Motixazotide-camptothecin conjugate as well as preparation method and application thereof

Abstract

The invention provides Motixazotide-camptothecin conjugates as well as a preparation method and application of the Motixazotide-camptothecin conjugates, and belongs to the fields of polypeptide preparation and biological medicines. According to the invention, a series of novel Motixafortide-camptothecin conjugates are synthesized, and the Motixafortide-camptothecin conjugates are formed by connecting Motixafortide with different quantities of camptothecin through a linking group. An anti-tumor proliferation and migration experiment, cellular uptake and an aging curve show that the Motixazotide-camptothecin conjugate can improve the membrane penetrability and the anti-tumor activity of the camptothecin, improve the selectivity of the camptothecin to tumor cells and reduce the toxicity of the camptothecin to normal cells. Meanwhile, the solubility of the Motixazotide-camptothecin conjugate is more than 1000 times of that of the camptothecin. In addition, according to the intramolecular self-quenching Motixazotide-camptothecin conjugate prepared by the preparation method disclosed by the invention, the real-time monitoring on the release condition of the camptothecin anti-tumor drugs is realized. Therefore, the method has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide preparation and disease treatment, and specifically relates to a targeting peptide-camptothecin conjugate based on CXCR4 receptor and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Camptothecin (CPT), a pyrroloquinoline cytotoxic alkaloid, was isolated from Camptotheca acuminata introduced in my country by Wall et al. in 1966 and has a wide range of antitumor activity. As a DNA topoisomerase I (TopoI) inhibitor, camptothecin binds to this enzyme and inhibits the formation of double helix of cellular DNA, leading to cell death. In particular, the representative derivative irinotecan has been included in the 2019 WHO Model List of Essential Medicines for Anticancer Therapy. However, its poor membrane permeability, high toxicity to normal cells and poor water solubility have greatly limited its widespread clinical application.

[0004] At present, a mainstream strategy to solve the problem of anti-tumor drug selectivity is to use targeting ligands to deliver cytotoxic drugs to cancer sites and then exert their corresponding anti-tumor activity. Tumor-specific targeting peptides are covalently coupled to small molecule anti-tumor drugs through linkers to obtain peptide-drug conjugates (PDC). PDC has broad application prospects in tumor targeted therapy. It has been approved for marketing for the treatment of tumors. PDC drugs modified by peptides have the advantages of high selectivity, strong tumor penetration, low immunogenicity, and low synthesis cost, and have attracted widespread attention.

[0005] High expression of chemokine receptor 4 (CXCR4) is associated with poor prognosis and chemotherapy resistance in many cancers. CXCR4 plays an important role in tumor growth, migration, invasion, metastasis, angiogenesis and tolerance to therapeutic drugs. Motixafortide (BL8040, BKT-140) is a cyclic peptide composed of 14 amino acids, which has high affinity with CXCR4 and is a specific inhibitor of CXCR4. The present invention covalently couples the anti-tumor drug camptothecin with the polypeptide Motixafrotide to increase its selective killing effect on tumor cells, and develops a new and efficient PDC compound for anti-tumor treatment. Summary of the invention

[0006] In view of the shortcomings of camptothecin, such as high toxicity to normal cells and poor water solubility, the present invention improves its water solubility and increases its anti-tumor selectivity by connecting CXCR4 specific antagonist Motixafortide (FXY-904) as a targeting peptide to camptothecin. In addition, the present invention obtains a series of conjugates of Motixafortide and different amounts of camptothecin by coupling different amounts of camptothecin to a polypeptide. The present invention couples Motixafortide and camptothecin together through a degradable ester bond, so that PDC can slowly and continuously release camptothecin and exert an anti-tumor effect. In addition, the present invention adopts the principle of fluorescence internal self-quenching to introduce the fluorescence quenching group dinitrophenyl (Dnp) into the conjugate, thereby achieving the purpose of real-time monitoring of camptothecin release from spatial and temporal levels.

[0007] Specifically, the present invention is implemented through the following technical solutions:

[0008] The first aspect of the present invention provides a Motixafortide-camptothecin conjugate, wherein the Motixafortide-camptothecin conjugate comprises the following amino acid residue sequence:

[0009]

[0010] The above-mentioned Motixafortide-camptothecin conjugate can greatly improve the water solubility of camptothecin, help it recognize and enter tumor cells, so that camptothecin can more easily reach the nucleus of tumor cells, bind to the complex formed by TopoⅠ-DNA, and prevent DNA replication and RNA synthesis. In addition, Motixafortide has the effect of inhibiting tumor cell migration and invasion. Camptothecin and Motixafortide exert anti-tumor effects through synergistic effects. At the same time, the release of camptothecin is monitored in real time from the time and space levels by utilizing the principle of fluorescence internal self-quenching based on camptothecin / Dnp.

[0011] In a second aspect of the present invention, a method for preparing the above-mentioned Mortixafortide-camptothecin conjugate is provided, wherein the preparation method comprises synthesizing Mortixafortide by a polypeptide solid phase synthesis method, and covalently linking the polypeptide and camptothecin by using linking groups such as lysine, succinic acid and AEEA to obtain a polypeptide drug conjugate.

