A method for radiolabeling compounds containing carboxylic acid, phosphoric acid, or sulfuric acid groups, radiolabeled substances, and their applications.

Abstract

This invention, entitled "A Method, Radiolabeled Compound, and Application for Radiolabeling of Compounds Containing Carboxylic Acid, Phosphoric Acid, or Sulfate Groups," belongs to the field of radioisotope labeling preparation technology. The technical problem to be solved is to reduce operational difficulty and simplify the preparation process; and to better monitor the metabolic processes of macromolecules in vivo. The key technical solution is: a method for radiolabeling compounds containing carboxylic acid, phosphate, or sulfate groups, comprising the steps of reacting the compound containing carboxylic acid, phosphate, or sulfate groups, a solvent, a catalyst, and a radioactive reagent, followed by purification.

Description

Technical Field

[0001] This invention provides a method for radiolabeling compounds containing carboxylic acid, phosphoric acid, or sulfuric acid groups, radiolabeled substances, and their applications, belonging to the field of radioisotope labeling preparation. Background Technology

[0002] For understanding the technical content of this invention:

[0003] RNA and DNA and their various derivatives are increasingly used in the prevention and treatment of human diseases and in the medical aesthetics industry. Before their products are approved for marketing by regulatory authorities, they must undergo toxicological and pharmacokinetic studies in animals and humans to clarify their absorption, distribution, metabolism, and excretion (ADME) characteristics in humans and animals, as well as their relationship with efficacy and potential toxicity.

[0004] However, because RNA and DNA are both endogenous substances and have poor stability or extremely low concentrations in vivo, they are not suitable for obtaining reliable ADME experimental results. Low-energy radioactivity ( 3 H and 14 C) Labeling technology has been widely used in ADME evaluation of small molecules and protein drugs, but due to the special structure of RNA and DNA, it is difficult to obtain radiolabeling methods that can track the entire molecule.

[0005] Site-directed radiolabeling for RNA and DNA synthesis is time-consuming and very expensive. Derivatization labeling at the 3' or 5' ends with biotin or fluorescein may significantly alter the metabolic properties of RNA and DNA, and relying on specialized detection methods increases operational complexity. 35 S or 32 Labeling P and other substances presents challenges such as labeling difficulties, high radioactivity, short half-life, and alteration of biological activity.

[0006] Relevant patent documents retrieved: This document, published in the United States (publication number US2016152656A1, publication date June 2, 2016), discloses a method for modifying label molecules on nucleic acids via phosphoramidation. The method involves indirect coupling modification of the label molecules on the nucleic acids, and includes the following steps: (1) Dissolve the nucleic acid and the activation reagent in the first buffer solution and react for 70-100 minutes. The 5' end of the nucleic acid is phosphate ester. (2) Purify and precipitate with ethanol to obtain the intermediate product; (3) Dissolve the intermediate product in a second buffer solution, add a nucleophilic crosslinking agent, and react for 2.5-3 hours. The second buffer solution contains EDTA. (4) Precipitate with ethanol and purify with urea-PAGE gel to obtain the nucleic acid conjugate modified by the nucleophilic crosslinking agent; (5) Dissolve the nucleic acid conjugate in the third buffer solution, add the labeling reagent, and react for 0.5-12 hours to obtain the final product.

[0007] The preparation method of this patent is complex and not conducive to large-scale application; compounds containing phosphate groups are generally hydrolyzed and metabolized in vivo from the 3' or 5' end, resulting in poor in vivo stability; the derivatization labeling with biotin or fluorescein at the 3' or 5' end introduces a large group that may significantly change the metabolic properties of RNA and DNA, and relying on special detection methods increases the complexity of operation.

