Nuclide imaging agent targeting PDPN as well as preparation method and application of nuclide imaging agent

Abstract

The invention belongs to the technical field of medical detection, and discloses a nuclide imaging agent targeting PDPN as well as a preparation method and application of the nuclide imaging agent. The nuclide imaging agent specifically comprises nuclide and small peptide, the small peptide specifically comprises an amino acid fragment with the sequence as shown in SEQ ID NO: 1, the small peptide has very excellent pharmacokinetics and can rapidly reach target tissues such as tumor tissues and inflammatory joints which are highly enriched with PDPN protein, imaging of the target tissues is achieved in a PDPN protein targeting mode, and the nuclide imaging agent has good application prospects. The small peptide has the advantages that the small peptide is small in molecular weight and extremely low in non-specific uptake in non-target tissues, so that a nuclide imaging agent taking the small peptide as a targeting probe shows unexpected targeting property on target tissues such as tumor tissues and inflammatory joints, specific imaging of the target tissues can be realized in a relatively short time, and the small peptide has excellent specific targeting effect and imaging time; the method has a very excellent application prospect in preparation of a tumor targeted color developing agent and / or an inflammatory joint targeted color developing agent.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical detection, and particularly relates to a nuclide imaging agent targeting PDPN and a preparation method and application thereof. BACKGROUND

[0002] The nuclide imaging technology, also known as the radionuclide imaging technology, utilizes the specific recognition and combination of a biological probe labeled with a radionuclide and a target point such as a metabolic substrate, a receptor or an antigen in a biological body, and through a ray detection and signal reconstruction technology, converts the spatial distribution and dynamic change of the biological probe into a visualized image, so as to realize the non-invasive or minimally invasive visualization and quantitative analysis of the molecular metabolism, physiological function, pathological change and drug distribution in the biological body. The nuclide imaging technology has excellent application prospects in early diagnosis, staging and typing of tumors and detection of the curative effect of anti-tumor drugs due to its high sensitivity, excellent target specificity, whole-body imaging capability and small detection trauma.

[0003] At present, the nuclide imaging agents commonly used for tumor imaging include 18 F-FDG, 11 C-MET, 18 F-FLT, 18 F-trastuzumab, 18 F-anti-TNF-ɑ antibody fragment, 64 Cu-anti-PD-L1 antibody and the like. Among them, 18 F-FDG, 11 C-MET, 18 F-FLT and the like target the tumor pangenesis characteristics such as glucose uptake and amino acid synthesis, are metabolic imaging agents, and are mainly used for pangenesis tumor screening, but have problems such as that the imaging effect is greatly affected by the transmembrane transport efficiency, the specificity is low, and false positive or false negative results are prone to occur. 18 F-trastuzumab, 18 F-anti-TNF-ɑ antibody fragment, 64 Cu-anti-PD-L1 antibody and the like target the antigens or receptors specifically expressed in tumor cells, tumor tissues and blood vessels thereof, are mainly used for detection and imaging of specific types of tumors, but have problems such as that the specific targeting effect is not ideal, the imaging time is long and the like, and have great limitations. SUMMARY

[0004] The first object of the present application is to solve the problems of the existing nuclide imaging agents such as that the specific targeting effect is not ideal and the imaging time is long, and to provide a nuclide imaging agent.

[0005] The second object of the present application is to provide a preparation method of the above-mentioned nuclide imaging agent.

[0006] The third object of the present application is to provide the use of the radionuclide imaging agent in the preparation of a tumor-targeting color developing agent.

[0007] The fourth object of the present application is to provide the use of the radionuclide imaging agent in the preparation of an inflammation joint-targeting color developing agent.

[0008] Specifically, the radionuclide imaging agent provided by the present application comprises a radionuclide and a small peptide, wherein the small peptide comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 1.

[0009] Further, the radionuclide is selected from one or more of 18 F, 11 C, 68 Ga, 111 In, 99m Tc and 64 Cu.

[0010] Further, the small peptide is modified with a chelating agent, the small peptide and the radionuclide are connected through the chelating agent, and the chelating agent is selected from one or more of DOTA, NOTA, DTPA, NOTA-Mal, DOTA-Mal and DTPA-NH2.

