CN108434468B - Radioiodinated protein binding ligand and application thereof - Google Patents

Abstract

The invention discloses a protein binding ligand labeled by radioactive iodine and application thereof, wherein the structural formula is as follows:

the I is radioactive iodine nuclide, R is OH or derived from PEG, folic acid, RGD, octreotide, epidermal growth factor, protein, nucleic acid or polysaccharide. The radiolabeled protein binding ligand labeling method provided by the invention is simple, low in cost and good in stability. The results of animal in-vivo experiments show that the albumin-bound ligand has higher uptake and longer retention time in blood, and has higher target/non-target ratio. The modified compound is modified on a targeting group or other functional groups, so that the pharmacokinetic property of the compound can be obviously improved, and the blood half-life of the compound is prolonged, so that the compound is suitable for being used as a blood pool, lymph and tumor diagnosis and treatment reagent.

Description

Radioiodinated protein binding ligand and application thereof

Technical Field

The invention belongs to the technical field of diagnostic reagents, and particularly relates to a protein binding ligand labeled by radioactive iodine and application thereof.

Background

Molecular imaging is known as medical imaging in the 21 st century because it can provide powerful guarantee for early detection, early diagnosis and early treatment of diseases. On one hand, development of imaging equipment promotes early diagnosis and treatment of diseases; on the other hand, the development of non-invasive probes with good targeting, strong specificity and high sensitivity has become a key element of molecular imaging. Only if a probe with good performance is obtained, the disease can be better diagnosed and treated and the prognosis effect can be better evaluated.

In recent years, cardiovascular diseases and malignancies have severely affected human health and have been the first two of the world population morbidity and mortality. Early diagnosis and treatment are important and urgent to reduce the risk of these two diseases and to reduce the burden on society. For cardiovascular diseases, it is necessary to effectively display blood vessels by using corresponding imaging means and to explore disease pathogenesis by combining immunohistochemical research. Early diagnosis, early treatment and individualized complex treatment of such diseases are the most effective measures to reduce mortality. One of the fundamental bases of the indications and the schemes of the early and individualized treatment is the diagnosis result of medical imaging, so that the sensitive and accurate imaging diagnosis technology is a key technology for realizing early diagnosis and early treatment and individualized treatment of cardiovascular diseases and tumors. The blood vessel developing method commonly used at present comprises an ink perfusion method, an immunohistochemical method, a tannic acid mordant method, an enzyme histochemical method and the like. However, these methods have disadvantages such as not being able to combine with immunohistochemical studies, complicated process or quenching of fluorescence. The use of radionuclide-labeled imaging agents for imaging the cardiovascular system is one of the hot spots in the study of radiopharmaceuticals today. Radionuclide-labeled blood pool imaging agents capable of noninvasive in vivo detectionThe method has the advantages of determining local or integral heart functions, providing certain heart function parameters, and having certain value for early diagnosis, healing and curative effect observation of heart diseases such as coronary heart disease, cardiomyopathy, valvular disease and the like. Most diagnostic and therapeutic drugs for cardiovascular diseases have the following problems: such as short blood half-life, rapid renal clearance, easy excretion from the body, high dosage or frequent administration, etc., which inevitably results in a series of toxic and side effects, thus limiting the use thereof. The most clinically used radiolabeled blood pool imaging agents are currently^99mTc-RBC (red blood cell) (Stedrova V, et al. Eur J Nucl Med Mol I2010, 37: S495-S495.);¹¹c or¹⁵O-labeled RBC (Kearfott KJ. Jnucl Med 1982, 23 (11): 1031-;⁶⁸ga-labeled Evans blue derivatives (Zhang JJ, et al.J. Nucl Med 2015, 56 (10): 1609-1614) and the like.

