PH-responsive octreotide-D-configuration oncolytic peptide conjugate as well as preparation method and application thereof

Abstract

The invention provides an octreotide-D-configuration oncolytic peptide conjugate as well as a preparation method and application thereof, and belongs to the technical field of polypeptide preparation and biological medicine. The invention designs and synthesizes a pH-responsive octreotide-D-configuration oncolytic peptide conjugate aiming at the defects that an L-configuration oncolytic peptide is poor in stability, low in anti-tumor activity, high in toxicity to normal cells, only capable of performing intratumor injection and the like. The pH-responsive octreotide-D-configuration oncolytic peptide conjugate can significantly improve the anti-tumor activity and stability of the oncolytic peptide, and shows a stronger tumor cell proliferation inhibition effect. The conjugate provided by the invention has the advantages of lower toxicity to normal cells, higher activity to high-expression somatostatin receptor tumor cell lines and the like, and meanwhile, the administration mode of the oncolytic peptide can be optimized, so that the conjugate has a 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 an octreotide-D-configuration oncolytic peptide conjugate and a preparation method and application thereof. Background Art

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

[0003] LTX-315 (amino acid sequence: H-KKWWKKW(Dip)K-NH2), which is modified from the antimicrobial peptide bovine lactoferrin sequence, is a cationic oncolytic peptide that shows significant antitumor activity against tumor cell lines. This oncolytic peptide can bind to the anionic tumor cell membrane through ionic interactions, destroy the cell membrane and mitochondrial membrane to allow the cell contents to flow out, and can also activate the autoimmune system to attack tumor cells, leading to tumor cell lysis and necrosis. Through dual anti-tumor mechanisms, LTX-315 can achieve efficient killing of tumor cells. Traditional oncolytic peptides such as LTX-315 are L-configuration polypeptides, which are easily degraded by proteases, have poor stability, short half-life, and potential immunogenicity. D-configuration polypeptides are composed of D-configuration amino acids and glycine, and can only be obtained through chemical synthesis. They have the advantages of low immunogenicity, strong stability, and not easily degraded by proteases. In addition, because LTX-315 binds to the cell membrane through the mutual attraction between positive and negative charges and kills tumor cells through the membrane-breaking mechanism, LTX-315 has a low ability to distinguish between tumor cells and normal cells and has greater toxic side effects. The high toxicity of LTX-315 limits its clinical application.

[0004] The pH value of normal human tissue is around 7.4, while the pH value of tumor tissue is between 6.0 and 6.8, which is weakly acidic. This is because the intense metabolism in tumor tissue promotes the consumption of glucose and the accumulation of lactic acid, so the pH conditions in tumor tissue are often 1.0 to 1.5 lower than those in healthy tissue. The weakly acidic pH of the tumor microenvironment provides conditions for the design of pH-responsive peptides.

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

[0006] The inventors found that although LTX-315 has advantages such as high anti-tumor activity, broad anti-cancer spectrum, and low induction of tumor resistance, it has disadvantages such as poor stability and large toxic side effects. Figure 1 As described above, since conventional oncolytic peptides are composed of L-configuration amino acids and are rich in basic amino acids such as lysine and arginine, they are easily degraded by proteases, have a short half-life, and are potentially immunogenic. At the same time, oncolytic peptides kill cells through a membrane-breaking mechanism, and have a low degree of differentiation between cancer cells and normal cells, so they are also highly toxic to normal cells. In addition, due to its unique anti-tumor mechanism, oncolytic peptides can only be injected intratumorally, and the route of administration is single. The above limits the widespread application of oncolytic peptides such as LTX-315. Summary of the invention

[0007] LTX-315 is easily degraded by proteases, has a short half-life, has potential immunogenicity, has poor selectivity for normal cells, can only be injected intratumorally, and has a single route of administration. Based on the above-mentioned deficiencies in the prior art, the present invention provides an octreotide-D-configuration oncolytic peptide conjugate and its preparation method and application. In view of the shortcomings of the L-configuration oncolytic peptide, such as short half-life and immunogenicity, the present invention obtains a D-configuration oncolytic peptide (YJF-606) composed of D-configuration amino acid residues through a series of structural modifications. In view of the poor selectivity of oncolytic peptides for tumor cells and the single route of administration, the present invention uses octreotide and D-configuration oncolytic peptides to covalently link to obtain an octreotide-D-configuration oncolytic peptide conjugate. In addition, in order to further reduce the toxicity of oncolytic peptides to normal cells, the present invention takes LTX-315 as an example, designs and synthesizes the anionic peptide segment corresponding to LTX-315, and synthesizes a pH-responsive D-configuration oncolytic peptide covalently bound by a cationic peptide and an anionic peptide segment, and uses octreotide and a pH-responsive D-configuration oncolytic peptide to covalently link to construct an oncolytic peptide conjugate that is pH-responsive and targets the somatostatin receptor.

