturation curve of receptor and ligand

RIN-m5f cells were cultured for about 3 days, the cells were confluent to about 80%, digested with 0.25% trypsin (Invitrogen Company), centrifuged, and after cell counting, transferred to a 24-well plate (Corning Company), the number of cells per well Approximately 5×10 ⁵ cells were cultured in a constant temperature incubator at 37°C. After 24 hours, the cell culture medium was aspirated and washed twice with 1×PBS. Different concentrations of ¹²⁵ I-exendin4 were added to each well, and the PBS buffer was adjusted to a volume of 500 μL so that the final concentrations were 0, 0.03, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 nmol/L. For each concentration, 3 replicate wells were set. To deduct non-specific binding, 5 μg of unlabeled exendin-4 was added to 4 wells of 0, 0.03, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 nmol/L ¹²⁵ I-exendin, respectively, with a volume of 500 μL. Three replicate wells were set for each concentration. The above two groups were placed in a constant temperature incubator at 37°C and incubated for 40 minutes. After the reaction, wash 3 times with cold PBS containing 0.5% BSA, suck out the supernatant, add 500 μL of cold NaOH with a concentration of 1 mol/L to each well, and blow down the cells in each well from the bottom of the culture dish after reacting for 5 minutes. Move it to a gamma immune tube, and measure its radioactive count per minute (count per minute) with a gamma counter (Shanghai Rihuan Company).

The obtained data were processed by the GraphPad Prism5 single-point binding saturation curve fitting program, and ^{the 125} I-exendin4 receptor binding saturation curve was drawn.

competition binding experiment

Transfer the cells to a 24-well plate, the number of cells per well is about 5×10 ⁵ , place them in a constant temperature incubator at 37°C for culture, suck out the cell culture medium after 24 hours, wash twice with 1×PBS; take ¹²⁵ I-exendin- 4 at a concentration of 0.4 nmol/L, unlabeled exendin-4 at a concentration of 0, 0.1, 1, 10, and 100 nmol/L was added to each well, and incubated in a constant temperature incubator at 37°C for 40 minutes. After the reaction, wash 3 times with cold PBS containing 0.5% BSA, aspirate the supernatant, add 500 μL of cold NaOH with a concentration of 1 mol/L to each well, react for 5 minutes, blow the cells from the culture dish and transfer them to the γ-immuno In the tube, the cpm was measured by a gamma counter.

The obtained data were processed by the GraphPad Prism5 single-site competitive binding curve fitting program, and the half-inhibition rate IC ₅₀ value of exendin-4 inhibiting ¹²⁵ I-exendin4 was obtained.

In vitro cell competition binding assay

Since the insulinoma cell line RIN-m5f highly expresses the GLP-1 receptor, competition binding experiments show that the half-inhibition IC ₅₀ value of exendin-4 inhibiting ¹²⁵ I-exendin4 is 0.35-2.81nmol/L (Figure 4), indicating that Labeled exendin-4 has higher affinity to GLP-1 receptor.

saturation curve

The equilibrium dissociation constant K _d of ¹²⁵ I-exendin-4 and GLP-1 receptors was calculated to be 56.64, and the maximum binding constant B _max was 4.651×10 ⁷ through Graphpad Prism5 program. It shows that ¹²⁵ I-SFB-exendin-4 can specifically bind to the GLP-1 receptor, has the characteristics of saturation, reversibility, and high affinity (Figure 5).

Embodiment 4, nude mouse model small animal PET/CT imaging

Establishment of Insulinoma Nude Mouse Model

Female BALB/C nude mice aged 4-5W (purchased from Shanghai Institute of Experimental Animals) were bred by the Experimental Animal Center of Shanghai Jiao Tong University School of Medicine, and the breeding conditions were SPF grade sterile conditions. The animal experiments were approved by the Ethics Committee of Shanghai Jiao Tong University School of Medicine, which complied with the ethical principles of animal protection, feeding and experimentation.

