Peptides targeting c-Met, pharmaceutical compositions comprising same, and uses thereof

Abstract

The present invention relates to c-Met targeting peptides and derivatives thereof, to pharmaceutical compositions, drug-peptide conjugates comprising the peptides or derivatives thereof, and their use in the detection, prevention or treatment of diseases (e.g., cancer). In addition, the peptide or the derivative thereof can increase the water solubility of the drug.

Description

Peptide targeting c-Met, pharmaceutical composition containing the peptide and use thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to application PCT / CN2023 / 102222 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 disclosure relates to the field of medicine, and in particular to a peptide targeting c-Met, a conjugate comprising the peptide, a pharmaceutical composition, and uses thereof. Background Art

[0004] Cancer, the world's leading killer, is claiming lives on an increasing scale. According to data released by my country's Ministry of Health, cancer has surpassed cardiovascular disease as the leading cause of death among Chinese residents, both in urban and rural areas.

[0005] With the development of medicine and healthcare in recent decades, significant progress has been made in various aspects of cancer treatment and control, and a variety of drugs and treatments have emerged. However, surgery, chemotherapy, and radiotherapy remain the three main treatment options, and these treatments are difficult to produce satisfactory therapeutic effects. While conventional drug treatments play an important role, they often cause significant damage to normal tissues and cells. The anti-cancer drug market is growing rapidly, with targeted anti-cancer drugs showing the fastest growth. Targeted drugs include conventional small molecule targeted drugs and monoclonal antibodies. They target cancer cells or tissues and deliver drugs to the cancer site, thereby minimizing damage to normal tissues and cells, thereby achieving the purpose of specific treatment.

[0006] c-Met, also known as hepatocyte growth factor receptor (HGFR), is a member of the receptor tyrosine kinase family. c-Met is overexpressed in primary epithelial cancers, including those of the breast, colon, stomach, kidney, liver, lung, and pancreas. Increased expression ranges from 35-65% in breast cancer, 30-70% in gastric cancer, 25-60% in pancreatic cancer, 25-60% in hepatocellular carcinoma, 15-80% in renal cell carcinoma, and 35-70% in non-small cell lung cancer (Biochim Biophys Acta Rev Cancer. 2020, 1874, 188425). In various cancers, c-Met binding to its ligand, hepatocyte growth factor (HGF), activates downstream signaling pathways, promoting cancer cell proliferation and migration, as well as invasion, metastasis, and angiogenesis. Therefore, targeting c-Met for cancer therapy and imaging is a hot research topic.

[0007] Currently, blocking c-Met-HGF signaling is an effective approach for cancer treatment. This approach can utilize small molecule kinase inhibitors, antibodies, and peptides to inhibit the specific binding of c-Met to HGF. Compared to small molecule or antibody drugs, peptides have the advantages of high affinity, virtually no immunogenicity, low production costs, and ease of modification. Peptides targeting c-Met have been disclosed in the prior art (e.g., CN112979753B), but their application in clinical treatment and diagnosis is limited due to their poor solubility in water and the need to improve their affinity for c-Met. Therefore, there is an urgent need to develop improved c-Met-targeting peptides, such as those with better water solubility and / or higher affinity for c-Met.

[0008] Summary of the Invention

[0009] Based on the above, the present disclosure provides peptides targeting c-Met that are simple in structure and easy to synthesize, isolate, and purify. These peptides exhibit specific binding to the c-Met protein in vivo and in vitro. The present disclosure also provides peptides with excellent c-Met protein targeting capabilities in vivo, which are enriched in cancer tissues that overexpress c-Met. The present disclosure further provides the aforementioned c-Met-targeting peptides, which have improved affinity for c-Met and / or water solubility. The peptides of the present disclosure also exhibit improved drug solubility after conjugation to drugs.

[0010] In a first aspect of the present disclosure, a peptide targeting c-Met or a derivative thereof is provided comprising the following Formula I or Formula II: (R)-(X1)-(X2)-(X3)-(X4)-(X5)-(X6) Formula I (Y1)-(Y2)-(Y3)-(Y4)-(A)-(N)-(Y5) Formula II

[0011] wherein X1-X6 independently represent any amino acid or a derivative thereof; Y1-Y5 independently represent any amino acid or a derivative thereof, and Formula II does not include the sequence (D)-(Q)-(I)-(I)-(A)-(N)-(N) of CM7-1; and the peptide or its derivative has similar or better binding affinity to c-Met than CM7-1.

[0012] In one embodiment of the present disclosure, X1 in Formula I represents S, H or E, X2 represents P, I or D, X3 represents any amino acid or a derivative thereof, X4 represents A, M or C, X5 represents N, C or P, and X6 represents any amino acid or a derivative thereof; Y1 in Formula II represents any amino acid or a derivative thereof, Y2 represents Q or A, Y3 represents I or A, Y4 represents I or A, and Y5 represents N or A, and Formula II does not include (D)-(Q)-(I)-(I)-(A)-(N)-(N).

[0013] In one embodiment of the present disclosure, the peptide targeting c-Met or its derivative comprises any one sequence selected from the following: SEQ ID NO: 1, SEQ ID NO: 3-6, SEQ ID NO: 8, SEQ ID NO: 9-144, SEQ ID NO: 148-150.

[0014] In one embodiment of the present disclosure, the peptide targeting c-Met or its derivative comprises any one sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3-6, SEQ ID NO: 8, SEQ ID NO: 9-20, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 35, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 79-81, SEQ ID NO: 83-85, SEQ ID NO: 90, SEQ ID NO: 96, SEQ ID NO: 105, SEQ ID NO: 111, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 135, SEQ ID NO: 144, SEQ ID NO: 148-150.

[0015] In one embodiment of the present disclosure, the peptide targeting c-Met or its derivative comprises Formula I, (R)-(X1)-(X2)-(X3)-(X4)-(X5)-(X6) Formula I

[0016] Wherein X1 is S, X2 is P, and X3-6 independently represent any amino acid or its derivative, and the peptide or its derivative has similar or better binding affinity to c-Met than P1-AI (SEQ ID NO: 1, RSPYANS).

[0017] In one embodiment of the present disclosure, the peptide targeting c-Met or its derivative comprises any one sequence selected from the following: SEQ ID NO: 148-150.

[0018] In a second aspect of the present disclosure, a peptide targeting c-Met or a derivative thereof is provided, comprising the following formula III: (R)-(S)-(P)-(X7)-(X8)-(X9)-(X10) Formula III

[0019] Wherein, X7-10 independently represent any one amino acid or its derivative, and the peptide or its derivative has similar or better binding affinity to c-Met than P1-AI.

[0020] In one embodiment of the present disclosure, the peptide targeting c-Met or its derivative comprises any one sequence selected from the following: SEQ ID NO: 148-150.

[0021] In a third aspect of the present disclosure, an isolated polynucleotide is provided, which encodes the peptide or derivative thereof according to the present disclosure.

[0022] In a fourth aspect of the present disclosure, an expression vector is provided, which expresses the polynucleotide according to the present disclosure.

[0023] In a fifth aspect of the present disclosure, a host cell is provided, comprising the polynucleotide according to the present disclosure or the expression vector according to the present disclosure.

[0024] In a sixth aspect of the present disclosure, a drug-peptide conjugate is provided, comprising a peptide or derivative thereof of the present disclosure and a drug. In some embodiments, the peptide or derivative thereof of the present disclosure is conjugated directly (e.g., via a covalent bond) or indirectly (e.g., via a linker) to the drug. In some preferred embodiments, the drug is selected from a compound, such as a cytotoxic agent, including but not limited to at least one of camptothecin, docetaxel, and paclitaxel.

[0025] In a seventh aspect of the present disclosure, a pharmaceutical composition is provided, comprising the peptide or its derivative, polynucleotide, expression vector, host cell or drug-peptide conjugate according to the present disclosure, and a pharmaceutically acceptable carrier or salt thereof.

