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CN119143843B - A CXCR4 targeting polypeptide and its preparation method and application - Google Patents

A CXCR4 targeting polypeptide and its preparation method and application Download PDF

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CN119143843B
CN119143843B CN202411596680.8A CN202411596680A CN119143843B CN 119143843 B CN119143843 B CN 119143843B CN 202411596680 A CN202411596680 A CN 202411596680A CN 119143843 B CN119143843 B CN 119143843B
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CN119143843A (en
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顾月清
尹显锐
贾胜岚
陈鑫
李傲
王鑫
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China Pharmaceutical University
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    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
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Abstract

本发明公开了一种CXCR4靶向多肽及其制备方法和应用。本发明设计并合成了一系列靶向趋化因子受体4（CXCR4）的多肽，通过SPR测定了所发现多肽与CXCR4蛋白的亲和力。本发明所述多肽能和多种肿瘤细胞特异性结合，优选黑色素瘤、淋巴癌、乳腺癌、肺癌、结直肠癌、脑胶质瘤、前列腺癌等。本发明也涉及用于制备所述多肽的方法；利用所述多肽直接或者通过配体偶联荧光染料或放射性核素，实现对肿瘤的治疗或诊断的用途。

The present invention discloses a CXCR4 targeting polypeptide and a preparation method and application thereof. The present invention designs and synthesizes a series of polypeptides targeting chemokine receptor 4 (CXCR4), and determines the affinity of the discovered polypeptides to the CXCR4 protein by SPR. The polypeptide of the present invention can specifically bind to a variety of tumor cells, preferably melanoma, lymphoma, breast cancer, lung cancer, colorectal cancer, glioma, prostate cancer, etc. The present invention also relates to a method for preparing the polypeptide; the use of the polypeptide directly or through ligand coupling fluorescent dyes or radionuclides to achieve the treatment or diagnosis of tumors.

Description

CXCR4 targeting polypeptide and preparation method and application thereof
Technical Field
The invention relates to CXCR4 targeting polypeptide, a preparation method and application thereof, and belongs to the field of polypeptides.
Background
Malignant tumors are one of the main causes of abnormal death of human beings worldwide, and pose a great threat to human health. Traditional tumor treatment methods mainly comprise operation, chemotherapy, radiotherapy and the like, but the traditional treatment methods generally bring great toxic and side effects to patients and increase the burden of the patients.
Tumor targeted therapies approach tumor inhibition at the cellular molecular level by interfering with molecular markers associated with tumor cell genesis, progression and metastasis.
Methods of treatment for inhibiting tumor progression by interfering with specific molecules involved in tumor cell development, progression and spread. Compared with the traditional treatment means, the tumor targeting treatment has high selectivity to tumor cells, good curative effect and small side effect, and has good prospect in the aspect of treating tumors. Researchers are also developing various tumor-targeted therapies. Among various targeted therapies, the use of targeted polypeptides for treating tumors is an ideal means, and has the advantages of 1) good penetrability, easy uptake by tumor cells, and 2) high plasma clearance rate, high selectivity and high affinity. 3) Easy chemical synthesis, low immunogenicity and can avoid the shortage of monoclonal antibody treatment. Thus, specific targeting polypeptides are ideal and attractive targets for medical research, clinical applications, and molecular imaging.
Chemokines are proteins of low relative molecular mass (8,000-10,000) belonging to the cytokine superfamily, and are key mediators of cell migration during development, homeostasis and immune monitoring. Chemokines are involved in regulating a variety of biological processes such as angiogenesis, morphogenesis, autoimmunity, tumor growth, and metastasis, which also direct the migration of various types of cells. Chemokine12 (CXCL 12) is also known as stromal CELL DERIVED factor-1 (SDF-1), and belongs to the family of chemokine proteins.
The type 4 chemokine receptor (CXCR 4), also known as a fusion or CD184, is a seven transmembrane G-protein coupled receptor (GPCR) that is activated upon binding of the extracellular domain to a ligand. CXCR4 is overexpressed in more than 20 human tumor types, including ovarian cancer, prostate cancer, esophageal cancer, lung cancer, melanoma, neuroblastoma, renal cell carcinoma, and the like.
CXCR4 (CXC motif chemokine receptor 4) is a specific receptor of CXCL12, and after the combination of the two, the specific receptor is involved in a series of physiological pathologies such as embryonic hematopoiesis, organogenesis, angiogenesis and the like, inflammation, tissue immune monitoring and the like. Activation of CXCL12/CXCR4 signaling pathways can lead to a range of biological processes such as tumor proliferation, angiogenesis, invasion and migration, tumor resistance, and the like.
At present, immunohistochemical (IHC) detection is required for tumor diagnosis, however, due to high time and space heterogeneity of tumor tissues, biopsy tissue sampling is not uniform, so that accurate pathological diagnosis of tumors cannot be performed. Therefore, a marker detection method that can reflect accurate diagnosis of a living body is urgently needed.
Disclosure of Invention
The invention aims to solve the technical problem of providing a CXCR4 targeting polypeptide, a preparation method and application thereof.
The invention provides a CXCR4 targeting polypeptide, which aims to solve the technical problems, and the amino acid sequence of the CXCR4 targeting polypeptide is as follows:
Gly-X 1—X2—X3—X4—X5—X6—X7—X8 or pharmaceutically acceptable salts thereof,
Wherein:
The polypeptide comprises X 1 selected from one of Ala, pro, DVal, DPro, HYP, OIC and alpha-Me-Pro, X 2 selected from one of Tyr, DTyr, ala, N-Me-Tyr, X 3 selected from one of Arg, HAR, N 2 -Me-Arg and Ala, X 4 selected from one of DVal and Ala, X 5 selected from one of Gly, ala, beta-Ala and Sar, X 6 selected from one of Arg, ala, D-2-Nal, HAR, N 2 -Me-Arg and Pro, X 7 selected from one of 2-Nal and D-2-Nal, ala, arg, and X 8 selected from one of Ala and Pro.
