A pH-responsive octreotide-D-configuration oncolytic peptide conjugate, its preparation method and application

Abstract

This invention provides an octreotide-D-configuration oncolytic peptide conjugate, its preparation method, and its applications, belonging to the fields of peptide preparation and biomedical technology. Addressing the shortcomings of L-configuration oncolytic peptides, such as poor stability, low antitumor activity, high cytotoxicity to normal cells, and limitation to intratumoral injection, this invention designs and synthesizes a pH-responsive octreotide-D-configuration oncolytic peptide conjugate. This pH-responsive octreotide-D-configuration oncolytic peptide conjugate significantly improves the antitumor activity and stability of the oncolytic peptide, exhibiting a stronger inhibitory effect on tumor cell proliferation. The conjugate provided by this invention has advantages such as lower toxicity to normal cells and stronger activity against tumor cell lines highly expressing somatostatin receptors. Furthermore, this invention can optimize the administration method of the oncolytic peptide, thus possessing good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide preparation and biomedical technology, specifically relating to an octreotide-D-configuration oncolytic peptide conjugate, its preparation method, and its application. Background Technology

[0002] Cancer poses a serious threat to human health. Chemotherapy is a conventional treatment for cancer, but it has many limitations due to poor water solubility, drug resistance, and strong systemic toxicity. Oncolytic peptides are bioactive peptides with tumor-suppressive effects. They can inhibit tumor growth through multiple mechanisms, including directly killing tumor cells, altering the tumor microenvironment, and activating the immune system, achieving highly efficient and rapid anti-tumor effects. Oncolytic peptides have advantages such as small molecular weight, simple structure, high activity, low side effects, diverse administration methods, and low likelihood of inducing drug resistance, and are receiving increasing attention in the field of cancer treatment.

[0003] LTX-315 (amino acid sequence: H-KKWWKKW(Dip)K-NH2), derived from the modified sequence of the antimicrobial peptide bovine lactoferrin, is a cationic oncolytic peptide exhibiting significant antitumor activity against tumor cell lines. This oncolytic peptide can bind to the anionic tumor cell membrane through ion interactions, disrupting the cell membrane and mitochondrial membrane, causing the cell contents to leak out. It can also activate the autoimmune system to attack tumor cells, leading to tumor cell lysis and necrosis. Through this dual antitumor mechanism, LTX-315 can achieve highly efficient killing of tumor cells. In contrast, traditional oncolytic peptides like LTX-315 are L-configuration polypeptides, which are easily degraded by proteases, have poor stability, short half-life, and potential immunogenicity. D-configuration polypeptides, composed of D-configuration amino acids and glycine, can only be obtained through chemical synthesis and have advantages such as low immunogenicity, high stability, and resistance to protease degradation. Furthermore, because LTX-315 binds to the cell membrane through the attraction of positive and negative charges, killing tumor cells via a membrane-breaking mechanism, it has low differentiation between tumor cells and normal cells, resulting in significant toxic side effects. The high toxicity of LTX-315 limits its clinical application.

[0004] The pH value of normal human tissues is around 7.4, while the pH value of tumor tissues is between 6.0 and 6.8, indicating a slightly acidic environment. This is because the intense metabolism in tumor tissues promotes glucose consumption and lactic acid accumulation, thus the pH conditions in tumor tissues are often 1.0 to 1.5 lower than those in healthy tissues. The slightly acidic pH of the tumor microenvironment provides a suitable environment for designing pH-responsive peptides.

[0005] Octreotide derivatives [Tyr] 3 Octreotate (amino acid sequence: SSTR2 is a cyclic octapeptide with somatostatin activity, containing two D-configuration amino acids, and exhibits high specificity and high affinity for the somatostatin receptor SSTR2. In January 2018, the FDA approved the first peptide-conjugate drug (PDC). 177 Lu-DOTA-[Tyr 3 Octreotate is primarily used to treat somatostatin receptor-positive gastrointestinal and pancreatic neuroendocrine tumors.

[0006] The inventors discovered that although LTX-315 possesses advantages such as high antitumor activity, a broad anticancer spectrum, and low susceptibility to inducing tumor drug resistance, it also suffers from drawbacks such as poor stability and significant toxic side effects. For example... Figure 1 As mentioned above, conventional oncolytic peptides are composed of L-configured amino acids and are rich in basic amino acids such as lysine and arginine, making them easily degraded by proteases, resulting in short half-lives and potential immunogenicity. Furthermore, oncolytic peptides kill cells through membrane permeation mechanisms, exhibiting low differentiation between cancer cells and normal cells, thus also showing strong toxicity to normal cells. In addition, due to their unique antitumor mechanism, oncolytic peptides can only be administered via intratumoral injection, limiting the route of administration. These limitations restrict the widespread application of oncolytic peptides such as LTX-315. Summary of the Invention

[0007] LTX-315 suffers from drawbacks such as easy degradation by proteases, short half-life, potential immunogenicity, poor selectivity for normal cells, and reliance on intratumoral injection, limiting its administration route. To address these shortcomings, this invention provides an octreotide-D-configuration oncolytic peptide conjugate, its preparation method, and its applications. To address the drawbacks of the short half-life and immunogenicity of L-configuration oncolytic peptides, this invention employs a series of structural modifications to obtain a D-configuration oncolytic peptide (YJF-606) composed of D-configuration amino acid residues. Furthermore, to address the issues of poor selectivity for tumor cells and limited administration routes, this invention uses octreotide and a D-configuration oncolytic peptide covalently linked to obtain the octreotide-D-configuration oncolytic peptide conjugate. In addition, in order to further reduce the toxicity of oncolytic peptides to normal cells, this invention takes LTX-315 as an example, designs and synthesizes the corresponding anionic peptide fragment of LTX-315, and synthesizes a pH-responsive D-configuration oncolytic peptide composed of a cationic peptide and an anionic peptide fragment covalently linked, and uses octreotide and the pH-responsive D-configuration oncolytic peptide covalently linked to construct an oncolytic peptide conjugate that is pH-responsive and targets the somatostatin receptor.