[0008] like Figure 2As shown in Figure 2, the octreotide-D-oncolytic peptide conjugate prepared in this experiment utilizes the characteristics of octreotide specifically binding to the somatostatin receptor of tumor cells to enhance the killing effect of LTX-315 on tumor cell lines that highly express somatostatin receptors. In addition, the pH-responsive octreotide-D-oncolytic peptide conjugate combines anionic peptide segments with cationic oncolytic peptides (such as Figure 3 ) covalently linked to mask the membrane-breaking effect of cationic oncolytic peptides on normal cells and reduce toxic side effects. In normal tissues, anionic peptides can mask the activity of oncolytic peptides. When the conjugate reaches the tumor tissue, the weakly acidic environment of the tumor microenvironment can neutralize the negative charge of the anionic peptide, and the cationic peptide is released, restoring the selective killing effect on tumor cells.

[0009] The antitumor dose-effect curve shows that the modified pH-responsive octreotide-oncolytic peptide conjugate has good pH sensitivity and improved antitumor activity, especially significantly improved inhibitory activity on tumor cells that highly express SSTR2 receptors, while significantly reducing toxicity to normal cells. Therefore, the pH-responsive octreotide-D-configuration oncolytic peptide conjugate of the present invention has good practical application value.

[0010] Specifically, the present invention is implemented through the following technical solutions:

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

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

[0013]

[0014] The above-mentioned octreotide-D-configuration oncolytic peptide conjugate greatly improves the anti-tumor activity, stability and selectivity of oncolytic peptides for tumor cells. YJF-608 will not be degraded by proteases while destroying cell membranes, exerting a stable anti-tumor effect, and has a stronger inhibitory activity on tumor cell lines that highly express SSTR2 somatostatin receptors.

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

[0016]

[0017] The above-mentioned pH-responsive D-configuration oncolytic peptide and pH-responsive octreotide-D-configuration oncolytic peptide conjugate have greatly improved selectivity for tumor cells and normal cells. In normal cells, the anionic peptide segments mask the membrane-breaking effect of the cationic oncolytic peptide on normal cells, reducing toxic side effects. The weakly acidic environment outside the tumor cells can neutralize the negative charge of the anionic peptide segments, so that the cationic oncolytic peptide is released to kill tumor cells. YJF-610 and YJF-611 reduce toxicity to normal cells while killing tumor cells. In addition, YJF-611 has a stronger inhibitory activity on tumor cell lines that highly express the SSTR2 somatostatin receptor, and is less toxic to normal cells.

[0018] The 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, the preparation method comprising a solid phase polypeptide synthesis technology used for synthesizing polypeptides, and a method for condensing the polypeptide with a linking group.

[0019] Specifically, the polypeptide is synthesized using 9-fluorenylmethoxycarbonyl-based solid phase peptide synthesis (Fmoc-SPPS).

[0020] The fourth aspect of the present invention provides use of the 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] The fifth aspect of the present invention provides the use of the pH-responsive octreotide D-configuration oncolytic peptide conjugate in the preparation of anticancer or antitumor drugs.

[0023] Preferably, the cancer or tumor can 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 carcinoma, thymic carcinoma, bladder cancer, papillary thyroid carcinoma, head and neck squamous cell carcinoma, testicular cancer, and gastric cancer.

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

[0025] In a sixth aspect of the present invention, a preparation is provided, which comprises a pH-responsive octreotide-D-configuration oncolytic peptide conjugate and a pharmaceutically acceptable excipient and / or carrier.

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

[0027] 1. The present invention can greatly improve the anti-tumor activity of oncolytic peptides. The octreotide-D-oncolytic peptide conjugate synthesized by the present invention uses very stable D-amino acids to synthesize novel D-oncolytic peptides, which have higher enzymatic stability than conventional oncolytic peptides composed of L-amino acids. Covalently linking octreotide and D-oncolytic peptides can improve the anti-tumor activity of oncolytic peptides and reduce their toxic side effects, which has great application value. Among them, the IC of each peptide for 24 hours is 50 ( Fig.10 ) were as follows: in MCF-7 cells, 43.6±2.8μM (YJF-606), 16.7±1.1μM (YJF-608), 42.4±3.3μM (YJF-610), 15.4±1.3μM (YJF-611), in HepG2 cells, 68.8±1.3μM (YJF-606), 17.4±2.9μM (YJF-608), 62.8 ±1.4μM (YJF-610), 19.7±2.5μM (YJF-611), and finally in BON-1 cells, 29.8±3.3μM (YJF-606), 32.5±1.7μM (YJF-608), 30.3±5.5μM (YJF-610), 33.3±2.2μM (YJF-611), which were significantly improved compared with the prototype oncolytic peptide LTX-315.

