A sr-b1-based tumor-targeting polypeptide drug conjugate, and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a tumor-targeting polypeptide drug conjugate based on SR-B1 and a preparation method and application thereof. The polypeptide drug conjugate of the application comprises an SR-B1 targeting polypeptide, a linker and an antitumor drug, and is connected by a covalent bond. Through specific binding of the high-affinity SR-B1 targeting polypeptide and the SR-B1 receptor, the polypeptide drug conjugate is highly enriched to tumor tissues, and the cytotoxic drug is rapidly released in the tumor cells by breaking, thereby significantly improving the antitumor efficacy and reducing the systemic toxic side effects. The polypeptide drug conjugate solves the core problems of the existing antitumor drugs, such as limited efficacy and large toxic side effects caused by the lack of targeting, and breaks through the technical bottlenecks of the existing polypeptide drug conjugate, such as poor targeting effect, poor stability in blood circulation and ineffective release of active drugs in tumor cells. The conjugate has excellent drug forming property, and can realize targeted treatment for various malignant tumors with high expression of SR-B1.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an SR-B1-based tumor-targeting polypeptide drug conjugate, its preparation method, and its application. Background Technology

[0002] Chemotherapy is the core treatment for intermediate and advanced solid tumors, and anti-tumor drugs occupy an irreplaceable position in chemotherapy due to their potent killing effect on tumor cells. However, most anti-tumor drugs lack tumor targeting, leading to two major problems: First, limited efficacy, as the drug concentration that can actually reach and accumulate at the tumor site is very limited, greatly weakening the treatment effect; second, significant toxic side effects, because drugs are distributed non-specifically in the body, killing normal tissue cells along with tumor cells, resulting in severe dose-limiting toxic side effects such as bone marrow suppression, hepatotoxicity, cardiotoxicity, and neurotoxicity. These not only severely reduce patients' quality of life but also often force treatment to be interrupted, and easily induce multidrug resistance in tumor cells, greatly limiting the clinical application of anti-tumor drugs.

[0003] Scavenger receptor type B 1 (SR-B1) is a membrane protein highly expressed on the surface of various tumor cells, including those in breast cancer, liver cancer, lung cancer, pancreatic cancer, and ovarian cancer. However, its expression level is extremely low in normal human tissue cells. This expression characteristic makes it a highly promising target for targeted cancer therapy. Targeted delivery systems based on the SR-B1 target can precisely deliver anti-tumor drugs to the tumor site through receptor-mediated efficient endocytosis, achieving tumor-targeted drug delivery, improving the targeting and effectiveness of cancer treatment, and reducing the toxic side effects of drugs on normal tissues. This represents a very promising tumor suppression strategy.

[0004] Currently, targeted cancer therapies mainly include monoclonal antibody drugs, small molecule targeted drugs, and antibody-drug conjugates (ADCs). However, antibody drugs and ADCs suffer from drawbacks such as large molecular weight, poor tumor penetration, strong immunogenicity, complex preparation processes, and high production costs. Small molecule targeted drugs still have problems such as wide systemic distribution, insufficient enrichment at tumor sites, and significant toxic side effects. These technological bottlenecks have led to the development of peptide-drug conjugates (PDCs), which are covalently coupled from three parts: a targeting peptide, a linker, and a payload. With advantages such as small molecular weight, strong tumor tissue penetration, low immunogenicity, simple synthesis, low production cost, and precise targeting, PDCs have become a core direction for overcoming the clinical bottlenecks of existing targeted drugs and anti-tumor drugs. PDC (Propionibacterial Drug Activation) can precisely bind to receptors specifically and highly expressed on the surface of tumor cells via targeted peptides, achieving active targeted accumulation of the drug at the tumor site. Then, through the tumor microenvironment responsiveness of the linker, it enables targeted release of the drug at the target site. This significantly enhances anti-tumor efficacy while greatly reducing systemic off-target toxicity, ultimately achieving the therapeutic goal of "reduced toxicity and increased efficacy." The world's first PDC drug was developed in 2018. 177 The approval of Lu-DOTATATE for the treatment of neuroendocrine tumors marks the entry of this field into the clinical validation stage. However, existing peptide drug conjugates still have many technical shortcomings: First, the binding affinity of existing targeting peptides to receptors is insufficient, resulting in poor drug-tumor targeting and enrichment effects; second, the linker design between peptides and anti-tumor drugs is unreasonable, leading to poor stability, premature drug leakage in the bloodstream and causing systemic toxicity, while the active drug cannot be effectively released into tumor cells, affecting the anti-tumor efficacy; third, the water solubility and in vivo metabolic stability of the conjugates are poor, resulting in low drug bioavailability; fourth, there is a lack of efficient, controllable, and industrially suitable conjugate synthesis processes, making it difficult to achieve large-scale preparation. Therefore, developing an SR-B1-based tumor-targeting peptide drug conjugate with strong targeting binding, good in vivo stability, precise and controllable drug release, low toxicity, excellent anti-tumor activity, and simple preparation process has significant clinical value and application prospects for overcoming the bottlenecks in the clinical application of existing targeted therapies and anti-tumor drugs. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies and the core bottlenecks in the clinical application of anti-tumor drugs by providing an SR-B1-based tumor-targeting peptide drug conjugate, its preparation method, and its application. The conjugate can achieve active targeted delivery of anti-tumor drugs by specifically recognizing the SR-B1 receptor highly expressed in tumor cells through the targeting peptide. Simultaneously, it achieves intracellular site-specific release of the drug through the tumor microenvironment response characteristics of different types of linkers. This fundamentally solves the technical problems of anti-tumor drugs lacking targeting, having strong toxic side effects, and existing PDC drugs having poor targeting, low stability, and complex preparation processes, thus achieving precise, efficient, and low-toxicity targeted therapy for tumors.

[0006] The specific technical solution is as follows: The first objective of this invention is to provide an SR-B1-based tumor-targeting peptide drug conjugate, comprising: an SR-B1-targeting peptide, a linker, and an antitumor drug, covalently linked together, wherein the structure of the peptide drug conjugate is shown in general formula I: SR-B1 Targeting Peptide-Linker-Antitumor Drug General formula I.

[0007] In this invention, the SR-B1 targeting peptide is directionally coupled to an antitumor active drug via a linker. The linker is specifically cleaved in the tumor cell microenvironment to release the antitumor drug.

[0008] Furthermore, the SR-B1 targeting peptide can specifically recognize and bind to the SR-B1 receptor, and the amino acid sequence of the SR-B1 targeting peptide is selected from SEQ ID NO:1 (CDGFAEKFKEAVKDYFAKFWD-NH2), SEQ ID NO:2 (Ac-CDDGFAEKFKEAVKDYFAKFWD), SEQ ID NO:3 (CPVLDLFRELLNELLEALKQKLK), SEQ ID NO:4 (CGVLESFKASFLSALEEWTKKLQ), or a variant that has at least 90% sequence homology with the amino acid sequence and retains SR-B1 binding activity.

[0009] The SR-B1 targeting peptide was verified by in vitro binding experiments to have a high affinity for the SR-B1 receptor, specifically recognize tumor cells that highly express SR-B1, and does not bind to normal tissue cells.

[0010] Furthermore, the N-terminus of the polypeptide shown in SEQ ID NO:1 is optionally linked to a C12-C18 fatty acid chain.

[0011] Furthermore, the fatty acid chain is selected from lauric acid (C12), myristic acid (C14), palmitic acid (C16), or stearic acid (C18).

[0012] The polypeptide shown in SEQ ID NO:1, after being modified with fatty acids, can significantly prolong its half-life in plasma and can still effectively exert its targeting function through the SR-B1 receptor, thereby enhancing its anti-tumor efficacy.

[0013] Furthermore, the linker is a tumor microenvironment-responsive linker, selected from any one of glutathione-responsive disulfide bonds, acid pH-responsive hydrazone bonds, and cathepsin B-sensitive peptide bonds.

[0014] Furthermore, the cathepsin B-sensitive peptide linker is preferably the Val-Cit-PAB linker.

[0015] The linker is stable in the bloodstream and can be rapidly cleaved by cathepsin B, which is highly expressed in tumor cells, to precisely release the active drug.

[0016] Furthermore, the antitumor drug is a small molecule cytotoxic antitumor drug, selected from any one of taxanes, anthracyclines, and camptothecins.

[0017] Furthermore, the taxanes are selected from any one of paclitaxel, docetaxel, and cabazitaxel; the anthracyclines are selected from any one of doxorubicin, epirubicin, and daunorubicin; and the camptothecins are selected from any one of irinotecan, SN-38, and topotecan.

[0018] The anti-tumor drug is directionally coupled to the SR-B1 targeting peptide via a linker without damaging the drug's own structure.

[0019] A second objective of this invention is to provide a method for preparing the above-mentioned SR-B1-based tumor-targeting polypeptide drug conjugate, comprising the following steps: (1) Synthesize SR-B1 targeting peptides using solid-phase peptide synthesis (SPPS); (2) The precursor compound of the linker is reacted with the antitumor drug in an organic solvent to obtain the linker-drug intermediate; (3) The SR-B1 targeting peptide obtained in step (1) and the linker-drug intermediate obtained in step (2) are reacted in the presence of a coupling agent to form a covalent link; (4) The product obtained in step (3) is purified by preparative high performance liquid chromatography to obtain the polypeptide drug conjugate.

[0020] The peptide drug conjugate of this invention possesses a high-expression SR-B1 receptor tumor cell targeting function. Through the specific binding of the high-affinity SR-B1 targeting peptide to the SR-B1 receptor, it is highly enriched in tumor tissue and rapidly releases cytotoxic drugs within tumor cells, significantly improving anti-tumor efficacy and reducing systemic toxicity. This solves the core problems of existing anti-tumor drugs, such as limited efficacy and high toxicity due to lack of targeting, and overcomes the technical bottlenecks of existing peptide drug conjugates, such as poor targeting effect, poor stability in blood circulation, and inability to effectively release active drugs within tumor cells. In addition, the peptide drug conjugate of this invention has a simple preparation process and is suitable for large-scale industrial production. This peptide drug conjugate has excellent drug-like properties and can achieve targeted therapy for various malignant tumors with high expression of SR-B1, and has extremely high clinical application value.

