Polypeptide conjugates of a chlorotoxin analogue and uses thereof

Abstract

The application belongs to the technical field of biological medicine polypeptide, and particularly relates to a polypeptide conjugate of chlorotoxin analog and application thereof in preparation of a medicine for preventing, treating and curing cancer related diseases. The polypeptide conjugate of chlorotoxin analog is shown as formula (I). The polypeptide drug conjugate of chlorotoxin analog provided by the application improves the solubility of the medicine, has the characteristics of high activity, strong specific binding with a target, obvious intracellular endocytosis, and the ability of increasing the penetration of the medicine through the blood-brain barrier.

Description

Technical Field

[0001] This invention belongs to the field of biomedical polypeptide technology, specifically relating to a polypeptide conjugate of a chlortoxin analog and its use in the preparation of drugs for the prevention, treatment and therapy of cancer-related diseases. Background Technology

[0002] Chlorotoxin (CTX) is a 36-amino acid peptide extracted from the venom of the Israeli golden scorpion (Leiurus quinquestriatus). It contains 36 amino acid residues, including 4 disulfide bonds, and its amino acid sequence is as follows:

[0003] Met 1 -Cys 2 -Met 3 -Pro 4 -Cys 5 -Phe 6 -Thr 7 -Thr 8 -Asp 9 -His 10 -Gln 11 -Met 12 -Ala 13 -Arg 14 -Lys 15 -Cys 16 -Asp 17 -Asp 18 -Cys 19 -Cys 20 -Gly 21 -Gly 22 -Lys 23 -Gly 24 -Arg 25 -Gly 26 -Lys 27 -Cys 28 -Tyr 29 -Gly 30 -Pro 31 -Gln 32 -Cys 33 -Leu 34 -Cys 35 -Arg 36 -NH2(DisuLfide bridge:Cys 2 -Cys 19 Cys 5 -Cys 28 Cys 16 -Cys 33Cys 20 -Cys 35 ).

[0004] Its molecular structural formula is:

[0005]

[0006] Previous studies have shown that chlorine toxin is a chloride channel blocker that can specifically bind to tumor cells. Furthermore, chlorine toxin has been shown to cross the blood-brain barrier. It also exhibits good biocompatibility and no significant toxicity to normal tissues and cells. In addition, chlorine toxin is metabolized slowly in vivo, providing researchers with ample time for imaging and treatment. Therefore, chlorine toxin can be used as a targeting agent to deliver cytotoxic agents and / or imaging agents to various tumors, including metastatic tumors and brain tumors such as malignant gliomas.

[0007] Currently, chloramphenicol is primarily used as a carrier to deliver radioactive isotopes, fluorescent molecules, and other substances into tumors, enabling tumor imaging. This allows for complete and accurate removal of tumor tissue during surgery, preserving normal brain tissue to the greatest extent possible. Chloramphenicol can also be used as a carrier to deliver nanoparticles and drugs into tumors, reducing drug damage to other organs and minimizing side effects. The binding properties of chloramphenicol to glioma cells were first studied with 125I-labeled small peptides (125I-CTX) and 131I-labeled small peptides (131I-CTX). Results showed that 125I-CTX could accumulate in tumors of tumor-bearing mice and specifically bind to glioma cells, but not to normal astrocytes. 131I-CTX is currently the most widely studied chloramphenicol complex; its emitted radiation can be detected to identify and locate brain tumors. Currently, 131I and indocyanine green (ICG)-labeled chloramphenicol have passed preclinical safety trials in the United States and have entered Phase II / I clinical trials, respectively. In another study, chloramphenicol was found to bind to neuroectodermal tumors (tumors sharing embryonic origin with cells of the central nervous system). It was also found that chloramphenicol conjugated with biotin could bind to biopsy samples of more than 200 types of malignant gliomas and other tumors at different stages, including melanoma, neuroblastoma, medulloblastoma, and small cell lung cancer, but not to normal tissues of the brain, skin, kidneys, and lungs. Simultaneously, some fluorescent dyes, such as Cy5.5, BLZ-100, and 800CW, when conjugated with chloramphenicol, could specifically target tumors in vivo. Furthermore, chloramphenicol could deliver nanoprobes, magnetic resonance imaging contrast agents, and therapeutic drugs to tumor tissues. Other chloramphenicol conjugates, including fusion proteins, such as the chloramphenicol-GST fusion protein conjugated with saponins, have also been shown to significantly and selectively kill tumor cells. To develop new tools for the diagnosis and treatment of gliomas, many CTX-based nanoparticles have been constructed. In addition, chloramphenicol has the potential to serve as a carrier for specifically delivering anticancer drugs to cancer cells. Chlorine toxins showed binding to glioma cells but not to normal rat astrocytes and human rhabdomyosarcoma cell lines. It has great potential as a selective targeted drug for human tumors and a specific marker for diagnosis (including grade determination).

[0008] Brain tumors, or more accurately, intracranial tumors, are classified into primary brain tumors and secondary malignant tumors originating from other organs. Malignant gliomas are a type of brain tumor and one of the most difficult types of cancer to treat. Common treatments include surgery, radiotherapy, chemotherapy, and targeted therapy, but if recurrence occurs, the average survival time is less than 12 months. The poor treatment outcomes for malignant brain tumors are due to two main reasons: first, surgery often fails to completely remove the tumor, raising concerns about potential damage to core brain functions; second, most anticancer drugs struggle to reach the tumor site because of a protective barrier called the blood-brain barrier (BBB). For example, paclitaxel (PTX), a common chemotherapy drug, cannot penetrate the brain due to the BBB and is therefore ineffective against gliomas.

[0009] Traditional treatments, lacking specificity for tumor cells, damage normal tissue cells while killing tumor cells. Furthermore, due to the presence of the brain tumor barrier (BBB), the search for novel targeted drug delivery systems has become a crucial direction in cancer treatment research. Therefore, there is a significant unmet clinical need for the treatment of brain tumors, making the development of novel brain tumor drugs urgent. Chinese patent CN102844044A discloses a lysine-reduced chloramphenicol polypeptide with no more than one usable binding site. In some embodiments, the provided lysine-reduced chloramphenicol polypeptide and / or its conjugates can be used in pharmaceuticals (e.g., in various therapeutic and / or diagnostic contexts). Based on the properties of chloramphenicol, this invention provides a novel polypeptide conjugate of a chloramphenicol analogue, which holds promise for developing a promising drug for brain tumors. Through artificial modification, its inherent advantages can be further enhanced, increasing its stability and efficacy while reducing toxicity. Summary of the Invention

[0010] The following is a brief overview of some aspects of the invention and is not intended to limit it. These aspects and other parts are described in more detail later. All references in this specification are incorporated herein by reference in their entirety. In the event of any discrepancy between the disclosure in this specification and the cited references, the disclosure in this specification shall prevail.

[0011] The purpose of this invention is to address the unmet clinical need in the treatment of malignant brain tumors by providing a novel polypeptide conjugate of chlortoxin analogues.

[0012] In a first aspect, the present invention provides a polypeptide conjugate of a chlortoxin analogue, comprising the structure shown in formula (I):

[0013] Peptide-(Linker-Drug)m

[0014] (I)

[0015] Where: Peptide is a polypeptide; Linker is a linker; Drug is an anticancer agent; m is 1, 2 or 3;

[0016] The amino acid sequence of the polypeptide is selected from one of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 8, and the amino acid sequence may or may not be modified.

[0017] SEQ ID NO: 1: MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR;

[0018] SEQ ID NO: 2: MCMPCFTTDHQMARRCDDCCGGRGRCYGPQCLCR;

[0019] SEQ ID NO: 3: MCMPCFTTDHQMARKCDDCCGGRGRGRCYGPQCLCR;

[0020] SEQ ID NO: 4: MCMPCFTTDHQMARRCDDCCGGKGRG RCYGPQCLCR;

[0021] SEQ ID NO: 5: MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR;

[0022] SEQ ID NO: 6: MCMPCFTTDHQMARRCDDCCGGKGRGKCYGPQCLCR;

[0023] SEQ ID NO: 7: MCMPCFTTDHQMARACDDCCGGKGRGKCYGPQCLCR;

[0024] SEQ ID NO: 8: NleCNlePCFTTDHQNleARRCDDCCGGRGRGKCYGPQCLCR.

[0025] Preferably, the amino acid sequence of the polypeptide is selected from one of the amino acid sequences shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and the amino acid sequence may or may not be modified.

[0026] In some embodiments, the amino acid sequence of the polypeptide may be modified, the modification being selected from N-terminal acetylation and / or C-terminal amidation.

[0027] In some embodiments, the amino acid sequence of the polypeptide has at least 85% overall sequence identity with SEQ ID NO: 1 to SEQ ID NO: 8.

[0028] In some embodiments, the amino acid sequence of the polypeptide has at least 90% sequence identity with SEQ ID NO: 1 to SEQ ID NO: 8.