The amino acid sequence of the CXCR4 targeting polypeptide is shown in any one of SEQ ID NO. 1-27.
The invention also provides a method for preparing the CXCR4 targeting polypeptide, which comprises the following specific steps:
(1) Coupling the amino acid to RINK AMIDE MBHA resin, dissolving the Fmoc-protected amino acid with DMF and activating with FastMoc chemistry HCTU/DIEA protocol, then removing the N-terminal Fmoc with 20% piperidine/DMF solution, then treating the polypeptide with a solution of TFA/H 2 O/TIS/anisole at room temperature while removing the side chain protection and cleaving the polypeptide from the resin, then drying the system with nitrogen, washing twice with methyl tert-butyl ether, centrifuging to give a precipitate;
(2) The amino acids are coupled to RINK AMIDE MBHA resin one by one, HCTU and Fmoc protected amino acids are dissolved in DMF containing DIPEA during the coupling process for more than 1H each time, deprotection process is carried out after Fmoc group is removed by using DMF solution containing piperidine, side chain protecting group is removed by reaction for 2-3H under the action of strong acid, and the strong acid comprises TFA 90-95%, H 2 O2-5%, TIS 2-5% and EDT 2-5% in parts by mass.
The invention also provides a molecular probe which is formed by coupling a fluorescent label or a radionuclide with the CXCR4 targeting polypeptide.
Wherein the fluorescent label comprises one or more of IRDye800, cy5, cy7, ICG, rhodamine and FITC.
Wherein the fluorescent label can be labeled by a click chemistry mode such as NHS, EDC, MAL.
Wherein the radionuclide is selected from 18F,68Ga,64Cu,99mTc,90Y,111In,125I,131 I or 177 Lu.
Wherein the chelator of the radionuclide comprises HYNIC, DOTA, NOTA or DTPA and derivatives thereof.
Wherein the radionuclide ligand comprises tris (hydroxymethyl) methylglycine or sodium m-sulfonate triphenylphosphine.
The invention also provides application of the CXCR4 targeting polypeptide or the molecular probe in preparing a reagent or a medicament for diagnosing tumors characterized by CXCR4 overexpression.
Wherein the tumor characterized by CXCR4 overexpression comprises one or more of melanoma, lymphoma, breast cancer, cervical cancer, lung cancer, colorectal cancer, glioma, prostate cancer, pancreatic cancer.
Further, the tumor is one or more of melanoma, breast cancer, cervical cancer, lung cancer and prostate cancer.
The invention also provides application of the CXCR4 targeting polypeptide or the molecular probe in preparation of fluorescent imaging or radioactive imaging reagents.
Wherein the imaging agent is one or more of a radionuclide, a radionuclide label, a magnetic resonance contrast agent, or a molecular imaging agent.
Wherein the imaging preparation further comprises a pharmaceutically acceptable carrier thereof. Such as PLGA polymers, dendrimer dendrimers, hydrogels, micelles, liposomes or inorganic nanoparticles, etc.
The invention also provides the use of the imaging preparation described above for the preparation of a medicament for CXCR4 positive tumor imaging diagnosis or detection of surgical navigation precise excision.
The positive tumor is all tumors characterized by CXCR4 overexpression, such as one or more of melanoma, lymphoma, breast cancer, cervical cancer, lung cancer, colorectal cancer, glioma, prostate cancer, pancreatic cancer.
The invention designs a novel CXCR4 targeting polypeptide which has high selectivity and affinity to CXCR4, can realize accurate excision of tumors in operation by coupling fluorescent dye and utilizing molecular image operation navigation equipment, and further, the CXCR4 targeting polypeptide can also realize nuclide imaging by coupling radionuclides so as to achieve the purposes of early diagnosis and treatment of tumors.
The targeted polypeptide method adopted by the invention creatively combines the diversity of the amino acid structure of the polypeptide and the diversity of the polypeptide sequence, and can accurately reflect the dynamic expression change of the living CXCR 4. And synthesis of polypeptides having excellent targeting ability due to the diversity of polypeptide sequences is not easy and hardly conceivable.
The principle of the invention is that a plurality of key amino acid residues in CXCR4 targeting polypeptide and a plurality of amino acid residues in a binding region of target CXCR4 are tightly combined through hydrogen bond formation, salt bond formation and van der Waals force formation, so that the polypeptide is targeted to the binding region. And by linking different chelators to the CXCR4 targeting polypeptide, different radionuclides such as 18F,68Ga,64 Cu, or 99mTc,90 Y, or 111In,125I,131 I or 177 Lu are linked. For tumors characterized by over-expression of CXCR4, the CXCR4 targeting polypeptide connected with the radionuclide is injected in an in vitro injection mode, and radioactive signals are detected through instruments such as positron emission computed tomography (PET-CT) and Single Photon Emission Computed Tomography (SPECT), so that tumor imaging diagnosis is carried out, and if the connected radionuclide has longer half-life and proper radiation quantity, the tumor area can be subjected to radioactive treatment.
Compared with the prior art, the invention has the advantages that a series of targeting polypeptides are designed, the targeting polypeptide has guiding significance on the development of CXCR4 receptor polypeptide antagonists and has a certain explanation on the research on the structure-activity relationship between CXCR4 and various amino acid residues, the invention constructs a diagnostic radioactive probe for specifically targeting CXCR4 positive tumors, provides a new and effective tool for diagnosing tumors in nuclear medicine, and has a certain guiding significance on the operation and the drug treatment of cancers.