[0021] A third object of the present invention is to provide a pharmaceutically acceptable salt of the SR-B1-based tumor-targeting peptide drug conjugate as described above.

[0022] A fourth object of the present invention is to provide an antitumor pharmaceutical composition comprising, as described above, a pharmaceutically acceptable salt of an SR-B1-based tumor-targeting peptide drug conjugate and / or as described above, and pharmaceutically acceptable excipients.

[0023] The pharmaceutically acceptable excipients are selected from at least one of the following: diluents, excipients, fillers, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, pH regulators, stabilizers, lyophilization protectants, antioxidants, antibacterial agents, and buffers.

[0024] A fifth object of the present invention is to provide the use of the SR-B1-based tumor-targeting peptide drug conjugates described above, the pharmaceutically acceptable salts of the SR-B1-based tumor-targeting peptide drug conjugates described above, or the antitumor drug compositions described above in the preparation of drugs for treating tumors with high SR-B1 expression.

[0025] Furthermore, the tumors with high SR-B1 expression include breast cancer, ovarian cancer, lung cancer, pancreatic cancer, liver cancer, colorectal cancer, prostate cancer, and glioma.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The targeted precision of the peptide drug conjugate provided by the present invention: The designed tumor-targeting peptide drug conjugate based on SR-B1 uses a high-affinity SR-B1 targeting peptide, which can specifically recognize tumor cells that highly express SR-B1. The drug is highly enriched at the tumor site, which greatly improves the anti-tumor effect and can significantly reduce damage to normal tissues and reduce systemic toxic side effects. (2) The drug release of the polypeptide drug conjugate provided by the present invention is controllable: the SR-B1 targeting polypeptide is conjugated to the anti-tumor drug through the tumor microenvironment responsive linker. The conjugate has good stability in blood circulation, which can avoid premature drug release. After entering the tumor cells, it can quickly lyse and release the drug to exert anti-tumor activity and significantly improve the treatment effect. (3) The peptide drug conjugate provided by the present invention has excellent drug-like properties: the peptide drug conjugate has a small molecular weight, strong tissue penetration ability, and can penetrate deep into the tumor tissue; it has good water solubility, stable metabolism in vivo, and its bioavailability is significantly higher than that of existing small molecule anti-tumor drugs. (4) The preparation process of the polypeptide drug conjugate provided by the present invention is simple: the preparation method has mild reaction conditions, simple steps, strong controllability, and high purification efficiency. It does not require complex equipment, is suitable for large-scale industrial production, and reduces production costs. (5) The peptide drug conjugate provided by the present invention has a wide range of applications: the peptide drug conjugate exhibits broad-spectrum and potent anti-tumor activity in tumors with high expression of SR-B1, and can achieve targeted therapy for a variety of malignant tumors with high expression of SR-B1 without relying on complex nano-formulation systems, thus broadening the application scope of tumor targeted therapy and having extremely high clinical translational value. Attached Figure Description

[0027] Figure 1 This is an ESI-MS image of the target peptide P2 synthesized in Example 2 of this invention; Figure 2 This is an HPLC chromatogram of the target peptide P2 synthesized in Example 2 of the present invention; Figure 3 The intermediate PTX-SS-Pyr synthesized in Example 2 of this invention 1 H NMR spectrum; Figure 4 ESI-MS image of intermediate PTX-SS-Pyr synthesized in Example 2 of this invention; Figure 5 This is an HPLC chromatogram of the intermediate PTX-SS-Pyr synthesized in Example 2 of the present invention; Figure 6 The image shown is an ESI-MS image of the P2-SS-PTX conjugate synthesized in Example 2 of this invention. Figure 7 This is an HPLC chromatogram of the P2-SS-PTX conjugate synthesized in Example 2 of this invention; Figure 8 This is an in vitro release diagram of the P2-SS-PTX conjugate synthesized in Example 2 of the present invention; Figure 9 This is an in vitro release diagram of the P4-Hyd-Dox conjugate synthesized in Example 4 of the present invention; Figure 10 This is an in vitro release diagram of the P3-Val-Cit-PAB-SN38 conjugate synthesized in Example 3 of the present invention; Figure 11 This is an in vitro cytotoxicity diagram of the Palm-P1-SS-PTX conjugate synthesized in Example 1 of this invention; Figure 12 This is a graph showing the stability results of the P2-SS-PTX conjugate synthesized in plasma in Example 2 of this invention; Figure 13 This is a diagram showing the cellular uptake results of the P2-SS-PTX conjugate synthesized in Example 2 of the present invention; Figure 14 The pharmacodynamic diagram of P2-SS-PTX synthesized in Example 2 of this invention in a 4T1 model; Figure 15 This is a graph showing the weight change of P2-SS-PTX synthesized in Example 2 of the present invention in a 4T1 model; Figure 16 The pharmacodynamic diagram of Palm-P1-SS-PTX synthesized in Example 1 of this invention in the SKOV-3 model; Figure 17 This is a graph showing the weight change of Palm-P1-SS-PTX synthesized in Example 1 of this invention in the SKOV-3 model; Figure 18 The pharmacodynamic diagram of Palm-P1-SS-PTX synthesized in Example 1 of this invention in the U87 model; Figure 19 This is a graph showing the weight change of Palm-P1-SS-PTX synthesized in Example 1 of the present invention in the U87 model; Figure 20 The pharmacodynamic diagram of P3-Val-Cit-PAB-SN38 synthesized in Example 3 of this invention in the PANC-1 model; Figure 21 This is a graph showing the weight change of P3-Val-Cit-PAB-SN38 synthesized in Example 3 of the present invention in the PANC-1 model; Figure 22 The pharmacodynamic diagram of P4-Hyd-Dox synthesized in Example 4 of this invention in the 4T1 model; Figure 23 This is a graph showing the weight change of P4-Hyd-Dox synthesized in Example 4 of the present invention in a 4T1 model. Detailed Implementation

[0028] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0029] Unless otherwise specified, all raw materials and reagents used in the following examples and test cases are commercially available products. RinkAmide resin, Wang resin, and various Fmoc protected amino acids were purchased from Jier Biochemical (Shanghai) Co., Ltd.; HBTU, HOBt, DIPEA, piperidine, trifluoroacetic acid (TFA), and triisopropylsilane (TIS) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; paclitaxel (PTX), doxorubicin hydrochloride (doxorubicin), and SN38 were purchased from Dalian Meilun Biotechnology Co., Ltd.; 2-(pyridin-2-yldithio)ethyl carbonate 4-nitrobenzene ester (PDEC-NB), MC-Val-Ci t-PAB-SN38 and 6-maleimide hexanoyl hydrazide (EMCH) were purchased from BroadPharm, Inc., USA; 4-dimethylaminopyridine (DMAP), triethylamine, anhydrous diethyl ether, dichloromethane, ethyl acetate, toluene, anhydrous sodium sulfate, dimethyl sulfoxide (DMSO), methanol, acetonitrile, and formic acid were purchased from Sinopharm Chemical Reagent Co., Ltd.; CCK-8 reagent was purchased from Dojin Chemical Research Institute, Japan; SR-B1 protein (catalog number: 50317-M08H) was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.; CM5 chips were purchased from GE Healthcare, Inc., USA; BALB / c nude mice were purchased from Shanghai Experimental Animal Center; docetaxel (internal standard) was purchased from China National Institutes for Food and Drug Control; PBS buffer, glycine-HCl buffer, HBS-EP+ buffer, and EDTA-K2 anticoagulant were purchased from Thermo Fisher Scientific (China) Co., Ltd.; N-ethylmaleimide (NEM) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0030] The Chinese names and English abbreviations of each substance are listed below: O-benzotriazole-tetramethylurea hexafluorophosphate: HBTU; 1-Hydroxybenzotriazole: HOBt; N,N-Diisopropylethylamine: DIPEA; DCM: Dichloromethane; N,N'-Diisopropylcarbodiimide: DIC; 1,2-Ethylenedithiol: EDT; N,N-Diisopropylethylamine: DIEA; Glutathione: GSH.

[0031] Example 1 Preparation of paclitaxel-targeting peptide conjugate (Palm-P1-SS-PTX) 1.1 Synthesis of Palmitate-Modified Targeting Peptide Palm-P1 (SEQ ID NO:1) (1) Resin swelling and pretreatment Weigh Rink Amide resin (degree of substitution 0.5 mmol / g, 1.0 g, 0.5 mmol) and add it to the synthesis reaction column. Add 10 mL of DMF, shake and swell at room temperature for 30 minutes, and then remove the solvent.

[0032] (2) Standard amino acid coupling cycle The amino acids were coupled sequentially from the C-terminus (Asp) to the N-terminus (Cys) according to the sequence Cys-Asp-Gly-Phe-Ala-Glu-Lys-Phe-Lys-Glu-Ala-Val-Lys-Asp-Tyr-Phe-Ala-Lys-Phe-Trp-Asp-NH2 (SEQ ID NO:1), with each amino acid coupled according to the following standard procedure: Step 1: Fmoc Deprotection Add 20% piperidine / DMF solution (v / v, 10 mL), shake for 5 minutes, and then dry under vacuum. Repeat once, reacting for 15 minutes. After drying, wash the resin 5 times with DMF (10 mL each time). The ninhydrin test should be positive (resin turns blue).

[0033] Step 2: Amino acid activation and coupling Fmoc-amino acids (1.5 mmol, 3 eq), HBTU (1.5 mmol, 3 eq), and HOBt (1.5 mmol, 3 eq) were dissolved in 5 mL of DMF, and activated with DIPEA (3.0 mmol, 6 eq). The activated solution was added to the reaction column and reacted with shaking at room temperature for 60 minutes. After drying, the column was washed five times with DMF. The ninhydrin test should be negative (the resin should be colorless, indicating complete coupling).

