Radiolabeled collagen hybridizing peptide and preparation method therefor and use thereof

Abstract

Disclosed in the present invention are a radiolabeled collagen hybridizing peptide and a preparation method therefor and the use thereof. The radiolabeled collagen hybridizing peptide of the present invention contains a radionuclide-chelator complex bound via a coordination bond, and a collagen hybridizing peptide. The radiolabeled collagen hybridizing peptide of the present invention has good stability, and an excellent definition and signal-to-noise ratio, can not only sensitively detect precancerous lesions of pancreatic ductal adenocarcinoma (e.g., pancreatic intraepithelial neoplasia), but can also reduce, after optimization, the uptake in non-target organs such as the liver and kidneys, thereby having good clinical application prospects.

Description

A radiolabeled collagen hybrid peptide and its preparation method and application

[0001] Cross-reference to related applications:

[0002] This application claims priority to Chinese patent application No. 2024102196335 filed on February 28, 2024, and priority to Chinese patent application No. 202510438663X filed on April 9, 2025, both of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biomedical technology, and in particular to a radioactively labeled collagen hybrid peptide and a preparation method and application thereof. Background Art

[0004] Fibrosis is a common pathological feature in the development of many chronic diseases. Its essence is the excessive deposition and abnormal distribution of the extracellular matrix (ECM) during the repair process after tissue damage. Studies have shown that not only is chronic fibrosis a risk factor for cancer, but the fibrotic matrix itself is also an important feature of malignant tumors such as pancreatic ductal adenocarcinoma (PDAC), breast cancer, prostate cancer, and colorectal cancer. Excessive proliferation of fibrotic matrix can even account for more than 90% of the tumor mass. Because the fibrotic matrix creates a dense barrier at the tumor site, it seriously hinders the penetration of drugs to kill tumor cells and also limits the detection of conventional molecular probes targeting tumor cells. Based on this, in order to detect lesions of fibrotic diseases earlier and achieve more accurate early diagnosis, the development of sensitive, stable, and low-background signal molecular probes has become an inevitable trend.

[0005] Radionuclide molecular probes are a key tool for early diagnosis. Currently, a growing number of clinical studies using radionuclide molecular probes are shifting their detection targets from tumor cells to the surrounding ECM and other disease microenvironments, transforming dense barriers that hinder probe penetration into opportunities for target binding. For example, Fibroblast Activation Protein Inhibitor (FAPI), which targets tumor-associated fibroblasts, has been extensively studied clinically. Various radionuclide-labeled FAPI probes have demonstrated excellent uptake and imaging in various tumors and are being extensively studied clinically in conditions such as pulmonary fibrosis and myocardial fibrosis. As the most abundant protein in the fibrotic ECM, collagen is a particularly attractive target. The basic sequence unit of the collagen molecule is Gly-Xaa-Yaa, where glycine (Gly, O) is the common starting amino acid in the cycle, while the Xaa and Yaa positions are typically proline (Pro, P) and hydroxyproline (Hyp, O), respectively. Three left-handed helical polyproline II (PPII) polypeptide chains, formed by periodic repetition of basic sequence units, are tightly wound together to form a stable right-handed triple helical structure under the action of interchain hydrogen bonds. In fibrotic lesions, due to the significant increase in the synthesis and metabolism of collagen in the tissue, under the degradation of abnormally elevated collagenases (such as matrix metalloproteinases, Matrix Metalloproteinase (MMP)), collagen undergoes triple helical conformation unfolding, leaving a large number of conformationally denatured collagen chains in pathological tissues. Based on the specific triple helical structure of collagen, the designed collagen hybridizing peptide (CHP) is composed of (GPO) n Or optimized modification probe (GfO) n CHP has a structure with a tendency to form triple helices. It can form triple helices by hybridizing with incomplete, degraded collagen peptide chains that have unwound, thereby marking damaged and denatured collagen in pathologically damaged tissues. At the same time, it will not interact with intact collagen or other proteins without triple helical structures. CHP is not only highly specific but also stable in vivo. It can be used for targeted imaging of fibrotic tissue and provide information on the location of lesions. However, in actual applications, it has been found that the currently used collagen hybrid peptide molecular probes still have problems such as poor labeling stability, low signal-to-noise ratio, and unsatisfactory biodistribution based on liver and kidney metabolism.

[0006] Based on this, there is an urgent need to develop a radiolabeled collagen hybrid peptide with good labeling stability and high signal-to-noise ratio to improve the early diagnosis of fibrosis-related diseases. Summary of the Invention

[0007] The first aspect of the present invention aims to provide a radiolabeled collagen hybrid peptide.

[0008] The second aspect of the present invention is to provide a method for preparing a radiolabeled collagen hybrid peptide.

[0009] The third aspect of the present invention is to provide a conjugate.

[0010] The fourth aspect of the present invention aims to provide a contrast agent.

[0011] The purpose of the fifth aspect of the present invention is to provide a radiolabeled collagen hybrid peptide for use in preparing products that specifically target structurally denatured collagen, preparing products for cancer diagnosis or lesion imaging characterized by fibrotic lesions, preparing products for detecting organ fibrotic diseases, and evaluating or screening anti-pancreatic ductal adenocarcinoma drugs or anti-fibrosis drugs.

[0012] The sixth aspect of the present invention aims to provide a kit for imaging tumor or organ fibrosis.

[0013] The seventh aspect of the present invention aims to provide a method for treating or diagnosing diseases related to organ fibrosis.

[0014] In order to achieve the above object, the technical solution adopted by the present invention is:

[0015] The first aspect of the present invention provides a radiolabeled collagen hybrid peptide comprising:

[0016] (a) a radionuclide-chelator complex bound by a coordinate bond; and

[0017] (b) collagen hybrid peptide;

[0018] wherein the radionuclide-chelator complex is connected to the collagen hybrid peptide via a linker molecule, and the linker molecule comprises at least one of a lysine residue, an oligoglycine residue, 6-aminohexanoic acid, and polyethylene glycol;

[0019] The amino acid sequence of the collagen hybrid peptide is (GfO) n or (GPO) n , wherein n is a positive integer between 6 and 10.

[0020] It is understood that collagen hybrid peptide (GfO) n or (GPO) n In the figure, G is glycine, f is fluoroproline, O is hydroxyproline, and P is proline.

[0021] The collagen hybrid peptide designed based on the specific triple helix structure of collagen is composed of (GPO)n Or optimized modification probe (GfO) n CHP has a propensity for triple helix formation, hybridizing with incomplete, degraded, and unwound collagen peptide chains to form triple helices, thereby marking damaged and denatured collagen in pathologically damaged tissues. However, it does not interact with intact collagen or other proteins without triple helical structures. CHP is not only highly specific but also stable in vivo, making it suitable for detecting and imaging disease-induced collagen remodeling and mechanical damage-induced collagen destruction, providing information on the location of collagen damage.

[0022] In some embodiments of the present invention, the amino acid sequence of the collagen hybrid peptide is (GfO) n Specifically, n is 8, 9 or 10.

[0023] (GfO) n and (GPO) n Both are collagen hybrid peptides, binding to denatured collagen via triple-helix hybridization. However, GPO itself is prone to trimerization, and in actual use, it must be heated to untwist it before hybridization with denatured collagen. GfO, on the other hand, cannot trimerize on its own, so it does not require heating and can be used directly.

[0024] In some embodiments of the present invention, the radionuclide in the radionuclide-chelator complex is selected from the group consisting of Al 18 F. 64 Cu, 67 Cu, 67 Ga, 68 Ga, 99 mTc, 89 Zr, 111 In, 177 Lu, 186 Re、 225 Any of Ac.

[0025] In some embodiments of the present invention, the radionuclide is selected from 68 Ga, 64 Cu and Al 18 Any of F.

[0026] In some more preferred embodiments of the present invention, the radionuclide is Al 18 F.

[0027] The present invention found that when the radionuclide is Al 18When F is used, the prepared probe can better realize the visualization and early diagnosis of PDAC in vivo. In the early diagnosis of PDAC, the detection of precancerous lesions, pancreatic intraepithelial neoplasia (PanIN), is the main goal. However, the lesion site of PanIN is extremely small, the target is relatively small, and the pancreas is very close to metabolic organs such as the liver, spleen, intestines, and kidneys in the abdomen. 68 Ga and 64 The probes made of radionuclides such as Cu, due to their inappropriate biodistribution, can easily cause the signals of metabolic organs such as the liver, spleen, intestines, and kidneys in the abdomen to block the signals of precancerous lesions, thereby affecting the detection results. However, the present invention has found that the use of radionuclides such as Al 18 When the probe was prepared by F, the biodistribution effect was the best and the signal-to-noise ratio was excellent, which could highlight the weak signals of the lesions.

[0028] In some embodiments of the present invention, the chelator in the radionuclide-chelator complex is selected from any one of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA), hydrazinonicotinamide (HYNIC), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and diethylenetriaminepentaacetic acid (DTPA), or a derivative thereof.

[0029] In some embodiments of the present invention, the derivative of NOTA includes NOTA-NH2 (abbreviated as NOTA2) or p-SCN-Bn-NOTA; wherein the NOTA-NH2 has a structure as shown in formula (a-1):

[0030] The p-SCN-Bn-NOTA has a structure as shown in formula (a-2):

[0031] In some preferred embodiments of the present invention, when the radionuclide is68 Ga, the derivative of NOTA has a structure as shown in formula (a-1), and when the connecting molecule is 6-aminohexanoic acid, the radiolabeled collagen hybrid peptide has excellent biodistribution characteristics with high uptake in target organs such as tumors and low uptake in non-target organs such as the liver and kidneys, and has good application prospects in clinical practice.

[0032] In some embodiments of the present invention, the NODAGA has the structure shown below:

[0033] In some embodiments of the present invention, the DOTA has the following structure:

[0034] In some embodiments of the present invention, the radiolabeled collagen hybrid peptide further comprises a fluorescent dye;

[0035] In some embodiments of the present invention, the fluorescent dye is linked to the linker molecule via an amino acid or an amino acid derivative;

[0036] In some embodiments of the present invention, the fluorescent dye is selected from at least one of anthocyanin, rhodamine, BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene), FITC (Fluorescein Isothiocyanate), erythrosine, phthalocyanine, phycocyanin, phycoerythrin and Alexa Fluor dye.

[0037] In some embodiments of the present invention, the anthocyanin is selected from any one of sulfo-Cy3, sulfo-Cy5, sulfo-Cy5.5, sulfo-Cy7 and sulfo-Cy7.5.

[0038] In some preferred embodiments of the present invention, the anthocyanin is sulfo-Cy3 or sulfo-Cy5.

[0039] In some embodiments of the present invention, the rhodamine is selected from at least one of 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylrhodamine, rhodamine isothiocyanate and tetramethylrhodamine.

[0040] In some embodiments of the present invention, the BODIPY is selected from at least one of BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, and BODIPY-TRX.

[0041] In some embodiments of the present invention, the phycocyanin is selected from at least one of allophycocyanin, phycocyanin C, and phycocyanin R.

[0042] In some embodiments of the present invention, the Alexa Fluor is selected from any one of Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647, Alexa Fluor 700, and Alexa Fluor 750.

[0043] In some embodiments of the invention, the amino acid derivative comprises 6-aminohexanoic acid.