[0008] like Figure 2As shown, the octreotide-D-configuration oncolytic peptide conjugate prepared in this experiment utilizes the specific binding characteristic of octreotide to the somatostatin receptor of tumor cells to enhance the killing effect of LTX-315 on tumor cell lines that highly express the somatostatin receptor. Furthermore, the pH-responsive octreotide-D-configuration oncolytic peptide conjugate binds the anionic peptide to the cationic oncolytic peptide (such as...) via D-configuration lysine (D-Lys). Figure 3 Covalent linkage masks the cell-permeable effect of cationic oncolytic peptides on normal cells, reducing toxic side effects. Within normal tissues, anionic peptides can mask the activity of oncolytic peptides. When the conjugate reaches tumor tissue, the weakly acidic environment of the tumor microenvironment neutralizes the negative charge of the anionic peptides, releasing the cationic peptides and restoring their selective killing effect on tumor cells.

[0009] The dose-response curves for antitumor activity showed that the modified pH-responsive octreotide-oncolytic peptide conjugate exhibited good pH sensitivity and enhanced antitumor activity, particularly with significantly improved inhibitory activity against tumor cells highly expressing the SSTR2 receptor, while simultaneously reducing toxicity to normal cells. Therefore, the pH-responsive octreotide-D-configuration oncolytic peptide conjugate of this invention has good practical application value.

[0010] Specifically, the present invention is achieved through the following technical solution:

[0011] In a first aspect, the present invention provides a D-configuration oncolytic peptide and an octreotide-D-configuration oncolytic peptide conjugate, wherein the D-configuration oncolytic peptide and the octreotide-D-configuration oncolytic peptide conjugate comprises the following amino acid residue sequence:

[0012] YJF-606H-kkwwkkw(dip)k-NH2

[0013]

[0014] The aforementioned octreotide-D-configuration oncolytic peptide conjugate significantly enhances the antitumor activity, stability, and selectivity of oncolytic peptides for tumor cells. YJF-608 disrupts cell membranes without being degraded by proteases, thus exerting a stable antitumor effect and exhibiting stronger inhibitory activity against tumor cell lines that highly express the SSTR2 somatostatin receptor.

[0015] A second aspect of the present invention provides a pH-responsive D-configuration oncolytic peptide and a pH-responsive octreotide-D-configuration oncolytic peptide conjugate, wherein the pH-responsive D-configuration oncolytic peptide and the pH-responsive octreotide-D-configuration oncolytic peptide conjugate comprise the following amino acid residue sequence:

[0016]

[0017] The aforementioned pH-responsive D-configuration oncolytic peptides and pH-responsive octreotide-D-configuration oncolytic peptide conjugates exhibit significantly improved selectivity for both tumor and normal cells. In normal cells, the anionic peptides mask the cell-permeable effect of the cationic oncolytic peptides, reducing toxicity. Meanwhile, the weakly acidic environment outside tumor cells neutralizes the negative charge of the anionic peptides, allowing the cationic oncolytic peptides to be released and kill tumor cells. YJF-610 and YJF-611 reduce toxicity to normal cells while killing tumor cells. Furthermore, YJF-611 exhibits stronger inhibitory activity against tumor cell lines highly expressing the SSTR2 somatostatin receptor, while showing lower toxicity to normal cells.

[0018] A third aspect of the present invention provides a method for preparing the above-mentioned octreotide-D-configuration oncolytic peptide, pH-responsive D-configuration oncolytic peptide, and pH-responsive octreotide-D-configuration oncolytic peptide conjugate, wherein the preparation method includes a solid-phase peptide synthesis technique used to synthesize the peptide, and a method for performing a condensation reaction between the peptide and a linker group.

[0019] Specifically, the above-mentioned polypeptides were synthesized using a solid-phase polypeptide synthesis method (Fmoc-SPPS) based on 9-fluorenemethyloxycarbonyl.

[0020] A fourth aspect of the present invention provides the use of the above-described pH-responsive octreotide-D-configuration oncolytic peptide conjugate in the preparation of a drug delivery system.

[0021] Preferably, the drug delivery system is a somatostatin receptor SSTR2 targeted drug delivery system.

[0022] A fifth aspect of the present invention provides the use of the above-described pH-responsive octreotide D-configuration oncolytic peptide conjugate in the preparation of anticancer or antitumor drugs.

[0023] Preferably, the cancer or tumor may be selected from breast cancer, prostate cancer, non-small cell lung cancer, pancreatic cancer, ovarian cancer, cervical cancer, colorectal cancer, neuroblastoma, osteosarcoma, leukemia, glioblastoma, bronchial cancer, thymic cancer, bladder cancer, papillary thyroid carcinoma, squamous cell carcinoma of the head and neck, testicular cancer, and gastric cancer.

[0024] More preferably, the cancer or tumor is a cancer or tumor that highly expresses the somatostatin receptor.

