An evans blue-modified epcam-targeting aptamer and preparation method and application thereof

Abstract

This invention discloses an Evans blue-modified nucleic acid aptamer targeting EpCAM, its preparation method, and its application. The Evans blue-modified nucleic acid aptamer EB-SYL3C is composed of SYL3C, a bifunctional chelating agent, and truncated Evans blue. Using EB-SYL3C as a ligand can prolong the blood circulation half-life of SYL3C, as well as improve tumor uptake and tumor retention time, thereby enabling its application in EpCAM-targeted radionuclide diagnosis or treatment.

Description

Technical Field

[0001] This invention belongs to the field of nuclear medicine and molecular imaging, and relates to radiolabeled complexes targeting EpCAM. Specifically, it relates to long-cycled half-life molecular probes with excellent kinetic properties in radionuclide diagnosis and treatment, obtained by using SYL3C as the targeting ligand and modifying it with Evans blue. Background Technology

[0002] Epithelial cell adhesion molecule (EpCAM) is a single-pass transmembrane glycoprotein involved in the Wnt signaling pathway and closely related to cell adhesion, migration, proliferation, and differentiation. Reports indicate that EpCAM is associated with the malignant progression of tumors and is an important target for early diagnosis and treatment of tumors.

[0003] Nucleic acid aptamers are a novel class of ligands based on nucleotide structures. Their affinity and specificity are comparable to antibodies, earning them the nickname "chemical antibodies." Compared to antibodies, nucleic acid aptamers offer several significant advantages: they can be screened in vitro; they have a broad target molecule range; they have lower molecular weights and lack immunogenicity and toxicity; they can be prepared, modified, and labeled through chemical synthesis; and they can undergo reversible denaturation and renaturation. These excellent biological and chemical properties have made the development of EpCAM-targeted probes using nucleic acid aptamers a new trend. However, nucleic acid aptamers are susceptible to degradation by nucleases in vivo, leading to problems such as poor in vivo stability, short circulating half-life, and unfavorable in vivo metabolic kinetics.

[0004] Evans blue (EB) is a commonly used azo dye formulation that binds to plasma albumin to form the Evans blue-albumin complex. Therefore, it is often used to modify drugs and prolong their circulation time in vivo by loading albumin onto the body. In addition, the Evans blue-albumin complex also exhibits an enhanced permeability and retention effect (EPR effect) similar to nanoparticles, allowing it to passively accumulate at tumor sites. Chinese patent CN113366064A indicates that truncated Evans blue derivatives have been used in radioligand therapy, but the truncation of Evans blue leads to a decrease in the derivative's binding affinity to albumin and its fluorescence emission. To address this, CN113366064A proposed an Evans blue derivative that enhances the modification of Evans blue in terms of quantity. However, the types of ligands that this Evans blue derivative can connect to do not include nucleic acid aptamers. Furthermore, because it adopts a structure in which one molecule of bifunctional chelator is directly linked to two truncated molecules of Evans blue, it is difficult to label it with radionuclides.

[0005] SYL3C is a 48-nucleotide DNA aptamer with nanomolar affinity for EpCAM. Chinese patent CN112843261A describes linking SYL3C to a bifunctional chelating agent for labeling radionuclides; however, preliminary experimental results show that... 68 SYL3C labeled with radionuclides such as Ga was completely excreted by the kidneys within 10 minutes of entering the mouse body, and the tumor uptake level of SYL3C was low. It is evident that solving the problem of easy degradation of SYL3C in vivo through modification remains a technical challenge. Summary of the Invention

[0006] The purpose of this invention is to provide an Evans blue-modified nucleic acid aptamer targeting EpCAM, its preparation method, and its application, thereby solving the problems of poor in vivo stability, rapid metabolism, and low tumor uptake levels of SYL3C in the radionuclide diagnosis or treatment of EpCAM-positive tumors.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] An Evans blue-modified nucleic acid aptamer (denoted as "EB-SYL3C"), wherein the nucleic acid aptamer EB-SYL3C is any one of a compound having the structure shown in Formula I, or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof:

[0009]

[0010] R1 is a bifunctional chelating agent residue used to label radionuclides, and R2 is a residue of SYL3C with a coupling group (for example, when the coupling group is a thiol, it is SYL3C with a thiol, which can be written as "SYL3C-SH"; SYL3C-SH is obtained by modifying the nucleic acid aptamer SYL3C, and the corresponding residue is SYL3C-S-). The dashed box contains groups (generally called linkers) used to connect the bifunctional chelating agent, the SYL3C with a coupling group, and the truncated Evans blue molecule respectively through chemical bonds (such as amide bonds, thiourea bonds, maleimide-thiol chemical bonds, etc.). Linkers have multiple functional groups (which can be provided by molecules such as lysine) to connect multiple branched structures.

[0011] Preferably, the linker further includes a maleimide functional group (e.g., bonded to the ε-amino group of lysine), and SYL3C with a coupling group (e.g., SYL3C-SH) is connected to the maleimide functional group (i.e., coupled).

[0012] Preferably, the nucleic acid aptamer EB-SYL3C is a compound having the structure shown in Formula II, or any one of the following: tautomer, stereoisomer, or pharmaceutically acceptable salt of the compound.