[0044] In some embodiments of the present invention, when the linker molecule of the radiolabeled collagen hybrid peptide comprises a lysine residue and 6-aminohexanoic acid, its chemical structure is as shown in formula (I):

[0045] In the formula, m is 3 or 5, and X is a radioactive nuclide selected from Al 18 F. 64 Cu, 67 Cu, 67 Ga, 68 Ga, 99 mTc, 89 Zr, 111 In, 177 Lu, 186 Re、 225 Any of Ac.

[0046] In some embodiments of the present invention, when the linker molecule of the radiolabeled collagen hybrid peptide comprises a lysine residue and 6-aminohexanoic acid, the radiolabeled collagen hybrid peptide is a compound represented by any one of Formula (I-1) to Formula (I-4):

[0047] In some embodiments of the present invention, when the linker molecule of the radiolabeled collagen hybrid peptide is 6-aminohexanoic acid, its chemical structure is shown in the following formula (II):

[0048] Wherein, the R is selected from any one of formula a-formula c:

[0049] Wherein, X is a radionuclide selected from Al 18 F. 64 Cu, 67 Cu, 67 Ga, 68 Ga, 99mTc, 89 Zr, 111 In, 177 Lu, 186 Re、 225 Any of Ac.

[0050] In some embodiments of the present invention, when the linker molecule of the radiolabeled collagen hybrid peptide is 6-aminohexanoic acid, the radiolabeled collagen hybrid peptide is a compound represented by any one of formula (II-1) to formula (II-4):

[0051] The second aspect of the present invention provides a method for preparing the radiolabeled collagen hybrid peptide according to the first aspect of the present invention, comprising the following steps:

[0052] A resin solid phase synthesis method is adopted to obtain a precursor probe containing the chelating agent and the collagen hybrid peptide, and then the precursor probe is labeled with the radioactive nuclide to obtain the product.

[0053] The preparation method of the radioactively labeled collagen hybrid peptide of the present invention is simple, and the prepared collagen hybrid peptide has good stability and high radiochemical yield.

[0054] In some embodiments of the present invention, obtaining the precursor probe further includes a step of removing the protecting group.

[0055] In some embodiments of the present invention, when the linker molecule of the radiolabeled collagen hybrid peptide is 6-aminohexanoic acid, the preparation method thereof comprises the following steps:

[0056] S1. Reacting NH2-Linker-CHP with NODAGA-tris (t-Bu ester), NOTA-bis (t-Bu ester) or DOTA-tris (t-Bu ester) to obtain NODAGA-Linker-CHP or NOTA2-Linker-CHP or DOTA-Linker-CHP, wherein the Linker is 6-aminohexanoic acid and the CHP has an amino acid sequence of (GfO) n or (GPO) n Collagen hybrid peptide, wherein n is any positive integer from 6 to 12;

[0057] S2. Label the NODAGA-Linker-CHP, the NOTA2-Linker-CHP or the DOTA-Linker-CHP with the radioactive nuclide and obtain the product after purification.

[0058] In some embodiments of the present invention, the NOTA2 has a structure as shown in formula (a-1):

[0059] In some embodiments of the present invention, the NH2-Linker-CHP is prepared by Fmoc chemical solid phase synthesis method.

[0060] In some embodiments of the present invention, when the radionuclide is 68 Ga, the labeling method comprises: in a solvent, 68 GaCl3 reaction, after purification, is obtained.

[0061] In some embodiments of the present invention, when the metal nuclide is Al 18 F, the labeling method comprises: in a solvent, 18 F- reacts with AlCl3 and is purified to obtain.

[0062] In some embodiments of the present invention, the solvent comprises a sodium acetate solution.

[0063] In some embodiments of the present invention, the CHP is a collagen hybrid peptide with an amino acid sequence of (GfO)9.

[0064] The third aspect of the present invention provides a conjugate comprising the radiolabeled collagen hybrid peptide according to the first aspect of the present invention, and a coupling moiety;

[0065] Wherein, the coupling part includes at least one of a protein, a drug or a detectable marker.

[0066] In some embodiments of the present invention, the protein includes a protein having improved biodistribution effect, such as albumin.

[0067] In some embodiments of the present invention, the medicament comprises a compound having therapeutic efficacy.

[0068] In some embodiments of the present invention, the detectable label comprises at least one of biotin, a spin label, an enzyme, gold nanoparticles, and magnetic nanoparticles.

[0069] In some embodiments of the present invention, the spin label includes but is not limited to deuterium.

[0070] In some embodiments of the present invention, the enzyme includes but is not limited to peroxidase, alkaline phosphatase, horseradish peroxidase or acetylcholinesterase.

[0071] The fourth aspect of the present invention provides a contrast agent comprising the radiolabeled collagen hybrid peptide probe according to any one of the first aspects of the present invention.

[0072] In some embodiments of the present invention, the contrast agent is used for positron emission tomography–computed tomography (PET-CT).

[0073] The fifth aspect of the present invention provides the use of the radiolabeled collagen hybrid peptide described in the first aspect of the present invention in any of the following:

[0074] A) preparing products that specifically target structurally denatured collagen;

[0075] B) preparing products for cancer diagnosis or imaging of lesions characterized by fibrotic lesions;

[0076] C) preparing products for detecting organ fibrosis diseases;

[0077] D) Evaluate or screen for anti-pancreatic ductal adenocarcinoma or anti-fibrosis drugs.

[0078] In some embodiments of the present invention, the structurally denatured collagen is structurally denatured collagen in the extracellular matrix (ECM).

[0079] Unlike the increase in ECM collagen, which can only reflect the static fact of ECM deposition, the production of structurally denatured collagen is related to both collagen production and collagen degradation. Therefore, structurally denatured collagen can also reflect the dynamic changes in ECM remodeling during disease progression. The detection of structurally denatured collagen can assist in the study of disease progression and drug development.

[0080] In some embodiments of the present invention, the uses of the product specifically targeting structurally denatured collagen include but are not limited to pancreatic cancer detection or early diagnosis, fibrotic disease detection, and inflammatory or cardiovascular disease detection.

[0081] In some embodiments of the present invention, the pancreatic cancer detection or early diagnosis includes but is not limited to the detection or early diagnosis of pancreatic ductal adenocarcinoma, the detection or early diagnosis of mucinous cystadenocarcinoma, the detection or early diagnosis of serous cystadenocarcinoma, etc.

[0082] In some embodiments of the present invention, the detection or early diagnosis of pancreatic cancer includes detecting early lesions PanIN lesions.

[0083] In some embodiments of the present invention, the fibrotic disease includes but is not limited to pulmonary fibrosis, alcoholic liver disease, liver fibrosis and renal fibrosis.

[0084] Pulmonary fibrosis is a group of diseases with different causes, including idiopathic pulmonary fibrosis (IPF), a progressive and fatal disease in which patients do not develop symptoms until the late stage of the disease. There is no treatment to prevent or reverse the progression, and patients will continue to have difficulty breathing and lung function decline until death. Although high-resolution computed tomography (HRCT) scans can help diagnose IPF, most suspected patients still need to undergo a risky lung biopsy to confirm the disease. Therefore, there is a need to develop more specific IPF diagnostic tools to guide treatment decisions. For example, the ability to non-invasively identify fibrosis through innovative molecular imaging technology can greatly improve the treatment of patients with fibrotic lung diseases. IPF is characterized by significant ECM deposition in the lungs, so the probes of the present invention that specifically target the pathological ECM can also be used for the precise detection and treatment of IPF.

[0085] In some embodiments of the present invention, the cancer characterized by fibrotic lesions includes but is not limited to pancreatic cancer, breast cancer, and colorectal cancer.

[0086] In some embodiments of the present invention, the organ fibrosis disease includes at least one of pulmonary fibrosis, pancreatic fibrosis, liver fibrosis, cardiac fibrosis, renal fibrosis and arterial hyperplasia.

[0087] In some embodiments of the present invention, the product includes a reagent, a kit, or an instrument.

[0088] In a sixth aspect, the present invention provides a kit for imaging tumors or organ fibrosis, comprising the radiolabeled collagen hybrid peptide according to the first aspect.

[0089] In some embodiments of the present invention, the tumor includes but is not limited to pancreatic tumors, breast tumors, prostate tumors, digestive tract tumors, lung tumors, and liver tumors. Specifically, the pancreatic tumor includes but is not limited to pancreatic ductal adenoma, pancreatic endocrine tumor, mucinous cystadenoma, serous cystadenoma, pancreatic angioleiomyoma, and pancreatic lymphoepithelial cyst.

[0090] In some embodiments of the present invention, the organ fibrosis comprises organ fibrosis collagen pathological remodeling.

[0091] In some embodiments of the present invention, the organ fibrosis includes but is not limited to pulmonary fibrosis, cardiac fibrosis, liver fibrosis, renal fibrosis, etc.

[0092] The seventh aspect of the present invention provides a method for treating or diagnosing organ fibrosis-related diseases, which comprises administering to a subject the radiolabeled collagen hybrid peptide described in any one of the first aspect of the present invention, the conjugate described in the third aspect of the present invention, or the contrast agent described in the fourth aspect of the present invention.

[0093] In some embodiments of the present invention, the organ fibrosis-related diseases include but are not limited to pancreatic tumors, breast tumors, prostate tumors, digestive tract tumors, lung tumors and liver tumors, as well as pulmonary fibrosis, cardiac fibrosis, liver fibrosis, renal fibrosis, etc.

[0094] In some embodiments of the present invention, the radiolabeled collagen hybrid peptide can be administered to a subject as a molecular probe by, for example, systemic, topical, and / or parenteral administration. These administration methods include, for example, injection, infusion, deposition, implantation, or topical administration, or any other administration method that allows the molecular probe to enter a tissue. In one example, the administration of the molecular probe can be by intravenous injection into the subject. The probe can be administered single or multiple times.

[0095] In some embodiments of the present invention, the radiolabeled collagen hybrid peptide can be used as a molecular probe and administered to a subject in the form of a pharmaceutical composition containing the molecular probe or a pharmaceutically acceptable water-soluble salt thereof in an amount detectable for the patient.

[0096] In some embodiments of the present invention, the preparation of the radiolabeled collagen hybrid peptide to be administered will vary according to the selected route of administration (e.g., solution, emulsion, capsule, etc.). Suitable pharmaceutically acceptable carriers may contain inert ingredients that do not excessively inhibit the biological activity of the compound. Pharmaceutically acceptable carriers should be biocompatible, for example, non-toxic, non-inflammatory, non-immunogenic, and no other undesirable reactions occur when administered to a subject. Standard pharmaceutical preparation techniques, such as those described in Remington's Pharmaceutical Sciences, can be used, ibid. Pharmaceutical carriers suitable for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg / ml benzyl alcohol), phosphate-buffered saline (PBS), Hank's solution, Ringer lactate solution, etc.

