< 68 > Ga-labeled double-target PET heterodimer polypeptide probe targeting FAP and VEGFR-2 and preparation method of < 68 > Ga-labeled double-target PET heterodimer polypeptide probe

Abstract

The invention discloses a < 68 > Ga labeled double-target PET (polyethylene terephthalate) heterodimer polypeptide probe targeting FAP (fibroblast activation protein) and VEGFR-2 (vascular endothelial growth factor receptor) and a preparation method of the < 68 > Ga labeled double-target PET heterodimer polypeptide probe. The double-target probe can be combined with tumor FAP and VEGFR-2 at the same time, the tumor tissue intake and residence time are increased, pharmacokinetic characteristics are optimized, tumor heterogeneity coverage is enhanced, diagnosis accuracy is improved, and the double-target probe has good clinical transformation potential and can be effectively applied to tumor PET imaging.

Description

Technical Field

[0001] This invention relates to the field of biomedicine; specifically, it relates to a... 68 Ga-labeled dual-target PET heterodimeric peptide probes targeting FAP and VEGFR-2 and their preparation method. Background Technology

[0002] The tumor microenvironment (TME) is the key "soil" for tumor development, progression, and metastasis, containing a variety of non-tumor cells and extracellular matrix. Cancer-associated fibroblasts (CAFs) are the most abundant stromal cell component of the TME, present in large quantities in over 90% of epithelial-derived tumors (such as breast cancer, colorectal cancer, and pancreatic cancer). Fibroblast activating protein (FAP) is a type II transmembrane serine protease specifically highly expressed on the surface of activated CAFs, but expressed at very low levels or absent in normal fibroblasts and most normal tissues, making it a highly attractive broad-spectrum target for tumor diagnosis and treatment. In recent years, PET probes based on FAP inhibitors (FAPI), such as... 68 The Ga-FAPI series has demonstrated excellent tumor targeting capabilities and imaging effects in preclinical and clinical studies.

[0003] On the other hand, angiogenesis is essential for tumor growth and metastasis. Vascular endothelial growth factor receptor 2 (VEGFR-2) is highly expressed on endothelial cells that generate new blood vessels in tumors and is a core receptor regulating angiogenesis, as well as an important target for anti-angiogenic therapy and imaging monitoring. The peptide A7R (ATWLPPR) has been shown to bind to VEGFR-2 with high affinity and specificity, and is therefore often used to construct molecular probes targeting tumor blood vessels.

[0004] While single-target PET probes (such as those targeting FAP or VEGFR-2) have achieved some success in tumor imaging, the high heterogeneity of tumors and the significant differences in target expression levels across different regions or patients mean that probes targeting only a single target may fail to capture images or lack sufficient sensitivity due to the absence or low expression of that target, making it difficult to comprehensively and accurately reflect the complex biological characteristics of tumors. Therefore, developing dual-target or multi-target PET probes capable of simultaneously identifying multiple key targets is of great significance for improving the sensitivity, specificity, and coverage of tumor heterogeneity in tumor detection. Summary of the Invention

[0005] This invention is achieved through the following technical solution: This invention provides 68 Ga-labeled dual-target PET heterodimeric peptide probes targeting FAP and VEGFR-2; as shown in Formula (I):

[0006] Formula (1).

[0007] The present invention also provides 68 The preparation method of Ga-labeled dual-target PET heterodimeric peptide probes targeting FAP and VEGFR-2 includes the following steps: (1) The target heterodimeric polypeptide DOTA-FAPI-A7R was synthesized using solid-phase synthesis. (2) 68 Ga-labeled heterodimeric peptide DOTA-FAPI-A7R yielded a dual-target PET heterodimeric peptide probe.

[0008] Preferably, the structure of the heterodimeric polypeptide DOTA-FAPI-A7R described in step (1) is as shown in formula (II):

[0009] Formula (II).

