A myocarditis nuclide molecular probe and its preparation method and application

Abstract

The present invention belongs to the technical field of radionuclide molecular probes, and in particular, relates to a myocarditis radionuclide molecular probe, a preparation method thereof, and an application thereof. The myocarditis radionuclide molecular probe comprises a DPP4 inhibitor, a bifunctional chelating agent covalently bonded to the amino group of the DPP4 inhibitor, and a metal radionuclide coordinated with the bifunctional chelating agent. The myocarditis radionuclide molecular probe provided by the present invention has high specificity and good targeting, has obvious high uptake in myocarditis inflammatory cells and low uptake in non-target tissues, and has the advantages of high resolution and high sensitivity in imaging. It can be applied to the early diagnosis of the degree of cardiac inflammatory infiltration, efficacy evaluation, and prognosis assessment, etc., and has good clinical application prospects in the fields of realizing non-invasive nuclear medicine diagnosis and treatment as a myocarditis imaging agent.

Description

Technical Field

[0001] The invention relates to the technical field of radionuclide molecular probes, and in particular to a myocarditis radionuclide molecular probe and a preparation method and application thereof. Background Art

[0002] Myocarditis refers to localized or diffuse inflammatory lesions of the myocardium caused by non-ischemic causes. Its histological characteristics are infiltration of inflammatory cells in the myocardial interstitium, accompanied by myocardial cell degeneration and necrosis. According to different causes, myocarditis can be divided into two categories: infectious and non-infectious. Among the above causes, viral infection is the most common, including Coxsackie A virus, Coxsackie B virus, adenovirus, cytomegalovirus, Japanese encephalitis virus, Epstein-Barr virus, influenza virus, parainfluenza virus and herpes simplex virus. Many viruses can cause or induce myocarditis. The clinical symptoms of myocarditis are atypical, mainly depending on the extent and severity of the lesions. A few may be completely asymptomatic. Mild cases may show non-specific symptoms such as fever, cough, diarrhea, etc. Severe cases may show severe arrhythmia, heart failure, dyspnea or fatigue. Patients with critical conditions may show cardiogenic shock and refractory arrhythmia or even death. Therefore, the diagnosis and treatment of myocarditis is difficult.

[0003] Traditional diagnostic methods include medical history, electrocardiogram, serum markers, cardiac ultrasound and cardiac magnetic resonance imaging, which lack sensitivity and specificity and cannot be used as a reliable basis for diagnosis. The gold standard for diagnosis is endomyocardial biopsy, which requires invasive methods to obtain myocardial tissue. Due to its high risk, it cannot be popularized and its clinical application is severely limited. In recent years, radionuclide imaging technology has emerged in biological imaging applications, which can provide real-time, on-site and non-invasive imaging modes. Some researchers have tried to use radioactive contrast agents for early diagnosis of myocarditis, including non-targeted probes commonly used in clinical practice. 18 F-fluorodeoxyglucose or targeted probe developed for translocation protein 18 F-fluoromethyl-PBR28, etc. However, the aforementioned radioactive contrast agents have shortcomings in the application of cardiac inflammation imaging, such as 18 F-fluorodeoxyglucose is a non-specific functional imaging probe that will be taken up in large quantities by myocardial cells, thus interfering with the detection of cardiac inflammatory foci. 18 The F-fluoromethyl-PBR28 probe has a lower sensitivity in detecting myocardial inflammation and is more suitable for detecting extracardiac inflammation. Therefore, it is urgent to find a non-invasive or low-invasive myocarditis detection method with high specificity and sensitivity for myocardial inflammation, which is of great significance for the clinical diagnosis and treatment of myocarditis. Summary of the invention

[0004] In view of the problems in the prior art of lacking myocardial inflammation focus targeting and specificity, high sensitivity and / or non-invasive myocarditis molecular probes, the present invention provides a myocarditis radionuclide molecular probe and a preparation method thereof, and provides the use of the myocarditis radionuclide molecular probe in a myocarditis contrast agent, as well as a myocarditis contrast agent containing the aforementioned myocarditis radionuclide molecular probe.

[0005] To achieve the above object, the present invention is specifically implemented through the following technical solutions:

[0006] The first aspect of the present invention provides a myocarditis radionuclide molecular probe, comprising a DPP4 inhibitor, a bifunctional chelating agent covalently bonded to the amino group of the DPP4 inhibitor, and a metal radionuclide coordinated with the bifunctional chelating agent, wherein the DPP4 inhibitor is selected from at least one of sitagliptin, saxagliptin, alogliptin, linagliptin and gemagliptin.

