A dabigatran polypeptide conjugate and use thereof

Abstract

The present application provides a kind of polypeptide conjugate and its application, especially to a kind of triclabendazole polypeptide conjugate and its application.The polypeptide conjugate provided in the present application is selectively released in tumor microenvironment, can solve the problem that the clinical application is limited due to the fact that triclabendazole drug does not have targeting and has strong side effects in the prior art, and a larger dose can be used in clinic to treat cancer, and the overall treatment effect is improved.

Description

Technical Field

[0001] This invention relates to a polypeptide conjugate and its application, and particularly to a tumor-targeting activated trabectedine polypeptide conjugate and its application. Background Technology

[0002] Trabectedin is a sucrose compound primarily used for unresectable or metastatic liposarcoma, leiomyosarcoma, and recurrent ovarian cancer that has been previously treated with anthracyclines.

[0003] Soft tissue sarcomas are a group of rare tumors originating from mesenchymal tissue, accounting for approximately 1% of adult cancers. There are more than 60 different histological subtypes, each with its own unique biological behavior and response to systemic therapy. Patients with metastatic soft tissue sarcomas have a poor prognosis, and available systemic treatment options are limited. For decades, the primary treatment has been trabected with or without ifosfamide. Several phase II trials have demonstrated the activity of trabectedin in anthracycline- and alkylating agent-resistant soft tissue sarcomas, suggesting its use as second- and third-line therapy, and showing significant activity in liposarcoma and leiomyosarcoma subtypes. Trabectedin has shown favorable toxicity profiles and has been approved for the treatment of metastatic soft tissue sarcomas in more than 70 countries.

[0004] Ovarian cancer can occur at any age, but it is more common in patients over 50. Patients typically present with nonspecific pelvic or abdominal symptoms. Prognosis is usually determined by cancer stage and grade, and is often not ideal, as 70% of cases are diagnosed at stage III or IV, thus associated with poor prognosis. Prophylactic visits provide an opportunity to identify and educate women at increased risk of ovarian cancer, but routine screening is not recommended. More robust solutions are needed to improve patient outcomes while optimizing treatment.

[0005] Trabectedin is a seaweed extract compound that exhibits broad antitumor activity against human tumor cells. However, its clinical application is limited due to certain side effects. Currently, there is no existing research on improving the targeting of trabectedin as an antitumor drug and reducing its side effects. Therefore, there is an urgent need to research a compound that can improve the targeting of trabectedin and reduce its side effects in order to achieve better cancer treatment. Summary of the Invention

[0006] This invention provides a tumor-targeting activated trabectedine peptide conjugate and its application, which solves the problem that the clinical application of trabectedine is limited due to its lack of targeting and strong side effects in existing technologies. The peptide conjugate of this invention transports trabectedine to tumor tissue via peptides. In the acidic tumor microenvironment, the trabectedine peptide conjugate can be activated by Legumin, a protein highly expressed by tumor cells and tumor-associated macrophages. This allows trabectedine to be selectively released in the tumor microenvironment, inducing immunogenic death of tumor cells, stimulating the body's anti-tumor immune function, and reducing the toxic side effects of trabectedine. It exhibits good targeting and achieves multiple effects, including inhibiting tumor growth, promoting anti-tumor immunity, and reducing drug toxicity.

[0007] This invention provides a polypeptide conjugate, the structural formula of which is as follows:

[0008] E-nPEG-L1-L2-L3-L4-L5-D

[0009] in,

[0010] E represents a maleimide group;

[0011] nPEG stands for n-polyethylene glycol, where n is an integer greater than or equal to 2 and less than or equal to 40.

[0012] L1 and L2 are glycine, alanine, or none;

[0013] L3 is either alanine or asparagine;

[0014] L4 is glycine, leucine, PAB (aminobenzyl alcohol), or absent;

[0015] L5 is an ethylenediamine derivative or is absent;

[0016] D represents trabectedin or its derivatives.

[0017] Furthermore, the structure of the D group in the polypeptide conjugate provided by the present invention is as follows:

[0018]

[0019] Furthermore, the structure of the E group in the polypeptide conjugate provided by the present invention is as follows:

[0020] Where n is an integer greater than or equal to 1 and less than or equal to 18.