Drawings
FIG. 1 is a synthetic diagram of polypeptide YQ-R-3;
FIG. 2 is a mass spectrum of HYNIC-NHS;
FIG. 3 shows the radiochemical purity of 99m Tc-HYNIC-YQ-R-3;
FIG. 4 is a SPECT-CT image of probe 99m Tc-HYNIC-YQ-R-3 in melanoma cell B16 tumor bearing mice;
FIG. 5 is a SPECT-CT image of probe 99m Tc-HYNIC-YQ-R-4 in melanoma cell B16 tumor bearing mice;
FIG. 6 is a SPECT-CT image of 99m Tc-HYNIC-YQ-R-11 in a melanoma cell B16 tumor-bearing mouse;
FIG. 7 is a SPECT-CT image of 99m Tc-HYNIC-YQ-R-12 in melanoma cell B16 tumor-bearing mice;
FIG. 8 is a SPECT-CT image of 99m Tc-HYNIC-YQ-R-13 in a human lung cancer cell H460 tumor-bearing mouse;
FIG. 9 is a SPECT-CT image of 99m Tc-HYNIC-YQ-R-14 in melanoma cell B16 tumor-bearing mice;
FIG. 10 is a SPECT-CT image of 99m Tc-HYNIC-YQ-R-17 in melanoma cell B16 tumor-bearing mice;
FIG. 11 is a SPECT-CT image of 99m Tc-HYNIC-YQ-R-17 in a breast cancer cell 4T1 tumor-bearing mouse;
FIG. 12 is a SPECT-CT image of 99m Tc-HYNIC-YQ-R-21 in melanoma cell B16 tumor-bearing mice;
FIG. 13 is a SPECT-CT image of 99m Tc-HYNIC-YQ-R-21 in a human lung cancer cell H460 tumor-bearing mouse;
FIG. 14 is a SPECT-CT image of 99m Tc-HYNIC-YQ-R-22 in melanoma cell B16 tumor-bearing mice;
FIG. 15 is a SPECT-CT image of 99m Tc-HYNIC-YQ-R-26 in melanoma cell B16 tumor-bearing mice;
FIG. 16 is a SPECT-CT image of 99m Tc-HYNIC-YQ-R-27 in a human lung cancer cell H460 tumor-bearing mouse;
FIG. 17 is a SPECT-CT image of 99m Tc-HYNIC-YQ-R-29 in melanoma cell B16 tumor-bearing mice;
FIG. 18 shows the cellular uptake of 99m Tc-HYNIC-YQ-R-11 in melanoma cells B16 and human colon carcinoma cells HCT 116;
FIG. 19 is a SPECT-CT image of 99m Tc-HYNIC-YQ-R-11 in melanoma cell B16 tumor-bearing mice and human colon cancer cell HCT116 tumor-bearing mice.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
N, N-Diisopropylethylamine (DIPEA), piperidine, trifluoroacetic acid (TFA), dichloromethane (DCM), N, N-Dimethylformamide (DMF), methanol, phenol, ninhydrin, anhydrous diethyl ether, resin, triisopropylsilane (TIS), 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU), 6-chlorobenzotriazole-1, 3-tetramethylurea Hexafluorophosphate (HCTU), dimethyl sulfoxide (DMSO), 4-toluamide resin (MBHA resin), 1, 2-Ethanedithiol (EDT), triphenylphosphine trimetaphosphate sodium salt (TPPTS), 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), 2- (N-morpholino) ethanesulfonic acid buffer (MES buffer), various Fmoc protected amino acids, polypeptide synthesis tubes, vacuum water pumps and other commercially available devices.
Example 1
The embodiment provides a synthetic method of polypeptide YQ-R-3, wherein the polypeptide sequence is Gly-Gly-Ala-DTyr-Arg-DVal-Gly-Arg-D-2-Nal-Pro (SEQ ID NO. 1), and the YQ-R-3 is synthesized by Fmoc solid phase synthesis method as shown in figure 1, and the specific synthetic method is as follows:
1. 400mg Rink Amide MBHA of resin (manufacturer: jiangsu Jitai peptide science and technology Co., ltd., product number: JT 230630-49101) was weighed into a solid phase synthesis tube, after which 6mL of DCM was added, and shaking was performed on a shaker for 20min, in order to swell the resin;
2. The reaction tube was then drained, washed twice with DMF by shaking, drained, 5mL of a mixed solution of piperidine and DMF (piperidine: dmf=2:8, v/v) was added, the reaction was repeated twice for 5min, and drained. The synthesis tube was washed five times with 5mL of DMF and drained, after which 1mmol of Fmoc-Ala, 0.4mL of DIPEA, 400mg of HCTU, 5mL of DMF were added and the mixture was allowed to react on a shaker for 1 hour;
3. repeating the step 2, sequentially adding Fmoc amino acids with equal molar mass from the carboxyl end to the amino end according to the amino acid sequence of the designed polypeptide, and keeping the rest components unchanged until all the amino acids of the polypeptide sequence are added;
4. After Fmoc protection of the last amino acid was removed with piperidine/DMF (2/8,v/v) solution, the system was washed twice with DMF, DCM, meOH each in sequence and drained, 5mL of the mixture (cleavage solution TFA 95%, H 2 O2%, TIS 1%, EDT 2%) was added and treated at room temperature for 3 hours, the cleavage solution volume was quickly evaporated to constant with nitrogen flow, 5mL of methyl tert-butyl ether was added, and after shaking, centrifugation, the precipitated peptide was obtained.
