A Motixafortide-camptothecin conjugate and its preparation method and application

Abstract

The present invention provides a class of Motixafortide-camptothecin conjugates and their preparation methods and applications, belonging to the fields of polypeptide preparation and biomedicine. The present invention synthesizes a series of novel Motixafortide-camptothecin conjugates, which are formed by connecting Motixafortide and different amounts of camptothecin through a linking group. Anti-tumor proliferation and migration experiments, cell uptake and time-effect curves show that Motixafortide-camptothecin conjugates can improve the membrane permeability and anti-tumor activity of camptothecin, improve the selectivity of camptothecin for tumor cells and reduce its toxicity to normal cells. At the same time, the solubility of Motixafortide-camptothecin conjugates is more than 1000 times that of camptothecin. In addition, the intramolecular self-quenching Motixafortide-camptothecin conjugates prepared by the present invention realize real-time monitoring of the release of camptothecin anti-tumor drugs. Therefore, it has good practical application value.

Description

Technical Field

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

[0002] The information disclosed in this background technology section is only intended to enhance 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 those skilled in the art.

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

[0004] At present, a mainstream strategy to solve the problem of selectivity of anti-tumor drugs is to use targeting ligands to deliver cytotoxic drugs to the cancer site, and then exert their corresponding anti-tumor activity. Tumor-specific targeting peptides are covalently coupled with 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 from peptides have attracted widespread attention due to their advantages such as high selectivity, strong tumor penetration, low immunogenicity, and low synthesis cost.

[0005] Overexpression of chemokine receptor 4 (CXCR4) is associated with poor prognosis and chemotherapy resistance in many cancers. CXCR4 plays a crucial role in tumor growth, migration, invasion, metastasis, angiogenesis, and tolerance to therapeutic drugs. Motixafortide (BL8040, BKT-140) is a 14-amino acid cyclic peptide that binds to CXCR4 with high affinity and is a specific inhibitor of CXCR4. This invention covalently couples the anti-tumor drug camptothecin with the peptide motixafrotide to enhance its selective killing effect on tumor cells, aiming to develop a novel and highly effective PDC compound for anti-tumor treatment. Summary of the Invention

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

[0007] Specifically, the present invention is achieved 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 motixafortide-camptothecin conjugate significantly increases the water solubility of camptothecin, helping it recognize and enter tumor cells. This allows camptothecin to more easily reach the nucleus of tumor cells, where it binds to the Topo I-DNA complex, inhibiting DNA replication and RNA synthesis. Furthermore, motixafortide inhibits tumor cell migration and invasion. Camptothecin and motixafortide exert their anti-tumor effects through a synergistic effect. Simultaneously, the release of camptothecin is monitored in real time, both temporally and spatially, utilizing the principle of intrinsic fluorescence 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 peptide solid-phase synthesis method, and covalently linking the peptide and camptothecin using linking groups such as lysine, succinic acid, and AEEA to obtain a peptide drug conjugate.

[0012] Specifically, the polypeptide was 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 cancer, thymic cancer, bladder cancer, bronchogenic carcinoma, non-small cell lung cancer, prostate cancer, ovarian cancer, neuroblastoma, ganglioneuroblastoma, gastric cancer, ganglioneuroma, papillary thyroid carcinoma, head and neck squamous cell carcinoma, and testicular cancer.

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

[0016] A fourth aspect of the present invention provides compounds for monitoring drug release from the aforementioned polypeptide-camptothecin conjugates. The motixafortide-camptothecin conjugates of the present invention, comprising camptothecin covalently linked to a DNP, exhibit no bright fluorescence when the camptothecin is covalently linked to the polypeptide. However, upon release of camptothecin from the polypeptide, the DNP is unable to absorb the free camptothecin fluorescence, resulting in the emission of brighter blue fluorescence. This enables real-time spatiotemporal monitoring of camptothecin release. The compounds 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 action 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), 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 A549 cells. ), 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 were significantly higher than those of 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 period of 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 to camptothecin monomer, the motixafortide-camptothecin conjugate can carry camptothecin to tumor cells and enter cells in a shorter time, greatly improving the selectivity and membrane permeability of camptothecin, thereby allowing a large amount of camptothecin to enter the cell and exert its anti-tumor effect.