[0008] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: Relying on special detection methods increases operational complexity; poor in vivo stability is evidenced by the fact that the nucleic acid in US patent US2016152656A1 has a phosphate ester at the 5' end for phosphoramidation. Compounds containing phosphate groups are generally hydrolyzed and metabolized in vivo from the 3' or 5' end, resulting in poor in vivo stability. Introducing a large group may also change the metabolic properties of RNA and DNA, and relying on special detection methods increases operational complexity. Summary of the Invention

[0009] The purpose of this invention is to provide: A method for radiolabeling compounds containing carboxylic acid, phosphoric acid, or sulfuric acid groups, radiolabeled substances and their applications, and related technologies, to solve technical problems such as reducing operational difficulty, simplifying preparation processes, and improving in vivo stability, yield, radioactivity reactivity, and radioactivity purity, or combinations thereof.

[0010] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0011] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0012] The definition of standard chemical terms can be found in the reference "Experimental Techniques in Modern Molecular Biology, 3rd Edition, Wei Chunhong, Higher Education Press".

[0013] Unless otherwise specified, conventional methods within the scope of the art, such as HPLC detection and LCS detection, shall be used.

[0014] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0015] As used herein, the term "room temperature" refers to ambient temperature, ranging from about 10°C to about 40°C. In some embodiments, "room temperature" refers to a temperature ranging from about 20°C to about 30°C; in other embodiments, "room temperature" refers to a temperature ranging from about 25°C to about 30°C; and in still other embodiments, "room temperature" refers to 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, etc.

[0016] In a first aspect, the present invention provides a method for radiolabeling a compound containing a carboxylic acid, phosphoric acid, or sulfuric acid group, comprising the following steps: reacting the compound containing a carboxylic acid, phosphoric acid, or sulfuric acid group, a solvent, a catalyst, and a radioactive reagent, and purifying the compound to obtain the radiolabeled compound.

[0017] Among them, the technical features of compounds containing carboxylic acid, phosphoric acid or sulfuric acid groups are selected from: macromolecular compounds containing carboxylic acid, phosphoric acid or sulfuric acid groups and small molecule compounds containing phosphoric acid or carboxylic acid groups.

[0018] The preferred macromolecular compounds for the technical features are: polydeoxyribonucleotides (PDRN), polynucleotides (PN), hyaluronic acid, heparin, and polypeptides or proteins that do not contain lysine but contain aspartic acid.

[0019] Among them, the small molecule compounds with technical characteristics are further preferably: small interfering RNA (siRNA), antisense oligonucleotide (ASO), and microRNA (miRNA).

[0020] The technical feature solvent is selected from at least one of the following: aprotic solvents or aqueous systems.

[0021] The preferred aprotic solvent is selected from at least one of dimethylacetamide, acetonitrile, tetrahydrofuran, dichloromethane, and DMF.

[0022] The preferred aqueous system for the technical feature is at least one of water, MES (2-morpholinoethanesulfonic acid), and PB (phosphate buffer).

[0023] The technical characteristic solvent is more preferably at least one of DMF and water, or DMF and MES.

[0024] The catalyst with technical characteristics is selected from at least one of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide).

[0025] The preferred catalysts for technical characteristics are EDC and NHS.

[0026] The technical characteristic reaction is selected from the following: the reaction temperature is 20-30℃ and the reaction time is 1-15h.

[0027] The technical feature of purification is ultrafiltration.

[0028] The preferred technical feature of ultrafiltration is: MWCO 10 kDa-50 kDa.

[0029] The technical feature includes a radioactive reagent selected from at least one of amino compounds, methanol, and ethanol.

[0030] The preferred amino compound is selected from at least one of methylamine, dimethylamine, diethylamine, and aliphatic amines.

[0031] The molar ratio of the compound containing carboxylic acid, phosphoric acid, or sulfuric acid groups to the catalyst is selected from: 0.002-0.004:0.02-0.07.

[0032] Among them, the molar ratio of the compound containing carboxylic acid, phosphoric acid or sulfuric acid groups to the radioactive reagent is (2-4):(0.002-0.004). Secondly, the present invention provides: a radiolabeled substance prepared by the above-described radiolabeling method, wherein the labeling site is located at a carboxylic acid, phosphate, or sulfate group.

[0033] Thirdly, the present invention provides the application of the above-described radioactive labeling method in improving the stability of radioactively labeled objects.

