Polypeptide coupling medicine of somatostatin receptor binding peptide coupling TLR agonist and preparation method of polypeptide coupling medicine

Abstract

The invention relates to the technical field of medicines, in particular to a polypeptide coupling medicine of a somatostatin receptor binding peptide coupling TLR agonist and a preparation method of the polypeptide coupling medicine. The polypeptide coupling medicine of the somatostatin receptor binding peptide coupling TLR agonist provided by the invention comprises at least one compound in a formula I-1 and a formula I-2 in the specification or a pharmaceutically acceptable salt of the compound. According to the polypeptide coupling drug based on the specific molecular structure provided by the invention, somatostatin receptor binding peptide is coupled with a TLR agonist, so that the polypeptide coupling drug not only has tumor tissue targeting property, but also can activate TLR7 and TLR8 receptors on cytoplasm endosomes in tumor tissues, exerts an anti-tumor immunostimulation effect, and has a good application prospect. Meanwhile, the immunotoxicity of blood and non-specific organs is lower.

Description

Technical Field

[0001] The present application belongs to the field of medical technology, and in particular relates to a somatostatin receptor binding peptide coupled to a TLR agonist polypeptide-coupled drug and a preparation method thereof. Background Art

[0002] Toll-like receptors (TLRs) are the innate immune system that monitors and recognizes various pathogen-associated molecular patterns (PAMPs). They are widely expressed in the immune cell spectrum, participate in nonspecific immunity and mediate specific immunity, and are the body's first barrier to resist infectious diseases. At present, 13 TLRs (TLR1-TLR13) have been identified in mammals, including TLR1-TLR11 in humans, TLR1-TLR9 and TLR11-TLR13 in mice. Activation of TLRs can induce MyD88 or TRIF-dependent signaling pathways, activate NF-κB, induce cytokine and chemokine secretion, activate innate immune responses, and mediate activation of acquired immune responses. They can be used as immune adjuvants for anti-tumor and infectious diseases.

[0003] TLR agonists include imidazoquinoline amine compounds, such as imidazomod (R837), resiquimod (R848), etc. For example, R848 is a dual agonist of TLR7 and TLR8 (i.e., TLR7 / 8 agonist), which is made into a topical gel for the treatment of skin lesions caused by herpes simplex virus and cutaneous T-cell lymphoma, and as an adjuvant to improve the effectiveness of vaccines, which can produce effective immunotherapy for tumors by immune stimulation of tumor-associated macrophages. Although TLR7 / 8 agonists are effective adjuvants for local anti-tumor therapy, TLR7 / 8 agonists as small molecule adjuvants do not have specific targeting, have a short half-life, and intravenous injection can lead to systemic immune hyperactivity, thereby producing cytokine release syndrome (CRS), which is difficult to tolerate in vivo. Therefore, the serious toxic side effects of TLR7 / 8 agonists remain an important problem facing current clinical applications.

[0004] Somatostatin (SST) is a neuropeptide hormone present in the gastrointestinal nervous system. Octreotide is a long-acting structural derivative of somatostatin and a synthetic peptide analog of somatostatin with high binding affinity to somatostatin receptors (SSTR) 2 and 5. Peptide drug conjugates (PDC) consist of three parts: targeting peptide, linker, and payload (also known as toxic drug). After the drug is combined with the peptide through the linker, the peptide and the drug have a bidirectional function, which can promote the killing or targeting effect of the drug. Different drugs require matching linkers to couple them to the targeting peptide, but there are no reports on peptide drug conjugates that combine somatostatin receptor binding peptides with TLR agonists. Summary of the invention

[0005] The purpose of the present application is to provide a somatostatin receptor binding peptide-coupled TLR agonist polypeptide-coupled drug and a preparation method thereof, aiming to solve the technical problem of how to improve the targeting of TLR7 / 8 agonists and reduce their side effects.

[0006] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0007] In a first aspect, the present application provides a somatostatin receptor binding peptide-coupled TLR agonist polypeptide-coupled drug, wherein the polypeptide-coupled drug comprises at least one compound of the following formula I-1 and formula I-2 or a pharmaceutically acceptable salt thereof:

[0008]

[0009] Wherein, R1, R2, R3, and R4 are independently selected from hydrogen, C 1-10 Alkyl, C 1-10 At least one of the alkoxy groups, n=1-48, and X is octreotide.

[0010] In a second aspect, the present application provides a method for preparing the above-mentioned somatostatin receptor binding peptide-coupled TLR agonist polypeptide-coupled drug, comprising:

[0011]

[0012] Provide compound A or compound B; wherein R1, R2, R3, and R4 in compound A are independently selected from hydrogen, C 1-10 Alkyl, C 1-10 at least one of alkoxy groups;

[0013] The resin linear peptide corresponding to octreotide was synthesized in the order from the C-terminus to the N-terminus of the polypeptide;

[0014] Connecting polyethylene glycol to the N-terminus of the resin straight-chain peptide corresponding to the octreotide, and then performing a condensation reaction with the compound A or the compound B to obtain an intermediate;

[0015] The intermediate is cleaved from the resin and then subjected to an oxidation reaction to obtain the compound of formula I-1 or the compound of formula I-2.

[0016] In a third aspect, the present application provides an application, namely, the application of the polypeptide conjugate drug of the present application and / or the polypeptide conjugate drug prepared by the preparation method of the present application in the preparation of anti-tumor drugs or immunostimulatory adjuvants.

[0017] In a fourth aspect, the present application provides a pharmaceutical composition, comprising the polypeptide-conjugated drug of the present application and / or the polypeptide-conjugated drug prepared by the preparation method of the present application.

[0018] The polypeptide conjugate drug of the somatostatin receptor binding peptide coupled to the TLR agonist provided in the first aspect of the present application utilizes a specific linker to connect the thiomethylated derivative of the TLR7 / 8 agonist (CAS accession number 1258457-59-8, referred to as 78A1) and the somatostatin receptor binding peptide, i.e., octreotide, and the polypeptide conjugate drug can deliver the small molecule 78A1 to the tumor site, and the level of inflammatory factors in the blood can be significantly reduced after intravenous injection, and the safety is significantly improved. Therefore, the polypeptide conjugate drug of the embodiment of the present application not only has tumor tissue targeting, but also can activate the TLR7 and TLR8 receptors on the cytoplasmic endosomes in the tumor tissue, exerting an anti-tumor immunostimulatory effect, and at the same time has lower blood and non-specific organ immunotoxicity.

[0019] The preparation method provided in the second aspect of the present application uses compound A or compound B, a resin straight-chain peptide corresponding to octreotide, and polyethylene glycol as raw materials, connects the N-terminus of the resin straight-chain peptide corresponding to octreotide to polyethylene glycol, and then performs a condensation reaction with compound A or compound B, and then further cuts and oxidizes from the resin to obtain a compound shown in formula I-1 or formula I-2. The preparation method of the present application has a high yield and few by-products, and the prepared polypeptide-coupled drug not only has tumor tissue targeting, but also can activate TLR7 and TLR8 receptors on the cytoplasmic endosomes in tumor tissues, exerting an anti-tumor immunostimulatory effect, and at the same time has lower blood and non-specific organ immunotoxicity.

[0020] The application provided in the third aspect of the present application is based on the above-mentioned advantages of the polypeptide-coupled drug unique to the present application, so the polypeptide-coupled drug of the present application can be used to prepare anti-tumor drugs and immunostimulatory adjuvants.