[0012] Specifically, the polypeptide is synthesized using 9-fluorenylmethoxycarbonyl-based solid phase peptide synthesis (Fmoc-SPPS).

[0013] The third aspect of the present invention provides the use of the above-mentioned Motixafortide-camptothecin conjugate in the preparation of anticancer or antitumor drugs.

[0014] Preferably, the cancer or tumor can be selected from breast cancer, nasopharyngeal carcinoma, thymic carcinoma, bladder cancer, bronchial lung cancer, non-small cell lung cancer, prostate cancer, ovarian cancer, neuroblastoma, ganglioneuroma, gastric cancer, ganglioneuroma, papillary thyroid carcinoma, head and neck squamous cell carcinoma, testicular cancer.

[0015] Further preferably, the cancer or tumor is a human cancer or tumor that highly expresses CXCR4.

[0016] The fourth aspect of the present invention provides a monitoring compound for the drug release of the above-mentioned polypeptide-camptothecin conjugate. The Motixafortide-camptothecin conjugate of the present invention, in which camptothecin is covalently linked to Dnp, has no bright fluorescence when camptothecin is covalently linked to the polypeptide, and when camptothecin is released from the polypeptide, Dnp cannot absorb the free camptothecin fluorescence, thereby emitting brighter blue fluorescence, thereby achieving real-time monitoring of camptothecin release at the spatiotemporal level, and can also be used for in vivo imaging of cells, tissues and animals, thereby exploring the distribution, anti-tumor activity and anti-tumor mechanism of the Motixafortide-camptothecin conjugate at the cellular, molecular, tissue and animal levels.

[0017] The beneficial effects of the above technical solution are as follows:

[0018] 1. The Mortixafortide-camptothecin conjugate synthesized by the present invention enhances the inhibitory effect of 24h camptothecin on tumor cell proliferation. 50(24h) were as follows: in B16-F10 cells, 12.8±1.7μM (FXY-922), 19.5±2.3μM (FXY-925), 65.8±4.5μM (FXY-926), 209.8±3.8μM (FXY-927), in MCF-7 cells, 56.2±2.7μM (FXY-922), 98.7± 3.5 μM (FXY-925), 184.1 ± 3.4 μM (FXY-926), >300 μM (FXY-927), in A549 cells, 51.3 ± 4.2 μM (FXY-922), 64.1 ± 5.6 μM (FXY-925), 162.8 ± 2.7 μM (FXY-926), >300 μM (FXY-927) ), in 4T1 cells, 18.9±3.8μM (FXY-922), 30.9±2.4μM (FXY-925), 84.9±3.4μM (FXY-926), >300μM (FXY-927), in Hep3B cells, 30.5±2.3μM (FXY-922), 41.4±5.1μM (FXY-925), 113.4±5.7μM (FXY-926), >300μM (FXY-927), and finally in Hepa1-6 cells, 37.9±1.8μM (FXY-922), 60.1±1.7μM (FXY-925), >300μM (FXY-926), >300μM (FXY-927), which is higher than the prototype anticancer drug camptothecin (IC 50 :>133μM) showed significant improvement.

[0019] 2. The above technical solution can improve the membrane permeability of camptothecin in a short time. This project uses the CXCR4 receptor antagonist Motixafortide to covalently link camptothecin. Motixafortide can specifically target CXCR4 on the surface of tumor cells and enter the cells. Compared with camptothecin monomers, Motixafortide-camptothecin conjugates can carry camptothecin to identify tumor cells and enter cells in a short time, greatly improving the selectivity and membrane permeability of camptothecin, thereby allowing a large amount of camptothecin to enter the cells to exert anti-tumor effects.

[0020] 3. The above technical solution can greatly improve the water solubility of camptothecin. The solubility of the Motixafortide-camptothecin conjugate designed and synthesized by the present invention in water is >3000 μM, which is greatly improved compared with the solubility of camptothecin monomer (2.83 μM).

[0021] 4. The above technical solution can realize real-time monitoring of camptothecin release through the principle of intramolecular self-quenching. Since the conjugate of the invention is coupled to the polypeptide through the active essential group hydroxyl of camptothecin, camptothecin needs to be released from the conjugate before it can exert its anti-tumor activity. Camptothecin itself emits blue fluorescence and is used in conjunction with the fluorescence quenching group Dnp. When camptothecin is released from the polypeptide, Dnp cannot absorb the fluorescence of free camptothecin, thereby emitting brighter blue fluorescence, thereby realizing the monitoring of camptothecin release. This technology can be applied to the imaging level of cells, tissues and living animals, and then explore the distribution and release of conjugates at the molecular, cellular, tissue and animal levels.

[0022] 5. The above technical solution can improve the selectivity of camptothecin. Camptothecin is a broad-spectrum cytotoxic drug with poor selectivity for various tumor cells and normal cells. The target of the conjugate obtained by the above technical solution is CXCR4. Therefore, this conjugate can be targeted and internalized into tumor cells with high expression of CXCR4 in large quantities, while less internalized into cells with low or no expression of CXCR4, thereby achieving the purpose of improving the selectivity of camptothecin for tumor cells and reducing toxicity.