[0029] In some embodiments, the amino acid sequence of the polypeptide has at least 95% sequence identity with SEQ ID NO: 1 to SEQ ID NO: 8.

[0030] In some embodiments, the amino acid sequence of the polypeptide has at least one site that can be used for linker linking, wherein the site that can be used for linker linking is -NH2 and / or -COOH in the amino acid sequence.

[0031] In some embodiments, the amino acid sequence of the polypeptide has at least one site that can be used for linker linking, wherein the -NH2 site that can be used for linker linking is the -NH2 of the lysine side chain.

[0032] In some embodiments, the amino acid sequence of the polypeptide has at least one site that can be used for linker linking, wherein the -NH2 site that can be used for linker linking is the -NH2 of the N-terminal methionine of the peptide chain;

[0033] Furthermore, the -NH2 site that can be used for linker linkage corresponds to the 1st, 15th, 23rd and / or 27th amino acid sequence of the polypeptide.

[0034] In some embodiments, the amino acid sequence of the polypeptide has at least one site that can be used for linker linking, wherein the -COOH site that can be used for linker linking is the -COOH of the aspartic acid or glutamic acid side chain.

[0035] In some embodiments, the connector is independently selected from the following structures or any combination of the following structures:

[0036] -GALGLPG-, where p is 1, 2, 3, 4 or 5.

[0037] Furthermore, the connector is independently selected from the following structures or any combination of the following structures. -GALGLPG-.

[0038] Furthermore, the connector is independently selected from the following structures or any combination of the following structures. -GALGLPG-.

[0039] Preferably, the present invention provides a polypeptide conjugate, characterized in that it has the structure shown in formula (II):

[0040]

[0041] Where: Peptide is a polypeptide, and Drug is an anticancer agent;

[0042] m is 1, 2, or 3;

[0043] The amino acid sequence of the polypeptide is selected from one of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 8, and the amino acid sequence may or may not be modified.

[0044] Preferably, the present invention provides a polypeptide conjugate, characterized in that it has the structure shown in formula (III):

[0045]

[0046] Where: Peptide is a polypeptide, and Drug is an anticancer agent;

[0047] m is 1, 2, or 3;

[0048] The amino acid sequence of the polypeptide is selected from one of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 8, and the amino acid sequence may or may not be modified.

[0049] Preferably, the present invention provides a polypeptide conjugate, characterized in that it has the structure shown in formula (IV):

[0050]

[0051] Where: Peptide is a polypeptide, and Drug is an anticancer agent;

[0052] m is 1, 2, or 3;

[0053] The amino acid sequence of the polypeptide is selected from one of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 8, and the amino acid sequence may or may not be modified.

[0054] Preferably, the present invention provides a polypeptide conjugate, characterized in that it has the structure shown in formula (V):

[0055]

[0056] Where: Peptide is a polypeptide, and Drug is an anticancer agent;

[0057] m is 1, 2, or 3;

[0058] The amino acid sequence of the polypeptide is selected from one of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 8, and the amino acid sequence may or may not be modified.

[0059] In some embodiments, the anticancer agent is selected from BCNU, cisplatin, gemcitabine, hydroxyurea, paclitaxel, temozolomide, topotecan, fluorouracil, vincristine, vinblastine, procarbazine, dacarbazine, hexamethylmelamine, methotrexate, mercaptopurine, thioguanine, fludarabine phosphate, cladribine, pentostatin, cytarabine, azacitidine, etoposide, teniposide, irinotecan, docetaxel, doxorubicin, daunorubicin, actinomycin D, idarubicin, procainoxine, etc. Mitomycin, bleomycin, tamoxifen, flutamide, leuprorelin, goserelin, aminoglutethimide, anastrozole, acridine, asparaginase, mitoxantrone, mitoxantrone, amifostine, oflambumab, bevacizumab, tosimomab, alemtuzumab, cetuximab, trastuzumab, gemtuzumab, oxozamicin, rituximab, panitumumab, tiimomab, maytansine, camptothecin, and / or their analogues (e.g., DM1) or combinations thereof.

[0060] In some embodiments, the anticancer agent in the polypeptide conjugate of the present invention is a poorly water-soluble compound. As those skilled in the art will recognize, a variety of poorly water-soluble anticancer agents are suitable for the present invention. For example, the anticancer agent may be further selected from taxanes, which are considered effective drugs for treating solid tumors refractory to many other antitumor agents. More preferably, the anticancer agent is paclitaxel, docetaxel, or a combination thereof.

[0061] In some embodiments, the anticancer agent in the polypeptide conjugate of the present invention is maytansine and / or its analogues (e.g., DM1).

[0062] In some embodiments, the polypeptide conjugates provided by the present invention are further substituted with lipophilic substituents, wherein the lipophilic substituents may be independently selected from the following groups: q is any integer from 0 to 20; n is 12, 13, 14, 15, 16, 17, 18, 19, or 20. Further, n is 12, 14, or 16.

[0063] Lipophilic substituents are directly linked to peptide conjugates or can be linked to peptide conjugates via linkers, and are independently selected from the following groups: The connector can be one of the connectors mentioned above or any combination thereof.

[0064] Preferably, the polypeptide conjugate provided by the present invention is further substituted, and the substituents may be independently selected from the following groups:

[0065]

[0066] This invention provides a polypeptide conjugate, the structure of which is selected from one of the following structures:

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] On the other hand, the present invention also relates to a pharmaceutical composition comprising any of the polypeptide conjugates described in the present invention.

[0078] In some embodiments, the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier and / or excipient.

[0079] On the other hand, the present invention relates to the use of the aforementioned polypeptide conjugate or pharmaceutical composition in the preparation of a medicament for the prevention, treatment, or relief of cancer, wherein the medicament is used for the prevention, treatment, or relief of cancer.

[0080] In some embodiments, the cancer includes, but is not limited to, breast cancer, lung cancer, prostate cancer, kidney cancer, leukemia, ovarian cancer, stomach cancer, uterine cancer, endometrial cancer, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, brain tumor, skin cancer, lymphoma, or multiple myeloma; preferably, the cancer is a brain tumor; further, the cancer is a glioma.

[0081] The advantages of this invention are:

[0082] (1) The chlorine toxin analogue peptide drug conjugate provided by the present invention utilizes hydrophilic peptides to modify hydrophobic antitumor drugs, thereby improving the solubility of the drugs.

[0083] (2) The polypeptide conjugate provided by the present invention has the characteristics of high activity, strong specific binding to the target site, and significant endocytosis. When the conjugate of the present invention is administered to patients, it can increase the specificity of the target cells (especially tumor cells), increase cell internalization, reduce cell degradation, increase the accumulation of the target site, reduce the accumulation of normal tissue, reduce its biotoxicity, overcome drug resistance, increase the bioactivity of the drug and / or prevent, limit or eliminate the problems of adverse side effects, toxicity and ineffectiveness compared with the administration of the therapeutic agent alone (i.e., not as part of the conjugate of the present invention).

[0084] (3) Chlorine toxin has a good ability to penetrate the blood-brain barrier. When anti-tumor drugs are coupled with it, the ability of the drugs to penetrate the blood-brain barrier can be greatly increased, and the effect on the indication of brain tumors can be better.

[0085] the term

[0086] The meaning of the terms "peptide" or "polypeptide" is 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 an adjacent amino acid. Polypeptides described herein may contain naturally occurring or non-naturally occurring amino acids. They can be modified into analogs, derivatives, functional mimics, pseudopeptides, and other compounds containing at least two amino acids. Unless a specific modification is specified at the N-terminus or C-terminus, a polypeptide containing a particular amino acid sequence includes both unmodified and modified amino and / or carboxyl terminals, as is well known to those skilled in the art. A polypeptide with a particular amino acid sequence may include modified amino acids and / or additional amino acids unless the N- and / or C-terminus contain modifications that prevent the further addition of amino acids. Such modifications include, for example, acetylation of the N-terminus and / or amidation of the C-terminus.

[0087] The peptides of this invention can be modified to form peptide derivatives. As is well known to those skilled in the art, various modifications can be made to the peptides. Typical 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 combinations of the above modifications. This invention includes any well-known modification of peptides. For example, peptide derivatives may include chemical modifications to the peptide, such as alkylation, acylation, carbamylation, iodination, or any other modification that produces peptide derivatives. The modification of the peptide may include modified amino acids, such as hydroxyproline or carboxyglutamic acid, and may include amino acids linked by non-peptide bonds.

[0088] 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 acids (Aad), 3-amino fatty acids (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), desmokinin (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), isodesin (Ide), isoleucine (aIle), N-methylglycine (MeGly), N-methylisoleucine (MeIle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), n-valine (Nva), n-leucine (Nle), and ornithine (Orn). Of course, all modified α-amino acids can be replaced by the corresponding β-, γ-, or ω-aminocarboxylic acids.

