CN115651063A - Radionuclide labeled PTP polypeptide and application thereof - Google Patents
Radionuclide labeled PTP polypeptide and application thereof Download PDFInfo
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- CN115651063A CN115651063A CN202210317132.1A CN202210317132A CN115651063A CN 115651063 A CN115651063 A CN 115651063A CN 202210317132 A CN202210317132 A CN 202210317132A CN 115651063 A CN115651063 A CN 115651063A
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Abstract
The invention belongs to the technical field of biomedicine, relates to a radiopharmaceutical for imaging and treatment, and more particularly relates to a nuclear medicine molecular probe or a therapeutic drug constructed by PTP polypeptide. In the radionuclide-labeled polypeptide of the present invention, the polypeptide is a reticulin-targeting polypeptide; the polypeptide marked by the radionuclide contains a reticulin targeting polypeptide and the radionuclide which are coupled by a bifunctional chelating agent. The invention also comprises a preparation method and application thereof. The PTP polypeptide has good tumor cell specificity; PTP polypeptide without marked radionuclide has good cell compatibility; the PTP polypeptide marked by the radionuclide shows good targeting and nuclear medicine imaging effects in various subcutaneous tumors, is quickly cleared in vivo, and has clinical potential in the aspects of nuclear medicine diagnosis and treatment.
Description
Technical Field
The invention discloses a PTP polypeptide labeled by radionuclide and application thereof, relates to the technical field of biomedicine, relates to a radiopharmaceutical for imaging and treatment, and more particularly relates to a nuclear medicine molecular probe or a therapeutic drug constructed for the PTP polypeptide. The nuclear medicine molecular probe or the therapeutic drug is obtained by PTP polypeptide, bifunctional chelating agent (chelator) and radionuclide (radionuclide), namely radionuclide-chelator-PTP; the PTP polypeptide has an amino acid sequence of KTLLPTP; the application comprises the application of the PTP polypeptide marked by the radionuclide in the preparation of an imaging agent for diagnosing Plectin protein high-expression tumors; the application also comprises the application of the PTP polypeptide marked by the radionuclide in the preparation of the medicine for the tumor with high expression of the biological target Plectin protein.
Background
Molecular imaging techniques can assess the disease development and progression at both the cellular and molecular level, and play an important role in the early, accurate diagnosis of disease. The key of the molecular imaging technology is the preparation of a molecular probe, the molecular probe generally consists of a ligand and an imaging agent, and the effect of active targeting molecular imaging is achieved by utilizing the specific binding of a ligand receptor.
Plectin (plectin) is a skeletal protein with a molecular weight of 500 KDa that is expressed in almost all mammalian tissues and cells and plays a crucial role in muscle, skin and nerve tissues. Plectin is highly expressed in the cell membrane and cytoplasm of pancreatic cancer, is also overexpressed in ovarian cancer, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, rectal cancer and the like, and can promote the growth, proliferation and migration of tumors. Therefore, plectin has the potential to become a diagnosis and treatment target of various malignant tumors.
The plectin-targeted peptide (PTP) is a heptapeptide (KTLLPTP, SEQ ID NO 1) selected from a phage display peptide library and can be specifically combined with plectin. Compared with an antibody, the PTP polypeptide has the characteristics of small molecular weight, low immunogenicity, simple modification and the like, and has obvious advantages in the aspect of developing molecular imaging probes. At present, modifications of various PTP polypeptides, such as fluorescein, drugs or drug delivery systems constructed by applying polymer carrier materials, are used for imaging and therapeutic research of various tumors. Although PTP polypeptide shows important potential in Plectin targeting, the research and development of the nuclear medicine in the existing tumor imaging and treatment system constructed by taking PTP as the targeting polypeptide has not been paid enough attention, and no related nuclear medicine imaging agent or radiotherapy medicine is reported.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a PTP polypeptide labeled by radionuclide.
It is a further object of the present invention to provide the use of the radionuclide-labeled PTP polypeptide in tumor imaging and therapy.