[0010] Preferably, step (1) specifically comprises: a) By sequentially coupling amino acid sequences through solid-phase synthesis, linear crude product 1: Hex-CPPTEFC and crude product 2: DOTA-CYS-PEG2-ATWLPPR were obtained; b) The crude product 1 was cyclized with 1,3,5-tris(bromomethyl)benzene under alkaline conditions to form a cyclic peptide structure; c) The cyclized product is coupled with crude product 2 via a thiol group to form a heterodimer structure; d) After purification, the precursor polypeptide DOTA-FAPI-A7R with a purity greater than 95% was obtained.

[0011] Preferably, step (2) specifically involves: mixing the precursor polypeptide obtained in step (1) with... 68 The GaCl3 solution was mixed in a buffer system, the pH was adjusted to 3.5-4.0, and the mixture was heated at 100℃ for 10-20 minutes to obtain a reaction mixture. The reaction mixture was purified by passing it through an activated C18 solid-phase extraction column, and the eluent was collected to obtain the desired product. 68 Ga-labeled dual-target PET heterodimer peptide probe 68 Ga-DOTA-FAPI-A7R.

[0012] The present invention has, but is not limited to, the following beneficial effects: Dual-target synergistic effect: This invention creatively integrates a FAP ligand targeting CAFs and a VEGFR-2 ligand targeting angiogenesis into a single molecule. This probe can simultaneously bind to two key cellular components in the tumor microenvironment (CAFs and vascular endothelial cells), achieving a "two-pronged approach." This not only increases the absolute uptake of the probe in tumor tissue but also prolongs its retention time within the tumor, resulting in a higher and more durable tumor / background contrast signal.

[0013] Overcoming tumor heterogeneity: Due to the potential spatial and temporal heterogeneity in the expression of FAP and VEGFR-2 within tumors, the dual-target design significantly increases the chances of probe binding to tumor tissue. Even in regions where one target is absent, the other target can still mediate probe uptake, thereby achieving more comprehensive and reliable detection of tumor lesions and reducing false negative results.

[0014] Excellent pharmacokinetics: The heterodimeric probe synthesized in this invention has a moderate molecular weight, exhibiting rapid blood clearance and efficient renal excretion in vivo. This property facilitates the acquisition of high-contrast tumor images within a short time after injection (e.g., 1-2 hours), while reducing radiation dose to non-target organs and background interference.

[0015] Simple preparation and easy conversion: This invention utilizes mature solid-phase peptide synthesis and efficient... 68 Ga-marking method. 68 Ga can be derived from readily available sources. 68 Ge / 68 The Ga generator has a moderate half-life (68 minutes), making it suitable for on-site labeling and use in clinical PET centers. The entire preparation process is rapid and stable, with high radiochemical yield and good purity, showing promising prospects for clinical translation. Attached Figure Description

[0016] To clearly illustrate the specific embodiments of the present invention and certain detection techniques used in the experiments, the implementation schemes and the techniques used will be described below, mainly through the accompanying drawings.

[0017] Figure 1 Schematic diagram of the chemical structure of the precursor polypeptide DOTA-FAPI-A7R.

[0018] Figure 2 : 68 Ga-labeled dual-target PET probe 68 Schematic diagram of Ga-DOTA-FAPI-A7R.

[0019] Figure 3 Example 2 68The radio-high performance liquid chromatography (Radio-HPLC) chromatogram of Ga-DOTA-FAPI-A7R showed a radiochemical purity of 100%, greater than 95%.

[0020] Figure 4 Example 3: Injection into tumor-bearing nude mice 68 Micro-PET / CT maximum intensity projection (MIP) images at different time points after Ga-DOTA-FAPI-A7R show the specific concentration of the probe at the tumor site and its changes over time.

[0021] The specific embodiments of the present invention are explained with the aid of examples. Except for the detection technology, which does not limit the present invention in any way, some of the solutions in the described embodiments are part of the embodiments of the invention. All embodiments obtained by ordinary people in the art without creative results are within the protection scope of the present invention. Example

[0022] The peptide DOTA-FAPI-A7R was synthesized using a solid-phase synthesis method. The specific steps are as follows: 1. Amino acid and resin condensation: Synthesis started from the C-terminus of the peptide. 0.195 mmol (1.3 eq) of a C-terminal amino acid was coupled to the resin using 0.5 mmol DIC + 0.5 mmol HOBT and 3 ml DMF as solvents. The reaction was carried out at room temperature for 1.5 h. Then, 0.5 mmol acetic anhydride and 10 ml DCM solution were added, followed by the addition of DIEA. The reaction was carried out for 20 min to block the unreacted amino sites on the resin.

