CN111228521B

CN111228521B - Dar2 polypeptide radiopharmaceutical and preparation method thereof

Info

Publication number: CN111228521B
Application number: CN202010050493.5A
Authority: CN
Inventors: 王凡; 史继云; 罗麒; 贾兵
Original assignee: Peking University; Institute of Biophysics of CAS
Current assignee: Peking University; Institute of Biophysics of CAS
Priority date: 2020-01-17
Filing date: 2020-01-17
Publication date: 2021-03-19
Anticipated expiration: 2040-01-17
Also published as: CN111228521A

Abstract

The invention discloses a Dar2 polypeptide radiopharmaceutical and a preparation method thereof, and the pharmaceutical bagThe radionuclide is used for marking the Dar polypeptide dimer through a bifunctional chelating agent; the Dar polypeptide dimer is synthesized by connecting GGG with Dar polypeptide monomers and dimerizing the two Dar polypeptide monomers connected with GGG; the Dar polypeptide monomer is D-type amino acid linear 7-membered polypeptide, and the sequence is as follows: anedywr. The medicine is prepared by labeling radionuclide onto Dar polypeptide dimer molecule with bifunctional chelating agent, in vivo labeling medicine concentrating to tumor part via targeting effect of Dar polypeptide, and performing single photon tomography (SPECT) or positron emission computed tomography (PET) to synthesize alpha of integrin₆And imaging and diagnosing the positive tumor.

Description

Dar2 polypeptide radiopharmaceutical and preparation method thereof

Technical Field

The invention relates to the technical field of radiopharmaceuticals, in particular to a novel Dar polypeptide-based radiopharmaceutical and a preparation method thereof.

Background

The integrin family is a class of heterodimeric transmembrane glycoproteins formed by the noncovalent association of two subunits, alpha and beta. 18 α subunits and 8 β subunits have been found in mammals. These subunits can combine to form 24 integrins. The distribution and physiological function of integrins vary with subunit combination. An integrin can have multiple ligands, and a ligand can bind to multiple receptors. Integrins are commonly involved in signal transduction inside and outside cells to regulate various important cellular functions such as adhesion, polarity, differentiation, migration, cell division, and the like. Integrin alpha₆As one member of the integrin family, predominantly and beta₁Or beta₄Subunit association, composition alpha₆β₁And alpha₆β₄Is layer adhesionSpecific receptors for zonulin. Integrin alpha₆It is up-regulated in breast cancer, liver cancer, nasopharyngeal carcinoma, cervical carcinoma and other tumors, and down-regulated in corresponding normal tissue. Integrin alpha₆Plays an important role in the occurrence, angiogenesis, invasion and metastasis of tumors. Furthermore, integrin α in tumors₆Is negatively correlated with its prognosis. Thus, integrin alpha₆Can be used as biomarker for tumor diagnosis and prognosis evaluation and is aimed at integrin alpha₆The development of tumor molecular imaging probes is particularly important.

The research proves that the polypeptide of the cCRWYDENAC sequence has good integrin alpha₆Targeting property, radionuclide-labeled probes can effectively treat integrin alpha of different tumor cells₆The expression was evaluated. However, the disulfide bond cyclization of two terminal cysteines of polypeptide is not favorable for the drug combination of polypeptide drugs. In the early research, an optimized cKiE polypeptide drug (cKRWYDENASOE) is synthesized, and the amido bonds are cyclized from head to tail, so that the kit of the drug is more facilitated. Meanwhile, the cKiE dimer polypeptide has higher affinity than the monomer, and the receptor-ligand affinity of the enhanced drug can achieve higher tumor uptake. However, in vivo metabolic stability experiments show that the cKiE dimer polypeptide probe marked by the radionuclide^99mTc-HYNIC-(GGG-cKiE)₂Has the problems of partial decomposition and instability in vivo.

Disclosure of Invention

The present invention aims to overcome the above technical drawbacks and to provide a novel Dar polypeptide-based radiopharmaceutical. Dar2 in the present invention is a polypeptide dimer (hereinafter Dar2 refers to Dar polypeptide dimers), in which three glycine molecules (GGG, G ═ Glutamic acid) are linked to the all-trans D-type polypeptide anedywr (Dar), and then dimerized so that two polypeptides in the dimer molecule have a sufficient distance to simultaneously bind to two integrin alpha-dimers₆The target improves the targeting property of the tumor while enhancing the stability in vivo and improving the pharmacokinetic property. The combination of the bivalent form can further enhance the medicine intake of the tumor, and achieve better diagnosis effect. The drug is chelated by dual functionsThe agent labels radionuclide onto Dar polypeptide dimer molecule, and the labeled drug in vivo is concentrated to tumor part via targeting effect of Dar polypeptide, and the integrin alpha is synthesized by nuclear single photon tomography (SPECT) or positron emission computed tomography (PET) technology₆And imaging and diagnosing the positive tumor.

The purpose of the invention is realized by the following technical scheme:

a Dar2 polypeptide radiopharmaceutical comprising a Dar polypeptide dimer and a radionuclide that labels said Dar polypeptide dimer with a bifunctional chelator; the Dar polypeptide dimer is a polypeptide dimer synthesized by linking GGG to a Dar polypeptide monomer and dimerizing two Dar polypeptide monomers linked to GGG to form (GGG-Dar)₂Represents; the Dar polypeptide monomer is D-type amino acid linear 7-membered polypeptide, and the sequence is as follows: anedywr (D-alanine-D-asparagine-D-glutamic acid-D-aspartic acid-D-tyrosine-D-tryptophan-D-arginine); the radionuclide is^99mTc、⁶⁸Ga、⁶⁴Cu、¹¹¹In、⁹⁰Y and¹⁷⁷lu, wherein the bifunctional chelating agent is any one of HYNIC, NOTA, DOTA and DTPA.

Further, the radionuclide is^99mAt the time of Tc, the temperature of the alloy is,^99mtc is formed by labeling a Dar polypeptide dimer connected with a pharmacokinetic linker through a bifunctional chelating agent HYNIC to form the Dar2 polypeptide radiopharmaceutical, wherein the pharmacokinetic linker is a polyethylene glycol molecule (PEG)_nPEG ═ Polyethylene glycol, n is not less than 4, preferably 4) or 8-aminocaprylic acid (Aoc, 8-aminoctanoic acid).