[0007] Furthermore, the DPP4 inhibitor is selected from linagliptin.

[0008] Further, the bifunctional chelating agent is selected from at least one of DOTA-NHS-ester, NOTA-NHS-ester, p-SCN-Bn-NOTA, p-SCN-Bn-DOTA, p-SCN-Bn-PCTA, p-SCN-Bn-HEHA, p-SCN-Bn-TCMC and p-SCN-Bn-DTPA.

[0009] Furthermore, the bifunctional chelating agent is selected from at least one of p-SCN-Bn-NOTA, p-SCN-Bn-DOTA, p-SCN-Bn-PCTA, p-SCN-Bn-HEHA, p-SCN-Bn-TCMC and p-SCN-Bn-DTPA.

[0010] Further, the bifunctional chelating agent is selected from p-SCN-Bn-DOTA.

[0011] Furthermore, the metal nuclide is selected from 68 Ga, 18 F-Al, 64 Cu, 89 Zr, 99m Tc, 111 In, 188 Re, 186 Re, 177 Lu and 90 At least one of Y.

[0012] Furthermore, the metal nuclide is selected from 68 Ga.

[0013] The second aspect of the present invention provides a method for preparing the myocarditis nuclide molecular probe as described above, comprising the following steps:

[0014] S1, dissolving the DPP4 inhibitor and the bifunctional chelator in an organic solvent, reacting under alkaline conditions, and purifying to obtain a precursor compound after the reaction is completed;

[0015] S2, adding the metal nuclide eluent and the precursor compound into a buffer solution to carry out a metal nuclide labeling reaction, and after the reaction is completed, separating and purifying to obtain a myocarditis nuclide molecular probe.

[0016] Furthermore, in step S1, the molar ratio of the DPP4 inhibitor to the bifunctional chelator is 1-2:1.

[0017] Furthermore, in step S1, the organic solvent is dimethyl sulfoxide, methanol or chloroform.

[0018] Furthermore, in step S1, the reaction pH is 8.5-9.5, and the reaction is carried out at 20-35°C in the dark with shaking for 1-3 hours or at 4°C overnight.

[0019] Furthermore, in step S1, high performance liquid chromatography is used to separate and purify the precursor compound.

[0020] Furthermore, in step S2, the dosage ratio of the metal nuclide to the precursor compound in the metal nuclide eluent is 30-50MBq:1nM.

[0021] Furthermore, in step S2, the buffer is 0.1-1M sodium acetate buffer.

[0022] Furthermore, in step S2, the reaction pH is 4.0-4.5, and the reaction is oscillated at 90-100° C. for 10-20 min.

[0023] Furthermore, in step S2, a solid phase extraction C18 column is used to separate and purify the myocarditis nuclide molecular probe.

[0024] The third aspect of the present invention provides the use of the myocarditis radionuclide molecular probe described above in the preparation of a myocarditis imaging agent.

[0025] Furthermore, the myocarditis is viral myocarditis.

[0026] A fourth aspect of the present invention provides a myocarditis imaging agent, comprising the myocarditis radionuclide molecular probe as described above.

[0027] The advantages and positive effects of the present invention are:

[0028] The myocarditis radionuclide molecular probe provided by the present invention has high specificity and good targeting, has obvious high uptake in myocarditis inflammatory cells and low uptake in non-target tissues, and imaging has the advantages of high resolution and high sensitivity. It can be applied to the early diagnosis of the degree of cardiac inflammatory infiltration, efficacy evaluation and prognosis assessment, etc., and has good clinical potential and application prospects in the field of non-invasive nuclear medicine diagnosis and treatment as a myocarditis imaging agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0030] Figure 1 The mass spectrum of the myocarditis nuclide molecular probe precursor compound synthesized in the embodiment of the present invention;

[0031] Figure 2 The ultraviolet spectrum of the myocarditis nuclide molecular probe precursor compound synthesized in the embodiment of the present invention;

[0032] Figure 3 The HPLC chromatograms of the myocarditis nuclide molecular probe before and after purification according to the embodiment of the present invention;

[0033] Figure 4 The results of cardiac pathological examination of myocarditis model mice in the embodiment of the present invention are shown in Figures A to C, HE staining, immunohistochemical staining, and immunofluorescence analysis, respectively;

