Bombesin polypeptide compounds and uses thereof

Abstract

The present invention belongs to the field of biopharmaceutical polypeptide technology, specifically relating to a bombesin-like polypeptide compound or a pharmaceutically acceptable salt thereof, and further relates to the use of such a polypeptide or a pharmaceutically acceptable salt thereof in pharmaceuticals for treating GRPR-related conditions. The polypeptides of the present invention exhibit high affinity, high plasma stability, and a long plasma half-life.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical polypeptides, and particularly relates to a bombesin polypeptide compound and uses thereof. Background Art

[0002] Gastrin-releasing peptide (GRP) is a mammalian neuropeptide composed of 27 amino acids. It is a small regulatory peptide homologous to caerulein that was first extracted from the skin of European frogs by Anastasi in 1971. GPR is similar to caerulein in structure and is very conserved at the C-terminus, which is the important part for its biological activity. Therefore, it plays similar biological roles in a variety of mammalian cells and tissues. GPR exerts its biological effects by binding to the GPR receptor (GRPreceptor, GRPR) with high affinity. GRPR also belongs to the G protein-coupled receptor family and contains typical 7 transmembrane domains. [1] GRPR is composed of 384 amino acids and is located on chromosomes Xp22.2-p22.13 (human), XF4 (mice), and Xq21 (rat) in humans, mice, and rats, respectively. Past studies have shown that the GRP / GRPR system widely affects physiological functions, including itch sensation, pain sensation, memory formation and expression, stress response, anxiety, fear, cell proliferation, and chemotaxis in the immune system. [2] .

[0003] GRP and its receptors are widely distributed in the human gastrointestinal tract and central nervous system. In the gastrointestinal tract, it is mainly distributed in the gastric antral G cells, gastric mucosal cells, pancreatic acinar cells, smooth muscle cells, etc. [3] . The distribution of GRP and its receptors in the brain is specific. Immunohistochemical studies have shown that GRPR is highly immunoreactive in the basolateral cerebral cortex, amygdala, hippocampus, hypothalamus, brainstem, nucleus tractus solitarius and many cerebral cortical areas of the mouse brain. Importantly, GRPR expression is limited to the cell bodies and dendrites of neurons and is not expressed in axons and glial cells. [4]. This indicates that GRPR is mainly involved in regulating synaptic transmission in the brain. A large number of studies have shown that GRPR in normal and tumor cells of humans and rodents is directly coupled to Gq protein in G protein, and participates in protein kinase signaling pathways, especially protein kinase C (PKC) / phospholipase C (PLC) and mitogen-activated protein kinase (MAPK) / extracellular signal-regulated protein kinase (ERK) pathways. After GPR binds to GRPR, it activates PLC, causing Ca stored in the cell to 2+ Release and extracellular Ca 2+ Influx, increasing intracellular free Ca 2+ concentration, activates PLC isoenzymes, causes the increase of PKC and MAPK activities, and finally exerts biological effects [5] Therefore, calcium flux test is one of the important indicators for GRPR activity detection.

[0004] Neurodegenerative Disease (NDD) is a type of disease that occurs in the nervous system and causes damage or dysfunction to neurons and their attached dendrites, axons and synapses, as well as glial cells throughout the nervous system. The World Health Organization predicts that by 2040, neurodegenerative diseases will replace cancer and become the second leading cause of death in humans. Currently, there is no drug in the world that can effectively treat neurodegenerative diseases. Parkinson's disease (PD) is a type of neurodegenerative disease. Studies on Parkinson's disease have found that the concentration of BLP (Bombesin-Like Peptides, BLP), a GRP analogue, in the caudate nucleus and globus pallidus of patients is significantly reduced. BLP density was found to be reduced in fibroblasts of Alzheimer's patients, and the Ca-induced 2+ The signal has changed. Pharmacological and genetic studies have confirmed that GRPR is also involved in regulating the pathogenesis of many other neurological and psychiatric diseases, such as anxiety, anorexia and bulimia. In addition, the GRP / GRPR system is also involved in other physiological effects, such as sexual function and itch sensation. Studies have also shown that the regulation of these two functions by GRP / GRPR comes from neurons in the same spinal region. [6] It can be seen that the development of highly active and stable GRP peptides has broad prospects in the treatment of neurodegenerative diseases.

[0005] Additionally, combined diagnostic and therapeutic (theranostic) approaches using radiolabeled molecules targeting molecular targets that are overexpressed in various tumor entities are gaining increasing interest in oncology. The gastrin-releasing peptide receptor (GRPr) is part of the bombesin family and has been shown to be overexpressed in a variety of tumors, and therefore, serves as a promising target for these theranostic applications. A large number of different radiolabeled bombs targeting GRPr derivatives have been evaluated in preclinical and clinical settings, showing rapid blood clearance and urinary excretion with selective GRPr binding. Most of the existing studies on GRPr-targeted imaging and therapy have evaluated theranostic approaches in prostate and breast cancer, applying theranostic agents labeled with the primary use of 68 Ga / 177 A bomb derivative of Lu.

[0006] Based on this, efforts are underway to identify active peptides that specifically target the gastrin-releasing peptide receptor (GRP) to address the current shortage of effective therapeutic drugs. However, peptide drugs suffer from poor membrane permeability and stability, which urgently need to be addressed. Therefore, the present invention aims to develop a highly active GRP agonist based on a bombesin analogue, through peptide sequence modification and performance evaluation, with high affinity, high plasma stability, and a long plasma half-life. This could be used to develop new drugs for the prevention, treatment, therapy, or alleviation of neurodegenerative diseases and cancer.

