Targeted MT1-MMP PET (Polyethylene Terephthalate) imaging agent as well as preparation method and application thereof

Abstract

The invention provides an MT1-MMP-targeted PET (Polyethylene Terephthalate) developer as well as a preparation method and application thereof, and relates to the technical field of medical imaging diagnosis. The structural formula of the PET developer for targeting MT1-MMP is as shown in formula I, and the PET developer is generated by reacting a precursor of the developer with salt of 68Ga. The PET imaging agent 68Ga-DOTA-BT1718 for targeting the MT1-MMP can be selectively taken in focuses of diseases with high expression of the MT1-MMP and rapidly removed from non-expression organs, and can play a significant role in diagnosis of diseases such as fibrosarcoma, pancreatic cancer and glioma.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical imaging diagnosis, and specifically relates to a PET imaging agent targeting tumor MT1-MMP, and a preparation method and application thereof. Background Art

[0002] Matrix metalloproteinases (MMPs) are a class of zinc-dependent proteases that play an important role in the degradation of the extracellular matrix (ECM) and pathological tissue remodeling. Although most MMPs are secreted as soluble zymogens, members of the membrane-type MMP subfamily (MT-MMPs) are expressed on the cell membrane and mediate pericellular proteolysis. Among MT-MMPs, MT1-MMP is the most important member and can activate MMP zymogens such as proMMP-2 and proMMP-13, which play an important role in tumor cell invasion and metastasis. In addition, MT1-MMP is also involved in the cleavage of cell surface receptors, including transglutaminase, CD44, pro-αv integrin, syndecan-1, and low-density lipoprotein receptor-related protein. Therefore, MT1-MMP processing of the above substrates can change the regulation of various cellular functions, such as motility, invasion, growth, differentiation, and apoptosis. Therefore, in patients with various cancers, the expression of MT1-MMP is often associated with poor prognosis, as increased expression of MT1-MMP in tumor cells promotes tumor growth, invasion, and metastasis. Therefore, in vivo monitoring of MT1-MMP activity in cancer is a valuable approach to control disease progression.

[0003] At present, the clinical method for evaluating the expression level of MT1-MMP in tumors is mainly to take tumor tissue specimens for immunohistochemistry (IHC) detection. This is a commonly used method that uses specific antibodies to stain fixed and embedded tissue or cell samples and then observe them under a microscope. This method can detect the expression level and localization of MT1-MMP in tumor tissues, but the IHC detection method has many limitations in actual clinical applications: IHC is an invasive examination, and the expression level of tumor MT1-MMP will change dynamically during the treatment process, making it difficult to dynamically monitor the expression level of tumor MT1-MMP by taking multiple samples during the treatment process; different parts of the tumor, different metastatic lesions and different stages of the disease will lead to spatial and temporal heterogeneity of the tumor. The biopsied tumor tissue is only a part of the whole tumor and cannot reflect the expression of MT1-MMP in the whole tumor, especially for patients with multiple metastatic lesions; after radiotherapy and chemotherapy, the tumor tissue may release immune antigens and change the immune status, affecting the MT1-MMP detection results; the types of antibodies, staining methods and the non-uniform MT1-MMP critical value standards used in various clinical laboratories will also affect the tumor MT1-MMP detection results.

[0004] In recent years, the use of MT1-MMP targeting peptides for tumor imaging and treatment has been widely studied in both experimental and clinical settings. However, the molecular probes targeting MT1-MMP currently developed mainly use biological macromolecules labeled with medium and long half-life radionuclides, especially monoclonal antibodies. This type of imaging agent has a large molecular weight, a long circulation time in the body, and a large radiation dose; the rich blood supply organs are clearly visualized, and the target / background ratio is low. In order to avoid the shortcomings of radionuclide-directed antibody imaging agents, such as large molecular weight, long circulation time in the body, and large radiation damage to normal tissues and organs, it is necessary to seek new molecular probes to make up for the shortcomings of the above imaging agents in evaluating the expression level of tumor MT1-MMP. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a PET imaging agent targeting MT1-MMP, which can selectively take up lesions of diseases with high MT1-MMP expression and quickly clear non-expressing organs, and can play a significant role in the diagnosis of diseases such as fibrosarcoma, pancreatic cancer, glioma, etc.