In the diagnosis and treatment of tumors, rapid blood clearance is generally considered to be an advantage in receptor-targeted imaging of tumors. Rapid blood clearance reduces normal tissue radiotoxicity and increases the target/background ratio of the probe in vivo, however, the short blood half-life also results in relatively less uptake and retention time of the molecular probe in tumor tissue. While a long blood half-life is very important for radionuclide targeted therapy of tumors. Fortunately, there are some methods available to prolong the half-life of blood, such as increasing the size of drug molecules, pegylation, glycosylation, microencapsulation, binding with albumin, etc., thereby reducing the renal clearance rate, resulting in a longer circulation time of the drug in the blood, which is beneficial for the diagnosis and treatment of malignant tumors. In addition, slowing the release rate of the drug over time may also allow for sustained uptake of the targeted organ or tissue, resulting in a therapeutic effect.

Human Serum Albumin (HSA) is a natural component in human blood, and is a single peptide chain macromolecular protein consisting of 576 amino acids, wherein the protein comprises 56 lysine residues, 17 tyrosine phenolic hydroxyl groups and a free sulfhydryl group, has a molecular weight of 68400, and has a very long biological half-life.^99mTc-HSA is useful as a blood pool imaging agent, generally direct labelingThe marking rate of the method is low, the stability of the marker is poor, so that the clearance rate of the marker in blood is high, and the imaging effect is poor. In recent years the conjugation of human serum albumin using bifunctional chelating agents has been carried out^99mTc labeling is reported more, including: DTPA (diethylenetriaminepentaacetic acid), DMP (2, 3-dimercaptopropionic acid) and other bifunctional chelating agents. But do not^99mTc-DTPA-HSA is cleared from blood quickly, needs to be imaged 5-10 min after injection, and is concentrated in the liver to be unfavorable for diagnosis of some diseases. More successfully have^99mTc-DMP-HSA (Cambier JP, et a1.NuclMed Commun 1997, 18: 31-37.), the preparation of which has been kit-packaged. In addition to the above^99mIn addition to Tc-labelled imaging agents⁶²Cu、⁶⁷Cu and⁶⁸HSA labeled with a radionuclide such as Ga and derivatives thereof. Although direct in vitro radionuclide labeling of albumin is a well-established method, it is a human extract, which is expensive, and it may cause immune rejection when injected into the body, and the protein is easily contaminated and easily denatured, which limits its application to some extent. Therefore, the prior successful serum albumin labeling method has the defects of complex preparation method, high price, easy virus infection, easy influence of other factors on the imaging effect and the like, and has wide application prospect if the radioactive marker which has simple preparation method, low cost and good effect and can be effectively combined with albumin can be provided.

Disclosure of Invention

The object of the present invention is to provide a radioiodinated protein binding ligand.

Another object of the present invention is to provide a method for producing the radioiodinated protein-binding ligand.

It is a further object of the present invention to provide the use of the radioiodinated protein binding ligands described above.

The technical scheme of the invention is as follows:

a radioiodinated protein-binding ligand of the formula:

the I is radioiodine nuclide, and R is OH or a group derived from PEG, folic acid, polypeptide, epidermal growth factor, protein, nucleic acid or polysaccharide.

In a preferred embodiment of the invention, the radioiodine species is¹³¹I、¹²⁵I、¹²⁴I or¹²³I。

When R is OH, the synthetic route of the preparation method of the protein binding ligand is as follows:

in a preferred embodiment of the present invention, the method comprises the following steps:

(1) dissolving 4- [ (4-boranophenyl) butyric acid 4-BBA in a reaction solvent, respectively adding dicyclohexylcarbodiimide DCC and N-hydroxysuccinimide NHS with the same molar weight, stirring at room temperature for reaction for 10-12 h, then filtering to remove byproducts, and concentrating the obtained filtrate to obtain white solid powder, namely activated ester 4-BBA-NHS of 4- [ (4-boranophenyl) butyric acid;

(2) adding a catalyst consisting of cuprous oxide and 1, 10-phenanthroline which are uniformly mixed into activated ester 4-BBA-NHS of 4- [ (4-boranophenyl) butyric acid;

(3) adding the material obtained in the step (2) into acetonitrile solution of radioactive iodine nuclide, and carrying out oscillation reaction to obtain a labeled product 4- [ I ] IBA-NHS;

(4) adding Na₂CO₃Or hydrolyzing the protein-binding ligand by NaOH, and adding hydrochloric acid solution for neutralization to obtain the protein-binding ligand.