The precipitated peptide was diluted with water and purification of the peptide was accomplished using standard preparative HPLC techniques using a reversed phase C18 column with a chromatography packing of 10 μm and gradient elution with a mobile phase system of 0.1% tfa in water with acetonitrile containing 0.1% tfa under 220nm uv detection. And (3) further determining a final product through mass spectrometry, and placing the collected eluent into a freeze dryer for concentration, and freeze-drying the eluent into white powder to obtain the polypeptide YQ-R-3.
Example 2
The sequence of the polypeptide YQ-R-4 is Gly-Gly-Pro-Ala-Arg-DVal-Gly-Arg-D-2-Nal-Pro (SEQ ID NO. 2)
The synthesis and preparation were performed as in example 1, except that Fmoc-Ala in the third position was replaced with Fmoc-Pro of equimolar mass, fmoc-D-Tyr (tBu) in the fourth position was replaced with Fmoc-Ala of equimolar mass, and the other starting materials and ratios were unchanged.
Polypeptide YQ-R-5-29 was synthesized according to the method of example 1.
Wherein the sequence of the polypeptide YQ-R-5 is Gly-Gly-Pro-DTyr-Ala-DVal-Gly-Arg-D-2-Nal-Pro (SEQ ID NO. 3)
The sequence of the polypeptide YQ-R-6 is Gly-Gly-Pro-DTyr-Arg-Ala-Gly-Arg-D-2-Nal-Pro (SEQ ID NO. 4)
The sequence of the polypeptide YQ-R-7 is Gly-Gly-Pro-DTyr-Arg-DVal-Ala-Arg-D-2-Nal-Pro (SEQ ID NO. 5)
The sequence of the polypeptide YQ-R-8 is Gly-Gly-Pro-DTyr-Arg-DVal-Gly-Ala-D-2-Nal-Pro (SEQ ID NO. 6)
The sequence of the polypeptide YQ-R-9 is Gly-Gly-Pro-DTyr-Arg-DVal-Gly-Arg-Ala-Pro (SEQ ID NO. 7)
The sequence of the polypeptide YQ-R-10 is Gly-Gly-Pro-DTyr-Arg-DVal-Gly-Arg-D-2-Nal-Ala (SEQ ID NO. 8)
The sequence of the polypeptide YQ-R-11 is Gly-Gly-Pro-Tyr-Arg-DVal-Gly-Arg-D-2-Nal-Pro (SEQ ID NO. 9)
The sequence of the polypeptide YQ-R-12 is Gly-Gly-DVal-DTyr-Arg-DVal-Gly-Arg-D-2-Nal-Pro (SEQ ID NO. 10)
The sequence of the polypeptide YQ-R-13 is Gly-Gly-Hyp-DTyr-Arg-DVal-Gly-Arg-D-2-Nal-Pro (SEQ ID NO. 11)
The sequence of the polypeptide YQ-R-14 is Gly-Gly-OIC- -DTyr-Arg-DVal-Gly-Arg-D-2-Nal-Pro (SEQ ID NO. 12)
The sequence of the polypeptide YQ-R-15 is Gly-Gly-Pro-DTyr-HAR-DVal-Gly-Arg-D-2-Nal-Pro (SEQ ID NO. 13)
The sequence of the polypeptide YQ-R-16 is Gly-Gly-Pro-DTyr-Arg-DVal-Gly-HAR-D-2-Nal-Pro (SEQ ID NO. 14)
The sequence of the polypeptide YQ-R-17 is Gly-Gly-Pro-DTyr-Arg-DVal-beta-Ala-Arg-D-2-Nal-Pro (SEQ ID NO. 15)
The sequence of the polypeptide YQ-R-18 is Gly-Gly-Pro-DTyr-N 2 -ME-Arg-DVal-Gly-Arg-D-2-Nal-Pro (SEQ ID NO. 16)
The sequence of the polypeptide YQ-R-19 is Gly-Gly-Pro-DTyr-Arg-DVal-Gly-N 2 -ME-Arg-D-2-Nal-Pro (SEQ ID NO. 17)
The sequence of the polypeptide YQ-R-20 is Gly-Gly-Pro-DTyr-Arg-DVal-beta-Ala-D-2-Nal-Arg-Pro (SEQ ID NO. 18)
The sequence of the polypeptide YQ-R-21 is Gly-Gly-Pro-N-ME-Tyr-Arg-DVal-beta-Ala-Arg-D-2-Nal-Pro (SEQ ID NO. 19)
The sequence of the polypeptide YQ-R-22 is Gly-Gly-Pro-DTyr-Arg-DVal-beta-Ala-Arg-2-Nal-Pro (SEQ ID NO. 20)
The sequence of the polypeptide YQ-R-23 is Gly-Gly-Pro-DTyr-Arg-DVal-Sar-Arg-D-2-Nal-Pro (SEQ ID NO. 21)
The sequence of the polypeptide YQ-R-24 is Gly-Gly-Pro-DTyr-Arg-DVal-beta-Ala-HAR-D-2-Nal-Pro (SEQ ID NO. 22)
The sequence of the polypeptide YQ-R-25 is Gly-Gly-Pro-Tyr-Arg-DVal-beta-Ala-Arg-D-2-Nal-Pro (SEQ ID NO. 23)
The sequence of the polypeptide YQ-R-26 is Gly-Gly-DPro-Tyr-Arg-DVal-beta-Ala-Arg-D-2-Nal-Pro (SEQ ID NO. 24)
The sequence of the polypeptide YQ-R-27 is Gly-Gly-DPro-DTyr-Arg-DVal-beta-Ala-Arg-D-2-Nal-Pro (SEQ ID NO. 25)
The sequence of the polypeptide YQ-R-28 is Gly-Gly-alpha-Me-Pro-DTyr-Arg-DVal-beta-Ala-Arg-D-2-Nal-Pro (SEQ ID NO. 26)
The sequence of the polypeptide YQ-R-29 is Gly-Gly-alpha-Me-Pro-Tyr-Arg-DVal-beta-Ala-Arg-D-2-Nal-Pro (SEQ ID NO. 27)
The mass spectrum characterization of the polypeptides YQ-R-n (n=3 to 29) is shown in table 1.
