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WO2024212817A1 - Conjugué polypeptidique d'analogue de chlorotoxine et utilisation associée - Google Patents

Conjugué polypeptidique d'analogue de chlorotoxine et utilisation associée Download PDF

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Publication number
WO2024212817A1
WO2024212817A1 PCT/CN2024/084351 CN2024084351W WO2024212817A1 WO 2024212817 A1 WO2024212817 A1 WO 2024212817A1 CN 2024084351 W CN2024084351 W CN 2024084351W WO 2024212817 A1 WO2024212817 A1 WO 2024212817A1
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Prior art keywords
cancer
polypeptide
peptide
amino acid
polypeptide conjugate
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Chinese (zh)
Inventor
李义龙
杨悌平
王登
刘文革
刘惠清
王新波
宫焕章
李向群
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Hunan Zonsen Peplib Biotech Co Ltd
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Hunan Zonsen Peplib Biotech Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids

Definitions

  • the present invention belongs to the technical field of biopharmaceutical polypeptides, and specifically relates to a polypeptide conjugate of a chlorotoxin analog and its use in preparing a drug for preventing, treating and curing cancer-related diseases.
  • Chlorotoxin 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:
  • chlorotoxin is a chloride channel blocker that can specifically bind to tumor cells.
  • chlorotoxin has been shown to pass through the blood-brain barrier. Again, chlorotoxin has good biocompatibility and has no obvious toxic effects on normal tissue cells.
  • chlorotoxin is slowly metabolized in the body, providing researchers with ample time for imaging and treatment. Therefore, chlorotoxin 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.
  • chlorotoxin is mainly developed as a carrier to carry radioactive isotopes, fluorescent molecules, etc. into the tumor to achieve tumor imaging, so that the tumor tissue can be completely and accurately removed during surgery, and normal brain tissue can be retained to the greatest extent.
  • Chlorotoxin as a carrier can also carry nanoparticles and drugs into the tumor, reducing the damage of drugs to other organs of the body and reducing side effects.
  • the characteristics of chlorotoxin binding to brain glioma cells were first studied on 125I-labeled small peptides (125I-CTX) and 131I-labeled small peptides (131I-CTX).
  • 125I-CTX can accumulate in the tumors of tumor-bearing mice, and 125I-CTX can specifically bind to glioma cells, but not to normal astrocytes.
  • 131I-CTX is the most widely studied chlorotoxin complex, and the radiation it emits can be detected to identify and locate brain tumors.
  • 131I and indocyanine green (ICG) labeled chlorotoxin have passed the US preclinical safety test and entered Phase II/I clinical trials respectively.
  • chlorotoxin was found to bind to tumors of neuroectodermal origin (tumors that share an embryonic origin with cells of the central nervous system).
  • chlorotoxin linked to biotin could bind to more than 200 biopsy samples of malignant gliomas and other tumors at different stages, including melanoma, neuroblastoma, medulloblastoma, and small cell lung cancer, but could not bind to normal tissues of the brain, skin, kidney, and lung.
  • some fluorescent dyes such as Cy5.5, BLZ-100, and 800CW, can specifically target tumors in vivo after being linked to chlorotoxin.
  • chlorotoxin can deliver nanoprobes, magnetic resonance imaging contrast agents, and therapeutic drugs to tumor tissues.
  • chlorotoxin conjugates including fusion proteins, such as chlorotoxin-GST fusion proteins linked to saporin, have also been shown to significantly and selectively kill tumor cells.
  • chlorotoxin-GST fusion proteins linked to saporin have also been shown to significantly and selectively kill tumor cells.
  • chlorotoxin has the potential to be a carrier for the specific delivery of anticancer drugs to cancer cells. Chlorotoxin was shown to bind to glioma cells, but not to normal rat astrocytes and human rhabdomyosarcoma cell lines, and has great application prospects as a specific marker for selectively targeting new drugs and diagnosis (including grade determination) of human tumors.
  • brain tumor should be intracranial tumor, which is divided into primary tumors in the brain and secondary tumors in other organs.
  • Malignant glioma is a type of brain tumor and one of the most difficult types of cancer to treat. Commonly used methods include surgery, radiotherapy, chemotherapy and targeted drugs, but if recurrence occurs, the average survival time of patients is less than 12 months.
  • BBB blood-brain barrier
  • PTX paclitaxel
  • BBB blood-brain barrier
  • Chinese patent CN102844044A discloses a lysine chlorotoxin polypeptide with no more than one binding site.
  • the provided lysine-reduced chlorotoxin polypeptide and/or its conjugate can be used in medicine (for example, in various treatment and/or diagnostic contexts).
  • the present invention Based on the characteristics of chlorotoxin, the present invention provides a new polypeptide conjugate of chlorotoxin analogs, which is expected to develop a drug for brain tumors with good development prospects. Through artificial modification, it can give full play to its own advantages, further increase its stability and efficacy, and reduce toxicity.
  • the purpose of the present invention is to provide a novel polypeptide conjugate of chlorotoxin analogues to address the unmet clinical needs in the treatment of malignant brain tumors.
  • the present invention provides a polypeptide conjugate of a chlorotoxin analog, comprising a structure as shown in the following formula (I): Peptide-(Linker-Drug) m (I)
  • Peptide is a polypeptide
  • Linker is a linker
  • Drug is an anticancer agent
  • m is 1, 2 or 3;
  • 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 be modified or unmodified.
  • SEQ ID NO: 1 MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR;
  • SEQ ID NO: 2 MCMPCFTTDHQMARRCDDCCGGRGRGRCYGPQCLCR;
  • SEQ ID NO: 3 MCMPCFTTDHQMARKCDDCCGGRGRGRCYGPQCLCR;
  • SEQ ID NO: 4 MCMPCFTTDHQMARRCDDCCGGKGRG RCYGPQCLCR;
  • SEQ ID NO: 5 MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR;
  • SEQ ID NO: 6 MCMPCFTTDHQMARRCDDCCGGKGRGKCYGPQCLCR;
  • SEQ ID NO: 7 MCMPCFTTDHQMARACDDCCGGKGRGKCYGPQCLCR;
  • SEQ ID NO: 8 NleCNlePCFTTDHQNleARRCDDCCGGRGRGKCYGPQCLCR.
