Polypeptide Conjugate Drug and Its Preparation Method and Application

Abstract

This application relates to the field of pharmaceutical technology, and particularly to a polypeptide conjugate drug, its preparation method and application. The polypeptide conjugate drug of this application includes at least one compound of Formula I-1 and Formula I-2 in the specification or a pharmaceutically acceptable salt thereof. Based on the polypeptide conjugate drug with a unique molecular structure provided by this application, it can bind to tumor cells through the integrin receptor-binding peptide via blood circulation or remain in the tumor tissue after intratumoral injection, activate the TLR7 and TLR8 receptors in the tumor tissue, and the level of inflammatory factors in the blood after injection is further reduced compared with the payload molecule 78A1, and the safety is significantly improved. Therefore, the polypeptide conjugate drug of this application has tumor tissue targeting, can activate the TLR7 and TLR8 receptors on the cytoplasmic endosomes in the tumor tissue, exert an anti-tumor immune stimulation effect, and at the same time has lower blood and non-specific organ immune toxicity.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical technology, and particularly relates to a polypeptide conjugate drug, its preparation method and application. Background Art

[0002] Toll-like receptors (TLR) are natural immune system receptors that monitor and recognize various different pathogen-associated molecular patterns (PAMP). They are widely expressed in the immune cell spectrum, participate in non-specific immunity and mediate specific immunity, and are the first line of defense for the body to resist infectious diseases. Currently, 13 TLRs (i.e., TLR1-TLR13) have been identified in mammals, including TLR1-TLR11 in humans, TLR1-TLR9 and TLR11-TLR13 in mice. Activation of TLR can induce MyD88- or TRIF-dependent signaling pathways, activate NF-κB, induce the secretion of cytokines and chemokines, activate innate immune responses and mediate the activation of acquired immune responses, and can be used as an immune adjuvant for anti-tumor and infectious diseases.

[0003] TLR agonists include imidazoquinolineamine compounds such as imiquimod (R837), resiquimod (R848), etc. For example, R-848 is a dual agonist of TLR7 and TLR8 (i.e., a TLR7 / 8 agonist). It is formulated as a topical gel for the treatment of skin lesions caused by herpes simplex virus and cutaneous T-cell lymphoma, and as an adjuvant to enhance the effectiveness of vaccines. It can produce effective immunotherapy for tumors through immune stimulation of tumor-associated macrophages. Although TLR7 / 8 agonists are effective adjuvants for local anti-tumor treatment, as small molecule adjuvants, TLR7 / 8 agonists do not have specific targeting, have a short half-life, and intravenous injection can lead to hyperactivity of the systemic immune system, thereby causing cytokine release syndrome (CRS), and it is difficult to tolerate in the body. Therefore, the severe side effects of TLR7 / 8 agonists are still an important problem faced in current clinical applications.

[0004] Peptide Drug Conjugate (PDC) consists of a targeting peptide, a linker, and a payload (also known as a toxic drug). After the drug is conjugated to the peptide through the linker, it endows the peptide and the drug with dual functions, and can promote the killing or targeting effect of the drug. Different drugs require matching linkers to conjugate them with the targeting peptide; however, currently, there are no related peptide conjugate drugs for TLR7 / 8 agonist small molecule compounds. Summary of the Invention

[0005] The object of the present application is to provide a polypeptide-conjugated drug, its preparation method and application, aiming to solve the technical problem of how to provide a polypeptide-conjugated drug related to TLR7 / 8 agonist small molecule compounds to improve the targeting of TLR7 / 8 agonists and reduce their side effects.

[0006] To achieve the above application object, the technical solution adopted in the present application is as follows:

[0007] In the first aspect, the present application provides a polypeptide-conjugated drug, and the polypeptide-conjugated drug includes at least one compound of formula I-1 and formula I-2 below or a pharmaceutically acceptable salt thereof:

[0008]

[0009] Wherein, R1, R2, R3, and R4 are independently selected from at least one of hydrogen, C 1-10 alkyl, C 1-10 alkoxy, and C 1-10 alkyl alcohol, n = 0 - 50, and X is an integrin receptor-binding peptide.

[0010] In the second aspect, the present application provides a preparation method of the above polypeptide-conjugated drug, including:

[0011] Condensing compound C with an integrin receptor-binding peptide to obtain intermediate S1;

[0012] Performing an amino deprotection reaction on the intermediate S1 to obtain intermediate S2;

[0013] Reacting the intermediate S2 with compound A and compound B1 to obtain the compound shown in formula I-1, or reacting the intermediate S2 with compound A and compound B2 to obtain the compound shown in formula I-2;

[0014]

[0015] In the third aspect, the present application provides an application, that is, the application of the polypeptide-conjugated drug of the present application and / or the polypeptide-conjugated drug prepared by the preparation method of the present application in the preparation of anti-tumor drugs, anti-viral drugs or immune-stimulating adjuvants.

[0016] In the fourth aspect, the present application provides a pharmaceutical composition, including the polypeptide-conjugated drug of the present application and / or the polypeptide-conjugated drug prepared by the preparation method of the present application.

[0017] The polypeptide conjugate drug provided in the first aspect of the present application uses a specific linker to connect a thiomethylated derivative of a TLR7 / 8 agonist (CAS registration number 1258457-59-8, referred to as 78A1) and an integrin receptor-binding peptide. This polypeptide conjugate drug can bind to tumor cells through the integrin receptor-binding peptide via blood circulation or remain in the tumor tissue after intratumoral injection, activate the TLR7 and TLR8 receptors in the tumor tissue, and the level of inflammatory factors in the blood after injection is further reduced compared to the payload molecule 78A1, and the safety is significantly improved. Therefore, the polypeptide conjugate drug of the present application has tumor tissue targeting, can activate the TLR7 and TLR8 receptors on the cytoplasmic endosomes in the tumor tissue, exert an anti-tumor immune stimulation effect, and at the same time has lower blood and non-specific organ immune toxicity.

[0018] The preparation method provided in the second aspect of the present application uses compound C, an integrin receptor-binding peptide, compound A and compound B1 or B2 as raw materials, and prepares the compound shown in formula I-1 or formula I-2 in the polypeptide conjugate drug through a series of steps. The preparation method of the present application has a high yield and few by-products, and the prepared polypeptide conjugate drug has tumor tissue targeting, can activate the TLR7 and TLR8 receptors on the cytoplasmic endosomes in the tumor tissue, exert an anti-tumor immune stimulation effect, and at the same time has lower blood and non-specific organ immune toxicity.

[0019] The application provided in the third aspect of the present application is based on the above advantages of the unique polypeptide conjugate drug of the present application. Therefore, the polypeptide conjugate drug of the present application can be used to prepare anti-tumor drugs, anti-viral drugs and immune-stimulating adjuvants.