[0006] Another object of the present invention is to provide a method for preparing a PET imaging agent targeting MT1-MMP.

[0007] Another object of the present invention is to provide an application of a PET imaging agent targeting MT1-MMP.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a PET imaging agent targeting MT1-MMP, and the structural formula of the PET imaging agent targeting MT1-MMP is shown in Formula I:

[0010]

[0011] Preferably, the PET imaging agent is composed of a precursor of the PET imaging agent DOTA-BT1718 and 68 The salt of Ga is generated by reaction.

[0012] More preferably, the structural formula of the precursor DOTA-BT1718 is as shown in Formula II:

[0013]

[0014] More preferably, the precursor DOTA-BT1718 is 68 The salt reaction of Ga is as follows:

[0015]

[0016] The present invention also provides a method for preparing the PET imaging agent, comprising the following steps:

[0017] Radioactive metal nuclides 68 Ga and PET imaging agent precursor DOTA-BT1718 are subjected to radioactive labeling reaction in sodium acetate buffer to obtain a crude product; the crude product is purified and eluted to obtain a PET imaging agent targeting MT1-MMP.

[0018] Preferably, the radiolabeling reaction is carried out under the conditions of heating at 90-100° C. for 10-15 min.

[0019] Preferably, the purification step comprises: purifying the crude product through a C-18-Sep-Pak column.

[0020] Preferably, the elution condition is to use 40-60% ethanol to elute the purified product.

[0021] The present invention also provides a use of the PET imaging agent in preparing a diagnostic product for MT1-MMP highly expressed tumors.

[0022] Preferably, the MT1-MMP highly expressed tumor includes one or more of fibrosarcoma, pancreatic cancer and glioma.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The small molecule polypeptide substance of the present invention is an inhibitor of membrane type 1 matrix metalloproteinase (MT1-MMP), named BT1718. The present invention can be used to label diagnostic nuclides by adding a modified chelating agent DOTA. 68 The tracer imaging agent prepared by Ga can be used to evaluate the expression level of MT1-MMP in tumors. 68 Ga-DOTA-BT1718 can selectively take up lesions of diseases with high expression of MT1-MMP (such as fibrosarcoma, pancreatic cancer, glioma, etc.) and quickly clear non-expressing organs, and can play an important role in the diagnosis of diseases such as fibrosarcoma, pancreatic cancer, glioma, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Prepared in Example 1 68 Ga-DOTA-BT1718HPLC test result diagram;

[0026] Figure 2 for 68 PET / CT images of the tail vein of a mouse model of human fibrosarcoma inoculated with Ga-DOTA-BT1718. From left to right, the images are at 30 minutes, 60 minutes, 120 minutes, and 240 minutes.

[0027] Figure 3 for 68 PET / CT images of the tail vein of pancreatic cancer model mice inoculated with Ga-DOTA-BT1718. From left to right, the images are at 30 minutes, 60 minutes, and 120 minutes.

[0028] Figure 4 for 68 PET / CT images of the tail vein of mice with glioma model inoculated with Ga-DOTA-BT1718. From left to right, the images are at 30 minutes, 60 minutes, and 120 minutes.

[0029] Figure 5 for 68 Biodistribution histogram of Ga-DOTA-BT1718 in tumor-bearing mice;

[0030] Figure 6 This is a graph showing the pharmacokinetic experimental results of Example 7. DETAILED DESCRIPTION

[0031] The present invention provides a PET imaging agent targeting MT1-MMP, and the structural formula of the PET imaging agent targeting MT1-MMP is shown in Formula I:

[0032]

[0033] In the present invention, the PET imaging agent is composed of a precursor of the PET imaging agent DOTA-BT1718 and 68 The salt of Ga is generated by reaction.