[0011] Further, the preparation method comprises: reacting the small peptide and the chelating agent to obtain a small peptide modified with a chelating agent; and reacting the small peptide modified with a chelating agent with a radionuclide solution to obtain the radionuclide imaging agent.

[0012] Further, the preparation method comprises one or more of the following technical features: (1) the method for realizing the reaction of the small peptide and the chelating agent is selected from one or more of an NHS ester activation method, a maleimide coupling method, an EDC / NHS carboxyl activation method and a CuAAC click chemistry method; (2) the addition ratio of the small peptide modified with a chelating agent to the radionuclide solution is (10-50) nmol:(0.5-2) GBq; and (3) the temperature for reacting the small peptide modified with a chelating agent with the radionuclide solution is 93℃-98℃, and the time is 5 min-15 min.

[0013] The present application provides the use of the radionuclide imaging agent in the preparation of a tumor-targeting color developing agent.

[0014] Further, the radionuclide imaging agent targets the PDPN protein highly expressed in tumor tissues.

[0015] Further, the tumor tissues are selected from one or more of perivascular cell tumors, primitive neuroectodermal tumors, synovial sarcomas, malignant peripheral nerve sheath tumors, squamous cell carcinomas, breast cancers, lung cancers, colorectal cancers and melanomas.

[0016] The application provides application of the radionuclide imaging agent in preparation of an inflammation joint targeting color developing agent.

[0017] Further, the radionuclide imaging agent targets PDPN protein highly expressed in an inflammation joint.

[0018] Beneficial effects: The radionuclide imaging agent provided by the application specifically comprises a radionuclide and a small peptide, and the small peptide specifically comprises an amino acid fragment with a sequence as shown in SEQ ID NO:1, and the small peptide has very excellent pharmacokinetics, can quickly reach target tissues such as tumor tissues and inflammation joints which are highly rich in PDPN protein, realizes imaging of the target tissues by targeting the PDPN protein, and has very low non-specific uptake in non-target tissues, so that the radionuclide imaging agent taking the small peptide (K D value of the PDPN protein is 1.4x10 -3 ) as a targeting probe has unexpected targeting for target tissues such as tumor tissues and inflammation joints, can realize specific imaging of the target tissues in a short time, has excellent specific targeting and imaging time, and has a very excellent application prospect in preparation of a tumor targeting color developing agent and / or an inflammation joint targeting color developing agent. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 An experimental result graph of a test on the binding force of the small peptide provided in example 1 in the application and human PDPN protein is shown; Figure 2 An experimental result graph of a test on the binding force of the small peptide provided in example 1 in the application and mouse PDPN protein is shown; Figure 3 A PET / CT image of a test on the tumor imaging performance of the radionuclide imaging agent provided in example 1 in the application is shown; Figure 4 An experimental result graph of a test on the uptake rate of each organ of a mouse when the radionuclide imaging agent provided in example 1 in the application is used for tumor imaging for 1h is shown; Figure 5 An experimental result graph of a test on the uptake rate of each organ of a mouse when the radionuclide imaging agent provided in example 1 in the application is used for tumor imaging for 2h is shown; Figure 6 An experimental result graph of a test on the biodistribution of the radionuclide imaging agent provided in example 1 in the application is shown (injection for 1h, non-competition group and competition group); Figure 7 An experimental result graph of a test on the biodistribution of the radionuclide imaging agent provided in example 1 in the application is shown (injection for 2h, non-competition group); Figure 8Figure 3 (tumor to tissue uptake ratio, 1h injection, non-competition group) of the experimental results of the biodistribution test of the nuclide imaging agent provided in Example 1 of the present application; Figure 9 Figure 4 (tumor to tissue uptake ratio, 2h injection, non-competition group) of the experimental results of the biodistribution test of the nuclide imaging agent provided in Example 1 of the present application; Figure 10 PET / CT images of the inflammation joint imaging performance test of the nuclide imaging agent provided in Example 2 of the present application; Figure 11 Experimental results figure of the inflammation joint uptake rate test of the nuclide imaging agent provided in Example 2 of the present application on normal group and model group-non-competition (score 4); Figure 12 Experimental results figure of the inflammation joint uptake rate test of the nuclide imaging agent provided in Example 2 of the present application on model group-non-competition and model group-competition at 2h imaging; Figure 13 Joint photos of different inflammation scores provided in Example 2 of the present application; Figure 14 Experimental results figure of the mouse joint inflammation score and uptake rate test provided in Example 2 of the present application. DETAILED DESCRIPTION

