A [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate and its fluorescence probe, preparation method and application

Abstract

The present invention provides a class of D-configured oncolytic peptides and [Tyr3]Octreotate-D-configured oncolytic peptide conjugates and their preparation methods and applications of fluorescent probes, belonging to the fields of polypeptide preparation and biomedicine. The present invention synthesizes a series of [Tyr3]Octreotate-D-configured oncolytic peptide conjugates and their fluorescent probes, which are prepared by solid-phase polypeptide synthesis. Through experimental verification, the [Tyr3]Octreotate-D-configured oncolytic peptide conjugates obtained in the present invention can significantly improve the stability of oncolytic peptides and enhance the selectivity of oncolytic peptides for tumor cells, showing stronger anti-tumor effects in vitro and in vivo. At the same time, the fluorescent probe of the D-configured oncolytic peptide conjugate prepared in the present invention realizes the spatio-temporal tracing and cell localization of oncolytic peptides, and thus has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptide preparation and biomedicine technology, and specifically relates to a [Tyr 3 ] Preparation method and application of Octreotate-D-configuration oncolytic peptide conjugate and its fluorescent probe. 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] Cancer is a major public health problem worldwide, with high morbidity and mortality. In recent years, tumor immunotherapy has attracted widespread attention. Tumor immunotherapy can reactivate and maintain tumor-immune balance, restore the body's anti-tumor immune response, thereby clearing tumors and preventing tumor regeneration. Oncolytic immunotherapy is one of the means of tumor immunotherapy, which directly kills tumor cells and stimulates the immune system to exert efficient anti-tumor effects, including oncolytic viruses, oncolytic peptides, etc.

[0004] Oncolytic peptides are a new type of polypeptide anticancer drugs, most of which are derived from natural host defense peptides. Oncolytic peptides are generally composed of positively charged amino acids and hydrophobic amino acids. Their amphiphilicity and cationicity make them have good tumor inhibitory effects as a whole. Oncolytic peptides have the characteristics of simple structure, easy synthesis, few side effects, and reduced risk of drug resistance. Unlike traditional anticancer drugs that act on various signal pathways or specific targets, oncolytic peptides target negatively charged membrane components and can directly bind to tumor cell membranes to induce membrane lysis, quickly induce cancer cell death, and then release tumor-specific antigens, which in turn induce systemic anticancer immune responses. By directly killing tumor cells and activating immune responses to indirectly kill tumor tissues, oncolytic peptides can achieve efficient tumor killing and reduce the risk of drug resistance.

[0005] Clip-71 (amino acid sequence: H-KFAKFAKKFAKFAKKFAK-NH2) is a linear amphipathic cationic oncolytic peptide that exhibits killing effects on a variety of tumor cell lines, has good synergy with standard anticancer drugs and can reverse drug resistance. The targeted anticancer peptide EP-100 composed of gonadotropin-releasing hormone and Clip-71 has broad prospects. EP-100 can specifically target cells such as ovarian cancer with high expression of LHRH receptors and produce anti-tumor effects.

[0006] Octreotide derivatives [Tyr 3 ]Octreotate(amino acid sequence: ) is an artificially synthesized octapeptide somatostatin analogue, which has high affinity and high specificity for somatostatin receptor 2 (SSTR2) in the G protein-coupled receptor family and is a long-acting specific agonist. [Tyr 3 Octreotate as a targeting peptide 177 Lu-DOTA-[Tyr 3 Octreotate polypeptide drug conjugate (PDC) was approved for marketing in 2018. In the clinical trial research stage, [Tyr 3 Octreotate was also used as the targeting peptide of a variety of PDC candidate drugs, loaded with other radionuclides, for the treatment of neuroendocrine tumors, non-small cell lung cancer, islet cell tumors and other diseases.

[0007] All known organisms on Earth have the characteristic of "chirally homogeneous". The central dogma followed by life is based on D-type DNA / RNA and L-type polypeptides / proteins. The 20 amino acids commonly found in nature are all L-type amino acids (note: glycine has no chirality). Natural polypeptides and proteins are almost all composed of L-type amino acids. Scientists' research on polypeptides and proteins focuses on L-type polypeptides and L-type proteins. The corresponding protein degrading enzymes in the human body can effectively recognize polypeptides or proteins in the natural configuration and cause their activity to be lost. Therefore, traditional L-configured polypeptides have problems such as short half-life, easy enzymatic degradation, and poor stability.

[0008] D-type polypeptides and D-type proteins are composed of D-configured amino acids. So far, there is no evidence that D-type polypeptides and D-type proteins have ever appeared in the evolution of life on Earth. Compared with natural L-type polypeptides and L-type proteins, D-type polypeptides and D-type proteins composed of D-amino acids have unique structures and functions. Due to reasons such as stereoselectivity, they are difficult to be recognized by the in vivo degradation enzyme system. Therefore, they have higher stability and lower immunogenicity under physiological conditions and retain the original protein binding ability.