[0020] The pharmaceutical composition provided in the fourth aspect of the present application includes the unique polypeptide conjugate drug of the present application. Based on its low blood toxicity, 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 the plasma, and thus achieves an anti-tumor effect while effectively reducing systemic immune toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0022] Figure 1 It is the mass spectrum of RGD-PEG12-OSSO-78A1 in the present application;

[0023] Figure 2Mass spectrum of RGD-PEG12-C2SS-78A1 in this application;

[0024] Figure 3 Mass spectrum of [RGD]2-PEG12-OSSO-78A1 in this application;

[0025] Figure 4 Mass spectrum of [RGD]2-PEG12-C2SS-78A1 in this application;

[0026] Figure 5 Mass spectrum of [RGD]2-PEG12-OC2SSC2O-78A1 in this application;

[0027] Figure 6 Schematic diagram of the detection results of the induced expression activities of 78A1, RGD-PEG12-OSSO-78A1, RGD-PEG12-C2SS-78A1, and [RGD]2-PEG12-OSSO-78A1 on TNF-α in mouse PBMC cells;

[0028] Figure 7 Schematic diagram of the detection results of the serum TNF-α and IL-6 contents after intravenous injection of 78A1, RGD-PEG12-OSSO-78A1, RGD-PEG12-C2SS-78A1, and [RGD]2-PEG12-OSSO-78A1, where A is the detection result of TNF-α and B is the detection result of IL-6;

[0029] Figure 8 Inhibitory effects of 78A1 and RGD-PEG12-OSSO-78A1 on tumors; among them, A: body weight change, B: tumor growth curve, C: tumor weighing, D: tumor size at the end of the experiment;

[0030] Figure 9 Antitumor inhibitory effects of RGD-SS-78A1, RGD-PEG12-SS-78A1, and RGD-PEG12-OSSO-78A1 on the 4T1 tumor model;

[0031] Figure 10 Antitumor inhibitory effects of RGD-PEG12-C2SS-78A1 and [RGD]2-PEG12-OSSO-78A1 on the CT26 tumor model; among them, A: body weight change, B: tumor growth curve, C: tumor weighing, D: tumor size at the end of the experiment;

[0032] Figure 11Schematic diagram of the detection results of the induced expression activities of [RGD]2-PEG12-C2SS-78A1 and [RGD]2-PEG12-OC2SSC2O-78A1 on TNF-α in mouse PBMC cells;

[0033] Figure 12 Schematic diagram of the detection results of serum TNF-α 1 h after intravenous injection of [RGD]2-PEG12-C2SS-78A1 and [RGD]2-PEG12-OC2SSC2O-78A1 in mice. Specific embodiments

[0034] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application clearer and more understandable, the following further details this application in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application.

[0035] In this application, the term "and / or" describes the association relationship of associated objects and indicates that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Among them, A and B can be singular or plural. The character " / " generally represents an "or" relationship between the front and back associated objects.

[0036] In this application, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following (items)" or its similar expression refers to any combination of these items, including any combination of single item (item) or plural items (items).

[0037] It should be understood that in various embodiments of this application, the size of the serial numbers of the above processes does not mean the order of execution. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of this application.

[0038] The terms used in the embodiments of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The singular forms of "a", "the" and "said" used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0039] In the specification of the embodiments of the present application, the weight of the relevant components mentioned not only can refer to the specific content of each component, but also can represent the proportional relationship of the weights between each component. Therefore, as long as the content of the relevant components in the specification of the embodiments of the present application is enlarged or reduced in proportion, it is within the scope disclosed in the specification of the embodiments of the present application. Specifically, the mass described in the specification of the embodiments of the present application can be mass units well-known in the chemical industry such as μg, mg, g, kg, etc.

[0040] The terms "first" and "second" are only used for descriptive purposes to distinguish objects such as substances from each other, and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX can also be referred to as the second XX. Similarly, the second XX can also be referred to as the first XX. Thus, the features defined with "first" and "second" can explicitly or implicitly include one or more of such features.

[0041] Antibody-drug conjugates (ADCs) can utilize the antibody drug HER2 antibody conjugated to ADC targeting tumor antigens to achieve targeted delivery of TLR7 / 8A. Although the antibody specifically binds to the antigen, the antigen distribution in tumors and normal cells is only different in quantity, which can only reduce but is difficult to solve the problem of systemic immunotoxicity. Peptide-drug conjugates (PDCs) consist of three parts: a targeting peptide, a linker, and a payload. After the drug binds to the peptide through the linker, it endows the peptide and the drug with two-way functions, which can promote the killing or targeting effect of the drug. The linker (Linker) among them is very crucial and can be divided into two major categories: cleavable and non-cleavable. Currently, most ADCs use acid-sensitive, protease-sensitive, or glutathione-sensitive cleavable linkers, which are degraded by highly expressed proteases and glutathione in tumors. However, there is no report on the linker for TLR7 / 8 agonists.

[0042] Due to the lack of targeting, short half-life, and systemic immunotoxicity of TLR7 / 8 agonist small molecule compounds, their efficacy and clinical applications are limited. Even when injected intratumorally, it is still difficult to overcome the systemic immunotoxicity caused by rapid absorption. Using antibody-drug conjugate (ADC) technology to target the delivery of TLR7 / 8 agonists to tumors can reduce their toxicity, but due to only quantitative differences in antigen distribution between tumors and normal cells, it is still difficult to overcome systemic immunotoxicity. In fact, in the cellular composition of the solid tumor microenvironment, in addition to a large number of tumor cells, there are also a large number of non-tumor cells, including various immune cells, fibroblasts, and stromal cells, etc. The target cells of TLR7 / 8 agonists are immune cells within the tumor (including the original immune cells within the tumor and the immune cells infiltrated induced by TLR7 / 8 agonists). Therefore, the design of novel TLR7 / 8 agonists needs to solve the problems of drug binding and anchoring to intratumoral cells, prolonging retention and reducing absorption, improving efficacy, and minimizing or overcoming systemic non-specific immunotoxicity. For this purpose, the embodiments of the present application design a polypeptide conjugate drug and its preparation method and application. The specific scheme is as follows.

[0043] In a first aspect, the embodiments of the present application provide a polypeptide conjugate drug, comprising 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:

[0044]

[0045] Wherein, R1, R2, R3, and R4 are independently selected from at least one of hydrogen, C 1-10 alkyl, C 1-10 alkoxy, and C 1-10 alkyl alcohol, n = 0 - 50, and X is an integrin receptor-binding peptide.

[0046] The polypeptide conjugate drug of the embodiments of the present application contains a unique compound of formula I-1 and / or formula I-2. After local injection of the polypeptide conjugate drug of the embodiments of the present application, the TLR7 / 8 agonist released acts on immune cells within the tumor, activates the TLR7 and TLR8 receptors on cytoplasmic endosomes, activates the expression of inflammatory cytokines through the MYD88-mediated NF-κB 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 polypeptide conjugate drug effectively reduces the content of TLR7 / 8 agonists in the blood, reduces or avoids 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.

[0047] Specifically, the polypeptide conjugate drug of the embodiments of the present application includes, according to its design principle: an imidazoquinoline-based small molecule TLR7 / 8 agonist, namely 78A1, an integrin receptor-binding peptide, and a cleavable linker that couples the two. This polypeptide conjugate drug can bind to the integrin receptor on the surface of tumor cells. After systemic injection, it can be enriched in tumor tissues through blood circulation, reducing the drug concentration in normal tissues. While after local injection, it can bind in tumor tissues, reducing the drug concentration in the blood while maintaining the anti-tumor activity of the drug, so that the polypeptide conjugate drug has lower immunotoxicity while having anti-tumor activity. The polypeptide conjugate drug of the embodiments of the present application can be used for intratumoral injection and / or intravenous injection immunotherapy of various solid tumors.

[0048] The cleavable linker is a commonly used design in PDC construction. It is stable for a long time during blood circulation and has the ability to rapidly and efficiently respond to release drugs in the tumor microenvironment. Disulfide bonds will break down in a reducing environment. Glutathione is a reducing agent present in the cytoplasm, and its intracellular concentration (0.5 - 10 mmol / L) is 1000 times higher than that in plasma (2 - 20 μmol / L). At the same time, the hypoxic environment caused by abnormal blood flow in tumor tissues further activates the activity of reductase, making the disulfide bond relatively stable in plasma, while it can be decomposed in tumor tissues to release the loaded cytotoxic molecules. However, the current single disulfide bond structure cannot achieve the perfect combination of high circulation stability and efficient intracellular release. PDC has a high tumor tissue penetration ability due to its small molecular weight and is more likely to penetrate the tumor stroma and enter tumor cells. However, the small molecular weight PDC is much smaller than the glomerular filtration threshold and can be rapidly cleared by the kidneys, limiting the effective accumulation of cytotoxins at the pharmacological site. Therefore, PDC must be further modified to ensure continuous exposure after administration. And the linker designed in the embodiments of the present application stably couples the TLR7 / 8 agonist, namely 78A1, and the integrin receptor-binding peptide well.

