Polypeptide conjugate, pharmaceutical composition containing polypeptide conjugate and application of polypeptide conjugate

Abstract

The invention discloses a polypeptide conjugate with a structure as shown in a formula (I): polypeptide-linker-compound formula (I), the polypeptide is mesenchymal-epithelial transition factor targeting peptide, and the compound is a taxane compound or an enantiomer, a diastereoisomer, a solvate or pharmaceutically acceptable salt thereof. Methods of making the polypeptide conjugates; a pharmaceutical composition comprising the polypeptide conjugate; the use of the polypeptide conjugate in the preparation of a medicament for the prevention or treatment of diseases; and a method for preventing or treating a disease using the polypeptide conjugate.

Description

Polypeptide conjugate, pharmaceutical composition containing the same and use thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to application PCT / CN2023 / 102220 filed on June 25, 2023. The prior application is deemed to be part of the disclosure of this application and is incorporated into this application in its entirety. Technical Field

[0003] The present application relates to a polypeptide conjugate, a pharmaceutical composition comprising the polypeptide conjugate, and uses thereof. Background Art

[0004] At the beginning of 2021, the World Health Organization's International Agency for Research on Cancer released the latest global cancer data for 2020. The data showed that China ranked first in the world in the number of new cancer cases and cancer deaths.

[0005] Currently, the main methods for treating cancer include surgery, chemotherapy, and radiotherapy. In chemotherapy, taxanes such as docetaxel and paclitaxel are classic microtubule protease inhibitors that inhibit tumor cell mitosis and trigger apoptosis by inhibiting microtubule depolymerization. Paclitaxel is considered one of the most widely used natural anticancer drugs and has been widely used in the treatment of diseases such as breast cancer, colorectal cancer, bladder squamous cell carcinoma, head and neck cancer, small cell and non-small cell lung cancer. Docetaxel has a broad antitumor spectrum and can be used to treat diseases such as advanced breast cancer, ovarian cancer, non-small cell lung cancer, head and neck cancer, pancreatic cancer, small cell lung cancer, gastric cancer, melanoma, and soft tissue sarcomas.

[0006] However, taxanes such as paclitaxel and docetaxel are known to significantly damage normal cells. With their widespread clinical use, the incidence of adverse reactions has increased significantly, dosages have been severely limited, and therapeutic efficacy has been less than satisfactory. Furthermore, taxanes such as paclitaxel and docetaxel are virtually insoluble in water, limiting their clinical application.

[0007] Therefore, it is of great significance to improve the efficacy of taxane anti-tumor drugs and reduce their toxic side effects.

[0008] Summary of the Invention

[0009] This application provides a polypeptide conjugate that covalently links a specific peptide sequence to a compound via a linker, enabling selective delivery of the compound to target cells, thereby enhancing the therapeutic efficacy of the cytotoxic compound and reducing systemic toxicity. Furthermore, through conjugation with the polypeptide of this application, the polypeptide conjugate and pharmaceutical composition of this application exhibit improved water solubility, thereby increasing the water solubility of taxane drugs such as paclitaxel and docetaxel.

[0010] In the first aspect, the present application provides a polypeptide conjugate having a structure of formula (I):

[0011] Polypeptide-linker-compound formula (I)

[0012] in,

[0013] The polypeptide is a mesenchymal-epithelial transition factor targeting peptide,

[0014] The compound is a taxane compound or an enantiomer, diastereomer, solvate or pharmaceutically acceptable salt thereof.

[0015] Mesenchymal-epithelial transition factor (C-Met) is a tyrosine kinase receptor activated by hepatocyte growth factor that regulates a variety of biological processes. C-Met targeting peptides can specifically bind to C-Met-positive tumor cells in various cancers, including gastric, liver, lung, and colorectal cancers. Therefore, taxanes conjugated to C-Met targeting peptides possess excellent targeting properties, potentially improving the efficacy of taxanes, reducing their dosage, and minimizing their toxic side effects, leading to more effective tumor treatment.

[0016] In a second aspect, the present application provides a pharmaceutical composition comprising the polypeptide conjugate as described herein and a pharmaceutically acceptable carrier.

[0017] In a third aspect, the present application provides a use of a polypeptide conjugate as described herein or a pharmaceutical composition as described herein in the preparation of a medicament for preventing or treating a disease, optionally, the disease comprises a tumor, more optionally, the tumor comprises one or more of thyroid cancer, esophageal cancer, lung cancer, non-small cell lung cancer, gastric cancer, liver cancer, bile duct cancer, kidney cancer, pancreatic cancer, intestinal cancer, colorectal cancer, bladder cancer, prostate cancer, breast cancer, uterine cancer, endometrial cancer, ovarian cancer, cervical cancer, fallopian tube cancer, skin cancer, brain tumor, glioma, astrocytoblastoma, melanoma, osteosarcoma, sarcoma, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia or chronic myeloid leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The following provides a brief description of the drawings, which are provided to illustrate exemplary embodiments disclosed herein and not to limit these embodiments.

[0019] Figure 1A shows the lateral migration diagram of tumor cells in Example 3, specifically showing the lateral migration diagrams of tumor cells in the blank group, positive control group and drug-treated group at 0h and 48h; Figure 1B shows the statistical diagram of the lateral migration rate of tumor cells in Example 3, specifically showing the statistical diagram of the lateral migration rate of tumor cells in the blank group, positive control group and drug-treated group.

[0020] Figure 2A shows the longitudinal migration diagram of tumor cells in Example 4, specifically showing the longitudinal migration diagram of tumor cells in the blank group, positive control group and drug-treated group at 48 hours; Figure 2B shows the statistical diagram of the longitudinal migration rate of tumor cells in Example 4, specifically showing the statistical diagram of the longitudinal migration rate of tumor cells in the blank group, positive control group and drug-treated group.

[0021] Figure 3A shows a visual diagram of the changes in tumor volume in nude mice inoculated with non-small cell lung cancer A549 cells after treatment with normal saline, DOC, and different concentrations of DOC-AI in Example 5; Figure 3B shows a curve diagram of the changes in tumor volume in nude mice inoculated with non-small cell lung cancer A549 cells after treatment with normal saline, DOC, and different concentrations of DOC-AI in Example 5.

[0022] FIG4 shows a curve diagram of the weight change of nude mice inoculated with non-small cell lung cancer A549 cells in Example 5 after being treated with normal saline, DOC, and different concentrations of DOC-AI.

[0023] FIG5 shows a statistical graph of tumor weights in nude mice inoculated with non-small cell lung cancer A549 cells in Example 5 after treatment with normal saline, DOC, and different concentrations of DOC-AI.

[0024] FIG6 shows the changes in tumor volume of nude mice inoculated with gastric cancer cells MKN-45 in Example 5 after being treated with normal saline, PTX, and different concentrations of PTX-AI.

[0025] FIG. 7 shows the hepatic microsomal metabolism curve of DOC-AI in Example 6. FIG.

[0026] FIG8 shows the hepatic microsomal metabolism curve of PTX-AI in Example 6.

[0027] Figure 9A shows a statistical graph of the tumor inhibition rate of nude mice inoculated with gastric cancer cells MKN-45 after treatment with DOC and DOC-AI in Example 5; Figure 9B shows a curve graph of the change in tumor volume of nude mice inoculated with gastric cancer cells MKN-45 after treatment with normal saline, DOC and DOC-AI in Example 5; Figure 9C shows a curve graph of the change in weight of nude mice inoculated with gastric cancer cells MKN-45 after treatment with normal saline, DOC and DOC-AI in Example 5.