[0023] 6. The above technical solution can inhibit the migration of tumor cells. Motixafortide has the effect of inhibiting the migration and invasion of tumor cells. The Motixafortide-camptothecin conjugate obtained by the above technical solution can inhibit the migration and invasion of tumor cells while killing tumor cells. Motixafortide and camptothecin work synergistically to better inhibit and kill tumor cells.

[0024] In summary, although camptothecin has a broad spectrum of anti-tumor activity, its low water solubility, low membrane permeability, and high toxicity limit its wide application. In view of the above shortcomings, the present invention designs and synthesizes a novel Motixafortide-camptothecin conjugate, improves the water solubility of camptothecin, improves the anti-tumor activity of camptothecin by targeting tumor cells with high expression of CXCR4, reduces the toxicity to normal cells, and improves the selectivity and membrane permeability to tumor cells. The preparation method of the present invention is simple and effective, and has good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0026] Figure 1 Schematic diagram of the solid phase synthesis method of peptides;

[0027] Figure 2The chemical structural formula, primary amino acid sequence, analytical reverse-phase high performance liquid chromatography and mass spectrum of FXY-904 of the present invention;

[0028] Figure 3 The chemical structural formula, primary amino acid sequence, analytical reverse-phase high performance liquid chromatography and mass spectrum of FXY-922 of the present invention;

[0029] Figure 4 The chemical structural formula, primary amino acid sequence, analytical reverse-phase high performance liquid chromatography and mass spectrum of FXY-923 of the present invention;

[0030] Figure 5 The chemical structural formula, primary amino acid sequence, analytical reverse-phase high performance liquid chromatography and mass spectrum of FXY-925 of the present invention;

[0031] Figure 6 The chemical structural formula, primary amino acid sequence, analytical reverse-phase high performance liquid chromatography and mass spectrum of FXY-926 of the present invention;

[0032] Figure 7 The chemical structural formula, primary amino acid sequence, analytical reverse-phase high performance liquid chromatography and mass spectrum of FXY-927 of the present invention;

[0033] Figure 8 The chemical structural formula, primary amino acid sequence, analytical reverse-phase high performance liquid chromatography and mass spectrum of FXY-928 of the present invention;

[0034] Fig. 9 To evaluate the inhibitory effect of the Motixafortide-camptothecin conjugate on tumor cell proliferation at the cell level of the present invention;

[0035] Fig.10 To evaluate the inhibitory effect of Motixafortide combined with camptothecin on tumor cell proliferation at the cellular level of the present invention;

[0036] Fig.11 This is the time-effect curve of the Motixafortide-camptothecin conjugate of the present invention in inhibiting tumor cell proliferation;

[0037] Fig.12 The uptake of FXY-922 by the cell B16-F10 of the present invention;

[0038] Fig.13 The schematic diagram of the release of camptothecin based on intramolecular self-quenching monitoring;

[0039] Fig.14 The present invention utilizes the intramolecular self-quenching principle to monitor the release of camptothecin;

[0040] Fig.15 The water solubility of Camptothecin and Motixafortide-Camptothecin conjugate determined in the present invention;

[0041] Fig.16 The present invention is used to determine the inhibition of tumor cell migration by Motixafortide-camptothecin conjugate;

[0042] Fig.17 Evaluation of the IC of Motixafortide-camptothecin conjugate at the cell level of the present invention 50 (μM); DETAILED DESCRIPTION

[0043] Below in conjunction with the accompanying drawings and specific embodiments, the specific embodiments of the present invention are described in further detail. In the specific embodiments, each original reagent and raw material all can be purchased. It should be pointed out that the following detailed description is all exemplary, and is intended to provide further description of the present invention. Unless otherwise indicated, all technical and scientific terms used in the present invention have the same meanings as those of ordinary skill in the art to which the present invention belongs.

[0044] As mentioned above, camptothecin is highly toxic to normal cells and has extremely poor water solubility. In addition, camptothecin has poor membrane permeability and is difficult to penetrate the cell membrane and enter the cytoplasm in a short period of time, so it takes a long time to exert its anti-tumor effect. These problems have greatly limited the further application of camptothecin in clinical practice.

[0045] In view of this, in a typical embodiment of the present invention, a Motixafortide-camptothecin conjugate is provided, wherein the Motixafortide-camptothecin conjugate comprises the following sequence:

[0046]

[0047] The above-mentioned Motixafortide-camptothecin conjugate can significantly improve the membrane permeability and water solubility of camptothecin, and improve its anti-tumor activity. By utilizing the different expression levels of CXCR4 on the tumor surface, the conjugate can be targeted to tumor cells and reduce the toxicity to normal cells. At the same time, by combining camptothecin with Dnp, the released camptothecin exhibits blue fluorescence, realizing the temporal and spatial monitoring of camptothecin release.

[0048] In another specific embodiment of the present invention, a method for synthesizing the above-mentioned Motixafortide-camptothecin conjugate is provided, and the synthesis method comprises: solid phase synthesis of the conjugate; covalently linking the polypeptide with a linker group and a small molecule drug group through a solid phase condensation reaction.