Further preferably, the reaction solvent includes dimethylformamide, tetrahydrofuran and dimethylsulfoxide.

In another method for preparing the protein-binding ligand, when R is a group derived from PEG, folic acid, polypeptide, epidermal growth factor, protein, nucleic acid or polysaccharide, the synthetic route is as follows:

in a preferred embodiment of the present invention, the method comprises the following steps:

(1) 4- [ (4-Borate phenyl) butyric acid 4-BBA is dissolved in a reaction solvent, dicyclohexylcarbodiimide DCC and N-hydroxysuccinimide NHS with the same molar weight are respectively added, and the mixture is stirred for reaction overnight. Filtering to remove by-products, and concentrating the filtrate to obtain white solid powder, namely the activated ester 4-BBA-NHS of 4- [ (4-boranophenyl) butyric acid;

(2) adding a catalyst consisting of cuprous oxide and 1, 10-phenanthroline which are uniformly mixed into activated ester 4-BBA-NHS of 4- [ (4-boranophenyl) butyric acid;

(3) adding the material obtained in the step (2) into acetonitrile solution of radioactive iodine nuclide, and carrying out oscillation reaction to obtain a labeled product 4- [ I ] IBA-NHS;

(4) reacting 4- [ I ] IBA-NHS and a molecule R with an amino structure in dimethylformamide, dimethyl sulfoxide or water at 20-70 ℃ for 30-60min, simultaneously adding a small amount of triethylamine, pyridine or N, N-diisopropylethylamine to promote the reaction to occur, drying and removing a solvent after the reaction is finished, and purifying to obtain the protein binding ligand.

Further preferably, the reaction solvent includes dimethylformamide, tetrahydrofuran and dimethylsulfoxide.

The protein binding ligand is applied to the preparation of diagnosis and treatment reagents.

The invention has the beneficial effects that: the radiolabeled protein binding ligand labeling method provided by the invention is simple, low in cost and good in stability. The results of animal in-vivo experiments show that the albumin-bound ligand has higher uptake and longer retention time in blood, and has higher target/non-target ratio. The modified compound is modified on a targeting group or other functional groups, so that the pharmacokinetic property of the compound can be obviously improved, and the blood half-life of the compound is prolonged, so that the compound is suitable for being used as a blood pool, lymph and tumor diagnosis and treatment reagent.

Drawings

FIG. 1 is the value of 4-, [ 2 ] in example 2 of the present invention¹³¹I]TLC analysis of IBA-NHS in physiological saline and acetonitrile over 24h, respectively.

FIG. 2 is the value of 4-, [ 2 ] in example 2 of the present invention¹³¹I]IBA and 4-¹³¹HPLC analysis of IBA-NHS.

FIG. 3 is the value of 4-, [ 2 ] in example 2 of the present invention¹³¹I]Figure showing the results of dialysis experiments with IBA in the presence and absence of human serum albumin, respectively.

FIG. 4 is the value of 4-, [ 2 ] in example 2 of the present invention¹³¹I]IBA and 4 [ [ alpha ] ]¹³¹I]Comparison graphs of dialysis experiment results of IBA-PEG in the presence or absence of bovine serum albumin respectively;

FIG. 5 is the value of 4-, [ 2 ] in example 2 of the present invention¹³¹I]IBA，4-[¹³¹I]IBA-PEG and 4-, [ 2 ]¹³¹I]Biodistribution result chart of IBA-FA in mice respectively;

FIG. 6 shows the cytotoxicity MTT assay after incubation of precursors with LO2 normal stem cells at different concentrations for 10 and 48h in example 2 of the present invention;

FIG. 7 is a graph showing the effect of varying amounts of labeled precursor on the binding of 4-IBA to albumin in example 2 of the present invention;

FIG. 8 is a Micro SPECT/CT micrograph of a protein-binding ligand according to example 2 of the present invention;

FIG. 9(a) is a graph of the trend of tumor volume in mice after treatment with different treatment regimens; (b) for the trend of the body weight of the mice during the treatment:

FIG. 10(a) is 4-, [ 2 ]¹³¹I]IC of IBA₅₀Graph: (b) IC of radioactive iodine labeled Evan blue₅₀Graph is shown.