[0021] Furthermore, the structure of the L5 group in the polypeptide conjugate provided by the present invention is as follows:

[0022] Where R1 and R2 are hydrogen or methyl, and n1 and n2 are integers greater than or equal to 0 and less than or equal to 20.

[0023] Furthermore, the polypeptide conjugate of the present invention may be selected from any of the following structures:

[0024]

[0025]

[0026] Among them, G: glycine, A: alanine, N: asparagine, PAB: aminobenzyl alcohol, and L: leucine.

[0027] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the polypeptide conjugate of the present invention and a pharmaceutically acceptable carrier.

[0028] The present invention also provides the use of the aforementioned polypeptide conjugate or the aforementioned pharmaceutical composition in the preparation of antitumor drugs.

[0029] The present invention also provides the use of the aforementioned polypeptide conjugate or pharmaceutical composition in the preparation of a medicament for treating cancer, wherein the cancers include gastrointestinal cancer, colorectal cancer, colon cancer, liver cancer, hepatocellular carcinoma, pancreatic cancer, biliary tract cancer, gastric cancer, genitourinary system cancers, bladder cancer, testicular cancer, cervical cancer, malignant mesothelioma, osteosarcoma, esophageal cancer, laryngeal cancer, prostate cancer, hormone-resistant prostate cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, triple-negative breast cancer, hematologic malignancies, leukemia, acute primitive lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, ovarian cancer, brain cancer, neuroblastoma, Ewing sarcoma, renal cancer, epidermoid carcinoma, skin cancer, melanoma, and oral cancer.

[0030] Compared with the prior art, the beneficial effects of the present invention include: (1) The tumor-targeting activated trabectedine polypeptide conjugate provided by the present invention has the general structural formula LD; after structural modification of the L group, the water solubility of the drug and its overall targeting to the tumor are improved. After the trabectedine polypeptide conjugate provided by the present invention enters the body, it selectively releases the prodrug trabectedine compound in the tumor microenvironment, allowing it to directly enter the tumor, thereby improving the drug's targeting to the tumor and significantly improving the killing effect on cancer cells. At the same time, while having high tumor targeting ability and good therapeutic effect, it also reduces the toxic side effects of trabectedine, and the drug safety is significantly improved. (2) The molecularly targeted and activated trabectedine polypeptide conjugate has a targeting effect of aggregation and retention at the sites on the tumor surface where desialyl glycoprotein receptor and aspartate endopeptidase are co-expressed, and has the characteristics of molecularly targeted tumor. (3) The molecularly targeted and activated trabectedine polypeptide conjugate can improve the activation efficiency through molecularly targeted targeting, and activate and release trabectedine at the tumor site. (4) In in vitro and in vivo metabolic experiments, dual-target activated trabectedine is not activated in the blood, exhibiting long-term blood stability and low toxicity to normal tissues and organs. (5) The toxicity of dual-target activated trabectedine peptide conjugates is significantly reduced compared to single-target trabectedine. (6) Due to the structure-activity relationship and polarity changes between chemical structures, molecularly targeted and activated trabectedine peptide conjugates have higher activation efficiency than single-target trabectedine, while they cannot be activated when linked to other toxicants. (7) Due to their high activation efficiency, molecularly targeted and activated trabectedine peptide conjugates can directly change the limitations of single-target indications and can be developed into anti-tumor drugs with a wider range of applications. (8) Tumor cells express a larger amount of the targeted bimolecule during metastasis, so molecularly targeted and activated trabectedine peptide conjugates have special efficacy in the treatment of tumor metastasis. (9) When trabectedine peptide conjugates with molecular targeting and activation are used in combination with targeted radiotherapy, radiotherapy leads to increased co-expression of the two target molecules. Experiments have shown that trabectedine peptide conjugates have synergistic therapeutic effects that the control compound does not possess. (10) Some tumor immunosuppressive cells express targeted bimolecules. Trabectedine peptide conjugates with molecular targeting and activation can enhance the effect of immunotherapy by killing tumor immunosuppressive cells. Unlike traditional chemotherapy drugs, they do not damage the overall immune system and can solve the problem that immunotherapy is difficult to combine with chemotherapy drugs. Attached Figure Description