[0049] In some embodiments, the integrin receptor-binding peptide in the polypeptide conjugate drug includes at least one of RGD monocyclic peptide and RGD bicyclic peptide, and the structure is as follows:

[0050]

[0051] In some embodiments, C 1-10 Alkyl represents a straight-chain alkyl or branched-chain alkyl containing 1 - 10 carbons, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, etc.; C 1-10 Alkoxy represents a straight-chain alkoxy or branched-chain alkoxy containing 1 - 10 carbons, such as methoxy, ethoxy, propoxy, butoxy, etc.; C 1-10 Alkyl alcohol represents a straight-chain alkyl alcohol or branched-chain alkyl alcohol containing 1 - 10 carbons, that is, a hydroxyl group substitutes C 1-10Alkyl groups, such as hydroxymethyl, hydroxyethyl, hydroxypropyl, etc. n = 0 - 50, where n is an integer, and can be typical but non-limiting values such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 16, 18, 20, 25, 26, 30, 35, 38, 40, 44, 45, 48, 50, etc. Further, n = 1 - 36, more further, n = 1 - 24, and still more further, n = 1 - 15. In some embodiments, R1, R2, R3, and R4 are independently selected from hydrogen and C 1-5 at least one of alkyl groups.

[0052] In some embodiments, the polypeptide conjugate drug includes at least one of the following formulas (1) - (4):

[0053]

[0054]

[0055] In some embodiments, in the compound of the above formula I-2, X is an RGD monocyclic peptide, R1, R2, R3, and R4 are hydrogen, and n = 12, that is, the linker includes a polyethylene glycol (PEG)-modified dithio bond carbamate. At this time, the chemical structure formula of the compound included in the polypeptide conjugate drug is as shown in formula I1, and is named RGD-PEG12-OSSO-78A1 in the present application:

[0056]

[0057] In some embodiments, in the compound of the above formula I-1, X is an RGD monocyclic peptide, R1 and R2 are hydrogen, R3 and R4 are methyl, and n = 12, that is, the linker includes a polyethylene glycol (PEG)-modified dithio bond. At this time, the chemical structure formula of the compound included in the polypeptide conjugate drug is as shown in formula I2, and is named RGD-PEG12-C2SS-78A1 in the present application:

[0058]

[0059] In some embodiments, in the compound of the above formula I-2, X is an RGD bicyclic peptide, R1, R2, R3, and R4 are hydrogen, and n = 12, that is, the linker includes a polyethylene glycol (PEG)-modified dithio bond carbamate. At this time, the chemical structure formula of the compound included in the polypeptide conjugate drug is as shown in formula I3, and is named [RGD]2-PEG12-OSSO-78A1 in the present application:

[0060]

[0061] In some embodiments, in the compound of formula I-1 as described above, X is an RGD bicyclic peptide, R1 and R2 are hydrogen, R3 and R4 are methyl, and n = 12, that is, the linker includes a polyethylene glycol (PEG)-modified disulfide bond. At this time, the structural formula of the polypeptide conjugate drug includes the compound shown in formula I4, which is named [RGD]2-PEG12-C2SS-78A1 in this application:

[0062]

[0063] In some embodiments, in the compound of formula I-2 as described above, X is an RGD bicyclic peptide, R1, R2, R3, and R4 are all methyl, and n = 12, that is, the linker includes a polyethylene glycol (PEG)-modified disulfide bond carbamate. At this time, the structural formula of the polypeptide conjugate drug includes the compound shown in formula I5, which is named [RGD]2-PEG12-OC2SSC2O-78A1 in this application:

[0064]

[0065] After administration, the above polypeptide conjugate drugs can target the tumor region, activate the TLR7 / 8 receptor, and then exhibit good anti-tumor activity. At the same time, the content of 78A1 released into the blood is low, resulting in low systemic immunotoxicity of these polypeptide conjugate drugs.

[0066] In a second aspect, an embodiment of the present application provides a method for preparing a polypeptide conjugate drug. The preparation method includes:

[0067] S01: Condensing compound C with an integrin receptor-binding peptide to obtain intermediate S1;

[0068] S02: Performing an amino deprotection reaction on intermediate S1 to obtain intermediate S2;

[0069] S03: Reacting intermediate S2 with compound A and compound B1 to obtain the compound shown in formula I-1, or reacting intermediate S2 with compound A and compound B2 to obtain the compound shown in formula I-2.

[0070] In some embodiments, the integrin receptor-binding peptide may include an RGD monocyclic peptide, i.e., compound D, and an RGD bicyclic peptide, i.e., compound E. The structural formulas of the provided raw materials: compound A, compound B1 / B2, compound C, compound D, and compound E are as follows:

[0071]

[0072] Specifically, condensing compound C with compound D or E in a reaction system to obtain intermediate S1; wherein, X is an RGD monocyclic peptide or an RGD bicyclic peptide.

[0073]

[0074] In some embodiments, a carbodiimide condensing agent and an acylation catalyst are used in the reaction system of the condensation reaction, so that the condensation reaction can be carried out under relatively mild conditions. Further, the condensation reaction is carried out in an N,N-dimethylformamide (DMF) solvent system.

[0075] In some embodiments, the carbodiimide condensing agent may include: dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), etc. One or more of them; the acylation catalyst may include 4-N,N-dimethylpyridine (DMAP), 1-hydroxybenzotriazole (HOBt), etc.

[0076] In some embodiments, the amino deprotection reaction of intermediate S1 is carried out under the condition of a deprotecting agent. Specifically, the deprotecting reagent added includes one or more of trifluoroacetic acid, hydrochloric acid solution, diethylamine, etc.

[0077] In some embodiments, diethylamine is added to the reaction solution of the condensation reaction to carry out the amino deprotection reaction to obtain intermediate S2.

[0078]

[0079] In some embodiments, at room temperature (25-30 °C), intermediate S2 and compound B1 or compound B2 are added to DMF solvent, and then N,N-diisopropylethylamine (DIPEA) is added and reacted to obtain intermediate S3. According to the structures of compound B1 or compound B2, the structural formula of the obtained intermediate S3 is as follows:

[0080]

[0081] Finally, at room temperature, in the reaction solution of intermediate S3, compound A is added for reaction, and the reaction obtains the compounds of formula I-1 and / or formula I-2 as the final product.

[0082] In a third aspect, the embodiments of the present application provide the use of the above polypeptide conjugate drug and / or the polypeptide conjugate drug prepared by the above preparation method in the preparation of anti-tumor drugs, anti-viral drugs or immune-stimulating adjuvants.

[0083] Based on the polypeptide conjugate drug of the embodiments of the present application, after local or intravenous injection, it can release TLR7 / 8 agonists in tumors, increase the content of inflammatory mediators in tumors, and effectively avoid the increase of pro-inflammatory cytokines such as TNF-α in plasma, making the polypeptide conjugate drug have great application potential in the preparation of anti-tumor, anti-viral and vaccine adjuvants.

[0084] 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 T-cell lymphoma, melanoma, breast cancer, rectal cancer, lung cancer, pancreatic cancer, prostate cancer, ovarian cancer, bone cancer, and brain tumor. Solid tumors are more likely to be administered by intratumoral injection, which can further improve the enrichment degree of TLR7 / 8 agonist molecules in the tumor, and is beneficial to further improving the anti-tumor effect of the polypeptide conjugate drug in the embodiments of the present application.

[0085] Fourthly, the embodiments of the present application provide a pharmaceutical composition, which includes the above-mentioned polypeptide conjugate drug and / or the polypeptide conjugate drug prepared by the above-mentioned preparation method.