[0089] The term "amino acid" refers to a molecule containing both an amino and a carboxyl group. Suitable amino acids include, but are not limited to, D- and L-isomers of naturally occurring amino acids, as well as non-naturally occurring amino acids prepared through organic synthesis or other metabolic pathways. As used herein, the term amino acid includes, but is not limited to, those containing, amino acids. α - Amino acids, natural amino acids, non-natural amino acids, and amino acid analogs.

[0090] The term "naturally occurring amino acid" refers to any one of the 20 L-amino acids commonly found in peptides synthesized in nature, namely, 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).

[0091] "Conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having 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), nonpolar side chains (e.g., A, V, L, I, P, F, M, W), β-branched side chains (e.g., T, V, I), and aromatic side chains (e.g., Y, F, W, H). Thus, for example, a predicted non-essential amino acid residue in a polypeptide is preferably replaced by another amino acid residue from the same side chain family. Other examples of acceptable substitutions are substitutions based on isosteric considerations (e.g., leucine replacing methionine) or other properties (e.g., 2-thienylalanine replacing phenylalanine).

[0092] 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 derivative contains one or more non-standard amino acids, it is highly likely that it was prepared by chemical synthesis. In addition to preparing polypeptides or their derivatives using chemical synthesis, they can also be prepared by expression encoding nucleic acids. This is particularly suitable for preparing polypeptides or their derivatives containing only natural amino acids, in which case well-known methods for preparing nucleic acid-encoded polypeptide sequences can be used (see Sambrook et al., Molecular Cloning: A La Borate 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 polypeptides can be expressed in organisms and purified using known purification techniques.

[0093] The term "analyte" refers to a substance that shares one or more specific structural features, elements, components, or portions with a reference substance. Typically, an "analyte" exhibits significant structural similarity to a reference substance, such as sharing a core or structure, and also differs in certain discrete manners. In some embodiments, an analogue is a substance that can be produced from a reference substance, for example, through chemical manipulation of the reference substance. In some embodiments, an analogue is a substance that can be produced by performing a synthetic process substantially similar to (e.g., sharing multiple steps) the synthetic process used to produce the reference substance. In some embodiments, an analogue is produced by, or can be produced by, a synthetic process different from the synthetic process used to produce the reference substance.

[0094] The term "chloramphenicol analogue" refers to a polypeptide whose amino acid sequence shows at least 45% identity with the amino acid sequence of a suitable reference chloramphenicol (e.g., the amino acid sequence of SEQ ID NO:1 or a related fragment thereof). In some embodiments, the chloramphenicol polypeptide has an amino acid sequence showing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:1 or a related fragment thereof. In some embodiments, the chloramphenicol analogue has the same amino acid sequence as SEQ ID NO:1. In some embodiments, the chloramphenicol analogue is a chloramphenicol variant because it has an amino acid sequence different from the amino acid sequence of SEQ ID NO:1 or a related fragment thereof. In some embodiments, the chloramphenicol variant has an amino acid sequence that differs from SEQ ID NO:1 or its associated fragment at no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions. In some embodiments, the associated fragment of SEQ ID NO:1 comprises at least 5 adjacent residues of SEQ ID NO:1. In some embodiments, the associated fragment of SEQ ID NO:1 comprises a range of 5 to 25 amino acids, said amino acid range having at least 45% sequence identity with the corresponding range of SEQ ID NO:1. Examples of chloramphenicol analogues suitable for use in the practice of this invention are described in International Application WO2003 / 101474 (the entire contents of which are incorporated herein by reference). Specific examples include polypeptides comprising or composed of SEQ ID NO.1 or SEQ ID NO.8, and variants thereof.

[0095] Regarding sequence identity, sequence identity is calculated through sequence alignment according to methods known in the art. To determine the percentage of identity between two amino acid sequences, the sequences are aligned for optimal alignment. For example, a vacancy may be introduced into the first amino acid sequence to achieve optimal alignment with the second amino acid sequence. The amino acid residues at the 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 percentage identity between two sequences is a function of the number of shared positions. Therefore, % identity = number of shared positions / total number of overlapping positions multiplied by 100. In this comparison, the sequences may be of the same length or may be of different lengths. The optimal sequence alignment for determining the comparison window can be performed using Smith and Waterman's local homology algorithm (J. Theor. Biol., 1981), Needleman and Wunsch's homology alignment algorithm (J. Mol. Biol., 1972), or Pearson and Lipman's method for finding similarity (Proc. Natl. Acad. Sci. USA., 1988). This can be achieved through computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetic Software Package version 7.0, Genetic Computer Group, 575, Science Drive, Madison, Wisconsin) or, for example, using publicly available computer software such as BLAST. When using such software, default parameters, such as gap penalties or extension penalties, are preferred. The optimal alignment produced by the various methods (i.e., the one that yields the highest percentage of identity across the entire comparison window) is selected.

[0096] The term "cancer" refers to a disease, condition, or disorder in which cells exhibit relatively abnormal, uncontrolled, and / or autonomous growth, resulting in an abnormally elevated rate of proliferation and / or an abnormal growth phenotype, characterized by a significant loss of control over cell proliferation. In some embodiments, cancer may be characterized by one or more tumors. In some embodiments, cancer may be or include precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic cells. In some embodiments, the associated cancer may be characterized by a solid tumor. In some embodiments, the associated cancer may be characterized by a hematologic malignancy. Typically, examples of different types of cancer known in this art include, for example, cancers of the hematopoietic system, including leukemia, lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma), myeloma and myeloproliferative disorders; sarcoma, melanoma, adenoma, solid tissue cancer, squamous cell carcinoma of the oral cavity, pharynx, larynx and lungs, liver cancer, cancers of the genitourinary system (such as prostate cancer, cervical cancer, bladder cancer, uterine cancer, endometrial cancer and renal cell carcinoma), bone cancer, pancreatic cancer, skin cancer, melanoma of the skin or eye, cancers of the endocrine system, thyroid cancer, parathyroid cancer, head and neck cancer, brain tumors, breast cancer, gastrointestinal cancer and cancers of the nervous system, benign lesions (such as papilloma), and so on.

[0097] The term "anticancer agent" has the meaning understood in its field of application, referring to one or more apoptosis-promoting agents, cell inhibitors, and / or cytotoxic agents, such as agents specifically included for and / or recommended for the treatment of one or more diseases, conditions, or disorders associated with unwanted cell proliferation. In some embodiments, an anticancer agent may be or include one or more alkyl agents, one or more anthracycline drugs, one or more cytoskeleton disruptors (e.g., microtubule-targeting moieties, such as taxanes, maytansine, and analogues thereof), one or more epothilosomatic agents, one or more histone deacetylase inhibitors (HDAC), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and / or one or more analogues of the following (i.e., sharing associated antiproliferative activity).In some specific implementations, the anticancer agent may be or include one or more of the following: BCNU, Actinomycin, all-trans retinoic acid, Auiristatin, Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Curcumin, Cytarabine, Daunorubicin, and Docetaxel. Doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, maytansine, and / or their analogues (e.g., DM1), mechlorethamine, mercaptopurine, methotrexate, mitoxantron e), Maytansinoid, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, and combinations thereof.

[0098] The term "pharmaceutical composition" refers to pharmaceutical compositions comprising therapeutically effective amounts of the polypeptides of the present invention and pharmaceutically acceptable carriers or excipients. As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antimicrobial 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, the composition preferably includes an isotonic agent, such as sugars, polyols, such as mannitol, sorbitol, or sodium chloride. Pharmaceutically acceptable substances may also be included, such as wetting amounts or trace amounts of excipients, such as wetting or emulsifying agents, preservatives, or buffers that improve the shelf life and effectiveness of antibodies or antibody moieties. Optionally, disintegrants may be included, such as cross-linked polyvinylpyrrolidone, agar, alginate, or salts thereof, such as sodium alginate. In addition to excipients, the pharmaceutical composition may also include one or more of the following: carrier proteins such as serum albumin, buffers, binders, sweeteners and other flavoring agents; colorants and polyethylene glycol.

[0099] The composition can be in many forms, such as liquids, semi-solids, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. Preferred forms will depend on the intended route of administration and therapeutic application. In one embodiment, the composition is in an injectable or infusionable liquid form, such as those used for passive immunization of humans with antibodies. In one embodiment, the route of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular); in another embodiment, the peptide is administered by intravenous injection or infusion. In yet another embodiment, the peptide is administered by intramuscular or subcutaneous injection.

[0100] Other suitable routes of administration for this pharmaceutical composition include, but are not limited to, rectal, transdermal, vaginal, transmucosal, or enteral administration.