[0028] Figure 10A shows the concentration-peak area standard curve of the DOC standard solution in Example 7; Figure 10B shows the concentration-peak area standard curve of the DOC-AI standard solution in Example 7; Figure 10C shows the concentration-peak area standard curve of the PTX standard solution in Example 7; Figure 10D shows the concentration-peak area standard curve of the PTX-AI standard solution in Example 7. DETAILED DESCRIPTION

[0029] Unless otherwise indicated, all numbers used in this specification and claims to indicate content, concentration, ratio, weight, particle size, percentage, technical effect, etc. should be understood as being modified by the term "about" or "approximately" in any case. Therefore, unless otherwise indicated, the numerical parameters listed in the following specification and the appended claims are approximate values.

[0030] Unless otherwise stated, the terms used herein have the usual understanding meaning to those skilled in the art. For those skilled in the art, it can vary according to the desired properties and effects sought to be obtained by the application, and each numerical parameter should be interpreted according to the number of significant digits and conventional rounding methods or the manner understood by those skilled in the art. In general, the nomenclature used herein and the experimental procedures of organic chemistry, medicinal chemistry, biology described herein are well known in the art and are generally adopted in the art. Unless otherwise defined, all technical and scientific terms used herein generally have the same meanings that are commonly understood by those of ordinary skill in the art of the application. Where there are multiple definitions for the terms used herein, unless otherwise stated, the definitions in this section shall prevail.

[0031] As used herein, the expression "A and / or B" includes three cases: (1) A; (2) B; and (3) A and B. The expression "A, B, and / or C" includes seven cases: (1) A;

[0032] (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B, and C. The meanings of similar expressions can be deduced analogously.

[0033] Peptide conjugates

[0034] In the first aspect, the present application provides a polypeptide conjugate having a structure of formula (I):

[0035] Polypeptide-linker-compound formula (I)

[0036] in,

[0037] The polypeptide is a mesenchymal-epithelial transition factor targeting peptide or a derivative thereof,

[0038] The compound is a taxane compound or an enantiomer, diastereomer, solvate or pharmaceutically acceptable salt thereof.

[0039] As used herein, the term "mesenchymal-epithelial transition factor" is a tyrosine kinase receptor activated by hepatocyte growth factor that regulates a variety of biological processes and can be represented by c-Met.

[0040] As used herein, unless otherwise indicated or contradicted by context, the term "polypeptide" or "peptide" or "targeting peptide" encompasses the referenced peptide itself as well as pharmaceutically acceptable salts, prodrugs, and metabolites thereof.

[0041] As used herein, the term "pharmaceutically acceptable salts" includes acid addition salts and base addition salts. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include, but are not limited to, acetate, adipate, aspartate, benzoate, benzenesulfonate, bicarbonate / carbonate, bisulfate / sulfate, borate, camphorsulfonate, citrate, cyclamates, edisylate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, 2-(4-hydroxybenzyl)benzoate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, 2-hydroxyethanesulfonate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthoate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, hexadecanoate, phosphate / hydrogenphosphate / dihydrogenphosphate, pyroglutamate, glucarate, stearate, salicylate, tannate, tartrate, toluenesulfonate, and trifluoroacetate. Suitable base addition salts are formed with bases which form non-toxic salts. Examples include, but are not limited to, aluminum, arginine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc salts. Hemi-salts of acids and bases, such as hemisulfate and hemicalcium salts, can also be formed. For a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection and Use by Stahl and Wermuth (Wiley-VCH, 2002).

[0042] In some embodiments, the term "prodrug" refers to all molecules that are converted into the peptide of formula I or its derivatives through oxidation, reduction, hydrolysis and the like reactions under physiological conditions in vivo, such as by enzymes, gastric acid and the like.

[0043] As used herein, the term "metabolites" refers to all molecules in a cell or organism (preferably a human) that are derived from a peptide of formula I or a derivative thereof.

[0044] As used herein, the term "enantiomer" refers to two compounds having the same planar structure and being non-superimposable stereoisomers that are mirror images of each other in terms of steric structure.

[0045] As used herein, the term "diastereomers" refers to two compounds that have identical planar structures but have multiple asymmetric atoms that differ in configuration, and the two compounds are not in a relationship of mirror images.

[0046] As used herein, the term "solvate" refers to an association of one or more solvent molecules with a compound of the present application or a salt thereof. Solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, ethyl acetate, or acetic acid.

[0047] In some embodiments, the mesenchymal-epithelial transition factor targeting peptide has an amino acid sequence shown in SEQ ID NO: 1.

[0048] As used herein, the term "amino acid" encompasses both L- and D-amino acids.

[0049] In some embodiments, the derivative comprises any one or more modifications selected from the group consisting of amino acid modification, conservative amino acid substitution, or substitution of hydrogen in an amino acid residue compared to the mesenchymal-epithelial transition factor targeting peptide.

[0050] As used herein, the term "plurality" means two or more than two.

[0051] As used herein, a "derivative" of a polypeptide refers to a product obtained by performing amino acid modification, conservative amino acid substitution and / or substitution of hydrogen in amino acid residues on the basis of the mesenchymal-epithelial transition factor targeting peptide.

[0052] As used herein, the term "amino acid modification" includes, but is not limited to, N-terminal modification, C-terminal modification, and side chain modification. Modifications include, but are not limited to, hydroxylation, carboxylation, alkylation, acylation, phosphorylation, sulfonation, amidation, aldehyde formation, alcoholization, mercaptoethylation, esterification, and glycosylation.

[0053] As used herein, the term "conservative amino acid substitution" refers to replacing one amino acid with another amino acid having similar structural and / or chemical properties, such as replacing leucine with isoleucine or valine, replacing aspartic acid with glutamic acid, or replacing threonine with serine. Thus, a "conservative amino acid substitution" means replacing an amino acid that is not important for the activity of the peptide, or replacing one amino acid with another amino acid having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that even replacing an important amino acid does not reduce the activity of the peptide. Conservative amino acid substitutions that provide functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0054] When used in this article with respect to polypeptides, "amino acid" and "amino acid residue" have the same meaning, which means that when amino acids are connected by chemical bonds, part of their groups are lost due to their participation in the formation of the connecting bonds, and the remaining amino acid part is the amino acid residue.

[0055] In some embodiments, the conservative amino acid substitutions include, for example, S in the amino acid sequence as shown in RSPYANS is replaced by A or T, Y in the amino acid sequence as shown in RSPYANS is replaced by F or W, and A in the amino acid sequence as shown in RSPYANS is replaced by S or T.

[0056] In some embodiments, the hydrogen in the amino acid residue is replaced by any conventional substituent known in the art, including but not limited to alkyl, alkenyl, alkynyl, aromatic, alkoxy, carboxyl, aldehyde, carbonyl, hydroxyl, halogen, cyano, acyl, sulfonic acid, amino, thiol or nitro groups.

[0057] In some embodiments, the taxane compound is one or more of a bicyclic taxane compound, a tricyclic taxane compound, a tetracyclic taxane compound, or a pentacyclic taxane compound.

