CN110227169B - Nuclear medicine of RGD polypeptide with modified structure - Google Patents

Abstract

A structurally modified RGD polypeptide, a complex formed by the polypeptide and a radionuclide, and a pharmaceutical composition comprising the complex for diagnosing or treating integrin α v β 3 positive tumors, the complex having the definition of A- (L) n-RGD polypeptide wherein A is the structure:

l represents a linker arm molecule having the structure:

wherein m is an integer of 1 to 8.

Description

Nuclear medicine of RGD polypeptide with modified structure

Technical Field

The invention relates to a nuclear medicine medicament modified by tumor-targeted RGD polypeptide, in particular to a radiopharmaceutical for diagnosing or treating the structure-modified RGD polypeptide.

Background

The molecular probes can be divided into nuclear medicine probes, optical probes, MRI probes and the like according to different imaging means, wherein the nuclear medicine molecular probes have unique advantages.

The sustained growth, invasion and metastasis of malignant tumors depend on tumor angiogenesis, integrin is one of important factors involved in regulating tumor angiogenesis and plays an important role, and the role of integrin α v β 3 is most important in integrin α v β 3 is highly expressed in endothelial cells of tumor new vessels and is not expressed or low expressed in normal cells or mature vessels.RGD can be specifically bound with integrin α v β, so RGD molecular probes designed by utilizing the specific binding of RGD and integrin 563 v β 3 are widely researched and applied.

However, many cyclic RGD monomeric peptide radiolabels have low tumor uptake, fast blood clearance rate, and high uptake by organs such as kidney and liver, which all limit the application of cyclic monomeric peptides as imaging agents; with the improvement of the polymerization degree, the uptake of the radioactive RGD polypeptide in organs such as kidney, liver, lung and the like is obviously increased, and the higher the polymerization degree is, the more complex the synthesis is, the higher the manufacturing cost is, and the radioactive RGD polypeptide is also a restriction factor for the development of the polymerization of the RGD probe; the advantages of simple multimerization or modification of the RGD probe with large molecular weight pharmacokinetic linkers are no longer evident.

Disclosure of Invention

In order to improve the above problems of the prior art, the present invention provides RGD polypeptides structurally modified by the following formula:

a- (L) n-RGD polypeptides

Wherein A is the following structure:

l represents a linker arm molecule having the structure:

wherein m is an integer from 1 to 8, such as from 2 to 6, preferably 5;

the L is linked by a carboxyl group thereof to an amino group in a, for example, by a carboxyl group marked with x in L to a bond;

n is 0 or 1;

when n is 0, the RGD polypeptide reacts with carboxyl in A through an amino group of the RGD polypeptide;

when n is 1, the RGD polypeptide is reactive linked through its amino group to another carboxyl group in L, for example when the carboxyl group marked as x in L is linked to a, the carboxyl group marked as x is reactive linked to the RGD polypeptide;

the RGD polypeptide is an RGD polypeptide selected from: c (RGDfV), c (RGDfK), c (RGDfE), c (RGDyk), E [ c (RGDyk)]₂、E[c(RGDfK)]₂、3PRGD₂。

The present invention also provides radionuclide-labelled complexes comprising the structurally modified RGD polypeptides as above, having the structure defined as follows:

Nu-BFC-A- (L) n-RGD polypeptide

Wherein:

nu is a radionuclide, such as a diagnostic imaging nuclide:¹¹¹In、⁶⁴Cu、^99mTc、⁶⁸ga, or therapeutic nuclides:⁹⁰Y、¹⁷⁷Lu、⁸⁹Sr、¹⁵³Sm、¹⁸⁸re; BFC is a bifunctional chelating agent (e.g., HYNIC (hydrazinium nicotinamide), MAG)₂(mercaptoacetyldiglycine), MAG₃(mercaptoacetyltriglycine), DTPA (diethyltriaminepentaacetic acid), DOTA (1,4,7, 10-tetraazacyclododecane-1, 4,7,10 tetraacetic acid), NOTA (1,4, 7-triazacyclononane-1, 4,7 tricarboxylic acid), TETA (1,4,8, 11-tetraazacyclotetradecane-1, 4,8,11 tetraacetic acid);

when n is 0, the bifunctional chelating agent is reacted with-NH in A through carboxyl in the structure₂A reactive linkage chain;

when n is 1, the bifunctional chelating agent is formed by carboxyl in the structure and-NH in L₂A chain of reactive bonds.

According to the invention, in the polypeptide RGD and the complex structurally modified as above:

the RGD polypeptide is selected from: c (RGDFK), 3PRGD₂；

Nu is selected from:⁹⁰Y、¹⁷⁷Lu、¹¹¹In、⁶⁴Cu、^99mTc；

when Nu is⁹⁰Y、¹⁷⁷When Lu, BFC is selected from DTPA, DOTA; when Nu is¹¹¹In、⁶⁴Cu、⁶⁸Ga、^99mAt Tc, BFC is selected from HYNIC and MAG₂、MAG₃DTPA, DOTA, TETA and NOTA.

According to the invention, Nu is⁹⁰Y is or¹⁷⁷Lu and BFC are selected from DTPA and DOTA; nu is¹¹¹When In, BFC is selected from DTPA, DOTA; nu is⁶⁴When Cu is adopted, BFC is selected from TETA and DOTA; nu is⁶⁸When Ga, BFC is selected from NOTA, DOTA; nu is^99mAt Tc, BFC is selected from HTNIC, DTPA, MAG₂、MAG₃；

By way of example, the complexes formed by the structurally modified polypeptides of the invention are as follows:

⁶⁸Ga-DOTA-A-c(RGDfk)；

^99mTc-HYNIC-A-3PRGD₂；

¹⁷⁷Lu-DOTA-A-L-3PRGD₂。

it is understood that all isomers, including enantiomers, diastereomers, and racemates, of the above-described structurally-modified polypeptides of the invention are within the scope of the invention. The present invention includes stereoisomers in optically pure form or as mixtures, and also includes racemic mixtures. For example, the amino group in structure A or L in the above polypeptides is present in L-or D-form.

It is well known to those skilled in the art that complexes as defined above also require a synergistic ligand when the bifunctional chelating agent as ligand cannot occupy all the coordination sites of the radionuclide. Radionuclides and bifunctional chelators requiring synergistic ligands for use in the present invention are well known to those skilled in the art. For example^99mWhen HYNIC is used as bifunctional chelating agent for Tc, which may be the same or different, as synergistic ligands, all of which are known in the art, wherein common synergistic ligands include water-soluble phosphines (e.g. triphenylphosphine sodium tri-meta-sulfonate TPPTS), N-tris (hydroxymethyl) methylglycine (Tricine), N-bis (hydroxyethyl) glycine, glucoheptonate, ethylenediamine-N, N' -diacetate (EDDA), 3-Benzoylpyridine (BP)Pyridine-2-azo-p-xylidine (PADA), and the like. For example,^99mwhen HYNIC is used as the bifunctional chelating agent, TPPTS and Tricine are used as synergistic ligands. But e.g.¹⁷⁷Lu or⁶⁸When the Ga takes DOTA as the bifunctional chelating agent, coordination of a coordinating ligand is not needed. By way of example, a complex of the structure:

the invention also provides a molecular probe with the complex structure.

The invention also provides a preparation method of the RGD polypeptide with the modified structure, which comprises the following steps:

optional step (1): coupling reaction of A and L;

step (2): coupling reaction of A- (L) n structural part and RGD polypeptide.

The preparation method further comprises a synthesis step of A, wherein the synthesis step comprises the following steps:

(3) formula 1:

and formula 2:

the coupling reaction of (1).

According to the preparation method of the present invention, the coupling reaction of steps (1), (2) and (3) is an amidation reaction of a carboxyl group with an amino group, which is well known in the art, and it is usually necessary to activate the carboxyl group and then react with the amino group. For example, N-hydroxybenzotriazole or N-hydroxysuccinimide (NHS) may be added to the solution of formula 1 to form an amide, and a condensing agent carbodiimide, such as DCC (dicyclohexylcarbodiimide) or DIC (N, N' -diisopropylcarbodiimide), may be added to promote the formation of the amide. The amide is then reacted with formula 2 in the presence of a condensing agent, such as EDCI, HOBT, DIEA, to give the desired compound, optionally in the presence of dichloromethane, DMF, etc. The reactions in the steps (2) and (1) are similar to those in the step (3), and since the carboxyl group is activated and then the amide is formed, the reactions can be carried out under the same conditions.