When the cultured RIN-m5f cells were confluent to 80%, aspirate the culture medium, digest with 0.25% trypsin, transfer to a centrifuge tube, centrifuge at 1500 r/min for 5 min, resuspend in PBS, centrifuge twice, and finally resuspend with 150 μl of PBS, The cell count was about 1×10 ⁷ . A total of 12 nude mice were subcutaneously injected with 150 μl of RIN-m5f cell suspension on the dorsal side of the right lower limb of each nude mouse. Thereafter, the nude mice were reared routinely aseptically, and the drinking water was fed with 5% glucose injection (Shanghai Pharmaceutical Co., Ltd.). lower than 3mmol/L. After feeding for about 2-3 weeks, the tumor grows to 0.5-1.0 cm, and is used for PET/CT imaging of small animals.

Nude mouse model small animal PET/CT imaging

Six RIN-m5f model mice were injected with [ ¹⁸ F]FB-exendin-4 with a volume of 100ul containing 3.7MBq (100uCi) through the tail vein, and each nude mouse was placed in an induction chamber before imaging after injection Inside, 5% isoflurane was injected for anesthesia; 30min and 120min after anesthesia, the nude mice were placed on the small animal PET/CT (Inveon mPET/CT, Siemens Preclinical Solution, Knoxville, Tennessee, USA) scanning bed for 10min minute data collection. During imaging acquisition, nude mice were anesthetized with 1-2% isoflurane through a custom face mask.

Nude mouse model small animal PET/CT block imaging

Three insulinoma nude mice were injected with 100 μl of unlabeled Exendin-4 containing 250 μg through the tail vein, and then injected with 100 μl of [ ¹⁸ F]FB-exendin-4 containing 3.7 MBq through the tail vein. The other three RIN-m5f model nude mice were injected with 100 μl of [ ¹⁸ F]FB-exendin-4 containing 3.7 MBq through the tail vein, and served as the control group. Before imaging, nude mice were anesthetized in an induction chamber, and 60 minutes after injection of [ ¹⁸ F]FB-exendin-4, nude mice were placed on a small animal and fixed on a PET/CT scanning bed for 10 minutes of image acquisition , nude mice were anesthetized by inhalation of 1-2% isoflurane through a breathing mask during imaging.

Small animal PET/CT image processing

All small animal PET images were reconstructed by the standard ordered-subset expectation maximization method. The reconstructed images were displayed in transverse, coronal and sagittal positions; a total of 512 continuous tomographic images were displayed, with a slice thickness of 0.11mm, and the transverse, coronal and sagittal images were evaluated visually. The CT imaging and PET imaging are fused together, and the main organs and tissues such as the liver, kidney, lung, stomach, spleen, and tumor are located by CT, and the region of interest (ROI) is drawn in the PET image, which is automatically given by the computer program. Get the corresponding maximum SUV (SUVmax) value of the tissue and organ.

Distribution of radioactivity in various organs in vitro of nude mice

After the dynamic collection of nude mice for 120 minutes, they were immediately dislocated and killed. First, blood was collected from the heart, and then the peritoneum was cut to separate the liver, spleen, stomach, heart, lung, pancreas, kidney, small intestine, colon, femur, muscle and other organs, and weighed. Put it in a centrifuge tube, and measure the radioactive count per minute by a gamma counter, and the result is expressed as %ID/g (the radioactivity per gram of tissue accounts for the percentage of the injected dose).

Statistical Analysis

±SD表示，平均值利用单向方差分析和Student’s t test处理，当P＜0.05有显著性差异。 All data ends with

±SD indicates that the mean value is processed by one-way analysis of variance and Student's t test, and there is a significant difference when P<0.05.

result

RIN-m5f model mouse small animal PET/CT imaging

RIN-m5f model rats were injected with [18F]FB-Exendin-4 into the tail vein of the coronal section of small animal PET/CT static imaging (Figure 6). It can be seen that the radioactivity in the tumor site was significantly concentrated at 30 minutes, and the lungs, liver, kidneys and Bladder and other organs were clearly visualized (Fig. 6B); the tumor image faded at 120 minutes, and the lungs were still visible. The liver, kidney, bladder and other organs were visualized, but the radioactivity decreased (Fig. 6D). Figures 6A and 6C are CT coronal tomographic images of nude mice at 30 minutes and 120 minutes respectively, showing that the tumor was located on the dorsal side of the right lower limb of the nude mice.