[0013]

[0014] Preferably, the structure of the SYL3C-SH is as follows:

[0015] 5'-SH-C X -CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3'

[0016] Among them, C X (CH2) X x = 3 to 9 (e.g., x = 6).

[0017] Preferably, the structure used to label the radionuclide in the bifunctional chelating agent is selected from any one of the ligands of crown ethers, cyclodextrins, porphyrins and their derivatives.

[0018] Preferably, the bifunctional chelating agent residues are selected from any of the following structures:

[0019]

[0020] Preferably, the nucleic acid aptamer EB-SYL3C is selected from NMEB-SYL3C or DMEB-SYL3C. The two are prepared using different bifunctional chelating agents; the former is based on NOA, and the latter is based on DOTA. NOA can chelate... 68 Ga、 18 F, 64 Cu and other nuclides, DOTA can chelate 177 Lu、 90 The two nuclides, such as Y, are suitable for labeling different types of nuclides.

[0021] The preparation method of the above-mentioned nucleic acid aptamer EB-SYL3C includes the following steps:

[0022] 1) The first intermediate is obtained by condensing 4,4'-diamino-3,3'-dimethylbiphenyl, a bifunctional chelating agent, a maleimide functional molecule, and lysine with a protecting group.

[0023] 2) The first intermediate was diazotized and then coupled with the monosodium salt of 1-amino-8-naphthol-2,4-disulfonic acid to obtain the second intermediate;

[0024] 3) The SYL3C with coupling group (e.g., SYL3C-SH) is coupled with the maleimide functional group in the second intermediate (formed by the maleimide functional molecule in condensation), and then purified by size exclusion chromatography, the target product (i.e. EB-SYL3C) is collected and dried.

[0025] Preferably, step 1 specifically includes the following steps:

[0026] 1.1 A 4,4'-diamino-3,3'-dimethylbiphenyl protected by a unilateral tert-butyloxycarbonyl (Boc) group is subjected to an amide condensation reaction with N-Fmoc-N'-[1-(4,4-dimethyl-2,6-dioxocyclohexylene)ethyl]-lysine, a condensing agent (e.g., 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), and an organic base (e.g., N,N-diisopropylethylamine or triethylamine, etc.) in a molar ratio (generally referring to the molar ratio) of 1:(1~2):(1~2):(2~3) (reaction time: 2~24h; reaction temperature: 25~30℃), thereby linking the amino-protected lysine to the unilaterally amino-protected 4,4'-diamino-3,3'-dimethylbiphenyl.

[0027] 1.2 The protecting group, 9-fluorenylmethoxycarbonyl (Fmoc), contained in the reaction product of step 1.1 was removed using a piperidine-containing reagent (reaction time: 20 min to 2 h; reaction temperature: 25 to 30 °C);

[0028] 1.3 The reaction product of step 1.2 is reacted with a bifunctional chelating agent and an organic base (such as N,N-diisopropylethylamine or triethylamine) in a ratio of 1:(1.2~2):(2~4) to carry out a condensation reaction (reaction time is 1~12h, reaction temperature is 25~30℃), so that the bifunctional chelating agent is attached to the exposed amino group (i.e. the α-amino group of lysine) in the reaction product of step 1.2;

[0029] 1.4 The protecting group, N-1-(4,4-dimethyl-2,6-dioxane-hexylene)ethyl (Dde), contained in the product of step 1.3 is removed using a hydrazine-hydrate reagent (reaction time: 3–30 min; reaction temperature: 25–30 °C). Then, it undergoes a condensation reaction with 1–2 times the amount of the product of step 1.3, of 3-(maleimide)propionic acid N-hydroxysuccinimide ester, and 2–4 times the amount of the product of step 1.3, of an organic base (e.g., N,N-diisopropylethylamine or triethylamine, etc.) (reaction time: 1–4 h; reaction temperature: 25–30 °C). This results in the maleimide functional group being attached to the amino group (i.e., the ε-amino group of lysine) exposed after the removal of Dde in the product of step 1.3.

[0030] 1.5 The protecting group, tert-butyloxycarbonyl (Boc), contained in the reaction product of step 1.4 was removed using trifluoroacetic acid (reaction time: 30 min to 1 h; reaction temperature: 25 to 30 °C).

[0031] Preferably, step 2 specifically includes the following steps:

[0032] Using an inorganic acid, sodium nitrite, and the product of step 1.4 (with the tert-butyloxycarbonyl group removed) as reactants, a diazotization reaction is carried out (the inorganic acid can be hydrochloric acid, sulfuric acid, perchloric acid, fluoroboric acid, etc.; the reaction time is 15 min to 1 h; the reaction temperature is -10 to 10 °C; the ratio of the product of step 1.4 to the inorganic acid is 1:2.5 to 4; the ratio of the product of step 1.4 to sodium nitrite is 1:1 to 1.5). Then, a coupling reaction is carried out with 1 to 2 times the amount of the product of step 1.4, using 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt (reaction time is 2 to 4 h; reaction temperature is 0 to 5 °C), thereby causing 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt to be linked to the corresponding unprotected (de-Boc) amino group in the product of step 1.4 through an N=N double bond.

[0033] Preferably, the reaction conditions for the linkage reaction in step 3 include: a reaction time of 2 to 4 hours; a reaction temperature of 25 to 30°C; and a ratio of SYL3C-SH to the second intermediate of 1:1 to 2.