[0007] Citation List:

[0008] [1]Jensen RT, Battey JF, Spindel ER, et al. International Union of Pharmacology. LXVIII. Mammalianbombesin receptors: nomenclature, distribution, pharmacy, signaling, and functions innormal and disease states [J]. PharmacolRev, 2008, 60 (1): 1-42.

[0009] [2]Roesler R,SchwartsmannG.Gastrin-releasingpeptide receptors in the central nervous system: role inbrain function and as a drug target[J].FrontEndocrinol(Lausanne),2012,3:159.

[0010] [3]Fleischmann A,Waser B,Gebbers JO,et al.Gastrin-relea sing peptidereceptors in normal and neoplastic human uterus:involvement of multiple tissuecompartments[J].J Clin Endo crinol Metab,2005,90(8):4722-4729.

[0011] [4]RoeslerR,KentP,Luft T,etal.Gastrin-releasingpeptide receptorsignalinginthe integration ofstress andmemory[J].Neurobiol LearnMem,2014,112:44-52.

[0012] [5]Petronilho f, vuolo f, galant ls, et al.gastrin-releasing peptidereceptor antagonisminduces protection from lethal sepsis:involvement oftoll-like receptor 4signaling[J].mol med,2012,18:1209-1219.

[0013] [6]RoeslerR, Henriques JA, Schwartsmann G.Gastrin-releasingpeptidereceptoras a molecular target forpsychiatric and neurological disorders[J].CNS Neurol Disord Drug Tar gets,2006,5(2):197-20. Summary of the Invention

[0014] The following is a summary of some aspects of the present invention and is not intended to be limiting. These and other aspects are described more fully below. All references in this specification are incorporated herein by reference in their entirety. In the event of a discrepancy between the disclosure of this specification and a reference, the disclosure of this specification shall prevail.

[0015] The first aspect of the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, characterized in that it comprises a sequence represented by the following formula (I):

[0016] Ser-Tyr-Gln-X 0 -Ala-Leu-βAla-X 1 -X 2 -Nle

[0017] (I)

[0018] in:

[0019] X 0 Selected from Ala, Trp or derivatives of said amino acids;

[0020] X 1 Selected from His, Lys, Arg or derivatives of said amino acids;

[0021] X 2 Selected from Ala, Leu, Phe, Val or derivatives of said amino acids;

[0022] The configuration of each amino acid in the sequence represented by the general formula (I) is independently selected from D-type or L-type;

[0023] The C-terminus and / or N-terminus of the sequence represented by the general formula (I) are modified or unmodified.

[0024] In some embodiments, the Ser, Tyr, X 1 and X 2 The configurations of the two are independently D-type.

[0025] In some embodiments, X0 is Nal or Trp; further, X1 is His or DHis; further, X2 is Cha, Leu, Phe or Nva.

[0026] In some embodiments, the C-terminus of the sequence represented by the general formula (I) is modified by the X-R1 structure.

[0027] in,

[0028] X is connected to the C-terminus of the sequence; X is selected from O, S, or NH; preferably X is O or NH.

[0029] R1 is selected from C1-6 alkyl, C1-6 cycloalkyl, C6-10 aryl or C6-10 heteroaryl; preferably, R1 is selected from methyl, ethyl, isopropyl, tert-butyl, n-pentyl, cyclopropane, phenyl, pyridyl.

[0030] In some embodiments, the polypeptide has one of the following structures or a pharmaceutically acceptable salt thereof:

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038] In some embodiments, the amino acid sequence of the polypeptide or a pharmaceutically acceptable salt thereof is selected from one of the following amino acid sequences: SEQ ID No. 1 to SEQ ID No. 16. DserDTyrGIn-Nal-Ala-Leu-βAla-His-Phe-Nle (SEQ ID No. 1),

[0039] Dser-DTyr-Gln-Trp-Ala-Leu-βAla-His-Phe-Nle(Ethylamide) (SEQ ID No. 2),

[0040] Dser-DTyr-Gln-Trp-Ala-Leu-βAla-His-Phe-Nle(Butylamide) (SEQ ID No. 3),

[0041] Dser-DTyr-Gln-Trp-Ala-Leu-βAla-His-Phe-Nle (Methyl ester) (SEQ ID No. 4),

[0042] Dser-DTyr-Gln-Trp-Ala-Leu-βAla-His-Phe-Nle (Ethyl ester) (SEQ ID No. 5),

[0043] Dser-DTyr-Gln-Trp-Ala-Leu-βAla-D-His-Phe-Nle (SEQ ID No. 6),

[0044] Dser-DTyr-Gln-Trp-Ala-Leu-βAla-His-D-Phe-Nle (SEQ ID No. 7),

[0045] Dser-DTyr-Gln-Trp-Ala-Leu-βAla-His-Leu-Nle (SEQ ID No. 8),

[0046] Dser-DTyr-Gln-Trp-Ala-Leu-βAla-His-D-Leu-Nle (SEQ ID No. 9),

[0047] Dser-DTyr-Gln-Trp-Ala-Leu-βAla-His-4-chloro-Phe-Nle (SEQ ID No. 10),

[0048] Dser-DTyr-Gln-Trp-Ala-Leu-βAla-His-4-methyl-phe-Nle (SEQ ID No. 11),

[0049] Dser-DTyr-Gln-Trp-Ala-Leu-βAla-His-3-chloro-Phe-Nle (SEQ ID No. 12),

[0050] Dser-DTyr-Gln-Trp-Ala-Leu-βAla-D-His-D-phe-Nle (SEQ ID No. 13),

[0051] Dser-DTyr-Gln-Trp-Ala-Leu-βAla-His-Cha-Nle (SEQ ID No. 14),

[0052] Dser-DTyr-Gln-Trp-Ala-Leu-βAla-His-Nva-Nle (SEQ ID No. 15),

[0053] Dser-DTyr-Gln-Trp-Ala-Leu-βAla-His-abu-Nle (SEQ ID No. 16).