[0049] Specifically, the above-mentioned polypeptide is synthesized by solid phase peptide synthesis (SPPS) based on 9-fluorenylmethoxycarbonyl.

[0050] Unless otherwise specified, the present invention selects Rink-AmideAM resin (substitution degree of 0.38 mmol / g) to synthesize polypeptides with amide at the carboxyl end. In the process of synthesizing the polypeptide part, the amino acids used are Fmoc-L-type amino acids except the eighth amino acid lysine (Lys, K), which is Fmoc-D-type amino acid.

[0051] More specifically, the synthesis method of the Motixafortide-camptothecin conjugate comprises:

[0052] Step 1, synthesizing the targeting peptide using a solid phase peptide synthesis method based on Fmoc (9-fluorenylmethoxycarbonyl);

[0053] Step 2: After the polypeptide is partially synthesized, the linking group and different amounts of camptothecin are connected to the targeting peptide through a solid phase condensation reaction;

[0054] Step 3, cyclizing the polypeptide on the resin to form a disulfide bond using a polypeptide solid phase cyclization reagent;

[0055] Step 4, adding a peptide cleavage reagent to the condensation product, cutting the condensation product from the resin and removing the side chain protecting groups of the amino acids, and precipitating with ether to obtain a crude product;

[0056] Step 5: The crude product obtained after peptide cleavage is separated, purified and freeze-dried to obtain Motixafortide-camptothecin conjugate.

[0057] The specific experimental method of step 1 is as follows:

[0058] Weigh the Rink-AmideAM resin, activate the resin, remove the Fmoc protecting group on the resin, couple the required amino acids to the resin in sequence, and then remove the Alloc protection of the side chain of D-lysine to prepare for the next step.

[0059] The specific activation process of the above resin is: the resin is rinsed with DMF and DCM alternately, and soaked with DMF at 28°C for 1 hour. Then, the resin is rinsed with DMF and DCM alternately, and activated in a thermostatic oscillator at 28°C with DMF:DCM=1:1 for 0.5-1 hour.

[0060] The Fmoc protecting group was removed by using a DMF solution (v:v) containing 20% ​​piperidine at 28°C for two times, with the first time lasting 5 minutes and the second time lasting 10 minutes.

[0061] The condensation reaction test method is: each amino acid is condensed twice at 28°C, the first time is 35 minutes, the second time is 45 minutes, and the ratio of the condensation reagents is Fmoc-Aa-OH:HCTU:DIEA=3:2.8:6 (molar ratio).

[0062] The cleaning method is: rinse with DMF and DCM alternately, and then use a water pump to drain the residual solvent.

[0063] The specific experimental method of step 2 is:

[0064] First, the Alloc side chain of lysine in the peptide synthesized in step 1 was removed by using Pd(PPh 3 ) 4 / PhSiH 3 The reaction was carried out in DMF solvent for 2-3 hours, and then Kaiser test was performed. After deprotection, the AEEA linker was coupled first, and then the camptothecin was coupled, and the reaction ratio was camptothecin / AEEA:HATU:HOAt:DIEA=2:1.8:2:4 (molar ratio).

[0065] The specific experimental method of step 3 is:

[0066] After the coupling of camptothecin is completed, an oxidation reaction is carried out on the resin. Thallium trifluoroacetate is dissolved in a DMF solution containing anisole, first ice-bathed for 5-10 minutes, and then added to the resin to react at room temperature for 1 hour. The reaction ratio is resin: thallium trifluoroacetate = 3:4 (molar ratio). While removing the Acm protecting group of Cys, a disulfide bond is formed in the molecule.

[0067] The specific experimental method of step 4 is:

[0068] After all the camptothecins are coupled, a peptide cleavage reagent is added to the prepared condensation product in a ratio of TFA (trifluoroacetic acid): water: TIPS = 95:2.5:2.5 (v:v:v). The reaction is carried out in a shaker at 28°C in the dark for 2-2.5 hours, the resin is washed twice with 0.5 mL of TFA, the reaction product is concentrated to 2-2.5 mL with high-purity nitrogen, and pre-cooled anhydrous ether is added to the concentrated solution to precipitate the target crude product. The crude peptide is then obtained by centrifugation.

[0069] The specific experimental method of step 5 is:

[0070] The crude peptide product obtained in the above step was dissolved in a mixed solution of acetonitrile and water containing 0.1% TFA, and then the crude peptide was separated and purified by RP-HPLC, and the molecular weight of the separated product was determined by ESI-MS. The separated pure peptide solution was stored at -80°C overnight. Finally, it was freeze-dried to obtain a flocculent powder, which was the target conjugate.

[0071] In another specific embodiment of the present invention, there is provided use of the above-mentioned Motixafortide-camptothecin conjugate in the preparation of anticancer or antitumor drugs.

[0072] In another embodiment of the present invention, the present invention can treat solid tumors and blood tumors. The solid tumors can be breast cancer, nasopharyngeal carcinoma, bladder cancer, bronchial lung cancer, non-small cell lung cancer, prostate cancer, ovarian cancer, neuroblastoma, gastric cancer, papillary thyroid cancer, head and neck squamous cell carcinoma, testicular cancer, liver cancer. The blood tumor can be acute myeloid leukemia, acute lymphocytic leukemia, granulocytic leukemia, malignant lymphoma.