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

[0021] 4. The above technical solution can achieve real-time monitoring of camptothecin release through the principle of intramolecular self-quenching. Since the conjugate of this invention is coupled to the polypeptide through the active essential group hydroxyl group of camptothecin, camptothecin needs to be released from the conjugate before it can exert its anti-tumor activity. Taking advantage of the blue fluorescence emitted by camptothecin itself, it 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 achieving 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 the conjugate 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 conjugate obtained by the above technical solution targets CXCR4. Therefore, this conjugate can be widely targeted and internalized into tumor cells with high CXCR4 expression, while being less internalized into cells with low or no CXCR4 expression, thereby achieving the purpose of improving the selectivity of camptothecin for tumor cells and reducing its toxicity.

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

[0024] In summary, while camptothecin exhibits broad-spectrum anti-tumor activity, its low water solubility, low membrane permeability, and high toxicity limit its widespread application. To address these deficiencies, the present invention designed and synthesized a novel motixafortide-camptothecin conjugate, which improves the water solubility of camptothecin. By targeting tumor cells with high CXCR4 expression, it enhances the anti-tumor activity of camptothecin, reduces toxicity to normal cells, and improves tumor cell selectivity and membrane permeability. The present preparation method is simple and effective, with promising application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings, 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 are shown;

[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 are shown;

[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 are shown;

[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 are shown;

[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 are shown;

[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 are shown;

[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 are shown;

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

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

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

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

[0038] Figure 13 Schematic diagram of the release of camptothecin based on intramolecular self-quenching;

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

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

[0041] Figure 16 The present invention measures the inhibition of tumor cell migration by Motixafortide-camptothecin conjugate;

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

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

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

[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 motixafortide-camptothecin conjugate significantly improves the membrane permeability and water solubility of camptothecin, enhancing its anti-tumor activity. By leveraging the varying expression of CXCR4 on tumor surfaces, the conjugate targets tumor cells while reducing toxicity to normal cells. Furthermore, by combining camptothecin with DNP, the released camptothecin exhibits blue fluorescence, enabling temporal and spatial monitoring of camptothecin release.

[0048] In another embodiment of the present invention, a method for synthesizing the above-mentioned Motixafortide-camptothecin conjugate is provided, which comprises: solid-phase synthesis of the conjugate; and covalently linking the polypeptide with a linker group and a small molecule drug group via 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, Rink-Amide AM resin (degree of substitution 0.38 mmol / g) was used in the present invention to synthesize peptides with amide carboxyl termini. All amino acids used in the peptide synthesis process, except for the eighth amino acid lysine (Lys, K), which was an Fmoc-D-type amino acid, were Fmoc-L-type amino acids.

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

[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 linker 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 using a polypeptide solid phase cyclization reagent to form a disulfide bond;

[0055] Step 4: adding a peptide cleavage reagent to the condensation product to cleave the condensation product from the resin and remove the side chain protecting groups of the amino acids, followed by precipitation with diethyl ether to obtain a crude product;

[0056] Step 5: The crude product obtained after peptide cleavage is separated, purified and freeze-dried to obtain a 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 as follows: the resin is alternately rinsed with DMF and DCM and soaked in DMF at 28°C for 1 hour. Then, the resin is alternately rinsed with DMF and DCM 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 containing 20% ​​piperidine (v:v) at 28° C. for two removal steps, with the first step lasting 5 minutes and the second step lasting 10 minutes.

[0061] The condensation reaction test method is as follows: each amino acid is condensed twice at 28°C, the first time is 35 minutes, and the second time is 45 minutes. 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, remove the Alloc side chain from the lysine in the peptide synthesized in Step 1 using Pd(PPh3)4 / PhSiH3 in DMF for 2-3 hours, followed by a Kaiser test. After deprotection, couple the AEEA linker first, followed by camptothecin, using a molar ratio of camptothecin / AEEA:HATU:HOAt:DIEA = 2:1.8:2:4.

[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 and reacted 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 camptothecins have been 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 allowed to proceed 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, and the reaction product is concentrated to 2-2.5 mL using high-purity nitrogen. Pre-cooled anhydrous ether is added to the concentrated solution to precipitate the desired 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 steps was dissolved in a mixture of acetonitrile and water containing 0.1% TFA. The crude peptide was then isolated and purified using RP-HPLC, and the molecular weight of the isolated product was determined using ESI-MS. The purified peptide solution was stored at -80°C overnight. Finally, it was freeze-dried to obtain a flocculent powder, which was the desired conjugate.

