阅读说明：本技术 一种肿瘤PET显像剂68Ga-NOTA-ADG及其制备方法和应用 (Tumor PET imaging agent68Ga-NOTA-ADG and preparation method and application thereof ) 是由 程登峰 林卿玉 石洪成 于 2021-09-16 设计创作，主要内容包括：本发明涉及一种肿瘤PET显像剂～(68)Ga-NOTA-ADG及其制备方法和应用,涉及PET显像剂～(68)Ga-NOTA-ADG的标记前体的合成和～(68)Ga-NOTA-ADG的放射性合成,其中,前体是第一次用于～(68)Ga-NOTA-ADG的标记,结果显示该制备方法放射化学产率接近100％。本发明的正电子显像剂～(68)Ga-NOTA-ADG能够作为靶向肿瘤的PET显像探针。因此,使得～(68)Ga-NOTA-ADG成为肿瘤的PET候选显像探针。本发明首次进行了～(68)Ga-NOTA-ADG放射性化学合成,制备方法简单快捷,为～(68)Ga-NOTA-ADG的科学研究和临床应用奠定了基础。(The invention relates to a PET imaging agent for tumors 68 Ga-NOTA-ADG, a preparation method and application thereof, relating to a PET imaging agent 68 Synthesis of labeled precursor of Ga-NOTA-ADG and 68 radioactive synthesis of Ga-NOTA-ADG, wherein the precursor is first used for 68 Ga-NOTA-ADG labeling shows that the preparation method has near 100% radiochemical yield. The positron imaging agent of the invention 68 Ga-NOTA-ADG can be used as a PET imaging probe for targeting tumors. Thus, make 68 Ga-NOTA-ADG becomes a PET candidate imaging probe for tumors. The invention is carried out for the first time 68 Ga-NOTA-ADG radiochemical synthesis, the preparation method is simple and rapid, and 68 the Ga-NOTA-ADG lays a foundation for scientific research and clinical application.)

1. Tumor PET imaging agent⁶⁸Ga-NOTA-ADG, wherein the imaging agent has the following structural formula:

2. the tumor PET imaging agent of claim 1⁶⁸A method for preparing Ga-NOTA-ADG, which is characterized by comprising the following steps: the precursor NOTA-Amino-DG solution was added to the NaOAc solution, and then added to the solution⁶⁸GaCl₃Culturing the reaction mixture to obtain the tumor PET imaging agent⁶⁸Ga-NOTA-ADG。

3. The PET tumor imaging agent of claim 2⁶⁸A process for producing Ga-NOTA-ADG, characterized in that the precursor NOTA-Amino-DG solution is prepared from⁶⁸GaCl₃The ratio of (A) to (B) is 50. mu.l of (0.70-0.74) GBq.

4. The PET tumor imaging agent of claim 2⁶⁸A method for producing Ga-NOTA-ADG, characterized in that the culture is carried out at a pH of 5 to 6 for a period of 4 to 8 min.

5. The PET tumor imaging agent of claim 2⁶⁸The preparation method of Ga-NOTA-ADG is characterized in that the precursor NOTA-Amino-DG has the following structural formula:

6. the PET tumor imaging agent of claim 2⁶⁸A method for producing Ga-NOTA-ADG, characterized in that the precursor NOTA-AmThe preparation method of ino-DG comprises the following steps: adding p-SCN-Bn-NOTA.3 HCl into the 2-Amino-2-deoxy-D-glucose and HEPES sodium salt solution, culturing the mixed solution to obtain a precursor NOTA-Amino-DG solution, and storing for later use.

7. The PET tumor imaging agent of claim 6⁶⁸The preparation method of Ga-NOTA-ADG is characterized in that the molar ratio of the 2-amino-2-deoxy-D-glucose to p-SCN-Bn-NOTA.3HCl is (40-50) to (10-12).

8. The PET tumor imaging agent of claim 6⁶⁸A process for producing Ga-NOTA-ADG, characterized in that the culture is carried out at a pH of 8 to 10 for a period of 9 to 12 hours.