The invention also provides a preparation method of the radionuclide coordination compound obtained from the RGD polypeptide with the modified structure, which comprises the following steps:

optional step (1): coupling reaction of A and L;

step (2): coupling reaction of A- (L) n structural part and RGD polypeptide;

and (3): coupling reaction of A- (L) n-RGD polypeptide and bifunctional chelating agent;

and (4): labeling the A- (L) n-RGD polypeptide linked to the bifunctional chelating agent with a radionuclide.

According to the invention, the preparation method of the complex further comprises a synthesis step of A, wherein the synthesis step comprises the following steps:

formula 1:

and formula 2:

the coupling reaction of (1).

According to the preparation method of the present invention, the coupling reaction of step (3) is also an amidation reaction of the amino group in the a- (L) n-RGD polypeptide with the carboxyl group of the bifunctional chelating agent, and thus the same reaction conditions as those of steps (1) and (2) can be employed, for example, in the presence of a condensing agent, including EDCI, HOBT, DIEA, using solvents such as dichloromethane, DMF, and the like. The labeling of the radionuclide in step (4) is well known to those skilled in the art, and is obtained, for example, by adding the radionuclide to a molecular probe precursor, i.e., a solution of an A- (L) n-RGD polypeptide linked to a bifunctional chelating agent, and heating. The heating temperature is 80-120 ℃, and the reaction time is 5-60 min. The label is usually purified and quality-controlled after the labeling is completed, and the labeling rate and the radiochemical purity are purified and measured, for example, by using a known Sep-Pak reverse phase chromatography column, Sephadex G-25 column, YMC-Pack ODS-A C18 analytical column, Radio-HPLC method, or the like.

The invention also provides a pharmaceutical composition, which comprises an effective amount of the marker complex Nu-BFC-A- (L) n-RGD polypeptide.

According to the present invention, when Nu is a diagnostic nuclide, the pharmaceutical composition of the present invention is a diagnostic drug, for example, said drug as an imaging agent for imaging diagnosis of integrin α v β 3-positive tumors, which is directly administered to an individual, diagnosed by detecting radiation emitted by the compound administered to the subject, and imaging information obtained based on the radiation, preferably, the diagnostic drug of the present invention is an injectable formulation comprising the above-mentioned labeled complex and an injectable carrier, preferably, said imaging agent is referred to as positron emission tomography PET, single photon emission computed tomography SPECT according to the present invention, when Nu is a therapeutic nuclide, the pharmaceutical composition of the present invention is a therapeutic drug for use in radiotargeted therapy of integrin α v β 3-positive tumors, which has specific affinity with integrin α v β 3, is directly administered to an individual, which is enriched in tumor tissue, which damages diseased tissue by generating biological effects of radiation by emitting pure β -rays or β -rays with gamma ionizing rays, preferably, the inventive drug is an injectable labeled complex, which comprises the above-mentioned injectable carrier.

Preferably, the pharmaceutical composition of the present invention is an intravenous injection, such as a colorless and transparent liquid injection. Excipients suitable for intravenous injection are well known in the art, and the pharmaceutical compositions may be formulated in aqueous solution, if desired using physiologically compatible buffers including, for example, phosphate, histidine, citrate and the like, for adjusting the pH of the formulation, tonicity agents such as sodium chloride, sucrose, glucose and the like, cosolvents such as polyethylene glycols, low toxicity surfactants such as polysorbates or poloxamers and the like.

Preferably, the pharmaceutical composition of the present invention further comprises an anti-adsorbent, such as physiological saline, a 1% cyclodextrin aqueous solution, and a tween-20-containing PBS solution (e.g., tween is 0.01 to 0.1 mass%), and when the pharmaceutical composition of the present invention is used, a surfactant, such as tween-20 (e.g., tween-20 mass% is 0.05 mass%), non-specific adsorption of the marker in the infusion solution can be effectively avoided, adsorption of the radioactive marker on the tube wall can be significantly prevented, accuracy of the administration dose can be achieved, and waste of the drug in the use process can be avoided. The medicinal composition containing the anti-adsorption agent is continuously transferred for 4 times in a series of new EP tubes, and the recovery rate of the marker can reach more than 97 percent.

According to the present invention, the drug is used for diagnosing or treating integrin α v β 3 positive tumor, which refers to solid tumor, such as malignant tumor in blood, liver, glands (e.g., breast, prostate, pancreas), intestine (e.g., colorectal), kidney, stomach, spleen, lung, muscle, bone, etc.

The present invention also provides a method of diagnosing or treating hematologic and solid malignancies in which integrin α v β 3 is highly expressed, comprising administering to an individual in need thereof an effective amount of the Nu-BFC-A- (L) n-RGD polypeptide as described above.

Advantageous effects

The invention designs and prepares the RGD polypeptide with modified structure, and prepares a series of novel RGD polypeptide molecular probes with improved pharmacokinetic property by the RGD polypeptide with modified structure. The invention discovers that the RGD polypeptide is modified and reformed through a structure, and the molecular probe formed by the RGD polypeptide modified by the structure and a radionuclide through a bifunctional chelating agent has higher in-vivo and in-vitro stability and binding rate with albumin (albumin), thereby obviously prolonging the half-life period; the molecular probe of the invention has higher tumor uptake rate, contrast ratio and safety, reduces the dosage and side effects, thereby improving the imaging effect of the RGD series probe as the imaging diagnosis molecular probe in SPECT and/or PET and the effect of radioactive targeted therapy as the therapeutic molecular probe.

An example of the novel molecular Probe of the present invention¹⁷⁷Lu-DOTA-A-L-3PRGD₂Has high blood intake, thereby improving the tumor intake, and the cumulative blood intake can reach the original valueThe cumulative uptake of the probe in the tumor about 8 times that of the original probe is 4

About twice as much. The tumor uptake reaches the highest 4 hours after injection, the percent injection dosage rate is 26.52 +/-0.58 percent ID/g, and the clear imaging can still be realized 48 hours after the injection. The property obviously improves the imaging effect of the RGD series probe as an imaging diagnosis molecular probe,

and the effect of radioactive targeting therapy as therapeutic molecular probes.

Definition and description of terms

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. All patents, patent applications, and publications cited herein are incorporated by reference in their entirety unless otherwise indicated. If there are multiple definitions of terms herein, the definition in this section controls.

RGD polypeptide: are all known in the art. RGD is a small molecule polypeptide containing an arginine-glycine-aspartic acid (Arg-Gly-Asp) amino acid sequence. D-phenylalanine and valine are added to synthesize an RGD cyclic pentapeptide structure-c (RGDFV), wherein c represents that the polypeptide is in a ring shape, R represents arginine, G represents glycine, D represents aspartic acid, f represents D-phenylglycine, and V represents valine. The 5-site amino acid of the cyclic pentapeptide structure-c (RGDFV) is substituted by other amino acids to obtain c (RGDFK), c (RGDFE) and c (RGDYK), wherein K is lysine, E is glutamic acid and y is D-tyrosine. For example, (rgdfk) has the following structure:

these cyclic peptide structures may form dimers, e.g. E [ c (RGDyk)]₂、E[c(RGDfK)]₂Two RGD cyclic peptides were connected with glutamate to form a dimer. 3PRGD₂Refers to RGD pentacyclic peptide dimer containing three polyethylene glycol modifications, namely PEG₄-[PEG₄-c(RGDXk)]₂And X is D-phenylglycine, D-tyrosine and the like. Illustratively, the structure schematic diagram is as follows:

bifunctional chelating agents: the bifunctional chelating agent (BFC) is a functional organic material which can be covalently connected with a biomolecule and can chelate a metal nuclide, the structure of the bifunctional chelating agent can ensure the firm combination with the metal nuclide, and the introduced metal nuclide is far away from the biomolecule to ensure that the bioactivity of the introduced metal nuclide is not lost, so that a stable nuclide-chelating agent-biomolecule marker is formed. Bifunctional chelating agents useful in the present invention are those known in the art, such as HYNIC (hydrazineniacinamide), MAG₂(mercaptoacetyldiglycine), MAG₃(mercaptoacetyltriglycine), DTPA (diethyltriaminepentaacetic acid), DOTA (1,4,7, 10-tetraazacyclododecane-1, 4,7,10 tetraacetic acid), NOTA (1,4, 7-triazacyclononane-1, 4,7 tricarboxylic acid), TETA (1,4,8, 11-tetraazacyclotetradecane-1, 4,8,11 tetraacetic acid), and the like.

