A PET probe targeting FDG-negative solid tumors, its preparation method and application

Abstract

This invention discloses a polypeptide PET probe targeting FDG-negative solid tumors, its preparation method, and its application. It belongs to the fields of tumor diagnosis and nuclear medicine imaging. The polypeptide probe primarily targets the CD44v6 protein, as tumors with low FDG uptake often highly express this important target. Specifically, the bifunctional chelating agent NOA is linked to the optimized targeting polypeptide sequence NRWHEDC, and then radioactive nuclide is used for further processing. 18 F or 68 The Ga labeling and purification steps yielded a radionuclide-labeled peptide probe. This probe preparation method is simple and efficient, suitable for large-scale production. More importantly, it possesses high affinity and specificity, enabling PET to identify FDG-negative solid tumors, facilitating early detection, staging, efficacy evaluation, and recurrence monitoring.

Description

Technical Field

[0001] This invention belongs to the field of nuclear medicine imaging, specifically relating to a PET probe targeting FDG-negative solid tumors, its preparation method, and its application. Background Technology

[0002] 18 F-FDG (fluorinated 18-deoxyglucose) PET / CT is an imaging method based on the hypermetabolic characteristics of tumor cells, primarily based on... 18 F-FDG glucose analogues are highly uptaken in tumor cells, thus enabling the identification and diagnosis of tumor lesions via PET / CT scans. However, some solid tumors, such as clear cell renal cell carcinoma, thyroid cancer, and gastric signet ring cell carcinoma, exhibit... 18 F-FDG imaging negative (no uptake or low uptake), providing clinical... 18 F-FDG PET imaging has brought considerable challenges to diagnosis, primarily in reducing the accuracy of tumor diagnosis and staging, and impacting efficacy evaluation, recurrence monitoring, and treatment decision-making.

[0003] The low FDG uptake in these solid tumors is mainly related to their unique metabolic characteristics and histological properties. For example, clear cell renal cell carcinoma relies more on fatty acid oxidation than glycolysis for energy, resulting in low FDG uptake; gastric signet ring cell carcinoma is a poorly differentiated tumor with abundant mucus, and its low GLUT-1 expression leads to low FDG metabolism; thyroid cancer, due to its slow growth and low glycolysis level, also exhibits low FDG uptake. Furthermore, the hypoxic state in the tumor microenvironment inhibits hexokinase HK2 activity, reducing FDG retention within cells, and treatment-resistant cancer stem cells (CSCs) are often in a metabolically quiescent state. These factors collectively contribute to false-negative results in FDG imaging.

[0004] Research has revealed that these tumors with low FDG uptake often highly express CD44v6, an important target, indicating a significant biological link between the two. CD44v6, a splice variant of CD44, is closely associated with tumor invasiveness and stem cell characteristics, rather than depending on glucose metabolism pathways. In FDG-low uptake tumors such as head and neck squamous cell carcinoma basaloid subtype and gastric signet ring cell carcinoma, CD44v6 promotes glutamine metabolism by activating the c-Met / PI3K signaling pathway. This alternative energy supply explains why these tumors maintain high malignancy while retaining low FDG uptake. More importantly, the specific high expression of CD44v6 at the tumor invasion front and metastatic lesions makes it an ideal target for scintigraphically negative but highly invasive tumors, providing a new breakthrough for clinical diagnosis and treatment.

