CN112237638B

CN112237638B - Combined probe for reducing radionuclide kidney condensation and preparation method thereof

Info

Publication number: CN112237638B
Application number: CN202011169328.8A
Authority: CN
Inventors: 张明如; 陈小元
Original assignee: Xi'an Huamu Biotechnology Co ltd
Current assignee: Xi'an Huamu Biotechnology Co ltd
Priority date: 2020-10-28
Filing date: 2020-10-28
Publication date: 2022-11-29
Anticipated expiration: 2040-10-28
Also published as: CN112237638A

Abstract

The invention relates to a combined probe for reducing radionuclide kidney condensation and a preparation method thereof, wherein the combined probe comprises a reagent 1 and a reagent 2; the reagent 1 is composed of a targeting ligand R ₃ And a bifunctional chelating agent R ₁ Is formed by connecting polypeptide sequences, wherein the general formula of the polypeptide sequences is Met-X-Lys, X is one of hydrophobic amino acids, and the tripeptide sequences can be specifically cut by renal neutral endopeptidase; reagent 2 comprises the same polypeptide sequence Met-X-Lys connected at one end to a non-targeting ligand R ₄ And the other end is connected with a group R ₅ ，R ₅ Being bifunctional chelating agents R ₁ Or an H atom. The two reagents of the combined probe both contain the same enzyme digestion sequence (namely Met-X-Lys, X is one of hydrophobic amino acids), one reagent is connected with a targeting ligand, and the other reagent is not connected with the targeting ligand.

Description

Combined probe for reducing radionuclide kidney condensation and preparation method thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a combined probe for reducing radionuclide kidney condensation and a preparation method thereof.

Background

Precision medicine, also known as individualized medicine or personalized medicine, is an individualized diagnosis and treatment strategy that matches the molecular biological pathological characteristics of a patient. Compared with the traditional medical treatment, the individual medical treatment has the advantages of improving the treatment quality, reducing the side effect and the like, and is a new trend and direction in the tumor medical treatment field. Molecular nuclear medicine is a subject which recognizes diseases from the molecular level by taking a nuclide tracing technology as a means, clarifies the change of the density and the function of a receptor of a pathological tissue, the abnormal expression of genes, the biochemical metabolic change, the cell information conduction and the like, and provides molecular level information for clinical diagnosis, treatment and the research of diseases.

With the wide application of molecular biology technology and the rapid development of radionuclide tracing technology, molecular nuclear medicine has developed in the fields of molecular functional imaging and molecular targeted therapy, so that it has an important place in the field of precise medical treatment. At this point itIn particular, nuclide diagnosis and treatment targeting Somatostatin receptors (SSTR) has been applied clinically for more than 20 years, and has drawn attention. The U.S. Food and Drug Administration (FDA) approved the first nuclide-targeting Drug in 2018 in 1 month ¹⁷⁷ Lu-DOTA-TATE (Lutathera,) is used for treating gastroenteropancreatic neuroendocrine tumors, and marks that the development of receptor-mediated radionuclide therapy enters a new era.

However, since many tumor-targeting ligands such as polypeptides, folic acid, aptamers, and the like are mainly metabolized in vivo via the kidney, and metabolites are continuously retained in the kidney due to renal reabsorption, radionuclides linked to the targeting ligands are excessively concentrated in the kidney for a long time. Thus, the kidney also becomes the primary dose-limiting organ for nuclide therapy with these targeting ligands. To protect the kidneys, the radioactive dose of the treatment is generally reduced or the number of courses of treatment is reduced, so that the effectiveness of the treatment is greatly limited. Therefore, how to reduce the accumulation and retention of radioactivity in the kidney becomes a key problem to be solved in the field of nuclide therapy.

At present, the scholars research two strategies of ligand structure modification and reabsorption inhibition to reduce the concentration of radioactivity in the kidney aiming at the reasons that the renal concentration is too high in the aspects of renal metabolism of radioactive targeting ligand and reabsorption in the kidney, but the two strategies have disadvantages:

(1) the structural modification of the radioligand has the disadvantages of unclear mechanism, uncertain effect and no universality, and the structural modification of the ligand may influence the affinity of the ligand to a receptor, so that the risk of reducing the tumor radioactive uptake is reduced.

(2) Inhibition of renal reabsorption of radioactive metabolic fragments can produce side effects. The continuous infusion of large doses of inhibitor can cause a number of serious toxic side effects, such as vomiting, nausea and hyperkalemia, some of which are lifelong. Whereas the use of succinylated gelatin results in transient urine proteins.

In summary, the two strategies of structural modification of the ligand and inhibition of reabsorption by using the inhibitor have the effect of reducing renal uptake, but still have various problems in terms of drug performance and toxic and side effects, and a solution with better effect and better safety needs to be explored.

The radioactive enzyme digestion removal strategy is a novel strategy for reducing kidney concentration, and separates radionuclides from metabolic fragments based on the enzyme digestion effect of enzyme highly expressed in kidney, so that the radionuclides are prevented from being reabsorbed, quickly enter bladder and are removed out of the body.

Because the enzyme digestion process is an enzymatic reaction, the relationship between the initial reaction speed and the substrate concentration conforms to the description of the Mie's equation, namely, under a certain enzyme concentration, when the substrate concentration is lower, the reaction is a first-order reaction relative to the substrate; and when the substrate concentration is in the middle range, the reaction is a mixed-stage reaction with respect to the substrate; as the substrate concentration continues to increase, the reaction transitions from a first order reaction to a zero order reaction. Although the relationship between the initial rate of the reaction and the concentration of the substrate is complicated, the initial rate of the enzymatic reaction can be increased by increasing the concentration of the substrate within a certain range. However, increasing the concentration of the enzyme-cleaved substrate results in an increase in the concentration of the targeting ligand linked thereto, thereby inhibiting tumor uptake and affecting the effect of nuclide diagnosis and treatment. The problem could be readily solved if a method could be provided to increase the concentration of the cleavage substrate without increasing the concentration of the targeting ligand.

Disclosure of Invention

The invention provides a combined probe, wherein two reagents of the combined probe both contain the same enzyme digestion sequence (namely Met-X-Lys, X is one of hydrophobic amino acids), one reagent is connected with a targeting ligand, and the other reagent is not connected with the targeting ligand.