[0023] 2. FMOC removal: FMOC was removed using a DMF solution containing 20% ​​piperidine, and the reaction was carried out at room temperature for 20 min. After removal, the resin was washed with DMF 4 times, 1 min each time, to obtain NH2-AA1- resin.

[0024] 3. Amino acid condensation: The remaining amino acids and resin peptides are dehydrated and condensed using a condensation reagent of 0.5 mmol DIC + 0.5 mmol HOBT and 3 ml DMF as solvent, and the reaction is carried out at room temperature for 1 hour. Steps 3-4 are repeated to condense the remaining amino acids sequentially (from C-terminus to N-terminus) to complete the peptide synthesis. 4. Peptide lysis: The lysis buffer composition is 95% TFA, 1% H2O, 2% EDT, and 2% TIS. The lysis time is 2 hours. After lysis, the lysate is filtered into ice-cold ether and centrifuged to obtain crude products. The crude products are then lyophilized to obtain crude product 1: Hex-CPPTEFC and crude product 2: DOTA-CYS-PEG2-ATWLPPR.

[0025] 5. Peptide cyclization: The lyophilized crude peptide 1: Hex-CPPTEFC was dissolved in 10% acetonitrile and water to a concentration of 2 mg / ml. An equimolar amount of 1,3,5-tris(bromomethyl)benzene was added, and the pH was adjusted to about 8 with ammonium bicarbonate. The mixture was stirred for 3 hours to form a cyclic peptide. Then, an equimolar amount of crude peptide 2: DOTA-CYS-PEG2-ATWLPPR was added, and the coupling was continued for 2 hours. The formation of the peptide was monitored by mass spectrometry.

[0026] 6. The final peptide product DOTA-FAPI-A7R with a purity greater than 95% was obtained through purification for subsequent applications. 68 Ga-labeled, specific molecular formula: Hex-CPPTEFC-(Cys,Cys-CYS,bridgeTBMB)-DOTA-CYS-PEG2-ATWLPPR; chemical structural formula as follows: Figure 1 As shown. In this invention, the FAP-targeting component, with FAP-2286 as its core (i.e., FAPI), is responsible for binding to tumor stromal cells. The VEGFR-2-targeting component, with A7R as its core, is responsible for binding to tumor vascular endothelial cells. Example

[0027] 68 Ga labeling to obtain PET heterodimeric peptide probes, the specific procedure is as follows: 1. The reaction system consists of 0.05M HCl and 0.25M sodium acetate solution, with a pH between 3.5 and 4.0.

[0028] 2. Activate the C18 column: Rinse the Sep-Pak light C18 column with 5ml of anhydrous ethanol, then rinse with 10ml of water, and then dry with 10ml of air.

[0029] 3. Preparation of precursor: Dissolve 0.5 mg of precursor DOTA-FAPI-A7R in 200 μL DMSO + 300 μL aqueous solution to obtain a 1 mg / mL precursor solution.

[0030] 4. Labeling: Add 100 μL of precursor solution to 4 ml of [precursor solution]. 68 After confirming the pH value to be 3.5-4.0 in GaCl3 solution, heat at 100℃ for 15 min. After the reaction is complete, pass the solution through an activated C18 column, dry it with air (2-3 times), elute the radiolabeled product with 1.0 mL of anhydrous ethanol, and transfer the product to a product vial to obtain the final product. 68 Ga-DOTA-FAPI-A7R. Chemical structure as follows: Figure 2 As shown.