[0028] 2. The present invention can significantly reduce the toxicity of oncolytic peptides to normal cells. The pH-responsive D-configuration oncolytic peptide conjugate and pH-responsive octreotide-D-configuration oncolytic peptide conjugate synthesized by the present invention reduce the toxicity of oncolytic peptides to normal cells. The 24h IC values ​​of YJF-610 and YJF-611 on normal MRC-5 cells are 50 ( Fig.10 ) were 177.3±8.7μM (YJF-610) and 181.2±7.5μM (YJF-611), respectively. The toxicity of pH-responsive D-configuration oncolytic peptide conjugate and pH-responsive octreotide-D-configuration oncolytic peptide conjugate to normal cells MRC-5 was significantly reduced compared with the prototype oncolytic peptide LTX-315.

[0029] 3. The present invention can improve the activity of oncolytic peptides on cells with high expression of SSTR2 receptors. The octreotide-D-configuration oncolytic peptide conjugates and pH-responsive octreotide-D-configuration oncolytic peptide conjugates synthesized by the present invention can be used to 3 ]Octreotate has a high affinity for the SSTR2 receptor, which targets the oncolytic peptide to tumor cells that highly express the SSTR2 receptor, specifically binds to the SSTR2 receptor, and improves the inhibitory activity of the oncolytic peptide against tumor cells that highly express the SSTR2 receptor.

[0030] In summary, the oncolytic peptide LTX-315 developed by predecessors is easily degraded by proteases, has a short half-life, has potential immunogenicity, has poor selectivity for normal cells, can only be injected intratumorally, and has a single route of administration. In view of the above shortcomings, the present invention first synthesized a D-configuration oncolytic peptide (YJF-606), which has the advantages of high enzymatic stability, long half-life, low immunogenicity, and stronger activity. The present invention utilizes the characteristics of octreotide specifically binding to somatostatin receptors, can target tumor cells that highly express SSTR2 somatostatin receptors, improve the efficacy of LTX-315, reduce toxicity to normal cells and improve the mode of administration. At the same time, the pH of normal tissues and tumor tissues is different, and pH-responsive oncolytic peptides are designed to improve the selectivity for tumor cells. The present invention designs and synthesizes a novel pH-responsive octreotide-D-configuration oncolytic peptide conjugate, which has the advantages of high enzymatic stability, stronger activity, and less toxicity to normal cells. The preparation method of the present invention is simple and effective, and has good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0032] Figure 1 This is a comparison chart of the characteristics of L-configuration polypeptide and D-configuration polypeptide;

[0033] Figure 2 Schematic diagram of the selectivity of the octreotide-D-configuration oncolytic peptide of the present invention;

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

[0035] Figure 4 Schematic diagram of the solid phase polypeptide synthesis method containing amide terminal polypeptides of the present invention;

[0036] Figure 5 Schematic diagram of the solid phase polypeptide synthesis method containing a carboxyl-terminal polypeptide of the present invention;

[0037] Figure 6 The chemical structural formula, primary amino acid sequence, analytical reverse-phase high performance liquid chromatogram and mass spectrum of YJF-606 of the present invention;

[0038] Figure 7 The chemical structural formula, primary amino acid sequence, analytical reverse-phase high performance liquid chromatography and mass spectrum of YJF-608 of the present invention;

[0039] Figure 8 The chemical structural formula, primary amino acid sequence, analytical reverse-phase high performance liquid chromatogram and mass spectrum of YJF-610 of the present invention;

[0040] Fig. 9 The chemical structural formula, primary amino acid sequence, analytical reverse-phase high performance liquid chromatogram and mass spectrum of YJF-611 of the present invention;

[0041] Fig.10 Evaluation of the IC of the D-oncolytic peptide and octreotide-D-oncolytic peptide conjugate at the cell level of the present invention 50 (μM);

[0042] Fig.11 This is a curve for evaluating the proliferation inhibition rate of the D-oncolytic peptide and octreotide-D-oncolytic peptide conjugate on tumor cells and normal cells at the cell level of the present invention;