As used herein, the term "treating" and other similar synonyms include alleviating, or ameliorating a symptom of a disease or disorder, preventing other symptoms, ameliorating, or preventing an underlying metabolic cause of a symptom, inhibiting a disease or disorder, e.g., arresting the development of a disease or disorder, alleviating a disease or disorder, ameliorating a disease or disorder, alleviating a symptom of a disease or disorder, or discontinuing a symptom of a disease or disorder, and further, the term encompasses prophylactic purposes. The term also includes obtaining a therapeutic effect and/or a prophylactic effect. The therapeutic effect refers to curing or ameliorating the underlying disease being treated. In addition, a cure or amelioration of one or more physiological symptoms associated with the underlying disease is also a therapeutic effect, e.g., an improvement in the condition of the patient is observed, although the patient may still be affected by the underlying disease. For prophylactic effect, the composition can be administered to a patient at risk of developing a particular disease, or to a patient presenting with one or more physiological symptoms of the disease, even if a diagnosis of the disease has not yet been made.

Drawings

FIG. 1: example 1 end product⁶⁸Of Ga-DOTA-A-c (RGDFk)A radioactive HPLC plot; FIG. 1a is before purification and FIG. 1b is after purification;

FIG. 2 is a drawing: the results of biodistribution of the molecular probes and the control probes of example 1 in the LLCC57BL/6J mouse;

FIG. 3: example 3 end product^99mTc-HYNIC-A-3PRGD₂A graph of the measurement of the labeling rate of (1);

FIG. 4 is a drawing: blood clearance test results plot of example 3;

FIG. 5: SPECT/CT visualization of U87 MG-bearing nude mice in example 3;

FIG. 6: FIG. A \ B \ C \ D are the comparison graphs of the ratio of the tumor% ID/g, tumor/kidney, tumor/muscle, tumor/liver of the molecular probe of the invention and the control probe of the SPECT/CT imaging result of example 12 in U87 respectively;

FIG. 7: the distribution result of the molecular probe of example 3 in the nude mouse with U87MG load is shown;

FIG. 8: end product of example 7¹⁷⁷Lu-DOTA-A-L-3PRGD₂Radioactive HPLC profile of (a);

FIG. 9: graph of the results of the molecular probe blood clearance experiment of example 7;

FIG. 10: SPECT/CT visualization of the molecular probe of example 7 with U87MG nude mice;

FIG. 11: biodistribution of the molecular probe of example 7 in U87 mice;

FIG. 12: trend plot of tumor volume versus time for molecular probe radiotherapy of example 7;

FIG. 13: biodistribution of the molecular probes of example 7 in the MC-38 mouse model, the SPECT/CT visualizations of the MC-38 nude mouse, and the trend plot of the volume of the radioactively treated tumor over time in the MC-38 mouse model.

Detailed Description

The compounds of the general formula and the preparation and use thereof according to the present invention will be described in further detail with reference to the following examples. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention. Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

Statistical analysis

The results of the experiment are expressed as mean ± standard deviation (mean ± SD). The differences between groups were statistically analyzed for results using analysis of variance and t-test. P <0.05, considered to have statistical differences (#).

Example 1⁶⁸Preparation of Ga-DOTA-A-c (RGDFk)

The synthetic route for DOTA-A-c (RGDFk) is as follows:

(1) synthesis of Compound 1

4- (4-iodophenyl) butanoic acid (9.8mg, 33.8mmol) was weighed into a single vial, dissolved in 400 μ L DMF and NHS (3.9mg, 33.9mmol) was added. Then 5.3. mu.L DIC was added. The reaction was stirred at 30 ℃ for 2h and monitored by TLC (ethyl acetate: petroleum ether: acetic acid 100:200:2) until the starting material disappeared. After the reaction is finished, the reaction solution is dissolved in ethyl acetate, the ethyl acetate phase is washed with water for three times, the ethyl acetate phase is dried by anhydrous sodium sulfate, the ethyl acetate phase is concentrated and then is separated by column chromatography (ethyl acetate: petroleum ether: acetic acid: 100:200:2), fractions are collected for detection, and the collected products are dried in a spinning mode to obtain a white solid 10.2mg, wherein the yield is 78%. The expected product is confirmed by MALDI-TOF mass spectrometry.

Ms (esi): m/z is 387.2137 (chemical formula: C)₁₄H₁₄INO₄And molecular weight 387.00 calculated). After a small amount of solid was redissolved, the purity was > 98% as determined by HPLC.

(2) Synthesis of Compound 2

Compound 1(20mg, 51.7mmol) was dissolved in a round-bottomed flask containing 500 μ L DMF, and Fmoc-D-lys.hcl (22.8mg, 56.3mmol) was added to the flask, and 12 μ L DIEA was added to adjust pH 8.5. The reaction was stirred at 30 ℃ for 0.5h and monitored by TLC (ethyl acetate: petroleum ether: acetic acid: 100:200:1) until the starting material disappeared. After completion of the reaction, the reaction mixture was dissolved in 20mL of ethyl acetate, and washed 3 times with 20mL of a saturated sodium chloride solution and 20mL of water, respectively. The ethyl acetate layer was dried over anhydrous magnesium sulfate and evaporated to dryness under reduced pressure to give 26.5mg of a yellow viscous liquid. The yield was 85%. The expected product is confirmed by MALDI-TOF mass spectrometry.

MALDI-TOF-MS：m/z＝1718.33(M+H)⁺，1741.02(M+Na)⁺(chemical formula: C)₈₀H₁₁₉N₂₅O₁₆S, calculating the molecular weight: 1719.02 Da.). A small amount of product was redissolved and analyzed by HPLC to a purity of 96.7%.

(3) Synthesis of Compound 3

Compound 2(10mg, 15.6mmol) was weighed into a single vial, dissolved in 500. mu.L DMF, NHS (1.83mg, 15.9mmol) was added, and DIC 4. mu.L was added to the single vial. The reaction was stirred at 35 ℃ for 35 minutes. When heated for 5min, the suspension was found to be clear. TLC (ethyl acetate: petroleum ether: acetic acid ═ 100:200:1) was monitored until the starting material disappeared. After completion of the reaction, the reaction mixture was dissolved in 20mL of ethyl acetate, and washed 3 times with 20mL of a saturated sodium chloride solution and 20mL of water, respectively. The ethyl acetate layer was dried over anhydrous magnesium sulfate, concentrated and chromatographed (ethyl acetate: petroleum ether: 1), fractions were collected and assayed, and the collected product was rotary-dried under reduced pressure and rotary-evaporated to dryness to give 8.02mg of a white solid with a yield of 70%. The expected product is confirmed by MALDI-TOF mass spectrometry.

MALDI-TOF-MS：m/z＝760.2(M+Na)⁺(chemical formula: C)₃₅H₃₆IN₃O₇Calculating the molecular weight: 737.16).

(4) Synthesis of Compound 4

Compound 3(11.5mg, 15.6mmol) was dissolved in 500 μ L DMF in a 1mL EP tube, c (rgdfk) (9.4mg, 15.6mmol) was added, DIEA was added to adjust pH 8.5, reaction was performed at 30 ℃ for 2 hours, and the reaction was monitored by HPLC (high performance liquid chromatography) method one, YMC-Pack ODS-A C18 semi-preparative column (10mm × 250mm,

poresize, particle size 5 μm), gradient elution for 25 minutes at a flow rate of 1mL/min, wherein mobile phase A is H₂O solution, B acetonitrile (containing 0.05% TFA). The elution gradient was set to 100% a and 0% B at the start, 0% a and 100% B at 20min, and 100% a and 0% B at 25 min. The product peaked at 14.854 min.

(5) Synthesis of Compound 5

125 mu.L of piperidine is added into the EP tube of the previous step reaction, the Fmoc protecting group is removed, 500 mu.L of diethyl ether is added into the reaction system, the mixture is subjected to high-speed centrifugal precipitation, the diethyl ether is discarded, and the product is obtained as a white solid, about 9.55mg and the yield is 61%. The expected product is confirmed by MALDI-TOF mass spectrometry.