[0054] In some embodiments, the polypeptide or a pharmaceutically acceptable salt thereof comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94% or 95% identical to the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 16.

[0055] On the other hand, the present invention also relates to a polynucleotide molecule encoding one or more polypeptide molecules.

[0056] In some embodiments, the polynucleotide molecule can encode one or more of the polypeptide molecules of SEQ ID NO. 1 to SEQ ID NO. 16.

[0057] On the other hand, the present invention also relates to a pharmaceutical composition comprising any one of the polypeptides of the present invention or a pharmaceutically acceptable salt thereof.

[0058] In some embodiments, the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier and / or excipient.

[0059] On the other hand, the present invention relates to use of the polypeptide or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of a medicament for preventing, treating, curing or alleviating neurodegenerative diseases.

[0060] In some embodiments, the neurodegenerative disease comprises cerebral ischemia, brain injury, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinocerebellar ataxia, or Pick's disease.

[0061] In another aspect, the present invention relates to use of the polypeptide or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of a medicament for preventing, treating, curing or alleviating cancer.

[0062] In some embodiments, the cancer comprises breast cancer, prostate cancer, pancreatic cancer, or lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 : Liquid phase detection diagram of compound 9;

[0064] Figure 2 : Mass spectrometry results of compound 9;

[0065] Figure 3 : Liquid phase detection diagram of compound 11;

[0066] Figure 4 : Mass spectrometry results of compound 11;

[0067] Figure 5 : Liquid phase detection diagram of compound 17;

[0068] Figure 6 : Mass spectrometry results of compound 17;

[0069] Figure 7 : Peptide activates GRPR cell EC 50 value;

[0070] Figure 8:Fluorescence imaging detected that the peptide induced GRPR receptor internalization in HEK293-GRPR-GFP cells;

[0071] Figure 9 : In vitro plasma stability of peptides;

[0072] Figure 10 :Fluorescence imaging detected that the peptide induced GRPR receptor internalization on tumor cells. DETAILED DESCRIPTION

[0073] The terms "peptide" or "polypeptide" have meanings well known to those skilled in the art. Generally, a peptide or polypeptide is two or more amino acids linked by an amide bond, which is formed by the amino group of one amino acid and the carboxyl group of the adjacent amino acid. The polypeptides described herein may contain naturally occurring amino acids or non-naturally occurring amino acids. They may be modified to form analogs, derivatives, functional mimetics, pseudopeptides, and the like, comprising at least two amino acids. Unless a specific modification is indicated at the N-terminus or C-terminus, a polypeptide comprising a specific amino acid sequence includes both unmodified and modified amino and / or carboxyl termini, as is well known to those skilled in the art. A polypeptide having a specific amino acid sequence may include modified amino acids and / or additional amino acids, unless the N- and / or C-termini contain modifications that prevent the addition of further amino acids. Such modifications include, for example, acetylation of the N-terminus and / or amidation of the C-terminus.

[0074] The term "amino acid" refers to a molecule containing an amino group and a carboxyl group. Suitable amino acids include, but are not limited to, the D- and L-isomers of naturally occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic pathways. As used herein, the term amino acid includes, but is not limited to, α-amino acids, natural amino acids, non-natural amino acids, and amino acid analogs.

[0075] The term "naturally occurring amino acid" refers to any of the 20 L-amino acids commonly found in peptides synthesized in nature, i.e., the L-isomers of alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glutamine (Glu or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0076] A "conservative amino acid substitution" is one in which the amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., K, R, H), acidic side chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S, T, Y, C), non-polar side chains (e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V, I), and aromatic side chains (e.g., Y, F, W, H). Thus, for example, a predicted nonessential amino acid residue in a polypeptide is preferably substituted with another amino acid residue from the same side chain family. Other examples of acceptable substitutions are substitutions based on isosteric considerations (e.g., norleucine for methionine) or other properties (e.g., 2-thienylalanine for phenylalanine).

[0077] The term "amino acid derivative" refers to a group that can be derived from a natural amino acid, as described and exemplified herein. Amino acid derivatives are obvious to those skilled in the art, and the polypeptides of the present invention can be modified by engineering to form polypeptide derivatives. As is well known to those skilled in the art, various engineering modifications can be made to polypeptides. Typical engineering modifications include, but are not limited to, N-terminal acetylation, C-terminal amidation, d-amino acid substitution, non-natural amino acid substitution, fatty acid modification, or a combination of the above various modifications. The present invention includes any well-known modification of polypeptides. For example, polypeptide derivatives can include chemical modifications to the polypeptide, such as alkylation, acylation, carbamylation, iodination, or any other engineering modifications that produce polypeptide derivatives. Engineering modifications of polypeptides can include engineered amino acids, for example, hydroxyproline or carboxyglutamate, and can include amino acids linked by non-peptide bonds.

