A gallium-68 / lutetium-177 / terbium-161 labeled cysteine-modified fapi complex, and a preparation method and application thereof

Abstract

The application provides a gallium-68 / lutetium-177 / terbium-161 labeled cysteine modified FAPI complex and a preparation method and application thereof, and belongs to the technical field of cysteine modified complexes. 68 Ga / 177 Lu / 161 Tb‑FAPI‑Cys, and two steps of preparation of the Tb‑FAPI‑Cys are carried out to obtain 68 Ga / 177 Lu / 161 Tb‑FAPI‑Cys complex. The complex is simple to prepare, has high radiochemical purity and good stability, has high tumor uptake, high target-to-background ratio in a fibroblast activation protein (FAP) high expression tumor model, can be specifically targeted to a tumor site, and is a new type of tumor radioactive drug with clinical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cysteine-modified complexes, in particular to a gallium-68 / lutetium-177 / terbium-161-labeled cysteine-modified FAPI complex as well as a preparation method and application thereof. BACKGROUND

[0002] Because in human tumors, CAFs expressing FAP may differ in origin, number and distribution, and expression level of FAP molecules in each cell. The current problem of FAPI molecules studied is that the blood half-life is relatively short, the clearance in vivo is fast, the tumor uptake is relatively low, and the intratumoral retention time is short. In terms of diagnostic imaging, rapid metabolism will lead to low tumor accumulation and low tumor / normal tissue contrast, resulting in short imaging window and difficult imaging quality guarantee, especially for small lesion detection. In terms of treatment, rapid metabolism and elution will lead to low effective dose at tumor site and short retention time, and high dose or more frequent administration is needed to meet the treatment needs, increasing the possibility of adverse reactions. Therefore, there is an urgent need in clinical to develop new FAP-targeted radio-diagnostic and therapeutic drugs, increase their in vivo circulation time, and improve tumor uptake and retention to achieve higher imaging contrast; at the same time, improve their binding affinity to FAP to improve their tumor targeting, improve their in vivo stability, have higher tumor uptake and faster whole-body background clearance rate, and thus obtain significantly higher tumor / normal tissue contrast to improve lesion detection rate. SUMMARY

[0003] The present application aims to provide a gallium-68 / lutetium-177 / terbium-161-labeled cysteine-modified FAPI complex as well as a preparation method and application thereof, to solve the above technical problems.

[0004] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0005] The present application provides a gallium-68 / lutetium-177 / terbium-161-labeled cysteine-modified FAPI complex, the structural general formula of which is 68 Ga / 177 Lu / 161 Tb-FAPI-Cys, the structural formula of which is shown in the following formula:

[0006]

[0007] In the above structural formula, 68 Ga、 177 Lu or 161 Tb forms a stable coordination bond with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid to obtain 68 Ga / 177 Lu / 161 Tb-FAPI-Cys complex.

[0008] The application further provides a preparation method of a gallium-68 / lutetium-177 / terbium-161 labeled cysteine-modified FAPI complex, comprising the following steps:

[0009] 1) dissolving compound 1 and compound 2 in a mixed solvent, adding a buffer solution with a pH of 7-7.5, and reacting at 20-40°C for 1-2 hours to obtain compound 3;

[0010] 2) dissolving compound 4 in an organic solvent, sequentially adding N,N'-dicyclohexyl carbodiimide and N-hydroxysuccinimide and reacting for 1-3 hours, then adding compound 3 and an organic base and continuing to react for 1-2 hours to obtain a FAPI-Cys ligand;

[0011] 3) dissolving the FAPI-Cys ligand in water, then adding a freshly eluted labeled compound hydrochloride solution and reacting, and purifying to obtain 68 Ga / 177 Lu / 161 Tb-FAPI-Cys complex;

[0012] The preparation process of the gallium-68 / lutetium-177 / terbium-161 labeled cysteine-modified FAPI complex is as follows:

[0013]

[0014] Further, the use amount ratio of the compound 1, the compound 2 and the mixed solvent is 0.5-2 mmol: 0.5-2 mmol: 10 mL; and the mixed solvent is a mixture of acetonitrile and water.