[0021] The pharmaceutical composition provided in the fourth aspect of the present application includes a polypeptide-coupled drug unique to the present application. On the basis of having anti-tumor activity, the pharmaceutical composition can reduce the effects on normal tissues and immune cells in the blood, thereby reducing the expression of inflammatory cytokines in plasma. This can achieve anti-tumor effects while effectively reducing systemic immunotoxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1This is the mass spectrum of the peptide-coupled drug OCT-PEG12-SS-78A1 synthesized in the examples of this application;

[0024] Figure 2 This is a chromatogram of the peptide-coupled drug OCT-PEG12-SS-78A1 synthesized in the examples of this application;

[0025] Figure 3 The mass spectrum of the peptide-coupled drug OCT-PEG24-SS-78A1 synthesized in the examples of the present application;

[0026] Figure 4 This is a chromatogram of the peptide-coupled drug OCT-PEG24-SS-78A1 synthesized in the examples of this application;

[0027] Figure 5 This is the mass spectrum of the peptide-coupled drug OCT-PEG24-C2SS-78A1 synthesized in the examples of this application;

[0028] Figure 6 This is a chromatogram of the peptide-coupled drug OCT-PEG24-C2SS-78A1 synthesized in the examples of this application;

[0029] Figure 7 This is the mass spectrum of the peptide-coupled drug OCT-PEG24-C2SSC2-78A1 synthesized in the examples of this application;

[0030] Figure 8 This is a chromatogram of the peptide-coupled drug OCT-PEG24-C2SSC2-78A1 synthesized in the examples of this application;

[0031] Fig. 9 This is the mass spectrum of the peptide-coupled drug OCT-PEG24-VC-78A1 synthesized in the examples of this application;

[0032] Fig.10 This is a chromatogram of the peptide-coupled drug OCT-PEG24-VC-78A1 synthesized in the examples of this application;

[0033] Fig.11 This is a schematic diagram of the test results of in vitro testing of the induction expression activity of OCT-PEG12-SS-78A1 on mouse PBMC cells TNF-α;

[0034] Fig.12A schematic diagram of the test results of in vitro testing of the induction expression activity of 78A1, OCT-PEG24-SS-78A1, OCT-PEG24-C2SS-78A1, OCT-PEG24-C2SSC2-78A1 and OCT-PEG24-VC-78A1 on mouse PBMC cells TNF-α in the examples of the present application;

[0035] Fig.13 This is a schematic diagram of the test results of serum TNF-α content after intravenous injection of different test substances in mice in the examples of this application;

[0036] Fig.14 This is a graph showing the anti-tumor inhibitory effect of OCT-PEG12-SS-78A1 in the present embodiment on a mouse colon cancer CT26 subcutaneous transplant tumor model; wherein A is a body weight change curve, and B is a tumor growth curve;

[0037] Fig.15 This is a graph showing the anti-tumor inhibitory effect of OCT-PEG24-VC-78A1 on a subcutaneous transplanted tumor model of mouse colon cancer CT26 in the example of the present application;

[0038] Fig.16 This is a graph showing the effect of the dose-effect relationship of OCT-PEG24-SS-78A1 on a mouse colon cancer CT26 subcutaneous transplant tumor model according to the example of the present application; wherein A is the body weight change curve, and B is the tumor growth curve. DETAILED DESCRIPTION

[0039] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0040] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0041] In the present application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0042] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0043] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms of "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0044] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0045] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0046] The discovery of somatostatin (SST) can be used to study diseases related to endocrine hypersecretion, such as acromegaly and gastrointestinal pancreatic neuroendocrine tumors (GEP-NETs). Octreotide is a long-acting structural derivative of somatostatin and a synthetic peptide analog of somatostatin with the same pharmacological effects. As a cyclic octapeptide, octreotide has a longer half-life in the circulation, higher efficacy, and good bioavailability after administration compared with somatostatin. Currently, radiolabeled octreotide has been successfully used for imaging (e.g., neuroendocrine, endocrine, breast cancer, small cell lung cancer, and prostate cancer), as well as for targeted radiotherapy. Peptide-drug conjugates (PDCs) consist of three parts: a targeting peptide, a linker, and a payload. After the drug is combined with the peptide through the linker, the peptide and the drug are given a bidirectional function, which can promote the killing or targeting effect of the drug. The linker is very critical.

[0047] Small molecule compounds based on TLR7 / 8 agonists have no targeting, short half-life, and systemic immunotoxicity, which limits their efficacy and clinical application. The present application embodiment uses somatostatin receptor-binding peptide drug coupling technology to target the delivery of TLR7 / 8 agonists, which promotes TLR7 / 8 agonists to improve efficacy and minimize or overcome systemic nonspecific immunotoxicity. The specific technical solution is as follows.

[0048] In the first aspect, the embodiments of the present application provide a somatostatin receptor binding peptide-coupled TLR agonist polypeptide-coupled drug, including a compound of formula I-1 and / or formula I-2, or a pharmaceutically acceptable salt corresponding to the compound of formula I-1 and / or formula I-2:

[0049]

[0050] Wherein, R1, R2, R3, and R4 are independently selected from hydrogen, C 1-10 Alkyl, C 1-10 At least one of the alkoxy groups, n=1-48, and X is octreotide.

[0051] The TLR7 / 8 agonist released after local or intravenous injection of the peptide-coupled drug in the embodiment of the present application acts on the immune cells in the tumor, activates the TLR7 and TLR8 receptors on the cytoplasmic endosomes, activates the expression of inflammatory cytokines through the MYD88-mediated NF-KB signaling pathway, induces the secretion of inflammatory cytokines such as TNF-α, and mediates antigen-specific T cell anti-tumor immunity. On this basis, the peptide-coupled drug effectively reduces the TLR7 / 8 agonist content in the blood, reduces the effect on immune cells in the blood, and minimizes the non-specific immunotoxicity caused by cytokine storms such as TNF-α while achieving local immune stimulation effects.

[0052] Specifically, the polypeptide conjugate drug of the embodiment of the present application includes, according to its design principle: an imidazoquinoline small molecule TLR7 / 8 agonist, namely 78A1, a somatostatin receptor binding peptide, namely octreotide, and a cleavable linker that couples the two. The polypeptide conjugate drug can bind to the somatostatin receptor on the surface of tumor cells, and can be enriched in tumor tissues through blood circulation after global injection, thereby reducing the concentration of TLR7 / 8 agonists in normal tissues, so that the polypeptide conjugate has lower non-specific immunotoxicity while having anti-tumor activity. In this way, it can be used for immunotherapy of intratumoral injection and / or intravenous injection of various solid tumors.

[0053] In some embodiments, C 1-10 Alkyl refers to a straight chain alkyl or branched chain alkyl containing 1 to 10 carbon atoms, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, etc.; 1-10Alkoxy represents a straight chain alkoxy or branched chain alkoxy containing 1-10 carbon atoms, such as methoxy, ethoxy, propoxy, butoxy, etc.; n=1-48, n is an integer, which can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 16, 18, 20, 25, 26, 30, 35, 38, 40, 44, 45, 48, etc., which are typical but non-limiting values. Further, n=10-36, and further, n=12-24. In some embodiments, R1, R2, R3, and R4 are independently selected from hydrogen and C 1-5 At least one of the alkyl groups.

[0054] After administration, the above-mentioned peptide-coupled drugs can target the tumor area and activate TLR7 / 8 receptors, thereby making these peptide-coupled drugs have good anti-tumor activity. At the same time, the content of 78A1 released into the blood is low, making the systemic immunotoxicity of these peptide-coupled drugs low.