Further, the radionuclide is⁶⁸In the case of Ga, the compound (I),⁶⁸ga labels Dar polypeptide dimers containing no pharmacokinetic linkers by the bifunctional chelating agent NOTA or DOTA to form the Dar2 polypeptide radiopharmaceutical.

A preparation method of the Dar2 polypeptide radiopharmaceutical,^99mtc is marked by bifunctional chelating agent HYNIC to form the Dar2 polypeptide radiopharmaceutical with Dar polypeptide dimer linked with pharmacokinetic linker,the method comprises the following steps:

a. preparation of HYNIC-PKM-COOH

Using PKM as the pharmacokinetic linking agent, dissolving Fmoc protected PKM-COOH in DMF solution with 20% piperidine concentration by volume fraction, reacting at room temperature for 15-30 min, adding ether to precipitate PKM-COOH, centrifuging, discarding supernatant, washing precipitate with ether, removing residual ether to obtain NH as expected product₂-PKM-COOH; HYNIC-NHS and NH₂-PKM-COOH in DMF, DIEA was added to adjust the pH to 8.5-9.0, the mixture was stirred overnight at room temperature, and the crude product was passed through YMC-Pack ODS-A C₁₈Separating and purifying by using a semi-preparative column HPLC, collecting fractions of a target substance, combining collected liquids and freeze-drying to obtain an expected product HYNIC-PKM-COOH;

b. preparation of HYNIC-PKM-OSu

Dissolving HYNIC-PKM-COOH in DMF, adding NHS and EDC & HCl, stirring at room temperature for 5-10 hr, adding 50% ACN aqueous solution to the reaction solution, filtering, and passing the filtrate through YMC-Pack ODS-AC₁₈Separating and purifying by using a semi-preparative column HPLC, collecting fractions of a target substance, combining collected liquid and freeze-drying to obtain an expected product HYNIC-PKM-OSu;

c、(GGG-Dar)₂preparation of-Glu

GGG-Dar and OSu₂Dissolving Glu-Boc in DMF, adding DIEA to adjust pH to 8.5-9.0, stirring at room temperature overnight, and subjecting the crude product to YMC-Pack ODS-A C₁₈Separating and purifying by semi-preparative column HPLC, collecting target fractions, combining the collected liquids, and lyophilizing to obtain the desired product (GGG-Dar)₂-Glu-Boc; freeze-dried product (GGG-Dar)₂Dissolving the-Glu-Boc in 1ml of TfTFA, reacting at room temperature for 5min, blowing the reaction solution with nitrogen to obtain the expected product (GGG-Dar)₂-Glu；

d、HYNIC-PKM-(GGG-Dar)₂Preparation of

Will (GGG-Dar)₂dissolving-Glu and HYNIC-PKM-OSu in DMF, adding DIEA to adjust pH to 8.5-9.0, stirring at room temperature overnight, and subjecting the crude product to YMC-Pack ODS-A C₁₈Separating and purifying by semi-preparative column HPLC, collecting target fraction, mixing the collected solutions, and lyophilizing to obtain the desired product HYNIC-PKM- (GGG-Dar)₂；

e、^99mTc-HYNIC-PKM-(GGG-Dar)₂Preparation of

Preparing a mixture containing triphenylphosphine sodium trisulfonate (TPPTS), tricine, disodium succinate, succinic acid and HYNIC-PKM- (GGG-Dar)₂500. mu.L of the mixture was put in a 10mL vial, and the mixture was lyophilized. Adding 1.0-1.5ml of LNa into the lyophilized powder^99mTcO₄Heating the solution in water bath at 100 deg.C for 20-25 min, cooling at room temperature for 10 min after the reaction is finished, and making into Dar2 polypeptide^99mTc radiopharmaceuticals. Analyzed by HPLC for further use.

Further, triphenylphosphine sodium trisulfonate, trihydroxymethyl glycine, disodium succinate, succinic acid and HYNIC-PKM- (GGG-Dar) in the step e₂The mixed solution of (1) comprises the following substances in parts by mass:

a method for preparing a Dar2 polypeptide radiopharmaceutical,⁶⁸ga labeling Dar polypeptide dimers not comprising a pharmacokinetic linker with a bifunctional chelating agent NOTA or DOTA to form said Dar2 polypeptide radiopharmaceutical, said method comprising the steps of:

A、(GGG-Dar)₂preparation of Glu:

B、NOTA(DOTA)-(GGG-Dar)₂The preparation of (1):

will (GGG-Dar)₂Dissolving Glu and NOTA-CNS in DMF, adjusting pH to 8.5-9.0 with DIEA, stirring at room temperature overnight, and subjecting the crude product to YMC-Pack ODS-A C₁₈Semi-preparationSeparating and purifying by column HPLC, collecting fractions of the target substance, combining the collected liquid and freeze-drying to obtain the expected product NOTA (DOTA) - (GGG-Dar)₂；

C、⁶⁸Ga-NOTA(DOTA)-(GGG-Dar)₂The preparation of (1):

fresh shower from germanium-gallium generator⁶⁸GaCl₃With 2.5M NH₄The pH was adjusted to 3.5 by OAc. Adding 50. mu.g of NOTA) - (GGG-Dar)₂Heating in water bath at 99 deg.C for 20 min, cooling at room temperature for 10 min after reaction to obtain Dar polypeptide⁶⁸Ga-radiopharmaceuticals. Analyzed by HPLC for further use.

The HPLC method was carried out using an Agilent 1260 HPLC system equipped with YMC-Pack ODS-A C18 semi-preparative columns (250X 10mm, I.D.S. -5 μm, 12nm) or analytical columns (250X 4.6mm, I.D.S. -5 μm, 12nm), gradient elution for 30 or 20 minutes, with mobile phase A being deionized water (containing 0.05% TFA) and mobile phase B being acetonitrile (containing 0.05% TFA). Step a: the semi-preparative column was equipped with a flow rate of 2.5mL/min and elution gradients set to 80% A and 20% B initially, 60% A and 40% B at 25 minutes, and 80% A and 20% B at 30 minutes. Step b: the analytical column was equipped with a flow rate of 1mL/min and elution gradients of 90% A and 10% B initially, 60% A and 40% B at 17.5 min and 90% A and 10% B at 20 min.

The Dar2 polypeptide radiopharmaceutical is used for imaging diagnosis of integrin alpha 6 positive tumor patients.