[0034] The beneficial effects of this invention are as follows: The present invention has at least the following beneficial effects: 1. Compared with the prior art, the present invention has better technical effects in terms of in vitro stability.

[0035] According to experimental tests, this invention improves in vitro stability from the previous technology where the label was removed after enzymatic digestion to the point where the radioactive label remains after enzymatic digestion. Even after enzymatic digestion, the labeled PDRN still shows detectable mass spectrometric peaks indicating the binding of nucleotides and reactants after digestion. Figure 5 and Figure 6 This demonstrates the stability of the PDRN marker.

[0036] Based on data from in vitro enzyme degradation and experimental results after administration to rats, radioactive peaks and mass spectrometry signals of single nucleotides bound to radiolabeled substances can be detected in vitro and in vivo after degradation.

[0037] 2. Selective modification: The hydroxyl groups of phosphate groups are modified by methylamine in aqueous or anhydrous solvents under EDC / NHS conditions.

[0038] 3. Enhanced sensitivity: Containing radioactive or fluorescein enhances the detection sensitivity of phosphate-containing compounds in vivo or in vitro.

[0039] 4. Unchanged stability: Compounds containing phosphate groups, such as oligonucleotides, are generally hydrolyzed from the 3' or 5' end in vivo. The labeled site is located on the phosphate group and does not affect the stability of the structure.

[0040] 5. Minimal impact on activity: The labeled site is located on the phosphate group, outside the nucleotide, and has no effect on activity.

[0041] Furthermore, based on the present invention: Based on the comparison between Example 1 and Comparative Examples 1-11, the present invention achieves new technical effects by combining different types of catalysts, solvents, and reaction conditions. The combined technical effect is superior to the sum of the effects of each individual technical means. Attached Figure Description

[0042] Figure 1 This is a radioactivity spectrum. The horizontal axis represents time (min), and the vertical axis represents radioactivity intensity (DPM).

[0043] Figure 2 This is a UV spectrum. The horizontal axis represents time (min), and the vertical axis represents UV (mV).

[0044] Figure 3 This is the liquid chromatogram after reaction with dimethylamine.

[0045] Figure 4 This is a liquid chromatogram of single nucleotides detected in the degradation solution.

[0046] Figure 5 This is a liquid chromatogram of mononucleotide and dimethylamine conjugates detected in the degradation solution.

[0047] Figure 6 The liquid chromatogram of the complex formed by the continued reaction of the degradation solution and dimethylamine.

[0048] Figure 7 This is a liquid chromatogram of experimental metabolites from rats. Detailed Implementation

[0049] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0050] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0051] Example 1 The reactants in Example 1 are shown in Table 1.

[0052] Table 1

[0053] Preparation method: (1) Dissolve PDRN in water (1 mg / mL, pH 6.0), add NHS and EDC dissolved in DMF, add a rotor, and stir at room temperature (25°C) for 1 h; (2) Ultrafiltration of the reaction solution (MWCO 10 kDa), take the filtrate to remove small molecule byproducts, and repeat the ultrafiltration step 3 times; (3) Add [ to the filtrate] dropwise 3 [H]dimethylamine, reacted overnight (12h) at room temperature; (4) Ultrafiltration of the reaction solution (MWCO 10 kDa) to remove small molecule byproducts and unreacted [ 3 [H]dimethylamine, to obtain the post-reaction solution; (5) Purity and labeling results were determined by HPLC.

[0054] Result: 20% yield (based on amines).

[0055] Example 2 Replace the water in step (1) of Example 1 with MES, and the rest is the same as in Example 1.

[0056] Result: 20% yield (based on amines).

[0057] Example 3 PDRN was replaced with equimolar amounts of sodium heparin and hyaluronic acid, respectively, with the rest being the same as in Example 1.

[0058] Result: 20% yield (based on amines).

[0059] Example 4 (1) Dissolve PDRN in water (1 mg / mL, pH 6.0), add DMF-dissolved NHS and EDC, add a rotor, and add [dropwise]. 3 [H]dimethylamine, reacted overnight (12h) at room temperature (25 degrees Celsius); (2) Ultrafiltration of the reaction solution (MWCO 10 kDa) to remove small molecule byproducts and unreacted [ 3 [H]dimethylamine, to obtain the post-reaction solution; (3) Purity and labeling results were determined by HPLC.