The technical scheme adopted by the invention is as follows: a combination probe for reducing renal concentration of a radionuclide, comprising:

the combined probe comprises a reagent 1 and a reagent 2;

the reagent 1 is composed of a targeting ligand R ₃ And a bifunctional chelating agent R ₁ Is formed by connecting polypeptide sequences, wherein the general formula of the polypeptide sequences is Met-X-Lys, X is one of hydrophobic amino acids, and the tripeptide sequences can be specifically cut by renal neutral endopeptidase; the structure of reagent 1 is:

reagent 2 comprises the same polypeptide sequence Met-X-Lys connected at one end to a non-targeting ligand R ₄ And the other end is connected with a connecting group R ₅ ，R ₅ Being bifunctional chelating agents R ₁ Or an H atom; the structure of reagent 2 is:

the bifunctional chelating agent R ₁ Is p-SCN-Bn-NOTA, NOTA-NHS-ester, p-SCN-Bn-DOTA, DOTA-NHS-ester, p-NCS-Bz-DFO, p-SCN-Bn-DTPA.

The hydrophobic amino acid is Gly, ala, val, leu, ile, phe, pro, tyr and Trp.

The targeting ligand R ₃ Is Cys ⁴⁰ -Exedin 4、Cys ⁴⁰ -Leu ¹⁴ -Exedin 4、Thiol-PSMA-617、Z _HER2:2891 、Z _HER2:2395 、Z _HER2:342 、Thiol-TATE、Thiol-folate acid。

The non-targeting ligand R ₄ The compounds are mPEG2K-SH, mPEG5K-SH and mPEG10K-SH.

A method of making a combination probe for reducing renal concentration of radionuclides as described herein, comprising:

the method comprises the following steps:

1) Mixing the protected lysine with 2-chloro triphenyl chloride resin at a molar ratio of 1.2, and connecting the protected lysine to the resin under the action of N, N-diisopropylethylamine;

2) Continuously connecting the amino acid X and the Boc-Met on the basis of the product obtained in the step 1) to obtain a second-step product;

3) Removing the protective agent Dde from the second-step product obtained in the step 2) under the action of 2% hydrazine hydrate to obtain a third-step product;

4) Cutting the third step product obtained in the step 3) from the resin under the action of 20% 1,1,1,3,3,3-hexafluoro-2-propanol to obtain a fourth step product;

5) Reacting the product obtained in the fourth step in the step 4) with 3- (maleimide) propionic acid N-hydroxysuccinimide ester under the action of triethylamine to obtain a reaction product in the fifth step;

6) Removing a protecting group Boc from the product obtained in the fifth step in the step 5) under the condition of trifluoroacetic acid to obtain a reaction product obtained in the sixth step;

7) Reacting the product obtained in the sixth step with a bifunctional chelating agent under the action of N, N-diisopropylethylamine to obtain a product obtained in the seventh step;

8) Reacting the product obtained in the seventh step in the step 7) with a receptor targeting ligand in PBS to obtain a reagent 1;

9) Reacting the product obtained in the seventh step in the step 8) with receptor targeting ligand replacing molecules in PBS to obtain a reagent 2, and using the reagent 1 and the reagent 2 together as a combined probe for reducing radionuclide kidney condensation.

The radiolabeled complex of a combination probe for reducing renal concentration of a radionuclide of claim wherein: the radioactive labeling complex is obtained by labeling the combined probe with radionuclide.

The radionuclide is selected from ¹⁸ F、 ⁶⁸ Ga、 ⁶⁴ Cu、 ⁶² Cu、 ⁸⁶ Y、 ⁸⁹ Zr、 ¹¹¹ In、 ¹⁷⁷ Lu、 ⁹⁰ Y、 ²²⁵ Ac、 ²¹³ Bi。

A method of making a radiolabeled complex injection of a combination probe for reducing radionuclide kidney accumulation as described herein, characterized by:

the methods include wet labeling and lyophilization labeling.

A combination probe for reducing renal concentration of a radionuclide, comprising:

the combined probe comprises two reagents which both contain the same enzyme cutting sequence, wherein one reagent is connected with a targeting ligand, and the other reagent is not connected with the targeting ligand, and the two reagents are combined.

The invention has the following advantages:

the combined probe can be applied to receptor-targeted nuclide diagnosis and treatment, has excellent imaging or treatment effect, can remarkably reduce the radioactive concentration and retention of the kidney under the condition of keeping the tumor uptake unchanged, has a very high target/non-target ratio, greatly reduces the radiation dose when being applied to human or animals, is safer and more effective, and has very high clinical popularization value.

Drawings

FIG. 1 is a HPLC analysis chart and LC-MS chart of Compound 5 prepared in example 1.

FIG. 2 is a HPLC analysis chart and LC-MS chart of Compound 8 prepared in example 2.

FIG. 3 is an HPLC analysis chart (radioactivity detection) of Compound 12 prepared in example 6.

FIG. 4 shows the PET imaging results of the combination probe (Compound 12 prepared in example 6 and Compound 6 prepared in example 1) in INS-1 tumor-bearing nude mice.

Fig. 5 is an HPLC analysis chart (radioactivity detection) of compound 13 prepared in example 7.

FIG. 6 shows the results of renal uptake of the combination probe (Compound 13 prepared in example 7 and Compound 9 prepared in example 2) in normal mice.

Detailed Description

The present invention will be described in detail with reference to specific embodiments.

The invention relates to a combined probe for reducing radionuclide kidney concentration, which comprises a reagent 1 and a reagent 2;

the reagent 1 is composed of a targeting ligand R ₃ And a bifunctional chelating agent R ₁ Is formed by connecting polypeptide sequences, wherein the general formula of the polypeptide sequences is Met-X-Lys, X is one of hydrophobic amino acids, and the tripeptide sequences can be used for being absorbed by kidneySpecific cleavage by neutral endopeptidase; the structure of reagent 1 is:

the invention creatively adopts a probe combination strategy, designs a combined probe, comprises two reagents which both contain the same enzyme cutting sequence, wherein one reagent is connected with a targeting ligand, and the other reagent is not connected with the targeting ligand, and the two reagents are combined, so that the aim of obviously reducing radionuclide kidney accumulation is fulfilled on the premise of ensuring that the tumor uptake is not influenced by a mode of directionally increasing the concentration of an enzyme cutting substrate without increasing the concentration of the ligand.

The hydrophobic amino acid is Gly, ala, val, leu, ile, phe, pro, tyr and Trp.

The method for preparing the combined probe for reducing the renal concentration of the radionuclide comprises the following steps:

4) Cutting the third step product obtained in the step 3) from the resin under the action of 20% of 1,1,1,3,3,3-hexafluoro-2-propanol to obtain a fourth step product;

9) Reacting the product obtained in the seventh step with receptor-targeted ligand substitute molecules in PBS to obtain a reagent 2, and using the reagent 1 and the reagent 2 together as a combined probe for reducing radionuclide nephroconcentration.