[0034] Figure 5 Figure 1 is a graph showing the results of a cellular uptake experiment of a radionuclide molecular probe according to an embodiment of the present invention; Figure A shows the expression level of DPP4 protein in 293T cells overexpressing DPP4, and Figure B shows the uptake value of the radionuclide molecular probe by 293T cells with high DPP4 expression;

[0035] Figure 6 The in vivo targeting experimental results of the radionuclide molecular probe of the embodiment of the present invention are shown in Figure A, wherein Figure A is an untreated mouse injected subcutaneously in the axilla, and Figure B is a mouse injected subcutaneously in the axilla with a 293T cell suspension overexpressing the DPP4 protein;

[0036] Figure 7 The PET / CT imaging results of the radionuclide molecular probe of the embodiment of the present invention in the myocarditis model mouse; wherein, Figure A is a mouse 3 days before virus infection, Figure B is a mouse 7 days after virus infection, and Figure C is a statistical graph of the radiation uptake values ​​of the heart in the mouse imaging before and after virus infection. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0038] Based on the information contained in the present invention, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for the purpose of illustrating specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0039] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers and other numerical values ​​used in the present invention to express the amount, percentage, etc. should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties to be obtained. Each numerical parameter should at least be regarded as obtained according to the reported significant figures and by conventional rounding methods.

[0040] In addition, it should be noted that, unless otherwise defined, in the context of the present invention, the scientific and technical terms used should have the meanings commonly understood by those of ordinary skill in the art. The meanings of the terms "comprise", "include", "contain", "have" and the like are non-restrictive, i.e., other steps and other ingredients that do not affect the result can be added. The term "and / or" should be regarded as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" will be regarded as including the following situations: (i) A, (ii) B, and (iii) A and B.

[0041] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is described in detail below.

[0042] Using targeted compounds to synthesize radioactive imaging agents, compared with 18 F-fluorodeoxyglucose or 18 Molecular probes such as F-fluoromethyl-PBR28 have the characteristics of strong tissue penetration and high specificity. However, in the research of radionuclide contrast agents for myocarditis, no radionuclide-labeled targeted small molecule compounds have been reported.

[0043] The present invention constructs a myocarditis model by infecting mice with Coxsackie B virus. In the study of viral myocarditis, it is found that dipeptidyl peptidase-4 (DPP4) protein is specifically and highly expressed in the inflammatory cell infiltration area of ​​myocarditis, while the intrinsic cardiomyocytes and myocardial fibroblasts of the heart hardly express DPP4 protein, which suggests that DPP4 can be used as a biological target for myocarditis imaging.

[0044] Based on the above findings, the present invention uses DPP4 inhibitors as the main structure. DPP4 inhibitors can be selected from sitagliptin, saxagliptin, alogliptin, linagliptin and gemigliptin. These chemicals are small molecule compounds with high targeting to DPP4. Taking linagliptin as an example, linagliptin belongs to a non-peptide DPP4 targeting compound with a xanthine structure, which can form a non-covalent bond with the group of the DPP4 active site. As a small molecule drug that has been used clinically for many years, the safety of linagliptin is beyond doubt. Its structural formula is as follows:

[0045]

[0046] The structural formula of sitagliptin is shown below:

[0047]

[0048] The structural formula of saxagliptin is shown below:

[0049]

[0050] The structural formula of alogliptin is shown below:

[0051]

[0052] The structural formula of Gemtagliptin is shown below:

[0053]

[0054] At the same time, the present invention forms a molecular probe by chelating radionuclides and connecting targeting molecules DPP4 inhibitors through bifunctional chelators. A bimolecular chelator is a chelator containing two reactive groups at the same time, one of which can covalently bind to -NH2, -SH or -OH groups of biological molecules such as polypeptides and proteins, such as carboxyl groups (-COOH), succinate groups (-NHS), thiocyanate groups (-NCS), etc., and the other reactive group is a coordinating atom on a heterocyclic ring, which can form a stable complex with metal nuclides (such as Ga, Cu).

[0055] Based on this, one embodiment of the present invention provides a myocarditis radionuclide molecular probe, comprising a DPP4 inhibitor, a bifunctional chelating agent covalently bonded to the amino group of the DPP4 inhibitor, and a metal radionuclide coordinated to the bifunctional chelating agent, wherein the DPP4 inhibitor is selected from at least one of sitagliptin, saxagliptin, alogliptin, linagliptin and gemagliptin.