[0078] For other modifications of the polypeptides of the present invention, non-natural amino acids can be used to replace the natural amino acids in the polypeptides. Non-natural amino acids include but are not limited to 2-amino fatty acid (Aad), 3-amino fatty acid (βAad), β-alanine, β-aminopropionic acid (βAla), 2-aminobutyric acid (Abu), 4-aminobutyric acid, piperidine carboxylic acid (4Abu), 6-aminohexanoic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (βAib), 2-aminopimelic acid (Apm), 2,4-diaminobutyric acid (Dbu), desmosine (Des), 2,2'-diaminopimelic acid (Dpm), 2,3-diaminopropionic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), hydroxylysine (Hyl), isohydroxylysine (aHyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isodesmosine (Ide), isoleucine (aIle), N-methylglycine (MeGly), N-methylisoleucine (MeIle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), norvaline (Nva), norleucine (Nle), and ornithine (Orn), cyclohexylalanine (Cha), 2-chlorophenylalanine (2-Cl-Phe), 3-chlorophenylalanine (3-Cl-Phe), 4-methylphenylalanine (4-Me-Phe). Of course, all modified α-amino acids can be replaced by the corresponding β-, γ-, or ω-aminocarboxylic acids.

[0079] The term "analog" refers to a substance that shares one or more specific structural features, elements, components or parts with a reference substance. Typically, an "analog" shows significant structural similarity to a reference substance, such as a shared core or shared structure, and also differs in some discrete ways. In some embodiments, an analog is a substance that can be produced from a reference substance, for example, by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be produced by conducting a synthetic process that is substantially similar to (e.g., shares multiple steps with) the synthetic process that produces the reference substance. In some embodiments, an analog is or can be produced by conducting a synthetic process that is different from the synthetic process used to produce the reference substance.

[0080] About sequence identity. Sequence identity is calculated by sequence alignment according to methods known in the art. In order to determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison. For example, a gap can be introduced in the sequence of a first amino acid sequence so as to optimally align with a second amino acid sequence. The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue at the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences. Therefore, % identity = number of identical positions / total number of overlapping positions multiplied by 100. In this comparison, the sequences can be the same length or can be different lengths. The optimal sequence alignment for determining the comparison window can be by the local homology algorithm (J.Theor.Biol., 1981) of Smith and Waterman, by the homology alignment algorithm (J.Mol.Biol, 1972) of Needleman and Wunsch, by the method for Pearson and Lipman, search similarity (Proc.Natl.Acad.Sci.USA, 1988) and carry out, by the computerized implementation (GAP, BESTFIT, FASTA and TFASTA in Wisconsin genetics software package 7.0 versions, Genetic Computer Group, 575, Science Drive, Madison, Wisconsin) of these algorithms or for example use public available computer software for example BLAST.When using this software, preferably use default parameters, for example gap penalty or extension penalty.Select the optimal comparison (i.e., producing the highest identity percentage in the whole comparison window range) produced by the whole bag of tricks.

[0081] The term "homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, if 6 out of 10 positions in the two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in the two sequences match or are homologous, then the two sequences are 95% homologous. Typically, when aligning two sequences, the comparison is performed to give the maximum percentage homology. For example, the comparison can be performed using the BLAST algorithm, where the algorithm parameters are selected to give the maximum match between the sequences over the entire length of each reference sequence. The following references relate to the BLAST algorithm commonly used for sequence analysis: BLAST algorithm (BLAST ALGORITHMS): Altschul, SF et al., (1990) J. Mol. Biol. 215: 403-410; Gish, W. et al., (1993) Nature Genet. 3: 266-272; Madden, TL. et al., (1996) Meth. Fnzymol. 266: 131-141; Altschul, SF et al., (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J. et al., (1997) Genome Res. 7: 649-656. Other conventional BLAST algorithms such as those provided by NCBI BLAST are also well known to those skilled in the art.

[0082] The term "pharmaceutical composition" refers to a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide of the present invention and a pharmaceutically acceptable carrier or excipient. As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and physiologically compatible analogs. Examples of pharmaceutically acceptable carriers or excipients include one or more of the following: water, saline, phosphate-buffered saline, glucose, glycerol, ethanol, and the like, and combinations thereof. In any case, preferably, the composition includes an isotonic agent, e.g., a sugar, a polyol, e.g., mannitol, sorbitol, or sodium chloride. Pharmaceutically acceptable substances may also be included, such as a wetting amount or a trace amount of an auxiliary substance, e.g., a wetting or emulsifying agent, preservative, or buffer that increases the shelf life and effectiveness of the antibody or antibody portion. Optionally, a disintegrant may be included, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof, e.g., sodium alginate. In addition to excipients, pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin, buffers, binders, sweeteners and other flavorings; colorants and polyethylene glycol.

[0083] The composition can be in many forms, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (such as injectable solutions and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form will depend on the established route of administration and therapeutic application. In one embodiment, the composition is in the form of an injectable or infusible liquid, for example, similar to those forms used for passive immunization of humans with antibodies. In one embodiment, the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular), in one embodiment, the polypeptide is administered by intravenous injection or infusion. In another embodiment, the polypeptide is administered by intramuscular or subcutaneous injection.

[0084] Other suitable routes of administration for the pharmaceutical composition include, but are not limited to, rectal, transdermal, vaginal, transmucosal or enteral administration.

[0085] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivering an antibody or antigen-binding fragment. A carrier can be an antiadhesive, a binder, a coating, a disintegrant, a filler or diluent, a preservative (such as an antioxidant, an antibacterial or antifungal agent), a sweetener, an absorption delaying agent, a wetting agent, an emulsifier, a buffer, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (such as olive oil), saline, buffer, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0086] The polypeptides of the present invention can be prepared using methods well known to those skilled in the art, including well-known chemical synthesis methods. Therefore, when a polypeptide or its derivatives contain one or more non-standard amino acids, it is very likely to be prepared by chemical synthesis. In addition to using chemical synthesis methods to prepare polypeptides or their derivatives, they can also be prepared by expressing encoding nucleic acids. This is particularly applicable to the preparation of polypeptides or their derivatives containing only natural amino acids. In this case, well-known methods for preparing nucleic acid encoding polypeptide sequences can be used (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)). The polypeptide can be expressed in an organism and purified using well-known purification techniques.