  • the amino acid sequence of the polypeptide is selected from one of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and the amino acid sequence may be modified or unmodified.
  • the amino acid sequence of the polypeptide may be modified, and the modification is selected from N-terminal acetylation and/or C-terminal amidation.
  • the amino acid sequence of the polypeptide has at least 85% overall sequence identity with SEQ ID NO: 1 to SEQ ID NO: 8.
  • the amino acid sequence of the polypeptide has at least 90% sequence identity with SEQ ID NO: 1 to SEQ ID NO: 8.
  • the amino acid sequence of the polypeptide has at least 95% sequence identity with SEQ ID NO: 1 to SEQ ID NO: 8.
  • the amino acid sequence of the polypeptide has at least one site that can be used for linking the sub-linker, and the site that can be used for linking the sub-linker is -NH2 and/or or -COOH.
  • the amino acid sequence of the polypeptide has at least one site that can be used for linking a linker, and the -NH2 site that can be used for linking a linker is the -NH2 of a lysine side chain.
  • the amino acid sequence of the polypeptide has at least one site that can be used for linking with a linker, and the -NH2 site that can be used for linking with a linker is the -NH2 of the methionine at the N-terminus of the peptide chain;
  • the -NH2 site available for linker linkage corresponds to the 1st, 15th, 23rd and/or 27th position of the amino acid sequence of the polypeptide.
  • the amino acid sequence of the polypeptide has at least one site that can be used for linking with a linker, and the -COOH site that can be used for linking with a linker is the -COOH of the side chain of aspartic acid or glutamic acid.
  • the linker is independently selected from the following structures or any combination of the following structures:
  • linker is independently selected from the following structures or any combination of the following structures -GALGLPG-.
  • linker is independently selected from the following structures or any combination of the following structures -GALGLPG-.
  • the present invention provides a polypeptide conjugate, characterized in that it has a structure as shown in the following formula (II):
  • Peptide is a polypeptide
  • Drug is an anticancer agent
  • n 1, 2 or 3;
  • 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 be modified or unmodified.
  • the present invention provides a polypeptide conjugate, characterized in that it has a structure as shown in the following formula (III):
  • Peptide is a polypeptide
  • Drug is an anticancer agent
  • n 1, 2 or 3;
  • 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 be modified or unmodified.
  • the present invention provides a polypeptide conjugate, characterized in that it has a structure as shown in the following formula (IV):
  • Peptide is a polypeptide
  • Drug is an anticancer agent
  • n 1, 2 or 3;
  • 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 be modified or unmodified.
  • the present invention provides a polypeptide conjugate, characterized in that it has a structure as shown in the following formula (V):
  • Peptide is a polypeptide
  • Drug is an anticancer agent
  • n 1, 2 or 3;
  • 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 be modified or unmodified.
  • 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, plicamycin, Mitomycin, bleomycin, tamoxifen, flutamide, leuprolide, goserelin, aminoglutethimide, anastrozole, amsacrine, asparaginase, mitoxantrone, mit
  • the anticancer agent in the polypeptide conjugate of the present invention is a poorly water-soluble compound.
  • the anticancer agent may be further selected from the taxanes, which are considered to be effective drugs for treating solid tumors that are refractory to many other anti-tumor agents.
  • the anticancer agent is preferably paclitaxel, docetaxel, or a combination thereof.
  • the anticancer agent in the polypeptide conjugates of the present invention is maytansine and/or its analogs (eg, DM1).
  • the polypeptide conjugate provided by the present invention is further substituted with a lipophilic substituent, wherein the lipophilic substituent can 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.
  • the lipophilic substituent is directly linked to the polypeptide conjugate or can be linked to the polypeptide conjugate via a linker, and is independently selected from the following groups:
  • the linker may be one of the above linkers or any combination thereof.
  • polypeptide conjugate provided by the present invention is further substituted, and the substituents can be independently selected from the following groups:
  • the present invention provides a polypeptide conjugate, wherein the structure of the polypeptide conjugate is selected from one of the following structures:
  • the present invention also relates to a pharmaceutical composition comprising any one of the polypeptide conjugates of the present invention.
  • the pharmaceutical composition described in the present invention further comprises at least one of a pharmaceutically acceptable carrier and/or excipient, a diluent, an adjuvant and a vehicle.
  • the present invention relates to the use of the polypeptide conjugate or pharmaceutical composition in the preparation of a drug for preventing, treating, curing or alleviating cancer, wherein the drug is used for preventing, treating, curing or alleviating cancer.
  • the cancer includes but is not limited to breast cancer, lung cancer, prostate cancer, kidney cancer, leukemia, ovarian cancer, gastric 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.
  • the polypeptide-drug conjugate of chlorotoxin analog provided by the present invention utilizes a hydrophilic polypeptide to modify a hydrophobic anti-tumor drug, thereby improving the solubility of the drug.
  • the polypeptide conjugate drug provided by the present invention has the characteristics of high activity, strong specific binding to the target, and obvious cellular endocytosis.
  • Administration of the conjugate of the present invention to patients can increase the specificity for target cells (especially tumor cells), increase cellular internalization of cells, reduce cellular degradation of cells, increase accumulation at target sites, reduce accumulation in normal tissues, reduce its biological toxicity, overcome drug resistance, increase the biological activity of the drug and/or prevent, limit or eliminate 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).
  • Chlorotoxin has a good ability to penetrate the blood-brain barrier. After coupling anti-tumor drugs with it, the ability of drugs to penetrate the blood-brain barrier can be greatly increased, and it has a better effect on brain tumor indications.
  • peptide or "polypeptide” is well known to those skilled in the art.
  • 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 into analogs, derivatives, functional mimetics, pseudopeptides, and the like containing at least two amino acids. Unless it is specified that the N-terminus or C-terminus has a specific modification, a polypeptide comprising a specific amino acid sequence includes unmodified and modified amino and/or carboxyl termini, which is well known to those skilled in the art.
  • a polypeptide of a specific amino acid sequence may include modified amino acids and/or additional amino acids, unless the N- and/or C-terminus contain modifications that prevent further addition of amino acids. Such modifications include, for example, acetylation of the N-terminus and/or amidation of the C-terminus.
  • polypeptides of the present invention can be modified to form polypeptide derivatives.
  • various modifications can be made to the polypeptides. 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 a combination of the above modifications.