[0101] Methods for synthesizing peptides (as described herein) that are analogues of chloramphenicol are known in the art. In some peptide synthesis methods, the amino group of one amino acid (or amino acid derivative) is attached to the carboxyl group of another amino acid (or amino acid derivative), which is activated by a reaction with a reagent such as dicyclohexylcarbodiimide (DCC). When the free amino group and the activated carboxyl group react chemically, a peptide bond is formed and dicyclohexylurea is released. In such methods, other potentially active groups (such as N-terminal amino acids or amino acid derivatives) can be blocked (“protected”). αProtecting groups (such as the amino group and the carboxyl group of the C-terminal amino acid or amino acid derivative) are used to prevent them from participating in chemical reactions. Thus, only specific active groups react to form the desired product. Protecting groups that can be used for this purpose include, but are not limited to, tert-butoxycarbonyl (t-Boc) and benzoyloxy groups for protecting the amino group; and simple esters (such as methyl and ethyl) and esters for protecting the carboxyl group. The process of protecting the reactive group (which should not react), coupling to form a peptide bond, and deprotecting the reactive group can usually be repeated by leaving intact peptide bonds (e.g., treatment with dilute acid). Peptides can be synthesized by sequentially adding amino acids to a growing peptide chain. According to the invention, both liquid-phase and solid-phase peptide synthesis methods are applicable. In solid-phase peptide synthesis methods, the growing peptide chain is typically attached to an insoluble matrix (e.g., polystyrene beads) by attaching the C-terminal amino acid to the matrix. At the end of the synthesis, the peptide can be released from the matrix using a cleaving agent that does not break the peptide bonds, such as hydrofluoric acid (HF). At this time, 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, for example, 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,” Curr0pin Drug Discov Devel., 11(6):762-70, all of which are incorporated herein by reference.

[0102] Furthermore, the polypeptides disclosed in this invention, including their salts, may also exist in their hydrated form or in the form of solvents containing them (e.g., ethanol, DMSO, etc.), and can be used for crystallization. The compounds disclosed in this invention can inherently or by design form solvates with pharmaceutically acceptable solvents (including water); therefore, the compounds of this invention include both solvated and unsolvated forms. Attached Figure Description

[0103] Figure 1 : Liquid chromatography detection results for compound 17;

[0104] Figure 2 Results of LC-MS / MS analysis of compound 17;

[0105] Figure 3 : Liquid chromatography detection results for compound 20;

[0106] Figure 4 Results of LC-MS / MS analysis of Compound 20;

[0107] Figure 5 : Liquid phase detection results of compound 23;

[0108] Figure 6 Results of LC-MS / MS analysis of compound 23;

[0109] Figure 7 Example 1: Killing curve of U87-MG glioma cells by the PDC molecules of this invention;

[0110] Figure 8 Example 1: Killing curve of U373 glioma cells using PDC molecules of the present invention;

[0111] Figure 9 Statistical results of the average fluorescence signal of U87-MG glioma cells 4 hours after endocytosis of polypeptide molecules in Test Example 2;

[0112] Figure 10 Statistical results of the average fluorescence signal of U373 glioma cells 4 hours after endocytosis of polypeptide molecules in Test Example 2;

[0113] Figure 11 Statistical results of the average fluorescence signal of U87-MG glioma cells 24 hours after endocytosis of polypeptide molecules in Test Example 2;

[0114] Figure 12 Statistical results of the average fluorescence signal of glioma cells U373 24 hours after endocytosis of polypeptide molecules in Test Example 2;

[0115] Figure 13 : Confocal imaging results of U87-MG glioma cells endocytosed with polypeptide molecules (3uM) 4 hours after test example 2;

[0116] Figure 14 : Confocal imaging results of U373 glioma cells endocytosed with polypeptide molecules (3uM) 4 hours after test example;

[0117] Figure 15 : Confocal imaging results of U87-MG glioma cells endocytized with a polypeptide molecule (1 uM) 24 h after test Example 2;

[0118] Figure 16 : Confocal imaging results of U373 glioma cells 24 hours after endocytosis of a polypeptide molecule (1 μM);

[0119] Figure 17 Example 3: Experimental results of the stability test of PDC molecules in human plasma according to the present invention;

[0120] Figure 18 Example 4: In vitro evaluation results of peptide molecules penetrating the blood-brain barrier;

[0121] Figure 19 : Drug-time curves for compound 24, where A is the drug-time curve for brain tissue and B is the drug-time curve for plasma;

[0122] Figure 20 Drug-time curves for compound 25, where A is the drug-time curve for brain tissue and B is the drug-time curve for plasma.

[0123] Figure 21 Drug-time curves for compound 26, where A is the drug-time curve for brain tissue and B is the drug-time curve for plasma.

[0124] Figure 22 : AUC blood-brain ratio calculation results. Detailed Implementation

[0125] The polypeptide compounds and their derivatives disclosed herein are synthesized using a solid-phase synthesis method, with Rink Amide-AM Resin resin as the synthesis support. During the synthesis process, the Rink Amide-AM Resin resin is first fully swollen in N,N-dimethylformamide (DMF). Then, the solid support is repeatedly subjected to condensation with the activated amino acid derivative, followed by washing, deprotection of the Fmoc, washing, and the next round of amino acid condensation to achieve the desired polypeptide chain length. N-terminal amidation is then performed on the solid phase, followed by coupling with AEEA and FITC or coupling with tetradecanoic acid. Subsequently, a mixed solution of trifluoroacetic acid:water:triisopropylsilane:aniline sulfide (90:2.5:2.5:5, v:v:v:v) is reacted with the resin to cleave the polypeptide from the solid support. The cleavage is then completed by precipitation with frozen methyl tert-butyl ether to obtain the crude solid product of the linear precursor. The crude linear precursor, after being cleaved, underwent disulfide bond oxidation in a weakly alkaline solution. The resulting product was then purified using a C-18 reversed-phase preparative chromatography column with a 0.1% trifluoroacetic acid in acetonitrile / water system to obtain the oxidized peptide. The oxidized peptide was then coupled with a PTX conjugate in the liquid phase. Following the reaction, the peptide was further purified using a C-18 reversed-phase preparative chromatography column with a 0.1% trifluoroacetic acid in acetonitrile / water system to obtain pure peptides and their derivatives.

[0126] Experimental reagents

[0127]

[0128]

[0129] Example 1. Preparation of Compound 1

[0130]

[0131] Step 1: Synthesis of linear precursor peptide chains

[0132] The linear precursor peptide chain of compound 1 is MCMPCFTTDHQMARKCDDCCG-GKGRGKCYGPQCLCR

[0133] 294 mg (0.2 mmol) of Rink Amide-AM Resin resin was fully swollen in DMF for 1 h. Then, the linear precursor was synthesized sequentially from the carboxyl terminus to the amino terminus according to the given sequence. Each coupling cycle was performed as follows:

[0134] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0135] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0136] • Dissolve 1.0 mmol Fmoc-AA, 1.0 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 2 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0137] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0138] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0139] Step 2: Cleavage of the linear precursor peptide chain

[0140] A freshly prepared cut cocktail (10 mL) of trifluoroacetic acid:water:triisopropylsilane:benzyl sulfide (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 1, and the mixture was shaken and reacted at room temperature for 2 hours. After the reaction was complete, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid. The mixture was combined with the reaction solution, and the crude product was precipitated with 4 times its volume of cold MTBE. The crude product was washed three times with MTBE and dried under vacuum.

[0141] Step 3: Intramolecular disulfide bond formation

[0142] The crude product obtained in step 2 was dissolved thoroughly in 20% (v:v) DMSO. Then, 2 mM GSH was added to 50 mM ammonium bicarbonate buffer (pH = 8.0, containing 30% acetonitrile). The dissolved peptide solution was slowly added dropwise to the buffer to a final concentration of 1 mg / mL. The mixture was shaken at room temperature for 16 h. The reaction results were monitored by LC-MS. After the reaction was completed, purification was performed directly.

[0143] Step 4: Preparation of Peptides

[0144] After filtration through a 0.45 μm membrane, the product was separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR C-18 (Saifen) reversed-phase column was used. During purification, the detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient was 20-40% acetonitrile in 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the pure peptide was obtained.

[0145] Step 5: Detection and Characterization Methods

[0146] The purity of the polypeptide from step 4 was determined by analytical high-performance liquid chromatography and liquid chromatography / mass spectrometry, and the formation of intramolecular disulfide bonds in the compound was also determined.

[0147] Example 2. Preparation of Compound 3

[0148]

[0149] Step 1: Synthesis of linear precursor peptide chains

[0150] The linear precursor peptide chain of compound 3 is M(Boc)-CMPCFTTDHQMARKCDDCCGG-KGRGK(Mtt)-CYGPQCLCR

[0151] 294 mg (0.2 mmol) of Rink Amide-AM Resin resin was fully swollen in DMF for 1 h. Then, the linear precursor was synthesized sequentially from the carboxyl terminus to the amino terminus according to the given sequence. Each coupling cycle was performed as follows:

[0152] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0153] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0154] • Dissolve 1.0 mmol Fmoc-AA, 1.0 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 2 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0155] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0156] After the linear polypeptide was synthesized, the resin was rinsed 5 times with DMF and 5 times with DCM.