[0087] Methods for synthesizing polypeptides (such as those described herein) are known in the art. In some peptide synthesis methods, the amino group of one amino acid (or amino acid derivative) is linked to the carboxyl group of another amino acid (or amino acid derivative), which is activated by reaction with a reagent such as dicyclohexylcarbodiimide (DCC). When the free amino group and the activated carboxyl group chemically react, a peptide bond is formed and dicyclohexylurea is released. In such methods, other potentially reactive groups (such as the α-amino group of the N-terminal amino acid or amino acid derivative and the carboxyl group of the C-terminal amino acid or amino acid derivative) can be blocked ("protected") to prevent them from participating in the chemical reaction. Thus, only specific reactive groups react to form the desired product. Protecting groups that can be used for this purpose include, but are not limited to, tert-butyloxycarbonyl (t-Boc) and benzoyloxy groups for protecting amine groups; and simple esters (such as methyl and ethyl) and esters for protecting carboxyl groups. The protecting groups can usually be subsequently removed by treatment that leaves the peptide bonds intact (e.g., treatment with dilute acid). The process of protecting reactive groups (which should not react), coupling to form peptide bonds, and deprotecting the reactive groups can be repeated. Peptides can be synthesized by sequentially adding amino acids to a growing peptide chain. According to the present invention, both liquid-phase and solid-phase peptide synthesis methods are applicable. In solid-phase peptide synthesis methods, the growing peptide chain is usually attached to an insoluble matrix (such as, for example, polystyrene beads) by attaching the C-terminal amino acid to the matrix. At the end of the synthesis, a shearing agent that does not destroy the peptide bonds, such as hydrofluoric acid (HF), can be used to release the peptide from the matrix. At this point, the protecting groups are also usually removed. According to the present invention, automated, high-throughput, and / or parallel peptide synthesis methods can also be used. For more information on peptide synthesis methods, see, e.g., Merrifield (1969) "Solid-phase peptide synthesis," Adv Enzymol Relat Areas Mol Biol., 32:221-96; Fridkin et al. (1974) Annu Rev Biochem., 43(0):419-43; Merrifield (1997) "Concept and Early Development of Solid Phase Peptide Synthesis," Methods in Enzymology, 289:3-13; Sabatino et al. (2009) "Advances in automatic, manual and microwave-assisted solid-phase peptide synthesis," Curr Opin Drug Discov Devel., 11(6):762-70, the entire contents of each of which are incorporated herein by reference.

[0088] In addition, the polypeptides disclosed in the present invention, including their salts, may also exist in the form of their hydrates or in the form of solvents (e.g., ethanol, DMSO, etc.) and may be used for crystallization. The compounds disclosed in the present invention may inherently or by design form solvates with pharmaceutically acceptable solvents (including water); therefore, the compounds of the present invention include both solvated and unsolvated forms.

[0089] Furthermore, the neurodegeneration-related disease disclosed in the present invention is not limited as long as it is a neurodegeneration-related disease.

[0090] The details of one or more embodiments of the present disclosure are set forth in the above description. Although any methods and materials similar or identical to those described herein can be used to implement or test the present invention, preferred methods and materials are described below. Other features, objects, and advantages of the present disclosure will be apparent from the description and claims. In the description and claims, unless the context clearly indicates otherwise, the singular includes the case of plural referents. Unless otherwise defined, all technical and scientific terms used herein have the common meaning understood by those of ordinary skill in the art to which the present invention belongs. All patents and publications cited in the description are incorporated by reference. The following examples are presented to more fully illustrate preferred embodiments of the present invention. These examples should not be construed as limiting the scope of the present invention in any way.

[0091] Example

[0092] The polypeptide compounds and derivatives provided herein are synthesized using a solid-phase synthesis method. The synthetic support is Fmoc-Cys(Trt)-2-Chlotrityl Resin resin. During the synthesis process, the Fmoc-Cys(Trt)-2-Chlotrityl Resin resin is first fully swollen in N,N-dimethylformamide (DMF). The solid-phase support is then subjected to repeated condensation with an activated amino acid derivative, followed by washing, Fmoc deprotection, washing, and the next round of amino acid condensation to achieve the desired polypeptide chain length. Finally, a mixed solution of trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) is reacted with the resin to cleave the polypeptide from the solid-phase support. The crude straight-chain precursor is then precipitated with chilled methyl tert-butyl ether to obtain a solid crude product. The crude polypeptide is then purified and separated using a C-18 reverse-phase preparative chromatography column in 0.1% trifluoroacetic acid in acetonitrile / water to obtain pure polypeptides and their derivatives. The following synthesis section is only an example.