[0015] Further, in the step 2), the use amount ratio of the compound 4, N,N'-dicyclohexyl carbodiimide, N-hydroxysuccinimide and the compound 3 is 0.5-2 mmol: 1-1.5 mmol: 1-1.5 mmol: 1-1.5 mmol; the organic solvent is N,N-dimethylformamide; and the reaction temperature is independently 20-40°C.

[0016] Further, the use amount ratio of the compound 3 and the organic base is 1 mmol: 2-4 mmol.

[0017] Further, the labeled compound hydrochloride solution is 68 a gallium chloride hydrochloride solution, 177 a lutetium chloride hydrochloride solution or 161 a terbium chloride hydrochloride solution;

[0018] The concentration of the hydrochloric acid solution of the labeling compound is 0.04-0.05 mol / L, and the radioactivity of the labeling compound is 1-10 mCi.

[0019] Further, the use amount ratio of the FAPI-Cys ligand and the hydrochloric acid solution of the labeling compound is 5-10 μg: 10 μL-2 mL, and the use amount ratio of the FAPI-Cys ligand and water is 5-10 μg: 1-2 mL.

[0020] Further, in the step 3), the reaction temperature is 80-100℃, and the reaction time is 10-20 min.

[0021] The application further provides a use of a gallium-68 / lutetium-177 / terbium-161 labeled cysteine-modified FAPI complex in preparation of a targeted radio-diagnosis and treatment drug.

[0022] The application has the following beneficial effects:

[0023] The application provides a gallium-68 / lutetium-177 / terbium-161 labeled cysteine-modified FAPI complex. By introducing cysteine as a connecting group structure, the application prolongs the blood circulation half-life of the FAPI probe, enhances the combination of the FAPI probe at a tumor site, and improves the target site uptake value. Results show that, compared with existing FAP targeted probes for diagnosis or treatment, the series of labeled compounds have enhanced tumor uptake and retention time, a high target / non-target ratio, and can achieve better diagnosis and treatment effects. The prepared radioactive drug has significantly excellent in-vivo stability, stronger tumor targeting, and higher contrast between tumor and non-target tissues. Meanwhile, the application also improves the biocompatibility of the probe and optimizes the pharmacokinetic properties.

[0024] Compared with prior art such as a typical molecular probe FAPI-04, the application has the following beneficial effects:

[0025] 1. The blood half-life can be adjusted according to different needs: the tumor uptake dose and retention time of the currently reported FAP targeted radioactive probes are very limited. The application appropriately prolongs the circulation half-life of the probe, so that the probe has appropriate metabolic kinetics, a higher tumor uptake dose, and a longer tumor retention time, and meets the needs of nuclide treatment and imaging.

[0026] 2. The tumor uptake is more efficient: the molecular probe of the application can simultaneously realize the simultaneous action of receptor-ligand and covalent binding, the probe taken up by the tumor through the action of receptor-ligand is fixed inside the tumor through the covalent binding mode, is not excreted, and has an absolute higher uptake value, which is not possessed by other FAPI probes.

[0027] 3. Excellent in vivo pharmacokinetic behavior: normal organ clearance and metabolism are faster, liver and spleen do not uptake, and can be metabolized and excreted through the kidney, so that the tumor / normal organ uptake ratio of the probe is higher, which is more conducive to nuclear medical imaging of tumors. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 High-resolution mass spectrum of the FAPI-Cys ligand prepared for Example 1 of the application;

[0029] Figure 2 HPLC spectrum of the FAPI-Cys ligand prepared for Example 1 of the application;

[0030] Figure 3 Dynamic imaging images of mice within 42 hours after injection of 68 Ga-FAPI-Cys, 68 Ga-FAPI-042 hours;