[0058] As used herein, the term "taxane compound" mainly includes compounds having a taxane skeleton structure.

[0059] As used herein, the term "m-ring taxane compound" refers to a taxane compound having a skeleton of m fused rings, for example, a bicyclic taxane compound refers to a taxane compound having a skeleton of two fused rings. The meanings of similar expressions can be deduced analogously.

[0060] As used herein, the term "skeleton" of a compound refers to the core structure of the compound, and multiple substituents may be substituted on the skeleton to form different compounds. Specifically in this application, different taxane compounds may be obtained.

[0061] As used herein, the term "fused" means that two rings share two or more atoms (including carbon atoms or heteroatoms).

[0062] As used herein, the term "ring" includes saturated rings and unsaturated rings.

[0063] In some embodiments, the bicyclic taxane compounds include 6-membered ring-fused and 12-membered ring taxane compounds. In some embodiments, the skeletons of the 6-membered ring-fused and 12-membered ring taxane compounds have the structure of Formula A. In some embodiments, the 6-membered ring-fused and 12-membered ring taxane compounds can be substituted with various substituents based on the skeleton of Formula A to form different types of 6-membered ring-fused and 12-membered ring taxane compounds.

[0064] In some embodiments, the tricyclic taxane compound comprises one or more of a 6-membered ring fused with an 8-membered ring fused with a 6-membered ring, a 6-membered ring fused with a 10-membered ring fused with a 6-membered ring, a 5-membered ring fused with a 7-membered ring fused with a 6-membered ring, or a 5-membered ring fused with a 6-membered ring fused with a 6-membered ring. In some embodiments, the backbone of the 6-membered ring fused with an 8-membered ring fused with a 6-membered ring taxane compound has a structure of Formula B. In some embodiments, the backbone of the 6-membered ring fused with an 10-membered ring fused with a 6-membered ring taxane compound has a structure of Formula C. In some embodiments, the backbone of the 5-membered ring fused with an 7-membered ring fused with a 6-membered ring taxane compound has a structure of Formula D. In some embodiments, the backbone of the 5-membered ring fused with an 6-membered ring fused with a 6-membered ring taxane compound has a structure of Formula E. In some embodiments, the taxane compound can be substituted with various substituents based on the backbone of Formula BE to form different types of tricyclic taxane compounds.

[0065] In some embodiments, the tetracyclic taxane compound comprises one or more of a 6-membered ring-fused, a 5-membered ring-fused, a 5-membered ring-fused, and a 6-membered ring, or a 6-membered ring-fused, an 8-membered ring-fused, a 6-membered ring-fused, and a 6-membered ring taxane compound. In some embodiments, the backbone of the 6-membered ring-fused, 5-membered ring-fused, and 5-membered ring-fused, and 6-membered ring taxane compound has a structure of Formula F. In some embodiments, the backbone of the 6-membered ring-fused, 8-membered ring-fused, and 6-membered ring taxane compound has a structure of Formula G. In some embodiments, the taxane compound can be substituted with various substituents based on the backbone of Formula F or G to form different types of tetracyclic taxane compounds.

[0066] In some embodiments, the pentacyclic taxane compounds include 6-membered ring-fused, 5-membered ring-fused, 5-membered ring-fused, 5-membered ring-fused, and 6-membered ring taxane compounds. In some embodiments, the backbone of the 6-membered ring-fused, 5-membered ring-fused, 5-membered ring-fused, 5-membered ring-fused, and 6-membered ring taxane compounds has a structure of Formula H. In some embodiments, the taxane compounds can be substituted with various substituents on the basis of the backbone of Formula H to form different types of pentacyclic taxane compounds.

[0067] In some embodiments, the taxane compound comprises any of the following structures:

[0068] For more information on taxane compounds, see Fu Yan et al. Research Progress of Taxane Compounds [J]. Natural Product Research and Development, 2004. Vol. 16, No. 3, P258-264; Huo Changhong et al. Nuclear Magnetic Resonance Proton Spectrum Characteristics of Natural Taxane Compounds [J]. Journal of Spectroscopy, 2007. Vol. 24, No. 1, P101-118; Wang Chunlin et al. Research Progress of Taxane Compounds [J]. Hebei Medicine, 2005. In some embodiments, taxane compounds known in the art can be incorporated into the present application to become the taxane compounds described herein.

[0069] In some embodiments, the taxane compound comprises one or more of paclitaxel, docetaxel, cabazitaxel, larotaxel, or tesetaxel. Alternatively, the taxane compound comprises one or more of paclitaxel or docetaxel. In some embodiments, the taxane compound is paclitaxel. In some embodiments, the taxane compound is docetaxel.

[0070] Paclitaxel, docetaxel, cabazitaxel, larotaxel and tesetaxel all have a skeleton structure of a 6-membered ring fused together with an 8-membered ring and a 6-membered ring. Compounds with the same skeleton have similar biological activities.

[0071] In some embodiments, the linker is CO(O)C(CH2) n C(O)OH, n is an integer from 2 to 6. In some embodiments, the linker is CO(O)C(CH2) n C(O)OH, n is selected from 2, 3, 4, 5 or 6.

[0072] In some embodiments, the linker is succinic acid, glutaric acid, adipic acid, pimelic acid, or suberic acid.

[0073] As used herein, the term "linker" refers to a structure connecting the polypeptide and the taxane compound.

[0074] As used herein, the term "succinic acid" refers to a compound having the chemical structure HOC(O)CH2CH2C(O)OH.

[0075] In some embodiments, the linker is linked to the polypeptide via an amide bond. In specific embodiments, the carboxyl group of the linker forms an amide bond with an amino group of the polypeptide, and the linker is linked to the polypeptide via an amide bond. In some embodiments, the carboxyl group of the linker forms an amide bond with an amino group of the N-terminal amino acid in the polypeptide, where the amino group of the N-terminal amino acid is not a side chain amino group. In some embodiments, the carboxyl group of the linker forms an amide bond with a non-side chain amino group of arginine in the polypeptide.

[0076] In some embodiments, the linker is linked to the taxane compound via an ester bond. In specific embodiments, the carboxyl group of the linker forms an ester bond with the hydroxyl group of the taxane compound, and the linker is linked to the taxane compound via an ester bond. In some embodiments, the carboxyl group of the linker forms an ester bond with the hydroxyl group at the 2' position of the taxane compound.

[0077] In some embodiments, the polypeptide conjugate has a structure of the following formula (II), (III) or (IV):

[0078] The polypeptide has an amino acid sequence shown in SEQ ID NO: 1 or a derivative thereof, and the carboxyl group of the linker is connected to the amino group of the nitrogen-terminal amino acid of the polypeptide by forming an amide bond.

[0079] Preparation method of polypeptide conjugate

[0080] According to one embodiment of the present application, the present application provides a method for preparing the polypeptide conjugate as described in the present application, the method comprising:

[0081] (1) providing the polypeptide;

[0082] (2) connecting the taxane compound and the linker to obtain the linker-taxane compound;

[0083] (3) Mixing the polypeptide and the linker-taxane compound to carry out a liquid phase reaction to obtain the polypeptide conjugate.

[0084] In some embodiments, in step (1), the polypeptide described in this application can be synthesized according to any method known in the art, for example, the polypeptide can be synthesized by solid phase reaction or by liquid phase reaction.