[0086] After the pharmaceutical composition in the embodiments of the present application is administered, the polypeptide conjugate drug contained in the pharmaceutical composition is used locally, and can 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, and has good targeting.

[0087] In some embodiments, the administration mode 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 rapid intratumoral injection, intratumoral controlled injection, and intravenous injection. Among them, rapid intratumoral injection and intravenous injection are to inject the drug into the tumor tissue in a short time with a conventional syringe at one time, and intratumoral controlled injection is to use a micro-injection pump to control the flow rate through an indwelling needle, so that the concentration of the drug in the tumor tissue is maintained within the effective concentration range during the continuous injection time.

[0088] The following will be described in conjunction with specific embodiments. In addition, the significance analysis of differences in the specification of the present application uses one-way / two-way ANOVA (ns: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0089] Example 1

[0090] A polypeptide conjugate drug, as shown in Formula I1 above, the polypeptide conjugate drug is RGD-PEG12-OSSO-78A1, and the preparation process is as follows:

[0091]

[0092] In a 50 mL single-necked flask at room temperature, add compound C (FmocNH-PEG12-CH2CH2COOH, Cas. 1952360-91-6, 800 mg, 0.952 mmol, 1.0 eq.), DMF (8 mL), add DCC (294.77 mg, 1.429 mmol, 1.5 eq.), DMAP (58.18 mg, 0.476 mmol, 0.5 eq.), and add compound D (RGD monocyclic peptide, Cas. 161552-03-0, 574.95 mg, 0.952 mmol, 1.0 eq.). Monitor the reaction by LCMS until completion, then stop the reaction to obtain intermediate S1, and directly proceed with the next reaction using the reaction solution.

[0093] At room temperature, add diethylamine (410.43 mg, 5.612 mmol, 10.0 eq.) to the above reaction solution containing intermediate S1, and react for 3 hours. Monitor the reaction by LCMS until completion, then stop the reaction to prepare intermediate S2 (130 mg). At room temperature, in a 10 mL single-necked flask, add compound B (94.29 mg, 0.216 mmol, 2.0 eq.), DMF (1 mL), DIPEA (27.93 mg, 0.216 mmol, 2.0 eq.), add intermediate S2 (130 mg, 0.108 mmol, 1.0 eq.). Monitor the reaction by LCMS until completion, then stop the reaction to obtain intermediate S3, and directly proceed with the next reaction using the reaction solution.

[0094] At room temperature, add compound A (91.95 mg, 0.256 mmol, 3.0 eq.) to the reaction solution containing intermediate S3, and react for 2 hours. Monitor the reaction by LCMS until completion, then stop the reaction. Purify the reaction solution to obtain 40 mg of the target product. The mass spectrometry results of RGD-PEG12-OSSO-78A1 are as Figure 1 shown.

[0095] Example 2

[0096] A polypeptide conjugate drug, as shown in formula I2 above, the polypeptide conjugate drug is RGD-PEG12-C2SS-78A1, and the preparation process is as follows:

[0097]

[0098] At room temperature, in a 1000 mL single-necked flask, add raw material 1 (100 g, 998.801 mmol, 1.0 eq.), add raw material 2 thioacetic acid (380.09 g, 4993.956 mmol, 5.0 eq.), and react at 98 °C overnight. Monitor the reaction by TLC until completion, stop the reaction, concentrate the reaction solution, and column chromatograph with PE:EA = 30:1 to 2:1 to obtain 38 g of crude product 3. At room temperature, in a 1000 mL single-necked flask, add crude product 3 (19 g, 107.814 mmol, 1.0 eq.), add water (200 mL), concentrated sulfuric acid (42.29 g, 431.223 mmol, 4.0 eq.), and react at 100 °C overnight. Monitor the reaction by TLC until completion, stop the reaction, add water and ethyl acetate and stir to separate the layers, separate the organic phase, and concentrate to obtain crude product 4 (13 g). At room temperature, in a 100 mL single-necked flask, add compound A (2 g, 5.564 mmol, 1.0 eq.), DMF (20 mL), add raw material 5 (Cas. 68181-17-9; 2.09 g, 6.691 mmol, 1.2 eq), DIEA (1.08 g, 8.372 mmol, 1.5 eq), and react for three hours; monitor the reaction by LCMS until completion, stop the reaction, and directly carry out the next step of the reaction with the reaction solution. At room temperature, in the above reaction solution, add crude product 4 (0.48 g, 3.577 mmol, 1.0 eq.), methanol (20 mL), and react for three hours; monitor the reaction by LCMS until completion, stop the reaction, and prepare 1.2 g of product 7. LCMS (ESI) calcd for C 30 H 37 N5O3S2, +[M+H]+ m / z 580.24, found 580.4. The structure of product 7 was verified to be correct by NMR (1H NMR (400 MHz, DMSO-d6) δ 8.45 (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).).

[0099] Weigh the product 7 (65 mg, 1.5 eq.) and HATU (43 mg, 1.5 eq.) separately into a reaction flask. After adding anhydrous DMF (5 mL) and stirring until dissolved, add DIEA (25 μL, 2 eq.) and stir for ten minutes. Then add the DMF (5 mL) solution of the intermediate S2 (90 mg, 1 eq.) in Example 1. The reaction solution reacts overnight at room temperature, and the molecular weight of the product is monitored by LCMS. The reaction solution is directly purified to obtain 36 mg of the target product with a purity of 97.08%. The mass spectrometry results of RGD-PEG12-C2SS-78A1 are as Figure 2 shown.

[0100] Example 3

[0101] A polypeptide conjugate drug, as shown in Formula I3 above, the polypeptide conjugate drug is [RGD]2-PEG12-OSSO-78A1, and the preparation process is as follows:

[0102]

[0103] Weigh compound 1 (1.0 g, 1.0 eq.) into a reaction tube, add anhydrous DMF (10 mL), compound 2 (1.39 g, 3.0 eq.), EDCI (2.33 g, 3.0 eq.), DIPEA (2.61 g, 5.0 eq.), and react at room temperature for 18 hours. Monitor the reaction by TLC and the molecular weight of the product by LCMS. Add water and ethyl acetate for extraction. The organic phase is dried over anhydrous sodium sulfate, the solvent is concentrated, and the product is obtained by column chromatography to get 300 mg of compound 3. The structure is verified to be correct by LCMS.

[0104] Weigh compound 3 (82 mg, 1.0 eq.) into a reaction flask, add anhydrous DMF (3.5 mL), compound D (280 mg, 2.5 eq.), potassium carbonate (65 mg, 2.5 eq.), and react at room temperature for 5 hours. Monitor the molecular weight of the product by LCMS. Prepare and purify to obtain 127 mg of compound 4.

[0105] Weigh compound 4 (40 mg, 1.0 eq.) into a reaction flask, add hydrochloric acid dioxane solution (3 mL, 4 M), and react at room temperature for 2 hours. Monitor the molecular weight of the product by LCMS. Concentrate the solvent to obtain 40 mg of compound E. Repeat this step to prepare compound E.

[0106] Compound C (79 mg, 1.1 eq.) was weighed into a reaction bottle, anhydrous DMF (3 mL) was added, and DIPEA (38 uL, 3.0 eq.) was added and stirred to dissolve. Then, a solution of compound E (100 mg, 1 eq.) in anhydrous DMF (2 mL) was added and reacted at room temperature for 2 hours. The molecular weight of the product compound 5 was monitored by LCMS. The reaction solution of compound 5 did not require post-treatment. Diethylamine (79 uL, 10 eq.) was directly added thereto and stirred to mix. The reaction solution was reacted at room temperature for 2 hours. The molecular weight of the product was monitored by LCMS. The reaction solution was directly sent for preparation and purification to obtain the target product compound 6, a total of 105 mg.

