A pet polypeptide probe for high signal-to-noise ratio identification of liver cancer and a preparation method and application thereof

Abstract

The application discloses a PET polypeptide probe for high signal-to-noise ratio identification of liver cancer and a preparation method and application thereof, and belongs to the field of liver cancer diagnosis and nuclear medical imaging. 18 The application can target and image the GPC3 high-expression liver cancer lesion area after F labeling, and the good water solubility can be rapidly metabolized through the kidney and will not be retained in normal liver tissue, so that the application can be used for high signal-to-noise ratio diagnosis of liver cancer. The development and application of the GPC3-targeted PET polypeptide probe overcome the fat-soluble problem of the conventional GPC3-targeted polypeptide, and then the liver cancer lesion can be specifically identified, the preparation method is simple, the specificity is high, the signal-to-noise ratio is high, and more accurate imaging information is provided for early diagnosis of primary liver cancer.

Description

Technical Field

[0001] This invention belongs to the fields of liver cancer diagnosis and nuclear medicine imaging, specifically relating to a PET polypeptide probe for high signal-to-noise ratio liver cancer identification, its preparation method, and its application. Background Technology

[0002] Liver cancer poses a serious threat to life and health. Although various treatment options are available clinically, the survival rate remains low because most liver cancer patients are diagnosed at an advanced stage. Early and accurate screening for liver cancer can help improve survival time. Currently, clinical diagnosis of liver cancer mainly relies on serum alpha-fetoprotein (AFP) levels for screening, but this method lacks sufficient sensitivity and specificity. Clinical CT and MRI imaging are also limited by spatial resolution and tissue specificity. Positron emission tomography (PET) has the advantages of high sensitivity and quantitative analysis capabilities, and... 18 F is the most commonly used radionuclide in clinical PET imaging and has a well-established labeling methodology. Therefore, it is worth considering developing targeted PET probes for the precise diagnosis of liver cancer.

[0003] Phosphatidylinositol proteoglycan 3 (GPC3) is a 70 kDa protein anchored to the cell membrane surface via glycosylphosphatidylinositol (GPI). It regulates cell growth and differentiation, and clinical pathological examination has revealed high expression of GPC3 in hepatocellular carcinoma (HCC), while it is absent or expressed at low levels in normal liver tissue, suggesting that GPC3 could serve as a tumor marker for specific diagnosis of HCC. Monoclonal antibodies against GPC3, after radionuclide labeling, can be used for PET diagnosis of HCC; however, the long in vivo circulation time and liver distribution of these antibodies limit their precise identification of primary HCC. Peptides possess advantages such as small molecular weight, low immunogenicity, and large-scale production capability. Several GPC3-targeting peptide sequences have been reported, such as RLNVGGTYFLTTRQ (Arch Pathol Lab Med 2008, 132, 1723), THVSPNQGGLPS (Macromol. Biosci. 2017, 17, 1600335), and FVGEFFTDV (Cancer Sci. 2011, 102, 918-925). However, while these peptides have demonstrated their targeting at the molecular biology level, the exploration and optimization of their in vivo distribution remains insufficient. In imaging experiments using a mouse model of liver cancer, the applicant discovered that these highly GPC3-targeting peptide sequences contain a large number of lipid-soluble amino acids. These lipid-soluble amino acids cause the peptide sequences to accumulate in the liver, making it impossible to distinguish between normal liver tissue and liver cancer lesions, i.e., failing to identify liver cancer regions with high specificity and high signal-to-noise ratio. Therefore, it is necessary to employ appropriate methods to reduce the retention of these targeting peptides in the liver. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a PET peptide probe for high signal-to-noise ratio identification of liver cancer, its preparation method, and its application. This polyethylene glycol-modified GPC3-targeting PET peptide probe provides effective imaging information for the high signal-to-noise ratio diagnosis of primary liver cancer.