[0034] (3) N-terminal palmitic acid coupling After coupling of the 21st amino acid (Cys), the Fmoc protection of Cys was removed using 20% ​​piperidine / DMF (v / v). Palmitic acid (384 mg, 1.5 mmol, 3 eq) was weighed and dissolved in 5 mL of a DMF / DCM mixed solvent (DMF:DCM = 1:1, v / v). DIC (190 μL, 1.5 mmol, 3 eq) and HOBt (203 mg, 1.5 mmol, 3 eq) were added, and the mixture was activated at room temperature for 10 min. The activated solution was added to the reaction column, followed by DIPEA (523 μL, 3.0 mmol, 6 eq), and the reaction was carried out with shaking at room temperature for 3 h. A small amount of resin was tested for ninhydrin; a negative result (colorless) confirmed that the N-terminal amino group had been completely palmitoylated.

[0035] (4) Pyrolysis and deprotection At a resin concentration of 10 mL / g, the resin obtained in step (3) was dried and added to the lysis buffer (TFA:phenol:EDT:H2O:TIS = 87.5:2.5:2.5:2.5:5, v / v). The mixture was stirred at room temperature for 2.5 hours, the resin was removed by filtration, and the filtrate was slowly added to 10 times its volume of ice-cold ether. A white precipitate was formed. The precipitate was centrifuged at 4°C (4000 rpm, 10 minutes), collected, washed three times with ether, and dried under vacuum to obtain crude peptide.

[0036] (5) Purification of crude peptides Purification was performed using preparative HPLC under the following conditions: Chromatographic column: C18 reversed-phase column (21.2×250 mm, 10 μm); mobile phase A: 0.1% TFA acetonitrile solution, mobile phase B: 0.1% TFA aqueous solution; detection wavelength: 220 nm; flow rate: 15 mL / min; gradient: 30% → 80% B, 30 min.

[0037] The target peak was collected, acetonitrile was removed by rotary evaporation, and the product was freeze-dried to obtain a white powder of pure Palm-P1, with the structural formula shown in formula (1).

[0038] ESI-MS and HPLC analysis showed that the molecular weight was consistent with the theoretical value and the purity was >95%, meeting the requirements for subsequent coupling reactions.

[0039]

[0040] Equation (1) 1.2 Synthesis of intermediate PTX-SS-Pyr Paclitaxel (5 g, 5.86 mmol, 1 eq) and 2-(pyridin-2-yldithio)ethyl carbonate 4-nitrobenzene ester (PDEC-NB, 2.27 g, 6.45 mmol, 1.1 eq) were weighed and dissolved in 50 mL of dichloromethane. 4-Dimethylaminopyridine (DMAP, 0.114 g, 2.34 mmol, 0.4 eq) was added as a catalyst, and the reaction was carried out at room temperature in the dark with stirring for about 8 h. After the reaction was completed, the reaction solution was washed successively with saturated sodium carbonate solution and saturated ammonium chloride solution. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was recrystallized from an ethyl acetate-toluene mixture and dried under vacuum to obtain PTX-SS-Pyr. The synthetic route is shown in formula (2).

[0041] through 1 The intermediate structure was confirmed to be correct and its purity >95% by ¹H NMR, ESI-MS and HPLC detection.

[0042]

[0043] Equation (2) 1.3 Synthesis of Palm-P1-SS-PTX At a molar ratio of Palm-P1 to PTX-SS-Pyr of 1:1.2, 0.018 mmol of the target peptide Palm-P1 obtained above was weighed and dissolved in 4 mL of dimethyl sulfoxide (DMSO). The solution was shaken until completely dissolved to obtain a polypeptide solution. 23.7 mg (0.022 mmol) of PTX-SS-Pyr was weighed and added to 1 mL of anhydrous DMSO. The solution was sonicated to obtain an intermediate solution. The reaction flask was placed on a constant-temperature magnetic stirrer, and the intermediate solution was slowly added dropwise to the polypeptide solution under stirring at room temperature. The reaction was carried out under a sealed environment at room temperature in the dark. After the reaction was completed, the solution was purified by preparative HPLC and freeze-dried under vacuum to obtain Palm-P1-SS-PTX. The synthetic route is shown in formula (3).

[0044] The structure was confirmed to be correct by ESI-MS and HPLC analysis, and the purity reached 95.36%.

[0045]

[0046] Equation (3) Example 2 Preparation of paclitaxel-targeting peptide conjugate (P2-SS-PTX) 2.1 Synthesis of targeting peptide P2 (SEQ ID NO.2) (1) Resin swelling and pretreatment Weigh out Fmoc-Asp(otbu)-Wang Resin resin (degree of substitution 0.5 mmol / g, 1.0 g, 0.5 mmol) and add it to the synthesis reaction column. Add 10 mL of DMF, shake and swell at room temperature for 30 minutes, and then remove the solvent.

[0047] (2) Standard amino acid coupling cycle The amino acids were coupled sequentially from the C-terminus (Asp) to the N-terminus (Cys) according to the sequence Cys-Asp-Asp-Gly-Phe-Ala-Glu-Lys-Phe-Lys-Glu-Ala-Val-Lys-Asp-Tyr-Phe-Ala-Lys-Phe-Trp-Asp (SEQ ID NO:2). The coupling of each amino acid was performed according to the standard procedure in Example 1.

[0048] (3) N-terminal acetylation After completing the Cys coupling of the last amino acid and removing the Fmoc group, 1.42 mL (15 mmol) of acetic anhydride and 5.23 mL (30 mmol) of DIEA were added to DMF at a molar ratio of α-amino (N-terminus of peptide chain): acetic anhydride: DIEA = 1:20:40, and the volume was adjusted to 30 mL. The mixture was stirred and mixed to prepare a mixed modification solution. The solution was then poured into a reaction column and stirred at room temperature for 30 min. After the reaction was completed, the reaction column was washed three times alternately with 15 mL of DMF and DCM to completely remove residual modification reagents and byproducts, and then dried under vacuum.

[0049] (4) Pyrolysis and deprotection Add the dried resin obtained in step (3) to the lysis buffer (TFA: TIS: H2O = 95:2.5:2.5, v / v) at a ratio of 10 mL / g resin, and stir at room temperature for 2.5 hours. Filter to remove the resin, and slowly add the filtrate to 10 times its volume of ice-cold diethyl ether. A white precipitate forms, which is centrifuged at 4°C (4000 rpm, 10 minutes). Collect the precipitate, wash it three times with diethyl ether, and dry it under vacuum to obtain the crude peptide.

[0050] (5) Purification of crude peptides Purification was performed using preparative HPLC under the following conditions: Chromatographic column: C18 reversed-phase column (21.2×250 mm, 10 μm); mobile phase A: 0.1% TFA acetonitrile solution, mobile phase B: 0.1% TFA aqueous solution; detection wavelength: 220 nm; flow rate: 15 mL / min; gradient: 20% → 60% B, 30 min.

[0051] The target peak was collected, acetonitrile was removed by rotary evaporation, and the product was freeze-dried to obtain a white powder of pure product P2, with the structural formula shown in formula (4).

[0052] Detected by ESI-MS and HPLC (see...) Figure 1-2 The molecular weight is consistent with the theoretical value, and the purity is >95%, which meets the requirements for subsequent coupling reactions.

[0053]

[0054] Equation (4) 2.2 Synthesis of intermediate PTX-SS-Pyr The synthesis of intermediate PTX-SS-Pyr is the same as in Example 1.

[0055] through 1 H NMR, ESI-MS and HPLC detection (see below) Figure 3-5 The intermediate structure was confirmed to be correct, with a purity >95%.

[0056] 2.3 Synthesis of P2-SS-PTX Weigh 50 mg (0.018 mmol) of the target peptide P2 obtained above at a molar ratio of 1:1.2 to PTX-SS-Pyr, dissolve it in 4 mL of dimethyl sulfoxide (DMSO), and shake until completely dissolved to obtain a polypeptide solution; weigh 23.7 mg (0.022 mmol) of PTX-SS-Pyr and add it to 1 mL of anhydrous DMSO, sonicate to dissolve, and obtain an intermediate solution. Place the reaction flask on a constant temperature magnetic stirrer, and slowly add the intermediate solution to the polypeptide solution while stirring at room temperature. Stir the mixture in a dark place at room temperature until the reaction is complete. After the reaction is complete, purify the mixture using preparative HPLC and freeze-dry it under vacuum to obtain P2-SS-PTX. The synthetic route is shown in formula (5).

[0057] Detected by ESI-MS and HPLC (see...) Figure 6-7 The structure was confirmed to be correct, and the purity reached 97.57%.

[0058]

[0059] Equation (5) Example 3 Preparation of SN38-targeting peptide conjugate (P3-Val-Cit-PAB-SN38) 3.1 Synthesis of targeting peptide P3 (SEQ ID NO:3) (1) Resin swelling and pretreatment Weigh out Wang resin (0.5 mmol / g, 1.0 g, 0.5 mmol) and add it to the synthesis reaction column. Add 10 mL of DMF, shake and swell at room temperature for 30 minutes, and then remove the solvent.

[0060] (2) Standard amino acid coupling cycle The amino acids were coupled sequentially from the C-terminus (Lys) to the N-terminus (Cys) according to the sequence Cys-Pro-Val-Leu-Asp-Leu-Phe-Arg-Glu-Leu-Leu-Asn-Glu-Leu-Leu-Glu-Ala-Leu-Lys-Gln-Lys-Leu-Lys (SEQ ID NO:3). The coupling of each amino acid was performed according to the standard procedure in Example 1.

[0061] (3) Pyrolysis and deprotection The resin obtained in step (2) was dried and added to the lysis buffer (TFA: TIS: H2O: EDT = 94:1:2.5:2.5, v / v) at a concentration of 10 mL / g resin. The mixture was stirred at room temperature for 2.5 hours. The resin was removed by filtration, and the filtrate was slowly added to 10 times its volume of ice-cold diethyl ether. A white precipitate was formed. The precipitate was centrifuged at 4°C (4000 rpm, 10 minutes), collected, washed three times with diethyl ether, and dried under vacuum to obtain the crude peptide.