After 30 minutes and 120 minutes of static imaging, the regions of interest of each organ were delineated from the PET image through CT positioning, and the corresponding SUVmax values were obtained (Figure 7). It can be seen that the SUV of kidney and liver was the highest at 30 minutes, followed by lung, stomach, small intestine and tumor; the ratio of SUV of tumor to muscle tissue was 4.57. After 120 minutes, the SUV of kidney and liver decreased, and the decrease of kidney and liver was the most obvious, but the kidney and liver still remained. Higher SUV value; lung, stomach and small intestine SUV decreased in varying degrees. The SUV in the skeletal muscle tissue was very low within 120 minutes. Because the pancreas is difficult to be clearly displayed on CT images, the SUV value of the pancreas was not determined. the

The above results show that [ ¹⁸ F]FB-Exendin-4 can effectively target tumors, and can be imaged by binding to tumor cell GLP-1 receptors. At the same time, [ ¹⁸ F]FB-Exendin-4 also binds to mouse lung tissue It was verified that the mouse lung tissue contained GLP-1 receptors, and the binding of [ ¹⁸ F]FB-Exendin-4 to GLP-1 receptors was further verified; [ ¹⁸ F]FB-Exendin-4 was mainly excreted through the kidney and liver , in line with the in vivo pharmacokinetic properties of the polypeptide.

Small Animal PET/CT Imaging of Model Mouse Exendin-4 Blocked

Model rats were injected with [ ¹⁸ F]FB-Exendin-4 10 minutes after tail vein injection of 250 μg Exendin-4, and small animal PET/CT imaging was performed 60 minutes later. The PET images showed no obvious radioactive uptake in the tumor site, and no development in the lungs. The liver was less developed, and both kidneys were significantly developed (Fig. 6F). The tumor site can be clearly located in the CT coronal tomographic image of nude mice, and the arrow indicates the tumor site (Fig. 6E).

The SUVmax value of the tumor and various organs was measured by PET imaging before and 60 minutes after Exendin-4 blockade (Figure 8), and it can be seen that the kidney, liver, stomach, small intestine, lung, and tumor all decreased to varying degrees. Among them, the reduction of tumor is the most obvious, followed by the lung, indicating that unlabeled Exendin-4 can specifically block the combination of [ ¹⁸ F]FB-Exendin-4 and tumor cells, and the biological characteristics of [ ¹⁸ F]FB-Exendin-4 are similar to those of Exendin- 4 is the same.

In vitro determination of radioactive biodistribution in various organs of model mice

After injecting 3.7MBq of [ ¹⁸ F]FB-Exendin-4 into the tail vein of RIN-m5f model mice for 120 minutes, the organs were isolated, and the measured results were expressed as %ID/g, as shown in FIG. 9 . It can be clearly seen that after 2 hours, the radioactivity count in the kidney was the highest at 0.27, followed by the liver at 0.18; the tumor was 0.15, which were higher than those in the lung, pancreas, small intestine, large intestine, stomach and spleen; the %ID/g in muscle tissue was the lowest , was 0.037; the ratio of tumor to muscle tissue was 7.1, and the radioactivity measurement results of each organ in nude mice were consistent with the imaging results in 120 minutes.

We performed indirect labeling by synthesizing N-succinimide-4-[ ¹⁸ F]fluorobenzoate ([ ¹⁸ F]SFB), an intermediate commonly used for ¹⁸ F-labeled peptides. Studies have shown that [ ¹⁸ F]SFB as an intermediate labeled Exendin-4 ( ¹⁸ F-SB-Exendin-4) has a radiochemical purity greater than 98%, a labeling rate of 35%, and good in vitro stability. In vitro binding experiments show that the IC50 value is 0.35-2.81nmol/L; the equilibrium dissociation constant Kd value is 56.64; the maximum binding constant Bmax is 4.651×107, indicating that 18F-SB-Exendin-4 has a high binding ability to the GLP-1 receptor specificity.