[0043] Fig.12 This is a curve for evaluating the proliferation inhibition rate of tumor cells and normal cells by the combined use of D-configuration oncolytic peptide and octreotide-D-configuration oncolytic peptide of the present invention at the cell level;

[0044] Fig.13 The present invention evaluates the inhibitory effect of pH-responsive octreotide-D-configuration oncolytic peptide conjugate YJF-611 on tumor cell proliferation and its toxicity to normal cells at the cellular level under different pH conditions (IC 50 μM);

[0045] Fig.14 The cytotoxicity (IC 50 μM);

[0046] Fig.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 of the present invention are shown. DETAILED DESCRIPTION

[0047] Below in conjunction with the accompanying drawings and specific embodiments, the specific embodiments of the present invention are described in further detail. In the specific embodiments, each original reagent and raw material all can be purchased. It should be pointed out that the following detailed description is all exemplary, and is intended to provide further description of the present invention. Unless otherwise indicated, all technical and scientific terms used in the present invention have the same meanings as those of ordinary skill in the art to which the present invention belongs.

[0048] As mentioned above, LTX-315 is composed of L-configuration amino acids, has poor enzymatic stability, a short half-life, is highly toxic to normal cells, and can only be injected intratumorally, with a single method of administration, which limits its application.

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

[0050] YJF-608

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

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

[0053]

[0054] The pH-responsive D-configuration oncolytic peptide and pH-responsive octreotide-D-configuration oncolytic peptide conjugate utilize the different pH between normal tissue and tumor tissue to design the pH-responsive oncolytic peptide YJF-610 to improve the selectivity for normal cells and reduce the toxicity to normal cells. At the same time, YJF-611 improves the anti-tumor activity against tumor cells that highly express the 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, and the synthesis method comprises: a solid phase peptide synthesis method used for synthesizing the polypeptide, and a method for condensing the polypeptide with a linker and the polypeptide.

[0056] Specifically, the polypeptide is synthesized using 9-fluorenylmethoxycarbonyl-based solid phase peptide synthesis (Fmoc-SPPS).

[0057] Unless otherwise specified, the present invention selects RinkAmide Am resin (substitution degree of 0.38 mmol / g) to synthesize polypeptides containing an amide terminal; selects Wang resin (substitution degree of 0.33 mmol / g) to synthesize polypeptides containing a carboxyl terminal; and the amino acids used in the synthesis of the oncolytic peptide part, except for glycine (Gly, G), are all Fmoc-D-configuration amino acids.

[0058] More specifically, the synthesis method of the octreotide-D-configuration oncolytic peptide conjugate comprises:

[0059] Step 1, synthesizing octreotide by solid phase peptide synthesis based on 9-fluorenylmethoxycarbonyl;

[0060] Step 2, attaching a linker group to the octreotide sequence;

[0061] Step 3, synthesizing a D-configuration polypeptide using a solid phase peptide synthesis method based on 9-fluorenylmethoxycarbonyl;

[0062] Step 4, solid phase cyclization of the synthesized polypeptide to form a disulfide bond through an oxidation reaction;

[0063] Step 5, adding a peptide cleavage reagent to the cyclization product to cleave the cyclization product from the resin;

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

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

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

[0067] In another specific embodiment of the present invention, the experimental methods of the above pre-activation and activation are respectively: the resin is alternately rinsed with DMF and DCM, and 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 oscillator at 28°C for 30 minutes.

[0068] The experimental method for removing the Fmoc protecting group is: at room temperature, use a DMF solution containing 20% ​​piperidine to remove the protecting group, and perform the removal twice. The Fmoc removal time of the second amino acid is 3 min and 5 min, and the removal time of other amino acids is 5 min and 10 min respectively.

[0069] The experimental method of the condensation reaction is: each amino acid is condensed twice at 28°C, the first amino acid condensation reaction time is 3h and 4h, and the ratio of reactants is amino acid: DIC: Oxyma: DAMP = 3 times equivalent: 6 times equivalent: 3 times equivalent: 0.5 times equivalent. The two condensation times of other amino acids are 30min and 40min respectively, and the ratio of reactants is amino acid: HCTU: DIEA = 3 times equivalent: 2.8 times equivalent: 6 times equivalent.

[0070] The cleaning experiment method is: use DMF and DCM to rinse the resin alternately, and use a water pump to drain the remaining solvent.