[0097] The beneficial effects of the present invention include:

[0098] (1) The present invention couples radionuclides with collagen hybrid peptide CHP to prepare radionuclide-labeled molecular probes that can detect collagen structural degeneration in ECM. The radionuclide-labeled molecular probes have excellent clarity and signal-to-noise ratio. They can not only detect PDAC in mouse pancreatic cancer models, but also sensitively detect early PanIN lesions and pulmonary fibrosis lesions in mouse bleomycin pulmonary fibrosis models. This is of great significance for achieving early diagnosis of PDAC and reflecting the dynamic changes of ECM remodeling. In addition, it is worth noting that ECM remodeling and ECM deposition are common characteristics of fibrosis, inflammation, cardiovascular disease, and various solid tumors. Traditional collagen-targeted probes such as CBP8-PET can usually only highlight changes in collagen content when detecting related diseases (such as pulmonary fibrosis), that is, they can only reflect the static fact of ECM deposition based on the increase in ECM collagen content. The radionuclide-labeled molecular probe of the present invention targets structurally denatured collagen. The production of structurally denatured collagen is related to both ECM collagen production and ECM collagen degradation. Therefore, it can not only reflect the static fact of ECM deposition, but also reflect the dynamic changes of ECM remodeling during disease progression. Compared with conventional molecular probes targeting collagen, it has a wider application value.

[0099] (2) The radiolabeled collagen hybrid peptide probe of the present invention has the characteristics of radiochemical labeling stability and excellent biodistribution characteristics with high uptake in target organs such as tumors and low uptake in non-target organs such as liver and / or kidneys, and has good application prospects in clinical practice.

[0100] In the present invention:

[0101] The terms "comprise," "include," "includes," "have," and "have" are used in an inclusive, open-ended sense to mean that additional elements may be included. As used herein, the terms "such as" and "for example" are non-limiting and are for illustrative purposes only. "Include" and "including but not limited to" are used interchangeably.

[0102] The term "agent" is used in the present invention to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract prepared from biological material.

[0103] The terms "peptide(s)", "protein(s)" and "polypeptide(s)" are used interchangeably herein. As used herein, "polypeptide" refers to any peptide or protein comprising two or more amino acids, wherein the two or more amino acids are linked to each other by peptide bonds or modified peptide bonds (i.e., peptide isomers). "Polypeptide(s)" refers to both short chains (often referred to as peptides, oligopeptides or oligomers) and longer chains (often referred to as proteins).

[0104] The term "treatment" refers to therapeutic treatment and preventive or protective measures, wherein the goal is to prevent or slow down (mitigate) undesirable physiological changes or disorders, such as cancer growth, development or spread. For example, useful or desired clinical results include but are not limited to symptom relief, disease severity alleviation, stable disease state (i.e., no worsening), disease progression delay or slowdown, disease state improvement and regression (whether partial or complete), whether or not detectable. "Treatment" can also refer to prolonged survival compared to survival expected if not receiving treatment. Those requiring treatment include those already having a disease or disorder and those tending to have or suspected of having the disease or disorder or those in which the disease or disorder is prevented.

[0105] The term "administering" refers to providing or delivering the radiolabeled collagen hybrid peptide in an amount and for a time effective to label cancer cells or fibrotic tissue in a subject.

[0106] Other features and advantages of the present invention will be set forth in the description that follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0108] Figure 1A-1B shows the structurally denatured collagen targeting probe of the present invention. 68 Schematic diagram of the synthesis reaction and chemical structure of Ga-Cym-CHP, where Figure 1A is 68 The synthetic reaction formula of Ga-Cym-CHP probe is shown in Figure 1B. 68 Chemical structure of the Ga-Cym-CHP probe.

[0109] Figure 2 is a schematic diagram of collagen decomposition and probe labeling in tissues during the development of tumors and fibrosis.

[0110] Figure 3 shows the free 68 GaCl3 solution and radiolabeled 68 Radioactive TLC detection results of Ga-Cy3-CHP.

[0111] Figures 4A-4B show 68 Radioactive TLC detection results of Ga-Cy3-CHP probe after incubation in different solutions for 0-2 h, wherein FIG4A is 1×PBS solution, and FIG4B is 10% FBS solution.

[0112] Figures 5A-5D show 68 The results of in vivo detection of PDAC subcutaneous tumors by Ga-Cy3-CHP probe, in which Figure 5A shows the results of subcutaneous tumor injection in mice. 68 Ga-Cy3-CHP or 68 Ga-Cy3-S After CHP and blocking, representative PET images were obtained at 30 and 60 minutes of 1-hour dynamic PET imaging. Figures 5B and 5C are the quantitative statistical results of tumor radioactivity, and Figure 5D is the ex vivo quantitative analysis result of tumor uptake in the tumor after 1-hour dynamic scanning.

[0113] Figure 6 is a schematic diagram of orthotopic tumor-bearing.

[0114] Figure 7A-7B shows the radionuclide probe injected into the tail vein of an in situ tumor 68 2h static scanning PET image and pancreatic ex vivo PET image result after Ga-Cy5-CHP, wherein FIG7A is a static scanning PET image, and FIG7B is a pancreatic ex vivo PET image result.

[0115] FIG8 is a statistical diagram showing the tumor probe uptake in the pancreatic tail (tumor) and the ratio of the pancreatic tail (tumor) to the pancreatic head, measured after mice were sacrificed after imaging.

[0116] FIG9 is a schematic diagram of the KPC mouse model.

[0117] Figure 10 68 Static scanning PET image of KPC lesions with Ga-Cy5-CHP probe targeting 2 hours.

[0118] Figures 11A-11B show 68 The in vitro PET and biodistribution results of the pancreas after Ga-Cy5-CHP probe targeted KPC lesions, where FIG11A shows the in vitro PET results of the pancreas, and FIG11B shows the biodistribution results.

[0119] Figure 12 64 Cu-Cy5-CHP 2h and 8h static scanning PET images and pancreatic ex vivo PET results.

[0120] Figures 13A-13D show Al 18 F-Cy5-CHP in vivo imaging of PDAC detection results of KPC model, where Figure 13A is Al 18 F-Cy5-CHP 3h static scan PET image, Figure 13B is the corresponding image of the same mouse 18 F-FDG 1h static scan PET image; Figure 13C is the injection of Al 18 The in vitro PET results of the pancreas 3 hours after F-Cy5-CHP administration are shown in Figure 13D as the biodistribution results.

[0121] Figures 14A-14C are Al 18 F-Cy5-CHP in vivo detection of PanIN lesion imaging results, where Figure 14A is Al 18Representative PET images of a 2-hour static scan of F-Cy5-CHP; FIG14B shows the ex vivo PET results of the pancreas; and FIG14C shows the quantification of radioactive uptake in the pancreas.

[0122] FIG15 shows the results of HE staining and CHP and CK19 fluorescence staining of pancreas frozen sections of low-age KPC mice.

[0123] Figures 16A-16D show 68 Ga-Cy5-CHP was used to detect and stage collagen remodeling during the progression of pulmonary fibrosis in mice. Figure 16A shows the results of the normal mice and the mice in the pulmonary fibrosis stage after bleomycin administration for 28 days. 68 Ga-Cy5-CHP or 68 Ga-Cy5- S Representative PET images of CHP; Figure 16B shows the quantitative statistical results of lung uptake; Figure 16C shows the normal mice and the mice injected with bleomycin in the inflammatory stage 7 days after administration. 68 Representative PET images of Ga-Cy5-CHP; Figure 16D shows the quantitative statistical results of lung uptake.

[0124] Figures 17A-17D are CHP hybridizations revealing the response of mice to anti-fibrotic therapy, wherein Figure 17A is the pirfenidone (PFD) dosing schedule; Figure 17B is a representative ex vivo fluorescence image of the lungs of mice from the normal, BM, and PFD groups, showing that the fluorescence signal in the PFD-treated lungs was reduced relative to the untreated BM-injured lungs; Figure 17C is the dihydrexidine (DHX) dosing schedule; Figure 17D is a representative ex vivo fluorescence image of the lungs of mice from the normal, BM, and DHX groups.

[0125] FIG18 is a probe of Example 5 of the present invention 68 Radioactive HPLC spectra of probe precursor and radiochemically labeled probe during the preparation of Ga-NODAGA-Ahx-(GfO)9.

[0126] FIG. 19 is a probe Al according to embodiment 6 of the present invention. 18 Radioactive HPLC profiles of the probe precursor and radiochemically labeled probe during the preparation of F-NODAGA-Ahx-(GfO)9.

[0127] FIG. 20 is a probe A1 of Example 7 of the present invention. 18 Radioactive HPLC profiles of the probe precursor and radiochemically labeled probe during the preparation of F-NOTA2-Ahx-(GfO)9.

[0128] FIG21 is a comparative example 4 of the present invention, the probe Cy5-Ahx-K (NOTA-Al 18F) Radioactive HPLC profiles of the probe precursor and radiochemically labeled probe during the preparation of K(NH2)-Ahx-(GfO)9.

[0129] FIG22 is a MALDI spectrum of the probe of Comparative Example 4 of the present invention.

[0130] FIG. 23 is a probe A1 of Comparative Example 6 of the present invention. 18 Radioactive HPLC spectra of the probe precursor and radiochemically labeled probe during the preparation of F-NOTA-Ahx-(GfO)9.

[0131] FIG24 shows the PET / CT results of the probe prepared in Example 5 of the present invention in subcutaneous pancreatic cancer-bearing mice for 1 hour.

[0132] FIG25 shows the PET / CT results of the probe prepared in Example 7 of the present invention in subcutaneous pancreatic cancer-bearing mice and normal mice for 1 hour.

[0133] FIG26 shows the PET / CT results of the probe prepared in Comparative Example 3 of the present invention in subcutaneous pancreatic cancer-bearing mice for 1 hour.

[0134] FIG27 shows the PET / CT results of the probe prepared in Comparative Example 4 of the present invention in normal mice for 1 hour.

[0135] FIG28 shows the PET / CT results of the probe prepared in Comparative Example 5 of the present invention in subcutaneous pancreatic cancer-bearing mice for 1 hour.

[0136] FIG29 shows the PET / CT results of the probe prepared in Comparative Example 6 of the present invention in subcutaneous pancreatic cancer-bearing mice for 1 hour. DETAILED DESCRIPTION

[0137] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0138] The terms "preferably," "more preferably," and the like, used herein refer to embodiments of the present invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the present invention.

[0139] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are understood to include any and all subranges subsumed therein.

[0140] In the description of the present invention, reference to terms such as "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the exemplary expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0141] In an embodiment of the present invention, GfO is a glycine-fluoroproline-hydroxyproline triplet, and n (GfO) together constitute a collagen hybridizing peptide (CHP), which has a tendency to form a triple helix and can form a triple helix by hybridizing with incomplete and degraded collagen peptide chains, thereby marking damaged and denatured collagen in pathologically damaged tissues, but will not interact with intact collagen or other proteins without a triple helix structure.

[0142] In an embodiment of the present invention, 68 The synthesis diagram of Ga-Cym-CHP probe is shown in Figure 1A-1B, where Figure 1A is 68 The synthesis reaction formula of Ga-Cym-CHP is shown in Figure 1B, which is a structurally denatured collagen targeting probe. 68 The chemical structure of Ga-Cym-CHP. A schematic diagram of the labeling of the radionuclide-labeled CHP probe in tissue dehelical denaturation is shown in Figure 2.

[0143] In an embodiment of the present invention, 68 Ga can be replaced by other radioactive elements, such as 64 Cu or 18 F, etc.; NOTA chelating agent can be replaced by other metal chelating agents, such as NODAGA; (GfO) n Can be replaced by (GPO) with the same triple helix forming propensity n .