[0062] (4) Purification of crude peptides Purification was performed using preparative HPLC under the chromatographic conditions of Example 2.

[0063] The target peak was collected, acetonitrile was removed by rotary evaporation, and the product was freeze-dried to obtain a white powder of pure product P3, with the structural formula shown in formula (6).

[0064] Analyzed by ESI-MS and HPLC, the HPLC purity was ≥95%, and the ESI-MS measured [M+H] content was [missing value]. + Consistent with theoretical values. The thiol content determined by the Ellman reagent method was 0.92 mol / mol peptide.

[0065]

[0066] Equation (6) 3.2 MC-Val-Cit-PAB-SN38 intermediate This embodiment uses commercially available MC-Val-Cit-PAB-SN38 (BroadPharm BP-40210), with the structural formula shown in formula (7), and a purity of ≥95%, which is directly used for coupling.

[0067]

[0068] Equation (7) 3.3 Conjugation of target peptide P3 with MC-Val-Cit-PAB-SN38 The target peptide P3 and MC-Val-Cit-PAB-SN38 were added at a peptide:intermediate molar ratio of 1:1.2 and stirred at room temperature in the dark for 4 hours. HPLC monitoring was performed until the peptide peak basically disappeared. The reaction solution was purified by preparative HPLC (C18 column, 0.1% TFA aqueous solution / acetonitrile gradient), the target peak was collected, and the solution was freeze-dried to obtain a pale yellow powder P3-Val-Cit-PAB-SN38. The synthetic route is shown in formula (8).

[0069] The HPLC purity was ≥95%. The molecular weight measured by MALDI-TOF MS was consistent with the theoretical value.

[0070]

[0071] Equation (8) Example 4: Preparation of doxorubicin-targeting peptide conjugate (P4-Hyd-Dox) 4.1 Synthesis of targeting peptide P4 (SEQ ID NO:4) (1) Resin swelling and pretreatment Weigh out Wang resin (0.5 mmol / g, 1.0 g, 0.5 mmol) and add it to the synthesis reaction column. Add 10 mL of DMF, shake and swell at room temperature for 30 minutes, and then remove the solvent.

[0072] (2) Standard amino acid coupling cycle The amino acids were coupled sequentially from the C-terminus (Gln) to the N-terminus (Cys) according to the sequence Cys-Gly-Val-Leu-Glu-Ser-Phe-Lys-Ala-Ser-Phe-Leu-Ser-Ala-Leu-Glu-Glu-Trp-Thr-Lys-Lys-Leu-Gln (SEQ ID NO:4). The coupling of each amino acid was performed according to the standard procedure in Example 1.

[0073] (3) Pyrolysis and deprotection The resin obtained in step (2) was dried and added to the lysis buffer (TFA:phenol:EDT:H2O:TIS = 87.5:2.5:2.5:2.5:5, v / v) at a concentration of 10 mL / g resin. The mixture was stirred at room temperature for 2.5 hours. The resin was removed by filtration, and the filtrate was slowly added to 10 times its volume of ice-cold diethyl ether. A white precipitate was formed. The precipitate was centrifuged at 4°C (4000 rpm, 10 minutes), collected, washed three times with diethyl ether, and dried under vacuum to obtain the crude peptide.

[0074] (4) Purification of crude peptides Purification was performed using preparative HPLC under the chromatographic conditions of Example 2.

[0075] The target peak was collected, acetonitrile was removed by rotary evaporation, and the product was freeze-dried to obtain a white powder of pure product P4, with the structural formula shown in formula (9).

[0076] ESI-MS and HPLC analysis showed that the HPLC purity was >95%, and the ESI-MS measured [M+H] content was [missing value]. + Consistent with the theoretical value.

[0077]

[0078] Equation (9) 4.2 Synthesis of EMCH-DOX intermediates Doxorubicin hydrochloride (100 mg, 0.172 mmol) was dissolved in anhydrous methanol (10 mL), and triethylamine (29 μL, 0.206 mmol) was added and stirred for 15 minutes. 6-maleimide hexanoyl hydrazide (EMCH, 156 mg, 0.688 mmol, 4.0 eq) was added, and the reaction was carried out at room temperature in the dark for 4 hours. TLC (dichloromethane:methanol = 10:1) was monitored until the doxorubicin spots essentially disappeared. The solution was concentrated by rotary evaporation and purified by silica gel column chromatography (dichloromethane:methanol gradient 10:1 → 5:1) to obtain a red solid EMCH-DOX in 68% yield. The synthetic route is shown in formula (10).

[0079] ESI-MS analysis showed that the [M+H] value was obtained. + The measured value is consistent with the theoretical value.

[0080]

[0081] Equation (10) 4.3 Synthesis of P4-Hyd-Dox The target peptide P4 (0.019 mmol, 1 eq) was dissolved in 10 mL of deoxy PBS buffer (pH 6.5, containing 10% DMF, pre-bubbled under nitrogen for 15 minutes to remove oxygen) to obtain a peptide solution. EMCH-DOX (0.023 mmol, 1.2 eq) was weighed and dissolved in 1 mL of DMF. The EMCH-DOX solution was added dropwise to the peptide solution, and the mixture was stirred at room temperature in the dark under nitrogen protection for 4 hours. HPLC monitoring showed that the peptide peak had essentially disappeared. The reaction solution was purified by preparative HPLC (C18 column, mobile phase A: 0.1% TFA aqueous solution, B: acetonitrile, gradient 20%→60% B for 30 minutes), the target peak was collected, and the solution was freeze-dried to obtain red powder P4-Hyd-Dox with a yield of 52%. The synthetic route is shown in formula (11).

[0082] The molecular weight measured by ESI-MS and HPLC was consistent with the theoretical value, and the purity of HPLC was 96.5%.

[0083]

[0084] Equation (11) Comparative Example 1: Preparation of P1-SS-PTX 1.1 Synthesis of P1 (1) Resin swelling and pretreatment Weigh Rink Amide resin (degree of substitution 0.5 mmol / g, 1.0 g, 0.5 mmol) and add it to the synthesis reaction column. Add 10 mL of DMF, shake and swell at room temperature for 30 minutes, and then remove the solvent.

[0085] (2) Standard amino acid coupling cycle The amino acids were coupled sequentially from the C-terminus (Asp) to the N-terminus (Cys) according to the sequence Cys-Asp-Gly-Phe-Ala-Glu-Lys-Phe-Lys-Glu-Ala-Val-Lys-Asp-Tyr-Phe-Ala-Lys-Phe-Trp-Asp-NH2 (SEQ ID NO:1). The coupling of each amino acid was performed according to the standard procedure in Example 1.

[0086] (3) Pyrolysis and deprotection At a resin concentration of 10 mL / g, the resin obtained in step (2) was dried and added to the lysis buffer (TFA:phenol:EDT:H2O:TIS = 87.5:2.5:2.5:2.5:5, v / v). The mixture was stirred at room temperature for 2.5 hours, the resin was removed by filtration, and the filtrate was slowly added to 10 times its volume of ice-cold diethyl ether. A white precipitate was formed. The precipitate was centrifuged at 4°C (4000 rpm, 10 minutes), collected, washed three times with diethyl ether, and dried under vacuum to obtain the crude peptide.

[0087] (4) Purification of crude peptides Purification was performed using preparative HPLC under the following conditions: Chromatographic column: C18 reversed-phase column (21.2×250 mm, 10 μm); mobile phase A: 0.1% TFA acetonitrile solution, mobile phase B: 0.1% TFA aqueous solution; detection wavelength: 220 nm; flow rate: 15 mL / min; gradient: 30% → 80% B, 30 min.

[0088] The target peak was collected, acetonitrile was removed by rotary evaporation, and the product was freeze-dried to obtain a white powder of pure product P1, with the structural formula shown in formula (12).

[0089] ESI-MS and HPLC analysis showed that the molecular weight was consistent with the theoretical value and the purity was >95%, meeting the requirements for subsequent coupling reactions.

[0090]

[0091] Equation (12) 1.2 Synthesis of intermediate PTX-SS-Pyr The synthesis of intermediate PTX-SS-Pyr is the same as in Example 1.

[0092] 1.3 Synthesis of P1-SS-PTX Weigh 0.018 mmol of the target peptide P1 obtained above at a molar ratio of 1:1.2 to PTX-SS-Pyr, dissolve it in 4 mL of dimethyl sulfoxide (DMSO), and shake until completely dissolved to obtain a polypeptide solution; weigh 23.7 mg (0.022 mmol) of PTX-SS-Pyr and add it to 1 mL of anhydrous DMSO, sonicate to dissolve, and obtain an intermediate solution. Place the reaction flask on a constant temperature magnetic stirrer, and slowly add the intermediate solution to the polypeptide solution with stirring at room temperature, and stir thoroughly in a dark place at room temperature. After the reaction is complete, purify by preparative HPLC and freeze-dry under vacuum to obtain P1-SS-PTX. The synthetic route is shown in formula (13).

[0093] The structure was confirmed to be correct by ESI-MS and HPLC analysis, and the purity reached 96.31%.

[0094]

[0095] Equation (13) Test Example 1: In vitro responsive release (1) Reduction-sensitive in vitro release Accurately weigh GSH and prepare 10 μM and 10 mM solutions with PBS buffer (pH 7.4).

[0096] The P2-SS-PTX conjugate was dissolved in DMSO to prepare a 10 mM stock solution. Immediately before use, it was diluted to 200 μM with PBS buffer (pH 7.4) (2% final DMSO concentration). 1 mL of the conjugate solution was added to a dialysis bag (molecular weight cutoff 1000), and both ends were sealed. The dialysis bag was then placed into separate Erlenmeyer flasks containing 50 mL of PBS buffer (pH 7.4) with different GSH concentrations (0, 10 μM, 10 mM). The flasks were incubated at 37°C with shaking at 100 rpm for release. 1 mL samples were taken at 0, 1, 2, 4, 8, 12, and 24 hours. 1 mL of fresh PBS buffer was added to each flask, and shaking continued. If the released sample contained GSH, 10 μL of 0.5 M NEM (approximately 5 mM final concentration) was immediately added to quench the thiol groups and prevent further reaction after sampling. The PTX concentration was determined using the following HPLC conditions, and the PTX release was calculated.