  • the present invention includes any well-known modification of the polypeptide.
  • the polypeptide derivatives may include chemical modifications to the polypeptide, such as alkylation, acylation, carbamylation, iodination, or any other modification that produces a polypeptide derivative.
  • the modification of the polypeptide may include modified amino acids, for example, hydroxyproline or carboxyglutamic acid, and may include amino acids connected by non-peptide bonds.
  • non-natural amino acids can be used to replace the natural amino acids in the polypeptide, including but not limited to 2-amino fatty acid (Aad), 3-amino fatty acid ( ⁇ Aad), ⁇ -alanine, ⁇ -aminopropionic acid ( ⁇ Ala), 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 ( ⁇ Aib), 2-aminopimelic acid (Apm), 2,4-diaminobutyric acid (Dbu), desmosine (Des), 2,2'- Diaminopimelate (Dpm), 2,3-diaminopropionic acid (Dpr), N-ethylglycine (EtGly), N
  • 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, and 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, ⁇ -amino acids, natural amino acids, non-natural amino acids, and amino acid analogs.
  • 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).
  • Constant amino acid substitutions are amino acid substitutions in which an 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), ⁇ -branched side chains (e.g., T, V, I), and aromatic side chains (e.g., Y, F, W, H).
  • 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,
  • 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., norleucine replacing methionine) or other properties (e.g., 2-thienylalanine replacing phenylalanine).
  • 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.
  • nucleic acid encoding polypeptide sequences can be used (see Sambrook et al., Molecula r Cloning: A Labora tory 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 by well-known purification techniques.
  • an 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 certain discrete ways.
  • an analog is a substance that can be produced from a reference substance, for example, by chemical manipulation of the reference substance.
  • an analog is a substance that can be produced by the conduction of a synthetic process that is substantially similar to (e.g., shares multiple steps with) the synthetic process that produces the reference substance.
  • an analog is or can be produced by the conduction of a synthetic process that is different from the synthetic process used to produce the reference substance.
  • chlorotoxin analog refers to a polypeptide having an amino acid sequence that exhibits at least 45% identity to the amino acid sequence of an appropriate reference chlorotoxin (e.g., the amino acid sequence of SEQ ID NO: 1 or a related fragment thereof).
  • the chlorotoxin polypeptide has an amino acid sequence that exhibits 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 to SEQ ID NO: 1 or a related fragment thereof.
  • the chlorotoxin analog has the same amino acid sequence as SEQ ID NO: 1. In some embodiments, the chlorotoxin analog is a chlorotoxin variant because it has an amino acid sequence different from the amino acid sequence of SEQ ID NO: 1 or its related fragments. In some embodiments, the chlorotoxin variant has an amino acid sequence that is different from SEQ ID NO: 1 or its related fragments at no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions. In some embodiments, the related fragments of SEQ ID NO: 1 contain at least 5 adjacent residues of SEQ ID NO: 1.
  • the related fragments of SEQ ID NO: 1 contain a range of 5 to 25 amino acids, and the range of amino acids has at least 45% sequence identity with the corresponding range of SEQ ID NO: 1.
  • chlorotoxin analogs suitable for use in the practice of the present invention are described in International Application WO2003/101474 (the entire contents of which are incorporated herein by reference). Specific examples include polypeptides comprising or consisting of SEQ ID NO. 1 or SEQ ID NO. 8, as well as variants thereof, etc.
  • sequence identity is calculated by sequence alignment.
  • sequence alignment 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 the first amino acid sequence so as to optimally align with the second amino acid sequence. The amino acid residues at the corresponding amino acid positions are then compared. When the 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 sequences can be the same length or can be different lengths.
  • the optimal sequence alignment for determining the comparison window can be performed by the local homology algorithm of Smith and Waterman (J. Theor. Biol., 1981), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol, 1972), by the method of Pearson and Lipman to find similarity (Proc. Natl. Acad. Sci. U.S.A., 1988), by computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics 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, it is preferred to use default parameters, such as gap penalties or extension penalties.
  • the optimal alignment produced by various methods i.e., the highest identity percentage produced within the entire comparison window range) is selected.
  • cancer refers to a disease, disorder or condition in which cells exhibit relatively abnormal, uncontrolled and/or autonomous growth, such that they exhibit abnormally elevated proliferation rates and/or abnormal growth phenotypes, characterized by a significant loss of control over cell proliferation.
  • the characteristic of cancer may be one or more tumors.
  • cancer may be or include precancerous (e.g., benign), malignant, pre-metastatic, metastatic and/or non-metastatic cells.
  • the characteristic of a related cancer may be a solid tumor.
  • the characteristic of a related cancer may be a blood tumor.
  • examples of different types of cancer known in the art include, for example, cancers of the hematopoietic system, including leukemias, lymphomas (Hodgkin's and non-Hodgkin's lymphomas), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, solid tissue cancers, squamous cell carcinomas of the oral cavity, pharynx, larynx and lung, liver cancer, genitourinary cancers (such as prostate cancer, cervical cancer, bladder cancer, uterine cancer, endometrial cancer and renal cell carcinoma), bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancers of the endocrine system, thyroid cancer, parathyroid cancer, head and neck cancer, brain tumors, breast cancer, gastrointestinal cancer and nervous system cancers, benign lesions (such as papilloma), and the like.
  • leukemias including leukemias, lymphomas (
  • anticancer agent has its art-understood meaning and refers to one or more pro-apoptotic, cytostatic and/or cytotoxic agents, for example, particularly including agents used and/or recommended for the treatment of one or more diseases, disorders or conditions associated with undesirable cell proliferation.
  • the anticancer agent can be or include one or more alkyl agents, one or more anthracyclines, one or more cytoskeletal disruptors (e.g., microtubule targeting moieties such as taxanes, maytansine and their analogs), one or more epothilones, 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 analogs of one or more of the following (i.e., sharing relevant antiproliferative activity).
  • cytoskeletal disruptors e.g., microtubule targeting moieties such as taxanes, maytansine and
  • the anticancer agent can be or include one or more of the following: BCNU, Actinomycin, all-trans retinoic acid, auristatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, ambucil), cyclophosphamide, curcumin, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Maytansine and/or their analogs (e.g., DM1), Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, Maytansine
  • the invention relates to vinblastine,
  • 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.
  • a 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.