[0055] In some embodiments, the cleavable linker is a glutathione-sensitive disulfide linker and / or a Val-Cit-PAB-type linker. These linkers can well connect the somatostatin structure derivative of the cyclic octapeptide-octreotide and the TLR7 / 8 agonist small molecule 78A1; among them, the glutathione-sensitive disulfide linker contains a disulfide bond, which uses the higher level of reduced glutathione GSH in tumor cells to decompose and release the drug; similarly, the Val-Cit-PAB cleavable linker uses the higher level of cathepsin B in tumor cells to decompose and release the drug. Both structures of polypeptide-coupled drugs have the characteristics of tumor-targeted drug release. Further, it can be selected from the Val-Cit-PAB-type linker.

[0056] In some embodiments, the somatostatin receptor binding peptide, octreotide, has the following structure.

[0057]

[0058] In some embodiments, X is octreotide, R 1、 R2, R3 and R4 are hydrogen, n=12, and in this case, the compound structure of the polypeptide-conjugated drug is as shown in Formula I1, and is named OCT-PEG12-SS-78A1 in this application;

[0059]

[0060] In some embodiments, X is octreotide, R1, R2, R3 and R4 are hydrogen, and n=24. In this case, the polypeptide-conjugated drug includes a compound having a structural formula as shown in Formula I2, which is named OCT-PEG24-SS-78A1 in this application;

[0061]

[0062] In some embodiments, X is octreotide, R1 and R2 are hydrogen, R3 and R4 are methyl, and n=24. In this case, the polypeptide-conjugated drug includes a compound having a structural formula as shown in Formula I3, which is named OCT-PEG24-C2SS-78A1 in this application;

[0063]

[0064] In some embodiments, X is octreotide, R1, R2, R3 and R4 are methyl groups, and n=24. In this case, the polypeptide-conjugated drug includes a compound having a structural formula as shown in Formula I4, which is named OCT-PEG24-C2SSC2-78A1 in this application;

[0065]

[0066] In some embodiments, X is octreotide, and the linker is a PEG24-val-cit-PAB cleavable linker. In this case, the polypeptide-conjugated drug includes a compound having a structural formula as shown in Formula I5, which is named OCT-PEG24-VC-78A1 in this application;

[0067]

[0068] In a second aspect, the present invention provides a method for preparing a somatostatin receptor binding peptide-coupled TLR agonist polypeptide-coupled drug. The preparation method comprises:

[0069] S01: providing compound A or compound B;

[0070] S02: Synthesizing the resin linear peptide corresponding to octreotide in the order from the C-terminus to the N-terminus of the polypeptide;

[0071] S03: connecting polyethylene glycol to the N-terminus of the resin straight-chain peptide corresponding to octreotide, and then performing a condensation reaction with compound A or compound B to obtain an intermediate;

[0072] S04: cutting the intermediate from the resin, and then performing an oxidation reaction to obtain the compound represented by Formula I-1 or the compound represented by Formula I-2.

[0073] In the present application, the octapeptide linear peptide of octreotide is condensed for PEG modification, and finally condensed and coupled with a TLR7 / 8 agonist to obtain a polypeptide-coupled drug in the form of formula I-1 and / or formula I-2. Wherein, when compound A is selected as the raw material, a polypeptide-coupled drug in the form of formula I-1 is synthesized; when compound B is selected as the raw material, a polypeptide-coupled drug in the form of formula I-2 is synthesized.

[0074]

[0075] In step S01, the molecular structures of compound A and compound B are as shown above.

[0076] In step S02, a conventional solid phase synthesis method is used to prepare a resin linear peptide corresponding to octreotide from the C-terminus to the N-terminus of the polypeptide; the resin linear peptide corresponding to octreotide, i.e., the octapeptide linear peptide constituting octreotide, is solid phase synthesized on a resin carrier.

[0077] The embodiment of the present application sequentially couples one amino acid from the C-terminus to the N-terminus of the polypeptide to obtain an octapeptide linear peptide of octreotide; including: performing an Fmoc deprotection reaction on Fmoc-Thr(tBu)-OL CTC resin to obtain H-Thr(tBu)-OL CTC resin, and then coupling with Fmoc-Cys(Trt)-OH to obtain Fmoc-Cys(Trt)-Thr(tBu)-OL CTC resin, and then deprotecting to obtain H-Cys(Trt)-Thr(tBu)-OL CTC resin, and then sequentially coupling all amino acids and removing Fmoc according to a similar principle to obtain HD-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-OL CTC resin, i.e., the resin linear peptide corresponding to octreotide. The specific reaction process is as follows.

[0078]

[0079] In step S03, the N-terminus of the synthesized resin linear peptide is connected to polyethylene glycol (PEG). For example, Fmoc-NH-PEG-COOH is added to carry out a dehydration condensation reaction on the basis of step S02, and polyethylene glycol is modified at the N-terminus of the linear peptide to obtain Fmoc-NH-PEG-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-OLCTC resin.

[0080] In some embodiments, the step of connecting the N-terminus of the resin linear peptide corresponding to octreotide to polyethylene glycol comprises: subjecting the N-terminus of the resin linear peptide corresponding to octreotide to a dehydration condensation reaction with Fmoc-NH-PEG-COOH, and then removing the amino protection for subsequent reactions. For example, N,N-dimethylformamide (DMF) is added to remove Fmoc, and then compound A or compound B is added to carry out a dehydration condensation reaction to obtain the following intermediate after coupling.

[0081] 1) Compound A-NH-PEG-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-OL CTC resin;

[0082] 2) Compound B: NH-PEG-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-OL CTC resin.

[0083] In step S04, the intermediate is cleaved from the resin and then subjected to an oxidation reaction to obtain the compound represented by formula I-1 or the compound represented by formula I-2.

[0084] For example, trifluoroacetic acid (TFA) is used to elute and cut from the resin to obtain an intermediate without a protecting group, which is then oxidized to obtain: Compound A-NH-PEG-D-Phe-(Cys-Phe-D-Trp-Lys-Thr-Cys)-Thr-OL(CC), i.e., the compound shown in Formula I-1; or Compound B-NH-PEG-D-Phe-(Cys-Phe-D-Trp-Lys-Thr-Cys)-Thr-OL(CC), i.e., the compound shown in Formula I-2;

[0085] In some implementations, the oxidation treatment includes: mixing the intermediate obtained by cleavage from the resin with oxygen for 10 to 24 hours.

[0086] The preparation method of the present application embodiment synthesizes octreotide octapeptide linear peptide by solid phase peptide synthesis technology, then performs PEG modification by condensation reaction, and finally performs condensation coupling with TLR7 / 8 agonist to obtain a polypeptide-coupled drug in the form of formula I-1 and / or formula I-2. The polypeptide-coupled drug prepared in the present application embodiment has good tumor selectivity, and reduces systemic immunotoxicity while ensuring local immunostimulatory effect.

[0087] In a third aspect, the embodiments of the present application provide the use of the above-mentioned polypeptide-coupled drug and / or the polypeptide-coupled drug prepared by the above-mentioned preparation method in the preparation of anti-tumor drugs or immunostimulatory adjuvants.

[0088] Based on the intravenous injection of the polypeptide-coupled drug in the embodiment of the present application, the TLR7 / 8 agonist is delivered to the tumor tissue through the blood circulation by the binding of octreotide to the somatostatin receptor, and the drug molecule is released. The drug molecule is a TLR7 / 8 agonist, which mainly acts on the immune cells in the tumor, mediates antigen-specific T cell anti-tumor immunity, achieves in situ immune activation, and effectively reduces the agonist activity of the TLR7 / 8 receptors of immune cells in the blood, thereby reducing systemic immunotoxicity, so that the polypeptide-coupled drug is used in anti-tumor drugs, and has the beneficial effect of low systemic immunotoxicity while having anti-tumor activity.