[0060] Result: 20% yield (based on amines).

[0061] Example 5 Without adding NHS, the rest is the same as in Example 1.

[0062] Comparative Example The reactants for Comparative Examples 1-11 are shown in Table 2.

[0063] Table 2

[0064] Note: The amounts of PDRN and dimethylamine (including [3H]dimethylamine) in Table 2 are the same as in Example 1.

[0065] Preparation method: (1) Dissolve PDRN and catalyst in a solvent, and add [ 3 [H]dimethylamine; (2) The reaction shall be carried out according to the reaction conditions in Table 2; (3) Ultrafiltration of the reaction solution (MWCO 10 kDa) to remove small molecule byproducts and unreacted methylamine to obtain the post-reaction solution; (4) Purity and labeling results were determined by HPLC.

[0066] Example 1: UV purity and radioactivity purity analysis HPLC was used for the purity analysis of PDRN. 5mM sodium chloride was used to analyze the chemical purity of PDRN after the reaction. At the same time, HPLC fractions were collected at 1-minute intervals using a fraction collector for LCS detection. The obtained UV retention time was consistent with the retention time of the radioactive peak detected by LCS. The UV purity and radioactive purity were qualified, indicating that the radioactive label was on the structure of PDRN and no side reactions were generated.

[0067] High performance liquid chromatography method: HPLC system: Shimadzu CBM20A Data processing system: Analyst 1.6.3 Liquid Scintillation Counter: Tricarb 4910TR Detectors: UV (260nm) and LSC Chromatographic column: Zenix SEC-150, 3μm 7.8×300mm Column temperature: 25°C Mobile phase: 5mM sodium chloride Analysis time: 30 min Flow rate: 0.5 mL / min LCS detection method: Radioactivity intensity was determined using a liquid scintillation counter. A suitable volume of sample was transferred from each test sample into a scintillation bottle, a certain volume of scintillation fluid was added and mixed thoroughly, and the sample was placed in the liquid scintillation counter to measure its radioactivity. Quenching correction was performed using the external standard method, and the radioactivity concentration of each sample was calculated based on the actual sample volume. The counting time for each sample was set to 5 minutes or 2% sigma as the termination standard. The scintillation counter automatically subtracted the instrument background (including the scintillation fluid background value) according to the method requirements and automatically converted the count value per minute (CPM) to the decay value per minute (DPM). When the coefficient of variation of the parallel sample test results was >10%, it was considered that there was a difference between the samples, and the measurement needed to be repeated. The use of the liquid scintillation counter and the determination of the sample radioactivity concentration strictly followed the relevant standard operating procedures (SOP) of Vantage.

[0068] use 3 The H calibration standard solution and background standard solution were used to evaluate the instrument stability of the liquid scintillation counter, determining parameters such as instrument background and scintillation counting efficiency. This validation was performed weekly. The resulting report after calibration was saved in the instrument log, and the results (instrument background and efficiency) were recorded in the instrument log.

[0069] The radiometric spectrum of Example 1 (obtained by HPLC and UV coupled with a radiometric detector) is shown below. Figure 1 See UV spectrum Figure 2 .

[0070] Example 2: In vitro degradation study Example 1 uses Nuclease P1 enzyme for in vitro degradation study.

[0071] The HPLC detection parameters are as shown in Example 1.

[0072] 1. Preparation of reference standards: dAMP, dTMP, dGMP, and dCMP standards were labeled using the same labeling method as in Example 1 and used as reference standards for analysis. (PDRN was replaced with equimolar amounts of dAMP, dTMP, dGMP, and dCMP, respectively; the remaining steps were the same as in Example 1.) The HPLC chromatograms after the reaction are shown in [Figure 1]. Figure 3 .