Table 1 mass spectrometric characterization of polypeptides YQ-R-n (n=3-29)
Example 3
The present embodiment provides a method for determining the affinity of polypeptide YQ-R-n (n=3 to 29) with human CXCR4 protein (manufacturer: associated biotechnology (martial arts) company, cat# PME 100834), mainly by surface ion resonance (SPR) technology, and selecting the commercial drug AMD3100 (manufacturer: shanghai pichia pharmaceutical technologies company, cat# BD 208340) as a reference, comprising the following steps:
1. The chip surface was rinsed with pure water, and after drying, the chip was immersed in 2.18 mg/mL (10 mmol/L) of 11-mercaptoundecanoic acid MUA (formulated with pure ethanol) and treated overnight, and the next day was rinsed with ethanol and pure water, respectively.
2. The CXCR4 protein is fixed on a gold film on the surface of the chip by adopting a covalent bonding method. The preparation method comprises the following steps of (1) setting the sample injection speed to be 10 mu L/min, starting to sample 150 mu L of EDC solution with the concentration of 0.4 mol/L and NHS solution with the concentration of 150 mu L and the concentration of 0.1 mol/L after a base line is stable (both prepared by MES buffer solution with the concentration and the pH of 0.1 mol/L respectively and pH=5.5), injecting for 10min to activate carboxyl groups on the gold membrane surface, (2) injecting CXCR4 protein solution with the concentration of 150 mu L and the concentration of 10 mu g/mL (prepared by sodium acetate solution with the concentration and the pH of 0.01 mol/L respectively and the pH=4.8) to enable the CXCR4 protein to flow through the surface of a chip to realize covalent bonding on the gold membrane surface, (3) flushing the gold membrane surface by introducing PBS after CXCR4 protein bonding is stable, (4) introducing BSA solution with the concentration of 10 mg/mL (prepared by sodium acetate solution with the concentration and the pH of 0.01 mol/L respectively and pH=4.8) to seal nonspecific sites.
3. After CXCR4 protein is fixed on the surface of the chip, PBS buffer solution is used as a working fluid to flow through the system, a blank channel is used as a control channel to eliminate nonspecific binding as much as possible, the sample injection speed is set to be 20 mu L/min, and the temperature is kept at 25 ℃. After the base line is stable, the injection of the polypeptide YQ-R-3-29 solutions with different concentrations (PBS buffer solution is prepared, the concentration of the PBS buffer solution is 0.01M, pH and 7.4, and the concentration of the PBS buffer solution is shown in Table 2) or the small molecule solution (AMD 3100 and the PBS buffer solution is prepared) is started, and the binding process is monitored in real time. And then PBS buffer solution is introduced to clean the surface of the chip. Data processing was then performed using GraphPad 9.0 software and KD values results are shown in table 3.
TABLE 2
TABLE 3 KD value results by SPR
As shown in Table 3, the binding capacity of the polypeptides YQ-R-3-29 to the target (CXCR 4 protein) was different, but most of them were superior to the marketed drug AMD3100.
EXAMPLE 4 preparation of prodrug HYNIC-YQ-R-n (n=3-29)
1. Synthesis of HYNIC-NHS:
The synthetic route is as follows:
1.79 g of 6-chloronicotinic acid (compound 1,11.36 mmol, 1 eq) was added to a 100 mL round bottom flask and reacted at reflux by heating with stirring at 100℃with 80% hydrazine hydrate 6.2 mL (124.98 mmol, 11 eq), 11 mL deionized water, 6 h. The solvent was removed by distillation under reduced pressure, ph=5.5 was adjusted with hydrochloric acid, concentrated by distillation under reduced pressure, allowed to stand overnight to precipitate, and dried by filtration to give 6-hydrazinonicotinic acid, which was then weighed for the next reaction.
0.55 G of 6-hydrazinonicotinic acid (compound 2,3.59 mmol,1 eq) and 0.54 g dimethylaminoterephthalaldehyde (compound 3,3.59 mmol,1 eq) were dissolved together in 8mL DMF and stirred at room temperature for 3 h to give compound 4, 0.62 g NHS (5.39 mmol,1.5 eq) and 1.8 g EDCI (9.39 mmol,2.5 eq) were added, 15:15 mL DMF was added and stirring was continued for 18: 18 h, DMF was concentrated by distillation under reduced pressure to 50: 50 mL deionized water to give a yellow precipitate which was filtered off with methanol and washed 3 times with suction, and then heated to reflux in ethyl acetate, filtered while hot, dried to give the product HYNIC-NHS and weighed. The product was confirmed by mass spectrometry. The mass spectrum is shown in figure 2. HYNIC-NHS: calculated [ M+H+ ]: 382.15, found [ M+H+ ]: 382.22.
2. The preparation of the prodrug HYNIC-YQ-R-n (n=3-29) comprises the following specific steps:
Purified 1 mg polypeptide YQ-R-n (n=3 to 29) and 1 mg HYNIC-NHS were dissolved in 0.1 mL DMSO, 1 μl DIPEA was added to react at 50 ℃ for 1.5 h, and after the reaction was completed, separation and purification were performed by preparing a high performance liquid phase (Agilent ZORBAX SB-C18,5 μm), and finally a product was obtained, taking HYNIC-YQ-R-3 as an example, and its structure was as follows:
calculated value of HYNIC-YQ-R-3 is [1/2M+H + ]: 697.86, actual value is 697.98. Purity of HYNIC-YQ-R-3 is 98%.