[0097] HPLC conditions: Column: Agilent ZORBAX Eclipse Plus C18 (2.1×50 mm, 1.8 μm); Mobile phase: Acetonitrile:Water = 60:40 (v / v); Flow rate: 1 mL / min; Column temperature: 30°C; Detection wavelength: 227 nm.

[0098] Reduced-sensitive in vitro release (see) Figure 8The results showed that the cumulative release rate of P2-SS-PTX under 10mM GSH conditions reached 88.3% in 24 hours, which was significantly higher than that of the 10μM GSH group (20.6%) and the group without GSH (9.5%).

[0099] (2) In vitro acid-sensitive release experiment P4-Hyd-Dox was dissolved in DMSO to prepare a 10 mM stock solution.

[0100] The 10mM P4-Hyd-Dox stock solution was diluted to 10μM (final DMSO concentration 0.1%) with PBS buffers of different pH values ​​(pH 7.4, pH 6.5, and pH 5.0), and incubated in a 37°C water bath in the dark. 200μL samples were taken at 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours, and immediately neutralized to pH 7-8 with 20μL of 1M NaOH to terminate the hydrazone bond hydrolysis reaction. The concentration of free doxorubicin was determined by fluorescence spectrophotometry: excitation wavelength 480nm, emission wavelength 590nm. The release concentration at each time point was calculated using the doxorubicin standard curve.

[0101] In vitro acid-sensitive release curve (see) Figure 9 The results showed that at pH 7.4, the cumulative release rate of the conjugate after 72 hours was only 22.5%, indicating that the hydrazone bond has good stability in the blood circulation environment. At pH 6.5, the release rate of the conjugate accelerated, reaching 78.4% after 24 hours and 88.6% after 72 hours. At pH 5.0, the conjugate exhibited rapid responsive release, with a release rate of 78.6% after 4 hours, 96.5% after 24 hours, and 99.0% after 72 hours. These results indicate that the drug release rate of the hydrazone bond linker is significantly pH-dependent: the stronger the acidity, the faster the release (pH 5.0 > pH 6.5 > pH 7.4).

[0102] The experimental results demonstrate that the hydrazone linker in the P4-Hyd-Dox designed in this invention exhibits significant pH-responsive release characteristics. It remains stable in a neutral physiological environment (pH 7.4) and achieves efficient drug release in the acidic tumor microenvironment (pH 6.5) and lysosomal environment (pH 5.0). This pH-selective release characteristic is a core functional validation of the hydrazone linker design and provides crucial technical evidence for the effectiveness of the PDC conjugate of this invention.

[0103] (3) In vitro enzyme-sensitive release experiment Dissolve P3-Val-Cit-PAB-SN38 in DMSO to prepare a 10 mM stock solution. Dilute with active buffer to a 200 μM working solution (final DMSO concentration 2%) before use.

[0104] Dissolve cathepsin B in ultrapure water to prepare a 1 mg / mL stock solution (approximately 20 U / mL). Dilute to 0.5 mg / mL (approximately 10 U / mL) with activity buffer immediately before use and pre-incubate at 37°C for 15 minutes to activate enzyme activity.

[0105] Take 50 μL of the working solution (200 μM) of the conjugate, add 50 μL of buffer with or without cathepsin B, and add activity buffer to 1 mL to make the final concentration of the conjugate 10 μM and the final concentration of the enzyme 0.5 U / mL. Incubate at 37°C in the dark. Take 50 μL samples at 0, 0.25, 0.5, 0.75, 1, 2, 4, 8, and 24 h, respectively. Immediately add 50 μL of ice-cold acetonitrile (containing 0.1% formic acid) to terminate the enzyme reaction and vortex mix. Centrifuge at 12000×g for 10 min at 4°C, and take the supernatant for HPLC-MS / MS analysis.

[0106] HPLC-MS / MS conditions: Agilent ZORBAX Eclipse Plus C18 column (2.1 × 50 mm, 1.8 μm); mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: 0.1% formic acid acetonitrile; gradient elution (0–1 min 20% B, 1–6 min 20% → 70% B); flow rate: 0.4 mL / min; injection volume: 10 μL; ESI + The mode was used to monitor ion m / z 393.1→349.1 (SN-38).

[0107] In vitro enzyme-sensitive release curve (see) Figure 10 The results showed that in the presence of cathepsin B, the conjugate exhibited rapid enzymatic release: a cumulative release rate of 78.5% within 1 hour, 94.6% within 4 hours, and 98.2% within 24 hours. This indicates that the Val-Cit-PAB linker can be efficiently and specifically cleaved by cathepsin B. In contrast, in the enzyme-free control group (buffer only, pH 5.0), the cumulative release rate after 24 hours was only 7.2%, indicating that the Val-Cit-PAB linker has excellent stability in acidic buffer and does not undergo nonspecific hydrolysis.

[0108] Test Example 2: In vitro cytotoxicity assay of Palm-P1-SS-PTX SR-B1-overexpressing HepG2 cells in logarithmic growth phase were digested with 0.25% trypsin to prepare a single-cell suspension, and the cell density was adjusted to 5 × 10⁻⁶ cells / cells. 4cells / mL (adjust according to cell growth rate). Seed cells into 96-well plates at 100 μL per well (i.e., 5000 cells / well) and incubate at 37°C in a 5% CO2 incubator for 24 hours. After the cells adhered, the old culture medium was removed, and fresh culture medium containing Palm-P1-SS-PTX conjugate (0.01, 0.1, 1, 10, 100, 1000 nM based on PTX equivalents), P1-SS-PTX conjugate, free PTX (paclitaxel), target peptide Palm-P1 (from 1.1 of Example 1), and a physical mixture of PTX and target peptide Palm-P1 (molar ratio 1:1) was added to each well. The concentration gradients of P1-SS-PTX conjugate, free PTX, target peptide Palm-P1, and the physical mixture of PTX and target peptide Palm-P1 were set the same as those of Palm-P1-SS-PTX conjugate, 200 μL per well. The cells were cultured for 48 h. After incubation, 10 μL of CCK-8 solution was added to each well, and the cells were incubated in the dark for another 2 h. The absorbance (OD value) at 450 nm was measured using a microplate reader to calculate cell viability and half-maximal inhibitory concentration (IC50). 50 ).

[0109] In vitro cytotoxicity (see) Figure 11 The results showed that free PTX, Palm-P1-SS-PTX conjugate, and P1-SS-PTX conjugate all exhibited significant inhibitory activity against the proliferation of HepG2 cells with high SR-B1 expression; the IC50 of Palm-P1-SS-PTX conjugate was [data missing]. 50 The concentration (25.6±0.9 nM) was close to that of free PTX (20.4±1.8 nM), retaining in vitro antitumor activity comparable to free PTX. The conjugate binds to the highly expressed SR-B1 receptor on the surface of HepG2 cells via the targeting peptide Palm-P1, and enters the cells efficiently through receptor-mediated endocytosis. After entering the cells, under the action of high concentrations of reducing substances, the disulfide bond (-SS-) breaks, releasing active free PTX. The inhibitory activity of Palm-P1-SS-PTX on the proliferation of HepG2 cells was superior to that of the P1-SS-PTX conjugate (43.3±2.6 nM), indicating that fatty acid modification can enhance the binding stability of the peptide to SR-B1. The inhibitory activity of the physical mixture PTX+Palm-P1 on the proliferation of HepG2 cells was significantly weakened, and its IC50 value was lower. 50 The IC50 value (150.1 ± 11.5 nM) was significantly higher than that of Palm-P1-SS-PTX conjugate and free PTX; the IC50 value of the free targeting peptide Palm-P1 was significantly higher. 50 A value >1000 nM indicates that the targeting peptide Palm-P1 itself has no significant cytotoxicity.

[0110] Test Example 3: Stability evaluation of the P2-SS-PTX conjugate in plasma Take 50 μL of 1 mM P2-SS-PTX conjugate stock solution and dilute it to 5 mL with human plasma (final conjugate concentration 10 μM, final DMSO concentration 1%), and vortex to mix. Aliquot the mixture into 1.5 mL low-adsorption centrifuge tubes (200 μL per time point), and incubate in a 37°C constant temperature water bath in the dark. Take samples at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours, and immediately add 400 μL of pre-cooled acetonitrile (containing 0.1% formic acid and 100 ng / mL internal standard docetaxel) to the centrifuge tubes, vortex for 1 minute. Centrifuge at 4°C, 12000×g for 10 minutes, and determine the content of intact conjugate and free paclitaxel in the samples using LC-MS / MS.

[0111] Chromatographic conditions: Agilent ZORBAX Eclipse Plus C18 column (2.1 × 50 mm, 1.8 μm); mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution; gradient elution: 0–1 min 30% B, 1–6 min 30% → 80% B, 6–8 min 80% → 95% B; flow rate 0.4 mL / min; column temperature 40°C; injection volume 10 μL.

[0112] Mass spectrometry conditions: ESI + Mode: Multiple Response Monitoring (MRM) mode, P2-SS-PTX[M+3H] 3+ m / z=1220.2, paclitaxel detection ion pair m / z 854.3→286.1 (CE 25 V); internal standard docetaxel detection ion pair m / z 808.3→226.1 (CE 28 V).

[0113] Stability results in plasma showed (see) Figure 12 The P2-SS-PTX conjugate exhibits excellent stability in human plasma. After 24 hours of incubation, the remaining percentage of intact conjugate in human plasma remains above 80%, with only a small amount of free paclitaxel (<20%) being detected. This indicates that the peptide-conjugated drug of the present invention has good stability in systemic circulation, effectively preventing premature drug leakage and significantly reducing systemic off-target toxicity.