[0107] Weigh compound B (Cas.1688598-83-5; 82 mg, 4 eq.) into a reaction bottle, add anhydrous DMF (3 mL), stir to dissolve, then add DIPEA (31 uL, 4.0 eq.) and stir to mix, then add a solution of compound 6 (90 mg, 1 eq.) in anhydrous DMF (2 mL), react at room temperature for 2 hours, LCMS monitors the molecular weight of the product compound 7, and the reaction solution is directly subjected to the next step of reaction without post-treatment. Add DIPEA (31 uL, 4.0 eq.) to the reaction solution of compound 7 and stir to mix, then add a solution of compound A (168 mg, 10 eq.) in anhydrous DMF (2 mL), react at room temperature for 2 hours, LCMS monitors the molecular weight of the product, and the reaction solution is directly purified to obtain the target product. The mass spectrometry results of [RGD]2-PEG12-OSSO-78A1 are shown in Figure 3 shown.

[0108] Example 4

[0109] A polypeptide-conjugated drug, as shown in the above formula I4, the polypeptide-conjugated drug is [RGD]2-PEG12-C2SS-78A1, and the preparation process is as follows.

[0110] Synthesis of [RGD]2-PEG12-C2SS-78A1: Weigh the product 7 (45.3 mg, 1.5 eq.) and HATU (29.7 mg, 1.5 eq.) in Example 2 respectively into a reaction bottle, add anhydrous DMF (5 mL) and stir to dissolve, then add DIEA (17.2 uL, 2 eq.) and mix and stir for ten minutes, then add a DMF (5 mL) solution of compound 6 (100 mg, 1 eq.) in Example 3, and react at room temperature for 3 h. LCMS monitors the molecular weight of the product. The reaction solution is directly purified to obtain 32 mg of the target product [RGD]2-PEG12-C2SS-78A1. The mass spectrometry results are as follows: Figure 4 shown.

[0111] Example 5

[0112] A polypeptide conjugate drug, as shown in Formula I5 above, is [RGD]2-PEG12-OC2SSC2O-78A1, and the preparation process is described as follows.

[0113] Synthesis of the used compound B2:

[0114] Among them, R1, R2, R3, and R4 are all methyl groups;

[0115] Weigh 2,2'-disulfanediyldibis(2-methylpropan-1-ol) (Cas. 132182-01-5, 0.5 g, 1.0 eq.) and N,N'-disuccinimidyl carbonate (Cas. 74124-79-1, 6.1 g, 10 eq.) into a reaction flask respectively. After adding anhydrous DCM (50 mL) and stirring to mix, then add pyridine (1.9 mL, 10 eq.) and stir the reaction overnight. The molecular weight of the product was monitored by LCMS. Water and DCM were added for extraction. The organic phase was dried over anhydrous sodium sulfate, and the solvent was concentrated to obtain 0.7 g of crude compound B2, and the structure was verified to be correct by LCMS.

[0116] Synthesis of [RGD]2-PEG12-OC2SSC2O-78A1: Weigh the above compound B2 (38.5 mg, 3 eq.) into a reaction flask. After adding anhydrous DMF (3 mL) and stirring to dissolve, then add DIEA (26 μL, 6 eq.) and stir to mix. Then add the DMF (2 mL) solution of compound 6 (50 mg, 1 eq.) in Example 3. The reaction solution was stirred at room temperature for 2 h. The molecular weight of the product was monitored by LCMS, and the reaction solution did not require post-treatment. Compound A (93.5 mg, 10 eq.) was added to the above reaction solution, and the reaction solution was reacted at room temperature for 2 h. The molecular weight of the product was monitored by LCMS, and the reaction solution was directly purified to obtain 24 mg of the target product [RGD]2-PEG12-OC2SSC2O-78A1. The mass spectrometry results are as Figure 5 shown.

[0117] Activity experiment of polypeptide conjugate drug

[0118] (1) Detection of the induced expression activity of TNF-α in mouse PBMC cells in vitro

[0119] SPF-grade BALB / C mice, 5 - 6 weeks old and weighing 18 - 20 grams, were used. Whole blood was extracted from the mice by enucleation for the extraction of PBMC from peripheral blood. An equal volume of whole blood diluent was added to the whole blood and mixed thoroughly, and then added to an equal volume of mouse peripheral blood lymphocyte separation solution. After centrifugation at 1000g for 20 min, the lymphocyte layer was carefully taken, washed with 10 mL of phosphate buffer solution (PBS), and then centrifuged at 250g for 10 min. The supernatant was discarded, and the lymphocytes were resuspended in complete RPMI-1640 cell culture medium. The mouse whole blood diluent and lymphocyte separation solution used in the experiment were purchased from Beijing Solarbio Science & Technology Co., Ltd. Among them, the product number of the mouse peripheral blood lymphocyte separation solution kit is P8620.

[0120] 78A1, namely compound A (control), RGD-PEG12-OSSO-78A1, RGD-PEG12-C2SS-78A1, and [RGD]2-PEG12-OSSO-78A1 were separately taken. Six concentrations of RPMI-1640 cell culture media were prepared for each compound. Using 78A1 as the positive control group, RGD-PEG12-OSSO-78A1, RGD-PEG12-C2SS-78A1, and [RGD]2-PEG12-OSSO-78A1 as the experimental groups, and RPMI-1640 cell culture medium as the blank control group. 40 μL of the RPMI-1640 cell culture media of each group above were separately added to 160 μL of 1.6×10 5 normal PBMC cell wells to prepare the peripheral lymphocyte culture solutions of each group, and the concentrations of different compounds in each peripheral lymphocyte culture solution were as shown in Table 1.

[0121] Table 1

[0122]

[0123] After incubating the peripheral lymphocyte culture solutions of each group in a 37°C, 5% CO2 cell culture incubator for 24 h, they were centrifuged at 1000 rpm for 5 min, and the cell supernatant was collected. The concentration of TNF-α in the supernatant of the peripheral blood monocyte culture solution was detected according to the ELISA instruction manual. Among them, the manufacturer of the mouse TNF-alpha double antibody sandwich ELISA detection kit used for detection is Wuhan Sanying, and the product number is KE10002.

[0124] The experimental results are as Figure 6As shown, within the tested dose range, compared with the blank control group, in the 78A1 group within the range of 0.0098 μM - 0.1563 μM, as the concentration of 78A1 increased, the concentration of TNF-α in the cell supernatant showed an upward trend. Subsequently, when the drug concentration was increased, the TNF-α level decreased instead; the immunostimulatory ability of RGD-PEG12-OSSO-78A1 and [RGD]2-PEG12-OSSO-78A1 at low concentrations was lower than that of the 78A1 control group. When the concentration reached 0.625 μM, an activation effect was exhibited. Subsequently, as the drug concentration increased, the concentration of TNF-α in the cell supernatant showed an upward trend. RGD-PEG12-C2SS-78A1 had the weakest in vitro stimulatory ability, and the content of TNF-α produced was significantly different from that of the positive control 78A1 group at low concentrations. When the concentration reached 2.5 μM, an activation effect was exhibited. Subsequently, as the drug concentration increased, the concentration of TNF-α in the cell supernatant showed an upward trend.

[0125] (2) Detection of the induced expression activity of TNF-α in the serum of mice by intravenous injection

[0126] Take 78A1, RGD-PEG12-OSSO-78A1, RGD-PEG12-C2SS-78A1 and [RGD]2-PEG12-OSSO-78A1, and use phosphate buffer solution (PBS) as the solvent to prepare PBS solutions containing 4 different components with appropriate concentrations respectively.

[0127] Take SPF-grade female BALB / C mice, 5 - 6 weeks old, weighing 18 - 20 grams. These mice were purchased from the Henan Experimental Animal Center. Divide these mice into 5 groups, namely the 1st group, the 2nd group, the 3rd group, the 4th group and the 5th group. Among them, there are 9 mice in each of the 1st group, the 2nd group, the 3rd group and the 4th group, and 3 mice in the 5th group.