Detailed Description

The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.

Example 1

1.4-[¹³¹I]Synthesis of IBA-NHS

1g of 4- [ (4-boranophenyl) butanoic acid (4-BBA) was dissolved in 15mL of DMF, and 981mg of dicyclohexylcarbodiimide and 548mg of N-hydroxysuccinimide were added, respectively, and the reaction was stirred at room temperature overnight. Filtering to remove by-products, and concentrating the filtrate to obtain white solid powder, namely the activated ester of 4- [ (4-boranophenyl) butyric acid;

taking 1mg of 4- [ (4-boranophenyl) butyric acid activated ester (4-BBA-NHS), and adding 50 mu L of uniformly mixed cuprous oxide and 1, 10-phenanthroline catalyst; adding the above solution into dried radioactive iodine, and performing concussion reaction at room temperature for 30min to obtain labeled product 4-, [ 2 ]¹³¹I]IBA-NHS; adding a proper amount of Na₂CO₃Hydrolyzing to obtain target product 4-, [ solution of ] or its salt¹³¹I]IBA. The labeled end product is identified by HPLC as having a radioactive peak time comparable to the stabilized albumin ligand 4-, and¹²⁷I]ultraviolet peak time matching of IBA (Stable ligand 4-, [ 2 ]¹²⁷I]The ultraviolet peak emergence time of IBA is 3.92min, 4-, [ 2 ]¹³¹I]The radioactive peak time of IBA was 4.16 min).

2.4-[¹³¹I]Synthesis of IBA-PEG

2-, [ 4 ]¹³¹I]IBA-NHS and NH2-PEG₅₀₀₀Reaction of-N3 in DMF at room temperature for 1h, and addition of 10. mu. L N, N-diisopropylethylamine to promote the reaction to obtain the target compound 4-, [ 2 ]¹³¹I]IBA-PEG：。

3.4-[¹³¹I]Synthesis of IBA-FA

2-, [ 4 ]¹³¹I]IBA-NHS and FA-PEG-NH₂The reaction was carried out in Dimethylformamide (DMF) at room temperature for 1h, and 10. mu. L N, N-diisopropylethylamine was added to accelerate the reaction. After the reaction is completed, purification is carried out by HPLC to finally obtain the target compound 4-, [ 2 ], [¹³¹I]IBA-FA。

The stable reference compound 4-, used in the above synthesis¹²⁷I]IBA, cuprous oxide, 1, 10-phenanthroline, dimethylformamide, N-diisopropylethylamine and Na₂CO₃And the common reagents are all obtained on the market.

Example 2

The following is the marker 4-, [ 2 ] synthesized by the method of example 1 above¹³¹I]IBA、4-[¹³¹I]IBA-PEG or 4-, [ 2 ]¹³¹I]Description of the performance measurements of IBA-FA:

analytical characterization by TLC and HPLC

4-[¹³¹I]The TLC analysis system for IBA is as follows: in the system of petroleum ether and ethyl acetate which are 1: 1, TLC (thin layer chromatography) is respectively carried out on a silica gel aluminum plate at different time points for analysis; the physiological saline system was analyzed by TLC with polyamide thin film assay at different time points. 4- [¹³¹I]The marking rate of IBA is fast, already exceeding 99% within 10 min; the radiochemical purity is high and is more than 95 percent; the nature is stable and not easy to decompose, and the TLC shows that only one single peak appears until 24h, and the result is shown in figure 1.

4-[¹³¹I]The HPLC analysis system for IBA is as follows: Perkin-Elmer Series 200LC was equipped with a Waters 2784 double absorption wavelength UV detector and a Bioscan radioactivity detector, a Waters Symmetry C18 analytical column (5 μm, 150 x3.9mm). Flow rate 1mL/min, elution gradient: 0-30 min: 80% methanol and 20% water, left unchanged. The HPLC result is shown in FIG. 2, and the result shows 4-, [ 2 ]¹³¹I]IBA-NHS and 4-, [ solution of A and S ]¹³¹I]The retention time of IBA was 5.23min and 4.16min, respectively, and the radiochemical purity was calculated to be greater than 95%.