[0073] In another embodiment of the present invention, the drug delivery system of the present invention can improve the membrane permeability of camptothecin, carry camptothecin into tumor cells, and exert its anti-tumor activity. It can also target CXCR4, thereby improving the selectivity of camptothecin for tumor cells and reducing its toxicity to normal cells.

[0074] In another specific embodiment of the present invention, the use of the above-mentioned Motixafortide-camptothecin conjugate as a drug release detection agent is provided. After camptothecin is taken into cells, the Dnp conjugate of the present invention is released from the conjugate to produce blue fluorescence, thereby realizing the spatiotemporal detection of camptothecin release, which can be applied to cell, tissue and living animal imaging, and further explore the anti-tumor activity and mechanism of action of Motixafortide-camptothecin conjugate at the molecular, cellular, tissue and animal levels.

[0075] In another specific embodiment of the present invention, the conjugate of the present invention greatly improves the water solubility of camptothecin, improves the administration method of camptothecin, and promotes the further clinical application of camptothecin.

[0076] In another specific embodiment of the present invention, the conjugate can not only kill tumors, but also inhibit tumor cell migration. The synergistic effect of the two greatly improves the ability of the conjugate to inhibit tumor growth.

[0077] In another embodiment of the present invention, the safety evaluation of the above conjugate as a drug is provided. The safety of the above conjugate is determined by conducting a cytotoxicity test on normal cells (293T, human embryonic kidney cells).

[0078] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0079] Example 1

[0080] In this example, all target conjugates were prepared using Fmoc (9-fluorenylmethoxycarbonyl)-based solid phase peptide synthesis (SPPS). Unless otherwise specified, the present invention selected Rink-AmideAM resin (substitution degree of 0.38 mmol / g) to synthesize peptides with amide at the C-terminus. In the process of synthesizing the peptide part, the amino acids used were all Fmoc-L-type amino acids except the eighth amino acid lysine (Lys, K), which was a D-type amino acid.

[0081] The scale of conjugate synthesis is generally 0.15 mmol. The basic process of conjugate synthesis is as follows: Figure 1 shown.

[0082] Solid-phase peptide synthesis: Weigh 400 mg of Rink-AmideAM resin (1 eq, 0.15 mmol), rinse the resin alternately with DCM and DMF, soak the resin in high-purity DMF for 1-2 hours, and then activate the resin by shaking in a constant temperature oscillator with DCM:DMF=1:1 for 0.5-1 hour. Then, remove the Fmoc protecting group on the resin at 28°C using a DMF solution (v:v) containing 20% ​​piperidine, remove twice, the first time for 5 minutes and the second time for 10 minutes, and use Kaiser reagent to detect whether Fmoc is successfully removed. Each amino acid is subjected to two condensation reactions at 28°C, the time is 35 minutes and 45 minutes, and the reaction system is Fmoc-Aa-OH:HCTU:DIEA=3:2.8:6 (molar ratio). After the first amino acid was condensed, the Fmoc protecting group of the amino acid was removed by using 20% ​​piperidine in DMF solution. The above operation was then repeated. After the last substance was condensed, the Alloc side chain of D-lysine was removed by adding Pd(PPh 3 ) 4 Dissolve in a mixed solution of DCM and DMF, and react for 3-4 hours in the dark. The reaction system is resin: Pd(PPh 3 ) 4 =5:3 (molar ratio). Kaiser test was then performed to detect whether the Alloc protecting group was successfully removed.

[0083] Synthesis of Motixafortide-camptothecin conjugate: On the side chain amino group of the exposed lysine in the previous step, a linker and camptothecin were introduced by amide bond reaction. The reaction system of the linker group is as follows: linker group: HATU: HOAt: DIEA = 2: 1.8: 2: 4 (molar ratio). For the synthesis of conjugates with different coupling amounts of camptothecin, the specific reaction system is as follows: the reaction ratio of FXY-922 is camptothecin: HATU: HOAt: DIEA = 2: 1.8: 2: 4 (molar ratio), the ratio of FXY-925 is camptothecin: HATU: HOAt: DIEA = 3: 2.8: 3: 6 (molar ratio), the ratio of FXY-926 is camptothecin: HATU: HOAt: DIEA = 4: 3.8: 4: 8 (molar ratio), and the ratio of FXY-927 is camptothecin: HATU: HOAt: DIEA = 5: 4.8: 5: 10 (molar ratio). When all the groups are coupled, the conjugate is cyclized. Thallium trifluoroacetate is added to the DMF solution containing anisole, the reagent is ice-bathed for 10 minutes, and then added to the resin for reaction for 1-2 hours. During the reaction, the temperature is kept below 30°C, and the specific reaction ratio is resin: thallium trifluoroacetate = 3:4 (molar ratio).