[0128] The mice in the 1st group were respectively intravenously injected with 200 μL of the above 78A1 solution, the mice in the 2nd group were respectively intravenously injected with 200 μL of the above RGD-PEG12-OSSO-78A1 solution, the mice in the 3rd group were respectively intravenously injected with 200 μL of the above RGD-PEG12-C2SS-78A1 solution, the mice in the 4th group were respectively intravenously injected with 200 μL of the above [RGD]2-PEG12-OSSO-78A1 solution, and the mice in the 5th group were respectively intravenously injected with 200 μL of PBS solution. And make the injection dosages of each group of mice as shown in Table 2.

[0129] Table 2

[0130] Grouping Injected compound Dosage administered Group 1 78A1 57 μg Group 2 RGD-PEG12-OSSO-78A1 Consistent with the molar amount of 57 μg of 78A1 Group 3 <![CDATA[RGD-PEG12-C2SS-78A1]]> Consistent with the molar amount of 57 μg of 78A1 Group 4 <![CDATA[[RGD]2-PEG12-OSSO-78A1]]> Consistent with the molar amount of 57 μg of 78A1 Group 5 PBS ——

[0131] Three mice from each of the first, second, third, and fourth groups were sacrificed by orbital blood collection at 30 min, 60 min, and 240 min after intravenous injection. Three mice from the fifth group were sacrificed by orbital blood collection 1 h after intravenous injection. After orbital blood collection, the mouse blood was centrifuged at 3800 rpm for 10 min to separate the serum, and the concentration of TNF-α in the serum was detected by the same method as in the above-mentioned polypeptide-conjugated drug activity experiment (1).

[0132] The experimental results are as Figure 7 shown in A below, indicating that in the first group of mice injected with 78A1 by rapid intravenous injection, the content of TNF-α in the serum increased significantly, and at 1 h after injection, the content of TNF-α in the mouse serum reached the peak, with an increase of 10 - 15 times. In the groups of mice injected with RGD-PEG12-OSSO-78A1, RGD-PEG12-C2SS-78A1, and [RGD]2-PEG12-OSSO-78A1 by rapid intravenous injection, at 1 h after injection, the level of TNF-α in the serum increased only 3 - 5 times, showing a significant difference compared with the first group injected with 78A1, and no significant difference compared with the fifth group of blank control. This indicates that RGD-PEG12-OSSO-78A1, RGD-PEG12-C2SS-78A1, and [RGD]2-PEG12-OSSO-78A1 are safer than an equimolar amount of 78A1 by intravenous injection.

[0133] (3) Detection of the induced expression activity of IL-6 in mouse serum by intravenous injection

[0134] Mice with the same experimental grouping as in the above-mentioned polypeptide-conjugated drug activity experiment (2) were used to detect the concentration of IL-6 in the serum. Among them, the manufacturer of the mouse IL-6 double-antibody sandwich ELISA detection kit used for detection was Wuhan Sanying, and the product number was KE10007.

[0135] The experimental results are as Figure 7 shown in B below, and the results indicate that at 4 h after administration in the group of mice injected with 78A1 by intravenous injection, the IL-6 in the mouse serum reached the peak, with an increase of about 20 times compared with the PBS group and showing a significant difference; the IL-6 levels in the RGD-PEG12-OSSO-78A1 and RGD-PEG12-C2SS-78A groups increased by about 6 times compared with the PBS group and showed no significant difference; the IL-6 in the [RGD]2-PEG12-OSSO-78A1 group increased by about 8 times compared with the PBS group and showed a slightly significant difference.

[0136] (4) Detection of the anti-tumor activity of RGD-PEG12-OSSO-78A1

[0137] Take the same mice as in the above polypeptide-conjugated drug activity experiment (2), and divide them into 5 groups, namely the 6th to 10th groups. Among them, there are 6 mice in each of the 6th to 9th groups, and the 10th group of 6 mice is the blank control group.

[0138] The CT-26 colon cancer cells purchased from ATCC were passaged and cultured in a 37°C, 5% CO2 incubator with DMEM medium containing 10% fetal bovine serum. The specific method was to discard the culture supernatant, and rinse the cells 1 - 2 times with PBS without calcium and magnesium ions. Add 3 - 5 ml of digestive solution (0.25% Trypsin - 0.53 mM EDTA) to the culture flask, place it in a 37°C incubator for digestion for 1 - 2 min, and then observe the cell digestion situation under a microscope. If most of the cells become round and detached, quickly take it back to the clean workbench and add 5 ml of complete medium containing 10% serum to terminate the digestion. Gently pipette the cells until they are completely detached and then aspirate them. Centrifuge at 250 g for 8 - 10 minutes, discard the supernatant, add 1 - 2 mL of culture medium and pipette evenly. Add culture medium at 5 - 6 ml / bottle, and divide the cell suspension into new T75 cm 2 cell culture flasks at a ratio of 1:2 to 1:4 into new T75 cm cell culture flasks containing 5 - 6 ml of culture medium. Select cells with good growth conditions in the logarithmic growth phase and graft tumors for the mice in the left axilla of the 6th to 10th groups of mice to construct a mouse tumor model. On the day of inoculation, centrifuge the above tumor cell solution at 1000 rpm for 5 min, discard the supernatant, and adjust the final cell concentration to 5×10 5 cells / 100 μL, and inject subcutaneously into the left axilla for mouse tumor grafting.

[0139] About one week after inoculation, when the tumor grew to 100 mm 3 , using PBS and sterile water as solvents respectively, take 78A1 and RGD-PEG12-OSSO-78A1 to prepare solutions with appropriate concentrations, and administer the drugs to the mice by peritumoral and intravenous injection. Among them, the 6th and 7th groups used 1 mL syringes to perform peritumoral and intravenous rapid injection of 78A1 respectively, the 8th and 9th groups used 1 mL syringes to perform peritumoral and intravenous rapid injection of RGD-PEG12-OSSO-78A1 respectively. The volume of the solution for peritumoral injection was 100 μL, and the volume of the solution for intravenous injection was 200 μL; the 10th group was injected with 100 μL of PBS solution peritumorally. The dosing doses of the drugs and compounds injected into the 6th to 10th groups of mice are shown in Table 3.

[0140] Table 3

[0141] Mouse Injection solution Dosage administered Group 6 Peritumoral injection of 78A1 solution 57 μg Group 7 Intravenous injection of 78A1 solution 57 μg Group 8 Peritumoral injection of RGD-PEG12-OSSO-78A1 Consistent with the molar amount of 57 μg of 78A1 Group 9 Intravenous injection of RGD-PEG12-OSSO-78A1 Consistent with the molar amount of 57 μg of 78A1 Group 10 Peritumoral injection of PBS solution ——

[0142] For the 6th to 10th groups of mice in this example, the drug was administered once every 3 days for a total of 3 times. The day of the first drug administration was defined as day 0. During the drug administration process, the body weight and tumor volume of the mice were measured every 2 - 3 days until 21 days after drug administration. Among them, the method for measuring the tumor volume was as follows: the long diameter (l) and short diameter (w) of the tumor were measured with a vernier caliper, and according to the volume formula: v = 0.5×l×w 2 After 21 days, the treatment ended, and the mice were euthanized. The tumors were dissected, photographed, and the results were analyzed.