2. Measurement of fat and Water distribution coefficient

¹³¹I-labeled albumin complex 4-, [ 2 ]¹³¹I]Determination of the lipid-water distribution coefficient (log P) of IBA and its derivatives was accomplished by the following steps:

mu.L of the diluted radio-injectate was added to a centrifuge tube containing 2.9mL of a mixture of ultrapure water and 3mL of n-octanol (PBS and n-octanol were mixed one day before the experiment and left to stand for the next day of the experiment), after vortex shaking for 2min, centrifuged at 6000rpm for 5min, and 100. mu.L of each of the liquid was taken from the aqueous phase and the n-octanol phase and counted by gamma-counter radioactivity. The experiment was repeated three times and the mean value was taken. The lipid-water distribution coefficient (log P) is calculated by the formula:

P＝(I_a-I)/(I_b-I)

wherein I_aRepresenting the measured radioactivity count in the organic phase, I_bRepresenting the radioactive counts measured in the aqueous phase, I representing the background counts.

By calculation, the lipid-water distribution coefficient of each radiolabeled targeting probe was finally determined.

The result indicates that the value of "4")¹³¹I]The fat-water distribution coefficient logP of IBA is 1.01 plus or minus 0.03, and the IBA is fat-soluble; 4- [¹³¹I]The logP of IBA-PEG is-1.438 +/-0.03, and the water solubility is shown, which indicates that the water solubility of the labeled probe modified by PEG is obviously improved; 4- [¹³¹I]IBA-FA log p 0.52 ± 0.03.

3. Determination of Albumin binding Performance

Respectively measuring 4-, [ 2 ] by dialysis test¹³¹I]IBA and 4 [ [ alpha ] ]¹³¹I]Albumin binding capacity of IBA-PEG. The cut-off molecular weight of the dialysis bag used was 8000-14000Da, and the dialysate was 1000mL of PBS7.4 solution. Accurately weighing 30mg of Human Serum Albumin (HSA) or Bovine Serum Albumin (BSA) and dissolving in 3mL of PBS7.4, adding labeled 4-, [ solution ] to the solution¹³¹I]IBA 18MBq, mixed well and added into a dialysis bag, and then placed in dialysate for dialysis. At various time points, 1mL of each dialysate was taken into a centrifuge tube and immediately supplemented with 1mL of PBS and the radioactivity count of the removed dialysate was measured using a gamma counter. Radioactivity in the dialysis bags was measured at different time points with a radioactivity meter. As a control, another tube of PBS7.4 without HSA or BSA was taken, and an equal dose of 4-, [ 2 ], [ solution ] was added¹³¹I]IBA, placed in dialysis bags for dialysis, was measured for radioactivity in the same manner. The above experiment was repeated three times to obtain an average value, and finally a concentration/radioactivity activity percentage curve was obtained, as shown in FIG. 3, and the results showed that in the presence of serum albumin, the radioactive marker was not easily dialyzed out of the dialysis bag until most of the radioactivity remained in the dialysis bag after 24h, while the radioactive marker was rapidly removed from the dialysis bag without albumin, indicating 4-, ", and¹³¹I]IBA can bind to serum albumin and is stable without decomposition within 24 h.

For comparison, the PEG-modified 4-, was¹³¹I]The change in binding ability of IBA to albumin was examined in Bovine Serum Albumin (BSA) by the same method as described above, and the results are shown in FIG. 4, and 4-, [¹³¹I]IBA comparison, after PEG modification, more radiation in the presence of bovine serum albuminRadioactivity trapped in the dialysis bag, but not in the dialysis bag with bovine serum albumin, was removed very quickly, indicating that the PEG-modified 4-, was¹³¹I]IBA-PEG can also be effectively combined with albumin.

