CN117586354A

CN117586354A - Targeting GPC3 polypeptide probe and application thereof in preparation of diagnosis and treatment radiopharmaceuticals

Info

Publication number: CN117586354A
Application number: CN202311559318.9A
Authority: CN
Inventors: 傅丽兰; 唐刚华; 莫纯威
Original assignee: Southern Hospital Southern Medical University
Current assignee: Southern Hospital Southern Medical University
Priority date: 2023-11-21
Filing date: 2023-11-21
Publication date: 2024-02-23

Abstract

The invention relates to the technical field of radiopharmaceuticals. The invention provides a target GPC3 polypeptide probe precursor, wherein the polypeptide probe precursor has a structure shown in a formula I. The invention also provides a method for obtaining the target GPC3 polypeptide probe precursorThe obtained target GPC3 polypeptide probe. The invention improves the targeting property and the pharmacokinetic property by optimizing the chemical structure, and overcomes the defects of lower tumor uptake, shorter tumor residence time, poorer pharmacokinetic property and the like of the existing targeted GPC3 polypeptide probe. The invention also provides a preparation method and application of the probe precursor or the probe.

Description

Targeting GPC3 polypeptide probe and application thereof in preparation of diagnosis and treatment radiopharmaceuticals

Technical Field

The invention relates to the technical field of radiopharmaceuticals, in particular to a targeted GPC3 polypeptide probe and application thereof in preparing a diagnosis and treatment radiopharmaceuticals.

Background

The occurrence and development of malignant tumors increasingly threatens the life of human beings, and becomes a serious social problem. Malignant tumors become the first or second cause of death of humans, malignant tumors in China are ranked second among various causes of death, and tumor mortality rate is obviously increased. Many malignant tumors are already in the middle and late stages at the time of diagnosis. Therefore, the early diagnosis and accurate treatment of malignant tumors are realized with profound clinical significance. Glypican-3 (GPC 3) is hardly expressed in normal tissue organs but is expressed in various malignant tumors (such as liver cancer, melanoma, lung squamous cell carcinoma, nephroblastoma, etc.) ^[1] . In the case of liver cancer (HCC), GPC3 is significantly highly expressed in HCC and highly correlated with the clinical prognosis of HCC ^[2] The method comprises the steps of carrying out a first treatment on the surface of the Studies have demonstrated that GPC3 mediates hepatocyte degeneration and promotes HCC growth by activating Wnt signaling pathways, highly correlated with HCC clinical prognosis ^[3] . Therefore, GPC3 is expected to become an important target for tumor visualization accurate diagnosis and treatment as a specific marker for positive expression of GPC3 tumors.

In recent years, radiolabeled probes targeting GPC3 have focused mainly on antibody-based probes and polypeptide-based probes ^[4-9] . GPC3 monoclonal antibody has good affinity and specificity for HCC, ⁸⁹ zr-labeled GPC3 monoclonal antibody can be shown in HCC tumor tissue ^[4-5] . However, antibodies have the disadvantages of large molecular weight, high price, difficult labeling, slow in vivo clearance, potential immunogenicity, etc. In contrast, polypeptides have the advantages of low molecular weight, low immunogenicity, ease of synthesis, modification and radiolabeling, good tumor penetration, etc. Reported probes targeting GPC3 polypeptides have ^99m Tc-HPG、[ ¹⁸ F]AlF-NOTA-TJ12P2 and [ ⁶⁸ Ga]DOTA-F3、[ ¹⁸ F]AlF-GP2633 and the like ^[6-9] The method comprises the steps of carrying out a first treatment on the surface of the These GPC 3-targeting polypeptide probes specifically target HCC tumors that are expressed positively for GPC3, but have lower tumor uptake, shorter tumor residence time, and poorer pharmacokinetic properties (e.g., abdominal)Non-specific uptake) in the area.

Previous studies have shown that the use of radiolabeled probes modified with plasma albumin and appropriate linkers can prolong blood circulation time and thereby increase tumor tissue uptake and tumor tissue retention, improving pharmacokinetic properties ^[10] . After the pharmacophore polypeptide GPC3P (GGGRDNRLNVGGTYFLTTRQ) is modified by combining a plasma albumin agent and a chelating agent, a plurality of radionuclide labels are used for preparing a targeted GPC3 polypeptide probe, so that the defects of the existing targeted GPC3 polypeptide probe in tumor diagnosis and treatment are overcome, and the purposes of improving the pharmacokinetic characteristics, improving the uptake of tumor focus and prolonging the residence time of the tumor focus are achieved.

Disclosure of Invention

In order to solve the prior art, the invention provides a brand-new target GPC3 polypeptide probe. The invention designs and constructs a novel targeted GPC3 polypeptide probe [ M ] with excellent pharmacokinetic properties by optimizing a chemical structure, improving targeting and improving pharmacokinetic properties, and overcoming the defects of low tumor uptake, short tumor residence time, poor pharmacokinetic properties and the like of the existing targeted GPC3 polypeptide probe ⁿ⁺ -Q]R-GPC3P, increases the uptake of tumor tissue positive for GPC3 expression and increases the residence time of tumor tissue. The invention provides a novel technology for synthesizing a targeted GPC3 polypeptide probe by automatic high-yield radiosynthesis, which has simple marking, and also provides a preparation method of the probe precursor raw materials; the present study further provides the use of these polypeptide probes in tumor diagnosis and treatment.

In one aspect, the invention provides a GPC 3-targeting polypeptide probe precursor, wherein the polypeptide probe precursor has a structure according to formula I:

wherein,

q is a chelating group;

r is 4- (p-methylphenyl) butanoyl or 4- (p-iodophenyl) butanoyl;

w is the pharmacophore polypeptide GPC3P.

In some embodiments, the chelating group is selected from: groups formed by 1,4,7, 10-tetraazacyclododecane-N ', N' -triacetoxy-N-acetyl (DOTA), 1,4,7, 10-tetraazacyclododecane, 1- (glutaric acid) -4,7, 10-triacetoxy (dotga), 1,4, 7-triazacyclononaalkyl-N ', N' -diacetoxy-N-acetyl (NOTA), 2S- (4-isothiocyanatobenzyl) -1,4, 7-triazacyclononane-1, 4, 7-triacetoxy (p-SCN-Bn-NOTA), 1,4, 7-triazacyclononane-N-glutarate-N ', N' -diacetic acid (nodagga) or 1,4, 7-triazacyclononane-1, 4-diacetic acid-methylphenylacetic acid (NODA-MPAA) chelating agents; preferably, the chelating group is selected from:

or p-SCN-Bn-NOTA.

In some embodiments, W is: pharmacophore polypeptide GPC3P: GGGRDNRLNVGGTYFLTTRQ.

In some embodiments, W is:

in another aspect, the invention also provides a target GPC3 polypeptide probe, which is obtained by chelating the polypeptide probe precursor of the invention with radioactive ions, wherein the radioactive ions have the definition of the invention.

In another aspect, the invention also provides a targeted GPC3 polypeptide probe having a structure represented by formula II,

wherein Q, W, R has the definition set forth herein;

M ⁿ⁺ is a radioactive ion.