[0005] Currently reported imaging probes for CD44v6 are mainly antibody-based, such as bivalizumab, which primarily targets the CD44v6 protein expressed on the surface of tumor cells for immunotherapy. However, their severe skin toxicity limits their clinical application. Furthermore, while antibodies have high affinity, their large molecular weight leads to prolonged in vivo retention, poor signal-to-noise ratio, and disruptions to the clinical workflow of rapid PET imaging. Peptides, on the other hand, have small molecular weights, are rapidly cleared from the blood, and have significantly lower immunogenicity and toxicity than antibodies, making them ideal for constructing targeted peptide probes. Most reported peptide sequences target CD44, while sequences targeting the CD44v6 subtype often suffer from insufficient in vivo affinity and specificity. Therefore, it is necessary to further develop high-affinity and specific peptide PET probes targeting CD44v6. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a PET probe targeting FDG-negative solid tumors, its preparation method, and its application. This invention primarily addresses the challenge of treating FDG-negative solid tumors by developing a peptide PET probe targeting CD44v6. Through peptide-protein docking and experimental verification, this invention has discovered that the HCAM peptide sequence exhibits good targeting specificity for the CD44v6 protein. Therefore, after analyzing its water-soluble physicochemical properties, targeted structural modifications were performed.

[0007] The method for preparing a peptide PET probe targeting FDG-negative solid tumors, as used in this invention, includes the following steps: 1) An unlabeled radionuclide-based targeting precursor compound was prepared by Fmoc solid-phase synthesis. The unlabeled radionuclide-based targeting precursor compound was obtained by coupling a bifunctional chelating agent to a polypeptide sequence having the function of targeting CD44v6, and the polypeptide sequence contained the HCAM sequence shown in SEQ ID No. 1 or a conserved modified variant containing the HCAM sequence. 2) Place the precursor compound in a buffer system, adjust the reaction conditions, and proceed. 68 Ga or 18 The radioactive labeling of F; 3) After the reactants cooled to room temperature, they were purified by C18 column chromatography to obtain the polypeptide PET molecular probe.

[0008] Preferably, the polypeptide sequence in the precursor compound in step 1) is an HCAM sequence, or its cyclized polypeptide, dimer, or trimer.

[0009] Preferably, the conserved modified variant is based on the HCAM sequence, with one or more amino acid substitutions, deletions, or additions, while retaining the CD44v6 targeting function.

[0010] Preferably, the chelating agent in the precursor compound of step 1) is 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA).

[0011] Preferably, the precursor compound in step 2) is subjected to... 68 When radiolabeling Ga, the buffer system is a solution containing [a certain substance] at pH 3.5-5.0. 68 The reaction temperature for Ga in acetate buffer is 37-100℃, and the reaction time is 5-20 minutes.

[0012] Preferably, the precursor compound in step 2) is subjected to... 18 When radiolabeling F, the buffer system is a pH 4-6 containing... 18 The reaction temperature is 70-105℃ and the reaction time is 5-30 minutes.

[0013] Preferably, step 3) involves separating the product using C18 Sep-PaK (WAT020515, Waters), eluting with 0.5 mL of ethanol, then purging the concentrated ethanol with nitrogen, and finally diluting the product with physiological saline.

[0014] The present invention also provides a polypeptide PET probe for targeting FDG-negative solid tumors prepared by the aforementioned method.

[0015] The present invention also provides the use of the aforementioned polypeptide PET molecular probe in the preparation of a positron emission tomography (PET) diagnostic imaging agent for FDG-negative solid tumors, wherein the FDG-negative solid tumors specifically highly express CD44v6 at the tumor invasion front and metastatic lesions, and wherein the FDG-negative solid tumors are head and neck squamous cell carcinoma, thyroid carcinoma, renal clear cell carcinoma, gastric signet ring cell carcinoma, or well-differentiated hepatocellular carcinoma.

[0016] This invention also provides the application of PET probes in the detection of primary tumor lesions, clinical staging, exploration of metastatic lesions, monitoring of treatment efficacy, or assessment of recurrence.

[0017] Compared with the prior art, the beneficial effects of the present invention include: This invention provides a targeting PET molecular probe based on the highly specific targeting polypeptide sequence HCAM. This probe has both high affinity and high specificity for the CD44v6 target. Its polypeptide structure can be optimized through strategies such as cyclization or multimerization, thereby significantly improving the probe's in vivo stability and tumor targeting efficiency.