9. The tumor PET imaging agent of claim 1⁶⁸Use of Ga-NOTA-ADG.

10. The tumor PET imaging agent according to claim 9⁶⁸The application of Ga-NOTA-ADG is characterized in that the imaging agent can be used as a tumor-targeted positron tracer and applied to imaging of animal model tumors.

Technical Field

The invention relates to the technical field of radiopharmaceutical chemistry, in particular to a tumor PET (polyethylene terephthalate) imaging agent⁶⁸Ga-NOTA-ADG and a preparation method and application thereof.

Background

Recently, the world health organization international agency for research on cancer (IARC) released the latest data on global cancer burden in 2020, estimating the latest morbidity, mortality and cancer development trends in 36 of 185 countries worldwide. According to the latest estimated data, 1929 ten thousand new cancer cases worldwide in 2020, wherein 1006 ten thousand cases are male, 923 ten thousand cases are female; by 2020, 996 million people worldwide will die of cancer, including 553 million men and 443 million women. Every year, many people die from cancer, and one of the important reasons for this is that cancer is not diagnosed and treated in a timely manner.

¹⁸F-FDG was synthesized as early as 1969 as a molecular probe reflecting glucose metabolism in tumor tissues. At present, the number of the current day,¹⁸F-FDG is the most popular radiopharmaceutical based on carbohydrate metabolism and is called the "century molecule" because of its remarkable imaging effect.¹⁸F-FDG PET/CT imaging has important values for diagnosis, staging, curative effect monitoring and prognosis evaluation of cancer patients. However, the production of F-18 nuclides requires positron cyclotron and advanced shielding fields, and the cost and initial investment are enormous, reaching millions of dollars.

However, the production of Ga-68 labeled PET probes is very simple and requires only a simple hot chamber. As a positron nuclide, its excellent nuclide properties make it very suitable for PET imaging (89% β +), especially for target molecules with short biological half-lives (67.6 min for T1/2). Ga-68 has many advantages such as good physicochemical properties, commercial generators, strong chemical labeling diversity, etc. Another significant advantage of Ga-68 is that it can be replaced by a therapeutic nuclide (e.g., Lu-177) in the study and development of radiopharmaceuticals, thus converting the imaging probe into a radiotherapeutic probe, which allows integration of diagnosis and therapy. This is described in detail in a number of documents, which strongly motivates researchers to develop new radiopharmaceuticals labeled with Ga-68. The importance and prevalence of Ga-68 can also be seen by comparing the number of clinical trials. The quantity of Ga-68 labeled molecular probes in clinical research is obviously higher than that of other nuclides.

In 2018, Zengwen and coworkers introduced glucose metabolism molecules labeled by various metal nuclides in detail. The imaging effect of partial molecular probe is even better than that of partial molecular probe¹⁸F-FDG, and entered the early clinical trial stage. From various aspectsGa-68 is considered to be a nuclide well suited for labeling molecular probes. One of the reasons is that Ga-68 is a positive electron nuclide, and the resolution of PET is higher than SPECT; another reason is for the preparation of Ga-68⁶⁸Ge-⁶⁸The service life of the Ga generator is as long as one year. If the Ga-68 labeled molecular probe PET tumor imaging can be applied to poor areas or areas with a small number of patients, the imaging method is beneficial to those critical patients and tumor patients who are not suitable for long-distance referral.

In 2012, Yankee and his colleagues labeled DOTA-ADG with Ga-68. In a microwave reactor, the purity of the obtained product is more than 98 percent after purification⁶⁸Ga-DOTA-ADG, labeling efficiency 85%. Using A431 cell (human skin squamous cell carcinoma) animal model, for⁶⁸Ga-DOTA-ADG has high tumor uptake rate when being biologically evaluated.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a tumor PET imaging agent⁶⁸Ga-NOTA-ADG and a preparation method and application thereof.