In some embodiments, the radioactive ion is selected from ¹⁸ F、 ⁵¹ Cr、 ⁶⁷ Ga、 ⁶⁸ Ga、 ¹¹¹ In、 ^99m Tc、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ¹³⁹ La、 ¹⁴⁰ La、 ¹⁷⁵ Yb、 ¹⁵³ Sm、 ¹⁶⁶ Ho、 ⁸⁸ Y、 ⁹⁰ Y、 ¹⁴⁹ Pm、 ¹⁶⁵ Dy、 ¹⁶⁹ Er、 ¹⁷⁷ Lu、 ⁴⁷ Sc、 ¹⁴² Pr、 ¹⁵⁹ Gd、 ²¹² Bi、 ²¹³ Bi、 ⁷² As、 ⁷² Se、 ⁹⁷ Ru、 ¹⁰⁹ Pd、 ¹⁰⁵ Rh、 ^101m Rh、 ¹¹⁹ Sb、 ¹²⁸ Ba、 ¹²³ I、 ¹²⁴ I、 ¹³¹ I、 ¹⁹⁷ Hg、 ²¹¹ At、 ¹⁵¹ Eu、 ¹⁵³ Eu、 ¹⁶⁹ Eu、 ²⁰¹ Tl、 ²⁰³ Pb、 ²¹² Pb、 ⁶⁴ Cu、 ⁶⁷ Cu、 ¹⁸⁸ Re、 ¹⁸⁶ Re、 ¹⁹⁸ Au、 ²²⁵ Ac、 ²²⁷ Th and ¹⁹⁹ ag forms ground ions.

In some embodiments, the radioactive element is selected from ¹⁸ F、 ⁶⁸ Ga、 ¹⁷⁷ Lu, and ⁶⁴ ions formed by Cu.

In some embodiments, the radioactive ion is selected from ⁶⁸ Ga ³⁺ 、[Al ¹⁸ F] ²⁺ 、 ⁶⁴ Cu ²⁺ And ¹⁷⁷ Lu ³⁺ 。

on the other hand, the invention also provides a preparation method of the target GPC3 polypeptide probe precursor, wherein GPC3P-Lys is formed after target GPC3 polypeptide is modified Lys, and then the target GPC3 polypeptide probe precursor is obtained through reaction with a chelating agent and a combined plasma albumin group, wherein the combined plasma albumin group is 4- (P-iodophenyl) butyric acid or 4- (P-methylphenyl) butyric acid; wherein the target GPC3 polypeptide probe precursors have the structures described herein.

And substitution of 4- (p-iodophenyl) butanoyl in combination with plasma albumin-based 4- (p-iodophenyl) butanoic acid (IPB) reaction; and substitution of 4- (p-methylphenyl) butanoyl in combination with plasma albumin-based 4- (p-methylphenyl) butanoic acid (MPB) reactions.

A GPC 3-targeting polypeptide probe precursor, wherein the polypeptide probe precursor has a structure according to formula I:

wherein Q, R, W has the definition set forth in the present invention.

The chelating agent is selected from 1,4,7, 10-tetraazacyclododecane-N ', N ' -triacetoxy-N-acetyl (DOTA), 1,4,7, 10-tetraazacyclododecane, 1- (glutarate) -4,7, 10-triacetic acid (DOTAGA), 1,4, 7-triazacyclononalkyl-N ', N ' -diacetoxy-N-acetyl (NOTA), 2S- (4-isothiocyanatobenzyl) -1,4, 7-triazacyclononane-1, 4, 7-triacetic acid (p-SCN-Bn-NOTA), 1,4, 7-triazacyclononane-N ' -glutarate-N ', N ' -diacetic acid (NODAGA) or 1,4, 7-triazacyclononane-1, 4-diacetic acid-methylphenylacetic acid (NODA-MPAA).

In another aspect, a method for preparing a target GPC3 polypeptide probe, wherein a target GPC3 polypeptide probe precursor is reacted with a radioactive ion salt in an acidic solvent under heating to obtain a target GPC3 polypeptide probe; wherein the radioactive ion salt is selected from the group consisting of ¹⁸ F、 ⁵¹ Cr、 ⁶⁷ Ga、 ⁶⁸ Ga、 ¹¹¹ In、 ^99m Tc、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ¹³⁹ La、 ¹⁴⁰ La、 ¹⁷⁵ Yb、 ¹⁵³ Sm、 ¹⁶⁶ Ho、 ⁸⁸ Y、 ⁹⁰ Y、 ¹⁴⁹ Pm、 ¹⁶⁵ Dy、 ¹⁶⁹ Er、 ¹⁷⁷ Lu、 ⁴⁷ Sc、 ¹⁴² Pr、 ¹⁵⁹ Gd、 ²¹² Bi、 ²¹³ Bi、 ⁷² As、 ⁷² Se、 ⁹⁷ Ru、 ¹⁰⁹ Pd、 ¹⁰⁵ Rh、 ^101m Rh、 ¹¹⁹ Sb、 ¹²⁸ Ba、 ¹²³ I、 ¹²⁴ I、 ¹³¹ I、 ¹⁹⁷ Hg、 ²¹¹ At、 ¹⁵¹ Eu、 ¹⁵³ Eu、 ¹⁶⁹ Eu、 ²⁰¹ Tl、 ²⁰³ Pb、 ²¹² Pb、 ⁶⁴ Cu、 ⁶⁷ Cu、 ¹⁸⁸ Re、 ¹⁸⁶ Re、 ¹⁹⁸ Au、 ²²⁵ Ac、 ²²⁷ Th and ¹⁹⁹ ag forms ground ions; preferably, the placementThe radioactive element is selected from ¹⁸ F、 ⁶⁸ Ga、 ¹⁷⁷ Lu, and ⁶⁴ salts of Cu-forming ions; preferably, the radioactive ion salt is selected from ⁶⁸ GaCl ₃ 、[Al ¹⁸ F] ²⁺ Salt(s), ⁶⁴ CuCl ₂ 、 ¹⁷⁷ Lu ion salt.

In another aspect, the invention provides a kit comprising the polypeptide probe of the invention.

In another aspect, the invention provides a tumor PET or SPECT imaging agent including the targeted GPC3 polypeptide probe of the invention.

On the other hand, the invention provides the application of the target GPC3 polypeptide probe in preparing tumor PET imaging agent and tumor SPECT imaging agent, or in evaluating tumor curative effect, or in preparing tumor peptide target radionuclide therapeutic probe.

The tumor peptide targeted radionuclide therapy probe is used for tumor therapy.

In some embodiments, the tumor comprises a tumor that is positively expressed by GPC3.

In some embodiments, the tumor that is positively expressed by GPC3 comprises liver cancer.

In some embodiments, the invention provides a probe [ M ] targeting a GPC3 polypeptide ⁿ⁺ ]NOTA-R-GPC3P and [ M ] ⁿ⁺ ]Use of DOTA-R-GPC3P in Positron Emission Tomography (PET) imaging of tumors, including [ M ] ⁿ⁺ ]NOTA-R-GPC3P and [ M ] ⁿ⁺ ]DOTA-R-GPC3P can be applied to PET imaging of tumors such as liver cancer, and can be further applied to PET imaging and curative effect evaluation of tumors such as liver cancer.