[0026] In an eighth aspect of the present disclosure, there is provided a peptide or derivative thereof of the present disclosure for use in increasing the water solubility of a drug. In one embodiment, the peptide or derivative thereof of the present disclosure is conjugated to a drug directly (e.g., via a covalent bond) or indirectly (e.g., via a linker). In some preferred embodiments, the drug is selected from a compound, such as a cytotoxic agent, including but not limited to at least one of camptothecin, docetaxel, and paclitaxel.

[0027] In a ninth aspect of the present disclosure, a method for increasing the water solubility of a drug is provided, comprising conjugating a peptide or derivative thereof of the present disclosure to a drug directly (e.g., via a covalent bond) or indirectly (e.g., via a linker). In some preferred embodiments, the drug is selected from a compound, such as a cytotoxic agent, including but not limited to at least one of camptothecin, docetaxel, and paclitaxel.

[0028] In the tenth aspect of the present disclosure, provided is the use of the peptide or its derivative, polynucleotide, expression vector, host cell, drug-peptide conjugate or pharmaceutical composition according to the present disclosure in the manufacture of an agent or drug for detecting, preventing or treating a disease.

[0029] In the eleventh aspect of the present disclosure, a method for detecting, preventing or treating a disease is provided, comprising administering to a subject in need thereof a detection-, prevention- or treatment-effective amount of a peptide or its derivative, polynucleotide, expression vector, host cell, drug-peptide conjugate or pharmaceutical composition according to the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The following provides a brief description of the drawings, which are provided to illustrate exemplary embodiments disclosed herein and not to limit these embodiments.

[0031] FIG1 shows the structure of polypeptide P1-AI.

[0032] FIG2A shows the HPLC detection results of P1-AI, FITC-P1-AI, CM7-1, FITC-CM7-1, FITC-CM7-1.1, and FITC-CM7-1.2 synthesized in Example 1.

[0033] FIG2B shows the HPLC detection results of FITC-CM7-1.3, FITC-CM7-1.4, FITC-CM7-1.5, FITC-CM7-1.6, and Cy5-P1-AI synthesized in Example 1.

[0034] Figure 3 shows the mass spectrometry analysis results of P1-AI, FITC-P1-AI, Cy5-P1-AI, CM7-1, FITC-CM7-1, FITC-CM7-1.1, FITC-CM7-1.2, FITC-CM7-1.3, FITC-CM7-1.4, FITC-CM7-1.5, and FITC-CM7-1.6 synthesized in Example 1.

[0035] Figure 4 shows the flow cytometry results for c-Met expression in different cells in Example 2, where the horizontal axis represents the fluorescence signal and the vertical axis represents the number of cells. The light-colored peak in the figure represents c-Met, and the dark-colored peak represents the FITC-c-Met isotype control IgG.

[0036] FIG5 shows the binding curve of the peptide disclosed in Example 3 with c-Met-positive cells MKN-45, wherein the horizontal axis represents log (peptide concentration), with the concentration unit being μM; and the vertical axis represents the binding rate (%).

[0037] Figure 6 shows the binding curves of the disclosed peptides in Example 3 for c-Met-positive MKN-45 cells, where the horizontal axis represents fluorescence signal and the vertical axis represents cell number. The leftmost curve in the figure represents a blank control group without peptide addition, the middle curve represents FITC-CM7-1, and the rightmost curve represents FITC-P1-A.

[0038] FIG7 shows a schematic diagram of the molecular docking of polypeptide CM7 and polypeptide P1-AI in Example 4, and their docking scores and average hydrophobicity values.

[0039] FIG8 shows the binding curve of the peptide disclosed in Example 5 to human hepatic stellate cells LX-2 that hardly express c-Met, wherein the horizontal axis represents the fluorescence signal and the vertical axis represents the cell number.

[0040] FIG9 shows the results of water solubility testing of the peptides disclosed in Example 6.

[0041] FIG10 shows the results of detecting the interaction between the peptides of the present disclosure and c-Met protein using MST in Example 7, wherein the horizontal axis represents the concentration of c-Met protein in μM; and the vertical axis represents the fluorescence intensity.

[0042] FIG11 shows the results of in vivo imaging of Cy5-P1-AI in tumor-bearing mice in Example 8.

[0043] Figure 12 shows the binding of the peptides P1-AI, P1-AI-1, P1-AI-2, P1-AI-3, P1-AI-4, P1-AI-6, and P1-AI-7 disclosed in Example 9 to c-Met-positive cells MKN-45, wherein the horizontal axis represents the fluorescence signal; the vertical axis represents the number of cells, the dark color represents the fluorescence chemical shift after the addition of the polypeptide, and the light color represents the blank control without the addition of the peptide.

[0044] Figure 13 shows the concentration-peak area standard curves of the CPT, CPT-P1-AI, DOC, DOC-P1-AI, PTX, and PTX-P1-AI series of standard solutions in Example 10, where the abscissa represents concentration and the ordinate represents peak area. DETAILED DESCRIPTION

[0045] 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.

[0046] 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.

[0047] 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; (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.

[0048] As used herein, the term "independently of one another" means that multiple events do not affect each other. For example, "X and Y are independently selected from any of a, b, c, d, e, f, and g" means that X can be any of a, b, c, d, e, f, and g, and Y can also be any of a, b, c, d, e, f, and g. The choice of X and the choice of Y can be the same or different, and the two do not interfere with each other.

[0049] As used herein, alanine scanning (Cunningham and Wells, Science 244, 1081-1085, 1989) is used to identify amino acids that are critical for the activity of the peptides of the invention (e.g., binding affinity to acetylcholine receptors) so that these amino acids are not substituted. Alanine scanning introduces mutations into every residue in the molecule and tests the biological activity of the resulting molecules to determine the amino acid residues that are critical for the activity of the molecule.

[0050] As used herein, the terms "comprises" and "includes" mean that in addition to the listed elements, other elements are not excluded.

[0051] Peptide or its derivatives

[0052] The present application provides a peptide targeting c-Met or a derivative thereof comprising the following Formula I or Formula II: (R)-(X1)-(X2)-(X3)-(X4)-(X5)-(X6) Formula I (Y1)-(Y2)-(Y3)-(Y4)-(A)-(N)-(Y5) Formula II

[0053] wherein X1-X6 independently represent any amino acid or a derivative thereof; Y1-Y5 independently represent any amino acid or a derivative thereof, and Formula II does not include the sequence (D)-(Q)-(I)-(I)-(A)-(N)-(N) of CM7-1; and the peptide or its derivative has similar or better binding affinity to c-Met than CM7-1.

[0054] In some embodiments, X1 in Formula I represents S, H, or E. In some embodiments, X2 in Formula I represents P, I, or D. In some embodiments, X3 in Formula I represents any amino acid or a derivative thereof. In some embodiments, X4 in Formula I represents A, M, or C. In some embodiments, X5 in Formula I represents N, C, or P. In some embodiments, X6 in Formula I represents any amino acid or a derivative thereof.

[0055] In some embodiments, Y1 in Formula II represents any amino acid or a derivative thereof. In some embodiments, Y2 in Formula II represents Q or A. In some embodiments, Y3 in Formula II represents I or A. In some embodiments, Y4 in Formula II represents I or A. In some embodiments, Y5 in Formula II represents N or A. In some embodiments, Formula II does not include (D)-(Q)-(I)-(I)-(A)-(N)-(N).

[0056] In some embodiments, the peptide targeting c-Met or its derivative comprises any one sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3-6, SEQ ID NO: 8, SEQ ID NO: 9-144, and SEQ ID NO: 148-150.

[0057] In some embodiments, the peptide targeting c-Met or its derivative comprises any one sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3-6, SEQ ID NO: 8, SEQ ID NO: 9-20, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 35, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 79-81, SEQ ID NO: 83-85, SEQ ID NO: 90, SEQ ID NO: 96, SEQ ID NO: 105, SEQ ID NO: 111, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 135, SEQ ID NO: 144, SEQ ID NO: 148-150.