[0018] The preparation method of this invention is for the most commonly used clinical applications. 68 Ga and 18The two nuclides, F and F, provide clear, efficient, and mild radiolabeling schemes with rapid preparation processes, facilitating clinical translation and application. They can provide a powerful new molecular imaging tool for the accurate staging, efficacy assessment, and recurrence monitoring of FDG-negative solid tumors. Attached Figure Description

[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Figure 1 Characterization of the NOTA-HCAM monomer of Example 1 of the present invention.

[0021] Figure 2 This is Embodiment 1 of the present invention. 68 Radiolabeled purity of Ga-NOTA-HCAM monomers.

[0022] Figure 3 This is Embodiment 1 of the present invention. 68 PET imaging of Ga-NOTA-HCAM monomer in the Caki-1 renal clear cell carcinoma subcutaneous tumor model.

[0023] Figure 4 Characterization of the NOTA-HCAM cyclic peptide in Example 2 of this invention.

[0024] Figure 5 Example 2 A1 of the present invention 18 Radiolabeled purity of F-NOTA-HCAM cyclic peptide.

[0025] Figure 6 Example 2 A1 of the present invention 18 PET imaging of F-NOTA-HCAM cyclic peptide in the FTC133 thyroid cancer subcutaneous tumor model. Detailed Implementation

[0026] Specific embodiments of the present invention are described below, but the implementation of the present invention is not limited thereto.

[0027] Example 1 68 Preparation of Ga-NOTA-HCAM monomers The polypeptide PET molecular probe of the present invention 68 The preparation of Ga-NOTA-HCAM monomers is as follows: S1. The Nota-HCAM monomer was obtained by solid-phase synthesis using a peptide synthesizer via Fmoc. The structural formula of the Nota-HCAM monomer is shown in Formula I below: Formula I The monomer was synthesized by Ganzhou Tanzhen Biomedical Co., Ltd., and characterized as follows: Figure 1 ; S2. Processing the monomer 68 Ga labeling: 30 mCi eluted 68 The GaCl3 solution was adjusted to pH 4.0 with acetate buffer, and then 20 μg of NOTA-HCAM monomer was added. The mixture was reacted at 95°C under sealed conditions for 10 min, then cooled to room temperature. The product was purified by solid-phase extraction using C18 Sep-PaK (WAT020515, Waters), eluted with 0.5 mL of ethanol, and then concentrated by purging with nitrogen. Finally, the product was diluted with physiological saline to obtain the final product. 68 Ga-NOTA-HCAM ( Figure 2 (for its radiochemical purity).

[0028] S3. Subsequently, a PET scan was performed after injecting the drug via the tail vein into a Caki-1 renal clear cell carcinoma subcutaneous tumor model. The same radiation dose was then injected. 18 F-FDG was used as a control group; the PET imaging images are as follows. Figure 3 As shown, its tumor area is affected by 18 F-FDG intake was almost nonexistent, but for 68 Ga-NOTA-HCAM exhibits significantly high uptake and a high signal-to-noise ratio. Here, Caki-1 cells are tumor cells that highly express CD44v6. These results demonstrate that this probe is helpful for PET imaging technology in identifying FDG-negative solid tumors.