[0031] Figure 1-1 , Figure 1-2 , Figure 1-3 , Figure 1-4 , Figure 1-5 This is a statistical chart of the toxicity test results of the trabectedine polypeptide conjugate in cells provided by the present invention;

[0032] Figure 2 This is a statistical chart of the toxicity test results of the trabectedine polypeptide conjugate provided by this invention in mice;

[0033] Figure 3 This is a statistical graph showing the results of the tumor homogenate activation experiment of the trabectedine polypeptide conjugate provided by the present invention.

[0034] [Symbol Explanation]

[0035] The symbols in the attached diagram are explained as follows:

[0036] C: Physiological saline;

[0037] X: Trabectin;

[0038] X1: Trabectin peptide conjugate X1 (without legumain);

[0039] X1leg: Trabectin polypeptide conjugate X1 (containing legumain);

[0040] X2: Trabectin peptide conjugate X2 (without legumain);

[0041] X2leg: Trabectedine polypeptide conjugate X2 (containing legumain);

[0042] X3: Trabectin peptide conjugate X3 (without legumain);

[0043] X3leg: Trabectin polypeptide conjugate X3 (containing legumain);

[0044] X4: Trabectin peptide conjugate X4 (without legumain);

[0045] X4leg: Trabectin polypeptide conjugate X4 (containing legumain);

[0046] X5: Trabectin peptide conjugate X5 (without legumain);

[0047] X5leg: Trabectin polypeptide conjugate X5 (containing legumain);

[0048] X6: Trabectin peptide conjugate X6 (excluding legumain);

[0049] X6leg: Trabectedine polypeptide conjugate X6 (containing legumain).

[0050] A: Human fibrosarcoma tissue; B: Human osteosarcoma tissue; C: Human ovarian cancer tissue; D: Human ovarian cancer; E: Human heart tissue. Detailed Implementation

[0051] The specific embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the materials, instruments, and reagents used in the following embodiments are all commercially available. Unless otherwise specified, the technical means used in the embodiments are all conventional means well known to those skilled in the art.

[0052]

Example 1

[0053] The synthetic method for the MI-6PEG-AAN-PAB-Ecteinascidin 743 peptide conjugate is as follows:

[0054]

[0055] The specific synthesis steps are as follows:

[0056] 1. Synthesis of Compound 1-II

[0057] Compound 1-I (500 mg, 0.83 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added. The reaction mixture was reacted at room temperature (25 °C) for 1 hour. The reaction was confirmed to be complete by thin-layer chromatography (TLC). The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 10:1) to give compound 1-II as a pale yellow solid (280 mg, yield 88.9%).

[0058] 2. Synthesis of Compound 1-IV

[0059] Compound 1-II (280 mg, 0.74 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (450 mg, 0.75 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 3 hours. Thin-layer chromatography (TLC) was used to detect the complete reaction. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 100:1 to 10:1) to give compound 1-IV as a yellow solid (310 mg, yield 48.4%).

[0060] 3. Synthesis of compounds 1-V

[0061] Compound 1-IV (310 mg, 0.36 mmol) was dissolved in N,N-dimethylformamide (15 mL), and di(p-nitrobenzene) carbonate (180 mg, 0.59 mmol) was added. The reaction mixture was reacted at room temperature (25 °C) for 3 hours. The reaction was confirmed to be complete by thin-layer chromatography (TLC). The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 100:1 to 12:1) to give compound 1-V as a yellow solid (190 mg, yield 51.2%).

[0062] 4. Synthesis of compound MI-6PEG-AAN-PAB-Ecteinascidin 743

[0063] Ecteinascidin 743 (130 mg, 0.17 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-V (190 mg, 0.18 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction mixture was heated to 60 °C and reacted for 1.5 hours. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by high pressure reverse phase preparation to obtain MI-6PEG-AAN-PAB-Ecteinascidin 743 as a pale yellow solid (8 mg, yield 2.8%).