[0114] Test Example 4: Affinity determination of Palm-P1-SS-PTX conjugate with SR-B1 receptor Using the Biacore T200 system, SR-B1 protein was immobilized on a CM5 chip via amine conjugation. The chip surface was activated for 7 minutes with 0.2 M EDC / 0.05 M NHS (EDC:NHS = 1:1, v / v). SR-B1 protein (20 μg / mL, 10 mM sodium acetate buffer, pH 5.0) was then conjugated at a flow rate of 10 μL / min, with a target conjugation volume of approximately 6500 RU. Blocking was performed for 7 minutes with 1 M ethanolamine (pH 8.5), with the reference channel being activated and blocked only. Palm-P1-SS-PTX conjugates and P1-SS-PTX conjugates were diluted with HBS-EP+ buffer to concentrations of 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, and 40 nM, respectively, and sequentially flowed through the chip surface at a flow rate of 30 μL / min. The binding time was 180 seconds, and the dissociation time was 300 seconds. After each cycle, the cells were regenerated with 10 mM glycine-HCl (pH 2.0) for 30 seconds. After subtracting the reference channel and blank signal, ka, kd, and KD were calculated using a 1:1 Langmuir binding model.

[0115] Affinity assays showed that the KD value of the Palm-P1-SS-PTX conjugate binding to SR-B1 was 12.4 nM, comparable to that of the blank targeting peptide Palm-P1 (KD = 7.8 nM), while the KD value of the P1-SS-PTX conjugate was 94.3 nM. Palmitic acid modification increased the affinity of the conjugate for SR-B1 by approximately 7.6 times, likely due to the interaction between the palmitic acid chain and the hydrophobic channels of SR-B1. The peptide-conjugated drug of this invention can efficiently and specifically recognize and bind to the SR-B1 receptor, laying the foundation for tumor-targeted delivery.

[0116] Test Example 5: Affinity Determination of P3-Val-Cit-PAB-SN38 Conjugate with SR-B1 Receptor Following the method described in Test Example 4, SR-B1 protein was immobilized on a CM5 chip via amine conjugation, with a target conjugation amount of approximately 6500 RU. The reference channel was only activated and blocked. The P3-Val-Cit-PAB-SN38 conjugate and free P3 peptide were diluted with HBS-EP+ buffer to concentrations of 1, 2.5, 5, 10, 25, and 50 nM, respectively, and sequentially flowed through the chip surface at a flow rate of 30 μL / min. The binding time was 180 seconds, and the dissociation time was 300 seconds. After each cycle, regeneration was performed for 30 seconds with 10 mM glycine-HCl (pH 2.0). After subtracting the reference channel and blank signal, a 1:1 Langmuir binding model was used to calculate ka, kd, and KD.

[0117] Affinity assays showed that the KD value of the P3-Val-Cit-PAB-SN38 conjugate bound to SR-B1 was 26.1 nM, while the KD value of the free P3 peptide was 15.3 nM. This indicates that the affinity of the P3 peptide for SR-B1 decreased slightly after being coupled to SN38 via the Val-Cit-PAB linker, but it still retained a high affinity for SR-B1.

[0118] Test Example 6: Affinity determination of P4-Hyd-Dox conjugate with SR-B1 receptor Following the method described in Test Example 4, SR-B1 protein was immobilized on a CM5 chip via amine conjugation, with a target conjugation amount of approximately 500 RU. The reference channel was only activated and blocked. The P4-Hyd-Dox conjugate and free P4 peptide were diluted with HBS-EP+ buffer to concentrations of 2.5, 5, 10, 25, 50, and 100 nM, respectively, and sequentially flowed through the chip surface at a flow rate of 30 μL / min. The binding time was 120 seconds, and the dissociation time was 300 seconds. After each cycle, regeneration was performed for 30 seconds with 10 mM glycine-HCl (pH 2.0). After subtracting the reference channel and blank signal, a 1:1 Langmuir binding model was used to calculate ka, kd, and KD.

[0119] Affinity assays showed that the KD value of P4-Hyd-Dox binding to the SR-B1 receptor was 15.5 nM, while the KD value of the free P4 peptide was 11.6 nM. Compared with the free P4 peptide, the conjugation of Hyd-Dox introduced some steric hindrance, resulting in a slight decrease in affinity. However, the conjugate still maintained nanomolar (nm) level receptor binding activity and retained its SR-B1 targeting ability.

[0120] Test Example 7: Cellular Uptake Assay of P2-SS-PTX Conjugate HepG2 human liver cancer cells with high SR-B1 expression were seeded in 24-well plates (1×10⁻⁶ cells / well). 5 Cells were cultured in wells until adherence and 70% confluence. The competitive inhibition group was pretreated with excess free targeting peptide P2 for 1 hour, followed by the addition of fluorescently labeled P2-SS-PTX (FITC-P2-SS-PTX). The experimental group received the FITC-P2-SS-PTX conjugate directly. The control group received both fluorescently labeled P2 (FITC-P2) and fluorescently labeled PTX (FITC-PTX), and were incubated at 37°C in a 5% CO2 incubator for 2 hours in the dark. After incubation, the culture medium was discarded, and the cells were washed with pre-cooled PBS and digested with 0.25% trypsin to collect the cell suspension. The cell pellet was collected by centrifugation, washed twice with PBS, resuspended in PBS, and the FITC fluorescence intensity was detected by flow cytometry (10,000 cells / sample).

[0121] Flow cytometry results (see) Figure 13 The results showed that the P2-SS-PTX conjugate was taken up in SR-B1-overexpressing HepG2 cells (MFI = 4.5 × 10⁻⁶). 3 The uptake level of the free target peptide P2 in cells was comparable (MFI = 5.0 × 10⁻⁶). 3 Both were significantly higher than the cellular uptake of free PTX (MFI = 1.2 × 10⁻⁶). 3 This indicates that conjugation with paclitaxel does not affect the SR-B1 targeting ability of the targeting peptide P2, and its cellular uptake efficiency is significantly better than that of passive diffusion of free PTX. The results of the competitive inhibition group showed that excess unlabeled free targeting peptide P2 significantly inhibited the uptake of the FITC-labeled conjugate, and the MFI value was significantly reduced (MFI = 0.9 × 10⁻⁶). 3 This demonstrates that drug uptake depends on specific binding to the SR-B1 receptor, rather than nonspecific pinocytosis.

[0122] Test Example 8: Cellular Uptake Assay of P3-Val-Cit-PAB-SN38 Conjugate U87 cells with high SR-B1 expression were used at a rate of 1×10⁻⁶. 5 Cells were seeded in 24-well plates and cultured until adherence and 70% confluence. The old culture medium was discarded, and the cells were washed once with PBS. The fluorescently labeled P3-Val-Cit-PAB-SN38 conjugate (Cy5-P3-Val-Cit-PAB-SN38) was diluted to a predetermined concentration (500 nM) with serum-free medium, with three replicates per group. A fluorescently labeled free SN38 control group (Cy5-SN38) and a fluorescently labeled P3 control group (Cy5-P3) were also included. After incubation at 37°C for 0.5, 2, 4, and 8 h, the supernatant was discarded, and the cells were washed three times with pre-cooled PBS to remove unbound fluorescent label. Cells were collected by trypsin digestion, centrifuged (1000 rpm, 5 min), the supernatant was discarded, and the cells were washed twice with PBS, finally resuspended in 500 μL PBS. Data were collected using flow cytometry, with 10,000 viable cells collected per sample. Fluorescence signals were detected in the Cy5 channel (excitation 640 nm / emission 670 nm), and the mean fluorescence intensity (MFI) was recorded.

[0123] Flow cytometry results (see Table 1) showed that the uptake of the Cy5-P3-Val-Cit-PAB-SN38 conjugate in SR-B1 positive target cells increased in a time-dependent manner. After incubation for 0.5 h, 2 h, 4 h, and 8 h, the mean fibroblast indices (MFI) were [1648±147], [4115±359], [5862±383], and [6096±506], respectively (n=3). At the same incubation time, the uptake of the P3-Val-Cit-PAB-SN38 conjugate in SR-B1-overexpressing U87 cells was comparable to that in free target peptide P3 cells (P>0.05), and significantly higher than that in free SN38 cells (P<0.05). The above results indicate that the P3-Val-Cit-PAB-SN38 conjugate can efficiently enter target cells via the receptor-specific endocytosis pathway mediated by the P3 peptide, and its uptake efficiency is significantly better than that of free SN38.

[0124] Table 1. P3-Val-Cit-PAB-SN38 conjugates and cell uptake in each group after different incubation times.

[0125] Test Example 9: Cellular Uptake Assay of P4-Hyd-Dox Conjugate HepG2 human liver cancer cells with high SR-B1 expression were seeded in 24-well plates (1×10⁻⁶ cells / well). 5 Cells were cultured in wells until adherence and 70% confluence. The culture medium was discarded, and the cells were washed once with PBS. The P4-Hyd-Dox conjugate was diluted with serum-free medium to preset concentrations (100, 250, 500, 1000 nM, based on Dox equivalents), with three replicates per group. The competitive inhibition group was pre-incubated with excess free targeting peptide P4 for 0.5 hours, followed by the addition of the P4-Hyd-Dox conjugate for uptake experiments. A free Dox control group and a blank culture medium control group were also included. Cells were incubated at 37°C for 4 hours. After incubation, the supernatant was discarded, and the cells were washed three times with pre-cooled PBS. Cells were collected by trypsin digestion, centrifuged, washed, and resuspended in 500 μL PBS. Intracellular Dox fluorescence signals (excitation wavelength 488 nm, emission wavelength 560 nm) were detected by flow cytometry, and the mean fluorescence intensity (MFI) was recorded.