[0009] The inventor found that Clip-71 (H-KFAKFAKKFAKFAKKFAK-NH2) is a linear polypeptide composed of L-type amino acids. There are eight lysine residues in the sequence, which are easily degraded by proteases, and have various deficiencies such as poor stability, short half-life, and lack of selectivity for tumor cells, which limit its further clinical application. Summary of the Invention

[0010] Clip-71 has deficiencies such as poor stability, weak ability to penetrate cell membranes, short half-life, and poor tumor tissue targeting, which are common in traditional oncolytic peptides. Based on the deficiencies of the above-mentioned prior art, the present invention provides a [Tyr3 Octreotate-D-configured oncolytic peptide conjugate, its preparation method and application. Through modification methods such as N / C-terminal modification, D-configured amino acid substitution, and polypeptide drug conjugation strategies, etc., the present invention synthesized D-configured oncolytic peptide and [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate. In vitro cytotoxicity experiments showed that the modified conjugate peptide could increase the anti-tumor selectivity of the oncolytic peptide, had extremely high enzymatic stability, and had a persistent and efficient inhibitory effect on the proliferation of tumor cells. Cellular uptake experiments showed that [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate penetrated the cell membrane and entered the cell faster than D-configured oncolytic peptide, indicating that the conjugate peptide could achieve precise killing of cancer cells by targeting the SSTR2 receptor, demonstrating the cytotoxicity of the targeting peptide mediated by SSTR2. In vivo anti-tumor experiments showed that [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate could effectively inhibit the growth of tumors in mice and significantly prolong the survival period of mice. In addition, by introducing the fluorescent group 7-DCCA into [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate, the tracing and subcellular localization of the conjugate were achieved. Therefore, the D-configured oncolytic peptide - [Tyr 3 Octreotate conjugate and its fluorescent probe of the present invention have good practical application value.

[0011] Specifically, the present invention is realized through the following technical solutions:

[0012] In the first aspect of the present invention, there is provided a D-configured oncolytic peptide and [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate, and the D-configured oncolytic peptide and [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate comprise the following amino acid residue sequences:

[0013]

[0014] The above D-configured oncolytic peptide and [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate greatly improve the anti-tumor activity and stability of the oncolytic peptide, are not easily degraded by proteases, etc. while destroying the tumor cell membrane, and the conjugate can enter the cell efficiently through specific receptors to play an anti-tumor role. At the same time, by covalently connecting the fluorescent group 7-DCCA with the D-configured oncolytic peptide or [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate, the intracellular tracing and spatio-temporal monitoring of the oncolytic peptide and the conjugate are realized.

[0015] In a second aspect of the present invention, there is provided a method for preparing the above-mentioned D-configured oncolytic peptide and [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate. The preparation method includes synthesizing the D-configured oncolytic peptide (such as Figure 1 ) by solid-phase peptide synthesis, and synthesizing the [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate (such as Figure 2 ) by solid-phase peptide synthesis, covalently linking the oncolytic peptide and the targeting peptide using the linking group AEEA, and constructing a disulfide bond on the resin.

[0016] Specifically, the above-mentioned polypeptides are synthesized by the 9-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis method (Fmoc-SPPS).

[0017] In a third aspect of the present invention, there is provided the use of the above-mentioned D-configured oncolytic peptide and [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate in the preparation of drugs for preventing and / or treating tumor-related diseases.

[0018] Preferably, the tumors include solid tumors and non-solid tumors, and are selected from breast cancer, cervical cancer, endometrial cancer, ovarian cancer, meningioma, head and neck glioma, non-small cell lung cancer, bronchogenic carcinoma, malignant lymphoma, thyroid cancer, thymic carcinoma, pancreatic cancer, bladder cancer, urothelial carcinoma, colorectal cancer, prostate cancer, gastric cancer, renal cancer, liver cancer, skin cancer, melanoma, neuroblastoma, pituitary adenoma, acute myeloid leukemia, acute lymphoblastic leukemia, granulocytic leukemia.

[0019] More preferably, the cancer or tumor is a human cancer or tumor with high expression of somatostatin receptor SSTR2.

[0020] In a fourth aspect of the present invention, there is provided the use of the above-mentioned D-configured oncolytic peptide fluorescent probe and [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate fluorescent probe in spatio-temporal localization and tracing. The fluorescent probe of the present invention can emit blue fluorescence under the irradiation of ultraviolet light, and can be applied to cell and tissue imaging, so as to explore the anti-tumor activity and mechanism of action of the D-configured oncolytic peptide and [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate at the molecular, cellular, tissue, and animal levels.