[0071] The specific experimental method of step 2 is:

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

[0073] The specific experimental method of step 3 is:

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

[0075] The specific experimental method of step 4 is:

[0076] The protecting group of the cysteine ​​side chain thiol in the amino acid sequence of octreotide is an ACM protecting group. The polypeptide synthesized in step 3 is cyclized under solid phase conditions at 28°C. The cyclization times are 1h and 1.5h respectively. The reactant ratio is amino acid: Tl(TFA)3=3 times equivalent: 2.34 times equivalent (molar ratio). Tl(TFA)3 is added to 2.5mL DMF containing 125μL anisole, and the cyclization is carried out at 28°C after ice bathing for 5min.

[0077] The specific experimental method of step 5 is:

[0078] Add peptide cleavage reagent to the prepared cyclization product in a ratio of TFA:TIPS:water = 95:2.5:2.5 (v:v:v) and react in a shaker at 28°C-30°C in the dark for 2.5h, wash the resin twice with 1mL of TFA, 0.5mL each time. Then concentrate the product to 2-3mL with high-purity nitrogen, add pre-cooled anhydrous ether to the concentrate to precipitate the target peptide. After centrifugation, add anhydrous ether again, repeat three times to obtain a crude peptide precipitate. Stand in a fume hood in the dark for more than 20min.

[0079] The specific experimental method of step 6 is:

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

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

[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, there is provided use of the above-mentioned octreotide-D-oncolytic peptide conjugate, pH-responsive D-oncolytic peptide and pH-responsive octreotide-D-oncolytic peptide conjugate in the preparation of anticancer or antitumor drugs.

[0084] In another embodiment of the present invention, the present invention can treat solid tumors and blood tumors. The solid tumors and blood tumors can be 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 cancer, head and neck squamous cell carcinoma, testicular cancer, and gastric cancer.

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

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

[0087] The above-mentioned pharmaceutical preparations can be any pharmaceutical dosage form acceptable in pharmaceutical science, 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), injections (including powder injections and injection solutions), etc.

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

[0089] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0090] Example 1

[0091] In this example, all target polypeptides were prepared using 9-fluorenylmethoxycarbonyl-based solid phase peptide synthesis technology (Fmoc-SPPS). Unless otherwise specified, the present invention selects RinkAmideAm resin (substitution degree of 0.38 mmol / g) to synthesize polypeptides containing amide termini; selects Wang resin (substitution degree of 0.33 mmol / g) to synthesize polypeptides containing carboxyl termini; and except for glycine (Gly, G), all other amino acids used in the synthesis of the oncolytic peptide part are Fmoc-D-configuration amino acids.

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

[0093] Solid-phase peptide synthesis experiment (D-configuration oncolytic peptide): Weigh 320 mg of RinkAmideAM resin (1 equivalent), rinse with DMF and DCM alternately, and soak the resin in DMF at room temperature for 1 to 2 hours to pre-activate the resin. After another alternate rinse, soak the resin in a DMF / DCM mixed solution (1:1), and shake it in a constant temperature oscillator at 28°C for 0.5 hours to activate the resin. Then use DMF containing 20% ​​piperidine to remove the Fmoc protecting group, with the first 5 minutes and the second 10 minutes. Each amino acid was condensed twice at 28°C, for 30 minutes and 40 minutes respectively, and the ratio of the reactants was Fmoc-D-configuration amino acid: HCTU: DIPEA = 3 equivalents: 2.8 equivalents: 6 equivalents. After the last amino acid was condensed, its Fmoc protecting group was removed and rinsed alternately with DMF and DCM.

[0094] Synthesis of octreotide-D-configuration oncolytic peptide conjugate: Weigh 450 mg of Wang resin (1 equivalent), rinse with DMF and DCM alternately, and pre-activate the resin by soaking in DMF at room temperature for 1 to 2 hours. After alternately rinsing again, use a DMF / DCM mixed solution (3:1) to shake in a constant temperature oscillator at 28°C for 0.5h to activate the resin. Then, amino acid condensation was performed, and each amino acid was condensed twice at 28°C. The first amino acid was condensed for 3h and 4h, and the ratio of the reactants was amino acid: DIC: Oxyma: DAMP = 3 equivalents: 6 equivalents: 3 equivalents: 0.5 equivalents. The two condensation times of other amino acids were 30min and 40min respectively, and the ratio of the reactants was amino acid: HCTU: DIEA = 3 equivalents: 2.8 equivalents: 6 equivalents. The synthesized linear octreotide sequence was subjected to condensation reaction with the linking group through an amide bond, and the reactant ratio was Fmoc-AEEA-OH: HATU: HOAT: DIEA = 3 times equivalents: 2.8 times equivalents: 3 times equivalents: 6 times equivalents. Then the linking group was subjected to subsequent condensation with the amino acid through an amide bond. After the last amino acid was condensed, its Fmoc protecting group was removed and washed alternately with DMF and DCM. The condensed polypeptide was cyclized under solid phase conditions at 28°C, and the cyclization time was 1h and 1.5h respectively. The reactant ratio was amino acid: Tl(TFA)3 = 3 times equivalents: 2.34 times equivalents. Tl(TFA)3 was added to 2.5mL DMF containing 125μL anisole, and the mixture was first ice-bathed for 5min and then cyclized at 28°C.