[0144] The present invention describes methods involving conventional molecular biology techniques. Such techniques are well known in the art and are described in detail in methodological papers, such as Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992. Unless otherwise defined, all technical terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which this application belongs. Commonly understood definitions of molecular biology terms can be found, for example, in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th Edition, Springer-Verlag: New York, 1991, and Lewin, Genes V, Oxford University Press: New York, 1994.

[0145] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0146] Example 1: 68 Synthesis of Ga-Cy3-CHP probe

[0147] In this embodiment, a radiolabeled collagen hybrid peptide probe was designed that can specifically target the lesion ECM to unwind collagen. The sequence of the probe is Cy3-Ahx-K (NOTA- 68 Ga)-K(NH2)-Ahx-(GfO)9, referred to as 68 Ga-Cy3-CHP, where Ahx is 6-aminohexanoic acid, K is lysine Lys, Cy3 is the cyanine dye sulfo-Cy3, and NOTA is the chelating agent p-SCN-Bn-NOTA, used to chelate radioactive metal nuclides 68 Ga, GfO are glycine-fluoroproline-hydroxyproline triplet, and 9 (GfO) together constitute collagen hybrid peptide.

[0148] In this embodiment 68 The structural formula of Ga-Cy3-CHP probe is shown in formula (I-1):

[0149] The synthesis of the probe specifically includes the following steps:

[0150] (1) Synthesis of the precursor probe NOTA-Cy3-CHP

[0151] First, Ahx-K(Boc)-K(Dde)-Ahx-(GfO)9-Rink Amide AM resin was synthesized using standard Fmoc chemistry on a PurePep Chorus peptide synthesizer. The Fmoc amino acids included Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Hyp-(tBu)-OH, Fmoc-Ahx-OH, Fmoc-Lys(Dde)-OH, and Fmoc-Lys(Boc)-OH.

[0152] Sulfonated Cy3 cyanine dye was coupled to the N-terminal amino group of a peptide on a resin via Fmoc solid-phase synthesis. The peptide Cy3-Ahx-K(NH2)-K(Dde)-Ahx-(GfO)9 was cleaved from the resin, purified by high-performance liquid chromatography (HPLC), and lyophilized. The lyophilized peptide was reacted with p-SCN-Bn-NOTA for 24 hours, after which the Dde protecting group of the lysine was removed using 3% hydrazine hydrate. The resulting peptide was purified by HPLC and lyophilized to obtain the precursor probe Cy3-Ahx-K(NOTA)-K(NH2)-Ahx-(GfO)9, referred to as NOTA-Cy3-CHP.

[0153] (2) 68 Ga radiolabeling

[0154] 24 nmol of NOTA-Cy3-CHP was dissolved in about 600 μL of 0.5 M sodium acetate buffer (pH 5.5) and then mixed with 222 MBq of 68 GaCl3 was reacted at 50 °C for 10 minutes, and then the reaction mixture was passed through a 0.22 μm Millipore filter to obtain the final product 68 Ga-Cy3-CHP.

[0155] Example 2: 68 Synthesis of Ga-Cy5-CHP probe

[0156] In this embodiment, another radiolabeled collagen hybrid peptide probe was designed that can specifically target the unwinding collagen in the lesion ECM. The sequence of the probe is Cy5-Ahx-K (NOTA- 68 Ga)-K(NH2)-Ahx-(GfO)9, referred to as 68 Ga-Cy5-CHP, where Ahx is 6-aminohexanoic acid, K is lysine Lys, Cy5 is a cyanine dye, and NOTA is a chelating agent p-SCN-Bn-NOTA, used to chelate radioactive metal nuclides 68 Ga.

[0157] In this embodiment 68 The structural formula of the Ga-Cy5-CHP probe is shown in formula (I-2):

[0158] The synthesis of this probe is similar to that of Example 1. 68 The synthesis of Ga-Cy3-CHP probe is similar and includes the following steps:

[0159] (1) Synthesis of the precursor probe NOTA-Cy5-CHP

[0160] Ahx-K(Boc)-K(Dde)-Ahx-(GfO)9-Rink Amide AM resin was synthesized using standard Fmoc chemistry on Rink Amide AM resin using a PurePep Chorus peptide synthesizer. Fmoc amino acids include Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Hyp-(tBu)-OH, Fmoc-Ahx-OH, Fmoc-Lys(Dde)-OH, and Fmoc-Lys(Boc)-OH.

[0161] Sulfonated Cy5 cyanine dye was coupled to the N-terminal amino group of a peptide on a resin via Fmoc solid-phase synthesis. The peptide Cy5-Ahx-K(NH2)-K(Dde)-Ahx-(GfO)9 was cleaved from the resin, purified by HPLC, and lyophilized. The lyophilized peptide was reacted with p-SCN-Bn-NOTA for 24 hours, followed by removal of the Dde protecting group of the lysine using 3% hydrazine hydrate. The resulting peptide was purified by HPLC and lyophilized to obtain the precursor probe Cy5-Ahx-K(NOTA)-K(NH2)-Ahx-(GfO)9, referred to as NOTA-Cy5-CHP.

[0162] (2) 68 Ga radiolabeling

[0163] 24 nmol of NOTA-Cy5-CHP was dissolved in about 600 μL of 0.5 M sodium acetate buffer (pH 5.5) and then mixed with 222 MBq of 68 GaCl3 was reacted at 50 °C for 10 minutes, and then the reaction mixture was passed through a 0.22 μm Millipore filter to obtain the final product 68 Ga-Cy5-CHP.

[0164] Example 3: 64 Synthesis of Cu-Cy5-CHP probe

[0165] This embodiment 68Ga-Cy5-CHP was improved and another metal nuclide was tried 64 Cu, Cy5-Ahx-K(NOTA- 64 Cu)-K(NH2)-Ahx-(GfO)9, hereinafter referred to as 64 The Cu-Cy5-CHP probe has a structural formula as shown in formula (I-3):

[0166] The probe was prepared using the same precursor probe NOTA-Cy5-CHP as in Example 2. 64 Cu is radiolabeled, specifically comprising the following steps:

[0167] (1) Synthesis of the precursor probe NOTA-Cy5-CHP

[0168] Ahx-K(Boc)-K(Dde)-Ahx-(GfO)9-Rink Amide AM resin was synthesized using standard Fmoc chemistry on Rink Amide AM resin using a PurePep Chorus peptide synthesizer. Fmoc amino acids include Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Hyp-(tBu)-OH, Fmoc-Ahx-OH, Fmoc-Lys(Dde)-OH, and Fmoc-Lys(Boc)-OH.

[0169] Sulfonated Cy5 cyanine dye was coupled to the N-terminal amino group of a peptide on a resin via Fmoc solid-phase synthesis. The peptide Cy5-Ahx-K(NH2)-K(Dde)-Ahx-(GfO)9 was cleaved from the resin, purified by HPLC, and lyophilized. The lyophilized peptide was reacted with p-SCN-Bn-NOTA for 24 hours, followed by removal of the Dde protecting group of lysine using 3% hydrazine hydrate, and purified by HPLC and lyophilized to obtain the precursor probe Cy5-Ahx-K(NOTA)-K(NH2)-Ahx-(GfO)9, hereafter referred to as NOTA-Cy5-CHP.

[0170] (2) 64 Cu radiolabeling

[0171] 24 nmol of NOTA-Cy5-CHP was dissolved in about 600 μL of 0.5 M sodium acetate buffer (pH 5.5) and then mixed with 222 MBq of 64 CuCl2 was reacted at 50 °C for 10 minutes, and then the reaction mixture was passed through a 0.22 μm Millipore filter to obtain the final product 64 Cu-Cy5-CHP.

[0172] Example 4: Al 18 Synthesis of F-Cy5-CHP probe

[0173] This example provides another radiolabeled collagen hybrid peptide probe Cy5-Ahx-K (NOTA-Al) that can specifically target the unwinding collagen in the lesion ECM. 18 F)-K(NH2)-Ahx-(GfO)9, hereinafter referred to as Al 18 F-Cy5-CHP is different from Example 2 in that the nuclide is changed to Al 18 F, whose structural formula is shown in formula (I-4):

[0174] The specific preparation process of the probe is as follows:

[0175] (1) Synthesis of the precursor probe NOTA-Cy5-CHP

[0176] Ahx-K(Boc)-K(Dde)-Ahx-(GfO)9-Rink Amide AM resin was synthesized using standard Fmoc chemistry on Rink Amide AM resin using a PurePep Chorus peptide synthesizer. Fmoc amino acids include Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Hyp-(tBu)-OH, Fmoc-Ahx-OH, Fmoc-Lys(Dde)-OH, and Fmoc-Lys(Boc)-OH.

[0177] Sulfonated Cy5 cyanine dye was coupled to the N-terminal amino group of a peptide on a resin via Fmoc solid-phase synthesis. The peptide Cy5-Ahx-K(NH2)-K(Dde)-Ahx-(GfO)9 was cleaved from the resin, purified by HPLC, and lyophilized. The lyophilized peptide was reacted with p-SCN-Bn-NOTA for 24 hours, followed by removal of the Dde protecting group of lysine using 3% hydrazine hydrate, and purified by HPLC and lyophilized to obtain the precursor probe Cy5-Ahx-K(NOTA)-K(NH2)-Ahx-(GfO)9, hereafter referred to as NOTA-Cy5-CHP.

[0178] (2)Al 18 F radiolabeling

[0179] exist 18 10 μL AlCl3 sodium acetate buffer (pH 4.0) was added to F-sodium acetate buffer (100 μL, 30 mCi, pH 4.0), mixed and allowed to stand at room temperature for 5 min to form [Al 18 F] 2+Conjugation; then add 40 μL of the above-prepared NOTA-Cy5-CHP (1 mM, 40 nmol) and heat the mixture at 110 ° C for 30 min. After the reaction solution is cooled to room temperature, it is diluted with 3 mL of ultrapure water and passed through a Sep-Pak C18-Light column. It is then washed with 10-20 mL of ultrapure water to remove unreacted Al 18 F. Finally, the product was eluted with anhydrous ethanol and concentrated by heating to obtain Al 18 F-Cy5-CHP.

[0180] Example 5: 68 Ga-NODAGA-Ahx-(GfO)9

[0181] This example provides a radiolabeled collagen hybrid peptide 68 Ga-NODAGA-Ahx-(GfO)9, wherein NODAGA is a chelating agent, Ahx is 6-aminohexanoic acid, and (GfO)9 is a collagen hybrid peptide. The chemical structure of the probe is shown in Formula (II-1):

[0182] above 68 The preparation method of Ga-NODAGA-Ahx-(GfO)9 is as follows:

[0183] (1) Synthesis of the probe precursor NODAGA-Ahx-(GfO)9: NH2-Ahx-(GfO)9 was obtained by solid-phase synthesis using standard Fmoc chemistry on Rink Amide AM resin. After further reaction with NODAGA-tris(t-Bu ester) (CAS: 1190101-34-8) for 24 h, the polypeptide was cleaved from the resin, purified by HPLC and lyophilized to obtain the probe precursor NODAGA-Ahx-(GfO)9.

[0184] (2) 68 Ga radiolabeling: 50 nmol of the probe precursor NODAGA-Ahx-(GfO)9 was dissolved in 300 mL of 0.5 M sodium acetate solution and then reacted with 666 MBq of 68 GaCl3 was reacted at 50°C for 10 minutes, and then the resulting reaction mixture was purified using a Sep-Pak C18 column to obtain.