[0021] The beneficial technical effects of the above technical solutions are as follows:

[0022] 1. The D-configured oncolytic peptide and [Tyr 3Octreotate-D-configured oncolytic peptide conjugate, which enhances the inhibitory activity of oncolytic peptide against tumor cells. Among them, the IC of each peptide at 24 h is 50 (such as Figure 16 ) as follows: In A549 cells, 36.6 ± 6.1 μM (QYM-1), 28.5 ± 5.1 μM (QYM-2), 71.2 ± 8.4 μM (QYM-3), 17.9 ± 1.8 μM (QYM-4), 36.6 ± 2.9 μM (QYM-5), 21.2 ± 3.6 μM (QYM-6), 18.1 ± 2.3 μM (QYM-7); in 4T1 cells, 74.6 ± 5.6 μM (QYM-1), 13.4 ± 2.8 μM (QYM-2), 46.1 ± 4.9 μM (QYM-3), 14.1 ± 2.9 μM (QYM-4), 11.6 ± 2.4 μM (QYM-5), 4.4 ± 3.7 μM (QYM-6), 5.8 ± 1.1 μM (QYM-7); in A20 cells, 20.9 ± 4.9 μM (QYM-1), 3.4 ± 1.1 μM (QYM-2), 22.3 ± 1.2 μM (QYM-3), 14.3 ± 2.3 μM (QYM-4), 7.4 ± 2.8 μM (QYM-5), 2.5 ± 0.3 μM (QYM-6), 6.2 ± 2.9 μM (QYM-7); in Daudi cells, 57.1 ± 2.2 μM (QYM-1), 25.2 ± 4.1 μM (QYM-2), 35.5 ± 2.7 μM (QYM-3), 29.1 ± 0.8 μM (QYM-4), 14.9 ± 1.8 μM (QYM-5), 3.0 ± 0.6 μM (QYM-6), 3.4 ± 0.3 μM (QYM-7); finally in MCF-7 cells, 52.9 ± 1.2 μM (QYM-1), 20.8 ± 2.3 μM (QYM-2), 46.1 ± 2.0 μM (QYM-3), 11.6 ± 2.1 μM (QYM-5), 4.4 ± 1.9 μM (QYM-6), showing a significant improvement compared with the prototype oncolytic peptide Clip-1. The inhibitory effect of QYM-2 combined with [Tyr 3 Octreotate on MCF-7 is significantly weaker than that of their covalent bond connection.

[0023] 2. The above technical solution can improve the stability of oncolytic peptides. Clip-71 developed by predecessors is an oncolytic peptide composed of L-amino acids, rich in lysine residues, easily degraded by proteases in the body, with poor stability and a short half-life, which limits its wide application. The present invention has carried out a series of structural optimizations, including C / N-terminal modification, amino acid sequence substitution, etc., and used stable D-configuration amino acids to synthesize a new D-configuration oncolytic peptide. The D-configuration oncolytic peptide has extremely high anti-proteolytic ability and a long half-life, can rapidly disrupt cell membranes, and has a wide range of anti-cancer abilities.

[0024] 3. The above technical solution can improve the transmembrane ability of oncolytic peptides in a short time. The present invention uses [Tyr 3 Octreotate targeting the SSTR2 receptor to condense with Clip-71. The [Tyr 3 Octreotate-D-configuration oncolytic peptide conjugate can carry oncolytic peptides in a short time, specifically target and recognize the SSTR2 receptor on the surface of tumor cells and rapidly internalize into cells, greatly improving the selectivity and membrane permeability of oncolytic peptides, and then enabling a large amount of oncolytic peptides to enter cells to play an anti-tumor role.

[0025] 4. The above technical solution can greatly improve the selectivity of oncolytic peptides. A large number of negative charges are carried on the surface of tumor cells, and oncolytic peptides directly cause cell lysis through electrostatic interaction, so the selectivity for normal cells and various tumor cells is poor. The expression levels of the SSTR2 receptor are different in tumor tissues and normal tissues. Therefore, the [Tyr 3 Octreotate-D-configuration oncolytic peptide conjugate can accumulate in tumor cells with high expression of SSTR2, thereby achieving the purpose of improving the selectivity of oncolytic peptides for tumor cells and reducing toxic and side effects.

[0026] 5. The above technical solution can significantly improve the anti-tumor activity of oncolytic peptides in mice. After intraperitoneal injection of D-configuration oncolytic peptides and [Tyr 3 Octreotate-D-configuration oncolytic peptide conjugates, the tumor weight and volume of Balb / c tumor-bearing mice decreased significantly, and the anti-tumor effect of the [Tyr 3 Octreotate-D-configuration oncolytic peptide conjugate was more significant. While the body weight of the mice remained basically stable, the tumor growth was significantly inhibited, and the survival period of the mice was significantly prolonged.

[0027] In summary, traditional oncolytic peptides have deficiencies such as lack of cell selectivity and poor enzymatic stability, which hinder their clinical application. To avoid the above disadvantages, the present invention designed and synthesized a new [Tyr 3Octreotate-D-configured oncolytic peptide conjugate, which has advantages such as extremely high enzymatic stability, stronger cell selectivity, and more excellent anti-cancer activity in vitro and in vivo. At the same time, through the fluorescence group 7-DCCA in combination with the D-configured oncolytic peptide and 3 Octreotate-D-configured oncolytic peptide conjugate, after entering the cell, emits blue fluorescence, realizing the tracing and spatial level monitoring of the polypeptide entering the cell, and thus has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings forming a part of this invention are used to provide a further understanding of the invention. The schematic embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0029] Figure 1 Schematic diagram of the solid-phase polypeptide synthesis method of the oncolytic peptide of the present invention;

