Radiopharmaceutical for targeting CD44v6 as well as preparation method and application of radiopharmaceutical

Abstract

The invention belongs to the technical field of radionuclide labeled radiochemistry and clinical nuclear medicine, and relates to a radionuclide labeled targeted CD44v6 antagonist as well as a preparation method and application thereof. The invention discloses a polypeptide radiopharmaceutical for CD44v6 targeting and a preparation method of the polypeptide radiopharmaceutical. The radiopharmaceutical is radionuclide-bifunctional chelating agent-CDA6, the radionuclide is labeled with polypeptide through the bifunctional chelating agent-CDA6, and the CDA6 polypeptide is of a straight chain structure composed of six amino acids. The radiopharmaceutical realizes specific nuclear medicine positron emission computed tomography (PET) or single photon emission computed tomography (SPECT) imaging diagnosis and / or radioactive therapy of CD44v6 positive tumors or CD44v6 positive lesions (autoimmune diseases, cardiovascular and cerebrovascular system diseases and the like) through specific recognition of the polypeptide CDA6 on CD44V6 in a living body.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and particularly relates to a radioactive drug for targeting CD44v6, its preparation method and uses. Background Art

[0002] In the prior art, CD44 is a group of complex transmembrane adhesion glycoproteins, including an extracellular region, a transmembrane region and a juxtamembrane region, and is a key regulator of epithelial-mesenchymal transition (EMT). In addition, CD44 is also involved in the signal transduction of multiple receptor tyrosine kinase pathways such as C-Met, VEGFR-2, and RON. As a type I transmembrane glycoprotein, CD44 first binds to hyaluronic acid, triggering an intracellular signaling pathway, which in turn leads to a series of cell shedding, metastasis, and invasion responses. CD44 is expressed in a variety of human cells, including embryonic stem cells, differentiated cells, and cancer cells. Different alternative splicing during transcription of CD44 generates two subtypes of CD44, including the standard subtype (CD44s) and the CD44 variant subtype (CD44v). CD44 is a recognized marker of cancer stem cells, and CD44v6 is one of the variants, which is involved in the adhesion with surrounding cells and stromal components, lymphocyte homing, and T lymphocyte activation. Existing research reports have shown that CD44v6 is highly expressed in glioblastoma, breast cancer, colorectal cancer, liver cancer, lung cancer, pancreatic cancer, ovarian cancer, etc., and is closely related to the occurrence, development, and metastasis of tumors.

[0003] Although conventional biopsy, immunohistochemical staining, hematological biomarkers and other detection methods have been widely used in the detection of CD44v6 expression, due to the high heterogeneity of CD44v6 expression in primary tumors and metastases and the dynamic changes in spatio-temporal expression, these conventional detection methods cannot completely accurately and comprehensively evaluate the true expression changes of CD44v6. In recent years, with the rapid development of nuclear medicine molecular imaging technology, this technology can non-invasively, quantitatively and visually monitor the expression of related immune checkpoints at the in vivo molecular level, provide more accurate, real-time and comprehensive information on the CD44v6 expression level, and thus screen patients suitable for CD44v6 immunotherapy and predict the curative effect.

[0004] Currently reported imaging and / or therapeutic probes targeting CD44v6 are only radionuclide-labeled antibody probes 68 Ga-HBED-CC and 89 Zr-anti-CD44. Antibody probes mainly have a long in vivo circulation time and a long irradiation time for patients, which is not conducive to CD44v6 targeted diagnosis and treatment. Therefore, developing polypeptide and small molecule imaging probes targeting CD44v6 with better physicochemical properties and imaging and / or therapeutic effects has important clinical significance and social value. Summary of the Invention

[0005] The object of the present invention is to provide a polypeptide radiopharmaceutical for specific imaging of CD44v6, its preparation method and use. The drug has a low preparation cost, good stability in vivo and in vitro, and has good tumor imaging effect.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] The first aspect of the present invention provides a polypeptide radiopharmaceutical for targeting CD44V6, which is formed by labeling a polypeptide with a radionuclide through a bifunctional chelating agent.