[0143] The experimental results are shown in Figure 8 as shown in A. Compared with the group injected with PBS, the body weight of the mice in the peritumoral and intravenous rapid injection 78A1 groups showed an obvious downward trend in the early stage of treatment, while the body weight of the mice in the peritumoral and intravenous rapid injection RGD - PEG12 - OSSO - 78A1 groups showed no obvious change compared with the group injected with PBS. As shown in Figure 8 from B to D, for the peritumoral rapid injection of 78A1 with a dose of 57 μg, the tumors of 6 mice completely disappeared, while among the 6 mice in the intravenous rapid injection 78A1 group, the tumors of 3 mice disappeared. Among the 6 mice in the peritumoral rapid injection RGD - PEG12 - OSSO - 78A1 group, the tumors of 3 mice disappeared, while the tumors of all 6 mice in the intravenous rapid injection RGD - PEG12 - OSSO - 78A1 group completely disappeared. Compared with the experimental results of injecting 78A1, intravenous injection of RGD - PEG12 - OSSO - 78A1 has lower blood immunotoxicity without affecting the drug efficacy, indicating that RGD - PEG12 - OSSO - 78A1 has practical clinical research value.

[0144] (5) Antitumor activity detection on the 4T1 tumor model

[0145] Take the same mice as in the above polypeptide conjugate drug activity experiment (4) (SPF - grade BALB / C, female mice, 5 - 6 weeks old, 18 - 20 grams), divide them into 5 groups, namely the 11th to 15th groups, with 6 mice in each group, and use the same modeling method as above to establish a tumor model for the mice. Adjust the 4T1 cell concentration to 5×10 5 cells / 100 μL, and subcutaneously inject it into the right axilla to graft tumors for the mice. Approximately one week after inoculation, when the tumor grew to 100 mm 3 , using PBS as the solvent, solutions with appropriate concentrations of 78A1, RGD - SS - 78A1, RGD - PEG12 - SS - 78A1, and RGD - PEG12 - OSSO - 78A1 were respectively prepared and intravenously injected into the mice. Among them, the injection volume of the solution was 200 μL, and the dosing doses of the drugs and compounds injected into each group of mice were as shown in Table 4.

[0146] Table 4

[0147] Mouse Injection solution Dosage administered Group 11 78A1 solution 57 ug Group 12 RGD-SS-78A1 solution Consistent with the molar amount of 57 μg of 78A1 Group 13 RGD-PEG12-SS-78A1 solution Consistent with the molar amount of 57 μg of 78A1 Group 14 RGD-PEG12-OSSO-78A1 solution Consistent with the molar amount of 57 μg of 78A1 Group 15 PBS solution ——

[0148] For the 11th to 15th groups of mice in this example, the drug was administered twice a week for 2 consecutive weeks. The first day of drug administration was defined as day 0. During the drug administration process, the body weight and tumor volume of the mice were measured every 2 - 3 days until 21 days after drug administration. Among them, the method for measuring the tumor volume was: using a vernier caliper to measure the major axis (l) and minor axis (w) of the tumor, and according to the volume formula: v = 0.5×l×w 2 , calculate the tumor volume. After 20 days, the treatment ended, and the mice were euthanized, and the tumors were dissected, photographed, and the results were analyzed.

[0149] Using a 4T1 cell subcutaneous tumor model, the anti - tumor activities of RGD - PEG12 - OSSO - 78A1 were compared with those of RGD - SS - 78A1 and RGD - PEG12 - SS - 78A1 with conventional linker configurations. As Figure 9 shown, the body weight of the mice in the 78A1 injection group showed an obvious downward trend in the early stage of drug administration, and the body weights of the mice in the other experimental groups did not show obvious abnormalities during the whole treatment process. The tumor inhibition rates of the mice in the RGD - SS - 78A1 and RGD - PEG12 - SS - 78A1 treatment groups were both lower than 30% at the experimental end - point, while the tumor inhibition rate of the mice in the RGD - PEG12 - OSSO - 78A1 treatment group reached more than 60%, and the anti - tumor effect was significantly better than that of RGD - SS - 78A1 and RGD - PEG12 - SS - 78A1. Although the tumor inhibition rate of the RGD - PEG12 - OSSO - 78A1 group was comparable to that of the 78A1 alone group, in terms of the body weight performance of the mice, RGD - PEG12 - OSSO - 78A1 had better safety than 78A1.

[0150] Due to its excessive hydrophobicity, RGD-SS-78A1 is prone to poor in vivo stability caused by hydrophobic aggregation, which in turn leads to a decrease in drug efficacy, and at the same time, strong systemic toxicity and immune side reactions are produced. Although RGD-PEG12-SS-78A1 has an additional PEG modification on the basis of RGD-SS-78A1 to improve its water solubility, the test results show that the water solubility does not reach the expected level, and turbidity appears after mixing with serum in vitro, which also results in no significant improvement in the efficacy of intravenous injection of RGD-PEG12-SS-78A1 compared with RGD-SS-78A1. RGD-PEG12-OSSO-78A1 belongs to a disulfide carbamate linker, which is different from a single disulfide linker. After improvement, its water solubility is significantly increased, and its solubility in the water system can reach 20 mg / ml, and it is clear after mixing with serum in vitro. Therefore, intravenous injection of RGD-PEG12-OSSO-78A1 has good stability during the systemic circulation process before reaching the target site, avoiding systemic toxicity caused by drug release at non-pharmacological sites, thereby increasing the anti-tumor efficacy.

[0151] In the above experiment, the structural formulas of RGD-SS-78A1 and RGD-PEG12-SS-78A1 are shown as follows:

[0152]

[0153] (6) Detection of anti-tumor activity on CT26 tumor model

[0154] Take the same mice as in the polypeptide conjugate drug activity experiment (4) (SPF-grade BALB / C, 5-6 weeks old, female mice weighing 18-20 g), divide them into 5 groups, namely groups 16 to 20, with 6 mice in each group, and use the same method for constructing mouse tumor models as above to establish mouse tumors. Approximately one week after inoculation, the tumors grew to 100 mm 3 , using PBS as the solvent, respectively prepare solutions with appropriate concentrations of 78A1, RGD-PEG12-OSSO-78A1, RGD-PEG12-C2SS-78A1, and [RGD]2-PEG12-OSSO-78A1, and administer them to the mice by intravenous injection. Among them, the volume of the injected solution is 200 μL, and the dosing doses of the drugs and compounds injected into each group of mice are shown in Table 5.

[0155] Table 5

[0156] Mouse Injection solution Dosage administered Group 16 78A1 solution 10 μg Group 17 RGD-PEG12-OSSO-78A1 solution Consistent with the molar amount of 10 μg of 78A1 Group 18 <![CDATA[[RGD]2-PEG12-OSSO-78A1 solution]]> Consistent with the molar amount of 10 μg of 78A1 Group 19 <![CDATA[RGD-PEG12-C2SS-78A1 solution]]> Consistent with the molar amount of 10 μg of 78A1 Group 20 PBS solution ——

[0157] For the 16th to 20th groups of mice in this example, the drug was administered once every 3 days for a total of 3 times. The day of the first drug administration was defined as day 0. During the drug administration process, the body weight and tumor volume of the mice were measured every 2 - 3 days until 20 days after drug administration. Among them, the method for measuring the tumor volume was: using a vernier caliper to measure the major axis (l) and minor axis (w) of the tumor, and according to the volume formula: v = 0.5×l×w 2 , calculate the tumor volume. After 20 days, the treatment ended, the mice were euthanized, the tumors were dissected, photographed, and the results were analyzed.

[0158] As Figure 10 shown in A therein, the body weight of the mice in the 78A1 injection group decreased significantly transiently after the first drug administration, and the body weights of the mice in the other experimental groups showed varying degrees of increase. From Figure 10 B to D therein, it can be seen that the tumors of the mice in the PBS control group grew rapidly, the volume continued to increase, and the tumor volume reached ~3000 mm 3 at the end of the experiment. The tumor inhibition rate of the mice in the 78A1 injection group was about 70%, while the tumor volumes of the mice in the treatment groups of RGD-PEG12-OSSO-78A1, RGD-PEG12-C2SS-78A1, and [RGD]2-PEG12-OSSO-78A1 grew slowly, and the tumor inhibition rates all exceeded 90%. In particular, 5 out of 6 mice in the [RGD]2-PEG12-OSSO-78A1 injection group had their tumors disappear, indicating that [RGD]2-PEG12-OSSO-78A1 has better clinical application value than RGD-PEG12-OSSO-78A1 and RGD-PEG12-C2SS-78A1.