The purpose of the invention can be realized by the following technical scheme:

the inventors have appreciated that NOTA is a more suitable chelator for labeling Ga-68 than DOTA, because the ionic radius of Ga-68 is smaller,⁶⁸thermodynamically stable constant ratio of Ga-NOTA⁶⁸Ga-DOTA is about 10 orders of magnitude higher. Thus, the present application attempts to synthesize NOTA-ADG and study its biological behavior, as follows:

tumor PET imaging agent⁶⁸Ga-NOTA-ADG, the structural formula of the imaging agent is as follows:

the tumor PET imaging agent⁶⁸A preparation method of Ga-NOTA-ADG comprises the following steps: the precursor NOTA-Amino-DG solution was added to the NaOAc solution, and then added to the solution⁶⁸GaCl₃Standing the reaction mixture at room temperature for several minutes to obtain the tumor PET imaging agent⁶⁸Ga-NOTA-ADG。

Further, the precursor NOTA-Amino-DG solution and⁶⁸GaCl₃the ratio of (A) to (B) is 50. mu.l of (0.70-0.74) GBq.

Further, the culture has pH of 5-6 and time of 4-8 min. The product does not need further purification, and the radiochemical purity is more than 95 percent.

Further, the precursor

The structural formula of 2,2' - (2- (4- (3- ((3R,4R,5S,6R) -2,4,5-trihydroxy-6- (hydroxymethy) tetrahydro-2H-pyran-3-yl) thioureido) benzyl) -1,4, 7-triazine-1, 4,7-triyl) triacetic acid (named NOTA-Amino-DG) is as follows:

further, the preparation method of the precursor NOTA-Amino-DG comprises the following steps: adding p-SCN-Bn-NOTA.3 HCl into 2-Amino-2-deoxy-D-glucose and HEPES sodium salt solution, culturing the mixed solution to obtain a precursor NOTA-Amino-DG solution, and storing at-20 ℃ for later use. The preparation method is a conventional organic synthesis reaction, and the product does not need to be purified by HPLC.

Further, the molar ratio of the 2-amino-2-deoxy-D-glucose to the p-SCN-Bn-NOTA.3HCl is (40-50): 10-12).

Further, the culture has pH of 8-10 and time of 9-12 h.

The tumor PET imaging agent⁶⁸Use of Ga-NOTA-ADG.

Furthermore, the imaging agent can be used as a positron tracer agent of a targeted tumor and applied to imaging of tumors of animal models.

Compared with the prior art, the invention has the following advantages:

(1) the precursors of the present invention were used for the first time⁶⁸The Ga-NOTA-ADG is marked, and the result shows that the radiochemical yield of the preparation method is close to 100 percent, and the positron imaging agent of the invention⁶⁸Ga-NOTA-ADG can be used as a PET imaging probe for targeting tumors, so that⁶⁸Ga-NOTA-ADG as a PET candidate for tumorsSelecting a development probe, the invention is carried out for the first time⁶⁸Ga-NOTA-ADG radiochemical synthesis, the preparation method is simple and rapid, and⁶⁸the basis is laid for the scientific research and clinical application of Ga-NOTA-ADG;

(2) in the target PET probe⁶⁸In the preparation method of Ga-NOTA-ADG, NaOAc solution is selected as a reaction solvent,⁶⁸GaCl₃the leacheate is selected from 0.05M HCl to obtain higher labeling rate, the labeling step is simple and convenient, the radiochemical purity is more than 95 percent, and the total radiochemical yield is nearly 100 percent (attenuation correction). The preparation method has the advantages of simple synthetic route, short synthetic time, high radiochemical purity and specific activity meeting the requirements.

Drawings

FIG. 1 is a drawing of⁶⁸Biodistribution of Ga-NOTA-ADG in ICR mice;

FIG. 2 is an ICR mouse PET image;

FIG. 3 is a tumor model mouse⁶⁸Ga-NOTA-ADG PET imaging.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

Example 1

Synthesis of precursor NOTA-Amino-DG:

to a solution (1mL) of 2-amino-2-deoxy-D-glucose (10mg, 46.4. mu. mol, 4.3 equivalents) and HEPES sodium salt (30mg, 115. mu. mol) was added p-SCN-Bn-NOTA.3 HCl (6mg, 10.7. mu. mol, 1.0 equivalents) at room temperature. The solution was incubated at room temperature for 10 hours and then stored for use at-20 ℃ according to the following reaction scheme:

target compound PET probe⁶⁸Synthesis of Ga-NOTA-ADG:

add 50. mu.l of precursor to NaOAc solution (0.25M, 950. mu.l)And (3) solution. Adding to the solution⁶⁸GaCl₃(eluent 0.05M HCl, 0.70-0.74GBq, 4 mL). I.e. after dissolving the precursor in the sodium acetate solution, the Ge/Ga generator was rinsed with 0.05M HCl and the rinse was added directly to the sterile bottle of sodium acetate solution containing the precursor.