[0071] In another 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 hematologic tumors. The solid tumors may be breast cancer, nasopharyngeal carcinoma, bladder cancer, bronchogenic carcinoma, non-small cell lung cancer, prostate cancer, ovarian cancer, neuroblastoma, gastric cancer, papillary thyroid carcinoma, head and neck squamous cell carcinoma, testicular cancer, or liver cancer. The hematologic tumors may be acute myeloid leukemia, acute lymphocytic leukemia, granulocytic leukemia, or malignant lymphoma.

[0073] In another embodiment of the present invention, the drug delivery system can enhance the membrane permeability of camptothecin, allowing it to enter tumor cells and exert its anti-tumor activity. It can also target CXCR4, thereby increasing camptothecin's selectivity for tumor cells and reducing its toxicity to normal cells.

[0074] In another embodiment of the present invention, the motixafortide-camptothecin conjugate is used as a drug release detector. After camptothecin is incorporated into cells, the DNP-camptothecin conjugate is released from the conjugate, generating blue fluorescence. This allows for spatiotemporal detection of camptothecin release. This can be applied to imaging of cells, tissues, and living animals, further exploring the anti-tumor activity and mechanism of action of the motixafortide-camptothecin conjugate at the molecular, cellular, tissue, and animal levels.

[0075] In another 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 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 enhances 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 was 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 is not intended to limit the present invention. It should be understood that these examples are only intended 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, Rink-Amide AM resin (degree of substitution 0.38 mmol / g) was used to synthesize C-terminal amide peptides. All amino acids used in the peptide synthesis process, except for the eighth amino acid lysine (Lys, K), which is D-type, were Fmoc-L-type.

[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-Amide AM resin (1 eq, 0.15 mmol) and rinse the resin alternately with DCM and DMF. Soak the resin in high-purity DMF for 1-2 hours, then activate it by shaking it in a thermostatic oscillator with a 1:1 ratio of DCM:DMF for 0.5-1 hour. The Fmoc protecting group on the resin was then removed twice at 28°C using a DMF solution containing 20% ​​piperidine (v:v). The first removal was for 5 minutes, and the second for 10 minutes. Kaiser reagent was used to detect successful Fmoc removal. Each amino acid was condensed twice at 28°C for 35 and 45 minutes, using a reaction system of Fmoc-Aa-OH:HCTU:DIEA = 3:2.8:6 (molar ratio). After condensing the first amino acid, remove the Fmoc protecting group from this amino acid using a 20% piperidine solution in DMF. Repeat the above steps to remove the Alloc side chain from D-lysine after condensing the last product. This is done by dissolving Pd(PPh3)4 in a mixture of DCM and DMF and reacting in the dark for 3-4 hours. The reaction system is a 5:3 molar ratio of resin to Pd(PPh3)4. A Kaiser test is then performed to determine if the Alloc protecting group has been successfully removed.

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

[0084] Peptide cleavage and purification: This process involves cleaving the conjugate from the resin and obtaining the target conjugate. A peptide cleavage reagent (TFA:TIPS:water = 95:2.5:2.5 (v:v:v)) is added to the resin. The reaction is allowed to proceed at 28°C in a thermostatic shaker for 2.5-3 hours. The resin is then washed twice with 0.5 mL of TFA. The reaction is concentrated to approximately 2 mL using high-purity nitrogen. Pre-cooled anhydrous ether is added to the concentrate, mixed thoroughly, and centrifuged. This process is repeated three times. The solid at the bottom is dried in a fume hood. The crude peptide is then dissolved in 0.1% acetonitrile and water to obtain a crude peptide solution. The crude peptide solution is analyzed and identified using ESI-MS. The crude peptide solution is purified by RP-HPLC, and the purified peptide solution is freeze-dried to obtain the target conjugate.