[0064]

Example 2

[0065] The synthetic method for the MI-6PEG-AANL-Ecteinascidin 743 peptide conjugate is as follows:

[0066]

[0067] The specific synthesis steps are as follows:

[0068] 1. Synthesis of compound 2-II

[0069] Compound Ecteinascidin 743 (500 mg, 0.66 mmol) and compound 2-I (1 g, 1.64 mmol) were dissolved in N,N-dimethylformamide (8 mL). 1-n-propylphosphonic anhydride T3P (3.5 g, 5.50 mmol, 50% ethyl acetate solution) was added under ice-bath cooling. The reaction mixture was heated to 60 °C and reacted for 4.5 h. The reaction mixture was then added to water (80 mL), and the aqueous phase was extracted with ethyl acetate (50 mL x 5). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 100:1 to 10:1) to give compound 2-II as a yellow solid (62 mg, yield 6.9%).

[0070] 2. Synthesis of Compound 2-III

[0071] Compound 2-II (62 mg, 0.046 mmol) was dissolved in N,N-dimethylformamide (10 mL), and piperidine (0.2 mL) was added. The reaction mixture was reacted at room temperature (25 °C) for 1 hour. Thin-layer chromatography (TLC) was used to detect the complete reaction. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 100:1 to 5:1) to give compound 2-III as a yellow solid (35 mg, yield 67.3%).

[0072] 3. Synthesis of compound MI-6PEG-AANL-Ecteinascidin 743

[0073] Compound 2-III (35 mg, 0.031 mmol) was dissolved in N,N-dimethylformamide (5 mL), and compound 1-III (50 mg, 0.083 mmol) and diisopropylethylamine (25 mg, 0.19 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography to give compound MI-6PEG-AANL-Ecteinascidin 743, a white solid (1.2 mg, yield 2.4%).

[0074]

Example 3

[0075] The synthetic method for the MI-6PEG-AANG-Ecteinascidin 743 peptide conjugate is as follows:

[0076]

[0077] The specific synthesis steps are as follows:

[0078] 1. Synthesis of compound 3-II

[0079] Compound Ecteinascidin 743 (500 mg, 0.66 mmol) and compound 3-I (900 mg, 0.66 mmol) were mixed.

[0080] 1.63 mmol) was dissolved in N,N-dimethylformamide (8 mL), and 1-n-propylphosphonic anhydride T3P (3.5 g, 5.50 mmol, 50% ethyl acetate solution) was added under ice bath cooling. The reaction solution was heated to 60 °C and reacted for 4.5 h. The reaction solution was added to water (80 mL), and the aqueous phase was extracted with ethyl acetate (50 mL * 5). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 100:1 to 10:1) to give compound 3-II as a yellow solid (71 mg, yield 8.3%).

[0081] 2. Synthesis of Compound 3-III

[0082] Compound 3-II (71 mg, 0.055 mmol) was dissolved in N,N-dimethylformamide (10 mL), and piperidine (0.2 mL) was added. The reaction mixture was reacted at room temperature (25 °C) for 1 hour. Thin-layer chromatography (TLC) was used to detect the complete reaction. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 100:1 to 5:1) to give compound 3-III as a yellow solid (53 mg, yield 89.6%).

[0083] 3. Synthesis of compound MI-6PEG-AANG-Ecteinascidin 743

[0084] Compound 3-III (53 mg, 0.049 mmol) was dissolved in N,N-dimethylformamide (5 mL), and compound 1-III (50 mg, 0.083 mmol) and diisopropylethylamine (25 mg, 0.19 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography to give compound MI-6PEG-AANG-Ecteinascidin 743, a yellow solid (3.2 mg, yield 4.2%).