[0056] The present invention uses a bifunctional chelating agent to react with the primary amino group of a DPP4 inhibitor to form a stable covalent bond, so as to place the complete structure of the DPP4 inhibitor outside, retain its activity of specifically binding to DPP4, and at the same time, the bifunctional chelating agent forms a coordination bond with a metal nuclide to obtain a nuclide molecular probe. The nuclide molecular probe of the present invention has good affinity and targeting for inflammatory cells of myocarditis with high expression of DPP4 protein. The nuclide molecular probe of the present invention is injected into a mouse model of viral myocarditis induced by coxsackie virus B3 (CVB3) and imaging is performed. It is found that the probe has obvious high uptake in the myocardial inflammation focus of the myocarditis model mouse and low uptake in non-target tissues, indicating that it has strong specificity and targeting, and the imaging has the advantages of high resolution and high sensitivity, etc., and can be applied to the early diagnosis of the degree of cardiac inflammatory infiltration, efficacy evaluation and prognosis evaluation, etc., and has good clinical application prospects in the fields of non-invasive nuclear medicine diagnosis and treatment as a myocarditis imaging agent. In addition, the DPP4 inhibitor selected in the present invention has been proven to have good biosafety in many years of drug application, and the prepared molecular probe has a safe metabolic pathway, does not damage organ functions, and has good biosafety.

[0057] Preferably, the DPP4 inhibitor is selected from linagliptin. The bifunctional chelating agent that can be used in the present invention is selected from DOTA, NOTA, NETA, PCTA, HEHA, TCMC, DTPA, hynic and their derivatives, specifically, including but not limited to: 1) DOTA-NHS-ester, NOTA-NHS-ester, this type of chelating agent has a succinate group (-NHS), which is an activatable esterification agent. When reacting with a molecule containing an amino group, NHS The ester will form an amide bond with the amino group, thereby connecting the DOTA and NOTA ester groups to the DPP4 inhibitor molecules; 2) p-SCN-Bn-NOTA, p-SCN-Bn-DOTA, p-SCN-Bn-PCTA, p-SCN-Bn-HEHA, p-SCN-Bn-TCMC and p-SCN-Bn-DTPA. This type of chelating agent has a reactive electrophilic group - thiocyanate group (-NCS), which can undergo nucleophilic substitution reaction with the amino group of the DPP4 inhibitor under alkaline conditions (pH 8.5 to pH 9.5) to form a stable thiourea bond (-NH-C(=S)-NH-).

[0058] Preferably, the bifunctional chelating agent is selected from at least one of p-SCN-Bn-NOTA, p-SCN-Bn-DOTA, p-SCN-Bn-PCTA, p-SCN-Bn-HEHA, p-SCN-Bn-TCMC and p-SCN-Bn-DTPA.

[0059] Radioactive metal nuclides are used as tracers. By using the targeted distribution of nuclide molecular probes in the myocardial inflammatory focus cells in vivo, the tissue-penetrating rays emitted by the radioactive nuclides during nuclear decay are detected and recorded in vitro by instruments, so as to achieve the purpose of locating and qualitatively diagnosing myocarditis. Therefore, the present invention does not specifically limit the type of metal nuclides, and any nuclides that can spontaneously emit tissue-penetrating rays are sufficient, including but not limited to: beta rays and gamma rays.

[0060] The metal nuclides that can be used in the present invention include but are not limited to: 68 Ga, 18 F-Al, 64 Cu, 89 Zr, 99m Tc, 111 In, 188 Re, 186 Re, 177 Lu and 90 Y.

[0061] In a preferred embodiment, the bifunctional chelating agent is p-SCN-Bn-DOTA, and the metal nuclide is 68 Ga.

[0062] Imaging methods include, but are not limited to, positron emission tomography / X-ray computed tomography (PET / CT), positron emission tomography and magnetic resonance imaging (PET / MR), positron emission tomography (PET), single photon emission computed tomography (SPECT / CT), single photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI).

[0063] Another embodiment of the present invention provides a method for preparing the myocarditis nuclide molecular probe as described above, comprising the following steps:

[0064] S1, dissolving the DPP4 inhibitor and the bifunctional chelator in an organic solvent, reacting under alkaline conditions, and purifying to obtain a precursor compound after the reaction is completed;

[0065] S2, adding the metal nuclide eluent and the precursor compound into a buffer solution to carry out a metal nuclide labeling reaction, and after the reaction is completed, separating and purifying to obtain a myocarditis nuclide molecular probe.