[0025] A sixth aspect of the invention provides a formulation comprising a pH-responsive octreotide-D-configuration oncolytic peptide conjugate and pharmaceutically acceptable excipients and / or carriers.

[0026] The beneficial technical effects of the present invention are as follows:

[0027] 1. This invention can significantly improve the antitumor activity of oncolytic peptides. The octreotide-D-configuration oncolytic peptide conjugate synthesized in this invention uses highly stable D-configuration amino acids to synthesize a novel D-configuration oncolytic peptide, exhibiting higher enzymatic stability compared to conventional oncolytic peptides composed of L-configuration amino acids. Covalently linking octreotide and the D-configuration oncolytic peptide can enhance the antitumor activity of the oncolytic peptide and reduce its toxic side effects, possessing significant application value. The IC50 of each peptide over 24 hours is [not specified in the original text]. 50 ( Figure 10 The values ​​are as follows: In MCF-7 cells, 43.6±2.8 μM (YJF-606), 16.7±1.1 μM (YJF-608), 42.4±3.3 μM (YJF-610), 15.4±1.3 μM (YJF-611); in HepG2 cells, 68.8±1.3 μM (YJF-606), 17.4±2.9 μM (YJF-608), 62.8... The concentrations of YJF-610, YJF-611, and LTX-311 in BON-1 cells were 19.7±1.4μM, 29.8±3.3μM, 32.5±1.7μM, 30.3±5.5μM, and 33.3±2.2μM, respectively, all of which were significantly improved compared to the original oncolytic peptide LTX-315.

[0028] 2. This invention can significantly reduce the toxicity of oncolytic peptides to normal cells. The pH-responsive D-configuration oncolytic peptide conjugates and pH-responsive octreotide-D-configuration oncolytic peptide conjugates synthesized in this invention reduce the toxicity of oncolytic peptides to normal cells. YJF-610 and YJF-611 showed IC50 values ​​of 24h against normal MRC-5 cells. 50 ( Figure 10 The pH values ​​were 177.3 ± 8.7 μM (YJF-610) and 181.2 ± 7.5 μM (YJF-611), respectively. The pH-responsive D-configuration oncolytic peptide conjugate and the pH-responsive octreotide-D-configuration oncolytic peptide conjugate showed significantly reduced toxicity to normal MRC-5 cells compared to the original oncolytic peptide LTX-315.

[0029] 3. This invention can enhance the activity of oncolytic peptides on cells highly expressing the SSTR2 receptor. The octreotide-D-configuration oncolytic peptide conjugate and the pH-responsive octreotide-D-configuration oncolytic peptide conjugate synthesized in this invention can utilize octreotide derivatives [Tyr 3 Octreotate's high affinity for the SSTR2 receptor allows the oncolytic peptide to target tumor cells that highly express the SSTR2 receptor, specifically binding to the SSTR2 receptor and enhancing the inhibitory activity of the oncolytic peptide against tumor cells that highly express the SSTR2 receptor.

[0030] In summary, the previously developed oncolytic peptide LTX-315 is easily degraded by proteases, has a short half-life, possesses potential immunogenicity, exhibits poor selectivity for normal cells, and can only be administered via intratumoral injection, limiting its route of administration. To address these shortcomings, this invention first synthesizes a D-configuration oncolytic peptide (YJF-606), which possesses advantages such as high enzymatic stability, long half-life, low immunogenicity, and enhanced activity. This invention utilizes the specific binding characteristic of octreotide to the somatostatin receptor to target tumor cells highly expressing the SSTR2 somatostatin receptor, improving the efficacy of LTX-315, reducing toxicity to normal cells, and improving the administration method. Simultaneously, leveraging the pH difference between normal and tumor tissues, a pH-responsive oncolytic peptide is designed to enhance selectivity for tumor cells. This invention designs and synthesizes a novel pH-responsive octreotide-D-configuration oncolytic peptide conjugate, which exhibits advantages such as high enzymatic stability, enhanced activity, and lower toxicity to normal cells. The preparation method of this invention is simple and effective, possessing significant application value. Attached Figure Description

[0031] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0032] Figure 1 A comparison diagram of the characteristics of L-configuration and D-configuration polypeptides;

[0033] Figure 2 This is a schematic diagram illustrating the selectivity of the octreotide-D-configuration oncolytic peptide of the present invention;

[0034] Figure 3 This is a schematic diagram illustrating the principle of the pH-responsive octreotide-D-configuration oncolytic peptide of the present invention;

[0035] Figure 4 This is a schematic diagram of the solid-phase polypeptide synthesis method containing amide-terminated polypeptides of the present invention;

[0036] Figure 5 This is a schematic diagram of the solid-phase polypeptide synthesis method containing carboxyl-terminated polypeptides of the present invention;

[0037] Figure 6 The chemical structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrum of YJF-606 of this invention are shown below.

[0038] Figure 7 The chemical structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrum of YJF-608 of this invention are shown below.

[0039] Figure 8 The chemical structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrum of YJF-610 of this invention are shown below.

[0040] Figure 9 The chemical structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrum of YJF-611 of this invention are shown below.

[0041] Figure 10 This invention provides an intracellular evaluation of the IC50 of D-configuration oncolytic peptides and octreotide-D-configuration oncolytic peptide conjugates. 50 (μM);

[0042] Figure 11 The present invention provides a cellular-level evaluation of the inhibition rate curves of D-configuration oncolytic peptide and octreotide-D-configuration oncolytic peptide conjugates on the proliferation of tumor cells and normal cells.