MALDI-TOF-MS：m/z[C43H62IN11O9]⁺(M+H)⁺1004.38; found 1004.88. After dissolving a small amount of solid, the purity was 94.4% by HPLC.

(6) Synthesis of Compound 6

Compound 5(5mg, 4.98mmol), DOTA-NHS-ester 3.79mg were weighed out and dissolved in 400 μ L dmf, DIEA was added to adjust pH 8.5, shaking reaction was performed at 30 ℃ for 30min, semi-preparative HPLC separation and purification, HPLC (high performance liquid chromatography) method two, YMC-Pack ODS-A C18 semi-preparative column (10mm × 250mm,

poresize, particle size 5 μm), gradient elution for 25 minutes at a flow rate of 1mL/min, wherein mobile phase A is H₂O solution, B acetonitrile (containing 0.05% TFA). The elution gradient was set to 80% a and 20% B at the start, 50% a and 50% B at 20min, and 80% a and 20% B at 25 min. Fractions at a retention time of 15.7 minutes were collected, pooled and lyophilized to give 3.2mg of a white powder. The yield is 46.3%, purity>95 percent. The expected product is confirmed by MALDI-TOF mass spectrometry.

MALDI-TOF-MS：m/z[C55H80IN15O16]⁺(M+H)⁺1390.50; found 1390.7040. Purity was 98% by HPLC.

(7)⁶⁸Preparation, purification and quality control of Ga-DOTA-A-c (RGDFk)

Accurately weighing quantitative DOTA-A-c (RGDFk), dissolving with water, packaging into 20 μ g/tube, and storing in a refrigerator at-80 deg.C. When the compound is to be radiolabeled, the dispensed compound is removed and thawed at room temperature for 30 minutes.

Preparation: leaching with 0.05M HCl⁶⁸Ge-⁶⁸Ga generator, obtaining 12.4mCi (469.9MBq)⁶⁸Ga liquid. Collecting 500 μ L⁶⁸Ga eluate, placed in a clean EP tube, was added 12. mu.L of 1.25M NaOAc solution, and the pH was adjusted to 4.0. To make this⁶⁸Ga solution (4.0mCi, 148MBq) was added to the polypeptide-filled EP tube. The metal bath was heated to 100 ℃ and reacted for 10 minutes.

And (3) purification: purification was carried out using a Sep-Pak C-18 cartridge, which was first activated with 10mL of absolute ethanol, followed by 10mLH₂And washing the column by using the solvent. The radioactive sample was passed through a Sep-Pak C-18 column, the column was washed with 10mL of physiological saline, and the free fractions were removed⁶⁸Ga, and finally washing the column with 0.4mL of 80% ethanol to collect the radioactive label. Sterilizing with 0.22 μm microporous filter membrane, and performing in vivo experiment.

Quality control: after the radioactive probe was left at room temperature for 10 minutes, the purity was monitored by radioactive HPLC. The labeling rate and radiochemical purity were determined by means of radioactive HPLC. Using an HPLC system, equipped with an on-line radioactivity detector and a Zorbax C18 analytical column (4.6mm x 250mm,

pore size), gradient elution for 30 minutes at a flow rate of 1.0mL/min, wherein the mobile phase A is H₂O solution, B acetonitrile (containing 0.05% TFA). The elution gradient was set to 100% a and 0% B at the start, 95% a and 5% B at 5min, 78% a and 22% B at 30min, and a return to the baseline gradient of 100% a and 0% B at 30-35 min.

Before labeling, both water and the buffer used were treated with Chelex 100column to remove metal ions.⁶⁸Ga-DOTA-A-C (RGDFk) is marked by a one-step method, the preparation process is simple and quick, the marking rate is 85 percent, and the radiochemical purity of the marker is more than 99 percent after the marker is purified by a C-18Sep-Pak column. After calibration calculation, the marker yield was 80%, see FIG. 1.

Example 2: example 1 in vivo biodistribution assay of molecular probes

Taking tumor-bearing C57BL/6J mouse 36, randomly divided into 9 groups of 4. Four groups of tail veins are injected with 0.1mL of each⁶⁸Ga-DOTA-A-c (RGDFk) (about 1.85MBq), four groups of tail vein injections⁶⁸Ga-DOTA-c (RGDFk) (about 1.85MBq), killed after blood is taken for 0.5h, 1h, 2h and 4h respectively, and dissected to take core, liver, spleen, lung, kidney, intestine, stomach, bone, meat and tumor. The mass and radioactivity counts were measured, the cpm radioactive counts were weighed and measured, and the percent injected dose per gram of tissue (% ID/g) was calculated after decay correction. The remaining group of tail veins were injected with 0.1mL simultaneously⁶⁸Ga-DOTA-A-c (RGDFk) and 0.05mL c (RGDFk) solution (0.5mg) were sacrificed one hour later, organs were weighed and radioactivity cpm was measured and counted, and the percent injected dose per gram of tissue (% ID/g) was calculated after decay correction.

Injection of drugs⁶⁸Ga-DOTA-A-c (RGDFk) reached 33.32 + -5.49% ID/g in blood after 0.5h, tumor uptake was 7.52 + -0.99% ID/g, in contrast,⁶⁸the blood uptake value after 0.5h of Ga-DOTA-c (RGDFk) is 1.49 +/-0.49% ID/g, and the uptake value in tumors is 2.74 +/-0.73% ID/g. At a later point in time, the system will,⁶⁸Ga-DOTA-A-c (RGDFk) tumor uptake was consistently higher than⁶⁸Ga-DOTA-c (RGDFk), after injection for 4h⁶⁸Ga-DOTA-A-c (RGDFk) tumor uptake value is 6.57 +/-0.89% ID/g, is⁶⁸Ga-DOTA-c (RGDFk) is 3.5 times, which shows that the novel probe can be effectively combined with serum protein, the blood half-life period of the probe is improved, and the tumor uptake value (P) is effectively improved<0.01, n ═ 4). The probe is metabolized by the kidney. The biodistribution data is shown in figure 2.

Example 3:^99mTc-HYNIC-A-3PRGD₂preparation of

HYNIC-A-3PRGD₂The synthetic route is schematically shown as follows:

R＝3PRGD₂

(1) synthesis of Compounds 1, 2, 3

The synthesis of the compounds is described in reference to example 1.

(2) Synthesis of Compound 7

Compound 3(5.64mg, 7.65mmol) was weighed into a 1mL EP tube, dissolved in 500. mu.L DMF, and 3PRGD was added₂(15.75mg, 7.8mmol), DIEA was added thereto to adjust pH to 8.5, and the reaction was allowed to proceed overnight at room temperature. The reaction was monitored using HPLC. The product peaked at 13.17min using HPLC (high Performance liquid chromatography) method one. HPLC collection, mass spectrum identification, identified as the expected product.

MALDI-TOF-MS: m/z is 2680.89 (chemical formula: C)₁₂₃H₁₈₁IN₂₄O₃₅Calculating the molecular weight: 2681.22 Da.).

(3) Synthesis of Compound 8

125 mu.L of piperidine is added into the EP tube of the previous step reaction, the Fmoc protecting group is removed, 500 mu.L of diethyl ether is added into the reaction system for high-speed centrifugal precipitation, the diethyl ether is discarded, and the product is obtained as a white solid, about 10.6mg and the yield is 56%. The expected product is confirmed by MALDI-TOF mass spectrometry.

MALDI-TOF-MS：m/z＝2460.85(M+H)⁺，2482.87(M+Na)⁺(chemical formula: C)₁₀₈H₁₇₁IN₂₄O₃₃Calculating the molecular weight: 2459.15 Da.).

A small amount of solid was redissolved and analyzed for purity by HPLC to 92.5%.