[0089] In some embodiments, the tumor can be controlled to be a solid tumor. In further embodiments, the solid tumor can include at least one of gastrointestinal pancreatic neuroendocrine tumors, colorectal cancer, breast cancer, small cell lung cancer and prostate cancer. Solid tumors are more easily administered by intratumoral injection, further increasing the enrichment of TLR7 / 8 agonist molecules in the tumor, which is conducive to further improving the anti-tumor effect of the polypeptide-coupled drug of the embodiment of the present application.

[0090] Based on the embodiments of the present application, the polypeptide-coupled drug can release TLR7 / 8 agonists in the tumor after local or intravenous injection, increase the content of inflammatory mediators in the tumor, and effectively reduce the release of pro-inflammatory cytokines such as TNF-α in plasma, so that the polypeptide-coupled drug has great application potential in the preparation of anti-tumor and vaccine adjuvants.

[0091] In a fourth aspect, an embodiment of the present application provides a pharmaceutical composition, which includes the above-mentioned polypeptide-conjugated drug and / or the polypeptide-conjugated drug prepared by the above-mentioned preparation method.

[0092] The pharmaceutical composition of the embodiment of the present application is based on the polypeptide-coupled drug of the embodiment of the present application with lower blood toxicity, so that the pharmaceutical composition reduces the effect on normal tissues and immune cells in the blood on the basis of anti-tumor activity, reduces the expression of inflammatory cytokines in plasma, achieves anti-tumor effect, and effectively reduces systemic immunotoxicity.

[0093] After administration of the pharmaceutical composition of the embodiment of the present application, the polypeptide-coupled drug contained in the pharmaceutical composition is used locally to release TLR7 / 8 agonists in the tumor area, activate immune cells in the tumor, and promote the increase of pro-inflammatory cytokines such as TNF-α in the tumor, with good targeting.

[0094] In some embodiments, the administration of the pharmaceutical composition can be controlled to be injection administration, further, it can be systemic injection administration and / or local injection administration, further, it can include at least one of intratumoral rapid injection, subcutaneous injection, and intravenous injection. The following uses multiple specific embodiments to illustrate the polypeptide-conjugated drug and its preparation method, application, and pharmaceutical composition of the present application.

[0095] The following is an explanation with reference to specific examples. In addition, the significance analysis of the differences in the specification of this application uses one-way / two-way ANOVA (ns: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0096] Example 1

[0097] A polypeptide-conjugated drug, as shown in the above formula I1, is OCT-PEG12-SS-78A1, and the preparation process is as follows.

[0098] 1) Synthesis of Compound A1

[0099]

[0100] In a 100 ml three-necked flask protected by nitrogen with a magnetic stirrer, EDCI (0.75 g, 3.9 mmol) and HOBT (0.352 g, 2.6 mmol) were added respectively, and then anhydrous DMF (30 mL) was added and stirred and mixed, and then 3,3'-dihydrooxolinic acid (2.7 g, 12.9 mmol) and DIPEA (0.65 mL, 3.9 mmol) were added and stirred for 10 min, and then N-hydroxysuccinimide (0.3 g, 2.6 mmol) was added. After the addition was completed, the reaction solution was stirred at room temperature overnight. After TLC confirmed that the reaction was completed, the reaction solution was directly subjected to the next step without treatment. Take 10 mL of the above reaction solution, add DIPEA (206 mg, 1.6 mmol) and stir and mix, then add 78A1 (Cas.1258457-59-8, 300 mg, 0.8 mmol) and stir and mix to react. After LCMS confirmed the completion of the reaction, the reaction solution was directly sent for preparation and purification without treatment to obtain 140 mg of the target product.

[0101] The structure was confirmed to be correct by NMR (1H NMR (400MHz, DMSO-d6) δ8.41 (t, J = 5.9 Hz, 1H), 7.79 (d, J = 8.2 Hz, 1H), 7.58 (d, J = 8.3 Hz, 1H), 7.34 (t, J = 7.7 Hz, 1H), 7.20 (d, J = 7.9 Hz, 2H), 7.04 (t, J = 7.6 Hz, 1H), 6.98 (d, J = 8.0 Hz, 1H). z,2H),6.60(s,2H),5.84(s,2H),4.22(d,J=5.9Hz,2H),2.88(m,J=14.7,7.2Hz,7H),2.6 0(t,J=6.9Hz,3H),1.72(m,J=7.6Hz,2H),1.38(m,J=7.4Hz,2H),0.88(t,J=7.3Hz,3H)).

[0102] 2) Synthesis of OCT-PEG12-SS-78A1

[0103] The resin linear peptide of octreotide was prepared from the C-terminus to the N-terminus of the polypeptide by solid phase synthesis: 0.6 mmol of Fmoc-Thr(tBu)-OL CTC resin was subjected to Fmoc deprotection reaction (reaction system: 20% pip / DMF 10 mL; time 30 min) to obtain H-Thr(tBu)-OL CTC resin, which was then coupled with 1.8 mmol of Fmoc-Cys(Trt)-OH (reaction system: 1.8 mmol of HBTU and 3.0 mmol of DIEA; time 60 min) to obtain Fmoc-Cys(Trt)-Thr(tBu)-OL CTC resin, and then deprotection reaction (reaction system: 20% pip / DMF 10mL; time 30min) to obtain H-Cys(Trt)-Thr(tBu)-OLCTC resin, and then all amino acids are coupled in sequence according to a similar principle and Fmoc is removed to obtain HD-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-OL CTC resin, which is the resin linear peptide corresponding to octreotide.

[0104] The above-synthesized resin linear peptide was subjected to a dehydration condensation reaction with 0.9 mmol of FmocNH-PEG12-COOH (reaction system: 0.9 mmol of HOBT and 0.9 mmol of DIC; time 20 h), wherein n in polyethylene glycol is 12; Fmoc-NH-PEG12-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-OLCTC resin was obtained, and then Fmoc deprotection reaction was carried out (reaction system: 20% pip / DMF 10 mL; time 30 min). After deprotection, 0.9 mmol of compound A1 was added for dehydration condensation reaction (reaction system: 0.9 mmol of HOBT and 0.9 mmol of DIC; time 20 h) to obtain the intermediate:

[0105] Compound A-NH-PEG12-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-OL CTC resin. The intermediate was eluted and cut from the resin (elution system: 95% TFA+2.5% Tis+2.5% H2O, time 3h) to obtain the intermediate without protecting group: compound A-NH-PEG12-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-OL; finally, it was mixed with oxygen for reaction (20h) to obtain the target product OCT-PEG12-SS-78A1.

[0106] The mass spectrometry results of OCT-PEG12-SS-78A1 are as follows Figure 1 The chromatographic results are shown in Figure 2 shown.

[0107] Example 2

[0108] A polypeptide-conjugated drug, as shown in the above formula I2, is OCT-PEG24-SS-78A1, and the preparation process is as follows.

[0109] The difference from Example 1 is that n in the polyethylene glycol is 24 (ie, PEG24), and the other steps are the same as in Example 1.

[0110] The mass spectrometry results of OCT-PEG24-SS-78A1 are as follows Figure 3 The chromatographic results are shown in Figure 4 shown.