[0126] Flow cytometry results (see Table 2) showed that the uptake of the P4-Hyd-Dox conjugate in target cells increased in a concentration-dependent manner. At the same concentration, the cellular uptake in the P4-Hyd-Dox conjugate group was significantly higher than that in the free Dox group (P<0.05). The lower level of cellular uptake in the free Dox group indicates that free Dox has weak passive transmembrane ability, and P4 peptide-mediated targeted delivery significantly enhances Dox accumulation in target cells. The results of the competitive inhibition group showed that after competition with free P4, the Dox fluorescence signal of P4-Hyd-Dox was significantly reduced (P<0.05), but still slightly higher than that in the free Dox group. These results indicate that the cell binding and uptake of P4-Hyd-Dox mainly rely on target-specific binding mediated by the P4 peptide.

[0127] Table 2. P4-Hyd-Dox conjugate at different concentrations and cellular uptake in each group.

[0128] Test Example 10: In vivo tissue distribution assay of the Palm-P1-SS-PTX conjugate HepG2 cells were subcutaneously injected into the right axilla of BALB / c nude mice, and tumors were allowed to grow to approximately 150-250 mm in size. 3 Tissue distribution experiments were conducted. Tumor-bearing nude mice were randomly divided into three groups (n=21 per group), and injected intravenously via tail vein with Palm-P1-SS-PTX conjugate, P1-SS-PTX conjugate, or free PTX, respectively, at a dose of 10 mg / kg (based on PTX equivalent). Blood and tissue samples were collected at 0.5, 1, 2, 4, 8, 12, and 24 h post-administration (n=3 mice per time point). Blood samples were collected via the orbital venous plexus into EDTA-K2 anticoagulant tubes and centrifuged at 3000×g for 10 minutes at 4°C to separate plasma. Tissues (tumor, liver, spleen, kidney, lung, and heart) were collected, weighed, flash-frozen in liquid nitrogen, and stored at -80°C.

[0129] Plasma sample: Take 100 μL of plasma, add 50 μL of internal standard (docetaxel), add 300 μL of acetonitrile to precipitate proteins, and vortex for 5 minutes. Centrifuge at 12000×g for 10 minutes at 4°C, take 200 μL of supernatant, blow dry with nitrogen, and reconstitute the residue with 100 μL of acetonitrile-water (1:1, v / v). Analyze using LC-MS / MS.

[0130] Tissue sample: Weigh approximately 50 mg of tissue and homogenize it with 3 times its volume of physiological saline. Take 100 μL of the homogenate, perform protein precipitation, reconstitution, and injection using the same method as for plasma.

[0131] The AUC of exposure in each tissue, the tissue distribution data of Palm-P1-SS-PTX conjugate and each group of drugs in tumor-bearing mice are calculated, as shown in Table 3.

[0132] Table 3. Tissue distribution of Palm-P1-SS-PTX conjugates and each drug group in tumor-bearing mice.

[0133] The results of in vivo tissue distribution assays (see Table 3) showed that after intravenous injection of the Palm-P1-SS-PTX conjugate, the plasma AUC (2.65 μg·h / g) was significantly higher than that of the P1-SS-PTX group (0.82 μg·h / g) and the free PTX group (0.52 μg·h / g). Palmitic acid modification enabled the conjugate to reversibly bind to plasma albumin, significantly prolonging the blood circulation half-life and reducing rapid renal clearance. The tumor AUC in the Palm-P1-SS-PTX conjugate group was significantly higher than that in the P1-SS-PTX group. 0-24 The concentration was 9.87 μg·h / g, which was 3.36 and 7.83 times higher than that of the P1-SS-PTX group (2.94 μg·h / g) and the free PTX group (1.26 μg·h / g), respectively. The Palm-P1-SS-PTX conjugate achieved selective enrichment in tumor tissue through SR-B1-mediated active targeting, significantly increasing the intratumoral drug concentration, and exhibited superior targeting performance compared to P1-SS-PTX and free PTX. Besides tumor tissue, the Palm-P1-SS-PTX conjugate was mainly distributed in the liver and spleen, with lower distribution in normal tissues of the heart, kidneys, and lungs compared to free PTX. This indicates that the Palm-P1-SS-PTX conjugate can also reduce exposure in normal tissues such as the heart, kidneys, and lungs, demonstrating its excellent tumor targeting and safety.

[0134] Test Example 11: Pharmacodynamic Study of P2-SS-PTX in a Mouse 4T1 Subcutaneous Xenograft Model of Breast Cancer Mouse breast cancer 4T1 cells were subcutaneously in the right axilla of BALB / c nude mice (purchased from Shanghai Laboratory Animal Center), with a cell seeding volume of 5 × 10⁶ cells. 6 Each tumor, individually or individually, forms a xenograft. Tumor growth is monitored regularly until the tumor reaches approximately 80-120 mm in size. 3 Animals were randomly divided into four groups (n=5 per group): P2-SS-PTX conjugate 20 mg / kg (based on PTX), paclitaxel (PTX) albumin nanoparticles 20 mg / kg, paclitaxel micelles 20 mg / kg, and a negative control PBS group. The drugs were administered via tail vein injection weekly, once every 3 days, for a total of 5 times. Mouse weight was measured every 3 days, and a weight change curve was plotted to evaluate drug safety in vivo. Simultaneously, the long axis (a) and short axis (b) of tumors in each nude mouse were measured using calipers, and the results were calculated as follows: (a×b...) 2The tumor volume is calculated using the formula (V / 2). The relative tumor volume (RTV) is calculated based on the measurement results using the formula: RTV = V / 2. t / V0, where V0 is the tumor volume measured when the drug is administered in separate cages (i.e., d0), V t This represents the tumor volume at each measurement time point. Based on the relative tumor volume results, the relative tumor proliferation rate T / C (%) is calculated, where T / C (%) = T RTV / C RTV ×100%, where T RTV The relative tumor volume at the endpoint of the experiment in the treatment group; C RTV The relative tumor volume represents the endpoint of the PBS group experiment.

[0135] Tumor growth changes and tumor-suppressive activity in different groups of the mouse 4T1 subcutaneous xenograft model of breast cancer are shown in Table 4. Figure 14 As shown.

[0136] Table 4. Pharmacodynamics of P2-SS-PTX and other drugs in mouse 4T1 mammary carcinoma xenografts in nude mice.

[0137] Note: V0 is the tumor volume measured when the drug is administered in separate cages (i.e., d0); V 15 Day 15 (d) 15 The tumor volume was measured.

[0138] In a mouse 4T1 subcutaneous xenograft model of breast cancer, the P2-SS-PTX 20 mg / kg group (calculated as PTX) administered via tail vein injection every 3 days for 5 consecutive times significantly inhibited the growth of mouse 4T1 subcutaneous xenografts of breast cancer (see [link to relevant documentation]). Figure 14 Furthermore, at the same dosage, its tumor-suppressing effect (T / C value of 21.80%) was superior to that of the PTX albumin nanoparticle group (T / C value of 65.95%) and the PTX micelle group (T / C value of 54.04%). During the experiment, the mice in the P2-SS-PTX group were in good condition and no significant weight loss was observed (see...). Figure 15 This indicates that P2-SS-PTX has good in vivo tolerability and safety in a mouse 4T1 subcutaneous xenograft model of breast cancer.

[0139] Test Example 12: Pharmacodynamics of Palm-P1-SS-PTX in a SKOV-3 nude mouse xenograft model of human ovarian cancer Human ovarian cancer SKOV-3 cells were subcutaneously inoculated into the backs of nude mice at a seeding density of 1 × 10⁻⁶ cells. 7 Each nude mouse will have a tumor volume of 500±50 mm. 3At that time, the mice were euthanized and the tumor tissue was aseptically dissected and cut into 2-3 mm pieces. 3 A tumor mass, uniformly distributed on both sides, was subcutaneously injected into the back of recipient nude mice under sterile conditions using a puncture cannula. The tumor volume in the tumor-bearing nude mice was increased to 50 mm. 3 Mice were randomly divided into 5 groups (4 mice per group): Palm-P1-SS-PTX conjugate 20 mg / kg (based on PTX), P1-SS-PTX conjugate 20 mg / kg (based on PTX), paclitaxel (PTX) albumin nanoparticles 20 mg / kg, paclitaxel micelles 20 mg / kg, and a negative control PBS group. All mice were administered via tail vein injection every 3 days for a total of 5 administrations. Similar to test case 11, tumor volume was measured every 3 days, relative tumor volume (RTV) and relative tumor proliferation rate (T / C) (%) were calculated, and mouse body weight was measured to plot body weight change curves and evaluate in vivo drug safety.

[0140] Table 5 shows the tumor growth changes and tumor-suppressive activity in different groups of the human ovarian cancer SKOV-3 subcutaneous xenograft model. Figure 16 As shown.

[0141] Table 5. Pharmacodynamics of Palm-P1-SS-PTX and other drugs in human ovarian cancer SKOV-3 nude mouse xenografts.

[0142] Note: V0 is the tumor volume measured when the drug is administered in separate cages (i.e., d0); V 42 Day 42 (d) 42 The tumor volume was measured.

[0143] In a human ovarian cancer SKOV-3 xenograft model in nude mice, the Palm-P1-SS-PTX group showed an excellent inhibitory effect on the growth of human ovarian cancer SKOV-3 subcutaneous xenografts (see...). Figure 16 The T / C percentage obtained on day 42 was 10.51%. At the same dose (20 mg / kg), the tumor inhibition effect was significantly better than that of the paclitaxel albumin nanoparticle group (T / C value of 49.36%), the paclitaxel micelle group (T / C value of 43.11%) and the P1-SS-PTX group (T / C value of 21.54%).

[0144] During the experiment, the mice in the Palm-P1-SS-PTX group were in good condition and no significant weight loss was observed (see...). Figure 17 This indicates that Palm-P1-SS-PTX has good in vivo tolerability and safety in the human ovarian cancer SKOV-3 subcutaneous xenograft model.