The optimized Dar polypeptide drug (anedywr) and the full-reverse D-type polypeptide are synthesized in the experiment, the polypeptide synthesis process is simplified, and the drug kit is easier to realize. Meanwhile, the D-type polypeptide sequence cannot be identified by in vivo protease, and the in vivo metabolic stability can be effectively improved, so that the uptake of tumor tissues is improved. In addition, the Dar polypeptide dimer introduces a linker between the two polypeptides that is long enough that the two polypeptides in the dimer molecule are sufficiently distant to simultaneously bind two integrins α₆Target site, higher integrin alpha than monomer₆Binding force, and enhancing the receptor-ligand affinity of the polypeptide to achieve higher tumor uptake. The pharmacokinetic modifying molecule PKM is added between the bifunctional chelating agent HYNIC or DOTA for labeling radionuclide and Dar dimer polypeptideAnd the pharmacokinetic property is optimized to achieve better tumor diagnosis effect.

The invention has the beneficial effects that:

1. the Dar2 polypeptide radiopharmaceutical has a totally-inverted D-type polypeptide sequence which can not be identified by in-vivo protease, and can effectively improve in-vivo metabolic stability, thereby improving the uptake of tumor tissues.

2. The Dar2 polypeptide radiopharmaceutical of the present invention is prepared by first linking three glycine molecules (GGG) to Dar polypeptide monomers, and then dimerizing the GGG to allow two polypeptides in a dimeric molecule to have a distance long enough to simultaneously bind two integrins alpha₆The combination of the bivalent form can further enhance the medicine intake of the tumor, and achieve better diagnosis effect.

3. The invention relates to a bifunctional chelating agent HYNIC and integrin alpha for radionuclide labeling₆The pharmacokinetic modification molecule PKM, namely HYNIC-PKM- (GGG-Dar), is introduced between the targeted Dar2 polypeptides₂The biocompatibility of the probe is improved, and the pharmacokinetic properties, in particular the clearance kinetics from non-tumor tissues, are optimized.

4. In the present invention, HYNIC is used as a bifunctional chelating agent, and TPPTS and tricine are used as synergistic ligands, so that "^99mTc-HYNIC nucleus has better in-vivo and in-vitro stability.

5. The Dar2 polypeptide radiopharmaceutical of the invention is a brand new integrin alpha₆The targeted molecular imaging probe can be applied to various integrins alpha₆The nuclear medicine molecular imaging of the high expression tumor realizes the early diagnosis and screening of the tumor.

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

Drawings

FIG. 1 (A) Dar polypeptide monomer, (B) Dar2 polypeptide (i.e. Dar polypeptide dimer), (C) HYNIC-PEG₄-(GGG-Dar)₂，(D)HYNIC-Aoc-(GGG-Dar)₂，(E)NOTA-(GGG-Dar)₂，(F)DOTA-(GGG-Dar)₂Schematic structural diagram of (1).

FIG. 2 is a fluorescent staining pattern of Cy5.5-Dar2 on HepG2 cells and tumor tissue sections.

FIG. 3 (A) Biotin-Dar2 and human integrin alpha₆β₄A binding affinity assay profile for the protein; (B) dar and Dar2 polypeptides were plotted in vitro for competitive binding.

Fig. 4.^99mTc-HYNIC-PKM-(GGG-Dar)₂Schematic structure of the tag.

FIG. 5 (A)^99mTc-HYNIC-PEG₄-(GGG-Dar)₂And (B)^99mTc-HYNIC-PEG₄-(GGG-cKiE)₂Metabolic stability profile in a normal ICR mouse model.

Fig. 6.^99mTc-HYNIC-PEG₄-(GGG-Dar)₂And^99mTc-HYNIC-PEG₄-(GGG-cKiE)₂blood clearance rate in a normal ICR mouse model is plotted against. (dotted circles indicate tumor sites)

FIG. 7 injection in HepG2 hepatocellular carcinoma model^99mTc-HYNIC-PEG₄-(GGG-Dar)₂(A) SPECT/CT visualizations after 0.5, 1 and 2 h; (B) SPECT/CT visualization of cold peptide blocking group at 0.5 h; (C) injection in HepG2 hepatocellular carcinoma model^99mTc-HYNIC-PEG₄-(GGG-cKiE)₂SPECT/CT visualization after 0.5 h.

FIG. 8 (A) injection^99mTc-HYNIC-PEG₄-(GGG-Dar)₂After 0.5, 1 and 2h, the results were distributed in vivo in the HepG2 tumor model; (B) injection of drugs^99mTc-HYNIC-PEG₄-(GGG-Dar)₂After 0.5h, the in vivo distribution results in HepG2 tumor model and HepG2 tumor model blocking experiments were compared; (C) injection of drugs^99mTc-HYNIC-PEG₄-(GGG-Dar)₂And^99mTc-HYNIC-PEG₄-(GGG-cKiE)₂after 0.5h, the results of in vivo distribution in the HepG2 tumor model were compared.

FIG. 9 (A) injection in HepG2 liver carcinoma in situ model^99mTc-HYNIC-PEG₄-(GGG-Dar)₂SPECT/CT visualization after 0.5 h; (B) ex vivo SPECT/CT visualization after dissection. (white arrows indicate tumor sites)

Detailed Description

The materials used in the examples of the present invention:

1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl, 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), N-hydroxysuccinimide (NHS, N-hydroxysuccinimide), succinic acid (succinic acid), sodium succinate (disodium succinate), trisodium triphenylphosphine-3,3' -trisufonate (TPPTS, sodium triphenylphospine), N, N-dimethylformide (DMF, N, N-dimethylformamide), tricine (trihydroxymethylglycine) were all available from Sigma-Aldrich, USA. HYNIC-NHS (hydrazinium nicotinamide), NOTA-CNS, DOTA-NHS were purchased from Noca-biochem, USA. GGG-anedywr (GGG-Dar) polypeptide monomers were purchased from Gell Biochemical, China. Na (Na)^99mTcO₄The eluent was purchased from Beijing atomic Kogaku corporation.

Example 1:

this example illustrates the in vitro specificity and affinity determination of Dar2 polypeptide (i.e., Dar polypeptide dimer).

Fluorescent staining of Cy5.5-Dar2 in HepG2 cells and tumor tissue sections: fluorescein Cy5.5 is coupled with Dar2 polypeptide to synthesize Cy5.5-Dar 2.Cy5.5-Dar2 were incubated with HepG2 cells and tumor tissue sections, respectively, and significant fluorescence signals were observed on cell membranes of HepG2 cells and tumor tissue sections, and were significantly decreased in blocking experiments (FIG. 2), indicating that Dar2 polypeptide was able to specifically interact with integrin alpha₆And (4) combining.