[0008] Wherein, the polypeptide structure is CDA6, as shown in formula (I):

[0009]

[0010] Wherein, the radionuclide is selected from 18 F, 64 Cu, 68 Ga, 89 Zr, 177 Lu or 225 Ac, any one or more of them;

[0011] The bifunctional chelating agent is selected from any one or more of DOTA, NOTA, TETA, DOTAGA, DOTMA, DOTAM, NOTAGA, p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, DOTA-NHS, NOTA-NHS, TETA-NHS, DOTAGA-NHS, NOTAGA-NHS or its derivatives;

[0012] The second aspect of the present invention provides a preparation method of the polypeptide radiopharmaceutical for targeting CD44v6 as described above, including the following steps:

[0013] S1 Preparation of bifunctional chelating agent-CDA6: Dissolve the polypeptide CDA6 in a suitable solvent, then add an appropriate amount of alkaline reagent to adjust the pH to weakly alkaline, add 1.1-20 times the equivalent amount of the bifunctional chelating agent based on the mass of the polypeptide, mix well and react at room temperature for 2-24 h. Separate and purify the reaction mixture solution by HPLC, collect the product peak, and lyophilize the liquid of the collected product peak to obtain a white powder, namely bifunctional chelating agent-CDA6;

[0014] Preparation of radionuclide-bifunctional chelator-CDA6: Dissolve the bifunctional chelator-CDA6 obtained in step S1 in a suitable solvent, add a weak acidic buffer to adjust the pH to weakly acidic, then add 1 mCi - 2 Ci of radionuclide thereto, and heat in a water bath at 80 - 120 °C for 10 - 30 min to prepare radionuclide-bifunctional chelator-CDA6;

[0015] Further, in step S1 or S2, the suitable solvent is selected from any one or more of water for injection, ethanol, DMSO, and DMF;

[0016] Further, in step S1, the basic reagent is triethanolamine (TEA), N,N'-diisopropylethylamine (DIEA), or dimethylaminopyrimidine (DMAP);

[0017] Further, in step S1, the HPLC is a semi-preparative HPLC method, and the chromatographic conditions include: mobile phase: the organic phase of phase A is acetonitrile containing 0.1% v / v trifluoroacetic acid; the aqueous phase of phase B is an aqueous solution of 0.1% w / w trifluoroacetic acid; elution conditions: at the 0th minute: phase A is 0% v / v, phase B is 100% v / v; at the 2nd minute: phase A is 0% v / v, phase B is 100% v / v; at the 12th minute: phase A is 98% v / v, phase B is 2% v / v; at the 13.5th minute: phase A is 98% v / v, phase B is 2% v / v; at the 14th minute: phase A is 0% v / v, phase B is 100% v / v; stop elution at the 15th minute;

[0018] Further, in step S1, the retention time range of the product peak is 5 - 11 minutes;

[0019] Further, in step S2, the weak acidic reagent is a NaAc buffer solution with a concentration of 0.5 - 2 mol / L;

[0020] Further, when the radionuclide is 64 Cu, 68 Ga, 89 Zr, 177 Lu or 225 Ac, step S2 includes the following steps: Dissolve the bifunctional chelator-CDA6 obtained in step S1 in an appropriate amount of sterile water for injection, add a NaAc buffer solution with a concentration of 0.5 - 2 mol / L to adjust the pH to 4.0 - 6.5, then add 5 mCi - 2 Ci of radionuclide ions thereto, heat in a water bath at 80 - 120 °C for 10 - 30 min, and cool to room temperature to prepare radionuclide 64 Cu, 68 Ga, 89 Zr, 177 Lu or 225Ac-Bifunctional Chelator-CDA6;

[0021] Alternatively, further, when the radionuclide is 18 F, step S2 includes the following steps: Mix the bifunctional chelator-CDA6 obtained in S1 with AlCl 3 solution and 1 mCi to 2 Ci radionuclide ions, adjust the pH to 3.8 to 6.5, react at 80 to 120 °C for 10 to 30 min, cool to room temperature, and prepare the radionuclide 18 F-bifunctional chelator-CDA6, wherein the bifunctional chelator-CDA6 and AlCl 3 solution are proportioned according to 20 to 300 μg bifunctional chelator-CDA6: 0.004 to 0.04 mmol of AlCl 3 ;

