CN103861129B - A kind of Research of Recombinant Human Endostatin developer and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of molecular targeting imaging agent prepared by recombinant human endostatin. Specifically, to provide a new type of positron radionuclide or gadolinium-labeled recombinant human endostatin imaging agent and its non-invasive and dynamic observation of neovascularization in various tumors, and to evaluate the efficacy of anti-angiogenesis therapy and its application in therapeutic effect prediction. The invention also provides a preparation method of the imaging agent, which combines the positron radioactive nuclide or gadolinium with the recombinant human vascular endostatin through a bifunctional chelating agent.

Description

A recombinant human endostatin imaging agent and its preparation method

technical field

The invention belongs to the field of medicine, and in particular relates to a novel angiogenesis imaging agent prepared by recombinant human vascular endostatin and its application in tumor angiogenesis imaging diagnosis.

Background technique

Tumor angiogenesis plays an important role in tumor growth and metastasis. As early as the 1970s, Professor Folkman put forward the theory that "tumor growth and metastasis depend on the formation of new blood vessels". According to this theory, the anti-tumor angiogenesis therapy—dormancy therapy (dormancy therapy) has become one of the hot research fields of tumor therapy today. Some anti-angiogenic targeted drugs have been used clinically, such as bevacizumab, endostar, etc. A large number of clinical studies have confirmed that these drugs combined with chemotherapy or radiotherapy have brought higher treatment efficiency to patients. According to the mechanism of drug action, anti-angiogenesis therapy does not directly kill tumor cells, and its treatment does not rapidly shrink the tumor, but inhibits its growth and metastasis. The current RECIST criteria and WHO criteria used to evaluate the efficacy of chemotherapy are based on traditional imaging observations of tumor size changes before and after treatment to determine the efficacy. Such curative effect evaluation criteria obviously cannot evaluate anti-angiogenic therapy in a timely and accurate manner. As more and more tumor anti-angiogenesis therapies are applied clinically, there is an urgent need for a new method to predict and evaluate anti-angiogenesis therapies.

Positron Emission Tomography (PET) uses positron radionuclide-labeled drugs as imaging agents, which can display and measure the distribution of the labeled imaging agents in the living body. It can display different physiological and pathological metabolic changes by labeling different biomolecules, such as the metabolism of glucose, protein, fat and nucleic acid in the living body. effect. The invention of PET/CT combines PET's high-sensitivity metabolic images with CT's high-resolution anatomical images, making it more accurate in disease diagnosis. Using positron radionuclides to mark molecules related to tumor angiogenesis pathways can directly reflect the status of tumor neovascularization in vivo through PET or PET/CT, making molecular imaging of tumor angiogenesis possible.

Magnetic resonance imaging (MRI) is an imaging technique based on the principle of nuclear magnetic resonance, which has been widely used in the diagnosis of various diseases. MR imaging is noninvasive and harmless, especially for its extremely high resolution of soft tissues. Gadolinium (Gd) complexes have been used clinically as MR contrast agents in the 1980s. Gd contrast agents commonly used in clinical practice are: gadopentetate dimeglu-mine (gadolinium diethylenetriaminoopenta-acetic acid-Gd-DTPA), gadoteridol (Gd-HP-DO3A), gadoterate meglumine (gadoliniumtetra-azacy-clododecanetre-acetic acid-Gd-DOTA) and gadodiamide (Gd-DTPA-bismethylamide). The application of these Gd contrast agents can increase the detection rate of tumor lesions. However, the Gd contrast agent currently used in clinical practice has no tumor and organ specificity, and the use of Gd to label certain antibodies or biomolecules for specific targeted MR molecular imaging has become a new research direction. Recently, Tan et al. reported the use of an MR molecular imaging agent that specifically targets the fibrin-fibronectin complex in the tumor stroma, CLT1-G2-(Gd-DOTA), to perform human prostate cancer tumor-bearing nude mice. Research on MR imaging (Pharm Res.2012;29(4):953-60.). This proves that it is scientifically feasible to use Gd complexes to label biomolecules for targeted MR molecular imaging.