[0020] The nuclide imaging agent provided in the present application specifically comprises a nuclide and a small peptide. The small peptide comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 1, and the small peptide has a Kd value of 1.4x10-8M with the human PDPN protein. D The Kd value is only 1.6x10-8M. -4 The Kd value is only 1.4x10-8M. D The Kd value is only 1.4x10-8M. -3 The Kd value is only 1.4x10-8M. However, the excellent pharmacokinetics and extremely low non-target tissue non-specific uptake of the small peptide in vivo unexpectedly endow the resulting nuclide imaging agent with excellent specific targeting and imaging time, which can better achieve the imaging effect of the target tissues such as tumor tissues, inflammation joints, etc. that are highly enriched with PDPN protein, and has a very excellent application prospect in the preparation of tumor targeting color developing agents and / or inflammation joint targeting color developing agents.

[0021] In the present application, the nuclide in the nuclide imaging agent refers to a nuclide with radioactivity, which serves as a signal source to enable the enrichment of the small peptide in the target tissue to be detected, thereby realizing the localization and quantitative imaging of the target tissue. The present application does not particularly limit the existing various choices, and specific examples include but are not limited to: 18 F, 11 C, 68 Ga,111 In、 99m Tc and 64 One or more of Cu.

[0022] In some specific embodiments, the radionuclide in the radionuclide imaging agent is preferably... 68 Ga. At this time, the... 68 The half-life of Ga is well-suited to the pharmacokinetics of small peptides, thus endowing radionuclide imaging agents with better specific targeting imaging effects.

[0023] In this invention, the small peptide in the radionuclide imaging agent is modified with a chelating agent, and the small peptide and the radionuclide are specifically linked through the chelating agent. The chelating agent refers to an organic compound containing two or more coordinating atoms that can provide lone pairs of electrons, capable of binding with the radionuclide through coordination bonds to form a stable mononuclear or polynuclear complex with a cyclic structure. Various existing options are possible, and this invention does not impose any particular limitation on them. Specific examples include, but are not limited to, one or more of DOTA, NOTA, DTPA, NOTA-Mal, DOTA-Mal, and DTPA-NH2.

[0024] In some specific embodiments, the chelating agent modified on the small peptide is preferably DOTA. In this case, the introduction of DOTA into the small peptide structure has little impact on the small peptide structure and its function, and can effectively achieve a stable connection between the small peptide and the radionuclide, thereby endowing the radionuclide imaging agent with better specific targeting imaging effect.

[0025] The method for preparing the above-mentioned radionuclide imaging agent provided by the present invention specifically includes: reacting the small peptide with a chelating agent to obtain a small peptide modified with a chelating agent; reacting the small peptide modified with a chelating agent with a radionuclide solution to obtain the radionuclide imaging agent.

[0026] In this invention, the method for reacting the small peptide with the chelating agent is determined based on the modifiable groups of the small peptide and the chelating agent to be modified. Various existing options can be used, and this invention does not particularly limit them. Specific examples include, but are not limited to, one or more of the following: NHS ester activation method, maleimide coupling method, EDC / NHS activated carboxyl group method, and CuAAC click chemistry method.

[0027] In the present application, the addition ratio of the small peptide modified with the chelating agent to the radionuclide solution is preferably (10-50) nmol:(0.5-2) GBq, and specifically can be 10 nmol:0.5 GBq, 10 nmol:1 GBq, 10 nmol:2 GBq, 20 nmol:1 GBq, 25.4 nmol:1.3 GBq, 30 nmol:2 GBq, 40 nmol:2 GBq, 50 nmol:2 GBq, or any value therebetween.