  • 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.
  • compositions preferably in the composition it includes an isotonic agent, for example, a sugar, a polyol, such as mannitol, sorbitol, or sodium chloride.
  • an isotonic agent for example, a sugar, a polyol, such as mannitol, sorbitol, or sodium chloride.
  • Pharmaceutically acceptable substances may also be included, such as a wetting amount or a trace amount of auxiliary substances, such as a wetting or emulsifier, preservative, or buffer that increases the shelf life and effectiveness of the antibody or antibody portion.
  • a disintegrant may be included, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof, such as sodium alginate.
  • pharmaceutical compositions 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.
  • 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.
  • liquid solutions such as injectable solutions and infusible solutions
  • dispersions or suspensions tablets, pills, powders, liposomes and suppositories.
  • 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.
  • Suitable routes of administration for the pharmaceutical composition include, but are not limited to, rectal, transdermal, vaginal, transmucosal or enteral administration.
  • 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).
  • Peptides can be synthesized by sequentially adding amino acids to a growing peptide chain.
  • liquid and solid phase peptide synthesis methods are applicable.
  • solid phase peptide synthesis methods the growing peptide chain is usually connected to an insoluble matrix (such as, for example, polystyrene beads) by connecting the C-terminal amino acid to the matrix.
  • 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.
  • HF hydrofluoric acid
  • automated, high throughput, and/or parallel peptide synthesis methods can also be used.
  • 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 inautomatic, manual and microwave-assisted solid-phase peptide synthesis," CurrOpin Drug Discov Devel., 11(6): 762-70, the entire contents of each of which are incorporated herein by reference.
  • polypeptides disclosed in the present invention may also exist in the form of their hydrates or in the form of containing their solvents (e.g., ethanol, DMSO, etc.), and may be used for crystallization.
  • solvents e.g., ethanol, DMSO, etc.
  • 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 solvated and unsolvated forms.
  • Figure 11 Killing curve of some PDC molecules of the present invention on brain glioma cells U87-MG in Test Example 1;
  • Figure 12 Killing curve of U373 glioma cells of some PDC molecules of the present invention in Test Example 1;
  • Figure 13 Statistical results of the average fluorescence signal of the cells in the brain glioma cells U87-MG 4 hours after the internalization of the polypeptide molecules in Test Example 2;
  • Figure 14 Statistical results of the average fluorescence signal of the cells in U373 glioma cells of test example 2 after endocytosis of the polypeptide molecule for 4 hours;
  • Figure 15 Statistical results of the average fluorescence signal of the cells in the brain glioma cells U87-MG after 24 hours of endocytosis of the polypeptide molecules in Test Example 2;
  • Figure 16 Statistical results of the average fluorescence signal of the cells in U373 glioma cells of test example 2 after 24 hours of endocytosis of the polypeptide molecule;
  • Figure 17 Confocal imaging results of the endocytosis of polypeptide molecules (3uM) by U87-MG glioma cells in Test Example 2 after 4 hours;
  • Figure 18 Confocal imaging results of U373 glioma cells in test example 2 after endocytosis of polypeptide molecules (3uM) for 4 hours;
  • Figure 19 Confocal imaging results of the endocytosis of polypeptide molecules (1uM) by U87-MG glioma cells in Test Example 2 after 24 hours;
  • Figure 20 Confocal imaging results of U373 glioma cells in test example 2 after endocytosis of polypeptide molecules (1uM) for 24 hours;
  • Test Example 3 Results of the human plasma stability test of some PDC molecules of the present invention
  • FIG22 Test Example 4: Results of mouse plasma stability test of some PDC molecules of the present invention.
  • Figure 23 In vitro evaluation results of peptide molecules penetrating the blood-brain barrier in Test Example 5;
  • Figure 24 Drug-time curve of compound 24, wherein A is the drug-time curve of brain tissue and B is the drug-time curve of plasma;
  • Figure 25 Drug-time curve of compound 25, wherein A is the drug-time curve of brain tissue and B is the drug-time curve of plasma;
  • Figure 26 Drug-time curve of compound 26, wherein A is the drug-time curve of brain tissue and B is the drug-time curve of plasma;
  • Figure 27 Drug-time curve of compound 27, wherein A is the drug-time curve of brain tissue and B is the drug-time curve of plasma;
  • Figure 28 AUC blood-brain ratio calculation results.
  • the polypeptide compounds and derivatives provided by the present invention are synthesized by solid phase synthesis method to synthesize their linear precursors, and the synthetic carrier is Rink Amide-AM Resin resin.
  • the Rink Amide-AM Resin resin is first fully swollen in N,N-dimethylformamide (DMF), and then the solid phase carrier and the activated amino acid derivative are repeatedly condensed ⁇ washed ⁇ Fmoc protection ⁇ washed ⁇ the next round of amino acid condensation to achieve the desired length of the synthesized polypeptide chain, and then the N-terminal amidation is completed on the solid phase, AEEA and FITC are coupled or tetradecanedioic acid is coupled, and then a mixed solution of trifluoroacetic acid: water: triisopropylsilane: anisyl thioether (90: 2.5: 2.5: 5:, v: v: v: v) is reacted with the resin to cleave the polypeptid
  • the crude straight-chain precursor after cleavage is subjected to disulfide bond oxidation in a weakly alkaline solution, and then purified and separated by a C-18 reverse phase preparative chromatography column using a 0.1% trifluoroacetic acid acetonitrile/water system to obtain an oxidized polypeptide.
  • the obtained oxidized polypeptide is coupled with a PTX conjugate in a liquid phase, and after the reaction, it is purified and separated by a C-18 reverse phase preparative chromatography column using a 0.1% trifluoroacetic acid acetonitrile/water system to obtain a pure polypeptide and its derivatives.
  • Step 1 Synthesis of linear precursor peptide chain
  • the linear precursor peptide chain of compound 1 is M-C-M-P-C-F-T-T-D-H-Q-M-A-R-K-C-D-D-C-C-G-G-K-G-R-G-K-C-Y-G-P-Q-C-L-C-R
  • Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.
  • the resin was washed 6-8 times with DMF until neutral pH.
  • the resin was washed 4-6 times with DMF before coupling the next amino acid.
