D-proline glycine polypeptide modified double-FAPI derivative and application thereof

Abstract

The invention relates to the technical field of radiopharmaceutical chemistry and clinical nuclear medicine, in particular to a D-proline glycine polypeptide modified double-FAPI derivative and application thereof. The FAPI derivative is a DOTA-modified FAPI derivative with a structure as shown in a general formula (I) and containing D-proline glycine peptides with different lengths, and a radioactive preparation obtained by labeling the DOTA-modified FAPI derivative with radionuclide is high in uptake in tumors, good in tumor / non-target ratio and good in tumor retention and is a novel tumor radiopharmaceutical worthy of popularization. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of radiopharmaceutical chemistry and clinical nuclear medicine, and in particular to a double FAPI derivative modified with a D-proline-glycine polypeptide and application thereof. Background Art

[0002] The tumor microenvironment plays an extremely important role in the occurrence and development of tumors. Tumor-associated fibroblasts are an important component of the tumor microenvironment and play an important role in tumor cell proliferation, migration, immune escape, resistance to radiotherapy and chemotherapy, and energy metabolism. Fibroblast activation protein (FAP) is an important molecular marker on the surface of tumor-associated fibroblasts. Because it is selectively expressed on the cell membrane of fibroblasts in the matrix of most solid tumors, it has become an important target for tumor detection and treatment.

[0003] The FAP small molecule inhibitor (FAPI) based on (4-quinolinyl)-glycyl-2-cyanopyrrolidine has high affinity and high selectivity. Through continuous modification and improvement of the structure, a series of 68 Ga-labeled FAPI complexes such as [ 68 Ga]Ga-FAPI-04 and [ 68 Ga]Ga-FAPI-46 and others have been reported and used for tumor imaging of primary lesions and metastatic lesions, but this type of probe has disadvantages such as rapid clearance from tumors. Although FAPI-46 has been improved on the FAPI-04 linker, its retention time in the blood and tumors has increased only slightly, and has not significantly improved the therapeutic effect. Related studies have shown that the introduction of dual targeting groups into the complex can effectively increase tumor uptake and retention; in addition, D-proline glycine peptides as linkers are also expected to increase the uptake of molecules in tumors. Macrocyclic polyamine bifunctional linkers (such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTA) can bind to 68 Ga, 64 Cu, 67 Cu, 89 Zr, 111 In, 177 Lu or 225 Based on the above background, the present invention uses FAPI-46 as a raw material, modifies its structure, and converts it into a double FAPI derivative containing a DOTA group modified with a D-proline glycine polypeptide (abbreviated as: DOTA-PG n -bisFAPI), and then use the DOTA group to coordinate with different nuclides to obtain a radioactive FAPI complex with good stability for tumor diagnosis or treatment, which has important scientific significance and broad application prospects. Summary of the invention

[0004] The present invention provides a bis-FAPI derivative modified with a D-proline-glycine polypeptide and an application thereof. The bis-FAPI derivative has good stability and is easy to prepare. After being radioactively labeled, it can be used for tumor diagnosis and treatment. It has high tumor uptake and a good target / non-target ratio, and a good tumor retention effect. It has important scientific significance and application prospects in the field of tumor diagnosis and treatment.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] A D-proline-glycine peptide-modified bis-FAPI derivative DOTA-PG n -bisFAPI, the structural formula is (I):

[0007]

[0008] In the formula, n represents an integer of 1 or more.

[0009] Preferably, in the above-mentioned D-proline-glycine polypeptide-modified bis-FAPI derivative, when n=2, the bis-FAPI derivative (DOTA-PG 2 -bisFAPI) has the structural formula (II):

[0010]

[0011] The present invention also provides a radioactive preparation, which comprises the above-mentioned bis-FAPI derivative labeled with a radionuclide.

[0012] Preferably, in the above radioactive preparation, the radioactive nuclide portion is a metal radioactive nuclide.

[0013] Preferably, in the above radioactive preparation, the metal radionuclide is 68 Ga, 64 Cu, 67 Cu, 89 Zr, 111 In, 177 Lu or 225 Ac.

[0014] Preferably, 68 Ga-labeled radioactive agents [ 68 Ga]Ga-DOTA-PG 2 -bisFAPI has a structural formula of (III). The complex has very low uptake in non-target organs, high tumor uptake values, satisfactory tumor / blood and tumor / muscle ratios, and good tumor retention effects.

[0015]

[0016] The present invention also provides the use of the above radioactive preparation in the field of tumor diagnosis and / or tumor treatment.