[0095] Peptide cleavage and separation and purification: Add a peptide cleavage reagent to the prepared condensation product. The ratio of the peptide cleavage reagent for the D-configuration oncolytic peptide is TFA: phenol: water: TIPS = 88: 5: 5: 2 (v: v: v: v). The ratio of the peptide cleavage reagent for the octreotide-D-configuration oncolytic peptide conjugate is TFA: TIPS: water = 95: 2.5: 2.5 (v: v: v). React in a 28°C-30°C shaker in the dark for 2.5 hours, and wash the resin twice with 1mL of TFA, 0.5mL each time. Subsequently, the product was concentrated to 2-3mL with high-purity nitrogen, and pre-cooled anhydrous ether was added to the concentrate to precipitate the target polypeptide. After centrifugation, anhydrous ether was added again, and the crude peptide precipitate was obtained by repeating three times. Stand in a fume hood in the dark for more than 20 minutes. Subsequently, a mixed solution of acetonitrile and water containing 1‰ TFA was used to dissolve the obtained crude peptide product, and then analytical reverse HPLC and ESI-MS were used to analyze and identify the obtained crude peptide. The dissolved crude peptide was placed at -80°C overnight. Freeze-dried with a freeze dryer to obtain a flocculent crude peptide. The prepared crude peptide was further dissolved in a mixed solution of acetonitrile and water containing 1‰ TFA, and separated and purified using semi-preparative reverse phase high performance liquid chromatography (RP-HPLC). The collected pure peptide solution was placed at -80°C overnight, and then freeze-dried with a freeze dryer to obtain a pure peptide.

[0096] The chemical structural formula, primary amino acid sequence, analytical reverse phase high performance liquid chromatography and ESI-MS mass spectra of the four polypeptides prepared by the present invention are as follows: Figure 6-9 shown.

[0097] MTT assay to detect tumor cell proliferation inhibition experiment:

[0098] The SSTR2 receptor high-expressing tumor cells MCF-7 and HepG2, low-expressing tumor cells BON-1 and normal cells MRC-5 in the logarithmic growth phase were selected, and the cells were collected and counted before inoculating the cells in a 96-well plate. After inoculation, the cells were sterilized and placed in an incubator for 24 hours, and 50 μL of YJF-606, YJF-608, YJF-610, YJF-611, LTX-315, and [Tyr 3 ]Octreotate, set up 3 replica wells for each group, and continue to culture cells in the incubator for 24 hours. After 24 hours, add 15μL MTT to each well in the dark, mix gently, and incubate in the incubator for 4 hours to terminate the culture. After aspirating the residual liquid in the well, add 150μL DMSO to each well and place it in the incubator for 1 hour. Measure the absorbance value at a wavelength of 492nm, repeat the measurement three times, record the results, and perform subsequent calculations according to the formula.

[0099] like Fig.11 As shown in Figure 2, the novel D-oncolytic peptide and octreotide-D-oncolytic peptide conjugate can kill MCF-7, HepG2 and BON-1 tumor cells in a concentration-dependent manner. The IC values ​​of the novel D-oncolytic peptide and octreotide-D-oncolytic peptide conjugate are 50 The values ​​were greatly improved compared with LTX-315, and the anti-tumor activity was significantly improved, among which YJF-610 and YJF-611 had less toxicity to normal cells. In addition, the novel octreotide-D-oncolytic peptide conjugates YJF-608 and YJF-611 had significantly improved inhibitory activity against SSTR2 receptor-highly expressed tumor cells MCF-7 and HepG2, while the pH-responsive octreotide-D-oncolytic peptide conjugate YJF-611 significantly improved its anti-tumor activity while reducing its toxicity to normal cells.