[0022] The third aspect of the present invention provides a detection reagent comprising any one of the above-mentioned polypeptide radiopharmaceuticals for targeting CD44v6;

[0023] The fourth aspect of the present invention provides the use of any one of the above-mentioned polypeptide radiopharmaceuticals for targeting CD44v6 in the preparation of a PET or SPECT imaging probe for treating CD44v6 targeting or / and a therapeutic radiopharmaceutical.

[0024] For a further vivid description of the present invention, the radionuclide labels the CDA6 polypeptide through a bifunctional chelator (the CDA6 polypeptide is a linear structure composed of 6 amino acids). This radiopharmaceutical realizes specific nuclear medicine positron emission tomography (PET) imaging diagnosis of CD44v6-positive tumors or CD44v6-positive lesions (autoimmune diseases, cardiovascular and cerebrovascular system diseases, etc.) in vivo through the specific recognition of CD44v6 by the polypeptide CDA6. The schematic diagram of the structure of the radionuclide-bifunctional chelator-labeled CDA6 polypeptide is as Figure 1 shown, wherein the polypeptide CDA6 is obtained by reacting with DOTA, NOTA or their derivatives and a radionuclide, ○ represents a DOTA or NOTA derivative, represents a radionuclide.

[0025] Beneficial Effects

[0026] The CD44v6-targeting polypeptide CDA6 of the present invention is a polypeptide composed of a total of 6 amino acids. Through structural modification, an amino group capable of condensing with a bifunctional chelating group is reserved to achieve a change in function, transforming a conventional chemotherapeutic drug into a nuclear medicine molecular imaging probe, thereby expanding its application field. After the polypeptide CDA6 is coupled with specific DOTA, NOTA or their derivatives through the reserved amino group and uses them as bifunctional chelating agents, the radionuclide 18 F, 64 Cu, 68 Ga, 89 Zr, 177 Lu or 225 Ac is labeled onto the polypeptide molecule. Thus, in vivo, through the specific recognition of the polypeptide and CD44v6, the radionuclide is carried to the lesion sites highly expressing CD44v6, such as the sites of tumors, inflammation, trauma, fibrosis, etc., and the nuclear medicine PET or SPECT imaging technology is used for non-invasive and real-time dynamic imaging diagnosis of the lesions.

[0027] Through this diagnosis, patients with high expression of CD44v6 can be screened out, which has a positive guiding role for the treatment of this disease with antibody drugs. It not only reduces the blindness of patients in using drugs, but also collects case data of patients with high expression of CD44v6 from a clinical perspective, which has a positive guiding significance for the further research of this target and the development of new therapeutic drugs. Brief Description of the Drawings

[0028] Figure 1 It is a schematic structural diagram of a polypeptide radiopharmaceutical for targeting CD44v6 formed by labeling the CDA6 polypeptide with a radionuclide through a bifunctional chelating agent.

[0029] Figure 2 It is the HPLC result of 68 Ga-DOTA-CDA6 prepared in Example 1.

[0030] Figure 3 It is the TLC result of 68 Ga-NOTA-CDA6 prepared in Example 2.

[0031] Figure 4 It is the TLC result of 18 FAl-NOTA-CDA6 prepared in Example 3.

[0032] Figure 5 It is the in vitro stability of 68 Ga-DOTA-CDA6 prepared in Example 1.

[0033] Figure 6 It is the 68Stability of Ga-DOTA-CDA6 in urine in vivo.

[0034] Figure 7 Prepared for Example 1 68 Uptake of Ga-DOTA-CDA6 in B16F10 cells.

[0035] Figure 8 For injection 68 PET / CT imaging diagrams of Balb / c mice bearing B16F10 tumors (i.e., CD44V6 positive) at 30, 60, and 90 min after injection of Ga-DOTA-CDA6.