[0005] The technical solution adopted in this invention includes the following steps:

[0006] This invention first provides a method for preparing a PET peptide probe for high signal-to-noise ratio identification of liver cancer, which includes the following steps:

[0007] 1) A polyethylene glycol-modified GPC3-targeting polypeptide sequence PEG-GPC3 polypeptide was prepared;

[0008] 2) Its fluorinated ligand Nota-NHS was coupled in dimethylformamide containing triethylamine and purified to obtain the Nota-PEG-GPC3 polypeptide;

[0009] 3) Add the NOA-PEG-GPC3 peptide to an acetate-sodium acetate buffer solution containing AlCl3, adjust the pH of the reaction solution, and then add radioactive Na. 18 Solution F is heated and reacted in a sealed environment;

[0010] 4) After the reactants were cooled to room temperature, they were purified to obtain Al. 18 The F-PEG-GPC3 polypeptide is the PET polypeptide probe.

[0011] Preferably, the polyethylene glycol-modified GPC3-targeting peptide is obtained by Fmoc solid-phase synthesis using a peptide synthesizer, wherein the number of PEGs is 3 to 4, and the GPC3-targeting peptide sequence is one or more of RLNVGGTYFLTTRQ (SEQ ID NO.1), THVSPNQGGLPS (SEQ ID NO.2), and FVGEFFTDV (SEQ ID NO.3).

[0012] Preferably, in step 2), the molar ratio of the PEG-GPC3 peptide, NOA-NHS, and triethylamine is 1:1 to 2:1 to 5.

[0013] Preferably, in step 2), the coupling reaction is carried out at room temperature, and the purification method is high performance liquid chromatography (HPLC) separation and purification.

[0014] Preferably, in step 3), each milligram of Nota-PEG-GPC3 peptide is supplemented with Na 18The radioactivity of F is 100–5000 mCi, and the mass ratio of NOTA-PEG-GPC3 to AlCl3 is 1:0.005–0.2.

[0015] Preferably, in step 3), the pH of the solution is adjusted to control the pH value between 4 and 6; the temperature range of the heating and sealed reaction is 50-105℃, and the reaction time is 3-30 minutes.

[0016] Preferably, the purification method in step 4) is solid-phase extraction purification.

[0017] The present invention also provides a PET polypeptide probe for high signal-to-noise ratio identification of liver cancer, which is prepared by the above method.

[0018] This invention also provides the application of PET probes in the preparation of reagents for early diagnosis or treatment monitoring of primary liver cancer.

[0019] The advantages and features of this invention are as follows:

[0020] 1. This invention is the first to discover the problem of GPC3-targeting peptide sequences accumulating in the liver in vivo, hindering the identification of liver cancer regions with high specificity and high signal-to-noise ratio. This invention creatively employs a chemical modification method to reduce the retention of the targeting peptide in the liver. The prepared polyethylene glycol-modified GPC3-targeting PET peptide probe exhibits high liver cancer targeting specificity and renal metabolic characteristics. By modifying the PET peptide probe with an appropriate length of polyethylene glycol and combining it with hydrophilic groups in the ligand, this invention effectively improves the water solubility of the PET peptide probe, enabling it to be rapidly metabolized by the kidneys without remaining in normal liver tissue. Therefore, it allows for the quantitative assessment of liver cancer with a high signal-to-noise ratio.

[0021] 2. The molecular modification strategy of the polyethylene glycol-modified GPC3-targeting PET peptide prepared by this invention is simple, has high labeling efficiency, and is easy to label and promote in clinical automated modules. Attached Figure Description

[0022] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] Figure 1 This is the structural formula of NOTA-PEG4-RLNVGGTYFLTTRQ in this invention.

[0024] Figure 2 Example 1A1 of the present invention 18 Radiolabeled purity of F-PEG4-RLNVGGTYFLTTRQ.

[0025] Figure 3 Example 1A1 of the present invention 18PET imaging of F-PEG4-RLNVGGTYFLTTRQ in a Hep3B subcutaneous hepatocellular carcinoma model.

[0026] Figure 4 This is the structural formula of NOTA-PEG3-THVSPNQGGLPS in this invention.

[0027] Figure 5 Example 1A1 of the present invention 18 Radiolabeled purity of F-PEG3-THVSPNQGGLPS.

[0028] Figure 6 Example 1A1 of the present invention 18 PET imaging of F-PEG3-THVSPNQGGLPS in a Hep3B subcutaneous hepatocellular carcinoma model.

[0029] Figure 7 This is the structural formula of NOTA-PEG4-FVGEFFTDV in this invention.