4. Biodistribution test

18-20g of female BALB/c mice were selected for the experiment. 3 mice per group, each mouse was sacrificed at different time points by injecting a radiotracer 370KBq via tail vein, dissected, collected blood and its organs, measured for weight, measured for radioactivity by gamma-counter, and calculated for the amount of radioactivity contained in the organs per unit mass at different time points, expressed as% ID/g. The results are shown in FIG. 5. The result showed that the radioactive marker 4 was injected¹³¹I]The amount of radioactivity in the blood 30min after IBA was 10.51. + -. 2.58% ID/g. While the radioactivity content in the major organs such as liver, lung and kidney is 2.93 + -0.11%, 4.47 + -0.13% and 5.68 + -0.40% respectively, which is the highest in comparison with the blood. Furthermore, thyroid, the most sensitive gland to iodide ions, has very low uptake at all times, indicating that the tracer is very stable in vivo and no deiodination occurs. The ratio of the radioactivity in blood/organ is the highest in each observation period, and a high target/non-target ratio is achieved. The experimental result shows that the tracer 4-, [ 2 ]¹³¹I]IBA effectively prolongs the half-life period of blood and reduces the clearance rate of kidney, and is an ideal blood pool developer.

In order to better improve the 4-, [ 2 ]¹³¹I]The pharmacokinetic property of IBA is that PEG is introduced to modify IBA, so as to enhance the water solubility and further prolong the half-life of blood. 4- [¹³¹I]IBA-PEG is injected into mice through tail vein for biodistribution experiment, after 30min, the radioactive content in blood is 24.79 +/-0.89% ID/g, which is far higher than other tissue organs. After a longer time point, e.g. 24h, the blood still contains a higher retention of radioactivity.

After introduction of the folate targeting group, the circulation time of the probe in the blood is maintained. The kidney tissue highly expresses the folate receptor, so the uptake of the probe in the kidney is higher, and the targeting of the folate group is further explained. In other organs such as liver and lung, the radioactivity retention is low, and the tumor imaging is more facilitated.

5. Cytotoxicity MTT assay

Normal hepatocytes LO2 were selected for the experiment, cells in log phase were collected after incubation, cell suspension concentration was adjusted, and plating was performed with a 96-well plate with cell density of 10000 cells/well. Adding prodrugs of 4-IBA with different concentration gradients in sequence after cells adhere to the wall, arranging 5 multiple wells, respectively incubating for 10h at 37 ℃, adding 10 mu L of 5mg/mL MTT solution after 24h, continuing to culture for 4h, terminating the culture, sucking out culture solution in each well, adding 150 mu L dimethyl sulfoxide into each well, dissolving formazan in the cells, and detecting an absorbance value at 490am wavelength by using an enzyme linked immunosorbent assay detector, wherein the absorbance value can indirectly reflect the number of living cells. The results are shown in fig. 6, where the addition of different concentrations of prodrug did not have a significant effect on cell activity.

6. Effect of different concentrations of precursor on 4-IBA binding to proteins

Taking the radioactive labeled tracer 4-, [ 2 ]¹³¹I]Mixing IBA (immunoglobulin A) with the concentration of about 5 mu Ci and human serum albumin (2 mg/mL), adding 4-IBA precursors with different concentration gradients, incubating for 30-60min, centrifuging in a 10kDa ultrafiltration centrifuge tube at the set rotation speed of 10000r/min for 10min, collecting the liquid on the upper layer and the liquid on the lower layer of the centrifuge tube respectively, and measuring the radioactivity count in a gamma-counter detector. As can be seen from FIG. 7, precursors of different concentrations were added to 4-, [ 2 ]¹³¹I]In the reaction system in which IBA binds to albumin, 4 [ - ] [ (])¹³¹I]The amount of IBA bound to albumin was not affected.

7. MicroSPECT imaging of mice

Compound 4-, [ solution ] having a radiochemical purity of greater than 95% is prepared by way of example¹³¹I]IBA-PEG, 0.1mL (about 3.7MBq) was injected via tail vein into healthy female BALB/c mice (approximately 18-20 grams body weight) and immediately subjected to static SPECT/CT image acquisition. The radiotracer was scanned separately at different time points after injection and the imaging results are shown in figure 8. 4- [¹³¹I]IBA-PEG can be retained in blood vessel and heart of mice, and can be retained for 4hThe clear image in the blood pool is seen, which shows that the structure has longer blood half-life period in the organism and provides possibility for the diagnosis, curative effect and prognosis evaluation of cardiovascular system diseases.