Aiming at the existing problems, the invention provides a PTP polypeptide labeled by radionuclide and application thereof in tumor imaging and treatment. The invention comprises a nuclear medicine molecular probe or a therapeutic drug constructed by PTP polypeptide; the nuclear medicine molecular probe or the therapeutic drug is obtained by PTP polypeptide, bifunctional chelating agent (chelator) and radionuclide (radionuclide), namely radionuclide-chelator-PTP; the amino acid sequence of the PTP polypeptide is KTLLPTP; the application comprises the application of PTP polypeptide marked by radionuclide in the preparation of imaging agent for diagnosing Plectin protein high-expression tumor; the application also comprises the application of the PTP polypeptide marked by the radionuclide in the preparation of the medicine for the biological targeting Plectin protein high expression tumor.
In one aspect, the invention provides a radionuclide-labeled polypeptide, which is a reticulin targeting polypeptide; the polypeptide marked by the radionuclide contains a reticulin targeting polypeptide coupled with a bifunctional chelating agent and the radionuclide.
Preferably, the radionuclide-labeled PTP polypeptide is obtained by coupling the PTP polypeptide with a bifunctional chelating agent and labeling the radionuclide.
Preferably, the amino acid sequence of the polypeptide is KTLLPTP. Preferably, the structure is as follows:
preferably, the bifunctional chelating agent is selected from, but not limited to, HYNIC, DTPA, DOTA or NOTA. Wherein, the first and the second end of the pipe are connected with each other,
HYNIC refers to 6-hydrazinonicotinic acid, and has a structure as follows:
DTPA, which is diethyltriaminepentaacetic acid, has the structure:
DOTA, which refers to 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid, having the structure:
NOTA, which refers to 2, 2'' - (1, 4, 7-triazacyclononane-1, 4, 7-triyl) triacetic acid, has the structure:
preferably, the radionuclide is selected from 68 Ga、 18 F[AlF]、 111 In、 67 Ga、 64 Cu、 188 Re、 177 Lu or 90 One or more of Y. Wherein the content of the first and second substances, 111 in and 67 ga can be used for nuclear medicine SPECT imaging and tumor diagnosis; 18 F[AlF]、 68 ga and 64 cu can be used for nuclear medicine PET imaging and used for tumor diagnosis; 188 Re、 177 lu and 90 y can be used as nuclear medicine nuclide therapeutic medicine for treating tumor.
In another aspect, the present invention provides a method for preparing the radionuclide-labeled polypeptide, comprising:
(a) A bifunctional chelator-conjugated reticulin targeting polypeptide; and
(b) Targeting the polypeptide using the bifunctional chelator-conjugated reticulin obtained in radionuclide labeling (a).
Preferably, the network protein targeting polypeptide is a PTP polypeptide. When the bifunctional chelating agent is coupled with the PTP polypeptide, the PTP polypeptide is dissolved in a solvent which is conventionally used in the modification process of the chelating agent, and then the bifunctional chelating agent is added. The molar ratio of PTP polypeptide to bifunctional chelator may be 1: (0.5-2), for example, 1:0.5,1:0.8,1:1,1:1.2,1:1.4,1:1.6,1:1.8,1:2, etc. The coupling reaction may be carried out at room temperature, for example, 15 to 30 ℃ and the reaction time may be adjusted depending on the temperature, or the time and temperature may be adjusted depending on the composition and amount of the reaction components, so that the coupling reaction may be terminated when the chemical purity thereof is confirmed to be more than 95% (for example, using an HPLC method). The radionuclide may then be selected according to the chelating agent used and the purpose of labeling and the labeling reaction may be carried out, and the labeling process may be carried out by configuring the relevant reagents and adjusting the reaction conditions according to a conventional labeling method to terminate the labeling reaction at a radiochemical purity of 95% or more.
Specifically, the preparation method can be completed according to the following steps:
(1) Dissolving PTP polypeptide in N, N-dimethylformamide, and adding a bifunctional chelating agent for polypeptide modification; the molar ratio of PTP polypeptide to bifunctional chelating agent is 1: (0.5-2); after reacting for 2-6 hours at room temperature, separating and purifying by using a high performance liquid chromatograph, freeze-drying, and confirming that the chemical purity is more than 95%.