The radiolabeled complex of a combination probe for reducing renal concentration of a radionuclide derived from said combination probe by radionuclide labeling. The radionuclide is selected from ¹⁸ F、 ⁶⁸ Ga、 ⁶⁴ Cu、 ⁶² Cu、 ⁸⁶ Y、 ⁸⁹ Zr、 ¹¹¹ In、 ¹⁷⁷ Lu、 ⁹⁰ Y、 ²²⁵ Ac、 ²¹³ And (4) Bi. The preparation method of the radiolabeled complex injection comprises a wet labeling method and a freeze-drying labeling method.

The technical solution of the present invention is further described in detail below:

the invention designs a combined probeThe probe is a combined polypeptide probe, and the general formula of the probe is shown as the following formula (I), wherein R is ₁ Refers to a bifunctional chelating agent group, R ₂ Refers to the side chain residue of a hydrophobic amino acid, R ₃ Refers to a targeting ligand, R ₄ Is a reaction with R ₃ Different alternative molecules, R ₅ Is a reaction with R ₁ The same bifunctional chelating agent or an H atom.

In the scheme of the invention, the bifunctional chelating agent can be various bifunctional connecting agents which can be used for chelating radionuclide in the prior art; the preferred bifunctional chelating agents of the present invention are p-SCN-Bn-NOTA and DOTA-NHS-ester; namely R in the formula (I) ₁ Is p-SCN-Bn-NOTA with the structure shown in formula (II) and DOTA-NHS-ester with the structure shown in formula (III);

in the scheme of the invention, the hydrophobic amino acids comprise glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L) isoleucine (Ile, I), phenylalanine (Phe, F), proline (Pro, P), tyrosine (Tyr, Y) and tryptophan (Trp, W); preferred hydrophobic amino acids according to the invention are Val, phe, tyr, trp, i.e.R in said formula (I) ₂ Val with a structure shown in a formula (IV), phe with a structure shown in a formula (V), trp with a structure shown in a formula (VI) or Trp with a structure shown in a formula (VII);

in the embodiment of the present invention, the targeting ligand may be various receptor targeting molecules in the prior art, such as polypeptide, affibody, folic acid, aptamer, etc.;preferred targeting ligands of the invention are exendin4 polypeptide, PSMA-617 polypeptide, HER2 affibody, octreotide analog, folic acid, i.e. R in said formula (I) ₃ Is exendin4 with a structure shown in a formula (VIII), PSMA-617 with a structure shown in a formula (IX), HER2 affibody with a structure shown in a formula (X), octreotide analogue with a structure shown in a formula (XI) or folic acid with a structure shown in a formula (XII);

the exendin4 sequence shown in the formula (VIII) is GEGTFTSDLSKQLEEEAVRLFIEWLK NGGPSSGAPPPSC-NH ₂ ；

HER2 affibody is a targeting molecule Z with a structure shown in a formula (X) _HER2:2891 The sequence of which is NH ₂ -AEAKYAKEMRNAYWEIALLPNLTNQQKRAFIRKLYDDPSQSSELLSEAKKLNDSQAPKC-COOH；

In the embodiment of the present invention, the targeting ligand replacement molecule may be other than R ₃ Of (4) other targeting ligands of (2),

or PEG molecules with different forms and different molecular weights, such as mPEG-SH and the like, which have good biological safety and low cost; preferred targeting ligand replacement molecules of the present invention are mPEG2K-SH, mPEG5K-SH, mPEG10K-SH, etc., i.e., R in said formula (I) ₄ Is mPEG-SH (the molecular mass depends on the value of n;)

In the scheme of the invention, R is ₅ Is a reaction with R ₁ The same bifunctional chelating agent or H atom; preferred in the present invention are H atoms;

reagent 1 in the combined probe of the invention can be labeled with radionuclide to carry out the mission of nuclide diagnosis and treatment; the reagent 2 contains a structural skeleton the same as that of the reagent 1, and mainly has the effects of increasing the concentration of enzyme digestion substrates, improving the enzyme digestion efficiency and accelerating the removal of radioactivity on the kidney. The use of the combined probe can skillfully realize that only the concentration of the enzyme digestion sequence is increased, but the concentration of the targeting ligand is not increased, thereby obviously reducing the radioactive concentration and retention in the kidney under the condition of not influencing the tumor uptake, greatly improving the clinical diagnosis and treatment value of the radioactive probe, and having wide clinical application prospect.

The invention also provides a method for preparing the combined probe, which comprises the following synthetic route:

as shown in the above synthetic route, the preparation method specifically comprises the following steps:

1) Mixing the protected lysine with 2-chloro triphenyl chloride resin at a molar ratio of 1.2, and connecting the protected lysine to the resin under the action of N, N-Diisopropylethylamine (DIPEA);

3) Removing the protective agent Dde from the second-step product obtained in the step (2) under the action of 2% hydrazine hydrate to obtain a third-step product;

4) Cutting the third step product obtained in the step (3) from the resin under the action of 20% of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to obtain a fourth step product;

5) Reacting the fourth step product obtained in the step (4) with 3- (maleimide group) propionic acid N-hydroxysuccinimide ester under the action of triethylamine to obtain a fifth step reaction product;

6) Removing a protecting group Boc from the fifth step product obtained in the step (5) under the condition of trifluoroacetic acid (TFA) to obtain a sixth step reaction product;

7) Reacting the product obtained in the sixth step in the step (6) with a bifunctional chelating agent under the action of DIPEA to obtain a product obtained in the seventh step;

8) And (5) reacting the product obtained in the seventh step in the step (7) with a receptor targeting ligand in PBS to obtain the reagent 1.

9) And (3) reacting the product obtained in the seventh step in the step (8) with receptor targeting ligand replacing molecules in PBS to obtain the reagent 2.

In the scheme of the invention, the amino acid X in the step (2) is selected from hydrophobic amino acids Gly, ala, val, leu, ile, phe, pro, tyr and Trp; preferred hydrophobic amino acids in the present invention are Val, phe, tyr, trp.

In the scheme of the invention, the bifunctional chelating agent in the step (7) can be selected from p-SCN-Bn-NOTA, NOTA-NHS-ester, p-SCN-Bn-DOTA, DOTA-NHS-ester, p-NCS-Bz-DFO or p-SCN-Bn-DTPA; p-SCN-Bn-NOTA and DOTA-NHS-ester are preferred.

In the embodiment of the present invention, the receptor targeting ligand exendin4 in step (8) may be selected from Cys ⁴⁰ -Exedin 4、Cys ⁴⁰ -Leu ¹⁴ Exedin 4, preferably Cys ⁴⁰ -Leu ¹⁴ -Exedin 4; PSMA-617 may be selected from Thiol-PSMA-617; HER2 affibody may be selected from Z _HER2:2891 、Z _HER2:2395 Or Z _HER2:342 Preferably Z _HER2:2891 (ii) a The octreotide analog may be selected from Thiol-TATE, thiol-NOC, thiol-TOC, preferably Thiol-TATE; the folic acid compound may be selected from Thiol-folate acid.