[0017] The beneficial effects of the present invention are as follows: the present invention provides a double FAPI derivative modified with a D-proline glycine polypeptide, and the radioactive preparation obtained by labeling the derivative with a radioactive nuclide has high uptake in tumors, and at the same time has a good tumor / non-target ratio and a good tumor retention effect, and is a new tumor radioactive drug with promotion significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Attached Figure 1 :[ 68 Ga]Ga-DOTA-PG 2 -bisFAPI and [ 68 Ga]Ga-FAPI-46 was imaged 1 h after injection in U87MG mice. DETAILED DESCRIPTION The present invention provides a double FAPI derivative modified with a D-proline glycine polypeptide and its application, wherein the radioactive nuclide in the radioactive preparation is 68 Ga, 64 Cu, 67 Cu, 89 Zr, 111 In, 177 Lu or 225 Ac.

[0019] The preparation steps are as follows:

[0020] (1) The synthetic route of the ligand is:

[0021]

[0022] In the formula, n represents an integer of 1 or more.

[0023] (2) Preparation of radiolabeled complexes

[0024] The solution containing the radionuclide is adjusted to a suitable pH, and the FAPI ligand dissolved in a buffer solution or deionized water is added, and the reaction is carried out at room temperature to 100°C for 10 to 30 minutes to generate a radionuclide-labeled complex injection solution.

[0025] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product instructions are used.

[0026] In the present invention, the instruments used without indicating the manufacturer are all conventional products that can be purchased through regular channels. The methods described are conventional methods unless otherwise specified, and the raw materials described can be obtained from public commercial channels unless otherwise specified.

[0027] Example 1

[0028] This embodiment provides a 68 Ga-labeled D-proline-glycine-containing peptide modified bis-FAPI derivative, referred to as [ 68 Ga]Ga-DOTA-PG 2 -bisFAPI, the structural formula is as follows:

[0029] The preparation steps are as follows:

[0030] 1. DOTA-PG 2 The synthesis of -bisFAPI is as follows:

[0031]

[0032] Compound 1 (200 mg) and compound 2 (170 mg) were dissolved in 10 mL DMF, and HATU (182 mg) and DIPEA (215 μL) were added, and the mixture was stirred and reacted at room temperature for 1 h. The reaction solution was dried by rotary evaporation, and 10 mL TFA was added, and the reaction was reacted at room temperature for 30 min. The reaction solution was then added to 100 mL icy ether, and a large amount of solid was precipitated. The solid was centrifuged and purified by reverse phase preparative liquid phase to obtain 219 mg of compound 3 (Yield: 68%). Compound Boc-β-Glu (30 mg) was dissolved in 5 mL DMF, and EDCI (29 mg) and HOBt (19 mg) were added, and the reaction was reacted at room temperature for 10 min. Compound 3 (219 mg) and NMM (40 μL) were added, and the reaction was reacted at room temperature for 2 h. The solvent was dried by rotary evaporation, and the mixture was dissolved in 5 mL The reaction mixture was added to TFA and reacted at room temperature for 30 min. The reaction mixture was then added to 50 mL of icy ether. A large amount of solid precipitated and centrifuged. The solid was purified by reverse phase preparative liquid chromatography to obtain 134 mg of compound 4 (Yield: 64%). Compound 4 (131 mg) was dissolved in 5 mL of DMF, TEA (42 μL) was added, and finally compound DOTA-NHS (39 mg) was added. The mixture was reacted at room temperature for 6 h. The solvent was dried by spin drying and separated and purified by reverse phase preparative liquid chromatography to obtain 80 mg of DOTA-PG 2 -bisFAPI(Yield:49%). MS(ESI)for C 99 H 133 F 4 N 27 O 21 calcd2113.31,[M+2H]2+ :found 1057.3; [M+3H] 3+ :found705.3.

[0033] 2. 68 Ga]Ga-DOTA-PG 2 -Synthesis of bisFAPI:

[0034] Take 0.5mL[ 68 Ga]GaCl 3 , 200 μL sodium acetate buffer solution (pH 4.0, 1 M), adjust the pH to about 4, add 30 μg of ligand, and react at 100°C for 10 min to obtain the [ 68 Ga]Ga-DOTA-PG 2 -bisFAPI.

[0035]

[0036] Test example

[0037] 1. Chromatographic Identification of the Radioactive Preparation Provided in Example 1

[0038] High performance liquid chromatography (HPLC) was used to identify the radiochemical purity of the marker: SHIMADZU high performance liquid chromatograph (CL-20AVP), Kromasil C18 reverse phase column (5μm, 250×4.6mm), Gabi raytest radioactivity detector. The elution gradient is shown in Table 1, the flow rate is 1mL / min, phase A is pure water containing 0.1% trifluoroacetic acid, and phase B is acetonitrile containing 0.1% trifluoroacetic acid. HPLC identification results show that [ 68 Ga]Ga-DOTA-PG 2 The retention time of -bisFAPI is 9.6 min.