[0034] In the present invention, the structural formula of the precursor DOTA-BT1718 is shown in Formula II:

[0035]

[0036] In the present invention, the precursor DOTA-BT1718 and 68 The salt reaction of Ga is as follows:

[0037]

[0038] The present invention also provides a method for preparing the PET imaging agent, comprising the following steps:

[0039] Radioactive metal nuclides 68 Ga and PET imaging agent precursor DOTA-BT1718 are subjected to radioactive labeling reaction in sodium acetate buffer to obtain a crude product; the crude product is purified and eluted to obtain a PET imaging agent targeting MT1-MMP.

[0040] In the present invention, the radioactive metal nuclide 68 Ga is preferably obtained by fractionation from 68 Ge / 68 The Ga generator is eluted with HCl solution. The concentration of the HCl solution is preferably 0.01-0.1 mol / L, more preferably 0.05 mol / L, 0.02-0.08 mol / L, or 0.04-0.06 mol / L. 68Ga is transferred to a sodium acetate buffer containing a PET imaging agent precursor DOTA-BT1718 precursor for radiolabeling reaction. The concentration of the sodium acetate buffer is preferably 0.1-0.5 mol / L, more preferably 0.25 mol / L or 0.2-0.4 mol / L; the pH of the sodium acetate buffer is 9-11, more preferably 10.4 or 9.8-10.5. In the present invention, the conditions of the radiolabeling reaction are preferably heated at 90-100°C for 10-15 min; more preferably, heated at 95°C for 12 min. After the radiolabeling reaction is completed, a crude product is obtained, and the crude product is preferably tested for radiolabeling rate by HPLC (high performance liquid chromatography).

[0041] In the present invention, the crude product after cooling is purified by a pretreated C-18-Sep-Pak column, and the target product of the reaction is adsorbed on the C-18-Sep-Pak column, and then the target product is eluted with ethanol and rinsed with ultrapure water. After the rinsing is completed, the target product remaining on the column and the sterile filter membrane is blown into the product bottle by air injection to reduce the residue and obtain the target product PET imaging agent. After obtaining the target product, the radiochemical purity of the purified PET imaging agent is measured by HPLC analysis. The concentration of ethanol in the present invention is preferably 40-60%, more preferably 50% or 45-55%.

[0042] The present invention also provides a use of the PET imaging agent in preparing a diagnostic product for MT1-MMP highly expressed tumors.

[0043] In the present invention, the MT1-MMP highly expressed tumors include but are not limited to one or more of fibrosarcoma, pancreatic cancer and glioma.

[0044] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0046] Example 1

[0047] This embodiment discloses a PET imaging agent 68 The preparation method of Ga-DOTA-BT1718, the specific steps are as follows:

[0048] Radioactive Metal Nuclides 68 Ga was extracted from 68 Ge / 68The Ga generator was eluted with 4 mL of 0.05 mol / L HCl solution and transferred to a reaction bottle containing 25 μL of 1 mg / mL PET imaging agent DOTA-BT1718 precursor and 1 mL of 0.25 mol / L sodium acetate buffer (pH 10.4). The final pH of the radiolabeling was 4.5. The radiolabeling reaction was heated at 95 ° C for 12 min without stirring. After the reaction, a crude product was obtained, and a 10 μL crude product sample was taken to detect the radiolabeling rate by high performance liquid chromatography (HPLC).

[0049] The specific steps of pretreating the C-18-Sep-Pak column are as follows: use a disposable syringe to draw 5 mL of medical disinfectant alcohol with a concentration of 75%, connect it to the inlet of the C-18-Sep-Pak column, slowly push the syringe to allow the alcohol to completely pass through the column, and after pushing the alcohol, use the syringe to push 10 mL of air into the column; then use a disposable syringe to draw 10 mL of ultrapure water, slowly push it through the C-18 column, and after the injection is completed, push 10 mL of air into the column again to complete the activation of the C-18Sep-Pak column to obtain a pretreated C-18-Sep-Pak column.