In the scheme of the invention, the receptor targeting ligand substitute in the step (9) can be selected from PEG molecules with different forms and different molecular weights, preferably mPEG2K-SH, mPEG5K-SH and mPEG10K-SH.

In the scheme of the invention, R is ₅ Is a reaction with R ₁ Same double workA chelating agent or an H atom; preferred in the present invention is an H atom.

On this basis, the present invention further provides a process for the preparation of the radiolabeled complex of reagent 1. The radioactive labeling complex can be used as a novel tumor radioactive diagnosis and treatment probe, namely a radionuclide diagnostic probe or a radionuclide therapeutic probe.

In the radiolabeled complex of the present invention, the radiodiagnostic nuclides may be selected from nuclides ¹⁸ F、 ⁶⁸ Ga、 ⁶⁴ Cu、 ⁶² Cu、 ⁸⁶ Y is or ⁸⁹ Any one of Zr, preferably Zr ¹⁸ F、 ⁶⁸ Ga、 ⁶⁴ Any one of Cu; the radiotherapeutic nuclides may be selected ¹¹¹ In、 ¹⁷⁷ Lu、 ⁹⁰ Y、 ²²⁵ Ac or ²¹³ Any one of Bi, preferably Bi ¹⁷⁷ Lu、 ⁹⁰ Any one of Y.

The radioactive labeling complex can be prepared by a compound containing radioactive nuclide and the reagent 1 according to various existing labeling methods; the preferred labeling method of the present invention is the following wet or freeze-drying method:

a wet marking scheme comprising: dissolving a proper amount of the reagent 1 in a buffer solution or deionized water; adding a radionuclide solution into the obtained solution, and carrying out closed reaction for 5-40min to generate a radionuclide-labeled complex;

alternatively, a lyophilization labeling protocol comprising: dissolving a proper amount of the reagent 1 in a buffer solution or deionized water; the obtained solution is aseptically filtered, subpackaged in containers, freeze-dried, plugged and sealed to obtain a freeze-dried medicine box; adding a proper amount of acetic acid solution or buffer solution into the freeze-dried medicine box for dissolving, then adding corresponding radionuclide solution, and carrying out closed reaction for 5-40min to generate the radionuclide-labeled complex. Wherein, the container for split charging is preferably a freezing storage tube or a tube-type antibiotic bottle. Excipients, such as mannitol, ascorbic acid and the like, can be added into the medicine box according to the forming condition of the freeze-dried powder of the medicine box, and the forming of the medicine box can be optimized by adjusting the dosage of the polypeptide compound and the excipients.

The products obtained by the wet labeling scheme and the freeze-drying labeling scheme can be further prepared into injection by conventional treatment (such as chromatographic separation and purification, solvent removal by rotary evaporation, residue dissolution with PBS or water or physiological saline, sterile filtration and the like).

A preferred preparation method of the radioactive labeling complex is a wet labeling method, and comprises the following steps: dissolving the reagent 1 of claim 1 in a buffer solution or deionized water; adding fresh radioactive solution, sealing at 37-90 deg.C for 5-40min, and cooling; diluting the reaction solution with water, separating and purifying by a Sep-Pak C18 chromatographic column, washing the chromatographic column with buffer solution or water to remove unreacted radioactive ions, leaching with hydrochloric acid ethanol solution or ethanol solution, diluting with normal saline or PBS, and performing sterile filtration to obtain the injection of the radioactive labeled complex with the structure as shown in formula (XIV); wherein R is ₁ Residues that are hydrophobic amino acids, such as Val, phe, tyr, and Trp; r ₂ Are targeting ligands, e.g. Cys ⁴⁰ -Leu ¹⁴ -Exedin 4、PSMA-617、Z _HER2:2891 Thiol-TATE or Thiol-folate acid, etc.; the radionuclide M is ⁶⁸ Ga、 ⁶⁴ Cu、 ¹⁸ F、 ¹⁷⁷ Lu、 ⁹⁰ Y, and the like.

Another preferred preparation method of the radioactive labeling complex is a freeze-drying labeling method, which comprises the following steps: dissolving the reagent 1 and other necessary reagents according to claim 1 in a buffer solution, aseptically filtering the obtained solution, subpackaging the sterile solution in a cryopreservation tube, freeze-drying the sterile solution, and sealing the lyophilized solution to obtain a lyophilized kit; adding appropriate amount of buffer solution into the lyophilized kit, dissolving, adding fresh radioactive solution, sealing, reacting at 37-120 deg.C for 5-40min, and cooling; diluting the reaction solution with water, separating and purifying with Sep-Pak C18 chromatographic column, washing the chromatographic column with buffer solution or water to remove unreacted radioactive ions, and adding hydrochloric acidLeaching the mixture with ethanol solution or ethanol solution, diluting with normal saline or PBS, and performing aseptic filtration to obtain the injection of the radiolabeled complex with the structure shown in formula (XIV); wherein R is ₁ Is a residue of a hydrophobic amino acid, such as Val, phe, tyr, and Trp; r2 is a targeting ligand, such as Cys ⁴⁰ -Leu ¹⁴ -Exedin4、PSMA-617、Z _HER2:2891 Thiol-TATE or Thiol-folate acid, etc.; the radionuclide M is ⁶⁸ Ga、 ⁶⁴ Cu、 ¹⁸ F、 ¹⁷⁷ Lu、 ⁹⁰ Y, and the like.

In the above method, the buffer solution is a substance that stabilizes the pH of the reaction solution, and may be any one or a mixture of two or more of acetate, lactate, tartrate, malate, maleate, succinate, ascorbate, carbonate, or phosphate.

The invention also provides the application of the combined probe in receptor-targeted radiation therapy. Compared with the prior art, the combined probe has excellent imaging or treatment effect when being applied to human or animals, can obviously reduce radioactive concentration and detention of the kidney, has very high target/non-target ratio, greatly reduces the radiation dose when being applied to the human or animals, is safer and more effective, and has very high clinical popularization value.

Example 1

Preparing a combined probe of exendin4, wherein the synthetic route is as follows:

the preparation process comprises the following steps:

1. synthesis of Compound 2:

0.5g of Fmoc-Lys (Dde) -Resin (amount of substance of Fmoc-Lys (Dde) -OH is 0.4mmol,1 eq) was added to a solid phase synthesizer, DMF was used as a reaction solvent, DIPEA/DMF at 0.8M and HBTU/DMF at 0.4M were used as activators, and 20% piperidine in DMF was used as an eluent for Fmoc, and Fmoc-Val-OH (0.542g, 4 eq) and Boc-Met-OH (0.399g, 4 eq) were added in this order to extend the peptide chain, according to the sequence of the polypeptide. After the solid phase synthesis is finished, the crude polypeptide product connected with the resin is placed in 3mL of DMF solution of 2% hydrazine hydrate, stirred for 1.5h at room temperature, filtered, and washed with dichloromethane, methanol and DMF in sequence. Then, the resin was placed in 3mL of 20% HFIP solution in methylene chloride, stirred at room temperature for 2 hours, filtered, and the filtrate was recrystallized from ether to obtain Compound 1 with a yield of about 70-84%.

