Polypeptide compound and preparation method and application thereof, and PET imaging agent and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a polypeptide compound and a preparation method and application thereof, and a PET imaging agent and a preparation method and application thereof. The polypeptide compound (DOTA-EP-1) with the structure as shown in the formula I is derived from EpCAM targeted peptide EP-1, and compared with antibody drugs, the polypeptide compound is smaller in molecular weight, faster in blood removal, lower in immunogenicity, higher in labeling efficiency and easier to realize diagnosis and treatment integration. The 68Ga-DOTA-EP-1 prepared by the invention can realize selective uptake at focuses of diseases (such as prostatic cancer and pancreatic cancer) with high expression of EpCAM, and can be quickly cleared in non-expression organs, so that the 68Ga-DOTA-EP-1 has important application value in diagnosis of diseases such as prostatic cancer and pancreatic cancer due to the characteristic of the 68Ga-DOTA-EP-1.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polypeptide compound and its preparation method and application, and a PET imaging agent and its preparation method and application. Background Technology

[0002] Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that plays multiple roles in tumor biology. It mediates calcium-independent homologous cell adhesion during cell signaling, migration, proliferation, and differentiation. EpCAM overexpression has been reported in most tumors, including colorectal adenocarcinoma, lung adenocarcinoma, breast cancer, and prostate cancer. To date, due to its high antigenicity and expression, EpCAM has become a target for monoclonal antibody therapy in colorectal cancer. Previous studies have shown that EpCAM is involved in processes such as tumor stem cells, cell proliferation, metabolism, angiogenesis, epithelial-to-mesenchymal transition (EMT), metastasis, chemotherapy and radioresistance, and immune regulation. It is expressed in cancer stem cells and circulating tumor cells (CTCs) and is closely associated with tumor recurrence and metastasis, leading the U.S. Food and Drug Administration (FDA) to approve EpCAM as a diagnostic marker for CTCs in breast cancer, prostate cancer, and colorectal cancer.

[0003] Currently, many antibodies targeting EpCAM have been developed both domestically and internationally for the treatment of cancer, such as calcizobactam, M701 (a bispecific antibody targeting EpCAM / CD3), CD40 / EpCAM bispecific antibody, and EpAb2-6 neutralizing monoclonal antibody. However, their efficacy is poor due to reasons such as low tolerability or excessive targeting toxicity. Summary of the Invention

[0004] The purpose of this invention is to provide a polypeptide compound and its preparation method and application, and a PET imaging agent and its preparation method and application. The polypeptide compound provided by this invention can specifically bind to epithelial cell adhesion molecule (EpCAM) expressed by prostate cancer, breast cancer, esophageal cancer, ovarian cancer, colorectal cancer, pancreatic cancer or other epithelial-derived tumors.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a polypeptide compound having the structure shown in Formula I:

[0007]

[0008] Preferably, the polypeptide compound is synthesized by Fmoc solid-phase synthesis.

[0009] The present invention also provides the application of the polypeptide compound described in the above technical solution in the preparation of PET imaging agents.

[0010] The present invention also provides a PET developer having the structure shown in Formula II:

[0011]

[0012] The present invention also provides a method for preparing the PET developer described in the above technical solution, comprising the following steps:

[0013] The polypeptide compound, buffer solution, and described in the above technical solution 68 The Ga eluent is mixed, and the resulting mixture is subjected to a radioactive labeling reaction under acidic conditions to obtain the PET imaging agent.

[0014] Preferably, the buffer solution is sodium acetate buffer solution.

[0015] Preferably, the pH value of the acidic conditions is 4 to 5.

[0016] Preferably, the temperature of the radiolabeling reaction is 75–85°C, and the holding time is 10–15 min.

[0017] The present invention also provides the application of the PET imaging agent described in the above technical solution or the PET imaging agent prepared by the preparation method described in the above technical solution in the preparation of tumor diagnostic products.