Targeting GPC3 polypeptide probes [ M ] ⁿ⁺ -Q]-R-GPC3P (formula II), targeting polypeptide (GPC 3P), linker (lysine), plasma albumin binding group (R), metal ion chelating group (Q) and radionuclides (M) ⁿ⁺ ) The composition is formed. Wherein the pharmacophore polypeptide GPC3P is GGGRDNRLNVGGTYFLTTRQ; the linking group is lysine; plasma albumin binding group R is 4- (p-methylphenyl) butyryl (MPB formation) or 4- (p-iodophenyl) butyryl (IPB formation), metal ion binding chelating group Q is 1,4,7,10-tetraazacyclododecane-N ', N "N '" -triacetoxy-N-acetyl (i.e., -DOTA) or 1,4, 7-triazacyclononalkyl-N ', N "-diacetoxy-N-acetyl (i.e., -NOTA) and its analog 2-S- (4-isothiocyanatobenzyl) -1,4, 7-triazacyclononane-1, 4, 7-triacetic acid (i.e., p-SCN-Bn-NOTA); radionuclides (M) ⁿ⁺ ) Is that ⁶⁸ Ga ³⁺ 、[Al ¹⁸ F] ²⁺ 、 ⁶⁴ Cu ²⁺ 、 ¹⁷⁷ Lu ³⁺ Or other radioactive metal ions. Introduction of 4- (p-methylphenyl) butyryl (MPB formation) or 4- (p-iodophenyl) butyryl (IPB formation) can optimize targeting of GPC3 polypeptide probes [ M ] ⁿ⁺ -Q]The pharmacokinetic properties of R-GPC3P can improve the aggregation degree of the targeted GPC3 polypeptide probe in tumor tissues and prolong the tumor residence time, thereby being beneficial to tumor diagnosis and treatment.

The invention relates to a target GPC3 polypeptide probe [ M ] ⁿ⁺ ]NOTA-R-GPC3P and [ M ] ⁿ⁺ ]DOTA-R-GPC3P also has the following advantages: (1) [ M ] ⁿ⁺ ]NOTA-R-GPC3P and [ M ] ⁿ⁺ ]DOTA-R-GPC3P exploits the plasmapheresis properties of tumor tissue by introducing a plasma albumin binding group (4- (P-methylphenyl) butyryl or 4- (P-iodophenyl) butyryl), thus targeting GPC3P polypeptide probes shows higher tumor uptake and longer tumor residence time. The probe can be taken up more highly after 30min after administration, so that early imaging of tumor is facilitated; in addition, the retention time of tumor tissues is longer, and high tumor uptake still exists in the tumor tissues 120min after administration. (2) [ M ] ⁿ⁺ ]NOTA-R-GPC3P and [ M ] ⁿ⁺ ]DOTA-R-GPC3P shows good pharmacokinetic properties in tumor-bearing mice, and the liver background is obviously reduced, which is beneficial to improving the tumor target/non-target ratio. (3) [ M ] ⁿ⁺ ]The higher tumor uptake and longer tumor retention of DOTA-R-GPC3P facilitates the use of longer half-life therapeutic nuclides ¹⁷⁷ Lu ³⁺ The mark is prepared into [ ¹⁷⁷ Lu]DOTA-R-GPC3P provides a new idea and direction for tumor peptide targeted radionuclide therapy targeting GPC3. (4) [ M ] ⁿ⁺ ]NOTA-R-GPC3P and [ M ] ⁿ⁺ ]DOTA-R-GPC3P may be derived from a plurality of positron species (e.g ⁶⁸ Ga ³⁺ 、[Al ¹⁸ F] ²⁺ 、 ⁶⁴ Cu ²⁺ Etc.) and the precursor, and separating and purifying by a small column; the preparation method is simple, has high yield and high purity of automatic synthesis, and is favorable for clinical popularization and application.

Description of the terms

Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying structural and chemical formulas. The invention is intended to cover all alternatives, modifications and equivalents, which may be included within the scope of the invention as defined by the appended claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described herein. In the event of one or more of the incorporated references, patents and similar materials differing from or contradictory to the present application (including but not limited to defined terms, term application, described techniques, etc.), the present application controls.

It should further be appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entirety.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

In the following, all numbers disclosed herein are approximate, whether or not the word "about" or "about" is used. The numerical value of each number may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15% or 20%. Whenever a number is disclosed having a value of N, any number having a value of N+/-1%, N+/-2%, N+/-3%, N+/-5%, N+/-7%, N+/-8%, N+/-10%, N+/-15% or N+/-20% is explicitly disclosed, where "+/-" means plus or minus.

The chelating group is part of a compound of the invention, wherein the chelating group is attached directly or indirectly (e.g., through a linker) to the compound of the invention. Preferred chelating groups are chelating agents that form metal chelates, preferably the metal is at least one radioactive metal. The at least one radiometal is preferably useful or adaptable for diagnostic and/or therapeutic and/or theranostic use, more preferably useful or adaptable for imaging and/or radiation therapy.

The term "chelator" or "chelate" is used interchangeably in the context of the present invention to refer to a molecule, typically an organic molecule, typically a lewis base, having two or more unshared electron pairs that can be provided to a metal ion. The metal ion is typically coordinated to the chelator by two or more electron pairs. The terms "bidentate chelator", "tridentate chelator" and "tetradentate chelator" refer to chelators having two, three and four electron pairs, respectively, that are readily available for simultaneous supply of metal ions coordinated by the chelator. Typically, the electron pair of the chelator forms a coordination bond with a single metal ion. However, in some examples, the chelating agent may form coordinate bonds with more than one metal ion, and a variety of binding means are possible.

Those skilled in the art will further appreciate that the presence of the chelating agent in the compounds of the invention, if not otherwise stated, includes the possibility of complexing the chelating agent with any metal complex partner (i.e., any metal that may be complexed by the chelating agent in theory). The explicit mention of chelating agents of the compounds of the invention or the generic term chelating agents in connection with the compounds of the invention refers to the uncomplexed chelating agent itself or to the chelating agent binding any metal complex partner, wherein the metal complex partner is any radioactive or non-radioactive metal complex partner. Preferably the chelator metal complex, i.e. the chelator to which the metal complex partner binds, is a stable chelator metal complex.

"plasma albumin binding" refers to a structure that is capable of stable non-covalent binding to human or mouse serum albumin. For example, it may be IPB or MPB-forming group, 4- (p-iodophenyl) butanoyl or 4- (p-methylphenyl) butanoyl.

Drawings

FIG. 1 is [ ¹⁸ F]HPLC analysis pattern of AlF-NOTA-IPB-GPC3P injection.

FIG. 2 is [ ¹⁸ F]HPLC analysis patterns of AlF-NOTA-IPB-GPC3P injection stability in vivo serum 1h, in vitro serum 2h and in vitro PBS buffer 2 h.

FIG. 3 is [ ¹⁸ F]Cell uptake patterns of AlF-NOTA-IPB-GPC3P injection in Huh7 hepatoma cells with high expression of GPC3 and PC3 prostate cancer cells with low expression of GPC3 for 30min, 60min, 90min and 120 min.

FIG. 4 is [ ¹⁸ F]Graph of uptake and inhibition of AlF-NOTA-IPB-GPC3P injection in GPC3 highly expressed Huh7 hepatoma cells for 60 min.

FIG. 5 is [ ¹⁸ F]Biological distribution map of AlF-NOTA-IPB-GPC3P injection in Huh7 liver cancer model, huh7 liver cancer inhibition model and PC3 prostate cancer model for 60 min.

