Targeting integrin α v Radiopharmaceuticals for β3 and PD-L1, their labeled precursors, preparation methods, and applications

Abstract

This invention belongs to the field of biomedicine and discloses a method for targeting integrin α. v β3 radiopharmaceuticals, their labeled precursors, preparation methods, and applications. Experimental verification revealed that the prepared radiopharmaceuticals are compatible with integrin α. v β3 has good binding specificity, is mainly metabolized via the pararenal pathway, and has low hepatotoxicity. It is found in areas with high expression of integrin α. v β3 has important clinical application value in the integrated diagnosis and treatment of diseases such as tumors, osteoporosis, eye diseases, and atherosclerosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to targeting integrin α. v Radiopharmaceuticals for β3 and / or PD-L1, their labeled precursors, preparation methods, and applications. Background Technology

[0002] With the development of nuclear medicine, especially the enormous application value of novel radiopharmaceuticals in clinical practice, novel radiopharmaceuticals have been widely used in the diagnosis and targeted therapy of cancer, neurodegenerative diseases, cardiovascular diseases, etc.

[0003] Integrin α v β3 is a transmembrane protein receptor, composed of α v It consists of two subunits, β1 and β2. It is expressed in various cell types, including endothelial cells, osteoblasts, and tumor cells. α v Integrin β3 plays a crucial role in physiological and pathological processes such as cell adhesion, migration, proliferation, and angiogenesis. Currently, integrin αvβ3 has been found to be abnormally highly expressed in various diseases, including tumors, osteoporosis, eye diseases, and atherosclerosis. Therefore, integrin α... v β3 is an important target for developing novel targeted radiotherapeutic drugs. Since Folkman first elucidated the concept in 1971 that the growth of solid tumors was still limited to 2-3 mm in diameter until angiogenesis began, subsequent studies have identified more than 20 angiogenic growth factors, their receptors, and signal transduction pathways.

[0004] Research on PD-1 / PD-L1 immune checkpoint inhibitors has received considerable attention. PD-L1 is overexpressed on the surface of many types of tumor cells, such as melanoma, non-small cell lung cancer, and gastric cancer. This activates the PD-1 / PD-L1 immune checkpoint signaling pathway, inactivating the anti-tumor response of T lymphocytes, enabling cancer cells to escape immune responses, and causing malignant proliferation. Therefore, by blocking the binding of PD-1 and PD-L1 and inhibiting the activation of the PD-1 / PD-L1 signaling pathway, the immune response of T cells can be activated, thereby restoring the body's normal immune response and controlling and eliminating cancer cells.

[0005] Studies have shown that α v β3-integrin depletion impairs tumor growth and triggers immunotherapy protection. Secondly, αvβ3-integrin blockade enables tumors to be used for anti-PD-1 therapy and induces durable anti-cancer immune protection when used in combination with anti-PD-1 therapy.

[0006] Therefore, a target α is designed. v It is essential to conduct dual-targeting imaging studies on tumors using dual-targeting molecules of β3 and PD-L1, based on [64 The Cu]-PEG-TPP-1 molecule was designed, and its effects on α were verified in a tumor model. v Targeting of β3 and PD-L1. We evaluated the radiolabeled peptide as an α-targeting agent using cellular uptake and binding assays, mouse PET imaging, and in vitro distribution experiments. v The role of β3 and PD-L1 targeting tracers in tumor imaging and treatment. Summary of the Invention

[0007] The purpose of this invention is to provide targeted integrin α v Radiotherapeutic agents of β3 and / or PD-L1, their precursors, preparation methods, and applications, to address the problems existing in the prior art, wherein the radiotherapeutic agent is related to integrin α. v β3 and / or PD-L1 have good binding specificity, are mainly metabolized via the pararenal pathway, have low hepatotoxicity, and are found in areas with high expression of integrin α. v It has important clinical application value in the integrated diagnosis and treatment of diseases such as tumors, liver fibrosis, central ischemic diseases, and atherosclerosis involving β3 and / or PD-L1.

[0008] To achieve the above objectives, the present invention provides the following solution:

[0009] This invention provides a targeted integrin α v A labeled precursor of a radiotherapeutic agent for β3 and / or PD-L1, characterized in that the labeled precursor has an XFRG structure, wherein X is a chelating group, R is a linker arm, F and G are polypeptide compounds, wherein the sequence of polypeptide compound F is SGQYASYHCWCWRDPGRSGGSK, and the sequence of polypeptide compound G is RGDyC.