[0030] Figure 8 Example 1A1 of the present invention 18 Radiolabeled purity of F-PEG4-FVGEFFTDV.

[0031] Figure 9 Example 1A1 of the present invention 18 PET imaging of F-PEG4-FVGEFFTDV in a Hep3B subcutaneous hepatocellular carcinoma model. Detailed Implementation

[0032] Specific embodiments of the present invention are described below, but the implementation of the present invention is not limited thereto.

[0033] Example 1

[0034] The polyethylene glycol-modified GPC3-targeting peptide sequence PEG4-RLNVGGTYFLTTRQ was obtained using a Fmoc solid-phase synthesis method. Then, 1 mmol of the synthesized peptide sequence was coupled with 1.2 mmol of NOTA-NHS in dimethylformamide containing 1.5 mmol of triethylamine. The product was then purified by high-performance liquid chromatography (HPLC) to obtain NOTA-PEG4-RLNVGGTYFLTTRQ. The structural formula of the obtained product is attached. Figure 1 As shown.

[0035] Dissolve 0.2 mg NOTA-PEG4-RLNVGGTYFLTTRQ in 1 ml of pure water, then add 20 μL of AlCl3 solution (1 mg / mL) and 300 μL of acetate-sodium acetate buffer (pH 4), followed by 200 mL of CiNa. 18 Solution F was reacted at 100℃ under sealed conditions for 10 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 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-PEG4-RLNVGGTYFLTTRQ injection ( Figure 2 (to determine its radiolabeled purity). Then Al... 18 F-PEG4-RLNVGGTYFLTTRQ was injected into a Hep3B subcutaneous hepatocellular carcinoma model, followed by PET scanning. PET images are shown below. Figure 3 As shown, Figure 3 Two PEG-modified probes Al prepared using the same method as in this embodiment were also compared. 18 F-PEG2-RLNVGGTYFLTTRQ and 6 PEG-modified probes Al 18 PET imaging images of F-PEG6-RLNVGGTYFLTTRQ under the same conditions. As can be seen from the images, the two PEG-modified peptides are distributed in both normal liver tissue and hepatocellular carcinoma lesions, making it impossible to distinguish between the two, i.e., failing to identify hepatocellular carcinoma areas with high specificity and high signal-to-noise ratio. When the number of PEGs is increased to four, there is no enrichment in the liver and the tumor signal is stronger; when the number of PEGs is increased to six, the rapid metabolism by the kidneys results in a weaker tumor signal, which is also detrimental to the accurate identification of lesion areas. The applicant's research found that modifying the RLNVGGTYFLTTRQ peptide sequence with three or four PEG repeat units can image tumors with high specificity and without liver retention. This high signal-to-noise ratio imaging can be used for accurate diagnosis of hepatocellular carcinoma.

[0036] Example 2

[0037] The polyethylene glycol-modified GPC3-targeting peptide sequence PEG3-THVSPNQGGLPS was obtained using a Fmoc solid-phase synthesis method. Then, 1 mmol of PEG3-THVSPNQGGLPS was coupled with 1.2 mmol of NOTA-NHS in dimethylformamide containing 1.5 mmol of triethylamine. The resulting product was purified by high-performance liquid chromatography (HPLC) to obtain NOTA-PEG3-THVSPNQGGLPS. The structural formula and mass spectrum of the obtained product are attached. Figure 4 As shown.

[0038] Dissolve 0.5 mg NOTA-PEG3-THVSPNQGGLPS 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 mL of CiNa 18 Solution F was reacted at 95°C under sealed conditions 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 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-PEG3-THVSPNQGGLPS injection ( Figure 5 (to determine its radiolabeled purity). Then Al... 18 F-PEG3-THVSPNQGGLPS were injected into a Hep3B subcutaneous hepatocellular carcinoma model, followed by PET scanning. PET images are shown below. Figure 6 As shown, modifying the THVSPNQGGLPS peptide sequence with three PEG repeat units can image tumors with high specificity and without liver retention. The probe can be highly specifically enriched at the tumor site without liver retention. This high signal-to-noise ratio imaging can be used to accurately diagnose liver cancer.