[0111] Example 3

[0112] A polypeptide-coupled drug, as shown in the above formula I3, is OCT-PEG24-C2SS-78A1, and the preparation process is as follows:

[0113] 1) Synthesis of Compound A2

[0114]

[0115] At room temperature, 3,3-dimethylacrylic acid (CAS No.: 541-47-9, 100 g, 998.801 mmol, 1.0 eq.) and thioacetic acid (380.09 g, 4993.956 mmol, 5.0 eq.) were added to a 1000 mL single-mouth bottle. The reaction was allowed to proceed overnight at 98 °C. After the reaction was complete as monitored by TLC, the reaction was stopped, the reaction solution was concentrated, and the mixture was filtered through a column using PE:EA=30:1-2:1 to obtain a crude product 1 (38 g). Under room temperature, crude product 1 (19 g, 107.814 mmol, 1.0 eq.) was added to a 1000 mL single-necked bottle. Water (200 mL) and concentrated sulfuric acid (42.29 g, 431.223 mmol, 4.0 eq.) were added. The reaction was allowed to proceed at 100 °C overnight. The reaction was completed after monitoring by TLC. The reaction was stopped. Water and ethyl acetate were added for stirring and separation. The organic phase was separated and concentrated to obtain crude product 2 (13 g). At room temperature, in a 100mL single-mouth bottle, add raw material 78A1 (2g, 5.564mmol, 1.0eq.), DMF (20mL), add 3-(2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (Cas.68181-17-9, 2.09g, 6.691mmol, 1.2eq.), DIEA (1.08g, 8.372mmol, 1.5eq) and react for three hours; LCMS monitors the reaction to be complete, stop the reaction, and the reaction solution is directly used for the next step. At room temperature, add crude product 2 (0.48g, 3.577mmol, 1.0eq.) and methanol (20mL) to the reaction solution of the previous step, react for three hours; LCMS monitors the reaction to be complete, stop the reaction, and prepare 1.2 grams of the target product.

[0116] The structure was confirmed to be correct by NMR (1H NMR (400 MHz, DMSO-d6) δ8.45 (t, J=5.9 Hz, 1H), 7.78 (dd, J=8.3, 1.4 Hz, 1H), 7.58 (dd, J=8.4, 1.3 Hz, 1H), 7.33 (ddd, J=8.4, 7.0, 1.3 Hz, 1H), 7.20 (d, J=8.0 Hz, 2H), 7.06–6.94 (m, 3H), 5.84 (s, 2H), 4.21 (d, J=5.8 Hz, 2H), 2.95–2.86 (m, 4H), 2.46 (d, J=18.4 Hz, 6H), 1.75–1.66 (m, 2H), 1.37 (s, 8H), 0.87 (t, J=7.4 Hz, 3H)).

[0117] 2) Synthesis of OCT-PEG24-C2SS-78A1

[0118] The difference from Example 2 is that Compound A1 is replaced by Compound A2, and the other steps are the same as Example 2.

[0119] The mass spectrometry results of OCT-PEG24-C2SS-78A1 are as follows Figure 5 The chromatographic results are shown in Figure 6 shown.

[0120] Example 4

[0121] A polypeptide-conjugated drug, as shown in the above formula I4, is OCT-PEG24-C2SSC2-78A1, and the preparation process is as follows.

[0122] 1) Synthesis of Compound A3

[0123]

[0124] Weigh 3-methyl-3-mercaptobutyric acid (Cas.59729-24-7, 10 g, 1.0 eq.) into a reaction bottle, add water (48 mL), methanol (66 mL), iodine (11.35 g, 0.6 eq.), react at room temperature for 2 hours, monitor with a spot plate, and there are points with increased polarity (iodine color development), add water and ethyl acetate for extraction, dry the organic phase over anhydrous sodium sulfate, concentrate the solvent, and pass through a column to obtain compound 1 (10 g). The structure was verified to be correct by NMR and used for the next step reaction. Weigh N-hydroxysuccinimide (Cas.6066-82-6, 800 mg, 1.0 eq.) in a reaction tube, add anhydrous DMF (25 mL), compound 1 (9.25 g, 5.0 eq.), EDCI (1.33 g, 1.0 eq.), HOBt (940 mg, 1.0 eq.), DIPEA (1.35 g, 1.5 eq.), react at room temperature for 1 hour, spot plate monitoring shows good reaction, add DIPEA (1.8 g, 2.0 eq.), 78A1 (1.5 g, 0.6 eq.), react at room temperature for 1 hour, LCMS monitors the product molecular weight. Add water and ethyl acetate to extract, dry the organic phase over anhydrous sodium sulfate, concentrate the solvent, and prepare and purify to obtain 700 mg of the target product.

[0125] The structure was confirmed to be correct by NMR (1H NMR (400MHz, DMSO-d6) δ8.40 (t, J = 5.9 Hz, 1H), 7.78 (dd, J = 8.3, 1.4 Hz, 1H), 7.59 (dd, J = 8.5, 1.3 Hz, 1H), 7.35 (ddd, J = 8.4, 6.9, 1.3 Hz, 1H), 7.21 (d, J = 8.0 Hz, 2H), 7.09-6.95 (m,3H),6.78(s,2H),5.84(s,2H),4.20(d,J=5.8Hz,2H),2.90(t,J=7.7Hz,2H),2.46( s,2H),2.38(s,2H),1.71(p,J=7.6Hz,2H),1.44-1.26(m,14H),0.87(t,J=7.4Hz,3H)).

[0126] 2) Synthesis of OCT-PEG24-C2SSC2-78A1

[0127] The difference from Example 2 is that Compound A1 is replaced by Compound A3, and the other steps are the same as Example 2.

[0128] The mass spectrometry results of OCT-PEG24-C2SSC2-78A1 are shown in Figure 7 The chromatographic results are shown in Figure 8 shown.

[0129] Example 5

[0130] A polypeptide-conjugated drug, as shown in the above formula I5, is OCT-PEG24-VC-78A1, and the preparation process is as follows.

[0131] 1) Synthesis of Compound B

[0132]

[0133] Weigh 4-(tert-Butoxy)-4-oxobutanoic acid (Cas.15026-17-2, 2.2 g, 1.0 eq.) into a reaction tube, add anhydrous DMF (20 mL), N-hydroxysuccinimide (Cas.6066-82-6, 2.18 g, 1.5 eq.), EDCI (3.63 g, 1.5 eq.), HOBt (1.71 g, 1.0 eq.), DIPEA (2.45 g, 1.5 eq.), react at room temperature for 1 hour, the spot plate monitoring reaction is good, add Val-Cit-PAB-OH (Cas.159857-79-1, 4.79 g, 1.0 eq.), react at room temperature for 1 hour, LCMS monitors the product molecular weight, add ethyl acetate, solid precipitates, filter and then slurry with dichloromethane to obtain 7.0 grams of compound 1. Weigh compound 1 (7.0 g, 1.0 eq.) in a reaction flask, add THF (70 mL), triethylamine (1.72 g, 1.3 eq.), DMAP (320 mg, 0.2 eq.), p-nitrophenyl chloroformate (Cas.7693-46-1, 3.03 g, 1.15 eq.), react at room temperature for 3 hours, LCMS monitors the product molecular weight, add water and ethyl acetate to extract, the organic phase is dried over anhydrous sodium sulfate, and the solvent is concentrated to obtain 7.0 grams of crude compound 2. Weigh compound 2 (7.0 g, 1.0 eq.) in a reaction flask, add anhydrous DMF (25 mL), triethylamine (810 mg, 0.8 eq.), 78A1 (2.15 g, 0.6 eq.), react at room temperature for 3 hours, LCMS monitors the product molecular weight. Add water and ethyl acetate to extract, the organic phase is dried over anhydrous sodium sulfate, the solvent is concentrated, and column purification is performed to obtain 3.8 grams of compound 3. Compound 3 (3.7 g, 1.0 eq.) was weighed into a reaction bottle, DCM (74 mL) was added, trifluoroacetic acid (19 mL) was added under ice bath, and the mixture was reacted at room temperature for 1 hour. The molecular weight of the product was monitored by LCMS. The solvent was concentrated and purified to obtain 925.06 mg of the target product.