(2) The following materials were prepared:
1 mg/mL of the PTP polypeptide aqueous solution modified by the bifunctional chelating agent obtained in the step (1),
SnCl 2 solution 1 mg/mL in 0.1M HCl, and
radionuclide 50-200 mCi/mL.
(3) Mixing 50-200 mu L of PTP polypeptide aqueous solution modified by bifunctional chelating agent with 0.5 mL of EDDA solution and 0.5 mL of Tricine solution, and then adding 25 mu L of SnCl 2 Reacting the solution with 0.5-1 mL of radionuclide for 10-20 minutes at 95-110 ℃; the reaction was stopped when the radiochemical purity was greater than 95%.
Alternatively, the above bifunctional chelator-modified PTP polypeptide may be prepared as follows:
(1) Dissolving PTP polypeptide in N, N-dimethylformamide, and adding a bifunctional chelating agent for polypeptide modification; the molar ratio of the PTP polypeptide to the bifunctional chelating agent is 1; (0.5-2); reacting at room temperature for 2-6 hr, separating and purifying with high performance liquid chromatograph, lyophilizing, and determining chemical purity of the extract to be greater than 95%;
(2) The following materials were prepared:
1 mg/m of the PTP polypeptide aqueous solution modified by the bifunctional chelating agent obtained in the step (1),
sodium acetate buffer 0.1 m, ph = 4.6-5.1, and
radionuclide 50-200 mCi/mL;
(3) 20-50 mu L of PTP polypeptide aqueous solution modified by the bifunctional chelating agent is mixed with 65-100 mu L of sodium acetate buffer solution, 0.5-1 mL of radionuclide reacts for 15-60 minutes at 85 ℃; after the completion of the reaction, the product was purified, and it was confirmed that the radiochemical purity of the purified radionuclide-labeled polypeptide was 95% or more.
Wherein EDDA
PBS is phosphate buffered saline and conventional PBS formulations commonly used in radionuclide labeling procedures or to solubilize Tricine can be used. For example, the following formulations may be used:
the invention also provides a preparation method of the PTP polypeptide marked by the radionuclide, which comprises the following steps:
(1) Dissolving 100 mu mol of PTP polypeptide in 100 mu L of N, N-dimethylformamide, adding 120 mu mol of bifunctional chelating agent, and performing polypeptide modification; reacting at room temperature for 4 hours, separating and purifying by using a High Performance Liquid Chromatograph (HPLC), freeze-drying, and finally identifying by using a high performance liquid chromatograph-mass spectrometer (HPLC-MS), and confirming that the chemical purity is more than 95%;
(2) Preparing 1 mg/mL of the bifunctional chelating agent modified PTP polypeptide aqueous solution obtained in the step (1), 20 mg/mL of EDDA solution (0.1M NaOH solution), 40 mg/mL of Tricine solution (0.2M PBS, pH = 6.0), snCl 2 Solution 1 mg/mL (0.1M HCl solution), radionuclide 50-200 mCi/mL;
(3) 50-200 mu L of PTP polypeptide aqueous solution modified by bifunctional chelating agent, 0.5 mL of EDDA solution and 0.5 mL of Tricine solution, and then 25 mu L of SnCl 2 Reacting the solution with 0.5-1 mL of radionuclide at 100 ℃ for 15 minutes; and (4) after the reaction is finished, measuring the radioactive chemical purity by Radio-HPLC to be more than 95 percent, and obtaining the product.
Preferably, the bifunctional chelating agent in the step (1) is HYNIC or DTPA, and the PTP polypeptide modified by the bifunctional chelating agent is HYNIC-PTP or DTPA-PTP.