In vitro affinity assay for Dar2 polypeptide: biotin was coupled to Dar2 polypeptide to synthesize Biotin-Dar 2. Biotin-Dar2 with different concentrations and human integrin alpha₆β₄Protein binding was determined to be Biotin-Dar2 and human integrin alpha₆β₄Binding affinity K of proteins_dThe value was 294.8+42.50nM (FIG. 3A). Competitive binding curve results show the IC of Dar2 and Dar polypeptide₅₀The values were 231.3+1.43nM, 1.3+ 0.01. mu.M, respectively (FIG. 3B), indicating that the polypeptide dimerizes such that it binds to integrin α₆The binding affinity of (a) is significantly higher than that of the monomeric polypeptide.

Example 2:

this embodiment is as follows^99mTc-HYNIC-PEG₄-(GGG-Dar)₂Polypeptide radiopharmaceuticals and methods of making the same are exemplified.

^99mTc-HYNIC-PEG₄-(GGG-Dar)₂In the preparation method, the Dar polypeptide monomer is D-type polypeptide anedywr, the Dar polypeptide dimer is formed by connecting a connecting agent GGG with the Dar polypeptide monomer and dimerizing two Dar polypeptide monomers connected with the GGG, and the radionuclide^99mTc is marked with the Dar polypeptide dimer through a bifunctional chelating agent HYNIC, and a pharmacokinetic modifying molecule PEG is connected between the Dar polypeptide dimer and the bifunctional chelating agent HYNIC₄The Dar2 polypeptide radiopharmaceutical is^99mTc-HYNIC-PEG₄-(GGG-Dar)₂The Dar2 polypeptide radiopharmaceutical is a colorless transparent liquid injection.

^99mTc-HYNIC-PEG₄-(GGG-Dar)₂The preparation method comprises the following steps:

HYNIC-PEG₄preparation of-COOH: fmoc protected PEG₄-COOH in DMF, piperidine to a final concentration of 20%, reaction at room temperature for 20 minutes, and PEG by addition of 10mL of diethyl ether at 4 deg.C₄-COOH precipitate, centrifuging at 4000rpm for 5min at 4 deg.C, discarding supernatant, washing precipitate with diethyl ether at 4 deg.C for 3 times, and removing residual diethyl ether by rotary evaporation to obtain NH₂-PEG₄-COOH; HYNIC-NHS and NH₂-PEG₄-COOH was dissolved in DMF, DIEA was added to adjust the pH to 8.5-9.0, the mixture was stirred overnight at room temperature, and the crude product was purified by HPLC on YMC-Pack ODS-A semi-preparative column. HPLC method YMC-Pack ODS-A C was prepared using Agilent 1260 HPLC System₁₈Semi-preparative columns (250X 10mm, I.D.S. -5 μm, 12nm), gradient elution for 30 min, flow rate 2.5mL/min, mobile phase A being deionized water (with 0.05% TFA) and mobile phase B being acetonitrile (with 0.05% TFA). The elution gradient was set to 80% a and 20% B at the start, 60% a and 40% B at 25 min, and 80% a and 20% B at 30 min. Collecting target fractions, combining the collected solutions, lyophilizing, and analyzing by MALDI-TOF-MS mass spectrometry to obtain product with M/z of 568.60([ M + H ]]⁺) Confirming to be the expected product HYNIC-PEG₄-COOH。

HYNIC-PEG₄Preparation of OSu: mixing HYNIC-PEG₄-COOH was dissolved in DMF, NHS and EDC. HCl were added, the mixture was stirred at room temperature for 7 hours, an aqueous solution of ACN at A volume fraction of 50% was added to the reaction mixture and filtered, and the filtrate was subjected to HPLC separation and purification using YMC-Pack ODS-A semi-preparative column. HPLC method YMC-Pack ODS-A C was prepared using Agilent 1260 HPLC System₁₈Semi-preparative columns (250X 10mm, I.D.S. -5 μm, 12nm), gradient elution for 30 min, flow rate 2.5mL/min, mobile phase A being deionized water (with 0.05% TFA) and mobile phase B being acetonitrile (with 0.05% TFA). The elution gradient was set to 80% a and 20% B at the start, 60% a and 40% B at 25 min, and 80% a and 20% B at 30 min. Collecting target fractions, combining the collected solutions, lyophilizing, and analyzing by MALDI-TOF-MS mass spectrometry to obtain product with M/z of 665.67([ M + H ]]⁺) Confirming to be the expected product HYNIC-PEG₄-OSu。

(GGG-Dar)₂Preparation of Glu: GGG-Dar and OSu₂Dissolving Glu-Boc in DMF, adding DIEA to adjust pH to 8.5-9.0, stirring at room temperature overnight, and separating and purifying the crude product by YMC-Pack ODS-A semi-preparative column HPLC. HPLC method YMC-Pack ODS-A C was prepared using Agilent 1260 HPLC System₁₈Semi-preparative columns (250X 10mmL. D.S. -5 μm, 12nm), gradient elution for 30 min, flow rate 2.5mL/min, mobile phase A being deionized water (with 0.05% TFA) and mobile phase B being acetonitrile (with 0.05% TFA). The elution gradient was set to 80% a and 20% B at the start, 60% a and 40% B at 25 min, and 80% a and 20% B at 30 min. Collecting target fractions, combining the collected solutions, lyophilizing, and analyzing by MALDI-TOF-MS mass spectrometry to obtain product with M/z of 2459.49([ M + H ]]⁺) Confirmed as the expected product (GGG-Dar)₂-Glu-Boc; freeze-dried product (GGG-Dar)₂Dissolving the (E) -Glu-Boc in 1mL of TFA, reacting at room temperature for 5min, blowing the reaction solution with nitrogen, and analyzing the M/z-2359.37 ([ M + H ] by MALDI-TOF-MS mass spectrometry]⁺) Confirmed as the expected product (GGG-Dar)₂Glu (FIG. 1B).