[0084] Peptide cleavage and separation and purification: This process is to cut the conjugate from the resin and obtain the target conjugate. Add a peptide cleavage reagent to the above resin, the peptide cleavage reagent is TFA:TIPS:water=95:2.5:2.5 (v:v:v). React for 2.5-3h at 28°C in a constant temperature oscillator, then wash the resin twice with 0.5mL of TFA, and then concentrate the reactant to about 2mL with high-purity nitrogen. Add pre-cooled anhydrous ether to the concentrate, mix thoroughly and centrifuge, repeat the operation three times, and place the bottom solid in a fume hood to dry. Then dissolve the crude peptide in acetonitrile and water containing 0.1% to obtain a crude peptide solution, and use ESI-MS to analyze and identify the crude peptide solution. Separate and purify the crude peptide solution by RP-HPLC, and freeze-dry the separated pure peptide solution to obtain the target conjugate.

[0085] The chemical structural formula, primary amino acid sequence, analytical RP-HPLC and ESI-MS of the Motixafortide-camptothecin conjugate prepared by the present invention are as follows: Figure 2-Figure 8 shown.

[0086] MTT assay was used to detect the inhibitory effect of the conjugate on tumor cells and its toxicity on normal cells:

[0087] B16-F10, MCF-7, A549, 4T1, Hep3B, Hepa 1-6, PC-3 tumor cells and a normal cell 293T in the logarithmic growth phase were selected and the cells were inoculated in a 96-well plate and allowed to adhere overnight. The next day, camptothecin and conjugates (FXY-904, FXY-922, FXY-925, FXY-926 and FXY-927) were diluted and added to the 96-well plate using the corresponding serum-free medium to make the final concentrations of 100μM, 50μM, 25μM, 12.5μM, 6.25μM, 3.125μM, respectively. Three replicate wells were set for each group and then placed in an incubator for incubation for 24h. Subsequently, 15μL MTT was added to each well and placed in an incubator for incubation. After 4 hours, all the liquid in each well was aspirated, 150 μL DMSO was added and placed in an incubator for 1 hour, and then the absorbance in each well was measured at a wavelength of 492 nm to calculate the inhibitory effect of the conjugate or camptothecin on tumor cells. The parallel experiment was repeated three times.

[0088] like Fig.17 As shown in Figure 2, the prototype targeting peptide, FXY-904, had no inhibitory effect on tumor cell proliferation. All tumor cells were insensitive to camptothecin. 50 The value is greater than 130 μM. In contrast, the entire cancer cell line is sensitive to the conjugate prepared by the present invention. The relationship between the drug loading of the targeting peptide and the anti-tumor activity was explored. The experimental results showed that the anti-tumor activity of FXY-922 obtained by coupling a camptothecin was the best, and the IC 50 The values ​​were between 12.8 and 56.2 μM. The activity of the FXY-925 conjugate with two camptothecins decreased, and the FXY-927 conjugated with four camptothecins almost lost its antitumor activity. The new conjugate inhibited tumor cell growth in a concentration-dependent manner, and its antitumor activity was significantly improved compared with camptothecin.

[0089] 293T is a human embryonic kidney cell. MTT test shows that camptothecin is highly toxic to 293T cells. IC 50 The value was 14.6 μM, while the IC 50 The values ​​were between 62 μM and 101 μM. Compared with camptothecin, the cytotoxicity of the conjugates to 293T cells was reduced by 4.2-6.9 times. Even the conjugate with the best antitumor activity, FXY-922, had an IC 50 The value is only 62.8 μM. Therefore, the conjugate improves the selectivity for tumor cells while reducing the toxicity to normal cells.

[0090] Motixafortide combined with camptothecin conjugate inhibits tumor cell growth:

[0091] B16-F10 cells in logarithmic growth were selected and inoculated in 96-well plates and allowed to adhere overnight. Three groups of combined drug regimens were set up. In the first group, the final concentration of camptothecin was 20 μM, and the conjugate FXY-922 was added at a final concentration of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM; in the second group, the final concentration of camptothecin was 40 μM, and the conjugate FXY-922 was added at a final concentration of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM; in the third group, the mixed solution of the conjugate FXY-904 and camptothecin was 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM. According to the above different combined administration methods, they were co-incubated with B16-F10 tumor cells for 24 hours. MTT was then added to terminate the effect. The absorbance of each well was measured at an absorption wavelength of 492 nm according to the same method as above, and the measurement was repeated three times. The results were recorded and the inhibition rate was calculated according to the formula.

[0092] like Fig. 9 , Fig.10 and Fig.17 As shown in Figure 2, the inhibitory effect of the three combined drugs on tumors was not much improved compared with FXY-904 or camptothecin alone, and more than half of the combination indexes (CI) were greater than 1 ( Fig.10 B), indicating that there is no synergistic effect between the two. The combined use of FXY-904 and camptothecin IC 50 The value is also greater than that of covalently linked FXY-922, indicating that non-covalent bonding cannot effectively inhibit tumor cell growth, indicating that covalent bonds play a vital role in exerting anti-cancer effects.

[0093] Determination of the time-effect of Motixafortide-camptothecin conjugate in inhibiting tumor cell proliferation:

[0094] B16-F10 cells in logarithmic growth were selected and inoculated into six 96-well plates and allowed to adhere overnight. Camptothecin 15μM FXY-904, 15μM FXY-922, 15μM FXY-925, 15μM FXY-926, 15μM FXY-927, 15μM FXY-904+10μM camptothecin and 15μM FXY-904+20μM camptothecin were added, and three replicate test wells were set for each group. The conjugate or camptothecin was incubated with the cells for different times (4h, 12h, 24h, 36h, 48h, 72h), and the cells were treated according to the above method at each time point. The absorbance of each well was measured at a wavelength of 492nm. The experiment was repeated three times in parallel.