0.08mmol of Compound 1 was weighed into a 10mL glass bottle, and 1.2 equivalents of N-hydroxysuccinimide 3- (maleimido) propionate, 2.6 equivalents of triethylamine, and 500. Mu.L of DMSO were sequentially added thereto, and the reaction was stirred at room temperature. The progress of the reaction was monitored by HPLC, and when the disappearance of the starting material was observed, the reaction was terminated by adding 500. Mu.L of 0.1% aqueous TFA solution. The product was isolated and purified by preparative HPLC to give compound 2 in about 45-56% yield. The molecular weight of Compound 2, identified by LC-MS, is 626[ M-H ]] ^- 。

2. Synthesis of Compound 3:

weigh 20mg of compound 2 into a 10mL glass vial, add 500. Mu. LTFA, stand at room temperature for 20min, check the progress of the reaction by HPLC, and stop the reaction when all starting materials are converted to the product. TFA was blown dry with nitrogen, water was added and freeze dried.

3. Synthesis of Compound 4:

to a reaction flask were added 500. Mu.L of DMSO,1 equivalent of p-SCN-Bn-NOTA, 1.05 equivalents of Compound 3, and 5 equivalents of triethylamine, and the reaction was stirred at room temperature. The progress of the reaction was checked by HPLC, and when the disappearance of the starting material was observed, the reaction was terminated by adding 500. Mu.L of 0.1% aqueous TFA solution. The product was isolated and purified by preparative HPLC to give compound 4 in about 46-50% yield. Compound 4 has a molecular weight of 976[ m-H ] as identified by LC-MS] ^- 。

4. Synthesis of Compound 5:

compound 4 (0.66. Mu. Mol,1.5 equivalents) was added to Cys ⁴⁰ -Leu ¹⁴ Exedin 4 (commercially available, 0.44. Mu. Mol,

1 eq) at room temperature for two hours and then separated and purified by HPLC to give compound 5 in about 50-59% yield. By LCThe molecular weight of compound 5 is 5268[ deg. ] M + H by-MS identification] ⁺ (chelate of sodium). The HPLC analysis chart and LC-MS chart of compound 5 are shown in FIG. 1.

5. Synthesis of Compound 6:

compound 3 (0.66. Mu. Mol,1.5 equivalents) was added to a solution of mPEG5K-SH (commercially available, 0.44. Mu. Mol,1 equivalent) in PBS, reacted at room temperature for two hours, and then isolated and purified using a NAP-5 column to give compound 6 in about 55-60% yield.

Example 2

The preparation method of the PSMA polypeptide compound containing the enzyme digestion sequence comprises the following steps:

the preparation process comprises the following steps:

1. synthesis of Compounds 1-3 the same as in example one;

2. synthesis of compound 7:

0.03mmol of Compound 2 was weighed into a 10mL glass vial, 500. Mu.L TFA was added, the mixture was allowed to stand at room temperature for 20min, the progress of the reaction was checked by HPLC, and the reaction was stopped when all starting materials were converted to the product. TFA was blown dry with nitrogen. To the reaction flask was added 500. Mu.L of DMSO,1 equivalent of p-SCN-Bn-DOTA and 5 equivalents of triethylamine, and the reaction was stirred at room temperature. The progress of the reaction was checked by HPLC, and when the disappearance of the starting material was observed, the reaction was terminated by adding 500. Mu.L of 0.1% aqueous TFA solution. The product was isolated and purified by preparative HPLC to give compound 7 in about 44-49% yield. The molecular weight of the compound 7 is 1079.46[ m + h ] respectively by LC-MS identification] ⁺ The calculated value (m/z) was 1078.45 (C) ₄₇ H ₇₀ N ₁₀ O ₁₅ S ₂ 。)

3. Synthesis of reagent 8:

compound 7 (0.66. Mu. Mol,1.5 equivalents) was added to a solution of Thiol-PSMA-617 (commercially available, 3mg, 0.44. Mu. Mol,1 equivalent) in PBS, reacted at room temperature for two hours, and then isolated and purified by HPLC to give compound 8 in about 43-50% yield. The molecular weight of the compound 8 is 18 respectively through LC-MS identification08.75[M+H] ⁺ The calculated value (m/z) was 1807.75 (C) ₈₂ H ₁₁₇ N ₁₅ O ₂₅ S ₃ . ) The HPLC analysis chart and LC-MS chart of compound 8 are shown in FIG. 2.

4. Synthesis of compound 9:

compound 7 (0.66. Mu. Mol,1.5 equivalents) was added to a solution of mPEG5K-SH (commercially available, 0.44. Mu. Mol,1 equivalent) in PBS, reacted at room temperature for two hours, and then isolated and purified using a NAP-5 column to give compound 9 in about 55-60% yield.

EXAMPLE 3 preparation of lyophilized kit for radiolabelling

1) Preparation of lyophilized kits for radioactive Ga-68 and Cu-64 labeling (example preparation of 100)

Weighing 4mg of the compound 5 prepared in example 1, dissolving in 40mL of 0.5mol/L acetic acid-sodium acetate buffer solution (pH = 4), aseptically filtering, dispensing into 400 freezing tubes, freeze-drying in a freeze-drying agent for 24 hours, plugging, and sealing to obtain a freeze-dried kit i. According to the forming condition of the freeze-dried powder injection of the medicine box, excipients, such as mannitol, ascorbic acid and the like, can be added into the medicine box, and the dosage of the compound 5 and the excipients can be adjusted so as to ensure that the forming of the medicine box is optimal.

2) Preparation of lyophilized kit for Radioactive F-18 labeling (example of 100 preparations)

Weighing 4mg of the compound 5 prepared in example 1 was dissolved in 10mL of 0.5mol/L tartaric acid-potassium sodium tartrate buffer solution (pH = 4), and 0.04mg of aluminum chloride (AlCl) ₃ ) Dissolved in 10mL of 0.5mol/L tartaric acid-potassium sodium tartrate buffer solution (pH = 4), and the two solutions were mixed well. And (3) sterile filtering, subpackaging in 100 freezing tubes, freeze-drying in a freeze-drying agent for 24 hours, plugging and sealing to obtain a freeze-dried medicine box II. According to the different requirements of the medicine box output and the component content in each medicine box, the dosage of the compound 5 and the aluminum chloride can be adjusted to ensure that the weight ratio of the compound 5 to the aluminum chloride falls between (20-100): 1, in the range of.