[0185] Example 6: Al 18 F-NODAGA-Ahx-(GfO)9

[0186] This example provides a radiolabeled collagen hybrid peptide Al 18F-NODAGA-Ahx-(GfO)9, wherein NODAGA is a chelating agent, Ahx is 6-aminohexanoic acid, and (GfO)9 is a collagen hybrid peptide. The chemical structure of the probe is shown in Formula (II-2):

[0187] The above Al 18 The preparation method of F-NODAGA-Ahx-(GfO)9 is as follows:

[0188] (1) Synthesis of the probe precursor NODAGA-Ahx-(GfO)9: NH2-Ahx-(GfO)9 was obtained by solid-phase synthesis using standard Fmoc chemistry on Rink Amide AM resin. After further reaction with NODAGA-tris(t-Bu ester) (CAS: 1190101-34-8) for 24 h, the polypeptide was cleaved from the resin, purified by HPLC and lyophilized to obtain the probe precursor NODAGA-Ahx-(GfO)9.

[0189] (2)Al 18 F radiolabeling: 18 F- (0.1 mL, 2.5 GBq) was mixed with AlCl3 (10 μL, 2 mM) and sodium acetate buffer (0.1 mL, 0.5 M, pH 4.0) at room temperature for 10 minutes. 50 nmol of the probe precursor NODAGA-Ahx-(GfO)9 was then added, and the mixture was heated at 110°C for 30 minutes. The reaction mixture was then purified on a Sep-Pak C18 column to obtain the product.

[0190] Example 7: Al 18 F-NOTA2-Ahx-(GfO)9

[0191] This example provides a radiolabeled collagen hybrid peptide Al 18 F-NOTA2-Ahx-(GfO)9, wherein NOTA2 is a chelating agent, Ahx is 6-aminohexanoic acid, and (GfO)9 is a collagen hybrid peptide. The chemical structure of the probe is shown in Formula (II-3):

[0192] The above Al 18 The preparation method of F-NOTA2-Ahx-(GfO)9 is as follows:

[0193] (1) Synthesis of the probe precursor NOTA2-Ahx-(GfO)9: NH2-Ahx-(GfO)9 was obtained by solid-phase synthesis using standard Fmoc chemistry on Rink Amide AM resin. The product was then reacted with NOTA-bis(t-Bu ester) (CAS: 1161415-28-6) for 24 h. The peptide was then cleaved from the resin, purified by HPLC and lyophilized to obtain the probe precursor NOTA2-Ahx-(GfO)9.

[0194] (2)Al 18 F radiolabeling: 18 F- (0.1 mL, 2.5 GBq) was mixed with AlCl3 (10 μL, 2 mM) and sodium acetate buffer (0.1 mL, 0.5 M, pH 4.0) at room temperature for 10 minutes. 50 nmol of the probe precursor NOTA2-Ahx-(GfO)9 was then added, and the mixture was heated at 110°C for 30 minutes. The reaction mixture was then purified on a Sep-Pak C18 column to obtain the product.

[0195] Comparative Example 1: Control Probe 68 Ga-Cy3- S CHP

[0196] This comparative example provides a non-targeted control probe Cy3-Ahx-K (NOTA- 68 Ga)-K(NH2)-Ahx- S (GfO)9, abbreviated as 68 Ga-Cy3- S CHP, where S The sequence of (GfO)9 is OfGGOfGfGfOfOGOfGOOfGGOOffG (SEQ ID NO: 1), wherein O is hydroxyproline, f is fluoroproline, and G is glycine. This sequence is obtained by randomly disrupting the target sequence (GfO)9.

[0197] 68 Ga-Cy3- S The preparation process of the CHP probe differs from that of Example 1 in that the target sequence is different, and the other conditions are the same.

[0198] Comparative Example 2: Control Probe 68 Ga-Cy5- S CHP

[0199] This comparative example provides a non-targeted control probe Cy5-Ahx-K (NOTA- 68 Ga)-K(NH2)-Ahx- S (GfO)9, abbreviated as 68Ga-Cy5- S CHP, where S The sequence of (GfO)9 is OfGGOfGfGfOfOGOfGOOfGGOOffG (SEQ ID NO: 1), wherein O is hydroxyproline, f is fluoroproline, and G is glycine. This sequence is obtained by randomly disrupting the target sequence (GfO)9.

[0200] 68 Ga-Cy5- S The preparation process of the CHP probe differs from that of Example 2 in that the target sequence is different, and the other conditions are the same.

[0201] Comparative Example 3: Cy5-Ahx-K(NOTA- 68 Ga)-K(NH2)-Ahx-(GfO)9

[0202] This comparative example provides a fluorescent-radioactive labeled collagen hybrid peptide Cy5-Ahx-K (NOTA- 68 Ga)-K(NH2)-Ahx-(GfO)9, where Cy5 is a fluorescent dye gene, Ahx is 6-aminohexanoic acid, K is a lysine residue, NOTA is a chelating agent, and (GfO)9 is a collagen hybrid peptide. The chemical structure of the probe is as follows:

[0203] The above Cy5-Ahx-K(NOTA- 68 The preparation method of Ga)-K(NH2)-Ahx-(GfO)9 is as follows:

[0204] (1) Synthesis of the probe precursor Cy5-Ahx-K(NOTA)-K(NH2)-Ahx-(GfO)9: Ahx-K(Boc)-K(Dde)-Ahx-(GfO)9-Rink Amide AM resin was obtained by solid-phase synthesis using standard Fmoc chemistry on Rink Amide AM resin using a PurePep Chorus peptide synthesizer. After reacting the Cy5 cyanine dye onto the resin, the peptide was cleaved from the resin, purified by HPLC, and lyophilized to obtain Cy5-Ahx-K(NH2)-K(Dde)-Ahx-(GfO)9. It was then reacted with p-SCN-Bn-NOTA (CAS: 147597-66-8) for 24 h, and then the Dde protecting group of lysine was removed using hydrazine hydrate. The product was then purified by HPLC and freeze-dried to obtain the probe precursor Cy5-Ahx-K(NOTA)-K(NH2)-Ahx-(GfO)9.

[0205] (2) 68Ga radiolabeling: 50 nmol of the probe precursor Cy5-Ahx-K(NOTA)-K(NH2)-Ahx-(GfO)9 was dissolved in 300 mL of 0.5 M sodium acetate solution and then reacted with 666 MBq of 68 GaCl3 was reacted at 50°C for 10 minutes, and then the resulting reaction mixture was purified using a Sep-Pak C18 column to obtain.

[0206] Comparative Example 4: Cy5-Ahx-K(NOTA-Al 18 F)-K(NH2)-Ahx-(GfO)9

[0207] This comparative example provides a fluorescent-radioactive labeled collagen hybrid peptide Cy5-Ahx-K (NOTA-Al 18 F)-K(NH2)-Ahx-(GfO)9, where Cy5 is a fluorescent dye gene, Ahx is 6-aminohexanoic acid, K is a lysine residue, NOTA is a chelating agent, and (GfO)9 is a collagen hybrid peptide. The chemical structure of the probe is as follows:

[0208] The above Cy5-Ahx-K(NOTA-Al 18 F)-K(NH2)-Ahx-(GfO)9 preparation method is as follows:

[0209] (1) Synthesis of the probe precursor Cy5-Ahx-K(NOTA)-K(NH2)-Ahx-(GfO)9: Ahx-K(Boc)-K(Dde)-Ahx-(GfO)9-Rink Amide AM resin was obtained by solid-phase synthesis using standard Fmoc chemistry on Rink Amide AM resin using a PurePep Chorus peptide synthesizer. After reacting the Cy5 cyanine dye onto the resin, the peptide was cleaved from the resin, purified by HPLC, and lyophilized to obtain Cy5-Ahx-K(NH2)-K(Dde)-Ahx-(GfO)9. It was then reacted with p-SCN-Bn-NOTA (CAS: 147597-66-8) for 24 h, and then the Dde protecting group of lysine was removed with hydrazine hydrate. The product was then purified by HPLC and freeze-dried to obtain the probe precursor Cy5-Ahx-K(NOTA)-K(NH2)-Ahx-(GfO)9.

[0210] (2)Al 18 F radiolabeling: 18F- (0.1 mL, 2.5 GBq) was mixed with AlCl3 (10 μL, 2 mM) and sodium acetate buffer (0.1 mL, 0.5 M, pH 4.0) at room temperature for 10 minutes. Then, 50 nmol of the probe precursor Cy5-Ahx-K(NOTA)-K(NH2)-Ahx-(GfO)9 was added, and the mixture was heated at 110°C for 30 minutes. The reaction mixture was then purified on a Sep-Pak C18 column to obtain the product.

[0211] Comparative Example 5: Cy5-Ahx-K(NOTA- 68 Ga)-Ahx-(GfO)9

[0212] This comparative example provides a fluorescent-radioactive labeled collagen hybrid peptide Cy5-Ahx-K (NOTA- 68 Ga)-Ahx-(GfO)9, where Cy5 is a fluorescent dye gene, Ahx is 6-aminohexanoic acid, K is a lysine residue, NOTA is a chelating agent, and (GfO)9 is a collagen hybrid peptide. The chemical structure of the probe is as follows:

[0213] The above Cy5-Ahx-K(NOTA- 68 The preparation method of Ga)-Ahx-(GfO)9 is as follows:

[0214] (1) Synthesis of the probe precursor Cy5-Ahx-K(NOTA)-Ahx-(GfO)9: Standard Fmoc chemistry was used for solid-phase synthesis and cleavage on Rink Amide AM resin, followed by HPLC purification to obtain Cy5-Ahx-K(NH2)-Ahx-(GfO)9. The lyophilized peptide was reacted with p-SCN-Bn-NOTA (CAS: 147597-66-8) for 24 h, followed by HPLC purification and lyophilization to obtain the probe precursor Cy5-Ahx-K(NOTA)-Ahx-(GfO)9.

[0215] (2) 68 Ga radiolabeling: 50 nmol of the probe precursor Cy5-Ahx-K(NOTA)-Ahx-(GfO)9 was dissolved in 300 mL of 0.5 M sodium acetate solution and then mixed with 666 MBq of 68 GaCl3 was reacted at 50°C for 10 minutes, and then the resulting reaction mixture was purified using a Sep-Pak C18 column to obtain.

[0216] Comparative Example 6: Al 18 F-NOTA-Ahx-(GfO)9

[0217] This comparative example provides a radiolabeled collagen hybrid peptide Al18 F-NOTA-Ahx-(GfO)9, where NOTA is a chelating agent, Ahx is 6-aminohexanoic acid, and (GfO)9 is a collagen hybrid peptide. The chemical structure of the probe is as follows:

[0218] The above Al 18 The preparation method of F-NOTA-Ahx-(GfO)9 is as follows:

[0219] (1) Synthesis of the probe precursor NOTA-Ahx-(GfO)9: Standard Fmoc chemistry was used for solid-phase synthesis and shearing on Rink Amide AM resin, followed by HPLC purification to obtain NH2-Ahx-(GfO)9. The lyophilized peptide was reacted with p-SCN-Bn-NOTA (CAS: 147597-66-8) for 24 h, followed by HPLC purification and lyophilization to obtain the probe precursor NOTA-Ahx-(GfO)9.