[0029] Example 2 Al 18 Preparation of F-NOTA-HCAM cyclic peptide The polypeptide PET molecular probe Al of the present invention 18 The preparation of F-NOTA-HCAM cyclic peptide is as follows: S1. The NOTA-HCAM cyclic peptide was obtained using a peptide synthesizer via Fmoc solid-phase synthesis. The structural formula of the NOTA-HCAM cyclic peptide is shown in Formula II below: Formula II The monomer was synthesized by Ganzhou Tanzhen Biomedical Co., Ltd., and characterized as follows: Figure 4 ; S2. Processing the monomer 18 F-labeling: Dissolve 0.1 mg NOTA-HCAM cyclic peptide in 1 ml of pure water, add 50 μL of AlCl3 solution (1 mg / mL) and 300 μL of acetate-sodium acetate buffer at pH 4, then add 1000 mCi Na 18Solution F was reacted in a sealed environment at 100℃ for 20 min, then cooled to room temperature. The product was then coated onto a solid-phase extraction column, washed with water to remove the acidic solvent, followed by elution with ethanol. Physiological saline was then added to dilute the product to a concentration of ethanol (<10%). Finally, the diluted solution was pressurized and passed through a pre-activated Sep-Pak alumina column to obtain Al. 18 F-NOTA-HCAM cyclic peptide injection ( Figure 3 (To determine its radioactive labeling purity).

[0030] S3. Subsequently, the tumor was injected via the tail vein into the FTC133 subcutaneous thyroid cancer model, followed by a PET scan. The same radiation dose was then injected. 18 F-FDG was used as a control group; the PET imaging images are as follows. Figure 4 As shown, its tumor area is affected by 18 F-FDG intake was almost nonexistent, but Al 18 The F-NOTA-HCAM cyclic peptide exhibits significantly high uptake and a high signal-to-noise ratio. Here, FTC133 cells are tumor cells that highly express CD44v6. These results demonstrate that this probe is helpful for PET imaging technology in identifying FDG-negative solid tumors.

[0031] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for preparing a polypeptide PET probe targeting FDG-negative solid tumors, characterized in that, The preparation method includes the following steps: 1) An unlabeled radionuclide-based targeting precursor compound was prepared by Fmoc solid-phase synthesis. The unlabeled radionuclide-based targeting precursor compound was obtained by coupling a bifunctional chelating agent to a polypeptide sequence having the function of targeting CD44v6, and the polypeptide sequence contained the HCAM sequence shown in SEQ ID No. 1 or a conserved modified variant containing the HCAM sequence. 2) Place the precursor compound in a buffer system, adjust the reaction conditions, and proceed. 68 Ga or 18 The radioactive labeling of F; 3) After the reactants cooled to room temperature, they were purified by C18 column chromatography to obtain the polypeptide PET molecular probe.

2. The preparation method according to claim 1, characterized in that, Step 1) The polypeptide sequence in the precursor compound is an HCAM sequence, or its cyclized polypeptide, dimer, or trimer.

3. The preparation method according to claim 1, characterized in that, The conserved modified variant is based on the HCAM sequence, with one or more amino acid substitutions, deletions, or additions, while retaining the CD44v6 targeting function.

4. The preparation method according to claim 1, characterized in that, Step 1) The chelating agent in the precursor compound is 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA).

5. The preparation method according to claim 1, characterized in that, In step 2) 68 When radiolabeling Ga, the buffer system is a solution containing [a certain substance] at pH 3.5-5.

0. 68 The reaction temperature for Ga in acetate buffer is 37-100℃, and the reaction time is 5-20 minutes.

6. The preparation method according to claim 1, characterized in that, In step 2) 18 When radiolabeling F, the buffer system is a pH 4-6 containing... 18 The reaction temperature is 70-105℃ and the reaction time is 5-30 minutes.

7. The preparation method according to claim 1, characterized in that, Step 3) The product is separated using C18 Sep-PaK, eluted with ethanol, then concentrated by purging with nitrogen, and finally diluted with physiological saline.

8. A polypeptide PET probe for targeting FDG-negative solid tumors prepared by the method of any one of claims 1-7.

9. Use of the polypeptide PET molecular probe of claim 8 in the preparation of a positron emission tomography (PET) diagnostic imaging agent for FDG-negative solid tumors, wherein the FDG-negative solid tumors are head and neck squamous cell carcinoma, thyroid carcinoma, clear cell renal cell carcinoma, gastric signet ring cell carcinoma, or well-differentiated hepatocellular carcinoma.

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