[0043] Figure 12 The cell-level evaluation curves of the combined use of D-configuration oncolytic peptide and octreotide-D-configuration oncolytic peptide were used to assess the proliferation inhibition rate of tumor cells and normal cells.

[0044] Figure 13 To evaluate the inhibitory effect of the pH-responsive octreotide-D-configuration oncolytic peptide conjugate YJF-611 on tumor cell proliferation and its toxicity (IC50) to normal cells at the cellular level under different pH conditions, this invention aims to provide an experimental study. 50 μM);

[0045] Figure 14 To evaluate the cytotoxicity (IC50) of the pH-responsive octreotide-D-configuration oncolytic peptide conjugate YJF-611 to high- and low-expressing cells after inhibition of somatostatin receptor at the cellular level, this invention provides an experimental method for evaluating the toxicity (IC50) of the somatostatin receptor-responsive octreotide-D-configuration oncolytic peptide conjugate YJF-611 to cells with high and low expression of somato 50 μM);

[0046] Figure 15 The stability test curves of the L-configuration oncolytic peptide LTX-315 and the pH-responsive octreotide-D-configuration oncolytic peptide conjugate YJF-611 in serum are shown. Detailed Implementation

[0047] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and specific examples. In the specific embodiments, all original reagents and raw materials are commercially available. It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0048] As mentioned earlier, LTX-315 is composed of L-configured amino acids, which have poor enzymatic stability, short half-life, and strong toxicity to normal cells. It can only be administered via intratumoral injection, limiting its application.

[0049] In view of this, in a typical embodiment of the present invention, an octreotide-D-configuration oncolytic peptide conjugate is provided, wherein the octreotide-D-configuration oncolytic peptide conjugate comprises the following amino acid residue sequence:

[0050] YJF-608

[0051] The above-mentioned octreotide-D-configuration oncolytic peptide conjugate has advantages such as high enzymatic stability and stronger activity. Utilizing the characteristic of octreotide derivatives specifically binding to the somatostatin receptor SSTR2, it can target tumor cells that highly express the somatostatin receptor and improve the efficacy of LTX-315.

[0052] In another specific embodiment of the present invention, a pH-responsive D-configuration oncolytic peptide and a pH-responsive octreotide-D-configuration oncolytic peptide conjugate are provided, wherein the pH-responsive D-configuration oncolytic peptide and the pH-responsive octreotide-D-configuration oncolytic peptide conjugate comprise the following amino acid residue sequence:

[0053]

[0054] The aforementioned pH-responsive D-configuration oncolytic peptide and pH-responsive octreotide-D-configuration oncolytic peptide conjugate utilize the pH difference between normal tissues and tumor tissues to design a pH-responsive oncolytic peptide YJF-610 to improve selectivity for normal cells and reduce toxicity to normal cells. At the same time, YJF-611 enhances antitumor activity against tumor cells that highly express somatostatin receptor SSTR2 and improves the administration method.

[0055] In another specific embodiment of the present invention, a method for synthesizing the above-mentioned D-configuration oncolytic peptide and octreotide-D-configuration oncolytic peptide conjugate is provided. The synthesis method includes: a solid-phase peptide synthesis method for synthesizing peptides, and a method for condensing peptides with linking groups and peptides.

[0056] Specifically, the above-mentioned polypeptides were synthesized using a solid-phase polypeptide synthesis method (Fmoc-SPPS) based on 9-fluorenemethyloxycarbonyl.

[0057] Unless otherwise specified, this invention uses RinkAmide Am resin (with a degree of substitution of 0.38 mmol / g) to synthesize polypeptides containing amide terminals; and uses Wang resin (with a degree of substitution of 0.33 mmol / g) to synthesize polypeptides containing carboxyl terminals. In the synthesis of the oncolytic peptide, all amino acids used except glycine (Gly, G) are Fmoc-D-configured amino acids.

[0058] More specifically, the method for synthesizing the octreotide-D-configuration oncolytic peptide conjugate includes:

[0059] Step 1: Synthesize octreotide using a solid-phase polypeptide synthesis method based on 9-fluorenemethyloxycarbonyl.

[0060] Step 2: Attach a linker group to the octreotide sequence;

[0061] Step 3: Synthesize D-configuration peptides using a solid-phase peptide synthesis method based on 9-fluorenemethyloxycarbonyl.

[0062] Step 4: The synthesized polypeptide is cyclized in a solid phase through an oxidation reaction to form disulfide bonds;

[0063] Step 5: Add a peptide cleavage reagent to the cyclization product to cleave the cyclization product off the resin;

[0064] Step 6: The peptide cleavage product was separated, purified, and freeze-dried to obtain the octreotide-D-configuration oncolytic peptide conjugate.

[0065] The specific experimental method for step 1 is as follows:

[0066] Weigh the Wang resin, pre-activate and activate the resin, perform amino acid condensation, wash after all amino acid condensation is completed, remove the last Fmoc protecting group and wash again.

[0067] In another specific embodiment of the present invention, the above-mentioned pre-activation and activation experimental methods are as follows: the resin is alternately rinsed with DMF and DCM, and then soaked in DMF at room temperature for more than 2 hours. After the resin is alternately rinsed with DMF and DCM, the resin is soaked in a DMF / DCM mixed solution (3:1) and shaken in a constant temperature shaker at 28°C for 30 minutes.