Preferably, the radionuclide in step (2) is selected from 188 Re or 111 In。
The invention also provides a second preparation method of the radionuclide-labeled PTP polypeptide, which comprises the following steps:
(1) Dissolving 100 mu mol of PTP polypeptide in 100 mu L of N, N-dimethylformamide, adding 120 mu mol of bifunctional chelating agent and 120 mu mol of diisopropylethylamine, and performing polypeptide modification; reacting at room temperature for 2 hours, separating and purifying by using a High Performance Liquid Chromatograph (HPLC), freeze-drying, and finally identifying by using a high performance liquid chromatograph-mass spectrometer (HPLC-MS), and confirming that the chemical purity is more than 95%;
(2) Preparing 1 mg/mL of the PTP polypeptide aqueous solution modified by the bifunctional chelating agent obtained in the step (1), 0.1M of sodium acetate buffer (pH = 4.8) and 50-200 mCi/mL of radionuclide;
(3) 20-50 mu L of PTP polypeptide aqueous solution modified by the bifunctional chelating agent is mixed with 65-100 mu L of sodium acetate buffer solution, 0.5-1 mL of radionuclide reacts for 15-60 minutes at 85 ℃; and after the reaction is finished, purifying by using Radio-HPLC, wherein the radioactive chemical purity is more than 95 percent after purification, and thus obtaining the product.
Specifically, the bifunctional chelating agent in the step (1) is DOTA or NOTA, and the PTP polypeptide modified by the bifunctional chelating agent is DOTA-PTP or NOTA-PTP.
Specifically, the radionuclide in the step (2) is selected from 111 In、 90 Y、 177 Lu、 18 F[AlF]、 67 Ga、 68 Ga or 64 Cu。
The preparation method of the PTP polypeptide labeled by the radionuclide can be prepared by conventional means in the field. For example, a solid phase synthesis method is adopted to prepare PTP polypeptide, bifunctional chelating agents HYNIC, DTPA, DOTA or NOTA are all conventional applications in the field of radiopharmaceuticals, and radionuclides are purchased from professional companies.
In another aspect, the present invention also provides the application of the polypeptide labeled by the radionuclide in constructing nuclear medicine molecular probes or preparing medicines for diagnosis or treatment.
The PTP polypeptide of the invention is subjected to targeted qualitative and quantitative research by using a flow cytometer and a laser confocal microscope, and the finding that the PTP has targeting property on various tumors is realized. The CCK-8 experiment evaluated the cellular compatibility of PTP polypeptides with unlabeled radionuclide. After the PTP polypeptide is labeled by the radionuclide, a nuclear medicine molecular probe or a therapeutic drug is obtained. Establishing a nude mouse subcutaneous tumor model, carrying out a SPECT imaging experiment, and evaluating the tumor imaging effect of the PTP polypeptide marked by the radionuclide. The result shows that the PTP polypeptide has better tumor cell specificity; PTP polypeptide without marked radionuclide has good cell compatibility; the PTP polypeptide marked by the radionuclide shows good targeting and nuclear medicine imaging effects in various subcutaneous tumors, is quickly cleared in vivo, and has clinical potential in the aspects of nuclear medicine diagnosis and treatment.
When the reflecting nuclear species is 111 In or 67 Ga, the application is the application of the polypeptide marked by the radionuclide in SPECT imaging in nuclear medicine or the application in preparing medicaments for diagnosing tumors.
When the reflecting nuclear species is 18 F[AlF]、 68 Ga and 64 when Cu is adopted, the polypeptide marked by the radionuclide is applied to nuclear medicine PET imaging, or the application in the preparation of drugs for diagnosing tumors is realized.
When the reflecting nuclear species is 188 Re、 177 Lu and 90 the application is the application of the polypeptide marked by the radioactive nuclide in preparing the medicine for treating nuclear medicine nuclide and/or tumor.
In yet another aspect, the present invention provides a nuclear medicine molecular probe comprising the polypeptide labeled with a radionuclide.
The invention also provides an imaging agent, which is an imaging agent for diagnosing diseases caused by high expression of Plectin protein; the imaging agent comprises the polypeptide labeled with the radionuclide. The application comprises the application in the preparation of a tumor imaging agent for diagnosing high expression of Plectin protein; or in the preparation of biological targeting Plectin protein high expression tumor radiopharmaceuticals.
The invention also provides a medicament for treating tumors caused by high expression of Plectin protein; the medicament contains the polypeptide marked by the radionuclide. Preferably, the medicine is a therapeutic medicine for biological targeting Plectin protein high expression tumor, and contains the PTP polypeptide marked by the radionuclide of the invention.