[0030] Figure 2 For the present invention 3 Schematic diagram of the solid-phase polypeptide synthesis method of Octreotate-oncolytic peptide conjugate of the present invention;

[0031] Figure 3 Chemical structural formula, primary amino acid sequence, analytical reverse-phase high performance liquid chromatography chart and mass spectrometry chart of QYM-1 of the present invention;

[0032] Figure 4 Chemical structural formula, primary amino acid sequence, analytical reverse-phase high performance liquid chromatography chart and mass spectrometry chart of QYM-2 of the present invention;

[0033] Figure 5 Chemical structural formula, primary amino acid sequence, analytical reverse-phase high performance liquid chromatography chart and mass spectrometry chart of QYM-3 of the present invention;

[0034] Figure 6 Chemical structural formula, primary amino acid sequence, analytical reverse-phase high performance liquid chromatography chart and mass spectrometry chart of QYM-4 of the present invention;

[0035] Figure 7 Chemical structural formula, primary amino acid sequence, analytical reverse-phase high performance liquid chromatography chart and mass spectrometry chart of QYM-5 of the present invention;

[0036] Figure 8 Chemical structural formula, primary amino acid sequence, analytical reverse-phase high performance liquid chromatography chart and mass spectrometry chart of QYM-6 of the present invention;

[0037] Figure 9 Chemical structural formula, primary amino acid sequence, analytical reverse-phase high performance liquid chromatography chart and mass spectrometry chart of QYM-7 of the present invention;

[0038] Figure 10 For the evaluation of D-configured oncolytic peptides and [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate and the combined use of drugs on the proliferation inhibition rate curve of tumor cells;

[0039] Figure 11 For the evaluation of D-configured oncolytic peptides and [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate on the time-effect curve of the inhibitory effect on 4T1 and MCF-7 tumor cells;

[0040] Figure 12 For the L-configured oncolytic peptide, D-configured oncolytic peptide and [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate in the stability detection curve in human serum;

[0041] Figure 13 For the D-configured oncolytic peptide fluorescence probe and [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate fluorescence probe uptake map in MCF-7;

[0042] Figure 14 For the D-configured oncolytic peptide and [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate on the schematic diagram of tumor inhibition effect on tumor-bearing mice;

[0043] Figure 15 For the D-configured oncolytic peptide and [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate on the treatment survival curve of tumor-bearing mice, the broken line graph of tumor volume and weight, and the broken line graph of mouse body weight;

[0044] Figure 16 For the evaluation of D-configured oncolytic peptides and [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate and the combined use of drugs on the IC 50 (μM); Detailed implementation manners

[0045] The following further describes in detail the specific implementation manners of the present invention in conjunction with the accompanying drawings and specific implementation manners. In the specific implementation manners, all the original reagents and raw materials can be purchased. It should be noted that the following detailed descriptions are all exemplary and are intended to provide further explanations of the present invention. Unless otherwise specified, all the technical and scientific terms used in the present invention have the same meanings as those commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0046] As described above, the oncolytic peptide Clip-71 is composed of L-amino acids and has the disadvantages of high cytotoxicity, low enzymatic stability, high immunogenicity, and a single administration method, which greatly limits its wide clinical application.

[0047] In view of this, in a typical specific embodiment of the present invention, a D-configured oncolytic peptide and a [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate are provided. The oncolytic peptide-polypeptide conjugate comprises the following amino acid residue sequence:

[0048]

[0049] The above D-configured oncolytic peptide and the [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate greatly improve the enzymatic stability and in vitro and in vivo anti-tumor activities of the oncolytic peptide. By utilizing the different expression levels of the SSTR2 receptor on the tumor surface, it can target and enter tumor cells and reduce the toxicity to normal cells, thereby efficiently exerting the anti-tumor effect. At the same time, by using the fluorescent group 7-DCCA in combination with the D-configured oncolytic peptide or the [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate, polypeptide tracing and spatio-temporal monitoring are achieved.

[0050] In another specific embodiment of the present invention, a synthesis method of the above D-configured oncolytic peptide and the [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate and its fluorescent probe is provided. The synthesis method includes: synthesizing the oncolytic peptide by solid-phase peptide synthesis; connecting the oncolytic peptide with a linking group, a fluorescent group, and a targeting peptide through a condensation reaction, and constructing a disulfide bond on the resin.

[0051] Specifically, the above polypeptide is synthesized by the 9-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis method (Fmoc-SPPS).

[0052] Unless otherwise specified, the present invention selects Wang resin (substitution degree of 0.33 mmol / g) to synthesize polypeptides containing a carboxyl terminus; selects Rink-Amide AM resin (substitution degree of 0.38 mmol / g) to synthesize polypeptides containing an amide terminus; and the amino acids used in the synthesis process of D-type polypeptides are all Fmoc-D-configured amino acids except glycine (Gly, G).

[0053] More specifically, the synthesis method of the D-configured oncolytic peptide and the [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate and its fluorescent probe includes:

[0054] Step 1: Synthesize D-configured oncolytic peptide by solid-phase peptide synthesis method based on 9-fluorenylmethoxycarbonyl;

[0055] Step 2: Connect the linker group and fluorescent group to the synthesized D-configured oncolytic peptide through dehydration condensation reaction between amino acids;

[0056] Step 3: Add solid-phase cyclization reagent to the resin and cyclize through oxidation reaction to form disulfide bonds.