[0031] 5. Analyze using high performance liquid chromatography (HPLC). 68Radiochemical purity of Ga-FAPI-A7R. System conditions were set as follows: flow rate 1 ml / min; solvent gradient: solvent A was an aqueous solution containing 0.1% trifluoroacetic acid (TFA), and solvent B was an acetonitrile solution containing 0.1% TFA; flow time: 25 min, with solvent B increasing from 1% to 50%. Results are as follows. Figure 3 As shown, HPLC analysis results indicate that the radiochemical purity is 100%. The results are as follows... Figure 3 As shown. Example

[0032] To verify 68 The tumor tracing effect of Ga-FAPI-A7R in vivo was investigated by constructing U87Mg and A549 tumor-bearing nude mouse models with high and low expression of FAP+VEGFR-2, until the tumor volume reached 100-250 mm. 3 Micro PET / CT imaging was performed on the mice (3 mice per group). Nude mice bearing tumors in good growth condition were selected, placed in a restraint device, and their tail veins were wiped with alcohol to dilate them. 0.1 ml of a radioactive dose of approximately 5-10 MBq was injected via the tail vein. 68 Ga-FAPI-A7R solution was used. Scans were performed at 0.5, 1, 2, and 4 hours post-injection. Gas anesthesia was administered 10 minutes before imaging; successful anesthesia was indicated by limb paralysis. The weight of the tumor-bearing mice was weighed and recorded. The mice were then positioned prone, with their limbs and tails flattened and secured to the examination table. Heating pads were placed under the mice to prevent frostbite or hypothermia-induced death. Static scans were performed for 10 minutes at the corresponding time points using Micro PET / CT and a suitable preset scanning program. The radioactivity of the syringes before and after injection was entered before the scan. After the scan, PMOD 4.0 software was used to quantitatively analyze the tissue radioactive uptake within the regions of interest (ROI), expressed as a percentage of the injected dose per gram of tissue (%ID / g). Results are as follows: Figure 4 As shown.

[0033] The study evaluated the results using static PET scans at multiple time points. 68 Tumor targeting efficacy of the Ga-FAPI-A7R probe in U87MG and A549 tumor-bearing nude mice (n=3). Representative decay-corrected coronal images at different time points after injection are shown below. Figure 4 As shown in Figure A.

[0034] 68The uptake of Ga-FAPI-A7R in U87MG tumors was clearly visible from 0.5 h after injection and reached its peak after 1 h (the uptake values ​​at 0.5 h, 1 h, 2 h, and 4 h were 4.7 ± 0.4, 4.9 ± 0.4, 3.4 ± 0.2, and 2.4 ± 0.4 %ID / g, respectively). Figure 4 B). Although tumor uptake decreased over time, the tumor-to-muscle uptake ratio (T / N) remained high due to the clearance of the tracer from normal tissue (7.7 ± 0.6, 8.0 ± 0.3, 7.9 ± 0.8, and 7.1 ± 0.5 %ID / g at 0.5h, 1h, 2h, and 4h, respectively). On the other hand, 68 Ga-FAPI-A7R was poorly taken up in A549 tumors and was rapidly cleared from the tumors (highest takeup at 0.5 h, 2.6 ± 0.3 %ID / g); Figure 4 B), resulting in a low T / N ratio (the ratio was highest at 1h, at 4.8 ± 0.2 %ID / g).

[0035] The uptake by the kidneys and liver is as follows: Figure 4 As shown in D and E, renal uptake in both tumor-bearing mouse species was highly significant (20.3 ± 1.8 and 19.0 ± 1.5 %ID / g in U87MG and A549 tumor-bearing nude mice, respectively, at 0.5 h), and decreased significantly over time (5.0 ± 0.5 and 5.6 ± 0.2 %ID / g, respectively, at 4 h). This indicates that... 68 Ga-FAPI-A7R is primarily excreted via the urinary system. Liver uptake was also observed at early time points (5.1 ± 0.2 and 5.3 ± 0.5 %ID / g in U87MG and A549 tumor-bearing nude mice at 0.5 h), gradually decreasing over time (1.2 ± 0.1 and 1.4 ± 0.1 %ID / g in U87MG and A549 tumor-bearing nude mice at 4 h, respectively), and gradually decreasing over time (1.2 ± 0.1 and 1.4 ± 0.1 %ID / g in U87MG and A549 tumor-bearing nude mice at 4 h, respectively).