In 1997, O'Reilly et al. isolated an angiogenesis inhibitor different from angiostatin from the culture serum of mouse hemangioendothelioma cells (EOMA), and named it Endostatin (vascular endostatin). The amino acid sequence analysis of Endostatin shows that it is an amino acid fragment of the C-terminal non-collagen region of collagen XVIII, with a molecular weight of 20kD and 184 amino acids. The direct target of Endostatin is the endothelial cells of neovascularization, and it has no inhibitory effect on other cells and normal quiescent endothelial cells. In 2005, recombinant human endostatin (Endostar, Endostar) was launched in my country. It is used in combination with chemotherapy drugs for the treatment of stage III and IV non-small cell lung cancer. Advantages of tumor metastasis. Endostatin has added 9 amino acids to the N-terminal of the original Endostatin sequence to make its properties more stable and maintain its activity in vivo. Zhang Jinhe et al. used ¹³¹ I to label Endostatin, and the results showed that Endostatin could be successfully labeled with ¹³¹ I, and the inhibition rate of the product on endothelial cells was higher than that of pure Endostatin, and it also had certain stability in vitro. However, ¹³¹ I can only be used for SPECT imaging, and its imaging resolution is low, which limits its clinical application.

Therefore, in view of the traditional imaging observation and treatment of tumor efficacy evaluation and the current defects of ¹³¹ I-labeled Endostatin as an imaging agent, as well as the urgent need for predicting and evaluating the effect of anti-angiogenic therapy in clinical practice, a new molecular target of recombinant human endostatin was developed. It is very necessary to perform PET, PET/CT imaging or MR imaging with the imaging agent.

Contents of the invention

The purpose of the present invention is to provide a novel molecular targeting imaging agent for non-invasive and dynamic observation of tumor neovascularization in vivo, which can be used for PET, PET/CT and MR imaging. Another object of the present invention is to provide the preparation method and application of the imaging agent. The positron radioactive nuclide or gadolinium is combined with recombinant human endostatin through a bifunctional chelating agent to make a imaging agent for PET and PET/CT imaging. It is a molecularly targeted imaging agent for imaging or MR imaging, which is used for non-invasive and dynamic observation of tumor angiogenesis in vivo in humans or other mammals.

The invention provides a recombinant human endostatin imaging agent, characterized in that the imaging agent has the general structure of the following formula:

R-Bifunctional Chelator-X,

Wherein R is a positron radionuclide or gadolinium, and X is recombinant human endostatin.

In the recombinant human endostatin imaging agent of the present invention, the R is selected from positron radionuclides or gadolinium (Gd), wherein positron radionuclides are selected from: ¹⁸ F, ⁵⁵ Co, ⁶⁰ Cu, ⁶¹ Cu, ⁶² Cu, ⁶⁴ Cu, ⁶⁶ Ga, ⁶⁸ Ga, ⁸² Rb, and ⁸⁶ Y; the R-bifunctional chelating agent is selected from: [ ¹⁸ F]SFB, [ ¹⁸ F]SFBS, [ ¹⁸ F]SFMB, [ ¹⁸ F]FPA, [ ¹⁸ F]FPPD, [ ¹⁸ F]FBEM, [ ¹⁸ F]FBBO, [ ¹⁸ F]NPFP, R-DOTA-NHS, R-NOTA-NHS; the recombinant human endostatin is preferably for Endostar.

The above R-DOTA-NHS is selected from: [ ⁵⁵ Co]DOTA-NHS, [ ⁶⁰ Cu]DOTA-NHS, [ ⁶¹ Cu]DOTA-NHS, [ ⁶² Cu]DOTA-NHS, [ ⁶⁴ Cu]DOTA-NHS, [ ⁶⁶ Ga]DOTA-NHS, [ ⁶⁸ Ga]DOTA-NHS, [ ⁸⁶ Y]DOTA-NHS, [Gd]DOTA-NHS; the R-NOTA-NHS is selected from: [Al ¹⁸ F]NOTA-NHS, [ ^66Ga ]NOTA-NHS, [ ^68Ga ]NOTA-NHS, [Gd]NOTA-NHS.