[0093] Experimental reagents

[0094] Serial number Reagents source 1 RinkAmide-AMResin Jill Biochemical 2 Fmoc-Nle-OH Jill Biochemical 3 Fmoc-Phe-OH Jill Biochemical 4 Fmoc-D-His(Trt)-OH Jill Biochemical 5 Fmoc-β-Ala-OH Jill Biochemical 6 Fmoc-Leu-OH Jill Biochemical 7 Fmoc-Ala-OH Jill Biochemical 8 Fmoc-Trp(Boc)-OH Jill Biochemical 9 Fmoc-Gln(Trt)-OH Jill Biochemical 10 Fmoc-D-Tyr(tBu)-OH Jill Biochemical 11 Fmoc-D-Ser(tBu)-OH Jill Biochemical 12 Fmoc-His(Trt)-OH Jill Biochemical 13 Fmoc-Cha-OH Jill Biochemical 14 Fmoc-Nva-OH Jill Biochemical 15 6-Chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) Aladdin 16 N,N-Dimethylformamide (DMF) Anaiji 17 Dichloromethane (DCM) Maclean 18 Trifluoroacetic acid (TFA) Aladdin 19 Triisopropylsilane TCI 20 Acetonitrile Sigma-Aldrich 21 4-Methylpiperidine Maclean 22 Methyl tert-butyl ether TCI 23 4-Methylmorpholine (NMM) TCI

[0095] Example 1. Synthesis of Compound 9

[0096]

[0097] Step 1: Peptide chain synthesis

[0098] 294 mg (0.2 mmol) of RinkAmide-AM Resin was fully swelled in DMF for 1 hour. The following sequence was then synthesized from the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0099] Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min;

[0100] Rinse the resin with DMF 6-8 times until the pH is neutral;

[0101] Dissolve 1.0 mmol Fmoc-AA, 1.0 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour;

[0102] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0103] After peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM, and then dried in vacuo.

[0104] Step 2: Peptide chain cleavage

[0105] Add 10 mL of freshly prepared cleavage cocktail (trifluoroacetic acid: water: triisopropylsilane (95:2.5:2.5, v:v:v)) to the resin obtained in step 1 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate the crude product with 4 volumes of cold MTBE. Wash the crude product three times with MTBE and dry it in a vacuum.

[0106] Step 3: Peptide purification

[0107] The crude polypeptide obtained in step 2 was dissolved in 20% acetonitrile aqueous solution, filtered through a 0.45um membrane, and separated using a reverse-phase high-performance liquid chromatography system. The buffers were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BRC-18 (Saifen) reverse-phase chromatographic column. During the purification process, the chromatograph detection wavelength was set to 230nm, the flow rate was 15mL / min, and the gradient was 35-55% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure polypeptide. The liquid phase mass spectrometry detection results are as follows: Figure 1 、 Figure 2 shown.

[0108] Example 2. Synthesis of Compound 11

[0109]

[0110] Step 1: Peptide chain synthesis

[0111] 294 mg (0.2 mmol) of RinkAmide-AM Resin was fully swelled in DMF for 1 hour. The following sequence was then synthesized from the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0112] Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min;

[0113] Rinse the resin with DMF 6-8 times until the pH is neutral;

[0114] Dissolve 1.0 mmol Fmoc-AA, 1.0 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour;

[0115] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0116] After peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM, and then dried in vacuo.

[0117] Step 2: Peptide chain cleavage

[0118] Add 10 mL of freshly prepared cleavage cocktail (trifluoroacetic acid: water: triisopropylsilane (95:2.5:2.5, v:v:v)) to the resin obtained in step 1 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate the crude product with 4 volumes of cold MTBE. Wash the crude product three times with MTBE and dry it in a vacuum.

[0119] Step 3: Peptide purification

[0120] The crude polypeptide obtained in step 2 was dissolved in 20% acetonitrile aqueous solution, filtered through a 0.45um membrane, and separated using a reverse-phase high-performance liquid chromatography system. The buffers were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BRC-18 (Saifen) reverse-phase chromatographic column. During the purification process, the chromatograph detection wavelength was set to 230nm, the flow rate was 15mL / min, and the gradient was 35-55% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure polypeptide. The liquid phase mass spectrometry detection results are as follows: Figure 3 、 Figure 4 shown.

[0121] Example 3. Synthesis of Compound 17

[0122]

[0123] Step 1: Peptide chain synthesis

[0124] 294 mg (0.2 mmol) of RinkAmide-AM Resin was fully swelled in DMF for 1 hour. The following sequence was then synthesized from the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0125] Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min;

[0126] Rinse the resin with DMF 6-8 times until the pH is neutral;

[0127] Dissolve 1.0 mmol Fmoc-AA, 1.0 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour;

[0128] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0129] After peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM, and then dried in vacuo.

[0130] Step 2: Peptide chain cleavage

[0131] Add 10 mL of freshly prepared cleavage cocktail (trifluoroacetic acid: water: triisopropylsilane (95:2.5:2.5, v:v:v)) to the resin obtained in step 1 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate the crude product with 4 volumes of cold MTBE. Wash the crude product three times with MTBE and dry it in a vacuum.

[0132] Step 3: Peptide purification

[0133] The crude polypeptide obtained in step 2 was dissolved in 20% acetonitrile aqueous solution, filtered through a 0.45um membrane, and separated using a reverse-phase high-performance liquid chromatography system. The buffers were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR C-18 (Saifen) reverse-phase chromatographic column. During the purification process, the chromatograph detection wavelength was set to 230nm, the flow rate was 15mL / min, and the gradient was 35-55% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure polypeptide. The liquid phase mass spectrometry detection results are as follows: Figure 5 、 Figure 6 shown.

[0134] The polypeptide of the present invention can be synthesized by referring to the synthesis method of the above examples.

[0135] Biological evaluation

[0136] Example 4. FLIPR test polypeptide sample activates GRPR receptor to induce cellular calcium flux

[0137] 1) Main experimental materials

[0138] Serial number Material Name source 1 Calcium5assaykit:componentA Molecular Devices 2 probenecid SIGMA 3 CHO-K1 / BB2 cells GenScript 4 CHO-K1 cells Subcon (Shanghai) Biotechnology Co., Ltd.