[0034] A radiolabeled complex targeting EpCAM, comprising a ligand and a radionuclide; wherein the ligand is the Evans blue-modified nucleic acid aptamer EB-SYL3C.

[0035] Preferably, the radionuclide is selected from... 68 Ga、 18 F, 64 Cu、 177 Lu、 111 In、 90 Any one of Y, based on the intended use of the nuclide and the ease of its acquisition, can be labeled to form corresponding radiolabeled complexes, such as... 68 Ga-EB-SYL3C, 64 Cu-EB-SYL3C 177 Lu-EB-SYL3C, 90 Y-EB-SYL3C, etc.

[0036] A method for preparing a radiolabeled complex targeting EpCAM includes the following steps:

[0037] Using the Evans blue-modified nucleic acid aptamer EB-SYL3C as a ligand, radionuclides were labeled on the ligand to obtain radiolabeled complexes targeting EpCAM.

[0038] Preferably, the radiolabeled complex can be prepared by EB-SYL3C with radionuclides using various labeling methods such as wet labeling or freeze-drying labeling.

[0039] Preferably, the wet labeling method specifically includes the following steps: reacting EB-SYL3C (e.g., the compound shown in Formula Ⅱ) with a solution containing a radionuclide under certain pH conditions (reaction temperature of 37-100℃; reaction time of 5-40 min), diluting the reaction system after the reaction is completed, and then purifying and collecting the target product (i.e., the radiolabeled complex) using a size exclusion chromatography column.

[0040] Preferably, the lyophilization labeling method specifically includes the following steps: sterilely filtering and lyophilizing the EB-SYL3C solution (e.g., the compound shown in Formula 1I), then reacting it with a solution containing a radionuclide under certain pH conditions (reaction temperature 37–100°C; reaction time 5–40 min), diluting the reaction system after the reaction, and then purifying and collecting the target product (i.e., the radiolabeled complex) using a size exclusion chromatography column.

[0041] Preferably, the mobile phase used for eluting the size exclusion column is physiological saline or water.

[0042] Preferably, the target product is sequentially eluted with a mobile phase, diluted with physiological saline or PBS (pH=6-8), and then sterile filtered to obtain a radiolabeled complex injection solution.

[0043] The above-mentioned Evans blue-modified nucleic acid aptamer EB-SYL3C and radiolabeled complexes targeting EpCAM (radiolabeled complexes with EB-SYL3C as ligand) are used in radionuclide imaging of EpCAM-targeted tumors (e.g., EpCAM-overexpressing tumors, EpCAM-positive tumors).

[0044] The above-mentioned Evans blue-modified nucleic acid aptamer EB-SYL3C and radiolabeled complexes targeting EpCAM (radiolabeled complexes with EB-SYL3C as ligand) are used in the preparation of radionuclide diagnostic reagents or therapeutic reagents targeting EpCAM (e.g., EpCAM-highly expressed tumors, EpCAM-positive tumors).

[0045] Preferably, the diagnostic or therapeutic reagent is an injection or infusion containing a radiolabeled complex targeting EpCAM, administered via intravenous injection or infusion for tumor patients with high EpCAM expression.

[0046] The beneficial effects of this invention are reflected in:

[0047] The Evans blue-modified nucleic acid aptamer EB-SYL3C of this invention is composed of SYL3C, a bifunctional chelating agent, and truncated Evans blue. The truncated Evans blue carries albumin in vivo and forms an effective protection for the EpCAM-targeting nucleic acid aptamer SYL3C, enhancing the stability of SYL3C in vivo (EB-SYL3C significantly increases the blood circulation half-life of SYL3C), making the radiolabeled SYL3C more suitable for radionuclide diagnosis and treatment of EpCAM-high expression tumors, EpCAM-positive tumors, etc.

[0048] Furthermore, in vivo bioevaluation results showed that Evans blue-modified nucleic acid aptamers (such as NMEB-SYL3C and DMEB-SYL3C with Formula II structure) not only significantly prolonged the circulating half-life, but also significantly improved tumor uptake and tumor retention time. Attached Figure Description

[0049] Figure 1A The synthetic route of compound 6 prepared for the example is shown.

[0050] Figure 1B The mass spectrometry identification chromatogram of compound 6 prepared for example.

[0051] Figure 2 Radiometric thin-layer chromatography (iTLC) chromatogram of compound 7 prepared for example.

[0052] Figure 3 Compound 7(A) and control group probe prepared for the example 68 Stability evaluation results of Ga-NOTA-SYL3C(B) in PBS and mouse serum.

[0053] Figure 4 The results of uptake and internalization of compound 7 prepared for the example in EpCAM-positive (4T1) and negative (293T) cells.

[0054] Figure 5 Compound 7(A) and control group probe prepared for the example 68 PET imaging results of Ga-NOTA-SYL3C(B) in mice bearing 4T1 tumors.

[0055] Figure 6A Synthetic route diagram of compound 11 prepared in the examples.

[0056] Figure 6B The mass spectrometry identification chromatogram of compound 11 prepared for the example.

[0057] Figure 7 Radiometric thin-layer chromatography (iTLC) chromatogram of compound 12 prepared for example.