[0036] Figure 9 Prepared for Example 1 68 Blood clearance rate of Ga-DOTA-CDA6 in Balb / c mice.

[0037] Figure 10 Prepared for Example 1 68 Biodistribution of Ga-DOTA-CDA6 in Balb / c mice in vivo. Specific implementation method:

[0038] The present invention will be further described below in conjunction with the accompanying drawings and examples.

[0039] Example 1: 68 Synthesis and labeling of Ga-DOTA-CDA6

[0040] (1) For the polypeptide radiopharmaceutical targeting CD44v6, the bifunctional chelating agent (DOTA)-CDA6 is DOTA-NHS, and its structure is shown in formula (II):

[0041]

[0042]

[0043] (2) Preparation method of the polypeptide radiopharmaceutical targeting CD44v6:

[0044] S1: Weigh 1.0 mg of polypeptide CDA6 and 1.1 - 20 equivalents of the bifunctional chelating agent and dissolve them in water for injection. Among them, the bifunctional chelating agent is DOTA-NHS. Add an appropriate amount of triethanolamine (TEA) or N,N'-diisopropylethylamine (DIEA) to adjust the pH to 8.5 - 9.0. After mixing, react at room temperature for 12 h. After the reaction is completed, dilute the reaction solution with water for injection and separate and purify it by semi-preparative HPLC. Collect the product peak. The chromatographic conditions include: chromatographic column: Phenomenex Gemini C 18Column (specification: 250×4.6 mm, 5 μm); mobile phase: phase A organic phase is acetonitrile containing 0.1% v / v trifluoroacetic acid; phase B aqueous phase is aqueous solution containing 0.1% w / w trifluoroacetic acid; elution conditions: at the 0th minute: phase A is 0% v / v, phase B is 100% v / v; at the 2nd minute: phase A is 0% v / v, phase B is 100% v / v; at the 12th minute: phase A is 98% v / v, phase B is 2% v / v; at the 13.5th minute: phase A is 98% v / v, phase B is 2% v / v; at the 14th minute: phase A is 0% v / v, phase B is 100% v / v; stop elution at the 15th minute; the flow rate is 1.0 mL / min. The retention time t of the product R is 7.2 minutes. Rotary evaporation and concentration, and then freeze-drying with a freeze dryer to obtain a white powder, namely bifunctional chelating agent - CDA6;

[0045] S2: Dissolve 1 mg of the bifunctional chelating agent - CDA6 obtained in S1 in 1 mL of sterile injection water. Take about 50 μL of the dissolved solution, add 800 μL of 1M NaAc, and 5 mL of 0.1M HCl 68 Ga eluent, and react at 115 °C for 10 min. Activate the Sep-PakC 18 column with 5 ml of anhydrous ethanol and 10 mL of sterilized water. After heating is completed, dilute the reactant to 10 mL with sterile injection water and then inject it into the C 18 column, wash it with 10 mL of sterile injection water, and finally collect the product with 1 mL of alcohol, namely radionuclide - bifunctional chelating agent - polypeptide.

[0046] The determination of the labeling rate adopts the Radio-HPLC method (the same as the separation and purification method in Example 1), and its radiochemical purity is 98.6%. See Figure 2 .

