WO2023165476A1 - 针对sort1的多肽化合物及其药物偶联物 - Google Patents

针对sort1的多肽化合物及其药物偶联物 Download PDF

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WO2023165476A1
WO2023165476A1 PCT/CN2023/078789 CN2023078789W WO2023165476A1 WO 2023165476 A1 WO2023165476 A1 WO 2023165476A1 CN 2023078789 W CN2023078789 W CN 2023078789W WO 2023165476 A1 WO2023165476 A1 WO 2023165476A1
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polypeptide
drug conjugate
arg
peptide
cys
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PCT/CN2023/078789
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English (en)
French (fr)
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董志超
陈昌发
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上海智肽生物科技有限公司
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Priority to CN202380010846.0A priority Critical patent/CN117098770A/zh
Publication of WO2023165476A1 publication Critical patent/WO2023165476A1/zh

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    • 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
    • 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
    • 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
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the present invention relates to the technical field of biomedicine, in particular to a polypeptide compound targeting the Sort1 target and drug-coupled drugs thereof, as well as their preparation method and application.
  • Cancer is the number one killer threatening human health. According to data released by the International Agency for Research on Cancer (IARC) of the World Health Organization, there will be 19.3 million newly diagnosed cancers in the world in 2020, of which 4.57 million are newly diagnosed in my country, ranking first in the world. Although surgery can remove localized tumors in the early stage, it has little effect on advanced or systemic cancers; radiotherapy and chemotherapy can cause side effects such as hair loss and vomiting.
  • IARC International Agency for Research on Cancer
  • Targeted drugs are the "rising stars" in the antineoplastic drug market.
  • a major feature of targeted drugs is that they act on specific targets. Each patient's situation is different, and the targeted drugs that can be selected are also different. To a certain extent, individualized treatment of tumors can be achieved. Judging from the demand trend of drugs, obvious curative effect and less side effects are the main demand direction of future product development. Driven by this market demand, the research and development and clinical application of anti-tumor targeted drugs will be the main development direction of the anti-tumor drug industry in the future one.
  • Sortilin (NP_002950.3), also known as neurotensin receptor 3 (NTSR3), is a 100kDa type 1 membrane glycoprotein encoded by the SORT1 gene (Gene ID: 6272), which is located on human chromosome 1P13. 3.
  • the SORT1 gene encodes five variants of Sortilin, including the full-length sequence of 831 amino acids (aa), four truncated splice variants 694, 118, 60, and 20 aa (Fatemeh Ghaemimanesh et al., 2021). Sortilin belongs to the vacuolar protein sorting 10 protein (VPS10) receptor family.
  • VPS10 vacuolar protein sorting 10 protein
  • the Vps10 protein family includes heterologous type 1 transmembrane receptors, including Sortilin (100kDa), SorLA, SorCS1, SorCS2 and SorCS3, and mature Sortilin is differentiated from the precursor prepro-Sortilin (Munck Petersen et al., 1999). Sortilin can not only be located on the Golgi membrane as a sorting protein, but also be located on the cell membrane as a clearance receptor.
  • Sortilin consists of a pre-peptide propeptide (common signal sequence for proteins that insert into the cell membrane; 1-33aa), a pro-peptide propeptide (34-77aa), a large luminal/extracellular region (Vps10p domain; 78–755aa), 756–778aa of the transmembrane segment and 779–831aa of the intracellular domain (Figure 1) (Fatemeh Ghaemimanesh et al., 2021).
  • Sortilin was first discovered for its ability to bind neurotensin, and has since been found to bind a range of ligands, including the neurotrophin precursor. More and more studies have confirmed that Sortilin can interact with many molecules and affect their intracellular distribution. As in neurons and B lymphocytes, Sortilin regulates the intracellular transport of neurotrophic factors. In particular, the precursor form of Sortilin After being cleaved off the N-terminal precursor part, it becomes mature Sortilin and is continuously secreted to the cell membrane. This process exposes Sortilin to the cell membrane and binds to its ligand, thereby promoting its binding to the ligand and transducing its intracellular The signal either directly mediates the cellular internalization of the ligand.
  • Sortilin has dual functions in cells: it not only participates in the regulation of cellular transport of substances, but also acts as a receptor to transduce signals of extracellular ligands in cells (Fatemeh Ghaemimanesh et al., 2021).
  • ADCs Antibody-drug conjugates
  • ADC drugs are a class of targeted biologics consisting of antibodies, linkers, and cytotoxic drugs.
  • the antibody used by ADC has a very high affinity to the surface antigen of the tumor cell, and the antibody has a long half-life (1-3 weeks) for normal cells containing the same target, so it will continue to kill normal cells during the time it remains in the body , which greatly increases the toxic and side effects of the drug.
  • the antibody is a clumsy means of transportation, and it is difficult to walk through the small alleys of the tumor. It is estimated that only 0.1% of the drug can reach the tumor tissue. In order to ensure that the other 99% of the highly toxic The warhead should not bring systemic toxicity, and the chemical link connecting the warhead to the antibody must be sufficiently stable.
  • ADCs are also prone to aggregation, which leads to modifications that reduce their ability to bind antigen.
  • Protein aggregation is a major hurdle in ADC development, and aggregation can occur at every stage as well as during shipping and long-term storage. Aggregates are immunogenic. Additionally, protein aggregation can lead to product loss. Overall, any chemical or physical degradation could lead to structural changes of ADCs and lead to excessive protein aggregation. There are also various other factors that can cause aggregation, such as frequent freeze/thaw, high protein and salt concentrations, elevated temperature or low pH. In addition, most payloads are hydrophobic, and binding payloads with high DAR on protein surfaces can lead to excessive protein aggregation, hindering the successful development of ADCs.
  • ADCs antibody-conjugated drugs
  • ADCs have the same immunogenicity as antibody drugs, and the risk of immunogenicity affects the safety and effectiveness of drugs for patients, and may even be caused by cross-talk between ADA and endogenous proteins. Bring deadly new diseases to patients.
  • PDC Peptide-Drug Conjugate
  • PDC drugs have small molecular weight, strong tumor penetration, Low immunogenicity, large-scale synthesis by solid-phase synthesis, low production cost, relatively good pharmacokinetics and other characteristics, it has become the next generation of targeted drugs after small molecule targeted drugs, monoclonal antibodies, and ADCs. antineoplastic drugs.
  • the object of the present invention is to provide a targeting peptide with non-natural amino acid or cyclic peptide targeting tumor highly expressed target protein Sortilin, and a targeting peptide-drug conjugate containing the targeting peptide.
  • a first aspect of the present invention provides an isolated polypeptide or a pharmaceutically acceptable salt thereof, the polypeptide comprising modified or unmodified unnatural amino acids, and/or through an intro-chain linker The two cysteine residues connected by cyclization, and the polypeptide specifically targets the SORT1 protein;
  • the in-chain linker is or disulfide bonds.
  • polypeptide is a linear peptide or a cyclic peptide.
  • the polypeptide is a cyclic peptide
  • the cyclic peptide is formed by cyclization of two cysteine residues separated in the polypeptide sequence, wherein the cysteine residues are respectively located at Within the range of 1-6 positions from the N-terminal of the polypeptide sequence, and within the range of 1-6 positions from the C-terminal of the polypeptide sequence, and the interval between the two cysteine residues is not less than 3 amino acids . .
  • amino acid sequence structure of the polypeptide is shown in Formula I: X 0 -X 1 -X 2 -X 3 -X 4 -X 5 -Ala-X 7 -Val-Arg-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 (I)
  • X 0 is acetylation (Ac) modified or unmodified Cys or none, or Z 0 -Cys, wherein Z 0 is 1-3 amino acid residues;
  • X is acetylated (Ac) modified or unmodified Gly, D-Ala, Val, Asn, Arg, Gln or none;
  • X2 is Val, 1Nal, 2Nal, D-2-Nal, or Tyr;
  • X3 is Arg or hArg
  • X 4 is Ala, Arg or none
  • X5 is Lys, hArg, Arg or Cys
  • X7 is Gly, Arg, D-Ala, Leu, Phe, or Cys;
  • X 10 is Asp or Asn
  • X 11 is Val or Nle
  • X 12 is Phe, 4-Cl-Phe, 4-F-Phe, Aib or 1Nal;
  • X 13 is Lys, hArg, Arg or Cys
  • X 14 is Ser, Aib, Asn or Cys
  • X 15 is Glu, Arg, Lys or Aib
  • X 16 is Ser, Arg or Aib
  • X 17 is Tyr or Aib
  • X 18 is Cys or none, or Cys-Z 1 , wherein Z 1 is 1-3 amino acid residues.
  • X 1 when X 0 is nothing, X 1 is Gly, D-Ala, Val, Asn, Arg or Gln modified by acetylation (Ac); when X 0 is not nothing, X 1 is not Modified Gly, D-Ala, Val, Asn, Arg or Gln.
  • the polypeptide is a cyclic peptide, which comprises two cysteine residues connected by cyclization of an intro-chain linker, wherein the cysteine residues They are respectively located in the range of X 0 -X 5 positions and X 13 -X 18 positions of the polypeptide sequence shown in formula (I).
  • the intra-chain linker is N-chain linker
  • polypeptide is selected from the following group:
  • polypeptide is a synthetic polypeptide.
  • a drug conjugate is provided, and the drug conjugate has the structure shown in the following formula II: (D) n -LP (II)
  • D is the effective load
  • P is targeting peptide, and described targeting peptide is the polypeptide as claimed in claim 1;
  • the payload is selected from the group consisting of docetaxel, paclitaxel, or cabazitaxel, or derivatives thereof.
  • the linker is selected from the group consisting of succinic acid or dimethylglutaric acid.
  • the linker is succinic acid.
  • the targeting peptide is selected from the polypeptides shown in Table A.
  • the drug conjugate is selected from the following group:
  • the payload is connected to the lysine residue of the targeting peptide through a linker.
  • the third aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (a) the drug conjugate according to claim 2; and (b) a pharmaceutically acceptable carrier.
  • the pharmaceutical composition also contains other drugs for treating tumors.
  • the fourth aspect of the present invention provides the use of the polypeptide according to the first aspect of the present invention, or the drug conjugate according to the second aspect of the present invention in the preparation of a drug for treating tumor or cancer.
  • the tumor or cancer includes solid tumors and blood tumors.
  • the tumor or cancer highly expresses SORT1 protein.
  • the tumor or cancer includes but is not limited to acute myeloid leukemia, chronic myelogenous leukemia, multiple myelopathy, non-Hodgkin's lymphoma, colorectal cancer, breast cancer, colorectal cancer, gastric cancer , liver cancer, leukemia, kidney tumors, lung cancer, small bowel cancer, bone cancer, prostate cancer, cervical cancer, lymphoma, adrenal gland tumors, or bladder tumors.
  • the fifth aspect of the present invention provides a method for preparing the polypeptide according to the first aspect of the present invention, the method comprising the following steps:
  • step (1) the linear polypeptide obtained in step (1) is subjected to a cyclization reaction with a cyclization reagent to obtain a cyclic peptide.
  • the sixth aspect of the present invention provides a method for preparing the drug conjugate as described in the second aspect of the present invention, the method comprising the following steps:
  • step (C) performing an activated ester reaction of the polypeptide according to claim 1 with the intermediate of step (A), thereby obtaining the drug conjugate;
  • step (D) purifying the drug conjugate in step (C).
  • the seventh aspect of the present invention provides the use of the polypeptide according to the first aspect of the present invention in the preparation of a detection reagent or kit for detecting SORT1 protein.
  • the kit is used to detect SORT1 protein in a sample.
  • the sample includes a blood sample, a body fluid sample or a tissue sample.
  • the kit further includes instructions, which describe that the kit is used for non-invasive detection of SORT1 protein expression in the subject.