The invention has the following beneficial effects:
(1) The invention provides a radionuclide-labeled PTP polypeptide with a target Plectin protein; the PTP polypeptide has targeting property, good tumor cell specificity and cell compatibility to various tumors.
(2) The PTP polypeptide marked by the radionuclide provided by the invention can be combined with tumor Plectin protein in vivo, is gathered in tumor tissues, and realizes the purpose of tumor nuclear medicine imaging through nuclear medicine diagnostic nuclide. The PTP polypeptide marked by the radionuclide of the invention has good targeting and nuclear medicine imaging effects in various subcutaneous tumors, is cleared quickly in vivo, and can become a new developer.
(3) The PTP polypeptide marked by the radionuclide provided by the invention can also realize the aim of tumor nuclear medicine treatment through nuclear medicine therapeutic nuclides. The PTP polypeptide marked by the radionuclide of the invention has quick clearance in vivo and has clinical potential in the aspects of nuclear medicine treatment and tumor resistance.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required to be used in the embodiments will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph showing the results of PTP polypeptide targeting determination by flow cytometry;
FIG. 2 is a graph showing the results of PTP polypeptide targeting by confocal laser microscopy;
FIG. 3 is a result of CCK-8 determining the cytocompatibility of PTP polypeptide;
FIG. 4 is 68 A Radio-HPLC chromatogram of Ga-DOTA-PTP;
FIG. 5 is a drawing 68 A TLC spectrogram of Ga-DOTA-PTP;
FIG. 6 is 68 Ga-DOTA-PTP in vitro stability;
FIG. 7 is 68 Ga-DOTA-PTP was visualized in subcutaneous U87 glioma and BxPC-3 pancreatic tumor small animal PET/CT.
Detailed Description
The invention provides the PTP polypeptide marked by the radionuclide obtained by the preparation method. Also relates to a nuclear medicine molecular probe or a therapeutic drug constructed by the PTP polypeptide. The nuclear medicine molecular probe or the therapeutic drug is obtained by PTP polypeptide, bifunctional chelating agent (chelator) and radionuclide (radionuclide), namely radionuclide-chelator-PTP; the PTP polypeptide has an amino acid sequence of KTLLPTP.
The application of the PTP polypeptide marked by the radionuclide comprises the application of the PTP polypeptide marked by the radionuclide in preparing an imaging agent for diagnosing Plectin protein high-expression tumors; the application also comprises the application of the PTP polypeptide marked by the radionuclide in the preparation of the medicine for the tumor with high expression of the biological target Plectin protein.
The technical solutions in the embodiments of the present application will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Example 1
PTP targeting determination by flow cytometry:
the C6 cell (rat glioma) suspension was added to six well plates at 2x10 per well 5 one/mL in a sterile cell incubator (37 ℃,5% CO) 2 ) The cells were cultured for 24 hours. Old medium in each well was discarded, 1mL of fresh incomplete medium containing fluorescein isothiocyanate (FITC-PTP) labeled PTP polypeptide (FITC-PTP) was added to C6 cells, the concentration of FITC-PTP was set at 0, 0.5,1, 2, 5, 10, 20 and 40. Mu.M (three secondary wells per concentration), PBS was used as a control group, and after the addition of the sample was completed, the cells were placed in an incubator for further incubation for 4 hours. After the cells were collected, the mean fluorescence signal intensity of each group of cells was measured on a flow cytometer.
The results showed that the relative fluorescence intensity of C6 cells increased proportionally with increasing concentration of FITC-PTP. When the concentration of FITC-PTP was 40. Mu.M, the relative fluorescence intensity of C6 cells reached 95%, indicating that FITC-PTP was saturated with C6 cells. Therefore, FITC-PTP has strong targeting property with C6 cells, see FIG. 1.