[0047] Example 2: 68 Ga / 177 Lu / 89 Synthesis and labeling of Zr-NOTA-CDA6

[0048] S1: Weigh 1.0 mg of polypeptide CDA6 and 1.1 - 30 equivalents of bifunctional chelating agent and dissolve them in water for injection. The bifunctional chelating agent is DOTA-NHS. Add an appropriate amount of triethanolamine (TEA) or triethylenediamine TEAB to adjust the pH to weakly alkaline 8.5 - 9.0. After mixing, react at 110 °C for 60 min. After the reaction, dilute the reaction solution with water for injection and separate and purify it by HPLC. Collect the product peak. The chromatographic conditions include: chromatographic column: Phenomenex Gemini C18 column (250×4.6 mm, 5 μm); mobile phase: phase A, the organic phase is acetonitrile containing 0.1% v / v trifluoroacetic acid; phase B, the aqueous phase is an aqueous solution containing 0.1% w / w trifluoroacetic acid; elution conditions: at the 0th minute: phase A is 0% v / v, phase B is 100% v / v; at the 2nd minute: phase A is 0% v / v, phase B is 100% v / v; at the 12th minute: phase A is 98% v / v, phase B is 2% v / v; at the 13.5th minute: phase A is 98% v / v, phase B is 2% v / v; at the 14th minute: phase A is 0% v / v, phase B is 100% v / v; stop elution at the 15th minute; the flow rate is 1.0 mL / min. The retention time t of the product R is 7.8 minutes. Rotate and evaporate to concentrate and freeze-dry with a freeze dryer into a white powder, namely bifunctional chelating agent - CDA6, whose structure is shown in formula (III):

[0049]

[0050] S2: Dissolve the bifunctional chelating agent - CDA6 obtained in S1 in an appropriate amount of sterile water for injection. The mass ratio of the bifunctional chelating agent - CDA6 to sterile water for injection is 1:1. Take 50 - 100 μL of the bifunctional chelating agent - CDA6 aqueous solution, add NaAc buffer solution to adjust the pH to 4.0 - 6.5, and then add 5 mCi - 1 Ci of radionuclide 68 Ga ions, heat at 38 °C for 10 - 30 min to prepare radionuclide - bifunctional chelating agent - polypeptide.

[0051] The determination of the labeling rate adopts the method of instant thin layer chromatography (iTLC): the stationary phase is glass fiber silica gel chromatographic paper, the mobile phase is a mixed solution of 1 mol / L ammonium acetate: methanol with a volume ratio of 1:1, and the detection equipment is a Mini - Scan radioactive TLC thin layer scanner. Its radiochemical purity is 96.83%, see Figure 3 .

[0052] Example 3: 18 Synthesis and labeling of FAl - NOTA - CDA6

[0053] (1) A multi-shaped polypeptide radiopharmaceutical for CD44v6 targeting, wherein the bifunctional chelating agent is NOTA or its derivative, for example, the compound shown in formula (III), and the radionuclide is 18 F:

[0054]

[0055]

[0056] (2) Preparation method of a polypeptide radiopharmaceutical for CD44v6 targeting:

[0057] S1: Weigh 1.0 mg of polypeptide CDA6 and 1.2 - 3 times the mass of the polypeptide of the bifunctional chelating agent and dissolve them in water for injection. Among them, the bifunctional chelating agent is NOTA-NHS. Add an appropriate amount of triethanolamine (TEA) or N,N'-diisopropylethylamine (DIEA) to adjust the pH to weakly alkaline 8.5 - 9.0. After mixing, react at room temperature for 12 h. After the reaction is completed, dilute the reaction solution with water for injection and separate and purify it by semi-preparative HPLC (the purification conditions are the same as those in Example 2). Rotate and evaporate to concentrate and freeze-dry with a freeze-dryer into a white powder, namely bifunctional chelating agent-CDA6;

[0058] S2: Dissolve the bifunctional chelating agent-CDA6 obtained in S1 in an appropriate amount of sterile water for injection. The mass ratio of the bifunctional chelating agent-CDA6 to sterile water for injection is 1:1. Take 50 μL of the bifunctional chelating agent-CDA6 aqueous solution and mix it with 3 μL of a solution with a concentration of 0.1 mol / L AlCl 3 solution and 5 mCi - 2 Ci of radionuclide ions. Adjust the pH to 4.0 - 6.5 with 100 μL of 0.1 mol / L acetic acid buffer solution. React at 95 °C for 30 min and cool to room temperature to prepare the radionuclide 18 F-bifunctional chelating agent-CDA6.

[0059] The determination of the labeling rate adopts the thin layer chromatography (TLC) method: The stationary phase is a glass silica gel chromatographic plate, the mobile phase is a mixed solution with a volume ratio of acetonitrile:water of 95:5, and the detection equipment is a Mini-Scan radioactive TLC thin layer scanner, and its radiochemical purity is 98.8% (see Figure 4 ).