[0050] The cooled crude product is passed through a pretreated C-18-Sep-Pak column. The target product of the reaction will be adsorbed on the C-18-Sep-Pak column. The target product is eluted with 1 mL of 50% ethanol and rinsed with 5 mL of ultrapure water. After the rinse is completed, 10 mL of air is injected to blow the residual target product on the column and the sterile filter membrane into the product bottle to reduce the residue.

[0051] After the reaction is completed, the PET imaging agent is obtained 68 Ga-DOTA-BT1718, measure the activity of radioactive drugs, calculate its specific activity, and analyze 10 μL of sample by HPLC to determine the radiochemical purity of the purified drug. The HPLC column parameters are as follows: Eclipse Plus C-18 column (5 μm, 4.6×250 mm Agilent, California, USA), mobile phase (A) is acetonitrile (containing 0.1% trifluoroacetic acid), mobile phase (B) is water (containing 0.1% fluoroacetic acid), flow rate is 1 mL / min, linear gradient is used for detection, 0-15 min mobile phase (A) concentration changes from 10% to 90%, mobile phase (B) concentration changes from 90% to 10%; 15-20 min mobile phase (A) concentration changes from 90% to 10%, mobile phase (B) concentration changes from 10% to 90%.

[0052] The radioactive gamma counter monitor detects the radioactivity of the drug in the HPLC system. The radiolabeling rate and radiochemical purity of the drug can be expressed by the area under the curve.

[0053] according to Figure 1 The results show that according to the radioactivity detector, the peak time Rt = 8.6min is 68 The emission peak of Ga-DOTA-BT1718.

[0054] Example 2

[0055] In this example, the PET imaging agent prepared in Example 1 68 Ga-DOTA-BT1718 was injected into the tail vein of human fibrosarcoma model mice for imaging observation. The specific steps are as follows:

[0056] The tumor size is about 500 mm 3 HT1080 human fibrosarcoma mice were injected with 3.7-5.55MBq (100-150μCi) via the tail vein. 68 Ga-DOTA-BT1718 imaging agent, whole-body PET / CT images of mice were collected at 30min, 60min, 120min and 240min after injection. Before imaging, mice were placed in a sealed anesthesia box and anesthetized with a 2% isoflurane / oxygen mixture at a flow rate of 4-5L / min. After the mice were fully anesthetized, they were placed in a micro PET / CT scanner for scanning. The anesthesia airway was connected to the scanner and the flow rate was changed to 1.5-2L / min to maintain continuous anesthesia. A Siemens small animal PET / CT scanner (Siemens Medical Solutions USA Inc.) was used, and the image acquisition parameters were as follows: CT scan first, tube voltage of 80kV, tube current of 0.5mAs, exposure time of 200ms, 3 beds, and a total acquisition time of 10min; PET scan after, resolution of 3mm, 1 bed, acquisition time of 10min. PET / CT image post-processing used Inveon ResearchWorkplace 4.2.0.8 software (Siemens Medical Solutions USA Inc.).

[0057] according to Figure 2 The results showed that HT1080 human fibrosarcoma mice were injected with tail vein 68 Whole-body micro-PET / CT imaging of Ga-DOTA-BT1718 after 30 min, 60 min, 120 min and 240 min, respectively, showed that Formula I was highly uptaken in fibrosarcoma.

[0058] Example 3

[0059] In this example, the PET imaging agent prepared in Example 1 68Ga-DOTA-BT1718 was injected into the tail vein of pancreatic cancer model mice for imaging observation. The specific steps are as follows:

[0060] The tumor size is about 500 mm 3 3.7-5.55MBq (100-150μCi) were injected into the tail vein of mice with Capan-2 pancreatic cancer. 68 Ga-DOTA-BT1718 imaging agent, whole-body PET / CT images of mice were collected at 30min, 60min and 120min after injection. Before imaging, mice were placed in a sealed anesthesia box and anesthetized with a 2% isoflurane / oxygen mixture at a flow rate of 4-5L / min. After the mice were fully anesthetized, they were placed in a micro PET / CT scanner for scanning. The anesthesia airway was connected to the scanner and the flow rate was changed to 1.5-2L / min to maintain continuous anesthesia. A Siemens small animal PET / CT scanner (Siemens Medical Solutions USA Inc.) was used, and the image acquisition parameters were as follows: CT scan first, tube voltage of 80kV, tube current of 0.5mAs, exposure time of 200ms, 3 beds, and a total acquisition time of 10min; PET scan after, resolution of 3mm, 1 bed, acquisition time of 10min. PET / CT image post-processing used Inveon ResearchWorkplace4.2.0.8 software (Siemens Medical Solutions USA Inc.).