[0066] The preparation method of the myocarditis nuclide molecular probe has the same advantages as the myocarditis nuclide molecular probe described above over the prior art, which will not be described in detail. In addition, the preparation method adopted by the present invention is simple, has a high yield, and has a fast reaction speed and low requirements on reaction equipment.

[0067] Optionally, the DPP4 inhibitor is selected from at least one of sitagliptin, saxagliptin, alogliptin, linagliptin and gemagliptin; preferably linagliptin.

[0068] Optionally, in step S1, the molar ratio of the DPP4 inhibitor to the bifunctional chelator is 1-2:1.

[0069] Optionally, in step S1, the organic solvent is dimethyl sulfoxide (DMSO), methanol or chloroform.

[0070] Optionally, in step S1, the reaction pH is 8.5-9.5, and the reaction is carried out at 20-35°C in the dark for 1-3 hours or at 4°C overnight, specifically at pH 7.5 and 25°C in the dark for 2 hours. The method for separating and purifying the precursor compound preferably adopts high performance liquid chromatography separation and purification, and the product at the corresponding separation peak is collected to obtain the precursor compound.

[0071] In step S2, the metal nuclide eluent may vary slightly depending on the nuclide selected. 68 For example, 68 The eluent contains Ga 68The hydrochloric acid solution of GaCl3 is specifically obtained by reacting the germanium gallium generator eluted with hydrochloric acid to obtain radioactive 68 The concentration of HCl used for elution in the mixture of GaCl3 and hydrochloric acid is adjusted according to the demand, and can be 0.01-0.1 M, specifically, 0.05 M. After obtaining the metal nuclide elution solution, the pH is adjusted to an acidic environment (pH 4.0-4.5) by a buffer solution, and then the precursor compound is added to form a chelating reaction system.

[0072] The preparation method of different metal nuclide eluents is a conventional technique in the art, and the present invention will not repeat it in detail. In addition, it should be noted that the reaction mechanism of different metal nuclide eluents and the precursor compound of the present invention is basically the same, both of which are through the coordination of metal and bifunctional chelating agent, therefore, different eluents have little effect on the chelation reaction.

[0073] Optionally, in step S2, the ratio of the metal nuclide to the precursor compound is 30-50MBq:1nM. In other words, the amount of the metal nuclide added is based on activity, and 30-50MBq of the metal nuclide activity is added per nM of the precursor compound, preferably 37MBq.

[0074] Optionally, in step S2, the buffer is sodium acetate buffer with a concentration of 0.1-1M.

[0075] Optionally, in step S2, during the chelation reaction, the reaction pH is 4.0-4.5, and the reaction is oscillated at 90-100°C for 10-20 minutes, specifically, the reaction can be oscillated at pH 4.0 and 95°C for 15 minutes. The method for separating and purifying the myocarditis nuclide molecular probe preferably uses solid phase extraction C18 column purification, and further verifies the structure of the synthesized product by mass spectrometry and ultraviolet spectroscopy.

[0076] Another embodiment of the present invention provides the use of the myocarditis radionuclide molecular probe as described above in the preparation of a myocarditis contrast agent, especially in the preparation of a viral myocarditis contrast agent.

[0077] The application of the myocarditis radionuclide molecular probe in the preparation of myocarditis imaging agent has the same advantages as those of the myocarditis radionuclide molecular probe described above over the prior art, which will not be described in detail here.

[0078] Based on the same inventive concept as above, another embodiment of the present invention provides a myocarditis contrast agent, comprising the myocarditis nuclide molecular probe as described above.

[0079] The advantages of the myocarditis contrast agent are the same as those of the myocarditis radionuclide molecular probe described above over existing techniques, which will not be described in detail here.

[0080] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples without specifying specific conditions are generally carried out according to the conditions recommended by the manufacturer.

[0081] In the following embodiments, the DPP4 inhibitor is selected from linagliptin, and the metal nuclide is selected from 68 Ga, bifunctional chelating agent selected p-SCN-Bn-DOTA (purchased from Macrocyclics America, product number B-205), 68 The synthetic route of Ga-p-SCN-Bn-DOTA-linagliptin nuclide molecular probe is as follows:

[0082]

[0083] The reaction processes of DPP4 inhibitors sitagliptin, saxagliptin, alogliptin and gemagliptin, and bifunctional chelators p-SCN-Bn-NOTA, p-SCN-Bn-DOTA, p-SCN-Bn-PCTA, p-SCN-Bn-HEHA, p-SCN-Bn-TCMC and p-SCN-Bn-DTPA are the same as above.