[0220] (2)Al 18 F radiolabeling: 18F- (0.1 mL, 2.5 GBq) was mixed with AlCl3 (10 μL, 2 mM) and sodium acetate buffer (0.1 mL, 0.5 M, pH 4.0) at room temperature for 10 minutes. 50 nmol of the probe precursor NOTA-Ahx-(GfO)9 was then added, and the mixture was heated at 110°C for 30 minutes. The reaction mixture was then purified on a Sep-Pak C18 column to obtain the product.

[0221] Test Example 1: Radioactivity and In Vitro Stability Analysis

[0222] This test example is based on the 68 Ga-Cy3-CHP is used as a detection probe, and the radioactivity and stability of the probe prepared by the scheme of the present invention are analyzed.

[0223] (1) Radioactive TLC analysis

[0224] The final product was characterized by radioactive TLC (ITLC, 10 mM EDTA, pH 7). 68 The radiochemical purity of Ga-Cy3-CHP was analyzed, where Rf 68 Ga / EDTA=1.0,Rf 68 Ga-Cy3-CHP=0.0. The results are shown in Figure 3. There is no free radioactive nuclide in the product. 68 Ga, indicating that the probe prepared by the present invention 68 Ga-Cy3-CHP can be obtained in high radiochemical yields without HPLC purification, with specific activities ranging from 2.69 to 7.34 GBq / μmol.

[0225] (2) In vitro stability analysis

[0226] The prepared 68 The Ga-Cy3-CHP probe was placed in 1×PBS and 10% fetal bovine serum (FBS) solution and incubated at 37°C for 2 h. Radioactive TLC detection was performed every 30 min.

[0227] The test results are shown in Figures 4A-4B. 68 There was no significant decrease after Ga labeling for about 2 hours, indicating that the probe prepared by the present invention exhibited good in vitro stability.

[0228] The probes prepared in other embodiments of the present invention are the same as those prepared in Example 1. 68 Ga-Cy3-CHP has considerable radioactivity and in vitro stability.

[0229] Test Example 2: In vivo targeting of denatured collagen in PDAC subcutaneous tumor lesions

[0230] This test example is based on the 68 Ga-Cy3-CHP was used as a sample to evaluate the in vivo targeting specificity of the radiolabeled CHP-PET probe constructed in the present invention on denatured collagen. The specific method is as follows:

[0231] First, a mouse subcutaneous tumor model was established using the human pancreatic cancer cell line CFPAC-1 (3×106 CFPAC-1 cells were injected into the right shoulder of 6-week-old female Balb / c-nu nude mice, and the subcutaneous tumor was allowed to grow to 200 mm). 3 The experiment was conducted at 37 ℃ and 12 ℃ respectively. The mice were divided into three groups, two of which were injected with about 8.42 MBq of denatured collagen targeting probe into the tail vein of subcutaneous tumor mice. 68 Ga-Cy3-CHP or non-targeted probes 68 Ga-Cy3- S CHP (i.e., the probe prepared in Comparative Example 1), another group of mice were injected with a blocking dose of the non-radioactive precursor probe NOTA-Cy3-CHP in advance, and then injected 68 Ga-Cy3-CHP (blocking group) Dynamic PET-CT imaging was performed for 1 hour.

[0232] The test results are shown in Figures 5A-5D, where Figure 5A shows subcutaneous tumor injection in mice 68 Ga-Cy3-CHP or 68 Ga-Cy3- SAfter CHP and blocking, representative PET images were obtained at 30 and 60 minutes of 1-hour dynamic PET imaging. Figures 5B and 5C are the quantitative statistical results of tumor radioactivity, and Figure 5D is the in vitro quantitative analysis of tumor uptake after 1-hour dynamic scanning. The results show that the tumor with the correct targeting sequence 68 Ga-Cy3-CHP can clearly observe subcutaneous tumors at 0.5h and 1h with good tumor-to-background contrast, but 68 Ga-Cy3- S The in vivo PET signal during 1 h dynamic PET imaging was quantified as the PET standardized uptake value (SUV), which was compared with the 68 Ga-Cy3- S Compared with the rapid clearance of CHP and the persistent low signal of the blocking group, 68 Ga-Cy3-CHP was slowly washed out of the tumor and maintained a significantly high signal during dynamic imaging (as shown in Figures 5B and 5C). Quantification of the PET signal of the tumor at 30 and 60 minutes showed statistically significant differences between the normal sequence, scrambled sequence, and blocked groups.

[0233] To verify the PET imaging results, mice were euthanized after 1 h of dynamic PET imaging, and tumors were harvested for in vitro biodistribution analysis, calculated as the percentage of injected dose per gram (% ID / g). The results are shown in Figure 5D, which are consistent with the PET results. 68 Ga-Cy3-CHP has significantly higher tumor uptake, further indicating that the denatured collagen targeting probe of the present invention can be used to detect PDAC in vivo.

[0234] Detection Example 3: In vivo detection of pancreatic in situ tumors in deep anatomical locations

[0235] One reason why PDAC is difficult to detect is that it is located in a deep anatomical location. Therefore, this detection example uses an in situ tumor-bearing model prepared in Example 2 above. 68 Ga-Cy5-CHP, and the comparative example 2 prepared 68 Ga-Cy5- S The CHP probe was used as a sample to evaluate the ability of the radiolabeled CHP-PET probe constructed in this invention to detect pancreatic in situ tumors in deep anatomical locations in vivo. The specific process included the following:

[0236] 1×106 human pancreatic cancer cell line CFPAC-1-LUC was injected into the pancreatic tail of 6-week-old female Balb / c-nu nude mice to establish an orthotopic model. The successful establishment of the model was confirmed by bioluminescence (as shown in Figure 6). 68About 8.42 MBq of Ga-Cy5-CHP was injected into the tail vein of mice with orthotopic tumors. 68 Ga-Cy5- S CHP (i.e., the probe prepared in Comparative Example 2), another group of mice were injected with a blocking dose of the non-radioactive precursor probe NOTA-Cy5-CHP in advance, and then injected 68 Ga-Cy5-CHP (blocking group) Static PET-CT imaging was performed for 2 hours.

[0237] The test results are shown in Figures 7A-7B, which are consistent with the results of subcutaneous tumors. The static PET-CT imaging results of 2 hours showed that the targeted sequence 68 The Ga-Cy5-CHP probe allowed in vivo visualization of pancreatic tumors, whereas no signal was observed in the pancreas of normal mice or in model mice using scrambled probes or blocking (Figure 7A). After static scanning, the mice were sacrificed and the pancreas was removed for ex vivo PET imaging. The ex vivo PET results further supported the in vivo PET imaging (Figure 7B).

[0238] Furthermore, since the in situ tumor model is to implant the tumor in the pancreatic tail, the pancreas was divided into two parts after sampling, the pancreatic tail (tumor) and the pancreatic head, and the radioactive uptake of the two parts and the ratio of pancreatic tail to pancreatic head were calculated. The results are shown in Figure 8. 68 The significantly high uptake of Ga-Cy5-CHP in the pancreatic tail tumor site further proves that the probe of the present invention can target the denatured collagen of abdominal pancreatic in situ tumors in vivo and has the ability to detect them in the abdomen.

[0239] Detection Example 4: In vivo Visualization Detection of Pancreatic Tumors

[0240] Since the probe targets the ECM, the tumor ECM induced by the injection of human tumor cells is quite different from the real tumor ECM, whether in subcutaneous or orthotopic tumor-bearing models. This test case uses a transgenic KPC mouse model (LSL-Kras) that can spontaneously form tumors and has a microenvironment closer to that of human pancreatic cancer lesions. G12D / + ;LSL-Trp53 R172H / + ; Pdx-1-Cre) was used as the experimental material, and the probe prepared in the embodiment of the present invention was used to detect PDAC lesions in the KPC model (>16 weeks old), and the priority targeting probe was further screened.

[0241] 1. 68 Ga-Cy5-CHP probe

[0242] This part is prepared in Example 2 68 Ga-Cy5-CHP probe (which has been shown to detect abdominal pancreatic orthotopic tumors in vivo) was used as a detection marker to detect and label PDAC lesions in the KPC model (>16 weeks old).

[0243] The KPC mouse model is the most commonly used genetically engineered mouse model for studying the PDAC tumor microenvironment. This model has conditional mutations in the oncogene Kras and the tumor suppressor gene TP53. KPC disease progression is very similar to that in humans (developing into PanIN at 8-10 weeks and into PDAC at 16 weeks) and recapitulates many of the significant clinical features of human PDAC (such as cachexia, ascites, bile duct obstruction, and other complications) and histopathological characteristics, as shown in Figure 9.

[0244] The specific experimental method is as follows: KPC mice and normal mice of the same age were taken and injected with about 10MBq into the tail vein of each mouse. 68 Ga-Cy5-CHP was used for 2h static scanning. After the static scanning, the mice were killed, the pancreas was taken for ex vivo PET, and the biodistribution of radioactivity in all organs was determined.

[0245] The test results are shown in Figures 10 and 11A-11B, where Figure 10 is 68 PET images of Ga-Cy5-CHP 2h static scanning, Figure 11A shows the pancreas ex vivo PET results, and Figure 11B shows the biodistribution results. Although the ex vivo PET and organ biodistribution results show that the probe is significantly enriched in the tumor site of KPC mice, this is different from the orthotopic tumor-bearing mice where the tumor is concentrated in the pancreatic tail (with a clear anatomical location and larger tumors). Since the tumors in KPC mice are scattered throughout the pancreas and are small tumors, 68 Ga-Cy5-CHP probe detection is affected by organs surrounding the pancreas, such as the liver, spleen, and kidney, which interferes with the in vivo visualization of PDAC to some extent.

[0246] These results indicate that different radionuclides can lead to variations in the biodistribution of probes in vivo, which can interfere with the signal-to-noise ratio of the probes in the target organs. Further improvement and optimization of the probes are needed to achieve in vivo tumor visualization in KPC mice that better reflects the actual clinical characteristics (dispersed distribution of tumors throughout the pancreas).

[0247] 2. 64 Cu-Cy5-CHP probe detection

[0248] In order to adjust the biodistribution of radionuclide-labeled molecular probes to improve the in vivo imaging of pancreatic tumors, this section 68 Ga-Cy5-CHP was improved and another metal nuclide was tried 64 Cu, i.e., obtained using the above-mentioned Example 3 64 Cu-Cy5-CHP is further tested. The specific method is as follows:

[0249] About 10 MBq was injected into the tail vein of KPC mice (>16 weeks old) and normal mice of the same age. 64 Cu-Cy5-CHP, static scanning was performed at multiple time points of 2h, 4h, 6h, and 8h, and the pancreas was taken for ex vivo PET after 8h.

[0250] The test results are shown in Figure 12, which shows 68 Ga-Cy5-CHP PET imaging results are similar, although ex vivo PET showed 64 The Cu-Cy5-CHP probe was enriched at the tumor site, but the strong signal in the abdominal intestine interfered with the in vivo visualization of PDAC.

[0251] 3. Al 18 F-Cy5-CHP probe detection

[0252] This section further attempts to change the nuclide to Al 18 F, namely the Al prepared in Example 4 18 In vivo imaging of pancreatic tumors was performed using the F-Cy5-CHP probe. The specific method was as follows: KPC mice (>16 weeks old) and normal mice of the same age were injected with about 10 MBq Al 18 F-Cy5-CHP, 3h static scan.