[0058] In some embodiments, the sequence of the peptide targeting c-Met or its derivative comprises SEQ ID NO: 1. In some embodiments, the sequence of the peptide targeting c-Met or its derivative comprises SEQ ID NO: 10. In some embodiments, the sequence of the peptide targeting c-Met or its derivative comprises SEQ ID NO: 90. In some embodiments, the sequence of the peptide targeting c-Met or its derivative comprises SEQ ID NO: 144.

[0059] In some embodiments, the peptide targeting c-Met or its derivative comprises the following Formula I, (R)-(X1)-(X2)-(X3)-(X4)-(X5)-(X6) Formula I

[0060] wherein X1 is S, X2 is P, and X3-6 independently represent any amino acid or derivative thereof, and the peptide or derivative thereof has similar or better binding affinity for c-Met than P1-AI (SEQ ID NO: 1, RSPYANS). In some embodiments, the peptide or derivative thereof comprises any one sequence selected from the group consisting of SEQ ID NOs: 148-150.

[0061] In some embodiments, the derivative comprises any one or more modifications selected from the group consisting of amino acid modification, conservative amino acid substitution, and substitution of hydrogen in an amino acid residue compared to the peptide.

[0062] As used herein, the term "peptide" refers to a compound formed by amino acids linked together with peptide bonds. A peptide composed of three or more amino acid molecules is a polypeptide. As used herein, unless otherwise specified or inconsistent with the context, the term "peptide" encompasses the peptide itself and its pharmaceutically acceptable salts, prodrugs and metabolites. The peptides of the present application are produced by conventional techniques in the art, including but not limited to solid phase synthesis (such as Fmoc peptide synthesis) and liquid phase synthesis.

[0063] As used herein, the term "prodrug" refers to all molecules that are converted into the peptide of the present disclosure or its derivatives through oxidation, reduction, hydrolysis, etc. reactions in vivo under physiological conditions, for example, by the action of enzymes, gastric acid, etc.

[0064] 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 the present disclosure or a derivative thereof.

[0065] When used in this article with respect to peptides, "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.

[0066] As used herein, unless otherwise indicated or contradicted, "amino acid" encompasses both L- and D-amino acids.

[0067] As used herein, "polar amino acids" refer to glycine (G), tyrosine (Y), serine (S), cysteine ​​(C), asparagine (N), glutamine (Q), threonine (T), aspartic acid (D), glutamate (E), lysine (K), arginine (R), and histidine (H).

[0068] As used herein, a "derivative" of a peptide refers to a product obtained by modifying the peptide, making conservative amino acid substitutions, and / or replacing hydrogen atoms in amino acid residues.

[0069] As used herein, the term "amino acid modification" includes, but is not limited to, N-terminal modification, C-terminal modification, and side chain modification. Methods of "amino acid modification" include, but are not limited to, hydroxylation, carboxylation, alkylation, acylation, phosphorylation, sulfonation, amidation, aldehyde formation, alcoholization, mercaptoethylation, esterification, and glycosylation.

[0070] 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).

[0071] As used herein, the replacement of a hydrogen atom in an amino acid residue may be a replacement of a hydrogen atom in an amino acid residue by any conventional substituent known in the art, including but not limited to deuterated, alkyl, alkenyl, alkynyl, aromatic, alkoxy, carboxyl, aldehyde, carbonyl, hydroxyl, halogen, cyano, acyl, sulfonic acid, amino, thiol or nitro groups.

[0072] The present application provides a peptide targeting c-Met or a derivative thereof comprising the following formula III, (R)-(S)-(P)-(X7)-(X8)-(X9)-(X10) Formula III

[0073] Wherein, X7-10 independently represent any one amino acid or its derivative, and the peptide or its derivative has similar or better binding affinity to c-Met than P1-AI.

[0074] In some embodiments, the peptide targeting c-Met or its derivative comprises any one sequence selected from the group consisting of SEQ ID NOs: 148 to 150. In some embodiments, the peptide targeting c-Met or its derivative comprises SEQ ID NO: 148.

[0075] In some embodiments, the derivative comprises any one or more modifications selected from the group consisting of amino acid modification, conservative amino acid substitution, and substitution of hydrogen in an amino acid residue compared to the peptide.

[0076] Polynucleotides, expression vectors, host cells

[0077] The present application provides an isolated polynucleotide encoding the peptide or its derivatives described in the present application.

[0078] As used herein, "polynucleotide" refers to a polymer of nucleotides (nucleotides or deoxynucleotides) of any length. The term refers to the primary structure of the molecule. Thus, it includes both double-stranded and single-stranded RNA, as well as double-stranded and single-stranded DNA. It also includes polynucleotides modified, such as by methylation and / or end-capping, as well as unmodified forms of polypeptide nucleotides. The polynucleotides described herein need not be obtained by physical methods and may be produced by any means, including, for example, chemical synthesis or DNA replication, reverse transcription, or transcription.

[0079] The present application provides an expression vector, which can express the polynucleotide described in the present application.

[0080] The present application provides a host cell comprising the polynucleotide described herein or the expression vector described herein. In some embodiments, the host cell is selected from at least one of Escherichia coli cells, Saccharomyces cerevisiae cells, Bacillus subtilis cells, and animal cells, such as CHO cells and HEK cells.

[0081] As used herein, a "host cell" is a cell that serves as a recipient for an expression vector or other transforming genetic fragment. These include the original cell that has been transfected and its progeny. Host cells include prokaryotes, yeast, or other eukaryotic cells.

[0082] Drug-peptide conjugates

[0083] The present application provides a drug-peptide conjugate comprising a peptide or derivative thereof of the present disclosure and a drug. In some embodiments, the peptide or derivative thereof of the present disclosure is conjugated to the drug directly (e.g., via a covalent bond) or indirectly (e.g., via a linker). In some embodiments, the linker is selected from at least one of a cleavable linker and a non-cleavable linker. In some embodiments, the linker comprises ((G)n(S))m, n can be an integer from 1 to 8, and m can be an integer from 1 to 8. In some embodiments, the linker comprises 1 to 8 amino acids, such as Val-Cit, Glu-Val-Cit. In some embodiments, the linker comprises CO(O)C(CH2) n C(O)OH, n may be an integer from 2 to 8. In some embodiments, the linker comprises at least one of succinic acid, fumaric acid, glutaric acid, adipic acid, pimelic acid, and suberic acid.

[0084] In some embodiments, medicine is selected from at least one of small molecule drugs, antibodies, polypeptides, proteins, chemicals, chemotherapeutics, fluorescent markers. In some embodiments, medicine includes but is not limited to cytotoxic agents, and cytotoxic agents can be any compound that causes cell death or induces cell death or reduces cell viability in a certain way. Exemplary cytotoxic agents include, for example, maytansine and maytansine analogs, taxanes, such as paclitaxel and docetaxel, CC-1065 and CC-1065 analogs, dolastatin and dolastatin analogs, methotrexate, daunorubicin, adriamycin, vincristine, vinblastine, mevalonate, mitomycin C, chloramphenicol, calicheamicin (calicheamicin), trichomoniasis and trichomoniasis analogs, erythromycin and erythromycin analogs. In some preferred embodiments, medicine is selected from cytotoxic agents, such as at least one of camptothecin, docetaxel, paclitaxel.

[0085] Pharmaceutical composition

[0086] The present application provides a pharmaceutical composition comprising a peptide or derivative thereof, a drug-peptide conjugate according to the present disclosure, and a pharmaceutically acceptable carrier or salt thereof. The present application provides a pharmaceutical composition comprising a peptide or derivative thereof, a polynucleotide, an expression vector, a host cell, or a drug-peptide conjugate according to the present disclosure, and a pharmaceutically acceptable carrier or salt thereof.

[0087] As used herein, the term "pharmaceutical composition" refers to a mixture of a peptide of the present disclosure and other chemical components such as a carrier, stabilizer, diluent, dispersant, suspending agent, thickener, and / or excipient. Pharmaceutical compositions facilitate administration of the peptide to an organism. Various techniques for administering peptides exist in the art, including, but not limited to, subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral administration, transdermal administration, transpulmonary administration, ocular administration, and topical administration.