Example 4. Preparation of a Ga-68-labeled radioactive diagnostic probe (Compound 10):

1) And (2) wet method: about 18.5 to 1850 million beike keli (MBq) ⁶⁸ GaCl ₃ The hydrochloric acid solution (rinsed from the germanium gallium generator) was added to a centrifuge tube containing 0.5mL of the acetic acid-acetate solution of Compound 5 prepared in example 1 (1.0 g/L) and allowed to react at 37 ℃ for 20min. A C18 separation column was taken and rinsed slowly with 10mL of absolute ethanol and then 10mL of water. The labeling solution was diluted with 10mL of water and applied to a column, and the unlabeled fraction was removed with 10mL of water ⁶⁸ Ga ions are leached by 0.3mL of 10Mm HCl ethanol solution to obtain a compound 10. The leacheate is diluted by normal saline and is subjected to sterile filtration to obtain the compound 10 injection.

2) The freeze-drying method comprises the following steps: about 18.5 to 1850 million beike keli (MBq) ⁶⁸ GaCl ₃ Hydrochloric acid solution (eluted from a germanium gallium generator) is added into the freeze-dried medicine box I containing the compound 5, mixed evenly and reacted for 20min at 37 ℃. A C18 separation column was taken and rinsed slowly with 10mL of absolute ethanol and then 10mL of water. The labeling solution was diluted with 10mL of water and applied to a column, and the unlabeled fraction was removed with 10mL of water ⁶⁸ Ga ions, and then 0.3mL of 10mM HCl in ethanol to obtain a compound 10. The leacheate is diluted by normal saline and is subjected to sterile filtration to obtain the compound 10 injection.

Example 5 preparation of an F-18 labeled radioactive diagnostic probe (Compound 11):

1) And (2) wet method: in a 1mL centrifuge tube, 3 μ L of a 2mM acetic acid-acetate solution (0.5 mol/L, pH = 4) and 6 μ L of a 3mM acetic acid-acetate solution of compound 5 prepared in example 1 (0.5 mol/L, pH = 4) were added, followed by 0.13mL of acetonitrile and 0.05mL of about 37MBq of acetonitrile ¹⁸ F ^- Mixing the water solution, and reacting in boiling water bath for 10min. A C18 separation column was taken and rinsed slowly with 10mL of absolute ethanol and then 10mL of water. The cooled labeling solution was diluted with 10mL of water and transferred to a separation column, where the unlabeled solution was removed with 10mL of water ¹⁸ F ion, and then 0.3mL10mMHCl solution is used for leaching to obtain a compound 11. Diluting the eluate with normal saline, and sterile filtering to obtain compound 11 injection.

2) The freeze-drying method comprises the following steps: to lyophilized kit II containing Compound 5 was added 0.5mL of a 0.5mol/L solution of acetic acid-acetate (pH = 4), and after all was dissolved, about37～3700MBq ¹⁸ F ^- Acetonitrile leacheate (obtained from anion trap column QMA), sealing and reacting at 120 ℃ for 5min, and cooling. A C18 separation column was taken and rinsed slowly with 10mL of absolute ethanol and then 10mL of water. The cooled labeling solution was diluted with 10mL of water and transferred to a separation column, where the unlabeled solution was removed with 10mL of water ¹⁸ F ion, and then 0.3mL of 10mMHCl solution is used for leaching to obtain a compound 11. Diluting the eluate with normal saline, and sterile filtering to obtain compound 11 injection.

Example 6. Preparation of a Cu-64 labeled radioactive diagnostic probe (Compound 12):

1) And (2) wet method: about 18.5 to 1850MBq ⁶⁴ CuCl ₂ The sodium acetate solution was added to a centrifuge tube containing 0.5mL of an acetic acid-acetate solution (4.0 g/L) of Compound 5 prepared in example 1, and the mixture was allowed to react at 37 ℃ for 20min. A C18 separation column was taken and rinsed slowly with 10mL of absolute ethanol and then 10mL of water. The labeling solution was diluted with 10mL of water and applied to a column, and the unlabeled fraction was removed with 10mL of water ⁶⁴ And leaching Cu ions with 0.3mL of 10mMHCl ethanol solution to obtain a compound 12. The leacheate is diluted by normal saline and is subjected to sterile filtration to obtain the compound 12 injection.

2) The freeze-drying method comprises the following steps: about 18.5 to 1850MBq ⁶⁴ CuCl ₂ Adding the sodium acetate solution into the freeze-dried medicine box I containing the compound 5, uniformly mixing, and reacting at 37 ℃ for 20min. A C18 separation column was taken and rinsed slowly with 10mL of absolute ethanol and then 10mL of water. The labeling solution was diluted with 10mL of water and applied to a column, and the unlabeled fraction was removed with 10mL of water ⁶⁴ Cu ions were eluted with 0.3mL of 10mM HCl in ethanol to obtain Compound 12. The leacheate is diluted by normal saline and is subjected to sterile filtration to obtain the compound 12 injection.

Example 7 preparation of a Ga-68-labeled radioactive diagnostic probe (Compound 13):

1) And (2) wet method: about 18.5 to 1850MBq ⁶⁸ GaCl ₃ The hydrochloric acid solution (eluted from the germanium gallium generator) was added to a centrifuge tube containing 0.5mL of the acetic acid-acetate solution of Compound 8 prepared in example 2 (1.0 g/L) and placed at 9The reaction was carried out at 0 ℃ for 20min. A C18 separation column is taken, and is rinsed slowly with 10mL of absolute ethyl alcohol and then 10mL of water.

The labeling solution was diluted with 10mL of water and applied to a column, and the unlabeled fraction was removed with 10mL of water ⁶⁸ Ga ions are leached by 0.3mL of 10Mm HCl ethanol solution to obtain a compound 13. Diluting the eluate with normal saline, and sterile filtering to obtain compound 13 injection.

2) The freeze-drying method comprises the following steps: about 18.5 to 1850MBq ⁶⁸ GaCl ₃ Adding hydrochloric acid solution (eluted from a germanium-gallium generator) into the freeze-dried medicine box II containing the compound 8, uniformly mixing, and reacting at 90 ℃ for 20min. A C18 separation column was taken and rinsed slowly with 10mL of absolute ethanol and then 10mL of water. The labeling solution was diluted with 10mL of water and applied to a column, and the unlabeled fraction was removed with 10mL of water ⁶⁸ Ga ions are eluted with 0.3mL of a 10mM HCl solution in ethanol to obtain a compound 13. Diluting the eluate with normal saline, and sterile filtering to obtain compound 13 injection.