[0073] 2. [3H-DMA]PDRN was degraded in vitro using Nuclease P1 enzyme under the following conditions: 1 mg [ 3 H-DMA]PDRN (Example 1) was dissolved in 1.05 mL of water, and 200 μL of 5 mM ZnCl2, 300 μL of 50 mM pH 5.5 sodium acetate buffer, and 2 U of nuclease P1 (Roche) were added, for a total volume of 1.55 mL (containing 1.3 U / mL enzyme, 0.65 mM ZnCl2, and 9.7 mM sodium acetate). After reacting at 37°C–38°C for 2 hours, the solution was obtained by centrifugation and ultrafiltration (YM-10 ultrafiltration tube). The degradation solution was analyzed by HPLC. The mononucleotides detected in the degradation solution were as follows: Figure 4 Mononucleotides and dimethylamine conjugates detected in the degradation solution, such as Figure 5 No dimethylamine signal peak was detected, indicating that the label was not detached after enzymatic digestion, and the in vitro stability of Example 1 is good.

[0074] 3. The degradation solution was reacted again according to the preparation method of Example 1 to demonstrate the possible degradation binding products. The degraded solution can continue to react with […]. 3 The reaction with dimethylamine (H) indicates that the degradation products are small molecule nucleotides that can react with dimethylamine and other substances, and also serves as a standard control for detection.

[0075] The complex formed by the continued reaction of the degradation solution and dimethylamine, such as Figure 6 .

[0076] Example 3: In vivo degradation study Example 1 was used for animal administration (n=3), and the administration regimen is as follows: A single dorsal injection was administered, specifically an intradermal injection along both sides of the midline of the rat's back, using a syringe equipped with an injection needle. The injected drug was: Example 1, with a specific activity of 1 mg / 30 μCi, dissolved in 1 mL of physiological saline, resulting in a concentration of 30 μCi / mL. The dosage was 15 μCi / kg, and the administration volume was 0.5 mL / kg. For example, if the rat weighed 260 g, the radioactivity administered to the rat would be 4 μCi.

[0077] The dosing regimen and sampling method are shown in Table 3.

[0078] Table 3

[0079] a: Approximately 0.4 mL of blood was collected by jugular vein puncture before and within 24 hours after drug administration. After euthanasia by inhaling excessive carbon dioxide at the last time point, approximately 7 mL of blood was collected by cardiac puncture, and plasma was separated at low temperature.

[0080] Plasma and feces were separated and extracted using acetonitrile, and then detected using the method described in Example 1. Urine was detected using the method described in Example 1. The radioactive metabolite profile of rats is as follows. Figure 7 .

[0081] Methods for detecting radioactive recovery rate: (1) Blood plasma, urine Sample: Collected plasma or urine.

[0082] After the sample is vortexed and mixed, one portion is weighed into a 20 mL scintillation bottle, 5 mL of scintillation liquid is added and mixed evenly, and the sample is measured using a liquid scintillation counter.

[0083] (2) Cage flushing fluid Sample: Rinse the cage with water and collect the rinsing solution (80 mL).

[0084] After the sample is shaken or stirred to mix, 1 g is weighed and added to a 20 mL scintillation bottle. 10 mL of scintillation liquid is added and mixed evenly. The sample is then measured using a liquid scintillation counter.

[0085] (3) Feces Soak the feces in a 50% isopropanol aqueous solution and store in a refrigerator at 4°C.

[0086] Weigh the total weight and record it. Homogenize the homogenate for 2 min using a homogenizer. Weigh two parallel portions of the homogenate (0.1 g) into a scintillation bottle, add 1 mL of 1 N KOH and heat until completely dissolved. Cool to room temperature, add 10 mL of scintillation solution and mix well. Measure the homogenate using a liquid scintillation counter.

[0087] (4) Animal carcasses Add approximately twice the volume of the animal carcass to a 6 N KOH solution, heat at 90 °C to dissolve the carcass, then weigh two portions (0.2 g) into 20 mL scintillation bottles, add 10 mL of scintillation liquid, mix thoroughly, and measure using a liquid scintillation counter.