[0159] (7) Detection of the induced expression activity of TNF-α in mouse PBMC cells by [RGD]2-PEG12-C2SS-78A1 and [RGD]2-PEG12-OC2SSC2O-78A1

[0160] Mouse PBMC was prepared according to the same experimental method as in the above-mentioned polypeptide conjugate drug activity experiment (1). According to the results of the above-mentioned polypeptide conjugate drug activity experiment (1), in vitro data showed that the immune activation effective dose of RGD-PEG12-C2SS-78A1 was relatively high. On this basis, 78A1 (positive control 1), RGD-PEG12-C2SS-78A1 (positive control 2), [RGD]2-PEG12-C2SS-78A1, and [RGD]2-PEG12-OC2SSC2O-78A1 were taken respectively, and RPMI-1640 cell culture media with 6 concentrations of each compound were prepared. The RPMI-1640 cell culture medium was used as the blank control group.

[0161] Add 40 μL of the RPMI-1640 cell culture medium of each of the above groups to 160 μL of 1.6×10 5 normal PBMC cell wells to prepare the peripheral lymphocyte culture solutions of each group, and make the concentrations of different compounds in each peripheral lymphocyte culture solution as shown in Table 6.

[0162] Table 6

[0163]

[0164] After incubating each group of peripheral lymphocyte culture solutions in a 37 °C, 5% CO2 cell culture incubator for 24 h, centrifuge at 1000 rpm for 5 min, collect the cell supernatant, and detect the TNF-α concentration in the supernatant of the peripheral blood mononuclear cell culture solution according to the ELISA instruction manual. Among them, the manufacturer of the mouse TNF-alpha double antibody sandwich ELISA detection kit used for detection is Wuhan Sanying, and the product number is KE10002.

[0165] The experimental results are as Figure 11 shown. Within the tested dose range, compared with the blank control group, in the 78A1 group within the range of 0.0098 μM - 0.6250 μM, as the concentration of 78A1 increased, the TNF-α concentration in the cell supernatant showed an upward trend, and then the TNF-α level decreased instead when the drug concentration was increased; the TNF-α content produced by in vitro stimulation of [RGD]2-PEG12-C2SS-78A1 and [RGD]2-PEG12-OC2SSC2O-78A1 was significantly different from that of the positive control 78A1 group at low concentrations, showed an activation effect when the concentration reached 2.5 μM, and then as the drug addition concentration increased, the TNF-α concentration in the cell supernatant showed an upward trend, and the overall response curve was comparable to that of RGD-PEG12-C2SS-78A1.

[0166] (8) Detection of the induced expression activity of TNF-α in mouse serum by intravenous injection

[0167] Take [RGD]2-PEG12-OSSO-78A1, [RGD]2-PEG12-C2SS-78A1 and [RGD]2-PEG12-OC2SSC2O-78A1 respectively, and use phosphate buffer solution (PBS) as the solvent to prepare PBS solutions containing 3 different components with appropriate concentrations. Take SPF-grade BALB / C female mice at 5 - 6 weeks old and weighing 18 - 20 g. These mice are purchased from the Henan Experimental Animal Center. Divide these mice into 4 groups, namely the 21st group to the 24th group, with 3 mice in each group.

[0168] The 21st group of mice were respectively intravenously injected with 200 μL of the above-mentioned [RGD]2-PEG12-OSSO-78A1 solution, the 22nd group of mice were respectively intravenously injected with 200 μL of the above-mentioned [RGD]2-PEG12-C2SS-78A solution, the 23rd group of mice were respectively intravenously injected with 200 μL of the above-mentioned [RGD]2-PEG12-OC2SSC2O-78A1 solution, and the 24th group of mice were respectively intravenously injected with 200 μL of PBS solution. And the injection dosages of each group of mice are shown in Table 7.

[0169] Table 7

[0170] Grouping Injected compound Dosage administered Group 21 <![CDATA[[RGD]2-PEG12-OSSO-78A1]]> Consistent with the molar amount of 57 μg of 78A1 Group 22 <![CDATA[[RGD]2-PEG12-C2SS-78A]]> Consistent with the molar amount of 57 μg of 78A1 Group 23 <![CDATA[[RGD]2-PEG12-OC2SSC2O-78A1]]> Consistent with the molar amount of 57 μg of 78A1 Group 24 PBS ——

[0171] At 60 min after intravenous injection, 3 mice in each group were taken to collect orbital blood. The obtained mouse blood was centrifuged at 3800 rpm for 10 min to separate the serum, and the concentration of TNF-α in the serum was detected by the same method as in the polypeptide-conjugated drug activity experiment (1).

[0172] From Figure 12 The results showed that in the group of mice intravenously injected with [RGD]2-PEG12-OSSO-78A1, at 1 h after injection, the level of TNF-α in the serum increased by 6-8 times compared with the PBS control group, and the difference was significant. The contents of TNF-α in the serum of the mice in the groups intravenously injected with [RGD]2-PEG12-C2SS-78A1 and [RGD]2-PEG12-OC2SSC2O-78A1 were basically the same at 1 h, and both had only a 2-3-fold increase compared with the PBS control group and there was no significant difference. The results indicated that intravenous injection of the same molar amount of [RGD]2-PEG12-C2SS-78A1 and [RGD]2-PEG12-OC2SSC2O-78A1 showed better safety than [RGD]2-PEG12-OSSO-78A1.

[0173] The above are only the preferred embodiments of the present application, and are not intended to limit the present application. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A polypeptide-conjugated 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: in, In formula I-1, R1 and R2 are selected from hydrogen, R3 and R4 are selected from C 1-5 Alkyl, n=8-15, X is an integrin receptor binding peptide, and the integrin receptor binding peptide includes at least one of an RGD single-ring peptide and an RGD double-ring peptide; In formula I-2, R1, R2, R3, and R4 are selected from hydrogen or C 1-5 Alkyl, n=8-15, X is an integrin receptor binding peptide, and the integrin receptor binding peptide includes at least one of RGD single-ring peptide and RGD double-ring peptide.

2. The polypeptide-conjugated drug according to claim 1, characterized in that: The polypeptide-conjugated drug comprises at least one of the following formulas (1) to (3): 。 3. A method for preparing a polypeptide-coupled drug according to any one of claims 1 to 2, characterized in that: include: Compound C is subjected to a condensation reaction with an integrin receptor binding peptide to obtain an intermediate S1; The intermediate S1 is subjected to an amine deprotection reaction to obtain an intermediate S2; The intermediate S2 is reacted with compound A and compound B1 to obtain the compound of formula I-1, or the intermediate S2 is reacted with compound A and compound B2 to obtain the compound of formula I-2; 。 4. The preparation method according to claim 3, characterized in that: The compound C and the integrin receptor binding peptide undergo the condensation reaction under the conditions of a carbodiimide condensation agent and an acylation catalyst.

5. The preparation method according to claim 3 or 4, characterized in that: The intermediate S1 undergoes the amine deprotection reaction under the conditions of a deprotecting agent.

6. Use of the polypeptide conjugate drug according to any one of claims 1 to 2 and / or the polypeptide conjugate drug prepared by the preparation method according to any one of claims 3 to 5 in the preparation of an anti-tumor drug, wherein the tumor targeted by the anti-tumor drug is rectal cancer or breast cancer.

7. A pharmaceutical composition, characterized in that It includes the polypeptide conjugate drug according to any one of claims 1-2 and / or the polypeptide conjugate drug prepared by the preparation method according to any one of claims 3-5.

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