[0068] The experimental method for removing the Fmoc protecting group was as follows: at room temperature, the protecting group was removed using a DMF solution containing 20% ​​piperidine. The removal was performed twice. The Fmoc removal time for the second amino acid was 3 min and 5 min, and the removal time for the other amino acids was 5 min and 10 min, respectively.

[0069] The condensation reaction experiment was conducted as follows: each amino acid was condensed twice at 28℃. The condensation reaction time for the first amino acid was 3 h and 4 h, and the reactant ratio was amino acid:DIC:Oxyma:DAMP = 3 equivalents: 6 equivalents: 3 equivalents: 0.5 equivalents. The condensation times for the other amino acids were 30 min and 40 min, respectively, and the reactant ratio was amino acid:HCTU:DIEA = 3 equivalents: 2.8 equivalents: 6 equivalents.

[0070] The cleaning experiment method was as follows: the resin was rinsed alternately with DMF and DCM, and the remaining solvent was pumped out with water.

[0071] The specific experimental method for step 2 is as follows:

[0072] The polypeptide synthesized in step 1 was condensed with the AEEA linker group. The reactant ratio of AEEA was AEEA:HATU:HOAT:DIEA = 3 equivalents: 2.8 equivalents: 3 equivalents: 6 equivalents.

[0073] The specific experimental method for step 3 is as follows:

[0074] Amino acid condensation was carried out on the prepared condensation product at 28℃. Each amino acid was condensed twice, for 30 minutes and 40 minutes respectively. The ratio of reactants was glycine or Fmoc-D-configured amino acid: HCTU: DIPEA = 3 equivalents: 2.8 equivalents: 6 equivalents.

[0075] The specific experimental method for step 4 is as follows:

[0076] The cysteine ​​side chain thiol group in the amino acid sequence of octreotide is protected by an ACM protecting group. The polypeptide synthesized in step 3 was cyclized twice under solid-phase conditions at 28°C for 1 h and 1.5 h, respectively, with a reactant ratio of amino acid:Tl(TFA)3 = 3 equivalents: 2.34 equivalents (molar ratio). Tl(TFA)3 was added to 2.5 mL of LDMF containing 125 μL of anisole, and the mixture was first incubated on ice for 5 min before cyclization at 28°C.

[0077] The specific experimental method for step 5 is as follows:

[0078] Add peptide-cleaving reagent to the prepared cyclization product at a ratio of TFA:TIPS:water = 95:2.5:2.5 (v:v:v). Incubate in a shaker at 28℃-30℃ in the dark for 2.5 h. Wash the resin twice with 1 mL of TFA, 0.5 mL each time. Then concentrate the product to 2-3 mL with high-purity nitrogen. Add pre-cooled anhydrous diethyl ether to the concentrate to precipitate the target peptide. After centrifugation, add anhydrous diethyl ether again, repeating this process three times to obtain a crude peptide precipitate. Let it stand in a fume hood in the dark for at least 20 min.

[0079] The specific experimental method for step 6 is as follows:

[0080] The crude peptide product obtained in step 5 was dissolved in a mixed solution of acetonitrile and water containing 1‰ TFA, and then analyzed and identified by analytical reversed-phase high-performance liquid chromatography (RP-HPLC) and ESI-MS. The dissolved crude peptide was incubated at -80°C overnight. It was then freeze-dried to obtain flocculent crude peptide. The crude peptide was further dissolved in a mixed solution of acetonitrile and water containing 1‰ TFA, and purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The collected pure peptide solution was incubated at -80°C overnight, and then freeze-dried to obtain pure peptide.

[0081] In another specific embodiment of the present invention, the application of the above-mentioned octreotide-D-configuration oncolytic peptide conjugate in the preparation of a drug delivery system is provided.

[0082] In another specific embodiment of the present invention, the drug delivery system of the present invention can be a somatostatin receptor SSTR2 targeted drug delivery system.

[0083] In another specific embodiment of the present invention, the above-mentioned octreotide-D-configuration oncolytic peptide conjugate, pH-responsive D-configuration oncolytic peptide, and pH-responsive octreotide-D-configuration oncolytic peptide conjugate are provided for use in the preparation of anticancer or antitumor drugs.

[0084] In another specific embodiment of the present invention, the present invention can treat solid tumors and hematologic malignancies. The solid tumors and hematologic malignancies may include breast cancer, prostate cancer, non-small cell lung cancer, pancreatic cancer, ovarian cancer, cervical cancer, colorectal cancer, neuroblastoma, osteosarcoma, leukemia, glioblastoma, bronchial cancer, thymic cancer, bladder cancer, papillary thyroid carcinoma, squamous cell carcinoma of the head and neck, testicular cancer, and gastric cancer.

[0085] In another specific embodiment of the present invention, the present invention can treat cancers or tumors with high expression of the statin receptor SSTR2.

[0086] In another specific embodiment of the present invention, a pharmaceutical formulation is provided, comprising an octreotide-D-configuration oncolytic peptide conjugate, a pH-responsive D-configuration oncolytic peptide, a pH-responsive octreotide-D-configuration oncolytic peptide conjugate, and a pharmaceutically acceptable carrier.

[0087] The aforementioned pharmaceutical preparations can be any pharmaceutically acceptable dosage form, such as tablets (including enteric-coated tablets, dispersible tablets, orally disintegrating tablets, effervescent tablets, etc.), soft capsules (including gastric-coated and enteric-coated soft capsules), granules, dry suspensions, powders, oral liquids (including solutions, suspensions, emulsions), and injections (including powders for injection and solutions for injection).