[0088] In the present application, the pharmaceutical composition can be configured into a dosage form suitable for administration to a subject via a desired route of administration, including but not limited to tablets, capsules, caplets, pills, lozenges, powders, syrups, brews, suspensions, solutions, emulsions, transdermal patches, suppositories, inhalants, creams, ointments, lotions, pastes, sprays, freeze-dried solutions, injections, and gels.

[0089] 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).

[0090] The term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable materials, compositions or vehicles, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials, which are involved in carrying or transporting the peptides of the present disclosure within a subject or carrying or transporting the peptides of the present disclosure to a subject so that it can perform its intended function. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate; Esters and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; diluents; granulating agents; lubricants; binders; disintegrants; wetting agents; emulsifiers; colorants; release agents; coating agents; sweeteners; flavorings; perfuming agents; preservatives; antioxidants; plasticizers; gelling agents; thickeners; hardening agents; setting agents; suspending agents; surfactants; humectants; carriers; stabilizers; and other nontoxic, compatible substances used in pharmaceutical formulations, or any combination thereof.

[0091] Purpose and method

[0092] The present application provides a use of the peptide or derivative thereof disclosed herein for increasing the water solubility of a drug. In one embodiment, the peptide or derivative thereof disclosed herein is conjugated to the drug directly (eg, via a covalent bond) or indirectly (eg, via a linker).

[0093] The present application provides a method for increasing the water solubility of a drug, comprising conjugating the peptide or derivative thereof disclosed herein to the drug directly (eg, via a covalent bond) or indirectly (eg, via a linker).

[0094] In some embodiments, the linker is selected from at least one of a cleavable linker and a non-cleavable linker. In some embodiments, the linker comprises ((G)n(S))m, where n can be an integer from 1 to 8 and m can be an integer from 1 to 8. In some embodiments, the linker comprises 1 to 8 amino acids, such as Val-Cit, Glu-Val-Cit. In some embodiments, the linker comprises CO(O)C(CH2) n C(O)OH, n may be an integer from 2 to 8. In some embodiments, the linker comprises at least one of succinic acid, fumaric acid, glutaric acid, adipic acid, pimelic acid, and suberic acid.

[0095] In some embodiments, the medicine is selected from at least one of small molecule drugs, antibodies, polypeptides, proteins, chemical drugs, chemotherapeutics, fluorescent markers. In some embodiments, the medicine includes but is not limited to cytotoxic agents, and cytotoxic agents can be any compound that causes cell death or induces cell death or reduces cell viability in some way. Exemplary cytotoxic agents include, for example, maytansine and maytansine analogs, taxanes, such as paclitaxel and docetaxel, CC-1065 and CC-1065 analogs, dolastatin and dolastatin analogs, methotrexate, daunorubicin, adriamycin, vincristine, vinblastine, mevalonate, mitomycin C, chloramphenicol, calicheamicin (calicheamicin), trichomoniasis and trichomoniasis analogs, erythromycin and erythromycin analogs. In some preferred embodiments, the medicine is selected from cytotoxic agents, such as at least one of camptothecin, docetaxel, paclitaxel. The application provides the purposes of the disclosed peptide or its derivatives, drug-peptide conjugates or pharmaceutical compositions in the manufacture of reagents or medicines for detecting, preventing or treating diseases. The present application provides use of the peptide or derivatives thereof, polynucleotides, expression vectors, host cells, drug-peptide conjugates or pharmaceutical compositions disclosed herein in the manufacture of reagents or drugs for detecting, preventing or treating diseases.

[0096] In some embodiments, the disease comprises cancer. In some embodiments, the disease comprises solid tumors and / or hematologic malignancies. In some preferred embodiments, the disease comprises one or more of thyroid cancer, esophageal cancer, lung cancer, gastric cancer, liver cancer (e.g., high metastasis liver cancer), bile duct cancer, kidney cancer (e.g., renal cell adenocarcinoma, human renal clear cell adenocarcinoma), 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, melanoma, osteosarcoma, sarcoma, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, or leukemia. 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.

[0097] 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.

[0098] As used herein, the term "treat," ...

[0099] The present application provides a method for detecting, preventing or treating a disease, comprising administering to a subject in need thereof a detection, prevention or treatment effective amount of a peptide or derivative thereof, a drug-peptide conjugate or a pharmaceutical composition of the present disclosure. The present application also provides a method for detecting, preventing or treating a disease, comprising administering to a subject in need thereof a detection, prevention or treatment effective amount of a peptide or derivative thereof, a polynucleotide, an expression vector, a host cell, a drug-peptide conjugate or a pharmaceutical composition of the present disclosure.

[0100] In some embodiments, the disease comprises cancer. In some embodiments, the disease comprises solid tumors and / or hematologic malignancies. In some preferred embodiments, the disease comprises one or more of thyroid cancer, esophageal cancer, lung cancer, gastric cancer, liver cancer (e.g., high metastasis liver cancer), bile duct cancer, kidney cancer (e.g., renal cell adenocarcinoma, human renal clear cell adenocarcinoma), 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, melanoma, osteosarcoma, sarcoma, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, or leukemia. 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, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, or lymphoma.

[0101] As used herein, the term "subject" includes animals, such as vertebrates, preferably mammals, such as dogs, cats, pigs, cows, sheep, horses, rodents (e.g., mice, rats, or guinea pigs), or primates (e.g., gorillas, chimpanzees, and humans).

[0102] As used herein, the term "detection, prevention or treatment effective dose" refers to an amount that results in a benefit in the detection, prevention or treatment of a disease compared to a corresponding subject not receiving that amount, but which is sufficiently low within the scope of sound medical judgment to avoid serious side effects. The detection, prevention or treatment effective dose of the peptide or its derivative, polynucleotide, expression vector, host cell, drug-peptide conjugate or pharmaceutical composition described herein will vary with the selected peptide or its derivative, polynucleotide, expression vector, host cell, drug-peptide conjugate or pharmaceutical composition; the route of administration; the severity of the disease; the age, size, weight and physical condition of the patient; the patient's medical history; the duration of treatment; the nature of concurrent treatment; the desired detection, prevention or treatment effect, etc., but can still be determined in a routine manner by those skilled in the art.

[0103] The various embodiments described above for the peptides or derivatives thereof, polynucleotides, expression vectors and host cells of the present disclosure are also applicable to the drug-peptide conjugates, pharmaceutical compositions, uses and methods of the present disclosure (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 disclosure.

[0104] Example

[0105] The following describes exemplary embodiments of the present application in conjunction with the accompanying drawings, including various details of the embodiments of the present application to facilitate understanding. It should be understood that they are considered merely exemplary 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, it should be appreciated by those skilled in the art 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.

[0106] 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.

[0107] Example 1: Preparation of peptides

[0108] 1. Preparation of P1-AI Peptide

[0109] Weigh 1 g of Wang resin (CAS: 1365700-43-1) with a degree of substitution of 1.1 mmol / g and add it to a polypeptide solid phase reactor (glass instrument, Shanghao Jikun Chemical Technology Co., Ltd.), add DCM (Annaiji) and use nitrogen to continuously bubbling and swell for 10-20 minutes, then drain. Accurately weigh 0.345 g of Fmoc-S(tBu)-OH (Sichuan Zhengyuan Biotechnology Co., Ltd.), 130 mg of DMAP, and 0.405 g of HOBT. Use 5-10 ml of DMF as solvent and add 2 ml of DIC (all purchased from Anaiji unless otherwise specified). At room temperature, use nitrogen to blow the reaction for 4-8 hours, drain, and wash twice with DMF.