  • Step 2 Cleavage of the linear precursor peptide chain
  • Step 5 Detection and characterization methods
  • the purified polypeptide from step 4 was subjected to analytical high performance liquid chromatography and liquid chromatography/mass spectrometry to determine the purity and intramolecular disulfide bond formation of the compound.
  • Step 1 Synthesis of linear precursor peptide chain
  • the linear precursor peptide chain of compound 3 is M(Boc)-C-M-P-C-F-T-T-D-H-Q-M-A-R-K-C-D-D-C-C-G-G-K-G-R-G-K(Mtt)-C-Y-G-P-Q-C-L-C-R
  • Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.
  • the resin was washed 6-8 times with DMF until neutral pH.
  • the resin was washed 4-6 times with DMF before coupling the next amino acid.
  • the resin was washed 5 times with DMF and 5 times with DCM.
  • Step 2 Coupling of AA27 lysine side chain with AEEA and FITC
  • Removal of the Mtt protecting group of the lysine side chain After swelling the resin with DCM for 1 h, add a mixed solution of hexafluoroisopropanol/dichloromethane (30% v/v, 10 mL) to the resin, shake the reaction at room temperature for 45 minutes and then remove the solution. Repeat the operation once. After the reaction, rinse the resin with DCM 5 times and DMF 6 times.
  • Lysine side chain coupling AEEA Weigh 1.0mmol Fmoc-AEEA-OH, 1.0mmol ethyl 2-oximecyanoacetate and dissolve in 8mL DMF, then add 160uLDIC for pre-activation for 3min, then add the mixed solution to the resin obtained in the previous step and shake for 3h. After the reaction, drain the reaction solution and wash with DMF 4-5 times.
  • N-terminal coupling of AEEA with FITC Weigh 0.4 mmol FITC and dissolve it in 5 mL DMF, add 1.0 mmol DIEA, and then add the mixed solution to the resin obtained in the previous step, and shake it in the dark for 5 hours. After the reaction, drain the reaction solution, rinse the resin with DMF 6-8 times until the discharged liquid is colorless, and rinse the resin with DCM 5 times. The resin is drained in a vacuum.
  • Step 3 Cleavage of the linear precursor peptide chain
  • Step 6 Detection and characterization methods
  • the purified peptide from step 6 was subjected to analytical HPLC and LC/MS to determine the purity and compound completion of K27 side-linking AEEA and FITC, as well as the formation of intramolecular disulfide bonds.
  • Step 1 Synthesis of linear precursor peptide chain
  • the linear precursor peptide chain of compound 17 is M-C-M-P-C-F-T-T-D-H-Q-M-A-R-R-C-D-D-C-C-G-G-R-G-R-G-K-C-Y-G-P-Q-C-L-C-R
  • Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.
  • the resin was washed 6-8 times with DMF until neutral pH.
  • the resin was washed 4-6 times with DMF before coupling the next amino acid.
  • acetylation reagent Dissolve 500uL of acetic anhydride and 500uL of DIEA in 9mL of DMF. Add 10mL of the prepared acetylation reagent to the resin obtained in step 1, shake well, and oscillate for 10 minutes. After the reaction, drain the reaction solution, rinse the resin with DMF 6-8 times, and rinse the resin with DCM 5 times. Drain the resin in a vacuum.
  • Step 3 Cleavage of the linear precursor peptide chain
  • Step 5 Purification and preparation of oxidized peptides
  • the product was separated using a reversed-phase high-performance liquid chromatography system, with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile).
  • the chromatographic column was a BR C-18 (Saifen) reversed-phase column, and during the purification process, the chromatograph detection wavelength was set at 230nm, the flow rate was 15mL/min, and the gradient was 20-40% acetonitrile in 40min.
  • the product-related fractions were collected, and after HPLC identification of the purity, the fractions >75% were combined and freeze-dried to obtain the oxidized peptide.
  • Step 8 Peptide coupling to PTX conjugate
  • Step 9 Purification of target peptide
  • the purified peptide from step 9 was subjected to analytical high performance liquid chromatography and liquid chromatography/mass spectrometry to determine the purity and compound completion of N-terminal acetylation, K27 side-linked PTX conjugate, and formation of intramolecular disulfide bonds.
  • the test results are shown in Figures 1 and 2.
  • Step 1 Synthesis of linear precursor peptide chain
  • the linear precursor peptide chain of compound 20 is M-C-M-P-C-F-T-T-D-H-Q-M-A-R-R-C-D-D-C-C-G-G-K-G-R-G-K-C-Y-G-P-Q-C-L-C-R
  • Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.
  • the resin was washed 6-8 times with DMF until neutral pH.
  • the resin was washed 4-6 times with DMF before coupling the next amino acid.
  • Step 3 Cleavage of the linear precursor peptide chain
  • Step 5 Purification and preparation of oxidized peptides
  • the product was separated using a reversed-phase high-performance liquid chromatography system, with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile).
  • the chromatographic column was a BR C-18 (Saifen) reversed-phase column, and during the purification process, the chromatograph detection wavelength was set at 230nm, the flow rate was 15mL/min, and the gradient was 20-50% acetonitrile in 40min.
  • the product-related fractions were collected, and after HPLC identification of the purity, the fractions >75% were combined and freeze-dried to obtain the oxidized peptide.
  • Purification method dissolve the sample in DMSO: acetonitrile 1:1, purify it with a C18 reverse phase column, and purify it with a gradient of 50-90% (the mobile phase is pure water and pure acetonitrile, without TFA)
  • Step 8 Peptide coupling to PTX conjugate
  • Step 9 Purification of target peptide
  • step 9 The purity of the peptide in step 9 was determined by analytical high performance liquid chromatography and liquid chromatography/mass spectrometry to confirm that the compound completed N-terminal acetylation, K23, K27 side-linked PTX conjugates, and formed intramolecular disulfide bonds.
  • the test results are shown in Figures 3 and 4.
  • Step 1 Synthesis of linear precursor peptide chain
  • the linear precursor peptide chain of compound 21 is M-C-M-P-C-F-T-T-D-H-Q-M-A-R-K-C-D-D-C-C-G-G-K-G-R-G-K-C-Y-G-P-Q-C-L-C-R
  • Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.
  • the resin was washed 6-8 times with DMF until neutral pH.
  • the resin was washed 4-6 times with DMF before coupling the next amino acid.