[0057] Step 4: Add peptide cleavage reagent to the obtained condensation product, cleave the condensation product from the resin, precipitate with ice-cold diethyl ether, and centrifuge to obtain the crude peptide product;

[0058] Step 5: The crude peptide product is separated, purified and freeze-dried to obtain D-configured oncolytic peptide and [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate.

[0059] The specific experimental method of Step 1 is as follows:

[0060] Weigh a certain amount of Rink-Amide AM resin or Wang resin according to the reaction amount, first wash it once standardly, soak and swell it with 3 - 4 mL of analytical pure DMF solvent at room temperature for 1 - 2 h, pre-activate the resin, then soak the resin with 3 - 4 mL of analytical pure DMF / DCM mixed solvent (3:1, v:v), shake it in a constant temperature shaker at 28 °C for 30 min to completely activate the resin. After activation, remove the Fmoc protecting group and perform condensation according to the amino acid sequence. After all condensations are completed, perform standard washing.

[0061] The method for removing the Fmoc protecting group is: Under the condition of 28 °C, use 2 - 3 mL of DMF solution containing 20% piperidine to remove the Fmoc protecting group twice, and the removal times are 5 min and 10 min respectively.

[0062] The method of amino acid condensation reaction is as follows: Dissolve amino acids and condensation reagents with analytical pure DMF, and uniformly add them to the resin. Shake and condense twice at 28 °C. The reaction time of the second condensation reaction can be slightly extended to ensure a complete reaction. When using Wang resin for condensation, the reactant ratio for the condensation of the first amino acid is amino acid: DIC: Oxyma: DAMP = 3 equivalent: 6 equivalent: 3 equivalent: 0.4 equivalent (molar ratio), and the reaction times are 6 h and 10 h; when other amino acids are condensed, the reactant ratio is amino acid: HCTU: DIEA = 3 equivalent: 2.8 equivalent: 6 equivalent (molar ratio), and the reaction times are 30 min and 40 min. After the two reactions are completed, wash once according to the standard. The standard washing method is: Alternately rinse with DMF and DCM. The specific steps are to wash twice with DMF, twice with DCM, once with DMF, once with DCM, and three times with DMF to thoroughly remove impurities in the resin, and then pump dry the residual solvent with a water pump.

[0063] The specific experimental method for step 2 is as follows:

[0064] Condense the D-configured oncolytic peptide synthesized in step 1 with a linking group and a fluorescent group. The reactant ratio is AEEA / 7-DCCA: HATU: HOAt: DIEA = 3 equivalent: 2.8 equivalent: 3 equivalent: 6 equivalent (molar ratio).

[0065] The specific experimental method for step 3 is as follows:

[0066] Add a polypeptide solid-phase cyclization reagent to the condensation product synthesized in step 2 under a constant temperature condition of 28 °C to remove the Acm protecting group of cysteine and form an intramolecular disulfide bond for the thiol group. The reaction ratio of the cyclization reagent is resin: Tl(TFA)3 = 3 equivalent: 2.34 equivalent (molar ratio). Dissolve thallium trifluoroacetate in a DMF solution containing anisole, ice-bath for 5 min, and then add it to the resin and react at room temperature for 3 h.

[0067] The specific experimental method for step 4 is as follows:

[0068] Add a peptide cleavage reagent to the prepared cyclization product. When synthesizing the D-configured oncolytic peptide, the ratio of the peptide cleavage reagent is TFA: phenol: water: TIPS = 88:5:5:2 (v:v:v:v). When synthesizing a targeting peptide containing a disulfide bond, the ratio of the peptide cleavage reagent is TFA

[0069] (Trifluoroacetic acid): TIPS: water = 95:2.5:2.5 (v:v:v). React in the dark in a shaker at 28 °C for 1.5 - 2 h. After the reaction, flow the reaction solution into a three-necked flask through a synthesis tube, and rinse the residual resin 2 times with 0.5 mL of TFA. Bubble-concentrate with high-purity nitrogen to remove excess TFA. When the reaction solution is concentrated to 2 - 3 mL, add pre-cooled anhydrous ether to the concentrated solution, and the target polypeptide forms a flocculent precipitate in the solution. Centrifuge the suspension for 3 min, discard the supernatant to obtain the crude peptide product. Precipitate and centrifuge three times repeatedly, and dry the crude product in a fume hood.

[0070] The specific experimental method of step 5 is as follows:

[0071] Add the crude peptide product prepared in step 4 to a mixed solution of acetonitrile and water containing 1‰ TFA, and ultrasonically assist to completely dissolve it. Analyze and identify the obtained polypeptide using analytical RP-HPLC and ESI-MS. The crude peptide solution is freeze-dried by a freeze dryer, and the freeze-dried powder is dissolved again and separated and purified using semi-preparative RP-HPLC, and then obtained the target pure peptide after another round of freeze-drying.