[0010] Preferably, the connecting arm R is selected from one of the following compounds:

[0011]

[0012] Where n is an integer in the range of 1 to 20.

[0013] Preferably, the chelating group X is selected from one of the following structures:

[0014]

[0015] This invention also provides a targeted integrin α v A method for preparing labeled precursors of β3 and / or PD-L1 radiopharmaceuticals, characterized by comprising the following steps:

[0016] Synthesize the polypeptide compound;

[0017] The polypeptide compound was coupled with the linker arm and the chelating group to obtain a crude product of the labeled precursor.

[0018] The crude product of the labeled precursor was purified to obtain the labeled precursor.

[0019] Preferably, the molar ratio of the polypeptide compound, the linker arm, and the chelating group is 1:(1-5):(1-5).

[0020] Preferably, the purification reagent is a mixture of diethyl ether and n-hexane, wherein the volume ratio of diethyl ether to n-hexane is 1:1.

[0021] This invention also provides a targeted integrin α v Radiotherapeutic agents for β3 and / or PD-L1, characterized in that the radiotherapeutic agent is obtained by radiolabeling the aforementioned labeled precursor with a radionuclide, wherein the radionuclide is selected from... 18 F, 64 Cu、 67 Cu、 68 Ga、 89 Zr、 99m Tc, 111 In、 177 Lu、 186 Re、 188 Re、 203 Pb, 212 Pb, 213 Bi、 225 Ac、 227 Th、 153 Gd, 161 Tb, 227 Th.

[0022] This invention also provides a targeted integrin α v A method for preparing radiotherapeutic agents of β3 and / or PD-L1, characterized in that the preparation method includes the following steps:

[0023] Preferably, the labeled precursor is mixed with a radionuclide solution and incubated to prepare the targeted integrin α. v The radiopharmaceuticals for β3 and / or PD-L1; the labeled precursor is dissolved in a buffer solution before being mixed with the radionuclide solution; wherein the buffer solution is one or a mixture of sodium acetate, water, ethanol, phosphate buffer solution or dimethyl sulfoxide, with a pH of 4.0-10.0. The incubation conditions are 20-110°C for 5 minutes to overnight.

[0024] The present invention also provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises targeting integrin α. v β3-labeled precursor or the targeted integrin α v Radiopharmaceuticals containing β3 and / or PD-L1.

[0025] The present invention also provides the use of the labeled precursor, the radiopharmaceutical, or the pharmaceutical composition in any of the following:

[0026] (1) Preparation of integrin α for diagnosis v Drugs for tumors, osteoporosis, eye diseases, and atherosclerosis involving β3 and / or PD-L1;

[0027] (2) Preparation for the prevention, treatment or improvement of integrin α expression v Drugs for tumors, osteoporosis, eye diseases, and atherosclerosis involving β3 and / or PD-L1.

[0028] The tumor is characterized by one or more of them expressing α v Tumors or benign tumors with β3 or PD-L1, selected from lung cancer, squamous cell carcinoma, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, bladder cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, thyroid cancer, female reproductive tract cancer, lymphoma, neurofibroma, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, leukemia, glioma, or sarcoma. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 for[ 64 A study on the correlation between time and cellular uptake of Cu]PEG-RGD-TPP-1 in B16F10 cells;

[0031] Figure 2 for[ 64 A study on the correlation between the concentration of Cu]PEG-RGD-TPP-1 and cell affinity in B16F10 cells;

[0032] Figure 3 for[ 64Cu]PEG-RGD-TPP-1, [ 18 F]AlF-PEG-RGD-TPP-1, [ 64 Dynamic PET imaging study of Cu]TPP-1 in normal mice;

[0033] Figure 4 for[ 64 Cu]PEG-RGD-TPP-1, [ 64 PET imaging study of Cu]PEG-TPP-1 in B16F10 tumor-bearing mice;

[0034] Figure 5 for[ 64 Cu]PEG-RGD-TPP-1, [ 64 Biodistribution of Cu]PEG-TPP-1 in tumor-bearing mice at different time points (A); and quantitative study of four organs: tumor, blood, liver, and kidney (B), where ** / *** indicate differences between the two sets of data. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] In this invention 64 Cu、 18 F was obtained via a cyclotron, and all radioactive diagnostic and therapeutic drugs were analyzed by high-performance liquid chromatography using an HPLC system (Shimadzu, Japan).