[0157] Step 2: AA27 lysine side chain conjugation of AEEA and FITC

[0158] Removal of the Mtt protecting group from the lysine side chain: After swelling the resin with DCM for 1 hour, add a mixed solution of hexafluoroisopropanol / dichloromethane (30% v / v, 10 mL) to the resin, shake and react at room temperature for 45 minutes, then remove the Mtt protecting group. Repeat the operation once. After the reaction is complete, wash the resin with DCM 5 times and DMF 6 times.

[0159] Lysine side chain coupling with AEEA: Weigh 1.0 mmol Fmoc-AEEA-OH and 1.0 mmol ethyl 2-oxime cyanoacetate and dissolve them in 8 mL DMF. Add 160 μL LDI for pre-activation for 3 min. Then add the mixed solution to the resin obtained in the previous step and shake to react for 3 h. After the reaction, drain the reaction solution and wash with DMF 4-5 times.

[0160] Deprotection of Fmoc: Perform Fmoc deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL). The first deprotection reaction lasts 5 min, and the second deprotection reaction lasts 20 min. After the reaction, drain the reaction solution and wash the resin with DMF 6-8 times until neutral pH is reached.

[0161] N-terminal coupling of FITC with AEEA: Weigh 0.4 mmol of FITC and dissolve it in 5 mL of DMF. Add 1.0 mmol of DIEA, then add the mixture to the resin obtained in the previous step. Shake and react in the dark for 5 hours. After the reaction, drain the reaction solution and wash the resin 6-8 times with DMF until the discharged liquid is colorless. Wash the resin 5 times with DCM. Dry the resin under vacuum.

[0162] Step 3: Cleavage of the linear precursor peptide chain

[0163] A freshly prepared cut cocktail (10 mL) of trifluoroacetic acid:water:triisopropylsilane:benzyl sulfide (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 2, and the mixture was shaken and reacted at room temperature for 2 hours. After the reaction was complete, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid. The mixture was combined with the reaction solution, and the crude product was precipitated with 4 times its volume of cold MTBE. The crude product was washed three times with MTBE and dried under vacuum.

[0164] Step 4: Intramolecular disulfide bond formation

[0165] The crude product obtained in step 3 was dissolved thoroughly in 20% (v:v) DMSO. Then, 2mM GSH was added to 50mM ammonium bicarbonate buffer (pH=8.0, containing 30% acetonitrile). The dissolved peptide solution was slowly added dropwise to the buffer to a final concentration of 0.5mg / mL, and the mixture was shaken at room temperature for 16h. The reaction results were monitored by LC-MS. After the reaction was completed, purification was performed directly.

[0166] Step 5: Preparation of Peptides

[0167] After filtration through a 0.45 μm membrane, the product was separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR C-18 (Saifen) reversed-phase column was used. During purification, the detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient was 20-40% acetonitrile in 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the pure peptide was obtained.

[0168] Step 6: Detection and Characterization Methods

[0169] The purity of the peptide from step 6 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry, and the K27 side linkages of AEEA and FITC were completed, as well as the formation of intramolecular disulfide bonds.

[0170] Example 3. Preparation of Compound 17

[0171]

[0172] Step 1: Synthesis of linear precursor peptide chains

[0173] The linear precursor peptide chain of compound 17 is MCMPCFTTDHQMARRCDDCCG-GRGRGKCYGPQCLCR

[0174] 294 mg (0.2 mmol) of Rink Amide-AM Resin resin was fully swollen in DMF for 1 h. Then, the linear precursor was synthesized sequentially from the carboxyl terminus to the amino terminus according to the given sequence. Each coupling cycle was performed as follows:

[0175] Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0176] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0177] Dissolve 1.0 mmol Fmoc-AA, 1.0 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 2 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0178] Rinse the resin with DMF 4-6 times before the next amino acid coupling.

[0179] After the linear polypeptide was synthesized, the resin was rinsed five times with DMF.

[0180] Step 2: N-terminal acetylation

[0181] Prepare 10 mL of acetylation reagent: Dissolve 500 μL of acetic anhydride and 500 μL of DIEA in 9 mL of DMF. Add 10 mL of the prepared acetylation reagent to the resin obtained in step 1, shake well, and agitate for 10 min. After the reaction, drain the reaction solution, rinse the resin 6-8 times with DMF, and rinse the resin 5 times with DCM. Dry the resin under vacuum.

[0182] Step 3: Cleavage of the linear precursor peptide chain

[0183] A freshly prepared cut cocktail (10 mL) of trifluoroacetic acid:water:triisopropylsilane:benzyl sulfide (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 2, and the mixture was shaken and reacted at room temperature for 2 hours. After the reaction was complete, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid. The mixture was combined with the reaction solution, and the crude product was precipitated with 4 times its volume of cold MTBE. The crude product was washed three times with MTBE and dried under vacuum.

[0184] Step 4: Intramolecular disulfide bond formation

[0185] The crude product obtained in step 3 was dissolved thoroughly in 20% (v:v) DMSO. Then, 2mM GSH was added to 50mM ammonium bicarbonate buffer (pH=8.0, containing 30% acetonitrile). The dissolved peptide solution was slowly added dropwise to the buffer to a final concentration of 1mg / mL. The mixture was shaken at room temperature for 16h. The reaction results were monitored by LC-MS. After the reaction was completed, purification was performed directly.

[0186] Step 5: Purification and preparation of oxidized peptides

[0187] After filtration through a 0.45 μm membrane, separation was performed using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR C-18 (Saifen) reversed-phase column was used. During purification, the chromatographic detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient to 20-40% acetonitrile in 40 min. The relevant fractions were collected, and after HPLC purity identification, fractions >75% were combined, lyophilized, and the oxidized peptides were obtained.

[0188] Step 6: Synthesis of 2-Succinyl PTX

[0189] Paclitaxel (2 g, 2.3 mmol) and succinic anhydride (1.875 g, 18.6 mmol) were dissolved in 20 mL of anhydrous pyridine and stirred at room temperature for 24 h. After the reaction was complete, the solvent was removed under reduced pressure, water was added and stirred for 1 h, and a large amount of white solid appeared. The solid was filtered and dried overnight at 50 °C to obtain succinylated paclitaxel.

[0190] Step 7: Synthesis of 2-NHS-Succinyl PTX

[0191] The 2-Succinyl PTX (2146.5 mg, 2.25 mmol) obtained in step 6 and N-hydroxysuccinimide (388.5 mg, 3.375 mmol) were mixed and dissolved completely in 65 mL of LDCM (CH2Cl2). DCC (696.4 mg, 3.375 mmol) was then added to the reaction system. After stirring at room temperature for 4.5 h, the solvent was removed under reduced pressure to obtain a white solid. The white solid was then extracted 3-5 times with saturated brine and ethyl acetate, and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate and filtered. The solvent was removed under reduced pressure, resulting in a large amount of white solid, yielding crude 2-NHS-PTX. Purification was performed using a C18 reversed-phase column, followed by lyophilization to obtain pure 2-NHS-PTX. Purification method: The sample was dissolved in DMSO:acetonitrile at a 1:1 ratio, and purified using a C18 reversed-phase column with a gradient of 50-90% (mobile phase consisted of pure water and pure acetonitrile, free of TFA).

[0192] Step 8: Peptide conjugation with PTX conjugate

[0193] Weigh 20 mg of the peptide obtained in step 5 and dissolve it in 6.8 mL of DMSO. Weigh 3 eq of 2-NHS-PTX obtained in step 7 and dissolve it completely in 6.8 mL of DMSO. Then, slowly add 6.4 mL of 1×PBS to the solution, at which point the solution will exhibit significant exothermic reaction. Cool the solution at 4°C for 5-10 min. After cooling, slowly add the peptide solution to the 2-NHS-PTX buffer to a final concentration of 1 mg / mL, shake well, and react at room temperature for 1-2 h. Monitor the reaction results by LC-MS. After the reaction is complete, proceed directly to purification.

[0194] Step 9: Purification and preparation of the target peptide

[0195] After filtration through a 0.45 μm membrane, the product was separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR C-18 (Saifen) reversed-phase column was used. During purification, the detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient was 30-60% acetonitrile in 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the pure peptide was obtained.

[0196] Step 10: Detection and Characterization Methods

[0197] The purity of the peptide obtained in step 9 was determined by analytical high-performance liquid chromatography (HPLC) and liquid chromatography / mass spectrometry (LC / MS). The N-terminal acetylation, K27-side linking of the PTX conjugate, and formation of an intramolecular disulfide bond were also confirmed. The detection results are as follows: Figure 1 , Figure 2 As shown.

[0198] Example 4. Preparation of Compound 20

[0199]

[0200] Step 1: Synthesis of linear precursor peptide chains

[0201] The linear precursor peptide chain of compound 20 is MCMPCFTTDHQMARRCDDCCG-GKGRGKCYGPQCLCR

[0202] 294 mg (0.2 mmol) of Rink Amide-AM Resin resin was fully swollen in DMF for 1 h. Then, the linear precursor was synthesized sequentially from the carboxyl terminus to the amino terminus according to the given sequence. Each coupling cycle was performed as follows:

[0203] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0204] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0205] • Dissolve 1.0 mmol Fmoc-AA, 1.0 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 2 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0206] Rinse the resin with DMF 4-6 times before the next amino acid coupling.