FIG. 6 is [ ¹⁸ F]PET/CT images of animals with AlF-NOTA-IPB-GPC3P injection at different time points (30 min, 60min, 90min and 120 min) in Huh7 liver cancer model (upper row) and PC3 prostate cancer model (lower row).

FIG. 7 is [ ¹⁸ F]AlF-NOTA-IPB-GPC3P injection in Huh7 liver cancer model and PC3 prostate cancer modelChange profile of tumor tissue uptake values (% ID values) over time.

FIG. 8 is [ ¹⁸ F]AlF-NOTA-IPB-GPC3P injection was used in a small animal PET/CT image of Huh7 liver cancer model (left image) and Huh7 liver cancer inhibition model (right image) for 60 min.

FIG. 9 is [ ⁶⁸ Ga]HPLC analysis pattern of DOTA-MPB-GPC3P injection.

FIG. 10 is [ ⁶⁸ Ga]HPLC analysis patterns of DOTA-MPB-GPC3P injection stability in vivo serum for 1h, in vitro serum for 2h, and in vitro PBS buffer for 2 h.

FIG. 11 is [ ⁶⁸ Ga]Cell uptake patterns of Huh7 hepatoma cells with high expression of GPC3 and PC3 prostate cancer cells with low expression of GPC3 in DOTA-MPB-GPC3P injection for 30min, 60min and 120 min.

FIG. 12 is [ ⁶⁸ Ga]PET/CT images of small animals of DOTA-MPB-GPC3P injection at different time points (30 min, 60min, 90min and 120 min) in Huh7 liver cancer model.

FIG. 13 is an immunohistochemical view of Huh7 liver cancer tissue and PC3 prostate cancer tissue.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications.

The reagents used in the present invention are all commercially available or can be prepared by the methods described herein.

The precursor NOTA-R-GPC3P may be prepared by solid-phase polypeptide synthesis. GPC3P is modified to form GPC3P-Lys, plasma albumin base (4- (P-iodophenyl) butyric acid or 4- (P-methylphenyl) butyric acid) and metal chelating group (-NOTA or analogue P-SCN-Bn-NOTA) are further modified, and product peaks are collected by separation and purification through preparative HPLC, so that a purified precursor raw material NOTA-R-GPC3P can be obtained.

Preparing a precursor DOTA-R-GPC3P, wherein GPC3P is modified to form GPC3P-Lys, and the GPC3P is respectively reacted with a conjugated plasma albumin group (4- (P-iodophenyl) butyric acid or 4- (P-methylphenyl) butyric acid) and a conjugated metal chelating group (-DOTA), and the precursor DOTA-R-GPC3P is obtained by separating and purifying the purified precursor raw material through preparative HPLC.

The invention takes NOTA-R-GPC3P or DOTA-R-GPC3P as precursor raw materials, respectively and ⁶⁸ Ga ³⁺ 、[Al ¹⁸ F] ²⁺ 、 ⁶⁴ Cu ² ⁺ 、 ¹⁷⁷ Lu ³⁺ or other radioactive metal ions, and adjusting the pH of the reaction solution to a proper value to finish ⁶⁸ Ga ³⁺ 、[Al ¹⁸ F] ²⁺ 、 ⁶⁴ Cu ²⁺ 、 ¹⁷⁷ Lu ³⁺ Or chelating other radioactive metal ions, separating and purifying by small column to obtain [ M ] ⁿ⁺ -Q]-R-GPC3P polypeptide probe.

The invention relates to [ M ] ⁿ⁺ -Q]Preparation of the R-GPC3P polypeptide probe precursors NOTA-R-GPC3P and DOTA-R-GPC3P. GPC3P is modified to form GPC3P-Lys, plasma albumin base (4- (P-iodophenyl) butyric acid or 4- (P-methylphenyl) butyric acid) and metal chelating group (-NOTA or analogue thereof, P-SCN-Bn-NOTA, -DOTA) are further modified, product peaks are collected through separation and purification by preparative HPLC, and initial products NOTA-R-GPC3P and DOTA-R-GPC3P are obtained after freeze drying. The chemical yields of NOTA-R-GPC3P and DOTA-R-GPC3P are relatively high, and the purity is greater than 95%. The implementation of the invention solves the preparation problems of precursor NOTA-R-GPC3P and DOTA-R-GPC3P, and further solves the one-step automatic synthesis [ M ] ⁿ⁺ -Q]The R-GPC3P polypeptide probe lays a foundation.

The present study also relates to [ M ⁿ⁺ ]And (3) amplifying and synthesizing a NOTA-R-GPC3P polypeptide probe. With NOTA-R-GPC3P as precursor under weakly acidic (pH 3.5-4.5) and 85-110deg.C conditions, preferably at pH4.0 and 100deg.C ⁶⁸ GaCl ₃ After chelation reaction, separating and purifying with HLB column or SEP-PAK C18 column to obtain the final product ⁶⁸ Ga]NOTA-R-GPC3P injection (the reaction scheme is shown in the specification)Scheme 1). With NOTA-R-GPC3P as precursor, under weakly acidic (pH 3.6-4.4) and 90-105deg.C conditions, preferably at pH4.0 and 100deg.C, with [ Al ¹⁸ F] ²⁺ After chelation reaction, separating and purifying by HLB column or SEP-PAK C18 column to obtain the final product ¹⁸ F]AlF-NOTA-R-GPC3P injection (reaction scheme 2). With NOTA-R-GPC3P as precursor under weakly acidic (pH 4.0-5.6) and 80-105deg.C conditions, preferably at pH 4.5 and 85deg.C ⁶⁴ CuCl ₂ After chelation reaction, separating and purifying by HLB column or SEP-PAK C18 column to obtain the final product ⁶⁴ Cu]NOTA-R-GPC3P injection (equation is shown in scheme 3).

Synthetic route 1: [ ⁶⁸ Ga]Synthesis of NOTA-R-GPC 3P:

synthetic route 2: [ ¹⁸ F]Synthesis of AlF-NOTA-R-GPC 3P:

synthetic route 3: [ ⁶⁴ Cu]Synthesis of NOTA-R-GPC 3P:

the invention details the experimental results in vitro and in vivo.