[0060] Example 4 68 In vitro and in vivo stability determination of Ga-DOTA-CDA6

[0061] Respectively take 200 μL of normal saline (Saline) solution, PBS (pH 7.4) and fetal bovine serum (FBS) and add them into EP tubes (n = 3). Add about 4 μCi of the prepared 68The Ga-DOTA-CDA6 probe was incubated at 37 °C for 30, 60, and 120 min, and then the radiochemical purity was determined by iTLC analysis. Approximately 200 μCi of the 68 Ga-DOTA-CDA6 probe was injected into Balb / c mice via the tail vein. Urine samples were collected at 30, 60, and 90 min after administration to determine its metabolic stability. The results showed that 68 the radiochemical purity of the Ga-DOTA-CDA6 probe was greater than 95% within 2 h of incubation in saline, PBS, and FBS, indicating that the probe could maintain good stability in different environments ( Figure 5 ). In addition, there was no significant change in the peak time ( Figure 6 ) in the urine HPLC results of mice at different time points compared with the probe ( Figure 2 ), indicating that the probe 68 Ga-DOTA-CDA6 maintained good stability in vivo, which was beneficial for further in vivo nuclear medicine imaging studies.

[0062] Example 5 68 In vivo targeting study of Ga-DOTA-CDA6

[0063] Cell uptake experiment: B16F10 cells were pre-seeded in 24-well plates at a density of approximately 105 cells per well. After adding 1 mL of complete DMEM medium to each well, the plates were placed in a humidified air incubator (37 °C, 5.0% CO 2 2) and cultured overnight. Then, the DMEM culture medium was aspirated, and the cells were washed three times with 500 μL of PBS. Approximately 4 μCi of the 68 Ga-DOTA-CDA6 probe prepared in Example 1 and 1 mL of serum-free DMEM medium were added to each well, and the cells were incubated in the incubator for 30, 60, and 90 min, respectively. Near the end of the incubation time, the drug was removed using a pipette, and the cells were washed three times with 500 μL of PBS. All the above were added to the same test tube, and then 500 μL of trypsin was added and allowed to stand for a period of time to detach the cells from the wall or lyse them. Then, the cells were washed three times with 500 μL of PBS, and the above liquids were added to another test tube (each addition was along the wall). Finally, the cpm value was measured using a gamma counter (see Figure 7 ). The results showed that the uptake of the 68Ga-DOTA-CDA6 probe by B16F10 cells gradually increased within 1 h, then tended to be stable, and slightly decreased after 120 min. This indicated that the probe had good targeting ability to B16F10 cells.

[0064] Example 6 68 Biological evaluation of Ga-DOTA-CDA6

[0065] The following describes the PET / CT imaging performance of the CD44v6-targeted probe prepared by the method of Example 1 of the present invention 68 Ga-DOTA-CDA6:

[0066] (1) Preparation of a mouse B16F10 melanoma model

[0067] Taking melanoma cell B16F10 as an example, a mouse tumor model was established. The B16F10 cells were rinsed with 3 mL of PBS, and after precipitation, 3 mL of digestive solution (0.25 wt% trypsin / 0.02 wt% EDTA) was added for digestion for about 2 min. After adding 3 mL of DMEM medium, the cells were collected in a 15 mL centrifuge tube and centrifuged at 1000 r / min for 3 min. The cells were resuspended in PBS to prepare a cell suspension of 5×10 4 / μL. Four- to five-week-old C57BL / 6 mice were subcutaneously inoculated with 5×10 6 cells / mouse (about 100 μL) in the axilla of the right forelimb and raised in a SPF-class animal house. After 2-3 weeks, when the average diameter of the tumor reached 0.8-1.0 cm, it was used for the experiment.