[0110] Use acetic anhydride to seal the head. Specifically, use 5 ml of DCM as the solvent, and add 2 ml of DIPEA and 2 ml of acetic anhydride (all purchased from Anaiji) to the reaction system. At room temperature, use nitrogen to blow the reaction for 20-30 minutes. After the reaction is completed, use DMF to wash 4 times;

[0111] Fmoc removal was performed twice using a commercially available 20% piperidine / DMF solution (100 ml / 400 ml) at room temperature with nitrogen purging for 10 min and 5 min, respectively. After the reaction, the resin was washed with DMF five times. The resin was detected using a commercially available ninhydrin:phenol:piperidin (2:1:1) for 30-60 s. If the resin turned dark blue, the Fmoc removal was successful.

[0112] 1.612 g of Fmoc-N(trt)-OH (Sichuan Zhengyuan Biotechnology Co., Ltd.) and 1.023 g of commercially available HBTU were weighed and added to a peptide solid phase reactor. 5 ml of DMF was used as the solvent, and 1 ml of DIPEA was added. The reaction was allowed to proceed at room temperature with nitrogen bubbling for 40-60 min. The mixture was then drained and washed four times with DMF. The mixture was tested with ninhydrin:phenol:piperidin (2:1:1) for 2-3 min. If the resin was colorless, the coupling was successful.

[0113] Remove Fmoc twice using 20% ​​piperidine / DMF solution (100 ml / 400 ml) at room temperature with nitrogen purging for 10 min and 5 min, respectively. Wash with DMF five times after the reaction. Detect with ninhydrin:phenol:piperidin (2:1:1) for 30-60 s. If the resin turns dark blue, Fmoc removal is successful.

[0114] Repeat the above steps to add each amino acid in the peptide sequence. Fmoc-A-OH, Fmoc-Y(tBu)-OH, Fmoc-P-OH, Fmoc-S(tBu)-OH, and Fmoc-R(pbf)-OH (all purchased from Sichuan Zhengyuan Biotechnology Co., Ltd.) were coupled in sequence according to the peptide sequence. The Fmoc protecting group was then removed using a 20% piperidine / DMF solution (100 ml / 400 ml). The solution was washed 6 times with DMF, shrunk twice with methanol, and drained to obtain the P1-AI protected peptide resin.

[0115] 15.2 ml of commercially available TFA, 0.8 ml of H2O, and 0.8 ml of commercially available Tis were used as a lysis solution. The P1-AI protected peptide resin and the lysis solution were placed in a centrifuge tube together. After reacting for 1.5-2 h, the mixture was settled using icy ether and centrifuged three times to obtain a white solid. The solid was dried in a vacuum drying oven to obtain a crude product with a purity of 90%. The crude product was purified by HPLC and lyophilized to obtain a white powder peptide P1-AI (SEQ ID NO: 1), the structure of which is shown in Figure 1.

[0116] 2. Preparation of FITC-P1-AI Peptide

[0117] Weigh 0.1 g of Wang resin (with a degree of substitution of 1.1 mmol / g) and add it to a peptide solid phase reactor. Add 5 ml of DCM and swell with nitrogen bubbling for 10 min. Drain and dry. Accurately weigh 0.0348 g of Fmoc-S(tBu)-OH, 13 mg of DMAP, and 0.0408 g of HOBT. Use DMF as the solvent and add 0.2 ml of DIC. React at room temperature with nitrogen bubbling for 4-8 h. Drain and dry, and rinse twice with DMF.

[0118] Use acetic anhydride to seal the head. Specifically, use 5 ml of DCM as solvent, add 0.2 ml of DIPEA and 0.2 ml of acetic anhydride, and react with nitrogen at room temperature for 20-30 minutes. After the reaction is completed, use DMF to wash 4 times;

[0119] Remove Fmoc twice using 20% ​​piperidine / DMF solution at room temperature with nitrogen bubbling for 10 min and 5 min respectively. Wash with DMF five times after the reaction. Detect with ninhydrin:phenol:piperidin (2:1:1) for 30-60 s. If the resin turns dark blue, it indicates that Fmoc has been successfully removed.

[0120] Weigh 0.1161 g of Fmoc-N(trt)-OH and 0.1023 g of HBTU into a reactor. Use DMF as solvent and add 0.1 ml of DIPEA. React at room temperature with nitrogen bubbling for 60 min. Drain dry, rinse four times with DMF, and detect with ninhydrin:phenol:piperidin (2:1:1) for 2-3 min. If the resin is colorless, the coupling is successful.

[0121] Remove Fmoc twice using 20% ​​piperidine / DMF solution at room temperature with nitrogen bubbling for 10 min and 5 min respectively. Wash with DMF five times after the reaction. Detect with ninhydrin:phenol:piperidin (2:1:1) for 30-60 s. If the resin turns dark blue, it indicates that Fmoc has been successfully removed.

[0122] Repeat the above steps to add each amino acid in the peptide sequence. Fmoc-A-OH, Fmoc-Y(tBu)-OH, Fmoc-P-OH, Fmoc-S(tBu)-OH and Fmoc-R(pbf)-OH are coupled in sequence according to the peptide sequence. The Fmoc protecting group is then removed using a 20% piperidine / DMF solution.

[0123] Weigh 0.381 g of Fmoc-Acp-OH and 0.1023 g of HBTU into a reactor. Use DMF as solvent and add 0.1 ml of DIPEA. React at room temperature with nitrogen bubbling for 3 h. Then drain and rinse with DMF four times. Detect with ninhydrin:phenol:piperidin (2:1:1) for 2-3 min. If the resin is colorless, the coupling is successful.

[0124] Remove Fmoc twice using 20% ​​piperidine / DMF solution for 10 min and 5 min respectively. Wash with DMF 5 times after the reaction. Detect with ninhydrin:phenol:piperidin (2:1:1) for 30-60 s. If the resin turns dark blue, it indicates that Fmoc has been successfully removed.

[0125] Weigh 0.14 g of FITC and add it to the reactor. Use DMF as solvent and add 1 ml of triethylamine. After reacting for 4 h in the dark, the solution is washed 6 times with DMF, shrunk twice with methanol, and dried to obtain FITC-P1-AI protected peptide resin.

[0126] Use 7.6 ml of TFA, 0.4 ml of H2O and 0.4 ml of Tis as the lysis solution, place the FITC-P1-AI protected peptide resin and the lysis solution in a centrifuge tube together, react for 1.5-2 hours, use ice ether for precipitation and centrifugation three times to obtain a yellow solid, and use a vacuum drying oven to obtain a crude product with a purity of 90%. Purify by HPLC and lyophilize to obtain the peptide FITC-P1-AI in a yellow powder.

[0127] 3. Preparation of Cy5-P1-AI Peptide

[0128] Weigh 0.1 g of Wang resin (with a degree of substitution of 1.1 mmol / g) and add it to a peptide solid phase reactor. Add 5 ml of DCM and swell with nitrogen bubbling for 10 min. Drain and dry. Accurately weigh 0.0348 g of Fmoc-S(tBu)-OH, 13 mg of DMAP, and 0.0408 g of HOBT. Use DMF as the solvent and add 0.2 ml of DIC. React at room temperature with nitrogen bubbling for 4-8 h. Drain and dry, and rinse twice with DMF.

[0129] Use acetic anhydride to seal the end. Specifically, use DCM as solvent, add 0.2 ml of DIPEA and 0.2 ml of acetic anhydride, and use nitrogen to blow the reaction at room temperature for 20-30 minutes. After the reaction is completed, use DMF to wash 4 times;

[0130] Remove Fmoc twice using 20% ​​piperidine / DMF solution at room temperature with nitrogen bubbling for 10 min and 5 min respectively. Wash with DMF five times after the reaction. Detect with ninhydrin:phenol:piperidin (2:1:1) for 30-60 s. If the resin turns dark blue, it indicates that Fmoc has been successfully removed.

[0131] Weigh 0.1161 g of Fmoc-N(trt)-OH and 0.1023 g of HBTU into a reactor. Use DMF as solvent and add 0.1 ml of DIPEA. React at room temperature with nitrogen bubbling for 60 min. Drain dry, rinse four times with DMF, and detect with ninhydrin:phenol:piperidin (2:1:1) for 2-3 min. If the resin is colorless, the coupling is successful.