[0058] Figure 8 Biodistribution results of compound 12 prepared for the example in mice bearing 4T1 tumors. Detailed Implementation

[0059] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments described are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0060] This invention prepares the Evans blue (EB) modified nucleic acid aptamer EB-SYL3C through chemical synthesis. Based on fully utilizing the targeting effect of SYL3C and effectively protecting it during in vivo circulation, it achieves the purpose of good radionuclide diagnosis and treatment by labeling with imaging radionuclides and therapeutic radionuclides.

[0061] (I) Preparation of unilaterally Boc-protected 4,4'-diamino-3,3'-dimethylbiphenyl (compound 1)

[0062] 4,4'-diamino-3,3'-dimethylbiphenyl (10 mmol, 1 eq.), di-tert-butyl dicarbonate (1 eq.), N,N-diisopropylethylamine (DIPEA, 1 eq.), and 20 mL of dichloromethane were added sequentially to a reaction flask. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by high-performance liquid chromatography (HPLC). The reaction was stopped when no new product was formed (i.e., the product increased no further). At this point, the reaction solution (referring to the reaction system) was separated and purified by HPLC, and the collected liquid was concentrated and dried using a lyophilizer to obtain compound 1, with a yield of 55%.

[0063] (II) Preparation of NMEB-SYL3C (synthetic route as follows) Figure 1A (As shown)

[0064] (a) Compound 1 (10 mmol, 1 eq.), N-Fmoc-N'-[1-(4,4-dimethyl-2,6-dioxocyclohexylene)ethyl]-lysine (Fmoc-Lys(Dde)-OH, 1.2 eq.), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 1 eq.), DIPEA (2 eq.), and 20 mL of dimethylformamide (DMF) were added sequentially to a reaction flask. The reaction was stirred at room temperature, and the reaction progress was monitored by HPLC until no new product was formed. The products were separated and purified by HPLC using a Phenomen Titank C18 column (250 × 10 mm, 5 u), with water containing 0.5% trifluoroacetic acid and acetonitrile as the mobile phases, and a flow rate of 5 mL / min.

[0065] (b) The collected solution obtained from the separation and purification in step a was concentrated and dried using a lyophilizer. Then, 3 mL of a DMF solution containing 20% ​​(v / v) piperidine (the product from step a was dissolved in this solution) was added. The mixture was stirred at room temperature to remove the protecting group of the α-amino group of lysine in the product from step a, namely the 9-fluorenylmethoxycarbonyl group (Fmoc). The reaction was monitored by HPLC until all Fmoc was removed. At this point, the reaction solution was separated and purified by HPLC (under the same conditions as step a). The collected solution was concentrated and dried using a lyophilizer to obtain compound 2, with a yield of 43%.

[0066] (c) Compound 2 (10 mmol, 1 eq.), 1H-1,4,7-triazine-1,4,7-triacetic acid, hexahydro, 1-(2,5-dioxa-1--1-pyrrolyl) ester (NOTA-NHS, 1.2 eq.), DIPEA (2 eq.), and 20 mmol DMF (compound 2 dissolved in DMF) were added sequentially to a reaction flask. The mixture was stirred at room temperature, and the reaction progress was monitored by HPLC until no new product was formed. At this point, the reaction solution was separated and purified by HPLC (under the same conditions as step a). The collected solution was concentrated and dried using a lyophilizer to obtain compound 3, with a yield of 47%.

[0067] (d) Compound 3 (10 mmol, 1 eq.) was added to 3 mL of DMF solution containing 2% (v / v) hydrazine hydrate (i.e., compound 3 was dissolved in this solution in a reaction flask). The reaction was stirred at room temperature to remove the protecting group of the lysine ε-amine group in compound 3, namely N-1-(4,4-dimethyl-2,6-dioxane-hexylene)ethyl (Dde). The reaction was monitored by HPLC until Dde was completely removed. After removing the solvent by vacuum filtration, the mixture was transferred to another reaction flask.

[0068] (e) To the other reaction flask from step d, add 12 mmol (1.2 eq.) of 3-(maleimide)propionic acid N-hydroxysuccinimide ester, 2 eq. of DIPEA, and 20 mL of DMF (the product from step d was dissolved in DMF). Stir the reaction at room temperature and monitor the reaction progress using HPLC until no new product is formed. At this point, separate and purify the reaction solution by HPLC (under the same conditions as step a). Concentrate and dry the collected solution using a lyophilizer to obtain compound 4, with a yield of 45%.

[0069] (f) Compound 4 (10 mmol, 1 eq.) and 3 mL of trifluoroacetic acid (TFA; compound 4 is dissolved in trifluoroacetic acid) were added sequentially to the reaction flask. The mixture was stirred at room temperature to remove Boc from compound 4. The reaction was monitored by HPLC until all Boc was removed. Trifluoroacetic acid was removed by purging nitrogen gas into the reaction flask.