[0061] according to Figure 3 The results showed that Capan-2 pancreatic cancer mice were injected with tail vein 68 Whole-body micro-PET / CT imaging of Ga-DOTA-BT1718 after 30 min, 60 min and 120 min, respectively, showed that Formula I was highly uptaken in pancreatic cancer.

[0062] Example 4

[0063] In this example, the PET imaging agent prepared in Example 1 68 Ga-DOTA-BT1718 was injected into the tail vein of glioma model mice for imaging observation. The specific steps are as follows:

[0064] The tumor size is about 500 mm 3 3.7-5.55MBq (100-150μCi) were injected into the tail vein of U87 glioma mice. 68Ga-DOTA-BT1718 imaging agent, whole-body PET / CT images of mice were collected at 30min, 60min and 120min after injection. Before imaging, mice were placed in a sealed anesthesia box and anesthetized with a 2% isoflurane / oxygen mixture at a flow rate of 4-5L / min. After the mice were fully anesthetized, they were placed in a micro PET / CT scanner for scanning. The anesthesia airway was connected to the scanner and the flow rate was changed to 1.5-2L / min to maintain continuous anesthesia. A Siemens small animal PET / CT scanner (Siemens Medical Solutions USA Inc.) was used, and the image acquisition parameters were as follows: CT scan first, tube voltage of 80kV, tube current of 0.5mAs, exposure time of 200ms, 3 beds, and a total acquisition time of 10min; PET scan after, resolution of 3mm, 1 bed, acquisition time of 10min. PET / CT image post-processing used Inveon ResearchWorkplace4.2.0.8 software (Siemens Medical Solutions USA Inc.).

[0065] according to Figure 4 The results showed that the tail vein injection of U87 glioma mice 68 Whole-body micro-PET / CT imaging of Ga-DOTA-BT1718 after 30 min, 60 min and 120 min, respectively, showed that Formula I was highly uptaken in glioma.

[0066] Example 5

[0067] In this example, the PET imaging agent prepared in Example 1 68 Ga-DOTA-BT1718 was injected into HT1080 nude mice for biodistribution analysis. The specific steps are as follows:

[0068] Prepare 15 C57BL / 6 and BALB / c-nu mice aged 5 to 8 weeks that were inoculated with HT1080 human fibrosarcoma model and inject 3.7 to 5.55 MBq (100 to 150 μCi) of 68 Ga-DOTA-BT1718 molecular probe. 68The mice were killed at 30min, 60min, 120min and 240min after Ga-DOTA-BT1718 injection and dissected to collect samples of organs and tissues of interest, including blood, tumors, heart, lungs, liver, spleen, stomach, small intestine, kidneys, muscles, bones, brain and salivary glands, weighed and measured for radioactivity counts using a gamma counter (CAPRAC-t, CAPINTEC, USA). The radioactivity counts of each tissue sample were determined, the measured counts were corrected for time decay, and then the %ID / g (Injected Dose Per Gram of Tissue, percentage of injected dose per gram of tissue) value of each organ of interest was calculated, and the %ID / g value was used to represent the radioactivity count of each organ and tissue. 68 The absorption of Ga-DOTA-BT1718 is plotted 68 The distribution histogram of Ga-DOTA-BT1718 in tumor-bearing mice shows its biodistribution in vivo.

[0069] according to Figure 5 The results showed that 68 After intravenous injection, Ga-DOTA-BT1718 is rapidly distributed into various tissues and organs, and then excreted from the body through the excretory organs (urinary system).