  • the eighth aspect of the present invention provides a method for detecting SORT1 protein in a sample, the method comprising the steps of: (i) contacting the sample with the polypeptide according to the first aspect of the present invention; (ii) detecting whether the polypeptide is formed - SORT1 protein complex, wherein formation of the complex indicates the presence of SORT1 protein in the sample.
  • the detection is non-diagnostic and non-therapeutic.
  • the detection is used for the diagnosis or prognosis of tumor or cancer.
  • the ninth aspect of the present invention provides a kit comprising the polypeptide according to the first aspect of the present invention.
  • the kit is used to detect SORT1 protein in a sample.
  • the sample includes a blood sample, a body fluid sample or a tissue sample.
  • the kit further includes instructions, which describe that the kit is used for non-invasive detection of SORT1 protein expression in the subject.
  • the tenth aspect of the present invention provides a method for treating tumor or cancer, the method comprising administering to a subject in need a therapeutically effective amount of the drug conjugate of the second aspect of the present invention or the third aspect of the present invention Aspect pharmaceutical composition.
  • the tumor or cancer includes solid tumors and blood tumors.
  • the tumor or cancer highly expresses SORT1 protein.
  • the tumor or cancer includes but is not limited to acute myeloid leukemia, chronic myelogenous leukemia, multiple myelopathy, non-Hodgkin's lymphoma, colorectal cancer, breast cancer, colorectal cancer, gastric cancer , liver cancer, leukemia, kidney tumors, lung cancer, small bowel cancer, bone cancer, prostate cancer, cervical cancer, lymphoma, adrenal gland tumors, or bladder tumors.
  • Figure 1 shows a schematic diagram of the structure of Sortilin.
  • Figure 2 shows the MS diagram of SMTB01 linear peptide.
  • Figure 3 shows the MS map of SMTB01.
  • Figure 4 shows the high-resolution mass spectrometry results of the SMTB01-docetaxel conjugate.
  • Figure 5 shows the relative body weight change (%) of MDA-MB-231 tumor-bearing female BALB/c nude mice after being treated with the test substance. Relative body weight changes were calculated based on animal body weight at the start of dosing. Data points represent mean percent change in body weight for each group, and error bars represent standard error (SEM).
  • FIG 6 shows the content of neutrophils in MDA-MB-231 tumor-bearing female BALB/c nude mice on the 4th day after being treated with the test substance. Data represent group means and error bars represent standard error (SEM).
  • FIG 7 shows the content of neutrophils in MDA-MB-231 tumor-bearing female BALB/c nude mice on the 18th day after being treated with the test substance. Data represent group means and error bars represent standard error (SEM).
  • Figure 8 shows the tumor growth curve of MDA-MB-231 tumor-bearing female BALB/c nude mice after being treated with the test substance. Data points represent mean tumor volumes for each group and error bars represent standard error (SEM).
  • the inventors unexpectedly developed a class of targeting peptides that specifically target Sortilin for the first time, and the targeting peptides include linear peptides or cyclic peptides containing unnatural amino acids , and using the targeting peptide to prepare a targeting peptide-drug conjugate.
  • the targeting peptide prepared by the present invention is a linear peptide or a cyclic peptide containing non-natural chemical modification, which can improve the half-life and stability of the targeting peptide-drug conjugate.
  • the targeting peptide-drug conjugate provided by the invention can specifically bind the target protein Sortilin with high affinity, thereby efficiently killing tumors expressing Sortilin, and provides a new type of anti-tumor targeting drug for the field of tumor treatment.
  • SORT1 protein and “Sortilin” are used interchangeably and both refer to the protein encoded by the SORT1 gene (Gene ID: 6272).
  • isolated polypeptide As used herein, the terms “isolated polypeptide”, “targeting peptide” and “peptide compound” are used interchangeably, and all refer to the synthetic polypeptide targeting SORT1 protein of the first aspect of the present invention.
  • drug conjugate As used herein, the terms “drug conjugate”, “targeting peptide-drug conjugate”, and “conjugated compound” are used interchangeably, and all refer to the polypeptide of the first aspect of the present invention and one or more therapeutic agent molecules.
  • small molecular compounds such as docetaxel, paclitaxel or cabazitaxel
  • linkers linkers
  • an isolated polypeptide is provided, and the polypeptide is a targeting peptide targeting Sortilin.
  • the polypeptide may be a linear peptide or a cyclic peptide comprising modified or unmodified unnatural amino acids, and/or two cysteine residues linked by cyclization of an intro-chain linker.
  • the term "intro-chain linker” refers to a linker used to cyclize two cysteine residues in a polypeptide chain, in a preferred embodiment of the invention, said chain The intro-chain linker is or disulfide bonds, preferably
  • the present invention also includes active fragments, derivatives and analogs of the polypeptide of the first aspect of the present invention.
  • fragment refers to a polypeptide that substantially retains the function or activity of binding Sortilin.
  • polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) A polypeptide having substituent groups in amino acid residues, or (iii) a polypeptide formed by fusing the polypeptide described in the first aspect of the present invention with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or ( iv) A polypeptide formed by fusing an additional amino acid sequence to this polypeptide sequence (a protein fused with a leader sequence, a secretory sequence, or a tag sequence such as 6His).
  • polypeptide formed by fusing an additional amino acid sequence to this polypeptide sequence a protein fused with a leader sequence, a secretory sequence, or a tag sequence such as 6His.
  • One class of preferred active derivatives refers to that compared with the amino acid sequence of formula I, there are at most 6, preferably at most 3, more preferably at most 2, and most preferably 1 amino acid is replaced by an amino acid with similar or similar properties. substitution to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.
  • the present invention also provides analogs of the polypeptides described in the first aspect of the present invention.
  • the difference between these analogs and the polypeptide described in the first aspect of the present invention may be the difference in amino acid sequence, or the difference in the modified form that does not affect the sequence, or both.
  • Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, ⁇ , ⁇ -amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.
  • Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications of polypeptides during synthesis and processing or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.
  • chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications of polypeptides during
  • the polypeptide of the present invention can also be used in the form of a salt derived from a pharmaceutically or physiologically acceptable acid or base.
  • salts include, but are not limited to, those formed with the following acids: hydrochloric, hydrobromic, sulfuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, succinic, oxalic, fumaric, maleic, acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid.
  • Other salts include those formed with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, as well as in the form of esters, carbamates or other conventional "prodrugs".
  • a targeting peptide-drug conjugate comprising the targeting peptide described in the first aspect of the present invention, which has the structure shown in the following formula II: (D) n -LP (II)
  • D is the effective load
  • P is the targeting peptide described in the first aspect of the present invention.
  • the payload is a small molecule compound, including but not limited to docetaxel, paclitaxel, or cabazitaxel.
  • docetaxel is as follows:
  • the linker is selected from succinic acid or dimethylglutaric acid, preferably succinic acid.
  • composition of the targeting peptide-drug conjugate of the present application is shown in the following table:
  • a method for preparing the isolated polypeptide (targeting peptide) of the first aspect of the present invention includes the following steps:
  • the initial polypeptide is synthesized by a solid-phase synthesis method
  • step (2) cracking the product of step (1) with a strong acid; adding a side chain protecting group scavenger, filtering, adding an appropriate amount of organic solvent to precipitate, centrifuging, washing the precipitate with an organic solvent, and drying to obtain a crude peptide;
  • step (3) the crude peptide obtained in step (2) is subjected to a cyclization reaction with a cyclization reagent to obtain a cyclized polypeptide.
  • the resin carrier used in the solid-phase synthesis in the step (1) is selected from Wang resin or 2-CTC resin.
  • the step (1) includes the following sub-steps:
  • the amino protecting group refers to a chemical group introduced to protect the amino group participating in the condensation reaction.
  • the amino protecting group can be selected from tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Z) or 9-fluorenyl-methoxycarbonyl (Fmoc), preferably 9-fluorenyl-methoxycarbonyl (Fmoc).
  • the solvent used in the step (a) is selected from: dimethylformamide (DMF), dichloromethane (DCM) or N-methylpyrrolidone (NMP), preferably DMF or DCM.
  • DMF dimethylformamide
  • DCM dichloromethane
  • NMP N-methylpyrrolidone
  • the removal agent for removing the amino protecting group in the step (a) is selected from the piperidine/DMF (PIP) with a concentration of 10-40%, preferably the piperidine/DMF (PIP) with a concentration of 20-25%;
  • the time is 20-50min, preferably the removal time is 25-35min.
  • the step of amino acid condensation in the step (a) needs to add a condensation reagent, and the condensation reagent is selected from carbodiimide type reagents, benzotriazolium salt type reagents or 1-hydroxybenzotriazole (HOBt), or a combination thereof.
  • the condensation reagent is selected from carbodiimide type reagents, benzotriazolium salt type reagents or 1-hydroxybenzotriazole (HOBt), or a combination thereof.
  • the carbodiimide type reagent is selected from dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) or N-diaminopropyl-N-ethylcarbodiimide (EDC ) in one.
  • DCC dicyclohexylcarbodiimide
  • DIC diisopropylcarbodiimide
  • EDC N-diaminopropyl-N-ethylcarbodiimide
  • the benzotriazolium salt-type reagent is selected from 2-(1H-benzotriazol L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) , O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), hexafluorophosphate benzotriazole-1-oxytris(dimethylamino)phosphorus ( BOP) or benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP).
  • TBTU 2-(1H-benzotriazol L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
  • HBTU O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate
  • BOP he
  • the coupling reagent is selected from diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt), or 2-(1H-benzotrisazo L-1-yl)-1, 1,3,3-Tetramethyluronium tetrafluoroborate (TBTU) and 1-hydroxybenzotriazole (HOBt), more preferably DIC (diisopropylcarbodiimide) and 1-hydroxybenzotriazole azole (HOBt).
  • the "monitoring" in step (a) uses ninhydrin detection to monitor the condensation reaction of the polypeptide.
  • the sequential coupling of remaining amino acids in the step (a) refers to connecting amino acids one by one from the C-terminal to the N-terminal according to the amino acid sequence of the polypeptide.
  • the side chain protecting group scavenger described in step (2) is selected from thioanisole, triisopropylsilane, phenol, water, 1,2-ethanedithiol or m-cresol One, two or a combination of several.
  • the cyclization reaction described in step (3) needs to add a cyclization reagent and ammonium bicarbonate (NH4HCO3), wherein the cyclization reagent is selected from 1,2-(dibromomethyl)benzene, 1, 3-(Dibromomethyl One or more combinations of benzene, 1,4-(dibromomethyl)benzene or other bromoalkyl-substituted benzene reagents, preferably 1,2-(dibromomethyl)benzene, 1,3- (Dibromomethyl)benzene or 1,4-(dibromomethyl)benzene, more preferably 1,3-(dibromomethyl)benzene.
  • a cyclization reagent and ammonium bicarbonate (NH4HCO3)
  • the polypeptide preparation method provided by the present invention may further include a purification step after obtaining the crude peptide from step (2).
  • Purification methods employed include but are not limited to reverse phase chromatography or ion exchange chromatography, preferably reverse phase chromatography.