[0085] In some embodiments, the solid phase reaction is an Fmoc solid phase peptide reaction. In some embodiments, the Fmoc solid phase peptide reaction is an existing technology for peptide synthesis in the art, and those skilled in the art can perform synthesis according to actual conditions.

[0086] In some embodiments, the carbon-terminal amino acid, such as Fmoc-carbon-terminal amino acid-OH, is coupled to Wang resin according to the amino acid sequence of the polypeptide. After Fmoc removal, the remaining amino acids are coupled sequentially according to the amino acid sequence of the polypeptide. The Fmoc residue is removed after each coupling of a new amino acid to obtain a polypeptide resin. In some embodiments, the remaining amino acids are added in the form of Fmoc-amino acid-OH.

[0087] In some embodiments, the side chains of the amino acids may be optionally protected with protecting groups. Specifically, amino acid side chain protecting groups include tert-butyl (t-Bu), trityl (Trt), or Pbf. In some embodiments, serine (Ser), tyrosine (Tyr), and threonine (Thr) may be protected with t-Bu. In some embodiments, asparagine (Asn) and glutamine (Gln) may be protected with Trt. In some embodiments, arginine (Arg) may be protected with Pbf.

[0088] In some embodiments, a cleavage agent is added to the polypeptide resin to obtain the polypeptide. In some embodiments, the cleavage agent includes one or both of trifluoroacetic acid (TFA) and triisopropylsilane (Tis).

[0089] In some embodiments, in step (2), the reaction temperature of the taxane compound and the linker is room temperature, and the reaction time is ≥3 h.

[0090] As used herein, the term "room temperature" refers to normal temperature or indoor temperature, for example, the room temperature may be 25°C±5°C, 23°C±2°C or 20°C±5°C.

[0091] In some embodiments, in step (3), the temperature for reacting the linker-taxane compound with the polypeptide is room temperature, and the reaction time is 3 h to 8 h or 4 h to 6 h.

[0092] In some embodiments, the obtained polypeptide conjugate can be verified by HPLC, thin layer chromatography, MR, melting point, mass spectrometry. In some embodiments, the obtained polypeptide conjugate can be verified by mass spectrometry and SDS-polyacrylamide gel electrophoresis.

[0093] Pharmaceutical composition

[0094] In a second aspect, the present application provides a pharmaceutical composition comprising the polypeptide conjugate as described herein and a pharmaceutically acceptable carrier.

[0095] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable raw material, component, or vehicle that imparts the form or consistency of the pharmaceutical composition. When combined, each carrier is compatible with the other components of the pharmaceutical composition, thereby avoiding significant reduction in the efficacy of the polypeptide conjugate of the present application upon administration to a subject and avoiding interactions with the drug components that would render the composition pharmaceutically unacceptable.

[0096] In some embodiments, the pharmaceutically acceptable carrier may include, but is not limited to, diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, taste-masking agents, colorants, anti-caking agents, humectants, chelating agents, plasticizers, viscosity-increasing agents, antioxidants, preservatives, stabilizers, surfactants, or buffers, etc. Those skilled in the art can selectively use the above-mentioned pharmaceutically acceptable carriers according to the dosage form of the pharmaceutical composition.

[0097] In some embodiments, the polypeptide conjugates of the present application and one or more pharmaceutically acceptable carriers are formulated into a dosage form suitable for administration to a subject via a desired route of administration. In some embodiments, the dosage form of the pharmaceutical composition includes, but is not limited to, tablets, capsules, caplets, pills, lozenges, powders, syrups, brews, suspensions, solutions, emulsions, transdermal patches, suppositories, inhalants, creams, ointments, lotions, pastes, sprays, injections, or gels.

[0098] use

[0099] In a third aspect, the present application provides use of the polypeptide conjugate as described herein or the pharmaceutical composition as described herein in the preparation of a medicament for preventing or treating a disease.

[0100] As used herein, the term "disease" refers to any change in the state of the body or some organ that interrupts or interferes with the performance of functions and / or causes symptoms (such as discomfort, dysfunction, adverse stress or even death) in the person suffering from the disease or those who come into contact with it.

[0101] As used herein, the term "treat," ...

[0102] In some embodiments, the disease is a tumor.

[0103] In some embodiments, the tumor comprises a solid tumor and / or a hematological tumor.

[0104] In some embodiments, the tumor comprises one or more of thyroid cancer, esophageal cancer, lung cancer, non-small cell lung cancer, gastric cancer, liver cancer, bile duct cancer, kidney cancer, pancreatic cancer, intestinal cancer, colorectal cancer, bladder cancer, prostate cancer, breast cancer, uterine cancer, endometrial cancer, ovarian cancer, cervical cancer, fallopian tube cancer, skin cancer, brain tumor, glioma, glioblastoma, melanoma, osteosarcoma, sarcoma, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia or chronic myeloid leukemia.

[0105] In some embodiments, the tumor comprises one or more of lung cancer, gastric cancer, liver cancer, ovarian cancer, prostate cancer, breast cancer, kidney cancer, uterine cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, bile duct cancer, skin cancer, lymphoma, multiple myeloma, glioma, melanoma, endometrial cancer, osteosarcoma, cervical cancer, bladder cancer, or leukemia.

[0106] In some embodiments, the tumor comprises one or more of non-small cell lung cancer, giant cell lung cancer, gastric cancer, liver cancer, ovarian cancer, esophageal cancer, colon cancer, breast cancer, bile duct cancer, osteosarcoma, cervical cancer, melanoma, bladder transitional cell carcinoma, pancreatic cancer, glioblastoma, chronic myeloid leukemia cells, or lymphoma.

[0107] Treatment

[0108] According to one embodiment of the present application, the present application provides a method for preventing or treating a disease, comprising administering the polypeptide conjugate as described herein or the pharmaceutical composition as described herein to a subject in need thereof.

[0109] As used herein, the term "subject" refers to primates (e.g., humans), dogs, rabbits, guinea pigs, pigs, rats, and mice. In some embodiments, the subject is a primate. In some specific embodiments, the subject is a human.

[0110] As used herein, a subject is "in need of" a treatment if the subject would benefit biologically, medically, or in quality of life from such treatment.

[0111] As used herein, the term "therapeutically effective dose" refers to an amount that results in a benefit or treatment of a disease compared to a corresponding subject not receiving that amount, but is sufficiently low within the scope of sound medical judgment to avoid serious side effects. The therapeutically effective dose of a polypeptide conjugate will vary with the specific polypeptide conjugate selected (e.g., taking into account the potency, efficacy, and half-life of the polypeptide conjugate); the route of administration selected; the disease being treated; the severity of the disease being treated; the age, size, weight, and physical condition of the patient being treated; the medical history of the patient being treated; the duration of treatment; the nature of concurrent treatment; the desired therapeutic effect, and the like, but can still be determined in a routine manner by those skilled in the art.

[0112] In some embodiments, the disease is a tumor.

[0113] In some embodiments, the tumor comprises a solid tumor and / or a hematological tumor.

[0114] In some embodiments, the tumor comprises one or more of thyroid cancer, esophageal cancer, lung cancer, non-small cell lung cancer, gastric cancer, liver cancer, bile duct cancer, kidney cancer, pancreatic cancer, intestinal cancer, colorectal cancer, bladder cancer, prostate cancer, breast cancer, uterine cancer, endometrial cancer, ovarian cancer, cervical cancer, fallopian tube cancer, skin cancer, brain tumor, glioma, glioblastoma, melanoma, osteosarcoma, sarcoma, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia or chronic myeloid leukemia.