(4) Synthesis of Compound 9

Compound 8(5mg, 2.03mmol), SBZH-HYNIC 0.9mg were weighed and dissolved in 500 μ L DMF, DIEA was added to adjust pH 8.5, shaking reaction was performed at 30 ℃ for 10 minutes, separation and purification by semi-preparative HPLC, method iii of HPLC (high performance liquid chromatography) method iii of YMC-Pack ODS-A C18 semi-preparative column (10mm × 250mm,

poresize, particle size 5 μm), gradient elution for 30 minutes at a flow rate of 1mL/min, wherein mobile phase A is H₂O solution, B acetonitrile (containing 0.05% TFA). The elution gradient was set to 100% a and 0% B at the start, 75% a and 25% B at 5min, 50% a and 50% B at 25min, and a return to the baseline gradient of 100% a and 0% B at 25-30 min. Collecting the fractions with retention time of 15.2 min, combining the collected liquids and freezingDry to give 1.95mg of a white powder. The yield is 34.8%, purity>95 percent. The expected product is confirmed by MALDI-TOF mass spectrometry.

MALDI-TOF-MS：m/z＝2763.07(M+H)⁺，2785.21(M+Na)⁺(chemical formula: C)₁₂₁H₁₈₀IN₂₇O₃₇S, calculating the molecular weight: 2762.18 Da.).

A small amount of solid is taken to be dissolved and analyzed by HPLC, and the purity is more than 99 percent.

(5)^99mTc-HYNIC-A-3PRGD₂Preparation and quality control of

Sequentially adding 20 μ L HYNIC-A-3PRGD into EP tube₂(1mg/mL in purified water), 100. mu.L tricine solution (100mg/mL in 25mM succinate buffer, pH 5.0), 100. mu.L TPPTS (60mg/mL in 25mM succinate buffer, pH 5.0), 100. mu.L Na^99mTcO₄(10 mCi). Mixing, and heating in 100 deg.C water bath for 25 min. After cooling the labeled product, it was analyzed by HPLC (HP1100 high performance liquid chromatography, equipped with LB-509 radioactive detector) for its labeling rate and radiochemical purity.

^99mTc-HYNIC-A-3PRGD₂Using non-SnCl₂And (3) preparing by a one-step method. The label was analyzed by radioactive HPLC,^99mTc-HYNIC-A-3PRGD₂the retention time was 11.8min, and the marking rate was found to be > 99%. As shown in fig. 3.

Example 4: example 3 molecular Probe blood removal assay

Taking 14 Kunming female mice of 4-5 weeks old, randomly dividing into two groups, and injecting 0.1mL each group^99mTc-HYNIC-A-3PRGD₂，^99mTc-HYNIC-3PRGD₂(about 1.85MBq), blood is taken at 1min, 3min, 5min, 7min, 10min, 15min, 20min, 30min, 60min, 90min and 120min after injection, the radioactive cpm count is measured, and the percentage injection dose (% ID/g) of the two probes in the blood is calculated after decay correction.

Through blood clearance experiments, we can see the 3PRGD after the structural modification₂The in vivo properties of the probe are significantly changed compared with those of the original probe.^99mTc-HYNIC-A-3PRGD₂The rapid half-life of the drug is 4.57min; the slow half-life was 93.32 min. While^99mTc-HYNIC-3PRGD₂(structurally unmodified) drug fast half-life of 0.72 min; the slow half-life was 17.91 min. The fast half-life period of the medicine is improved by 6.3 times, and the slow half-life period of the medicine is improved by 5.2 times. The injection time is 1min, the blood uptake value of the unmodified molecular probe is 17.07 +/-11.77 percent ID/g, and^99mTc-HYNIC-A-3PRGD2 uptake was 44.76. + -. 11.83% ID/g, which was about 2.6 times higher than the former. The former uptake value is only 4.39 +/-2.55% ID/g at 5min, while the latter, namely the molecular probe uptake of the invention is 33.63 +/-7.83% ID/g, which is 7.6 times of the former. The former ingested 1.99. + -. 1.54% ID/g and the latter ingested 19.06. + -. 6.51% ID/g at 10 min. The former is taken 60min later and reduced to below 0.5% ID/g, and at 240min, the intake value is 0.34 + -0.18% ID/g; in contrast, the uptake of the latter was 2.81. + -. 0.83% ID/g, which is 8 times that of the original probe. It can be seen that the structural modification greatly prolongs the 3PRGD₂Blood retention time (P)<0.01, and n is 7), which is beneficial to increase the uptake value of the probe at the tumor site. The blood clearance results are shown in FIG. 4.

Example 5: SPECT/CT imaging of tumor-bearing nude mice with molecular probes of example 3

^99mTc-HYNIC-A-3PRGD₂(hereinafter, referred to as the molecular probe of the present invention) was prepared by the method of 1.2.3. Simultaneously according to 3PRGD₂Marking method of^99mTc-HYNIC-3PRGD₂(hereinafter referred to as "control probe") was prepared. After performing radioactive HPLC detection, diluting to 2mCi/100 μ L with physiological saline, and injecting 100 μ L of nude mice with U87MG tumor into tail vein of each mouse^99mTc-HYNIC-A-3PRGD₂. The specific uptake of the drug in each tissue and organ of tumor-bearing mice was verified by a blocking experiment, and 100 μ L of 1mg 3PRGD was injected via tail vein into blocking mice₂Cold peptide, and immediately injected in 100. mu.L^99mTc-HYNIC-A-3PRGD₂At the same time, 50. mu. Ci of each drug was taken for quantification. SPECT/CT image acquisition is carried out at 0.5, 1, 2, 4,8, 12 and 24h respectively, and the result is shown in a SPECT/CT image of a mouse with U87MG load in 5.

FIG. 6A is^99mTc-HYNIC-A-3PRGD₂And^99mTc-HYNIC-3PRGD₂% ID/g at U87 tumor; 6B, 6C and 6D are respectively^99mTc-HYNIC-A-3PRGD₂And^99mTc-HYNIC-3PRGD₂tumor/kidney, tumor/muscle, tumor/liver ratios. The results are expressed as means ± SD, (n ═ 3).

We found that the new probe was able to image tumors well at each time point of acquisition, even 24 hours after injection. This is associated with a longer blood residence time of the modified probe, which in turn enhances tumor uptake. As shown in the quantitative analysis of FIG. 6A, the molecular probe of the present invention has slow tumor clearance, 11.85 + -2.18% ID/g, 21.17 + -0.49% ID/g and 22.27 + -1.64% ID/g at 0.5, 8 and 24h after injection. As shown in fig. 6B, the tumor/kidney ratio of the modified new probe was improved compared to the control probe, 1h after injection, the tumor/kidney ratio of the control probe was 1.14 ± 0.26, and 24h after injection was 1.21 ± 0.17; the corresponding uptake of the molecular probe is 1.57 +/-0.26 and 1.92 +/-0.17 respectively. Post-retrofit 3PRGD₂The tumor/non-tumor ratio of (a) was also improved, as shown in fig. 6C, 6D. This indicates that the modified molecular probe has better biodistribution and pharmacokinetic properties than those before modification.

Example 6: molecular Probe biodistribution of example 3

24 nude mice bearing U87 were randomly divided into 6 groups of 4 mice each, and time points were set at 1h, 2h, 4h, 8h, and 12 h. Each group of nude mice was injected with 20. mu. Ci via tail vein^99mTc-HYNIC-A-3PRGD₂One group of injections of 3PRGD₂Used as a Block group. And animals were sacrificed after injection, blood and major organs were removed, weighed and measured for radioactive cpm counts. The percent injection dose rate per gram of tissue (% ID/g) was calculated after decay correction.

Preparing a biodistribution standard sample: adding 100 mu L of marker solution for a biodistribution experiment into a 100mL volumetric flask by using an injector, adding water to a constant volume of 100mL, fully and uniformly mixing, accurately taking out 1mL by using a pipette, measuring radioactivity count by using a gamma counter, amplifying the count by 100 times, using the count as a standard injection dose, preparing three parallel samples, and taking an average value.

As shown in FIG. 7, tail vein injection^99mTc--HYNIC-A-3PRGD₂Later, the uptake of the imaging agent in the U87 tumor increased and decreased with time, and the uptake in the tumor was (22.38. + -. 3.68% ID/g, 21.71. + -. 3.61% ID/g, 25.96. + -. 1.69% ID/g, 23.53. + -. 3.40% ID/g) at four time points of 1h, 2h, 4h and 8h, respectively. The organ with the highest uptake was the kidney, always above 30% ID/g, so the radioactive probe should be metabolized through the kidney, consistent with SPECT/CT imaging results. The uptake of other organs after injection is higher than that of the control probe, because the polypeptide structure of the invention is modified to lead the polypeptide to circulate in the body for a longer time along with blood, so that the uptake value of each organ is improved.