  • the present invention also provides a method for preparing the targeting peptide-drug conjugate of the present invention, the method comprising:
  • step (C) performing an activated ester reaction between the targeting peptide of the present invention and the intermediate of step (A), thereby obtaining the targeting peptide-drug conjugate;
  • step (D) purifying the targeting peptide-drug conjugate in step (C).
  • the linker used in the step (A) is selected from succinic acid or dimethylglutaric acid.
  • the payload in step (A) is a small molecule compound, including but not limited to docetaxel, paclitaxel, or cabazitaxel.
  • the organic solvent in step (A) is selected from DMSO, DCM, DMF, or THF, preferably DMSO.
  • the organic base in step (A) is selected from one or a combination of DIEA, TEA, and DMAP; preferably DMAP/triethylamine.
  • the activating reagent in step (B) is selected from 2-(1H-benzotrisazo L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), benzotriazole-1-oxytri(dimethylamino)hexafluorophosphate ) phosphorus (BOP) or benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP).
  • TBTU 2-(1H-benzotrisazo L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
  • HBTU O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate
  • BOP benzotriazole
  • the therapeutic agent in step (C) is linked to the targeting peptide at a lysine residue, and wherein the targeting peptide comprises 1, 2, 3 or 4 molecules of the therapeutic agent linked thereto superior.
  • the purification method used in the step (D) includes but not limited to reverse phase chromatography or ion exchange chromatography, preferably reverse phase chromatography.
  • compositions and methods of administration are provided.
  • the present invention also provides a pharmaceutical composition, which contains (a) a safe and effective amount of the present The drug conjugate described in the second aspect of the invention; and (b) a pharmaceutically acceptable carrier.
  • the amount of the drug conjugate of the present invention in the pharmaceutical composition is usually 10 ⁇ g-100 mg/dose, preferably 100-1000 ⁇ g/dose.
  • an effective dosage is about 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg body weight of the drug conjugate of the present invention administered to an individual.
  • the drug conjugates of the present invention can be used alone or together with other therapeutic agents (eg formulated in the same pharmaceutical composition).
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent.
  • pharmaceutical carriers which do not, by themselves, induce the production of antibodies deleterious to the individual receiving the composition and which are not unduly toxic upon administration. These vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
  • Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • Pharmaceutically acceptable carriers in therapeutic compositions can contain liquids, such as water, saline, glycerol and ethanol.
  • liquids such as water, saline, glycerol and ethanol.
  • auxiliary substances in these carriers such as wetting agents or emulsifying agents, pH buffering substances and the like.
  • therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution, or suspension, in liquid carriers prior to injection can also be prepared.
  • compositions of the present invention may be administered by conventional routes including, but not limited to, intramuscular, intravenous, subcutaneous, intradermal or topical administration.
  • the subject to be prevented or treated may be an animal; especially a human.
  • various dosage forms of the pharmaceutical composition can be used according to the usage conditions.
  • freeze-dried powder injections, injections, oral preparations, etc. can be exemplified.
  • compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents, etc. (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a conventional manner depending on the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents, etc. (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a conventional manner depending on the dosage form.
  • the present invention also provides the use of the polypeptide and drug conjugates in the preparation of drugs for treating tumors or cancers.
  • the present invention also provides a method for treating tumor or cancer, the method comprising administering a therapeutically effective amount of the drug conjugate of the second aspect of the present invention to a subject in need.
  • the tumor or cancer includes solid tumors and blood tumors, and the tumor or cancer highly expresses SORT1 protein.
  • Tumors or cancers that can be treated with the drug conjugates of the present invention include, but are not limited to, acute myeloid leukemia, chronic myelogenous leukemia, multiple myelopathy, non-Hodgkin's lymphoma, colorectal cancer, breast cancer, large intestine Cancer, stomach cancer, liver cancer, leukemia, kidney tumors, lung cancer, small bowel cancer, bone cancer, prostate cancer, prostate cancer, cervical cancer, lymphoma, adrenal gland tumors, or bladder tumors.
  • the targeting peptide in the drug conjugate of the present invention is a linear peptide or a cyclic peptide containing an unnatural amino acid, which can improve the half-life of the conjugate and improve stability.
  • the conjugated drug can bypass its drug resistance pathway and avoid drug resistance during treatment.
  • the compounds of the present application can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and the methods well known to those skilled in the art In an equivalent alternative, preferred implementations include but are not limited to the examples of the present application.
  • Embodiment 1 Preparation and purification of SMTB01
  • amino acids are: Fmoc-Cys(Trt)-OH, Fmoc-Tyt(tbu)-OH, Fmoc-Ser(tbu)-OH, Fmoc-Glu(otbu)-OH, Fmoc-Lys(boc)-OH, Fmoc- Phe-OH, Fmoc-Nle-OH, Fmoc-Asp(tbu)-OH, Fmoc-Arg(pbf)-OH, Fmoc-Val-OH, Fmoc-Gly-OH, Fmoc-Ala-OH.
  • Phase A 0.1% HAc/water solution
  • Phase B 0.1% HAc/acetonitrile
  • Chromatographic column Kromasil C18 analytical column (4.6mm ⁇ 250mm, 5 ⁇ m)
  • Phase A 0.05% TFA/water
  • Phase B 0.05% TFA/acetonitrile
  • the target components with a purity greater than 90% were collected, acetonitrile was removed by rotary evaporation, and vacuum freeze-dried.
  • the affinity between the targeting peptides prepared in Example 1 and Example 2 and SORT1 protein was studied using SPR technology, and the kinetic parameters Ka(M -1 s -1 ), Kd(s -1 ), KD(M) were determined.
  • the affinity of SORT1 protein targeting peptides SMTB53 (SEQ ID NO:53) and SMTB54 (SEQ ID NO:54) targeting SORT1 protein in the prior art was determined.
  • Biacore 8K instrument was used to immobilize SORT1 protein on the CM5 chip by standard amino coupling method, and the coupling level was 1200-3000RU.
  • a series of peptides were dissolved in PBST (0.02% Tween) to make a mother solution with a concentration of 100 nM, and concentration gradient samples were prepared by 2-fold dilution with PBST.
  • PBST was used as the running buffer
  • 10mMGly (PH4.0) was used as the regeneration buffer
  • SPR was run.
  • the binding time was 60S
  • the dissociation time was 60-100S.
  • Corresponding software was used to process the data and simulate kinetics. combine.
  • NA means not determined or no binding.
  • SMTB01 a targeting peptide-drug conjugate was prepared.
  • SMTB01 was dissolved in 1% TEA DMSO, the activated ester solution was added dropwise to it, the reaction was stirred at room temperature, the reaction was detected by HPLC until it was completed, and an appropriate amount of acetic acid was added to terminate the reaction.
  • Phase A 0.1% TFA/water solution
  • Phase B 0.1% TFA/acetonitrile
  • the collected products were analyzed by Thermo U3000 HPLC.
  • Chromatographic column Kromasil C4 analytical column (4.6mm ⁇ 250mm, 5 ⁇ m)
  • Phase A 0.05% TFA/water
  • Phase B 0.05% TFA/acetonitrile
  • the target components with a purity greater than 90% were collected, acetonitrile was removed by rotary evaporation, and vacuum freeze-dried.
  • the structure of the prepared SMTB011 is as follows:
  • Paclitaxel and cabazitaxel drug conjugates were prepared in a similar manner.
  • the prepared partial conjugates are as follows:
  • SMTB051 (SMTB05-docetaxel conjugated)
  • Embodiment 5 Cytotoxicity experiment of SMTB011
  • Test cell lines human ovarian clear cell carcinoma cell ES-2, breast ductal carcinoma cell HCC-70, human ovarian carcinoma cell OV90, human breast cancer cell MDA-MB-231.
  • Tumor cell lines were cultured in an incubator at 37 °C, 5% CO2 . Passage regularly, and take cells in logarithmic growth phase for plating.
  • SMTB531 The structure of SMTB531 is as follows:
  • SMTB011 can kill tumor cells or inhibit growth of ES-2, HCC-70, OV90, MDA-MB-231 tumor cells, IC50 in some cell lines (such as HCC70, MDA-MB-231) The value is lower than that of Docetaxel, and lower than that of SMTB531 in the above four cell lines.
  • Embodiment 6 Cytotoxicity experiment of SMTB051, SMTB191, SMTB461
  • Test cell lines human ovarian clear cell carcinoma cell ES-2, breast ductal carcinoma cell HCC-70, human ovarian carcinoma cell OV90, human breast cancer cell MDA-MB-231.
  • Tumor cell lines were cultured in an incubator at 37 °C, 5% CO2 . Passage regularly, and take cells in logarithmic growth phase for plating.
  • SMTB051, SMTB191, SMTB461 have the effect of killing tumor cells or inhibiting the growth of ES-2, HCC-70, OV90, MDA-MB-231 tumor cells.
  • SMTB191 has lower IC50 values than SMTB531 in the above four cell lines, and its IC50 values are lower than Docetaxel in HCC70 and MDA-MB-231 cell lines.
  • Example 7 Pharmacodynamic study of targeting peptide-drug conjugates in animal models
  • the experiments were started after the animals arrived in the experimental environment for 7 days.
  • the animals were kept in IVC (independent ventilation system) cages (5 per cage) in an SPF animal room.
  • the animal information card for each cage indicates the number of animals in the cage, sex, strain, date of receipt, dosing regimen, experiment number, group and date of start of the experiment. All cages, bedding and drinking water were sterilized before use. Cages, feed and drinking water were changed twice a week.
  • the feeding environment and light conditions are as follows:
  • Photoperiod 12 hours of light, 12 hours of no light (turn on the light at 8 am to turn off the light at 8 pm)
  • Cage Made of polycarbonate, volume 300mm x 180mm x 150mm.
  • the bedding is corn cobs, which are changed twice a week.
  • Drinking water Experimental animals can freely drink sterilized water.
  • the animal information card for each cage should indicate the number of animals in the cage, sex, strain, date of receipt, dosing regimen, experiment number, group and date of start of the experiment.
  • MDA-MB-231 cells were cultured on the wall in vitro, and the culture conditions were L-15 medium plus 10% fetal bovine serum and 1% PS, cultured at 37°C and 0% CO2. Routine passage 2 times a week. Cells were harvested, counted, and seeded while the cells were maintained in the exponential growth phase.
  • 0.2 mL (10 ⁇ 10 6 cells + Matrigel) of MDA-MB-231 cells were inoculated on the right side of the nape of each BALB/c nude mouse.
  • the experimental animals were marked with ear tags as the only confirmation mark for subsequent experiments. Waiting for tumor growth, randomized group administration was started when the average tumor volume reached 132 mm 3 on day 27 after inoculation. See Table 9 for specific grouping information.
  • the experimental index is to investigate whether tumor growth is inhibited, delayed or cured.
  • Tumor diameters were measured twice a week with vernier calipers.
  • TGI (%) reflects tumor growth inhibition rate.
  • TGI (%) [1-(Average tumor volume at the end of administration of a certain treatment group-Average tumor volume at the beginning of administration of this treatment group)/(Average tumor volume at the end of treatment of the same type control group- The average tumor volume at the beginning of treatment in the same type control group)] ⁇ 100.
  • T/C% average tumor volume at the end of administration of a certain treatment group/average tumor volume at the end of treatment of solvent control group ⁇ 100.
  • mice in the Docetaxel 15mg/kg high-dose group began to lose weight on the 25th day after group administration, and on the 28th day after group administration, the mice in this group were given drug withdrawal (administration 4 times), and other groups were administered for 5 days. The drug was discontinued after that time. The result is shown in Figure 5.
  • SMT011, SMTB051, SMTB191, and SMTB461 showed sustained anti-tumor effect in the in vivo drug efficacy experiment of the MDA-MB-231 model, and the weight loss and neutrophil decline were significantly improved. Especially SMT011 showed significantly better tumor inhibitory effect than the positive control SMTB531. The SMT011 high-dose group continued to inhibit tumor growth up to 70 days after drug withdrawal.