[0085] The chemical structure, 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 line 293T in the logarithmic growth phase were selected and seeded into 96-well plates and allowed to adhere overnight. The next day, camptothecin and conjugates (FXY-904, FXY-922, FXY-925, FXY-926, and FXY-927) were diluted in the corresponding serum-free medium and added to the 96-well plates to a final concentration of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM, respectively. Three replicate wells were set up for each group and then placed in an incubator for incubation for 24 hours. Subsequently, 15 μL of MTT was added to each well and the cells were placed in an incubator for incubation. After 4 hours, all the liquid in each well was aspirated, 150 μL DMSO was added and the wells were placed in an incubator for 1 hour. The absorbance in each well was then measured at a wavelength of 492 nm to calculate the inhibitory effect of the conjugate or camptothecin on tumor cells. The experiment was repeated three times in parallel.

[0088] like Figure 17 As shown in Figure 2, the prototype targeting peptide, FXY-904, had no effect on inhibiting tumor cell proliferation. All tumor cells were insensitive to camptothecin, and IC 50 The value is greater than 130μM. 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 with a camptothecin was the best, and the IC 50 The values ​​ranged from 12.8 to 56.2 μM. The activity of the FXY-925 conjugate, derived from two camptothecins, decreased, while FXY-927, derived from four camptothecins, nearly lost its antitumor activity. The new conjugate inhibited tumor cell growth in a concentration-dependent manner, demonstrating significantly enhanced antitumor activity compared to camptothecin.

[0089] 293T is a human embryonic kidney cell line. MTT test showed that camptothecin has a high toxicity to 293T cells. IC 50 The value was 14.6 μM, while the IC 50 The values ​​ranged from 62 μM to 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 anti-tumor 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] Logarithmically growing B16-F10 cells were seeded into 96-well plates and allowed to adhere overnight. Three drug combinations were developed: in the first group, the final concentration of camptothecin was 20 μM, and the conjugate FXY-922 was added at final concentrations 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 final concentrations of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM; and in the third group, a mixed solution of the conjugate FXY-904 and camptothecin was added at final concentrations of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM. The cells were incubated with B16-F10 tumor cells for 24 hours using the various combined administration methods described above. MTT was then added to terminate the effect. Following the same treatment method as above, the absorbance of each well was measured at a wavelength of 492 nm. The measurements were repeated three times, and the results were recorded. The inhibition rate was calculated according to the formula.

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

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

[0094] Logarithmically growing B16-F10 cells were seeded into six 96-well plates and allowed to adhere overnight. Three replicate wells were set up for each group of cells, each containing 15 μM FXY-904, 15 μM FXY-922, 15 μM FXY-925, 15 μM FXY-926, 15 μM FXY-927, 15 μM FXY-904 + 10 μM camptothecin, and 15 μM FXY-904 + 20 μM camptothecin. The cells were incubated with the conjugate or camptothecin for various times (4, 12, 24, 36, 48, and 72 hours) and treated as described above at each time point. The absorbance of each well was measured at 492 nm. Three replicates were performed.

[0095] The results are as follows Figure 11As shown, at 4 hours, the FXY-922 conjugate exhibited a high tumor cell inhibition ability of approximately 30%. After 24 hours, the inhibition rate of FXY-922 approached 80%, while the inhibition rates of FXY-904, camptothecin, or the combination were all below 60%. The inhibition rate of FXY-922 stabilized at approximately 90% between 48 and 72 hours, while the inhibition rates of camptothecin and FXY-904 remained lower than those of FXY-922 at 72 hours (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 24 hours and stabilized at approximately 70% between 48 and 72 hours, but were still lower than those of FXY-922, obtained by coupling a single camptothecin. These results demonstrate that the FXY-922 conjugate maintained high antitumor activity for both 24 and 72 hours, and was superior to those of the camptothecin and combination groups. Uptake of the conjugate by CXCR4 high-expressing cells B16-F10:

[0096] Logarithmically growing B16-F10 cells were seeded at 30,000 cells per well in a 12-well plate and allowed to adhere overnight. The next day, 1 mL of phenol red-free 1640 medium containing SYBR Green I was added to each well and incubated for 30 minutes. The medium was then 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 plus 12.5 μM camptothecin, and 12.5 μM FXY-904 plus diethylaminocoumarin was added to different wells and incubated with the cells in an incubator. At different time points (2, 4, 6, and 12 hours), the medium in the wells was discarded and the cells were washed three times with PBS. 1 mL of phenol red-free 1640 medium was then added for observation under a fluorescence microscope. Cell 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 statistical analysis.