[0028] In the present application, the reaction conditions of the small peptide modified with the chelating agent and the radionuclide solution specifically include that the temperature is preferably 93-98°C, and specifically can be 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, or any value therebetween; and the time is preferably 5-15 min, and specifically can be 5 min, 8 min, 10 min, 12 min, 14 min, 15 min, or any value therebetween.

[0029] The present application provides the use of the radionuclide imaging agent in the preparation of a tumor-targeting developing agent. More specifically, the radionuclide imaging agent is mainly taken up by PDPN protein-dependent specificity, and realizes rapid target enrichment in tumor tissues with high expression of PDPN protein, and has good specific targeting effect and imaging time.

[0030] In the present application, the tumor tissue has high expression of PDPN protein, and specific examples thereof include, but are not limited to, one or more of perivascular cell tumors, primitive neuroectodermal tumors, synovial sarcomas, malignant peripheral nerve sheath tumors, squamous cell carcinomas, breast cancers, lung cancers, colorectal cancers, and melanomas.

[0031] The present application provides the use of the radionuclide imaging agent in the preparation of an inflammation joint-targeting developing agent. More specifically, the radionuclide imaging agent is mainly taken up by PDPN protein-dependent specificity, and realizes rapid target enrichment in inflammation joint tissues with high expression of PDPN protein, and has good specific targeting effect and imaging time.

[0032] The embodiments of the present application are described in detail below, and the examples of the embodiments are intended to explain the present application, and cannot be understood as a limitation of the present application. The specific techniques or conditions not mentioned in the embodiments are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be obtained by purchase in the market.

[0033] Example 1 This example is used to illustrate the preparation of the radionuclide imaging agent and its application in tumor imaging, and specifically includes: 1. Preparation of the radionuclide imaging agent (1) The amino acid sequence of the small peptide molecule based on the sequence shown in SEQ ID NO: 1 was sent to Shengong Biotechnology Co., Ltd. for synthesis and modification to obtain a small peptide with DOTA modification at the N-terminus.

[0034] (2) 40 μg of the small peptide was dissolved in 1 mL of 0.25 M sodium acetate aqueous solution, followed by the addition of 2 mL of 68 GaCl3solution (prepared by dissolving 1.1 GBq of 68 GaCl3in a 0.1 M hydrochloric acid solution), and reacted at 95°C for 10 min to obtain a radionuclide imaging agent.

[0035] 2, Binding capacity of the small peptide to PDPN protein (1) Human PDPN protein (purchased from R&D) and mouse PDPN protein (purchased from R&D) were immobilized on a CM5 chip (purchased from Cytiva) according to the instructions to obtain a test chip, which was installed on a BIAcore 8K (Cytiva) machine.

[0036] (2) The small peptide and PBS buffer (100 mM, pH = 7.4) were mixed to prepare a small peptide solution with a concentration of 6.25 μM to 100 μM.

[0037] (3) The small peptide solution was passed through the test chip at a temperature of 27°C and a flow rate of 30 μL / min, and the SPR response value (RU) was detected. The average value of three parallel tests was taken, and the results are shown in Figure 1 and 2 .

[0038] As shown by the results in Figure 1 and 2 , the K D value of the small peptide to human PDPN protein was 1.6 x 10 -4 , and the K D value to mouse PDPN protein was 1.4 x 10 -3 .

[0039] 3, Test of tumor imaging performance of radionuclide imaging agent (1) Construction of tumor-bearing mouse model: 6-8 week old female C57 / B6 mice were used as experimental animals, and were adaptively fed under standard dietary feeding conditions for 5 days. Then, 5 x 10 5 6 cells per mouse (injection volume of 100 μL) were injected subcutaneously into the left shoulder of the mice, and the mice were further fed under standard dietary feeding conditions for 10 days to obtain a tumor-bearing mouse model. The tumor-bearing mouse model was randomly divided into two groups and treated as follows: (i) Non-competitive group (n=6): The radionuclide imaging agent was injected into the tail vein of the tumor-bearing mouse model at a dose of 225 μCi / mouse; (ii) Competition group (n=3): The radionuclide imaging agent was injected into the tail vein of the tumor-bearing mouse model at a dose of 225 μCi / mouse, and the small peptide was injected into the tail vein of the tumor-bearing mouse model at a dose of 30 nmol / mouse.