[0253] The test results are shown in Figures 13A-13D, wherein Figure 13A is Al 18 F-Cy5-CHP 3h static scan PET image, Figure 13B is the corresponding image of mice of the same age 18 F-FDG 1h static scan PET image; Figure 13C is the injection of Al 18 Figure 13D shows the biodistribution results of the pancreas after 3 hours of F-Cy5-CHP in vitro. 18 The uptake of F-Cy5-CHP in the liver and spleen was significantly reduced, which allowed accurate detection of PDAC lesions in the KPC model with higher clarity and signal-to-noise ratio, and was superior to traditional 18 F-FDG. Ex vivo PET results and biodistribution further supported the PET data.

[0254] In summary, compared with 68 Ga-Cy5-CHP probe and 64 Cu-Cy5-CHP probe, Al 18 The uptake of F-Cy5-CHP in the liver is further reduced, and the uptake in the pancreas is further increased, so that the pancreatic tumor in the abdomen can be clearly exposed in the PET image. 18The F-Cy5-CHP probe significantly improved the signal-to-noise ratio of pancreatic tumor detection, making it possible to detect precancerous lesions.

[0255] Test Example 5: In vivo detection of PanIN lesions

[0256] In order to explore whether the radionuclide-labeled CHP probe can detect pancreatic intraepithelial neoplasia PanIN lesions in vivo, this test case used Al 18 F-Cy5-CHP probe was used. Younger KPC mice were used. Theoretically, 8-week-old KPC mice have not yet progressed to the PDAC stage. They were injected with about 10 MBq Al through the tail vein of normal mice of the same age. 18 F-Cy5-CHP, 2h PET-CT static scan.

[0257] The test results are shown in Figures 14A-14C, wherein Figure 14A is Al 18 Representative PET images of a 2-hour static scan of F-Cy5-CHP. Figure 14B shows the pancreatic ex vivo PET results, and Figure 14C shows the quantitative uptake of pancreatic radioactivity. The results show the presence of high signal points in the abdominal pancreas. After imaging, the pancreas was taken for ex vivo PET and its radioactivity was measured. The results showed that KPC pancreas indeed had a stronger presence of Al 18 F-Cy5-CHP uptake.

[0258] Furthermore, the pancreas was frozen and stained with HE and CHP. The results are shown in Figure 15. The results showed that the pancreas of the low-age KPC had high-grade PanIN and a large amount of denatured collagen around the PanIN. This is because a large amount of unwound collagen existed in the PanIN lesions as early as the evolution of PDAC. Therefore, the pathological results confirmed that Al 18 The signal detected by F-Cy5-CHP in vivo is indeed PanIN lesion. 18 The F-Cy5-CHP probe has an excellent signal-to-noise ratio, enabling it to successfully detect tiny lesions such as PanIN, providing a new tool for the early detection of PDAC.

[0259] In summary, it can be seen from the partial detection results of pancreatic cancer that the present invention is based on (GfO) n Based on the CHP sequence, a class of radionuclide-labeled molecular probes (CHP-PET probes) that can specifically target structurally denatured collagen in lesion ECM were designed. 68Ga-labeled CHP-PET probes can detect denatured collagen in PDAC in subcutaneous and orthotopic mouse models with excellent specificity. In addition, we used a transgenic KPC mouse model that can spontaneously develop PDAC. This model recapitulates the different stages of PDAC progression (including PanIN) and has a tumor microenvironment that is closer to human PDAC, thus providing results that are more consistent with actual clinical applications. Unfortunately, 68 Ga and 64 Cu-labeled CHP-PET probes cannot achieve in vivo visualization of pancreatic tumors in KPC mice due to inappropriate biodistribution. In order to improve the signal-to-noise ratio of CHP-PET probes at pancreatic tumor lesions, the present invention optimizes the CHP-PET probes. 18 The F-labeled CHP-PET probe was able to detect not only PDAC in the KPC model but also PanIN lesions with excellent clarity and signal-to-noise ratio, surpassing conventional FDG PET / CT imaging, providing a new and powerful tool for the early detection of pancreatic cancer.

[0260] Test Example 6: Detecting and staging collagen remodeling during progression of idiopathic pulmonary fibrosis

[0261] To further validate the versatility of CHP-PET probes for disease detection, this study used a bleomycin-induced pulmonary fibrosis mouse model to explore the potential of CHP-PET probes for detecting pulmonary fibrosis. The specific methods are as follows:

[0262] Bleomycin (BM) was dissolved in PBS and administered as a single dose of 5.0 mg / kg to each male C57BL / 6J mouse (6-8 weeks old). The control group received no treatment. After inhalation of bleomycin, the animals were rotated up and down to evenly distribute the drug solution in the lungs. The mice were observed for transient respiratory distress during the nasal drip process. The mice were placed in a cage and returned to the SPF animal room for breeding after their breathing stabilized. During the 28-day period of drug-induced pulmonary fibrosis, the mice were intravenously injected with approximately 9.67 MBq of 68 Ga-Cy5-CHP or 68 Ga-Cy5- S CHP.

[0263] The PET images 2 hours after injection are shown in Figure 16A, showing 68 Ga-Cy5-CHP accumulated specifically in the fibrotic lungs of BM-treated mice for 28 days, but not in the healthy lungs of control mice, whereas non-targeted 68 Ga-Cy5- SCHP did not preferentially enter the lungs of BM-treated mice. Immediately after PET / CT scanning, lungs were collected and radioactivity was measured. The quantitative results further supported the PET imaging (as shown in Figure 16B). Subsequently, PET-CT imaging was performed 7 days after drug induction during the inflammatory period. The imaging and quantitative results showed that the lungs 68 The uptake of Ga-Cy5-CHP was also significantly higher than that of the normal control (as shown in Figures 16C and 16D). 68 Ga-Cy5-CHP can detect lung lesions in vivo and has the potential to non-invasively detect early-stage pulmonary fibrosis.

[0264] The above results show that 68 In addition to detecting pancreatic cancer, Ga-Cy5-CHP can also detect and stage collagen remodeling during the progression of idiopathic pulmonary fibrosis. This result demonstrates that due to the conservation of structurally denatured collagen in multiple fibrotic diseases, the CHP-PET probe developed in this invention for detecting collagen structural denaturation can be used as a platform technology for the detection of other fibrotic diseases.

[0265] Test Example 7: CHP hybridization reveals mouse response to anti-fibrosis therapy

[0266] Unlike the static fact that increases and decreases in ECM collagen only reflect ECM deposition or regression, the production of structurally denatured collagen is related to both collagen production and collagen degradation. Therefore, detecting denatured collagen can also characterize the dynamic changes in ECM remodeling during disease progression and explore the underlying mechanisms. Given the current lack of therapies that can prevent or reverse organ fibrosis, the lack of effective drug efficacy testing is considered a major factor limiting the development of anti-fibrotic therapies. Therefore, detecting denatured collagen can assist in the study of disease progression and drug development.

[0267] This test example uses the 68 The Ga-Cy5-CHP probe was used to characterize the in vivo responses of mice to two different anti-fibrotic treatments, further demonstrating that the probe developed in this invention can not only be used for disease detection imaging, but also reflect the dynamic changes in ECM remodeling, guiding the study of the mechanisms of disease progression and the efficacy of drug development. The specific characterization method for this detection example is as follows:

[0268] (1) For efficacy testing of pirfenidone (PFD)

[0269] As one of the two treatments for IPF that are conditionally recommended in clinical practice, PFD not only inhibits the production of TGF-β1 and collagen synthesis, but also exerts an anti-fibrotic effect by inhibiting fibroblast proliferation and differentiation. PFD inhibits the synthesis and degradation of collagen. Therefore, this test case predicts that the lungs of BM-injured mice treated with PFD should have less denatured collagen. The specific PFD treatment method is shown in Figure 17A. PFD treatment was continuously given 1 day after bleomycin modeling, and the mice were injected into the tail vein on the 21st day after modeling. 68 Ga-Cy5-CHP, lungs were harvested for fluorescence imaging at 4 h.

[0270] The results showed that the PDF-treated group significantly reduced lung uptake of the probe in vivo compared with the untreated BM control (as shown in Figure 17B), indicating that hybridization of CHP with denatured collagen caused by lung fibrosis remodeling can reflect the response of mice to anti-fibrotic treatment.

[0271] (2) Research on dihydroxetine treatment pathways

[0272] In addition to inhibiting inflammation and collagen synthesis, there are also new strategies targeting collagen degradation pathways to promote ECM absorption in IPF. For example, agonism of the dopamine receptor D1 in lung fibroblasts using dihydrexidine (DHX) has been shown to accelerate the clearance of excess ECM in mouse lung fibrosis tissue by upregulating cathepsin K. Thus, DHX promotes collagen degradation, thereby reducing fibrosis.

[0273] We hypothesized that fibrotic lungs treated with DHX would show less collagen accumulation but higher levels of collagen degradation and denaturation compared to untreated BM controls. This hypothesis was further confirmed using animal experiments. The specific DHX treatment method is shown in Figure 17C. DHX treatment was continued for 10 days after bleomycin modeling. On day 24 after modeling, mice were injected into the tail vein with DHX. 68 Ga-Cy5-CHP, lungs were harvested for fluorescence imaging at 4 h.

[0274] The results are shown in Figure 17D, which show that the lungs of the DHX-treated group had significantly higher probe uptake than the untreated BM disease group. These results indicate that collagen hybridization can reveal subtle differences in different therapeutic pathways related to collagen metabolism (inhibition of synthesis vs. promotion of degradation).

[0275] From the test results of the above-mentioned test examples 1-7, it can be seen that the radionuclide-labeled molecular probe designed by the present invention, which can specifically target the denatured collagen in the pathological ECM structure, provides a new and powerful tool for the early detection of pancreatic cancer. Moreover, due to the conservation of ECM proteins, it can be applied as a platform technology in the detection and treatment of various other diseases characterized by ECM deposition. At the same time, since denatured collagen reflects the dynamic changes in ECM remodeling during disease progression, the probe developed by the present invention can be used not only for disease detection imaging, but also for guiding the study of the mechanism of disease progression and the efficacy of drug development.

[0276] Test Example 8: Chemical Stability Assessment

[0277] Based on the presence of only a very small amount of nuclides (such as 666MBq 68 Ga is only 0.00648 nmol; 2.5 GBq 18 F is only 0.03944 nmol). The amount of precursor added to each reaction (50 nmol) is significantly in excess compared to the nuclide atoms. Therefore, if the probe molecular structure is stable during radiochemical labeling, a sufficient amount of precursor molecules should be present in the reaction solution after labeling. In this test example, the reaction solutions after radiochemical labeling of different probes prepared in Examples 5-7 and Comparative Examples 4 and 6 were subjected to radioactive HPLC analysis and compared with the HPLC spectra of the corresponding probe precursors to determine their stability.

[0278] Judgment criteria: If there is a peak shape in the labeled reaction solution that is consistent with the corresponding probe precursor peak time, it can be considered that there is excess precursor in the reaction solution, indicating that the probe precursor structure is stable and has not undergone radiation deterioration.

[0279] Figure 18 shows radiolabeled collagen hybrid peptides 68 During the preparation of Ga-NODAGA-Ahx-(GfO)9, the HPLC spectrum of the labeled reaction solution and its probe precursor was shown in 68 After the Ga nuclide labeling was completed, there were still sufficient precursor molecules and only one radiation peak, indicating that the structural probe had good stability and no radiolysis occurred.