[0070] (g) Add 5 mL of acetonitrile to the reaction flask of step f, and add 1.5 mL of 2 M hydrochloric acid solution dropwise under ice bath conditions. Stir the reaction for 15 min, then add an aqueous solution of sodium nitrite (10 mmol, 1 eq.), and continue stirring for half an hour. This solution is prepared as solution A. In another reaction flask, add 10 mmol, 1 eq. of monosodium 1-amino-8-naphthol-2,4-disulfonic acid, sodium carbonate (1 eq.; adjust pH), and 5 mL of water (1-amino-8-naphthol-2,4-disulfonic acid monosodium salt and sodium carbonate dissolved in water, prepared as solution B). Slowly add solution A to solution B under ice bath conditions, and continue stirring under ice bath conditions. Monitor the reaction progress with HPLC until no new product is formed. At this point, separate and purify the reaction solution by HPLC (under the same conditions as step a). Concentrate and dry the collected solution using a freeze dryer to obtain compound 5 (blue-purple), with a yield of 25%.

[0071] (h) Add SYL3C-SH (100 μmol, 1 eq.), compound 5 (1.2 eq.), triethylamine (2 eq.), and 0.5 mL of ultrapure water sequentially to a reaction flask, and stir at room temperature for 4 h; wherein, the specific structure of SYL3C-SH is as follows: 5'-SH-C6-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3'

[0072] The reaction solution was loaded onto a NAP-5 desalting column and eluted with ultrapure water as the mobile phase. The eluent was concentrated and dried by a freeze dryer to obtain compound 6 (i.e., NMEB-SYL3C).

[0073] The molecular weight of NMEB-SYL3C, as determined by mass spectrometry, is 16159.7 [M+Na]. + ( Figure 1B The theoretical value is 16135.8.

[0074] (three) 68 Preparation of Ga-NMEB-SYL3C (Compound 7)

[0075] Wet labeling protocol: Dissolve 50 μg of compound 6 (as a labeling precursor) in 50 μL of deionized water, then add 0.05 M sodium acetate solution and freshly rinsed water sequentially. 68 GaCl3 radioactive solution (labeled precursor relative to radionuclide) 68 Ga is in excess. 68GaCl3 is soluble in hydrochloric acid; the pH was adjusted to 3.5–4 by adding sodium acetate solution, and the reaction was carried out in a sealed environment at 37°C for 20 min. After diluting the reaction solution with deionized water, it was purified by separation using a PD-10 desalting column. The diluted reaction solution was loaded onto the PD-10 desalting column, and the column was washed with deionized water as the mobile phase. The eluent containing the labeled product (compound 7) was collected. The eluent was diluted with physiological saline and then aseptically filtered to obtain... 68 Ga-labeled NMEB-SYL3C injection (i.e.) 68 Ga-NMEB-SYL3C injection solution).

[0076] Lyophilization labeling scheme: Dissolve 50 μg of compound 6 in deionized water; after sterile filtration, dispense the resulting solution into containers, freeze-dry, and then seal to obtain lyophilized kits (lyophilization is for extending shelf life and facilitating commercial sale); add an appropriate amount of 0.05M sodium acetate solution to the lyophilized kits to dissolve the lyophilized powder, and then add freshly rinsed... 68 GaCl3 radioactive solution (labeled precursor relative to radionuclide) 68 Ga was in excess; the pH was adjusted to 3.5-4, and the reaction was carried out in a sealed environment at 37°C for 20 min. After diluting the reaction solution with deionized water, it was purified by separation using a PD-10 desalting column. The diluted reaction solution was loaded onto the PD-10 desalting column, washed with deionized water, and the eluent containing the labeled product (compound 7) was collected. The eluent was then diluted with physiological saline and sterilely filtered to obtain the product. 68 Ga-labeled NMEB-SYL3C injection.

[0077] (Four) 68 Performance determination of Ga-NMEB-SYL3C (compound 7)

[0078] 1. iTLC analysis and identification

[0079] Support system: Xinhua No. 1 paper; Development system: physiological saline. The performance of the injection solution prepared by the wet-labeling method was determined, and the R of compound 7 was... f The value is 0–0.1, and based on this, the chemical purity is calculated to be greater than 95%. iTLC analysis results are shown below. Figure 2 .

[0080] 2. Stability Evaluation

[0081] Stability assay in PBS: Take 20 μL (7.4 MBq) of freshly prepared PBS. 68 Ga-NMEB-SYL3C injection solution or control group probe 68The injection solution of Ga-NOTA-SYL3C (preparation process according to CN112843261A) was added to 200 μL of PBS (pH=7) and incubated at room temperature. At 1 h and 2 h, 50 μL of the incubation mixture was taken with a microsyringe and the radiochemical purity was determined by radio-HPLC.

[0082] Stability assay in mouse serum: Take 20 μL (7.4 MBq) of freshly prepared serum. 68 Ga-NMEB-SYL3C injection solution or control group probe 68 The injection solution of Ga-NOTA-SYL3C (preparation process according to CN112843261A) was added to 200 μL of fresh mouse serum solution and incubated at 37 °C. At 1 h and 2 h, 50 μL of the incubation mixture was taken with a microsyringe and an equal volume of acetonitrile was added to precipitate the protein. Then, the mixture was centrifuged at 12000 rpm for 5 min using a high-speed centrifuge. The supernatant was carefully obtained and the radiochemical purity was determined by radio-HPLC.

[0083] The purpose of PBS (or saline) stability evaluation is to assess whether the drug remains stable from preparation to injection. If it remains stable for 2 hours, it means it can be injected within 2 hours. If stability is poor, it needs to be injected immediately after preparation. Results showed ( Figure 3 ), 68 Ga-NMEB-SYL3C remained stable in PBS for 2 hours, while unmodified Ga-NMEB-SYL3C... 68 Ga-NOTA-SYL3C exhibited poor stability in PBS, with 25% of its activity decreasing by 1 hour. 68 Ga-NOTA-SYL3C was decomposed.