[0132] Remove Fmoc twice using 20% ​​piperidine / DMF solution at room temperature with nitrogen bubbling for 10 min and 5 min respectively. Wash with DMF five times after the reaction. Detect with ninhydrin:phenol:piperidin (2:1:1) for 20-30 s. If the resin turns dark blue for 20-30 s, Fmoc removal is successful.

[0133] Repeat the above steps, adding each amino acid in the peptide sequence. Fmoc-A-OH, Fmoc-Y(tBu)-OH, Fmoc-P-OH, Fmoc-S(tBu)-OH, and Fmoc-R(pbf)-OH are coupled sequentially in the peptide sequence. The Fmoc protecting group is then removed using a 20% piperidine / DMF solution. The solution is washed six times with DMF, condensed twice with methanol, and drained to obtain the P1-AI protected peptide resin.

[0134] Weigh 100 mg of sulfonic acid-Cy5 carboxylic acid, an excess of P1-AI protected peptide resin, and 0.1023 g of HBTU, add them into a reactor, use DMF as solvent, and add 0.1 ml of DIPEA using DMF as solvent. After reacting for 2 hours in the dark, the solution is washed 6 times with DMF, shrunk twice with methanol, and dried to obtain Cy5-P1-AI protected peptide resin.

[0135] Use 7.6 ml of TFA, 0.4 ml of H2O and 0.4 ml of Tis as the lysis solution, place the Cy5-P1 protected peptide resin and the lysis solution in a centrifuge tube together, react for 1.5-2 hours, use ice ether for precipitation and centrifugation three times to obtain a blue solid, and use a vacuum drying oven to obtain a crude product with a purity of 90%. Purify it using HPLC and lyophilize it to obtain the peptide Cy5-P1-AI in a blue powder.

[0136] 4. Preparation of other peptides

[0137] The peptides P1-AI (RSPYANS, molecular weight 793.82), FITC-P1-AI (FITC-RSPYANS, molecular weight 1296.36), Cy5-P1-AI (Cy5-RSPYANS, molecular weight 1432.63), CM7-1 (DQIIANN, molecular weight 786.83, corresponding to CN 112979753B), FITC-CM7-1 (FITC-DQIIANN, molecular weight 1289.36), FITC-CM7-1.1 (FITC-AQIIANN, molecular weight 1245.35), FITC-CM7-1.2 (FITC-DAIIANN, molecular weight 1232.31), FITC-CM7-1.3 (FITC-DQAIANN, molecular weight 1247.28), FITC-CM7-1.4 (FITC-DQIAANN, molecular weight 1247.28), FITC-CM7-1.5 (FITC-DQIIAAN, molecular weight 1246.34), FITC-CM7-1.6 (FITC-DQIIANA, molecular weight 1246.34). The peptides and their sequences involved herein are shown in Table 1. The HPLC test results are shown in Figures 2A and 2B, and the mass spectrometry analysis results are shown in Figure 3.

[0138] Table 1. Peptides and their sequences in Example 1

[0139] Example 2: Flow cytometry detection of c-Met expression in different cells

[0140] Human gastric cancer cells MKN-45 (Beijing Beina Chuanglian Biotechnology Research Institute), human highly metastatic hepatocellular carcinoma cells HCCLM3, human colon cancer cells HT29 (Beijing Beina Chuanglian Biotechnology Research Institute), renal cell adenocarcinoma cells ACHN, human renal clear cell adenocarcinoma cells 786-O, and human hepatic stellate cells LX-2 (all cells were purchased from Nanjing Saihongrui Biotechnology Co., Ltd. unless otherwise specified) were revived and cultured, and passaged for more than three times for later use; after three generations of cell culture, the cells were washed several times with 4°C pre-cooled commercial PBS at an appropriate density to remove cells in poor condition and floating objects, and then digested with trypsin solution; after the cells became round, an equal volume of 16 Digestion was terminated with 40% or DMEM medium and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and the cells were blocked with 2% commercially available BSA blocking solution at 4°C for 1 hour. 100 μl of the cells at the above concentrations were placed in 1.5 ml centrifuge tubes, and then anti-human FITC-c-Met antibody (purchased from eBioscience) and FITC-c-Met isotype control IgG (purchased from eBioscience) were added at 4°C and incubated in the dark for 30 minutes. The cells were centrifuged at 2000 rpm for 5 minutes at 4°C, washed three times with PBS, and then analyzed using a flow cytometer (CytoFLEX). The results are shown in Figure 4. Figure 4 shows the flow cytometric detection results of c-Met expression in different cells, where the horizontal axis represents the fluorescence signal and the vertical axis represents the cell number.

[0141] The results showed that HCCLM3, HT29, MKN-45, ACHN, and 786-O cells significantly overexpressed c-Met protein, while human hepatic stellate cells LX-2 cells hardly expressed c-Met protein.

[0142] Example 3: Affinity detection of peptides to c-Met positive cells MKN45

[0143] MKN-45 cells containing 1640 culture medium (Nanjing Senbega Biotechnology Co., Ltd.) were collected and washed twice with pre-cooled PBS. 1 mL of 1% BSA solution was added, fixed on a rotating mixer (Lichen Technology Co., Ltd.), and mixed at 4°C for 30 minutes. Different concentrations of FITC-P1-AI, FITC-CM7-1, FITC-CM7-1.1, FITC-CM7-1.2, FITC-CM7-1.3, FITC-CM7-1.4, FITC-CM7-1.5, and FITC-CM7-1.6 peptide solutions were added under light-proof conditions to obtain multiple groups, which were fixed on a rotating mixer and mixed at 4°C for 1 hour in the dark. After incubation, the supernatant was discarded after centrifugation at 800 rpm for 5 minutes. Wash twice with pre-cooled PBS, and adjust the cell concentration to 1×10 6cells / mL. Three replicate wells were prepared for each cell group, and 0.5 mL of each sample was prepared. Flow cytometry was used to detect the binding of the peptide to c-Met-positive MKN-45 cells, and EC50 data and binding curves were generated. The results are shown in Table 2 and Figure 5. Figure 5 shows the binding curve of the disclosed peptide to c-Met-positive MKN-45 cells, where the horizontal axis represents log(peptide concentration) in μM, and the vertical axis represents the binding rate (%).

[0144] Table 2. Binding affinity of peptides to MKN-45

[0145] The results showed that the EC50s of CM7-1.1, CM 7-1.2, CM 7-1.3, CM7-1.4, and CM7-1.6 were lower than that of CM 7-1 (the optimal peptide designed in CN 112979753 B), indicating that CM7-1.1, CM 7-1.2, CM 7-1.3, CM7-1.4, and CM7-1.6 have improved affinity for c-Met-positive MKN45 cells relative to CM7-1. In particular, the EC50s of CM7-1.2 and CM7-1.3 were significantly lower than that of CM 7-1, indicating that CM7-1.2 and CM7-1.3 have significantly improved affinity for c-Met-positive MKN45 cells relative to CM7-1. Furthermore, CM7-1.2 and CM7-1.3 bind to c-Met-positive MKN45 cells in a dose-dependent manner.

[0146] Collect MKN-45 cells containing 1640 culture medium and wash twice with pre-cooled PBS. Add 1mL of 1% BSA solution, fix on a rotating mixer, and mix at 4°C for 30-60min. Add 10μM FITC-P1-AI peptide solution and FITC-CM7-1 to form two groups under light-proof conditions, fix on a rotating mixer, and mix at 4°C for 1h. After incubation, centrifuge at 800rpm for 5min and discard the supernatant. Wash 1-2 times with pre-cooled PBS and adjust the cell concentration to 1×10 6 cells / mL. Three replicate wells were prepared for each group of cells, and 0.5 mL of each sample was prepared. The binding of the peptide to c-Met-positive cells was detected by flow cytometry, and the results are shown in Figure 6. Figure 6 shows the binding curve of the peptide of the present disclosure to c-Met-positive cells MKN-45, where the horizontal axis represents the fluorescence signal and the vertical axis represents the number of cells.