[0115] In some embodiments, the tumor comprises one or more of lung cancer, gastric cancer, liver cancer, ovarian cancer, prostate cancer, breast cancer, kidney cancer, uterine cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, bile duct cancer, skin cancer, lymphoma, multiple myeloma, glioma, melanoma, endometrial cancer, osteosarcoma, cervical cancer, bladder cancer, or leukemia.

[0116] In some embodiments, the tumor comprises one or more of non-small cell lung cancer, giant cell lung cancer, gastric cancer, liver cancer, ovarian cancer, esophageal cancer, colon cancer, breast cancer, bile duct cancer, osteosarcoma, cervical cancer, melanoma, bladder transitional cell carcinoma, pancreatic cancer, glioblastoma, chronic myeloid leukemia cells, or lymphoma.

[0117] The various embodiments described above for the polypeptide conjugates, polypeptides, taxane compounds, and linkers of the present application are also applicable to the pharmaceutical compositions, uses, and methods of the present application (as long as they are not inherently inconsistent with each other), and the various embodiments formed by such combination are considered part of the present application.

[0118] The technical solutions of the present application will be more clearly and specifically described below in conjunction with embodiments in an illustrative manner. It should be understood that these embodiments are for illustrative purposes only and are in no way intended to limit the scope of protection of the present application. The scope of protection of the present application is defined solely by the claims. Therefore, those skilled in the art will recognize that various changes and modifications may be made to the embodiments described herein without departing from the scope of the present application. Similarly, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.

[0119] Example

[0120] Unless otherwise stated, all reagents and instruments used in the following examples are commercially available conventional products. Unless otherwise stated, experiments were performed under conventional conditions or conditions recommended by the manufacturer.

[0121] Example 1: Preparation of Peptide-Conjugated Paclitaxel (PTX-AI)

[0122] 1. Preparation of P1-AI Peptide

[0123] Weigh 10g of Wang resin (CAS: 1365700-43-1) with a degree of substitution of 0.91mmol / g, add it to a peptide solid phase reactor (glass apparatus, custom-made by the manufacturer), and wash it twice with DMF. After swelling the Wang resin with DMF for 60 minutes, take 12.71g of Fmoc-Ser(tBu)-OH, 1.35g of HOBt, 1.73g of DIC, and 0.11g of DMAP, use 60mL of DMF as solvent, add it to the solid phase reactor, and react at room temperature for 3 hours. After the reaction is completed, wash it with DMF 3 times and DCM 3 times;

[0124] Add a capping solution (9.29 g acetic anhydride, 3.53 g DIEA, and 0.22 g DMAP dissolved in 60 mL DMF) and cap for 1.5 h. After capping, wash with DMF three times, DCM three times, and methanol three times, shrink and drain to obtain Fmoc-Ser(tBu)-Wang resin with a substitution value of 0.68 mmol / g.

[0125] Weigh 2 g (1.36 mmol) of Fmoc-Ser(tBu)-Wang resin with a substitution value of 0.68 mmol / g into a solid-phase reactor and wash twice with DMF. Swell the resin with DMF for 30 minutes, drain, and then wash four more times with DMF. Remove the Fmoc residue twice by adding 20% ​​piperidine / DMF (100 mL / 400 mL). The removal times are 3 and 7 minutes, respectively. After the reaction, wash the resin six times with DMF.

[0126] Weigh Fmoc-Asn(Trt)-OH (3 eq) and HOBt (3.6 eq) and dissolve them in DMF. Add DIC (3.6 eq) and activate for 4 min in an ice bath at 0°C. After activation, add the mixture to a solid phase reactor and react for 1 h at 25°C under nitrogen protection. Use the ninhydrin method (ninhydrin:phenol:piperidine = 2:1:1) to detect amino groups. If the resin is colorless and transparent, the reaction is complete. If the resin shows color, the reaction is incomplete and the coupling reaction time needs to be extended, for example, 0.5-1 h, until the resin is colorless and transparent using the ninhydrin method. Drain the DMF and wash four times. Add 20% piperidine / DMF (100 mL / 400 mL) to remove Fmoc twice, with the removal times of 3 min and 7 min, respectively. After the reaction, wash six times with DMF.

[0127] Repeat the above steps and add each amino acid in sequence according to the polypeptide sequence. According to the amino acid sequence of SEQ ID NO: 1, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Arg(pbf)-OH were coupled in sequence. HOBt / DIC was used as a condensing agent for the coupling of amino acids, and each reaction was 0.5 h. After each coupling, the resin was colorless and transparent using the ninhydrin method, and the Fmoc protecting group was then removed using a 20% piperidine / DMF solution (100 mL / 400 mL). The resulting product was washed with DMF 4 times, DCM 3 times, and MeOH 3 times, and dried to obtain the P1-AI peptide resin.

[0128] The P1-AI peptide resin was placed in a cleavage reactor. The cleavage reagent was added at a ratio of 10 mL / g resin, with a ratio of TFA: TiS: water = 95:2.5:2.5 (v / v / v). After stirring at room temperature for 2.0 h, the product was filtered using a sand filter, the filtrate collected, and then washed three times with a small amount of TFA. The combined filtrates were concentrated under reduced pressure. The concentrate was added to cooled methyl tert-butyl ether to allow the solid to precipitate. The solid was then centrifuged, washed again with methyl tert-butyl ether, and centrifuged four times. The crude linear P1-AI peptide was obtained by vacuum drying with a purity of 60.8% and a yield of 37.21%. The product was purified by HPLC using a reversed-phase C18 column with a detection wavelength of 220 nm. Mobile phases A and B were 0.1% (v / v) TFA in water and acetonitrile. The eluate from the target peak was collected, concentrated by rotary evaporation, and lyophilized to obtain the P1-AI peptide with a purity of >99%.

[0129] 2. Preparation of Linker-Paclitaxel

[0130] 1.20 g of paclitaxel (PTX, Anhui Zesheng Technology Co., Ltd.) and 0.21 g of succinic anhydride pyrimidine (Anhui Zesheng Technology Co., Ltd.) were reacted at room temperature for 3 hours to obtain succinyl-paclitaxel.

[0131] 3. Peptide conjugates

[0132] 0.88 g of succinyl-paclitaxel was reacted with 0.53 g of EDC (Anhui Zesheng Technology Co., Ltd.) and 0.21 g of NHS (Shanghai Aladdin Biochemical Technology Co., Ltd.) dissolved in DCM at room temperature for 9 h to produce succinyl-paclitaxel-NHS activated ester. 0.51 g of succinyl-paclitaxel-NHS activated ester and 0.58 g of P1-AI polypeptide were dissolved in a solvent (water:nitromethylpyrrolidone = 1:2.5, v:v) and triethylamine was added to adjust the pH to a weak base. The reaction was continued at room temperature for 4 h to produce 0.32 g of PTX-AI, which was then lyophilized. The purity of the PTX-AI was 96.7% and the yield was 37.21%. The extent of the coupling reaction was monitored by HPLC.