Example 2
PTP targeting of laser confocal microscope determination:
normal lung epithelium BESB-2B cell, pancreatic cancer BxPC-3 cell, glioma C6, U87 cell, non-small cell lung cancer A549 cell and three negativeAdding sexual breast cancer 4T1 cell suspension into six-hole plate, adding 2x10 of the suspension into each hole 5 one/mL in a sterile cell incubator (37 ℃,5% CO) 2 ) And (5) standing and culturing for 24 hours. Old medium was aspirated from each dish, 1mL of fresh incomplete culture medium containing 10. Mu.M FITC-PTP was added to the six different cell confocal dishes, and 1mL of PBS was added to the six different cell confocal dishes as a negative control. After the sample is added, the cells are placed in an incubator and are incubated for 4 hours. 200. Mu.L of 4% paraformaldehyde was dropped onto the bottom of each dish, and the mixture was fixed for about 20 minutes. After PBST cleaning, 200. Mu.L of DAPI was dropped on the bottom of each dish in a dark environment, the cell nuclei were stained for 5 minutes, and finally the fluorescence brightness of the cells in each dish was observed under a confocal laser microscope and fluorescence images were collected.
The results show that glioma C6 and U87 cells, breast cancer 4T1 cells, pancreatic cancer BxPC-3 and non-small cell lung cancer A549 cells have higher green fluorescence signals compared with normal lung epithelial BESA-2B cells. FITC-PTP was shown to be specifically taken up by five different malignancies, see FIG. 2.
Example 3
CCK-8 determination of PTP polypeptide cytocompatibility:
BESB-2B and C6 cells were plated evenly into two 96-well plates and incubated overnight in an incubator. Removing old culture medium in each hole of a 96-well plate, adding 10 mu L of bifunctional chelating agent HYNIC modified PTP polypeptide (HYNIC-PTP polypeptide) with different concentrations, wherein the concentration gradient is 0, 1, 5, 10, 20, 50, 100 and 200 mu g/mL, simultaneously, setting up a PBS control group, setting up 6 auxiliary holes in each group, and placing in an incubator for standing culture for 24 hours after the sample addition is finished. Old medium in each well was discarded, and 100. Mu.L of fresh medium solution containing 10. Mu.L of LCCK-8 solution was added to each well in a 96-well plate. After further incubation for 1.5-2 hours, the absorbance of each well in the 96-well plate was measured with a microplate reader (450 nm).
The results show that after the treatment of HYNIC-PTP with different concentration gradients for 24 hours, the survival rates of normal lung epithelial BESA-2B cells and C6 cells are both more than 95%. Therefore, the HYNIC-PTP polypeptide has good cell compatibility when the concentration of the polypeptide is within 0-200 mug/mL, as shown in figure 3.
Example 4
Preparation and radiochemical purity determination of radionuclide-labeled PTP polypeptide:
68 the specific steps of Ga-labeled PTP polypeptide are as follows:
dissolving PTP polypeptide (DOTA-PTP polypeptide) modified by bifunctional chelating agent DOTA by using sodium acetate solution, wherein the concentration is 1 mg/mL; from 68 Ge/ 68 Fresh under the leaching of Ga generator 68 GaCl 3 Mixing 2 mL of the mixture with 130 mu L of DOTA-PTP polypeptide solution, and reacting for 10 minutes at 100 ℃ to obtain 68 Ga-DOTA-PTP; radio-HPLC and TLC were used to determine radiochemical purity.
The results show that 68 The Ga-DOTA-PTP preparation method is simple and efficient, and the radioactive chemical purity is greater than 95%, see FIG. 4.
Example 5
In vitro stability evaluation of radionuclide-labeled PTP polypeptides:
respectively taking 100 mu L 68 Ga-DOTA-PTP was mixed with 900. Mu.L PBS solution (pH = 7.4) at room temperature or 900. Mu.L serum at 37 ℃, assayed for 1, 2, 4 and 6 hour radiochemical purity, and evaluated for in vitro stability; the TLC method is a solution spotting silica gel plate and develops by using 50% acetonitrile as a mobile phase.
The results show that it is possible to display, 68 the radioactive chemical purity of Ga-DOTA-PTP in PBS and serum at 37 ℃ at room temperature is not obviously changed after 3 hours, which shows that 68 Ga-DOTA-PTP was stable in vitro, see FIG. 5.