[0134] The structure was confirmed by NMR (1H NMR (400MHz, DMSO-d6) δ12.24 (s, 1H), 9.90 (s, 1H), 8.15 (d, J = 7.4 Hz, 1H), 7.98 (d, J = 8.3 Hz, 1H), 7.76 (q, J = 7.3, 6.2 Hz, 2H), 7.60 (dd, J = 8.2, 5.1 Hz, 3H), 7.45-7.24 (m, 3H), 7.20 (d, J = 7.9 Hz, 2H), 7.11-6.92 (m, 3H), 6.76 (s, 2H), 6.09 (s, 1H),5.84(s,2H),5.49(s,2H),4.94(s,2H),4.34(d,J=7.1Hz,1H),4.26-4.05(m,3H),2.98(dq,J=13.1,6.8Hz,2H),2. 90(t,J=7.8Hz,2H),2.43(s,4H),2.00(h,J=6.5Hz,1H),1.78-1.54(m,4H),1.38(h,J=7.5Hz,4H),0.98-0.72(m,9H)).

[0135] 2) Synthesis of OCT-PEG24-VC-78A1

[0136] The difference from Example 2 is that compound A1 is replaced by compound B, and the other steps are the same as Example 2.

[0137] The mass spectrometry results of OCT-PEG24-VC-78A1 are as follows Fig. 9 The chromatographic results are shown in Fig.10 shown.

[0138] Peptide-conjugated drug activity assay

[0139] (1) Detection of the induction activity of OCT-PEG12-SS-78A1 on TNF-α expression in mouse PBMC cells in vitro

[0140] Take SPF-grade BALB / C mice, 5-6 weeks old, 18-20 grams, and extract the whole blood of the mice by enucleation method for the extraction of PBMC from peripheral blood. Add the same volume of whole blood diluent to the whole blood and mix thoroughly, and add it to the same volume of mouse peripheral blood lymphocyte separation solution, centrifuge at 1000g for 20min, carefully take the lymphocyte layer, wash with 10mL phosphate buffer (PBS), and centrifuge at 250g for 10min. Discard the supernatant and resuspend the lymphocytes in complete RPMI-1640 cell culture medium. The mouse whole blood diluent and lymphocyte separation solution used in the experiment were purchased from Beijing Solebow Technology Co., Ltd., among which the mouse peripheral blood lymphocyte separation solution kit item number is P8620.

[0141] 78A1 (control) and OCT-PEG12-SS-78A1 were taken respectively, and 6 concentrations of each compound were prepared in RPMI-1640 cell culture medium, 78A1 was used as the positive control group, and RPMI-1640 cell culture medium was used as the blank control group.

[0142] Among them, the structural formula of 78A1 is as follows:

[0143]

[0144] 100 μL of RPMI-1640 cell culture medium from each group was added to 100 μL 1.6*10 5 Each group of peripheral lymphocyte culture solutions were prepared in each normal PBMC cell well, and the concentrations of different compounds in each peripheral lymphocyte culture solution were as shown in Table 1.

[0145] Table 1

[0146]

[0147] The peripheral lymphocyte culture solution of each group was placed in a cell culture incubator at 37°C and 5% CO2 for 24 hours, and then centrifuged at 1000rpm for 5 minutes. The cell supernatant was collected and the TNF-α concentration in the peripheral blood monocyte culture supernatant was detected according to the ELISA instructions. The manufacturer of the mouse TNF-alpha double antibody sandwich ELISA detection kit used for detection was Wuhan Mitaka, and the product number was KE10002.

[0148] The experimental results are as follows Fig.11 As shown, within the tested dosage range, relative to the blank control group, in the 78A1 group within the range of 0.08μM-50μM, with the increase of 78A1 concentration, the TNF-α concentration in the cell supernatant showed an upward trend, and then the TNF-α level decreased with the increase of drug concentration; the in vitro immunostimulatory ability of OCT-PEG12-SS-78A1 was significantly lower than that of the 78A1 control group at low concentrations, and exhibited an activation effect equivalent to that of the 78A1 group when the concentration reached 50uM.

[0149] (2) In vitro detection of TNF-α expression activity in mouse PBMC cells

[0150] PBMCs were prepared by the same method as above. 78A1, OCT-PEG24-SS-78A1, OCT-PEG24-C2SS-78A1, OCT-PEG24-C2SSC2-78A1 and OCT-PEG24-VC-78A1 were prepared, and 6 concentrations of each compound were prepared in RPMI-1640 cell culture medium. 78A1 was used as the positive control group, and RPMI-1640 cell culture medium was used as the blank control group.

[0151] 40 μL of RPMI-1640 cell culture medium from each group was added to 160 μL 1.6*10 5 Each group of peripheral lymphocyte culture solutions were prepared in each normal PBMC cell well, and the concentrations of different compounds in each peripheral lymphocyte culture solution were as shown in Table 2.

[0152] Table 2

[0153]

[0154]

[0155] The peripheral lymphocyte culture solution of each group was placed in a cell culture incubator at 37°C and 5% CO2 for 24 hours, and then centrifuged at 1000rpm for 5 minutes. The cell supernatant was collected and the TNF-α concentration in the peripheral blood monocyte culture supernatant was detected according to the ELISA instructions. The manufacturer of the mouse TNF-alpha double antibody sandwich ELISA detection kit used for detection was Wuhan Mitaka, and the product number was KE10002.

[0156] The experimental results are as follows Fig.12 As shown, within the tested dose range, relative to the blank control group, in the 78A1 group, within the range of 0.0098 μM-10 μM, as the concentration of 78A1 increased, the concentration of TNF-α in the cell supernatant showed an upward trend, and the TNF-α level basically reached a peak at 2.5 uM; the in vitro immunostimulatory abilities of OCT-PEG24-SS-78A1, OCT-PEG24-C2SS-78A1, OCT-PEG24-C2SSC2-78A1 and OCT-PEG24-VC-78A1 were significantly lower than those of the 78A1 control group, and no obvious stimulatory effect was shown at low concentrations. When the concentration reached 0.625 uM and above, an activation effect comparable to that of the 78A1 group was shown. The results showed that the lowest effective dose of the peptide-modified molecules for immunostimulating PBMC in vitro was higher than that of the 78A1 molecules.

[0157] (3) Detection of TNF-α expression activity in mouse serum after intravenous injection

[0158] Take 78A1, OCT-PEG12-SS-78A1, OCT-PEG24-SS-78A1, OCT-PEG24-C2SS-78A1, OCT-PEG24-C2SSC2-78A1 and OCT-PEG24-VC-78A1 respectively, use phosphate buffered saline (PBS) as solvent, and prepare PBS solutions containing 6 different components with appropriate concentrations.

[0159] SPF-grade BALB / C female mice, 5-6 weeks old, 18-20 grams, were purchased from the Henan Provincial Experimental Animal Center and divided into 7 groups, namely, Group 1 to Group 7, with 3 mice in each group.

[0160] The mice in group 1 were intravenously injected with 200 μL of the 78A1 solution, the mice in group 2 were intravenously injected with 200 μL of the OCT-PEG12-SS-78A1 solution, the mice in group 3 were intravenously injected with 200 μL of the OCT-PEG24-SS-78A1 solution, the mice in group 4 were intravenously injected with 200 μL of the OCT-PEG24-C2SS-78A1 solution, the mice in group 5 were intravenously injected with 200 μL of the OCT-PEG24-C2SSC2-78A1 solution, the mice in group 6 were intravenously injected with 200 μL of the OCT-PEG24-VC-78A1 solution, and the mice in group 7 were intravenously injected with 200 μL of the PBS solution. The injection dosage of each group of mice is shown in Table 3.