HYNIC-PEG₄-(GGG-Dar)₂The preparation of (1): will (GGG-Dar)₂-Glu and HYNIC-PEG₄Dissolving OSu in DMF, adding DIEA to adjust pH to 8.5-9.0, stirring at room temperature overnight, and separating and purifying the crude product by HPLC with YMC-Pack ODS-A semi-preparative columnEquipping with YMC-Pack ODS-A C for use with Agilent 1260 HPLC system₁₈Semi-preparative columns (250X 10mm, I.D.S. -5 μm, 12nm), gradient elution for 30 min, flow rate 2.5mL/min, mobile phase A being deionized water (with 0.05% TFA) and mobile phase B being acetonitrile (with 0.05% TFA). The elution gradient was set to 80% a and 20% B at the start, 60% a and 40% B at 25 min, and 80% a and 20% B at 30 min. Collecting target fractions, combining the collected solutions, lyophilizing, and analyzing by MALDI-TOF-MS mass spectrometry to obtain product with M/z of 2909.96([ M + H ]]⁺) Confirming to be the expected product HYNIC-PEG₄-(GGG-Dar)₂(FIG. 1C).

^99mTc-HYNIC-PEG₄-(GGG-Dar)₂The preparation of (1): 5.0mg of triphenylphosphine sodium trisulfonate (TPPTS), 6.5mg of trihydroxymethyl glycine (tricine), 38.5mg of disodium succinate, 12.7mg of succinic acid and 50 mu g of HYNIC-PEG are prepared₄-(GGG-Dar)₂500. mu.L of the mixture was put in a 10mL vial, and the mixture was lyophilized. Adding 1.0-1.5mL of Na into the lyophilized powder^99mTcO₄Heating the solution (10-35mCi) in a water bath at 100 ℃ to react for 20-25 minutes, and cooling at room temperature for 10 minutes after the reaction is finished to prepare the Dar2 polypeptide radiopharmaceutical.

Samples of Dar2 polypeptide radiopharmaceutical were subjected to radioactive HPLC analysis. HPLC method YMC-Pack ODS-A C was prepared using Agilent 1260 HPLC System₁₈Analytical column (250X 4.6mm, I.D.S. -5 μm, 12nm), gradient elution for 20 min, flow rate 1mL/min, mobile phase A being deionized water (with 0.05% TFA) and mobile phase B being acetonitrile (with 0.05% TFA). The elution gradient was set to 90% a and 10% B at the start, 60% a and 40% B at 17.5 min, and 90% a and 10% B at 20 min.^99mTc-HYNIC-PEG₄-(GGG-Dar)₂Marking rate of>95% radiochemical purity>98% (fig. 5A).

^99mTc-HYNIC-PEG₄-(GGG-Dar)₂And^99mTc-HYNIC-PEG₄-(GGG-cKiE)₂in vivo metabolic stability in a normal ICR mouse model: 2 ICR mice were selected, and one mouse was injected via tail vein with 100 μ L (. about.37 MBq)^99mTc-HYNIC-PEG₄-(GGG-Dar)₂And the other mouse is through the tailIntravenous injection 100 μ L (37 MBq)^99mTc-HYNIC-PEG₄-(GGG-cKiE)₂At 2 hours post-injection, mouse urine was collected and samples of urine were subjected to radioactive HPLC analysis. HPLC method YMC-Pack ODS-A C was prepared using Agilent 1260 HPLC System₁₈Analytical column (250X 4.6mm, I.D.S. -5 μm, 12nm), gradient elution for 20 min, flow rate 1mL/min, mobile phase A being deionized water (with 0.05% TFA) and mobile phase B being acetonitrile (with 0.05% TFA). The elution gradient was set to 90% a and 10% B at the start, 60% a and 40% B at 17.5 min, and 90% a and 10% B at 20 min. The experimental results show that^99mTc-HYNIC-PEG₄-(GGG-Dar)₂Good stability in mice, the drug retained the prototype form in vivo without degradation, which was more favorable for drug tumor uptake (fig. 5A). While^99mTc-HYNIC-PEG₄-(GGG-cKiE)₂Will be partially degraded in mice (fig. 5B).

^99mTc-HYNIC-PEG₄-(GGG-Dar)₂And^99mTc-HYNIC-PEG₄-(GGG-cKiE)₂blood clearance rate in normal ICR mouse model: ICR mice were divided into 2 groups of 5 mice each. One group of mice was injected via tail vein with 100 μ L (-101 kBq)^99mTc-HYNIC-PEG₄-(GGG-Dar)₂Another group of mice was injected via tail vein with 100 μ L (. about.101 kBq)^99mTc-HYNIC-PEG₄-(GGG-cKiE)₂Blood was taken at 1, 5, 15, 30, 60, 90, 120, 150 and 180 minutes post-injection, weighed and radioactivity counts were measured, and the percent dose rate per gram of tissue (% ID/g) was calculated after decay correction. Experimental results show that both drugs are rapidly cleared from the blood,^99mTc-HYNIC-PEG₄-(GGG-Dar)₂has a blood half-life which is higher than that reported before^99mTc-HYNIC-PEG₄-(GGG-cKiE)₂Slightly longer, more favorable for SPECT imaging (fig. 6).

^99mTc-HYNIC-PEG₄-(GGG-Dar)₂SPECT/CT imaging in tumor-bearing mice: in the HepG2 hepatocellular carcinoma tumor model,^99mTc-HYNIC-PEG₄-(GGG-Dar)₂the tumor uptake was clearly visible except for the kidney which was higherBesides the uptake, the background of other organs is low, and the retention time of the polypeptide at the tumor part is longer, which is more beneficial to the diagnosis of the tumor (fig. 7A). In the blocking group, tumor uptake was significantly reduced (FIG. 7B), indicating that the polypeptide and integrin α are present₆Specific binding of the site. In addition to this, the present invention is,^99mTc-HYNIC-PEG₄-(GGG-cKiE)₂SPECT/CT imaging in tumor-bearing mice is shown in FIG. 7C,^99mTc-HYNIC-PEG₄-(GGG-cKiE)₂there is also uptake in the tumor, but too high uptake in the kidney results in reduced contrast of the image, making it impossible to clearly image the tumor.