[0068] (2) 68 MicroPET / CT imaging of Ga-DOTA-CDA6 in the melanoma model

[0069] The tumor-bearing mice (n = 3) raised in (1) were pre-anesthetized for 3 min, and then 0.2 mL of the probe of about 150 μCi was injected via the tail vein. PET / CT imaging was performed 30, 60, and 90 min after the injection of the probe. The image clarity, tumor uptake rate, retention time in the tumor, and uptake in normal tissues, especially the kidneys, liver, and muscles, of the molecular probe were investigated (see Figure 8 ). The results showed that: 68 The Ga-DOTA-CDA6 probe could be taken up by the tumor 30 min after injection in the two tumor models. The uptake of the tumor was the highest at 30 min after injection, and the tumor had the highest target / non-target ratio with the contralateral muscle tissue at 60 min after injection. In addition, the probe was mainly cleared through the kidneys. It was shown that the investigated probe could bind well to tumor CD44v6 and could be used for the molecular imaging monitoring of tumor CD44v6 expression.

[0070] (3) 68 Blood clearance rate of Ga-DOTA-CDA6 in BALB / c mice

[0071] Six groups of radioimmunoassay tubes were prepared in advance, with 8 tubes in each group. They were numbered and weighed. The 68The Ga-DOTA-CDA6 probe was diluted with physiological saline to approximately 600 μCi / mL. Six normal BALB / c mice were taken, and after injecting 200 μL of the drug, approximately 0.3 cm of the tail was cut off. Blood was taken with a capillary tube at the time points of 1, 3, 5, 15, 30, 60, 90, and 120 min and placed in a radioimmunoassay tube. The glass tubes with the collected samples were measured in a gamma counter and finally weighed again. By calculating %ID / g at each time point, the clearance of the probe in the blood could be obtained (see Figure 9 ). The results showed that: 68 The blood half-life of the Ga-DOTA-CDA6 probe was 21.9 min, and it could be basically metabolized completely from the body approximately 120 min after injection, causing relatively low damage to the organs of the mice and having good biosafety.

[0072] (4) 68 Biodistribution of Ga-DOTA-CDA6 in a melanoma model

[0073] Three groups of radioimmunoassay tubes were prepared in advance, with 14 tubes in each group, numbered and weighed. The 68 Ga-DOTA-CDA6 probe prepared in Example 1 was diluted with physiological saline to approximately 400 μCi / mL. After injecting approximately 200 μL of the diluted solution into the tail vein of the tumor-bearing mice (n = 3) cultured in (1), blood was taken and the mice were dissected at the time points of 30, 60, and 90 min. The heart, liver, spleen, lung, kidney, stomach, large intestine, small intestine, bladder, muscle, bone, brain, blood, and tumor of the mice were sequentially added to the radioimmunoassay tubes. The glass tubes with the collected samples were measured in a gamma counter and finally weighed again. By calculating %ID / g of different organs, the distribution of the probe in the organism could be obtained (see Figure 10 ). The results showed that the uptake of the Ga-DOTA-CDA6 probe by each organ at the three time points decreased sequentially and was basically metabolized completely at 90 min. 68 ​

Claims

1. A polypeptide radiopharmaceutical for targeting CD44v6, characterized in that: The drug is formed by labeling a polypeptide with a radionuclide via a bifunctional chelator. Wherein, the polypeptide structure is CDA6, as shown in formula (I): Wherein, the radionuclide is selected from 18 F. 64 Cu, 68 Ga, 89 Zr, 177 Lu or 225 Ac, the bifunctional chelating agent is selected from any one or more of DOTA, NOTA, TETA, DOTAGA, DOTMA, DOTAM, NOTAGA, p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, DOTA-NHS, NOTA-NHS, TETA-NHS, DOTAGA-NHS, NOTAGA-NHS or any one or more of their derivatives.