[0040] (2) Tumor imaging: PET / CT imaging was performed on the tumor-bearing mouse model at 1h and 2h after injection. A whole-body single-bed PET scan was performed for 5 minutes, followed by a low-dose CT scan (parameters: tube voltage 120kV, tube current 35mA, pitch 1.0, reconstruction slice thickness 1mm). The obtained images were processed and analyzed using the PMOD system. The results are as follows: Figures 3-5 As shown.

[0041] Depend on Figures 3-5 The results show that, at 1 hour after injection of the radionuclide imaging agent and / or small peptide, 68 Besides being concentrated in the kidneys and liver—organs related to the metabolism of radionuclide imaging agents—Ga exhibits specific uptake in tumor tissues, resulting in high imaging efficiency. Furthermore, a comparison of uptake rates in tumor tissues between the non-competitive and competitive groups reveals that tumor uptake of radionuclide imaging agents depends on the specific binding of small peptide molecules to the PDPN protein. Normal tissues such as the lungs and muscles show low signal intensity, demonstrating excellent targeting specificity for tumor tissues. Additionally, a comparison of data from the 1st and 2nd hour after injection shows that the uptake of radionuclide imaging agents did not fluctuate significantly over two hours, indicating relatively stable distribution in the body. This suggests promising applications in the preparation of tumor-targeting chromogenic agents.

[0042] (3) Biodistribution: After PET / CT imaging, the tumor-bearing mouse model was euthanized, and tumor, blood, heart, liver, spleen, lung, kidney, muscle, pancreas, and brain tissue were weighed and radioactive counts were measured using a gamma counter. The data were standardized to the uptake (in %ID / g) based on 1% of the total count. The results are as follows: Figures 6-9 As shown.

[0043] Depend on Figures 6-9 The results shown indicate that, 68 Besides being concentrated in organs or tissues related to the transport and metabolism of radionuclide imaging agents, Ga exhibits specific uptake by PDPN protein in tumor tissues, showing promising application prospects in the preparation of tumor-targeting chromogenic agents.

[0044] Example 2 This example is to illustrate the preparation of a radionuclide imaging agent and its application in imaging of inflammatory joints, the radionuclide imaging agent is the radionuclide imaging agent provided in Example 1, which specifically comprises: 1. Construction of CIA mouse model: 8-week-old, 18g-22g male DBA-1 mice were used as experimental animals, and after adaptive feeding for 5 days under standard dietary feeding conditions, the mouse model was randomly divided into groups and treated as follows: (i) Normal group (n=6): 0.1 mL of the mixed solution was injected into the tail intradermally for immunization, and 0.1 mL was injected again on the 21st day after injection.

[0045] (ii) Model group (n=12): The type II collagen (Chondrex.Inc., item number 20021) and the 0.05M acetic acid solution were mixed to obtain a collagen solution with a final concentration of 2mg / mL; 0.1 mL of the mixed solution was injected into the tail intradermally for immunization, and 0.1 mL was injected again on the 21st day after injection, and 24 hours later, the mice showed redness and swelling of the paws, emotional agitation, licking of the paws, and lameness, which was considered as successful modeling. According to the following standards, each joint of the mouse was scored: 0 points (normal), 1 point (single paw or single finger / toe swelling), 2 points (two joints involved), 3 points (full paw redness with severe edema), and 4 points (severe arthritis or loss of limb function).