The present invention also provides a preparation method for preparing recombinant human endostatin imaging agent, comprising the following steps: eluting the R-bifunctional chelating agent with acetonitrile, blowing dry with nitrogen, and then mixing with recombinant human endostatin The buffer solution reacts, and after the reaction is completed, the reaction product is eluted and separated to obtain the target product. Wherein the R-bifunctional chelating agent is selected from: [ ¹⁸ F]SFB, [ ¹⁸ F]SFBS, [ ¹⁸ F]SFMB, [ ¹⁸ F]FPA, [ ¹⁸ F]FPPD, [ ¹⁸ F]FBEM, [ ¹⁸ F] FBBO, [ ¹⁸ F]NPFP.

The present invention also provides another preparation method for preparing recombinant human endostatin imaging agent, comprising the following steps: first reacting the bifunctional chelating agent with recombinant human endostatin buffer solution to prepare bifunctional chelating agent-recombinant Human vascular endostatin, and then use R to combine with bifunctional chelating agent-recombinant human vascular endostatin. After the reaction is completed, the reaction product is eluted and separated to obtain the target product. Here the R-bifunctional chelating agent is selected from: R-DOTA-NHS, R-NOTA-NHS, wherein the R-DOTA-NHS is selected from: [ ⁵⁵ Co]DOTA-NHS, [ ⁶⁰ Cu]DOTA-NHS, [ ⁶¹ Cu]DOTA-NHS, [ ⁶² Cu]DOTA-NHS, [ ⁶⁴ Cu]DOTA-NHS, [ ⁶⁶ Ga]DOTA-NHS, [ ⁶⁸ Ga]DOTA-NHS, [ ⁸⁶ Y]DOTA-NHS, [Gd ]DOTA-NHS; the R-NOTA-NHS is selected from: [Al ¹⁸ F]NOTA-NHS, [ ⁶⁶ Ga]NOTA-NHS, [ ⁶⁸ Ga]NOTA-NHS, [Gd]NOTA-NHS.

In the above two labeling preparation methods, the molar ratio of the bifunctional chelating agent to recombinant human endostatin is 10-40:1, preferably 20:1, the reaction temperature is 30-45°C, and the reaction time is 10min-60min. The temperature is 37°C, and the preferred reaction time is 30 minutes. After the reaction is completed, the reaction product is eluted and separated with a desalting column. Wherein the recombinant human endostatin is preferably Endostar.

The present invention also provides a recombinant human endostatin imaging agent used in live imaging for observing tumor angiogenesis, wherein the imaging agent labeled with a positron radionuclide is applied to PET or PET/CT imaging, and gadolinium-labeled The imaging agent used in MR imaging. The tumors include: lung cancer, glioma, endometriosis, angiogenic fundus lesions, neuroendocrine tumors, colon cancer, bone cancer, liver cancer, gastric cancer, pancreatic cancer, oral cancer, breast cancer, prostate cancer , lymphoma, esophageal cancer, nasopharyngeal cancer, sarcoma, kidney cancer, gallbladder cancer, malignant melanoma.

After the targeted imaging agent of the present invention enters the body of a normal mouse via vein, it is quickly distributed from the blood to various organs, mainly distributed in the kidney, and is metabolized through the kidney.

After the targeted imaging agent of the present invention enters the tumor-bearing nude mice through veins, in addition to the above-mentioned physiological distribution in normal mice, the tumor also has obvious uptake of the targeted imaging agent.

The advantages and positive effects of the present invention are that it is proposed and proved for the first time that recombinant human endostatin labeled with positron radionuclides such as ¹⁸ F can be used as a targeted molecular imaging agent for molecular imaging of PET or PET/CT; Gd-labeled recombinant human vascular endostatin can be used as a targeted molecular imaging agent for MR molecular imaging, non-invasive and dynamic observation of tumor neovascularization in vivo, and is expected to be used in the evaluation and prediction of the efficacy of tumor anti-angiogenesis therapy .