Example 8 preparation of a Lu-177-labeled radioactive diagnostic probe (Compound 14):

1) And (2) wet method: about 18.5 to 1850MBq ¹⁷⁷ LuCl ₃ The sodium acetate solution was added to a centrifuge tube containing 0.5mL of an acetic acid-acetate solution (1.0 g/L) of Compound 8 prepared in example 2, and the mixture was allowed to react at 90 ℃ for 20min. A C18 separation column is taken, and is rinsed slowly with 10mL of absolute ethyl alcohol and then 10mL of water. The labeling solution was diluted with 10mL of water and applied to a column, and the unlabeled fraction was removed with 10mL of water ¹⁷⁷ Lu ion, then 0.3mL of 10mM HCl in ethanol to obtain compound 14. The leacheate is diluted by normal saline and is subjected to sterile filtration to obtain the compound 14 injection.

2) The freeze-drying method comprises the following steps: about 18.5 to 1850MBq ⁶⁴ CuCl ₂ Adding the sodium acetate solution into a freeze-dried medicine box II containing the compound 8, uniformly mixing, and reacting at 90 ℃ for 20min. A C18 separation column was taken and rinsed slowly with 10mL of absolute ethanol and then 10mL of water. The labeling solution was diluted with 10mL of water and applied to a column, and the unlabeled fraction was removed with 10mL of water ¹⁷⁷ Lu ion, thenElution with 0.3mL of 10mM HCl in ethanol afforded Compound 14. The leacheate is diluted by normal saline and is subjected to sterile filtration to obtain the compound 14 injection.

EXAMPLE 9 Effect of analysis and application

The following Compound 6 prepared in example 1 was combined with the radioactivity prepared in example 6 ⁶⁴ The Cu-labeled probe (Compound 12) is an example, and the performance measurement is described below:

1. HPLC analytical identification

The HPLC system is as follows: agilent 1100; a C18 column (ZORBAX, 5 μm, 4.6X 250 mm) was used for polypeptide analysis. The retention time of compound 12 was 15.67min and the calculated chemical purity was greater than 95%. The HPLC results are shown in FIG. 3.

Elution gradient: 0-3 minutes: 15% acetonitrile (0.1% tfa) and 85% water (0.1% tfa) were kept unchanged; 3-20 minutes: increase to 70% acetonitrile (0.1% TFA) and 30% water (0.1% TFA).

2. MicroPET imaging test of compound 12 and compound 6 combined probe on INS-1 tumor-bearing nude mice

1) Control probes with radiochemical purity greater than 95% were prepared as in example 6, ⁶⁴ Cu-NOTA-Exendin 4. The INS-1 tumor-bearing nude mice were randomly divided into two groups (5 mice each) and injected with 100. Mu.L (about 3.7 MBq) of the probe at different specific activities via the tail vein. The ligand content for the first set of injections was 0.2nmol and for the second set 0.8nmol. PET image acquisition was performed at 1, 4, 24 and 48h post dose, respectively. The results are shown in figure 4, where the ligand dose was 0.8nmol, the tumor uptake at each time point was significantly lower than in the 0.2nmol group, since the radioactive probe competed with the nonradioactive ligand for binding to the receptor site, and when the nonradioactive ligand increased, binding of the radioligand to the receptor was inhibited, as evidenced by a decrease in the radioactive signal at the tumor site. Comparison shows that the kidney uptake of the two groups does not have significant difference, and the suggestion is that the increase of the ligand amount does not promote the excretion of the radionuclide in the kidney.

2) Control probes with radiochemical purity greater than 95% were prepared as in example 6 ⁶⁴ Cu-NOTA-Exendin 4 and experimental group probes ⁶⁴ Cu-NOTA-MVK-Exendin4 (compound 12). Putting lotus in the airINS-1 tumor nude mice were randomly divided into five groups (5 mice each) and injected with 100. Mu.L (about 3.7 MBq) of control group probes and experimental group probes of different specific activities, respectively, via tail vein. The first group is injected with a control group probe with the ligand content of 0.2nmol, and the second group to the fifth group are respectively injected with an experimental group probe with the ligand content of 0.2, 0.8, 1.0 and 1.4 nmol. PET image acquisition was performed at 1, 4, 24 and 48h post dose, respectively.

Results as shown in fig. 4, when the dose of both the injected control probe and the experimental probe was 0.2nmol, the tumor uptake levels of both at each time point were substantially the same, while the renal uptake of the experimental group was significantly lower than that of the control group at 1h and 4h after administration, demonstrating the effectiveness of enzymatic clearance based strategies to reduce radioactive renal accumulation. When the ligand dosage of the probe in the experimental group is increased from 0.2nmol to 0.8nmol and 1.0nmol in sequence, the radioactive renal concentration at the same time point shows a gradually reduced trend, for example, after administration for 1h, the renal uptake of the experimental group of 0.2, 0.8 and 1.0nmol is 64%, 45% and 17% of that of the control group respectively; while the renal uptake 4h after administration was 78%, 41% and 17% of the control group, respectively. This indicates that the rate of cleavage is gradually increasing with increasing substrate concentration. It is worth noting that when the ligand dose of the probe in the experimental group is further increased to 1.4nmol, the radioactive kidney concentration level of the group is on the contrary leveled with that of the 1.0nmol group, which suggests that when the substrate concentration of the enzyme is increased to a certain extent, the enzyme digestion rate reaches a plateau, i.e. the enzyme digestion rate no longer changes with the increase of the substrate concentration. However, with the increase of the ligand dose in the experimental group, the level of tumor radioactivity uptake is continuously reduced, for example, after 1h of administration, the tumor uptake of the 0.8nmol and 1.0nmol groups is reduced by 55% and 71% respectively compared with that of the 0.2nmol group; and after 4h of administration, the reduction was 59% and 71%, respectively. Therefore, a combined reagent is sought, the concentration of the enzyme digestion sequence is increased, the concentration of the targeting ligand is not increased, and the radioactive concentration of the kidney can be greatly reduced under the condition of keeping the tumor uptake unchanged.

2) Control probes with radiochemical purity greater than 95% were prepared as in example 6 ⁶⁴ Cu-NOTA-Exendin 4 and experimental group probes ⁶⁴ Cu-NOTA-MVK-Exendin4 (compound 12). INS of lotus-1 tumor nude mice were randomly divided into four groups (5 mice each) and 100. Mu.L (about 3.7 MBq) of control group probes and experimental group probes of different specific activities were injected through tail vein, respectively. The first group of control group probes with the injection ligand content of 0.2nmol, the second group of experimental group probes with the injection ligand content of 0.2nmol, and the third group and the fourth group are respectively injected with 1.3nmol or 2.6nmol of MVK-PEG5K besides the experimental group probes with the injection ligand content of 0.2 nmol. PET image acquisition was performed at 1, 4, 24 and 48h post dose, respectively.