[0133] 4. Preparation of other peptide conjugates

[0134] The above method was used to obtain peptide-conjugated docetaxel (DOC-AI), the only difference being that paclitaxel was replaced by docetaxel (Anhui Zesheng Technology Co., Ltd.).

[0135] The peptide PTX-5-AI was obtained using the above method, except that the succinic acid in PTX-AI was replaced by glutaric acid (Anhui Zesheng Technology Co., Ltd.).

[0136] Example 2: Detection of tumor proliferation inhibition effect of polypeptide conjugates

[0137] The experiments were conducted using highly metastatic human hepatocellular carcinoma cells (HCCLM3), highly metastatic human gastric cancer cells (MKN45), human gastric cancer cells (BGC-823 and MGC-803), non-small cell lung cancer cells (A549), human pancreatic cancer cells (ASPC-1), human colon cancer cells (LoVo), human ovarian cancer cells (A2780), human cervical cancer cells (SiHa), human malignant melanoma cells (SK-mel-2 and M14), human osteosarcoma cells (Saos-2), human bladder transitional cell carcinoma cells (T24), human astrocytic glioblastoma cells (U87-MG), human lymphoma cells (Raji), and human epidermal cancer cells (A431). HCLM3, MKN45, BGC-823, MGC-803, A431, and A549 cells were provided by Nanjing Anji Biotechnology Co., Ltd., and other cells were provided by Nanjing Ouji Pharmaceutical Technology Service Co., Ltd.

[0138] The tumor cells were maintained in a culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin and incubated at 37°C, 5% CO2, and 95% humidity. The tumor cells were cultured in T75 flasks at a cell density of 80%. The tumor cell line was mycoplasma negative.

[0139] The logarithmic growth phase tumor cells were used for the experiment. The cells were digested, counted, and made into 1×10 5 Cell suspensions of 100 μL / well were inoculated into 96-well plates (100 μL / well), and the 96-well plates were placed in a 37°C, 5% CO2 incubator for 24 hours. For different tumor cells, 10 μM, 1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM of PTX-AI, or DOC-AI, or PTX-5-AI were added to each well of the drug-treated group, and 10 μM, 1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM of DOC-AI, or PTX-5-AI were added to each well of the positive control group. 1 nM, 0.1 nM, and 0.01 nM PTX or DOC were added. 100 μL of basal culture medium was added to each well of the blank group, and three replicates were set up for each group. After the plate was placed in an incubator for 72 hours, the cell morphology of each group was observed under a microscope. 10 μL of CCK8 solution (Nanjing Enjing Biotechnology Co., Ltd.) was added to each well and incubated in the cell culture incubator for another 4 hours. After the incubation, the absorbance was measured at 450 nm, and the tumor cell proliferation inhibition rate was calculated. The efficacy ratio was the IC of the free drug. 50 IC value of peptide conjugates 50 The ratio of values.

[0140] Table 1 Tumor cell proliferation inhibition rate of peptide conjugates

[0141] Table 1

[0142] The results show that: as shown in Table 1, the polypeptide conjugates DOC-AI, PTX-AI, and PXT-5-AI of the present application have good tumor proliferation inhibitory effects on various tumor cells.

[0143] Example 3: Detection of the inhibitory effect of polypeptide conjugates on tumor lateral migration

[0144] A549 cells in the logarithmic growth phase were obtained and digested with trypsin to prepare a cell suspension. 1 mL of the cell suspension was inoculated into a 24-well plate, with 3.5 × 10 cells per well. 5 cells. After incubation for 24 hours in a 37°C, 5% CO2 incubator, a 24-well plate was scratched perpendicular to the horizontal line on the bottom of the plate using a 200 μL pipette tip. Each well was then washed twice with 1 mL of PBS. Blank, positive drug, and drug-treated groups were set up. The blank group received 1 mL of F-12K medium containing 5% FBS, the positive drug group received 1 mL of 100 nM DOC, and the drug-treated groups received 1 mL of 100 nM, 10 nM, and 1 nM DOC-AI, respectively. Cell migration was imaged at 100× magnification using a microscope (model: CKX31SF, manufacturer: OLYMPUS CORPORATION) at 0 and 48 hours. The number of migrated tumor cells was quantified using ImageJ 1.74V, and the results are expressed as mean ± SD (n = 3). Statistical analysis was performed using a t-test at each time point (**** indicates a P value < 0.001 for differences between the blank group and other conditions).

[0145] The results show that, as shown in Figures 1A and B, DOC-AI effectively inhibited the lateral migration of A549 cells compared to the blank control group in a concentration-dependent manner. Furthermore, the effective concentration of DOC-AI was much lower than that of DOC. For example, the lateral migration rate of 1 nM DOC-AI was lower than that of 100 nM DOC, demonstrating that the polypeptide conjugates of the present application can significantly enhance the efficacy of the compound.

[0146] Example 4: Detection of the inhibitory effect of polypeptide conjugates on tumor longitudinal migration

[0147] A549 cells in the logarithmic growth phase were obtained and digested with trypsin to prepare a cell suspension. 100 μL of the cell suspension was inoculated into the Transwell chamber, with 3.5×10 cells per well. 4Blank, positive drug, and drug-treated groups were set up. The blank group received 100 μL of F-12K medium without FBS, the positive drug group received 100 μL of 10 nM DOC, and the drug-treated group received 100 μL of each of 1 μM, 100 nM, 10 nM, and 1 nM DOC-AI. 600 μL of F12-K medium containing 10% FBS was added to a 24-well plate containing a chamber. The cells were incubated at 37°C, 5% CO₂ in a humidified incubator for 48 hours. The cells were then fixed with methanol for 30 minutes and stained with crystal violet for 10 minutes. After staining, the cells were photographed at 100× magnification under a microscope (model: CKX31SF, manufacturer: OLYMPUS CORPORATION). The number of migrating tumor cells was quantified using Photoshop 20.0.1. Results are expressed as mean ± SD (n = 3). Statistical analysis was performed using the t-test at each time point (**** indicates a p-value < 0.001 for differences between the blank group and other conditions).

[0148] The results show that, as shown in Figures 2A and 2B, DOC-AI effectively inhibited the longitudinal migration of A549 cells compared to the blank group in a concentration-dependent manner, with the effective concentration of DOC-AI as low as 1 nM. Furthermore, the effect of DOC-AI was superior to that of DOC. For example, the longitudinal migration rate of 10 nM DOC-AI was lower than that of 10 nM DOC, demonstrating that the polypeptide conjugates of the present application can greatly enhance the efficacy of the compound.

[0149] Example 5: In vivo anti-tumor activity detection of polypeptide conjugates

[0150] Non-small cell lung cancer A549 cells and human gastric cancer cell lines MKN-45 were maintained in F12-K medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin and incubated at 37°C, 5% CO2, and 95% humidity. These cells were cultured in T175 flasks at a cell density of 80%. These cell lines were mycoplasma negative. A549 and MKN-45 cells were provided by Nanjing Anji Biotechnology Co., Ltd.