[0084] Example 1 Myocarditis radionuclide molecular probe and its preparation

[0085] A myocarditis nuclide molecular probe, comprising linagliptin, a bifunctional chelating agent p-SCN-Bn-DOTA covalently bonded to the amino group of linagliptin, and a metal nuclide coordinated and bonded to the bifunctional chelating agent 68 Ga, its structural formula is as follows:

[0086]

[0087] The preparation method comprises the following steps:

[0088] S1. Dissolve linagliptin and bifunctional chelating agent p-SCN-Bn-DOTA in an organic solvent DMSO at a molar ratio of 1.2:1, adjust the reaction pH to 7.5, and react at 25°C in the dark for 2 h. After the reaction is completed, separate and purify by high performance liquid chromatography to obtain a precursor compound.

[0089] The separation and purification steps of the high performance liquid chromatography method include: a chromatographic column is a C18 light Sep-Pak cartridge (Waters, USA), a mobile phase is 15-35% acetonitrile, and a linear gradient elution is adopted. From 0 to 20 minutes, the acetonitrile concentration is increased from 15% to 35% at a flow rate of 1 mL / min, and then the concentration is maintained and eluted to 30 minutes; the eluate of 26-28 minutes is collected, and the precursor compound is obtained by drying with nitrogen.

[0090] The liquid chromatography mass spectrometry (LC-MS) of the precursor compound is shown in Figure 1 As shown, it can be seen that the molecular weight of the target product is 1023.4385. If it carries one charge, the molecular weight displayed by LC-MS is 1024.44. If it carries two charges, the molecular weight displayed by LC-MS is 512.73. If it carries three charges, the molecular weight displayed by LC-MS is 342.15. If it carries four charges, the molecular weight displayed by LC-MS is 256.87. The mass spectrum shows that the molecular weight of the target product is 1023.44, but there is a slight difference in the charge, indicating that the target product was successfully synthesized.

[0091] The UV spectrum of the precursor compound is shown in Figure 2 As shown, DOTA represents p-SCN-Bn-DOTA, HY19285 represents linagliptin, and DOTA-HY19285 represents DOTA-linagliptin. It can be seen from the figure that the purified product DOTA-linagliptin contains the characteristic ultraviolet absorption peaks of DOTA and linagliptin.

[0092] S2, through the hydrochloric acid elution of the germanium gallium generator reaction to obtain 68 GaCl3 hydrochloric acid solution, as 68 The GaCl3 eluent was adjusted to pH 4.0 by 0.25M sodium acetate buffer, and the purified precursor compound was added and mixed to obtain a reaction mixture containing 111MBq of active 68 GaCl3 and 3 nM precursor compound were shaken at 95°C for 15 min. After the reaction was completed, it was separated and purified by solid phase extraction C18 column to obtain the myocarditis nuclide molecular probe.

[0093] The solid phase extraction C18 column separation and purification steps include: 1) C18 column activation: 10 mL of anhydrous ethanol and 10 mL of ultrapure water were dripped in sequence; 2) C18 column purification: the reaction solution was loaded onto the C18 column, and after the dripping was completed, 10 mL of ultrapure water was dripped to remove free 68 Ga, then 0.5 mL of anhydrous ethanol was quickly pushed to obtain the product 68 Ga-p-SCN-Bn-DOTA-linagliptin nuclide molecular probe, hereinafter referred to as 68 Ga-DOTA-linagliptin.

[0094] 68 The HPLC chromatograms of Ga-DOTA-linagliptin probe before and after purification are shown in Figure 2. Figure 3 As shown, peak ① is free 68 Ga, peak ④ is 68 Ga-DOTA-linagliptin, peaks ②③ are impurities. It can be seen from the figure that after purification by C18 column, most of the obtained 68Ga-DOTA-linagliptin.

[0095] Example 2 Analysis of DPP4 protein expression in cardiac tissue of mice with myocarditis

[0096] Myocarditis mouse model: The Coxsackie B virus type 3 (CVB3) used in viral myocarditis modeling was obtained from the Institute of Virology, Wuhan University, and was amplified and passaged in Hela cells. Referring to the relevant literature "Chen Liang. Study on the inhibition of CVB3-induced viral myocarditis by CYP2J2 / EETs and its mechanism [D]. Huazhong University of Science and Technology, 2018.", 4-week-old Balbc male mice (from Jiangsu Jicui Pharmaceutical Co., Ltd.) were selected and intraperitoneally injected with 10 5 The model was established with 150 μL of virus solution at a TCID50 dose. The mice in the control group were intraperitoneally injected with 150 μL of PBS solution.