[0095] The results are as follows Fig.11As shown, at 4h, the conjugate FXY-922 showed a high ability to inhibit tumor cells, about 30%. After 24h, the inhibition rate of FXY-922 was close to 80%, while the inhibition rates of FXY-904, camptothecin or combined drugs on tumor cells were all less than 60%. The inhibition rate of FXY-922 tended to be stable at 48h-72h, about 90%, while the inhibition rates of camptothecin and FXY-904 at 72h were still lower than FXY-922 (65% and 41%, respectively). The inhibition rates of the conjugates FXY-925, FXY-926 and FXY-927 obtained by coupling two or more camptothecins gradually increased after 24h, and gradually stabilized at 48-72h, about 70%, but still lower than FXY-922 obtained by coupling one camptothecin. The above results show that the conjugate FXY-922 can maintain a high anti-tumor activity whether it is 24h or 72h, and the effect is better than that of camptothecin and combined drug groups. Uptake of the conjugate by CXCR4 high-expressing cells B16-F10:

[0096] B16-F10 cells in logarithmic growth were selected and seeded in 12-well plates at 30,000 cells per well and allowed to adhere to the wall and grow overnight. On the second day, 1 mL of phenol red-free 1640 medium containing SYBR Green I was added to each well and incubated for 30 minutes, then the medium was discarded and 1 mL of phenol red-free 1640 medium containing 12.5 μM FXY-922, 12.5 μM FXY-923, 12.5 μM FXY-904+12.5 μM camptothecin and 12.5 μM FXY-904+diethylaminocoumarin was added to different wells and co-incubated with the cells in the incubator. At different time points (2h, 4h, 6h and 12h), the medium in the wells was discarded and washed three times with PBS, followed by the addition of 1mL of phenol red-free 1640 medium. The cells were observed under a fluorescence microscope, and the nuclei were stained green, while the conjugates FXY-922, FXY-923, coumarin or camptothecin showed blue fluorescence. Three different fields of view were selected for each treatment group for the final statistical analysis.

[0097] like Fig.12As shown in A and 12B, after only 2 hours, the conjugate FXY-922 entered more than 50% of the tumor cells. Similarly, the conjugate FXY-923 obtained by coupling the targeting peptide to coumarin (a widely used fluorescent group) also had a similar uptake within 2 hours. After 12 hours, the conjugates FXY-922 and FXY-923 entered more than 80% of the tumor cells in the field of view. At the same time, the targeting peptide FXY-904 non-covalently linked to camptothecin or coumarin, and the proportion of camptothecin or coumarin entering the cells within 12 hours was less than 10%, indicating that the targeting peptide could not help the non-covalently bound camptothecin or coumarin enter the tumor cells. The above results show that the CXCR4 high-expressing cell B16-F10 has a higher uptake rate of the conjugate FXY-922, while the cell uptake rate of the non-covalent application of the targeting peptide FXY-904 and camptothecin is very low. The cell uptake experiment proves the key role of covalent bonds in exerting anti-tumor activity.

[0098] Real-time monitoring of camptothecin release using the principle of intramolecular self-quenching:

[0099] Dissolve human serum in PBS, weigh an appropriate amount of FXY-928 or a mixture of camptothecin and H-Lys(Dnp)-OH, dissolve it in PBS and add it to the PBS solution containing serum, use a pipette to mix well, so that the final concentration of the sample is 100μM and the final concentration of the serum is 10%. At time zero, immediately take 80μL of the reaction solution, add 20μL of acetonitrile containing 0.1% TFA, mix well by pipetting, and place on ice for 5 minutes, then add 400μL of water containing 0.1% TFA and 60μL of acetonitrile, mix well by pipetting, and store at -80℃. Subsequently, place the mixed sample in a 37℃ water bath, take samples at 4h, 8h, 12h, 24h, 36h, 48h and 72h, and repeat the above steps. Fig.13 As shown, when all the time points are taken, the fluorescence intensity at different time points is measured by a fluorescence spectrophotometer, and the real-time monitoring of camptothecin release is achieved by using the principle of intramolecular self-quenching.

[0100] like Fig.14 As shown, as time goes by, the fluorescence intensity of FXY-928 gradually increases, indicating that the free camptothecin gradually increases, indicating that camptothecin can be released from the conjugate at the human serum level, and at 72 hours, it almost reaches the same fluorescence intensity as the free camptothecin of the same concentration. At the same time, the fluorescence intensity is quantified, and it is found that the release of camptothecin exceeds 80% after 72 hours. The above results show that the conjugate FXY-928 obtained by introducing Dnp into FXY-922 can monitor the release of camptothecin in real time.