Example 6
In vivo biodistribution assay of radionuclide-labeled PTP polypeptide:
subcutaneous C6 tumor nude mouse tail intravenous injection 99m Tc-HYNIC-PTP (100 μ L,20 μ Ci), nude mice were sacrificed at 0.5,1, 2 and 4 hours post-injection, respectively, 3 per time point, blood, tumor, heart, lung, meat, spleen, small intestine, liver and kidney were taken, weighed, and the percent injection dose rate per gram of tissue (% ID/g) was calculated; in the same way, measure 99m The biodistribution of Tc-HYNIC-PTP in subcutaneous 4T tumor nude mice.
The results show that it is possible to display, 99m the biodistribution of Tc-HYNIC-PTP in 4T1 and C6 subcutaneous tumor nude mice is similar, and the tumor tissue has obvious uptake; radioactivity mainly accumulates in the kidney and bladder, which indicates that the imaging agent is excreted through the urinary system, while the liver, spleen, heart and soft tissue and the like are weakly imaged; blood clearance is fast, facilitating short imaging times and reducing in vivo radiation, see fig. 6.
Example 7
Subcutaneous tumor small animal PET/CT imaging:
subcutaneous pancreatic cancer BxPC-3 and glioma U87 nude mice 3 per group were injected via tail vein 68 Ga-DOTA-PTP (200. Mu.L, 200. Mu. Ci), was subjected to PET/CT imaging of small animals 1 hour after injection.
The results show that it is possible to display, 68 the biological distribution of Ga-DOTA-PTP in U87 glioma nude mice and BxPC-3 pancreatic cancer nude mice is good, and the tumors are obviously taken; 68 Ga-DOTA-PTP has concentration mainly in kidney and bladder, which prompts that the imaging agent is excreted through urinary system, while the development of liver, spleen, heart and soft tissue is weak, and no radioactive uptake is seen in thyroid gland area, indicating that 68 Ga-DOTA-PTP was effective in displaying tumor tissues, see FIG. 7.
The above description is only for the specific embodiments of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present disclosure should be covered within the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.
SEQUENCE LISTING
<110> first-person hospital in Shanghai City; ningxia medical university; ningxia medical university general hospital
PTP polypeptide marked by <120> radionuclide and application thereof
<130> 20220224
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 1
Lys Thr Leu Leu Pro Thr Pro
1 5
Claims (11)
1. A radionuclide-labeled polypeptide, wherein said polypeptide is a reticulin targeting polypeptide; the polypeptide marked by the radionuclide contains a reticulin targeting polypeptide coupled with a bifunctional chelating agent and the radionuclide.
2. The radionuclide-labeled polypeptide of claim 1, wherein the amino acid sequence of the polypeptide is KTLLPTP; or
The bifunctional chelating agent is selected from HYNIC, DTPA, DOTA or NOTA;
the radionuclide is selected from 68 Ga、 18 F[AlF]、 111 In、 67 Ga、 64 Cu、 188 Re、 177 Lu or 90 One or more of Y.
3. The radionuclide-labeled polypeptide according to claim 1,
the polypeptide is PTP polypeptide, and the structure is:
or
The bifunctional chelating agent is selected from the following substances: HYNIC refers to 6-hydrazinonicotinic acid, and has the structure as follows:
or
DTPA, refers to diethyltriaminepentaacetic acid, whose structure is:
or
DOTA, which refers to 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid, having the structure:
or
NOTA, which refers to 2, 2'' - (1, 4, 7-triazacyclononane-1, 4, 7-triyl) triacetic acid, has the structure:
4. the method of any of claims 1 to 3, wherein the method comprises:
(a) A bifunctional chelator-conjugated reticulin targeting polypeptide; and
(b) The bifunctional chelator-conjugated reticulin targeting polypeptide obtained using radionuclide labeling (a).