[0097] Heart tissues of model mice were obtained on day 7 (d) after CVB3 virus infection, paraffin sections were prepared, and HE staining, immunohistochemical staining and immunofluorescence analysis were performed (DPP4 antibody was purchased from RD Company, catalog number AF954). The results are shown in Figure 4 , where Figures AC are HE staining, immunohistochemistry staining and immunofluorescence results, respectively. The myocarditis group in the figure represents model mice.

[0098] from Figure 4 It can be seen that in the paraffin sections of the hearts of mice in the control group, no inflammatory cell infiltration was observed in the myocardium, and immunohistochemistry and immunofluorescence analysis showed that DPP4 was extremely low in expression. In the heart sections of model mice on the 7th day after CVB3 virus infection, a large number of inflammatory cells were observed to infiltrate (see the black solid arrows), and immunohistochemistry and immunofluorescence showed that the expression of DPP4 protein was significantly increased. The above results show that DPP4 protein is specifically highly expressed in the inflammatory cell infiltration area of ​​viral myocarditis, while myocardial cells and myocardial fibroblasts in non-inflammatory areas hardly express DPP4 protein. Therefore, DPP4 has the potential to become a specific target for myocarditis imaging.

[0099] Example 3 Affinity test of myocarditis nuclide molecular probe and 293T cells overexpressing DPP4 protein

[0100] Ordinary 293T cells (purchased from ATCC) have very low DPP4 protein expression and can be used as experimental control (293T-Control). Overexpression of DPP4 by lentiviral transfection can obtain a 293T cell line with high DPP4 protein expression (293T-DPP4 cells); among them, the DPP4 transcript sequence ID is NM001935.4, the protein sequence ID is NP001926.2, and the coding sequence is 2301bp.

[0101] The targeting and specificity of the nuclide molecular probe were verified by in vitro cell uptake; the myocarditis nuclide molecular probe synthesized in Example 1 was added to the culture medium in conventional in vitro culture control 293T cells and 293T-DPP4 cells, incubated for an appropriate time, and the cell radioactivity uptake value was detected to evaluate the binding affinity of the probe with the DPP4 protein in vitro. The specific steps are as follows: the frozen 293T-Control and 293T-DPP4 cell lines were revived and routinely cultured and expanded, the two cells with good morphology were digested and resuspended, and then inoculated in a 24-well plate, and when the cells were attached and the density was about 70-80%, the supernatant was discarded and starved for 2 hours with serum-free high-glucose DMEM medium. The nuclide molecular probe was dissolved in serum-free high-glucose DMEM medium, and 1 mL of serum-free medium containing 2 μCi of nuclide molecular probe was added to each well of the 24-well plate. At 30min and 60min, the supernatant and the cell layer lysed by NaOH were taken, and the radioactivity counts of the supernatant and the cell layer were determined using a γ-counter, and the uptake rate was calculated.

[0102] Figure 5 The present invention shows the nuclide molecular probe 68 The results of the cell uptake experiment of Ga-DOTA-linagliptin, Figure A shows the expression level of DPP4 protein in 293T cells overexpressing DPP4, detected by Western Blot method, DPP4 antibody purchased from RD company, item number AF954, GAPDH protein was used as an internal reference, GAPDH antibody purchased from Proteintech company, item number 60004-1, Figure B shows the uptake value of radionuclide molecular probe by 293T cells with high DPP4 expression, in the figure, Control represents the control group 293T cells, and DPP4 represents the 293T cells overexpressing DPP4. It can be seen from the figure that the expression of DPP4 protein in 293T cells (293T-Control) is extremely low. After transfection and overexpression of DPP4, a 293T cell line with high DPP4 expression (293T-DPP4) can be obtained. Compared with the control group 293T cells, the uptake rate of 293T-DPP4 cells for radionuclide molecular probes is significantly higher, indicating that the probe has a higher in vitro binding affinity.

[0103] In addition, each mouse in the DPP4 group was subcutaneously injected with 4.5×10 6 The mice were imaged with the radionuclide molecular probe 2 hours after injection to determine the in vivo affinity of the probe to DPP4-293T cells.