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Abstract

一种特异性靶向Sortilin/SORT1蛋白的靶向肽，以及包含所述靶向肽的靶向肽-药物偶联物。该靶向肽为含非天然化学修饰的线性肽或环肽，可以改善靶向肽-药物偶联物的半衰期并提高其稳定性。靶向肽-药物偶联物能够以高亲和力特异性结合靶蛋白Sortilin/SORT1，从而高效杀伤表达Sortilin/SORT1的肿瘤，为肿瘤治疗领域提供了一种新型抗肿瘤靶向药。

Description

针对SORT1的多肽化合物及其药物偶联物 技术领域
本发明涉及生物医药技术领域，具体涉及一种针对Sort1靶点的多肽化合物及其药物偶联药物，以及它们的制备方法和用途。
背景技术
癌症，是威胁人类健康的头号杀手。世界卫生组织国际癌症研究机构(IARC)公布的数据显示，2020年全球新诊断癌症1930万例，其中我国新发癌症457万人，位居全球第一。手术虽然可以切除早期的局限性肿瘤，但对于晚期或全身性癌症来说收效甚微；而放疗和化疗会带来脱发、呕吐等副作用。
靶向药是抗肿瘤药市场的“明日之星”。靶向药的一大特点是针对特定靶点产生作用，每个病人的情况各不相同，可以选用的靶向药也各有不同，一定程度上实现对肿瘤的个体化治疗。从药品的需求趋势来看，疗效明显、副作用小是未来产品发展的主要需求方向，在这种市场需求驱动下，抗肿瘤靶向药的研发与临床应用将是抗肿瘤药物行业未来主要发展方向之一。
Sortilin(NP_002950.3)也称为神经降压素受体3(NTSR3)，是一种100kDa的1型膜糖蛋白，它由SORT1基因(Gene ID:6272)编码，该基因位于人类染色体1P13.3。SORT1基因编码5种Sortilin的变体，包括831个氨基酸(aa)的全长序列、4个截短剪接变体694、118、60、和20aa(Fatemeh Ghaemimanesh等，2021)。Sortilin属于空泡蛋白分选10蛋白(VPS10)受体家族。Vps10蛋白家族包括异源1型跨膜受体，包括Sortilin(100kDa)、SorLA、SorCS1、SorCS2和SorCS3，成熟的Sortilin是由前体prepro-Sortilin分化而来(Munck Petersen等，1999)。Sortilin不仅可定位在高尔基体膜上作为分拣蛋白，还可定位于细胞膜上作为清除受体。
Sortilin由pre-peptide前肽(插入细胞膜的蛋白质的常见信号序列；1-33aa)、pro-peptide前肽(34-77aa)、大管腔/胞外区(Vps10p结构域；78–755aa)、756–778aa的跨膜片段和779–831aa的胞内结构域组成(图1)(Fatemeh Ghaemimanesh等，2021)。信号肽酶和furin蛋白转化酶两种酶负责从pre-peptide前肽切割位点切割前肽和pro-peptide前肽以产生成熟形式的蛋白质(Munck Petersen等，1999)。
Sortilin因为其能够结合神经降压素的能力而首次被发现，而后被发现可以结合神经营养因子前体等一系列配体。越来越多的研究证实了Sortilin能够与许多分子相互作用并影响它们的细胞内分布。如在神经元和B淋巴细胞内，Sortilin调节神经营养因子的细胞内的转运。特别的是，Sortilin的前体形式 被剪切掉N端的前体部分后，变成成熟的Sortilin后被持续分泌到细胞膜，这一过程使得Sortilin暴露于细胞膜上并结合其配体，进而促进其结合配体并转导其胞内信号或者直接介导配体的细胞内化。现有的研究表明Sortilin在细胞内具有双重的功能：不仅参与物质的细胞转运的调节，而且作为受体在胞内发挥转导细胞外配体的信号的作用(Fatemeh Ghaemimanesh等，2021)。
Sortilin的过度表达及其在肿瘤学中的临床病理学意义已在各种类型的人类实体癌中陆续报道(例如：神经内分泌，Kim等，2018年；乳腺癌，Demont等，2012年；Rhost等，2018年；Roselli等，2015年；结肠直肠癌，Akil等,2011年；卵巢癌，Ghaemimanesh等，2014年；Hemmati等，2009年)和血液系统恶性肿瘤(慢性淋巴细胞白血病，Lia Farahi等，2019年)。
抗体药物偶联物(ADC)主要应用于在靶向肿瘤治疗领域。ADC药物是一类由抗体、连接子和细胞毒性药物组成的靶向生物制剂。
ADC所使用的抗体与肿瘤细胞表面抗原有极高的亲和力，对含有相同靶点的正常细胞由于抗体半衰期较长(1-3周)，因此其在体内留存的时间内会不断地杀伤正常细胞，极大的增大了药物的毒副作用，另外抗体是个比较笨拙的运输工具、在肿瘤的小胡同穿行比较困难，据估计只有0.1％的药物能到达肿瘤组织，为了保证其它99％的剧毒弹头不要带来系统毒性，连接弹头与抗体的化学链接必须足够稳定。但为了能在细胞内释放弹头又不能太稳定，这显然为药物设计带来麻烦。由于分子量的巨大差异，对于相同摩尔数的有效载荷(payload)，ADC药物所用的质量是有效载荷的300倍左右，因此副作用相对较大。
ADC还易于聚集，ADC聚集会导致修饰从而降低其与抗原的结合能力。蛋白质聚集是ADC开发的主要障碍，聚集可以发生在每个阶段以及运输和长期储存期间。聚集体具有免疫原性。此外，蛋白质聚集会导致产品损失。总体而言，任何化学或物理降解都可能导致ADC的结构改变并导致蛋白质过度聚集。还有各种其他因素会导致聚集，例如频繁的冷冻/解冻、高浓度蛋白和盐浓度、升高的温度或低pH值。此外，大多数有效载荷是疏水的，在蛋白质表面以高DAR结合有效载荷会导致蛋白质过度聚集，从而阻碍ADC的成功开发。
另外抗体偶联药物(ADC)与抗体药物一样也存在免疫原性，其免疫原性风险对病人来说，免疫原性影响了药物的安全性和有效性、甚至会因为ADA和内源蛋白交叉给病人带来致命的新疾病。
PDC(Peptide-Drug Conjugate)即多肽偶联药物，由连接子(linker)、归巢肽(homing peptide)以及具有细胞毒性的有效载荷(payload)构成，归巢肽可以特异性靶向肿瘤细胞表面过表达的蛋白受体从而传递细胞毒素诱导肿瘤细胞凋亡。相比于目前ADC药物，PDC药物具有分子量小、肿瘤穿透性强、 免疫原性低、利用固相合成法可大规模合成、生产成本较低、相对较好的药代动力学等特点，成为继小分子靶向药、单克隆抗体、ADC之后的下一代靶向抗肿瘤药。
发明内容
本发明的目的在于提供一种具有非天然氨基酸或环肽的靶向肿瘤高表达靶蛋白Sortilin的靶向肽，以及含有所述靶向肽的靶向肽-药物偶联物。
本发明的第一方面，提供了一种分离的多肽或其药学上可接受的盐，所述多肽包含修饰或未修饰的非天然氨基酸，和/或通过链内连接子(intro-chain linker)环化连接的两个半胱氨酸残基，并且所述多肽特异性靶向SORT1蛋白；
其中，所述链内连接子为或二硫键。
在另一优选例中，所述多肽为线性肽或环肽。
在另一优选例中，所述多肽为环肽，所述环肽是由多肽序列中隔开的两个半胱氨酸残基环化形成，其中，所述半胱氨酸残基分别位于多肽序列N末端起第1-6位的范围内，和多肽序列C末端起第1-6位的范围内，且所述两个半胱氨酸残基之间的间隔不少于3个氨基酸。。
在另一优选例中，所述多肽的氨基酸序列结构如式I所示：
X0-X1-X2-X3-X4-X5-Ala-X7-Val-Arg-X10-X11-X12-X13-X14-X15-X16-X17-X18    (I)
其中，X0为乙酰化(Ac)修饰或未修饰的Cys或无，或Z0-Cys，其中Z0为1-3个氨基酸残基；
X1为乙酰化(Ac)修饰或未修饰的Gly、D-Ala、Val、Asn、Arg、Gln或无；
X2为Val、1Nal、2Nal、D-2-Nal、或Tyr；
X3为Arg或hArg；
X4为Ala、Arg或无；
X5为Lys、hArg、Arg或Cys；
X7为Gly、Arg、D-Ala、Leu、Phe、或Cys；
X10为Asp或Asn
X11为Val或Nle；
X12为Phe、4-Cl-Phe、4-F-Phe、Aib或1Nal；
X13为Lys、hArg、Arg或Cys；
X14为Ser、Aib、Asn或Cys；
X15为Glu、Arg、Lys或Aib；
X16为Ser、Arg或Aib；
X17为Tyr或Aib；
X18为Cys或无，或Cys-Z1，其中Z1为1-3个氨基酸残基。
在另一优选例中，当X0为无时，X1为乙酰化(Ac)修饰的Gly、D-Ala、Val、Asn、Arg或Gln；当X0不为无时，X1为未修饰的Gly、D-Ala、Val、Asn、Arg或Gln。
在另一优选例中，所述多肽为环肽，其包含通过链内连接子(intro-chain linker)环化连接的两个半胱氨酸残基，其中，所述半胱氨酸残基分别位于式(I)所示多肽序列的X0-X5位范围内和X13-X18位范围内。
在另一优选例中，所述链内连接子为
在另一优选例中，所述多肽选自下组：
表A