^99mTc-HYNIC-PEG₄-(GGG-Dar)₂Biodistribution in tumor-bearing mice: BALB/c Nude mouse lotus HepG2 hepatocellular carcinoma tumors were divided into 4 groups of 4 tumors each. Three groups of mice were injected via tail vein with 100 μ L (-74 kBq)^99mTc-HYNIC-PEG₄-(GGG-Dar)₂Sacrificed at 0.5, 1 and 2 hours post injection; another group of mice were co-injected via tail vein with 100 μ L (. about.74 kBq)^99mTc-HYNIC-PEG₄-(GGG-Dar)₂And 500 μ g Dar polypeptide, sacrificed at 0.5 hours post injection; blood and major organs were taken, weighed and radioactivity counts measured, and the percent injection dose rate per gram of tissue (% ID/g) was calculated after decay correction. Experimental results confirmed the imaging results and showed the distribution of the probes in various tissues and organs (fig. 8A, 8B); in addition, with^99mTc-HYNIC-PEG₄-(GGG-cKiE)₂Compared with the biological distribution in tumor-bearing mice,^99mTc-HYNIC-PEG₄-(GGG-Dar)₂uptake in tumors is similar to that, but^99mTc-HYNIC-PEG₄-(GGG-Dar)₂The renal uptake of the probe was significantly reduced, which resulted in higher contrast imaging of the probe and better tumor imaging (fig. 8C).

^99mTc-HYNIC-PEG₄-(GGG-Dar)₂SPECT/CT imaging in HepG2 liver in situ tumor model: in the HepG2 liver in situ tumor model,^99mTc-HYNIC-PEG₄-(GGG-Dar)₂it also has high tumor uptake (FIG. 9A), and can be used for SPECT imaging of liver carcinoma in situ. SPECT/CT imaging knot for each organ in vitroThe results showed that there was a significant signal for radioactivity uptake by the tumor in the liver (fig. 9B), consistent with in vivo imaging.

Example 3:

this embodiment is as follows^99mTc-HYNIC-Aoc-(GGG-Dar)₂Polypeptide radiopharmaceuticals and methods of making the same are exemplified.

^99mTc-HYNIC-Aoc-(GGG-Dar)₂In the preparation method, the Dar polypeptide monomer is D-type polypeptide anedywr, the Dar polypeptide dimer is formed by connecting a connecting agent GGG with the Dar polypeptide monomer and dimerizing two Dar polypeptide monomers connected with the GGG, and the radionuclide^99mTc is marked with the Dar polypeptide dimer through a bifunctional chelating agent HYNIC, a pharmacokinetic modifying molecule Aoc is connected between the Dar polypeptide dimer and the bifunctional chelating agent, and the Dar2 polypeptide radiopharmaceutical is^99mTc-HYNIC-Aoc-(GGG-Dar)₂The Dar2 polypeptide radiopharmaceutical is a colorless transparent liquid injection.

^99mTc-HYNIC-Aoc-(GGG-Dar)₂The preparation method comprises the following steps:

preparation of HYNIC-Aoc-COOH: dissolving Fmoc-protected Aoc-COOH in DMF, adding piperidine to a final concentration of 20%, reacting at room temperature for 20 minutes, adding 10mL of diethyl ether at 4 ℃ to precipitate Aoc-COOH, centrifuging at 4000rpm at 4 ℃ for 5 minutes, discarding the supernatant, washing the precipitate with diethyl ether at 4 ℃ for 3 times, and performing rotary evaporation to remove the residual diethyl ether to obtain a product, namely NH₂-Aoc-COOH; HYNIC-NHS and NH₂-Aoc-COOH in DMF, DIEA was added to adjust the pH to 8.5-9.0, the mixture was stirred overnight at room temperature, and the crude product was passed through YMC-Pack ODS-A C₁₈And (5) separating and purifying by using semi-preparative column HPLC. HPLC method YMC-Pack ODS-A C was prepared using Agilent 1260 HPLC System₁₈Semi-preparative columns (250X 10mm, I.D.S. -5 μm, 12nm), gradient elution for 30 min, flow rate 2.5mL/min, mobile phase A being deionized water (with 0.05% TFA) and mobile phase B being acetonitrile (with 0.05% TFA). The elution gradient was set to 80% a and 20% B at the start, 60% a and 40% B at 25 min, and 80% a and 20% B at 30 min. Collecting target fractions, combining the collected solutions, lyophilizing, and analyzing by MALDI-TOF-MS mass spectrometry to obtain product with M/z of 462.52([ M + H ]]⁺) Confirmed as the expected product HYNIC-Aoc-COOH。

Preparation of HYNIC-Aoc-OSu: dissolving HYNIC-Aoc-COOH in DMF, adding NHS and EDC & HCl, stirring at room temperature for 7 hr, adding 50% ACN aqueous solution to the reaction solution, filtering, and passing the filtrate through YMC-Pack ODS-A C₁₈And (5) separating and purifying by using semi-preparative column HPLC. HPLC method YMC-Pack ODS-A C was prepared using Agilent 1260 HPLC System₁₈Semi-preparative columns (250X 10mm, I.D.S. -5 μm, 12nm), gradient elution for 30 min, flow rate 2.5mL/min, mobile phase A being deionized water (with 0.05% TFA) and mobile phase B being acetonitrile (with 0.05% TFA). The elution gradient was set to 80% a and 20% B at the start, 60% a and 40% B at 25 min, and 80% a and 20% B at 30 min. Collecting target fractions, combining the collected solutions, lyophilizing, and analyzing by MALDI-TOF-MS mass spectrometry to obtain product with M/z of 559.59([ M + H ]]⁺) The product was identified as HYNIC-Aoc-OSu.

(GGG-Dar)₂Preparation of Glu: the preparation method is the same as above.

HYNIC-Aoc-(GGG-Dar)₂The preparation of (1): will (GGG-Dar)₂Dissolving Glu and HYNIC-Aoc-OSu in DMF, adding DIEA to adjust pH to 8.5-9.0, stirring at room temperature overnight, separating and purifying the crude product by HPLC using YMC-Pack ODS-A semi-preparative column equipped with YMC-Pack ODS-A C HPLC system using Agilent 1260 HPLC₁₈Semi-preparative columns (250X 10mm, I.D.S. -5 μm, 12nm), gradient elution for 30 min, flow rate 2.5mL/min, mobile phase A being deionized water (with 0.05% TFA) and mobile phase B being acetonitrile (with 0.05% TFA). The elution gradient was set to 80% a and 20% B at the start, 60% a and 40% B at 25 min, and 80% a and 20% B at 30 min. Collecting target fractions, combining the collected solutions, lyophilizing, and analyzing by MALDI-TOF-MS mass spectrometry to obtain product with M/z of 2803.88([ M + H ]]⁺) And confirmed to be the expected product HYNIC-Aoc- (GGG-Dar)₂(FIG. 1D).