[ ¹⁸ F]The radiochemical purity of AlF-NOTA-IPB-GPC3P is greater than 95% (FIG. 1); HPLC analysis maps of the probe in vitro and in vivo show a single main peak, obvious defluorination and decomposition imaging are not seen, and the radiochemical purity is more than 95 percent (figure 2); the above results suggest that the probe has excellent in vivo and in vitro stability. [ ¹⁸ F]AlF-NOTA-IPB-GPC3P showed high uptake in GPC 3-highly expressed Huh7 hepatoma cells and had a tendency to increase the uptake over time, and showed significant low uptake in Huh7 hepatoma cell inhibition test and in GPC 3-low expressed PC3 prostate cancer cellsFetch (fig. 3, fig. 4); the result suggests [ ¹⁸ F]AlF-NOTA-IPB-GPC3P may specifically bind GPC3.[ ¹⁸ F]The biodistribution of AlF-NOTA-IPB-GPC3P in the tumor-bearing murine model for 60min (FIG. 5) showed that the probe had higher uptake of radioactivity in the kidneys, suggesting that the probe was excreted primarily through the kidneys; the probe has low uptake in the liver, spleen, gastrointestinal tract and other abdominal organs, which suggests that the probe has excellent pharmacokinetic properties; furthermore, the higher radioactive uptake of the probe in the blood pool suggests that the probe blood pool clearly is slower, associated with the introduction of bound plasma albumin-based IPB; the probe shows high uptake in Huh7 liver cancer tumor tissues, the uptake of Huh7 tumor tissues is obviously reduced after the inhibition of imaging, and meanwhile, the uptake of PC3 tumor tissues is lower, so that the probe can specifically show GPC3 positive expression tumor tissues. [ ¹⁸ F]The PET/CT results of the small animals and the time-activity curve of the tumor tissues (shown in fig. 6, 7 and 8) of AlF-NOTA-IPB-GPC3P in the tumor-bearing murine model show that the Huh7 tumor tissues have higher uptake, the PC3 tumor tissues have almost no uptake, the uptake of the Huh7 tumor tissues in the inhibition imaging is obviously reduced, and the probe can be specifically targeted to GPC3 of the tumor tissues; furthermore, huh7 tumor tissue maintained relatively high uptake from 30min to 120min after injection. It can be seen that [ ¹⁸ F]AlF-NOTA-IPB-GPC3P has excellent pharmacokinetic specificity, has higher tumor uptake and long tumor retention time in tumors positively expressed by GPC3 such as liver cancer, and can be used for PET imaging of tumors positively expressed by GPC3.

The invention also relates to [ M ] ⁿ⁺ ]And (3) amplifying and synthesizing the DOTA-R-GPC3P polypeptide probe. DOTA-R-GPC3P is used as precursor and is reacted with the precursor under weak acidity (pH 3.5-4.5) and at 85-110deg.C, preferably at pH4.0 and 100deg.C ⁶⁸ GaCl ₃ After chelation reaction, separating and purifying with HLB column or SEP-PAK C18 column to obtain the final product ⁶⁸ Ga]DOTA-R-GPC3P injection (equation is shown in scheme 4). DOTA-R-GPC3P is used as precursor and is reacted with the precursor under weak acidity (pH 4.0-5.6) and at 80-105deg.C, preferably at pH 4.5 and 85deg.C ⁶⁴ CuCl ₂ After chelation reaction, separating and purifying by HLB column or SEP-PAK C18 column to obtain the final product ⁶⁸ Cu]DOTA-R-GPC3P injection (reaction scheme 5). DOTA-R-GPC3P is used as precursor and is reacted with the precursor under weak acidity (pH 5.2-5.7) and at 90-100deg.C, preferably at pH 5.5 and 95deg.C ¹⁷⁷ After the chelation reaction of Lu ion hydrochloric acid solution, separating and purifying by HLB small column or SEP-PAK C18 small column to obtain the final product ¹⁷⁷ Lu]DOTA-R-GPC3P injection (equation is shown in scheme 6).

Synthetic route 4: [ ⁶⁸ Ga]Synthesis of DOTA-R-GPC 3P:

synthetic route 5: [ ⁶⁴ Cu]Synthesis of DOTA-R-GPC 3P:

synthetic route 6: [ ¹⁷⁷ Lu]Synthesis of DOTA-R-GPC 3P:

the invention details the experimental results in vivo and in vitro:

[ ⁶⁸ Ga]the degree of amplification purity of DOTA-MPB-GPC3P was greater than 95% (FIG. 9); HPLC analysis maps of the probe in vitro and in vivo show a single main peak, obvious defluorination and decomposition imaging are not seen, and the radiochemical purity is more than 95 percent (figure 10); the result suggests [ ⁶⁸ Ga]DOTA-MPB-GPC3P has excellent in vivo and in vitro stability. [ ⁶⁸ Ga]DOTA-MPB-GPC3P showed high uptake in Huh7 liver cancer cells highly expressed in GPC3 and had a tendency to increase the degree of uptake with time, and showed low uptake in PC3 prostate cancer cells low in GPC3 (FIG. 11); the above results suggest that the probe shows specific uptake in tumor cells positively expressed by GPC3.[ ⁶⁸ Ga]The PET/CT results (fig. 12) of DOTA-MPB-GPC3P in the tumor-bearing murine model showed that Huh7 tumor tissue had higher uptake; furthermore, huh7 tumor tissue from 30min to 120min after injectionRelatively high uptake of imaging agent is maintained. It can be seen that [ ⁶⁸ Ga]DOTA-MPB-GPC3P has excellent pharmacokinetic specificity, has higher tumor uptake and long tumor retention time in tumors positively expressed by GPC3 such as liver cancer, and can be used for PET imaging of tumors positively expressed by GPC3.

EXAMPLE 1 preparation of precursors NOTA-R-GPC3P and DOTA-R-GPC3P

The precursors NOTA-R-GPC3P and DOTA-R-GPC3P were prepared by the following methods: GPC3P is modified to form GPC3P-Lys, plasma albumin base (4- (P-iodophenyl) butyric acid or 4- (P-methylphenyl) butyric acid) and metal chelating group (-NOTA or analogue thereof, P-SCN-Bn-NOTA, -DOTA) are further modified, product peaks are collected through separation and purification by preparative HPLC, and initial products NOTA-R-GPC3P and DOTA-R-GPC3P are obtained after freeze drying. The chemical yields of NOTA-R-GPC3P and DOTA-R-GPC3P are relatively high, and the purities are both greater than 95%. Determination of the retention time t of NOTA-IPB-GPC3P by Mass Spectrometry MS (m/z) _R =10.19 min, molecular weight (mr.) 2867.95; determination of the retention time t of DOTA-MPB-GPC3P by Mass Spectrometry MS (m/z) _R = 9.926min, molecular weight (mr.) 2857.14.

Example 2[ ¹⁸ F]Synthesis of AlF-NOTA-R-GPC3P

In a reaction flask containing NOTA-R-GPC3P (50. Mu.g/. Mu.L, 50. Mu.L), 2mM MAlCl was added in sequence ₃ 6. Mu.L of the solution, 5. Mu.L of glacial acetic acid and 300. Mu.L of acetonitrile are mixed. By cyclotrons ¹⁸ O(p,n) ¹⁸ Produced by F nuclear reaction ¹⁸ F ^- At N ₂ Under the carrier, the mixture is trapped in a Sep-Pak QMA anion small column, ¹⁸ the O-water was collected in a recovery bottle. QMA was packed in a column with 0.3 to 0.4mL of physiological saline ¹⁸ F ^- Eluting into small bottle, and adding 50 μl into the reaction bottle. Stirring and mixing evenly, and then heating and reacting for 10-15 min at 100 ℃. Cooling, adding 6-8 mL of water into a reaction bottle, uniformly mixing, and transferring into an HLB small column or an SEP-PAK C18 small column. After the transfer of the solution in the reaction flask was completed, the column was rinsed with 10ml×3 water for injection and dried. Finally, 1.5mL of ethanol is used for eluting the product, the product is collected in a receiving bottle after passing through a sterile filter membrane, and the product is diluted into a product solution containing 5 percent of ethanol by normal saline, thus obtaining the product solution meeting the requirements[ of ] ¹⁸ F]AlF-NOTA-R-GPC3P injection. [ ¹⁸ F]The uncorrected radiochemical yield of AlF-NOTA-R-GPC3P was 10-20% with a total radiosynthesis time of about 35min.