[0039] Example 3

[0040] The polyethylene glycol-modified GPC3-targeting peptide sequence PEG4-FVGEFFTDV was obtained using a Fmoc solid-phase synthesis method via a peptide synthesizer. Then, 1 mmol of the synthesized peptide sequence was coupled with 1.2 mmol of NOTA-NHS in dimethylformamide containing 1.5 mmol of triethylamine. The product was then purified by high-performance liquid chromatography (HPLC) to obtain NOTA-PEG4-FVGEFFTDV. The structural formula and mass spectrum of the obtained product are attached. Figure 7 As shown.

[0041] Dissolve 0.3 mg NOTA-PEG4-FVGEFFTDV in 1 ml of pure water, add 30 μL of AlCl3 solution (1 mg / mL) and 300 μL of acetate-sodium acetate buffer solution at pH 4, then add 500 mL of CiNa 18 Solution F was reacted at 105℃ under sealed conditions for 15 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-PEG4-FVGEFFTDV injection ( Figure 8(to determine its radiolabeled purity). Then Al... 18 F-PEG4-FVGEFFTDV was injected into a Hep3B subcutaneous hepatocellular carcinoma model, followed by PET scanning. PET images are shown below. Figure 9 As shown, modification of the FVGEFFTDV polypeptide sequence with four PEG repeat units can image tumors with high specificity and without liver retention. The probe can be highly specifically enriched at the tumor site without liver retention. This high signal-to-noise ratio imaging can be used to accurately diagnose liver cancer.

[0042] 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 PET polypeptide probe for high signal-to-noise ratio identification of liver cancer, characterized in that, Includes the following steps: 1) Preparation of polyethylene glycol-modified GPC3-targeting peptide sequence PEG-GPC3 peptide; The polyethylene glycol-modified GPC3-targeting peptide was obtained by Fmoc solid-phase synthesis, wherein the number of repeating units of PEG is 3~4, and the GPC3-targeting peptide sequence is one or more of RLNVGGTYFLTTRQ, THVSPNQGGLPS, and FVGEFFTDV. 2) The PEG-GPC3 peptide and Nota-NHS were coupled in dimethylformamide containing triethylamine and purified to obtain the Nota-PEG-GPC3 peptide. 3) Add the NOA-PEG-GPC3 peptide to an acetate-sodium acetate buffer solution containing AlCl3, adjust the pH of the reaction solution, and then add radioactive Na. 18 Solution F is heated and reacted in a sealed environment; 4) After the reactants were cooled to room temperature, they were purified to obtain Al. 18 The F-PEG-GPC3 polypeptide is the PET polypeptide probe.

2. The method for preparing a PET polypeptide probe for high signal-to-noise ratio liver cancer identification according to claim 1, characterized in that, In step 2), the molar ratio of PEG-GPC3 peptide, NOA-NHS and triethylamine is 1:1~2:1~5.

3. The method for preparing a PET polypeptide probe for high signal-to-noise ratio liver cancer identification according to claim 1, characterized in that, In step 2), the coupling reaction is carried out at room temperature, and the purification method is high performance liquid chromatography (HPLC) separation and purification.

4. The method for preparing a PET polypeptide probe for high signal-to-noise ratio liver cancer identification according to claim 1, characterized in that, In step 3), Na is added per milligram of Nota-PEG-GPC3 peptide. 18 The radioactivity of F is 100~5000 mCi, and the mass ratio of NOTA-PEG-GPC3 to AlCl3 is 1:0.005~0.

2.

5. The method for preparing a PET polypeptide probe for high signal-to-noise ratio liver cancer identification according to claim 1, characterized in that, In step 3), the pH of the reaction solution is adjusted to be controlled between 4 and 6; the temperature range of the heated and sealed reaction is 50 to 105°C, and the reaction time is 3 to 30 minutes.

6. The method for preparing a PET polypeptide probe for high signal-to-noise ratio liver cancer identification according to claim 1, characterized in that, The purification method described in step 4) is solid-phase extraction.

7. A PET polypeptide probe for high signal-to-noise ratio identification of liver cancer, characterized in that, Prepared using the method described in any one of claims 1-6.

8. The application of the PET polypeptide probe for high signal-to-noise ratio identification of liver cancer as described in claim 7 in the preparation of a reagent for early diagnosis or treatment monitoring of primary liver cancer.

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