[0280] Figure 19 shows radiolabeled collagen hybrid peptide Al 18 During the preparation of F-NODAGA-Ahx-(GfO)9, the HPLC spectrum of the labeled reaction solution and its probe precursor showed that the probe 18 The F labeling process was very stable, with only one radioactive peak. In addition, there was an excess of unchelated and structurally intact precursor in the reaction product solution, indicating that the probe precursor was structurally stable and had not undergone radiation deterioration.

[0281] Figure 20 shows radiolabeled collagen hybrid peptide Al 18 During the preparation of F-NOTA2-Ahx-(GfO)9, the HPLC spectrum of the labeled reaction solution and its probe precursor showed that there was only one radioactive peak and there was an excess of unchelated and structurally intact precursor, which proved that the structural probe was in the presence of Al 18 The structure is stable during the F labeling process and no radiolysis occurs.

[0282] FIG21 shows the fluorescence-radioactive labeled collagen hybrid peptide Cy5-Ahx-K (NOTA-Al 18 F) -K (NH2) -Ahx - (GfO) 9 preparation process before and after the HPLC spectrum of the radionuclide labeling, the results show that there is no ultraviolet peak (red line marked position) consistent with the precursor peak time in the solution after radionuclide labeling, indicating that the excess probe precursor compound in the reaction solution is decomposed. Further, the reaction solution was subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) analysis for molecular weight detection. The results are shown in Figure 22, showing that the molecular weight of the substances in the reaction solution is less than that of the probe precursor, among which the molecular weight of the substances in the reaction solution is 3733.95Da and 4016.08Da, and the molecular weight of the probe precursor is 4140.01Da, indicating that the precursor molecule is in Al 18 The F labeling process was unstable and radiolysis occurred.

[0283] Figure 23 shows radiolabeled collagen hybrid peptide Al 18 During the preparation of F-NOTA-Ahx-(GfO)9, the HPLC spectrum of the reaction solution after labeling and its probe precursor showed that the solution after radionuclide labeling showed multiple radioactive peaks (pink line, 17.5-20.5min), indicating that there were multiple radiochemically labeled substances in the solution. It can be seen that the structure is in the Al 18 The F labeling process was unstable and radiolysis occurred.

[0284] The above results show that the radiolabeled collagen hybrid peptide of the present invention has a stable structure during the radiochemical labeling process, and the probe precursor hardly undergoes radiolysis after radionuclide labeling, which helps to improve its synthetic stability and radiochemical labeling yield and purity.

[0285] Test Example 9: Biodistribution Characterization Assessment

[0286] In order to intuitively evaluate the denatured collagen targeting specificity and in vivo biodistribution effects of the various constructed CHP-PET probes, this test example used the human pancreatic cancer cell line CFPAC-1 to construct a mouse subcutaneous tumor model. Then, approximately 8.42 MBq of the collagen hybrid peptide probes prepared in Examples 5-7 and Comparative Examples 3-6 were injected through the tail vein, and PET-CT imaging was performed for a specified time.

[0287] The specific method for establishing the mouse subcutaneous tumor model was as follows: 3×10 6 CFPAC-1 cells, wait until the subcutaneous tumor grows to 200mm 3 Follow-up experiments were conducted.

[0288] Figure 24 shows radiolabeled collagen hybrid peptides 68 PET / CT results of Ga-NODAGA-Ahx-(GfO)9 in mice bearing subcutaneous pancreatic cancer tumors demonstrated excellent biodistribution of the probe within these mice, demonstrated by significant signal in the subcutaneous tumor target organ, while uptake was low in metabolic non-target organs such as the liver and kidneys. By imaging one hour after tail vein injection, most of the probe had been excreted from the kidneys into the bladder. The overall low background signal resulted in a high signal-to-noise ratio at the tumor site. This excellent biodistribution profile of the probe opens the door to potential clinical translation.

[0289] Figure 25 shows radiolabeled collagen hybrid peptide Al 18 The PET / CT results of F-NOTA2-Ahx-(GfO)9 in pancreatic cancer subcutaneous tumor-bearing mice and normal mice for 1 hour showed that the biodistribution effect of the probe in subcutaneous tumor-bearing mice or normal mice was similar to that of the probe prepared in Example 1. 68 Ga-NODAGA-Ahx-(GfO)9 is similar to Ga-NODAGA-Ahx-(GfO)9 and also has an excellent tumor signal-to-noise ratio, especially in important non-target organs such as the liver and heart, where it has extremely low signals, which helps to improve the accuracy of in vivo imaging.

[0290] Figure 26 shows the Cy5-Ahx-K (NOTA- 68 The PET / CT results of the probe (Ga)-K(NH2)-Ahx-(GfO)9 in subcutaneous pancreatic cancer-bearing mice showed that after 1 hour, the probe detected strong signals in the liver, kidney, and pancreas of subcutaneous pancreatic cancer-bearing mice, especially in the kidney, which caused strong signal interference to the in vivo imaging of pancreatic lesions.

[0291] Figure 27 shows the Cy5-Ahx-K (NOTA-Al 18 The 1-hour PET / CT results of F)-K(NH2)-Ahx-(GfO)9 in mice showed that although the probe had low liver uptake, it had strong kidney uptake, which still interfered with the imaging of organs around the kidney, such as the pancreas.

[0292] Figure 28 shows the Cy5-Ahx-K (NOTA- 68The 1-hour PET / CT results of Ga)-Ahx-(GfO)9 in subcutaneous pancreatic cancer tumor-bearing mice showed that the probe differed from the probe in Comparative Example 1 in that the lysine (K(NH2)) residue was removed. The test results showed that the biodistribution of the probe changed significantly, with extremely strong uptake in the liver, kidneys, etc., as well as in non-target organs such as the heart. The high background noise made the tumor signal almost invisible.

[0293] Figure 29 shows the radiolabeled collagen hybrid peptide Al prepared in Comparative Example 6 18 The 1-hour PET / CT results of F-NOTA-Ahx-(GfO)9 in subcutaneous pancreatic cancer tumor-bearing mice showed that the probe differed from the probe in Comparative Example 4 in that the fluorescent dye group and other redundant amino acids were removed. The results showed that although the probe had signal accumulation in the subcutaneous tumor, it was strongly taken up in the liver, kidneys, etc., and its biodistribution characteristics were worse than those of the probe in Comparative Example 4.

[0294] Furthermore, a comparison of the probes prepared in Comparative Examples 5 and 6 demonstrates that simple structural deletions from the previously investigated structure alone can improve the radiochemical labeling stability and biodistribution characteristics of the probe, enabling the preparation of probes for in vivo PET imaging and even clinical research. Even subtle differences in the functional groups of the probe structure can lead to significant differences in in vivo biodistribution characteristics. Currently, there is no method to accurately predict the relationship between chemical structure and biodistribution characteristics, necessitating systematic optimization and validation of different probe structures.

[0295] The above results show that the radiolabeled collagen hybrid peptide prepared by the present invention has excellent biodistribution characteristics with high uptake in target organs such as tumors and low uptake in non-target organs such as liver and kidneys, and has important clinical translation value.

[0296] As can be seen from the above test examples 8-9, the present invention further screens out radiolabeled collagen hybrid peptides by optimizing the structure of the radiolabeled collagen hybrid peptides, which not only have the characteristics of radiochemical labeling stability, but also have excellent biodistribution characteristics such as high uptake in target organs such as tumors and low uptake in non-target organs such as the liver and kidneys.

[0297] While the embodiments of the present invention have been described in detail above, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A radiolabeled collagen hybrid peptide, characterized in that: The radiolabeled collagen hybrid peptide comprises: (a) a radionuclide-chelator complex bound by a coordinate bond; and (b) collagen hybrid peptide; wherein the radionuclide-chelator complex is connected to the collagen hybrid peptide via a linker molecule, and the linker molecule comprises at least one of a lysine residue, an oligoglycine residue, 6-aminohexanoic acid, and polyethylene glycol; The amino acid sequence of the collagen hybrid peptide is (GfO) n or (GPO) n , wherein n is a positive integer between 6 and 10.

2. The radiolabeled collagen hybrid peptide according to claim 1, characterized in that The radionuclide in the radionuclide-chelator complex is selected from Al 18 F. 64 Cu, 67 Cu, 67 Ga, 68 Ga, 99 mTc, 89 Zr, 111 In, 177 Lu, 186 Re、 225 Any of Ac.

3. The radiolabeled collagen hybrid peptide according to claim 1, characterized in that The chelating agent in the radionuclide-chelating agent complex is selected from any one of NOTA, NODAGA, HYNIC, DOTA and DTPA or a derivative thereof; The derivatives of NOTA include NOTA-NH2 or p-SCN-Bn-NOTA; wherein the NOTA-NH2 has a structure as shown in formula (a-1): The p-SCN-Bn-NOTA has a structure as shown in formula (a-2): The NODAGA has the structure shown below: The DOTA has the following structure:

4. The radiolabeled collagen hybrid peptide according to claim 1, characterized in that The radiolabeled collagen hybrid peptide further comprises a fluorescent dye; The fluorescent dye is connected to the linker molecule via an amino acid or an amino acid derivative; The fluorescent dye is selected from any one of anthocyanin, rhodamine, BODIPY, FITC, erythrosine, phthalocyanine, phycocyanin, phycoerythrin and Alexa Fluor dyes.

5. The radiolabeled collagen hybrid peptide according to any one of claims 1 to 4, characterized in that When the linker molecule of the radiolabeled collagen hybrid peptide comprises a lysine residue and 6-aminohexanoic acid, the radiolabeled collagen hybrid peptide is a compound represented by any one of formula (I-1) to formula (I-4): Alternatively, when the linker molecule of the radiolabeled collagen hybrid peptide is 6-aminohexanoic acid, the radiolabeled collagen hybrid peptide is a compound represented by any one of formula (II-1) to formula (II-4):

6. A method for preparing the radiolabeled collagen hybrid peptide according to any one of claims 1 to 5, characterized in that: The following steps are involved: A resin solid phase synthesis method is adopted to obtain a precursor probe containing the chelating agent and the collagen hybrid peptide, and then the precursor probe is labeled with the radioactive nuclide to obtain the product.

7. A conjugate, characterized in that Comprising: the radiolabeled collagen hybrid peptide according to any one of claims 1 to 5, and a coupling portion; Wherein, the coupling part includes at least one of a protein, a drug or a detectable marker.

8. A contrast agent, characterized in that A method comprising the radiolabeled collagen hybrid peptide according to any one of claims 1 to 5.

9. Use of the radiolabeled collagen hybrid peptide according to any one of claims 1 to 5 in any one of the following: A) preparing products that specifically target structurally denatured collagen; B) preparing products for cancer diagnosis or imaging of lesions characterized by fibrotic lesions; C) preparing products for detecting organ fibrosis diseases; D) Evaluate or screen for anti-pancreatic ductal adenocarcinoma or anti-fibrosis drugs.

10. A kit for imaging tumors or organ fibrosis, characterized in that: A method comprising the radiolabeled collagen hybrid peptide according to any one of claims 1 to 5.

11. A method for treating or diagnosing organ fibrosis-related diseases, characterized in that: The method comprises administering the radiolabeled collagen hybrid peptide according to any one of claims 1 to 5, the conjugate according to claim 7, or the contrast agent according to claim 8 to a subject.

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