[0147] The results showed that P1-AI and CM7-1 bound to c-Met-positive MKN45 cells. Furthermore, the affinity of P1-AI peptide for c-Met-positive MKN45 cells was significantly higher than that of CM7-1.

[0148] Example 4: Molecular docking

[0149] The amino acids at various positions of the CM7-1 peptide sequence were replaced and docked using a computer and alanine scanning, and the replaced amino acid sequences shown in Table 3 were obtained. The docking scores of these peptide sequences with c-met are also presented in Table 3.

[0150] Table 3. Molecular docking analysis

[0151] The peptides P1-AI (Arg-Ser-Pro-Tyr-Ala-Asn-Ser) and CM7-1 (Asp-Gln-Ile-Ile-Ala-Asn-Asn) were docked with the c-Met protein. As shown in Figure 7, P1-AI had a docking score of -10.162 and a mean hydrophobicity (GRAVY; negative values ​​indicate hydrophilicity, with smaller GRAVY values ​​indicating stronger hydrophilicity) of -0.46. CM7-1 had a docking score of -3.724 and a GRAVY of -1.53. These results indicate that P1-AI has a significantly better docking score than CM7-1 and also exhibits better theoretical water solubility.

[0152] Example 5: Detection of specific binding of peptides to c-Met protein

[0153] Collect human hepatic stellate cells LX-2 containing 1640 culture medium and wash twice with pre-cooled PBS. Add 1 mL of 1% BSA solution, fix on a rotating mixer, and mix at 4°C for 30 minutes. Add 10 μM FITC-P1-AI peptide solution and FITC-CM7-1 to form two groups under light-proof conditions, fix on a rotating mixer, and mix at 4°C for 1 hour. After incubation, centrifuge at 800 rpm for 5 minutes and discard the supernatant. Wash twice with pre-cooled PBS and adjust the cell concentration to 1×10 6 cells / mL. Three replicate wells were prepared for each group of cells at concentrations of 1, 5, and 10 μM, with 0.5 mL of each sample prepared. Flow cytometry was used to detect the binding of the peptides to LX-2 cells, and the results are shown in Figure 8. Figure 8 shows the binding curves of the disclosed peptides to human hepatic stellate cells (LX-2) that barely express c-Met, where the horizontal axis represents the fluorescence signal and the vertical axis represents the cell number.

[0154] As demonstrated in Example 2, human hepatic stellate cells (LX-2) barely express c-Met. The results in Figure 8 show that FITC-P1-AI and FITC-CM7-1 barely bind to LX-2 cells at concentrations up to 10 μM. Taking the results presented in Figures 6-8 together, it is clear that both P1-AI and CM7-1 peptides specifically bind to c-Met, with P1-AI exhibiting superior binding compared to CM7-1.

[0155] Example 6: Peptide solubility test

[0156] 13.4 mg of CM7-1 and 15 mg of P1-AI were weighed and dissolved in ultrapure (UP) water (prepared using the UPR-II-10T Ultrapure Water Manufacturing System). The results are shown in Figure 9. Figure 9 shows the results of the water solubility test of the peptides disclosed herein.

[0157] The results showed that at a concentration of 6 mM, CM7-1 appeared completely turbid in water, while at a concentration of 19 mM, P1-AI formed a clear solution in water, identical to water when observed with the naked eye. This indicates that the water solubility of peptide P1-AI is much higher than that of CM7-1, making it more beneficial for subsequent drug development and production.

[0158] Example 7: Detection of the interaction between peptides and c-Met protein using microscale thermophoresis (MST)

[0159] FITC-P1-AI and FITC-CM7-1 were prepared into liquids with different concentrations, the peptide solution was siphoned using a capillary tube, and the fluorescence absorption value was detected using an MST instrument (OCTED RED 96e).

[0160] Dilute c-Met protein with PBS by 2-fold to form 12 concentration gradients, namely 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.32 μM, 0.16 μM, 0.08 μM, 0.04 μM, 0.02 μM, 0.01 μM, and 0.005 μM. Mix the c-Met protein dilution with FITC-P1-AI or FITC-CM7-1 at a 1:1 volume ratio and let it stand.

[0161] The affinity of FITC-P1-AI or FITC-CM7-1 for c-Met protein was calculated. Data fitting was performed using NT Analysis software, and the results are shown in Figure 10. Figure 10 shows the results of MST detection of the interaction between the peptides of the present disclosure and c-Met protein, wherein the horizontal axis represents the concentration of c-Met protein in μM; the vertical axis represents the fluorescence intensity.

[0162] The results showed that the affinity of P1-AI for c-Met protein was Kd = 41.097 nM, while the affinity of CM7-1 for c-Met protein was only Kd = 1270.355 nM. Therefore, compared with CM7-1, the affinity of P1-AI for c-Met protein was increased by 30.9 times.

[0163] Example 8: In vivo imaging study of subcutaneous transplanted tumors in nude mice

[0164] In MKN-45 subcutaneous transplanted tumor mice (nude mice purchased from Changzhou Cavens Laboratory Animal Co., Ltd.), 5×10 6 MKN45 cells were implanted into the subcutaneous tissue of nude mice and the tumor volume was 400 mm 3 Around 24 hours after injection, 5 mg / kg of Cy5-P1-AI was injected into the tail vein, and its tissue distribution was analyzed using an in vivo imaging instrument. The results are shown in Figure 11. Figure 11 shows the results of in vivo imaging of Cy5-P1-AI in tumor-bearing mice. The circled area in the figure indicates the tumor site.

[0165] The results showed that Cy5-P1-AI was clearly enriched in cancer sites with high c-Met expression.

[0166] Example 9: P1-AI Alanine Scanning Analysis

[0167] Alanine scanning was used to generate peptides as listed in Table 4 using P1-AI as the starting peptide. MKN-45 cells containing 1640 culture medium were collected and washed twice with pre-cooled PBS. 1 mL of 1% BSA solution was added, fixed on a rotating mixer, and mixed at 4°C for 30 minutes. Under light-proof conditions, FITC-P1-AI, FITC-P1-AI-1, FITC-P1-AI-2, FITC-P1-AI-3, FITC-P1-AI-4, FITC-P1-AI-6, and FITC-P1-AI-7 peptide solutions of different concentrations (10, 5, 2.5, 1.25, 0.625 μM) were added to form 7 groups, fixed on a rotating mixer, and mixed at 4°C for 1 hour in the dark. After incubation, centrifuge at 800 rpm for 5 minutes and discard the supernatant. Wash 1-2 times with pre-cooled PBS, and adjust the cell concentration to 1×10 6 cells / mL. Three replicate wells were prepared for each cell group, with 0.5 mL of each sample prepared. Flow cytometry was used to detect the binding of the peptide to c-Met-positive MKN-45 cells. Figure 12 shows the binding curve of the disclosed peptides to c-Met-positive MKN-45 cells, where the horizontal axis represents the fluorescence signal and the vertical axis represents the number of cells. The darker color represents the fluorescence chemical shift after the addition of the peptide, and the lighter color represents the blank control without the addition of the peptide.

[0168] Table 4. Peptide sequences

[0169] Figure 12 shows that P1-AI-4 has a stronger binding affinity for c-Met-positive MKN45 cells than P1-AI, while P1-AI-6 and P1-AI-7 have similar binding affinities to P1-AI. P1-AI-1, P1-AI-2, and P1-AI-3 have lower binding affinities than P1-AI. Sequence alignment reveals that the first three positions (R, S, and P) of the P1-AI peptide sequence are key for maintaining c-Met binding.

[0170] In summary, the binding affinities of P1-AI, P1-AI-4, P1-AI-6 and P1-AI-7 to c-Met are better than that of CM7-1.