[0041] This invention uses the mouse skin melanoma B16F10 cell line as a cell model. The culture medium is DMEM high glucose medium + 10% fetal bovine serum + 1% penicillin / streptomycin. The incubator is set at 37°C and 5% CO2. The medium is changed once a day, and passages are performed every two days.

[0042] All animal experiments used in this invention were approved by the Animal Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences. All animal experiments adhered to the 3R principle and were conducted under the guidance of the Laboratory Animal Ethics Committee. A B16F10 mouse model was established via subcutaneous injection; each mouse received a unilateral axillary injection of 1×10⁻⁶ ppm. 6 One tumor was constructed per mouse using 100 cells. After 2 weeks, when the tumor tissue grew to approximately 100-300 mm... 3 At that time, mice were given the drug and PET / CT imaging studies were performed.

[0043] The English and Chinese meanings of the abbreviations in this invention are as follows:

[0044] Fmoc: 9-Shuthylmethoxycarbonyl;

[0045] DMF: N,N-dimethylformamide;

[0046] DCM: Dichloromethane;

[0047] NMP: N-methylpyrrolidone;

[0048] TFA: Trifluoroacetic acid;

[0049] TIS: Triisopropylsilane;

[0050] NHS: Sodium N-hydroxysuccinimide sulfonate;

[0051] EDCI: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0052] NOTA: 1,4,7-triazacyclononane-1,4,7-triacetic acid;

[0053] Rink Amide-MBHAResin: 4-Methyldiphenylmethylamine resin hydrochloride;

[0054] HCTU: 6-Chlorobenzotriazole-1,1,3,3-Tetramethylurea hexafluorophosphate;

[0055] DIPEA: N,N-diisopropylethylamine;

[0056] PEG: Polyethylene glycol;

[0057] Trt: Triphenylmethyl;

[0058] tBu: tert-butyl;

[0059] Pbf: 2,2,4,6,7-pentamethylbenzofuran-5-sulfonyl;

[0060] PBS: Phosphate-buffered saline solution.

[0061] Example 1 Targeting integrin α v The radiolabeled prodrug PEG-RGD-TPP-1 of β3 and / or PD-L1 and the radiopharmaceutical [ 64 Synthesis of Cu]PEG-RGD-TPP-1

[0062] The F and G fragments of the polypeptide precursor were commercially available. They were then conjugated to a linker arm and purified by HPLC to obtain pure PEG-RGD-TPP-1. The synthetic route is shown below:

[0063] The specific synthesis steps are as follows:

[0064]

[0065] (1) Reaction of 4-PEG-4 succinimide carbonate with G fragment: 4-PEG-4 succinimide carbonate (20 mg) was added to the reaction tube, followed by c(RGDyC) (2 eq, 21.83 mg), and the reaction was carried out overnight in PBS solution (pH = 6.5-7.5).

[0066] (2) Reaction of 4-PEG-4 succinimide carbonate-c(RGDyC)2 with F fragment: 4-PEG-4 succinimide carbonate-c(RGDyC)2 (10 mg) was added to the reaction tube, followed by the F fragment (2 eq, 21.85 mg), and the reaction was carried out overnight in PBS solution (pH = 6.5-7.5).

[0067] (3) Purification: Add 3 mL of a mixed solution of acetonitrile / water (acetonitrile:water = 1:1) to the reaction system and purify it by HPLC.

[0068] The structure of the synthesized radiopharmaceutical labeled precursor PEG-RGD-TPP-1 is shown below:

[0069]

[0070] (5) 64 Cu nuclide labeling: 20 μg PEG-RGD-TPP-1 was dissolved in sodium acetate solution (0.25 mol / L, 250 μL, pH 4.2), and then added... 64 1 mL of CuCl2 solution was incubated at 100 °C for 10 minutes; the reaction solution was then purified using a Sep-Pak C18 column to obtain [ 64 Cu]PEG-RGD-TPP-1. Analysis was performed using radio-HPLC with a YMC-tria-C18 column. 64 The radiochemical purity of Cu]PEG-RGD-TPP-1 was determined under the following analytical conditions:

[0071] Solvent gradient: Solvent A, deionized water; Solvent B, acetonitrile (0.1% TFA); Flow time: 15 minutes, solvent B from 0% to 100%, flow rate 1.0 mL / min.