在另一优选例中，所述多肽为人工合成的多肽。
本发明的第二方面，提供了一种药物偶联物，所述药物偶联物具有如下式II所示结构：
(D)n-L-P    (II)
其中，D为有效荷载；
L为接头；
P为靶向肽，所述靶向肽为如权利要求1所述的多肽；
n为≥1的正整数，较佳地，n＝1至4，最佳地，n＝1或2。
在另一优选例中，所述有效荷载选自下组：多西他赛、紫杉醇、或卡巴他赛，或其衍生物。
在另一优选例中，所述接头选自下组：琥珀酸或二甲基戊二酸。
在另一优选例中，所述接头为琥珀酸。
在另一优选例中，所述靶向肽选自表A所示的多肽。
在另一优选例中，所述药物偶联物选自下组：
(Z1)(多西他赛)n-琥珀酸-SMTB05；
(Z2)(多西他赛)n-琥珀酸-SMTB01；
(Z3)(多西他赛)n-琥珀酸-SMTB19；
(Z4)(多西他赛)n-琥珀酸-SMTB46；
(Z5)(紫杉醇)n-琥珀酸-SMTB05；
(Z6)(紫杉醇)n-琥珀酸-SMTB01；
(Z7)(紫杉醇)n-琥珀酸-SMTB19；
(Z8)(紫杉醇)n-琥珀酸-SMTB46；
(Z9)(卡巴他赛)n-琥珀酸-SMTB05；
(Z10)(卡巴他赛)n-琥珀酸-SMTB01；
(Z11)(卡巴他赛)n-琥珀酸-SMTB19；或
(Z12)(卡巴他赛)n-琥珀酸-SMTB46。
在另一优选例中，所述有效荷载通过接头连接在所述靶向肽的赖氨酸残基上。
本发明的第三方面，提供了一种药物组合物，所述药物组合物包含：(a)如权利要求2所述的药物偶联物；和(b)药学上可接受的载体。
在另一优选例中，所述药物组合物中还含有治疗肿瘤的其他药物。
本发明的第四方面，提供了如本发明第一方面所述的多肽，或如本发明第二方面所述的药物偶联物在制备用于治疗肿瘤或癌症的药物中的用途。
在另一优选例中，所述肿瘤或癌症包括实体肿瘤和血液肿瘤。
在另一优选例中，所述肿瘤或癌症高表达SORT1蛋白。
在另一优选例中，所述肿瘤或癌症包括但不限于急性髓细胞白血病、慢性粒细胞性白血病、多发性骨髓病、非霍奇金淋巴瘤、结直肠癌、乳腺癌、大肠癌、胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、宫颈癌、淋巴癌、肾上腺肿瘤、或膀胱肿瘤。
本发明的第五方面，提供了一种制备如本发明第一方面所述的多肽的方法，所述方法包括以下步骤：
(1)采用固相合成方法合成线性多肽；和
(2)可选择的，将步骤(1)所得线性多肽与环化试剂进行环化反应，从而得到环肽。
本发明的第六方面，提供了一种制备如本发明第二方面所述的药物偶联物的方法，所述方法包括以下步骤：
(A)提供一接头和有效荷载，使所述接头与有效荷载在有机溶剂中反应以获得中间体；
(B)使用活化试剂对中间体进行活化；
(C)将如权利要求1所述的多肽与步骤(A)的中间体进行活化酯反应，从而获得所述药物偶联物；
(D)对步骤(C)中的药物偶联物进行纯化。
本发明的第七方面，提供了如本发明第一方面所述的多肽在制备用于检测SORT1蛋白的检测试剂或试剂盒中的用途。
在另一优选例中，所述试剂盒用于检测样品中的SORT1蛋白。
在另一优选例中，所述样品包括血液样本、体液样本或组织样本。
在另一优选例中，所述试剂盒还包含说明书，所述说明书记载所述的试剂盒用于非侵入性地检测待测对象的SORT1蛋白表达。
本发明的第八方面，提供了一种检测样品中SORT1蛋白的方法，所述方法包括步骤：(i)将样品与如本发明第一方面所述的多肽接触；(ii)检测是否形成多肽-SORT1蛋白复合物，其中形成复合物就表示样品中存在SORT1蛋白。
在另一优选例中，所述的检测为非诊断和非治疗性的。
在另一优选例中，所述检测用于肿瘤或癌症的诊断或预后。
本发明的第九方面，提供了一种试剂盒，所述试剂盒包含如本发明第一方面所述的多肽。
在另一优选例中，所述试剂盒用于检测样品中的SORT1蛋白。
在另一优选例中，所述样品包括血液样本、体液样本或组织样本。
在另一优选例中，所述试剂盒还包含说明书，所述说明书记载所述的试剂盒用于非侵入性地检测待测对象的SORT1蛋白表达。
本发明的第十方面，提供了一种治疗肿瘤或癌症的方法，所述方法包括向有需要的受试者施用治疗有效量的本发明第二方面所述药物偶联物或本发明第三方面的药物组合物。
在另一优选例中，所述肿瘤或癌症包括实体肿瘤和血液肿瘤。
在另一优选例中，所述肿瘤或癌症高表达SORT1蛋白。
在另一优选例中，所述肿瘤或癌症包括但不限于急性髓细胞白血病、慢性粒细胞性白血病、多发性骨髓病、非霍奇金淋巴瘤、结直肠癌、乳腺癌、大肠癌、胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、宫颈癌、淋巴癌、肾上腺肿瘤、或膀胱肿瘤。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了Sortilin的结构示意图。
图2显示了SMTB01直链肽MS图。
图3显示了SMTB01的MS图。
图4显示了SMTB01-多西他赛偶联物的高分辨质谱结果。
图5显示了MDA-MB-231荷瘤雌性BALB/c nude鼠在给予受试物治疗后相对体重变化(％)。相对体重变化基于开始给药时动物体重计算得出。数据点代表各组平均体重变化百分比，误差线代表标准误差(SEM)。
图6显示了MDA-MB-231荷瘤雌性BALB/c nude鼠在给予受试物治疗后第4天的中性粒细胞含量。数据代表各组平均值，误差线代表标准误差(SEM)。
图7显示了MDA-MB-231荷瘤雌性BALB/c nude鼠在给予受试物治疗后第18天的中性粒细胞含量。数据代表各组平均值，误差线代表标准误差(SEM)。
图8显示了MDA-MB-231荷瘤雌性BALB/c nude鼠在给予受试物治疗后的肿瘤生长曲线。数据点代表各组平均肿瘤体积，误差线代表标准误差(SEM)。
具体实施方式
本发明人经过广泛而深入的的研究，通过大量的实验筛选，首次意外地研发了一类特异性靶向Sortilin的靶向肽，所述靶向肽包括含有非天然氨基酸的线性肽或环肽，并利用所述靶向肽制备了靶向肽-药物偶联物。本发明制备的靶向肽为含非天然化学修饰的线性肽或环肽，可以改善靶向肽-药物偶联物的半衰期并提高其稳定性。本发明提供的靶向肽-药物偶联物能够以高亲和力特异性结合靶蛋白Sortilin，从而高效杀伤表达Sortilin的肿瘤，为肿瘤治疗领域提供了一种新型抗肿瘤靶向药。
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
如本文所用，术语“SORT1蛋白”、“Sortilin”可互换使用，均指由SORT1基因(Gene ID:6272)编码的蛋白。
如本文所用，术语“分离的多肽”、“靶向肽”、“肽化合物”可互换使用，均指本发明第一方面的靶向SORT1蛋白的合成多肽。
如本文所用，术语“药物偶联物”、“靶向肽-药物偶联物”、“缀合化合物”可互换使用，均指本发明第一方面的多肽与一个或多个治疗剂分子(例 如多西他赛、紫杉醇或卡巴他赛等小分子化合物)通过接头(连接子)缀合形成的偶联物。
本发明的靶向肽
在本发明的一个方面，提供了一种分离的多肽，所述多肽为靶向Sortilin的靶向肽。所述多肽可以为线性肽或环肽，其包含修饰或未修饰的非天然氨基酸，和/或通过链内连接子(intro-chain linker)环化连接的两个半胱氨酸残基。如本文所用，术语“链内连接子(intro-chain linker)”是指用于环化连接多肽链内两个半胱氨酸残基的接头，在本发明的优选实施方式中，所述链内连接子(intro-chain linker)为或二硫键，优选为
本发明还包括本发明第一方面所述多肽的活性片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持结合Sortilin的功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)本发明第一方面所述多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合于此多肽序列而形成的多肽(与前导序列、分泌序列或6His等标签序列融合而形成的蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
一类优选的活性衍生物指与式I的氨基酸序列相比，有至多6个，较佳地至多3个，更佳地至多2个，最佳地1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A