The results are shown in FIG. 4, when the third group is used in combination with 0.2nmol ⁶⁴ Renal uptake in tumor-bearing mice after Cu-NOTA-MVK-Exendin4 and 1.3nmol of MVK-PEG5K compared to 0.2nmol injected alone ⁶⁴ The Cu-NOTA-MVK-Exendin4 shows remarkable reduction, and the reduction values are 49 percent, 61 percent, 45 percent and 72 percent at 1 hour, 4 hours, 24 hours and 48 hours respectively. This indicates that the use of a combination probe containing a restriction enzyme sequence increases the concentration of the restriction enzyme sequence and accelerates the rate of the enzymatic reaction, thereby allowing the radioactivity to be cleared more quickly from the body. Meanwhile, the combined probe does not increase the concentration of the targeting ligand, so that the tumor uptake of the two groups has no obvious difference. When 2.6nmol of MVK-PEG5K was used in combination in the fourth group, the decrease in renal uptake in tumor-bearing mice was 52%,56%,40% and 70% at 1, 4, 24 and 48h, respectively, compared to the second group of imaging probes injected alone. The level of amplitude reduction is comparable to that of the third group, which shows that when 1.3nmol of MVK-PEG5K is added, the concentration of the enzyme digestion sequence is saturated, and the enzyme digestion rate enters the plateau phase. The tumor uptake levels of the fourth group were identical to those of the first three groups, again demonstrating that tumor uptake was only related to the concentration of the targeting ligand, not to the concentration of the enzyme digestion sequences.

EXAMPLE 10 analytical and application effects

The following compound 9 prepared in example 2 was combined with the radioactivity prepared in example 7 ⁶⁸ The Ga-labeled probe (Compound 13) is exemplified, and the measurement of the properties thereof is described below:

1. HPLC analysis identification

The HPLC system is as follows: agilent 1100; c18 column (ZORBAX, 5 μm, 4.6X 250 mm) was used for polypeptide analysis. The retention time of compound 10 was 14.89min and the calculated chemical purity was greater than 95%. The HPLC results are shown in FIG. 5.

Elution gradient: 0-3 minutes: 5% acetonitrile (0.1% TFA) and 95% water (0.1% TFA) were kept constant; 3-20 minutes: increase to 95% acetonitrile (0.1% TFA) and 5% water (0.1% TFA).

2. MicroPET imaging test of compound 13 and compound 9 combined probe in normal mice

First, a probe having a radiochemical purity of greater than 95% was prepared as in example 7 ⁶⁸ Ga-DOTA-NCS-MVK-PSMA (Compound 13), and DOTA-NCS-MVK-PEG5K (Compound 9) were prepared as in example 2. Normal mice were randomly divided into two groups (3 per group), and injected with 100 μ L (about 3.7 MBq) of compound 9 via tail vein, respectively, and the second group was injected with 2nmol of compound 3 in addition to compound 13. Dynamic PET image acquisition was performed for 1h after dosing. During post-treatment, 1 frame is reconstructed every 1min for the first 5min, one frame is reconstructed every 5min for the last 5min, 15 min to 20min, 25 min to 30min, 35 min to 40min, 45 min to 50 min and 55 min to 60min, the double kidneys are selected as interested areas, the radioactivity count (kBq/mL) of the kidneys is obtained by delineating, and the radioactivity count of the kidneys is plotted against time to obtain a radioactivity uptake-time curve of the kidneys (figure 6). As shown in fig. 6, the radioactive renal uptake using the combined probe set was significantly lower than the radioactive renal uptake using the individual probe set. The renal uptake of the combination injection group was about 60% of that of the single injection group within the first 30min, and about 60-73% of that of the single injection group within the last 30 min.

According to the above, compared with the single use of radioactive probes, the combined probe containing the enzyme digestion sequence can remarkably reduce the radioactive concentration of the kidney while not changing the radioactive uptake of the tumor, thereby not only ensuring the radioactive diagnosis and treatment effect, but also greatly improving the application safety. Moreover, because the concentration of the kidney is reduced, the initial treatment dose can be increased or the treatment cycle can be increased according to the tolerance dose of the kidney, so that better treatment effect can be obtained.

In conclusion, the method using the combined probe can obviously reduce the renal concentration of the radioactive ligand, thereby having extremely strong application value and prospect in receptor-targeted nuclide diagnosis and treatment.

The invention is not limited to the embodiment examples, and any equivalent changes of the technical solution of the invention by the person skilled in the art after reading the description of the invention are covered by the claims of the present invention.

Claims

1. A combination probe for reducing renal concentration of a radionuclide, comprising:

the combined probe comprises a reagent 1 and a reagent 2;

the reagent 1 is formed by connecting a targeting ligand R3 and a bifunctional chelating agent R1 through a polypeptide sequence, wherein the general formula of the polypeptide sequence is Met-X-Lys, X is one of hydrophobic amino acids, and the polypeptide sequence can be specifically cut by renal neutral endopeptidase; the structure of reagent 1 is:

reagent 2 comprises the same polypeptide sequence Met-X-Lys connected at one end to a non-targeting ligand R4 and at the other end to a group R ₅ ，R ₅ Being bifunctional chelating agents R ₁ Or an H atom; the structure of reagent 2 is:

the hydrophobic amino acid is Ala, val, leu, ile, phe, tyr and Trp;

the non-targeting ligand R4 is mPEG2K-SH, mPEG5K-SH or mPEG10K-SH.

2. The combination probe for reducing radionuclide renal concentration according to claim 1, characterized in that:

the bifunctional chelating agent R1 is p-SCN-Bn-NOTA, NOTA-NHS-ester, p-SCN-Bn-DOTA, DOTA-NHS-ester, p-NCS-Bz-DFO or p-SCN-Bn-DTPA.

3. The combination probe for reducing radionuclide renal concentration according to claim 2, characterized in that:

the targeting ligand R ₃ Is Cys ⁴⁰ -Exedin4、Cys ⁴⁰ -Leu ¹⁴ -Exedin4、Thiol-PSMA-617、Z _HER2:2891 、Z _HER2:2395 、Z _HER2:342 、Thiol-TATE、THiol-folate acid。

4. A method of making a combination probe for reducing renal concentration of a radionuclide as in claim 3, characterized by: the method comprises the following steps:

5. The radiolabeled complex prepared according to the combination probe of claim 3, wherein:

the radioactive labeling complex is obtained by labeling the combined probe with radionuclide.

6. The radiolabeled complex of combination probes according to claim 5, wherein:

7. A method of preparing a radiolabeled complex injection of a combined probe for reducing renal accumulation of radionuclides as in claim 6 wherein:

the methods include wet labeling and lyophilization labeling.