[0018] Preferably, the tumor includes one or more of the following: prostate cancer, breast cancer, esophageal cancer, ovarian cancer, colorectal cancer, pancreatic cancer, and glioma.

[0019] The polypeptide compound (DOTA-EP-1) with the structure shown in Formula I of this invention is derived from the EpCAM-targeting peptide EP-1. Compared with antibody drugs, it has a smaller molecular weight, faster blood clearance, lower immunogenicity, and higher labeling efficiency, making it easier to achieve integrated diagnosis and treatment. In addition, its small molecular weight (mostly metabolized by the kidneys) results in lower metabolic stress, a cleaner background, and less uptake by other tissues, thus reducing targeted toxicity.

[0020] In this invention, EP-1 is modified with the chelating agent DOTA to form the precursor DOTA-EP-1, which can be used to label diagnostic radionuclides. This chelating agent is then used to label diagnostic radionuclides. 68 PET developer prepared from Ga ( 68 Ga-DOTA-EP-1, exhibiting low background signal and enhanced tumor-specific uptake, is capable of assessing EpCAM expression levels in tumors. Experiments show that the product prepared in this invention... 68 Ga-DOTA-EP-1 can selectively take up EpCAM at lesions of diseases with high EpCAM expression (such as prostate cancer and pancreatic cancer) and rapidly clear it from non-expressing organs. This characteristic makes it of great value in the diagnosis of diseases such as prostate cancer and pancreatic cancer. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is the synthetic route diagram for Example 1, DOTA-EP-1;

[0023] Figure 2 The mass spectrum of DOTA-EP-1 in Example 1 is shown below.

[0024] Figure 3 Example 2 68 Synthesis roadmap for Ga-DOTA-EP-1;

[0025] Figure 4 Example 2 68 Radio-HPLC detection results of Ga-DOTA-EP-1;

[0026] Figure 5 Injecting PC3-bearing tumor model mice via tail vein 68 Images taken at 0.5h, 1h, 2h, and 4h after using Ga-DOTA-EP-1200uci;

[0027] Figure 6 In a BXPC3-bearing tumor model mouse, the tumor was injected via the tail vein. 68 Images taken at 0.5h, 1h, 2h, and 4h after using Ga-DOTA-EP-1200uci;

[0028] Figure 7 In a mouse model of OE19 tumor, an injection was administered via the tail vein. 68 Images taken at 0.5h, 1h, and 2h after using Ga-DOTA-EP-1200uci;

[0029] Figure 8 for 68 Radio-HPLC detection results were obtained after Ga-DOTA-EP-1 labeling and incubation under NS, FBS, and PBS conditions for 0.5h, 1h, and 2h, respectively. Detailed Implementation

[0030] This invention provides a polypeptide compound having the structure shown in Formula I:

[0031]

[0032] In one embodiment of the present invention, the polypeptide compound is synthesized by Fmoc solid-phase synthesis.

[0033] The present invention also provides the application of the polypeptide compound described in the above technical solution in the preparation of PET imaging agents.

[0034] This polypeptide compound can be chemically coupled with diagnostic radionuclides (such as...) 99m Tc, 68 Ga) or therapeutic radionuclides (such as Ga) 177 Lu、 161 By combining with Tb, a dual-modal probe with both diagnostic and therapeutic functions can be formed, which is suitable for the precision diagnosis and treatment of malignant tumors with high EpCAM expression.

[0035] The present invention also provides a PET developer having the structure shown in Formula II:

[0036]

[0037] The present invention also provides a method for preparing the PET developer described in the above technical solution, comprising the following steps:

[0038] The polypeptide compound, buffer solution, and described in the above technical solution 68 The Ga eluent is mixed, and the resulting mixture is subjected to a radioactive labeling reaction under acidic conditions to obtain the PET imaging agent.

[0039] In one embodiment of the present invention, the pH value under the acidic conditions can be 4 to 5.

[0040] In one embodiment of the present invention, the buffer solution may be sodium acetate buffer solution, and the concentration of the sodium acetate buffer solution may be 0.25M.