[0088] Depending on the dosage form, other suitable pharmaceutical carriers may be selectively included, such as diluents, fillers, disintegrants, surfactants, suspending agents, binders, lubricants, colorants, flavorings, etc.

[0089] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0090] Example 1

[0091] In this embodiment, all target peptides were prepared using solid-phase peptide synthesis technology based on 9-fluorene methoxycarbonyl (Fmoc-SPPS). Unless otherwise specified, RinkAmideAm resin (degree of substitution 0.38 mmol / g) was selected to synthesize peptides containing amide terminals; Wang resin (degree of substitution 0.33 mmol / g) was selected to synthesize peptides containing carboxyl terminals; and all amino acids used in the synthesis of the oncolytic peptide moiety, except for glycine (Gly, G), were Fmoc-D-configuration amino acids.

[0092] The scale of peptide synthesis is typically 0.15 mmol. The basic process of peptide synthesis is as follows: Figure 4 and Figure 5 As shown.

[0093] Solid-phase peptide synthesis experiment (D-configuration oncolytic peptide): 320 mg of RinkAmide AM resin (1 equivalent) was weighed and rinsed alternately with DMF and DCM. The resin was pre-activated by soaking in DMF at room temperature for 1–2 h. After another alternating rinse, the resin was soaked in a DMF / DCM mixed solution (1:1) and shaken at 28 °C in a constant-temperature shaker for 0.5 h to activate the resin. Then, the Fmoc protecting group was removed using DMF containing 20% ​​piperidine, with two removal cycles of 5 min and 10 min respectively. Each amino acid underwent two condensation cycles at 28 °C for 30 min and 40 min respectively. The reactant ratio was Fmoc-D-configuration amino acid: HCTU: DIPEA = 3 equivalents: 2.8 equivalents: 6 equivalents. After the condensation of the last amino acid, its Fmoc protecting group was removed, and the resin was rinsed alternately with DMF and DCM.

[0094] Synthesis of octreotide-D-configuration oncolytic peptide conjugate: 450 mg of Wang resin (1 equivalent) was weighed and rinsed alternately with DMF and DCM, and pre-activated by soaking in DMF at room temperature for 1–2 h. After another alternating rinse, the resin was activated by shaking in a DMF / DCM mixture (3:1) at 28 °C in a constant temperature shaker for 0.5 h. Then, amino acid condensation was performed, with each amino acid condensed twice at 28 °C. The first amino acid was condensed for 3 h and 4 h, and the reactant ratio was amino acid:DIC:Oxyma:DAMP = 3 equivalent:6 equivalent:3 equivalent:0.5 equivalent. The condensation times for other amino acids were 30 min and 40 min, respectively, and the reactant ratio was amino acid:HCTU:DIEA = 3 equivalent:2.8 equivalent:6 equivalent. The synthesized linear octreotide sequence was condensed with a linker group via an amide bond. The reactant ratio was Fmoc-AEEA-OH:HATU:HOAT:DIEA = 3 equivalents: 2.8 equivalents: 3 equivalents: 6 equivalents. The linker group was then further condensed with an amino acid via an amide bond. After the last amino acid condensation, the Fmoc protecting group was removed, and the mixture was washed alternately with DMF and DCM. The condensed peptide was then cyclized under solid-phase conditions at 28°C for two cycles of 1 h and 1.5 h, with the reactant ratio being amino acid:Tl(TFA)3 = 3 equivalents: 2.34 equivalents. Tl(TFA)3 was added to 2.5 mL of DMF containing 125 μL anisole, and the mixture was incubated on ice for 5 min before cyclization at 28°C.

[0095] Peptide cleavage and purification: Peptide cleavage reagents were added to the prepared condensation product. The ratio of peptide cleavage reagents for the D-configuration oncolytic peptide was TFA:phenol:water:TIPS = 88:5:5:2 (v:v:v:v). The ratio of peptide cleavage reagents for the octreotide-D-configuration oncolytic peptide conjugate was TFA:TIPS:water = 95:2.5:2.5 (v:v:v). The reaction was carried out in a shaker at 28℃-30℃ in the dark for 2.5 h. The resin was washed twice with 1 mL of TFA, 0.5 mL each time. The product was then concentrated to 2-3 mL with high-purity nitrogen. Pre-cooled anhydrous diethyl ether was added to the concentrate to precipitate the target peptide. After centrifugation, anhydrous diethyl ether was added again, and the process was repeated three times to obtain a crude peptide precipitate. The precipitate was then allowed to stand in a fume hood in the dark for at least 20 min. The crude peptide product was then dissolved in a mixed solution of acetonitrile and water containing 1‰ TFA, and the crude peptide was analyzed and identified by analytical reversed-phase high-performance liquid chromatography (RP-HPLC) and ESI-MS. The dissolved crude peptide was incubated at -80°C overnight. It was then freeze-dried to obtain flocculent crude peptide. The crude peptide was further dissolved in a mixed solution of acetonitrile and water containing 1‰ TFA, and purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The collected pure peptide solution was incubated at -80°C overnight, and then freeze-dried to obtain pure peptide.

[0096] The chemical structural formulas, primary amino acid sequences, analytical reversed-phase high-performance liquid chromatography (RP-HPLC) chromatograms, and ESI-MS mass spectra of the four polypeptides obtained in this invention are shown below. Figure 6-9 As shown.