[0145] Test Example 13: Pharmacodynamics of Palm-P1-SS-PTX in a Human Glioma U87 Nude Mouse Xenograft Model Human glioma U87 cells were subcutaneously inoculated into the back of nude mice at a seeding density of 1 × 10⁻⁶ cells. 7 Each nude mouse will have a tumor volume of 500±50 mm. 3 At that time, the mice were euthanized and the tumor tissue was aseptically dissected and cut into 2-3 mm pieces. 3 A uniform tumor mass was implanted subcutaneously into the back of a recipient nude mouse under sterile conditions using a puncture cannula. The tumor in the tumor-bearing nude mouse grew to approximately 100 mm in size. 3 Mice were randomly divided into 5 groups (5 mice per group): Palm-P1-SS-PTX conjugate 20 mg / kg (based on PTX), P1-SS-PTX conjugate 20 mg / kg (based on PTX), paclitaxel (PTX) albumin nanoparticles 20 mg / kg, paclitaxel micelles 20 mg / kg, and a negative control PBS group. All mice were administered via tail vein injection every 3 days for a total of 5 administrations. Similar to test case 11, tumor volume was measured every 3 days, relative tumor volume (RTV) and relative tumor proliferation rate (T / C) (%) were calculated, and mouse body weight was measured to plot body weight change curves and evaluate in vivo drug safety.

[0146] Table 6 shows the tumor growth changes and tumor-suppressive activity in different groups of the human glioma U87 subcutaneous xenograft model. Figure 18 As shown.

[0147] In a nude mouse model of human glioma U87, the Palm-P1-SS-PTX group significantly inhibited the growth of subcutaneous human glioma U87 xenografts (see...). Figure 18 At a dose of 20 mg / kg, Palm-P1-SS-PTX showed significantly better tumor-suppressive effects (T / C value of 9.07%) than the PTX albumin nanoparticle group (T / C value of 46.35%), PTX micelle group (T / C value of 28.66%), and P1-SS-PTX group (T / C value of 22.94%) at the same dose. During the experiment, mice in the Palm-P1-SS-PTX group were in good condition and showed no significant weight loss (see...). Figure 19 This indicates that Palm-P1-SS-PTX has good in vivo tolerability and safety in the human glioma U87 subcutaneous xenograft model.

[0148] Table 6. Pharmacodynamics of Palm-P1-SS-PTX and other drugs in human glioma U87 nude mouse xenografts.

[0149] Note: V0 is the tumor volume measured when the drug is administered in separate cages (i.e., d0); V 15 Day 15 (d) 15 The tumor volume was measured.

[0150] Test Example 14: Pharmacodynamics of P3-Val-Cit-PAB-SN38 in a human pancreatic cancer PANC-1 xenograft model in nude mice Human pancreatic cancer PANC-1 cells were used at a rate of 1×10⁻⁶. 7 Cells were subcutaneously injected into the right back of BALB / c (nu / nu) mice. Tumors in the injected nude mice were allowed to grow to 100-150 mm in size. 3 Mice were randomly divided into four groups (n=4 per group): P3-Val-Cit-PAB-SN38 conjugate 5 mg / kg (based on SN38), P3-Val-Cit-PAB-SN38 conjugate 10 mg / kg (based on SN38), irinotecan injection 15 mg / kg, and a negative control PBS group. All mice were administered via tail vein injection, once every four days, for a total of four administrations. Similar to test case 11, tumor volume was measured every three days, and relative tumor volume (RTV) and relative tumor proliferation rate (T / C) (%) were calculated. Mouse body weight was also measured, and a mouse body weight change curve was plotted to evaluate the in vivo safety of the drug.

[0151] Tumor growth changes and tumor-suppressive activity in different groups of the human pancreatic cancer PANC-1 subcutaneous xenograft model are shown in Table 7. Figure 20 As shown.

[0152] The results showed that P3-Val-Cit-PAB-SN38 dose-dependently inhibited the growth of human pancreatic cancer PANC-1 subcutaneous xenografts (see...). Figure 20 On day 21, the T / C percentages of the P3-Val-Cit-PAB-SN38 5 mg / kg (based on SN38) group and the 10 mg / kg (based on SN38) group were 46.10% and 28.71%, respectively, showing significantly better tumor-suppressing effects than the positive control irinotecan injection 15 mg / kg group (T / C value of 64.64%). This may be due to the effective release of the P3 peptide-mediated SR-B1 targeted endocytosis and Val-Cit linker into the lysosome, which significantly inhibits tumor growth.

[0153] During the experiment, the mice in the P3-Val-Cit-PAB-SN38 group were in good condition and no significant weight loss was observed (see...). Figure 21 This indicates that P3-Val-Cit-PAB-SN38 has good in vivo tolerability and safety in the human pancreatic cancer PANC-1 subcutaneous xenograft model.

[0154] Table 7. Pharmacodynamics of P3-Val-Cit-PAB-SN38 and other drugs in human pancreatic cancer PANC-1 xenografts in nude mice.

[0155] Note: V0 is the tumor volume measured when the drug is administered in separate cages (i.e., d0); V 21 Day 21 (d) 21 The tumor volume was measured.

[0156] Test Example 15: Pharmacodynamics of P4-Hyd-Dox in a mouse 4T1 subcutaneous xenograft model of breast cancer Mouse breast cancer 4T1 cells were subcutaneously in the right axilla of BALB / c nude mice (purchased from Shanghai Laboratory Animal Center), with a cell seeding volume of 5 × 10⁶ cells. 6 Each tumor, individually or individually, forms a xenograft. Tumor growth is monitored regularly until the tumor reaches approximately 80-120 mm in size. 3 Animals were randomly divided into four groups (n=5 per group): P4-Hyd-Dox conjugate 5 mg / kg (based on Dox), P4-Hyd-Dox conjugate 10 mg / kg (based on Dox), Dox injection 5 mg / kg, and negative control PBS. All animals were administered the drug via tail vein injection every 3 days for a total of 5 administrations. Similar to test case 11, tumor volume was measured every 3 days, and relative tumor volume (RTV) and relative tumor proliferation rate (T / C) were calculated. Mouse body weight was also measured, and a weight change curve was plotted to evaluate the drug's safety in vivo.

[0157] Table 8 shows the tumor growth changes and tumor-suppressive activity in different groups of the mouse 4T1 subcutaneous xenograft model of breast cancer. Figure 22 As shown.

[0158] Table 8. Pharmacodynamics of P4-Hyd-Dox and each drug group in mouse 4T1 mammary carcinoma xenografts in nude mice.

[0159] Note: V0 is the tumor volume measured when the drug is administered in separate cages (i.e., d0); V 15 Day 15 (d) 15 The tumor volume was measured.

[0160] In a mouse 4T1 subcutaneous xenograft model of breast cancer, the P4-Hyd-Dox conjugate at 5 mg / kg and 10 mg / kg (based on Dox) was administered via tail vein injection every 3 days for 5 consecutive days, significantly inhibiting the growth of 4T1 subcutaneous xenografts of breast cancer in mice (see...). Figure 22The effect was dose-dependent, and at the same dose (5 mg / kg), its tumor-suppressing effect (T / C value of 33.73%) was significantly better than that of Dox injection (T / C value of 44.55%). Furthermore, during the experiment, the mice in the P4-Hyd-Dox group were in good condition and no significant weight loss was observed (see...). Figure 23 The Dox injection group showed a significant decrease in body weight, indicating that P4-Hyd-Dox has good in vivo tolerability and safety in the mouse 4T1 subcutaneous xenograft model of breast cancer.

[0161] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A tumor-targeting peptide drug conjugate based on SR-B1, characterized in that, include: An SR-B1 targeting peptide, a linker, and an antitumor drug are covalently linked together, and the structure of the peptide-drug conjugate is shown in general formula I: SR-B1 Targeting Peptide-Linker-Antitumor Drug General Formula I The SR-B1 targeting peptide can specifically recognize and bind to the SR-B1 receptor. The amino acid sequence of the SR-B1 targeting peptide is selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or a variant that has at least 90% sequence homology with the amino acid sequence and retains SR-B1 binding activity.

2. The SR-B1-based tumor-targeting polypeptide drug conjugate according to claim 1, characterized in that, The N-terminus of the polypeptide shown in SEQ ID NO:1 is optionally linked to a C12-C18 fatty acid chain.

3. The SR-B1-based tumor-targeting polypeptide drug conjugate according to claim 2, characterized in that, The fatty acid chain is selected from lauric acid, myristic acid, palmitic acid or stearic acid.

4. The SR-B1-based tumor-targeting polypeptide drug conjugate according to claim 1, characterized in that, The linker is a tumor microenvironment-responsive linker, selected from any one of glutathione-responsive disulfide bonds, acid pH-responsive hydrazone bonds, and cathepsin B-sensitive peptide bonds.

5. The SR-B1-based tumor-targeting polypeptide drug conjugate according to claim 1, characterized in that, The antitumor drug is selected from any one of taxanes, anthracyclines, and camptothecins.

6. A method for preparing an SR-B1-based tumor-targeting polypeptide drug conjugate as described in any one of claims 1 to 5, characterized in that, Includes the following steps: (1) SR-B1 targeting peptides were synthesized by SPPS solid-phase peptide synthesis method; (2) The precursor compound of the linker is reacted with the antitumor drug in an organic solvent to obtain the linker-drug intermediate; (3) The SR-B1 targeting peptide obtained in step (1) and the linker-drug intermediate obtained in step (2) are reacted in the presence of a coupling agent to form a covalent link; (4) The product obtained in step (3) is purified by preparative high performance liquid chromatography to obtain the polypeptide drug conjugate.

7. A pharmaceutically acceptable salt of the SR-B1-based tumor-targeting peptide drug conjugate as described in any one of claims 1 to 5.

8. An antitumor drug composition, characterized in that, It includes pharmaceutically acceptable salts of the SR-B1-based tumor-targeting peptide drug conjugate as described in any one of claims 1 to 5 and / or the SR-B1-based tumor-targeting peptide drug conjugate as described in claim 7, as well as pharmaceutically acceptable excipients.

9. The use of the SR-B1-based tumor-targeting peptide drug conjugate as described in any one of claims 1 to 5, the pharmaceutically acceptable salt of the SR-B1-based tumor-targeting peptide drug conjugate as described in claim 7, or the antitumor drug composition as described in claim 8 in the preparation of a drug for treating tumors with high SR-B1 expression.

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