Wherein the separation and purification steps are as follows, elution is carried out for 35 minutes by using a preparation high performance liquid phase gradient, the flow rate is 1 mL/min, wherein the mobile phase A is ultrapure water (containing 0.1% of TFA), and the mobile phase B is acetonitrile (containing 0.1% of TFA). The gradient of the elution was set to 85% A and 15% B to 60% A and 40% B at 0-20 minutes, 50% A and 50% B at 25 minutes, and 10% A and 90% B at 25-35 minutes. HYNIC-YQ-R-4-29 is also prepared according to the method. All products had a chemical purity of greater than 95%.
Example 5 preparation of 99m Tc-HYNIC-YQ-R-n (n=3-29)
Preparing 100.0 mg/mL of TPPTS solution, 130.0 mg/mL of Tricine (trimethylglycine) and 102.4 mg/mL of succinic acid-sodium succinate buffer (wherein the mass ratio of succinic acid to sodium succinate is 77.0:25.4), respectively taking 10.0 mu L of TPPTS solution, 10.0 mu L of Tricine solution, 10.0 mu L of succinic acid-sodium succinate buffer and 10.0 mu L (1.0 mg/mL) of the prodrug described in example 4, respectively mixing in a penicillin bottle, then respectively adding 10 mCi Na 99mTcO4 (Nanjing) of Tricine (America medical technology Co., ltd.) into the penicillin bottle, heating the mixture for 20 minutes at 100 ℃, cooling the mixture to room temperature after the reaction is finished, and respectively obtaining the polypeptide radioactive drug 99m Tc-HYNIC-YQ-R-n (n=3-29), and analyzing and identifying the product by Agilent ZORBAX SB-Aq analysis column. The HPLC method used was an Agilent 1220 Infinity II series HPLC system equipped with a radioactive online detector (Flow-RAM) and Agilent ZORBAX SB-Aq analytical column (4.6X1250 mm,5 μm). Gradient elution was carried out for 45 minutes at a flow rate of 1 mL/min, where mobile phase A was ultrapure water (0.01% TFA) and B was acetonitrile (0.01% TFA). The gradient of the elution was set to 95% A and 5% B at 0-5 minutes, 70% A and 30% B at 15 minutes, 65% A and 35% B at 20 minutes, 45% A and 55% B at 25 minutes, 5%A and 95% B at 45 minutes. 99m The radiochemical purity of Tc-HYNIC-YQ-R-n (n=3-29) is not lower than 95%. Taking polypeptide 99m Tc-HYNIC-YQ-R-3 as an example, the radiochemical purity is shown in FIG. 3.
EXAMPLE 6 SPECT-CT imaging of radioactive probe 99m Tc-HYNIC-YQ-R-3 in tumor-bearing mice
18.5 MBq/200. Mu.L of probe 99m Tc-HYNIC-YQ-R-3 prepared in example 5 was prepared as a physiological saline solution, and 500. Mu. Ci was injected through the tail vein, and at least three tumor-bearing nude mice (Jiangsu Hua Xinnuo medical science Co., ltd.) were injected with melanoma cells B16. And SPECT signal acquisition was performed at 0.5 h, 1 h, 2 h, 3h, 4 h post-dose. The imaging result after administration is shown in figure 4, and the probe 99m Tc-HYNIC-YQ-R-3 has obvious uptake in tumor sites, which shows that the probe has good targeting.
EXAMPLE 7 SPECT-CT imaging of radioactive probe 99m Tc-HYNIC-YQ-R-4 in tumor-bearing mice
18.5 MBq/200. Mu.L of probe 99m Tc-HYNIC-YQ-R-4 prepared in example 5 was formulated as a physiological saline solution and 500. Mu. Ci was injected via the tail vein, at least three tumor-bearing nude tail veins with melanoma cells B16. And SPECT signal acquisition was performed at 0.5 h,1h,2h, 3h, 4 h post-dose. The imaging result after administration is shown in figure 5, and the probe 99m Tc-HYNIC-YQ-R-4 has obvious uptake at the tumor site, which shows that the probe has good targeting.
EXAMPLE 8 SPECT-CT imaging of radioactive probe 99m Tc-HYNIC-YQ-R-11 in tumor-bearing mice
18.5 MBq/200. Mu.L of probe 99m Tc-HYNIC-YQ-R-11 prepared in example 5 was formulated as a physiological saline solution and 500. Mu. Ci was injected via the tail vein, at least three tumor-bearing nude tail veins with melanoma cells B16. And SPECT signal acquisition was performed at 0.5 h, 1h, 2h, 3h, 4h post-dose. The imaging result after administration is shown in fig. 6, and the probe 99m Tc-HYNIC-YQ-R-11 has obvious uptake in tumor sites, which indicates that the probe has good targeting.
EXAMPLE 9 SPECT-CT imaging of radioactive probe 99m Tc-HYNIC-YQ-R-12 in tumor-bearing mice
18.5 MBq/200. Mu.L of probe 99m Tc-HYNIC-YQ-R-12 prepared in example 5 was prepared as a physiological saline solution, 500. Mu. Ci was injected through the tail vein, and at least three of each of the tumor-bearing mice (purchased from Jiangsu Hua Xinnuo medical science Co., ltd.) were injected with melanoma cells B16. And SPECT signal acquisition was performed at 0.5 h, 1 h, 2h, 3h, 4h post-dose. The imaging result is shown in figure 7, and the probe 99m Tc-HYNIC-YQ-R-12 has obvious uptake in tumor sites, which shows that the probe has good targeting property.