^99mTc-HYNIC-Aoc-(GGG-Dar)₂The preparation of (1): 5.0mg of triphenylphosphine trisulfonate (TPPTS), 6.5mg of tricine, 38.5mg of disodium succinate, 12.7mg of succinic acid and 50 mu g of HYNIC-Aoc- (GGG-Dar) are prepared₂500. mu.L of the mixture was put in a 10mL vial, and the mixture was lyophilized. Adding 1.0-1 part of the lyophilized powder5mL of Na^99mTcO₄Heating the solution (10-35mCi) in a water bath at 100 ℃ to react for 20-25 minutes, and cooling at room temperature for 10 minutes after the reaction is finished to prepare the Dar2 polypeptide radiopharmaceutical.

Samples of Dar2 polypeptide radiopharmaceutical were subjected to radioactive HPLC analysis. HPLC method YMC-Pack ODS-A C was prepared using Agilent 1260 HPLC System₁₈Analytical column (250X 4.6mm, I.D.S. -5 μm, 12nm), gradient elution for 20 min, flow rate 1mL/min, mobile phase A being deionized water (with 0.05% TFA) and mobile phase B being acetonitrile (with 0.05% TFA). The elution gradient was set to 90% a and 10% B at the start, 60% a and 40% B at 17.5 min, and 90% a and 10% B at 20 min.^99mTc-HYNIC-Aoc-(GGG-Dar)₂Marking rate of>95% radiochemical purity>98％。

^99mTc-HYNIC-Aoc-(GGG-Dar)₂SPECT/CT imaging in tumor-bearing mice: in the HepG2 hepatocellular carcinoma tumor model,^99mTc-HYNIC-Aoc-(GGG-Dar)₂the uptake of tumors is clearly visible, but there is significant uptake by the gallbladder and liver. In the blocking experimental group, tumor uptake was significantly reduced, indicating that polypeptide and integrin α₆Specific binding of the site.

Example 4:

this embodiment is as follows⁶⁸Ga-NOTA-(GGG-Dar)₂Polypeptide radiopharmaceuticals and methods of making the same are exemplified.

⁶⁸Ga-NOTA-(GGG-Dar)₂The Dar polypeptide is D-type polypeptide anedywr, the Dar polypeptide dimer is formed by connecting a connecting agent GGG with a Dar polypeptide monomer and dimerizing two Dar polypeptide monomers connected with the GGG, and the radionuclide⁶⁸Ga labels the Dar polypeptide dimer through a chelating agent NOTA, and the Dar2 polypeptide radiopharmaceutical is⁶⁸Ga-NOTA-(GGG-Dar)₂The Dar2 polypeptide radiopharmaceutical is a colorless transparent liquid injection.

⁶⁸Ga-NOTA-(GGG-Dar)₂The preparation method comprises the following steps:

(GGG-Dar)₂preparation of Glu: the preparation method is the same as above.

NOTA-(GGG-Dar)₂The preparation of (1): will (GGG-Dar)₂Dissolving Glu and NOTA-CNS in DMF, adjusting pH to 8.5-9.0 with DIEA, stirring at room temperature overnight, and subjecting the crude product to YMC-Pack ODS-A C₁₈Semi-preparative column HPLC separation and purification is carried out by preparing YMC-Pack ODS-A C using Agilent 1260 HPLC system₁₈Semi-preparative columns (250X 10mm, I.D.S. -5 μm, 12nm), gradient elution for 30 min, flow rate 2.5mL/min, mobile phase A being deionized water (with 0.05% TFA) and mobile phase B being acetonitrile (with 0.05% TFA). The elution gradient was set to 80% a and 20% B at the start, 60% a and 40% B at 25 min, and 80% a and 20% B at 30 min. Collecting target fractions, combining the collected solutions, lyophilizing, and analyzing by MALDI-TOF-MS mass spectrometry to obtain product with M/z of 2809.88([ M + H ]]⁺) Confirmed to be the expected product NOTA- (GGG-Dar)₂(FIG. 1E).

⁶⁸Ga-NOTA-(GGG-Dar)₂The preparation of (1): elution from the Ge-Ga generator with 0.05M HCl one fraction per 1mL⁶⁸GaCl₃5mL of the solution is rinsed. Selecting 1-2mL⁶⁸GaCl₃With 2.5M NH₄The pH was adjusted to 3.5 by OAc. Add 50. mu.g DOTA-PEG₄-(GGG-Dar)₂Heating in water bath at 99 deg.c for 20 min, and cooling at room temperature for 5min to prepare the Dar polypeptide radioactive medicine.

Samples of Dar2 polypeptide radiopharmaceutical were subjected to radioactive HPLC analysis. HPLC method YMC-Pack ODS-A C was prepared using Agilent 1260 HPLC System₁₈Analytical column (250X 4.6mm, I.D.S. -5 μm, 12nm), gradient elution for 20 min, flow rate 1mL/min, mobile phase A being deionized water (with 0.05% TFA) and mobile phase B being acetonitrile (with 0.05% TFA). The elution gradient was set to 90% a and 10% B at the start, 60% a and 40% B at 17.5 min, and 90% a and 10% B at 20 min.⁶⁸Ga-NOTA-(GGG-Dar)₂Marking rate of>95% radiochemical purity>98％。

⁶⁸Ga-NOTA-(GGG-Dar)₂PET/CT imaging in tumor-bearing mice: in the HepG2 hepatocellular carcinoma tumor model,⁶⁸Ga-NOTA-(GGG-Dar)₂the tumor uptake is clearly visible, except for the kidney, the background of other organs is low, and probing is performedThe residence time of the needle at the tumor part is long, which is beneficial to the diagnosis of the tumor. In the blocking group, tumor uptake was significantly reduced, indicating that the probe and integrin α were present₆Specific binding of the site.⁶⁸Ga-NOTA-(GGG-Dar)₂The liver cancer imaging kit also has higher tumor uptake in a HepG2 liver in-situ tumor model, and can carry out PET imaging on liver in-situ cancer.