[0207] After the linear polypeptide was synthesized, the resin was rinsed five times with DMF.

[0208] Step 2: N-terminal acetylation

[0209] Prepare 10 ml of acetylation reagent: Dissolve 500 μL of acetic anhydride and 500 μL of DIEA in 9 ml of DMF. Add 10 ml of the prepared acetylation reagent to the resin obtained in step 1, shake well, and agitate for 10 min. After the reaction, drain the reaction solution, rinse the resin 6-8 times with DMF, and rinse the resin 5 times with DCM. Dry the resin under vacuum.

[0210] Step 3: Cleavage of the linear precursor peptide chain

[0211] Add 10 mL of freshly prepared cut cocktail (trifluoroacetic acid:water:triisopropylsilane:aniline sulfide) (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 2, and react with shaking at room temperature for 2 hours. After the reaction is complete, filter the reaction solution, wash the resin with trifluoroacetic acid, combine the washings with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain the crude product. Wash the crude product three times with MTBE and dry it under vacuum.

[0212] Step 4: Intramolecular disulfide bond formation

[0213] The crude product obtained in step 3 was dissolved thoroughly in DMSO (DMSO volume was 20% of the total reaction volume). Then, 2 mM GSH was added to 50 mM ammonium bicarbonate buffer (pH = 8.0, containing 30% acetonitrile). The dissolved peptide solution was slowly added dropwise to the buffer to a final concentration of 1 mg / ml. The mixture was shaken at room temperature for 16 hours. The reaction results were monitored by LC-MS. After the reaction was completed, purification was performed directly.

[0214] Step 5: Purification and preparation of oxidized peptides

[0215] After filtration through a 0.45 μm membrane, separation was performed using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR C-18 (Saifen) reversed-phase column was used. During purification, the detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient was 20-50% acetonitrile in 40 min. The relevant fractions were collected, and after HPLC purity identification, fractions >75% were combined, lyophilized, and the oxidized peptides were obtained.

[0216] Step 6: Synthesis of 2-Succinyl PTX

[0217] Paclitaxel (2 g, 2.3 mmol) and succinic anhydride (1.875 g, 18.6 mmol) were dissolved in 20 mL of anhydrous pyridine and stirred at room temperature for 24 hours. After the reaction was complete, the solvent was removed under reduced pressure, water was added and stirred for 1 hour, resulting in a large amount of white solid. The solid was filtered and dried overnight at 50 °C to obtain succinylated paclitaxel.

[0218] Step 7: Synthesis of 2-NHS-Succinyl PTX

[0219] The 2-Succinyl PTX (2146.5 mg, 2.25 mmol) obtained in step 6 and N-hydroxysuccinimide (388.5 mg, 3.375 mmol) were mixed and dissolved completely in 65 mL of LDCM (CHCl2). Then, DCC (696.4 mg, 3.375 mmol) was added to the reaction system, and the mixture was stirred at room temperature for 4.5 h. The solvent was removed under reduced pressure to obtain a white solid. The white solid was then extracted 3-5 times with saturated brine and ethyl acetate, and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate and filtered. The solvent was removed under reduced pressure, resulting in a large amount of white solid, yielding crude 2-NHS-PTX. Purification was performed using a C18 reversed-phase column, followed by lyophilization to obtain pure 2-NHS-PTX. Purification method: The sample was dissolved in DMSO:acetonitrile at a 1:1 ratio, and purified using a C18 reversed-phase column with a gradient of 50-90% (mobile phase consisted of pure water and pure acetonitrile, free of TFA).

[0220] Step 8: Peptide conjugation with PTX conjugate

[0221] Weigh 20 mg of the peptide obtained in step 5 and dissolve it in 13.6 ml of DMSO. Weigh 6 eq of 2-NHS-PTX obtained in step 7 and dissolve it completely in 13.6 ml of DMSO. Then, slowly add 12.8 ml of 1×PBS to the solution, at which point the solution will exhibit significant exothermic reaction. Cool the solution at 4°C for 5-10 min. After cooling, slowly add the peptide solution to the 2-NHS-PTX buffer to a final concentration of 0.5 mg / ml, shake well, and react overnight at room temperature. Monitor the reaction results by LC-MS. After the reaction is complete, proceed directly to purification.

[0222] Step 9: Purification and preparation of the target peptide

[0223] After filtration through a 0.45 μm membrane, the product was separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR-C18 (Saifen) reversed-phase column was used. During purification, the detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient was 40-80% acetonitrile in 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the purified peptide was obtained.

[0224] Step 10: Detection and Characterization Methods

[0225] The purity of the peptide obtained in step 9 was determined by analytical high-performance liquid chromatography (HPLC) and liquid chromatography / mass spectrometry (LC / MS). The N-terminal acetylation, K23 and K27 side linkage with PTX conjugates, and intramolecular disulfide bond formation were also confirmed. The detection results are as follows: Figure 3 , Figure 4 As shown.

[0226] Example 5. Preparation of Compound 23

[0227]

[0228] Step 1: Synthesis of linear precursor peptide chains

[0229] The linear precursor peptide chain of compound 23 is MCMPCFTTDHQMARRCDDCCG-GRGRGKCYGPQCLCR

[0230] 294 mg (0.2 mmol) of Rink Amide-AM Resin resin was fully swollen in DMF for 1 h. Then, the linear precursor was synthesized sequentially from the carboxyl terminus to the amino terminus according to the given sequence. Each coupling cycle was performed as follows:

[0231] Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0232] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0233] Dissolve 1.0 mmol Fmoc-AA, 1.0 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 2 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0234] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0235] After the linear polypeptide was synthesized, the resin was rinsed five times with DMF.

[0236] Step 2: N-terminal acetylation

[0237] Prepare 10 mL of acetylation reagent: Dissolve 500 μL of acetic anhydride and 500 μL of DIEA in 9 mL of DMF. Add the prepared acetylation reagent to the resin obtained in step 1, shake well, and agitate for 10 min. After the reaction, drain the reaction solution, wash the resin 6-8 times with DMF, and wash the resin 5 times with DCM. Dry the resin under vacuum.

[0238] Step 3: Cleavage of the linear precursor peptide chain

[0239] A freshly prepared cut cocktail (10 mL) of trifluoroacetic acid:water:triisopropylsilane:benzyl sulfide (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 2, and the mixture was shaken and reacted at room temperature for 2 hours. After the reaction was complete, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid. The mixture was combined with the reaction solution, and the crude product was precipitated with 4 times its volume of cold MTBE. The crude product was washed three times with MTBE and dried under vacuum.

[0240] Step 4: Intramolecular disulfide bond formation

[0241] The crude product obtained in step 3 was dissolved thoroughly in 20% (v:v) DMSO. Then, 2mM GSH was added to 50mM ammonium bicarbonate buffer (pH=8.0, containing 30% acetonitrile). The dissolved peptide solution was slowly added dropwise to the buffer to a final concentration of 1mg / mL. The mixture was shaken at room temperature for 16h. The reaction results were monitored by LC-MS. After the reaction was completed, purification was performed directly.

[0242] Step 5: Purification and preparation of oxidized peptides

[0243] After filtration through a 0.45 μm membrane, separation was performed using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR C-18 (Saifen) reversed-phase column was used. During purification, the chromatographic detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient to 20-40% acetonitrile in 40 min. The relevant fractions were collected, and after HPLC purity identification, fractions >75% were combined, lyophilized, and the oxidized peptides were obtained.

[0244] Step 6: Synthesize Compound 1

[0245]

[0246] Paclitaxel (0.950 g, 1.8 mmol) and 4-nitrophenyl carbonate (2.28 g, 7.49 mmol) were dissolved in 5 mL of LDMF, and then DIEA (1.63 mL, 9.36 mmol) was added and stirred at room temperature for 1 h. The reaction results were monitored by LC-MS. After the reaction was completed, the product was purified by C18 reversed-phase chromatography and lyophilized to obtain Compound 1. Purification method: The sample was dissolved in DMSO:acetonitrile at a ratio of 1:1, and purified by C18 reversed-phase chromatography with a gradient of 40-90% (the mobile phase was pure water and pure acetonitrile, without TFA).

[0247] Step 7: Peptide conjugation with PTX conjugate

[0248] Weigh 20 mg of the peptide obtained in step 5, add 2 mL of DMF to dissolve the peptide, and then add DIEA (6 eq). Weigh 2 eq of Compound 1 obtained in step 6, add 2 mL of DMF to dissolve it completely. Slowly add the Compound 1 solution dropwise to the peptide mixture to a final concentration of 5 mg / mL. Under N2 protection, stir in a 37°C water bath overnight. Monitor the reaction results by LC-MS. After the reaction is complete, proceed directly to purification preparation.