[0072] In another specific embodiment of the present invention, there is provided the above D-configured oncolytic peptide and the application of [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate in the preparation of drugs for preventing and / or treating tumor-related diseases, and its application in anti-cancer or anti-tumor drugs.

[0073] In another specific embodiment of the present invention, the present invention can treat solid tumors and non-solid tumors, including breast cancer, cervical cancer, endometrial cancer, ovarian cancer, meningioma, head and neck glioma, non-small cell lung cancer, bronchogenic carcinoma, thyroid cancer, thymoma, pancreatic cancer, bladder cancer, urothelial carcinoma, colorectal cancer, prostate cancer, gastric cancer, renal cancer, liver cancer, skin cancer, melanoma, neuroblastoma, pituitary adenoma, acute myeloid leukemia, malignant lymphoma, acute lymphoblastic leukemia, granulocytic leukemia.

[0074] In another specific embodiment of the present invention, the treatable cancer or tumor is a cancer or tumor with high expression of somatostatin receptor SSTR2.

[0075] In another specific embodiment of the present invention, there is provided the above D-configured oncolytic peptide fluorescent probe and the application of [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate fluorescent probe as a tumor cell proliferation inhibitor and / or tracer for non-therapeutic purposes. The "non-therapeutic purpose" is, for example, to inhibit tumor cell proliferation and promote tumor cell death in vitro.

[0076] The present invention will be further explained and illustrated by the following examples, but it does not constitute a limitation to the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.

[0077] Example 1

[0078] In this example, all target polypeptides were prepared using 9-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis technology (Fmoc-SPPS). Unless otherwise specified, Wang resin (substitution degree of 0.33 mmol / g) was selected in the present invention to synthesize polypeptides containing carboxyl termini; Rink Amide Am resin (substitution degree of 0.38 mmol / g) was selected to synthesize polypeptides containing amide termini; in the synthesis process of D-type polypeptides, except for glycine (Gly, G), the amino acids used were all Fmoc-D-configured amino acids.

[0079] The scale of polypeptide synthesis was generally 0.15 mmol. The basic synthesis process of polypeptides containing amide termini is as Figure 1 shown, and the basic synthesis process of polypeptides containing carboxyl termini is as Figure 2 shown.

[0080] Solid-phase synthesis experiment of D-configured oncolytic peptide: Take the resin out of the 4°C refrigerator and let it return to room temperature. Weigh 400 mg of Rink-Amide AM resin (1 eq, 0.15 mmol), wash it once with standard DMF and DCM, and pre-activate the resin by soaking it in analytical grade DMF at room temperature for 1 - 2 h. Then soak it at a constant temperature of 28°C in DMF:DCM = 1:1 (v:v) and shake to activate the resin for 0.5 - 1 h. Add 2 - 3 mL of 20% piperidine in DMF solution (v:v) to the activated resin, react twice, the first reaction for 5 min and the second reaction for 10 min, to remove the Fmoc protecting group, and set it aside after standard washing.

[0081] Dissolve the amino acid and the condensation reagent in analytical grade DMF, mix them evenly, and add them to the resin. Each amino acid is condensed twice to ensure sufficient condensation of the amino acid. After the two reactions are completed, wash it once with the standard method and dry it with a water pump for standby. The ratio of the reactants is amino acid: HCTU: DIEA = 3-fold equivalent: 2.8-fold equivalent: 6-fold equivalent (molar ratio).

[0082] [Tyr 3 Synthesis of Octreotate-D-configured oncolytic peptide conjugate: Condense the above-synthesized oncolytic peptide with the linking group through an amide bond, and condense the D-configured oncolytic peptide synthesized in step 1 with the AEEA linking group and the fluorescent group. The ratio of the reactants is AEEA / 7-DCCA: HATU: HOAt: DIEA = 3-fold equivalent: 2.8-fold equivalent: 3-fold equivalent: 6-fold equivalent (molar ratio).

[0083] Cyclization, peptide cleavage, and separation and purification:

[0084] After all amino acids are linked, thallium trifluoroacetate is added to a DMF solution containing anisole, and the mixture is ice-bathed for 5 min. Then the thallium trifluoroacetate solution is added to the solid-phase peptide synthesis tube, and the reaction is shaken at a constant temperature of 28 °C for 3 h. The reaction ratio of the cyclization reagent is resin: Tl(TFA)3 = 3 eq: 2.34 eq (molar ratio).

[0085] 20% piperidine solution is added to the cyclization product to remove the Fmoc protecting group, and it is washed once according to the standard. Then it is washed five times with DCM, dried by suction, and the residual solvent in the resin is removed. The peptide cleavage reagent is added, and the reaction is shaken at a constant temperature in a shaker at 28 °C for 2 - 3 h. After the reaction is completed, the solution in the peptide synthesis tube is poured into a three-necked flask, and the residual peptide is rinsed with 0.5 mL of TFA twice. The solutions are combined, and bubbling concentration is carried out using high-purity nitrogen to remove the excess TFA. When the reaction solution is concentrated to 2 - 3 mL, pre-cooled anhydrous ether is added to the concentrated solution, and the target peptide forms a flocculent precipitate in the solution. The suspension is centrifuged for 3 min, and the supernatant is discarded to obtain the crude peptide product. The precipitate is centrifuged three times repeatedly, and the product is placed in a fume hood to dry.