Example 3[ ⁶⁸ Ga]NOTA-R-GPC3P and [ [ ⁶⁴ Cu]Synthesis of NOTA-R-GPC3P

To a reaction tube containing precursor NOTA-R-GPC3P (50. Mu.g/. Mu.L, 50. Mu.L) was added 200. Mu.L of 1.25M sodium acetate solution. From the slave ⁶⁸ Ge/ ⁶⁸ Elution with 4mL of 0.05M hydrochloric acid in Ga generator ⁶⁸ GaCl ₃ Mixing the materials in the reaction tube, adjusting the pH of the solution to 4.0, and heating the solution at 100 ℃ for reaction for about 10-15 min. Cooling, adding 4mL of physiological saline into a reaction bottle, uniformly mixing, and transferring into an HLB column or an SEP-PAK C18 column. After the reaction flask had been completely transferred, the column was rinsed with 10ml×2 water for injection and dried. Then eluting the product with 1.5mL of ethanol, collecting the eluted product in a receiving bottle after passing through a sterile filter membrane, and diluting the eluted product into a product solution containing 5% ethanol by using physiological saline to obtain the product solution meeting the requirements ⁶⁸ Ga]NOTA-R-GPC3P injection. [ ⁶⁸ Ga]The uncorrected radiochemical yield of NOTA-R-GPC3P was 30-50% with a total radiosynthesis time of about 30min.

1000. Mu.L of a precursor-containing NOTA-R-GPC3P (50. Mu.g/. Mu.L) and were sequentially added to a reaction tube ⁶⁴ CuCl ₂ 0.100-1.000mL of solution, adjusting the pH to 4.0-5.6 by using sodium acetate solution, and reacting for 10-15 min at 85 ℃. Finally, the mixture is diluted by normal saline and filtered by a sterile filter membrane and then is collected in a receiving bottle, thus obtaining the product meeting the requirements ⁶⁴ Cu]NOTA-R-GPC3P injection. [ ⁶⁴ Cu]The uncorrected radiochemical yield of NOTA-R-GPC3P was 40-70%. The total radiosynthesis time was about 25min.

Example 4[ ⁶⁸ Ga]DOTA-R-GPC3P and [ [ ⁶⁴ Cu]Discharge synthesis of DOTA-R-GPC3P

DOTA-R-GPC3P (50. Mu.g/. Mu.L, 50. Mu.L) and 200. Mu.L of 1.25M sodium acetate solution were sequentially added to the reaction tube. From the slave ⁶⁸ Ge/ ⁶⁸ Elution with 4mL of 0.05M hydrochloric acid in Ga generator ⁶⁸ GaCl ₃ Mixing the materials in the reaction tube, adjusting the pH of the solution to 4.0, and heating at 100 ℃ for reaction for about 10 min. Cooling, adding 4mL of physiological saline into the reaction bottle, and mixingTransferred to HLB pillars. After the reaction flask had been completely transferred, the column was rinsed with 10ml×2 water for injection and dried. Then eluting the product with 1.5mL of ethanol, collecting the eluted product in a receiving bottle after passing through a sterile filter membrane, diluting the eluted product into a product solution containing 5% ethanol by using physiological saline to obtain the product solution meeting the requirements ⁶⁸ Ga]DOTA-R-GPC3P injection. [ ⁶⁸ Ga]The uncorrected radiochemical yield of DOTA-R-GPC3P was 40-55% with a total radiosynthesis time of about 30min.

DOTA-R-GPC3P (50. Mu.g/. Mu.L, 50. Mu.L) and DOTA-R-GPC3P were sequentially added to the reaction tube ⁶⁴ CuCl ₂ 0.100-1.0mL of solution, adjusting the pH to 4.0-5.6 by using sodium acetate solution, and reacting for 10-15 min at 85 ℃. Finally, the mixture is diluted by normal saline and filtered by a sterile filter membrane and then is collected in a receiving bottle, thus obtaining the product meeting the requirements ⁶⁴ Cu]DOTA-R-GPC3P injection. [ ⁶⁴ Cu]The uncorrected radiochemical yield of DOTA-R-GPC3P was 30-70%. The total radiosynthesis time was about 25min.

Example 5[ ¹⁷⁷ Lu]Discharge synthesis of DOTA-R-GPC3P

DOTA-R-GPC3P (50. Mu.g/. Mu.L, 50. Mu.L) and were added to the reaction tube ¹⁷⁷ Mixing Lu ion hydrochloric acid solution, adding NaOAc buffer solution to adjust pH to 5.2-5.7, reacting at 95deg.C for 15min, separating and purifying with HLB column or SEP-PAK C18 column to obtain the final product ¹⁷⁷ Lu]DOTA-R-GPC3P injection. [ ¹⁷⁷ Lu]The uncorrected radiochemical yield of DOTA-R-GPC3P can reach more than 90%, and the total radiosynthesis time is about 30min.

EXAMPLE 6 determination of the product radiochemical purity and stability

The radiochemical purity of the drug injection was determined by High Performance Liquid Chromatography (HPLC). HPLC analysis conditions: the analytical column was Zorbax eclipse xdb-c 18. Acetonitrile solution of 0.1% trifluoroacetic acid (TFA) in mobile phase: aqueous 0.1% tfa, gradient elution: at 0min, acetonitrile solution containing 0.1% tfa/aqueous solution of 0.1% tfa: 10/90 (v/v); gradually rise to 10min, 0.1% TFA in acetonitrile/0.1% TFA in water: 80/20 (v/v). The flow rate was 1mL/min, and the UV detection wavelengths were 210nm and 254nm. By non-radioactive standard of defined structure ¹⁹ F]AlF-NOTA-R-GPC3P、[Ga ³⁺ ]NOTA-R-GPC3P、[Cu ²⁺ ]NOTA-R-GPC3P、[Ga ³⁺ ]DOTA-R-GPC3P、[Cu ²⁺ ]DOTA-R-GPC3P and [ Lu ] ³⁺ ]DOTA-R-GPC3P, respectively associated with the corresponding radioactive probes [ ¹⁸ F]AlF-NOTA-R-GPC3P、[ ⁶⁸ Ga]NOTA-R-GPC3P、[ ⁶⁴ Cu]NOTA-R-GPC3P、[ ⁶⁸ Ga]DOTA-R-GPC3P、[ ⁶⁴ Cu]DOTA-R-GPC3P and [ [ ¹⁷⁷ Lu]DOTA-R-GPC3P injections were co-injected into HPLC to determine whether the retention times (Rt) were consistent and confirm the authenticity of the prepared probes. The radiochemical purity of the compounds is more than 95% as measured by an HPLC method. Representative [ ¹⁸ F]AlF-NOTA-IPB-GPC3P injection and [ ⁶⁸ Ga]The results of the radioactive HPLC analysis of DOTA-MPB-GPC3P injection are shown in FIG. 1 and FIG. 9, respectively, with radiochemical purity greater than 95%.