Claims

1. A Dar2 polypeptide radiopharmaceutical, which is characterized in that: comprises a Dar polypeptide dimer and a radionuclide, wherein the radionuclide labels the Dar polypeptide dimer through a bifunctional chelating agent; the Dar polypeptide dimer is synthesized by connecting GGG (GGGGG) with Dar polypeptide monomers and dimerizing two Dar polypeptide monomers connected with GGG, wherein the GGG refers to three continuous glycine molecules; the Dar polypeptide monomer is a full-reverse D-type amino acid linear 7-membered polypeptide, and the sequence is as follows: anedywr; the radionuclide is^99mTc、⁶⁸Ga、⁶⁴Cu、¹¹¹In、⁹⁰Y and¹⁷⁷lu, wherein the bifunctional chelating agent is any one of HYNIC, NOTA, DOTA and DTPA.

2. The Dar2 polypeptide radiopharmaceutical of claim 1, wherein: and a pharmacokinetic linking agent is connected between the Dar polypeptide dimer and the bifunctional chelating agent, and the pharmacokinetic linking agent is a polyethylene glycol molecule or 8-aminocaprylic acid.

3. The Dar2 polypeptide radiopharmaceutical of claim 1, wherein: the radionuclide is^99mTc，^99mTc is marked by bifunctional chelating agent HYNIC to be connected with Dar polypeptide dimer of pharmacokinetic linker, and the pharmacokinetic linker is polyethylene glycol molecule or 8-amino caprylic acid.

4. The Dar2 polypeptide radiopharmaceutical of claim 3, which is characterized in that: the polymerization degree of the polyethylene glycol molecule is 4.

5. The Dar2 polypeptide radiopharmaceutical of claim 1, wherein: the radionuclide is⁶⁸Ga，⁶⁸Ga labels Dar polypeptide dimers containing no pharmacokinetic linkers by the bifunctional chelating agent NOTA or DOTA.

6. The method for preparing a Dar2 polypeptide radiopharmaceutical of claim 1,^99mtc is marked by bifunctional chelating agent HYNIC to form the Dar2 polypeptide radiopharmaceutical with Dar polypeptide dimer linked with pharmacokinetic linker, and is characterized in that: the method comprises the following steps:

a. preparing HYNIC-PKM-COOH:

b. preparation of HYNIC-PKM-OSu:

dissolving HYNIC-PKM-COOH in DMF, adding NHS and EDC & HCl, stirring at room temperature for 5-10 hr, adding 50% ACN aqueous solution to the reaction solution, filtering, and passing the filtrate through YMC-Pack ODS-A C₁₈Separating and purifying by using a semi-preparative column HPLC, collecting fractions of a target substance, combining collected liquid and freeze-drying to obtain an expected product HYNIC-PKM-OSu;

c、(GGG-Dar)₂preparation of Glu:

GGG-Dar and OSu₂Dissolving Glu-Boc in DMF, adding DIEA to adjust pH to 8.5-9.0, stirring at room temperature overnight, and subjecting the crude product to YMC-Pack ODS-A C₁₈Separating and purifying by semi-preparative column HPLC, collecting target fractions, combining the collected liquids, and lyophilizing to obtain the desired product (GGG-Dar)₂-Glu-Boc; will freezeDry product (GGG-Dar)₂Dissolving the-Glu-Boc in TFA, reacting at room temperature for 5min, blowing the reaction solution with nitrogen to obtain the expected product (GGG-Dar)₂-Glu；

d、HYNIC-PKM-(GGG-Dar)₂The preparation of (1):

e、^99mTc-HYNIC-PKM-(GGG-Dar)₂The preparation of (1):

preparing the mixture containing triphenylphosphine sodium trisulfonate, trihydroxymethyl glycine, disodium succinate, succinic acid and HYNIC-PKM- (GGG-Dar)₂The mixed solution of (1), freeze-drying the mixed solution; adding Na into lyophilized powder^99mTcO₄Heating the solution in water bath at 100 ℃ for reaction for 20-25 minutes, and cooling at room temperature after the reaction is finished to prepare the Dar2 polypeptide radiopharmaceutical.

7. The method of preparing a Dar2 polypeptide radiopharmaceutical of claim 6, which comprises: the triphenylphosphine sodium trisulfonate, the trihydroxymethyl glycine, the succinic acid disodium, the succinic acid and the HYNIC-PKM- (GGG-Dar) in the step e₂The mixed solution of (1) comprises the following substances in parts by mass:

8. the method for preparing a Dar2 polypeptide radiopharmaceutical of claim 1,⁶⁸ga labels the Dar polypeptide dimer without pharmacokinetic linker through the bifunctional chelating agent NOTA to form the Dar2 polypeptide radiopharmaceutical, wherein: the method comprises the following steps:

A、(GGG-Dar)₂preparation of Glu:

GGG-Dar and OSu₂Dissolving Glu-Boc in DMF, adding DIEA to adjust pH to 8.5-9.0, stirring at room temperature overnight, and subjecting the crude product to YMC-Pack ODS-A C₁₈Separating and purifying by semi-preparative column HPLC, collecting target fractions, combining the collected liquids, and lyophilizing to obtain the desired product (GGG-Dar)₂-Glu-Boc; freeze-dried product (GGG-Dar)₂Dissolving the-Glu-Boc in TFA, reacting at room temperature for 5min, blowing the reaction solution with nitrogen to obtain the expected product (GGG-Dar)₂-Glu；

B、NOTA-(GGG-Dar)₂The preparation of (1):

will (GGG-Dar)₂Dissolving Glu and NOTA-CNS in DMF, adjusting pH to 8.5-9.0 with DIEA, stirring at room temperature overnight, and subjecting the crude product to YMC-Pack ODS-A C₁₈Separating and purifying by semi-preparative column HPLC, collecting target fractions, combining the collected liquids, and lyophilizing to obtain the desired product NOTA- (GGG-Dar)₂；

C、⁶⁸Ga-NOTA-(GGG-Dar)₂The preparation of (1):

fresh shower from germanium-gallium generator⁶⁸GaCl₃By NH₄Adjusting pH to 3.5 with OAc, adding NOTA- (GGG-Dar)₂And heating in water bath at 99 ℃, and cooling at room temperature after the reaction is finished to prepare the Dar2 polypeptide radiopharmaceutical.

9. The use of Dar2 polypeptide radiopharmaceutical of claim 1 wherein: the Dar2 polypeptide radiopharmaceutical can be used for preparing integrin alpha₆Positive tumor imaging diagnostic radiopharmaceuticals.

10. The use of Dar2 polypeptide radiopharmaceutical of claim 9, wherein: the integrin alpha₆Positive tumors include hepatocellular carcinoma, breast cancer, lung cancer, pancreatic cancer, colorectal cancer, and head and neck cancer.