[0031] Figure 4 Dynamic imaging images of mice within 42 hours after injection of 161 Tb-FAPI-Cys, 161 Tb-FAPI-0424 hours. DETAILED DESCRIPTION

[0032] The application provides a gallium-68 / lutetium-177 / terbium-161 labeled cysteine modified FAPI complex, which has a general structure as shown in the following formula: 68 Ga / 177 Lu / 161 Tb-FAPI-Cys, and has a structure as shown in the following formula:

[0033]

[0034] In the above structural formula, 68 Ga, 177 Lu or 161 Tb forms a stable coordination bond with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid to obtain 68 Ga / 177 Lu / 161 Tb-FAPI-Cys complex.

[0035] The application also provides a preparation method of a gallium-68 / lutetium-177 / terbium-161 labeled cysteine modified FAPI complex, which comprises the following steps:

[0036] 1) Dissolve compound 1 and compound 2 in a mixed solvent, add a buffer solution with a pH of 7-7.5, and react at 20-40℃ for 1-2h to obtain compound 3.

[0037] 2) Compound 4 is dissolved in an organic solvent, N,N'-dicyclohexyl carbodiimide and N-hydroxysuccinimide are added in sequence and reacted for 1-3 h, then compound 3 and an organic base are added and reacted for 1-2 h to obtain a FAPI-Cys ligand;

[0038] 3) The FAPI-Cys ligand is dissolved in water, then a freshly eluted hydrochloric acid solution of a labeling compound is added and reacted, and after purification a 68 Ga / 177 Lu / 161 Tb-FAPI-Cys complex is obtained.

[0039] The preparation process of the gallium-68 / lutetium-177 / terbium-161 labeled cysteine-modified FAPI complex is as shown below:

[0040]

[0041] In the present application, the amount ratio of compound 1, compound 2 and mixed solvent is 0.5-2 mmol: 0.5-2 mmol: 10 mL; preferably 1 mmol: 1 mmol: 10 mL; the mixed solvent is a mixture of acetonitrile and water, and the volume ratio of acetonitrile to water is preferably 1:1.

[0042] In the present application, in step 2), the amount ratio of compound 4, N,N'-dicyclohexyl carbodiimide, N-hydroxysuccinimide and compound 3 is 0.5-2 mmol: 1-1.5 mmol: 1-1.5 mmol: 1-1.5 mmol, preferably 1 mmol: 1.2 mmol: 1.2 mmol: 1 mmol; the organic solvent is N,N-dimethylformamide; the reaction temperature is independently 20-40°C, preferably 25-35°C, and further preferably 28-32°C.

[0043] In the present application, the amount ratio of compound 3 and organic base is 1 mmol: 2-4 mmol, preferably 1 mmol: 3 mmol.

[0044] In the present application, the hydrochloric acid solution of the labeling compound is 68 a gallium-68 chloride hydrochloric acid solution, 177 a lutetium-177 chloride hydrochloric acid solution, or 161 a terbium-161 chloride hydrochloric acid solution.

[0045] The concentration of the hydrochloric acid solution of the labeling compound is 0.04-0.05 mol / L, preferably 0.04 mol / L; the radioactivity of the labeling compound is 1-10 mCi, and when the labeling compound is 68Ga, the radioactivity is 5-10 mCi; when the labeled compound is 177 Lu, the radioactivity is 5-10 mCi; when the labeled compound is 161 Tb, the radioactivity is 1 mCi.

[0046] In the present application, the use ratio of the hydrochloric acid solution of the FAPI-Cys ligand and the labeled compound is 5-10 μg: 10 μL-2 mL, preferably 6-8 μg: 20 μL-1 mL;

[0047] The use ratio of the FAPI-Cys ligand and water is 5-10 μg: 1-2 mL, preferably 6-8 μg: 2 mL.

[0048] In the present application, in the step 3), the reaction temperature is 80-100℃, preferably 95℃; the reaction time is 10-20 min, preferably 15 min.