[0046] 2. Inflammatory joint imaging: (1) After the successful construction of the normal group and the model group mice, the mice were treated as follows: (i) Normal group (n=6): The radionuclide imaging agent was injected into the tail vein of the mice at a dose of 225μCi per mouse; (ii) Model group-non-competitive (n=6): 6 mice from the model group were randomly selected and injected with the radionuclide imaging agent at a dose of 225μCi per mouse; (iii) Model group-competitive (n=6): The radionuclide imaging agent was injected into the tail vein of the remaining mice in the model group at a dose of 225μCi per mouse, and the small peptide was injected into the tail vein of the mice at a dose of 30nmol per mouse.

[0047] (2) At 1h and 2h after injection, the mice were subjected to PET / CT imaging: first whole body single bed 5min PET acquisition, then low dose CT scan (parameters: tube voltage 120kV, tube current 35mA, pitch 1.0, reconstruction layer thickness 1mm), and the obtained images were processed and analyzed using PMOD system, and the results are shown in Figures 10-14

[0048] As can be seen from the results shown in Figures 10-14 Compared with normal mice, the uptake value of the inflammatory joint of the inflammatory model mice was significantly increased, the uptake rate increased with the increase of the arthritis inflammation score, and the joint uptake rate in the model group-competition mice was significantly decreased, indicating that the radionuclide imaging agent had good targeting imaging effect on the inflammatory joint, and had good application prospect in the preparation of the inflammatory joint targeting color developing agent.

[0049] Although the embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments without departing from the principles and purposes of the present application within the scope of the present application.

[0050] The amino acid sequence involved in the present application is specifically shown in Table 1.

[0051] Table 1. ​

Claims

1. A radionuclide imaging agent, characterized in that, The radionuclide imaging agent comprises a radionuclide and a small peptide, wherein the small peptide comprises an amino acid fragment with the sequence shown in SEQ ID NO:

1.

2. The radionuclide imaging agent according to claim 1, characterized in that, The nuclide is selected from 18 F, 11 C 68 Ga、 111 In、 99m Tc and 64 One or more of Cu.

3. The radionuclide imaging agent according to claim 1, characterized in that, The small peptide is modified with a chelating agent, and the small peptide and the nuclide are linked by the chelating agent, which is selected from one or more of DOTA, NOTA, DTPA, NOTA-Mal, DOTA-Mal and DTPA-NH2.

4. The method for preparing the radionuclide imaging agent according to any one of claims 1 to 3, characterized in that, The preparation method includes: reacting the small peptide with a chelating agent to obtain a small peptide modified with a chelating agent; reacting the small peptide modified with a chelating agent with a radionuclide solution to obtain the radionuclide imaging agent.

5. The method for preparing the radionuclide imaging agent according to claim 4, characterized in that, The preparation method includes one or more of the following technical features: (1) The method for realizing the reaction between the small peptide and the chelating agent is selected from one or more of the following: NHS ester activation method, maleimide coupling method, EDC / NHS activated carboxyl group method and CuAAC click chemistry method; (2) The ratio of the modified chelating peptide to the radionuclide solution is (10~50) nmol: (0.5~2) GBq; (3) The temperature for reacting the modified chelating peptide with the radionuclide solution is 93℃~98℃ and the time is 5min~15min.

6. The use of the radionuclide imaging agent according to any one of claims 1 to 3 in the preparation of tumor-targeting chromogenic agents.

7. The application of the radionuclide imaging agent according to claim 6 in the preparation of tumor-targeting chromogenic agents, characterized in that, The radionuclide imaging agent targets the PDPN protein, which is highly expressed in tumor tissue.

8. The application of the radionuclide imaging agent according to claim 6 in the preparation of tumor-targeting chromogenic agents, characterized in that, The tumor tissue is selected from one or more of the following: hemangiopericytoma, primitive neuroectodermal tumor, synovial sarcoma, malignant peripheral nerve sheath tumor, squamous cell carcinoma, breast cancer, lung cancer, colorectal cancer, and melanoma.

9. The use of the radionuclide imaging agent according to any one of claims 1 to 3 in the preparation of an inflammatory joint-targeting chromogenic agent.

10. The application of the radionuclide imaging agent according to claim 9 in the preparation of an inflammatory joint-targeting chromogenic agent, characterized in that, The radionuclide imaging agent targets the PDPN protein, which is highly expressed in inflamed joints.

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