[0151] 5.1 In vivo tumor inhibitory activity assay of DOC-AI

[0152] 5.1.1 Non-small cell lung cancer A549 cells

[0153] Non-small cell lung cancer A549 cells in the vigorous growth stage were inoculated subcutaneously in the right axilla of BALB / c nude mice (Nanjing Zhonghua Biotechnology Co., Ltd.) under sterile conditions. The cell inoculation volume was 1×10 7 The diameter of the transplanted tumor in nude mice was measured with a vernier caliper. 330 nude mice with good growth and uniform tumor size were selected and divided into 5 groups of 6 mice each. The specific grouping and administration method are shown in Table 2 below. The administration frequency was 4 consecutive days followed by 3 days of interval and this cycle was repeated for 21 days. The anti-tumor effect of nude mice was dynamically observed by measuring the tumor diameter. The tumor diameter was measured every other day, and the nude mice were weighed at the same time as the tumor diameter was measured. After 21 days of administration, the nude mice were euthanized, the tumor inhibition rate (%) was calculated, and the tumor mass was removed and weighed.

[0154] Table 2 In vivo tumor inhibition activity detection of DOC-AI Note: *P<0.05, **P<0.01 vs model group, # P<0.05, ## P<0.01 vs DOC group

[0155] The results show that, as shown in Table 2, Figures 3, and 5, DOC-AI significantly inhibited the growth of non-small cell lung cancer A549 xenograft tumors in nude mice. At equivalent doses, for example, 6.20 μM DOC achieved a tumor inhibition rate of 59.55%, while DOC-AI achieved a high inhibition rate of 90.40%. This demonstrates that the peptide conjugate DOC-AI exhibits significantly superior tumor inhibition compared to the active agent DOC. Furthermore, the inhibition rate of DOC-AI increased significantly with increasing dose, demonstrating a dose-dependent pattern. Furthermore, as shown in Figure 4, no significant weight loss was observed in any of the nude mice groups during the dosing period.

[0156] 5.1.2 Gastric Cancer MKN45 Cells

[0157] MKN45 gastric cancer cells in the vigorous growth stage were obtained and inoculated subcutaneously in the right axilla of BALB / c nude mice (Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.) under sterile conditions. The cell inoculation volume was 1×10 7 The diameter of the transplanted tumor in nude mice was measured with a vernier caliper. 3 Eighteen nude mice bearing tumors with good growth and uniform tumor size were selected and divided into three groups of six mice each. The specific grouping and dosing method are shown in Table 7 below. The dosing frequency was once every three days and this cycle lasted for 21 days. The anti-tumor effect of nude mice was dynamically observed by measuring the tumor diameter. The tumor diameter was measured every other day, and the nude mice were weighed at the same time as the tumor diameter was measured. After 21 days of dosing, the nude mice were euthanized, the tumor inhibition rate (%) was calculated, and the tumor mass was removed and weighed.

[0158] Table 7 Results of in vivo tumor inhibition activity test of DOC-AI Note: *P<0.05, **P<0.01 vs. model group; # P<0.05, ##P<0.01 vs DOC group

[0159] The results show that, as shown in Table 7 and Figures 9A and B, DOC-AI significantly inhibited the growth of MKN45 gastric cancer xenografts in nude mice. At equivalent doses, for example, the tumor inhibition rate of 6.20 μmol / kg DOC was 73.76±13.15%, while that of DOC-AI was as high as 84.25±9.15%, demonstrating that the peptide conjugate DOC-AI exhibited significantly superior tumor inhibition compared to the active agent DOC. Furthermore, as shown in Figure 9C, the nude mice in the DOC-AI group showed no significant weight loss during the dosing period, while the nude mice in the active agent DOC group experienced weight loss, demonstrating the safety of DOC-AI.

[0160] 5.2 In vivo tumor inhibition activity assay of PTX-AI

[0161] Human gastric cancer cells MKN-45 at the vigorous growth stage were inoculated subcutaneously in the right axilla of BALB / c nude mice under sterile conditions. The cell inoculation volume was 5×10 6 The diameter of the transplanted tumor in nude mice was measured with a vernier caliper. 3 Sixty-eight tumor-bearing nude mice with good growth status and uniform tumor size were selected and divided into six groups, including a model group of 10 mice and the remaining groups of 8 mice each. The specific grouping and dosing methods are shown in Table 3 below. The anti-tumor effect of the test substance was dynamically observed by measuring tumor diameter. Tumor diameter was measured every other day, and the nude mice were weighed at the same time. After 21 days of dosing, the nude mice were euthanized, the tumor inhibition rate (%) was calculated, and the tumor mass was removed and weighed.

[0162] Table 3 In vivo tumor inhibition activity detection of PTX-AI Note: *P<0.05, **P<0.01 vs. model group

[0163] The results show that: as shown in Table 3 and Figure 6, the polypeptide conjugate PTX-AI has a significant inhibitory effect on the growth of human gastric cancer cell MKN45 nude mouse xenograft tumors. As the dose increases, the inhibition rate of PTX-AI increases significantly, showing a dose-dependent manner. When the dose is 7.92 μmol / Kg, the tumor inhibition rate of PTX-AI is as high as 60.8%. At the same time, it can also be seen that at the same dose, the tumor inhibition activity of the polypeptide conjugate PTX-AI is better than that of free PTX. Among them, the dosing frequency of free PTX is different from that of the polypeptide conjugate drug because free PTX has greater toxic side effects on the body. At the dosing frequency of the polypeptide conjugate, it will cause irreversible damage to rats, affecting the measurement of subsequent data. This phenomenon also proves that the polypeptide conjugate of the present application has better safety due to its targeting.

[0164] Example 6: Detection of Hepatic Microsomal Metabolism of Peptide Conjugates

[0165] Accurately weigh 1.85 mg of DOC-AI (or 1.81 mg of PTX-AI) and dissolve in 1 mL of DMSO to obtain a 1 mM stock solution. Dilute the 1 mM stock solution with methanol to a 100 μM working solution for sample incubation. 12.5 μL of 20 mg / mL liver microsomes (mouse, Shanghai Quanyang Trading Co., Ltd., batch number: MIC255038; rat, Shanghai Quanyang Trading Co., Ltd., batch number: MIC254034; dog, Shanghai Quanyang Trading Co., Ltd., batch number: MIC257015; monkey, Red Liver Disease Research (Shanghai) Co., Ltd., batch number: SYXS; human, Shanghai Quanyang Trading Co., Ltd., batch number: 1210097) were added to a 96-well plate, mixed, and preincubated at 37°C for 5 min. The reaction was initiated by adding 5 μL of the 100 μM working solution to the 96-well plate. At the designated time points, 50 μL of the reaction sample was transferred to an EP tube containing 100 μL of glacial acetonitrile, vortexed to inactivate the sample, and stored at -90°C until assayed. LC-MS / MS was used to quantitatively detect the concentrations of peptide conjugate DOC-AI and dissociated DOC.

[0166] Table 4 Results of DOC-AI liver microsomal metabolism test

[0167] Table 5 PTX-AI liver microsomal metabolism test results

[0168] The metabolic stability of the peptide conjugates DOC-AI and PTX-AI in mice, rats, dogs, monkeys, and humans was studied using an in vitro liver microsomal metabolic incubation system. Their metabolic half-lives and clearance rates in liver microsomal enzymes from different species were analyzed to inform the selection of animals for subsequent preclinical in vivo studies. The results in Tables 4-5 and Figures 7-8 show that DOC-AI is a highly cleared drug in mice and monkeys, and moderately cleared in rats, dogs, and humans. PTX-AI is a highly cleared drug in mice, dogs, and monkeys, and moderately cleared in rats and humans. Moderately cleared drugs exhibit good in vivo stability, are not rapidly degraded, and do not accumulate excessively, thus preventing excessive damage.