We established a RIN-m5F BALB/c nude mouse insulinoma model, and the tumor can be clearly visualized by small animal PET/CT imaging, and the tumor imaging can be blocked by unlabeled Exendin-4, indicating that [ ¹⁸ F] FB-Exendin-4 can specifically bind to insulinoma cells. Simultaneous imaging showed that the kidney and liver were significantly developed, and Exendin-4 blocking imaging still showed liver and kidney development. It can be seen that [ ¹⁸ F]FB-Exendin-4 is mainly metabolized by the kidney and liver. From the SUVmax analysis of PET/CT imaging of [ ¹⁸ F]FB-Exendin-4 nude mice, it can be seen that in addition to organs such as liver and kidney, the SUVmax of organs such as lung, stomach and small intestine is higher, and Exendin-4 blocking imaging It can be seen that the SUVmax of the above organs was significantly reduced. Gotthardt et al. showed that GLP-1 receptors were expressed in lung, stomach, pancreas and other organs through biodistribution and imaging studies in mice and rats. But for humans, in addition to the high expression of GLP-1 receptors in the islets and duodenum (Brunner's gland), there are only a small amount of GLP-1 receptors in organs such as the ileum, colon, kidney, lung and pancreas (acinus) Expression, but no GLP-1 receptor expression in tissues such as liver, spleen, skeletal muscle and fat. At the same time, PET/CT imaging can avoid the influence of overlapping organs, especially through the accurate positioning of CT, which reduces the radiation influence of surrounding organs, and has a good clinical application prospect.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications should also be considered Be the protection scope of the present invention. the

Claims (2)

1. one kind [ ¹⁸F] preparation method of FB-Exendin-4, may further comprise the steps:

(1) ¹⁸F ^-With the reaction of 4-Trimethylamine 99 ethyl benzoate three fluoro sulfonates, preparation intermediate N succinimide 4-[ ¹⁸F] the fluorobenzoic acid ester [ ¹⁸F] SFB;

(2) [ ¹⁸F] SFB and Eexdin-4 coupling obtain [ ¹⁸F] FB-Exendin-4,

It is characterized in that step (1) comprising: by magnetic resonance acceleator PET trace preparation [ ¹⁸F] F ^-, to fill activation [ ¹⁸F] F ^-Reaction flask in add 4-Trimethylamine 99 ethyl benzoate three fluoro sulfonates, with NaOH, HCl reaction, reaction solution is by the drip washing of Sep-Pak C18 post hydrochloric acid, the N of activation successively ₂Dry up, obtain 4-[with the acetonitrile wash-out again ¹⁸F] fluorobenzoic acid; Add hydroxide tetrapropyl amine, azeotropic drying adds O-(N-succinimide)-1,1,3, and acidifying with acetic acid is used in 3-tetramethyl-urea Tetrafluoroboric acid ester TSTU reaction then; With the Sep-Pak C18 post of above-mentioned solution by activation, use successively V: VN is used in=1/7 acetonitrile/water drip washing ₂Dry up Sep-Pak C18 post, use the methylene dichloride wash-out; Chemical purity is penetrated in TLC, HPLC measuring, and activity instrumentation activity is calculated putting productive rate.

According to claim 1 [ ¹⁸F] preparation method of FB-Exendin-4, it is characterized in that step (2) comprising: will [ ¹⁸F] SFB is dissolved in the acetonitrile, adds Exendin-4, and it is 0.1M borax-borate buffer of 8.5 that Exendin-4 is dissolved in the pH value, reacts 30min under the room temperature; Separate by HPLC behind the reacting liquid filtering, used chromatographic column is ZORBAX SB C18,5 μ m, 9.4 mm * 150 mm, the HPLC system adopts following gradient with the speed drip washing of 3 mL/min: wherein A mutually for the acetonitrile that contains 0.1% trifluoroacetic acid, B mutually for containing the water of 0.1% trifluoroacetic acid; 0-20 min, A is raised to 90% from 16%; 20-30 min, A are 90%; Collect t _RThe peak of=18-19.5 min, products therefrom solution, the rotation evaporate to dryness dissolves with 0.9% NaCl injection liquid again, get the target product compound [ ¹⁸F] FB-Exendin-4 is through Radio-TLC, and selecting developping agent for use is ethyl acetate and methylene dichloride, and both volume ratios are 3:1, and Radio-HPLC characterizes, and the Radio-HPLC analytical procedure is identical with the preparation method, but flow velocity is 1mL/min, chromatographic column is μ Bondapak ^TMC18,5 μ m, 300 mm * 3.9 mm.

Images