[0101] Water solubility test of conjugate FXY-922 and camptothecin:

[0102] First, saturated solutions of FXY-922 and camptothecin were prepared with water as solvent, and their absorbances were measured by ultraviolet spectrophotometer, and the absorbances at a wavelength of 366 nm were recorded. Subsequently, aqueous solutions of the above two substances with known concentrations were prepared respectively (600 μg / mL, 300 μg / mL, 150 μg / mL, 75 μg / mL, 37.5 μg / mL, 18.75 μg / mL for FXY-922; 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL for camptothecin), and the absorption spectra of the above known concentrations were measured respectively, and the absorbances of the corresponding concentrations at 366 nm were recorded. Finally, a concentration-absorbance standard curve was established with concentration as the horizontal axis and absorbance at 366 nm as the vertical axis. The saturated solutions of FXY-922 and camptothecin were introduced into the standard curve, and the concentrations of the saturated solutions of the two were calculated respectively.

[0103] like Fig.15 As shown, the solubility of camptothecin in water is 2.8 μM, while the solubility of FXY-922 in water is greater than 3000 μM. The above results show that the conjugate FXY-922 has better water solubility, and the modification of the targeting peptide greatly improves the water solubility of camptothecin, providing strong help for the wider clinical application of camptothecin.

[0104] Inhibitory cell migration assay of the conjugate FXY-922:

[0105] The well-growing B16-F10 cells were inoculated in a 6-well plate at a cell density of 400,000 / well and allowed to adhere to the wall and grow overnight. When the fusion rate was close to 90%, scratches were made with the tip of a sterile pipette, and then washed twice with PBS, and then treated with low-concentration serum medium containing different concentrations of drugs (12.5μM FXY-904, 12.5μM camptothecin, 12.5μM FXY-922 and 6.25μM FXY-922) for 12 or 24h. Subsequently, the cells were observed under a microscope at different times and quantified.

[0106] like Fig.16 As shown in the figure, compared with the control group, FXY-904, FXY-922, and camptothecin all inhibited the migration of tumor cells. The inhibitory effect of FXY-922 on tumor cells showed a dose-effect relationship, and the inhibitory effect of 12.5μM concentration was significantly stronger than that of 6.25μM concentration. Whether it was 12h or 24h, 12.5μM FXY-922 had a strong inhibitory effect on the migration of tumor cells, which was stronger than FXY-904 and camptothecin at the same concentration, and there was a large statistical difference among the three.

[0107] Finally, it should be noted that the above is only a preferred implementation case of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned implementation case, it is still possible for a technician in the field to modify the technical solution recorded in the aforementioned implementation case, or to perform equivalent replacement on part of it. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Although the above describes the specific implementation methods of the present invention, it is not a limitation on the protection scope of the present invention. A technician in the relevant field should understand that on the basis of the technical solution of the present invention, various modifications or deformations that can be made by those skilled in the art without creative labor are still within the protection scope of the present invention.

Claims

1. A type of Motixafortide-camptothecin conjugate, characterized in that The Motixafortide-camptothecin conjugate comprises the following amino acid sequence 2. The method for preparing the Motixafortide-camptothecin conjugate according to claim 1, characterized in that: The preparation method comprises synthesizing a polypeptide by solid phase polypeptide synthesis, solid phase connecting the polypeptide with a linking group and a small molecule drug group, and constructing a disulfide bond on a resin; Preferably, the polypeptide synthesis method is 9-fluorenylmethoxycarbonyl-based solid phase polypeptide synthesis (Fmoc-SPPS); Preferably, the synthesis method of the Motixafortide-drug conjugate is as follows: The polypeptide is synthesized by a solid phase polypeptide synthesis method based on 9-fluorenylmethoxycarbonyl, and then the synthesized polypeptide is connected with a linking group and a small molecule drug group through a solid phase condensation reaction, a disulfide bond is constructed on the resin, a peptide cleavage reagent is added to the condensation product, the condensation product is cleaved from the resin, and the product after peptide cleavage is separated and purified to obtain a Motixafortide-drug conjugate.

3. Use of the Motixafortide-camptothecin conjugate according to claim 1 in the preparation of drugs or health products for preventing and / or treating tumor-related diseases; Preferably, the tumor is selected from breast cancer, nasopharyngeal carcinoma, bladder cancer, bronchial lung cancer, non-small cell lung cancer, prostate cancer, ovarian cancer, neuroblastoma, gastric cancer, head and neck squamous cell carcinoma, testicular cancer, liver cancer, acute myeloid leukemia, acute lymphocytic leukemia, and malignant lymphoma.

4. A pharmaceutical composition, characterized in that The composition comprises the Motixafortide-camptothecin conjugate according to claim 1.

5. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the polypeptide according to claim 1 and a pharmaceutically acceptable excipient and / or carrier.

6. Use of the Motixafortide-camptothecin conjugate according to claim 1 as a drug release monitoring agent.

7. Use of the polypeptide according to claim 1 as an inhibitor of tumor cell proliferation and / or migration for non-therapeutic purposes.

8. A method for inhibiting tumor cell proliferation and / or migration in vitro, characterized in that: The method comprises administering the polypeptide of claim 1, the pharmaceutical composition of claim 4 or the pharmaceutical preparation of claim 5 to tumor cells cultured in vitro.

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