EXAMPLE 10 SPECT-CT imaging of radioactive probe 99m Tc-HYNIC-YQ-R-13 in tumor-bearing mice
18.5 MBq/200. Mu.L of probe 99m Tc-HYNIC-YQ-R-13 prepared in example 5 was prepared as a physiological saline solution, and 500. Mu. Ci was injected through the tail vein, and at least three human lung cancer cell H460 tumor-bearing mice (purchased from Jiangsu Hua Xinnuo medical science Co., ltd.) were injected through the tail vein. And SPECT signal acquisition was performed at 0.5h, 1 h, 2h post-dose. The imaging result is shown in figure 8, and the probe 99m Tc-HYNIC-YQ-R-13 has obvious uptake in tumor sites, which indicates that the probe has good targeting.
EXAMPLE 11 SPECT-CT imaging of radioactive probe 99m Tc-HYNIC-YQ-R-14 in tumor-bearing mice
18.5 MBq/200. Mu.L of probe 99m Tc-HYNIC-YQ-R-14 prepared in example 5 was formulated as a physiological saline solution, and 500. Mu. Ci was injected via the tail vein, and at least three of each was injected into the tail vein of a melanoma cell B16 tumor-bearing mouse (purchased from Jiangsu Shu Chuan Xinnuo medical science Co.). And SPECT signal acquisition was performed at 0.5 h, 1h, 2 h, 3 h post-dose. The imaging result is shown in figure 9, and the probe 99m Tc-HYNIC-YQ-R-14 has obvious uptake in tumor sites, which shows that the probe has good targeting property.
EXAMPLE 12 SPECT-CT imaging of radioactive probe 99m Tc-HYNIC-YQ-R-17 in tumor-bearing mice
18.5 MBq/200. Mu.L of probe 99m Tc-HYNIC-YQ-R-17 prepared in example 5 was prepared into physiological saline solution, and 500. Mu. Ci was injected through tail vein, and tumor-bearing nude mice of melanoma cell B16 and tumor-bearing nude mice of breast cancer cell 4T1 (purchased from Jiangsu Hua Xinnuo medical science Co., ltd.) were injected respectively, at least three of each. And SPECT signal acquisition was performed at 0.5 h, 1h, 2 h, 3 h, 4 h, respectively, after dosing. The imaging result of the tumor-bearing nude mice injected with the melanoma cells B16 is shown in fig. 10, the imaging result of the tumor-bearing nude mice injected with the breast cancer cells 4T1 is shown in fig. 11, and the probe 99m Tc-HYNIC-YQ-R-17 has obvious uptake at the tumor part, which indicates that the probe has good targeting.
EXAMPLE 13 SPECT-CT imaging of radioactive probe 99m Tc-HYNIC-YQ-R-21 in tumor-bearing mice
18.5 MBq/200. Mu.L of probe 99m Tc-HYNIC-YQ-R-21 prepared in example 5 was prepared as a physiological saline solution, and 500. Mu. Ci was injected through the tail vein, and at least three of each of the tail veins of a melanoma cell B16 tumor-bearing nude mouse and a human lung cancer cell H460 tumor-bearing nude mouse (purchased from Jiangsu Hua Xinnuo medical science Co., ltd.) were injected respectively. And SPECT signal acquisition was performed at 0.5h, 1h, 2h, 3h, 4 h, respectively, after dosing. The imaging result of the injection of melanoma cell B16 tumor-bearing nude mice is shown in figure 12, the imaging result of the injection of human lung cancer cell H460 tumor-bearing nude mice is shown in figure 13, and the probe 99m Tc-HYNIC-YQ-R-21 has obvious uptake at tumor sites, which indicates that the probe has good targeting.
EXAMPLE 14 SPECT-CT imaging of radioactive probe 99m Tc-HYNIC-YQ-R-22 in tumor-bearing mice
18.5 MBq/200. Mu.L of probe 99m Tc-HYNIC-YQ-R-22 prepared in example 5 was formulated as a physiological saline solution and 500. Mu. Ci was injected via the tail vein, and at least three of the tumor-bearing tail veins of melanoma cells B16 were injected. And SPECT signal acquisition was performed at 0.5h, 1h, 2h, 3 h post-dose. The imaging result is shown in figure 14, and the probe 99m Tc-HYNIC-YQ-R-22 has obvious uptake in tumor sites, which shows that the probe has good targeting property.
EXAMPLE 15 SPECT-CT imaging of radioactive probe 99m Tc-HYNIC-YQ-R-26 in tumor-bearing mice
18.5 MBq/200. Mu.L of probe 99m Tc-HYNIC-YQ-R-26 prepared in example 5 was formulated as a physiological saline solution and 500. Mu. Ci was injected via the tail vein, and at least three of the tumor-bearing tail veins of melanoma cell B16 were injected. And SPECT signal acquisition at rows 0.5h, 2, h after dosing. The imaging result is shown in figure 15, and the probe 99m Tc-HYNIC-YQ-R-26 has obvious uptake in tumor sites, which shows that the probe has good targeting property.
EXAMPLE 16 SPECT-CT imaging of radioactive probe 99m Tc-HYNIC-YQ-R-27 in tumor-bearing mice
18.5 MBq/200. Mu.L of probe 99m Tc-HYNIC-YQ-R-27 prepared in example 5 was prepared as a physiological saline solution and 500. Mu. Ci was injected via the tail vein, and at least three human lung cancer cells H460 were injected into the tumor-bearing tail veins of the nude mice. And SPECT signal acquisition was performed at post-dose 2 h. The imaging result is shown in figure 16, and the probe 99m Tc-HYNIC-YQ-R-27 has obvious uptake in tumor sites, which shows that the probe has good targeting property.