[0085]

Example 4

[0086] The synthetic method for the MI-6PEG-AAN-PAB-EA(2PEG)-Ecteinascidin 743 peptide conjugate is as follows:

[0087]

[0088] The specific synthesis steps are as follows:

[0089] 1. Synthesis of compound 4-I

[0090] Ecteinascidin 743 (200 mg, 0.26 mmol) was dissolved in N,N-dimethylformamide (10 mL), and p-nitrobenzene chloroformate (75 mg, 0.37 mmol) and diisopropylethylamine (100 mg, 0.78 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 3 hours. The reaction was confirmed to be complete by thin-layer chromatography (TLC). The reaction mixture was evaporated to dryness under reduced pressure. The residue was slurried with methyl tert-butyl ether and then purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 100:1 to 10:1) to give compound 4-I as a yellow solid (126 mg, yield 52.3%).

[0091] 2. Synthesis of Compound 4-III

[0092] Compound 4-I (126 mg, 0.14 mmol) was dissolved in N,N-dimethylformamide (10 mL), and compound 4-II (58 mg, 0.22 mmol) and diisopropylethylamine (100 mg, 0.77 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 3 hours. The reaction was confirmed to be complete by thin-layer chromatography (TLC). The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 100:1 to 10:1) to give compound 4-III as a yellow solid (110 mg, yield 74.8%).

[0093] 3. Synthesis of compound MI-6PEG-AAN-PAB-EA(2PEG)-Ecteinascidin 743

[0094] Compound 4-III (110 mg, 0.10 mmol) was added to dichloromethane (15 mL), followed by trifluoroacetic acid (4 mL). The reaction mixture was reacted at room temperature (25 °C) for 2 hours. The reaction was confirmed to be complete by thin-layer chromatography (TLC). The reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in N,N-dimethylformamide (15 mL), and compound 1-V (150 mg, 0.14 mmol) and diisopropylethylamine (150 mg, 1.16 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 2 hours. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase chromatography under high pressure to give MI-6PEG-AAN-PAB-EA(2PEG)-Ecteinascidin 743, a pale yellow solid (22 mg, yield 11.4%).

[0095]

Example 5

[0096] The synthetic method for the MI-6PEG-AAA-PAB-EA(2PEG)-Ecteinascidin 743 peptide conjugate is as follows:

[0097]

[0098] The specific synthesis steps are as follows:

[0099] 1. Synthesis of compound MI-6PEG-AAA-PAB-EA(2PEG)-Ecteinascidin 743

[0100] Compound 4-III (110 mg, 0.10 mmol) was added to dichloromethane (15 mL), followed by trifluoroacetic acid (4 mL). The reaction mixture was reacted at room temperature (25 °C) for 2 hours. The reaction was confirmed to be complete by thin-layer chromatography (TLC). The reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in N,N-dimethylformamide (15 mL), and compound 5-I (150 mg, 0.15 mmol) and diisopropylethylamine (150 mg, 1.16 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 2 hours. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase chromatography under high pressure to give MI-6PEG-AAA-PAB-EA(2PEG)-Ecteinascidin 743, a pale yellow solid (36 mg, yield 13.2%).

[0101]

Example 6

[0102] The synthetic method for the MI-6PEG-GAN-PAB-EA(2PEG)-Ecteinascidin 743 peptide conjugate is as follows:

[0103]

[0104] The specific synthesis steps are as follows:

[0105] 1. Synthesis of compound MI-6PEG-GAN-PAB-EA(2PEG)-Ecteinascidin 743

[0106] Compound 4-III (110 mg, 0.10 mmol) was added to dichloromethane (15 mL), followed by trifluoroacetic acid (4 mL). The reaction mixture was reacted at room temperature (25 °C) for 2 hours. The reaction was confirmed to be complete by thin-layer chromatography (TLC). The reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in N,N-dimethylformamide (15 mL), and compound 6-I (150 mg, 0.15 mmol) and diisopropylethylamine (150 mg, 1.16 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 2 hours. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase chromatography under high pressure to give MI-6PEG-GAN-PAB-EA(2PEG)-Ecteinascidin 743, a pale yellow solid (33 mg, yield 12.3%).