Example 7:¹⁷⁷Lu-DOTA-A-L-3PRGD₂preparation of

(1) Preparation of Compound 2 As in example 1

(2) Preparation of Compound 11

100.0mg (250. mu. mol) of Compound 10, 76.4mg (400. mu. mol) of EDC.HCL and 46.0mg (400. mu. mol) of NHS were weighed out and dissolved in 5mL of dichloromethane and stirred at room temperature overnight. The mixture was separated and purified by chromatography on silica gel, eluting with 2% methanol in dichloromethane. The solvent was distilled off under reduced pressure to obtain 85.2mg of a white powdery solid. The HPLC gradient and time method was: 0 minutes 50% mobile phase a and 50% mobile phase B; 10% mobile phase a and 90% mobile phase B at 25 minutes; 50% mobile phase a and 50% mobile phase B at 30 minutes. A small amount of the product was checked for purity by HPLC (retention time 17.7 min) and then identified by MALDI-TOF mass spectrometry.

(3) Preparation of Compound 12

85.2mg (147. mu. mol) of Fmoc-Cys (tBu) -NHS compound 11 and 61.0mg (145. mu. mol) of Compound 2 were weighed, 400. mu.L of LDMF and 20. mu.L of DIEA were added, and the mixture was sonicated in a water bath to form a suspension. The solid in the solution was completely dissolved by adding 400. mu.L of purified water, and stirred at room temperature overnight. The mixture was separated and purified by HPLC method and the elution peak with retention time of 20.9 min was collected. The HPLC gradient and time method was: 0 minutes 50% mobile phase a and 50% mobile phase B; 10% mobile phase a and 90% mobile phase B at 25 minutes; 50% mobile phase a and 50% mobile phase B at 30 minutes. Freeze-drying to obtain white powdery solid 67.2 mg. A small amount of the product was purified by HPLC and then identified by MALDI-TOF mass spectrometry.

(4) Preparation of Compound 13

67.2mg (84.1. mu. mol) of Compound 12 was weighed out, dissolved in 20% piperidine-DMF, and reacted at room temperature for 10 minutes. The mixture was separated and purified by HPLC method and the peak eluted with a retention time of 24.1 minutes was collected. The HPLC gradient and time method was: 0min 85% mobile phase a and 15% mobile phase B; 45% mobile phase a and 55% mobile phase B at 25 minutes; at 30 minutes 85% mobile phase a and 15% mobile phase B. Freeze-drying to obtain white powdery solid 27.8 mg. A small amount of the product was purified by HPLC and then identified by MALDI-TOF mass spectrometry.

(5) Preparation of Compound 14

10.0mg (12.5. mu. mol) of Compound 13, 10mg (13.1. mu. mol) DOTA-NHS were weighed out in 200. mu.L DMF. 10 μ LDIEA was added and the insoluble material was sonicated to break up the suspension. The solid in the solution was completely dissolved by adding 200. mu.L of purified water, and stirred at room temperature overnight. The mixture was separated and purified by HPLC method, and the peak eluted with a retention time of 21.7 minutes was collected. The HPLC gradient and time method was: 0min 85% mobile phase a and 15% mobile phase B; 45% mobile phase a and 55% mobile phase B at 25 minutes; at 30 minutes 85% mobile phase a and 15% mobile phase B. Freeze-drying to obtain white powdery solid 8.5 mg. The product was purified by HPLC and then identified by MALDI-TOF mass spectrometry.

(6) Preparation of Compound 15

8.5mg (8.8. mu. mol) of Compound 14 was weighed out, and 200. mu.L of 20% TFMSA-TFA was added to react for 30 seconds, and 400. mu.L of DMF was immediately added to prevent acidolysis of the product. The mixture was separated and purified by HPLC method and the elution peaks with retention time of 26.6 and 26.9 minutes were collected. The HPLC gradient and time method was: 0min 85% mobile phase a and 15% mobile phase B; 45% mobile phase a and 55% mobile phase B at 25 minutes; at 30 minutes 85% mobile phase a and 15% mobile phase B. Freeze-drying to obtain white powdery solid 1.2 mg. The product was purified by HPLC and then identified by MALDI-TOF mass spectrometry.

(7)MAL-3PRGD₂Preparation of (L7)

10mg (4.8. mu. mol) of 3PRGD2, 2.0mg (6.5. mu. mol) of Mal-NHS were weighed out and 200. mu. LDMF and 10. mu. LDIEA were added. The mixture was stirred at room temperature overnight. The mixture was separated and purified by HPLC method and the peak eluted with a retention time of 22.1 minutes was collected. The HPLC gradient and time method was: 0min 90% mobile phase a and 10% mobile phase B; 60% mobile phase a and 40% mobile phase B at 25 minutes; at 30 minutes 90% mobile phase a and 10% mobile phase B. Freeze-drying to obtain white powdery solid 6.7 mg. A small amount of the product was purified by HPLC and then identified by MALDI-TOF mass spectrometry.

(8) Preparation of Compound 16

0.7mg (0.8. mu. mol) of compound 15 was weighed, 1.8mg (0.8. mu. mol) of MAL-3PRGD2 was added, and 0.1M phosphate buffer (pH 7.0) was added, followed by shaking at room temperature overnight. The product was isolated and purified by HPLC method and the peak eluted with a retention time of 25.6 min was collected. The HPLC gradient and time method was: 0min 90% mobile phase a and 10% mobile phase B; 60% mobile phase a and 40% mobile phase B at 25 minutes; at 30 minutes 90% mobile phase a and 10% mobile phase B. The product peak eluates were combined and lyophilized to obtain 1.2mg of a white powdery solid. A small amount of the product was purified by HPLC and then identified by MALDI-TOF mass spectrometry. The product purity by HPLC analysis was > 98%, and MALDI-TOF mass spectrometry showed m/z 3160.84. The experimental results show that [ M + H ] + is consistent with the theoretical molecular weight M3161.36 for C137H215IN30O 45S.

(9)¹⁷⁷Lu-DOTA-A-L-3PRGD₂Preparation, purification and quality control of

Weighing 20 μ g of compound 16, DOTA-3PRGD₂Or DOTA-A-L, and then 200. mu.L of ammonium acetate buffer (0.1M, pH 4.8) and 5-25 mCi 177LuCl₃. And (3) placing the mixed solution in an air bath heater at 99 ℃ for reaction for 20 minutes, and naturally cooling to obtain the catalyst. To prevent the label from undergoing radiation self-decomposition, 200. mu.L of gentisic acid aqueous solution (1mg/mL) was added. The marker can maintain room temperature stability for more than 6 hours.

¹⁷⁷Lu-DOTA-A-L-3PRGD2、¹⁷⁷Lu-3PRGD2 and¹⁷⁷radiochemical purity (RCP) measurement of Lu-DOTA-A-L an Agilent HPLC-1260Infinity liquid chromatography system with a radioactivity detector and an Agilent ZORBAXExtend-C18(250X 4.6mm, 5um) chromatography column (250X 10mm, 5 μm) at a flow rate of 1mL/min were used. Mobile phase a was water (with 0.05% TFA) and mobile phase B was acetonitrile (with 0.05% TFA). The elution gradient and time method is as follows: 90% of mobile phase A and 10% of mobile phase B in 0-5 minutes; 60% mobile phase a and 40% mobile phase B at 25 minutes; at 30 minutes 90% mobile phase a and 10% mobile phase B.

The final product¹⁷⁷Lu-DOTA-A-L-3PRGD₂FIG. 8 shows a radioactive HPLC chart

Example 8: blood distribution of molecular probes of example 7

10 Kunming mice were divided into two groups and injected with 0.1mL, and 15 Kunming mice were randomly divided into three groups (n ═ 5), and 100. mu.L (740KBq) of the present invention obtained in example 14 was injected via the tail vein¹⁷⁷Lu-DOTA-A-L-3PRGD₂And as a control¹⁷⁷Lu-3PRGD2 or¹⁷⁷Lu-DOTA-A-L. Appropriate amounts of blood were taken from the inner canthus at time points after injection of the mice, weighed and measured for radioactivity counts cpm. The percent injection dose rate (% ID/g) of the radiopharmaceutical per gram of tissue in the blood was calculated. Experimental results non-linear regression analysis was performed using GraphPad Prism 7.0 software to calculate the fast half-life and slow half-life in a dual chamber model of blood metabolism (Twophase decay).