[0161] Table 3

[0162]

[0163]

[0164] 60 minutes after intravenous injection, orbital blood was collected from 3 mice in each group, and the blood was centrifuged at 3800 rpm for 10 minutes to separate serum. The concentration of TNF-α in serum was detected by the same method as above.

[0165] The experimental results are as follows Fig.13 As shown, the significance analysis of the differences in the specification of this application used one-way / two-way ANOVA (ns: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0166] Depend on Fig.13The results showed that the serum TNF-α level of mice in the intravenous injection of 78A1 group increased by 10-12 times compared with the PBS control group 1 hour after injection, and the difference was significant; the serum TNF-α level of mice in the intravenous injection of OCT-PEG12-SS-78A1, OCT-PEG24-C2SS-78A1, OCT-PEG24-C2SSC2-78A1 and OCT-PEG24-VC-78A1 groups increased to varying degrees compared with the PBS group at 1 hour, but was lower than that of the 78A1 group to a certain extent; the serum TNF-α level of mice in the intravenous injection of OCT-PEG24-SS-78A1 group increased by only 3-4 times compared with the PBS group at 1 hour, and the difference was not significant, but was the most obvious decrease compared with the 78A1 injection group, and the difference was significant. The TNF-α content stimulated by OCT-PEG12-SS-78A1 and OCT-PEG24-VC-78A1 was also relatively low, second only to OCT-PEG24-SS-78A1. The results showed that intravenous injection of the same molar amount of peptide-coupled drugs reduced the immune activation side effects in the serum compared with 78A1 alone because of its increased tumor targeting, and systemic administration showed better safety.

[0167] (4) Detection of antitumor activity of OCT-PEG12-SS-78A1

[0168] The same mice as above were divided into 3 groups, namely Group 8 to Group 10, wherein Group 8 and Group 9 each had 6 mice, and Group 10 had 6 mice as a blank control group.

[0169] CT-26 colon cancer cells purchased from ATCC were subcultured in a 37°C, 5% CO2 incubator with DMEM medium containing 10% fetal bovine serum. The specific method is to discard the culture supernatant and rinse the cells 1-2 times with PBS without calcium and magnesium ions. Add 3-5ml of digestion solution (0.25% Trypsin-0.53mM EDTA) to the culture bottle, place it in a 37°C incubator for digestion for 1-2 minutes, and then observe the cell digestion under a microscope. If most of the cells become round and fall off, quickly return to the clean workbench and add 5ml of complete culture medium containing 10% serum to terminate the digestion. Gently blow the cells until they are completely detached and then aspirate them, centrifuge at 250g for 8-10 minutes, discard the supernatant, add 1-2mL of culture medium and blow evenly. Add 5-6ml of culture medium per bottle, and divide the cell suspension into a new T75 cm containing 5-6ml of culture medium at a ratio of 1:2 to 1:4. 2Cell culture flasks were placed. Cells with good growth conditions in the logarithmic growth phase were selected to implant tumors in the left armpits of mice in groups 8 to 10 to construct mouse tumor models. On the day of inoculation, the tumor cell solution was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded to adjust the final cell concentration to 5×10 5 cells / 100 μL, injected subcutaneously in the left armpit to engraft tumors in mice.

[0170] About one week after inoculation, the tumor grew to 80-100 mm 3 PBS was used as solvent to prepare solutions of appropriate concentrations of 78A1 and OCT-PEG12-SS-78A1, and the mice were intravenously injected. Group 8 was intravenously injected with 78A1 solution using a 1 mL syringe, and Group 9 was intravenously injected with OCT-PEG12-SS-78A1 solution using a 1 mL syringe. The volume of the intravenous solution was 200 μL; Group 10 was intravenously injected with 200 μL PBS solution. The dosages of drugs and compounds injected into mice from Group 8 to Group 10 are shown in Table 4.

[0171] Table 4

[0172] Mouse Injection Dosage Group 8 Intravenous injection of 78A1 solution 57 μg Group 9 Intravenous injection of OCT-PEG12-SS-78A1 solution This is consistent with the molar amount of 57 μg 78A1 Group 10 Intravenous injection of PBS solution ——

[0173] The mice in groups 8 to 10 of this example were administered once every 3 days, for a total of 3 times. The day of the first administration was defined as day 0. During the administration process, the changes in the weight and tumor volume of the mice were measured every 2 to 3 days until 20 days after administration. The tumor volume was measured by measuring the long diameter (l) and short diameter (w) of the tumor with a vernier caliper, according to the volume formula: v = 0.5 × l × w 2 After 20 days, the treatment was completed, the mice were euthanized, the tumors were dissected, photographed and analyzed.

[0174] The experimental results are from Fig.14 As shown in A, the body weight of mice in the intravenous 78A1 group showed a significant downward trend after the first injection, while the body weight of mice in the OCT-PEG12-SS-78A1 group showed a steady increase during the entire experimental period. Fig.14 As shown in Figure B, at the end of the experiment, the mean tumor volume of mice in the model control group (Group 10) was 2539±227mm 3 The mean tumor volume of mice in the 78A1 group injected with 57 μg intravenously was 581±335 mm 3 , TGI (%) = 81.28%; the mean tumor volume of mice in the OCT-PEG12-SS-78A1 group was 918 ± 197 mm 3, TGI (%) = 66.88%. The results of this study showed that the anti-tumor activity of the peptide-conjugated drug was not as ideal as that of 78A1. This was considered to be due to the poor plasma stability caused by the poor hydrophilicity of the peptide and the reduced efficacy caused by the rapid clearance of the drug. However, from the perspective of the weight of mice and the release of the toxic factor TNF-α in the body, the intravenous injection of OCT-PEG12-SS-78A1 with the same molar amount as 78A1 had little effect on the weight of mice, and the overall safety was improved. Therefore, in order to improve the water solubility of the molecule, the PEG chain length was increased in order to obtain a peptide conjugate with better efficacy and lower toxicity.

[0175] (5) Detection of antitumor activity of OCT-PEG24-VC-78A1

[0176] The same mice (SPF BALB / C, 5-6 weeks old, 18-20 g female mice) were divided into 3 groups, namely Group 11 to Group 13, 6 mice in each group, and the same modeling method was used to establish mouse tumor models. About one week after inoculation, the tumor grew to 80-100 mm 3 PBS was used as solvent to prepare a solution of appropriate concentration of 78A1 and OCT-PEG24-VC-78A1, and the solution was intravenously injected into mice. The volume of the injected solution was 200 μL, and the dosage of drugs and compounds injected into each group of mice was as shown in Table 5.

[0177] Table 5

[0178] Mouse Injection Dosage Group 11 78A1 solution 57ug Group 12 OCT-PEG24-VC-78A1 solution This is consistent with the molar amount of 57 μg 78A1 Group 13 PBS solution ——

[0179] The mice in groups 11 to 13 of this example were administered once every 3 days, for a total of 3 times. The day of the first administration was defined as day 0. During the administration process, the changes in the weight and tumor volume of the mice were measured every 2 to 3 days until 21 days after administration. The tumor volume was measured by measuring the long diameter (l) and short diameter (w) of the tumor with a vernier caliper, according to the volume formula: v = 0.5 × l × w 2 After 21 days, the treatment was terminated, the mice were euthanized, the tumors were dissected, photographed and analyzed.

[0180] like Fig.15 As shown, at the end of the experiment, the mean tumor volume of mice in the 78A1 group was 36±36mm 3 , tumor volume inhibition rate TGI (%) = 98.96%; the mean tumor volume of mice in the OCT-PEG24-VC-78A1 group was 29±29mm 3The average tumor volume was smaller than the tumor volume at the initial administration, TGI (%) = 99.89%. Although the tumor inhibition rate of the OCT-PEG24-VC-78A1 group was equivalent to that of the 78A1 group alone, considering the level of serum toxic factor TNF-α in the body, OCT-PEG24-VC-78A1 had better safety than 78A1 intravenous injection.