  • acetylation reagent Dissolve 500uL of acetic anhydride and 500uL of DIEA in 9mL of DMF. Add 10mL of the prepared acetylation reagent to the resin obtained in step 1, shake well, and oscillate for 10 minutes. After the reaction, drain the reaction solution, rinse the resin with DMF 6-8 times, and rinse the resin with DCM 5 times. Drain the resin in a vacuum.
  • Step 3 Cleavage of the linear precursor peptide chain
  • Step 5 Purification and preparation of oxidized peptides
  • the product was separated using a reversed-phase high-performance liquid chromatography system, with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile).
  • the chromatographic column was a BR C-18 (Saifen) reversed-phase column, and during the purification process, the chromatograph detection wavelength was set at 230nm, the flow rate was 15mL/min, and the gradient was 20-50% acetonitrile in 40min.
  • the product-related fractions were collected, and after HPLC identification of the purity, the fractions >75% were combined and freeze-dried to obtain the oxidized peptide.
  • Step 8 Peptide coupling to PTX conjugate
  • Step 9 Purification of target peptide
  • the purified peptide from step 9 was subjected to analytical high performance liquid chromatography and liquid chromatography/mass spectrometry to determine the purity and compound completion of N-terminal acetylation, K15, K23, K27 side-linked PTX conjugates, and formation of intramolecular disulfide bonds.
  • the detection spectra are shown in Figures 5 and 6.
  • Step 1 Synthesis of linear precursor peptide chain
  • the linear precursor peptide chain of compound 23 is M-C-M-P-C-F-T-T-D-H-Q-M-A-R-R-C-D-D-C-C-G-G-R-G-R-G-K-C-Y-G-P-Q-C-L-C-R
  • Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.
  • the resin was washed 6-8 times with DMF until neutral pH.
  • the resin was washed 4-6 times with DMF before coupling the next amino acid.
  • acetylation reagent 500uL of acetic anhydride and 500uL of DIEA dissolved in 9mL of DMF. Add the prepared acetylation reagent to the resin obtained in step 1, shake well, and oscillate for 10 minutes. After the reaction, drain the reaction solution, rinse the resin with DMF 6-8 times, and rinse the resin with DCM 5 times. Drain the resin in a vacuum.
  • Step 3 Cleavage of the linear precursor peptide chain
  • Step 5 Purification and preparation of oxidized peptides
  • the product was separated using a reversed-phase high-performance liquid chromatography system, with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile).
  • the chromatographic column was a BR C-18 (Saifen) reversed-phase column, and during the purification process, the chromatograph detection wavelength was set at 230nm, the flow rate was 15mL/min, and the gradient was 20-40% acetonitrile in 40min.
  • the product-related fractions were collected, and after HPLC identification of the purity, the fractions >75% were combined and freeze-dried to obtain the oxidized peptide.
  • Paclitaxel (0.950 g, 1.8 mmol) and 4-nitrophenyl carbonate (2.28 g, 7.49 mmol) were dissolved in 5 mL DMF, and 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.
  • the product was purified by C18 reverse phase chromatography column and freeze-dried to obtain Compound 1. Purification method: The sample was dissolved in DMSO: acetonitrile in a ratio of 1:1, and purified by C18 reverse phase chromatography column with a gradient of 40-90% (the mobile phase was pure water and pure acetonitrile without TFA).
  • Step 7 Peptide coupling to PTX conjugate
  • Step 8 Purification of target peptide
  • the purified peptide from step 9 was subjected to analytical high performance liquid chromatography and liquid chromatography/mass spectrometry to determine the purity and compound completion of N-terminal acetylation, K27 side-linked PTX conjugate, and formation of intramolecular disulfide bonds.
  • the test results are shown in Figures 7 and 8.
  • Step 1 Synthesis of linear precursor peptide chain
  • Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.
  • the resin was washed 6-8 times with DMF until neutral pH.
  • the resin was washed 4-6 times with DMF before coupling the next amino acid.
  • acetylation reagent Dissolve 500uL of acetic anhydride and 500uL of DIEA in 9mL of DMF. Add 10mL of the prepared acetylation reagent to the resin obtained in step 1, shake well, and oscillate for 10 minutes. After the reaction, drain the reaction solution, rinse the resin with DMF 6-8 times, and rinse the resin with DCM 5 times. Drain the resin in a vacuum.
  • Step 3 Coupling of AA15 lysine side chain to tetradecanedioic acid
  • Removal of Mtt protecting group of lysine side chain After swelling the resin with DCM for 1h, add hexafluoroisopropanol/dichloromethane mixed solution (30% v/v, 10mL) to the resin, shake and react at room temperature for 45 minutes, then remove, repeat the operation once, and wash the resin with DMF 6 times after the reaction.
  • Coupling of lysine side chain with tetradecanedioic acid weigh 1.0mmol tetradecanedioic acid, 1.0mmol ethyl 2-oximecyanoacetate and dissolve in 8mL DM, then add 160uL DIC for pre-activation for 3min, then add the mixed solution to the resin obtained in the previous step, shake and react for 3h. After the reaction, drain the reaction solution and wash with DMF 4-5 times.
  • Step 4 Cleavage of the linear precursor peptide chain
  • Step 6 Purification and preparation of oxidized peptides
  • the product was separated using a reversed-phase high-performance liquid chromatography system, with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile).
  • the chromatographic column was a BR C-18 (Saifen) reversed-phase column.
  • the chromatograph detection wavelength was set at 230nm, the flow rate was 15mL/min, and the gradient was 25-50% acetonitrile in 40min.
  • the product-related fractions were collected, and after HPLC identification of the purity, the fractions >75% were combined and freeze-dried to obtain the oxidized peptide.
  • Step 8 Synthesis of 2-NHS-Succinyl PTX
  • Purification method dissolve the sample in DMSO: acetonitrile 1:1, purify it with a C18 reverse phase column, and purify it with a gradient of 50-90% (the mobile phase is pure water and pure acetonitrile, without TFA)
  • Step 9 Peptide coupling to PTX conjugate
  • Step 10 Purification of target peptide
  • step 9 The purity of the peptide in step 9 was determined by analytical high performance liquid chromatography and liquid chromatography/mass spectrometry to confirm that the compound had completed N-terminal acetylation, K15 was linked to tetradecanedioic acid, K23, K27 were linked to PTX conjugates, and intramolecular disulfide bonds were formed.