According to the experimental results, the results are shown in fig. 9:¹⁷⁷Lu-DOTA-A-L-3PRGD₂the fast half-life and the slow half-life of (A) are 6.909min and 77.15min, respectively, and¹⁷⁷Lu--3PRGD₂the fast half-life period and the slow half-life period are respectively 1.231min and 20.83min, and the invention can be seen¹⁷⁷Lu-DOTA-A-L-3PRGD₂And¹⁷⁷Lu--3PRGD₂compared with the prior art, the retention time of blood is obviously prolonged, and the structural modification of the polypeptide can really prolong the retention time of c (RGDFk) in the blood. This shows that the RGD probe modified by the invention has strong binding capacity with MSA, and the probe can circulate in vivo for a longer time along with blood, so that the concentration of radioactive probe in the blood is increased. The probe is in the bloodThe integral of the percent injected dose rate per gram of tissue in the fluid over time (area under the curve, AUC) can represent the effect of the drug in the blood.¹⁷⁷Lu-DOTA-A-L-3PRGD₂、¹⁷⁷Lu--3PRGD₂AUC (% ID/g-h) values at 0 to 72 hours after administration were 208.9 and 27.0 in this order. The experimental results show that the invention is novel¹⁷⁷Lu-DOTA-A-L-3PRGD₂Has a ratio of¹⁷⁷Lu--3PRGD₂Blood pharmacokinetics 7.7 times higher. Although it is used for¹⁷⁷Lu-DOTA-A-L has a longer half-life in blood at the slowest rate than the molecular probe of the present invention, but its other properties as a molecular probe, such as retention and uptake in tumors, are much inferior to those of the molecular probe of the present invention, as shown in the following examples.

Example 9: SPECT/CT imaging of tumor-bearing nude mice with molecular probes of example 7

The SPECT/CT imaging system (Mediso Inc.) has 4 probes and parallel-hole collimators. U87-MG tumor-bearing mice were injected 100. mu.L (20MBq) via tail vein¹⁷⁷Lu-DOTA-A-L-3PRGD₂、¹⁷⁷Lu-3PRGD₂Or¹⁷⁷Lu-DOTA-A-L. SPECT imaging was then performed 1,4,8, 12, 24, 48, and 72 hours after injection. Mice were anesthetized with 1.5% isoflurane-oxygen during imaging, keeping them stationary in the prone position on the bed of the small animal. The blocked mice were mixed with 1.0mg of the labeling agent

Cold peptides. The SPECT image was fused with the CT image, and then the region of interest (ROI) in the SPECT image was outlined in a 3D visualization map, and the percent injection dose rate per gram of tissue (% ID/cc) of tissues and organs was calculated. Imaging and quantification results included physical and biological half-lives without decay correction. The results are shown in FIG. 10.

We have found that the probes of the invention¹⁷⁷Lu-DOTA-A-L-3PRGD₂Has proper pharmacokinetics, high tumor uptake and tumor contrast in tumors, and more importantly, the uptake in tumors is always significantly higher than that in other normal tissues and organs in the whole body. After injection in mice bearing tumorsThe quantitative result of the tumor within 1-72 hours shows that,¹⁷⁷Lu-DOTA-A-L-3PRGD₂the cumulative uptake value in the tumor was 662.0 ID/cc-h;¹⁷⁷Lu-3PRGD₂the cumulative uptake value in the tumor was 158.7 ID/cc-h;¹⁷⁷the cumulative uptake value of Lu-DOTA-A-L in the tumor is 266.3 ID/cc-h.¹⁷⁷Lu-DOTA-A-L-3PRGD₂At each acquisition time point, the tumor can be well imaged, even the tumor is clearly imaged after 48 hours of injection, and the contrast probe has high background, low contrast and less enrichment at the tumor. As shown in the quantitative analysis of FIG. 10, the molecular probe of the present invention has the highest uptake value and slow elimination at the tumor, and the tumor uptake reaches the highest 4 hours after the administration of the tumor-bearing mice, and the percent injection dosage rate is 26.52 + -0.58% ID/g. This shows that the molecular probe of the present invention has better biodistribution and pharmacokinetic properties than before modification.

Example 10: example 7 in vivo biodistribution assay of molecular probes

16U 87-MG-bearing mice were injected via tail vein with 100. mu.L (0.74MBq)¹⁷⁷Lu-DOTA-A-L-3PRGD₂. Mice were sacrificed 1,4, 24 and 72 hours after injection (n-4); 4 tumor-bearing mice were injected via tail vein with 100. mu.L of 740KBq cocktail¹⁷⁷Lu-DOTA-A-L-3PRGD₂And 0.5mg of 3PRGD₂Cold peptide, mice sacrificed 1 hour post injection; 12 tumor-bearing mice were randomly divided into 3 groups (n-4) and injected with 100. mu.L (0.74MBq) via tail vein¹⁷⁷Lu-DOTA-A-L-3PRGD₂、¹⁷⁷Lu-3PRGD2 or¹⁷⁷Lu-DOTA-A-L, mice were sacrificed 4 hours after injection. Blood and other major tissues and organs were taken, weighed and their radioactivity counts (cpm) measured, and the percent injection dose rate per gram of tissue (% ID/g) was calculated.

As shown in fig. 11a, 4 hours after injection,¹⁷⁷Lu-DOTA-A-L-3PRGD₂the tumor uptake value of (a) is 26.52 +/-0.58% ID/g, the tumor uptake value of 177Lu-3PRGD2 is 4.91 +/-0.92% ID/g,¹⁷⁷the tumor uptake value of Lu-DOTA-A-L is 4.80 +/-1.19% ID/g. The uptake of 177Lu-k428-3PRGD2 in tumors was significantly higher than 177Lu-3PRGD2 (P)<0.0001) and 177Lu-k428 (P)<0.0001)。

As shown in the graph b, the tumor uptake values at 1,4, 24 and 72 hours after administration of the drug to tumor-bearing mice were 19.93. + -. 1.99%, 28.57. + -. 5.27%, 11.67. + -. 2.80% and 2.66. + -. 1.14%, respectively. The uptake of this probe is highest in the kidney, except for the tumor, and the radioactive probe should be metabolized through the kidney, consistent with the SPECT/CT imaging results. At 4 hours, the tumor/kidney ratio was more than 2-fold (28.57/13.70% ID/g).

As shown in the figure c, the tumor uptake value 1 hour after the administration of the closed group was 6.41. + -. 1.52% ID/g, and the tumor uptake value 1 hour after the administration of the normal group was 19.93. + -. 1.98% ID/g. The experimental results show that the tumor uptake of the blocking group is significantly lower than that of the non-blocking group (P <0.005), and the uptake of 177Lu-k428-3PRGD2 in the U87-MG tumor is specific.

Example 11: radioactive targeting therapy with the molecular probes of example 7

35U 87-MG tumor-bearing mice were randomly divided into 5 groups (n ═ 7): group 1 mice were injected with 100. mu.L (18MBq) via tail vein¹⁷⁷Lu-DOTA-A-L-3PRGD₂Group 2 mice were injected with 100. mu.L (18MBq) via tail vein¹⁷⁷Lu--3PRGD₂Group 3 mice were injected with 100. mu.L (18MBq) via tail vein¹⁷⁷Lu-DOTA-A-L; group 4 mice were injected with 100. mu.L (9MBq) via tail vein¹⁷⁷Lu-DOTA-A-L-3PRGD₂Group 5 tumor-bearing mice were injected with 100. mu.L of PBS as a control. Monitoring tumor volume and body weight change every 2 days after injection, wherein the tumor volume reaches 1000mm³The kindness endpoint was set.

See figure 12 for results. FIGS. A and C are views showing the molecular probes of the present invention¹⁷⁷Lu-DOTA-A-L-3PRGD₂And a control group¹⁷⁷Lu--3PRGD₂、¹⁷⁷Trend plot of tumor volume over time for Lu-DOTA-A-L at the same dose (18Mbq), and for the molecular probes of the invention at half dose (9 Mbq).