[0049] The present application also provides a use of a gallium-68 / lutetium-177 / terbium-161 labeled cysteine-modified FAPI complex in the preparation of a targeted radio-diagnosis and treatment drug.

[0050] The technical solutions provided by the present application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0051] Example 1

[0052] Synthesis of the cysteine-modified FAPI derivative:

[0053] (1) 1 mmol of compound 1 and 1 mmol of compound 2 were dissolved in 5 mL of acetonitrile and 5 mL of H2O, 2.5 mL of buffer solution with pH=7.2 was added, and the reaction was carried out at 25℃ for 1 h, LC-MS was used to monitor the completion of the reaction, centrifugal extraction was performed, and compound 3 was obtained after reversed-phase preparative liquid chromatography purification, with a yield of 71%.

[0054] (2) 1 mmol of compound 4 was dissolved in 10 mL of DMF, 1.2 mmol of N,N'-dicyclohexyl carbodiimide and 1.2 mmol of N-hydroxy succinimide were added, and the reaction was carried out at 20℃ for 2 h, then 1 mmol of compound 3 and 3 mmol of N,N-diisopropyl ethylamine were added, and the reaction was carried out at 25℃ for 1 h, LC-MS was used to monitor the completion of the reaction, the DMF was removed, and the FAPI-Cys ligand was obtained after reversed-phase preparative liquid chromatography purification.

[0055] The synthesis route is as follows:

[0056]

[0057] 68Preparation of Ga-FAPI-Cys complex:

[0058] (1) 68 Preparation of Ga-FAPI-Cys complex: 10 μg of FAPI-Cys ligand was weighed into 2 mL of ultrapure water, and fresh eluted 68 GaCl3 hydrochloride solution (0.05 M HCl, 10 mCi, 2 mL) was added thereto, and the mixture was reacted at 95°C for 15 minutes and then cooled to room temperature. An HLB separation cartridge was taken, and the Ga-FAPI-Cys complex was obtained after purification. 68

[0059] Example 2

[0060] 177 Preparation of Lu-FAPI-Cys complex: 10 μg of FAPI-Cys ligand was weighed into 2 mL of ultrapure water, and fresh eluted 177 LuCl3 hydrochloride solution (0.05 M HCl, 10 mCi, 15 μL) was added thereto, and the mixture was reacted at 95°C for 15 minutes and then cooled to room temperature. An HLB separation cartridge was taken, and the Lu-FAPI-Cys complex was obtained after purification. 177

[0061] The FAPI-Cys ligand was prepared according to Example 1.

[0062] Example 3

[0063] 161 Preparation of Tb-FAPI-Cys complex: 10 μg of FAPI-Cys ligand was weighed into 1.5 mL of ultrapure water, and fresh eluted 161 TbCl3 hydrochloride solution (0.04 M HCl, 1 mCi, 15 μL) was added thereto, and the mixture was reacted at 95°C for 15 minutes and then cooled to room temperature. An HLB separation cartridge was taken, and the Tb-FAPI-Cys complex was obtained after purification. 161

[0064] The FAPI-Cys ligand was prepared according to Example 1.

[0065] Application Example 1

[0066] Construction of HT1080-FAP tumor model mice: HT1080-FAP cells (purchased from Hunan Fenghui Biotechnology Co., Ltd.) were subcutaneously inoculated into the right axillary part of Balb / c nude mice (purchased from Shanghai Slek Experimental Animal Co., Ltd.) at an inoculation amount of 2 million cells per mouse, to obtain HT1080-FAP tumor model mice.

[0067] ​​​The labeled compound obtained by the method of this invention was injected into mice via tail vein. 0.2 mL (approximately 11 MBq) of the compound was then injected into the mouse tail vein. 68 Ga-FAPI-Cys complex (sample group), 0.2 mL 68 Ga-FAPI-04 (control group, prepared according to the preparation method of the complex in Example 1, using FAPI 04 purchased from Ganzhou Tanzhen Biomedical Co., Ltd. as the ligand) 68 Ga-FAPI-04 was injected into the constructed HT1080-FAP tumor model mice. MicroPET dynamic imaging was performed 2 hours after injection, and regions of interest (ROIs) were delineated on the obtained whole-body images. The probe distribution values ​​were calculated, and the results are as follows: Figure 3 As shown.