[0169] Example 7: Solubility determination of polypeptide conjugates

[0170] Preparation of a series of standard solutions of DOC, DOC-AI, PTX and PTX-AI:

[0171] Weigh 2 mg each of DOC, DOC-AI, PTX, and PTX-AI into a 2 mL volumetric flask, add methanol to the mark, and shake to obtain a 1 mg / mL stock solution. Accurately measure 25, 50, 100, 200, 300, and 400 μL of these stock solutions into a 1 mL volumetric flask and dilute to the mark with methanol to obtain a series of standard solutions with concentrations of 25, 50, 100, 200, 300, and 400 μg / mL, respectively. Detect the peak areas of these standard solutions by HPLC (mobile phase: 0.065% TFA in water + 0.05% TFA; column: YMC-Triart C18; wavelength: 220 nm). Construct a concentration-peak area standard curve, where the abscissa represents concentration and the ordinate represents peak area.

[0172] Preparation of supersaturated aqueous solutions of DOC, DOC-AI, PTX and PTX-AI:

[0173] Weigh 10 mg each of DOC, DOC-AI, PTX, and PTX-AI into 5 mL of ultrapure water, add 1 mL of ultrapure water, sonicate for 15 min, filter with an organic filter membrane into a liquid phase vial, and detect the peak area of ​​the sample by HPLC. The saturation in water was calculated using the concentration-peak area standard curve. The results are shown in Table 6.

[0174] Table 6 Solubility test results of polypeptide conjugates in water

[0175] As shown in Figure 10, the concentration-peak area standard curves of DOC, DOC-AI, PTX, and PTX-AI are R 2 >0.99, indicating a good fit. As shown in Table 6, the solubility of DOC and PTX in water is 2.74×10 -5 mg / mL, 1.2×10 -4 mg / mL, and is almost insoluble in water. DOC-AI has good solubility in water, which is 4.43 mg / mL, which is 1.62×10 higher than that of the positive drug DOC. 5 times, and the solubility of PTX-AI was increased by 46.33 times compared with the positive drug PTX.

[0176] Machulkin et al. coupled the targeted PSMA ligand to DOC via a linker dipeptide to obtain PSMA-DOC, whose solubility in water was 130 μg / mL (Machulkin AE, Uspenskaya AA, Zyk NY, et al. PSMA-targeted small-molecule docetaxel conjugate: Synthesis and preclinical evaluation. Eur J Med Chem. 2022; 227: 113936.), which still has great limitations on the application of PSMA-DOC. The solubility of the polypeptide conjugate in water of the present application is 4.43 mg / mL, which is 1.62×10 higher than the solubility of DOC alone. 5 times, which can greatly improve the application of DOC. The compound SNG-1005, which is formed by coupling the PTX molecule with the 19-amino acid peptide Angiopep-2, has completed Phase III clinical trials. However, SNG1005 still does not dissolve in water, which makes it difficult to produce in batches and the relevant clinical trials are forced to be postponed. It can be seen that the polypeptide conjugate of this application greatly increases the water solubility of DOC and PTX.

[0177] Although specific embodiments have been described, it is possible that alternatives, modifications, variations, improvements, and substantial equivalents of the above embodiments may exist or are not currently foreseeable to the applicant or other skilled in the art. Therefore, the appended claims, as filed and as they may be amended, are intended to cover all such alternatives, modifications, variations, improvements, and substantial equivalents.

Claims

1. A polypeptide conjugate having a structure of formula (I): polypeptide-linker-compound formula (I) in, The polypeptide is a mesenchymal-epithelial transition factor targeting peptide or a derivative thereof, The compound is a taxane compound or an enantiomer, diastereomer, solvate or pharmaceutically acceptable salt thereof.

2. The polypeptide conjugate according to claim 1, wherein The mesenchymal-epithelial transition factor targeting peptide has an amino acid sequence shown in SEQ ID NO:

1.

3. The polypeptide conjugate according to claim 1 or 2, wherein: Compared with the mesenchymal-epithelial transition factor targeting peptide, the derivative comprises any one or more modifications selected from the following: amino acid modification, conservative amino acid substitution, or substitution of hydrogen in an amino acid residue.

4. The polypeptide conjugate according to any one of claims 1 to 3, wherein The taxane compound is one or more of a bicyclic taxane compound, a tricyclic taxane compound, a tetracyclic taxane compound or a pentacyclic taxane compound. Optionally, the bicyclic taxane compound includes a 6-membered ring-fused and a 12-membered ring taxane compound, the tricyclic taxane compound includes one or more of a 6-membered ring-fused and an 8-membered ring-fused and a 6-membered ring, a 6-membered ring-fused and a 10-membered ring-fused and a 6-membered ring, a 5-membered ring-fused and a 7-membered ring-fused and a 6-membered ring, or a 5-membered ring-fused and a 6-membered ring taxane compound, the tetracyclic taxane compound includes one or more of a 6-membered ring-fused and a 5-membered ring-fused and a 5-membered ring-fused and a 6-membered ring, or a 6-membered ring-fused and an 8-membered ring-fused and a 6-membered ring taxane compound, and the pentacyclic taxane compound includes a 6-membered ring-fused and a 5-membered ring-fused and a 5-membered ring-fused and a 5-membered ring-fused and a 6-membered ring taxane compound.

5. The polypeptide conjugate according to any one of claims 1 to 4, wherein The taxane compound includes one or more of paclitaxel, docetaxel, cabazitaxel, larotaxel or tesetaxel. Optionally, the taxane compound includes one or more of paclitaxel or docetaxel.

6. The polypeptide conjugate according to any one of claims 1 to 5, wherein The linker is CO(O)C(CH2) n C(O)OH, n is an integer of 2-6, and optionally, the linker is succinic acid, glutaric acid, adipic acid, pimelic acid or suberic acid.

7. The polypeptide conjugate according to any one of claims 1 to 6, wherein The linker is connected to the polypeptide by forming an amide bond, and the linker is connected to the taxane compound by forming an ester bond.

8. The polypeptide conjugate according to any one of claims 1 to 7, wherein The polypeptide conjugate has the structure of the following formula (II), (III) or (IV): The polypeptide has an amino acid sequence shown in SEQ ID NO: 1 or a derivative thereof, and the carboxyl group of the linker is connected to the amino group of the amino acid at the nitrogen terminal of the polypeptide by forming an amide bond.

9. A pharmaceutical composition comprising the polypeptide conjugate according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier.

10. Use of the polypeptide conjugate according to any one of claims 1 to 8 or the drug combination according to claim 9 in the preparation of a medicament for preventing or treating a disease, wherein the disease comprises a tumor, and more preferably, the tumor comprises one or more of thyroid cancer, esophageal cancer, lung cancer, non-small cell lung cancer, gastric cancer, liver cancer, bile duct cancer, kidney cancer, pancreatic cancer, intestinal cancer, colorectal cancer, bladder cancer, prostate cancer, breast cancer, uterine cancer, endometrial cancer, ovarian cancer, cervical cancer, fallopian tube cancer, skin cancer, brain tumor, glioma, glioblastoma, melanoma, osteosarcoma, sarcoma, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia or chronic myeloid leukemia.

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