In addition, recombinant human vascular endostatin is labeled with positron radionuclides or gadolinium through a bifunctional chelator to form a targeted imaging agent. The targeted imaging agent obtained under different pH labeling conditions, biological There is no significant difference in activity between recombinant human endostatin stock solution.

Description of drawings

Figure 1 ¹⁸ F-SFB marker Endostar roadmap.

Figure 2 R-TLC analysis spectrum.

Figure 3 Comparison of the activity of the targeted imaging agent and the original drug of Endostar under different pH conditions. (P1 peak is ¹⁹ F-FB-Endostar).

Fig. 4 Time-biodistribution curve of ¹⁸ F-FB-Endostar in normal mice.

Fig. 5 PET/CT imaging of normal mouse ¹⁸ F-FB-Endostar small animal.

Figure 6 Typical pictures of ¹⁸ F-FB-Endostar imaging of nude mice bearing human lung adenocarcinoma A549 cells ("ten" in the figure indicates the tumor site).

Fig. 7 Typical pictures of ¹⁸ F-FB-Endostar imaging of nude mice bearing human malignant glioma U87-MG cells ("ten" in the figure indicates the tumor site).

Figure 8 The results of the inhibition experiment in nude mice bearing human lung adenocarcinoma A549 cells.

Fig. 9 The results of inhibition experiments on nude mice bearing human brain malignant glioma U87-MG cells.

Detailed ways

The specific implementation cases are to further illustrate the present invention but do not constitute a limitation to the protection scope of the present invention.

Material: Recombinant human endostatin injection--Endostar is provided by Shandong Xiansheng Mai Dejin Biopharmaceutical Co., Ltd., 15mg/bottle. ¹⁸ F ions were produced by the RDS111 accelerator (CTI Corporation, USA). All other materials were commercially available.

Example 1: Synthesis experiment of ¹⁸ F-FB-Endostar labeling

The synthesis steps are as follows:

1. The 800mCi ¹⁸ F ion synthesized by the accelerator, 20mg _K2.2.2 , 5mg K2CO3 and 5mg ₄ -trimethylaminobenzoic acid ethyl trifluoromethanesulfonate were reacted at 120°C for 20min, and then added 1ml of 0.5mmol/L NaOH solution was hydrolyzed, and then 15ml of 0.1mmol/L HCL solution was added to generate 4-[ ¹⁸ F]fluorobenzoic acid ( ¹⁸ F-FBA);

2. The aforementioned ¹⁸ F-FBA and 17mg TSTU (2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate) were heated for 15min, and then 3ml of 5% acetic acid and 10ml of H The acetic acid solution obtained by mixing with ₂ O was acidified and purified by a C-18 column to obtain ¹⁸ F-SFB ( ¹⁸ FN-succinimide-4-fluorobenzoate).

3. The ¹⁸ F-SFB obtained in the above reaction was eluted with acetonitrile, blown dry with nitrogen, then dissolved 1mg Endostar in 0.5ml phosphate buffer (pH8.2) into the reaction tube, and reacted at 37°C for 30min. The final reaction product was separated by a P-10 gel column (GE Company) with neutral phosphate buffer (pH 7.0) as the mobile phase to obtain the product ¹⁸ F-FB-Endostar.

Example 2. Stability of markers in human serum

Mix the obtained 500μCi ¹⁸ F-FB-Endostar with 1ml of normal human serum and place it at 37°C, take samples at 1h and 2h respectively for R-TLC analysis (radioactive chromatography analysis), and observe the stability of the marker in human serum .

The results are shown in Figure 2 for analysis. When mixed with normal human serum for 1 hour and 2 hours, the position of the radiation peak of the marker did not change, and no other radioactive peaks appeared. It shows that the marker has good stability in serum, and there is no obvious degradation in serum at 37°C for 2 hours.