8. Radionuclide therapy experiments

2-, [ 2 ]¹³¹I]The effect of IBA on tumor treatment was examined. Female BALB/c mice (weighing about 18-20 g) were inoculated subcutaneously in the right hind leg with 4T1 tumor cells and treatment was initiated after one week after the tumor diameter had grown to about 0.5 cm. Mixing 15MBq 4-, [ 2 ]¹³¹I]IBA was injected into mice via the tail vein and the tumor size of the mice was measured every other day by a vernier caliper, while the body weight change of the mice was measured. Tumor volume V ═ a × b²/2. In the treatment group of two treatment courses, 15MBq 4-, [ 2 ] is injected for the first time¹³¹I]On the 5 th day after IBA, 15MBq 4-, [ 2 ] is injected again¹³¹I]IBA. As a control, physiological saline and Na were added¹³¹Control group I, 200. mu.L of physiological saline and 15MBq Na were injected¹³¹And (I) solution. The results of the treatment are shown in FIG. 9. 4-¹³¹The growth trend of the tumors in the IBA-treated group was significantly slowed down. The tumor growth inhibition effect of the double-treatment course group is more obvious. Physiological saline group and Na¹³¹The tumor growth rate was significantly higher in group I than in the treated group. After the injection of the radionuclide, the body weight of the mice slightly decreased and quickly returned to normal, and there was no death of the mice during the treatment.

4-[¹³¹I]IBA is a new radiolabeled albumin complex, has strong radioactive ray penetrability compared with the existing common blood pool imaging agent, and can carry out in-vivo noninvasive blood pool imaging. Compared with the existing radioactive blood pool developer^99mTc-RBC and^99mTc-HSA differs significantly in that both are biological extracts, the labeling process is complex, the labeled product is unstable, and multiple purification steps are required after synthesis, which inevitably results in loss of product and radioactivity; and the protein is easy to denature, possibly generates immunological rejection reaction, is expensive, and is easy to infect viruses. Radionuclide-labeled evans blue has been extensively studied as a superior blood pool imaging agent (CN 201310157168.9). Measurement of 4-, by a method of competitive binding¹³¹I]IC of IBA and radioiodinated Evans blue₅₀The values are respectively: at 46.5. mu.M and 25.1. mu.M (FIG. 10), Evans blue exhibits higher affinity for the protein. However, in tumor and blood pool imaging, very high affinity is not the optimal choice, and it is desirable to find a dynamic balance that allows higher uptake at the focal site after binding of the imaging agent to the protein, and faster clearance of radioactivity trapped in normal tissues and blood, which increases both target to non-target contrast and reduces radiopharmaceutical damage to normal tissues. Therefore, the level of affinity is not the most important index for evaluating the quality of the developer, and the advantages and disadvantages of the developer need to be comprehensively considered in combination with other factors. A series of experiments prove that 4-, is¹³¹I]The binding capacity of IBA to proteins is sufficient to allow its use as an imaging agent for blood pools, lymph and tumours. Furthermore, the preparation process of the evan blue marker requires harsh conditions and cumbersome steps: 1) a bifunctional chelating agent (NOTA or DOTA) is required to be connected for chemical modification so as to mark nuclide; 2) most of the applied positive electron nuclides need accelerators for preparation, so that the operation is complicated and the operation cost is high; 3) the half-life of nuclides is short, and long-distance transport cannot be realized; 4) after marking, the high performance liquid chromatography is needed for purification to meet the application requirements. In contrast, radioiodinated 4-IBA has the advantages not possessed by radiolabeled Evan's blue probe: 1) the reaction is simple and rapid, the marking can be completed in a short time, and high yield is achieved; 2) the labeling rate is high, purification is not needed, the requirement of a large amount of preparation in hospitals can be met, the toxicity of redundant labeled precursors is low, and the combination of 4-IBA and albumin is not influenced; 3) the molecular structure of the compound can be further modified through chemical reaction to obtain a radioactive tracer or a therapeutic drug with better performance without additionally introducing a bifunctional chelating agent; 4) radioactive iodine has a variety of isotopes,¹³¹I、¹²⁴i can be used as the imaging nuclide of SPECT and PET respectively,¹³¹i and¹²⁵the I can be used as a radioactive therapeutic nuclide and can meet different use requirements. In general, 4-¹³¹I]IBA has advantages not possessed by common blood pool imaging agents: the compound is small organic molecule, such asThe complex has the advantages of simple substructure, mild labeling condition, reaction at room temperature, simple and easy labeling method, high labeling rate, easy obtainment of a complex with high radiochemical purity, long storage time, good stability, effective prolongation of the half-life period of blood and reduction of the clearance rate of kidney.