[0084] The purpose of mouse serum stability evaluation is to assess whether a drug is stable in the presence of nucleases (serum contains various nucleases). Results showed ( Figure 3 ), 68 Ga-NMEB-SYL3C remained stable in mouse serum for 2 hours, while unmodified Ga-NMEB-SYL3C... 68 Ga-NOTA-SYL3C exhibits poor stability in mouse serum, with 35% loss occurring by 1 hour. 68 Ga-NOTA-SYL3C is broken down. This may be because the truncated EB carries serum albumin, forming... 68 The Ga-NMEB-SYL3C-albumin complex, due to the large size of albumin, objectively forms a "protective umbrella," thereby preventing the nucleic acid aptamer SYL3C from being degraded by nucleases.

[0085] In summary, the in vitro and in vivo stability of SYL3C is significantly improved after modification with truncated EB, laying the foundation for enhancing the in vivo biological properties of SYL3C.

[0086] 3. Uptake assay in cells with different EpCAM expression levels

[0087] Pick 68 The injection solution of Ga-NMEB-SYL3C was mixed with serum-free culture medium.

[0088] One day in advance, EpCAM strongly positive cells (4T1) and negative control cells (293T) were separately divided into groups of 10... 5 Cells were seeded per well in 24-well plates. At the start of the experiment, the culture medium was removed, and the cells were washed twice with pre-cooled PBS. 500 μL of serum-free medium containing 37 kBq compound 7 was added to each well, and the plates were incubated at 37°C for 15, 30, 90, and 210 min, respectively. At each time point, the culture medium was removed, the cells were washed twice with pre-cooled PBS, and then 0.2 mL of 0.1 M NaOH was added to lyse the cells. The cell lysates from each well were collected, and the radioactivity count was performed.

[0089] Experimental results show that ( Figure 4 ), 68 Ga-NMEB-SYL3C showed significantly higher total binding and internalization in EpCAM-positive cells than in negative control cells, indicating that... 68 Ga-NMEB-SYL3C is a specific molecular probe for EpCAM targeting.

[0090] 4. Animal PET Imaging Studies

[0091] Tumor-bearing mice with 4T1 tumors were injected with 3.7 MBq via the tail vein. 68 Ga-NMEB-SYL3C or 68 Ga-NOTA-SYL3C was used, and PET images (Nano PET / CT, Mediso) were acquired at different time points. Experimental results showed that, without truncated EB modification, 68 Ga-NOTA-SYL3C is rapidly metabolized in tumor-bearing mice, with almost no uptake at the tumor site. Figure 5 B). And in the case of modified truncated EB. 68 Ga-NMEB-SYL3C exhibits significantly enhanced in vivo stability and a significantly improved level of tumor visualization. From... Figure 5As shown in Figure A, the tumor can be clearly visualized within 0.5 hours, and the uptake value gradually increases with time, reaching its highest level at 3 hours. This indicates that the albumin-carrying strategy can effectively improve the in vivo stability of SYL3C, prolong its circulation time, thereby improving the metabolic kinetics of SYL3C in vivo and thus increasing the tumor's uptake of SYL3C.

[0092] (V) Preparation of DMEB-SYL3C (synthetic route as follows) Figure 6A (As shown)

[0093] (a) and (b) Preparation of compound 2 as described in (II)

[0094] (c) Compound 2 (10 mmol, 1 eq.), hydroxysuccinimide-tetraazacyclododecanetetraacetic acid (DOTA-NHS, 1.2 eq.), DIPEA (2 eq.), and 20 mL of dimethylformamide (DMF) were added sequentially to the reaction flask. The mixture was stirred at room temperature, and the reaction progress was monitored by HPLC until no new product was formed. At this point, the reaction solution was separated and purified by HPLC (under the same conditions as step (a) in (II). The collected solution was concentrated and dried using a lyophilizer to obtain compound 8, with a yield of 45%.

[0095] (d) Compound 8 (10 mmol, 1 eq.) was added to 3 mL of DMF solution containing 2% (v / v) hydrazine hydrate (i.e., compound 8 was dissolved in this solution in a reaction flask). The reaction was stirred at room temperature to remove the protecting group of the lysine ε-amine group in compound 8, namely N-1-(4,4-dimethyl-2,6-dioxane-hexylene)ethyl (Dde). The reaction was monitored by HPLC until Dde was completely removed. After removing the solvent by vacuum filtration, the mixture was transferred to another reaction flask.

[0096] (e) To the other reaction flask from step d, add 12 mmol (1.2 eq.) of 3-(maleimide)propionic acid N-hydroxysuccinimide ester, 2 eq. of DIPEA, and 20 mL of DMF (the product from step d was dissolved in DMF). Stir the reaction at room temperature and monitor the reaction progress using HPLC until no new product is formed. At this point, separate and purify the reaction solution by HPLC (under the same conditions as step a in (ii)). Concentrate and dry the collected solution using a lyophilizer to obtain compound 9, with a yield of 42%.