2. A method for preparing a polypeptide radiopharmaceutical targeting CD44v6 according to claim 1, characterized in that: The following steps are involved: S1 Preparation of bifunctional chelating agent-CDA6: Dissolve the polypeptide CDA6 in a suitable solvent, then add an appropriate amount of alkaline reagent to adjust the pH to a weak alkaline state, add a bifunctional chelating agent of 1.1 to 20 times the mass of the polypeptide, mix well, and react at room temperature for 2 to 24 hours, separate and purify the reaction mixture by HPLC, collect the product peak, and freeze-dry the collected product peak liquid to obtain a white powder, namely, bifunctional chelating agent-CDA6; S2 Preparation of radionuclide-bifunctional chelator-CDA6: dissolve the bifunctional chelator-CDA6 obtained in step S1 in a suitable solvent, add a weak acidic buffer to adjust the pH to weak acidity, then add 1mCi~2Ci of radionuclide thereto, heat in a water bath at 80~120℃ for 10~30min to prepare radionuclide-bifunctional chelator-CDA6.

3. The method for preparing a polypeptide radiopharmaceutical targeting CD44v6 according to claim 2, characterized in that: In step S1 or S2, the suitable solvent is any one or more of water for injection, ethanol, DMSO, DMF, and DCM.

4. The method for preparing a polypeptide radiopharmaceutical targeting CD44v6 according to claim 2, characterized in that: In step S1, the alkaline reagent is triethanolamine (TEA), N,N'-diisopropylethylamine (DIEA) or dimethylaminopyrimidine (DMAP).

5. The method for preparing a polypeptide radiopharmaceutical targeting CD44v6 according to claim 2, characterized in that: In step S1, the HPLC is a semi-preparative HPLC method, and the chromatographic conditions include: mobile phase: phase A organic phase is acetonitrile containing 0.1% v / v trifluoroacetic acid; phase B aqueous phase is 0.1% w / w trifluoroacetic acid aqueous solution; elution conditions: at the 0th minute: phase A is 0% v / v, phase B is 100% v / v; at the 2nd minute: phase A is 0% v / v, phase B is 100% v / v; at the 12th minute: phase A is 98% v / v, phase B is 2% v / v; at the 13.5th minute: phase A is 98% v / v, phase B is 2% v / v; at the 14th minute: phase A is 0% v / v, phase B is 100% v / v; and elution is stopped at the 15th minute.

6. The method for preparing a polypeptide radiopharmaceutical targeting CD44v6 according to claim 2, characterized in that: In step S1, the retention time of the product peak is in the range of 5 to 11 minutes.

7. The method for preparing a polypeptide radiopharmaceutical targeting CD44v6 according to claim 2, characterized in that: In step S2, the weakly acidic reagent is a NaAc buffer solution with a concentration of 0.5 to 2 mol / L.

8. The method for preparing a polypeptide radiopharmaceutical targeting CD44v6 according to claim 2, characterized in that: When the radionuclide is 64 Cu, 68 Ga, 89 Zr, 177 Lu or 225 Ac, the step S2 comprises the following steps: dissolving the bifunctional chelating agent CDA6 obtained in step S1 in an appropriate amount of sterile water for injection, adding a 0.5-2 mol / L NaAc buffer to adjust the pH to 4.0-6.5, then adding 5 mCi-2 Ci of radioactive nuclide ions thereto, heating in a water bath at 80-120°C for 10-30 min, and cooling to room temperature to prepare a radioactive nuclide 64 Cu, 68 Ga, 89 Zr, 177 Lu or 225 Ac-bifunctional chelator-CDA6; Or when the radionuclide is 18 F, the step S2 comprises the following steps: mixing the bifunctional chelating agent-CDA6 obtained in S1 with an AlCl3 solution and 1mCi-2Ci radioactive nuclide ions, adjusting the pH to 3.8-6.5, reacting at 80-120°C for 10-30min, cooling to room temperature, and preparing the radioactive nuclide 18F-bifunctional chelating agent-CDA6, wherein the bifunctional chelating agent-CDA6 and the AlCl3 solution are mixed in a ratio of 20-300μg bifunctional chelating agent-CDA6: 0.004-0.04mmol AlCl3.

9. A detection reagent, characterized in that: A polypeptide radioactive drug for targeting CD44v6 as claimed in claim 1.

10. Use of the polypeptide radiopharmaceutical targeting CD44v6 according to claim 1 in the preparation of diagnostic PET / SPECT imaging probes and / or therapeutic radiopharmaceuticals targeting CD44v6.

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