5. The method of claim 4, comprising the steps of:
(1) Dissolving PTP polypeptide in N, N-dimethylformamide, and adding a bifunctional chelating agent for polypeptide modification; the mol ratio of the PTP polypeptide to the bifunctional chelating agent is 1 (0.5-2); reacting at room temperature for 2-6 hr, separating and purifying with high performance liquid chromatograph, lyophilizing, and determining chemical purity of the product to be greater than 95%;
(2) The following materials were prepared:
1 mg/mL of the PTP polypeptide aqueous solution modified by the bifunctional chelating agent obtained in the step (1),
EDDA solution 20 mg/mL, 0.1M NaOH as solution,
tricine solution 40 mg/mL, 0.2M PBS as solution, pH = 5.8-6.3,
SnCl 2 solution 1 mg/mL in 0.1M HCl, and
radionuclide 50-200 mCi/mL;
(3) Mixing 50-200 mu L of PTP polypeptide aqueous solution modified by bifunctional chelating agent with 0.5 mL of EDDA solution and 0.5 mL of Tricine solution, and then adding 25 mu L of SnCl 2 Reacting the solution with 0.5-1 mL of radionuclide for 10-20 minutes at 95-110 ℃; and terminating the reaction when the radiochemical purity of the target product is greater than or equal to 95%.
6. The method of claim 4, comprising the steps of:
(1) Dissolving PTP polypeptide in N, N-dimethylformamide, and adding a bifunctional chelating agent for polypeptide modification; the molar ratio of PTP polypeptide to bifunctional chelating agent is 1: (0.5-2); reacting at room temperature for 2-6 hr, separating and purifying with high performance liquid chromatograph, lyophilizing, and determining chemical purity of the product to be greater than 95%;
(2) The following materials were prepared:
1 mg/m of the PTP polypeptide aqueous solution modified by the bifunctional chelating agent obtained in the step (1),
sodium acetate buffer 0.1 m, ph = 4.6-5.1, and
radionuclide 50-200 mCi/mL;
(3) 20-50 mu L of PTP polypeptide aqueous solution modified by the bifunctional chelating agent is mixed with 65-100 mu L of sodium acetate buffer solution, 0.5-1 mL of radionuclide reacts for 15-60 minutes at 85 ℃; after the completion of the reaction, the product was purified, and it was confirmed that the radiochemical purity of the purified radionuclide-labeled polypeptide was 95% or more.
7. Use of the radionuclide-labeled polypeptide of any of claims 1 to 3, for constructing a nuclear medicine molecular probe or for preparing a diagnostic or therapeutic drug.
8. The use according to claim 7,
when the radionuclide is 111 In or 67 Ga, the application is the application of the polypeptide marked by the radionuclide in SPECT imaging in nuclear medicine or the application in preparing medicaments for diagnosing tumors;
when the radionuclide is 18 F[AlF]、 68 Ga and 64 when Cu is adopted, the polypeptide marked by the radionuclide is used for nuclear medicine PET imaging, or is applied to the preparation of a medicament for diagnosing tumors; or
When the reflecting nuclear species is 188 Re、 177 Lu and 90 the application is the application of the polypeptide marked by the radioactive nuclide in preparing the medicine for treating nuclear medicine nuclide and/or tumor.
9. A nuclear medicine molecular probe comprising the radionuclide-labeled polypeptide according to any of claims 1 to 3.
10. An imaging agent, wherein said imaging agent is an imaging agent for diagnosing a disease caused by high expression of Plectin protein; the imaging agent comprising the radionuclide-labeled polypeptide according to any of claims 1 to 3.
11. A medicament for treating tumors caused by high expression of Plectin protein, said medicament comprising the radionuclide-labeled polypeptide of any one of claims 1 to 3.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116790759A (en) * | 2023-08-08 | 2023-09-22 | 潍坊医学院 | Application of PLEC in early diagnosis and treatment of epithelial ovarian cancer |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116790759A (en) * | 2023-08-08 | 2023-09-22 | 潍坊医学院 | Application of PLEC in early diagnosis and treatment of epithelial ovarian cancer |
| CN116790759B (en) * | 2023-08-08 | 2023-12-01 | 潍坊医学院 | Application of PLEC in early diagnosis and treatment of epithelial ovarian cancer |
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