[0072] (6) 18 F-nucleolabeling: using 18 O-H2O is produced by using the CYPRIS-18 cyclotron. 18 F nuclear reaction produces 18 F. Elute with 0.5 mL of 0.4 M KHCO3 and purify using a Sep-Pak Light QMA filter. Collect 200 μL containing... 18 F active fraction. The pH was adjusted to 4.0 with 12 μL of metal-free glacial acetic acid solution. After cooling to room temperature, 100 μL of PEG-RGD-TPP-1 dissolved in sodium acetate buffer was added, and the mixture was heated at 80 °C for 15 min to chelate with Nota and AlF. Separation was performed by RP-HPLC. The fraction containing... 18 The fraction of the F-labeled polypeptide was dried using a nitrogen apparatus.

[0073] The radiopharmaceutical prepared in this embodiment [ 64 Cu]PEG-RGD-TPP-1 and [ 18 F]AlF-PEG-RGD-TPP-1 was analyzed by Radio-HPLC, and the results were as follows: 64 The radiochemical purity of Cu]PEG-RGD-TPP-1 is >98% (Table 1), and the retention time is approximately 9.3 minutes (Table 1); 18The radiochemical purity of F]AlF-PEG-RGD-TPP-1 is >95% (Table 1), and the retention time is approximately 9.2 minutes (Table 1).

[0074] Table 1 [ 64 Cu]PEG-RGD-TPP-1 and [ 18 Quality control data of F]AlF-PEG-RGD-TPP-1

[0075]

[0076] Example 2

[0077] This invention relates to compounds [ 64 Cu]PEG-RGD-TPP-1, [ 18 Cellular uptake experiments were conducted using F]AlF-PEG-RGD-TPP-1, specifically as follows:

[0078] B16F10 cells were seeded into 12-well plates, and then [ ] were added to each well. 64 Cu]PEG-RGD-TPP-1, [ 18 F]AlF-PEG-RGD-TPP-1 was added, and cells were incubated at 37℃ for 5 min, 10 min, 30 min, and 60 min. After incubation, the culture medium was removed, and the cells were washed three times with cold PBS. Then, 300 μL of 0.2M NaOH was added, and the cell lysate was collected in a 1.5 mL radioimmunoassay tube. The radioactivity was detected using an automated gamma counter. Figure 1 ).

[0079] Example 3

[0080] This invention relates to compounds [ 64 Cu]PEG-RGD-TPP-1 was used in a cell competition experiment, specifically:

[0081] Unlabeled TPP-1-NOTA is used as [ 64 A competitive inhibitor of Cu]PEG-RGD-TPP-1 was detected and co-incubated at different concentrations. After 2 hours of incubation, its binding affinity was simulated using Prism 8.0 software. Figure 2 ).

[0082] Example 4

[0083] The radiopharmaceutical of the present invention [ 64 Cu]PEG-RGD-TPP-1, [ 18 F]AlF-PEG-RGD-TPP-1, [ 64 The PET imaging study of Cu]TPP-1 in normal mice is as follows:

[0084] PET imaging was performed using Inveon Micro-PET / CT (Siemens Medical Solutions, Knoxville, Munich, Germany). Each mouse was injected with 200 μCi via the tail vein. 64 Cu]PEG-RGD-TPP-1, [ 18 F]AlF-PEG-RGD-TPP-1 was used for PET imaging at 10 seconds, 30 minutes, 60 minutes, 120 minutes, and 16 hours post-injection (n=3). PET imaging results showed that... 64 Cu]PEG-RGD-TPP-1, [ 18 F]AlF-PEG-RGD-TPP-1, [ 64 Cu]TPP-1 is mainly metabolized via pararenal metabolism, with low hepatic uptake and good safety profile. Figure 3 ).

[0085] Example 5

[0086] The radiopharmaceutical of the present invention [ 64 Cu]PEG-RGD-TPP-1, [ 64 The PET imaging study of Cu]PEG-TPP-1 in B16F10 tumor-bearing mice is as follows:

[0087] Each mouse was injected with 200 μCi via the tail vein. 64 Cu]PEG-RGD-TPP-1, [ 64 Cu]PEG-TPP-1 was subjected to PET imaging (n=3) at 30, 60, 120, 240, 7, 15, 16, and 17 hours post-injection, and its distribution in tumors, liver, and kidneys was quantitatively analyzed using ROI. PET imaging results showed that... 64 Cu]PEG-RGD-TPP-1 uptake in tumors is relatively [ 64 Cu]PEG-TPP-1 and [ 64 Cu]TPP-1 high ( Figure 4 ).