[0097] MTT assay for tumor cell proliferation inhibition:

[0098] MCF-7 and HepG2 tumor cells with high SSTR2 receptor expression in logarithmic growth phase, BON-1 tumor cells with low SSTR2 receptor expression, and MRC-5 normal cells were selected. Cells were collected, counted, and seeded in 96-well plates. After seeding, the cells were sterilized and cultured for 24 hours. Then, 50 μL of YJF-606, YJF-608, YJF-610, YJF-611, LTX-315, and [Tyr] were added to the cells at final concentrations (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM). 3 Octreotate was added to each well, with three sub-wells. Cells were cultured in an incubator for 24 hours. After 24 hours, 15 μL of MTT was added to each well in the dark, gently mixed, and incubated for 4 hours to terminate the culture. Residual liquid in the wells was aspirated, and 150 μL of DMSO was added to each well. The cells were then incubated for 1 hour. The absorbance was measured at 492 nm, repeated three times, and the results were recorded. Subsequent calculations were performed using the formula.

[0099] like Figure 11 As shown, the novel D-configuration oncolytic peptide and octreotide-D-configuration oncolytic peptide conjugate can kill MCF-7, HepG2, and BON-1 tumor cells in a concentration-dependent manner. The IC50 of the novel D-configuration oncolytic peptide and octreotide-D-configuration oncolytic peptide conjugate is [not specified]. 50 Compared with LTX-315, the values ​​were significantly improved, and the antitumor activity was significantly enhanced. Among them, YJF-610 and YJF-611 showed less toxicity to normal cells. In addition, the novel octreotide-D-configuration oncolytic peptide conjugates YJF-608 and YJF-611 significantly enhanced the inhibitory activity against SSTR2 receptor-overexpressing tumor cells MCF-7 and HepG2, while the pH-responsive octreotide-D-configuration oncolytic peptide conjugate YJF-611 significantly enhanced antitumor activity while reducing cytotoxicity to normal cells.

[0100] Combination therapy of octreotide and D-configuration oncolytic peptide inhibits tumor cell proliferation:

[0101] MCF-7, HepG2, BON-1, and MRC-5 cells in logarithmic growth phase were selected, collected, and counted before being seeded in 96-well plates. After seeding, the cells were sterilized and incubated overnight. Six drug groups were set up, including the first group [Tyr...]. 3 A mixed solution of Octreotate and YJF-606 at a concentration ratio of 1:1 was prepared. In the second group, the final concentration of YJF-606 was fixed at 20 μM, and [Tyr] solutions of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM were added. 3 Octreotate solution, third group fixation [Tyr] 3 Octreotate was concentrated to a final concentration of 20 μM. YJF-606 solutions of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM were added. The fourth group [Tyr] 3 A mixed solution of Octreotate and YJF-610 at a concentration ratio of 1:1 was prepared. In the fifth group, the final concentration of YJF-610 was fixed at 20 μM, and [Tyr] solutions of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM were added. 3 Octreotate solution, sixth group fixation [Tyr 3 The final concentration of Octreotate was 20 μM. YJF-610 solutions of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM were added, with three replicates per group. Cells were cultured in an incubator for 24 h. After 24 h, 15 μL of MTT was added to each well in the dark, gently mixed, and incubated for 4 h to terminate the culture. After aspirating any residual liquid from the wells, 150 μL of LDMSO was added to each well, and the cells were shaken for 10 min. The absorbance was measured at a wavelength of 492 nm, repeated three times, and the results were recorded. Subsequent calculations were performed according to the formula.

[0102] like Figure 12 As shown, YJF-606, [Tyr] is covalently bonded. 3 Octreotate (YJF-608) exhibits stronger inhibitory effects on MCF-7 and HepG2 tumor cells that highly express the SSTR2 receptor than their non-covalently linked counterparts, while both show comparable toxicity to normal cells. Similarly, covalently linked YJF-610, [Tyr 3 Octreotate (YJF-611) has a stronger inhibitory effect on MCF-7 and HepG2 tumor cells that highly express the SSTR2 receptor than on their non-covalent linkage, while its toxicity to normal cells is comparable.

[0103] MTT assay for tumor cell proliferation inhibition under different pH conditions:

[0104] MCF-7, BON-1, and MRC-5 cells in logarithmic growth phase were selected, collected, and counted before being seeded into 96-well plates. After seeding, the plates were sterilized and incubated overnight. The culture medium was removed, and 150 μL of YJF-611 at final concentrations of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM were added. The pH was adjusted to 6.0 or 7.4, with three sub-wells for each group. Cells were cultured for another 24 hours in an incubator. After 24 hours, 15 μL of MTT was added to each well in the dark, gently mixed, and incubated for 4 hours to terminate the culture. After removing any residual liquid from the wells, 150 μL of LDMSO was added to each well, and the plates were shaken for 10 minutes. The absorbance was measured at 492 nm, repeated three times, and the results were recorded. Subsequent calculations were performed using the formula.

[0105] like Figure 13 As shown, the novel pH-responsive octreotide-D-configuration oncolytic peptide conjugate YJF-611 killed MCF-7 and BON-1 tumor cells in a concentration-dependent manner at pH 6.0, and was toxic to normal MRC-5 cells. At pH 7.4, YJF-611 showed weak activity against MCF-7 and BON-1 tumor cells, and almost no toxicity to normal MRC-5 cells (IC50). 50 (>200 μM). The above results indicate that the novel pH-responsive octreotide-D-configuration oncolytic peptide conjugate has good pH sensitivity.