[0139] 2) Experimental steps

[0140] Step 1: CHO-K1 / BB2 cells were cultured and grown in culture medium (F12K, 10% FBS, 400ug / ml G418). When the cell density reached 80-90% of the culture flask, the cells were rinsed with DPBS and then digested with 0.25% trypsin (containing 0.5mM EDTA). The cell suspension was collected into a centrifuge tube and centrifuged at 1000rpm for 3 minutes. The supernatant culture medium was removed. The cells were resuspended in 6-8ml of fresh growth medium and passaged at a ratio of 1:3 to 1:8. The cells were then cultured in a 37°C, 5% CO2 incubator. The medium was changed or passaged every 2-3 days after passage.

[0141] CHO-K1 cells were cultured and grown in F12K medium (10% FBS). When the cell density reached 80-90% of the flask, cells were rinsed with DPBS and then digested with 0.25% trypsin (containing 0.5 mM EDTA). The cell suspension was collected into a centrifuge tube and centrifuged at 1000 rpm for 3 minutes. The supernatant was removed. 6-8 ml of fresh growth medium was added to resuspend the cells and passaged at a ratio of 1:3 to 1:8. The cells were then cultured in a 37°C, 5% CO2 incubator. After passage, the medium was changed or the cells were subcultured every 2-3 days.

[0142] Step 2: CHO-K1 / BB23 cells and CHO-K1 cells were passaged and expanded to the desired cell number. After digestion and resuspending, the cells were incubated in a 384-well black clear-bottom cell culture plate at a cell density of 8000 cells / well and 25 μl / well of cell resuspension solution for 1 day before use in experimental detection.

[0143] Step 3: Dissolve the peptide in DMSO to 8 mM. Dilute the peptide to a concentration of 5 μM (5×) in 1× Loading buffer (HBSS + 1 g / L glucose + 20 mM HEPES, pH 7.4). Perform a 5-fold serial dilution in 1× Loading buffer for a total of 12 concentrations. Dissolve component A in 10 ml of 1× Loading buffer. Prepare the required amount of calcium dye solution at a ratio of component A: 1× Loading buffer: 250 mM probenecid = 10:18:2. Drain the medium from the overnight cell culture plate and add 50 μl / well of the calcium dye solution. Incubate at room temperature in the dark for 1.5 hours. Place the cell plate and peptide plate in the corresponding positions on the FLIPR and measure the calcium flux signal.

[0144] 3) Results

[0145] The activity of the peptide was evaluated by detecting the effect of the synthetic peptide on the release of calcium ions from the endoplasmic reticulum after activation of CHO-K1 / BB2 cells. The EC50 of the peptide invented by the present invention to activate GRPR cells is good. The specific values ​​are shown in Table 1. Figure 7 .

[0146] Table 1 EC50 values ​​of peptides activating GRPR cells

[0147] Compound number EC50 (nM) Test 1 EC50 (nM) Test 2 EC50 (nM) Test 3 Average value (nM) variance 1 2.85 3.55 3.55 3.32 0.33 2 5.01 3.53 2.81 3.78 0.92 7 1.45 2.88 2.88 2.40 0.67 8 0.54 1.12 1.18 0.95 0.29 9 3.8 3.24 4.36 3.80 0.46 11 0.64 0.66 0.54 0.61 0.05 17 0.46 0.21 0.29 0.32 0.10 18 1.96 0.75 0.53 1.08 0.63 19 12.42 17.63 9.68 13.24 3.30

[0148] Example 5. Fluorescence imaging detection of polypeptide-induced GRPR receptor endocytosis in HEK293-GRPR-GFP cells

[0149] 1) Main experimental materials

[0150] Serial number Material Name source 1 G418 Aladdin, 11811031 / ant-gn-5 / G110917-5 2 HEK293-GRPR-GFP cells self made 3 FBS Hyclone, SV30208.02 4 pancreatic enzymes Gibco, 27250-018 5 PEI40K Yisheng 6 pcDNA3.1(+)-GRPR-GFP plasmid Homemade, with G418 resistance

[0151] 2) Method

[0152] In order to further observe the interaction between the polypeptide of the present invention and GPRP-expressing cells at the cellular level, HEK293 cells were transfected with the pcDNA3.1(+)-GRPR-GFP plasmid (with G418 resistance) using PEI40K transfection reagent. The cells were then screened and cultured in a screening medium containing G418 (1000 μg / mL). After about two weeks of screening and culture, cells that were not transfected and could not stably express GRPR were killed, while cells that could stably express GRPR expanded due to their G418 resistance. Then, using the monoclonal screening method, the cells that grew after G418 screening were plated on 96-well plates at 0.5 cells / well and cultured. After about two weeks, monoclonal cells that all showed strong green fluorescence and were in good cell growth were selected from the 96-well plates for a second round of screening. After the second round of monoclonal screening, the cells that showed complete GFP green fluorescence and were in good condition were the desired HEK293 cells stably expressing GRPR. After obtaining HEK293-GRPR-GFP cells stably expressing GRPR and exhibiting green fluorescence, the HEK293-GRPR-GFP cells were incubated with peptide samples screened using FLIPR. Since GRPR-GFP is a fusion protein, if the peptide binds to the GRPR receptor and is internalized into the cell, the resulting green endocytic particles can be visualized under a fluorescence microscope. Before performing the peptide internalization experiment, a predetermined number of HEK293-GRPR-GFP cells were plated in a black, transparent-bottomed 96-well plate and cultured overnight. The next day, the peptide samples were diluted using serum-free basal medium. After dilution, the cell culture medium was aspirated from the 96-well plate, and the diluted peptide solution was transferred to the cell wells. The cells were then incubated at 37°C for 0.5 h. After incubation, the cells were washed three times with an appropriate amount of 1× DPBS to remove any peptide that was not internalized or remained on the cell surface. The cells were then fixed with 4% paraformaldehyde for 20 min at room temperature. After the cells are fixed, they need to be washed 1-2 times with 1×DPBS, then the nuclei are stained with DAPI nuclear staining solution. After the nuclear staining is completed, the cells are washed 2-3 times with 1×DPBS, and finally observed and photographed under a fluorescence microscope.