[0088] Example 6

[0089] This embodiment describes the compound [ 64 Cu]PEG-RGD-TPP-1, [ 64 The biodistribution of Cu]PEG-TPP-1 in B16F10 tumor-bearing mice was studied, specifically as follows:

[0090] Each mouse was injected via tail vein (50 μCi / mouse), and the distribution of the drug in nine organs was recorded at 5 min, 30 min, 60 min, and 120 min. The twelve organs were: blood, heart, liver, spleen, lung, kidney, small intestine, lymph nodes, and tumor. 64 Cu]PEG-RGD-TPP-1 compared to [ 64 Cu]PEG-TPP-1 increased uptake in tumors while decreasing uptake in the kidneys and liver, indicating that this tracer has low in vivo toxicity. Figure 5 ).

Claims

1. A targeted integrin α v A labeled precursor of a radiopharmaceutical for β3 and / or PD-L1, characterized in that, The structure of the labeled precursor is XFRG, wherein X is a chelating group, R is a linker arm, F and G are polypeptide compounds, the sequence of polypeptide compound F is SGQYASYHCWCWRDPGRSGGSK, and the sequence of polypeptide compound G is c(RGDyC).

2. The targeted integrin α according to claim 1 v A labeled precursor of a radiopharmaceutical for β3 and / or PD-L1, characterized in that, The connecting arm R is selected from one of the following compounds: Where n is an integer in the range of 1 to 20.

3. The targeted integrin α according to claim 1 v A labeled precursor of a radiopharmaceutical for β3 and / or PD-L1, characterized in that, The chelating group X is selected from one of the following structures:

4. A method for preparing the labeled precursor as described in any one of claims 1-3, characterized in that, Includes the following steps: Synthesize the polypeptide compound; The polypeptide compound was coupled with the linker arm and the chelating group to obtain the crude product of the labeled precursor. The crude product of the labeled precursor was purified to obtain the labeled precursor.

5. The method for preparing the labeled precursor according to claim 4, characterized in that, The molar ratio of the polypeptide compound, the linker arm, and the chelating group is 1:(1-5):(1-5); The purification reagent is a mixture of diethyl ether and n-hexane, wherein the volume ratio of diethyl ether to n-hexane is 1:

1.

6. A targeting integrin α v Radiotherapeutic agents of β3 and / or PD-L1, characterized in that, The radiopharmaceutical is obtained by radiolabeling the labeled precursors of claims 1-3 with a radionuclide, wherein the radionuclide is selected from... 18 F, 64 Cu、 67 Cu、 68 Ga、 89 Zr、 99m Tc, 111 In、 177 Lu、 186 Re、 188 Re、 203 Pb, 212 Pb, 213 Bi、 225 Ac、 227 Th、 153 Gd, 161 Tb, 227 Th.

7. A method for preparing a radiopharmaceutical as described in claim 6, characterized in that, The preparation method includes the following steps: The labeled precursor was mixed with a radionuclide solution and incubated to prepare the targeted integrin α. v The radiopharmaceuticals for β3 and / or PD-L1; the labeled precursor is dissolved in a buffer solution before being mixed with the radionuclide solution; wherein the buffer solution is one or a mixture of sodium acetate, water, ethanol, phosphate buffer solution or dimethyl sulfoxide, with a pH of 4.0-10.

0. The incubation conditions are 20-110°C for 5 minutes to overnight.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the labeled precursor as described in any one of claims 1-3 or the radiopharmaceutical as described in claim 6.

9. The use of the labeled precursor as described in any one of claims 1-3, the radiopharmaceutical as described in claim 6, or the pharmaceutical composition as described in claim 8 in any one of the following: (1) Preparation of integrin α for diagnosis v Drugs for tumors, osteoporosis, eye diseases, and atherosclerosis involving β3 and / or PD-L1; (2) Preparation for the prevention, treatment or improvement of integrin α expression v Drugs for tumors, osteoporosis, eye diseases, and atherosclerosis involving β3 and / or PD-L1.

10. The application according to claim 9, characterized in that, One or more of them express α v Tumors or benign tumors with β3 or PD-L1, selected from lung cancer, squamous cell carcinoma, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, bladder cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, thyroid cancer, female reproductive tract cancer, lymphoma, neurofibroma, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, leukemia, glioma, or sarcoma.

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