[0097] like Figure 12As shown in Figures 12A and 12B, after just 2 hours, the FXY-922 conjugate had entered over 50% of tumor cells. Similarly, FXY-923, a conjugate derived from a targeting peptide coupled to coumarin (a widely used fluorescent group), also achieved similar uptake within 2 hours. After 12 hours, the FXY-922 and FXY-923 conjugates had entered over 80% of the tumor cells within the field of view. Meanwhile, when the targeting peptide FXY-904 was non-covalently linked to camptothecin or coumarin, the percentage of camptothecin or coumarin entering cells within 12 hours was less than 10%, indicating that the targeting peptide was unable to facilitate the non-covalently bound camptothecin or coumarin entry into tumor cells. These results demonstrate that the FXY-922 conjugate has a high uptake rate in CXCR4-high-expressing B16-F10 cells, while the non-covalent uptake rates of the targeting peptide FXY-904 and camptothecin are very low. These cellular uptake experiments demonstrate the critical role of covalent bonding 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, 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 it by pipetting, and place it on ice for 5 minutes, then add 400μL of water containing 0.1% TFA and 60μL of acetonitrile, mix it by pipetting, and store it 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. Figure 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 utilizing the principle of intramolecular self-quenching.

[0100] like Figure 14 As shown, the fluorescence intensity of FXY-928 gradually increased over time, indicating a gradual increase in free camptothecin. This suggests that camptothecin can be released from the conjugate at human serum levels. At 72 hours, the fluorescence intensity reached nearly the same level as free camptothecin at the same concentration. Quantification of the fluorescence intensity revealed that over 80% of camptothecin was released after 72 hours. These results demonstrate that the conjugate FXY-928, obtained by introducing Dnp into FXY-922, can monitor camptothecin release in real time.

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

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

[0103] like Figure 15 As shown in the figure, 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. The modification of the targeting peptide greatly improves the water solubility of camptothecin, providing strong support for the wider clinical application of camptothecin.

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

[0105] Well-growing B16-F10 cells were seeded at a density of 400,000 cells / well in 6-well plates and allowed to adhere overnight. When the confluency approached 90%, the cells were scratched with a sterile pipette tip and washed twice with PBS. The cells were then treated with low-serum medium containing various drug concentrations (12.5 μM FXY-904, 12.5 μM camptothecin, 12.5 μM FXY-922, and 6.25 μM FXY-922) for 12 or 24 hours. The cells were then observed microscopically at various times for quantitative analysis.

[0106] like Figure 16 As shown, compared with the control group, FXY-904, FXY-922, and camptothecin all inhibited tumor cell migration. The inhibitory effect of FXY-922 on tumor cells showed a dose-response relationship, with the inhibitory effect at a concentration of 12.5 μM significantly greater than that at a concentration of 6.25 μM. At both 12 and 24 hours, 12.5 μM FXY-922 exhibited a strong inhibitory effect on tumor cell migration, surpassing that of FXY-904 and camptothecin at the same concentration, with significant statistical differences among the three.

[0107] Finally, it should be noted that the above is only a preferred embodiment 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 embodiment, those skilled in the art can still modify the technical solutions described in the aforementioned embodiment or make equivalent replacements for parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention. Although the above describes the specific embodiments of the present invention, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that, based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. A motixafortide-camptothecin conjugate, characterized in that: The Motixafortide-camptothecin conjugate is selected from the following amino acid sequences:

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 linking the polypeptide with a linker group and a small molecule drug group, and constructing a disulfide bond on a resin; The preparation method of the Motixafortide-camptothecin conjugate is as follows: The polypeptide is synthesized using a 9-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis method, and then the synthesized polypeptide is connected to a linker group and a small molecule drug group through a solid-phase condensation reaction. A disulfide bond is constructed on the resin, and a peptide cleavage reagent is added to the condensation product, which is cleaved from the resin. The peptide cleavage product is separated and purified to obtain a Motixafortide-camptothecin conjugate.

3. Use of the Motixafortide-camptothecin conjugate according to claim 1 in the preparation of a medicament for treating tumor-related diseases; The tumor-related disease is selected from breast cancer, nasopharyngeal cancer, bladder cancer, bronchogenic carcinoma, 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 Motixafortide-camptothecin conjugate according to claim 1 and pharmaceutically acceptable excipients and / or carriers.

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

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