The radionuclides used in the present invention may also include other radioiodine species, such as¹²⁵I、¹²⁴I, and the like, and the preparation method is similar.

The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.

Claims (8)

1. A radioiodinated protein-binding ligand, characterized in that: the structural formula is as follows:

the above I is radioactive iodine, R is OH or a group derived from PEG or folic acid; the radioactive iodine is¹³¹I、¹²⁵I、¹²⁴I or¹²³I；

When R is a group derived from PEG or folic acid, the synthetic route is as follows:

2. a method of preparing a protein binding ligand according to claim 1, wherein: when R is OH, the synthetic route is as follows:

3. the method of claim 2, wherein: the method comprises the following steps:

(1) dissolving 4- [ (4-boranophenyl) butyric acid 4-BBA in a reaction solvent, respectively adding dicyclohexylcarbodiimide DCC and N-hydroxysuccinimide NHS with the same molar weight, stirring at room temperature for reaction for 10-12 h, then filtering to remove byproducts, and concentrating the obtained filtrate to obtain white solid powder, namely activated ester 4-BBA-NHS of 4- [ (4-boranophenyl) butyric acid;

(2) adding a catalyst consisting of cuprous oxide and 1, 10-phenanthroline which are uniformly mixed into activated ester 4-BBA-NHS of 4- [ (4-boranophenyl) butyric acid;

(3) adding the material obtained in the step (2) into acetonitrile solution of radioactive iodine, and carrying out oscillation reaction to obtain a labeled product 4- [ I ] IBA-NHS;

(4) adding Na₂CO₃Or hydrolyzing the protein by NaOH, and adding hydrochloric acid solution for neutralization to obtain the protein binding ligand.

4. The method of claim 3, wherein: the reaction solvent is dimethylformamide, tetrahydrofuran or dimethyl sulfoxide.

5. A method of preparing a protein binding ligand according to claim 1, wherein: when R is a group derived from PEG or folic acid, the synthetic route is as follows:

6. the method of claim 5, wherein: the method comprises the following steps:

(1) dissolving 4- [ (4-boranophenyl) butyric acid 4-BBA in a reaction solvent, respectively adding dicyclohexylcarbodiimide DCC and N-hydroxysuccinimide NHS with the same molar weight, and stirring for reacting overnight; filtering to remove by-products, and concentrating the filtrate to obtain white solid powder, namely the activated ester 4-BBA-NHS of 4- [ (4-boranophenyl) butyric acid;

(2) adding a catalyst consisting of cuprous oxide and 1, 10-phenanthroline which are uniformly mixed into activated ester 4-BBA-NHS of 4- [ (4-boranophenyl) butyric acid;

(3) adding the material obtained in the step (2) into acetonitrile solution of radioactive iodine, and carrying out oscillation reaction to obtain a labeled product 4- [ I ] IBA-NHS;

(4) reacting 4- [ I ] IBA-NHS and a molecule R with an amino structure in dimethylformamide, dimethyl sulfoxide or water at 20-70 ℃ for 30-60min, simultaneously adding a small amount of triethylamine, pyridine or N, N-diisopropylethylamine to promote the reaction to occur, drying and removing a solvent after the reaction is finished, and purifying to obtain the protein binding ligand.

7. The method of claim 6, wherein: the reaction solvent is dimethylformamide, tetrahydrofuran or dimethyl sulfoxide.

8. Use of the protein binding ligand of claim 1 for the preparation of a diagnostic or therapeutic agent.

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