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

[0101] MCF-7, HepG2, BON-1 and MRC-5 cells in the logarithmic growth phase were selected, and the cells were collected and counted before inoculating the cells in a 96-well plate. After inoculation of the cells, the outer wall was disinfected and incubated overnight. Six drug groups were set up, namely, the first group [Tyr 3 ]Octreotate and YJF-606 concentration ratio of 1:1 mixed solution, the second group fixed YJF-606 final concentration of 20μM, added 100μM, 50μM, 25μM, 12.5μM, 6.25μM, 3.125μM [Tyr 3 ]Octreotate solution, the third group fixed [Tyr 3 The final concentration of octreotate was 20 μM, and 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM YJF-606 solutions were added. 3 ]Octreotate and YJF-610 concentration ratio of 1:1 mixed solution, the fifth group fixed YJF-610 final concentration of 20μM, added 100μM, 50μM, 25μM, 12.5μM, 6.25μM, 3.125μM [Tyr 3 ]Octreotate solution, group VI fixed [Tyr 3 ]The final concentration of Octreotate is 20μM. Add 100μM, 50μM, 25μM, 12.5μM, 6.25μM, and 3.125μM YJF-610 solutions. Set up three replicate wells in each group and continue to culture the cells in the incubator for 24 hours. After 24 hours, add 15μL MTT to each well in the dark, mix gently, and incubate in the incubator for 4 hours to terminate the culture. After aspirating the residual liquid in the well, add 150μL DMSO to each well and shake for 10 minutes. Measure the absorbance value at a wavelength of 492nm, repeat the measurement three times, record the results, and perform subsequent calculations according to the formula.

[0102] like Fig.12 As shown, covalently linked YJF-606, [Tyr 3 ]Octreotate (i.e. YJF-608) has a stronger inhibitory effect on MCF-7 and HepG2 tumor cells that highly express SSTR2 receptors than the non-covalently linked YJF-610, and the toxicity of the two to normal cells is comparable. 3 Octreotate (i.e. YJF-611) has a stronger inhibitory effect on MCF-7 and HepG2 tumor cells that highly express SSTR2 receptors than the non-covalent bond between the two, and has comparable toxicity to normal cells.

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

[0104] MCF-7, BON-1 and MRC-5 cells in the logarithmic growth phase were selected, and the cells were collected and counted before inoculating the cells in a 96-well plate. After inoculating the cells, the outer wall was disinfected and incubated overnight, the culture medium was removed, 150 μL of YJF-611 with a final concentration of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM was added, and the pH was adjusted to 6.0 or 7.4. Three sub-wells were set up in each group, and the cells were continued to be cultured in the incubator for 24 hours. After 24 hours, 15 μL of MTT was added to each well in the dark, mixed gently, and incubated in the incubator for 4 hours to terminate the culture. After aspirating the residual liquid in the well, 150 μL of DMSO was added to each well and shaken for 10 minutes. The absorbance value was measured at a wavelength of 492 nm, and the measurement was repeated three times. The results were recorded and the subsequent calculations were performed according to the formula.

[0105] like Fig.13 As shown in the figure, the novel pH-responsive octreotide-D-configuration oncolytic peptide conjugate YJF-611 kills MCF-7 and BON-1 tumor cells in a concentration-dependent manner at pH 6.0, and is toxic to normal cells MRC-5. At pH 7.4, YJF-611 has weak activity against MCF-7 and BON-1 tumor cells and is almost non-toxic to normal cells MRC-5 (IC 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 to detect proliferation inhibition of high-expressing and low-expressing cells after inhibiting somatostatin receptors:

[0107] MCF-7, HepG2 and BON-1 cells in the logarithmic growth phase were selected, and the cells were collected and counted before inoculating the cells in a 96-well plate. After inoculating the cells, the outer wall was disinfected and incubated overnight, the culture medium was removed, and two groups of 3 sub-wells were set up. 150 μL of Octreotate with a final concentration of 100 μM and complete culture medium were added respectively, and incubated in an incubator for 4 hours. After 4 hours, the culture medium was removed and 150 μL of YJF-611 with a final concentration of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM were added to each well, and the cells were continued to be cultured in the incubator for 24 hours. After 24 hours, 15 μL of MTT was added to each well in the dark, gently mixed, and placed in the incubator for incubation for 4 hours to terminate the culture. After aspirating the residual liquid in the well, 150 μL of DMSO was added to each well and shaken for 10 minutes. The absorbance value was measured at a wavelength of 492 nm, and the measurement was repeated three times. The results were recorded and the subsequent calculations were performed according to the formula.

[0108] like Fig.14 As shown in the figure, after the action of 100 μM Octreotide, the novel pH-responsive octreotide-D-oncolytic peptide conjugate YJF-611 significantly reduced the activity of tumor cells MCF-7 and HepG2 that highly expressed the SSTR2 receptor, but had no effect on the inhibitory activity of tumor cells BON-1 that lowly expressed the SSTR2 receptor. The above results indicate that the novel pH-responsive octreotide-D-oncolytic peptide conjugate has better binding and activity to tumor cells that highly expressed the SSTR2 receptor.