Example 3. ¹⁹ F-SFB-labeled Endostar experiment at different pH values and detection of Endostar activity after labeling

In order to verify whether Endostar still has activity after labeling, we carried out ¹⁹ F-SFB labeling Endostar experiment and labeling Endostar activity detection under different pH value conditions. In order to avoid radioactive contamination when detecting activity, non-radioactive ¹⁹ F should be used instead of ¹⁸ F for labeling, and ¹⁹ F and ¹⁸ F have exactly the same chemical properties. ¹⁹ F-SFB was synthesized by Changshu Huayi Chemical Co., Ltd.

¹⁹ F-SFB and Endostar were labeled at pH 6.5, 7.0 and 8.0 respectively, the molar ratio of ¹⁹ F-SFB and Endostar was 20:1, the reaction temperature was 37°C, and the reaction time was 30 minutes. After the reaction was completed, the reaction product was eluted and separated by a desalting column, and two peaks were obtained during the eluted separation. The first peak (p1) is the target product ¹⁹ F-FB-Endostar, and the second peak (p2) is the excluded protein. Because the labeling intensity of peak 2 protein is high, resulting in epitope loss, it was discarded. The separated target product ¹⁹ F-FB-Endostar was tested for its binding ability to the Endostar antibody by ELISA, and compared with the original drug of Endostar. The results are shown in Figure 3. It can be seen from the results in Figure 3 that ¹⁹ F-FB-Endostar has the same binding ability to Endostar antibody as the original drug of Endostar, while peak 2 cannot bind to Endostar antibody. The results show that the target product ¹⁹ F-FB-Endostar has the same activity as the original drug of Endostar.

Example 4: In vivo distribution experiment of ¹⁸ F-FB-Endostar in normal mice

20 normal Kunming mice were randomly divided into 5 groups, 4 in each group, injected with purified 80 Ci ¹⁸ F-FB-Endostar from the tail vein, at 15 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes after injection. Minutes were decapitated and dissected, and the percentage injection dose per gram of tissue of heart, liver, spleen, kidney, lung, brain, blood, muscle and other tissues and organs was measured, and the time-biodistribution curve was drawn. Observe the normal biodistribution data of ¹⁸ F-FB-Endostar in mice.

The normal biodistribution of ¹⁸ F-FB-Endostar in mice is shown in Figure 4. After ¹⁸ F-FB-Endostar was injected intravenously into the mice, it quickly distributed to various organs, mainly in the kidney, and had the lowest uptake in the brain. Considering that it was related to the blood-brain barrier, the uptake in the lungs was also low. Based on these data, future application of this imaging agent to brain and lung tumors may lead to clearer images.

One normal Kunming mouse from the same batch was injected with 1mCi ¹⁸ F-FB-Endostar through the tail vein for 1 hour, then anesthetized by intraperitoneal injection of 100mg/kg ketamine, the mouse was placed in prone position, the limbs were stretched, and the heat preservation was fixed for small animal PET/CT. Imaging (GE Explore VISTA Micro PET/CT in the United States), a total of two beds, each bed collected about 10 minutes. The ¹⁸ F-FB-Endostar imaging images of normal mice are shown in Figure 5.

Example 5: Imaging experiment on tumor-bearing nude mice

Tumor cells (lung adenocarcinoma A-549 cells, human brain malignant glioma U87-MG cells) were cultured routinely. The cells were purchased from the American Type Culture Collection (ATCC) and provided by the Central Laboratory of Shandong Cancer Hospital. Cultivate and preserve. Ten BALB/c nude mice, female, aged 5-6 weeks, weighing 15-20 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. The above-mentioned tumor cells in the logarithmic growth phase were collected, and the cell concentration was adjusted to 1×10 ⁶ cells/mL. 100 μl of the cell suspension was extracted from each nude mouse with a 1 ml syringe, and inoculated subcutaneously on the back of the right forelimb of the nude mouse. Five nude mice were inoculated with each cell. After the nude mice were inoculated with tumors, they were kept in constant temperature (25°C±2°C), constant humidity (45%-50%), and kept in a sterile purification barrier system, and the nude mice were regularly observed for spirit, diet, defecation, and tumor development. grow. Imaging experiments were started when the tumor diameter grew to more than 1 cm. Each tumor-bearing nude mouse was injected with 1mCi of imaging agent through the tail vein, and small animal PET/CT imaging was performed 1 hour after the injection (imaging conditions were the same as those of normal mice).