[0107] [Example 7] Legumain enzyme cleavage activation experiment of trabectin polypeptide conjugate

[0108] Buffer preparation: 50 mM MES, 250 mM sodium chloride, pH adjusted to 5.0 with 0.5 M sodium hydroxide. Legumain was selected at a concentration of 1 mg / mL. The compound was prepared to a concentration of 0.5 μmol / mL using the buffer. 50 μL of the 0.5 μmol / mL compound and 50 μL of the buffer were accurately transferred to a centrifuge tube, and 100 μL of Legumain was added. The reaction was incubated at 37°C for 2 h. The reaction solution was analyzed by LC-MS.

[0109] MI-6PEG-AAN-PAB-Ecteinascidin 743 Ecteinascidin 743 MI-6PEG-AANL-Ecteinascidin 743 L-Ecteinascidin 743 MI-6PEG-AANG-Ecteinascidin 743 G-Ecteinascidin 743 MI-6PEG-AAN-PAB-EA(2PEG)-Ecteinascidin 743 Ecteinascidin 743 MI-6PEG-AAA-PAB-EA(2PEG)-Ecteinascidin 743 Ecteinascidin 743 MI-6PEG-GAN-PAB-EA(2PEG)-Ecteinascidin 743 Ecteinascidin 743

[0110] As shown in the table above, the trabectedin polypeptide conjugates of the present invention can be cleaved by legumain enzyme, which is highly expressed in tumor cells and tumor-associated macrophages, releasing trabectedin. Specifically, for the two polypeptide conjugates MI-6PEG-AANL-Ecteinascidin 743 and MI-6PEG-AANG-Ecteinascidin 743, legumain enzyme can only cleave the AAN group; the L (leucine) and G (glycine) groups are not cleaved and remain on the cleavage product.

[0111] [Example 8] Comparative toxicity study of trabectedine peptide conjugate in sarcoma and ovarian cancer cell lines (CCK8)

[0112] Sarcoma and breast cancer cell lines were cultured in complete medium (RPMI 1640 (DMEM high-glucose medium) + 10% fetal bovine serum + penicillin-streptomycin mixture 1X P / S + sodium pyruvate 1mM solution) and incubated at 37°C in a 5% CO2 incubator until the cells reached a sufficient number. Cells were collected, centrifuged at 1000g for 5 min, and resuspended in an appropriate volume of 10% RPMI 1640 (DMEM high-glucose medium), adjusting the density to 4,000,000 cells / mL. 100 μL of cell culture medium containing different concentrations of the drug was added to each 96-well culture plate. Control wells (0.1% DMSO) containing only the corresponding drug solvent and blank wells (medium-only medium) were also included, with three parallel wells in each group. After cell counting, the cells were seeded onto 96-well plates at a concentration of 100 μL of cell suspension per well, resulting in a seeding concentration of 5000 cells (100 μL) / well. The plate was then incubated at 37°C in a 5% CO2 incubator for 48 hours. After 48 hours, 10 μL of CCK8 reagent (water-soluble tetrazolium salt) (concentration 5 mg / ml) was added to each well, and the plate was incubated in a cell culture incubator for about 2 hours. The absorbance at 450 nm was then measured.

[0113] Calculate cell viability and the half-maximal inhibitory concentration (IC50) of the drug on cells. Cell viability % = (OD assay - OD blank control) / (OD assay control - OD blank control) * 100%. Viability (%) was calculated using Excel software, and the dose-response curve of the drug on cells was plotted using Prism 5 software. All indicators are expressed as means, and the coefficient of variation (CV) was used to assess the consistency of the data.

[0114] Based on the above experimental method, the maximum initial concentration of the test drug was set to 1000 μM, and the initial concentration of the Legumin-activated complex was set to 100 μM. Nine dose groups were serially diluted at a ratio of 1:4 (three replicates per group). The concentration of the drug solvent (DMSO) in all wells was controlled at 0.1%. A control group (Control) was formed by adding only the drug solvent (0.1% DMSO), and a blank group (Blank) was formed by adding only culture medium and no cells. The tumor cell survival rate (%) of each dose group relative to the control group (Control) was then calculated using the following method:

[0115] Cell viability = [(Experimental group absorbance - Blank control absorbance) / (Control group absorbance - Blank control absorbance)] × 100%

[0116] The specific experimental results are shown in the following table:

[0117]

[0118] Table 1. Toxicity of trabectedin and trabectedin peptide conjugate in different cell lines (IC50)

[0119] As shown in Table 1 and Figures 1-1 to 1-5 As shown, the IC50 (half-maximal inhibitory concentration) of the trabectedin peptide conjugates in the above cells was higher than that of trabectedin, while the IC50 decreased significantly after activation with Legumin. This indicates that the trabectedin peptide conjugates provided in Table 1 are less toxic than the trabectedin compound itself. In the tumor microenvironment, after activation by the Legumin enzyme, they enter and are released from tumor cells. The trabectedin drug provided in Table 1 is more toxic after activation by the Legumin enzyme, thus exhibiting stronger toxicity after targeting tumor cells, making it easier to kill tumor cells without affecting cells outside the tumor microenvironment. Patients treated with trabectedin peptide conjugates show significantly improved treatment safety and tumor targeting.

[0120] [Example 9] Pharmacodynamic study of trabectedine peptide conjugate in the treatment of U2OS human osteosarcoma cell tumor model

[0121] Objective: To investigate the antitumor efficacy of the above compounds in the U2OS tumor model.

[0122] Test drugs: trabectedin polypeptide conjugate, trabectedin, and saline control group.

[0123] Experimental animals: 6-8 week old BALB / c mice, all of which were female.

[0124] Preparation of tumor models:

[0125] U2OS cells were purchased from ATCC and cultured in DMEM (1640) medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells were passaged every three days, and cells up to passage 15 were used. 8 × 10⁸ cells were then cultured. 6 The corresponding cells were subcutaneously injected into the back of nude mice. The tumor reached at least 100 mm. 3 Mice were then randomly divided into groups of six. Treatment began on day one. The trabectedine experimental group and the trabectedine peptide conjugate experimental group were administered the same molar dose of 0.16 μmol / kg. The control group received saline. Administration was once weekly for four weeks.

[0126] physiological saline 3762.71 0 20.33％ Trabectin 2736.53 27.27 2.32％ Trabectin Peptide Conjugate X1 1977.57 47.44 11.71％ Trabectin Peptide Conjugate X2 1862.54 50.5 10.54％ Trabectin Peptide Conjugate X3 2025.21 46.17 14.75％ Trabectin peptide conjugate X4 1751.82 53.44 13.17％ Trabectin Peptide Conjugate X5 1547.16 58.88 9.47％ Trabectin Peptide Conjugate X6 1625.25 56.8 12.87％

[0127] Table 2. Statistical results of toxicity experiments on trabectedine and trabectedine peptide conjugate in mice.

[0128] Based on the data in Table 2 and Figure 2It can be seen that the trabectedine peptide conjugate has a stronger inhibitory efficiency and is safer than trabectedine.

[0129] [Example 10] Detection of the Maximum Dead Concentration (MTD) of Trabectedine Peptide Conjugate

[0130] Experimental animals: 6-8 week old mice, all female, were randomly divided into groups of six. Mice were administered the drug at doses of 0.1, 0.2, 0.4, 0.8, and 1.0 μmol / kg, respectively, and monitored for 14 days. Mice were euthanized when they lost 20% of their initial body weight, which was considered death due to toxicity. The maximum dose level (MTD) was defined as the highest dose level in which none of the six mice died from the drug, and the weight loss of any single mouse did not exceed 20%, or the average weight loss within the group did not exceed 15%. The final statistical results showed that the MTD of trabectedine peptide conjugate X1 was 0.4 μmol / kg; the MTD of trabectedine peptide conjugate X2 was 0.4 μmol / kg; the MTD of trabectedine peptide conjugate X3 was 0.6 μmol / kg; the MTD of trabectedine peptide conjugate X4 was 0.4 μmol / kg; the MTD of trabectedine peptide conjugate X5 was 0.6 μmol / kg; and the MTD of trabectedine peptide conjugate X6 was 0.6 μmol / kg.

[0131] [Example 11] Experiment on the activation efficiency of trabectin peptide conjugate in tumor homogenate

[0132] Buffer preparation: 50mM MES, 250mM sodium chloride, pH adjusted to 5.0 with 0.5M sodium hydroxide.