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

[0110] Dissolve the serum in PBS to prepare a 5% serum solution, weigh an appropriate amount of LTX-315 and YJF-611 polypeptide solids, add water to dissolve, add the prepared serum solution to the polypeptide solution, prepare a mixed solution with a final concentration of 200μM, and shake up and down for 3 times. Immediately take 80μl of the reaction solution, add 20μl of acetonitrile containing 0.1% TFA, shake three times, let stand on ice for 2 minutes, then add 60μl acetonitrile and 400μl deionized water containing 0.1% TFA, shake three times, and immediately store at -80℃. The reaction tube is sealed and placed in a constant temperature water bath at 37℃ for incubation, and samples are taken at 0.5h, 1h, 2h, 4h, 8h, 16h, 24h, 36h, 48h, and 72h according to the above method. The remaining polypeptide amount is counted and analyzed by RP-HPLC.

[0111] like Fig.15 As shown in the figure, as time goes by, the remaining percentage of LTX-315 polypeptide gradually decreases, and is almost completely hydrolyzed at 48h (remaining polypeptide <10%). However, the YJF-611 conjugate is not easily hydrolyzed, and the remaining percentage at 72h is 75%. The above results show that the pH-responsive octreotide-D-configuration oncolytic peptide conjugate has extremely strong serum stability.

[0112] Finally, it should be noted that the above is only a preferred implementation case of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned implementation case, it is still possible for a technician in the field to modify the technical solution recorded in the aforementioned implementation case, or to perform equivalent replacement on part of it. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Although the above describes the specific implementation methods of the present invention, it is not a limitation on the protection scope of the present invention. A technician in the relevant field should understand that on the basis of the technical solution of the present invention, various modifications or deformations that can be made by those skilled in the art without creative labor are still within the protection scope of the present invention.

Claims

1. A class of octreotide-D-configuration oncolytic peptide conjugates, characterized in that: The octreotide-D-configuration oncolytic peptide conjugate comprises the following amino acid residue sequence:

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 comprise the following amino acid residue sequence:

3. The method for preparing the octreotide-D-oncolytic peptide conjugate, the pH-responsive D-oncolytic peptide and the pH-responsive octreotide-D-oncolytic peptide conjugate according to claim 1 and claim 2, characterized in that: The preparation method includes a solid phase peptide synthesis method for synthesizing the peptide, and a method for selectively covalently linking octreotide with a linking group and an oncolytic peptide; Preferably, the polypeptide synthesis method is a solid phase polypeptide synthesis method based on 9-fluorenylmethoxycarbonyl; Preferably, the synthesis method of the octreotide-D-configuration oncolytic peptide conjugate is as follows: Octreotide is synthesized by a solid phase peptide synthesis method based on 9-fluorenylmethoxycarbonyl, and then the synthesized polypeptide is connected to a linker group and an oncolytic peptide through a condensation reaction. A peptide cleavage reagent is added to the condensation product, and the condensation product is cleaved from the resin. The product after peptide cleavage is separated and purified to obtain an octreotide-D-configuration oncolytic peptide conjugate.

4. Use of the octreotide-D-configuration oncolytic peptide conjugate according to claim 1 and claim 2 in the preparation of a drug delivery system; Preferably, the drug delivery system is a somatostatin receptor SSTR2 targeted drug delivery system.

5. Use of the octreotide-D-oncolytic peptide conjugate, the pH-responsive D-oncolytic peptide and the pH-responsive octreotide-D-oncolytic peptide conjugate according to claim 1 and claim 2 in the preparation of anticancer or antitumor drugs; Preferably, the cancer or tumor can 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 carcinoma, thymic carcinoma, bladder cancer, papillary thyroid carcinoma, head and neck squamous cell carcinoma, testicular cancer, gastric cancer, ganglioneuroma; Further preferably, the cancer or tumor is a cancer or tumor that highly expresses the somatostatin receptor SSTR2.

6. A pharmaceutical preparation, characterized in that It comprises the octreotide-D-configuration oncolytic peptide conjugate according to claim 1 and claim 2, the pH-responsive D-configuration oncolytic peptide, the pH-responsive octreotide-D-configuration oncolytic peptide conjugate and a pharmaceutically acceptable carrier; Preferably, the carrier includes a filler, a disintegrant, a diluent, a surfactant, a binder, a lubricant, a colorant, a suspending agent and a flavoring agent.

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