[0070] Example 6

[0071] The lipid-water partition coefficient (logP) is one of the important physicochemical properties in drug development and is related to the solubility, distribution characteristics, and metabolic rate of the drug in the body. 68 The lipid-water partition coefficient of Ga-DOTA-BT1718 is analyzed, and the specific steps are as follows:

[0072] Take 3 test tubes, add 0.5mL saturated n-octanol and 0.5mL phosphate buffer PBS to each test tube, add 20μL (about 10μCi) freshly prepared nuclear medicine, use an ultrasonic oscillator to shake the mixture for 30min, and centrifuge at 5000r / min for 5min. Take 6 test tubes and divide them into two groups, A and B, numbered A-1, B-1, A-2, B-2, A-3, B-3, respectively. After the two phases are separated, 100μL of the organic phase (upper liquid) is collected from group A and the radioactivity count is measured, and 100μL of the aqueous phase (lower liquid) is collected from group B and the radioactivity count is measured. Calculate the lipid-water partition coefficient (Lipid-water partition, logP), logP = log [(organic phase count - background count) / (aqueous phase count - background count)], and take the average value after distribution measurement to express it as mean ± standard deviation.

[0073] 68The lipid-water partition coefficient of Ga-DOTA-BT1718 describes its distribution degree between pure water and saturated n-octanol. The experimental results show that 68 The logP value of Ga-DOTA-BT1718 is -3.025±0.09, indicating that it has good hydrophilicity and is mainly metabolized by the kidneys in the body.

[0074] Example 7

[0075] Pharmacokinetics is used to study the relationship between the absorption, distribution, metabolism, excretion and other processes of drugs in the body and time. 68 About 100 μCi of Ga-DOTA-BT1718 was injected into the tail vein of three 8-week-old male KM mice. Orbital blood was collected at different time points after injection (1, 3, 5, 10, 30, 60, and 120 min). About 5 μL of blood samples were taken with capillaries and placed at the bottom of a counting tube. The radioactivity counts of the blood samples at each time point were measured, and the measured counts were corrected for time decay. Then, the blood drug concentration (%ID / g) of each blood sample was calculated. The blood drug concentration-time curve was drawn. The results are shown in Figure 6 As shown, the results show 68 Ga-DOTA-BT1718 is rapidly metabolized after intravenous injection and then excreted from the excretory organs (urinary system) with low blood concentration.

[0076] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A PET imaging agent targeting MT1-MMP, characterized in that: The structural formula of the PET imaging agent targeting MT1-MMP is shown in Formula I:

2. The PET imaging agent according to claim 1, characterized in that The PET imaging agent is composed of a precursor of the PET imaging agent DOTA-BT1718 and 68 The salt of Ga is generated by reaction.

3. The PET imaging agent according to claim 2, characterized in that The structural formula of the precursor DOTA-BT1718 is shown in Formula II:

4. The PET imaging agent according to claim 2, characterized in that The precursor DOTA-BT1718 and 68 The salt reaction of Ga is as follows:

5. The method for preparing the PET imaging agent according to any one of claims 1 to 4, characterized in that: The steps include: Radioactive metal nuclides 68 Ga and PET imaging agent precursor DOTA-BT1718 are subjected to radioactive labeling reaction in sodium acetate buffer to obtain a crude product; the crude product is purified and eluted to obtain a PET imaging agent targeting MT1-MMP.

6. The preparation method according to claim 5, characterized in that: The radioactive labeling reaction is carried out under the conditions of heating at 90-100° C. for 10-15 min.

7. The preparation method according to claim 5, characterized in that: The purification step comprises: purifying the crude product through a C-18-Sep-Pak column.

8. The preparation method according to claim 5, characterized in that: The elution condition is to use 40-60% ethanol to elute the purified product.

9. Use of the PET imaging agent according to any one of claims 1 to 4 in the preparation of a diagnostic product for tumors with high MT1-MMP expression.

10. The use according to claim 9, characterized in that: The MT1-MMP highly expressed tumors include one or more of fibrosarcoma, pancreatic cancer and glioma.

Images