[0171] Example 10: Solubility of drug-peptide conjugates

[0172] Preparation of the conjugates of camptothecin (CPT) and P1-AI, i.e., CPT-P1-AI, the conjugates of docetaxel (DOC) and P1-AI, i.e., DOC-P1-AI, and the conjugates of paclitaxel (PTX) and P1-AI, i.e., PTX-P1-AI: 1.20 g of paclitaxel, camptothecin, or docetaxel was reacted with 0.21 g of succinic anhydride (Anhui Zesheng Technology Co., Ltd.) at room temperature for 3 hours to obtain butyrate-paclitaxel, butyrate-camptothecin, and butyrate-docetaxel. 0.88g of paclitaxel butyrate, camptothecin butyrate, or docetaxel butyrate was reacted with 0.53g of EDC (Anhui Zesheng Technology Co., Ltd.) / 0.21g of NHS (Shanghai Aladdin Biochemical Technology Co., Ltd.) dissolved in DCM at room temperature for 9h to obtain paclitaxel butyrate-NHS activated ester, camptothecin butyrate-NHS activated ester, or succinyl-docetaxel-NHS activated ester. 0.51g of each of the three NHS activated esters was dissolved with 0.58g of the P1-AI peptide 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 4h to obtain CPT-P1-AI, DOC-P1-AI, and PTX-P1-AI.

[0173] Preparation of a series of standard solutions of CPT, CPT-P1-AI, DOC, DOC-P1-AI, PTX, and PTX-P1-AI: Weigh 2 mg each of CPT, CPT-P1-AI, DOC, DOC-P1-AI, PTX, and PTX-P1-AI into a 2 mL volumetric flask. Add methanol to the mark and shake well to obtain 1 mg / mL stock solutions. 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. Peak areas of the standard solutions were measured by HPLC (mobile phase: 0.065% TFA in water + 0.05% TFA; column: YMC-Triart C18; wavelength: 220 nm). A concentration-peak area calibration curve was constructed, as shown in Figure 13. In FIG13 , the abscissa represents concentration, and the ordinate represents peak area.

[0174] Supersaturated aqueous solutions of CPT, CPT-P1-AI, DOC, DOC-P1-AI, PTX, and PTX-P1-AI were prepared, sonicated for 15 minutes, and filtered through an organic membrane into liquid phase vials. The peak areas of the samples were determined by HPLC, and their solubility in water was calculated using a concentration-peak area calibration curve. The results are shown in Table 5.

[0175] Table 5. Solubility values ​​of samples in water

[0176] As shown in Figure 13, the concentration-peak area standard curves of CPT, CPT-P1-AI, DOC, DOC-P1-AI, PTX, and PTX-P1-AI have good fitting. As shown in Table 5, CPT, DOC, and PTX are almost insoluble in water, with solubility in water of 8.4×10 -5 , 2.74×10 -5 , 1.2×10 -4 CPT-P1-AI has good solubility in water, which is 913.37 mg / mL, which is 1.09×10 higher than that of the positive drug CPT. 7 times; the solubility of DOC-P1-AI in water is 4.43 mg / mL, which is 1.62×10 higher than that of the positive drug DOC. 5 times, and PTX-P1-AI increased by 46.33 times compared with the positive drug PTX.

[0177] 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, SNG-1005 still does not dissolve in water, which makes it difficult to produce in batches and the relevant clinical trials are forced to be postponed. Machulkin et al. coupled the targeted PSMA ligand with DOC through a linker dipeptide to obtain PSMA-DOC, which has a solubility of 130 μg / mL in water (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 DOC-P1-AI in water is 4.43 mg / mL, which is 1.62×10 higher than the solubility of DOC alone. 5 times, which could greatly improve the application of DOC. Camptothecin's poor water solubility hinders drug absorption. Researchers are currently overcoming this drawback through nano- and micron-scale formulation systems. However, this approach suffers from issues such as low drug loading and low solvent solubility. Chemical group modification of camptothecin has also been used to improve its water solubility. Fan et al. synthesized 7-ethyl-10-[2-oxy-2(piperidinyl)ethoxy]camptothecin by attaching a saturated carbon atom to camptothecin, and its solubility in water is about 6.22 μg / mL (FAN S, CAO YX, LI GY, et al. F10, a new camptothecin derivative, was identified as a new orally-bioavailable, potent antitumor agent[J]. Eur J Med Chem, 2020, 202: 112528.). This solubility still needs to be improved. The solubility of the peptide conjugate CPT-P1-AI in water is 913.37 mg / mL, which is 1.09×10 higher than that of CPT alone. 7 The peptide conjugates of the present invention greatly increase the water solubility of CPT, DOC and PTX.

[0178] 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 peptide targeting c-Met or a derivative thereof comprising the following formula I or formula II, (R)-(X1)-(X2)-(X3)-(X4)-(X5)-(X6) Formula I (Y1)-(Y2)-(Y3)-(Y4)-(A)-(N)-(Y5) Formula II in, X1-X6 independently represent any amino acid or its derivative; Y1-Y5 independently represent any amino acid or a derivative thereof, and Formula II does not include the sequence (D)-(Q)-(I)-(I)-(A)-(N)-(N) of CM7-1; The peptide or its derivative has similar or better binding affinity to c-Met than CM7-1.

2. The peptide or derivative thereof according to claim 1, wherein X1 represents S, H or E, X2 represents P, I or D, X3 represents any amino acid or a derivative thereof, X4 represents A, M or C, X5 represents N, C or P, and X6 represents any amino acid or a derivative thereof; Y1 represents any amino acid or its derivative, Y2 represents Q or A, Y3 represents I or A, Y4 represents I or A, Y5 represents N or A, and formula II does not include (D)-(Q)-(I)-(I)-(A)-(N)-(N).

3. The peptide or derivative thereof according to claim 1 or 2, comprising any one sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3-6, SEQ ID NO: 8, SEQ ID NO: 9-144, SEQ ID NO: 148-150.

4. The peptide or derivative thereof according to claim 1 or 2, wherein the peptide or derivative thereof comprises any one sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3-6, SEQ ID NO: 8, SEQ ID NO: 9-20, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 35, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 79-81, SEQ ID NO: 83-85, SEQ ID NO: 90, SEQ ID NO: 96, SEQ ID NO: 105, SEQ ID NO: 111, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 135, SEQ ID NO: 144, SEQ ID NO: 148-150.

5. The peptide or derivative thereof according to claim 1, comprising the following formula I (R)-(X1)-(X2)-(X3)-(X4)-(X5)-(X6) Formula I in, X1 is S, X2 is P, and X3-6 independently represent any amino acid or a derivative thereof, and the peptide or its derivative has a similar or better binding affinity to c-Met than P1-AI.

6. The peptide or derivative thereof according to claim 5, wherein the peptide or derivative thereof comprises any one sequence selected from the group consisting of SEQ ID NOs: 148-150.

7. The peptide or derivative thereof according to any one of claims 1 to 6, wherein the derivative comprises any one or more modifications selected from the following compared to the peptide: amino acid modification, conservative amino acid substitution, and substitution of hydrogen in an amino acid residue.

8. A drug-peptide conjugate comprising a peptide or a derivative thereof according to any one of claims 1 to 7 and a drug, preferably, wherein the peptide or a derivative thereof is conjugated to the drug directly or through a linker, preferably, the drug is selected from cytotoxic agents, such as at least one of camptothecin, docetaxel, and paclitaxel.

9. A pharmaceutical composition comprising the peptide or derivative thereof according to any one of claims 1 to 7 or the drug-peptide conjugate according to claim 8, and a pharmaceutically acceptable carrier or salt.

10. Use of the peptide or derivative thereof according to any one of claims 1 to 7 for increasing the water solubility of a drug, wherein the peptide or derivative thereof is directly or indirectly conjugated to the drug.

11. A method for increasing the water solubility of a drug, comprising directly or indirectly conjugating the peptide or derivative thereof according to any one of claims 1 to 7 to the drug.

12. A method for detecting, preventing or treating a disease, the method comprising administering to a subject in need thereof a detection, prevention or treatment effective amount of a peptide or derivative thereof according to any one of claims 1 to 7, a drug-peptide conjugate according to claim 8 or a pharmaceutical composition according to claim 9, preferably, wherein the disease comprises cancer, such as a solid tumor and / or a blood tumor, preferably, the disease 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.

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