[0039] Table 1 Gradient elution conditions of the complexes

[0040]

[0041] 2. Determination of lipid-water partition coefficient

[0042] Take 100 μL (10 μCi) of the labeled solution and place it in a 5 mL centrifuge tube, then add 1 mL of n-octanol and 900 μL of PBS (0.025 M, pH 7.4), vortex for 3 min (2500 rpm), let the solution stand until it is separated, centrifuge it in a centrifuge for 5 min (9000 rpm), take out 3 portions of 100 μL from each phase, and measure their radioactivity counts in a γ-counter. The lipid-water partition coefficient D = organic phase radioactivity count / aqueous phase radioactivity count, usually expressed as log D. After determination [ 68 Ga]Ga-DOTA-PG 2 The logD value of -bisFAPI was -3.29 ± 0.06, indicating that it is a water-soluble substance.

[0043] 3. Stability determination

[0044] Will[ 68 Ga]Ga-DOTA-PG 2 The radiochemical purity of -bisFAPI was determined by HPLC after it was placed in normal saline at room temperature and in mouse serum at 37°C for 2 hours. The experimental results showed that the radiochemical purity of -bisFAPI was greater than 90% after it was placed in normal saline at room temperature and in mouse serum at 37°C for 2 hours, indicating that it has good in vitro stability.

[0045] 4. PET imaging of tumor-bearing mice

[0046] Radiolabeled complex (0.1mL, 3.7MBq) was injected into mice with U87MG tumors via the tail vein. PET imaging was performed at the corresponding time points after injection under isoflurane anesthesia. The inhibition group was injected with 50μg (0.1mL, 100nmol) FAPI ligand solution 30min before injection of radiolabeled complex. At the corresponding time points, imaging was performed under isoflurane anesthesia. The imaging results are shown in the attached figure. max See Table 2 for values.

[0047] Table 2 68 Ga]Ga-DOTA-PG 2 -bisFAPI in PET imaging of tumor SUV in mice bearing U87MG tumors max Value (n=3)

[0048]

[0049] From the results, it can be seen that compared with [ 68 Ga]Ga-FAPI-46, [ 68 Ga]Ga-DOTA-PG 2 -bisFAPI SUV in tumors maxIt was higher at each time point, and the retention effect was good as time went on. After adding the FAP inhibitor, the tumor uptake was significantly reduced (0.71 vs 0.33), indicating that [ 68 Ga]Ga-DOTA-PG 2 -bisFAPI uptake in tumors has high FAP specificity.

[0050] 5. Biodistribution determination in tumor-bearing mice

[0051] Will[ 68 Ga]Ga-DOTA-PG 2 -bisFAPI labeling solution (0.1mL, 370kBq) was injected into the U87MG tumor-bearing mice through the tail vein. The mice were anesthetized and killed 1 hour after the injection (3 mice in each phase). After dissection, the heart, liver, lung, kidney, spleen, bone, muscle, small intestine, blood and tumor tissues or organs of interest were removed. The radioactivity count of each organ was measured by γ-counter, and the uptake value of each organ was obtained by conversion of the mass of each organ (in %ID / g). The biodistribution results of the marker in tumor-bearing mice are shown in Table 3.

[0052] Table 3 68 Ga]Ga-DOTA-PG 2 -BisFAPI biodistribution results in U87MG tumor-bearing mice (n=3, mean±SD, %ID / g)

[0053]

[0054] From the results, it can be seen that [ 68 Ga]Ga-DOTA-PG 2 -bisFAPI uptake in the tumor was high (32.71±0.98% ID / g) 1 hour after injection, and the tumor / muscle (11.24) and tumor / blood (6.74) ratios were high, consistent with the imaging results.

[0055] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention, and the radioactive preparations obtained by labeling the double FAPI derivative modified with D-proline glycine polypeptide with radionuclides, all fall within the scope of protection claimed by the present invention.

Claims

1. A D-proline-glycine polypeptide-modified bis-FAPI derivative, characterized in that: The structural formula of the FAPI derivative is (I): In the formula, n represents an integer of 1 or more.

2. A radioactive preparation, characterized in that: The radioactive preparation comprises the FAPI derivative according to any one of claims 1 labeled with a radionuclide.

3. The radioactive preparation according to claim 2, characterized in that The radionuclide is 68 Ga, 64 Cu, 67 Cu, 89 Zr, 111 In, 177 Lu or 225 Ac.

4. The radioactive preparation according to claim 3, characterized in that When the radionuclide is 68 When Ga, the structural formula of the radioactive agent is (II): In the formula, n represents an integer of 1 or more.

5. Use of the radioactive preparation according to any one of claims 2 to 4 in the field of tumor diagnosis and / or tumor treatment.

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