本发明还提供本发明第一方面所述多肽的类似物。这些类似物与本发明第一方面所述多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
一些常用的非天然氨基酸列于下表B。
表B
修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
本发明多肽还可以以由药学上或生理学可接受的酸或碱衍生的盐形式使 用。这些盐包括(但不限于)与如下酸形成的盐：氢氯酸、氢溴酸、硫酸、柠檬酸、酒石酸、磷酸、乳酸、丙酮酸、乙酸、琥珀酸、草酸、富马酸、马来酸、草酰乙酸、甲磺酸、乙磺酸、苯磺酸或羟乙磺酸。其他盐包括：与碱金属或碱土金属(如钠、钾、钙或镁)形成的盐，以及以酯、氨基甲酸酯或其他常规的“前体药物”的形式。
本发明的靶向肽-药物偶联物
在本发明的一个方面，提供了一种包含本发明第一方面所述的靶向肽的靶向肽-药物偶联物，其具有如下式II所示的结构：
(D)n-L-P    (II)
其中，D为有效荷载；
L为接头；
P为本发明第一方面所述的靶向肽；
n为≥1的正整数，较佳地，n＝1至4，最佳地，n＝1或2。
在一些实施方式中，所述有效荷载为小分子化合物，包括但不限于多西他赛、紫杉醇、或卡巴他赛。
其中，多西他赛的化学结构如下所示：
紫杉醇的化学结构如下所示：
卡巴他赛的化学结构如下所示：
在一些实施方式中，所述接头选自琥珀酸或二甲基戊二酸，优选琥珀酸。
在一些实施方式中，本申请的靶向肽-药物偶联物组成如下表所示：
本发明的靶向肽制备方法
在本发明的另一个方面，提供了本发明第一方面的分离的多肽(靶向肽)的制备方法，具体地，所述方法包括以下步骤：
(1)采用固相合成方法合成初始多肽；
(2)将步骤(1)的产物用强酸进行裂解；加入侧链保护基清除剂，过滤后，加入适量有机溶剂沉淀后离心，用有机溶剂洗涤沉淀，经干燥后得到粗肽；
(3)可选择的，将步骤(2)所得粗肽与环化试剂进行环化反应，得到环化多肽。
在一些实施方式中，所述步骤(1)中所述固相合成所用的树脂载体选自Wang树脂或2-CTC树脂。
在一些实施方式中，所述步骤(1)包括如下子步骤：
(a)树脂溶胀—投料(首个氨基酸/缩合试剂)—测定树脂取代值—脱除氨基保护基—溶剂洗涤—监测—偶联氨基酸—监测—溶剂洗涤—脱除氨基保护基—顺序偶联剩余氨基酸—直到最后一个氨基酸脱除氨基保护基并洗涤；
(b)加入乙酸酐/DIEA进行乙酰化反应，洗涤；
其中，所述的氨基保护基是指为保护参与缩合反应的氨基而引入的化学基团。
所述的氨基保护基可选自叔丁氧羰基(Boc)、苄氧羰基(Z)或9-芴基-甲氧羰基(Fmoc)，优选9-芴基-甲氧羰基(Fmoc)。
所述步骤(a)所用溶剂选自：二甲基甲酰胺(DMF)、二氯甲烷(DCM)或N-甲基吡咯烷酮(NMP)，优选DMF或DCM。
所述步骤(a)中脱除氨基保护基的脱除剂选自浓度10-40％的哌啶/DMF(PIP)，优选浓度为20-25％的哌啶/DMF(PIP)；脱除时间为20-50min，优选脱除时间为25-35min。
所述步骤(a)中氨基酸缩合的步骤需要加入缩合试剂，所述缩合试剂选自碳二亚胺型试剂、苯并三氮唑鎓盐型试剂或1-羟基苯并三唑(HOBt)、或其组合。
所述碳二亚胺型试剂选自二环己基碳二亚胺(DCC)、二异丙基碳二亚胺(DIC)或N-二氨基丙基-N-乙基碳二亚胺(EDC)中的一种。
所述苯并三氮唑鎓盐型试剂选自2-(1H-苯并三偶氮L-1-基)-1,1,3,3-四甲基脲四氟硼酸酯(TBTU)、O-苯并三唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU)、六氟磷酸苯并三唑-1-氧基三(二甲氨基)磷(BOP)或六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷(PyBOP)中的一种。
所述偶联试剂选自二异丙基碳二亚胺(DIC)和1-羟基苯并三唑(HOBt)，或2-(1H-苯并三偶氮L-1-基)-1,1,3,3-四甲基脲四氟硼酸酯(TBTU)和1-羟基苯并三唑(HOBt)，进一步优选DIC(二异丙基碳二亚胺)和1-羟基苯并三唑(HOBt)。在一些实施方式中，步骤(a)中所述的“监测”采用茚三酮检测法监测多肽的缩合反应。
所述步骤(a)中所述的顺序偶联剩余氨基酸是指根据多肽氨基酸序列从C端向N端逐个连接氨基酸。
在一些实施方式中，步骤(2)中所述的侧链保护基清除剂选自茴香硫醚、三异丙基硅烷、苯酚、水、1,2-乙二硫醇或间甲酚中的一种、两种或几种组合。
在一些具体的实施方式中，步骤(2)中所述的侧链保护基清除剂选自三氟乙酸(TFA)：三异丙基硅烷：水＝95:2.5:2.5(V/V/V)。
在一些实施方式中，步骤(3)中所述的环化反应需加入环化试剂和碳酸氢铵(NH4HCO3)，其中环化试剂选自1，2-(二溴甲基)苯、1，3-(二溴甲 基)苯、1，4-(二溴甲基)苯或其他溴烷基取代的苯试剂中的一种或几种组合，优选1，2-(二溴甲基)苯、1，3-(二溴甲基)苯或1，4-(二溴甲基)苯，进一步优选1，3-(二溴甲基)苯。
本发明所提供的多肽制备方法从步骤(2)获得粗肽后还可以进一步包括纯化步骤。所采用的纯化方法包括但不限于反相色谱法或离子交换色谱法，优选反相色谱法。
本发明的靶向肽-药物偶联物制备方法
在另一方面，本发明还提供了用于制备本发明所述的靶向肽-药物偶联物的方法，所述的方法包括：
(A)提供一接头和有效荷载，使所述接头与有效荷载在含有机碱的有机溶剂中反应以获得中间体；
(B)使用活化试剂对中间体进行活化；
(C)将本发明的靶向肽与步骤(A)的中间体进行活化酯反应，从而获得所述靶向肽-药物偶联物；
(D)对步骤(C)中的靶向肽-药物偶联物进行纯化。
在一些实施方式中，所述步骤(A)中使用的接头选自琥珀酸或者二甲基戊二酸。
在一些实施方式中，所述步骤(A)中有效荷载为小分子化合物，包括但不限于多西他赛、紫杉醇、或卡巴他赛。
在一些实施方式中，步骤(A)中的有机溶剂选自DMSO、DCM、DMF、或THF，优选DMSO。
在一些实施方式中，步骤(A)中的有机碱选自DIEA、TEA、DMAP其中的一种或者组合；优选DMAP/三乙胺。
在一些实施方式中，步骤(B)中的活化试剂选自2-(1H-苯并三偶氮L-1-基)-1,1,3,3-四甲基脲四氟硼酸酯(TBTU)、O-苯并三唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU)、六氟磷酸苯并三唑-1-氧基三(二甲氨基)磷(BOP)或六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷(PyBOP)中的一种。
在一些实施方式中，所述步骤(C)中治疗剂在赖氨酸残基处与靶向肽相连，并且其中靶向肽包含与其连接的1、2、3或者4个治疗剂分子连接其上。
在一些实施方式中，所述步骤(D)中所采用的纯化方法包括但不限于反相色谱法或离子交换色谱法，优选反相色谱法。
药物组合物和施用方法
另一方面，本发明还提供了一种药物组合物，它含有(a)安全有效量的本 发明第二方面所述的药物偶联物；以及(b)药学上可接受的载体。本发明药物偶联物在药物组合物中的量通常为10微克-100毫克/剂，较佳地为100-1000微克/剂。
为了本发明的目的，有效的剂量为给予个体约0.01毫克/千克至50毫克/千克，较佳地0.05毫克/千克至10毫克/千克体重的本发明的药物偶联物。此外，本发明的药物偶联物可以单用，也可与其他治疗剂一起使用(如配制在同一药物组合物中)。
药物组合物还可含有药学上可接受的载体。术语“药学上可接受的载体”指用于治疗剂给药的载体。该术语指这样一些药剂载体：它们本身不诱导产生对接受该组合物的个体有害的抗体，且给药后没有过分的毒性。这些载体是本领域普通技术人员所熟知的。在Remington's Pharmaceutical Sciences(Mack Pub.Co.，N.J.1991)中可找到关于药学上可接受的赋形剂的充分讨论。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、佐剂及其组合。
治疗性组合物中药学上可接受的载体可含有液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如润湿剂或乳化剂、pH缓冲物质等。
通常，可将治疗性组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液中、液体载体的固体形式。
一旦配成本发明的组合物，可将其通过常规途径进行给药，其中包括(但并不限于)：肌内、静脉内、皮下、皮内或局部给药。待预防或治疗的对象可以是动物；尤其是人。
当本发明的药物组合物被用于实际治疗时，可根据使用情况而采用各种不同剂型的药物组合物。较佳地，可以例举的有冻干粉针剂、注射剂、口服制剂等。
这些药物组合物可根据常规方法通过混合、稀释或溶解而进行配制，并且偶尔添加合适的药物添加剂，如赋形剂、崩解剂、粘合剂、润滑剂、稀释剂、缓冲剂、等渗剂(isotonicities)、防腐剂、润湿剂、乳化剂、分散剂、稳定剂和助溶剂，而且该配制过程可根据剂型用惯常方式进行。
治疗性应用
本发明还提供了所述多肽和药物偶联物在制备用于治疗肿瘤或癌症的药物中的用途。本发明还提供了一种治疗肿瘤或癌症的方法，所述方法包括向有需要的受试者施用治疗有效量的本发明第二方面所述药物偶联物。
其中，所述肿瘤或癌症包括实体肿瘤和血液肿瘤，所述肿瘤或癌症高表达 SORT1蛋白。可使用本发明的药物偶联物进行治疗的肿瘤或癌症包括但不限于急性髓细胞白血病、慢性粒细胞性白血病、多发性骨髓病、非霍奇金淋巴瘤、结直肠癌、乳腺癌、大肠癌、胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、前列腺癌、宫颈癌、淋巴癌、肾上腺肿瘤、或膀胱肿瘤。
本发明的主要优点包括：
(1)本发明的药物偶联物中的靶向肽是含非天然氨基酸的线性肽或环肽，该所述靶向肽可以改善偶联物的半衰期并提高稳定性，此外与靶向肽偶联后的药物能够绕过其耐药途径，避免治疗过程中的耐药性。
(2)本发明的药物偶联物血浆稳定性显著提高。
(3)多肽偶联物对有效载荷耐药细胞株同样有效。
本申请的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本申请的实施例。
本申请具体实施方式的化学反应是在合适的溶剂中完成的，所述的溶剂须适合于本申请的化学变化及其所需的试剂和物料。为了获得本申请的化合物，有时需要本领域技术人员在已有实施方式的基础上对合成步骤或者反应流程进行修改或选择。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1：SMTB01的制备与纯化
按照以下方法制备SMTB01靶向肽：
SMTB01结构式：
1.1材料及试剂
Wang树脂，取代值0.31mmol/g。
氨基酸为：Fmoc-Cys(Trt)-OH，Fmoc-Tyt(tbu)-OH，Fmoc-Ser(tbu)-OH、Fmoc-Glu(otbu)-OH、Fmoc-Lys(boc)-OH、Fmoc-Phe-OH、Fmoc-Nle-OH、Fmoc-Asp(tbu)-OH、Fmoc-Arg(pbf)-OH、Fmoc-Val-OH、Fmoc-Gly-OH、Fmoc-Ala-OH。
合成试剂：HOBt，DIC，DMF，DCM，哌啶
1.2仪器
CS-BIO型多肽合成仪、Waters600半制备型高效液相色谱仪、Beckman离心机、Buchi旋蒸仪。
1.3操作步骤(以0.15mmol为例)
a.固相化学合成多肽
称取Fmoc-Cys(Trt)-Wang树脂0.5g，置于多肽合成仪反应器中，加入10ml DCM，浸泡2h，加入20％PIP/DMF溶液15ml，混合25min脱除氨基保护基，DCM洗涤树脂6次；称取三倍量的Fmoc-Tyt(tbu)-OH，DIC，HOBT加入10ml DMF溶解，加入反应器中进行反应，反应温度为室温，以茚三酮反应监测反应进程。监测为蓝紫色则表示缩合反应不完全，无色则为反应完成，然后用DCM洗涤树脂6次；随后加入20％PIP/DMF溶液15ml，混合20min脱除氨基保护基，DMF洗涤树脂6次，此时茚三酮检测为蓝紫色。
按照上述方法继续进行下一个氨基酸的偶联反应，如此循环，直至全部氨基酸偶联完成，最后一个氨基酸脱保护后洗涤，加入乙酸酐(1.2eq)、DIEA(3.6eq)的DMF溶液，反应30分钟，用DCM洗涤树脂6次。
b.裂解及沉淀
真空干燥树脂肽，称重。按照1g树脂加入10ml裂解试剂的比例配置裂解试剂，试剂配比为TFA:三异丙基硅烷:水＝95:2.5:2.5(V:V:V)，裂解试剂加入树脂中，室温搅拌反应3小时，抽滤，旋蒸除去部分TFA，向抽滤液中加入10倍体积的冰乙醚沉淀多肽，离心，沉淀用冰乙醚反复洗涤4-5次，真空干燥，称重。
c.液相中反应制备环肽
称取干燥后的粗肽0.05mmol，加入1ml水溶解，用碳酸氢铵调pH至8.0，称取0.05mmol环化试剂1，3-二(溴甲基)苯溶于乙腈中，将环化试剂的乙腈溶液加入到粗肽溶液汇总，室温下反应0.5h～1h。
d.分离纯化
用半制备型RP-HPLC，对粗肽进行纯化。
(1)纯化
色谱柱：YMC-pack ODS-A-HG C18制备柱(10mm×250mm,10μm)
流速：5ml/min
检测波长：215nm、280nm
流动相：A相：0.1％HAc/水溶液；B相：0.1％HAc/乙腈
梯度洗脱程序如下表1：
表1
(2)分析
用Thermo U3000型HPLC，对收集产物进行分析
色谱柱：Kromasil C18分析柱(4.6mm×250mm,5μm)
流速：1ml/min
检测波长：215nm
流动相：A相：0.05％TFA/水；B相：0.05％TFA/乙腈
梯度洗脱程序如下表2:
表2
收集纯度大于90％的目标组分，旋蒸除去乙腈，真空冷冻干燥。经ESI-MS进行分子量确证，M/Z＝1117.52(M+H)2+，与理论分子量相符。
实施例2：其余靶向肽的制备与纯化
按照实施例1中所述的方法，制备其余靶向肽化合物，经ESI-MS进行分子量确证：
表3