  • the detection spectra are shown in Figures 9 and 10.
  • the invention evaluates the killing effect of PDC molecules on two types of brain glioma cells, U87-MG and U373, by measuring the killing experiment IC50.
  • U373 cells (Fenghui Biotechnology); U87-MG cells (Punosai); 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).
  • U87-MG cells were cultured and grown in culture medium (MEM, 10% FBS, 1% P/S).
  • culture medium MEM, 10% FBS, 1% P/S.
  • the cells were first rinsed with DPBS, and then digested with 0.25% trypsin (containing 0.5mM EDTA); the cell suspension was collected into a centrifuge tube, centrifuged at 1000rpm for 3min, and the supernatant culture medium was removed; 6-8mL of fresh growth medium was added to resuspend the cells, and the cells were passaged at a ratio of 1:3 to 1:8, and cultured in a 37°C, 5% CO 2 incubator. After passage, the medium was changed or passaged every 2-3 days.
  • U373 cells were cultured and grown in culture medium (DMEM, 10% FBS, 1% P/S). When the cell growth density reaches 80-90% of the culture flask, rinse the cells with DPBS first, then digest the cells with 0.25% trypsin (containing 0.5mM EDTA); collect the cell suspension into a centrifuge tube, centrifuge at 1000rpm for 3min, remove the supernatant medium; add 6-8mL of fresh growth medium to resuspend the cells, subculture at a ratio of 1:3 to 1:8, and culture in a 37°C, 5% CO2 incubator. After subculture, change the medium or subculture every 2-3 days.
  • culture medium DMEM, 10% FBS, 1% P/S.
  • U373 cells and U87-MG cells were subcultured 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 medium, the cell viability was detected and counted, and the cell concentration was adjusted to 2 ⁇ 104 cells/mL with complete medium.
  • 100 ⁇ L/well was inoculated into the middle well of the 96-well plate, and the edge wells were filled with the same volume of 100 ⁇ L/well DPBS, and incubated in a 37°C, 5% CO 2 incubator overnight.
  • PDC drug was dissolved to 1 mM using sterile water or DMSO.
  • the PDC drug was diluted with the growth medium of U373 cells and U87-MG cells to a concentration of 1 ⁇ M (2 ⁇ concentration), and then diluted 5-fold using the growth medium of the corresponding cells, for a total of 9 concentrations (500 nM-0.00128 nM).
  • the IC50 results of the tumor cell killing experiment of some PDC molecules of the present invention are shown in Table 1, and the killing curves of two types of brain glioma cells, U87-MG and U373, are shown in Figures 11 and 12.
  • 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-related protein (LRP1) transport system and penetrate malignant cells.
  • LRP1 low-density lipoprotein receptor-related protein
  • the experimental results show that the PDC molecule of the present invention has a good killing effect on tumor cells U87-MG and U373.
  • Test Example 2 Fluorescence imaging to detect the endocytosis effect of peptides in U87-MG and U373 cells
  • peptides designed and synthesized by the company 96-well black transparent cell plates (Aligent); fixative (Biyuntian); DAPI (Biyuntian); DPBS (homemade).
  • 4405, 24, 25, 26, and 27 are Cy5-labeled polypeptides, of which 4405 is Cy5-labeled Angiopep-2, and the PDC molecules corresponding to 24, 25, 26, and 27 are 17, 18, 20, and 21, respectively.
  • U87-MG or U373 cells were subcultured 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 medium, the cell viability was detected and counted, and the cell concentration was adjusted to 4 ⁇ 104 cells/mL with complete medium.
  • 100 ⁇ L/well was inoculated into the middle well of the 96-well plate, and the edge wells were filled with the same volume of 100 ⁇ L/well DPBS, and incubated in a 37°C, 5% CO2 incubator overnight.
  • the peptide was dissolved in DMSO to 1 mM. During the experiment, the peptide was diluted with each cell growth medium to a concentration of 10 ⁇ M. The peptide was diluted 3-fold with each cell growth medium, for a total of 3 concentrations (10 ⁇ M-1.11 ⁇ M).
  • the confocal imaging results are shown in Figures 13 to 20.
  • the endocytosis effect of the polypeptides in U87-MG and U373 cells is 27>26>25>24>4405, and the corresponding endocytosis effect of PDC molecules is 21>20>18>17>ANG-1005.
  • test samples Dissolve the peptide to be tested in DMSO or other organic solvents to 100 times the final concentration of 1mM, and place it at -20°C for use.
  • Plasma thawing Take out human plasma (sample number * 2.1) mL from the -80°C refrigerator and quickly thaw it in a 37°C water bath.
  • MIX mixture
  • Oscillate on a vortex oscillator for 30s divide 100 ⁇ L into each according to the time gradient, and incubate, all on ice. Incubation: Incubate in a 37°C water bath at six time points: 0min, 15min, 30min, 60min, 90min, and 120min. Stop the reaction: After incubation, add 4 times the volume of precipitant. Mixing: Oscillate on a vortex oscillator for 30s. Centrifugation: 4°C, 15000r/min for 10min. Take the supernatant, transfer to a small injection tube, and send to LC-MS/MS for analysis.
  • the dot-line graph with the original drug remaining rate (%) as the ordinate and time as the abscissa shows a trend of sample degradation in in vitro plasma over time, and the sample stability result is obtained.
  • the elimination rate constant (Ke) was calculated based on the first-order kinetic formula, and the T 1/2 (min) of the compound in plasma was further calculated based on the formula.
  • Mouse plasma (heparin sodium) was obtained by Slack.
  • Samples were diluted to 0.1 mM using DMSO.
  • Termination reaction After incubation, different pretreatment methods are performed according to the pretreatment methods required for biological analysis.
  • Centrifugation Centrifuge at 4°C, 13,000 rpm for 10 min in a low-temperature high-speed centrifuge. Take 70 ⁇ L of the supernatant, transfer it to a sample injection bottle, and analyze it by LC-MS/MS.
  • Result evaluation The peak area of the peptide at different time points was detected by LC-MS/MS method, and the results were expressed as the percentage of the original drug remaining rate. The results are shown in Table 3, Table 4, and Figure 22. Table 3 shows the fitting results of the prism software. The half-life of compound 21 and the half-life of compound 32 were 1021 min and 304.5 min, respectively, using the prism software.