[0106] MTT assay for proliferation inhibition of cells with high and low expression of somatostatin receptors:

[0107] MCF-7, HepG2, and BON-1 cells in logarithmic growth phase were selected, collected, and counted before being seeded into 96-well plates. After seeding, the plates were sterilized and incubated overnight. The culture medium was removed, and two groups were set up with three accessory wells in each group. 150 μL of Octreotate (final concentration 100 μM) and complete culture medium were added to each well, respectively, and the cells were incubated for 4 h. After 4 h, the culture medium was removed, and 150 μL of YJF-611 (final concentrations 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM) were added to each well, respectively. The cells were cultured for another 24 h. After 24 h, 15 μL of MTT was added to each well in the dark, gently mixed, and incubated for 4 h to terminate the culture. After removing any residual liquid from the wells, 150 μL of DMSO was added to each well, and the plates were shaken for 10 min. The absorbance value was measured at a wavelength of 492 nm. The measurement was repeated three times, and the results were recorded. Subsequent calculations were performed according to the formula.

[0108] like Figure 14 As shown, after treatment with 100 μM octreotide, the novel pH-responsive octreotide-D-configuration oncolytic peptide conjugate YJF-611 significantly reduced the activity of MCF-7 and HepG2 tumor cells that highly express the SSTR2 receptor, but had no effect on the inhibitory activity of BON-1 tumor cells that lowly express the SSTR2 receptor. These results indicate that the novel pH-responsive octreotide-D-configuration oncolytic peptide conjugate has better binding affinity and activity against tumor cells that highly express the SSTR2 receptor.

[0109] Serum stability assay of pH-responsive octreotide-D-configuration oncolytic peptide conjugate:

[0110] Prepare a 5% serum solution by dissolving serum in PBS. Weigh appropriate amounts of LTX-315 and YJF-611 peptide solids, dissolve them in water, and add the prepared serum solution to the peptide solution to prepare a mixed solution with a final concentration of 200 μM. Shake and stir three times. Immediately take 80 μl of the reaction solution, add 20 μl of acetonitrile containing 0.1% TFA, shake three times, and let stand on ice for 2 minutes. Then add 60 μl of acetonitrile and 400 μl of deionized water, each containing 0.1% TFA, shake three times, and immediately store at -80℃. Seal the reaction tube and incubate it in a constant temperature water bath at 37℃. Take samples at 0.5h, 1h, 2h, 4h, 8h, 16h, 24h, 36h, 48h, and 72h according to the above method. Analyze the remaining peptide amount by RP-HPLC.

[0111] like Figure 15 As shown, the remaining percentage of LTX-315 peptide gradually decreased with increasing time, and almost all of it was hydrolyzed (remaining peptide <10%) at 48 h. In contrast, the YJF-611 conjugate was not easily hydrolyzed, with a remaining percentage of 75% at 72 h. These results indicate that the pH-responsive octreotide-D-configuration oncolytic peptide conjugate possesses extremely strong serum stability.

[0112] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended 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 make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. Although the specific embodiments of the present invention have been described above, they are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A class of octreotide-D-configuration oncolytic peptide conjugates, characterized in that, The octreotide-D-configuration oncolytic peptide conjugate has the following amino acid residue sequence: YJF-608 2. A class of pH-responsive D-configuration oncolytic peptides and pH-responsive octreotide-D-configuration oncolytic peptide conjugates, characterized in that, The pH-responsive D-configuration oncolytic peptide and the pH-responsive octreotide-D-configuration oncolytic peptide conjugate have the following amino acid residue sequences: YJF-610 YJF-611 3. The method for preparing the octreotide-D-configuration oncolytic peptide conjugate of claim 1 and the pH-responsive D-configuration oncolytic peptide and pH-responsive octreotide-D-configuration oncolytic peptide conjugate of claim 2, characterized in that, The preparation method includes a solid-phase polypeptide synthesis method for synthesizing polypeptides, and a method for selectively covalently linking octreotide with linking groups and oncolytic peptides.

4. The preparation method according to claim 3, characterized in that, The polypeptide synthesis method described is a solid-phase polypeptide synthesis method based on 9-fluorenemethyloxycarbonyl.

5. The preparation method according to claim 3, characterized in that, The method for synthesizing the octreotide-D-configuration oncolytic peptide conjugate is as follows: Octreotide was synthesized using a solid-phase polypeptide synthesis method based on 9-fluorenemethyloxycarbonyl. The synthesized polypeptide was then linked to a linker group, an oncolytic peptide, via a condensation reaction. A peptide-cleaving reagent was added to the condensation product to cleave the product from the resin. The cleaved product was then separated and purified to obtain an octreotide-D-configuration oncolytic peptide conjugate.

6. The use of the octreotide-D-configuration oncolytic peptide conjugate of claim 1 and the pH-responsive D-configuration oncolytic peptide and pH-responsive octreotide-D-configuration oncolytic peptide conjugate of claim 2 in the preparation of anticancer drugs, characterized in that, The cancers mentioned are selected from breast cancer, pancreatic cancer, and liver cancer.

7. A pharmaceutical preparation, characterized in that, It comprises the octreotide-D-configuration oncolytic peptide conjugate of claim 1 and the pH-responsive D-configuration oncolytic peptide, pH-responsive octreotide-D-configuration oncolytic peptide conjugate of claim 2, and a pharmaceutically acceptable carrier.

8. The pharmaceutical preparation according to claim 7, characterized in that, The carrier includes fillers, disintegrants, surfactants, binders, lubricants, colorants, suspending agents, and flavoring agents.

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