实施例3：表面等离子共振(SPR)亲和力测定
使用SPR技术研究实施例1和实施例2中制备的靶向肽与SORT1蛋白的亲和力，确定动力学参数Ka(M-1s-1)、Kd(s-1)、KD(M)。同时测定了现有技术中靶向SORT1蛋白的靶向肽SMTB53(SEQ ID NO:53)和SMTB54(SEQ ID NO:54)与SORT1蛋白的亲和力。
采用Biacore 8K仪器，通过标准的氨基偶联方式将SORT1蛋白固定在CM5芯片上，偶联水平1200～3000RU水平。采用PBST(0.02％Tween)溶解系列肽，制成浓度为100nM的母液，用PBST以2倍稀释法制备浓度梯度样品。在25℃下以PBST为运行缓冲液，10mMGly(PH4.0)为再生缓冲液，运行SPR，结合时间为60S，解离时间为60～100S，采用相应的软件对数据进行处理和动力学拟合。
所述靶向肽的亲和力测定结果如下表4所示：
表4


其中，NA表示未测定或无结合。
实施例4：制备靶向肽-药物偶联物
以SMTB01为例，制备靶向肽-药物偶联物。
4.1制备多西他赛琥珀酸酯
搅拌下将DMAP(2.5eq)加入到多西他赛的DMSO溶液中(100mg/ml)，搅拌30分钟后，将琥珀酸酐(1.2eq)的DMSO溶液5ml滴加入上述溶液中，30分钟内加完，搅拌反应12小时；反应结束后，加入等体积的碳酸氢钠溶液，搅拌30分钟，向溶液中滴加HCl，至大量白色沉淀产生，离心，沉淀用水洗涤两次后冻干。
4.2制备多西他赛琥珀酸酯活化酯
搅拌下将TBTU(2eq)加入到多西他赛琥珀酸酯的DMSO溶液中，将DIEA(6eq)加入到上述溶液中，活化30分钟。
4.3 SMTB01与多西他赛缀合的合成
SMTB01溶解在1％TEA DMSO中，向其滴加活化酯溶液，室温下搅拌反应，HPLC检测反应直至完成，加入适量醋酸终止反应。
4.4 SMTB011(SMTB01-多西他赛缀合)的纯化
(1)纯化
色谱柱：YMC-pack C4制备柱(10mm×250mm,10μm)
流速：5ml/min
检测波长：215nm、280nm
流动相：A相：0.1％TFA/水溶液；B相：0.1％TFA/乙腈
梯度洗脱程序如下表5：
表5
(2)分析
用Thermo U3000型HPLC，对收集产物进行分析。
色谱柱：Kromasil C4分析柱(4.6mm×250mm,5μm)
流速：1ml/min
检测波长：215nm
流动相：A相：0.05％TFA/水；B相：0.05％TFA/乙腈
梯度洗脱程序如下表6:
表6
收集纯度大于90％的目标组分，旋蒸除去乙腈，真空冷冻干燥。
制备得到的SMTB011的结构如下所示：
紫杉醇与卡巴他赛药物偶联物采用类似方法制备。
其他靶向肽与多西他赛、紫杉醇或卡巴他赛的偶联物均采用类似方法制备，在此不再赘述。
制备得到的部分偶联物如下所示：
SMTB051(SMTB05-多西他赛缀合)
SMTB191(SMTB19-多西他赛缀合)
SMTB461(SMTB46-多西他赛缀合)
实施例5：SMTB011的细胞毒性实验
测试样品：SMTB011
对照样品：多西他赛
实验方法：
测试细胞系：人卵巢透明细胞癌细胞ES-2，乳腺导管癌细胞HCC-70，人卵巢癌细胞OV90，人乳腺癌细胞MDA-MB-231。
将肿瘤细胞系在37℃，5％CO2的培养箱中进行培养。定期传代，取处于对数生长期的细胞用于铺板。
1.细胞铺板
(1)用台盼兰进行细胞染色并计数活细胞。
(2)将细胞浓度调整至合适浓度。
(3)在培养板中每孔加入90μL细胞悬液，在空白对照孔中加入不含细胞的培养液。
(4)将培养板在37℃，5％CO2,及100％相对湿度的培养箱中培养过夜。
2.SMTB011存储板制备
制备1000X SMTB011存储板：将SMTB011用DMSO从最高浓度梯度稀释至最低浓度。
3. 10X偶联物工作液的配制及PDC处理细胞
(1)10X偶联物工作液的配制：在V形底的96孔板中加入990μL细胞培养液，从1000X SMTB011存储板中吸取10μL SMTB011加入96孔板的细胞培养液中。在溶媒对照和空白对照中加入10μL相对应的溶剂，用排枪吹打混匀。
(2)加药：取10μL的10X SMTB011工作加入到细胞培养板中。在溶媒对照和空白对照中加入10μL相对应的溶剂。
(3)将96孔细胞板放回培养箱中培养72小时。
4.CellTiter-Glo发光法细胞活性检测
以下步骤按照Promega CellTiter-Glo发光法细胞活性检测试剂盒的说明书来进行。
(1)将CellTiter-Glo缓冲液融化并放置至室温。
(2)将CellTiter-Glo底物放置至室温。
(3)在一瓶CellTiter-Glo底物中加入CellTiter-Glo缓冲液以溶解底物，从而配制CellTiter-Glo工作液。
(4)缓慢涡旋震荡使充分溶解。
(5)取出细胞培养板放置30分钟使其平衡至室温。
(6)在每孔中加入50μL(等于每孔中细胞培养液一半体积)的CellTiter-Glo工作液。用铝箔纸包裹细胞板以避光。剩余CellTiter-Glo工作液分装至50mL离心管中，-20度避光保存，并在一个月内使用完毕。
(7)将培养板在轨道摇床上振摇2分钟以诱导细胞裂解。
(8)培养板在室温放置10分钟以稳定发光信号。
(9)在2104 EnVision读板器上检测发光信号。
同时，以现有技术中靶向SORT1蛋白的靶向肽-药物偶联物(SMTB531)作为阳性对照，按照上述方法，测定了其细胞毒性。SMTB531的结构如下所示：
根据检测结果分析数据，如下表7所示。
表7 SMTB011的细胞毒性实验结果
结果与讨论：SMTB011对ES-2，HCC-70，OV90，MDA-MB-231肿瘤细胞都有杀死肿瘤细胞或抑制生长作用，在部分细胞系(例如HCC70，MDA-MB-231)中IC50值低于Docetaxel，且在上述4种细胞系中均低于SMTB531。
实施例6：SMTB051、SMTB191、SMTB461的细胞毒性实验
测试样品：SMTB051、SMTB191、SMTB461
对照样品：多西他赛
实验方法：
测试细胞系：人卵巢透明细胞癌细胞ES-2，乳腺导管癌细胞HCC-70，人卵巢癌细胞OV90，人乳腺癌细胞MDA-MB-231。
将肿瘤细胞系在37℃，5％CO2的培养箱中进行培养。定期传代，取处于对数生长期的细胞用于铺板。
1.细胞铺板
(1)用台盼兰进行细胞染色并计数活细胞。
(2)将细胞浓度调整至合适浓度。
(3)在培养板中每孔加入90μL细胞悬液，在空白对照孔中加入不含细胞的培养液。
(4)将培养板在37℃，5％CO2,及100％相对湿度的培养箱中培养过夜。
2.SMTB051、SMTB191、SMTB461存储板制备
制备1000X SMTB051、SMTB191、SMTB461存储板：将SMTB051、SMTB191、SMTB461用DMSO从最高浓度梯度稀释至最低浓度。
3. 10X偶联物工作液的配制及PDC处理细胞
(1)10X偶联物工作液的配制：在V形底的96孔板中加入990μL细胞培养液，从1000X SMTB051、SMTB191、SMTB461存储板中分别吸取10μL SMTB051、SMTB191、SMTB461加入96孔板的细胞培养液中。在溶媒对照和空白对照中加入10μL相对应的溶剂，用排枪吹打混匀。
(2)加药：取10μL的10X SMTB051、SMTB191、SMTB461工作加入到细胞培养板中。在溶媒对照和空白对照中加入10μL相对应的溶剂。
(3)将96孔细胞板放回培养箱中培养72小时。
4.CellTiter-Glo发光法细胞活性检测
以下步骤按照Promega CellTiter-Glo发光法细胞活性检测试剂盒的说明书来进行。
(1)将CellTiter-Glo缓冲液融化并放置至室温。
(2)将CellTiter-Glo底物放置至室温。
(3)在一瓶CellTiter-Glo底物中加入CellTiter-Glo缓冲液以溶解底物，从而配制CellTiter-Glo工作液。
(4)缓慢涡旋震荡使充分溶解。
(5)取出细胞培养板放置30分钟使其平衡至室温。
(6)在每孔中加入50μL(等于每孔中细胞培养液一半体积)的CellTiter-Glo工作液。用铝箔纸包裹细胞板以避光。剩余CellTiter-Glo工作液分装至50mL离心管中，-20度避光保存，并在一个月内使用完毕。
(7)将培养板在轨道摇床上振摇2分钟以诱导细胞裂解。
(8)培养板在室温放置10分钟以稳定发光信号。
(9)在2104 EnVision读板器上检测发光信号。
同时，以前述SMTB531作为阳性对照，按照上述方法，测定了其细胞毒性。
根据检测结果分析数据，如下表8所示。
表8 SMTB051、SMTB191、SMTB461的细胞毒性实验结果

结果与讨论：SMTB051、SMTB191、SMTB461对ES-2，HCC-70，OV90,MDA-MB-231肿瘤细胞都有杀死肿瘤细胞或抑制生长作用。其中，SMTB191在上述4种细胞系中IC50值均低于SMTB531，且在HCC70和MDA-MB-231细胞系中其IC50值低于Docetaxel。
实施例7：靶向肽-药物偶联物动物模型药效研究
1.实验目的:评价受试物在BALB/c nude小鼠MDA-MB-231皮下移植瘤模型中的体内药效。
2.实验设计：如下表9所示
表9体内药效实验动物分组及给药方案