[0153] 3) Results

[0154] From the endocytosis photos of HEK293-GRPR-GFP cells incubated with these polypeptides, compared with blank cells, it can be clearly seen that the polypeptides of the present invention are endocytosed in HEK293-GRPR-GFP cells to form fluorescent endocytic particles, and the endocytosis results are obvious. Figure 8 shown.

[0155] Example 6. In vitro plasma stability test

[0156] 1) Control experimental materials

[0157] Serial number Material Name sequence source 1 Negative control (d-TAT) rrrqrrkkrGy Nanjing GenScript Biotechnology Co., Ltd. 2 Positive control (BA3) Dser-DTyr-Gln-Trp-Ala-Val-βAla-His-Phe-Nle self made

[0158] 2) Experimental steps

[0159] Preparation of test samples: Dissolve the peptide to be tested in DMSO or other organic solvents to a final concentration of 100 times 1 mM and store at -20°C until use.

[0160] Plasma thawing: Take out human plasma (sample number * 2.1) mL from the -80℃ freezer and thaw rapidly in a 37℃ water bath.

[0161] Prepare the MIX mixture: Add 693 μL of plasma to a 1.5 mL EP tube. Prepare three replicates for each time point, and prepare three MIX tubes. Add 7 μL of the test sample to each tube to a final concentration of 10 μM, which is detectable or an in vivo drug concentration. Vortex for 30 seconds. Aliquot 100 μL of the aliquots at each time point and incubate. Keep on ice throughout the entire process.

[0162] Incubation: Incubate in a 37°C water bath at six time points: 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min.

[0163] Stop the reaction: After incubation, add 4 volumes of precipitant.

[0164] Mixing: Vortex on a vortex shaker for 30 seconds.

[0165] Centrifuge at 4°C, 15,000 rpm for 10 min. Transfer the supernatant to a vial and analyze by LC-MS / MS.

[0166] 3) Calculation formula

[0167] The dot-line graph with the original drug remaining rate (%) on the ordinate and time on the abscissa shows the degradation trend of the sample in plasma in vitro over time, providing the results of the sample stability.

[0168]

[0169] Calculate the half-life T1 / 2 of a drug in plasma

[0170] The elimination rate constant (Ke) was calculated based on the first-order kinetic formula, and the T1 / 2 (min) of the compound in plasma was further calculated based on the formula.

[0171] T 1 / 2 =-0.693 / Ke

[0172] 4) Results

[0173] From these polypeptide stability tests, it can be seen that the polypeptide of the present invention has good stability in plasma, among which compound 11 has a T 1 / 2 Up to 1229min (see Figure 9 ), the specific values ​​are shown in Table 2. At the same time, from T 1 / 2 The results show that the stability of the polypeptide of the present invention in plasma is much better than that of the positive control.

[0174] Table 2 T of peptides in plasma in vitro 1 / 2

[0175] Compound <![CDATA[T 1 / 2 (min)]]> Negative control (d-TAT) 6652 Positive control (BA3) 8.721 Compound 7 111.0 Compound 8 117.8 Compound 9 318.9 Compound 11 1229 Compound 17 303.7 Compound 18 84.66

[0176] Example 7. Fluorescence imaging detection of polypeptide-induced GRPR receptor endocytosis in tumor cells

[0177] 1) Materials

[0178] Peptide: CY5-labeled peptide sample: compound 17-CY5 (self-made).

[0179] Cells: HPAF-II (Punosai), HuTu-80 (Punosai), and Hs 766T (Nanjing Kebai Biotechnology).

[0180] Reagents and consumables related to endocytosis experiments: 96-well black transparent cell plates (Agilent), fixative (Beyotime); DAPI (Beyotime); 1× DPBS (homemade).

[0181] 2) Method

[0182] To further investigate the interaction between positive peptides and tumor cells, we incubated tumor cells with CY5-labeled positive peptides and observed the internalization of the peptides by tumor cells using fluorescence. The procedure for tumor cell internalization was similar to that for HEK293-GRPR cells in Example 5.

[0183] 3) Results

[0184] Endocytosis imaging images Figure 10 As shown in the figure, we can see that the positive peptide labeled with CY5 can be observed as obvious endocytic red fluorescent particles in HPAF-II, Hs 766T, and HuTu-80 cells respectively.

[0185] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all within the scope defined by the claims of this application.

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof, characterized in that: The polypeptide is one of the following structures: 。 2. A polynucleotide molecule encoding any one of the polypeptides of claim 1.

3. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

4. Use of the polypeptide or pharmaceutically acceptable salt thereof according to claim 1, the polynucleotide molecule according to claim 2, or the pharmaceutical composition according to claim 3 in the preparation of a medicament for preventing or treating cancer; the cancer is selected from at least one of breast cancer, prostate cancer, pancreatic cancer, or lung cancer.

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