[0181] (6) Dose-effect relationship study of OCT-PEG24-SS-78A1 in the mouse colon cancer CT26 subcutaneous tumor model

[0182] The same mice (SPF BALB / C, 5-6 weeks old, 18-20 g female mice) were divided into 5 groups, namely Group 14 to Group 18, with 6 mice in each group, and the same mouse tumor model construction method was used to establish the mouse tumor model. About one week after inoculation, the tumor grew to 80-100 mm 3 PBS was used as solvent to prepare a solution of appropriate concentration of 78A1 and OCT-PEG24-SS-78A1, and the solution was intravenously injected into mice. The volume of the injected solution was 200 μL, and the dosage of drugs and compounds injected into each group of mice was as shown in Table 6.

[0183] Table 6

[0184]

[0185] The mice in groups 14 to 18 of this example were administered once every 3 days, for a total of 3 times. The day of the first administration was defined as day 0. During the administration process, the changes in the weight and tumor volume of the mice were measured every 2 to 3 days until 20 days after administration. The tumor volume was measured by measuring the long diameter (l) and short diameter (w) of the tumor with a vernier caliper, according to the volume formula: v = 0.5 × l × w 2 After 20 days, the treatment was completed, the mice were euthanized, the tumors were dissected, photographed and analyzed.

[0186] like Fig.16 As shown in A, the weight of mice in the 78A1 injection group and the OCT-PEG24-SS-78A1 high-dose group decreased significantly after the first administration, and the weight change trend of the two groups of mice was basically the same during the whole experiment. The weight of mice in the OCT-PEG24-SS-78A1 medium-dose and low-dose groups showed different degrees of increase. Fig.16 As shown in Figure B, the tumors of mice in the PBS control group grew rapidly. At the end of the experiment, the mean tumor volume of mice in the model control group (PBS) was 2134±410mm 3The mean tumor volume of mice in the low-dose OCT-PEG24-SS-78A1 group was 1522±160mm 3 , tumor inhibition rate TGI (%) = 28.65%; the mean tumor volume of mice in the OCT-PEG24-SS-78A1 medium-dose group was 298±158mm 3 , tumor inhibition rate TGI (%) = 86.01%; the mean tumor volume of mice in the OCT-PEG24-SS-78A1 high-dose group was 110±58mm 3 , tumor inhibition rate TGI (%) = 94.82%; the mean tumor volume of mice in group 78A1 was 667±227mm 3 , tumor inhibition rate TGI (%) = 68.73%. It shows that OCT-PEG24-SS-78A1 has better anti-tumor effect than 78A1 of the same molar amount in the CT26 subcutaneous tumor model, has significant anti-tumor effect, and shows a dose-effect relationship. Combined with the previous research data, the disulfide linker and VC-PABC linker in the peptide conjugate both show the characteristics of specific drug release at the tumor site, making the intravenous injection of PDC have the effect of reduced toxicity and increased efficacy compared with 78A1 alone; it also shows that the water solubility of the peptide conjugate has a significant effect on the efficacy and toxicity improvement of intravenous injection.

[0187] In order to observe the effect of cleavable linkers and non-cleavable linkers on the activity of the peptide-coupled drug, a non-cleavable linker structure (OCT-PEG24-GMBS-78A1) was designed as a control. OCT-PEG24-GMBS-78A1 has good water solubility and is comparable to OCT-PEG24-SS-78A1 in dissolution. The solubility in water can reach 20 mg / ml. The same modeling method was used to construct a mouse tumor model, and the tumor grew to 80-100 mm. 3 At the same time, the control group PBS, and the drug administration group 78A1 (10ug / mouse) and OCT-PEG24-GMBS-78A1 (equimolar amount to the 78A1 group) were intravenously injected once every 3 days, for a total of 3 times. During the drug administration process, the changes in mouse body weight and tumor volume were measured every 2 to 3 days until 20 days after administration. At the end of the experiment, the results showed that the tumor inhibition rate of the 78A1 injection group was 69.71%, while the OCT-PEG24-GMBS-78A1 injection group had almost no therapeutic effect on tumor-bearing mice, and the tumor growth curve was basically consistent with the PBS control group. It can be seen that the effect of connecting the small molecule 78A1 to octreotide through a cleavable linker to form PDC for intravenous injection is significantly better than that of the non-cleavable linker structure.

[0188] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A somatostatin receptor binding peptide coupled to a TLR agonist polypeptide-coupled drug, characterized in that: The polypeptide-coupled drug comprises at least one compound of the following formula I-1 and formula I-2 or a pharmaceutically acceptable salt thereof: Wherein, R1, R2, R3, and R4 are independently selected from hydrogen, C 1-10 Alkyl, C 1-10 At least one of the alkoxy groups, n=1-48, and X is octreotide.

2. The polypeptide-conjugated drug according to claim 1, characterized in that: R1, R2, R3, R4 are independently selected from hydrogen and C 1-5 At least one of the alkyl groups.

3. The polypeptide-conjugated drug according to claim 1, characterized in that: n＝10-36。 4. The polypeptide-conjugated drug according to any one of claims 1 to 3, characterized in that: In formula I-1: R1, R2, R3, and R4 are all hydrogen, and n=12; or, R1, R2, R3, and R4 are all hydrogen, and n=24; or, R1 and R2 are hydrogen, and R3 and R4 are methyl, and n=24; or, R1, R2, R3, and R4 are all methyl, and n=24; Alternatively, in formula I-2: n=24.

5. A method for preparing a polypeptide-coupled drug according to any one of claims 1 to 4, characterized in that: include: Provide compound A or compound B; wherein R1, R2, R3, and R4 in compound A are independently selected from hydrogen, C 1-10 Alkyl, C 1-10 at least one of alkoxy groups; The resin linear peptide corresponding to octreotide was synthesized in the order from the C-terminus to the N-terminus of the polypeptide; Connecting polyethylene glycol to the N-terminus of the resin straight-chain peptide corresponding to the octreotide, and then performing a condensation reaction with the compound A or the compound B to obtain an intermediate; The intermediate is cleaved from the resin and then subjected to an oxidation reaction to obtain the compound of formula I-1 or the compound of formula I-2.

6. The preparation method according to claim 5, characterized in that: The step of connecting the N-terminus of the resin straight-chain peptide corresponding to the octreotide to polyethylene glycol comprises: subjecting the N-terminus of the resin straight-chain peptide corresponding to the octreotide to a dehydration condensation reaction with Fmoc-NH-PEG-COOH, and then removing the amino protection.

7. The preparation method according to claim 5 or 6, characterized in that: The oxidation treatment comprises: mixing the intermediate obtained by cutting from the resin with oxygen for 10 to 24 hours.

8. Use of the polypeptide conjugate drug according to any one of claims 1 to 4 and / or the polypeptide conjugate drug prepared by the preparation method according to any one of claims 5 to 7 in the preparation of anti-tumor drugs or immunostimulatory adjuvants.

9. The use according to claim 8, characterized in that The tumors targeted by the anti-tumor drug include at least one of gastrointestinal pancreatic neuroendocrine tumors, colorectal cancer, breast cancer, small cell lung cancer and prostate cancer.

10. A pharmaceutical composition, characterized in that It includes the polypeptide conjugate drug according to any one of claims 1 to 4 and / or the polypeptide conjugate drug prepared by the preparation method according to any one of claims 5 to 7.

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