注：
a.N:每组小鼠数目
b.给药容积：根据小鼠体重10μl/g。如果体重下降超过15％，给药方案应做出相应调整。
c.如果体重下降超过15％(相比于D0)，暂时停药，待小鼠体重恢复到下降<10％后再继续给药。
3.实验材料
3.1实验动物
种属：小鼠
品系：BALB/c nude
周龄：6～8周龄
性别：雌性
体重：18～22克
数量：65只，不包括分组剩余鼠
3.2饲养环境
动物到达后在实验环境饲养7天后方开始实验。动物在SPF级动物房以IVC(独立送风系统)笼具饲养(每笼5只)。每笼动物信息卡注明笼内动物数目，性别，品系，接收日期，给药方案，实验编号，组别以及实验开始日期。所有笼具、垫料及饮水在使用前均灭菌。笼具、饲料及饮水每周更换两次。饲养环境及光照情况如下：
温度：20～26℃
湿度：40～70％
光照周期：12小时光照，12小时无光照(上午8点开灯～下午8点关灯)
笼具：以聚碳酸酯制成，体积300mm x 180mm x 150mm。垫料为玉米芯，每周更换两次。
食物：实验动物在整个实验阶段中可自由进食(照射灭菌，干颗粒状食物)。
饮水：实验动物可自由饮用灭菌水。
笼具标识：每笼动物信息卡应注明笼内动物数目，性别，品系，接收日期，给药方案，实验编号，组别以及实验开始日期。
4.实验方法与步骤
4.1细胞培养
MDA-MB-231细胞体外贴壁培养，培养条件为L-15培养基中加10％胎牛血清和1％PS，37℃，0％CO2培养。一周常规传代2次。当细胞维持在指数增长期时，收取细胞，计数，接种。
4.2肿瘤细胞接种
在每只BALB/c nude小鼠的右侧颈背部接种0.2mL(10×106个+Matrigel)MDA-MB-231细胞。接种同时将实验动物进行耳标号标记，作为后续实验的唯一确认标志。等待肿瘤生长，接种后第27天肿瘤平均体积达到132mm3时开始进行随机分组给药。具体分组后的信息见表9。
4.3实验动物日常观察
本实验方案的拟定及任何修改均通过了南通药明康德新药开发有限公司实验动物管理与使用委员会(IACUC)的评估核准。实验动物的使用及福利遵照国际实验动物评估和认可委员会(AAALAC)的规定执行。每天监测动物的健康状况及死亡情况，例行检查包括观察肿瘤生长和药物治疗对动物日常行为表现的影响如行为活动，摄食摄水量(仅目测)，体重变化(每周测量两次体重)，外观体征或其它不正常情况。
4.4肿瘤测量和实验指标
实验指标是考察肿瘤生长是否被抑制、延缓或治愈。每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。
化合物的抑瘤疗效用TGI(％)或相对肿瘤增殖率T/C(％)评价。TGI(％)，反映肿瘤生长抑制率。TGI(％)的计算：TGI(％)＝[1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积)/(同型对照组治疗结束时平均瘤体积－同型对照组开始治疗时平均瘤体积)]×100。
相对肿瘤增殖率T/C(％)的计算：T/C％＝某处理组给药结束时平均肿瘤体积/溶剂对照组治疗结束时平均肿瘤体积×100。
4.5统计分析
统计分析基于开始给药后第35天每组肿瘤体积的平均值和标准误(SEM)。三组或多组间比较用one-way ANOVA。使用GraphPad Prism软件进行所有数据分析，p<0.05认为有显著性差异。
5.实验结果
5.1体重变化情况
Docetaxel 15mg/kg高剂量组小鼠在分组给药后第25天体重开始断崖式下降，在分组给药后第28天给予该组小鼠停药(给药4次)，其它组给药5次后停药。结果如图5所示。
5.2中性粒细胞(CBC)检测结果分析
SMT011、SMTB051、SMTB191、SMTB461在给药后4天，18天测定中性粒细胞含量。结果如图6和图7所示。
5.3肿瘤体积变化情况
给予MDA-MB-231荷瘤雌性BALB/c nude鼠受试物治疗后各组肿瘤体积变化结果如图8所示。
5.4抗肿瘤药效评价指标
基于分组给药后第35天肿瘤体积计算，获得受试物对MDA-MB-231肿瘤模型的抑瘤药效评价，如下表10所示。
表10受试物对MDA-MB-231肿瘤模型的抑瘤药效评价

注：
a.平均值±SEM
b.肿瘤生长抑制评价指标根据公式T/C％＝Ttreament/TVehicle×100和TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100计算。
c.各治疗组与Vechile组间p值采用one-way ANOVA方法计算。
结果与讨论：SMT011、SMTB051、SMTB191、SMTB461在MDA-MB-231模型体内药效实验中显示持续抑瘤效果，体重下降、中性粒细胞下降情况明显改善。尤其是SMT011，显示出显著优于阳性对照SMTB531的抑瘤效果。SMT011高剂量组在停药后直到70天仍能持续抑制肿瘤生长。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种分离的多肽或其药学上可接受的盐，其特征在于，所述多肽包含修饰或未修饰的非天然氨基酸，和/或通过链内连接子(intro-chain linker)环化连接的两个半胱氨酸残基，并且所述多肽特异性靶向SORT1蛋白；
    其中，所述链内连接子为或二硫键。
  2. 如权利要求1所述的多肽，其特征在于，所述多肽为线性肽或环肽。
  3. 如权利要求1所述的多肽，其特征在于，所述多肽为环肽，所述环肽是由多肽序列中隔开的两个半胱氨酸残基环化形成，其中，所述半胱氨酸残基分别位于多肽序列N末端起第1-6位的范围内，和多肽序列C末端起第1-6位的范围内，且所述两个半胱氨酸残基之间的间隔不少于3个氨基酸。
  4. 如权利要求1所述的多肽，其特征在于，所述多肽的氨基酸序列结构如式I所示：
    X0-X1-X2-X3-X4-X5-Ala-X7-Val-Arg-X10-X11-X12-X13-X14-X15-X16-X17-X18  (I)
    其中，X0为乙酰化(Ac)修饰或未修饰的Cys或无，或Z0-Cys，其中Z0为1-3个氨基酸残基；
    X1为乙酰化(Ac)修饰或未修饰的Gly、D-Ala、Val、Asn、Arg、Gln或无；
    X2为Val、1Nal、2Nal、D-2-Nal、或Tyr；
    X3为Arg或hArg；
    X4为Ala、Arg或无；
    X5为Lys、hArg、Arg或Cys；
    X7为Gly、Arg、D-Ala、Leu、Phe、或Cys；
    X10为Asp或Asn
    X11为Val或Nle；
    X12为Phe、4-Cl-Phe、4-F-Phe、Aib或1Nal；
    X13为Lys、hArg、Arg或Cys；
    X14为Ser、Aib、Asn或Cys；
    X15为Glu、Arg、Lys或Aib；
    X16为Ser、Arg或Aib；
    X17为Tyr或Aib；
    X18为Cys或无，或Cys-Z1，其中Z1为1-3个氨基酸残基；
    并且，当X0为无时，X1为乙酰化(Ac)修饰的Gly、D-Ala、Val、Asn、Arg或Gln；当X0不为无时，X1为未修饰的Gly、D-Ala、Val、Asn、Arg或Gln。
  5. 如权利要求1所述的多肽，其特征在于，所述多肽的氨基酸序列如SEQ ID NO:1或19所示。
  6. 如权利要求1所述的多肽，其特征在于，所述多肽的氨基酸序列如SEQ ID NO:5或46所示。
  7. 一种药物偶联物，其特征在于，所述药物偶联物具有如下式II所示结构：
    (D)n-L-P  (II)
    其中，D为有效荷载；
    L为接头；
    P为靶向肽，所述靶向肽为如权利要求1-6任一项所述的多肽；
    n为≥1的正整数，较佳地，n＝1至4，最佳地，n＝1或2。
  8. 如权利要求7所述的药物偶联物，其特征在于，所述有效荷载选自下组：多西他赛、紫杉醇、或卡巴他赛，或其衍生物。
  9. 一种药物组合物，其特征在于，所述药物组合物包含：(a)如权利要求7所述的药物偶联物；和(b)药学上可接受的载体。
  10. 如权利要求1-6任一项所述的多肽，或如权利要求7所述的药物偶联物在制备用于治疗肿瘤或癌症的药物中的用途。
  11. 一种制备如权利要求1所述的多肽的方法，所述方法包括以下步骤：
    (1)采用固相合成方法合成线性多肽；和
    (2)可选择的，将步骤(1)所得线性多肽与环化试剂进行环化反应，从而得到环肽。
  12. 一种制备如权利要求7所述的药物偶联物的方法，所述方法包括以下步骤：
    (A)提供一接头和有效荷载，使所述接头与有效荷载在有机溶剂中反应以获得中间体；
    (B)使用活化试剂对中间体进行活化；
    (C)将如权利要求1-6任一项所述的多肽与步骤(A)的中间体进行活化酯反应，从而获得所述药物偶联物；
    (D)对步骤(C)中的药物偶联物进行纯化。
  13. 一种检测样品中SORT1蛋白的方法，所述方法包括步骤：(i)将样品与如权利要求1所述的多肽接触；(ii)检测是否形成多肽-SORT1蛋白复合物，其中形成复合物就表示样品中存在SORT1蛋白。
  14. 一种用于检测样品中的SORT1蛋白的试剂盒，所述试剂盒包含如权利要求1所述的多肽。
  15. 一种治疗肿瘤或癌症的方法，所述方法包括向有需要的受试者施用治疗有效量的如权利要求7所述药物偶联物或权利要求9的药物组合物。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117531021A (zh) * 2024-01-10 2024-02-09 哈尔滨吉象隆生物技术有限公司 一种刺五加苷e-靶向肽偶联物及其应用
WO2024103161A1 (en) * 2022-11-14 2024-05-23 Theratechnologies Inc. Drug conjugate compounds for stimulating the antitumor immune response

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2954934A1 (en) * 2014-06-11 2015-12-16 3B Pharmaceuticals GmbH Conjugate comprising a neurotensin receptor ligand and use thereof
CN106334194A (zh) * 2015-07-14 2017-01-18 中国科学院化学研究所 缀合物及其制备方法和用途
CN108473541A (zh) * 2015-11-24 2018-08-31 特拉斯福特普拉斯公司 通过受体介导的化学疗法治疗癌症的肽化合物和肽缀合物
WO2021108929A1 (en) * 2019-12-06 2021-06-10 Theratechnologies Inc. Sortilin binding conjugate compounds, compositions and uses thereof for treating cancer
CN113286820A (zh) * 2018-08-24 2021-08-20 特拉斯福特普拉斯公司 用于靶向分拣蛋白受体和抑制血管生成拟态的方法和化合物

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2954934A1 (en) * 2014-06-11 2015-12-16 3B Pharmaceuticals GmbH Conjugate comprising a neurotensin receptor ligand and use thereof
CN106334194A (zh) * 2015-07-14 2017-01-18 中国科学院化学研究所 缀合物及其制备方法和用途
CN108473541A (zh) * 2015-11-24 2018-08-31 特拉斯福特普拉斯公司 通过受体介导的化学疗法治疗癌症的肽化合物和肽缀合物
CN113286820A (zh) * 2018-08-24 2021-08-20 特拉斯福特普拉斯公司 用于靶向分拣蛋白受体和抑制血管生成拟态的方法和化合物
WO2021108929A1 (en) * 2019-12-06 2021-06-10 Theratechnologies Inc. Sortilin binding conjugate compounds, compositions and uses thereof for treating cancer

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DEMEULE MICHEL, CHARFI CYNDIA, CURRIE JEAN‐CHRISTOPHE, LAROCQUE ALAIN, ZGHEIB ALAIN, KOZELKO SOPHIE, BÉLIVEAU RICHARD, MARSOLAIS C: "TH1902, a new docetaxel‐peptide conjugate for the treatment of sortilin‐positive triple‐negative breast cancer", CANCER SCIENCE, JAPANESE CANCER ASSOCIATION, TOKYO, JP, vol. 112, no. 10, 1 October 2021 (2021-10-01), JP , pages 4317 - 4334, XP093087938, ISSN: 1347-9032, DOI: 10.1111/cas.15086 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024103161A1 (en) * 2022-11-14 2024-05-23 Theratechnologies Inc. Drug conjugate compounds for stimulating the antitumor immune response
CN117531021A (zh) * 2024-01-10 2024-02-09 哈尔滨吉象隆生物技术有限公司 一种刺五加苷e-靶向肽偶联物及其应用
CN117531021B (zh) * 2024-01-10 2024-04-12 哈尔滨吉象隆生物技术有限公司 一种刺五加苷e-靶向肽偶联物及其应用

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