[0133] Trabectin peptide conjugate was prepared at a concentration of 1 mg / ml using pH 5.0 buffer. 100 μg of tumor tissue homogenate (the tumor tissue homogenate was prepared from various tumor tissues using a Jingxin F6 / 10 handheld homogenizer) was added, and the solution was incubated at 37°C for 2 hours. The tumor tissue homogenate effectively activated the trabectin peptide conjugate and released trabectin. High-performance liquid chromatography (HPLC) was used to detect the decrease in the concentration of the compound and the increase in trabectin, allowing for comparison of the drug's activation efficiency in tumor tissue.

[0134] Release percentage = peak area of ​​trabectedine / total peak area of ​​trabectedine and all other trabectedine-containing compounds. Results are shown in the table below:

[0135]

[0136] Table 3. Release efficiency percentage of trabectedine peptide conjugate in different tissues (%)

[0137] From the data in Table 3 and Figure 3It can be seen that the trabectedin peptide conjugate has good release efficiency in different cancer tissue homogenates, but no large release was observed in the heart, indicating good tumor targeting.

[0138] In the treatment of cancer, the following cancers, such as gastrointestinal cancer, colorectal cancer, colon cancer, liver cancer, hepatocellular carcinoma, pancreatic cancer, biliary tract cancer, stomach cancer, genitourinary system cancers, bladder cancer, testicular cancer, cervical cancer, malignant mesothelioma, osteosarcoma, esophageal cancer, laryngeal cancer, prostate cancer, hormone-resistant prostate cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, triple-negative breast cancer, hematologic malignancies, leukemia, acute primitive lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, ovarian cancer, brain cancer, neuroblastoma, Ewing sarcoma, renal cancer, epidermoid carcinoma, skin cancer, melanoma, and oral cancer, currently use cisplatin, carboplatin, or oxaliplatin in combination with other drugs in clinical practice. In treating the aforementioned cancers, the combination of the trabectedine polypeptide conjugate, its pharmaceutically acceptable salt, and its pharmaceutically acceptable carrier provided by this invention with the aforementioned drugs can significantly improve the therapeutic effect. Furthermore, the trabectedine polypeptide conjugate provided by this invention has low toxicity and strong tumor targeting, thereby further enhancing the therapeutic effect.

[0139] In summary, the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A trabectedin polypeptide conjugate, wherein the structural formula of the polypeptide conjugate is as follows: E-nPEG-L1-L2-L3-L4-L5-D in, E represents a maleimide group; nPEG stands for n-polyethylene glycol, where n is an integer greater than or equal to 2 and less than or equal to 40. L1 is either glycine or alanine; L2 is either glycine or alanine; L3 is either alanine or asparagine; L4 is glycine, leucine, or aminobenzyl alcohol (PAB); L5 is an ethylenediamine derivative with the following structure: Where R1 and R2 are hydrogen or methyl, and n1 and n2 are both integers greater than or equal to 0 and less than or equal to 20; D is trabectedine, and its structure is as follows: ； The conjugate can be specifically cleaved by the Legumain enzyme, releasing trabectedine at specific sites in the tumor microenvironment.

2. The polypeptide conjugate according to claim 1, characterized in that, The structure of the E group is as follows: , where n is an integer greater than or equal to 1 and less than or equal to 18.

3. The polypeptide conjugate according to claim 1, characterized in that, The polypeptide conjugate is selected from any of the following structures: （X1）、 （X2）、 （X3）。 4. A pharmaceutical composition, characterized in that, It comprises the polypeptide conjugate according to any one of claims 1-3 and a pharmaceutically acceptable carrier.

5. Use of the polypeptide conjugate according to any one of claims 1-3 or the pharmaceutical composition according to claim 4 in the preparation of a Legumain-overexpressing tumor therapeutic agent, wherein the Legumain-overexpressing tumor is selected from: sarcoma, ovarian cancer, liver cancer, pancreatic cancer, gastric cancer, breast cancer, or lung cancer.

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