HPLC detection [ M ] ⁿ⁺ ]NOTA-R-GPC3P and [ M ] ⁿ⁺ ]In vivo and in vitro stability of DOTA-R-GPC3P. A representative example is detection by HPLC ¹⁸ F]AlF-NOTA-IPB-GPC3P injection had stability in vivo serum for 1 hr, in vitro serum for 2 hr and in vitro PBS buffer for 2 hr (FIG. 2) and [ ⁶⁸ Ga]DOTA-MPB-GPC3P injection was stable in vivo serum for 1 hour, in vitro serum for 2 hours, and in vitro PBS buffer for 2 hours (FIG. 10). In vivo serum 60min after injection [ ¹⁸ F]AlF-NOTA-IPB-GPC3P and [ ⁶⁸ Ga]DOTA-MPB-GPC3P showed only a single main peak without significant defluorination and decomposition, suggesting good in vivo stability. Incubation in PBS buffer for 2 hours and in serum for 2 hours [ ¹⁸ F]AlF-NOTA-IPB-GPC3P and [ ⁶⁸ Ga]The radiochemical purity of DOTA-MPB-GPC3P is more than 95%, and obvious defluorination and decomposition phenomena do not occur, which indicates that the in vitro stability is good.

Example 7 in vitro cell uptake and inhibition experiments and competitive binding experiments

Tumor cell lines Huh7 cells (human liver cancer cell lines) and PC3 cells (human prostate cancer cells) of Shanghai cell banks of China academy of sciences are routinely cultured. Taking cancer cells in logarithmic growth phase, digesting the cancer cells with 0.25% pancreatin, and washing the cells twice with PBS; centrifuging at 1000 rpm for 5min, collecting cells, and regulating cell density to about 6×10 ⁶ And each mL. Add 0 to each well in 24-well plate1mL of cell suspension is cultured for 24h, and fresh culture solution is replaced for cell experiments after the cells are attached. Cells useful in cell experiments, five random A, B, C, D, E groups, were added [ ¹⁸ F]Culturing AlF-NOTA-IPB-GPC3P, washing with PBS for 3 times after 30min, 60min, 90min and 120min respectively, adding inhibitor simultaneously into the inhibition group, washing with PBS for 3 times after 60min, adding sodium dodecyl sulfate to make cells shed, collecting cells of each hole, measuring cell count, and taking in labeled probe in cytoplasm after cell count. Cells useful in cell experiments, in random A, B, C groups, were added [ ⁶⁸ Ga]Continuing culturing DOTA-MPB-GPC3P, respectively washing with PBS for 3 times after 30min, 60min and 120min, adding sodium dodecyl sulfate to make cells fall off, collecting cells of each hole, measuring cell count, and taking in labeled probe in cytoplasm.

Cell uptake and inhibition assay results: [ ¹⁸ F]AlF-NOTA-IPB-GPC3P showed that the high uptake of Huh7 hepatoma cells highly expressed by GPC3 reached 10.77.+ -. 1.39% ID/million cells at 30min, and also remained at 15.13.+ -. 2.29% ID/million cells at 120min, and showed lower uptake values in both PC3 prostate and Huh7 cell inhibition groups, which were low-expressed by GPC3. The above results indicate that [ [ ¹⁸ F]AlF-NOTA-IPB-GPC3P showed a specific high uptake in Huh7 hepatoma cells in which GPC3 was highly expressed and had a tendency to increase the uptake over time (FIGS. 3 and 4).

Cell uptake and inhibition assay results: [ ⁶⁸ Ga]DOTA-MPB-GPC3P showed that Huh7 liver cancer cells highly expressed in GPC3 had high uptake of 4.51.+ -. 0.20% ID/million cells at 30min and also maintained at 4.39.+ -. 0.19% ID/million cells at 120min, and showed lower uptake in PC3 prostate with low expression of GPC3. The above results indicate that [ [ ⁶⁸ Ga]DOTA-MPB-GPC3P had a relatively high specific uptake in Huh7 cells and had a tendency to increase the extent of uptake over time (FIG. 11).

Example 8 in vivo biodistribution experiment

The biodistribution experimental method of the tumor-bearing mouse animal model is as follows, each group of tumor-bearing animal models is provided with tumor3 mice are selected from animal models (Huh 7 liver cancer model, huh7 liver cancer inhibition model, PC3 prostate cancer model), and each tumor-bearing mouse is injected with 0.1-0.2mL of the drug containing 30-50 μCi via tail vein ¹⁸ F]AlF-NOTA-IPB-GPC3P injection, mice were sacrificed 60min after removing blood from the eyeballs after injection. Dissecting and taking tissue samples of tumor tissues and organs of interest (blood, brain, lung, liver, gall bladder, kidney, spleen, stomach, small intestine, muscle, femur and other tissues), weighing, measuring the radioactivity counts of the tissue samples, and calculating the percentage of radioactive injection dose (% ID/g) of each gram of tissue at different time points.

Biodistribution results: [ ¹⁸ F]The biological distribution of AlF-NOTA-IPB-GPC3P 60min after injection of Huh7 liver cancer model, huh7 liver cancer inhibition model and PC3 prostate cancer model (figure 5) shows that the radioactive uptake of the probe in the kidney is higher, which indicates that the probe is mainly excreted through the kidney; the probe has low uptake in the liver, spleen, gastrointestinal tract and other abdominal organs, which suggests that the probe has excellent pharmacokinetic properties; furthermore, the higher radioactive uptake of the probe in the blood pool suggests that the probe blood pool clearly is slower, associated with the introduction of bound plasma albumin-based IPB; the probe shows high uptake in Huh7 liver cancer tumor tissues, the uptake of Huh7 tumor tissues is obviously reduced after the inhibition of imaging, and meanwhile, the uptake of PC3 tumor tissues is lower, so that the probe can specifically show GPC3 positive expression tumor tissues.

Example 9 PET/CT imaging study of Small animals in tumor-bearing murine model animals

Micro-PET/CT imaging studies utilize Siemens Inveon Micro-PET/CT (resolution approximately 1.4mm, aperture 12cm, axial field 12.7 cm), acquisition workstation InveonAcquirision workplace (IRW) 2.0, new Workflow (including CT acquisition, reconstruction, PET acquisition, PET histogram, PET Recon.) was established prior to data acquisition, and nude mice animals were tested as underarm transplants of Huh7 liver cancer cell line and PC3 prostate cancer cell line, etc., to make an underarm tumor model of nude mice. The PET/CT scan was performed after anesthesia with 10% chloral hydrate. Collecting developer [ ¹⁸ F]Dynamic PET/CT images 120min after injection of AlF-NOTA-IPB-GPC3P 200-300 μCi (Huh 7 liver cancer model, PC3 prostate cancer model); simultaneously carrying out 60min Huh7 tumor-bearing miceStatic imaging and inhibition imaging of (a). Collecting developer ⁶⁸ Ga]PET/CT images of small animals at various time points (30 min, 60min, 90min and 120 min) of Huh7 liver cancer model after 200-300 μCi of DOTA-MPB-GPC3P injection.