[0041] In one embodiment of the present invention, the temperature of the radiolabeling reaction can be 75–85°C, specifically 75°C, 80°C, or 85°C, and the holding time can be 10–15 min, specifically 10 min, 12 min, or 15 min. In another embodiment of the present invention, after the radiolabeling reaction, the crude product obtained from the reaction is further purified by HLB column chromatography; the eluent for the HLB column purification is a 40–60 vol.% aqueous ethanol solution.

[0042] As one embodiment of the present invention, the 68 Ga eluent includes 68 GaCl3.

[0043] As one embodiment of the present invention, the 68The preparation method of Ga rinsing solution includes the following steps: using an acid solution to... 68 Ge- 68 The Ga generator is rinsed to obtain 68 Ga eluent. With 68 Taking GaCl3 as an example, the acid solution can be a hydrochloric acid solution; as one embodiment of the present invention, the molar concentration of the hydrochloric acid solution can be 0.05-0.5M, specifically 0.1-0.4M, and 0.25M is used as an example in the embodiment.

[0044] In one embodiment of the present invention, the polypeptide compound can be used in the form of an aqueous solution of the polypeptide compound; the mass ratio of the polypeptide compound to water in the aqueous solution of the polypeptide compound can be 1:1 to 2, specifically 1:1 or 1:2. In one embodiment of the present invention, the polypeptide compound and... 68 The volume ratio of Ga elution solution can be 5–25 μg: 3 mL. 68 The volume ratio of Ga elution buffer to buffer solution can be 1 to 4:1, specifically 2 to 3:1.

[0045] This invention also provides the application of the PET imaging agent described in the above-mentioned technical solution in the preparation of tumor diagnostic products. As one embodiment of this invention, the tumor includes one or more of prostate cancer, breast cancer, esophageal cancer, ovarian cancer, colorectal cancer, pancreatic cancer, and glioma.

[0046] Experiments show that the imaging agent exhibits excellent stability, controllable pharmacokinetic characteristics, and significant tumor tissue retention effect in vivo.

[0047] To further illustrate the present invention, the following detailed description of the invention's solutions, in conjunction with the accompanying drawings and embodiments, is provided, but should not be construed as limiting the scope of protection of the present invention.

[0048] Example 1

[0049] The polypeptide compound having the structure shown in Formula I (denoted as DOTA-EP-1) was synthesized by Peptide Library Biotechnology Co., Ltd. using the Fmoc solid-phase synthesis method. The synthetic route is shown below. Figure 1 Mass spectrum can be found Figure 2 .

[0050] Example 2

[0051] PET developer with the structure shown in Formula II ( 68 Crafting Ga-DOTA-EP-1:

[0052] Dissolve 20 μg of DOTA-EP-1 in 20 μL of distilled water in a reaction vessel. Add 1.5 mL of 0.25 M sodium acetate solution to the reaction vessel, mix well, and then add 3 mL of freshly rinsed sodium acetate solution. 68 Ga rinsing solution (rinsing with 0.05M hydrochloric acid solution) 68 Ge- 68 (Ga was obtained from a Ga generator). The pH of the reaction system was adjusted to 4.0, and after thorough mixing, the radiolabeling reaction was carried out at 85℃ for 15 minutes. After the reaction, the reaction product was purified using an Oasis HLB column, with 1 mL of 50 vol.% ethanol aqueous solution as the eluent. The reaction equation is as follows: Figure 3 As shown, Example 2 68 The Radio-HPLC detection results for Ga-DOTA-EP-1 are shown below. Figure 4 .

[0053] Example 3

[0054] Take the preparation of Example 2 68 Approximately 100 μCi of Ga-DOTA-EP-1 was placed in 100 μL of 0.9% physiological saline, fetal bovine serum (FBS), and phosphate-buffered saline (PBS), respectively, and thoroughly mixed before being stored at 37°C. Samples were taken at 0.5 h, 1 h, and 2 h, and changes in radiochemical purity were detected by analytical radiometric high-performance liquid chromatography (Radio-HPLC).