[0086] The crude peptide product is dissolved in a mixed solution of acetonitrile and water containing 1‰ TFA, and the obtained crude peptide is analyzed and identified by analytical RP-HPLC and ESI-MS. The identified crude peptide is freeze-dried by a freeze dryer, dissolved, and separated and purified by semi-preparative reverse-phase high-performance liquid chromatography, and the target pure peptide is obtained by freeze-drying.

[0087] The chemical structural formulas, primary amino acid sequences, analytical reverse-phase high-performance liquid chromatography diagrams, and ESI-MS mass spectrometry diagrams of the 7 polypeptides prepared by the present invention are as Figures 3 - 9 shown.

[0088] CCK-8 method for detecting tumor cell proliferation inhibition experiment:

[0089] A549, 4T1, A20, Daudi, and MCF-7 cells in the logarithmic growth phase are selected, collected, and inoculated on a 96-well plate at a density of about 1×10 4 cells / well. After inoculating the cells, they are pre-cultured in a cell incubator at 37 °C and 5% CO2 for 24 h, and 50 μL of QYM-1, QYM-2, QYM-3, QYM-4, QYM-5, QYM-6, QYM-7, and [Tyr 3Octreotate, with 3 replicate wells set in each group and the DMSO content in all groups ≤ 0.1%, was continuously cultured in an incubator for 24 h. After incubation, CCK-8 solution was added to each well to a final concentration of 5 μmol·L -1 . After incubating in the incubator for 3 - 4 h, the absorbance value was measured using a microplate reader at a wavelength of 450 nm, and the measurement was repeated three times.

[0090] As Figure 10 shown, the synthesized novel D-configured oncolytic peptide and [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate have significant anti-tumor activities. The L-configured prototype peptide QYM-1 is insensitive to other tumor cells except A20 cells; the prototype targeting peptide [Tyr 3 Octreotate has no inhibitory effect on the proliferation of all tumor cells. The activity of the peptide QYM-3 modified with acetylated and hydrazide-terminated ends has no significant change, while the D-configured oncolytic peptide QYM-2 has a significant enhancement, indicating that D-configured polypeptides have higher anti-tumor activities compared to L-configured polypeptides. [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate has a strong killing effect on all tumor cells, and its activity against suspension cells is higher than that against adherent cells. The anti-tumor activity of the non-covalently linked combination drug is basically equivalent to that of the D-configured oncolytic peptide, but weaker than that of the covalently linked QYM-6 conjugate, indicating that the formation of covalent bonds plays an important role in inhibiting tumor growth.

[0091] Determination of the 72 h time effect of inhibiting tumor cell proliferation:

[0092] 4T1 and MCF-7 cells in the logarithmic growth phase were selected, centrifuged to collect the cells, and seeded in a 96-well plate at a density of about 1×10 4 cells / well, 100 μL per well. After incubating in the incubator for 24 h, wells with good adherent status were observed, and 50 μL of QYM-1, QYM-2, QYM-3, QYM-5, and QYM-6 were added to each well respectively, with 3 replicate wells set in each group to a final concentration of 10 μM. After continuously culturing the cells in the incubator for 4 h, 8 h, 24 h, 36 h, 48 h, and 72 h, 15 μL of CCK-8 solution was added to each well to a final concentration of 5 μmol·L -1 . After shaking well, it was placed in the incubator for incubation for 3 - 4 h, and the absorbance value was measured at a wavelength of 450 nm, and the measurement was repeated three times.

[0093] The results are as Figure 11 shown, [Tyr 3The octreotate-D-configured oncolytic peptide conjugate, namely QYM-6, already exhibited strong tumor cell killing ability at 12 h, with an inhibitory effect reaching 80%, and it tended to be stable within 12 - 72 h, showing a long-lasting anti-tumor effect. The two L-configured polypeptides, QYM-1 and QYM-3, reached their peaks at around 24 h, but showed a continuous downward trend after 24 h, indicating that the terminal modification strategy could only slightly improve the anti-tumor activity but could not maintain a lasting killing effect. Compared with the L-configured polypeptides, the anti-tumor abilities of QYM-2 and QYM-5 polypeptides were significantly improved, and the inhibitory effect within 24 h exceeded 50%, but there was still a decline to varying degrees subsequently.

[0094] Serum stability experiment:

[0095] Take fresh peripheral blood from normal people. After standing and centrifuging, take the supernatant to obtain serum and store it in a -80 °C refrigerator for later use. Accurately weigh the polypeptide powders of QYM-1, QYM-2, QYM-3, QYM-5, and QYM-6 and dissolve them in PBS solution to prepare a 100 μM polypeptide solution. Add an equal volume of 10% serum solution and incubate at 37 °C in a constant temperature water bath. At 0, 4, 8, 24, 36, 48, and 72 h of the reaction, respectively, pipette 50 μL of the reaction solution, add 50 μL of acetonitrile and 250 μL of deionized water solution (both containing 0.1% TFA) to terminate the reaction. Store the reaction solution at -20 °C for later use. Before use, thaw the reaction solution, centrifuge it, and take the supernatant for RP-HPLC analysis.