[0068] Depend on Figure 3 As can be seen, the area indicated by the circle is the location of the tumor. 68 Ga-FAPI-04 shows minimal uptake and rapid clearance at tumor sites. This invention... 68 The Ga-labeled probe showed significant uptake and retention at tumor sites at all imaging time points (30 min–120 min), and was rapidly cleared from normal tissues such as muscle, liver, and kidneys. A high radioactive signal was observed in the bladder, indicating that the probe was metabolized and excreted in urine. Therefore, the labeling compound of this invention… 68 Ga-FAPI-Cys exhibits excellent tumor uptake. This advantage gives the markers of the present invention significant advantages in practical applications. On the one hand, it facilitates longer imaging periods, and with the guarantee of high imaging contrast, the diagnosis of primary micro-lesions will be more accurate, which is beneficial for target delineation. On the other hand, the high uptake and long retention of the radionuclide-labeled probe at the tumor site will lay the foundation for radionuclide-targeted therapy.

[0069] In summary, the FAP-targeted radiolabeled complex provided by this invention... 68 Ga-FAPI-Cys can significantly prolong its circulating half-life and enhance tumor uptake and retention time. This novel property is not found in other FAPI imaging agents and is expected to be used for radionuclide imaging of tumors with high FAPI expression.

[0070] Application Example 2

[0071] Construction of HT1080-FAP tumor model mice: HT1080-FAP cells (purchased from Hunan Fenghui Biotechnology Co., Ltd.) were subcutaneously injected into the right axilla of Balb / c nude mice (purchased from Shanghai Slack Laboratory Animal Co., Ltd.) at an inoculation rate of 2 million cells / mouse to obtain HT1080-FAP tumor model mice.

[0072] 0.4 mL (about 22 MBq) of the labeled compound obtained by the method of the present application was injected into the tail vein of a mouse 161 Tb-FAPI-Cys complex (sample group), 0.4 mL 161 Tb-FAPI-04 (control group, FAPI 04 purchased from Ganzhou Taizhen Biomedical Co., Ltd. was prepared into a complex as a ligand according to the preparation method of the complex in Example 1 161 Tb-FAPI-04) were injected into the constructed HT1080-FAP tumor model mice. MicroSPECT dynamic imaging was performed within 24 hours after injection, and the region of interest (ROI) was outlined on the whole body image obtained by scanning, and the probe distribution value was calculated, and the results are shown in Figure 4 .

[0073] As can be seen from Figure 4 , the area indicated by the circle is the tumor position, 161 Tb-FAPI-04 is rapidly cleared at the tumor site. The 161 Tb-labeled probe has significant uptake and retention at the tumor site at each imaging time point (1h-24h), and is rapidly cleared in normal tissues such as muscle, liver, kidney, etc. There is a higher radioactive signal at the bladder position, which means that the probe is metabolized in the body and excreted out of the body through urine. Therefore, the labeled compound of the present application 161 Tb-FAPI-Cys has good tumor uptake and helps the tumor to stay longer, laying a foundation for efficient nuclide targeted therapy.

[0074] In summary, the FAP-targeted radiolabeled complex 161 Tb-FAPI-Cys can significantly prolong its circulation half-life, enhance tumor uptake and retention time, and is expected to be used for nuclide therapy of FAP high-expression tumors.

[0075] As can be seen from the above examples, the present application provides a gallium-68 / lutetium-177 / terbium-161 labeled cysteine modified FAPI complex, a preparation method and application thereof. By introducing cysteine as a linking group structure, the present application provides a new FAPI based on quinolinic acid derivative and a radiolabeled complex thereof, prolongs the blood circulation half-life of the FAPI probe and improves the target site uptake value, and the prepared radiopharmaceutical has excellent in vivo stability, stronger tumor targeting and longer tumor retention time.