Tumor volumes after 14 days of dosing were: 18MBq¹⁷⁷Lu-DOTA-A-L-3PRGD₂The tumor volume of the treatment group was 401.3 + -195.5 mm3, 9MBq¹⁷⁷The tumor volumes of the Lu-DOTA-A-L treatment group are 691.3 +/-195.9 mm3 and 18MBq¹⁷⁷Lu--3PRGD₂Treatment ofThe tumor volumes of the groups were 1122.4 + -189.6 mm3, 18MBq¹⁷⁷The tumor volume of the Lu-DOTA-A-L treatment group was 897.4 + -178.0 mm3, and the tumor volume of the PBS control group was 1336.3 + -315.4 mm 3. The results of the treatment experiments show that under the condition that the injection dose is 18MBq,¹⁷⁷Lu-DOTA-A-L-3PRGD₂the treatment effect of the treatment group is obviously better than that of the treatment group¹⁷⁷Lu-3PRGD₂Treatment group (p)<0.0001) and¹⁷⁷Lu-DOTA-A-L treatment group (p)<0.05). In the treatment process, the weight change and survival index of the three groups of mice are in a normal range. The change ratio of the body weight of the mice injected with the three probes is within a normal range, and no obvious acute toxicity occurs. More importantly, reduce¹⁷⁷Lu-DOTA-A-L-3PRGD₂To 9MBq, the therapeutic effect is still significantly higher than that of the double dose¹⁷⁷Lu-3PRGD₂(p<0.01). The experimental results show that the traditional method is similar to the traditional method¹⁷⁷Lu-3PRGD₂In contrast to the above-mentioned results,¹⁷⁷Lu-DOTA-A-L-3PRGD₂has better treatment effect. The experiment proves that the method has the advantages that,¹⁷⁷Lu-DOTA-A-L-3PRGD₂the targeted nuclide treatment in U87-MG tumor-bearing mice can significantly inhibit tumor growth within 10 days after injection. The initial volume ratio (V/V0) on day 10 of the treatment group and the control group was 1.4 and 8.0.

FIG. B is a view of the molecular probe of the present invention¹⁷⁷Lu-DOTA-A-L-3PRGD₂And a control group¹⁷⁷Lu--3PRGD₂、¹⁷⁷Trend graph of mouse body weight over time for Lu-DOTA-A-L at the same dose (18Mbq), and for the molecular probes of the invention at a half-reduced dose (9 Mbq). The experimental results show that BALB/c nude mice dose 18MBq¹⁷⁷Lu-DOTA-A-L-3PRGD₂Tolerance, and mice in the administration group recover to normal after the body weight and blood conventional indexes are reduced. Mice after the 18MBq dose had the greatest rate of weight loss at day 6 and returned to normal at day 14 after dosing. The initial weight percentages of the day 6 dosing group and the control group were 90.8 ± 3.7% and 98.7 ± 5.0%; the initial weight percentages of the day 14 dosing group were 100.5 + -4.1% and 100.7 + -3.4%. Compared with the control group, the weight of the mouse does not change obviously in the period of 14 days by using the molecular probe, so that the molecular probe can be seenHas less side effect and high safety.

Example 12: example 7 testing of molecular probes in the MC-38 mouse model

(1) Example 7 in vivo biodistribution assay of molecular probes

The results are shown in FIG. 13A. The distribution of the molecular probe of the invention is 1,4, 24 and 72 hours. From the results, it can be seen that the molecular probe of the present invention shows the increased and decreased uptake of the imaging agent in the MC-38 tumor with time, which reaches the maximum uptake at 4 hours, and the percent injection dose rate is 32.51 + -4.95% ID/g. And is the highest of all organs. As can be seen, the molecular probe of the present invention has a high uptake ratio of tumor/other organs.

(2) Molecular Probe MC-38 SPECT/CT imaging of nude mice of example 7

The results are shown in FIG. 13B. The probe provided by the invention can be used for well imaging tumors at the collection time points of 1,4 and 24 hours, and the tumors are clear in imaging, almost have no background and high in contrast. This shows that the molecular probe of the present invention has better biodistribution and pharmacokinetic properties than before modification.

(3) Tumor volume and mouse body weight changes after injection of the molecular Probe of example 7

The results are shown in FIGS. 13C and 13D. FIG. 13C shows a molecular probe of the present invention¹⁷⁷Lu-DOTA-A-L-3PRGD₂Trend plot of tumor size over time at doses of 18Mbq, 9 Mbq. As can be seen, the molecular probe of the present invention completely eliminated the tumor from day 12 to day 20 at the 18Mbq dose, which is not the case in FIG. 12A of example 11. Compared with the mouse with the load U87 in example 18, the MC-38 mouse of the present invention has autoimmune function, and the mouse with the load U87 is an immunodeficient mouse, so the MC-38 mouse is closer to the body state of human patients, which can show that the molecular probe of the present invention can stimulate the self immune function of the patients to be treated when treating tumors, so that the molecular probe has better treatment effect, and can effectively inhibit and eliminate the tumors. FIG. 13D is a graph showing the time course of the body weight of mice with the molecular probe of the present invention at doses of 18Mbq and 9 Mbq. As can be seen, the molecular probe of the present invention has unknown change in the body weight of the mouse during the 20-day periodObviously, the molecular probe of the invention has small side effect and high safety.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement and the like, which are within the spirit and principle of the present invention, should be included in the protection scope of the present invention.

Claims (13)

1. A radionuclide-labelled complex having the following definitions:

Nu-BFC-A- (L) n-RGD polypeptide

Wherein:

nu is selected from:¹⁷⁷Lu、⁶⁸Ga、^99mTc；

BFC is a bifunctional chelating agent (selected from HYNIC, DOTA);

wherein A is the following structure:

n is 0 or 1;

l represents a linker arm molecule having the structure:

wherein m is an integer from 2 to 6;

the RGD polypeptide is an RGD polypeptide selected from: c (RGDfV), c (RGDfK), c (RGDfE), c (RGDyk), E [ c (RGDyk)]₂、E[c(RGDfK)]₂、3PRGD₂；

With the following conditions:

when n is 0, Nu is⁶⁸Ga or^99mTc, and the RGD polypeptide reacts with carboxyl in A through an amino group thereof; the bifunctional chelating agent is used for chelating a carboxyl group in the structure of the bifunctional chelating agent and-NH in A₂A reactive linkage chain;

when n is 1, Nu is¹⁷⁷Lu, and the L reacts with the amino group in A through the carboxyl group, the RGD polypeptide reacts with the carboxyl group in L through the amino group, and the bifunctional chelating agent reacts with the carboxyl group in L through the carboxyl group in the structureOf (2) is-NH₂A chain of reactive bonds.

2. The complex of claim 1:

the RGD polypeptide is selected from: c (RGDFK), 3PRGD₂；

m is 5;

when Nu is¹⁷⁷When Lu, BFC is selected from DOTA;

when Nu is⁶⁸When Ga, BFC is selected from DOTA;

when Nu is^99mAt Tc, BFC is selected from HYNIC.

3. The complex according to claim 2, which is as follows:

⁶⁸Ga-DOTA-A-c(RGDfk)；

^99mTc-HYNIC-A-3PRGD₂；

¹⁷⁷Lu-DOTA-A-L-3PRGD₂。

4. a complex according to claim 3, having the structure:

5. a pharmaceutical composition comprising an effective amount of a marker complex according to any one of claims 1 to 4.

6. The pharmaceutical composition according to claim 5, which is a diagnostic drug for imaging diagnosis of integrin α v β 3 positive tumor when Nu is a diagnostic nuclide.

7. The pharmaceutical composition according to claim 5, which is a therapeutic drug for the radiotargeted treatment of integrin α v β 3 positive tumors when Nu is a therapeutic nuclide.

8. A pharmaceutical composition according to any one of claims 5 to 7, which is an injectable formulation comprising the marker complex and an injectable carrier.

9. The pharmaceutical composition according to claim 8, which is an intravenous injection.

10. The pharmaceutical composition according to claim 9, which contains 0.05% tween-20 in PBS.

11. Use of a complex of any one of claims 1-4 in the preparation of a medicament for the diagnosis or treatment of integrin α v β 3 positive tumors.

12. The use according to claim 11, wherein the tumor is a malignant tumor in the blood, liver, gland, intestine, kidney, stomach, spleen, lung, muscle, bone.

13. The use according to claim 12, wherein the gland is a breast, prostate, or pancreas; the intestinal tumor is colorectal cancer.

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