[0104] Figure 6 The present invention shows the nuclide molecular probe 68The results of the in vivo targeting experiment of Ga-DOTA-linagliptin, where Figures AB are images of mice without treatment and injected with 293T-DPP4 cell suspension in the axilla, respectively. As shown in the figure, the corresponding preparation was injected subcutaneously in the axilla at the point indicated by the white arrow, and then imaged with a radionuclide molecular probe, and it can be seen that there is obvious radiation concentration at the injection point of 293T-DPP4 cells, while there is no obvious concentration point in the axilla of the control group.

[0105] Example 4 Imaging Effect and Biodistribution of Myocarditis Nuclide Molecular Probe in Myocarditis Model Mice

[0106] The method for modeling viral myocarditis in mice was the same as in Example 2. PET / CT imaging monitoring was performed using radionuclide molecular probes on the same mouse 3 days before and 7 days after virus infection, and 30 minutes after the injection of radionuclide molecular probes in other mice treated during the same period, the main organs were taken out to detect the quantitative biodistribution in a γ-counter.

[0107] Figure 7 The same mouse before and after modeling is shown. 68 The tracking imaging results of Ga-DOTA-linagliptin, where Figure A is the imaging results of mice (Control group) 3 days before virus infection, Figure B is the imaging results of mice (Myocarditis group) 7 days after virus infection, and Figure C is a statistical chart of the radioactive uptake values ​​of the heart in the imaging of mice before and after virus infection. The results showed that the radioactive uptake values ​​of the mouse heart were low before infection with the CVB3 virus, and when a large area of ​​inflammatory infiltration appeared in the heart on the 7th day after virus infection, the radioactive uptake values ​​of the mouse heart increased significantly.

[0108] Table 1 shows the biodistribution of the radionuclide molecular probe, in units of %ID / g, and the data are expressed as mean ± standard deviation. The quantitative results of the biodistribution of the radionuclide molecular probe are basically consistent with the imaging results. It can be seen that the probe has a high affinity for myocardial inflammatory foci with high expression of DPP4. The heart uptake in the myocarditis group was 0.94±0.15, while the heart of the control group was only 0.41±0.21 (p<0.05). There was a specific high uptake and low uptake in non-target tissues. The radioactivity concentration was significantly different from that in adjacent tissues, and the detection sensitivity was high, which could achieve early diagnosis and efficacy evaluation of myocarditis.

[0109] Table 1 Biodistribution of the radionuclide molecular probe of the present invention in the living body of myocarditis model mice

[0110]

[0111] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A myocarditis nuclide molecular probe, characterized in that: The invention comprises a DPP4 inhibitor, a bifunctional chelating agent covalently bonded to an amino group of the DPP4 inhibitor, and a metal nuclide coordinated and bonded to the bifunctional chelating agent, wherein the DPP4 inhibitor is selected from linagliptin, the bifunctional chelating agent is selected from p-SCN-Bn-DOTA, and the metal nuclide is selected from 68 Ga.

2. A method for preparing a myocarditis nuclide molecular probe, characterized in that: The method for preparing the myocarditis nuclide molecular probe according to claim 1 comprises the following steps: S1, dissolving the DPP4 inhibitor and the bifunctional chelator in an organic solvent, reacting under alkaline conditions, and purifying to obtain a precursor compound after the reaction is completed; S2, adding the metal nuclide eluent and the precursor compound into a buffer solution to carry out a metal nuclide labeling reaction, and after the reaction is completed, separating and purifying to obtain a myocarditis nuclide molecular probe.

3. The method for preparing the myocarditis nuclide molecular probe according to claim 2, characterized in that: In step S1, the molar ratio of the DPP4 inhibitor to the bifunctional chelator is 1-2:1; The organic solvent is dimethyl sulfoxide, methanol or chloroform; The reaction pH is 8.5-9.5, and the reaction is carried out at 20-35°C in the dark with shaking for 1-3 hours or at 4°C overnight.

4. The method for preparing the myocarditis nuclide molecular probe according to claim 2, characterized in that: In step S2, the ratio of the amount of the metal nuclide to the amount of the precursor compound in the metal nuclide eluent is 30-50MBq:1nM; The buffer is 0.1-1M sodium acetate buffer; The reaction pH is 4.0-4.5, and the reaction is oscillated at 90-100°C for 10-20 minutes.

5. Use of the myocarditis nuclide molecular probe as claimed in claim 1 in the preparation of a myocarditis contrast agent.

6. A myocarditis contrast agent, characterized in that: It comprises the myocarditis nuclide molecular probe as claimed in claim 1.

Images