Typical ¹⁸ F-FB-Endostar imaging images of tumor-bearing nude mice are shown in Figure 6 and Figure 7 . Figure 6 is a typical picture of ¹⁸ F-FB-Endostar imaging of nude mice bearing human lung adenocarcinoma A549 cells. Fig. 7 is a typical picture of ¹⁸ F-FB-Endostar imaging of nude mice bearing human brain malignant glioma U87-MG cells. It can be seen from the figure that the tumor has obvious uptake of ¹⁸ F-FB-Endostar. Meanwhile, the uptake of human brain malignant glioma U87-MG cell xenografts with rich angiogenesis was significantly higher than that of lung adenocarcinoma A-549 cell xenografts. The center of lung adenocarcinoma A-549 cell xenograft tumor had obvious necrosis, and the imaging agent was mainly distributed around the tumor. The above results indicate that the ¹⁸ F-FB-Endostar imaging agent can perform tumor vessel imaging.

Example 6: Imaging experiment of "competitive inhibition" in tumor-bearing nude mice

Two nude mice bearing the above-mentioned tumors were taken, and one tumor of lung adenocarcinoma A-549 cells and one tumor of human brain malignant glioma U87-MG cells were transplanted. Each nude mouse was imaged twice, and the imaging conditions on the first day were the same as in Example 4. On the second day, 1 hour before the injection of the ¹⁸ F-FB-Endostar imaging agent, each nude mouse was injected with 1 mg of Endostar original drug through the tail vein, and the imaging time and conditions after the injection of the imaging agent were exactly the same as those on the first day .

Figure 8 shows the results of the inhibition experiment on nude mice bearing human lung adenocarcinoma A549 cells. Figure 9 shows the results of the inhibition experiment on nude mice bearing human brain malignant glioma U87-MG cells. From the results in Figure 8 and Figure 9, it can be seen that after injection of the original drug of Endostar, the uptake of ¹⁸ F-FB-Endostar imaging agent in tumor-bearing nude mice was significantly inhibited. It shows that the ¹⁸ F-FB-Endostar imaging agent has the same target in vivo as the original drug of Endostar, which can faithfully reflect the distribution of the original drug of Endostar in tumor-bearing nude mice.

Claims (8)

1. a recombinant human endostatin imaging agent, characterized in that, the imaging agent has the general structure of the following formula: R-Bifunctional Chelator-X, Wherein R is a positron radioactive nuclide, X is a recombinant human endostatin selected from Endostar, and the R-bifunctional chelating agent is selected from [ ¹⁸ F]SFB. 2. The imaging agent according to claim 1, whose structural formula is ¹⁸ F-FB-Endostar. 3. a preparation method of imaging agent as claimed in claim 1 or 2, is characterized in that, comprises the steps: The R-bifunctional chelating agent is eluted with acetonitrile, dried with nitrogen, and then reacted with recombinant human endostatin buffer solution. After the reaction, the reaction product is eluted and separated to obtain the target product. 4. The preparation method according to claim 3, characterized in that the molar ratio of the R-bifunctional chelating agent to recombinant human endostatin is 10-40:1. 5. The preparation method according to claim 3, wherein the molar ratio of the bifunctional chelating agent to recombinant human endostatin is 20:1. 6. The preparation method according to claim 3, characterized in that the reaction temperature for the combination of the bifunctional chelating agent and recombinant human endostatin is 30-45°C, and the reaction time is 10-60 minutes. 7. The preparation method according to claim 6, characterized in that the reaction temperature of the combination of the bifunctional chelating agent and recombinant human endostatin is 37° C., and the reaction time is 30 minutes. 8. The application of the recombinant human vascular endostatin imaging agent according to claim 1 or 2 in the preparation of an in vivo detection imaging agent of tumor neovascularization status, the application is PET or PET/CT imaging, and the tumor Lung cancer, glioma.