[0097] (f) Compound 9 (10 mmol, 1 eq.) and 3 mL of trifluoroacetic acid (TFA; compound 9 is dissolved in trifluoroacetic acid) were added sequentially to the reaction flask. The mixture was stirred at room temperature to remove Boc from compound 9. The reaction was monitored by HPLC until all Boc was removed. Trifluoroacetic acid was removed by purging nitrogen gas into the reaction flask.

[0098] (g) Add 5 mL of acetonitrile to the reaction flask in step f, and add 1.5 mL of 2 M hydrochloric acid solution dropwise under ice bath conditions. After stirring for 15 min, add an aqueous solution of sodium nitrite (10 mmol, 1 eq.) dropwise, and continue stirring for half an hour. This solution is prepared as solution A. Take another reaction flask and add 10 mmol, 1 eq. of monosodium 1-amino-8-naphthol-2,4-disulfonic acid, sodium carbonate (1 eq.), and 5 mL of water (1-amino-8-naphthol-2,4-disulfonic acid monosodium salt and sodium carbonate are dissolved in water, prepared as solution B). Add solution A slowly dropwise to solution B under ice bath conditions, and stir the reaction under ice bath conditions. Monitor the reaction progress with HPLC until no new product is formed. At this point, separate and purify the reaction solution by HPLC (under the same conditions as step a in (II)). The collected solution is concentrated and dried by a freeze dryer to obtain compound 10 (blue-purple), with a yield of 22%.

[0099] (h) Add SYL3C-SH (100 μmol, 1 eq.), compound 10 (1.2 eq.), triethylamine (2 eq.), and 0.5 mL of ultrapure water sequentially to a reaction flask, and stir at room temperature for 4 h; wherein, the specific structure of SYL3C-SH is as follows: 5'-SH-C6-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3'

[0100] The reaction solution was loaded onto a NAP-5 desalting column and eluted with ultrapure water as the mobile phase. The eluent was concentrated and dried using a freeze dryer to obtain compound 11 (i.e., DMEB-SYL3C).

[0101] The molecular weight of DMEB-SYL3C, as determined by mass spectrometry, is 16263.1 [M+Na]. + ( Figure 6B The theoretical value is 16239.

[0102] (six) 177 Preparation of Lu-DMEB-SYL3C (Compound 12)

[0103] Wet labeling protocol: Dissolve 50 μg of compound 11 (as a labeling precursor) in 50 μL of deionized water, then add 0.4 M sodium acetate solution and freshly rinsed water sequentially. 177 LuCl3 radioactive solution (labeled precursor relative to radionuclide) 177 Lu is in excess. 177LuCl3 is soluble in hydrochloric acid; the pH was adjusted to 5.6 by adding sodium acetate solution, and the reaction was carried out in a sealed environment at 90°C for 20 min. After diluting the reaction solution with deionized water, it was purified by separation using a PD-10 desalting column. The diluted reaction solution was loaded onto the PD-10 desalting column, and the column was washed with deionized water as the mobile phase. The eluent containing the labeled product (compound 12) was collected, diluted with physiological saline, and sterilely filtered to obtain... 177 Lu-labeled DMEB-SYL3C injection (i.e.) 177 Lu-DMEB-SYL3C injection solution).

[0104] Lyophilization labeling scheme: Dissolve 50 μg of compound 11 in deionized water; after sterile filtration, dispense the resulting solution into containers, freeze-dry, and then seal to obtain lyophilized kits; add an appropriate amount of 0.4 M sodium acetate solution to the lyophilized kits to dissolve the lyophilized powder, and then add freshly rinsed... 177 LuCl3 radioactive solution (labeled precursor relative to radionuclide) 177 LuCl3 was used in excess (pH adjusted to 5.6), and the reaction was carried out at 90℃ in a sealed environment for 20 min. After diluting the reaction solution with deionized water, it was purified by separation using a PD-10 desalting column. The diluted reaction solution was loaded onto the PD-10 desalting column, which was then rinsed with deionized water. The eluent containing the labeled product (compound 12) was collected, diluted with physiological saline, and sterilely filtered to obtain the final product. 177 Lu-labeled DMEB-SYL3C injection.

[0105] (seven) 177 Performance determination of Lu-DMEB-SYL3C (compound 12)

[0106] 1. iTLC analysis and identification

[0107] Support system: Xinhua No. 1 paper; Development system: physiological saline. The performance of the injection solution prepared by the wet-labeling method was determined, and the R of compound 12 was... f The value is approximately 0–0.1, and based on this, the chemical purity is calculated to be greater than 95%. iTLC analysis results are shown below. Figure 7 .

[0108] 2. 177 Biodistribution of Lu-DMEB-SYL3C in nude mice bearing 4T1

[0109] 0.1 mL was injected into the tail vein of a mouse carrying a 4T1 tumor. 177The Lu-DMEB-SYL3C injection solution (370 KBq) was administered to mice, which were then sacrificed at 1, 4, 24, and 48 hours post-injection, with five mice per time step. The euthanized mice were dissected, and relevant tissues and organs, including the heart, liver, lung, kidney, spleen, bone, muscle, tumor, and blood, were collected, cleaned, weighed, and their radioactivity counts were determined using a gamma counter. The percentage dose per gram (%ID / g) for each tissue or organ was calculated.