[0096] The results are as Figure 12 shown. After 72 h of incubation, the L-configured polypeptides QYM-1, QYM-3, and QYM-5 were completely degraded in serum, while the D-configured polypeptides QYM-2 and QYM-6 were relatively resistant to hydrolysis, and about 80% and 40% still remained in serum after 72 h. The above results indicate that the stability of D-configured polypeptides is much higher than that of L-configured polypeptides.

[0097] Uptake experiment of MCF-7 cells with high SSTR2 expression:

[0098] Select MCF-7 cells in logarithmic growth phase, centrifuge to collect the cells and at about 5×10 4The cells were seeded at a density of

[0099] per well onto a 12-well plate and allowed to adhere overnight. After incubating in an incubator for 24 h, 500 μL of DMEM medium containing 1 μL of SYBR Green I (10,000×) staining solution was added to fluorescently label the cell nuclei for 30 min. After labeling, 5 μM QYM-4, 5 μM QYM-7, and 5 μM QYM-2 + 5 μM 7-DCCA were added to the wells and co-incubated. At 10 min, 30 min, 60 min, and 90 min, the medium was discarded, the cells were washed three times with PBS, and then DMEM medium without phenol red was added. Fluorescence microscopy was used to observe and take pictures. Figure 13 As shown in 3 the results, as time increased, the fluorescence intensities detected by QYM-4 and QYM-7 both gradually increased, indicating that both were taken up by tumor cells. Medium blue fluorescence was detected near the cell nuclei after incubating with QYM-7 for 10 min, and the ratio of blue to green fluorescence (about 2) increased significantly. After 90 min of treatment, bright blue fluorescence was shown around the cell nuclei, indicating that a large amount of the hybrid peptide had entered the cells. At the same time, almost no blue fluorescence was observed for the non-covalently linked QYM-2 and coumarin within 90 min, indicating that the non-covalently bound oncolytic peptide and coumarin could not enter tumor cells. The above results indicate that SSTR2-high-expressing cells MCF-7 have a high uptake rate for the [Tyr

[0100] Antitumor experiment in mice:

[0101] A tumor-bearing mouse model was constructed by injecting approximately 10 7 4T1-LUC cells into the right axilla of the abdomen of 6-week-old male Balb / c mice. The mice were randomly divided into 4 groups of 5 each and received saline, 5 μmol / kg of QYM-2, 2.5 μmol / kg of QYM-6, and 5 μmol / kg of QYM-6, respectively, by intraperitoneal injection every other day for a total of two weeks. During the experiment, the body weights of the mice and the volumes of the tumors (volume = length × width 2 × 0.5) were measured and recorded every two days. After the experiment, all the mice were euthanized, and the tumor tissues were dissected and weighed.

[0102] The results are shown in Figure 14 and Figure 15As shown, from the in vitro tumor images, intraperitoneal injection of QYM-2 did not achieve a good tumor suppression effect, and the tumor volume only decreased slightly. The QYM-6 group showed a strong effect in inhibiting tumor growth in vivo. When injected at a low dose of 2.5 μmol / kg, trends such as a decrease in tumor weight and volume were observed. In mice injected with a high dose of 5 μmol / kg, obvious tumor necrosis and volume reduction occurred on the 10th day, and the inhibitory effect was significantly stronger than that of the low-dose group.

[0103] Treatment with QYM-2 and QYM-6 significantly prolonged the survival time of the mice. Mice in all three treatment groups survived until 30 days. In contrast, due to the large tumor growth, mice in the control group were all euthanized within 15 days. During the entire experiment, the body weights of the mice did not decrease significantly, indicating that the mice had good tolerance to the polypeptides at the therapeutic dose.

[0104] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of them. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention. Although the specific implementation manners of the present invention have been described above, it is not a limitation on the protection scope of the present invention. Those skilled in the art should understand that various modifications or deformations that can be made without creative labor on the basis of the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate, characterized in that, Its structure is as follows:

2. A [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate fluorescent probe, characterized in that, Its structure is as follows:

3. The application of the Octreotate-D-configured oncolytic peptide conjugate according to claim 1 or the Octreotate-D-configured oncolytic peptide conjugate fluorescent probe according to claim 2 in the preparation of a drug for treating tumor diseases, characterized in that, 3 3 The tumor disease is one or more of breast cancer, non-small cell lung cancer or malignant lymphoma.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate described in claim 1 or the [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate fluorescent probe described in claim 2.

5. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation contains the pharmaceutical composition described in claim 4 and pharmaceutically acceptable excipients; The excipients include buffering agents, solubilizers, fillers, disintegrants, diluents, binders, lubricants, emulsifiers, surfactants, colorants, suspending agents and flavoring agents.

6. Use of the [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate or the [Tyr 3 Octreotate-D-configured oncolytic peptide conjugate fluorescent probe for preparing a tracer.

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