[0036] In summary, compared with A549 tumors, 68 Ga-FAPI-A7R exhibits better tumor imaging performance in U87MG tumors that highly express FAP and VEGFR-2. Example

[0037] To verify the Micro PET / CT imaging results, after 4 hours of imaging, [the following was observed]... 68Biodistribution experiments were conducted on U87MG and A549 tumor-bearing nude mice using Ga-FAPI-A7R. Before the experiment, a sufficient number of gamma counters were prepared, labeled, and weighed, with the weight recorded. After 4 hours of imaging, the nude mice were euthanized. The heart was quickly dissected, and a suitable amount of blood was immediately drawn from the heart using a 1 ml syringe. The remaining necessary tissues and organs (including brain, heart, lungs, liver, pancreas, spleen, stomach, small intestine, kidneys, bladder, testes, muscles, bones, and tumors) were dissected. Note that residual blood in the heart and lungs should be rinsed with physiological saline, and the contents of the stomach and small intestine should be squeezed out. The removed tissues and organs were then placed sequentially into the corresponding labeled gamma counters.

[0038] Before gamma counting, the gamma counting tubes containing tissues and organs were weighed on a balance, the weight was recorded, and the net weight of the counting tubes was subtracted to obtain the weight of each tissue and organ. Additionally, 0.01 ml was used for tail vein injection in nude mice. 68 The Ga-FAPI-A7R solution was placed in a new gamma counter tube for radioactivity attenuation correction. The gamma counter tube was placed in a gamma counter for radioactivity counting, and the counting and measurement times were recorded. %ID / g was calculated after attenuation correction. The results are shown in Table 1. 68 The uptake value of Ga-FAPI-A7R in U87MG tumors was 3.11 ± 0.52 %ID / g, significantly higher than that in the A549 group (1.13 ± 0.53 %ID / g). P <0.05). High levels of radiotracer retention were observed in the kidneys and bladder. Other major organs showed low uptake and rapid clearance. Overall, the results are consistent with those of the Micro PET / CT studies, further confirming the high specificity of the tracer. .

Claims

1. A kind 68 A Ga-labeled dual-target PET heterodimeric peptide probe targeting FAP and VEGFR-2; characterized in that... As shown in equation (1): Formula (1).

2. A kind 68 The method for preparing Ga-labeled dual-target PET heterodimeric peptide probes targeting FAP and VEGFR-2 is characterized by... Includes the following steps: (1) The target heterodimeric polypeptide DOTA-FAPI-A7R was synthesized using solid-phase synthesis. (2) 68 Ga-labeled heterodimeric peptide DOTA-FAPI-A7R yielded a dual-target PET heterodimeric peptide probe.

3. The preparation method according to claim 2, characterized in that: The structure of the heterodimeric polypeptide DOTA-FAPI-A7R described in step (1) is shown in formula (II): Formula (II).

4. The preparation method according to claim 2, characterized in that: Step (1) is as follows: a) By sequentially coupling amino acid sequences through solid-phase synthesis, linear crude product 1: Hex-CPPTEFC and crude product 2: DOTA-CYS-PEG2-ATWLPPR were obtained; b) The crude product 1 was cyclized with 1,3,5-tris(bromomethyl)benzene under alkaline conditions to form a cyclic peptide structure; c) The cyclized product is coupled with crude product 2 via a thiol group to form a heterodimer structure; d) After purification, the precursor polypeptide DOTA-FAPI-A7R with a purity greater than 95% was obtained.

5. The preparation method according to claim 2, characterized in that: Step (2) specifically involves: mixing the precursor peptide obtained in step (1) with... 68 The GaCl3 solution was mixed in a buffer system, the pH was adjusted to 3.5-4.0, and the mixture was heated at 100℃ for 10-20 minutes to obtain a reaction mixture. The reaction mixture was purified by passing it through an activated C18 solid-phase extraction column, and the eluent was collected to obtain the desired product. 68 Ga-labeled dual-target PET heterodimer peptide probe 68 Ga-DOTA-FAPI-A7R.

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