[0055] Experimental results show that, under the above experimental conditions, 68 Ga-DOTA-EP-1 maintained a radiochemical purity above 98% after 2 hours, exhibiting good stability with almost no decomposition. The results are as follows... Figure 8 As shown.

[0056] Example 4

[0057] The PET developer prepared in Example 2 68 Ga-DOTA-EP-1 was injected into PC3 tumor-bearing mice, BXPC3 tumor-bearing mice, and OE19 tumor-bearing mouse models, respectively, as follows:

[0058] Each model mouse was placed on a Micro PET bed and anesthetized by inhalation of isoflurane, then fixed in position with tape. 0.1 mL of [a specific drug / method] was injected via the tail vein into each model mouse. 68Ga-DOTA-EP-1 was administered in a saline solution (radioactivity 200 uCi), followed by static PET / CT imaging of small animals at 0.5h, 1h, 2h, and 4h post-injection (excluding the OE19 model, which did not have images acquired at 4 hours). The relevant imaging results are as follows: Figures 5-7 As shown.

[0059] Example 5

[0060] The PET developer prepared in Example 2 68 The lipid-water partition coefficient of Ga-DOTA-EP-1 was analyzed. Six test tubes were used, and 0.5 mL of saturated n-octanol and 0.45 mL of purified water were added to each tube. 50 μL (approximately 1 μCi) of freshly prepared radiopharmaceutical was added, and the mixture was agitated for 2 min using an ultrasonic vibrator and then centrifuged at 5000 r / min for 5 min. Six more test tubes were taken and labeled A1, A2, A3, B1, B2, and B3. Background counts were measured first, and then the two phases were separated. For group A, 100 μL of liquid was precisely drawn from the upper organic phase for radioactivity counting; for group B, 100 μL of liquid was drawn from the lower aqueous phase for the same radioactivity counting. The lipid-water partition coefficient (logP) was calculated as logP = log[(organic phase count - background count) / (aqueous phase count - background count)]. The results are shown in Table 1. The lipid-water partition coefficient describes... 68 The partitioning degree of Ga-DOTA-EP-1 between pure water and saturated n-octanol. Experimental results show that... 68 The average logP of Ga-DOTA-EP-1 is -3.01, indicating that it has good hydrophilicity and is mainly metabolized by the kidneys in vivo.

[0061] Table 1 68 The lipid-water partition coefficient of Ga-DOTA-EP-1

[0062] fat water Background logP 360 147828 188 -2.93367559 330 146016 188 -3.011552575 306 142427 188 -3.081136683

[0063] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A polypeptide compound having the structure shown in Formula I:

2. The polypeptide compound according to claim 1, characterized in that, The polypeptide compound was synthesized using the Fmoc solid-phase synthesis method.

3. The use of the polypeptide compound according to claim 1 or 2 in the preparation of PET imaging agents.

4. A PET developer having the structure shown in Formula II:

5. The method for preparing the PET developer according to claim 4, comprising the following steps: The polypeptide compound, buffer solution, and as described in claim 1 68 The Ga eluent is mixed, and the resulting mixture is subjected to a radioactive labeling reaction under acidic conditions to obtain the PET imaging agent.

6. The preparation method according to claim 5, characterized in that, The buffer solution is sodium acetate buffer.

7. The preparation method according to claim 5, characterized in that, The pH value of the acidic conditions is 4 to 5.

8. The preparation method according to claim 5, characterized in that, The temperature of the radiolabeling reaction is 75–85°C, and the holding time is 10–15 min.

9. The use of the PET imaging agent according to claim 4 or the PET imaging agent prepared by the preparation method according to any one of claims 5 to 9 in the preparation of tumor diagnostic products.

10. The application as described in claim 9, characterized in that, The tumors include one or more of the following: prostate cancer, breast cancer, esophageal cancer, ovarian cancer, colorectal cancer, pancreatic cancer, and glioma.

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