The reaction mixture was incubated at room temperature for 5 minutes. The product does not need further purification, the radiochemical purity is more than 95 percent, and the reaction formula is as follows:

application example 1:⁶⁸in vitro stability assay of Ga-NOTA-ADG:

obtained as in example 1⁶⁸Ga-NOTA-ADG about 1mCi is respectively put in 100 mu L of 0.9% physiological saline, fully mixed and stored at 37 ℃. Samples were taken at 0.5h, 1h, 2h, and 3h, respectively, and checked for purity change on analytical HPLC. HPLC results show that the purity of the PET probe after 3h is the same as that of the PET probe immediately after marking, which indicates that the molecular probe is very stable and hardly decomposed.

Application example 2:⁶⁸biodistribution experiments of Ga-NOTA-ADG:

obtained as in example 1⁶⁸Ga-NOTA-ADG about 100 mu Ci tail vein is injected into 9 male nude mice of 8 weeks, under anesthesia, eyeball-picking blood-taking is adopted, 3 nude mice are killed respectively at 15min, 30min and 60min, blood, heart, lung, liver, stomach, large intestine, spleen, kidney, muscle, bone and brain tissues are collected for weighing and radioactive counting. After decay correction, the counts of each tissue sample were compared to standard counts and the results expressed as% ID/g (percentage of radioactivity per gram of sample tissue in the injected dose) were assigned to each organ pair⁶⁸The relative absorption values of Ga-NOTA-ADG are shown in FIG. 1.

Application example 3:⁶⁸dynamic in vivo imaging assay for Ga-NOTA-ADG:

obtained as in example 1⁶⁸Ga-NOTA-ADG approximately 200. mu. Ci/8 week male ICR mice injected by tail vein alone were group A,¹⁸F-FDG about 100. mu.Ci/8 week male ICR mice injected by tail vein alone were group B.

Results are shown in FIG. 2, which is an ICR mouse⁶⁸Ga-NOTA-ADG PET imaging (A) and¹⁸F-FDG PET imaging (B) found in normal ICR mouse PET/CT images⁶⁸Ga-NOTA-ADG can be concentrated in kidney and urine (A in FIG. 2); imaging agent¹⁸F-FDG is mainly concentrated in the heart and brain (B in FIG. 2).

Application example 4:⁶⁸dynamic in vivo imaging assay for Ga-NOTA-ADG:

obtained as in example 1⁶⁸Ga-NOTA-ADG was injected into 4 groups of 8-week-old male tumor model mice (in FIG. 3, A is a gastric cancer tumor model, C is a liver cancer tumor model, E is a colon cancer tumor model, G is a lung cancer tumor model) through the tail vein,¹⁸F-FDG was injected into 4 groups of 8-week male tumor model mice (in FIG. 3, B is a gastric cancer tumor model, D is a liver cancer tumor model, F is a colon cancer tumor model, and H is a lung cancer tumor model) via tail vein at about 100. mu. Ci/mouse.

Injection of drugs⁶⁸Ga-NOTA-ADG or¹⁸F-FDG(¹⁸The F-FDG group needs to be fasted for 2h) in advance, and then PET/CT imaging is carried out for 15min under a continuous anesthesia state after 30 min.

As shown in FIG. 3, AB is a gastric cancer tumor model mouse, CD is a liver cancer tumor model mouse, EF is a colon cancer tumor model mouse, and GH is a lung cancer tumor model mouse. A, C, E, G are⁶⁸Imaging with Ga-NOTA-ADG PET; b, D, F, H are¹⁸F-FDG PET imaging. In the tumor model mice of groups A, C, E and G, the imaging agent was significantly taken up in tumor tissues in addition to the kidney and urine. These results illustrate that⁶⁸Ga-NOTA-ADG is effective as a PET imaging probe for targeting tumors.

The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.