  • Test Example 5 In vitro evaluation of peptide molecules penetrating the blood-brain barrier
  • 25, 26, and 27 are Cy5-labeled polypeptides, of which 4405 is Cy5-labeled Angiopep-2, and the PDC molecules corresponding to 25, 26, and 27 are 18, 20, and 21, respectively.
  • the original culture medium was aspirated and discarded, and room temperature sterile HBSS was added for rinsing once, and replaced with EBM-2 culture medium containing 3 ⁇ g/mL puromycin, and the medium was changed every two days thereafter (no puromycin was added for subsequent changes).
  • EBM-2 culture medium containing 3 ⁇ g/mL puromycin
  • the medium was changed every two days thereafter (no puromycin was added for subsequent changes).
  • the above cells were transferred to a Transwell covered with rat tail glue for culture, and continued to be cultured for 7-8 days, and the medium was changed every two days and the resistance value was tested.
  • the rate at which drugs permeate through rat primary brain endothelial cells is expressed by the apparent permeability coefficient (Papp, unit: ⁇ 10-6cm/s).
  • VR is the volume of the receiving solution (0.6 mL)
  • Area is the membrane area of the Transwell-24 well plate chamber (0.33 cm 2 )
  • Time is the incubation time (unit: s)
  • CR is the drug concentration at the sample receiving end
  • C0 is the drug concentration at the initial time 0 of the sample.
  • the experimental results are shown in FIG23 .
  • the experimental results show that in the in vitro blood-brain barrier model, the peptide penetration is 27>26>25>4405, and the corresponding PDC molecule penetration is 21>20>18>ANG-1005.
  • mice 6-8 week old male ICR mice (purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.), DMSO (dimethyl sulfoxide) (purchased from Aladdin).
  • 24, 25, 26, and 27 are Cy5-labeled polypeptides, and the PDC molecules corresponding to 24, 25, 26, and 27 are 17, 18, 20, and 21, respectively.
  • Preparation of test samples and sample processing label the peptide to be tested with cy5, prepare the marker working solution with DMSO, plot the fluorescence value and the marker curve corresponding to the concentration of the labeled peptide in plasma and brain homogenate, respectively, and prepare the injection dose according to the 1/2000 ratio of the marker curve detection line.
  • Tail vein administration while the animal is awake, use a 1mL syringe with a 27# needle to administer 200uL into the tail vein of the mouse.
  • Blood collection and centrifugation of sampled serum under isoflurane anesthesia, cut open the heart and ears at fixed time points, and collect blood with a pipette. The blood collection volume is not less than 0.4ml.
  • the collected sampled blood is centrifuged at 4°C and 4000g for 5min. Take the supernatant. Take the brain and grind the brain tissue homogenate: perfuse the whole body of the mouse with 20mL PBS from the heart, then cut off the head and take the brain. Take half of the brain tissue, weigh it, and homogenize it at 1:2 (0.1g brain tissue plus 100uL PBS). The homogenization speed is 60hz, 60s, and two 2mm magnetic beads per tube. After homogenization, take the middle homogenate, and put the remaining brain tissue in a culture dish and store it in a -80°C refrigerator.
  • Sample detection After the brain tissue is taken out and before homogenization, use the IVIS system imaging scan, take 100 ⁇ L of serum and brain homogenate, add it to a 96-well plate to read the fluorescence value, and the sample to be tested and the standard curve are detected on the enzyme reader at the same time (640/670Cy5).
  • the concentrations in different matrices at different time points were obtained according to the standard curves.
  • the drug-time curves were plotted using the drug concentrations in brain tissue and plasma as the ordinate and time as the abscissa.
  • the areas under the curves (AUCs) of the brain tissue and plasma drug-time curves were calculated respectively, and the AUC blood-brain ratio was calculated based on the AUCs.
  • AUC blood-brain ratio AUCBrain/AUCPlasma, where AUCBrain refers to the area under the curve of the brain tissue drug-time curve, and AUCplasma refers to the area under the curve of the plasma drug-time curve.
  • the experimental results are shown in Table 5 and Figures 24 to 28.
  • the PDC molecules of the present invention can penetrate the blood-brain barrier in mice and reach the brain tissue, and have a good penetration effect.
  • the peptide was dissolved in acetonitrile/water solution, it was analyzed by Agilent HPLC 1260, and the buffer was A (90% H2O (20mM Na 2 HPO4 + 20mM NaH2PO4) / 10% ACN) and B (70% ACN + 30% H2O).
  • the chromatographic column was Yuexu C18 reverse phase column (4.6*150mm, 5um, ), the chromatograph detection wavelength was set at 214 nm, the flow rate was 1 mL/min, and the gradient was 10% B-95% B in 26 min.
  • mice ICR mice, 18, male, 6-7 weeks old, purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.
  • mice aged 6-7 weeks were raised under standard conditions. After one week of adaptive feeding, 8 mice of similar weight were randomly divided into 2 groups, 4 mice in each group. The compounds were prepared at 1.25 mg/mL, and the tail vein injection volume was 10 mL/kg. After the administration, about 200uL of blood was collected from the submandibular vein of the mice at 40s, 5min, 15min, 30min, 60min, 120min, 240min, and 360min. The blood samples were placed in EDTA anticoagulant tubes, centrifuged, separated, and collected plasma, which was placed in a -80°C refrigerator for testing.
  • the PDC molecules of the present invention have good pharmacokinetic properties.

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Abstract

L'invention concerne un conjugué polypeptidique d'un analogue de chlorotoxine et son utilisation dans la préparation d'un médicament pour la prévention, le soin et le traitement de maladies associées au cancer. Le conjugué polypeptidique d'un analogue de chlorotoxine est tel que représenté dans la formule (I). Le conjugué de médicament polypeptidique d'un analogue de chlorotoxine améliore la solubilité du médicament, et présente les caractéristiques d'une activité élevée, d'une capacité de liaison hautement spécifique à une cible, d'une endocytose significative, étant capable d'augmenter la capacité du médicament à pénétrer à travers la barrière hémato-encéphalique, etc.
PCT/CN2024/084351 2023-04-10 2024-03-28 Conjugué polypeptidique d'analogue de chlorotoxine et utilisation associée Pending WO2024212817A1 (fr)

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