EXAMPLE 17 SPECT-CT imaging of radioactive probe 99m Tc-HYNIC-YQ-R-29 in tumor-bearing mice
18.5 MBq/200. Mu.L of probe 99m Tc-HYNIC-YQ-R-29 prepared in example 5 was formulated as a physiological saline solution and 500. Mu. Ci was injected via the tail vein, and at least three of the tumor-bearing tail veins of melanoma cells B16 were injected. And SPECT signal acquisition was performed at 1h, 2 h, 4 h post-dose. The imaging result is shown in figure 17, and the probe 99m Tc-HYNIC-YQ-R-29 has obvious uptake in the tumor part, which shows that the probe has good targeting property.
Example 18 cell uptake comparative experiment
This example compares the cellular uptake rates of 99m Tc-HYNIC-YQ-R-11 in CXCR4 high expressing cells B16F10 and CXCR4 low expressing cells HCT 116.
HCT116 cells and B16F10 cells were seeded in 2 12-well plates (2×10 5 cells per well), HC116 cells were added to 1.5 mL medium per well (DMEM medium with 10% fetal bovine serum), and B16F10 cells were added to 1.5 mL medium per well (1640 medium with 10% fetal bovine serum). Cells 24h were incubated at 37 ℃. The plates were removed and replaced with the corresponding fresh medium (1 mL per well). 37 KBq of 99m Tc-HYNIC-YQ-R-11 was added to each well and incubated at 37℃for 1h, 2 h, respectively. After incubation, the supernatant was aspirated, the cells were washed twice with PBS, and the supernatant was also incorporated after aspiration of PBS. Then digested with pancreatin, aspirated as pellet, and the residual cells washed with PBS are also aspirated and incorporated into pellet. Supernatant and cell gamma Counts (CPM) were measured using WIZARD 2470 gamma counter and cell uptake was calculated. The formula is as follows:
as shown in fig. 18, the uptake rate was found to be positively correlated with the expression level of CXCR4 in cells. The expression quantity of the nuclide probe in B16F10 cells is obviously higher than that in HC116 cells, and the binding capacity and the specificity of the probe and CXCR4 high-expression cells are proved.
EXAMPLE 19 99m Tc-HYNIC-YQ-R-11 imaging in tumor-bearing mice
To evaluate the imaging ability and metabolic profile of 99m Tc-HYNIC-YQ-R-11 probe in vivo, a B16F10 melanoma model mouse and HCT116 tumor bearing mouse (available from Jiangsu Hua Xinnuo pharmaceutical technologies Co., ltd.) were used for the course imaging experiments. Each mouse was injected with 18 MBq of 99m Tc-HYNIC-YQ-R-11 in physiological saline. SPECT/CT tomography was started 30min after injection. After each scan and by software, the data is reconstructed automatically to obtain a specific SPECT/CT image. Mice were then scanned at different time points to observe changes in vivo distribution. As shown in FIG. 19, 99m Tc-HYNIC-YQ-R-11 had good imaging effect in B16F10 (upper panel in FIG. 19) mice, and tumor uptake was evident. The nuclide probe shows good effects in B16F10 tumor-bearing mice from 30min to 5h 30min after injection, and particularly at 30min and 1h30min, the probe is mainly metabolized by the kidney and excreted through the bladder, and the uptake of other organs is very low. TBR was calculated to be 14.2.+ -. 2.2 at maximum by processing images of B16F10 tumor bearing mice in software for 1h30 min. In vivo imaging results of xenograft subcutaneous tumor mice with HCT116 (lower panel in fig. 19, left and right panels are parallel experiments of two mice) with low CXCR4 expression served as controls, and tumor uptake was found to be very low and almost invisible, demonstrating significant specificity of the nuclide probe. Tumor imaging experiments with 99m Tc-HYNIC-YQ-R-11 were continued in more B16F10 tumor-bearing mice, demonstrating the reproducibility of the imaging ability of the nuclear probe, as shown in FIG. 6.

Claims (8)

1. The CXCR4 targeting polypeptide or pharmaceutically acceptable salt thereof is characterized in that the amino acid sequence of the CXCR4 targeting polypeptide is shown in any one of SEQ ID NO.5, SEQ ID NO.15, SEQ ID NO.20 and SEQ ID NO. 23-27.
2. A molecular probe, wherein the molecular probe is formed by coupling a fluorescent label or a radionuclide with the CXCR4 targeting polypeptide of claim 1.
3. The molecular probe of claim 2, wherein the fluorescent label comprises one or more of IRDye800, cy5, cy7, ICG, rhodamine, FITC.
4. The molecular probe of claim 2, wherein the radionuclide is selected from 18F,68Ga,64Cu,99mTc,90Y,111In,125I,131 I or 177 Lu.
5. The molecular probe of claim 2, wherein the chelator of radionuclides comprises HYNIC, DOTA, NOTA or DTPA.
6. The molecular probe of claim 2, wherein the radionuclide ligand comprises tris (hydroxymethyl) methylglycine or sodium m-sulfonate triphenylphosphine.
7. Use of a CXCR4 targeting polypeptide of claim 1 or a molecular probe of any one of claims 2-6 in the preparation of a reagent for diagnosing a tumor characterized by CXCR4 overexpression.
8. Use of a CXCR4 targeting polypeptide of claim 1 or a molecular probe of any one of claims 2-6 in the preparation of a fluorescence imaging or radiological imaging reagent.
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CN116023438A (en) * 2023-02-06 2023-04-28 中国药科大学 A CXCR4 targeting polypeptide and its application

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