[0076] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A gallium-68 / lutetium-177 / terbium-161-labeled cysteine-modified FAPI complex, characterized in that, The general structural formula is 68 Ga / 177 Lu / 161 The structure of Tb-FAPI-Cys is shown in the following formula: In the above structural formula, 68 Ga、 177 Lu or 161 Tb forms a stable coordination bond with the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid structure, yielding 68 Ga / 177 Lu / 161 Tb-FAPI-Cys complex.

2. The method for preparing the gallium-68 / lutetium-177 / terbium-161-labeled cysteine-modified FAPI complex according to claim 1, characterized in that, Includes the following steps: 1) Compound 1 and Compound 2 were dissolved in a mixed solvent, a buffer solution with a pH of 7 to 7.5 was added, and the mixture was reacted at 20 to 40 °C for 1 to 2 h to obtain Compound 3; 2) Compound 4 was dissolved in an organic solvent, and N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide were added sequentially and reacted for 1-3 h. Then, compound 3 and an organic base were added and the reaction was continued for 1-2 h to obtain the FAPI-Cys ligand. 3) The FAPI-Cys ligand was dissolved in water, and then a freshly rinsed hydrochloric acid solution of the labeled compound was added to react with the solution. After purification, the following was obtained: 68 Ga / 177 Lu / 161 Tb-FAPI-Cys complex; The preparation process of the gallium-68 / lutetium-177 / terbium-161 labeled cysteine-modified FAPI complex is as follows:

3. The method for preparing the gallium-68 / lutetium-177 / terbium-161 labeled cysteine-modified FAPI complex according to claim 2, characterized in that, The ratio of compound 1, compound 2 and the mixed solvent is 0.5–2 mmol: 0.5–2 mmol: 10 mL; the mixed solvent is a mixture of acetonitrile and water.

4. The method for preparing the gallium-68 / lutetium-177 / terbium-161-labeled cysteine-modified FAPI complex according to claim 2 or 3, characterized in that, In step 2), the ratio of compound 4, N,N'-dicyclohexylcarbodiimide, N-hydroxysuccinimide and compound 3 is 0.5–2 mmol: 1–1.5 mmol: 1–1.5 mmol: 1–1.5 mmol; the organic solvent is N,N-dimethylformamide; and the reaction temperature is independently 20–40 °C.

5. The method for preparing the gallium-68 / lutetium-177 / terbium-161-labeled cysteine-modified FAPI complex according to claim 4, characterized in that, The ratio of compound 3 to organic base is 1 mmol: 2-4 mmol.

6. The method for preparing the gallium-68 / lutetium-177 / terbium-161-labeled cysteine-modified FAPI complex according to claim 2, 3, or 5, characterized in that, The hydrochloric acid solution of the labeled compound is 68 GaCl3 hydrochloric acid solution, 177 LuCl3 hydrochloric acid solution or 161 TbCl3 hydrochloric acid solution; The concentration of the hydrochloric acid solution containing the labeled compound is 0.04–0.05 mol / L, and the radioactivity of the labeled compound is 1–10 mCi.

7. The method for preparing the gallium-68 / lutetium-177 / terbium-161 labeled cysteine-modified FAPI complex according to claim 6, characterized in that, The ratio of the FAPI-Cys ligand to the hydrochloric acid solution of the labeled compound is 5–10 μg: 10 μL–2 mL, and the ratio of the FAPI-Cys ligand to water is 5–10 μg: 1–2 mL.

8. The method for preparing the gallium-68 / lutetium-177 / terbium-161 labeled cysteine-modified FAPI complex according to claim 7, characterized in that, In step 3), the reaction temperature is 80–100°C and the reaction time is 10–20 min.

9. The use of the gallium-68 / lutetium-177 / terbium-161 labeled cysteine-modified FAPI complex according to claim 1 in the preparation of targeted radiotherapeutic drugs.

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