PET/CT imaging results show that: [ ¹⁸ F]AlF-NOTA-IPB-GPC3P had higher tumor uptake in Huh7 hepatoma tumors (upper row of FIG. 6), and immunohistochemical results confirmed that Huh7 hepatoma tissues expressed GPC3 positively (FIG. 13); [ ¹⁸ F]AlF-NOTA-IPB-GPC3P was hardly taken in PC3 prostate cancer tumor tissue (lower row of FIG. 6), and the immunohistochemical results showed that PC3 prostate cancer tissue low-expressed GPC3 (FIG. 13). FIG. 8 shows that inhibition of Huh7 tumor tissue uptake was significantly reduced in imaging suggesting [ [ ¹⁸ F]AlF-NOTA-IPB-GPC3P can specifically target GPC3 of tumor tissues. Tumor tissue in Huh7 liver cancer model [ ¹⁸ F]The profile of AlF-NOTA-IPB-GPC3P uptake (%ID) over time (FIG. 7) shows that Huh7 tumor tissue has a higher imaging agent uptake (4.05.+ -. 0.27% ID/g at 30 min) and a relatively high tumor uptake (5.05.+ -. 0.23% ID/g) is maintained for 120min [ it can be seen that ¹⁸ F]AlF-NOTA-IPB-GPC3 probes showed higher tumor uptake and longer tumor residence time.

PET/CT imaging results show that: [ ⁶⁸ Ga]DOTA-MPB-GPC3P showed high tumor uptake in Huh7 hepatoma tumor (FIG. 12), and relatively high tumor uptake (4.65% ID/g) was maintained up to 120min after administration [ see ] ⁶⁸ Ga]DOTA-MPB-GPC3P probes exhibit higher tumor uptake and longer tumor residence time.

[ ¹⁸ F]AlF-NOTA-IPB-GPC3P and [ ⁶⁸ Ga]DOTA-MPB-GPC3P has high tumor uptake and long tumor residence time in GPC3 positive expression tumor such as liver cancer, and can be used for PET imaging of GPC3 positive expression tumor. While further improvements in the pharmacokinetic properties may be required by structural optimisation thereof, modifications and alterations to the structure may be made by those skilled in the art in light of the foregoing description, and all such modifications and alterations are intended to be within the scope of the present invention as defined in the appended claims.

Reference to the literature

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While the methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and combinations of the methods and applications described herein can be made and applied within the spirit and scope of the invention. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included within the present invention.

Claims

1. A GPC 3-targeting polypeptide probe precursor, wherein the polypeptide probe precursor has a structure according to formula I:

wherein,

q is a chelating group;

r is 4- (p-methylphenyl) butanoyl or 4- (p-iodophenyl) butanoyl;

w is the pharmacophore polypeptide GPC3P.

2. The polypeptide probe precursor according to claim 1, wherein the chelating group is selected from the group consisting of: groups formed by 1,4,7, 10-tetraazacyclododecane-N ', N' -triacetoxy-N-acetyl (DOTA), 1,4,7, 10-tetraazacyclododecane, 1- (glutaric acid) -4,7, 10-triacetoxy (dotga), 1,4, 7-triazacyclononaalkyl-N ', N' -diacetoxy-N-acetyl (NOTA), 2S- (4-isothiocyanatobenzyl) -1,4, 7-triazacyclononane-1, 4, 7-triacetoxy (p-SCN-Bn-NOTA), 1,4, 7-triazacyclononane-N-glutarate-N ', N' -diacetic acid (nodagga) or 1,4, 7-triazacyclononane-1, 4-diacetic acid-methylphenylacetic acid (NODA-MPAA) chelating agents; preferably, the chelating group is selected from:

or p-SCN-Bn-NOTA.

3. The polypeptide probe precursor according to claim 1,

w is:

4. a targeted GPC3 polypeptide probe having a structure represented by formula II,

wherein Q, W, R has the definition of any one of claims 1 to 3;

M ⁿ⁺ is a radioactive ion.

5. The polypeptide probe of claim 5, wherein the probe comprises a probe fragment,

the radioactive ions are selected from ¹⁸ F、 ⁵¹ Cr、 ⁶⁷ Ga、 ⁶⁸ Ga、 ¹¹¹ In、 ^99m Tc、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ¹³⁹ La、 ¹⁴⁰ La、 ¹⁷⁵ Yb、 ¹⁵³ Sm、 ¹⁶⁶ Ho、 ⁸⁸ Y、 ⁹⁰ Y、 ¹⁴⁹ Pm、 ¹⁶⁵ Dy、 ¹⁶⁹ Er、 ¹⁷⁷ Lu、 ⁴⁷ Sc、 ¹⁴² Pr、 ¹⁵⁹ Gd、 ²¹² Bi、 ²¹³ Bi、 ⁷² As、 ⁷² Se、 ⁹⁷ Ru、 ¹⁰⁹ Pd、 ¹⁰⁵ Rh、 ^101m Rh、 ¹¹⁹ Sb、 ¹²⁸ Ba、 ¹²³ I、 ¹²⁴ I、 ¹³¹ I、 ¹⁹⁷ Hg、 ²¹¹ At、 ¹⁵¹ Eu、 ¹⁵³ Eu、 ¹⁶⁹ Eu、 ²⁰¹ Tl、 ²⁰³ Pb、 ²¹² Pb、 ⁶⁴ Cu、 ⁶⁷ Cu、 ¹⁸⁸ Re、 ¹⁸⁶ Re、 ¹⁹⁸ Au、 ²²⁵ Ac、 ²²⁷ Th and ¹⁹⁹ ag forms ground ions; preferably, the radioactive element is selected from ¹⁸ F、 ⁶⁸ Ga、 ¹⁷⁷ Lu, and ⁶⁴ ions formed by Cu; preferably, the radioactive ions are selected from ⁶⁸ Ga ³⁺ 、[Al ¹⁸ F] ²⁺ 、 ⁶⁴ Cu ²⁺ And ¹⁷⁷ Lu ³⁺ 。

6. a preparation method of a target GPC3 polypeptide probe precursor, wherein, GPC3P-Lys is formed after target GPC3 polypeptide is modified by Lys, and the target GPC3 polypeptide probe precursor is obtained by respectively reacting with a chelating agent and a combined plasma albumin group, wherein the combined plasma albumin group is 4- (P-iodophenyl) butyric acid or 4- (P-methylphenyl) butyric acid; wherein the targeted GPC3 polypeptide probe precursor has the structure of any of claims 1 to 3; the chelating agent as claimed in claim 2.

7. The preparation method of the target GPC3 polypeptide probe comprises the steps of heating and reacting a target GPC3 polypeptide probe precursor and radioactive ion salt in an acidic solvent to obtain the target GPC3 polypeptide probe; wherein the radioactive ion salt is a salt of a radioactive ion according to claim 5; preferably, the radioactive ion salt is selected from ⁶⁸ GaCl ₃ 、[Al ¹⁸ F] ²⁺ Salt(s), ⁶⁴ CuCl ₂ 、 ¹⁷⁷ Lu ionAnd (3) salt.

8. A kit comprising the polypeptide probe of any one of claims 4 to 5.

9. A tumor PET or SPECT imaging agent comprising the targeted GPC3 polypeptide probe of any of claims 4-5.

10. The use of a target GPC3 polypeptide probe according to any one of claims 4 to 5 in the preparation of a tumor PET imaging agent, a tumor SPECT imaging agent, or for evaluation of tumor efficacy, or in the preparation of a tumor peptide-targeted radionuclide therapy probe; preferably, the tumor comprises a tumor that is positively expressed by GPC3 (such as liver cancer, etc.).