[0249] Step 8: Purification and preparation of the target peptide

[0250] After filtration through a 0.45 μm membrane, the product was separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR C-18 (Saifen) reversed-phase column was used. During purification, the detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient was 30-45% acetonitrile in 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the pure peptide was obtained.

[0251] Step 9: Detection and Characterization Methods

[0252] The purity of the peptide obtained in step 9 was determined by analytical high-performance liquid chromatography (HPLC) and liquid chromatography / mass spectrometry (LC / MS). The N-terminal acetylation, K27-side linking of the PTX conjugate, and formation of an intramolecular disulfide bond were also confirmed. The detection results are as follows: Figure 5 , Figure 6 As shown.

[0253] Other compounds of the present invention can be synthesized with reference to the above synthesis examples.

[0254] Biological evaluation

[0255] Test Example 1: PDC molecule killing experiment on tumor cells

[0256] This invention evaluates the in vitro killing effect of PDC molecules on two types of glioma cells, U87-MG and U373, by measuring the IC50 of PDC molecules.

[0257] Materials: U373 cells (Fenghui Biotechnology); U87-MG cells (Pronosai); FBS (EXCELL); DMEM (SIGMA); MEM (SIGMA); P / S solution 100x (homemade); trypsin (Gibco); EDTA (SIGMA); DPBS (homemade); 96-well black transparent bottom cell plate (Aligent); Celltiter-blue (Promega); DMSO (aladdin).

[0258] Experimental steps:

[0259] U87-MG cells were cultured and grown in medium (MEM, 10% FBS, 1% P / S). When the cell density reached 80-90% of the culture flask, the cells were first washed with DPBS, then digested with 0.25% trypsin (containing 0.5 mM EDTA); the cell suspension was collected into centrifuge tubes, centrifuged at 1000 rpm for 3 min, and the supernatant was removed; the cells were resuspended in 6-8 mL of fresh growth medium and passaged at a ratio of 1:3 to 1:8, and cultured in a 37°C, 5% CO2 incubator. After passage, the medium was changed or passaged every 2-3 days. U373 cells were cultured and grown in medium (DMEM, 10% FBS, 1% P / S). When the cell density reaches 80-90% of the culture flask, rinse the cells with DPBS, then digest them with 0.25% trypsin (containing 0.5mM EDTA). Collect the cell suspension into a centrifuge tube, centrifuge at 1000 rpm for 3 min, and remove the supernatant. Resuspend the cells in 6-8 mL of fresh growth medium and passage them at a ratio of 1:3 to 1:8. Incubate at 37°C in a 5% CO2 incubator. Change the medium or passage again every 2-3 days after passage.

[0260] 16-24 hours before the experiment, U373 and U87-MG cells were passaged and expanded to the required cell number. The cells were digested and centrifuged to collect the cell pellet. The cells were resuspended in an appropriate amount of complete culture medium, and cell viability was assessed and counted. The cell concentration was then adjusted to 2 × 10⁴ cells / mL with complete culture medium. 100 μL / well was seeded into the center wells of a 96-well plate, and the edge wells were filled with the same volume of 100 μL / well of DPBS. The plates were incubated overnight at 37°C in a 5% CO₂ incubator.

[0261] PDC drug was dissolved to 1 mM using sterile water or DMSO. In the experiment, PDC drug was diluted to 1 μM (2× concentration) using the growth medium of U373 cells and U87-MG cells, and then serially diluted 5-fold with the corresponding cell growth medium, resulting in a total of 9 concentrations (500 nM-0.00128 nM).

[0262] Remove cells that have been seeded overnight, discard 50 μL of culture medium from each well, add 50 μL of PDC working solution per well, and incubate at 37°C, 5% CO2 for 48 h. Then add 20 μL of Celltiter-Blue staining solution equilibrated to room temperature to each well, incubate at 37°C, 5% CO2 for 1 h, and then detect 560EX / 590EM.

[0263] The IC50 results of the tumor cell killing experiments of some PDC molecules of this invention are shown in Table 1. The killing curves against two types of glioma cells, U87-MG and U373, are shown in Table 1. Figure 7 , Figure 8 As shown.

[0264] ANG-1005 is a brain-penetrating peptide drug conjugate. ANG-1005 is a taxane derivative consisting of three paclitaxel molecules covalently linked to Angiopep-2 (structure shown below), designed to cross the blood-brain and blood-brain-spinal cord barriers via the low-density lipoprotein receptor-associated protein (LRP1) transport system and penetrate malignant cells.

[0265]

[0266] Table 1. Experimental IC50 values ​​of some PDC molecules of the present invention against tumor cells U87-MG and U373. 50 result

[0267]

[0268] Experimental results show that the PDC molecule of the present invention has a good killing effect on tumor cells U87-MG and U373.

[0269] Test Example 2: Fluorescence Imaging Detection of Peptide Internalization Effect in U87-MG and U373 Cells

[0270] Materials: Polypeptides designed and synthesized by the company; 96-well black transparent bottom cell plates (Aligent); fixative (Beyotime); DAPI (Beyotime); DPBS (self-made).

[0271] 4405, 24, 25, and 26 are Cy5-labeled peptides, where 4405 is a Cy5-labeled Angiopep-2, and 24, 25, and 26 correspond to PDC molecules 17, 18, and 20, respectively.

[0272] Experimental steps:

[0273] U87-MG or U373 cells were incubated with Cy5-labeled peptides, and the locations of the FITC-peptide-labeled cells were then monitored using confocal microscopy.

[0274] 16-24 hours before the experiment, U87-MG or U373 cells were passaged and expanded to the required number of cells. The cells were digested and centrifuged to collect the cell pellet. The cells were resuspended in an appropriate amount of complete culture medium, and cell viability was tested and counted. The cell concentration was then adjusted to 4 × 10⁴ cells / mL with complete culture medium. 100 μL / well was seeded into the center wells of a 96-well plate, and the edge wells were filled with the same volume of 100 μL / well of DPBS. The plates were incubated overnight at 37°C in a 5% CO₂ incubator.

[0275] The peptides were dissolved in DMSO to a concentration of 1 mM. During the experiment, the peptides were diluted with the respective cell growth media to a concentration of 10 μM. The peptides were then serially diluted 3-fold with the respective cell growth media to obtain 3 concentrations (10 μM-1.11 μM).

[0276] Remove the cell plate that has been inoculated overnight, aspirate the old culture medium from the wells, and replace it with 100 μL of diluted peptide per well. Continue...

[0277] After incubation at 37°C and 5% CO2 for 4 hours / 24 hours, the samples were fixed with fixative, stained with DAPI, and observed under a confocal microscope using fluorescence imaging.

[0278] Confocal imaging results as follows Figures 9-16 As shown, the endocytosis efficiency of peptides on U87-MG and U373 cells was 26>25>24>4405, and the corresponding endocytosis efficiency of PDC molecules was 20>18>17>ANG-1005.

[0279] Test Example 3: Plasma Stability

[0280] Experimental materials: methanol (purchased from Sigma); formic acid (purchased from Aladdin); DMSO (dimethyl sulfoxide) (purchased from Aladdin).

[0281] Experimental Procedure: Sample Preparation: Dissolve the test peptide in DMSO or other organic solvent to a final concentration of 1mM (100 times the final concentration) and store at -20℃. Plasma Thawing: Remove human plasma (sample number * 2.1) mL from the -80℃ freezer and thaw rapidly in a 37℃ water bath. MIX Preparation: Add 693 μL of plasma to a 1.5 mL EP tube, with 3 parallel samples at each time point, preparing 3 MIX tubes. Add 7 μL of the test sample to each tube to achieve a final concentration of 10 μM, which is the detectable concentration or the in vivo drug concentration. Vortex for 30 s, aliquot 100 μL at each time point, and incubate on ice throughout. Incubation: Incubate in a 37℃ water bath at six time points: 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min. Termination of Reaction: After incubation, add 4 times the volume of precipitant. Mixing: Vortex for 30 s. Centrifugation: Centrifuge at 15000 rpm for 10 min at 4℃. Collect the supernatant, transfer it to a sample injection tube, and send it to LC-MS / MS for analysis.

[0282] Experimental results:

[0283] A line graph with the ordinate representing the residual percentage of the original drug (%) and the abscissa representing time shows the trend of sample degradation in in vitro plasma over time, thus providing the results of sample stability.

[0284]

[0285] Calculate the half-life T of the drug in plasma 1 / 2 .

[0286] The elimination rate constant (Ke) was calculated using first-order kinetics. Furthermore, the Ti of the compound in plasma was determined using the same formula. 1 / 2 (min).

[0287] T 1 / 2 =-0.693 / Ke

[0288] The plasma stability test results of some PDC molecules in this invention are shown in Table 2. Figure 17 As shown:

[0289] Table 2. Experimental results of human plasma stability of some PDC molecules in this invention.

[0290]

[0291]

[0292] Experimental results show that the PDC molecule of the present invention has good plasma stability, which is helpful for clinical application.