[0110] The results show ( Figure 8 ), 177 Lu-DMEB-SYL3C was absorbed most abundantly in the liver of mice, followed by the spleen and kidney. 177 Lu-DMEB-SYL3C also exhibits higher uptake in the blood and heart due to the modification of truncated EB, which prolongs its circulation time in the body. Meanwhile, 177 The uptake of Lu-DMEB-SYL3C in tumors gradually increased over time, with uptake values ​​of 1.12±0.23, 1.37±0.72, 1.85±0.53 and 3.43±0.44%ID / g at 1, 4, 24 and 48 h, respectively.

[0111] In summary, this invention, through modification of truncated EB, can effectively improve the in vivo stability of the nucleic acid aptamer SYL3C, improve its in vivo metabolic kinetic properties, and thus significantly enhance the tumor uptake level, making the radioactive EpCAM targeting probe obtained by radiolabeling EB-SYL3C a promising candidate for tumor diagnosis and treatment.

Claims

1. An Evans blue-modified nucleic acid aptamer, characterized in that: The modified nucleic acid aptamer is any one of a compound having the structure shown in Formula II or a pharmaceutically acceptable salt thereof: Formula II Wherein, R1 is a bifunctional chelating agent residue, and R2 is a residue of SYL3C with a coupling group, namely SYL3C-S-, wherein the SYL3C with the coupling group is SYL3C-SH. The specific structure of the SYL3C with the coupling group is as follows: 5'-SH-C X -CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3' Among them, C X (CH2) X x = 3~9; The bifunctional chelating agent residues are selected from any of the following structures: 。 2. A method for preparing the Evans blue-modified nucleic acid aptamer as described in claim 1, characterized in that: Includes the following steps: 1) The first intermediate is obtained by condensing 4,4'-diamino-3,3'-dimethylbiphenyl, a bifunctional chelating agent, a maleimide functional molecule, and lysine with a protecting group; wherein the maleimide functional molecule is 3-(maleimide)propionic acid N-hydroxysuccinimide ester. 2) The first intermediate was diazotized and then coupled with the monosodium salt of 1-amino-8-naphthol-2,4-disulfonic acid to obtain the second intermediate; 3) Connect SYL3C with a coupling group to the maleimide functional group formed by the maleimide functional molecule in the second intermediate during condensation to obtain the Evans blue modified nucleic acid aptamer; Step 1 specifically includes the following steps: 1.1 A 4,4'-diamino-3,3'-dimethylbiphenyl with unilateral Boc protection is subjected to an amide condensation reaction with N-Fmoc-N'-[1-(4,4-dimethyl-2,6-dioxocyclohexylene)ethyl]-lysine, the condensing agent 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and the organic base N,N-diisopropylethylamine or triethylamine in a molar ratio of 1:(1~2):(1~2):(2~3). The reaction time is 2~24 h and the reaction temperature is 25~30 ℃, thereby connecting the amino-protected lysine with the unilaterally amino-protected 4,4'-diamino-3,3'-dimethylbiphenyl. 1.2 The protecting group Fmoc contained in the reaction product of step 1.1 was removed using a piperidine-containing reagent at a reaction time of 20 min to 2 h and a reaction temperature of 25 to 30 °C; 1.3 The reaction product of step 1.2 is condensed with a bifunctional chelating agent and an organic base N,N-diisopropylethylamine or triethylamine at a molar ratio of 1:(1.2~2):(2~4) for 1~12 h at a reaction temperature of 25~30 ℃, thereby attaching the bifunctional chelating agent to the exposed amino group of lysine α-amino group in the reaction product of step 1.

2. 1.4 The protecting group N-1-(4,4-dimethyl-2,6-dioxane-hexylene)ethyl in the product of step 1.3 is removed using a hydrazine-hydrate reagent at a reaction time of 3-30 min and a reaction temperature of 25-30 °C. Then, it undergoes a condensation reaction with 1-2 times the amount of 3-(maleimide)propionic acid N-hydroxysuccinimide ester and 2-4 times the amount of organic base N,N-diisopropylethylamine or triethylamine at a reaction time of 1-4 h and a reaction temperature of 25-30 °C. This results in the maleimide functional group being attached to the ε-amino group of lysine, the amino group exposed after the removal of N-1-(4,4-dimethyl-2,6-dioxane-hexylene)ethyl in the product of step 1.

3. 1.5 The protecting group Boc contained in the reaction product of step 1.4 was removed by using trifluoroacetic acid at a reaction time of 30 min to 1 h and a reaction temperature of 25 to 30 °C.

3. A radiolabeled complex targeting EpCAM, characterized in that: The radiolabeled complex includes a ligand and a radionuclide; the ligand is an Evans blue-modified nucleic acid aptamer as described in claim 1.

4. A method for preparing a radiolabeled complex targeting EpCAM as described in claim 3, characterized in that: Includes the following steps: The modified nucleic acid aptamer is labeled with a radionuclide to obtain a radiolabeled complex targeting EpCAM.

5. The use of an Evans blue-modified nucleic acid aptamer as described in claim 1 in the preparation of radionuclide diagnostic reagents or therapeutic reagents.

6. The use of a radiolabeled complex targeting EpCAM as described in claim 3 in the preparation of radionuclide diagnostic reagents or therapeutic reagents.

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