[0088] Detection of radioactivity intensity using a liquid scintillation counter The radioactivity intensity in biological samples was determined using a liquid scintillation counter, and the radioactivity concentration was calculated based on the actual sample volume. The termination criterion for counting each sample was 5 minutes or 2% 2 Sigma. The measured value of the blank sample was used as the background value, and the actual radioactivity concentration of the biological samples in the experimental group was reduced by the background value of the blank sample.

[0089] The liquid scintillation counter uses the external standard method for quenching calibration. The scintillation counting standard refers to the relevant standard operating procedures (SOPs) of Jiangsu Wanlue. The scintillation counter is set to automatically convert the count value per minute (CPM) to the decay value per minute (DPM). The parameter requirements for the sample measurement report are as follows: a. The external quenching effect factor (LUM) should be less than 10%; b. When the error between the measured values ​​of two parallel samples and their average value is within 10%, the analytical results are acceptable, and the radioactivity concentration of each analysis is >200 DPM; for samples with an actual radioactivity concentration of less than 200 DPM, the difference between samples is not considered.

[0090] Calculation formula: .

[0091] The plasma detection data of M03 rats are shown in Table 4.

[0092] Table 4

[0093] Note: DPM stands for decays per minute, representing the number of decays that occur within one minute. DPM / g refers to the number of decays per gram of sample per minute. Actual DPM is DPM minus the background value (53 DPM).

[0094] The results of the excretion data experiment are shown in Table 5, where URN represents urine, FCS represents feces, and CRS / CWS represents cage flushing fluid.

[0095] The total radioactivity recovery rate from 1.0 to 168 hours is approximately 65%, with excretion mainly concentrated within 48 hours. This is primarily through urine; partly through feces; and locally from corpses.

[0096] Table 5

[0097] Note: / represents data that was not detected.

[0098] The experiments of this invention can well illustrate the excretion pattern and degradation trend of PDRN in rats, which is slowly degraded from local tissues and mainly excreted in urine.

[0099] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for radiolabeling compounds containing carboxylic acid, phosphoric acid, or sulfate groups, characterized in that, The process includes the following steps: reacting a compound containing carboxylic acid, phosphoric acid, or sulfuric acid groups, a solvent, a catalyst, and a radioactive reagent, followed by purification.

2. The radioactive labeling method according to claim 1, characterized in that, The compounds containing carboxylic acid, phosphoric acid, or sulfuric acid groups are: macromolecular compounds containing carboxylic acid, phosphoric acid, or sulfuric acid groups and small molecule compounds containing phosphoric acid or carboxylic acid groups.

3. The radioactive labeling method according to claim 2, characterized in that, The macromolecular compounds are polydeoxyribonucleotides (PDRN), polynucleotides (PN), hyaluronic acid, heparin, and polypeptides or proteins that do not contain lysine but contain aspartic acid.

4. The radioactive labeling method according to claim 2, characterized in that, The small molecule compound is further preferably: small interfering RNA (siRNA), antisense oligonucleotide (ASO), or microRNA (miRNA).

5. The radioactive labeling method according to claim 1, characterized in that, The solvent is at least one of an aprotic solvent or an aqueous system.

6. The radioactive labeling method according to claim 1, characterized in that, The molar ratio of the compound containing carboxylic acid, phosphoric acid, or sulfuric acid groups to the catalyst is (0.002-0.004):(0.02-0.07); the molar ratio of the compound containing carboxylic acid, phosphoric acid, or sulfuric acid groups to the radioactive reagent is (2-4):(0.002-0.004).

7. The radioactive labeling method according to claim 6, characterized in that, The catalyst is EDC or a combination of EDC and NHS; the combination of EDC and NHS is such that the molar ratio of EDC to NHS is (1-2):(1-2).

8. The radioactive labeling method according to claim 1, characterized in that, The reaction temperature is 20-30℃, and the reaction time is 1-15h.

9. A radiolabeled substance prepared by the radiolabeling method according to any one of claims 1-8.

10. The application of the radiolabeling method according to any one of claims 1-8 in improving the stability of radiolabeled objects.

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