[0293] Test Example 4: In vitro assessment of peptide molecules' penetration of the blood-brain barrier

[0294] Experimental materials: methanol (purchased from Sigma); formic acid (purchased from Aladdin); DMSO (dimethyl sulfoxide) (purchased from Aladdin); balanced salt solution (HBSS) prepared in-house.

[0295] 4405, 25, and 26 are Cy5-labeled peptides, where 4405 is a Cy5-labeled Angiopep-2, and 25 and 26 correspond to PDC molecules 18 and 20, respectively.

[0296] Experimental steps:

[0297] Prepare the following solutions: precipitant (1% formic acid in methanol), balanced salt solution (HBSS): 137mM NaCl, 5.4mM KCl, 0.44mM KH2PO4, 0.5mM NaH2PO4, 4.2mM Na2HPO4, 4.2mM NaHCO3, 5.5mM D-glucose.

[0298] Male SD rats aged 3-4 weeks were sacrificed and placed in a clean bench. The skull was cut open along the midline, and the whole brain was removed and placed in buffer solution. The cerebellum and diencephalon were removed, microvessels were harvested, and cerebral parenchyma was removed, preserving the cerebral cortex. The cerebral cortex was laid flat on sterile paper and rolled downwards once, repeating the above steps until the vascular mucosa was completely removed. The cells were digested, purified, and the microvascular cell layer was transferred to a T75 culture flask for culture. On the second day, the original culture medium was aspirated, and the cells were rinsed once with sterile HBSS at room temperature. The medium was then replaced with EBM-2 medium containing 3 μg / mL puromycin. The medium was changed every two days thereafter (without adding puromycin in subsequent medium changes). When the cells reached approximately 80% confluence, they were transferred to Transwell culture medium coated with rat tail gel and cultured for another 7-8 days, with the medium changed every two days and electrical resistance measured.

[0299] Dilute each peptide sample to a final concentration of 10 μM using HBSS and vortex for 30 s. For the primary rat endothelial cell model that meets experimental requirements, discard the old culture medium from both sides of the chambers and wash each well once with preheated (37°C) transport buffer. Discard the buffer. Add 100 μL of the drug-end solution to each well of the upper chamber (Apical, AP) and 600 μL of HBSS to each well of the lower chamber (Basolateral, BL). Incubate at 37°C for 1 h. After incubation, aspirate 10 μL of the drug-end sample from the upper chamber (AP) and add 90 μL of HBSS to the microplate; aspirate 100 μL of the sample from the lower chamber (BL) and add 90 μL of HBSS to the microplate. At time T0, aspirate 10 μL of the sample and add 90 μL of HBSS to the microplate, then vortex to mix.

[0300] All peptide samples were CY5 labeled, and the OD values ​​were detected using a microplate reader (excitation wavelength: OD640nm, emission wavelength: OD681nm).

[0301] The rate at which the drug passes through primary rat brain endothelial cells is expressed as the apparent permeability coefficient (Papp, unit: ×10-6 cm / s).

[0302]

[0303] VR is the volume of the receiving solution (0.6 mL), and Area is the membrane area of ​​the Transwell-24 well plate chamber (0.33 cm²). 2 Time is the incubation time (in seconds), CR is the drug concentration at the sample receiving end, and C0 is the drug concentration at the initial time 0 of the sample.

[0304] Experimental results are as follows Figure 18 As shown in the figure, the experimental results show that in the in vitro blood-brain barrier model, the penetration power of the polypeptide is 26>25>4405, and the corresponding penetration power of the PDC molecule is 20>18>ANG-1005.

[0305] Test Example 5: Evaluation of peptide molecules penetrating the blood-brain barrier in mice

[0306] Experimental materials: 6-8 week old male ICR mice (purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.), DMSO (dimethyl sulfoxide) (purchased from Aladdin).

[0307] 24, 25, and 26 are Cy5-labeled peptides, and the PDC molecules corresponding to 24, 25, and 26 are 17, 18, and 20, respectively.

[0308] Experimental steps:

[0309] Sample preparation and processing: The test peptide was labeled with Cy5, and a standard curve working solution was prepared with DMSO. Standard curves were plotted in plasma and brain homogenate, showing the fluorescence value and corresponding labeled peptide concentration. The injection dose was prepared at 1 / 2000 of the lower limit of the standard curve detection. Tail vein administration: While the animal was awake, 200uL was administered via the tail vein of the mouse using a 1mL syringe with a 27# needle. Blood collection and centrifugation of serum samples: Under isoflurane anesthesia, the open auricle was opened at fixed time points, and blood was collected using a pipette, with a blood volume of not less than 0.4ml. The collected serum samples were centrifuged at 4℃ and 4000g for 5min, and the supernatant was collected. Brain tissue collection and homogenization: Mice were perfused with 20 mL PBS from the heart, then decapitated and the brain was collected. Half of the brain tissue was collected, weighed, and homogenized at a 1:2 ratio (0.1 g brain tissue to 100 μL PBS) at 60 Hz for 60 seconds, using two 2 mm magnetic beads per tube. The middle homogenate was collected after homogenization, and the remaining brain tissue was placed in a culture dish and stored at -80°C. Sample detection: Before homogenization, the brain tissue was scanned using an IVIS system. 100 μL each of serum and brain homogenate were added to a 96-well plate, and fluorescence values ​​were read. The test samples and standard curves were simultaneously detected on a microplate reader (640 / 670 Cy5).

[0310] Based on the standard curve, the drug concentrations in different matrices at different time points were obtained. Drug-time curves were plotted using the drug concentrations in brain tissue and plasma as the ordinate and time as the abscissa. The area under the curve (AUC) of the drug-time curves in brain tissue and plasma were calculated respectively, and the AUC blood-brain ratio was calculated based on the AUC.

[0311] AUC blood-brain ratio = AUCBrain / AUCPlasma, where AUCBrain refers to the area under the curve of the drug-time curve in brain tissue, and AUCplasma refers to the area under the curve of the drug-time curve in plasma.

[0312] The experimental results are shown in Table 3. Figures 19-22 As shown, the PDC molecules of this invention can penetrate the blood-brain barrier and reach brain tissue in mice, and have a good penetration effect.

[0313] Table 3. Calculation results of AUC blood-brain ratio

[0314] compound 24 25 26 AUC Brain / AUC Plasma 0.007533 0.006877 0.010333

[0315] Test Example 6: Pharmacokinetic Evaluation of Peptide Molecules in Mice

[0316] Experimental materials: PDC drugs (self-made synthetic polypeptide combination drugs).

[0317] Relevant reagents and consumables: 1mL insulin injection, 1.5mL EDTA anticoagulant tube, 0.3mL EP tube, EDTA, protease inhibitor.

[0318] Experimental animals: 18 male ICR mice, 6-7 weeks old, purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.

[0319] Experimental methods:

[0320] Six- to seven-week-old female ICR mice were housed under standard conditions. After one week of acclimatization, eight mice of similar weight were randomly divided into two groups of four. The compound was prepared at a concentration of 1.25 mg / mL, and administered via tail vein injection at a volume of 10 mL / kg. After administration, approximately 200 μL of blood was collected from the submandibular vein at 40 s, 5 min, 15 min, 30 min, 60 min, 120 min, 240 min, and 360 min. The blood samples were placed in EDTA anticoagulant tubes, centrifuged, and the plasma was collected and stored at -80°C for analysis.

[0321] Take a plasma sample, thaw it, vortex it to mix, take 50 uL into a 1.5 mL EP tube, add 50 uL of 50% methanol PBS containing 1% FA, vortex for 3 min, add 200 uL of 0.1% FA 75% acetonitrile, vortex for 3 min, centrifuge for 10 min (13000 rpm), and take the supernatant for LC-MS analysis.

[0322] Experimental results

[0323] After intravenous administration of the compound to mice, the results at each time point were measured to create a blood drug concentration-time curve.

[0324] The pharmacokinetic parameters of different PDC drugs after intravenous administration to mice were calculated using PKslover 2.0 pharmacokinetic software. The results are shown in the table (t1 / 2: elimination half-life; C0: initial concentration; CL: clearance rate; AUC: area under the curve; Vss: volume of distribution at steady state concentration; Vz: volume of distribution).

[0325] The PDC molecule of the present invention has good pharmacokinetic properties.

[0326] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A polypeptide conjugate of a chlorotoxin analog, characterized in that, the polypeptide conjugate structure is selected from one of the following structures, 、 、 。 2. A pharmaceutical composition, characterized by, the pharmaceutical composition comprises the polypeptide conjugate of claim 1 and a pharmaceutically acceptable excipient.

3. A pharmaceutical composition, characterized by, the pharmaceutical composition comprises the polypeptide conjugate of claim 1 and a pharmaceutically acceptable carrier.

4. Use of the polypeptide conjugate of claim 1, or the pharmaceutical composition of claim 2 or 3, in the manufacture of a medicament for treating a cancer, wherein the cancer is a brain tumor.

5. Use according to claim 4, characterized in that, the cancer is a glioma.

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